National Poultry

Improvement Plan 

and Auxiliary 

Provisions

United States

Department of

Agriculture

Animal and

Plant Health

Inspection

Service

APHIS 91–55–088

June 2009

U.S.

MONITORED

NPIP

H5/H7 Avian Influenza

U.S.

CLEAN

NPIP

S. Enteritidis
Issued April 2007

Slightly Revised June 2009

The U.S. Department of Agriculture (USDA) prohibits discrimination in all its programs 

and activities on the basis of race, color, national origin, age, disability, and where applicable, sex, marital status, familial status, parental status, religion, sexual orientation, 

genetic information, political beliefs, reprisal, or because all or part of an individual’s 

income is derived from any public assistance program. (Not all prohibited bases apply 

to all programs.) Persons with disabilities who require alternative means for communication of program information (Braille, large print, audiotape, etc.) should contact 

USDA’s TARGET Center at (202) 720–2600 (voice and TDD). To file a complaint of 

discrimination, write to USDA, Director, Office of Civil Rights, 1400 Independence 

Avenue, S.W., Washington, D.C. 20250–9410, or call (800) 795–3272 (voice) or (202) 

720–6382 (TDD). USDA is an equal opportunity provider and employer.
Part 145—National Poultry Improvement Plan for Breeding Poultry

Table of Contents

Subpart A—General Provisions

Section

145.1 Definitions.

145.2 Administration.

145.3 Participation.

145.4 General provisions for all participants.

145.5 Specific provisions for participating flocks.

145.6 Specific provisions for participating hatcheries.

145.7 Specific provisions for participating dealers.

145.8 Terminology and classification; general.

145.9 Terminology and classification; hatcheries and dealers.

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

145.11 Supervision.

145.12 Inspections.

145.13 Debarment from participation.

145.14 Testing.

145.15 Diagnostic surveillance program for low pathogenic avian influenza.

 

Subpart B—Special Provisions for Multiplier Egg-Type Chicken Breeding Flocks 

and Products

 

145.21 Definitions.

145.22 Participation.

145.23 Terminology and classification; flocks and products.

145.24 Terminology and classification; States.

 

Subpart C—Special Provisions for Multiplier Meat-Type Chicken Breeding Flocks 

and Products

 

145.31 Definitions.

145.32 Participation.

145.33 Terminology and classification; flocks and products.

145.34 Terminology and classification; States.

 

Subpart D—Special Provisions for Turkey Breeding Flocks and Products

 

145.41 Definitions.

145.42 Participation.

145.43 Terminology and classification; flocks and products.

145.44 Terminology and classification; States.

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Subpart E—Special Provisions for Waterfowl, Exhibition Poultry, and 

Game Bird Breeding Flocks and Products

 

145.51 Definitions.

145.52 Participation.

145.53 Terminology and classification; flocks and products.

145.54 Terminology and classification; States.

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

Breeding Flocks and Products

145.61 Definitions.

145.62 Participation.

145.63 Terminology and classification; flocks and products.

Subpart G—Special Provisions for Primary Egg-Type Chicken Breeding 

Flocks and Products

145.71 Definitions.

145.72 Participation.

145.73 Terminology and classification; flocks and products.

Subpart H—Special Provisions for Primary Meat-Type Chicken Breeding 

Flocks and Products

145.81 Definitions.

145.82 Participation.

145.83 Terminology and classification; flocks and products.

Authority: 7 U.S.C. 8301–8317; 7 CFR 2.22, 2.80, and 371.4.

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Subpart A—General Provisions

§145.1 Definitions.

Words used in this part in the singular form shall be deemed to import the plural, and 

vice versa, as the case may demand. Except where the context otherwise requires, for 

the purposes of this part the following terms shall be construed, respectively, to mean:

The Administrator, Animal and Plant Health Inspection Service, or any person 

authorized to act for the Administrator.

A flockowner who is participating in the Plan through an agreement with a participating 

hatchery.

The Animal and Plant Health Inspection Service of the U.S. Department of Agriculture.

Any person designated under § 145.11(a) to collect official samples for submission to 

an authorized laboratory as described in §§ 147.1(a) and 147.12 of this subchapter.

An authorized laboratory is a laboratory that meets the requirements of § 147.51 and 

is thus qualified to perform the assays described in part 147 of this subchapter.

Any person designated under § 145.11(a) to collect official samples for submission to 

an authorized laboratory as described in §§ 147.1(a) and 147.12 of this subchapter 

and to perform the stained antigen, rapid whole blood test for pullorum typhoid. 

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

For the purpose of this chapter, those organisms which are gram negative, non sporeforming bacilli, which ferment lactose with gas formation, and serve as an index of 

fecal contamination.

An individual or business that deals in commerce in hatching eggs, newly-hatched 

poultry, and started poultry obtained from breeding flocks and hatcheries. This does 

not include an individual or business that deals in commerce in buying and selling 

poultry for slaughter only.

The U.S. Department of Agriculture.

Propagated and maintained under the control of a person.

Requirements which are equal to or exceed the program, conditions, criteria, or 

classifications with which they are compared, as determined by the Official State 

Agency and with the concurrence of the Service.

Contact with birds, equipment, personnel, supplies, or any article infected with, or 

contaminated by, communicable poultry disease organisms.

Affiliated flockowner 

Administrator

Authorized laboratory 

Dealer

Domesticated

Animal and Plant Health 

Inspection Service

Department

Authorized Agent

Baby poultry 

Colon bacilli

Equivalent or equivalent 

requirements 

Authorized testing agent

Exposed (Exposure)
2

(1) As applied to breeding. All poultry of one kind of mating (breed and variety or 

combination of stocks) and of one classification on one farm;

(2) As applied to disease control. All of the poultry on one farm except that, at the 

discretion of the Official State Agency, any group of poultry which is segregated 

from another group and has been so segregated for a period of at least 21 days 

may be considered as a separate flock.

Feathers, shell membrane, and other debris resulting from the hatching of poultry.

A disease of poultry caused by Salmonella gallinarum.

A breeder who normally sells products under a specific strain or trade name and who 

authorizes other hatcheries to produce and sell products under this same strain or 

trade name.

A hatchery which has been authorized by a franchise breeder to produce and sell 

products under the breeder’s strain or trade name.

Hatchery equipment on one premises operated or controlled by any person for the 

production of baby poultry.

A flock that produces hatching eggs and that has no ownership affiliation with a 

specific hatchery.

A flock in which an authorized laboratory has discovered one or more birds infected 

with a communicable poultry disease for which a program has been established under 

the Plan.

Approximately 2–3 months after a flock begins to lay or after a molted flock is put back 

into production.

A flock that is intended for the production of hatching eggs used for the purpose of 

producing progeny for commercial egg or meat production or for other non-breeding 

purposes. 

A committee made up of technical experts on poultry health, biosecurity, surveillance, 

and diagnostics. The committee consists of representatives from the poultry and egg 

industries, universities, and State and Federal governments and is appointed by the 

Senior Coordinator and approved by the General Conference Committee.

A committee made up of technical experts on poultry health, biosecurity, surveillance, 

and diagnostics. The committee consists of representatives from the poultry and egg 

industries, universities, and State and Federal governments and is appointed by the 

Senior Coordinator and approved by the General Conference Committee.

The State authority recognized by the Department to cooperate in the administration of 

the Plan.

Franchise breeder

Infected flock

Fowl typhoid or typhoid

Flock

Fluff sample

Franchise hatchery

Hatchery

Midlay

Multiplier breeding flock

Official State Agency

Independent flock

Multiplier breeding flock

NPIP Technical 

Committee 
3

(1) As applied to Plan programs. The direction, inspection, and critical evaluation by 

the Official State Agency of compliance with the provisions of the Plan;

(2) As applied to non-Plan but equivalent State poultry improvement programs. The 

direction, inspection, and critical evaluation by an officer or agency of a State 

government, of compliance with a publicly announced State poultry improvement 

program.

A natural person, firm, or corporation.

The provisions of the National Poultry Improvement Plan contained in this part.

Domesticated fowl, including chickens, turkeys, ostriches, emus, rheas, cassowaries, 

waterfowl, and game birds, except doves and pigeons, which are bred for the primary 

purpose of producing eggs or meat.

A flock composed of one or more generations that is maintained for the purpose of 

establishing, continuing, or improving parent lines.

Poultry breeding stock and hatching eggs, baby poultry, and started poultry.

Management, sanitation, testing, and monitoring procedures which, if complied with, 

will qualify, and maintain qualification for, designation of a flock, products produced 

from the flock, or a state by an official Plan classification and illustrative design, as 

described in § 145.10 of this part.

A public show of poultry.

A disease of poultry caused by Salmonella pullorum.

A bird that has a positive reaction to a test, required or recommended in Parts 145 or 

147 of this chapter, for any poultry disease for which a program has been established 

under the Plan.

Any bacteria belonging to the genus Salmonella, including the arizona group.

To treat with a product which is registered by the Environmental Protection Agency 

as germicidal, fungicidal, pseudomonocidal, or tuberculocidal, in accordance with 

the specifications for use as shown on the label of each product. The Official State 

Agency, with the concurrence of the Service, shall approve each product or procedure 

according to its specified usage.

An employee of the Service whose duties may include, but will not necessarily be 

limited to: 

(1) Serving as executive secretary of the General Conference Committee; 

(2) Serving as chairperson of the Plan Conference described in § 147.47;

(3) Planning, organizing, and conducting the Plan Conference; 

Primary breeding flock

Pullorum disease or 

pullorum

Reactor

Salmonella

Sanitize

Products

Person

Official supervision

Plan

Program

Poultry

Public exhibitiion

Senior Coordinator
4

Strain

Succeeding flock

Trade name or number

Suspect flock

(4) Reviewing NPIP authorized laboratories as described in § 147.51;

(5) Coordinating the State administration of the NPIP through periodic reviews of the 

administrative procedures of the Official State Agencies, according to the 

applicable provisions of the Plan and the Memorandum of Understanding;

(6) Coordinating rulemaking to incorporate the proposed changes of the provisions

approved at the Plan conference into the regulations in parts 145, 146, and 147 of 

this subchapter;

(7) Directing the production of official NPIP publications;

(8) Proposing an annual budget for plan activities and the General Conference

Committee; and

(9) Providing overall administration of the NPIP. 

The total quantity of completed product which has been thoroughly mixed in a single 

container and identified by a serial number.

The Animal and Plant Health Inspection Service, Veterinary Services, of the 

Department.

The average age at which a species of poultry is biologically capable of reproduction.

Young poultry (chicks, pullets, cockerels, capons, poults, ducklings, goslings, keets, 

etc.) that have been fed and watered and are less than 6 months of age.

Any State, the District of Columbia, or Puerto Rico.

Any person employed or authorized under § 145.11(b) to perform functions under this 

part.

A term used to identify the progeny of a specific breeding combination within a species 

of poultry. These breeding combinations may include pure strains, strain crosses, 

breed crosses, or combinations thereof.

Poultry breeding stock bearing a given name produced by a breeder through at least 

five generations of closed flock breeding.

A flock brought onto a premises during the 12 months following removal of an infected 

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.

A name or number compatible with State and Federal laws and regulations applied to 

a specified stock or product thereof. 

Stock

State Inspector

State

Started poultry

Sexual maturity

Service

Serial
5

§145.2 Administration. 

(a) The Department cooperates through a Memorandum of Understanding with 

Official State Agencies in the administration of the Plan. In the Memorandum of 

Understanding, the Official State Agency must designate a contact representative 

to serve as a liaison between the Service and the Official State Agency.

(b) The administrative procedures and decisions of the Official State Agency are 

subject to review by the Service. The Official State Agency shall carry out the 

administration of the Plan within the State according to the applicable provisions 

of the Plan and the Memorandum of Understanding.

(c) An Official State Agency may accept for participation an affiliated flock located 

in another State under a mutual understanding and agreement, in writing, 

between the two Official State Agencies regarding conditions of participation and 

supervision.

(d) The Official State Agency of any State may, except as limited by § 145.3(d), 

adopt regulations applicable to the administration of the Plan in such State further 

defining the provisions of the Plan or establishing higher standards compatible 

with the Plan. 

(e) An authorized laboratory of the National Poultry Improvement Plan will follow the 

laboratory protocols outlined in part 147 of this chapter when determining the 

status of a participating flock with respect to an official Plan classification.

(Approved by the Office of Management and Budget under control number 0579-

0007) 

§ 145.3 Participation. 

(a) Any person producing or dealing in products may participate in the Plan when 

he has demonstrated, to the satisfaction of the Official State Agency, that his 

facilities, personnel, and practices are adequate for carrying out the applicable 

provisions of the Plan, and has signed an agreement with the Official State 

Agency to comply with the general and the applicable specific provisions of the 

Plan and any regulations of the Official State Agency under § 145.2. Affiliated 

flockowners may participate without signing an agreement with the Official State 

Agency.

(b) Each participant shall comply with the Plan throughout the operating year of the 

Official State Agency, or until released by such Agency.

(c) A participant in any State shall participate with all of his poultry hatching egg 

supply flocks and hatchery operations within such State. He shall report to the 

Official State Agency on VS Form 9-2 (formerly NPIP Form 3B) or through other 

appropriate means each breeding flock before the birds reach 24 weeks of age 

or, in the case of ostriches, emus, rheas, cassowaries before the birds reach 20 

months of age.

 This report will include:

(1) Name and address of flockowner;
6

(2) Flock location and designation;

(3) Type: Primary or Multiplier;

(4) Breed, variety, strain, or trade name of stock;

(5) Source of males;

(6) Source of females;

(7) Number of birds in the flock; and

(8) Intended classification of flock.

(d) No person shall be compelled by the Official State Agency to qualify products for 

any of the other classifications described in § 145.10 as a condition of qualification 

for the U.S. Pullorum-Typhoid Clean classification.

(e) Participation in the Plan shall entitle the participant to use the Plan emblem 

reproduced below: 

 

(Approved by the Office of Management and Budget under control number 0579-0007) 

§ 145.4 General provisions for all participants.

(a) Records of purchases and sales and the identity of products handled shall be 

maintained in a manner satisfactory to the Official State Agency.

(b) Products, records of sales and purchase of products, and material used to 

advertise products shall be subject to inspection by the Official State Agency at 

any time.

Figure 1
7

(c) Advertising must be in accordance with the Plan, and applicable rules and 

regulations of the Official State Agency and the Federal Trade Commission. A 

participant advertising products as being of any official classification may include 

in his advertising reference to associated or franchised hatcheries only when such 

hatcheries produce the same kind of products of the same classification.

(d) Except as provided by this paragraph, participants in the Plan may not buy or 

receive products for any purpose from nonparticipants unless they are part of an 

equivalent program, as determined by the Official State Agency. Participants in 

the Plan may buy or receive products from flocks that are neither participants nor 

part of an equivalent program, for use in breeding flocks or for experimental 

purposes, under the following conditions only:

(1) With the permission of the Official State Agency and the concurrence of the 

Service; and

(2) By segregation of all birds before introduction into the breeding flock. Upon 

reaching sexual maturity, the segregated birds must be tested and found 

negative for pullorum-typhoid. The Official State Agency may require a second 

test at its discretion.

(e) Each participant shall be assigned a permanent approval number by the Service. 

This number, prefaced by the numerical code of the State, will be the official 

approval number of the participant and may be used on each certificate, invoice, 

shipping label, or other document used by the participant in the sale of his 

products. Each Official State Agency which requires an approval or permit number 

for out-of-State participants to ship into its State should honor this number. The 

approval number shall be withdrawn when the participant no longer qualifies for 

participation in the Plan. 

(Approved by the Office of Management and Budget under control number 0579-0057) 

§ 145.5 Specific provisions for participating flocks.

(a) Poultry equipment, and poultry houses and the land in the immediate vicinity 

thereof, shall be kept in sanitary condition as recommended in §§ 147.21 

and 147.22(a) and (e) of this chapter. The participating flock, its eggs, and 

all equipment used in connection with the flock shall be separated from 

nonparticipating flocks, in a manner acceptable to the Official State Agency.

(b) All flocks shall consist of healthy, normal individuals characteristic of the breed, 

variety, cross, or other combination which they are stated to represent.

(c) A flock shall be deemed to be a participating flock at any time only if it has 

qualified for the U.S. Pullorum-Typhoid Clean classification, as prescribed in 

Subparts B, C, D, E, or F of this part.

(d) Each bird shall be identified with a sealed and numbered band obtained through or 

approved by the Official State Agency: Provided, That exception may be made at 

the discretion of the Official State Agency. 
8

§ 145.6 Specific provisions for participating hatcheries. 

(a) Hatcheries must be kept in sanitary condition, acceptable to the Official State 

Agency. The procedures outlined in §§ 147.22 through 147.25 of this chapter 

will be considered as a guide in determining compliance with this provision. The 

minimum requirements with respect to sanitation shall include the following:

(1) Egg room walls, ceilings, floors, air filters, drains, and humidifiers should 

be cleaned and disinfected at least two times per week. Cleaning and 

disinfection procedures should be as outlined in § 147.24 of this chapter.

(2) Incubator room walls, ceilings, floors, doors, fan grills, vents, and ducts should 

be cleaned and disinfected after each set or transfer. Incubator rooms should 

not be used for storage. Plenums should be cleaned at least weekly. Egg 

trays and buggies should be cleaned and disinfected after each transfer. 

Cleaning and disinfection procedures should be as outlined in § 147.24 of this 

chapter.

(3) Hatcher walls, ceilings, floors, doors, fans, vents, and ducts should be cleaned 

and disinfected after each hatch. Hatcher rooms should be cleaned and 

disinfected after each hatch and should not be used for storage. Plenums 

should be cleaned after each hatch. Cleaning and disinfection procedures 

should be as outlined in § 147.24 of this chapter.

(4) Chick/poult processing equipment and rooms should be thoroughly cleaned 

and disinfected after each hatch. Chick/poult boxes should be cleaned and 

disinfected before being reused. Vaccination equipment should be cleaned 

and disinfected after each use. Cleaning and disinfection procedures should 

be as outlined in § 147.24 of this chapter.

(5) Hatchery residue, such as chick/poult down, eggshells, infertile eggs, and 

dead germs, should be disposed of promptly and in a manner satisfactory to 

the Official State Agency.

(6) The entire hatchery should be kept in a neat, orderly condition and cleaned 

and disinfected after each hatch.

(7) Effective insect and rodent control programs should be implemented.

(b) A hatchery that keeps started poultry must keep such poultry separated from the 

incubator room in a manner satisfactory to the Official State Agency.

(c) All baby and started poultry offered for sale under Plan terminology should be 

normal and typical of the breed, variety, cross, or other combination represented.

(d) Eggs incubated should be sound in shell, typical for the breed, variety, strain, or 

cross thereof and reasonably uniform in shape. Hatching eggs should be trayed 

and the baby poultry boxed with a view to uniformity of size.

(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.

(f) If a person is responsibly connected with more than one hatchery, all of such 

hatcheries must participate in the Plan if any of them participate. A person is 

deemed to be responsibly connected with a hatchery if he or she is a partner, 

officer, director, holder, owner of 10 percent or more of the voting stock, or an 

employee in a managerial or executive capacity.
9

§ 145.7 Specific provisions for participating dealers. 

Dealers in poultry breeding stock, hatching eggs, or baby or started poultry shall 

comply with all provisions in this part which apply to their operations.

§ 145.8 Terminology and classification; general. 

(a) The official classification terms defined in §§ 145.9 and 145.10 and the various 

designs illustrative of the official classifications reproduced in § 145.10 may be 

used only by participants and to describe products that have met all the specific 

requirements of such classifications.

(b) Products produced under the Plan shall lose their identity under Plan terminology 

when they are purchased for resale by or consigned to nonparticipants.

(c) Participating flocks, their eggs, and the baby and started poultry produced from 

them may be designated by their strain or trade name. When a breeder’s trade 

name or strain designation is used, the participant shall be able by records to 

substantiate that the products so designated are from flocks that are composed 

of either birds hatched from eggs produced under the direct supervision of the 

breeder of such strain, or stock multiplied by persons designated and so reported 

by the breeder to each Official State Agency concerned.

§ 145.9 Terminology and classification; hatcheries and dealers. 

Participating hatcheries and dealers shall be designated as “National Plan Hatchery” 

and “National Plan Dealer”, respectively. All Official State Agencies shall be notified by 

the Service of additions, withdrawals, and changes in classification. 

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

Participating flocks, products produced from them, and States which have met 

the respective requirements specified in Part 145 Subpart B, C, D, E, or F may be 

designated by the following terms or illustrative designs:
10

(a) [Reserved]

(b) U.S. PullorumTyphoid Clean. (See 

§ 145.23(b), §145.33(b), 

§ 145.43(b), § 145.53(b), 

§ 145.63(a), § 145.73 (b), 

and § 145.83 (b).)

(c) U.S. M. Gallisepticum

Clean. (See § 145.23(c), 

§ 145.23(f), § 145.33(c), 

§ 145.33(f), § 145.43(c), 

§ 145.53(c), § 145.73(c) 

and § 145.83 (c).)

U.S.

CLEAN

NPIP

M. Gallisepticum

(d) (e) (f)

Figure 5 Figure 6 Figure 7

(d) U.S. Sanitation 

Monitored. (See 

§ 145.33(d).)

(e) U.S. M. Synoviae 

Clean. (See § 145.23(e), 

§ 145.23(g), § 145.33(e), 

§ 145.33(g), § 145.43(e), 

§ 145.53(d), § 145.73(e) 

and § 145.83 (d).)

(f) U.S. M. Meleagridis 

Clean. (See § 145.43(d).)

U.S.

MONITORED

NPIP

Sanitation

U.S.

CLEAN

NPIP

M. Synoviae

U.S.

CLEAN

NPIP

M. Meleagridis

(g) (h) (i)

Figure 8 Figure 9 Figure 10

(g) U.S. PullorumTyphoid Clean State. 

(See § 145.24(a), 

§145.34(a), § 145.44(a) 

and § 145.54(a).) 

(h) U.S. PullorumTyphoid Clean State, 

Turkeys. (See § 

145.44(b).) 

(i) U.S. M. Gallisepticum 

Clean State, Turkeys. 

(See § 145.44(c).)

NATIONAL POULTRY IMPROVEMENT PLAN

U.S.

PULLORUM-TYPHOID

CLEAN STATE

TURKEYS

U.S. DEPARTMENT OF AGRICULTURE

NATIONAL POULTRY IMPROVEMENT PLAN

U.S.

M. GALLISEPTICUM

CLEAN STATE

TURKEYS

U.S. DEPARTMENT OF AGRICULTURE

NATIONAL POULTRY IMPROVEMENT PLAN

U.S.

PULLORUM-TYPHOID

CLEAN STATE

U.S. DEPARTMENT OF AGRICULTURE

(b) (c)

Figure 3 Figure 4

U.S.

 CLEAN

NPIP

Pullorum-Typhoid
11

(j) (k)

Figure 11 Figure 12

(j) U.S. M. Gallisepticum 

Clean State, MeatType Chickens. (See § 

145.34(b).)

(k) U.S. Sanitation 

Monitored, Turkeys. (See 

§ 145.43(f).)

(m) (n) (o)

Figure 14 Figure 15 Figure 16

(m) U.S. S. Enteritidis 

Clean. (See § 145.23(d) 

and § 145.33(h), 

§ 145.73(d), and 

§ 145.83 (e).)

(n) U.S. M. Synoviae 

Clean State, Turkeys. 

(See § 145.44(d).)

(o) U.S. Salmonella 

Monitored. (See 

§ 145.33(i) and 

§ 145.83 (f).)

NATIONAL POULTRY IMPROVEMENT PLAN

U.S.

M. SYNOVIAE

CLEAN STATE

TURKEYS

U.S. DEPARTMENT OF AGRICULTURE

(p) (q) (r)

Figure 17 Figure 18 Figure 19

(p) U.S. M. Gallisepticum 

Monitored. (See 

§ 145.33(j).)

(q) U.S. M. Synoviae 

Monitored. (See 

§ 145.33(k).)

(r) U.S. Avian Influenza 

Clean. (See § 145.23(h), 

§ 145.33(l), § 145.63(b), 

§ 145.73 (f) and 

§ 145.83 (g).)

U.S.

CLEAN

NPIP

S. Enteritidis

U.S.

CLEAN

NPIP

Avian Influenza

U.S.

MONITORED

TURKEYS

NPIP

Sanitation

U.S.

MONITORED

NPIP

Salmonella

U.S. 

MONITORED 

NPIP 

M. Synoviae

U.S. 

MONITORED 

NPIP 

M. Gallisepticum
12

Figure 20

(s) (s) U.S. M. Meleagridis 

Clean State, Turkeys. 

(See § 145.44(e).)

NATIONAL POULTRY IMPROVEMENT PLAN

U.S.

M. MELEAGRIDIS

CLEAN STATE

TURKEYS

U.S. DEPARTMENT OF AGRICULTURE

(t) U.S. H5/H7 Avian 

Influenza Clean. 

(See § 145.43(g) and

§ 145.53 (e).) 

Figure 21

(t)
13

§ 145.11 Supervision. 

(a) The Official State Agency may designate qualified persons as Authorized Agents 

to do the sample collecting provided for in § 145.14 and may designate qualified 

persons as Authorized Testing Agents to do the sample collecting and blood 

testing provided for in § 145.14.

(b) The Official State Agency shall employ or authorize qualified persons as State 

Inspectors to perform the qualification testing of participating flocks, and to perform 

the official inspections necessary to verify compliance with the requirements of the 

Plan.

(c) Authorities issued under the provisions of this section shall be subject to 

cancellation by the Official State Agency on the grounds of incompetence or 

failure to comply with the provisions of the Plan or regulations of the Official State 

Agency. Such actions shall not be taken until a thorough investigation has been 

made by the Official State Agency and the authorized person has been given 

notice of the proposed action and the basis therefor and an opportunity to present 

his views. 

§ 145.12 Inspections. 

(a) Each participating hatchery shall be audited at least one time annually or a 

sufficient number of times each year to satisfy the Official State Agency that the 

operations of the hatchery are in compliance with the provisions of the Plan.

(b) The records of all flocks maintained primarily for production of hatching eggs shall 

be examined annually by a State Inspector. 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. On-site inspections 

of flocks and premises will be conducted if the State Inspector determines that 

a breach of sanitation, blood testing, or other provisions has occurred for Plan 

programs for which the flocks have or are being qualified. 

§ 145.13 Debarment from participation. 

Participants in the Plan, who after investigation by the Official State Agency or its 

representative, are notified in writing of their apparent noncompliance with the Plan 

provisions or regulations of the Official State Agency, shall be afforded a reasonable 

time, as specified by the Official State Agency, within which to demonstrate or achieve 

compliance. If compliance is not demonstrated or achieved within the specified 

time, the Official State Agency may debar the participant from further participation in 

the Plan for such period, or indefinitely, as the Agency may deem appropriate. The 

debarred participant shall be afforded notice of the bases for the debarment and 

opportunity to present his views with respect to the debarment in accordance with 
14

procedures adopted by the Official State Agency. The Official State Agency shall 

thereupon decide whether the debarment order shall continue in effect. Such decision 

shall be final unless the debarred participant, within 30 days after the issuance of the 

debarment order, requests the Administrator to determine the eligibility of the debarred 

participant for participation in the Plan. In such event the Administrator shall determine 

the matter de novo in accordance with the rules of practice in 7 CFR Part 50, which 

are hereby made applicable to proceedings before the Administrator under this 

section. The definitions in 7 CFR 50.10 and the following definitions shall apply with 

respect to terms used in such rules of practice:

(a) “Administrator” means the Administrator, Animal and Plant Health Inspection 

Service of the U.S. Department of Agriculture or any officer or employee to whom 

authority has heretofore been delegated or to whom authority may hereafter be 

delegated to act in his stead. 

§ 145.14 Testing. 

Poultry must be more than 4 months of age when blood tested for an official 

classification: Provided, That turkey candidates under subpart D of this part may be 

blood tested at more than 12 weeks of age; game bird candidates under subpart E 

of this part may be blood tested when more than 4 months of age or upon reaching 

sexual maturity, whichever comes first; and ostrich, emu, rhea, and cassowary 

candidates under subpart F of this part may be blood tested when more than 12 

months of age. Blood samples for official tests shall be drawn by an Authorized Agent 

Blood samples for official tests shall be drawn by an Authorized Agent, Authorized 

Testing Agent, or State Inspector and tested by an authorized laboratory, except that 

the stained antigen, rapid whole-blood test for pullorum-typhoid may be conducted 

by an Authorized Testing Agent or State Inspector. For Plan programs in which a 

representative sample may be tested in lieu of an entire flock, except the ostrich, emu, 

rhea, and cassowary program in § 145.63(a), the minimum number tested shall be 30 

birds per house, with at least 1 bird taken from each pen and unit in the house. The 

ratio of male to female birds in representative samples of birds from meat-type chicken, 

waterfowl, exhibition poultry, and game bird flocks must be the same as the ratio of 

male to female birds in the flock. In houses containing fewer than 30 birds other than 

ostriches, emus, rheas, and cassowaries, all birds in the house must be tested.

(1) The official blood tests for pullorum-typhoid shall be the standard tube agglutination 

test, the microagglutination test, the enzyme-labeled immunosorbent assay test 

(ELISA), or the rapid serum test for all poultry; and the stained antigen, rapid 

whole-blood test for all poultry except turkeys. The procedures for conducting 

official blood tests are set forth in §§ 147.1, 147.2, 147.3, and 147.5 of this 

chapter and referenced in footnote 3 of this section or in literature provided by the 

producer. Only antigens approved by the Department and of the polyvalent type 

shall be used for the rapid whole-blood 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 

(a) For PullorumTyphoid
15

that serial continues to be made available for use. All microtest antigens and 

enzyme-labeled immunosorbent assay reagents shall also be approved by the 

Department.1 

(2) [Reserved]

(3) There shall be an interval of at least 21 days between any official blood test and 

any previous test with pullorum-typhoid antigen.

(4) [Reserved]

(5) The official blood test shall include the testing of a sample of blood from each bird 

in the flock: Provided, That under specified conditions (see applicable provisions 

of §§ 145.23, 145.33, 145.43, 145.53 and 145.63) the testing of a portion or 

sample of the birds may be used in lieu of testing each bird.

(6) Poultry from flocks undergoing qualification testing for participation in the Plan that 

have a positive reaction to an official blood test named in paragraph (a)(1) of this 

section shall be evaluated for pullorum-typhoid as follows: 

 (i) Serum samples that react on rapid serum test or enzyme-labeled 

immunosorbent assay test (ELISA), or blood from birds that react on the 

stained antigen, rapid whole-blood test for all birds except turkeys, shall be 

tested with either the standard tube agglutination test or the microagglutination 

test. 

 (ii) Reactors to the standard tube agglutination test (in dilutions of 1:50 or 

greater) or the microagglutination test (in dilutions of 1:40 or greater) shall 

be submitted to an authorized laboratory for bacteriological examination. 

If there are more than four reactors in a flock, a minimum of four reactors 

shall be submitted to the authorized laboratory; if the flock has four or fewer 

reactors, all of the reactors must be submitted. The approved procedure for 

bacteriological examination is set forth in § 147.11 of this chapter. When 

reactors are submitted to the authorized laboratory within 10 days of the date 

of reading an official blood test named in paragraph (a)(6)(i) of this section, 

and the bacteriological examination fails to demonstrate pullorum-typhoid 

infection, the Official State Agency shall presume that the flock has no 

pullorum-typhoid reactors. 

(iii) If a flock owner does not wish to submit reactors for bacteriological 

examination, then the reactors shall be isolated and retested within 30 days 

using an official blood test named in paragraph (a)(1) of this section. If this 

retest is positive, additional examination of the reactors and flock will be 

performed in accordance with paragraph (a)(6)(ii) of this section. During this 

30-day period, the flock must be maintained under a security system, specified 

or approved by the Official State Agency, that will prevent physical contact with 

other birds and assure that personnel, equipment, and supplies that could be 

a source of pullorum-typhoid spread are sanitized. 

(7) When S. pullorum or S. gallinarum organisms are isolated by an authorized 

1 The criteria and procedures for Department approval of antigens and reagents 

may be obtained from the Animal and Plant Health Inspection Service, Veterinary 

Services, Center for Veterinary Biologics, 510 South 17th Street, Suite 104, Ames, 

IA 50010–8197.
16

laboratory from baby poultry, or from fluff samples produced by hatching eggs, 

the infected flock shall qualify for participation in the Plan with two consecutive 

negative results to an official blood test named in paragraph (a)(1) of this section. 

A succeeding flock must be qualified for participation in the Plan's pullorumtyphoid program with a negative result to an official blood test named in paragraph 

(a)(1) of this section. 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. If the 

State Inspector determines that a primary breeding flock has been exposed to S. 

pullorum or S. gallinarum,2

 the Official State Agency shall require: 

 (i) The taking of blood samples—performed by or in the presence of a State 

Inspector—from all birds on premises exposed to birds, equipment, supplies, 

or personnel from the primary breeding flock during the period when the State 

Inspector determined that exposure to S. pullorum or S. gallinarum occurred.2

(ii) The banding of all birds of these premises—performed or physically supervised 

by a State Inspector—in order to identify any bird that tests positive; and 

(iii) The testing of blood samples at an authorized laboratory using an official blood 

test named in paragraph (a)(1) of this section. 

(8) All domesticated fowl, except waterfowl, on the farm of the participant shall either 

be properly tested to meet the same standards as the participating flock or these 

birds and their eggs shall be separated from the participating flock and its eggs.

(9) All tests for pullorum-typhoid in flocks participating in or candidates for participation 

in the Plan shall be reported to the Official State Agency within 10 days following 

the completion of such tests. All reactors shall be considered in determining the 

classification of the flock.

(10) Any drug, for which there is scientific evidence of masking the test reaction or 

hindering the bacteriological recovery of Salmonella organisms, shall not be 

fed or administered to poultry within 3 weeks prior to a test or bacteriological 

examination upon which a Salmonella classification is based.

2

In making determinations of exposure, the State Inspector shall evaluate both 

evidence proving that exposure occurred and circumstances indicating a high 

probability of contacts with: infected wild birds; contaminated feed or waste; or 

birds, equipment, supplies, or persons from or exposed to flocks infected with 

S. pullorum or S. gallinarum. 
17

(11) When suitable evidence, as determined by the Official State Agency or the State 

Animal Disease Control Official, indicates that baby or started poultry produced 

by participating hatcheries are infected with organisms for which the parent flock 

received an official control classification and this evidence indicates that the 

infection was transmitted from the parent flock, the Official State Agency may, at 

its discretion, require additional testing of the flock involved. If infection is found in 

the parent flock, its classification shall be suspended until the flock is requalified 

under the requirements for the classification. Furthermore, the Official State 

Agency may require that the hatching eggs from such flocks be removed from 

the incubator and destroyed prior to hatching. When Salmonella organisms are 

isolated from a specimen which originated in a participating hatchery, the Official 

State Agency shall attempt to locate the source of the infection. The results of the 

investigation and the action taken to eliminate the infection shall be reported by 

the Official State Agency to the Service.

(1) The official blood tests for M. gallisepticum and M. synoviae shall be the serum 

plate agglutination test, the tube agglutination test, the hemagglutination 

inhibition (HI) test, the microhemagglutination inhibition test, the enzyme-labeled 

immunosorbent assay (ELISA) test3 or a combination of two or more of these tests. 

The HI test, the microhemagglutination inhibition test, and the ELISA test shall be 

used to confirm the positive results of other serological tests. HI titers of 1:40 or 

less may be interpreted as equivocal, and final judgment may be based on further 

samplings and/or culture of reactors. 

(2) The tests shall be conducted using M. gallisepticum or M. synoviae antigens 

approved by the Department or the Official State Agency and shall be performed in 

accordance with the recommendations of the producer of the antigen.

(3) When reactors to the test for which the flock was tested are submitted to a 

laboratory as prescribed by the Official State Agency, the criteria found in § 147.6 

shall be used in determining the final status of the flock.

(4) Any drug, for which there is scientific evidence of masking the test reaction or 

hindering the bacteriological recovery of mycoplasma organisms, shall not be 

fed or administered to poultry within three weeks prior to a test or bacteriological 

examination upon which a Mycoplasma classification is based.

(5) The official molecular examination procedures for Mycoplasma gallisepticum and 

M. synoviae are the polymerase chain reaction (PCR) test described in 

§ 147.30 of this subchapter and the real-time PCR test described in § 147.31 of 

this subchapter.

(b) For M. gallisepticum 

and M. synoviae

3

Procedures for the enzyme-labeled immunosorbent assay (ELISA) test are set forth 

in the following publications:

A.A. Ansari, R.F. Taylor, T.S. Chang, “Application of Enzyme-Linked Immunosorbent 

Assay for Detecting Antibody to Mycoplasma gallisepticum Infections in Poultry,” Avian 

Diseases, Vol. 27, No. 1, pp. 21–35, January–March 1983; and

H.M. Opitz, J.B. Duplessis, and M.J. Cyr, “Indirect Micro-Enzyme-Linked 

Immunosorbent Assay for the Detection of Antibodies to Mycoplasma synoviae and M. 

gallisepticum,” Avian Diseases, Vol. 27, No. 3, pp. 773–786, July–September 1983; 

and 

H.B. Ortmayer and R. Yamamoto, “Mycoplasma meleagridis Antibody Detection by 

Enzyme-Linked Immunosorbent Assay (ELISA),” Proceedings, 30th Western Poultry 

Disease Conference, pp. 63–66, March 1981. 
18

The official blood tests for M. meleagridis are specified in § 145.43(d)(2). 

The official tests for avian influenza are described in paragraphs (d)(1) and (d)(2) of

this section.

(1) Antibody detection tests. 

 (i) Enzyme-linked immunosorbent assay (ELISA). ELISA must be conducted 

 using test kits approved by the Department and the Official State Agency 

 and must be conducted in accordance with the recommendations of the 

 producer or manufacturer.

(ii) The agar gel immunodiffusion (AGID) test. 

 (A) The AGID test must be conducted on all ELISA-positive samples. 

 (B) The AGID test must be conducted using reagents approved by the 

 Department and the Official State Agency. 

 (C) Standard test procedures for the AGID test for avian influenza are set 

 forth in § 147.9 of this subchapter. The test can be conducted on egg yolk 

 or blood samples. 

 (D) Positive tests for the AGID must be further tested by Federal Reference 

 Laboratories using appropriate tests for confirmation. Final judgment may 

 be based upon further sampling and appropriate tests for confirmation. 

(2) Agent detection tests. Agent detection tests may be used to detect influenza A 

matrix gene or protein but not to determine hemagglutinin or neuraminidase 

subtypes. Samples for agent detection testing should be collected from naturally 

occurring flock mortality or clinically ill birds.

(i) The real time reverse transcriptase/polymerase chain reaction (RRT-PCR) 

 assay. 

 (A) The RRT-PCR tests must be conducted using reagents approved by the

 Department and the Official State Agency. The RRT-PCR must be 

 conducted using the National Veterinary Services Laboratories (NVSL) 

 official protocol for RRT-PCR (AVPR01510) and must be conducted by 

 personnel who have passed an NVSL proficiency test. 

 (B) Positive results from the RRT-PCR must be further tested by Federal 

 Reference Laboratories using appropriate tests for confirmation. Final 

 judgment may be based upon further sampling and appropriate tests for 

 confirmation. 

(ii) USDA-licensed type A influenza antigen capture immunoassay (ACIA). 

 (A) The USDA-licensed type A influenza ACIA must be conducted using test

 kits approved by the Department and the Official State Agency and 

 must be conducted in accordance with the recommendations of the 

 producer or manufacturer.

 (B) Positive results from the ACIA must be further tested by Federal Reference

 Laboratories using appropriate tests for confirmation. Final judgment may

 be based upon further sampling and appropriate tests for confirmation.

(3) The official determination of a flock as positive for the H5 or H7 subtypes of avian 

influenza may be made only by NVSL.

(d) For avian influenza

(c) For M. meleagridis
19

§ 145.15. Diagnostic surveillance program for low pathogenic 

avian influenza. 

(a) The Official State Agency must develop a diagnostic surveillance program for 

H5/H7 low pathogenic avian influenza for all poultry in the State. The exact 

provisions of the program are at the discretion of the States. The Service will use 

the standards in paragraph (b) of this section in assessing individual State plans 

for adequacy, including the specific provisions that the State developed. The 

standards should be used by States in developing those plans.

 (b) Avian influenza must be a disease reportable to the responsible State authority 

(State veterinarian, etc.) by all licensed veterinarians. To accomplish this, all 

laboratories (private, State, and university laboratories) that perform diagnostic 

procedures on poultry must examine all submitted cases of unexplained 

respiratory disease, egg production drops, and mortality for avian influenza by both 

an approved serological test and an approved antigen detection test. Memoranda 

of understanding or other means must be used to establish testing and reporting 

criteria (including criteria that provide for reporting H5 and H7 low pathogenic 

avian influenza directly to the Service) and approved testing methods. In addition, 

States should conduct outreach to poultry producers, especially owners of 

smaller flocks, regarding the importance of prompt reporting of clinical symptoms 

consistent with avian influenza.

(Approved by the Office of Management and Budget under control number 0579-0007)

[ 36 FR 23112, Dec. 3, 1971]

Editorial Note: For Federal Register citations affecting §145.14, see the list of CFR

sections Affected, which appears in the Finding Aids section of the printed volume and

on GPO Access. 
20

Subpart B—Special Provisions for Multiplier Egg-Type Chicken Breeding 

Flocks and Products

§ 145.21 Definitions. 

Except where the context otherwise requires, for the purposes of this subpart the 

following terms shall be construed, respectively, to mean:

Newly hatched chickens.

Flocks that are composed of stock that has been developed for egg production and are 

maintained for the principal purpose of producing chicks for the ultimate production of 

eggs for human consumption.

