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

Veterinary Microbiology 215 (2018) 57–65

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

T

Michał. K. Krzysiaka,b, Artur Jabłońskic, Wojciech Iwaniakd, Monika Krajewskad, ⁎ Julia Kęsik-Maliszewskae, Magdalena Larskae, a

Białowieża National Park, Park Pałacowy 11, 17-230, Białowieża, Poland Department of Epizootiology and Clinic of Infectious Diseases, Faculty of Veterinary Medicine, University of Life Sciences, 20-612, Lublin, Poland c Department of Swine Diseases, National Veterinary Research Institute (NVRI), Al. Partyzantów 57, 24-100, Puławy, Poland d Department of Microbiology, National Veterinary Research Institute (NVRI), Al. Partyzantów 57, 24-100, Puławy, Poland e Department of Virology, National Veterinary Research Institute (NVRI), Al. Partyzantów 57, 24-100 Puławy, Poland b

A R T I C L E I N F O

A B S T R A C T

Keywords: BVDV BoHV-1 PIV-3 BRSV BAdV-3 Leptospira spp. Toxoplasma gondii Brucella spp. Mycoplasma spp. Mycobacterium spp. European bison

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.

1. Introduction The European bison (Bison bonasus) has been reintroduced to Poland after its complete extinction from the wild since 1929. The number of European bison founders, which survived in zoos and private parks was limited, therefore present population is largely inbred. Initially, the animals were bred and maintained in captivity, and first released into the wild in 1952 (Dackiewicz, 2009). The present world population of European bison is approx. 5000 individuals. In the restitution breeding of endangered species, monitoring of health threats, especially those of an infectious and invasive nature, in addition to conventional

⁎

procedures is crucial. First reports on infectious diseases derive from the nineteenth century, when clinical cases or specific lesions characteristic for contagious bovine pleuropneumonia (mycoplasmosis) or pasteurellosis and hemorrhagic septicaemia among European bison were described (Wróblewski, 1927; Kita et al., 2003). The second half of the twentieth century has brought new international and intercontinental threats to the species such as bovine tuberculosis (TB), footand-mouth-disease and Q fever (Kita et al., 2003; Krajewska et al., 2015a,b). Over the last 20 years, five TB outbreaks caused by Mycobacterium caprae or bovis have been confirmed in European bison from different regions of Poland. The eradication of the first outbreak in the

Corresponding author. E-mail address: [email protected] (M. Larska).

https://doi.org/10.1016/j.vetmic.2018.01.005 Received 29 September 2017; Received in revised form 8 January 2018; Accepted 14 January 2018 0378-1135/ © 2018 Elsevier B.V. All rights reserved.

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lead to abortions and weak newborns, while ubiquitous and also zoonotic protozoa Toxoplasma gondii appears in a variety of warm-blooded animal species, moreover felids are considered the definite host, necessary for the parasite reproduction. T. gondii may cause abortions, stillbirths and fetal abnormalities, however bovids are considered rather resistant to T. gondii infection (Costa et al., 2011). The aim of the study was to screen the exposure of European bison from the main populations in Poland to the potentially most threatening pathogens, which may influence the condition and development of the endangered species reared in wild and in captivity. Due to the uniqueness of the research material (the total Polish European bison population is around 1500 animals), such studies bring new insights for the efforts to protect the species of those rare animals. Moreover, since European bison inhabit various environments, they may be also indicate the exposure risk for other wild ruminants in the country.

