Introduction
The global population of European bison (Bison bonasus) currently stands at over 10 000 individuals, with the largest groups inhabiting Poland, where the population was estimated at over 2300 free-living individuals and over 200 animals kept in captivity at the end of 2022 (Raczyński, Reference Raczyński2023). However, the species remains near-threatened (ICUN), with its main threats being infectious diseases and parasite infestation (Kita and Anusz, Reference Kita and Anusz2006; Krasińska and Krasiński, Reference Krasińska and Krasiński2013). European bison are known to host a wide diversity of parasites: studies have recorded 88 species, with many being typical for cattle, small ruminants and cervids, but also few species specific to European bison (Karbowiak et al., Reference Karbowiak, Demiaszkiewicz, Pyziel, Wita, Moskwa, Werszko, Bień, Goździk, Lachowicz and Cabaj2014a). Of these, the most numerous group in European bison are the nematodes, represented by 43 species, with gastrointestinal nematodes (GINs) being the most prevalent (Karbowiak et al., Reference Karbowiak, Demiaszkiewicz, Pyziel, Wita, Moskwa, Werszko, Bień, Goździk, Lachowicz and Cabaj2014b). Among the GINs, the blood-sucking nematodes of the Haemonchinae subfamily are considered to be of considerable pathogenic and economic importance in both domestic and wild ruminants worldwide (Osińska et al., Reference Osińska, Demiaszkiewicz and Lachowicz2010; Gilleard, Reference Gilleard2013); 2 taxa of note include the alien species Ashworthius sidemi, brought to Poland with Asiatic deer, and Haemonchus contortus, which quickly develops resistance to anthelmintics used in its control (Gilleard, Reference Gilleard2013; Demiaszkiewicz et al., Reference Demiaszkiewicz, Merta, Kobielski, Filip and Pyziel2017; Pyziel et al., Reference Pyziel, Björck, Wiklund, Skarin, Demiaszkiewicz and Höglund2018). Serious infestation can be fatal for the host, due to the blood loss from the mucosal lesions associated with parasite feeding causing haemorrhagic anaemia (Gilleard, Reference Gilleard2013; Kołodziej-Sobocińska et al., Reference Kołodziej-Sobocińska, Demiaszkiewicz, Lachowicz, Borowik and Kowalczyk2016a, Reference Kołodziej-Sobocińska, Demiaszkiewicz, Pyziel, Marczuk and Kowalczyk2016b).
While the morphological aspects of the parasitofauna of free-ranging European bison are relatively well understood, little molecular data exists regarding their GINs. Also, little is known of the diversity of abomasal nematodes of captive wisents, as most studies of parasitic infections in European bison concern free-roaming individuals. In fact, these are the captive wisents that serve as a genetic reservoir for the species, and are more often translocated, i.e. between breeding centres, zoos or enclosures in Poland and abroad. Furthermore, while considerable data concerning the morphological identification of GINs is available for many ruminant species (Lichtenfels and Pilitt, Reference Lichtenfels and Pilitt1991; Pyziel-Serafin et al., Reference Pyziel-Serafin, Vetter, Klich and Anusz2023), the morphology of the GIN flora of European bison remains sparse. A thorough understanding of the composition of the parasitofauna of captive animals will help limit the health risks associated with translocation of animals, prevent the spread of alien parasite species and monitor the efficacy of deworming of captive animals (Vadlejch et al., Reference Vadlejch, Kyriánová, Rylková, Zikmund and Langrová2017; Gałązka et al., Reference Gałązka, Klich, Anusz and Pyziel-Serafin2022, Reference Gałązka, Klich, Filip-Hutsch, Olech, Anusz and Pyziel2023).
The present study examines the infection level and composition of GINs in the abomasa of European bison, and compares the nematode species present in captive European bison with those in free-ranging animals. It also describes the morphometric characteristics of the identified nematodes to enable more accurate morphological diagnosis.
Materials and methods
Material collection and area of the study
Abomasa were collected during post-mortem examinations of 30 European bison, viz. 23 captive and 7 free-ranging individuals, in Poland during the years 2018–2023. All animals had died due to natural causes or culling for breeding and health reasons.
