[Federal Register Volume 59, Number 53 (Friday, March 18, 1994)]
[Unknown Section]
[Page 0]
From the Federal Register Online via the Government Publishing Office [www.gpo.gov]
[FR Doc No: 94-6187]


[[Page Unknown]]

[Federal Register: March 18, 1994]


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DEPARTMENT OF AGRICULTURE
9 CFR Parts 145 and 147

[Docket No. 92-151-2]

 

National Poultry Improvement Plan and Auxiliary Provisions

AGENCY: Animal and Plant Health Inspection Service, USDA.

ACTION: Final rule.

-----------------------------------------------------------------------

SUMMARY: We are amending the National Poultry Improvement Plan (the 
Plan) and its auxiliary provisions by providing new administrative and 
laboratory procedures for examining and testing participating flocks 
and preventing and responding to disease outbreaks. The changes, which 
were voted on and approved by the voting delegates at the Plan's 1992 
Biennial Conference, will keep the provisions of the Plan current with 
changes in the poultry industry, allow the use of state-of-the-art 
laboratory procedures, and allow the Plan to better respond to disease 
emergencies.

EFFECTIVE DATE: April 18, 1994.

FOR FURTHER INFORMATION CONTACT: Mr. Andrew R. Rhorer, Senior 
Coordinator, Poultry Improvement Staff, National Poultry Improvement 
Plan, Veterinary Services, APHIS, USDA, room 205, Presidential 
Building, 6525 Belcrest Road, Hyattsville, MD 20782, (301) 436-7768.

SUPPLEMENTARY INFORMATION:

