Cytokine inflammatory response in dairy cows with mastitis caused by Streptococcus agalactiae.

Introduction: The aim of the study was evaluation of the concentrations of interleukin (IL)-1ß, IL-8, IL-12ß and tumour necrosis factor alpha (TNF-α) in the serum and milk of cows with mastitis caused by Streptococcus agalactiae. Material and Methods: A total of 60 milk samples from diseased cows and 30 milk samples from healthy cows were included in the study. Blood and milk samples were taken from Holstein-Friesian cows from three herds (two in tie-stall and one in a free-stall housing system) in Lublin Province in Poland. The concentrations of cytokines in blood serum and quarter milk samples were determined by ELISA. Results: The levels of IL-1ß, IL-8, IL-12ß and TNF-α were significantly higher in the milk of cows suffering from mastitis caused by S. agalactiae compared to the milk of healthy cows (263.03 vs 55.36 pg/mL, 298.34 vs 131.82 pg/mL, 604.10 vs 139.17 pg/mL and 460.86 vs 78.82 pg/mL, respectively). In the group of sick cows, cytokine levels were significantly higher in milk than in serum (263.03 vs 55.25 pg/mL for IL-1ß, 298.34 vs 164.22 pg/mL for IL-8, 604.10 vs 70.34 pg/mL for IL-12ß and 460.86 vs 104.78 pg/mL for TNF-α). Conclusion: The results confirm the involvement of the entire bovine immune system to protect against the bacteria first locally in the udder. The response of the mammary gland to infection caused by S. agalactiae is rapid and already very strong at the beginning of the infection.

Mycobacterial Interspersed Repeat Unit-Variable Number Tandem Repeat Typing of Mycobacterium avium Strains Isolated from the Lymph Nodes of Free-Living Carnivorous Animals in Poland.

Non-tuberculous mycobacteria (NTM) are ubiquitous organisms, of which some, especially those of the Mycobacterium avium complex (MAC), may be opportunistic animal and human pathogens. Infection with NTM can interfere with tuberculosis (TB) diagnosis and induce zoonoses, especially in immunocompromised individuals. Diseases caused by NTM have become more readily recognized; however, they are likely still underestimated. In this study, we identified and genotyped Mycobacterium avium strains that were isolated during TB monitoring among free-living carnivorous animals from southeastern Poland. In 2011-2020, lymph node samples from 192 such animals were tested for mycobacteria. A total of 41 isolates of M. avium strains were detected with the use of IS901, IS900, IS1245, and mycobacterial interspersed repeat unit-variable number tandem repeat (MIRU-VNTR) identification. Thirty-three were identified as M. avium subsp. avium. These strains were derived from 1 beech marten (Martes foina), 1 common buzzard (Buteo buteo), 2 European badgers (Meles meles), 3 wolves (Canis lupus), and 26 red foxes (Vulpes vulpes). One strain isolated from a wolf was identified as M. avium subsp. hominissuis. The results show the widespread occurrence of MAC bacilli in the studied environment and additionally comprise new data on the molecular characteristics of M. avium subspecies carried by free-living southeastern Polish carnivores.

No evidence of Mycobacterium tuberculosis complex infection in samples from cervids in various regions of Poland.

INTRODUCTION: Tuberculosis (TB) is a widespread disease known to affect livestock and wildlife, as well as humans. However, its incidence in wildlife remains poorly recognized on the global level. In Europe, the majority of TB cases have been confirmed in red deer, badgers and wild boar. OBJECTIVE: The aim of the study was to examine the occurrence of TB in Cervidae in Poland, in areas where TB has been detected in cattle and wildlife. MATERIAL AND METHODS: Head and thoracic lymph nodes were collected from a total of 76 free-living red deer (Cervus elaphus) and roe deer (Capreolus capreolus), from nine Polish provinces during a single hunting season (autumn - winter 2018-19). Samples were subjected to conventional microbiological procedures to isolate mycobacteria. RESULTS: No Mycobacteria was isolated in the material collected from red or roe deer. CONCLUSIONS: There is a need to continue monitoring the presence of TB in cattle and other animal species to ensure the protection of public health.

Ten Years of Animal Tuberculosis Monitoring in Free-Living European Bison (Bison bonasus) in Poland.