free-ranging European bison herd in Bieszczady Mountains (south of Poland) took over 16 years (1997–2013) (Krajewska et al., 2015a,b). In 2013, another three outbreaks of TB caused by Mycobacterium caprae were identified in European bison (Anusz et al., 2017). Molecular analysis has confirmed the high homology of the strains indicating TB spread between the bison from the three different breeding centres. Interspecies transmission from local cattle kept on pastures adjacent to bison territory is suspected to be the main source of the tuberculosis in bison. Fortunately, the largest population of European bison in Białowieża Primeval Forest remains free of TB, perhaps because of less contact with livestock and rigorous sanitary measures applied. Since the European bison is closely related to cattle (Bos taurus), the starting point for this study was to investigate the exposure to the pathogens known to affect bovids, endemic to Poland and Europe. The respiratory and reproductive infectious diseases remain a problem for domestic and wild ruminants, which may also affect the condition and size of European bison population (Golbert et al., 2013). In the study, the distribution of infections with the key pathogens prevalent to cattle such as bovine viral diarrhea virus (BVDV), bovine herpesvirus type 1 (BoHV-1), parainfluenza virus type 3 (PIV-3), bovine adenovirus (BAdV-3), bovine respiratory syncytial virus (BRSV), Mycobacterium tuberculosis, Mycoplasma spp. involved in respiratory diseases, as well as Brucella abortus, Toxpolasma gondii and Leptospira spp. producing reproductive losses were investigated. BVDV belongs to a genus of highly variable pestiviruses, which are highly damaging and widely spread in domestic and wild ruminants and pigs all over the world. Due to the immunosuppressive nature of the virus, acute infections often confounded by other infections are manifested by respiratory disease and fertility failure. BVDV persistent infections (PI) of cattle infected in utero lead to death of the animal within the first 24 months of life after developing mucosal disease. BoHV-1 is the most important and best characterized member of a large group of alphaherpesviruses (Roizmann et al., 1992). BoHV-1 causes infectious bovine rhinotracheitis (IBR), infectious pustular vulvovaginitis (IPV), conjunctivitis, fatal multisystemic infection of newborn calves, encephalitis and abortions (Muylkens et al., 2007). Cervid herpesvirus 1 (CvHV-1) and 2 (CVHV-2), alphaherpesvirus species closely related to BoHV-1 are endemic in wild ruminants (das Neves et al., 2010). In Poland, animallevel BoHV-1 seroprevalence in cattle was estimated at 30%, while BoHV-1 infections occurred in over 70% of herds (Rypuła et al., 2012). Worldwide endemic PIV-3, BAdV and BRSV usually occur as co-infections and are described as etiological agents of bovine respiratory disease (BRD), which also involve BVDV, BoHV-1 and Mycoplasma bovis (Taylor et al., 2010). Mycoplasmas are conditional pathogens responsible for pneumonia, arthritis and mastitis in cattle also in Poland (Dudek and Bednarek, 2012; Nicholas, 2011). Mycoplasma mycoides subspecies mycoides small colony (MmmSC) causes the most devastating contagious bovine pleuropneumonia (CBPP) OIE-listed hazard for international trade and subject to compulsory eradication. Despite the zoonotic character of Mycobacterium tuberculosis, the decision making and eradication of TB in European bison is complicated because of the nature of the species. Since the bacteria may be spread between susceptible species also in sylvatic cycle, the exposure of bison is constant and should be monitored. Other pathogens of possible threat to European bison reproduction include Brucellae, which are gram-negative, facultative, intracellular bacteria causing zoonosis of worldwide public health and economic importance (Godfroid et al., 2005; Franco et al., 2007). B. abortus, responsible for bovine brucellosis, B. melitensis, the main agent of ovine and caprine brucellosis, and B. suis, which causes brucellosis in pigs, play the main role in brucellosis epidemiology. While the national brucellosis monitoring program does not include wildlife, the disease is reported in wild animals in Europe (Cvetnic et al., 2004; Szulowski and Pilaszek, 2001). The animal testing is based almost entirely on serological assays, however unequivocal diagnosis of Brucella infection can be made only by the culture and identification of the agent. Leptospira spp. infection occurring in most mammals may

2. Methods 2.1. Sample collection A total of 240 serum samples was obtained from European bison between 2011 and 2015. The tested European bison originated from locations: Białowieża Primeval Forest (n = 115); Bieszczady Mountains (n = 14); Gołuchów (n = 5); Niepołomice (n = 26); Pszczyna (n = 46); Borecka Forest (n = 10); Smardzewice (n = 20); and Warsaw ZOO (n = 4) (Fig. 1). The population sizes are presented at Table S1. Captive (kept in fenced reserves) and free-ranging European bison were 150 (62.5%) and 90 (37.5%), respectively. The free-ranging animals originated from Białowieża (n = 75), Bieszczady (n = 7) and Borecka (n = 8; the remaining two individuals were kept in fenced quarantine for few months during the sampling, therefore were included as captive). No vaccinations are given to either free-living or captive European bison in Poland. Slightly more female European bison (n = 127) were sampled than males (n = 108). The age of the animals ranged between a few months and 27 years, with mean of 6.6 years (median 4.0; 95% CI: 5.8; 7.5). For further statistical analysis, the animals were divided into three age categories using the key of Krasińska and Krasiński (2013) as follows: 1) calves ≤ 1 year of age (n = 53); 2) young animals between 2 and 3 years of age (n = 52); and 3) adult animals ≥ 4 years of age which have reached sexual maturity (n = 125). Most of samples (n = 165; 68.8%) originated from European bison pharmacologically immobilized for diagnostic purposes, transportation or collaring according to the previously described protocols (Krzysiak and Larska, 2014). The rest of the samples were collected postmortem from animals which had been selectively eliminated due to poor condition (n = 64); fallen (n = 9); or killed in a traffic accident (n = 2). Due to the limited volume or quality of some samples not all sera were suitable for simultaneous testing for all pathogens, therefore the number of observations was given for each analysis (Tables 1, 2, 5, 6). Heparinized blood samples from 78 bison immobilized between 2012 and 2015, delivered to the laboratory within 24 h from collection were tested for bovine tuberculosis. The tested European bison originated from eight locations: Bałtów (n = 3); Białowieża Primeval Forest (n = 13); Bieszczady Mountains (n = 3); Gołuchów (n = 5); Niepołomice (n = 7); Pszczyna (n = 14); Wałcz (n = 1); and Smardzewice (n = 32). 2.2. Serological methods To detect BoHV-1 and BVDV antibodies, blocking IBR gB ×3 Antibody Test kit and indirect IDEXX BVDV Ab Test (IDEXX Laboratories, Inc., Liebefeld-Bern, Switzerland) were used. The tests use cut-off values of S/N = 55% and S/P = 0.3 respectively. According to the manufacturer’s brochure, the tests provide 99.8% and 96.3% sensitivity and 100% and 95.0 % specificity respectively. For the detection 58

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Fig. 1. Map of distribution of European bison populations included in the study. The sizes of the populations are given at Table S1.

