9 C.F.R. Subpart B—Bacteriological Examination Procedure
Title 9 - Animals and Animal Products
Birds selected for bacteriological examination from egg-type breeding flocks positive for Salmonella enteritidis after environmental monitoring should be examined as described in §147.11(a) of this subpart, with the following exceptions and modifications allowed due to the high number of birds required for examination: (a) Except when visibly pathological tissues are present, direct culture, §147.11(a)(1) of this subpart, may be omitted; and (b) Enrichment culture of organ (non-intestinal) tissues using a non- selective broth, §147.11(a)(2) of this subpart, may be omitted. [59 FR 12801, Mar. 18, 1994] (a) For egg- and meat-type chickens, 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 (paragraph (a)(1)) and selective enrichment (paragraph (a)(2)) procedures described in this section. Careful aseptic technique should be used when collecting all tissue samples. (1) Direct culture (refer to illustration 1). Grossly normal or diseased liver, heart, pericardial sac, spleen, lung, kidney, peritoneum, gallbladder, oviduct, misshapen ova or testes, inflamed or unabsorbed yolk sac, and other visibly pathological tissues where purulent, necrotic, or proliferative lesions are seen (including cysts, abscesses, hypopyon, and inflamed serosal surfaces) should be sampled for direct culture using either flamed wire loops or sterile swabs. Since some strains may not dependably survive and grow in certain selective media, inoculate non-selective plates (such as blood or nutrient agar) and selective plates (such as MacConkey [MAC] and brilliant green novobiocin [BGN] for pullorum-typhoid and MAC, BGN, and xylose-lysine-tergitol 4 [XLT 4] for SE). After inoculating the plates, pool the swabs from the various organs into a tube of non-selective broth (such as nutrient or brain-heart infusion). Refer to illustration 1 for recommended bacteriological recovery and identification procedures.7 7 Biochemical identification charts may be obtained from “A Laboratory Manual for the Isolation and Identification of Avian Pathogens,” chapter 2, Salmonellosis. Fourth edition, 1998, American Association of Avian Pathologists, Inc., Kennett Square, PA 19348. (2) Selective enrichment culture (refer to illustration 1). Collect and culture organ samples separately from intestinal samples, with intestinal tissues collected last to prevent cross-contamination. Samples from the following organs or sites should be collected for culture in selective enrichment broth: (i) Heart (apex, pericardial sac, and contents if present); (ii) Liver (portions exhibiting lesions or, in grossly normal organs, the drained gallbladder and adjacent liver tissues); (iii) Ovary-Testes (entire inactive ovary or testes, but if ovary is active, include any atypical ova); (iv) Oviduct (if active, include any debris and dehydrated ova); (v) Kidneys and spleen; and (vi) Other visibly pathological sites where purulent, necrotic, or proliferative lesions are seen. (3) From each bird, aseptically collect 10 to 15 grams of each organ or site listed in paragraph (a)(2) of this section. Mince, grind, or blend and place in a sterile plastic bag. All the organs or sites listed in paragraph (a)(2) of this section from the same bird may be pooled into one bag. Do not pool samples from more than one bird. Add sufficient tetrathionate enrichment broth to give a 1:10 (sample to enrichment) ratio. Follow the procedure outlined in illustration 1 for the isolation and identification of Salmonella. (4) From each bird, aseptically collect 10 to 15 grams of each of the following parts of the digestive tract: Crop wall, duodenum, jejunum (including remnant of yolk sac), both ceca, cecal tonsils, and rectum-cloaca. Mince, grind, or blend tissues and pool them into a sterile plastic bag. Do not pool tissues from different birds into the same sample. Add sufficient tetrathionate enrichment broth to give a 1:10 (sample to enrichment) ratio. Follow the procedure outlined in illustration 1 for the isolation and identification of Salmonella. (5) After selective enrichment, inoculate selective plates (such as MAC and BGN for pullorum-typhoid and MAC, BGN, and XLT 4) for SE. Inoculate three to five Salmonella-suspect colonies from plates into triple sugar iron (TSI) and lysine iron agar (LIA) slants. Screen colonies by serological (i.e., serogroup) and biochemical procedures (e.g., the Analytical Profile Index for Enterobacteriaceae [API]) as shown in illustration 1. As a supplement to screening three to five Salmonella-suspect colonies on TSI and LIA slants, a group D colony lift assay may be utilized to signal the presence of hard-to-detect group D Salmonella colonies on agar plates. (6) If the initial selective enrichment is negative for Salmonella, a delayed secondary enrichment (DSE) procedure is used. Leave the tetrathionate-enriched sample at room temperature for 5 to 7 days. Transfer 1 mL of the culture into 10 mL of fresh tetrathionate enrichment broth, incubate at 37 C for 20 to 24 hours, and plate as before. (7) Serogroup all isolates identified as salmonellae and serotype all serogroup D1 isolates. Phage-type all SE isolates. (b) For turkeys—(1) Bacteriological examination of Salmonella reactors and necropsy specimens. Grossly normal or diseased liver, heart, pericardial sac, spleen, lung, kidney, pancreas, peritoneum, drained gallbladder, oviduct, misshapen ova, testes, inflamed or unabsorbed yolk sac, and other visibly pathological tissues where purulent, necrotic, or proliferative lesions are seen (including cysts, abscesses, hypopyon, and inflamed serosal surfaces), should be directly cultured by means of a flamed wire loop or with sterile swabs.8 8 Culture media preparation and biochemical identification charts can be obtained from Culture Methods for the Detection of Animal Salmonellosis and Arizonosis, Committee on Salmonellosis and Arizonosis, AAVLD, 1976 Iowa State University Press, Ames, IA 50010. (2) Bacteriologic examination of environmental and other contaminated specimens. (i) Culture a representative sample of the