United States

Department of Agriculture Animal and Plant Health Inspection Service **Veterinary Services**  **July 2012**  

# National Poultry **Improvement Plan Program Standards**  **Jul y**  **2012**"The U.S. Department of Agriculture (USDA) prohibits discrimination in all of its programs and activities on the basis of race, color, national origin, age, disability, and where applicable, sex (including gender identity and expression ), marital status, familial status, parental status, religion, sexual orientation, political beliefs, genetic information, reprisal, or because all or part of an individual's income is derived from any public assistance program. (Not all prohibited bases a pply 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 d iscrimination, write USDA, Director, Office of Civil Rights, 1400 Independence Avenu e, SW , W ashington, DC 20250 - 9410 or call (800) 795 - 3272 or (202) 720 - 6382 (TDD). USDA is an equal opportunity provider and employer.National Poultry Improvement Plan Program Standards

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### PROGRAM STANDARDS OF THE NATIONAL POULTRY IMPROVEMENT PLAN **Table of Contents** Pa ge 4 - Introduction Pa ge 6 - Definitions **Subpart A —**  **Blood Testing Procedures Page**  **Section** 11 (1) The standard tube agglutina tion test. 6 12 (2) The rapid serum test. 7 13 (3) The stained - antigen, rapid, whole - blood test. 8 13 (4) The microagglutination test for pullorum - t yphoid. 14 (5) Pr ocedure for determining the status of flocks reacting to tests for Mycoplasma gallisepticum , Mycoplasma synoviae , and Mycoplasma meleagridis . 

### 15 (6) Standard test procedures for mycoplasma. 9 

### 22 (7) Pr ocedures for preparing egg yolk samples for diagnostic tests. 

### 23 (8) Standard test procedures for avian influenza. **Subpart B —**  **Bacteriological Examination Procedure** 25 (1) Laboratory procedure recommended for the bacteriological examination of egg - ty pe breeding flocks with salmonella enteritidis positive environments. 25 (2) Laboratory procedure recommended for t he bacteriological examination of s almon ella. 27 (3) Procedures for collection, isolation, and identification of Salmonella from environment al samples, cloacal swabs, chick box papers, and meconium samples. 31 (4) Pr ocedure for bacteriological culturing of eggshells for colon bacilli organisms. 

### 3 1 (5) Pr ocedures to determine status and effect iveness of sanitation monitored program. 

### 3 1 (6) Laboratory procedure recommended for the bacteriological examination of mycoplasma reactors. 17 

### 34 (7) Pr ocedure for the e valuation of myco p lasma reactors by in vivo bio - assay (enrichment). 

### 35 (8) Laboratory procedure recommended for the bacteriological examination of cull chicks and poults for salmonella. **Subpart C —**  **Sanitation Procedures** 37 (1) Flock sanitation. 37 (2) Hatching egg sanitation. 38 (3) Hatchery sanitation. 38 (4) Cleaning and disinfecting. 3 9 (5) Fumigation. 3 9 (6) Pr ocedures for establishing isolation and maintaining sanitation and good management practices

for the control of Salmonella and Mycoplasma infections. 

### 40 (7) Pr ocedures recommended to prevent the spread of disease by artificial insemination of turkeys. 40 (8) Hygiene and Biosecurity Procedures for Poultry Primary Breeding Flocks and Hatcheries **Subpart D —**  **Molecular Exam ination Procedures**  43 (1) Laboratory procedure recommended for the polymerase chain reaction (PCR) test for My coplasma gallisepticum and M. synoviae. 43 (2) Laboratory procedures recommended for the real - time polymerase chain reacti on test for My coplasma gallisepticum (MGLP ReTi). 45 (3) Laboratory procedures recommended for the conventional polymerase chain reaction test for Salmonella Enteritidis. 45 (4) Laboratory procedures recommended for the real - tim e polymerase chain reaction test for Salmonella sp. Group DNational Poultry Improvement Plan Program Standards

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### Introduction These program standards describe specific tests and sanitation procedures. Formerl y, these tests and procedures were outlined in our regulations at title 9, Code of Federal Regulations (9 CFR) part 147. We have amended our regulations to require that tests to be performed and sanitation to be maintained in a manner approved b y the Administrator . The recommended tests and sanitation procedures are contained in this document. These changes streamline the provisions of the Plan, keep those provisions current w ith changes in the poultry industry , and provide for the use of new sampling and testing procedures. Changes to this document are voted on and approved by the voting delegates at the Plan's Biennial National Plan Conference. The National Poultry

I mprovem ent Plan (NPIP, also referred to as “ the Plan ” ) is a cooperative Federal - State - industry

mechanism for controlling certain poultry diseases. The Plan consists of a variet y of programs intended to prevent and control poultry diseases. Participation in all Pl an programs is voluntary , but breeding flocks, hatcheries, and dealers must first qualify as "U.S. Pullorum - Ty

phoid Clean" as a condition for participating in the other Plan programs. The Plan identifies States, flocks, hatcheries, dealers, and slaughte r plants that meet certain disease control standards specified in the Plan's various programs. As a result, customers can bu y poultry that has tested clean of certain diseases or that has been produced under disease - prevention conditions. The NPI P regulati ons in 9 CFR parts 145 and 146 contain requirements that must be observed b y flocks that participate in the Plan. These requirements include requirements to test poultry for the specific disease addressed by each classification in which the flock participa tes. The procedures b y which that testing is conducted have historically been contained in 9 CFR part 147, subparts A, B, and D. Subpart A set out blood testing procedures, subpart B set out bacteriological examination procedures, and subpart D set out molecular examination procedures, which currently include poly merase chain reacti on (PCR) tests. In addition, subpart C of part 147 contained santiation procedures. These test and sanitation procedures are now incorporated into this P rogram S tandards document. We have prepared this program standards document for several reasons. First , there are constant changes in the science and technology that go into developing effective, efficient tests. T o have a successful voluntary

program to reduce the incidence of disease in poultry , we need to be able to update the NPIP testing procedures wh en new scientific evidence indicates that different procedures can increase the reliability of a test, or when new technology is developed to make a test more efficient or accurate. In addition, new tests are also continually developed that can provide va luable alternatives to existing approved tests. For example, there has been a great deal of progress in developing PCR tests in recent ye ars. Adding such tests allows NPI P participants to take advantage of the latest testing technology . Similarly , the s anitation procedures used as best practices to prevent the introduction or spread of disease in a poultry

flock are constantly

changing, as more information becomes available about possible sources of infection and about the effectiveness of various means of preventing infection. In the past, we have updated the regulations once every 2 years, following the biennial PlanNational Poultry Improvement Plan Program Standards

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### Conference. However, with the continual changes in diagnostic science and testing technology , and in best practices for maintaining san itation, the biennial update schedule has resulted in the regulations becoming out of date between updates. When this happens, sometimes the Plan's General Conference Committee (GCC) approves interim changes to the tests or sanitation procedures in accorda nce with the process outlined in 9 CFR 147.43(d)(5)(iii). We hope to make the program more effective by making all participants aware of the new tests and sanitation procedures immediately b y updating this document which we intend to be recognized in the re gulations as a resource for tests and sanitation procedures. Finally , tests can be difficult to render in the regulations. The current regulations in 9 CFR 147.11 and 9 CFR 147.12, for example, contain diagrams and flow charts that are part of larger proc esses, all of which require several pages to describe in narrative format. These program standards allow us to lay out our tests in an easier - to - understand format .National Poultry Improvement Plan Program Standards

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### D **efinitions** Administrator . 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. Affiliated flock. 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. Affiliated flockowner . A flockowner who is participating in the Plan through an agreement with a participating hatchery. Animal and Plant Health Inspection Service (APHIS , the Service ) . The Animal and Plant Health Inspection Service of the U.S. Department of Agriculture. Authorized agent . Any person designated under title 9, Code of Federal Regulations (9 CFR) 145.11(a) to collect official samples for submission to an authorized laboratory as described in 9 CFR 147.1(a) and 9 CFR 147.12. An authorized agent is also a person designated to perform functions under 9 CFR 146.10(a). Authorized laboratory . An authorized laboratory is a laboratory that meets the requirements of 9 CFR 147.51 and is thus qualifi ed to perform the assays described in 9 CFR part 147. Authorized testing agent . Any person designated under 9 CFR 145.11(a) to collect official samples for submission to an authorized laboratory as described in 9 CFR 147.1(a) and 9 CFR 147.12 and to perform the stained antigen, rapid whole blood test for pullorum typhoid. Avian influenza . An infection or disease of poultry caused by viruses in the family Orthomyxoviridae , genus Influenzavirus A. Baby poultry . Newly hatched poultry (chi cks, poults, ducklings, goslings, keets, etc.). Classification. A designation earned by participation in a Plan program. Colon bacilli. For the purpose of this chapter, those organisms which are gram negative, non spore - forming bacilli, which ferment la ctose with gas formation, and serve as an index of fecal contamination. Commercial meat - type flock . 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 Offi cial 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. Commercial t able - egg layer flock . All table - egg layers of common age or pullet source on one premises. Commercial table - egg layer premises . A farm containing contiguous flocks of commercial table - egg layers under common ownership. Commercial table - egg layer pullet f lock . A table - egg layer flock prior to the onset of egg production. Cooperating State Agency . Any State authority recognized by the Department to cooperate in the administration of the provisions of part 56 of this chapter. This may include the State animal health authority or the Official State Agency. Dealer . An individual or business that deals in commerce in hatching eggs, newly - hatched poultry, and startedNational Poultry Improvement Plan Program Standards

