Prevalence and diversity of Bartonella species in small rodents from coastal and continental areas

Worldwide, Bartonella infections are known to inflict a wide range of mammals and, within rodents alone, more than 20 Bartonella species have been detected. There is, however, a lack of studies on the presence of Bartonella spp. in rodents in the Baltic region. We analysed 580 individuals belonging to eight small rodent species trapped in coastal and continental areas of Lithuania during 2015–2016. The presence of Bartonella DNA was examined by real-time PCR targeting the ssrA gene. The molecular characterization of the bacteria strains was based on sequence analysis of two housekeeping genes (rpoB, groEL) and the intergenic spacer region (ITS). For the rodents overall, the prevalence of Bartonella spp. was 54.8%, while the prevalence figures for each of the individual species were 8.3% in M. musculus, 15.8% in A. agrarius, 33.3% in M. arvalis, 42.4% in M. glareolus, 53.4% in M. oeconomus, 57.5% in M. minutus, 79.6% in A. flavicollis to 80% in M. agrestis. Sequence analysis revealed that the Bartonella strains belonged to the B. grahamii, B. taylorii, B. rochalimae, B. tribocorum, B. coopersplainsensis and B. doshiae genogroups. The highest Bartonella infection rates and the highest species diversity were both detected in rodents captured in the coastal area. To our knowledge, these are the first reports of the presence of B. coopersplainsensis, B. doshiae and B. tribocorum in Lithuania.