In the period 1996-2012, two outbreaks of animal tuberculosis were noted in the population of free-living European bison (Bison bonasus caucasicus) in the Bieszczady Mountains, Southern Poland. As the European bison is an endangered species and particularly susceptible to tuberculosis, not to mention a national icon, the decision was made to test all deceased bison for TB in Poland. The screened bison were obtained by elimination due to poor health or natural death. A total of 159 European bison have been examined over the last 10 years. The individuals came from four regions of Poland (Bialowieza Forest, Bieszczady Mountains, Borecka Forest, Knyszynska Forest), not only from the area where tuberculosis is still endemic. Mycobacterium bovis and Mycobacterium avium spp. hominisuis were identified in two different herds. The isolation of M. bovis from European bison was the first case described in Poland. So far, the only causative agent of tuberculosis identified in European bison in Poland, both in the wild and in captive herds, was Mycobacterium caprae. The isolated M. bovis spoligotype has not previously been registered in international spoligotype databases so far. The obtained results highlight the need to monitor TB in European bison in Poland.

The Eradication of M. Caprae Tuberculosis in Wild Boar (Sus Scrofa) in the Bieszczady Mountains, Southern Poland - An Administrative Perspective.

Introduction: Animal tuberculosis (TB) is a zoonotic disease caused by acid-fast bacteria belonging to the Mycobacterium tuberculosis complex (MTBC). Both animals and humans are susceptible to infection by the MTBC. Interspecies transmission is also possible, including to livestock and humans. In the years 1997-2013, many tuberculosis cases were recorded in European bison in the Bieszczady Mountains; more alarmingly, TB was also recorded in wild boar in the years 2013-2020. Material and Methods: In the years 2013-2020, 104 wild boar from the Bieszczady Mountains were tested for TB through necropsy, mycobacterial culture, strain identification and spoligotyping. Results: The microbiological examination confirmed TB in 46 wild boar; these infections were identified as M. caprae, spoligotype SB2391. Conclusion: Free-living European bison are at risk of TB infection from wild boar carrying M. caprae. This situation also poses a risk to local cattle. There is a need for further activities aimed at monitoring the disease, preventing further transmission, and minimising the risk to public health.

Mycobacterial Infections in Invasive Turtle Species in Poland.

Over the last 30 years, the number of invasive turtle species living in the wild has significantly increased in Poland. This proliferation carries many threats, which mainly include the displacement of native species of animals from their natural habitats. Turtles can also be reservoirs for pathogens, including bacteria from the Mycobacterium genus. In order to confirm or rule out the presence of acid-fast mycobacteria in the population of invasive turtle species, samples from carapace, plastron, internal organs and mouth cavity swabs from 125 animals were tested. Twenty-eight mycobacterial strains were isolated in culture, which were classified as atypical following multiplex-PCR reactions. The GenoType Mycobacterium Common Mycobacteria (CM) test, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, PCR-restriction fragment length polymorphism (PRA)-hsp65 and DNA sequencing were used to identify the species of isolates. Of the 28 strains, 11 were identified as M. fortuitum, 10 as M. chelonae, 3 as M. avium ssp. avium, 2 as M. nonchromogenicum and 1 each of M. neoaurum and M. scrofulaceum. The results of the research will also strengthen the understanding that these animals can be vectors for pathogens when living in the wild.

Is Serological Monitoring a Fit-for-purpose Tool to Assess the Epidemiological Situation of Tuberculosis in the Sylvatic Species of European Bison (Bison Bonasus) in Poland?

Introduction: Bovine tuberculosis is one of the most dangerous zoonotic diseases. Despite the near-complete elimination of the disease from cattle breeding in Poland achieved in 2009, its re-emergence is now observed. Globally, the number of human cases is underestimated and the importance of free-living animals as reservoirs of tuberculosis is growing. As a species highly susceptible to Mycobacterium tuberculosis complex infection, the European bison (Bison bonasus) has a role in the transmission of the disease in Poland. The purpose of the investigation was to assess the epidemiological situation of tuberculosis in Polish European bison serologically. Material and Methods: A total of 460 serum samples were collected from 436 European bison from 15 out of 26 national populations between 2013 and 2020. An M. bovis ELISA was used, and its sensitivity and specificity were assessed with an eyelid tuberculin skin test (TST) and interferon gamma release assay (IGRA). Results: Mycobacterium bovis antibodies were detected in nine serum samples. The presence of antibodies was found in two animals from the Bialowieza Forest (1.2% of the population), and one each from the Borecka Forest (2.4%) and the Warsaw Zoo (14.3%). One European bison among the 14 sampled (7.1%) from Smardzewice was positive on five occasions. Other samples from Smardzewice and the Bieszczady Mountains, where tuberculosis had previously been reported, were negative. Conclusion: ELISA testing is an effective, easy and cost-efficient tool for monitoring of tuberculosis-naïve populations. Serological testing in tuberculosis control programmes can significantly improve the detection of infected herds. Antibody ELISAs may supplement TST and IGRA, but cannot replace them.