Background

    The National Poultry Improvement Plan (referred to below as ``the 
Plan'') is a cooperative Federal-State-industry mechanism for 
controlling certain poultry diseases. The Plan consists of a variety of 
programs intended to prevent and control egg-transmitted, hatchery-
disseminated poultry diseases. Participation in all Plan programs is 
voluntary, but flocks, hatcheries, and dealers must qualify as ``U.S. 
Pullorum-Typhoid Clean'' before participating in any other Plan 
program. Also, regulations in 9 CFR part 82.34 require that no hatching 
eggs or newly hatched chicks from egg-type chicken breeding flocks may 
be moved interstate unless they are classified ``U.S. Sanitation 
Monitored'' under the Plan or they meet the requirements of a State 
classification plan determined by the Administrator of the Animal and 
Plant Health Inspection Service (APHIS) to be equivalent to the Plan, 
in accordance with 9 CFR 145.23(d).
    The Plan identifies States, flocks, hatcheries, and dealers that 
meet certain disease control standards specified in the Plan's various 
programs. As a result, customers can buy poultry that has tested clean 
of certain diseases or that has been produced under disease-prevention 
conditions.
    The regulations in 9 CFR parts 145 and 147 (referred to below as 
``the regulations'') contain the provisions of the Plan. APHIS amends 
these provisions from time to time to incorporate new scientific 
information and technologies within the Plan.
    On August 25, 1993, we published in the Federal Register (58 FR 
44782-44793, Docket No. 92-151-1) a proposal to amend the regulations 
by:
    1. Adding definitions of Administrator, Animal and Plant Health 
Inspection Service, serial, and suspect flock;
    2. Clarifying the recordkeeping requirements for flocks maintained 
primarily for the production of hatching eggs;
    3. Providing for U.S. Department of Agriculture (USDA) approval of 
pullorum-typhoid tube agglutination antigens;
    4. Allowing a sample of at least 500 birds, in lieu of the entire 
flock, to be tested by the State Inspector to qualify certain 
succeeding flocks for participation in the Plan's pullorum-typhoid 
program;
    5. Removing provisions that allow two consecutive generations in 
egg-type chicken breeding flocks, meat-type chicken breeding flocks, 
and waterfowl, exhibition poultry, and game bird breeding flocks to go 
without testing for pullorum-typhoid;
    6. Providing for the Plan to investigate any multi-State outbreak 
of a Plan disease;
    7. Allowing the use of a federally licensed Salmonella enteritidis 
bacterin to vaccinate birds in egg-type chicken multiplier breeding 
flocks;
    8. Providing for various sample sizes of live birds for 
bacteriological examination under the U.S. Sanitation Monitored program 
for egg-type chickens;
    9. Changing the name of the U.S. Sanitation Monitored program for 
egg- type chickens to U.S. S. enteritidis Monitored;
    10. Adding a USDA-approved polymerase chain reaction (PCR)-based 
DNA procedure as a method of diagnosing mycoplasma;
    11. Adding the enzyme-linked immunosorbent assay (ELISA) as a basic 
screening test for mycoplasma;
    12. Adding an alternative laboratory procedure for mycoplasma 
hemagglutination inhibition (HI) testing using a microtiter technique;
    13. Providing for the most contemporary laboratory methods for use 
in environmental sample selection, Salmonella isolation, examination of 
Salmonella reactors, and program monitoring procedures for egg-type 
chicken breeding flocks, meat-type chicken breeding flocks, and 
waterfowl, exhibition poultry, and game bird breeding flocks; and
    14. Amending the procedure for determining the status and 
effectiveness of sanitation monitored programs.
    In addition to the changes discussed above, we also proposed to 
redesignate, revise, or amend certain footnotes in the regulations and 
remove paragraph designations where they appeared before individual 
definitions.
    We solicited comments concerning our proposal for a 30-day comment 
period ending September 24, 1993. We received three comments by that 
date, from a State department of agriculture, a college of veterinary 
medicine, and a veterinary research laboratory. These comments are 
addressed below.
    One comment referred to our proposal to amend Sec. 145.23(d)(1) to 
allow the use of a federally licensed Salmonella enteritidis bacterin 
to vaccinate birds in egg-type chicken multiplier breeding flocks 
following the bacteriological examination of environmental samples 
collected when the birds were 2 to 4 weeks of age. The commenter asked 
if there was a decrease in the efficacy of the bacterin when older 
birds were vaccinated. The label on the licensed bacterin calls for 
birds to be vaccinated twice, once at 10 to 12 weeks of age, and again 
at 17 to 18 weeks of age; there are no instructions regarding older 
birds. Because the bacterin must be used in accordance with the label 
instructions, we believe that the regulations need not address the 
vaccination of older birds.
    In our proposed amendment to Sec. 147.7, ``Standard test procedures 
for mycoplasma,'' the second sentence of paragraph (e)(2)(ii)(C) states 
that the dilution required to give four hemagglutination (HA) units is 
calculated by dividing the stock antigen HA titer by 8. One commenter 
stated that the stock antigen HA titer should be divided by 4 instead 
of 8. We disagree. The antigen titration is done with volumes of 50 
L. In the HI test, 25 L of antigen is added to 25 
L of serum dilution. The antigen, then, must contain 4 HA 
units in 25 L; the 4 HA units would then be doubled for 50 
L, so dividing by 8 is correct. Therefore, we did not make any 
changes in response to the comment.
    Also in our proposed amendment to Sec. 147.7, paragraph 
(e)(2)(iii)(E) calls for the serial dilution of 25 L from a 
specified number of wells. One commenter suggested that such multiple 
transfers of volumes as small as 25 L may be difficult using a 
multichannel pipettor due to incomplete volume transfer. We believe 
that no change in the regulations is necessary because multichannel 
pipettors calibrated to deliver the proper volume are readily available 
from commercial sources.
    Proposed paragraph (e)(iv)(B)(3) of Sec. 147.7 states that for the 
assay described in the paragraph to be valid, the backtitration of the 
antigen must be 1:4 or 1:8. One commenter suggested that the latter 
number should be omitted because a backtitration of 1:8 would result in 
potentially suppressed HI titers. We believe that the 4-HA to 8-HA 
range allows for realistic performance variation within the test while 
maintaining stringent quality control. As proposed, the protocol stated 
that the positive control must be within one dilution of the previously 
determined titer, so any loss of sensitivity would be detected if a 
backtitration approaching 8 HA units was suppressing the HI titers of 
samples. Therefore, we did not make any changes in response to the 
comment.
    One commenter pointed out that the 1:5 serum dilution referred to 
in paragraph (e)(2)(v)(D)(1) of the proposed amendment to Sec. 147.7 
should actually be a 1:5.5 serum dilution. While 1:5.5 is actually 
correct, the ultimate serial dilutions of the sample would be 1:11, 
1:22, 1:44, etc., each of which can be presented as the nearest 
standard dilution (1:10, 1:20, 1:40, etc.) without a loss of accuracy 
in the test. Therefore, we did not make any changes in response to the 
comment.
    Proposed new paragraph (a)(5) of Sec. 147.11 stated that the 
Analytical Profile Index for Enterobacteriaceae (API) system may be 
used to aid cultural identifications. One commenter noted that API is 
not the only such system that could be used. We agree and have changed 
Sec. 147.11(a)(5) to indicate that systems other than API are 
available.
    Two of the comments encouraged us to amend illustration 1 in 
Sec. 147.11 to accurately reflect the procedures called for in the text 
of proposed new paragraph (a)(1) of Sec. 147.11. As proposed, the text 
of Sec. 147.11(a)(1) required the inoculation of non-selective plates 
in addition to two selective plating media. The commenters pointed out 
that the upper right-hand block of illustration 1 did not include the 
inoculation of non-selective plates. We agree, and have added the 
inoculation of non-selective plates to the upper right-hand block of 
illustration 1. The probability of isolating Salmonella from organ 
tissues will be enhanced if non-selective plating media are used in 
addition to selective plating media.
    One of the commenters suggested that a reference to footnote 2 be 
added to the upper left-hand block of illustration 1, which refers to 
non-selective enrichment broths. Because footnote 2 to illustration 1 
contains pertinent information concerning non-selective enrichment, we 
agree and have added a reference to footnote 2 in the upper left-hand 
block of illustration 1. The same commenter noted that we had omitted 
the word ``broths'' after the word ``enrichment'' in footnote 1 to 
illustration 1, and also suggested that the first sentence of footnote 
2 be revised for the sake of clarity. We agree with both of these 
points and have added the word ``broths'' to footnote 1 and have 
revised the first sentence of footnote 2 to read ``Beef extract or 
infusion broths and plates are preferred.''
    Another commenter suggested that illustrations 1 and 2 are 
difficult to follow and that wording should be added to the 
illustrations to indicate that Salmonella pullorum is a slow grower and 
produces a smaller colony than other salmonellae, that the production 
of H2S is delayed or absent, and that the production of gas is 
weak or absent. We believe that the illustrations are easily understood 
and that the additional information suggested by the commenter is 
unnecessary. Each illustration contains a block referring to the use of 
``additional identification media and diagnostic systems,'' which 
includes means of biochemical identification and differentiation of 
bacteria. Further, we believe that a person conducting such tests would 
be familiar with the isolation of Salmonella, including the 
identification of characteristic colonies of pullorum and other 
salmonellae on various media. Therefore, we have made no changes in 
response to the comment.
    Finally, paragraph (a)(2) of our proposed amendment to Sec. 147.14 
stated that culturing for the dependable recovery of salmonellae should 
include the use of preenrichment broths supplemented with ferrous 
sulfate. One of the commenters noted that there is debate regarding the 
usefulness of adding ferrous sulfate to overcome the inhibitory effects 
of conalbumin, and pointed out that the egg culture protocol included 
in recently published APHIS regulations (``Chicken Disease Caused by 
Salmonella Enteritidis'') does not include the addition of ferrous 
sulfate. The ``regulations'' to which the commenter referred were 
actually proposed regulations published in the Federal Register on 
August 2, 1993 (58 FR 41048-41061, Docket No. 91-016-1) and, as such, 
have no regulatory effect. The protocols included in that proposed rule 
are still under review and will not become effective until a final rule 
is published. We believe that the ability of conalbumin to chelate 
metallic ions such as Fe3+ or Cu2+ has been clearly 
demonstrated by both Gelb and Harris (1980) and Tan and Woodworth 
(1969). Additionally, Board et al. (1991) demonstrated that the 
addition of iron to preenrichment broth aided in the recovery of 
Salmonella enteritidis from eggs. Therefore, we have made no changes in 
response to the comment.
    In addition to the changes discussed above, we are making two other 
changes. First, we are adding Office of Management and Budget (OMB) 
control numbers to Secs. 147.1, 147.2, 147.3, 147.5, 147.11, 147.12, 
147.13, and 147.21. The existing paperwork requirements contained in 
those sections--not any new requirements that may be contained in this 
final rule--were approved by OMB after the proposed rule was published, 
so the control numbers must be added to the end of each of those 
sections. Second, we are correcting an out-of-date reference in 
Sec. 147.43, which contains provisions regarding the Plan's General 
Conference Committee. In that section, there is a reference to the 
Assistant Secretary of Agriculture for Marketing and Transportation 
Services. In 1982, the Marketing and Transportation Services division 
was reorganized and renamed Marketing and Inspection Services, so we 
have corrected the reference in Sec. 147.43 to reflect the current 
organization.
    Therefore, based on the rationale set forth in the proposed rule 
and in this document, we are adopting the provisions of the proposal as 
a final rule, with the changes discussed in this document.

