147.16—Procedure for the evaluation of mycoplasma reactors by in vivo bio-assay (enrichment).
This procedure has been shown to be sensitive enough to detect less than 100 mycoplasma organisms under proper conditions. 19 Proper conditions are defined in this section.
Code of Federal Regulations
Footnote(s): 19 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.
(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 1/2 ml of inoculum per site.
(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.
(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.