Prevalence of coronaviruses in European bison (Bison bonasus) in Poland.

Coronaviruses have been confirmed to infect a variety of species, but only one case of associated winter dysentery of European bison has been described. The study aimed to analyze the prevalence, and define the impact on the species conservation, the source of coronavirus infection, and the role of the European bison in the transmission of the pathogen in Poland. Molecular and serological screening was performed on 409 European bison from 6 free-ranging and 14 captive herds over the period of 6 years (2017-2023). Presence of coronavirus was confirmed in one nasal swab by pancoronavirus RT-PCR and in 3 nasal swab samples by bovine coronavirus (BCoV) specific real time RT-PCR. The detected virus showed high (> 98%) homology in both RdRp and Spike genes to BCoV strains characterised recently in Polish cattle and strains isolated from wild cervids in Italy. Antibodies specific to BCoV were found in 6.4% of tested samples, all originating from free-ranging animals. Seroprevalence was higher in adult animals over 5 years of age (p = 0.0015) and in females (p = 0.09). Our results suggest that European bison play only a limited role as reservoirs of bovine-like coronaviruses. Although the most probable source of infections in the European bison population in Poland is cattle, other wild ruminants could also be involved. In addition, the zoonotic potential of bovine coronaviruses is quite low.

Immunoexpression of Spexin in Selected Segments of the Bovine (Bos taurus taurus) Gastrointestinal Tract.

In the expansive domain of neuropeptide investigation, spexin (SPX) has emerged as a captivating subject, exerting a significant impact on diverse physiological processes. Initially identified in mice, SPX's distribution transcends various organs, suggesting its potential regulatory roles. Despite extensive research in smaller species, a notable gap exists in our comprehension of SPX in larger mammals, particularly ruminants. Our study meticulously explores the immunolocalization of SPX within the gastrointestinal organs of bovines, with a specific focus on the abomasum, jejunum, and colon. Tissue samples from Holstein-Friesian cattle underwent careful processing, and gene mRNA expression levels, particularly GALR2 and SPX, were assessed. Intriguingly, our findings revealed that GALR2 expression was highest in the jejunum, signifying a potentially critical role in this digestive segment. Immunohistochemistry further unveiled distinct patterns of SPX immunoreactivity in each examined region-abomasum, jejunum, and colon-highlighting nuanced, region-specific responses. Notably, the abomasum and jejunum predominantly exhibited positive immunoreactivity in the submucosal plexus, while the colon, in contrast, demonstrated a higher degree of immunoreactivity in myenteric plexus neurons. Our investigation, grounded in the hypothesis of ubiquitous SPX distribution in ruminants, delves deeper into the intricate role of SPX within the enteric nervous system. This study meticulously explores the spatial distribution of SPX within the myenteric and submucosal plexuses, integral components of the enteric nervous system. These findings significantly enhance our understanding of SPX's potential roles in gastrointestinal regulation in bovines, providing a unique perspective on larger mammals and enriching our comprehension of this intriguing neuropeptide's significance in various physiological processes.

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.

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.

Acute-phase protein concentrations in serum of clinically healthy and diseased European bison (Bison bonasus) - preliminary study.

BACKGROUND: This is the first report describing levels of APPs in European bison. Serum concentration of acute phase proteins (APPs) may be helpful to assess general health status in wildlife and potentially useful in selecting animals for elimination. Since there is a lack of literature data regarding concentration of APPs in European bisons, establishment of the reference values is also needed. METHODS: A total of 87 European bison from Polish populations were divided into two groups: (1) healthy: immobilized for transportation, placing a telemetry collar and routine diagnostic purposes; and (2) selectively culled due to the poor health condition. The serum concentration of haptoglobin, serum amyloid A and α1-acid-glycoprotein were determined using commercial quantitative ELISA assays. Since none of the variables met the normality assumptions, non-parametric Mann-Whitney U test was used for all comparisons. Statistical significance was set at p < 0.05. Statistical analyses were performed using Statistica 13.3 (Tibco, USA). RESULTS: The concentration of haptoglobin and serum amyloid A was significantly higher in animals culled (euthanised) due to the poor condition in respect to the clinically healthy European bison. The levels of α1-acid-glycoprotein did not show statistical difference between healthy and sick animals. CONCLUSIONS: Correlation between APPs concertation and health status was proven, therefore the determination of selected APPs may be considered in future as auxiliary predictive tool in assessing European bison health condition.

The European bison (Bison bonasus) as an indicatory species for the circulation of tick-borne encephalitis virus (TBEV) in natural foci in Poland.