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### 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. Department . The U.S. Department of Agriculture. Domesticated . Propagated and maintained under the control of a person. Equivalent or equivalent requirements . Requiremen ts 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. Exposed (Exposure) . Contact with birds, equipment, pers onnel, supplies, or any article infected with, or contaminated by, communicable poultry disease organisms. Flock : (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 f arm . (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. Fluff sample . Feathers, shell membrane, and other debris resulting from the hatching of poultry. Fowl typhoid or typhoid . A disease of poultry caused by Salmonella gallinarum . Franchise breeder . 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. Franchise hatchery . A hatchery which has been authorized by a franchise breeder to produce and sell products under the breeder's strain or trade name. H5/H7 low pathogenic avian influenza (LPAI). An infection of poultry caused by an influenza A virus of H5 or H7 subtype that has an intravenous pathogenicity index in 6 - week - old chickens less than 1.2 or less than 75 percent mortality in 4 - to 8 - week - old chickens infected intravenously, or an infect ion with influenza A viruses of H5 or H7 subtype with a cleavage site that is not consistent with a previously identified highly pathogenic avian influenza virus. H5/H7 LPAI virus infection (infected). (1) Poultry will be considered to be infected with H5 /H7 LPAI for the purposes of this part if: (i) H5/H7 LPAI virus has been isolated and identified as such from poultry; or (ii) Viral antigen or viral RNA specific to the H5 or H7 subtype of AI virus has been detected in poultry; or (iii) 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, as determined by APHIS. (2) The official determination that H5 /H7 LPAI virus has been isolated and identified, viral antigen or viral RNA specific to the H5 or H7 subtype of AI virus has been detected, or antibodies to the H5 or H7 subtype of AI virus have been detected may only be made by the National Veterinary Ser vices Laboratories. Hatchery . Hatchery equipment on one premises operated or controlled by any person for the production ofNational Poultry Improvement Plan Program Standards

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### baby poultry. Independent flock . A flock that produces hatching eggs and that has no ownership affiliation with a specific hatcher y. Infected flock . 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. Midlay . Approximately 2

– 3 months after a flock begins to lay or after a molted flock is put back into production. Multiplier breeding flock . 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 nonbreeding purpo ses. NPIP . The National Poultry Improvement Plan. NPIP Program Standards . A document that contains tests and sanitation procedures approved by the Administrator. This document may be obtained from the NPIP Web site at http://www.aphis.usda.gov/animal_health/animal_dis_spec/poultry/ or by writing to the Service at National Poultry Improvement Plan, APHIS, USDA, 1506 Klondike Road, Suite 300, Conyers, GA 30094. NPIP Technical Committee . A committee made up of technical e xperts 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. Official State Agency . The State authority recognized by the Department to cooperate in the administration of the Plan. Official supervision : (1) As applied to Plan programs. The direction, inspection, and critical evalu ation 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. Person . A natural person, firm, or corporation. Plan . The provisions of the National Poultry Improvement Plan. Poultry . Domesticated fowl, including chickens, turkeys, ostriche s, emus, rheas, cassowaries, waterfowl, and ga me birds, except doves and pigeons, which are bred for the primary purpose of producing eggs or meat. Primary breeding flock . A flock composed of one or more generations that is maintained for the purpose of e stablishing, continuing, or improving parent lines. Products . Poultry breeding stock and hatching eggs, baby poultry, and started poultry. Program . Management, sanitation, testing, and monitoring procedures which, if complied with, will qualify, and main tain 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 9 CFR 145.10 of this part. Public exhibition . A public show of poultry.National Poultry Improvement Plan Program Standards

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### Pullorum disease or pullorum . A disease of poultry caused by Salmonella pullorum . Reactor . A bird that has a positive reaction to a test, required or recommended in 9 CFR parts 145 or 147, for any poultry disease for which a program has been established under the Plan. Salmonella . Any bacteria belonging to the genus Salmonella , including the arizona group. Sanitize . 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. Senior Coordinator . 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 9 CFR 147.47 . (3) Plannin g, organizing, and conducting the Plan Conference . (4) Reviewing NPIP authorized laboratories as described in 9 CFR 147.51 . (5) Coordinating the State administration of the NPIP through periodic reviews of the administrative procedures of the Official Stat e 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 9 CFR parts 145, 1 46, and 147. (7) Directing the production of official NPIP publications . (8) Proposing an annual budget for plan activities and the General Conference Committee . (9) Providing overall administration of the NPIP. Service. The Animal and Plant Health Inspec tion Service of the U.S. Department of Agriculture. Serial . The total quantity of completed product which has been thoroughly mixed in a single container and identified by a serial number. Sexual maturity . The average age at which a species of poultry is biologically capable of reproduction. Started poultry . 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. State. 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. State Inspector . Any perso n employed or authorized to perform functions under 9 CFR 145.11(b) or 9 CFR 146.10(b) . Stock . 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. Strain . Poultry breeding stock bearing a given name produced by a breeder through at least five generations of closed flock breeding. Succeeding flock . A flock brought onto a premises during the 12 months following removal of an infected flock.National Poultry Improvement Plan Program Standards

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### Suspect flock . A flock shall be considered, for the purposes of the Plan, to be a suspect flock if any evidence exists that it has been exposed to a communicable poultry disease. Trade name or number . A name or num ber compatible with State and Federal laws and regulations applied to a specified stock or product thereof. United States . All of the States.National Poultry Improvement Plan Program Standards

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### Subpart A

**— Blood Testing Procedures**  **(1) The standard tube agglutination te**  **st. 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, preferably3/8by 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 incis ion 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 gage needle) which is properly cleansed between bleedings with physiological saline solution. To facili tate 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 sa mples 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 sy stem which w ill 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 labora tory. (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 tra

nsferring 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. (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 a ntigen 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 t

hrough 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) Thiosulfat e - Gly cerin (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, Re cent 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 a ntigen from a given amount of medium.

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.National Poultry Improvement Plan Program Standards

[DRAFT] 12 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 antigen - growing tubes or bottles should be incu bated 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 B uchner 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 mas s 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 (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 d ay and kept cold. The diluted antigen may be employed in 2 cc. quantities in 4 by1/2

– 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 b e 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 effici ent. 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 seru m - 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 incomple te. 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 allow ance 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 conside ration. 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.)National Poultry Improvement Plan Program Standards

13 **(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 Section A.1 (a) of these standards . (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 ste rile 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 antigen s 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 t hat 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 anti gen 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 h asten agglutination. Before reading, the plate should be rotated several times. (h) The results shall be recorded as described in Section 1(h). **( 3**  **) The stained**  **- antigen, rapid, whole**  **- blood test**  **. 3**  (a) The description of the preparation of antigen is n ot 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 satisfact ory loop may be made from a piece of No. 20 gage nichrome wire, 21/2inches 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 me dicine 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 loopful of blood should be taken up from the wing vein. When s ubmerged 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 m ixture 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

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). 3 The procedure described is a modification of the method reported by Schaffer, MacDonald, Hall, and Bunyea, Jour. Amer. Vet. M ed. Assoc. 79 (N. S. 32): 236

– 240 (1931).National Poultry Improvement Plan Program Standards

[DRAFT] 14 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 desirabl e to examine representative birds bacteriologically to determine the presence or absence of S. pullorum.) **(4) The microagglutination test for pullorum**  **- typhoid.**  Routinely, the microagglutination test is applied as a single - dilution test and only a singl e 18

– 24 hour reading is made. (a) The procedure for the collection and delivery of b lood samples in the microagglutination test is the same as that described in Section 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 - ty phoid is su pplied 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 n ot 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 sh all 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 ser um 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 deliver y 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 se aler 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 recomm ended 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 the microplate. 2 . To the wells representative of the lowest dilution in the titration, add an a dditional 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 (4) (d)2 of these Standards 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 . Dil ute 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. f or 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:

4 In formation as to criteria and procedures for approval of con centrated stock suspension of stained microtest antigens may be obtained from the National Poultry I

mprovement Plan, Veterinary Services, APHIS, USDA, 1506 Klondike Road, Suite 300, Conyers, GA 30094.National Poultry Improvement Plan Program Standards

15 1 . N, or − (negative) when the microplate well has a large, distinc t 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. **(5) Procedure for determining the status of flocks reacting to tests for Mycoplasma gallisepticum,**  **Mycoplasma synoviae, and Mycoplasma meleagridis.** 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; Kleven, S.H., F.T.W. Jordan, and J.M. Bradbury, Avian Mycoplasmosis ( My coplasma gallisepticum ), Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, Fifth Ed., Office International des Epizooties, pp 842

