[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.
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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
