Rickettsia asembonensis Characterization by Multilocus Sequence Typing of Complete Genes, Peru.

While studying rickettsial infections in Peru, we detected Rickettsia asembonensis in fleas from domestic animals. We characterized 5 complete genomic regions (17kDa, gltA, ompA, ompB, and sca4) and conducted multilocus sequence typing and phylogenetic analyses. The molecular isolate from Peru is distinct from the original R. asembonensis strain from Kenya.

Isolation and characterization of a novel Rickettsia species (Rickettsia asembonensis sp. nov.) obtained from cat fleas (Ctenocephalides felis).

A novel rickettsial agent, 'Candidatus Rickettsia asembonensis' strain NMRCiiT, was isolated from cat fleas, Ctenocephalides felis, from Kenya. Genotypic characterization of the new isolate based on sequence analysis of five rickettsial genes, rrs, gltA, ompA, ompB and sca4, indicated that this isolate clustered with Rickettsia felis URRWXCal2. The degree of nucleotide similarity demonstrated that isolate NMRCiiT belongs within the genus Rickettsia and fulfils the criteria for classification as a representative of a novel species. The name Rickettsia asembonensis sp. nov. is proposed, with NMRCiiT (=DSM 100172T=CDC CRIRC RAS001T=ATCC VR-1827T) as the type strain.

Identification of critical host mitochondrion-associated genes during Ehrlichia chaffeensis infections.

Ehrlichia chaffeensis is an obligate intracellular bacterium that causes human monocytic ehrlichiosis (HME). To determine what host components are important for bacterial replication, we performed microarray analysis on Drosophila melanogaster S2 cells by comparing host gene transcript levels between permissive and nonpermissive conditions for E. chaffeensis growth. Five-hundred twenty-seven genes had increased transcript levels unique to permissive growth conditions 24 h postinfection. We screened adult flies that were mutants for several of the "permissive" genes for the ability to support Ehrlichia replication. Three additional D. melanogaster fly lines with putative mutations in pyrimidine metabolism were also tested. Ten fly lines carrying mutations in the genes CG6479, separation anxiety, chitinase 11, CG6364 (Uck2), CG6543 (Echs1), withered (whd), CG15881 (Ccdc58), CG14806 (Apop1), CG11875 (Nup37), and dumpy (dp) had increased resistance to infection with Ehrlichia. Analysis of RNA by quantitative real-time reverse transcription-PCR (qRT-PCR) confirmed that the bacterial load was decreased in these mutant flies compared to wild-type infected control flies. Seven of these genes (san, Cht11, Uck2, Echs1, whd, Ccdc58, and Apop1) encoded proteins that had mitochondrial functions or could be associated with proteins with mitochondrial functions. Treatment of THP-1 cells with double-stranded RNA to silence the human UCK2 gene indicates that the disruption of the uridine-cytidine kinase affects E. chaffeensis replication in human macrophages. Experiments with cyclopentenyl cytosine (CPEC), a CTP synthetase inhibitor and cytosine, suggest that the nucleotide salvage pathway is essential for E. chaffeensis replication and that it may be important for the provision of CTP, uridine, and cytidine nucleotides.

Comparison of Lethal and Nonlethal Mouse Models of Orientia tsutsugamushi Infection Reveals T-Cell Population-Associated Cytokine Signatures Correlated with Lethality and Protection.