mycoides biotype small colony, M. bovis and M. agalactiae using MmmSC antibody ELISA test (IDEXX, Montpellier, France), Mycoplasma bovis ELISA kit (Bio-X Diagnostics, Rochefort, Belgium) and Mycoplasma agalactiae ELISA (IDEXX, Montpellier, France), respectively. To verify MmmSC positive reactions, complement fixation test (CFT) (CIRAD, Monpellier, France) was performed according to the manufacturer’s protocol. For detecting antibodies for B. abortus, the Rose Bengal test (RBT), complement fixation test (CFT), serum agglutination test (SAT) were used. The methods were performed according to the national regulations and in accordance with guidelines included in OIE Manual (2016). Tests were performed using the commercial antigenic preparation (Biowet, Pulawy, Poland). Regarding the RBT, any agglutination was considered as positive result. In SAT, positive results have been recognized when tested sera showed more than 30 international units/ml (IU/ml) of serum and respectively for CFT more than 20 international complement fixation units/ml (icftu/ml). All ELISA tests were conducted and calculated according to manufacturer’s protocols The tests are dedicated to test individual bovine or small ruminant sera notwithstanding some were used previously in multiple ruminant species serosurveys (Krzysiak et al., 2014b; Mingliang et al., 2015; Rodríguez-Prieto et al., 2016). Intravital diagnosis of bovine tuberculosis was performed using of the gamma-interferon Bovigam test (Prionics, Switzerland) (Wood and Jones, 2001). This method detects gamma interferon (γIFN) released in

of PIV-3, BAdV and BRSV antibodies Trivalent Antibody Test kit (IDEXX Montpelier SAS, Montpellier, France) was used. The samples with c S/P (Corrected Sample to Positive percentage) greater or equal to 20% were considered positive. The results was also graded accordingly to c S/P values between weak positive (+) to very strong positive (+ + + + + ). Sensitivity and specificity for PIV-3 and BRSV were 98.85%, 75% and 86.67%, 100% respectively; for BAdV- not provided. The sera were also tested for Toxoplasma gondii antibodies using a commercial ELISA (IDEXX, Liebefeld-Bern, Switzerland) and screened against a panel of 13 leptospiral serovars using the microscopic agglutination test (MAT). The panel represented the following serovars of Leptospira interrogans: Icterohaemorrhagiae (strain RGA), Grippotyphosa (strain Moskva V), Sejroe (strain M84), Tarrasovi (strain Perepelicyn), Pomona (strain Pomona), Canicola (strain Hond Utrecht IV), Bratislava (strain Jez Bratislava), Ballum (strain Mus 127), Zanoni (strain Zanoni), Hebdomadis (strain Hebdomadis), Hardjo type Bovis (strain Hardjo Bovis), Saxkoebing (strain MUS 24) and Poi (strain Poi). The end-point sera dilution was 1:100. The reference strains were provided by the WHO/FAO Collaborating Centre for Reference and Research on Leptospirosis of the Royal Tropical Institute, Amsterdam. Using reference sera, MAT was described as highly specific (99.8%) and sensitive (95%) for leptospirosis, however it lacks sensitivity in an early detection of the acute stage of infection. The sera were screened for the presence of antibodies against Mycoplasma mycoides subspecies 59

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Table 1 The frequencies of seropositive to Parainfluenza type 3 (PIV-3), bovine adenovirus type 3 (BAdV3) and bovine respiratory syncytial virus (BRSV) European bison in regard to their different characteristics and their associations according to the univariate statistical analysis by Fisher’s exact test. Variable

PIV-3 seroprevalence

Origin (N = 240) Białowieża Forest Bieszczady Mountains Gołuchów Niepołomice Pszczyna Borecka Forest Smardzewice Warsaw ZOO Population type (N = 240) Captive Free-living Sex (N = 235) Female Male Age group (N = 230) ≤ 1 year old 2–3 years old ≥ 4 years old Health status (N = 234) Immobilized (apparently healthy) Eliminated Fallen Traffic accident a b c

n/Na

% (95% CIb)

71/115 0/14 0/5 0/26 1/46 3/10 7/20 0/4

61.2 (52.2–70.2) 0 (0–23.2) 0 (0–52.2) 0 (0–13.2) 2.2 (0.05–11.5) 30.0 (6.6–65.2) 35.0 (15.4–59.2) 0 (0–60.2)

22/150 60/90

14.7 (9.4–21.4) 66.7 (55.9–76.2)

41/127 39/108

32.3 (24.3–41.1) 36.1 (27.1–45.9)

5/53 10/52 61/125

9.4 (3.1–20.7) 19.2 (9.6–32.5) 48.8 (39.8–57.9)

24/165 50/64 6/9 2/2

14.5 (9.5–20.9) 78.1 (66.0–87.5) 66.7 (29.9–92.5) 100 (15.8–100)

BAdV3 seroprevalence Pc

n/Na

% (95% CIb)

82/115 1/14 0/5 21/26 32/46 4/10 4/20 1/4

71.3 (62.1–79.3) 7.1 (0.2–33.9) 0 (0–5.2) 80.8 (60.6–93.4) 69.6 (54.2–82.2) 40.0 (12.1–73.7) 20.0 (5.7–43.7) 25.0 (0.6–80.6)

79/150 66/90

52.7 (44.5–60.7) 73.3 (64.0–82.6)

85/127 58/108

66.9 (58.6–75.2) 53.7 (44.1–63.2)

29/53 26/52 84/125

54.7 (40.8–68.6) 50.0 (35.9–64.0) 67.2 (58.9–75.5)

85/165 53/64 5/9 2/2

51.5 (43.8–59.2) 82.8 (71.3–91.0) 55.6 (21.2–86.3) 100 (15.8–100)

BRSV seroprevalece Pc

< 0.001

n/Na

% (95% CIb)

18/115 0/14 3/5 0/26 0/46 1/10 1/20 2/4 14/150 11/90

15.6 (9.5–23.6) 0 (0–23.1) 60.0 (14.7–94.7) 0 (0–13.2) 0 (0–7.7))