Executive Order 12866 and Regulatory Flexibility Act

    This rule has been determined to be not significant for purposes of 
Executive Order 12866 and, therefore, has not been reviewed by the 
Office of Management and Budget.
    The changes contained in this document are based on the 
recommendations of representatives of member States, hatcheries, 
dealers, flockowners, and breeders who took part in the Plan's 31st 
Biennial Conference. Because participation in the Plan is voluntary, 
individuals are likely to remain in the program as long as the costs of 
implementing the program are lower than the added benefits they receive 
from the program. The changes in this final rule will keep the 
provisions of the Plan current with changes in the poultry industry, 
will allow the use of state-of-the-art laboratory and testing 
procedures, and will allow the Plan to better respond to disease 
emergencies.
    Of the changes contained in this final rule, only two are expected 
to have more than a negligible economic effect on Plan participants. 
The amendment that will allow, in certain cases, a 500-bird sample to 
be tested in lieu of the entire flock will result in a cost savings for 
affected Plan participants because fewer tests will be required to 
qualify certain multiplier breeding flocks and succeeding flocks for 
participation in the Plan's pullorum-typhoid program. It is likely, 
however, that those savings will be offset by the amendment that 
increases testing requirements by removing, for all poultry except 
turkeys, provisions that allow two consecutive generations of breeding 
flocks to go without testing for pullorum-typhoid. The remaining items, 
because they are either administrative or procedural in nature, will 
not have a significant economic impact.
    Under these circumstances, the Administrator of the Animal and 
Plant Health Inspection Service has determined that this action will 
not have a significant economic impact on a substantial number of small 
entities.

Executive Order 12372

    This program/activity is listed in the Catalog of Federal Domestic 
Assistance under No. 10.025 and is subject to Executive Order 12372, 
which requires intergovernmental consultation with State and local 
officials. (See 7 CFR part 3015, subpart V.)

Executive Order 12778

    This final rule has been reviewed under Executive Order 12778, 
Civil Justice Reform. This rule: (1) Preempts all State and local laws 
and regulations that are in conflict with this rule; (2) has no 
retroactive effect; and (3) does not require administrative proceedings 
before parties may file suit in court challenging this rule.

Paperwork Reduction Act

    In accordance with the Paperwork Reduction Act of 1980 (44 U.S.C. 
3501 et seq.), the information collection or recordkeeping requirements 
included in this final rule have been submitted for approval to the 
Office of Management and Budget.

List of Subjects in 9 CFR Parts 145 and 147

    Animal diseases, Poultry and poultry products, Reporting and 
recordkeeping requirements.

    Accordingly, 9 CFR parts 145 and 147 are amended as follows:

PART 145--NATIONAL POULTRY IMPROVEMENT PLAN

    1. The authority citation for part 145 continues to read as 
follows:

    Authority: 7 U.S.C. 429; 7 CFR 2.17, 2.51, and 371.2(d).

    2. Section 145.1 is amended by adding, in alphabetical order, four 
new definitions to read as follows:


Sec. 145.1  Definitions.

* * * * *
    Administrator. The Administrator, Animal and Plant Health 
Inspection Service, or any person authorized to act for the 
Administrator.
* * * * *
    Animal and Plant Health Inspection Service. The Animal and Plant 
Health Inspection Service of the U.S. Department of Agriculture.
* * * * *
    Serial. The total quantity of completed product which has been 
thoroughly mixed in a single container and identified by a serial 
number.
* * * * *
    Suspect Flock. A flock shall be considered, for the purposes of the 
Plan, to be a suspect flock if any evidence exists that it has been 
exposed to a communicable poultry disease.
* * * * *
    3. In Sec. 145.10, paragraph (d), the words ``Sec. 145.23(d) and'' 
are removed.
    4. In Sec. 145.10, a new paragraph (l) is added to read as follows:


Sec. 145.10  Terminology and classification; flocks, products, and 
States.

* * * * *
    (l) U.S. S. Enteritidis Monitored. (See Sec. 145.23(d).)

BILLING CODE 3410-34-P

TR18MR94.002

                                  Figure 13

BILLING CODE 3410-34-C

    5. In Sec. 145.12, paragraph (b), two new sentences are added after 
the first sentence to read as set forth below.


Sec. 145.12  Inspections.

* * * * *
    (b) * * * Records shall include VS Form 9-2, ``Flock Selecting and 
Testing Report''; VS Form 9-3, ``Report of Sales of Hatching Eggs, 
Chicks, and Poults''; set and hatch records; egg receipts; and egg/
chick orders or invoices. Records shall be maintained for 3 years. * * 
*
    6. In Sec. 145.14, paragraph (a)(1), at the end of the third 
sentence, the word ``test.'' is removed and the words ``and tube 
agglutination tests. Each serial of tube antigen shall be submitted by 
the antigen producer to the Department for approval upon manufacture 
and once a year thereafter as long as antigen from that serial 
continues to be made available for use.'' are added in its place.
    7. In Sec. 145.14, the introductory text of paragraph (a)(6), the 
third sentence is revised to read as follows:


Sec. 145.14  Blood testing.

