Andrea Varela-Stokes

Isolation of Rickettsia parkeri and Identification of a Novel Spotted Fever Group Rickettsia sp. from Gulf Coast Ticks (Amblyomma maculatum) in the United States

ABSTRACT Until recently, Amblyomma maculatum (the Gulf Coast tick) had garnered little attention compared to other species of human-biting ticks in the United States. A. maculatum is now recognized as the principal vector of Rickettsia parkeri, a pathogenic spotted fever group rickettsia (SFGR) that causes an eschar-associated illness in humans that resembles Rocky Mountain spotted fever. A novel SFGR, distinct from other recognized Rickettsia spp., has also been detected recently in A. maculatum specimens collected in several regions of the southeastern United States. In this study, 198 questing adult Gulf Coast ticks were collected at 4 locations in Florida and Mississippi; 28% of these ticks were infected with R. parkeri, and 2% of these were infected with a novel SFGR. Seventeen isolates of R. parkeri from individual specimens of A. maculatum were cultivated in Vero E6 cells; however, all attempts to isolate the novel SFGR were unsuccessful. Partial genetic characterization of the novel SFGR revealed identity with several recently described, incompletely characterized, and noncultivated SFGR, including “Candidatus Rickettsia andeanae” and Rickettsia sp. Argentina detected in several species of Neotropical ticks from Argentina and Peru. These findings suggest that each of these “novel” rickettsiae represent the same species. This study considerably expanded the number of low-passage, A. maculatum-derived isolates of R. parkeri and characterized a second, sympatric Rickettsia sp. found in Gulf Coast ticks.

Role of the lone star tick, Amblyomma americanum (L.), in human and animal diseases.

We reviewed scientific literature pertaining to known and putative disease agents associated with the lone star tick, Amblyomma americanum. Reports in the literature concerning the role of the lone star tick in the transmission of pathogens of human and animal diseases have sometimes been unclear and even contradictory. This overview has indicated that A. americanum is involved in the ecology of several disease agents of humans and other animals, and the role of this tick as a vector of these diseases ranges from incidental to significant. Probably the clearest relationship is that of Ehrlichia chaffeensis and A. americanum. Also, there is a definite association between A. americanum and tularemia, as well as between the lone star tick and Theileria cervi to white-tailed deer. Evidence of Babesia cervi (= odocoilei) being transmitted to deer by A. americanum is largely circumstantial at this time. The role of A. americanum in cases of southern tick-associated rash illness (STARI) is currently a subject of intensive investigations with important implications. The lone star tick has been historically reported to be a vector of Rocky Mountain spotted fever rickettsiae, but current opinions are to the contrary. Evidence incriminated A. americanum as the vector of Bullis fever in the 1940s, but the disease apparently has disappeared. Q fever virus has been found in unfed A. americanum, but the vector potential, if any, is poorly understood at this time. Typhus fever and toxoplasmosis have been studied in the lone star tick, and several non-pathogenic organisms have been recovered. Implications of these tick-disease relationships are discussed.

Rickettsia parkeri Rickettsiosis, Argentina.

Rickettsia parkeri, a recently identified cause of spotted fever rickettsiosis in the United States, has been found in Amblyomma triste ticks in several countries of South America, including Argentina, where it is believed to cause disease in humans. We describe the clinical and epidemiologic characteristics of 2 patients in Argentina with confirmed R. parkeri infection and 7 additional patients with suspected R. parkeri rickettsiosis identified at 1 hospital during 2004–2009. The frequency and character of clinical signs and symptoms among these 9 patients closely resembled those described for patients in the United States (presence of an inoculation eschar, maculopapular rash often associated with pustules or vesicles, infrequent gastrointestinal manifestations, and relatively benign clinical course). Many R. parkeri infections in South America are likely to be misdiagnosed as other infectious diseases, including Rocky Mountain spotted fever, dengue, or leptospirosis.

Rickettsia parkeri in Amblyomma maculatum Ticks, North Carolina, USA, 2009–2010

We detected Rickettsia parkeri in 20%−33% of Amblyomma maculatum ticks sampled in North Carolina. Results highlight the high frequencies of R. parkeri–infected ticks in the state with the highest annual incidence of Rocky Mountain spotted fever. Epidemiologic studies are needed to definitively link R. parkeri to cases of spotted fever rickettsiosis.

Rickettsia parkeri and Candidatus Rickettsia andeanae in Gulf Coast ticks, Mississippi, USA.