– 855, 2004; and §§147.15 and 147.16. (a) The status of a flock for Mycoplasma shall be determined according to the following criteria: 1 . If the tube agglutination test, enzyme - labeled immunosorbent assay (ELISA), official molecular examination procedure, or serum plate test is negative, the flock qualifies for the classification for which it was tested. 2 . If the tube agglutination, ELISA, or serum plate test is positive, the hemaglutination inhibition (HI) test or a molecular examination procedure shall be conducted: Provided, for the HI test, that if more than 50 percent of the samples are positive for M. gal lisepticum, M. meleagridis, or M. synoviae, the HI test shall be conducted on 10 percent of the positive samples or 25 positive samples, whichever is greater. HI titers of 1:40 or more may be interpreted as suspicious and appropriate antigen detection samp les should be taken promptly (within 7 days of the original sampling) from 30 clinically affected birds and examined by an approved cultural technique individually, or pooled (up to 5 swabs per test) and used in a molecular examination procedure or in vivo bioassay. 3 . If the in vivo bioassay, molecular examination procedure, or culture procedure is negative, the Official State Agency may qualify the flock for the classification for which it was tested. In the event of contaminated cultures, the molecular examination technique must be used to make a final determination. 4 . If the in vivo bioassay, molecular examination procedure, or culture procedure is positive, the flock will be considered infected. **(6) Standard test procedures for**  **mycoplasma .**  **5** The serum plate agglutination test, the tube agglutination test, and the ELISA test 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 an d turkey sera are considered positive, while a 1:40 or 1:20 titer would be strongly suspicious and additional tests should be required. (a) Serum plate agglutination test **.** 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 1⁄2 inch squares on a ruled glass plate. Limit the number of samples (no more than 25) to be set up at one time

5 For additional information on mycoplasma test procedu res, refer to the following references: Proc. 77th Annual Meeting, U.S. Animal Health Association, 1973; I solation and I dentification of Avian Pathogens, 3rd Edition; Methods for Examining Poultry Biologics and for I dentifying and Quantifying Avian Pathoge ns, 1991.National Poultry Improvement Plan Program Standards

[DRAFT] 16 ac cording 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 and read the results in accordance with the product insert. 2 . A positive reaction is characterized by the formation of definite clumps, usually starting at the perip hery 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. Highquality an tigen 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. (b) Serum plate dilution 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, or to evaluate th e 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), m ixing 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 as sist 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. (vii) Interpretation of the SPD test results should be based on the criteria in Section ( 6 ) (a). (c) Tube agglutination test. 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: **Grams** Sodium hydroxide (C.P.) 0.15 Sodium chloride (C.P.) 8.5 Potassium dihydrogen phosphate (KH 2 PO 4 ) (C.P.) 0.68 Phenol (Crystal) (C.P.) 2.5 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 acco rding 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 pro per 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 dilut ed 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 aNational Poultry Improvement Plan Program Standards

17 cl earing 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 positi ve. 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. (d) Hemagglutination Inhibition (HI) test. The mycoplas ma HI test is conducted by the constant - antigen, decreasing - serum method. This method requires using a 4 - hemagglutination (HA) unit of diluted antigen. 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 (P BS) as follows: **Grams** Sodium hydroxide (C.P.) 0.15 Sodium chloride (C.P.) 8.5 Potassium dihydrogen phosphate (KH 2 PO 4 ) (C.P.) 0.68 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: **Grams** Sodium citrate 8.0 Sodium chloride 4.2 Dextrose 20.5 Distilled water 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 s odium chloride solution. (iii) From turkeys or chickens known to be free of the mycoplasma being tested, withdraw sufficient blood with a sy ringe containing Alsever's solution to give a ratio of 1 part blood to 5 parts Alsever's solution (e.g., 8 ml blo od 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 RBCs 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 RBCs is added to 99 ml of buffered saline to make a 0.25 percent RBC suspension. 2. Hemagglutination (HA) antigen titration. The HA stock antigen is stored at −70 °C in PBS buffer containing 25 perc ent glycerin (vol/vol) in a concentrated suspension ( i.e. , 320

– 640 HA units/ml) in screw - type 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 paragraphs (d)(2)(i) through (x) of this section.National Poultry Improvement Plan Program Standards

[DRAFT] 18 (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 wil l 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 RBCs 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 paragraphs (d)(2)(i) through (vii) of this section, using 0.25 percent turkey RBC's. (ix) The end point of the titrati on is the highest dilution of antigen that produces complete agglutination of the RBCs, 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 dil ution 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 varyi ng HI titers should be used if turkey sera is also to be tested.National Poultry Improvement Plan Program Standards

19 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 RBCs. 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 o f 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 1 0. 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 1/4units of antigen. (vii i) 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.National Poultry Improvement Plan Program Standards

[DRAFT] 20 (ix) In this test system, positive serum should inhibit the HA activity o f the antigen, while negative serum should have no effect. Inhibition will be evidenced by the formation of a free - flowing bottom of cells in the botto m of the tube. The titer of the serum can be calculated as the reciprocal of the highest dilution of seru m 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 . (xi) If serological results from agglutination tests complemented by the HI test are inconclusive, cultural examinat ion, bio - assay, or retesting of samples after an interval of at least 21 days may be indicated. (e) Procedure for mycolplasma hemagglutination inhibition tests using microtiter technique

— 1. Procedure No. 1 **.** The microtiter mycoplasma HI test was developed from the tube HI test described in Section (6) (d). Refer to these procedures for preparation of materials not listed below. (i) Materials needed. (A) Microtiter equipment (minimal); i.e., microplates, microdiluters, mic ropipettes, 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 (RBCs) suspension 0.5 percent (2 ml of 25 percent RBCs to 98 ml of PBS) obtained from birds free of the mycoplasma to be tested. (See paragraphs (d)(1)(ii) through (v) of this section for preparation of RBCs.) (ii) Microtiter hemagglutination (HA) antigen titr ation. (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 mi x, 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. **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**  Se rum A (HI n eg.)

− + + + + + + + + + Serum B (HI 1:40)

− − − − + + + + + + Serum C (HI 1:160)

− − − − − − + + + + Serum D (HI 1:20)

− − − + + + + + + + +, HA; −, no HA or HINational Poultry Improvement Plan Program Standards

21 (F) Add 0.05 ml of 0.5 percent RBC suspension to all wells using a 0.05 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 sera 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 d ropper 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 well 3 through 8 for each row. (G) For each serum to be tested, load 0.05 ml diluter with 1:5 dilution as prepar ed in paragraph (iii) 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; wel l 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 conta ins 1 unit; well 5 contains1/2 unit; and well 6 contains1/4 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 m l of a 0.5 percent suspension of RBCs 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 ser um dilution exhibiting complete inhibition of hemagglutination 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. 2. Procedure No. 2 **.** Purpose: To test for antibodies to avian mycoplasma by hemagglutination inhibition (HI). The test uses the constant antigen, titered - sera method for measuring antibodies to M. gallisepticum, M. synoviae, or M. meleagridis. (i) Materials needed. (A) M. gallisepticum, M. synoviae, and/or M. meleagridis HI antigens. (B) Positive and negative control sera. (C) Phosphate buffered saline (PBS). (D) Microtiter plates, 96 - well, U - bottom. (E) 12 - channel pipettor (Titerek). (F) 50 μL pipettor (Pipetman P200). (G) Pipette tips. (H) 0.5 percent homologous red blood cells (RBCs) in PBS (use RBCs 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 o f 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.National Poultry Improvement Plan Program Standards

[DRAFT] 22 ( 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 RB C control. (B) Incubate at room temperature (approximately 30 minutes) until the control RBC's give tight buttons. The HA titer is read as the last well to give a complete lawn (hemagglutination). (C) Dilute stock antigen to 4 HA units for the HI t est. 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 unit 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. (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 inappr opriately 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 RBCs to all wells. Note: Do not agitate afte r RBCs 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 RBCs 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 RBCs. The final dilution scheme includes the antigen in the dilution calculation and i s 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 neg ative, report as “negative with non - specific agglutination or non - specific hemolysis” or “unable to evaluate due to nonspecific 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 agglutina tion

— (A) Purpose. Treatment of serum to remove non - specific agglutination that is interfering with HI assays. (B) Specimen. Serum. (C) Materials. Homologous RBC's (chicken or turkey), 50 percent solution PBS, centrifuge, incubator, 4C (refrigerator). (D) Procedure. ( 1 ) Prepare a 1:5 dilution of test serum by adding 50 μL of serum to 200 μL of PBS. ( 2 ) Prepare a 50 percent solution of RBC's by adding equal volumes of p acked 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. ( 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 1:640 in rows B through H.National Poultry Improvement Plan Program Standards

