Institute of Parasitology, Slovak Academy of Sciences, Hlinkova 3, Košice, Slovakia. *Corresponding author:

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1 DOI: /s W. Stefański Institute of Parasitology, PAS Acta Parasitologica, 2013, 58(3), ; ISSN The occurrence of Borrelia burgdorferi sensu lato in certain ectoparasites (Mesostigmata, Siphonaptera) of Apodemus flavicollis and Myodes glareolus in chosen localities in the Czech Republic Jakub Netušil 1, Alena Žákovská 1, Karel Vostal 1 *, Adam Norek 1 and Michal Stanko 2, 3 1 Institute of Experimental Biology, Faculty of Science, Masaryk University, Kotlářská 2, Brno, Czech Republic 2 Institute of Zoology, Slovak Academy of Sciences, Löfflerova 10, Košice, Slovakia 3 Institute of Parasitology, Slovak Academy of Sciences, Hlinkova 3, Košice, Slovakia Abstract Ticks represent the primary vectors of several serious diseases, including the Lyme disease caused by Borrelia burgdorferi sensu lato (Bbsl). In this study two dominant ectoparasitic groups of arthropods (Mesostigmata, Siphonaptera) were investigated for the presence of borrelian DNA in order to determine their potential role of vectors (or carriers) of this bacterium. All individuals (235) were collected from wild-living rodents obtained in three localities in the Czech Republic (Bažantula, Baba and Křižovice). The majority of parasites were members of the families Parasitidae and Dermanyssidae (Mesostigmata) and families Hystrichopsyllidae and Ceratophyllidae (Siphonaptera). The rodent host species was almost exclusively the yellow-necked mouse (Apodemus flavicollis). Bbsl was detected by the PCR method in the following ectoparasite species: Euryparasitus emarginatus (1), Eulaelaps stabularis (1), Haemogamassus nidi (1), Laelaps agilis (5), Myonyssus gigas (1) (Mesostigmata) and Ctenophthalmus agyrtes (1), C. solutus (3) (Siphonaptera). Keywords Borrelia burgdorferi, Acarina, Siphonaptera, PCR, rodents Introduction Ectoparasites generally participate in spreading of various pathogens in the nature. These pathogens include Borrelia burgdorferi sensu lato (Bbsl), an etiological agent of the Lyme disease. Ticks (especially those of the genus Ixodes) play a very important role in the Bbsl transmission. Ixodes pacificus Cooley et Kohls, 1943 and I. scapularis (formerly I. dammini) Say, 1821 transfer borreliae in the United States, I. persulcatus Schulze, 1930 in Asia and partially in Europe (Gray 1998). I. ricinus is the vector that transmits Bbsl in Europe and parasitizes over 300 species of animals (148 mammals, 20 reptiles and 149 birds, as reported by Bennett 1995). The presence of Bbsl was also confirmed in several other bloodsucking arthropods, such as mosquitoes of the family Culicidae, the sucking lice Anoplura and the fleas Siphonaptera (Magnarelli et al. 1986, Halouzka 1993, Žákovská et al. 2002). Various species of reptiles, birds and mammals serve as the reservoir for this bacterium (Anderson 1989, Tarageľová et al. 2008). Small rodents seem to be really important in that way (Gern 2008, Hanincová et al. 2003). Ixodes ricinus is characterized as a competent vector of the Bbsl in Europe (Gern 2008). Besides the places of general occurrence, its presence was also detected in high attitudes (Mačička 1955, Danielová et al. 2006). Pre-adult stages of this common tick parasitize rodents and shrews, mainly in the late spring and summer (Stanko et al. 2007). Two additional arthropod parasite groups, fleas and mites (dominant components of the parasite communities), parasitize small mammals during the whole year (Haitlinger 1997, Stanko and Miklisova 2000, 2002). Fleas (Siphonaptera) are holometabolous insects and their adults survive as obligate ectoparasites on the mammalian or avian hosts. Not all flea larvae are parasitic and those that are not feed on organic matter found in the nests or burrows of avian or mammalian hosts. Flea larval and pupal development is entirely off-host. However, relationships be- *Corresponding author: karel.vostal@seznam.cz

