Recent reports of natural paratuberculosis (or Johne's disease) in rabbits, foxes, and stoats has focused debate on the presence and importance of wildlife reservoirs in the epidemiology of this disease. This paper describes an extensive study investigating 18 nonruminant wildlife species for evidence of paratuberculosis. Using both culture and histopathological analysis, fox, stoat, weasel, crow, rook, jackdaw, rat, wood mouse, hare, and badger were found to harbor Mycobacterium avium subsp. paratuberculosis, the causative organism of paratuberculosis, suggesting that the epidemiology of this disease is more complex than previously realized.

BackgroundMycobacterium avium subspecies paratuberculosis (Map) causes an infectious chronic enteritis (paratuberculosis or Johne's disease) principally of ruminants. The epidemiology of Map is poorly understood, particularly with respect to the role of wildlife reservoirs and the controversial issue of zoonotic potential (Crohn's disease). Genotypic discrimination of Map isolates is pivotal to descriptive epidemiology and resolving these issues. This study was undertaken to determine the genetic diversity of Map, enhance our understanding of the host range and distribution and assess the potential for interspecies transmission.Results164 Map isolates from seven European countries representing 19 different host species were genotyped by standardized IS900 - restriction fragment length polymorphism (IS900-RFLP), pulsed-field gel electrophoresis (PFGE), amplified fragment length polymorphisms (AFLP) and mycobacterial interspersed repeat unit-variable number tandem repeat (MIRU-VNTR) analyses. Six PstI and 17 BstEII IS900-RFLP, 31 multiplex [SnaBI-SpeI] PFGE profiles and 23 MIRU-VNTR profiles were detected. AFLP gave insufficient discrimination of isolates for meaningful genetic analysis. Point estimates for Simpson's index of diversity calculated for the individual typing techniques were in the range of 0.636 to 0.664 but a combination of all three methods increased the discriminating power to 0.879, sufficient for investigating transmission dynamics. Two predominant strain types were detected across Europe with all three typing techniques. Evidence for interspecies transmission between wildlife and domestic ruminants on the same property was demonstrated in four cases, between wildlife species on the same property in two cases and between different species of domestic livestock on one property.ConclusionThe results of this study showed that it is necessary to use multiple genotyping techniques targeting different sources of genetic variation to obtain the level of discrimination necessary to investigate transmission dynamics and trace the source of Map infections. Furthermore, the combination of genotyping techniques may depend on the geographical location of the population to be tested. Identical genotypes were obtained from Map isolated from different host species co-habiting on the same property strongly suggesting that interspecies transmission occurs. Interspecies transmission of Map between wildlife species and domestic livestock on the same property provides further evidence to support a role for wildlife reservoirs of infection.

A survey of wild rabbits in Tayside, Scotland revealed that 67 per cent were infected with Mycobacterium avium subspecies paratuberculosis. In general, the infected rabbits had histopathological changes within the lymph nodes and intestines which were consistent with the changes due to paratuberculosis in ruminants. The survey raises the possibility that rabbits and other wildlife may be involved in the epidemiology of paratuberculosis, a possibility which has important implications for the control of the disease.

A survey of 22 farms confirmed the presence of paratuberculosis in wild rabbits in Scotland. Regional differences were apparent in the prevalence of the disease in rabbits, with a significantly higher incidence occurring in the Tayside region. Statistical analysis showed a significant relationship between a previous history or current problem of paratuberculosis in cattle and the presence of paratuberculosis in rabbits on the farms. Molecular genetic typing techniques could not discriminate between selected rabbit and cattle isolates from the same or different farms, suggesting that the same strain may infect and cause disease in both species and that interspecies transmission may occur. The possibility of interspecies transmission and the involvement of wildlife in the epidemiology of paratuberculosis have important implications for the control of the disease.