specimen in tetrathionate Hajna (TTH) selective broth (TT Mueller-Kauffmann or selenite-cystine is also acceptable) as a temperature of 41–42 °C for 24 hours. Note: Do not use selenite-cystine if double strength skim milk is used as a preservative for the sample. (ii) Inoculate an agar late of brilliant green novobiocin (BGN) and an agar plate of xylose-lysine-tergitol 4 (XLT4), incubate at 37 °C for 24 hours, and retain culture tubes at room temperature for 5–7 days for possible reculturing of the negative tubes using 0.25 ml in TTH. (iii) Inoculate Salmonella suspect colonies to slants of triple sugar-iron (TSI) and lysine-iron (LI) agar and incubate at 37 °C for 24 hours. Five colony picks per plate should be taken unless 50 percent or more of the plates have Salmonella- like colonies. In that case, the number of picks may be reduced to three per plate. A group D colony lift assay may be utilized to signal the presence of the hard-to-detect group D salmonella colonies on agar culture plates. (iv) Conduct serologic screening of cultures revealing typical reactions of Salmonella on TSI and LI agar slants using somatic O-group antisera agglutination or transfer for further identification to appropriate biochemical tests such as: Dextrose, lactose, sucrose, mannitol, maltose, dulcitol, malonate, gelatin, urea broth, citrate, lysine decarboxylase, ornithine decarboxylase, methyl red and Voges-Proskauer, KCN, salicin broths, indole, and hydrogen sulfide. Motility or non-motility is demonstrated by inoculating a suitable semisolid medium. The Analytical Profile Index API 20E)9 9 We use trade names solely for the purpose of providing specific information. Mention of a trade name does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture or an endorsement over other products not mentioned. (v) Serotype all Salmonella group D cultures. (3) The following organs should be aseptically collected for culture: (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, use own judgment and include any atypical ova.); (iv) Oviduct (if active, include any debris and dehydrated ova.); (v) Pancreas and kidneys; and (vi) Spleen. (4) Aseptically collect 10–15 g or whatever lesser amount is available of each organ or site listed in paragraph (b)(3) of this section from each reactor, and grind or blend them completely in 10 times their volume of VI broth. Organs may be processed individually or in combinations where appropriate. Suspensions should be transferred in 10-ml aliquots to 100-ml of both VI and tetrathionate brilliant green (TBG) broth and incubated at 37 °C for 24 hours. Plate the VI broth on VI and BG agar and plate the TBG broth on BG agar and incubate at 37 °C. Examine these plates after 24 and 48 hours of incubation. The contents of the gallbladder can be cultured separately by inoculating 10-ml of VI and TBG broth with cotton swabs and incubating at 37 °C for 24 hours. Plate on BG agar and incubate at 37 °C. Examine these plates after 24 and 48 hours of incubation. If contamination with pseudomonas or proteus is a problem, make platings on BG sulfapyridine (BGS) agar. (5) Where field samples are directly inoculated into enrichment broths and a delay of several days occurs before they reach a laboratory, or if recovery of low numbers or organisms is expected from a primary culture, a secondary enrichment culture should be prepared. Subculture a week-old primary culture by transferring 1-ml of inoculum into a fresh tube 10-ml of enrichment broth. This secondary enrichment should be incubated at 37 °C for 24 hours before plating. (See paragraph (b)(1) of this section.) TBG broth is recommended for this procedure. (6) Make a composite sample of the following parts of grossly normal or diseased tissues from the digestive tract: Crop wall, duodenum, jejunum (including remnant of yolk-sac attachment), both ceca, cecal tonsils, and rectum-cloaca. Aseptically collect 10–15 g of each organ or tissue, or whatever lesser amount is available, and grind or blend them completely in 10 times their volume of TBG broth. Transfer 10-ml of a composite sample of a suspension from the digestive tract into 100-ml of TBG broth, and incubate flasks at 42 °C for 24 hours. Cultures may be incubated at 37 °C if 42 °C incubators are not available. The higher incubation temperatures for TBG broth reduce populations of competitive contaminants common in gut tissue. Plate on BG agar and incubate at 37 °C. Examine the plates after 24 and 48 hours of incubation. If contamination with pseudomonas or proteus is a problem make platings on BGS agar. (7) If preferred, individual cotton swab samples may also be taken from the upper, middle, and lower intestinal tract (including both ceca and the rectum-cloaca). Deposit swabs in 10-ml of TBG broth and incubate and plate as described in paragraph (b)(6) of this section. (8) Transfer suspect colonies to triple-sugar-iron (TSI) agar and lysine-iron (LI) agar and incubate at 37 °C for 24 hours. (9) Cultures revealing typical reactions of salmonellae on TSI and LI agar slants should be transferred to any of the following appropriate biochemical tests for final identification: Dextrose, lactose, sucrose, mannitol maltose, dulcitol, malonate, gelatin, urea broth, citrate, lysine decarboxylase, ornithine decarboxylase, methyl red and Voges-Proskauer, KCN, salicin broths, indole, and hydrogen sulfide. Motility or non-motility is demonstrated by inoculating a suitable semisolid medium.10 10 Formulation for the semisolid motility medium can be obtained from: Isolation and Identification of Avian Pathogens, American Association of Avian Pathologists, University of Pennsylvania, New Bolton Center, Kennett Square, Pennsylvania 19348–1692, 1980. 