Mycobacterium bovis Transmission between Cattle and a Farmer in Central Poland.

INTRODUCTION: Zoonoses have recently become an increasing public health problem. Zoonoses are estimated to account for 60% of all emerging infectious diseases. One particularly important zoonosis is human tuberculosis, especially tuberculosis due to Mycobacterium bovis (M. bovis), which is naturally resistant to pyrazinamide (PZA). MATERIAL AND METHODS: The patient had a pulmonary form of tuberculosis accompanied by a cough and fever. At the same time, the disease was also confirmed in 20 out of 25 cattle on the farm. The clinical specimen (sputum) was examined in accordance with the European Union (EU) laboratories' methodology. Tissue materials from cattle were verified in the National Veterinary Research Institute (NVRI), in the Bovine tuberculosis (BTB) Reference Laboratory, Pulawy, Poland and tested in accordance with the guidelines for the laboratory diagnosis of BTB. RESULTS: All M. bovis isolates represented one spoligotype, SB0120. The results of mycobacterial interspersed repetitive unit variable number tandem repeat (MIRU-VNTR) evaluation showed the same genetic pattern. CONCLUSIONS: Findings from this study suggest the first confirmed interspecific transmission of Mycobacterium bovis, between a farmer and his cattle, in Poland. Present findings support the increasing concern regarding zoonotic TB that has been highlighted elsewhere.

Pulmonary mycobacteriosis of sitatunga antelope caused by M. avium ssp. hominissuis.

INTRODUCTION AND OBJECTIVE: Mycobacteriosis are diseases caused by acid-fast mycobacteria other than M. leprae and tuberculous mycobacteria. Animal mycobacteriosis is often caused by M. avium ssp. hominissuis. Many species of animals are susceptible to infection with this bacterium, even those kept in Zoological Gardens. The aim of the study was to determine the species of bacterium responsible for causing the disease in the tested animals. MATERIAL AND METHODS: Tissue samples of two male sitatunga antelopes (Tragelaphus spekii) were analyzed. Lymph node and lung samples were subjected to anatomical examination and Ziehl-Neelsen staining. Real-time PCR was performed to confirm or rule out tuberculosis mycobacteria infection. In order to isolate the bacterial strain, tissue samples were inoculated on both solid and liquid media. HainLifescience CM tests, mass spectrometry and New Generation Sequencing were used to determine the mycobacterial species. RESULTS: Results showed that atypical mycobacteria are responsible for the antelope disease. The results of the HainLifescience CM test and mass spectrometry indicated that the mycobacterium responsible for causing mycobacteriosis was M. avium. New Generation Sequencing helped to identified a subspecies that was M. avium ssp. hominissuis. CONCLUSIONS: The sitatunga antelope is an animal susceptible to infection by M. avium ssp. hominissuis. Considering the wide range of hosts and the easiness of interspecies transmission of the pathogen, as well as its zoonotic nature, the mycobacteriosis induced by this microorganism should not be underestimated.

Pregnancy in European bison (Bison bonaus) with generalized tuberculosis - no evidence of vertical transmission.

INTRODUCTION AND OBJECTIVE: One of the main health threats to the endangered European bison (Bison bonsasus) is bovine tuberculosis, the pathogenesis of which in this species is not fully known. The aim of the study was to confirm a possible case of vertical transmission from a pregnant European bison with generalized tuberculosis to its 12-week-old foetus. MATERIAL AND METHODS: During the autopsy it was found that the bison had become pregnant, despite an advanced stage of tuberculosis. Material collected from the organs and foetus was placed on Lowenstein and Stonebrink media and incubated at 37 °C for 12 weeks. RESULTS: Mycobacteria were isolated from the lungs and lymph nodes; however, the tissue of the foetus and fragments of the reproductive system were negative. CONCLUSIONS: Vertical transmission was excluded, although it cannot be ruled out that infection could occur as pregnancy progresses.

The Potential Risk of International Spread of Mycobacterium Bovis Associated with Movement of Alpacas.