Bartonellae are facultative intracellular, fastidious, gram-negative bacteria that are transmitted to humans and other mammals by bloodsucking arthropod vectors such as fleas, mites, sand fleas and ticks 1,2 . The most important vectors for the maintenance and transmission routes of the Bartonella species are fleas, that feed on mammalian hosts such as cats, dogs, rodents, insectivores and rabbits 3 . There are currently 35 Bartonella species and three subspecies with standing in the Taxonomy Database of the National Center for Biotechnology Information (http://www.bacterio.net/Bartonella.html). However, the number of species in genus Bartonella is growing, as not all Bartonella species have been validated. Currently, 45 official and candidate Bartonella species have been detected in vertebrates, and at least fifteen of them have been related to human diseases 4 . During their evolution, Bartonella spp. have adapted to a variety of reservoir hosts and have become pathogenic when introduced into an incidental host 5,6 . With more than 20 Bartonella species associated with rodents, this group of mammals represents an important group of potential reservoirs for many Bartonella infections that have been reported worldwide 7 . Human pathogenic Bartonella species such as B. elizabethae, B. tribocorum, B. grahamii, B. rochalimae, B. vinsonii and B. washoensis have been isolated from various rodent species 3,5,7 . More than one Bartonella species can circulate in rodent communities, and multiple Bartonella genotypes in the same rodent host have been reported 8,9 .
The yellow-necked mouse (Apodemus flavicollis), wood mouse (Apodemus sylvaticus), striped field mouse (Apodemus agrarius), bank vole (Myodes glareolus), common vole (Microtis arvalis) field vole (Microtus agrestis) and root vole (Microtus oeconomus) are rodent species that play important roles in the maintenance and circulation of Bartonella infections in Europe 5,8 . Bartonella pathogens have been reported in several rodent populations in Poland, Germany, Denmark and Sweden (reviewed by Gutierrez et al. 8 ). However, Bartonella epidemiology and host-pathogen associations in Europe, including in the Baltic region, are insufficiently characterized. www.nature.com/scientificreports www.nature.com/scientificreports/ Diversity of Bartonella species in rodents. Eighty-seven Bartonella-positive PCR products of partial rpoB, groEL genes and ITS region derived from 56 different rodent specimens of eight species were subjected to sequence analysis (Table 2). A total of 73 good-quality sequences of rpoB (n = 43), groEL (n = 9) genes, and ITS region (n = 21) were obtained and analyzed.
Worldwide, the prevalence of Bartonella spp. is ranging from 25 to 80%. The high rate of Bartonella infection in rodent communities suggests a reciprocal adaptation between the bacteria and their reservoirs 8 . We found the overall prevalence of Bartonella species in different rodent species in Lithuania ranging from 8.3 to 80% with the highest Bartonella-infection rates in M. agrestis (80%) and A. flavicollis (79.6%) ( Table 1). Our findings are similar to those reported in A. flavicollis from the eastern Germany (84.4% 10 ) but higher than those from Slovakia (63.0%; 244/387 11 ), Slovenia (62.7%; 27/43 12 ), Denmark (53.3%; 8/15 13   www.nature.com/scientificreports www.nature.com/scientificreports/ In this study, the 52.9% of sequences derived from small rodents were ascribable to B. taylorii. In general, B. taylorii strains demonstrate high diversity and are frequently found in mice, as well as in Myodes and Microtus voles, inhabiting boreal forests of the Eurasian continent (reviewed by Buffet et al. 5 ). In line with this, the present study showed high diversity of B. taylorii strains in Lithuanian rodents. Phylogenetic analysis of rpoB gene revealed the presence of twelve B. taylorii genotypes associated with A. flavicollis mice (four out of twelve) and three species of voles M. glareolus, M. agrestis and M. oeconomus (eight out of twelve) (Fig. 1). The high variability of B. taylorii strains in rodents could be explained by a potential accelerated evolution of the Bartonella genus in small rodents as a result of frequent recombination events, horizontal gene acquisitions, and accumulation of mutations (reviewed by Gutierrez et al. 8 ).
In  Table 2). Similar results have been observed in some European countries, while higher polymorphism of B. grahamii has been observed in Asia 17 . The low diversity of B. grahamii has been explained by the spread of these bacteria from Asia to Europe by the introduction of its hosts and/ or a severe bottleneck relatively with too little time having elapsed for polymorphisms to reaccumulate 17 .
B. rochalimae is typically associated with carnivores (cats, dogs, foxes and raccoons), and Eremeeva et al. 18 reported clinical case of bacteremia, fever, and splenomegaly in a patient who traveled to Peru. In Europe, Bartonella spp. related to B. rochalimae group have been detected in A. agrarius, A. flavicollis and M. arvalis from Slovakia 11,19 . We report Bartonella spp. from B. rochalimae group in M. oeconomus for the first time (Fig. 1).
The main vectors for the maintenance and transmission of B. grahamii, B. taylorii and B. rochalimae among populations of small mammals are fleas 5 , these also having been identified as a risk factor for the transmission of Bartonella pathogens to humans. In Lithuania, strains identical or similar to B. grahamii, B. taylorii and B. rochalimae were detected in five flea species with an overall prevalence of 29.1%. B. grahamii was also detected in Ixodes ricinus ticks 20 .
This study is the first report of B. doshiae infection in M. agrestis from Lithuania. In Europe, this species has been found with very low prevalence and genetic diversity in Apodemus spp., M. glareolus and M. agrestis 5 .
Previous studies strongly supported the association of B. tribocorum with rats of the genus Rattus 5 . However, Ko et al. 21 detected B. tribocorum in A. agrarius in South Korea. In the current study, the human pathogenic B. tribocorum was identified in A. agrarius in coastal (site 6) and continental (site 8) areas with 100% similarity to the B. tribocorum isolate from South Korea (Fig. 1). Kraljik et al. 19   coopersplainsensis in A. agrarius in the coastal area of the Nemunas River Delta. This result is unexpected, as B. coopersplainsensis is distributed in Asia and Australia, and the species spread in Europe is associated with rats. The detection of B. tribocorum and B. coopersplainsensis in A. agrarius could be explained through the accidental contact between A. agrarius and rats in agricultural habitats.
The highest Bartonella species diversity was detected in the specific habitats of flooded meadow and flooded forest within the Nemunas River Delta: five rodent species trapped in this area harbored six different Bartonella species ( Table 2). The high Bartonella species diversity detected in the Nemunas River Delta could be explained by another particular features of this area, namely that it is the most important stopover area for migratory birds in Lithuania, with about 200 species also breeding 22 . Migratory birds are important carriers for a variety of ectoparasites with a great potential to assist in their spread. An alternative explanation for the high Bartonella species diversity in the Nemunas Delta is that the area is also characterized by a high intensity of local shipping, thereby also providing a suitable environment for rats and consequently for the transmission of rat-related Bartonella spp.
In conclusion, the present study demonstrates that small rodents from both coastal and continental areas of Lithuania are frequently infected with Bartonella spp. Our findings provide evidence of a high diversity of Bartonella species and the presence of strains identical or closely related to the humans pathogenic B. grahamii, B. tribocorum, and B. rochalimae. This study provides the first detection of B. coopersplainsensis, B. doshiae and B. tribocorum in Lithuania. To our knowledge, it is also the first report of Bartonella spp. in the agriculture-related rodent species A. agrarius and indoor rodent species such as M. musculus in Lithuania.

Material and Methods
Ethical statement. Rodents Table 1).
In the western part of the country rodents were trapped on the Curonian Spit (sites 1-5, close to the Baltic Sea) and in the Nemunas River Delta (sites 6-7). The Curonian Spit is a narrow sand peninsula (2 km wide and 98 km