Trypanosomes in Eastern and Central European bats

Bats are presumed primary hosts of trypanosomes of the subgenus Schizotrypanum, including the human pathogen Trypanosoma cruzi. As such, research on bat trypanosomes has been focused on South America, where Chagas disease is a serious issue. While the majority of European studies have been performed in the United Kingdom, there is virtually no data available for Eastern and Central parts of Europe. To address this, the present study aims to identify and assess the prevalence and pathogenicity of trypanosomes in bats sampled in the Czech Republic, Bulgaria, and Poland. Blood collected from 381 adult bats of eight species was tested for presence of trypanosomes using nested polymerase chain reactions. To assess possible impacts of trypanosome parasites on the health status of their hosts, haematological and biochemical analyses were performed for 56 greater mouse-eared bats (Myotis myotis) emerging from hibernacula and 36 females of the same species from summer colonies. The overall prevalence of the two trypanosome species detected (T. dionisii and T. vespertilionis) was 27%, with a significantly higher prevalence in the Czech Republic compared to the other countries studied. Significant differences in bat trypanosome prevalence in different European countries appear to be connected with presence or absence of possible vectors in summer roosts. No impact of trypanosomes on haematology and blood chemistry parameters was detected in Trypanosoma-positive greater mouse-eared bats. Though T. dionisii infection in bats appears asymptomatic, long-term health consequences still need to be studied in greater detail. Blood parasites, Schizotrypanum, Trypanosoma dionisii, Trypanosoma vespertilionis, Chiroptera, health status Bats host several trypanosome species of the subgenus Schizotrypanum, including the important human pathogen Trypanosoma cruzi that causes Chagas disease, a serious issue in Latin America. As such, most studies on bat trypanosomes and their host-parasite relationships have been focused on South American bat and trypanosome species (Lisboa et al. 2008; Cottontail et al. 2014; Ramírez et al. 2014). In Europe, research concerning bat trypanosomes was mainly performed in the UK in the early part of the twentieth century (Petrie 1905; Coles 1914) and the 1970s and 80s (Baker et al. 1972; Gardner and Molyneux 1988). These classic morphological studies were later followed by molecular research (Lord 2010; Hamilton et al. 2012). Knowledge on the presence of bat trypanosomes in the rest of Europe, however, is limited ACTA VET. BRNO 2020, 89: 69–78; https://doi.org/10.2754/avb202089010069 Address for correspondence: Hana Bandouchova Department of Ecology and Diseases of Zoo Animals, Game, Fish and Bees University of Veterinary and Pharmaceutical Sciences Palackeho tr. 1946/1, 612 42, Brno, Czech Republic Phone: +420 541 562 653 E-mail: bandouchovah@vfu.cz http://actavet.vfu.cz/ and/or anecdotal. While it is presumed that bat trypanosomes are highly prevalent in European bat species, only the UK data are available, meaning that we lack evidence on the infection status in Eastern and Central European bats. Likewise, there is a lack of data on trypanosome pathogenicity in bats. As no adverse effects associated with this infection have yet been reported, trypanosomes of the subgenus Schizotrypanum are considered non-pathogenic for bats (Lord and Brooks 2014). On the other hand, bats are exposed to many stressors during their lifetime, e.g. allocation of resources during physiological states such as hibernation torpor or lactation, and these could influence the outcome of any infection (Bandouchova et al. 2009; Kopp et al. 2018). During hibernation, immune system functions are limited (Bouma et al. 2010), while lactation imposes high energy demands on the female (Harshman and Zera 2007). The cryptic lifestyle of nocturnal mammals has also contributed to the lack of knowledge regarding the impact of infection on bat health. To date, no studies have yet been published on the impact of trypanosomes on bats during hibernation or lactation. The aim of the present study, therefore, is a) to improve our knowledge of the prevalence of bat trypanosomes in European countries, and b) to analyse haematology and blood chemistry parameters in order to assess the impact of trypanosomes on hibernating and lactating greater mouse-eared bats (Myotis myotis). Materials and Methods Animals Between 2015 and 2019, blood samples of 381 bats of eight vespertilionid bat species were collected in the Czech Republic, Bulgaria, and Poland. Bats were netted at swarming sites, maternity roosts or while emerging from hibernacula at the end of the hibernation period. The number of blood samples collected from the different species were as follows: the greater mouse-eared bat (M. myotis, n = 180), the noctule bat (Nyctalus noctula, n = 100), the lesser mouse-eared bat (Myotis blythii, n = 42), the Daubenton’s bat (Myotis daubentonii, n = 25), the Geoffroy’s bat (Myotis emarginatus, n = 8), the Natterer’s bat (Myotis nattereri, n = 13), the brown long-eared bat (Plecotus auritus, n = 7) and the Barbastelle bat (Barbastella barbastellus, n = 6). A detailed overview of the sampling sites, periods of sampling and site-specific number of individuals is summarised in Table 1. Sampling sites Bats were sampled at five localities in the Czech Republic, two in Bulgaria and one in Poland. Sampling in the Czech Republic was performed in the Moravian Karst including one swarming site (the Kateřinská cave [49.3607006N, 16.7102508E]), two hibernacula (the Sloupsko-Šošůvské caves [49.4104556N, 16.7390147E] and the Býčí skála cave [49.3074614N, 16.6947844E]), and in the Czech Karst (Malá Amerika mine [49.9545178N, 14.1760375E]). Sampling at maternity roosts was performed on two summer colonies (church attics in Doubravník [49.4256094N, 16.3518378E] and Otaslavice [49.3848658N, 17.0676067E]). Ambient temperatures ranged from 5.5 to 8.8 °C at the hibernacula, 35 to 55 °C in the church attics containing summer colonies, and 12 to 20 °C at the swarming sites. Hibernating noctules were sampled in captivity when held in an artificial hibernaculum (temperature 8 °C) and swarming noctules were caught in Brno while emerging from tree holes. In Poland, sampling was performed at the end of hibernation in the Nietoperek bat reserve (underground corridors of an abandoned German military fortification from the central sector of the Międzyrzecz Fortified Front in western Poland [52.3956606N, 15.5120972E]) with ambient temperatures ranging from 6.1 to 9.9 °C. Bulgarian swarming sites were in the Rhodope Mountains (Lednitzata ice cave [41.6497364N, 24.5339131E]) and in the Danubian Plain, north-eastern Bulgaria (the Orlova chuka cave with a constant year-round temperature of 14 °C [43.5899N, 25.9603E]). Collection of blood samples Blood samples were collected using methods described by Bandouchova et al. (2018) and Pikula et al. (2017). Polymerase chain reaction (PCR) analysis required 20 μl of blood, while additional 100 μl of blood were collected from 56 greater mouse-eared bats emerging from hibernacula and 36 lactating females of the same species from two maternity colonies for haematology and biochemistry measurements. Prior to release, the bats were provided with fluids and energy by oral administration of glucose and saline. All animals were handled so as to minimise stress. Both the sampling of bats and blood collection were performed in accordance with Czech Law No. 114/1992 on Nature and Landscape Protection, based on permits 1662/MK/2012S/00775/MK/2012, 866/JS/2012 and 00356/KK/2008/ AOPK issued by the Agency for Nature Conservation and Landscape Protection of the Czech Republic. Collection and sampling of bats in Bulgaria and Poland was authorized through permit Nos. 645/13.08/2015, 153/11.07/2016, WPN-I-6205.10.2015.AI, WPN-I-6205.13.2019.MZ and Resolution Nr. 45/2015 and 14/2018. The authors of the study 70

