Comparison of immunomagnetic bead separation-polymerase chain reaction andfaecal culture for the detection of Mycobacterium avium subspparatuberculosis in sheep faeces

摘要

Paratuberculosis or Johne's disease is a chronic enteropathy of both domestic and wild ruminants caused by Mycobacterium avium subsp paratuberculosis. The clinical disease in sheep is characterised by progressive loss of weight and some animals have intermittent diarrhoea. The disease has been recognised in cattle in Australia since 1925 but only recently has been reported in sheep. The first report of paratuberculosis in sheep in Australia was in 1980 and the first outbreak reported was in New South Wales in 1981. The prevalence of ovine paratuberculosis in Australia has been increasing ever since. The most sensitive method currently available for diagnosis of paratuberculosis in sheep flocks is the c. ulture of M a paratuberculosis from faecal or tissue samples. The main difficulty however, is that the procedure requires lengthy incubation periods, for example, 12 weeks to report a negative result. Consequently there is a quest for a rapid test. Alternative tests such as enzyme-linked immunosorbent assay, agar gel immuno-diffusion, complement fixation and the gamma interferon assay lack sensitivity, specificity or practicality. The potential for molecular methods such as PCR to detect paratuberculosis has been recognised due to the promise of increased sensitivity and specificity but there is a difficulty in processing samples such as faeces, which contain substances inhibitory to PCR. The use of immunomagnetic beads to separate substances of interest from complex mixtures has been extended to the recovery of specific target bacteria including mycobacteria from heterogenous suspensions. The method relies on the interaction between specific antibodies attached to magnetic polystyrene beads and cell-surface antigens on the bacteria of interest. The bacteria are concentrated and substances, which may be inhibitory to subsequent reactions, are removed.