11 ONPG discs are available from: Baltimore Biological Laboratories, Cockeysville, MD 21030. (10) All salmonella cultures should be serologically typed. [36 FR 23121, Dec. 3, 1971. Redesignated at 44 FR 61586, Oct. 26, 1979, and amended at 47 FR 21994, May 20, 1982; 50 FR 19900, May 13, 1985; 57 FR 57342, Dec. 4, 1992; 59 FR 12801, Mar. 18, 1994; 61 FR 11521, Mar. 21, 1996; 63 FR 3, Jan. 2, 1998; 65 FR 8019, 8023, Feb. 17, 2000; 67 FR 8469, Feb. 25, 2002] Information concerning the pen arrangement and number of birds per pen should be obtained from the owner so that the required number of samples per pen and per flock can be determined. A means of identifying each sample by pen of origin should be provided. The vehicle transporting the personnel taking the samples should be left as far as practical from the poultry pens. Sanitary precautions, including personal cleanliness, should be observed during the sampling procedure. The hands should be carefully washed with a sanitizing soap prior to the sampling. Outer clothing, including gloves, should be changed between visits to different premises so that clean clothing is worn upon entering each premises. The used and clean apparel should be kept separate. Boots or footwear should be cleaned and disinfected between visits to different premises. Disposable caps should be provided and discarded after use on each premises. After collection, the samples should be protected from drying, light, and excessive temperatures and delivered to the laboratory within one day. If delivery is delayed, samples should be refrigerated. (a) For egg- and meat-type chickens, waterfowl, exhibition poultry, and game birds. All samples and swabs described in this paragraph should be cultured in accordance with illustration 2 of §147.11, including delayed secondary enrichment. All salmonellae recovered shall be serogrouped or serotyped. (1) Environmental samples. Fecal material, litter, dust, or floor litter surface or nest box drag swab samples to be submitted for bacteriological examination shall be collected in accordance with the procedures described in paragraphs (a)(1), (a)(2), or (a)(3) of this section: (i) Procedure for sampling in broth. Authorized laboratories will provide capped tubes 1 to 2 cm in diameter and 15 to 20 cm in length that are two-thirds full of a recently made, refrigerated, sterile enrichment broth for each sample. Sufficient tubes shall be taken to the premises to provide at least one tube per pen or one tube per 500 birds, whichever is greater. At least one sterile, cotton-tipped applicator will be needed for each tube. The dry applicator is first placed in or drawn through fresh manure (under roost, near water troughs, fecal droppings, or diarrhetic droppings). After each streaking, place the cotton-tipped applicator in the tube of broth and swirl the applicator to remove the collected material. Withdraw the applicator from the tube and use it to take additional specimens by streaking on or through areas where defecation, trampling of feces, or settling of dust is common; e.g., on or near waterers, feeders, nests, or rafters, etc. When the volume of material collected equals approximately 10 percent of the volume of the broth (usually 10–12 streakings), place the applicator in the tube and break the stick in half, leaving the lower or cotton-tipped half in the broth and retaining the upper half for future disposal. Replace the cap on the inoculated tube and continue the sampling procedure in other areas of the pen. (ii) Procedure for sampling in dry containers. Place a sample of fecal material, litter, or dust in a sterile, sealable container. The sample shall consist of several specimens of material taken from a representative location in the pen or house. Collect at least 10 g (approximately a heaping tablespoonful) of material for each sample. Collect the specimens in each sample with a sterile tongue depressor or similar uncontaminated instrument. The samples shall vary in type and consistency. Half of the samples shall be comprised of material representing defecated matter from a large portion of the flock; i.e., trampled, caked material near waterers and feeders. The minimum number of samples to be taken shall be determined by the following: Five samples from pens or houses of up to 500 birds; Ten samples from pens or houses of 500 to 2,500 birds; Fifteen samples from pens or houses with more than 2,500 birds. The samples may be pooled to not fewer than five samples at the laboratory as long as the volume of material collected equals approximately 10 percent of the volume of the broth. (2) Cloacal swabs. Cloacal swabs for bacteriological examination shall be taken from each bird in the flock or from a minimum of 500 birds in accordance with the procedure described in paragraph (a)(2)(i) of this section. (i) Procedure for taking cloacal swabs. The authorized laboratory will provide sterile capped tubes or other suitable containers and cotton-tipped applicators for use in taking the cloacal swabs. Insert the cotton-tipped applicator into the cloaca and rectum in such a manner as to ensure the collection of fecal material. Place the swab and adhering fecal material in the tube and break the stick in half, keeping the upper half of the stick for future disposal. The cloacal swabs may be combined in the sterile tubes in multiples of five or in combinations specified by the authorized laboratory. (ii) [Reserved] (3) Drag-swabs. Utilization of drag swabs (DS) involves the exposure of gauze pads (or commercially available sponges designed for this purpose), a key component of a DS sampler, to the surface of random, flock-representative floor litter and nest box areas. The sampler pads shall be sterile and slightly moist to promote adherence of particulate material, and impregnated with double-strength skim milk12 12 Obtain procedure for preparing double strength skim milk from USDA-APHIS “Recommended Sample Collection Methods for Environmental Samples,” available from the National Poultry Improvement Plan, Veterinary Services, APHIS, USDA, 1498 Klondike Road, Suite 200, Conyers, GA 30094. (i) Drag-swab sampler assembly. Drag-swab (DS) samplers may be assembled using two 3- by 3-inch sterile gauze pads; size 20 wrapping twine; and paper clips, staples, or similar fasteners. Fold each gauze pad in half and attach one pad to a 2-foot-long (60 cm) piece of twine and the other to a 1-foot-long (30 cm) piece of twine. To attach a pad to the twine with a paper clip, bend the end wires of the paper clip slightly