title: 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.
authors.affiliation: Faculty of Veterinary Science, University of Sydney, Camden, Australia.
authors.forename: Karren M
authors.initials: KM
authors.lastname: Plain
authors.forename: Ian B
authors.initials: IB
authors.lastname: Marsh
authors.forename: Anna M
authors.initials: AM
authors.lastname: Waldron
authors.forename: Francesca
authors.initials: F
authors.lastname: Galea
authors.forename: Ann-Michele
authors.initials: AM
authors.lastname: Whittington
authors.forename: Vanessa F
authors.initials: VF
authors.lastname: Saunders
authors.forename: Douglas J
authors.initials: DJ
authors.lastname: Begg
authors.forename: Kumudika
authors.initials: K
authors.lastname: de Silva
authors.forename: Auriol C
authors.initials: AC
authors.lastname: Purdie
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Whittington
mesh_terms: D000818:Animals; D001315:Australia; D001431:Bacteriological Techniques / Q000379:methods; D002417:Cattle; D002418:Cattle Diseases / Q000175:diagnosis* / Q000382:microbiology; D005243:Feces / Q000382:microbiology*; D025202:Molecular Diagnostic Techniques / Q000379:methods; D016927:Mycobacterium avium subsp. paratuberculosis / Q000302:isolation & purification*; D009520:New Zealand; D010283:Paratuberculosis / Q000175:diagnosis* / Q000382:microbiology; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012756:Sheep; D012757:Sheep Diseases / Q000175:diagnosis* / Q000382:microbiology; D014730:Veterinary Medicine / Q000379:methods*