To the Editor: Rickettsia parkeri, a spotted fever group Rickettsia (SFGR) bacterium, is transmitted by Amblyomma maculatum, the Gulf Coast tick (1). The prevalence of R. parkeri in Gulf Coast ticks has been reported as <42% in the United States, which is higher than reported rates of R. rickettsii (the cause of Rocky Mountain spotted fever) in Dermacentor species ticks. Misdiagnosis among SFGR infections is not uncommon, and R. parkeri rickettsiosis can cause symptoms similar to those for mild Rocky Mountain spotted fever (1). We evaluated infection rates of R. parkeri and Candidatus Rickettsia andeanae, a recently identified but incompletely characterized SFGR, in Gulf Coast ticks in Mississippi, USA. During May–September 2008–2010, we collected adult Gulf Coast ticks from vegetation at 10 sites in Mississippi. We extracted genomic DNA from the ticks using the illustra tissue and cells genomicPrep Mini Spin Kit (GE Healthcare Life Sciences, Piscataway, NJ, USA). We tested amplifiable tick DNA by PCR of the tick mitochondrial 16S rRNA gene (2). We tested for molecular evidence of any SFGR species by nested PCR of rompA (rickettsial outer membrane protein A gene) (1). Samples positive for SFGR were subsequently tested by using species-specific rompA PCR for R. parkeri (3) and Candidatus R. andeanae (4). All PCRs included 1) a positive control of DNA from cultured R. parkeri– (Tate’s Hell strain) or Candidatus R. andeanae–infected Gulf Coast ticks and 2) a negative control of water (nontemplate). PCR products were purified by using Montage PCR Centrifugal Filter Devices (Millipore, Bedford, MA, USA) and sequenced by using Eurofins MWG Operon (Huntsville, AL, USA). We generated consensus sequences using ClustalW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/) alignment and identified the sequences using GenBank BLAST searches (www.ebi.ac.uk/Tools/clustalw2/). Proportions of ticks infected with SFGR, by region and year, were compared separately by using Fisher exact test followed by pairwise comparisons with a Bonferroni adjustment (PROC FREQ, SAS for Windows, V9.2; SAS Institute, Cary, NC, USA). For all analyses, p<0.05 was considered significant. An index of co-infection was calculated by using the formula IC = ([O – E]/N) × 100, in which IC is index of co-infection, O is number of co-infections, E is expected occurrence of co-infection caused by chance alone, and N is total number of ticks infected by either or both Rickettsia species. A χ2 test was used to determine statistical significance (5). A total of 707 adult Gulf Coast ticks were collected during the 3 years (350 in 2008, 194 in 2009, and 163 in 2010). Tick mitochondrial 16S rRNA gene was detected in 698 (98.7%), of which 128 (18.3%) were positive for SFGR DNA, comprising 106 (15.2%) positive only for R. parkeri, 10 (1.4%) positive only for Candidatus R. andeanae, and 12 (1.7%) co-infected with R. parkeri and Candidatus R. andeanae (Table). Positive test results from 22 ticks singly or co-infected with Candidatus R. andeanae were confirmed by sequencing. Table PCR results for adult Rickettsia parkeri– and Candidatus Rickettsia andeanae–infected Gulf Coast ticks (Amblyomma maculatum) collected from 10 sites in Mississippi, USA, 2008–2010* Most (94.6%) ticks were from northern (n = 260) and southern (n = 409) Mississippi (Technical Appendix Figure). No significant difference in the number of R. parkeri–infected ticks between northern and southern Mississippi was observed (p = 0.13) (Table). However, significantly more ticks were singly infected with Candidatus R. andeanae in southern sites than in northern sites (p = 0.03). The infection rate for co-infected ticks in southern sites was higher than that in northern sites (p = 0.06). Among the 3 collection years for northern and southern sites, only the prevalence of R. parkeri in singly infected ticks differed significantly (p = 0.01) (data not shown); the infection rate was significantly greater during 2010 than during 2009 (p = 0.003, α/3 = 0.02). The overall index of co-infection with R. parkeri and Candidatus R. andeanae was 6.5, statistically higher than expected by chance alone (Table) (p<0.0001). The overall prevalence of infection with SFGR species in Gulf Coast ticks sampled was 18.3%; 15.2% of ticks were singly infected with R. parkeri, and 1.7% were infected with R. parkeri and Candidatus R. andeanae. As reported, the frequency of R. parkeri in Gulf Coast ticks is generally high, ranging from ≈10% to 40% (3,4,6–8). We found approximately 1 R. parkeri-infected Gulf Coast tick for every 6 ticks tested, suggesting that infected Gulf Coast ticks are commonly encountered in Mississippi. Because Gulf Coast ticks are among the most common human-biting ticks in Mississippi (9), awareness of R. parkeri rickettsiosis should be increased in this state. We identified Candidatus R. andeanae in ≈3% of Gulf Coast ticks in Mississippi; this frequency is similar to those reported in other studies of Gulf Coast ticks in the southern United States (4,6). Our finding of co-infected Gulf Coast ticks is at a frequency significantly higher than expected from chance alone. The biologic role of co-infections of Gulf Coast ticks with R. parkeri and Candidatus R. andeanae remains to be determined. Technical Appendix: Distribution of Gulf Coast ticks and cases of rickettsiosis. Click here to view.(117K, pdf)

