First Molecular Detection of Bartonella bovis and Bartonella schoenbuchensis in European Bison (Bison bonasus)

Simple Summary The European bison is the largest ruminant in Europe. The population of European bison in Lithuania is increasing and several free-ranging herds exist. Owing to their protected status, studies of vector-borne pathogens in European bison are still lacking. By analyzing European bison and ticks collected from them in Lithuania, we checked for the presence of Bartonella spp. and found a low frequency (7.9%) of positive animals. This study showed that European bison may be infected by at least two Bartonella species, namely B. bovis and B. schoenbuchensis. Our results demonstrated that further research is needed to determine the distribution of Bartonella species in wild and domestic ruminants, and it is important to identify the transmission route. Abstract Bartonella bacteria infect the erythrocytes and endothelial cells of mammalians. The spread of the Bartonella infection occurs mainly via bloodsucking arthropod vectors. Studies on Bartonella infection in European bison, the largest wild ruminant in Europe, are lacking. They are needed to clarify their role in the maintenance and transmission of Bartonella spp. The aim of this study was to investigate the presence of the Bartonella pathogen in European bison and their ticks in Lithuania. A total of 38 spleen samples from bison and 258 ticks belonging to the Ixodes ricinus and Dermacentor reticulatus species were examined. The bison and tick samples were subjected to ssrA, 16S–23S rRNA ITS, gltA, and rpoB partial gene fragment amplification using various variants of PCR. Bartonella DNA was detected in 7.9% of the tissue samples of European bison. All tick samples were negative for Bartonella spp. The phylogenetic analysis of 16S–23S rRNA ITS, gltA, and rpoB partial gene fragment revealed that European bison were infected by B. bovis (2.6%) and B. schoenbuchensis (5.3%). This is the first report addressing the occurrence of B. bovis and B. schoenbuchensis in European bison in Europe.

In Lithuania, various species of Bartonella have been detected in domestic and wild animals and their ectoparasites. Bartonella grahamii, B. tailorii, B. rochalimae, B. tribocorum, B. coopersplainsensis, B. doshiae, and B. washoensis have been identified in several species of small rodents and in the fleas, ticks or mites collected from them [10][11][12][13]. Furthermore, B. henselae, B. clarridgeiae and the Bartonella sp., closely related to B. schoenbuchensis have been detected in domestic cats and their fleas [14]. However, there is no data on the presence of Bartonella pathogens in domestic or wild ruminants in Lithuania.
The European bison (Bison bonasus), the largest ruminant in Europe, is known for its status as an animal under species protection. Recent studies have demonstrated that bison may play a role as a natural reservoir of Anaplasma phagocytophilum, Babesia divergens, and Babesia venatorum [15,16]. However, there is no information about Bartonella infection in European bison in Europe. In the present study, we aimed to investigate the presence of the Bartonella pathogen in European bison and their ticks (Ixodes ricinus and Dermacentor reticulatus) in Lithuania using real-time PCR targeting the ssrA gene, and to characterize Bartonella strains by the PCR and sequence analysis of the 16S-23S rRNA ITS (the 16S-23S rRNA intergenic species region), gltA (citrate synthase gene) and rpoB (RNA polymerase β-subunit) genes.

Sample Collecting
A total of 38 spleen samples of European bison (21 males and 17 females) were collected in the period 2019 to 2022 in Central and Northern Lithuania (Kėdainiai and Panevėžys regions). Bison samples were collected in all seasons (n = 4 spring, n = 1 summer, n = 11 autumn, and n = 22 winter). Due to the status of animals under species protection, the tissue samples of European bison were taken from animals found dead in the field (n = 6), accidentally killed on roads (n = 10), and eliminated from nature under a protection plan due to diseases or genetic disorders (n = 22). The bison sampling was conducted with permission from the Environmental Protection Department under the Ministry of Environment (permit No. AAA 2019-04-01 use protected species No. 12, in accordance with the protection plan for Bison bonasus L.). A total of 258 ticks were collected from 6 animals that had died due to unknown causes. Ticks were placed in 1.5 mL tubes with 70% ethanol and kept at 4 • C until investigation. Tick species were identified based on morphological criteria [17]. Two species of ticks were identified: Ixodes ricinus (40 male, 88 female, and 1 nymph) and Dermacentor reticulatus (95 male, 34 female). A total of 116 ticks were engorged (85 I. ricinus and 31 D. reticulatus) and 143 non-engorged (45 I. ricinus and 98 D. reticulatus).

