Rickettsia rickettsii subsp. californica subsp. nov., the etiologic agent of Pacific Coast tick fever.

The etiologic agent of Pacific Coast tick fever (PCTF), a moderately severe tickborne illness that resembles Rocky Mountain spotted fever (RMSF), was first isolated in 1966 from specimens of Dermacentor occidentalis (the Pacific Coast tick) obtained in California. For several decades, this bacterium was identified ambiguously as the unclassified spotted fever group Rickettsia species 364-D, Rickettsia 364, or Rickettsia philipii. However, none of these epithets satisfied criteria of formal bacterial nomenclature. Data developed from mouse serotyping studies performed 45 years ago, and multi-locus sequence typing several decades later, indicated that this bacterium was similar to, but distinct from isolates of Rickettsia rickettsii, the etiological agent of RMSF. We applied an integrative taxonomic approach, combining phenotypic, ecological, and clinical data with whole genome sequencing of 11 contemporary isolates of this pathogen to identify it as a distinct subspecies of R. rickettsii, and propose the name Rickettsia rickettsii subsp. californica subsp. nov.

Applying MALDI-TOF MS to resolve morphologic and genetic similarities between two Dermacentor tick species of public health importance.

Hard ticks (Acari: Ixodidae) have been historically identified by morphological methods which require highly specialized expertise and more recently by DNA-based molecular assays that involve high costs. Although both approaches provide complementary data for tick identification, each method has limitations which restrict their use on large-scale settings such as regional or national tick surveillance programs. To overcome those obstacles, the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been introduced as a cost-efficient method for the identification of various organisms, as it balances performance, speed, and high data output. Here we describe the use of this technology to validate the distinction of two closely related Dermacentor tick species based on the development of the first nationwide MALDI-TOF MS reference database described to date. The dataset obtained from this protein-based approach confirms that tick specimens collected from United States regions west of the Rocky Mountains and identified previously as Dermacentor variabilis are the recently described species, Dermacentor similis. Therefore, we propose that this integrative taxonomic tool can facilitate vector and vector-borne pathogen surveillance programs in the United States and elsewhere.

Phylogenetic Differentiation of Rickettsia parkeri Reveals Broad Dispersal and Distinct Clustering within North American Strains.

The tick-borne pathogen Rickettsia parkeri causes a mild rickettsiosis, with cases reported from several countries to its known distribution in the Americas. Molecular analyses have identified a clear distinction between strains of R. parkeri sensu stricto (s. s.) and R. parkeri sensu lato (s. l.) as well as separation between North American and South American R. parkeri s. s. strains. To expand on this previous work, we developed a multilocus sequence typing analysis with two aims: first, to investigate the genetic diversity within strains of North American R. parkeri s. s., and second, to further the understanding of the genetic relationships between R. parkeri s. s. and R. parkeri s. l. Sixty-four R. parkeri isolates and 12 R. parkeri-positive tick lysates were analyzed using a novel typing scheme consisting of four coding regions and two intergenic regions. A concatenated Bayesian phylogeny that identified eight clades was constructed: three represent the R. parkeri s. l. strains, and five represent the R. parkeri s. s. strains. The clades appear to be generally phylogeographically organized and associated with specific tick vectors. However, while one of the four R. parkeri s. s. North American clades appears to be limited to the southwestern United States, the other North American clades exhibit broad dispersal, most notably seen in the largest group, which includes representative samples extending from northern Mexico to Delaware. This work highlights the increasingly recognized geographic range of R. parkeri in the Americas and suggests a potential public health risk for these areas. IMPORTANCE Since 1937, when Rickettsia parkeri was originally identified in Amblyomma maculatum group ticks, the recognized range and associated vectors for this pathogen have expanded significantly. In recent years, R. parkeri has been identified in 12 tick species from seven countries in the Americas. Herein, we provide evidence that the greatest genetic diversity within R. parkeri exists in North America, where one R. parkeri sensu lato and four R. parkeri sensu stricto genotypes are present. While one distinct R. parkeri sensu stricto genotype exists only in the southwestern United States, three genotypes are broadly distributed in the eastern United States, with the largest of these found across the known range of R. parkeri in North America. In contrast, the South American R. parkeri sensu stricto samples represent a single genotype and are completely clonal at the loci analyzed, irrespective of their country of origin.

Isolate-Dependent Differences in Clinical, Pathological, and Transcriptional Profiles following In Vitro and In Vivo Infections with Rickettsia rickettsii.