23 **(7) 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 9 CF R 145.23(c)(1)(ii)(C) and 9 CF R 145.33(c)(1)(ii)(C), for retaining the classification U.S. M. Synoviae Clean under 9 CFR 145.23(e)(1)(ii)( b ) and 9 CFR 145.33(e)(1)(ii)( b ), and for retaining the class ification U.S. H5/H7 Avian Influenza Monitored under 9 CFR 146.23(a), 9 CFR 146.33(a), and 9 CFR 146.44(a). (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 preparati on 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 Section A(6) (for these tests the resultant supernatant would be substituted for serum); except that a single 1:20 dilution hemaggl utination inhibition (HI) test may be used as a screening test in accordance with the procedures set forth in Section A(6) . (ii) For egg yolk samples being tested to retain the U.S. H5/H7 Avian Influenza Monitored classification, test the resultant supe rnatant in accordance with the requirements in 9 CF R 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. **(8) Standard test procedure**  **s 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 antige ns, namely the ribonucleoprotein (RNP) and matrix (M) proteins. Therefore, this test will detect antibodies to all influenza A viruses, regardless of subtype. The AGID test can also be used as a group - specific test to identify isolates as Type A influenza viruses. The method used is similar to that described by Beard. 6 The basis for the AGID test is the concurrent migration of antigen and antibodies toward each other through an agar gel matrix. When the antigen and specific antibodies come in contact, they combine to form a precipitate that is trapped in the gel matrix and produces a visible line. The precipitin line forms where the concentration of antigen and antibodies is optimum. Differences in the relative concentration of the antigen or antibodies will shift the location of the line towards the well with the lowest concentration or result in the absence of a precipitin line. Electrolyte concentration, pH, temperature, and other variables also affect precipitate formation. 1 . Materials needed. (i) Refrigerator (4 °C). (ii) Freezer ( −20 °C). (iii) Incubator or airtight container for room temperature (approximately 25 °C) incubations. (iv) Autoclave. (v) Hot plate/stirrer and magnetic stir bar

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

– 785. 1970.National Poultry Improvement Plan Program Standards

[DRAFT] 24 (optional). (vi) Vacuum pump. (vii) Microscope illuminator or other appropriate light source for viewing re sults. (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) Pipetti ng device capable of delivering 50μl portions. (xi) Common laboratory supplies and glassware

— Erlenmeyer flasks, graduated cylinders, pipettes, 100 × 15 mm or 60 × 15 mm petri dishes, flexible vacuum tubing, side - arm flask (500 mL or larger), and a 12 - o r 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 opti onal). 3 . Preparing the avian influenza AGID agar. (i) Weigh 9 gm of agarose and 80 gm of NaCl and add to 1 liter of PBS (0.01 M, pH 7.2) in a 2 liter Erlenmeyer flask. (ii) To mix the agar, either: (A) Autoclave the mixture for 10 minutes and mix the contents by swirling after removing from the autoclave to ensure a homogeneous mixture of ingredients; or (B) Dissolve the mixture by bringing to a boil on a hot plate using a magnetic stir bar to mix the contents in the flask while heating. After boiling, allow the agar to cool at room temperature (approximately 25 °C) for 10 to 15 minutes before dispensing into petri plates. (iii) Agar can be dispensed into small quantities (daily working volumes) and stored in airtight containers at 4 °C for several weeks, and melted and dispensed into plates as needed. Note: Do not use agar if microbial contamination or precipitate is observed. 4 . Performing the AGID

— (i) Detection of serum antibodies. (A) Dispense 15 to 17 mL of melted agar into a 100 × 15 mm petri plate or 5 to 6 mL agar into a 60 × 15 mm petri plate using a 25 mL pipette. The agar thickness should be approximately 2.8 mm. (B) Allow plates to cool in a relatively dust - free environment with the lids off to permit the escape of water vap or. The lids should be left off for at least 15 minutes, but not longer than 30 minutes, as electrolyte concentration of the agar may change due to evaporation and adversely affect formation of precipitin lines. Note: Plates should be used within 24 hours after they are poured. (C) Record the sample identification, reagent lot numbers, test date, and identification of personnel performing and reading the test. (D) Using the template, cut the agar after it has hardened. Up to seven template patterns can be cut in a 100×15 mm plate and two patterns can be cut in a 60×15 mm plate. (E) Remove the agar plugs by aspiration with a 12 - to 14 - gauge cannula connected to a side arm flask with a piece of silicone or rubber tubing that is connected to a vacuu m 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 pipett e 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 prov iding 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 f igure 2). (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 plates 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 positiveNational Poultry Improvement Plan Program Standards

25 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 te st 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 te st 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 lin e 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 - s pecific line does not form a continuous line with positive control lines.

(b) The 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.National Poultry Improvement Plan Program Standards

[DRAFT] 26 

### Subpart B **— Bacteriological**  **Examination Procedure (**  **1 )**  **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 and meat - ty pe chicken breeding flocks positive for Salmonella enteritidis after environmental monitoring should be examined as described in Section B(2) (a) of th ese Program Standards , with the following exceptions and modifications allowed due to the high number of birds required for examination: Except when visibly pathological tissues are present, direct culture, Section B(2)(a)1 of th e s e standards , may be omitted . **(2) Laboratory procedure recommended for the bacteriological examination of salmonella**  **from birds .**  (a) For egg

- and meat

- type chickens, turkeys, waterfowl, exhibition poultry, and game birds. 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 se ction: Provided, That in turkeys, if there are more than four reactors to the pullorum - typhoid tests in the flock, a minimum of four reactors as provided for in 9 CFR 145.14(a)(6)(ii) 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, missh apen ova or testes, inflamed or unabsorbed yolk sac, and other visibly pathological tissues where purulent, necrotic, or proliferative lesions are seen (including cysts, abscesses, hy popy on, and inflamed serosal surfaces) should be sampled for direct cultu re using either flamed wire loops or sterile swabs. Since some strains may not dependably survive and grow in certain selective media, inoculate nonselective plates (such as blood or nutrient agar) and selective plates (such as MacConkey [MAC] and brillia nt green novobiocin [BGN] for pullorum - typhoid and MAC, BGN, and xylose - lysine - tergitol 4 [XLT 4] for SE). Refer to illustration 1 for recommended bacteriological recovery and identification procedures. 7 Pr oceed immediately with collection of organs and ti ssues for selective enrichment culture. 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 t he 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 pat

hological sites where purulent, necrotic, or proliferative lesions are seen. 3 . Fro m 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. Incubate the sample at 37 C or 42.0 C for 20 - 24 hours. Follow the procedure outlined in illustration 1 for the isolation and identification of Salmonella. 4 . Fro m each bird, aseptically collect 10 to 15 grams of each of the following parts of the digestive trac t: Crop wall, duodenum, jejunum (including remnant of yolk sac), both ceca, cecal tonsils, and rectum - cloaca. Mince, grind, or

7 Biochemical identification charts may be obtained from “A Laboratory Manual for the Is olation and I dentification of Avian Pathogens,” chapter 2, Salmonellosis. Fourth edition, 1998, American Association of Avian Pathologists, I nc., Kennett Square, PA 19348.National Poultry Improvement Plan Program Standards

27 blend tissues and pool them into a sterile plastic bag. Do not pool tissues from different birds into the same sample. Add suffic ient tetrathionate enrichment broth to give a 1:10 (sample to enrichment) ratio. Incubate the sample at 37 C or 42.0 C for 20 - 24 hours. Fo llow the procedure outlined in illustration 1 for the isolation and identification of Salmonella. 5 . After selectiv e enrichment, inoculate selective plates (such as MAC and BGN for S. pullorum and S. gallinarum and MAC, BGN, and XLT 4) for SE. Incubate the plates at 37C for 20 - 24 hours . Inoculate three to five Salmonella suspect colonies from plates into triple sugar iron (TSI) and lysine iron agar (LIA) slants. If there are no suspect colonies after 24 hours of incubation, incubate the plates an additional 24 hours before considering negative. Screen colonies by serologi cal ( i.e. , serogroup) and biochemical procedures (e.g., the Analytical Profile Index for Enterobacteriaceae [API]) as shown in illustration 1. 6 . If the initial selective enrichment is negative for Salmonella, a delayed secondary enrichment (DSE) procedu re is used. Leave the tetrathionate - enriched sample at room temperature for 5 to 7 days. Transfer 1 mL of the culture into a tube containing 10 mL of fresh tetrathionate enrichment broth, incubate at 37 C for 20 to 24 hours, and plate as in Section B(5) . 7 . Serogroup all isolates identified as salmonellae and serotype all serogroup D1 isolates. Phage - ty pe all SE isolates.National Poultry Improvement Plan Program Standards

[DRAFT] 28National Poultry Improvement Plan Program Standards

29

**(3)Procedures for collection, isolation, and identification of Salmonella**  **from house environmen tal samples,**  **cloacal swabs, and hatchery 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 identifyin g 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. Biosecurity precautions, including using disinfected sampling equipment, sterile sampling supplies, and personal cleanliness, should be followed. Hands should be carefully washed with a sanitizing soap before sampling. Outer clothing, including gloves, should be changed between visits to different premises so that clean clothing is worn upon e ntering each premises. A minimum size of 3 inches by 3 inches should be used for the sterile gauze pads. Used and clean apparel and sampling materials should be kept separate. Boots or footwear should be cleaned and disinfected between visits to different premises. Disposable caps or hairnets 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 1 day. If delivery is delay ed, samples should be refrigerated. (a) **For egg**

