[From the U.S. Government Printing Office, www.gpo.gov]



                \@ Id



           NOAA Technical Report NMFS I I I                                            October 1992


                            Control of Disease in Aquaculture

                            Proceedings of the Nineteenth
                            U.S. -Japan Meeting on Aquaculture
                            Ise, Mie Prefecture, Japan
                            29-30 October 1-990-


                            Ralph S. Svrjcek (editor)
























                            U.S. Department of Commerce

    SHII
     A44672
   no - 3IL11






                                                        NOAA Technical Reports NMFS
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                                             Recently Atblished NOAA Technical Reports NMFS

                     98.   Marine mammal strandings in the United States.                   the North Pacific, 1973-88, by Michael A. Perez and
                           proceedings of the second marine mammal                          Thomas R. Loughlin. December 1991, 57 p.
                           stranding workshop; Miami, Florida, 3-5 Decem-
                           ber, 1987, edited by John E. Reynolds III and Daniel       105.  Biology, oceanography, and fisheries of the North
                           K. Odell. January 1991, 157 p.                                   Pacific transition zone and subarctic frontal zone,
                                                                                            edited byJerry A. Wetherall. December 1991, 92 p.
                     99.   Marine flora and fauna of the northeastern United
                           States: erect Bryozoa, by John S. Ryland and Peter         106.  Marine ranching: proceedings of the eighteenth
                           J. Hayward, February 1991, 48 p.                                 U.S.-Japan meeting on aquaculture; Port.Ludlow,
                                                                                            Washington, 18-19 September 1989, edited by Ralph
                     100.  Marine flora and fauna of the eastern United States:             S. Svijcck. February 1992, 136 p.
                           Dicyerridda, by Robert B. Short. February 1991, 16 p.
                     101.  Larvae of nearshore fishes in oceanic waters near          107.  Field guide to the searobins (P@ionotus and Bel-
                                                                                            lator) in the western North Atlantic, by Mike Russell,
                           Oahu, Hawaii, by Thomas A. Clarke. March 1991,                   Mark Grace, and Elmerj. Gutherz. March 1992, 26 p.
                           19 P.
                     102.  Marine ranching: proceedings of the seventeenth            108.  Marine debris survey manual, by Christine A. Ribic,
                           U.S.-Japan meeting on aquaculture; Ise, Mie                      Trevor R. Dixon, and Ivan Vining. April 1992, 92 p.
                           Prefecture, Japan, 16-18 October 1988, edited by
                           Ralph S. Svdcek. May 1991, 180 p.                          109.  Seasonal climatologies and variability of eastern
                                                                                            tropical Pacific surface waters, by Paul C. Fiedler.
                     103.  Benthic macrofauna of the New York Bight,                        April 1992, 65 p.
                           1979-89, by Robert N. Reid, David J. Radosh, Ann B.
                           Frame, and Steven A. Fromm. December 1991, 50 p.           110.  The distribution of Kemp's ridley sea turtles
                                                                                            (Lepidockelys kempt) along the Texas coast: an
                     104.  Incidental catch of marine mammals by foreign                    adas, by Sharon A. Manzella and Jo A. Williams. May
                           and joint venture trawl vessels in the U.S. EEZ of               1992, 52 p.











                                                NQAA Technical Report NMFS 111


                                                Control of Disease in Aquaculture


                                                Proceedings of the Nineteenth
                                                U.S. -Japan Meeting on Aquaculture
                                                Ise, Mie Prefecture, Japan
                                                29-30 October 1990
                                                Satellite Symposium: 2 November


                                                Ralph S. Svijcek
                                                Publications Unit
                                                Northwest and Alaska Fisheries Science Centers



                                                Panel Chairmen:
                                                Conrad Mahnken, United States
                                                Seiji Sakaguchi, Japan


                                                Under the U.S. -japan Cooperative Progam.
                                                in Natural Resources (UJNR)



                                                October 1992





                                                U.S. DEPARTMENT OF COMMERCE
                                                Barbara Hackman Franklin, Secretary
                                                National Oceanic and Atmospheric Administration
                                                John A. Knauss, Under Secretary for Oceans and Atmosphere
                                                National Marine Fisheries Service
                                                William W. Foxjr., Assistant Administrator for Fisheries
     Q






               C"
               C"
               V-
                                                             LIB-RAPY
                                                           NOAA/CCEH
                                                       1990 HOBSON AVE.
                                                     CHA-3- SC 29408-2623







                                                                                   PREFACE




                                       The United States and Japanese counterpart panels on aquaculture were formed in 1969 under the
                                       United States-Japan Cooperative Program in Natural Resources (UJNR). The panels currently in-
                                       clude specialists drawn from the federal departments most concerned with aquaculture. Charged
                                       with exploring and developing bilateral cooperation, the panels have focused their efforts on ex-
                                       changing information related to aquaculture which could be of benefit to both countries.
                                            The LJNR was begun during the Third Cabinet-Level Meeting of the joint United States-Japan
                                       Committee on Trade and Economic Affairs in January 1964. In addition to aquaculture, current
                                       subjects in the program include desalination of seawater, toxic microorganisms, air pollution, energy,
                                       forage crops, national park management, my@oplasmosis, wind and seismic effects, protein resources,
                                       forestry, and several joint panels and committees in marine resources research, development, and
                                       utilization.
                                            Accomplishments include increased communication and cooperation among technical special-
                                       ists; exchanges of information, data, and research findings; annual meetings of the panels, a policy-
                                       coordinative body; administrative staff meetings; exchanges of equipment, materials, and samples;
                                       several major technical conferences; and beneficial effects on international relations.

                                                                                                                   Conrad Mahnken-United States
                                                                                                                              Seiji Sakaguchi-Japan























                                                            The National Marine Fisheries Service (NMFS) does not approve, rec-
                                                            ommend or endone any proprietary product or proprietary Material
                                                            mentioned in this publication. No reference shall be made to NMTS,
                                                            or to this publication furnished by NMFS, in any advertising or sales
                                                            promotion which would indicate or imply that NMFS approves, recom-
                                                            mends or endorses any proprietary product or proprietary material
                                                            mentioned herein, or which has as its purpose an intent to cause
                                                            directly or indirectly the advertised product to be used or purchased
                                                            because of this NMFS publication. The U.S.-Japan subseries of NOAA
                                                            Technical Reports on aquaculture is used to communicate preliminary
                                                            results, interim reports, and similar timely information. It is not subject
                                                            to formal peer review.







               CONTENTS





                         0. BERGH        Studies on diseases of cultured Atlantic halibut                                                1
                     G. H. HANSEN
                           1. HUSE
                       A.JELMERT

                       T. AKIYAMA        Scoliosis of fishes caused by tryptophan deficiency                                             7

                         T. MEYERS       Control of IHN virus in sockeye salmon culture                                                13

                       R.J. BARRIE       Identification of a conserved antigenic domain in the major                                   15
                      C. L. "ON          capsid protein of infectious pancreatic necrosis virus
                       J. C. LEONG

                           T. AOKI       Cloning of hemolysin genes of aeromonads                                                      21
                         I. HIRONO


                         T. HONJO        Harmful red tides of Heteros@gma akashiwo                                                     27

              J. L. BARTHOLOMEW          Impact of the myxosporean parasite Ceratomyxa shasta on survival                              33
                         J. L. FRYER     of migrating Columbia River Basin salmonids
                    J. S. ROHOVEC

                    M. YOSHIMIZU         Viral infections of cultured fish injapan                                                     43
                         1 KIMURA


                   K. MOMOYAMA.          Some important infectious diseases of kuruma shrimp,                                          49
                                         Penaeusjaponicus, injapan

                     J. R- WINTON        The application of molecular biology to the detection of                                      53
                                         infectious hematopoietic necrosis virus

                       K. MUROGA         Bacterial and viral diseases of marine fish during seed production                            57

                    T. YOSHINAGA         An ecological study of the, parasitic nematode Hysterothylacium haze in theJapanese           63
                                         common goby Acanthogubiusfiat4manus, in a brackish inlet

                       H. ISHIOKA        Epidemiology of marine fish diseases in the warm waters along the Kuroshio Current            69

                         P. W. RENO      Characterization of hematic neoplasia in the softshell clam Mya arenafia                      85
                 A. ILLINGWORTH
                         M. DORITY


                       M.OTOTAKE         Kinetics of bovine serum albumin administered by the immersion method                         95
                     T. NAKANISHI        in fishes acclimatized to seawater and to fresh water

                       T. NOMURA         The epidemiological study of furunculosis in salmon propagation                              101
                    M. YOSHIMIZU
                         T. KIMURA









                      I SUZUKI         Functions of hemocytes during the wound healing process in the pearl oyster                     109

                      Y MAENO          Skeletal abnormalities of fishes caused by parasitism of Myxosporea                             113
                 M. SORIMACHI


             E. READ-CONNOLE           Presence of oncogenes in fish tissues and in fish cell lines                                    119
                     C. A. SMITH
                  R M. HETRICK


                        H. SAKO        Streptococcal infection in cultured yellowtail                                                  125

                    G. MOBERG          Stress induced pathologies in fish: the cost of stress                                          131

                     A. MURATA         Control of fish disease injapan                                                                 135






                          Studies on Diseases of Cultured juvenile Atlantic Halibut


                        01VIND BERGH-, GEIR HOVIK HANSEN'                     INGVAR HUSE* and ANDERS JELMERT*
                                                               * Institute of Marine Research
                                                          Austevoll Aquarulture Research Station
                                                                 N-5392 Storebo, Norway
                                                       Department of Micro&iology and Plant Physiology
                                                            University of Bergen, Jahnebakken 5
                                                                 N-5007 Bergen, Norway




                                                                     ABSTRACT


                                  Bacterial infections by Flexibact& and Vibrio species are major causes of mortalities in
                                Atlantic halibut (Hippoglossus hippoglossus L.) larviculture. Egg surface disinfection is a
                                possible prophylactic treatment. This article summarizes and reviews several experiments
                                concerning causes of mortality of Atlantic halibut eggs and larvae.



               Introduction                                                    groups was infected with 200 @LL of a suspension of
                                                                               an accenic culture of one of the following bacteria:
               Cultivation of* Atlantic halibut (Hippoglossus                  Flexibacter sp. strains NCIMB 13128 and NCIMB
               hippoglossus L.) is presently at the verge of a commer-         13127" (National Collection of Industrial and Ma-
               cial breakthrough in Norway. However, as is the case            rine Bacteria, Aberdeen, Scotland) which were
               with all cultivated species, there are problems emerg-          isolated from two different groups of halibut eggs
               ing concerning diseases related to opportunistic                and otherwise seemingly identical (Hansen and
               microorganisms (Bergh et al. 1992; Bergh and                    Bergh et al. 1992), Vibrio strain HI-10448 (Institute of
               Jelmert 1990; Opstad and Bergh 1990; Pittman et al.             Marine Research, Bergen, Norway) and Vibrio
               1990). The purpose of this work is to summarize sev-            anguillarum NCMB 6 (National Collection of Marine
               eral experiments by studying the effects of microorganisms      Bacteria, Aberdeen, Scotland); and Vibrio fischeri
               on mortalities of halibut eggs and yolk sac larvae,             strain ATCC 7744 (American Type Culture Collec-
               possible prophylactic treatment procedures, and ef-             tion, Rockville, MD). Final total counts of bacteria in
               fects of some physical stressors.                               the wells were measured by staining with DAPI (Por-
                                                                               ter and Feig 1980) and counting in a Nikon
                                                                               epiflourescence microscope at 60OX to be in the or-
               Methods and Materials                                           der of 2-3 X 101 bacteria x mU'. One group of 60
                                                                               eggs was not infected, serving as the control. Within
                 Eggs from one female were artificially stripped and           24 hours after hatching, visible remnants of the egg-
               fertilized with sperm from two males and reared in              shell were removed along with 10 mL of the water,
               250-L upstream incubators between 6 and 7' C until              and 10 mL of sterile seawater were immediately
               further processing. ,                                           added. Mortality was recorded until Day 37 after
                 The eggs were transferred to polystyrene muldwell             hatching. For a further description of the infection
               dishes (NUNC, Roskilde, Denmark) for the disinfec-              experiment, see Bergh et al. (1992).
               tion trials and the infection experiment. Each well               For the disinfection experiment another egg group
               contained one egg and 11 mL of sterilized seawater.             was disinfected one day before hatching. The follow-
               The dishes were     "incubated in darkness between 5            ing procedure was followed. Eggs were divided into
               and 6' C for the duration of the experiment.                    four groups, which were exposed to different concen-
                 For the infection experiment, eggs were divided               trations of the iodophor disinfectant Buffodine
               into 6 groups, each containing 60 eggs. Four days               (Evans Vanodine, Preston, England): 0.5, 0.05, and
               before hatching,. each well of each of the 5 treatment          0.005%, plus one untreated control group. Applica-







           2       NOAA Technical Report NMEFS I I I


                                      77









                                                                                                                           Figure I
                                                                                                                   Halibut egg showing
                                                                                                                   large surface wounds.
                                                                                                                   This appearance is
                                                                                                                   typical of eggs in-
                                                                                                                   fected with FL-xibacter
                                                                                                                   sp. Egg diameter is
                                                                                                                   approximately 3 mm.
                                                                                                                   The photograph was
                                                           't                                                      taken from a Wild
                                                                                                                   zoom binocular mi-
                                                                                                                   croscope operated in
                                                                                                                   the dark field mode.
                                                                                                                   Photo by Guri Grung
                                                                                                                   and Vibeke Valkner.


           tion time was 10 minutes. Immediately following the                 The infection experiment revealed three different
           disinfection, the solution was carefully pipetted off             types of mortality patterns:
           the-eggs, and more sterile seawater was added. This
           procedure was repeated three times. The control                   1  The uninfected control group showed very low
           group was washed the same way as the other groups.                   mortality throughout the experiment, as only 5
           Thereafter, 60 randomly chosen eggs from each                        out of 60 larvae died.
           group were incubated in polystyrene multiwell dishes              2  The two groups infected with Flexibacter sp.
           as previously described. Within 24 hours after hatch-                showed high mortalities at hatching; out of 60 lar-
           ing, visible remnants of the eggshell were removed                   vae per group, 40 and 49 had died in the NUMB
           along with 10 mL of water, and 10 mL of sterile sea-                 13127  T and NUMB 13128 groups, respectively. At
           water was added. Mortality was recorded until Day 37                 Day 18, these groups -were terminated in order to
           after hatching. The remaining living larvae were ex-                 gain material for, re-isolation of bacteria. Eighty
           amined microscopically under a dissection                            and 93% of the larvae were dead in Groups
           microscope for developmental, disorders. A further                   NUMB 13127      T and NUMB 13128, respectively.
           description of this experiment is given by Bergh and
           jelmert (1990).                                                   3  The groups infected with V anguillarum strains or
                                                                                with V fische7i showed an intermediate mortality
           Results                                                              pattern. Only 1-4 larvae died per group at hatch-
                                                                                ing, but high mortalities occurred throughout the
                                                                                rest of the experiment. When the experiment was
           Figure I  shows a halibut egg with severe surface dam-               terminated at Day 37, mortalities had risen to
           age, an   appearance typical for eggs infected with                  95% in the V anguillarum NCMB 6 group, 78% in
           Flexibacter sp. Figure 2 demonstrates a normal egg,                  the V fischeri ATCC 7744 group, and 67% in the
           without visible damage.                                              group infected with V anguillarum 651.
             Scanning electron microscopy of infected eggs re-
           vealed the chorion to be completely dissolved over                  In the disinfection experiment, 7 out of 56 remain-
           large areas (up to 206 @Lrn in diameter), whereas the             ing larvae in the group treated with 0.5% Buff6dine
           zona radiata was severely damaged. Isolation of                   were dead when the experiment was terminated, 9
           epibiotic bacteria from this egg group revealed an                out of 59 were dead in the 0.05% Buffodine group,
           epiflora totally dominated (99% of colony-forming                 24 out of 59 were dead in the 0.005% Buffodine
           units) by Flexibacter sp. (Bergh et al. 1992)                     group, and 19 out of 58 in the untreated group.







                                                                      Bergh et al.: Studies on Diseases of Cultured Atlantic, Halibut  3















                                                                                                                          Figure 2
                                                                                                                   A normal halibut
                                                                                                                   egg. Diameter is ap-
                                                                                                                   proximately 3 mm.
                                                                                                                   The photograph is
                                                                                                                   taken from a Wild
                                                                                                                   zoom binocular mi-
                                                                                                                   croscope operated in
                                                                                                                   the dark field mode.
                                                                                                                   Photo     by    Guri
                                                                                                                   Grung and Vibeke
                                                                                                                   Valkner.


                 The groups that were disinfected with 0.5% and               been shown that bacteria are able to cause at least
               0.05% Buffodine could not be significantly distin-             minor destruction to the chorion (Hansen and
               guished for cumulative mortality at the end of the             Olafsen 1989), but we are not aware of other reports
               experiment (P>0.05, t-test with arcus sinus transfor-          showing that bacteria are able to dissolve the chorion
               mation of proportions). The two remaining groups               completely. It could not be deduced from the scan-
               were not statistically separable, but they both had sig-       ning electron micrographs that the zona radiata was
               nificantly higher mortalities than the two groups that         completely destroyed; however, evidence of severe de-
               were treated with the highest Buffodine concentra-             struction was clear. This biotype is commonly isolated
               tions (P<0.05%, t-test with arcus sinus transformation         from Atlantic halibut eggs and could be considered a
               of proportions).                                               major problem in halibut larviculture.
                 With respect to developmental disorders, 5 out of              With the two Vibyio species that were used in the
               49 living larvae treated with 0.5% Buffodine were              infection experiment, the situation is different.
               found to posess at least one kind of disorder when             These bacteria seemed to be harmless to the egg
               the experiment was terminated in the group. In the             stage, causing no significant mortality. However, the
               other groups, scores were 16 out of 50 (0.05%                  profound mortality of the infected groups through-
               Buffodine), 22 out of 36 (0.005% Buffodine) and 23             out the yolk-sac stage indicates that Vibfio-infections
               out of 39 (control). The. most common disorder was             during the yolk-sac stage may help to explain the
               the presence of black, probably necrotic, tissue in the        high mortality rates experienced so far.
               gill, heart, or frontal yolk-sac region. Work is in            . Recent observations indicate that infectious pan-
               progress to characterize the ultrastructural changes           creatic necrosis virus (IPNV), serotype Nl, is
               associated with these kinds of disorders.                      prevalent in juvenile stages of the Atlantic halibut
                                                                              (Mortensen et al. 1990). The IPNV is associated with
                                                                              high mortalities, but it is not verified whether the
               Discussion                                                     virus is the principal lethal agent. The time of infec-
                                                                              tion has so far not been shown. Also, nematodes
               We have presented initial results showing that bacte-          could be present in relatively large amounts (G.
               rial infections. may be closely involved in the                Bristow, Zoological Laboratory, University of Bergen,
               mortality of cultured halibut eggs and yolk-sac larvae.        pers. commun. 1991); these are probably introduced
               The infection experiment proves that Flexibacter sp. is        by feeding the larvae with collected zooplankton.
               a causative agent of mortality at the egg stage, at              The disinfection experiment demonstrated that
               hatching, and at the early yolk-sac stage. It has earlier      the bacterial epiflora on halibut eggs is at least partly







             4       NOAA Technical Report NKIN I I I


             responsible for many of the develo          pmental disorders              although four critical periods of high mortality were
             commonly occuring in halibut yolk-sac larvae. Sur-                         identified for aquaculture systems at temperatures
             face disinfection of eggs with iodophors should be an                      between 3 and 9' C: hatching, 10-14, 25-35, and
             adequate prophylactic treatment, as they have good                         45-60 days after hatching. The latter was probably
             pathogen/host differential of toxicity (Amend and                          due to starvation (I. Opstad and A.B. Skiftesvik,
             Pietsch 1972; Ross and Smith 1972; Amend 1974).                            Austevoll Aquaculture Research Station, pers.
             However, more work is needed to establish reliable                         commun. 1991). For the three other critical periods
             disinfection procedures.                                                   of the yolk-sac stage, evidence presented here indi-
                Mortality rates could be augmented by several sub-                      cates that effects of bacteria could not be ruled out.
             lethal factors (Rosenthal and Alderdice 1976).                               There are not yet any data available ranking the
             Sublethal physical stressors might increase egg and                        quantitative importance of the different causes of
             larvae sensitivity to infectious microorganisms, rather                    death of Atlantic halibut eggs and larvae, although
             than per se be the causative agent of death. The effects                   early-life stage mortalities are still a major factor lim-
             of sublethal stressors to early life stages of halibut                     iting the commercial success of halibut aquaculture.
             have been investigated in several studies.                                 The data presented here, however, give evidence that
                Jelmert and Naas (1990) reported that lowered 02                        pathogenic or opportunistic microorganisms are
             concentrations, exposure to H2S and exposure to                            closely involved in some typical mortality and devel-
             high light levels led to a higher prevalence of de-                        opmental disorder patterns of eggs and yolk-sac
             formed yolk-sac larvae.                                                    larvae.
                Sensitivity of halibut eggs to physical shocks was
             investigated by Holmeflord and Bolla (1988), who                           Acknowledgments
             found that eggs were most sensitive before the clo-
             sure of the blastopore. In a more extensive study,
             eggs of halibut were compared with several other ma-                       This work has been supported by the Royal Norwe-
             rine fishes, Opstad (L Opstad, Austevoll Aquaculture                       gian Council for Scientific and Industrial Research
             Research Station, pers. commun. 1991) found similar                        (NTNF), by the Norwegian Council for Fisheries Re-
             results, with the addition that eggs during the hatch-                     search (NFFR), and by Mowi a/s. We are grateful to
             ing period were highly sensitive to physical stress.                       Guri Grung and Vibeke Valkner who kindly let us use
             Effects of water flow on yolk-sac larvae were studied                      their photographs.
             by Opstad and Bergh (1990), who concluded that
             high rates of water exchange in upstream incubators                        Citations
             significantly increased mortality. Yolk-sac utilization
             was inversely related to rate of flow.
                Absence of flow, however, caused rapid increase in                      Amend, D. F.
                                                                                            1974. Comparative toxicity of two iodophors to rainbow
             the amount of bacteria in the incubators (Opstad                                  trout eggs. Trans Am. Fish. Soc. 103:73-78.
             and Bergh 1990; Skiftesvik et al. 1990), and subse-                        Amend, D. F., andj. P. Pietsch.
             quent larval mortality. Thus, these two effects must                           1972. Virucidal activity of two iodophors to salmonid
             be carefully weighed against each other. Although                                 viruses. J. Fish. Res. Board Can. 29:6165.
             normally not regarded as a sublethal stressor or caus-                     Bergh, 0., and A. Jelmert.
             ative agent of diseases, extreme light regimes have                            1990. Antibacterial treatment procedures of eggs of halibut
                                                                                               (Hippoglossus hippoglossus L.). Presented at council meeting,
             been shown to induce reduced yolk-sac utilization                                 International Council for the Exploration of the Sea. ICES-
             and increased mortality of halibut yolk-sac larvae                                CM-1990/17:39, 6 p.
             (Skiftesvik et al. 1990).                                                  Bergh, 0., G. H. Hansen, and R. E. Taxt.
                Studying development and mortality of Atlantic                              1992. Experimental infection of eggs and yolk sac larvae of
             halibut eggs and larvae at different temperatures,                                halibut, Hippoglossus hippoglossus L. J. Fish Dis. 15. (In
             Pittman et al. (1990) concluded that 3' C is near the                             press.)
                                                                                        Hansen, G. H., andj. Olafsen.
             lower limit for development of halibut eggs and lar-                           1989. Bacterial colonization of cod (Gadus morhua L.) and
             vae. At this temperature, the larvae often showed                                 halibut (Hippoglossus hippoglossus) eggs in marine
             incomplete caudal development and suffered higher                                 aquaculture. Appl. Environ. Microbiol. 55(6):1435-1446.
             mortality than those reared at 6' C. The groups                            Hansen, G. H., 0 Bergh, J. Michaelsen, and D. Knappskog.
             reared at 9' C had high egg mortality and quickly                              1992. Flexibacter ovolyticus sp. nov., a pathogen of eggs and
                                                                                               larvae of Atlantic halibut, HippoglossushippoglossusL. Int.J.
             ,developed abnormalities, such as small hearts and                                System. Bacteriol. 42(3). (In press.)
             livers, and large peritoneal and pericardial spaces,                       Holmeflord, I., and S. Bolla'
             indicating that this temperature was sublethal. No                             1988. Effects of mechanical stress on Atlantic halibut eggs at
             primary cause of larval death could be identified,                                different times after fertilization. Aquaculture68:369-371.







                                                                                           Bergh et aL: Studies on Diseases of Cultured Atlantic Halibut                      5


                     jelmert, A., and K E. Naas.                                                     Porter, K G., and Y S. Feig.
                          1990. Induced deformities of the Atlantic halibut                               1980. The use of DAPI for identifying and counting aquatic
                            (Hippoglossus hippoglossus L.) yolk sac larvae. A new experi-                   microflora. Limnol. Oceanogr. 25:943-948.
                            mental approach. Presented at council meeting, Interna-                  Rosenthal, H., and D. F. Alderdice.
                            tional Council for the Exploration of the Sea. ICES-CM-                       1976. Sublethal effects of environmental stressors, natural
                            1990/F:45, 8 p.                                                                 and pollutional on marine fish eggs and larvae. J. Fish Res.
                     Mortensen, S. H., B. Hjeltnes, 0. Rodseth, J. Krogsrud, and K. E.                      Board Can. 33:2047-2065.
                       Christie.                                                                     Ross, A. J., and C. A. Smith.
                          1990. Infectious pancreatic necrosis virus, serotype NI, iso-                   1972. Effect of two iodophors on bacterial and fungal fish
                            lated from Norwegian halibut (Hippoglossus hippoglossus),                       pathogens. J. Fish Res. Board Can. 29:1359-1361.
                            turbot (Scophthalmus maximus), and scallops (Pecten                      Skiftesvik, A. B., 1. Opstad, 0. Bergh, K Pittman, and L. Skjoldclal.
                            maximus). Bull. Eur. Assoc. Fish. Pathol. 10(2):42.                           1990. Effects of light on the development, activity and mor-
                     Opstad, L, and 0. Bergh.                                                               tality of halibut (Hippoglossus hippoglossus L.) yolk sac
                          1990. Effects of continuous flow rate on development and                          larvae. Presented at council meeting, International Coun-
                            mortality of halibut yolk sac larvae. Presented at council                      cil for the Exploration of the Sea. ICES-CM-1990/F:43, 16 p.
                            meeting, International Council for the Exploration of the
                            Sea. ICES-CM-1990/F:41, 11 p.
                     Pittman, K_ 0. Bergh, 1. Opstad, A. B. Skiftesvik, L. Skjolddal, and
                       H. Strand.
                          1990. Development of eggs and yolk sac larvae of halibut
                            (Hippoglossus hippogiossus L.). J. Appl. Ichthyol. 6:142-160.






                                 Scoliosis of Fishes Caused by Tryptophan Deficiency



                                                                    TOSHIO AKIYAMA

                                                             National Research Institute of Aquaculture
                                                                         Fisheries Agency
                                                                    Thmaki, Mie 519-04, Japan




                                                                         ABSTRACT


                                     Scoliosis, caused by dietary tryptophan (Trp) deficiency, has been reported mainly in
                                   salmonids. Neither abnormality in the vertebra per se nor microscopically visible damage
                                   in the surrounding tissues was detected in the scoliotic fish, most of which returned to
                                   normal shape within a short period of time after restoration of Trp to the diet. There-
                                   fore, serotonin (5-HT), which is one of the Trp metabolites and a known
                                   neurotransmitter, was suspected as a key substance responsible for the symptom. This
                                   paper reviews several feeding studies where purified diets containing various combina-
                                   tions of L-Trp, 5-hydroxy-L- tryptophan (5-HTP, direct precursor of 5-HT), MK486 (local
                                   inhibitor of 5-HT synthesis only in periphery) and DL-p-chlorophenylalanine (PCPA,
                                   general inhibitor of 5-HT synthesis) were fed to chum salmon fry (Oncorhynchus keta).
                                   The findings indicate that occurrence of the spinal deformity is related to depletion of 5-
                                   HT in the central nervous system. In addition, the relationship between water
                                   temperature during rearing period and incidence of the scoliosis is also discussed.



                 Introduction                                                       Characteristics of Spinal Deformity

                 Although it is known that 10 amino acids are essen-                The spinal deformity caused by Trp deficiency is
                 tial for normal fish growth, all of the quantitative               mainly scoliotic or slightly lorcloscoliotic, and neither
                 requirements for essential amino acids have been                   lordosis or kyphosis has been noted. Scoliosis occurs
                 determined only for chinook salmon, Oncorhynchus                   after 1-2 weeks of feeding a Trp-deficient diet in
                 tschawytscha, coho salmon, 0. kisutsch, carp,                      rainbow trout (Kitamura 1969; Kloppel and Post
                 Cyprinus carpio, channel catfish, Ictalurus punctatus,             1975) and chum salmon (Akiyama et al. 1986a). Most
                 Japanese eel, Anguilla japonica, and Nile tilapia,                 scoliotic fish return to normal shape after restoration
                 Oreochromis niloticus. The author conducted a series               of Trp to their diet; therefore, this symptom is revers-
                 of dietary studies to determine the amino acid re-                 ible. However, most spinal deformities caused by
                 quirements for the fry of chum salmon, 0. keta,                    nutrient deficiency are not reversed even by restoring
                 which is one of the most important species in the                  nutrients to their optimum level in the diet. For ex-
                 salmon enhancement project in Japan. In these ex-                  ample, ascorbic acid-deficient fish form thermally
                 periments, spinal deformity was observed in many                   labile underhydroxylated collagen which is dena-
                 of the fish fed a, tryptophan (Trp) -deficient diet                tured and digested at higher temperatures; this
                 (Fig.1; Akiyama et al. 1985). Si     'nce the abnormality          results in connective tissues with a low collagen con-
                 was first attributed to Tip, deficiency in sockeye                 tent and in the development of a fragile bone
                 salmon by Halver and Shanks (1960), the same de-                   structure, which finally results in irreversible symp-
                 ficiency symptom has been reported in rainbow                      toms of scurvy, such as lorclosis and scoliosis (Sato et
                 trout (Shanks et al. 1962; Kitamura 1969; Kloppel                  al. 1983; Ikeda et al. 1983). Although Kloppel and
                 and Post 1975; Poston and Rumsey 1983; Walton et                   Post (1975) observed some minor abnormalities such
                 al. 1984) and coho salmon (Ogata and Arai 1981).                   as protrusions of the fibrous matrix sheath investing
                 So far, the biochemical pathway resulting in the                   the notochord of scoliotic rainbow trout that were
                 occurrence of spinal deformity dule to Trp defi-                   caused by Trp deficiency, serious lesions of the verte-
                 ciency has not been elucidated.                                    brae and microscopically visible damage in the

                                                                                                                                                7







           8       NOAA Technical Report NAUS I I I



                    100









                  0
                  U

                  @N 50
                  0


                  Z
                  PQ








                      0

                     M



                     150




                     100




                     50                       Requirement
                                                 0.29%
                                                                                                             Figure I
                      0                                                                      Relationships between tryptophan level
                                        0.2                0.4               0.6             in diet and weight gain or incidence of
                                          TRYPTOPHAN % IN DIET                               scoliosis; average value of duplicate tanks
                                                                                             of 35 fish, each group fed for 4 weeks at
                                                                                             16.0' C (Akiyama et al. 1985).


           surrounding tissues have not been recognized in                 vous system. Many reports on spinal deformity caused
           rainbow trout (Kitamura 1969) and chum salmon                   by a metabolic disfunction or lesion in the nervous
           (Akiyama et al. 1986b).                                         system are available. In mammals, scoliosis develops
                                                                           in bipedal rats with brain-stem lesions (Tamura 1974)
                                                                           and in rabbits whose dorsal root in the spinal cord
           Construction of Hypothesis                                      was removed (MacEwen 1973). In fish, yellowtail
                                                                           parasitized by cysts of Myxobolus in the 4th ventricle
           Trptophan is not only an essential structural element           of the brain (Egusa 1985; Sakaguchi et al. 1987)
           of protein but also the precursor of nicotinamide ad-           showed scoliosis. Vertebral deformity was reported to
           enine dinucleotide (NAD) and niacin in higher                   occur in yellowtail whose brain was infected by beta-
           vertebrates (Fig.2). Therefore, attention was focused           hemolytic streptococcaI bacterium (Shiomitsu 1982;
           on the role of dietary niacin in the early studies of           Kaige et al. 1984). Spinal deformities caused by an
           Trp metabolism. Poston and Combs (1980), however,               abnormality in the peripheral nervous system are
           reported that dietary Trp is not an efficient precursor         well known in fishes exposed to pesticides such as
           of niacin in salmonids. Moreover, Poston and Rumsey             diazinon, Which develop severe spinal curvature to-
           (1983) showed that the deletion of dietary niacin did           gether with fracturing (Hirose and Kitsukawa 1976;
           not significantly increase the incidence of scoliosis in        Hirose et al. 1979). It is speculated that these symp-
           rainbow trout fed a diet containing a low level of Trp.         toms are induced by excess accumulati6n of
             It is possible that the symptom of scoliosis may be           acetylcholine in the neuromuscular junction. Thus,
                            I I       I
                          /            X;74@6




           induced by an abnormal and involuntary contraction              abnormality in the nervous system is one of the most
           or relaxation of muscle due to a defect in the net-             important factors for occurrence of spinal deformity.






                                                                              Akiyama: Scoliosis of Fishes Caused by Tryptophan Deficiency              9


                         Protein 4        Tryptophan         05- ydroxy-ltryptophan              Serotonin
                                                                      (5-M)                       (5-HT)


                                          Kynurenine                                            Melatonin


                     Xanthurenic aci
                                          3-Hydroxyanthranic acid



                                                                 Guinolinic acid


                                          C02,'H20                   D                                                            Figure 2
                                                                                                                    Map of tryptophan metabolism.


                   Among the various Trp metabolites, serotonin (5-                     neuron. From these           facts, I hypothesized that
                 HT) is known to function as a brain neurotransmitter                   scoliosis caused by Trp      deficiency would be induced
                 or modulator and is involved in the regulation of                      by an imbalance of muscular tension due to a de-
                 sleep, body heat, sexual behavior, appetite, pain rec-                 creased 5-HT level in the nervous system.
                 ognition, secretion of growth hormone                       and
                 prolactin, besides classical functions such as the          con-
                 traction of smooth muscle of blood vessels, the                        5-HT Involvement in the Occurrence
                 uterus, and the digestive tract. In addition,              it is       of Scoliosis
                 known that torticollis and abnormal posture can be
                 induced by the destruction of rat midbrain in which                    In the first experiment which tested the involvement
                 both serotoninergic and dopaminergic neurons are                       of 5-HT in the occurrence of scoliosis an oral admin-
                 located (Tanaka and Kimura'1984), and that 5-HT                        istration of 5-HT to Trp-deficient chum salmon fry
                 modulates.the central pattern generator for locomo-                    decreased the incidence of scoliosis, but did not com-
                 tion in the spinal cords of the lampreys Ichthyomyzon                  pletely inhibit its occurrence (Akiyama et al. 1986a).
                 unicuspis and Petramyzon ma-yinus (Harris-Warrich et                   Therefore, we fed fry a Trp-deficient diet supple-
                 al. 1985). These reports suggest that muscular func-                   mented with 5-hydroxy@tryptophan (5-HTP, 100-130
                 tion can be partly controlled by a serotoninergic                      mg/100 g diet), which is a direct precursor of 5-HT


                          (Z)             Trp 0.051
                           60             Trp 0.05%    + Kynurenine
                                 0-0 Trp 0.05Z         + 5-HTP
                                 0-0 Trp 0.29Z
                                                                                                                                      Figure 3
                                          Trp 0.29Z    + PCPA
                                                                                                                             Effects of oral administra-
                                                                                                                             tion of tryptophan(Trp)
                           40
                                                                                                                             metabolites to Trp-defi-
                     Cn
                                                                                                                             cient or sufficient chum
                                                                                                                             salmon fry. Administra-
                     U                                                                                                       tion of 5-hydroxy-L-
                     0                                                                                                                    1 HTP) to Trp-
                     PA                                                                                                      tryptophan(5-
                           20
                                                                                                                             deficient fish completely
                                                                                                                             prevented scoliosis, where-
                                                                                                                             as kynurenine failed to in-
                                                                                                                             hibit the occurrence.
                                                                                                                             DL-p-Chlorophenylala-
                            0                                                                                                nine (PCPA) developed
                                                                    2                                       4                scoliosis even in the fish
                                                       FEEDING PERIOD (WEEK)                                                 fed Trp-sufficient diet
                                                                                                                             (Akiyama et al. 1986b).






            io        NOAA Technical Report NIM I I I


                                                                                                                            Figure 4
                                                                                                                   Functions of adminis-
                                                                                                                   trated drugs on serotonin
                                                                                                                   (5-HT) pathway. DL-p-
                            Tryptophan hydroxylase               Arowatic L-amino acid                             Chlorophenylalanine
                                                                      decarboxylase                                (PCPA) is an inhibitor of
                                                                                                                   trypto'phan-hydroxylase
                         PCPA                                                             NK486                    (TrpOHase) and inhibits
                                                                                                                   a biosynthesis of 5-hy-
                                                                                                                   droxy-L-tryptophan
                                                                                                                   (5-HTP) from Trp. L-2-
                              COOH              OH,-t:::)@@ COOH                                                   hydrazino-alfa-methyl-
                        "@Y                                 .1    NH                 OH               N            b e t a- (3,4- d i h y d r o xy-
                            n2                             N        2                           N      H2
                      N                                                                                            phenylpropionic acid);
                      H                                    H                                    H
                Tryptophan                     5-Hydroxy-L-tryptophan                                              (MK486) is an inhibitor
                                                                                        Serotonin                  of L-amino acid decar-
                   (Trp)                               (5-HTP)                             (5-HT)                  boxylase only in periphery
                                                                                                                   and consequently induces
                                                                                                                   a conversion of exogenous
                                                                                                                   5-HTP to 5-HT in central
                                                                                                                   nervous system.



            and can easily pass through a blood-brain barrier in                5-HT and 5-hydroxyindoleacetic acid than did adding
            contrast to 5-HT (Akiyama et al. 1986b, 1989). The                  5-HTP alone (Akiyama and Murai, unpubl. data).
            treatment completely prevented the occurrence of                    The experiment suggests that the deficiency of 5-HT
            scoliosis and increased the brain 5-HT level in Trp-                in the central nervous system was related to the oc-
            deficient fish (Fig.3). Kynurenine, a precursor of                  currence of scoliosis.
            niacin, NAD, and xanthurenic acid was fed at 120 mg
            (as L-kynurenine)/100 g diet to Trp-deficient fish,
            but failed to prevent scoliosis. Moreover, both                     Effect of Temperature on the Occurrence
            scoliosis and decreased brain 5-HT levels were ob-                  of Scoliosis
            served in fish fed a Trp-sufficient diet supplemented
            with DL-p-chlorophenylalanine (PCPA) (Akiyama et                    Scoliosis caused by Trp deficiency has been reported
            al. 1986a; 1986b), which is an inhibitor of trypto-                 only in salmonids such as sockeye salmon, rainbow
            phan-hydroxylase (TrpOHase) and a potent depletor                   trout, coho salmon and chum salmon, although it
            of both brain and peripheral stores of 5-HT (Fig. 4).               has also been studied in chinook salmon (Halver et
            TrpOHase is a rate-limiting enzyme on the 5-HT                      al. 1957), channel catfish (Dupree and Halver 1970),
            pathway. These findings indicated a relationship be-                eel (Arai et al. 1972), carp (Nose et al. 1974; Nose
            tween the occurrence of scoliosis and 5-HT levels.                  1979), red sea bream, Pagrus major (Yone 1976) and
            The reduced ability of orally administrated 5-HT to                 tilapia, Tilapia zilhi (Mazid et al. 1978). At first I con-
            inhibit the development of scoliosis compared with                  sidered. scoliosis to be a characteristic Trp deficiency
            complete prevention with the use of 5-HTP suggested                 symptom of all salmonids except chinook salmon.
            involvement of the serotoninergic neuron in the cen-                Arai et al. (1986), however, reported development of
            tral nervous system. Therefore, the author prepared                 scoliosis in Trp-deficient Ayu fish (Plecoglossidae)
            Trp-deficient diets (0.05%) containing various com-                 reared at 16' C, although the incidence was low. I
            binations of 5-HTP (10 or 50 mg/100 g diet) with or                 also found one scoliotic fish when a Trp-deficient
            without        L-2-hydrazino-alpha-methyl-beta-(3,4-                diet was fed to yellowtail at 20' C (Akiyama, unpubl.
            dihydroxyphenylpropionic acid) (MK486, I or 5 mg/                   data), even though the brain 'was not infected by
            100 g diet). MK486 functions hs an inhibitor of aro-                Streptococcus and not parasitized by cysts of Myxobolus.
            matic L-amino acid decarboxylase only in periphery                  These facts suggest that scoliosis due to Trp defi-
            and inhibits 5-HT biosynthesis from 5-HTP. Thus, it                 ciency is unlikely to be peculiar to salmonids.
            consequently functions to promote the conversion of                 Because salmonids are coldwater fish, and because
            exogenous 5-HTP to 5-HT in the central nervous sys-                 most of the fishes in which scoliosis was not observed
            tem. Feeding a Trp-deficient diet supplemented with                 as a symptom of Trp deficiency are warmwater fish
            both 5-HTP and MK486 resulted in a significantly                    (except chinook salmon), I focused my attention on
            lower incidence of scoliosis and higher levels of brain             the influence of environmental temperature. It is







                                                                                     Akiyaxna: Scoliosis of Fishes Caused by Tryptophan Deficiency                   I I


                    likely that among the fishes developing scoliosis, inci-                     HalverjE., and W.E. Shanks.
                    dences of scoliosis decrease as the optimum                                       1960. Nutrition of salmonoid fishes. VIII. Indispensable
                    temperature for each species rises. Moreover, spinal                                amino acids for sockeye salmon. J. Nutr. 72:340-346.
                    deformity has not been observed to be an external                            HalverjE., D.C. Delong, and E.T. Mertz.
                                                                                                      1957. Nutrition of salmonoid fishes. V. Classification of es-
                    symptom of dietary Trp deficiency in mammals and                                    sential amino acids for chinook salmon. J. Nutr. 63:95-
                    birds, which are warm-blooded animals. In fact, the                                 105.
                    author presumed that the occurrence and incidence                            Harris-Warrick, R.M.,J.C. McPhee, andj.A. Filler.
                    of scoliosis might be influenced by rearing tempera-                              1985. Distribution of serotonergic neurons and processes in
                    tures, and therefore fed the Trp-deficient diet to                                  the lamprey spinal cord. Neuroscience 14(4):Il      127-1140.
                    chum salmon fry at three different temperatures: 10,                         Hirose, K., and M. Kitsukawa.
                                                                                                      1976. Acute toxicity of agricultural chemicals to seawater te-
                    16,* and 200 C. The experiment showed that as the                                   leosts, with special respect to TLM and the vertebral
                    rearing temperature was lowered, the incidence of                                   abnormality. Bull. Tokai Reg. Fish. Res. Lab. 84:11-20. (In
                    scoliotic fish increased and brain 5-HT levels de-                                  Japanese; English abstr.)
                    creased (Akiyama and Murai, unpubl. data). It is still                       Hirose, K_ M. Kitsukawa, and A. Ishikawa.
                    unknown why the brain 5-HT level in Trp-deficient                                 1979. Effects of water temperature on median lethal concen-
                    fish varied under different temperature conditions.                                 trations (LC50) of a few pesticides to seawater teleosts. Bull.
                                                                                                        Tokai Reg. Fish. Res. Lab. 98:45-53. (In Japanese; English
                                                                                                        abstr.)
                                                                                                 Ikeda, S., M. Sato, and R. Yoshinaka.
                    Conclusion                                                                        1983. Role of vitamin C in collagen formation of fish. Vita-
                                                                                                        mins Uapan) 57(8):433-449. (Injapanese, English abstr.)
                    All of these findings described above indicate that                          Kaige, N., T Miyazaki., and S. Kubota.
                    scoliosis caused by Trp deficiency is related to the                              1984. The pathogen and the histopathology of vertebral de-
                                                                                                        formity in cultured yellowtail. Fish. Pathol. 19(3):173-179
                    level of 5-HT in the central nervous system. In fishes,                             (Injapanese, English abstr.)
                    hereafter, the central nervous system, especially the                        Kitamura, S.
                    5-HT neuron, should be considered as one of the                                   1969. Summary on the hypovitaminosis C of rainbow trout,
                    important factors in an occurrence of idiopathic spi-                               Salmo gairdneri. Fish Pathol. 3:73-85 (In Japanese.)
                    nal deformity.                                                               KJoppel, T.M., and G. Post.
                                                                                                      1975. Histological alterations in tryptophan-deficient rain-
                                                                                                        bow trout. J. Nutr. 105:861-866.
                                                                                                 MacEwen, G.D.
                    Citations                                                                         1973.. Experimental scoliosis. Clin. Ortfiop. 93:69-74.
                                                                                                 Mazid, M.A., Y. Tanaka, T. Katayama, K.L. Simpson, and C.O.
                    Akiyama, T., S. Arai, T. Murai, and T. Nose.                                   Chichester.
                        .1985. Tryptophan requirement of chum salmon fry. Bull.                       1978. Metabolism of amino acids in aquatic animals-Ill Indis-
                           Jpn. Soc. Sci. Fish. 51(6):1005-1008.                                        pensable amino acids for Tilapia zillii. Bull. Jpn. Soc. Sci.
                    Akiyama, T., T. Murai, and T. Nose.                                                 Fish. 44(7):739-742.
                        1986a. Oral administration of serotonin against spinal defor-            Nose, T.
                           mity of chum salmon fry induced by tryptophan                              1979. Summary report on the requiremenis of essential
                           deficiency. Bull. Jpn. Soc. Sci. Fish. 52(7):1249-1254.                      amino acids for carp. In Finfish nutrition and fishfeed
                    Akiyama, T., T. Murai, K. Mori..                                                    technology, Vol. I (J. E. Halver and K. Tiews, eds.), p. 146-
                        1986b. Role of tryptophan metabolites in inhibition of spi-                     156. Heenemann Verlagsgesellschaft GmbH, Berlin.
                           nal deformity of chum salmon fry caused by Tryptophan                 Nose, T., S. Arai, D. Lee, and Y. Hashimoto.
                           deficiency. Bull. Jpn. Soc. Sci. Fish. 52(7):1255-1259.                    1974. A note on amino acids essential for growth of young
                    Akiyama, T., H. Kabuto, M. Hiramatsu, T. Murai, and K. Mori.                        carp. Bull.jpn. Soc. Sci. Fish. 40(9):903-908.
                        1989. Effect of dietary 5-hydroxy-L-tryptophan for preven-               Ogata, H., and S. Arai.
                           tion of scoliosis in tryptophan-deficient chum salmon                      1981. Essential amino acid requirements for coho salmon 11 .
                           fry. Nippon Suisan Gakkaishi 55(1):99-104.                                   Leucine,     isoleucine,     tryptophan      and      histidine
                    Arai, S., T. Nose, and Y. Hashimoto.                                                requirements. Abstract of the Spring Meeting of Jpn. Soc.
                        1972. Amino acids essential for the growth of eels, Anguilla                    Sci. Fish., p.44. (Injapanese).
                           anguillaa-ndA.japonica. Bull. jpn. Soc. Sci. Fish. 38(7):753-         Poston, H.A., and G.F. Combs, Jr.
                           759.                                                                       1980. Nutritional implications of tryptophan catabolizing en-
                    Arai, S., A. Nakazawa, and Y Deguchi.                                               zymes in several species of trout and salmon. Proc. Soc.
                        1986. Effects of each essential and non-essential amino acids                   Exp. Biol. Med. 163:452-454.
                           on free amino acids in whole body of ayu fish. Abstr. of the          Poston, H.A., and G.L. Rumsey.
                           Autumn Meeting ofJpn. Soc. Sci. Fish., p.147. (Injapanese.)                1983. Factors affecting dietary requirement and deficiency
                    Dupree, H.K., andj.E. Halver.
                        1970. Amino acids essential for the growth of channel cat-                      signs of L-tryptophan in rainbow trout. J. Nutr. 113:2568-
                           fish, Ictaluruspunctatus. Trans. Am. Fish. Soc. 99(1):90-@92.                2577.
                    Egusa, S.                                 4                                  Sakaguchi, S., T. Hara, T. Matsusato, T. Shibahara, Y. Yamagata, H.
                        1985. Myxobolus buii sp. n. (Myxosporea: Bivaivulida) parasitic            Kawai, and Y. Maeno.
                           in the brain of Seriola quinqueradiata TEMMINCK et                         1987. Scoliosis of cultured yellowtail caused by parasitic
                           SCHLEGEL. Fish Pathol. 19 (4):239-244. (In Japanese; En-                     Myxobolus buri. Bull. Natl. Res. Inst. Aquaculture 12:79-86.
                           glish abstr.)                                                                (Injapanese; English abstr.)







             12        NOAA Technical Report NNEM I I I


             Sato, M., T. Kondo, R. Yoshinaka, and S. Ikeda.                             Tanaka, C., and M. Kimura.
                  1983. Effect of water temperature on the skeletal deformity                 1984. Serotonin(5-hydroxytryptamin). In Neuro transmitters
                    in ascorbic acid-deficient rainbow trout. Bull. Jpn. Soc.                    (G. Takagaki and T. Nagatsu, eds.), p.156-191. Kodansha,
                    Sci. Fish. 49(3):443-446.                                                    Tokyo. (Injapanese.)
             Shanks, W.E., G.D. Gahimer, andj.E. Halver.                                 Walton, MJ., R.M. Coloso, C.B. CoweyJ.W. Adron, and D. Knox.
                  1962. The indispensable amino acids for rainbow trout. Prog.                1984. The effects of dietary tryptophan levels on growth and
                    Fish-Cult. 24:68-73.                                                         metabolism of rainbow trout (Salmo gairdnen). Br. J. Nutr.
             Shiomitsu, K.                                                                       51:279-287.
                  1982. Isolation of Streptococrus sp. from the brain of cultured        Yone, Y.
                    yellowtail. Fish Pathol. 17(l):27-31. (In Japanese; English               1976. Nutritional studies of red sea bream. Rep. Fish. Res.
                    abstr.)                                                                      Lab. Kyushu Univ., 3:87-101.
             Tamura, T.
                  1974. An experimental study on scoliosis in bipedal rats with
                    brainstern lesion. J. Jpn. Orthop. Assoc. 48(3):137-158.
                     Injapanese. English abstr.)






                            Control of IHN Virus in Alaskan Sockeye Salmon                                              Culture



                                                                  THEODORE MEYERS

                                                               Alaska Department ofFish and Game
                                                                        ERE.D. Division
                                                                         RO. Box 25526
                                                                     Juneau, AK 99802-5526




                                    A recent review of trends in the prevalence and risk management of Infectious Hemato-
                                  poietic Necrosis Virus (IHNV) in Alaskan sockeye salmon Oncorhynchus net*a culture has
                                  been reported by Meyers et al. (1990). The reader is referred to this report for further
                                  details, discussion, and references.                    0
                                    Prior to 1980, IHNV prevented successful culture of sockeye salmon in Alaska. This led
                                  the Fisheries Rehabilitation, Enhancement, and Development Division (FRED) of the
                                  Alaska Department of Fish and Game to develop a policy to control the negative effects
                                  of the virus in sockeye salmon culture. This policy included procedures for the collection
                                  and incubation of eggs and for the rearing of fry that were based upon the known and
                                  suspected biological characteristics of the virus-host relationship. Many of these criteria
                                  are common sense approaches such as: use of a virus-free water supply; disinfection of
                                  utensils, facilities, and external surfaces of broodfish; separate fertilization of eggs from
                                  each female using 1 or 2 males; separate water hardening of each family of eggs in a 100
                                  ppm iodine disinfectant for I hour; compartmentalization of families into Kitoi Box
                                  incubators or into stacks of Nopad trays at egg densities of 250,000-300,000 (80-100
                                  females), or into modified Bams Boxes used at one facility that are each loaded with
                                  500,000 eggs; physical isolation of each sockeye stock and isolation of all sockeye stocks
                                  from any nonsockeye species; and release of fry unfed or after short-term rearing (4-6
                                  weeks) with pooling of fry in raceways or start tanks according to the date of eggtake.
                                  These criteria nearly eliminate opportunities for horizontal virus transmission from the
                                  parents to offspring or from the water supply. They also further reduce the rare occur-
                                  rence of vertical transmission of the virus within the egg and allow for
                                  compartmentalization of eggs and fry so that the occasional incubators or raceways of
                                  fish developing IHN can be destroyed and the virus contained to protect the remaining
                                  fish inventory. This "sockeye culture policy" has allowed Alaskan hatcheries a great mea-
                                  sure of success in controlling IHNV @t several different facilities around the state for
                                  nearly 10 years. Based on these guidelines, an average of 2-3 million. sockeye salmon eggs
                                  can be spawned in a day and totals of 20-36 million eggs may be taken at certain
                                  facilities. Although vertical transmission of the virus generally occurs in fry almost every
                                  year at certain facilities, total losses are minimized to between I and 3% of the statewide
                                  fry production. In 1990 only 1% of the sockeye fry were destroyed owing to IHN of 68
                                  million healthy fish that were released. Production data from various Alaskan sockeye
                                  salmon hatcheries suggest that vertical transmission of IHNV is more dependent upon
                                  the proportion of high virus-titered fernale fish rather than total virus prevalence. Also,
                                  as one would. expect, the risk of vertical virus transmission increases with increasing
                                  numbers of eggs taken from females of a high-titered stock. Hence, IHN outbreaks are
                                  more common at the larger eggtake facilities that have greater prevalences of high-
                                  titered broodfish.
                                    During the past 14 years, yearly monitoring of sockeye salmon stocks by.FRED has
                                  resulted in a data base summarizing IHNV occurrence in over 96 wild and hatchery
                                  sockeye salmon stocks in Alaska. Yearly prevalence of IHNV ranges from 0 to 100% in
                                  both ripe and postspawned females with as many as 92% within a stock having high titers
                                  (@tlO% Repeated yearly sampling has shown that all anadromous sockeye salmon tested
                                  in Alaska are positive for IHNV. The data base has been useful for examining general
                                  trends and has shown some differences from previously reported IHNV-sockeye salmon
                                  relationships. For example, no significant differences in mean IHNV prevalence were


                                                                                                                                                13







           14       NOAA Technical Report NXM I I I


                             found in ovarian fluids from postspawned female sockeye salmon vs. those from ripe
                             females. Furthermore there were no significant differences between geometric mean
                             virus titers of postspawned vs. ripe female fish, but postspawners did have a significantly
                             greater mean proportion of high-titered fish. The log value of 10' was selected as the
                             breakpoint for high virus titers owing to the tendency for bimodality of lHNV titers to
                             occur at this level in most stocks of Alaskan sockeye salmon. The significance of this
                             phenomenon needs further investigation. As found by other investigators, the mean virus
                             prevalence in male fish from 27 stocks of sockeye salmon was significantly less (9%) than
                             in female fish (40.1
                                This data base is a useful tool for examining general trends for IHNV within a geo-
                             graphic area or statewide. However, these    'trends may not always be true for certain
                             individual sockeye salmon stocks that may be unique due to genetic reasons, the strain
                             of the indigenous virus, or environmental factors affecting natural virus exposure and
                             transmission.



            Citation


            Meyers, T.R.J.B. ThomasJE. Follett, and R.R. Saft.
                1990. Infectious hernatopoietic necrosis virus: trends in
                 pievalence and the risk management approach in Alaskan
                 sockeye salmon culture. J. Aquat. Anim. Health 2:85-98.






                              Identification of a Conserved Antigenic Domain in the
                        Major Capsid Protein of Infectious Pancreatic Necrosis Virus


                                                   RJ. BARRIE, C.L. "ON, andj.C. LEONG*
                                                                   Department of Microbiology
                                                                     Oregon State University
                                                                  Corvallis, Oregon 97331-3804




                                                                         ABSTRACT

                                    The gene for the major capsid protein, VP2, of the Sp serotype of infectious pancreatic
                                  necrosis virus (IPW) was cloned and expressed in Escherichia coli. Nonoverlapping frag-
                                  ments of the VP2 gene were recloned in trpE fusion vectors of the pATH series and the
                                  expressed fusion proteins were characterized for reactivity with antisera to three different
                                  serotypes of IPNV. One clone, pBI0, which contained an insert encoding amino acids 99
                                  to 206 of the VP2 protein, produced a fusion protein recognized by antisera for all three
                                  serotypes. In contrast, the pA43 clone, which contained an adjacent region on the VP2
                                  gene encoding for amino acids 207 to 315, produced a fusion protein that was only
                                  recognized by homologous antisera in Western immunoblots. A comparison of the de-
                                  rived amino acid sequence for each clone with that reported for two other IPNV clones
                                  indicates that the pB 10 region is conserved and the pA43 region is very heterogeneous.



                Introduction                                                       recognized only three major serotypes characterized
                                                                                   by the following virus isolates: VR299, a North Ameri-
                Infectious pancreatic necrosis virus (IPNV) is a                   can strain; Sp, a European strain which is pathogenic
                birnavirus that causes one of the most serious dis-                for trout; and AB, a European strain which is
                eases in trout and salmon farms in North America,                  nonpathogenic for trout (Wolf 1988). In our paper
                Europe, and Asia. It can also kill a number of                     we review recent efforts to unravel the mechanisms
                nonsalmonid fish species including striped bass                    that biologically distinguish the different IPNV iso-
                (Morone saxatilis), turbot, menhaden, and eels (Wolf               lates by characterizing the immunoreactive regions of
                1988), and it has been isolated from a variety of ma-              the major capsid protein of the Sp serotype of 1PNV.
                rine fish and molluscs. The ubiquitous nature of this
                birnavirus and its ability to infect such a wide variety
                of hosts make this virus'important for scientific study.           Methods
                Most IPN-V isolates are closely related antigenically
                and yet, they exhibit marked differences in host-                  The viral genome is         composed of two double-
                range in vivo and in vitro, pathogenicity, and                     stranded RNA segments, A and B. The B segment
                temperature of replication.                                        encodes the viral RNA polymerase, VPI. The A seg-
                  The most extensive study of the antigenic relation-              ment encodes the virion proteins, VP2 and VP3. The
                ships of the aquatic birnaviruses compared 175 virus               major capsid protein, VP2, is responsible for the in-
                isolates from 44 fish and shellfish species from 11                cluction of neutralizing antibodies (Lipipun et al.
                countries by reciprocal plaque reduction tests using               1989 ). In addition, there is a nonstructural protein,
                polyclonal sera (Hill and Way 1983). From these re-                NS, which is an autocatalytic protease responsible for
                sults, it was proposed that there are 2 major                      cleavage of the polyprotein, VP2-NS-VP3, encoded by
                serogroups: I containing 9 serotypes which includes                the viral genome A segment (Duncan et al. 1987;
                171 isolates from fish, and the other containing I                 Manning et al. 1990).
                serotype which includes those viruses isolated from
                molluscs. These virus isolates also contain common
                immunoreactive determinant(s). Other studies have                  *Send correspondence to this author

                                                                                                                                               15







                16           NOAA Technical Report NNM I I I



                                                                                                                       smal
                                                                                             TrPE p                    Bamm
                                                      KW                                                               XbW
                                                                                                                       son
                                                NS                                                                     Pal
                    Pst      VP2                                             Arm R                         TV          HIndill
                    A
                   Lao P          pUC19/A+SAK                                             PATH 1, 2, or 3
                                       4.1 Kb                                                  3.78 Kb








                                          JV Pstvpni                                                 BaMHYCIP

                             ATG-1
                                        Ndel



                                          1,5 Kb


                           Pat
                             6MEMMIMTEI                             SaU3A fregmeM
                             225 105 321     227    183 235
                                                  27    81   37



                                   GEL ISOLATION OF
                                   EACH FRAGMENT
                                                                   I   T4 DNA Ugese                                                                             Figure I
                                                      COLONYIMMUNOBLOT                                                                     Construction of trp&VP2 gene fusions.
                                                                                                                                           The 1.5 Kb cloned insert containing
                                  T@pE P                                                                                                   the IPNV-Sp VP2 gene was restricted
                                              TtPE                        Sau3A                                                            with Sau3Al; the fragments were puri-
                                                                          froWnerd                                                         fied and subcloned into the trpE
                                                                                                                                           fusion expression vectors, pATH 1, 2,
                                                                                                                                           or 3. Recombinants containing frag-
                                                                                                                                           ments in the correct orientation and
                                                                                                                                           .in the appropriate reading frame were
                                                                                                                                           selected by colony immunoblot as pre-
                                                                                                                                           viously described (Gilmore et al. 1988).



                    Since the entire VP2 gene has been expressed in                                           Results and Discussion
                Escheyichia coli as part of a trpE fusion protein (Man-
                ning and Leong 1990), it was possible to examine                                              Two recombinant plasmids were identified from the
                different regions of the VP2 gene for immunoreactiv-                                          clones derived from the pATH1 vector/insert ligation
                ity with a panel of rabbit antisera and monoclo                                  nal          mixture, and the trpFVP2 fusion proteins expressed by
                antibodies to different serotypes of IPNV. The VP2                                            these plasmids were characterized by Western
                gene was excised from the plasmid pUC19/A+SAK                                                 immunoblot analysis. The antisera used to detect the
                and cut with the restriction enzyme Sau3A1 which                                              VP2-specific protein in the bacteria had been pre-
                generated seven DNA fragments (Fig. 1). These frag-                                           pared against purified virions of IPNV-Sp. The
                ments were inserted in-frame with the trpE gene in                                            recombinant plasmid, pB10, was found to produce a
                one of the three pATH vectors, pATH 1, pATH2, or                                              47 kDa fusion protein and the recombinant plasmid,
                pATH3, which put the resulting trpFVP2 fusion gene                                            pA43, was found to produce a 52 kDa fusion protein
                under the control of the tryptophan operator and                                              (Fig. 2). The VP2 derivation of the IPNV sequence in.
                promoter (Dieckmann and Tzagaloff 1985). Recom-                                               the fusion proteins was verified by Western
                binants expressing a portion of the VP2 gene were                                             immunoblot using anti-VP2-specific antisera prepared
                detected by direct colony immunoblot with anti-                                               against purified VP2 virion protein from the IPNV-Sp
                IPNV-Sp sera (Gilmore et al. 1988).                                                           strain (Huang et al. 1986). The cell lysate prepared







                                                         Barrie et al.: Antigenic Domain in Protein of Infectious Pancreatic Necrosis Virus              17

                                                                                                                               Figure 2
                                    1     2    3     4     5    6     7    8      9                          Immunoblot showing reactivity of 17p&
                                                                                                             VP2 fusion proteins with anti-IPNV-Sp
                                                                                                             sera. E. coli cells containing the recombi-
                                                M                                                            nant plasmids (pB10 or pA43) were
                   75 kDa --- Mw
                                                                                         O-Vpl
                                                                                                             grown to mid-log phase before induction
                                                                                                             with 15 @ig/ml indoleacrylic acid. The
                                                                                                             cultures were then grown to stationary
                                                                                             VP2
                                                                                                             phase before the cells were harvested by
                       5 0
                                                                                                             centrifugation for protein analysis. The
                                                                                                             cells were lysed and the proteins sepa-
                                                                                                             rated on an SDS-polyacrylamide gel as
                       39 ---W                                                           -4-VP3
                                                                                                             previously described (Gilmore et al.
                                                                                            VP3a
                       27 0                                                                                  1988). The proteins were transferred to
                                                                                                             nitrocellulose and then exposed to anti-
                       17                                                                                    IPNV-Sp antisera. Lane i contains
                                                                                                             IPNV-Buhl infected fish tissue culture cell
                                                                                                             lysate; lane 2, prestained low molecular
                                                                                                             weight markers from BioRad at 75 kDa,
                                                                                                             50 kDa, 39 kDa, 27 kDa, and 17 kDa;
                                                                                                             lanes 3 and 4, pB10 induced bacterial cell
                 from induced cells containing pBlO or pA43 was                                              lysate; lane 5, pA43 induced bacterial cell
                 found to contain protein bands that were strongly re-                                       lysate; lane 6, bacterial cells containing
                 active with the anti-IPNV-Sp/VP2 sera (Fig. 3).                                             the pATH vector with no insert; lane 7,
                   The DNA sequence of the viral insert in pB10 and                                          bacterial cells without a plasmid; lane,8,
                 in pA43 was determined after subcloning of the in-                                          low molecular weight markers; and lane
                                                                                                             9, purified IPNV-Buhl. The arrow in lane
                 sert into the M13 sequencing vectors mp18 and                                               5 indicates the position of the trpFVP2
                 mp19. Sequence analysis was performed by the modi-                                          fusion protein encoded by the recombi-
                 fied Sanger dideoxy chain termination method                                                nant plasmid, pA43. The computed
                 (Davis et al. 1986). The pB10 insert comprised 323                                          molecular weight of the typ&VP2 fusion
                 nucleotides encoding 108 amino acids and mapped                                             protein was 56.5 kDa.
                 to amino acid number 99 to 206 of the VP2 protein
                 of IPNV-Sp (Mason and Leong, unpubl. data). The
                 pA43 insert c6mprised two neighboring Sau3A frag-                       proteins produced by each plasmid were noted. Care-
                 ments of 297 and 27 nucleoticles. This insert                           ful analysis of the 3' terminal sequence of both
                 probably originated from a partial cleavage product                     plasmids by direct sequence analysis from the recom-
                 (Fig. 1) and it was mapped to the adjacent region of                    binant pATH fusion plasmids themselves (Wang et al.
                 the VP2 protein at amino acid number 207 to 315 of                      1988) indicated that the translational termination
                 the VP2 protein. Although both p1310 and pA43 con-                      codon, TAG, was present immediately after the end
                 tained inserts encoding 108 amino acids, striking                       of both VP2 cDNA inserts. The calculated isoelectric
                 differences in the observed migration of the fusion                     points for both fusion proteins was 6.6, and there was



                                 1     2     3      4     5       6 7

                                                                                                                         Figure 3
                                                                                                 Immunoblot of trp&VP2 fusion proteins with antisera
                                                                            of     75 kDa        to the VP2 protein of IPNV-Sp. E. coli cells containing
                                                                                                 the recombinant plasmid, pBlO or pA43, were grown
                                                                                                 and prepared as described in Figure 2. Lane I con-
                 56.5kDa 10*0                                                      50            tains purified IPNV-Sp; lanes 2 and 3, lysates from
                                                                     'T                          uninduced and induced cells containing pBlO; lane
                                                                                                 4, lysate from uninduced cells containing the pATH1
                                                                            44     39            vector without any VP2 insert; lane 5, prestained low
                                                                                                 molecular weight markers from BioRad at 75 kDa, 50
                                                                                                 kDa, and 39 kDa; lanes 6 and 7, lysates from
                                       U      I     U             U    I                         uninduced and induced cells containing pA43. The
                                                                                                 photograph taken for lanes 6 and 7 was a lighter ex-
                                >                                    CO
                                z                                    Itr                         posure of the immunoblot and these lanes contained
                                          ra        <     Y)         <
                                LL        0.        CL    0.         CL                          -five times as much bacterial lysate as that used in
                                                                                                 lanes 2, 3, and 4.