Introduction: The study highlights the transboundary nature of tuberculosis (TB) in alpacas and the failure of current ante-mortem testing protocols (the tuberculin skin and Enferplex Camelid TB tests) to identify TB-free alpaca herds and individuals for export. Our research and the available literature indicate that the alpaca (Vicugna pacos) is extremely susceptible to Mycobacterium bovis infection, and that testing periodicity fails to take into account that animals do not manifest disease symptoms for a long time. The skin test failed to identify Mycobacterium bovis infection in two alpacas prior to their movement from the UK to Poland. The animals were purchased by a breeding centre in Poland, and were then shown at an international animal exhibition. The last owner of the alpacas before their deaths from TB bought the infected animals unwittingly in order to run rehabilitation activities with disabled children on his farm. Material and Methods: Thoracic lymph node, lung and liver tissue samples obtained at necropsy were examined histopathologically after Ziehl-Neelsen staining. Tissue samples were homogenised and mycobacteria present there were cultured on Stonebrink's medium during a 6-week incubation. A commercial test using polymorphism of the chromosomal direct repeat region provided species identification and additional identification was by spacer oligonucleotide typing and mycobacteria interspersed repetitive unit-variable number tandem repeat analysis with a gel electrophoresis protocol. Results: The microbiological examination confirmed multiorgan TB caused by the SB0666 spoligotype of Mycobacterium bovis. Conclusion: Due to the suboptimal performance of current diagnostic tests for TB in alpacas, there is a risk that infected animals may be moved unwittingly. A risk of TB spread associated with the international movement of alpacas is implied by this study.

Infection of a Free-Living Wild Boar (Sus scrofa) with a Bacterium from the Mycobacterium kansasii Complex.

The most numerous group of bacteria in the genus Mycobacterium is the nontuberculous mycobacteria. Currently, over 200 species of bacteria have been classified as belonging to this group, of which approximately 30 are pathogenic to humans and animals. Mycobacterium kansasii complex numbers among these pathogenic species. The submandibular lymph nodes of a wild boar shot by a hunter were examined in order to confirm or exclude infection with bacteria of the genus Mycobacterium. In culture, a bacterial isolate was obtained after 12 days of incubation on Petragnani and Stonebrink media. A multiplex PCR clearly indicated that the isolate was a nontuberculous mycobacterium. The results of species identification attempts via both molecular biology methods and mass spectrometry confirmed that the isolated strain belonged to MKC. The described case of a wild boar infection with MKC is the first documented case in Poland and only the second in Europe, and in confirming the presence of this pathogen among free-living animals, this report implies that MKC is of great concern. Our research elucidates some specifics of wild boar mycobacteriosis and may be used to instill awareness in the public of the dangers of dressing hunt prey or consuming its meat in ignorance of safe procedures, which can contribute to the transmission of the pathogen to humans.

Mixed Infection of Mycobacterium szulgai, M. lentiflavum, and Gram-Negative Bacteria as a Cause of Death in a Brown Caiman Caiman crocodylus: A Case Report.

This paper describes a fatal case of nontuberculosis mycobacteriosis in a four-year-old brown caiman kept in captivity. Although the clinical signs were asymptomatic, severe gross lesions were observed, namely necrotic inflammation of the intestines and granulomatous hepatitis. Microbiological and histopathological examination performed on the tissues collected postmortem revealed a mixed infection of Mycobacterium lentiflavum and Mycobacterium szulgai, secondarily mimicked with Salmonella Coeln, Aeromonas hydrofila, Citrobacter freundii, and Providencia rettgeri. Those microorganisms are not only potentially pathogenic to reptiles, but also have a zoonotic importance for humans. Our findings clearly demonstrate the importance of educating owners and maintaining hygiene rules when handling reptiles.

Intra-Palpebral Tuberculin Skin Test and Interferon Gamma Release Assay in Diagnosing Tuberculosis Due to Mycobacterium caprae in European Bison (Bison bonasus).

Despite the threat posed by tuberculosis (TB) to the protected European bison (Bison bonasus), no validated TB tests exist for this species. This pilot study evaluates two tests based on detecting cellular immunity for this purpose: interferon gamma release assay (IGRA) and tuberculin skin test (TST). Ten animals were subjected to ante-mortem and post-mortem examinations. IGRA was performed using a commercial test, and the comparative TST was performed in the eyelids. The lesions were assessed post-mortem and material was collected for mycobacterial culture. The isolated strains were subjected to genotyping. At post-mortem examination, five out of ten individuals demonstrated both tuberculous lesions and positive culture results (Mycobacterium caprae). Compared to the palpebral TST, the findings of the IGRA are easier to interpret when diagnosing tuberculosis in European bison.

