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Validation of an in-house real-time PCR fecal assay and comparison with two commercial assays for the antemortem detection of Mycobacterium avium subsp. paratuberculosis infection in culled sheep.

ABSTRACT: Paratuberculosis is a chronic infectious enteritis of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). In sheep, the antemortem detection of the infection is challenging given the slow progression of the disease and the lack of sensitive, specific, and cost-effective validated tests. We adapted an in-house real-time PCR (rtPCR) assay targeting the multi-copy IS 900 element of MAP. The sensitivity and specificity of this essay for the detection of MAP infection were estimated in a convenience sample of culled ewes from 7 infected flocks and compared to a commercial fecal rtPCR, a commercial ELISA, and fecal culture. An infected ewe was defined as a ewe with a positive culture of the ileum and/or mesenteric lymph node. A non-infected ewe was defined as a ewe negative in intestinal tissue culture, negative in fecal culture, and with no lesions consistent with paratuberculosis. The in-house rtPCR had a sensitivity estimate of 84% (95% confidence interval [CI]: 59%, 97%) among the 44 infected ewes, which was significantly higher ( p ? 0.05) than the sensitivity of a commercial fecal rtPCR (52%, 95% CI: 27%, 76%; or 63%, 95% CI: 35%, 87% depending on the cutoff used), an ELISA (14%, 95% CI:2.0%, 41%), and fecal culture (21%, 95% CI: 2.7%, 59%). No statistical difference in assay specificities was observed for the 30 non-infected ewes. The in-house rtPCR is a promising tool that could be used advantageously for the antemortem detection of MAP infection in sheep.

SUBMITTER: Arsenault J

PROVIDER: S-EPMC6505751 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Validation of an in-house real-time PCR fecal assay and comparison with two commercial assays for the antemortem detection of Mycobacterium avium subsp. paratuberculosis infection in culled sheep.

Arsenault Julie J Singh Sohal Jagdip J Leboeuf Anne A Hélie Pierre P Fecteau Gilles G Robinson Yves Y L'Homme Yvan Y

Journal of veterinary diagnostic investigation : official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc 20181102 1

Paratuberculosis is a chronic infectious enteritis of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). In sheep, the antemortem detection of the infection is challenging given the slow progression of the disease and the lack of sensitive, specific, and cost-effective validated tests. We adapted an in-house real-time PCR (rtPCR) assay targeting the multi-copy IS 900 element of MAP. The sensitivity and specificity of this essay for the detection of MAP infection were estimate ...[more]

PMID: 30387705

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Project description:Johne's disease (JD) is a chronic enteric disease caused by Mycobacterium avium subsp. paratuberculosis that affects ruminants. Transmission occurs by the fecal-oral route. A commonly used antemortem diagnostic test for the detection of M. avium subsp. paratuberculosis in feces is liquid culture; however, a major constraint is the 2- to 3-month incubation period needed for this method. Rapid methods for the detection of M. avium subsp. paratuberculosis based on PCR have been reported, but comprehensive validation data are lacking. We describe here a new test, the high-throughput-Johnes (HT-J), to detect M. avium subsp. paratuberculosis in feces. Its diagnostic accuracy was compared with that of liquid radiometric (Bactec) fecal culture using samples from cattle (1,330 samples from 23 herds) and sheep (596 samples from 16 flocks). The multistage protocol involves the recovery of M. avium subsp. paratuberculosis cells from a fecal suspension, cell rupture by bead beating, extraction of DNA using magnetic beads, and IS900 quantitative PCR. The limit of detection of the assay was 0.0005 pg, and the limit of quantification was 0.005 pg M. avium subsp. paratuberculosis genomic DNA. Only M. avium subsp. paratuberculosis was detected from a panel of 51 mycobacterial isolates, including 10 with IS900-like sequences. Of the 549 culture-negative fecal samples from unexposed herds and flocks, 99% were negative in the HT-J test, while 60% of the bovine- and 84% of the ovine-culture-positive samples were positive in the HT-J test. As similar total numbers of samples from M. avium subsp. paratuberculosis-exposed animals were positive in culture and HT-J tests in both species, and as the results of a McNemar's test were not significant, these methods probably have similar sensitivities, but the true diagnostic sensitivities of these tests are unknown. These validation data meet the consensus-based reporting standards for diagnostic test accuracy studies for paratuberculosis and the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines (S. A. Bustin et al., Clin. Chem. 55:611-622, 2009, doi:10.1373/clinchem.2008.112797). The HT-J assay has been approved for use in JD control programs in Australia and New Zealand.

Project description:The cross-reactivity of mycobacterial antigens in immune-based diagnostic assays has been a major concern and a criticism of the current tests that are used for the detection of paratuberculosis. In the present study, Mycobacterium avium subsp. paratuberculosis recombinant proteins were evaluated for antigenic specificity compared to a whole-cell sonicate preparation (MPS). Measures of cell-mediated immunity to M. avium subsp. paratuberculosis antigens were compared in calves inoculated with live M. avium subsp. paratuberculosis, M. avium subsp. avium (M. avium), Mycobacterium kansasii, or Mycobacterium bovis. Gamma interferon (IFN-γ) responses to MPS were observed in all calves that were exposed to mycobacteria compared to control calves at 4 months postinfection. Pooled recombinant M. avium subsp. paratuberculosis proteins also elicited nonspecific IFN-γ responses in inoculated calves, with the exception of calves infected with M. bovis. M. avium subsp. paratuberculosis proteins failed to elicit antigen-specific responses for the majority of immune measures; however, the expression of CD25 and CD26 was upregulated on CD4, CD8, gamma/delta (γδ) T, and B cells for the calves that were inoculated with either M. avium subsp. paratuberculosis or M. avium after antigen stimulation of the cells. Stimulation with MPS also resulted in the increased expression of CD26 on CD45RO(+) CD25(+) T cells from calves inoculated with M. avium subsp. paratuberculosis and M. avium. Although recombinant proteins failed to elicit specific responses for the calves inoculated with M. avium subsp. paratuberculosis, the differences in immune responses to M. avium subsp. paratuberculosis antigens were dependent upon mycobacterial exposure. The results demonstrated a close alignment in immune responses between calves inoculated with M. avium subsp. paratuberculosis and those inoculated with M. avium that were somewhat disparate from the responses in calves infected with M. bovis, suggesting that the biology of mycobacterial infection plays an important role in diagnosis.

Dataset Information

Validation of an in-house real-time PCR fecal assay and comparison with two commercial assays for the antemortem detection of Mycobacterium avium subsp. paratuberculosis infection in culled sheep.

Publications

Validation of an in-house real-time PCR fecal assay and comparison with two commercial assays for the antemortem detection of Mycobacterium avium subsp. paratuberculosis infection in culled sheep.

Similar Datasets

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