Young chickens (chicks, pullets, cockerels, capons) which have been fed and watered 

and are less than 6 months of age. 

§ 145.22 Participation. 

Participating flocks of multiplier egg-type chickens, and the eggs and chicks produced 

from them, shall comply with the applicable general provisions of Subpart A of this part 

and the special provisions of this Subpart B.

(a) Started chickens shall lose their identity under Plan terminology when not 

maintained by Plan participants under the conditions prescribed in § 145.5(a).

(b) Hatching eggs produced by multiplier breeding flocks shall be fumigated (see 

§ 147.25 of this chapter) or otherwise sanitized. 

(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.

§ 145.23 Terminology and classification; flocks and products. 

Participating flocks, and the eggs and chicks produced from them, which have met the 

respective requirements specified in this section may be designated by the following 

terms and the corresponding designs illustrated in § 145.10:

A flock in which freedom from pullorum and typhoid has been demonstrated to the 

official State agency under the criteria in one of the following paragraphs (b)(1) 

through (5) of this section: Provided, That a flock qualifying by means of a blood test 

shall be tested within the past 12 months, except that the retesting of a participating 

flock which is retained for more than 12 months shall be conducted a minimum of 

4 weeks after the induction of molt. (See § 145.14 relating to the official blood test 

where applicable.)

(1) It has been officially blood tested with no reactors.

Started chickens

(b) U.S. PullorumTyphoid Clean 

(a) [Reserved]

Egg-type chicken 

breeding flocks

Chicks
21

(2) It is a multiplier breeding flock and meets the following specifications as 

determined by the Official State Agency and the Service:

(i) The flock is located in a State where 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 poultry specimens from which 

S. pullorum or S. gallinarum is isolated;

(ii) The flock is composed entirely of birds that originated from U.S. PullorumTyphoid Clean breeding flocks or from flocks that met equivalent requirements 

under official supervision; and

(iii) The flock is located on a premises where either no poultry or a flock not 

classified as U.S. Pullorum-Typhoid Clean were located the previous year: 

Provided, That an Authorized Testing Agent must blood test up to 300 birds 

per flock, as described in § 145.14, if the Official State Agency determines 

that the flock has been exposed to pullorum-typhoid. In making determinations 

of exposure and setting the number of birds to be blood tested, the Official 

State Agency shall evaluate the results of any blood tests, described in § 

145.14(a)(1) of this part, that were performed on an unclassified flock located 

on the premises during the previous year; the origins of the unclassified flock; 

and the probability of contacts between the flock for which qualification is 

being sought and (a) infected wild birds, (b) contaminated feed or waste, or 

(c) birds, equipment, supplies, or personnel from flocks infected with pullorumtyphoid.

(3) It is a multiplier breeding flock that originated from U.S. Pullorum-Typhoid Clean 

breeding flocks or from flocks that met equivalent requirements under official 

supervision, and is located in a State in which it has been determined by the 

Service that: 

(i) All hatcheries within the State are qualified as “National Plan Hatcheries” or 

have met equivalent requirements for pullorum-typhoid control under official 

supervision;

(ii) All hatchery supply flocks within the State are qualified as U.S. PullorumTyphoid Clean or have met equivalent requirements for pullorum-typhoid 

control under official supervision: Provided, That if other domesticated fowl, 

except waterfowl, are maintained on the same premises as the participating 

flock, freedom from pullorum-typhoid infection shall be demonstrated by an 

official blood test of each of these fowl;

(iii) All shipments of products other than U.S. Pullorum-Typhoid Clean, or 

equivalent, into the State are prohibited;

(iv) 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 

poultry specimens from which S. pullorum or S. gallinarium is isolated;

(v) All reports of any disease outbreak involving a disease covered under the 

Plan are promptly followed by an investigation by the Official State Agency 

to determine the origin of the infection: 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; 
22

(vi) All flocks found to be infected with pullorum or typhoid are quarantined until 

marketed or destroyed under the supervision of the Official State Agency, or 

until subsequently blood tested, following the procedure for reacting flocks 

as contained in § 145.14(a)(5), and all birds fail to demonstrate pullorum or 

typhoid infection;

(vii) All poultry, including exhibition, exotic, and game birds, but excluding 

waterfowl, going to public exhibition shall come from U.S. Pullorum-Typhoid 

Clean or equivalent flocks, or have had a negative pullorum- typhoid test 

within 90 days of going to public exhibition;

(viii) Discontinuation of any of the conditions or procedures described in 

paragraphs (b)(3)(i), (ii), (iii), (iv), (v), (vi), and (vii) of this section, or the 

occurrence of repeated outbreaks of pullorum or typhoid in poultry breeding 

flocks within or originating within the State shall be grounds for the Service to 

revoke its determination that such conditions and procedures have been met 

or complied with. Such action shall not be taken until a thorough investigation 

has been made by the Service and the Official State Agency has been given 

an opportunity to present its views.

(4) It is a multiplier breeding flock located in a State which has been determined by 

the Service to be in compliance with the provisions of (b)(3) of this section, and in 

which pullorum disease or fowl typhoid is not known to exist nor to have existed in 

hatchery supply flocks within the State during the preceding 12 months.

(1) A flock maintained in compliance with the provisions of § 147.26 of this chapter 

and in which freedom from M. gallisepticum has been demonstrated under the 

criteria specified in paragraph (c)(1)(i) or (ii) of this section.

(i) [Reserved]

(ii) It is a multiplier breeding flock which originated as U.S. M. Gallisepticum Clean 

chicks from primary breeding flocks and from which a sample comprised 

of a minimum of 150 birds per flock has been tested for M. gallisepticum as 

provided in § 145.14(b) when more than 4 months of age: Provided, That to 

retain this classification, the flock shall be subjected to one of the following 

procedures:

(A) At intervals of not more than 90 days, 75 birds from the flock shall be 

tested, Provided, that fewer than 75 birds from the flock may be tested at 

any one time if all pens are equally represented and a total of at least 75 

birds from the flock is tested within each 90-day period; or

(B) At intervals of not more than 30 days, a sample of 25 cull chicks produced 

from the flock shall be subjected to laboratory procedures acceptable to 

the Official State Agency and approved by the Service, for the detection 

and recovery of M. Gallisepticum; or

(C) At intervals of not more than 30 days, egg yolk testing shall be conducted 

in accordance with § 147.8.

(c) U.S. M. Gallisepticum 

Clean
23

(2) A participant handling U.S. M. Gallisepticum Clean products shall keep these 

products separate from other products in a manner satisfactory to the Official 

State Agency. 

(3) U.S. M. Gallisepticum Clean chicks shall be boxed in clean boxes and delivered in 

trucks that have been cleaned and disinfected as described in § 147.24 (a) of this 

chapter.

This classification is intended for egg-type breeders wishing to assure their customers

that the hatching eggs and chicks produced are certified free of Salmonella enteritidis.

(1) A flock and the hatching eggs and chicks produced from it which have met the 

following requirements as determined by the Official State Agency:

(i) The flock originated from a U.S. S. Enteritidis Clean flock, or meconium from 

the chick boxes and a sample of chicks that died within 7 days after hatching 

are examined bacteriologically for salmonella at an authorized laboratory. 

Cultures from positive samples shall be serotyped.

(ii) All feed fed to the flock shall meet the following requirements:

(A) Pelletized feed shall contain either no animal protein or only animal protein 

products produced under the Animal Protein Products Industry (APPI) 

Salmonella Education/Reduction Program*. The protein products must 

have a minimum moisture content of 14.5 percent and must have been 

heated throughout to a minimum temperature of 190 °F, or above, or to a 

minimum temperature of 165 °F. for at least 20 minutes, or to a minimum 

temperature of 184 °F. under 70 lbs. pressure during the manufacturing 

process.

(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.

(iii) Feed shall be stored and transported in such a manner as to prevent possible 

contamination;

(iv) The flock is maintained in compliance with §§ 147.21, 147.24(a), and 147.26 

of this chapter. Rodents and other pests should be effectively controlled;

(v) Environmental samples shall be collected from the flock by an Authorized 

Agent, as described in § 147.12 of this chapter, when the flock is 2 to 4 

weeks of age. The Authorized Agent shall also collect samples every 30 days 

after the first sample has been collected. The samples shall be examined 

bacteriologically for group D salmonella at an authorized laboratory. Cultures 

from positive samples shall be serotyped.

(d) U.S. S. Enteritidis 

Clean

*Documents concerning the APPI/Salmonella Education Reduction Program may be obtained 

from Mr. A. R. Rhorer; National Animal Health Programs; VS, APHIS, USDA; 1498 Klondike 

Road, Suite 101, Conyers, GA 30094.
24

(vi) If a Salmonella vaccine is used that causes positive reactions with pullorumtyphoid antigen, one of the following options must be utilized:

(A) Administer the vaccine after the pullorum-typhoid testing is done as 

described in paragraph (d)(1)(vii) of this section.

(B) If an injectable bacterin or live vaccine that does not spread is used, 

keep a sample of 350 birds unvaccinated and banded for identification 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, vaccinate the banded, non-vaccinated birds.

(vii) Blood samples from 300 non-vaccinated birds as described in paragraph 

(d)(1)(vi) of this section shall be tested with either pullorum antigen or by 

a federally licensed Salmonella enteritidis enzyme-linked immunosorbent 

assay (ELISA) test when the flock is more than 4 months of age. All birds 

with positive or inconclusive reactions, up to a maximum of 25 birds, shall be 

submitted to an authorized laboratory and examined for the presence of group 

D salmonella, as described in § 147.11 of this chapter. Cultures from positive 

samples shall be serotyped.

(viii) Hatching eggs are collected as quickly as possible and are handled as 

described in § 147.22 of this chapter and are sanitized or fumigated (see 

§ 147.25 of this chapter).

(ix) Hatching eggs produced by the flock are incubated in a hatchery that is in 

compliance with the recommendations in §§ 147.23 and 147.24(b) of this 

chapter, and sanitized either by a procedure approved by the Official State 

Agency or fumigated (see § 147.25 of this chapter).

(2) A flock shall not be eligible for this classification if Salmonella enteritidis ser 

enteritidis (SE) is isolated from a specimen taken from a bird in the flock. Isolation 

of SE from an environmental or other specimen as described in section (d)(1)(v) 

of this paragraph will require bacteriological examination for SE in an authorized 

laboratory, as described in § 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. If no SE is recovered from any of the specimens in the 

second sample, the flock will be eligible for the classification.

(3) A non-vaccinated flock shall be eligible for this classification if Salmonella

enteritidis (S. enteritidis ser Enteritidis) is isolated from an environmental sample 

collected from the flock in accordance with paragraph (d)(1)(v) of this section: 

Provided, That testing is conducted in accordance with paragraph (d)(1)(vii) of this 

section each 30 days and no positive samples are found.

(4) In order for a hatchery to sell products of this classification, all products handled 

shall meet the requirements of the classification.

(5) This classification may be revoked by the Official State Agency if the participant 

fails to follow recommended corrective measures.
25

(1) A flock maintained in compliance with the provisions of § 147.26 and in which 

freedom from M. synoviae has been demonstrated under the criteria specified in 

paragraph (e)(1)(i) or (ii) of this section.

(i) [Reserved]

(ii) It is a multiplier breeding flock which originated as U.S. M. Synoviae Clean 

chicks from primary breeding flocks and from which a sample comprised 

of a minimum of 150 birds has been tested for M. synoviae as provided in 

§ 145.14(b) when more than 4 months of age: Provided, That to retain this 

classification, the flock shall be subjected to one of the following procedures:

(A) At intervals of not more than 90 days, 75 birds from the flock shall be 

tested: Provided, That fewer than 75 birds from the flock may be tested at 

any one time if all pens are equally represented and a total of at least 75 

birds from the flock is tested within each 90-day period; or

(B) At intervals of not more than 30 days, egg yolk testing shall be conducted 

in accordance with § 147.8.

(2) A participant handling U.S. M. Synoviae Clean products shall keep these products 

separate from other products in a manner satisfactory to the Official State Agency. 

(3) U.S. M. Synoviae Clean chicks shall be boxed in clean boxes and delivered in 

trucks that have been cleaned and disinfected as described in § 147.24(a).

(1) A flock which originated from U.S. M. Gallisepticum Clean breeding flocks and was 

hatched in a hatchery approved by the Official State Agency for the production of 

U.S. M. Gallisepticum Clean chicks.

(2) All other poultry on the premises of the candidate flock must originate from 

U.S. M. Gallisepticum Clean sources.

(3) The flock is maintained in compliance with the provisions of § 147.26 of this 

chapter.

(4) The flock’s freedom from M. gallisepticum is demonstrated by a negative blood 

test, as provided in § 145.14(b), of a sample of 75 birds, with a minimum of 50 

birds per poultry house, between 15–20 days prior to the flock being moved to 

laying quarters.

(5) Started poultry shall be delivered to and from the farm premises in crates and 

vehicles which have been cleaned and disinfected as described in § 145.24(a) of 

this chapter.

(1) A flock which originated from U.S. M. Synoviae Clean breeding flocks and was 

hatched in a hatchery approved by the Official State Agency for production of 

U.S. M. Synoviae Clean chicks.

(2) All other poultry on the premises of the candidate flock must originate from 

U.S. M. Synoviae Clean sources.

(3) The flock is maintained in compliance with the provisions of § 147.26 of this 

chapter.

(e) U.S. M. Synoviae 

Clean

(g) U.S. M. Synoviae

Clean Started Poultry 

(f) U.S. M. Gallisepticum 

Clean Started Poultry 
26

(4) The flocks’ freedom from M. synoviae is demonstrated by a negative blood test, 

as provided in § 145.14(b), of a sample of 75 birds, with a minimum of 50 birds 

per poultry house, between 15–20 days prior to the flock being moved to laying 

quarters.

(5) Started poultry shall be delivered to and from the farm premises in crates and 

vehicles which have been cleaned and disinfected as described in § 147.24(a) of 

this chapter. 

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) [Reserved]

(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 90 days; 

or Provided: That multiplier spent fowl must be tested within 30 days prior to 

movement to disposal; 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.

(Approved by the Office of Management and Budget under control number 0579-0007)

§ 145.24 Terminology and classification; States. 

(1) A State will be declared a U.S. Pullorum-Typhoid Clean State when it has been 

determined by the Service that:

(i) The State is in compliance with the provisions contained in 

§ 145.23(b)(3)(i) through (vii), § 145.33(b)(3)(i) through (vii), 

§ 145.43(b)(3)(i) through (vi), § 145.53(b)(3)(i) through (vii), § 145.73(b)(2)(i) 

and § 145.83(b)(2)(i).

(ii) No pullorum disease or fowl typhoid is known to exist nor to have existed 

in hatchery supply flocks within the State during the preceding 12 months: 

Provided, That pullorum disease or fowl typhoid found within the preceding 

24 months in waterfowl, exhibition poultry, and game bird breeding flocks will 

not prevent a State, which is otherwise eligible, from qualifying.

(a) U.S. PullorumTyphoid Clean State 

(h) U.S. Avian Influenza 

Clean
27

(2) Discontinuation of any of the conditions described in paragraph (a)(1)(i) of this 

section, or repeated outbreaks of pullorum or typhoid occur in hatchery supply 

flocks described in paragraph (a)(1)(ii) of this section, or if an infection spreads 

from the originating premises, the Service shall have grounds to revoke its 

determination that the State is entitled to this classification. Such action shall not 

be taken until a thorough investigation has been made by the Service and the 

Official State Agency has been given an opportunity for a hearing in accordance 

with rules of practice adopted by the Administrator.

(b) [Reserved]
28

Subpart C—Special Provisions for Multiplier Meat-Type Chicken Breeding Flocks and Products

§ 145.31 Definitions. 

Except where the context otherwise requires, for the purposes of this subpart the 

following terms shall be construed, respectively, to mean:

Newly hatched chickens.

Flocks that are composed of stock that has been developed for meat production 

and are maintained for the principal purpose of producing chicks for the ultimate 

production of meat.

Young chickens (chicks, pullets, cockerels, capons) which have been fed and watered 

and are less than 6 months of age. 

§ 145.32 Participation. 

Participating flocks of multiplier meat type chickens, and the eggs and chicks 

produced from them, shall comply with the applicable general provisions of Subpart A 

of this part and the special provisions of this Subpart C.

(a) Started chickens shall lose their identity under Plan terminology when not 

maintained by Plan participants under the conditions prescribed in § 145.5(a).

(b) Hatching eggs produced by multiplier breeding flocks shall be fumigated (see 

 § 147.25 of this chapter) or otherwise sanitized. 

(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.

§ 145.33 Terminology and classification; flocks and products. 

Participating flocks, and the eggs and chicks produced from them, which have met the 

respective requirements specified in this section may be designated by the following 

terms and the corresponding designs illustrated in § 145.10:

A flock in which freedom from pullorum and typhoid has been demonstrated to the 

official State agency under the criteria in one of paragraphs (b)(1) through (5) of this 

section: Provided, That a flock qualifying by means of a blood test shall be tested 

within the past 12 months, except that the retesting of a participating flock which is 

retained for more than 12 months shall be conducted a minimum of 4 weeks after the 

induction of molt. (See § 145.14 relating to the official blood test where applicable.)

(1) It has been officially blood tested with no reactors.

Started chickens 

(a) [Reserved]

(b) U.S. PullorumTyphoid Clean 

Meat-type chicken 

breeding flocks 

Chicks 
29

(2) It is a multiplier breeding flock and meets the following specifications as 

determined by the Official State Agency and the Service:

(i) The flock is located in a State where 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 poultry specimens from which 

S. pullorum or S. gallinarum is isolated;

(ii) The flock is composed entirely of birds that originated from U.S. PullorumTyphoid Clean breeding flocks or from flocks that met equivalent requirements 

under official supervision; and

(iii) The flock is located on a premises where either no poultry or a flock not 

classified as U.S. Pullorum-Typhoid Clean were located the previous year; 

Provided, That an Authorized Testing Agent must blood test up to 300 birds 

per flock, as described in § 145.14, if the Official State Agency determines that 

the flock has been exposed to pullorum-typhoid. In making determinations of 

exposure and setting the number of birds to be blood tested, the Official State 

Agency shall evaluate the results of any blood tests, described in § 145.14(a)

(1) of this part, that were performed on an unclassified flock located on the 

premises during the previous year; the origins of the unclassified flock; and 

the probability of contacts between the flock for which qualification is being 

sought and (a) infected wild birds, (b) contaminated feed or waste, or (c) birds, 

equipment, supplies, or personnel from flocks infected with pullorum-typhoid.

(3) It is a multiplier breeding flock that originated from U.S. Pullorum-Typhoid Clean 

breeding flocks or from flocks that met equivalent requirements under official 

supervision, and is located in a State in which it has been determined by the 

Service that:

(i) All hatcheries within the State are qualified as “National Plan Hatcheries” or 

have met equivalent requirements for pullorum-typhoid control under official 

supervision;

(ii) All hatchery supply flocks within the State, are qualified as U.S. PullorumTyphoid Clean or have met equivalent requirements for pullorum-typhoid 

control under official supervision: Provided, That if other domesticated fowl, 

except waterfowl, are maintained on the same premises as the participating 

flock, freedom from pullorum-typhoid infection shall be demonstrated by an 

official blood test of each of these fowl;

(iii) All shipments of products other than U.S. Pullorum-Typhoid Clean, or 

equivalent, into the State are prohibited;

(iv) 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 

poultry specimens from which S. pullorum or S. gallinarum is isolated;

(v) All reports of any disease outbreak involving a disease covered under the 

Plan are promptly followed by an investigation by the Official State Agency 

to determine the origin of the infection: 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;
30

(vi) All flocks found to be infected with pullorum or typhoid are quarantined until 

marketed or destroyed under the supervision of the Official State Agency, or 

until subsequently blood tested following the procedure for reacting flocks 

as contained in § 145.14(a)(5), and all birds fail to demonstrate pullorum or 

typhoid infection;

(vii) All poultry, including exhibition, exotic, and game birds, but excluding 

waterfowl, going to public exhibition shall come from U.S. Pullorum-Typhoid 

Clean or equivalent flocks, or have had a negative pullorum-typhoid test within 

90 days of going to public exhibition;

(viii) Discontinuation of any of the conditions or procedures described in 

paragraphs (b)(3)(i), (ii), (iii), (iv), (v), (vi), and (vii) of this section, or the 

occurrence of repeated outbreaks of pullorum or typhoid in poultry breeding 

flocks within or originating within the State shall be grounds for the Service to 

revoke its determination that such conditions and procedures have been met 

or complied with. Such action shall not be taken until a thorough investigation 

has been made by the Service and the Official State Agency has been given 

an opportunity to present its views.

(4) It is a multiplier breeding flock located in a State which has been determined by the 

Service to be in compliance with the provisions of paragraph (b)(3) of this section, 

and in which pullorum disease or fowl typhoid is not known to exist nor to have 

existed in hatchery supply flocks within the State during the preceding 12 months.

(1) A flock maintained in compliance with the provisions of § 147.26 of this chapter 

and in which freedom from M. gallisepticum has been demonstrated under the 

criteria specified in paragraph (c)(1)(i) or (ii) of this section.

(i) [Reserved]

(ii) It is a multiplier breeding flock which originated as U.S. M. Gallisepticum Clean 

chicks from primary breeding flocks and from which a sample comprised 

of a minimum of 150 birds per flock has been tested for M. gallisepticum as 

provided in §145.14(b) when more than 4 months of age: Provided, That to 

retain this classification, the flock shall be subjected to one of the following 

procedures:

(A) At intervals of not more than 90 days, 75 birds from the flock shall be 

tested, Provided, that fewer than 75 birds from the flock may be tested at 

any one time if all pens are equally represented and a total of at least 75 

birds from the flock is tested within each 90-day period; or

(B) At intervals of not more than 30 days, a sample of 25 cull chicks produced 

from the flock shall be subjected to laboratory procedures acceptable to 

the Official State Agency and approved by the Service, for the detection 

and recovery of M. gallisepticum; or

(C) At intervals of not more than 30 days, egg yolk testing shall be conducted 

in accordance with § 147.8 of this chapter.

(c) U.S. M. Gallisepticum 

Clean 
31

(2) A participant handling U.S. M. Gallisepticum Clean products must keep these 

products separate from other products through the use of separate hatchers and 

incubators, separate hatch days, and proper hatchery sanitation and biosecurity 

(see §§ 147.22, 147.23, and 147.24) in a manner satisfactory to the Official State 

Agency. 

(3) U.S. M. Gallisepticum Clean chicks shall be boxed in clean boxes and delivered in 

trucks that have been cleaned and disinfected as described in § 147.24(a) of this 

chapter.

(4) Before male breeding birds may be added to a participating multiplier breeding 

flock, a sample of at least 30 birds to be added, with a minimum of 10 birds 

per pen, shall be tested for M. gallisepticum as provided in § 145.14(b) or by a 

polymerase chain reaction (PCR)-based procedure approved by the Department. 

If few than 30 male breeding birds are being added, all the birds shall be tested 

as described above. The male birds shall be tested no more than 14 days prior to 

their intended introduction into the flock. If the serologic testing of the birds yields 

hemagglutination inhibition titers of 1:40 or higher as provided in § 145.14 (b), or if 

the PCR testing is positive for M. gallisepticum, the male birds may not be added 

to the flock and must be either retested or destroyed.

This program is intended to be the basis from which the breeding-hatching industry 

may conduct a program for the prevention and control of Salmonellosis. It is intended 

to reduce the incidence of Salmonella organisms in hatching eggs and chicks through 

an effective and practical sanitation program at the breeder farm and in the hatchery. 

This will afford other segments of the poultry industry an opportunity to reduce the 

incidence of Salmonella in their products.

(1) A flock and the hatching eggs and chicks produced from it which have met the 

following requirements as determined by the Official State Agency:

(i) The flock shall originate from a source where sanitation and management 

practices, as outlined in § 145.33(d)(1) of this paragraph, are conducted;

(ii) The flock is maintained in compliance with §§ 147.21, 147.24 (a), and 147.26 

of this chapter;

(iii) If pelletized feed contains animal protein, the protein products shall be 

purchased from participants in the Animal Protein Products Industry (APPI) 

Salmonella Education/Reduction Program or the Fishmeal Inspection Program 

of the National Marine Fisheries Service. The protein products must have 

a minimum moisture content of 14.5 percent and must have been heated 

throughout to a minimum temperature of 190 °F. or above, or to a minimum 

temperature of 165 °F. for at least 20 minutes, or to a minimum temperature of 

184 °F. under 70 lbs. pressure during the manufacturing process;

(iv) If mash feed contains animal protein, the protein products shall be purchased 

from participants in the Animal Protein Products Industry (APPI) Salmonella 

Education/Reduction Program or the Fishmeal Inspection Program of the 

National Marine Fisheries Service.

(v) Feed shall be stored and transported in such a manner as to prevent possible 

contamination;

(d) U.S. Sanitation 

Monitored 
32

(vi) Chicks shall be hatched in a hatchery meeting the requirements of §§ 147.23 

and 147.24(b) and sanitized or fumigated (see § 147.25 of this chapter);

(vii) An Authorized Agent shall take environmental samples, as described in 

§ 147.12 of this chapter, from each flock at 4 months of age and every 

90 days thereafter. An authorized laboratory for Salmonella shall examine the 

environmental samples bacteriologically;

(viii) Owners of flocks found infected with a paratyphoid Salmonella may vaccinate 

these flocks with an autogenous bacterin with a potentiating agent.4 

(2) The Official State Agency may use the procedures described in § 147.14 of this 

chapter to monitor the effectiveness of the sanitation practices.

(3) In order for a hatchery to sell products of this classification, all products handled 

shall meet the requirements of the classification.

(4) This classification may be revoked by the Official State Agency if the participant 

fails to follow recommended corrective measures.

(1) A flock maintained in compliance with the provisions of § 147.26 and in which 

freedom from M. synoviae has been demonstrated under the criteria specified in 

paragraph (e)(1)(i) or (ii) of this section.

(i) [Reserved]

(ii) It is a multiplier breeding flock which originated as U.S. M. Synoviae Clean 

chicks from primary breeding flocks and from which a sample comprised 

of a minimum of 150 birds has been tested for M. synoviae as provided in 

§ 145.14(b) when more than 4 months of age: Provided, That to retain this 

classification, the flock shall be subjected to one of the following procedures:

(A) At intervals of not more than 90 days, 75 birds from the flock shall be tested: 

Provided, That fewer than 75 birds from the flock may be tested at any one 

time if all pens are equally represented and a total of at least 75 birds from the 

flock is tested within each 90-day period; or

(B) At intervals of not more than 30 days, egg yolk testing shall be conducted in 

accordance with § 147.8.

(2) A participant handling U.S. M. Synoviae Clean products shall keep these products 

separate from other products in a manner satisfactory to the Official State Agency. 

(3) U.S. M. Synoviae Clean chicks shall be boxed in clean boxes and delivered in 

trucks that have been cleaned and disinfected as described in § 147.24(a) of this 

chapter.

(4) Before male breeding birds may be added to a participating multiplier breeding 

flock, a sample of at least 30 birds to be added, with a minimum of 10 birds per 

pen, shall be tested for M. synoviae as provided in § 145.14(b) or by a polymerase 

chain reaction (PCR)-based procedure approved by the Department. If fewer 

than 30 male breeding birds are being added, all the birds shall be tested as 

described above. The male birds shall be tested no more than 14 days prior to 

their intended introduction into the flock. If the serologic testing of the birds yields 

hemagglutination inhibition titers of 1:40 or higher as provided in § 145.14 (b), or if 

the PCR testing is positive for M. synoviae, the male birds may not be added to the 

flock and must be either retested or destroyed.

(e) U.S. M. Synoviae 

Clean

4

Preparation and use of this type of vaccine may be regulated by State statutes. 
33

(1) A flock which originated from U.S. M. Gallisepticum Clean breeding flocks and was 

hatched in a hatchery approved by the Official State Agency for the production of 

U.S. M. Gallisepticum Clean chicks.

(2) All other poultry on the premises of the candidate flock must originate from 

U.S. M. Gallisepticum Clean sources.

(3) The flock is maintained in compliance with the provisions of § 147.26 of this 

chapter.

(4) The flock’s freedom from M. gallisepticum is demonstrated by a negative blood 

test, as provided in § 145.14(b), of a sample of 75 birds, with a minimum of 

50 birds per poultry house, between 15–20 days prior to the flock being moved to 

laying quarters.

(5) Started poultry shall be delivered to and from the farm premises in crates and 

vehicles which have been cleaned and disinfected as described in § 147.24(a) of 

this chapter.

(1) A flock which originated from U.S. M. Synoviae Clean breeding flocks and was 

hatched in a hatchery approved by the Official State Agency for the production of 

U.S. M. Synoviae Clean chicks.

(2) All other poultry on the premises of the candidate flock must originate from U.S. 

M. Synoviae Clean sources.

(3) The flock is maintained in compliance with the provisions of § 147.26 of this 

chapter.

(4) The flock’s freedom from M. synoviae is demonstrated by a negative blood test, 

as provided in § 145.14(b), of a sample of 75 birds, with a minimum of 50 birds 

per poultry house, between 15–20 days prior to the flock being moved to laying 

quarters.

(5) Started poultry shall be delivered to and from the farm premises in crates and 

vehicles which have been cleaned and disinfected as described in § 147.24(a) of 

this chapter. 

(h) U.S. S. Enteritidis 

Clean [Reserved] 

(g) U.S. M. Synoviae 

Clean Started Poultry

(f) U.S. M. Gallisepticum 

Clean Started Poultry 

(i) U.S. Salmonella 

Monitored [Reserved]
34

(1) A multiplier breeding flock in which all birds or a sample of at least 30 birds per 

house has been tested for M. gallisepticum as provided in §145.14(b) when more 

than 4 months of age: Provided, That to retain this classification, a minimum of 

30 birds per house shall be tested again at 36 to 38 weeks and at 48 to 50 weeks 

at a minimum: And provided further, That each 30-bird sample should come from 

2 locations within the house (15 from the front half of the house and 15 from the 

back half of the house). A representative sample of males and females shall be 

sampled. The samples shall be marked "male" or "female."

(2) A participant handling U.S. M. Gallisepticum Monitored products shall keep these 

products separate from other products in a manner satisfactory to the Official State 

Agency: Provided, That U.S. M. Gallisepticum Monitored chicks from multiplier 

breeding flocks shall be produced in incubators and hatchers in which only eggs 

from flocks qualified under paragraph (j)(1) of this section are set. Eggs from U.S. 

M. Gallisepticum Monitored multiplier breeding flocks shall not be set in hatchers 

or incubators in which eggs from U.S. M. Gallisepticum Clean primary breeding 

flocks qualified under paragraph (c)(1)(i) of this section are set.

(3) U.S. M. Gallisepticum Monitored chicks shall be boxed in clean boxes and delivered 

in trucks that have been cleaned and disinfected as described in § 147.24(a) of 

this chapter.

(1) A multiplier breeding flock in which all birds or a sample of at least 30 birds per 

house has been tested for M. synoviae as provided in § 145.14(b) when more 

than 4 months of age: Provided, That to retain this classification, a minimum of 

30 birds per house shall be tested again at 36 to 38 weeks and at 48 to 50 weeks 

at a minimum: And provided further, That each 30-bird sample should come from 

2 locations within the house (15 from the front half of the house and 15 from the 

back half of the house). A representative sample of males and females should be 

sampled. The samples shall be marked "male" or "female."

(2) A participant handling U.S. M. Synoviae Monitored products shall keep these 

products separate from other products in a manner satisfactory to the Official State 

Agency: Provided, That U.S. M. Synoviae Monitored chicks from multiplier breeding 

flocks shall be produced in incubators and hatchers in which only eggs from flocks 

qualified under paragraph (k)(1) of this section are set. Eggs from U.S. M. Synoviae 

Monitored multiplier breeding flocks shall not be set in hatchers or incubators in 

which eggs from U.S. M. Synoviae Clean primary breeding flocks qualified under 

paragraph (e)(1)(i) of this section are set.

(3) U.S. M. Synoviae Monitored chicks shall be boxed in clean boxes and delivered in 

trucks that have been cleaned and disinfected as described in § 147.24(a) of this 

chapter.

(k) U.S. M. Synoviae 

Monitored

(j) U.S. M. Gallisepticum 

Monitored 
35

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) [Reserved]

(2) It is a multiplier breeding flock in which a minimum of 15 birds have been tested 

negative for antibodies to avian influenza when more than 4 months of age and 

prior to the onset of egg production. To retain this classification:

(i) A sample of at least 15 birds must be tested negative at intervals of 90 days; 

Provided: That multiplier spent fowl must be tested within 30 days prior to 

movement to slaughter; or

 (ii) A sample of fewer than 15 birds may be tested, and found to be negative, 

at any one time if all pens are equally represented and a total of 15 birds is 

tested within each 90-day period.

(Approved by the Office of Management and Budget under control number 0579-0007) 

§ 145.34 Terminology and classification; States. 

(1) A State will be declared a U.S. Pullorum-Typhoid Clean State when it has been 

determined by the Service that:

(i) The State is in compliance with the provisions contained in § 145.23(b)(3)(i)

 through (vii), § 145.33(b)(3)(i) through (vii), § 145.43(b)(3)(i) through (vi), 

 § 145.53(b)(3)(i) through (vii), § 145.73(b)(2)(i), and § 145.83(b)(2)(i).

(ii) No pullorum disease or fowl typhoid is known to exist nor to have existed 

in hatchery supply flocks within the State during the preceding 12 months:

Provided, That pullorum disease or fowl typhoid found within the preceding 

24 months in waterfowl, exhibition poultry, and game bird breeding flocks will 

not prevent a State, which is otherwise eligible from qualifying.

(2) Discontinuation of any of the conditions described in paragraph (a)(1)(i) of this 

section, or repeated outbreaks of pullorum or typhoid occur in hatchery supply 

flocks described in paragraph (a)(1)(ii) of this section, or if an infection spreads 

from the originating premises, the Service shall have grounds to revoke its 

determination that the State is entitled to this classification. Such action shall not 

be taken until a thorough investigation has been made by the Service and the 

Official State Agency has been given an opportunity for a hearing in accordance 

with rules of practice adopted by the Administrator.

(l) U.S. Avian Influenza 

Clean

(a) U.S. PullorumTyphoid Clean State 
36

(1) A State will be declared a U.S. M. Gallisepticum Clean State, Meat-Type Chickens, 

when it has been determined by the Service that:

(i) No M. gallisepticum is known to exist nor to have existed in meat-type chicken 

breeding flocks in production within the State during the preceding 12 months;

(ii) All meat-type chicken breeding flocks in production are classified as U.S. 

M. Gallisepticum Clean in accordance with §§ 145.33(c) and 145.83(c) or 

have met equivalent requirements for M. gallisepticum control under official 

supervision;

(iii) All hatcheries within the State which handle products from meat-type 

chicken breeding flocks only handle products which are classified as U.S. M. 

Gallisepticum Clean or have met equivalent requirements for M. gallisepticum 

control under official supervision;

(iv) All shipments of products from meat-type chicken breeding flocks other than 

those classified as U.S. M. Gallisepticum 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 

specimens from chickens from meat-type chicken breeding flocks that have 

been identified as being infected with M. gallisepticum;

(vi) All reports of M. gallisepticum infection in chickens from meat-type chicken 

breeding flocks are promptly followed by an investigation by the Official State 

Agency to determine the origin of the infection;

(vii) All chickens from meat-type chicken breeding flocks found to be infected 

with M. gallisepticum are quarantined until marketed under supervision of the 

Official State Agency.

(2) Discontinuation of any of the conditions described in paragraph (b)(1) of this 

section, or if repeated outbreaks of M. gallisepticum occur in meat-type chicken 

breeding flocks described in paragraph (b)(1)(ii) of this section, or if an infection 

spreads from the originating premises, the Service shall have grounds to revoke 

its determination that the State is entitled to this classification. Such action shall 

not be taken until a thorough investigation has been made by the Service and the 

Official State Agency has been given an opportunity for a hearing in accordance 

with rules of practice adopted by the Administrator.

(b) U.S. M. Gallisepticum 

Clean State, Meat-Type 

Chickens 
37

Subpart D—Special Provisions for Turkey Breeding Flocks and Products

§ 145.41 Definitions. 

Except where the context otherwise requires, for the purposes of this subpart the 

following terms shall be construed, respectively, to mean:

Newly hatched turkeys. 

§ 145.42 Participation. 

(a) Participating turkey flocks, and the eggs and poults produced from them, shall 

comply with the applicable general provisions of Subpart A of this part and the 

special provisions of this Subpart D.

(b) Hatching eggs shall be fumigated (see § 147.25 of this chapter) or otherwise 

sanitized. 

(c) 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.

§ 145.43 Terminology and classification; flocks and products. 

Participating flocks, and the eggs and poults produced from them, which have met the 

respective requirements specified in this section may be designated by the following 

terms and the corresponding designs illustrated in § 145.10:

A flock in which freedom from pullorum and typhoid has been demonstrated to the 

Official State Agency under the criteria in one of the following paragraphs (b)(1) 

through (5) of this section: Provided, That a flock qualifying by means of a blood test 

shall be tested within the past 12 months, except that the retesting of a participating 

flock which is retained for more than 12 months shall be conducted a minimum of 4 

weeks after the induction of molt. (See § 145.14 relating to the official blood test where 

applicable.)

(1) It has been officially blood tested with no reactors.

(2) It is a multiplier breeding flock, or a breeding flock composed of progeny of a 

primary breeding flock which is intended solely for the production of multiplier 

breeding flocks, and meets the following specifications as determined by the 

Official State Agency and the Service:

(i) The flock is located in a State where 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 poultry specimens from which 

S. pullorum or S. gallinarum is isolated;

Poults 

(a) [Reserved]

(b) U.S. PullorumTyphoid Clean 
38

(ii) The flock is composed entirely of birds that originated from U.S. PullorumTyphoid Clean breeding flocks or from flocks that met equivalent requirements 

under official supervision; and

(iii) The flock is located on a premises where either no poultry or a flock not 

classified as U.S. Pullorum-Typhoid Clean were located the previous year; 

Provided, That an Authorized Testing Agent must blood test up to 300 birds 

per flock, as described in § 145.14, if the Official State Agency determines that 

the flock has been exposed to pullorum-typhoid. In making determinations of 

exposure and setting the number of birds to be blood tested, the Official State 

Agency shall evaluate the results of any blood tests, described in § 145.14(a)

(1) of this part, that were performed on an unclassified flock located on the 

premises during the previous year; the origins of the unclassified flock; and 

the probability of contacts between the flock for which qualification is being 

sought and (a) infected wild birds, (b) contaminated feed or waste, or (c) birds, 

equipment, supplies, or personnel from flocks infected with pullorum-typhoid.

(3) It is a multiplier breeding flock, or a breeding flock composed of progeny of a 

primary breeding flock which is intended solely for the production of multiplier 

breeding flocks, that originated from U.S. Pullorum-Typhoid Clean breeding flocks 

or from flocks that met equivalent requirements under official supervision, and is 

located in a State in which it has been determined by the Service that:

(i) All turkey hatcheries within the State are qualified as “National Plan 

Hatcheries” or have met equivalent requirements for pullorum-typhoid control 

under official supervision;

(ii) All turkey hatchery supply flocks within the State are qualified as 

U.S. Pullorum-Typhoid Clean or have met equivalent requirements for 

pullorum- typhoid control under official supervision: Provided, That if other 

domesticated fowl, except waterfowl, are maintained on the same premises 

as the participating flock, freedom from pullorum-typhoid infection shall be 

demonstrated by an official blood test of each of these fowl;

(iii) All shipments of products other than U.S. Pullorum-Typhoid Clean, or 

equivalent, into the State are prohibited;

(iv) 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 

poultry specimens from which S. pullorum or S. gallinarum is isolated;

(v) All reports of any disease outbreak involving a disease covered under the 

Plan are promptly followed by an investigation by the Official State Agency to 

determine the origin of the infection: 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;

(vi) All flocks found to be infected with pullorum or typhoid are quarantined until 

marketed or destroyed under the supervision of the Official State Agency, or 

until subsequently blood tested, following the procedure for reacting flocks 

as contained in § 145.14(a)(5), and all birds fail to demonstrate pullorum or 

typhoid infection;

(vii) [Reserved]
39

(viii) Discontinuation of any of the conditions or procedures described in 

paragraphs (b)(3)(i), (ii), (iii), (iv), (v), and (vi) of this section, or the occurrence 

of repeated outbreaks of pullorum or typhoid in turkey breeding flocks within 

or originating within the State shall be grounds for the Service to revoke 

its determination that such conditions and procedures have been met or 

complied with. Such action shall not be taken until a thorough investigation has 

been made by the Service and the Official State Agency has been given an 

opportunity to present its views.

(4) It is a multiplier breeding flock located in a State which has been determined by 

the Service to be in compliance with the provisions of paragraph (b)(3) of this 

section and in which pullorum disease or fowl typhoid is not known to exist nor to 

have existed in turkey hatchery supply flocks within the State during the preceding 

24 months.

(5) It is a primary breeding flock located in a State determined to be in compliance 

with the provisions of paragraph (b)(4), of this section and in which a sample of 

300 birds from flocks of more than 300, and each bird in flocks of 300 or less, 

has been officially tested for pullorum-typhoid with no reactors: Provided, That a 

bacteriological examination monitoring program acceptable to the Official State 

Agency and approved by the Service may be used in lieu of blood testing.

(1) A flock maintained in accordance with the conditions and procedures described 

in § 147.26 of this chapter, and in which no reactors are found when a random 

sample of at least 10 percent of the birds in the flock, or 300 birds in flocks of more 

than 300 and each bird in flocks of 300 or less, is tested when more than 12 weeks 

of age, in accordance with the procedures described in § 145.14(b): Provided, That 

to retain this classification, a minimum of 30 samples from male flocks and 60 

samples from female flocks shall be retested at 28–30 weeks of age and at 4–6 

week intervals thereafter.