* * * * *
    (a) * * *
    (6) * * * Testing to qualify flocks for Plan participation must 
include the testing of all birds in infected flocks and succeeding 
flocks for a 12-month period, and shall be performed or physically 
supervised by a State Inspector; Provided, That at the discretion of 
the Official State Agency, a sample of at least 500 birds, rather than 
all birds in the flock, may be tested by the State Inspector if it is 
agreed upon by the Official State Agency, the flockowner, and the 
Administrator.* * *
* * * * *


Sec. 145.21  [Amended]

    8. Section 145.21 is amended by removing all paragraph designations 
and rearranging the definitions in alphabetical order.
    9. Section 145.23 is amended as follows:
    a. In the introductory text of paragraph (b)(3), the words ``, or a 
breeding flock composed of progeny of a primary breeding flock which is 
intended solely for the production of multiplier breeding flocks,'' are 
removed.
    b. Paragraph (b)(3)(v) is amended by removing the words ``S. 
pullorum or S. gallinarum isolations from poultry'' and adding the 
words ``any disease outbreak involving a disease covered under the 
Plan'' in their place, and by adding a proviso at the end of the 
paragraph to read as set forth below.
    c. In paragraph (d), the paragraph heading and the first sentence 
of paragraph (d)(1)(i) are amended by removing the word ``Sanitation'' 
and adding the words ``S. enteritidis'' in its place.
    d. In paragraph (d)(1)(v), the first sentence is amended by 
removing the words ``more than 4 months'' and replacing them with the 
words ``2 to 4 weeks''.
    e. Paragraphs (d)(1)(vi), (d)(1)(vii), and (d)(1)(viii) are 
redesignated as paragraphs (d)(1)(vii), (d)(1)(viii), and (d)(1)(ix), 
respectively, and a new paragraph (d)(1)(vi) is added to read as set 
forth below.
    f. In newly redesignated paragraph (d)(1)(vii), the first sentence 
is amended by removing the word ``birds'' and replacing it with the 
words ``non-vaccinated birds as described in paragraph (d)(1)(vi) of 
this section''.
    g. In paragraph (d)(2), the second and third sentences are revised 
to read as set forth below.
    h. Paragraph (d)(3) is amended by removing the words ``A flock'' 
and adding the words ``A non-vaccinated flock'' in their place; by 
removing the reference ``(d)(v)'' and adding the reference 
``(d)(1)(v)'' in its place; and by removing the reference 
``(d)(1)(vi)'' and adding the reference ``(d)(1)(vii)'' in its place.
    i. Paragraphs (e)(1)(ii) (a) and (b) are redesignated as paragraphs 
(e)(1)(ii) (A) and (B).


Sec. 145.23  Terminology and classification: flocks and products.

* * * * *
    (b) * * *
    (3) * * *
    (v) * * * Provided, That if the origin of the infection involves 
another State, or if there is exposure to poultry in another State from 
the infected flock, then the National Poultry Improvement Plan will 
conduct an investigation;
* * * * *
    (d) * * *
    (1) * * *
    (vi) A federally licensed Salmonella enteritidis bacterin may be 
used in multiplier breeding flocks that are negative for Salmonella 
enteritidis upon bacteriological examination as described in paragraph 
(d)(1)(v) of this section: Provided, that a sample of 350 birds, which 
will be banded for identification, shall remain unvaccinated until the 
flock reaches at least 4 months of age. Following negative serological 
and bacteriological examinations as described in paragraph (d)(1)(vii) 
of this section, the banded, non-vaccinated birds shall be vaccinated.
* * * * *
    (2) * * * Isolation of SE from an environmental or other specimen, 
as described in paragraph (d)(1)(v) of this section, will require 
bacteriological examination for SE in an authorized laboratory, as 
described in Sec. 147.11(a) of this chapter, of a random sample of 60 
live birds from a flock of 5,000 birds or more, or 30 live birds from a 
flock with fewer than 5,000 birds. If only one specimen is found 
positive for SE, the participant may request bacteriological 
examination of a second sample, equal in size to the first sample, from 
the flock. * * *
* * * * *


Sec. 145.31  [Amended]

    10. Section 145.31 is amended by removing all paragraph 
designations and rearranging the definitions in alphabetical order.
    11. Section 145.33 is amended as follows:
    a. The introductory text of paragraph (b)(3) is amended by removing 
the words ``, or a breeding flock composed of progeny of a primary 
breeding flock which is intended solely for the production of 
multiplier breeding flocks,''.
    b. Paragraph (b)(3)(v) is amended by removing the words ``S. 
pullorum or S. gallinarum isolations from poultry'' and adding the 
words ``any disease outbreak involving a disease covered under the 
Plan'' in their place, and by adding a proviso at the end of the 
paragraph to read as set forth below.
    c. In paragraph (d)(1)(viii), footnote 4a and its reference in the 
text are redesignated as footnote 4.
    d. Paragraphs (e)(1)(ii) (a) and (b) are redesignated as paragraphs 
(e)(1)(ii) (A) and (B).


Sec. 145.33  Terminology and classification: flocks and products.

* * * * *
    (b) * * *
    (3) * * *
    (v) * * * Provided, That if the origin of the infection involves 
another State, or if there is exposure to poultry in another State from 
the infected flock, then the National Poultry Improvement Plan will 
conduct an investigation;
* * * * *


Sec. 145.41  [Amended]

    12. In Sec. 145.41, the paragraph designation ``(a)'' assigned to 
the definition of the term poults is removed.
    13. Section 145.43 is amended as follows:
    a. Paragraph (b)(3)(v) is amended by removing the words ``S. 
pullorum or S. gallinarum isolations from poultry'' and adding the 
words ``any disease outbreak involving a disease covered under the 
Plan'' in their place, and by adding a proviso at the end of the 
paragraph to read as set forth below.
    b. In paragraph (f)(3)(ii), the words ``Industry/Education 
Salmonella Reduction'' are removed and the words ``Industry (APPI) 
Salmonella Education/Reduction'' added in their place, and the footnote 
reference ``4'' is removed.


Sec. 145.43  Terminology and classification; flocks and products.