            18         NOAA Technical Report NN[FS I I I





                       p810 insert


                       SP ISRKYDIQSSTLPAGLYALNGTLNAATFEGSLSEVESLTYNSIMSLTTNPODKA
                       jA ISRKYDIQSSTLPAGLYALNGTLNAATFEGSLSEVESLTYNSLMSLTTNPQDKV
                       mi ISRKYDIQSSTLPAGLYALNGTLNAATFEGSLSEVESLTYt4LMSLTTNPQDKV



                       sp NNQLVTKGVTVLNLPTGFDKPYVRLEDETPQGLQSMNGARMRCTAAIAPRRYEI
                       ix UNQLVTKGITVLNLPTGFDKPYVRLEDETPQGPQSMNGAPMRCTAAIAPRRYEI
                       Ni NNQLVTKGVTVLNLPTGFDKPYVRLEDETPQGLOSMNGAKMRCTAAIAPRRYEI


                       pA43 insert

                                                                                                                          Figure 4
                       sp DLPSQSLPPVPATGTLTTLYEGNADIVNSTTVTGDINFSLAEQPAMTRFDFQL
                       aA DLPSERLPTVAATGTPTTIYEGNADIVNSTAVTGDITFQLEAEPVNETRFDFIL                     A comparison of the derived amino acid se-
                       al DLPSQRLPPVPATGTLTTLYEGNADIVNSTTVTGDINFSLMQPANETKFDFQL                      quence of IPNV-Sp, IPNV- jasper, and IPNV-NI
                                         * * * *      * *                                            cDNA inserts present in pBIO and in pA43. The
                       sp DLMGIJ)NDVPVVTVVS SVLAnMNYRGVSAK4TQS IPTENI TKP ITRVKLSYKI
                       jx QFLGIJ)NDVPVV7VTSSTLVTADNYRGASAKFTQSIPTFMITKPITRVKLAYQL                    asterisks indicate differences in amino acids.
                       KI DFMGLDLDVPVVTVVSSVIATNDNYRGASAKMTQSIPTENITKPITRVKLSYKI                     The IPNV-Jasper Ua) sequence was taken from
                                                                                                     Duncan and Dobos (1986). The IPNV-NI se-
                                                                                                     quence was taken from Havarstein et al. (1990).


                    A                                                                                    B
                                  1 2      3 4       5 6       7           8 9     10                             1 2          3   4


                 130 kDa Iw
                       75 --ill                                                                                                                 50 kDa

                                                                                   mow,-4- 56.5 kDa
                       50 -10,                                                                                                            -4-39




                       39 -0,




                                 U         U  I      U I                   U 1                                     U          U   I

                                             C)       0                           >                                           M
                                                                                   Z
                                                       im     CO            M
                                   CL        M         a                     CL                                     CL         CL



                                                                             Figure 5
              Immunoblots of trpFVP2 fusion proteins with antisera to the heterologous IPNV strains, IPNV-Buhl and IPNV-EVE. E. coli
              cells containing trpFVP2 fusion proteins encoded by the recombinant plasmids, pBlO or pA43, were analyzed for reactiv-
              ity with antisera prepared to purified virus of the two heterologous IPNV strains. (A) Reactivity with antisera to IPNV-Buhl.
              Lanes I and 2 contain lysates from uninduced and induced cells containing the pATHI expression vector with no viral
              insert; lanes 3 and 4, lysates from uninduced and induced cells containing pA43; lanes 5 and 6, lysates from uninduced
              and induced cells containing pBlO; lane 7, prestained low molecular weight markers from BioRad; lanes 8 and 9, lysates
              from uninduced and induced cells containing pBlO; lane 10, purified IPNV. (B) Reactivity with antisera to IPNV-EVE.
              Lanes 1 and 2 contain lysates from uninduced and induced cells containing pBIO; lanes 3 and 4, lysates from uninduced
              and induced cells containing pA43.


            no dramatic difference in the amino acid composi-                         gous VP2       proteins in Western immunoblots (R.
            tion. Thus far, the only possible explanation for the                     Barrie andJ. Leong, unpubl. data). Thus, there are
            slower migration of the pA43 fusion protein might be                      conserved linear epitopes among the IPNV strains.
            the series of four prolines found towards the amino                       When the expressed trpE-VP2 fusion proteins were
            terminus of this insert (Fig. 4).                                         examined for reactivity with polyclonal anti-IPNV
               Polyclonal rabbit antisera prepared to the different                   sera prepared to three different lPNV serotypes, only
            serotypes of lPNV will crossreact with the heterolo-                      pBlO reacted with the heterologous antisera in West-
 





                                                             Barrie et al.: Antigenic Domain in Protein of Infectious Pancreatic Necm@is Virus                     19

                  ern immunoblots (Fig. 5, A and B). The anti-IPNV-                            Acknowledgments
                  Buhl sera was prepared against purified virions of
                  the Buhl virus isolate which had been previously                             This publication is the result of research sponsored
                  characterized as a member of the IPNV-VR299 sero-                            by the Oregon Sea Grant with funds from the Na-
                  type found in rainbow trout (Oncorhynchus mykiss)                            tional Oceanic and Atmospheric Administration,
                  in North America (Hill and Way 1983). The IPNV-                              Office of Sea Grant, Department of Commerce, un-
                  EVE isolate was obtained from Japanese eels                                  der grant no. NA85AA-D-SGO95 (project no. R/
                  (Anguilla japonica) suffering from branchio-                                 FSD-11) and the United States Department of Agri-
                  nephritis in Japan (Sano et al. 1981); it has been                           culture to the Western Regional Aquaculture
                  antigenically grouped with the AB serotype of                                Consortium under grant nos. 87-CRSR-2-2319 and 88-
                  IPNV, which is nonpathogenic for rainbow trout.                              38500-4027. Oregon Agricultural Experiment Station
                  The fusion protein encoded by pA43 was com-                                  Technical Paper No. 9327. We thank L. Bootland for
                  pletely nonreactive with the heterologous antisera.                          reviewing the manuscript.
                  Thus, it appears that the VP2 gene region encoded
                  by pBI0 contains an antigenic determinant(s)
                  which is conserved among the IPNV strains exam-                              Citations
                  ined and the insert in pA43 encodes a region
                  which is highly variable.                                                    Christie, K.E., L.S. Havarstein, H.O. Djupvik, S. Ness, and C.
                     A comparison of the, nucleotide sequence and its                            Endresen.
                  derived amino acid sequence of 'each insert. with                                1988. Characterization of a new serotype of infectious pan-
                                                                                                      creatic necrosis virus isolated from Atlantic salmon. . Arch.
                  that of other published sequences of the VP2 gene                                   Virol. 103:167-177.
                  showed that the pBI0 region was highly conserved                             Davis, L.C., M.D. Dibner, andj.F. Batty (eds.),
                  at the nucleotide and amino acid level (Fig. 4;                                  1986.. Basic methods in molecular biology. Elsevier Science
                  Christie et al. 1988; Havarstein et al. 1990). There                                Pub. Co., NY, 377 p.
                  were three amino acid differences between the Sp                             Dieckmarm, C.L., and A. Tzagaloff.
                  and the jasper isolate (a member of the VR299 sero-                              1985. Assembly of the mitochondrial membrane system.
                                                                                                      Biol. Chem. 260:1513-1520.
                  type), and there were only two amino acid                                    Duncan, R., and P. Dobos.
                  differences between Sp and NI isolates, the latter of                            1986. The nucleotide sequence of in      fectious pancreatic ne-
                  which is another IPNV isolate from the Sp serotype                                  crosis virus (IPNV) dsRNA segment A reveals one large ORF
                  (Havarstein et al. 1990). In contrast, 27 amino acid                                encoding a precursor protein. Nucl. Acids Res, 14:5934-
                                                                                                      5935.
                  changes between the Sp and jasper isolates were ob-                          Duncan, R., E. Nagy, P.J. Krell, and P. Dobos.
                  served for the pA43 gene fragment. Only five amino                               1987. Synthesis of the infectious paricreatic necrosis virus
                  acid   changes were found between isolates Sp and                                   polyprotein, detection of virus-encoded protease, and fine
                  NI. The similarity between the Sp and NI genomes                                    structure mapping of genome segment A coding regions.
                  indicates that these two isolates are highly related, a                             J. Virol. 61:3655-3644.
                                                                                               Gilmore, R.D.Jr, H.M. Engelking, D.S. Manning, andj.C. Leong.
                  finding that is consistent with the findings of                                  1988. Expression in Eschefichia coli of an epitope of the
                  Christie et al. (1988).                                                             glycoprotein of infectious hematopoietic necrosis virus
                                                                                                      protects against viral challenge. Bio/Technology 6:295-
                                                                                                      300.
                                                                                               Havarstein, L.S., K.H. Kalland, KE. Christie, and C. Endresen.
                  Conclusion                                                                       1990. Sequence of the large double-stranded RNA segment
                                                                                                      of the NI strain of infectious pancreatic necrosis virus: A
                  In summary, two immunoreactive regions of the viral                                 comparison with other Birnaviridae. J. Gen. Virol. 71:299-
                  major capsid protein, VP2, have been identified. One                                308.
                  region from amino acids 99 to 206 contains a very                            Hill, BJ., and K Way.                                             -
                                                                                                   1983. Serological classification of fish and shellfish
                  conserved epitope(s) which was recognized by neu-                                   birnaviruses. Abstract, First international conference of
                  tralizing antisera to three different IPNV serotypes.                               european association of fish pathology, Plymouth, England,
                  Another region from amino acids 207 to 315 contains                                 October, 1983.
                  a highly divergent epitope(s) that may encode the                            Huang, M.T.F., D.S. Manning, M. Warner, E.B. Stephens, andj.C.
                  serotype-specific epitope(s) of an IPNV strain. A sug-                         Leong.
                                                                                                   1986. A physical map of the viral genome for infectious pan-
                  gestion that the amino acid region from 206 to 350                                  creatic necrosis virus Sp: Analysis of cell-free translation
                  encoded the serotype-specific epitope(s) of IPNV was                                products derived from viral cDNA clones. J. Virol.
                  made by Havarstein et al. (1990) when a comparison                                  60(3):1002-1011.
                  of the deduced amino acid sequence of IPNV-Nl and                            Lipipun, V., P. Caswell-Reno, Y-L. Hsu, J.L. Wu, M.-C. Tung, P.W.
                  IPNV-Jasper capsid proteins revealed that this region                          Reno, W. Wattanarijarn, and B.L. Nicholson.
                                                                                                   1989. Antigenic analysis of Asian aquatic birnavirus isolates us-
                  was very heterogeneous.                                                             ing monoclonal antibodies. Fish Pathology 24(3):155-160.







            20        NOAA Technical Report NNM I I I


            Manning, D.S., andj.C. Leong.                                             Wang, L.M., DX Weber, T. Johnson, and AX Sakaguchi.
                1990. Expression in Escherichia coli of the large genomic seg-             1988. Supercoil sequencing using unpurified templates pro-
                   ment of infectious pancreatic necrosis virus. J. Virol.                   duced by rapid boiling. Biotechniques 6:839-843.
                   179:16-25.                                                         Wolf, K
            Manning, D.S., C.L. Mason, andj.C. Leong.                                      1988. Infectious pancreatic necrosis virus. In Fish viruses
                1990. Cell-free translational analysis of the processing of in-              and fish viral diseases (K. Wolf, ed), p. 115-157. Cornell
                   fectious pancreatic necrosis virus polyprotein. 1. Virol.                 Univ. Press, NY.
                   179:9-15.
            Sano, T., N. Okamoto, and T. Nishimura.
                1981. Anew viral epizootic of Anguilla japonica. J.Fish.Dis.
                   4:127-139.






                                       Cloning of Hemolysin Genes of Aeromonads



                                                        TAKASHI AOKI and IKU0 HIRONO

                                                               Department of Biological Resources
                                                                     Faculty of Agriculture
                                                                      Miyazaki University
                                                                    Miyazaki 889-21, Japan




                                                                        ABSTRACT


                                    The role of extracellular products is critical in the pathogenic mechanisms of bacterial
                                  infections. In Aeromonas spp., hemolysins may be the most important of these products in
                                  establishing and maintaining infections. This report reviews our knowledge of the struc-
                                  ture and expression of hemolysin genes in Aeromonas and discusses preliminary results on
                                  gene homology and ancestry among various Aeromonas spp.



                  The related species Aeromonas hydrophila (Ljungh                 erythrocytes (T-lysin) (Rockey et al. 1988). H-lysin
                and Wadstrï¿½m 1982) and A. salmonicida (Titball and                 contains GCAT. Nomura et al. (1988) purified
                Munn 1985a) produce several extracellular proteins                 salmolysin, an extracellular hemo lytic toxin from A.
                that are virulence factors. In the study of pathogenic             salmonicida. Salmolysin was lethal to rainbow trout
                mechanisms.of these bacteria, there has been interest              Oncorhynchus mykiss when it was injected intramuscu-
                in the role of these extracellular substances as toxins.           larly.
                A. hydrophila produces a variety of extracellular prod-              Almost -all isolates of A. hydrophila and A.
                ucts, including a protease, glycerophospholipid                    salmonicida produce aerolysin, a substance with he-
                cholesterol acyltransferase (GCAT), cytotoxin, an en-              molytic activity. The level of aerolysin production is
                terotoxin, acetylcholinesterase (Nieto et al. 1991),               known to vary under different growth conditions,
                and hemolysins (Ljungh and Wadstr6m 1982). Extra-                  and individual isolates can alternate between high
                cellular products of A. salmonicida include hemolytic,             and low level phases of production. When this hemo-
                leukocytolytic, proteolytic, and GCAT activities, (Ellis           lysin gene is cloned into E. coli, the gene's
                et al. 1981, 1988). The virulence of A. hydrophila and             characteristics can be more easily studied.
                A. salmonicida is significantly enhanced by their abil-              For this reason, we cloned the two hemolysin
                ity to secrete hemolysin. Hemolysin may be the most                genes from A. hydrophila and the one hemolysin
                important of these products in causing tissue damage               gene from A. salmonicida to study their structure
                and in establishing and maintaining infections with                and expression (Aoki and Hirono 1991; Hirono and
                Aeromonas.                                                         Aoki 1992, a and b).
                   There are many reports describing the number                      We have previously reported the cloning of two he-
                and nature of hemolysins found in A. hydrophila                    molysin genes (for aerolysins AHH-1 and AHH-2)
                (Ljungh et al. 1981; Thune and Johnson 1986; Asao                  from A. hydraphila ATCC7966 into a plasmid vector in
                et al. 1986) and A. salmonicida (Titball and Munn                  E. coli K-12 (Aoki and Hirono 1991; Hirono and Aoki
                1985a; Rockey et al. 1988). Asao et al. (1986) purified            1991, Table 1). Open reading frames (ORF) of the
                and characterized two hemolysins from A. hydrophila                AHH-1 and AHH-2 genes were 1,734 and 981 base
                which were biologically similar but immunologically                pairs (bp), respectively. The sequences included the
                distinct. Both hemolysins caused fluid accumulation                -10 region and the -35 region of a promoter and a
                in infant mouse intestines    'and rabbit ileal loops and          ribosome binding site (Shine-Dalgarno sequences)
                elicited a cytotoxic effect on Vero cells. Two distinct            upstream from the ORF. Two palindromic sequences
                hemolysins have also been found in A. salmonicida.                 were found immediately following thetermination
                One is a broad-spectrum hemolysin with maximum                     site. Analysis of the deduced amino acid sequences
                activity against horse erythrocytes (H-lysin) (Titball             indicated a highly hydrophobic N-terminal region in
                et al. 1985b), and the other is active against trout               the AHH-1 gene with the characteristics of a leader

                                                                                                                                              21







                    22              NOAA Technical Report NWS 111





                                    10          20          so         40          50          60          70         so          so          100
                               OCATGCMAATCATC(AWTTAGATOOTATMMATccnwTemchAuATAAATVATTCDCMCCACACTGTtAATT('ACGMGAATAATGAATGTCA

                               Ito        120        130         Ito         150        150         170         180         190        too         210
                               TGACAPGCAAGCAGAAUACGC=AAATATAA                      ffTM=ATTMTTCATTOMAAATAGCMCMMr=C&AGOAGAT.AA
                                                                                                                            45
                                    220         230         240        250         260         270         290        290         Soo         310
                               WAAAAAUAAAAACCACGCAAATTrATCAMCAAG=CCAM7MTCTCGMCG=WMMZCAGGCAGMTGCAAGOCOAAGATATTOGOGA
                               -to
                               320        330        340         $50         260        370         $80         390         400        410         420
                               ACGTACCGACCMCAM=7G=MGCCrGCAATCCOAACAMMMTTTA=CAATG===MA&MCAGGGIMMACG=CT
                                          so         MetLeuAlaSerLeuGInSerG 10MG lyLeu I leTYrLetL&snAlaAspYalTrpLedysGIrGinGlyAI aThrProLe

                                    430         440         450        460         470         480         190        500         510         520
                               CATGACCMGGATCACCrGMMAGMGGTG=CAOGGGGTGAGMTCMTT(ATOGATrUGC=TCADMACrAGATMAGMACAACAGOOCAGAAA
                               uMetThrAr&AspGlnLeuArgGluArgValLauAlaArgGlyGlukrW.wPhaI IeAspPheSerA1aValThrAspG1nI leGlaAr&G1nGInAlaArgLy

                               530        540        550         560         570        580         590         goo         610        $20         630
                               GGCCATOWCAGCTGGCMGGATCrCMTOATCOGGACTOGGTGCTWMCMGCTAUAGOOV@AGMCMTTCAC=MGGAGOMTOGATGACCC
                               &A)&NetGluGlnLonAlaGlylleSerPbeAspA]aAspTrpYalLeuVa]SerGlyTyrLysG]yG]uLeuLeuPboThyPrdeuGlyGlyValAspAspPr

                                    640         650         Soo        STO         Soo         690         100        710         120         730
                               =MCTATCAGCMATMMGGTMAGCMGAAGOGUMGCAACDGCCACAAGMCrCGCTGACMAGOMOMCMCCGAGGCCGGTCTGCM
                               oAlaPheTyrOInLeuMtGluArgYalGluSerLeW luGlyGInGlYA8nGlyRieLyaArgSarLwThrGInProProAlaAlaOluAlaGlyLeuProilI

                               740        750        780         770         780        790         800         $to         820        830         840
                               TGTGG=MACCTCAAMTUA=CAAGATCAGMATCCMAGTOCACM=M=CGTACMMCCMMA=GMITMCGATrCOCC
                               &V&IAIaPheTyrLaaAenValAsnArgLyalleSerAspAlaOluCyaThrPhaProArgSorArgThrtrpSerArgGlyAsWgLouPheCYaAspSerPr

                                    850         860         870        980         890         goo         910        920         930         940
                               GMCAT=CTCOTCTACDOWCAAM7MGCOCTCWrWMTTTGGCAACAMOMMMAOGCCOGATOML4uTAGTOMOAT=CTGGAMA
                               oAsalleSerLeuVallyrArgValAsriLau0luArgSarLeuGInPheOlyAsnThrAlaSerAlaThrProAspAIaLysileValArglieSerLeuAspGI

                               950        goo        270         980         990        1000        1010        1020        1030       1040        1050
                               AGAOTCGGCMGTGC=ATOCAOCTUACGAGGATCMAGCTOGAGMAAAACATMMCTATCTGCTW=CM=G=MACTATGOCACMA
                               tiGiuSerAlaGlyAlaGlylleGInLeubnGluAspLeuSerTrpSerGluAsulleAlaAspTyrLeaLsuLeuAspGly7rpAlaArgAepTyrAIRThrAs

                                    1060        1070        1080       1090        1100        1110        1120       1130        1140        1150
                               TGOCATCGCCCAGGATTATMCMCOACMAGGMTMAACACUMGCMMGMCTCAAGAGOMMMOCAAOCTCAAuGcuffAcoAcCATOOMA
                               pAlaileAlaGInAspTyrArsPhoThrThrOluAlaSerAenThrLysAlaAlaVelLeuLyeSerLeuProThrA&nLwAgnSerLYsTyrOluBigAr&iGI

                               1160       1170       1180        1190        1200       1210        1220        1230        1240       1250        1200
                               GATCTM'MMGMMGTCA=MGGGTMAGGTGAACAACOATGGCCCAAGGCUAGCMAGWCGGCCAAMCAGMAGCAGMCCAGCTC
                               alleSerGlyPheGluValGlyValThrGlyGlyValGluValAenLyeAspOlyProArsProSerTrpArgAr&ArsProSerSerAlaSerSerAlaSerSe

                               .    1270        1280        1290       1800        1310        1320        1330       1340        1356        logo
                               GCCTACAACACCrAGGATTACCGGGTrGAACGC==AGMMGAAGGMAOTTT(AGC=MMMATCAATAOGMACCGUGAMWWCT
                               rProThrThrPr*Argl leThrOlyLeuAsnAlaProMaCluArgProOluGlyGluPheGtrLLeuDlYAlaArgSerI leArgAsPArgAr&ValProAlaLe

                               1370       1390       1390        1400        1410       1420        1430        1440        1450       1460        1470
                               OCTCCAACAMOCCAMTCMCATGGCTAMAOffGGATCACAACMCATMW=CTCAGCTACAAGGGGTTMTGCMMTCMAMTCATCTACAAGOC
                               uLeuGInAspGlyHi8ValTrpAlaTrpLeuArgArgOlySerGInProHisProAlaAlaGlnLeuGIrLGIyVaICY&AIsGiuSerAspVaI I I e7yrLysA I


                                    1480        1490        1500       1610        . 1520      15SO        1540       1550        1560        1570
                               GGCGCMGAMAGAMGGCAGCACCOAGTTCAAGATCOACTCCMTCAACATCOOCCCCATCrAcAa=UTLTACAAGCACTACtAMMMGGGC=
                               aAl droAspOluThrGlySerThrOluPhdyalleAspUrSerValAorLIleArgProlleTYrThrGlYlleTyrLysH[sTyrTyrValValGlyAlaUt

                               1580       1590       1600        1610        1620       1630        1640        1650        1660       1670        1680
                               TG7CMCTrWAGGGMMMOATACCGAUMCGCAr,AMGGTGGCGGCGTCUCCAGCrTUAGGTGOACTMUCCACCMGMMACMGC(;GTCO=
                               sValSerPhaOIROlyPhoOluAspThrAspLy&ArgArgAr&ValAlaAlaSerThrSer?heLysValAepTrpAsngleProValPhoThrGlyGlyArgPr

                                    1690        1700        1710       1720        1730        1740        1750       1760        1770        1780
                               COMA 'OMCAGCMGGGUMACMCMMCCMAGMMOATOCCAACCATMTCrGAGOGCGGTGACCMMAGAMM=CMAGTCM
                               oValAinLeuGinLouG[YGIYPheAspAoMrgCysLeuSerA[aAW laAanHisGlyLeu$erAlaValThrPheAspGluThrSerAlaAlaGInSerSe

                               1790       1800       1810        1820        logo       1840        1950        1860        1870       1880        1890
                               CAlWATGAMMAMGCMCrAMUGCGCGCAGGATA=CMCMWMATGGUACAAWnMMGMCAGAGCTGCAGCCMAG=M=
                               rileTyrAspOluTyrOlyArgryrValSerAlaGInAaMrLauArgCnLeuAspGlyAsMsnLeuGlYGlnLeuGInSerCysSarLouSerLeuGlyGI

                                    1900        1910        1920       1930        1940        1950        1960       1970        1980        logo
                               GOG=GAffOGAMGMGACAGCGATGCGCTCAGCAAWrGAGTGCMAMAGCTGmGmcAmAcAACUaAGMGGGCOMMGCTCTACOACGACAA
                               nArgTrPGIUTrPLY&AlaAspSerAspAiaLeuSerAonLeaSerAlaNiSCInLeuLeuVallisAspLyaGInSerOlYAtaLeuDlyLeuTyrAspOluAs                                                             Figure I
                               2000       2010       2020        2080        2040       2050        2060        2070        2090       2090        2100                   Nucleotide sequence and deduced
                               =UAT=CWAA7MOMTACGGACCMA==ArAC=CATCnMMccAmm=MUC7r,ACAGAGCAMGWMMMAGACAACC
                               nGlyAsnProG]nAsnValSerVa]ArgThrLeuThrSerTyrThrArgl]ePbeGlyProProAlaSerRi9                                                                  amino acid sequence of hemolysin
                                    2110        2120        2130       2140        2150        2160        2170       2180        2190        2200                        gene AHH-1 from Aeromonas hydrophila
                               ACDACGCCAAGMCMGMTTTMATGAGGGGOGGATCAGGI)CMTTTTCrAMAGMMGOGAGCACOTCM=GCMAGAAGGTCTGCAC==G                                                      ATCC7966. The deduced amino acid
                               PWWMM BUWUG                                                                                                                                sequence is given under the nucleo-
                               2210       2220       2230        2240        2250       2260        2270        2280        2290       2300        2310
                               MCMGGCGCCACACCAG=ATCTMCGGTAGTGAMMCMGCAGMMATMACCAGAT=MTMTMAGGATC=CATCGATMCrAICTGe                                                           tide sequence. A putative promoter is
                                    2S20        2830        2340       2350        2360        2S70        2380       2390        2400        2410                        indicated by the areas marked -35 and
                               GGCAGGMCGTMMAG=GCGCMACCATMCACCAGGMMCAGOCTGMMC,WMGGTMATCMMMTCA=CAG=CAGGGC                                                                   -10, and ribosome binding site is indi-
                               2420       2430       2440        2450        2460       2470        2480        2490        NO         2510        2520                   cated by SD. The palindromic structure
                               rACCGWMn=GTGAGAGAGTCGTCCCOCTOCAOCAGGAAGATACTCfGGTMCAGCTMTMACTMAAM=mTr,cArGrMG(rAmo.Tc4w,G                                                  sequence of a terminator-like region is
                                                                                                                                                                          also indicated downstream from the
                                                                                                                                                                          AHH-1 coding region.






                                                                                      Aoki and Hirono: Cloning of Heniolysin Genes of Aeronionads                        23


                                                                                                    of E. coli and the plasmid-encoded proteins examined
                                                    Table I                                         by polyacrylamide gel electrophoresis (Sancar et al.
                         Maximum matching comparison of amino acid se-.                             1979). Strains containing pAHH-1 and pASH-1 pro-
                         cluences of cloned hcmolysin genes of Aeromonas                            duced proteins of 62 kDa and 56 kDa, respectively
                         hydrophila and A. salmonicida.                                             (Hirono and Aoki 1991, 1992a). These results are in
                                             AHH-1        AHH-2       ASH-1         aer             agreement with the size of the proteins predicted by
                                                                                                    the DNA-sequenced ORK However, the molecular size
                         AHH-1                                                                      of novel protein synthesized by cells harboring the
                         AHH-2               17.4%                                                  pAHH-2 plasmid was 26 kDa., The molecular size of
                         ASH-1               17.0            17.0                                   the protein was clearly different from the size pre-
                         aer*                17.1            16.9        18.5                       dicted by the hemolysin AHH-2 gene sequence which
                          Howard and Buckley 1986.                                                  inferred a protein with a molecular weight of 37.7
                                                                                                    kDa. The products of transcrip6on or translation in
                                                                                                    AHH-2 may be either greatly truncated or reduced in
                                                                                                    quantity. As mentioned above, analysis of the deduced
                                                                                                    amino acid sequences indicated that the N-terminal
                     peptide (Figure 1). However, the               N-terminal region.              region of the AHH-2 gene was not hydrophobic. As
                     of the AHH-2 gene was not hydrophobic.                                         yet, it is difficult to explain the difference in the mo-
                         Two aerolysin genes were also cloned from A.                               lecular size of the final protein.
                     hydrophila, Ah65 (Howard and.Buckley 1986; Howard                                Hemolysin was released by the E. coli cells contain-
                     et al. 1987) and AH2 (Chakraborty et al. 1986). The                            ing the AHH-1 gene and those containing the ASH-1
                     nucleoticle sequence of the Ah65 aerolysin gene                                gene. The hemolytic activity in a supernatant was in-
                     was 1,458 bp. There was very low homology between                              activated by heating to 70' C for 10 minutes.
                     the abrolysin gene from Ah65 and each of the                                   However, the hemolysin was expressed but was not
                     ATCC7966 genes, and there were no indications of                               secreted from E. coli cells carrying the recombinant
                     similarity in the predicted amino acid sequences                               plasmid containing the AHH-2 gene. The aerolysin
                     (Table 1).                                                                     cloned by Howard and Buckley (1986) similarly was
                         We also cloned one hernolysin gene (ASH-1) from                            not released from the E. coli cells in which they were
                     A. salnionicida ATCC14174 (Hirono and Aoki 1992,                               cultured. The E. coli hemolysin requires four cistrons
                     a and b) (Table 2) which had an ORF of 1,716 bp. It                            encoded with the hemolysin structured protein for
                     had the -10 region and the -35 region of a putative                            full expr   .ession of active protein to be achieved
                     promoter and a ribosome-bin ding site up stream                                (Felmlee et al. 1985). These proteins are associated
                     from the CIRF, and the termination codon and                                   with the cell envelope and are involved in transport-
                     palindromic sequences downstream from the ORK                                  ing the hemolysins out of the cells. This complex
                     The N-terminal region of the ASH-1 gene was highly                             system is unlike the one producing the extracellular
                     hydrophobic. Comparative analysis of the fundamen-                             hemolysins in A. hydrophila and A. salmonicida. It is
                     tal molecular structures of our cloned AHH-I,                                  very interesting that the hemolysin release mecha-
                     AHH-2, and ASH-1 genes, and the previously re-                                 nism produced by E. coli cells containing the hemo-
                     ported aerolysin gene from Ah65 (Howard et al.                                 lysin gene is different from Aeromonas spp.
                     1987) suggests that they have not descended from a                               Using colony hybridization analysis, the cloned he-
                     common ancestor.                                                               molysin genes were tested for presence of
                         The recombinant plasmids pAHH-1, pAHH-2, and                               homologous regions in isolates A. hydrophila and A.
                     pASH-I were introduced into a maxicell strain CSR603                           salmonicida from humans and fish in Japan (Hirono




                                                                                             Table 2
                                                      Cloned hemolysin genes from Aeromonas hydrophila and A. salmonicida.

                         Hemolysin                                     Open reading                 predicted molecular size                  Molecular size
                         genes                       Sources            frame (bp)                from DNA sequences (Da)                  from Maxicell (kDa)

                         AHH-1                    A. hydrophila             1,734                            63,658                    62 (Hirono and Aoki 1991)
                         AHH-2                    A. hydrophila               981                            37,797                    26 (Aoki and Hirono 1991)
                         ASH-1                    A. salmonicida            1,716                            64,780                    56 (Hirono and Aoki 1992a)







             24         NOAA Technical Report NXIFS I I I


             and Aoki 1992, a and b). Ten of 15 strains of A.                               of A. hydrophila and A. salmonicida isolated in the
             hydraphila isolated from humans and 14 of 33 strains                           United States for cloning the hemolysin gene and
             from fish possessed sequences homologous to the he-                            tested for homology with strains isolated in Japan.
             molysin gene of AHH-1 (Table 3). The AHH-1 gene                                Homology of the tested strains with the cloned hemo-
             was detected in all 38 strains of A. salmonicida. The                          lysin was low. We shall attempt to survey the
             AHH-2 gene was detected only in the original strain                            distribution of our cloned hemolysin genes in more
             ATCC7966 and not in the other strains of A.                                    isolates of aeromonads. The presented data, however,
             hydrophila and A. salmonicida tested. The ASH-1 gene                           indicate that the hemolysin genes having different
             was detected.in two strains of A. hydrophila from fish                         structures occur in A. hydrophila and A. salmonicida
             and the original ATCC14174 strain. We used strains                             isolates from different geographical locations.






                                                                                    Table 3
                 Detection of DNA sequences homologous to the hemolysin genes from Aeromonas hydrophila and A. salmonicida by
                 colony hybridization. (Hirono and Aoki 1991, 1992b.)

                        Strains                             Sources                         AHH-I                  AHH-2                      ASH-1


                        A. hydrophila                       Human                           10/15*                   0/4                      0/15,
                                                            Fish                            14/33                    0/5                      2/33
                                                            ATCC7966                          1/1                    1/1                      0/1
                        A. salmonicida                      Fish                            38/38                    0/19                     0/38
                                                            ATCC14174                         1/1                    0/1                      1/1

                   Number of strains containing hemolysin genes/Number of tested strains.
                   Hemolysin genes of the AHH-1 and AHH-2 of A. hydrophila ATCC7966 and the ASH-I gene of A. salmonicida ATCC14174 were
                   cloned into plasmid vectors in E. coli K-1 2.



             Citations                                                                            ings of the Symposium on Diseases in Asian Aquaculture.
                                                                                                  Asian Fisheries Society@ (In press.)
             Aoki, 1, and 1. Hirono.                                                            1992b. Nucleotide sequence, expression, and characteriza-
                   1991. Cloning and characterization of the hemolysin deter-                     tion of a hemolysin gene from Aeromonas salmonicida.
                    minants from Aeromonas hydrophila. J. Fish Dis. 14:303-312.                   Submitted to infection Immun.
             Asao, T., S. Kozaki, K. Kato, Y. Kinoshita, K_ Otsu, T. Umemura,               Howard, S.P., andj.T. Buckley.
               and G. Sakaguchi.                                                                1986. Molecular cloning and expression in Escherichia coli of
                   1986. Purification and characterization of an Aeromonas                        the structural gene for the hemolytic toxin aerolysin from
                    hydrophila hemolysin. J. Clin. Microbiol. 24:228-232.                         Aeromonas hydrophila. Mol. & Gen. Genet. 204:289-295.
             Chakraborty, T., B. Huhle, H. Bergbauer, and W. Goebel.                        Howard, S.P., W.J. Garland, M.J. Green, andj.T. Buckley.
                   1986. Cloning, expression, and mapping of the Aeromonas                      1987. Nucleotide sequence of the gene for the hole-forming
                                                                                                  toxin aerolysin of Aeromonas hydrophila. J. Bacteriol.
                    hydraphila aerolysin gene determinant in Escherichia coli K-                  169:2869-2871.
                    12. J. Bacteriol. 167:368-374.                                          LJungh A., and T. Wadstr6m.
             Ellis, A.E., T.S. Hastings, and A.L.S. Munro.                                      1982. Aeromonas toxins. Pharmacol. & Ther. 15:339-354.
                   1981. The role of Aeromonas salmonicida extracellular prod-              LJungh, A.,-B. Wretlind, and R. M611by.
                    ucts in the pathology of furunculosis. J. Fish Dis. 4:41-51.                1981. Separation and characterization of enterotoxin and
             Ellis, A.E., A.S. Burrows, and K-J. Stapleton.                                       two haemolysins from Aermnonas hydrophila. Acta Pathol.
                   1988. Lack of relationship between       virulence of Aermonas                 Microbiol. Scand. Sect. B. Microbiol. Immunol. 89:387-397.
                    salmonicida and the putative virulence factors: A-layer, extra-         Nieto, T.P., Y Santos, L.A. Rodriguez, and A.E. Ellis.
                    cellular proteases and extracellular haemolysins. J. Fish                   1991. An extracellular ace tylcholinesterase produced by
                    Dis. 11:309-323.                                                              Aeromonas hydrophila is a major lethal toxin for fish. Microb.
             Felmlee, T., S. Pellett, and R.A. Welch.                                             Pa thog. 11:101-110.
                   1985. Nucleotide sequence of an Escherichia coli chromo-                 Nomura, S., Fujino, M., Yamakawa, M., and E. Kawahara.
                    somal hemolysin. J. Bacteriol. 163:94-105.                                  1988. Purification and characterization of salmolysin, and
             Hirono, L, and T. Aoki.                                                              extracellular      hemolysin * toxin        from     Aeromonas
                   1991. Nucleotide sequence and expression of a hemolysin gene                   sabnonicida. J. Bacteriol. 170:3694-3702.
                    from Aeromonas hydrophila. Microb. Pathog. 11:189-197.                  Rockey, D.D.J.L. Fryer, andj.S. Rohovec.
             Hirono, I., and T. Aoki.                                                           1988. Separation and in vivo analysis of two extracelluar
                   1992a. Molecular cloning, expression, and nucleotide sequence                  proteases and the T-hemolysin from Aeromonas
                    of a hemolysin gene from Aeramonas salmanicida. The Proceed-                  salmonicida. Dis. Aquat. Org. 5:197-204.






                                                                                 Aoki and Hirono: Cloning of Hemolysin Genes of Aerontonads                    25


                  Sancar, A., A.M. Hack, and W.D. Rupp.                                      Titball, R.W., and C.B. Munn.
                       1979. Simple method for identification of plasmid-coded                    1985a. Interrelationships of extracellular products from
                         proteins. J. Bacteriol. 137:692-693.                                       Aeromonas salmonicida. In Fish and shellfish pathology, (A.E.
                  Thune, R.L., and M.C. Johnson.                                                    Ellis, ed.), p. 61-68. Acad. Press, NY
                       1986. Aeromonas hydrophila B-haemolysin: purification and ex-         Titball, R.W., and C.B. Munn.
                         amination of its role in virulence in O-group channel cat-               1985b. The purification and some properties of H-lysin from
                         fish, Ictaluruspunctatus (Rafinesque). J. Fish Dis. 9:55-61.               Aeromonassalmonicida. J. Gen. Microbiol..131:1603-1609.






                                         Harmful Red Tides. of Heterosigma akashiwo



                                                                     TSUNEO HONJO

                                                            Nansei National Fisheries Research Institute
                                                                         Fisheries Agency
                                                               Ohno, Saeki, Hiroshima'739-04, Japan




                                                                         ABSTRACT


                                     The raphidophyte Heterosigma akashiwo is one of several species of flagellates that
                                   cause harmful red tides. This paper reviews the distribution of H. akashiwo, the relation-
                                   ship between eutrophication and the occurrence of red tides in the Seto Inland Sea, and,
                                   environmental and biological features of red tide development (life cycle, growth dy-
                                   namics, and allelopathy).




                 Introduction                                                       Distribution

                 The raphidophyte Heterosig7na akashiwo is one of sev-              Heterosigma akashiwo occurs in the temperate and sub-
                 eral species of flagellates causing prodigious. red                tropical embayments in Japan (Hara and Chihara
                 tides. The total damage by H. akashiwo red tides has               1987), Singapore (Taylor 1990), New Zealand (Taylor
                 amounted to about 2 billion yen over a period of 16                1990), England (Lackey and Lackey 1963), and Bel-
                 years (1972-87) in the Seto Inland Sea of Japan.                   gium (Conrad and Kufferath 1954), and in the
                 Most of this damage affected fish culture operations.              eastern (Tomas 1982) and western areas (Lackey and
                 This paper seeks to advance research on red tides by               Clendenning 1965; Taylor 1990) of North America,
                 revie*wing the known ecological features of Hetero-                Bermuda (Tomas 1982), and Chile (Taylor 1990)
                 sigma red tides.                                                   (Fig. 1). Damages to fish have been recorded for the
                                                                                    Seto Inland Sea (yellowtail, Seriola quinqueradiata and
                                                                                    red sea brearn, Pagrus'major), New Zealand (salmon),
                 Morphology                                                         British Columbia (salmon), and Chile (salmon) (Tay@
                                                                                    lor 1990).
                 The cells of Heterosigma      akashiwo (HADA) Hara et
                 Chihara are yellow-brown'and ovoid, and slightly
                 compressed dorso-ventrally. The cell size is 8-25 X                Eutrophication and Red Tides
                 6-15 pm. This organism is thought to lack a cell                   In the Seto Inland Sea, nitrate, inorganic nitrogen
                 wall and be limited only by a single membrane. The                 (ammonia + nitrite + nitrate), and phosphorous con-
                 delicate structure of the cell has hindered the study              centrations have increased rapidly since the
                 of its surface Morphology, although the existence of               mid-1960s (Fig. 2). Nutrient concentrations reached
                 an external structure, the glycocalyx, has been dem-               a maximum in the mid-1970s then gradually de-
                 onstrated (Yokote et al. 1985). In Japan, H. akashiwo              creased thereafter. The total number of red tide
                 has been referred to as Entornosigma sp. and H.                    occurrence  .s in the Seto Inland Sea before 1965 was
                 inlandica; in many other countries it has been con-                less than 50 cases per year. Beginning in 1968, the
                 fused with the chrysophyte Olisthodiscus luteus                    number increased year by year, reaching a peak of
                 CARTER. Recently, Hara and Chihara (1987) re-                      299 cases in 1976. After 1976, the number decreased
                 ported that Entornosigma sp. and H. inlandica are                  to 160-170 cases per year. The increase in the 1-970s
                 synonymous with H. akashiwo, and that most red                     coincided with a rapid development in the Japanese
                 tides ascribed to 0. Iuteus actually involve H.                    economy, with a time lag of 2-3 years. The decrease
                 akashiwo.                                                          in frequency after the first oil shock (197a-1974) cor-

                                                                                                                                               27







            28       NOAA Technical Report NNIFS 111



                                                                      cog


























                                                                        Figure I
            Distribution of Heterosig7na akashiwo red tide (dotted areas). Fish indicate places where cultured fish have sustained damage.





                                                                                                           ri


                300
                        \Z
                       WZ+ z
                             E                                                                           C A@
                           ff E
                         =4 !E + -k                                              Nitrate, nitrite and
                        -                                                       arnrnonia in Bisan Strait
                       4- 8-                                                                              2 -0.8

                                                                              0   Phosphate in Bisan
                200- 3- 6-                                                          ral
                                                                                               0
                                                                                                 0
                                                                                      .0
                                                                                        0 0/
                       2 4                                                                                tO - 0.4
                a                                  te.                                                                          Figure 2
                                               inHi hima Bay                                                              Total number of
                E                                                                                                         red tide cases (open
                       1- 2                                                                              *5 - 02
               z             0        Phosphate        ....... 6-1                                                        barsl and of Hetero-
                100 - , @-O-- - -*-* ---- in Hiuchi Nada ....                                                             sigma akashiwo red
                       0 L o@-O   0                                                                      10   0           tides (black bars)
                                                                                                                          and concentrations
                                                                                                                          of nitrogen (open
                                                                                                                          circles and solid
                                                                                                                          lines) and phospho-
                                               lad                                                                        rous (black circles
                                                                                                                          and dashed lines)
                  0             n
                                         1960                 WX                   IM                                     in the Seto Inland
                                                                                                                          Sea (1950-89).







                                                                                                       Honjo: Red Tides of Hekirosigma akashiwo                29


                                                                                                  Red Tide -summer ----O@Autumn


                          0
                                                                                                                                                  Affl
                                    0                                                                                                                 .............
                                                                                              t
                                                      iatoms                           le
                                                                   Ivertical migrat@ion
                                  0       0                                          j                          div day
                                                                             1-2div/dqy      1-2div/da
                                        0                                    (exponential)
                                  0         0              2-5div/day                                                                               0   0
                                                                apid                         lysis
                                 overwintering                                                                                                         0
                                                                                                                      lysis
                     -5         vegetative cells     et     ama                                                                                         0
                     CL                             - -----   - -----
                     0   7m        0      0                                                      ------ 0 --------------        --- Pycnocline     0
                     C3
                                                                      Utilization of nutrients                                                         0
                                          0                           and growth-promoting         \    0  Water mass with
                                     0        germination             substances Veand org       nics       low oxygen content                        0
                                                150C
                                                             _,Release from bottom sediment
                                   esting spores
                         15m-                                                                                                                     Bottom
                                                                                                     resting spores



                                                                                     Figure 3
                                          Schematic diagram of ecological features during the period of red tide development.




                 responds with the switchover of the Japanese economy                        middle layer (Fig. 3). Oxygen conce                'ntrations i    n
                 to a lower rate of developryient. The patterns of                           bottom water decrease rapidly after the formation
                 change-in the total number of tides and the number                          of the pycnocline. The pH of the anoxic bottom
                 of Heterosigma red tides were similar to that of nutrient                   water decreases to about 7. As a result, nutritive
                 concentrations. Thus, the frequency of red tide occur-                      substances (inorganic nutrients, metals, and or-
                 rences is closely related to eutrophication.                                ganic constituents including growth-promoting
                                                                                             substances) are released from the bottom sedi-
                                                                                             ments and these substances accumulate to high
                 Environmental Features During the Period                                    levels in anoxic bottom waters (Honjo 1974). The
                 of Red Tide Development                                                     Heterosigma population has access to these sub-
                                                                                             stances in the bottom water through diurnal
                 Terriperatures suitable for the growth of H. akashiwo                       vertical migration at night.
                 have been reported fr@rri culture experiments to be
                 in the range of 15-30' C (Tomas 1978a; Mori et al.
                 1982; Yamochi 1989). This helps to explain why                              The Development of Heterosigma
                 Heterosig7na red tides tend to occur in coastal waters                      Red Tides
                 from May through late June. However, the range of
                 suitable salinity differs among culture strains: 30%o
                 for a Fukuyama strain (Iwasaki et al. 1968); 10%o for                       Growth originates from cell stocks that overwintered
                 a Gokasho strain (Iwasaki and Sasada 1969); 27-28%o                         as motile forms (Yamochi 1989) and/or that germi-
                 for a Hakata strain (Honjo, and Hanaoka 1973); 12-                          nated from resting cells (Imai et al. Nansei National
                 40%o for a Narragansett strain (Tomas 1978a); and                           Fisheries Research Institute pers. commun. 15 Octo-
                 12-28%o for a Tanig4wa strain (Mori et al. 1982).                           ber, 1990). Tomas (1978b) and Yamochi (1989)
                 This suggests that these strains have developed a                           found that motile cells aggregate and change to
                 physiological acclimation to the range of salinity in                       nonmotile cells. Population growth can be divided
                 each habitat.                                                               into two phases: rapid and exponential (Fig. 3; Honjo
                                    0


                                  0



                                  0
                                          0


                                        0


                                  verwi  ntering
                                  ge tive cell
                                ve 5ta
                                                s
                                          0




                                              g Or
                                                15








































                   Growth of H. akashiwo is usually initiated in early                       and Tabata 1985). The rapid phase is defined as the
                 summer when a pycnocline is formed in the                                   period when cells increase more than four-fold each







           30      NOAA Technical Report NWN I I I

           day and the exponential phase as that period. when           ARelopathy
           the correlation coefficient between log cell number
           and time is greater than or equal to 0.95 and there          The study of allelopathy is important for elucidating
           is an obvious increase in the number of cells for            the mechanisms of temporal succession of phyto-
           more than 4 days. The duration of Heterosig7na red           plankton and of monospecific bloom events.
           tides is shorter than for other flagellates. The             Allelopathic interactions in which organic metabo-
           abrupt disappearance of Heterosigma cells requires           lites of one plant or microorganism suppress or
           further study.                                               enhance the growth of other plants or microorgan-
                                                                        isms are different from. competitive interactions
                                                                        which involve the removal or reduction of certain fac-
                                                                        tors such as water, minerals, foods, and light. The
           Growth Rate and Division Periodicity                         interactions among marine and freshwater phyto-
                                                                        plankton have been well studied and have been
           Blooms of thi .s organism develop so rapidly and dra-        reviewed by Smayda (1980), Maestrini and Bonin
           matically that many workers have studied the growth          (1981), and Rice (1984). Some of the most dramatic
           of H. akashiwo in vitro and in situ as an a proach to        changes of species composition in marine phyto-
                                                       p                plankton have been observed during Heterosigma red
           understanding the enigma of red tide formation.              tide blooms, and allelopathic interactions have been
           Their results, however, have conflicted (Honjo and           observed between H. akashiwo and the centric diatom
           Hanaoka 1973; Tomas 1978a).                                  Skeletonema costatum during in vitro experiments
             Honjo and Tabata (1985) studied Heterosigma                (Pratt 1966; Honjo et al. 1978). Their studies imply
           growth dynamics and division periodicity in outdoor          that an allelopathic relationship between these or-
           tanks. They found that H. akashiwo has a potential for       ganisms occurs during high cell densities of H.
           high growth rates (2-5 divisions per day). There was         akashiwo. Stuart (1972) and Sakshaug (1977) exam-
           a strong tendency for large cells to dominate at the         ined physical features of the allelopathic substances
           beginning of this fast growth phase and to be re-            but experienced difficulty in determining their mo-
           placed by small cells toward the end of the phase.           lecular weight.
           During culture experiments, a portion of a H.                  Recently Honjo et al. (unpubl. data) investigated
           akashiwo patch in a tank was cultured in  a I-liter volu-
           metric flask hung in the tank. The growth rate in this       the biological and chemical features of allelopathic
           experiment was 2.3 divisions per day. Other H.               substances from H. akashiwo (Fig. 4). During a bloom
           akashiwo cells were collected from the tank and indi-        of H. akashiwo in an outdoor tank, an abrupt de-
                                                                        crease in cell numbers of centric diatoms (dominant
           vidually cultured in small tissue chambers. In these         species, S. costatum) and an increase in the dinophyte
           chambers the highest growth rate was 3.3 divisions           Prorocentrum triestinum occurred when high concen-
           per day; the growth rates of large cells inoculated          trations of dissolved carbohydrates were detected in
           into these chambers were much higher than those of           the water. Crude polysaccharide extracts from
           small cells.                                                 Heterosigma cells and from filtrates of the bloom sea-
             In the tank environment, H. akashiwo prefers dark-         water were both separated into two main fractions. by
           associated cell division. Cell division first occurred a     gel chromatography. In bioassay experiments, a mac-
           little before sunset and continued through the night         romolecular fraction from Heterosigma cells greatly
           until the next morning. Similarly, in the small cham-        suppressed the growth of S. costatum at concentra-
           bers cell division began just before the onset of the        tions above 1.0 jig glucose per mL, whereas this same
           dark period and continued for 4-5 hours into the             fraction enhanced the growth of P triestinum and
           next light period. Seven of 24 H. akashiwo cells di-         H. akashiwo and had no effect on the growth of
           vided three times during the night in the small              Phaeodactylum sp. The other fraction caused moder-
           chambers, with a cell division interval of about 6           ate suppression of S. costatum growth. Results of
           hours. Puiseux-Dao (1981) comprehensively reviewed           bioassays using S. costatum and the two fractions from
           the events of the cell cycle and Chisholm (1981) de-         H. akashiwo bloom water were similar to those using
           scribed the chronobiology of cell division in                the fractions from Heterosigma cells. Histochernical
           unicellular algae. In their reviews, the cell division       analysis of the crude polysaccharide extracts from
           cycle was discussed in terms of the length of the cir-       Heterosigma cells indicated a polysaccharide-protein
           cadian period (I division per day), but no                   complex with features analogous to the glycocalyx on
           consideration was given to the rapid division cycles of      the cell surface of the organism. Results suggest that
           H. akashiwo.                                                 a polysaccharide-protein complex exfoliated from







                                                                                                                         Honjo: Red Tides of Hekmsigma akashiwo                           31







                                                                                                                                            fn
                                                                                                                                                           growth
                                                                                                                                      cell                 suppresAion



                                                                         lycocalyx


                                                                                    (seawater)
                                                                                                                                   Heterosi ma           growth
                                                                          excreti  n     polysaccha ride - --a,                    cell                  enhancement
                                                                          exfoliation protein complex




                                                Heterosigme
                                                 red tide



                                                                                                                              Pro ocentrum            growth
                                                                                                                                                      enhancement


                                    I


                                                                                                  Figure 4
                                    General scheme of allelopathic relationships between Heterosigma akashiwo and other phytoplankton.



                    and/or excreted by H. akashiwo is a species-specific                                    Honjo, T., and T. Hanaoka
                                                                                                                  1973. Studies on the mechanisms of red tide occurrence in
                    allelopathic substance and plays an important role in                                           Hakata Bay. 11. General features of the red tide flagellate,
                    causing dramatic changes in cell numbers of other                                               Heterosigma sp. Bull. Plankton Soc. Jpn. 19:75-81.(In
                    phytoplankton during Heterosigma blooms.                                                        Japanese.)
                                                                                                            Honjo, T., and I- Tabata.
                                                                                                                  1985. Growth dynamics of 01isthodiscus luteus in outdoor
                                                                                                                    tanks with flowing coastal water and in small vessels.
                    Citations                                                                                       Limnol. Oceanogr. 30:653-664.
                                                                                                            Honjo, T., T. Shimouse, N. Ueda, and T. Hanaoka.
                    Chisholm, S.W.                                                                                1978. Changes of phytoplankton composition and its charac-
                         1981. Temporal patterns of cell division in unicellular                                    teristics during red tide season. Bull. Plankton Soc. Jpn.
                            algae. In Physiological bases of phytoplankton ecology (T.                              25:13-19. (Injapanese.)
                            Platt, ed.) p.150-181. Can. Bull. Fish. Aquat. Sci. 210.                        Iwasaki, H. and K. Sasada.
                    Conrad, W., and H. Kufferath.                                                                 1969. Studies on the red tide dinoflagellites .11. On Hetero-
                         1954. Recherches sur les eau saumatres des                  environs de                    sig7na inlandica appeared in Gokasho Bay, Shima Penin-
                            Lilloo. Perti 11. Descrictive. Inst. R. Sci. Nat. Belg. Mem.                            sula. Bull.jpn. Soc. Sci. Fish. 35:943-947. (Injapanese.)
                            127:1-346.                                                                      Iwasaki, H., T. Fujiyama, and E. Yamashita.
                    Hara, Y., and M. Chihara.                                                                     1968. Studies on the red tide dinoflagellates I. On
                         1987. Morphology, ultrastructure and taxonomy of the                                       Entomosigma sp. appeared in coastal area of Fukuyama. J.
                            raphidophycean alga Heterosi&a ahashiwo. Bot. Mag. To-                                  Fac. Fish. Anim. Husb. Hiroshima Univ. 7:259-267. (In Japa-
                            kyo 100:151-163.                                                                        nese.)
                    Honjo, T.                                                                               Lackey, J.B., and E. Lackey.
                         1974. Studies on the mechanisms of red tide occurrence in                                1963. Microscopic algae and protozoa in the waters near Ply-
                            Hakata Bay. IV. Environmental conditions during the                                     mouth in August 1962. J. Mar. Biol. Assoc. UK 43:797-805.
                            blooming season and essential factors of red tide                               Lackey, J.B. and K-A. Clendenning.
                                                                                                                                      cell
                                                                                                                               geteros', m2
                                                                                                                                   cell












                                                                                                                                   r






























                            occurrence. Bull. Tokai Reg, Fish. Res. Lab. 79: 77-121.                              1965. Ecology of the microbiota of San Diego Bay,
                            (Injapanese.)                                                                           California. Trans. San Diego Soc. Nat. Hist. 14:9-40.







              32         NOAA Technical Report NMB I I I


              Maestrini, S.Y, and Dj. Bonin.                                                  Stuart, M.
                   1981. Allelopathic relationships between phytoplankton spe-                      1972. The effect of 01isthodiscus luteus Carter upon the
                     cies. In Physiological bases of phytoplankton ecology (T.                        growth of SkeLetonema costatum (Grev.) Cleve. M.S. thesis,
                     Platt, ed.), p.323-338. Can. Bull. Fish. Aquat. Sci. 210.                        Univ. Rhode Island, Kingston, RI, 82p.
              Mori, S., Y. Nakamura, M. Watanabe, S. Yamochi, and M.                          Taylor, F.J.R.
                Watanabe.                                                                           1990. Red tides, brown tides and other harmful algal
                   1982. The effect of various environmental factors on the                           blooms: the view into the 1990s. In Toxic marine phyto-
                     growth yield of red tide algae. 11. 01isthodiscus luteus. Res.                   plankton (E. Grameli, B. Sundstr6m, L. Edler, and D.M.
                     Rep. Nad. Inst. Environ. Stud. 30:71-86. (Injapanese.)                           Anderson, eds.), p.527-533. Elsevier, NY.
              Pratt, D.M.                                                                     Tomas, C.R.
                   1966. Competition between Skeletonema costatum                   and             1978a. 01isthodiscus luteus (Chrysophyceae) 1. Effects of salin-
                     01isthodiscus luteus in Narragansett Bay and in                cul-              ity and temperature on growth, motility and survival. J.
                     ture. Limnol. Oceanogr. 11:447-455.                                              Phycol. 14:309-313.
              Puiseux-Dao, S.                                                                       1978b. 01isthodiscus luteus (Chrysophyceae) II. Formation
                   1981. Cell-cycle events in unicellular algae. In Physiological                     and survival of a benthic stage. J. Phycol, 14:314-319.
                     bases of phytoplankton ecology (Platt, T. ed.), p.130-                         1982. 01isthodiscus luteus (Chrysophyceae) V. Its occurrence,
                     149.. Can. Bull. Fish. Aquat. Sci. 210.                                          abundance and dynamics in Narragansett Bay, Rhode
              Rice, E. L.                                                                             Island. J. Phycol. 16:157-166.
                   1984. Allelopathy. Acad. Press. London, 422p.                              Yamochi, S.
              Sakshaug, E.                                                                          1989. Mechanisms for outbreak of Heterosigina akashiwo red
                   1977. Limiting nutrients and maximum growth rates for dia-                         tide in Osaka Bay, Japan. Bull. Osaka Pref. Fish. Exp. Stat.
                     toms in Narragansett Bay. J. Exp. Mar. Biol. Ecol. 28:109-123.                   8:1-110. (In Japanese.)
              Smayda, T. J.                                                                   Yokote, M., T. Honjo, and M. Asakawa.
                   1980. Phytoplankton species succession. In The'physiologi-                       1985. Histochernical demonstration of a glycocalyx on the
                     cal ecology of phytoplankton (I. Morris, ed.), p.493-                            cell surface of Heterosigma ahashiwo. Mar. Biol. 88:295-299.
                     570. Blackwell, London.






                   Impact of the Myxosporean Parasite Ceratomyxa shasta on Survival
                                      of Migrating Columbia River Basin Salmonids


                                            J.L. BARTHOLOMEW, J.L. FRYER, andj.S. ROHOVEC*
                                                                    Department of Microbiology
                                                                      Oregon State University
                                                                   Corvallis, Oregon 97331-3804




                                                                          ABSTRACT


                                    Columbia River Basin salmonids are exposed to the parasite Ceratomyxa shasta during
                                  both their seaward and return migrations. The impact of ceratomyxosis on the survival of
                                  migrating fish is difficult to assess because there are few data on causes of fish mortality
                                  once they are released from the hatchery. This study examines the impact of this disease
                                  on juvenile salmonids by 1) sampling the downstream migrants to determine what per-
                                  cent of fish leaving the basin are infected with the parasite and by 2) determining the
                                  effects of entering salt water on the progress of the infection. Results of comparisons of
                                  methods for diagnosing a C. shasta infection indicate that serological techniques using
                                  monoclonal antibodies are more sensitive than techniques for examination of spores.



               Introduction                                                         distribution Uohnson et al. 1979; Hoffmaster et al.
                                                                                    1988) indicate that the parasite has spread within the
               The Columbia River Basin has long supported an im-                   Columbia River Basin, but the extent and cause of
               portant Pacific salmon fishery for Oregon,                           expansion is not yet known. The degree of resistance
               Washington, and Idaho. However, the resource has                     to infection among salmonid fish within the basin has
               steadily declined even with supplementation of fish                  also been examined and compared with resistance
               from hatcheries. As research efforts are directed to-                among fish populations from watersheds where the
               ward determining the causes behind the depletion of                  parasite is not found (Zinn et al. 1977; Buchanan et
               Columbia River Basin salmonid stocks, the impact of                  al. 1983). All groups of fish tested from the Columbia
               diseases must be considered. One disease that is enzo-               River Basin were relatively resistant. However, the
               otic to the basin is caused by the myxosporean parasite              ability of many wild and upriver stocks to resist infec-
               Ceratomyxa shasta. Although this parasite has devas-                 tion has not been examined.
               tated certain hatchery productions, it is not normally a                Further investigations of the impact of
               disease of hatchery fish; therefore, its impact on sur-              ceratomyxosis on migrating          Columbia River salmo-
               vival of fish in the wild is difficult to evaluate. The              nids were designed to answer 1) how many Columbia
               parasite first infects and multiplies in the intestinal              River salmonids become infected during their down-
               tract of the fish and from that site spreads to other                stream migration, 2) how the disease progresses after
               tissues. The infection results in tissue necrosis accom-             the fish enter salt water, and 3) how effective are the
               panied by a severe host inflammatory reaction. Signs                 methods currently used to diagnose ceratomyxosis.
               of infection may include lethargy, darkening of the
               body surface, abdominal distension, and hemorrhag-
               ing in the area of the vent (Bartholomew et al. 1989a).              Materials and Methods
               In addition, the disease has a prolonged incubation
               period and current diagnostic methods (Amos 1985)
               detect the parasite only in heavily infected fish.                   Examination of Downstream Migrants for
                 Assessing the impact of ceratomyxosis requires ex-                 Infection
               amining a variety of factors. Studies of geographic
                                                                                    To estimate how many salmonids become infected
                Send correspondence to this author                                  during their downstream migration, outmigrants

                                                                                                                                                   33







            34      NOAA Technical Report NWS I I I




                                                       Cross section
                                                       Rock                                                                  Sand
                                                      Cliff                      Puget               Ship                    beach
                                                                                 Island              channel
                                                       A             7 m               7 rn    9M 11ml4mlim 7m                  B


                                                                -*-600 rn-i-I                         1200 rn I
                                                                                                   950 rn ------------- V,-




                                                     X.
                                             C11


                                                                             A
                                                                                           WASHINGTON
                     Puget Island
                                                                                                                                River
                                                                                                                                flow
                                                                                              Cape
                                                                                              -.Horn'
                                                                                                                      A-
                                                                                                              coo oll
                                                                           Jetty
                                                                                 v e  r
                                              C 0     U M
                               0.0 cy,
                                           14mdepth 200rnwidth
                                                                        7i-C. p in
                                  - - - - - - - - - - - - - -

                                                                                                         n
                                                                                                    e:                        U
                                                                                                                           10
                                                                                                                       e
                                                                                                                      C
                                                                                                               W a
                                                     'Fish proceisin
                                                     'facility
                                                                                                               U.


