DOI: 10.1002/vms3.1314
OR I G I N A L A RT I C L E
Antibodies against theMycobacterium tuberculosis complex
and Brucella spp. in captive and free-living European bison
(Bison bonasus) in Poland
AnnaDidkowska1 Elisa Ferreras Colino2 WandaOlech3
Hugguette Gloddy2,4 Krzysztof Anusz1 José Antonio Infantes-Lorenzo5
Christian Gortázar2
1Department of FoodHygiene and Public
Health Protection, Institute of Veterinary
Medicine,WarsawUniversity of Life Sciences
(SGGW),Warsaw, Poland
2SaBio Instituto de Investigación en Recursos
Cinegéticos IREC, Consejo Superior de
Investigaciones Científicas, Universidad de
Castilla – LaMancha, Ciudad Real, Spain
3Department of Animal Genetics and
Conservation, Institute of Animal Sciences,
University of Life Sciences (SGGW),Warsaw,
Poland
4Centre de Recherche en Sciences Naturelles
de Lwiro (CRSN Lwiro), Lwiro, South Kivu,
Democratic Republic of Congo
5Unidad de InmunologíaMicrobiana, Centro
Nacional deMicrobiología, Instituto de Salud
Carlos III, Majadahonda,Madrid, Spain
Correspondence
AnnaDidkowska, Department of Food
Hygiene and Public Health Protection,
Institute of VeterinaryMedicine,Warsaw
University of Life Sciences (SGGW),
Nowoursynowska 159, 02-776Warsaw,
Poland.
Email: anna_didkowska@sggw.edu.pl
Funding information
Complex project of European bison
conservation by State Forests, Forest Fund
(Poland), Grant/Award Number: contract no
OR.271.3.10.2017
Abstract
Background: The European bison (Bison bonasus), a symbol of Polish nature, is a pro-
tected species that requires active health monitoring. However, conservation efforts
aremade 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 theMycobacterium tuberculosis complex (MTC) and Brucella spp.
Methods:A total of 323 free-living and captive European bison from13 localitieswere
tested serologically for antibodies against theM. bovis P22 multi-protein complex (in-
house ELISA) and against Brucella spp. (commercial ELISA).
Results:Antibodies against theMTC (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 pop-
ulations sampled, always in populations with larger sample sizes including the four
free-livingones.Antibodies againstBrucella spp.weredetected in36% (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 enterocoliticaO:9) in the case of Brucella spp.
KEYWORDS
Brucella spp, ELISA, European bison, P22, serology, tuberculosis
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© 2023 The Authors. Veterinary Medicine and Science published by JohnWiley & Sons Ltd.
Vet Med Sci. 2023;1–12. wileyonlinelibrary.com/journal/vms3 1
2 DIDKOWSKA ET AL.
1 INTRODUCTION
The European bison (Bison bonasus) is an iconic Polish wildlife species,
withmore than aquarter of the global Europeanbisonpopulation living
in Poland. The species is currently safe from extinction, having recently
changed its conservation status to ‘Near Threatened’, according to the
International Union for Conservation of Nature (IUCN) (Plumb et al.,
2020).
The number of European bison in Poland at the end of 2021 was
206 in captivity (most herds are of a low number) and 2223 in free-
living populations located in eight different herds (Raczynski & Bołbot,
2022). TheEuropeanbison inPoland is undergoing dynamic population
development (Olech & Perzanowski, 2022) that results in high local
population densities; therefore, it is important to monitor infectious
diseases and emerging threats (Klich et al., 2020, 2021).
Tuberculosis (TB), caused by members of the Mycobacterium tuber-
culosis complex (MTC), and brucellosis, caused by the genus Brucella,
not only have a considerable impact on cattle herds worldwide but
also are bacterial zoonoses with significant potential impact on public
health. Although Poland is officially free of TB and brucellosis in cat-
tle (Regulation [(EU) 2021/620]) both are occasionally noted in Polish
wildlife (Szulowski et al., 2013a; Krajewska et al., 2014). It has been
suggested that European bison are highly sensitive to Mycobacterium
spp. infections (Didkowska, Orłowska, et al., 2021); indeed, TB has
been confirmed in both captive (Didkowska, Orłowska, et al., 2021)
and free-living herds of European bison in Poland (Welz et al., 2005).
Although paratuberculosis does not seem to be an emerging problem
in Polish European bison herds (Didkowska, Ptak et al., 2021), atypical
mycobacteria (Mycobacterium avium and Mycobacterium xenopi) have
been isolated from them (Didkowska, Orłowska, et al., 2021).
