A serological survey of pathogens associated with the respiratory and digestive system in the Polish European bison (Bison bonasus) population in 2017-2022.

BACKGROUND: The European bison (Bison bonasus) is a near threatened species and requires health monitoring. The aim of the present study was to determine the prevalence of antibodies to pathogens known to cause respiratory and digestive illness in ruminants. RESULTS: In the studied 328 European bison, the highest seroprevalence was observed for Bovine herpesvirus-1 (BoHV-1) (50.27%), Bovine Coronavirus (BCoV) (26.36%), and Bluetongue Virus (BTV) (12.83%). For Mycoplasma bovis strains and Bovine Viral Diarrhea Virus (BVDV), positive results were rare. Interestingly, a higher prevalence of BTV antibodies was noted in the northeastern populations and older animals. CONCLUSIONS: Our findings indicate that the Polish European bison population appears to have considerable contact with BoHV-1; however, this does not appear to be of great significance, as clinical symptoms and post-mortem lesions are rarely noted in Polish European bison population. The high seroprevalence of BTV in the north-east of Poland is an ongoing trend, also noted in previous studies. It is possible that European bison may perpetuate the virus in this region. This is the first report of antibodies for BCoV in European bison.

The influence of Percoll® density gradient centrifugation before cryopreservation on the quality of frozen wisent (Bison bonasus) epididymal spermatozoa.

BACKGROUND: The wisent (Bison bonasus) is a species that has undergone a population bottleneck. Homozygosity is prevalent within the population and may have a negative impact on semen quality in wisent bulls. Semen samples containing a large amount of functionally and morphologically impaired or dead spermatozoa have lower tolerance for cryopreservation process. Such samples are prone to involve damage acrosomes, to produce and release reactive oxygen which negatively affects proper function of spermatozoas. It is a good practice to select intact and viable gametes before subjecting the sample to cryopreservation to improve the efficiency of this process. The aim of this study was to assess the ability of Percoll® density gradient centrifugation in order to improve the quality of wisent spermatozoa after cryopreservation. Spermatozoa samples were analysed with computer-assisted semen analysis system and flow cytometry. RESULTS: Percoll® density gradient centrifugation resulted in increased percentage of motile spermatozoa, higher proportion of spermatozoa with normal morphology and proper functionality but also in a significant reduction of the total number of gametes. Nevertheless, the concentration of frozen spermatoza was still sufficient for obtaining a few complete insemination doses suggested for cattle from each epididymis. CONCLUSIONS: While creating a high-quality genetic reserve, for in vitro fertilisation purposes, eliminating detritus and improving the overall quality of samples is more important than total number of spermatozoa. For these reasons, the achievement of higher post thaw quality of spermatozoa justifies the purification of samples by centrifugation in a Percoll® density gradient prior to the cryopreservation process.

Pathogens with potential impact on reproduction in captive and free-ranging European bison (Bison bonasus) in Poland - a serological survey.

BACKGROUND: The European bison is an endangered species, and as such it is extremely important to monitor herds for pathogens which can lead to reproductive failure. The aim of the present study was to determine the current prevalence of antibodies to pathogens known to potentially influence reproduction in European bison. Serum samples from 183 bison, originating from different parts of Poland, were tested using commercial ELISA tests for antibodies to Chlamydia spp., Coxiella burnetti, Leptospira interrogans, Neospora caninum and Toxoplasma gondii; the findings were compared between captive and main free-ranging herds, and with regard to the influence of demographic factors such as age and sex. The prevalence of seropositivity was also checked with regard to location and the animal species sharing it. RESULTS: Chlamydia spp. antibodies were present in 48 out of 130 (36.9%) tested samples. Coxiella burnetii was found in one sample out of 178 (0.58%). N. caninum in 36 out of 172 (20.9%) and T. gondii in 23 out of 172 (13.4%). No sample was positive for leptospirosis. Neither sex nor age appeared to have a significant effect on the occurrence of antibodies to the identified species. The prevalence of Chlamydia spp. in the samples varied significantly according to location; however, similar frequency ranges were observed between free ranging and captive herds. In contrast, antibodies to N. caninum were more common in free-ranging herds than captive herds, with the highest frequency observed in the Bieszczady Mountains. CONCLUSIONS: Chlamydia spp., N. caninum and T. gondii might have a similar impact on the reproductive potential of European bison as they have on cattle. The high occurrence of antibodies to N. caninum in bison from the Bieszczady Mountains may be associated with the relatively high density of the wolf population in the area.

