147.11—Laboratory procedure recommended for the bacteriological examination of salmonella.
(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 section: 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 § 145.14(a)(6)(ii) of this subchapter shall be submitted to the authorized laboratory for bacteriological examination. Careful aseptic technique should be used when collecting all tissue samples.
(1)
Direct culture (refer to illustration 1). Grossly normal or diseased liver, heart, pericardial sac, spleen, lung, kidney, peritoneum, gallbladder, oviduct, misshapen ova or testes, inflamed or unabsorbed yolk sac, and other visibly pathological tissues where purulent, necrotic, or proliferative lesions are seen (including cysts, abscesses, hypopyon, and inflamed serosal surfaces) should be sampled for direct culture using either flamed wire loops or sterile swabs. Since some strains may not dependably survive and grow in certain selective media, inoculate non-selective plates (such as blood or nutrient agar) and selective plates (such as MacConkey [MAC] and brilliant green novobiocin [BGN] for pullorum-typhoid and MAC, BGN, and xylose-lysine-tergitol 4 [XLT 4] for SE). After inoculating the plates, pool the swabs from the various organs into a tube of non-selective broth (such as nutrient or brain-heart infusion). Refer to illustration 1 for recommended bacteriological recovery and identification procedures. 7 Proceed immediately with collection of organs and tissues for selective enrichment culture.
Code of Federal Regulations
Footnote(s): 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:
(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);
(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.
Code of Federal Regulations
Code of Federal Regulations
945
(Approved by the Office of Management and Budget under control number 0579-0007)