European brown hare (Lepus europaeus Pallas 1778) is a broadly distributed lagomorph species in Europe, recognized as a host for Ixodes ricinus and reservoir of a wide range of pathogens with zoonotic potential. Even though Lepus europaeus represents an important game animal in Central Europe, the data available on Anaplasma phagocytophilum in this lagomorph are scarce. In this study, three populations of brown hare from distinct localities in the Czech Republic were analysed for the presence of Anaplasma phagocytophilum DNA. We used standard qPCR, targeting the msp2 gene and adapted the same assay also for digital droplet PCR. Out of 91 samples, these two methods identified 9 and 12 as positive, respectively. For taxonomic analysis, we amplified the groEL gene from five of six samples that were found positive by both methods. In phylogenetic analyses, this haplotype belongs to ecotype 1, and to the subclade with isolates from cervids and I. ricinus. Our findings underline the importance of correct result interpretation and positivity cut-off set-up for different detection methods of A. phagocytophilum. This bacterium is characterized by a high intraspecific variability and highly sensitive detection itself, is not enough. Detailed molecular typing is necessary to define the zoonotic potential of different strains and their natural reservoirs.

Biology Centre Institute of Parasitology Czech Academy of Sciences České Budějovice Czech Republic

Biomedical Center Faculty of Medicine in Pilsen Charles University Plzeň Czech Republic

CEITEC Central European Institute of Technology University of Veterinary Sciences Brno Brno Czech Republic

Department of Botany and Zoology Faculty of Science Masaryk University Brno Czech Republic

Department of Ecology and Diseases of Zoo Animals Game Fish and Bees University of Veterinary Sciences Brno Brno Czech Republic

Department of Pathology and Parasitology University of Veterinary Sciences Brno Brno Czech Republic

Department of Veterinary Sciences CINeZ Faculty of Agrobiology Food and Natural Resources Czech University of Life Sciences Prague Prague Czech Republic

Laboratory for Zoonoses and Environmental Microbiology National Institute for Public Health and the Environment Bilthoven The Netherlands

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European Brown hare (Lepus europaeus) as an accidental host of Trypanosoma pestanai