(2) A flock qualified as U.S. M. Gallisepticum Clean may retain the classification 

through its first egg-laying cycle, provided it is maintained in isolation and no 

evidence of M. gallisepticum infection is revealed. A flock which is molted following 

completion of an egg-laying cycle and subsequently brought back into production, 

shall be retested within 2 weeks prior to production, as described in paragraph (c)

(1) of this section. A State inspector shall visit with the owner or manager of each 

flock at least once during each laying cycle to discuss and ascertain whether the 

applicable conditions outlined in § 147.26 of this chapter are being met. If a flock 

proves to be infected with M. gallisepticum, it shall lose this classification.

(3) In order to sell hatching eggs or poults of this classification, all hatching eggs and 

poults handled by the participant must be of this classification.

(c) U.S. M. Gallisepticum 

Clean 
40

(1) A flock in which freedom from M. meleagridis has been demonstrated under the 

following criteria:

(i) A sample of 100 birds from each flock has been tested for M. meleagridis when 

more than 12 weeks of age: Provided, That to retain this classification, a minimum of 30 

samples from male flocks and 60 samples from female flocks

shall be retested at 28–30 weeks of age and at 4–6 week intervals thereafter.

(2) The official blood tests for M. meleagridis shall be the serum plate agglutination test, 

the tube agglutination test, or the microagglutination test. The hemagglutination 

inhibition (HI) test, microhemagglutination inhibition test, serum plate dilution test, 

microagglutination test and the enzyme-labeled immunosorbent assay (ELISA)5 test 

may be used as supplemental tests to determine the status of the flock, in accordance 

with § 147.6(b) of this chapter. 

(3) The tests shall be conducted using M. meleagridis antigens and the protocols for testing 

approved by the Department or the Official State Agency.

(4) When reactors to the official test are found and can be identified, 10 tracheal swabs 

and/or vaginal or phallus swabs and their corresponding blood samples shall be 

submitted to a laboratory for serological and cultural examination. If reactors cannot 

be identified, at least 30 tracheal swabs and/or vaginal or phallus swabs and their 

corresponding blood samples shall be submitted. In a flock with a low reactor rate (less 

than 5 reactors), the reactors may be submitted to the laboratory within 10 days for 

serology, necropsy, and thorough bacteriological examination.

(5) If a mycoplasma is isolated, the organism must be serotyped. If M. meleagridis is 

isolated, the flock shall be considered infected.

(1) All birds, or a sample of at least 100 birds from flocks of more than 100 and each bird 

in flocks of 100 or less, have been tested for M. synoviae when more than 12 weeks 

of age in accordance with the procedures in § 145.14(b): Provided, That to retain this 

classification a minimum of 30 samples from male flocks and 60 samples from female 

flocks shall be retested at 28–30 weeks of age and at 4–6 week intervals thereafter.

(2) When reactors to the official test are found and can be identified, tracheal swabs and 

their corresponding blood samples from 10 (all if fewer than 10) reacting birds shall 

be submitted to an authorized laboratory for serological and cultural examination. If 

reactors cannot be identified, at least 30 tracheal swabs and their corresponding blood 

samples shall be submitted. In a flock with a low reactor rate (less than five reactors) 

the reactors may be submitted to the laboratory within 10 days for serology, necropsy, 

and thorough bacteriological examination. When reactors to the official test are found, 

the procedures outlined in § 147.6 will be used to determine the status of the flock.

(3) Flocks located on premises which, during 3 consecutive years, have contained 

breeding flocks qualified as U.S. M. Synoviae Clean, as described in paragraph (e)(1) 

above, may qualify for this classification by a negative blood test of at least 100 birds 

from flocks of more than 100 and each bird in flocks of 100 or less, when more than 

12 weeks of age, and by testing a minimum of 30 samples from male flocks and 60 

samples from female flocks at 28–30 weeks of age and at 45 weeks of age.

(e) U.S. M. Synoviae 

Clean 

5

See footnote 3 to § 145.14(b)(1).

(d) U.S. M. Meleagridis 

Clean 
41

A flock or hatchery whose owner is controlling or reducing the level of salmonella 

through compliance with sanitation and management practices as described in 

Subpart C of Part 147 of this chapter, and where the following monitoring, testing, and 

management practices are conducted:

((1) Hatchery debris (dead germ hatching eggs, fluff, and meconium collected by 

sexors), swabs collected from hatch debris in hatcher trays, a sample of all the 

poults that died within 10 days after hatching up to 10 poults, or a combination of 

2 or all 3 of the above, from each hatch or a candidate breeding flock produced by 

a primary breeder, are examined bacteriologically at an authorized laboratory for 

Salmonella.

(2) The poults for the candidate breeding flock are placed in a building that has been 

cleaned and disinfected. An Authorized Agent must collect environmental samples 

from the building and submit them to an authorized laboratory for a bacteriological 

examination for the presence of Salmonella, as described in § 147.12 of this 

chapter.

 (3) Feed for turkeys in the candidate breeding flock shall meet the following 

requirements:

(i) All feed manufactured in pellet form must have a maximum moisture content of 

13.5 percent upon delivery to the farm. It should have been preconditioned to 

the minimum of one of the following parameters before pelleting: 

(A) Feed is to reach a minimum temperature of 185 °F for a minimum of 6 

minutes of retention in the conditioning chamber. The conditioned mash 

feed moisture must be a minimum of 16 percent during the conditioning 

process. This method utilizes time retention to allow permeation to the 

center core of each feed particle; or 

(B) The feed is to be pressurized in order to expedite the transfer of the 

heat and moisture to the core of each feed particle. The feed should be 

conditioned to the parameters of a minimum of 16 percent moisture and 

200 °F; or 

(C) The feed should be submitted to pressurization to the extent that the initial 

feed temperature rises to 235 °F for 4 seconds; or 

(D) The feed should be submitted to an equivalent thermal lethality treatment; 

or 

(E) A Food and Drug Administration (FDA)-approved product for Salmonella

control may be added to either unfinished or finished feed. 

(ii) Mash feed should be treated with an FDA-approved Salmonella control product. 

(iii) All feed is to be stored and transported in such a manner as to prevent possible 

contamination with pathogenic bacteria. 

(iv) FDA-approved products for Salmonella control may be added to either 

unfinished or finished feed. 

(4) Environmental samples shall be taken by an Authorized Agent, as described in 

§ 147.12 of this chapter, from each flock at 12–20 weeks of age and examined 

bacteriologically at an authorized laboratory for Salmonella.

(5) Owners of flocks found infected with a paratyphoid Salmonella may vaccinate these 

flocks with an autogenous bacterin with a potentiating agent.6

(f) U.S. Sanitation 

Monitored, Turkeys 

6

Preparation and use of this type of vaccine may be regulated by state statutes. 
42

(6) Environmental samples shall be taken by an Authorized Agent, as described in 

§ 147.12 of this chapter, from each flock at 35–50 weeks of age and from each 

molted flock at midlay, and examined bacteriologically at an authorized laboratory 

for Salmonella.

(7) Hatchery debris (dead germ hatching eggs, fluff, and meconium collected by 

sexors), swabs collected from hatch debris in hatcher trays, a sample of all the 

poults that died within 10 days after hatching up to 10 poults, or a combination of 2 

or all 3 of the above, shall be cultured as a means of evaluating the effectiveness 

of the control procedures. 

This program is intended to be the basis from which the turkey breeding industry 

may conduct a program for the prevention and control of the H5 and H7 subtypes of 

avian influenza. It is intended to determine the presence of the H5 and H7 subtypes 

of avian influenza in breeding turkeys through routine serological surveillance of each 

participating breeding flock. A flock, and the hatching eggs and poults produced from 

it, will qualify for this classification when the Official State Agency determines that it 

has met one of the following requirements: 

(1) It is a primary breeding flock in which a minimum of 30 birds has been 

tested negative for antibodies to type A avian influenza virus by the agar gel 

immunodiffusion test specified in § 147.9 of this chapter. Positive samples shall 

be further tested by an authorized laboratory using the hemagglutination inhibition 

test to detect antibodies to the hemagglutinin subtypes H5 and H7 when more 

than 4 months of age and prior to the onset of egg production. To retain this 

classification:

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

Provided, that primary spent fowl be tested within 30 days prior to movement 

to disposal; 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 are 

tested within each 90-day period. 

(2) It is a multiplier breeding flock in which a minimum of 30 birds has been 

tested negative for antibodies to type A avian influenza virus by the agar gel 

immunodiffusion test specified in § 147.9 of this chapter. Positive samples shall 

be further tested by an authorized laboratory using the hemagglutination inhibition 

test to detect antibodies to the hemagglutinin subtypes H5 and H7 when more 

than 4 months of age and prior to the onset of egg production. To retain this 

classification:

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

Provided, that multiplier spent fowl be tested within 30 days prior to movement 

to disposal; 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 are 

tested within each 90-day period. 

(g) U.S. H5/H7 Avian

Influenza Clean
43

(3) For both primary and multiplier breeding flocks, if a killed influenza vaccine against 

avian influenza subtypes other than H5 and H7 is used, then the hemagglutinin 

and the neuraminidase subtypes of the vaccine must be reported to the Official 

State Agency for laboratory and reporting purposes.

(Approved by the Office of Management and Budget under control number 0579-0007) 

§ 145.44 Terminology and classification; States. 

(1) A State will be declared a U.S. Pullorum-Typhoid Clean State when it has been 

determined by the Service that:

(i) The State is in compliance with the provisions contained in 

§ 145.23(b)(3)(i) through (vii), § 145.33(b)(3)(i) through (vii), 

§ 145.43(b)(3)(i) through (vi), and § 145.53(b)(3)(i) through (vii).

(ii) No pullorum disease or fowl typhoid is known to exist nor to have existed 

in hatchery supply flocks within the State during the preceding 12 months: 

Provided, That pullorum disease or fowl typhoid found within the preceding 

24 months in waterfowl, exhibition poultry, and game bird breeding flocks will 

not prevent a State, which is otherwise eligible, from qualifying.

(2) Discontinuation of any of the conditions described in paragraph (a)(1)(i) of this 

section, or repeated outbreaks of pullorum or typhoid occur in hatchery supply 

flocks described in paragraph (a)(1)(ii) of this section, or if an infection spreads 

from the originating premises, the Service shall have grounds to revoke its 

determination that the State is entitled to this classification. Such action shall not 

be taken until a thorough investigation has been made by the Service and the 

Official State Agency has been given an opportunity for a hearing in accordance 

with rules of practice adopted by the Administrator.

(1) A State will be declared a U.S. Pullorum-Typhoid Clean State, Turkeys, when it has 

been determined by the Service that:

(i) The State is in compliance with the provisions contained in

 §145.43(b)(3)(i)through (vi).

(ii) No pullorum disease or fowl typhoid is known to exist nor to have existed in 

turkey hatchery supply flocks within the State during the preceding 24 months.

(2) Discontinuation of any of the conditions described in paragraph (b)(1)(i) of 

this section, or repeated outbreaks of pullorum or typhoid occur in hatchery 

supply flocks described in paragraph (b)(1)(ii) of this section, or if an infection 

spreads from the originating premises, Service shall have grounds to revoke its 

determination that the State is entitled to this classification. Such action shall not 

be taken until a thorough investigation has been made by the Service and the 

Official State Agency has been given an opportunity for a hearing in accordance 

with rules of practice adopted by the Administrator.

(a) U.S. PullorumTyphoid Clean State 

(b) U.S. PullorumTyphoid Clean State, 

Turkeys 
44

(1) A State will be declared a U.S. M. Gallisepticum Clean State, Turkeys when it has 

been determined by the Service that:

 (i) No M. gallisepticum 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 classified as U.S. M. Gallisepticum 

Clean or have met equivalent requirements for M. gallisepticum control under 

official supervision.

(iii) All turkey hatcheries within the State handle products which are classified 

as U.S. M. Gallisepticum Clean or have met equivalent requirements for 

M. gallisepticum control under official supervision.

(iv) All shipments of turkey products other than those classified as 

 U.S. M. Gallisepticum 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. gallisepticum.

(vi) All reports of M. gallisepticum infection in turkeys are promptly followed by 

an investigation by the Official State Agency to determine the origin of the 

infection.

(vii) All turkey flocks found to be infected with M. gallisepticum are quarantined 

until marketed under supervision of the Official State Agency.

(2) Discontinuation of any of the conditions described in paragraph (c)(1) of this 

section, or if repeated outbreaks of M. gallisepticum occur in turkey breeding flocks 

described in paragraph (c)(1)(ii) of this section, or if an infection spreads from the 

originating premises, the Service shall have grounds to revoke its determination 

that the State is entitled to this classification. Such action shall not be taken until 

a thorough investigation has been made by the Service and the Official State 

Agency has been given an opportunity for a hearing in accordance with rules of 

practice adopted by the Administrator.

(3) If a State retains this status for 2 or more years, individual breeding flocks in the 

State may qualify for an M. gallisepticum classification based on a negative test of 

a sample of 100 birds.

(1) A State will be declared a U.S. M. Synoviae Clean State, Turkeys, if the Service 

determines that:

(i) No Mycoplasma synoviae 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. 

Synoviae Clean or have met equivalent requirements for M. synoviae control 

under official supervision;

(iii) All turkey hatcheries within the State only handle products that are classified 

as U.S. M. Synoviae Clean or have met equivalent requirements for 

M. synoviae control under official supervision;

(iv) All shipments of products from turkey breeding flocks other than those 

classified as U.S. M. Synoviae Clean, or equivalent, into the State are 

prohibited;

(c) U.S. M. Gallisepticum 

Clean State, Turkeys 

(d) U.S. M. Synoviae

Clean State, Turkeys 
45

(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. synoviae;

(vi) All reports of M. synoviae 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. synoviae 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. Synoviae Clean 

State, Turkeys, if any of the conditions described in paragraph (d)(1) of this section 

are discontinued. The Service shall not revoke the State’s classification as a U.S. 

M. Synoviae 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.

(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.

(Approved by the Office of Management and Budget under control number 0579-0007) 

(e) U.S. M. Meleagridis 

Clean State, Turkeys 
46

Subpart E—Special Provisions for Waterfowl, Exhibition Poultry, and 

Game Bird Breeding Flocks and Products

§ 145.51 Definitions. 

Except where the context otherwise requires, for the purposes of this subpart the 

following terms shall be construed, respectively, to mean:

Domesticated fowl which are bred for the combined purposes of meat or egg 

production and competitive showing.

Domesticated fowl such as pheasants, partridge, quail, grouse, and guineas, but not 

doves and pigeons.

Domesticated fowl that normally swim, such as ducks and geese.

§ 145.52 Participation. 

Participating flocks of waterfowl, exhibition poultry, and game birds, and the eggs and 

baby poultry produced from them shall comply with the applicable general provisions 

of Subpart A of this part and the special provisions of this Subpart E.

(a) Started poultry shall lose their identity under Plan terminology when not maintained 

by Plan participants under the conditions prescribed in § 145.5(a).

(b) Hatching eggs produced by primary breeding flocks shall be fumigated (see 

§ 147.25 of this chapter) or otherwise sanitized.

(c) Subject to the approval of the Service and the Official State Agencies in the 

importing and exporting States, participating flocks may report poultry sales to 

importing States by using either VS Form 9–3, “Report of Sales of Hatching Eggs, 

Chicks, and Poults,” or by using a hatchery invoice form (9-3I) approved by the 

Official State Agency and the Service to identify poultry sales to clients. If the 

selling hatchery uses the 9-3I form, the following information must be included on 

the form:

(1) The form number "9-3I", printed or stamped on the invoice;

(2) The hatchery name and address;

(3) The date of shipment;

(4) The hatchery invoice number;

(5) The purchaser name and address;

(6) The quantity of products sold;

(7) Identification of the products by bird variety or by NPIP stock code as listed in 

the NPIP APHIS 91-55-078 appendix; and

(8) The appropriate NPIP illustrative design in § 145.10. One of the designs in 

§ 145.10(b) or (g) must be used. The following information must be provided 

in or near the NPIP design:

 (i) The NPIP State number and NPIP hatchery approval number; and

 (ii) The NPIP classification for which product is qualified (e.g., U.S. Pullorum-

 Typhoid Clean).

(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.

Waterfowl 

Exhibition Poultry 

Game birds 
47

§ 145.53 Terminology and classification; flocks and products. 

Participating flocks, and the eggs and baby poultry produced from them, which have 

met the respective requirements specified in this section may be designated by the 

following terms and the corresponding designs illustrated in § 145.10.

A flock in which freedom from pullorum and typhoid has been demonstrated to 

the Official State Agency under the criteria in one of the following paragraphs 

(b)(1) through (5) of this section (See § 145.14 relating to the official blood test where 

applicable.):

(1) It has been officially blood tested within the past 12 months with no reactors.

(2) It is a multiplier breeding flock, or a breeding flock composed of progeny of a 

primary breeding flock which is intended solely for the production of multiplier 

breeding flocks, and meets the following specifications as determined by the 

Official State Agency and the Service:

(i) The flock is located in a State where 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 poultry specimens from which 

S. pullorum or S. gallinarum is isolated;

(ii) The flock is composed entirely of birds that originated from U.S. PullorumTyphoid Clean breeding flocks or from flocks that met equivalent requirements 

under official supervision; and

(iii) The flock is located on a premises where either no poultry or a flock not 

classified as U.S. Pullorum-Typhoid Clean were located the previous year: 

Provided, That an Authorized Testing Agent must blood test up to 300 birds 

per flock, as described in § 145.14, if the Official State Agency determines 

that the flock has been exposed to pullorum-typhoid. In making determinations 

of exposure and setting the number of birds to be blood tested, the Official 

State Agency shall evaluate the results of any blood tests, described in § 

145.14(a)(1), that were performed on an unclassified flock located on the 

premises during the previous year; the origins of the unclassified flock; and 

the probability of contacts between the flock for which qualification is being 

sought and (a) infected wild birds, (b) contaminated feed or waste, or (c) birds, 

equipment, supplies, or personnel from flocks infected with pullorum-typhoid.

(3) It is a multiplier breeding flock that originated from U.S. Pullorum-Typhoid Clean 

breeding flocks or from flocks that met equivalent requirements under official 

supervision, and is located in a State in which it has been determined by the 

Service that:

(i) All hatcheries within the State are qualified as “National Plan Hatcheries” or 

have met equivalent requirements for pullorum-typhoid control under official 

supervision;

(b) U.S. PullorumTyphoid Clean 

(a) [Reserved]
48

(ii) All hatchery supply flocks within the State are qualified as U.S. PullorumTyphoid Clean or have met equivalent requirements for pullorum-typhoid 

control under official supervision: Provided, That if other domesticated fowl, 

except waterfowl, are maintained on the same premises as the participating 

flock, freedom from pullorum-typhoid infection shall be demonstrated by an 

official blood test of each of these fowl;

(iii) All shipments of products other than U.S. Pullorum-Typhoid Clean, or 

equivalent, into the State are prohibited;

(iv) 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 

poultry specimens from which S. pullorum or S. gallinarum is isolated;

(v) All reports of any disease outbreak involving a disease covered under the 

Plan are promptly followed by an investigation by the Official State Agency to 

determine the origin of the infection: 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;

(vi) All flocks found to be infected with pullorum or typhoid are quarantined until 

marketed or destroyed under the supervision of the Official State Agency, or 

until subsequently blood tested, following the procedure for reacting flocks 

as contained in § 145.14(a)(5), and all birds fail to demonstrate pullorum or 

typhoid infection;

(vii) All poultry, including exhibition, exotic, and game birds, but excluding 

waterfowl, going to public exhibition shall come from U.S. Pullorum-Typhoid 

Clean or equivalent flocks, or have had a negative pullorum-typhoid test within 

90 days of going to public exhibition;

(viii) Discontinuation of any of the conditions or procedures described in 

paragraphs (b)(3)(i), (ii), (iii), (iv), (v), (vi), and (vii) of this section, or the 

occurrence of repeated outbreaks of pullorum or typhoid in poultry breeding 

flocks within or originating within the State shall be grounds for the Service to 

revoke its determination that such conditions and procedures have been met 

or complied with. Such action shall not be taken until a thorough investigation 

has been made by the Service and the Official State Agency has been given 

an opportunity to present its views.

(4) It is a multiplier breeding flock located in a State which has been determined by 

the Service to be in compliance with the provisions of paragraph (b)(3) of this 

section, and in which pullorum disease or fowl typhoid is not known to exist nor 

to have existed in hatchery supply flocks within the State during the preceding 24 

months.

(5) It is a primary breeding flock located in a State determined to be in compliance 

with the provisions of paragraph (b)(4) of this section, and in which a sample of 

300 birds from flocks of more than 300, and each bird in flocks of 300 or less, 

has been officially tested for pullorum-typhoid within the past 12 months with no 

reactors: Provided, That a bacteriological examination monitoring program or 

serological examination monitoring program for game birds acceptable to the 

Official State Agency and approved by the Service may be used in lieu of annual 
49

blood testing: And provided further, That when a flock is a waterfowl or exhibition 

poultry primary breeding flock located in a State which has been deemed to be a 

U.S. Pullorum-Typhoid Clean State for the past three years, and during which time 

no isolation of pullorum or typhoid has been made that can be traced to a source 

in that State, a bacteriological examination monitoring program or a serological 

examination monitoring program acceptable to the Official State Agency and 

approved by the Service may be used in lieu of annual blood testing.

(1) A flock maintained in compliance with the provisions of § 147.26 of this chapter 

and in which freedom from M. gallisepticum has been demonstrated under the 

criteria specified in paragraph (c)(1)(i) or (ii) of this section.

 (i) It is a flock in which all birds or a sample of at least 300 birds has been 

tested for M. gallisepticum as provided in § 145.14(b) when more than 4 

months of age or upon reaching sexual maturity: Provided, That to retain this 

classification, a random sample of serum or egg yolk from at least 5 percent 

of the birds in the flock, but at least 30 birds, shall be tested at intervals of not 

more than 90 days: And provided further, That a sample comprised of less 

than 5 percent may be tested at any one time, with the approval of the Official 

State Agency and the concurrence of the Service, provided that a total of at 

least 5 percent of the birds in the flock, but at least 30 birds, is tested within 

each 90-day period; or

(ii) It is a multiplier breeding flock which originated as U.S. M. Gallisepticum Clean 

baby poultry from primary breeding flocks and a random sample comprised 

of 50 percent of the birds in the flock, with a maximum of 200 birds and 

a minimum of 30 birds per flock, has been tested for M. gallisepticum as 

provided in § 145.14(b) when more than 4 months of age or upon reaching 

sexual maturity: Provided, That to retain this classification, the flock shall be 

subjected to one of the following procedures:

(A) At intervals of not more than 90 days, a random sample of serum or egg 

yolk from at least 2 percent of the birds in the flock, with a minimum of 

30 birds per pen, shall be tested; or

(B) At intervals of not more than 30 days, a sample of 25 cull baby poultry 

produced from the flock shall be subjected to laboratory procedures 

acceptable to the Official State Agency and approved by the Service, for 

the detection and recovery of M. gallisepticum.

(2) A participant handling U.S. M. Gallisepticum Clean products shall keep these 

products separate from other products in a manner satisfactory to the Official 

State Agency: Provided, That U.S. M. Gallisepticum Clean baby poultry from 

primary breeding flocks shall be produced in incubators and hatchers in which only 

eggs from flocks qualified under paragraph (c)(1)(i) of this section are set.

(3) U.S. M. Gallisepticum Clean baby poultry shall be boxed in clean boxes and 

delivered in trucks that have been cleaned and disinfected as described in 

§ 147.24(a) of this chapter. 

(c) U.S. M. Gallisepticum 

Clean 
50

(1) A flock maintained in compliance with the provisions of § 147.26 of this chapter 

and in which freedom from Mycoplasma synoviae has been demonstrated under 

the criteria specified in paragraph (d)(1)(i) or (d)(1)(ii) of this section.

 (i) It is a flock in which a minimum of 300 birds has been tested for M. synoviae 

as provided in § 145.14(b) when more than 4 months of age: Provided, That 

to retain this classification, a sample of at least 150 birds shall be tested 

at intervals of not more than 90 days: And provided further, That a sample 

comprised of fewer than 150 birds may be tested at any one time with the 

approval of the Official State Agency and the concurrence of the Service, 

provided that a minimum of 150 birds is tested within each 90-day period; or

(ii) It is a multiplier breeding flock that originated as U.S. M. Synoviae Clean 

chicks from primary breeding flocks and from which a sample comprised 

of a minimum of 75 birds has been tested for M. synoviae as provided in 

§ 145.14(b) when more than 4 months of age: Provided, That to retain this 

classification, the flock shall be subjected to one of the following procedures:

(A) At intervals of not more than 90 days, a sample of 50 birds shall be tested: 

Provided, That a sample of fewer than 50 birds may be tested at any one 

time, provided that a minimum of 30 birds per flock with a minimum of 15 

birds per pen, whichever is greater, is tested each time and a total of at 

least 50 birds is tested within each 90-day period; or

(B) At intervals of not more than 30 days, egg yolk testing shall be conducted 

in accordance with § 147.8 of this chapter.

(2) A participant handling U.S. M. Synoviae Clean products shall keep those products 

separate from other products in a manner satisfactory to the Official State Agency: 

Provided, That U.S. M. Synoviae Clean chicks from primary breeding flocks shall 

be produced in incubators and hatchers in which only eggs from flocks qualified 

under paragraph (d)(1)(i) or (d)(1)(ii) of this section are set.

(3) U.S. M. Synoviae Clean chicks shall be boxed in clean boxes and delivered in 

trucks that have been cleaned and disinfected as described in § 147.24(a) of this 

chapter.

This program is intended to be the basis from which the breeding-hatchery industry 

may conduct a program for the prevention and control of the H5 and H7 subtypes of 

avian influenza. It is intended to determine the presence of the H5 and H7 subtypes of 

avian influenza in waterfowl, exhibition poultry, and game bird breeding flocks 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 it has met one of the following requirements: 

(1) It is a primary breeding flock in which a minimum of 30 birds has been tested 

negative for antibodies to the H5 and H7 subtypes of avian influenza by the agar 

gel immunodiffusion test specified in § 147.9 of this chapter 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 are 

tested within each 90-day period. 

(e) U.S. H5/H7 Avian

Influenza Clean 

(d) U.S. M. Synoviae 

Clean
51

(2) It is a multiplier breeding flock in which a minimum of 30 birds has been tested 

negative for antibodies to the H5 and H7 subtypes of avian influenza by the agar 

gel immunodiffusion test specified in § 147.9 of this chapter 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 unvaccinated 

sentinel birds are tested within each 180-day period.

(Approved by the Office of Management and Budget under control number 0579-0007)

§ 145.54 Terminology and classification; States. 

(1) A State will be declared a U.S. Pullorum-Typhoid Clean State when it has been 

determined by the Service that:

(i) The State is in compliance with the provisions contained in § 145.23(b)(3)(i) 

through (vii), § 145.33(b)(3)(i) through (vii), § 145.43(b)(3)(i) through (vi), and 

§ 145.53(b)(3)(i) through (vii);

(ii) No pullorum disease or fowl typhoid is known to exist nor to have existed 

in hatchery supply flocks within the State during the preceding 12 months: 

Provided, That pullorum disease or fowl typhoid found within the preceding 

24 months in waterfowl, exhibition poultry, and game bird breeding flocks will 

not prevent a State, which is otherwise eligible, from qualifying.

(2) Discontinuation of any of the conditions described in paragraph (a)(1)(i) of this 

section, or repeated outbreaks of pullorum or typhoid occur in hatchery supply 

flocks described in paragraph (a)(1)(ii) of this section, or if an infection spreads 

from the originating premises, the Service shall have grounds to revoke its 

determination that the State is entitled to this classification. Such action shall not 

be taken until a thorough investigation has been made by the Service and the 

Official State Agency has been given an opportunity for a hearing in accordance 

with rules of practice adopted by the Administrator.

(a) U.S. PullorumTyphoid Clean State
52

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

Breeding Flocks and Products

§ 145.61 Definitions.

Except where the context otherwise requires, for the purposes of this subpart the 

following terms shall be construed, respectively, to mean:

Newly hatched ostriches, emus, rheas, or cassowaries.

Birds of the species Struthio camelus, including all subspecies and subspecies 

hybrids. 

§ 145.62 Participation.

Participating flocks of ostriches, emus, rheas, and cassowaries, and the eggs and 

chicks produced from them, shall comply with the applicable general provisions of 

subpart A of this part and the special provisions of this subpart.

(a) Started poultry shall lose their identity under Plan terminolgy when not maintained 

by Plan participants under the conditions prescribed in § 145.5 (a).

(b) Hatching eggs produced by primary breeding flocks shall be fumigated or 

otherwise sanitized (see § 147.22 of this chapter).

(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 Terminology and classification; flocks and products.

Participating flocks, and the eggs and baby poultry produced from them, that have 

met the respective requirements specified in this section may be designated by the 

following terms and their corresponding designs illustrated in § 145.10.

A flock in which freedom from pullorum and typhoid has been demonstrated to the 

Official State Agency under the criteria in paragraph (a)(1) or (a)(2) of this section. 

(See § 145.14(a) relating to the official blood test for pullorum-typhoid where 

applicable.)

(1) It has been officially blood tested within the past 12 months with no reactors.

(2) It is a breeding flock that meets one of the following criteria:

(i) (A) It is a multiplier or primary breeding flock of fewer than 300 birds in which 

a sample of 10 percent of the birds in a flock or at least 1 bird from each pen, 

whichever is more, has been officially tested for pullorum-typhoid within the 

past 12 months with no reactors; or

(B) It is a multiplier or primary breeding flock of 300 birds or more in which a 

sample of a minimum of 30 birds has been officially tested for pullorumtyphoid within the past 12 months with no reactors.

Ostrich

Chicks

(a) U.S. PullorumTyphoid Clean
53

(ii) It is a flock that has already been designated U.S. Pullorum-Typhoid Clean 

and uses a subsequent bacteriological examination monitoring program of 

hatcher debris or eggs for ostriches, emus, rheas, or cassowaries acceptable 

to the Official State Agency and approved by the Service in lieu of annual 

blood testing.

(iii) It is a multiplier breeding flock located in a State that has been deemed to 

be a U.S. Pullorum-Typhoid Clean State for the past 3 years, and during which 

time no isolation of pullorum or typhoid has been made that can be traced to 

a source in that State, that uses a bacteriological examination monitoring 

program of hatcher debris or eggs or a serological examination monitoring 

program acceptable to the Official State Agency and approved by the Service 

in lieu of annual blood testing.

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 all ostrich, emu, rhea,

and cassowary breeding flocks through routine serological surveillance of each

participating breeding flock. Acceptable tests include antigen and antibody detection

tests, as approved by the Official State Agency. A flock, and the hatching eggs and

chicks produced from it, will qualify for this classification when the Official State

Agency determines that it has met one of the following requirements: 

(1) It is a primary breeding flock in which 10 percent of the flock, up to a maximum

of 30 birds, has been tested negative for type A influenza virus with all pens 

represented equally and when the tested birds are more than 4 months of age. 

Positive samples shall be further tested by an authorized laboratory. 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 less than 10 percent of the birds, up to a maximum of 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 are tested within each 180-day period.

 (2) It is a multiplier breeding flock in which a minimum of 30 birds has been tested 

negative to type A influenza virus with all pens represented equally and when the 

tested birds are more than 4 months of age. Positive samples shall be further 

tested by an authorized laboratory. 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 at least 10 percent of birds from each pen with all pens being 

represented must be tested negative at intervals of 180 days; or

(iii) A sample of less than 10 percent of the birds may be tested, and found to 

be negative, at any one time if all pens are equally represented and a total of 

10 percent of the birds are tested within each 180-day period.

(Approved by the Office of Management and Budget under control number 0579-0007)

(b) U.S. Avian Influenza 

Clean 
54

Subpart G—Special Provisions for Primary Egg-Type Chicken Breeding 

Flocks and Products

§ 145.71 Definitions.

Except where the context otherwise requires, for the purposes of this subpart the 

following terms shall be construed, respectively, to mean: 

Newly hatched chickens. 

Foundation flocks that are composed of pedigree, great-grandparent, and grandparent 

stock that has been developed for egg production and are maintained for the principal 

purpose of producing multiplier breeding chicks used to produce table egg layers. 

Young chickens (chicks, pullets, cockerels, capons) which have been fed and watered 

and are less than 6 months of age. 

§ 145.72 Participation.

Participating flocks of primary egg-type chickens, and the eggs and chicks produced 

from them, shall comply with the applicable general provisions of subpart A of this part 

and the special provisions of this subpart G. 

(a) Started chickens shall lose their identity under Plan terminology when not 

maintained by Plan participants under the conditions prescribed in § 145.5(a). 

(b) Hatching eggs produced by primary breeding flocks shall be fumigated (see 

§ 147.25 of this chapter) or otherwise sanitized.

(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.

§ 145.73 Terminology and classification; flocks and products.

Participating flocks, and the eggs and chicks produced from them, which have met the 

respective requirements specified in this section, may be designated by the following 

terms and the corresponding designs illustrated in § 145.10:

A flock in which freedom from pullorum and typhoid has been demonstrated to the 

Official State Agency under the criteria in paragraph (b)(1) or (b)(2) of this section: 

Provided, That a flock qualifying by means of a blood test shall be tested within the 

past 12 months, except that the retesting of a participating flock which is retained for 

more than 12 months shall be conducted a minimum of 4 weeks after the induction of 

molt. (See § 145.14 relating to the official blood test where applicable.)

Chicks 

Primary egg-type 

chicken breeding flocks

Started chickens

(a) [Reserved]

(b) U.S. PullorumTyphoid Clean.
55

(1) It has been officially blood tested with no reactors. 

(2) It is a primary breeding flock that meets the following criteria:

(i) The primary breeding flock is located in a State in which pullorum disease or 

fowl typhoid is not known to exist nor to have existed in hatchery supply 

flocks during the preceding 12 months and in which it has been determined by 

the Service that:

(A) All hatcheries within the State are qualified as “National Plan Hatcheries” 

or have met equivalent requirements for pullorum-typhoid control under 

official supervision; 

(B) All hatchery supply flocks within the State, are qualified as U.S.

Pullorum-Typhoid Clean or have met equivalent requirements for 

pullorum-typhoid control under official supervision: Provided, That if other

domesticated fowl, except waterfowl, are maintained on the same 

premises as the participating flock, freedom from pullorum-typhoid 

infection shall be demonstrated by an official blood test of each of these 

fowl; 

(C) All shipments of products other than U.S. Pullorum-Typhoid Clean, or 

equivalent, into the State are prohibited; 

(D) 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 poultry specimens from which S. pullorum or S. gallinarum is 

isolated;

(E) All reports of any disease outbreak involving a disease covered under 

the Plan are promptly followed by an investigation by the Official State 

Agency to determine the origin of the infection; 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 officials 

administering the National Poultry Improvement Plan will conduct an

investigation; 

(F) All flocks found to be infected with pullorum or typhoid are quarantined 

until marketed or destroyed under the supervision of the Official State 

Agency, or until subsequently blood tested following the procedure 

for reacting flocks as contained in § 145.14(a)(5), and all birds fail to 

demonstrate pullorum or typhoid infection; 

(G) All poultry, including exhibition, exotic, and game birds, but excluding

waterfowl, going to public exhibition shall come from U.S. PullorumTyphoid Clean or equivalent flocks, or have had a negative pullorumtyphoid test within 90 days of going to public exhibition; and

(H) Discontinuation of any of the conditions or procedures described 

in paragraphs (b)(2)(i)(A) through (b)(2)(i)(G) of this section, or the 

occurrence of repeated outbreaks of pullorum or typhoid in poultry 

breeding flocks within or originating within the State shall be grounds

for the Service to revoke its determination that such conditions and 

procedures have been met or complied with. Such action shall not be

taken until a thorough investigation has been made by the Service and the 

Official State Agency has been given an opportunity to present its views; 

and 
56

(ii) In the primary breeding flock, a sample of 300 birds from flocks of more than 

 300, and each bird in flocks of 300 or less, has been officially tested for 

 pullorum-typhoid with no reactors: Provided, That a bacteriological 

examination monitoring program acceptable to the Official State Agency and 

approved by the Service may be used in lieu of blood testing. 

(1) A flock maintained in compliance with the provisions of § 147.26 of this chapter 

and in which freedom from M. gallisepticum has been demonstrated under the 

criteria specified in paragraph (c)(1)(i) of this section. 

 (i) It is a flock in which all birds or a sample of at least 300 birds has been tested 

for M. gallisepticum as provided in §145.14(b) when more than 4 months of 

age: Provided, That to retain this classification, a minimum of 150 birds shall 

be tested at intervals of not more than 90 days: And provided further, That a 

sample comprised of fewer than 150 birds may be tested at any one time, if all 

pens are equally represented and a total of 150 birds is tested within each 90-

 day period 

 (ii) [Reserved] 

(2) A participant handling U.S. M. Gallisepticum Clean products shall handle only

products of equivalent status. 

(3) U.S. M. Gallisepticum Clean chicks shall be boxed in clean boxes and delivered in 

trucks that have been cleaned and disinfected as described in §147.24(a) of this 

chapter. 

This classification is intended for primary egg-type breeders wishing to assure their 

customers that the hatching eggs and multiplier chicks produced are certified free of 

Salmonella enteritidis. 

(1) A flock and the hatching eggs and chicks produced from it which have met the 

following requirements as determined by the Official State Agency: 

 (i) The flock originated from a U.S. S. Enteritidis Clean flock, or 

meconium from the chick boxes and a sample of chicks that died within 

 7 days after hatching are examined bacteriologically for salmonella at an 

authorized laboratory. Cultures from positive samples shall be serotyped. 

(ii) All feed fed to the flock shall meet the following requirements: 

(A) Pelletized feed shall contain either no animal protein or only animal protein 

products produced under the Animal Protein Products Industry (APPI) 

Salmonella Education/Reduction Program. The protein products must 

have a minimum moisture content of 14.5 percent and must have been 

heated throughout to a minimum temperature of 190 °F, or above, or to a 

minimum temperature of 165 °F for at least 20 minutes, or to a minimum 

temperature of 184 °F under 70 lbs. pressure during the manufacturing 

process. 

 (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. 

(c) U.S. M. Gallisepticum 

Clean.

(d) U.S. S. Enteritidis 

Clean.
57

(iii) Feed shall be stored and transported in such a manner as to prevent possible 

 contamination; 

(iv) The flock is maintained in compliance with §§ 147.21, 147.24(a), and 147.26

 of this chapter. Rodents and other pests should be effectively controlled;

(v) Environmental samples shall be collected from the flock by an Authorized 

Agent, as described in § 147.12 of this chapter, when the flock is 2 to 4 weeks

 of age. The samples shall be examined bacteriologically for group D

 salmonella at an authorized laboratory. Cultures from positive samples shall

 be serotyped. The Authorized Agent shall also collect samples every 30 days

 after the first sample has been collected.

(vi) If a Salmonella vaccine is used that causes positive reactions with pullorum-

 typhoid antigen, one of the following options must be utilized: 

(A) Administer the vaccine after the pullorum-typhoid testing is done as

 described in paragraph (d)(1)(vii) of this section. 

(B) If an injectable bacterin or live vaccine that does not spread is used, keep

 a sample of 350 birds unvaccinated and banded for identification 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, vaccinate the banded, non-vaccinated birds.

(vii) Blood samples from 300 non-vaccinated birds as described in paragraph 

(d)(1)(vi) of this section shall be tested with either pullorum antigen or by 

a federally licensed Salmonella enteritidis enzyme-linked immunosorbent 

assay (ELISA) test when the flock is more than 4 months of age. All birds 

with positive or inconclusive reactions, up to a maximum of 25 birds, shall be 

submitted to an authorized laboratory and examined for the presence of group 

D salmonella, as described in § 147.11 of this chapter. Cultures from positive 

samples shall be serotyped. 

 (viii) Hatching eggs are collected as quickly as possible and are handled as 

described in § 147.22 of this chapter and are sanitized or fumigated (see 

 § 147.25 of this chapter). 

(ix) Hatching eggs produced by the flock are incubated in a hatchery that is in

 compliance with the recommendations in §§ 147.23 and 147.24(b) of this

 chapter, and sanitized either by a procedure approved by the Official State

 Agency or fumigated (see § 147.25 of this chapter).

(2) A flock shall not be eligible for this classification if Salmonella enteritidis serotype 

enteritidis (SE) is isolated from a specimen taken from a bird in the flock. 

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 § 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. If no SE is recovered from any of 

the specimens in the second sample, the flock will be eligible for the classification.
58

(3) A non-vaccinated flock shall be eligible for this classification if SE is isolated from 

an environmental sample collected from the flock in accordance with paragraph 

(d)(1)(v) of this section: Provided, That testing is conducted in accordance with 

paragraph (d)(1)(vii) of this section each 30 days and no positive samples are 

found.

(4) In order for a hatchery to sell products of this classification, all products handled

shall meet the requirements of the classification.

(5) This classification may be revoked by the Official State Agency if the participant

fails to follow recommended corrective measures. The Official State Agency shall 

not revoke the participant’s classification until the participant has been given an 

opportunity for a hearing in accordance with rules of practice adopted by the 

Official State Agency. 

(1) A flock maintained in compliance with the provisions of § 147.26 of this chapter 

and in which freedom from M. synoviae has been demonstrated under the criteria 

specified in paragraph (e)(1)(i) of this section.