DNA Extraction and PCR Amplification
Genomic DNA was extracted from bison spleens and engorged ticks using a Genomic DNA Purification Kit (Thermo Fisher Scientific, Vilnius, Lithuania) according to the manufacturer's recommendations. DNA from non-engorged ticks was extracted using 2.5% ammonium hydroxide [18].
Screening for the presence of Bartonella DNA (124 bp product of ssrA gene) was conducted by using TaqMan real-time PCR with ssrA-F1 and ssrA-R1 primers and a ssrA-P1 probe, as previously described [11]. Bartonella-positive samples in real-time PCR were further analyzed using nested PCR assays that amplify partial sequences of the 16S-23S rRNA ITS region (external primers WITS-F and WITS-R; internal primers Bh311-332F and ITS-R) and conventional PCR assays of gltA (primers BhCS.781p and BhCS.1137n) and rpoB (primers 1400 F and 2300 R) genes [19][20][21][22] (Table S1). Positive (DNA of Bartonella-infected rodents, confirmed by sequencing) and negative (sterile, double-distilled water) controls were included in each PCR run. PCR products were identified by electrophoresis on 1.5 % agarose gel.

DNA Sequencing and Sequence Analysis
PCR products of all Bartonella-positive samples were extracted from agarose gel and purified using the GeneJET™ Gel Extraction Kit (Thermo Fisher Scientific, Vilnius, Lithuania). The obtained partial 16S-23S rRNA ITS region, gltA, and rpoB gene sequences were analyzed using the MegaX software package [23] and were aligned with the previously published sequences in GenBank using BLASTn. The phylogenetic trees were constructed using the maximum-likelihood method and Tamura-Nei model. Partial 16S-23S rRNA ITS region, gltA, and rpoB gene sequences for representative samples obtained in this study were submitted to the GenBank database under accession numbers OP888096 for the ITS region, OP894362-OP894364 for the gltA gene, and OP894359-OP894361 for the rpoB gene.

Frequency of Bartonella Infection
Bartonella DNA was detected in three out of the thirty-eight (7.9%) tissue samples of European bison. Positive results were obtained in each animal when applying all three PCR techniques (real-time PCR, and conventional and nested PCR). The Bartonella infection was detected in samples collected during winter. All ticks (n = 258) collected from the six European bison specimens not infected with Bartonella were negative for Bartonella spp. All positive samples were subjected to sequence analysis.

16. S-23S rRNA ITS Region
Only one sample of European bison demonstrated the partial 16S-23S rRNA ITS fragment. The obtained sequence (GenBank: OP888096) was 100% identical to B. bovis sequences isolated from domestic cattle from France, Guatemala, and Israel (GenBank: KF218230, KF218232, KM371094) ( Figure 1). Animals 2022, 12, x Lithuania). The obtained partial 16S-23S rRNA ITS region, gltA, and rpoB ge were analyzed using the MegaX software package [23] and were aligned w ously published sequences in GenBank using BLASTn. The phylogenetic tr structed using the maximum-likelihood method and Tamura-Nei model. Partial 16S-23S rRNA ITS region, gltA, and rpoB gene sequences for r samples obtained in this study were submitted to the GenBank database un numbers OP888096 for the ITS region, OP894362-OP894364 for the glt OP894359-OP894361 for the rpoB gene.

Frequency of Bartonella Infection
Bartonella DNA was detected in three out of the thirty-eight (7.9%) tiss European bison. Positive results were obtained in each animal when appl PCR techniques (real-time PCR, and conventional and nested PCR). The Ba tion was detected in samples collected during winter. All ticks (n = 258) colle six European bison specimens not infected with Bartonella were negative for B All positive samples were subjected to sequence analysis.

gltA Gene
In total, three gltA partial gene fragments were obtained from three Eu samples. The sequence analysis revealed that the Bartonella strain (GenBan derived from one individual European bison clustered with B. bovis isolate from various countries (GenBank: KJ909846, MN615927, JX094278) with an 100%. The other two sequences (GenBank: OP894363, OP894364) demonst

gltA Gene
In total, three gltA partial gene fragments were obtained from three European bison samples. The sequence analysis revealed that the Bartonella strain (GenBank: OP894362) derived from one individual European bison clustered with B. bovis isolated from cattle from various countries (GenBank: KJ909846, MN615927, JX094278) with an identity score 100%. The other two sequences (GenBank: OP894363, OP894364) demonstrated a 100% similarity with B. schoenbuchensis isolated from roe deer (GenBank: AJ278183, CP019789, FN645507), deer ked from Germany (GenBank: AJ564632), and a human sample from France (GenBank: HG977196) (Figure 2).

rpoB Gene
The rpoB gene sequence (GenBank: OP894361) clustered with other B. bovis sequences. The highest similarity (99.9%) was shared with B. bovis, previously isolated from cattle from France (GenBank: KF218217). Meanwhile, two sequences derived from two bison specimens (GenBank: OP894659, OP894360) were 99.1% similar to the B. schoenbuchensis sequences isolated from roe deer from Germany (GenBank: CP019789) and human (GenBank: HG977196), differing at six nucleotide positions (Figure 3). Additionally, these sequences shared a 98.5% similarity with B. capreoli isolated from red deer from USA (GenBank: HM167505), differing at fifteen nucleotide positions.