Tick-borne encephalitis (TBE) is one of the most common zoonotic diseases in Europe transmitted by Ixodidae vectors. While small mammals such as bank voles and ticks constitute the main reservoirs for virus transmission, large sylvatic species act as a food source for ticks. Cervids such as roe deer and red deer are considered sentinel species for TBE in natural foci. In addition, an increase of the population size and density of large wild mammals in an area corresponds to an increase in the tick burden and may potentially increase the prevalence of TBE virus (TBEV) in ticks and tick hosts and further exposure risk in humans. Humans are considered accidental hosts. The prevalence of TBE relies on interactions between host, vector and environment. The present study examines the exposure of the largest European herbivore, the European bison (Bison bonasus) to TBEV infection. Assessed using the IMMUNOZYM FSME ELISA (PROGEN), the overall TBEV seroprevalence was 62.7% in the 335 European bison that were studied. ELISA results were confirmed by the gold-standard virus neutralization test (VNT) with 98.7% sensitivity and thus giving a true prevalence of 63.5%. TBEV seroprevalence was significantly correlated to the origin, age group, sex, population type (free living/captive) and sanitary status (healthy/selectively eliminated/found dead/killed in accident) of the European bison in the univariable analysis. The highest seroprevalences were observed in the three largest north-eastern wild populations (Bialowieska, Borecka and Knyszynska forests), which corresponded with the highest incidence of human cases reported in the country. The risk of TBEV seropositivity increased with age and was higher in female and free-ranging European bison. Additionally, to the epidemiological investigation, the continuous detection of TBEV antibodies was studied by repetitive testing of animals over the course of 34 months. Two of six seropositive animals remained seropositive throughout the study. The presence of antibodies was followed throughout the study in seropositive European bison and for at least a year in animals that seroconverted during the observation period.

A Freedom of Coxiella burnetii Infection Survey in European Bison (Bison bonasus) in Poland.

Q fever is an important zoonosis caused by the intracellular Gram-negative bacteria Coxiella burnetii. The source of infection are numerous species of mammals, birds, reptiles and amphibians, as well as ticks. The disease is widespread throughout Europe, but the role of wildlife in its epidemiology is poorly understood. The European bison (Bison bonasus) population has been growing European-wide quite dynamically over the last few years. The aim of this study was to determine whether C. burnetii infection occurs in European bison and whether it can be considered an important bacterial reservoir in the natural environment. Five hundred and twenty three samples of European bison sera originating from 14 (out of the 26 existing) Polish populations were examined for the presence of specific antibodies using an ID Screen Q Fever Indirect Multi-species ELISA test. Only one (0.19%) serum sample was positive in ELISA, and two other samples were doubtful. The only seropositive animal found in this study was a free-living bull. It suggests possible transmission from domestic cattle by sharing pastures. The transmission of C. burnetii into the European bison was rather accidental in the country and its role as an important wild reservoir is unlikely. Since no tests are available for wildlife ruminants there is a need for the adaptation of the available tests.

The First Report of Immunoglobulin G, M, and A Concentrations in Serum of European Bison and Their Changes with Age.

The age-specific reference values for immunoglobulin (Ig) serum concentrations in European bison (Bison bonasus) are lacking. Identification of immune alterations that accompany normal physiological aging will help assist in development of better monitoring health programs. In the present study, the age-associated changes in concentration of IgG, IgM, and IgA in serum of apparently healthy European bison of various ages were studied. The quantities of IgA, IgM, and IgG were measured by the use of a commercial ELISA kit. The serum samples originating from apparently healthy European bison (n = 206) were divided into the following age categories: (1) <1 year of age; (2) animals between 1 and 3 years of age; and (3) animals which have reached sexual maturity: (3a) animals between 4 and 8 years of age, (3b) animals between 9 and 15 years of age, and (3c) animals > 15 years of age. IgG was found to be predominant Ig in the serum regardless of the age of the animals. The significant positive correlation between IgG absolute and relative concentration and the age of animals was found. The absolute concentration of IgM did not differ significantly during the lifespan; however, the negative correlation was observed between percentage of IgM and European bison's age. IgA represented the least class of serum Ig. Total serum concentration of analyzed Ig also significantly increased with age. No gender-related differences were detected. Our findings represent a meaningful contribution to the studies on the immunity of European bison and effect of age on the immunoglobulin level. Our results would be useful for veterinarians and researchers in the studies with this animal's species.

Parasitological Monitoring of European Bison (Bison Bonasus) from Three Forests of North-eastern Poland between 2014 and 2016.

