00-3832. National Poultry Improvement Plan and Auxiliary Provisions

AGENCY:

Animal and Plant Health Inspection Service, USDA.

ACTION:

Final rule.

SUMMARY:

We are amending the National Poultry Improvement Plan (the Plan) and its auxiliary provisions by establishing new program classifications and providing new or modified sampling and testing procedures for Plan participants and participating flocks. These changes were voted on and approved by the voting delegates at the Plan's 1998 National Plan Conference. These changes will keep the provisions of the Plan current with changes in the poultry industry and provide for the use of new sampling and testing procedures.

EFFECTIVE DATE:

March 20, 2000.

FOR FURTHER INFORMATION CONTACT:

Mr. Andrew R. Rhorer, Senior Coordinator, Poultry Improvement Staff, National Poultry Improvement Plan, Veterinary Services, APHIS, USDA, 1498 Klondike Road, Suite 200, Conyers, GA 30094-5104; [phone].

SUPPLEMENTARY INFORMATION:

Background

The National Poultry Improvement Plan (NPIP, also 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, the regulations in 9 CFR part 82, subpart C, which provide for certain testing, restrictions on movement, and other restrictions on certain chickens, eggs, and other articles due to the presence of Salmonella enteritidis, 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. S. Enteritidis Monitored” under the Plan or have met equivalent requirements for S. enteritidis control, in accordance with 9 CFR 145.23(d), under official supervision.

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. The Animal and Plant Health Inspection Service (APHIS) amends these provisions from time to time to incorporate new scientific information and technologies within the Plan.

On August 10, 1999, we published in the Federal Register (64 FR 43301-43314, Docket No. 98-096-1) a proposal to amend the regulations to:

1. Establish two new classifications: “U.S. Avian Influenza Clean” for primary and multiplier egg- and meat-type breeding chicken flocks and “U.S. Mycoplasma Meleagridis Clean State, Turkeys.”

2. Identify the agar gel immunodiffusion (AGID) test and the enzyme-linked immunosorbent assay (ELISA) as official tests for avian influenza in the Plan.

3. Allow the use of Food and Drug Administration (FDA) approved feed sanitizing agents or salmonella control products in certain chicken and turkey breeding flocks.

4. Eliminate references to Salmonella typhimurium throughout the regulations.

5. Add the colony lift assay for group D salmonella and eliminate the referral of all group D salmonella to APHIS' National Veterinary Services Laboratories (NVSL) in the laboratory protocol for isolation and identification of salmonella in breeding turkeys.

6. Make several changes to the duties of the General Conference Committee of the NPIP.

7. Establish technical protocol for culturing chick meconium.

8. Provide for the use of either chick papers or meconium as testing samples in the “U.S. Salmonella Monitored” program of meat-type breeding chickens.

9. Amend the procedure for determining the status of a flock reacting to tests for Mycoplasma gallisepticum, M. synoviae, and M. meleagridis.

10. Provide for the participation of emu, rhea, and cassowary breeding flocks in the provisions of the Plan.

11. Remove exceptions to the requirements for pullorum typhoid clean States that pertain to turkey hatcheries or supply flocks.

12. Add or amend several definitions.

We solicited comments concerning our proposal for 60 days ending on October 12, 1999. We received one comment by that date. The comment was from a retired State animal health official. The commenter suggested that the 35 °C plate incubation temperature called for in paragraph (f) of proposed § 147.18, “Chick meconium testing procedure for salmonella,” be changed to 37 °C, which is the temperature used for the incubation of plates in the procedure set forth in the current regulations in paragraph (a) of § 147.11, “Laboratory procedure recommended for the bacteriological examination of salmonella.” We agree that the incubation temperature in §§ 147.11(a) and 147.18(f) should be consistent and have made the commenter's suggested change in § 147.18(f) of this final rule.

Therefore, for the reasons given in the proposed rule and in this document, we are adopting the proposed rule as a final rule, with the change discussed in this document.