The examined captive animals were kept in 4 breeding centres (Smardzewice Breeding Center, Niepołomice Breeding Center, Pszczyna-Jankowice Breeding Center and Wolisko Breeding Center), 3 zoos (Bydgoszcz zoo, Warsaw zoo and Ustroń zoo) and 2 enclosures (Kiermusy enclosure and Muczne enclosure) (Fig. 1). The free-ranging animals inhabited the Borecka Forest, Białowieska Forest, Knyszyńska Forest and Bieszczady Mountains (Fig. 1). Three management types were considered in the study: enclosures and zoos with smaller paddock area; breeding centres with larger pastures and free-living herds (Table 1). Detailed information about the number of animals examined in each location is available in the online Supplement (Tables S1, S2).
Location of examined European bison in Poland
Prevalence of abomasal nematodes of examined European bison according to management type
n, number of examined animals.
Parasitological dissections were performed in the field, according to the principles of dissecting technique and parasitological procedures (Dróżdż, Reference Dróżdż1966; Malicka, Reference Malicka2008). In each case, the abomasa were removed from the abdominal cavity, ligated at both ends and secured for examination. The material was transported to the laboratory and examined immediately, or frozen at −20°C and examined after thawing.
Each abomasum was placed in standing water. Its wall was cut along the greater curvature and the contents were then decantated and a sample of 1/10 of the total volume of the sediment was prepared. The sediment was poured into Petri dishes (in portions) and investigated under a Delta Optical SZ-450 T stereomicroscope (Delta Optical, Mińsk Mazowiecki, Poland) using 40 × magnification. The observed GINs were isolated with a use of a dissecting needle and counted. Afterwards, the male and female individuals were separated and each group was preserved in 70% ethanol.
Morphological identification of nematodes
Male nematodes were isolated from 1/10 of the abomasal contents. These were used for morphological species identification due to the species-specific structure of the copulatory bursa. The caudal part of the body was cut off with scalpel and placed in Amman's lactophenol (10 g of phenol + 10 g of lactic acid + 20 g of glycerol + 10 g of distilled water) for 20 min to increase the translucency of the cuticle and improve the visibility of the spicules. The rest of the body was placed in a labelled vial filled with 70% ethanol for subsequent species verification using molecular biology techniques. When removed from lactophenol, the caudal body parts of male nematodes were placed on a basal slide and identified to the species level; identification was based on the shape and length of the spicules and copulatory sac under a LAB40 microscope (OPTA-TECH, Warsaw, Poland) under 100 × to 400 × magnification (Dróżdż, Reference Dróżdż1966, Reference Dróżdż1995; Dróżdż et al., Reference Dróżdż, Demiaszkiewicz and Lachowicz1998). The spicules were photographed and measured with a digital camera and OPTA View-15 2019 software (OPTA-TECH). The female nematodes were identified to the subfamily level and counted.
Molecular identification of nematodes
Genomic DNA was extracted individually from the ethanol-preserved anterior male parts of A. sidemi, Cooperia oncophora, H. contortus, O. leptospicularis, O. kolchida (a minor morph of O. leptospicularis), O. ostertagi, O. lyrata (a minor morph of O. ostertagi), Spiculopteragia boehmi and Teladorsagia circumcincta. The extraction was performed using a NucleoSpic Tissue DNA extraction kit (Macherey-Nagel, Düren, Germany) according to the manufacturer's protocol.
Identification was performed by PCR using various combinations of primer sets. A partial region of the internal transcribe spacer 2 (ITS-2) and large subunit (LSU) of the ribosomal DNA was amplified using the following set of primers: forward-NC1 (5′-ACG TCT GGT TCA GGG TTG TT-3′) and reverse-NC2 (5′-TTA GTT TCT TTT CCT CCG CT-3′) according to Gasser et al. (Reference Gasser, Chilton, Hoste and Beveridge1993). Additionally, 2 sets of primers amplifying a partial region of the small subunit ribosomal rDNA (SSU) were designed for the purpose of this study, namely: forward-N380F (5′-AAG CGA GCA GGC GCG AAA C-3′) and reverse-N1690R (5′-ACC CGG TTC AAG CCA TTG CGA-3′); forward-N350F (5′-GAG CCT TAG AAA CGG CTA CCA CAT CCA-3′) and reverse-N1287R (5′-AGC AGG CTA GAG TCT CGC TCG T-3′).
Other combinations of primer sets targeting SSU were also used, including: forward SSU07 (5′-AAA GAT TAA GCC ATG CAT G-3′) and reverse BNR1 (5′-ACC TAC AGA TAC CTT GTT ACG AC-3′); forward SSU07 and reverse N1070R (5′-TTG CAA CCA TAC TAC CCC AGG AAC CGA A-3′); forward N800F (5′-GGG CAT TCG TAT CCC TGC GCG AGA G-3′) and reverse NBR1; forward NF50 (5′-TGA AAC TGC GAA CGG CTC AT-3′) and reverse BNR.