The antigenic diversity of Orientia tsutsugamushi as well as the interstrain difference(s) associated with virulence in mice impose the necessity to dissect the host immune response. In this study we compared the host response in lethal and non-lethal murine models of O. tsutsugamushi infection using the two strains, Karp (New Guinea) and Woods (Australia). The models included the lethal model: Karp intraperitoneal (IP) challenge; and the nonlethal models: Karp intradermal (ID), Woods IP, and Woods ID challenges. We monitored bacterial trafficking to the liver, lung, spleen, kidney, heart, and blood, and seroconversion during the 21-day challenge. Bacterial trafficking to all organs was observed in both the lethal and nonlethal models of infection, with significant increases in average bacterial loads observed in the livers and hearts of the lethal model. Multicolor flow cytometry was utilized to analyze the CD4+ and CD8+ T cell populations and their intracellular production of the cytokines IFNγ, TNF, and IL2 (single, double, and triple combinations) associated with both the lethal and nonlethal murine models of infection. The lethal model was defined by a cytokine signature of double- (IFNγ-IL2) and triple-producing (IL2-TNF-IFNγ) CD4+ T-cell populations; no multifunctional signature was identified in the CD8+ T-cell populations associated with the lethal model. In the nonlethal model, the cytokine signature was predominated by CD4+ and CD8+ T-cell populations associated with single (IL2) and/or double (IL2-TNF) populations of producers. The cytokine signatures associated with our lethal model will become depletion targets in future experiments; those signatures associated with our nonlethal model are hypothesized to be related to the protective nature of the nonlethal challenges.

Pathogen Carriage by Peri-Domestic Fleas in Western Kenya.

Fleas are carriers for many largely understudied zoonotic, endemic, emerging, and re-emerging infectious disease agents, but little is known about their prevalence and role as a vector in Africa. The aim of this study was to determine the diversity of fleas and the prevalence of infectious agents in them collected from human dwellings in western Kenya. A total of 306 fleas were collected using light traps from 33 human dwellings; 170 (55.56%) were identified as Ctenocephalides spp., 121 (39.54%) as Echidnophaga gallinacea, 13 (4.25%) as Pulex irritans, and 2 (0.65%) as Xenopsylla cheopis. Of the 306 individual fleas tested, 168 (54.9%) tested positive for rickettsial DNA by a genus-specific quantitative real-time PCR (qPCR) assay based on the 17-kDa antigen gene. Species-specific qPCR assays and sequencing revealed presence of Rickettsia asembonensis in 166 (54.2%) and Rickettsia felis in 2 (0.7%) fleas. Borrelia burgdorferi, normally known to be carried by ticks, was detected in four (1.3%) flea DNA preparations. We found no evidence of Yersinia pestis, Bartonella spp., or Orientia spp. Not only were Ctenocephalides spp. the most predominant flea species in the human dwellings, but also almost all of them were harboring R. asembonensis.

Ehrlichia chaffeensis infections in Drosophila melanogaster.

Ehrlichia chaffeensis is an obligate, intracellular bacterium, transmitted by the tick Amblyomma americanum, and is the causative agent of human monocytic ehrlichiosis infections. We previously demonstrated that E. chaffeensis is capable of growing in Drosophila S2 cells. Therefore, we tested the hypothesis that E. chaffeensis can infect adult Drosophila melanogaster. Adult Drosophila organisms were experimentally challenged with intra-abdominal injections of bacteria. Ehrlichia-infected flies showed decreased survival compared to wild-type flies, and bacteria isolated from flies could reinfect mammalian macrophages. Ehrlichia infections activated both the cellular and humoral immune responses in the fly. Hemocytes phagocytosed bacteria after injection, and antimicrobial peptide pathways were induced following infection. Increased pathogenicity in flies carrying mutations in genes in both the Toll and Imd pathways suggests that both immune defense pathways participate in host defense. Induction of Drosophila cellular and humoral responses and the in vivo replication of E. chaffeensis suggests that D. melanogaster is a suitable host for E. chaffeensis. In the future, it will be a useful tool to unlock some of the in vivo mysteries of this arthropod-borne bacterium.

Use of Drosophila S2 cells as a model for studying Ehrlichia chaffeensis infections.