**- and meat** 

**- type chickens, turkeys, waterfowl, exhibition poultry, and game birds**  . All samples and swabs described in this paragraph should be cultured in accordance with illustration 2. All Salmonellae recovered shall be serogrouped or serotyped. (1) **Poultry house e**  **nvironmental samples:** (i) Fecal material, litter or dust . With a clean gloved hand or sterile collection device, collect fecal material, litter, or dust from several locations representing all areas of the pen or house into a sterile bag or container. A suggested number of samples would be 5 samples from pens or houses with less than 500 birds; 10 samples from pens or houses with 500 to 2,500 birds; and 15 samples from pens or h ouses with more than 2,500 birds. (ii) Drag swabs (DS). DS, which consist of gauze pads or commercially available sponges, enable the sampling of large areas of the pen or house. (A) Preparation. DS may be purchased commercially or prepared by the u ser. One suggested method of making the DS assemblies is as follows: A sterile gauze pad is folded in half and a 2 - foot - long (60 cm) piece of twine is securely attached to the folded pad using a paper clip, staple, or similar device. A second sterile gauze pad is similarly fastened to a 5 - foot (150 cm) long piece of twine. The shorter piece of twine is then tied to the longer piece producing a DS sample set of 2 swabs arranged in a Y - shaped configuration. Alternatively, two separate DS samplers may be prepa red. The twine is wrapped around the swabs, and the swabs are moistened with double - strength skim milk (DSSM). The moistened swabs are placed in an instrument package. The sterilized swabs contained in the instrument pack may be frozen to prevent drying un til use. (B) Procedure. At the farm the thawed DS assemblies are unraveled and the ends of the twine held in gloved hands. The swabs are dragged across the environmental surfaces of the house for 15 min utes or the length of the house (down and back). One set of swabs ( two individual pads) is dragged across the center of the house floor and another set of swabs ( two individual pads) is dragged across the inside perimeter of the house floor. The four pads are individually placed in labeled, sterile bags . If necessary to prevent drying out, additional DSSM (evaporated skim milk) may be added to the bags. The bags should be protected from excessive heat and submitted as soon as possible to the authorized laboratory for testing. If the samples cannot be sub mitted to the laboratory the same day, they should be stored at 2 ° to 4 °C or placed in a cooler with ice or ice packs for no more than 5 days before culturing. (iii) Shoe cover swabs. Absorbable fabric shoe covers involve the exposure of the bottom sur face of shoe covers toNational Poultry Improvement Plan Program Standards

30 the surface of floor litter and slat areas. Wearing clean gloves, place the shoe covers over footwear that is only worn inside the poultry house. This can be footwear dedicated to the facility or disposable overshoes. Each pair of s hoe covers should be worn while walking at a normal pace over a distance of 1,000 feet (305 meters). For flocks with fewer than 500 breeders, at least 1 pair of shoe covers should be worn to sample the floor of the bird area. For flocks with 500 or more br eeders, at least 2 pairs of shoe covers should be worn to sample the floor of the bird area. After sampling, place each shoe cover in a sterile container with 30 ml of double strength skim milk, unless pre - moistened swabs are used. Seal the sterile contai ners and promptly refrigerate them at 2 to 4 °C or place in a cooler with ice or ice packs. Do not freeze. Samples should be stored at refrigerator temperatures of 2 to 4 °C no more than 5 days prior to culturing. (iv) Nest box or Egg belt swabs as a lternative sampling source (A) Two sterile premoistened (ex. DSSM) gauze pads or sponges are swabbed inside approximately 10 percent of the nest boxes. Each swab or sponge is placed into a separate sterile bag and submitted to the authorized laborato ry. (B) Two sterile premoistened (ex. DSSM) gauze pads or sponges are used to swab the egg belts. At least 30 feet of belt material is swabbed with each swab. Each swab is place d into a separate sterile bag and submitted to the authorized laboratory. **(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 this section. A sterile cotton - tipped applicator or swab is inserted into the cloaca and rectum of the bird in a manner that ensure s the collection of fecal material. The applicator may be broken off into a sterile tube. The cloacal swabs may be combined in multiples of five or in combinations specified by the authorized laboratory. **(3 ) Hatchery samples**  . Hatchery - related samples, such as chick box papers, meconium, and fluff, may be examined for the presence of Salmonella to indicate the transfer of Salmonella from parent to offspring. (i) Chick box papers . Chick box paper samples may be collected by an authorized agent according to paragraph (a)(3)(i)(A) of this section or may be submitted directly to an authorized laboratory for testing according to paragraph (a)(3)(ii)(B) of this section. It is important to remove the paper from the chick box before the box is placed in the brooding house. (A) Instructions for sampling chick box papers. One chick box paper is collected for every 10 boxes of chick s placed in a house. With sanitized and gloved hands lay out the papers on a clean, disinfected surface. Saturate a sterile gauze pad or sponge with DSSM and swab the surface of five chick box papers. The pad should be rubbed over approximately 75 percent of each paper with sufficient pressure to remove any dried meconium. Addition of more DSSM may facilitate sampling. The process is repeated with a second swab and the other five chick box papers. Both swabs may be added to a single sterile, labeled plasti c bag and submitted to the authorized laboratory. Promptly refrigerate the Whirl - Pak bags containing the samples and transport them, on ice or otherwise refrigerated, to a laboratory to be cultured within 5 days of collection. (ii) The Plan participant may send chick box papers directly to a laboratory, where samples may be collected as described in paragraph (a)(3)(i)(A) of this section. To send chick box papers directly to a laboratory: (A) Collect 1 chick box paper for each 10 boxes of chicks plac ed in a house and place the chick papers immediately into large plastic bags and label 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.National Poultry Improvement Plan Program Standards

31

(iii) Chick meconium . After collection, the container of meconium is mixed to obtain a uniform consistency. In the laboratory a 25 - gram sample will be removed for bacteriological examination. (iv) Fluff . Fluff samples may be collected from the floor of the hatcher y or from the tray following hatching. The fluff sample may be collected by either swabbing the floor or tray with a premoistened gauze pad or sponge or by placing fluff material directly into a sterile bag. **(b) Isolation and identification of Salmonella**  . There are two enrichment procedures approved for the isolation of Salmonella as described in this section ( See Illustration 2). Alternatively, approved rapid methods may be used to detect the presence of Salmonella , positive samples of which must then be isolated. (1) Direct tetrathionate enrichment followed by MSRV enrichment ( Illustration 2). (i) Fresh t etrathionate enrichment broth is added to the sample to give a 1:10 (sample to en richment) ratio. Incubate the samples at 37 ° C or 42 ° C for 20 to 24 hours. (ii) After incubation transfer approximately 100 microliters (3 drops ) of the enriched culture into (subsurface) a n MSRV plate. Incubate the plate right side up at 42 ° C for 2 4 hours. (iii) Observe the MSRV plate for growth migrating from the point of inoculation. If present, insert a sterile loop into the outer edge of the zone of growth and inoculate selective agar plates, such as BGN and XLT4. (iv) If no zone of grow

th is present, incubate the MSRV plate at 42 ° C for another 24 hours. Observe the MSRV plate for growth migrating from the point of inoculation. If growth is present, insert a sterile loop into the outer edge of the zone of growth and inoculate selective agar plates, such as BGN and XLT4. If still no zone, insert the loop into the point of inoculation and inoculate selective agar plates. This ensures that weakl y or non - motile strains of Salmonella will not be missed. (v) Incubate the selective agar plates at 37 ° C for 20 to 24 hours. Observe the plates for Salmonella suspect colonies. Screen 3 to 5 colonies by inoculating them individually into triple sugar iron agar (TSI) and lysine iron agar (LIA) slants or equivalent method. Incubate the slants at 37 ° C for 20 to 24 hours. Screen the colonies by serological (i.e., serogroup) or biochemical (e.g. , API) procedures as shown in Illustration 2. (vi) Serogro up all isolates identified as Salmonella and serotype all serogroup D isolates. Phage type one SE isolate per flock per submission. (2) Pre - enrichment followed by selective enrichment. (Illustration 2.) (i) Pre - enrichment broth (e.g. buffered peptone water, BPW) is added to the sample to give a 1:10 (sample to enrichment) ratio. Incubate the sample at 37 ° C for 20 to 24 hours. (ii) Transfer 1 ml of the pre - enriched sample into a tube containing 10 ml of tetrathionate enrichment broth and tr ansfer 0.1 ml into either a tube containing 10 ml of Rappaport - Vassiliadis (RV) enrichment broth or into a MSRV plate. Incubate at 42 ° C for 20 to 24 hours. (iii) After incubation inoculate the tetrathionate and RV enrichments onto separate selective a gar plates, such as BGN and XLT4. If the MSRV media was inoculated then follow the steps in (1)(iii) and (1) (iv). (iv) Screen the selective agar plates for Salmonella as described in (1)(v) and (1)(vi). ( 3) Approved rapid methods for the detection of Salmonella . (i) Rapid methods may be approved for detecting Salmonella by the NPI P as set forth in 9 CF R 147.52.National Poultry Improvement Plan Program Standards

32 (ii) The enrichment and testing procedures used for the respective rapid method are those recommended by the manufacturer and approved by the NPIP. (iii) Samples that are positive with the rapid method must be confirmed as Salmonella by inoculation of selective plating media, such as BGN and XLT4, from the enrichment broths used in the detection process. (iv) Follow the iso lation procedures in (b)(1)(v) of this section.National Poultry Improvement Plan Program Standards

33



### Il lustration 2. Culture procedures for house environmental samples, cloacal swabs, and hatchery samplesNational Poultry Improvement Plan Program Standards