(i) It is a flock in which a minimum of 300 birds has been tested for M. synoviae 

as provided in §145.14(b) when more than 4 months of age: Provided, That 

to retain this classification, a sample of at least 150 birds shall be tested 

at intervals of not more than 90 days: And provided further, That a sample 

comprised of fewer than 150 birds may be tested at any one time if all pens 

are equally represented and a total of 150 birds is tested within each 90-day 

period.

(ii) [Reserved]

(2) A participant handling U.S. M. Synoviae Clean products shall handle only products 

of equivalent status.

(3) U.S. M. Synoviae Clean chicks shall be boxed in clean boxes and delivered in 

trucks that have been cleaned and disinfected as described in § 147.24(a) of this 

chapter. 

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 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: Provided, That primary spent fowl must be tested within 30 days prior to 

movement to disposal; 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) [Reserved] 

(e) U.S. M. Synoviae 

Clean.

(f) U.S. Avian Influenza 

Clean.
59

Subpart H—Special Provisions for Primary Meat-Type Chicken Breeding 

Flocks and Products

§ 145.81 Definitions.

Except where the context otherwise requires, for the purposes of this subpart the

following terms shall be construed, respectively, to mean: 

Newly hatched chickens. 

Foundation flocks that are composed of pedigree, great-grandparent, and grandparent

stock that has been developed for meat production and are maintained for the principal

purpose of producing multiplier breeding chicks used to produce commercial broilers. 

Young chickens (chicks, pullets, cockerels, capons) which have been fed and watered

and are less than 6 months of age. 

§ 145.82 Participation.

Participating flocks of primary meat-type chickens, and the eggs and chicks produced

from them, shall comply with the applicable general provisions of subpart A of this part

and the special provisions of this subpart H. 

(a) Started chickens shall lose their identity under Plan terminology when not

maintained by Plan participants under the conditions prescribed in § 145.5(a). 

(b) Hatching eggs produced by primary breeding flocks shall be fumigated (see 

§ 147.25 of this chapter) or otherwise sanitized.

(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.

§ 145.83 Terminology and classification; flocks and products.

Participating flocks, and the eggs and chicks produced from them, which have met

the respective requirements specified in this section, may be designated by the

following terms and the corresponding designs illustrated in §145.10: 

A flock in which freedom from pullorum and typhoid has been demonstrated to the

Official State Agency under the criteria in paragraph (b)(1) or (b)(2) of this section: 

Provided, That a flock qualifying by means of a blood test shall be tested within the

past 12 months, except that the retesting of a participating flock which is retained for 

more than 12 months shall be conducted a minimum of 4 weeks after the induction of

molt. (See §145.14 relating to the official blood test where applicable.) 

Chicks

Started chickens

(a) [Reserved]

(b) U.S. PullorumTyphoid Clean

Primary meat-type 

chicken breeding flocks
60

(1) It has been officially blood tested with no reactors. 

(2) It is a primary breeding flock that meets the following criteria:

(i) The primary breeding flock is located in a State in which pullorum disease 

or fowl typhoid is not known to exist nor to have existed in hatchery supply 

flocks within the State during the preceding 12 months and in which it has 

been determined by the Service that:

(A) All hatcheries within the State are qualified as “National Plan 

Hatcheries”or have met equivalent requirements for pullorum-typhoid 

control under official supervision; 

(B) All hatchery supply flocks within the State, are qualified as U.S. PullorumTyphoid Clean or have met equivalent requirements for pullorum-typhoid 

control under official supervision: Provided, That if other domesticated 

fowl, except waterfowl, are maintained on the same premises as the 

participating flock, freedom from pullorum-typhoid infection shall be 

demonstrated by an official blood test of each of these fowl; 

(C) All shipments of products other than U.S. Pullorum-Typhoid Clean, or 

equivalent, into the State are prohibited; 

(D) 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 poultry specimens from which S. pullorum or S. gallinarum is 

isolated; 

(E) All reports of any disease outbreak involving a disease covered under 

the Plan are promptly followed by an investigation by the Official State 

Agency to determine the origin of the infection; 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 officials administering the 

National Poultry Improvement Plan will conduct an investigation; 

(F) All flocks found to be infected with pullorum or typhoid are quarantined 

until marketed or destroyed under the supervision of the Official State 

Agency, or until subsequently blood tested following the procedure for 

reacting flocks as contained in § 145.14(a)(5) of this chapter, and all birds 

fail to demonstrate pullorum or typhoid infection; 

(G) All poultry, including exhibition, exotic, and game birds, but excluding 

waterfowl, going to public exhibition shall come from U.S. PullorumTyphoid Clean or equivalent flocks, or have had a negative pullorumtyphoid test within 90 days of going to public exhibition; and

(H) Discontinuation of any of the conditions or procedures described 

in paragraphs (b)(2)(i)(A) through (b)(2)(i)(G) of this section, or the 

occurrence of repeated outbreaks of pullorum or typhoid in poultry 

breeding flocks within or originating within the State shall be grounds 

for the Service to revoke its determination that such conditions and 

procedures have been met or complied with. Such action shall not be 

taken until a thorough investigation has been made by the Service and the 

Official State Agency has been given an opportunity to present its views; 

and
61

(ii) In the primary breeding flock, a sample of 300 birds from flocks of more than 

300, and each bird in flocks of 300 or less, has been officially tested for 

 pullorum-typhoid with no reactors: Provided, That a bacteriological 

examination monitoring program acceptable to the Official State Agency and 

 approved by the Service may be used in lieu of blood testing. 

(1) A flock maintained in compliance with the provisions of § 147.26 of this chapter 

and in which freedom from M. gallisepticum has been demonstrated under the 

criteria specified in paragraph (c)(1)(i) of this section. 

(i) It is a flock in which all birds or a sample of at least 300 birds has been tested 

for M. gallisepticum as provided in § 145.14(b) of this chapter when more than 

4 months of age: Provided, That to retain this classification, a minimum of 40 

birds shall be tested at intervals of not more than 28 days, and a total of at 

least 150 birds shall be tested within each 90-day period.

(ii) [Reserved]

(2) A participant handling U.S. M. Gallisepticum Clean products must handle only 

products of equivalent status. 

(3) U.S. M. Gallisepticum Clean chicks shall be boxed in clean boxes and delivered in 

trucks that have been cleaned and disinfected as described in § 147.24(a) of this 

chapter. 

(1) A flock maintained in compliance with the provisions of § 147.26 of this chapter 

and in which freedom from M. synoviae has been demonstrated under the criteria 

specified in paragraph (d)(1)(i) of this section. 

(i) It is a flock in which all birds or a sample of at least 300 birds has been tested 

for M. synoviae as provided in § 145.14(b) of this chapter when more than 4 

months of age: Provided, That to retain this classification, a sample of at least 

40 birds shall be tested at intervals of not more than 28 days, and a total of at 

least 150 birds shall be tested within each 90-day period.

(ii) [Reserved]

(2) A participant handling U.S. M. Synoviae Clean products shall handle only products 

of equivalent status. 

(3) U.S. M. Synoviae Clean chicks shall be boxed in clean boxes and delivered in 

trucks that have been cleaned and disinfected as described in §147.24(a) of this 

chapter. 

This classification is intended for primary meat-type breeders wishing to assure their

customers that the chicks produced are certified free of Salmonella enteritidis. 

(c) U.S. M. 

Gallisepticum Clean. 

(d) U.S. M. Synoviae 

Clean.

(e) U.S. S. Enteritidis 

Clean. 
62

(1) A flock and the hatching eggs and chicks produced from it shall be eligible for this 

classification if they meet the following requirements, as determined by the Official 

State Agency: 

(i) The flock originated from a U.S. S. Enteritidis Clean flock, or one of the 

following samples has been examined bacteriologically for S. enteritidis at an 

 authorized laboratory and any group D Salmonella samples have been 

serotyped: 

(A) A 25-gram sample of meconium from the chicks in the flock collected and 

cultured as described in § 147.12(a)(5) of this chapter; or 

(B) A sample of chick papers collected and cultured as described in 

§ 147.12(c) of this chapter; or 

(C) A sample of 10 chicks that died within 7 days after hatching. 

(ii) All feed fed to the flock meets the following requirements: 

(A) Pelletized feed must have a minimum moisture content of 14.5 percent and

must have been heated throughout to a minimum temperature of 190 °F, 

or to a minimum temperature of 165 °F for at least 20 minutes, or to 

a minimum temperature of 184 °F under 70 lbs. pressure during the 

manufacturing process;

 (B) Mash feed may contain animal protein if the finished feed is treated with a

 salmonella control product approved by the Food and Drug 

 Administration. 

 (C) All feed is stored and transported in such a manner as to prevent possible

 contamination. 

(iii) The flock is maintained in compliance with §§ 147.21, 147.24(a), and 147.26 of 

this chapter. 

(iv) Environmental samples are collected from the flock by or under the 

supervision of an Authorized Agent, as described in § 147.12 of this chapter, 

when the flock reaches 4 months of age and every 30 days thereafter. The 

environmental samples shall be examined bacteriologically for group D 

salmonella at an authorized laboratory, and cultures from group D positive 

samples shall be serotyped. 

(v) Blood samples from 300 birds from the flock are officially tested with pullorum

antigen when the flock is at least 4 months of age. All birds with positive or

inconclusive reactions, up to a maximum of 25 birds, shall be submitted to an

authorized laboratory and examined for the presence of group D salmonella in

accordance with §§ 147.10 and 147.11 of this chapter. Cultures from group D 

positive samples shall be serotyped. 

(vi) Hatching eggs produced by the flock are collected as quickly as possible and 

are handled as described in § 147.22 of this chapter.

(vii) Hatching eggs produced by the flock are incubated in a hatchery that is in

compliance with the recommendations in §§ 147.23 and 147.24(b) of this 

chapter, and the hatchery must have been sanitized either by a procedure 

approved by the Official State Agency or by fumigation. 
63

(2) If Salmonella enteritidis serotype enteritidis (SE) is isolated from a specimen taken 

from a bird in the flock, except as provided in paragraph (e)(3) of this section, the 

flock shall not be eligible for this classification. 

(3) If SE is isolated from an environmental sample collected from the flock in

accordance with paragraph (e)(1)(iv) of this section, 25 randomly selected live

birds from the flock and/or 500 cloacal swabs collected in accordance with 

§147.12(a)(2) of this chapter must be bacteriologically examined for SE as 

described in § 147.11 of this chapter. If only 1 bird from the 25-bird sample is 

found positive for SE, the participant may request bacteriological examination of 

a second 25-bird sample from the flock. If no SE is recovered from any of the 

specimens in the second sample, the flock will be eligible for the classification 

and will remain eligible for this classification if the flock is tested in accordance 

with paragraph (e)(1)(v) of this section each 30 days and no positive samples are 

found. 

(4) In order for a hatchery to sell products of this classification, all products handled 

by the hatchery must meet the requirements of this paragraph. 

(5) This classification may be revoked by the Official State Agency if the participant

fails to follow recommended corrective measures. The Official State Agency shall

not revoke the participant’s classification until the participant has been given an

opportunity for a hearing in accordance with rules of practice adopted by the 

Official State Agency. 

(6) A pedigree, experimental, or great-grandparent flock that is removed from the U.S.

S. Enteritidis Clean program may be reinstated whenever the following conditions 

are met: 

(i) The owner attests that corrective measures have been implemented, which 

may include one or more of the following: 

(A) Test and slaughter infected birds based on blood tests of every bird in the 

flock, with either pullorum antigen or by a federally licensed Salmonella 

enteritidis enzyme linked immunosorbent assay (ELISA) test when the 

flock is more than 4 months of age. 

(B) Perform other corrective actions including, but not limited to, vaccination,

medication, cleaning and disinfection of houses, rodent control, and 

movement of uninfected birds to premises that have been determined to 

be environmentally negative for S. enteritidis as described in § 147.12(a) 

of this chapter. 

(C) One hundred percent of blood samples from the birds moved to the clean

premises are tested negative for Salmonella pullorum and group D 

Salmonella. All birds with positive or inconclusive reactions, up to a 

maximum of 25 birds, shall be submitted to an authorized laboratory and 

examined for the presence of group D Salmonella, as described in 

§ 147.11 of this chapter. Cultures from positive samples shall be 

serotyped. 

(D) Two consecutive environmental drag swabs taken at the clean premises 

collected as specified in § 147.12(a) of this chapter 4 weeks apart are 

negative for S. enteritidis.

 (E) Other corrective measures at the discretion of the Official State Agency. 
64

(ii) Following reinstatement, a flock will remain eligible for this classification if the

flock is tested in accordance with paragraph (e)(1)(v) of this section every 30 

days and no positive samples are found and the flock meets the requirements 

set forth in §145.83(e). 

This program is intended to be the basis from which the breeding-hatching industry

may conduct a program for the prevention and control of salmonellosis. It is intended to

reduce the incidence of Salmonella organisms in hatching eggs and chicks through an

effective and practical sanitation program at the breeder farm and in the hatchery. This

will afford other segments of the poultry industry an opportunity to reduce the 

incidence of Salmonella in their products. 

(1) A flock and the hatching eggs and chicks produced from it that have met the 

following requirements, as determined by the Official State Agency: 

(i) The flock is maintained in compliance with §§ 147.21, 147.24(a), and 147.26 

of this chapter;

(ii) If feed contains animal protein, the protein products must have a minimum 

moisture content of 14.5 percent and must have been heated throughout to 

a minimum temperature of 190 °F or above, or to a minimum temperature of 

165 °F for at least 20 minutes, or to a minimum temperature of 184 °F under 

70 lbs. pressure during the manufacturing process; 

(iii) Feed shall be stored and transported in a manner to prevent possible 

contamination;

(iv) Chicks shall be hatched in a hatchery meeting the requirements of §§ 147.23 

and 147.24(b) of this chapter and sanitized or fumigated (see § 147.25 of this 

chapter). 

(v) An Authorized Agent shall take environmental samples from the hatchery 

every 30 days; i.e., meconium or chick papers. An authorized laboratory for 

Salmonella shall examine the samples bacteriologically; 

(vi) An Authorized Agent shall take environmental samples as described in 

§ 147.12 of this chapter from each flock at 4 months of age and every 30 

days thereafter. An authorized laboratory for Salmonella shall examine the 

environmental samples bacteriologically; 

(vii) Owners of flocks may vaccinate with a paratyphoid vaccine: 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. 

(2) The Official State Agency may use the procedures described in § 147.14 of this

chapter to monitor the effectiveness of the egg sanitation practices. 

(3) In order for a hatchery to sell products of this classification, all products handled

shall meet the requirements of the classification. 

(4) This classification may be revoked by the Official State Agency if the participant

fails to follow recommended corrective measures. 

(f) U.S. Salmonella 

Monitored
65

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 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 and 

prior to the onset of egg production. To retain this classification: 

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

 Provided, that primary spent fowl be tested within 30 days prior to movement 

to slaughter; 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) [Reserved] 

(g) U.S. Avian Influenza 

Clean
66
67

PART 146–National Poultry Improvement Plan For Commercial Poultry

Table of Contents

Subpart A–General Provisions

Section

146.1 Definitions.

146.2 Administration.

146.3 Participation.

146.4 General provisions for all participating flocks and slaughter plants.

146.5 Specific provisions for all participating flocks.

146.6 Specific provisions for participating slaughter plants.

146.7 Terminology and classification; general.

146.8 Terminology and classification; slaughter plants.

146.9 Terminology and classification; flocks, products, and States.

146.10 Supervision.

146.11 Inspections.

146.12 Debarment from participation.

146.13 Testing.

146.14 Diagnostic surveillance program for H5/H7 low pathogenic avian 

influenza.

Subpart B–Special Provisions for Commercial Table-Egg Layer Flocks

146.21 Definitions.

146.22 Participation.

146.23 Terminology and classification; flocks and products.

146.24 Terminology and classification; States.

Subpart C–Special Provisions for Meat-Type Chicken Slaughter Plants

146.31 Definitions.

146.32 Participation.

146.33 Terminology and classification; meat-type chicken slaughter plants.

Subpart D–Special Provisions for Meat-Type Turkey Slaughter Plants

146.41 Definitions.

146.42 Participation.

146.43 Terminology and classification; meat-type turkey slaughter plants.

146.44 Terminology and classification; States.

Page

69

71

71

72

73

73

73

73

74

75

75

76

76

78

79

79

79

80

81

81

81

83

83

83

84
68

Subpart E–Special Provisions for Commercial Upland Game Birds, Commercial Waterfowl, Raised-for-Release Upland Game Birds, and Raisedfor-Release Waterfowl 

146.51 Definitions.

146.52 Participation.

146.53 Terminology and classification; slaughter plants and premises.

Authority: 7 U.S.C. 8301–8317; 7 CFR 2.22, 2.80, and 371.4.

85

85

85
69

Administrator 

Affiliated flock 

Animal and Plant Health 

Inspection Service 

(APHIS)

Authorized Agent

Authorized laboratory

Classification

Commercial meat-type 

flock 

Commercial table-egg 

layer flock

Commercial table-egg 

layer premises 

Department

Domesticated 

Equivalent 

Subpart A–General Provisions

§ 146.1 Definitions.

Except where the context otherwise requires, for the purposes of this subpart the 

following terms shall be construed, respectively, to mean: 

The Administrator, Animal and Plant Health Inspection Service, or any person 

authorized to act for the Administrator.

A meat-type flock that is owned by or has an agreement to participate in the Plan with 

a slaughter plant and that participates in the Plan through that slaughter plant.

The Animal and Plant Health Inspection Service of the U.S. Department of Agriculture.

Any person designated under § 146.10(a) to perform functions under this part.

An authorized laboratory is a laboratory that meets the requirements of § 147.51 and 

is thus qualified to perform the assays described in part 147 of this subchapter.

A designation earned by participation in a Plan program.

All of the meat-type chickens, meat-type turkeys, commercial upland game birds, or 

commercial waterfowl on one farm. However, at the discretion of the Official State 

Agency, any group of poultry which is segregated from another group in a manner 

sufficient to prevent the transmission of H5/H7 LPAI and has been so segregated for a 

period of at least 21 days may be considered as a separate flock.

All table-egg layers of one classification in one barn or house.

A farm containing contiguous flocks of commercial table-egg layers under common 

ownership.

The U.S. Department of Agriculture.

Propagated and maintained under the control of a person.

Requirements which are equal to or exceed the program, conditions, criteria, or 

classifications with which they are compared, as determined by the Official State 

Agency and with the concurrence of the Service.
70

An infection of poultry caused by an influenza A virus of H5 or H7 subtype that has 

an intravenous pathogenicity index test in 6-week-old chickens less than 1.2 or any 

infection with influenza A viruses of H5 or H7 subtype for which nucleotide sequencing 

has not demonstrated the presence of multiple basic amino acids at the cleavage site 

of the hemagglutinin.

Poultry will be considered to be infected with H5/H7 LPAI for the purposes of this part 

if: 

(1) H5/H7 LPAI virus has been isolated and identified as such from poultry; or 

(2) Viral antigen or viral RNA specific to the H5 or H7 subtype of AI virus has been

detected in poultry; or 

(3) Antibodies to the H5 or H7 subtype of the AI virus that are not a consequence of

vaccination have been detected in poultry. If vaccine is used, methods should 

be used to distinguish vaccinated birds from birds that are both vaccinated and 

infected. In the case of isolated serological positive results, H5/H7 LPAI infection 

may be ruled out on the basis of a thorough epidemiological investigation that 

does not demonstrate further evidence of H5/H7 LPAI infection.

The State authority recognized by the Department to cooperate in the administration of 

the Plan.

 A natural person, firm, or corporation. 

The provisions of the National Poultry Improvement Plan contained in this part.

Domesticated chickens and turkeys that are bred for the primary purpose of producing 

eggs or meat. 

Management, sanitation, testing, and monitoring procedures which, if complied with, 

will qualify, and maintain qualification for, designation of a flock, a slaughter plant, or a 

State by an official Plan classification and illustrative design, as described in § 146.9 of 

this part. 

The Animal and Plant Health Inspection Service of the U.S. Department of Agriculture.

Any of the States, the District of Columbia, the Commonwealth of Puerto Rico, Guam, 

the Commonwealth of the Northern Mariana Islands, the Virgin Islands of the United 

States, or any territory or possession of the United States. 

Any person employed or authorized under § 146.10(b) to perform functions under this 

part. 

All of the States. 

H5/H7 low pathogenic 

avian influenza (LPAI)

H5/H7 LPAI virus 

infection (infected) 

Official State Agency 

Person

Plan

Poultry

Program

Service

State

State Inspector

United States
71

§ 146.2 Administration.

(a) The Department cooperates through a Memorandum of Understanding with the 

Official State Agency in the administration of the Plan. In the Memorandum of 

Understanding, the Official State Agency must designate a contact representative 

to serve as a liaison between the Service and the Official State Agency.

(b) The administrative procedures and decisions of the Official State Agency are 

subject to review by the Service. The Official State Agency shall carry out the 

administration of the Plan within the State according to the applicable provisions of 

the Plan and the Memorandum of Understanding.

(c) (1) An Official State Agency may accept for participation a commercial table-egg 

 layer flock or a commercial meat-type flock (including an affiliated flock)

 located in another participating State under a mutual understanding and 

agreement, in writing, between the two Official State Agencies regarding 

conditions of participation and supervision.

(2) An Official State Agency may accept for participation a commercial tableegg layer flock or a commercial meat-type flock (including an affiliated 

flock) located in a State that does not participate in the Plan under a mutual 

understanding and agreement, in writing, between the owner of the flock and 

the Official State Agency regarding conditions of participation and supervision.

(d) The Official State Agency of any State may adopt regulations applicable to the 

administration of the Plan in such State further defining the provisions of the Plan 

or establishing higher standards, compatible with the Plan.

(e) An authorized laboratory will follow the laboratory protocols outlined in part 147 of 

this chapter when determining the status of a participating flock with respect to an 

official Plan classification.

(f) States will be responsible for making the determination to request Federal 

assistance under part 56 of this chapter in the event of an outbreak of H5/H7 LPAI.

§ 146.3 Participation.

(a) Any table-egg producer, raised-for-release upland game bird premises, and 

raised-for-release waterfowl premises and any commercial upland game bird, 

commercial waterfowl, meat-type chicken or meat-type turkey slaughter plant, 

including its affiliated flocks, may participate in the Plan when the producer or plant 

has demonstrated, to the satisfaction of the Official State Agency, that its facilities, 

personnel, and practices are adequate for carrying out the relevant special 

provisions of this part and has signed an agreement with the Official State Agency 

to comply with the relevant special provisions of this part.

(b) Each participant shall comply with the Plan throughout the operating year, or until 

released by the Official State Agency.
72

(c) A participating slaughter plant shall participate with all of the commercial upland 

game bird, commercial waterfowl, meat-type chicken and/or meat-type turkey 

flocks that are processed at the facility, including affiliated flocks. Affiliated flocks 

must participate through a written agreement with a participating slaughter plant 

that is approved by the Official State Agency.

(d) Participation in the Plan shall entitle the participant to use the Plan emblem 

reproduced as follows:

(e) Participation in the NPIP by commercial table-egg layers will cease after 

September 28, 2008, unless the majority of the commercial table-egg layer 

delegates vote to continue the program in accordance with subpart E of part 147 

of this chapter at a National Plan Conference.

§ 146.4 General provisions for all participating flocks and slaughter 

plants.

(a) Records that establish the identity of products handled shall be maintained in a 

manner satisfactory to the Official State Agency.

(b) Material that is used to advertise products shall be subject to inspection by the 

Official State Agency at any time.

(c) Advertising must be in accordance with the Plan, and applicable rules and 

regulations of the Official State Agency and the Federal Trade Commission. A 

participant advertising products as being of any official classification may include 

in their advertising reference to associated or franchised slaughter or production 

facilities only when such facilities produce products of the same classification.

(d) Each participant shall be assigned a permanent approval number by the Service. 

This number, prefaced by the numerical code of the State, will be the official 

approval number of the participant and may be used on each certificate, invoice, 

shipping label, or other document used by the participant in the sale of the 

participant’s products. Each Official State Agency which requires an approval 

number for out-of-State participants to ship into its State shall honor this number. 
73

§ 146.5 Specific provisions for all participating flocks.

(a) Participating flocks, and all equipment used in connection with the flocks, shall be 

separated from non-participating flocks in a manner acceptable to the Official State 

Agency.

(b) Poultry equipment, and poultry houses and the land in the immediate vicinity 

thereof, shall be kept in sanitary condition as recommended in § 147.21(c) of this 

subchapter. 

§ 146.6 Specific provisions for participating slaughter plants. 

(a) Only commercial upland game bird, commercial waterfowl, meat-type chicken, 

and meat-type turkey slaughter plants that are under continuous inspection by 

the Food Safety and Inspection Service of the Department or under State 

inspection that the Food Safety and Inspection Service has recognized as 

equivalent to Federal inspection may participate in the Plan.

(b) To participate in the Plan, meat-type chicken, meat-type turkey, and commercial 

upland game bird and commercial waterfowl slaughter plants must follow the 

relevant special provisions in §§ 146.33(a), 146.43(a), and 146.53(a), respectively, 

for sample collection and flock monitoring, unless they are exempted from the 

special provisions under §§ 146.32(b), 146.42(b), or 146.52(b), respectively.

§ 146.7 Terminology and classification; general.

The official classification terms defined in §§ 146.8 and 146.9 and the various designs 

illustrative of the official classifications reproduced in §146.9 may be used only by 

participants and to describe products that have met all of the specific requirements of 

such classifications.

§ 146.8 Terminology and classification; slaughter plants.

Participating slaughter plants shall be designated as “U.S. H5/H7 Avian Influenza 

Monitored.” All Official State Agencies shall be notified by the Service of additions, 

withdrawals, and changes in classification.
74

§ 146.9 Terminology and classification; flocks, products, and States.

Participating flocks (including affiliated flocks), products produced from them, and 

States which have met the respective requirements specified in subparts B, C, or D of 

this part may be designated by the following terms or illustrative designs:

(a) U.S. H5/H7 Avian Influenza Monitored. (See §§ 146.23(a), 146.33(a), 146.43(a), 

and 146.53(a) and (b).) 

Figure 2.

(b) U.S. H5/H7 Avian Influenza Monitored State, Layers. (See § 146.24.)

Figure 3.

(c) U.S. H5/H7 Avian Influenza Monitored State, Turkeys. (See § 146.44.)

Figure 4.

U.S.

MONITORED

NPIP

H5/H7 Avian Influenza
75

§ 146.10 Supervision.

(a) The Official State Agency may designate qualified persons as Authorized Agents 

to do the sample collecting provided for in § 146.13 of this part.

(b) The Official State Agency shall employ or authorize qualified persons as 

State Inspectors to perform the selecting and testing of participating flocks 

and to perform the official inspections necessary to verify compliance with the 

requirements of the Plan.

(c) Authorities issued to Authorized Agents or State Inspectors under the provisions 

of this section shall be subject to cancellation by the Official State Agency on 

the grounds of incompetence or failure to comply with the provisions of the Plan 

or regulations of the Official State Agency. Such actions shall not be taken until 

thorough investigation has been made by the Official State Agency and the 

authorized person has been given notice of the proposed action and the basis 

thereof and an opportunity to present his or her views.

§ 146.11 Inspections. 

(a) Each participating slaughter plant shall be audited at least once annually or a 

sufficient number of times each year to satisfy the Official State Agency that the 

participating slaughter plant is in compliance with the provisions of this part. The 

yearly audit will consist of an evaluation of 2 weeks' worth of records, selected at 

random, of the following data: 

(1) The actual flock slaughter date for each flock. This information must come 

 from a verifiable source. Verifiable sources include electronic record systems 

 that have oversight from the Department's Grain Inspectors, Packers and 

 Stockyards Administration or Food Safety and Inspection Service (FSIS) 

 documents such as FSIS Form 9061-2. 

(2) Laboratory test results for each flock slaughtered with the sample collection 

 date and test result. The test must be NPIP-approved and performed in an 

 authorized laboratory of the NPIP. 

(b) A flock will be considered to be not conforming to protocol if there are no test 

results available, if the flock was not tested within 21 days before slaughter, or if 

the test results for the flocks were not returned before slaughter.

(c) Two or more flocks that are found to be not conforming to protocol in the yearly 

audit for a slaughter plant shall be cause for a deficiency rating for that 

plant. However, if the root cause for the deficiency was identified, corrected, and 

documented, the plant will be eligible for an immediate reevaluation of 2 additional 

weeks' worth of records, again selected at random. If no more than one missed 

flock is identified in this reevaluation, the plant will be considered in compliance 

and no further action will be required. Plants found to be deficient must provide 

a written corrective action plan to the auditor within 2 weeks of receipt of the 

deficiency rating. A followup audit on the information in paragraphs (a)(1) and (a)

(2) of this section will occur within 90 days from the receipt of the corrective action 
76

plan. Slaughter plants will retain their classification and may continue to use the 

Plan emblem in § 146.9(a) during this process. A failure on the followup audit may 

result in disbarment from participation according to the procedures in § 146.12.

(d) On-site inspections of any participating flocks and premises will be conducted if 

a State Inspector determines that a breach of testing has occurred for the Plan 

programs for which the flocks are certified.

(e) The official H5/H7 LPAI testing records of all participating flocks and slaughter 

plants shall be examined annually by a State Inspector. Official H5/H7 LPAI 

testing records shall be maintained for 3 years.

§ 146.12 Debarment from participation. 

Participants in the Plan who, after investigation by the Official State Agency or its 

representative, are notified in writing of their apparent noncompliance with the Plan 

provisions or regulations of the Official State Agency shall be afforded a reasonable 

time, as specified by the Official State Agency, within which to demonstrate or achieve 

compliance. If compliance is not demonstrated or achieved within the specified 

time, the Official State Agency may debar the participant from further participation 

in the Plan for such period, or indefinitely, as the Official State Agency may deem 

appropriate. The debarred participant shall be afforded notice of the bases for the 

debarment and opportunity to present his or her views with respect to the debarment 

in accordance with procedures adopted by the Official State Agency. The Official 

State Agency shall thereupon decide whether the debarment order shall continue in 

effect. Such decision shall be final unless the debarred participant, within 30 days 

after the issuance of the debarment order, requests the Administrator to determine 

the eligibility of the debarred participant for participation in the Plan. In such an event, 

the Administrator shall determine the matter de novo in accordance with the rules of 

practice in 7 CFR part 50, which are herby made applicable to proceedings before 

the Administrator under this section. The definitions in 7 CFR 50.10 and the following 

definitions shall apply with respect to terms used in such rules of practice:

The Administrator, Animal and Plant Health Inspection Service of the U.S. Department 

of Agriculture, or any officer or employee to whom authority has heretofore been 

delegated or to who authority may hereafter be delegated to act in his or her stead.

§ 146.13 Testing.

Either egg or blood samples may be used for testing. Samples must be collected in 

accordance with the following requirements:

(1) Egg samples. Egg samples must be collected and prepared in accordance with 

the requirements in § 147.8 of this subchapter.

(a) Administrator

(b) [Reserved]

(a) Samples
77

(b) Avian influenza 

(2) Blood samples. Blood samples obtained in the slaughter plant should be collected 

after the kill cut with birds remaining on the kill line. Hold an open 1.5 mL snap cap 

micro-centrifuge tube under the neck of the bird directly after the kill cut and collect 

drips of blood until the tube is half full. Keep the blood tubes at room temperature 

for the clot to form, which should require a minimum of 4 hours and a maximum of 

12 hours. Refrigerate the tube after the clot has formed. Put tubes in a container 

and label it with plant name, date, shift (A.M. or Day, P.M. or Night), and flock 

number. After the clot is formed, the clot should be removed by the Authorized 

Agent in order to ensure good-quality sera. Prepare a laboratory submission form 

and ship samples with submission forms to the laboratory in a polystyrene foam 

cooler with frozen ice packs. Submission forms and the manner of submission 

must be approved by the Official State Agency and the authorized laboratory to 

ensure that there is sufficient information to identify the samples and that the 

samples are received in an acceptable condition for further tests to be reliably 

performed. Blood samples should be shipped routinely to the laboratory. Special 

arrangements should be developed for samples held over the weekend to ensure 

that the samples can be reliably tested. Blood samples for official tests shall be 

drawn by an Authorized Agent or State Inspector. 

The official tests for avian influenza are described in paragraphs (b)(1) and (b)(2) of 

this section:

(1) Antibody detection tests. 

 (i) Enzyme-linked immunosorbent assay (ELISA). ELISA must be conducted 

 using test kits approved by the Department and the Official State Agency 

 and must be conducted in accordance with the recommendations of the 

 producer or manufacturer.

(ii) The agar gel immunodiffusion (AGID) test. 

 (A) The AGID test must be conducted on all ELISA-positive samples. 

 (B) The AGID test must be conducted using reagents approved by the 

 Department and the Official State Agency. 

 (C) Standard test procedures for the AGID test for avian influenza are set 

 forth in § 147.9 of this subchapter. The test can be conducted on egg yolk 

 or blood samples. 

 (D) Positive tests for the AGID must be further tested by Federal Reference 

 Laboratories using appropriate tests for confirmation. Final judgment may 

 be based upon further sampling and appropriate tests for confirmation. 

(2) Agent detection tests. Agent detection tests may be used to detect influenza A 

matrix gene or protein but not to determine hemagglutinin or neuraminidase 

subtypes. Samples for this testing should be collected from naturally occurring 

flock mortality or clinically ill birds.

(i) The real time reverse transcriptase/polymerase chain reaction (RRT-PCR) 

 assay. 

 (A) The RRT-PCR tests must be conducted using reagents approved by the 

 Department and the Official State Agency. The RRT-PCR must be 

 conducted using the National Veterinary Services Laboratories (NVSL) 

 official protocol for RRT-PCR (AVPR01510) and must be conducted by 

 personnel who have passed an NVSL proficiency test. 
78

 (B) Positive results from the RRT-PCR must be further tested by Federal 

 Reference Laboratories using appropriate tests for confirmation. Final 

 judgment may be based upon further sampling and appropriate tests for 

 confirmation.

(ii) USDA-licensed type A influenza antigen capture immunoassay (ACIA). 

 (A) The USDA-licensed type A influenza ACIA must be conducted using test 

 kits approved by the Department and the Official State Agency and must 

 be conducted in accordance with the recommendations of the producer or 

 manufacturer.

 (B) Positive results from the ACIA must be further tested by Federal Reference

 Laboratories using appropriate tests for confirmation. Final judgment may 

 be based upon further sampling and appropriate tests for confirmation.

(3) The official determination of a flock as positive for the H5 or H7 subtypes 

avian influenza may be made only by NVSL.

§ 146.14 Diagnostic surveillance program for H5/H7 low pathogenic avian 

influenza.

The Official State Agency must develop a diagnostic surveillance program for H5/H7

low pathogenic avian influenza for all poultry in the State. The exact provisions of 

the program are at the discretion of the States. The Service will use the standards 

in paragraph (b) of this section in assessing individual State plans for adequacy, 

including the specific provisions that the State developed. The standards should be 

used by States in developing those plans.

Avian influenza must be a disease reportable to the responsible State authority (State 

veterinarian, etc.) by all licensed veterinarians. To accomplish this, all laboratories 

(private, State, and university laboratories) that perform diagnostic procedures on 

poultry must examine all submitted cases of unexplained respiratory disease, egg 

production drops, and mortality for avian influenza by both an approved serological 

test and an approved antigen detection test. Memoranda of understanding or other 

means must be used to establish testing and reporting criteria (including criteria that 

provide for reporting H5 and H7 low pathogenic avian influenza directly to the Service) 

and approved testing methods. In addition, States should conduct outreach to poultry 

producers, especially owners of smaller flocks, regarding the importance of prompt 

reporting of clinical symptoms consistent with avian influenza.

(b) Avian influenza is a 

reportable disease.

(a) Role of the Official 

State Agency in 

developing a diagnostic 

surveillance program for 

H5/H7 LPAI.
79

Table-egg layer

(a) U.S. H5/H7 Avian 

Influenza Monitored

(b) [Reserved]

Subpart B–Special Provisions for Commercial Table-Egg Layer Flocks 

§ 146.21 Definitions.

A domesticated chicken grown for the primary purpose of producing eggs for human 

consumption.

§ 146.22 Participation.

(a) Participating commercial table-egg layer flocks shall comply with the applicable 

general provisions of subpart A of this part and the special provisions of subpart B 

of this part.

(b) Commercial table-egg laying premises with fewer than 75,000 birds are exempt 

from the special provisions of subpart B of this part.

§ 146.23 Terminology and classification; flocks and products.

Participating flocks which have met the respective requirements specified in this 

section may be designated by the following terms and the corresponding designs 

illustrated in § 146.9 of this part:

This program is intended to be the basis from which the table-egg layer industry may 

conduct a program to monitor for the H5/H7 subtypes of avian influenza. It is intended 

to determine the presence of the H5/H7 subtypes of avian influenza in table-egg layers 

through routine serological surveillance of each participating commercial table-egg 

layer flock. A flock will qualify for this classification when the Official State Agency 

determines that it has met one of the following requirements:

(1) It is a meat-type turkey slaughter plant that accepts only meat-type turkeys from 

flocks where a minimum of 6 birds per flock has tested negative for antibodies to 

type A avian influenza virus with an approved test no more than 21 days prior to 

slaughter. 

(2) It is a commercial table-egg layer flock in which a minimum of 11 birds or egg 

samples have been tested negative for antibodies to the H5/H7 subtypes of avian 

influenza within a 12-month period; or

(3) It is a commercial table-egg layer flock that has an ongoing active and diagnostic 

surveillance program for the H5/H7 subtypes of avian influenza in which the 

number of birds or egg samples tested is equivalent to the number required in 

paragraph (a)(1) or (a)(2) and that is approved by the Official State Agency and the 

Service.
80

§ 146.24 Terminology and classification; States.

(1) A State will be declared a U.S. H5/H7 Avian Influenza Monitored State, Layers 

when it has been determined by the Service that:

(i) All commercial table-egg layer flocks in production within the State that are 

not exempt from the special provisions of this subpart B under § 146.22 are 

classified as U.S. H5/H7 Avian Influenza Monitored under § 146.23(a) of this 

part;

(ii) All egg-type chicken breeding flocks in production within the State are classifid 

as U.S. Avian Influenza Clean under § 145.23(h) of this subchapter;

(iii) All persons performing poultry disease diagnostic services within the State are 

required to report to the Official State Agency, within 24 hours, the source of 

all table-egg layer specimens that were deemed positive on an official test for 

avian influenza, as designated in § 146.13(a) of this chapter;

(iv) All table-egg layer specimens that were deemed positive on an official test for 

avian influenza, as designated in § 146.13(a) of this chapter, are sent to an 

authorized laboratory for subtyping; and

(v) All table-egg layer flocks within the State that are found to be infected with 

the H5/H7 subtypes of avian influenza are quarantined, in accordance with 

an initial State response and containment plan as described in part 56 of this 

chapter and under the supervision of the Official State Agency.

(2) If there is a discontinuation of any of the conditions described in paragraph (a)(1) 

of this section, or if repeated outbreaks of the H5/H7 subtypes of avian influenza 

occur in commercial table-egg layer flocks as described in paragraph 

(a) (1)(i) of this section, or if an infection spreads from the originating premises, 

the Service shall have grounds to revoke its determination that the State is 

entitled to this classification. Such action shall not be taken until a thorough 

investigation has been made by the Service and the Official State Agency has 

been given an opportunity for a hearing in accordance with rules of practice 

adopted by the Administrator.

(a) U.S. H5/H7 Avian 

Influenza Monitored 

State, Layers

(b) [Reserved]
81

Meat-type chicken 

Meat-type chicken 

slaughter plant 

Shift 

(a) U.S. H5/H7 Avian 

Influenza Monitored 

Subpart C–Special Provisions for Meat-Type Chicken Slaughter Plants 

§ 146.31 Definitions.

A domesticated chicken grown for the primary purpose of producing meat, including 

but not limited to broilers, roasters, fryers, and cornish.

A meat-type chicken slaughter plant that is federally inspected or under State 

inspection that the Food Safety Inspection Service has recognized as equivalent to 

federal inspection.

The working period of a group of employees who are on duty at the same time.

§ 146.32 Participation.

(a) Participating meat-type chicken slaughter plants shall comply with applicable 

general provisions of subpart A of this part and the special provisions of this 

subpart C.

(b) Meat-type chicken slaughter plants that slaughter fewer than 200,000 meat-type 

chickens in an operating week are exempt from the special provisions of this 

subpart C.

§ 146.33 Terminology and classification; meat-type chicken slaughter 

plants.

Participating meat-type chicken slaughter plants that have met the respective 

requirements specified in this section may be designated by the following terms and 

the corresponding designs illustrated in § 146.9 of this part:

This program is intended to be the basis from which the meat-type chicken industry 

may conduct a program to monitor for the H5/H7 subtypes of avian influenza. It is 

intended to determine the presence of the H5/H7 subtypes of avian influenza in meattype chickens through routine surveillance of each participating meat-type chicken 

slaughter plant. A meat-type chicken slaughter plant will qualify for this classification 

when the Official State Agency determines that it has met one of the following 

requirements:

(1) It is a meat-type chicken slaughter plant where a minimum of 11 birds per shift 

are tested negative for antibodies to the H5/H7 subtypes of avian influenza at 

slaughter; Provided, that with the approval of the Official State Agency, fewer 

than 11 birds per shift may be tested on any given shift if the total number of birds 

tested during the operating month is equivalent to testing 11 birds per shift; or
82

(2) It is a meat-type chicken slaughter plant which accepts only meat-type chickens 

from flocks where a minimum of 11 birds have been tested negative for antibodies 

to the H5/H7 subtypes of avian influenza no more than 21 days prior to slaughter; 

or

(3) It is a meat-type chicken slaughter plant that has an ongoing active and diagnostic 

surveillance program for the H5/H7 subtypes of avian influenza in which the 

number of birds tested is equivalent to the number required in paragraph (a)(1) or 

(a)(2) and that is approved by the Official State Agency and the Service. 

