Borrelia burgdorferi not confirmed in human-biting Amblyomma americanum ticks from the southeastern United States

Abstract

The predominant human-biting tick throughout the southeastern United States is Amblyomma americanum. Its ability to transmit pathogens causing Lyme disease-like illnesses is a subject of ongoing controversy. Results of previous testing by the Department of Defense Human Tick Test Kit Program and other laboratories indicated that it is highly unlikely that A. americanum transmits any pathogen that causes Lyme disease. In contrast, a recent publication by Clark and colleagues (K. L. Clark, B. Leydet, and S. Hartman, Int. J. Med. Sci. 10:915-931, 2013) reported detection of Lyme group Borrelia in A. americanum using a nested-flagellin-gene PCR. We evaluated this assay by using it and other assays to test 1,097 A. americanum ticks collected from humans. Using the Clark assay, in most samples we observed nonspecific amplification and nonrepeatability of results on subsequent testing of samples. Lack of reaction specificity and repeatability is consistent with mispriming, likely due to high primer concentrations and low annealing temperatures in this protocol. In six suspect-positive samples, Borrelia lonestari was identified by sequencing of an independent gene region; this is not a Lyme group spirochete and is not considered zoonotic. B. burgdorferi was weakly amplified from one pool using some assays, but not others, and attempts to sequence the amplicon of this pool failed, as did attempts to amplify and sequence B. burgdorferi from the five individual samples comprising this pool. Therefore, B. burgdorferi was not confirmed in any sample. Our results do not support the hypothesis that A. americanum ticks are a vector for Lyme group Borrelia infections.

FIG 1

Agarose gels after nested PCR to amplify the Borrelia flaB gene (26) in Amblyomma americanum ticks. Nonspecific binding was present in all tick samples but absent in the negative-control lane (no. 2) and the B. burgdorferi B31 positive-control lane (no. 12). Only samples with bright bands at 437 bp were considered suspect positive in our study. (A) Lanes 3 to 11 correspond to pooled samples of ticks. (B) Lanes 3 to 8 correspond to individual tick samples from previously tested pools, and lanes 9 to 11 are retests of pooled samples. Pooled sample P123 (gel A, lane 7) and individual sample 131114 from P123 (gel B, lane 8) were confirmed to be infected with B. lonestari by PCR and sequencing of the IGS PCR product and by IA/PCR/ESI-MS (Table 2).