Rickettsia Peacockii Research Articles

Exotic ticks removed from German travelers.

We report the finding of five nymphs and three adult ticks attached to German tourists while traveling the American continents. All eight specimens were morphologically identified and confirmed genetically using the 16S rRNA gene and screened for Rickettsia spp. infections. Five tick species were identified: one Amblyomma mixtum nymph from Ecuador, one Amblyomma varium nymph from Colombia, three Amblyomma coelebs nymphs from Costa Rica, one Amblyomma americanum male from the USA, one Dermacentor andersoni female and one D. andersoni male from Canada. Tick-borne microorganisms screening using the pan-Rickettsia-PCR resulted in two positive and six negative ticks. The A. mixtum nymph was positive for Rickettsia amblyommatis, while the D. andersoni female was positive for Rickettsia peacockii.

Identification of Rickettsia spp. and Babesia conradae in Dermacentor spp. Collected from Dogs and Cats Across the United States.

In the United States, Dermacentor variabilis and Dermacentor andersoni are considered key vectors for Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever. Through regional surveillance, a wide diversity of Rickettsia spp. have been documented in D. variabilis, and Dermacentor spp. has been suggested as potential vectors for various other pathogens, including Babesia spp. and Ehrlichia canis. To better define the prevalence and diversity of pathogens in Dermacentor spp. across the United States, 848 ticks collected from dogs and cats in 44/50 states in 2018–2019 were tested by PCR for Rickettsia spp.-specific 17 kDa and ompA gene fragments; a subset of Dermacentor spp. was also tested with PCR, targeting fragments of the 18S and large subunit region rRNA genes of Babesia spp. and 16S rRNA genes of E. canis. Rickettsia spp. was identified in 12.5% (106/848) of ticks. Species detected include Rickettsia montanensis (n = 64 ticks), Rickettsia bellii (n = 15 ticks), Rickettsia rhipicephali (n = 13 ticks), Rickettsia peacockii (n = 8 ticks), Rickettsia amblyommatis (n = 3 ticks), Rickettsia cooleyi (n = 1 tick), and unclassified Rickettsia spp. (n = 2 ticks). Ticks with R. montanensis and R. bellii were submitted from every U.S. region; R. rhipicephali was predominantly detected in ticks from the southern half of the United States, and all R. peacockii-positive ticks were D. andersoni that originated from the Rocky Mountain states. Ehrlichia canis was not detected in any Dermacentor spp., and Babesia conradae was detected in two Dermacentor albipictus. Because most ticks had fed on dogs or cats before submission, these findings do not implicate a given Dermacentor sp. as a primary vector of these agents, but in regard to Rickettsia spp., the data do support other published work showing D. variabilis harbors a diversity of Rickettsia species with unknown implications for animal and human health.

Rickettsia and Anaplasma species in Dermacentor andersoni ticks from Washington

Dermacentor andersoni, the Rocky Mountain wood tick, occurs predominantly in the northwestern United States and southwestern Canada. There are relatively few contemporary data to evaluate the occurrence of Rickettsia and Anaplasma species in D. andersoni in western North America, and even less information about these associations in the state of Washington, where this tick species is widely distributed and often bites humans. We used PCR assays to detect DNA of Rickettsia and Anaplasmataceae bacteria in 203 adult D. andersoni ticks collected from 17 sites in 9 counties of Washington between May 2012 and May 2015. Of these, 56 (27.6 %) were infected with a Rickettsia species and 3 (5.4 %) with a member of the Anaplasmataceae family. Rickettsia peacockii, R. bellii and R. rhipicephali were found in 17.7 %, 4.9 %, and 4.4 % of the Rickettsia positive ticks, respectively. Coinfections of R. bellii with R. peacockii or R. rhipicephali were identified in 6 ticks. Of the Anaplasmataceae-positive ticks, one was identified as being infected with Anaplasma phagocytophilum AP-Variant 1. No ticks were infected with a recognized human or animal pathogen, including R. rickettsii, A. phagocytophilum-ha, A. bovis, or A. marginale.

Identification of bacteria in the Rocky Mountain wood tick, Dermacentor andersoni, using single-strand conformation polymorphism (SSCP) and DNA sequencing.

