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