TB has also been identified in European bison in a Brazilian zoo
(Zimpel et al., 2017) and is endemic in American bison (Bison bison)
in some North American populations (Himsworth et al., 2010; Nishi
et al., 2002; Shury et al., 2015; Tessaro et al., 1990). In recent years,
due to the emerging cases of TB in bison, diagnostic procedures have
been extended to incorporate newmethods, including serological tests
(Didkowska, Dziekan, et al., 2021).
One antigen that has recently been studied for use in serological
tests in various mammalian species is P22 (Bezos et al., 2018; Casal
et al., 2017; Infantes-Lorenzo et al., 2018; Infantes-Lorenzo, Dave
et al., 2019; Infantes-Lorenzo, Moreno et al., 2019; Thomas et al 2019;
Arrieta-Villegas et al., 2020; Ferreras-Colino et al., 2022). P22 is a
multi-protein complex obtained fromMycobacteriumbovispurified pro-
tein derivative (B-PPD) by affinity chromatography (Infantes-Lorenzo
et al., 2017). P22 has demonstrated high sensitivity (Se) and specificity
(Sp), depending on the animal species and epidemiological situation
(Infantes-Lorenzo, Moreno et al., 2019). In the case of cattle Se was
87% (Casal et al., 2017) and Sp was 92.5-99.4 depending on cut-off
and epidemiological situation (Infantes-Lorenzo, Moreno et al., 2019).
However, no data about the antibody response to P22 in European
bison are available.
Another disease of livestock and wildlife that has significant con-
sequences for animal and public health, as well as international trade,
is brucellosis (Khurana et al., 2021). Brucella abortus is the primary
cause of brucellosis in cattle, but it also affects wildlife, including bison
(Mackintosh et al., 2002; Rhyan et al., 2001; Tessaro et al., 1990).
A recent meta-analysis found that among wild Bovidae, the highest
prevalence of brucellosis was noted in American bison (Bison bison)
(39.5%) (Dadar et al., 2021). Highly similar B. abortus variable number
tandem repeat patterns have been identified in cattle and American
bison, indicating possible transmission between wildlife and livestock
(O’Brien et al., 2017; Rhyan et al., 2013). As B. abortus was also
noted in American bison in northern Canada (Wobeser 2009; Shury
et al., 2016), it would be advisable to monitor European bison for
brucellosis.
Although only a few studies have been performed on brucellosis in
European bison, their findings indicate that antibodies to B. abortus
are generally absent or only present in small numbers (Kita & Anusz,
1991; Krzysiak et al., 2018). It is important that endangered species
are monitored for diseases causing abortions. To this end, recent stud-
ies have attempted to evaluate the potential of serological tests against
brucellosis (e.g. Sánchez-Sarmiento et al., 2020).
The aim of this article was to describe the serum antibody response
of European bison to the P22 protein complex and to Brucella spp. and
related factors: gender, age and locality.
2 MATERIALS AND METHODS
2.1 Materials
Between October 2017 and April 2022, a total of 323 blood samples
were collected from captive (n = 152) and free-living (n = 159) Euro-
pean bison. Captive European bisonwere those living in zoos/breeding
centres, and free-living, living in different herds in thewild. The animals
were derived from different locations, presented in Figure 1. The age
of the sampled bison ranged from three months to 25 years (mean 6.5
years). The age of the bison was assessed by experienced veterinari-
ans based on the appearance of horns and tooth eruption (Krasińska
& Krasiński, 2017). Age was categorized into three groups: calves (<1-
year old), juveniles (1–3-year old), and adults (>3-year old) as described
previously (Krasińska & Krasiński, 2017). The samples originated from
males (n = 136) and females (n = 175). The material was collected
from living animals following immobilization, or dead ones that had
been culled or found dead. Immobilization was performed pharmaco-
logicallywith the use of a PalmerCap-Chur tranquilization gun (Palmer
Cap-Chur Equipment, Inc.) as described previously (Didkowska et al.,
2022). For some animals, it was not possible to acquire data regarding
age, locality, or sex; therefore, these animalswere not consideredwhen
performing statistics.