PCR-based single-strand conformation polymorphism (SSCP) analyses combined with DNA sequencing of the prokaryotic 16S ribosomal (r) RNA gene encompassing the hypervariable V4 region was used to determine the bacterial composition of Rocky Mountain wood ticks (Dermacentor andersoni) attached to Richardson's ground squirrels (Urocitellus richardsonii) and questing on vegetation in southern Saskatchewan, Canada. The bacteria present in questing adult ticks from Saskatchewan Landing Provincial Park included Rickettsia peacockii, a Francisella-like endosymbiont (FLE) and an Arsenophonus-like endosymbiont. Bacteria in the adult and nymphal ticks attached to U. richardsonii collected from Beechy included R. peacockii, a FLE, and several other genera (e.g., Ralstonia, Sphingobium, Comamonas and Pseudomonas). The bacteria detected in D. andersoni in the present study are consistent with the findings of other studies that have characterized the microbiome of this tick species in the USA using next generation sequencing. This result demonstrates that the SSCP-based approach used in this study is cost- and time-effective for examining bacterial composition in ticks.

‘Candidatus Rickettsia nicoyana’: A novel Rickettsia species isolated from Ornithodoros knoxjonesi in Costa Rica

Rickettsiae are intracellular bacteria commonly associated with hematophagous arthropods. Most of them have been described in hard ticks, but some have been found in soft ticks. Here we report the detection and isolation of a new Rickettsia from Ornithodoros knoxjonesi larvae collected from Balantiopteryx plicata (Emballonuridae) in Nicoya, Costa Rica. Two ticks were processed to detect Rickettsia spp. genes gltA, ompA, ompB, and htrA by PCR. Part of the macerate was also inoculated into Vero E6 and C6/36 cell lines, and cells were evaluated by Giménez stain, indirect immunofluorescence assay (IFA), and PCR. Both ticks were positive by PCR and rickettsial growth was successful in Vero E6 cells. Amplification and sequencing of near full length rrs, gltA, sca4 genes, and fragments of ompA and ompB showed that the Rickettsia sp. was different from described species. The highest homologies were with ‘Candidatus Rickettsia wissemanii’ and Rickettsia peacockii: 99.70% (1321/1325) with both sequences for rrs, 99.58% (1172/1177) and 99.76% (1246/1249) for gltA, 99.26% with both sequences (2948/2970 and 2957/2979) for sca4, 98.78% (485/491) and 98.39% (2069/2115) for ompA, and 98.58 (1453/1474) and 98.92% (1459/1475) for ompB; respectively. Bat blood, spleen, liver, and lung samples analyzed for Rickettsia detection were negative. Results demonstrate that the Rickettsia isolated from O. knoxjonesi is probably an undescribed species that belongs to the spotted fever group, for which ‘Candidatus Rickettsia nicoyana’ is proposed. Considering that B. plicata inhabits areas where contact with humans may occur and that human parasitism by Ornithodoros has been reported in the country, it will be important to continue with the characterization of this species and its pathogenic potential.

PCR denaturing gradient gel electrophoresis as a useful method to identify of intestinal bacteria flora in Haemaphysalis flava ticks.

Ticks are blood-sucking ectoparasites of great medical and veterinary significance that can transmit bacteria, protozoa, fungi and viruses, causing a variety of human and animal diseases worldwide. In the present study, the intestinal bacterial flora associated with Haemaphysalis flava ticks in different developmental stages were analyzed using polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE). Eleven distinct DGGE bands were found using PCR-DGGE method. Sequences analyses indicated that they belonged to Bacillus cereus, Candidatus rickettsia, Erwinia sp., Klebsiella pneumoniae, Pectobacterium carotovorum, Pseudomonas aeruginosa, Rickettsia peacockii, Rickettsia helvetica, Rickettsia slovaca, Staphylococcus simulans and Uncultured bacterium clone. Our find that the K. pneumoniae and P. aeruginosa isolates were presented in all H. flava ticks in different developmental stages. The present results indicated that zoonotic pathogens are present in H. flava ticks in Henan province, China. To our knowledge, this is the first report on intestinal bacterial flora associated with H. flava ticks in China.