All PCRs were performed in a T100 thermal cycler (Bio-Rad, Hercules, CA, USA) in a volume of 50 μL. Each 50 μL PCR reaction contained 20 μL of Molecular Biology Reagent Water (Sigma-Aldrich, St. Louis, MO, USA), 25 μL of AccuStart II PCR ToughMix (×2 concentation) (Quantabio, Beverly, MA, USA), 1 μL of GelTracl Loading Dye (×50 concentration) (Quantabio), 1 μL of forward primer (20 mm), 1 μL of reverse primer (20 mm), and 2 μL of template DNA. The conditions for PCRs were as follows: 94°C for 2 min to denature the DNA, 35 cycles at 94°C for 45 s, 60°C (for ITS-2 and LSU)/57°C (for SSU) for 60 s, and 72°C for 45 s, and a final extension of 10 min at 72°C to ensure complete amplification.
The PCR products were verified on 1% agarose gel containing ethidium bromide (0.5 μg mL−1), with Gene Ruler 100 bp DNA Ladder (Thermo Fisher Scientific, Waltham, MA, USA) loaded as a reference. The products were identified by exposure to UV light using a Gel Doc XR+ (Bio-Rad) gel documentation system, equipped with Image Lab 6.1 Software (Bio-Rad Laboratories, USA). The PCR products were purified using the NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel), eluted with 30 μL of Molecular Biology Reagent Water (Sigma-Aldrich) and sequenced in both directions by Genomed S.A. (Warsaw, Poland) using the primers previously used for amplification (5 mm). The sequences were assembled into contigs using CodonCode Aligner version 8.0 (CodonCode Corporation, Centerville, MA, USA). The obtained nucleotide sequences were compared to the NCBI database of sequences using the basic local alignment search tool (BLAST) (http://www.ncbi.nlm.nih.gov/BLAST/) and submitted to the GenBank database.
Statistical analysis
Due to the low intensity of nematode infection in the examined European bison, statistical analysis allowing for the identification of factors explaining the intensity of nematode infection was limited. Therefore, the analysis was restricted to 2 areas. The first evaluated the number of nematode species according to European bison maintenance type (enclosures (including zoos), breeding centres and free-living herds) using the Kruskall-Wallis test with Dunn's post-hoc test with Bonferroni correction.
In addition, the number of nematodes of each selected nematode species was analysed. Briefly, after analysing the number of nematodes present, 3 species were subjected for detailed statistical analysis: H. contortus, O. ostertagi and O. lyrata. The numbers of these species were assessed for collinearity using Pearson's r and Kendall's b correlation coefficients. Following this, the number of nematodes in each species was analysed separately based on a generalized linear model with a negative binomial distribution and a log link function. The number of nematodes in the European bison was used as the dependent variable, while bison sex, maintenance type [enclosures (including zoos), breeding centres and free-living herds], and northern gradient were used as explanatory variables. The northern gradient variable was introduced into the analysis to account for the effect of the north-south gradient on the occurrence of various parasite species (e.g. Thieltges et al., Reference Thieltges, Hof, Dehling, Brändle, Brandl and Poulin2011; Klich et al., Reference Klich, Nowicki, Didkowska, Bełkot, Popczyk, Wiśniewski and Anusz2022) and the distribution of samples from the north to the south of Poland (Fig. 1). Each model underwent a selection process comparing all model variants (variable composition, including a null model), and the highest ranked model was selected based on the Akaike information criterion, i.e. the highest AIC value (Burnham and Anderson, Reference Burnham and Anderson2002). All statistical analyses were performed with IBM SPSS v29.0 (Armonk, New York).
Results
Morphological identification of nematodes
In the study, 11 GIN species were identified: H. contortus, A. sidemi, O. ostertagi, O. lyrata (a minor morph of O. ostertagi), O. leptocpicularis, O. kolchida (a minor morph of O. leptospicularis), T. circumcinta, C. oncophora, C. surnabada, S. boehmi and S. asymmetrica (Fig. 2). Among these, H. contortus, A. sidemi, O. ostertagi, O. lyrata, O. kolchida, C. oncophora, S. boehmi and S. asymmetrica were noted in both free-roaming and captive animals. However, T. circumcincta and C. surnabada were exclusively found in captive bison and O. leptospicularis in free-roaming animals. The overall prevalence of GIN infection in examined European bison was 90% (i.e. 27 positive out of 30 examined abomasa). The median and mean intensity of GIN infection were respectively 725 and 5065.7 per animal (range 0–47 030 nematodes per animal). The general prevalence was the highest in breeding centres (100%), followed by free-ranging herds (85.7%) and enclosures including zoos (77.8%).