The blood was collected into 6–9 mL sterile tubes with a clot acti-
vator with a needle of a diameter of 1.2 mm from a jugular vein (vena
jugularis externa), or sometimes from the tail vein (vena caudalis medi-
ana). In the case of dead animals, blood was collected from the heart
and body cavities. The tubes containing the blood were refrigerated
and transported immediately to the laboratory. In the laboratory, the
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DIDKOWSKA ET AL. 3
F IGURE 1 Map of Poland indicating the distribution of European bison (Bison bonasus) sampling localities included in the study. The Białowieża
Forest, BieszczadyMountains and Borecka Forest locations include both captive and free-living herds, whereas Knyszyńska Forest only includes a
free-living herd. All other localities include only captive herds.
blood was centrifuged (3000 g, 10 min) and the serum was separated.
To minimize the risk of false positive results, all extensively hemolysed
samples were excluded from the study. The samples were stored at
−20◦C until analysis.
2.2 Enzyme-linked immunosorbent assays (ELISA)
Serum samples were brought to room temperature (RT) and then
subjected to two serological tests: an in-house ELISA for anti-P22 anti-
bodies and a commercial test for detecting antibodies against Brucella
spp.
To detect anti-P22 antibodies, the serawere testedwith an in-house
indirect P22-ELISA as described previously (Thomas et al., 2019).
Positive controls were derived from TB-positive cervids (compatible
lesions, M. bovis positive culture, OD > 1 at P22-ELISA). Negative
controls were derived from TB-negative cervids (the absence of com-
patible lesions, negative culture, OD < 0.2 at P22-ELISA). Briefly, the
ELISA plate was coated with P22 at 10 µg/mL in phosphate-buffered
saline solution (PBS; Panreac Química S.L.U.) and incubated overnight
at 4◦C. After washing with PBS solution containing 0.05% Tween-20
(PBST) (Tween 20; Sigma-Aldrich Quimica SA), the wells were blocked
with 5% skimmed milk powder solution in PBS (SM) for 1 h at RT.
Then tested sera and controls were added in 1:10 dilution in SM.
After 1-h incubation at 37◦C, the plates were washed three times
with PBST. Next, protein G horseradish peroxidase (HRP)-conjugated
(Sigma-Aldrich Química SA) at a concentration of 0.002 mg/mL in PBS
was added. After incubation for 1 h at RT, the plates were washed with
PBST, and o-phenylenediamine-dihydrochloride substrate (FASTOPD;
Sigma-Aldrich Química SA) was added. After incubation for 20 min at
RT, the reaction was stopped by adding H2SO4⋅3N. The optical density
(OD) was read in a spectrophotometer at a wavelength of 492 nm. The
result for each samplewasexpressedasELISApercentage (E%).E%was
calculated according to the following formula: [sample E% = (sample
OD/2 ×mean of negative control OD)x 100]. The cut-off value of 110
was chosen based on the plotted results (Figure 2). A conservative cut-
off of 110 E% was chosen to maximize specificity (Infantes-Lorenzo,
Moreno et al., 2019).
To detect antibodies against Brucella spp., a commercial ELISA
R.10.BRU.K3 INgezim BRUCELLA Compacis (Ingenasa) test based on
a blocking enzyme immunoassay was performed according to the
manufacturer’s instructions. The test allows the detection of specific
antibodies to the LPS of Brucella spp. inmultiple animal species (Muñoz
et al. 2010). The cut-off value was 40, as instructed. Absorbance was
read with a spectrophotometer at 450 nm. According to manufactur-
ers’ instructions, the sensitivity and specificity of the test for cattle are
98% and 99.9%.
2.3 Statistical analysis
The effects of sex, age, captive/free, year of collecting material and
locality were assessed by homogeneity tests (two-tailed Fisher’s exact
test and chi-square test where needed). In addition, 95% confidence
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4 DIDKOWSKA ET AL.
F IGURE 2 ELISA percentage (E%) results of Polish European bison (Bison bonasus) antibodies against protein complex P22 by age category
(calves, juveniles and adults). The solid red line indicates the E%value above which the result was considered positive (i.e. cut-off); E%= 110.
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DIDKOWSKA ET AL. 5
intervals (95%CI) were calculated for seroprevalence using Sterne’s
exact method.
2.4 Ethical statement
No animal was culled or immobilized specifically for this study. Ante-
mortem sampling (e.g. during translocation, putting the collar) was
performed as part of standard veterinary care. The procedures did not
require ethical approval according to II Local Ethical Committee for
Animal Experiments (Warsaw, Poland).