Genomic Footprints of Recovery in the European Bison.

After extinction in the wild in the beginning of the 20th century, the European bison has been successfully recovered in 2 distinct genetic lines from only 12 and 7 captive founders. We here aimed at characterizing the levels of realized inbreeding in these 2 restored lines to provide empirical insights into the genomic footprints left by population recovery from a small number of founders. To that end, we genotyped 183 European bison born over the last 40 years with the Illumina BovineHD beadchip that contained 22 602 informative autosomal single-nucleotide polymorphisms after data filtering. We then identified homozygous-by-descent (HBD) segments and classified them into different age-related classes relying on a model-based approach. As expected, we observed that the strong and recent founder effect experienced by the 2 lines resulted in very high levels of recent inbreeding and in the presence of long HBD tracks (up to 120 Mb). These long HBD tracks were associated with ancestors living approximately from 4 to 32 generations in the past, suggesting that inbreeding accumulated over multiple generations after the bottleneck. The contribution to inbreeding of the most recent groups of ancestors was however found to be decreasing in both lines. In addition, comparison of Lowland individuals born at different time periods showed that the levels of inbreeding tended to stabilize, HBD segments being shorter in animals born more recently which indicates efficient control of inbreeding. Monitoring HBD segment lengths over generations may thus be viewed as a valuable genomic diagnostic tool for populations in conservation or recovery programs.

Antibodies against the Mycobacterium tuberculosis complex and Brucella spp. in captive and free-living European bison (Bison bonasus) in Poland.

BACKGROUND: The European bison (Bison bonasus), a symbol of Polish nature, is a protected species that requires active health monitoring. However, conservation efforts are made difficult by the zoonotic diseases such as brucellosis and tuberculosis. OBJECTIVE: The aim of this study was to screen the Polish European bison population for exposure to the Mycobacterium tuberculosis complex (MTC) and Brucella spp. METHODS: A total of 323 free-living and captive European bison from 13 localities were tested serologically for antibodies against the M. bovis P22 multi-protein complex (in-house ELISA) and against Brucella spp. (commercial ELISA). RESULTS: Antibodies against the MTC (P22) were detected in 7% (22/323) of the tested European bison. Anti-MTC antibody positivity was not significantly different by sex, age, and captive/free range status. Anti-MTC antibodies were found in six of 13 populations sampled, always in populations with larger sample sizes including the four free-living ones. Antibodies against Brucella spp. were detected in 36% (116/323) of the tested bison. While Brucella spp. antibody prevalence was not different by sex, it was significantly different by age (lower in adults) and captive/free-living status. Brucella spp. seroprevalence decreased with sample size and seropositive bison were found in 12 of 13 sampling populations. CONCLUSIONS: Our findings identify potential emerging threats to the European bison population and confirm the first serological response to P22 in European bison. As Poland is currently officially free of brucellosis and bovine tuberculosis, our results require careful interpretation. Further studies are needed to establish the presence of cross-reactions with atypical mycobacteria in the case of MTC and other bacteria (e.g. Yersinia enterocolitica O:9) in the case of Brucella spp.

Determination of hematological and biochemical values blood parameters for European bison (Bison bonasus).

Hematological and biochemical blood parameters are important tools for evaluating animals' health. They might be crucial in assessing the health of entire populations of wild animals, such as European bison (Bison bonasus). The aim of this study was to establish hematological and biochemical values for healthy European bison and to determine whether there were significant relations with age and sex. Blood samples were collected from 79 animals and tested according to generally accepted standards and the results were subjected to statistical analysis. Most of the age and gender-related correlations found in our study were predictable based on previous reports. Due to bone growth, juvenile animals have typically higher ALP and P concentrations relative to adults. Several age-related dependencies were surprising, like higher Na concentration in younger European bison. Determination of hematological and biochemical blood parameters of healthy European bison may significantly contribute to the further restitution of this endangered species.

Over 300 km Dispersion of Wild Boar during Hot Summer, from Central Poland to Ukraine.