Bats host several trypanosome species of the subgenus Schizotrypanum, including the important human pathogen Trypanosoma cruzi that causes Chagas disease, a serious issue in Latin America. As such, most studies on bat trypanosomes and their host-parasite relationships have been focused on South American bat and trypanosome species (Lisboa et al. 2008;Cottontail et al. 2014;Ramírez et al. 2014).
In Europe, research concerning bat trypanosomes was mainly performed in the UK in the early part of the twentieth century (Petrie 1905;Coles 1914) and the 1970s and 80s (Baker et al. 1972;Gardner and Molyneux 1988). These classic morphological studies were later followed by molecular research (Lord 2010;Hamilton et al. 2012). Knowledge on the presence of bat trypanosomes in the rest of Europe, however, is limited and/or anecdotal. While it is presumed that bat trypanosomes are highly prevalent in European bat species, only the UK data are available, meaning that we lack evidence on the infection status in Eastern and Central European bats. Likewise, there is a lack of data on trypanosome pathogenicity in bats. As no adverse effects associated with this infection have yet been reported, trypanosomes of the subgenus Schizotrypanum are considered non-pathogenic for bats (Lord and Brooks 2014). On the other hand, bats are exposed to many stressors during their lifetime, e.g. allocation of resources during physiological states such as hibernation torpor or lactation, and these could influence the outcome of any infection (Bandouchova et al. 2009;Kopp et al. 2018). During hibernation, immune system functions are limited (Bouma et al. 2010), while lactation imposes high energy demands on the female (Harshman and Zera 2007). The cryptic lifestyle of nocturnal mammals has also contributed to the lack of knowledge regarding the impact of infection on bat health. To date, no studies have yet been published on the impact of trypanosomes on bats during hibernation or lactation.
The aim of the present study, therefore, is a) to improve our knowledge of the prevalence of bat trypanosomes in European countries, and b) to analyse haematology and blood chemistry parameters in order to assess the impact of trypanosomes on hibernating and lactating greater mouse-eared bats (Myotis myotis).