3 RESULTS
3.1 Antibodies against the M. tuberculosis complex
Antibodies against P22 were detected in 6.81% (95% CI: 4.44–10.17;
22/323; Table 1) of the tested European bison. The anti-MTC antibody
positivity was not significantly different by sex (Fisher’s exact test,
p = 0.085), nor age category (chi-square test, chi-square value = 3.45,
degree of freedom = 2, p = 0.18). E% results had a similar distribution
for each age category. For each age category, a few individuals showed
high antibody responses (6 of 51 calves, 11.8%; 7 of 83 juveniles,
8.4%; 8 of 170 adults, 4.7%) (Figure 2). The MTC antibody positivity
was not significantly different by captive/free-living status (Fisher’s
exact test, p = 1). Data on seroprevalence by year are presented in
Table 1.
The seropositive bison were found in 6 of the 13 sampling localities
(46.15%), including the four free-living ones (Table 1). In these six local-
ities, at least 19 bison samples were tested (range 19–72); in contrast,
in the remaining eight localities, only 1–8 samples were tested. Thus,
comparisons among localities were only performed for localities with
>19 samples (chi-square test, chi-square value = 3.3, degree of free-
dom = 5, p = 0.65). Seroprevalence slightly increased with sample size
(Figure 3).
3.2 Brucella spp. ELISA results
The general seroprevalence for Brucella spp. was 35.91% (95% CI:
30.79–41.32; 116/323; Table2). Anti-Brucella spp. antibodyprevalence
was not significantly different by sex (Fisher’s exact test, p = 0.64);
however, it was significantly different by both captive/free-living sta-
tus (Fisher’s exact test, p < 0.001) and age (chi square test, chi-square
value = 16.18, degree of freedom = 2, p = 0.003). All age categories
demonstrated a similar E% distribution, as shown in Figure 4. For each
age category, a significant number of individualswere positive: 23of 51
calves (45.1%), 42 of 83 juveniles (50.6%), and45of 170 adults (26.5%).
Data on seroprevalence by year are presented in Table 2.
Seropositive bison were found in 12 of the 13 sampling localities
(92.31%) (Table 2). The comparisons among localities only included the
localities where at least 19 bison samples, that is larger groups, were
TABLE 1 The frequencies of seropositive toMTC European bison
(Bison bonasus) regarding their different characteristics.
Variable
Number seropositive to all
samples tested
Population type 21/311 (6.8%; 95%CI: 4.37–10.08)
Captive 10/152 (6.6%; 95%CI: 3.52–11.75)
Free-living 11/159 (6.9%, 95%CI: 3.62–12.16)
Sex 18/311 (5.8%, 95%CI: 3.66–8.96)
Female 14/175 (8%, 95%CI: 4.76–13.06)
Male 4/136 (2.9%, 95%CI: 1.02–7.25)
Age group 21/304 (6.9%, 95%CI: 4.47–10.32)
Calves (<1-year old) 6/51 (11.8%, 95%CI: 5.25–23.36)
Juveniles (1–3-year old) 7/83 (8.4%, 95%CI: 4.03–16.69)
Adults (>3-year old) 8/170 (4.7%, 95%CI: 2.22–9.03)
Sampling year
2017 0/12 (0%)
2018 10/97 (10.3%, 95%CI: 5.51–17.93)
2019 4/47 (8.5%, 95%CI: 2.96–19.99)
2020 2/45 (4.4%, 95%CI: 0.8–15.20)
2021 2/84 (2.4%, 95%CI: 0.43–8.16)
2022 2/38 (5.3%, 95%CI: 0.95–17.99)
Localities
Bałtów (captive) 0/2 (0%)
Białowiska Forest 5/72 (6.9%, 95%CI: 2.78–15.13)
Captive 3/38 (7.9%, 95%CI: 2.19–20.79)
Free-living 2/34 (5.9%, 95%CI: 1.06–20.11)
BieszczadyMountains 5/67 (7.5%, 95%CI: 2.99–16.25)
Captive 2/16 (12.5%, 95%CI: 2.27–37.16)
Free-living 3/51 (5.9%, 95%CI: 1.63–16.36)
Borecka Forest 2/45 (4.4%, 95%CI: 0.8–15.2)
Captive 0/8 (0%)
Free-living 2/37 (5.4%, 95%CI: 0.97–18.48)
Gdańsk zoo (captive) 0/2 (0%)
Gołuchów (captive) 0/7 (0%)
Kiermusy (captive) 0/8 (0%)
Międzyzdroje (captive) 0/1 (0%)
Niepołomice (captive) 3/19 (15.8%, 95%CI: 4.45–39.19)
Pszczyna (captive) 2/38 (5.3%, 95%CI: 0.95–17.99)
Knyszyńska Forest (free-living) 4/37 (10.8%, 95%CI: 3.78–25.38)
Ustroń (captive) 0/4 (0%)
Warszawa zoo (captive) 0/8 (0%)
tested (range 19–72): If the sample size was small, it was impossible
to draw reliable conclusions. The anti-Brucella spp. seroprevalencewas
significantly different by locality (chi-square test, chi-square value =
16.383, degree of freedom = 5, p = 0.006). The seroprevalence rela-
tion to sample size is shown in Figure 3. Brucella spp. seroprevalence
decreasedwith sample size.