Rickettsia rickettsii, the etiological agent of Rocky Mountain spotted fever (RMSF), a life-threatening tick-borne disease that affects humans and various animal species, has been recognized in medicine and science for more than 100 years. Isolate-dependent differences in virulence of R. rickettsii have been documented for many decades; nonetheless, the specific genetic and phenotypic factors responsible for these differences have not been characterized. Using in vivo and in vitro methods, we identified multiple phenotypic differences among six geographically distinct isolates of R. rickettsii, representing isolates from the United States, Costa Rica, and Brazil. Aggregate phenotypic data, derived from growth in Vero E6 cells and from clinical and pathological characteristics following infection of male guinea pigs (Cavia porcellus), allowed separation of these isolates into three categories: nonvirulent (Iowa), mildly virulent (Sawtooth and Gila), and highly virulent (Sheila SmithT, Costa Rica, and Taiaçu). Transcriptional profiles of 11 recognized or putative virulence factors confirmed the isolate-dependent differences between mildly and highly virulent isolates. These data corroborate previous qualitative assessments of strain virulence and suggest further that a critical and previously underappreciated balance between bacterial growth and host immune response could leverage strain pathogenicity. Also, this work provides insight into isolate-specific microbiological factors that contribute to the outcome of RMSF and confirms the hypothesis that distinct rickettsial isolates also differ phenotypically, which could influence the severity of disease in vertebrate hosts.

Reproductive incompatibility between Amblyomma maculatum (Acari: Ixodidae) group ticks from two disjunct geographical regions within the USA.

The Amblyomma maculatum Koch group of ixodid ticks consists of three species: A. maculatum, A. triste, and A. tigrinum. However, since Koch described this group in 1844, the systematics of its members has been the subject of ongoing debate. This is especially true of A. maculatum and A. triste; recent molecular analyses reveal insufficient genetic divergence to separate these as distinct species. Further confounding this issue is the discovery in 2014 of A. maculatum group ticks in southern Arizona (AZ), USA, that share morphological characteristics with both A. triste and A. maculatum. To biologically evaluate the identity of A. maculatum group ticks from southern Arizona, we analyzed the reproductive compatibility between specimens of A. maculatum group ticks collected from Georgia (GA), USA, and southern Arizona. Female ticks from both Arizona and Georgia were mated with males from both the Georgia and Arizona Amblyomma populations, creating two homologous and two heterologous F1 cohorts of ticks: GA ♀/GA ♂, AZ ♀/AZ ♂, GA ♀/AZ ♂, and AZ ♀/GA ♂. Each cohort was maintained separately into the F2 generation with F1 females mating only with F1 males from their same cohort. Survival and fecundity parameters were measured for all developmental stages. The observed survival parameters for heterologous cohorts were comparable to those of the homologous cohorts through the F1 generation. However, the F1 heterologous females produced F2 egg clutches that did not hatch, thus indicating that the Arizona and Georgia populations of A. maculatum group ticks tested here represent different biological species.

Distribution and Occurrence of Amblyomma maculatum sensu lato (Acari: Ixodidae) and Rickettsia parkeri (Rickettsiales: Rickettsiaceae), Arizona and New Mexico, 2017-2019.

Amblyomma maculatum Koch sensu lato (s.l.) ticks are the vector of Rickettsia parkeri in Arizona, where nine cases of R. parkeri rickettsiosis have been identified since the initial case in 2014. The current study sought to better define the geographic ranges of the vector and pathogen and to assess the potential public health risk posed by R. parkeri in this region of the southwestern United States. A total of 275 A. maculatum s.l. ticks were collected from 34 locations in four counties in Arizona and one county in New Mexico and screened for DNA of Rickettsia species. Rickettsia parkeri was detected in 20.4% of the ticks, including one specimen collected from New Mexico, the first report of R. parkeri in A. maculatum s.l. from this state. This work demonstrates a broader distribution of A. maculatum s.l. ticks and R. parkeri in the southwestern United States than appreciated previously to suggest that R. parkeri rickettsiosis is underrecognized in this region.

Rickettsia parkeri (Rickettsiales: Rickettsiaceae) in the Sky Islands of West Texas.