(b) [Reserved]
83

Meat-type turkey

Meat-type turkey 

slaughter plant 

(a) U.S. H5/H7 Avian 

Influenza Monitored 

Subpart D–Special Provisions for Meat-Type Turkey Slaughter Plants 

§ 146.41 Definitions.

A domesticated turkey grown for the primary purpose of producing meat.

A meat-type turkey slaughter plant that is federally inspected or under State inspection 

that the Food Safety and Inspection Service has recognized as equivalent to federal 

inspection.

§ 146.42 Participation.

(a) Participating meat-type turkey slaughter plants shall comply with applicable 

general provisions of subpart A of this part and the special provisions of this 

subpart D.

(b) Meat-type turkey slaughter plants that slaughter fewer than 2 million meat-type 

turkeys in a 12-month period are exempt from the special provisions of this subpart 

D.

§ 146.43 Terminology and classification; meat-type turkey slaughter 

plants.

Participating meat-type turkey slaughter plants which have met the respective 

requirements specified in this section may be designated by the following terms and 

the corresponding designs illustrated in § 146.9 of this part:

This program is intended to be the basis from which the meat-type turkey industry may 

conduct a program to monitor for the H5/H7 subtypes of avian influenza. It is intended 

to determine the presence of avian influenza in meat-type turkeys through routine 

surveillance of each participating meat-type turkey slaughter plant. A participating 

meat-type turkey slaughter plant will qualify for this classification when the Official 

State Agency determines that it has met one of the following requirements:

(1) It is a meat-type turkey slaughter plant that accepts only meat-type turkeys from 

flocks where a minimum of 6 birds per flock has tested negative for antibodies

to type A avian influenza virus with an approved test no more than 21 days prior to 

slaughter. Positive samples shall be further tested by an authorized laboratory 

using the hemagglutination inhibition test to detect antibodies to the hemagglutinin 

subtypes H5 and H7. It is recommended that samples be collected from flocks 

over 10 weeks of age with respiratory signs such as coughing, sneezing, snicking, 

sinusitis, or rales; depression; or decreases in food or water intake. 

(2) It is a meat-type turkey slaughter plant that has an ongoing active and diagnostic

surveillance program for the H5/H7 subtypes of avian influenza in which the 

number of birds tested is equivalent to the number required in paragraph (a)(1) 

and that is approved by the Official State Agency and the Service. 

(b) [Reserved]
84

§ 146.44 Terminology and classification; States.

(1) A State will be declared a U.S. H5/H7 Avian Influenza Monitored State, Turkeys 

when it has been determined by the Service that:

(i) All meat-type turkey slaughter plants within the State that are not exempt from 

the special provisions of this subpart D under § 146.42 are classified as U.S. 

H5/H7 Avian Influenza Monitored under § 146.43(a) of this part;

(ii) All turkey breeding flocks in production within the State are classified as U.S. 

H5/H7 Avian Influenza Clean under § 145.43(g) of this subchapter;

(iii) All persons performing poultry disease diagnostic services within the State are 

required to report to the Official State Agency, within 24 hours, the source of 

all meat-type turkey specimens that were deemed positive on an official test 

for avian influenza, as designated in § 146.13(a) of this chapter;

(iv) All meat-type turkey specimens that were deemed positive on an official test 

for avian influenza, as designated in § 146.13(a) of this chapter, are sent to an 

authorized laboratory for subtyping; and

(v) All meat-type turkey flocks within the State that are found to be infected with 

the H5/H7 subtypes of avian influenza are quarantined, in accordance with 

an initial State response and containment plan as described in part 56 of this 

chapter, and under the supervision of the Official State Agency.

(2) If there is a discontinuation of any of the conditions described in paragraph (a)(1) 

of this section, or if repeated outbreaks of the H5/H7 subtypes of avian influenza 

occur in meat-type turkey flocks as described in paragraph (a)(1)(i) of this section, 

or if an infection spreads from the originating premises, the Service shall have 

grounds to revoke its determination that the State is entitled to this classification. 

Such action shall not be taken until a thorough investigation has been made by the 

Service and the Official State Agency has been given an opportunity for a hearing 

in accordance with rules of practice adopted by the Administrator.

(a) U.S. H5/H7 Avian 

Influenza Monitored 

State, Turkeys.
85

Subpart E—Special Provisions for Commercial Upland Game Birds, 

Commercial Waterfowl, Raised-for-Release Upland Game Birds, and 

Raised-for-Release Waterfowl

§ 146.51 Definitions.

A commercial upland game bird slaughter plant that is federally inspected or under 

State inspection that the U.S. Department of Agriculture's Food Safety and Inspection 

Service has recognized as equivalent to Federal inspection.

Upland game bird pheasants, quail, or partridges grown under confinement for the 

primary purpose of producing meat for human consumption.

Domesticated ducks or geese grown under confinement for the primary purpose of 

producing meat for human consumption. 

A commercial waterfowl slaughter plant that is federally inspected or under State inspection that the U.S. Department of Agriculture's Food Safety and Inspection Service 

has recognized as equivalent to Federal inspection.

Pheasants, quail, and partridge that are raised under confinement for release in game 

preserves and are not breeding stock.

Waterfowl that are raised under confinement for release in game preserves and are not 

breeding stock.

The working period of a group of employees who are on duty at the same time.

§ 146.52 Participation.

(a) Participating commercial upland game bird slaughter plants, commercial waterfowl 

slaughter plants, raised-for-release upland game bird premises, and raised-forrelease waterfowl premises shall comply with the applicable general provisions of 

Subpart A of this part and the special provisions of this subpart E.

(b) Commercial waterfowl and commercial upland game bird slaughter plants that 

slaughter fewer than 50,000 birds annually are exempt from the special provisions 

of this subpart E.

(c) Raised-for-release upland game bird premises and raised-for-release waterfowl 

premises that raise fewer than 25,000 birds annually are exempt from the special 

provisions of this subpart E.

§ 146.53 Terminology and classification; slaughter plants and premises.

Participating flocks which have met the respective requirements specified in this section may be designated by the following terms and the corresponding designs illustrated in § 146.9 of this part:

This program is intended to be the basis from which the commercial waterfowl and 

commercial upland game bird industry may conduct a program to monitor for the H5/

H7 subtypes of avian influenza. It is intended to determine the presence of the H5/H7 

subtypes of avian influenza in commercial waterfowl and commercial upland game 

Commercial upland 

game bird slaughter 

plant 

Commercial upland 

game birds

Commercial waterfowl

Commercial waterfowl 

slaughter plant

Raised-for-release 

upland game birds

Raised-for-release waterfowl

Shift

(a) U.S. H5/H7 Avian 

Influenza Monitored 

(Slaughter Plants)
86

birds through routine surveillance of each participating slaughter plant. A 

slaughter plant will qualify for this classification when the Official State Agency 

determines that it has met one of the following requirements:

(1) It is a commercial upland game bird slaughter plant or commercial waterfowl 

 slaughter plant where a minimum of 11 birds per shift are tested negative for 

 the H5/H7 subtypes of avian influenza at slaughter;

(2) It is a commercial upland game bird slaughter plant or commercial waterfowl 

 slaughter plant that only accepts commercial upland game birds or 

 commercial waterfowl from flocks where a minimum of 11 birds per flock have 

 been tested negative for antibodies to the H5/H7 subtypes of avian influenza 

 no more than 21 days prior to slaughter; or

(3) It is a commercial upland game bird slaughter plant or commercial waterfowl 

 slaughter plant that has an ongoing active and passive surveillance program 

 for H5/H7 subtypes of avian influenza that is approved by the Official State 

 Agency and the Service.

This program is intended to be the basis from which the raised-for-release upland 

game bird and raised-for-release waterfowl industries may conduct a program to monitor for the H5/H7 subtypes of avian influenza. It is intended to determine the presence of the H5/H7 subtypes of avian influenza through routine surveillance of each 

participating premises. A premises will qualify for the classification when the Official 

State Agency determines that a representative sample of 30 birds from the participating premises has been tested with negative results for the H5/H7 subtypes of avian 

influenza every 90 days.

(b) U.S. H5/H7 Avian 

Influenza Monitored 

(Premises)
87

PART 147—Auxiliary Provisions On 

National Poultry Improvement Plan

Table of Contents

Subpart A—Blood Testing Procedures

 

Section 

147.1 The standard tube agglutination test. 

147.2 The rapid serum test. 

147.3 The stained-antigen, rapid, whole-blood test. 

147.4 [Reserved] 

147.5 The microagglutination test for pullorum-typhoid. 

147.6 Procedure for determining the status of flocks reacting to tests for 

 Mycoplasma gallisepticum, Mycoplasma synoviae, and Mycoplasma

 meleagridis. 

147.7 Standard test procedures for Mycoplasma. 

147.8 Procedures for preparing egg yolk samples for diagnostic tests. 

147.9 Standard test procedures for avian influenza.

Subpart B—Bacteriological Examination Procedure

 

147.10 Laboratory procedure recommended for the bacteriological examination of 

egg-type breeding flocks with Salmonella enterditis positive environments.

147.11 Laboratory procedure recommended for the bacteriological examination of 

salmonella. 

147.12 Procedures for collection, isolation, and idenitication of Salmonella from 

environmental samples, cloacal swabs, chick box papers, and meconium 

samples. 

147.13 Procedure for bacteriological culturing of eggshells for colon bacilli 

organisms. 

147.14 Procedures to determine status and effectiveness of sanitation 

monitored program. 

147.15 Laboratory procedure recommended for the bacteriological examination of 

mycoplasma reactors. 

147.16 Procedure for the evaluation of mycoplasma reactors by in vivo bioassay (enrichment). 

147.17 Laboratory procedure recommended for the bacteriological examination of 

cull chicks and poults for salmonella. 

Subpart C—Sanitation Procedures

 

147.21 Flock sanitation. 

147.22 Hatching egg sanitation. 

147.23 Hatchery sanitation. 

147.24 Cleaning and disinfecting. 

147.25 Fumigation. 

Page

89

92

93

94

94

95

97

108

109

113

113

116

124

125

125

128

129

131

132

133

133

135
88

147.26 Procedures for establishing isolation and maintaining sanitation and good 

 management practices for the control of Salmonella and Mycoplasma 

 infections. 

147.27 Procedures recommended to prevent the spread of disease by artificial 

 insemination of turkeys. 

Subpart D—Molecular Examination Procedures

147.30 Laboratory procedure recommended for the polymerase chain reaction 

 (PCR) test for Mycoplasma gallisepticum and M. synoviae.

147.31 Laboratory procedures recommended for the real-time polymerase chain 

 reaction test for Mycoplasma gallisepticum (MGLP ReTi).

Subpart E—Procedure for Changing National Poultry 

Improvement Plan

 

147.41 Definitions. 

147.42 General. 

147.43 General Conference Committee. 

147.44 Submitting, compiling, and distributing proposed changes. 

147.45 Official delegates. 

147.46 Committee consideration of proposed changes. 

147.47 Conference consideration of proposed changes. 

147.48 Approval of conference recommendations by the Department. 

Subpart F—Authorized Laboratories and Approved Tests

147.51 Authorized laboratory minimum requirements.

147.52 Approved tests.

Authority: 7 U.S.C. 8301–8317; 7 CFR 2.22, 2.80, and 371.4. 

139

140

140

142

142

142

147

144

135

136

137

138

145

146
89

Subpart A—Blood Testing Procedures

§ 147.1 The standard tube agglutination test.1

 

(a) The blood samples should be collected and delivered as follows:

(1) The blood samples should be taken by properly qualified and authorized 

persons only, and in containers provided by the laboratory. The containers 

should be stout-walled test tubes, preferably 3/8 by 3 inches, without lip, or 

small well-selected medicine vials, which have been thoroughly cleaned and 

dried in a hot-air drying oven. If stoppers are used, they should be thoroughly 

cleaned and dried.

(2) Sufficient blood should be procured by making a small incision in the large 

median wing vein with a small sharp lancet and allowing the blood to run into 

the tube, or by the use of a small syringe (with 20 or 21 gauge needle) which 

is properly cleansed between bleedings with physiological saline solution. To 

facilitate the separation of the serum, the tubes should be placed in a slanted 

position until the blood has solidified. After the blood has completely clotted, 

they should be packed and shipped by mail (special delivery), rapid express, 

or by messenger, to the laboratory. All labeling must be clear and permanent, 

and may be done with a suitable pencil on etched portions of the tube, or by 

means of fast-gum labels.

(3) The blood samples must reach the laboratory in a fresh and unhemolyzed 

condition. Hemolyzed samples should be rejected. It is imperative, therefore, 

to cool the tubes immediately after slanting and clotting, and unless they reach 

the laboratory within a few hours, to pack them with ice in special containers, 

or use some other cooling system which will insure their preservation during 

transportation. In severe cold seasons, extreme precautions must be exercised 

to prevent freezing and consequent laking. The samples must be placed in 

cold (5 to 10 °C.) storage, immediately upon arrival at the laboratory.

(b) The antigen shall consist of representative strains of S. pullorum which are of 

known antigenic composition, high agglutinability, but are not sensitive to negative 

and nonspecific sera. The stock cultures may be maintained satisfactorily by 

transferring to new sloped agar at least once a month and keeping at 18 to 25 °C. 

(average room temperature) in a dark closet or chest, following incubation for from 

24 to 36 hours at 37 °C. The antigenic composition and purity of the stock cultures 

should be checked consistently.

(c) A medium which has been used satisfactorily has the following composition: 

Water 1,000 cc.

Difco beef extract 4 gm. (0.4 percent)

Difco Bacto-peptone 10 gm. (1.0 percent)

Difco dry-granular agar 20 gm. (2.0 percent)

Reaction—pH 6.8 to 7.2

1

The procedure described is a modification of the method reported in the Proceedings of the 

U.S. Live Stock Sanitary Association, November 30 to December 2, 1932, pp. 487 to 491. 
90

(d) Large 1-inch test tubes, Kolle flasks, or Blake bottles should be streaked liberally 

over the entire agar surface with inoculum from 48-hour slant agar cultures 

prepared from the stock cultures of the selected strains. The antigen-growing tubes 

or bottles should be incubated 48 hours at 37 °C., and the surface growth washed 

off with sufficient phenolized (0.5 percent) saline (0.85 percent) solution to make a 

heavy suspension. The suspension should be filtered free of clumps through a thin 

layer of absorbent cotton in a Buchner funnel with the aid of suction. The antigens 

of the separate strains should be combined in equal volume-density and stored in 

the refrigerator (5 to 10 °C.) in tightly stoppered bottles.

(e) Thiosulfate-Glycerin (TG) medium may be used as an alternate medium for the 

preparation of tube agglutination antigen. The TG medium, formerly used for 

the preparation of stained, whole-blood antigen, is described in more detail in 

the article by A. D. MacDonald, Recent Developments in Pullorum Antigen for 

the Rapid, Whole-Blood Test, Report of the Conference of the National Poultry 

Improvement Plan, pages 122–127, 1941. This medium provides a tube antigen of 

excellent specificity and greatly increases the yield of antigen from a given amount 

of medium. The TG medium has the following composition:

Beef infusion 1,000 cc.

Difco Bacto-peptone 20 gm. (2.0 percent)

Sodium thiosulfate 5 gm. (0.5 percent) 

Ammonium chloride 5 gm. (0.5 percent)

Glycerin, U.S.P.(95 percent) 20 cc.(2.0 percent)

Difco dry-granular agar 30 gm. (3.0 percent)

Reaction—pH 6.8 to 7.2

Large 1-inch test tubes, Kolle flasks, Blake bottles, or Erlenmeyer flasks should be 

seeded over the entire agar surface with inoculum from 24-hour beef infusion broth 

cultures prepared from the stock cultures of the selected strains. The antigengrowing tubes or bottles should be incubated 96 hours at 37 °C., and the surface 

growth washed off with sufficient phenolized (0.5 percent) saline (0.85 percent) 

solution to make a heavy suspension. The suspension should be filtered free of 

clumps through a thin layer of absorbent cotton in a Buchner funnel with the aid 

of suction. The antigen then should be centrifuged. The mass of bacteria should 

be removed from the centrifuge tubes or bowl and resuspended in saline (0.85 

percent) solution containing 0.5 percent phenol. After the bacterial mass has been 

uniformly suspended in the diluent, it should be again passed through a cotton pad 

in a Buchner funnel without the aid of suction. The antigens of the separate strains 

should be combined in equal volume-density and stored in the refrigerator
91

(5 to 10 °C.) in tightly stoppered bottles.

(f) The diluted antigen to be used in the routine testing should be prepared from 

the stock antigen by dilution of the latter with physiological (0.85 percent) saline 

solution containing 0.25 percent of phenol to a turbidity corresponding to 0.75–

1.00 on the McFarland nephelometer scale. The hydrogen-ion concentration of 

the diluted antigen should be corrected to pH 8.2 to 8.5 by the addition of dilute 

sodium hydroxide. New diluted antigen should be prepared each day and kept 

cold. The diluted antigen may be employed in 2 cc. quantities in 4 by 1/2-inch 

test tubes, or 1 cc. quantities in smaller tubes, in which the final serum-antigen 

mixtures are made and incubated. The distribution of the antigen in the tubes may 

be accomplished by the use of long burettes, or special filling devices made for the 

purpose.

(g) The maximum serum dilution employed must not exceed 1:50 for chickens, nor 

1:25 for turkeys. The available data indicate that 1:25 dilution is the most efficient. 

In all official reports on the blood test, the serum dilutions shall be indicated. The 

sera should be introduced into the agglutination tubes in the desired amounts with 

well-cleaned serological pipettes or special serum-delivery devices which do not 

permit the mixing of different sera. The antigen and serum should be well mixed 

before incubation. The serum and antigen mixture must be incubated for at least 

20 hours at 37 °C.

(h) The results shall be recorded as: 

N, or –(negative) when the serum-antigen mixture remains uniformly turbid. 

P, or + (positive) when there is a distinct clumping of the antigen, and the

liquid between the agglutinated particles is clear. 

S, or ? (suspicious) when the agglutination is only partial or incomplete. 

M, or missing, when samples listed on the original record sheet are missing. 

H, or hemolyzed, when blood samples are hemolyzed and cannot be tested. 

B, or broken, when sample tubes are broken and no serum can be obtained. 

(Some allowance must always be made for the difference in sensitiveness of different 

antigens and different set-ups, and therefore, a certain amount of independent, 

intelligent judgment must be exercised at all times. Also, the histories of the flocks 

require consideration. In flocks where individuals show a suspicious agglutination, it is 

desirable to examine representative birds bacteriologically to determine the presence 

or absence of S. pullorum.)

(Approved by the Office of Management and Budget under control number 0579-0007) 
92

§ 147.2 The rapid serum test.2 

(a) The procedure for the collection and delivery of blood samples in the rapid serum 

test is the same as that described in § 147.1(a).

(b) The selection and maintenance of suitable strains of S. pullorum and the 

composition of a satisfactory medium are described in § 147.1(b) and (c).

(c) Large 1-inch test tubes, Kolle flasks, or Blake bottles are streaked liberally from 

48-hour slant-agar cultures prepared from stock cultures of the selected strains.

(d) The antigen-growing tubes or bottles should be incubated 48 hours at 37 °C., and 

the surface growth washed off with a very slight amount of 12 percent solution 

of sodium chloride containing 0.25 to 0.5 percent phenol, filtered through lightly 

packed sterile absorbent cotton placed in the apex of a sterile funnel.

(e) The washings should be adjusted (using 12 percent sodium chloride containing 

0.25 to 0.5 percent phenol) so that the turbidity is 50 times greater than tube 0.75 

of McFarland’s nephelometer, or to a reading of 7 mm. by the Gates nephelometer.

(f) The individual strain antigens should be tested with negative sera for their 

insensitivity and with positive sera for high agglutinability in comparison with 

known satisfactory antigen. The antigens of the separate strains should be 

combined in equal volume-density and stored in the refrigerator (5 to 10 °C.) in 

tightly stoppered bottles.

(g) The tests should be conducted on a suitable, smooth plate. The serum-antigen 

dilution should be made so that the dilution will not exceed 1:50 when compared 

to the standard tube agglutination test. When testing turkey blood samples, it 

is desirable to use a serum-antigen dilution equivalent to the 1:25 in the tube 

method. The serum should be added to the antigen and mixed thoroughly by use 

of the tip of the serum pipette. Most strong positive reactions will be plainly evident 

within 15 to 20 seconds. The final reading should be made at the end of 2 or 

3 minutes. Heating the plate at approximately 37 °C. will hasten agglutination. 

Before reading, the plate should be rotated several times.

(h) The results shall be recorded as described in § 147.1(h).

(Approved by the Office of Management and Budget under control number 0579-0007) 

2

The procedure described is a modification of the method reported by Runnels, Coon, 

Farley, and Thorpe, Amer. Vet. Med. Assoc. Jour. 70 (N.S. 23): 660–662 (1927). 
93

§ 147.3 The stained-antigen, rapid, whole-blood test.3 

(a) The description of the preparation of antigen is not herein included because the 

antigen is a proprietary product produced only under license from the Secretary of 

Agriculture.

(b) A loop for measuring the correct quantity of blood can usually be obtained from 

the manufacturer of the antigen. A satisfactory loop may be made from a piece of 

No. 20 gauge nichrome wire, 2 1/2 inches long, at the end of which is fashioned a 

loop three-sixteenths of an inch in diameter. Such a loop, when filled with blood so 

that the blood appears to bulge, delivers 0.02 cc. A medicine dropper whose tip is 

adjusted to deliver 0.05 cc. is used to measure the antigen. A glass plate about 15 

inches square, providing space for 48 tests, has proved satisfactory for this work. 

The use of such a plate enables the tester to have a number of successive test 

mixtures under observation without holding up the work to wait for results before 

proceeding to the next bird.

(c) A drop of antigen should be placed on the testing plate. A loop full of blood should 

be taken up from the wing vein. When submerged in the blood and then carefully 

withdrawn, the loop becomes properly filled. On looking down edgewise at the 

filled loop, one observes that the blood appears to bulge. The loopful of blood then 

should be stirred into the drop of antigen, and the mixture spread to a diameter of 

about 1 inch. The loop then should be rinsed in clean water and dried by touching 

it to a piece of clean blotting paper, if necessary. The test plate should be rocked 

from side to side a few times to mix the antigen and blood thoroughly, and to 

facilitate agglutination. The antigen should be used according to the directions of 

the producer.

(d) Various degrees of reaction are observed in this as in other agglutination tests. 

The greater the agglutinating ability of the blood, the more rapid the clumping and 

the larger the clumps. A positive reaction consists of a definite clumping of the 

antigen surrounded by clear spaces. Such reaction is easily distinguished against 

a white background. A somewhat weaker reaction consists of small but still clearly 

visible clumps of antigen surrounded by spaces only partially clear. Between this 

point and a negative or homogeneous smear, there sometimes occurs a very fine 

granulation barely visible to the naked eye; this should be disregarded in making a 

diagnosis. The very fine marginal clumping which may occur just before drying up 

is also regarded as negative. In a nonreactor, the smear remains homogeneous.

(Allowance should be made for differences in the sensitiveness of different antigens 

and different set-ups, and therefore, a certain amount of independent, intelligent 

judgment must be exercised at all times. Also, the histories of the flocks require 

consideration. In flocks where individuals show a suspicious agglutination, it is 

desirable to examine representative birds bacteriologically to determine the presence 

or absence of S. pullorum.)

(Approved by the Office of Management and Budget under control number 0579-0007) 

↓

3The procedure described is a modification of the method reported by Schaffer, 

MacDonald, Hall, and Bunyea, Jour. Amer. Vet. Med. Assoc. 79 (N. S. 32): 236–240 (1931). 
94

§ 147.4 [Reserved]

§ 147.5 The microagglutination test for pullorum-typhoid. 

Routinely, the microagglutination test is applied as a single-dilution test and only a 

single 18–24 hour reading is made.

(a) The procedure for the collection and delivery of blood samples in the 

microagglutination test is the same as that described in § 147.1(a). A method that 

has proven advantageous is to transfer the serum samples from the blood clot to a 

microplate as described in “Applied Microbiology,” volume 24, No. 4, October 1972, 

pages 671–672. The dilutions are then performed according to paragraphs (d) or 

(e) of this section.

(b) Stained microtest antigen for pullorum-typhoid is supplied as concentrated stock 

suspension and must be approved by the Department.4

 Directions for diluting will 

be provided with the antigen. The stock as well as the diluted antigen prepared 

each day should be kept sealed in the dark at 5 to 10 °C. when not in use. 

(c) Available data indicate that a 1:40 dilution for the microagglutination test is 

most efficient for the detection of pullorum-typhoid agglutinins in both chickens 

and turkeys. In all official reports on the blood test, the serum dilutions shall be 

indicated.

(d) The recommended procedure for the 1:40 dilution in the microagglutination test is 

as follows:

(1) Add 100 microliters (0.10 cc.) of 0.85 percent physiological saline to each well 

of the microplate.

(2) Using a microdiluter or a multimicrodiluter handle fitted with twelve 10 

microliter microdiluters, transfer 5 microliters (0.005 cc.) of the serum sample 

from the collected specimen to the corresponding well of the microplate. 

This is accomplished by touching the surface of the serum sample with the 

microdiluter and then transferring and mixing with the diluent in the microplate 

well. The microdiluter is removed, blotted, touched to the surface of the distilled 

water wash, and again blotted. Other acceptable methods of serum delivery 

are described in “Applied Microbiology,” volume 21, No. 3, March 1971, pages 

394–399.

(3) Dilute the microtest antigens with 0.50 percent phenolized saline and add 

100 microliters (0.1 cc.) to each microplate well.

(4) Seal each plate with a plastic sealer or place unsealed in a tight incubation 

box as described in “Applied Microbiology,” volume 23, No. 5, May 1972, pages 

931–937. Incubate at 37 °C. for 18–24 hours.

(5) Read the test results as described in paragraph (f) of this section.

(e) The recommended procedure for a microagglutination test titration is as follows:

(1) Add 50 microliters (0.05cc.) of 0.85 percent physiological saline to each well of 

4

Information as to criteria and procedures for approval of concentrated stock 

suspension of stained microtest antigens may be obtained from the National Poultry 

Improvement Plan, Veterinary Services, APHIS, USDA, 1498 Klondike Road, 

Suite 101, Conyers, GA 30094.
95

the microplate.

(2) To the wells representative of the lowest dilution in the titration, add an 

additional 50 microliters (0.05 cc.) of 0.85 percent physiological saline making 

a total of 100 microliters in these wells.

(3) Transfer each serum sample as described in § 147.5(d)(2) of this section to the 

first well containing 100 microliters (0.10cc.) in the titration, which represents 

the lowest dilution.

(4) Make twofold serial dilutions of each serum by transferring 50 microliters 

(0.05cc.) of diluted serum from one well to the next using twelve 50 microliter 

microdiluters fitted in a multimicrodiluter handle. When transfers have been 

made to all of the wells of the desired series, the 50 microliters remaining in 

the microdiluters are removed by blotting, touching the microdiluters to the 

surface of the distilled water wash, and blotting again.

(5) Dilute the desired microtest antigen with 0.50 percent phenolized saline and 

add 50 microliters (0.05 cc.) to each microplate well.

(6) Seal each plate with a plastic sealer or place the unsealed microplates in a 

tight incubation box and incubate at 37 °C. for 18–24 hours.

(7) Read the test results as described in paragraph (f) of this section.

(f) Read the test results with the aid of a reading mirror. Results are interpreted as 

follows:

(1) N, or – (negative) when the microplate well has a large, distinct button of 

stained cells; or

(2) P, or + (positive) when the microplate well reveals no antigen button; or

(3) S, or ? (suspicious) when the microplate well has a small button. Suspicious 

reactions may tend to be more positive than negative [±] or vice versa [ ] and 

can be so noted if desired. 

(Approved by the Office of Management and Budget under control number 0579-0007) 

§ 147.6 Procedure for determining the status of flocks reacting to tests 

for Mycoplasma gallisepticum, Mycoplasma synoviae, and Mycoplasma 

meleagridis. 

The macroagglutination tests for Mycoplasma antibodies, as described in "Standard 

Methods for Testing Avian Sera for the Presence of Mycoplasma Gallisepticum 

Antibodies" published by the Agricultural Research Service, USDA, March 1966, and 

the microagglutination tests, as reported in the Proceedings, Sixteenth Annual Meeting 

of the American Association of Veterinary Laboratory Diagnosticians, 1973, shall be 

the official tests. Procedures for isolation and identification of Mycoplasma may be 

found in Isolation and Identification of Avian Pathogens, published by the American 

Association of Avian Pathologists and §§ 147.15 and 147.16 of this part.

(a) The status of a flock for Mycoplasma shall be determined according to the following 

criteria:
96

(1) If the tube agglutination or the serum plate test is negative, the flock qualifies.

(2) If the tube agglutination or the serum plate test is positive, the hemaglutination 

inhibition (HI) test and/or the Serum Plate Dilution (SPD) test shall be 

conducted. Provided, that for egg-type and meat-type chicken and waterfowl, 

exhibition poultry, and game bird flocks, if more than 50 percent of the samples 

are positive for either Mycoplasma gallisepticum, M. synoviae, or both, the HI 

and/or the SPD test shall be conducted on 10 percent of the positive samples 

or 25 positive samples, whichever is greater. The results of the HI and/or SPD 

tests must be followed by the action prescribed in paragraphs (a)(3), (a)(4), 

and (a)(5) of this section.

(3) If the tube agglutination or serum plate tests are positive and HI and/or 

the SPD tests are negative, the flock shall be retested in accordance with 

paragraph (a)(6) of this section.

(4) If HI titers of 1:40 or SPD titers of 1:5 are found, the flock shall be considered 

suspicious and shall be retested in accordance with paragraph (a)(6) of this 

section.

(5) If HI titers of 1:80, positive enzyme-labeled immunosorbent assay (ELISA) 

titers, or SPD titers of 1:10 or higher are found, the Official State Agency shall 

presume the flock to be infected. If the indicated titers are found, tracheal 

swabs from 30 randomly selected birds shall be taken promptly and cultured 

individually or a PCR-based procedure conducted on these specimens for 

Mycoplasma, and additional tests conducted in accordance with paragraph 

(a)(6) of this section before final determination of the flock status is made.

(6) Fourteen days after the previous bleeding date, all birds or a random sample 

comprised of 75 birds shall be tested by the serum plate or tube agglutination 

test. Tested birds shall be identified by numbered bands.

(7) If the tube agglutination test or serum plate test is negative for the Mycoplasma 

for which the flock was tested, the flock qualifies.

(8) If the tube agglutination or serum plate test is positive on the retest, the HI 

and/or SPD test shall be conducted on the reacting samples.

(9) On the retest, if the tube agglutination or serum plate tests are positive at the 

same or higher rate and the HI or SPD tests are negative, the flock shall be 

considered suspicious and shall be retested in accordance with paragraph 

(a)(6) of this section.

(10) On the retest if HI titers of 1:80 and/or SPD titers of 1:10 or higher are found, 

the flock shall be considered infected: Provided, That, at the discretion of the 

Official State Agency, additional tests may be conducted in accordance with 

paragraph (a)(6) of this section before final determination of the flock status is 

made.

(11) If HI titers of 1:80 and/or SPD titers of 1:10 or higher are found on the second 

retest, the flock shall be considered infected for the Mycoplasma for which it 

was tested.

(12) If the tube agglutination or serum plate tests are found on the second retest 

to be positive at the same or higher rate and the HI and/or SPD tests are 

negative, the flock should be considered infected: Provided, That if the status 

of the flock is considered to be equivocal, the Official State Agency may 

examine reactors by the in vivo bio-assay, PCR-based procedures, and/or 
97

culture procedures before final determination of the flock status is made.

(13) If the in vivo bio-assay, PCR-based procedures, and culture procedures are 

negative, the Official State Agency may qualify the flock for the classification 

for which it was tested. 

(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 bioassay, PCR-based procedures, or cultural examinations may be conducted 

by the Official State Agency before final determination of the flock's status 

is made.

(15) If the in vivo bio-assay, PCR-based procedures, or cultures are positive on 

retest, the flock shall be considered infected for the Mycoplasma for which it 

was tested.

(b) [Reserved]

§ 147.7 Standard Test Procedures for Mycoplasma.5 

The serum plate agglutination test, tube agglutination test, and the enzyme-linked 

immunosorbent assay (ELISA) should be considered basic screening tests for 

Mycoplasma antibodies. The test selected will depend on preference, laboratory 

facilities, and availability of antigen. These three tests, though quite accurate, 

determine flock status rather than individual bird status, since occasional reactions are 

nonspecific. Under normal circumstances, the rate of such nonspecific reactions is low. 

Nonspecific reactions may occasionally be high, particularly after the use of erysipelas 

bacterin in turkeys and where Mycoplasma antibodies are present for closely related 

Mycoplasma other than for the species being tested. The hemagglutination inhibition 

(HI) test is too cumbersome for routine screening use. Positive reactions are extremely 

accurate however, and are useful in evaluating serum samples that react with the 

ELISA, plate, and/or tube antigens. The test should be conducted with 4 HA units. 

Titers of 1:80 or greater for both chicken and turkey sera are considered positive, 

while a 1:40 or 1:20 titer would be strongly suspicious and additional tests should be 

required.

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, 3rd Edition; Methods for Examining 

Poultry Biologics and for Identifying and Quantifying Avian Pathogens, 1991. 
98

(1) The serum plate agglutination test for Mycoplasma is conducted by contacting 

and mixing 0.02 ml of test serum with 0.03 ml of serum plate antigen on a glass at 

room temperature. The standard procedure is:

 (i) Allow antigen and test serums to warm up to room temperature before use.

(ii) Dispense test serums in 0.02 ml amounts with a pipette or standardized loop 

(rinsed between samples) to 1 inch squares on a ruled glass plate. Limit the 

number of samples (no more than 25) to be set up at one time according to 

the speed of the operator. Serum should not dry out before being mixed with 

antigen.

(iii) Dispense 0.03 ml of antigen beside the test serum on each square. Hold 

antigen dispensing bottle vertically.

(iv) Mix the serum and antigen, using a multimixing device if large numbers are to 

be run at one time.

(v) Rotate the plate for 5 seconds. At the end of the first minute, rotate the plate 

again for 5 seconds and read 55 seconds later.

(2) A positive reaction is characterized by the formation of definite clumps, usually 

starting at the periphery of the mixture. Most samples that are highly positive 

will react well within the 2-minute test period. Reactions thereafter should be 

considered negative, although partial agglutination at 3 and 5 minutes may 

warrant further retesting. High-quality antigen contacted with negative serum will 

usually dry up on the plate without visible clumping. Whenever samples are run, 

the antigen should be tested against known positive and negative control serums. 

Standard reference antigens and negative and positive titered sera are available 

from the National Veterinary Services Laboratories (NVSL), P.O. Box 884, Ames, 

Iowa 50010.

(3) Since it is difficult to measure uniform amounts of serum with a calibrated loop, this 

technique should not be used in conducting an official test.

(1) The serum plate dilution (SPD) test may be used to evaluate possible nonspecific 

reactions, gain additional information to evaluate positive plate tests occurring in 

an unexpected manner, and/or to evaluate the level of Mycoplasma antibodies 

present in the serum sample. If sufficient serum is available, the following method 

would provide the dilutions required to conduct the test.

 (i) Rack three tubes and put 0.8 ml of phosphate-buffered saline (PBS) in tube 1

and 0.5 ml of PBS in tubes 2 and 3.

 (ii) Pipette 0.2 ml of the test serum into tube 1 and discard the pipette.

 (iii) With a pipette, mix the serum and PBS in tube 1 and withdraw 0.5 ml and add 

to tube 2.

(iv) Repeat the process in step (iii), mixing the contents of tube 2 and transferring 

0.5 ml to tube 3.

 (v) Conduct the test, as described for the serum plate test in paragraph (a), on 

the undiluted sample and on samples in tubes 1, 2, and 3 after proper mixing 

of each dilution.

(vi) To assist in the evaluation of the test, conduct concurrent SPD tests using 

both positive 1:80 and positive 1:160 HI sera for the Mycoplasma being tested. 

The antigen should be pretested for reactivity with standard serum at the 1:5 

and 1:10 dilution.

(b) Serum plate dilution 

test 

(a) Serum plate 

agglutination test
99

(vii) Interpretation of the SPD test results should be based on the criteria in 

§ 147.6(a) of this part.

(1) The Mycoplasma tube agglutination test is conducted by mixing 0.08 ml of test 

serum with 1.0 ml of diluted (1:20) antigen in a tube and allowing the mixture to 

react for 18–24 hours at 37 °C. The diluent will be the standard phosphate-buffered 

saline with phenol. This solution is made up as follows:

Sodium hydroxide (C.P.) 0.15 g

Sodium chloride (C.P.) 8.50 g

Potassium dihydrogen phosphate

(KH2

PO4)(C.P.) 0.68 g

Phenol (Crystal) (C.P.) 2.50 g

Distilled water to make 1,000 ml

The pH of the buffered phenolized saline will be 7.1–7.2 if all reagents are 

accurately measured. The stock tube antigen is diluted 1:20 with buffered 

phenolized saline. The procedures for the tube test are as follows:

(i) Rack 12 × 75 mm clean tubes and identify the tubes according to the sample to 

be tested.

(ii) Add 0.08 ml of the individual test serum to each tube. This will create 

approximately a 1:12.5 screening dilution of test serum when 1.0 ml of diluted 

antigen is added. The use of a pipetting device will insure proper mixing of 

serum and antigen.

(iii) To interpret positive reactions to the 1:12.5 dilution, two additional dilutions 

may be made by adding 0.04 ml of serum for 1:25 dilution and 0.02 ml of 

serum for 1:50 dilution, with the addition of 1.0 ml of diluted antigen as 

indicated in paragraph (c)(1)(ii) of this section.

(iv) Shake racks and incubate test systems for 18–24 hours at 37 °C.

(2) Tests are read against a dark background under indirect fluorescent light. 

Regarded as a positive reaction is a clearing of the supernatant fluid, with visible 

sediment in the bottom of the tube. Incomplete reactions are suspect. Positive 

and negative control serums should be incorporated into each day’s run of tests. 

Reactions at 1:25 or greater are considered positive. They should be confirmed 

by the HI test. Incubation for periods greater than 24 hours may be helpful in 

evaluating suspicious reactions and need for possible retesting or other diagnostic 

tests.

The Mycoplasma HI test is conducted by the constant-antigen, decreasing-serum 

method. This method requires using a 4-hemagglutination (HA) unit of diluted antigen. 

(d) Hemagglutination 

Inhibition (HI) test 

(c) Tube agglutination 

test 
100

Differences in the number of HA units used will change the titers of positive sera 

markedly. Standard HA antigens for Mycoplasma gallisepticum, M. synoviae, and 

M. meleagridis are available from NVSL. The antigen has been titrated and diluted to 

approximately 1:640. The HA titration of each sample should be checked as described 

in paragraph (d)(2) on initial use or after long storage. To maintain HA activity, the 

undiluted HA antigen should be stored at –60 to –70 °C. The test procedures are 

illustrated in Tables 2 and 3 of this paragraph.

(1) Preparation of materials

(i) Prepare phosphate-buffered saline (PBS) as follows: 

 Sodium hydroxide (C.P.) 0.15 g 

 Sodium chloride (C.P.) 8.5 g 

 Potassium dihydrogen phosphate (KH2PO4) (C.P.) 0.68 g 

 Distilled water to make 1,000 ml 

The pH of the PBS will be 7.1–7.2 if all reagents are accurately measured.

(ii) Collect the turkey or chicken red blood cells (RBC’s) in Alsever’s solution which 

has been prepared as follows: 

 Sodium citrate 8.0 g 

 Sodium chloride 4.2 g 

 Dextrose 20.5 g 

 Distilled to make 1,000 ml 

The sodium citrate and sodium chloride are dissolved in 800 ml distilled water 

and sterilized at 15 lbs. pressure for 15 minutes. Dissolve the dextrose in 

200 ml distilled water, sterilize by Seitz or other type of filtration and then add 

aseptically to the sterile sodium citrate and sodium chloride solution.

(iii) From a turkey(s) or chicken(s) known to be free of the Mycoplasma being 

tested, withdraw sufficient blood with a syringe containing Alsever’s solution 

to give a ratio of 1 part blood to 5 parts Alsever’s solution (e.g., 8 ml blood in 

40 ml of Alsever’s solution). Centrifuge the blood suspension at 1,000 rpm for 

10 minutes and remove the Alsever’s solution or supernatant with a pipette.

(iv) Wash the RBC’s two times in 10 or more parts of Alsever’s solution, 

centrifuging after each washing. Centrifugation is at 1,000 rpm for 10 minutes. 

The supernatant fluid is removed and the RBC deposit resuspended to give a 

25 percent suspension of packed RBC’s in Alsever’s solution. (In testing either 

chicken or turkey sera, the homologous RBC system must be used; i.e., use 

chicken cells when testing chicken serum and turkey cells when testing turkey 

serum.) If this suspension is kept refrigerated, it should keep for 7 or 8 days 

after the blood has been collected.

(v) For the test, 1 ml of the 25 percent RBC’s is added to 99 ml of buffered saline 

to make a 0.25 percent RBC suspension.
101

(2) Hemagglutination (HA) antigen titration

The HA stock antigen is stored at –70 °C in PBS buffer containing 25 percent 

glycerin (vol/vol) in a concentrated suspension (i.e., 320–640 HA units/ml) 

in screwtype vials. Under such conditions, potency will be retained for years. 

There will be a rapid loss of titer if improperly stored. The titer of HA antigen is 

determined as illustrated in Table 1 and described in subparagraphs (d)(2)(i) 

through (x) of this paragraph. 