Male caudal body parts of abomasal nematodes found in European bison: 1 – Ashworthius sidemi (×10); 2 – Haemonchus contortus (×10); 3 a, b – Ostertagia ostertagi (×20); 4 a, b– Ostertagia lyrata (×20); 5 – Ostertagia leptospicularis (×20); 6 – Ostertagia kolchida (×20); 7 a, b– Teladorsagia circumcincta (×10); 8 – Cooperia oncophora (×20); 9 – Cooperia surnabada (×20); 10 a, b – Spiculopteragia boehmi (×20); 11 – Spiculopteragia asymmetrica (×20).
The most common, and also most abundant, species was Ostertagia ostertagi (prevalence: 80%), followed by H. contortus, O. lyrata and O. kolchida (prevalence: 63.3, 43.3 and 30%, respectively) (Table 1). The rarest nematode species was C. surnabada (prevalence: 3.3%) (Table 1). Regarding maintenance type, O. ostertagi and H. contortus were found to have similar prevalences in free-roaming and captive European bison (both types of maintenance), whereas the greatest difference between maintenance types was noted for A. sidemi: the second most prevalent species in free-ranging animals (prevalence: 57.1%) but one of the rarest in captive wisents (prevalence in enclosures and zoos 11.1%, breeding centres 0%) (Table 1).
GIN composition was found to be related to the management type of the European bison. Ashworthius sidemi occurred in most free-ranging herds, but was noted in only one enclosure, where the species was also present in the wild population (Table 1, online Supplementary Table S1). Teladorsagia circumcincta and C. surnabada were only found in captive animals; O. leptospicularis was noted only in free-ranging animals; all other GINs were present in both captive and free-ranging animals. In addition, mixed infections comprising 2 to 8 GIN species were noted in 25 cases; however, no relationship was found between the number of species and the type of maintenance.
The parasite loads differed regarding the maintenance type. The European bison in breeding centres presented higher intensity of infection with GINs than in enclosures and zoos (Fig. 3). Free ranging animals did not differ significantly from those in enclosures and zoos. Regarding the most prevalent GINs, the highest average and median values, as well as the widest range of specimens, were observed for O. ostertagi in breeding centres (351.1; 87.5; 10–21 230, respectively). In free-ranging European bison the highest intensity of infection was noted also for O. ostertagi (40–800), with the highest average and median (25.6 and 5, respectively) (Table 2).
Boxplot of intensity of infection with GINs in European bison with regard to maintenance type. In Kruskall–Wallis test only animals in breeding centres presented higher intensity of infection with GINs than in enclosures and zoos (enclosures) (Dunn's test statistic = 10.38, P = 0.017). Free ranging animals didn't differ significantly from those in enclosures and zoos (Dunn's test statistic = 5.67, P = 0.604) nor from breeding centres (Dunn's test statistic = 4.71, P = 0.741, for Kruskal–Wallis test: P = 0.022) (n, number of examined animals).
Intensity of abomasal nematodes of examined European bison according to management type
n, number of examined animals.
a Nematodes found only in 1 examined European bison.
Ostertagia ostertagi and O. lyrata were found at significantly higher intensities in animals kept in breeding centres than in enclosures or in wild herds (Figs 4 and 5), but such a difference was not observed in H. contortus (online Supplementary Table S3). Free ranging animals did not differ significantly from animals in enclosures for all 3 nematodes species. Haemonchus contortus was present at significantly higher intensity in cows than in bulls (Fig. 6), but for O. ostertagi and O. lyrata no sex differences were found (online Supplementary Table S3). Interestingly, the intensity of H. contortus infection decreased as the location became more northerly, regardless of maintenance type; for O. ostertagi and O. lyrata northern gradient was not significant in the model (online Supplementary Table S3). More detailed data about the prevalence and intensity of parasitic infections in the examined European bison in all locations is given in the online Supplementary material (Tables S1 and S2).
