First broad-range molecular screening of tick-borne pathogens in Ixodes (Pholeoixodes) kaiseri, with special emphasis on piroplasms

Recently, the occurrence of Ixodes (Pholeoixodes) kaiseri has been reported for the first time in several European countries, but data on the molecular analysis of this hard tick species are still lacking. Therefore, in this study DNA extracts of 28 I. kaiseri (collected from dogs and red foxes in Germany, Hungary and Romania) were screened with reverse line blot hybridisation (RLB), PCR and sequencing for the presence of 43 tick-borne pathogens or other members of their families from the categories of Anaplasmataceae, piroplasms, rickettsiae and borreliae. Rickettsia helvetica DNA was detected in one I. kaiseri female (from a red fox, Romania), for the first time in this tick species. Six ticks (from red foxes, Romania) contained the DNA of Babesia vulpes, also for the first time in the case of I. kaiseri. Molecular evidence of R. helvetica and B. vulpes in engorged I. kaiseri does not prove that this tick species is a vector of the above two pathogens, because they might have been taken up by the ticks from the blood of foxes. In addition, one I. kaiseri female (from a dog, Hungary) harboured Babesia sp. badger type-B, identified for the first time in Hungary and Central Europe (i.e. it has been reported previously from Western Europe and China). The latter finding can be explained by either the susceptibility of dogs to Babesia sp. badger type-B, or by transstadial survival of this piroplasm in I. kaiseri. The subgenus Pholeoixodes belongs to the most species-rich genus of hard ticks (Acari: Ixodidae: Ixodes). Pholeoixodes species are usually associated with ‘pholeophilic’ mammals and birds, which are named as such because they prefer to hide in cavities (implying burrow-dwelling mammals as well as terrestrial birds that nest in tree holes or burrows: Hornok et al., 2017). In the Western Palaearctic, five species of this subgenus feed on domestic and wild carnivores (mainly Canidae, Mustelidae), i.e. Ixodes canisuga Johnston, 1849, I. kaiseri Arthur, 1957, I. crenulatus Koch, 1844, I. hexagonus Leach, 1815 and I. rugicollis Schulze and Schlottke, 1929. Among these, I. rugicollis is regarded as very rare, and data on the occurrence of I. crenulatus in Europe appear to be either historical or uncertain (Hornok et al., 2017). On the other hand, I. canisuga, I. hexagonus and I. kaiseri commonly infest dogs and foxes in many European countries (Hornok et al., 2017; Sándor, 2017a,b). Considering these three species, several molecular studies have been conducted to screen pathogens in I. canisuga and I. hexagonus (reviewed in Sándor, 2017a,b; Hornok et al., 2018a). However, reports on PCR-based screening of pathogens in I. kaiseri are missing (Estrada-Peña, 2017), also taking into account that in a report from Poland ticks resembling I. kaiseri were later shown to be I. canisuga (Wodecka et al., 2016; in GenBank: KF471772). In this study, whole body DNA extracts of 28 I. kaiseri specimens were used (Table 1). These ticks (originally collected from four red foxes in Germany, from eight dogs in Hungary, and from one dog and 15 red foxes in Romania) were molecularly identified following morphological comparison to type specimens (Hornok et al., 2017). In order to screen these samples for a broad range of tick-borne pathogens, reverse line blot hybridisation (RLB) was performed (Kirstein et al., 1997), modified as previously published (Schötta et al., 2017). The oligonucleotides included group-level (catch-all) probes for Anaplasma/Ehrlichia spp., Theileria/Babesia spp., Borrelia burgdorferi sensu lato and Rickettsia spp. The species-specific probes targeted eight species from Anaplasmataceae, 17 species of piroplasms, eight species of borreliae and ten Rickettsia species (Schötta et al., 2017).