Molecular epidemiological confirmation and circumstances of occurrence of sheep (S) strains of Mycobacterium avium subsp. paratuberculosis in cases of paratuberculosis in cattle in Australia and sheep and cattle in Iceland.

Distinct strains of Mycobacterium avium subsp. paratuberculosis with a tendency to segregate in either sheep, or cattle and other ruminants, have been described and are known as S and C strains, respectively. These strains can be distinguished by a polymorphism in the IS1311 element and other DNA-based methods. C strains are relatively easy to culture from tissues and faeces of animals with paratuberculosis but S strains are difficult to culture. A retrospective survey of archival formalin-fixed paraffin-embedded tissue samples from culture negative Australian paratuberculous cattle was undertaken to determine whether infection in these cases was due to S strains. Polymerase chain reaction and restriction endonuclease analysis of the amplified product was used to identify the polymorphism in IS1311. Three cases of bovine paratuberculosis due to S strain were confirmed from three different farms. A serological survey led to the identification of a further two cases on one of these farms. S strains were also identified in archival tissues from paratuberculous sheep and cattle from Iceland, confirming epidemiological and microbiological evidence that paratuberculosis in Iceland was due to S strain following importation of infected sheep from Europe. In each bovine case in both Iceland and Australia there had been direct or indirect contact of calves with paratuberculous sheep. We were unable to determine whether S strains had established endemic infection in cattle or whether repeated infection from sheep had occurred. Limited epidemiological evidence suggests that transmission of S strains to cattle in Australia has been uncommon under extensive grazing conditions. In Iceland, different husbandry practices appear to have favoured transmission of S strains to cattle.

Molecular epidemiology of Mycobacterium avium subsp. paratuberculosis: IS900 restriction fragment length polymorphism and IS1311 polymorphism analyses of isolates from animals and a human in Australia.

The distribution and prevalence of strains of Mycobacterium avium subsp. paratuberculosis were determined among sheep, cattle, and other species with Johne's disease in Australia. A total of 328 isolates were evaluated from numerous farms in New South Wales, Victoria, Tasmania, and South Australia, Australia. Restriction fragment length polymorphism (RFLP) analysis of genomic DNA using BstEII and an IS900 probe and IS1311 polymorphism analysis using PCR and restriction endonuclease analysis (PCR-REA) was used to classify isolates as cattle (C) or sheep (S) strains. IS1311 PCR-REA provided similar information to IS900 RFLP analysis but was more useful than RFLP analysis where DNA was degraded or scarce. Twelve IS900 RFLP types were found. Johne's disease in sheep was always due to S strains, while cattle were infected only with C strains. RFLP type S1 was the dominant strain in sheep in New South Wales (97% of isolates) and was the only strain found in sheep from Victoria. Seven RFLP types were present in cattle. RFLP types C3 and C1 were most common (collectively, 85% of isolates), but C1 was not found in New South Wales and C3 was present in dairy cattle but not in beef cattle in Victoria. These differences may be explained by restricted livestock trading patterns between different segments of the cattle industry. Up to five RFLP types were present in some geographic regions in Victoria, while up to three RFLP types were found among cattle on some farms. Individual cattle usually were infected with only one RFLP type, but one animal was infected with both C5 and CU4. Two isolates from goats were C type as were three from alpacas, one from a rhinoceros, and two from a human with Crohn's disease. The prevalences of specific RFLP types in Australia differ from those reported in Europe and elsewhere. Given the existence of geographical and farm enterprise differences in IS900 RFLP type, this technique may be applied selectively to trace the spread of Johne's disease, at least in the cattle industries. As these observations reflect past exposure of livestock to M. avium subsp. paratuberculosis, the monitoring of strains present in animals in Australia is continuing.

Use of pooled fecal culture for sensitive and economic detection of mycobacterium avium subsp. paratuberculosis infection in flocks of sheep.

Ovine Johne's disease, or paratuberculosis, occurs in many countries. In Australia, surveillance using serology is used as part of a control program, but the testing regime is costly relative to its sensitivity. For this reason, culturing of Mycobacterium avium subsp. paratuberculosis in fecal samples pooled from a number of sheep was evaluated. Initially, the effect of pooling on the sensitivity of fecal culture was evaluated using samples from 20 sheep with multibacillary paratuberculosis and 20 sheep with paucibacillary paratuberculosis, each confirmed histologically. All multibacillary cases and 50% of paucibacillary cases were detected by culturing of feces at a pooling rate of 1 infected plus 49 uninfected sheep. In a pilot-scale study in 1997, M. avium subsp. paratuberculosis was detected by pooled fecal culture on 93% of 27 infected farms which were identified originally based on history, clinical signs, and one or more rounds of testing using serologic and histopathologic examinations. Pooled fecal culture was compared with serologic examination for submissions from 335 farms where both tests had been conducted on the same sheep and was significantly more sensitive (P<0.001). Computer simulation of random sampling indicated that the testing of 6 pools of 50 sheep would provide 95% confidence in detecting > or =2% prevalence of infection. The estimated laboratory cost of pooled fecal culture when applied as a flock test is approximately 30% that of serologic examination, and sample collection costs are lower. It is recommended that pooled fecal culture replace serologic examination for detection of M. avium subsp. paratuberculosis infection at the flock level.