Rickettsia parkeri, a tick-borne pathogen distributed throughout several countries of the Americas, causes a mild to moderately severe, eschar-associated spotted fever rickettsiosis. Although most U.S. cases of R. parkeri rickettsiosis are reported from southeastern states, some have been reported recently from remote regions of southern Arizona. These cases are linked to R. parkeri-infected ticks of the Amblyomma maculatum (Acari: Ixodidae) group found in several isolated mountain ranges of southern Arizona and New Mexico, referred to as 'sky islands'. Archival records also document ticks of the A. maculatum group collected from domestic and wild animals in West Texas. We surveyed sites in two sky island chains of Jeff Davis and Brewster counties to document the off-host occurrence of these ticks and identify the presence of R. parkeri in the Trans-Pecos region of Texas. During August 2019, 43 adult A. maculatum group ticks were flagged from vegetation or removed from a road-killed, female mule deer. Of 39 samples evaluated by PCR, eight contained a partial sca0 sequence with complete identity to R. parkeri and two with complete identity to 'Candidatus Rickettsia andeanae', a species of undetermined pathogenicity. Four isolates of R. parkeri were obtained using cell culture. Persons at risk for R. parkeri rickettsiosis include those who work or recreate in these mountains, such as hikers, backpackers, research scientists, foresters, and border enforcement personnel. Additional investigations are needed to define the distribution of these medically important arthropods in other parts of the southwestern United States and northern Mexico.

Rickettsia parkeri and "Candidatus Rickettsia andeanae" in Amblyomma maculatum (Acari: Ixodidae) collected from the Atlanta metropolitan area, Georgia, United States.

Rickettsia parkeri is a recently recognized human pathogen transmitted in the southeastern United States by Amblyomma maculatum, the Gulf Coast tick. Since R. parkeri was conclusively identified as a human pathogen in 2004, over 40 cases of R. parkeri rickettsiosis have been identified in the United States, most of which occur in the southeastern states. During 2012-2014, five of these cases were identified by a single urgent care practice in Coweta County, a Georgia county within the Atlanta metropolitan area. To investigate the occurrence of R. parkeri-infected A. maculatum in the Atlanta metropolitan area, ticks were collected from 6 counties around the city of Atlanta and evaluated for infection with a Rickettsia species. A total of 263 questing adult A. maculatum were collected during 2015 and 2016. Of these, 93 (35%) were PCR-positive for DNA of R. parkeri and an additional 46 (17%) were PCR-positive for DNA of "Candidatus Rickettsia andeanae," a spotted fever group Rickettsia species of unknown pathogenicity. No co-infections of these two rickettsiae were detected; however four of the six counties sampled showed presence of both rickettsial organisms. The high frequency of R. parkeri in these tick populations indicates a potential risk for those living, working, or recreating in A. maculatum-infested habitats within these six counties in the Atlanta metropolitan area.

Multistate Survey of American Dog Ticks (Dermacentor variabilis) for Rickettsia Species.

Dermacentor variabilis, a common human-biting tick found throughout the eastern half and along the west coast of the United States, is a vector of multiple bacterial pathogens. Historically, D. variabilis has been considered a primary vector of Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever. A total of 883 adult D. variabilis, collected between 2012 and 2017 from various locations in 12 states across the United States, were screened for rickettsial DNA. Tick extracts were evaluated using three real-time PCR assays; an R. rickettsii-specific assay, a Rickettsia bellii-specific assay, and a Rickettsia genus-specific assay. Sequencing of ompA gene amplicons generated using a seminested PCR assay was used to determine the rickettsial species present in positive samples not already identified by species-specific real-time assays. A total of 87 (9.9%) tick extracts contained R. bellii DNA and 203 (23%) contained DNA of other rickettsial species, including 47 (5.3%) with Rickettsia montanensis, 11 (1.2%) with Rickettsia amblyommatis, 2 (0.2%) with Rickettsia rhipicephali, and 3 (0.3%) with Rickettsia parkeri. Only 1 (0.1%) tick extract contained DNA of R. rickettsii. These data support multiple other contemporary studies that indicate infrequent detection of R. rickettsii in D. variabilis in North America.

Genotypic Characterization of Rickettsia bellii Reveals Distinct Lineages in the United States and South America.

The bacterium Rickettsia bellii belongs to a basal group of rickettsiae that diverged prior to the pathogenic spotted fever group and typhus group Rickettsia species. Despite a diverse representation of R. bellii across more than 25 species of hard and soft ticks in the American continent, phylogeographical relationships among strains of this basal group-Rickettsia species are unknown; the work described here explores these relationships. DNA was extracted from 30 R. bellii tick isolates: 15 from the United States, 14 from Brazil, and 1 from Argentina. A total of 2,269 aligned nucleotide sites of 3 protein coding genes (gltA, atpA, and coxA) and 2 intergenic regions (rpmE-tRNAfmet and RC1027-xthA2) were concatenated and subjected to phylogenetic analysis by Bayesian methods. Results showed a separation of almost all isolates between North and South Americas, suggesting that they have radiated within their respective continents. Phylogenetic positions of the 30 isolates could be a result of not only their geographical origin but also the tick hosts they have coevolved with. Whether R. bellii originated with ticks in North or South America remains obscure, as our analyses did not show evidence for greater genetic divergence of R. bellii in either continent.