Fluorescent Protein Expressing Rickettsia buchneri and Rickettsia peacockii for Tracking Symbiont-Tick Cell Interactions.

Rickettsiae of indeterminate pathogenicity are widely associated with ticks. The presence of these endosymbionts can confound a One Health approach to combatting tick-borne diseases. Genomic analyses of symbiotic rickettsiae have revealed that they harbor mutations in gene coding for proteins involved in rickettsial pathogenicity and motility. We have isolated and characterized two rickettsial symbionts—Rickettsia peacockii and R. buchneri—both from ticks using tick cell cultures. To better track these enigmatic rickettsiae in ticks and at the tick-mammal interface we transformed the rickettsiae to express fluorescent proteins using shuttle vectors based on rickettsial plasmids or a transposition system driving insertional mutagenesis. Fluorescent protein expressing R. buchneri and R. peacockii will enable us to elucidate their interactions with tick and mammalian cells, and track their location and movement within individual cells, vector ticks, and host animals.

Prevalence of Rickettsia species in Dermacentor variabilis ticks from Ontario, Canada

Relatively little is known about the prevalence of rickettsial species in Dermacentor ticks in eastern Canada. In this study, Dermacentor ticks from the province of Ontario, Canada, were tested for the presence of spotted fever group rickettsial (SFGR) species, Coxiella burnetii and Francisella tularensis. Rickettsia rickettsii was not detected in any ticks tested, but R. montanensis was detected at a prevalence of 2.2% in D. variabilis (17/778). Two other SFGR species, R. parkeri and Candidatus R. andeanae, were detected individually in 2 Amblyomma maculatum ticks. Rickettsia peacockii, a non-pathogenic endosymbiont, was detected in two D. andersonii ticks. Given the highly abundant nature of D. variabilis, surveillance for human pathogens in this species of tick has important public health implications, but the lack of detection of known human pathogens indicates a low risk of infection via this tick species in Ontario. However, the detection of R. parkeri in an adventive A. maculatum tick indicates that health care providers should be aware of the possibility of spotted fever rickettsioses in individuals with a history of travel outside of Ontario and symptoms compatible with a spotted fever rickettsiosis. Coxiella burnetii and Francisella tularensis, human pathogens also potentially transmitted by D. variabilis, were not detected in a subset of the ticks.

Identification of intestinal bacterial flora in Rhipicephalus microplus ticks by conventional methods and PCR-DGGE analysis.

In this study, we have analyzed the intestinal microbial flora associated with Rhipicephalus microplus ticks using both culture-dependent and independent methods based on PCR and denaturing gradient gel electrophoresis (PCR-DGGE). The R. microplus ticks were collected from cattle and goats in Jiangxi, Hunan and Guizhou Provinces of China. Three distinct strains of bacteria were isolated using culture-dependent methods: Staphylococcus simulans, Bacillus subtilis and Bacillus flexus strain. Nineteen distinct DGGE bands were found using PCR-DGGE analysis, and their search for identity shows that they belonged to Rickettsiaceae, Xanthomonadaceae, Coxiella sp., Ehrlichia sp., Pseudomonas sp., Ehrlichia sp., Orphnebius sp., Rickettsia peacockii, Bacillus flexus. Rickettsia peacockii and Coxiella genus were the dominant strain of the R. microplus ticks from cattle, Pseudomonas sp. and B. flexus strain were the most common species in all tick samples from goats. Ehrlichia canis were detected only in R. microplus ticks from Yongshun area in Hunan Province. The results indicate that the intestinal microbial diversity of R. microplus ticks was influenced by tick hosts and local differences in the sampling location and these two aspects may affect transmission of pathogen to humans and animals.