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6 DIDKOWSKA ET AL.
F IGURE 3 TheMycobacterium tuberculosis complex (MTC) and Brucella spp. seroprevalence in Polish European bison (Bison bonasus) in relation
to sample sizes. Blue colour: Brucella spp. seroprevalence; red colour: MTC (protein complex P22) seroprevalence; triangle: captive herd; square:
free-living herd. Dashed regression lines are plotted forMTC and Brucella spp. antibody prevalence.
4 DISCUSSION
Our findings indicate a 6.81% prevalence of anti-MTC antibodies
and a 35.91% prevalence of anti-Brucella spp. antibodies in Euro-
pean bison from Poland. This is the first confirmation of an antibody
response to P22 in bison. The correct diagnosis of TB still presents a
challenge in this species, so every new diagnostic tool is desirable (Did-
kowska, Krajewska-Wędzina et al., 2021). In protected species, there
is also a greater need to monitor diseases that can cause reproductive
alterations, such as brucellosis, as these can reduce the size of the pop-
ulation and thus its genetic variability, which is already low in European
bison (Druet et al., 2020). It should be emphasized that our findings
are particularly valuable as they were obtained from a large group
of animals over several years; this was necessary as the material was
taken from a protected species. This study indicates the current trends
regarding the diagnostic value of P22 in European bison and micro-
biological tests toward Brucella spp., highlighting the need for further
research.
To confront the problem posed by TB in Polish European bison
herds, efforts have been made to find cheap, convenient, and highly
sensitive diagnostic tools by improving sample collection (Didkowska
et al., 2020) and imaging methods (Didkowska et al., 2021d). Even
though culture remains the gold standard for TB diagnosis, it is not a
convenient tool for motoring wildlife (Thomas et al. 2021). Microbi-
ological tests are quite expensive, require long wait times and entail
difficulties in collecting suitable antemortem material due to peri-
odic shedding (Cousins & Florisson, 2005). Therefore, some studies
suggest using serological TB monitoring as an alternative (Thomas
et al., 2021). However, few have evaluated the potential of TB sero-
logical assays in European bison. Didkowska, Krajewska-Wędzina
et al. (2021) found Mycobacterium caprae-infected European bison to
demonstrate responses to the antibodies ESAT-6, CFP-10, MPB70,
MPB83, CFP10/ESAT-6, MPB70/MPB83, DID65 and B-PPD in dual-
path platform, multi-antigen print immunoassay (MAPIA) and IDEXX
M. bovis Ab ELISA (IDEXX Laboratories, Inc.); however, this study (Did-
kowska, Krajewska-Wędzina, et al., 2021) was performed on a limited
number of samples, including only four positives. More recently, sero-
logicalmonitoringbasedonMPB70andMPB83has shownmuch lower
MTC antibody seroprevalence in European bison (1.96%; Krzysiak
et al., 2022).
This is the first serological study to use P22 in European bison. Con-
sidering Poland that is generally free of brucellosis and TB in cattle,
we used caution when interpreting the results. We also considered
the lack of bison reference sera and that field samples, as opposed
to experimental infection derived ones, can yield lower specificity
(Ferreras-Colino et al., 2022). Therefore, we used a conservative cut-
off value of 110 instead of the commonly used cut-off value of 100
(Thomas, Infantes-Lorenzo, Moreno, Cano-Terriza, et al., 2019). The
cut-off value of 110was chosen based on the plotted results (Figure 2).
With a cut-off of 110, the MTC seroprevalence in European bison
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DIDKOWSKA ET AL. 7
TABLE 2 The frequencies of seropositive to Brucella spp. European
bison (Bison bonasus) regarding their different characteristics.