Ehrlichia chaffeensis is an obligate intracellular bacterium and the causative agent of human monocytic ehrlichiosis. Although this pathogen grows in several mammalian cell lines, no general model for eukaryotic cellular requirements for bacteria replication has yet been proposed. We found that Drosophila S2 cells are permissive for the growth of E. chaffeensis. We saw morulae (aggregates of bacteria) by microscopy, detected the E. chaffeensis 16S rRNA gene by reverse transcriptase PCR, and used immunocytochemistry to detect E. chaffeensis in S2 and mammalian cells. Bacteria grown in S2 cells reinfected mammalian macrophages. S2 cells were made nonpermissive for E. chaffeensis through incubation with lipopolysaccharide. Our results demonstrate that S2 cells are an appropriate system for studying the pathogenesis of E. chaffeensis. The use of a Drosophila system has the potential to serve as a model system for studying Ehrlichia due to its completed genome, ease of genetic manipulation, and the availability of mutants.

Worldwide Presence and Features of Flea-Borne Rickettsia asembonensis.

Rickettsia asembonensis, the most well-characterized rickettsia of the Rickettsia felis-like organisms (RFLO), is relatively unknown within the vector-borne diseases research community. The agent was initially identified in peri-domestic fleas from Asembo, Kenya in an area in which R. felis was associated with fever patients. Local fleas collected from domestic animals and within homes were predominately infected with R. asembonensis with < 10% infected with R. felis. Since the identification of R. asembonensis in Kenya, it has been reported in other locations within Africa, Asia, the Middle East, Europe, North America, and South America. With the description of R. asembonensis-like genotypes across the globe, a need exists to isolate these R. asembonensis genotypes in cell culture, conduct microscopic, and biological analysis, as well as whole genome sequencing to ascertain whether they are the same species. Additionally, interest has been building on the potential of R. asembonensis in infecting vertebrate hosts including humans, non-human primates, dogs, and other animals. The current knowledge of the presence, prevalence, and distribution of R. asembonensis worldwide, as well as its arthropod hosts and potential as a pathogen are discussed in this manuscript.

A Review of Scrub Typhus (Orientia tsutsugamushi and Related Organisms): Then, Now, and Tomorrow.

Scrub typhus and the rickettsial diseases represent some of the oldest recognized vector-transmitted diseases, fraught with a rich historical aspect, particularly as applied to military/wartime situations. The vectors of Orientia tsutsugamushi were once thought to be confined to an area designated as the Tsutsugamushi Triangle. However, recent reports of scrub typhus caused by Orientia species other than O. tsutsugamushi well beyond the limits of the Tsutsugamushi Triangle have triggered concerns about the worldwide presence of scrub typhus. It is not known whether the vectors of O. tsutsugamushi will be the same for the new Orientia species, and this should be a consideration during outbreak/surveillance investigations. Additionally, concerns surrounding the antibiotic resistance of O. tsutsugamushi have led to considerations for the amendment of treatment protocols, and the need for enhanced public health awareness in both the civilian and medical professional communities. In this review, we discuss the history, outbreaks, antibiotic resistance, and burgeoning genomic advances associated with one of the world's oldest recognized vector-borne pathogens, O. tsutsugamushi.

Outer Membrane Protein A Conservation among Orientia tsutsugamushi Isolates Suggests Its Potential as a Protective Antigen and Diagnostic Target.

Scrub typhus threatens one billion people in the Asia-Pacific area and cases have emerged outside this region. It is caused by infection with any of the multitude of strains of the bacterium Orientia tsutsugamushi. A vaccine that affords heterologous protection and a commercially-available molecular diagnostic assay are lacking. Herein, we determined that the nucleotide and translated amino acid sequences of outer membrane protein A (OmpA) are highly conserved among 51 O. tsutsugamushi isolates. Molecular modeling revealed the predicted tertiary structure of O. tsutsugamushi OmpA to be very similar to that of the phylogenetically-related pathogen, Anaplasma phagocytophilum, including the location of a helix that contains residues functionally essential for A. phagocytophilum infection. PCR primers were developed that amplified ompA DNA from all O. tsutsugamushi strains, but not from negative control bacteria. Using these primers in quantitative PCR enabled sensitive detection and quantitation of O. tsutsugamushi ompA DNA from organs and blood of mice that had been experimentally infected with the Karp or Gilliam strains. The high degree of OmpA conservation among O. tsutsugamushi strains evidences its potential to serve as a molecular diagnostic target and justifies its consideration as a candidate for developing a broadly-protective scrub typhus vaccine.