Genome Sequence of the Endosymbiont Rickettsia peacockii and Comparison with Virulent Rickettsia rickettsii: Identification of Virulence Factors

Rickettsia peacockii, also known as the East Side Agent, is a non-pathogenic obligate intracellular bacterium found as an endosymbiont in Dermacentor andersoni ticks in the western USA and Canada. Its presence in ticks is correlated with reduced prevalence of Rickettsia rickettsii, the agent of Rocky Mountain Spotted Fever. It has been proposed that a virulent SFG rickettsia underwent changes to become the East Side Agent. We determined the genome sequence of R. peacockii and provide a comparison to a closely related virulent R. rickettsii. The presence of 42 chromosomal copies of the ISRpe1 transposon in the genome of R. peacockii is associated with a lack of synteny with the genome of R. rickettsii and numerous deletions via recombination between transposon copies. The plasmid contains a number of genes from distantly related organisms, such as part of the glycosylation island of Pseudomonas aeruginosa. Genes deleted or mutated in R. peacockii which may relate to loss of virulence include those coding for an ankyrin repeat containing protein, DsbA, RickA, protease II, OmpA, ScaI, and a putative phosphoethanolamine transferase. The gene coding for the ankyrin repeat containing protein is especially implicated as it is mutated in R. rickettsii strain Iowa, which has attenuated virulence. Presence of numerous copies of the ISRpe1 transposon, likely acquired by lateral transfer from a Cardinium species, are associated with extensive genomic reorganization and deletions. The deletion and mutation of genes possibly involved in loss of virulence have been identified by this genomic comparison. It also illustrates that the introduction of a transposon into the genome can have varied effects; either correlating with an increase in pathogenicity as in Francisella tularensis or a loss of pathogenicity as in R. peacockii and the recombination enabled by multiple transposon copies can cause significant deletions in some genomes while not in others.

Restriction of the growth of typhus group rickettsiae in tick cells

The vectors of typhus group rickettsiae (TGR) are lice andfleas that belong to Insecta. Those of spotted fever group rickettsiae (SFGR), however, are ticks classified as Arachnida. Until recently, the host dependency of the growth of TGR and SFGR in cells derived from arthropods had not been well documented. As for insect cells, it was reported by Rovery et al. that the transcription of spoT gene paralogues was suppressed during the infection of Rickettsia conorii in Aedes albopictus (C6 ⁄ 36) cells at 10 C for 38 days. Shifting the temperature to 37 C was followedby a rapidupregulationof spoT1 gene expression [1]. Uchiyama reported that some SFGR (Rickettsia japonica and Rickettsia montanensis) do not grow in the AeAl2 cell line from A. albopictus, even though the rickettsiae achieved adherence and invasion [2]. On the contrary, Sakamoto et al. demonstrated that some non-pathogenic SFGR, R. montanensis and Rickettsia peacockii successfully grew in two mosquito cell lines (A. albopictus cell line Aa23 and Anopheles gambie cell line Sua5B) [3]. Concerning tick cell lines, several reports regarding the growth of SFGR in the cells have been published [4,5]. In this study, the growth of TGR in tick cells was examined. The DALBE3 cell line from Dermacentor albipictus (a generous gift of U.G. Munderloh) and the ISE6 cell line from Ixodes scapuralis (generously donated by I. Takashima under agreement of U.G. Munderloh) were used as tick cells. As mammalian cells, Vero and ECV304 cells were used. These cells were inoculated with R. japonica, strain YH and R. conorii, strain Malish 7 as SFGR and Rickettsia prowazekii, strain Breinl and Rickettsia typhi, strain Wilmington as TGR at a multiplicity of infection (MOI) of 0.1 PFU ⁄ cell. The infected cells were cultured at 34 C in a medium described previously [4,5]. The medium was changed every 3 days and the yields of rickettsiae were plaqueassayed on Vero cell monolayers. As shown in Fig. 1(a), SFGR grew well in DALBE3 and ISE6 cells as well as in Vero and ECV304 cells (data from ISE6, Vero and ECV304 cells are not shown). However, growth of TGR was restricted in these tick cells contrary to their growth in Vero and ECV304 mammalian cells (data from ISE6, Vero and ECV304 cells are not shown). Rickettsial infectivity decreased to an undetectable level up to 2 weeks after infection. Because the growth of TGR in these cells was restricted, the adherence step was analysed to determine if it was completed. We inoculated the tick cells with rickettsiae at an MOI of 1.0 PFU ⁄ cell and incubated these cells for 5, 10, 60 or 180 min at 25 C. As shown in Fig. 1b, the adherence of these cells by TGR was successfully completed as were those in other combinations of rickettsiae and host cells, although the extent of adherence differed among the combinations tested. We also performed Gimenez staining of the tick cells inoculated with either group of rickettsiae at an MOI of 50 PFU ⁄mL and found that the cells showed adherence and ⁄ or invasion by rickettsiae (data not shown). Transmission electron microscopy was performed to confirm these findings and examine morphological changes. DALBE3 cells were inoculated with TGR and SFGR at an MOI of 50 PFU ⁄ cell and incubated at 25 C for 10 min, 60 min, and 180 min, and then cultured at 34 C for 24 h and 72 h until fixed. Thin sections of the samples were observed under a Hitachi H-7650 transmission electron microscope. DALBE3 cells demonstrated adherence and invasion by these rickettsiae independent of their group (data not shown). Rickettsiae under the process of escaping from phagosomes to the cytoplasm after being engulfed by DALBE3 cells were also shown. Thus, it is suggested that restriction occurred after these steps. Further analyses of the restrictive steps Corresponding author and reprint requests: T. Uchiyama, Department of Virology, Institute of Health Biosciences, The University of Tokushima Graduate School, 3-18-15 Kuramoto, Tokushima 770–8503, Japan E-mail: uchiyama@basic.med.tokushima-u.ac.jp