Variable
Number seropositive to all samples
tested
Population type 113/311 (36.3%; 95%CI: 31.84–40.99)
Captive 75/152 (49.3%; 95%CI: 41.44–57.52)
Free-living 38/159 (23.9%; 95%CI: 17.87–31.09)
Sex 112/311 (36%, 95%CI: 30.86–41.63)
Female 61/175 (34.9%; 95%CI: 27.96–42.27)
Male 51/136 (37.5%; 95%CI: 29.74–45.94)
Age group 110/304 (36.2%, 95%CI: 30.91–41.76)
Calves (≤1-year old) 23/51 (45.1%; 95%CI: 31.82–58.87)
Juveniles (2–3-year old) 42/83 (50.6%; 95%CI: 39.72–61.49)
Adults (≥4-year old) 45/170 (26.5%; 95%CI: 20.24–33.78)
Year of colleting blood
2017 7/12 (58.3%, 95%CI: 29.40–81.89)
2018 45/97 (46.4%, 95%CI: 36.56–56.72)
2019 15/47 (31.9%, 95%CI: 20–46.78)
2020 10/45 (22.2%, 95%CI: 11.84–36.56)
2021 28/84 (33.3%, 95%CI: 26.39–44.02)
2022 11/38 (28.9%, 95%CI: 16.67–45.29)
Localities
Bałtów (captive) 2/2 (100%)
Białowieża Forest 35/72 (48.6%, 95%CI: 36.95–60.74)
Captive 24/38 (63.2%, 95%CI: 46.53–77.38)
Free-living 11/34 (32.4%, 95%CI: 18.85–50)
BieszczadyMountains 17/67 (25.4%, 95%CI: 16.26–37.24)
Captive
Free-living
8/16 (50%, 95%CI: 27.20–72.80)
9/51 (17.5%. 95%CI 9.26–30.27)
Borecka Forest 9/45 (20%, 95%CI: 10.57–34.30)
Captive 3/8 (37.5%, 95%CI: 11.12–71.07)
Free-living 6/37 (16.2%, 95%CI: 7.31–32.19)
Gdańsk-zoo (captive) 0/2 (0%)
Gołuchów (captive) 0/2 (0%)
Kiermusy (captive) 1/8 (12.5%, 95%CI: 0.64–50)
Międzyzdroje (captive) 1/1 (100%)
Niepołomice (captive) 8/19 (42.1%, 95%CI: 22.19–65.51)
Pszczyna (captive) 18/38 (47.4%, 95%CI: 31.35–63.28
Knyszyńska Forest
(free-living)
12/37 (32.4%, 95%CI: 18.49–48.64)
Ustroń (captive) 2/4 (50%, 95%CI: 9.77 to 90.23)
Warszawa-zoo
(captive)
2/8 (25%, 95%CI: 4.64–63.53)
was 6.81% (95% CI: 4.44–10.17; 22/323) and varied from 0% to 8.8%
depending on the sampling year (Table 1).
A previous study on TBmicrobiological monitoring carried out from
2017 to 2019 isolated M. caprae from 4.44% (4/90) European bison;
however, all animals in the study derived from a single captive herd in
central Poland (Didkowska, Orłowska, et al., 2021). Animals from this
herd have not been tested in the present study. In another region of
Polandwhere TB is being confirmed inwildlife—BieszczadyMountains
(Krajewska-Wędzina et al., 2023), there was no higher seroprevalence
than in other free-living sites (Table 2). As such, our findings are higher
than expected, and the occurrence of MTC in different localizations is
surprising. This can be explained in several ways. The first explanation
may be that the P22-ELISA was characterized by high Se; however, it
would be necessary to examine the selected animals microbiologically
to confirm this. If the test turned out to be very sensitive and specific, it
would be a great diagnostic tool for European bison.
Alternatively, a cross-reaction may be present with atypical
mycobacteria, a well-known phenomenon in TB diagnostics (Jenkins
et al., 2018; Raffo et al., 2020). However, Didkowska, Orłowska et al.
(2021) reported that atypical mycobacteria were isolated from only
2.22% (2/90) of tested European bison. It should be highlighted that
P22-ELISA offers fair to good specificity (Sp) in detecting antibodies
against MTC, depending on the species, and so it also would not
necessarily be perfect for European bison. In other ruminants, the
tested Sp was 92.5%–99.4% in cattle, 30.9%–78% in goats, 94.4%–
100% in sheep and 99.0% in red deer (Thomas, Infantes-Lorenzo,
Moreno, Romero, et al, 2019). Another hypothesis is that bison could
have contact with other mycobacteria belonging to the MTC, such as
Mycobacterium microti. M. microti infections are increasingly reported
in free-living wild animals in Europe (Ghielmetti et al., 2021; Michelet
et al., 2021; Pérez de Val et al., 2019; Tagliapietra et al., 2021).