The movement of wild boars is a complex process influenced by both internal conditions and external factors. Despite their typically sedentary lifestyle, dispersion constitutes an integral element of the wild boar's behavior. This report documents the longest observed wild boar dispersal, involving a collared two-year-old male near Warsaw, Poland. The aim of this study was to present the characteristics of movement during the "nomadic phase", drawing comparisons with the "sedentary phase". The other aim was to evaluate the influence of meteorological factors on the minimum daily travel distance of the wild boar. We collected data from two-year-old males. The first exhibited long-distance dispersal and the second only demonstrated local movements. We calculated the minimum daily distance of both wild boars based on collar locations and calculated basic statistics of movement. We used a generalized linear model with a gamma distribution and log link function to assess the potential impact of weather conditions on the minimum daily distance of wild boars. We tested maximum daily temperature, average daily temperature, and the sum of daily precipitation. The wild boar during a "nomadic phase" covered a total of 922 km with a mean minimum daily movement of 6 km. The dispersion distance was 307 km. The highest value of the minimum daily distance reached 31.8 km/day. The second wild boar (near Warsaw) covered a mean minimum daily distance of 1.4 km; the highest value of the minimum daily distance was 3.9 km. Both wild boars exhibited no dependence of minimum daily distance on weather conditions. However, when intensive and non-intensive dispersion were analyzed separately, it was demonstrated that the maximum daily temperature positively influenced the minimum daily distance. We speculate that the wild boar was forced to search for water sources after dark on hot days, which induced a longer traveling distance in an unfamiliar environment. This study highlights the significant spatial capabilities of wild boar in the transmission of genes or pathogens. We speculate that extended daily distances during the initial "nomadic phase" might suggest a panicked escape from a perceived threat. It is plausible that the wild boar found improved shelter within tall cereal crops in July and August, which resulted in lower daily distances.

An assessment of the habitat preferences of European bison with airborne laser scanning data in forest ecosystem.

Research on habitat preferences is an important part of contemporary ecology. For the European bison, the classic approach to distinguishing habitat features is still being followed, but the limitations of this approach cannot provide the standard features of optimal habitats for this species. The study consisted in comparing analyses of the habitat preferences of European bison that were based on either classic forest typology (habitat types) or airborne laser scanning data. The data for these analyses were collected from telemetry collars on European bison in Bialowieza Forest. The model based on airborne laser scanning features presented better parameters (percent of correctly classified cases and ROC) than the model based on habitat types. The results show that it is possible to find universal indicators of European bison's preferences that are independent of local forest classification methodology. The indicators used suggest that European bison have a preference for forest habitats with low canopy cover and a small share of woody plants in the lower parts of the forest. Low canopy cover itself is not necessarily beneficial for European bison. Our study also indicates that airborne laser scanning is also useful in the assessment of habitat suitability for European bison in forest ecosystems.

Endoparasite loads and the efficacy of conventional anthelmintics against gastrointestinal nematodes in captive European bison.

Although little information exists on the efficacy of deworming in wild ruminants, gastrointestinal nematodes have been found to demonstrate increasing drug resistance. The spread of drug-resistant strains may be increased by transmission among livestock and susceptible wildlife species, thus posing a potential threat to endangered species, such as the European bison. The aim of the study was twofold: to identify the parasite loads in captive European bison with the use of coprological techniques, and to test the influence of other nearby ungulates on the richness of bison parasitofauna. Additionally, the efficacy of deworming procedures against gastrointestinal nematodes in bison was evaluated. The survey was based on a coprological investigation of 285 fecal samples from 156 European bison in 15 enclosures. The parasitofauna of the captive European bison was consistent with those of free-ranging populations. The highest prevalence was noted for Eimeria spp. oocysts (60.7%), strongyle eggs (50.9%), Fasciola hepatica eggs (13.1%), Dictyocaulus viviparus larvae (12.3%) and Trichuris sp. Eggs (9.47%). Moreover, the close proximity of other ungulate species resulted in a higher diversity of parasite species. In all cases, deworming with albendazole, fenbendazole and ivermectin proved to be ineffective against strongylids and Trichuris sp. The results of fecal egg count reduction test (FECRT) ranged from 37.2 to 99.6% (95% CI <90%) for albendazole; values >95% (95% CI = 41-100) were noted for fenbendazole, and FECRT ranged from 63.2 to 97.5 (95% CI = 0-99) for ivermectin. As the results of anthelmintic treatment are unsatisfactory, it seems justified to continue study in this area. Our study is the first large-scale attempt to evaluate the efficacy of anthelminthics in captive European bison. The potential sharing of parasite species between bison and other ungulates should also be further investigated from the perspective of minimizing the risk of the spread of drug-resistant parasite strains.