                                                      OREGON
                                                                                                  0          1
                                                                                                  I
                                                                                                    Kitometers




                                                                      Figure I
             Jones Beach sampling site. The beach and purse seining areas are indicated by the two asterisks on the main map. From
             Dawley et al. (1984a).


            were collected just prior to entering the estuary and             tember in 1984. In 1984, fish were collected by beach
            were maintained in fresh water to monitor disease                 seine only. All fish were transported to the Round
            development. Outmigrating juvenile salmonids of dif-              Butte Hatchery Isolation Facility on the Deschutes
            ferent species and year classes were obtained by                  River, a facility operated by the Oregon Department
            beach and purse seine from a collection facility oper-            of Fish and Wildlife. Holding tanks were I-m
            ated by the National Marine Fisheries Service at                  circulars with a 375-L capacity. The water supply was
            Jones Beach (75 river km, measured upstream from                  free of pathogens and the water temperature was
            the mouth of the Columbia River) on the Oregon                    10' C. All groups of fish were fed an Oregon Moist
            side of the Columbia River (Fig. 1). Seining proce-               Pellet diet containing 3% terramycin in the form of
            dures were those described by Dawley et al. (1984a).              TM50 (Pfizer) as a prophylactic measure against bac-
            A purse seine 206 m long and 11 m deep with a mesh                terial fish pathogens (Udey et al. 1975). Groups were
            of 1-2 cm was used in water about 9 m deep. The                   held for at least 150 days and observed for develop-
            seine was set drifting with the current, then towed               ment of ceratomyxosis. Fish deaths occurring within
            upstream for 5 minutes before closing and pursing.                10 days aft@r transportation were attributed to han-
            Beach seining with a net 95 m long, 5 m deep, with a              dling mortality and were not included in the results.
            mesh of 1-2 cm, was done in water about 6 m deep at                  In all experiments, dead fish were collected daily,
            the outer end of the net sweep. Fish were collected               and either immediately necropsied or frozen for later
            on 12 occasions between 20 May and 8 September in                 examination. All fish held in tanks that remained at
            1983 and on six occasions between 5 July and 20 Sep-              the end of the observation period were killed and







                                                                Bartholomew et al: Impact of Cera"xa shasto on Salmonid Survival         35

                examined for C. shasta. Wet mounts of intestinal tract           supplied with 126 C pathogen-free water until they
                scrapings were examined microscopically (as detailed             reached smolt stage. Fish were exposed to the infec-
                later) for up to 5 minutes, and samples containing               tious stage of C shasta in     'the Willamette Ri@,er    at
                spores of C shasta were considered infected.                     Corvallis, Oregon, on three occasions. On the first
                                                                                 occasion, two groups of 50 Alsea steelhead trout
                                                                                 were exposed: one group for 3 days and the other
                Determining Saltwater Effects                                    for 5 days. On the second occasion, groups of 50
                                                                                 Alsea steelhead trout and 50 Big Creek coho salmon'
                Three strains of salmonid smolts were used to deter-             were held for 5 days. On the third *occasion, 100
                mine the effects of salt water on the progress of                Alsea steelhead trout and 100 Round Bu       tte chinook
                ceratomyxosis.        Big      Creek    coho       salmon        salmon were exposed for 3 days. Control groups of
                (Oncorhynchus kisutch) were from Big Creek Hatch-                an equal number of each species were not exposed
                ery located on the lower Columbia River and Round                in the river. After exposure, the groups were divided
                Butte chinook salmon (0. tshawytscha) Were from                  and half of the fish were transported to freshwater
                the Round Butte Hatchery on the Deschutes River, a               holding facilities at the OSU-FDL and the other half
                Columbia River tributary. Both strains migrate                   to ultraviolet-treated saltwater facilities at the Mark
                through waters enzootic for C. shasta and are rela-              0. Hatfield Marine Science Center in Newport, Or-
                tively resistant to the parasite (Zinn et al. 1977;              egon. The fish were fed Oregon Moist Pellets
                Johnson e.t al. 1979). Alsea steelhead trout (0.                 containing 3% terramycin in the form of TM               50
                mykiss) were taken from the Alsea Hatchery. The                  (Pfizer). All groups were held for at least 100 days.
                Alsea River empties directly into the Pacific Ocean
                and does not harbor the infectious stage of C
                shasta. Salmonids from this system have not devel-               Comparison of Detection Methods
                oped resistance. All fish were held at the Oregon
                State University-Fish Disease Laboratory (OSU-FDL)               The sensitivity of the standard detection method of
                in 1-m diameter tanks of 375 L capacity and were                 examining wet mounts of material from the lower





                                                                           Table I
                   Prevalence of Ceratomyxa shasta in chinook salmon smolts beach seined from the Columbia River at Jones Beach,
                    Oregon.

                                                                                                                        Percent of
                      Date                                                                      Mortalities           fish collected
                      collected                   Number                   Holding             infected with          infected with
                      (1983)                      collected,             mortalities              C  shasta              C shasta


                      May  20                        91                       3                      3                       3
                           27                        81                       6                      1                       1


                      June    3                      75                       8                      2                       3
                           10                        53                       1                      1                       2
                           17                        65                     36                       3                       5
                           '24                       130                    21                       3                       2


                      July    1                      113                    24                     17                      15
                           15                        141                    46                     17                      12
                           29                        68                     33                       2                       3


                      Aug. 12                        109                    36                     13                      12
                           26                        34                     20                       8                     24


                      Sept. 8                        112                    39                     25                      22


                      Total                          1072                  273                     95                        9

                   aNumber collected minus holding mortality during the first 10 days.







             36              NOAA Technical Report NNM I I I



                                                                                    Table 2
                Prevalence of Ceratomyxa shasta in chinook salmon smolts purse seined from the Columbia River at Jones Beach,
                 Oregon.

                                                                                                                                           Percent of
                    Date                                                                                      Mortalities                fish collected
                    collected                          Number                       Holding                  infected with               infected with
                    (1983)                             collecteda                mortalities                   C shasta                     C shasta

                    May      .20                             9                          5                           3                          33
                             27                           37                            6                           2                            5


                    June     10                           58                          17                            0                            0
                             17                           47                          18                            4                            9
                             24                           98                          25                            8                            8


                    July     1                            128                         46                          21                           16
                             15                           43                          38                            5                          12
                             29                           47                          33                          11                           23


                    Aug.     12                           47                          45                            4                            9
                             26                           21                          18                            1                            5


                    Sept.    8                            14                          12                            2                          14


                    Total                                 549                       263                           61                           11


                 Number collected minus holding mortality during the first 10 days.




                                                                                    Table 3
                Prevalence of Ceralomyxa shasta in coho salmon smolts purse seined from the Columbia River atJones Beach, Oregon.

                                                                                                                                           Percent of
                    Date                                                                                      Mortalities                fish collected
                    collected                          Number                       Holding                  infected with               infected with
                    (1983)                             collected"                 mortalities                   G shasta                    C. shasta

                    May      20                           82                          13                            4                            5
                             27                           38                            4                           3                            8


                    June     3                            23                            1                           0                            0
                             10                              5                          4                           1                          20
                             17                              1                          2                           0                            0
                             24                           12                            2                           1                            8


                    July     1                               1                          0                           0                            0
                             15                              1                          0                           0                            0


                    Aug. 26                                  2                          0                           0                            0

                    Total                                 179                         26                            9                            5


                  Number collected minus holding mortality during the first 10 days.



             intestinal wall (Amos 1985) was compared with detec-                           fish. The first smear was examined for spores as a
             tion of C. shasta by serological methods (Bartholo-                            wet mount at 40OX magnification for a maximum of
             mew et al. 1989b). The lower portion of the intestine                          5 minutes. The second smear was air-dried, fixed in
             was excised from 121 adult salmon that had died                                1:1 acetone:xylene solution, and incubated for 15
             prior to spawning. Two smears were made from each                              minutes with a monoclonal antibody specific for







                                                                               Bartholomew et al: Impact of Ceratomyza shasta on Salmonid Survival                       37



                                                                                           Table 4
                        Prevalence of Ceratomyxa shasta in steelhead trout smolts purse seined from the Columbia River at Jones Beach,
                        Oregon.

                                                                                                                                                   Percent of
                            Date                                                                                      Mortalities                 fish collected
                            collected                          Number                      Holding                   infected with                infected with
                            (1983)                            collecteda                  mortalities                    C  shasta                  C shasia

                            May      20                           11                             5                          2                          18
                                     27                           13                             4                          0                            0


                            June     3                            45                          13                            6                          13
                                     10                              4                           2                          0                            0
                                     24                              1                           1                          1                          100


                            July     I                               I                           1                          0                            0


                            Total                                 75                          26                            9                          12


                        'Number collected minus holding mortality during the first 10 days.






                                                                                           Table 5
                        Prevalence of Ceratomyxa shasta in chinook salmon smolts beach seined from the Columbia River at Jones B,Zch,
                        Oregon.

                                                                                                                                                   Percent of
                            Date                                                                                      Mortalities                 fish collected
                            collected                         Number                       Holding                   infected with                infected with
                            (1984)                            collected'                  mortalities                   C shasta                    C shasta

                            July     5                            88                          76                            6      1                     7
                                     26                           75                          22                          12                           16
                                     31                           84                          31.                         14                           17


                            Aug.     16                           82                          34                          11                           13
                                     23                           87                          74                          13                           15


                            Sept.    20                           47                          29                            9                          19


                            Total                                 463                        266                          65                           14


                        'Number collected minus holding mortality during the first 10 days.




                    prespore stages of C shasta. Specific antibodies were                           Results
                    detected using biotinylated horse anti-mouse IgG
                    and fluorescein-conjugated avidin D (Vector Labora-
                    tories, Burlingame, CA). Methyl green dye (1% in                                Infections in Downstream Migrants
                    distilled water) was used as a counterstain. Smears
                    were examined with a Zeiss standard microscope                                  Chinook salmon were the largest group collected in
                    containing an IV epifluorescence condensor at                                   1983; 1072 subyearling smolts were captured in
                    250X magnification until prespore or spore stages                               beach seines and 549 yearling chinook salmon were
                    of C shasta were detected or for three minutes. All                             taken in purse seines. Smaller numbers of coho
                    microscopic examinations were made by the same                                  salmon (179) and steelhead trout (75) were also cap-
                    individual.                                                                     tured in purse seines. Ceratomy'pca shasta was present in
                                                                                                                                          A







             38         NOAA Technical Report NWS I I I



                                                                                      Table 6
                  Effects of salt water on steelhead trout and coho and chinook salmon exposed to the infectious stage of Ceratomyxa
                  shasta.


                                                                                      Fresh water                                   Salt water
                                                    Exposure

                                                     length       N6. offish          No. of fish   Percent        No. of fish     No. of fish       Percent
                  Salmonid                           (days)       recovered'          infected      infected       recovered,       infected         infected


                  Alsea steelhead trout                 36             21                21            100              6               3                50
                                                        31             24                24            100              37              37               100
                                                     controld          25                0              0               11              0                0
                                                        5b             23                23            100              13              7                54
                                                        5`             18                18            100              9               8                89
                                                     con trold         25                0              0               16              0                0


                  Big Creek coho salmon                 5b             25                1              4               25              0                0
                                                     controld          25                0              0               25              0                0


                  Round Butte                           31             27                0              0               30              0                0
                  chinook salmon                     controld          25                0              0               27              0                0


                  aNumber of fish exposed minus number of fish that died before spores were detected.
                  'Fifty fish exposed; 25 were transferred to fresh water and 25 to salt water.
                  One hundred fish exposed; 50 were transferred to fresh water and 50 to salt water.
                  d Control fish were not exposed to the infectious stage of C shasta.








             1-24% of the individuals of the chinook salmon                                  Effects of Salt Water
             groups caught by beach seine (Table 1). The preva-
             lence of infection was between 1-3% in groups                                   All Alsea steelhead trout held in fresh water after ex-
             collected from May'through June but increased to                                posure to C shasta died from ceratomyxosis (Table
             12-24% from July to the final sample period in Sep-                             6)'. In groups of Alsea trout transferred to salt water,
             tember. The prevalence of infection among yearling,                             between 26 and 76% of the fish died prior to devel-
             purse-seined chinook salmon also showed a tendency                              opment of the disease; losses of 36 to 56% of
             to increase during the later collection periods (Table                          unexposed, control fish held in salt water indicated
             2). Among the yearling chinook salmon, the preva-                               that these deaths were caused by inability.of the fish
             lence of infection in groups collected from May                                 to adjust to saltwater conditions. The prevalence of
             through June was generally less than 10%, except for                            infection among fish surviving the prepatent losses in
             a 20 May collection where three of. nine fish devel-                            salt water was lower in two of             'the four groups than
             oped ceratomyxosis. Infection incidence after July                              among fish transferred to fresh water. Big Creek
             averaged 15%. The total incidence of C. shasta infec-                           coho and Round Butte chinook salmon were resis-
             tion among chinook salmon was 9% for beach-seined                               tant to infection when held in either fresh or salt
             groups and 11% for purse-seined groups, Smaller                                 water after exposure to C. shasta.
             numbers of coho salmon and steelhead trout were
             collected in May and early June, during the peak of
             their migration. The prevalence of infection for these                          Sensitivity of Detection Methods
             species was 5 and 12%, respect             'ively (Tables 3 and 4).
                  In 1984, 463 chinook salmon smolts were caught in                             Detection of C shasta infections by standard wet
             beach seines between 5 July and 20 September. Infec-                            mount examination of intestinal tract scrapings was
             tion incidence among these subyearling salmon                                   less sensitive than detection by indirect fluorescent
             averaged 14% (Table 5).                                                         antibody techniques (IFATs) in which a monoclonal






                                                                    Bartholomew et al: Impact of Ceratomyza shasta on Salmonid Survival           39



                                                                               Table 7
                    Comparison of detection sensitivity between wet mount examination for spores and indirect fluorescent antibody
                    techniques (IFAT) using monoclonal antibodies against Ceratomyxa shasta prespore stages.

                                                                                      Wet mount                                IFAT
                    Sample origin'                     No. samples           No. positive       % positive        No. positive        % positive

                    Spring chinook salmon
                      Willamette Hatchery                   20                    8                 40                 19                 95
                      Clackamas Hatchery                    27                    16                59                 25                 93

                    Fall chinook salmon
                      Bonneville Hatchery                   40                    7                 17                 14                 35

                    Coho salmon
                      Big Creek Hatchery                    14                    14                100                14                 100
                      Cascade Hatchery                      20                    17                85                 20                 100

                    Total                                   121                   62                51                 92                 76


                     Adult prespawning mortalities.




                 antibody was used. Of 121 intestinal samples exam-                   among Columbia River strains of chinook salmon
                 ined where both methods were used, 51% of the                        and steelhead trout was generally less than 5%, even
                 samples were diagnosed as positive for the presence                  when fish were exposed to the infectious stage for
                 of spores by the wet mount technique and 76% were                    120 days. However, of 2084 chinook salmon smolts
                 diagnosed positive for the presence of prespore and                  collected ationes Beach during 1983-84, 221 fish or
                 spore stages using IFATs (Table 7).                                  10.6% died from ceratomyxosis. The number of fish
                                                                                      infected did not vary significantly between year
                                                                                      classes, as the prevalence in yearling purse-seined
                 Discussion                                                           chinook salmon paralleled that of the subyearling
                                                                                      chinook salmon collected in beach seines. The preva-
                 Evaluating the impact of ceratomyxosis on salmonid                   lence also did not vary significantly between the 2
                 populations requires consideration of a variety.of fac-              years of this study, although the number and type of
                 tors. Two of these, geographic distribution of the                   samples collected were limited during 1984, but
                 parasite and resistance to infection of resident salmo-              there was a trend toward increasing pumbers of in-
                 nids, have been examined in previous studies (Zinn                   fected fish from the later collection dates.
                 et al. 1977; Johnson et al. 1979; Buchanan et al. 1983;                The dates of peak migration at Jones Beach were
                 Hoffmaster et al. 1988). Past studies have demon-                    determined by Dawley et al. (1984b) for each species.
                 strated the presence of the infectious stage of C.                   Coho salmon and yearling chinook salmon and steel-
                 shasta in the mainstem of the Columbia and Snake                     head trout migrated through the lower Columbia
                 Rivers and have shown that parasite presence and                     River during May and early June. Migration of
                 concentration vary between locations. There is also                  subyearling chinook salmon past Jones Beach began
                 evidence that its distribution has increased within the              to increase during May but reached its peak from
                 basin. Because all salmonids migrating in the Colum-                 early June to mid-July. This means that the majority
                 bia River Basin are exposed to C. shasta, native strains             of subyearling chinook salmon were migrating dur-
                 have developed some resistance to infection. How-                    ing periods of increasing water temperature when
                 ever, resistance is not complete in the' fish                        the impact from C shasta is greater (Udey et al.
                 populations studied and little is known about the sus-               1975). Dawley et al. (1984b) also determined that
                 ceptibility of feral and upper Columbia and Snake                    migration rates were 22, 18, 17, and 35 km/day for
                 River salmonids to infection by C. shasta.                           subyearling chinook, yearling chinook and coho
                    The prevalence of infection among outmigrating                    salmon, and steelhead trout, respectively. Therefore,
                 fish was higher than the incidence predicted from                    the duration of exposure to C. shasta for subyearling
                 susceptibility studies. In studies by Zinn et al. (1977)             and yearling salmon and steelhead trout migrating
                 and Buchanan et al. (1983), the infection incidence                  from Oxbow Dam on the Snake River, a distance of







           40      NOAA Technical Report NWS I I I

           over 1000 river miles from the Columbia Estuary,.               ity was 100% in both groups. Similar experiments
           could be as long as 43, 53, and 27 days, respectively.          were conducted in this study using shorter exposure
           Coho salmon originate from the lower Columbia                   lengths and both susceptible and resistant strains of
           River and therefore have a shorter time of exposure             salmonids. Although survival was poor, in C. shasta-
           to the parasite. Long periods of exposure to C. shasta          susceptible steelhead trout transferred to salt water
           combined with the stress of migration and                       after exposure, two of four groups had a lower inci-
           smoltification may explain why the numbers of                   dence of infection than groups transferred to fresh
           chinook salmon and steelhead.trout infected in our              water. It appears that migration to salt water may re-
           study are higher than numbers predicted from sus-               duce the progress of ceratomyxosis if the fish are not
           ceptibility studies. Also, it was not possible in this          overwhelmed by a large infectious dose. Entry into
           study to determine the strain origin of the infected            salt water did not impair the ability of resistant fish to
           fish so their resistance status cannot be assumed.              block the infection.
           Studies by Ratliff (1981) and Ching and Munday                    While all of the studies described here show the
           (1984) demonstrate that fish originating from rivers            impact of ceratomyxosis on captured fish held under
           enzootic for the parasite can still be highly suscep-           laboratory conditions, many field studies rely on sam-
           tible to infection. Ching and Munday exposed                    pling, fish prior to overt signs of ceratomyxosis.
           chinook salmon representing six stocks from the                 Reports by Yasutake et al. (1986) and Bartholomew et
           Fraser River, British Columbia, to the infectious stage         al. (1989a) indicate that spores do not form until late
           of C shasta in the lower Fraser River. Between 87 and           in the infection and that, in cases of acute
           100% of the fish in each group died from                        ceratomyxosis, fish may die before spore develop-
           ceratomyxosis. Ratliff (1981) further demonstrated              ment. Therefore, diagnosis made on the basis of
           that prevalence of infection may increase with in-              identifying spores is likely to underestimate the
           creasing length of exposure to C shasta and he                  prevalence of the parasite. To demonstrate this, an
           calculated that 50-70% of all chinook salmon re-                IFAT utilizing monoclonal antibodies specific for
           leased into the Deschutes River may become                      prespore stages of C shasta was compared with ex-
           infected. Prior to changes in the Columbia River Ba-            amination by standard wet mount procedures. When
           sin that resulted from the building of dams, upriver            both methods were used to examine samples col-
           salmonids may also have avoided ceratomyxosis by                lected from prespawning adult salmon mortalities,
           migrating through infectious areas before parasite              there was a 25% increase in the number of infections
           concentrations were high. However, the presence of              detected by using the serological method. An even
           dams and reservoirs has impeded outmigration,                   greater increase in sensitivity could be expected
           raised water temperatures (Raymond 1979) and may                when techniques are developed which can detect the
           have created conditions favorable for proliferation of          earlier stages of C. shasta infection. Monoclonal anti-
           C. shasta (Ratliff 1981). These changes may have                bodies and DNA probes that specifically recognize all
           caused the range and numbers of the parasite to in-             life stages of the parasite are necessary for an accu-
           crease more rapidly in the upper portions of the                rate evaluation of the numbers of fish infected with
           basin than fish could adapt by developing resistance            C shasta.
           or avoidance strategies. Therefore, strains of salmo-
           nids from the upper Columbia and Snake Rivers may
           have lower or more variable levels of resistance than           Acknowledgments
           the strains that have been examined.
             Because ceratomyxosis has a long incubation pe-               The authors acknowledge the technical help of J.E.
           riod, most salmonids that become infected during                Sanders, C.A. Arakawa, W. Moynihan, D.E. Ratliff,
           their downstream migration enter the ocean before               and E.M. Dawley. This publication is the result of re-
           the disease results in death. Acute ceratomyxosis has           search sponsored by the Bonneville Power
           been reported in juvenile chum salmon (0. keta) cap-            Administration under contract No. DE-A179-83 BP
           tured off the coast of British Columbia (Margolis and           11987; G.R. Bouck, Contracting Officer's Technical
           Evelyn 1975). This finding indicates that the disease           Representative; and, in part, by Oregon Sea Grant
           is not attenuated when fish enter salt water. To dem-           with funds from the National Oceanic and Atmo-
           onstrate this under laboratory conditions, Ching and            spheric Administration, Office of Sea 'Grant,
           Munday (1984) exposed chinook salmon to the infec-              Department of Commerce under grant No. NA89AA-
           tious stage of C. shasta for 10 days, then held the fish        D-SGIO8. This is Oregon Agricultural Experiment
           in either fresh or salt water. They found that mortal-          Station Technical Paper No. 9491.






                                                                            Bartholomew et al- Impact of Ceratomyxa shasta on Salmonid Survival                    41
                                                                                                                         r

                   Citations                                                                    Hoffmaster, J.L., J.E. Sanders, J.S. Rohovec, J.L. Fryer, and D.G.
                                                                                                  Stevens.
                   Amos, KH., (ed.).                                                                1988. Geographic distribution of the myxosporean parasite,
                        1985. Procedures for the detection and identification of cer-                  Ceratomyxa shasta Noble, 1950, in the Columbia River basin,
                           tain fish pathogens, 3rd ed. Am. Fish. Soc. Fish Health                     U.S.A. J. Fish Dis. 11:97-100.
                                                                                                Johnson, ILA.,J.E. Sanders, and].L. Fryer.
                           Section, 114 p..
                   Bartholomew, J.L., C.E. Smith,J.S. Rohovec, andj.L. Fryer.                       1979. Ceratomyxa shasta in salmonids. U.S. Fish Wildl. Serv.,
                        1989a. Characterization of a host response to the                              Fish Dis. Leafl. 58, 11 p.
                                                                                                Margolis, L., and T.P.T. Evelyn.
                           myxosporean parasite, Ceratomyxa shasta (Noble), by histol-              1975. Ceratonyxa shasta (Myxosporida) disease in chum
                           ogy, scanning electron microscopy and immunological
                           techniques. J. Fish Dis. 12:509-522.                                        salmon (Oncorhynchus keta) in British Columbia. J. Fish.
                   Bartholomew, J.L.,J.S. Rohovec, andj.L. Fryer.                                      Res. Board Can. 32:1640-1643.
                        1989b. Development, characterization, and use of mono-                  Ratliff, D.E.
                           clonal and polyclonal antibodies against the myxosporean,                1981. Ceralmnym shasta: epizootiology in chinook salmon of
                           Ceratomyxa shasta. J. Protozool. 36:397-401.                                centralOregon. Trans. Am. Fish. Soc. 110:507-513.
                   Buchanan, D.V.,J.E. Sanders,J.L. Zinn, andj.L. Fryer.                        Raymond, H.L.
                        1983. Relative susceptibility of four strains of summer steel-              19 .79. Effects of dams and impoundments on migrations of
                           head to infection by Ceratomyxa shasia. Trans. Am. Fish.                    juvenile chinook salmon and steelhead from the Snake
                           Soc. 112:541-543.                                                           River, 1966 to 1975. Trans. Am. Fish. Soc. 108:505-529.
                                                                                                Udey, L.R.,J.L. Fryer, and ILS. Pilcher.
                   Ching, H.L., and D.R. Munday.
                        1984. Susceptibility of six Fraser chinook salmon stocks        to          1975. Relation of water temperature to ceratomyxosis in
                           Ceratom xa shasta and the effects of salinity on                            rainbow trout (Salmo gairdneri) and coho salmon
                                   Y                                                                   (Oncorhynchus kisutch). J. Fish. Res. Board Can. 32:1545-
                           ceratomyxosis. Can.J. Zool. 62:1081-1083.                                   1551.
                   Dawley, E.M., R.D. Ledgerwood, T.H. Blahm, R.A. Kirn, A.E.                   Yasutake, W.T., J.D. McIntyre, and A.R. Hemmingsen.
                      Rankis, and F.J. Ossiander.                                                   1986. Parasite burdens in experimental families of coho
                        1984a. Migrational characteristics and survival of juvenile                    salmon. Trans. Am. Fish. Soc. 115:636-640.
                           salmonids entering the Columbia River estuary during                 Zinn,J.L., ICA. Johnson, J.E. Sanders, andl.L. Fryer.
                           1982. Annual report of research by the Coastal Zone and                  1977. Susceptibilty of salmonid species and hatchery strains
                           Estuarine Studies Division, NMFS, January 1984, 49 p.                       of chinook salmon (Oncorhynchus tshauytscha) to infections
                   Dawley, E.M., R.D. Ledgerwood, TH. Blahm, R.A. Kirn, and A.E.                       by Geratomyxa shasta. J. Fish. Res. Board Can. 34:933-936.
                      Rankis.
                        1984b. Migrational characteristics and survival of juvenile
                           salmonids entering,the Columbia River estuary during
                           1983. Annual report of research by the Coastal Zone and
                           Estuarine Studies Division, NMFS, July 1984, 88 p.






                                           Viral Infections of Cultured Fish in Japan



                                                 MAMORU YOSHIMIZU and TAKALHISA KIMURA

                                                                     Laboratory of Microbiology
                                                                        Fa cu Ity of Fisheries
                                                                 Hokkaido University, Minato 3-1-1
                                                                  Hakodate, Hokkaido 041,japan





                                                                          ABSTRACT


                                     Since infectious pancreatic necrosis virus and infectious hematopoietic necrosis virus
                                   were first isolated in the 1970s, more than 20 fish viruses have been isolated and at least 5
                                   viruses have been observed by electron microscopy. Viral diseases are major problems
                                   and cause economic losses among cultured fishes in Japan and other countries. This
                                   paper reports our current understanding of the extent of viral infection in the cultured
                                   fishes of Japan.


                Introduction                                                         and masu salmon (0. masou). Recently, the fry of
                                                                                     rainbow trout were observed to be less susceptible to
                  A virological   study of cultured fishes in Japan was              IPN-V and, consequently, damage attributed to IPN
                initiated when    an unknown disease occurred. among                 has decreased (Okamoto et al. 1987).
                rainbow trout    Oncorhynchus mykiss in the 1960s. The
                causative agent was identified as infectious pancreatic
                necrosis virus (IPNV) (Sano 1971). Subsequently, in-                 Infectious Hematopoietic Necrosis
                fectious hematopoietic necrosis virus .(IHNV) was
                isolated from kokanee salmon 0. nerka (Kimura and                    Infectious hematopoietic necrosis is an acute sys-
                Awakura 1977). Since then, various viral infections                  temic disease which mainly affects the fry of rainbow
                of fish have been reported. At present, more than 20                 trout and masu and kokanee salmon but which has
                fish viruses have been isolated and at least 5 viruses               also been isolated from moribund ayu Plecoglossus
                have been observed by electron microscopy studies                    altivelis (Yoshimizu et al. 1987c). The characteristic
                (Table 1).                                                           sign of IHNV infection is V-shaped hemorrhages
                                                                                     located in muscle tissue. Recently, a large rainbow
                                                                                     trout, with a body weight of 50-80 g was found to be
                Viral Diseases of Salmonid Fishes                                    infected with IHNV and subsequently died (Mori et
                                                                                     al. 1987). In this case, petechiae were observed in the
                                                                                     fatty tissues and on the wall of the body cavity. This
                Infectious Pancreatic Necrosis                                       virus is widespread and especially prevalent in the
                                                                                     central part of Honshu, the Japanese mainland (Sano
                Infectious pancreatic necrosis is an acute systemic                  et al. 1977). In several districts, river waters have
                disease affecting the fry and fingerlings of rainbow                 been contaminated with IHNV and are now unsuit-
                trout. Its occurrence is widespread in Japan. Suscep-                able for rainbow trout culture. Although the vertical
                tibility of fish to IPNV depends on body weight;                     transmission of IHNV is doubtful (Yoshimizu et al.
                smaller fry are more susceptible. The signs of this                  1988, a and b), it can be controlled by disinfecting
                disease are darkening of body coloring, moderate                     eggs with iodine during the early eyed stage. Fish at
                exophthalmia, and abdominal distention. Internally,                  the fry stage are very. susceptible to IHNV. They
                the spleen, heart, liver, and kidneys are pale and the               should be reared in either well water or ultra-violet
                digestive tract is almost always devoid of food. Rain-               irradiated river water. When fish are past this sensi-
                bow trout is the fish most affected by IPNV, but the                 tive stage, they can be transferred to the usual
                virus has also been isolated from amago (0. rhodurus)                rearing ponds.


                                                                                                                                                  43







              44        NOAA Technical Report NWS I I I

                                             Table I                                         showed that OMV is enzootic in the northern part,of
                       Viral infection in cultured fishes in Japan.                          Japan (Yoshimizu et al. 1988b) and that the charac-
                                                                                             terist.ics of these three herpesviruses are similar
                 Isolated virus                                     Host                     except that NeVTA lacks oncogenicity (Hedrick et al.
                                                                                             1987; Sano et al. 1988). In 1983, we recommended
                 DNA virus                                                                   the disinfection of fish eggs with iodine at the early
                   Nerkavirus in Towada Lake,                                                eyed stage in Hokkaido. Now OMV cannot be de-
                     Aomori and Akita                                                        tected in most of the hatcheries in this area
                     Prefecture (NeVTA)                   Kokanee salmon                     (Yoshimizu et al. 1988b). Although the host species
                   Oncorhynchus masou virus (OMV)         Masu salmon                        of this virus is primarily masu salmon, OMV has also
                   Yarname tumor virus (M)                Yamame (masu salmon)
                   lcosahedral cytoplasmic                                                   been isolated from the tumor tissues of pen-cultured
                     deoxyribovirus (ICDV)                Japanese eel                       coho salmon, 0. kisutch.
                   He?pesvims typyini                     Fancy carp
                   Herpesvirus                            Japanese eel
                   Unidentified small virus               Tiger puffer                       Chum Salmon Virus Infection
                 RNA virus
                   Infectious pancreatic necrosis
                     virus (IPNV)                         Salmonid fish                      In 1978, a reovirus was isolated from an apparently
                   Infectious hematopoietic                                                  normal adult chum salmon, 0. keta returning to its
                     necrosis virus (IHNV)                Salmonid fish                      hatchery in Hokkaido (Winton et al. 1981). After ini-
                   Chum salmon virus (CSV)                Masu salmon
                   Yellowtail ascitic virus (YAV)         Yellowtail                         tial isolation and characterization, it was named
                   Rhabdovirus olivaceus (HRV)            Various marine fish                chum salmon virus (CSV). This virus was not ob-
                   Eel virus from European eel (EVE)      European eel                       served again until 1986, during an episode of mass
                   Eel virus of America (EVA)             American eel                       mortalities of masu salmon fry for which it was re-
                   Eel virus of Europe X (EVEX)           European eel                       sponsible. Since then, the virus has been detected in
                   Papovavirus                            Japanese eel                       stocks of adult masu salmon at new locations in
                   Birnavirus                             Yellowtail
                   Birnavirus                             Japanese flounder                  Hokkaido (Yoshimizu 1988). Artificial infection stud-
                   Birnavirus                             Red sea brearn                     ies of this virus showed no significant mortality in
                   Coronavirus cypTini virus (CACV)       Common carp                        several species of salmonid fishes (Winton et al.
                   Picornavirus                           Japanese eel                       1989).
                   Reovirus                               Common carp
                   Reovirus                               Japanese eel
                 Observed by electron microscopy
                   Viral erythrocytic necrosis virus      Various marine fish                Viral ]Fxythrocydc Necrosis
                   Lymphocystis virus                     Various marine fish
                   Paramyxovirus                          Black rockfish                     Inclusion bodies stained with Giemsa were observed
                   Herpesvirus                            Japanese flounder                  in the erythrocytes of churn and pink salmon, 0.
                   Picornavirus                           Ishidai
                                                                                             gorbuscha, collected in Okhotsuku and along the
                                                                                             north Pacific coast of Hokkaido. The causative agent
                                                                                             of viral erythrocytic necrosis (VEN), an iridovirus,
              Herpesvirus Infection                                                          was subsequently observed by electron microscopy
                                                                                             (Yoshimizu et al. 1988b).
              A herpesvirus, nerkavirus in Towada Lake Akita and
              Aomori Prefecture (NeVTA), was first isolated from
              diseased kokanee salmon in Towada Lake (Sano                                   Viral Infections of Eels
              1976). In 1978, another herpesvirus was isolated
              from the ovarian fluid of apparently normal mature                             Many viruses have been isolated from cultured eels
              masu salmon (Kimura et al. 1980). This virus was                               (Anguilla anguilla, A. japonicus, and A. rostrata) by
              named Oncorhynchus masou virus (0MV) from the sci-                         .   Sano (1976) and Sano and Fukuda (1987). They in-
              entific name of the host fish. Oncorhynchus masou                              clude a birnavirus, eel virus from the European eel
              virus was found to be pathogenic and significantly                             (EVE); the rhabdoviruses, eel virus of America (EVA)
              more oncogenic in young masu salmon and several                                and eel virus of Europe X (EVEX); papovavirus; her-
              other salmonid fish (Kimura et al. 1981, a and b;                              pesvirus; picornavirus; and a reovirus. These viruses
              Yoshimizu. et al. 1987a). In 1983, a similar herpesvi-                         are not recognized as pathogenic against eel except
              rus 'were isolated from tumor tissue.of yamame                                 for EVE (Nishimura et al. 1981). Sorimachi (1982,
              (landlocked 0. masou) and was named yamame tu-                                 1984) reported having isolated icosahedral. cytoplas-
              m  .or virus (Sano et al. 1983). Subsequent study                              mic deoxyribovirus (ICDV) from a diseased eel. This







                                                                     Muroga and Yoshimizu: Viral Infections of Ctiltured Fish in Japan        45

               virus was shown to be pathogenic against Japanese                  Sebastes inermis, and also for salmonid species, espe-
               eels following artificial infection. Mortality was 40-             cially rainbow trout and masu salmon (Yoshimizu et
               75% at water temperatures of 14.5-18.50 C, 15% at                  al. J987b). Signs of HRV infection are gonadal con-
               22.8' C, and 0% at 24.1' C. Infected fish showed                   gest@ion, focal hemorrhage of skeletal muscle and
               signs of decoloration; congestion of the anal, pecto-              fins, and accumulation of ascitic fluid. Hirame rhab-
               ral, and dorsal fins; and an increase of mucus on the              dovirus. is distributed widely from Hokkaido to
               body surface.                                                      Honshu in Japan.


               Viral Infections of Carp                                           Ruchishiroshou of Tiger Puffer

               Herpesvirus cyprini was isolated from papilloma tissue             From cultured tiger puffer, Fugu rubripes, an uniden-
               of cultured fancy carp (Cyprinus carpio, also called               tified, small virus was isolated (Inoue et al. 1986).
               common or asagi carp) and confirmed as the agent                   The epizootic period is from May to June during wa-
               of infection by induction of epithelia] tumors by arti-            ter temperatures of 18-22' C. Moribund fish had
               ficial infection (Sano et al. 1985). Coronavirus cyprini,          necrosis around the mouth and had been observed
               carp coronavirus (CACV), was isolated from diseased                to be fighting with each other. From the signs of this
               common carp raised in the laboratory. Fish infected                infection, the disease was named "Kuchishiroshou",
               with CACV showed acute mortality showing no exter-                 from the Japanese words "kuchi", meaning mouth,
               nal signs except erythematous skin on the abdomen.                 "shiro", meaning white, and "shou", meaning disease.
               Experimentally, CACV was virulent for carp fry at                  Viral particles were observed in the brain by electron
               20' C. Cumulative mortality for 3-week-old fry was                 microscopy. Kuchichiroshou occurs in southwest Ja-
               72.5%. The affected fish manifested swollen and                    pan where tiger puffer are cultured.
               hemorrhagic abdomens filled with ascites and even-
               tually died. Reovirus was also isolated from common
               carp (Sano and Fukuda 1987).                                       Epidermal Hyperplasia of Japanese Flounder.

                                                                                  Outbreaks of a disease resulting in mass mortalities
               Viral Infection of Other Marine Fishes-                            of larval and juvenile Japanese flounder was reported
                                                                                  by Iida et al. (1989). Once the disease occurs in a
                                                                                  pond, the resident fish populations usually become
               Viral Pancreatic-Hepatic Necrosis of Yellowtail                    extinct within one month. Affected fish are character-
                                                                                  ized by opaque fins. Histopathologically, hyperplasia
               A yellowtail ascites virus (a birnavirus) was isolated             is observed in the epidermal layer of the fins and
               from the fry of yellowtail Seriola quinqueradiata                  skin. In the epidermal tissues of infected fish, hex-
               (Sorimachi and Hara 1985). This epizootic is an                    agonal virus particles were observed by electron
               acute viral infection of both naturally grown and                  microscopy. Japanese flounder larvae experimentally
               hatchery-raised fry. The epizootic period occurs from              exposed to the filtrate of infected tissue homogenate
               May to June at water temperatures of 18 to 22' C.                  suffered 18-50% mortalities with 93-100% of the sur-
               The moribund fry typically show anemic gills, hemor-               vivors exhibiting epidermal hyperplasia. This virus
               rhaging in the liver, and ascites and petechiae in the             has not been isolated in any of the 33 fish cell lines in
               .pyloric caea. The disease name, viral pancreatic-he-              which culture has been attempted, including that of
               patic necrosis, was proposed by Egusa and Sorimachi                the host species.
               (1986).                                                              Birnaviruses were also isolated from Japanese
                                                                                  flounder and red sea bream (Yoshimizu and Kimura,
                                                                                  unpubl.. data), These viruses were neutralized with
               Rhabdovirus Infection of Japanese Flounder                         antibody against IPNV; the pathogenicities of these
                                                                                  birnaviruses have not been clarified.
               The rhabdovirus, Rhabdovirus olivaceus-also referred
               to as hirame rhabdovirus (HRV)-was isolated from
               diseased hirame (Japanese flounder), Paralichthys                  Lyinphocystis Disease
               olivaceus, and black sea bream, Milio macrocephalus
               (Gorie et al. 1985; Kimura et al. 1986). This virus is             In several species of marine fishes, suzuki, Lateolabrax
               pathogenic for marine fish such as hirame, black sea               japonicus, yellowtail, red sea bream, Japanese
               bream, red sea bream Pagrus major and black rockfish               flounder, and others, lymphocystis disease was re-







              46         NOAA Technical Report MM I I I


              ported and iridovirus was observed by electron mi-                            Kimura, T., M. Yoshimizu, and S. Gorie.
              croscopy (Matsusato 1975; Miyazaki and Egusa 1972;                                 1986. A new rhabdovirus isolate in Japan from cultured
              Tanaka et al. 1984). Seasonal variation in the preva-                                hirame (Japanese flounder) Paratichthys olivaceus and ayu
                                                                                                   Plecoglossus altivelis. Dis. Aquat. Org. 1:209-217.
              lence of lymphocystis was noted with increased                                Matsusato, T.
              prevalence in summer. Lymphocystis cells were ob-                                  1975. On the lymphocystis disease in cultured yellow-
              served mainly on the fins or body sur--ace. The virus                                tail. Fish Pathol. 10:90-93. (Injapanese.)
              particles were polyhedral, presenting hexagonal or                            Miyazaki, T., and S. Egusa.
              pentagonal profiles in tissue sections. They may be                                1972. A histopathological observation on lymphocystis dis-
                                                                                                   ease in sea bass Lateolabrax japonicus (Cuvier and
              seen in a crystalline array and are always located in                                Valenciennes), Fish PaLhol. 6:83-87. (In Japanese.)
              the cytoplasm.                                                                Miyazaki, T., K. Fujiwara,J. Kobara, and N. Matumoto.
                                                                                                 1989. HistopaLhology associated with two viral diseases of lar-
                                                                                                   val and juvenile fishes: epidermal necrosis of Japanese
                                                                                                   flounder Paratichthys olivaceus epithelial necrosis of black sea
              Other Diseases                                                                       bream Acanthopagrus schlegeli. J. Aquatic Animal Health
                                                                                                   1:85-93.
              Yoshikoshi and        Inoue (1988) reported picornavirus                      Mori, S., F. Iketani, T. Komatsu, and T. Nishimura.
                                                                                                 1987. IHN of large size of rainbow trout. Proceedings of
              in moribund fry of ishidai, Oplegnathus fasciatus, and                               the Annual Meeting of Japanese Society of Fish Pathology,
              Miyazaki et al. (1989) reported herpesvirus in the                                   p.9. (Injapanese.)
              epidermal necrosis of Japanese flounder and                                   Nishimura, T., M. Toba, F. Ban, N. Okamoto, and I Sano.
              paramyxovirus in the epithelial necrosis of black sea                              1981. Eel rhabdovirus, EVA, EVEX and their infectivity to
              bream. To date, these viruses have not been isolated.                                fishes. Fish Pathol. 15:173-183. (In Japanese.)
                                                                                            Okamoto, N., T. Matsumoto, N. Kato, S. Tazaki, M. Tanaka, N. Ai,
                                                                                              H. Hanada, Y Suzuki, C. Takamatsu, T. Tayama, and T. Sano.
                                                                                                 1987. Difference in susceptibility to IPN virus among rain-
                                                                                                   bow trout populations from three hatcheries in
              Citations                                                                            Japan. Nippon Suisan Gakkaishi 58:1121-1124. (In
                                                                                                   Japanese.)
              Egusa, S., and M. Sorimachi.                                                  Sano, T.
                   1986. A histopathological study of yellowtail ascites virus                   1971. Studies on viral diseases of Japanese fishes-1. Infectious
                     (YAV) infection of fingerling yellowtail, Seriola                             pancreatic necrosis of rainbow trout; first isolation from
                     quinqueradiata. Fish Pathol. 21:113-122.                                      epizootics in Japan. Bull. Jpn. Soc. Sci. Fish. 37:405-408.
              Gorie, S., K. Nakamoto, K. Katashima.                                                (InJapanese.)
                   1985. Disease of cultured hirame Uapanese flounder,                           1976. Viral diseases of cultured fishes in Japan. FishPathol.
                     Paralichthys olivaceus)-I. Preliminary report on a disease of                 10:221-226.
                     marine pen cultured hirame may be caused by viral                      Sano, T., and H. Fukuda.
                     infection. Bull. Hyogo Pref. Fish. Exp. Sm. 23:66-68. (In                   1987. Principal microbial diseases of mariculture in
                     Japanese.)                                                                 I Japan. Aquaculture 67, Vol. 1:59-69.
              Hedrick, R.P., T. McDowell, W.D. Eaton, T. Kimura, and T. Sano.               Sano, T., H. Fukuda, and M. Furukawa.
                   1987. Serological relationships of five herpesviruses isolated                1985. Herpesvirus cyprini: Biological and oncogenic
                     from salmonid fishes. J. Appl. Ichthyol. 3:87-92.                             properties. Fish Pathol. 20:381-388.
              lida, Y, IL Masumura, T. Nakai, M. Sorimachi, and H. Matsuda.                 Sano, T., N. Fukuda, N. Okamoto, and F. Kaneko.
                   1989. A viral disease occurred in larvae and juveniles ofJapa-                1983. Yamame tumor virus: lethality and oncogenicity@ Bull.
                     nese flounder, Paralichthys olivaceus. Aquatic Animal                         Jpn. Soc. Sci. Fish. 49:1159-1163.
                     Health 1: 7-12.                                                        Sano, T., H. Fukuda, T. Inokari, F. Tichiya, and H. Hosoya.
              Inoue, K., S. Yasumoto, N. Yasunaga, and I. Takami.                                1988. Comparison of three representative strains of salmo-
                   1986. Isolation of a virus from cultured tiger puffer, Tahifugu                 nid herpesvirus. Proceedings of the Annual Meeting of
                     ruhripes, infected with "Kuchishiro-sho" and it's patho-                      Japanese Society of Fish Pathol, p.21.
                     genicity. Fish Pathol. 21:129-130.                                     Sano, T., T. Nishimura, N. Okamoto, T. Yamazaki, H. Hanada, and
              Kimura, T., and T. Awakura.                                                     Y. Watanabe.
                   1977. Current status of disease of cultured salmonids in                      1977. Studies on viral disease of Japanese fish. IV. Infectious
                     Hokkaido, Japan. In Proceedings from the international                        hematopoietic necrosis (IHN) of salmonids in the mainland
                     symposium on disease of cultured salmonids, sponsored by                      of Japan. J. Tokyo Univ. Fish. 63:81-85.
                     Tavolek Inc., Seattle, WA, p. 124-160.                                 Sorimachi, M.
              Kimura, T., M. Yoshimizu, and M. Tanaka.                                           1982. Characteristics and distribution       'of viruses isolated
                   1980. Salmonid viruses: a syncytium-forming herpesvirus                         from pond-cultured eels. Bull. Nad. Inst. Aquaculture
                     from landlocked Oncorhynchus masou. Fish Health News                          3:97-105. (Injapanese.)
                     9:iii.                                                                      1984. Pathogenicity of ICD virus isolated from Japanese
                   1981a. Studies on a new virus (OMV) from Oncorhynchus                           eel. Bull. Natl. Inst. Aquaculture 6:71-75. (In Japanese.)
                     masou-1. Characteristics and pathogenicity. Fish Pathol.               Sorimachi, M., and T. Hara.
                     .15:143-147.                                                                19M. Characteristics and pathogenicity of a virus isolated
                   1981b. Studies on a new virus (OMV) from Oncorhynchus                           from yellowtail fingerling showing ascites. Fish Pathol.
                     masou-11. Oncogenic nature. Fish Pathol. 15:149-153.                          19:231-238.







                                                                                  Muroga and Yoshimizu: Viral Infections of Cultured Fish in Japan                      47


                    Tanaka, M., M. Yoshimizu, M. Kusakari, and T Kimura.                          Yoshimizu, M., M. Tanaka, and T. Kimura.
                         1984. Lymphocystis disease in kurosoi Sebastes schlegeli and                  1987a. Oncorhynchus masou virus (OMV): incidence of tumor
                           hirame Paralichthys olivaceus in Hokkaido, Japan. Bull. Jpn.                  development among experimentally infected representative
                           Soc. Sci. Fish. 50:37-42. (Injapanese.)                                       salmonid species. Fish Pathol. 22:7-10.
                    Winton, J. R., C. N. Lannan, J. L. Fryer, and T Kimura.                       Yoshimizu, M., N. Oseko, T Nishizawa, and T Kimura.
                         1981. Isolation of a new reovirus from chum salmon in                         1987b. Rhabdovirus disease of Japanese flounder. Fish
                           Japan. Fish Pathol. 15:155-162.                                               Pathol. 22:54-55.
                    Winton, J. R., C. N. Larman, M. Yoshimizu, and T Kimura.                      Yoshimizu, M., M. Sami, T. Kimura, and M. Ugazhin.
                         1989. Response of salmonid fish to artificial infection with                  1987c. Characteristics of the virus isolated from cultured ayu
                           chum salmon virus. In Viruses of lower vertebrates (W.                        (PL-coglossus altivelis). Proceedings of the Annual Meeting
                           Ahne and E. Kurstak, eds.), p. 270-278. Springer-Verlag,                      of Japanese Society of Fish Pathology, p. 12.
                           Berlin.                                                                Yoshimizu, M., M. Sami, and T. Kimura.
                    Yoshikoshi, K., and K Inoue.                                                       1988a. Survival and inactivation of infectious hernatopoietic
                         1ï¿½88. Viral neuron necrosis of ishidai. Proceedings of the                      necrosis virus (IHNV) in fertilized eggs of masu salmon
                           Annual Meeting of Nippon Suisan Gakkai, p.121.                                Oncorhynchus masou and chum salmon 0. keta. Nippon
                    Yoshimizu, M.                                                                        Suisan Gakkaishi 54:2089-2097.
                         1988. Chum salmon virus isolated from masu salmon,                       Yoshimizu, M., T. Nomura, T Awakura, and I Kimura.
                           Oncorhynchus masou. Fish and Eggs, Techn. Rep. Hokkaido                     1988b. Incidence of fish pathogenic viruses among anadro-
                           Salmon Hatchery 157:36-38.                                                    mous salmonid in northern part of Japan. Sci. Rep.
                                                                                                         Hokkaido Salmon Hatchery 42:1-20.






                           Some Important Infectious Diseases of Kuruma Shrimp,
                                                      Penaeusjaponicus, in Japan



                                                                KAZUO MOMOYAMA

                                                  Yamaguchi Prefectural Naikai Fisheries Experimental Station
                                                                   Yamaguchi 754, Japan



                                                                      ABSTRACT


                                   The kuruma shrimp, Penaeus japonicus, is the most widely cultured crustacean in Japan.
                                Shrimp farming operations for this species have been greatly affected by several infec-
                                tious diseases including baculoviral midgut gland necrosis (BMN), vibriosis, and black
                                gill disease. This paper discusses our knowledge of the BMN virus in detail, and brieflly
                                reviews the latter two diseases with respect to the culture of crustaceans.



               Introduction                                                     al. 1987; Johnson and Lightner 1988). They exhibit
                                                                                higher pathogenicity to larval or postlarval shrimp
               It may safely be said that kuruma shrimp, Penaeus                than to adults. Baculoviral midgut gland necrosis was
               japonicus, is the only crustacean species cultured on an         first noticed in 1971, and since then it has often
               industrial scale in Japan. About six to seven hundred            caused mortalities of over 190% during the mass pro-
               million post-larval kuruma shrimp are produced at                duction of kuruma shrimp larvae in Japan
               public and private hatcheries every year for stocking            (Momoyama 1981; Sano et al. 1981). Heavy losses
               and farming (Japan Sea Farming Assoc. 1990). Annual              due to baculovirus infection during the larval pro-
               production of cultured kuruma shrimp reached 3,020               duction of P. aztecus (Couch 1978) and R monodon
               metric tons in 1988 (DSI 1990).                                  (Lightner et al. 1983) have also been reported in
                 The infectious diseases responsible for high mortali-          other countries.
               ties to cultured kuruma. shrimp injapan are baculoviral             The results of our histological and epizootiological
               midgut gland necrosis (BMN), vibriosis, and black gill           studies on BMN in kuruma shrimp *are summarized
               disease. Baculoviral midgut gland necrosis is a very se-         as follows.
               vere viral disease that occurs during the seed
               production process which usually causes 90% or higher
               mortalities in the population of the rearing tank. Re-           Histopathology
               cently, the frequency of BMN outbreaks has decreased
               greatly, probably because of the preventive measures ex-         Midgut glands of diseased shrimp become soft and
               ecuted at hatcheries. Vibriosis is a bacterial disease           develop a white turbid appearance at the advanced
               affecting the lymphoid organ that has been responsible           stage of infection, whereas those of healthy
               for a great deal of damage to the shrimp farming indus-          postlarvae appear brown or colorless during the early
               try over the past several years. Black gill disease is           developmental stages (Momoyama 1981). From histo-
               caused by Fusarium solani, a ubiquitous pathogen of              logical examinations, it was confirmed that only the
               shrimp and lobster, invading the gills, appendages, and          midgut gland and the intestine are affected by the
               various parts of the exoskeleton.                                disease. Disarrangement and exfoliation of epithelial
                 In the present paper BMN is discussed in detail, and           cells are remarkable in the midgut gland of the dis-
               vibriosis and black gill disease are touched briefly.            eased shrimp. Nuclear hypertrophy and chroma-
                                                                                tolysis of infected epithelia] cells are the most
                                                                                characteristic cytop ath o logical changes in BMN
               Baculoviral Midgut Gland Necrosis                                (Momoyama 1981). In contrast with other penaeid
                                                                                shrimp baculovirus infections, no inclusion bodies
               To date, four baculoviruses have been recorded in                are present in the hypertrophied nuclei (Lightner
               penaeid shrimp worldwide (Lightner 1985; Lester et               1985; Lester et al. 1987; Johnson and Lightner 1988).

                                                                                                                                           49







           50      NOAA Technical Report NMB I 11


             Electron microscopy observations of the hypertro-           zoeae, mysis larvae, and postlarvae were exposed,
           phied nuclei and the midgut gland lumen have                  some or all of the shrimp were diagnosed to be in-
           revealed many rod-shaped particles having outer and           fected on the final day of the test period (6 to 16
           inner envelopes, which represent virions of the               days). Susceptibility to infection tended to decrease
           baculovirus group. The average length and diameter            with the advance in developmental stages from zoea
           of the virions are 310 nm and 72 nm, respectively             to P-10 (10-day-old postlarva). Cumulative mortalities
           (Sano et al. 1981).                                           decreased from 100% in zoeal stage larvae to 0% in
             Baculoviral midgut gland necrosis can be diag-              P-6 larvae. The growth rates of test shrimp inoculated
           nosed by detecting the hypertrophied nuclei of                at stages P-2 through P-6 were lower than those of
           affected midgut gland epithelial cells in both fresh          controls' but there were no differences in mortality
           and stained squash preparations. In stained squash            or growth rates between test shrimp and controls in-
           preparations homogeneous hypertrophied nuclei                 oculated at stages P-8 and P-10. These results indicate
           about 20 to 30 @Lrn in diameter appear among the              that kuruma shrimp from the zoeal to the P-6 stage
           smaller normal ones which are about 10 @Lm in diam-           are highly susceptible to BMN virus, but stage P-8 or
           eter. The Feulgen reaction makes the difference               older postlarvae become refractory to this disease.
           clearer between hypertrophied and normal nuclei.                The route of baculovirus infection in shrimp has
           The diagnostic technique of using a dark field micro-         generally been considered to be by oral ingestion of
           scope with a wet-type condenser has the advantages            virus contaminated sediments or by cannibalism of
           of precision and rapidity. Hypertrophied nuclei are           diseased shrimp (Lightner et al. 1983; Couch 1974).
           clearly seen as white bodies of 10 to 30 pm diameter          In the previously mentioned study on waterborne sus-
           in fresh squash preparations from diseased samples            ceptibility, kuruma shrimp were not administered any
           under the dark field microscope. The reason why the           food during the inoculation period. However, peri-
           infected nuclei appear white is thought to result from        staltic movements were frequently observed in the
           an increased number of reflected or diffracted rays           oesophageal part of the shrimp, suggesting that test
           due to the numerous virus particles in the nucleus            shrimp ingest the seawater containing the virus Par-
           (Momoyama 1983).                                              ticles through the mouth. This hypothesis was
                                                                         supported by an observation that azocarmine G accu-
                                                                         mulated in the stomach and midgut gland lumen of
           Source of Infection                                           shrimp when they were dipped in seawater contain-
                                                                         ing this dye (Momoyama and Sano 1989).
           Epizootiological investigations indicate that mature
           female kuruma shrimp spawners with latent BMN-vi-
           rus infections and cultured young kuruma shrimp               Inactivation and Survival of BMN Virus
           that have recovered from infection with the virus are
           the main source of infection in hatchery epizootics           The effects of disinfectants, heating, and ultraviolet
           (Momoyama 1988). Histological examinations reveal             irradiation on BMN virus, and the survival time of
           nuclear hypertrophy of the midgut gland epithelial            the virus in seawater at different temperatures were
           cells in both spawners and young cultured survivors           examined by waterborne infectivity experiments us-
           of the disease, Fluorescent antibody techniques have          ing larval and postlarval kuruma shrimp. The virus
           been used to reveal the presence of BMN-specific vi-          was inactivated by 10-minute exposure at 25' C to any
           rus antigen in the hypertrophied nuclei of spawners           of the following chemicals: 5 ppm chlorine, 25 ppm
           (Momoyama 1988).                                              iodine, 100 ppm benzalkonium chloride and
                                                                         benzethonium chloride, 30% ethyl alcohol, and 0.5%
                                                                         formalin (Momoyama 1989a). The virus was still ac-
           The Effect of Developmental Stage of the Host                 tive after 2 hours of heat treatment at 35 and 40' C,
           on the Susceptibility to BMN Virus                            but was inactivated within 2 hours at 45' C, 30 min-
                                                                         utes at 50 and 55' C, and 5 minutes at 60' C
           The susceptibility of kuruma shrimp to BMN virus by           (Momoyama 1989b). The virus'was also inactivated
           waterborne infection (Momoyama and Sano 1988)                 within 20 minutes by ultraviolet irradiation with a 15-
           was determined for fertilized eggs, nauplii, zoeae,           watt ultraviolet lamp at a distance of 30 cm
           mysis larvae, and postlarvae (2, 4, 6, 8, and 10 day          (Momoyama 1989b). In seawater, the virus could not
           old) (Momoyama and Sano 1989). When the fertil-               survive longer than 4 days at 30' C, -7 days at 25' C,'
           ized eggs and nauplii were exposed to the virus, they         12 days at 20' C, and 20 days at 15' C (Momoyama
           showed no evidence of infection. But when the                 1989c).







                                                                                             Momoyarna: Infectious Diseases of Penaeusjaponicus               51

                   Prevention                                                                  Black gill disease is caused by Fusafium solani,
                                                                                             which is a member of the imperfect fungi. This fun-
                   The following two preventive measures are now used                        gus is a ubiquitous pathogen and infects penaeid
                   against this epizootic in some hatcheries. One pre-                       shrimp (Cook 1971; Lightner 1975) as well as lobster
                   vents vertical infection from spawners by rinsing the                     (Lightner and Fontaine 1975; Alderman 1981) and
                   fertilized eggs with virus-free seawater then transfer-                   freshwater shrimp (Burns et al. 1979). The fungus
                   ring them to a disinfected rearing tank. The other                        usually invades the gills, appendages, and various
                   prevents horizontal infection by adding chlorine in                       parts of the exoskeleton.
                   the rearing tank to kill infected populations. Since                        In kuruma shrimp, gills are most susceptible to the
                   1985, the measure to prevent vertical infection has                       fungus and infected gills always become black
                   been carried out on an industrial scale, and BMN has                      (Ishikawa'1968; Bian and Egusa 1981). In the degen-
                   never occurred in the hatcheries where this treat-                        erating gill filaments, hemal channels are 'found
                   ment has been practiced.                                                  extremely congested with hemocytes, encapsulated
                                                                                             hyphae, and tissue debris. The fungus also often pen-
                                                                                             etrates into the thoracic central nerve and sometimes
                   Vibriosis                                                                 into the ventral thoracic artery (Momoyama 1987).
                                                                                             Tissue destruction, cellular inflammation, afid
                   Vibriosis has caused a great deal of damage to the                        hyphae encapsulated by multiple layers of hemocytes
                   shrimp farming industry over the last several years.                      are always observed in the lesion. Failure to ex-
                     Although some dead shrimp infected with vibriosis                       change gas in the gills and damage to the central
                   develop white turbid muscle at the 6th abdominal                          nerve and ventral thoracic artery are thought to be
                   segment, shrimp suffering from this disease do not                        responsible for death.
                   usually show any specific external clinical signs.                          As blacke   ning of the gills in, shrimp is often i            n-
                     The lymph6id organ (Oka 1969) is intensively in-                        duced. by various causes, detection of macro- and
                   vaded by the causative bacterium resulting in                             micro-conidiospores is necessary for diagnosis of this
                   extensive necrosis and nodule formation (Egusa et                         disease (Egusa and Ueda 1972).
                   al. 1988). The nodules are seen by the unassisted eye
                   as very small black spots and are composed of a bac-
                   terial colony in the center, a melanized zone around
                   the bacterial colony, and multiple layers of hemocytes                    Citations
                   encapsulating the melanized zone. Small nodules are
                   also frequently observed in other organs such as the                      Alderman, DJ.
                   gills, heart, midgut gland, and abdominal muscula-                            1981. Fusarium solani causing an exoskeletal pathology in
                   ture, but extensive necrotic lesions can not be found                           cultured lobsters, Homarus vulga7is. Trans. Brit. Mycol.
                   in these organs.                                                                Soc. 76:25-27.
                     The Vib7io sp. isolated from kuruma shrimp has                          Bian, B.Z., and S. Egusa.
                                                                                                 .1981. Histopa!hology of black gill disease caused byFusalium
                   been identified as a new species (Takahashi et al.                              solani (Ma*rtius) infection in the kuruma prawn, Penaeus
                   1985a), and was tentatively named Vib7io sp. PJ (PJ is                          japonicus Bate. J. Fish. Dis. 4:19@-201.
                   the abbreviation of the scientific name of kuruma                         Burns, C.D., M.E. Berrigan, and G.E. Henderson.
                   shrimp Penaeus japonicus). Vibyio sp. PJ has very high                        1979. Fusmium sp. infections in the freshwater prawn Maao-
                   pathogenicity to kuruma shrimp. LD,, values ob-                                 brachium rosenbergii (De Mann). Aquaculture 16:193-198.
                                                                                             Cook, H.L.
                   tained by intramascular injection were about 20 to                            1971. Fungi parasitic on shrimp. FA0 Aquaculture Bull.
                   100 cells/g body weight of shrimp.                                              3:13.
                     Now, two antibiotics, oxytetracycline (Takahashi et                     Couch, J.A.
                   al. 1985b) and oxolinic acid, are on the market with                          1974. An enzootic nuclear polyhedrosis virus of pink shrimp:
                                                                                                   ultrastructure, prevalence and enhancement. J. Invertebr.
                   the government's approval. Although mortalities are                             Pathol. 24:311-331.
                   decreased significantly by administering these antibi-                        1978. Diseases, parasites and toxic responses of  . commercial
                   otics, disease often returns shortly after treatment.                           penaeid shrimps of the Gulf of Mexico and South Atlantic
                                                                                                   coasts of North America. Fishery Bull, U.S. 76:1-44.
                                                                                             DSI (Department of Statistics Information Division).
                   Black Gill Disease                                                            1990. Annual statistics on the fishery and culture production
                                                                                                   1988. DSI, Ministry of Agriculture, Forestry, and Fisheries,
                                                                                                   Tokyo, 296 p. (In Japanese.)
                   Black gill disease often occurs in the intensive culture                  Egusa, S., and T Ueda.
                   systems in the Okinawa and Kagoshima districts, bui                           1972. A Fusafium sp. associated with black gill disease of the
                   rarely in those of the Chugoku district.                                        kuruma prawn, Penaeus japonicus Bate. Bull. Jpn. Soc. Sci.
                                                                                                   Fish. 38:1253-1260.







             52         NOAA Technical Report NMFS I I I


             Egusa, S., Y. Takahashi, T. Itami, and K Momoyama.                                  1983. Studies on baculoviral mid-gut gland necrosis of
                  1988. Histopathology of vibriosis in the kuruma prawn,                           kuruma shrimp (Penaeus japonicus)-III        Presumptive diag-
                     Penaeus japonicus Bate. Fish Pathol. 23:59-65. (In Japa-                      nostic techniques. Fish. Pathol. 17:263-268. (In Japanese;
                     nese; English abstr.)                                                         English abstr.)
             Ishikawa, Y                                                                         1987. Distributions of the hyphae in kuruma shrimp, Penaeus
                  1968. Preliminary report on black gill disease of the kuruma                     japonicus, infected with Fusarium solani. Fish Pathol.
                     prawn, Penaeus japonicus Bate. Fish Pathol. 3:34-38. (In                      22:15-23. (Injapanese; English abstr.)
                     Japanese.)                                                                  1988. Infection source of baculoviral mid-gut gland necrosis
             Japan Sea Farming Association.                                                        in mass production of kuruma shrimp larvae, Penaeus
                  1990. Annual seed production and stocking record for sea                         japonicus. Fish Pathol. 23:105-110. (In Japanese; English
                     farming 1988, p421. (Injapanese.)                                             abstr.)
             Johnson, P.T., and D.V. Lightner.                                                   1989a. Virucidal effect of some disinfectants on baculoviral
                  1988. Rod-shaped nuclear viruses of crustaceans: gut-infect-                     mid-gut gland necrosis (BMN) virus. Fish Pathol. 24:47-
                     ing species. Dis. Aquat. Org. 5:123-141.                                      49. (Injapanese; English abstr.)
             Lester, R.G., A. Doubrovsky, J.L. Paynter, S.K. Sambhi, and                         1989b. Inactivation of baculoviral mid-gut gland necrosis
               J.G. Atherton.                                                                      (BMN) virus by ultraviolet irradiation, sunlight exposure,
                  1987. Light and 0ectron microscope evidence of baculovirus                       heating and drying. Fish Pathol. 24:115-118. (In Japa-
                     infection in the prawn Penaeus plebejus. Dis. Aquat.                          nese; English abstr.)
                     Org.3:217-219.                                                              1989c. Survival of baculoviral mid-gut gland necrosis virus
             Lightner, D.V.                                                                        (BMNV) in infected tissues and in seawater. Fish Pathol.
                  1975. Some potentially serious disease problems in the cul-                      24:1797181. (Injapanese; English abstr.)
                     ture of penaeid shrimp in North America. In Proceedings                Momoyama, YL, and T. Sano.
                     of the third U.S.japan meeting on aquaculture; 15-16 Oct.                   1988. A method of experimental infection of kuruma shrimp
                     1974, Tokyo, Japan, p. 75-97. Special Publ. Fish. Agency                      larvae, Penaeus japonicus Bate, with baculoviral mid-gut
                     Japanese Government and Japan Sea Reg. Fish. Res. Lab.,                       gland necrosis (BMN) virus. J. Fish Dis. 11:105-111.
                     Niigata.                                                                    1989. Developmental stages of kuruma shrimp, Penaeus
                  1985. A review of the diseases of cultured penaeid shrimps                       japonicus Bate, susceptible to baculoviral mid-gut gland ne-
                     and prawns with emphasis on recent discoveries and devel-                     crosis (BMN) virus. J. Fish Dis. 12:585-589.
                     opments. In Proceedings of the first international confer-             Oka, M.
                     ence on the culture of penaeid prawns/shrimps; 4-7 De-                      1969. Studies on Penaeus orientafts Kishinoue-VIII. Structure
                     cember 1984, Iloilo City, Philippines (Y. Taki, J.H.                          of the newly found lymphoid organ. Bull. Jpn. Soc. Sci.
                     Primavera, J.A. Llobera, eds.), p. 79-103.                                    Fish. 35:245-250.
             Lightner, D.V., and C.T. Fontaine.                                             Sano, T., T. Nishimura, K_ Oguma, K Momoyama, and N. Takeno.
                  1975. A mycosis of the American lobster, Homarus                               1981. Baculovirus infection of kuruma shrimp, Penaeus
                     ameficanus, caused by Fusafium sp. J. Invertebr. Pathol.                      japonicus in Japan. Fish Pathol. 15:185-19 1.
                     25:239-245.                                                            Takahashi, Y, Y. Shimoyama, and K. Momoyama.
             Lightner, D.V., R.M. Redman, and T.A. Bell.                                         1985a. Pathogenicity and characteristics of Vib7io sp. isolated
                  1983. Observations on the geographic distribution, patho-                        from cultured kuruma prawn Penaeusjaponivus Bate. Bull.
                     genesis and morphology of the baculovirus from Penaeus                        Jpn. Soc. Sci. Fish. 51:721-730. (Injapanese; English abstr.)
                     monodon Fabricius. Aquaculture 32:209-233.                             Takahashi, Y, T. Itami, A. Nakagawa, H. Nishimura, and T. Abe.
             Momoyama, K.                                                                        1985b. Therapeutic effects of oxytetracycline trial tablets
                  1981. Studies on infectious mid-gut gland necrosis of                            against vibriosis in cultured kuruma prawns Penaeus
                     kuruma shrimp (Penaeus japonicus)-I. Occurrences and                          japonicusBate. Bull. Jpn. Soc: Sci. Fish. 51:1639-1643.
                     symptoms. Bull. Yamaguchi Pref. Naikai Fish. Exper. St.
                     8:1-11. (Injapanese.)






