Evaluation of a modified next-generation sequencing assay to detect Borrelia, Anaplasma, and Babesia in blood and ear tissue samples from field-collected rodents.

Efforts to monitor the expanding geographic distribution of pathogens associated with Ixodes scapularis Say (Acari: Ixodidae), and to evaluate the outcomes of environmental control interventions aiming to reduce the intensity of enzootic transmission of these pathogens, are dependent on sensitive and specific molecular assays for detection of pathogens in ticks and their rodent hosts. In this study, we modified a highly sensitive and specific multiplex PCR amplicon sequencing assay developed to identify pathogens in Ixodes ticks for use in screening blood or ear tissue from rodents (Peromyscus leucopus Rafinesque (Rodentia: Cricetidae)) to detect Borrelia burgdorferi sensu stricto Johnson, Schmid, Hyde, Steigerwalt and Brenner (Spirochaetales: Spirochaetaceae), Borrelia miyamotoi Fukunaga (Spirochaetales: Spirochaetaceae), Anaplasma phagocytophilum (Foggie, 1949) Dumler (Rickettsiales: Anaplasmataceae), and Babesia microti Franca, 1910 (Piroplasmida: Babesiidae). The modified assay retained similarly high sensitivity to the original assay for detection of Bo. burgdorferi sensu stricto, Bo. miyamotoi, A. phagocytophilum, and Ba. microti. However, the probability of detecting each of the pathogens was dependent on the sample type (ear tissue or blood) tested. Generally, pathogens were detected in either ear tissue or blood samples from an individual mouse, but not in both sample types highlighting the need to test both blood and ear tissue from all individual animals to accurately categorize their infection status.