Dataset Information

High-throughput direct fecal PCR assay for detection of Mycobacterium avium subsp. paratuberculosis in sheep and cattle.

ABSTRACT: 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.

SUBMITTER: Plain KM

PROVIDER: S-EPMC3957769 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature

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High-throughput direct fecal PCR assay for detection of Mycobacterium avium subsp. paratuberculosis in sheep and cattle.

Plain Karren M KM Marsh Ian B IB Waldron Anna M AM Galea Francesca F Whittington Ann-Michele AM Saunders Vanessa F VF Begg Douglas J DJ de Silva Kumudika K Purdie Auriol C AC Whittington Richard J RJ

Journal of clinical microbiology 20131218 3

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 compre ...[more]

PMID: 24352996

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Project description:The sensitivity of current diagnostics for Johne's disease, a slow, progressing enteritis in ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP), is too low to reliably detect all infected animals in the subclinical stage. The objective was to identify individual metabolites or metabolite profiles that could be used as biomarkers of early MAP infection in ruminants. In a monthly follow-up for 17 months, calves infected at 2 weeks of age were compared with aged-matched controls. Sera from all animals were analyzed by 1H nuclear magnetic resonance spectrometry. Spectra were acquired, processed, and quantified for analysis. The concentration of many metabolites changed over time in all calves, but some metabolites only changed over time in either infected or non-infected groups and the change in others was impacted by the infection. Hierarchical multivariate statistical analysis achieved best separation between groups between 300 and 400 days after infection. Therefore, a cross-sectional comparison between 1-year-old calves experimentally infected at various ages with either a high- or a low-dose and age-matched non-infected controls was performed. Orthogonal Projection to Latent Structures Discriminant Analysis (OPLS DA) yielded distinct separation of non-infected from infected cattle, regardless of dose and time (3, 6, 9 or 12 months) after infection. Receiver Operating Curves demonstrated that constructed models were high quality. Increased isobutyrate in the infected cattle was the most important agreement between the longitudinal and cross-sectional analysis. In general, high- and low-dose cattle responded similarly to infection. Differences in acetone, citrate, glycerol and iso-butyrate concentrations indicated energy shortages and increased fat metabolism in infected cattle, whereas changes in urea and several amino acids (AA), including the branched chain AA, indicated increased protein turnover. In conclusion, metabolomics was a sensitive method for detecting MAP infection much sooner than with current diagnostic methods, with individual metabolites significantly distinguishing infected from non-infected cattle.

Dataset Information

High-throughput direct fecal PCR assay for detection of Mycobacterium avium subsp. paratuberculosis in sheep and cattle.

Publications

High-throughput direct fecal PCR assay for detection of Mycobacterium avium subsp. paratuberculosis in sheep and cattle.

Similar Datasets

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