                  The Application of Molecular Biology to the Detection of Infectious
                                                    Hematopoietic Necrosis Virus



                                                                   JAMES R. WINTON
                                                                   U.S. Fish and Wildlife Service
                                                                National Fisheries Research Center
                                                                   Building 204, Naval Station
                                                                    Seattle, Washington 98115




                                                                         ABSTRACT


                                    Traditionally, the detection of most fish viruses, including infectious hematopoietic
                                  necrosis virus (IHNV), has relied upon isolation of the virus in cell culture and identifi-
                                  cation by serum neutralization or fluorescent antibody assays using polyclonA rabbit
                                  antisera. In recent years, the techniques of molecular biology have provided new strate-
                                  gies for rapid and sensitive detection of antigens, antibodies, and nucleic acids. This
                                  paper reviews the application of these powerful new methods for the detection of IHNV.
                                  Included, are discussions on the creation of monoclonal antibodies specific for viral
                                  proteins, development of enzyme-linlked immunoassays for detection of viral antigens
                                  and antibodies, electrophoretic identification of radiolabeled viral proteins', a nonradio-
                                  active DNA probe specific for the nucleoprotein (N) gene messenger RNA of IHNV, and
                                  a polymerase chain reaction (PCR) based method for amplification of genomic or mes-
                                  senger RNA of the virus. As these methods become more widely accepted, they will result
                                  in significant improvements in the speed, precision, and sensitivity of IHNV detection.



                Introduction                                                         Virological examinations are regularly conducted
                                                                                   on stocks of trout and salmon. These include routine
                Infectious hematopoietic necrosis (IHN) is the most                health checks, diagnostic examinations to identify
                important viral disease of trout and salmon in west-               causes of mortality, and pathogen-free certification
                ern North America. Depending upon the species,                     examinations, which are required prior to moving
                stock, and size of the fish, strain of the virus, and              fish from one location to another. Traditional meth-
                environmental conditions such as temperature, an                   ods for isolation and identifi@ation of IHNV rely
                outbreak of IHN may result in losses approaching                   upon cell cultures for recovering the virus and serum
                100% when fish are infected at a small size. Among                 neutralization or fluorescent antibody assays to iden-
                groups of larger fish, mortality is reduced, often be-             tify the agent, a time-consuming and expensive
                coming chronic, with a typical loss of 25% or less                 process (Amos 1985). Recently, new techniques from
                (Wolf 1988). Presently, no control measure for IHN is              molecular biology utilizing monoclonal antibodies,
                available other than avoidance of exposure to the                  enzyme-linked immunosorbent assays, nonradioactive
                causative rhabdovirus, infectious hematopoietic ne-                DNA probes, and the polymerase chain reaction,
                crosis virus (IHNV). This has not proven practical at              promise to provide new tools for detection and iden-
                many large hatcheries with open water supplies or at               tification of IHNV that are more rapid, sensitive,
                commercial trout operations where. production of                   specific, and cost-effective.
                fish occurs on a continuous basis. The chemical,
                physical, and serological characteristics of IHNV, es-
                sential features of the biology of the virus, and a                Electrophoresis of Structural Proteins
                description of the host and geographic range, have
                been extensively reviewed (McAllister 1979; Pilcher                Leong et al. (1981) described a method for identifi-
                and Fryer 1980; Nicholson 1982; Wolf 1988).                        cation of different strains of lHNV using

                                                                                                                                               53









           54      NOAA Technical Re ort NWS I I I
                                      I P

           polyacryramide gel electrophoresis (PAGE) and auto-               Another application of monoclonal antibodies for
           radiography of the virion structural proteins that had          detection of IHNV includes an immunohistochemical
           been radioactively labeled with 'IS-methionine. The             staining method developed by Yam           'amoto et al.
           method required approximately 48 hours and pro-                 (1989). The, method proved useful for in situ detec-
           vided a type of fingerprint that would not only                 tion of fish cells initially infected with IHNV
           confirm the presence of IHNV, but yielded data                  following waterborne exposure to the virus
           about the strain of the virus, giving researchers addi-         (Yamamoto et al. 1990). Monoclonal antibodies have
           tional epizootiological information. Hsu and Leong              also been use to detect neutralization variants among
           (1985) compared this electrophoretic method with                strains of IHNV (Winton et al. 1988) and in deter-
           two immunoblotting techniques using either 1211-la-             mining important epizootiological information about
           beled Protein A or peroxidase to detect the rabbit              the distribution of IHNV strains (Ristow and Arnzen
           anti-IHNV serum bound to structural proteins sepa-              1989).
           rated by PAGE and transferred to nitrocellulose
           membranes. When used to detect and confirm the                  Enzyme-Linked Immunosorbent Assay
           presence of IHNV, the direct electrophoretic method
           was the most sensitive and rapid of the three. Al-              The enzyme-linked immunosorbent assay (ELISA) is
           though the need to use radioactive materials has
           limited the field application of this method, it has            an important tool for the rapid and sensitive detec-
           proven useful in analyzing the structural proteins of           tion of antigens. An ELISA was used to detect IHNV
           IHNV (Hsu et al. 1985) and in identifying strains of            antigens present in infected cell cultures and fish tis-
           the virus (Hsu et al. 1986). This electropherotypic             sues (Dixon and Hill 1984; Way and Dixon 1988).
           analysis (based upon variation in'the mdlecular                 Because the assays used polyclonal antisera, low level
           weights of structural proteins) has become widely               cross-reactions were observed with other fish rhab-
           used in various epizootiological studies of IHNV.               doviruses and with uninfected fish cell cultures or
                                                                           fish tissue extracts. While differences between anti-
                                                                           sera affected the results, the IHNV ELISA appeared
           Monoclonal Antibodies                                           to be quite specific and could detect infections in cell
                                                                           culture as early as 48 hours after infection at the time
           Monoclonal antibodies (MAb.s) are useful for both               that cytopathic effect was first noted. The assay was
           research and diagnostic applications as they are                also used to detect IHNV in acutely infected fry.
           highly reactive, very consistent, and do not cross-re-            A modification of the ELISA, the dot blot, where
           act with other a@tigens to any significant extent               IHNV antigens were spotted onto nitrocellulose pa-
                                                                           per and detected by labeled antisera, was reported by
           (Harlow and Lane 1988). The first monoclonal anti-              McAllister and Schill (1986). This assay was rapid and
           body against IHNV was developed by Schultz et al.               required no special instrumentation, but the
           (1985). While the antibody lacked neutralizing activ-           polyclonal IHNV antiserum required extensive ad-
           ity, it could be biotinylated and used to develop an            sorption with both uninfected cells and fetal bovine
           immunoblot assay for IHNV (Schultz et al. 1989). For            serum to remove cross-reacting antibodies before
           maximum sensitivity,  'the assay required initi2il ampli-       use. While the assay was not suitable for use with tis-
           fication of the virus in cell culture where as few as           sues or fluids because the filter matrix became
           100 plaque forming units of IHNV could provide a                clogged, it was able to detect I-HNV in cell culture
           poIsitive diagnosis of IHNV as early as 36 hours after          supernatant fluids at concentrations       of 105 to 106
           infection.                                                      plaque forming units (PFU)/mL when initial cyto-
             A monoclonal antibody was compared with                       pathic effect was observed.
           polyclonal rabbit serum in a rapid fluorescent antibody           Although fish lack the complex immune system of
           test (FAT) for IHNV by L   'aPatra et al. (1989). The FAT       the higher vertebrates, it is possible to confirm past
           used fish cell cultures grown on coverslips and infected        infection with IHNV using methods that detect the
           with IHNV for approximately 48 hours at which time              presence of antibodies in serum. Amend and Smith
           the test approached the plaque assay in sensitivity and         (1974) reported that rainbow trout Oncorhynchus
           offered a significant improvement in speed. One advan-          mykiss developed good titers of neutralizing antibod-
           tage of the MAb over the polyclonal serum was that it           ies that could be detected at least 90 days after
           did not require extensive adsorption before use.                immunization with live virus. Techniques that have
           Arnzen et al. (1991) 'Used a monoclonal antibody that           been used to assay for antibodies to IHNV in fish
           was conjugated with fluorescein isothiocyanate to pro-          serum include neutralization, FAT, and ELISA
           duce a direct FAT that detected IHNV antigens in cell           (Amend and Smith 1974; Hattenberger-Baudouy et
           cultures within 6-8 hours after infection.                      al. 1989; Jorgensen et al. 1991). Of these, the ELISA






                                                                             Winton: Molecular Biological Methods for Detection of EON               55

                  appeared to have the best combination of sensitivity                  able to detect IHNV infections in rainbow trout for
                  and ease of processing the numerous samples needed                    up to 32 weeks post-infection.
                  for serological screening of fish populations.
                  Nonradioactive DNA Probe                                              Discussion
                  A biotinylated DNA probe for            rapid detection of            Control of IHNV continues to rest upon avoidance of
                  IHNV was developed by Deering et al. (1991). This                     the virus through use of virus-free fish reared in vi-
                  probe was designed to detect the nucleoprotein (N)                    rus-free water supplies (Wolf 1988). Currently,
                                                                                        assurance of the virus-free status of salmonid fish re-
                  gene messenger RNA (mRNA) of IHNV because this                        quires a series of time-consuming, expensive, and
                  molecule is synthesized early and in high abundance                   labor-intensive procedures (Amos 1985). Many of the
                  during viral replication. A 30 nucleotide target site                 methods reviewed in this paper will be important im-
                  for the probe was chosen by computer search of the                    provements in the speed, sensitivity, and precision of
                  published sequence of the N gene of IHNV (Gilmore                     these assays. These novel assays and procedures will
                  and Leong 1088). A synthetic oligonucleotide,                         find increasing use in examination of fish and fish
                  complementary to this sequence, was made by auto-                     eggs to be moved to new locations and in providing
                  mated chemical synthesis, coupled with biotin, and                    early diagnosis of IHNV outbreaks allowing prompt
                  detected with a streptavidin-peroxidase conjugate.                    management actions.
                  The probe was shown to be specific for IHNV mRNA
                                                                                          One area of concern has been the ability to trans-
                  and detected as little as one picogram of target se-                  fer these new methods to field stations where much
                  quence. It did not react with mRNA of -viral                          of the fish diagnostic work is being performed. In
                  hemorrhagic septicemia virus or hirame rhabdovirus,                   this regard, the use of radioactive material, expensive
                  but it did recognize strains of IHNV representing                     equipment, or technically demanding procedures
                  each of the electropherotypes described by Hsu et al.                 have not proven attractive. While it seems likely that
                  (1986). The method required initial amplification of                  improvements in speed and sensitivity of diagnostic
                  the virus mRNA in fish cell cultures, but at high rnul-               methods are still possible, they must be developed
                  tiplicities of infection, detectable levels of mRNA                   with these limitations in mind.
                  could be extracted from these cells after less than 24                  In the future, additional methods will become
                  hours yielding a positive diagnosis in less than 48                   available for control of IHNV. These include the use
                  hours.                                                                of antiviral drugs (Hasobe and Saneyoshi 1985;
                                                                                        Kimura et al. .1990), chemicals (Batts et al. 1991), or
                                                                                        modem vaccines based upon recombinant DNA tech-
                  Polymerase Chain Reaction                                             nology (Leong et al. 1988). The combination of
                                                                                        newer detection methods and improved control strat-
                  The polymerase chain reaction (PCR) uses two DNA                      egies promises to provide - substantial reduction in the
                  primers and repeated cycles of DNA synthesis per-                     losses caused by this important virus.
                  formed by a thermostable polymerase to amplify low
                  copy numbers of a specific nucleic acid sequence to
                  levels that can be easily detected (Erlich 1989; Innis                Acknowledgment
                  et al. 1990). Arakawa et al. (1990) used PCR to am-
                  plify a 252 nucleofide portion of the N gene of IHNV
                  that included the site for the DNA probe used by                      Some of the research appearing in this review was
                  Deering et al. (1991). The primers directed the syn-                  supported by the Cooperative State Research Service,
                  thesis of large amounts of DNA from all strains of                    U.S. Department of Agriculture to the Western Re-
                  IHNV tested. The DNA produced was confirmed to                        gional Aquaculture Consortium under Agreements
                  be of the appropriate sequence by Southern and dot                    87-CSRS-2-3219, 88-38500-4027, 89-385004287, 90-
                  blot assays. Sufficient messenger RNA for cDNA syn-                   38500-5025, or 91-38500-6078.
                  thesis using reverse transcriptase and subse             quent
                  PCR amplification could be extracted from cell cul-                   Citations
                  tures infected for as little as 24 hours. The PCR.was
                  able to amplify target sequences from IHNV-infected                   Amend, D.F., and L.S. Smith.
                  rainbow trout to levels that were easily detected with                    1974. Pathophysiology of infectious hematopoietic necrosis
                  a biotinylated probe. Recent tests using clinical mate-                    virus disease in rainbow trout (Salmo gairdneri): early
                                                                                             changes in blood and aspects of the immune response after
                  rial from IHNV-infected fish revealed that the                             injection of IHN virus. J. Fish. Res. Board Can. 21:1371-
                  method approached cell culture in sensitivity and was                      1378.







             56         NOAA Technical Report NMB I I I


             Amos, K.H.                                                                       LaPatra, S.E., K.A. Roberti, J.S. Rohovec, andj.L. Fryer.
                  1985. Procedures for the detection and identification of cer-                    1989. Fluorescent antibody test for the rapid diagnosis of
                     tain fish pathogens, 3rd ed. Fish Health Section, American                       infectious hematopoietic necrosis. J. Aquat. Anim. Health
                     Fisheries Society@ Corvallis, Oregon, 114 p.                                     1:29-36.
             Arakawa, C.K., R,E. Deering, K-H. Higman, K.H. Oshima, P.J.                      Leong, J., YL. Hsu, H.M. Engelking, and D. Mulcahy.
                O'Hara, andj.R. Winton.                                                            1981. Strains of infectious hematopoietic necrosis (IHN)
                  1990. Polymerase chain reaction (PCR) amplification of a                            virus may be identified by structural protein dif-
                     nucleoprotein gene sequence of infectious hematopoietic                          ferences.   Dev. Biol. Stand. 49:43-55.
                     necrosis virus. Dis. Aquat, Org. 8:165-170.                              Leong,J.C.,J.L. Fryer, andj.R. Winton,
             Arnzen,J.M., S.S. Ristow, C.P. Hesson, andj. Lientz.                                  1988. Vaccination against infectious hematopoietic necrosis.
                  1991. Rapid fluorescent antibody tests for infectious hemato-                       In Fish vaccination (A.E. Ellis, ed.), p. 193-203. Acad.
                     poietic necrosis virus (IHNV) utilizing monoclonal antibod-                      Press, London.
                     ies to the nucleoprotein and glycoprotein. J. Aquat. Anim.               McAllister, P.E.
                     Health 3:109-113.                                                             1979. Fish viruses and viral infections. In Comprehensive
             Batts, W.N, M.L. Landolt, andj.R. Winton.                                                virology, Vol. 14 (H. Fraenkel-Conrat and R.R. Wagner,
                  1991. Inactivation of infectious hematopoietic necrosis virus                       eds.), p. 401-470. Plenum, New York, NY.
                     by low levels of iodine, Appl. Environ. Microbiol. 57:1379-              McAllister, P.E., and W.B. Schill.
                     1385.                                                                         1986. Immunoblot assay: a rapid and sensitive method for
             Deering, R.E, C.K. Arakawa, K.H. Oshima, P.J. O'Hara, M.L.                               identification of salmonid viruses. J. Wildl. Dis. 22:468-
                Landolt, andj.R. Winton.                                                              474.
                  1991. Development of a biotinylated DNA probe for detec-                    Nicholson, B.L.
                     tion and identification of infectious hematopoietic necrosis                  1982. Infectious hematopoietic necrosis (I.H.N.). In Anti-
                     virus. Dis. Aquat. Org. 11:57-65.                                                gens of fish pathogens: Development and production for
             Dixon, P.F., and BJ. Hill.                                                               vaccines and serodiagnostics (Anderson, D.P., M. Dorson,
                  1984. Rapid detection of fish rhabdoviruses by the enzyme-                          and P. Dubourget, eds.), p. 63-69. Merieux, Lyon.
                     linked immunosorbent assay (ELISA). Aquaculture. 42:                     Pitcher, K.S., andj.L. Fryer.
                     1-12.                                                                         1980. The viral diseases of fish: a review through 1978. Part
             Erlich, H.A. (ed.).                                                                      1: Diseases of proven viral etiology. CRC Crit. Rev.
                  1989. PCR technology. Stockton Press, NY.                                           Microbiol. 7:287-364.
             Gilmore, R.D., andj.C. Leong.                                                    Ristow, S.S., andj.M. Arnzen.
                  1988. The nucleocapsid gene of infectious hematopoietic                          1989. Development of monoclonal antibodies that recognize
                     necrosis virus, a fish rhabdovirus. Virology 167:644-648.                        a type-2 specific and a common epitope on the nucleopro-
             Harlow, E., and D. Lane.                                                                 tein of infectious hematopoietic necrosis virus. J. Aquat.
                  1988. Antibodies, a laboratory manual. Cold Spring Har-                             Anim. Health 1:119-125.
                     bor Laboratory, Cold Spring Harbor, NY.                                  Schultz, C.L., B.C. Lidgerding, P.E. McAllister, and F.M. Hetrick.
             Hasobe, M., and M. Saneyoshi.                                                         1985. Production and characterization of monoclonal anti-
                  1985. On the approach to the viral chemotherapy against                             body against infectious hematopoictic necrosis virus. Fish
                     infectious hematopoittic necrosis virus (IHNV) in vitro and                      Pathol. 20:339-341.
                     in vivo on salmonid fishes. Fish Pathol. 20:343-351.                     Schultz, C.L., P.E. McAllister, W.B. Schill, B.C. Lidgerding, and
             Hattenberger-Baudouy, A.-M., M. Danton, G. Merle, C. Torchy, and                   F.M. Hetrick.
                P. de Kinkelin.                                                                    1989. Detection of infectious hernatopoietic necrosis virus in
                  1989. Serological evidence of infectious hematopoietic ne-                          cell culture fluid using immunoblot assay and biotinylated
                     crosis in rainbow trout from a French outbreak of                                monoclonal antibody. Dis. Aquat. Org. 7:31-37.
                     disease. J. Aquat. Anim. Health 1:126-134.                               Way, K., and RE Dixon.
             Hsu, YL., andj.C. Leong.                                                              1988. Rapid detection of VHS and IHN viruses by the en-
                  1985. A comparison of detection methods for infectious he-                          zyme-linked immunosorbetit assay (ELISA). j. Appl.
                     matopoietic necrosis virus. J. Fish Dis. 8:1-12.                                 Ichthyol. 4:182-189.
             Hsu, YL., H.M. Engelking, andj.C. Leong.                                         WintonjR., CX Arakawa, C.N. Larman, andj.L. Fryer.
                  1985. Analysis of the quantity and synthesis of the virion pro-                  198@8. Neutralizing monoclonal antibodies recognize anti-
                     teins of infectious hematopoietic necrosis virus. Fish                           genic variants among isolates of infectious hematopoietic
                     Pathol. 20:331-338.                                                              necrosis virus. Dis. Aquat. Org. 4:199-204.
                  1986. Occurrence of different types of infectious hematopoi-                Wolf, K_
                     etic necrosis virus in fish. Appl. Environ. Microbiol.                        1988. Infectious hematopoietic necrosis. In Fish viruses and
                     52:1353-1361.                                                                    fish viral diseases, p. 83-114. Cornell Univ. Press, Ithaca,
             Innis, M.A., D.H. GelfandjJ. Sninsky, and T.J. White (eds.).                             NY.
                  1990. PCR Protocols. Acad. Press, NY.                                       Yamamoto, T., C.K. Arakawa, W.N. Batts, andj.R. Winton.
             Jorgensen, P.E.V., NJ. Olesen, N. Lorenzen, J.R. Winton, and S.                       1989. Comparison of infectious hematopoietic necrosis in
                Ristow.                                                                               natural and experimental infections of spawning salmonids
                  1991. Infectious hematopoietic necrosis virus: detection of                         by infectivity and immunohistochemistry. In Viruses of
                     humoral antibodies in rainbow trout by plaque neutraliza-                        lower vertebrates (W. Ahne and E. Kurstak, eds.), p. 411-
                     tion, immunofluorescence, and enzyme-linked immuno-                              429. Springer, Berlin.
                     sorbent assays. J. Aquat. Anim. Health 3:100-108.                        Yamamoto, T., W.N. Batts, C.K. Arakawa, andj.R. Winton.
             Kimura, T., M. Yoshimizu, Y Ezura, and Y. Kamei.                                      1990. Multiplication of infectious hematopoictic necrosis vi-
                  1990. An antiviral agent (46NW-04A) produced by Pseudo-                             rus in rainbow trout following immersion infection: Whole
                     monas sp. and its activity against fish viruses. J. Aquat.                       body assay and immunohistochemistry. J. Aquat. Anim.
                     Anim. Health 2:12-20.                                                            Health 2:271-280..







                                        Bacterial and Viral Diseases of Marine Fish
                                                         During Seed Production



                                                                  KIYOKUNI MUROGA

                                                               Faculty of Applied Biological Science
                                                                      Hiroshima University
                                                                 Higashi-Hiroshima 724, Japan




                                                                         ABSTRACT


                                    In Japan, seed production techniques have been developed for about 40 species of
                                  marine fishes; however, mass mortalities due to infectious and noninfectious diseases
                                  have often occurred. Among these problems are the bacterial and viral diseases reviewed
                                  in the present paper. Vibriosis and other bacterial diseases have occurred in various
                                  marine fishes during theirjuvenile stage. These diseases have essentially the same pathol-
                                  ogy in both juvenile and adult fish in their tendency to terminate in systemic infection,
                                  except for Flexibacter infections. On the other hand, larvae most frequently develop intes-
                                  tinal infections, such as bacterial enteritis with Vibrio sp. INFL in Japanese flounder. It
                                  has been suspected that live diets contaminated with pathogenic bacteria serve as an
                                  important source of these intestinal infections. Recently, some new viral infections, such
                                  as viral epidermal hyperplasia and viral nervous necrosis, have been reported in larval
                                  and juvenile marine fishes. Although isolation of the causative viruses in cultured cells
                                  has not been successful, except for yellowtail ascites virus (YAV), these diseases were
                                  confirmed to occur only in the larval, or larval and juvenile, stages by infection experi-
                                  ments and epidemiological surveys.




                Introduction                                                       Bacterial Diseases


                In Japan, demand for coastal fisheries and aquacul-                A wide range of pathogenic bacteria have been iso-
                ture products has been increasing drastically over the             lated from cultured fishes during theirjuvenile stage
                past, few decades, especially since the establishment              (Table 1). Vibrio anguillarum infection has been re-
                of the 200-mile economic zone. Consequently, gov-                  ported in red seabream, tiger puffer (Takifugu
                ernmental programs have concentrated on the                        ruhripes), and Japanese flounder (Muroga and Tatani
                improvement and development of coastal fisheries                   1982; Muroga et al. 1987b ; and Yamanoi et al. 1988;
                stocks, including mass release programs for impor-                 respectively) and Vibrio ardalii infection has been ob-
                tant species. At present, more than 100 million                    served in juvenile rockfish (Sebastes schlegeli) (Muroga
                juveniles of about 40 marine finfish species ate pro-              et al. 1986). Pasteurella piscicida was isolated from ju-
                duced annually at national, prefectural, and private               veniles of black seabrearn and red grouper
                hatcheries. Red seabrearn (PagTus major), Japanese                 (Epinephelus akaara) as a disease agent (Muroga et al.
                flounder (Paralichthys olivaceus), and black seabream              1977; Ueki et al. 1990; respectively). Flexibacter
                (Acanthopagrus schlegeli) are the representative species           maritimus infection has been reported in red
                produced in southern Japan. Fish production tech-                  seabream, black seabream, and flounder (Waka-
                niques, particularly for the three species mentioned               bayashi et al. 1984, 1986; Baxa et al. 1986). Except
                above, are apparently well established (Fukuhara                   for Flexibacter infections, these bacterial infections ter-
                1987). However, during fish production, difficulties               minate in septicemi'a, or systemic infection in
                are often encountered in controlling diseases of                   juvenile fishes as they do in adult fishes. On the other
                known and unknown etiology. The present paper will                 hand, these systemic infections have rarely been re-
                review bacterial and viral diseases of marine fishes in            ported during the larval stage. Instead, high
                the course of seed production in Japan.                            mortalities during the larval stage are usually associ-

                                                                                                                                              57







               58         NOAA Technical Report NAM 111


                                                                                             Table 1
                                                             Bacterial diseases in larval andjuvenile marine fishes.

                   Stage                               Name of disease                                       Pathogen                                       Host fish

                   Larval stage                      Abdominal swelling                            Vib7io alginolyticus                                Red seabrearn
                                                                                                   Other pathogenic organisms                          Black seabream

                                                     Bacterial enteritis                           Vibrio sp. INFL                                     Japanese flounder

                   juvenile stage                    Vibriosis                                     Vibrio anguillarum                                  Red seabream
                                                                                                                                                       Japanese flounder
                                                                                                                                                       Tiger puffer
                                                                                                   Vib7io ordahi                                       Rockfish,


                                                     Pasteurellosis                                Pasteurella piscicida                               Black seabream
                                                                                                                                                       Red grouper

                                                     Flexibacter infection                         Flexibacter ma7itimus                               Red seabream
                                                                                                                                                       Black seabream
                                                                                                                                                       Japanese flounder

                   'Sebastes schlegeli.



               ated with intestinal infections. These intestinal infec-                              gram-negative, short rod, which is motile by its single
               tions occur in the larvae of various marine fishes.                                   polar flagellum. It grows well on ZoBell's 2216e agar
                   Abdominal swelling ("Fukubu-boman-sho" in Japa-                                   and heart infusion or brain heart infusion agar with
               nese) occurs in red and black seabreams (Iwata et al.                                 3% NaCl, forming small, circular, gray@colored colo-
               1978; Kusuda et al. 1986; respectively). Vibrio                                       nies. Based on biochemical characteristics and GC
               alginolyticus has been reported to be the causative                                   (guanine and cytosine) value (44.1 mol%) of DNA,
               agent or to be associated with the disease in both fish                               the organism was placed in the genus Vibrio. How-
               species. In an investigation of this disease in red                                   ever, the characteristics of this pathogen differ from
               seabream reared in extensive nursery ponds, various                                   those of previously described fish-pathogenic Vib7io
               species of the genus Vib7io, including V alginolyticus                                species. The pathogen was tentatively named Vibrio
               and V vulnificus, were the predominant isolates from                                  sp. INFL after the disease it causes: intestinal necrosis
               different batches       -of diseased larvae. The role of these                        of flounder larvae (Masumura et al. 1989b).
               vibricis was not made clear, however, because signifi-                                   Infection experiments using Vib7io sp. INFL were
               cant mortality or abdominal swelling could not be                                     carried out by oral administ                    'ration via rotifers
               produced by oral challenge in red seabream larvae                                     (Brachionus plicatilis) and brine shrimp (Artemia
               with these isolates (Yasunobu et al. 1988). A marine                                  salina) nauplii, by addition to rearing tanks, and by
               turbellarian (Allostoma sp.) was also reported to be                                  intraperitoneal injection (Masumura et al. 1989b).
               associated with abdominal swelling of black seabream                                  The disease was reproduced in flounder larvae only
               (Yamaguti 1987). At present, abdominal swelling of                                    by oral challenge. On the contrary, V anguillarum,
               seabreams can only be interpreted as being a syn-                                     which was used for comparison, killed flounder juve-
               drome caused by ingesting some pathogenic or                                          niles by intraperitoneal injection, but did not kill
               toxicogenic organisms.                                                                flounder larvae by oral challenge. The pathogenicity
                   A disease called "Chokan-hakudaku-sho" in Japa-                                   of ViMo sp. INFL therefore seems to be                      quite differ-
               nese, which means a disease condition characterized                                   ent from that of V anguillarum.
               by opaque intestine, occurs in larval Japanese                                           Six different age groups of flounder (11, 16, 17,
               flounder. The causative agent is thought to be a Vibrio                               27, 41, and 60 day olds) were submitted to an oral
               species isolated from an affected intestine (Murata                                   challenge test with Vibrio sp. INFL (Muroga et al.
               1987). According to Murata (1987), this disease oc-                                   1990). The bacteria were incorporated into brine
               curs in 14 to 30- or 40-day-old larval flounder;                                      shrimp nauplii or rotifers and were given for three
               symptoms include darkening of body color and                                          days. As a result, mortality of the test groups was sig-
               opaqueness and shrinkage of the intestine. Mortali-                                   nificantly higher than that of the respective controls
               ties sometimes reach 90% or higher, especially when                                   in the groups of 16, M and 27-day-old fish. The char-
               it occurs in younger fish. The causative bacterium is a                               acteristic clinical sign of the disease, an opaque






                                                                       Muroga: Bacterial and Viral Diseases of Marine Fish During Seed Production                            59

                    intestine, occurred and subsequently Vibrio sp. INFL                              and Muroga 1989b), and recently, it was also re-
                    was isolated from the intestine in these test groups.                             ported that the bacterial contamination of rotifers
                    In the older fish groups (41- and 60-day olds), no                                and brine shrimp was significantly reduced by freez-
                    apparent changes were observed in either the test                                 ing at -15' C for I month (Yamanoi and Katayama
                    groups or the controls. Based on these results and                                1989).
                    the before-mentioned epidemiological data on natu-
                    ral outbreaks of the disease, it was concluded that
                    this bacterial enteritis is confined to the larval stage                          Viral Diseases
                    of flounder. Histopathological and electron micro-
                    scopic examinations revealed that pathogen                                        Recently, several viral diseases have been reported                     in
                    multiplication and resultant pathological changes oc-                             the larvae and juveniles of marine fishes (Table 2).
                    curred only in the intestine (Miyazaki et al. 1990;                               These include viral ascites of yellowtail (Seriola
                    Muroga et al. 1990). Although pili-like structures                                quinqueradiata), viral epidermal hyperplasia of Japa-
                    were not observed on the cells of the pathogen, an                                nese flounder, viral epithelial necrosis of black
                    adhesive property was demonstrated on a chinook                                   seabream, and viral nervous necrosis of Japanese
                    salmon embryo (CHSE-214) cell line. These findings                                parrotfish (OpL-gwathus fasciatus), red grouper and
                    led us to the conclusion that the necrotic enteritis                              striped jack (Pseudocaranx dentex). Except for the first
                    caused by Vihiio sp. INFL in flounder larvae is similar                           virus mentioned, the causative agents have not been
                    to enteric colibacillosis of young mammals (Muroga                                isolated in cultured cells.
                    et al. 1990).                                                                       In 1983, an acute disease characterized by ascites
                       Investigations of the intestinal bacterial flora of lar-                       occurred among yellowtail juveniles cultured in a
                    val and juvenile marine fishes seem to be essential to                            hatchery, and an IPNV (infectious pancreatic necro-
                    elucidate the pathogenesis of the above-mentioned                                 sis virus) -like virus, which was named YAV (yellowtail
                    intestinal infections in larval fishes. A method for iso-                         ascites virus), was isolated inseveral'cell lines includ-
                    lating and enumerating the aerobic intestinal                                     ing RTG-2 and CHSE-214. It was confirmed by an
                    bacteria of larval and juvenile fish was devised by                               immersion challenge that the disease could be repro-
                    Muroga et al.:(1987a); the intestinal bacterial flora of                          duced in yellowtail juveniles and that the mortality
                    seabreams, flounder, and other fishes was subse-                                  was higher in smaller fish (Sorimachi and Hara 1985;
                    quently investigated (Tanasomwang and Muroga                                      Sorimachi and Egusa 1986). The same disease oc-
                    1988, 1989a). The compositions of the intestinal                                  curred among. wild juveniles of yellowtail which were
                    flora of these marine fishes were characterized by two                            caught as seedlings for net cage culture (Isshiki et al.
                    predominating groups, Vibrio and Pseudomonas, which                               1989). Histopathological observations of naturally
                    were thought to be derived from the diets of live roti-                           and experimentally infected fish suggest that acinous
                    fers and brine shrimp nauplii. The same genera,                                   tissues of the pancreas and parenchymal tissue of the
                    Vib7io and Pseudomonas, were most frequently isolated                             liver are the primary tissues involved in YAV infection
                    from these live diets (Tanasomwang and Muroga                                     (Egusa and Sorimachi 1986).
                    1990). Sodium nifurstyrenate bath proved effective in                               Since 1985, outbreaks of a disease resulting in mass
                    reducing bacterial contamination of rotifers (Hayashi                             mortalities have occurred in larvae and juveniles of
                    et al. 1976; Yamanoi and Sugiyama 1987; Tanasomwang                               the Japanese flounder cultured at several hatcheries.



                                                                                              Table 2
                                                                  Viral diseases in larval and juvenile marine fishes.

                       Stage                                   Name of disease                             Pathogen                                       Host fish

                       Larval stage                      Viral epidermal hyperplasia               Herpesvirus                                       Japanese flounder
                                                         (Viral epidermal necrosis)
                                                         Viral epithelial necrosis                 Paramyxovirus-like virus                          Black seabrearn
                                                         Viral nervous necrosis                    Picornavirus-like virus                           Japanese parrotfish
                                                                                                                                                     Red grouper
                                                                                                                                                     Stripedjack
                                                                                                                                                     Seabass,
                       juvenile stage                    Viral ascites                             YAV (Yellowtail ascites                           Yellowtail
                                                                                                      virus=IPW-like)

                       "Lates calcalif-







             60        NOAA Technical Report NNM I I I


             The disease occurred in 10- to 30-day-old fish reared                       system for marine fishes from the standpoints of
             at 18-20' C, and mortality usually reached 80-90% in                        quarantine and hygiene, both of which have almost
             .a few weeks. The affected fish had opaque fins and a                       been established for salmon hatcheries.
             hyperplastic epidermis on the fins and skin. Electron
             microscopy revealed hexagonal virus particles in the
             nucleus and cytoplasm (diameter in enveloped state:                         Citations
             200 nm) of the affected epidermal cells. Although                           Baxa, D.V., K. Kawai, and R. Kusuda.
             isolation of the causative agent by the use of several                            1986. Characteristics of gliding bacteria isolated from dis-
             fish-cell cultures was not successful, the disease was                             eased cultured flounder, Paralichthys olivaceus. Fish Pathol.
             transmitted to healthy larval flounder by exposing                                 21:251-258.
             them to a 0.45 @Lni filtrate of diseased fish                               Egusa, S., and M. Sorimachi.
             homogenate. Morphological and physiological char-                                 1986. A histopathological study of yellowtail ascites virus
             acteristics of the virus indicate that the agent is a                              (YAV) infection of fingerling yellowtail, Seriola quin-
                                                                                                queradiata. Fish Pathol. 21:113-121. (in Japanese; English
             herpesvirus (lida et al. 1989). When immersion chal-                               abstr.)
             lenge tests of flounder larvae were made at three                           Fukuhara, 0.
             different temperatures (15, 20, and 25' Q, the dis-                               1987. Seed. production of red sea        bream Pagrus major
             ease progress was apparently delayed at the lowest                                 (Sparidae) injapan. In Reproduction, maturation, and seed
             temperature (15' Q, though the cumulative mortal-                                  production of cultured species: proceedings of the twelfth
             ity was the same for all three temperatures. Larvae                                U.S.-Japan meeting on aquaculture (CJ. Sindermann, ed.),
                                                                                                p. 13-20. U.S. Dep. Commer., NOAA Tech. Rep. NMFS 47.
             younger than 20 days old (smaller than 9.5 min in                           Glazebrook, J.S., M.P. Heasman, and S.W. de Beer.
             total length) were highly susceptible, but the suscep-                            1990. Picorna-like viral particles associated with mass mor-
             tibility was significantly decreased in fish 23 days old                           talities in larval barramundi, Lates calcarifer Bloch. J. Fish
             or older (larger than 11.0 mm). Therefore, this dis-                               Dis. 13:245-249.
             ease proved specific to the larval stage of flounder                        Hayashi, K., T. Kimura, and 1. Sugahara.
                                                                                               1976. Microbiological studies on the artificial seedling pro-
             (Masumura et al. 1989a). A similar disease was re-                                 duction of ayu fish, Plecoglossus altivelis (Temminck et
             ported in Japanese flounder larvae and juveniles                                   Schlegel)-rV. On the elimination of bacteria contaminated
             under a name of viral epidermal necrosis (Miyazaki                                 in Brachionusplicatilis and Moina macrocopa. Bull.Fac.Fish.
             et al. 1989); however, a precise comparison of these                               Mie Univ. 3:87-99. (in Japanese; English abstr.)
                                                                                         lida, Y, K Masumura, T. Nakai, M. Sorimachi, and H. Matsuda.
             herpesvirus infection's has not been made owing to                                1989. A viral disease in larvae and juveniles of Japanese
             the unsuccessful in-vitro culture of the agents. Aii-                              flounder Paralichthys olivaceus. J. Aquatic Animal Health
             other epithelia] necrosis due to a paramyxovirus-like                              1;7-12.
             virus was reported from larval black seabream                               Isshiki, T., K. Kawai, and R. Kusuda.
             (Miyazaki et al. 1989).                                                           1.989. Incidence of yellowtail ascites virus (YAV) in wild yel-
                                                                                                lowtail fingerling. Nippon Suisan Gakkaishi 55:633-637.
                Mass mortalities of hatchery-reared Japanese                                    (InJapanese; English abstr.)
             parrotfish larvae and juveniles have occurred in                            Iwata, K_ Y Yanohara, and 0. Ishibashi.
             Nagasaki Prefecture, the westernmost part of Japan.                               1978. Studies on factors related to mortality of young red
             Light and electron microscopic examinations re-                                    seabream (Pagrus major) in the artificial seed pro-
             vealed an extensive necrosis of the nervous system in                              duction. Fish Pathol. 13:97-102. .(In Japanese; English
             the spinal cord, spinal ganglia, and brain. Numerous                               abstr.)
                                                                                         Kusuda, R.,J. Yokoyarna, and K. Kawai.
             nonenveloped virus particles, icosahedral in mor-                                 1986. Bacteriological study on cause of mass mortalities in
             phology and measuring 34 nrn in diameter, were                                     cultured black sea bream fry. Nippon Suisan Gakkaishi
             found in the cytoplasm of the affected neurones and                                52:1745-1751. (InJapanese; English abstr.)
             glial cells. The disease was named viral nervous ne-                        Masumura, K., Y lida, T. Nakai, and T. Mekuchi.
             crosis (Yoshikoshi and Inoue 1990). A similar disease                             1989a. The effects of water temperature and fish age on a
                                                                                                herpesvirus infection of Japanese flounder larvae,
             was found in red grouper larvae in 1989 and striped                                Paralichthys olivaceus. Fish Pathol. 24:111-114. (In Japa-
             jack larvae in 1990 (Yoshikoshi 1990). Similar                                     nese; English abstr.)
             picorna-like viral particles were observed in degen-                        Masumura, K, H. Yasunobu, N. Okada, and K Muroga,
             erative areas of the brain and retina in seabass (Lates                           1989b. Isolation of a Vibrio sp., the causative bacterium of
             calcafifff) larvae cultured in Australia (Glazebrook et                            intestinal necrosis of Japanese flounder larvae. Fish
                                                                                                Pathol. 24:135-141. (InJapanese; English abstr.)
             al. 1990). A comparative study on these new disease                         Miyazaki, T., K. Fujiwara, J. Kobara, N. Matsumoto, M. Abe, and
             agents is a matter of great interest because traffic of                       T. Nagano.
             live fishes has been increasing among south and                                   1989. Histopathology associated with two viral diseases of lar-
             southeast Asian countries. The frequent occurrences                                val and juvenile fishes: epidermal necrosis of the Japanese
             of these new viral diseases remind us of the necessity                             flounder Paralichthys olivaceus and epithelial necrosis of
                                                                                                black sea bream Acanthopagrus schlegeli J. Aquatic Animal
             of rapid improvement in the current seed production                                Health 1:85-93.







                                                                     Muroga: Bacterial and Viral Diseases of Marine Fish During Seed Production                         61


                   Miyazaki, T., N. Kajihara, K. Fujiwara, and S. Egusa.                               1989b. Effects of sodium nifurstyrenate and tetracycline on
                        1990. A histopathological study on intestinal necrosis of lar-                    the bacterial flora of rotifer (Brachionus plicatilis). Fish
                          val Japanese flounder. Fish Pathol. 25:7-13. (In Japanese;                      Pathol. 24:29-35.
                          English abstr.)                                                              1990. Intestinal microflora of marine fishes at their larval
                   Murata, 0.                                                                             and juvenile stages. In Proceedings of the second Asian
                        1987. Infectious intestinal necrosis in flounder. Fish                            fisheries forum (R. Hirano, and I. Hanyu, eds.), p.647-
                          Pathol. 22:59-61. (Injapanese.)                                                 650. Asian Fisheries Soc., Manila, Philippines.
                   Muroga, YL, and M. Tatani.                                                     Ueki, N., Y Kayano, and K- Muroga.
                        1982. Isolation of Vibrio.anguillarum from juvenile red                        1990. Pasteurella piscicida infection in juvenile red grouper
                          seabrearn (Pagrus major). Fish Pathol. 16:211-214. (in                          (Epinephelus akaara). Fish Pathol. 25:43-44. (In Japanese.)
                          Japanese; English abstr.)                                               Wakabayashi, H., M. Hikida, and K Masumura.
                   Muroga, IC, T. Sugiyama, and N. Ueki.                                               1984. Flexibacter infection in cultured marine fish in
                        1977. Pasteurellosis in cultured black seabream (Mylio                            Japan. Helgol. Meeresunters. 37:587-593.
                          macrocephalus). J. Fac. Fish. Anim. Husb. Hiroshima Univ.                    1986. Flexibacter maritimus sp. nov., a pathogen of marine
                          16:17-21.                                                                       fishes. Int.J. Syst. Bacteriol. 36:396-398.
                   Muroga, K, Y Jo, and K Masumura.                                               Yamaguti, M.
                        1986. Vibrio ordahi isolated from diseased ayu (Plecoglossus                   1987. Abdominal swelling in black seabream. Fish Pathol.
                          altivelis) and rockfish (Sebastes schlegeli). Fish Pathol.                      22:58-59. (Injapanese.)
                          21:239-243. (InJapanese; English abstr.)                                Yamanoi, H., and I Sugiyama.
                   Muroga, K, M. Higashi, and H. Keitoku.                                              1987. Effects of sodium nifurstyrenate bath and ultraviolet
                        1987a. The isolation of intestinal microflora of farmed red-                      irradiation on the elimination of bacteria associated with
                          seabrearn (Pagrus major) and black seabream (Acanthopagrus                      rotifer. Suisanzoshoku (Aquiculture) 25:191-195. (InJapa-
                          schlegeli) at larval andjuvenile stages. Aquaculture, 65:79-                    nese.)
                          88.                                                                     Yamanoi, H., and K Katayama.
                   Muroga, K., V. Tanasomwang, and K Momoyama.                                         1989. Effects of freezing on bacterial flora of rotifer and
                        1987b. Vib7io anguillarum infection in tiger puffer (Takifugu                     brine shrimp nauplii. Nippon Suisan Gakkaishi 55:2207.
                          rubripes) fingerlings. Fish Pathol. 22:29-30. (Injapanese.)                     (Injapanese.)
                   Muroga, K, H. Yasunobu, N. Okada, and K Masumura.                              Yamanoi, H., Y, Momoyama, H. Yasunobu, and K. Muroga.
                        1990. A bacterial enteritis of cultured flounder Paralichthys                  1988. Vibrio anguillarum infection in flounder (Paralichthys
                          olivaceus larvae. Dis. Aquat. Org. 9:121-125.                                   olivaceus) fingerlings. Fish Pathol. 23:69-70. (Injapanese.)
                   Sorimachi, M., and T. Hara.                                                    Yasunobu, H., K Muroga, and K Maruyarna.
                        1985. Characteristics and pathogenicity of a        virus isolated             1988. A bacteriological investigation on the mass mortalities
                          from yellowtail fingerlings showing ascites. Fish.Pathol.                       of red seabream -Pagrus major larvae with intestinal
                          19:231-238. (InJapanese; English abstr.)                                        swelling. Suisanzoshoku (Aquiculture), 36:11-20. (InJapa-
                   Sorimachi, M., and S. Egusa.                                                           nese; English abstr.)
                        1986. Experimental infection of yellowtail ascites virus (YAV)            Yoshikoshi, K
                          to yellowtail fingerlings. Fish Pathol. 21:133-134. (In Japa-                1990. A virus found in larval marine fishes asssociated with
                          nese.)                                                                          mass mortalities. In Abstracts of workshop of the Japanese
                   Tanasomwang, V., and K. Muroga.                                                        Society of Fish Pathology, p.5. Japanese Society of Fish Pa-
                        1988. Intestinal microflora     of larval and juvenile stages in                  thology, Tokyo. (Injapanese.)
                          Japanese flounder (Paralichthys olivaceus). Fish Pathol.                Yoshikoshi, K-, and Y- Inoue.
                          23:77-83.                                                                    1990. Viral nervous necrosis in hatchery-reared larvae and
                        1989a. Intestinal microflora of rockfish Sebastes schlegeli, tiger                juveniles of Japanese parrotfish, Oplegnathus fasciatus
                          puffer Takifugu rub7ipes and red grouper Epinephelus akaara                     (Temminck & Schlegel). J. Fish Dis. 13:69-77.
                          at their larval and juvenile stages. Nippon Suisan
                          Gakkaishi 55:1371-1377.






                                     An Ecological Study of the Parasitic Nematode
                                Hysterothylacium haze in the Japanese Common Goby
                                        Acanthogobiusflavi.manus, in a Brackish Inlet



                                                              TOMOYOSHI YOSHINAGA

                                                          National Research Institute ofFisheries Science
                                                                      Kachidoki, Chou-ku
                                                                        Tokyo 104, Japan




                                                                         ABSTRACT


                                    The unique life cycle of the parasitic nematode. Hysterothylacium haze in the Japanese
                                  common goby Acanthogobius flavimanus is presented. It is a direct cycle in which the fish
                                  become infected by ingesting fully developed eggs or hatched larvae of the nematode or
                                  by ingesting invertebrates that can act as transport hosts. Infections by the nematode
                                  were investigated monthly in a wild population of common goby in.a brackish inlet over
                                  a period of five years. Seasonally, the rate of infection in the body cavity peaked in
                                  November in fish age 0+ and in May in fish'age I+. Annually, the infection rate declined
                                  during the investigation period. The intensity of infection in individual fish tended to
                                  fluctuate with the population's overall infection rate for both age 0+ and age I+ fish. The
                                  influence of the infection on the host population is briefly discussed.



                Introduction                                                       harmful to the host goby, heavy infections causing
                                                                                   the deaths of some hosts. There is no other known
                Mass'mortalities of the Japanese common goby                       case in which an anisakid nematode infection is so
                Acanthogobiusflavimanus (yellowfin goby) occurred in               harmful as to kill the host fish. From the extensive
                the inner part of Tokyo Bay every summer from 1973                 damage observed in heavily infected fish, parasitism
                to 1975. The body cavities of the dead fish were                   by the nematode may even reduce the host popula-
                heavily infected with the eggs, larvae, and adults of a            tion size.
                nematode; visceral adhesions were often noted                        A previous study of the biology of H. haze has al-
                (Takahashi et a]. 1976; Takahashi et al. 1977).                    ready elucidated its peculiar life cycle (Yoshinaga et
                Takahashi et al. (1977) demonstrated that the heavily              al. 1989). An ecological investigation was made for
                infected fish were intolerant of low dissc4ved oxygen              five years. In this paper, the seasonal and annual
                concentrations and concluded that the mass mortali-                changes in H. haze infections in goby inhabiting a
                ties of the goby were caused by the nematode                       brackish inlet and the life cycle of this parasite are
                infections in combination with the scarcity of dis-                described. In addition, the influence of its infection
                solved oxygen. The nematode was identified as a new                on the population of the host is discussed briefly.
                anisakid, Thynnasca7is haze, by Machida et al. (1978)
                and later assigned to the genus Hysterothylacium as H.
                haze, a new combination by Deardorff and Overstreet                Life Cycle
                (1981).
                  This nematode possesses unique features among                    In general, nematodes molt four times prior to be-
                the anisakid nematodes. Adult worms and eggs of H.                 coming adults. The first-stage larvae are worms
                haze were found in the body cavity of the host fish,               before the first molting. Following the first, second,
                whereas the adults of other anisakid nematodes har-                and third moltings, the worms are called the second,
                bor and deposit their eggs in the' digestive tract of              third, and fourth-stage larvae, respectively, and after
                their definitive hosts. It also appears that H. haze has           the fourth molting (the fifth stage), they are adults.
                a unique life cycle and that infection is apparently               It was reported by Yoshinaga et al. (1988) that all

                                                                                                                                              63







              64       NOAA Technical Report NAM I I I

                                                                                      Ecological Study

                                                                                      An ecological study of H. haze was carried out in
                            EGG                Invertebrates                          Lake Shonai, a brackish inlet of Lake Hamana, an
                                            (transport host)                          estuary located on the Pacific coast of central Ja-
                                                                                      pan (Fig. 2). TheJapanese common goby has a life
                                                                                      span of one year and spawns in the spring
                                                                                      (Miyazaki 1940); however, some fish do not be-
                                                                                      come fully mature during the spawning season,
                                                                                      probably because of delayed growth. These fish sur-
                          ADULT -o- L4.*-L3           L34-L2                          vive to become age 1+ in some areas. Thus, the
                             I              +
                          EGG (Ll--o- L2) "P-12                     _4*_j             goby population in Lake Shonai consists of two
                                                    digestive                         year classes from late spring to autumn.
                              body cavity           tract wall                          Hysterothylacium haze infections in the Japanese
                                                                                      common goby were investigated monthly from June
                               Japanese conunon goby                                  1981 to November 1982 and from December 1983 to
                                  (definitive host)                                   September 1986. Fish captured commercially by trap
                                                                                      net were sampled. They were examined for H. haze
                                          Figure I                                    infection in the body cavity or in the digestive tract
              Life cycle of Hysterothylacium haze. L,   L 2, L3, and L 4 repre-       wall, or both, after age determination by scale read-
              sent the first-, second-, third-, and     fourth-stage larvae,          ings. Infections in the body cavity were examined
              respectively. The development from L       2 to L. in the body
              cavity of the Japanese common goby        (dotted arrow) is un-
              certain. (From Yoshinaga et al. 1989.)



              stages of H. haze are harbored in the body cavity of
              heavily infected fish; this is an exceptional feature
              among anisakid nematodes. Detailed observations of
              H. haze infections in goby and experiments with in-
              fected goby and invertebrates resulted in the                                                                              R. liana
              elucidation of the H. haze life cycle (Yoshinaga et al.
              1989). It is summarized as follows (Fig. 1): when a
              goby eats fully developed worm eggs, second-stage                                      Th.e Paci-fiIC.            ..2.m
              larvae in the eggs hatch and penetrate the digestive
              tract wall of the fish. The fish may also become in-
              fected by eating invertebrate transport hosts
              containing second-stage larvae in their body cavities;                                                                    R. Isaji
              various invertebrates (e.g., polychaetes, amphipods,                                                      .2
              and mysids) can act as transport hosts. Second-stage
              larvae develop to the third stage in the fissue of the
              goby's digestive tract wall, then migrate into the body                                            2
              cavity. In the body cavity, third-stage larvae develop
                                                                                                                                       R. Shin.
              through the fourth stage to become adults, that ma-
              ture and deposit eggs in the body cavity of thg host.
              These eggs are released into the water after the death                                  0   1    2 kn
              of heavily infected goby. It should be noted that the
              deaths of heavily infected goby are essential for egg
              dispersion and subsequent invasion of new hosts. The
              eggs hatch in the body cavity of the host. Although it
              is uncertain whether the second-stage larvae can de-                                                Figure 2
              velop to third-stage larvae in the body cavity, it seems                    Map of Lake Hamana (A) and Lake Shonai (B).
              that a large number of larvae are harmful to the host                       Lake Shonai is a brackish water inlet of Lake
              and contribute to its death even when the host is                           Hamana, an estuary located on the Pacific Coast in
              infected with only a small number of adults.                                Shizuoka Prefecture, Honshu.







                                                             Yoshinaga: Ecological Study of the para itic nematode Hysterolftylacium haze         65



                       100   1980                           1983


                        50




                          0-
                  dP   100
                             1981                           1984



                        50


                   U
                          OL *7           . . . . .......
                   H

                       100
                             1982                           1985



                        50

                                                                                                                        Figure 3
                          OL                                                                        Monthly changes          in    the    level   of
                          L-L  'L'-.L' L' _L'A' L L' I
                          M J  A S 0 N D J F I . M J @ A S 0 N C1 1986                              Hysterothylacium haze infection in the body
                                     Month                                                          cavities of the Japanese common goby by year
                          []ID 1: lightly infected                                                  class from 1980 to 1986. Asterisks indicate
                          IMID 2: moderately infected                                               months when sample sizes were less than 30.
                                                                                                    ID'= intensity index determined by macro-
                             ID 3: heavily infected                                                 scopical observation; ID 1 = lightly infe   'cted
                                                          M I J A S 0 N 0 J F M A M J J A S 0 N D   with 1-9 worms: ID 2 = moderately infected
                                                                     Month                          with 10-99 worms; ID 3         heavily infected
                                                                                                    with more than 100 worms.


                with a stereomicroscope during the entire investiga-
                tion period. Infections in the digestive tract wall were                        100 -   1984
                examined microscopically by compressing the tract
                between glass slides.                                                             50-

                                                                                            &      0.
                Seasonal Changes                                                                 100-   1985
                Infections in the body cavities occurred in a clear                               ;0-
                seasonal pattern, although there were slight differ-
                ences in the pattern between. years (Fig. 3).                                      0.
                Infections in age 0+ fish were first noticed in early                       H    100-
                summer. The incidence of infected fish peaked in                                        1986
                November and subsequently declined to a minimum                                                                 BC
                level. between January and March. A second peak oc-                               50.                        IMBC+DTW
                curred in May in age I+ fish that had survived after                               0-                        EDTW
                their spawning season, and then the peak declined.
                When the prevalence of. the nematode was                 . high,                     MJ JASOND JFMAMJ JASON D
                heavily infected fish containing worm eggs in the                                                 Month
                body cavity were frequently found.
                  Although infection of the digestivetract walls was                                             Figure 4
                investigated for only two years, it was observed that                    Monthly changes in the incidence of infection with
                the year when the infection level in the body cavity                     Hysterothylacium haze in the two infection sites in the
                was lower and showed a less clear seasonal pattern, a                    1984-86 year classes of the Japanese common goby.
                seasonal pattern was still discernible in the incidence                  Blank, semidark, and dark areas represent fish in-
                                                                                         fected only in the body cavity (BC), both in the body
                in the two sites of infection (Fig. 4). Characteristi-                   cavity and digestive tract wall (BC+DTW), and only
                cally, infection in the digestive tract wall preceded                    in the digestive tract wall (DTW), respectively.






            66      NOAA Te4knical Report NMFS I 11,

            that in the body cavity. Infection in the digestive tract        age 1+ to age 0+ fish occurred in summer, suggesting
            wall began in June or July prior to or in the same               that the survival of part of the goby population after
            month as the infection in the body cavity. The inci-             the spawning season is indispensable to the nema-
            dence of nematode infection in the digestive tract               tode life cycle.
            walls peaked in August and declined to its lowest lev-
            els in November and December (1985 year class)
            when the incidence of infection in the body cavities             AnntW Changes
            began to peak. The incidence of nematodes in the
            digestive tract walls continued to rise until April and          During this 5-year investigation, the prevalence and
            declined in May, when it reached a second peak in                intensities of H. haze infection in goby body cavities
            the body cavity.                                                 showed sharp annual declines when infection peaks
                                                                             were observed (Fig. 6). The declines were observed
                                                                             over the whole life span of the goby (Fig. 3). Also the
               Age of                                                        beginning of the invasion of the body cavity was de-
               the goby         0+                         1+                layed to July in 1985 and to August in 1986, whereas
                                                                             it first occurred in June in the 1982 and 1984 year
               Invasion to                                                   classes.
               the digestive
               tract vall


               migration to
               the body cavity                                                  0100                  2    100                  2
                                                                                                0+                       1+
               Depo ition of              ONE            ON11                   OP
               the :ggs

                                                                                                                                   4J
               infection level                                                                                                     r4
               in the body cavj                                                 H  50                       so-                 11
                                                                                >
                                                                                                                                   4J
                                . . . . . . . . . . ..                                                                             ri
                              M J J 0, S 0 N D J F M A M J J
                                     Figure 5                                       J                 J o     J.              Jo
              A diagram of the seasonal pattern of Hysterothylacium                  '81182'83'841 85          '81'82'83'84'85
              haze infection in the Japanese common goby in Lake
              Shonai.                                                                 Year class                Year class

                                                                                                      Figure 6
              The seasonal pattern of H. haze infection is summa-            Annual changes in the level of Hysterothylacium haze in the
            rized from these results as follows (Fig. 5):                    body cavities of the Japanese common goby by year class
            seIcond-stage larvae invade the digestive tract wall of          from 1981 to 1985. The incidence and mean intensity in-
            age 0+ goby in summer and develop to the third                   dex (total intensity indexes/number of fish examined) are
            stage there. They migrate to the body cavity from                shown for the first and second peaks in age 0+ and age 1+
            summer to autumn, develop to the adult stage, ma-                goby, respectively. See Figure 3 caption for definition of
            ture, and then deposit their eggs there in late                  intensity index. Vertical bars are 95% confidence limits.
            autumn through early winter when the infection level
            in the body cavity in the body cavity first peaks. Be-              If H. haze infection had regulated the host popula-
            tween December and March, new invasions into the                 tion size, some relationship would have been found
            digestive tract wall occur again and the infection               between the levels of the host population and the
            level in the body cavity reaches a second peak in May            nematode population. Figure 7 shows that the goby
            in age 1+ fish.                                                  catch in Lake Shonai from 1982 to 1986 (data from
              Hysterothylacium haze has never been reported to oc-           the Shirasu Branch, Hamana Fisheries Cooperation).
            cur in any fish other than the Japanese common                   Though the total catch of goby showed considerable
            goby; therefore it appears that the biology of the               annual fluctuation, there was no apparent relation-
            goby is closely related to the biology of the nema-              ship between the infection levels and catch. It seems
            tode. It is also apparent, from the seasonal pattern             likely that the H. haze infection has little influence
            above, that the transmission of the nematode from                upon the population's size, despite the lethal damage






                                                                    Yosbinaga: Ecological Study of the parasitic nematode Hysterothykxium haze                   67

                                                                                                 Another question arose from the annual decline of
                              2-                                                              the H. haze infection level. Why did the nematode
                                 1982                                                         population decrease during the investigation? Al-
                                                                                              though there were no data available to. interpret it,
                                                                                              two possible reasons may be assumed from the biol-
                                                                                              ogy of the nematode: one is that the populations of
                              0                        n      m     n             J
                                    A " M           J        J      A       S                 the invertebrate transport hosts may have decreased,
                                                                                              and another is that the biological conditions of the
                              2- 1983                                                         Japanese common goby in Lake Shonai (e.g., spawn-
                                                                                              ing season, survival after the spawning season, and
                                                                                              growth) may have changed. Nevertheless, some eco-
                                                                                              logical changes should have occurred in Lake
                                 0                    n n Q n                                 Shonai, based on Price's (1980) contention that para-
                              0-                                        ra        -
                                    A      M        J        J      A       S                 sites generally exist in nonequilibrium` states, where
                                                                                              the departure of any one element in the patch may
                                                                                              render the population inviable.
                              2- 1984

                                                                                              Citations
                      U

                                                                                              Deardorff, T.L., and R.M. Overstreet.
                              0                                            S                       1981. Reviews of Hyste,rothylacium and 1heringascafis (both pre-
                                                                                                     viously = Thynnascafis) (Nematoda: Anisakidae) from the
                      x       2-                                                                     northern Gulf of Mexico. Proc. Biol. Soc. Wash 93:1035-
                                 1985                                                                1079.
                                                                                              Machida, M., YL Takahashi, and S. Masuuchi.
                      U_      1                                                                    1978. Thynnascaris haze n. sp. (Nematoda, Anisakidae) from
                                                                                                     goby in the Bay of Tokyo. Bull. Natl. Sci. Mus., Ser. A
                                                 -AIMMMMAM                                           (Zool.) 4:241-'244.
                                                   i         J      A       S                 Miyazaki, 1.
                                                                                                   1940. Studies on the Japanese common goby, Acanth6gobius
                              2-                                                                     flavimanus (Temminck et Schlegel). Bull. Jpn. Soc. Sci.
                                 1986                                                                Fish. 9:159-180. (Injapanese; English abstr.)
                                                                                              Price, P.W.
                              1 -                                                                  1980. Evolutionary biology of parasites. Princeton Univ.
                                                                                                     Press, Princeton, NJ.
                                                                                              Takahashi, K_ S. Masuuchi, T. Nakamura, and M. Saito.
                              0-                    J        J      A       S                      1976. On A nematode from Acanthogobius flavimanus. In
                                                                                                     Annual report of ecological survey of fisheries and shell-
                                                   MONTH                                             fishes in the inner area of Tokyo Bay (1974). Publication
                                                                                                     of the Tokyo Metropolitan Fisheries Experimental Station
                                                                                                     267:42-50. (Injapanese.)
                                                Figure 7                                      Takahashi, K, S. Masuuchi, T. Nakamura, M. Ogura, and T. Arima.
                  Changes in the catch (metric tons)       of the Japanese common                  1977. On a nematode from Acanfhogobiusflavimanus (2). In
                  goby in Lake Shonai from 1982 to 1986 (based on data from                          Annual report of ecological survey of fisheries and shell-
                  Shirasu Branch, Hamana Fisheries Cooperation). Blank and                           fishes in the inner area of Tokyo Bay (1975). Publication
                  dark areas represent age 0+ and age I+ fish respectively. In                       of the Tokyo Metropolitan Fisheries Experimental Station
                  the semidark area, age was not determined.                                         276:89-93. (Injapanese.)
                                                                                              Tokyo Metropolitan Fisheries Experimental Station.
                                                                                                   1981. Annual report of ecological survey of fisheries and
                                                                                                     shell-fishes in the inner area of Tokyo Bay (1976-
                  to heavily infected hosts. This is probably due to the                             1979). Publication of the Tokyo Metropolitan Fisheries Ex-
                  fact that the Japanese common goby has a life span of                              perimental Station 30, 73 p. (Injapanese.)
                  only I year and the influence of the infection does                         Yoshinaga, T., K Ogawa, and H. Wakabayashi.
                  not accumulate year by year, and because the goby                                1988. Developmental morphology of Hysterothylacium haze
                  population is regulated more by environmental fac-                                 (Nematoda: Anisakidae). Fish Pathol. 23:19-28.
                  tors than by the population size of the previous                            Yoshinaga, T., YL Ogawa, and H. Wakabayashi.
                                                                                                   1989. Life cycle of Hysterothylacium haze (Nematoda:
                  generation (Tokyo Metropolitan Fisheries Experi-                                   Anisakidae: Raphidascaridinae). J. Parasitol. 75:756-763.
                  mental Station 1981).
                                 LAMI.







                                               Epidemiology of Marine Fish Diseases
                                               MP
                                      in the Warm Waters Along the'Kuroshio Current



                                                                      HIROKO ISHIOKA

                                                              Nansei National Fisheries Research Institute
                                                               2-17-5 Maruishi, Ohno-Cho, Saeki-Gun
                                                                      Hiroshima, 739-04,japan




                                                                           ABSTRACT


                                      In Japan, epidemiological studies have been carried out on the diseases of marine fish
                                   for many years. From 1981 to 1989, daily clinical record cards were used in every prefec-
                                   ture to obtain information on the outbreak of diseases in cultured marine fish.
                                   Observations were recorded on a standard form, translated into codes, and stored on a
                                   data base. During the analysis of this data, fish diseases were classified into six groups
                                   according to the causal agent: bacterial, viral, fungal, parasitic, nutritional, or unidenti-
                                   fied disease. The most frequently reported group was bacterial disease, while the least
                                   common one was fungal disease. This paper describes the species-specific features and
                                   seasonal variations in outbreaks of these disease groups and discusses the relationship
                                   between fish size and diseases. Also, the relationship between locality and diseases are
                                   analyzed for yellowtail, Seriola quinqueradiata. From this epidemiological examination, it is
                                   clear that disease outbreaks by bacteria that are normally nonvirulent, such as strepto-
                                   cocci and gliding bacteria, suggest that fish cultivation itself disturbs the natural
                                   relationship between fish and the environment. In other words, deteriorated environ-
                                   mental quality and poor fish health led to a beneficial situation for the growth of
                                   pathogens.