9.3 (4.6–14.0) 12.2 (5.3–19.1)

15/127 8/108

11.8 (6.1–17.5) 7.4 (2.4–14.4)

3/53 6/52 12/125

5.6 (−0.8–12.1) 11.5 (2.6–20.5) 9.7 (43.6–14.8)

12/165 12/64 1/9 0/2

7.2 (3.8–12.3) 18.8 (10.0–30.4) 11.1 (0.2–48.2) 0 (0–84.0)

< 0.001

< 0.001

0.002

0.6

< 0.001

0.04

< 0.001

0.5

0.2

0.06

< 0.001

Pc

0.6

< 0.001

0.07

number seropositive to all samples tested. the Clopper-Pearson 95% confidence interval for binomial distribution (one-sided 97.5% for the mean = 0 and 100). P ≤ 0.05 considered significant.

vitro by the leukocytes of the infected animal. The test sensitivity between 81.8% and 100% for culture-confirmed bovine TB and specificity between 94% and 100% was reported (Wood and Jones, 2001). To perform the assay, full blood collected to lithium heparin or another anticoagulant is used. The blood is incubated with bovine and avian tuberculin purified protein derivatives (PPDs) in approx. 37 °C for 24 h, and then the level of released cytokine is measured using sandwich ELISA. In the European Union, the test is currently used as ancillary, being especially useful when examining wild ruminants, for which no standard tuberculin test reading has been established.

0.3; Spearman ρ > 0.5 ; P < 0.05) between the predictors was considered when building up the multivariable model. Possible confounding and clustering was analysed as described by Dohoo et al. (2010). To account for clustering, models including random intercept were assessed by checking the variance of the component and other covariates. The model with Akaike Information Criterion (AIC) and Bayesian Information Criterion (BIC) lowest and highest values respectively was considered better fitting. The analyses were preformed using STATA v.13.0 software (StataCorp LP, Texas, USA). The P value ≤ 0.05 was considered significant in all the analysis.

2.3. Statistical analysis

3. Results

The sample size for the study was calculated using forZ2pq

3.1. Descriptive statistics

Z2pq

mula: = L2 n = L2 , where Z is percentile of a standard distribution (1.96); p–estimate of true proportion; q = 1-p; L–the precision of the estimate (0.05) (Dohoo et al., 2010). With the assumed seroprevalence at 10%, the minimum sample size was 138. Next, the numbers were 1 adjusted for small populations using n′ = 1 / n+1 /N , where N is the size of population detailed in S1. Since only 78 animals were tested in the tuberculosis Bovigam test, the results were excluded from the statistical analysis. Primarily, the univariate associations between the seropositivities to studied pathogens, environmental (origin, population type: captive; free-ranging; health status) and individual-level (sex; age) variables were estimated using Fisher’s exact test. The correlations between all the variables were assessed by Cramér's V or Spearman rank tests. Since, the sample numbers collected from European bison in Bieszczady Mountains, Gołuchów and Borecka Forest has been assessed as not representative for their populations (Table S1), these observations were removed in the preparation of the final multivariable models. The generalized linear mixed models (GLMMs) were developed by backward elimination of insignificant (with P > 0.05) predictor variables one-by-one. The logit link function was used to model the probability of detection of a seropositive animal. The collinearity (Cramér's V above

3.1.1. Significant association between seroprevalence and origin, population type, age and health status were observed mostly for PIV-3 and BAdV-3 Significant percentages of the two hundred forty European bison from eight Polish populations sampled during four subsequent years had antibodies against two respiratory viruses: PIV-3 and BAdV-3. The infection with BRSV was less frequent. The highest seroprevalence was found for BAdV-3 (145/240; 60.4%) and PIV-3 (82/240; 34.2%), while BRSV antibodies were found only in 10.4% (25/240) samples. The descriptive statistics of the viral exposure are presented in Table 1. PIV3 and BAdV-3 seroprevalences were associated with the origin, population type and health status (P < 0.05). PIV-3 seropositivity was increasing with the age of European bison, while this increase was not that evident in respect to BAdV-3. The similar association may be observed at Fig. 2. The significant differences in BRSV seroprevalences were found only between the animals from different locations. Additionally, the trivalent ELISA used allowed semi-quantitative evaluation of the antibody level. PIV-3 and BAdV-3 antibody levels (from + to +++++) were distributed evenly in the seropositive samples, while the majority (72.0%) of BRSV seropositive samples showed the lowest antibody level (+) (Table S2). 60

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Saxkoebing/Hardjo/Hebdomadis/Zanoni, Hebdomadis/Hardjo and Zanoni/Bratislava. Stronger MAT reactions (titres ≥ 1:200) were detected in most of the positive results to serovars Saxkoebing, Zanoni, Pomona, Grippotyphosa, Hebdomadis. Relatively, the lowest mean titers among the positive results were observed for serovars Canicola, Bratislava and Hardjo. The detailed distribution of T. gondii and Leptospira spp. seroprevalence is presented at Table 3. No associations between the variables have been observed and therefore no multivariate models were built. The specific ELISA results showed European bison exposure to Mycoplasma bovis (6/240; 2.5%) and MmmSC (4/240; 1.7%), while no sera were M. agalactiae seropositive. None of MmmSC seropositive sera was confirmed in CFT. Mycoplasma bovis seropositive animals originated from three populations including Białowieża (Table 4). Out of 240 samples examined for B. abortus antibodies, most positive reactions were 63 (26.3%) found by SAT (in the range from 31 to 143 IU/ml of serum), however only two were confirmed (0.8%) in CFT with a rather low (20 IU/ml) but still positive antibody levels. None of the tested animals developed anti-Brucella antibodies detectable in RBT.