Mean (±s.e.) number of O. ostertagi specimens in European bison with regard to maintenance type. Animals in breeding centres differed significantly from those in enclosures and free-ranging animals (P < 0.001 in both cases) in generalized linear model. Free ranging animals did not differ significantly from animals in enclosures (P = 0.336) (for whole model: χ 2 = 50.81, P < 0.001) (n, number of examined animals).
Mean (±s.e.) number of O. lyrata specimens in European bison with regard to maintenance type. Animals in breeding centres differed significantly from those in enclosures and free-ranging animals (P < 0.01 in both cases) in generalized linear model. Free ranging animals did not differ significantly from animals in enclosures (P = 0.529) (for whole model: χ 2 = 54.18, P < 0.001) (n, number of examined animals).
Mean (±s.e.) number of H. contortus specimens in European bison with regard to sex of animals. Females differed significantly from males (P = 0.039) in generalized linear model (for whole model: χ 2 = 26.51, P < 0.001) (n, number of examined animals).
Nematode species were identified based on the morphology and length of the male copulatory bursa and the spicules; the morphological differences between H. contortus and A. sidemi can be seen in Fig. 2. In H. contortus, the lateral rib is clearly visible in the spicules. This difference allows accurate identification of this species by light microscopy, as confirmed by our present molecular analysis (Fig. 2.2). However, all species demonstrated a wide range of spicule lengths. In A. sidemi, T. circumcinta and S. boehmi, the median spicule length was significantly higher than the mean length due to the occurrence of significantly lower values (Table 3).
Comparison of the spicule length of abomasal nematodes of European bison
s.d., standard deviation.
Molecular identification of nematodes
Nucleotide sequences were obtained for 5 species of abomasal nematodes, including A. sidemi, H. contortus, O. ostertagi, O. kolchida and S. boehmi (Table 4). None of the primer sets used in the investigation provided a PCR product for all nematode species included in the study. The following primer pars proved to be completely unsuccessful for the purpose of this research: SSU07 + BNR1, SSU07 + N1070R, N800F + NBR1 and NF50 + BNR1. The reason for this is the fact that no PCR product was produced by these sets of primers. However, the remaining primer sets were selectively useful, i.e. they could accurately identify only certain nematode species.
Obtained nucleotide sequences of abomasal nematodes of captive European bison
*NC1: 5′-ACGTCTGGTTCAGGGTTGTT-3′; NC2: 5′-TTAGTTTCTTTTCCTCCGCT-3′; N380F: 5′-AAGCGAGCAGGCGCGAAAC-3′; N1690R: 5′-ACCCGGTTCAAGCCATTGCGA-3′; N350F: 5′-GAGCCTTAGAAACGGCTACCACATCCA-3′; N1287R: 5′-AGCAGGCTAGAGTCTCGCTCGT-3′
The general primer set NC1 + NC2 obtained sequences for ITS2 and LSU from A. sidemi, H. contortus and O. ostertagi; the products varied in length from 239 to 257 bp. Partial SSU sequences were obtained for A. sidemi, H. contortus, O. kolchida and S. boehmi; these varied in length from 830 bp (for O. kolchida) to 1211 bp (for S. boehmi). The 350F + N1287R set was useful for only O. kolchida, whereas primer set 380F + N1690R obtained sequences from the 3 remaining nematode species.
Discussion
In total, 11 GIN species of the family Trichostrongylidae were isolated from the alimentary tracts of European bison. Although our study concerned mostly captive animals, the GIN species composition was similar to that of free-living European bison obtained by other authors worldwide (Dróżdż et al., Reference Dróżdż, Demiaszkiewicz and Lachowicz1994; Karbowiak et al., Reference Karbowiak, Demiaszkiewicz, Pyziel, Wita, Moskwa, Werszko, Bień, Goździk, Lachowicz and Cabaj2014a, Reference Karbowiak, Demiaszkiewicz, Pyziel, Wita, Moskwa, Werszko, Bień, Goździk, Lachowicz and Cabaj2014b; Demiaszkiewicz et al., Reference Demiaszkiewicz, Pyziel, Filip-Hutsch, Januszczak and Olech2020). GINs are the most significant parasites of European bison and other grazing ruminants (Karbowiak et al., Reference Karbowiak, Demiaszkiewicz, Pyziel, Wita, Moskwa, Werszko, Bień, Goździk, Lachowicz and Cabaj2014b) and the general prevalence of parasitic infections in our research was high, reaching 90%.