2 338 Jakub Netušil et al. tween the mesostigmatid mites (Acarina, Mesostigmata) and rodents are more complicated. Diet of the majority of mite species consists not only of blood or of other vertebrate host s body fluids but also of small arthropods, including other mites or larvae and insect eggs. Some mesostigmatid mites (e.g. mites of the genus Laelaps, Hyperlaelaps) are blood-sucking parasitic mites permanently occurring on the body of rodents. Other mites, the facultative parasites of rodents (e.g. mites of genus Haemogamasus or Eulalelaps), more frequently occur in the nests of rodents or on rodents themselves. A certain group of these facultative parasites (e.g. families Parasitidae, Rhodacaridae, Macrochelidae) is present in the rodent nests/on rodents bodies rather because these parasites are predators that feed on organic mater in the nests; in some cases their presence is a result of phoretic relations (Mrciak et al. 1966, Daniel et al. 1970). Fleas have already been tested for presence of pathogenic borreliae. They were found to be rarely infected (Lindsay et al. 1991). There are very few studies addressing the issue of the role of mites as vectors of selected groups of bacterial pathogens (Borrelia burgdorferi, Anaplasma phagocytophilum Skoracki et al. 2006, Literák et al. 2008). However, there are too few informations about relationship between mesostigmatid mites and borreliae. The main aim of this study was to answer the question whether other parasite groups (fleas, mesostigmatid mites), which frequently occur on small mammals, can harbour borreliae and thus play any role in circulation of Bbsl. Therefore the range of ectoparasite species on small mammals was studied and an attempt to confirm presence of borreliae in arthropods was made. Materials and Methods Character of localities Ectoparasites were collected from the hair of rodents caught by snapping traps. The traps were baited with a piece of lardfried wick. 100 pieces of them were laid into scrubs overnight and checked in the early morning. The trapping was carried out in three localities in the Czech Republic (Bažantula, Baba and Křižovice). Bažantula (49 44 N, E) represents a typical area with deciduous forest where elms and oaks predominate. It is a relatively small area covering about 200 x 300 m, situated approximately 2 km northeast from the town of Studénka. This locality was chosen because of soil moisture, that creates a very favourable environment for mites. Křižovice (49 26 N, E) is situated in the Bohemian-Moravian Highlands at 500 meters above the sea level. The local woods are mostly mixed, with the predominance of beeches, hornbeams and spruces predominating in this area. The soil moisture in this region is lower in comparison with Bažantula. As the third locality a mixed wood, near the protected landscape area Baba (49 14 N, E) on the edge of Brno City, was chosen. This area lies 365 meters above the sea level and is characteristic of a high diversity of tree species oaks, beeches, hornbeams, birches as well as coniferous, such as pines. Animal material processing A two-step procedure was adopted for processing the animal material. Immediately after collection, the collected ectoparasites were dissected on a sterile glass slide under a microscope using sterile preparation needles. The abdomen of each individual was separated and its contents initially examined for presence of spirochaetes (spiral shaped cells) using the darkfield microscopy (DFM). Only the positive specimens were further tested for presence of the borrelian DNA by the PCR method. The dissected mites were kept in 70% ethanol (Novák and Povolný 1969) and later identified. This DFM process, however, showed rather time consuming, not allowing examination of large number of specimens. Therefore the parasites were classified as the first step and then concentrated into pools containing more than one individual of the same species. Contents of each pool were triturated and such-obtained samples directly tested for presence of borreliae using the PCR method. Fleas (Siphonaptera) were determined by Rosický (1957), mites (Mesostigmata) by Karg (1993) and Mašán and Fenďa Dark-field microscopy The midgut content of each sample examined by the DFM was individually triturated in 200 μl of BSK H medium (Sigma- Aldrich) and observed with a magnification of 400X. DNA purification Borrelian DNA samples were isolated from homogenates using the DNA isolation kit QIAamp DNA Blood Mini Kit (Qiagen, Germany). DNA was eluted from QIAamp membrane in 40 l of AE buffer (10 mm Tris-Cl; 0.5 mm EDTA; ph 9.0.). A volume of 4 l of this preparation was used for amplification. Polymerase chain reaction PCR assay based on the specific flagellin sequence amplification for detection of B. burgdorferi sensu lato was performed (Picken et al. 1996). 40 µl of PCR master mix contained: 1x HotStarTaq Master Mix (Qiagen, Germany), 0.1 pmol of each FL3 and FL5 primers, and 20 pmol of FL6 and FL7 primers, 100 mm of dutp (Sigma, USA), 10 2 internal competitive standard, gene for flagellin of B. afzelii cloned into plasmid with concentration of 100 copies per 1 ml (Genex CZ, Czech Republic) and 4 ml of template DNA received after standard DNA isolation. All PCR runs were performed on a thermocycler (PTC-200, MJ Research) with the following profile: an initial activation step at 96 C for 12 min, 30 cycles consisting