Executive Order 12866 and Regulatory Flexibility Act

This rule has been reviewed under Executive Order 12866. The rule has been determined to be not significant for the 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 1998 National Plan Conference. This rule amends the Plan and its auxiliary provisions by establishing new program classifications and providing new or modified sampling and testing procedures for Plan participants and participating flocks. These changes, which were voted on and approved by the voting delegates at the Plan's 1998 National Plan Conference, will keep the provisions of the Plan current with changes in the poultry industry and provide for the use of new sampling and testing procedures.

The Plan serves as a “seal of approval” for egg and poultry producers in the sense that tests and procedures recommended by the Plan are considered optimal for the industry. In all cases, the changes made by this rule have been generated by the industry itself with the goal of reducing disease risk and increasing product marketability. 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.

Assuming they wished to voluntarily remain in the program, the cost to comply with this rule's protocols, tests, classification schemes, etc. will be borne primarily by the approximately 12 primary breeders in NPIP. However, the net economic effect of the changes on those breeders is expected to be positive over the long term. This is because the breeders' compliance costs should be more than offset by the expected benefits resulting from compliance (i.e., increased U.S. poultry exports). U.S. exports are expected to increase because, by serving to reduce disease risk, the protocols and procedures should make domestic poultry more marketable in foreign markets. That the net economic effect of the changes on the poultry industry is expected to be positive is evidenced by the fact that it was the NPIP's industry participants who initiated the changes.

The precise dollar amount of the costs that the breeders will incur to comply with this rule is not available. However, those costs are not expected to be significant, especially since many of the changes are no more than technical corrections to the provisions of the Plan or are intended to bring those provisions into conformity with current developments in the scientific community. In 1997, the dollar value of U.S. exports of meat and edible offal of poultry (fresh, chilled, and frozen) totaled $2.2 billion (World Trade Atlas, September 1998 edition). Even if exports increase by only 1 percent as a result of this rule, the benefit would be $22 million.

In any event, the breeder participants in NPIP always have the option of withdrawing from the Plan, in which case they would not be subject to this rule. As indicated above, industry participation in the NPIP is voluntary.

Economic Effects on Small Entities

The Regulatory Flexibility Act requires that agencies consider the economic effects of their rules on small entities (i.e., small businesses, organizations, and governmental jurisdictions). This rule is not expected to have a significant economic effect on a substantial number of small entities, if for no other reason than few, if any, of those entities most affected by its provisions—NPIP-participating breeders and producers—are small in size. The U.S. Small Business Administration's small entity threshold for almost all standard industrial classification categories for poultry and egg producers is annual revenues of $0.5 million or less. We believe that most, if not all, breeders and producers participating in the Plan generate annual revenues in excess $0.5 million.

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 12988

This rule has been reviewed under Executive Order 12988, 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 1995 (44 U.S.C. 3501 et seq.), the information collection or recordkeeping requirements included in this rule have been approved by the Office of Management and Budget (OMB) under OMB control number 0579-0007.

List of Subjects in 9 CFR Parts 145 and 147

• Animal diseases
• Poultry and poultry products
• Reporting and recordkeeping requirements

Accordingly, we are amending 9 CFR parts 145 and 147 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 2.22, 2.80, and 371.2(d).

2. Section 145.1 is amended as follows:

a. The definition of authorized laboratory is revised to read as set forth below.

b. The definition of baby poultry is revised to read as set forth below.

c. A new definition of independent flock is added, in alphabetical order, to read as set forth below.

d. The definition of poultry is amended by adding the words “emus, rheas, cassowaries,” immediately after the word “ostriches,”.

e. The definition of S. typhimurium infection or typhimurium is removed.

§ 145.1

Definitions.

* * * * *

Authorized laboratory. A laboratory designated by an Official State Agency, subject to review by the Service, to perform the blood testing and bacteriological examinations provided for in this part. The Service's review will include, but will not necessarily be limited to, checking records, laboratory protocol, check-test proficiency, periodic duplicate samples, and peer review. A satisfactory review will result in the authorized laboratory being recognized by the Service as a nationally approved laboratory qualified to perform the blood testing and bacteriological examinations provided for in this part.