Our morphometric analysis found that most GIN species demonstrated high size ranges, and this corresponded with values obtained in other ruminant species (Lichtenfels and Pilitt, Reference Lichtenfels and Pilitt1991; Jacquiet et al., Reference Jacquiet, Cabaret, Cheikh and Thiam1997; Pyziel-Serafin et al., Reference Pyziel-Serafin, Vetter, Klich and Anusz2023). The morphological identification was made possible by the structure of the nematode spicules, and these results were confirmed in the molecular analysis. Our results represent the first available set of specular lengths of GINs in European bison. Nevertheless, due to the limited sample size the descriptions may not be comprehensive for all possible sizes and it is advised to perform further measurements of nematode spicules.
Unfortunately, limited data is available on the nucleotide sequences of abomasal nematodes of ruminants, and as it was not possible to acquire many of the sequences of the SSU and ITS genetic markers in the studied GIN from European bison. Even so, a nucleotide sequence was acquired from O. kolchida (GenBank: OP320518.1), and this is the first to be made available in GenBank regarding any genetic marker gene of the species. Similarly, the obtained SSU sequence of S. boehmi (GenBank: OP320506.1) is the first SSU sequence of the species available, and first molecular data from S. boehmi isolated from European bison. In addition, the acquired second sequence of ITS2 and LSU of O. ostertagi (GenBank: OP787667.1) is the second to be derived from European bison. The previous molecular data on the species were obtained from Lowland-Caucasian wisent from Avesta Visentpark in Sweden (GenBank: KX358862.1) (Pyziel et al., Reference Pyziel, Björck, Wiklund, Skarin, Demiaszkiewicz and Höglund2018).
In contrast, sequences of blood-sucking H. contortus and A. sidemi are more available. In addition to the data on H. contortus obtained in this study (GenBank: OP577478.1, OP320502.1), sequences have been derived from European bison inhabiting Almindingen in Danish island, Bornholm (GenBank: ON677956.1, ON677957.1, ON677958.1) and Avesta Visentpark in Sweden (GenBank: KX358860.1). Our data regarding the molecular features of an alien A. sidemi isolated from European bison are the third set to be published (GenBank: OP787564.1, OP320470.1). This issue was discussed previously by Moskwa et al. (Reference Moskwa, Bień, Goździk and Cabaj2014) in Poland (GenBank: KF414629.1) and by Vadlejch et al. (Reference Vadlejch, Kyriánová, Rylková, Zikmund and Langrová2017) in the Czech Republic (GenBank: KX228148.1, KX228149.1).
The most prevalent GIN was O. ostertagi, a parasite typical for bovids and considered one of the most common GINs of European bison. It has been observed in both captive and free-living animals since the beginning of the European bison restitution (Karbowiak et al., Reference Karbowiak, Demiaszkiewicz, Pyziel, Wita, Moskwa, Werszko, Bień, Goździk, Lachowicz and Cabaj2014b). Other parasites typical of bovines, like H. contortus and O. lyrata were also very common, followed by O. kolchida, characteristic for cervids. Indeed, during its reintroduction, the European bison has become a host for many parasite species originally characteristic for other mammals (Dróżdż et al., Reference Dróżdż, Demiaszkiewicz and Lachowicz1998, Reference Dróżdż, Demiaszkiewicz and Lachowicz2002), which is consistent with our study.
As the development and spread of parasitic infections are affected by many environmental and individual factors (Kołodziej-Sobocińska, Reference Kołodziej-Sobocińska2019), some differences between captive and free-living animals might be expected. In our study, 2 nematode species, T. circumcincta and C. surnabada, were isolated only from European bison in captivity. Both nematodes are considered typical parasites of domestic ruminants and have been rarely reported in free-living animals (Demiaszkiewicz and Pyziel, Reference Demiaszkiewicz, Pyziel, Kowalczyk, Ławreszuk and Wójcik2010; Karbowiak et al., Reference Karbowiak, Demiaszkiewicz, Pyziel, Wita, Moskwa, Werszko, Bień, Goździk, Lachowicz and Cabaj2014b). In contrast, O. leptospicularis was not present in captive animals but isolated only from free-living ones. Ostertagia leptospicularis is primarily a parasite of cervids (Wyrobisz-Papiewska et al., Reference Wyrobisz-Papiewska, Kowal, Łopieńska-Biernat, Nosal, Polak, Paukszto and Rehbein2021), but it has been adapted by free-living European bison as a result of inter-species parasite transmission (Dróżdż et al., Reference Dróżdż, Demiaszkiewicz and Lachowicz1989; Karbowiak et al., Reference Karbowiak, Demiaszkiewicz, Pyziel, Wita, Moskwa, Werszko, Bień, Goździk, Lachowicz and Cabaj2014b). Previous studies indicate that the parasitofauna of free-ranging bison might be enriched with species typical for cervids which are rarely present in captive individuals (Dróżdż et al., Reference Dróżdż, Demiaszkiewicz and Lachowicz2002).
