title: Evaluation of radiometric faecal culture and direct PCR on pooled faeces for detection of Mycobacterium avium subsp. paratuberculosis in cattle.
abstract: Dilution rates for pooled faecal culture (PFC) and direct IS900 polymerase chain reaction (D-PCR) tests were evaluated on faecal samples from infected cows mixed with uninfected faeces in dilutions from 1 in 5 to 1 in 50. PFC was performed by radiometric culture, with confirmation by IS900 PCR and restriction endonuclease analysis (PCR/REA) on growth, and by mycobactin dependency testing on solid medium. Using 37 culture positive faecal samples from 12 subclinical cows, 83.8% and 94.6% of samples were confirmed positive in the PFC assay at dilutions of 1 in 50 and 1 in 30, respectively. Lower dilutions (1 in 5 to 1 in 20) provided only marginally better sensitivity, and confirmation of PFC growth by PCR/REA was significantly more sensitive than mycobactin dependency. D-PCR had significantly lower sensitivity than PFC confirmed by PCR/REA, with pools of 1 in 50, 30, 10 and 5 yielding positive results in 64.9%, 70.3%, 78.4% and 83.8% of samples, respectively. Cattle considered to be shedding 1.5 x 10(6) viable M. avium subsp. paratuberculosis (Map)/g faeces (on the basis of estimated losses in processing and growth rates in radiometric broth) were positive at dilutions up to 1 in 50 in the PFC and D-PCR. Those shedding 5 x 10(5) viable Map/g were positive in the PFC at dilutions up to 1 in 40, but required a 1 in 10 dilution or less for D-PCR. The results suggest that for cattle shedding relatively high concentrations of Map in faeces (>2 x 10(5) viable Map/g), maximal dilutions of 1 in 30 for PFC and 1 in 10 for D-PCR would be applicable.
authors.affiliation: Elizabeth Macarthur Agricultural Institute, NSW Department of Primary Industries, Menangle, NSW, Australia. graeme.eamens@dpi.nsw.gov.au
authors.forename: Graeme J
authors.initials: GJ
authors.lastname: Eamens
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Whittington
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Turner
authors.forename: Susan L
authors.initials: SL
authors.lastname: Austin
authors.forename: Shayne A
authors.initials: SA
authors.lastname: Fell
authors.forename: Ian B
authors.initials: IB
authors.lastname: Marsh
mesh_terms: D000818:Animals; D002417:Cattle; D002418:Cattle Diseases / Q000175:diagnosis / Q000382:microbiology*; D004251:DNA Transposable Elements / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D005243:Feces / Q000382:microbiology; D005260:Female; D016927:Mycobacterium avium subsp. paratuberculosis / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D010283:Paratuberculosis / Q000175:diagnosis / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000662:veterinary; D015183:Restriction Mapping / Q000662:veterinary
chemical_list: D004251:DNA Transposable Elements; D004269:DNA, Bacterial