Prevalence ofRickettsiaSpecies in Canadian Populations ofDermacentor andersoniandD. variabilis

We determined the prevalence of rickettsiae in Dermacentor adults at 15 localities in Canada. Rickettsia rickettsii was not detected in any tick, whereas Rickettsia peacockii was present in 76% of Dermacentor andersoni adults and Rickettsia montanensis in 8% of Dermacentor variabilis adults. This host specificity was maintained in localities where both tick species occurred in sympatry.

Phagocytosis of the Lyme Disease Spirochete,Borrelia burgdorferi, by Cells from the Ticks,Ixodes scapularisandDermacentor andersoni, Infected with An Endosymbiont,Rickettsia peacockii

Tick cell lines were used to model the effects of endosymbiont infection on phagocytic immune responses. The lines tested for their ability to phagocytose the Lyme disease spirochete, Borrelia burgdorferi (Spirochaetales: Spirochaetaceae), were ISE6 and IDE12 from the black-legged tick, Ixodes scapularis Say (Acari: Ixodidae) and DAE15 from the Rocky Mountain wood tick, Dermacentor andersoni Stiles. Rickettsia peacockii (Rickettsiales: Rickettsiaceae), an endosymbiont of D. andersoni, was used as a representative tick endosymbiont. 70–80% of uninfected or R. peacocciz-infected IDE12 and DAE15 cells phagocytosed heat-killed borreliae and 80–90% of IDE12 and DAE15 cells phagocytosed viable spirochetes. ISE6 cells were permissive of spirochetes; less than 1% of these cells phagocytosed borreliae, and spirochetes remained adherent to the cells seven days after inoculation. Cytochalasin B blocked phagocytosis of killed and viable borreliae by IDE12 cells, and prevented phagocytosis of killed spirochetes by DAE15 cells, whereas viable spirochetes successfully invaded cytochalasin-treated DAE15. IDE12 and DAE15 cells degraded borreliae within phagolysosome-like compartments. Time-lapse microscopy showed that DAE15 cells phagocytosed borreliae more rapidly than IDE12 cells. IDE12 and DAE15 cells eliminated most adherent spirochetes within 7 days of inoculation. Thus, endosymbiont infection does not significantly interfere with the phagocytic activity of immunocompetent tick cells.

