National Poultry Improvement Plan and Auxiliary Provisions

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\

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\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.

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TR18MR94.003

TR18MR94.004

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(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