Similar differences between captive and free-ranging European bison were observed in relation to nematodes from Haemonchiinae subfamily, including H. contortus and A. sidemi. Ashworthius sidemi was imported to Poland with sika deer (Cervus nippon) from Asia in the end of XXth century. It has since become a common parasite of free-living European bison (Dróżdż et al., Reference Dróżdż, Demiaszkiewicz and Lachowicz1998; Demiaszkiewicz et al., Reference Demiaszkiewicz, Lachowicz and Osińska2009; Kołodziej-Sobocińska et al., Reference Kołodziej-Sobocińska, Demiaszkiewicz, Lachowicz, Borowik and Kowalczyk2016a, Reference Kołodziej-Sobocińska, Demiaszkiewicz, Pyziel, Marczuk and Kowalczyk2016b); however, in the present study, it was isolated only from one individual in enclosure, and the predominant Haemonchinae species in most captive European bison was H. contortus. Other authors attribute the presence of A. sidemi in captive European bison to accidental contact with infected wild ruminants in the study area, as enclosure is not always a sufficient barrier to transmission of infectious diseases (Kowal et al., Reference Kowal, Nosal, Bonczar and Wajdzik2012; Rendón-Franco et al., Reference Rendón-Franco, Romero-Callejas, Villanueva-García, Osorio-Sarabia and Muñoz-García2013; Mazur et al., Reference Mazur, Tkacz, Staniszewski and Barański2018).
Overall, the median and mean intensity of infection (725; 5065.7 nematodes per animal, respectively) were lower than those reported in previous studies of European bison in Poland (Dróżdż et al., Reference Dróżdż, Demiaszkiewicz and Lachowicz1989; Demiaszkiewicz et al., Reference Demiaszkiewicz, Bielecki, Rodo, Pyziel and Filip2018; Kołodziej-Sobocińska et al., Reference Kołodziej-Sobocińska, Demiaszkiewicz, Pyziel and Kowalczyk2018). The lower parasite burden may be related to management type, e.g. captive animals are dewormed biannually to prevent the spread of parasitic disease (Viggers et al., Reference Viggers, Lindenmayer and Spratt1993), while the free-ranging European bison are not receiving anthelmintics due to lack of control over the drug intake and a potential development of anthelmintic resistance. Nonetheless, the infection intensity was higher in animals in breeding centres compared with animals in enclosures and zoos and free-living herds; this may be due to the other factors faced by captive animals, such as higher population density, close proximity to other animals (Gałązka et al., Reference Gałązka, Klich, Filip-Hutsch, Olech, Anusz and Pyziel2023), higher stress and specific environmental conditions (Papini et al., Reference Papini, Girivetto, Marangi, Mancianti and Giangaspero2012). Moreover, herds living in breeding centres were larger than those enclosures and zoos, which may pose a potential difficulty in estimating the body mass of all animals and administering a correct dosage of anthelmintic drugs to a group, which may lead to inefficient deworming and development of drug resistance in parasites (Gałązka et al., Reference Gałązka, Klich, Filip-Hutsch, Olech, Anusz and Pyziel2023). However, only a relatively small number of samples, especially from free-living individuals were examined and further study with emphasis on management type in European bison should be undertaken.
Among the examined European bison, a higher infection intensity of H. contortus was found among females than males; however, this pattern was not noted for other GINs. Female-biased parasitism has been reported in European bison before (Krasińska et al., Reference Krasińska, Krasiński and Bunevich2000; Pyziel et al., Reference Pyziel, Kowalczyk and Demiaszkiewicz2011; Kołodziej-Sobocińska, Reference Kołodziej-Sobocińska2019; Filip-Hutsch et al., Reference Filip-Hutsch, Laskowski, Myczka, Czopowicz, Moskwa and Demiaszkiewicz2022). It has been attributed to the higher exposure of females to parasites due to their living among other animals, which favours transmission of diseases between individuals. Their susceptibility to parasitic infection may also be enhanced by immunosuppression caused by pregnancy and lactation (Lloyd, Reference Lloyd1983).