34 3 . Approved rapid methods f or the detection of Salmonella. (i) Rapid methods may be approved for detecting Salmonella by the NPIP under guidelines in 9 CF R 147.52 . (ii) The enrichment and testing procedures used for the respective rapid method are those recommended by the manufacturer and approved by the NPIP. (iii) Samples that are positive with the rapid method must be confirmed as Salmonella by inoculation of selective plating media, such as BGN and XLT4, from the enrichment broths used in the detection process. (iv) Follow the isolation procedures in (b)(1)(v) of this section. **(4) 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 collectio n 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 ma intain a record showing that the required instruction was given to each State Inspector. (a) Sample selection. Forty eggs in the top flats of each of three randomly selected cases of sanitized eggs from each flock will be u s ed for each sampling. (b) Swab procedure. 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 . Fro m the tube containing four swabs and lactose broth or other suitable media, 1 ml. will be transferred to 10 ml. lactose in a f

ermentation 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. **(5) Procedures to**  **determine status and effectiveness of sanitation monitored program.** The following monitoring procedures 8 ma y be applied at the discretion of the Official State Agency: (a) Monitor effectiveness of sanitation program. 1 . Culture the surface of cased egg s periodically for fecal contaminating organisms as described in Section B(4). 2 . Culture a sample of dead - in - shell eggs periodically from each breeding flock for coliforms. Such eggs should also be cultured for the dependable recovery of salmonellae. Cu lturing for the dependable recovery of salmonellae should include the use of: (i) Pre - enrichment broths supplemented with 35 mg ferrous sulfate per 1,000 ml pre - enrichment 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 these Program Standards . **(6) Laboratory procedure recommended for the bacteriological examination of mycoplasma reactors.**  **9** (a) Turbinates, trachea, air sacs, sinuses, nasal passages, respiratory exudates, synovial fluid, eggs (including yolk, yolk sacs, membranes and allantoic f luid), should be directly sampled with sterile swabs **.** 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 s ites listed above and occasionally from the oviduct

8 La boratory procedures for monitoring operations proposed here are described in the following two publications: I solation and I dentification of Avian Pathogens, American Association of Avian Pathologists, University of Pennsylvania, New Bolton Center, Kennett Square, Pennsyl vania 19348

– 1692, 1980, and Culture Methods for the Detection of Animal Salmonellosis and Arizonosis, I owa State University Press, Ames, I owa 50010, 1976. 9 Yoder, H. W., Jr., “Mycoplasmosis.” In: Isolation and I dentification of Avian Pathogens. (Stephen B. Hitchner, Chairman, Char les H. Domermuth, H. Graham Purcha se, James E. Williams.) 1980, pp. 40

– 42, Creative Printing Company, I nc., Endwell, NY 13760.National Poultry Improvement Plan Program Standards

35

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 o rder of preference for recovery of the mycoplasma organisms are: live birds, refrigerated fresh tissues, and tissue specimens packed with dry ice. (b) Inoculate 5

– 10 ml of MBM with a swab, wire loop or 0.1 ml of the tissue suspension. When evidence of gro wth 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. Incubate plates 3

– 5 days at 37 °C in a high humidity chamber. If preferred, 5 percent CO 2 ma y 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 microsco pe are recommended for observation of the colonies as they are rarely more than 0.2

– 0.3 mm in diameter. (c) Isolates must be serotyped. 1 . Isolates may be shipped in MBM with ice packs if shipment will be in transit less than 2 to 3 days. Longer shipmen ts 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 sever al years. (d) Alternate method of culture: An overlay enrichment culture for fastidious and sensitive mycoplasma, especially for M. meleagridis should be included. 1 . Po ur 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 t

he culture as indicated in paragraph (b) of this section. 4 . Incubate and examine the overlay as indicated in paragraph (b) of this section. (e) Preparation of media components: 10 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. 11 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. 12 Thallium acetate is added to deionized distilled water first, except as noted i n 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. 13 Thallium acetate an d 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. S wine 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. 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. 14 9 . Cy steine hydrochloride, prepared as a 1 percent solution, is used to reduce the NAD for M. synoviae growth.

10 Trade names are used in these procedures solely for the purpose of providing specific information. Mention of a trade name do es not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture or an endorsement over other products not mentioned. 11 Alcojet is available from: Alconox, Inc., New York, NY 10003. 12 Thallium acetate may be obtained from Fischer Scientific Company. 13 Myco plasma Broth Base may be obtained from: (a) Product #M 33600, Gibco Diagnostics, 2801 I ndustrial Drive, Madison, WI 53711. (b ) Product #3900

– 3212, Scott Laboratories, I nc., 8 Westchester Plaza, Elmsford, NY 10523. 14 NAD Grade III may be obtained from: Sig ma Chemical Company, P.O. Box 14508, St. Louis, MO 63178.National Poultry Improvement Plan Program Standards

36 1 0. A purified agar product such as Nobel (Spec ial agar) is used in the MAM. 15 1 1. Adjust the pH with NaOH. 1 2. Sterilization may be accomplished by two methods: (i) Filtration sterilization through a 0.20 micron filter is the recommended method. Aseptic techniques must be u s ed. (ii) Autoclav e 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. meleagri dis and M. gallinarum. 14 . When culturing for M. meleagridis from contaminated samples include 100 units/ml of Polymyxin B in MBM. (f) Mycoplasma Broth Medium (Frey) is prepared as follows: To 850

– 880 ml of deionized distilled water , a dd:  Thallium ace tate (ml)

— 2.5 (1:4000)  Potentially contaminated samples (ml)

— 5.0 (1:2000)  My coplasma 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  Cy steine hy drochloride (ml)

— 12.5  Dextrose (g)

— 1.0

– 1.5 Adjust pH to 7.8 . Filter sterilize . Broth may be stored at 4 °C for at least 2 weeks or at −40 °C for longer periods. (g) Mycoplasma Agar Medium (Frey) is prepared as follows: To 850

– 880 ml of deionized distilled water , a dd: My coplasma 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 Cy steine hy drochloride (ml)

— 12.5 1 . Rotat e flask gently and pour about 15 ml of media into each petri dish. 2 . Stack petri dishes only 2 to 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 day s. (h) New component or media batches should be monitored to compensate for changes in formulation due to alterations of purity, concentration, preparation, etc. 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 . 16

15 Noble Agar may be obtained from: Difco Laboratories, Box 1058

– A, Detroit, MI

48201. 16 “L aboratory Procedures and Medium For The I solation Of Mycoplasma From Clinical Materials.” Laboratory Diagn osis 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.National Poultry Improvement Plan Program Standards

37

**( 7**  **) 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. 17 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 anim als. 3 . Personnel caring for the test birds should take the necessary precautions (see Section C(6) (b)) to prevent the mechanical transfer of infectious avian diseases from other sources. (b) Test birds to be used for inoculation with contaminated tissu es 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 M ycoplasma Broth Medium (Frey) (see Section B(6) (f)). Suspensions may be made from tissue pools. Inoculate test birds within 30 minutes for preparation of suspensions. 1 . Inoculate at least four test birds for each suspension pool via the abdominal air sa c and infraorbital sinus, with up to1/2ml 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 mycoplas ma attempted (see Section B(6) ). 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 bir ds 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 b irds. **(8) 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 - ty

pe and meat - ty

pe chicken flocks and waterfowl, exhibition poultry, and game bird flocks and poults from turkey flocks for salmon ella. (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 with in 10 days after hatching, prepare organ pools, yolk pools, and intestinal pools as follows: 1 . Organ pool: Fro m 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 w all of the bursa of Fabricius for chicks only. 2 . Yolk pool: Fro m 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. 3 . Intest inal pool: Fro m each of five chicks or two poults, composite and mince approximately 0.5 cm 2 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 brot h (Hajna or Mueller - Kauffmann) at a ratio of 1 part

17 Research results are described in the f ollowing 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 Respir atory Tract of the Fowl.” I

n: The Veterinary Record, September 23, 1967, pp. 317

– 320.National Poultry Improvement Plan Program Standards

38 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 enri chment in illustration 2 of these Standards . Incubate the organ and yolk pools for 24 hours at 37 °C and the intestinal pools at 41.5 °C. Plate as described in illustration 2 of these Standards 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. A colony lift assay may also be u s ed as a s upplement to TSI and LI agar picks of suspect colonies.National Poultry Improvement Plan Program Standards

39 

### Subpart C **— Sanitation Procedures**  **( 1**  **) 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 footwea

r and change of outer clothing, to prevent the introduction of infection through droppings that ma y adhere to the shoes, clothing, or hands. (See Section C 4(a).) (b) Range used for growing young stock should not have been used for poultry the preceding ye ar. 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, sh eep, 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 Section C4 (a).) Feed and water containers should be situated where they cannot be contaminated by droppings and should be frequently cleaned and disinfected. D ropping 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 to permit free passage of droppings and to prevent the birds from coming i n 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 laborat ory

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) Introdu ction 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. (h) In rearing broiler or rep lacement 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. **( 2**  **) 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 hatc hing. Egg handlers should thoroughly wash their hands with soap and water before 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 th e nest eggs. Slightly soiled nest 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 Section C(4) of these Standards . (d) The egg processing area should be cleaned and disinfected daily. (e) Effective rodent and insect control programs should be implemented.National Poultry Improvement Plan Program Standards