* * * * *
    (b) * * *
    (3) * * *
    (v) * * * Provided, That if the origin of the infection involves 
another State, or if there is exposure to poultry in another State from 
the infected flock, then the National Poultry Improvement Plan will 
conduct an investigation;
* * * * *


Sec. 145.51  [Amended]

    14. Section 145.51 is amended by removing all paragraph 
designations and rearranging the definitions in alphabetical order.
    15. Section 145.53 is amended as follows:
    a. In paragraph (a), footnote 1 and its reference in the text are 
redesignated as footnote 7.
    b. The introductory text of paragraph (b)(3) is amended by removing 
the words ``, or a breeding flock composed of progeny of a primary 
breeding flock which is intended solely for the production of 
multiplier breeding flocks,''.
    c. Paragraph (b)(3)(v) is amended by removing the words ``S. 
pullorum or S. gallinarum isolations from poultry'' and adding the 
words ``any disease outbreak involving a disease covered under the 
Plan'' in their place, and by adding a proviso at the end of the 
paragraph to read as set forth below.


Sec. 145.53  Terminology and classification: flocks and products.

* * * * *
    (b) * * *
    (3) * * *
    (v) * * * Provided, That if the origin of the infection involves 
another State, or if there is exposure to poultry in another State from 
the infected flock, then the National Poultry Improvement Plan will 
conduct an investigation;
* * * * *

PART 147--AUXILIARY PROVISIONS ON NATIONAL POULTRY IMPROVEMENT PLAN

    16. The authority citation for part 147 continues to read as 
follows:

    Authority: 7 U.S.C. 429; 7 CFR 2.17, 2.51, and 371.2(d).


Secs. 147.1, 147.2, and 147.3  [Amended]

    17. In Secs. 147.1, 147.2, and 147.3, at the end of the regulatory 
text of each section, the words ``(Approved by the Office of Management 
and Budget under control number 0579-0007)'' are added.


Sec. 147.5  [Amended]

    18. In Sec. 147.5, paragraph (b), footnote 1 and its reference in 
the text are redesignated as footnote 4, and the footnote is amended by 
removing the words ``Federal Building,'' and adding the words 
``Presidential Building, 6525 Belcrest Road,'' in their place.
    19. In Sec. 147.5, at the end of the regulatory text, the words 
``(Approved by the Office of Management and Budget under control number 
0579-0007)'' are added.


Sec. 147.6  [Amended]

    20. In Sec. 147.6, the introductory text of paragraph (b), the 
second sentence, the words ``or identified as infected by a polymerase 
chain reaction (PCR)-based procedure approved by the Department'' are 
added after the word ``bacteriologically''.
    21. In Sec. 147.6, paragraph (b)(5), the second sentence, the words 
``or a PCR-based procedure conducted on these specimens'' are added 
after the word ``individually''.
    22. In Sec. 147.6, in paragraphs (b)(12) through (b)(15), the words 
``, PCR-based procedures,'' are added after the words ``in vivo bio-
assay'' each time they appear.
    23. Section 147.7 is amended as follows:
    a. In the section heading, footnote 1 and its reference are 
redesignated as footnote 5.
    b. In the introductory text, the first sentence is amended by 
removing the words ``plate of the tube agglutination'' and adding the 
words ``plate agglutination test, the tube agglutination test, and the 
enzyme-linked immunosorbent assay (ELISA)'' in their place.
    c. In the introductory text, the beginning of the third sentence is 
amended by removing the word ``Both'' and adding the words ``These 
three'' in its place.
    d. In the introductory text, the seventh sentence is amended by 
removing the words ``the plate and/or'' and adding the words ``the 
ELISA, plate, and/or'' in their place.
    e. In paragraph (a), the paragraph heading and the first sentence 
of the introductory text of paragraph (a)(1) is amended by removing the 
words ``plate test'' and adding the words ``plate agglutination test'' 
in their place.
    f. Paragraph (e) is amended as follows:
    i. In the paragraph heading, the word ``test'' is removed and the 
word ``tests'' added in its place.
    ii. Paragraphs (e)(1) introductory text through (e)(3)(xi) are 
redesignated as follows: 

------------------------------------------------------------------------
            Old section                          New section            
------------------------------------------------------------------------
147.7(e)(1) introductory text......  147.7(e)(1)(i) introductory text.  
147.7(e)(1)(i).....................  147.7(e)(1)(i)(A).                 
147.7(e)(1)(ii)....................  147.7(e)(1)(i)(B).                 
147.7(e)(1)(iii)...................  147.7(e)(1)(i)(C).                 
147.7(e)(1)(iv)....................  147.7(e)(1)(i)(D).                 
147.7(e)(2) introductory text......  147.7(e)(1)(ii) introductory text. 
147.7(e)(2)(i).....................  147.7(e)(1)(ii)(A).                
147.7(e)(2)(ii)....................  147.7(e)(1)(ii)(B).                
147.7(e)(2)(iii)...................  147.7(e)(1)(ii)(C).                
147.7(e)(2)(iv)....................  147.7(e)(1)(ii)(D).                
147.7(e)(2)(v).....................  147.7(e)(1)(ii)(E).                
147.7(e)(2)(vi)....................  147.7(e)(1)(ii)(F).                
147.7(e)(2)(vii)...................  147.7(e)(1)(ii)(G).                
147.7(e)(2)(viii)..................  147.7(e)(1)(ii)(H).                
147.7(e)(3) introductory text......  147.7(e)(1)(iii) introductory text.
147.7(e)(3)(i).....................  147.7(e)(1)(iii)(A).               
147.7(e)(3)(ii)....................  147.7(e)(1)(iii)(B).               
147.7(e)(3)(iii)...................  147.7(e)(1)(iii)(C).               
147.7(e)(3)(iv)....................  147.7(e)(1)(iii)(D).               
147.7(e)(3)(v).....................  147.7(e)(1)(iii)(E).               
147.7(e)(3)(vi)....................  147.7(e)(1)(iii)(F).               
147.7(e)(3)(vii)...................  147.7(e)(1)(iii)(G).               
147.7(e)(3)(viii)..................  147.7(e)(1)(iii)(H).               
147.7(e)(3)(ix)....................  147.7(e)(1)(iii)(I).               
147.7(e)(3)(x) introductory text...  147.7(e)(1)(iii)(J) introductory   
                                      text.                             
147.7(e)(3)(x)(A)..................  147.7(e)(1)(iii)(J)(1).            
147.7(e)(3)(x)(B)..................  147.7(e)(1)(iii)(J)(2).            
147.7(e)(3)(x)(C)..................  147.7(e)(1)(iii)(J)(3).            
147.7(e)(3)(x)(D)..................  147.7(e)(1)(iii)(J)(4).            
147.7(e)(3)(x)(E)..................  147.7(e)(1)(iii)(J)(5).            
147.7(e)(3)(x)(F)..................  147.7(e)(1)(iii)(J)(6).            
147.7(e)(3)(x)(G)..................  147.7(e)(1)(iii)(J)(7).            
147.7(e)(3)(x)(H)..................  147.7(e)(1)(iii)(J)(8).            
147.7(e)(3)(x)(I)..................  147.7(e)(1)(iii)(J)(9).            
147.7(e)(3)(xi)....................  147.7(e)(1)(iii)(K).               
------------------------------------------------------------------------