Animals
Between 2015 and 2019, blood samples of 381 bats of eight vespertilionid bat species were collected in the Czech Republic, Bulgaria, and Poland. Bats were netted at swarming sites, maternity roosts or while emerging from hibernacula at the end of the hibernation period. The number of blood samples collected from the different species were as follows: the greater mouse-eared bat (M. myotis, n = 180), the noctule bat (Nyctalus noctula, n = 100), the lesser mouse-eared bat (Myotis blythii, n = 42), the Daubenton's bat (Myotis daubentonii, n = 25), the Geoffroy's bat (Myotis emarginatus, n = 8), the Natterer's bat (Myotis nattereri, n = 13), the brown long-eared bat (Plecotus auritus, n = 7) and the Barbastelle bat (Barbastella barbastellus, n = 6). A detailed overview of the sampling sites, periods of sampling and site-specific number of individuals is summarised in Table 1.

Sampling sites
Bats were sampled at five localities in the Czech Republic, two in Bulgaria and one in Poland.

Collection of blood samples
Blood samples were collected using methods described by Bandouchova et al. (2018) and Pikula et al. (2017). Polymerase chain reaction (PCR) analysis required 20 µl of blood, while additional 100 µl of blood were collected from 56 greater mouse-eared bats emerging from hibernacula and 36 lactating females of the same species from two maternity colonies for haematology and biochemistry measurements. Prior to release, the bats were provided with fluids and energy by oral administration of glucose and saline. All animals were handled so as to minimise stress.

Detection of trypanosomes in blood samples
Total genomic deoxyribonucleic acid (DNA) was isolated from the blood samples using a DNA isolation kit (High Pure PCR Template Preparation Kit, Roche, Switzerland), according to the protocol recommended by the manufacturer. We used the nested PCR analysis protocol for Trypanosoma spp. detection described in Seward et al. (2017). The PCR was performed using Mini Opticon (Bio-Rad, USA), with reactions undertaken in a 20 μl reaction mixture containing 10 μl 2 × EmeraldAmp Max PCR Master Mix (Takara, Japan), 4 μl water, 0.5 μl of each primer (10 pmol/μl) and a 5 μl aliquot of isolated DNA in the first round, and 5 μl of the PCR product from the first round instead of DNA in the second round.