                 Introduction                                                        tured, 3) produce readily available seedlings, and 4)
                                                                                     must be commercially valuable as sashimi (raw fish).
                 Japan is surrounded by two major oceanic currents:                    Among the 30 species now being cultured in Japan
                 the Kuroshio Uapan) Current and the Oyashio                         are Seriola quinqueradiata (yellowtail), Pagrus major
                 (Okhotsk) Current. The Kuroshio Current is warmer                   (red sea bream), Paralichthys olivaceus (Japanese
                 and its surface temperature varies from about 10' C                 flounder), and Takifugu ru@rripes (tiger puffer). Yel-
                 in winter to about 30' C in summer (Fig. 1). Most                   lowtail. is the most popular and familiar species
                 marine fish cultures in Japan are carried out in the                among these and accounts for about 70% of the total
                 coastal waters along this current. The marine envi-                 production of cultured marine fish.
                 ronment in these coastal waters is suitable for marine                The production of marine fish by artificial cultures
                 fish cultivation because of its warmer temperature,                 and traditional methods is shown for the period
                 milder weather, abundant sunlight, and lower chlo-                  1976-86 in Figure 2 (SID 1981-90). Recently, the an-
                 rinity. Nitrogenous and phosphorous nutrients                       nual catch by marine fisheries (excluding shellfish
                 supplied by many rivers make this area highly fertile.              and seaweeds) has amounted to about 10 million
                 These factors support the high production of phyto-                 metric tons (t) yielding a gross income of about 210
                 plankton and zooplankton, which in turn assure food                 billion yen. The harvest from marine cultivation has
                 for fish and shellfish larvae. Also, the rias coastline is          totaled about 0.2 million t valued at 20 billion yen.
                 well suited for the culturing of marine fish because                Although the net production of fish both by tradi-
                 culture equipment can remain set up throughout the                  tional marine fisheries (A) and marine cultures (B)
                 year and the daily work routine of fish culturing can               has increased slightly since the early 1980s, the ratio
                 be easily performed in the small gulfs and bays.                    between them (B/A) has remained fairly constant at
                   Fish species chosen for cultivation must be 1)                    about the 2% level. In contrast, the ratio of gross
                 adaptable to the culture environment, 2) easily cul-                income (b/a, Fig. 2) has steadily increased and

                                                                                                                                                  69







           70       NOAA Technical Report NPM I I I


                                                                           and are fed nonliving raw food, such as minced sand
                                                                           eels.
                                                                              In the case of fish species such as red sea breams,
                                                                           where seedling production is done mainly in land-
                                                                           based ponds, fertilized eggs are collected by net and
                                                       41                  then transferred into a rearing tank. After hatching,
                                                      4@   I               fish are fed with live feed suitable for their size, such
                                                                           as oyster eggs, rotifers, brine shrimp, and zooplank-
                                                                           ton, which are collected from the sea. As they
                                                      W                    develop, they are eventually fed nonliving feed such
                                                                           as minced raw fish, chopped shrimp, or shellfish until
                         G                                                 development to the young fish stage is almost com-
                     W
                            C                         W                    pleted.
                                                   I'-,                       Intensive rearing in the marine net pen is then
                                       1  C                                carried out for about 1 or 2 years until the fish grow
                                    j 2               41 PREFECTURES       to a marketable size. During this rearing period, fish
                                               W 'I
                                                      1. MIE               are fed principally with sliced or cut, raw or frozen
                            4 1                       2. WAKAYAMA          fish meat.
                                                      3. HYOGO
                                                      4YAMAGUCHI              Intensive culture techniques have an adverse effect
                              09        W
                                                      5KAGAWA
                                                      6TOKUSIMA            not only on fish health, but also on the conditions of
                                                      7KOCHI
                                                      8. EHIME             the surrounding environment. Food containing high
                                                      9.MIYAZAKI
                                                      10. KUMAMOTO         levels of protein and fat, such as raw fish meat, is
                                                      11. KAGOS I MA       supplied in large amounts in expectation of more
                                                      12. NAGASAKI
                                                      13, OITA             rapid growth. The leftover food and fish excrement
                                                      14.SAGA
                                                                           deposited on the sea bottom cause a deterioration of
                                                      CCold wafer          water quality, which is believed to be a cause of red
                                                      WWarm water          tides and diseases. In addition, excessive feeding and
                                                                           restricted activity in the'limited confines of the rear-
                                                                           ing pens also make fish fatty.
                                    Figure 1
                             Currents around Japan.
                                                                           Investigating Disease Outbreak
           amounted to about 10% in 1986. This suggests that
           the market not only demands cultured fish as a pro-             At the request of an aquaculturist fish diseases are
           tein source but also holds their specie s-spe cific             diagnosed at the prefectural experimental stations,
           quality and freshness in high regard.                           where daily clinical records are maintained. Diagnos-
              Because of the importance of cultured marine                 tic data are recorded on a standard form (Table 1),
           fishes to the Japanese economy, an epidemiological              translated into codes, and stored in data bases of ma-
           study of the fish diseases affecting the industry was           rine fish diseases at the Nansei National Fisheries
           undertaken. Data compiled by the various prefectural            Research Institute. These data can then be entered
           fisheries institutes from 1981 to 1989 were examined            into a personal computer (NEC 9801) and be statisti-
           to establish trends in disease outbreaks and relation-          cally sorted and calculated using DATA BASE IV
           ships between fish, their culture environment, and              software (Nihon Integrated Software Co., Ltd.). The
           disease organisms.                                              program for compiling, sorting, calculating, and
                                                                           printing disease case data was previously reported
                                                                           (Umezawa and Ishioka 1988). The data base of the
           Marine Fish Cultivation:                                        present study represents diagnoses conducted by 20
           Technical Features                                              prefectures during the period from 1981 to 1989.
                                                                              The standard form consists of five parts, as shown
           The techniques used to culture marine fishes in Ja-             in Table 1. The first part includes information essen-
           pan vary little between species and localities. In the          tial for registering one diagnostic case: fish species
           popular case of yellowtail, fry or seed fish migrating          name, region where the disease occurred, name of
           with drifting algae in the surface of the Kuroshio              the fish farm or fish culturist, diagnosis date, and the
           Current are captured by a small purse seine. The fish           disease name. Diagnosis relies upon physical observa-
           are transferred to floating net pens located in a bay           tions, parasitology, and bacteriology of the diseased






                                                                                        Ishioka: Epidemiology of Mar ine Fish Diseases     71



                                        A: GROSS CATCH QUANTITY
                                                 OF SEA FISHERIES


                       110-

                       _100-
                       Z
                       0go-                                 ------
                                                                                        .3
                        so.
                                         B: PRODUCTION BY
                       >-' 70                                          RATIO B/A
                                           MARINE FISH CULTURE
                                                                                        -2

                       L)                                                               0
                                                                               'd5  'd6
                                              W


                        25-              a:GROSS MARINE FISHERIES INCOME


                       2-
                                               .0
                        20-


                       0                 b:MARINE CULTURE INCOME
                       0
                       Z
                                                                   RATIO    b/a         .10.
                       W
                       Cr                                                               8
                       W
                                                                                        .6
                       LL
                          2-                                                            .4
                       0                                                                2
                       a:
                          0                                    t2'  "43' "61 __@85      0                           Figure 2
                                                  YEARS                                            Catch quantity and gross income by ma-
                                                                                             I     rine fisheries.




                 fish. Because fish are cultured in a group, one disease          years of fish cultivation experience accumulated by
                 is defined as one diagnostic case of one species in              the aquaculturist.
                 one fish culture establishment in a region.                        The fifth part is a memorandum for recording
                   The second part of the form contains         specific in-      other information, such as the results of bacterial
                 formation on the diseased fish; date of observation of           checks, symptoms of unidentified diseases, or infor-
                 unusual behavior or other abnormal symptoms, such                mation on the occurrence of red tide.
                 as body color change or appetite loss; date of appear-
                 ance of mortality; number of dead fish per pen;
                 whether the progress towards death was sudden or                 Results
                 prolonged; fish size (body length, and weight); and
                 any treatment administrated to the diseased fish.                Number of diseases reported
                   The third part details cultivation conditions such
                 as food type and species, region of food production,             The rate of fish farms requesting disease diagnosis in
                 size of healthy fish in the pen, the basic information           each prefecture was calculated by summing the total
                 necessary for calculating fish density, pen type, and            number of establishments requesting diagnosis by using
                 seed information.                                                the standard forms for that prefecture and dividing it
                   The fourth part contains environmental informa-                by the total number of marine fish culture establish-
                 tion on the cultivation area, that is, water depth,              ments quoted from governmental statistics (SID
                 weather, degree of rain, temperlature conditions,                1981-90). While both the number of disease cases re-
                 number of years the area was used as a fish farm, and            ported and the. diagnostic rate differed from one







              72        NOAA Ted@riiical Report NAM I I I

                                                                                               During the 9 years from 1981 to 1989, 56 species
                                              Table I                                       fish were reported to be diseased and over 20,000
                 Standard data form used to compile disease diagnosis                       disease cases were registered in 20 prefectures. About
                 information.                                                               three quarters of these cases involved yellowtail. Gov-
                                                                                            ernmental statistics (SID 1981-90) indicate an
                    Category                  Content                                       increased number of fish species in marine cultiva-
                    Essential                 Species name                                  tion. This increase in cultivation may contribute to
                    Information               Region                                        the increased number of fish with disease; an espe-
                                              Name of fish farm                             cially high incidence of disease originated from the
                                              Date of investigation                         recent increase in the culture ofjapanese flounder.
                                              Name of disease
                    Information               Date abnormality was detected
                    on the                    Date dead fish was observed
                    diseased fish             Number of dead fish per pen                   Disease Diversity
                                              Was death sudden or prolonged?
                                              Size of dead fish (cm)                        Fish disease was classified into six groups according
                                              Weight of dead fish (g)                       to the causal agent: bacteria, parasite, virus, nutri-
                                              Treatment used
                    Information on            Foodtype
                    cultivation               Region of food production                                    UNIDENTIFIED            OTHERS
                    conditions                Age of the fish                                  NUTRITIONAL    (8.4%)                10.6%)
                                              Size of healthy fish (cm)                          (6.5%)
                                              Weight of healthy fish (g)
                                              Number of the fish per pen
                                              Square measure of the pen(ml)                        VIRAL
                                                                                                    (3.9%)
                                              Length of the pen (m)
                                              Pen type
                                              Days after pen renewing
                                              Place of seed production                                          . . . . .... ..
                                                                                                                                                      BACTERIAL
                                              Age of seed introduced                                                                                    (78.5%)
                                              Date of the seed supplied                               F
                                                                                                   PARASITIC
                    Information               Water depth (m)                                       (8.3%)
                    on the                    Weather
                    cultivation               Degree of rain
                    environment               Temperature conditions
                                              Period the area was applied
                                                for fish cultivation (years)
                                              Length of experience of                                                      Figure 3
                                                aquaculturist                               Disease groups observed in marine fish cultures located
                    Memorandum                Other information such as                     along the Kuroshio Current.
                                                bacterial checks, symptoms
                                                of unidentified diseases,
                                                occurrence of red tide, etc ...             tion, fungus, and unidentified disease. When the dis-
                                                                                            eases occurred simultaneously, the causal agents were
                                                                                            classified into both partitions, so the total amount of
                                                                                            relative frequencies were over            * 100%. Bacterial dis-
              prefecture to another, a considerably high correlation                        eases were most frequently reported, accounting for
              was calculated between the numbers of disease cases                           approximately 80% of all disease cases (Fig. 3). Para-
              and numbers of marine fish culture establishments in                          sitic (8.3%), viral (3.9%), nutritional (6.5%), and
              each prefecture. Spearman's rank correlation coeffi-                          unidentified (8.4%) diseases were less frequent. The
              cient between number of establishments requesting                             partitioning of these values seems to be closely re-
              diagnoses by their prefectural experimental station and                       lated to the fact that diagnoses were made during the
              the total number of establishments in each prefecture                         advanced levels of disease progression. The number
              was about 0.9 for yellowtail, and 0.7 for red sea brearn                      of dead fish recorded in diagnostic cards suggests
              ,in 1985. These results may suggest that collected data                       that the diagnoses had been performed after a con-
              represent the trend of disease outbreaks in the fish of                       siderable number of dead fish were noticed.
              the warm current on a smaller scale. Mean diagnostic
              rates were about 20% for yellowtail, about 8% for red
              sea bream, and about 15% for total marine fishes cul-                         Species-specificity of diseases
              tured. Based on these values red sea breams appear to
              be less sensitive to disease than yellowtail.                                 The relative frequencies of the six disease groups var-






                                                                                  Ishioka: Epidemiology of Marine Fish Diseases     73




                          BACrERIAL     VIRAL            UNIDENTIFIED
                          PARASITIC El NUTRITIONALEM OTHER
                   N=2388     E    M-illi 1111111111                     Paralichthys olivaceus

                                        . ..... ..
                   N=37                                                  Sebastes schleseli
                                      ..... ........



                   N=901                                                 Takifugu rubripes
                                              MWW
                             E&TO

                   N=103                                                 Seriola duserili



                   N=428                                                 Seriola aureavittata



                   N=14689                                               Seriola quinqUeradiata



                   N = 171                                               Caranx delicatissimus



                   N=206                                                 Trachuruef japmicus


                            S@.
                   N=1857                                                pagrus major


                   N =104                                                Ar-anthopagrus schlewli


                   N=108                                                 Lateolabrax japonicus


                   N=59
                                                                         oplegnathus puctatus



                   N =209
                              IFS     W@N::                              oplegnathus fasciatus
                          120 110100 90 80 70  60  5@O 4'0 A 2'0. 10 0'
                                        Relative Frequency                                                         Figure 4
                                                                                                        Composition of disease variet-
                                                                                                        ies in each species.


               ied with fish species (Fig. 4). Over 90% of the dis-          pathogen -has not yet identified officially. The rate of
               eased yellowtail experienced bacterial disease. In red        unidentified disease was highest among the 13 fish
               sea brearn, a high rate (22.1%) of parasitic disease          species listed in Figure 4.
               resulted from parasitism by Bivagina tai on the gill, by        In Japanese flounder, bacterial disease accounted
               Longicollum pagrosom in the rectum, and                       for about 67% of all the cases registered, while the
               by other parasites. The high rate (33.4%) of nutri-           rate of parasitic disease, mainly due to a! Tyichodina
               tional disease involves yellow fat disease and peroxi-        sp. and Cryptocaryon irritans, was 17%.
               dative lipid intoxication caused by lipid metabolism            The relative frequency of the six disease groups
               disorders.                                                    was not related to any taxonomical order nor to the
                 In'tiger puffer, several serious parasitic diseases         ecological habitat of the cultured fish species.
               (Heterobothrium tetradonis, Trichodina sp., and                 The relative frequency of diseases caused by differ-
               Cryptocaryon i mitans) break out frequently (39.2%).          ent pathogenic bacteria, are shown in Figure 5 for 13
               The specific viral disease (9.0%), named kuchi-jiro           fish species. Yellowtail, purple yellowtail (Seriola
               sho (white mouth symptom) is often reported; its              aureovittata), and gold striped yellowtail (Seriola








           74      NOAA Technical Report NMFS I I I

                 ORDER                            Percifornes          Tetr    tifo@s
                 SUBORDER                    Percoidei  --I
                                              dae         Percichthyidae       Pleuronectif6ree--
                 FAMILY                                                
                                                Oplegrathidae      SPa id" Scarpe ifonles
                 SUBFAMILY                            I
                               Seriala           Opiegmthus
                 GENUS


                 120-

                 110-
                5100-

                   go-
                r.
                :4) 80-
                   70-

                   60-

                   50-
                ca
                   40-

                   30-

                   20-

                   10

                   0
                                                              cc
                                                                           C
                                                                           A
                                                                           1  z
                                                to  U
                                               4d
                                                              A            E
                                                                                    M   0

                              0
                                               V                                         4J
                                    0                                                    U






                               Vibi.ig       ftdtuherculasis          StreptDcoccosis
                               Edwardsiellosis 0 Glidig bct.ri.1 Di..e                                             Figure 5
                                                                                                          Composition of bacterial
                                                                                                          diseases in each species.





           dumefili), showed higher rates of pseudotuberculosis            bacteria and Streptococcus spp.
           and streptococcosis diagnoses, whereas jack mackerel              Tiger puffer suffered mainly from vibri.osis and
           (Tachurusjaponicus), taxonomically closely related to          gliding bacteria infections.
           yellowtail, had high rates of vibriosis and                       With some exceptions, the relative frequency of
           streptococcosis.                                                bacterial disease seems to be related to the host by
             Red sea bream were frequently infected with Vibrio            the host's ecological niche and related physiological
           spp., Edwardsiella tarda, and gliding bacteria (mainly          characteristics. As shown in Figure 5, pelagic or mi-
           Flexibacter sp.), but less so with Streptococcus spp. and       gratory fish species such as ye'llowtail were more
           Pasteurella piscicida. No edwardsiellosis was reported          susceptible to Ptisteurella piscicida, while bottom fishes
           from black sea bream (Acanthagrus schlegeli), which           like tiger puffer, Japanese flounder, and Japanese
           are related to red sea bream.                                   rockfish (Sebastes schlegeli), were more susceptible to
             Japanese flounder were seriously infected with                gliding bacteria. Other species in Figure 5 fell be-
           Edwardsiella tarda and suffered heavy damage; the               tween these two extremes.
           relative frequency of edwardsiellosis in Japanese                 A species' ecological habits are closely related to its
           flounder amounted to 41.8% of all bacterial disease             anatomical, physiological, and biochemical character-
           cases. They were affected to a lesser extent by gliding         istics. Pelagic and migratory fishes have a typical
 





                                                                                                  Ishi oka: Epidemiology of Marine Fish Diseases              75



                           35-


                           30-


                      i@   25-


                           20-


                                                                                      ..............
                           15                                     ........... .. .........


                      04
                           10"


                            5
                             01    .............                                                                  ...........
                                JAN    FEB     MAR      APR     MAY     JUN     JILY     AUG     SEP OCT         NOV     DEC
                                                                     MONTH                                                                      Figure 6
                                                                                                                                         Seasonal changes in
                                -YELLOWTAIL        ---- RED SEA BREAM         .....  JAPANESE     -TIGER PUFFER                          number of diseases
                                                                                   FLOUNDER                                              registered between
                                                                                                                                         1981 and 1989.




                   spindle shape suitable for swimming, developed cer-                       the pathogen) are very important to understand the
                   ebellum and vascular system, abundant superficial                         phenomenon or mechanism of bacterial disease out-
                   dark muscle, and an abundance of myoglobin in                             break.
                   their ordinary muscle. On the other hand, the bot-
                   tom fishes of coastal areas, such asjapanese flounder,
                   rockfish, puffers, and sea breams have flat or                            Seasonal Variations of Disease Outbreaks
                   rounded shapes, less-developed cerebellums, and
                   characteristics that indicate that these fishes are gen-                  Monthly variation in the relative frequency of disease
                   erally less active. Their white ordinary muscle has less                  cases reported is shown in Figure 6, for four fish spe-
                   myoglobin and hemoglobin, and their superficial                           cies: over 900 cases for each species were reported
                   dark muscle is not as developed.                                          during the 9-year period 1981-89.
                      Because Pasteurella piscicida, a causative bacterium for                 The highest evidence of disease occurred during
                   pseuclotuberculosis, migrates in the bloodstream of in-                   July for yellowtail (30%) and tiger puffer (17%), dur-
                   fected fish, active fish, with well-developed vascular                    ing August for Japanese flounder (15.5%), and
                   systems, may be more likely to reach an exhausted state.                  during September for red sea bream (13%). Reports
                      Infections by gliding bacteria tended to break out                     of disease for the latter three species peaked in the
                   in benthic fish or fish living near the beach. Because                    summer fforn June through September when the wa-
                   these fish are less active than the pelagic species and                   ter temperature is higher than 20' C. These peaks
                   may have a susceptible body surface, gliding bacteria,                    were not as steep as that of yellowtail, yet the summer
                   which move by means of surface mucus, may have an                         peaks suggest that fish under cultivation are exposed
                   advantage in expanding their living area. Also, be-                       to physiological and environmental conditions favor-
                   cause these fish are more likely to come into contact                     able to infection by pathogens, in spite of possible
                   with other individuals via their body surface, new in-                    efforts to promote the defensive activities of fish,
                   fections and propagation of gliding bacteria are                          such as immunological resistance.
                   prevalent.                                                                   In yellowtail, viral and bacterial diseases, especially
                      It is believed that the accumulation and integra-                      pseudotuberculosis, show significantly sharp seasonal
                   tion of information on fish species and pathogens                         changes (Fig. 7). Since the official identification
                   (such as cell or tissue susceptibility to pathogens; ana-                 (Sorimachi and Hara 1985) of the viral disease viral
                   tomical, histological, and physiological aspects of                       ascites (VA), the rate of this viral disease has in-
                   fish; environmental conditions; and characteristics of                    creased. VA disease breaks out in late May, reaches a







                 76            NOAA Technical Report NMFS 111



                               30-
                                                                                                                        12-
                               25-  -VIRAL                                                                              11-
                                         NUTRITIONAL                                                                    lo-
                               20 . . ......                                                                            9-                                                    %%
                                                                                                                   Z
                                                                                                                        8-
                               15-                                                                                 0    7-
                                                                                                                   W
                               10                                                                                       6-
                                                                                                                   w    5-
                               5                                                                                        4                                ............
                                                                                                                        3-                                         ..... ...............%
                               01                                                                                       2-
                       L)       I                      I    I     I           I          I     I
                       Z                                                                                                17
                               1.2-
                                                                                                                        0
                               .I-       VIRAL                                                                            JAN FES MAP APR MAY JUN              JLY AUG SEP OCT NOV DEC
                               0.9. ...... PARASITIC                                                                                                  MONTH
                       LU      0.8-
                       >                -NUTRITIONAL                                                                    -VIRAL        PARASITIC - - - NUTRITIONAL -UNIDENTIFIED         BACTERIAL
                               0.7-
                                         UNIDENTIFIED
                               0.6-
                       UJ      0.5-                               ........
                               0.4-
                                                                                                                                                     Figure 9
                               0.3-                                           ...........                               Seasonal changes in diseases ofjapanese flounder.
                               0.2-
                               0.1-    . ....... .

                               JAN  FEB MAR APR MAY JUN JLY AUG SEP OCT NOV DEC
                                                           MONTH


                                                      Figure 7                                                  October. Parasitic, nutritional,. and unidentified dis-
                               Seasonal changes in yellowtail diseases.                                         eases showed gentle seasonal changes in their
                                                                                                                occurrence. Parasitic and unidentified diseases broke
                                                                                                                out during summer, and the level of nutritional dis-
                                                                                                                eases peaked in autumn.
                         6-                                                                                        In red sea bream, viral diseases represented only
                                                                                                                3.6% of the total disease cases reported (Fig. 8), and
                     -Z@ 5-                                                                                     for the most part were observed as lymphocystis dis-
                                                                                                                ease every month of the year. Parasitic diseases were
                      .1 4-
                                                                                                                reported least in February and March and most fre-
                         3-                                                                                     quently between May and December. Nutritional
                     >                              . ........
                                                                                                                disease outbreaks appeared between May and Sep-
                         2-
                                                                                                                tember, indicating that they are chronic in nature.
                         1                                                                                      Half of the bacterial disease cases reported were
                         0                                                                                      vibriosis (53.2%), which occurs all year round with a
                               JAN FEB MAR    AM MAY JUN JLY        d* 41' 0&         NOrV OTC                  peak at winter (see Fig. 14). Gliding bacterial disease
                                                       MONTH                                                    also occurred all year round but the higher rates
                               VIRAL ..... PARASITIC --- NUTRITIONAL - UNIDENTIFIED   -BACTERIAL                were found in January and from May through July
                                                                                                                (see Fig. 15). Edwardsiellosis accounted for about
                                                      Figure 8                                                  10% of all disease cases in red sea bream and was
                               Seasonal changes in diseases of red sea bream.                                   prevalent between July and November (see Fig. 13).
                                                                                                                   Since the successful development of seedling pro-
                                                                                                                duction methods, production of the Japanese
                                                                                                                flounder has gradually increased. The main viral dis-
                 peak in June, and ends in mid-July. The rate of VA                                             ease observed in cultural flounder was epidermal
                 disease diagnoses was very low and account for on                                   ly         necrosis, which occurred in spring during the fry
                 1% of yellowtail disease reported in June; its occur-                                          stage and accounted for only 0.,8% of all disease cases
                 rence fluctuates yearly. The rate at which the major                                           (Fig. 9). Japanese flounder suffered from parasitism
                 bacterial diseases were reported showed similar sea-                                           by protozoa such as Cryptocaryon spp., Trichodina spp.,
                 sonal variations. Vibriosis accounted for about 10%                                            and other Ciliata. Because there are no effective
                 of all the bacterial diseases reported and the number                                          treatments for these parasites, infected fish are left
                                        V'    A

                                              A
                                              L
                                        @
                                              S'TIC
                                          AR  '110-Al
                                              NIT @nM
                                        IN




                                                                              ...........
                                     . ..........

































                 of cases peaked in June. Cases of pseudotuberculosis                                           untreated, resulting in a great deal of infection and
                 peaked sharply in July (see.Fig. 11), and strepto-                                             death. A trial is now being conducted to see if condi-
                 coccosis epidemics seem to occur between July and                                              tions leading to infection can be effectively removed







                                                                                                                     Ishioka: Epidemiology of Marine Fish Diseases                           77

                     by increasing the water supply to rearing areas. It is
                     unknown why nutritional diseases are rare in Japa-                                                25 -
                     nese flounder. Bacterial diseases are serious for this                                            20 -
                     fish accounting for about 67% of all disease cases
                     reported. The most virulent, edwardsiellosis,                                                     15-
                     amounts to about 28% of all disease cases.                                                      >_ 10-
                                                                                                                     0
                                                                                                                     Z
                     Edwardsiellosis is most prevalent during the summer,                                            W
                     from June through October, with the number of re-                                                   5-
                     ports peaking in August. Streptococcosis is most
                                                                                                                     UJ  0.1                          -----------------
                     prevalent from July to October and vibriosis and glid-                                          ?!
                                                                                                                       0.5-
                     ing bacterial disease are both most prevalent between
                     April and August. Unidentified disease accounts for                                             cc 0.4-
                     16% of all disease cases reported.
                                                                                                                       0.3-


                                                                                                                       0.2

                           6-
                                                                                                                       0.1                                 A
                           5-
                                                                              .. .........                               DI                           V N         ................
                        Z
                        w: 4-                                                                                              JAN FEB MAR APR MAY JUN JLY AUGG SEP           OCT NOV DEC
                                                                                                                                                    MONTH
                           3-                   ................                                                                        YELLOWTAIL             RED SEA    BREAM

                                                                                                                                   ..... JAPANESE              TIGER PUFFER
                           2-                                                                                                           FLOUNDER



                                                                                                                                                Figure I I
                             JAN FEB     MAR APR    MAY    JUN JLY     AUG   ShP  OL    N6V   DEIC              Seasonal changes in pseudotuberculosis for each species.

                                                          MONTH
                               VIRAL .... PARASITIC--- NUTRITIONAL   - UNIDENTIFIED


                                                      Figure 10                                                      10
                                                                                                                     9-
                               Seasonal changes in diseases of tiger puffer.                                         K
                                                                                                                     8-

                                                                                                                     Z
                                                                                                                     7-
                                                                                                                     W
                                                                                                                     36-
                       In tiger puffer (Fig. 10), kuchi-
                                                                         jiro sho was regis                          ff 5-
                     tered often throughout the year. Parasitic disease                                              4-
                     amounted to about 39% of all disease cases (Fig. 10).                                           3-
                                                                                                                     2-
                     Fatal parasites included a Dichodina spp., a protozoa
                     which was prevalent between April and October, and                                              0-
                     Heterobothrium tetrodonis, a monogenea prevalent                                                  JAN FEB MAR A6 MAY JUN JLY AUG SEP OCT NOV DEC
                     mainly from April through August. Nutritional dis-                                                                             MONTH
                     ease was also prevalent from spring through autumn.                                               -YELLOWTAIL   ----- RED SEA BREAM --JAPANESE -TIGER PUFFER
                                                                                                                                                            FLOUNDER
                     Higher levels of bacterial diseases were diagnosed
                     from June to September: most were cases of vibriosis
                     and gliding bacterial disease.                                                                                             Figure 12
                       Pasteurella piscicida was a virulent pathogen for                                             Seasonal changes in streptococco-sis for each species.
                     pseudotuberculosis in yellowtail and to a less extent
                     in other cultured fish species (Fig. 11). This disease
                     broke out seasonally, appearing during warmer
                     months in all fish species.                                                             June through October; the level. of reported cases
                       Streptococcus sp. was most common in yellowtail and                                   peaked during August in Japanese flounder and
                     Japanese flounder and occurred less frequently in                                       showed a plateau from July to November in red sea
                     red sea bream and tiger puffer, showing similar sea-                                    brearn (Fig. 13). The optimal water temperature
                     sonal variation patterns that peaked in September                                       ranges from 15 to 24' C for Japanese flounder and is
                     (Fig. 12). Edwardsiella tarda was virulent for Japanese                                 slightly broader from 13 to 28' C for red sea brearn.
                     flounder and red sea brearn and most prevalent from                                     Cultured Japanese flounder suffer from exposure to







                78          NOAA Tectinical Report NMFS I I I

                                                                                                        high temperatures above 25' C in summer and become
                           7-                                                                           more susceptible to bacterial infection. On the other
                           6-                                                                           hand, red sea brearn grow wdll during summer and suf-
                                                                                                        fer from nutritional disorders in autumn. Because
                           5-
                                                                                                        EdwardsieHa tarda can multiply more actively during the
                      0    4-
                                                                                                        summer season, Japanese flounder, which are less toler-
                           3-                                                                           ant of higher temperatures, are more susceptible to this
                                                                                                        pathogen. Conversely, red sea bream, which are more
                           2-
                      W                                                    ----------
                                                             ----------
                                                                                                        tolerant of higher temperatures, are less susceptible to
                                                                                                        this pathogen thanjapanese flounder.
                           0.                 .............. .                                             Seasonal variations in the relative frequency of
                           JAN FEB MAR APR MAY         JUN   III AUG SEP OCT      NOV DEC
                                                     MONTH                                              vibriosis are complicated and differ according to fish
                                       ---- @REO SEA BREAM     JAPANESE FLOUNDER                        species (Fig. 14). This complexity may result not only
                                                                                                        from the difference in susceptibility of each fish spe-
                                                                                                        cies but also from the presence of different varieties of
                                                  Figure 13                                             Vibfio. Vibrio anguillarum is known to have varied sero-
                      Seasonal changes in edwardsiello-sis for each species.                            types. Susceptibility of fish to this pathogen would vary
                                                                                                        with the serotype and fish species involved.
                                                                                                         . The term "gliding bacteria" implies several bacte-
                                                                                                        rial species. The gliding bacteria resulting in marine
                           5                                                                            fish diseases are mostly Flexibacter, Cytophaga, and
                                                                                                        Sporocytophaga spp. The unique characteristics of each
                           4-                                                                           gliding bacterium may contribute to the complicated
                      0
                      Z                                                                                 patterns of seasonal variations in reported infection
                           3-
                                                                                                        among the different fish species (Fig. 15).

                      w    2-


                                                                                                        Fish size and disease
                                                                        ............V

                           0
                           JAN FEB MAR      APFI MAY JUN JLY     AUG   SEP OCT NOV     DEC              The variation in relative frequency of disease cases
                                                    MONTH                                               reported in yellowtail is illustrated according to fish
                           -YELLOWTAIL   ----- RED SEA BREAM   JAPANESE -TIGER PUFFER                   size (body weight) in Figure 16. For this analysis, rela-
                                                               FLOUNDER
                                                                                                        tive frequency was calculated by dividing the total
                                                                                                        number of reports for each disease in the defined
                                                  Figure 14                                             size class by the total number of disease cases with
                           Seasonal changes in vibriosis for each species.                              body weight (bw) data for each species. Viral disease
                                                                                                        was more frequent in younger fish (<50g bw), while
                                                                                                        nutritional diseases were more prevalent in older fish
                                                                                                        (>250g bw) and season also an influencing factor. Oc-
                           6-                                                                           currence of bacterial disease appears closely related
                           5-                                                                           to body weight. Yellowtail seemed to be successively
                                                                                                        attacked by VA virus first, then Vibrio and Pasteurella
                           4-
                                                                                                        piscicida, and finally Streptococcus. This sequence of in-
                      a                                                                                 fection suggests that susceptibility of yellowtail to
                           3-
                                                                                                        different pathogens varies with body weight or
                           2
                                                                                                        growth. This may imply not only the development of
                                                                                                        defense mechanisms to the pathogens but may also
                           01                                       ---------- --                       represent the changes in the overall physiology of the
                           JAN FEB MAR APR MAY         JUN   JLY A6G SEP OCT NOV DE'C                   fish under cultivation.
                                                       MONTH                                               In red sea bream, parasitic diseases occurred more
                           -YELLOWTAIL ---- RED SEA BREAM    JAPANESE       TIGER PUFFER                frequently in fish between 50 and 250 g bw while
                                                             FLOUNDER                                   reports of nutritional disease increased in fish over
                                                                                                        250 g bw (Fig. 17). The rate of gliding bacteria diag-
                                                  Figure 15                                             noses was highest in young fish below 10 g bw and
                 Seasonal changes in gliding bacterial diseases for each                                that of vibriosis was highest in fish between 50 and
                  species.                                                                              100 g bw. The diagnostic rate of edwardsiellosis was






                                                                                                                         Ishioka: Epidemiology of Marine Fish Diseases                            79


                                2.4-                                       VIRAL
                                2.2-
                                2-                                         PARASITIC

                                                                           NUTRITIONAL
                                IA-
                                1.4-                                       UNIDENTIFIED



                                            M



                            W
                                0.2
                            Uj
                            cr
                                                     0                                                    AGE
                            LLJ 20-
                                                                           VIBRIOSIS
                            F-
                                                                           PSEUDOTUBERCULOSIS
                            W   Is-
                                14-                                        STREPTOCOCCOSIS

                                12 -






                                4-

                                2


                                   =@Ill     =@Ioo     =<511     =@Illl =<2110       =<4110 =<1000        =<1010
                                        =<51      =<250      =<750    =<1511      =<3111   =<5110    =<7000                                              Figure I    .6
                                                                 BODY W    .EIGHT (9)                                         Relationship between fish size and disease
                                                                                                                      I       outbreaks in yellowtail.




                                13-
                                12-
                                11-                                                  13 VIRAL

                                                                                     10 PARASITIC

                                                                                     0 NUTRITIONAL
                                6-                                                   0 UNIDENTIFIED
                                5-
                                4-

                          Z
                          w     2-

                          W
                          LL
                          LU                                                             AGE
                          >


                          LU
                          X     5-                                                   0   VIBRIOSIS
                                                                                     0   PSEUDOTUSERCULOSIS
                                4-
                                                                                     0   STREPTOCOCCOSIS
                                                                                     0   EDWARDSIELLOSIS

                                                                                         GLIDING BACTERIA




                                                [email protected]
                                                             =<580           loll        =@2000
                                                                     =-751        =<1590       =@31109
                                                              BODY    WEIGHT (9)                                                                         Figure 17
                                                                                                                              Relationship between fish size and disease
                                                                                                                              outbreaks in red sea bream.







          80      NOAA Tectinical Report NNM I I I




                                                      VIRAL


                 I-                                   PARASITIC

                 11-
                                                      NUTRITIONAL


                 4-
                                                      UNIDENTIFIED

                 3-



             Z


             0
             LU          0                                 AGE
             LL  21@
                                                 El VIBRIOSIS
             Uj

             <   14-                                  PSEUDOTUBERCULOSIS

             LU  12-
                                                      STREPTOCOCCOSIS
                 Is-

                 11-
                                                 0    EDWARDSIELLOSIS


                 4                                    GLIDING BACTERIAL

                 2


                               =@111 -251   511 =@110 =@1111 =@1610 =<2110
                               BODY WEIGHT(g)                                                    Figure 18
                                                                               Relationship between fish size and disease
                                                                          I    outbreaks in Japanese flounder.




              4 0-





                                                      VIRAL

                                                      PARASITIC

                                                      NUTRITIONAL

                                                      UNIDENTIFIED
             L)  16-
             Z


             0

             U.  0-
                      0




             W   4-
             a:
                                                  El VIBRIOSIS

                 3-                                   PSEUDOTU13ERCULOSIS

                                                      STREPTOCOCCOSIS
                 2-
                                                  E3 GLIDING BACTERIA





                                                                                                Figure 19
                                     BODY WEIGHT (9)                           Relationship between fish size and disease
                                                                               outbreaks in tiger puffer.







                                                                                                                                                                                                Ishioka: Epidemiology of Marine Fish Diseases                                                                         81

                                   higher in larger fish, especially those over 250 g bw.                                                                                               streptococcosis were synchronized with nutritional
                                         All ages of Japanese flounder experienced out-                                                                                                 disorders.
                                   breaks of parasitic diseases, while viral diseases were
                                   found only in fish below 10 g bw (usually herpes vi-
                                   rus) (Fig. 18). Vibriosis and gliding bacterial disease                                                                                              Locality and Disease
                                   outbreaks were more common in younger fish
                                   (<50g). Although streptococcosis and edwardsiellosis                                                                                                 As shown in Figure 20, for yellowtail in 1986, some
                                   were both more prevalent in larger fish (>250 g),                                                                                                    characteristics are common to all the prefectures
                                   edwardsiellosis was more common than streptococcosis                                                                                                 listed. Although the relative frequency of bacterial
                                   in the fish of 50-100 g bw.                                                                                                                          disease was about 80% in all prefectures, the compo-
                                         In tiger puffer, a unique feature was found: fish                                                                                              sitions of different bacterial diseases were to some
                                   250 to 500 g bw appear to be most susceptible to                                                                                                     extent different by locality. Mie Prefecture is the
                                   pathogens (Fig. 19). This may be dependent on the                                                                                                    most northern of all the prefectures listed in Figure
                                   method used to culture this species. Though this                                                                                                     20, while Kagoshima Prefecture @is located in most
                                   species is in great demand by the market, its cultiva-                                                                                               southern part of Japan. Interprefectural analysis sug-
                                   tion from the egg through the spawning stage is                                                                                                      gests that the composition of different bacterial
                                   very difficult. The eggs for seedling production are                                                                                                 diseases seems not to be related to geographical lo-
                                   generally raised by the public sector and are in most                                                                                                cality. A possible similarity exists between the
                                   cases supplied from the natural resource by                                                                                                          composition of bacterial infections observed and the
                                   fisheries, which also provides some large sized seed-                                                                                                type of cultivation management used in some prefec-
                                   lings. Because the aquaculturist cultures mainly                                                                                                     tures. There are two types of management:
                                   large-size seedlings, diagnoses may concentrate on                                                                                                   cultivation of large seedlings and production of fish
                                   the larger sized fish.                                                                                                                               of marketable size. Streptococcosis tended to be re-
                                         In all species, nutritional disease tended to occur                                                                                            ported at a higher rate in the prefectures managing
                                   in larger fish, probably as a result of prolonged nutri-                                                                                             the larger fish (2-year-olds) of marketable size.
                                   tional treatment. Outbreaks of edwardsiellosis and                                                                                                     No characteristic difference in @seasonal outbreak





                                                             783            129       288           100           111          164         327           4 9               139          154     45           72           19          23 (N)
                                                  too-                      S-d
                                                                            0 C
                                                                            )0                                                                                                                                                                                            UNIDENTIFIED
                                                                            0 C
                                                                                                                                                                           N:
                                                                            )0
                                                                                                                                                                    ......                                                                                                OTHERS
                                                                            XX

                                                                            ...:.:.X*.                                                                                                                                                                                    NUTRITIONAL
                                                                                                                                                                           . ........
                                                                                                                                                                                                                                    -X -:::j
                                                                                                                                                                                        ....               .......
                                                                                                                                                                    .......                                     ...
                                                                                                  ........                                                          ......
                                                                                                                                                                                                                                                                          VIRAL
                                                                                                                                                                                                                                                          F0
                                                                                                                                                                                                                                                              nn]

                                                  so
                                                                            ..N                                                                                                                                                                                           PARASITIC
                                         Z
                                                                                                                                                                                                                                                                          OTHER
                                                                                                                                                                                                                                                                          BACTERIAL DISEASE
                                                                                                                                                                           ...                             .......

                                         W
                                         LL                                                                                                                                                                                                                               NOCARDIOSIS
                                                  40
                                         Uj
                                                                                     .. ...                                                              . . . ......
                                                                                                                                                                                                                                                                          PSEUDOTUBERCULOSIS


                                                                                                                                                         ....                                                                                                             VIBRIOSIS
                                         LU
                                                  20-                                                                                                                                                                                                                     COMB NED DISEASES
                                                                                                                                                                                                                                                                          WITH STREPTOCOC-
                                                                                                                                                                                                                                                                          costs
                                                                                                                                                                                                                                                                          STREPTOCOCCOSIS



                                                             LU             0                                      0                        W                                                   <            <            <
                                                                            0                                                                            <                 <                                                           U
                                                             T                                                                                                                                  <            7            <
                                                             LU                       0             ro                                                                                  0       A0           &0
                                                                                      @4            <              <                                     <                 <
                                                                                                                               0                                                                             D            <            <
                                                                                                                                                                           <
                                                                                                                                                                           Z                                 0            <


                                                                                                                    PREFECTURE
                                                            X. -X














                                                                                    X










                                                                                                                                                                    Figure 20
                                                                                                      Composition of diseases of yellowtail by prefecture during 1986,







             82        NOAA Technical Report NNn I I I



                       501 M I E                             40' KOCHI
                       40-    N=327                          30-    N=288
                       30-                                   20-

                       20-                                   10

                       10
                                                                SAGA
                       0
                       20 WAKAYAMA                           20.    N= 45
                       1.0                                   to-
                              N= 19
                       0 1 . .
                       30- HYOGO                             59 N AGASAK I
                       20-                                   40
                              N=129                                 N=139
                       10-                                   0
                Z
                W                            In              20
                D 0
                a      20,
                W         YAMAGUCHI                          1
                CC     10,
                LL            N=23                           01. . .
                W 0                          01@113 30          KUMAMOTO
                >      40- TOKUSIMA           -              20
                       30-                                          N=81
                              N= 72                          1
                W
                cc     20-                                                       r,3
                       10,                                   39 OITA
                                                             2
                       0.       . . . .
                       40 -KAGAWA                            10     N=154
                       30-    N=100
                       20-                                   48, MIYAZAKI
                       10.                                   30-    N= 49
                       0'                     0              20-
                       40 EHIME                              10.
                       30     N= 783                         4
                       20                                    20 KAGOSIMA
                       10                                    10
                       0                            ri       0      N=164
                       > 0 Z    co cr cc      -11   CL          6 @i io d:  C*C >       a 0.                           Figure 21
                       0 W <    W     a.    D :)    W 0      0  W < W             5 M :3 LU
                       Z 0 n    LL      M   1) -3<  9) 0     Z  a , LL                  < rn 0            Patterns   of the outbreak of
                                                                                                          pseudo tuberculosis in yellow-tail
                                                                                                          in each prefecture in 1986.


             of virulent disease was found according to geographi-              gests that all fish culture fields along the warm cur-
             cal location. In the example of 1986 shown in Figure               rent are in an area favorable to the multiplication
             21, pseudotuberculosis broke out earlier in warmer                 and propagation of pathogens.
             areas. This disease was reported to occur at a higher
             rate in June and July in Kagoshima, Miyazaki, and
             Kochi Prefectures, while the rates were higher in July             Discussions
             and August in other prefectures. Because the optimal
             range (20 to 25' Q for multiplication of Pasteurella               From this epidemiological examination, it is clear
             piscicida is strict, the location of outbreaks of this dis-        that the natural and delicate relationship between
             ease could be determined by calendar and locality or               fish and their environment is disturbed on a local
                                                             0


                                                             0
                                                             0
                                                             "1                     0
                                                             01"
                       LN















             latitude.                                                          basis by fish cultivation in coastal areas and induces
               As a whole, no characteristic local feature was ob-              the propagation of pathogens and the outbreak of
             served that could explain particular disease                       disease in the cultured animals. As shown in Figure
             outbreaks in the area surrounded by warm oceanic                   22, the means and methods of fish culture affect not
             current, except perhaps slight differences. This sug-              only fish health but also water quality and pathogen






                                                                                               Ishioka: Epidemiology of Marine Fish Diseases              83

                                                                                          tal factor. Higher temperature is beneficial in activat-
                                                                                          ing various physiological functions, including
                                          0 LI T BREAK                                    immunological processes in healthy fish. But this fac-
                                              0 F                                         tor can also be a harmful to unhealthy fish under
                                          D ISE A SES                                     poor environmental conditions.
                                                                                             The results of this study help us to direct future
                                                                                          study and research in this field. Unidentified diseases
                        PATHOGEN         INFECT ION         F I S H                       must be clarified as soon as possible, especially in
                                                                                          new fish species introduced for cultivation. In this
                                                                                          type of research, it is most important to have a thor-
                                                                                          ough knowledge of the anatomy, physiology, ecology,
                                        ENVIRONMENTAL                                     pathology, and immunology of the fish and patho-
                                         CONDITION                                        gens. This may lead to the development of improved
                                                                                          cultivation techniques.
                                                                                             Effective methods to diagnose fish health should
                                   CULTIVATION       TECHNIQUE
                                                                                          be developed, not only to prevent mass mortality by
                                                                                          pathogens that are generally nonvirulent, but also to
                                             Figure 22                                    regulate cultivation techniques. A large amount of
                 Disease outbreak flowchart for marine fish cultured in the               research is needed in this area, for example: physi-
                 Kuroshio Current.                                                        ological studies on poikilothermal fish, pathological
                                                                                          studies that clarify the mode of action of pathogen
                 distribution; major factors include fish density in a                    within their hosts during infection, and immunologi-
                 pen, location of the pen, pen density, handling, ex-                     cal .studies to explain fish defense mechanisms
                 cess food supply, food quality, sanitary treatment of                    against pathogens.
                 nets, type of food and other materials, and treatment                       Epidemiological research in a small area such as
                 of dead fish. Supplying unsuitable or unsanitary food                    one bay unit or a fisheries cooperative association
                 induces the degeneration of fat in fish, whereas sup-                    unit should be carried out in detail to clarify the di-
                                                                                          rect relationships among disease outbreaks,
                 plying excessive food makes them generate fat.                           environmental factors, and cultivation techniques.
                 Unsuitable handling techniques injure the body sur-                      Such research could provide knowledge for a more
                 face, especially in younger fish, and allows pathogens                   successful cultivation of healthy fish.
                 to invade. Fish are also susceptible to pathogens un-
                 der some nutritional conditions and during certain
                 developmental stages. Culturing fish under high den-                     Citations
                 sity adds additional stress.
                    A high density of pens interrupts the water current                   Sorimachi, M., and T. Hara.
                 and causes water quality to deteriorate. As excess                           1985. Characteristics and pathogenecity of a virus isolated
                 food and fish excrement are deposited on the bot-                              from yellowtail fingerlings showing ascites. Fish Pathol.
                 tom and dissolve into seawater, the quality of water in                        19:231-238. (Injapanese.)
                 the cultivation area degrades. These changes in envi-                    SID (Statistics and Information Department)..
                                                                                              1981-90. Annual statistical report on fisheries and culture
                 ronmental conditions are beneficial for pathogen                               production of Japan. SID, Jpn. Min. Agri. Forest. Fish.,
                 multiplication, which, in turn, lead to infection of                           Tokyo. (InJapanese)
                 the fish and to outbreak of disease.                                     Umezawa, S., and H. Ishioka.
                    As previously described, the seasonal changes of                          1988. Program for compiling disease cases diagnosed in pre-
                                                                                                fectures. In Personal computer programs for fish popula-
                 disease cases are very remarkable, and it is thought                           tion dynamics, vol. 1, p. 314-339. Tokai Regional Fisheries
                 that temperature is the most influential environmen-                           Research Laboratory, Tokyo. (Injapanese.)






                                          Characterization of Hematic Neoplasia in the
                                                        Softshell Clain Mya arenaria



                                 PAUL W. RENO*, ANDREA ILLINGWORTH**                        ' and MICHAEL DORITY***

                                                      *Coastal Oregon Marine Experiment Station/Microbiology
                                                              Mark 0. Ha0eld Marine Science Center
                                                                    2030 S. Marine Science Dr
                                                                   Newport, Oregon 97365-5296

                                                                  "Institutefor Cellular Research
                                                                           345 State St.
                                                                        Bangor, ME 04401

                                                                   ***Department of Microbiology
                                                                        181 Hitchner Hall
                                                                        University of Maine
                                                                     Orono, ME 04469LO131





                                                                          ABSTRACT


                                     A leukemia-like disease, variously termed hemic, hematopoietic, or hematic neoplasia,
                                   (HN) has been detected in bivalves from diverse geographic locales. The wide geo-
                                   graphic range of the disease, coupled with mortalities due putatively to HN, makes it
                                   important to determine the nature of the abnormal cells characteristic of the disease.
                                   The studies reported here determined certain biochemical and genomic characteristics
                                   of HN in the softshell clam M@a arenaria. No significant difference in the total amount of
                                   lipid of normal and abnormal hemocytes was noted. Fatty acids of the omega 3 and
                                   omega 6 families were elevated in clams with the highest HN intensities. Similarly, in
                                   vitro incorporation of acetate into phospholipids and neutral lipids by normal lympho-
                                   cytes was significantly different from HN hemocytes but unlike the pattern seen in
                                   mammalian leukemias. Examination of the DNA content of normal and HN cells by flow
                                   cytometry indicated a high degree of aneuploidy in the HN cells. This consisted of clams
                                   with hyperdiploid DNA content of 1.6 to 2.0 times normal, and a hypodiploid population
                                   -With DNA 0.85 times normal. Hyperdiploid cells had significantly higher DNA synthesis
                                   than diploid cells. In this respect, HN cells are similar to true leukemias of mammals, but
                                   the combination of hypodiploid and hyperdiploid cells in one individual is uncharacter-
                                   istic of mammalian neoplasia. In sunimary, hemocytes of M. arenaria affected with HN
                                   have some features coincident with mammalian leukemias but also several unique char-
                                   acteristics.




                 Introduction                                                       diseases of cultured and feral bivalves in order to pre-
                                                                                    dict sustainable harvests of this significant marine
                 The harvest of bivalves from commercial and cul-                   resource accurately.
                 tured sources is of considerable economic im-                        Among the most widespread diseases of bivalves
                 portance on both worldwide and national scales. Dis-               are the putative neoplastic diseases which have been
                 ease affects natural as well as artificially propagated            detected in a wide variety of commercially important
                 populations. Unfortunately, our knowledge of dis-                  species and from a geographically disparate range
                 eases in bivalves is quite restricted in comparison to
                 the information base available for finfish. It is impor-
                 tant that we establish a more detailed database for                *Send correspondence to this author.


                                                                                                                                               85







            86      NOAA Technical Report NMEFS I I I

            (Couch 1969; Farley 1969; Brown et al. 1977). The                 Materials and Methods
            primary tissue distribution of these diseases ap-
            pears to be gonadal ("germinomas") (Yevich and
            Barszcz 1976, 1977) and hernatic (variously termed                Sample Collection, Evaluation of HN, and
            hernatopoietic neoplasms, hernatic neoplasms, or                  Animal Maintenence
            hernic neoplasms). Hernatic neoplasia (HN) has
            been associated with elevated levels of mortality in              Softshelled clams ranging in size from 45 to 120-mm
            field and laboratory studies with the softshell clam              valve length were collected from Long Cove,
            Mya arenaria (Appeldoorn and Oprandy 1980; Coo-                   Searsport, Maine, during the summer, placed on ice,
            per et al. 1982) and the edible oyster Ostrea edulis              and returned to the laboratory for evaluation of HN
            (Alderman et al. 1977). The prevalence rates for                  levels. When necessary, animals were held in the wet
            HN in M. arena?ia range from 0.02 to 84% , and                    laboratory at. the University of Maine in a re-
            intensities of HN in individuals span the range                   circulating artificial seawater system at 14' C until
            from less than 0.01% to greater than 99% of hemo-                 needed. Animals were generally evaluated within I
            cytes affected (Couch 1969; Mix 1986; Reno,                       week of collection. Evaluation of HN was performed
            unpubl. observations). The pervasive nature of this               by a modification of the method of Cooper (1982).
            disease and its potential for killing feral and cul-              Approximately 100 @LL of hemolymph was removed
            tured bivalves makes it essential to understand the               from the cardiac or anterior adductor sinus, 50 IiL
            nature of the disease and its etiology. The results               was placed in one well of a 96-well, flat-bottomed
            reported here reflect an initial attempt to define                microtitration plate, and hemocytes were allowed to
            mo re clearly the nature of the disease in M.                     adhere for 20 to 30 minutes. Samples were then
            arena,ria.                                                        evaluated for the levels of HN by microscopic exami-
              Hernatic neoplasia is characterized by its prolif-              nation under phase optics at 20OX      'with an inverted
            erative nature and the resulting cytological mor-                 cell culture microscope. The number of nonadherant
            phological abnormalities assessed by microscopic                  or loosely adherant, rounded cells in the hemolymph
            examination of either unstained, or fixed and                     (HN cells) was compared to the number of fully ad-
            stained cells (Couch 1969; Farley 1969; Cooper et                 herent spreading normal hemocytes by counting 500
            al. 1982). These criteria, however, are not adequate              to 1000 cells and converting the proportion to
            for accurately defining HN in bivalves as neoplastic              percentages.
            because these invertebrates are evolutionarily dis-
            tant from the mammals, the phylum on which                        Lipid Analysis
            virtually all definitive work has been done on the
            characteristics of neoplasia. This lack of definition is          The major lipids that are altered in neoplasia are the
            noted in two prominent reviews of presumed neo-                   fatty acids, the neutral lipids, and the phospholipids.
            plasms of bivalves: "Some of the neoplasms may be                 Several methods were used to evaluate lipid changes
            of questionable validity ... and little is known about            in HN cells, including gravimetric total lipid content,
            the exact nature of the cells involved but, in gen-               fatty acid methyl ester (FAME) profiles, and in vitro
            eral, they are considered neoplastic" (Mix 1986);                 incorporation of radiolabelled acetate into neutral
            and "In spite of intensive study, the true nature of              lipids and phospholipids.
            these neoplastic cells is not yet quite clear."                     Gravimetric analysis was carried out on washed he-
            (Lauckner 1983). There are scores of structural,                  mocytes from normal and abnormal clams using the
            biochemical, immunological, and genomic alter-                    method of Sasaki and Capuzzo (1984). This involved
            ations in mammalian cells associated with the                     a modified Folch procedure employing several ex-
            transformed or neoplastic state (reviewed in Wood                 traction cycles with chloroform: methanol. Total lipid
            1972; Marchesi 1976; Vasiliev and Gelfand 1981;                   by weight was obtained using an ultramicrobalance
            Heim and Mitelman 1987; Iversen 1988). Among                      (Cahn model 25 automatic electrobalance). Fatty ac-
            the most well characterized biochemical alterations               ids were esterified to fatty acid methyl esters (FAMEs)
            that occur in transformed cells are those occurring               to stabilize them prior to analysis, which was carried
            in two fundamental classes of molecules: the nucleic              out on hemocytes from clams with various levels of
            acids and the lipids. The present work was designed               HN and clams with no evidence of HN. Fatty acid
            to monitor potential differences between normal                   separation was carried out using the modified Bligh
            hemocytes'and HN cells from M. arenaria compared                  and Dyer (1959) method of Jerkofsky and DeSiervo
            by flow cytometric analysis and in vivo and in vitro              (1986). Washed, pelleted hemocytes were extracted
            lipid ccinstitution.                                              with chloroform:methanol followed by a saline phase






                                                                   Reno et al.: Hematic Neophisia in the Softshell Clam Mya amuwia        87

                separation, and final suspension in chloroform. A                Cells were washed in buffered saline with 0.1% bo-
                known quantity of a 19-carbon fatty acid was added to            vine serum albumin, counted, and suspended in a
                the cell pellet prior to extraction to serve as an inter-        solution of I jig/mL RNAse and 0.01% NP-40 deter-
                nal standard for the extraction process. Samples were            gent to increase membrane permeability. This was
                transesterified with a methanolic base reagent fol-              followed by the addition of propidium iodide
                lowed by methylene chloride and final suspension in              fluorochrome at the ratio of logg/106 cells in a solu-
                hexane. Esterified samples were then applied to a gas            tion of NP-40 and 0.1% polyethylene glycol 8000 at
                chromatograph (Hewlett Packard model HP5890A)                    pH 5.0. Examination of the cells under a ultraviolet
                equipped with a 30 m DB carbowax capillary column.               light microscope confirmed that nuclear morphology
                Peak areas were calculated with a disc integrator, and           was normal and that no artifacts were present. The
                retention times were compared with known standard                cells were examined in a flow cytometer (Becton-
                FAMEs.                                                           Dickinson, EPICS model CS) and the relative
                  In vitro assimilation of radiolabelled acetate into            fluorescence (DNA content) and cell cycle analysis
                neutral and polar lipids was carried out using hemo-             was performed using a Modfit analysis program (Ver-
                cytes that were removed from the clams within, 8                 ity So ftware Corp.), which integrated the area under
                hours of capture. To each of four replicate tubes con-           the various peaks and estimated the percentage of
                taining the hemocytes, an equal volume of 0.45                   total area for each peak, as well as their coefficients
                @Lrn-filtered sterile seawater containing   10 RCi of 14C_       of variation.
                acetate was added. The cell-acetate mixture was
                incubated at 15' C for 24 hours and extracted as de-
                scribed for FAMEs, except that, in order to improve              Results
                the precipitation of labelled lipid, non-labelled car-
                rier lipid was added. The carrier lipid was prepared
                from two shucked clams by removing the digestive                 Lipid Analysis
                diverticula and skin of the siphons, blending them in
                a blender, and extracting as previously described.               Comparison of hemocytes from HN-affected and nor-
                The lipid extract was weighed and resuspended in                 mal clams by gravimetric analysis indicated that the     're
                chloroform to a concentration of 300 mg/mL; 30 mg                was no significant difference between the total ex-
                of carrier lipid was added to each sample. A final               tractable lipid in the cells (normal cells = 15.97ï¿½8.51
                suspension in solvent was divided into 2 aliquots                pg lipid/cell; HN cells = 23.21ï¿½11.88 pg lipid/cell,
                which were then evaluated for either neutral lipids by           P = 0.223). In order to more fully define any differ-
                one-dimensional silicic acid paper chromatography                ences that might exist between the two cell types,
                (Wuthier 1966) or for polar lipids by two-dimen-                 both the basic fatty acid components, as well as the
                sional silicic acid paper chromatography, followed by            complex lipid moieties derived from them were ana-
                autoradiography Uerkofsky and DeSiervo 1986).                    lyzed.
                                                                                   Gas chromatographic analysis of FAMEs from he-
                                                                                 mocytes taken from HN-affected clams was compared
                Flow Cytometric Analysis                                         with that of normal clams as well as with the pattern
                                                                                 found in normal and SV40 virus-transformed WI-38
                In order to determine the configuration of the DNA               cells (human diploid fibroblasts) (Table 1). When
                in the genome of HN cells versus normal hemocytes,               compared to normal clams, only clams with rates of
                flow cytometric analysis of the total DNA content of             HN in excess of 90% abnormal cells exhibited signifi-
                the cells as well as the cell cycle status of the cells was      cant alterations in FAME composition. The saturated
                carried out. Preliminary experiments were per-                   class of FAMEs remained stable in affected clams,
                formed to determine whether live or fixed hemocytes              with the exception of a decrease in palmitic acid and
                were optimal for flow cytometric analysis. Fixation of           in stearic acid. Abnormal cells had significantly lower
                HN hemocytes in either 2% formalin-seawater or 2%                levels of monounsaturated fatty acids, as well as sig-
                glutaraldehyde-seawater led to reduced resolution of             nificantly higher levels of the longer chain fatty acids
                DNA peaks compared to unfixed materials sus-                     (20 and 22 carbon polyunsaturated groups). The
                pended in the anticoagulant 0.01 M cysteine in                   changes found in the FAME profiles of HN cells were
                seawater. Consequently, fresh, unfixed, washed hemo-             opposite in direction from those found in SV40-trans-
                cytes were used for the flow cytometric analysis in all          formed human cells.
                further studies. The processing of hemocytes for flow              Evaluation of the in vitro incorporation of lipid
                cytometric analysis of DNA followed standard meth-               precursors (acetate)    into the more complex lipids
                ods used in human clinical oncology (Raber 1988).                also indicated significant changes in hemocytes from







             88         NOAA Technical Report NKB 111



                                                                                   Table I
                    A summary comparison of lipid composition of normal and SV40@virus transformed mammalian cells and normal
                                              and HN cells from Mya arenaria. Levels of HN ranged from 5 to >95%.


                                                                                                SV-40
                                                                                                infected             Normal                         HN
                                 Lipid class                             WI-38a                 WI-38a              hemocytes                   hemocytes

                 Total lipid/cell (pg)                                     65.0                 59.0                   16.0                 23.2 (P--0.223)
                 Neutral lipids (% of total cpm)                           29.0                 41.0*                   5.0                  8.0 (P<0.05)
                    Triacylglycerides (% of neutral)                       27.0                 25.0*                  40.0                 62.0 (P<0.005)
                    Free fatty acids (% of neutral)                        21.0                 26.0*                  36.0                 22.0 (P<0.01)
                    Cholesterol (% of neutral)                             35.0                 35.0                    6.2                  4.2 (P<0.05)
                    Cholesterol esters (% of neutral)                        5.0                  4.0                  18.0                  8.0 (P<0.05)
                 Phospholipids (% of total cpm)                            71.0                 59.0*                  95.0                 92.0 (P<0.001)
                    Cardiolipin (% of phospholipids)                         6.1                  2.7*                  6.0                  2.0 (P<0.001)
                    Phosphotidylethanolamine                               12.0                 13.0                   75.0                 71.0 (P<0.001)
                    (% of phospholipids)
                    Phosphotidylinositol                                   13.0                 10.0*                   1.0                  2.0 (P<0.001)
                    (% of phospholipids)
                    Phosphotidylcholine                                    57.0                 57.0                    2.0                  8.0 (P<0.005)
                    (% of phospholipids)
                 Fatty acid methyl esters
                    Palmitic acid (16:0) (% of FAME)                       44.0                 50.0*                  70.0                 59.0 (P<0.01)
                    Stearic acid (18:0 ) (% of FAME)                       37.0                 30.0*                  20.0                 25.0 (P<0.05)
                    18:1 Fatty acids (% of FAME)                           63.0                 78.0*                  25.6                 22.5 (P<0.05)
                    20:4 Fatty acids (% of FAME)                           37.0                 17.5*                   1.2                  4.0 (P<0.01)


                 a Data from Howard et al. 1977.
                 *Significant difference between WI-38 and SV40-transformed WI-38.



             diseased clams relative to normal ones. The percent-                          and the phosphotidylcholines(P<0.001 and P<0.05 re-
             age of labelled acetate incorporated into the total                           spectively). Many of the changes occurring in the
             neutral lipid fraction of the HN cells was significantly                      lipids of HN hemocytes differ with those documented
             higher, especially the triacylglycerols (P<0.005), the                        in mammalian neoplastic cells (Howard et al. 1973).
             primary storage form of lipids (Table 1). In fact,
             while the level of the other classes of neutral lipids
             evaluated, including free fatty acids, cholesterol, and                       Flow Cytometric Analysis
             cholesterol esters, decreased significantly, the eleva-'
             tion in triacylglycerides masked the depressed                                Preliminary experiments indicated that using forma-
             synthesis of these compounds in the total neutral                             lin or glutaraldehyde fixatives for flow cytometric
             lipid fraction. Oil red 0 staining, which preferen-                           analysis of hemocytes altered the chromosomal mate-
             tially stains triacylglycerols (Luna 1986), specifically                      rial andjesulted in poor separation of the normal
             stained the vacuoles or "droplets" seen in HN cells by                        diploid and aneuploid peaks in HN-positive clams.
             phase contrast microscopy; these droplets are not ap-                         Therefore, for all of the experiments reported here,
             parent in normal hemocytes.                                                   unfixed hemocytes were rapidly (within 5 minutes of
                 As with the neutral lipids, there were also signifi-                      removal from the clam) processed for flow
             cant changes in the incorporation of acetate into the                         cytometric analysis. This resulted in a clear separa-
             polar lipids, which consist almost entirely of the                            fion of DNA peaks in the cells.
             membrane-associated phospholipids. The overall                                     Flow cytometric analysis indicated that the DNA
             level of incorporation into the total phospholipid                            content of the abnormal hemocytes was distinctly
             class decreased significantly (P<0.001). Likewise, de-                        aneupoid. As shown in Figure 1, there was a strong
             creases occurred in the amount of labelled                                    positive linear correlation (r = 0.95) between the ab-
             phosphatidylglycerol and cardiolipin, which is de-                            normal microscopic appearance of the nonadherent
             rived from phosphatidylglycerol (P<0.001) (Table 1).                          cells and abnormal DNA content as detected by flow
             By contrast, there were significant increases in the                          cytometry. Two forms of aneuploid cells were de-
             levels of incorporation into the phosphoddylinositols                         tected: those with a hypodiploid DNA content and







                                                                Reno et al.: Heinatic Neoplasia in the Softshell Clam Mya arenatia  89

               those with a hyperdiploid DNA content. Examples of            higher (mean = 16.4%, P = 0.0001) proportion of
               the DNA contents of normal and abnormal hemo-                 cells which were either undergoing DNA synthesis, in
               cytes are shown in Figure 2. The amount of DNA in             preparation for mitosis, or actually in the process of
               diploid clam hemocytes was approximately 3.2 pg,              mitosis (Fig. 3). The proportion of FIN cells in some
               compared with 6.8 pg in a human lymphocyte stan-              phase of replicative activity ranged from 10 to 25%;
               dard. In contrast to diploid clam hemocytes, DNA              most of this activity was in the hyperdiploid popula-
               profiles of cells from abnormal clams invariably had a        tion, but the hypodiploid and even the diploid
               population of cells containing hyperdiploid DNA               populations also were undergoing more extensive
               comparable in number to the percentage of abnor-              multiplication than normal.
               mal cells as assessed by microscopic observation. The
               DNA index (DI)-the quantity of aneuploid DNA
               relative to the DNA content of the normal diploid             Discussion
               hemocyte-of clams showing hyperdiploidy varied
               from 1.6 to 2.0 in 12 HN-positive individuals. In most,       The present study was designed to help characterize
               but not all clams that were HN-positive, a hypodip-           the abnormal hemocytes in M. arenayia with hematic
               loid cell population also occurred which comprised            neoplasia. This disease, and similar diseases in a vari-
               between 0.5 and 10% of the cells (Fig. 2). The DI of          ety of other bivalves, have attracted much attention
               the hypodiploid cell population, ranging from 0.82            in the scientific community for two reasons. In the
               to 0.86, was more constant than the DI of the                 marine community, they can cause significant mor-
               hyperdiploid population. This hypodiploid peak was            talities in the affected animals (Cooper et al. 1982;
               not noted in the hemocytes of normal clams.                   Brousseau and Baglivo 1991; Reno, Leavitt, and
                 One of the hallmarks of neoplastic transformation           Capuzzo, unpubl. observations). In addition, these
               is the proliferative nature of the cells. Cell cycle          diseases have become the focus of epidemiological
               analysis of hemocytes from HN-positive clams indi-            investigations assessing the- potential causal interac-
               cated that this was indeed the case for this particular       tion between anthrop    ogenic pollutants in marine
               malady. Normal cells showed some indication of pro-           environments and the generation of neoplasia, and
               liferative activity; 2 to 5% (mean = 4.4%) were in the        the potential for using FIN     Idisease in bivalves as
               S (DNA synthesis) phase and an approximately equal            sentinals in appraising the potential carcinogenic ac-
               percentage were in the G2/M (premitotic and mi-               tivity of these pollutants in humans. While the
               totic) phases of the cell cycle. Hemocytes from clams         rationale of the first reason still holds'true irrespec-
               that were aneuploid, however, had a significantly             tive of the true nature of the disease, the assumption




                                  y  1.1029 + 0.91207x RA2   0.907


                         1W_




                         so-




                         60,



                    CL

                         40,




                         20




                          0
                                     20        40         60        so        100                            Figure I
                                            % HN (microscopy)                                Relationship between the percentage of HN
                                                                                             by microscopic evaluation and the percent-
                                                                                             age of DNA aneuploidy by flow cytometry.