Microbiological and molecular monitoring for bovine tuberculosis in the Polish population of European bison (Bison bonasus).

INTRODUCTION AND OBJECTIVE: In recent years, bovine tuberculosis (BTB) has become one of the major health hazards facing the European bison (EB, Bison bonasus), a vulnerable species that requires active protection, including regular and effective health monitoring. Monitoring of zoonotic disease in wildlife is also an important part of public health protection. The aim of the study was to determine whether BTB still influences the EB population in Poland. MATERIAL AND METHODS: During 2017-2019, mandibular, retropharyngeal and mediastinal lymph nodes were collected from 90 EB during post-mortem examination, and then cultivated on Lowenstein-Jensen and Stonebrink media. Isolated strains were subjected to molecular analysis to determine the species, spoligotype and MIRU-VNTR pattern. RESULTS: Lesions were found in lymph nodes originating from eight EB (8.89%). Positive microbiological cultures for mycobacteria were obtained in samples from six (6.67%) EB. The isolated strains were identified as Mycobacterium caprae (material from four EB) and atypical mycobacteria (material from two EB). For M. caprae strains spoligotype M. bovis 4_CA 1600 was identified and the MIRU-VNTR pattern was identified as 345751355413232. CONCLUSIONS: It is recommended that this potentially dangerous disease should be monitored in EB via a comprehensive strategy based on a combination of microbiological and molecular methods. Such monitoring will protect the health of both animals and humans.

The Risk of False-Positive Serological Results for Paratuberculosis in Mycobacterium bovis-Infected Cattle.

Both bovine tuberculosis (BTB) and paratuberculosis (paraTB) continue to cause significant economic losses in cattle breeding; in addition, their etiological agents have zoonotic potential. Although the diagnostics of both diseases are still being improved, problems still remain, such as the potential for cross-reactivity to the antigens used in tests. The aim of the present study was to confirm whether animals known to harbor Mycobacterium bovis antibodies are at increased risk of yielding positive results in paraTB serotesting and, additionally, to verify the accuracy of three commonly used methods for confirming M. bovis infection: ELISA, the tuberculin skin test (TST), and the presence of gross lesions. Material was collected from 98 dairy cattle suspected of BTB due to TST-positive results. During postmortem examination, gross lesions were assessed visually. Blood, lymph nodes, and TB-suspected organs were collected. Serum was obtained from the collected blood and tested serologically for TB and paraTB. The tissues underwent standard microbiological testing for M. tuberculosis complex. Among the 98 TST-positive individuals, tuberculous gross lesions were detected in 57 (58.1%), MTBC were isolated in 83 (84.7%), and the ELISA test was positive for 21 (21.4%). None of the lesions characteristic for paraTB were detected. The chance of obtaining a positive TB result by ELISA was seven times higher using the ELISA-paraTB method; hence, there is a significant risk of obtaining false-positive serological results for paraTB in M. bovis-infected cattle. However, the hypothesis that infection of M. bovis or prior TST performance may have boosted the host immune response and therefore increased the sensitivity of the paraTB-ELISA cannot be excluded.

The first visually-guided bronchoscopy in European bison (Bison bonasus) - An additional tool in the diagnosis of bovine tuberculosis?

The European bison (Bison bonasus, EB) is an endangered species, and as about 1/3 of its global population is found in Poland, it is particularly important that Polish herds should be monitored. One particular concern is tuberculosis, which is not a marginal problem in wildlife in Poland, and has been microbiologically confirmed in EB, wolves (Canis lupus) and wild boar (Sus scrofa). However, ante mortem diagnosis of tuberculosis in EB is troublesome. Therefore, the present paper evaluates the potential of bronchoscopy as a diagnostic tool. Seven EB were studied, four of which were found to be naturally infected with M. caprae; in two of these, endoscopy identified abnormalities in the respiratory tract ante mortem. Therefore, despite some limitations, endoscopy can be an additional tool for diagnosing tuberculosis in EB, especially in highly valuable animals, and to assess the stage of the disease.

Antibody responses in European bison (Bison bonasus) naturally infected with Mycobacterium caprae.