40 (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 easil y, 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 Section C(4) . **( 3**  **) 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 ar eas ( 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, shou ld 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 d estroyed. (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. **( 4**  **) Cleaning and**  **disinfecting.** The following procedures are recommended: (a) In the poultry houses: 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 decontam inate 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 sp ray (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. 4 . Spray with a d isinfectant 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. (b) In the hatchers and hatc hery rooms: 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 swee ping, 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 (minimu m water temperature of 140 °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 fat ty deposits. Allow the chemical to cling to treatedNational Poultry Improvement Plan Program Standards

41

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 nea r 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 th e trays, preferably still wet from cleaning, and bring the incubator to normal operating temperature. 3 . The hatcher should be fumigated ( see Section C(5) ) or otherwise disinfected before transferring the eggs. 4 . If the same machine is used for incuba ting 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 Section C( 5 ) ) or otherwise sanitize d. (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 or poults: 1 . Spray truck tires thoroughly with disinfectant before leaving the main road a nd 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 or coveralls and place into a 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 boo ts 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. **( 5**  **) Fumigation.**  Fu migation ma y 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, 29 CFR 1910.1048. **(6) Procedures for establishing isolation and maintaining sanitation and good management practices for the**  **control of Salmonella and Mycoplasma infections.** (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: 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 proced ures as approved by the Official State Agency . 3 . Dispose of all dead birds by locally approved methods. (b) Recommended procedures **:** 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 . Minimiz e 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 ea ch 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 .National Poultry Improvement Plan Program Standards

42 12 . Seek services of a veterinary diagnostician if unaccountable mortality or signs of disease occur . 13 . Adopt and maintain a clean - egg program. 14 . Use only crates and vehicles that have been cleaned and disinfected in accordance with Section C(4)(a) to h aul live poultry t o and from the premises. **( 7**  **) 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 o f 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 on 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 m oved 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 collect ion 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. 

### ( **8) Hygiene and Biosecurity Procedures for Poultry Primary Breeding Flocks and Hatcheries**  (a) Recommendations applicable to breeding establishments 1 . The choice of a suitably isolated geographical location, taking into account the direction of the prevailing winds, facilitates hy giene and disease control. The establishment should be surrounded by a security fence and a gateway to control traffic and access to the site. A sign indicating restricted entry should be posted at the entrance. If a vehicle must enter the biosecure area, it must be cleaned and disinfected before entry. 2 . Poultry breeding establishments should be single - purpose. Single - species enterprises, and ideally an all - in, all out single age group principle, should be adopted whenever possible. 3 . Where several f

locks are maintained on one premises, the individual flocks should be managed as separate entities. 4 . Buildings housing poultry or used to store feed or eggs should be free of vermin and not accessible to wild birds. 5 . Poultry houses should be constr ucted so that all surfaces inside the buildings can be cleaned and disinfected. 6 . The area immediately surrounding the poultry houses should be free from debris and ideally this should consist of an area of concrete, gravel, or a similar material. Grass surrounding the poultry houses should be kept short. 7 . Domestic animals should not be permitted access to poultry houses. 8 . Appropriate disease security precautions should be adopted for all visitors to the premises and for all staff entering indivi dual poultry houses. 9 . When a poultry house or premises is depopulated, all manure should be removed from the houses and effective cleaning and disinfection procedures applied. Bacteriological monitoring of the efficacy of disinfection procedures is rec ommended. When necessary, rodent and insect control procedures should also be carried out. 10 . Repopulation of poultry houses or premises should only be made from poultry flocks that are of known high health status and that are regularly monitored for Sa lmonella and other poultry pathogens. 11 . All feed used in poultry houses should be manufactured and stored in such a manner that prevents exposure to wild birds, waterfowl, and rodents. 12 . The water supply to poultry houses should be of a satisfactory potable status. 13 . Sick and dead birds should be removed from poultry houses as soon as possible and effective and safe disposal procedures implemented.National Poultry Improvement Plan Program Standards

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### 14 . Full records relating to morta lity, disease diagnosis, treatments, and vaccinations should be maintained on an individual flock basis. Such records should be readily available for inspection. (b) Recommendations applicable to hatching egg hygiene and transport 1 . The litter in the laying house should be kept dry and in good condition. 2 . Eggs should be collected at frequent intervals of not less than twice per day and placed in clean disinfected containers. 3 . Dirty, broken, cracked, leaking, and dented eggs should be collected in a separate container and should not be used for hatching purposes. 4 . The clean eggs should be sanitized as soon as possible after collection. The methods of sanitation are described in Section D( 7 ) . 5 . The sanitized eggs should be stored in a clean, dust - free room used exclusively for this purpose. 6 . The eggs should be transported to the hatchery in new or clean containers that have been fumigated or sanitized with a liquid disinfectant. The cleaning and disinfection of vehic les must be a regular part of the hatchery routine. (c) Recommendations applicable to hatchery buildings 1 . The design of the hatchery should be based on suitable work flow and air circulation principles. It should be constructed so that there is a one - way flow for the movement of eggs and chicks. The air flow should follow the same one - way direction. 2 . The hatchery buildings should include physical separation of all work areas. If possible, separate ventilation should be provided for these work are as, namely, the rooms for: (i) Egg receiving and egg storage . (ii) Egg traying . (iii) Fumigation . (iv) Setting or initial incubation . (v) Hatching . (vi) Sorting, sexing, and placing chicks in boxes . (vii) Material storage, including eg g and chick boxes, egg flats, box pads, chemicals and other items . (viii) Facilities for washing equipment and disposal of waste . (ix) Employee meals . (x) Other office functions. 3 . Openable windows, ventilators, and other open areas should be screened against insects and vermin. (d) Recommendations applicable to hatchery building hygiene 1 . The area adjacent to the hatchery buildings should have limited access to traffic, and access into the building should be restricted. 2 . Wild birds and domestic and wild animals must be excluded from the hatchery area. When necessary, a specific program for fly control should be implemented. 3 . The hatchery area should be maintained free from all hatchery waste, garbage of all kinds, and discarded equipment. 4 . Approved disposal methods and adequate drainage must be available. 5 . All hatchery equipment, tables, and horizontal surfaces in roo ms must be promptly and thoroughly cleaned of organic material, washed, scrubbed, rinsed with clean water, and finally disinfected with an approved disinfectant. ( e) Requirements applicable to personnel and visitors 1 . Clean clothing and footwear must b e provided for all personnel and visitors entering the premise or the hatchery. 2 . Clean and disinfected footwear or a means to clean and disinfect footwear should be available at each entrance. Washing the hands in disinfectant solution or with soap and water should be required. 3 . Personnel and visitors should have no direct contact with other poultry or poultry products. (f) Hygiene measures during the handling of eggs and day - old birds 1 . Day - old poultry must be delivered or distributed in new ch ick boxes; or in used boxes made of suitable material which have been thoroughly cleaned and disinfected or fumigated. 2 . The day - old poultry should be delivered directly from the hatchery by personnel wearing clean, disinfectedNational Poultry Improvement Plan Program Standards

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### outer clothing. Outer clo thing should be changed or disinfected between each delivery. 3 . The delivery truck must be cleaned and disinfected before loading each consignment of day - old poultry. (g) Sanitation of hatching eggs and hatchery equipment Sanitation means: (i) Fumig ation with formaldehyde, or (ii) Spraying with or immersion in an egg shell disinfectant in accordance with the manufacturer’s instructions, or (iii) made hygienic by another method approved by the veterinary authority. (iv) Should notifiable avia n influenza (NAI) be detected within a S tate where a primary breeder has production facilities then the biosecurity procedures for breeding flocks and hatcheries will be implemented in accordance with the USDA - approved S tate response and containment plan f or NAI.National Poultry Improvement Plan Program Standards

45 

### Subpart D **— Molecular Examination Procedures**  **(1) Laboratory procedure recommended**  **for the polymerase chain reaction (PCR) test for Mycoplasma gallisepticum and M. synoviae.**  (a) DNA isolation **.** Isolate DNA from 1 mL of eluate from tracheal swabs in PBS or 1 mL of broth culture by a nonphenolic 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 spectrop hotometric reading at 260 nm and 280 nm. (b) Primer selection **.** 1 . M. gallisepticum. The primer for M. gallisepticum should consist of the following sequences: 2 . M. synoviae. The primer for M. synoviae should consist of the following sequences: (c ) Polymerase chain reaction. (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 (50 μM), 4 μl MgCl 2 (25 mM), 1 μl t aq - polymerase (5 U), 32 μl DEP water. (ii) 18 μl water, 25 μl 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. 18 The optimized PCR program is :

**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). (d) Electrophoresis. 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) am plicons can be visualized under an ultraviolet transilluminator along with the PCR marker (50 to 2000 bp; Sigma). **(2) Laboratory procedures recommended for the real**  **- time polymerase chain reaction test for Mycoplasma**  **gallisepticum (MGLP ReTi).** (a) DNA extraction . Use Qiagen Qiamp Mini Kit for DNA extraction or equivalent validated technique/procedure. This kit u s es the following methods: 100 μl of swab suspension incubates with 10 μl of proteinase K and 400 μl of lysis buffer at 56 °C for 10 minutes. Fo llowing incubation, 100 μl of 100 percent ethanol is added to lysate. Wash and centrifuge following extraction kit recommendations.