3 Bbsl in certain ectoparasites of wild rodents 339 of a denaturation step for 10 sec at 96 C, an annealing step for 10 sec at 68 C, an extension step for 40 sec at 72 C, and additional 45 cycles consisting of 10 sec at 96 C of denaturation, 10 sec at 54 C of annealing, and of extension step at 72 C for 30 sec. Agarose gel electrophoresis Amplified products were separated on 2% of agarose gel containing ethidium bromide (5 g/1 ml) and visualized using the UV illumination. In case of a positive finding, both the 276 bp long amplification product and the product of internal standard amplification (420 bp) were detectable. In case of sample negativity, only the internal standard was detected. Internal standard served as the control of the reaction. It was visible when the reaction was not inhibited. No amplification product was detectable in case of inhibition of the PCR reaction. Results All ectoparasites collected from the hair of wild living rodents were determined as members of the orders Mesostigmata and Siphonaptera. The study focused on the most prevalent species of rodents in the chosen localities: the bank vole Myodes (Clethrionomys) glareolus Schreber, 1780 and the yellownecked mouse Apodemus flavicollis Melchior, Mite communities constituted five species of the Dermanyssidae family: Laelaps agilis Koch, 1836, Eulaelaps stabularis Koch, 1836, Haemogamasus nidi Michael, 1892, Haemogamasus hirsutus Berlese, 1889, Myonyssus gigas (= gigas) Oudemans, 1912; two species of family Parasitidae: Parasitus (Eugamasus) lunuatus Miller, 1859, Poecilochirus sp., and one species of family Rhodacaridae Euryparasitus emarginatus Koch, Altogether, five flea species (Siphonaptera) of two families were confirmed on the hosts: Nosopsyllus fasciatus Bosc, 1800, Megabothris turbidus Rothschild, 1909 (family Ceratophyllidae) and three species of family Hystrichopsyllidae: Hystrichopsylla talpae Curtis, 1826, Ctenoph-thalmus agyrtes Heller, 1896, Ctenophthalmus solutus Jordan et Rothschild, All the observed flea species were characterised as oligoxenous or euryxenous parasites, i.e. they ei-ther infested a narrower or wider host spectrum (Rosický 1957) or fleas generalist (Krasnov 2008). In 2004, a total of 64 parasitic mites were examined, collected from wild rodents during three collections in the localities Křižovice and Bažantula. From these collections, 6 species of ectoparasites and 2 host rodent species were obtained and the yellow-necked mouse (Apodemus flavicollis) was their predominant host. Presence of spirochaetes was observed in 13 samples by DFM, while the borrelian DNA confirmed by the PCR method was found just in one case (E. stabularis) (Table I). In 2005, the samples were tested directly for the presence of Bbsl DNA using PCR, utilised to examine the parasites in pools. During two collections in the localities Křižovice and Baba, 171 ectoparasites (157 mites and 14 fleas) were obtained; both the mites and fleas belonged to 5 species. The proportional representation of most species was, however, very small. Only 1 host rodent species (Apodemus flavicollis) was caught. Borreliae were detected in 12 samples (Table I). During both years of this study, a total of 235 ectoparasites of small mammals (221 mites and 14 fleas) were collected and examined, mostly obtained from the yellow-necked mouse A. flavicollis. Altogether, 8 mite species and 5 flea spe- Table I. DFM and/or PCR positivity of examined mites and fleas species Species Locality number of individuals Křižovice Bažantula Baba Number of samples (individuals in pool) DFM positivity (locality abbreviation) PCR positivity (locality abbreviation) E. emarginatus (Bab) E. stabularis (4) 6 (Kr) 1 (Kr) H. hirsutus (Kr) 0 H. nidi (3) + 1(5) 4 (Kr) 1 (Bab) L. agilis (10) + 3(11) + 4(10) 2 (Baz) 5 (2 Kr, 3 Bab) M. gigas (Bab) P. lunulatus *Poecilochirus sp C. agyrtes (Bab) C. solutus (2) (Bab) H. talpae M. turbidus (2) 0 0 N. fasciatus (3) 0 0 Total *The only one parasite species caught from the hair of M. glareolus.