Fig. 2. LOWESS (locally weighted scatterplot smoothing) regression lines of the association between seroprevalence to parainfluenza virus type 1 (PIV-3), bovine adenovirus (BAdV-3), bovine respiratory syncytial virus (BRSV), Toxpolasma gondii and Leptospira spp. and European bison age.

3.2. Detection of one TB outbreak 3.1.2. Low seroprevalences to BVDV, BoHV-1, Toxoplasma gondii, Leptospira spp., Mycoplasma bovis and Brucella spp. suggested limited exposure For six out of ten pathogens identified as the major risk factors for the species health and population survival, the seroprevalences were low which suggested infrequent exposure or lower susceptibility of European bison to the infections and invasions with dose microorganisms. Only 2 (0.8%) and 1 (0.4%) out of 240 animals were found positive against BVDV and BoHV-1. BVDV seropositive European bison originated from wild populations of Białowieża Primeval Forest (16years old male) and Bieszczady Mountains (3-years old male). BoHV-1 seropositive result was obtained for a serum obtained from wild bull from Białowieża which had to be eliminated due to the frequent incursions into farms and aggression towards humans. Relatively small percentage −10.7% serum samples (95% CI: 6.6–14.8) gave positive reactions against Toxoplasma gondii in ELISA. Antibodies against Leptospira spp. were found in 20 (8.9%; 95% CI: 5.2–12.7) serum samples. The sera reacted positively in MAT against 8 different Leptospira serovars resulting in 26 positive results (Table 2). Cross-reactivity of some serovars was observed in 4 sera. The most frequently established Leptospira spp. serovars were Zanoni, Hardjo, and Bratislava. Some positive single reactions for serovars Grippotyposa, Pomona and Canicola were also detected. Cross-reacting serovars were

Under the suspicion of bovine tuberculosis (TB) or for the diagnostic purposes, 78 European bison were additionally tested in the γIFN assay. Six animals, all originating from a single herd located in Smardzewice sampled on 2015 were tested positive. Subsequently, the animals were culled and TB was confirmed microbiologally by Mycobacterium caprae identification. 3.3. Multivariable models The correlations between the individual variables were studied (Table S3) and confirmed previous observations presented in Tables 1 and 2. Some dependence between the explanatory variables was observed, however the collinearity was confirmed only between population type and health status. Most certainly, this was due to the fact that over 90% of immobilized European bison were captive, while 80% of those selectively eliminated were free-ranging animals. Additionally, BAdV-3 seropositivity was positively associated with PIV-3 with 75% of animals carrying antibodies against both viruses. Significant association between PIV-3 and BAdV-3 antibody levels (Table S2) was also observed (Spearman ρ = 0.2; P = 0.0001). Overall, European bison which originated from the world’s largest wild population of Białowieża Primeval Forest were exposed to all studied pathogens except Mycobacterium spp. and B. abortus (Table 4). Interestingly, the animals (3/5, in the age of 1–3 years) from fenced reserve in Gołuchów had antibodies only to BRSV. Except for one BVDV seropositive animal, additionally only one 6-years old European bison from wild population in Bieszczady Mountains had antibodies against BAdV3 at ++++ level (Tables 1 and 4). Two separate generalized linear mixed models (GLMMS) could be developed to study risk factors of PIV-3 and BAdV3 seropositivity (Table 5 and 6). The clustering of data at the origin variable was controlled by including it as a random effect in both models. The odds ratio of PIV-3 seropositive result was higher in the free-living European bison in comparison to the captive ones; in older age groups in respect to the animals at the age of one year or less (Table 5). Interestingly, the odds ratio for the eliminated PIV-3 seropositive versus chemically immobilized (apparently healthy) bison was significantly higher. Similarly to PIV-3, the odds ratio of the risk of seropositivity was higher in free-living bison in reference to captive. In the model, a small but significant negative effect of male gender was also observed.

Table 2 Leptospira spp. antibody titers according to microscopic agglutination test (MAT). Leptospira

Number of positive sera (%a)

Mean titer (SDb)

Min-Max

Icterrohaemorhagiae Grippotyphosa Sejroe Tarassovi Pomona Canicola Bratislava Ballum Zanoni Hebdomadis Hardjo Poi Saxkoebing Total

0 1 (0.4) 0 0 1 (0.4) 1 (0.4) 4 (1.7) 0 11 (4.6) 2 (0.8) 5 (2.1) 0 1(0.4) 26c

– 200 – – 400 100 125 (50.0) – 145.5 (93.4) 150 (70.7) 120 (44.7) – 800 173.1 (87.2)

– – – – – – 100–200 – 100–400 100–200 100–200 – – 100–800

a b c

4. Discussion

percentage of all 240 serum tested. standard deviation. four sera had detectable antibodies to more than one Leptosira species.

