Novel PCR exclusion assay to detect spotted fever group rickettsiae in the lone star tick (Amblyomma americanum)

The lone star tick (Amblyomma americanum) is the most common and abundant human-biting tick in the southeastern United States where spotted fever rickettsioses frequently occur. However, the role of this tick in transmitting and maintaining pathogenic and non-pathogenic spotted fever group rickettsiae (SFGR) remains poorly defined. This is partially due to the high prevalence and abundance of Rickettsia amblyommatis in most populations of A. americanum. Many molecular assays commonly employed to detect rickettsiae use PCR primers that target highly conserved regions in the SFGR so low abundance rickettsia may not be detected when R. amblyommatis is present. It is costly and inefficient to test for low abundance rickettsial agents with multiple individual specific assays even when they are multiplexed, as most samples will be negative. Real time PCR assays may also be hampered by inadequate limits of detection (LODs) for low abundance agents. We exploited the absence of an otherwise relatively SFGR-conserved genome region in R. amblyommatis to design a hemi-nested PCR-assay which has a sensitivity of 10 copies in detecting the presence of most SFGR, but not R. amblyommatis in DNA of infected lone star ticks. This deletion is conserved in 21 isolates of R. amblyommatis obtained from multiple states. We demonstrated the assay’s utility by detecting a pathogenic SFGR, Rickettsia parkeri, in 15/50 (30 %) of field collected A. americanum ticks that were previously screened with conventional assays and found to be positive for R. amblyommatis. These co-infected ticks included 1 questing female, 6 questing nymphs, and 8 attached males. The high prevalence of R. parkeri among host-attached ticks may be due to several variables and does not necessarily reflect the risk of disease transmission from attached ticks to vertebrate hosts. This novel assay can provide accurate estimates of the prevalence of less common SFGR in A. americanum and thus improve our understanding of the role of this tick in the maintenance and transmission of the SFGR commonly responsible for human rickettsioses.