4 340 Jakub Netušil et al. cies were registered. Bbsl was detected in 13 samples out of which 9 were the pooled specimens created by a higher number of individuals (Table I). Discussion This study focused on ectoparasites obtained from small rodents found in chosen localities throughout Moravia region (Czech Republic). The purpose was to determine which arthropod species parasitize on small mammals in these localities and to detect presence of pathogenic spirochaete Bbsl in these parasites. So far, Bbsl has been found in various species of arthropods-mites (Acarina), flies (Diptera) and fleas (Siphonaptera) (Pokorný 1989, Literák et al. 2008). Ticks are known as the most significant vectors of borreliae. Other groups of bloodsucking arthropods have not yet been much investigated, even though they occur on small rodents throughout the year (e.g. Laelaps genus). Trophic niches of the mentioned mites are diverse. The most numerous species, L. agilis, is an obligate haematophagous parasite with regular occurrence in the hair of its host. E. stabularis, H. nidi and H. hirsutus are facultative parasites which mainly occur in mammalian nests. While L. agilis is a host-specialist concentrating on the Apodemus genus, mites like E. stabularis and the genus Haemogamasus, are typical parasites on many rodent species. They, however, occur more frequently in the nests (or hair) of vole sof the family Microtidae (Karg, 1993). The other determined mites are non-parasitic species and their trophic niches are characterised as predatory (P. lunulatus, E. emarginatus), or free living mites in the soils with nymphal stages with a frequent phoretic relationships to necrophagous beetles and rodents (Poecilochirus sp.) (Haitlinger 1977). The most predominant mite species was Laelaps agilis, an obligate parasitic mite infesting a very wide host range (Mrciak et al. 1966, Haitlinger 1977). In comparison with some other studies carried out for example in Slovakia (Stanko and Miklisova 2000, 2002) its dominating position in the scale of arthropods parasitizing small mammals in the central Europe was confirmed. Haemogamasus nidi and Eulaelaps stabularis were the next most frequent species, while other mite species were rare. The role of some mesostigmatid mites as vectors of various pathogens or parasites has already been investigated and successfully confirmed. Pathogenic Francisella tularensis causing tularemia was isolated from Haemogamasus nidi and Laelaps muris (Lysý et al. 1979; Zuevskij 1976) and rickettsiae were observed in a small number of species of nest ectoparasites (Kocianová 1989). Laelaps agilis was examined in connection with Hepatozoon sylvatici and its potential importance in transmission of this parasite was also confirmed (Frank 1977). Some mesostigmatid mites contained developmental stages of coccidia (Mohamed et al. 1987). This study attempted to enhance the up-to-date knowledge by testing mesostigmatid mites for presence of Bbsl using a PCR method. The lower positivity detected in 2004 by the PCR compared to the positivity detected by the DFM was probably caused by an unequal amount of DNA in the samples. This was also the main reason for the pool analysis in Bbsl was detected in Laelaps agilis, while 5 pooled samples (each with a content of 10 individuals) were positive. Other PCR positive species were Haemogamasus nidi (pool with contents of 5 individuals) and 3 samples coming from single mites Myonyssus gigas, Eulaelaps stabularis and Euryparasitus emarginatus. Occurrence of borreliae in mites that are bloodsucking parasites (Laelaps agilis, Myonyssus gigas) is not surprising. It might be beneficial to artificially infect other species/genera (Eulaelaps, Haemogamasus, Euryparasitus) of parasitic mites by pathogenic borreliae, either by making the mites suck on infected rodents or by predation. 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