Since the most common pathological lesions in European bison 61

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Table 3 The frequencies of seropositive to Toxoplasma gondii and Leptospira spp. (see Table 2 for details) European bison in respect to their different characteristics and the associations according to the univariate statistical analysis by Fisher’s exact test. Variable

Toxoplasma gondii seroprevalence

Origin (N = 224) Białowieża Forest Bieszczady Mountains Gołuchów Niepołomice Pszczyna Borecka Forest Smardzewice Warsaw ZOO Population type (N = 224) Captive Free-living Sex (N = 219) Female Male Age group (N = 214) ≤ 1 year old 2–3 years old ≥ 4 years old Health status (N = 224) Immobilized (apparently healthy) Eliminated Fallen Traffic accident a b c

n/Na

% (95% CI)b

10/99 0/14 0/5 6/26 4/46 0/10 3/20 1/4

10.1 (5.0–17.8) 0 (0–23.2) 0 (0–52.2) 23.1 (8.9–43.6) 8.7 (2.4–20.8) 0 (0–30.8) 15.0 (3.237.9) 25.0 (0.6–80.6)

19/140 5/84

13.5 (8.4–20.4) 6.0 (2.0–13.3)

14/116 10/103

12.1 (6.8–19.4) 9.7 (4.8–17.1)

7/49 4/48 13/117

14.2 (5.9–27.2) 8.3 (2.3–20.0) 11.1 (6.0–18.2)

19/152 5/61 0/9 0/2

12.5 (7.7–18.8) 8.2 (2.7–18.1) 0 (0–33.6) 0 (0–84.2)

Leptospira spp. seroprevalece Pc

n/Na

% (95% CI)

b

12/99 0/14 0/5 2/26 3/46 0/10 3/20 0/4

12.1 (6.4–20.2) 0 (0–23.1) 0 (0–52.2) 7.7 (0.9–25.1) 6.5 (13.6–17.9) 0 (0–30.8) 15 (3.2–37.9) 0 (0–60.2)

13/140 7/84

9.3 (5.0–15.3) 8.3 (3.4–16.4)

11/116 9/103

9.5 (4.9–16.3) 8.7 (4.1–15.9)

2/49 4/44 13/117

4.1 (−1.6–9.8) 8.3 (0.2–16.4) 11.1 (5.3–16.9)

10/152 8/61 2/9 0/2

6.6 (3.2–11.8) 13.1 (5.8–24.2) 22.2 (2.8–60.0) 0 (0–84.2)

Pc

0.3

0.8

0.06

0.5

0.4

0.5

0.6

0.4

0.6

0.2

number seropositive to all samples tested. the Clopper-Pearson 95% confidence interval for binomial distribution (one-sided 97.5% for the mean = 0 and 100). P ≤ 0.05 considered significant.

Table 4 Evidence of exposure to the studied pathogens in the eight populations of European bison in Poland. Origin/pathogen

BVDV

BoHV-1

PIV-3

BAdV-3

BRSV

Brucella abortusa

Toxoplasma gondii

Mycoplasma bovis

Leptospira spp.

Białowieża Forest Bieszczady Mountains Gołuchów Niepołomice Pszczyna Borecka Forest Smardzewice Warsaw ZOO

+ + – – – – – –

+ – – – – – – –

+ – – – + + + –

+ + – + + + + +

+ – + – – + + +

– – – – – – – –

+ – – + + – + +

+ – – + – – + –

+ – – + + – + –

a

based on Rose-Bengal test (RBT).

ELISA results, however these were not confirmed by the virus neutralization test. Borchers et al. (2002) have found some additional BoHV-2 weak seropositive animals. In our study, the only BVDV and BoHV-1 seropositive European bison were free-ranging bulls, which may have contracted the infection from domestic ruminants. Wild male bison may more frequently be exposed to cattle since they dwell on larger acreages, significantly further away from the forest compared with the females and often graze the pastures of neighboring farmland (Krasińska and Krasiński, 2013). The demonstration of the antibodies specific to PIV-3 and BRSV in European bison was not unexpected, since the pathogens were reported in almost all Polish dairy herds in Poland (Rypuła and WojewodaKotwica, 2008; Socha and Rola, 2013). Moreover, PIV-3 antibodies have been reported in 14% of European bison from Białowieża Primeval Forest previously (Salwa et al., 2007). Lower PIV-3 and BRSV seroprevalences in European bison in this study compared to Polish cattle suggest that these infections may be a spillover from domestic ruminants. Furthermore, the interspecies contacts such as simultaneous grazing of European bison and cattle was observed in Białowieża National Park (Krasińska et al., 2000; Krasiński, 1978). This seems to be also supported by the absence of BRSV antibodies in the populations of

population in Białowieża Primeval Forest in recent years were pathological changes of the respiratory system (Krzysiak et al., 2014a), the study was designed to investigate the distribution of the known bovine respiratory pathogens to verify their possible involvement. Other important, especially to protected species are reproductive alterations, which may reduce the population size, leading to limiting of the gene pool and possible extinction. It needs to be acknowledged that while designing the study, we have confronted some limitations, which included the inability to provide random sampling which may have biased some of the statistical analysis. Furthermore, the high sensitivities of the tests used refer mostly to cattle, therefore some alterations in detecting serologic response in European bison may be expected. Nevertheless, the obtained data is unique for such a small population, and many of the presented findings are original or even pioneering. As reported previously, BVDV and BoHV-1 infections in European bison have been shown to be rather sporadic (Borchers et al., 2002; Kita and Anusz, 1991; Salwa et al., 2007). BoHV-1 was wrongly suspected to be involved in the etiology of balanoposthitis in Polish and Belarusian European bison, which has been affecting the health and reproductive disposition of bulls since the 1980′s (Jakob et al., 2000; Kita et al., 2003). Only Kita and Anusz (1991) observed some seropositive BoHV-1 62