Baby poultry. Newly hatched poultry (chicks, poults, ducklings, goslings, keets, etc.).

* * * * *

Independent flock. A flock that produces hatching eggs and that has no ownership affiliation with a specific hatchery.

* * * * *

§ 145.3

[Amended]

3. In § 145.3, the introductory text of paragraph (c) is amended by adding the words “emus, rheas, cassowaries,” immediately after the word “ostriches,”.

4. In § 145.6, paragraph (e) is redesignated as paragraph (f) and a new paragraph (e) is added to read as follows:

§ 145.6

Specific provisions for participating hatcheries.

* * * * *

(e) Any nutritive material provided to baby poultry must be free of the avian pathogens that are officially represented in the Plan disease classifications listed in § 145.10.

* * * * *

5. In § 145.10, new paragraphs (r) and (s) are added to read as follows:

§ 145.10

Terminology and classification; flocks, products, and States.

* * * * *

(r) U.S. Avian Influenza Clean. (See §§ 145.23(h) and 145.33(l).)

(s) U.S. M. Meleagridis Clean State, Turkeys. (See § 145.44(e).)

6. Section 145.14 is amended as follows:

a. In the introductory text, at the end of the first sentence, the words “and ostriches blood tested under subpart F must be more than 12 months of age” are removed and the words “and ostrich, emu, rhea, and cassowary candidates must be blood tested when at least 12 months of age or upon reaching sexual maturity, depending upon the species and at the discretion of the Official State Agency” are added in their place.

b. A new paragraph (d) is added to read as follows:

§ 145.14

Blood testing.

* * * * *

(d) For avian influenza. The official blood tests for avian influenza are the agar gel immunodiffusion (AGID) test and the enzyme-linked immunosorbent assay (ELISA).

(1) The AGID test must be conducted on all ELISA-positive samples. Positive tests by AGID or ELISA must be further tested by Federal Reference Laboratories. Final judgment may be based upon further sampling or culture results.

(2) The tests must be conducted using antigens or test kits approved by the Department and the Official State Agency and must be performed in accordance with the recommendations of the producer or manufacturer.

* * * * *

7. In § 145.21, the definition of chicks is revised to read as follows:

§ 145.21

Definitions.

* * * * *

Chicks. Newly hatched chickens.

* * * * *

8. In § 145.22, a new paragraph (e) is added to read as follows:

§ 145.22

Participation.

* * * * *

(e) Any nutritive material provided to chicks must be free of the avian pathogens that are officially represented in the Plan disease classifications listed in § 145.10.

9. Section 145.23 is amended as follows:

a. In paragraph (b)(3)(i), the words “, except turkey hatcheries,” are removed.

b. In paragraph (b)(3)(ii), the words “, except turkey flocks,” are removed.

c. In paragraph (b)(3)(viii), the words “, other than turkey flocks,” are removed.

d. In paragraph (b)(4), the words “, other than turkey, waterfowl, exhibition poultry, and game bird supply flocks,” are removed.

e. Paragraph (d)(1)(ii)(B) is revised to read as follows.

§ 145.23

Terminology and classification; flocks and products.

* * * * *

(d) * * *

(1) * * *

(ii) * * *

(B) Mash feed may contain no animal protein other than an APPI animal protein product supplement manufactured in pellet form and crumbled: Provided, that mash feed may contain nonpelleted APPI animal protein product supplements if the finished feed is treated with a salmonella control product approved by the Food and Drug Administration.

