Bovine Tuberculosis in European Bison as Possible Zoonotic Impact in Poland

Kita Jerzy2*, Anusz Krzysztof1*, Salwa Andrzej3, Welz Miroslaw4, Orlowska Blanka1 and Zaleska Magdalena5 1Department of Food Hygiene and Public Health Protection, Faculty of Veterinary Medicine, Warsaw University of Life Sciences – SGGW 2Faculty of Veterinary Medicine, Warsaw University of Life Sciences – SGGW 3Veterinary Hygiene Institute, Gdansk 4Voivodeship Veterinary Inspectorate, Krosno 5Department of Pathology and Veterinary Diagnostics, Faculty of Veterinary Medicine, Warsaw University of Life Sciences – SGGW, Nowoursynowska, Warsaw, Poland


Introduction
Tuberculosis remains the most prevailing disease worldwide. This infectious disease is caused by various strains of mycobacteria -Mycobacterium tuberculosis, M. bovis, M. bovis BCG, M. microti and M. africanum all known under one name Mycobacterium tuberculosis complex. Each of the aforementioned agents is recognized as dangerous both to humans and animals. Bovine tuberculosis (bTB) results from infection by M. bovis.
Suspicions of tuberculosis incidents in wildlife of Bieszczady Mountains (Poland, Podkarpackie Province) have a long history, however, first records on the confirmed bTB cases date back 1996 and concern European bison found dead within Brzegi Dolne Forest Inspectorate ( Fig. 1, 3, 4) ( urawski et al.1997). Culling of the European bison for diagnostic purposes, carried out in the period 1997-2001, revealed 13 bTB cases of 18 individuals tested (Welz et al.2006). In 2005In -2008 in Bieszczady Mountains the next study was conducted. 5 strains of M. bovis were isolated -from 2 European bison (of 3 investigated), 2 cows and badger (of 2 investigated).

Molecular epidemiology of M. bovis isolates
The whole study covered 215 free -ranging animals: red deer, wild boar, roe deer, a few examples of European bison, badger, wolf and lynx (Fig. 2). All of the animals were examined for mycobacterial infections. The studied material also involved 4 domestic cows. The examination of the collected material resulted in isolation of 14 strains of acid-resistant mycobacteria. The strains were distinguished as follows: 4 strains of M. tuberculosis (3 wolfs and a cow), 5 strains of M. bovis (2 European bison, 2 cows and badger), 4 strains of M.avium (4 red deer), and a strain of M. species (1 red deer) (Salwa et al. in prep.).    The examination toward the isolation and identification of tuberculosis mycobacteria was carried out by culture technique with the use of biological tests recommended by the Microbiology Unit of State Veterinary Institute in Pulawy. Lymph nodes were www.intechopen.com microsectioned and chosen tissue fragments were homogenized. The inoculation was carried out in solid medium according to Lowenstein-Jansen and Stonebrink. Identification of isolated bacteria was carried out by the culture and biochemical methods.
Parallel to bacteriological identification of isolated Mycobacterium strains, a confirmation via PCR methods was performed. DNA isolation from examined strains was carried out by the column method using Genomic Mini AX Tissue set (A&A Biotechnology, Gdynia), according to the producer's instructions. The amplification of the selected fragment of region 16S-23 rRNA ITS was carried out utilizing 2 starters complementary to 5' and 3' with the following sequences: ITS-F1-1 5'-TTG ATC CGA CGA AGT CGT AAC AAG g-3' Mycom-3 5'-ATG CTC GCA ACC ACT ATC CA-3' Oligonucleotides were synthesized by using a DNA Synthesizer (Genmed Synthesis, Inc.-USA). After finished reaction, the amplification products were analysed electrophoretically in 2% agarose gel (Sigma, Chemical Co.) with addition of ethydine bromide. After electrophoresis the gel was photographed wit an UV transiluminator (Fotodyne Inc.).
Restriction analysis based on the enzyme Sau 961 (Fermentas, Lithuania) to digest bacterial DNA, identifying and cutting the DNA chain in the region of a specific nucleotide sequence. The electrophoresis was carried out in polyacrylamide gel electrophoresis apparatus filled with TAE buffer. The electrophoresis was carried out at voltage 100V/3 hours.
Sequencing was carried out using an automatic sequencer ABI PRISM 3100 Avant Genetic Analyzer, Applied Biosystem. The analysis involved a chosen area of the 16S-23 ITS region of all strains included in the examination. The obtained DNA sequences were then subjected to comparative analysis and subsequently a phylogenetic tree image was created by the Tree View program (version: 1.6.6).
The following strains were used for comparative examination: M. tuberculosis isolated from humans (strain PBG/28) and M. bovis isolated from a cow (strain DG/358-9). The strains were supplied by the Tuberculosis Diagnostic Pomeranian Center of Infectious Diseases and Tuberculosis in Gdansk, Poland and from the Tuberculosis Diagnostic Laboratory Veterinary Hygiene Institute in Bydgoszcz, Poland.
Among the compared M. bovis strains, the highest affinity was displayed by strain derived from two European bisons. Sequence comparision of the analyzed fragment 16S-23S rDNA revealed that it was homologous except for only two nucleotides. What is important, the animals came from two herds living in different regions of Bieszczady. A high genetic differentiation was demonstrated comparing nucleotide sequences od M. bovis strains isolated from the badger and cattle. These evidence indicate that M. bovis spread within the Bieszczady region is represented by different variants (Salwa et al. in prep.). Consistent with this observation, studies of others authors have shown DNA diversity of M. bovis. (Skuce et al. 1996, Sechi et al.1999 The result of the aforementioned study suggest that tuberculosis had been transmitted to European bison from the cattle when grazing the same pastures. The role of badgers in the spreading of M. bovis in Bieszczady Mountains is not yet fully recognized. It is necessary to underline that M. bovis was not isolated from deer and boars in Bieszczady Mountains.