For one of the most common GINs, H. contortus, infection intensity was found to change depending on the latitude. While other authors also report a tendency for increasing parasite species richness when moving away from the equator, there is no strong evidence indicating that latitude has a direct effect on mammal parasitofauna (Kamiya et al., Reference Kamiya, O'Dwyer, Nakagawa and Poulin2014). It is more likely that affect the latitudinal patterns of helminth diversity are influenced indirectly by several additional factors, such as temperature, precipitation and host availability (Villalobos-Segura et al., Reference Villalobos-Segura, García-Prieto and Rico-Chávez2020). Moreover, most sheep and goat farming occurs in the south of Poland, while more cattle farming occurs in the north, which might be taken under consideration in explanation of the higher intensity of infection with nematodes commonly associated with small ruminants (H. contortus) in the southern enclosure locations (Łączyński et al., Reference Łączyński, Kuliś, Przypaśniak, Wieczorkowski, Dach-Oleszek and Tylkowska-Siek2022). However, this issue is more complex, as indicated by regional differences in the observed contacts between European bison and cattle which were not always corresponding to cattle numbers in given region (Klich et al., Reference Klich, Didkowska, Pyziel-Serafin, Perlińska-Teresiak, Wołoszyn-Gałęza, Żoch, Balcerak and Olech2023). Due to limited access to invasive sampling from endangered species the sampling was not randomized, and since further research ought to be conducted to investigate potential correlations, the sampling protocol requires to consider this methodological aspect in the future.
As captive-bred animals are widely used in reintroduction programmes (Mathews et al., Reference Mathews, Moro, Strachan, Gelling and Buller2006; Kołodziej-Sobocińska et al., Reference Kołodziej-Sobocińska, Demiaszkiewicz, Pyziel and Kowalczyk2018), it is especially important to monitor their health status. Despite this, few studies have examined the extent and nature of parasitic infections in captive European bison. Our study has implications for managing the captive European bison reintroduction process. Although individuals from breeding centres are better adapted to natural conditions, they may constitute a significant reservoir of nematodes. However, the intensity of GIN infection is not high in all individuals, so a preliminary assessment of this intensity should be carried out before selecting specimens for transportation. Moreover, animals in breeding centres may be reservoirs of nematode species that occur in the local environment, even though they do not live in the wild. Therefore, if a species of nematode unknown to the target place for reintroduction was found in the environment in the source region a thorough analysis of the nematode species composition of animals from breeding centre before transportation is highly justified. This study provides new data about GINs in captive European bison in Poland, which might simplify the morphometrical and molecular identification of Trichostrongylidae species, and help develop new management strategies for the European bison population, such as, preventing from transporting alien or potentially threatening species to new locations and free-living populations.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S003118202400088X
Acknowledgements
The authors would like to thank the following individuals for assistance throughout all aspects of our study: Wojciech Bielecki, Mieczysław Hławiczka, Stanisław Kaczor, Elwira Plis-Kuprianowicz, Zbigniew Pawliński, Patryk Gałązka. This study was conducted as part of the Doctoral thesis of Mrs. Marta Gałązka.
Authors’ contributions
MG conceived and designed the study, collected the abomasa of European bison, conducted molecular investigation. MG, KFH conducted morphological investigation, wrote the article. DK performed statistical analysis and created the visualization. WO, KA supervised the study. AMP conducted molecular investigation, supervised the study and edited the article.
Financial support
This work was supported by the Forest Fund, Poland as part of ‘Complex project of European bison conservation by State Forests’ [contract no. OR.271.3.10.2017].
Competing interests
None.
Ethical standards
No individual was killed for research purposes; samples were only collected on occasion. Culling of European bison was carried out by designated persons and approved by the General Directorate for Environmental Protection in Poland, based on the Act Of 16 April 2004 on The Protection of Nature. All methods were carried out in accordance with relevant guidelines and regulations. Namely, the collection and storage of samples of dead individuals for the study were based on the decision of the Regional Director of Environmental Protection in Warsaw. According to this decision, the collection of dead animals for scientific purposes does not need any permit. Permission was given for the storage of the biological samples of the European bison without any time limit.
Data availability statement
The data that support the findings of this study are available from the corresponding author (MG).
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