* * * * *

f. A new paragraph (h) is added to read as follows:

(h) U.S. Avian Influenza Clean. This program is intended to be the basis from which the breeding-hatchery industry may conduct a program for the prevention and control of avian influenza. It is intended to determine the presence of avian influenza in breeding chickens through routine serological surveillance of each participating breeding flock. A flock and the hatching eggs and chicks produced from it will qualify for this classification when the Official State Agency determines that they have met one of the following requirements:

(1) It is a primary breeding flock in which a minimum of 30 birds have been tested negative for antibodies to avian influenza when more than 4 months of age. To retain this classification:

(i) A sample of at least 30 birds must be tested negative at intervals of 90 days; or

(ii) A sample of fewer than 30 birds may be tested, and found to be negative, at any one time if all pens are equally represented and a total of 30 birds is tested within each 90-day period.

(2) It is a multiplier breeding flock in which a minimum of 30 birds have been tested negative for antibodies to avian influenza when more than 4 months of age. To retain this classification:

(i) A sample of at least 30 birds must be tested negative at intervals of 180 days; or

(ii) A sample of fewer than 30 birds may be tested, and found to be negative, at any one time if all pens are equally represented and a total of 30 birds is tested within each 180-day period.

* * * * *

10. In § 145.31, the definition of chicks is revised to read as follows:

§ 145.31

Definitions.

* * * * *

Chicks. Newly hatched chickens.

* * * * *

11. In § 145.32, a new paragraph (d) is added to read as follows:

§ 145.32

Participation.

* * * * *

(d) Any nutritive material provided to chicks must be free of the avian pathogens that are officially represented in the Plan disease classifications listed in § 145.10.

12. Section 145.33 is amended as follows:

a. In paragraph (b)(3)(i), the words “, except turkey hatcheries,” are removed.

b. In paragraph (b)(3)(ii), the words “, except turkey flocks,” are removed.

c. In paragraph (b)(3)(viii), the words “, other than turkey flocks,” are removed.

d. In paragraph (b)(4), the words “, other than turkey, waterfowl, exhibition poultry, and game bird supply flocks,” are removed.

e. In paragraph (h)(1)(ii)(A), at the end of the first sentence, the acronym “(NMFS)” is added after the word “Service”.

f. Paragraph (h)(1)(ii)(B) is revised to read as set forth below.

g. Paragraph (i)(1)(vi) is amended by removing the words “meconium and” and adding the words “meconium or” in their place.

h. A new paragraph (l) is added to read as follows.

§ 145.33

Terminology and classification; flocks and products.

* * * * *

(h) * * *

(1) * * *

(ii) * * *

(B) Mash feed may contain no animal protein other than an APPI/NMFS animal protein product supplement manufactured in pellet form and crumbled: Provided, that mash feed may contain nonpelleted APPI/NMFS animal protein product supplements if the finished feed is treated with a salmonella control product approved by the Food and Drug Administration.

* * * * *

(l) U.S. Avian Influenza Clean. This program is intended to be the basis from which the breeding-hatchery industry may conduct a program for the prevention and control of avian influenza. It is intended to determine the presence of avian influenza in primary breeding chickens through routine serological surveillance of each participating breeding flock. A flock and the hatching eggs and chicks produced from it will qualify for this classification when the Official State Agency determines that they have met one of the following requirements:

(1) It is a primary breeding flock in which a minimum of 30 birds have been tested negative for antibodies to avian influenza when more than 4 months of age. To retain this classification:

(i) A sample of at least 30 birds must be tested negative at intervals of 90 days; or

(ii) A sample of fewer than 30 birds may be tested, and found to be negative, at any one time if all pens are equally represented and a total of 30 birds is tested within each 90-day period.

(2) It is a multiplier breeding flock in which a minimum of 30 birds have been tested negative for antibodies to avian influenza when more than 4 months of age. To retain this classification:

(i) A sample of at least 30 birds must be tested negative at intervals of 180 days; or

(ii) A sample of fewer than 30 birds may be tested, and found to be negative, at any one time if all pens are equally represented and a total of 30 birds is tested within each 180-day period.

* * * * *

13. In § 145.41, the definition of poults is revised to read as follows:

§ 145.41

Definitions.

* * * * *

Poults. Newly hatched turkeys.

14. In § 145.42, a new paragraph (d) is added to read as follows:

§ 145.42

Participation.