Paratuberculosis (Johne's disease) was long considered only a disease of ruminants. Recently non-ruminant wildlife species have been shown to harbor Mycobacterium avium subsp. paratuberculosis, the causative organism of paratuberculosis. We review the known non-ruminant wildlife host range of M. avium subsp. paratuberculosis and consider their role in the epidemiology of paratuberculosis in domestic ruminant livestock. Mycobacterium avium subsp. paratuberculosis has been isolated from lagomorph, canid, mustelid, corvid, and murid species. In agricultural environments domestic ruminants may contact wildlife and/or their excreta when grazing or feeding on farm-stored feed contaminated with wildlife feces, opening up the possibility of inter-species transmission. Of the wildlife species known to harbor M. avium subsp. paratuberculosis in Scotland, the rabbit is likely to pose the greatest risk to grazing livestock. Paratuberculosis in domestic ruminants is a notoriously difficult disease to control; the participation of non-ruminant wildlife in the epidemiology of the disease may partially account for this difficulty.

Livestock feed is susceptible to contamination from wildlife excreta during on farm storage. Pathogens associated with diseases such as paratuberculosis, salmonella and cryptosporidiosis are present in wild rodent and bird excreta. Feed stores on four farms in the east of Scotland were monitored monthly over the winter of 1998/9 to quantify the levels of wildlife faecal contamination. A mean of 79·9 rodent (95% confidence interval: 37·5–165·9) and 24·9 (14·3–41·7) bird faeces were deposited per m2 of stored feed per month. It was estimated that individual cattle and sheep could encounter 1626 and 814 wildlife faeces over the winter.A model based on the numbers of infected faeces consumed per annum was used to estimate ‘infectious probabilities’ (Pinf) required to account for the reported prevalence of paratuberculosis, salmonella and cryptosporidiosis in sheep and cattle in the east of Scotland in 1998. Based on empirical data for input variables [the number of faeces encountered (Fe), the number ingested (Fi) and the prevalence of infection in wildlife species (Ip)], Pinf estimates ranged from 1·6×10−8 for cryptosporidiosis in sheep to 8·2×10−6 for paratuberculosis in cattle. The model suggested that ingestion of feed contaminated by wildlife faeces could account for the prevalence of all three diseases. Wildlife faecal contamination of stored feed should be given serious consideration as a potential source of infection to livestock.

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FIG 1: Ziehl-Neelsen stained section of mesenteric lymph node from a fox, showing intracellular acid-fast rods. x 1000 JOHNE'S disease, or paratuberculosis, is a chronic enteritis of ruminants caused by Mycobacterium avium subspecies paratuberculosis (M a paratuberculosis). Clinical signs of Johne's disease include weight loss, decreased production, and eventual death. There is no effective treatment and no cure. Until recently, M a paratuberculosis was thought to cause natural infection in ruminants only. Johne's disease is found worldwide in domestic ruminants, and has also been reported in wild and captive ruminants, including saiga antelope (Saiga tatarica) (Dukes and others 1992), bighorn sheep (Ovis canadensis) and Rocky Mountain goats (Oreamnos americanus) (Williams and others 1983), and wild deer (Riemann and others 1979, Sharp and others 1996). Although Johne's disease has been present in the UK sheep and cattle population for many years, there is still relatively little known about the epidemiology of this important disease. There is mounting evidence for a much wider host range than had been prevously recognised, including non-ruminants. Natural infection has now been documented in macaque monkeys (McClure and others 1987) and rabbits. Recent studies in Scotland (Greig and others 1997) have found M a paratuberculosis infecting the intestinal tract of rabbits. A strong statistical association was found between Johne's disease in rabbits and a history of Johne's disease in cattle on affected farms (Greig and others 1999). This evidence suggests that rabbits may play a role as wildlife reservoirs of the disease. This short communication reports preliminary findings from a wider study investigating other wildlife, especially carnivorous species, to delineate further the host range of Johne's disease. Nine foxes (Vulpes vulpes) from four farms (A, B, C and D), and five stoats (Mustela erminea) from a separate farm (E) were collected as part of the farm vermin control programme. Farms A, B and E had Johne's disease in both the ruminant livestock and rabbit populations; farms C and D were in the close vicinity of an affected farm. Farms A, B and D were in the Perth and Kinross region, and farms C and E in Angus. Postmortem examination was carried out, and samples of liver, jejunum, terminal ileum, ileocaecal junction, caecum, colon and mesenteric lymph node were collected for culture and histopathology. The samples for histopathology were fixed in 10 per cent buffered formol saline for a minimum of 48 hours, trimmed, dehydrated through graded alcohols,