             90       NOAA Technical Report NMFS I 11




                      1000-
                 A                      a


                        800-                                                   HN-




                        600-


                0
                        400-




                        200.



                          0
                            20     40      60      80      100     120      140     160     180
                                             IRFL (DNA)                                                                   Figure 2
                                                                                                             Examples of DNA distrib ution
                      1000-                                                                                  and cell-cycle analysis of normal
                 B                                        a                                                  and abnormal hemocytes. (A)
                                                                                                             Normal clam; (B) clam with ap-
                        800-                                                  78% HN+                        proximately 78% abnormal
                                                                                                             cells. IRFL = integrated red
                                                                                                             fluorescence (DNA content).
                        60o-                                                                                 Key to letters on graphs:
                                                                                                             a: resting stage of cell cycle (G,
                                                                                                                and G, phases) for diploid
                        400-                                                                                    cells
                                                                                                             b: DNA synthesis phase of cell
                                          a                                                                     cycle (S phase)
                        200-                                                                                 c: Premitotic and mitotic phases
                                                                                                                of cell. cycle (G, and M
                                   A                                                                            phases) for diploid cells.
                          0 -                                                                                d: Hypodiploid DNA peak
                            20     40       60       80     100      120      140     160      180           e: Hyperdiploid DNA peak
                                                      IRFL (DNA)                                             f: S phase for hyperdiploid cells
                                                                                                             g: G,/M phases for hyper-
                                                                                                                diploid cells
             on which the second is based is subject to invalida-                 1979; Lauckner 1983), prudently note that the crite-
             tion if the disease is not a tr      'ue neoplasm, but is            ria presently used to determine the nature of the
             rather a hyperplastic or anaplastic disease-or even                  disease are inadequate when compared with the
             due to an exogenous source such as a protozooan                      manifold criteria that are demonstrable in mammals,
             parasite.                                                            and that it may be premature or unwarranted to de-
               Information is readily available in the literature                 scribe the leukernia-like disease in bivalves as a true
             with respect to the characteristics of neoplasia and                 neoplasm without further investigations into the na-
             transformed cells in mammals and birds. Relatively                   ture of the disease.
             little is present that deals stringently with the disease              Mammalian and avian neoplastic cells derived from
             and nature of the altered cell in poikilothermic verte-              tumor tissue or transformed by biological, physical,
             brates; virtually none is available with respect to the              or chemical agents have several dozen characteristics
             "diseased cell" in invertebrates. Mix's comprehensive                in common that differ significantly from normal
             review (1986) of the nature of the interaction be-                   cells. These characteristics are most readily evaluated
             tween anthropogenic pollutants and "neoplastic"                      when the cells are separated from their normal resi-
                                                     L78*/* HN+


             diseases in aquatic vertebrates and invertebrates rep-               dence in vivo and are cultured in vitro, if only for
             resents the most critical analysis of the extant data.               short periods. This process removes the major con-
             The author, as well as others (Harshbarger et al.                    founding factor of the large number and wide variety






                                                                         Reno et al.: Heinatic Neoplasia in the Softshell Clam Mya arenaria            91

                                                                                        of normal cells that are present in tumor tissue               in
                                               E %S                                     vivo. Affected cells of the circulatory system are most
                                               M % G2-M                                 readily divested of these contaminants by simply
                                               0 % GO-GI                                drawing off hematic fluids which contain the abnor-
                                                                                        mal cells and placing them in culture for further
                      A       100-                                                      analysis. This is the approach taken in the current
                                                                                        investigations.
                              080-                                                         Lipids are the major constituents of cellular mem-
                                                                                        branes, as well as a source of stored energy, and the
                              Q60-
                              M                                                         specific lipid components that are present have a
                              .2 40-                                                    marked effect on the properties of the membrane
                              20-                                                       (Wood 1972; Shinitzky 1984). Because a wide variety
                              A                                                         of the alterations occurring in neoplastically trans-
                                                                                        formed cells are associated with the plasma
                              0-
                                   0     62     88     92     94     95                 membrane surface, it is not surprising that many al-
                                                                                        terations in the lipid composition of neoplastic cells
                                                 % HN                                   have been documented in mammalian systems. Wood
                      B                                                                 (1972) and others have published a variety of papers
                              100-                                                      dealing with the alterations in lipid metabolism and
                                                                                        biochemistry in mammalian neoplasia (e.g., Awad
                              So-                                                       and Spector 1976; Yau et al. 1976; Montaudon et al.
                                -                                                       1981; Schroeder and Gardiner 1984; Kier et al. 1988;
                              60-                                                       Calorini et al. 1989). The SV40-induced transforma-
                                                                                        tion of human WI-38 lung fibroblast cells (Howard et

                              0
                              40-                                                       al. 1973; Perkins and Scott 1978) is an in vitro system
                              CL                                                        resembling HN of M. arenafia because transforma-
                              20-                                                       tion by viruses produces a homogeneous population
                                                                                        of altered cells, as is found with HN hemocytes, and
                              0                                                         because it has been indicated that FIN in M. arenafia
                                   0     62     88     92    94     95                  is also viral-induced (Oprandy et al. 1981).
                                                                                          There,are significant alterations in more than one
                                              % HN                                      half of the lipid moieties examined in the SV40-WI-38
                      C                                                                 cells in culture. With the essential building blocks of
                              100-                                                      lipids, the fatty acids, there are increases in the levels
                                                                                        of the saturated fatty acid palmitic acid (16:0), the
                              80-
                                                                                        monounsaturated fatty acid oleic acid (18:IA9), and
                              V
                              '3 60-                                                    the diunsaturated fatty acid linoleic acid (18:2M). In
                              CL                                                        contrast, in M. arenaria FIN cells show a decrease in
                              40-                                                       palmitic acid but a similar increase in the two unsat-
                                                                                        urated fatty acids. In the virally transformed WI-38
                              CL
                              20-
                                                                                        cells, a significant decrease occurs in the levels of
                              ge 0-                                                     stearic (18:0), palmitoleic (16:lA7), linolenic
                                  0     62     88     92     94    95                   (18:3M), and arachidonic (20:4A6) acids. In M.
                                                                                        arenaria HN, however, there is a marked increase in
                                                 % HN                                   the levels of stearic acid in conjunction with de-
                                                                                        creases in the levels of 16:IA7, 18:3A3, and 20AA6
                                                                                        fatty acids. The changes seen in mammalian and bi-
                                           Figure 3                                     valve cells are at variance in their levels of two major
                 Percent of diploid and aneuploid cells in various phases of            essential fatty acids derived from de novo sources:
                 the cell cycle. (A) Aiploid cells; (B) hypodiploid cells;              palmitic and stearic acids. These variations may re-
                 (C) hyperdiploid cells. % S = cells in DNA synthesis phase             flect a difference in the process of synthesis of the
                 of cell cycle; G2/M = cells in premitotic and mitotic phases           fatty acids in invertebrates compared with mammals
                 of the cell cycle; GO/G, = cells in the resting stages of the
                 cell cycle.                                                            or may reflect a basic difference in the nature of the
                                                                                        abnormal HN cell and the transformed WI-38 cell.
                                                                                        With respect to the more complex lipids, a significant







             92        NOAA Technical Report NWS I I I


             increase is seen in the levels of cholesterol in both                      The available data on the etiology of HN in
             virally transformed WI-38 and in murine hepatoma                        bivalves is scant. There is evidence that the abnormal
             cells (Steele and Jenkin 1977). Cholesterol is one of                   hemocytes of HN clams possess unique surface anti-
             the most important lipids in the cellular membrane                      gens as detected with both polyclonal and
             and contributes much to the attributes of the cell                      monoclonal antibodies (Reinish et al. 1983; Miosky et
             surface. In studies with transformed mammalian cells                    al. 1989). In Smolowitz et al. (1989), this same group
             there is a significant increase in the levels of choles-                has also produced a monoclonal antibody that reacts
             terol in the cytoplasmic membrane (Howard et al.                        both with a subpopulation of cells in the gut of nor-
             1973). In our study, the incorporation of labelled ac-                  mal clams and with HN cells, indicating an internal
             etate into cholesterol, and its derivatives, the                        source for HN cells. However, with the well-known
             cholesterol esters, of HN cells was significantly lower                 possibility of epitopic crossreactivity, or even with ac-
             than the levels of incorporation seen in normal he-                     tive mimicry of epitopes by parasites, the reaction of
             mocytes. This is in contrast to uptake observed in                      a single monoclonal antibody with a subpopulation
             mammalian neoplastic cells. Likewise, the changes in                    of M. arenafia hemocytes is insufficient to confirm
             another class of neutral lipids, the free fatty acids,                  the endogenous source of the HN cells. As men-
             remained unchanged while in mammalian cells there                       tioned above, several authors have noted that the
             is an increase in free fatty acids. Taken together,                     available information on the origin of the HN cell in
             these major differences between the lipid biochemis-                    clams does not preclude the possibility that the cells
             try of HN cells and that of neoplastically transformed                  are parasitic rather than neoplastic in nature
             mammalian cells indicate that it would be                               (Lauckner 1983; Mix 1986). Unfortunately, the data
             presumptious to declare HN cells neoplastic 6n the                      presented here does not alleviate this problem be-
             basis of their lipid biochemistry.                                      cause, for example, the heteroploid cells may be
                A fundamental change that occurs in neoplastically                   parasitic protozooans or even algae, which would
             transformed cells is the alteration of the genome of                    have a considerably different chromosomal makeup
             the cell, which is heritable and leads to the initiation                than clam hemocytes. Further work on genetic analy@
             and progression of tumors. These alterations may be                     sis of these cells using cytogenetic techniques, as well
             "considerable, such as heteroploidy, which can be eas-                  as molecular biological techniques, must be carried
             ily detected by flow cytometric analysis, or more                       out before the resolution of this problem can be
             subtle, such as chromosomal rearrangements or alter-                    achieved.
             ations in specific gene sequences which must be
             detected by karyotyping or molecular DNA analysis.
             In this study, flow cytometric analysis was used -to de-
             termine the potential lesions in the DNA content of                     Citations
             HN cells and also to assess the proliferative potential
             of the disease. It was found that HN cells were dis-                    Alderman, D.G., P. Vin Banning, and A. Perez-Colomer.
             tinctly aneuploid - in nature and that the most                              1977. Two European oyster (Ostrea edulis) mortalities associ-
             consistent change in the DNA content in HN cells                               ated with abnormal hemocytic condition. Aquaculture.
                                                                                            10:35-340.
             was to a hyperdiploid state. The level of this                          Appledoorn, R.S., andj.J. Oprandy.
             hyperdiploid population correlated well with the pro-                        1980. Tumors in soft-shell clams and the role played by a
             portion of cells which appeared abnormal by                                    virus. Maritimes. August, 1980:4-6.
             microscopic examination. There was significant varia-                   Awad, A.B., and A.A. Spector.
             tion, however, in the amount of aberrant DNA in the                          1976. Modification of the fatty acid composition of Erhlich
             cells, which ranged from 1.6 to 2.0 times normal.                              ascites tumor cell plasma membranes. Biochim. Biophys.
                                                                                            Acta. 426:723-731.
             This would be consistent with the clonal develop-                       Bligh, E.G., and W.J. Dyer.
             ment of the transformed cell (Iversen 1988).                                 1959. A rapid method of total lipid extraction and
             However, it was also found that most of the clams that                         *purification. Can. J. Biochem. Physiol. 37:911-917.
             were HN positive had a second, markedly stable hy-                      Brousseau, D.J., andj.A. Baglivo.
                                                                                          1991. Field and laboratory comparisons of mortality in nor-
             podiploid population of cells, a finding that is                               mal and neoplastic Mya arenmia. J. Invertebra. Pathol.
             inconsistent with the clonal development of trans-                             57:59-65.
             formed cells. This finding is problematic if one is                     Brown, R.S., R.E. Wolke, S.B.N. Saila, an&C.W. Brown.
             making the assumption that HN in M. arenari                .a is a           1977. Prevalence of neoplasia in 10 New England popula-
             true neoplastic disease because it is unlikely that a                          tions of the soft shell clam (Mya arenaiia). Ann. N.Y Acad.
                                                                                            Sci. 198:522-532.
             stable hypodiploid population would coexist with a                      Calorini, L., A. Fallani, D.Tombaccini, E. Barletta, G.Magnai, M. Di
             more variable hyperdiploid population in the same                          Renzo, P. Comoglio, and S. Ruggieri.
             animal if this were true.                                                    1989. Lipid characteristics of RSV-Lransformed Balb/C 3T3






                                                                                    Reno et al.: Hematic Neoplasia in the Softshell Chun Mya arenaria                          99

                          cell lines with different spontaneous metastatic                            Montaudon, D.J.C. Louis, andj. Rober.
                          potentials. Lipids. 24:685-690.                                                  1981. Phospholipid acyl group composition in normal and
                  Cooper, K-R., R.S.Brown, and P.W. Chang.                                                   tumoral nerve cells in culture. Lipids. 16:293-297.
                        1982. Accuracy of blood cytological screening techniques for                  Oprandy, jj., P.W. Chang, A.D. Pronovost, K.R. Cooper, R.S.
                          the diagnosis of a possible hematopoietic neoplasm in the                     Brown, and V.J. Yates.
                          bivalve mollusc, Mya arena7ia. J. Invertebra. Pathol.                            1981. Isolation of a viral agent causing hernatopoietic
                          39:281-289.                                                                        neoplasia in the soft-shell clam, Mya arenaiia. J. Invertebra.
                  Couch, J.A.                                                                                Pathol. 38:45-51.
                        1969. Sarcoma-like disease in a single specimen of the                        Perkins, R.G., and R.E Scott.
                          American oyster. Comparative Leukemia Research Bibliog-                          1978. Differences in the phospholipid, cholesterol, and fatty
                          raphy: Haernatology. No. 36.                                                       acyl composition of 3T3 and SV3T3 plasma
                  Farley, C.A.                                                                               membranes. Lipids. 13:653-657.
                        1969. Probable neoplastic disease of the hematopoietic Sys-                   Raber, M.
                          tem in oysters, Crassostrea virginica and Crassostrea                            1988. Clinical applications of flow cytometry. Oncology.
                          gigas. Nad, Cancer Inst. Monogr. 31:541-555.                                       2:35-43.
                  "arshbarger,J.C., S.V. Otto, and S.C. Chang.                                        Reinisch, C.L., A.M. Charles, andj. Troutner.
                        1979. Proliferative disorders in Crassostrea virginica and Mya                     1983. Unique antigens on neoplastic cells of the soft shell
                          arenafia from the Chesapeake bay and intranuclear virus-                           clam, Mya arenaria. Dev. Comp. Immunol. 7:33-39.
                          like inclusions in Mya arenaria with germinomas from a                      Sasaki, G.C., andj.M. Capuzzo.
                          Maine oil-spill site. Haliotis. 8:243-248.                                       1984. Degradation of Artemia lipids; under storage. Comp.
                  Heim, S., and F. Mitelman.                                                                 Biochem. Physiol. B. Comp. Biochem. 78:525-531.
                        1987. Cancer Cytogenetics. Alan R. Liss, Inc., New York,                      Schroeder, F., andj.M. Gardiner.
                          NY                                                                               1984. Membrane lipids and enzymes of cultured high and
                  Howard, B.V.J.D. Butler, andj.M. Bailey.                                                   low-metastatic B16 melanoma variants. Cancer Res.
                        1973. Lipid metabolism in normal           and tumor cells in cul-                   44:3262-3269.
                          ture. In Tumor lipids: biochemistry and metabolism (R.                      Shinitzky, M.
                          Wood, ed.), p. 200-214. American Oil Chemists' Society                           1984. Membrane fluidity in malignancy, adversative and
                          Press, IL.                                                                         recuperative. Biochim. Biophys. Acta. 738:251-261.
                  Iversen, O.H.                                                                       Smolowitz, R.M., D. Miosky, and C.L. Reinisch.
                        1988. Theories of carcinogenesis: facts, fashion or                                1989. Ontogeny of leukemic cells of the soft shell clam. J.
                          fiction? Hemisphere Publishing Co., Washington, DC,                                Invertebra. Pathol. 53:41-51.
                          327 p.                                                                      Steele, W., and H.M.Jenkin.
                  jerkofsky, M., and Aj. DeSiervo.                                                         1977. Lipids and lipid metabolism of Novikoff rat hepatoma
                        1986. Differentiation of strains of varicella-zoster virus by                        cells. In Tumor lipids: biochemistry and metabolism (R.
                          changes of neutral lipid metabolism of infected cells. J.                          Wood, ed.), p. 215-225. American Oil Chemists' -Society
                          Virol. 57:809-815.                                                                 Press, IL.
                  Kier, A.B., M.T. Parker, and F. Schroeder.                                          VaseleivJ.M., and I.M. Gelfand.
                        1988. Local and metastatic tumor growth and membrane                               1981. Neoplastic and, Normal Cells in Culture. Cambridge
                          properties of LM fibroblasts in athymic (nude)                                     Univ. Press, Cambridge, UX
                          mice. Biochim. Biophys. Acta. 938:434-446.                                  Wood, R., ed.
                  Lauckner, G.                                                                             1972. Tumor lipids: biochemistry and metabolism. American
                        1983. Diseases of Molluska:Bivalvia. In Diseases of marine                           Oil Chemists' Society Press, Champaign, IL.
                          animals. (0. Kinne, ed.), p. 477-962. Biol. Anst.                           Wuthier, R.E..
                          Helgoland, Hamburg, FRG. Vol 11.                                                 1966. Two-dimensional chromatography on silica gel-loaded
                  Luna, L.G.                                                                                 paper for the microanalysis of polar lipids. J. Lipid Res.
                        1968. Manual of histological staining methods of the Armed                           7:544-550.
                          Forces Institute of Pathology, 3rd ed. McGraw-Hill Inc.,                    Yau, T.M., T. Buckman, A.H. Hale, and M.J. Weber.
                          New York, NY.                                                                    1976. Alterations in lipid acyl group composition and mem-
                  Marchesi, V.T. (ed.)                                                                       brane structure in cells transformed by Rous sarcoma
                        1976, Membranes and neoplasia: new approaches and strate-                            -virus. Biochemistry. 15:3212-3219.
                          gies. Alan R. Liss, Inc., New York, NY                                      Yevich, P., and C. Barszcz.
                  Miosky, D.L., R.M. Smolowitz, and C.L. Reinisch.                                         1976. Gonadal and hematopoietic neoplasms in Mya
                        1989. Leukemia cell-specific protein of the bivalve mollusc,                         arenatia. U.S. Natl. Mar. Fish. Serv. Mar. Fish. Rev. 38:
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                  Mix, M.C.                                                                                1977. Neoplasia in soft-shell clams, Mya arenairia, collected
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                          review. Mar. Environ. Res. 20:1-92.






                   Kinetics of Bovine Serum Albumin Administered by the Immersion
                       Method in Fishes Acclimatized to Seawater and to Fresh Water



                                                MITSURU OTOTAKE and TERUYUKI NAKANISHI

                                                     Inland Station of National Research Institute ofAquaculture
                                                                    Tamaki, Mie 519-04, Japan




                                                                            ABSTRACT


                                     To clarify plasma BSA kinetics in.marine fish after hyperosmotic infiltration (HI) treat-
                                  ment, chum salmon (Oncorhynchus keta) and blue tilapia (Tilapia aurea) were acclimatized
                                  to seawater or to fresh water, and were administered bovine serum albumin (BSA) by HI
                                  treatment or intravenous injection. First, by using a least squares program, regression
                                  curves were computed from the plasma BSA leve       Is of fish injected with BSA to obtain the
                                  clearance rate of BSA from plasma. Then, by using a deconvolution method, the BSA
                                  release rate from the organ of BSA entry to plasma after HI treatment was calculated from
                                  plasma BSA levels after HI treatment and the clearance rate..The results showed that the
                                  clearance rates of both fishes acclimatized to seawater were higher than those of the fish
                                  acclimatized to fresh water. On the other hand, the release rates of both fishes acclimatized
                                  to seawater were much.lower than those of the respective fish acclimatized to fresh water.
                                  Thus, it is quite plausible that the high clearance rates and low release rates of fish in
                                  seawater work synergistically, resulting in lower plasma BSA levels of fish in seawater com-
                                  pared with fish in fresh water after HI treatment.



                Introduction                                                         plasma BSA levels of these animals, the rate of BSA
                                                                                     release into plasma and BSA clearance from the
                The immersion method, first reported by Amend and                    plasma were calculated.
                Fender (1976), is a useful method for mass immuni-
                zation in aquaculture. Many papers have shown the
                effectiveness of this method on freshwater fish (Ellis               Materials and Methods
                1988). On the other hand, there is a little knowledge
                as to the immqrsion method's effectiveness on ma-
                rine fish. In a preliminary study (unpubl. data), the                Experimental Animals
                authors used hyperosmotic infiltration (HI) treat-
                ment to administer bovine serum albumin (BSA) to                     Six-month-old chum salmon, 16 ï¿½ 1 g (mean ï¿½ stan-
                yellowtail Seriola quinqueradiata, which is the most im-             dard deviation) in body weight, and 1-year-old blue
                portant aquaculture species in Japan, and rainbow                    tilapia, 157 ï¿½ 20 g in body weight, were used in this
                trout Oncorhynchus mykiss, and then measured the                     experiment. Tilapia were fed commercial floating dry
                BSA levels in their plasma. We found that plasma BSA                 pellets in a I-ton tank supplied with running fresh
                levels were much lower in yellowtail than in rainbow                 water at 25' C, whereas chum salmon were kept in a
                trout after HI treatment. This difference may have                   I-ton tank supplied with running fresh water at
                been caused by two factors: species specificity and the              15' C, and fed commercial dry pellets for rainbow
                type of environmental water-fresh water or seawater.                 trout. These tanks were kept indoors.
                To analyze this difference it is necessary to compare
                the kinetics of BSA in the plasma of several species of
                fish that can reside in both seawater and fresh water.               Acclimatization to Seawater
                Thus, in the present study, chum salmon (0. keta)
                and blue tilapia (Tilapia aurea) were acclimatized- to               Before the administration of        BSA, one half of the
                seawater or to fresh water, and then administered                    tilapia and chum salmon were acclimatized to seawa-
                BSA by HI treatment or injection. By using the                       ter, and the other half were kept in running fresh

                                                                                                                                                  95







            96      NOAA Technical Report NWS I I I


            water throughout the experiment. Fish were acclima-              1978). The gel plate was run at I V/mm and 15' C
            tized to seawater (salinity: 34 ppt) by increasing the           for 3 hours. The exact amounts of BSA in normal fish
            proportion of seawater day by day to 50% on the first            plasma were run in parallel with all tests for standard
            day, 75% on the second day, 87.5% on the third day,              reference.
            and 100% on the fourth day. Afterwards the fish were
            kept in the tank supplied with running seawater for 3
            weeks before the experiment. During the acclimatiz-              Calculations
            ing period, no fish died or showed abnormal
            behavior. Experiments were carried out with four                 Plasma BSA Clearance By means of an unweighted
            groups of fishes: chum salmon in fresh water (fresh-             least squares program, the plasma BSA levels Cpi"i(t)
            water salmon); chum salmon acclimatized to seawater              at each time t after injection were fitted to following
            (seawater salmon); tilapia in fresh water (freshwater            exponential equations:
            tilapia); and tilapia acclimatized to seawater (seawa-
            ter tilapia). Water temperature was maintained at                               Cp ini(t) = a, - exp (- bt); or
            15' C for freshwater or seawater salmon and at 25' C
            for freshwater or seawater tilapia.                                     Cp'-i(t) = a, . exp(-b,.t) + a 2 *exp (-b 2*  0.

                                                                             For the index of BSA clearance from plasma, the
            Administration of BSA by HI Treatment                            mean residence time (MRT) of each experimental
                                                                             group was given by
            Fifty fish of each group were administered BSA (frac-
            tion V, Sigma Co.) by the two-step hyperosmotic                                       MRT = l1b,; or
            infiltration technique according to the method of
            Amend and Fender (1976) with slight modification.                   MRT = l1bi + l1b     2- (a, + a,)I(a, - b 2+ a 2 -b).
            Distilled water was used to prepare the two bath solu-
            tions; 5.3% NaCl solution and 2% BSA solution. Fish              The BSA distribution volume V of each experimental
            were immersed in 5.3% NaCl solution for 3 minutes                group was calculated by
            and then placed directly into 2% BSA solution for 3
            minutes. After the bath treatment, the treated fish                                    V = D/ Cp i-j(O),
            were placed in tanks supplied with either running                where D is the amount of BSA administered by injec-
            seawater or fresh water for 3 minutes. Afterwards
            they were returned to their original tanks. Blood                tion (I mg/kg body weight).
            samples were taken 0.5, 1, 2, 4, and 8 hours after the           BSA Release into Plasma-The BSA release rate into
            bath treatment. At each sampling time six fish were              the plasma U(t) at each time t was given by'
            sacrificed and the blood was withdrawn from the cau-
            dal blood vessels using heparinized syringes.                                 CpHl(t) = f.U(t) - Cpili(t-r)   dT,

                                                                             where Cp(t) is the plasma BSA level after the HI
            Administration of BSA by Injection                               treatment and r is the time constant. The gross BSA
                                                                             release into the plasma after the HI treatment was
            Fifty fish of each group were administered BSA by                calculated from U(t) by integration.
            intravenous injection. Bovine serum albumin was dis-
            solved with sterilized 0.85% NaCl solution to 2 mg/
            mL and then injected into fish via the caudal blood              Statistics
            vessels at the dose of 1 mg/kg body weight. Blood
            samples were taken 0.5, 1, 2, 4, 8, and 24 hours after           Data were analyzed using the F test, and then by the
            the administration by the same method as mentioned               Student's t test to detect significant differences.
            above.


                                                                             Results
            Immunoelectrophoresis
                                                                             Plasma BSA Levels After the HI Treatment
            Bovine serum albumin content in the plasma was
            quantitatively assayed by rocket immunoelectro-                  Concentration of plasma BSA in the freshwater
            phoresis (Laurell 1966; Wallenborg and Andersson                 salmon increased until 4 hours after the HI treatment







                                                                                 Ototake and Nakanishi: Kinetics of Bovine Serum in Fish             97



                                         A                                        B

                    120-                                         20

                   -100-
                                                              E15
                      80
                                                                                     ----------
                                                             M
                   C< 60                                      <io.                                                           Figure I
                   n
                   co 40    1110                                                                             Changes in plasma concentrations of
                                                                                                             bovine serum albumin (BSA) after
                      20                                          5                                          hyperosmotic infiltration treatment in
                                                                    II                                       freshwater-acclimatized fish (A) and
                        041                                      0 il Ll@* -                                 seawater-acclimatized fish (B). Key:
                         0      2       4       6       8           0     2       4        6       8         (o) = tilapia; (*) = chum salmon. Data
                              HOURS AFTER BATH                          HOURS AFTER BATH                     are presented as means ï¿½ standard
                                                                                                             errors.




                and reached a maximum of 90.6 ï¿½ 6.5 @Lg/mL (mean                       was adopted for express plasma BSA              concentration
                standard error), and then decreased gradually (Fig.                    for freshwater salmon after injection.         On the other
                IA). On the other hand, plasma BSA levels of the sea-                  hand,
                water salmon (Fig. 1B) were always significantly lower
                (NO.01) than those of freshwater salmon.'A maximum                                       Cpi@i(t) = a, - exp(-b,-t)
                plasma BSA level of 18.8 ï¿½ 2.6 @Lg/mL was observed in
                the seawater salmon 2 hours after the bath; from then                  was adopted     as the regression equation for seawater
                on, plasma BSA stayed at a rather constant level of I I-               salmon, freshwater tilapia, and seawater tilapia
                19 I.Lg/mL. The plasma BSA level of freshwater tilapia                 (Fig. 2). The MRT and the distribution volume of
                increased until 4 hours following' the bath when it                    BSA after the injection are shown in Table 1. Both in
                reached a maximum of 119.0 ï¿½ 8.1 @tg/mL and subse-
                quently decreased to 64.1 ï¿½ 2.8, @Lg/mL 8 hours after
                the treatment (Fig. IA). In contrast, plasma BSA levels
                of seawater tilapia (Fig. IB) were always significantly                            60-
                lower (P<0.001) than those of freshwater tilapia. A low                                                                   A
                level of BSA (0.09-1     `1 I.Lg/mL) was detected in the                           50
                plasma of the seawater tilapi.a for the initial 2 hours,                           40@
                but no BSA was detected in the plasma after 4 hours                                30    p'=31 OexP(- I -20-0+26-9-exp(-0.027-t)
                (minimum detectable level, 0.01 pg/mL).                                            20
                                                                                                 E
                                                                                                   10
                                                                                                   OL      Cp"--29-3-exp(-0.088-
                Plasma BSA Levels After the Injection                                           <
                                                                                                co 40-
                                                                                                          C0=26.9-exV-0.964-0             B
                Following BSA injection, the plasma BSA levels were                                30.
                always lower in seawater salmon than in freshwater                                 20
                salmon, and the difference was significant (NO.01)                                 10-               "=36-6-exW0.145-0
                0.5, 1, 2, 8, and 24 hours after the injection (Fig. 2A).                          0
                The difference between seawater and freshwater ac-                                    0    4    8     12    16 20 24
                climatized fish was more distinct in tilapia than in                                       HOURS AFTER INJECTION
                salmon, and the plasma BSA levels of seawater tilapia
                were always significantly lower (P<0.001) than those                                              Figure 2
                of freshwater tilapia (Fig. 2B).                                           Changes in plasma concentrations of bovine serum
                                                                                           albumin (BSA) after injection of BSA into chum
                Plasma BSA Clearance                                                       salmon (A) and tilapia (B)."Open circles indicate the
                                                                                           fish in fresh water and closed circles indicate the fish
                The regression equation                                                    acclimatized to seawater. Data are presente        d as
                                                                                                       C






















                                                                                           means      standard errors. Regression equations were
                         Cp         a,  exp (-b I - t) + a2 - exp (-b.2- t)                calculated with a least squares program:







              98        NOAA Technical Report NWS I I I



                                                                                   Table I
                                          Kinetics of plasma BSA administered by hyperosmoticinfiltration treatment.

                  Experimental                            Maximum BSA                Mean residence               Gross release              Distribution
                  group                                   level in plasma              time (MRT)                  into plasma                 volume
                                                             (Rg/mL)                       (hours)                  (pg/mL)                (mL/100gb.w.    a

                  Freshwater salmon                              84                           36                        167                      1.7
                                                                                           (37) b                       (95) b                  (3.7) b
                  Seawater salmon                                19                           11                        24                       3.4
                  Freshwater tilapia                             119                          6                         146                      2.7
                  Seawater tilapia                               1                            1                         14                       3.7

                  'b.w. = body weight.
                  bCalculated by the modified regression equation.




              chum salmon and in tilapia, the plasma BSA clear-
              ance, as represented by the MRT, was more rapid in                                      60-                                          A
              seawater acclimatized fish than in respective freshwa-
                                                                                                  3   40-
              ter acclimatized fish. The MRT of seawater salmon
              and seawater. tilapia were 1/3 and 1/6 of that of                                       20.
              respective freshwater acclimatized fish. The distribu-
              tion volume of freshwater salmon was smaller than                                   a      0
                                                                                                  UJ  100
              those of other experimental groups.                                                 tR                                               B
                                                                                                  cc  80
                                                                                                  ul
                                                                                                  co
                                                                                                  <   60
              BSA Release into the Plasma
                                                                                                  LU
                                                                                                  Ir  40.
              The BSA release rate into the plasma, calculated                                    <
                                                                                                  co  20.
              from the clearance rate and the plasma BSA levels,
              is shown in Figure 3. The BSA release rate was al-                                        00
              ways lower in seawater acclimatized fish than in                                            0      1      2       3      4      5       6
              freshwater acclimatized fish. In all experimental                                                     HOURS AFTER BATH
              groups, the BSA release rate was high during the                                                          Figure 3
              early period, and the peak of the rate was observed                             Changes in the BSA release rate into plasma after
              within one hour after the bath treatment. The rate                              hyperosmotic infiltration treatment in chum
              rapidly decreased after one hour and became low                                 salmon (A) and tilapia (B). Open circles indicate
              six hours after the bath. The gross BSA release into                            the fish in fresh water and closed circles the fish
              plasma, which is given by the area under the curve                              acclimatized to seawater. Data are presented as
              in Figure 3, is shown in Table 1. Both in chum                                  mean ï¿½ standard errors.
              salmon and tilapia, the gross BSA release of freshwa-
              ter acclimatized fish was larger than that of seawater
              acclimatized fish.
                                                                                           (1982) reported that plasma BSA administered by the
                                                                                           immersion method was trapped in the spleen and the
              Discussion                                                                   kidney of rainbow trout. Similarly, the trapping of
                                                                                           plasma BSA by the spleen and the kidney has been
              In the present study, BSA clearance from the plasma                          observed in plaice administered BSA by intravenous
              after the intravenous injection differed greatly within                      injection (Ellis 1980). The kidney is one of the most
              the same species; in each species, the BSA clearance                         important organs for osmoregulation in fish, and its
              was much more rapid in seawater-acclimatized fish                            function changes following seawater acclimatization
              than in freshwater-acclimatized fish. These results                          (Hickman and Trump 1969). Thus it is possible that
              strongly suggest that the environmental water affects                        the seawater acclimatization mechanism influenced
              fish physiology and consequently, by some mechan-                            the BSA trapping in the kidney and affected the
              ism(s), changes the plasma BSA clearance. Smith                              plasma BSA clearance. The plasma BSA clearance was








                                                                             Ototake and Nakanishi: Kinietics of Bovine Serum in Fish        99


                also different between species. Tilapia showed a rapid            rates in freshwater salmon were examined by modify-
                BSA clearance, while the chum salmon showed a slow                ing the regression equation to ignore the first term,
                BSA clearance. This difference may not only be at-                which is the main component for the two hours after
                tributed to species specificity, but also to the                  the injection. The distribution volume, the MRT, and
                temperature of the rearing water.                                 the gross BSA release were recalculated by using this
                  The estimated gross BSA release into the plasma of              modified regression equation and the deconvolution
                seawater salmon and seawater tilapia was much                     method. Recalculation with the modified regression
                smaller than the amount released into respective                  equation also showed more rapid BSA clearance and
                freshwater acclimatized fish. This indicates that the             less gross BSA release in seawater salmon than in
                physiological changes that resulted from seawater                 freshwater salmon (Table 1).
                residence reduced the gross BSA release after the HI                It has been reported that the plasma kinetics of
                treatment. Amend and Fender (1976) suggested that                 several ions, such as Na', Ca2', Mg2+, and Cl-, are
                hyperosmotic pretreatment had a hydrating effect on               different in seawater- and freshwater-acclimatized fish
                membranes of fish tissues, and thus made BSA infil-               (Hirano and Uchida 1972; Bentley 1971). There have
                trate into the fish more easily. However, fish residing           also been reports comparing the kinetics of medi-
                in seawater are adapted to a hyperosmotic environ-                cines in plasma of seawater- and freshwater-
                ment, and their membranes are more resistant to                   acclimatized fish, but the results are contradictory:
                hydration than those of fish in fresh water. There-               among the chemicals. Endo and Onozawa (1987)
                fore, it is reasonable to consider that hyperosmotic              and Ishida (1990) reported that when oxolinic acid
                pretreatment stimulates BSA uptake more strongly in               was administered to ayu, Plecoglossus altivelis, and
                fish in fresh water than in fish in seawater. In any              coho salmon, 0. kisutch, by bath and oral methods
                case, it is quite plausible that some factors in seawater         the seawater-acclimatized fish of both species showed
                influenced the fish physiology and induced the low                lower oxolinic acid concentration and shorter reten-
                BSA release rate and high BSA clearance rate. These               tion time than freshwater-acclimatized fish. On the
                two rates of seawater-acclimatized fish worked syner-             other hand, BergsJo and Bergsjo (1978) reported
                gistically to make their plasma BSA levels after the HI           that when sulfanilamide or sulfadimidine was admin-
                treatment much lower than those of -freshwater-                   istered by bath treatment to rainbow trout, the
                acclimatized fish.                                                plasma concentration of seawater-acclimatized fish
                  The BSA distribution volume after the intravenous               was higher than that of freshwater-acclimatized fish.
                injection is expected to agree with the whole plasma              It still remains unknown why there are such differ-
                volume estimated by tracer-dilution methods using                 ences between medicines. In any case, from the view
                Evans blue dye, serum albumin, or red blood cells, all            of vaccine application to marine fishes, it is quite im-
                of which have been widely used to determine whole                 portant to clarify whether the phenomenon observed
                plasma (or blood) volumes. Using these methods,                   in the present study for BSA (seawater-acclimatized
                whole plasma volumes of fish have been determined                 fish have a lower release rate and higher clearance
                to be 2.5-4.7 mL/100 g body weight in salmonids                   rate) is common to protective antigens such as li-
                (Conte et al. 1963; Smith 1966; Huggel et al. 1969,               popolysaccharides and proteases.
                Nikinmaa et al. 1981; Gingerich et al. 1987) and 3.5
                mL/100 g body weight in yellowtail (Itazawa et al.
                1983). The distribution volumes of the present study              Acknowledgments
                for seawater salmon, freshwater tilapia, and seawater
                tilapia agree well with those reported values. In fresh-          Financial assistance for this study was provided in part
                water salmon, however, the distribution volumes in                by a grant-in-aid (Bio-Media Program) from the Minis-
                the present study were smaller than the whole plasma              try of Agriculture, Forestry, and Fisheries (BMP-91-
                volumes reported so far in the literature. Smith                  IV-1-9). We would like to thank Yasuo Inui     'for his criti-
                (1966) suggested that blood sampling periods of less              cal review of this manuscript. We also acknowledge the
                                                                                  staff of the Hokkaido  Salmon Hatchery for supplying us
                than two hours after tracer injection tended to pro-              with the chum salmon eggs used in this study.
                duce low plasma volume and high clearance rate
                estimates in salmonids because these periods did not
                allow for adequate circulation of tracers in the vascu-           Citations
                lar system. Therefore, in the case of freshwater
                salmon in the present study, there is a possibility that
                                                                                  Amend, DY, and D.C Fender.
                the plasma BSA clearance rate was overestimated. In                   1976. Uptake of bovine serum albumin by rainbow trout
                such a case, the gross BSA release is likely to be over-                from hyperosmotic infiltration: a model for vaccinating
                estimated. Therefore, the BSA release and clearance                     fish. Science 192:793-794.







             100        NOAA Technical Report NAM I I I


             Bentley, P.J.                                                                     par la method de la dilution isoto-pique de 1'1311 et du
                 1971. Endocrines and osmoregulation. Springer-Verlag, Ber-                    5ICr. J. Physiol. (Paris), 61:145-154. (In French.)
                   lin/Heidelberg/New York, 300 p.                                      Ishida, N.
             Bergsjo, T., and T.H Bergsjo.                                                   1990. Comparison of tissue level of oxohnic acid in fresh
                 1978. Absorption from water as an alternative method for                      and seawater fishes after the oral administration. Bull. jap.
                   the administration of sulphonamides to rainbow trout,                       Soc. Sci. Fish. 56(2):281-286. (Injapanese; English abstr.)
                   Salmo gairdne7i. Acta. Vet. Scand. 19:102-109.                       Itazawa, Y, T. Takeda, I- Yamamoto, and T. Azuma.
             Conte, F.P., H.H. Wagner, and T.O. Haris.                                       1983. Determination of circulating blood volume in three
                 1963. Measurement of blood volume in the fish (Salmo                          teleosts, carp, yellowtail and porgy. jpn. J. Ichthyol.
                   gairdnerigairdne7i). Am.J.Physiol.205:533-540.                              30(l):94-101. (Injapanese.)
             Ellis, A.E.                                                                Laurell, C.B.
                 1980. Antigen trapping in the spleen and kidney of the pla-                 1966. Quantitative estimation of proteins by electrophoresis
                   ice Pleuranedes platessa L. J. Fish Dis. 3:413- 426.                        in agarose gel containing antibodies. Anal. Biochem.
                 1988. Current aspects of fish vaccination. Dis. Aquat. Org.                   15:45-52.
                   4(2)-.159-164.                                                       Nikinmaa, M., A. Soivio, and E. Aiolo.
             Endo, T., and M. Onozawa.                                                       1981. Blood volume of Salmo gairdneri: influence of ambient
                 1987. Effects of bath salinity and number of fish on the up-                  temperature. Comp. Biochem. Physiol. A Comp. Physiol.
                   take of oxolinic acid by ayu. Bull. jap. Soc. Sci. Fish                     69A(4):767-769.
                   53(4):557-562. (Injapanese; English abstr.)                          Smith, L.S.
             Gingerich, W.H., R.A. Pityer, andjj. Rach.                                      1966. Blood volumes of three salmonids. J. Fish. Res. Board
                 1987. Estimates of plasma, packed cell and total blood vol-                   Can. 23:1439-1446.
                   ume in tissues of the rainbow trout. Comp. Biochem.                  Smith, P.D.
                   Physiol. A Comp. Physiol., 87A(2):251-256.                                1982. Analysis of the hyperosmotic and bath methods for
             Hickman, C.P.Jr. and B.F. Trump.                                                  fish vaccination: Comparison of uptake of particulate and
                 1969. The kidney. In Fish physiology I (W.S. Hoar and Dj.                     nonparticulate antigens. Dev. Comp. Immunol., Suppl.
                   Randall, eds.) p. 91-241. Acad. Press, New York/ L6ndon.                    2:81-186.
             Hirano, T., and S.'Uchida.                                                 Wallenborg, B., and U.B. Andersson.
                 1972. Water and ion movements in the intestine and the uri-                 1978. Immunoelectrophore tic techniques with the LKB 2117
                   nary bladder of the teleosts and their hormonal                             multiphor. LKB Application Note, 249:1-12. (Pharmacia
                   control. Medical Science 23(2):56-68. (Injapanese.)                         LKB Biotechnology Inc., 800 Centennial Ave., Piscataway, NJ
             Huggel, Hj., H.C. Lane, and C.G. Ducret.                                          08854.)
                 1969. Determination de la courve D'homogeneisation et du
                   volume sanguin circulant de la truite Salmo gairdneri Rich.






                                          The Epidemiological Study of Furunculosis
                                                            m Salmon Propagation


                                   TETSUICHI NOMURA*, MAMORU YOSHIMIZU**, and TAKAHISA KIMURA**

                                                            *Hokkaido Salmon Hatchery, Fisheries Agency
                                                                  Nakanoshima 2-2, Toyohiraku
                                                                         Sapporo, Japan.

                                                             "Faculty, ofFisheries, Hokkaido University
                                                                        Minatomachi 3-1
                                                                         Hakodate, Japan




                                                                         ABSTRACT


                                     The authors attempted to determine the distribution and prevalence of Aeromonas
                                   salmdnicida in mature chum (Oncorhynchus keta), pink (0. gorbuscha), and masu
                                   salmon (0. masou) in Hokkaido that showed no apparent clinical signs of furunculo-
                                   sis. From September 1979 to November 1989, a total of 12,891 chum, pink, and masu
                                   salmon were collected from 30 rivers. The changing pattern of the annual prevalence
                                   of A. salmonicida in salmon was closely related to changes in fish density in the
                                   holding ponds: the prevalence of A. salmonicida increased in proportion to the in-
                                   crease in the number of fish in the ponds. We concluded from the results of
                                   histological and bacteriological examinations that fish with A. salmonicida in the kid-
                                   ney were not diseased but were carriers of A. salmonicida. The agent could not be
                                   isolated from the immature fish examined. A. salmonicida was also isolated from the
                                   ovarian fluid of fish showing no apparent clinical sign of furunctilosis. Few A.
                                   salmonicida were found on the surface of,the eggs one hour         ' after fertilization. A
                                   survey of agglutination titers against A. salmonicida in sera of chum, pink, and masu
                                   salmon showed great variability within the species. The isolated strains were identi-
                                   fled as A. salmonicida subsp. salmonicida and were pathogenic to salmonids. We
                                   concluded that the A. salmonicida carrier state in fish poses a serious problem in the
                                   prevention of furunculosis and its reduction plays a key role in salmon propagation.
                                   Both maturation of fish under conditions of low density in ponds, and disinfection of
                                   their eggs, are necessary to prevent fish furunculosis during artificial propagation of
                                   salmon.




                 Introduction                                                       documented in juvenile amago salmon (0. rho-
                                                                                    durus), and masu salmon (0. masou) in accordance
                 Furunculosis of salmonid fishes, caused by Aeromonas               with increased production of these fish. In
                 salmonicida, was first reported in 1890s by Emmerich               Hokkaido, outbreaks of furunculosis have been re-
                 and Weibel (1890, a and b). Since these first reports,             ported to occur in chum salmon (0. keta) by
                 furunculosis has been reported in virtually all parts              Nishino (1967), and in masu and pink salmon (0.
                 of the world where wild or cultured salmonids occur                gorbuscha) by Kimura (1970) during the maturation
                 (Smith 1960; Herman 1968; Snieszko 1972; Austin                    of these species in holding ponds. Nomura and
                 and Austin 1987).                                                  Kimura (1981), Nomura (1983), and Nomura et al.
                   Furunculosis is not a serious problem in rainbow                 (1983, 1991, a and b) reported isolating A.
                 trout (Oncorhynchus mykiss) culture in Japan be-                   salmonicida from the kidneys of mature chum, pink,
                 cause this species is resistant to the causative agent             and masu salmon that showed no apparent clinical
                 of the disease. However, serious mortality has been                signs of furunculosis.


                                                                                                                                              101







               102         NOAA Technical Report NNM I 11,




                                              10 11               Sea of Okhotsk
                                     9            12
                                                   13 14 is
                 Sea of Japan                               16 17  19
                                   7 8                          18    20 21      Nernuro Strait
                                                                          22 23 24
                                   6                                        25                                                          Figure 1
                        4 5                                                                                Rivers where chum, pink, and masu salmon were col-
                                                          27  26                                           lected (after Nomura et al. 1991a).
                            30             29                                                               1 Moheji        2 Hekirichi       3 Shiriuchi       4 Shubuto
                                              28                                                            5 Shiribetsu    6 Ishikari        7 Shokanbetsu     8 Nobusha
                                                                                                            9 Teshio       10 Tonbetsu       11 Hrobetsu       12 Tokushibetsu
                             2 1                Cape Erimo               Pacific Ocean                     13 Horonai      14 Shokotsu       15 Yuubetsu       16 Tokoro
                            3                                                                              17 Abashiri     18 Shari          19 lwaobeLsu      20 Ichani
                     Tsupru Strait                                                                         21 Shibetsu     22 Tohoro         23 Nishibetsu     24 Furen
                                                                                                           25 Bettouga     26 Kushiro        27 Tokachi        28 Shizunai
                                                                                                           29 Niikappu     30 Yurappu


                  Recently, no systematic epidemiological studies                                      We isolated A. salmonicida from -chum salmon in 11 of
               have been done to establish control measures for fu-                                 the 22 rivers examined; the percent occurrence of the
               runculosis in salmonids from which quarantine and                                    bacterium in this species of fish ranged from 0.6 to
               disease control policies could be based.                                             49.2%. Populations Of pink salmon, from 13 rivers were
                  In this paper, we report the recent epidemiological                               tested and A. salmonicida was isolated from 6 of these
               study of A. salmonicida which was carried out for the                                rivers with percent occurrence ranging from 0.2 to
               purpose of establishing control measures for the dis-                                13.3%. In masu salmon the bacterium was isolated from
               ease.                                                                                5 of 10 rivers examined and the percent occurrence
                                                                                                    ranged from 1.0 to 5.6%. Hence, A. salmonicida was de-
                                                                                                    termined to be distributed widely in the salmonid
               Distribution of A. salmonicida                                                       populations of Hokkaido, except those of rivers located
               in Salmonids in Hokkaido                                                             between Tsugaru Strait and Cape Erimo (Fig. 2).
                                                                                                       In the Ishikari, Shari, Iwaobetsu, Shibetsu, and
               We attempted to determine the distribution and                                       Tokachi rivers, the prevalence of A. salmonicida was
               prevalence of A. salmonicida in mature chum, pink,                                   found to vary yearly. In the Ishikari river, the preva'-
               and masu salmon populations in Hokkaido that                                         lence of A. salmonicida in chum salmon was high from
               showed no apparent clinical signs of furunculosis                                    1979 to 1984 but has gradually been decreasing since
               (Nomura et al. 1991 a).                                                              1985 (Fig. 3). In the chum salmon of the Tokachi
                  From September 1979 to November 1989, a total of                                  River and in all three species in the Shibetsu River,
               12,891 chum, pink, and masu salmon were collected                                    the prevalence of the bacterium remained high
               from 30 rivers (Fig. 1). At each sampling, a total of 60                             throughout the examination period. In the Shari
               fish of each species were randomly selected from the                                 river from 1979 to 1988, A. salmonicida was not iso-
               rivers' salmonid populations in accordance with Amos                                 lated from any of the fish examined; however, it was
               (1985). The fish were separated by species and river                                 isolated from 4 of 60 fish examined in 1989.
               and held in individual ponds at each river for about I                                  From 1979 to 1984, changes in the monthly preva-
               month until maturity.- After spawning, they Were pro-                                lence of the agent could be observed in fishes in the
               cessed for examination. Kidney materials were                                        Ishikari river. The incidence of A. salmonicida in-
               streaked onto nutrient agar plates (Eiken Co., Tokyo,                                creased until the middle of October and then
               Japan) and cultured at 20'C for 7 days. No clinical                                  decreased thereafter. The pattern of change was
               signs of furunculosis were observed in the examined                                  closely related with changes in fish density in the
               fish. Bacterial colonies that produced a soluble brown                               holding pond; the prevalence of the bacterium ap-
               pigment and showed the following characteristics were                                peared to increase proportionately as density of fish
               classified as A. salmonicida: Gram-negative staining,                                in the pond increased (Fig. 4).
               lack of motility, failure to grow at 37'C, tested positive                              The number of A. salmonicida bacteria found in
               for cytochrome oxidase, and had the ability to ferment                               kidney tissues ranged from 10' to                 105  colony forming
               on oxidative fermentative basal medium.                                              units per gram (cfu/g) (Nomura et al. 1991 a).







                                                                                                            Nomura et al.: Furuculosis in Salmon Propagation                          103





                                                        10 c               Sea of Okhotsk
                                                  9 c       12 CPM
                                                            13 CP
                                                                         16 CP
                        Sea of Japan                                     17 P
                                                  8 M                    18       20 cp Nemuro Strait
                                                                                  21 CPM
                                                                                      23 C
                                                  6 C                                                                                             Figure 2
                                                                                                                    Rivers where Aeromonas salmonicida was isolated from
                                 M                                       26 C                                       chum, pink, and iasu salmon from 1979 to 1989. C:
                                                                         27U                                       isolated from chum salmon; P: isolated from pink
                                                                                                                    salmon; M: isolated from masu salmon (after'
                                                                           Pacific Ocean                            Nomura et al. 1991a).
                                                        Cape Frimo                                                   5 Shiribetsu              6 Ishikari               8 Nobusha
                                                                                                                     Teshio                 10 Tonbetsu              12 Tokushibetsu
                             Tsugarn Strait                                                                         13 Horonai                16 Tokoro                17 Abashiri
                                                                                                                    18 Shari                  20 Ichani                21 Shibetsu
                                                                                                                    23 Nishibetsu             26 Kushiro               27 Tokachi



                                      Yew                                Year
                                      1979              n=263            1984         n=240                    The kidney materials of chum salmon in which A.
                                                                                                            salmonicida was isolated were fixed with Bouin's solu-
                                 50 -             0                      -                                  tion for histopathological examination. The kidney
                                                  0                                                         organs were dehydrated and embedded in Paraplast,
                                                  0                          0                              and sections of the samples were made and stained
                                                  a     , i              I I-  %                            with HE and Gimsa stain.
                                      1980              n7--360          1985         n=274                    Histopathological examination of the infected fish
                                                                                                            did not, however, reveal colonies of A. salmonicida
                                 50-                                     -                                  typically observed in fishes with furunculosis. Also,
                                                  000                                                       no outbreaks of furunculosis were recorded in the
                                                  0                                                         examined populations during the research period
                                      10          R I       I            ocb I          I                   (Nomura et al. 1991a).
                                      1981        0     n=274            1986         n--240                   There are few reports examining the prevalence
                                 50 -             0.                     -                                  of A. salmonicida in the organs of apparently nor-
                                                  0                                                         mal mature fish. In fact, as far as we know, there is
                                                  0
                                                                               0                            only one report by Daly and Stevenson (1985).
                                      _L          011       1            1 10     Ino                       They reported that A. salmonicida was detected in
                                      19112             n=300            1987         n=300                 31 of 286 brown trout (Salmo trutta) sampled from
                                                                                                            spawning runs in the Ganaraska River, Ontario,
                                 50   -                                                                     Canada, over a period of two years. Our results
                                                  0                                                         showed that the incidence of this agent in appar-
                                      _L 00,                             -dcbO b- 1                         ently normal chum salmon was higher compared to
                                      1983              0n=469           1988         n=541                 that of Daly and Stevenson's (1985) estimated for
                                                                                                            brown trout, and that A. salmonicida is distributed
                                 50   -           0                      -                                  widely in the river populations of salmonids in
                                                                                                            Hokkaido.
                                                  000                                                          Morikawa. et al. (1981) reported that the number
                                                  001       1                                               of A. salmonicida in the kidneys of moribund amago
                                                  Sep. Oct. Nov.         Sep. Oct Nov.                      salmon was 10' to 101 cfu/g. The reason why diseased
                                                             Month                                          fish were not found in the population we examined,
                                                                                                            even though they had A. salmonicida in their kidneys,
                                                        Figure 3                                            was that the degree of infection was not high
                     Changes in the monthly incidence of Aeromonas salmonicida                              enough. From the results of histological and bacte-
                                                                                                            riological examinations, we'conclude that fish with A.
                                                        'o C                 ea 0'0
                                                    @12
                                                                M
                                                               cp
                                                  9 C
                               @Japa                                     '6 CP17,PS
                                                                         18
                                                  8 M                             20 C
                                                                                  2,

                                                  6 C


                                 M


                                                                         27



                                                                           Par
                                                                               @iflc
                                                                                  '0

                                                            pe Fri..





















































                     in chum salmon collected from the Ishikari river, and held
                     for maturation periods during the period September to                                  salm  .onicida in the kidney are not                diseased fish but
                     November, 1979-88 (Modified from Nomura et al. 1991a).                                 carriers of A. salmonicida.
 






            104        NOAA Technical Report NMB I I I


                                                                                   in ovarian fluids ranged from 101 to 101 cfu/mL in
                                                                                   the populations from the Shibetsu and Teshio rivers.
                                                                                     Ovarian fluid containing A. salmonicida flows out of
                                                                 01981             the fish at the time eggs are stripped or during the
                      40                                                           process of maturation in the pond. Consequently, in-
                                                                                   fected water and infected ovarian fluid are expelled
                                                                                   into the river because the sewage from the egg strip-
                                                 0 1980                            ping areas and the holding ponds is not disinfected
                                                   0 im                            in Hokkaido. We suspect from these results that the
                      20                          0 im                             agent drained from the fish may be a source of infec-
                                                                                   tion for other anadromous salmon that ascend the
                                                01982
                                    0 1984                                         river for spawning.
                                      01M
                                                  0190                               Horne and Maj (1928), McCraw (1952), and
                                     19W , -0 IM                                   Hastein and Lindstad (1991) stated that the most im-
                                        0                                          portant source of A. salmonicida in the spread of
                        0                2             4              6
                                                                                   furunculosis is the existence of fish carrying this
                                   Fish stock density (flsh/m@)                    agent. Fish carrying the bacterium pose a serious
                                                                                   problem to the prevention of furunculosis, and their
                                       Figure 4                                    reduction in fish plays a key role in salmon propaga-
            The relationship between average density of the fish in                tion.
            hol 'ding ponds and the incidence (% occurre@ce) of
            Aeromonas salmonicida in fish take n from the Ishikari River,
            Numbers in the figure indicate the year of examination                 A. salmonicida on Egg Surfaces
            (unpubl. data).
                                                                                   The existence of the bacterium in the ovarian fluid
                                                                                   suggests that the surface of eggs taken from the fish
            A. salmonicida in Immature Fish                                        will also be contaminated. Contaminated eggs may
                                                                                   spread the agent to areas where the eggs will be
            We attempted to isolate A. salmonicida from immature                   transplanted. We studied the existence of A.
            chum and masu salmon (Nomura et al. 1991b). A                          salmonicida on the surface of eggs by artificially con-
            total of 680 fish were collected in four coastal set nets              taminating chum salmon eggs with A. salmonicida.
            off Hokkaido, and a total of 1,200 juvenile masu                         The A. salmonicida 20-1, strain, which was isolated
            salmon and chum salmon fry were collected from I I                     from the kidneys of chum salmon in the Tokachi
            hatcheries of the Hokkaido Salmon Hatchery system.                     River, was used as innoculum. The strain was cultured
                The bacterium was not isolated from any of the                     and harvested, then was suspended in phosphate
            examined fish.                                                         buffer saline (PBS). The chum salmon eggs were
                                                                                   bathed in PBS containing the agent for an hour. The
                                                                                   eggs were incubated in well water at 8'C in the labo-
            A. salmonicida in Ovarian Fluid                                        ratory. At one hour and at 24 hours after fertilization,
                                                                                   we took 20 eggs from the incubator and put them
            In 1989 i   *n Hokkaido, we attempted to isolate A.                    into sterilized water. The flask was shaken strongly for
            salmonicida from the ovarian fluids of mature chum,                    5 minutes, we then measured the viable number of A.
            pink, and masu salmon.                                                 salmonicida in the water according to the method of
                Ovarian fluids were collected according to the                     Nomura et al. (1991b).
            method of Yoshimizu et al. (1985). The ovarian fluids                    Egg surfaces were initially bathed with 1.1 x 101 to
            were streaked onto nutrient agar plates (Eiken Co.,                    4.3 x 101 cfu/egg of A. salmonicida. The number of A.
            Tokyo, Japan) and the plates were cultured at 20'C                     salmonicida present on the egg surfaces decreased
            for 7 days. Number of viable counts of A. salmonicida                  from 68 to 4.6 cfu/egg an hour after fertilization and
            in ovarian fluid and kidney were measured in accor-                    A. salmonicida could not be isolated from the eggs
            dance with the method of Nomura et al. (1991a).                        cultured on plates 24 hours after the initial bath
                A. salmonicida was i solated from the ovarian fluid of             treatment.
            fish showing no apparent clinical signs of furunculo-                    We also attempted to isolate A. salmonicida from
            sis. For example, A. salmonicida was isolated from the                 eggs in the incubation boxes at the Satsunai,
            ovarian fluid of 22 of 120 fish examined from the                      Nakagawa, Nemuro, and Tokachi hatcheries. These
            Tokachi River. The number of A. salmonicida bacteria                   eggs were taken from brood fish in which the preva-






                                                                                               Nomura et at.: Furunculosis in Salmon Propagation                 105

                   lence of A. salmonicida was high (Nomura et al.                                It is believed that A. salmonicida is not able to exist
                   1991a). Fortunately, A. salmonicida was not isolated                        for long time in water without fish, but McCarthy
                   from any of the 15 hatcheries' eggs (Nomura et al.                          (1980) studied the survival of the agent in water us-
                   1991b).                                                                     ing an antibiotic-resistant strain of A. salmonicida and
                       In Hokkaido, fertilized eggs are transported to a                       found the agent could survive for 8 days in water.
                   hatchery from the egg collection location I hour af-                           The results of McCarthy (1980) and this study indi-
                   ter fertilization. From the results of our experiment,                      cate that A. salmonicida survives long enough to infect
                   it appears that A. w1monicida is able to exist on the                       other fish in the water.
                   surface of an egg. This makes us concerned that we
                   may be transporting the bacteria to the hatchery with
                   the fertilized egg. We believe that it is necessary to                      Variation of ALmdutination Titer
                   prevent the transfer of A. salmonicida via eggs in or-                      Against A. salmonicida in the Serum
                   der to control furunculosis.
                                                                                               A serological survey of adult salmon was -made from
                                                                                               blood samples collected in 1988 in Hokkaido, from
                   Survival of A. salmonicida in Water                                         mature chum, pink, and masu salmon. Blood was
                                                                                               aseptically extracted from the donsal artery with 10
                   By definition, A. salmonicida is considered to be an                        mL of Vacteinor (Terumo Co., Tokyo, Japan). The
                   obliga  te pathogen (Popoff 1984) and is never found                        resulting serum was separated from the blood-cell
                   in surface water. Its ability to survive and remain in-                     clot by centrifugation and was stored at -90'C until
                   fectious in the external environment may be a major                         assayed. The serum was tested for agglutinating anti-
                   determinant in the spread of furunculosis. We stud-                         body titers individually, by test-tube methods with A.
                   ied the viability of A. salmonicida in nonsterile, sterile                  salmonicida ATCC 14174.
                   filtered, and autoclaved fresh water and in salt water.                        Agglutinin titers against A. salmonicida in the se-
                       A. salmonicida strain 20-1 isolated from chum                           rum of mature chum, pink, and masu salmon in
                   salmon in the Tokchi River was used in this experi-                         Hokkaido in 1988 are shown in Table 1.
                   ment. The strain was cultured at 20'C and harvested,                           Of a total of 75 serum samples taken from mature
                   then suspended in fresh water or in sea water. The                          chum salmon, 73.3% did not have 'the agglutinin,
                   suspended cells were inoculated into 200 mL of                              and the range of titers was 8 to 32. In pink salmon,
                   nonsterile, sterile filtered, and autoclaved fresh water                    10% of the sample did not have the agglutinin, and
                   and saltwater and were incubated at 10'C.                                   the range was 4 to 32. In masu salmon, 16.6% of the
                       In sterilized fresh water, A. salmonicida survived for                  examined serum did not have the agglutinin, and the
                   60 days and in nonsterile water, only 4 days. The sur-                      range was 4 to 128.
                   vival of A. salmonicida in sterile salt water was 8 days;                      The diversity in the incidence of agglutination titer
                   this was a shorter survival period than that in sterile                     within each of the three species indicates a continu-
                   fresh water.                                                                ous, widespread interaction between individuals of




                                                                                        Table 1
                       Prevalence and ranges of agglutinin titers against A. salmonicida in the serum of mature chum, pink, and masu salmon
                       ascending various rivers    in Hokkaido.