Mycobacterium caprae, a member of the Mycobacterium tuberculosis complex, infects humans and animals causing lesions and disease like that of Mycobacterium bovis. The aim of this study was to evaluate antibody responses in European Bison (EB, Bison bonasus; a vulnerable species) naturally infected with M. caprae using dual path platform (DPP) BovidTB test and multi-antigen print immunoassay (MAPIA). Study cohorts consisted of naturally M. caprae-infected EB (n = 4), M. caprae-exposed but uninfected (n = 3), EB infected with non-tuberculous mycobacteria or other respiratory pathogens (n = 3), and negative controls (n = 19). M. caprae-infected EB were seropositive by both DPP and MAPIA; 3/4 were seropositive by DPP; and 4/4 were seropositive by MAPIA. One M. caprae-infected animal that developed generalized disease with most advanced gross lesions in the group produced the most robust antibody response. All 25 EB with no culture-confirmed M. caprae infection, including three animals exposed to M. caprae and three other animals infected with non-tuberculous pathogens, were seronegative on both tests. Antibody responses to M. caprae infection included IgM antibodies against MPB70/MPB83 and IgG antibodies to both MPB70/MPB83 and CFP10/ESAT-6. This study demonstrates the potential for use of serological assays in the ante-mortem diagnosis of M. caprae infection in EB.

Biopsy and Tracheobronchial Aspirates as Additional Tools for the Diagnosis of Bovine Tuberculosis in Living European Bison (Bison bonasus).

The diagnosis of bovine tuberculosis (BTB) in living wildlife remains a complex problem, and one of particular importance in endangered species like European bison (Bison bonasus). To identify infection and avoid the unnecessary culling of such valuable individuals, current best practice requires the collection and culture of material from living animals, as mycobacteria isolation remains the gold standard in BTB diagnosis. However, such isolation is challenging due to the need for the immobilization and collection of appropriate clinical material, and because of the sporadic shedding of mycobacteria. In the present study, we evaluated the potential of sampling for the detection of BTB in a group of seven living European bison suspected of being infected with Mycobacterium caprae. The specimens were collected both as swabs from the nasal and pharyngeal cavities, tracheobronchial aspirates (TBA), ultrasound-guided biopsies from lateral retropharyngeal lymph nodes, and post mortem, from mandibular, retropharyngeal and mediastinal lymph nodes. Clinical samples were tested for mycobacterial species via mycobacteriological culture and PCR. M. caprae was isolated from collected material in two out of four living infected individuals (TBA, biopsy) and mycobacterial DNA was detected in three out of four (TBA, pharyngeal swab) bison. This is the first report of isolation of M. caprae in living European bison. Our findings demonstrate the value of diagnostic tests based on both molecular testing and culture in European bison and confirm the respiratory shedding of viable M. caprae in this host species.

Epidemiological characterization of Mycobacterium caprae strains isolated from wildlife in the Bieszczady Mountains, on the border of Southeast Poland.

BACKGROUND: The majority of animal tuberculosis (TB) cases reported in wildlife in Poland over the past 20 years have concerned the European bison inhabiting the Bieszczady Mountains in Southeast Poland: an area running along the border of Southeast Poland. As no TB cases have been reported in domestic animals in this region since 2005, any occurrence of TB in the free-living animals inhabiting this area might pose a real threat to local livestock and result in the loss of disease-free status. The aim of the study was to describe the occurrence of tuberculosis in the wildlife of the Bieszczady Mountains and determine the microbiological and molecular characteristics of any cultured strains. Lymph node samples were collected for analysis from 274 free-living animals, including European bison, red foxes, badgers, red deer, wild boar and roe deer between 2011 and 2017. Löwenstein-Jensen and Stonebrink media were used for culture. Molecular identification of strains was performed based on hsp65 sequence analysis, the GenoType®MTBC (Hain Lifescience, Germany) test, spoligotyping and MIRU-VNTR analysis. RESULTS: Mycobacterium caprae was isolated from the lymph nodes of 21 out of 55 wild boar (38.2%; CI 95%: 26.5%, 51.4%) and one roe deer. Since 2014, no new TB cases have been reported in the Bieszczady European bison population. CONCLUSIONS: The identification of TB in wild boar in the Bieszczady is an alarming phenomenon, which requires further investigation. The Bieszczady mountains are a precious, unique area, home to many protected species. However, it is also the only area in Poland where TB cases have been reported in free-living animals. The occurrence of TB in wild boar inhabiting this area might pose a real threat to local livestock and many of the protected species (for example European bison that can share feeding places with wild boar). Given this situation, ongoing monitoring of the prevalence of TB should be conducted, and protective measures should be considered.