Rickettsia peacockii, an endosymbiont of Dermacentor andersoni, does not elicit or inhibit humoral immune responses from immunocompetent D. andersoni or Ixodes scapularis cell lines

Ixodes scapularis and Dermacentor andersoni cell lines were stimulated with heat-killed Escherichia coli and Micrococcus luteus to investigate whether infection by Rickettsia peacockii, an endosymbiont of D. andersoni, modifies humoral immune responses. Radial diffusion assays, western blotting, flow cytometry, and quantitative reverse-transcription PCR were used to determine if expression of bacteriolytic peptides, including lysozyme and defensin, was upregulated by bacterial stimulation or infection with R. peacockii. The I. scapularis line IDE12 upregulated expression of lysozyme and defensin following stimulation. The D. andersoni cell line DAE15 also expressed defensin and lysozyme, but only lysozyme was upregulated by bacterial stimulation. R. peacockii infection alone, or in cells stimulated with bacteria, did not modify defensin or lysozyme expression in either cell line. These results suggest tick endosymbionts may avoid recognition by the tick immune system, and infection may not affect humoral immune responses to bacteria not normally associated with ticks.

Factors influencing in vitro infectivity and growth of Rickettsia peacockii (Rickettsiales: Rickettsiaceae), an endosymbiont of the Rocky Mountain wood tick, Dermacentor andersoni (Acari, Ixodidae)

Rickettsia peacockii, a spotted fever group rickettsia, is a transovarially transmitted endosymbiont of Rocky Mountain wood ticks, Dermacentor andersoni. This rickettsia, formerly known as the East Side Agent and restricted to female ticks, was detected in a chronically infected embryonic cell line, DAE100, from D. andersoni. We examined infectivity, ability to induce cytopathic effect (CPE) and host cell specificity of R. peacockii using cultured arthropod and mammalian cells. Aposymbiotic DAE100 cells were obtained using oxytetracycline or incubation at 37 °C. Uninfected DAE100 sublines grew faster than the parent line, indicating R. peacockii regulation of host cell growth. Nevertheless, DAE100 cellular defenses exerted partial control over R. peacockii growth. Rickettsiae existed free in the cytosol of DAE100 cells or within autophagolysosomes. Exocytosed rickettsiae accumulated in the medium and were occasionally contained within host membranes. R. peacockii multiplied in other cell lines from the hard ticks D. andersoni, Dermacentor albipictus, Ixodes scapularis, and Ixodes ricinus; the soft tick Carios capensis; and the lepidopteran Trichoplusia ni. Lines from the tick Amblyomma americanum, the mosquito Aedes albopictus, and two mammalian cell lines were non-permissive to R. peacockii. High cell densities facilitated rickettsial spread within permissive cell cultures, and an inoculum of one infected to nine uninfected cells resulted in the greatest yield of infected tick cells. Cell-free R. peacockii also were infectious for tick cells and centrifugation onto cell layers enhanced infectivity approximately 100-fold. The ability of R. peacockii to cause mild CPE suggests that its pathogenicity is not completely muted. An analysis of R. peacockii–cell interactions in comparison to pathogenic rickettsiae will provide insights into host cell colonization mechanisms.

A novel and naturally occurring transposon, ISRpe1 in the Rickettsia peacockii genome disrupting the rickA gene involved in actin‐based motility

While examining the molecular basis for the lack of actin-based motility for the non-pathogenic spotted fever group (SFG) R. peacockii, we identified a novel insertion sequence (IS) element, ISRpe1, disrupting the coding sequence of rickA, demonstrated to induce actin-tail polymerization for the SFG rickettsiae. This rickettsial IS element appears to be active in that complete terminal inverted repeat and recombinase/transposase open reading frame sequences are present and the transposase is transcriptionally expressed. Phylogenetically, ISRpe1 belongs to a new IS family that is most closely related to those transposable elements of other intracellular bacteria like Wolbachia spp. ISRpe1 was demonstrated to be present in at least 10 locations throughout the R. peacockii genome, including one that disrupted the putative cell surface antigen encoding gene, sca1 considered to be involved in adhesion and virulence of the rickettsiae. Additionally, three IS sites demonstrated rearrangements/relocations of the R. peacockii genome when compared to those of other SFG rickettsiae. Our findings of the disruptions of rickA and sca1 along with the comparative genomic reassortments associated with ISRpe1 in the non-virulent R. peacockii provides opportunities to uncover molecular mechanisms underlying the pathogenesis and evolution of rickettsiae as well as its potential to be used in rickettsial transposon-based mutagenesis.