INTRODUCTION: Common parasites of the European bison include gastro-intestinal and pulmonary nematodes, liver flukes (Fasciola hepatica), tapeworms, and protozoa of the genus Coccidia. This study compared the extensiveness and intensities of European bison parasitic invasions in three north-eastern Polish forests in different seasons and queried the role of parasitological monitoring in sanitary and hygienic control of feeding places. MATERIAL AND METHODS: Faecal samples were collected in the Bialowieza, Knyszynska, and Borecka Forests between 2014 and 2016, as were some from an area neighbouring the Bialowieza Forest outside the Natura 2000 protected area. Parasites were detected in individual samples with the flotation, decanting and Baermann methods. RESULTS: The eggs of Trichostrongylidae, Aonchotheca sp., Nematodirus sp., Strongyloides spp., Trichuris sp., Moniezia spp., and Fasciola hepatica; the larvae of Dictyocaulus viviparus; and the oocytes of Eimeria spp. were identified. Significant variation in invasion intensity and diversity was seen by origin and season. The relationships were assessed first by univariable tests and next multivariately, when origin and season emerged as the major risk factors for exposure to most of the parasites. CONCLUSION: The differences in the level of parasitic infection between the forests did not have implications for its sufficiency to cause clinical symptoms. However, the associations and risk factors found enable the necessary preventive measures to be taken to protect the E. bison from exposure or decrease the risks. Additionally, parasitological monitoring is appropriate as the method of sanitary and hygienic control of European bison winter feeding places. Threats to public health through adventitious invasions by zoonotic factors such as F. hepatica have been identified.

Seroprevalence and risk factors for selected respiratory and reproductive tract pathogen exposure in European bison (Bison bonasus) in Poland.

After the complete extinction from the wild of European bison (Bison bonasus) at the beginning of the twentieth century, the worldwide species population was restored to approximately 5500 individuals, with the species however remaining endangered. Despite numerous studies on the ecology and genetics of European bison, the threats of infectious diseases have been largely unexamined. The aim of this study was to screen the exposure of the world's largest population of European bison to the pathogens, which may influence the condition and development of the endangered species. A total of 240 free-ranging and captive European bison from eight main Polish populations sampled were tested for the presence of specific antibodies against ten different viruses, bacteria or protozoan. The samples were collected from chemically immobilized, selectively culled or found dead animals. Based on serology, the exposure to bovine viral diarrhea virus (BVDV), bovine herpesvirus type 1 (BoHV-1), Mycoplasma and Brucella spp. was determined as rather accidental. Using gamma-interferon assay followed by Mycobacterium tuberculosis subs. caprae detection in tissues, diagnosis of bovine tuberculosis was made for 6 out of 78 (7.7%) bison from one captive herd. The highest seroprevalence was found for bovine adenovirus type 3 (BAdV-3) -60.2% and bovine parainfluenza type 3 (PIV-3) -34.0%, while the antibodies against bovine respiratory syncytial virus (BRSV), Toxoplasma gondii and Leptospira spp. were found in 10.4%, 10.4% and 8.7% of samples, respectively. In the multivariable statistical analysis using generalized linear mixed models (GLMMS), the risk factors for PIV-3 seropositivity included population type (free-living/captive), age and health status (apparently healthy/eliminated due to the poor condition). Higher risk of BAdV-3 seropositive result was observed in free-living female European bison. The high BAdV-3 and PIV-3 seroprevalences may suggest involvement of these pathogens in the most frequently observed respiratory disorders in European bison. Moreover, this is the first study demonstrating BAdV-3 exposure in the species.

EPIDEMIOLOGY OF SCHMALLENBERG VIRUS IN EUROPEAN BISON ( BISON BONASUS) IN POLAND.

Schmallenberg virus (SBV), an emerging arbovirus in Europe, is an important pathogen in domestic ruminants; however, its impact on free-ranging wild ruminants is not well studied. Three hundred and forty-seven serum samples collected between 2011 and 2016 from 302 European bison ( Bison bonasus) from 12 different sites in Poland were tested for the presence of SBV antibodies. In addition, 86 sera were collected between 2013 and 2016 from three species of cervids for testing for SBV antibodies. After the first detection of the virus in Poland in October 2012, the proportion of SBV-seropositive European bison reached 81% (95% confidence interval [CI]: 77.1-85.8%), whereas in cervids seroprevalence was 34% (95% CI: 23.5-43.9%). There was an increase in seroprevalence in European bison from 2012 to 2014. Biting midges ( Culicoides spp.), the primary vectors of SBV, were monitored entomologically for the identification of the biting midge populations and virologically for SBV infections in the Bialowieza Forest region, which contains the world's largest European bison population. We detected SBV by PCR in 3% of Culicoides pools from 2015. In addition, seven fetal brain samples from European bison or cervids were tested and were negative for SBV RNA. Our results indicate a high seroprevalence with reduced transmission of SBV in subsequent years in the European bison populations and lower seroprevalence in cervids.

First Toxoplasma gondii isolate from an aborted foetus of European bison (Bison bonasus bonasus L.).