Evaluation of Gulf Coast Ticks (Acari: Ixodidae) for Ehrlichia and Anaplasma Species.

Amblyomma maculatum Koch (the Gulf Coast tick) is an aggressive, human-biting ixodid tick distributed throughout much of the southeastern United States and is the primary vector for Rickettsia parkeri, an emerging human pathogen. Amblyomma maculatum has diverse host preferences that include white-tailed deer, a known reservoir for Ehrlichia and Anaplasma species, including the human pathogens E. ewingii and E. chaffeensis. To examine more closely the potential role of A. maculatum in the maintenance of various pathogenic Ehrlichia and Anaplasma species, we screened DNA samples from 493 questing adult A. maculatum collected from six U.S. states using broad-range Anaplasmataceae and Ehrlichia genus-specific PCR assays. Of the samples tested, four (0.8%) were positive for DNA of Ehrlichia ewingii, one (0.2%) was positive for Anaplasma platys, and one (0.2%) was positive for a previously unreported Ehrlichia species closely related to Ehrlichia muris and an uncultivated Ehrlichia species from Haemaphysalis longicornis ticks in Japan. No ticks contained DNA of Ehrlichia chaffeensis, Ehrlichia canis, the Panola Mountain Ehrlichia, or Anaplasma phagocytophilum. This is the first identification of E. ewingii, A. platys, and the novel Ehrlichia in questing Gulf Coast ticks; nonetheless the low prevalence of these agents suggests that A. maculatum is not likely an important vector of these zoonotic pathogens.

Development of a Rickettsia bellii-Specific TaqMan Assay Targeting the Citrate Synthase Gene.

Rickettsia bellii is a rickettsial species of unknown pathogenicity that infects argasid and ixodid ticks throughout the Americas. Many molecular assays used to detect spotted fever group (SFG) Rickettsia species do not detect R. bellii, so that infection with this bacterium may be concealed in tick populations when assays are used that screen specifically for SFG rickettsiae. We describe the development and validation of a R. bellii-specific, quantitative, real-time PCR TaqMan assay that targets a segment of the citrate synthase (gltA) gene. The specificity of this assay was validated against a panel of DNA samples that included 26 species of Rickettsia, Orientia, Ehrlichia, Anaplasma, and Bartonella, five samples of tick and human DNA, and DNA from 20 isolates of R. bellii, including 11 from North America and nine from South America. A R. bellii control plasmid was constructed, and serial dilutions of the plasmid were used to determine the limit of detection of the assay to be one copy per 4 µl of template DNA. This assay can be used to better determine the role of R. bellii in the epidemiology of tick-borne rickettsioses in the Western Hemisphere.

High prevalence of "Candidatus Rickettsia andeanae" and apparent exclusion of Rickettsia parkeri in adult Amblyomma maculatum (Acari: Ixodidae) from Kansas and Oklahoma.

Amblyomma maculatum (the Gulf Coast tick), an aggressive, human-biting, Nearctic and Neotropical tick, is the principal vector of Rickettsia parkeri in the United States. This pathogenic spotted fever group Rickettsia species has been identified in 8-52% of questing adult Gulf Coast ticks in the southeastern United States. To our knowledge, R. parkeri has not been reported previously from adult specimens of A. maculatum collected in Kansas or Oklahoma. A total of 216 adult A. maculatum ticks were collected from 18 counties in Kansas and Oklahoma during 2011-2014 and evaluated by molecular methods for evidence of infection with R. parkeri. No infections with this agent were identified; however, 47% of 94 ticks collected from Kansas and 73% of 122 ticks from Oklahoma were infected with "Candidatus Rickettsia andeanae" a spotted fever group Rickettsia species of undetermined pathogenicity. These preliminary data suggest that "Ca. R. andeanae" is well-adapted to survival in populations of A. maculatum in Kansas and Oklahoma, and that its ubiquity in Gulf Coast ticks in these states may effectively exclude R. parkeri from their shared arthropod host, which could diminish markedly or preclude entirely the occurrence of R. parkeri rickettsiosis in this region of the United States.