Multiple strain infections and high genotypic diversity among Mycobacterium avium subsp. paratuberculosis field isolates from diseased wild and domestic ruminant species in the eastern Alpine region of Austria
Introduction
Many wild and domestic mammal species are susceptible hosts for Mycobacterium avium complex (MAC) species. The subspecies M. avium subspecies paratuberculosis (MAP) is the etiological agent of a chronic fatal ruminant gastroenteritis called Johne’s disease or paratuberculosis. Occurring worldwide, the disease mainly affects both domestic and wild ruminants. However, MAP has also been isolated from various non-ruminant wildlife species (Motiwala et al., 2006, Stevenson et al., 2009).
Paratuberculosis is regarded as one of the most important livestock diseases causing more than 250 million USD estimated yearly losses in the dairy industry in the USA alone, even though accurate estimates are difficult to obtain (Ott et al., 1999, Smith et al., 2009; for review see Hasonova and Pavlik, 2006). The association of MAP with Crohn’s disease (Behr and Kapur, 2008, Chiodini et al., 2012, Naser et al., 2004), Diabetes type I (Rani et al., 2010) and, more recently, Multiple sclerosis (Cossu et al., 2012) in humans highlights the zoonotic potential of the agent, even though a clear causative relation is still under debate (Chiodini et al., 2012, Gitlin et al., 2012). Since the isolation of MAP from animals does not necessarily coincide with clinical symptoms (Begg et al., 2005, Deutz et al., 2005, Mackintosh et al., 2010) consumption of both wild and domestic animal products poses a potential public health threat (Chiodini et al., 2012, Hermon-Taylor, 2009, Wynne et al., 2011). This holds especially true as MAP may disseminate throughout the body during infection and has been detected in muscle tissue (Antognoli et al., 2008, Brady et al., 2008). Also MAP is particularly resilient to heat inactivation and can be found in high-temperature, short-time pasteurized milk. (For review see Collins, 2011 and Eltholth et al. 2009).
The role of wildlife in the epizootiology of paratuberculosis is controversial with respect to its function as a spill-over host versus a reservoir (Carta et al., 2011, Deutz et al., 2005, Fritsch et al., 2012, Manning, 2011) and clinical manifestation in wildlife ruminant species can differ considerably from livestock (Manning, 2011). Nevertheless previous studies have shown that domestic livestock and wild mammals infected with MAP can share highly similar strains (Fritsch et al., 2012, Greig et al., 1999, Motiwala et al., 2004).
While the costs of wildlife infection itself are arguably economically negligible, it should not be overlooked when paratuberculosis eradication in livestock herds is attempted. This is especially true in areas where domestic cattle and wildlife share habitat, as is the case in virtually all pasture associated farming systems.
Due to a substantial increase in Austrian cattle serologically positive for MAP between 1999 and 2003, paratuberculosis in livestock ruminants was declared a notifiable disease in Austria in 2006 (Khol et al., 2007). Free-living wild ruminants however, were not included in this regulation. During the same period (2002–2008) an increase in clinically manifest MAP infections in the local wild ruminant population in the Austrian state of Styria was reported (Deutz et al., 2005). In response to the increase in MAP infections we initiated a genetic investigation into the potential epizootiologic association of MAP isolates from all five, locally occurring, wild ruminant species and domestic cattle sharing the mountainous habitat in this area. The growing INRA–Nouzilly list (Thibault et al., 2007) of known MAP strains, as classified by mycobacterial interspersed repetitive units (MIRUs) and variable number tandem repeat (VNTR) patterns, highlighted MIRU-VNTR typing and subsequent product sequencing as an efficient approach to detect the sharing of strains between regions and species. Our goal was to use this typing method to establish if wildlife species represent a reservoir for consequent MAP infection of domestic cattle or whether, alternatively, MAP strains are restricted to individual species within the mountainous habitat of Styria.
Section snippets
Animals
A total of 39 MAP isolates from domestic cattle (n = 7), roe deer (Capreolus capreoulus) (n = 5), red deer (Cervus elaphus) (n = 18), chamois (Rupicapra rupicapra) (n = 1), European mouflon (Ovis orientalis musimon) (n = 4), and Alpine ibex (Capra ibex) (n = 3) were included in the study. Of these isolates 34 originated from the Austrian province of Styria. The remaining 4 isolates were recovered from districts in Lower Austria and Burgenland in immediate proximity to Styria, and a single isolate was
MAP identification and MIRU-VNTR patterndiversity
All 39 isolates were positive for the IS900 element and considered to be MAP isolates (Table 1). Within the 39 isolates, all loci showed variability (Table 2). via standard MIRU-VNTR analysis, mixed infections were identified in five isolates (13%), three of which yielded two bands at one locus only and were classed as multiple strain infections and hence mixed cultures of at least two MAP strains. A sample of a roe deer showed double bands at two loci and finally one sample of domestic cattle
Discussion
The role of wildlife as a reservoir for MAP has been discussed in numerous studies (Beard et al., 2001, Deutz et al., 2005, Fritsch et al., 2012, Stevenson et al., 2009). In areas with overlapping habitats, transmission of MAP from wildlife to domestic livestock or vice versa has been suggested (Fritsch et al., 2012, Greig et al., 1999). However, proof or identification of a uni- or bi-directional infection pattern is still pending. MAP has recently been isolated from various soil and water
Conclusion
Identification of at least 15 MAP genotypes across multiple host species in the Styrian wild and domestic ruminant populations reinforces the view that wild ruminants can act as reservoirs in the transmission of MAP to domestic herds. As such, infection rates need to be monitored and managed in wild ruminant populations concurrently with control in domestic populations. Furthermore, the detection of 6 multiple strain infections during the rise of clinically apparent MAP cases raises concerns
Acknowledgments
This work was supported by grant PARARISK of the Fund of the Austrian National Bank OeNB. We are indebted to A. Haiden and S. Wildmann for constant lab-work assistance.
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2016, Molecular and Cellular ProbesCitation Excerpt :Sequencing of the same locus from MAP and MA isolates confirmed the in silico findings and revealed two additional tandem repeats that had not been described previously (Fig. 3). In accordance with our results, additional nucleotides at the VNTR 7 locus were reported previously for three MAP S strains (type III) from sheep in Germany [11] and one MAP isolate from a red deer in Austria [12]. However, none of these studies provided precise sequence data.