Rickettsial Infections among Ctenocephalides felis and Host Animals during a Flea-Borne Rickettsioses Outbreak in Orange County, California.

Due to a resurgence of flea-borne rickettsioses in Orange County, California, we investigated the etiologies of rickettsial infections of Ctenocephalides felis, the predominant fleas species obtained from opossums (Didelphis virginiana) and domestic cats (Felis catus), collected from case exposure sites and other areas in Orange County. In addition, we assessed the prevalence of IgG antibodies against spotted fever group (SFGR) and typhus group (TGR) rickettsiae in opossum sera. Of the 597 flea specimens collected from opossums and cats, 37.2% tested positive for Rickettsia. PCR and sequencing of rickettsial genes obtained from C. felis flea DNA preparations revealed the presence of R. typhi (1.3%), R. felis (28.0%) and R. felis-like organisms (7.5%). Sera from opossums contained TGR-specific (40.84%), but not SFGR-specific antibodies. The detection of R. felis and R. typhi in the C. felis fleas in Orange County highlights the potential risk for human infection with either of these pathogens, and underscores the need for further investigations incorporating specimens from humans, animal hosts, and invertebrate vectors in endemic areas. Such studies will be essential for establishing a link in the ongoing flea-borne rickettsioses outbreaks.

Whole-Genome Sequence of "Candidatus Rickettsia asemboensis" Strain NMRCii, Isolated from Fleas of Western Kenya.

Herein we present the draft genome sequence and annotation of "Candidatus Rickettsia asemboensis" strain NMRCii. "Ca. Rickettsia asemboensis" is phylogenetically related to but distinct from the flea-borne spotted fever pathogen Rickettsia felis. "Ca. Rickettsia asemboensis" was initially identified in and subsequently isolated from Ctenocephalides cat and dog fleas from Kenya.

Strategies for detecting rickettsiae and diagnosing rickettsial diseases.

Rickettsial diseases and scrub typhus constitute a group of the oldest known vector-borne diseases. The cosmopolitan distribution of the vectors that transmit rickettsiae and orientiae leads to a worldwide prevalence of these diseases. Despite their significant historical status, detection and diagnosis of these diseases are still evolving today. Serological methods remain among the most prevalent techniques used for the detection/diagnosis of rickettsial diseases and scrub typhus. Molecular techniques have been instrumental in increasing the sensitivity/specificity of diagnosis, identifying new Rickettsia and Orientia species and have enhanced epidemiological capabilities when used in combination with serological methods. In this review, we discuss these techniques and their associated pros and cons.

Isolation of Candidatus Rickettsia asemboensis from Ctenocephalides Fleas.

Candidatus Rickettsia asemboensis was identified molecularly in fleas collected in 2009 from Asembo, Kenya. Multilocus sequence typing using the 17-kD antigen gene, rrs, gltA, ompA, ompB, and sca4 demonstrated that Candidatus R. asemboensis is closely related to Rickettsia felis but distinct enough to be considered for separate species classification. Following this molecular characterization of Candidatus R. asemboensis, the in vitro cultivation of this bacterium was then performed. We used Ctenocephalides canis and Ctenocephalides felis fleas removed from dogs in Kenya to initiate the in vitro isolation of Candidatus R. asemboensis. Successful cultures were obtained using Drosophila melanogaster S2 and Aedes albopictus C6/36 cell lines. Cytological staining and quantitative real-time PCR (qPCR) assays were used to visualize/confirm the culture of the bacteria in both cell lines. Sequencing of fragments of the 17-kD antigen gene, gltA, and ompB genes confirmed the identity of our Candidatus R. asemboensis isolates. To date, we have passaged Candidatus R. asemboensis 12 times through S2 and C6/36 cells, and active and frozen cultures are currently being maintained. This is the first time that a R. felis-like organism has been grown and maintained in culture and is therefore the first time that one of them, Candidatus R. asemboensis, has been characterized beyond molecular typing.