Isolation of “Candidatus Rickettsia andeanae” (Rickettsiales: Rickettsiaceae) in Embryonic Cells of Naturally Infected Amblyomma maculatum (Ixodida: Ixodidae)

ABSTRACT The Gulf Coast tick, Amblyomma maculatum Koch, has become increasingly important in public health for its role as a vector of the recently recognized human pathogen, Rickettsia parkeri. More recently, these ticks were also found to harbor a novel spotted fever group rickettsia, “Candidatus Rickettsia andeanae.” First identified in Peru, and subsequently reported in ticks collected in the United States, Chile, and Argentina, “Ca. R. andeanae” remains largely uncharacterized, in part because of the lack of a stable isolate. Although the isolation of “Ca, R. andeanae” was recently described in DH82, Vero, and Drosophila S2 cells, its stability in these cell lines was not shown. To evaluate “Ca. R. andeanae” transmission and pathogenicity in vertebrates, as well as further describe biological characteristics of this candidate species to fulfill criteria for its establishment as a new species, availability of a stable isolate is essential. Here we describe the propagation of “Ca. R. andeanae” by using a primary culture derived from naturally infected A. maculatum embryos. Subsequent passage of the “Ca. R. andeanae” isolate to ISE6 (Ixodes scapularis embryonic) and Vero (African green monkey kidney epithelial) cell lines demonstrated limited propagation of the rickettsiae. Treatment of the infected primary cells with tetracycline resulted in cultures negative for “Ca. R. andeanae” by polymerase chain reaction and microscopy. Establishment of an isolate of “Ca. R. andeanae” will promote further investigation into the significance of this tick-associated rickettsia, including its role in spotted fever and interactions with the sympatric species, R. parkeri in A. maculatum.

Detection of Vector-Borne Agents in Lone Star Ticks, Amblyomma americanum (Acari: Ixodidae), from Mississippi

ABSTRACT In this study, we evaluated Amblyomma americanum (lone star tick) in Mississippi for the presence of Ehrlichia chaffeensis, causative agent of human monocytic ehrlichiosis; Ehrlichia ewingii, causative agent of human and canine granulocytic ehrlichiosis; Borrelia lonestari, putative agent of southern tick-associated rash illness; Francisella tularensis, the agent of tularemia; and Rickettsia spp., particularly R. amblyommii, a suspected pathogen, We collected adult A. americanum from four regions of Mississippi: Northeast, Northwest, Southeast, and East. Of the ticks collected, 192 were dissected and DNA was extracted for nested polymerase chain reaction (PCR) assays to detect the above bacteria. In all, 3% of tick extracts had evidence of Borrelia sp., 4% for E. chaffeensis, 6% for E. ewingii, and 44% for a Rickettsia species. As determined by sequencing, most Rickettsia spp, were R. amblyommii. In addition, extracts from 42 pools (total of 950) of larval A. americanum collected in Southwest Mississippi were tested for the presence of E. chaffeensis and Rickettsia species. Of these extracts from pools, nine of 37 (24%) were PCR positive for a Rickettsia sp., most often, R. amblyommii; none had evidence of E. chaffeensis, supporting the ability of lone star ticks to transovarially transmit R. amblyommii, but not E. chaffeensis. This study demonstrates E. chaffeensis, E. ewingii, “B. lonestari”, and R. amblyommii in A. americanum by PCR for the first time in Mississippi. Understanding the prevalence and epidemiology of these agents in Mississippi should increase awareness of tick-borne disease in the medical community.

Evidence of antibodies to spotted fever group rickettsiae in small mammals and quail from Mississippi.