                       Species                        River          No. of fish examined                              Agglutinin titers
                                                                                               Negative                     Modes                   Range


                       Chum salmon                    lchani                  15                    80.0                       32                   16- 32
                                                      Tokachi                 30                    76.6                         8                  8-  32
                                                      Shizunai                30                    66.6                       16                   8-  32

                       Pink salmon                    Shokotsu                30                    16.6                       16                   4-  32
                                                      lwaobetsu               30                      6.7                      16                   4-  32
                                                      Nishibetsu              30                      6.7                      16                   4-  32

                       Masu salmon                    Shiribetsu              39                    15.4                         8                  4-128
                                                      Shari                   18                     0                         16                   4-128
                                                      Nishibetsu              27                    29.6                         8                  4- 32







          106      NOAA Technical Report NNM I I I


          the host populations and A. salmonicida. The differ7           suspected that A. salmonicida can survive for a long
          ence in the amount of agglutination titer is                   time in the river water in Hokkaido after leaving the
          proportionate to the period of A. salmonicida infec-           fish and that its existence may be a source of infec-
          tion.                                                          tion to salmonid fish. The results suggest that such
             In general, the percentage of serologically reactive        bacteriophage could be very useful for studying the
          salmon increased as their length of freshwater resi-           existence of the agent in water.
          dency increased. Weber and Zwicker (1979) reported
          that of a total of 43 serum sampled from Atlantic
          salmon (Salmo salar) in the Miramichi or Margareen                                     Table 2
          rivers in Canada, none had A. salmonicida aggluti-               Isolation of Aeromonas salmonicida bacteriophage
          nin, but of 27 Restigouche River salmon, four had a              from the samples of river water and hatchery water.
          titer of 10, five had a titer of 20, and one had a titer
          of 640. They confirmed that Atlantic salmon have                 Sample                         Samples
          previously contacted A. salmonicida in, the                                       Numbers examined       Isolated
          Restigouche River.                                                                                   containing phage
             In our study, agglutinate titers in the serums were           River water              4                 2
          low. It was suggested that the fish were infected with           Hatchery water           9                 2
          A. salmonicida shortly before their eggs were stripped.          Sewage of hatchery       13                3
          Kimura (1970) reported that the immunological
          method of preventing furunculosis was useful in
          adult masu salmon during the holding period be-
          cause these salmon stay in fresh water for a long              Pathogenicity of the
          enough period to allow them to produce antibodies              Isolated A. salmonicida
          after antigen inoculation. In chum salmon, however,
          the freshwater residency period is short, so this              The isolated strains were identified as A. salmonicida,
          method of prevention would not be practical.                   subspecies salmonicida, by their biological, biochemi-
                                                                         cal, and immunological characteristics. All of the
                                                                         isolated A. salmonicida strains showed auto-agglutina-
          Isolation of the Bacteriophage                                 tion and produced protease in the medium, so we
          of A. salmonicida from Water                                   also expected them to be pathogenic.
                                                                           In order to examine the pathogenicity of the iso-
          There is no sensitive medium for selecting A.                  lated strain, we injected it into chum and masu
          salmonicida. This means that when the number of A.             salmon fry and adult chum salmon.
          salmonicida in water is low, the isolation of A.                 A. salmonicida 20-1 was cultured for 48 hours at
          salmonicida from the water will be difficult. This is          20'C. The cells were washed three time in PBS and
          because A. salmonicida cannot grow on the culture              were suspended in PBS. The strain was injected into
          medium under competitive conditions with other                 chum salmon fry, yearling masu salmon, and chum
          natural bacteria populations. We attempted to isolate          salmon brood fish at concentrations of 1.7 X 102, 1.8
          the bacteriophage of A. salmonicida to ascertain the           X 101 and 6.0 X 101 cfu/fish, respectively.
          existence of A. salmonicida in the water.                        All of the examined fish showed typical signs of
             Water samples, from 11 hatcheries and 4 rivers              furunculosis 3 to 4 days after injection. The number
          were examined. Nutrient agar (Eiken Co., Tokyo, Ja-            of A. salmonicida in the kidneys of moribund fish was
          pan) was employed for the routine culture, dilution,           around 108 cfu/g kidney tisstie, the same number re-
          and enumeration of A. salmonicida and its phage                ported by Morikawa. et al. (1981) in the kidneys of
          strain. One hundred mL of sample was added to 500              moribund amago salmon. On the basis of these re-
          mL'of cultured A. salmonicida Ar-32, Ar-43, Ar-71, and         sults, we suspect the isolate is a pathogenic strain.
          H-70 strains in the logarithmic phase. Detection and
          enumeration of phage were achieved using the me-
          dium and double agar layer technique (Paterson et              Control of A. salmonicida
          al. 1969). The results are shown in Table 2.                   on the Surface of Egg
             McCraw (1952) stated that when the bacteriophage
          of A. salmonicida exists, its presence may indicate the        To establish a method of controlling A. salmonicida
          existence of the bacterium. The bacteriophage was              on the eggs, the bactericidal effect of popidon-iodine
          isolated from two samples of river water and five              (Isodine), and the toxicity of this agent to the chum
          samples from hatchery water. From this result, it was          salmon egg were studied.






                                                                                                        Nomura et al.: Furunctdosis in Salmon Propagation                        107

                       The bactericidal effects of popidon-iodine to A.                                     Chum salmon in the Ishikari River were randomly
                    salmonicida were determined in accordance with the                                  assigned to experimental holding ponds.and held un-
                    method of Amend and Fryer (1972).                                                   der low (4.9 fish/M2         ) and high density (14.7 fish/M2)
                       A. salmonicida was completely killed by treatment                                conditions until maturation. The kidney tissues of all
                    with 25 ppm isodine for five minutes and this solu-                                 the fish used in experiment were cultured on nutri-
                    tion was not toxic to the chum salmon eggs for                                      ent agar in accordance with the method of Nomura
                    treatments lasting up to one hour. Thus, the authors                                et al. (1991).
                    confirmed that isodine solution has a sanitizing ef-                                   As we expected, we found that 12.4% of the fish
                    fect on the agent, and thatit.does not have adverse                                 examined harbored A. salmonicida when they were
                    effects on chum salmon eggs.                                                        stocked at a high density, but no examined fish con-
                                                                                                        tained the agent when they we're stocked at a low
                                                                                                        density (Fig. 5A). The incidence of the bacterium in
                    Method for Decreasing the Prevalence                                                fish that were held under low dissolved. oxygen condi-
                    of A. salmonicida in Chum Salmon                                                    tions was higher than that of fish held under high
                                                                                                        dissolved oxygen levels (Fig. 51)). These results
                    From the results of our epidemiological study, we sus-                              clearly indicate that high stocking densities and low
                    pected that the incidence of A. salmonicida was                                     dissolved oxygen levels in holding ponds have a
                    affected by the density of fish during their maturation                             marked effect on the prevalence of the agent in the
                    period in the holding ponds; as the average density of                              fish. We concluded that fish maturation in the pond
                    brood fish stocked in ponds decreased, the incidence                                under low density conditions and disinfection of the
                    of A. salmonicida in examined fish also decreased (Fig.                             eggs, are necessary to prevent fish furunculosis in the
                    4). Therefore, we examined the relationship between                                 artificial propagation of salmon.
                    the stocking density of fish in the pond and the preva-
                    lence of A. salmonicida in,the fish.
                                                                                                        Citation

                                       FiSh/M2  A FUh stock density                                     Amend, D.F., andj.L. Fryer.
                                             15                                                               1972. Virucidal activity of two iodo.-phors to salmonid
                                                                                                                virus. J. Fish. Res. Board Can. 29:61-65.
                                             10                                                         Amos, ILH., ed.
                                                                                                              1985. Fish health blue book-procedures for the detection of
                                             5                                                                  certain fish pathogens. Fish health section, Am. Fish. Soc.,
                                                                                                                114 p.
                                             0                                                          Austin, B., and D.A. Austin.
                                             PPM 11'  Dissolved oxygen                                        1987. Bacterial fish pathogens: disease in farmed and wild
                                             10 -                    0                                          fish. Ellis Horwood Ltd., Chichester, West Sussex, En
                                                                                                                gland, 195 p.
                                             5  -                                                       Daly, J.G., and R.M.W. Stevenson.
                                                                                                              1985. Importance of culturing several organs to detect
                                             0                                                                  Aeromonas salmonicida in salmonid fish. Trans. Am. Fish.
                                             % C        Survival rate                                           Soc. 114:909-910.
                                          100   -                                                       Emmerich, R., and C. Weibel.
                                                                                                              1890a. Ober eine durch Bacterien verursachte Infection-
                                             50 -                                                               skrankheit der Forellen. Allg. Fish. Zgt. 15:73-77.
                                                      1             1                                         1890b. Ober eine durch Bacterien verursachte Infection-
                                             0  D         Incidence                                             skrankheit der Forellen. Allg. Fish. Zgt. 15:85-92.
                                             %                                                          Hastein, T., and T. Lindstad.
                                             2                                                                1991. Disease in wild and cultured salmon: possible
                                             0                                                                  interaction. Aquaculture. 98:277-288.
                                                                                                        Herman, R.L.
                                             0                      1
                                             0                                                                1968. Fish furunculosis 1952-1966. Trans. Am. Fish. Soc.
                                                    Lot A           Lot B                                       97:221-230.
                                                                                                        Hornej.H., and I.M.S. Maj.
                                                     Figure 5                                                 1928. Furunculosis in trout and the importance of carriers
                                  The relationship between the density                                          in the spread of the disease. J. Hyg. 28:67-78.
                                  of fish stock in holding ponds, the                                   Kimura, T.
                                  concentration of dissolved oxygen in                                        1970. Studies on a bacterial disease occurred in the adult
                                  the water, survival rate, and the inci-                                       "Sakuramasu" (Oncorhynchus masou) and pink salmon (0.
                                                                                                                gorbuscha) rearing for maturity. Sci. Rep. Hokkaido Salmon
                                  dence (% occurrence) of Aeromonas                                             Hatchery. 24:9-100. Onjapanese; English abstr.)
                                  salmonicida (unpubl. data).







             108         NOAA Technical Report NNM I I I


             McCraw, B.M.                                                                  Nomura, T., M. Yoshimizu and T. Kimura.
                  1952. Furunculosis of fish. U.S. Fish and Wildl. Serv. Spec.                  1991b. Prevalence of Aeromonas salmonicida in the kidney of
                    Sci. Rep. 84:87 p.                                                             chum salmon (Oncorhynchus kela) and masu salmon (0.
             McCarthy, D.H.                                                                        masou) at various life stages. Fish Pathol., 26:149-
                  1980. Some ecological aspects of the bacterial fish patho-                       153. (InJapanese; English abstr).
                    gen-Aeromonas salmonicida. In Aquatic microbiology: sym-               Paterson, W.D., RJ. Douglas, I. Grinyer, and L.A. McDermott.
                    posium of the Society of Applied Bacteriology 6:299-324.                    .1969. Isolation and preliminary characterization of some
             Morikawa, S., S. Miki, and F. Tashiro.                                                Aeromonas salmonicida bacteriophages. J. Fish. Res. Board
                  1981. Changes in hemato logical properties and viable cell                       Can. 26:629-632.
                    number of bacteria in amago salmon artificially infected               Popoff, M.
                    with Aeromonas salmonicida. Fish Pathol. 16:43-49. (In                      1984. Family 11. Vibrionaceae. In Bergey's manual of system-
                    Japanese; English abstj%)                                                      atic bacteriology, Vol. I (Noel R. Krieg and John G. Holt
             Nishino, K.                                                                           eds.), p. 545-550. Williams and Wilkins, Baltimore.
                  1967. On a bacterial disease of adult "sake" Oncorhynchus                Smith, I.W.
                    kela, in captivity. Fish Pathol. 2(l):73-74. (InJapanese.)                  1960. Furunculosis in salmon kelts. Nature 186:733-734.
             Nomura, T.-                                                                   Snieszko, S.F.
                  1983. Incidence of Aeromonas salmonicida among anadro-                        1972. Panel review on furunculosis of salmonidae. In Report
                    mous salmonids, 1980-1982. Sci. Rep. Hokkai- do Salmon                         of the symposium on the major communicabable fish dis-
                    Hatchery. 37:63-65. (InJapanese; English abstr.)                               eases in Europe and their control; 20-22 April 1972,
             Nomura, T., T. Kimura.                                                                Amsterdam (William A. Dill, ed.): p 157-163. European In-
                  1981. Incidence of Aeromonas salmonicida among anadro-                           land Fisheries Advisory Commitee EIFA Technical paper 17
                    moussalmonids. Fish Pathol. 16:69-74. (Injapanese;En-                          (suppl. 2).
                    glish abstr.)                                                          Yoshimizu, M., T Kimura, andJ.R. Winton.
             Nomura, T., I Kimura, I. Shimizu, and K. Nara.                                     1985. An improved technique for collecting reproductive
                  1983. Incidence of Aeromonas salmonicida among chum                              fluid samples from salmonid fish. Prog. Fish-Cult. 47:199-
                    salmon, Oncorhynchus keta, in Chitose River. Sci. Rep.                         200.
                    Hokkaido Salmon Hatchery. 37:53-61. (In Japanese; En-                  Weber, J.M. and B.M. Zwicker.
                    glish abstr.)                                                               1979. Aeromonas salmonicida in Atlantic salmon (Salmo
             Nomura@ T., M. Yoshimizu, and T. Kimura.                                              salar). J. Fish. Res. Board Can. 36:1102-4107.
                  1991a. Prevalence of Aeromonas salmonicida in the chum
                    salmon (Oncorhynchus keta), pink salmon (0. garbuscha) and
                    masu salmon (0. masou) returning to rivers in
                    Hokkaido. Fish Pathol. 28, 139-147. (In Japanese; En-
                    glish abstr.)






                          Functions of Hemocytes During the Wound Healing Process
                                                             in the Pearl Oyster



                                                                    TOHRU SUZUKI

                                                           National Research Institute of Aquaculture
                                                                  Nansei, Mie 516-01, Japan




                                                                       ABSTRACT


                                   Morphological evidence from previous studies suggests that bivalve hemocytes function
                                 in hemostasis and extracellular matrix production during the wound healing process.
                                 The present paper describes the use of an in vitro cell culture system to show that the
                                 agranular hemocytes of the. pearl oyster Pinctada jucata have the ability to perform these
                                 healing functions. A possible wound healing system is also presented.



               Introduction                                                       muscle with a plastic syringe attached to a 24 gauge
                                                                                  needle (Fig. 1). Approximately 2 mL of blood was
               Defense reactions against infection and the removal of             collected from each animal. Blood pooled from five
               tissue debris are the responses of the immune system               animals was centrifuged at 100 X g for 8 minutes and
               that occur after wounding. On the other hand, tissue               a pellet of hemocytes was obtained. The pellet was
               regeneration at the wound site is an organogenetic                 washed twice with a balanced salt solution for marine
               event in which cell-cell and extracellular matrix (ECM)-           molluscs (MMBSS) prepared according to Machii
               cell interactions occur. Thus, the wound healing                   and Wada (1989). The pellet was suspended on 2 mL
               process can be a useful model to study the functions of            of culture medium Pf35 (Machii and Wada 1989),
               proteins and other cell components in both the im-                 developed specifically. for pearl oyster tissue. A 0.5-
               mune and organogenesis systems. Because bivalves have              mL sample of the suspension was pipetted into a
               .no humoral clotting factors, platelets, or capillary ves-         plastic culture flask (50 mL: Nunc). After an hour, to
               sels, it is assumed that their wound healing. system               allow the hemocytes to adhere to the plastic surface,
               differs from that of vertebrates and is probably less com-         2.0 mL of P05 was added to the flask. The cells were
               plex. The wound healing process has been described                 then incubated at 25'C. The culture medium was re-
               for several bivalves at the morphological level. It is             newed on the fourth day of culture.
               known that the hemocytes prevent blood loss at the
               wound site by forming a cellular sheath and producing              Microscopy
               an ECM (DesVoigne and Sparks 1968; Pauley and
               Heaton 1969; Ruddell 1971). The healing process in                 The cultured cells were fixed in Karnovsky's fixative
               the pearl oyster Pinctadafucata and the associated func-           containing 8% sucrose at VC for I hour. After wash-
               tions of hemocytes have been demonstrated (Suzuki et               ing in 0. 1 M phosphate buffer (PB; pH 7.2), they were
               al. 1991). In this paper, the author describes a study             post-fixed in 1% osmic acid in PB for I hour, washed
               using cultured hemocytes from the pearl oyster in                  tv@ice in PB, dehydrated through graded alcohols, and
               which agranular hemocytes show the ability to form a               embedded in Taab 812 resin. Ultrathin sections were
               cellular sheath and to produce an ECM. A possible                  stained with uranyl acetate and lead acetate. All trans-
               wound healing system is also presented.                            mission electron microscopy was performed with a
                                                                                  JEOLJEM- 1200EX electron microscope.
               Methods                                                            Results and Discussion
               Hemocyte Culture                                                   Aggregate Formation
               While the valves of R fucata were held slightly open               The hemocytes formed cellular aggregates during
               with a wedge, blood was drawn from the adductor                    blood collection and rinsing. Three hours after the

                                                                                                                                            109







            Ito       NOAA Technical Report NNUS I I I


                                                                             viewed under phase-contrast microscopy (Fig. 5).
                      Blood collection                                       The matrix continued to develop until about the sev-
                                                                             enth day of culture, after which time the number of
                                                                             living hemocytes rapidly decreased, Agranular hemo-
                                                                             cytes of 1-3 layers thickness were attached to the
                                                                             matrix, which was composed of fine fibrils and
                                                                             flocculent substances (Figs. 6 and 7). The fibrils were
                         Washing                                             20 nm in diameter and showed a faint banding pat-
                                                                             tern indicating that they are collagenous fibrils. The
                                                                             flocculent substances are probably proteoglycans. It
                                                                             has also been ascertained at the biochemical level
                        Suspension                                           that the matrix includes collagen (Suzuki et al.
                                                                             1991).


                      Standing for 1h                                        Wound Healing System
                                                                             In the pearl oyster, four noticeable cellular reactions
                                                                             occur during the healing process (Suzuki et al.
                      Culture at 25*C                                        1991): 1) removal of tissue debris, 2) cellular sheath
                                                                             formation, 3) ECM deposition, and 4) epithelial re-
                                                                             generation. The first reaction is attributed to the
                                    Figure I                                 phagocytic ability of -agranular hemocytes. In addi-
                      Procedure used for hemocyte culture.                   tion, these cells also perform the second and third of
                                                                             these healing reactions, cellular sheath formation
                                                                             and ECM deposition, as suggested by the results of
            beginning of culture, the aggregates were 80-200 Rm              this paper.
            in diameter and adhered well to the plastic surface    . of        Based on these results, a possible wound healing
            the culture flask (Fig. 2). In bivalves, two categories          system in the pearl oyster is presented in Figure 8.
            of hemocytes occur-agranular. and granular hemo-                 When the pearl oyster.is wounded, agranular hemo-
            cytes (Feng et al. 1971). It has been demonstrated               cytes infiltrate the injured area to remove tissue
            that the former are macrophage-like cells with active            debris (and possibly foreign particles) and then form
            phagocytic capabilities (Reade and Reade 1972;                   a cellular sheath to prevent blood loss. After sheath
            Moore and Lowe 1977). One hemocyte of the pearl                  formation, they begin to produce an ECM, which
            oyster is able to take in 1-9 vertebrate erythrocytes            functions as a template for the regenerating epithe-
            (Suzuki and Mori 1990). As shown by transmission                 lium. It is possible that the wound site is repaired
            electron microscopy, the aggregate is formed only by             through this sequence of processes. Assuming this ex-
            agranular hemocytes (Fig. 3). They adhere to one                 planation is accurate, the wound healing system of
            another at an adhesion plaque from which microfila-              this bivalve is simple compared with that of verte-
            ments can be seen running into the cytoplasm (Fig.               brates.
            4). Thus, agranular hemocytes can form a cellular                  It is well known that in vertebrates growth factors
            aggregate in a relatively short time (perhaps several            secreted from the platelets, macrophages, and
            minutes if stimulation is provided), although it is not          fibroblasts are the humoral factors that control cell
            understood what factors stimulate the reaction. It is            growth and ECM production at the wound site. In
            possible that this aggregate formation is a homolo-              bivalves, however, neither a growth factor nor its
            gous reaction to the cellular sheath formation at the            production cell has been identified. The agranular
            wound site.                                                      hemocyte is the most likely candidate to be a growth
                                                                             factor production cell, because, like macrophages and
                                                                             fibroblasts in vertebrates, it is the primary cell type
            ECM Production                                                   in bivalves that appears at the wound site. In addi-
                                                                             tion, platelets are absent from bivalve hemocytes.
            During the first 2-3 days of culture, deposition of an           Future studies with in vitro cultures will improve our
            ECM started in the agranular hemocyte aggregate.                 understanding of both the wound healing system
            The matrix was observed as a transparent gel when                and organogenesis in bivalves.







                                                                      Suzuki: Function of Hemocytes During Wound Healing in the Pearl Oyster                         III


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                                                  9;0 11M                                                                                   'K                    M
                      45


                     Figure 2    Hemocyte aggregate after 3 hours of culture. X 135.
                     Figure 3    Electron microscopy of aggregate after I day of culture. X 2,000.
                     Figure 4    Adhesion plaque formed at contacting site of agranular hemocytes. X 6,700.
                     Figure 5    Matrix (arrowhead) formed in a hemocyte aggregate after 7 days of culture. X 135.
                     Figure 6    Electron microscopy of matrix and agranular-hemocyte layer. X 2500.
                     Figure 7    Fibrils in the matrix at high magnification. X 31,000.




                   Citation


                   DesVoigne, D.M., and A.K. Sparks.                                             Moore, M.N., and D.M. Lowe.
                        1968. The process of wound healing in the pacific oyster,                     1977. The cytology and cytochemistry of the hemocytes of
                          Crassostrea gigas. J. Invertebr. Pathol. 12:53-65.                            Mytilus edulis and their responses to experimentally injected
                   Feng, S.Y.JS. Feng., C.N. Burke, and L.H. Khairallah.                                carbon particles. J. Invertebr. Pathol. 29:18-30.
                        1971. Light and electron microscopy of the leucocytes of                 Pauley, G.B., and L.H. Heaton.
                          Crassostrea virginica (mollusca: pelecypoda). Z. Zellforsch.                1969. Experimental wound repair in the freshwater muisel
                          120:222-245.                                                                  Anotionta oregonensis.    Invertebr. Pathol. 13:241-249.
                   Machii, A., and K.T. Wada.                                                    Reade, P., and E. Reade.
                        1989. Some marine invertebrates tissue culture. In Inverte-                   1972. Phagocytosis in invertebrates 11-the clearance of car-
                          brate cell system application, Vol. 11 U. Mitsuhashi, ed.) p.                 bon particles by the clam, Didacna maxima.
                          225-223. CRC Press, Boca Raton, Florida.                                      Reticuloendothel. Soc. 12:349-360.







              112           NOAA Technical Report NMFS I I I

                  9000                                                                              0
                                     '.. O/V                              9SE
                                                                                0






                     Infiltration of agranular hemocytes                Cellular sheath formation
                     Removal of tissue debris by them                   by agranular hemocytes





                                                                    00

                     ><-









                         Regeneration of epithelium                       Secretion of ECIVI
                        along the new ECM                                 by agranular hemocytes                                                Figure 8
                                                                          Growth factors?                          A possible wound healing system in the pearl oys-
                                                                                                                   ter: ECM=extracellular matrix.




              Ruddell, C.L.                                                                            Suzuki, T., R. Yoshinaka, S. Mizuta, S. Funakoshi, and F, Wada.
                     1971. The fine structure of oyster agranular amebocytes                                1991. Extracellular matrix formation by amebocytes during
                       from regenerating mantle wounds in the paciU oyster,                                    epithelial regeneration in the pearl oyster Pinctada
                       Crassostrea gigas. J.. Invertebr. Pathol. 18:260-268.                                   fucata. Cell Tissue Res. 266:75-82.
              Suzuki, T., and Y, Mori.
                     1990. Hemolymph lectin of the pearl oyster, Pincladafucata
                       martensii: a possible non-self recognition system. Dev.
                       Comp. Immunol. 14:161-173.
                                                                                    0;1






                                         Skeletal Abnormalities of Fishes Caused by
                                                        Parasitism of Myxosporea



                                                      YUKIO MAENO and MINORU SORIMACHI
                                                            National Research Institute of Aquaculture
                                                                        Fisheries Agency
                                                                 Nansei-cho, Mie 516-01, Japan




                                                                         ABSTRACT


                                    The relationship between skeletal abnormalities and parasitic infection was studied for
                                 deformed cultured yellowtail, Seriola quinqueradiata, Japanese bluefish, Scombrops boops,
                                 and mullet, Mugil cephalus. Soft radiographic observations indicated that the deformities
                                 were due to skeletal abnormalities: deformed yellowtail, Japanese bluefish, and mullet
                                 were characterized by scoliosis, lordosis, and lordo-scoliosis, respectively. Myx6sporean
                                 cysts were found in various parts of the brain of deformed fish such as the fourth ven-
                                 tricle' the cavity of the optic tectum, the surface of the olfactory lobe and bulb, and the
                                 optic lobe. The cysts were observed in the fourth ventricle in all deformed fish, but not in
                                 normal fish. From morphological characteristics, myxosporeans from the deformed yel-
                                 lowtail and Japanese bluefish were identified as Myxobolus buri, and that from deformed
                                 mullet was identified as M. spinacurvatura. These results suggest that Myxobolus species are
                                 responsible for the skeletal abnormalities observed in these deformed fish.



               Introduction                                                        tu re from 1987 to 1989.Four deformed Japanese
                                                                                   bluefish and four deformed mullet were captured by
               Skeletal abnormalities of fish have been reported to                a set net in Mie Prefecture in 1989.
               be caused for various reasons, such as dietary defi-                  After external observation, radiography of all fish
               ciencies (Halver and Shanks 1960), pesticide                        was performed with Fuji Lx film at 40 milliamps and
               exposure (Couch et al. 1977), heavy metal exposure                  50kV for 20 seconds and subsequently examined for
               (Holcombe et al. 1976), and bacterial (Kaige et al.                 parasitism in viscera by necropsy. From these animals,
               1984) and parasitic infections (Halliday 1976). Many                three deformed and three normal yellowtail, three
               studies report sporulation of myxosporeans in fish                  Japanese bluefish, and three mullet were used for
               brains. However, only a few studies have described a                subsequent histological examination.
               possible correlation between skeletal abnormalities                   The brain, liver, kidney, and spleen were excised
               and myxosporean parasitism in the brain. While                      from each fish and fixed in 10% formalin, embedded
               studying skeletal abnormalities in three species of                 in paraffin, cut serially at 4 gm, stained with Giemsa
               fish, cultured yellowtail (Se7iola quinqueradiata), Japa-           or hematoxylin and eosin, and then observed for the
               nese bluefish (Scombrops boops), and mullet (Mugil                  presence and location of cysts.
               cephalus), we found spores of a myxosporean in the                    For the study of spore morphology, some myxospor-
               brains of these fish. The present paper elaborates on               ean cysts were removed from the brain and crushed in
               the possible relationship between skeletal abnormali-               phosphate buffer saline to get a suspension of fresh
               ties and parasitism by myxosporeans with special                    spores. Light microscopic observations were made both
               reference to parasitized regions of the brain.                      on fresh spores and those stained with Giemsa.
                                                                                     For the scanning electron microscopy, spore-sus-
                                                                                   pensions were fixed in Karnovsky's fixative for 2
               Materials and Methods                                               hours at room temperature. They were then dehy-
                                                                                   drated in a graded ethanol series, treated with
               Seventeen yellowtail (age I+), including        7 deformed          isoamyl acetate three times, each for 15 minutes, pro-
               ones, were collected from a fish farm in        Mie Prefec-         cessed in a critical point dryer, sputter-coated with

                                                                                                                                              113







            114       NOAA Technical Report NMFS I I I



















                                                       Pw""
















                             IIN#



                                                A*



                                                                tow                                                          Figure I
                                                                                                                   Scoliosis found in yellowtail
                                                                                                 W                 Se7iola quinqueradiata (dor-
                                                                                                                   sal view).
                                                                                                  4'
                                                                                                                             Figure 2
                                                                                                                   A cyst of Myxobolus bu7i found
                                                                                                                   n the brain of yellowtail
                                                                                                                   (Seriold quinqueradiata) Gi-
                                                                                                                   entsa stain (X300).




            gold, and examined with the JEOL Uapan Electron-                     curvatures consisting of a smooth S-like bend in the
            ics Optical Limited) T220A scanning electron                         lateral plane, scoliosis (Fig. 1).
            microscope.                                                             In the seven deformed yellowtail, myxosporean
                                                                                 cysts were found in one or more of the following
                                                                                 regions: beneath the meninges of the olfactory bulb
            Results                                                              and lobe, surface of optic lobe, cerebellum, medulla
                                                                                 oblongata, inferior lobe, the cavity of optic tecturn,
            Cultured Yellowtail                                                  and the fourth venticle. In particular, the cysts were
                                                                                 found in the fourth ventricle of the brain in all de-
            The deformities were externally apparent as curva-                   formed fish examined (Table 1). In the normal fish,
            tures from the trunk to caudal region. Radiography                   on the other hand, no cysts were found in the fourth
            of the deformed specimens revealed extreme skeletal                  ventricle of three fish, even though seven fish had






                                                          Maeno and Sorimachi: Skeletal Abi@ormalities of Fishes Caused by Myxosporea                115




                                                                                        V





                                                                            V11




                                                                           III
                                                                                   IV(







                                                                                 Table I
                    Distribution of cysts in the brains of deformed (n--7) and apparently normal (n=10) yellowtail Seriola quinqueradiata.
                    (I=olfactory bulb; Il=olfactory lobe; III=optic lobe; IV=the cavity of optic tectum; V=cerebellum; VI=f6urth ventricle;
                    VII=inferior lobe.)


                                                       I             II            III            W              V             VI            VII


                    Deformed fish                    5/7            5/7            2/7            3/7           1/7            7/7           2/7
                    Normal fish
                      Infected                        2/5           3/5            2/5            1/5           0/5            0/5           1/5
                      Non-infected                   0/5            0/5            0/5            0/5           0/5            0/5           0/5





                cysts in some other region of the brain. No cysts were                    In deformed Japanese bluefish, cysts were always
                observed in regions other than the brain.                               found in the fourth ventricle and less frequently
                   The cysts range from 0.5 to 3 mm in diameter and                     found in the cavity of the optic tectum. In some cases
                were enveloped by a collagenous capsule with                            cysts were found attached to the surface of the olfac-
                fibroblasts (Fig. 2). Most cysts were filled with nearly                tory bulb and olfactory lobe and in the cavity of the
                mature or mature myxosporean spores. Morphologi-                        optic tecturn. No cysts were found in tissue other
                cal observations showed spores to be broadly                            than the brain. Both light and scanning electron mi-
                ellipsoidal and symmetrical in shape and to possess a                   croscopy revealed that the spores from these fish
                prominent sutural ridge in the front view. In the side                I were morphologically similar to those obtained from
                view they were lenticular and symmetrical and con-                      deformed yellowtail. The spores were broadly ellip-
                sisted of two shell valves. The polar capsules were                     soidal in frontal view and broadly lenticular in side
                pyriform and nearly equal in size, and a small                          view; the shell valve of the spore had a sutural ridge
                intercapsular appendix was clearly seen. The length,                    with fold; each spore had two polar capsules nearly
                width, and thickness of spores were 10.5, 9.0, and 6.3                  equal in shape and size; and an intercapsular appen-
                gm, respectively. From these morphological charac-                      dix was present (Figs. 4 and 5). The average length of
                teristics they were identified as Myxobolus buyi Egusa,                 fresh spores was 10.4 gm, width 9.1 9m, and their
                1985.                                                                   thickness 6.2 gm.


                Japanese Bluefish                                                       Mullet

                Radiographic observations demonstrated that the                         The four deformed mullet showed spinal curvature
                abnormality in the four Japanese bluefish was                           and lordo-scoliosis in radiography (Figs. 6 and 7).
                dorso-ventral deformity of the vertebral column                         Histological        examination          revealed         many
                and flexure of the neural spine and hernal spine                        myxosporean cysts (in the surface of the gut and in
                (Fig. 3).                                                               various visceral organs such as the liver, kidney,







            1.16     NOAA Technical Report NNM I I I








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                              W."




                                                   1. VW
                                                     X-
                                                                                 Q':






                                                           Njl@'


                                                                                                               Figure 3
            V                                                                              Soft radiograph of lordosis in a Japanese blue-
                                                                                           fish (Scombrops boops) (side view).

                                                                                                               Fig ure 4
                                                                                           Fresh spore of Myxobolus sp. obtained from the
                                                                                           brain of Japanese bluefish. (a) frontal view (ar-
                                                                                           row indicates intercapsular appendix); (b) side
                                                                                           view (bar=10 lim). Phase contrast microscopy.

                                                                                                                Figure 5
                                                                                           Scanning electron micrographs of spores of
                                                                                           Myxobolus sp.obtained from the brain of Japa-
                                                                                           nese bluefish. (a) frontal view; (b) side view.



            spleen, pancreas, and brain of the deformed mullet.                  tural plane, and lack of an intercapsular appendix
            As in yellowtail and Japanese bluefish, cysts we@e                   (Fig. 9), these myxosporean spores were considered
            found in various regions of the brain, such as the                   to belong to the genus Myxobolus (Lom and Noble
            fourth ventricle, the cavity of the optic tecturn, the               1984).
            surface of the olfactory lobe and bulb, and the optic
            lobe. Cysts were observed in the fourth ventricle of
            all four fish examined (Fig. 8).The average length of                Discussion
            fresh spores was 11.5 gm, their width was 9.8 gm, and
            thickness 6.7 gm. From the morphological character-                  The morphological characteristics of the spores
                     ;eool@@
                     ,e                                          "@7@




            istics of the spores, such as their rounded frontal                  clearly indicate that the myxosporean found in de-
            view, lenticular side view, two smooth shell valves, two             formed yellowtail of the present study is M. bwri. The
            teardrop-shaped polar capsules situated on the su-                   light and scanning electron microscopic examina-






                                                       Maeno and Sorimachi: Skeletal Abnormalities of Fishes Caused by Myxosporea          117














































                                                                                                                    Figure 6
                                                                                                 Soft radiograph of lordosis in a mullet (Mugil
                                                                                                 cephalus) (side view).

                                                                                                                    Figure 7
                                                                                                 Soft radiograph of scoliosis in a mullet (Mugil
                                                                                                 cephalus) (dorsal view).

                                                                                                                    Figure 8
                                                                                                 Diagrammatic view of the distributions of the
                                                   T
                                                 & r
                                                  4'r                                            cysts in the brain of mullet Mugil cephalus
                                                                                                 (*=loci of the cyst).

                                                                                                                    Figure 9
                L             T-WW'L









                                                                                                 Fresh spore of Myxobolus spinacurvatura ob-
                                                                                                 tained from the brain of mullet. (a) frontal
                                                                                                 view; (b) side view (bar=10 gm).







              118         NOAA Technical Report NNOS 111


              tions also demonstrated a complete similarity in the                          These studies, as well as the present study, indicate
              morphology between the spores from the deformed                               some correlation between skeletal abnormalities and
              Japanese bluefish and those from deformed yel-                                myxosporean parasitism in the brains of fish. In addi-
              lowtail. Thus, the spores parasitic in the brain of the                       tion, the present study strongly suggests that skeletal
              deformed Japanese bluefish are considered to be                               abnormalities occur when cysts infect particular re-
              those of M. bu7i.                                                             gions of the brain such as the fourth ventricle. Thus,
                 To date, more than 400 species of Myxobolus have                           it is quite possible that myxosporean infection in this
              been described. The morphological characteristics of                          region of the brain mechanically affects central ner-
              Myxobolus from the deformed mullet differ from                                vous system function to produce skeletal
              those of most species described. Only three Myxobolus                         abnormalities in fish.
              species, M. achmerovi Shulman, 1966, M. bu?i, and M.
              spinacurvatura Maeno et al., 1990, have morphologi-
              cal similarities to those found in deformed mullet.                           Citations
              However, M. achmerovi, which has been found from
              the gill, fin, and mesentery of common carp                                   Couch, J.A., J.T. Winstead, L.R. Goodman.
              (Cyprinus carpio) and mullet (Mugil cephalus)                                      1977. Kepone-induced scoliosis and its histological
              (Shulman 1966) is different from the present                                          cosequences in fish. Science 197:585-w587.
                                                                                            HalverjE., and W.E. Shanks.
              Mymbolus in that the former spore has an ellipsoidal                               1960. Nutrition of salmonid fishes VIII. Indispensable
              frontal view, and a distinct intercapsular appendix.                                  aminoacids for sockeye salmon. J. Nutrition 72:340-346.
              M. buri also differs from the present Myxobolus in                            Halliday, M.M.
              mullet because the spore of M. buyi has a broadly                                  1976. The biology of Myxosoma cerebralis: the causative organ-
              ellipsoidal frontal view, distinct folds around the                                   ism of whirling disease of salmonids. J. Fish Biol. 9:339-
                                                                                                    357.
              edge, an intercapsular appendix, and a distinct polar                         Holcombe, G.W., D.A. Benoit, E.N. Leonard, andj.M. McKim.
              filament inside the polar capsule. On the other hand,                              1976. Long-term effects of lead exposure on three genera-
              the spores from the deformed mullet were quite simi-                                  tions of brook trout (Salvelinus fontinalis). J. Fish. Res.
              lar to those of M. spinacurvatura in both                                             Board Can. 33:1731-1741.
              morphological characteristics and in spore dimen-                             Hoshina, T.
                                                                                                 1952. Notes on some myxosporidian parasites on fish of
              sions. Thus, the Myxobolus found in deformed mullet                                   Japan. J. Tokyo Univ. Fish. 39:69-89.
              in the present study is considered to be M.                                   Kaige, N., T. Miyazaki, and S. Kubota.
              spinacurvatura.                                                                    1984. The pathogen and the histopathology of vertebral de-
                 In the present study the myxosporean cysts were                                    formity in cultured yellowtail. Fish Pathol. 19:173-179. (In
              invariably observed in the fourth ventricle of the                                    Japanese; English abstr.)
                                                                                            .Langdon,J.S.
              brain in all deformed fishes examined. Myxosporean                                 1987. Spinal curvatures and encephalotropic myxosporean,
              parasitism in the central nervous system of fish have                                 Triangula percae sp. nov. (Myxozoa: Ortholineidae), enzootic
              been studied in the bullbead Cottus gobio (Lom et al.                                 in redfin perch, Percafluviatilis L., in Australia. J. Fish Dis.
              1989), the redfin perch Perca fluviatilis (Langdon                                    10:425-434.
              1987), the yellowtail Sefiola quinqueradiata (Sakaguchi                       Lom, J., and E.R. Noble.
                                                                                                 1984. Revised classification of the class Myxosporea Butschi,
              et al. 1987), the fathead minnow Pintephales promelas                                 1881. Folia Parasitologia (Praha). al:193-205.
              (Mitchell et al. 1985), and the Japanese river goby                           Lomj, S.W. Feist, 1. Dykova, and T. Kerp.
              Acanthogobius flavimanus (Hoshina 1952). However,                                  1989. Brain myxoboliasis of bullhead, Coitus gobio L., due to
              even in a heavily myxosporean-infected bullhead, mo-                                  Myxobolus firoved sp. nov.: light and electron microscope
              tor or sensory disturbances were not observed. Also,                                  observation. J. Fish Dis. 12:15-27.
                                                                                            Mitchell, L.G., C.L. Seymour, andj.M. Gamble.
              in myxosporean-infected fathead minnow and Japa-                                   1985. Light and electron microscopy of Myxobolus
              nese river goby, no clear external signs of deformity                                 hendiicksoni sp. nov. (Myxozoa: Myxobolidae) infecting the
              were found. On the other hand, skeletal abnormali-                                    brain of the fathead minnow, Pimephales promelas
              ties were observed in the myxosporean-infected                                        Rafinesque. J. Fish Dis. 8:75-89.
              redfin perch and yellowtail. In the redfin perch, cysts                       Sakaguchi, S., T. Hara, T. Matsusato, I Shibahara, Y Yamagata, H.
                                                                                               Kawai, Y. Maeno.
              were located in the regions of the mesencephalon,                                  1987. Scoliosis of cultured yellowtail caused by parasitic
              diencephalon, third ventricle, and medulla oblon-                                     Myxobolus bu7i. Bull. Natf. Res. Inst. Aquaculture. 12:79-86.
              gata, while in the yellowtail, cysts were found in the                                (Injapanese; English abstr.)
              cavity of the optic tectum, the surface of the optic                          Shulman, S.S.
              and olfactory lobes, and in the fourth ventricle.                                  1966 Myxosporidian fauna of the USSR. Nauka, Moscow and
                                                                                                    Leningrad, 251 p.






                              Presence of Oncogenes in Fish Tissues and in Fish CeR Unes



                                              E. READ-CONNOLE, C. A. SMITH, and F. A HETRICK*
                                                                   Department of Microbiology
                                                                     University of Maryland
                                                                    College Park, MO 20742




                                                                        ABSTRACr


                                    Work during the past decade has shown that avian and mammalian proto-oncogenes
                                  are centrally involved in cell transformation in vitro and in the formation of tumors in
                                  vivo. The fish systems in which oncogenes have been described are reviewed as are the
                                  general methodologies used to detect oncogenes and their gene products. Also discussed
                                  is preliminary work on the development of a test system that measures oncogene activa-
                                  tion in fish cell lines in order to evaluate carcinogenic chemicals in the environment.
                                  Sequences related to the rask, raP, v-raf, v-erb-B, c-src, c-myc, c-abl, and c-fos proto-
                                  oncogenes, and to the p53 suppressor gene, were detected by Southern, Northern, and
                                  Western blots. Taken together, the above findings indicate that proto-oncogenes are well
                                  conserved evolutionarily in vertebrates and may be responsible for development of the
                                  transformed phenotype in fish.


                Introduction                                                      1990). However, wild type ras protein can be trans-
                                                                                  formed in a variety of carcinogen-induced anim"al
                Evidence has been accumulating in recent years that               tumor model systems. For example, in rats almost
                in mammals, particularly humans, tumor develop-                   90% of mammary carcinomas induced by a single
                me nt and progression are correlated with changes in              dose of nitrosomethylurea possess a c-H-ras-I
                the structure or expression of cellular genes. Yet in a           oncogene, which is the activated form of the *c-H-ras
                number of higher vertebrates, oncogenic retroviruses              gene having a G to A transition at codon 12
                have long been known to be causally involved in the               (Barbacid 1986, 1987). In humans, highly amplified
                initial appearance and subsequent growth of a variety             N-ras DNA sequences were found in small cell lung
                of naturally arising tumors (Bishop 1985; Klein and               cancer, and this amplification seems to correlate with
                Klein 1985). These two statements are not contradic-              tumor progression and prognosis Uolinson et al.
                tory. The retroviral oncogenes (v-oncs) responsible               1987). Changes in the structure (mutation) of one or
                for the transformation event are now recognized to                more oncogenes, their amplification, and the level of
                have arisen from the capture and processing of whole              expression of their mRNA, are all potential indicators
                or portions of essential cellular genes termed proto-             of tumorigenic initiation and progression.
                oncogenes or c-oncs (Bishop 1983; Stehelin et al.                   In view of the widespread occurrence of neoplasia
                1976). It is now considered important to understand               in freshwater and saltwater fish species, and because
                the extent of evolutionary conservation of these cel-             fish are becoming recognized as convenient model
                lular genes in order to fully characterize their normal           systems for the study of natural and environmentally
                function and delineate their involvement in the                   induced diseases (Powers 1989), the search for proto-
                tumorigenic conversion of normal cells either di-                 oncogene-like sequences in teleosts is warranted.
                rectly or through retrovirus-induced transformation.                Experimental evidence indicates that some fish
                  The,family of ras proto-oncogenes, rash (Harvey),               species, when compared with rodents, are less sensi-
                ras' (Kirsten), and N-ras are highly conserved from               tive to the toxic effects of chemical agents and more
                mammals to yeast. The 21 kilo dalton ras protein is               susceptible to their carcinogenic effects. Because of
                expressed in a wide variety of nontransformed cells,              their aquatic habitat, fish are fully exposed to chemi-
                suggesting a normal cellular function. Xenopus laevis             cals in the water at the gill, eye, skin, and gut levels,
                oocytes espress the ras protein throughout oogenesis
                and embryonic development (Baum and Beberriitz                    *Send correspondence to this author.







          120       NOAA Technical Report N?M I I I


          and therefore may be good indicators of carcinogens             are detected. Comparisons of the number and size of
          in the environment. Thus, it is worth investigating             oncogene homologous fragments from the Southern
          oncogenes, and their expression in tumorigenesis in             blots with the more quantifiable data from the dot
          fish, especially when they are induced by chemicals.            blot experiments should reveal whether any of the
          Such studies have now begun. Here we describe the               fish genes detected have been rearranged or ampli-
          general methodology used in detecting oncogenes                 fied in the tumor tissues under study.
          and their expression and review those systems where
          oncogenes have been found in fish tissues and in
          continuously cultivated fish cell lines.                        Detection of Oncogene Expression

                                                                          For any gene to exert an effect on cells and tissues,
          Methodology                                                     mRNA must be transcribed and eventually translated
                                                                          into protein. One can therefore screen RNA isolated
                                                                          from the various sources for the presence of tran-
          Oncogene Detection                                              scription products homologous to those of
                                                                          mammalian oncogenes. Enhanced expression of vari-
          For the detection of oncogenes at the DNA level, it is          ous oncogenes following the amplification of DNA
          important to isolate high molecular weight genomic              may contribute to malignant progression as is sug-
          DNA with a minimal size of 100-200 kb. Standardized             gested by the human c-N-myc gene involvement in the
          procedures are available for the rapid isolation of             progression of human neuroblastomas (Schwab et al.
          good quality DNA from either cell cultures or directly          1984), or it may involve the loss of a gene and its
          from tissues excised from organisms. One procedure              expression as has been observed in the case of the
          (Smith et al. 1988) employs a gentle sodium dodecyl             human retinoblastoma susceptibility gene (Friend et
          sulfate (SDS) and proteinase-K lysis step followed by           al. 1986; Lee et al. 1987).
          phenol extraction, ethanol precipitation, and spool-               Described briefly, cells and tissues being examined
          ing of DNA (contaminating RNA can be removed by                 for oncogene transcription products are first pro-
          RNAse treatment), Tissue samples are processed by               cessed by the guanidinium thiocyanate/hot phenol
          this method by including liquid nitrogen freezing               procedure to extract the total cellular RNA. The total
          and tissue crushing steps prior to the lysis. Methods           cellular RNA obtained is subjected to oligo-dT cellu-
          for isolating genomic DNA from fish cell lines are              lose affinity chromatography to purify or enrich the
          described in Smith et al. (1988) and Read-Connole et            poly A+ RNA fraction (putative mRNA). The poly A+
          al. (1990).                                                     RNA species are then separated by size by denaturing
             Samples of DNA from the various sources are then             (formaldehyde) agarose gel electrophoresis. After
          digested to completion with one or more various re-             transfer of the RNA to nitrocellulose or ilylon mem-
          striction endonucleases (e.g., EcoRl, Hind III and Bam          branes, mature mRNA species with homology to
          HI), and the resulting DNA fragments are resolved               oncogene probes can be detected by northern blot-
          by size by agarose gel electrophoresis. After transfer          ting procedures. In addition, RNA dot blot
          of the DNA fragments to nitrocellulose or nylon                 experiments can also be performed to quantify the
          membrane supports, the DNA samples can then be                  levels of mature mRNA hybridizing with the
          screened by Southern blotting for the presence of               oncogene probes (Smith et al. 1987; Louis et al.
          sequences homologous to nick-translated labelled                1988). The mRNA studies give valuable information
          DNA probes specific for mammalian and viral                     on whether any of the specific oncogenes detected in
          oncogenes. In addition, serial dilutions of samples of          the various tumor tissue   .preparations are of different
          restriction-endonuclease-treated DNA can be directly            sizes (suggesting either truncation of the gene or re-
          spotted onto nitrocellulose or nylon membranes.                 arrangements) and whether they are overexpressed
          These dot blots can then be probed in parallel with             or aberrantly expressed when compared with the nor-
          the Southern blots to aid in the quantification of              mal counterparts.
          hybridizable DNA in each sample, and to estimate
          any gene amplification that may occur in the tumor
          tissues.                                                        Detection of Oncogene Products
             Comparisons between normal and tumor tissues
          can be made from the banding patterns obtained in               The third approach to monitor oncogenes in cells is
          Southern blots probed by the different oncogenes. In            to assay for their gene products (e.g., tyrosine kinase
          this way one can obtain some idea on the similarity of          for c-src expression, epidermal growth factor receptor
          the gross structure of the various fish oncogenes that          for c-erb-B expression). Monoclonal antibodies






                                                                       Read-Connole et al.: Oncogenes in Fish Tissues and Cell Lines       121

                 (Mabs) have been raised against synthetic peptides               gene. It was also reported (van Beneden et al. 1988)
                 spanning the active site regions of the src and ras              that the blots, when rehybridized to other viral
                 gene families and are available commercially.                    oncogenes, gave no indication of gross amplification
                 Nonidet P-40 (non-ionic detergent) lysates of cells              or rearrangement. Clearly, cloning and sequencing
                 are cleared by high speed centrifugation and the pro-            of the entire gene is necessary to show putative dif-
                 teins imm,unoprecitated with Mab mixtures.                       ferences.
                 Following electrophoresis on 10% SDS-polyacrylam-                   Similarly c-ras-related sequences were cloned from
                 ide gels, the bands are transferred to nitrocellulose            the genomic libraries of the goldfish (Cerassius
                 membranes, and immunoblotted with an appropriate                 auratus) by Nemoto et al. (1986). Comparison of the
                 antibody. These western hybridizations, in which                 nucleotide sequences of one of these clones with
                 Mabs are used against oncogene products, can reveal              those in mammalian c-ras genes showed extensive ho-
                 the size and level of expression of these proteins.              mology to the- gene coding for the mammalian p2l.
                                                                                  protein (96% homology to the c-ras' protein). Subse-
                                                                                  quent work (Nemoto et al. 1987) involved
                 Oncogenes Detected in Fish Tissues                               sequencing and comparison of the first exon and its
                                                                                  flanking regions in the c-ras-related genes from nor-
                 A certain population of platyfish (Xiphophorus                   mal goldfish liver tissues with those from goldfish
                 maculatus) carry special genes (Tu or tumor genes)               erythrophoroma cells cultured in vitro. No. differ-
                 for macrome Ian oph ores. When they are inter-                   ences were apparent in the first 245 nucleotides
                 specifically hybridized with swordtails (X. hellmi), the         which covered the first exonic region and whose
                 F, offspring carrying these genes develop a                      length was identical to the first exonic region in
                 preneoplastic state. When the F, offspring are back-             mammalian c-ras genes. Enhanced expression of c-ras
                 crossed with swordtails, a certain percentage of the             gene was seen in the erythrophoroma cells.
                 backcrossed offspring develop a. heritable form of                 The high incidence of hepatomas present in winter
                 melanoma with a characteristic inheritance pattern               flounder Pseudopleuronectes ame7icanus in Boston har-
                 (Ozato and Wakamatsu 1983; Anders et al. 1984). Ex-              bor has been associated with the high levels of
                 pansion of these classic and elegant genetic studies             polycyclic aromatic hydrocarbons (PAHs) found in
                 involves the use of modern biotechnology techniques              the harbor's sediment. Genomic DNA from the liver
                 to define the molecular basis of the melanoma for-               tumors produced foci in the NIH-3T3 transfection
                 mation. For example, elevated levels of cellular src             assay and a c-rask oncogene was identified in a
                 mRNA and phosphoprotein (pp 60) kinase activity                  transformant derived from one of the tumors
                 are detected in melanomas or tumors induced by car-              (McMahon et al. 1988). Sequencing of c-ras@ se-
                 cinogens in Xiphophorus (Schard et al. 1985) and                 quences from the tumor and from the transformants
                 more recent research indicates that the signal to start          indicated the oncogene to be of fl6under origin. Se-
                 the events leading to melanoma development may                   quencing of the tumor cell DNA amplified by
                 come from the platyfish erbB region (Zechel et al.               polymerase chain reaction (McMahon et al. 1987)
                 1988). They have named this gene Xmrk. An addi-                  showed GC to AT or GC to TA conversions in               ,the
                 tional copy of the Xmrk gene is found linked to either           12th codon of this gene (McMahon et al. 1990). Such
                 sex chromosome of the platyfish when hybridized to               mutations in the 12th codon can activate c-ras proto-
                 a probe specific for this region (Schartl 1990). The             oncogenes to oncogenic forms (Barbacid 1987).
                 cellular myc (c-myc) gene has been cloned from rain-             DNA samples from livers taken from fish at a less
                 bow trout Oncorhynchus mykiss and sequenced (van                 polluted site did not transform NIH-3T3 cells, and
                 Beneden et al. 1986). The results of this work were              only wild-type sequences (GGT) were seen at the 12
                 used for evolutionary comparison with other verte-               codon of c-rask.
                 brate c-myc genes. Using the rainbow trout c-?nyc                  A cellular 53 kilo dalton nuclear phosphoprotein,
                 clone as a -probe, DNA was examined from various                 denoted p53, was discovered more than a decade ago
                 fish tumors, including hepatocellular carcinomas in-             in SV40 virus transformed mouse cell lines (Chang et
                 duced in the medaka Oryzias latipes by di-                       al. 1979). Because p53 was found complexed to the
                 methylnitrosamine. Compared with DNA isolated                    transformation antigen (T antigen) of SV-40 virus,
                 from normal tissues, there was no apparent increase              and because the p53 gene was thought by many inves-
                 in the intensity of hybridizing bands or differences in          tigators to function as a dominantly acting oncogene,
                 restriction patterns noted in Southern blots (van                it has been intensively investigated during the past
                 Beneden et al. 1988). However such experiments                   decade. Current work on mutations in the p53 gene
                 would not detect mutations, which could be detected              indicates that the wild-type gene product actually
                 only by cloning and sequencing the entire c-myc                  functions like a tumor suppressor gene (Finlay et al.







              122          NOAA Technical Report NNEFS I I I



                                                                                            Table I
                                                                       Oncogenes Detected in Fish Tissues.


                                                                                                          Transforms
                  Fish Species                          Tissue Tested                   Oncogene          NIH-3T3 Cells                     Reference

                  Platyfish - swordtail                 Hereditary melanoma             Xmrk (tu)                                           Schard et al. 1985
                  hybrid (Xiphophorus sp.)                                                                                                  Wittbrodt et al. 1989

                                                                                        erb-B                                               Zechel et al. 1988

                  Rainbow Trout                         Normal liver                    c MYC                                               van Beneden et al. 1986
                  (Oncorhyncus mykiss)

                  Goldfish                              Normal liver                    ras                                                 Nemoto et al. 1986
                  (Carassius auratus)

                  Winter flounder                       Liver tumor                     ras'              +                                 McMahon et al. 1988, 1990
                  (Pseudopleuronectes
                  americanus)

                  Tomcod                                Liver tumor                     raSk              +                                 Wirgin et al. 1989
                  (Microgadus tomcod)

                  Northern pike                         External                        ?                 +                                 van Beneden et al. 1990
                  (Esox lucius)                         lymphomas





              1989). Mutations in the p53 gene are frequently de-                                  Southern blots with v-erb-b, v-src, and v-ras' revealed a
              tected in diverse human tumor types (Nigro et al.                                    striking similarity in the banding patterns of homolo-
              1989), and currently it is believed that p53 gene mu-                                gous sequences between the rainbow trout 0. mykiss
              tations play an important role in the development of                                 and the chinook salmon 0. tshawytscha cell line. This
              many common human cell malignancies. The hy-                                         suggests that, as expected, these genes in the rainbow
              pothesis is that the mutated p53, which develops                                     trout and chinook salmon are more closely linked in
              during the process of tumorigenesis, binds to the                                    an evolutionary sense than they are to the homolo-
              wild-type p53 gene product, creating an inactive com-                                gous genes in the other fish species studied. A very
              plex. Further loss'of growth control can occur when                                  high degree of homology has been shown in the pro-
              the wild-type allele is deleted. Thus, p53, like the                                 tamine genes of chum salmon (0. keta) and rainbow
              retinoblastoma gene, is under intensive study by                                     trout (Moir and Dixon 1988).
              many investigators as a potential tumor suppressor                                      Many of the monoclonal antibodies raised against
              gene (Sager 1989). The tumor suppressor genes are                                    mammalian viral or cellular oncogene protein prod-
              wild-type alleles of genes that play regulatory roles in                             ucts recognized fish proteins in Western blots.
              cell proliferation and differentiation, and it is their                              Immunoprecipitation followed by Western blotting is
              loss or inactivation that is oncogenic. The p53 gene is                              considered a highly sensitive and discriminatory pro-
              highly conserved in vertebrates including fish (Smith                                cedure, especially when monoclonal antibodies are
              et al. 1988).                                                                        used that recognize discrete and specific epitopes on
                                                                                                   the proteins of interest. The apparent recognition of
                                                                                                   fish proteins by this technique shows that these re-
              -Oncogenes Retected- iry Fish-Cell-Lines-                                            gions of the oncoproteins may be well -conserved
                                                                                                   from viruses to mammals and fish.
              A number of oncogenes have been described in some                                       Both the c-ras' and c-rask gene products were de-
              of the more commonly used fish cell lines (Read-                                     tected in all the fish cells, showing that these proteins
              Connole et al. 1990). These have been detected by                                    are conserved in vertebrates and may be essential. It
              Southern, Northern, and Western blots (Table 2).                                     is possible, however, that the c-ras' and c-ras' antibod-
                  The presence of many bands in Southern blots sug-                                ies recognized the same fish proteins, but the
              gests that, as in mammals, some of the fish                                          monoclonal antibodies used were raised against dif-
              proto-oncogenes may exist as families of homologous                                  ferent pepticles representing regions of the two viral
              sequences that share common functions. Probing                                       proteins and did not cross-react.






                                                                                                         Read-Connole et al.: Oncogenes in Fish Tissues and Cell Lines                                         123



                                                                                                                 Table 2
                                                                                        Oncogenes Detected in Fish Cell Lines.


                             Detection Method                                                   Oncogene(s) Detected                                                              Cell line (s)

                             Southern blots                                                     rash, raf,                                                        BB, RTG-2,
                                Using mammalian                                                 erb-B, src                                                        EPC, CHSE-214
                                viral onc probes                                                p53                                                               EPC

                             Western blots                                                                                                                        BB, EPC,
                                Using Mabs against                                              c-myc, c-ab4 c-fos,                                               CHSE-214
                                human and viral                                                 v-ras, p53 (murine), v-ras,
                                oncogene products                                               v-raf v-sre
                             Northern blots                                                     rash                                                              High expression in BB          and EPC
                                Using mammalian                                                                                                                   Moderate expression in RTG
                                viral onc probes                                                                                                                  Low in CHSE-214

                                                                                                abl                                                               High expression in CHSE-214
                                                                                                                                                                  Moderate in RTG

                                                                                                src, abl, myc,   rask                                             BB, EPC,
                                                                                                                                                                  RTG-2, CHSE-214

                                                                                                p53                                                               EPC, CHSE-214

                             *BB = Brown bullhead catfish line (Wolf and Quimby 1969); RTG-2                                       rainbow trout gonad           (Wolf and Quimby 1962); EPC
                             epithelioma papulosurn cyprini (Fijan et al. 1983); and CHSE-214                          chinook salmon embryo (Fryer et al. 1965).



                         Significance                                                                                      ing to establish tissue culture cell lines from these
                                                                                                                           tumors with the intent of comparing the genomic
                         The mechanisms by which normal cells acquire the                                                  DNA of the tumor cells to that of the normal skin
                         malignant phenotype form the central focus of can-                                                tissue and the brown bullhead (BB) cell line to deter-
                         cer research. Transforming genes have been detected                                               mine if any of the potential fish oncogenes are
                         in a variety of tumors, including human tumors, and                                               altered or expressed differently in the tumor cells. If
                         in cell lines derived from tumors by their ability to                                             mutations identified in specific fish oncogenes can be
                         tranform NIH-3T3 mouse cells in culture. Genetic al-                                              shown to result from chemically-induced DNA dam-
                         ternations also accompany tumorigenesis in feral                                                  age, then such mutations could serve as indicators
                         populations of aquatic organisms exposed to environ-                                              for environmental disease.
                         mental carcinogens. Any unambigious signal
                         regarding oncogene or tumor-repressor-gene expres-
                         sion that correlates with tumor formation could serve                                             Citations
                         as the basis for a test system for evaluating carcino-
                         genic chemicals in the environment. Changes in the                                                Anders, F., M. Schard, A. Barkenow, and A. Anders.
                         structure (mutation) of one or more oncogenes,                                                         1984. Xiphophorus as an in vivo model for studies on normal
                         their amplification, or the level of expression of their                                                  and defective control of oncogenes. Adv. Cancer Res. 42:
                                                                                                                                   191-275.
                         mRNA are all potential indicators of tumorigenic ini-                                             -Barbacid, M.
                         tiation and progression.                                                                               1986. Involvement of ras oncogenes in the initiation of car-
                             The number of genes homologous to either the                                                          cinogen-induced tumors. Int. Symp. Princess Takamatsu
                         viral oncogenes or the mammalian cell counterparts                                                        Cancer Research Fund. 17:43-53.
                         found in fish genomes suggests that these genes are                                                    1987. Ras genes. Annu. Rev. Biochem. 56:779-827.
                                                                                                                           Baum, EX, and G.A. Berbernitz.
                         highly conserved evolutionarily, and therefore their                                                   1990. K-ras oncogene expression in Xenopus laevis. Oncogene
                         role in both normal cells and those cells in various                                                      5:763-767.
                         stages of tumorigenic transformation may be com-                                                  Bishop,J.M.
                         mon to all vertebrates. The brown bullhead Ictalurus                                                   1983. Cellular oncogenes and retroviruses. Annu. Rev.
                         nebulosus is of particular interest to us because of the                                                  Biochem. 52:301-354.
                                                                                                                                1985. Viral oncogenes. Cell 42:23-38.
                         high prevalenceof tumors that occur in them during                                                Chang, C., D.T. Simmons, M.A. Martin, and P.T. Mora.
                         the late spring and summer in certain areas of the                                                     1979. Identification and characterization of new antigens
                         Chesapeake Bay (unpubl. data). We are now attempt-                                                        from SV,-transformed mouse cells. J. Virol. 31:463-471.







             124          NOAA Technical Report NWS I I I


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                S. Chilmonezykj Vautherot, and P. de Kinkelin.                                     1990. Nucleotide sequence homologous to mammalian
                  1983. Some properties of the epithelioma papulosum cyprini                          proto-oncogenes and their expression in fish cell lines. J.
                     (EPC) cell line from carp. Annales Virologie (Institut                           Aquat. Animal Health 2:77-84.
                     Pasteur) 134:207-220.                                                    Sager, R.
             Finlay, C.A., P.W. Hinds, and Aj. Levine.                                             1989. Tumor suppressor genes: the puzzle                   and the
                  1989. The p53 Proto-oncogene can act as a suppressor of                             promise. Science 246:1406-1412.
                     transformation. Cell 57:1083-1090.                                       Schard, M.
             Friend, S.H., R. Bernards, S. Roge1j, R.E. Weinberg, J.M. Rapaport,                   1990. Homology of melanoma-inducing loci in Xiphophorus.
                D.M. Albert, and T.P. Drya.                                                           Genetics 126:1083-1091.
                  1986. A human DNA segment with properties of the gene                       Schard, M., C.R. Schmidt, A. Anders, and A. Barkenow.
                     that predisposes to retinoblastoma and osteosar-                              1985. Elevated expression of the cellular sre gene in tumors
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             Fryer, J.L., A. Yusha, and K-S. Pilcher.                                                 207.
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                  1987. Myc family oncogene amplification in tumor cell lines                         sequent to amplification of DNA may contribute to malig-
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                     tionship to clinical status and course. J. Clin. Invest.                         81:4940-4944.
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             Klein, G. and E. Klein.                                                               1989. Amplified DNA in cells of genetic melanoma of
                  1985. Evolution of tumors         and the impact of molecular                       Xiphophorus. Oncogene 4:139-144.
                     biology. Nature 315:190-195.                                             Smith, C.A., M.J. Louis, and F.M. Hetrick.
             Lee, W.H., R. Bookstein, F. Hong, LJ. Young, JX Shew, LY.M.P.                         1988. A sequence homologous to the mammalian p53
                Lee.                                                                                  oncogene in fish cell lines. J. Fish. Dis. 11:525 -530.
                  1987. Human retinoblastorna susceptibility gene: cloning,                   Smith, C.A.D., Dj. Winterbourne, VW. McFarland, and P.T. Mora.
                     identification, and sequence. Science 235:1394-1399.                          1987. Changes in Heparan Sulfate pattern but not in
             Louis, Mj., VW. McFarland, P. May, and P.T. Mora.                                        oncogene expression correlate with tumor growth in spon-
                  1988. The phosphoprotein p53 is down-regulated post-                                taneous transformation of cells. Oncogene Research
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                     brates. Biochimica. Biophysica Acta. 956:395-402.                        Stehelin, D., H.E. Varmus,J.M. Bishop, and P. Vogut.
             McMahon, G., E. Davis, and G.N. Wogan.                                                1976. DNA related to the transforming genes of avian sar-
                  1987. Characterization of c-Ki-ras oncogene alleles by direct                       coma virus is present in normal avian DNA. Nature
                     sequencing of enzymatically amplified DNA from carcino-                          260:170-173.
                     gen-induced tumors. Proc. Natl. Acad. Sci. (USA),                        van Beneden, R.J., D. R. Watson, T.T. Chen, J.A. Lautenberger, and
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             McMahon, G., Lj. Huber, JJ. Stegeman, and G.N. Wogan.                                 1986. Cellular myc (c-?nyc) in fish (rainbow trout): its rela-
                  1988. Identification of a c-Ki-ras oncogene in a neoplasm iso-                      tionship to other vertebrate myc genes and to the transform-
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                  1988. Characterization       of a protamine gene from chum                          neoplasms. Cancer Res. 50:5671s-5674s.
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                                        Streptococcal Infection in Cultured Yellowtail



                                                                     HIROSHISAKO

                                                             Nansei National Fisheries Research Institute
                                                                            Ohno, Saeki
                                                                     Hiroshima, 739@04, Japan




                                                                         ABSTRACT


                                    Streptococcal infection is widespread in yellowtail (Seriola quinqueradiata) cultured in
                                  southwestern Japan and results in extensive losses. It is urgent that control measures be
                                  established against this disease. This paper presents the etiology, prophylaxis, and treat-
                                  ment of streptococcal infections in cultured yellowtail.