Table 1 Titration of Hemagglutination (HA) Antigen

Tube No.

Reagents (ml) 1 2 3.... 8 9 10 11a

PBS 0.5 0.5 0.5... 0.5 0.5 0.5 0.5

Antigen 0.2

Transfer 0.5→ 0.5→ 0.5...→ 0.5→ 0.5→ 0.5 c

0.25% RBC 0.5 0.5 0.5... 0.5 0.5 0.5 0.5

Ant. dilution 1:5 1:10 1:20... 1:640 1:1,280 1:2,560

Resultsb + + +... + – –

a

Tube 11, PBS/RBC control.

b

+ = HA; –= no HA (sample titer 1:640). c

Discard 0.5 ml.

 

(i) Rack a series of 11 chemically clean 12 × 75 mm test tubes. Label the tubes 

1–11 left to right.

(ii) Put 0.8 ml of PBS in tube 1 and 0.5 ml of PBS in each of tubes 2–11.

(iii) Add 0.2 ml of antigen to tube 1. This will make a 1:5 dilution of antigen. 

Discard pipette.

(iv) Mix contents of tube 1 thoroughly with a clean pipette, and transfer 0.5 ml to 

tube 2. This will make a 1:10 dilution of antigen in tube 2. Discard pipette.

(v) Continue making serial twofold dilutions of antigen, changing pipettes after 

each transfer, through tube 10. This will result in a series of twofold dilutions 

ranging from 1:5 to 1:2560. Discard 0.5 ml of antigen dilution from tube 10.

(vi) Add 0.5 ml of 0.25 percent RBC’s to tubes 1–11. Tube 11 will serve as 

PBS/RBC control.

(vii) Shake the rack and incubate at room temperature until the cells in the 

PBS/RBC control tube have settled into a compact button at the bottom of 

the tube.

(viii) If turkey sera is also to be tested for HI titer, repeat steps outlined in 

(d)(2)(i) through (vii) of this paragraph, using 0.25 percent turkey RBC’s.

(ix) The end point of the titration is the highest dilution of antigen that produces 

↓
102

complete agglutination of the RBC’s, as evidenced by the formation of a thin 

sheet of cells covering the concave bottom of the tube. For example, if 

complete agglutination is produced through tube 8 (a dilution of 1:640 of 

antigen), the antigen would be said to titer 640, the reciprocal of the dilution.

(x) Specificity of HA antigen should be determined by conducting HI tests with 

specific chicken sera of variable HI titers. Specific turkey sera of varying HI 

titers should be used if turkey sera is also to be tested. 

Table 2 Hemagglutination Inhibition (HI) Test 

Tube No.

Reagents (ml) 1a 2 3.... 8 9 10 11b

PBS 0.8 0 0 0 0 0.5

8-unit antigen 0 0.5 0 0 0 0

4-unit antigen 0 0 0.5 0.5 0.5 0.5 0

Test serum 0.2 0 0 0 0 0 0

Transfer 0.5→ 0.5→ 0.5...→ 0.5→ 0.5→ 0.5 c

0.25% RBC 0.5 0.5 0.5 0.5 0.5 0.5 0.5

Serum dilution 1:5 1:10 1:20... 1:640 1:1280 1:2560 

a

Tube 1, serum control.

b

Tube 11, PBS/RBC control.

c

Discard 0.5 ml.

Table 3 Antigen Control 

Tube No.

Reagents (ml) 1 2 3 4 5

4-unit antigen 1.0 0 0 0 0

PBS 0 0.5 0.5 0.5 0.5

Transfer 0.5→ 0.5→ 0.5→ 0.5→ 0.5b

0.25% RBC 0.5 0.5 0.5 0.5 0.5

Unit Antigen/tube 4 2 1 1/2 1/4

Resultsa + + + – –

a+=HA; – = no HA. b

Discard 0.5 ml.

↓

↓
103

(3) Reagents for mycoplasma HI test

(i) Eight-unit antigen (Dilution factor for stock antigen is established by dividing 

titer by 8; i.e., 640 antigen is diluted 1:80 in PBS to make 8-unit antigen.)

(ii) Four-unit antigen (made by diluting surplus 8-unit antigen 1:2 with PBS).

(iii) PBS at pH 7.0.

(iv) Unknown test serums.

(v) Positive control serum of known titer (should be from the same species as the 

unknown).

(vi) Negative control serum (should be from the same species as the unknown).

(vii) Solution of 0.25 percent washed RBC’s.

(4) Test outline

(i) Rack 10 chemically clean 12 × 75 mm tubes for each serum, including controls, 

to be tested. Identify each row of tubes, and label tubes in each row 1–10, left 

to right. In row 1, add tube 11 for a PBS/RBC control.

(ii) Put 0.8 ml of PBS in tube 1 of each test row; put 0.5 ml of 8-unit antigen in 

tube 2 of each test row; put 0.5 ml of 4-unit antigen in each of tubes 3–10 in 

each test row; and put 0.5 ml of PBS in tube 11.

(iii) Add 0.2 ml of test serum to tube 1. This tube will be the serum control in the 

test system.

(iv) Mix and make 0.5 ml transfers from tube 1 through tube 10. This will result 

in serial twofold dilutions of serum starting with 1:5 and ending with 1:2560. 

Discard 0.5 ml from tube 10.

(v) Rack five tubes in which to set up an antigen control.

(vi) In tube 1, put 1.0 ml of 4-unit antigen; put 0.5 ml of PBS in tubes 2–5.

(vii) Make 0.5 ml serial transfers from tube 1 through tube 5, changing pipettes 

after each transfer. Discard 0.5 ml from tube 5. This will result in a series of 

tubes respectively containing 4, 2, 1, 1/2, and 1/4 units of antigen.

(viii) After 20–30 minutes at room temperature to permit antibody-antigen reaction, 

add 0.5 ml of 0.25 percent washed RBC’s to each tube. Shake racks and 

incubate as for HA titration.

(ix) In this test system, positive serum should inhibit the HA activity of the antigen, 

while negative serum should have no effect. Inhibition will be evidenced by the 

formation of a free-flowing button of cells in the bottom of the tube. The titer of 

the serum can be calculated as the reciprocal of the highest dilution of serum 

that produces complete HI. Controls should read as follows:

(A) Serum control (tube 1). Cells should settle out.

(B) PBS/RBC control (tube 11). Cells should settle out.

(C) Antigen control. HA in tubes 1–3. Cells should settle out in tubes 4–5.

(D) Positive and negative serum control. Positive control should inhibit to its 

known titer; negative control should have no inhibitory effect.

(x) With this test system and 4 units of antigen, HI titers of 80 or above are 

considered positive and titers of 40 are strongly suspicious. However, titers of 

10 or 20 are usually negative. Sample test results are illustrated in Table 4 in 

this paragraph.
104

(xi) If serological results from agglutination tests complemented by the HI test are 

inconclusive, cultural examination, bio-assay, or retesting of samples after an 

interval of at least 21 days may be indicated.

Table 4 Sample Results of HI Tests 

[Tube and Serum Dilution]

 1 2 3 4 5 6 7 8 9 10

 1:5 1:10 1:20 1:40 1:80 1:160 1:320 1:640 1:1280 1:2560

Serum A (HI neg.) – + + + + + + + + +

Serum B (HI 1:40) – – – – + + + + + +

Serum C (HI 1:60) – – – – – – + + + +

Serum D (HI 1:20) – – – + + + + + + +

+, HA.

–, no HA or HI.

(e) Procedure 

for Mycoplasma 

hemagglutination 

inhibition tests using 

microtiter technique

(1) Procedure No. 1. The microtiter Mycoplasma HI test was developed from the tube 

HI test described in § 147.7(d). Refer to these procedures for preparation of materials 

not listed below. 

(i) Materials needed

(A) Microtiter equipment (minimal); i.e., microplates, microdiluters, 

micropipettes, go–no-go diluter delivery tester, (0.05 ml).

(B) Phosphate-buffered saline (PBS).

(C) Reagents from NVSL; i.e., HA antigen and negative and positive titered 

sera for the Mycoplasma to be tested.

(D) Homologous red blood cells (RBC’s) suspension 0.5 percent (2 ml 

of 25 percent RBC’s to 98 ml of PBS) obtained from birds free of the 

Mycoplasma to be tested. (See paragraph (d)(1)(ii) through (v) of this 

section for preparation of RBC’s.)

(ii) Microtiter hemagglutination (HA) antigen titration 

(A) Mark off two rows of 10 wells each for antigen titer (HA is done in 

duplicate).

(B) Mark last well in each row for cell controls.

(C) Prepare in small test tube (12 × 75 mm) a starting dilution of antigen by 

combining 0.1 ml antigen with 0.9 ml PBS. This is a 1:10 dilution.

(D) Add 0.05 ml PBS to all wells, including cell controls.

(E) Add 0.05 ml antigen (1:10 dilution) with diluters to the first well in both 

rows, mix thoroughly, transfer diluter to second well of each row and mix, 

continuing through the 10th well of each row. With mixture in diluter from 

last well, check diluter on go–no-go card, then place diluter in distilled 

water. If diluter checks out, antigen dilution will be 1:20, 1:40, 1:80, 1:160, 

1:320, 1:640, 1:1280, 1:2560, 1:5120.
105

(F) Add 0.05 ml of 0.5 percent RBC suspension to all wells using a 0.05 ml 

dropper.

(G) Seal plate (if plate is to be held over 2 hours); shake and allow to stand at 

room temperature until cells in cell control gather in compact button. The 

titer is the highest dilution in which agglutination is complete. The dilution 

contains 1 HA unit in 0.05 ml.

(H) Prepare a dilution of antigen which contains 8 HA units in 0.05 ml. 

Example: if the antigen titer is 1:640, then that dilution contains 1 HA unit 

per 0.05 ml. Then 640÷8 = 80, or a dilution of 1:80 containing 8 HA units. 

Or 640÷4 = 160, a dilution of 1:160 containing 4 HA units per 0.05 ml.

(iii) Microtiter HI test 

(A) Prepare two dilutions of antigen, one containing 8 HA units per 0.05 ml 

and one containing 4 HA units per 0.05 ml. The 4-unit antigen can be prepared 

from the 8-unit antigen by mixing with equal parts of PBS.

(B) Mark off one row of 8 wells for each test.

(C) Prepare a 1:5 dilution of each serum to be tested in a small test tube (12×75 

mm): 0.1 ml serum plus 0.4 ml PBS or 0.05 ml serum plus 0.20 ml PBS.

(D) Add 0.05 ml PBS with the 0.05 ml dropper to the first well in each row.

(E) Add 0.05 ml of 8-unit antigen to well 2 in each row.

(F) Add 0.05 ml of 4-unit antigen to wells 3 through 8 for each row.

(G) For each serum to be tested, load 0.05 ml diluter with 1:5 dilution as prepared 

in paragraph (C) above and place in first well of row.

(H) Mix well and transfer loaded diluter to well 2. Continue serial twofold dilutions 

through well number 8.

(I) Well 1 (serum dilution of 1:10) is serum control. Well 2 = 1:20 dilution; 

well 3 = 1:40 dilution; well 4 = 1:80 dilution; well 5 = 1:160 dilution; 

well 6 = 1:320 dilution; well 7 = 1:640 dilution; and well 8 = 1:1280 dilution.

(J) Antigen control. 

(1) Mark off 6 wells for antigen controls.

(2) Add 0.05 ml PBS to wells 2, 3, 4, 5, and 6.

(3) Add 0.05 ml 8-unit antigen to wells 1 and 2.

(4) With empty diluter, mix contents of well 2. Continue serial twofold 

dilutions through well 6.

(5) Well 1 contains 8 units; well 2 contains 4 units; well 3 contains 2 units; 

well 4 contains 1 unit; well 5 contains 1/2 unit; and well 6 contains 

1/4 unit.

(6) Mark off two wells for cell controls and add 0.05 ml PBS to each.

(7) After 20–30 minutes at average room temperature (20–23 °C) to 

permit antibody-antigen reaction, add 0.05 ml of a 0.5 percent 

suspension of RBC’s to all wells.

(8) Seal all wells (if wells are to be held over 2 hours). Shake the plate 

thoroughly.

(9) Incubate at room temperature for 30–45 minutes.

(K) Interpretation: The HI titer is the highest serum dilution exhibiting complete 

inhibition of hemmagglutination as indicated by flowing of cells when 

the plate is tilted. Serum having a titer of 1:80 or greater is considered 

positive. A titer of 1:40 or 1:20 is suspicious. 
106

(2) Procedure No. 2. Purpose: To test for antibodies to avian mycoplasma by 

hemagglutination (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-button.

(E) 12-channel pipettor (Titerek).

(F) 50μL 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 μL of PBS into each well of 3 rows of a 96-well microtiter plate.

(2) Dispense 50 μL of stock antigen into the wells of 2 rows.

(3) Perform serial two-fold dilutions (50 μL) using a 12-channel pipettor. 

The dilution series will be from 1:2 to 1:4096.

(4) Add 50 μL 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’c give tight buttons. The HA titer is read as the last well to give a 

complete lawn (hemagglutination). 

(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 μL of PBS to the top row of wells (row A) of the plate.

(C) Add 25 μL of PBS to all remaining wells of the plate.

(D) Add 10 μL of each test sera to well A of each column (making a 1:5 sera 

dilution).

(E) Serially dilute 25 μL from well A through H using a 12-channel pipettor. 

Discard the final 25 μL. Row A = 1:5...row H = 1:640.

(F) With an Oxford doser, add 25 μL of 4 HA units antigen to wells B through H. 

Well A serves as sera control.

(G) Prepare an antigen backtitration by adding 25 μL of PBS to each well 

of one column. Add 25 μL of diluted antigen to well A and serially dilute 

25 μL from wells A to D. This prepares 1:2, 1:4, 1:8, and 1:16 dilutions. 
107

(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 μL 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 nonspecific 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, 4  ̊C (refrigerator).

(D) Procedure. 

(1) Prepare a 1:5 dilution of test serum by adding 50 μL of serum to 

200 μL 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 μL of 50 percent RBC solution to the serum dilutions.

(4) Vortex gently to mix.

(5) Incubate at 4  ̊C for 1 hour.
108

(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 μL of the 1:5 treated supernatant to row A. Serially dilute 25 μL 

from rows A through H. This prepares a serum dilution of 1:10 through 

1:60 in rows B through H. 

§ 147.8 Procedures for preparing egg yolk samples for diagnostic tests. 

The following testing provisions may be used for retaining the classification 

U.S. M. Gallisepticum Clean under § 145.23(c)(1)(ii)(C) and § 145.33(c)(1)(ii)(C); and 

for retaining the classification U.S. M. Synoviae Clean under § 145.23(e)(1)(ii)(B) and 

§ 145.33(e)(1)(ii)(B); and for retaining the classification U.S. H5/H7 Avian Influenza 

Monitored under § 146.23(a), § 146.33(a) and § 146.44(a) of this chapter.

(a) Under the supervision of an Authorized Agent or State Inspector, the eggs 

which are used in egg yolk testing must be selected from the premises where 

the breeding flock is located, must include a representative sample of 30 eggs 

collected from a single day’s production from the flock, must be identified as to flock 

of origin and pen, and must be delivered to an authorized laboratory for preparation 

for diagnostic testing.

(b) The authorized laboratory must identify each egg as to the breeding flock and pen 

from which it originated, and maintain this identity through each of the following:

(1) Crack the egg on the round end with a blunt instrument.

(2) Place the contents of the egg in an open dish (or a receptacle to expose the 

yolk) and prick the yolk with a needle.

(3) Using a 1 ml syringe without a needle, aspirate 0.5 ml of egg yolk from the 

opening in the yolk.

(4) Dispense the yolk material in a tube. Aspirate and dispense 0.5 ml of PBS 

(phosphate-buffered saline) into the same tube, and place in a rack.

(5) After all the eggs are sampled, place the rack of tubes on a vortex shaker for 

30 seconds.

(6) Centrifuge the samples at 2500 RPM (1000×g) for 30 minutes. 

(7) (i) For egg yolk samples being tested to retain the U.S. M. Gallisepticum

Clean and U.S. M. Synoviae Clean classifications, test the resultant 

supernatant for M. gallisepticum and M. synoviae by using test procedures 

specified for detecting IgG antibodies set forth for testing serum in § 147.7 (for 

these tests the resultant supernatant would be substituted for serum); except 

that a single 1:20 dilution hemagglutination inhibition (HI) test may be used as a 

screening test in accordance with the procedures set forth in § 147.7.

(ii) For egg yolk samples being tested to retain the U.S. H5/H7 Avian Influenza 

Monitored classification, test the resultant supernatant in accordance with 

requirements in § 146.13(b).

Note: For evaluating the test results of any egg yolk test, it should be remembered that 

a 1:2 dilution of the yolk in saline was made of the original specimen.
109

§ 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 Beard6. 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 x 15 mm or 60 x 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).

6

Beard, C.W. Demonstration of type-specific influenza antibody in mammalian and avian sera 

immunodiffusion. Bull. Wld. Hlth. Org. 42:779–785. 1970.
110

(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 x 15 mm petri plate or 5 to 

 6 mL agar into a 60 x 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 x 15 mm plate and two patterns can

 be cut in a 60 x 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, 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 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 1). 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 2).
111

(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.
112

Figure 1. —Immunodiffusion test that has AI AGID antigen in the center well; 

 AI-positive control serum in wells A, C, and E; and AI-negative test serum in 

 wells B, D, and F.

Figure 2.—Immunodiffusion test that has AI AGID antigen in the center well;

 AI-positive control serum in wells A, C, and E; AI-negative test serum in

 well B; AI-positive test serum in well D; and weak positive test serum in

 well F.

(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.

F B

A

D

E C

A

B

C

D

E

F
113

Subpart B—Bacteriological Examination Procedure

§ 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 § 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, §147.11(a)(1) 

of this subpart, may be omitted; and 

(b) Enrichment culture of organ (non-intestinal) tissues using a non-selective broth, 

§ 147.11(a)(2) of this subpart, may be omitted.

§ 147.11 Laboratory procedure recommended for the 

bacteriological examination of salmonella.

All reactors to the pullorum-typhoid tests, up to 25 birds, and birds from Salmonella 

enteritidis (SE) positive environments should be cultured in accordance with both 

the direct enrichment (paragraph (a)(1)) and selective enrichment (paragraph (a)(2)) 

procedures described in this section: Provided, That in turkeys, if there are more 

than 4 reactors to the pullorum-typhoid tests in the flock, a minimum of 4 reactors as 

provided for in § 145.14(a)(6)(ii) of this subchapter shall be submitted to the authorized 

laboratory for bacteriological examination. 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 (such as blood or nutrient agar) and selective plates (such as MacConkey 

[MAC] and brilliant green novobiocin [BGN] for pullorum-typhoid and MAC, BGN, 

and xylose-lysine-tergitol 4 [XLT 4] for SE). After inoculating the plates, pool the 

swabs from the various organs into a tube of non-selective broth (such as nutrient 

or brain-heart infusion). Refer to illustration 1 for recommended bacteriological 

recovery and identification procedures7. Proceed immediately with collection of 

organs and tissues for selective enrichment culture.

(a) For egg- and meattype chickens, turkeys, 

waterfowl, exhibition 

poultry, and game birds

7

 Biochemical identification charts may be obtained from “A Laboratory Manual for the 

Isolation and Identification of Avian Pathogens,” chapter 2, Salmonellosis. Fourth edition, 

1998, American Association of Avian Pathologists, Inc., Kennett Square, PA 19348.
114

(2) Selective enrichment culture (refer to illustration 1). 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:

(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 visibly pathological sites where purulent, necrotic, or proliferative 

lesions are seen.

(3) From each bird, aseptically collect 10 to 15 grams of each organ or site listed 

in paragraph (a)(2) of this section. Mince, grind, or blend and place in a sterile 

plastic bag. All the organs or sites listed in paragraph (a)(2) of this section from 

the same bird may be pooled into one bag. Do not pool samples from more than 

one bird. Add sufficient tetrathionate enrichment broth to give a 1:10 (sample to 

enrichment) ratio. Follow the procedure outlined in illustration 1 for the isolation 

and identification of Salmonella.

(4) From each bird, aseptically collect 10 to 15 grams of each of the following parts 

of the digestive tract: Crop wall, duodenum, jejunum (including remnant of yolk 

sac), both ceca, cecal tonsils, and rectum-cloaca. Mince, grind, or blend tissues 

and pool them into a sterile plastic bag. Do not pool tissues from different birds 

into the same sample. Add sufficient tetrathionate enrichment broth to give a 1:10 

(sample to enrichment) ratio. Follow the procedure outlined in illustration 1 for the 

isolation and identification of Salmonella.

(5) After selective enrichment, inoculate selective plates (such as MAC and BGN 

for pullorum-typhoid and MAC, BGN, and XLT 4) for SE. Inoculate three to five 

Salmonella-suspect colonies from plates into triple sugar iron (TSI) and lysine iron 

agar (LIA) slants. Screen colonies by serological (i.e., serogroup) and biochemical 

procedures (e.g., the Analytical Profile Index for Enterobacteriaceae [API]) as 

shown in illustration 1. As a supplement to screening three to five Salmonellasuspect colonies on TSI and LIA slants, a group D colony lift assay may be utilized 

to signal the presence of hard-to-detect group D Salmonella colonies on agar 

plates.

(6) If the initial selective enrichment is negative for Salmonella, a delayed secondary 

enrichment (DSE) procedure is used. Leave the tetrathionate-enriched sample at 

room temperature for 5 to 7 days. Transfer 1 mL of the culture into 10 mL of fresh 

tetrathionate enrichment broth, incubate at 37 o

C for 20 to 24 hours, and plate as 

before.

(7) Serogroup all isolates identified as salmonellae and serotype all serogroup D1 

isolates. Phage-type all SE isolates.

(b) [Reserved]
115

ILLUSTRATION 1—Procedure for culturing Pullorum-Typhoid reactors and birds from SE-Positive 

environments.

1. Non-selective plates such as blood or nutrient agar.

2. Selective plates such as MacConkey, Brillant Green Novobioccin (BGN) forpullorum-typhoid reactors and MacConkey, BGN, and xylose-lysine 

tergitol 4 (XLT 4) for SE.

3. Tetrathionate enrichment broth.

4. Reevaluate if epidemiologic, necropsy, or other information indicates the presences of an unusual strain of Salmonella.

5. If biochemical identification and serogroup procedures are inconclusive, restreak original colony onto non-selective plating media to check for 

purity. Repeat biochemical and serology tests.

Chicken

37 °C 24 and 48 hours

37 °C 24 and 48 hours

DSE

Organs

Pools swabs into Organ pools

non-selective broth Direct culture:

Inoculate

non-selective1

and selective 2

plates

 37 °C 20-24 hours

Non-selective1

and selective2

plates

 37 °C 20-24 hours

Inoculate TT3

enrichment

broths

(1:10 ratio)

37 or 41.5 °C 20-24 hours

Inoculate TT3

enrichment

broths

(1:10 ratio)

37 °C 20-24 hours

Inoculate selective plates2

Inoculate 3 to 5 suspect colonies into TSI and LIA slants

TSI and LI reactions: Both 

positive for Salmonella

TSI and LI reactions: One 

positive and one negative for 

Salmonella

TSI and LI reactions: Both 

negative for Salmonella

Biochemical

identification Discard4 Serogroup

Positive for 

Salmonella

Negative for 

Salmonella

Discard4 Serogroup

Positive for 

Salmonella

Negative for 

Salmonella

Reevaluate5

Positive for 

Salmonella

Negative for 

Salmonella

Positive for 

Salmonella

Negative for 

Salmonella

Discard4 Reevaluate5

Biochemical 

identification

Serogroup all 

Group D1

Serogroup all 

Group D1

Intestines

Intestinal pools
116

§ 147.12 Procedures for collection, isolation, and identification of Sal- monella from environmental samples, cloacal swabs, chick box papers, 

and meconium samples. 

Information concerning the pen arrangement and number of birds per pen should 

be obtained from the owner so that the required number of samples per pen and per 

flock can be determined. A means of identifying each sample by pen of origin should 

be provided. The vehicle transporting the personnel taking the samples should be 

left as far as practical from the poultry pens. Sanitary precautions, including personal 

cleanliness, should be observed during the sampling procedure. The hands should be 

carefully washed with a sanitizing soap prior to the sampling. Outer clothing, including 

gloves, should be changed between visits to different premises so that clean clothing is 

worn upon entering each premises.

The used and clean apparel should be kept separate. Boots or footwear should be 

cleaned and disinfected between visits to different premises. Disposable caps should 

be provided and discarded after use on each premises. After collection, the samples 

should be protected from drying, light, and excessive temperatures and delivered to 

the laboratory within one day. If delivery is delayed, samples should be refrigerated.

All samples and swabs described in this paragraph should be cultured in accordance 

with illustration 2 of § 147.11, including delayed secondary enrichment. All 

salmonellae recovered shall be serogrouped or serotyped.

(1) Environmental samples. Fecal material, litter, dust, or floor litter surface or nest 

box drag swab samples to be submitted for bacteriological examination shall be 

collected in accordance with the procedures described in paragraphs (a)(1), (a)(2), 

or (a)(3) of this section:

(i) Procedure for sampling in broth. Authorized laboratories will provide capped 

tubes 1 to 2 cm in diameter and 15 to 20 cm in length that are two-thirds full 

of a recently made, refrigerated, sterile enrichment broth for each sample. 

Sufficient tubes shall be taken to the premises to provide at least one tube 

per pen or one tube per 500 birds, whichever is greater. At least one sterile, 

cotton-tipped applicator will be needed for each tube. The dry applicator is 

first placed in or drawn through fresh manure (under roost, near water troughs, 

fecal droppings, or diarrhetic droppings). After each streaking, place the 

cotton-tipped applicator in the tube of broth and swirl the applicator to remove 

the collected material. Withdraw the applicator from the tube and use it to 

take additional specimens by streaking on or through areas where defecation, 

trampling of feces, or settling of dust is common; e.g., on or near waterers, 

feeders, nests, or rafters, etc. When the volume of material collected equals 

approximately 10 percent of the volume of the broth (usually 10-12 streakings), 

place the applicator in the tube and break the stick in half, leaving the lower or 

cotton-tipped half in the broth and retaining the upper half for future disposal. 

Replace the cap on the inoculated tube and continue the sampling procedure 

in other areas of the pen.

(a) For egg- and meattype chickens, turkeys, 

waterfowl, exhibition 

poultry, and game birds
117

(ii) Procedure for sampling in dry containers. Place a sample of fecal material, 

litter, or dust in a sterile, sealable container. The sample shall consist of 

several specimens of material taken from a representative location in the 

pen or house. Collect at least 10 g (approximately a heaping tablespoonful) 

of material for each sample. Collect the specimens in each sample with a 

sterile tongue depressor or similar uncontaminated instrument. The samples 

shall vary in type and consistency. Half of the samples shall be comprised of 

material representing defecated matter from a large portion of the flock; i.e., 

trampled, caked material near waterers and feeders. The minimum number of 

samples to be taken shall be determined by the following: Five samples from 

pens or houses of up to 500 birds; Ten samples from pens or houses of 500 to 

2,500 birds; Fifteen samples from pens or houses with more than 2,500 birds. 

The samples may be pooled to not fewer than five samples at the laboratory 

as long as the volume of material collected equals approximately 10 percent of 

the volume of the broth.

(2) Cloacal swabs. Cloacal swabs for bacteriological examination shall be taken 

from each bird in the flock or from a minimum of 500 birds in accordance with the 

procedure described in paragraph (a)(2)(i) of this section.

(i) Procedure for taking cloacal swabs. The authorized laboratory will provide 

sterile capped tubes or other suitable containers and cotton-tipped applicators 

for use in taking the cloacal swabs. Insert the cotton-tipped applicator into 

the cloaca and rectum in such a manner as to ensure the collection of fecal 

material. Place the swab and adhering fecal material in the tube and break 

the stick in half, keeping the upper half of the stick for future disposal. The 

cloacal swabs may be combined in the sterile tubes in multiples of five or in 

combinations specified by the authorized laboratory.

(ii) [Reserved].

(3) Drag-swabs. Utilization of drag swabs (DS) involves the exposure of gauze pads 

(or commercially available sponges designed for this purpose), a key component 

of a DS sampler, to the surface of random, flock-representative floor litter and 

nest box areas. The sampler pads shall be sterile and slightly moist to promote 

adherence of particulate material, and impregnated with double-strength skim 

milk8

 to protect salmonella viability during sample collection, batching, storage, 

and shipment. Floor litter surface DS sample results tend to reflect the salmonella 

carrier/shedder status of a flock. Nonetheless, other environmental samples as 

described in paragraphs (a)(1)(i), (a)(1)(ii), or (a)(3)(iv) of this section shall also be 

periodically collected.

(i) Drag-swab sampler assembly. Drag-swab (DS) samplers may be assembled 

using two 3- by 3-inch sterile gauze pads; size 20 wrapping twine; and paper 

clips, staples, or similar fasteners. Fold each gauze pad in half and attach 

one pad to a 2-foot-long (60 cm) piece of twine and the other to a 1-foot-long 

(30 cm) piece of twine. To attach a pad to the twine with a paper clip, bend 

the end wires of the paper clip slightly and push them through the fabric of 

8 Obtain procedure for preparing double strength skim milk from USDA–APHIS “Recommended Sample 

Collection Methods for Environmental Samples” available from the National Poultry Improvement Plan, 

Veterinary Services, APHIS, USDA, 1498 Klondike Road, Suite 101, Conyers, GA 30094.
118

the folded pad, thus securing the clips to the folded pad, then securely tie the 

twine to the free rounded end of the paper clip. To attach a pad to the twine 

with a staple, staple the twine to the pad near the center of the fold, applying 

the staple at a right angle to the twine and parallel to the fold. (A pre-tied knot 

in the free end of the twine will prevent the twine from slipping under the staple 

during use.) Once the pads and the twine have been attached, securely 

connect the free ends of both lengths of twine to a small loop tied at the end 

of a 5-foot-long piece of twine. The resulting assembly resembles the letter Y, 

with a long vertical stem and two diagonal branches of different lengths with 

a gauze pad securely attached to the end of each branch. Wrap the twine 

around each two-pad DS sampler to produce a small bundle. Autoclave the 

assembled DS sampler bundle and transfer it with sterile forceps or other 

aseptic method to a resealable sterile bag. Aseptically add 15 mL of doublestrength skim milk to the bag and massage the milk into the gauze pads. Seal 

the bags and store at −20 o

C. 

(ii) Procedures and applications for DS samplers. DS samplers shall be 

completely thawed prior to use. Complete pad/twine/fastener assemblies shall 

be used to sample floor litter surfaces; nest box surfaces may be sampled 

using 3- by 3-inch sterile gauze pads impregnated with double-strength skim 

milk in the manner described in paragraph (a)(3)(i) of this section. In either 

instance, the Plan participant collecting the samples shall wear a fresh pair of 

disposable sterile gloves for each flock or house sampled. Each sampler bag 

shall be marked with the type of sample (floor litter or nest box surface) and 

the identity of the house or flock from which the sample was taken. 

(iii) Floor litter sampling technique. For flocks with fewer than 500 breeders, 

at least one DS set (two DS pads) shall be dragged across the floor litter 

surface for a minimum of 15 minutes. For flocks with 500 or more breeders, 

a minimum of two DS sets (four DS pads) shall be dragged across the floor 

litter surface for a minimum of 15 minutes per DS set. Upon completion of 

dragging, lower each DS pad by its attached twine into a separate, resealable 

sterile bag. Alternatively, each DS set of two pads may be may be lowered by 

its attached twine into the storage/transport bag from which the DS set was 

originally taken. Remove the twine from the pad or DS set by grasping the 

pad or DS set through the sides of the bag with one hand while pulling on the 

twine with the other hand until the connection is broken. Seal the bags and 

promptly refrigerate them to between 2 and 4 o

C. Do not freeze. Discard the 

twine in an appropriate disposal bag.

(iv) Nest box or egg belt sampling technique. Collect nest-box or egg belt 

samples by using two 3- by 3-inch sterile gauze pads premoistened with 

double-strength skim milk and wiping the pads over assorted locations in 

about 10 percent of the total nesting area or the egg belt. Upon completion, 

place each pad in a separate, resealable sterile bag. Seal the bags and 

promptly refrigerate them to between 2 and 4 o

C. Do not freeze.

(v) Culturing of litter surface and nest box samples. When refrigerated to between 

2 and 4 oC, pads impregnated with double-strength skim milk may be stored 

or batched for 5 to 7 days prior to culturing. Pads shipped singly or paired in a 
119

single bag shall not be pooled for culturing but shall be separately inoculated 

into 60 mL of selective enrichment broth.

(4) Chick box papers. Samples from chick box papers may be bacteriologically 

examined for the presence of Salmonella. The Plan participant may collect the 

samples in accordance with paragraph (a)(4)(i) of this section or submit chick 

box papers directly to a laboratory in accordance with paragraph (a)(4)(ii) of this 

section. It is important that the paper be removed from the chick box before the 

box is placed in the brooding house.

(i) Instructions for collecting samples from chick box papers:

(A) Collect 1 chick box paper for each 10 boxes of chicks placed in a house 

and lay the papers on a clean surface.

(B) Clean your hands and put on latex gloves. Do not apply disinfectant to the 

gloves. Change gloves after collecting samples from 10 chick box papers 

or any time a glove is torn.

(C) Saturate a sterile 3-by-3 inch gauze pad with double-strength skim milk 

(see footnote 12 to this section) and rub the pad across the surface of five 

chick box papers. Rub the pad over at least 75 percent of each paper and 

use sufficient pressure to rub any dry meconium off the paper. Pouring a 

small amount of double-strength skim milk (1 to 2 tablespoons) on each 

paper will make it easier to collect samples.

(D) After collecting samples from 10 chick box papers, place the two gauze 

pads used to collect the samples (i.e., one pad per 5 chick box papers) 

into an 18 oz. Whirl-Pak bag and add 1 to 2 tablespoons of doublestrength skim milk.

(E) Promptly refrigerate the Whirl-Pak bags containing the samples and 

transport them, on ice or otherwise refrigerated, to a laboratory within 

48 hours of collection. The samples may be frozen for longer storage if 

the Plan participant is unable to transport them to a laboratory within 48 

hours.

(ii) The Plan participant may send chick box papers directly to a laboratory, where 

samples may be collected as described in paragraph (a)(4)(i) of this section. 

To send chick box papers directly to a laboratory:

(A) Collect 1 chick box paper for each 10 boxes of chicks placed in a house 

and place the chick papers immediately into large plastic bags and seal 

the bags.

(B) Place the plastic bags containing the chick box papers in a clean box and 

transport them within 48 hours to a laboratory. The plastic bags do not 

require refrigeration.

(iii) The laboratory must follow the procedure set forth in paragraph (a)(5) of this 

section for testing chick meconium for Salmonella.

(5) Chick meconium testing procedure for Salmonella. 

(i) Record the date, source, and flock destination on the "Meconium Worksheet."

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

(iii) 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.
120

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

(v) Streak the enriched sample onto brilliant green novobiocin (BGN) agar and 

xylose lysine-tergitol 4 (XLT4) agar.

(vi) Incubate both plates at 37 °C for 24 hours and process suspect Salmonella 

colonies according to paragraph (b) of this section. 

Either of the two enrichment procedures or the rapid detection method in this 

paragraph may be used.

(1) Tetrathionate enrichment with delayed secondary enrichment (DSE):

(i) Add tetrathionate enrichment broth to the sample to give a 1:10 (sample to 

enrichment) ratio. Incubate the sample at 37 or 41.5 oC for 20 to 24 hours as 

shown in illustration 2.

(ii) After selective enrichment, inoculate selective plates (such as BGN and 

XLT4). Incubate the plates at 37 o

C for 20 to 24 hours. Inoculate three to five 

Salmonella-suspect colonies from the plates into triple sugar iron (TSI) and 

lysine iron agar (LIA) slants. Incubate the slants at 37 oC for 20 to 24 hours. 

Screen colonies by serological (i.e., serogroup) and biochemical (e.g., API) 

procedures as shown in illustration 2. As a supplement to screening three 

to five Salmonella-suspect colonies on TSI and LIA slants, a group D colony 

lift assay may be utilized to signal the presence of hard-to-detect group D 

Salmonella colonies on agar plates.

(iii) If the initial selective enrichment is negative for Salmonella, use a DSE 

procedure. Leave the original tetrathionate-enriched sample at room 

temperature for 5 to 7 days. Transfer 1 mL of the culture into 10mL of fresh 

tetrathionate enrichment broth, incubate at 37 oC for 20 to 24 hours, and plate 

as in paragraph (b)(1)(ii) of this section.

(iv) Serogroup all isolates identified as Salmonella and serotype all serogroup D 

isolates. Phage-type all Salmonella enteritidis isolates.

(2) Pre-enrichment followed by selective enrichment. (See illustration 2.)

(3) Approved rapid detection method. After selective enrichment, using a PCRbased assay approved by the NPIP under § 145.15, a rapid ruthenium-labeled 

Salmonella sandwich immunoassay may be used to determine the presence 

of Salmonella. Positive samples from the immunoassay are then inoculated to 

selective plates (such as BGN and XLT4). Incubate the plates at 37 °C for 20 

to 24 hours. Inoculate three to five Salmonella-suspect colonies fromthe plates 

into triple sugar iron (TSI) and lysine iron agar (LIA) slants. Incubate theslants 

at 37 °C for 20 to 24 hours. Screen colonies by serological (i.e., serogroup)and 

biochemical (e.g., API) procedures as shown in illustration 2. As a supplementto 

screening three to five Salmonella-suspect colonies on TSI and LIA slants, agroup 

D colony lift assay may be utilized to signal the presence of hard-to-detect group D 

Salmonella colonies on agar plates. 

(b) Isolation and 

identification of 

Salmonella
121

(1) Environmental Samples. Fecal material, litter, or dust to be submitted for 

bacteriological examination should be collected in accordance with the procedures 

described in paragraphs (c)(1)(i) or (c)(1)(ii) of this section:

(i) Procedure for sampling in broth. Authorized laboratories will provide capped 

tubes 1–2 cm in diameter and 15–20 cm in length which are two-thirds full 

of a recently made, refrigerated, sterile enrichment broth (Selenite Brilliant 

Green Sulfapyridine or Tetrathionate Brilliant Green) for each sample. 

Sufficient tubes should be taken to the premises to provide at least one tube 

per pen or one tube per 500 birds, whichever is greater. At least one sterile, 

cotton-tipped applicator will be needed for each tube. The dry applicator is 

first placed or drawn through fresh manure (under roost, near water troughs, 

cecal droppings, or diarrhetic droppings). After this and each subsequent 

streaking, the cotton-tipped applicator is placed in the tube of broth and 

swirled to remove the collected material. The applicator is then withdrawn 

and is used for taking additional specimens by streaking on or through areas 

where defecation, trampling of feces, or settling of dust are common; i.e., on 

or near waterers, feeders, nests, or rafters, etc. When the volume of material 

collected equals approximately 10 percent of the volume of the broth (usually 

10–12 streakings), the applicator is placed in the tube and the stick is broken 

in half. The lower or cotton-tipped half is left in the broth, and the upper half is 

retained for future disposal. The cap is then replaced on the inoculated tube, 

and the sampling procedure is continued in other areas of the pen.

(ii) Procedure for sampling in dry containers. A sample of fecal material, litter, 

or dust is placed in a sterile, sealable container. The sample shall consist of 

several specimens of material taken from a representative location in the pen 

or house. At least 10 g (approximately a heaping tablespoonful) of material 

shall be collected for each sample. The specimens in each sample shall be 

collected with a sterile tongue depressor or similar uncontaminated instrument. 

The samples should vary in type and consistency. Half of the samples should 

be comprised of material representing defecated matter from a large portion 

of the flock; i.e., trampled, caked material near waterers and feeders. The 

minimum number of samples to be taken shall be determined by the following: 

Five samples from pens or houses of up to 500 birds;

Ten samples from pens or houses of 500 to 2,500 birds;

Fifteen samples from pens or houses with more than 2,500 birds. 

 The composite samples above may be pooled to not less than five samples at 

the laboratory as long as the volume of material collected equals approximately 

10 percent of the volume of the broth.

(c) For turkeys
122

Inoculate TT1

enrichment

broths

(1:10 ratio)

Serogroup

Positive for 

Salmonella

Reevalute4

Serogroup all 

Group D1

ILLUSTRATION 2—Culture procedures for environmental samples, chick papers, or meconium.

Environmental samples, chick papers, or meconium

37 °C 20-24 hours

Inoculate selective plates2

37 °C 20-24 hours

Pre-enrichment media (BPW)(1:10 ratio)

Inoculate TT1

enrichment

broths

(1:10 ratio)

Either MSRV or RV1

enrichment media

(1:100 ratio)

37 or 41.5 °C 20-24 hours

42 °C 20-24 hours

DSE

37 or 41.5 °C 20-24 hours

TSI and LIA reactions: 

Both negative for 

Salmonella

TSI and LI reactions: One 

positive and one negative for 

Salmonella

TSI and LIA reactions: 

Both positive for 

Salmonella

Biochemical

identification Discard3

Positive for 

Salmonella

Negative for 

Salmonella

Discard3 Serogroup

Positive for 

Salmonella

Negative for 

Salmonella

Reevaluate4

Positive for 

Salmonella

Negative for 

Salmonella

Discard3

Biochemical 

identification

Serogroup all 

Group D1

1. Tetrathionate enrichment broth, e.g., Rappaprt-Vassilades (RV) or modified semisolid RV (MSRV)

2. Selective plates such Brilliant Green Novobiocin (BGN) or xylose-lysine tergitol 4 (XLT 4).

3. Reevaluate if epidemiologic, necropsy, or other information indicates the presences of an unusual strain of Salmonella.

