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Lice are blood-sucking insects that are of medical and veterinary significance as parasites and vectors for various infectious agents. More than half of described blood-sucking lice species are found on rodents. Rodents are important hosts of several Bartonella and Rickettsia species, and some of these bacteria are characterized as human pathogens in Europe. Rodent ectoparasites, such as fleas and ticks, are important vectors of Bartonella spp. and Rickettsia spp., but knowledge about the presence of these bacteria in lice is limited. The aim of this study was to determine the prevalence of Bartonella and Rickettsia bacteria in lice collected from rodents in Slovakia. The ectoparasites were collected from small rodents captured from 2010 to 2015 at four different sites in eastern Slovakia. The presence of Bartonella and Rickettsia species in lice samples was screened by real-time PCR, targeting ssrA and gltA genes, respectively. The molecular characterization of the Bartonella strains was based on sequence analysis of partial rpoB and intergenic spacer (ITS) genes, and of the Rickettsia species on sequence analysis of the gltA gene. A total of 1074 lice of seven species were collected from six rodent species. Bartonella DNA was detected in Hoplopleura affinis (collected from Apodemus agrarius, Apodemus flavicollis, and Myodes glareolus), Polyplax serrata (from A. agrarius), and Hoplopleura sp. (from A. flavicollis). Sequence analysis revealed that the Bartonella strains belonged to the Bartonella coopersplainsensis, Bartonella tribocorum, and Bartonella taylorii genogroups. Rickettsia DNA was detected in H. affinis and P. serrata collected from A. agrarius. Sequence analysis revealed two Rickettsia species: Rickettsia helvetica and Rickettsia sp. The results of the study confirm the presence of Bartonella spp. and Rickettsia spp. in lice collected from rodents.