The morphology, morphometry and functionality of fresh and cryopreserved wisent (Bison bonasus) epididymal spermatozoa.

Epididymal spermatozoa obtained post mortem are considered a valuable source of genetic material which is often irrevocably lost. This makes these gametes constitute a key element in protection and restitution programs. The wisent (Bison bonasus, Linnaeus 1758) is a species that survived in zoos after extinction from its natural habitat. This resulted in a narrowing of the genetic pool of the whole population, which is at present derived from only 12 ancestors. Currently, wisent protection programs are aimed at preserving the genetic diversity by establishing a germplasm bank. The objective of this study was to comprehensively characterize the morphology, morphometry and functionality of wisent epididymal spermatozoa and evaluate the effectiveness of their cryopreservation in extender based on Tris buffer and chicken egg yolk. The median total number of spermatozoa obtained from one individual was 1985.0 × 106 (62.5 × 106-7452.0 × 106). These gametes were characterized by median: 40.0% (0.5-70.0%) subjective motility, 69.8% (32.5-90.0%) viability and 54.3% (10.5-83.3%) normal morphology. The sperm head had a median size of 5.0 µm (3.5-6.7 µm) width, 8.5 µm (6.4-11.3 µm) length and 36.9 µm2 (23.7-48.6 µm2) surface area. The viable population of the obtained gametes was characterized by median values 53.2% (4.5-80.3%) of intact sperm membrane, 50.8 (26.0-76.6%) of intact acrosome, 0.4% (0-98.7%) of fragmented chromatin, 5.9% (0.0-88.8%) of cells with high mitochondrial potential and 42.1% (8.3-63.7%) without lipid peroxidation. The viable population of the frozen/thawed gametes was characterized by median values: 18.4% (2.4-57.9%) of intact sperm membrane, 35.1 (11.9-56.7%) of intact acrosome, 0.07% (0-89.2%) of fragmented chromatin, 12.8% (0.0-49.7%) of cells with high mitochondrial potential and 16.3% (2.2-53.6%) without lipid peroxidation. Due to the material originating from a relatively large number of wild individuals, the research presented here contributed to the description of certain species standards for the assessment of wisent epididymal spermatozoa. The presented effect of cryopreservation on these gametes justifies the use of an extender based on Tris buffer with the addition of chicken egg yolk. The obtained effects are satisfactory from the point of view of preserving valuable genetic material and their use in ART.

A validated LC-MS/MS method for simultaneous determination of key glucocorticoids in animal hair for applications in conservation biology.

A new method for the determination of main glucocorticoids (cortisol, cortisone, and corticosterone) in hair by liquid chromatography-tandem mass spectrometry was developed. Glucocorticoids were extracted from hair shafts using methanol followed by solid-phase extraction. A validation test was performed using hair from three species of wild mammals with different body size (0.2-800 kg), lifestyle (terrestrial, burrowing and arboreal species), social organization (living in herds or solitary), and different predicted type of hair glucocorticoids: European bison (Bison bonasus), European hamster (Cricetus cricetus), and Eurasian red squirrel (Sciurus vulgaris). Regardless of the species evaluated, the method shows good linearity for all analytes accompanied by satisfactory accuracy (91-114%) and precision (RSD < 13%). Depending on the analyte and hair origin, the calculated limits of quantification were between 0.05 and 1.19 ng/mL, which corresponds to 1.28-31.51 pg/mg. Using cortisol and cortisone as examples, we have demonstrated that measuring multiple glucocorticoids simultaneously provides more comprehensive information than solely concentrating on one, thereby contributing to a more balanced and reliable interpretation of the acquired results. However, the utility of cortisol metabolites as markers of stress response in keratinized tissues should be substantiated by additional experimental studies on targeted animals. We posit that this paper could serve as a crucial catalyst to prompt such experiments.

Comorbidities and concentration of trace elements in livers of European bison from Bieszczady Mountains (Poland).