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in multivariable model. Possible influence of the infections on European bison health should not be disregarded and needs to be investigated further, since the infections with both pathogens may add up to the observed clinical picture caused by multiple infectious and invasive factors (Krzysiak et al., 2014a). While, the association between BAdV-3 and PIV-3 may be certainly explained by the co-occurrence of infections with those pathogens observed in cattle (Härtel et al., 2004). PIV-3, BAdV-3 and BRSV commonly implicated in BRD in cattle affect mostly the young animals causing high mortality in calves (Taylor et al., 2010). These infections in European bison probably remain subclinical in the youngest animals, since no higher mortality has been reported for this age group (Krasińska and Krasiński, 2013). The survival rates of calves (0.93), young bison (0.97) and adults (0.98) studied for the population of Carpathian bison including the Białowieża population did not differ and were comparable to the average mortality observed in adult cattle (Alvåsen et al., 2014; Kuemmerle et al., 2001). While the mortality of calves in cattle herds may range between 4.3% in herds with no BRD outbreaks to 33% in calves where three or more BRD episodes are observed (Pardon et al., 2013). Therefore, it is very unlikely that BRD occurs in European bison like in cattle, however the importance of respiratory viruses, especially the high prevalence of BAdV-3 should be further investigated. Previous studies on epidemiology of brucellosis, mycoplasmosis, leptospirosis and toxpoplasmosis in European bison in Poland were also rather limited. Obtained in a present study results suggest some possible exposure to Brucella, however considering the epizootic situation in Poland, most likely a cross-reaction with Yersinia enterocolitica O:9, Escherichia coli O:157, Salmonella urbana, Stenotrophomonas maltophilia or unspecific reactions associated with the physiological condition of the animal (pregnancy, perinatal period, natural antibodies, etc.) were observed (Gerbier et al., 1997; Nielsen et al., 2004). The contact of the bison with Brucella suis biovar 2, not with Brucella abortus, cannot be excluded. In Poland, bovine brucellosis was eradicated in 1980 and since then no cases of B. abortus infection have been reported. However, brucellosis still may occur in Polish wildlife, which is probably the main and only source of infection for domestic animals. In Poland, the most important and the most probable source of Brucella are the wild boars (Szulowski et al., 2015), which are infected with B. suis biovar 2 (Szulowski et al., 2013). Similarly, the results of serological testing against remaining pathogens: Mycoplasma spp., Leptospira spp. and Toxoplasma gondii have shown that the problem of those infections in European bison is rather marginal. As shown in previous studies (Krzysiak et al., 2014b), the occurrence of MmmSC and M. agalactiae is uncommon and it mirrors the epidemic situation in domestic ruminants in Poland (Dudek and Bednarek, 2012). A single animal seropositive to Mycoplasma bovis, which was the free-living bull suggests rare but possible transmission from cattle. The results of Leptospira antibody testing confirmed the average seroprevalence observed commonly in free-living herbivores and the occurrence of antibodies against eight pathogenic serovars of Leptospira spp. in clearly noticeable ratios. The percentage of seropositive bison was higher compared to the research

Table 5 The final generalized linear mixed model (GLMM) presenting risk factors of Parainfluenza virus type 3 (PIV-3) seropositivity in European bison (number of observations = 203). Variable

Category

Population type captive free-living

β (SE)a

zb

P > |z|

95% CIc

reference 5.3

3.1

2.9

0.004

1.7–16.7

reference 7.2 20.4

5.8 13.7

2.5 4.5

0.014 < 0.001

1.5–34.9 5.5–75.9

reference 4.3 5.6 Variance

2.6 6.1 β (SE)c

2.4 1.6 95% CI

0.015 0.106

1.5–34.9 6.9–46.2

4.7

5.1

0.6–39.2

Odds Ratio (OR)

Age group ≤ 1 year old 2–3 years old ≥ 4 years old Health status immobilized eliminated fallen Random effect Origin a b c

standard error for the estimate of the variance reported in the previous column. Wald z statistic. 95% confidence interval.

Bieszczady Mountains, Niepołomice and Pszczyna located at the south of the country, where BRSV was observed only in 30% of cattle reflecting the lowest densities of cattle in this area (Socha and Rola, 2013). A further indication may be the higher seroprevalences in the free-living European bison, which might have been exposed to domestic ruminants more frequently than the captive animals. PIV-3 seroprevalence increased with age of European bison, indicating increased risk of exposure over time, while the exposure to BRSV was similar in all age groups. More unexpected was the high seroprevalence of BAdV-3 in Polish European bison. The BAdV infections have never been reported in the country and little is known on the prevalence of adenoviruses in European wildlife. Some studies have shown involvement of BAdV-3 and BAdV-7 in the development of BRD in cattle (Härtel et al., 2004). In ungulates in Turkish ZOO, BAdV-1 and BAdV-3 antibodies were detected in 46.6%, 60.1% of animals, respectively (Yeşilbağ et al., 2011). BAdV-3 infections have been wide-spread in all European bison populations studied, except for Gołuchów, however similarly to PIV-3 and BRSV, BAdV-3 seroprevalence was significantly higher in wild populations than in captive. The higher seroprevalence of BAdV-3 in female European bison may be explained by the space use and behavioral differences between the sexes. In the wild, European bison cows and calves remain in larger so-called mixed groups, while adult bulls live separately or within bachelor groups consisting of 2–3 males. In the univariable analysis, BAdV-3 and PIV-3 seroprevalences were the highest in the eliminated due to poor condition European bison. The elimination was also confirmed as an risk factor for PIV-3 seropositivity

Table 6 The final generalized linear mixed model (GLMM) presenting risk factors of Bovine adenovirus type 3 (BAdV-3) seropositivity in European bison (number of observations = 212). Variable

Category

Odds Ratio (OR)

β (SE)a

zb

P > |z|

95% CIc

Population type

captive free-living

reference 7.1

3.3

4.2

< 0.001

2.8–17.9

female male

reference 0.3 Variance 1.8

0.1 β (SE)c 1.7

−3.0 95% CI 0.3–11.1

0.003

0.2–0.7

Sex

Random effect Origin a b c

standard error for the estimate of the variance reported in the previous column. Wald z statistic. confidence interval.

