Evaluation of radiometric faecal culture and direct PCR on pooled faeces for detection of Mycobacterium avium subsp. paratuberculosis in cattle

Eamens, G. J.; Whittington, Richard J.; Turner, Mark J.; Austin, Susan L.; Fell, Shayne A.; Marsh, Ian

doi:10.1016/j.vetmic.2007.04.043

Cited by 17 publications

(26 citation statements)

References 26 publications

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“…However, this procedure is laborious and slow (requiring up to 6 months) and has low sensitivity (17,18). Radiometric culture is an improvement over standard culture as results require at least 7 weeks for diagnosis (19,20). Liquid medium cultures such as Bactec MGIT 960 and TREK ESP pressure detection system, allow for detection of M. avium subsp.…”

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confidence: 99%

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

Mundo

Gilardoni

Hoffman

et al. 2013

Appl Environ Microbiol

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Paratuberculosis is an infectious, chronic, and incurable disease that affects ruminants, caused by Mycobacterium avium subsp. paratuberculosis. This bacterium is shed primarily through feces of infected cows but can be also excreted in colostrum and milk and might survive pasteurization. Since an association of genomic sequences of M. avium subsp. paratuberculosis in patients with Crohn's disease has been described; it is of interest to rapidly detect M. avium subsp. paratuberculosis in milk for human consumption. IS900 insertion is used as a target for PCR amplification to identify the presence of M. avium subsp. paratuberculosis in biological samples. Two target sequences were selected: IS1 (155 bp) and IS2 (94 bp). These fragments have a 100% identity among all M. avium subsp. paratuberculosis strains sequenced. M. avium subsp. paratuberculosis was specifically concentrated from milk samples by immunomagnetic separation prior to performing PCR. The amplicons were characterized using DNA methylase Genotyping, i.e., the amplicons were methylated with 6-methyl-adenine and digested with restriction enzymes to confirm their identity. The methylated amplicons from 100 CFU of M. avium subsp. paratuberculosis can be visualized in a Western blot format using an anti-6-methyl-adenine monoclonal antibody. The use of DNA methyltransferase genotyping coupled to a scintillation proximity assay allows for the detection of up to 10 CFU of M. avium subsp. paratuberculosis per ml of milk. This test is rapid and sensitive and allows for automation and thus multiple samples can be tested at the same time.

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confidence: 99%

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

Mundo

Gilardoni

Hoffman

et al. 2013

Appl Environ Microbiol

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“…Pooled fecal culture for sheep is now a routine procedure made available through animal health diagnostic laboratories in Australia. Pooled fecal culture is also used for cattle and goats, and a variation, culturing of pooled samples taken from the environment, also appears to be useful for herd-level detection (8,10,18,21,36,40). The aims of this study were to assess procedural factors that affect the isolation of M. avium subsp.…”

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

Whittington

2009

J Clin Microbiol

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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.

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“…To account for the demonstrated delayed onset of growth in samples containing few viable M. avium subsp. paratuberculosis organisms, a minimum incubation of 10 weeks has been recommended for bovine and caprine pooled fecal cultures, and 12 weeks is routinely used for sheep pooled fecal cultures (38)(39)(40). In this study, a few cultures were extended to 17 weeks incubation to confirm that there was no detrimental effect of prolonged incubation on PCR detection in the culture.…”

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confidence: 99%

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

Whittington

Waldron

et al. 2013

J Clin Microbiol

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Liquid culture of Mycobacterium avium subsp. paratuberculosis from clinical samples, such as feces, is the most sensitive antemortem test for the diagnosis of Johne's disease in ruminants. In Australia, New Zealand, the United States, and some other countries, the Bactec 460 system with modified Bactec 12B medium (Becton, Dickinson) has been the most commonly used liquid culture system, but it was discontinued in 2012. In this study, a new liquid culture medium, M7H9C, was developed. It consists of a Middlebrook 7H9 medium base with added Casitone, albumin, dextrose, catalase, egg yolk, mycobactin J, and a cocktail of antibiotics. We found that polyoxyethylene stearate (POES) was not essential for the cultivation of M. avium subsp. paratuberculosis in either the Bactec 12B or the M7H9C medium. The limit of detection determined using pure cultures of the C and S strains of M. avium subsp. paratuberculosis was 7 bacilli per 50 l inoculum in the two media. The new medium was validated using 784 fecal and tissue samples from sheep and cattle, >25% of which contained viable M. avium subsp. paratuberculosis. Discrepant results for the clinical samples between the two media were mostly associated with samples that contained <10 viable bacilli per gram, but these results were relatively uncommon, and the performances of the two media were not significantly different. M7H9C medium was less than half the cost of the Bactec 12B medium and did not require regular examination during incubation, but a confirmatory IS900 PCR test had to be performed on every culture after the predetermined incubation period.

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Evaluation of radiometric faecal culture and direct PCR on pooled faeces for detection of Mycobacterium avium subsp. paratuberculosis in cattle

Cited by 17 publications

References 26 publications

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

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

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

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

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