* * * * *

(d) Any nutritive material provided to poults must be free of the avian pathogens that are officially represented in the Plan disease classifications listed in § 145.10.

15. In § 145.43, paragraphs (f)(3)(ii) and (f)(3)(iii) are revised to read as follows:

§ 145.43

Terminology and classification; flocks and products.

* * * * *

(f) * * *

(3) * * *

(ii) Initial feed for poults to 2 weeks of age must be manufactured in pellet form. Initial feed may contain no animal protein other than animal protein products produced under the Animal Protein Products Industry (APPI) Salmonella Education/Reduction Program or the Fishmeal Inspection Program of the National Marine Fisheries Service (NMFS). Finished feed must be treated with a Food and Drug Administration (FDA) approved salmonella control product at FDA-approved levels.

(iii) Succeeding feed for turkeys 2 weeks or older must be either:

(A) Pelleted feed that meets the requirements of paragraph (f)(3)(ii) of this section; or

(B) Mash feed that contains no animal protein products; or

(C) Mash feed that contains an APPI/NMFS animal protein products supplement that has been manufactured in pellet form and crumbled. Finished feed must be treated with an FDA-approved salmonella control product at FDA-approved levels.

* * * * *

16. In § 145.44, a new paragraph (e) is added to read as follows:

§ 145.44

Terminology and classification; States.

* * * * *

(e) U.S. M. Meleagridis Clean State, Turkeys. (1) A State will be declared a U.S. M. Meleagridis Clean State, Turkeys, if the Service determines that:

(i) No Mycoplasma meleagridis is known to exist nor to have existed in turkey breeding flocks in production within the State during the preceding 12 months;

(ii) All turkey breeding flocks in production are tested and classified as U.S. M. Meleagridis Clean or have met equivalent requirements for M. meleagridis control under official supervision;

(iii) All turkey hatcheries within the State only handle products that are classified as U.S. M. Meleagridis Clean or have met equivalent requirements for M. meleagridis control under official supervision;

(iv) All shipments of products from turkey breeding flocks other than those classified as U.S. M. Meleagridis Clean, or equivalent, into the State are prohibited;

(v) All persons performing poultry disease diagnostic services within the State are required to report to the Official State Agency within 48 hours the source of all turkey specimens that have been identified as being infected with M. meleagridis;

(vi) All reports of M. meleagridis infection in turkeys are promptly followed by an investigation by the Official State Agency to determine the origin of the infection; and

(vii) All turkey breeding flocks found to be infected with M. meleagridis are quarantined until marketed under supervision of the Official State Agency.

(2) The Service may revoke the State's classification as a U.S. M. Meleagridis Clean State, Turkeys, if any of the conditions described in paragraph (d)(1) of this section are discontinued. The Service will not revoke the State's classification as a U.S. M. Meleagridis Clean State, Turkeys, until it has conducted an investigation and the Official State Agency has been given an opportunity for a hearing in accordance with rules of practice adopted by the Administrator.

* * * * *

17. In § 145.52, a new paragraph (d) is added to read as follows:

§ 145.52

Participation.

* * * * *

(d) Any nutritive material provided to baby poultry must be free of the avian pathogens that are officially represented in the Plan disease classifications listed in § 145.10.

§ 145.53

[Amended]

18. In § 145.53, paragraph (b) is amended as follows:

a. In paragraph (b)(3)(i), the words “, except turkey hatcheries,” are removed.

b. In paragraph (b)(3)(ii) the words “, except turkey flocks,” are removed.

c. In paragraph (b)(3)(viii), the words “, other than turkey flocks,” are removed.

d. In paragraph (b)(4), the words “, other than turkey flocks,” are removed.

19. The subpart heading for subpart F is revised to read as follows:

Subpart F—Special Provisions for Ostrich, Emu, Rhea, and Cassowary Breeding Flocks and Products

20. In 145.61, a definition of chicks is added, in alphabetical order, to read as follows:

§ 145.61

Definitions.