63

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sampling of European bison from Pszczyna and Niepołomice. Special thanks also to Małgorzata Głowacka and Elwira Orłowska for the technical assistance. This study was supported by the Polish National Science Centre (Project No. 2013/09/B/NZ7/02563). Sample collection and dataset preparation was performed primarily for the National Centre for Research and Development (NCBiR) project No PBS2/A8/ 24/2013.

results reported previously using the same cut-off vale (1:100) by Salwa et al. (2007), while it was lower in respect to the investigations of Leptospira antibodies in European and American bison when a lower level (< 1:100) was accepted as a positive signal (Kita and Anusz, 1991; Taylor et al., 1997). The most frequent exposure of European bison exposure to Zanoni serovar confirmed the distribution observed in roe, red and fallow deer (Żmudzki et al., 2016). Another most frequent serovar Bratislava identified was reported in wild boar in Poland previously (Żmudzki et al., 2016). The second most prevalent serovar Hardjo has been recovered elsewhere in Europe from domestic cattle (Ellis, 2015), which is considered to be the maintenance host. Whether European bison are persistently infected with serovar Hardjo remains to be addressed in further studies. As to T. gondii, the parasite has been recently isolated from an aborted European bison foetus (Moskwa et al., 2017), and the seroprevalence in the population of Białowieża was previously reported somehow higher (25%) than in this study (Majewska et al., 2014). However, whether this invasion may produce large loses in the population is rather doubtful, albeit it should be investigated further. In view of the Polish epidemiological situation of bovine tuberculosis in bison as well as the fact that this species is particularly vulnerable to Mycobacterium caprae and Mycobacterium bovis infection, introduction of continuous surveillance of this disease appears to be indispensable. All TB cases detected were further confirmed by the postmortem isolation of Mycobacterium caprae in these six bison (Krajewska et al., 2016). The results of Bovigam test in three animals from Bieszczady Mountains are an interesting case (Anusz et al., 2017). The said individuals were kept in the acclimatization enclosure and the previous contact between these bison and the TB-infected individuals from the herd stamped out in 2013 cannot be ruled out. According to the manufacturer’s instruction, the test results of these bison were qualified as negative. However, in two bison the values obtained in samples stimulated with bovine tuberculin are so high that the exposure of these animals to both avian and bovine mycobacteria cannot be excluded. The problems of TB diagnosis in American bison have been already discussed by Chapinal et al. (2012). Since the most common intradermal test is of limited value in bison, γIFN release assays are fit for purpose. Bovine tuberculosis surveillance in this area still appears to be necessary due to TB occurrence in other free-ranging species (Orłowska et al., 2017). In the majority of cases, the course of the disease in both cattle and wildlife is devoid of characteristic clinical signs, and thus the result of gamma-interferon test is instrumental to contain the spread of the disease in the environment. Gamma-interferon test fulfils its purpose in TB diagnosis of live bison.

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Borchers, K., Brackmann, J., Wolf, O., Rudolph, M., Glatzel, P., Krasińska, M., Krasiński, Z.A., Frölich, K., 2002. Virologic investigations of free-living European bison (Bison bonasus) from the Białowieża Primeval Forest, Poland. J. Wildl. Dis. 38, 533–538. Chapinal, N., Elkin, B.T., Joly, D.O., Schumaker, B.A., Stephen, C., 2012. Agreement between the caudal fold test and serological tests for the detection of Mycobacterium bovis infection in bison. Prev. Vet. Med. 105, 326–330. Costa, G.H., da Costa, A.J., Lopes, W.D., Bresciani, K.D., dos Santos, T.R., Esper, C.R., Santana, A.E., 2011. Toxoplasma gondi: infection natural congenital in cattle and an experimental inoculation of gestating cows with oocysts. Exp. Parasitol. 127, 277–281. Cvetnic, Z., Toncic, J., Spicic, S., Lojkic, M., Terzic, S., Jemersic, L., Humski, A., Curic, S., Mitak, M., Habrun, B., Brstilo, M., Ocepek, M., Krt, B., 2004. Brucellosis in wild boar (Sus scrofa) in the Republic of Croatia. Vet. Med. 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5. Conclusions The study presents epizootic situation of selected viral pathogens among European bison from eight locations in Poland. The possibility of some infections as BAdV and BRSV was presented in this species for the first time. The frequency of the studied pathogens were comparable or lower to those observed in domestic ruminants. Cattle are considered the main drivers of transmission of most infections in European bison, therefore the spill-over effect is probably more common than spill-back. This is apparent from the differences in population densities, within and between group contacts and the environment in domestic and wild animals. Competing interests The authors declare that they have no competing interests. Acknowledgements The authors would like to thank Jerzy Dackiewicz from BNP for collaboration and Mieczysław Hławiczka for collaboration and 64

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