Discussion
This study reports the first molecular detection of B. bovis and B. schoenbuchensis in European bison in Europe. Furthermore, we tested I. ricinus and D. reticulatus ticks collected from bison and did not detect the presence of Bartonella species. The role of ticks in the transmission of Bartonella is not fully understood [4,6]. Other vectors such as deer ked and other fly species may be involved in the transmission of Bartonella spp. in ruminants [6,24].
Bartonella bovis is mainly detected in cattle. Infection with B. bovis is usually asymptomatic, but it is known that it can be associated with bovine endocarditis [4,9]. The prevalence of B. bovis in domestic cattle ranges from 7% to 36% in Europe and from 5% to 90% in other continents [4,6,[25][26][27]. Furthermore, B. bovis infections also have been reported in wild ruminants such as moose, red deer, and roe deer [28,29]. A recent study reported that B. bovis DNA has been identified in 3.2% of ticks collected from cattle [6]. Meanwhile, in our study, Bartonella infection in ticks has not been detected. This study presents a low frequency of B. bovis (2.6%) in European bison in Lithuania. A previous study conducted in Poland has shown that 6.8% of cattle (Bos taurus), a closely related species to European bison, had asymptomatic B. bovis infection [26]. In order to estimate the prevalence of Bartonella in Lithuanian ruminants, an epidemiological study with a larger sample size is needed.
The present study revealed that two individuals of European bison (5.3%) were infected with B. schoenbuchensis. This result is not unexpected because previous studies reported cases of B. schoenbuchensis in domestic and wild ruminants from Georgia, Poland, France, and Norway [4,28,30,31]. Moreover, this species was determined in the deer keds collected from wild ruminants [31,32]. According to other studies, Bartonella spp. highly similar to B. capreoli, B. chomelii, and B. schoenbuchensis had been identified in deer ked distribution areas [29,31,33]. That indicates a potential role of deer ked for the transmission of Bartonella species. Furthermore, outside the deer ked distribution area, other vectors such as ticks or culicoides biting midges could also be involved in Bartonella transmission [29,34]. European bison are gregarious animals that live and travel in herds. Such a lifestyle provides a possibility for blood-sucking vectors to have a greater effect on the transmission of the diseases [35].

Discussion
This study reports the first molecular detection of B. bovis and B. schoenbuchensis in European bison in Europe. Furthermore, we tested I. ricinus and D. reticulatus ticks collected from bison and did not detect the presence of Bartonella species. The role of ticks in the transmission of Bartonella is not fully understood [4,6]. Other vectors such as deer ked and other fly species may be involved in the transmission of Bartonella spp. in ruminants [6,24].
Bartonella bovis is mainly detected in cattle. Infection with B. bovis is usually asymptomatic, but it is known that it can be associated with bovine endocarditis [4,9]. The prevalence of B. bovis in domestic cattle ranges from 7% to 36% in Europe and from 5% to 90% in other continents [4,6,[25][26][27]. Furthermore, B. bovis infections also have been reported in wild ruminants such as moose, red deer, and roe deer [28,29]. A recent study reported that B. bovis DNA has been identified in 3.2% of ticks collected from cattle [6]. Meanwhile, in our study, Bartonella infection in ticks has not been detected. This study presents a low frequency of B. bovis (2.6%) in European bison in Lithuania. A previous study conducted in Poland has shown that 6.8% of cattle (Bos taurus), a closely related species to European bison, had asymptomatic B. bovis infection [26]. In order to estimate the prevalence of Bartonella in Lithuanian ruminants, an epidemiological study with a larger sample size is needed.
The present study revealed that two individuals of European bison (5.3%) were infected with B. schoenbuchensis. This result is not unexpected because previous studies reported cases of B. schoenbuchensis in domestic and wild ruminants from Georgia, Poland, France, and Norway [4,28,30,31]. Moreover, this species was determined in the deer keds collected from wild ruminants [31,32]. According to other studies, Bartonella spp. highly similar to B. capreoli, B. chomelii, and B. schoenbuchensis had been identified in deer ked distribution areas [29,31,33]. That indicates a potential role of deer ked for the transmission of Bartonella species. Furthermore, outside the deer ked distribution area, other vectors such as ticks or culicoides biting midges could also be involved in Bartonella transmission [29,34]. European bison are gregarious animals that live and travel in herds. Such a lifestyle provides a possibility for blood-sucking vectors to have a greater effect on the transmission of the diseases [35].

Conclusions
The results of this study suggest that European bison may be infected by at least two Bartonella species, namely B. bovis and B. schoenbuchensis. Further research is needed to determine the vectors of transmission of Bartonella species among bison and other ruminant species.