In contrast to most previous studies (Joly & Messier, 2004; Varela-
Castro et al., 2020), our findings indicate that theMTC seroprevalence
is not significantly different by sex and age. This may be influenced
by the behaviour of bison and their gregarious nature (Krasińska &
Krasiński, 2017). In addition, the MTC seroprevalence is not signifi-
cantly different by status and locality. In the case of locality, this is sur-
prising as in previous years, TB was microbiologically confirmed only
in two captive herds, namely one herd in Smardzewice, central Poland
and another (Wolisko) in Borecka Forest, and in two free-living herds,
namely in the Bieszczady Mountains and Borecka Forest (Krajewska
et al., 2011, 2016; Welz et al., 2005); therefore, it was anticipated
that most positive results would be identified in these locations. In
addition, localities with the highest seroprevalence (Białowieża Forest,
Bieszczady Mountains, Niepołomice and Pszczyna) are not adjacent
to each other (Figure 1, Table 1). In Poland, free-living bison popu-
lations are rarely spatially connected with each other (Perzanowski
et al., 2019), and new areas are rarely colonised (Ziółkowska et al.,
2016); in such cases, therefore, there is probably no bison-driven
pathogen transmission between localities. Another important consid-
eration regarding the effect of locality is that, in the case of free-living
herds in Poland, the carrying capacity of forest complexes is often
exceeded, and so captive herds generally have higher densities (Olech
& Perzanowski, 2014). Alternatively, the increase of seroprevalence
with sample size (Figure 3) might just point at a higher probability of
finding cross-reactions in larger samples.
Previous reports confirmed a sporadic occurrence of B. abortus anti-
bodies in European bison (Kita & Anusz, 1991; Krzysiak et al., 2018).
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8 DIDKOWSKA ET AL.
F IGURE 4 ELISA percentage (E%) results of Brucella spp. antibodies by age category (calves, juveniles and adults) in European bison (Bison
bonasus) from Poland. The solid red line indicates the E%value above which the result was considered positive (i.e. cut-off): E%= 40.
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DIDKOWSKA ET AL. 9
The 35.91% (95% CI: 30.79–41.32; 116/323) seroprevalence of Bru-
cella spp. identified herein suggests that the Polish European bison
population has had contact with these bacteria. The annual seropreva-
lence has varied significantly over time. The period from 2017 to 2020
saw a downward trend, and this was then followed by a relatively con-
stant level (Table 2). Due to the epizootic situation in Poland, with no
case ofB. abortusbeing reported since its eradication in 1980, it is likely
that any such had been with other species of Brucella. Indeed, Brucella
suis biovar 2 has been isolated from Polish cattle with positive sero-
logical results (Szulowski et al., 2013a), and this variant has also been
confirmed in Polish wildlife, mainly wild boar (Sus scrofa) (Szulowski
et al., 2013b). It would be advisable that microbiological monitoring is
continued, as Brucella spp. can also be a potential threat to European
bison, especially in the localities where seroprevalence was high, such
as Pszczyna and Białowieża (Table 2).
Our findings indicate that the Brucella spp. seroprevalence was sig-
nificantly different by age; however, unlike most studies, (Alhamada
et al., 2017; Asgedom et al., 2016; Chaka et al., 2018), the highest
seropositivity was noted in juveniles, not in adults (Table 2). It is pos-
sible that in such cases, the animal may have made first contact with
the pathogen without the protection offered by colostrum antibod-
ies and these would demonstrate the highest serological response. In
American bison, most calves were seronegative by 5 months, and the
highest seroconversionwas noted at 2 years (Rhyan et al., 2009). In the
present study, seropositivity was also more likely in captive than free
ranging bison (Table 2). This may be due to the higher animal densities
characteristic of captive herds. Localities were also found to have a sig-
nificant effect, whichmight be related to a greater presence of bacteria
in the areas concerned. Finally, sex was found to have no such effect,
and this is in-line with previous studies on American bison, where sex
had little influence on the prevalence of B. abortus antibodies (Scurlock
& Edwards, 2010).