European bison is a species for which health monitoring is essential in conservation activities. So far, little research has been carried out on the concentration of elements in this species. Most previous studies did not associate the concentration of elements with susceptibility to diseases. In this study we investigate the relationship between comorbidities in European bison and concentrations of a wide spectrum of elements in the liver. Samples were collected during the monitoring of the European bison population in Bieszczady (southeast Poland) over the 2020-2022 period. Each individual was also visually inspected by a veterinarian in the field for the presence of lesions as a part of a post-mortem examination. The animals were divided into 3 groups: group A-one type of clinical sign; group B-two types of clinical signs; group C-three or more types of clinical signs. The ICP-OES method was applied to assess the concentration of 40 elements in livers. Discriminant analysis showed clear differences between the mineral status of individuals in the groups with one, two, and at least three types of clinical signs. Detailed analysis of selected elements showed that, in the case of eight elements, there was a relationship with age, sex, or comorbidities. Cu, Se, and Zn showed significant differences in relation to comorbidities, but only Cu concentration was lower when the frequency of lesions was higher. We concluded that in research on the mineral status of the population, apart from the availability of trace elements in the environment, the health condition of the studied individuals should also be considered. However, inferring the mineral status of the population on the basis of randomly obtained samples from dead individuals may give an incomplete view of the population, especially in the case of species susceptible to diseases, such as European bison.

Anaplasma phagocytophilum in European bison (Bison bonasus) and their ticks from Lithuania and Poland.

The increasing population of European bison (Bison bonasus) can contribute to the prevalence of zoonotic pathogens. The aim of the present study was to assess the presence of A. phagocytophilum infection in European bison tissues as well as ticks removed from European bison in Lithuania and Poland. A further objective of this work was to compare the detected A. phagocytophilum strains. A total of 85 tissue samples (spleen) of European bison and 560 ticks belonging to two species, Ixodes ricinus (n = 408) and Dermacentor reticulatus (n = 152) were tested. DNA of A. phagocytophilum was detected based on RT-PCR in 40% of the European bison samples, 8.8% of the I. ricinus and 5.9% of the D. reticulatus ticks. Analysis of the obtained partial 16S rRNA gene sequences of A. phagocytophilum revealed the presence of three variants with two polymorphic sites. Furthermore, phylogenetic analysis with partial msp4 gene sequences grouped A. phagocytophilum variants into three clusters. This study revealed that the groEL gene sequences of A. phagocytophilum from European bison and their ticks grouped into ecotype I and only one sequence from Lithuanian European bison belonged to ecotype II. The results of the present study indicated that European bison may play a role as a natural reservoir of A. phagocytophilum.

Contact between European bison and cattle from the cattle breeders' perspective, in the light of the risk of pathogen transmission.

Pathogens transmitted between wildlife and domestic animals can pose a threat to endangered species, undermine conservation efforts in wildlife, and affect productivity and parasite control in domestic animals. There are several examples of pathogen transmission between European bison and other animals. The present study surveyed breeders from the vicinity of four large wisent populations in eastern Poland about observed contacts between wisent and cattle. Such contacts were noted by 37% of breeders, indicating a significant risk of contact between European bison and cattle in the study areas, even in the areas where the European bison live mainly in a forest complex, i.e., in the Borecka Forest. A higher potential risk of contacts between European bison and cattle was noted in the Bialowieska Forest and the Bieszczady Mountains than in the Borecka and Knyszynska Forests. In the Bialowieska Forest, the risk of viral pathogen transmission resulting from contacts is higher (more direct contacts), and in the case of the Bieszczady Mountains, the probability of parasitic diseases is higher. The chance of contacts between European bison and cattle depended on the distance of cattle pastures from human settlements. Moreover, such contact was possible throughout the year, not only in spring and fall. It appears possible to minimize the risk of contacts between wisent and cattle by changing management practices for both species, such as keeping grazing areas as close as possible to settlements, and reducing the time cattle graze on pastures. However, the risk of contact is much greater if European bison populations are large and are dispersed beyond forest complexes.

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.

Evaluation of the Impact of the Peregrine Falcon (Falco peregrinus peregrinus) Reintroduction Process on Captive-Bred Population.

The main objective of this study was to determine the impact of increased demand for peregrine falcons via breeding (mainly Polish, Czech, German and Slovak) on the genetic structure of the birds. In the analysis, 374 specimens from six countries were sampled in 2008-2019 (omitting 2009), and all the birds analyzed were released into the wild as part of the Polish reintroduction program. The assessment of genetic variation was based on a well-known panel of 10 microsatellite markers described for the species. We calculated a fixation index for the samples from each year, and based on this, we determined the level of inbreeding. We also performed an analysis using the Bayesian cluster method, assuming that 1-19 hypothetical populations would define the division that best fit the samples. The most probable division was into two groups; in the first group, the samples from individuals delivered in 2013 were most often segregated; moreover, in this year, a jump in inbreeding, expressed by the fixation index, was observed.

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.