Susceptibility of Rickettsia monacensis and Rickettsia peacockii to Cecropin A, Ceratotoxin A, and Lysozyme

Ticks host obligate intracellular bacteria that range from benign symbiotes to virulent human pathogens. The effects on those bacteria of antimicrobial peptides (AMPs) involved in arthropod innate immunity to microbial infections are largely unknown. We evaluated effects of AMPs and a c-type lysozyme on host cell-free suspensions of the tick symbiotes Rickettsia monacensis and Rickettsia peacockii with stain-based infectivity and viability assays. Cecropin A at a concentration of 8 muM: had a lethal effect on both rickettsiae while ceratotoxin A was approximately 20-fold less effective. Toxicity of both AMPs was synergized by lysozyme, an enzyme expressed by ticks. Lactoferrin, a transferrin, had no effect on R. monacensis at up to 110 microM. The rickettsiae were less sensitive to the AMPs than is typical of bacteria that grow extracellularly. Our assays may be useful in the study of AMP activity against other obligate intracellular bacteria.

Rickettsia peacockii sp. nov., a new species infecting wood ticks, Dermacentor andersoni, in western Montana.

Rickettsia peacockii, a new species of spotted fever group rickettsiae, was identified from Rocky Mountain wood ticks (Dermacentor andersoni) collected in the Sapphire Mountain Range on the eastern side of Bitterroot Valley, Montana. DNA from R. peacockii SkalkahoT (T = type strain) in naturally infected tick tissue was amplified by a PCR assay with primer sets derived from eubacterial 16S ribosomal DNA (rDNA), rickettsial citrate synthase, and 190-kDa surface antigen (rOmpA) genes. Partial 16S rDNA and rOmpA gene sequences exhibited levels of similarity of 99.7 and 93.2%, respectively, with the sequences of the spotted fever agent Rickettsia rickettsii R. By using Gimenez staining, fluorescent antibody tests, a PCR assay, and a restriction fragment length polymorphism analysis, 76 of 115 female ticks (minimal field infection rate, 66.1%) collected between 1992 and 1995 were found to be infected. The organism is passed transstadially and transovarially (minimal vertical transmission rate, 73.3%), and infections are localized in ovarial tissues. Attempts to cultivate R. peacockii were unsuccessful.

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Rickettsiae are intracellular bacteria commonly associated with hematophagous arthropods. Most of them have been described in hard ticks, but some have been found in soft ticks. Here we report the detection and isolation of a new Rickettsia from Ornithodoros knoxjonesi larvae collected from Balantiopteryx plicata (Emballonuridae) in Nicoya, Costa Rica. Two ticks were processed to detect Rickettsia spp. genes gltA, ompA, ompB, and htrA by PCR. Part of the macerate was also inoculated into Vero E6 and C6/36 cell lines, and cells were evaluated by Giménez stain, indirect immunofluorescence assay (IFA), and PCR. Both ticks were positive by PCR and rickettsial growth was successful in Vero E6 cells. Amplification and sequencing of near full length rrs, gltA, sca4 genes, and fragments of ompA and ompB showed that the Rickettsia sp. was different from described species. The highest homologies were with ‘Candidatus Rickettsia wissemanii’ and Rickettsia peacockii: 99.70% (1321/1325) with both sequences for rrs, 99.58% (1172/1177) and 99.76% (1246/1249) for gltA, 99.26% with both sequences (2948/2970 and 2957/2979) for sca4, 98.78% (485/491) and 98.39% (2069/2115) for ompA, and 98.58 (1453/1474) and 98.92% (1459/1475) for ompB; respectively. Bat blood, spleen, liver, and lung samples analyzed for Rickettsia detection were negative. Results demonstrate that the Rickettsia isolated from O. knoxjonesi is probably an undescribed species that belongs to the spotted fever group, for which ‘Candidatus Rickettsia nicoyana’ is proposed. Considering that B. plicata inhabits areas where contact with humans may occur and that human parasitism by Ornithodoros has been reported in the country, it will be important to continue with the characterization of this species and its pathogenic potential.