    iii. The introductory text of paragraph (e) is redesignated as 
paragraph (e)(1) and a new paragraph heading for paragraph (e)(1) is 
added to read as set forth below.
    iv. A new paragraph (e)(2) is added to read as set forth below.


Sec. 147.7  Standard test procedures for mycoplasma.\5\
---------------------------------------------------------------------------

    \5\For additional information on mycoplasma test procedures, 
refer to the following references: Proc. 77th Annual Meeting, U.S. 
Animal Health Association, 1973; Isolation and Identification of 
Avian Pathogens, 2nd Edition; Methods for Examining Poultry 
Biologics and for Identifying and Quantifying Avian Pathogens, 1971.
---------------------------------------------------------------------------

* * * * *
    (e) * * *
    (1) Procedure No. 1. * * *
* * * * *
    (2) Procedure No. 2. Purpose: To test for antibodies to avian 
mycoplasma by hemagglutination inhibition (HI). The test uses the 
constant antigen, titered-sera method for measuring antibodies to M. 
gallisepticum, M. synoviae, or M. meleagridis.
    (i) Materials needed.
    (A) M. gallisepticum, M. synoviae, and/or M. meleagridis HI 
antigens.
    (B) Positive and negative control sera.
    (C) Phosphate buffered saline (PBS).
    (D) Microtiter plates, 96-well, U-bottom.
    (E) 12-channel pipettor (Titerek).
    (F) 50 L pipettor (Pipetman P200).
    (G) Pipette tips.
    (H) 0.5 percent homologous red blood cells (RBC's) in PBS (use 
RBC's from the same species being tested).
    (I) Plate-sealing tape.
    (J) Mirrored plate reader.
    (ii) Microtiter hemagglutination antigen (HA) titration.
    (A) Perform standard hemagglutination test (HA) on mycoplasma 
antigen to determine titer of antigen.
    (1) Dispense 50 L of PBS into each well of 3 rows of a 96-
well microtiter plate.
    (2) Dispense 50 L of stock antigen into the wells of 2 
rows.
    (3) Perform serial two-fold dilutions (50 L) using a 12-
channel pipettor. The dilution series will be from 1:2 to 1:4096.
    (4) Add 50 L of 0.5 percent homologous RBC's to each well 
of all 3 rows. The row with no antigen serves as an RBC control.
    (B) Incubate at room temperature (approximately 30 minutes) until 
the control RBC's give tight buttons. The HA titer is read as the last 
well to give a complete lawn (hemagglutination). The desired endpoint 
is 4 HA units. The well containing the 1:4 dilution should give a 
complete HA while the 1:8 dilution should show less than complete HA.
    (C) Dilute stock antigen to 4 HA units for the HI test. The 
dilution required to give 4 HA units is calculated by dividing the 
stock antigen HA titer by 8. (Example: 1:320 HA units  8 = 40, 
dilute stock antigen 1:40.)
    (iii) Hemagglutination inhibition assay.
    (A) Label one column (A to H) of a 96-well, U-bottom microtiter 
plate for each sample, each positive and negative control sera, antigen 
backtitration, and RBC control.
    (B) Add 40 L of PBS to the top row of wells (row A) of the 
plate.
    (C) Add 25 L of PBS to all remaining wells of the plate.
    (D) Add 10 L of each test sera to well A of each column 
(making a 1:5 sera dilution).
    (E) Serially dilute 25 L from well A through H using a 12-
channel pipettor. Discard the final 25 L. Row A = 1:5...row H 
= 1:640.
    (F) With an Oxford doser, add 25 L of 4 HA unit antigen to 
wells B through H. Well A serves as sera control.
    (G) Prepare an antigen backtitration by adding 25 L of PBS 
to each well of one column. Add 25 L of diluted antigen to 
well A and serially dilute 25 L from wells A to D. This 
prepares 1:2, 1:4, 1:8, and 1:16 dilutions. (It is recommended that the 
antigen control backtitration be performed before the diluted antigen 
is used in the assay. Dilution problems could be detected and corrected 
before the inappropriately diluted antigen is used in the assay.)
    (H) Leave a column of wells blank for an RBC control.
    (I) Agitate gently and incubate for 30 minutes at room temperature.
    (J) Add 50 L of 0.5 percent RBC's to all wells. Note: Do 
not agitate after RBC's have been added (agitation may result in false 
positive reactions by causing the RBC's to fall, resulting in ``false'' 
buttons).
    (K) Cover the plate with sealing tape. Incubate at room temperature 
for 30 minutes or until control RBC's give a tight button.
    (L) Read the reaction on a mirrored plate reader.
    (iv) Results.
    (A) The titer is reported as the reciprocal of the last dilution to 
give a tight button of RBC's. The final dilution scheme includes the 
antigen in the dilution calculation and is as follows: B=1:20, C=1:40, 
D=1:80, E=1:160, F=1:320, G=1:640, H=1:1,280.
    (B) For the assay to be valid:
    (1) The positive control sera must give a result within one 
dilution of the previously determined titer.
    (2) The negative control sera must be negative.
    (3) The backtitration of the antigen must be 1:4 or 1:8.
    (4) The RBC control must give tight, non-hemolyzed buttons.
    (5) Sera controls (well A of each test sera) must not have non-
specific agglutination or hemolysis. If negative, report as ``negative 
with non-specific agglutination or non-specific hemolysis'' or ``unable 
to evaluate due to non-specific agglutination or hemolysis'' or treat 
the serum to remove the non-specific agglutination and repeat the test. 
(See paragraph (e)(2)(v) of this section.)
    (v) Treatment to remove non-specific agglutination.
    (A) Purpose. Treatment of serum to remove non-specific 
agglutination that is interfering with HI assays.
    (B) Specimen. Serum.
    (C) Materials. Homologous RBC's (chicken or turkey), 50 percent 
solution PBS, centrifuge, incubator, 4C (refrigerator).
    (D) Procedure. (1) Prepare a 1:5 dilution of test serum by adding 
50 L of serum to 200 L of PBS.
    (2) Prepare a 50 percent solution of RBC's by adding equal volumes 
of packed RBC's to PBS. Mix well.
    (3) Add 25 L of 50 percent RBC solution to the serum 
dilutions.
    (4) Vortex gently to mix.
    (5) Incubate at 4  deg.C for 1 hour.
    (6) Centrifuge to pellet the RBC's.
    (7) Use the supernatant to perform the HI assay. Modify the 
dilution scheme in the assay to consider the initial 1:5 dilution 
prepared in the treatment. For the 1:5 dilution scheme, do not add PBS 
to row A. Add 50 L of the 1:5 treated supernatant to row A. 
Serially dilute 25 L from rows A through H. This prepares a 
serum dilution of 1:10 through 1:640 in rows B through H.
    24. In part 147, ``Subpart B--Bacteriological Examination 
Procedure,'' a new Sec. 147.10 is added to read as follows:


Sec. 147.10  Laboratory procedure recommended for the bacteriological 
examination of egg-type breeding flocks with salmonella enteritidis 
positive environments.

    Birds selected for bacteriological examination from egg-type 
breeding flocks positive for Salmonella enteritidis after environmental 
monitoring should be examined as described in Sec. 147.11(a) of this 
subpart, with the following exceptions and modifications allowed due to 
the high number of birds required for examination:
    (a) Except when visibly pathological tissues are present, direct 
culture, Sec. 147.11(a)(1) of this subpart, may be omitted; and
    (b) Enrichment culture of organ (non-intestinal) tissues using a 
non- selective broth, Sec. 147.11(a)(2) of this subpart, may be 
omitted.
    25. Section 147.11 is amended as follows:
    a. Footnotes 1 through 4 and their references in the regulatory 
text are redesignated as footnotes 7 through 10.
    b. Paragraphs (a) through (j) are redesignated as follows: 

------------------------------------------------------------------------
            Old section                          New section            
------------------------------------------------------------------------
147.11(a)..........................  147.11(b)(1).                      
147.11(b) introductory text........  147.11(b)(2) introductory tex.t    
147.11(b)(1).......................  147.11(b)(2)(i).                   
147.11(b)(2).......................  147.11(b)(2)(ii).                  
147.11(b)(3).......................  147.11(b)(2)(iii).                 
147.11(b)(4).......................  147.11(b)(2)(iv).                  
147.11(b)(5).......................  147.11(b)(2)(v).                   
147.11(c) introductory text........  147.11(b)(3) introductory text.    
147.11(c)(1).......................  147.11(b)(3)(i).                   
147.11(c)(2).......................  147.11(b)(3)(ii).                  
147.11(c)(3).......................  147.11(b)(3)(iii).                 
147.11(c)(4).......................  147.11(b)(3)(iv).                  
147.11(c)(5).......................  147.11(b)(3)(v).                   
147.11(c)(6).......................  147.11(b)(3)(vi).                  
147.11(d)..........................  147.11(b)(4).                      
147.11(e)..........................  147.11(b)(5).                      
147.11(f)..........................  147.11(b)(6).                      
147.11(g)..........................  147.11(b)(7).                      
147.11(h)..........................  147.11(b)(8).                      
147.11(i)..........................  147.11(b)(9).                      
147.11(j)..........................  147.11(b)(10).                     
------------------------------------------------------------------------

    c. A new paragraph (a) and a paragraph heading for paragraph (b) 
are added to read as set forth below.
    d. At the end of the regulatory text of the section, the words 
``(Approved by the Office of Management and Budget under control number 
0579- 0007)'' are added.


Sec. 147.11  Laboratory procedure recommended for the bacteriological 
examination of salmonella.

    (a) For egg- and meat-type chickens, waterfowl, exhibition poultry, 
and game birds. All reactors to the Pullorum-Typhoid tests, up to at 
least four birds, should be cultured in accordance with both direct 
(paragraph (a)(1)) and selective enrichment (paragraph (a)(2)) 
procedures described in this section. Careful aseptic technique should 
be used when collecting all tissue samples.
    (1) Direct culture (refer to illustration 1). Grossly normal or 
diseased liver, heart, pericardial sac, spleen, lung, kidney, 
peritoneum, gallbladder, oviduct, misshapen ova or testes, inflamed or 
unabsorbed yolk sac, and other visibly pathological tissues where 
purulent, necrotic, or proliferative lesions are seen (including cysts, 
abscesses, hypopyon, and inflamed serosal surfaces), should be sampled 
for direct culture using either flamed wire loops or sterile swabs. 
Since some strains may not dependably survive and grow in certain 
selective media, inoculate non-selective plates in addition to two 
selective plating media. Refer to illustration 1 for recommended 
bacteriological recovery and identification procedures.\6\ Proceed 
immediately with collection of organs and tissues for selective 
enrichment culture.
---------------------------------------------------------------------------

    \6\Biochemical identification charts may be obtained from ``A 
Laboratory Manual for the Isolation and Identification of Avian 
Pathogens,'' chapter 1, Salmonellosis. Third edition, 1989, American 
Association of Avian Pathologists, Inc., Kendall/Hunt Publishing 
Co., Dubuque, IA 52004-0539.
---------------------------------------------------------------------------

