A multiplex real-time PCR assay was developed for the simultaneous detection of Anaplasma phagocytophilum and Borrelia burgdorferi. The assay was tested on various Anaplasma, Borrelia, Erhlichia, and Rickettsia species, as well as on Bartonella henselae and Escherichia coli, and the assay was found to be highly specific for A. phagocytophilum and the Borrelia species tested (B. burgdorferi, B. parkeri, B. andersonii, and B. bissettii). The analytical sensitivity of the assay is comparable to that of previously described nested PCR assays (A. phagocytophilum, 16S rRNA; B. burgdorferi, fla gene), amplifying the equivalent of one-eighth of an A. phagocytophilum-infected cell and 50 borrelia spirochetes. The dynamic range of the assay for both A. phagocytophilum and B. burgdorferi was >4 logs of magnitude. Purified DNA from A. phagocytophilum and B. burgdorferi was spiked into DNA extracted from uninfected ticks and from negative control mouse and human bloods, and these background DNAs were shown to have no significant effect on sensitivity or specificity of the assay. The assay was tested on field-collected Ixodes scapularis ticks and shown to have 100% concordance compared to previously described non-probe-based PCR assays. To our knowledge, this is the first report of a real-time multiplex PCR assay that can be used for the simultaneous and rapid screening of samples for A. phagocytophilum and Borrelia species, two of the most common tick-borne infectious agents in the United States.Lyme disease and human anaplasmosis (formerly human granulocytic ehrlichiosis) have emerged as two of the most common vector-borne bacterial illnesses in the United States, where Borrelia burgdorferi and Anaplasma phagocytophilum have been identified as their respective etiologic agents (3)(4)(5)20). Both B. burgdorferi and A. phagocytophilum share common tick vectors and rodent reservoirs, with Ixodes scapularis characterized as the principal vector species and the white-footed mouse, Peromyscus leucopus, as a major reservoir species (3,14,19,22,25). Lyme disease and human anaplasmosis are found in overlapping regions of the United States, with most cases occurring in the northeast and upper midwest regions, areas that have large populations of both rodents and I. scapularis ticks.The screening of tick and rodent samples for B. burgdorferi and A. phagocytophilum has been used for studies designed to assess the prevalence of these agents in nature and the risk they pose to the human population. Numerous methods for the detection of B. burgdorferi and A. phagocytophilum have been described and include antibody and antigen detection assays (immunofluorescence assay, enzyme-linked immunosorbent assay, and Western blotting), culture isolation, and PCR-based assays. PCR assays have been shown to be both sensitive and specific for the detection of B. burgdorferi and A. phagocytophilum in clinical and field-collected samples (5,11,17,18,23,26). However, PCR assays can be time-consuming and laborintensive, particularly when testing for multi...
Primers were used to amplify a 561-bp region of the 16S rRNA gene of Ehrlichia phagocytophila from Ixodes scapularis ticks and small mammals collected in Rhode Island and Connecticut. DNA sequences for all 50 E. phagocytophila-positive samples collected from 1996 through 1998 in southwestern Connecticut were identical to the sequence reported for E. phagocytophila DNA from confirmed human cases. In contrast, the sequences from 92 of 123 E. phagocytophila-positive Rhode Island samples collected from 1996 through 1999 included several variants differing by 1-2 nucleotides from that in the agent infecting humans. While 11.9% of 67 E. phagocytophila-positive ticks collected during 1997 in Rhode Island harbored ehrlichiae with sequences identical to that of the human agent, 79.1% had a variant sequence not previously described. The low incidence of human ehrlichiosis in Rhode Island may in part result from interference by these variant ehrlichiae with maintenance and transmission of the true agent of human disease.
We examined the reservoir potential of white-tailed deer for Anaplasma phagocytophilum. Results suggest that white-tailed deer harbor a variant strain not associated with human infection, but contrary to published reports, white-tailed deer are not a reservoir for strains that cause human disease. These results will affect surveillance studies of vector and reservoir populations.
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