4. If biochemical identification and serogroup procedures are inconclusive, restreak original colony onto non-selective 

plating media to check for purity. Repeat biochemical and serology tests.

Inoculate 3 to 5 suspect colonies into TSI and LIA slants

Negative for 

Salmonella
123

(2) Cloacal swabs. Cloacal swabs for bacteriological examination are taken from each 

bird in the flock or from a minimum of 500 birds in accordance with the procedure 

described in paragraph (c)(2)(i) of this section.

(i) Procedure for taking cloacal swabs. The authorized laboratory will provide 

sterile capped tubes or other suitable containers and cotton-tipped applicators 

for use in taking the cloacal swabs. The cotton-tipped applicator is inserted 

into the cloaca and rectum in such a manner as to insure the collection of fecal 

material. The swab and adhering fecal material are then placed in the tube and 

the stick is broken in half, with the upper half retained for future disposal. The 

cloacal swabs may be combined in the sterile tubes in multiples of five or in 

combinations specified by the authorized laboratory.

(ii) [Reserved]

(3) Drag-swabs. Drag-swabs for bacteriological examination should involve the 

exposure of at least six unpooled pads per house to promote representative 

sampling and some element of quantification.

(i) Drag-swab assembly. Assemble drag-swab sampling sets from folded-once 

3-by-3-inch sterile gauze pads secured with paper clips. Bend end wires of 

each paper clip slightly to catch into the swab fabric, thus securing the clips 

to the folded pads. Use two pads, assembled as described to make each 

drag-swab sampling set. Securely connect one pad through the free rounded 

end of the paper clip to a 2-ft (0.6 m) length of size 20 fibrous wrapping twine. 

Similarly connect the other pad to a 1-ft (0.3 m) length of twine. Then securely 

connect the free ends of both lengths of twine to a small loop tied at the end 

of a similar 5-ft length of twine. The resulting assembly resembles the letter 

Y with a 5-ft long vertical stem and two diagonal branches (one 1 ft long and 

the other 2 ft long), with a folded swab securely attached at the end of each 

branch. After assembly, place each two-pad drag-swab sampling set into a 

sterile bag.

(ii) Procedure for taking drag-swab. 

(A) Floor litter: The Plan participants should collect two samples as follows: 

Drag four 3-by-3-inch sterile gauze pads premoistened with double 

strength skim milk9

 over the floor litter surface for 15 min minimally. Place 

the gauze pads used to collect the samples in 18-oz whirl-pack bags, two 

pads per bag with each bag containing 5 ml of double strength skim milk. 

This will maintain the moistness of the sample during transport. Mark the 

bags with the type of sample and the house identification. 

9 Obtain procedure for preparing double strength skim milk from USDA–APHIS 

“Recommended Sample Collection Methods for Environmental Samples,” available 

from the National Poultry Improvement Plan, Veterinary Services, APHIS, USDA, 1498 

Klondike Road, Suite 101, Conyers, GA 30094.
124

(B) Nest-boxes. The Plan participant should collect one nest-box sample 

by using two 3-by-3-inch sterile gauze pads premoistened with double 

strength skim milk. Wipe the two gauze pads used to collect the sample 

over assorted locations of about 10 percent of the total nesting area. Place 

the gauze pads used to collect the sample in an 18-oz whirl-pack bag 

containing 5 ml of double strength skim milk. Mark the bag with the type of 

sample and the house identification. 

(Approved by the Office of Management and Budget under control number 0579-0007)

§ 147.13 Procedure for bacteriological culturing of eggshells for colon 

bacilli organisms. 

Proper precautions to avoid environmental contamination of the samples during 

the collection and laboratory process, and proper handling of the samples following 

collection are essential. Each State Inspector involved in eggshell culture activities 

must receive instruction in the necessary sanitation procedures, sampling procedures, 

and sample handling by the authorized laboratory involved. The Official State Agency 

will maintain a record showing that the required instruction was given to each State 

Inspector.

Forty (40) eggs in the top flats of each of three randomly selected cases of sanitized 

eggs from each flock will be utilized for each sampling.

A 2.5 centimeter diameter circular area of the large end of each of the eggs will be 

rubbed with a sterile swab previously moistened with sterile lactose broth, or other 

suitable liquid media provided by the authorized laboratory. One swab will be used for 

five eggs, and four swabs will be pooled to each sterile, capped tube provided by the 

authorized laboratory.

(1) From the tube containing four swabs and lactose broth or other suitable media, 

1 ml will be transferred to 10 ml lactose in a fermentation tube.

(2) Incubate at 37 °C for 48 hours. The presence of acid, and gas in the amount of 

10 percent or more after 24 and 48 hours of incubation, provides a presumptive 

conclusion of the presence of colon bacilli organisms. 

(Approved by the Office of Management and Budget under control number 0579-0007) 

(a) Sample selection

(b) Swab procedure
125

§ 147.14 Procedures to determine status and effectiveness of 

sanitation monitored program. 

The following monitoring procedures10 may be applied at the discretion of the Official 

State Agency:

(1) Culture the surface of cased eggs periodically for fecal contaminating organisms 

as described in § 147.13.

(2) Culture a sample of dead-in-shell eggs periodically from each breeding flock for 

coliforms. The culture media will be designed to include detection of Salmonella 

species. Such eggs should also be cultured for the dependable recovery of 

Salmonella. 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.), delayed secondary enrichment procedures, and colony lift 

assays detailed in paragraph (a)(5) and illustration 2 of § 147.12. 

§ 147.15 Laboratory procedure recommended for the bacteriological 

examination of mycoplasma reactors.11

Aseptic techniques are very important as some organisms may not be suppressed 

by the antimicrobial agents used in this procedure. Tissue suspensions from large 

volumes are sometimes desirable from the sites listed above and occasionally from 

the oviduct and cloaca. Tissues should be ground or blended completely in 10 times 

their volume of Mycoplasma Broth Medium (MBM). (See paragraph (f) of this section.) 

Specimens submitted to referral laboratories in order of preference for recovery of 

the Mycoplasma organisms are: (1) live birds, (2) refrigerated fresh tissues, (3) tissue 

specimens packed with dry ice.

When evidence of growth is observed (lowered pH or turbidity of broth) transfer each 

broth culture as needed to maintain the original isolates. Incubate tubes at 37 °C 

for at least 21 days before discarding as negative. When growth is first observed or 

if no growth occurs by the 4th or 5th day of incubation, inoculate broth culture onto 

a plate of Mycoplasma Agar Medium (MAM). (See paragraph (g) of this section.) 

Several cultures may be inoculated on one plate by using a wire loop or a cotton swab. 

(b) Inoculate 5–10 ml of 

MBM with a swab, wire 

loop or 0.1 ml of the 

tissue suspension.

(a) Monitor effectiveness 

of sanitation program

(a) Turbinates, trachea, 

air sacs, sinuses, nasal 

passages, respiratory 

exudates, synovial fluid, 

eggs (including yolk, yolk 

sacs, membranes and 

allantoic fluid), should 

be directly sampled with 

sterile swabs.

10“Laboratory Procedures for monitoring operations pproposed here are described in the following 

two publicatons: isolation and identification of Avian Pathogens, American Association of Avian 

Pathoologists, University of Pennsylvania, New Bolton Center, Kenneth Square, Pennsylvania 

1934-1962, 1980, and Culture Methods for the Detection of Animal Salmonellosis and Arizonosis, 

Iowa State Univeristy Press, Ames, Iowa 50010, 1976. 

11Yoder, H.W., Jr., "Mycoplasmosis." In: Isolation and Identification of Avian Pathogens. (Stephen 

B. Hitchner, Chairman, Charles H. Domermuth, H. Graham Purchase, James E. Williams.) 1980, 

pp.40–42, Creative Printing Company, Inc., Endwell, NY 13760. 
126

Incubate plates 3–5 days at 37 °C in a high humidity chamber. If preferred, 5 percent 

CO2 may be added or a candle jar may be used. Tiny circular and translucent colonies 

with elevated centers are very suggestive of mycoplasma. Indirect lighting and a low 

power or dissecting microscope are recommended for observation of the colonies as 

they are rarely more than 0.2–0.3 mm in diameter.

(1) Isolates may be shipped in MBM with ice packs if shipment will be in transit less 

than 2–3 days. Longer shipments require freezing of the MBM with dry ice, or 

shipping MAM slants at room temperature. Isolates must have indications of 

growth before shipment is made.

(2) Isolates may be stored in MBM at –20 °C for 2–3 weeks, or they may be stored at 

–68 °C for several years.

(1) Pour 2–3 ml of MAM into a test tube and tilt the tube until a slant (approximately 

45 °) is obtained. Other containers are acceptable.

(2) Overlay the slant with sufficient MBM, so that the media (including inoculum) 

covers the agar slope.

(3) Inoculate the culture as indicated in paragraph (b) of this section.

(4) Incubate and examine the overlay as indicated in paragraph (b) of this section.

(1) Deionized distilled water suitable for cell culture fluids should be used.

(2) All glassware should be carefully washed with a nonresidue detergent such as 

Alcojet and rinsed three times in tap water and twice in deionized distilled water.13

(3) Thallium acetate in a 10 percent solution is added to an approximate final 

concentration of 1:4000; however, highly contaminated specimens may require 

a final concentration of 1:2000.14 Thallium acetate is added to deionized distilled 

water first, except as noted in paragraph (e)(4) of this section, to prevent the 

precipitation of proteins. 

(4) Mycoplasma Broth Base, dextrose, phenol red, and cysteine hydrochloride 

are added to deionized distilled water first if autoclave sterilization is used.15 

Thallium acetate and then the remaining components are added aseptically after 

cooling the autoclaved media to 45 °C or less. 

(5) Use sterile deionized distilled water to reconstitute penicillin.

(6) Sterile serum should be inactivated by heating at 56 °C for 30 minutes. Swine 

serum may be used for M. gallisepticum, M. synoviae, M. gallinarum, and 

M. meleagridis isolation; however, horse serum is usually recommended for 

M. meleagridis isolation.

12Trade names are used in these procedures solely for the purpose of 

providing specific information. Mention of a trade name does not constitute 

a guarantee or warranty of the product by the U.S. Department of 

Agriculture or an endorsement over other products not mentioned. 

13Alcojet is available from: Alconox, Inc., New York, NY 10003. 14Thallium acetate may be obtained from Fischer Scientific Company. 15Mycoplasma Broth Base may be obtained from: (a) Product #M 33600, 

Gibco Diagnostics, 2801 Industrial Drive, Madison, WI 53711. (b) Product 

#3900-3212, Scott Laboratories, Inc., 8 Westchester Plaza, Elmsford, NY 

10523. 

(d) Alternate method 

of culture: An overlay 

enrichment culture for 

fastidious and sensitive 

mycoplasma, especially 

for M. meleagridis

should be included.

(e) Preparation of media 

components:12

(c) Isolates must be 

serotyped.
127

(7) Phenol red should be prepared as a 1 percent solution.

(8) NAD (beta nicotinamide adenine dinucleotide or coenzyme I) should be prepared 

as a 1 percent solution.16 

(9) Cysteine hydrochloride, prepared as a 1 percent solution, is used to reduce the 

NAD for M. synoviae growth.

(10) A purified agar product such as Nobel (Special agar) is used in the MAM.17 

(11) Adjust the pH with NaOH. 

(12) Sterilization may be accomplished by two methods:

(i) Filtration sterilization through a 0.20 micron filter is the recommended method. 

Aseptic techniques must be utilized.

(ii) Autoclave sterilization at 120 °C, 15 pounds pressure (103 kPa), for 15 

minutes may be used, if preferred, when following the procedure described in 

paragraph (e)(4) of this section.

(13) Phenol red, dextrose, and NAD may be omitted when culturing for M. meleagridis 

 and M. gallinarum.

(14) When culturing for M. meleagridis from contaminated samples include 

 100 units/ml of Polymyxin B in MBM. 

To 850–880 ml of deionized distilled water;

Add: 

Thallium acetate (ml) .................................... 2.5 (1:4000) 

Potentially contaminated samples (ml)............................................ 5.0 (1:2000) 

Mycoplasma Broth Base (g) .......................................................... 22.5 

Aqueous penicillin (units)...................................................... 500,000 

Sterile serum (ml)............................................................. 120 to 150.0 

Phenol red plus (ml) ........................................................................ 2.5 

NAD (ml)........................................................................................ 12.5 

Cysteine hydrochloride (ml)........................................................... 12.5 

Dextrose (g)..................................................................................... 1.0–1.5 

Adjust pH to 7.8 

Filter sterilize 

(1) Broth may be stored at 4 °C for at least 2 weeks or at –40 °C for longer periods.

(f) Mycoplasma Broth 

Medium (Frey) is 

prepared as follows:

16NAD Grade III may be obtained from: Sigma Chemical Company, P.O. Box 14508, St. 

Louis, MO 63178. 

17Noble Agar may be obtained from: Difco Laboratories, Box 1058-A, Detroit, MI 48201. 
128

To 850–880 ml of deionized distilled water;

Add: 

Mycoplasma Broth Base (g) .......................................................... 22.5 

Adjust pH to 7.8 

Purified agar (g)............................................................................. 12.0 

Autoclave and cool in 45 °C water bath 

Thallium acetate (ml)....................................................................... 2.0 (1:4000) 

Sterile serum at 45 °C (ml).......................................................... 150.0 

Aqueous penicillin (units)...................................................... 400,000 

NAD (ml)........................................................................................ 12.5 

Cysteine hydrochloride (ml)........................................................... 12.5 

(1) Rotate flask gently and pour about 15 ml of media into each petri dish.

(2) Stack petri dishes only 2–3 high in a 37 °C incubator up to 2 hours to remove 

excess moisture.

(3) Wrap inverted plates in sealed bundles and store at 4 °C for not more than 15 

days.

A known series of titrations from a single culture should be made on both new and 

old media. The media should be compared on the basis of growth, colony size, and 

numbers of colonies which develop.18

§ 147.16 Procedure for the evaluation of mycoplasma reactors by in vivo 

bio-assay (enrichment). 

This procedure has been shown to be sensitive enough to detect less than 

100 Mycoplasma organisms under proper conditions.19 Proper conditions are defined 

in this section. 

(a) Obtain chickens or turkeys (test birds) which are at least 3 weeks of age and are 

free of M. gallisepticum, M. synoviae, and M. meleagridis and transport them in a 

manner to prevent their being contaminated by any infectious avian disease.

(1) Maintain test birds in an area that has been effectively cleaned and disinfected.

(2) The area should be isolated from other birds or animals.

(3) Personnel caring for the test birds should take the necessary precautions (see 

§ 147.26(b)) to prevent the mechanical transfer of infectious avian diseases 

from other sources.

(g) Mycoplasma Agar 

Medium (Frey) is 

prepared as follows:

(h) New component 

or media batches 

should be monitored 

to compensate for 

changes in formulation 

due to alterations of 

purity, concentration, 

preparation, etc.

18“Laboratory Procedures and Medium For The Isolation Of Mycoplasma From Clinical Materials.” 

Laboratory Diagnosis of Mycoplasma in Food Animals, Proceedings of Nineteenth Annual Meeting, 

The American Association of Veterinary Laboratory Diagnosticians, 1976, pp. 106–115, AAVLD, 

6101 Mineral Point Road, Madison, WI 53705. 

19Research results are described in the following two publications: (a) Bigland, C. H. and A. J. 

DaMassa, “A Bio-Assay for Mycoplasma gallisepticum.” In: United States Livestock Sanitary 

Association Proceedings, 67th, 1963, pp. 541–549. (b) McMartin, D. A., “Mycoplasma gallisepticum

in the Respiratory Tract of the Fowl.” In: The Veterinary Record, September 23, 1967, pp. 317–320. 
129

(b) Test birds to be used for inoculation with contaminated tissues should be 

serologically negative by the serum plate agglutination test.

(1) Inoculated test birds should be isolated from non-inoculated control birds for 

the length of any experiment.

(c) Aseptically obtain tracheal, turbinate, and sinus mucosa, lung and sinus exudates, 

cervical, thoracic, and abdominal airsac tissues (including lesions), and portions 

of oviduct and synovial fluid from at least four suspect, donor birds. In a sterile 

device, blend the tissues completely in four times their volume of Mycoplasma 

Broth Medium (Frey), (see § 147.15(f)). Suspensions may be made from tissue 

pools. Inoculate test birds within 30 minutes of preparation of suspensions.

(1) Inoculate at least four test birds for each suspension pool via the abdominal air 

sac and infraorbital sinus, with up to 1/2 ml of inoculum per site.

(2) Test birds should be bled every 7 days for 35 days to identify sero-converters.

(3) At 35 days, test birds should be sacrificed and bacteriologic isolation and 

identification of mycoplasma attempted (see § 147.15). Note especially the 

sites of inoculation for typical gross or microscopic mycoplasma lesions.

(d) Donor birds are considered infected when:

(1) Test birds have serum plate antibodies for the mycoplasma for which the 

donor birds were tested, regardless of HI test results, and control birds stay 

serologically negative; or

(2) Mycoplasma organisms are isolated from the test birds and serotyped positive 

for the mycoplasma for which the donor birds were tested, and control birds 

stay serologically and culturally negative.

(e) Laboratory findings may be verified by direct cultures of material from sick birds or 

by inoculating seronegative birds from the suspect flock and comparing serological 

findings with those from the test birds. 

§ 147.17 Laboratory procedure recommended for the bacteriological 

examination of cull chicks and poults for salmonella.

The laboratory procedure described in this section is recommended for the 

bacteriological examination of cull chicks from egg-type and meat-type chicken flocks

and waterfowl, exhibition poultry, and game bird flocks and poults from turkey flocks for

 salmonella.

(a) For cull chicks, from 25 randomly selected 1- to 5-day-old chicks that have not 

been placed in a brooding house, prepare 5 organ pools, 5 yolk pools, and 5 

intestinal tissue pools as follows. For poults, from a sample of 10 poults that died 

within 10 days after hatching, prepare organ pools, yolk pools, and intestinal pools 

as follows:

(1) Organ pool: From each of five chicks or two poults, composite and mince 1- to 

2-gram samples of heart, lung, liver, and spleen tissues. Include the proximal 

wall of the bursa of Fabricius for chicks only.

(2) Yolk pool: From each of five chicks or two poults, composite and mince 1- to 

 2-gram samples of the unabsorbed yolk sac or, if the yolk sac is essentially 

absent, the entire yolk stalk remnant.
130

(3) Intestinal pool: From each of five chicks or two poults, composite and mince 

approximately 0.5 cm2 sections of the crop wall and 5-mm-long sections of the 

duodenum, cecum, and ileocecal junction. 

(b) Transfer each pool to tetrathionate selective enrichment broth (Hajna or MuellerKauffmann) at a ratio of 1 part tissue pool to 10 parts broth.

(c) For cull chicks, repeat the steps in paragraphs (a) and (b) of this section for each 

5-chick group until all 25 chicks have been examined, producing a total of 15 pools 

(5 organ, 5 yolk, and 5 intestinal). For poults, repeat the steps in paragraphs (a) 

and (b) of this section for each two-poult group until all the poults in the sample 

have been examined.

(d) Culture the tetrathionate pools as outlined for selective enrichment in illustration 

2 of § 147.12. Incubate the organ and yolk pools for 24 hours at 37 o

C and the 

intestinal pools at 41.5 oC. Plate as described in illustration 2 of § 147.12 and 

examine after both 24 and 48 hours of incubation. Confirm suspect colonies 

as described. Further culture all salmonella-negative tetrathionate broths by 

delayed secondary enrichment procedures described for environmental, organ, 

and intestinal samples in illustration 2 of § 147.12. A colony lift assay may also be 

utilized as a supplement to TSI and LI agar picks of suspect colonies.
131

Subpart C—Sanitation Procedures

§ 147.21 Flock sanitation. 

To aid in the maintenance of healthy flocks, the following procedures should be 

practiced:

(a) Baby poultry should be started in a clean brooder house and maintained in 

constant isolation from older birds and other animals. Personnel that are in 

contact with older birds and other animals should take precautions, including 

disinfection of footwear and change of outer clothing, to prevent the introduction of 

infection through droppings that may adhere to the shoes, clothing, or hands (see 

§ 147.24(a)).

(b) Range used for growing young stock should not have been used for poultry the 

preceding year. Where broods of different ages must be kept on the same farm, 

there should be complete depopulation of brooder houses and other premises 

following infection of such premises by any contagious disease.

(c) Poultry houses should be screened and proofed against free-flying birds. An active 

rodent eradication campaign is an essential part of the general sanitation program. 

The area adjacent to the poultry house should be kept free from accumulated 

manure, rubbish, and unnecessary equipment. Dogs, cats, sheep, cattle, horses, 

and swine should never have access to poultry operations. Visitors should not be 

admitted to poultry areas, and authorized personnel should take the necessary 

precautions to prevent the introduction of disease.

(d) Poultry houses and equipment should be thoroughly cleaned and disinfected prior 

to use for a new lot of birds (see § 147.24(a)). Feed and water containers should 

be situated where they cannot be contaminated by droppings and should be 

frequently cleaned and disinfected. Dropping boards or pits should be constructed 

so birds do not have access to the droppings.

(e) Replacement breeders shall be housed at the proper density consistent with the 

type of building and locality and which will allow the litter to be maintained in a 

dry condition. Frequent stirring of the litter may be necessary to reduce excess 

moisture and prevent surface accumulation of droppings. Slat or wire floors should 

be constructed so as to permit free passage of droppings and to prevent the birds 

from coming in contact with the droppings. Nesting areas should be kept clean 

and, where appropriate, filled with clean nesting material.

(f) When an outbreak of disease occurs in a flock, dead or sick birds should be 

taken, by private carrier, to a diagnostic laboratory for complete examination. All 

Salmonella cultures isolated should be typed serologically, and complete records 

maintained by the laboratory as to types recovered from each flock within an 

area. Records on isolations and serological types should be made available to 

Official State Agencies or other animal disease control regulatory agencies in the 

respective States for followup of foci of infection. Such information is necessary for 

the development of an effective Salmonella control program.

(g) Introduction of started or mature birds should be avoided to reduce the possible 

hazard of introducing infectious diseases. If birds are to be introduced, the health 

status of both the flock and introduced birds should be evaluated.
132

(h) In rearing broiler or replacement stock, a sound and adequate immunization 

program should be adopted. Since different geographic areas may require certain 

specific recommendations, the program recommended by the State experiment 

station or other State agencies should be followed.

(i) Feed, pelleted by heat process, should be fed to all age groups. Proper 

feed pelleting procedures can destroy many disease producing organisms 

contaminating feedstuffs. 

(Approved by the Office of Management and Budget under control number 0579-0007) 

§ 147.22 Hatching egg sanitation. 

Hatching eggs should be collected from the nests at frequent intervals and, to aid 

in the prevention of contamination with disease-causing organisms, the following 

practices should be observed:

(a) Cleaned and disinfected containers, such as egg flats should be used in collecting 

the nest eggs for hatching. Egg handlers should thoroughly wash their hands with 

soap and water prior to and after egg collection. Clean outer garments should be 

worn.

(b) Dirty eggs should not be used for hatching purposes and should be collected in 

a separate container from the nest eggs. Slightly soiled eggs may be gently dry 

cleaned by hand. 

(c) Hatching eggs should be stored in a designated egg room under conditions that 

will minimize egg sweating. The egg room walls, ceiling, floor, door, heater, and 

humidifier should be cleaned and disinfected after every egg pickup. Cleaning and 

disinfection procedures should be as outlined in § 147.24.

(d) The egg processing area should be cleaned and disinfected daily.

(e) Effective rodent and insect control programs should be implemented.

(f) The egg processing building or area should be designed, located, and constructed 

of such materials as to assure that proper egg sanitation procedures can be 

carried out, and that the building itself can be easily, effectively, and routinely 

sanitized.

(g) All vehicles used for transporting eggs or chicks/poults should be cleaned and 

disinfected after use. Cleaning and disinfection procedures should be as outlined 

in § 147.24.
133

§ 147.23 Hatchery sanitation. 

An effective program for the prevention and control of Salmonella and other infections 

should include the following measures:

(a) An effective hatchery sanitation program should be designed and implemented.

(b) The hatchery building should be arranged so that separate rooms are provided for 

each of the four operations: Egg receiving, incubation and hatching, chick/poult 

processing, and egg tray and hatching basket washing. Traffic and airflow patterns 

in the hatchery should be from clean areas to dirty areas (i.e., from egg room to 

chick/poult processing rooms) and should avoid tracking from dirty areas back into 

clean areas.

(c) The hatchery rooms, and tables, racks, and other equipment in them should be 

thoroughly cleaned and disinfected frequently. All hatchery wastes and offal 

should be burned or otherwise properly disposed of, and the containers used to 

remove such materials should be cleaned and sanitized after each use.

(d) The hatching compartments of incubators, including the hatching trays, should be 

thoroughly cleaned and disinfected after each hatch.

(e) Only clean eggs should be used for hatching purposes.

(f) Only new or cleaned and disinfected egg cases should be used for transportation 

of hatching eggs. Soiled egg case fillers should be destroyed.

(g) Day-old chicks, poults, or other newly hatched poultry should be distributed 

in clean, new boxes and new chick papers. All crates and vehicles used for 

transporting birds should be cleaned and disinfected after each use.

§ 147.24 Cleaning and disinfecting. 

The following procedures are recommended:

(1) Remove all live "escaped" and dead birds from the building. Blow dust from 

equipment and other exposed surfaces. Empty the residual feed from the feed 

system and feed pans and remove it from the building. Disassemble feeding 

equipment and dump and scrape as needed to remove any and all feed cake and 

residue. Clean up spilled feed around the tank and clean out the tank. Rinse 

down and wash out the inside of the feed tank to decontaminate the surfaces and 

allow to dry. 

(2) Remove all litter and droppings to an isolated area where there is no opportunity 

for dissemination of any infectious disease organisms that may be present. 

Housing where poultry infected with a mycoplasmal disease were kept should 

remain closed for 7 days before removal of the litter.

(3) Wash down the entire inside surfaces of the building and all the installed equipment 

such as curtains, ventilation ducts and openings, fans, fan housings and shutters, 

feeding equipment, watering equipment, etc. Use high pressure and high volume 

water spray (for example 200 pounds per square inch and 10 gallons per minute 

or more) to soak into and remove the dirt to decontaminate the building. Scrub the 

walls, floors, and equipment with a hot soapy water solution. Rinse to remove soap.

(a) In the poultry 

houses:
134

(4) Spray with a disinfectant which is registered by the Environmental Protection 

Agency as germicidal, fungicidal, pseudomonocidal, and tuberculocidal, in 

accordance with the specifications for use, as shown on the label of such 

disinfectant.

(1) Use cleaning agents and sanitizers that are registered by the U.S. Environmental 

Protection Agency as germicidal, fungicidal, pseudomonocidal, and 

tuberculocidal. Use manufacturer's recommended dilution. Remove loose 

organic debris by sweeping, scraping, vacuuming, brushing, or scrubbing, or by 

hosing surface with high pressure water (for example 200 pounds per square 

inch and 10 gallons per minute or more). Remove trays and all controls and fans 

for separate cleaning. Use hot water (minimum water temperature of 140 o

F) 

for cleaning hatching trays and chick separator equipment. Thoroughly wet the 

ceiling, walls, and floors with a stream of water, then scrub with a hard bristle 

brush. Use a cleaner/sanitizer that can penetrate protein and fatty deposits. Allow 

the chemical to cling to treated surfaces at least 10 minutes before rinsing off. 

Manually scrub any remaining deposits of organic material until they are removed. 

Rinse until there is no longer any deposit on the walls, particularly near the fan 

opening, and apply disinfectant. Use a clean and sanitized squeegee to remove 

excess water, working down from ceilings to walls to floors and being careful not 

to recontaminate cleaned areas.

(2) Replace the cleaned fans and controls. Replace the trays, preferably still wet from 

cleaning, and bring the incubator to normal operating temperature.

(3) The hatcher should be fumigated (see § 147.25) or otherwise disinfected prior to 

the transfer of the eggs.

(4) If the same machine is used for incubating and hatching, the entire machine 

should be cleaned after each hatch. A vacuum cleaner should be used to remove 

dust and down from the egg trays; then the entire machine should be vacuumed, 

mopped, and fumigated (see § 147.25) or otherwise sanitized. 

(1) Spray truck tires thoroughly with disinfectant before leaving the main road and 

entering the farm driveway.

(2) Put on sturdy, disposable plastic boots or clean rubber boots before getting out of 

the truck cab. Put on a clean smock or coveralls and a hairnet before entering the 

poultry house.

(3) After loading eggs or unloading chicks/poults, remove the dirty smock/coveralls 

and place into plastic garbage bag before loading in the truck. Be sure to keep 

clean coveralls separate from dirty ones.

(4) Reenter the cab of the truck and remove boots before placing feet onto 

floorboards. Remove hairnet and leave with disposable boots on farm.

(5) Sanitize hands using appropriate hand sanitizer.

(6) Return to the hatchery or go to the next farm and repeat the process. 

(b) In the hatchers and 

hatchery rooms:

(c) The egg and chick/

poult delivery truck 

drivers and helpers 

should use the following 

good biosecurity 

practices while picking 

up eggs or delivering 

chicks/poults:
135

§ 147.25 Fumigation. 

Fumigation may be used for sanitizing eggs and hatchery equipment or rooms as a 

part of a sanitation program. APHIS disclaims any liability in the use of formaldehyde 

for failure on the part of the user to adhere to the Occupational Safety and Health 

Administration (OSHA) standards for formaldehyde fumigation, published in the Dec. 4, 

1987, Federal Register (52 FR 46168, Docket Nos. H-225, 225A, and 225B). 

§ 147.26 Procedures for establishing isolation and maintaining 

sanitation and good management practices for the control of

Salmonella and Mycoplasma infections. 

(1) Allow no visitors except under controlled conditions to minimize the introduction of 

Salmonella and Mycoplasma. Such conditions must be approved by the Official 

State Agency and the Service;

(2) Maintain breeder flocks on farms free from market birds and other domesticated 

fowl. Follow proper isolation procedures as approved by the Official State Agency;

(3) Dispose of all dead birds by locally approved methods.

(1) Avoid the introduction of Salmonella, Mycoplasma gallisepticum, or 

Mycoplasma synoviae infected poultry;

(2) Prevent indirect transmission from outside sources through contaminated 

equipment, footwear, clothing, vehicles, or other mechanical means;

(3) Provide adequate isolation of breeder flocks to avoid airborne transmission from 

infected flocks;

(4) Minimize contact of breeder flocks with free-flying birds;

(5) Establish a rodent control program to keep the rodent population and other pests 

under control;

(6) Tailor vaccination programs to needs of farm and area;

(7) Clean and disinfect equipment after each use;

(8) Provide clean footwear and provide an adequate security program;

(9) Clean and disinfect houses before introducing a new flock;

(10) Use clean, dry litter free of mold;

(11) Keep accurate records of death losses;

(12) Seek services of veterinary diagnostician if unaccountable mortality or signs of 

disease occur;

(13) Adopt and maintain a clean-egg program; and

(14) Use only crates and vehicles that have been cleaned and disinfected in 

accordance with the provisions of § 147.24(a) to haul live poultry to and from the 

premises. 

(Approved by the Office of Management and Budget under control number 0579-0007) 

(a) The following 

procedures are required 

for participation under 

the U.S. Sanitation 

Monitored, U.S. M. 

Gallisepticum Clean, U.S. 

M. Synoviae Clean, U.S. 

S. Enteritidis Monitored, 

and U.S. 

S. Enteritidis Clean 

classifications:

(b) Recommended 

procedures:
136

§ 147.27 Procedures recommended to prevent the spread of disease by 

artificial insemination of turkeys. 

(a) The vehicle transporting the insemination crew should be left as far as practical 

from the turkey pens.

(b) The personnel of the insemination crew should observe personal cleanliness, 

including the following sanitary procedures:

(1) Outer clothing should be changed between visits to different premises so 

that clean clothing is worn upon entering each premises. The used apparel 

should be kept separate until laundered. This also applies to gloves worn while 

handling turkeys;

(2) Boots or footwear should be cleaned and disinfected between visits to different 

premises;

(3) Disposable caps should be provided and discarded after use on each 

premises.

(c) The use of individual straw or similar technique is highly recommended. 

Insemination equipment which is to be reused should be cleaned and disinfected 

before reusing. Equipment used for the convenience of the workers should not be 

moved from premises to premises.

(d) No obviously diseased flock should be inseminated. If evidence of active disease is 

noted after insemination is begun, operations should be stopped and the hatchery 

notified.

(e) Care should be taken during the collection of semen to prevent fecal 

contamination. If fecal material is present, it should be removed before the semen 

is collected. Likewise, care should be taken not to introduce fecal material into the 

oviduct of the hen.
137

Subpart D— Molecular Examination Procedures

§ 147.30 Laboratory procedure recommended for the polymerase chain 

reaction (PCR) test for Mycoplasma gallisepticum and M. synoviae.

 

Isolate DNA from 1 mL of eluate from tracheal swabs in PBS or 1 mL of broth culture

by a non-phenolic procedure. Centrifuge samples at 14,000 x g for 5 to 10 minutes. 

Decant supernatant and wash the pellet with 1 mL of PBS. Centrifuge as above and 

re-suspend the pellet in 25-50 μl of 0.1 percent DEP (Diethyl Pyrocarbonate; Sigma) 

water. Boil at 120 °C for 10 minutes followed by 10 minutes incubation at 

4 °C. Centrifuge as above and transfer the supernatant DNA to a nuclease-free tube. 

Estimate the DNA concentration and purity by spectrophotometric reading at 260 nm 

and 280 nm.

 (1) M. gallisepticum. The primer for M. gallisepticum should consist of the following 

sequences:

MG–F 5’ GAG CTA ATC TGT AAA GTT GGT C

MG–R 5’ GCT TCC TTG CGG TTA GCA AC

(2) M. synoviae. The primer for M. synoviae should consist of the following sequences:

MS–F 5’ GAG AAG CAA AAT AGT GAT ATC A

MS–R 5’ CAG TCG TCT CCG AAG TTA ACA A

 (1) Treat each sample (100 to 2000 ng/5 μl) with one of the following 45 μl PCR 

cocktails:

(i) 5 μl 10x PCR buffer, 1 μl dNTP (10 mM), 1 μl of Reverse primer (50 μM), 1 μl 

of Forward primer (50μM), 4 μl MgCl2

 (25 mM), 1 μl taq-polymerase (5 U), 32 

μl DEP water.

(ii) 18 μl water, 25μl PCR mix (Promega), 1 μl Reverse primer (50 μM), 1 μl 

Forward primer (50 μM).

(2) Perform DNA amplification in a Perkin-Elmer 9600 thermocycler or in a Hybaid 

PCR Express thermocycler.20 The optimized PCR program is as follows:

 

Temperature (°C) Duration Cycles

 94 30 seconds 30–40

 55 30 seconds 30–40

 72 1 minute 30–40

 72 5 minutes 1 (final extension)

Mix PCR products (5 to 10 μl) with 2 μl loading buffer (Sigma) and electrophorese on 

a 2 percent agarose gel containing 0.5 μg/mL ethidium bromide in TAE buffer (40 mM 

tris; 2 mM EDTA; pH 8.0 with glacial acetic acid) for 30 minutes at 80 V. 

M. gallisepticum (185 bp) and M. synoviae (214 bp) amplicons can be visualized under 

an ultraviolet transilluminator along with the PCR marker (50 to 2000 bp; Sigma).

(a) DNA isolation.

(b) Primer selection.

(c) Polymerase chain 

reaction

(d) Electrophoresis 

20Trade names are used in these procedures solely for the purpose of providing 

specific information. Mention of a trade name does not constitute a guarantee or 

warranty of the product by the U.S. Department of Agriculture or an endorsement 

over other products not mentioned.
138

§ 147.31 Laboratory procedures recommended for the real-time polymerase chain reaction test for Mycoplasma gallisepticum (MGLP ReTi).

Use Qiagen Qiamp Mini Kit for DNA extraction or equivalent validated technique/procedure. This kit utilizes the following methods: 100 μl of swab suspension incubates 

with 10 μl of proteinase K and 400 μl of lysis buffer at 56 oC for 10 minutes. Following 

incubation, 100 μl of 100 percent ethanol is added to lysate. Wash and centrifuge following extraction kit recommendations.

A forward primer mglpU26 (5'-CTA GAG GGT TGG ACA GTT ATG-3’) located at 

nucleotide positions 765,566 to 765,586 of the M. gallisepticum R strain genome 

sequence; a reverse primer mglp164 (5'-GCT GCA CTA AAT GAT ACG TCA AA-3’) 

located at nucleotide positions 765,448 to 765,470 of the M. gallisepticum R strain genome sequence; and a Taqmandual-labeled probe mglpprobe (5'-FAM-CAG TCA TTA 

ACA ACT TAC CAC CAG AAT CTG-BHQ1-3’) located at nucleotide positions 765,491 

to 765,520 of the M. gallisepticum R strain genome should be used to amplify a 139-

bp fragment of the lp gene. 

Primers and probe should be utilized in a 25 μl reaction containing 12.5 μl of Quantitect Probe PCR 2X mix (Qiagen, Valencia, CA),21 primers to a final concentration of 

0.5 μmolar, and probe to a final concentration of 0.1 μmolar, 1μl of HK-UNG Thermolabile Uracil N-glycosylase (Epicentre, Madison, WI), 2 μl of water, and 5 μl of template. The reaction can be performed in a SmartCycler (Cepheid, Sunnyvale, CA) or 

other equivalent validated platform procedure for real-time thermocycler at 50 °C for 2 

minutes; 95 °C for 15 minutes with optics OFF; and 40 cycles of 94 °C for 15 seconds 

followed by 60 °C for 60 seconds with optics ON.

For each MGLP ReTi assay reaction, the threshold cycle number (CT value) was 

determined to be the PCR cycle number at which the fluorescence of the reaction 

exceeded 30 units of fluorescence. For all samples tested, any MGLP reaction that 

has a recorded CT value was considered positive, while any MGLP reaction that had 

no recorded CT value was considered negative.

 

Proper controls should be used when conducting the MGLP ReTi assay as an official 

test of the Plan. Positive, quantitative, extraction, and internal controls are commercially available from GTCAllison, LLC, Mocksville, NC.

(a) DNA extraction 

(b) Primer selection

(c) MGLP ReTi

(d) Determination of 

positive 

(e) Controls

21 Trade names are used in these procedures solely for the purpose of providing specific information. Mention of a trade name does not constitute a guarantee or warranty of the product by the U.S. Department of 

Agriculture or an endorsement over other products not mentioned.
139

Subpart E—Procedure for Changing National Poultry Improvement Plan

§ 147.41 Definitions. 

Except where the context otherwise requires, for the purposes of this subpart the 

following terms shall be construed, respectively, to mean:

The U.S. Department of Agriculture.

Chickens bred for the primary purpose of producing eggs for human consumption.

Domesticated fowl which are bred for the combined purposes of meat or egg 

production and competitive showing.

Domesticated fowl, such as pheasants, partridge, quail, grouse, and guineas, but not 

doves and pigeons. 

Chickens bred for the primary purpose of producing meat.

A meeting convened for the purpose of recommending changes in the provisions of 

the Plan.

The National Poultry Improvement Plan.

The Animal and Plant Health Inspection Service, Veterinary Services, of the 

Department.

Any State, the District of Columbia, or Puerto Rico.

Domesticated fowl that normally swim, such as ducks and geese.

Department 

Egg-type chickens 

Exhibition poultry 

Game birds 

Meat-type chickens 

Plan Conference 

Plan or NPIP

Service 

State

Waterfowl 
140

§ 147.42 General. 

Changes in this subchapter shall be made in accordance with the procedure described 

in this subpart: Provided, That the Department reserves the right to make changes in 

this subchapter without observance of such procedure when such action is deemed 

necessary in the public interest.

§ 147.43 General Conference Committee. 

A representative of the Animal and Plant Health Inspection Service will serve as 

Executive Secretary and will provide the necessary staff support for the General 

Conference Committee. The General Conference Committee shall consist of one 

member-at-large who is a participant in the National Poultry Improvement Plan and 

one member to be elected, as provided in paragraph (b) of this section, from each of 

the following regions:

(1) North Atlantic: Maine, New Hampshire, Vermont, Massachusetts, Rhode Island, 

Connecticut, New York, New Jersey, and Pennsylvania.

(2) East North Central: Ohio, Indiana, Illinois, Michigan, and Wisconsin.

(3) West North Central: Minnesota, Iowa, Missouri, North Dakota, South Dakota, 

Nebraska, and Kansas.

(4) South Atlantic: Delaware, District of Columbia, Maryland, Virginia, West Virginia, 

North Carolina, South Carolina, Georgia, Florida, and Puerto Rico.

(5) South Central: Kentucky, Tennessee, Alabama, Mississippi, Arkansas, Louisiana, 

Oklahoma, and Texas.

(6) Western: Montana, Idaho, Wyoming, Colorado, New Mexico, Arizona, Utah, 

Nevada, Washington, Oregon, California, Alaska, and Hawaii.

There must be at least two nominees for each position, the voting will be by secret 

ballot, and the results will be recorded. At least one nominee from each region must 

be from an underrepresented group (minorities, women, or persons with disabilities). 

The process for soliciting nominations for regional committee members will include, 

but not be limited to: Advertisements in at least two industry journals, such as the 

newsletters of the American Association of Avian Pathologists, the National Chicken 

Council, the United Egg Producers, and the National Turkey Federation; a Federal 

Register announcement; and special inquiries for nominations from universities or 

colleges with minority/disability enrollments and faculty members in poultry science or 

veterinary science.

(a) The General 

Conference Committee 

Chairperson and the 

Vice Chairperson 

shall be elected by the 

members of the General 

Conference Committee. 

