Mycobacteria distenct from Mycobacterium avium subsp. paratuberculosis isolated from the faeces of ruminants possess IS900-like sequences detectable IS900 polymerase chain reaction: implications for diagnosis.

PCR targeting the 5' end of IS 900 has been considered specific for identification of Mycobacterium avium subsp. paratuberculosis and is frequently applied to confirm the presence of this organism in the diagnosis of Johne's disease. IS 900 PCR has also been applied to studies of the aetiology of Crohn's disease. Mycobacterium spp. isolated from the faeces of 3 clinically normal animals in 2 Australian states appeared not to be M. paratuberculosis but were positive by IS 900 PCR. The isolates were characterized using mycobactin dependency, biochemical tests, IS 900 and 16 S rRNA sequencing and restriction fragment length polymorphism (RFLP) using IS 900 as probe. DNA sequencing confirmed that the isolates had between 71% and 79% homology with M. paratuberculosis in the region of IS 900 amplified, were most closely related to Mycobacterium scrofulaceum, and confirmed the usefulness of restriction enzyme analysis of amplified product to identify the false positive results. RFLP analysis with Bst Ell detected three to five copies of the IS 900 -like element in the isolates. These were located in molecular weight fragments that were clearly different to IS 900 in previously characterized strains of M. paratuberculosis. It is likely that these isolates are environmental mycobacteria. Southern blotting with an internal probe is unlikely to provide differentiation of M. paratuberculosis from these organisms. We recommend the adoption of restriction endonuclease analysis of IS 900 PCR product as a routine precaution to prevent the reporting of false positive IS 900 PCR results.

Cousins DV ，Whittington R ，Marsh I ，Masters A ，Evans RJ ，Kluver P ... -  《MOLECULAR AND CELLULAR PROBES》

PCR-restriction endonuclease analysis for identification and strain typing of Mycobacterium avium subsp. paratuberculosis and Mycobacterium avium subsp. avium based on polymorphisms in IS1311.

Point mutations in the IS 1311 sequences from sheep and cattle strains of Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) and M. avium subsp. avium (M. avium) were targeted to develop a polymerase chain reaction (PCR) that would be useful in the diagnosis and control of Johne's disease. Candidate PCR tests were evaluated for sensitivity, specificity and ease of interpretation of the restriction endonuclease analysis (REA) products. One IS 1311 PCR, amplifying a 608 base pair product, was shown to be suitable when the amplified product was digested with Hinf I and Mse I. The PCR detected 50 fg of template DNA from M. paratuberculosis strain 316 V, the equivalent of 10 organisms. The test was evaluated further using purified DNA from M. paratuberculosis and M. avium isolates and diagnostic samples including primary radiometric cultures. All 89 M. paratuberculosis samples were correctly identified and typed according to host species or IS 900 restriction fragment length polymorphism (RFLP) type. All 28 isolates of M. avium were also correctly identified. A second PCR/REA strategy based on a shorter fragment of IS 1311 was developed for formalin-fixed paraffin-embedded tissue samples. It correctly differentiated sheep and cattle strains of M. paratuberculosis in 27 tissue samples in which acid fast bacilli had been observed in Ziehl Neelsen stains and in which sufficient amplified product was present for REA with Hinf I. Both tests were specific for M. paratuberculosis when tested against 24 other mycobacterial species. These simple and rapid tests can be used on a range of diagnostic samples for the confirmation of Johne's disease and will be of benefit in control and eradication programmes for this disease.

Marsh I ，Whittington R ，Cousins D 《MOLECULAR AND CELLULAR PROBES》

Development of a nested PCR method targeting a unique multicopy element, ISMap02, for detection of Mycobacterium avium subsp. paratuberculosis in fecal samples.

Stabel JR ，Bannantine JP 《-》

Evaluation of radiometric faecal culture and direct PCR on pooled faeces for detection of Mycobacterium avium subsp. paratuberculosis in cattle.

Eamens GJ ，Whittington RJ ，Turner MJ ，Austin SL ，Fell SA ，Marsh IB ... -  《VETERINARY MICROBIOLOGY》

Mycobacterium porcinum strains isolated from bovine bulk milk: implications for Mycobacterium avium subsp. paratuberculosis detection by PCR and culture.

Taddei R ，Barbieri I ，Pacciarini ML ，Fallacara F ，Belletti GL ，Arrigoni N ... -  《VETERINARY MICROBIOLOGY》