The study was performed on a male European bison (Bison bonasus bonasus L.) foetus spontaneously aborted at the fourth or fifth month of pregnancy in the Bialowieza Forest. Serum samples from the foetus and mother revealed the presence of antibodies against T. gondii (S/P% = 88% and 75%, respectively). Mobile extracellular tachyzoites were first observed in a Vero cell culture, 110 days following inoculation of brain homogenate. PCR amplification with TGR1E1 and TGR1E2 primers confirmed the presence of T. gondii DNA, which was classified as Type I by PCR-RFLP genotyping. The sequences of 18S ribosomal RNA (18S rRNA) and 5.8S ribosomal RNA (5.8S rRNA) genes; internal transcribed spacer 1 (ITS1) and internal transcribed spacer 2 (ITS2), obtained from T. gondii isolate, have been deposited in GenBank (accession number KX459518.1). This is the first in vitro isolation and molecular identification of T. gondii from an aborted European bison foetus. The origin of this protozoan isolate indicates that the species is a significant threat to the European bison conservation program implemented in the Bialowieza Forest.

Cross-sectional study of Schmallenberg virus seroprevalence in wild ruminants in Poland at the end of the vector season of 2013.

BACKGROUND: In view of recurrent Schmallenberg virus (SBV) infections all over Europe between 2011 and 2013, a lively scientific debate over the importance of the sylvatic transmission cycle of the virus has emerged. The study presents results of serosurvey which included wild ruminants representing species of red deer (Cervus elaphus), roe deer (Capreolus capreolus), European bison (Bison bonasus), fallow deer (Dama dama), mouflon (Ovis orientalis musimon) hunted or immobilized at 34 different locations of Poland in the autumn/winter 2013. RESULTS: Out of 580 sera, 145 (25%) were considered positive for SBV antibodies. The overall SBV seroprevalence calculated using district probability weights was estimated at 27.7% (95% CI: 24.0-31.4). The seroprevalences at the district level varied between 0 and 80.0% (95% CI: 24.5-135.0%) with the mean within-district prevalence of 24.0% (95% CI: 16.5-31.4). Significantly higher seroprevalence was observed in animals from the Eastern provinces (36.6%) compared to the Western provinces (22.8%). SBV infection impact varied significantly between different species (higher SBV seroprevalence in larger species such as European bison), population type (free-ranging; captive), age, body weight, percent of the district forest area, part of Poland, and the densities of wild and domestic ruminants at the district and province level. Using statistical multivariable logistic model, population type, age, part of Poland and domestic ruminant density were identified as the main risk factors for SBV infection in wild ruminants in Poland. CONCLUSIONS: SBV seroprevalence in wild ruminants, similarly to the epizootic situation in domestic ruminants in the country, varied significantly between districts and provinces. Association between SBV seropositivity, species, animal body weight and age group expressed by a higher prevalence in larger ruminants may be explained by more frequent exposure to midge-vector bites of the latter, however it might also be related to the different species susceptibility to SBV infection. The positive effect of higher domestic ruminant density on the risk of SBV infection in wildlife and lower SBV seroprevalences in the latter suggested that the sylvatic cycle of SBV transmission is an effect of the pathogen spillover from the domestic animals.

Nematodes of the large intestine of the European bison of the Bialowieza National Park.

The present study was conducted on 31 males and females of the European bison, eliminated during the winter seasons 2007-2011 in the Bialowieza Primeval Forest, Poland. The caeca of 14 free-ranging bison, aged from 3 months up to 16 years, the most favorable infection site for the large intestine nematodes, were investigated during the winter of 2007/2008. The parasitological autopsies of the large intestines of 9 free-ranging bison aged from 5 months up to 10 years as well as 9 bison kept in the close reserves aged from one up to 20 years were done during the winter seasons of 2008-2011 to determine localization of nematodes in large intestine and the total intensity of parasite infection. Five species of nematodes (i.e., Trichuris ovis, Oesophagostomum venulosum, O. radiatum, Ashworthius sidemi, Nematodirus helvetianus) were found in dissected caecum of bison culled during the winter of 2007/2008. During the seasons of 2008-2010, 6 species of nematodes were found in the large intestine of bison (i.e., T. ovis, O. venulosum, O. radiatum, A. sidemi, N. roscidus and Bunostomum trigonocephalum). We did not find any nematodes in the lumen of the large intestine of captive bison culled during the seasons of 2010/2011. The results of the present study indicate that the intensity of infection by the large intestine nematodes of the European bison in the Bialowieza Primeval Forest has stayed at the comparable level throughout the last 20 years; however the number of nematode species has increased. The observed level of parasitic infection is typical of subclinical parasitoses.