Discussion
According to the report listing the local zoonotic disease in Bieszczady found during 1960-2008, in the sixties tuberculosis was common in cattle of Bieszczady. Although in the seventies the disease was believed to be eradicated, some isolated cases of tuberculosis positive cows were being found regularly every year thereafter. The report pointed at Daszówka as the location with the highest density of tuberculosis loci (Fig. 2). According to the study bTB is endemic in the herds of free-ranging European bison of Bieszczady Mountains. The transmission of M. bovis probably originated from the infected cattle. Despite the actions taken in order to eliminate infections, new cases of bTB in European bison are still being reported. Last two cases are quite recent (2011) and they legitimate further studies on the spread of tuberculosis also in many wildlife species, also from the orders Carnivora (wolf -Canis lupus, red fox -Vulpes vulpes, badger -Meles meles) and Rodentia.
M. bovis has been isolated from many free-ranging animals worldwide (Aranaz et al. 2004, Delahay et al. 2007). First world reports on tuberculosis in wildlife related to the kudu www.intechopen.com antelope in South Africa (Griffith 1929). Since then the disease have been observed in other species inhabiting different countries for example: the red deer, elks and coyotes from Texas (Perumaalla et al. 1996), red deer, elks, lynxes and hares from Spain (Aranaz et al. 2004), the African buffalo from Southern Africa (De Vos et al. 2001, Michel et al. 2006) and the bison from Canada (Schmit et al. 2002). Tuberculosis was also found in the baboon, lion, panther and cheetah (Michel et al. 2006). In Poland tuberculosis in wildlife was described for the first time in roe deer in the Gdańsk district (Czarnowski 1956). Later research showed quite frequent occurrence of Mycobacterium infection in the zoo animals (Dąbrowski 1974(Dąbrowski , urawski et al. 1980 Tuberculosis, one of the oldest recognized disease in humans and animals, still remains a serious health hazard. It is estimated that one third of the human world population is European bison in Bieszczady Mountains should also be undertaken (evaluation of vaccine efficacy by microbiological and molecular examination -samples from characteristic pathological lesions; gamma interferon and lymphocyte proliferation tests, ELISA -blood samples). Oral administration of vaccines has a number of advantages including ease of administration, low cost, and the avoidance of needles. Furthermore, oral immunization more effectively targets the mucosal immune response. Oral bait vaccines have been successfully used to prevent rabies in foxes and other wildlife carriers (Schneider 1995).
It is necessary to remind that the European bison (Bison bison) is the largest terrestrial mammal in Europe (Fig. 5.). Long since being exterminated, it has been eventually returned to the wild. Considering health threats to European bison, the fact that those alive today descent from just 12 individuals (5 males and 7 females) survived in zoos and animal parks should be taken into account. Limited genetic variability characterizes all the European bison living now. That may lead to the appearance of genetic defects, less-flexibility and tolerance to unfavorable environmental changes, or a lowering of resistance and consequent vulnerability to disease. The threats to the European bison not only concern infectious disease, but also genetically derived problems such as testicular anomalies and environmental limitation like lack of opportunity for migration between isolated populations, limitations on food resources, competition with other ungulates (

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Bieszczady Mountains is one of the most valuable Polish mainstays for many species. It houses about 230 species of vertebrates, including protected animals as European bison, the wolf, brown bear, lynx and badger.