Statistical analysis
Chi-square test was used to compare differences in the prevalence of trypanosomes between sexes, localities, periods of sampling and host species, as well as differences in prevalence between the noctule bats T. dionisii and T. vespertilionis.
Normal distribution of variables for the whole haematology and blood chemistry parameter datasets was tested using Kolmogorov-Smirnov and Shapiro-Wilk tests. All blood parameters were normally distributed with the exception of Na, K, BUN, pCO 2 and pH. The one-way analysis of variance (ANOVA) and post hoc least significant difference (LSD) tests were used to assess trypanosome impact and sampling period. As a significant impact of the sampling period was confirmed in two subsets, i.e. hibernation and lactation, these were analysed separately. Normality was then re-checked using Kolmogorov-Smirnov and Shapiro-Wilk tests. In hibernating animals, all parameters were normally distributed with the exception of K, BUN and pCO 2 , while K, CL, HCO 3 , BE and tCO 2 were non-normally distributed in lactating females. One-way ANOVA and post hoc LSD tests were used to assess differences in haematology and blood chemistry parameters between Trypanosoma-positive and -negative animals. Non-normally distributed parameters were tested for using Kruskal-Wallis ANOVA. All analyses were performed in Statistica v.13.2.

Results
Of the 381 individual blood samples collected from the eight bat species, 103 proved positive for Trypanosoma spp. Direct DNA sequencing of the amplified small subunit (SSU) revealed a predominance of T. dionisii. Prevalence of T. dionisii was 31.8, 28.0, and 9.5% in greater mouse-eared, noctule and lesser mouse-eared bats. A second parasite species, T. vespertilionis, was only confirmed in nine noctule bats. Sequence comparisons revealed a percentual identity of > 99.6% with T. dionisii and T. vespertilionis isolates in the GenBank. Phylogenetic relationships of Trypanosoma dionisii and Trypanosoma vespertilionis 18S rRNA sequences isolated from the greater mouse-eared bat (M. myotis) and the noctule bat (N. noctule) are illustrated in Fig. 1.
Chi-square tests confirmed significant differences between (i) T. dionisii prevalence in the Czech Republic (32.3%) and Bulgaria (8.3%; P < 0.001) and Poland (16.2%; P = 0.007); (ii) prevalence of T. dionisii (28%) and T. vespertilionis (9 %) in noctule bats (P < 0.001); (iii) swarming prevalence of T. dionisii in M. myotis from the Czech Republic (50.0%) compared to its sibling species lesser mouse-eared bat from Bulgaria (9.5%; P = 0.001); and (iv) T. dionisii prevalence in greater mouse-eared bats from the Czech Republic (35.1%) compared with Daubenton's bats from Poland (9.5%; P = 0.025) during hibernation. While there was no difference in infection prevalence between sexes during any of the sampling periods, a significantly higher prevalence was confirmed between hibernating bats (29.8%) and those caught during the swarming period (18.2%; P = 0.028). A detailed overview of Schizotrypanum prevalence in bat species, countries and sampling periods is provided in Table 2.
One-way ANOVA and post hoc LSD test undertaken on the whole dataset revealed a significant impact of the sampling period on all haematology and blood chemistry parameters except Cl, Hct, pCO 2 and Hb. Separate analyses of the hibernating and lactating subsets indicated no differences in haematology and blood chemistry parameters between Trypanosoma-positive and -negative bats. Infection intensity was very low with up to 4 trypomastigotes per the whole blood smear. The results of haematology and blood chemistry analysis on Trypanosoma-positive and -negative bats are summarised in Tables 3 and 4.

