Use of an exclusion assay to detect novel rickettsiae in field collected Amblyomma americanum.

In the south-central United States, several tick-borne diseases (TbDs) occur at or near their highest levels of incidence of anywhere in the U.S. The diversity of Rickettsia species found in Amblyomma americanum continues to be under-characterized in this region and throughout the U.S. and Canada where this tick species is expanding. One reason for this lack of knowledge about Rickettsia diversity is the high prevalence of the endosymbiont Rickettsia amblyommatis that obscures detection of other bacteria in this genus. Focusing on unknown rickettsial agents, we used a recently described R. amblyommatis exclusion assay to screen 1909 A. americanum collected in Oklahoma City, Oklahoma, which resulted in eight ticks that had unique rickettsial sequences. Through the process of characterizing primary and secondary rickettsiae, we identified ticks primarily infected with Rickettsia rhipicephali and a Rickettsia species (2019-CO-FNY) previously linked with a canine rickettsiosis case in Tulsa, Oklahoma. We also identified a Rickettsia agent that was 97% identical with an endosymbiont of Amblyomma tonelliae and which aligned with archaic rickettsial species. Through this study, we further demonstrate the usefulness of this exclusion assay for rapid screening in large cohort A. americanum studies to identify a small number of ticks that contain poorly described and previously undocumented rickettsiae.