Factors Affecting Isolation and Identification of Mycobacterium avium subsp. paratuberculosis from Fecal and Tissue Samples in a Liquid Culture System

Abstract

Culture of Mycobacterium avium subsp. paratuberculosis is the definitive diagnostic test for Johne's disease, a chronic granulomatous enteropathy of animals. Compared to solid media, the identification of all strains of the organism in liquid media can be more difficult because the appearance of colonies and mycobactin dependence are not observable, and the growth of other organisms needs to be distinguished, commonly by PCR. Factors affecting the isolation rate of S strains and the contamination rate in modified Middlebrook 7H9 broth (Bactec 12B) and 7H10 agar were studied using 11,598 fecal samples and 2,577 tissue samples from sheep from 1,421 farms over 10 years. Minimization of contamination in Bactec cultures required the avoidance of the carryover of fecal particles from the first sedimentation step in the double-incubation centrifugation method, and contamination was reduced significantly by incubating the sample in a solution containing vancomycin, amphotericin B, and nalidixic acid for 3 days compared to 2 days. The growth of irrelevant microorganisms confounded the identification of M. avium subsp. paratuberculosis in liquid culture by inhibiting IS900 PCR and in solid medium culture by inhibiting the growth of M. avium subsp. paratuberculosis or obscuring colonies. The contamination of samples was clustered in certain laboratory submissions and was reduced by including ampicillin in Bactec medium without affecting the odds of isolation of M. avium subsp. paratuberculosis. The long-term contamination rate for fecal cultures was about 7%, and that for tissue cultures was <0.2%. Liquid medium was more sensitive than solid medium culture for M. avium subsp. paratuberculosis.

Optimization of Hexadecylpyridinium Chloride Decontamination for Culture of Mycobacterium avium subsp. paratuberculosis from Milk

“…Media most commonly used for the culture of MAP from milk include Bactec 12B, para-JEM, and Herrold's egg yolk (HEY) (10,(12)(13)(14). These media vary greatly in composition, including the presence of growth supplements and antibiotics, as well as growth detection methods, and therefore vary in their ability to support MAP growth (15,16).…”

Rapid and Sensitive Method To Identify Mycobacterium avium subsp. paratuberculosis in Cow's Milk by DNA Methylase Genotyping

“…Liquid medium cultures such as Bactec MGIT 960 and TREK ESP pressure detection system, allow for detection of M. avium subsp. paratuberculosis in fecal samples with high sensitivity, although it takes 10 to 45 days to obtain a result (21). Identification of infected animals is accomplished indirectly by monitoring immune responses.…”

Development and Validation of a Liquid Medium (M7H9C) for Routine Culture of Mycobacterium avium subsp. paratuberculosis To Replace Modified Bactec 12B Medium

“…However, because solid media have lower analytical sensitivities than liquid media for M. avium subsp. paratuberculosis C and S strains, subculture from broth to solid medium has a false-negative culture rate of about 30% and requires an additional 6 weeks of incubation compared to liquid medium (28). Antigen-capture ELISA for the detection of secreted products in growing cultures is useful for confirming the presence of M. avium complex species but requires a specific PCR to confirm M. avium subsp.…”