(b) The regional 

committee members 

and their alternates 

will be elected by the 

official delegates of their 

respective regions, and 

the member-at-large will 

be elected by all official 

delegates. 
141

(c) Three regional 

members shall be 

elected at each Plan 

Conference. All members 

shall serve for a period 

of 4 years, subject to 

the continuation of 

the Committee by the 

Secretary of Agriculture, 

and may not succeed 

themselves:

(d) The duties and 

functions of the General 

Conference Committee 

shall be as follows:

Provided, That an alternate member who assumed a Committee member vacancy 

following mid-term would be eligible for re-election to a full term. When there is 

a vacancy for the member-at-large position, the General Conference Committee 

shall make an interim appointment and the appointee shall serve until the next Plan 

Conference at which time an election will be held. If a vacancy occurs due to both a 

regional member and alternate being unable to serve, the vacant position will be filled 

by an election at the earliest regularly scheduled national or regional Plan Conference, 

where members of the affected region have assembled.

(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.

(3) Assist the Department in planning, organizing, and conducting the biennial 

National Poultry Improvement Plan Conference.

(4) Consider each proposal submitted as provided in § 147.44 and make 

recommendations to subpart Committees and the Conference. Meet jointly with 

the NPIP Technical Committee and consider the technical aspects and accuracy of 

each proposal. Recommend whether new proposals (i.e., proposals that have not 

been submitted as provided in § 147.44) should be considered by the delegates to 

the Plan Conference.

(5) During the interim between Plan Conferences, represent the cooperating States in:

(i) Advising the Department with respect to administrative procedures and 

interpretations of the Plan provisions as contained in 9 CFR.

(ii) Assisting the Department in evaluating comments received from interested 

persons concerning proposed amendments to the Plan provisions.

(iii) Recommending to the Secretary of Agriculture any changes in the provisions 

of the Plan as may be necessitated by unforeseen conditions when 

postponement until the next Plan Conference would seriously impair the 

operation of the program. Such recommendations shall remain in effect only 

until confirmed or rejected by the next Plan Conference, or until rescinded by 

the committee.

(6) Serve as an official advisory committee for the study of problems relating to poultry 

health and as the need arises, to make specific recommendations to the Secretary 

of Agriculture concerning ways in which the Department may assist the industry in 

solving these problems. 

(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.
142

§ 147.44 Submitting, compiling, and distributing proposed changes. 

(a) Changes in this subchapter may be proposed by any participant, Official State 

Agency, the Department, or other interested person or industry organization.

(b) Except as provided in § 147.43(d)(2), proposed changes shall be submitted in 

writing so as to reach the Service not later than 150 days prior to the opening date 

of the Plan Conference, and participants in the Plan shall submit their proposed 

changes through their Official State Agency.

(c) The name of the proponent shall be indicated on each proposed change when 

submitted. Each proposal should be accompanied by a brief supporting statement.

(d) The Service will notify all persons on the NPIP mailing lists concerning the dates 

and general procedure of the conference. Hatchery and dealer participants will be 

reminded of their privilege to submit proposed changes and to request copies of 

all the published proposed changes.

(e) The proposed changes, together with the names of the proponents and supporting 

statements, will be compiled by the Service and issued in processed form. When 

two or more similar changes are submitted, the Service will endeavor to unify 

them into one proposal acceptable to each proponent. Copies will be distributed to 

officials of the Official State Agencies cooperating in the NPIP. Additional copies 

will be made available for meeting individual requests. 

§ 147.45 Official delegates. 

Each cooperating State shall be entitled to one official delegate for each of the 

programs prescribed in subparts B, C, D, E, F, G, and H of part 145 of this chapter 

and for each of the programs prescribed in subparts B, C, D, and E of part 146 of 

this chapter in which it has one or more participants at the time of the Conference. 

The official delegates shall be elected by a representative group of participating 

industry members and be certified by the Official State Agency. It is recommended 

but not required that the official delegates be Plan participants. Each official delegate 

shall endeavor to obtain, prior to the Conference, the recommendations of industry 

members of his State with respect to each proposed change. 

§ 147.46 Committee consideration of proposed changes. 

(a) The following committees shall be established to give preliminary consideration to 

the proposed changes falling in their respective fields:

(1) Egg-type breeding chickens.

(2) Meat-type breeding chickens.

(3) Breeding turkeys.

(4) Breeding waterfowl, exhibition poultry, and game birds.

(5) Breeding ostrices, emus, rheas, and cassowaries.

(6) Egg-type commercial chickens.
143

(7) Meat-type commercial chickens.

(8) Meat-type commercial turkeys.

(9) Commercial upland game birds and waterfowl and raised-for-release upland

 game birds and waterfowl.

(b) Each official delegate shall be appointed a voting member in one of the 

committees specified in paragraph (a) of this section.

(c) Since several of the proposals may be interrelated, the committees shall consider 

them as they may relate to others, and feel free to discuss related proposals with 

other committees.

(d) The committees shall make recommendations to the conference as a whole 

concerning each proposal. The committee report shall show any proposed 

change in wording and the record of the vote on each proposal, and suggest 

an effective date for each proposal recommended for adoption. The individual 

committee reports shall be submitted to the chairman of the conference, who will 

combine them into one report showing, in numerical sequence, the committee 

recommendations on each proposal.

(e) The committee meetings shall be open to any interested person. Advocates for or 

against any proposal should feel free to appear before the appropriate committee 

and present their views. 

§ 147.47 Conference consideration of proposed changes. 

(a) The chairperson of the conference shall be a representative of the Department.

(b) At the time designated for voting on proposed changes by the official delegates, 

the chairperson of the General Conference Committee and the four committee 

chairpersons shall sit at the speaker’s table and assist the chairperson of the 

conference.

(c) Each committee chairperson shall present the proposals which his/her committee 

approves or recommends for adoption as follows: “Mr. Chairman. The committee 

for Egg-type chickens recommends the adoption of Proposal No. ——, for the 

following reasons (stating the reasons): I move the adoption of Proposal No. ——.” 

A second will then be called for. If the recommendation is seconded, discussion 

and a formal vote will follow.

(d) Each committee chairman shall present the proposals which his committee does 

not approve as follows: “Mr. Chairperson. The Committee for Egg-type chickens 

does not approve Proposal No. ——.” The chairperson will then ask if any official 

delegate wishes to move for the adoption of the proposal. If moved and seconded, 

the proposal is subject to discussion and voted. If there is no motion for approval, 

or if moved but not seconded, there can be no discussion or vote.
144

(e) Discussion on any motion must be withheld until the motion has been properly 

seconded, except that the delegate making the motion is privileged, if he/she 

desires, to give reasons for his/her motion at the time of making it. To gain the floor 

for a motion or for discussion on a motion, the official delegate in the case of a 

motion, or anyone in case of discussion on a motion, shall rise, address the chair, 

give his/her name and State, and be recognized by the chair before proceeding 

further. While it is proper to accept motions only from official delegates and to limit 

voting only to such delegates, it is, however, equally proper to accept discussion 

from anyone interested. To conserve time, discussion should be pointed and 

limited to the pertinent features of the motion.

(f) Proposals that have not been submitted in accordance with § 147.44 will be 

considered by the Conference only with the unanimous consent of the General 

Conference Committee. Any such proposals must be referred to the appropriate 

committee for consideration before being presented for action by the Conference.

(g) Voting will be by States, and each official delegate, as determined by § 147.45, will 

be allowed one vote on each proposal pertaining to the program prescribed by the 

subpart which he/she represents.

(h) A roll call of States for a recorded vote will be used when requested by a delegate 

or at the discretion of the chairperson.

(i) All motions on proposed changes shall be for adoption.

(j) Proposed changes shall be adopted by a majority vote of the official delegates 

present and voting.

(k) The Conference shall be open to any interested person. 

§ 147.48 Approval of conference recommendations by the Department. 

Proposals adopted by the official delegates will be recommended to the Department

for incorporation into the provisions of the NPIP. The Department reserves the right to

approve or disapprove the recommendations of the Conference as an integral part of

its sponsorship of the National Poultry Improvement Plan.
145

Subpart F—Authorized Laboratories and Approved Tests

§ 147.51 Authorized laboratory minimum requirements.

These minimum requirements are intended to be the basis on which an authorized 

laboratory of the Plan can be evaluated to ensure that official Plan assays are performed and reported as described in this part A satisfactory evaluation will result in 

the laboratory being recognized by the NPIP office of the Service as an authorized 

laboratory qualified to perform the assays provided for in this part.

The laboratory must use a regularly scheduled check test for each assay that it performs. 

The testing procedures at the laboratory must be run or overseen by a laboratory 

technician who has attended and satisfactorily completed Service-approved laboratory 

workshops for Plan-specific diseases within the past 3 years. 

Official Plan assays must be performed and reported as described in this part.

The Official State Agency will conduct a site visit and recordkeeping audit annually. 

Authorized laboratories will be reviewed by the Service (NPIP staff) every 3 years. 

The Service’s review may include, but will not necessarily be limited to, checking records, laboratory protocol, check-test proficiency, technician training, and peer review. 

(1) A memorandum of understanding or other means shall be used to establish 

testing and reporting criteria to the Official State Agency, including criteria that 

provide for reporting H5 and H7 low pathogenic avian influenza directly to the 

Service. 

(2) Salmonella pullorum and Mycoplasma Plan disease reactors must be 

reported to the Official State Agency within 48 hours. 

Random samples may also be required to be submitted for verification as specified by 

the Official State Agency.

(a) Check-test 

proficiency

(b) Trained technicians

(c) Laboratory protocol

(d) State site visit 

(e) Service review 

(g) Verification 

(f) Reporting 
146

§ 147.52 Approved tests.

(a) The procedures for the bacteriological examination of poultry and poultry 

environments described in this part are approved tests for use in the NPIP. In 

addition, all tests that use veterinary biologics (e.g., antiserum and other products

of biological origin) that are licensed or produced by the Service and used as 

described in this part are approved for use in the NPIP.

(b) Diagnostic test kits that are not licensed by the Service (e.g., bacteriological 

culturing kits) may be approved through the following procedure:

(1) The sensitivity of the kit will be estimated in at least three authorized 

 laboratories selected by the Service by testing known positive samples, as 

 determined by the official NPIP procedures found in Subparts A, B, C, and 

 D of this part. If certain conditions or interfering substances are known to 

 affect the performance of the kit, appropriate samples will be included so that 

 the magnitude and significance of the effect(s) can be evaluated.

(2) The specificity of the kit will be estimated in at least three authorized 

 laboratories selected by the Service by testing known negative samples, as 

 determined by the official NPIP procedures found in this part. If certain 

 conditions or interfering substances are known to affect the performance of

 the kit, appropriate samples will be included so that the magnitude and 

 significance of the effect(s) can be evaluated.

(3) The kit will be provided to the cooperating laboratories in its final form and 

 include the instructions for use. The cooperating laboratories must perform 

 the assay exactly as stated in the supplied instructions. Each laboratory must 

 test a panel of at least 25 known positive clinical samples supplied by the

 manufacturer of the test kit. In addition, each laboratory will be asked to 

 test 50 known negative clinical samples obtained from several sources, to 

 provide a representative sampling of the general population. The identity of 

 the samples must be coded so that the cooperating laboratories are blinded to 

 identity and classification. Each sample must be provided in duplicate or 

 triplicate, so that error and repeatability data may be generated.

(4) Cooperating laboratories will submit to the kit manufacturer all raw data 

 regarding the assay response. Each sample tested will be reported as 

 positive or negative, and the official NPIP procedure used to classify the

 sample must be submitted in addition to the assay response value.

(5) The findings of the cooperating laboratories will be evaluated by the NPIP

 technical committee, and the technical committee will make a 

 recommendation regarding whether to approve the test kit to the General 

 Conference Committee. If the technical committee recommends approval, 

 the final approval will be granted in accordance with the procedures described 

 in §§ 147.46 and 147.47.
147

Part 56–Control of H5/H7 Low Pathogenic Avian Influenza

Table of Contents

Section

56.1 Definitions.

56.2 Cooperation with States.

56.3 Payment of indemnity.

56.4 Determination of indemnity amounts.

56.5 Destruction and disposal of poultry and cleaning and disinfection of premises,

conveyances, and materials.

56.6 Presentation of claims for indemnity.

56.7 Mortgage against poultry or eggs.

56.8 Conditions for payment.

56.9 Claims not allowed.

56.10 Initial State response and containment plan.

Authority: 7 U.S.C. 8301–8317; 7 CFR 2.22, 2.80, and 371.4.

Page

148

150

151

152

154

157

157

157

158

159
148

§ 56.1 Definitions.

The Administrator, Animal and Plant Health Inspection Service, or any other 

employee of the Animal and Plant Health Inspection Service delegated to act in the 

Administrator’s stead. 

The Animal and Plant Health Inspection Service of the U.S. Department of Agriculture.

A flock that is composed of stock that has been developed for commercial egg or meat 

production and is maintained for the principal purpose of producing chicks for the 

ultimate production of eggs or meat for human consumption.

A designation earned by participation in a Plan program.

All of the meat-type chickens or meat-type turkeys on one farm. However, at the 

discretion of the Official State Agency, any group of poultry which is segregated from 

another group in a manner sufficient to prevent the transmission of H5/H7 LPAI and 

has been so segregated for a period of at least 21 days may be considered as a 

separate flock.

All table-egg layers of one classification in one barn or house.

A farm containing contiguous flocks of commercial table-egg layers under common 

ownership.

Any State authority recognized by the Department to cooperate in the administration of 

the provisions of this part 56. This may include the State animal health authority or the 

Official State Agency.

The U.S. Department of Agriculture.

Propagated and maintained under the control of a person.

A written flock management agreement developed by APHIS and the Official State 

Agency with input from the flock owner and other affected parties. A flock plan sets 

out the steps to be taken to eradicate H5/H7 LPAI from a positive flock, or to prevent 

introduction of H5/H7 LPAI into another flock. A flock plan shall include, but is not 

necessarily limited to, poultry and poultry product movement and geographically 

appropriate infected and control/monitoring zones. Control measures in the flock plan 

should include detailed plans for safe handling of conveyances, containers, and other 

associated materials that could serve as fomites; disposal of flocks; cleaning and 

disinfection; downtime; and repopulation. 

Part 56–Control of H5/H7 Low Pathogenic Avian Influenza

Administrator 

Animal and Plant Health 

Inspection Service 

(APHIS) 

Breeding flock 

Classification 

Commercial meat-type 

flock 

Commercial table-egg 

layer flock 

Commercial table-egg 

layer premises 

Cooperating State 

Agency 

Department

Domesticated 

Flock plan
149

An infection of poultry caused by an influenza A virus of H5 or H7 subtype that has 

an intravenous pathogenicity index test in 6-week-old chickens less than 1.2 or any 

infection with influenza A viruses of H5 or H7 subtype for which nucleotide sequencing 

has not demonstrated the presence of multiple basic amino acids at the cleavage site 

of the hemagglutinin.

At risk of developing H5/H7 LPAI because of association with birds or poultry infected 

with H5/H7 LPAI, excrement from birds or poultry infected with H5/H7 LPAI, or other 

material touched by birds or poultry infected with H5/H7 LPAI, or because there is 

reason to believe that association has occurred with H5/H7 LPAI or vectors of H5/H7 

LPAI, as determined by the Cooperating State Agency and confirmed by APHIS. 

Poultry will be considered to be infected with H5/H7 LPAI for the purposes of this part 

if: 

(1) H5/H7 LPAI virus has been isolated and identified as such from poultry; or 

(2) Viral antigen or viral RNA specific to the H5 or H7 subtype of AI virus has been 

detected in poultry; or 

(3) Antibodies to the H5 or H7 subtype of the AI virus that are not a consequence of 

vaccination have been detected in poultry. If vaccine is used, methods should 

be used to distinguish vaccinated birds from birds that are both vaccinated and 

infected. In the case of isolated serological positive results, H5/H7 LPAI infection 

may be ruled out on the basis of a thorough epidemiological investigation that 

does not demonstrate further evidence of H5/H7 LPAI infection.

A domesticated chicken grown for the primary purpose of producing meat including but 

not limited to broilers, roasters, fryers, and cornish.

A domesticated turkey grown for the primary purpose of producing meat.

Any mortgage, lien, or other security or beneficial interest held by any person other 

than the one claiming indemnity for the destruction of poultry or eggs due to H5/H7 

LPAI.

A person authorized by APHIS to appraise poultry for the purposes of this part. A 

State official appraiser is selected by a State and authorized by APHIS.

The State authority recognized by the Department to cooperate in the administration of 

the Plan.

The provisions of the National Poultry Improvement Plan contained in parts 145, 146, 

and 147 of this chapter.

Domesticated fowl, including chickens, turkeys, ostriches, emus, rheas, cassowaries, 

waterfowl, and game birds, except doves and pigeons, which are bred for the primary 

purpose of producing eggs or meat. 

H5/H7 low pathogenic 

avian influenza (LPAI)

H5/H7 LPAI exposed

H5/H7 LPAI virus 

infection (infected) 

Meat-type chicken 

Meat-type turkey 

Mortgage 

Official appraiser (APHIS 

official appraiser, State 

official appraiser) 

Official State Agency 

Plan

Poultry 
150

The Secretary of the United States Department of Agriculture, or any officer or 

employee of the Department delegated to act in the Secretary’s stead.

Any of the States, the District of Columbia, the Commonwealth of Puerto Rico, Guam, 

the Commonwealth of the Northern Mariana Islands, the Virgin Islands of the United 

States, or any territory or possession of the United States. 

A domesticated chicken grown for the primary purpose of producing eggs for human 

consumption. 

All of the States.

§ 56.2 Cooperation with States.

(a) The Administrator has been delegated the authority to cooperate with Cooperating 

State Agencies in the eradication of H5/H7 LPAI. This cooperation may include, 

but is not necessarily limited to, the following activities:

(1) Payment to Cooperating State Agencies for surveillance and monitoring 

associated with poultry that have been infected with or exposed to H5/H7 

LPAI;

(2) Transfer of vaccine for H5/H7 LPAI to Cooperating State Agencies if provided 

for in the initial State response and containment plan developed by the Official 

State Agency and approved by APHIS under § 56.10; and

(3) Payment for vaccine administration by Cooperating State Agencies, if provided 

for in the initial State response and containment plan developed by the Official 

State Agency and approved by APHIS under § 56.10

(b) (1) Any payment made to a State or an Official State Agency for the activities 

listed in paragraphs (a)(1) and (a)(3) of this section must be made through a 

cooperative agreement between the Cooperating State Agency and APHIS. 

The payment for which the Cooperating State Agency is eligible will be 

determined in the cooperative agreement.

(i) For any Cooperating State Agency that participates in the National Poultry 

Improvement Plan diagnostic surveillance program for H5/H7 LPAI, as 

described in § 146.14 of this chapter, and has an initial State response 

and containment plan for H5/H7 LPAI that is approved by APHIS, as 

described in § 56.10 of this part, the cooperative agreement will provide 

that the Cooperating State Agency is eligible for payment of 100 percent 

of the costs of surveillance and monitoring and 100 percent of the costs of 

vaccine administration, as determined in the cooperative agreement.

(ii) For any Cooperating State Agency that does not meet the criteria in 

paragraph (b)(1)(i) of this section, the cooperative agreement will provide 

that the Cooperating State Agency is eligible for payment of 25 percent 

of the costs of surveillance and monitoring and 25 percent of the costs of 

vaccine administration, as determined in the cooperative agreement.

Secretary 

State 

Table-egg layer 

United States
151

(2) Transfer of vaccine under paragraph (a)(2) of this section must be 

accomplished through a cooperative agreement between the Cooperating 

State Agency and APHIS.

(c) States will be responsible for making the determination to request Federal 

assistance under this part in the event of an outbreak of H5/H7 LPAI.

§ 56.3 Payment of indemnity.

The Administrator may pay indemnity for the activities listed in paragraphs (a)(1) 

through (a)(3) of this section, as provided in paragraph (b) of this section:

(1) Destruction and disposal of poultry that were infected with or exposed to H5/H7 

LPAI; 

(2) Destruction of any eggs destroyed during testing of poultry for H5/H7 LPAI during 

an outbreak of H5/H7 LPAI; and

(3) Cleaning and disinfection of premises, conveyances, and materials that came into 

contact with poultry that were infected with or exposed to H5/H7 LPAI; or, in the 

case of materials, if the cost of cleaning and disinfection would exceed the value of 

the materials or cleaning and disinfection would be impracticable for any reason, 

the destruction and disposal of the materials.

Except for poultry that are described by the categories in paragraphs (b)(1) through 

(b)(7) of this section, the Administrator is authorized to pay 100 percent of the costs, 

as determined in accordance with § 56.4, of the activities described in paragraphs 

(a)(1) through (a)(3) of this section, regardless of whether the infected or exposed 

poultry participate in the Plan. For infected or exposed poultry that are described by 

the categories in paragraphs (b)(1) through (b)(7) of this section, the Administrator 

is authorized to pay 25 percent of the costs of the activities described in paragraphs 

(a)(1) through (a)(3) of this section:

(1) The poultry are egg-type breeding chickens from a flock that participates in any 

Plan program in part 145 of this chapter but that does not participate in the U.S. 

Avian Influenza Clean program of the Plan in § 145.23(h) of this chapter; or

(2) The poultry are meat-type breeding chickens from a flock that participates in any 

Plan program in part 145 of this chapter but that does not participate in the U.S. 

Avian Influenza Clean program of the Plan in § 145.33(l) of this chapter; or

(3) The poultry are breeding turkeys from a flock that participates in any Plan program 

in part 145 of this chapter but that does not participate in the U.S. H5/H7 Avian 

Influenza Clean program of the Plan in § 145.43(g) of this chapter; or

(4) The poultry are commercial table-egg layers from a premises that has 75,000 

or more birds and that does not participate in the U.S. H5/H7 Avian Influenza 

Monitored program of the Plan in § 146.23(a) of this chapter; or

(a) Activities eligible for 

indemnity 

(b) Percentage of costs 

eligible for indemnity 
152

(5) The poultry are commercial meat-type chickens that are associated with a 

slaughter plant that slaughters 200,000 or more meat-type chickens per operating 

week and that does not participate in the U.S. H5/H7 Avian Influenza Monitored 

program of the Plan in § 146.33(a) of this chapter; or

(6) The poultry are commercial meat-type turkeys that are associated with a slaughter 

plant that slaughters 2 million or more meat-type turkeys in a 12-month period and 

that does not participate in the U.S. H5/H7 Avian Influenza Monitored program of 

the Plan in § 146.43(a) of this chapter; or

(7) The poultry are associated with a flock or slaughter plant that participates in the 

Plan, but they are located in a State that does not participate in the National 

Poultry Improvement Plan diagnostic surveillance program for H5/H7 LPAI, 

as described in § 146.14 of this chapter, or that does not have an initial State 

response and containment plan for H5/H7 LPAI that is approved by APHIS, unless 

such poultry participate in the Plan with another State that does participate in the 

National Poultry Improvement Plan diagnostic surveillance program for H5/H7 

LPAI, as described in § 146.14 of this chapter, and has an initial State response 

and containment plan for H5/H7 LPAI that is approved by APHIS.

If the recipient of indemnity for any of the activities listed in paragraphs (a)(1) through 

(a)(3) of this section also receives payment for any of those activities from a State or 

from other sources, the indemnity provided under this part will be reduced by the total 

amount of payment received from the State or other sources.

§ 56.4 Determination of indemnity amounts.

(1) Indemnity for the destruction of poultry infected with or exposed to H5/H7 LPAI will be 

based on the fair market value of the poultry, as determined by an appraisal. Poultry 

infected with or exposed to H5/H7 LPAI that are removed by APHIS or a Cooperating 

State Agency from a flock will be appraised by an APHIS official appraiser and a State 

official appraiser jointly, or, if APHIS and State authorities agree, by either an APHIS 

official appraiser or a State official appraiser alone. For laying hens, the appraised 

value should include the hen’s projected future egg production. Appraisals 

of poultry must be reported on forms furnished by APHIS and signed by the 

appraisers and must be signed by the owners of the poultry to indicate agreement 

with the appraisal amount. Appraisals of poultry must be signed by the owners 

of the poultry prior to the destruction of the poultry, unless the owners, APHIS, 

and the Cooperating State Agency agree that the poultry may be destroyed 

immediately. Reports of appraisals must show the number of birds and the value 

per head.

(2) Indemnity for disposal of poultry infected with or exposed to H5/H7 LPAI will be 

based on receipts or other documentation maintained by the claimant verifying 

expenditures for disposal activities authorized by this part. Any disposal of poultry 

infected with or exposed to H5/H7 LPAI for which compensation is requested 

must be performed under a compliance agreement between the claimant, the 

(c) Other sources of 

payment

(a) Destruction and 

disposal of poultry
153

Cooperating State Agency, and APHIS. APHIS will review claims for compensation 

for disposal to ensure that all expenditures relate directly to activities described in 

§ 56.5 and in the initial State response and containment plan described in § 56.10. 

If disposal is performed by the Cooperating State Agency, APHIS will indemnify the 

Cooperating State Agency for disposal under a cooperative agreement. 

(3) The destruction and disposal of the indemnified poultry must be conducted in 

accordance with the initial State response and containment plan for H5/H7 LPAI, 

as described in § 56.10. 

Indemnity for eggs destroyed during an outbreak for testing for H5/H7 LPAI will be 

based on the fair market value of the eggs, as determined by an appraisal. Eggs 

destroyed for testing for H5/H7 LPAI will be appraised by an APHIS official appraiser 

and a State official appraiser jointly, or, if APHIS and State authorities agree, by either 

an APHIS official appraiser or a State official appraiser alone. Appraisals of eggs must 

be reported on forms furnished by APHIS and signed by the appraisers and must be 

signed by the owners of the eggs to indicate agreement with the appraisal amount. 

Appraisals of eggs must be signed by the owners of the eggs prior to the destruction of 

the poultry, unless the owners, APHIS, and the Cooperating State Agency agree that 

the eggs may be destroyed immediately. Reports of appraisals must show the number 

of eggs and the value per egg.

(1) Indemnity for cleaning and disinfection of premises, conveyances, and materials 

that came into contact with poultry that are infected with or exposed to H5/H7 

LPAI will be based on receipts or other documentation maintained by the claimant 

verifying expenditures for cleaning and disinfection activities authorized by this 

part. Any cleaning and disinfection of premises, conveyances, and materials for 

which indemnity is requested must be performed under a compliance agreement 

between the claimant, the Cooperating State Agency, and APHIS. APHIS 

will review claims for indemnity for cleaning and disinfection to ensure that all 

expenditures relate directly to activities described in § 56.5 and in the initial State 

response and containment plan described in § 56.10.

(2) In the case of materials, if the cost of cleaning and disinfection would exceed the 

value of the materials or cleaning and disinfection would be impracticable for any 

reason, indemnity for the destruction of the materials will be based on the fair 

market value of those materials, as determined by an appraisal. Materials will be 

appraised by an APHIS official appraiser and a State official appraiser jointly, or, if 

APHIS and State authorities agree, by either an APHIS official appraiser or a State 

official appraiser alone. Indemnity for disposal of the materials will be based on 

receipts or other documentation maintained by the claimant verifying expenditures 

for disposal activities authorized by this part. Any disposal of materials for which 

indemnity is requested must be performed under a compliance agreement 

between the claimant, the Cooperating State Agency, and APHIS. APHIS will 

review claims for compensation for disposal to ensure that all expenditures relate 

directly to activities described in § 56.5 and in the initial State response and 

containment plan described in § 56.10.

(b) Destruction of eggs 

(c) Cleaning and 

disinfection
154

§ 56.5 Destruction and disposal of poultry and cleaning and disinfection 

of premises, conveyances, and materials.

Poultry that are infected with or exposed to H5/H7 LPAI may be required to be 

destroyed at the discretion of the Cooperating State Agency and APHIS and in 

accordance with the initial State response and containment plan described in 

§ 56.10. The Cooperating State Agency and APHIS will select a method to use for 

the destruction of such poultry based on the following factors:

(1) The species, size, and number of the poultry to be destroyed;

(2) The environment in which the poultry are maintained;

(3) The risk to human health or safety of the method used;

(4) Whether the method requires specialized equipment or training;

(5) The risk that the method poses of spreading the H5/H7 LPAI virus;

(6) Any hazard the method could pose to the environment;

(7) The degree of bird control and restraint required to administer the destruction 

method; and

(8) The speed with which destruction must be conducted.

Carcasses of poultry that have died from H5/H7 LPAI infection or poultry that have 

been humanely slaughtered to fulfill depopulation requirements must be disposed of 

promptly and efficiently in accordance with the initial State response and containment 

plan described in § 56.10 to prevent the spread of H5/H7 LPAI infection. Disposal 

methods will be selected by the Cooperating State Agency and APHIS and may 

include one or more of the following: Burial, incineration, composting, or rendering. 

Regardless of the method used, strict biosecurity procedures must be implemented 

and enforced for all personnel and vehicular movement into and out of the area 

in accordance with the initial State response and containment plan to prevent 

dissemination of the H5/H7 LPAI virus.

(1) At the discretion of the Cooperating State Agency and APHIS, poultry that 

has been infected with or exposed to H5/H7 LPAI may be allowed to move for 

controlled marketing in accordance with the initial State response and containment 

plan described in § 56.10 and in accordance with the following requirements:

(i) Poultry infected with or exposed to H5/H7 LPAI must not be transported to 

a market for controlled marketing until 21 days after the acute phase of the 

infection has concluded, as determined by the Cooperating State Agency in 

accordance with the initial State response and containment plan described in 

§ 56.10; and

(ii) Within 7 days prior to slaughter, each flock to be moved for controlled 

marketing must be tested for H5/H7 LPAI using a test approved by the 

Cooperating State Agency and found to be free of the virus.

(2) Poultry moved for controlled marketing will not be eligible for indemnity under 

§ 56.3.

(a) Destruction of poultry 

(b) Disposal of poultry 

(c) Controlled marketing
155

Premises, conveyances, and materials that came into contact with poultry infected with 

or exposed to H5/H7 LPAI must be cleaned and disinfected; Provided, that materials 

for which the cost of cleaning and disinfection would exceed the value of the materials 

or for which cleaning and disinfection would be impracticable for any reason may be 

destroyed and disposed. Cleaning and disinfection must be performed in accordance 

with the initial State response and containment plan described in § 56.10, which must 

be approved by APHIS. This paragraph (d) provides guidelines for the development of 

a cleaning and disinfection plan for a premises and for the materials and conveyances 

on that premises.

(1) Preparation for cleaning and disinfection. Following the depopulation or controlled 

marketing of all poultry infected with or exposed to H5/H7 LPAI on a premises, the 

following procedures should be completed prior to cleaning and disinfection:

(i) Secure and remove all feathers that might blow around outside the house in 

which the infected or exposed poultry were held by raking them together and 

burning the pile;

(ii) Apply insecticides and rodenticides immediately after the removal of the birds, 

before the house cools;

(iii) Close the house in which the poultry were held, maintaining just enough 

ventilation to remove moisture. Leave the house undisturbed for a minimum 

of 21 days and for as long as possible thereafter, in order to allow as much 

H5/H7 LPAI virus as possible to die a natural death. 

(iv) Heat the house to 100 °F for the 72 hours prior to cleaning and disinfection. 

(2) Cleaning and disinfection. All premises, conveyances, and materials that came 

into contact with poultry that were infected with or exposed to H5/H7 LPAI must 

be cleaned and disinfected. Cleaning and disinfection must be performed on all 

buildings that came into contact with poultry that were infected with or exposed 

to H5/H7 LPAI within a premises, including pumphouses and service areas. 

To accomplish cleaning and disinfection, the following procedures should be 

completed:

(i) Disposal of manure, debris, and feed. Clean up all manure, debris, and 

feed. Compost manure, debris, and feed in the house if possible. If this is not 

possible, set up a system for hauling manure, debris, and feed to an approved 

site for burial, piling, or composting. Do not clean out the house or move 

or spread litter until any H5/H7 LPAI virus that may have contaminated the 

manure and litter is dead, as determined by the Cooperating State Agency and 

in accordance with the initial State response and containment plan described 

in § 56.10. If composting is used as a disposal method, manure and litter 

should be composted in accordance with State and local regulations. If litter 

is piled, the litter pile must be covered and allowed to sit undisturbed for an 

amount of time approved by the Cooperating State Agency and APHIS and 

in accordance the initial State response and containment plan described in 

§ 56.10. Drying and heat in situ over time are effective and may be used in 

place of composting if weather conditions or conditions in the building are 

favorable. After use, equipment used to clean out manure, debris, and feed 

must be washed, disinfected, and inspected at the site to which the manure 

(d) Cleaning and 

disinfection of premises, 

conveyances, and 

materials
156

and litter was transported. In the case of inclement weather, the equipment 

may be washed, disinfected, and inspected at off-site wash stations at the 

discretion of the Cooperating State Agency and APHIS. 

(ii) Cleaning of premises and materials. Cleaning and washing should be 

thorough to ensure that all materials or substances contaminated with H5/H7 

LPAI virus, especially manure, dried blood, and other organic materials, are 

removed from all surfaces. Spray all contaminated surfaces above the floor 

with soap and water to knock dust down to the floor, using no more water than 

necessary. Wash equipment and houses with soap and water. Disassemble 

equipment as required to clean all contaminated surfaces. Special attention 

should be given to automatic feeders and other closed areas to ensure 

adequate cleaning. Inspect houses and equipment to ensure that cleaning 

has removed all contaminated materials or substances and let houses and 

equipment dry completely before applying disinfectant. 

(iii) Disinfection of premises and materials. When cleaning has been completed 

and all surfaces are dry, all interior surfaces of the structure should be 

saturated with a disinfectant authorized in § 71.10(a) of this chapter. A power 

spray unit should be used to spray the disinfectant on all surfaces, making 

sure that the disinfectant gets into cracks and crevices. Special attention 

should be given to automatic feeders and other closed areas to ensure 

adequate disinfection.

(iv) Cleaning and disinfection of conveyances. Clean and disinfect all trucks and 

vehicles used in transporting affected poultry or materials before soil dries 

in place. Both exterior, including the undercarriage, and interior surfaces, 

including truck cabs, must be cleaned. The interior of the truck cabs should 

be washed with clean water and sponged with a disinfectant authorized in 

§ 71.10(a) of this chapter. Manure and litter removed from these vehicles 

should be handled in a manner similar to that described in paragraph (d)(2)(i) 

of this section. 

(3) Activities after cleaning and disinfection. Premises should be checked for virus 

before repopulation in accordance with the initial State response and containment 

plan described in § 56.10. The premises may not be restocked with poultry 

until after the date specified in the initial State response and containment plan 

described in § 56.10.

(4) Destruction and disposal of materials. In the case of materials for which the cost 

of cleaning and disinfection would exceed the value of the materials or for which 

cleaning and disinfection would be impracticable for any reason, the destruction 

and disposal of the materials must be conducted in accordance with the initial 

State response and containment plan described in § 56.10. 
157

§ 56.6 Presentation of claims for indemnity.

Claims for the following must be documented on a form furnished by APHIS and 

presented to an APHIS employee or the State representative authorized to accept the 

claims:

(a) Compensation for the value of poultry to be destroyed due to infection with or 

exposure to H5/H7 LPAI;

(b) Compensation for the value of eggs to be destroyed during testing for H5/H7 LPAI; 

and

(c) Compensation for the cost of cleaning and disinfection of premises, conveyances, 

and materials that came into contact with poultry infected with or exposed to 

H5/H7 LPAI, or, in the case of materials, if the cost of cleaning and disinfection 

would exceed the value of the materials or cleaning and disinfection would be 

impracticable for any reason, the cost of destruction and disposal for the materials.

§ 56.7 Mortgage against poultry or eggs.

When poultry or eggs have been destroyed under this part, any claim for indemnity 

must be presented on forms furnished by APHIS. The owner of the poultry or eggs 

must certify on the forms that the poultry or eggs covered are, or are not, subject to 

any mortgage as defined in this part. If the owner states there is a mortgage, the 

owner and each person holding a mortgage on the poultry or eggs must sign the 

APHIS-furnished form, consenting to the payment of indemnity to the person specified 

on the form. 

§ 56.8 Conditions for payment. 

(a) When poultry or eggs have been destroyed pursuant to this part, the Administrator 

may pay claims to any party with which the owner of the poultry or eggs has 

entered into a contract for the growing or care of the poultry or eggs. The 

indemnity the Administrator may pay to such a party or parties shall be determined 

as follows:

(1) Divide the value of the contract the owner of the poultry or eggs entered into 

with another party for the growing and care of the poultry or eggs in dollars by 

the duration of the contract as it was signed prior to the H5/H7 LPAI outbreak 

in days;

(2) Multiply this figure by the time in days between the date the other party began 

to provide services relating to the destroyed poultry or eggs under the contract 

and the date the birds were destroyed due to H5/H7 LPAI.
158

(b) (1) If indemnity for the destroyed poultry or eggs is being provided for 100 percent 

of eligible costs under § 56.3(b), the Administrator may pay contractors eligible 

for compensation under this section 100 percent of the indemnity determined 

in paragraph (a) of this section.

(2) If indemnity for the destroyed poultry or eggs is being provided for 25 percent 

of eligible costs under § 56.3(b), the Administrator may pay contractors eligible 

for compensation under this section 25 percent of the indemnity determined in 

paragraph (a) of this section.

(c) If indemnity is paid to a contractor under this section, the owner of the poultry or 

eggs will be eligible to receive the difference between the indemnity paid to the 

growers and the total amount of indemnity that may be paid for the poultry or eggs.

(d) In the event that determination of indemnity to a party with which the owner of 

destroyed poultry or eggs has entered into a contract for the growing or care of 

the poultry or eggs using the method described in paragraph (a) of this section is 

determined to be impractical or inappropriate, APHIS may use any other method 

that the Administrator deems appropriate to make that determination.

§ 56.9 Claims not allowed.

(a) The Department will not allow claims arising out of the destruction of poultry 

unless the poultry have been appraised as prescribed in this part and the owners 

have signed the appraisal form indicating agreement with the appraisal amount as 

required by § 56.4(a)(1).

(b) The Department will not allow claims arising out of the destruction of poultry 

unless the owners have signed a written agreement with APHIS in which they 

agree that if they maintain poultry in the future on the premises used for poultry for 

which indemnity is paid, they will maintain the poultry in accordance with a plan 

set forth by the Cooperating State Agency and will not introduce poultry onto the 

premises until after the date specified by the Cooperating State Agency. Persons 

who do not maintain their poultry and premises in accordance with this written 

agreement will not be eligible to receive indemnity under this part.

(c) The Department will not allow claims arising out of the destruction of poultry 

unless the poultry have been moved or handled by the owner in accordance with 

an agreement for the control and eradication of H5/H7 LPAI and in accordance 

with part 56, for any progeny of any poultry unless the poultry have been moved 

or handled by the owner in accordance with an agreement for the control and 

eradication of H5/H7 LPAI and in accordance with part 56, or for any poultry that 

become or have become infected with or exposed to H5/H7 LPAI because of 

actions not in accordance with an agreement for the control and eradication of 

H5/H7 LPAI or a violation of this part. 
159

§ 56.10 Initial State response and containment plan. 

(a) In order for poultry owners within a State to be eligible for indemnity for 100 

percent of eligible costs under § 56.3(b), the State in which the poultry participate 

in the Plan must have in place an initial State response and containment plan 

that has been approved by APHIS. The initial State response and containment 

plan must be developed by the Official State Agency and administered by the 

Cooperating State Agency of the relevant State. This plan must include:

(1) Provisions for a standing emergency disease management committee,

 regular meetings, and exercises, including coordination with any tribal 

governments that may be affected;

(2) A minimum biosecurity plan followed by all poultry producers;

(3) Provisions for adequate diagnostic resources;

(4) Detailed, specific procedures for initial handling and investigation of 

suspected cases of H5/H7 LPAI;

(5) Detailed, specific procedures for reporting test results to APHIS. These

 procedures must be developed after appropriate consultation with poultry

 producers in the State and must provide for the reporting only of confirmed

 cases of H5/H7 LPAI in accordance with § 146.13 of this chapter;

(6) Detailed, strict quarantine measures for presumptive and confirmed index

 cases;

(7) Provisions for developing flock plans for infected and exposed flocks;

(8) Detailed plans for disposal of infected flocks, including preexisting

 agreements with regulatory agencies and detailed plans for carcass disposal,

 disposal sites, and resources for conducting disposal, and detailed plans

 for disposal of materials that come into contact with poultry infected with or

 exposed to H5/H7 LPAI;

(9) Detailed plans for cleaning and disinfection of premises, repopulation, and

 monitoring after repopulation;

(10) Provisions for appropriate control/monitoring zones, contact surveys, and

 movement restrictions;

(11) Provisions for monitoring activities in control zones; 

(12) If vaccination is considered as an option, a written plan for use in place with

 proper controls and provisions for APHIS approval of any use of vaccine; 

(13) Plans for H5/H7 LPAI-negative flocks that provide for quarantine, testing, and

 controlled marketing; and

(14) Public awareness and education programs regarding avian influenza.

(b) If a State is designated a U.S. Avian Influenza Monitored State, Layers under 

§ 146.24(a) of this chapter or a U.S. Avian Influenza Monitored State, Turkeys 

under § 146.44(a) of this chapter, it will lose that status during any outbreak of 

H5/H7 LPAI and for 90 days after the destruction and disposal of all infected 

or exposed birds and cleaning and disinfection of all affected premises are 

completed.
160
161
162

United States

Department of Agriculture

APHIS–VS

National Poultry Improvement Plan

Suite 101

1498 Klondike Road

Conyers, GA 30094–5104

OFFICIAL BUSINESS

Penalty for Private Use, $300