    (2) Selective enrichment culture (refer to illustration 2). Collect 
and culture organ samples separately from intestinal samples, with 
intestinal tissues collected last to prevent cross-contamination. 
Samples from the following organs or sites should be collected for 
culture in selective enrichment broth. A non-selective broth culture 
(illustration 1) of pooled organs and sites should also be included as 
described in paragraph (a)(3) of this section.
    (i) Heart (apex, pericardial sac, and contents if present);
    (ii) Liver (portions exhibiting lesions or, in grossly normal 
organs, the drained gallbladder and adjacent liver tissues);
    (iii) Ovary-Testes (entire inactive ovary or testes, but if ovary 
is active, include any atypical ova);
    (iv) Oviduct (if active, include any debris and dehydrated ova);
    (v) Kidneys and spleen; and
    (vi) Other visible pathological sites where purulent, necrotic, or 
proliferative lesions are seen.
    (3) From each reactor, aseptically collect 10 to 15 g, or the 
nearest lesser amount available, from each organ or site listed in 
paragraph (a)(2) of this section and mince, grind, and blend them 
completely in 10 times their volume of beef extract broth or a 
comparable non-selective broth. Organs or sites listed in paragraph 
(a)(2) of this section may be pooled from the same individual bird. 
Suspensions should be transferred in 10-ml aliquots to 100 ml of both 
tetrathionate brilliant green (TBG) (Hajna or Mueller-Kauffmann) broth 
and a separate non-selective broth and incubated at 37  deg.C for 24 
hours. Refer to illustration 2 for recommended bacteriological recovery 
and identification procedures, including delayed secondary enrichment 
and combinations of plating media that significantly suppress the 
overgrowth of contaminants, such as brilliant green Novobiocin (BGN) 
and Xylose-Lysine- Tergitol 4 (XLT4).
    (4) From each reactor, make a composite sample of the following 
parts of grossly normal or diseased tissues from the digestive tract: 
Crop wall, duodenum (including portions of the pancreas), jejunum 
(including remnant of yolk-sac attachment), both ceca, cecal tonsils, 
and rectum-cloaca. Aseptically collect 10-15 g or the nearest lesser 
amount available from each specified digestive or intestinal tissue, 
and mince, grind, and blend them completely in 10 times their volume of 
TBG broth. The digestive/intestinal tissues may be pooled from the same 
individual bird. Do not pool tissues from different birds. Transfer 10 
ml of the described digestive TBG suspensions into 100 ml of TBG broth, 
and incubate at 41.5  deg.C for 24 hours. Cultures may be incubated at 
37  deg.C if 41.5  deg.C incubators are not available. The higher 
incubation temperatures for TBG broth reduce populations of competitive 
contaminants common in gut tissue. Refer to illustration 2 for 
recommended bacteriological recovery and identification procedures, 
including delayed secondary enrichment and combinations of plating 
media that significantly suppress the overgrowth of contaminants, such 
as BGN and XLT4.
    (5) A system such as the Analytical Profile Index for 
Enterobacteriaceae (API) may be utilized to aid cultural 
identifications.
    (6) All isolates culturally identified as salmonellae should be 
serogrouped or serotyped.

BILLING CODE 3410-34-P

TR18MR94.003


TR18MR94.004


BILLING CODE 3410-34-C
    (b) For turkeys. * * *
* * * * *


Sec. 147.12  [Amended]

    26. In Sec. 147.12, paragraph (c)(2), footnote 1 and its reference 
in the text are redesignated as footnote 11.
    27. In Sec. 147.12, at the end of the regulatory text, the words 
``(Approved by the Office of Management and Budget under control number 
0579-0007)'' are added.


Sec. 147.13  [Amended]

    28. In Sec. 147.13, at the end of the regulatory text, the words 
``(Approved by the Office of Management and Budget under control number 
0579-0007)'' are added.
    29. Section 147.14 is amended as follows:
    a. In the section heading, footnote 1 and its reference are 
redesignated as footnote 12; the reference is removed from the section 
heading and added to the introductory text of Sec. 147.14, immediately 
after the word ``procedures''; and the text of newly redesignated 
footnote 12 is amended by removing the designations ``(a)'' and ``(b)'' 
and by adding a comma after ``1980''.
    b. In the introductory text of paragraph (a)(2), the second 
sentence is revised and paragraphs (a)(2)(i) and (a)(2)(ii) added to 
read as set forth below.


Sec. 147.14  Procedures to determine status and effectiveness of 
sanitation monitored programs.

* * * * *
    (a) * * *
    (2) * * * Such eggs should also be cultured for the dependable 
recovery of salmonellae. Culturing for the dependable recovery of 
salmonellae should include the use of:
    (i) Preenrichment broths supplemented with 35 mg ferrous sulfate 
per 1,000 ml preenrichment to block iron-binding, Salmonella-inhibiting 
effects of egg conalbumin; and
    (ii) Tetrathionate selective enrichment broths, competitor-
controlling plating media (XLT4, BGN, etc.), and delayed secondary 
enrichment procedures detailed in illustration 2 of Sec. 147.11(a) of 
this part.


Secs. 147.15 and 147.16  [Amended]

    30. In Secs. 147.15 and 147.16, footnotes 4 through 12 and their 
references in the regulatory text are redesignated as footnotes 13 
through 21, respectively.


Sec. 147.21  [Amended]

    31. In Sec. 147.21, at the end of the regulatory text, the words 
``(Approved by the Office of Management and Budget under control number 
0579-0007)'' are added.


Sec. 147.41  [Amended]

    32. Section 147.41 is amended by removing all paragraph 
designations and rearranging the definitions in alphabetical order.


Sec. 147.43  [Amended]

    33. In Sec. 147.43, in the introductory text of paragraph (a), the 
words ``Transportation Services'' are removed and the words 
``Inspection Services'' added in their place.

    Done in Washington, DC, this 11th day of March 1994.
Patricia Jensen,
Acting Assistant Secretary, Marketing and Inspection Services.
[FR Doc. 94-6187 Filed 3-17-94; 8:45 am]
BILLING CODE 3410-34-P