                Introduction                                                       clearly distinguishable by their biochemical and
                                                                                   physiological characteristics (Table 1; Sako, unpubl.
                Yellowtail (Seriola quinqueradiata) culture developed              data). The species reported by Kusuda et al. is gener-
                rapidly in the 1960s, mainly in southwestern Japan.                ally called alpha-hemolytic Streptococcus sp. Almost all
                By 1988, the farming of yellowtail was being practiced             of the streptococci isolated from yellowtail were con-
                in 27 prefectures (Fig. 1), the total production was               firmed to be the alpha-hemolytic Streptococcus sp.
                about 166,000 metric tons, or 68% of the total pro-                (Kitao 1982). Recently, this species was transferred to
                duction of marine fish cultured in Japan (Fig. 2).                 genus Enterococcus (Riichi Kusuda, Kochi Univ., pers.
                The incidence of diseases in cultured yellowtail is                commun., April 1990). Another species isolated from
                gradually increasing as the culture of this fish be-               a yellowtail juvenile in 1976 by Minami et al. (1979)
                comes more popular. About 30 diseases have been                    displayed beta-hemolysis on sheep blood agar, so it
                identified so far. Figure 3 shows the loss of cultured             has been called beta-hemolytic Streptococcus sp. This
                yellowtail due to disease in 1988; the cost to fish                species is classified with pyogenic streptococci based
                farmers was about 11,300 million yen. Bacterial dis-               on its biochemical characteristics. Its biochemical
                eases are responsible for the majority of the losses,              characteristics also agreed well with those of S. iniae
                whereas those due to viruses and parasites are minor.              ATCC 29178 isolated from the Amazon freshwater
                               acterial diseases, streptococcosis was re-          dolphfin, Inia geof
                Among the b                                                                            'frensis (Pier and Madin 1976). The
                sponsible for the highest losses (64%), pasteurellosis             last species is a nonhemolytic Streptococcus reported
                ranking second'(21%). Thus, with yellowtail culture,               by Iida et al. (1986), which has biochemical charac-
                it is very important to control the incidence of strep-            teristics similar to those of S.agalactiae and has the
                tococcal infection.                                                Lancefield grouping antigen B, type lb.
                  The author describes here some aspects of strepto-                 A species that shows the same characteristics as al-
                coccal infection in cultured yellowtail with special               pha-hemolytic Streptococcus sp. was also isolated from
                emphasis on the etiology of the causative bacteria,                cultured yellowtail in the Republic of Korea (Park et
                prophylaxis, and methods used to treat the disease.                al. 1987). The nonhemolytic, group B, type Ib Strepto-
                                                                                   coccus was also isolated from several marine fishes in
                                                                                   the United States (Plumb et al. 1974).
                Causative Bacteria

                Streptococcal infection in cultured yellowtail was first           Host Range
                identified by Kusuda et al. in 1974 (Kusuda et al.
                1976). Since then, three species of fish pathogenic                Large numbers of Enterococcus sp. and S. iniae have
                Streptococcus have been reported. These bacteria are               been isolated from wild and cultured marine fishes as

                                                                                                                                             125







            126        NOAA Technical Report NPM I I I


                                                                                    their minced meats, are a major feed for yellowtail, it
                                                                                    is possible that they are the origin of streptococcal
                     Total Woduction                                                infection in cultured fish.
                   166,000 niet& tons                                               Pathogenicity

                                                                                    Eac h of the Streptococcus spp. and their strains ex-
                                                                                    hibit different degrees of virulence. Enterococcus sp.
                                                                                    produces both intra- and extracellular toxins
                                                                                    (Kimura and Kusuda 1979). In artificially induced in-
                                                                                    fections, even small amounts of Enterococcus sp. cells
                                           10                                       killed yellowtail (Taniguchi 1982a). However, S. iniae
                                                                                    killed yellowtail only occasionally although it fre-
                                                                                    quently caused vertebral deformity (Sako, unpubl.
                                                                                    data).
                                        0        10,000 - 40,000                      Streptococci is an indigenous bacteria in warm-
                                                  1,000 - 9,999                     blooded animals and grows rapidly at 30-35' C. S.
                                                          -       999               iniae isolated from yellowtail have been confirmed to
                                                         mtric tons                 be pathogenic to mice by intraperitoneal injection.
                                                                                    However, Enterococcus sp. and nonhemolytic group B,
                                                                                    type lb Streptococcus isolated from yellowtail were
                                        Figure I                                    nonpathogenic to mice (Sako, unpubl. data).
            Yellowtail production in Japan by prefecture, 1988. Source:
            Statistics and Information Department, Ministry of Agricul-
            ture I Forestry, and Fisheries, Japan.                                  Diagnosis

            well as in small quantities from cultured freshwater                    Major clinical signs caused by Enterococcus sp. are
            fishes in Japan (Table 2; Kusuda et al. 1976; Kusuda                    exophthalmos, petechiae, and ulceration on the in-
            et al. 1978; Minami 1979; Minami et al. 1979; Kitao et                  side of the opercula, and congestion of the pectoral
            al. 1981; Ohnishi and Jo 1981; Kusuda and Kawai                         and caudal fins. Signs such as congestion and
            1982; Kusuda et al. 1982; Ogawa et al. 1982; Yasunaga                   haernorrhagia are also found in the intestine, liver,
            1982; Hatai et al. 1983; Nakatsugawa 1983; Kaige et                     spleen, and kidney (Kusuda et al. 1976). Epicarditis is
            al. 1984; lida et al. 1986; Sakai et al. 1986; Atsuta et                also seen often. Pathogenic Streptococcus is frequently
            al. 1990). Because sardines and anchovies, as well as                   isolated from the brains of diseased yellowtail



                                                                 JAPANESE FLOUNDER

                 JAPANESE HORSE MACKEREL
                                                              @@the@rs


                    COHO SALMON
                                                                                  YELLOWTAILS
                                                       242,000
                                                       metric tons

                       SEA BREAMS                                                                                              Figure 2
                                                                                                                    Marine fish culture produc-
                                                                                                                    tion for 1988. Source: Sta-
                                                                                  166,000 metric tons
                                                                                                                    tistics   and     Information
                                                                                                                    Department, Ministry of Ag-
                                                                                                                    riculture, Forestry, and Fish-
                                                                                                                    eries,japan.







                                                                                                      Sako: Streptococcal Infection in Cultured YeHowtail                      127


                                                                                           Nocardlosis                    others
                                                                                      Vibriosis


                                                                             COMO' ion*


                                                                                                                  119300                           Streptococcosis
                                    Figure 3                                            F_                      million yen
                    Loss of cultured yellowtail to                       Pasteurellosis
                    diseases in 1988. *This classifi-
                    cation contains cases that the                                                                                                   7,270 million yen
                    cause of a disease are two or
                    more: e.g. mixed infection.
                    Source: Research Division, Fish-
                    eries Agency ofjapan.




                    (Shiomitsu 1982; Kaige et al. 1984; lida et al. 1986).                             Prevention
                        Immunological identification using fluorescent an-
                    tibody techniques is also useful for the rapid                                     It was reported that fish meats used as feed for yel-
                    identification of yellowtail pathogenic streptococci                               lowtail have fish pathogenic Streptococcus (Minami
                    (Kusuda and Kawahara 1987).                                                        1979; Yasunaga 1982). The organisms grow quickly in
                        A rapid identification system is also commercially                             the minced meat of fish,within several hours under
                    available for the identification of fish pathogenic                                optimal temperature (Fig. 4; Sako, unpubl. data). In
                    streptococci. Three species of streptococci from yel-                              laboratory experiments, it was demonstrated that yel-
                    lowtail can be easily distinguished using API 20 Strep                             lowtail can be infected orally by Streptococcus through
                    System (BIO MERIEUX S.A.) within 4 hours (Table                                    feed such as minced meat (Taniguchi 1982a). In or-
                    3; Sako, unpubl. data).                                                            der to prevent contamination by the pathogen and to





                                                                                               Table 1
                                        Biochemical characteristics of ot, P and nonhemolytic Streptococcus sp. isolated from yellowtail.


                                                                      ot-hemolytic                             P-hernolytic                           nonhernolytic
                                                                    Streptococcus sp.                        Streptococcus sp.                        Strepwoccus sp.
                        Characteristics                            (Enterococcus sp.)                            (S. iniae)                            (S. agalactiae)


                        Hemolysis                                         U, -
                        Voges-Proskauer reaction                             +                                                                                +
                        Esculin hydrolysis                                   +                                       +
                        Starch hydrolysis                                                                            +
                        Hippurate hydrolysis                                                                                                                  +
                        Pyrrolidonylarylarnidase                             +                                       +
                        P-galactosidase                                      -                                       +
                        Alkaline phosphatase                                 -                                       +                                        +
                        Sensitivity to bacitracin                            -                                       +                                        +
                        cAMP test                                            -                                       +
                        Lancefield group antigen                 not A, B, C, D, F, G                      not A, B, C, D, F, G,                            BIbb


                        'No reaction at least against the antisera of A-G.
                        'Blb denotes type of group antigen.


   





               128          NOAA Technical Report NMFS     I I I



                                                                                              Table 2
                   Fish -species in Japan from which a pathogenic streptococci was isolated (Kusuda et al. 1976; Kusuda et al. 1978;
                   Minami 1979; Minami et al. 1979; Ohnishi and Jo 1981; Kitao et al. 1981;    Yasunaga 1982; Kusuda and Kawai 1982;
                   Kusuda et al. 1982; Ogawa et al. 1982; Nakatsugawa 1983; Hatai et al. 1983; Kaige et al. 1984; Iida et al. 1986;
                   Sakai et al. 1986; Atsuta et al. 1990).


                                                                                Marine fish                                                                  Freshwater
                   Isolates                             Cultured                                                 Wild                                       cultured fish


                   Enterococcus  sp.   Yellowtail (Seriola quinqueradiata)                     Sardine (Sardinops melanosticta)                       Eel (Anguillajaponica)
                                      Japanese horse mackerel (Trachurusjaponicus)            Anchovy (Engraulis japonica)
                                      Purplish amberjack (Seriola dumerils)                    Round herring (Etrumeus microps)
                                      Goldstriped amberjack (Seriola lalandi)                 Sand lance (Ammodytes personatus)
                                      Largescale blackfish (Girella punctata)                 Chub mackerel (Scomber japonicus)
                                      Black scraper (Thamnaconus modestus)                    Moon dragonets (Repomucenus lunatus)
                                                                                              Black scraper (Thamnaconus modestus)
                                                                                              Multicolorfin (Halichoeres poecilopterus)
                                                                                              Silver  jewfish (Pennahia argentata)
                                                                                              Red sea bream (Pagrus major)

                    Streptococcus       Yellowtail (Seriola quinqueradiata)                     Yellowtail (Seriola quinqueradiata)                    Ayu (Plecoglossus altivelis)
                      iniae           Japanese horse mackerel (Trachurusjaponicus)            Japanese horse mackerel (Trachurus japonicus)            Rainbow trout
                                      Japanese flounder (Paralichthys olivaceus)              Chub mackerel (Scomber japonicus)                          (Oncorhynchus mykiss)
                                      Knifejaw (Oplegnathus fasciatus)                         Sardine (Sardinops melanosticta)                       Amago salmon
                                      Rabbit fish (Siganus fuscescens)                               Red sea bream (Pagrus major)                              (Oncorhynchus rhodurus)
                                      Largescale blackfish (Girella punctata)                                                                         Tilapia (Tilapia nilotica)
                                      Triggerfish (Aluteres monoceros)
                                      Jacopever (Sebastes schlegeli)   
                                      Coho salmon (Oncorhynchus kisutch)

                   Streptococus        Yellowtail (Seriola quinqueradiata)
                      agalactiae



               slow its growth, the following techniques are recom-                                    Therapy
               mended: 1) washing feed fish in uninfected water, 2)
               feeding fish without defrosting, and 3) feeding pro-                                    It is not easy to cure yellowtail that are in the ad-
               cessed pellets (Taniguchi 1982b).                                                       vanced stages of streptococcal infection. Minimal
                     Yellowtail can acquire immunity after streptococcal                                 inhibitory concentrations (MIC) of antimicrobial
               infection (Kusuda and Takagi 1983). Indeed, many                                        agents against Streptococcus from yellowtail are shown
               attempts have been made to prevent streptococcal in-                                    in Figure 5 (Sako, unpubl. data). Sensitivities to sev-
               fection using bacterin. lida et al. (1982) reported on                                  eral drugs are different between Enterococcus sp. and
               the efficacy of vaccination for control of streptococ-                                  S. iniae, but both species have high sensitivities to
               cal infection.                                                                          penicillins, macrolides, tetracyclines, and lincomycin.




                                                                                              Table 3
                   Differential characteristics among Enterococcus sp., Streptococcus iniae, and Streptococcus agalactiae isolated from
                   yellowtail by API 20 Strep System incubated at 35' C for four hours (Sako, unpubl. data). VP = Voges-Proskauer reaction;
                   HIP = Hippurate hydrolysis; ESC = Esculin hydrolysis; PYRA = Pyrrolidonylaryl amidase; PAL = Alkaline phosphatase.


                                                                                                    Test (characteristics)
                   Species                                   VP                         HIP                     ESC                        PYRA                        PAL


                   Enterococcus sp.                         +                                                   +                          +
                   Streptococcus iniae                                                                          +                          +                           +
                   Streptococcus agalactiae                 +                           +                                                                              +
 






                                                                                                                 Sako: Streptococcal Infection in Cultured Yellowtail                               129



                                                                                                                                                        Table 4
                                 7                                                     301C                              Drugs used to cure yellowtail streptococcal infection
                            a    6                                                                                             in Japan. Source: Fisheries Agency ofJapan.
                                 5                                                                                    Drugs                                  Daily dose (mg/kg body weight)
                                                                                        201C                          Alkyl trimethyl ammonium                                   50
                                 4                                                                                       calcium oxytetracycline
                                                                                                                      Doxycycline hydrochloride                               20-50
                                 3                                                     10 V                           Spiramycin embonate                                     25-40
                                                                                                                      Kitasamycin                                                80
                                 2                                                                                    Erythromycin                                            25-50
                                                                                                                      josamycin                                               30-50
                                        0      3      6       9                    is                                 Oleandomycin polystyrene                                   25
                                                                                        hr                               sutfonic acid
                                                                                                                      Lincomycin hydrochloride                                20-40
                                                                                                                      Florfenicol                                                40
                                                         Figure 4
                        Growth of Streptococcus iniae in minced meat of sand
                        lance (Sako, unpubl. data).


                     At present, two kinds of tetracyclines, five kinds of                                          Citations
                     macrolides, lincomycin, and florfenicol are permit-
                     ted as the chemotherapy of streptococcal infection in                                          Atsuta, S.j Yoshimoto, M. Sakai, and M. Kobayashi.
                                                                                                                         1990. Streptococcosis occurred in the pen-cultured coho
                     yellowtail in Japan (Table 4). Recently, streptococci                                                   salmon, Oncorhynchus kisutch. Suisanzoshoku 38:215-219.
                     that have aquired resistance to macrolides and tetra-                                                   (Injapanese.)
                     cyclines were found in many yellowtail farms (M.                                               Hatai,   K., S. Yasumoto, J. Tsukahara, E. Hirakawa, N. Yasunaga,
                     Fukudome, Kagoshima Pref. Fish. Exp. Sta., pers.                                                 and T. Ichiki.
                                                                                                                         1983. Drug sensitivity of various fish-pathogenic bacteria iso-
                     commun., July 1990). Therefore, chemotherapy                                                            lated from cultured fish in Nagasaki Prefecture,
                     treatments for yellowtail infected with drug-resistarit                                                 1982. Bull, Nagasaki Pref. Inst.            Fish, 9:13-22. (In Japa-
                     streptococci are likely to be ineffective.                                                              nese; English abstr.)



                                      Vx
                                                            :0.00625:  0.0125:  0.025     0.05    0.10     0.20     0.39     0.78    1.56     3.13     6.25    12.5      25        so     100    a200

                       Peni c i I I i n        G               0.
                       Ampi C i I I i n                                 0
                                                                                  0
                       Erythromycin                                                        0:
                                                                                                                    a 0
                       Oleandomycin
                       Kita,samycin                                                                        0        0
                       Spiramycin                                                                                   0        0       0
                       Jor.amycin                                                                          0                 49
                       Chloramphenicol                                                                                               0        0

                       Oxytetracycline
                                                                                                                    0        0

                       Doxycycline                                                               0 0
                       Streptomycin                                                                                               i                                             0 0:     0

                       Lincomycin
                                                                                        0 0::

                       Colistin                                                                                                                                                                  00
                       Sulfadimethoxine                                                                                                                                                          so,
                       Sodium nif                                              :.0                                                   0
                                             ar-
                                      styrenate
                       Trimethoprim                                                                        0        0                                                             0

                       Oxolinic acid
                                                                                                                                                                                               :49 0



                                                                                                         Figure 5
                                        z
















































                     Sensitivities of Enterococcus sp. and Streptococcus iniae isolated from yellowtail against various drugs: 0                                              Enterococcus sp.; 0
                     Streptococcus iniae (Sako, unpubl.data).







               130          NOAA Technical Report NAM I I I


               lida, T., H. Wakabayashi, and S. Egusa.                                               Minami, T.
                     1982. Vaccination for control of streptococcal disease in cul-                        1979. Streptococcus sp., pathogenic to culture7d yellowtail, iso-
                       tured yellowtail. Fish Pathol. 16:201-206. (In Japanese; En-                          lated from fishes for diets. Fish Pathol. 14:15-19. (In Japa-
                       glish abstn)                                                                          nese; English abstr.)
               lida, T., K_ Furukawa, M. Sakai, and H. Wakabayashi.                                  Minami, T., M. Nakamura, Y Ikeda, and H. Ozaki.
                     1986. Non-hemolytic Streptococcus isolated from the brain of                          1979. A beta-hemolytic Streptococcus isolated from cultured
                       the vertebral deformed yellowtail. Fish Pathol. 21:33-38.                             yellowtail. Fish Pathol. 14:33-38. (In Japanese; English
                       (Injapanese; English abstr.)                                                          abstr.)
               Kaige, N., M. Miyazaki, and S. Kubota.                                                Nakatsugawa, T.
                     1984. The pathogen and the histopathology of vertebral de-                            1983. A Streptococcal disease of cultured flounder. Fish
                       formity in cultured yellowtail. Fish Pathol. 19:173-179. (In                          Pathol. 17:281-285. (Injapanese; English abstr.)
                       Japanese; English abstr.)                                                     Ogawa, S., N. Yasunaga, EL Hatai, and E. Hirakawa.
               Kimura, H., and R. Kusuda.                                                                  1982. A beta-haemolytic Streptococcus isolated from epizootics
                     1979. Studies on the pathogenesis of streptococcal infection                            of cultured seawater fishes. Bull. Nagasaki Pref. Inst. Fish
                       in cultured yellowtail Se?iola spp.: effect of the cell-free cul-                     8:81-90. (Injapanese; English abstr.)
                       ture on experimental streptococcal infection. J. Fish Dis.                    Ohnishi, K., and Y. Jo.
                       2:501-510.                                                                          1981. Studies on streptococcal infection in pond-cultured
               Kitao, T.                                                                                     fishes-1. Characteristics of a beta-hemolytic streptococcus
                     1982. The methods for detection of Streptococcus sp., caus-                             isolated from cultured ayu and amago in 1977-1978. Fish
                       ative bacteria of streptococcal disease of cultured yellowtail                        Pathol. 16:63-67. (In Japanese; English abstr.)
                       (Seriola quinq  Iueradiata), especially their cultural, biochemi-             Park, S.W., D.S. Sim, and Y.C. Ybo.
                       cal and serological properties. Fish Pathol. 17:17-26. (In                          1987. Pathogenic Streptococcus sp. isolated from an epizootic
                       Japanese; English abstr.)                                                             of cultured yellowtail. Seriola quinqueradiata. Bull. Nat.
               Kitao, T., T. Aoki, and R. Sakoh.                                                             Fish. Res. Dev. Agency 40:75-79. (In Korean; English abstr.)
                     1981. Epizootic caused by 0-haemolytic Streptococcus species                    Pier, G.B., and S.H. Madin.
                       in cultured freshwater fish. Fish Pathol. 15:301-307. (In                           1976. Streptococcus iniae sp. nov., a beta-hemolytic streptococ-
                       Japanese; English abstr.)                                                             cus isolated from an Amazon freshwater dolphin, Inia
               Kusuda, R., and K. Kawai.                                                                     geofftensis. Int. J. Syst. Bacteriol. 26:545-553.
                     1982. Characteristics of Streptococcus sp. pathogenic to                        Plumb,J.A.,J.H. Schachte, and].L. Gaines.
                       yellowtail. Fish Pathol. 17:11-16. (In Japanese; English                            1974. Streptococcus sp. from marine fishes along the Alabama
                       abstr.)                                                                               and Northwest Florida Coast of the Gulf of Mexico. Trans.
               Kusuda, R., and S. Takagi.                                                                    Am. Fish. Soc. 103:358-361.
                     1983. Antibody production against Streplococcus sp. in natu-                    Sakai, M., S. Atsuta, and M. Kobayashi.
                       rally infected yellowuail Se?iola quinqueradiala. Rep. USA.                         1986. A streptococcal disease of cultured jacopever Sebastes
                       Mar. Biol. Inst. Kochi Univ. 5:21-28. (In Japanese; English                           schlegeli. Suisanzoshoku 34:171-177. (In Japanese; English
                       abstr.)                                                                               abstr.)
               Kusuda, R., and E. Kawahara.                                                          Shiomitsu, K.
                     1987. Direct and indirect fluorescent antibody identification                         1982. Isolation of Streptococcus sp. from the brain of cultured
                       of yellowtail pathogens. Bull. Jpn. Soc. Sci. Fish. 53:389-                           yellowtail. Fish Tathol. 17:27-31. (In Japanese; English
                       394. (Injapanese; English abstr.)                                                     abstr.)
               Kusuda, R., K Kawai, T. Toyoshima, and I. Komatsu.                                    Taniguchi, M.
                     1976. A new pathogenic bacterium belonging to the genus                               1982a. Experiment on peroral inoculation via food to in-
                       Streptococcus, isolated from an epizootic of cultured yel-                            duce yellowtail streptococcicosis. Bull. Jpn. Soc. Sci. Fish.
                       lowtail. Bull. Jpn. Soc. Sci. Fish. 42:1345-1352. (In Japanese;                       48:1717-1720. (Injapanese; English abstr.)
                       English abstr.)                                                                     1982b. Studies on the streptococcicosis of yellowtail and its
               Kusuda, R., 1. Komatsu, and K. Kawai.                                                         preventions in Kochi Prefecture. Fish Pathol. 17:55-59.
                     1978. Streptococcus sp. isolated from an epizootic of cultured                          (Injapanese; English abstr.)
                       eels. Buli.jpn. Soc. Sci. Fish. 44:295.                                       Yasunaga, N.
               Kusuda, R., K. Kawai, and T. Shirakawa.                                                     1982. Occurrence of Streptococcus sp., a pathogen of cultured
                     1982. Serological study of Streptococcus sp. pathogenic to cul-                         yellowtail, in muscle of sardine for diets. Fish Pathol.
                       tured yellowtail. Bull. Jpn. Soc. Sci. Fish. 48:1731-1738.                            17:195-198. (Injapanese; English abstr.)
                       (Injapanese; English abstr.)






                                 Stress Induced Pathologies in Fish: The Cost of Stress



                                                                      GARY P. MOBERG

                                                                     Department ofAnimal Science
                                                                                   and
                                                                           Fisheries Program
                                                                     University of California, Davis
                                                                           Davis, CA 95616





                                                                           ABSTRACT


                                     Pathology is the consequence of prolonged stress. Maintaining fish under aquaculture
                                   conditions intensifies the problems of stress by adding the impact of stressoIrs that are
                                   unique to culture conditions. Unlike terrestrial domestic animals, fish have not
                                   benefitted from the genetic pressures of generations of domestication that favor those
                                   individuals most suitable for culture. The physiological mechanisms by which stress in-
                                   duces the development of pathology remain unknown, although it is recognized that one
                                   of the major factors affecting health during stress is the response of the neuroendocrine
                                   system, a stress responsive system that directly regulated growth, reproduction, and the
                                   immune system. During stress, the neuroendocrine system shifts biological resources
                                   from pre-stress activities to new functions at a biological cost to the fish. As the biologi-
                                   cal cost of shifting these resources rises during stress, the fish is placed into a
                                   prepathological state, rendering it vulnerable to the development of pathology. It is by
                                   focusing on this biological cost of stress that it is possible to develop strategies to reduce
                                   stress in aquaculture and to understand the biological basis for the development of
                                   disease.





                Introduction                                                         Practical considerations have dictated most culture
                                                                                     conditions with emphasis on such factors as the ease
                Pathology is the consequence of prolonged stress.                    of cleaning, maximizing the number of fish, or using
                While we can not identify the exact mechanisms re-                   any holding facility 'that is readily available. Little
                sponsible for the development of pathologies during                  concern is given to the fish, at least until stress be-
                stress, there is indisputable evidence that stress can               comes so severe that pathology occurs. Certainly this
                result in the development of a diverse number of pa-                 is an area of aquaculture that needs to be addressed,
                thologies that include not only disease, but the loss                especially as increasingly intensified culture condi-
                of reproduction, the failure to grow normally, and                   tions are adopted.
                even the development of abnormal, deleterious be-
                haviors (Moberg 1985).
                   Fish in culture are especially at risk to the adverse             Problem of Defining Stress
                effects of stress. Unlike domesticated mammals, fish
                have not benefitted from generations of genetic se-                  Although there is considerable evidence demonstrat-
                lection for traits that would assist them in adapting to             ing the disruptive, effects of stress on the well-being
                the restraints of confinement. In essence, fish are still            of fish (Schreck 1982; Pickering and Pottinger 1987;
                wild animals, and when culture conditions do not du-                 Maule et al. 1989; Vijayan and Leatherland 1990), it
                plicate their natural habitat, they are forced to make               is surprising that there is not a better understanding
                biological adjustments to survive. Exacerbating this                 of how stress actually leads to the development of
                problem is the failure of aquaculturists to make any                 pathologies. One reason for the difficulty in discover-
                serious effort to systematically define those 'culture               ing the mechanisms involved is a lack of consistent
                conditions that the fish would find least stressful.                 responses to stress. Most fish do not develop a pathol-







         132       NOAA Technical Report NMFS I I I

         ogy during stress. In fact, it is not possible to predict         work see Pickering 1981 and Adams 1990). Because
         which fish will become vulnerable to a pathology due              these responses are so varied and complex, I will use
         to stress. This is because stress, per se, is not necessar-       a model of animal stress (Fig. 1) to organize current
         ily harmful to an individual. Animals have developed              concepts of stress biology and examine how the bio-
         defenses for coping with stress. Stress is a part of life,        logical cost of stress results in the development of
         and for a species to be successful, it must have                  pathology. The development of this model has been
         evolved biological defenses to stress. Therefore, stress          discussed elsewhere (Moberg 1985, 1987), but, in
         responses are not bad. In fact, they are desirable and            brief, the model divides the biological response to
         necessary. Pathology occurs only when the animal is               stress into three major components: recognition of a
         confronted with stress of such a magnitude that the               threat to homeostasis, the stress response, and the
         very biological responses that evolved for defense re-            consequences of stress. It is the central nervous sys-
         sult in a biological cost to the individual and renders           tem that perceives a threat to homeostasis and
         it vulnerable to pathology. To understand how the                 organizes the fish's biological defense. If a threat
         biological cost of stress leads to the development of             (also referred to as a stressor) is perceived, three gen-
         pathology, it is necessary to first examine how fish              eral types of biological responses are available to the
         respond to stress.                                                fish: behavioral, autonomic nervous system, and neu-
                                                                           roendocrine system responses. In the wild, behavior
                                                                           can be the most biologically efficient way for a fish
         Model of the Stress Response                                      to cope by simply allowing the animal to remove
                                                                           itself frorn'the stressor. However, under the confine-
         Numerous studies have identified the various biologi-             ment of culture conditions, this option is severely
         cal responses that fish use in responding t6 and                  limited. If the fish is unable to avoid the stressor,
         coping with stress (for monographs summarizing this               then the roles of the autonomic nervous and neu-
                                                                           roendocrine systems become critical. While
                                                                           activation of the autonomic nervous system repre-
                                                                           sents an important way for a fish to avoid or cope
                                                                           with a stressor, its effects are rapid in onset, of short
                                             STIMULU                       duration, and relatively specific. For this reason, the
                                                                           importance of the autonomic nervous system in in-
                RECOGNITION OF A                                           ducing stress-related pathologies is questionable. In
                            STASIS      CDM"4"A@@
              THREAT TO HOMEO
                                      t,                                   contrast to the autonomic nervous system, the by-
                                              PERCEPTIION                  pophyseal hormones of the neuroendocrine system
                                            OF sTRESSOR
                                                                           undoubtedly play an important role in stress-in-
                                                                           duced pathology (Moberg 1985). These hormones
                                                                           have a widespread action on the animal and long-
                                          ORGANIZATION OF
                                         BIOLOGICAL DEFENSE                lasting effects on such diverse biological functions
                                                                           as reproduction, growth, metabolism, resistance to
                                                                           diseases, and behavior. Each of these functions are
                                         BIOLOGICAL RESPONSE               vital to the fish's well-being.
                STRESS RESPONSIE         (Behavimal. Aoonoffk,                When the central nervous system organizes the
                                            NemwWocrlne)
                                                                           biological defense to a stressor, it is a combination of
                                                                           these three general biological systems that alters
                                             CHANGEIN                      function. In mammals, we have found that a number
                                         BIOLOGICAL FUNCTION               of factors influence the pattern of this biological re-
                                                I                          sponse, and we have found it impossible to predict
                    CONSEQUENCES                     GICA                  how the individual systems will respond (Moberg
                     OF STRESS            PREPATHOLO                       1985). While the data for fish is limited, there is evi-
                                               STATE                       dence indicating that in fish there are also a number
                                                4                          of factors that can alter the stress response. For ex-
                                     LEE                     I
                                                           I








                                            M'4ELOPMENT                    ample, water quality and temperature (Pickering and
                                            OF PATHOLOGY                   Pottinger 1987, 1989), genetics (Refstie 1982), and
                                                                           social interactions (Peters et al. 1988) have all been
                                   Figure I                                found to influence the stress response. Thus, mea-
           Model for the response of animals to a stressful event          surement of the three general biological systems have
           (from Moberg 1985).                                             not proved to be reliable as an indicator of stress






                                                                               Moberg- Suvss Induced Pathologies in Fish: The Cost of Su-ess               133

                    (Moberg 1987). However, what is important to the                           The concept that there is a biological cost associ-
                    fish is not the pattern of the biological response but                  ated with coping with stress also explains how the
                    the resulting change in biological function-the ulti-                   effects of subclinical stressors can accumulate, result-
                    mate consequence of stress.                                             ing in a significant stress that results in pathology
                      Regardless of which of the general biological sys-                    (Moberg 1985, 1992). Separately, none of the
                    tems the fish chooses in response to the stressor, the                  subclinical stressors would result in a significant ex-
                    result is a change in biological function. Depending                    penditure of the fish's resources, but combined, the
                    upon the appropriateness of the response, this                          subclinical stressors could cost the animal sufficient
                    change in biological function may alleviate, or even                    resources to induce a prepathological state and lead
                    eliminate the stressor. But the change in biological                    to the development of a pathology. For example,
                    function can also lead to the development of a                          Jarvi (1989) found a greater mortality rate in Atlantic
                    prepathological state which can eventually result in                    salmon smolts. (Salmo salar) that were exposed simul-
                    the development of a pathology (Fig. 1). For ex-                        taneously to both osmotic stress and the presence of
                    ample, if the change in biological function during                      predators than if the smolts experienced only one of
                    stress results in the suppression of the immune sys-                    the stressors. Because of the accumulation of biologi-
                    tem (a prepathological state), then the animal is                       cal costs, a series of apparently innocuous events can
                    vulnerable to pathogens, and the opportunity for dis-                   lead to' disease, loss in reproduction, diminished
                    ease (a pathological state) exists. The longer the fish                 growth, or mortality.
                    is stressed, the longer the immune system is sup-
                    pressed and the greater the opportunity for disease
                    to occur. However, disease is only one possible patho-                  Conclusion
                    logical state. Other examples would be the inability
                    to reproduce, abnormal behavior, or the failure to                      The biological cost of stress is the key to under-
                    grow normally.                                                          standing why fish expo@ed to stress develop
                                                                                            pathologies. For a fish to cope with a stressor, it-
                                                                                            must expend biological resources. The more pro-
                    Biological Cost of Stress                                               longed the stressor or the greater the effort needed
                                                                                            to cope with a severe stressor, the greater the diver-
                    The change in biological function that occurs as part                   sion of biological resources, and thus the greater
                    of the stress response results in a biological cost to                  the biological cost of the stress. This diversion of
                    the fish. The fish's resources are diverted from such                   resources occurs at the expense of other biological
                    pre-stress activities as growth to new activities. For ex-              functions, leading to a prepathological state where
                    ample, the glucocorticosteroid hormone cortisol                         the fish is at risk to the development of pathology.
                    secreted during stress will induce gluconeogenesis,                     For some fish, the biological cost of coping with a
                    diverting metabolic resources supporting such func-                     stressor is greater than for other fish and these are
                    tions as growth to the production of glucose. Barton                    the first to succumb to the stressor. As we manage
                    and Schreck (1987) found in juvenile steelhead                          fish in culture, every effort must be made to reduce
                    (Oncorhynchus mykiss) that acute stress reduced by                      stress and, as a result, lower the biological cost for
                    about 25% the amount of energy available for other                      fish living in culture.
                    activities. Likewise, the biological cost of responding
                    to a stressor may suppress the' ability of the immune
                    system to respond to pathogens, rendering the fish                      Citations
                    vulnerable to disease (Maule et al. 1989). It does not
                    matter which pattern of responses the animal                            Adams, S.M.
                    chooses; a change in biological function occurs that                        1990. Biological indicators of stress in fish. Am. Fish. Soc.
                    imposes, a cost, whether or not the change in func-                            Symposium 8, Bethesda, Maryland, 191 p.
                                                                                            Barton, B.A. and C.B. Schreck.
                    tion is effective in helping the animal to cope with                        1987. Metabolic cost of acute physical stress in juvenile
                    the stressor.                                                                  steelhead. Trans. Am. Fish. Soc. 116:257-263.
                      Fortunately, most stressors last for only a brief time                Jarvi, T.
                    and the biological cost of coping with them is rela-                        1989. Synergistic effect on mortality in Atlantic salmon,
                    tively small. However, if a stressor is severe, if it                          Salmo salar smolt caused by osmotic stress and presence of
                                                                                                   predators. Environ. Biol. Fishes 26:149-152.
                    persists, or if the fish experiences a series of stressors,             Maule, A.G., R.A. Tripp, S.L. Kaattari, and C.B. Schreck.
                    the resulting biological cost may be sufficient to in-                      1989. Stress alters immune function and disease resistance in
                    duce a prepathological state which in turn can lead                            chinook salmon (Oncorhynchus tshauytscha). J. Endocrinol.
                    to the development of pathology.                                               120:135-142.







            134         NOAA Technical Report NXB I 11


            Moberg, G.P.                                                                  Pickering, A.D. and T.G. Pottinger.
                 1985. Biological response to stress: key to assessment of ani-                1989. Stress responses and disease resistance in salmonid
                    mal well-being? In Animal stress (G.P. Moberg, ed.), p 24-                   fish: effects of chronic elevation of plasma cortisol. Fish
                    29. American Physiological Society, Bethesda, MD.                            Physiol. and Biochem. 7:253-258.
                 1987. Problems in defining stress and distress in animals. J.            Refstie, T.
                    Am. Vet. Med. Assoc. 19 1: 1207-1211.                                      1982. Preliminary results: differences between rainbow trout
                 1992. Stress: diagnosis, cost and management. In The well-                      families in resistance against vibriosis and stress. Dev.
                    being of agricultural animals in biomedical and agricultural                 Comp. Immunol. Supplement 2: 205-209.
                    research U.A. Mench, Sj. Mayer and L. Krulisch, eds.), p              Schreck, C.B.
                    M-61. Scientists Center for Animal Welfare, Bethesda,                      1982. Stress and rearing of salmonids. Aquaculture 28:241-
                    MD.                                                                       . 249.
            Peters, G., M. Faisal, T. Ung, and I. Ahmed.                                  Vijayan, M.M., andj.F. Leatherland.
                 1988. Stress caused by social interaction and its effect on                   1990. High stocking density affects cortisol secretion and tis-
                    susceptibility to Aeromonas hydrophila infection in rainbow                  sue distribution in brook charr, Salvelinus fontinalis. J,
                    trout Salmo gairdneri. Dis. Aquat. Org. 4:83-89.                             Endocrinol. 124:311-318.
            Pickering A.6.
                 1981. Stress and fish. Acad. Press. New York, NY, 354 p.
            Pickering, A.D., and T.G. Pottinger.
                 1987. Poor water quality suppresses the cortisol response of
                    salmonid fish to handling and confinement.           Fish Biol.
                    30:363-374.






                                                     Control of Fish Disease mijapan



                                                                       AKIRA MURATA

                                                                  Fisheries Agency, Research Division
                                                                    Kasumigaseki 1-2-1, Tiyoda-ku
                                                                             Tokyo, Japan




                                                                          ABSTRACT


                                     The damage to Japanese aquaculture by fish, disease in 1988 was estimated at 20,000
                                   metric tons, worth 144 million dollars, and amounted to 6% of the total aquaculture
                                   production. The Fisheries Agency of the Government of Japan financially supports fish
                                   disease control projects carried out by prefectural governments and fisheries cooperative
                                   associations. It also entrusts the Japan Fisheries Resource Conservation Association
                                   UFRCA) with the following tasks; 1) voluntary pathogen inspection of imported larval
                                   fish and fish eggs; 2) training of advisory personnel on fish disease control; 3) improve-
                                   ment of diagnostic methods; and 4) improvement of disease prevention methods and the
                                   publication of their findings. The main body of the Agency is augmented by the
                                   Shimonoseki University of Fisheries and nine national research institutes. One of these is
                                   the National Research Institute of Aquaculture, which has a Fish Pathology Division
                                   totalling 11 researchers. Each of the 47 prefectural governments in Japan has at least one
                                   Fishery Experimental Station. There are a total of 96 stations. Prefectural governments,
                                   with their institutions, undertake disease diagnosis and drug 1@esidue testing and provide
                                   guidance on fish disease control and drug use. Fisheries co-operative associations play
                                   important roles in fish disease prevention by giving guidance to fishermen and control-
                                   ling local fishery practices. There are 17 universities that have a fishery faculty and 16
                                   universities with a veterinary faculty. All these universities have curriculums for fish dis-
                                   ease and conduct research on it.





                Aquaculture and Fish Disease                                           There are two types of aquaculture; feeding aquac-
                                                                                     ulture (i.e., fish culture) and aquaculture without
                                                                                     feeding (i.e., shellfish culture). Production by feed-
                Fishery Products and Aquaculture Production                          ing aquaculture has grown markedly in recent years
                                                                                     and has reached 343,000 t or $2.5 billion in 1988-
                In 1988, fishery production in, Japan totaled                        about 7 and 19 times the same values for those in
                12,785,000 metric tons (t) valued at $19.4 billion (1                1965, respectively.
                dollar = 140 yen) (MAFF 1988). Changes in amount                       The production of marine fish by aquaculture in
                and value of aquaculture products over the last 23                   1988 was valued at $1,811 million and amounted to
                years are shown in Figure 1. Aquaculture production                  245,000 t. Yellowtail Seriola quinqueradiata production
                in 1988 reached 1,426,000 t or I I % of the total fish-              was greatest at $949 million (165,900 t), followed by
                ery output. These products were valued at $4.9                       red sea bream, Pagrus major, and coho salmon,
                billion or 25% of the total fishery value.                           Oncorhynchus kisutch. (Table 1). Coho salmon, which
                   Aquaculture has been rapidly developing and has                   is cultured in. fresh water until the smolt stage
                become more important because 1) consumers are                       and then transferred to seawater, is one of the
                demanding higher quality and more variety in fishery                 species the production of which has increased rapidly
                products, 2) aquaculture techniques have improved,                   during recent years. Likewise the production of
                and 3) the total amount of aquacultural grounds has                  horse mackerel, Trachurus japonicus, striped jack,
                increased owing to the development of new aquacul-                   Caranx delicatissimus, and tiger puffer, Takifugu
                tural facilities, and the expansion of older ones.                   rubripes, have increased markedly. Increased variety

                                                                                                                                                 135







              136        NOAA Technical Report NMOFS 111



                                                                             Total fishery




                        10-





                   E
                   C
                   0
                        5-




                                                                              Aquaculture

                        0                                                    Feeding aquaculture
                        1965      1970      1975       1980       1985
                                                  Year






                        20,                                                Total fishery


                   0



                        IUI



                                                                           Aquaculture
                                                                                                                                Figure I
                         I                                                 Feeding aquaculture            Cultured fish production (feeding aquacul-
                        0                                                                                 ture and aquaculture without feeding) and
                        1965     1970      1975  Year 1980      1985      1990                            the total fishery production since 1965
                                                                                                          (MAFF 1988) in metric tons and dollars. (1$=
                                                                                                   1      140 yen.)



              of cultured species is a current trend in Japanese                          decline in the health of the cultured fish owing to
              aquaculture.                                                                environmental deterioration and overpopulation,
                Production of freshwater fish by aquaculture was                          and partly by the recent entry of foreign pathogens.
              $732 million (97,800 t) in 1988. Japanese eel,                                  For example, infectious pancreatic necrosis (IPN),
              Anguilla japonica, production was largest at 39,600 t                       infectious hematopoietic necrosis (IFIN), and bacte-.
              ($417 million), followed by Ayu, Plecoglossus altivelis,                    rial kidney disease (BKD) are diseases considered to
              salmonids, and common carp, Cyprinus carpio. A di-                          have been introduced from abroad. Both IHN and
              versity of species is also seen in freshwater                               BKD are thought to have entered through eggs im-
              aquaculture. Among these are Japanese native salmo-                         ported for propagation, while IPN probably entered
              nids such as landlocked salmon, 0. masou, dwarf rill                        through eggs imported for culture. These diseases
              trout, 0. rhodurus, and mountain trout, Salvelinus                          have become prevalent in salmonid farms and are
              pluvius.                                                                    causing severe damage to salmonid production.
                                                                                              Based on a questionnaire sent to all aquaculture
                                                                                          farmers through the prefectural governments every
              Fish Disease and Its Damage                                                 year, the Fisheries Agency has estimated the annual
                                                                                          damage by fish diseases to feeding aquaculture. The
              The rapid development of aquaculture has also                               results are shown in Figure 2.
              brought about a problem, namely with the occur-                                 The damage       to aquaculture by fish disease in 1988
              rence of diseases. These are caused partly by the                           was estimated at 20,000 t or $144 million which is






                                                                                                                   Murata: Control of Fish Disease in Japan                   137



                                                                                                Table I
                                              Cultured fish production by species and damage due to disease in 1988 (MAFF 1988).

                                                                     Production                                  Damage                     Ratio (Damage/Production)

                                                             Amounta             Valueb                  Amount           Value                Amount              Value
                         Fish                                 (1,000 t)     (million dollars)            (1,000 t)   (million dollars)            M                  M

                         Marine fish                            245.0              1811                  17.3                119                     7                 7
                            yellowtail                          165.9               949                  14.4                81                      9                 9
                            red sea bream                        45.5               431                   0.5                  5                     1                 1
                            coho salmon                          16.5               109                   1.0                  7                     6                 6
                            flounder                              3.1                64                   0.4                  6                   13                10
                            horse mackerel                        6.5                37                   0.4                  2                     6                 6
                            tiger puffer                          1.2                37                   0.3                  7                   27                19
                            kuruma prawn                          3.0               140                   0.2                10                      7                 7
                            others                                3.3                44                   0.1                  1                     3                 4

                         Freshwater fish                         97.8               732                   2.8                25                      3                 3
                            eel                                  39.6               417                   1.1                14                      3                 3
                            ayu                                  13.6               120                   0.3                  3                     2                 3
                            salmonids                            19.1                73                   1.0                  5                     5                 7
                            common carp                          18.1                57                   0.2                  1                     1                 1
                            others                                7.4                65                   0.2                  2                     3                 4

                         Total                                  342.8             2,543                  20.1                144                     6                 6


                         1 in metric tons, t,
                         b 1$ = 140 yen.




                     about 6% of the total value of all aquaculture prod-                                culture generally showed low damage rates, while
                     ucts. In 1979 estimated damage was $137 million and                                 species with a short history of culture, such as tiger
                     increased year by year to reach a peak of $190 million                              puffer and japanese flounder, showed high damage
                     in 1982. It then decreased and has been maintained                                  rates. This difference may be caused by the lack of
                     at around $140 million since 1985. During the 1980s,                                experience and less-developed management tech-
                     the total value of aquaculture products increased rap-                              niques for culturing new species.
                     idly while the rate of damage by diseases either                                     In recent years, fish disease problems have become
                     decreased or remained nearly constant. It is thought                                more diversified and complicated. In yellowtail cul-
                     that two factors have worked effectively in reducing                                ture, streptococcosis and pseudotuberculosis are the
                     damage by diseases'in recent years. One is the prac-                                two major diseases. When an episode of mass mortal-
                     tice of control measures, such as disinfection,                                     ity occurs, both of these agents are often found in a
                     disposal of dead fish, and suitable layout of ponds.                                single fish. Nocardiosis, viral ascite disease (diseases
                     The other is the improvement of husbandry tech-                                     for which treatments are not yet established), and a
                     niques, such as improvement of food and the control                                 disease causing peduncle curvature have recently be-
                     of population levels and pen location with regard to                                come an additional problem.
                     the environmental capacity.
                        The rate of disease damage (estimated damage
                     value/total product value) for each species is shown                                Fish Disease Control
                     in Table 1. Disease reduced yellowtail production by
                     9% in 1988. The damage rates of tiger puffer, Japa-                                 System-Because of the complexity and variety of dis-
                     nese flounder (Paralichthys olivaceus) and kuruma                                   eases *occurring in Japanese aquaculture, the
                     prawn (Penaeus japonicus) were 19, 10, and 7%, re-                                  Fisheries Agency is carrying out systematic fish dis-
                     spectively, while only 1% of red sea brearn                                         ease control in cooperation with prefectural
                     production was affected. As for freshwater finfish,                                 governments, fisheries cooperative associations, and
                     rainbow trout (0. mykiss) showed a, high damage rate                                some universities in order both to decrease disease
                     of 7%, while that of common carp was only 1%. In                                    damage and to ensure the safety of cultured fish for
                     marine aquaculture, species with a long history of                                  human consumption. It is important for aquaculture







           138       NOAA Technical Report NNM 111


                                                                             tural governments and the fisheries cooperative asso-
                                  VALUE OF PRODUCTION                        ciations. It also entrusts the Japan Fisheries Resource
                                                                             Conservation Association UFRCA) with the following
                2WO                                                          responsibilities: 1) voluntary pathogen inspection of
                                                                             imported live fish and eggs; 2) training of advisory
             0                                                               personnel on fish disease control; 3) improvement of
                                                                             diagnosis methods; and 4) improvement of disease
                                                                             prevention methods and the publication of their
                 2250-
             -0                                                              findings (Fig. 4).
                                                                                The Fisheries Agency oversees nine national re-
                                                                             search institutes, the Shimonoseki University of
                                                                             Fisheries, and the Hokkaido Salmon Hatchery. In
                 2000                                                        these facilities either research or education in
                     1979    1981     1983       1985     1987               fisheries is carried out, but among these national re-
                                                                             search facilities, only the National Research Institute
                                   VALUE OF DAMAGE                           of Aquaculture, which was established in 1979 to
                                                                             carry out basic and leading research for aquaculture,
                                                                             has a fish pathology division. This division has 11 re-
                                                                             searchers and consists of 4 research sections;
                 175                                                         Pathogens, Pathophysiology, Pharmacology, and Im-
                                                                             munology (Fig. 5).
                 150-
              C
              a                                                                 Prefectural Government-Control measures on
                                                                             the aquaculture grounds are very important for dis-
                 125                                                         ease prevention. Prefectural governments play im-
                                                                             portant roles in guiding aquaculture farmers either
                 100,                                                        directly or indirectly through the fisheries coopera-
                     1979    1981      1983      1985      1987              tive associations.
                                                                                Prefectural governments and their research institu-
                                   DAMAGE RATE                               tions undertake disease diagnosis and drug residue
                                                                             testing, and provide guidance on fish disease control
                     8-                                                      and drug use (Fig. 6). ,                                  i
                                                                                There are 96 prefectural fisheries experimental sta-
                                                                             tions in Japan: at least one station in each prefecture.
                                                                             Investigation and research for aquaculture and fish
                     6.                                                      disease is carried out in their propagation and aqua-
                                                                             culture divisions. In most of these prefectures, @sh
                                                                             disease control centers (20 in total) have been estab-
                     4-                                                      lished, either as a part of the fishery experimental
                     1979    1981       1 @83     485       1987             station or as an independent establishment. Prefec-
                                          YEAR                               tural governments have also established fisheries
                                                                             extension offices to give technical guidance to aqua-
                                     Figure 2                                culture farmers. These offices and fishery
           Changes in cultured fish production (feeding aquaculture)         experimental stations undertake fish disease preven-
           and damage by fish disease. (I$= 140 yen.)                        tion measures in a cooperative manner.
                                                                                The number of staff engaged in disease control
                                                                             measures in prefectural governments was 470 in
           farmers to practice proper feeding and fish disease               1986. In addi  'tion, 30 staff members -from various cit-
           control. Thus, prefectural governments and fisheries              ies, towns, and villages were also engaged in these
           cooperative associations have been requested to es-               measures.
           tablish a system to assist the      aquaculture farmer's
           practice of disease control (Fig. 3).                                Fisheries Cooperative Association-Fisheries coop-
                                                                             erative associations play a very important role in fish
             Fisheries Agency@This agency financially supports               disease control by giving guidance to aquaculture
           fish disease control projects carried out by prefec-              farmers and controlling local fishery grounds. How-






                                                                                                                                   Murata: Control of Fish Disease in Japan                           139


                                                                    - Funding            Japan Fisheries
                                             @islheries           ---------              Resource
                                             Agency              -*-                     Conservation
                                                                     Report              Association

                                                                                              Information
                               Guidance                      Rq-Z
                               Financial support  I I                      - Guidance, Assistance
                                         Prefectural
                                         government

                                                                              Information


                               Guidance
                               Financial support             Rep.,                                 Farmer

                                         Fisheries
                                         Co-operative                        Guidance, Assistance
                                         Association
                                                                               Infognation                                                                      Figure 3
                                                                                                                                             Fish disease control system in Japan.


                                       Fisheries @g7ency7]                                                                 Fisheries Agency

                                                                                                                                             National Research institute of Aquaculture
                                     Funding                      Report                                                                     Fish Pathology Division

                                -Japan Fisheries Resource                                                                                    National Fisheries Research Institute (8)
                                  Conservation Association
                                                                                                                                             Shimonoseki University of Fisheries

                                 ï¿½ Voluntary pathogen examination of imported live fish and eggs                                                          Figure 5
                                ï¿½ Training on fish disease ctwacteristics                                                 Fish disease research system of the Fisheries Agency.

                                  Improvement of diagnosis and disease prevention methods
                                  Extension (Films, Video tape recordings, Textbooks)                                   Universities-Fish disease education is carried out
                                                                                                      . I            by 17 fishery faculties in universities (including the
                                                            Figure 4                                                 Shimonoseki University of Fisheries, Fisheries
                         Role of the Japan Fisheries Resource Conservation Asso-                                     Agency) and 16 veterinary faculties. All -*these facul-
                         ciation in the control of disease in aquaculture.                                           ties also have fish disease research facilities.


                                                                                                                     Prevention of the Introduction and Outbreak of
                       ever, many of these associations are small in scale,                                          Pathogens-In Japan, neither laws nor regulations
                       and the level of guidance varies widely. Only a few of                                        have been established concerning fish quarantine.
                       them carry out drug residue testing, diagnosis of fish                                        However, in order to avoid the introduction of fish
                       disease, and observation of environmental conditions                                          pathogens from foreign countries, the Fisheries
                       in their aquaculture grounds (Fig. 7). The number of                                          Agency requests importers (through prefectural gov-
                       staff engaging in disease control from those associa-                                         ernors) to take the following steps. Importers are
                       tions was only 80 in 1986.                                                                    requested 1) to import living eggs or fish in conjunc-







            140       NOAA Technical Report NNIFS I I I

            tion with health certificates issued by the government
            authority of the.exporting country, 2) to receive an                 Pref ural Governments (47)
            examination for pathogens in the fish or eggs at the
            time of import, and 3) to disinfect eggs immediately
            after importation.                                                                Fisheries Experimental Stations (96)
              Examination for pathogens at the time of import is
            carried out by the JFRCA as entrusted by the Fisheries
            Agency. Examination is done on viral diseases such as
            VHS (viral hemorrhagic septicemia), BKD and whirl-                                Fish Disease Control Centers (20)
            ing disease of salmonid fish (Mxyosoma cerebralis), and
            on viruses, bacteria, and parasites for eel. Examina-
            tions are also conducted for other species.                                       Fisheries Extension Offices
              To prevent the spread of fish diseases, prefectural
            fish disease control centers and fisheries experimen-
            tal stations examine live fish and eggs for pathogens                     Disease diagnosis
            upon the request of the aquaculture. farmer. The                          Drug residue testing
                                                                                      Providing guidance on fish disease control and drug use
            Fisheries Agency encourages farmers to transport live                     To survey and observe enviromnental conditions in aquaculture grounds
            fish and eggs with records of species, place of produc-                   Research
            tion, name and address of producer, dates of                                                Figure 6
            fertilization or hatching, history of fish disease, and           Role of the prefectural governments in the fish disease con-
            medication history.                                               trol system.






                                                 Guidance, Assistance
                         Prefectural
                Guidance Government              infortnation
                Financial support I I -Report
                         Fisheries               Guidance. Assistancet
                         Co-operative
                         Association             Inforniation

                         Drug residue testing
                         Diagnosis of fish disease                                                         Figure 7
                         Observation of enviromental conditions in aquaculture ground. Role of fisheries coopefartive associations in the con-
                                                                                    trol of fish disease.





            Training-Fish disease specialists are indispensable               There are various training courses offered by the
            for carrying out adequate control measures such as                JFRCA for fish disease technical workers. A basic class
            providing advice to the aquaculture farmers and di-               consists of 20 day-lectures and practical exercises
            agnosing and disinfecting facilities and equipment.               each year for 3 years. In 1989, there were 30 to 40
            Since 1973, the Fisheries Agency has been training                trainees for each year course; altogether, 100 people
            the staffs of prefectural governments-in 1984 staffs              have participated in the training. Other training on
            of fisheries cooperative associations were included in            fish disease is also carried out. Since 1974, thejapan
            the training program. This training was aimed at e.du-            Veterinary Medical Association has been providing a
            cating new fish disease specialists and at improving              fish disease course for veterinarians every year as
            the knowledge of the current specialists. In recent               postgraduate education. In Japan, training for fish
            years, the Fisheries Agency has entrusted this train-             disease technical workers is carried out separately in
            ing to JFRCA.                                                     the fishery and veterinary fields. On the other hand,







                                                                                                         Murata: Control of Fish Disease in Japan              141


                 aquacultural guidance and leadership are mainly car-
                 ried out in the fishery field.                                                          Fisheries Agency
                 Investigation and Extension-Under the present cir-                                      Japan Fisheries Resource
                 cumstances where fish disease can cause large-scale                                     Conservation Association
                 damage and is becoming more complicated to pre-                                                            textbooks
                 vent, diagnose, and treat, it is becoming more                                                             films
                 important to promote research on fish disease and to                                                       video tapes
                 give aquaculture farmers information and techniques
                 to control this threat.                                                                   Prefectural government
                    Studies on fish disease by the Fisheries Agency are
                 mainly conducted at the National Research Institute                                                        guidance
                 of Aquaculture in cooperation with six of the nine                                                         textbooks and pamphlets
                 regional national fisheries research institutes and the
                                                                                                                   Farmer]

                                                                                                                          Figure 9
                          Fisheries Agency                                                           The Fisheries Agency's extension system for
                            National Fisheries Research Institutes (8)                               providing fish disease informat    ion in Japan.
                             National Research institute of Aquaculture (1)


                          Universities            Fishery faculties (17)                     Drugs-Appropriate aquaculture management is im-
                                                  Veterinary faculties (16)                  portant to prevent or'to reduce damage by fish
                                                                                             disease. Drugs are also important to reduce damage
                                                                                             when disease breaks out.
                          Prefectural government                                                At present, there are 26 antibacterial medicines
                            Fisheries Experimental Stations                                  available for fish in Japan. In addition, there are vac-
                                                                                             cines for vibriosis, insecticides, anesthetics, and
                                               Figure 8                                      nutritive drugs. In 1988, all the fishery drugs sold in
                     Major organizations involved in fish disease research                   Japan amounted to 1,540 t, which was worth $55 mil-
                     in Japan.                                                               lion.
                                                                                                All  drugs are strictly evaluated for safety, effi-
                                                                                             cacy,   residue accumulation in fish tissues, and
                 Hokkaido Salmon Hatchery. The Agency entrusts                               other   properties by the Central Pharmaceutical Af-
                 some universities with fundamental research, such as                        fairs   Council prior to receiving manufacture
                 exploring the mechanisms of outbreak and infection                          approval from the Ministry of Agriculture, Forestry,
                 of various fish diseases. It also entrusts prefectural                      and Fisheries, based on the Pharmaceutical Affairs
                 fisheries experimental stations with applied research                       Law. Use of antibacterial drugs for fish as well as
                ,for fish disease control techniques, such as disinfec-                      livestock is regulated by "The Standard to be Ob-
                 don and vaccines (Fig. 8).                                                  served by User," which is based on the same law
                    Through its extension program, the Agency dis-                           (Table 2).
                 tributes textbooks about disease diagnosis and                                 Staffs of prefectural governments and fisheries co-
                 techniques of disease prevention and lends films                            operative association assist all aquaculture farmers to
                 and video tape recordings through the JFRCA to the                          use drugs properly by visiting, distributing pam-
                 prefectural staffs who are in charge of guiding                         .   phlets, or carrying out guidance courses at regular
                 aquaculture farmers. Prefectural governments also                           intervals. The public health divisions of prefectural
                 offer guidance courses and distribute textbooks or                          governments carry out drug residue testing by sam-
                 pamphlets on fish disease to the aquaculture farmer.                        pling cultured fish from shops. The fisheries divisions
                 These measures are used to impart knowledge on                              of prefectural governments and fisheries cooperative
                 fish disease characteristics, disease prevention,                           associations also examine aquaculture products sub-
                 aquaculture management, and use of drugs, and                               mitted on a voluntarily basis for drug residues before
                 how to put all of this knowledge into practice appro-                       harvest, in order to ensure that foods are safe for
                 priately (Fig. 9).                                                          human consumption.







               142        NOAA Technical Report NMOFS I I I



                                                                                       Table 2
                                                       The standard to be observed by user of drug (MAFF 1980).

                   Drug                            Sub ect animal                          Administration and dosage                         Withdrawal perioda

                   Feed additive                   Yellowtail                        Administer orally, mixing not more than                        20 days
                   containing                                                        50 g (potency) in I t of feed'
                   alkyltrimethyl-
                   ammoniumcalcium-
                   oxytetracycline
                   Feed additive                   Yellowtail                        Administer orally, mixing not more than                        5 days
                   containing                                                        20 g (potency) in I t of feed
                   ampicillin
                   Feed additive                   Yellowtail                        Administer orally, mixing not more than                        10 days
                   containing                                                        50 g (potency) in I t of feed
                   chlortetracycline
                   hydrochloride                   Eel                               Administer orally, mixing not more than                        15 days
                                                                                     50 g (potency) in I t of feed
                   Feed additive                   Yellowtail                        Administer orally, mixing not more than                        30 days
                   containing                                                        50 g (potency) in I t of feed
                   erythromycin
                   Feed additive                   Yellowtail                        Administer orally, mixing not more than                        5 days
                   containing                                                        10 g in 1 t of feed
                   florphenicol

                   Feed additive                   Yellowtail                        Administer orally, mixing not more than                        20 days
                   containing                                                        80 g (potency) in I t of feed
                   kitasamaycin

                   Feed additive                   Yellowtail                        Administer orally, mixing not more than                        20 days
                   containing                                                        50 g (potency) in I t of feed
                   oxytmaccycline
                   hydrochloride
                                                   Red sea brearn                    Administer orally, mixing not more than                        30 days
                                                                                     50 g (potency) in I t of feed
                                                   Coho salmon                       Administer orally, mixing not more than                        30 days
                                                                                     50 g (potency) in I t of feed

                                                   Eel                               Administer orally, mixing not more than                        30 days
                                                                                     50 g (potency) in I t of feed
                                                   Rainbow trout                     Administer orally, mixing not more than                        30 days
                                                                                     50 g (potency) in I t of feed
                   Feed additive                   Yellowtail                        Administer orally, mixing not more than                        16 days
                   containing                                                        30 g in I t of feed
                   oxolinic acid
                   (except liquid)                 Coho salmon                       Administer orally, mixing not more than                        21 days
                                                                                     20 g in I t of feed

                                                   Eel                               Administer orally, mixing not more than                        20 days
                                                                                     20 g in 1 t of feed
                                                   Rainbow trout                     Administer orally, mixing not more than                        21 days
                                                                                     20 g in 1 t of feed

                                                   Ayu                               Administer orally, mixing not more than                        14 days
                                                                                     20 g in I t of feed

                                                   Carp                              Administer orally, mixing not more than                        28 days
                                                                                     10 g in I t of feed
                   Feed additive                   Yellowtail                        Administer orally, mixing not more than                        16 days
                   containing                                                        20 g in I t of feed
                   oxolinic acid
                   (liquid)







                                                                                                                 Murata: Control of Fish Disease in Japan                  143



                                                                                     Table 2 (Continued)

                        Drug                              Subject animal                         Administration and dosage                          Withdrawal period    a

                        Bath agent                       Eel                              Give bath, dissolving not more than                            25 days
                        containing                                                        5 g in 1 t of water
                        oxolinic acid
                                                         Ayu                              Give bath, dissolving not more than                            14 days
                                                                                          10 g in I t of water

                        Feed additive                    Yellowtail                       Administer orally, mixing not more than                        30 days
                        containing                                                        40 g (potency) in 1 t of feed
                        spiramaycin
                        embonate

                        Feed additive                    Rainbow trout                    Administer orally, mixing not more than                        30 days
                        containing                                                        100 g in I t of feed
                        sulfadimethoxine
                        or its sodium salt

                        Feed additive                    Yellowtail                       Administer orally, mixing not more than                        15 days
                        containing                                                        200 g in I t of feed
                        sulfamonomethoxine
                        or its sodium salt               Eel                              Administer orally, mixing not more than                        30 days
                                                                                          200 g in I t of feed
                                                         Coho salmon                      Administer orally, mixing not more than                        30 days
                                                                                          100 g in I t of feed
                                                         Ayu                              Administer orally, mixing not more than                        15 days
                                                                                          100 g in I t of feed
                                                         Rainbow trout                    Admini@ter orally, mixing not more than                        30 days
                                                                                          150 g in 1 t of feed

                        Bath agent                       Rainbow trout                    Give bath, dissolving not more than                            15 days
                        containing                                                        10 g in I liter of salt water (less
                        sulfamonomethoxine                                                than 1% concentration)
                        or its sodium salt

                        Feed additive.                   Ayu                              Administer orally, mixing not more than                        15 days
                        containing                                                        15 g of sulfamonomethoxine and
                        sulfamonomethoxine                                                5 g of ormetoprim in I t of feed
                        and ormetoprim
                        (compound drug)

                        Feed additive                    Yellowtail                       Administer orally, mixing not more than                        15 days
                        containing                                                        50 g in I t of feed
                        thiamphenicol

                         Withdrawal period defined as the required waiting period following drug administration prior to use of fish for human consumption.
                        b t = metric ton.


                    I



                    Citations


                    MAFF (Ministry of Agriculture, Forestry, and Fisheries)
                        1980. Ministerial ordinance regarding the control and use of
                           drugs for animals. MAFF Ordinance No. 42, Sept. 30, 1980,
                           MAFF, Government ofJapan, Tokyo, Japan. (In English.)
                        1988. Annual statistics of fisheries and aquaculture produc-
                           tion, MAFF, Government of Japan. (In English.)








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