* * * * *

Chicks. Newly hatched ostriches, emus, rheas, or cassowaries.

* * * * *

21. In § 145.62, the introductory text of the section is amended by adding the words “emus, rheas, and cassowaries,” immediately after the word “ostriches,” and a new paragraph (c) is added to read as follows:

§ 145.62

Participation.

* * * * *

(c) Any nutritive material provided to chicks must be free of the avian pathogens that are officially represented in the Plan disease classifications listed in § 145.10.

§ 145.63

[Amended]

22. In § 145.63, paragraph (a)(2) is amended by adding the words “, emus, rheas, or cassowaries” immediately after the word “ostriches”.

PART 147—AUXILIARY PROVISIONS ON NATIONAL POULTRY IMPROVEMENT PLAN

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

Authority: 7 U.S.C. 429; 7 CFR 2.22, 2.80, and 371.2(d).

§ 147.4

[Removed and reserved]

24. Section 147.4 is removed and reserved.

25. In § 147.6, paragraph (a)(14) is revised to read as follows:

§ 147.6

Procedure for determining the status of flocks reacting to tests for Mycoplasma gallisepticum, Mycoplasma synoviae, and Mycoplasma meleagridis.

* * * * *

(a) * * *

(14) If the in vivo bio-assay, PCR-based procedures, or culture procedures are positive, the flock will be considered infected. However, the following considerations may apply:

(i) In PCR-positive flocks for which there are other negative mycoplasma test results, the flock's mycoplasma status should be confirmed through either seroconversion or culture isolation of the organism, or through both methods, before final determination of the flock's status is made.

(ii) In flocks for which only the bio-assay is positive, additional in vivo bio-assay, PCR-based procedures, or cultural examinations may be conducted by the Official State Agency before final determination of the flock's status is made.

* * * * *

§§ 147.11, 147.12, 147.14, 147.15, 147.16

[Footnotes redesignated]

26. In §§ 147.11, 147.12, 147.14, 147.15, 147.16, footnotes 6 through 22 and their references are redesignated as footnotes 7 through 23, respectively.

27. A new § 147.9 is added to read as follows:

§ 147.9

Standard test procedures for avian influenza.

(a) The agar gel immunodiffusion (AGID) test should be considered the basic screening test for antibodies to Type A influenza viruses. The AGID test is used to detect circulating antibodies to Type A influenza group-specific antigens, namely the ribonucleoprotein (RNP) and matrix (M) proteins. Therefore, this test will detect antibodies to all influenza A viruses, regardless of subtype. The AGID test can also be used as a group-specific test to identify isolates as Type A influenza viruses. The method used is similar to that described by Beard. ^[6] The basis for the AGID test is the concurrent migration of antigen and antibodies toward each other through an agar gel matrix. When the antigen and specific antibodies come in contact, they combine to form a precipitate that is trapped in the gel matrix and produces a visible line. The precipitin line forms where the concentration of antigen and antibodies is optimum. Differences in the relative concentration of the antigen or antibodies will shift the location of the line towards the well with the lowest concentration or result in the absence of a precipitin line. Electrolyte concentration, pH, temperature, and other variables also affect precipitate formation.

(1) Materials needed.

(i) Refrigerator (4 °C).

(ii) Freezer (−20 °C).

(iii) Incubator or airtight container for room temperature (approximately 25 °C) incubations.

(iv) Autoclave.

(v) Hot plate/stirrer and magnetic stir bar (optional).

(vi) Vacuum pump.

(vii) Microscope illuminator or other appropriate light source for viewing results.

(viii) Immunodiffusion template cutter, seven-well pattern (a center well surrounded by six evenly spaced wells). Wells are 5.3 mm in diameter and 2.4 mm apart.

(ix) Top loading balance (capable of measuring 0.1 gm differences).

(x) Pipetting device capable of delivering 50μl portions.

(xi) Common laboratory supplies and glassware—Erlenmeyer flasks, graduated cylinders, pipettes, 100 × 15 mm or 60 × 15 mm petri dishes, flexible vacuum tubing, side-arm flask (500 mL or larger), and a 12- or 14-gauge blunt-ended cannula.