This study has some limitations. First, randomsamplingwas not pos-
sible. In addition, the tests were not specially designed for European
bison. Furthermore, as the true status of the bison remains unknown,
further microbiological testing should be included in later studies.
In addition, it should be considered that the controls for MTC sero-
prevalence testing were taken from a different species (deer). What is
more, even though following the recommendations of the OIE-World
Organisation for Animal Health, complement fixation tests and serum
agglutination tests are being replaced by more sensitive and specific
tools for screening brucellosis in mammalian species, including indi-
rect ELISA and fluorescence polarization assay (Greiner et al., 2009;
Muñoz et al., 2010; Ragan et al., 2013); most brucellosis serological
tests intended for use in wildlife still need validation and standardiza-
tion (Dadar et al., 2021). One further limitation for Brucella sp. results
are cross-reactions with Yersinia enterocolitica O:9, Stenotrophomonas
maltophilia, Salmonella urbana, Escherichia coli O:157, or non-specific
reactions associated with the condition of the animal (Ducrotoy et al.,
2016; Nielsen et al., 2004).
The obtained results for the MTC and Brucella spp. serological sur-
veys raise another important problem. As European bison can be a
reservoir of other diseases, care should be taken when introducing
them into new areas, as in Spain (Castillo et al., 2019) or Romania
(Da˘nila˘ et al., 2022).
5 CONCLUSIONS
This study is the first to report an antibody response to the P22 multi-
protein complex in European bison. MTC serological survey showed
that 6.81% of the examined European bison are seropositive, which is
higher than noted in previous studies using specific M. bovis antigens.
In addition, more than 1/3 of the tested European bison were seropos-
itive against Brucella spp., which shows another direction for future
research. Our findings suggest that brucellosis and TB may represent
anemerging threat to the largestEuropeanbisonpopulation.AsPoland
is TB and brucellosis free, obtainingmany positive results might be due
to cross-reactions. In the future, it would be worth testing European
bison in Poland for pathogens that can cause false positive results.
AUTHOR CONTRIBUTIONS
Conceptualization; data curation; formal analysis; investigation; project
administration; resources; writing—original draft: Anna Didkowska. Data
curation; formal analysis; investigation; resources; visualization; writing—
review & editing: Elisa Ferreras. Data curation; funding acquisition;
project administration; resources; writing—review & editing: Wanda Olech.
Investigation: Huggette Gloddy. Supervision; writing—review & editing:
Krzysztof Anusz. Methodology; supervision; writing—review & editing:
Jose Antonio Infantes-Lorenzo. K conceptualization; funding acquisition;
methodology; supervision; visualization; writing—original draft; writing—
review & editing: Christian Gortazar.
ACKNOWLEDGEMENTS
We thank ‘Wildlife Conservation Network Grants’ for supporting HG.
CONFLICT OF INTEREST STATEMENT
Authors declared no conflicts of interest.
FUNDING INFORMATION
Complex project of European bison conservation by State Forests,
Forest Fund (Poland), contract noOR.271.3.10.2017
DATA AVAILABILITY STATEMENT
Data are available in Department of Food Hygiene and Public Health
Protection, Institute ofVeterinaryMedicine,WarsawUniversity of Life
Sciences (SGGW).
ETHICS STATEMENT
No animal was culled or immobilized specifically for this study. Ante-
mortem sampling (e.g. during translocation, putting the collar) was
performed as part of standard veterinary care. The procedures did not
require ethical approval according to II Local Ethical Committee for
Animal Experiments (Warsaw, Poland).
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iley Online Library on [14/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on W
iley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
10 DIDKOWSKA ET AL.
ORCID
AnnaDidkowska https://orcid.org/0000-0003-1073-174X
Elisa FerrerasColino https://orcid.org/0000-0002-4790-5259
WandaOlech https://orcid.org/0000-0002-6166-3954
Krzysztof Anusz https://orcid.org/0000-0002-8116-8491
JoséAntonio Infantes-Lorenzo https://orcid.org/0000-0002-0446-
7682
ChristianGortázar https://orcid.org/0000-0003-0012-4006
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How to cite this article: Didkowska, A., Colino, E. F., Olech,W.,
Gloddy, H., Anusz, K., Infantes-Lorenzo, J. A., & Gortázar, C.
(2023). Antibodies against theMycobacterium tuberculosis
complex and Brucella spp. in captive and free-living European
bison (Bison bonasus) in Poland. Veterinary Medicine and Science,
1–12. https://doi.org/10.1002/vms3.1314
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iley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License