18 Trade names are used in these procedures solely for the purpose of providing specific information. Mention of a trade name do es not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture or an endorsement over other products not mentioned.National Poultry Improvement Plan Program Standards

46 (b) Primer selection . 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 Taqman dual - labeled prob e mglpprobe (5' - FA M

– 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. (c) MGLP ReTi . Primers and probe sh ould be u s ed in a 25 μl reaction containing 12.5 μl of Quantitect Probe PCR 2X mix (Qiagen, Valencia, CA), 22 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 w ith optics OFF; and 40 cy cles of 94 °C for 15 seconds followed by 60 °C for 60 seconds with optics ON. (d) Determination of positive . For each MGLP ReTi assay reaction, the threshold cycle number (CT value) was determined to be the PCR cycle number at whi ch the fluorescence of the reaction exceeded 30 units of fluorescence. Fo r 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. (e) Controls . 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. 

### ( **3) Laboratory procedu res recommended for the conventional polymerase chain reaction test for**  **Salmonella Enteritidis.**  

### (a ) Sample Enrichment: Samples (drag swabs, chick paper swabs, etc.) are enriched in Tetrathionate enrichment for 18 - 24 hours 37 or 42.0°C and sub cultured onto Mo dified Semi - solid Rappaport - Vassiliadis (MSRV) Enrichment in accordance with procedures set forth in these program standards . 

### (b) Quality Control : A positive control, known Salmonella Enteritidis or ATCC strain, from BHI broth should be inoculated onto MSRV m edia, incubated for 18 - 24 hours and 1 - 3 plugs harvested from the zone of white precipitate growth outside the initial inoculation site. A negative control is harvested plugs from an area of uninoculated MSRV plate. (c) DNA extraction: DNA is extracted from 1 - 3 plugs (~100 L ) o f M S R V a g a r i n t h e z o n e o f m i g r a t i o n o u t s i d e o f i n i t i a l i n o c u l a t i o n site by boiling in 100  l PCR grade water for 10 minutes or by another DNA extraction method. Samples are cooled to room temperature before PCR us e or stored at 2 - 8  C, if PCR is not performed immediately. For the boiling method, the extracted samples are spun at 16,000 rcf for 3 minutes. The DNA is contained in the supernatant. (d) Primer Selection: The SE specific primers are: sdf I (forward)

– TG TGTTTTATCTGATGCAAGAGG sdf I (reverse)

– CGTTCTTCTGGTACTTACGATGAC. The internal control primers are: rpl I (forward)

– GGGTGATCAGGTTAACGTTAAAG rpl I (reverse)

– CTTCGTGTTCGCCAGTGGTACGC. (e) Polymerase chain reaction: The following multiplex - PCR reaction (2 sets of primers per reaction tube) should be set up in a 200  l PCR tube or a 25  l PCR tube, in a clean environment.National Poultry Improvement Plan Program Standards

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### Reaction Mix **50 L**  **V o**  **l u**  **m e**  **25 L**  **V o**  **l u**  **m e**  **Final Concentration** 10X PCR Gold Buffer 5  l 2.5  l 1X MgCl 2 

### (25mM) 5  l 2.5  l 2.5mM 10mM dNTP mix 3  l 1.5  l 150  M each sdf I Forward Primer 4  l 2.0  l 0.4  M sdf I Reverse Primer 4  l 2.0  l 0.4  M rpl I Forward Primer 4  l 2.0  l 0.4  M rpl I Reverse Primer 4  l 2.0  l 0.4  M Am plitaq Gold Polymerase 0.5  l 0. 25  l 2.5U Sterile PCR Grade Water 15.5  l 5.25  l DNA Template ***** 5.0  l 5.0  l ***** The DNA template is 5.0  l of the boiled MSRV solution or DNA from another DNA extraction method. (f) PCR amplification program: Polymerase Activation Step 95  C for 10 minutes 1 cycle Denaturation Step 95  C for 30 seconds Annealing Step 60  C for 30 seconds 35 cycles Extension Step 72  C for 1 minute Final Extension 72  C for 7 minutes 1 cycle 4  C Hold infinite hold until samples are refrigerated or frozen (g) Electrophoresis: After PCR is completed, samples should be analyzed by DNA electrophoresis. A 3 percent RAGE gel, or a 1 percent to 3 percent conventional gel with a sample volume of 3.5  l, in addition to 1.5  l of loading dye, is recommended. The sdf I p rimers will yield a 293bp band only in the presence of Salmonella Enteritidis DNA and the rpl I primers will yield a 343bp band in the presence of any bacterial DNA (the primers have worked well with every organism so far with the exception of Proteus mir abilis ). **Reference:** 1. Charlton BR, Walker RL, Kinde H, Bauer CR, Channing - Santiago SE, et al. (2005) Comparison of a Salmonella Enteritidis - Specific Polymerase Chain Reaction Assay to Delayed Secondary Enrichment Culture for the Detection of Salmonella En teritidis in Environmental Drag Swab Samples. Avian Diseases: Vol. 49, No. 3 pp. 418

– 422 . **(4) Laboratory procedures recommended for the real**  **- time polymerase chain reaction test for**  **Salmonella sp. Group D**  (a) Sample Enrichment: Samples (drag swabs, chick paper swabs, etc.) are enriched in Tetrathionate enrichment for 24 hours 37 or 42° C (see Section B(3) ) and subcultured onto Modified Semi - solid Rappaport - Vassiliadis (MSRV) Enrichment (see Section B(3) ). (b) Quality Control: A positive control, known Salmonella Enteritidis or ATCC strain, from BHI broth should be inoculated onto MSRV media, incubated for 18 - 24 hours and 1 - 3 plugs harvested from the zone of white precipitate growth outside the initial inoculation site. A negative control is harvested plugs from an area of uninoculated MSRV plate.National Poultry Improvement Plan Program Standards

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### (c) DNA extraction: DNA is extracted from 1 - 3 plugs (~100 L ) o f M S R V a g a r i n t h e z o n e o f m i g r a t i o n o u t s i d e o f i n i t i a l i n o c u l a t i o n site by boiling in 100  l PCR grade water for 10 minute s or by another DNA extraction method. Samples are cooled to room temperature before PCR use or stored at 2 - 8  C, if PCR is not performed immediately. For the boiling method, the extracted samples are spun at 16,000 rcf for 3 minutes. The DNA is contained in the supernatant. (d) Primer & Probe Selection: sefA F (forward): 22 bp sequence 5’GGCTTCGGTATCTGGTGGTGTA3’ (50 nM final concentration); (example: use volumes below if stock primer is 10  M concentration). sefA R (reverse): 24 bp sequence 5’GGTCATTAATATTGGCCCTGAATA3’ (900 nM concentration); (example: use volumes below if stock primer is 10  M concentration). sefA PR (probe): 25 bp sequence 5’FAM/CCACTGTCCCGTTCGTTGATGGACA/TAMRA’ or comparable quencher (250 nM concentration); (FAM = 3’ 5(6) - Carboxyfluorscein; TAMRA, 6 - carboxytetramethylrhodamine (quencher dye)); (example: use volumes below if stock primer is 10  M concentration). (e) PCR reactions: The following real - time PCR reaction (or equivalent) should be set up in a clean en vironment. The reaction should be assembled using aerosol resistant pipette tips to decrease the chance of contamination. Total volume for reaction is 50  l (Opticon) or 25  l (SmartCycler). **Reaction Mix 50**   **l Volume 25**   **l Volume Final Concentration**  10X P CR Gold Buffer 5  l 2.5  l 1X MgCl 2 

### (25mM) 5  l 2.5  l 2.5mM 10mM dNTP mix 4  l 2.0  l 200  M each Forward Primer ( sef

A F) 0.2  l 0.1  l 0.05uM Reverse Primer ( sefA R) 4.6  l 2.3  l 0.9 uM TaqMan probe ( sefA PR) 0.6  l 0.3  l 0.25uM Am plitaq Gold Polymerase 0.25  l 0.13  l 1.25U Sterile PCR Grade Water 25.35  l 10.17  l DNA Template ***** 5.0  l 5.0  l ***** The DNA template is 5.0  l of the boiled MSRV solution or DNA from another DNA extraction method. (f) PCR amplification program: Pre - incubation 50  C for 2 minutes 1 cycle Taq activation 95  C for 10 minutes 1 cycle Annealing Step 95  C for 15 seconds Extension Step 60  C for 60 seconds 40 cycles (g) Analysis of results: A sample with a Ct value less than 35 is considered positive. A sample with a Ct value between 35 and 40 is considered to be indeterminate. Positive and indeterminate results are to be further tested by culture methods .National Poultry Improvement Plan Program Standards

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### Reference: 1. Seto KH, Valentin - Bon IE, Brackett, RE, and Holt, PS. (2004) Rapid, Specific Detecti on of Salmonella Enteritidis in Pooled Eggs by Real - Tim e PCR. Journal Food Protection: Vol 67, No. 5, pp. 864 - 869