Approaches for Reverse Line Blot-Based Detection of Microbial Pathogens in Ixodes ricinus Ticks Collected in Austria and Impact of the Chosen Method

Ticks transmit a large number of pathogens capable of causing human disease. In this study, the PCR-reverse line blot (RLB) method was used to screen for pathogens in a total of 554Ixodes ricinus ticks collected from all provinces of Austria. These pathogens belong to the generaBorrelia ,Rickettsiae ,Anaplasma /Ehrlichia (including “Candidatus Neoehrlichia”),Babesia , andCoxiella . The pathogens with the highest detected prevalence were spirochetes of theBorrelia burgdorferi sensu lato complex, in 142 ticks (25.6%).Borrelia afzelii (80/142) was the most frequently detected species, followed byBorrelia burgdorferi sensu stricto (38/142) andBorrelia valaisiana (36/142).Borrelia garinii/Borrelia bavariensis ,Borrelia lusitaniae , andBorrelia spielmanii were found in 28 ticks, 5 ticks, and 1 tick, respectively.Rickettsia spp. were detected in 93 ticks (16.8%):R. helvetica (39/93),R. raoultii (38/93),R. monacensis (2/93), andR. slovaca (1/93). ThirteenRickettsia samples remain uncharacterized. “Candidatus Neoehrlichia mikurensis,”Babesia spp. (B. venatorum ,B. divergens ,B. microti ), andAnaplasma phagocytophilum were found in 4.5%, 2.7%, and 0.7%, respectively.Coxiella burnetii was not detected. Multiple microorganisms were detected in 40 ticks (7.2%), and the cooccurrence ofBabesia spp. and “Candidatus Neoehrlichia mikurensis” showed a significant positive correlation. We also compared different PCR-RLBs for detection ofBorrelia burgdorferi sensu lato andRickettsia spp. and showed that different detection approaches provide highly diverse results, indicating that analysis of environmental samples remains challenging.IMPORTANCE This study determined the wide spectrum of tick-borne bacterial and protozoal pathogens that can be encountered in Austria. Surveillance of (putative) pathogenic microorganisms occurring in the environment is of medical importance, especially when those agents can be transmitted by ticks and cause disease. The observation of significant coinfections of certain microorganisms in field-collected ticks is an initial step to an improved understanding of microbial interactions in ticks. In addition, we show that variations in molecular detection methods, such as in primer pairs and target genes, can considerably influence the final results. For instance, detection of certain genospecies of borreliae may be better or worse by one method or the other, a fact of great importance for future screening studies.