and push them through the fabric of the folded pad, thus securing the clips to the folded pads; then securely tie the twine to the free rounded end of the paper clip. To attach a pad to the twine with a staple, staple the twine to the pad near the center of the fold, applying the staple at a right angle to the twine and parallel to the fold. (A pre-tied knot in the free end of the twine will prevent the twine from slipping under the staple during use.) Once the pads and the twine have been attached, securely connect the free ends of both lengths of twine to a small loop tied at the end of a 5-foot-long piece of twine. The resulting assembly resembles the letter Y, with a long vertical stem and two diagonal branches of different lengths with a gauze pad securely attached to the end of each branch. Wrap the twine around each two-pad DS sampler to produce a small bundle. Autoclave the assembled DS sampler bundle and transfer it with sterile forceps or other aseptic method to a resealable sterile bag. Aseptically add 15 mL of double-strength skim milk to the bag and massage the milk into the gauze pads. Seal the bags and store at -20 °C. (ii) Procedures and applications for DS samplers. DS samplers shall be completely thawed prior to use. Complete pad/twine/fastener assemblies shall be used to sample floor litter surfaces; nest box surfaces may be sampled using 3- by 3-inch sterile gauze pads impregnated with double-strength skim milk in the manner described in paragraph (a)(3)(i) of this section. In either instance, the Plan participant collecting the samples shall wear a fresh pair of disposable sterile gloves for each flock or house sampled. Each sampler bag shall be marked with the type of sample (floor litter or nest box surface) and the identity of the house or flock from which the sample was taken. (iii) Floor litter sampling technique. For flocks with fewer than 500 breeders, at least one DS set (two DS pads) shall be dragged across the floor litter surface for a minimum of 15 minutes. For flocks with 500 or more breeders, a minimum of two DS sets (four DS pads) shall be dragged across the floor litter surface for a minimum of 15 minutes per DS set. Upon completion of dragging, lower each DS pad by its attached twine into a separate, resealable sterile bag. Alternatively, each DS set of two pads may be lowered by its attached twine into the storage/transport bag from which the DS set was originally taken. Remove the twine from the pad or DS set by grasping the pad or DS set through the sides of the bag with one hand while pulling on the twine with the other hand until the connection is broken. Seal the bags and promptly refrigerate them to between 2 and 4 °C. Do not freeze. Discard the twine in an appropriate disposal bag. (iv) Nest box or egg belt sampling technique. Collect nest box or egg belt samples by using two 3-by-3 inch sterile gauze pads premoistened with double-strength skim milk and wiping the pads over assorted locations in about 10 percent of the total nesting area or the egg belt. Upon completion, place each pad in a separate, resealable sterile bag. Seal the bags and promptly refrigerate them to between 2 and 4 °C. Do not freeze. (v) Culturing of litter surface and nest box samples. When refrigerated to between 2 and 4 °C, pads impregnated with double-strength skim milk may be stored or batched for 5 to 7 days prior to culturing. Pads shipped singly or paired in a single bag shall not be pooled for culturing but shall be separately inoculated into 60 mL of selective enrichment broth. (4) Chick box papers. Samples from chick box papers may be bacteriologically examined for the presence of Salmonella. The Plan participant may collect the samples in accordance with paragraph (a)(4)(i) of this section or submit chick box papers directly to a laboratory in accordance with paragraph (a)(4)(ii) of this section. It is important that the paper be removed from the chick box before the box is placed in the brooding house. (i) Instructions for collecting samples from chick box papers: (A) Collect 1 chick box paper for each 10 boxes of chicks placed in a house and lay the papers on a clean surface. (B) Clean your hands and put on latex gloves. Do not apply disinfectant to the gloves. Change gloves after collecting samples from 10 chick box papers or any time a glove is torn. (C) Saturate a sterile 3-by-3 inch gauze pad with double-strength skim milk (see footnote 12 to this section) and rub the pad across the surface of five chick box papers. Rub the pad over at least 75 percent of each paper and use sufficient pressure to rub any dry meconium off the paper. Pouring a small amount of double-strength skim milk (1 to 2 tablespoons) on each paper will make it easier to collect samples. (D) After collecting samples from 10 chick box papers, place the two gauze pads used to collect the samples (i.e., one pad per 5 chick box papers) into an 18 oz. Whirl-Pak bag and add 1 to 2 tablespoons of double-strength skim milk. (E) Promptly refrigerate the Whirl-Pak bags containing the samples and transport them, on ice or otherwise refrigerated, to a laboratory within 48 hours of collection. The samples may be frozen for longer storage if the Plan participant is unable to transport them to a laboratory within 48 hours. (ii) The Plan participant may send chick box papers directly to a laboratory, where samples may be collected as described in paragraph (a)(4)(i) of this section. To send chick box papers directly to a laboratory: (A) Collect 1 chick box paper for each 10 boxes of chicks placed in a house and place the chick papers immediately into large plastic bags and seal the bags. (B) Place the plastic bags containing the chick box papers in a clean box and transport them within 48 hours to a laboratory. The plastic bags do not require refrigeration. (iii) The laboratory must follow the procedure set forth in paragraph (a)(5) of this section for testing chick meconium for Salmonella. (5) Chick meconium testing procedure for Salmonella. (i) Record the date, source, and flock destination on the “Meconium Worksheet.” (ii) Shake each plastic bag of