(2) Reagents needed.

(i) Phosphate buffered saline (PBS), 0.01M, pH 7.2 (NVSL media #30054 or equivalent).

(ii) Agarose (Type II Medium grade, Sigma Chemical Co. Cat.# A-6877 or equivalent).

(iii) Avian influenza AGID antigen and positive control antiserum approved by the Department and the Official State Agency.

(iv) Strong positive, weak positive, and negative control antisera approved by the Department and the Official State Agency (negative control antisera optional).

(3) Preparing the avian influenza AGID agar.

(i) Weigh 9 gm of agarose and 80 gm of NaCl and add to 1 liter of PBS (0.01 M, pH 7.2) in a 2 liter Erlenmeyer flask.

(ii) To mix the agar, either:

(A) Autoclave the mixture for 10 minutes and mix the contents by swirling after removing from the autoclave to ensure a homogeneous mixture of ingredients; or

(B) Dissolve the mixture by bringing to a boil on a hot plate using a magnetic stir bar to mix the contents in the flask while heating. After boiling, allow the agar to cool at room temperature (approximately 25 °C) for 10 to 15 minutes before dispensing into petri plates.

(iii) Agar can be dispensed into small quantities (daily working volumes) and stored in airtight containers at 4 °C for several weeks, and melted and dispensed into plates as needed.

Note:

Do not use agar if microbial contamination or precipitate is observed.

(4) Performing the AGID. (i) Detection of serum antibodies.

(A) Dispense 15 to 17 mL of melted agar into a 100 × 15 mm petri plate or 5 to 6 mL agar into a 60 × 15 mm petri plate using a 25 mL pipette. The agar thickness should be approximately 2.8 mm.

(B) Allow plates to cool in a relatively dust-free environment with the lids off to permit the escape of water vapor. The lids should be left off for at least 15 minutes, but not longer than 30 minutes, as electrolyte concentration of the agar may change due to evaporation and adversely affect formation of precipitin lines.

Note:

Plates should be used within 24 hours after they are poured.

(C) Record the sample identification, reagent lot numbers, test date, and identification of personnel performing and reading the test.

(D) Using the template, cut the agar after it has hardened. Up to seven template patterns can be cut in a 100×15 mm plate and two patterns can be cut in a 60×15 mm plate.

(E) Remove the agar plugs by aspiration with a 12- to 14-gauge cannula connected to a side arm flask with a piece of silicone or rubber tubing that is connected to a vacuum pump with tubing. Adjust the vacuum so that the agar surrounding the wells is not disturbed when removing the plugs.

(F) To prepare the wells, either:

(1) Place 50 μl of avian influenza AGID antigen in the center well using a micropipette with an attached pipette tip. Place 50 μl AI AGID positive control antiserum in each of two opposite wells, and add 50 μl per well of test sera in the four remaining wells. This arrangement provides a positive control line on one side of the test serum, thus providing for the development of lines of identity (see figure 1); or

(2) Place 50 μl AI AGID positive control antiserum in each of three alternate peripheral wells, and add 50 μl per well of test sera in the three remaining wells. This arrangement provides a positive control line on each side of the test serum, thus providing for the development of lines of identity on both sides of each test serum (see figure 2).

Note:

A pattern can be included with positive, weak positive, and negative reference serum in the test sera wells to aid in the interpretation of results (see figure 3).

(G) Cover each plate after filling all wells and allow the plates to incubate for 24 hours at room temperature (approximately 25 °C) in a closed chamber to prevent evaporation. Humidity should be provided by placing a damp paper towel in the incubation chamber. Note: Temperature changes during migration may lead to artifacts.

(ii) Interpretation of test results.

(A) Remove the lid and examine reactions from above by placing the plate(s) over a black background, and illuminate the plate with a light source directed at an angle from below. A microscope illuminator works well and allows for varying intensities of light and positions.

