**Conflict of interest**

*Molecular Epidemiology Study of Mycobacterium Tuberculosis Complex*

in the Bieszczady region [63]. Even so, over the past 20 years, TB has been found in other species of wild animals in the Bieszczady Mountains, mostly in wild boars. The first case of TB in a wild boar was reported in 2012 in a four-year-old female from Nasiczne in the Bieszczady, which was found dead due to *Metastrongylus* spp. invasion. Postmortem examination showed small caseous, yellowish tubercules in submandibular lymph nodes, from which *M.caprae* was isolated (at that time *M. bovis* ssp. *caprae*). The strain was found to have the same spoligo pattern as those strains isolated in 2011 from European bison from the Bieszczady area [45], this being 200003777377400, or SB2391 as assigned by www.Mbovis.org [69].

Since then, a number of cases of TB have been found in the Bieszczady wild boar population each year. Between 2012 and 2017, *M. caprae* was isolated from the lymph nodes of 21 out of 55 investigated wild boar [63]. These strains were subjected to molecular analysis based on spoligotyping according to Kamerbeek et al. [70], and MIRU-VNTR typing, as given in the public protocol [71]. A total of 15 loci were investigated: MIRU 4, MIRU 10, MIRU 16, MIRU 26, MIRU 31, MIRU 40, VNTR 424, VNTR 577, VNTR 2165, VNTR 2401, VNTR 3690, VNTR 4156, VNTR 2163b, VNTR 1955 and VNTR 4052. All 21 isolated strains shared an identical spoligotype 200003777377400 – SB2391. From this group, 19 strains shared a single MIRU-VNTR pattern (464652364413423), while the other two had patterns that differed with regard to a single locus (464552364413423 and

To describe the occurrence of TB in wildlife other than European bison and wild boar, both within the Bieszczady Mountain region and elsewhere, lymph node samples were collected for analysis from red foxes, wolves, badgers, red deer, roe deer and brown bear between 2011 and 2017. *M. caprae* was isolated from the lymph nodes of one roe deer and three wolves. Those animals had no visible TB-like lesions

All molecular research of *M. caprae* strains isolated from wildlife in the Bieszczady Mountains has been performed based on hsp65 sequence analysis, the GenoType®MTBC (Hain Lifescience, Germany) test, spoligotyping and MIRU-VNTR analysis. Further studies to determine the epidemiological link and the possible route of transmission of the source of infection are needed based on whole

In conclusion, bovine tuberculosis remains a real threat in Poland, as indicated by the increasing number of cases observed in wildlife and the recent report of the first confirmed case of *M. caprae* infection in human. *M. caprae* is the main etiologi-

We recommend that in Poland, bovine tuberculosis should not only be monitored in cattle but also in wildlife. This is especially true in the European bison population, which seems to be highly sensitive to infection. This is highly important for protecting public health, maintaining the OTF status of Poland and of course, protecting the European bison themselves. In which case, particular attention should be paid to the free-living animal population in the Bieszczady Mountains. There is also a particular need to monitor alpacas, as TB-positive animals pose a particular risk to children and disabled people due to increased contact during

We recommend the more intensive use of molecular tests in monitoring and the proper archiving of the identified DNA. Such molecular methods play an essential role in epidemiological investigations, as these can accurately identify the source

cal agent of bovine tuberculosis in wildlife, and *M. bovis* in cattle.

**90**

463652364413423) [63].

genome sequencing.

**6. Conclusions**

animal therapy.

[48, 63].

The authors declare no conflict of interest.