meconium until a uniform consistency is achieved. (iii) Transfer a 25 gm sample of meconium to a sterile container. Add 225 mL of a preenrichment broth to each sample (this is a 1:10 dilution), mix gently, and incubate at 37 °C for 18–24 hours. (iv) Enrich the sample with selective enrichment broth for 24 hours at 42 °C. (v) Streak the enriched sample onto brilliant green novobiocin (BGN) agar and xylose-lysine-tergitol 4 (XLT4) agar. (vi) Incubate both plates at 37 °C for 24 hours and process suspect Salmonella colonies according to paragraph (b) of this section. (b) Isolation and identification of Salmonella. Either of the two enrichment procedures or the rapid detection method in this paragraph may be used. (1) Tetrathionate enrichment with delayed secondary enrichment (DSE): (i) Add tetrathionate enrichment broth to the sample to give a 1:10 (sample to enrichment) ratio. Incubate the sample at 37 or 41.5 °C for 20 to 24 hours as shown in illustration 2. (ii) After selective enrichment, inoculate selective plates (such as BGN and XLT4). Incubate the plates at 37 °C for 20 to 24 hours. Inoculate three to five Salmonella-suspect colonies from the plates into triple sugar iron (TSI) and lysine iron agar (LIA) slants. Incubate the slants at 37 °C for 20 to 24 hours. Screen colonies by serological (i.e., serogroup) and biochemical (e.g., API) procedures as shown in illustration 2. As a supplement to screening three to five Salmonella-suspect colonies on TSI and LIA slants, a group D colony lift assay may be utilized to signal the presence of hard-to-detect group D Salmonella colonies on agar plates. (iii) If the initial selective enrichment is negative for Salmonella, use a DSE procedure. Leave the original tetrathionate-enriched sample at room temperature for 5 to 7 days. Transfer 1 mL of the culture into 10 mL of fresh tetrathionate enrichment broth, incubate at 37 °C for 20 to 24 hours, and plate as in paragraph (b)(1)(ii) of this section. (iv) Serogroup all isolates identified as Salmonella and serotype all serogroup D isolates. Phage-type all Salmonella enteritidis isolates. (2) Pre-enrichment followed by selective enrichment. (See illustration 2.) (3) Approved rapid detection method. After selective enrichment, a rapid ruthenium-labeled Salmonella sandwich immunoassay may be used to determine the presence of Salmonella. Positive samples from the immunoassay are then inoculated to selective plates (such as BGN and XLT4). Incubate the plates at 37 °C for 20 to 24 hours. Inoculate three to five Salmonella-suspect colonies from the plates into triple sugar iron (TSI) and lysine iron agar (LIA) slants. Incubate the slants at 37 °C for 20 to 24 hours. Screen colonies by serological (i.e., serogroup) and biochemical (e.g., API) procedures as shown in illustration 2. As a supplement to screening three to five Salmonella-suspect colonies on TSI and LIA slants, a group D colony lift assay may be utilized to signal the presence of hard-to-detect group D Salmonella colonies on agar plates. (c) For turkeys—(1) Environmental samples. Fecal material, litter, or dust to be submitted for bacteriological examination should be collected in accordance with the procedures described in paragraphs (c)(1)(i) or (c)(1)(ii) of this section: (i) Procedure for sampling in broth. Authorized laboratories will provide capped tubes 1–2 cm in diameter and 15–20 cm in length which are two-thirds full of a recently made, refrigerated, sterile enrichment broth (Selenite Brilliant Green Sulfapyridine or Tetrathionate Brilliant Green) for each sample. Sufficient tubes should be taken to the premises to provide at least one tube per pen or one tube per 500 birds, whichever is greater. At least one sterile, cotton-tipped applicator will be needed for each tube. The dry applicator is first placed or drawn through fresh manure (under roost, near water troughs, cecal droppings, or diarrhetic droppings). After this and each subsequent streaking, the cotton-tipped applicator is placed in the tube of broth and swirled to remove the collected material. The applicator is then withdrawn and is used for taking additional specimens by streaking on or through areas where defecation, trampling of feces, or settling of dust are common; i.e., on or near waterers, feeders, nests, or rafters, etc. When the volume of material collected equals approximately 10 percent of the volume of the broth (usually 10–12 streakings), the applicator is placed in the tube and the stick is broken in half. The lower or cotton-tipped half is left in the broth, and the upper half is retained for future disposal. The cap is then replaced on the inoculated tube, and the sampling procedure is continued in other areas of the pen. (ii) Procedure for sampling in dry containers. A sample of fecal material, litter, or dust is placed in a sterile, sealable container. The sample shall consist of several specimens of material taken from a representative location in the pen or house. At least 10 g (approximately a heaping tablespoonful) of material shall be collected for each sample. The specimens in each sample shall be collected with a sterile tongue depressor or similar uncontaminated instrument. The samples should vary in type and consistency. Half of the samples should be comprised of material representing defecated matter from a large portion of the flock; i.e., trampled, caked material near waterers and feeders. The minimum number of samples to be taken shall be determined by the following:
Five samples from pens or houses of up to 500 birds; Ten samples from pens or houses of 500 to 2,500 birds; Fifteen samples from pens or houses with more than 2,500 birds. The composite samples above may be pooled to not less than five samples at the laboratory as long as the volume of material collected equals approximately 10 percent of the volume of the broth. (2) Cloacal swabs. Cloacal swabs for bacteriological examination are taken from each bird in the flock or from a minimum of 500 birds in accordance with the procedure described in paragraph (c)(2)(i) of this section. (i) Procedure for taking cloacal swabs. The authorized laboratory will provide sterile capped tubes or other suitable containers and cotton-tipped applicators for use in taking the cloacal swabs. The cotton-tipped applicator is inserted into the cloaca and rectum in such a manner as to insure the collection of fecal material. The swab and adhering fecal material is then placed in the tube and the stick is broken in half, with the upper half retained for future disposal. The cloacal swabs may be combined in the sterile tubes in multiples of five or in combinations specified by the authorized laboratory. (ii) [Reserved] (3) Drag-swabs. Drag-swabs for bacteriological examination should involve the exposure of at least six unpooled pads per house to promote representative sampling and some element of quantification. (i) Drag-swab assembly. Assemble drag-swab sampling sets from folded-once 3-by-3-inch sterile gauze pads secured with paper clips. Bend end wires of each paper clip slightly to catch into the swab fabric, thus securing the clips to the folded pads. Use two pads, assembled as described to make each drag-swab sampling set. Securely connect one pad through the free rounded end of the paper clip to a 2-ft (0.6 m) length of size 20 fibrous wrapping twine. Similarly connect the other pad to a 1-ft (0.3 m) length of twine. Then securely connect the free ends of both lengths of twine to a small loop tied at the end of a similar 5-ft length of twine. The resulting assembly resembles the letter Y with a 5-ft long vertical stem and two diagonal branches (one 1 ft long and the other 2 ft long), with a folded swab securely attached at the end of each branch. After assembly, place each two-pad drag-swab sampling set into a sterile bag. (ii) Procedure for taking drag-swab—(A) Floor litter: The Plan participants should collect two samples as follows: Drag four 3-by-3-inch sterile gauze pads premoistened with double strength skim milk13 13 Obtain procedure for preparing double strength skim milk from USDA-APHIS “Recommended Sample Collection Methods for Environmental Samples” available from the National Poultry Improvement Plan, Veterinary Services, APHIS, USDA, 1498 Klondike Road, Suite 200, Conyers, GA 30094. (B) Nest-boxes. The Plan participant should collect one nest-box sample by using two 3-by-3-inch sterile gauze pads premoistened with double strength skim milk. Wipe the two gauze pads used to collect the sample over assorted locations of about 10 percent of the total nesting area. Place the gauze pads used to collect the sample in an 18-oz whirl-pack bag containing 5 ml of double strength skim milk. Mark the bag with the type of sample and the house identification. [38 FR 13709, May 24, 1973. Redesignated at 44 FR 61586, Oct. 26, 1979, and amended at 57 FR 57342, Dec. 4, 1992; 59 FR 12805, Mar. 18, 1994; 59 FR 67617, Dec. 30, 1994; 61 FR 11524, Mar. 21, 1996; 62 FR 44070, Aug. 19, 1997; 63 FR 3, Jan. 2, 1998; 65 FR 8019, Feb. 17, 2000; 67 FR 8471, Feb. 25, 2002; 68 FR 64512, Nov. 14, 2003] Proper precautions to avoid environmental contamination of the samples during the collection and laboratory process, and proper handling of the samples following collection are essential. Each State Inspector involved in eggshell culture activities must receive instruction in the necessary sanitation procedures, sampling procedures, and sample handling by the authorized laboratory involved. The Official State Agency will maintain a record showing that the required instruction was given to each State Inspector. (a) Sample selection. Forty (40) eggs in the top flats of each of three randomly selected cases of sanitized eggs from each flock will be utilized for each sampling. (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) From the tube containing four swabs and lactose broth or other suitable media, 1 ml. will be transferred to 10 ml. lactose in a fermentation tube. (2) Incubate at 37 °C for 48 hours. The presence of acid, and gas in the amount of 10 percent or more after 24 and 48 hours of incubation, provides a presumptive conclusion of the presence of colon bacilli organisms. [41 FR 14256, Apr. 2, 1976. Redesignated at 44 FR 61586, Oct. 26, 1979, and amended at 59 FR 12805, Mar. 18, 1994] The following monitoring procedures14 14 Laboratory procedures for monitoring operations proposed here are described in the following two publications: Isolation and Identification of Avian Pathogens, American Association of Avian Pathologists, University of Pennsylvania, New Bolton Center, Kennett Square, Pennsylvania 19348–1692, 1980, and Culture Methods for the Detection of Animal Salmonellosis and Arizonosis, Iowa State University Press, Ames, Iowa 50010, 1976. (a) Monitor effectiveness of sanitation program. (1) Culture the surface of cased eggs periodically for fecal contaminating organisms as described in §147.13. (2) Culture a sample of dead-in-shell eggs periodically from each breeding flock for coliforms. Such eggs should also be cultured for the dependable recovery of salmonellae. Culturing for the dependable recovery of salmonellae should include the use of: (i) Preenrichment broths supplemented with 35 mg ferrous sulfate per 1,000 ml preenrichment to block iron-binding, Salmonella-inhibiting effects of egg conalbumin; and (ii) Tetrathionate selective enrichment broths, competitor-controlling plating media (XLT4, BGN, etc.), delayed secondary enrichment procedures, and colony lift assays detailed in paragraph (a)(5) and illustration 2 of §147.11. [41 FR 48726, Nov. 5, 1976. Redesignated at 44 FR 61586, Oct. 26, 1979, and amended at 57 FR 57343, Dec. 4, 1992; 59 FR 12805, Mar. 18, 1994; 59 FR 59640, Nov. 18, 1994; 61 FR 11524, 11525, Mar. 21, 1996; 65 FR 8019, Feb. 17, 2000] 15 Yoder, H. W., Jr., “Mycoplasmosis.” In: Isolation and Identification of Avian Pathogens. (Stephen B. Hitchner, Chairman, Charles H. Domermuth, H. Graham Purchase, James E. Williams.) 