In Europe, lice collected from rodents have never been examined before for the presence of these bacteria. This study is the first report on the prevalence and diversity of Bartonella and Rickettsia species in lice collected from rodents in Slovakia. Phylogenetic analysis based on the Bartonella rpoB gene and ITS region and the Rickettsia gltA gene revealed the presence of B. tribocorum, B. coopersplainsensis, B. taylorii, R. helvetica and Rickettsia sp. in rodent lice.
In this study, B. taylorii was detected in H. affinis lice collected from A. flavicollis. In previous studies, B. taylorii has been confirmed in the small mammals A. agrarius, A. flavicollis, M. glareolus, M. arvalis and Talpa europaea in Slovakia [13, 14]. B. taylorii strains in small mammals and their ectoparasites have also been reported in several studies conducted in Europe, including in Germany , England , Lithuania [11, 12], Slovenia , Poland  and Spain . B. taylorii can infect several sympatric woodland rodents at a given site. A high diversity of B. taylorii strains is frequently found in Apodemus mice and in Myodes and Microtus voles . The pathogenic potential of B. taylorii is as yet unknown [11, 40].
In this study, the B. tribocorum infection was detected in P. serrata and H. affinis lice collected from A. flavicollis, A. agrarius and M. glareolus. This Bartonella species is pathogenic to humans . Previous studies have strongly supported the association of B. tribocorum with rats of the genus Rattus. B. tribocorum has been detected in rats and their fleas in Thailand  and Bartonella strain closely related to B. tribocorum has been detected in louse (adult P. spinulosa) collected from rats in Egypt . In the striped field mouse A. agrarius, B. tribocorum was detected for the first time in South Korea  and closely related strains were later confirmed in A. agrarius from Slovakia  and Lithuania .
The present study is the first to detect the B. coopersplainsensis infection in Slovakia in H. affinis lice collected from A. agrarius. Previously, B. coopersplainsensis has been isolated in rats from Australia  and New Zealand  and in one louse pool (Hoplopleura spp.) collected from rats in Thailand . B. coopersplainsensis has also been reported in A. agrarius in Lithuania . There is a lack of information on B. coopersplainsensis, therefore the public health impact of this bacteria is unknown .
The present study is also the first to demonstrate the presence of R. helvetica and Rickettsia sp. in lice collected from rodents in Slovakia. Two R. helvetica strains were detected in H. affinis and P. serrata lice collected from A. agrarius. R. helvetica are considered to be agents of human rickettsioses . In recent studies conducted in Slovakia, R. helvetica has been identified in rodents and in fleas, mites and ticks collected from rodents [3, 19, 20, 21]. R. helvetica has also been reported in rodents and their ectoparasites in other European countries, such as the Netherlands , Hungary , Germany , Poland  and Lithuania [24, 25].
Based only on sequence analysis of the gltA gene, the Rickettsia sp. detected in this study in H. affinis lice pool collected from A. agrarius was not identified to species level. The obtained gltA sequence showed 100% identity with the corresponding sequences of R. aeschlimann, R. heilongjiangensis and R. raoultii in the GenBank database.
The presence of Bartonella spp. and Rickettsia spp. in lice may result from the acquisition of these bacteria via blood meals from infected rodents. Bartonella spp. are transmitted via horizontal transmission: arthropod vectors become infected with Bartonella bacteria while feeding on infected hosts, including rodents, and can then transfer the bacteria to another host . Worldwide, the prevalence of Bartonella spp. in rodents ranges from 25 to 80%, which suggests a reciprocal adaptation between the bacteria and their reservoirs . As a result of their blood-feeding habits, lice could transfer disease agents between closely-related host species [26, 29] and physical contact between individual rodents may promote the transmission of different Bartonella species .
Some SFG rickettsiae are thought to circulate in enzootic or epizootic cycles between wild vertebrates and arthropod vectors. The high prevalence of R. helvetica previously obtained in small rodents suggests that they may play an important role as potential natural reservoir hosts for this pathogen [18, 25].
The rodents from which the lice were collected have previously been tested for the presence of Bartonella and Rickettsia pathogens [3, 13, 14]. However, almost all the Bartonella-infected and Rickettsia-infected lice were derived from non-infected rodent hosts (except for two specimens of A. agrarius; data not shown). In this case, lice could become infected by parasitising on other infected hosts. Examined small rodents infested with lice also harbour other ectoparasites species such as mites, fleas and I. ricinus ticks [3, 20]. The presence of Bartonella pathogens in lice may also result from acquisition pathogens by co-feeding with Bartonella-infected fleas. Fleas are the main vectors for the maintenance and transmission of B. grahamii, B. taylorii and B. rochalimae among populations of small mammals . B. tribocorum has been detected in fleas and B. coopersplainsensis in ticks and lice . Lice that infest rodents could acquire Rickettsia pathogens by co-feeding with infected I. ricinus ticks, fleas and mites. Horizontal transmission through a shared blood meal has been demonstrated for some rickettsial pathogens . In a previous study conducted in Slovakia, Rickettsia spp. was detected in four species of mites, I. ricinus ticks and four flea species, with an overall prevalence of 9.3%, 17.2% and 3.5% respectively . R. helvetica has been identified in fleas, ticks and mites [3, 21].
Although, the results of the present study confirm the circulation of Bartonella spp. and Rickettsia spp. in lice, the role of lice in the transmission of Bartonella and Rickettsia species remains unknown. Thus, future studies should be performed to determine the specific roles of different species of lice parasitising small rodents in the transmission of Bartonella spp. and Rickettsia spp. bacteria in order to estimate the potential risks for other mammals (e.g. cats) and humans.