Rickettsia parkeri is a recently recognized human pathogen primarily associated with the Gulf Coast tick Amblyomma maculatum, with immature stages of this tick reported from wild vertebrates. To better understand the role of vertebrates in the natural history of this bacterium, we evaluated small mammals and ground-dwelling birds for evidence of infection with R. parkeri or exposure to the organism. We sampled small mammals (n=39) and passerines (n=47) in both north-central and southeast Mississippi, while northern bobwhite (Colinus virginianus) samples (n=31) were obtained from farms in central Mississippi. Blood from all sampled animals was tested using polymerase chain reaction (PCR) for spotted fever group rickettsiae (SFGR), and for antibodies to SFGR using R. parkeri antigen. Ectoparasite samples were removed from animals and included mites, lice, fleas, and immature ticks. Of 39 small mammal samples collected, 7 were positive for antibodies to SFGR; none tested positive by PCR for DNA of SFGR. Of 47 passerine blood samples collected, none were positive for DNA of SFGR by PCR, nor did any show serological evidence of exposure. Finally, none of 31 northern bobwhite samples tested were positive for SFGR DNA, while 7 were seropositive for rickettsial antibodies. Detection of seropositive rodents and quail suggests a role for these host species in the natural history of SFGR, possibly including R. parkeri, but the extent of their role has not yet been elucidated.

Cattle and the natural history of Rickettsia parkeri in Mississippi.

Cattle have been recognized as hosts for Amblyomma maculatum, the Gulf Coast tick, for over 100 years. For nearly as long, A. maculatum have been known to harbor the spotted fever group Rickettsia (SFGR), now known as Rickettsia parkeri. However, human infection with R. parkeri was not documented until 2004. Results presented herein describe a laboratory and a field study evaluating cattle and the natural history of A. maculatum and R. parkeri in Mississippi. In the laboratory study, seroconversion to R. parkeri antigen occurred in calves exposed to R. parkeri by injection or by feeding R. parkeri-infected A. maculatum, and two out of six animals were transiently rickettsemic. All calves remained clinically normal during the study, except for gotch ear-like lesions in all tick-infested calves, regardless of infection status of ticks, suggesting that R. parkeri is not involved in the condition. In the field study, A. maculatum (n=34) removed from Mississippi sale barn cattle (n=183) and the cattle hosts were tested for R. parkeri. Cattle were not rickettsemic by polymerase chain reaction, but 49.7% demonstrated low titers to R. parkeri antigen when tested by indirect fluorescent antibody for SFGR. Of ticks removed from cattle, 11.8% were hemolymph positive and 8.7% were indirect fluorescent antibody positive. Approximately 22% (5/23) and 4% (1/23) of harvested tick extracts were positive for R. parkeri by polymerase chain reaction of the 17 kDa antigen gene and ompA gene, respectively. An amplicon for the ompA gene from one tick was successfully sequenced and showed 100% similarity with the homologous sequence of R. parkeri. Thus, cattle may harbor R. parkeri-infected A. maculatum and produce antibodies to SFGR. Cattle may play a role in the natural history of R. parkeri infection by expanding populations of A. maculatum and transporting R. parkeri-infected ticks to various locations, rather than as a reservoir for R. parkeri.

Artificial infection of the bed bug with Rickettsia parkeri.

ABSTRACT Although a variety of disease agents have been reported from bed bugs, the mechanical and biological disease transmission potential of bed bugs remains unelucidated. In this study we assayed survivability of the mildly pathogenic spotted fever group rickettsia, Rickettsia parkeri, in bed bugs after feeding on R. parkeri-infected chicken blood. Two groups of 15 adult bed bugs each were fed on infected or noninfected blood, and two groups of fourth-instar bed bugs also were fed on either infected or noninfected blood. One group of 15 adult bed bugs received no bloodmeal and was included as an additional control. Two weeks postfeeding, two pools of five live bed bugs from each group were surface sterilized, macerated, and placed in Vero cell cultures in an attempt to grow live organism. The remaining five individual bed bugs from each group were dissected, their salivary glands were removed for immunofluorescence assay (IFA) staining, and the remaining body parts were processed for polymerase chain reaction (PCR) analysis. Results indicated that no immature (now molted to fifth instar) bed bugs were positive for R. parkeri by IFA or PCR, indicating that organisms did not survive the molting process. After 4 wk of cell culture, no organisms were seen in cultures from any of the treatment or control groups, nor were any cultures PCR positive. However, two of the adult bed bugs were IFA positive for rickettsia-like organisms, and these two specimens were also PCR positive using R. parkeri-specific primers. These IFA and PCR results indicate that remnants of Rickettsia parkeri (possibly whole organisms) survived in the bugs for 2 wk, but the viability of the organisms in these two specimens could not be determined.