1980, pp. 40–42, Creative Printing Company, Inc., Endwell, NY 13760. (a) Turbinates, trachea, air sacs, sinuses, nasal passages, respiratory exudates, synovial fluid, eggs (including yolk, yolk sacs, membranes and allantoic fluid), should be directly sampled with sterile swabs. Aseptic techniques are very important as some organisms may not be suppressed by the antimicrobial agents used in this procedure. Tissue suspensions from large volumes are sometimes desirable from the sites listed above and occasionally from the oviduct and cloaca. Tissues should be ground or blended completely in 10 times their volume of Mycoplasma Broth Medium (MBM). (See paragraph (f) of this section.) Specimens submitted to referral laboratories in order of preference for recovery of the mycoplasma organisms are: (1) live birds, (2) refrigerated fresh tissues, (3) tissue specimens packed with dry ice. (b) Inoculate 5–10 ml of MBM with a swab, wire loop or 0.1 ml of the tissue suspension. When evidence of growth is observed (lowered pH or turbidity of broth) transfer each broth culture as needed to maintain the original isolates. Incubate tubes at 37 °C for at least 21 days before discarding as negative. When growth is first observed or if no growth occurs by the 4th or 5th day of incubation, inoculate broth culture onto a plate of Mycoplasma Agar Medium (MAM). (See paragraph (g) of this section.) Several cultures may be inoculated on one plate by using a wire loop or a cotton swab. Incubate plates 3–5 days at 37 °C in a high humidity chamber. If preferred, 5 percent CO2 may be added or a candle jar may be used. Tiny circular and translucent colonies with elevated centers are very suggestive of mycoplasma. Indirect lighting and a low power or dissecting microscope are recommended for observation of the colonies as they are rarely more than 0.2–0.3 mm in diameter. (c) Isolates must be serotyped. (1) Isolates may be shipped in MBM with ice packs if shipment will be in transit less than 2–3 days. Longer shipments require freezing of the MBM with dry ice, or shipping MAM slants at room temperature. Isolates must have indications of growth before shipment is made. (2) Isolates may be stored in MBM at −20 °C for 2–3 weeks, or they may be stored at −68 °C for several years. (d) Alternate method of culture: An overlay enrichment culture for fastidious and sensitive mycoplasma, especially for M. meleagridis should be included. (1) Pour 2–3 ml of MAM into a test tube and tilt the tube until a slant (approximately 45°) is obtained. Other containers are acceptable. (2) Overlay the slant with sufficient MBM, so that the media (including inoculum) covers the agar slope. (3) Inoculate the culture as indicated in paragraph (b) of this section. (4) Incubate and examine the overlay as indicated in paragraph (b) of this section. (e) Preparation of media components:16 16 Trade names are used in these procedures solely for the purpose of providing specific information. Mention of a trade name does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture or an endorsement over other products not mentioned. (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.17 17 Alcojet is available from: Alconox, Inc., New York, NY 10003. (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 1:2000.18 18 Thallium acetate may be obtained from Fischer Scientific Company. (4) Mycoplasma Broth Base, dextrose, phenol red, and cysteine hydrochloride are added to deionized distilled water first if autoclave sterilization is used.19 19 Mycoplasma Broth Base may be obtained from: (a) Product #M 33600, Gibco Diagnostics, 2801 Industrial Drive, Madison, WI 53711. (b) Product #3900–3212, Scott Laboratories, Inc., 8 Westchester Plaza, Elmsford, NY 10523. (5) Use sterile deionized distilled water to reconstitute penicillin. (6) Sterile serum should be inactivated by heating at 56 °C for 30 minutes. Swine serum may be used for M. gallisepticum, M. synoviae, M. gallinarum, and M. meleagridis isolation; however, horse serum is usually recommended for M. meleagridis isolation. (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.20 20 NAD Grade III may be obtained from: Sigma Chemical Company, P.O. Box 14508, St. Louis, MO 63178. (9) Cysteine hydrochloride, prepared as a 1 percent solution, is used to reduce the NAD for M. synoviae growth. (10) A purified agar product such as Nobel (Special agar) is used in the MAM.21 21 Noble Agar may be obtained from: Difco Laboratories, Box 1058–A, Detroit, MI 48201. (11) Adjust the pH with NaOH. (12) Sterilization may be accomplished by two methods: (i) Filtration sterilization through a 0.20 micron filter is the recommended method. Aseptic techniques must be utilized. (ii) Autoclave sterilization at 120 °C, 15 pounds pressure (103 kPa), for 15 minutes may be used, if preferred, when following the procedure described in paragraph (e)(4) of this section. (13) Phenol red, dextrose, and NAD may be omitted when culturing for M. meleagridis and M. gallinarum. (14) When culturing for M. meleagridis from contaminated samples include 100 units/ml of Polymyxin B in MBM. (f) Mycoplasma Broth Medium (Frey) is prepared as follows: To 850–880 ml of deionized distilled water; Add: Thallium acetate (ml)—2.5 (1:4000) Potentially contaminated samples (ml)—5.0 (1:2000) Mycoplasma Broth Base (g)—22.5 Aqueous penicillin (units)—500,000 Sterile serum (ml)—120 to 150.0 Phenol red plus (ml)—2.5 NAD (ml)—12.5 Cysteine hydrochloride (ml)—12.5 Dextrose (g)—1.0–1.5 Adjust pH to 7.8 Filter sterilize (1) Broth may be stored at 4 °C for at least 2 weeks or at −40 °C for longer periods. (g) Mycoplasma Agar Medium (Frey) is prepared as follows: To 850–880 ml of deionized distilled water; Add: Mycoplasma Broth Base (g)—22.5 Adjust pH to 7.8 Purified agar (g)—12.0 Autoclave and cool in 45 °C water bath Thallium acetate (ml)—2.0; (1:4000) Sterile serum at 45 °C (ml)—150.0 Aqueous penicillin (units)—400,000 NAD (ml)—12.5 Cysteine hydrochloride (ml)—12.5 (1) Rotate flask gently and pour about 15 ml of media into each petri dish. (2) Stack petri dishes only 2–3 high in a 37 °C incubator up to 2 hours to remove excess moisture. (3) Wrap inverted plates in sealed bundles and store at 4 °C for not more than 15 days. (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.22 22 “Laboratory Procedures and Medium For The Isolation Of Mycoplasma From Clinical Materials.” Laboratory