Prevalence and Genetic Diversity of Trueperella pyogenes Isolated from Infections in European Bison (Bison bonasus).

Trueperella pyogenes is a Gram-positive bacterium causing purulent infections in many animal species, including the European bison. However, the data about the virulence and genetic relationships of T. pyogenes strains isolated from these wild ruminants are strongly limited. The aim of this study was to investigate the prevalence of T. pyogenes infections in the European bison, and to evaluate the genetic diversity of isolates from these animals. In the time span of 10 years, 328 European bison from 16 different locations were examined. The standard bacteriological methods were used for T. pyogenes isolation and identification from clinical specimens obtained from urogenital tract infections and abscesses of different locations. The presence of genes encoding known virulence factors was investigated by PCR, and the genetic diversity of T. pyogenes strains was examined with the RAPD-PCR method. The prevalence of T. pyogenes infections was 14.6%, and the pathogen was isolated from both female (47.9% of isolates) and male (52.1% of isolates) European bison. It should be highlighted that a considerable number of strains were isolated from the prepuce and penis infections. Therefore, the role of T. pyogenes in the pathogenesis of balanoposthitis should be seriously perceived. A total of 39 T. pyogenes strains were subjected to genetic characterization. All studied strains carried the plo gene, while the nanH (25.6%), nanP (23.1%), cbpA (7.7%), fimA (97.4%), fimC (69.2%), fimE (92.3%) and fimG (15.4%) genes were present with a variable frequency among the tested strains. The virulence genotype plo/fimA/fimC/fimE was dominant. RAPD-PCR typing showed a high level of genetic diversity among European bison T. pyogenes strains, and a total of 31 different RAPD profiles were distinguished. In a few cases, the same RAPD profile was found in strains obtained from animals living in the same area. This study provided the first data about the prevalence and genetic relationships of T. pyogenes in the Polish population of European bison. However, further epidemiological investigations are needed to understand the routes of transmission and dissemination of the pathogen in these wild animals.

Establishment of a Wisent (Bison bonasus) Germplasm Bank.

The wisent, or European bison (Bison bonasus), belongs to the same family (Bovidae) as the American bison and domestic cattle. The wisent is the largest mammal in Europe, and is called the "Forest Emperor". The wisent is listed as "Vulnerable" on the IUCN Red List, and is protected by international law. Achievements in reproductive biotechnology have opened new possibilities for the cryoconservation of the wisent germplasm. Therefore, this research aimed to improve a strategy for the protection and preservation of the European bison through the creation of a wisent germplasm bank, based on the following procedures: isolation and in vitro maturation (IVM) of oocytes, in vitro fertilization (IVF) of matured oocytes, in vitro embryo culture (IVC), and embryo cryopreservation. Wisent ovaries were isolated from females outside the reproductive season, and eliminated from breeding for reasons other than infertility. Cumulus-oocyte complexes (COCs) were isolated from follicles greater than 2 mm in diameter and matured for 24 h and 30 h. After IVM, COCs were fertilized in vitro with wisent sperm. The obtained wisent zygotes, based on oocytes matured for 24 h and 30 h, were cultured for 216 h. Embryos at the morula and early blastocyst stages were vitrified and then warmed and transferred to interspecies recipients (Bos taurus). USG and biochemical tests were used to monitor pregnancies. This study obtained embryos in the morula and early blastocyst stages only after oocytes were fertilized and matured for 30 h. On average, per oocyte donor, 12.33 ± 0.5 COCs were isolated, and only 9.33 ± 0.61 COCs were qualified for in vitro maturation (75.68%), while 9.16 ± 0.48 COCs were matured (84.32%). On average, per donor, 5.5 ± 0.34 embryos were cleaved (59.96%) after 48 h post-fertilization (hpf), and 3.33 ± 0.21 achieved the eight-cell stage (36.52%) after 96 hpf, while 1 ± 0.21 morula and early blastocyst stages (10.71%) were achieved after 216 hpf. A total of six embryos (one morula and five early blastocysts) were obtained and vitrified; after warming, five of them were interspecies transferred to cattle (Bos taurus). On day 41 after fertilization, 3 out of 5 pregnancies were detected based on USG, P4, and PAG tests. However, no pregnancy was observed on day 86 after fertilization, indicating embryo resorption. This study shows that obtaining wisent embryos in vitro, and subsequent cryopreservation to create a wisent embryo bank, can be applied and implemented for the wisent protection program.

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.