Discussion
Our results indicate a high prevalence rate for trypanosomes of the subgenus Schizotrypanum in the Czech Republic, Bulgaria and Poland, especially as regards T. dionisii, which was dominant in all the bat species tested. Our T. dionisii isolates from noctules (N. noctula) are closely related to T. dionisii B clade from the UK, previously confirmed to be more closely related with T. dionisii isolates from Brazil (Hamilton et al. 2012). This result supports the idea of connected population of noctules within Europe or migration of noctules across long distances within their range. A second species, T. vespertilionis, was only confirmed in noctule bats, with prevalence significantly lower than that for T. dionisii. Both T. dionisii and T. vespertilionis were confirmed in noctules from the same colony, though no case of co-infection was recorded. Differences in the prevalence of these trypanosome species may have resulted from interspecific competition within the bat hosts or within invertebrate vectors, as both species utilise the same ecological niche. A similar effect has previously been described in co-infection with different strains of T. brucei (Balmer et al. 2009).
Isolates of 18S rRNA obtained in the present study: tvnn -Trypanosoma vespertilionis isolate from Nyctalus noctula, Czech Republic (GenBank MN604082); tdmm -Trypanosoma dionisii isolate from Myotis myotis, Poland (GenBank MN604028) and tdnn -Trypanosoma dionisii isolate from Nyctalus noctula, Czech Republic (GenBank MN604041). of bat trypanosomes in Europe. In Central Europe, these nidicolous ectoparasites are strongly bound to summer maternity colonies of M. myotis and summer shelters or bat boxes used by N. noctula or Pipistrellus spp., with Cimex spp. having been confirmed in ca. 80% of summer M. myotis colonies in the Czech Republic (Balvín et al. 2014). We found approximately three times higher trypanosome prevalence in noctules and greater mouseeared bats in the Czech Republic compared to Bulgaria and Poland, suggesting that these two species play an important role in maintaining Shizotrypanum trypanosomes in Central 74  (58) 50.0 (14) 57.1 (7) 42.9 (7) 31.8 ( Europe. Noctules in particular play a crucial role as these migratory bats are thought to spread Cimex spp. between European bat colonies (Balvín et al. 2012). In comparison, Cimex spp. are relatively rare in bats from the Balkan region, probably due to sub-optimal conditions in their summer roosts, which are predominantly found in caves (Balvín et al. 2014). A similar situation also appears true for Poland, though it should be noted that the only survey of Polish Cimex spp. was undertaken during hibernation, hence no data are available for maternity roosts (Haitlinger and Łupicki 2008). On the other hand, a number of Polish greater mouse-eared bat maternity roosts are found in underground Table 3. Haematology and blood chemistry parameters of hibernating and lactating Trypanosoma-positive and negative greater mouse--eared bats (Myotis myotis) with normal distribution and results of one-way analysis of variance. As the lesser mouse-eared bat has similar ecological requirements to its sibling species the greater mouse-eared bat (Arlettaz at al. 1997), a similar trypanosome prevalence would be expected. Surprisingly, however, we recorded a significantly lower prevalence in lesser mouse-eared bats, again probably due to the absence of vectors in Bulgarian summer roosts (Balvín et al. 2014). We also noted significantly higher trypanosome prevalence in hibernating over swarming bats, though this was probably due to differences in the number of each species in the hibernation and swarming groups and their countries of origin.
As summer roosts comprise almost exclusively female bats and their offspring, females might be expected to be more exposed to vectors than males; however, we found no significant differences in T. dionisii prevalence between males and females. A possible explanation for this may be that bats are infected as juveniles or through vertical transmission from mother to offspring, as previously described for T. cruzi (Muñoz et al. 2009). Howard et al. (2014), for example, reported the likelihood of T. cruzi transmission from mother to foetus at around 5% (Howard et al. 2014). Like humans, bats have a haemochorial placenta (Carter and Mess 2008); hence, the barrier between maternal and foetal blood is similar in bats and humans, though minimal compared to some other mammalian species. Vertical transmission represents a long-term advantage for the pathogen in terms of its spread within host populations, with parasites undergoing vertical transmission generally benign towards their host (Ewald 1995). Nevertheless, we propose that a detailed survey be undertaken to confirm this hypothesis.
The hypothesis of benign behaviour by bat Schizotrypanum trypanosomes is also supported by the lack of any significant difference in haematology and blood chemistry parameters between Trypanosoma-positive and -negative animals. Neither hibernating nor lactating Trypanosoma-positive bats showed any impact of infection on the measured blood parameters. On the other hand, Schizotrypanum trypanosomes are known to form cystic structures in bat organs and tissues, including the heart and skeletal muscles (Molyneux 1991). This has also been confirmed in T. cruzi (Cardoso et al. 2016;Ponte-Sucre 2016), where chronic effects involving tissue damage caused by the host's own antibodies targeting encysted developmental trypanosome stages were the main pathogenic mechanism in Chagas disease (Lozano et al. 2017). The same effects may also be expected in long-lived bats as a consequence of long-term infection. We suggest that additional studies examining antibody-mediated effects of Schizotrypanum on bats will be needed to assess whether such a process affects particular species at the population level.