Diagnosis of Mycoplasma in Food Animals, Proceedings of Nineteenth Annual Meeting, The American Association of Veterinary Laboratory Diagnosticians, 1976, pp. 106–115, AAVLD, 6101 Mineral Point Road, Madison, WI 53705. [47 FR 21995, May 20, 1982, as amended at 57 FR 57343, Dec. 4, 1992; 59 FR 12805, Mar. 18, 1994; 61 FR 11524, Mar. 21, 1996; 65 FR 8019, Feb. 17, 2000] This procedure has been shown to be sensitive enough to detect less than 100 mycoplasma organisms under proper conditions.23 23 Research results are described in the following two publications: (a) Bigland, C. H. and A. J. DaMassa, “A Bio-Assay for Mycoplasma Gallisepticum.” In: United States Livestock Sanitary Association Proceedings, 67th, 1963, pp. 541–549. (b) McMartin, D. A., “Mycoplasma Gallisepticum in the Respiratory Tract of the Fowl.” In: The Veterinary Record, September 23, 1967, pp. 317–320. (a) Obtain chickens or turkeys (test birds) which are at least 3 weeks of age and are free of M. gallisepticum, M. synoviae, and M. meleagridis and transport them in a manner to prevent their being contaminated by any infectious avian disease. (1) Maintain test birds in an area that has been effectively cleaned and disinfected. (2) The area should be isolated from other birds or animals. (3) Personnel caring for the test birds should take the necessary precautions (see §147.26(b)) to prevent the mechanical transfer of infectious avian diseases from other sources. (b) Test birds to be used for inoculation with contaminated tissues should be serologically negative by the serum plate agglutination test. (1) Inoculated test birds should be isolated from non-inoculated control birds for the length of any experiment. (c) Aseptically obtain tracheal, turbinate, and sinus mucosa, lung and sinus exudates, cervical, thoracic, and abdominal airsac tissues (including lesions), and portions of oviduct and synovial fluid from at least four suspect, donor birds. In a sterile device, blend the tissues completely in four times their volume of Mycoplasma Broth Medium (Frey), (see §147.15(f)). Suspensions may be made from tissue pools. Inoculate test birds within 30 minutes for preparation of suspensions. (1) Inoculate at least four test birds for each suspension pool via the abdominal air sac and infraorbital sinus, with up to (2) Test birds should be bled every 7 days for 35 days to identify sero-converters. (3) At 35 days, test birds should be sacrificed and bacteriologic isolation and identification of mycoplasma attempted (see §147.15). Note especially the sites of inoculation for typical gross or microscopic mycoplasma lesions. (d) Donor birds are considered infected when: (1) Test birds have serum plate antibodies for the mycoplasma for which the donor birds were tested, regardless of HI test results, and control birds stay serologically negative; or (2) Mycoplasma organisms are isolated from the test birds and serotyped positive for the mycoplasma for which the donor birds were tested, and control birds stay serologically and culturally negative. (e) Laboratory findings may be verified by direct cultures of material from sick birds or by inoculating seronegative birds from the suspect flock and comparing serological findings with those from the test birds. [47 FR 21996, May 20, 1982, as amended at 57 FR 57343, Dec. 4, 1992; 59 FR 12805, Mar. 18, 1994; 61 FR 11524, Mar. 21, 1996; 65 FR 8019, Feb. 17, 2000] The laboratory procedure described in this section is recommended for the bacteriological examination of cull chicks from egg-type and meat-type chicken flocks and waterfowl, exhibition poultry, and game bird flocks for salmonella. (a) 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: (1) Organ pool: From each of five chicks, composite and mince 1- to 2-gram samples of heart, lung, liver, and spleen tissues and the proximal wall of the bursa of Fabricius. (2) Yolk pool: From each of five chicks, 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) Intestinal pool: From each of five chicks, composite and mince approximately 0.5 cm2 sections of the crop wall and 5-mm-long sections of the duodenum, cecum, and ileocecal junction. (b) Transfer each pool to tetrathionate selective enrichment broth (Hajna or Mueller-Kauffmann) at a ratio of 1 part tissue pool to 10 parts broth. (c) Repeat the steps in paragraphs (a) and (b) of this section for each five-chick group until all 25 chicks have been examined, producing a total of 15 pools (5 organ, 5 yolk, and 5 intestinal). (d) Culture the 15 tetrathionate pools as outlined for selective enrichment in illustration 2 of §147.11. 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 §147.11 and examine after both 24 and 48 hours of incubation. Confirm suspect colonies as described. Further culture all salmonella-negative tetrathionate broths by delayed secondary enrichment procedures described for environmental, organ, and intestinal samples in illustration 2 of §147.11. A colony lift assay may also be utilized as a supplement to TSI and LI agar picks of suspect colonies. [61 FR 11525, Mar. 21, 1996]
Title 9: Animals and Animal Products
PART 147—AUXILIARY PROVISIONS ON NATIONAL POULTRY IMPROVEMENT PLAN
Subpart B—Bacteriological Examination Procedure
§ 147.10 Laboratory procedure recommended for the bacteriological examination of egg-type breeding flocks with salmonella enteritidis positive environments.
§ 147.11 Laboratory procedure recommended for the bacteriological examination of salmonella.
§ 147.12 Procedures for collection, isolation, and identification of Salmonella from environmental samples, cloacal swabs, chick box papers, and meconium samples.
§ 147.13 Procedure for bacteriological culturing of eggshells for colon bacilli organisms.
§ 147.14 Procedures to determine status and effectiveness of sanitation monitored program.
§ 147.15 Laboratory procedure recommended for the bacteriological examination of mycoplasma reactors.15
§ 147.16 Procedure for the evaluation of mycoplasma reactors by in vivo bio-assay (enrichment).
§ 147.17 Laboratory procedure recommended for the bacteriological examination of cull chicks for salmonella.
