Rickettsia parkeri in Amblyomma americanum Ticks, Tennessee and Georgia, USA

To determine the geographic distribution of the newly recognized human pathogen Rickettsia parkeri, we looked for this organism in ticks from Tennessee and Georgia, USA. Using PCR and sequence analysis, we identified R. parkeri in 2 Amblyomma americanum ticks. This rickettsiosis may be underdiagnosed in the eastern United States.

T he most commonly reported rickettsial human patho- gen in the United States is Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever (RMSF).First identified in 1937 (1), R. parkeri has been recognized as a human pathogen only since 2004, when it was isolated from an eschar on a serviceman from Virginia (2), although an Ohio patient suspected to have RMSF died of R. parkeri rickettsiosis in 1990 (3).Little is known about the geographic distribution of R. parkeri in the United States or the epidemiology of the disease it causes.The primary vector of R. parkeri is thought to be the Gulf Coast tick (Amblyomma maculatum), and naturally infected Gulf Coast ticks have been reported in numerous southeastern states (4).Experimentally, A. americanum ticks can maintain and transmit R. parkeri (5); thus, this tick species, which is more abundant than A. maculatum and bites humans aggressively (6), might contribute to R. parkeri transmission.To determine the geographic distribution of R. parkeri, we examined ticks collected in Tennessee and Georgia.

The Study
In Georgia, during 2005-2006, residents were encouraged to submit ticks to the state's Division of Public Health for identification and testing for various tick-borne pathogens.We studied only ticks that had been attached to persons.Ticks were individually homogenized with metal beads and resuspended in 225 μL of phosphate-buffered saline.DNA was extracted from 100 μL of the homogenate by using a QIAamp DNA Micro Kit (QIAGEN Inc, Valencia, CA, USA) according to the manufacturer's instructions.We conducted a nested PCR targeting the 17-kDa gene (7) and used bidirectional sequencing at the Integrated Biotechnology Laboratories (University of Georgia, Athens, GA, USA) to confirm positive results.
In Tennessee, from April 2007 through September 2008, the United States Department of Agriculture, Animal and Plant Health Inspection Service, Wildlife Services, and the Tennessee Department of Health collected A. americanum and A. maculatum ticks from 31 counties by examining wild animals and dragging flannel sheets through vegetation.Ticks were stored in 100% ethanol and were sent to the Tennessee Department of Health Vector-Borne Diseases Laboratory for identification of species and life stage and detection of pathogens by molecular testing.A PCR targeting the outer membrane protein A (rOmpA) gene of spotted fever group (SGF) rickettsiae was conducted as previously described, using primers Rr190.602n and Rr190.70p(8).To identify the rickettsiae species, we subjected positive samples to a restriction fragment length polymorphism (RFLP) assay by digestion of the amplicons with RsaI (Promega, Madison, WI, USA) and PstI (Fermentas, Glen Burnie, MD, USA) enzymes at 37°C for 2 h.Digested fragments were subjected to electrophoresis on 10% polyacrylamide gels.To confirm species identification, we purified representative positive PCR products with Exosap-It (USB Corporation, Cleveland, OH, USA), sequenced at the Tennessee Department of Health Laboratory Services (Nashville, TN, USA) and entered into the National Center for Biotechnology Information BLAST database (www.blast.ncbi.nlm.nih.gov/Blast.cgi).
Of 611 A. americanum and 2 A. maculatum ticks collected in Tennessee, 446 A. americanum (164 adults and 282 nymphs) and 2 A. maculatum adults were individually tested for Rickettsia spp.An additional 103 A. americanum larvae were divided in 10 pools of 4-19 ticks each according to collection site.A single A. americanum adult male (0.2% of total) had a positive RFLP pattern that matched the previously described rOmpA gene pattern of R. parkeri (8).The sequence of this amplicon (532 bp, GenBank ac-cession no.FJ793521) was 99% similar to that of R. parkeri (EU715288).The positive tick had been collected from a coyote in Knox County, Tennessee, in July 2007.

Conclusions
We identified R. parkeri in ticks in Tennessee and Georgia.In Tennessee, our identification of only 2 A. maculatum ticks (the primary vector of R. parkeri) supports previous reports that this tick species is uncommon in Tennessee (9).In Georgia, the 19 A. maculatum ticks identified were <5% of the total number of ticks collected in Georgia.In contrast to these low numbers, A. americanum ticks are ubiquitous in high densities throughout the other southeastern states and readily feed on humans (8).Recently, the range of A. americanum ticks has expanded in the United States and now extends from west-central Texas to the Atlantic Coast, encompassing the entire Southeast and parts of the lower Midwest and coastal New England (10).The distribution of A. americanum ticks completely overlaps the suspected distribution of A. maculatum ticks (Figure ).The positive A. americanum tick from Tennessee was collected from a free-ranging coyote, and the detected R. parkeri may have been present in the blood meal taken by the tick.However, A. americanum ticks can maintain and transmit R. parkeri infection both transovarially and transtadially (6).R. parkeri has previously been identified in A. maculatum ticks from Georgia (4), but the identification of R. parkeri in A. americanum ticks renews concerns that this tick species may be involved in the natural history of another zoonotic pathogen.Additional study is needed to determine the extent of the role of A. americanum ticks as a natural vector for R. parkeri.
This study raises concerns about the serologic diagnosis of RMSF.R. parkeri may be the etiologic agent of some rickettsiosis cases in Tennessee and Georgia that have been misdiagnosed as RMSF.Because reliable clinical tests specific for different SFG rickettsiae are not readily available, several different rickettsioses may be serologically cross-reactive, leading to misdiagnosis of RMSF (11)(12)(13)(14).Reliable diagnosis requires PCR or culture of biopsy specimens from eschars, when present (15).Additional studies characterizing SFG rickettsioses, including development of rickettsial species-specific clinical tests, will assist in attributing rickettsiosis to R. rickettsii, R. parkeri, or other SFG rickettsial infections.

Figure .
Figure.Location of ticks, Rickettsia parkeri in ticks, and human cases of rickettsiosis in the United States.Green shading indicates approximate distribution of Amblyomma americanum ticks, which completely overlaps with the known or suspected distribution of A. maculatum.Yellow circles indicate locations where R. parkeri was detected in A. americanum ticks (this study).Red circles indicate locations of confirmed or suspected cases of R. parkeri infection in humans (11).
This research was supported by an appointment to the Emerging Infectious Diseases Fellowship Program administered by the Association of Public Health Laboratories and funded by the Centers for Disease Control and Prevention.Ms Cohen completed this work as part of an Emerging Infectious Diseases Laboratory Training Fellowship at the Tennessee Department of Health.She is entering a doctoral degree program at Cornell University to pursue her interests in infectious disease.Emerging Infectious Diseases • www.cdc.gov/eid• Vol. 15, No. 9, September 2009