(B) The type of reaction will vary with the concentration of antibody in the sample being tested. The positive control serum line is the basis for reading the test. If the line is not distinct, the test is not valid and must be repeated. The following types of reactions are observed (see figure 3):

(1) Negative reaction. The control lines continue into the test sample well without bending or with a slight bend away from the antigen well and toward the positive control serum well.

(2) Positive reaction. The control lines join with, and form a continuous line (line of identity) with, the line between the test serum and antigen. The location of the line will depend on the concentration of antibodies in the test serum. Weakly positive samples may not produce a complete line between the antigen and test serum but may only cause the tip or end of the control line to bend inward toward the test well.

(3) Non-specific lines. These lines occasionally are observed between the antigen and test serum well. The control lines will pass through the non-specific line and continue on into the test serum well. The non-specific line does not form a continuous line with positive control lines.

(b) The enzyme-linked immunosorbent assay (ELISA) may be used as a screening test for avian influenza. Use only federally licensed ELISA kits and follow the manufacturer's instructions. All ELISA-positive serum samples must be confirmed with the AGID test conducted in accordance with paragraph (a) of this section.

§ 147.11

[Amended]

28. Section 147.11 is amended as follows:

a. In paragraph (b)(2)(iii) the words “A group D colony lift assay may be utilized to signal the presence of the hard-to-detect group D salmonella colonies on agar culture plates.” are added after the final sentence.

b. In paragraph (b)(2)(v), the words “at the National Veterinary Services Laboratory” are removed.

29. A new § 147.18 is added to read as follows:

§ 147.18

Chick meconium testing procedure for salmonella.

Procedure:

(a) Record the date, source, and flock destination on the “Meconium Worksheet.”

(b) Shake each plastic bag of meconium until a uniform consistency is achieved.

(c) Transfer a 25 gm sample of meconium to a sterile container. Add 225 mL of a preenrichment broth to each sample (this is a 1:10 dilution), mix gently, and incubate at 37 °C for 18-24 hours.

(d) Enrich the sample with selective enrichment broth for 24 hours at 42 °C.

(e) Streak the enriched sample onto brilliant green-Novobiocin (BGN) agar and xylose-lysine-tergitol 4 (XLT4) agar.

(f) Incubate both plates at 37 °C for 24 hours and process suspect salmonella colonies according to § 147.11.

30. In § 147.43, paragraphs (d)(1) through (d)(4) are redesignated as paragraphs (d)(3) through (d)(6), respectively, and new paragraphs (d)(1), (d)(2), (d)(7), and (d)(8) are added to read as follows:

§ 147.43

General Conference Committee.

* * * * *

(d) * * *

(1) Advise and make recommendations to the Department on the relative importance of maintaining, at all times, adequate departmental funding for the NPIP to enable the Senior Coordinator and staff to fully administer the provisions of the Plan.

(2) Advise and make yearly recommendations to the Department with respect to the NPIP budget well in advance of the start of the budgetary process.

* * * * *

(7) Serve as a direct liaison between the NPIP and the United States Animal Health Association.

(8) Advise and make recommendations to the Department regarding NPIP involvement or representation at poultry industry functions and activities as deemed necessary or advisable for the purposes of the NPIP.

§ 147.45

[Amended]

31. Section 147.45 is amended by removing the words “and E” and adding the words “E, and F” in their place.

32. In § 147.46, the introductory text of paragraph (a) is amended by removing the word “four” and adding the word “five” in its place, and a new paragraph (a)(5) is added to read as follows:

§ 147.46

Committee consideration of proposed changes.

(a) * * *

(5) Ostriches, emus, rheas, and cassowaries.

* * * * *

Done in Washington, DC, this 11th day of February 2000.

Bobby R. Acord,

Acting Administrator, Animal and Plant Health Inspection Service.

Footnotes

6.  Beard, C.W. Demonstration of type-specific influenza antibody in mammalian and avian sera by immunodiffusion. Bull. Wld. Hlth. Org. 42:779-785. 1970.