Human granulocytic ehrlichiosis is an emerging infectious disease in the United States and Europe, and PCR methods have been shown to be effective for the diagnosis of acute infections. Numerous PCR assays and primer sets have been reported in the literature. The analytical sensitivities (limits of detection) of 13 published PCR primer sets were compared using DNA extracted from serial dilutions of Anaplasma phagocytophilum-infected HL-60 cells. The specificity of the assays that were able to detect <2.5 infected cells was tested by the use of template DNA extracted from Ehrlichia chaffeensis, Rickettsia rickettsii, and Bartonella henselae. The assays with the lowest limits of detection were shown to be a nested assay that amplifies the 16S rRNA gene (primer pairs ge3a-ge10 [primary] and ge9-ge3 [nested]; detects 0.25 infected cell), a direct assay that amplifies the major surface protein gene msp2 (primer pair msp2-3f-msp2-3r; detects 0.25 infected cell), and a direct assay that amplifies the 16S rRNA gene (primer pair ehr521-ehr790; detects 0.25 infected cell). The specificity and limit of detection of the MSP2 and 16S rRNA direct assays were further tested by use of A. phagocytophilum template DNA from both North America and Europe and from human, tick, white-footed mouse, equine, deer, bovine, and wood rat samples and of template DNA from closely related species (Anaplasma marginale, the white-tailed deer agent, and additional E. chaffeensis-positive samples). Three manufacturers' PCR kits were tested and showed distinct variations in the limit of detection, specificity, and nonspecific background amplification. The importance of these results for the molecular diagnosis of human granulocytic ehrlichiosis is discussed.Human ehrlichiosis is an acute, febrile illness that is caused by at least three known agents in the United States. Ehrlichia chaffeensis infects mononuclear white blood cells, while Ehrlichia ewingii and Anaplasma phagocytophilum (previously Ehrlichia phagocytophila, the human granulocytic ehrlichiosis [HGE] agent) infect granulocytic-lineage leukocytes (2, 6-9, 11). HGE was first described in 1994, and the number of confirmed cases has increased to the point where it is now the predominant form of ehrlichiosis (3,7,20; J. McQuiston, personal communication) and second to Lyme disease as the most common tick-borne infection in the United States. Tetracyclines are efficacious for the treatment of all forms of ehrlichiosis; however, the nonspecific symptoms of infection can make diagnosis problematic. Serological methods are most commonly used for diagnosis, but these assays are often negative in the early stages of an acute infection, as antibody will typically be absent during the first week of infection (1). Diagnosis prior to seroconversion has utilized molecular methods that are based on PCR amplification of the DNA of the agent, and numerous PCR-based assays and primer sets have been reported (7,13,16,19,(24)(25)(26)(27)29). The purpose of this study was to compare a selection of these ass...

In 1973, investigators isolated a rickettsial organism, designated strain WB-8-2T, from an adult Amblyomma americanum tick collected at Land Between the Lakes National Recreation Area, TN, USA. This organism is now recognized as highly prevalent in A. americanum, as well as several other Amblyomma species found throughout the Western hemisphere. It has been suggested that cross-reactivity to WB-8-2T and similar strains contributes to the increasing number of spotted fever cases reported in the USA. In 1995, investigators provided preliminary evidence that this strain, as well as another strain from Missouri, represented a distinct taxonomic unit within the genus Rickettsia by evaluating sequences of the 16S rRNA and 17 kDa protein genes. However, the bacterium was never formally named, despite the use of the designation ‘Rickettsia amblyommii’ and later ‘Candidatus Rickettsia amblyommii’, for more than 20 years in the scientific literature. Herein, we provide additional molecular evidence to identify strain WB-8-2T as a representative strain of a unique rickettsial species and present a formal description for the species, with the proposed name modified to Rickettsia amblyommatis sp. nov. to conform to the International Code of Nomenclature of Prokaryotes. We also establish a pure culture of strain WB-8-2T and designate it as the type strain for the species. The type strain is WB-8-2T (=CRIRC RAM004T=CSURP2882T).

In the north-central United States, the blacklegged tick (Ixodes scapularis) is currently known to vector seven human pathogens. These include five bacteria (Borrelia burgdorferi sensu stricto, Borrelia mayonii, Borrelia miyamotoi, Anaplasma phagocytophilum, Ehrlichia muris eauclairensis), one protozoan (Babesia microti) and one virus (Powassan). We sought to assess the prevalence and distribution of these pathogens in host-seeking nymphs collected throughout Minnesota, a state on the northwestern edge of the tick's expanding range, where reported cases of I. scapularis-borne diseases have increased in incidence and geographic range over the past decade. Among the 1240 host-seeking I. scapularis nymphs that we screened from 64 sites, we detected all seven pathogens at varying frequencies. Borrelia burgdorferi s.s. was the most prevalent and geographically widespread, found in 25.24% of all nymphs tested. Anaplasma phagocytophilum and Babesia microti were also geographically widespread, but they were less prevalent than Bo. burgdorferi s.s. (detected in 6.29% and 4.68% of ticks, respectively). Spatial clusters of sites with high prevalence for these three pathogens were identified in the north-central region of the state. Prevalence was less than 1.29% for each of the remaining pathogens. Two or more pathogens were detected in 90 nymphs (7.26%); coinfections with Bo. burgdorferi s.s. and either A. phagocytophilum (51 nymphs, 4.11%) or Ba. microti (43 nymphs, 3.47%) were the most common combinations. The distribution and density of infected ticks mirrors the distribution of notifiable tick-borne diseases in Minnesota and provides information on the distribution and prevalence of recently described human pathogens.