Growth of Rickettsia felis in Drosophila melanogaster S2 cells.

Rickettsia felis is an obligate, intracellular, Gram-negative bacterium and a member of the transitional group rickettsiae. This bacterium has been shown to grow in vitro in amphibian, tick, and mosquito cell lines. Here, we present data to show the growth of R. felis strain LSU in Drosophila melanogaster S2 cells, an embryonic, hemocytic cell line with phagocytic properties. R. felis LSU was isolated from Ixodes scapularis E6 (ISE6) cells and used to infect S2 cells, grown at 25°C. By 19 days postinfection, the S2 cells were 100% infected with R. felis as determined by Acridine Orange and Diff-Quik staining. A species-specific R. felis qPCR assay was used to demonstrate that the kinetics associated with the S2 cell culture infection involved a lag/adaptation phase, followed by continued growth to 20 days postinfection. Moreover, R. felis organisms were observed in the S2 cells using transmission electron microscopy and a polyclonal antibody against spotted fever rickettsiae. The ability to use D. melanogaster S2 cells for growing rickettsial agents is a useful tool owing to the ease of manipulation of the S2 cultures and the wide-ranging possibility of Drosophila resources available for future studies.

An intradermal inoculation model of scrub typhus in Swiss CD-1 mice demonstrates more rapid dissemination of virulent strains of Orientia tsutsugamushi.

Scrub typhus is an important endemic disease of the Asia-Pacific region caused by Orientia tsutsugamushi. To develop an effective vaccine to prevent scrub typhus infection, a better understanding of the initial host-pathogen interaction is needed. The objective of this study was to investigate early bacterial dissemination in a CD-1 Swiss outbred mouse model after intradermal injection of O. tsutsugamushi. Three human pathogenic strains of O. tsutsugamushi (Karp, Gilliam, and Woods) were chosen to investigate the early infection characteristics associated with bacterial virulence. Tissue biopsies of the intradermal injection site and draining lymph nodes were examined using histology and immunohistochemistry to characterize bacterial dissemination, and correlated with quantitative real-time PCR for O. tsutsugamushi in blood and tissue from major organs. Soluble adhesion molecules were measured to examine cellular activation in response to infection. No eschar formation was seen at the inoculation site and no clinical disease developed within the 7 day period of observation. However, O. tsutsugamushi was localized at the injection site and in the draining lymph nodes by day 7 post inoculation. Evidence of leukocyte and endothelial activation was present by day 7 with significantly raised levels of sL-selectin, sICAM-1 and sVCAM-1. Infection with the Karp strain was associated with earlier and higher bacterial loads and more extensive dissemination in various tissues than the less pathogenic Gilliam and Woods strains. The bacterial loads of O. tsutsugamushi were highest in the lungs and spleens of mice inoculated with Karp and Gilliam, but not Woods strains. Strains of higher virulence resulted in more rapid systemic infection and dissemination in this model. The CD-1 mouse intradermal inoculation model demonstrates features relevant to early scrub typhus infection in humans, including the development of regional lymphadenopathy, leukocyte activation and distant organ dissemination after low-dose intradermal injection with O. tsutsugamushi.

In vitro propagation of Candidatus Rickettsia andeanae isolated from Amblyomma maculatum.

Candidatus Rickettsia andeanae was identified during an investigation of a febrile outbreak in northwestern Peru (2002). DNA sequencing from two ticks (Amblyomma maculatum, Ixodes boliviensis) collected during the investigation revealed a novel Rickettsia agent with similarity to the spotted fever group rickettsiae. Since then, Candidatus R. andeanae has been detected in A. maculatum ticks collected in the southeastern and southcentral United States, Argentina, and Peru. To date, Candidatus R. andeanae has not been successfully cultivated in the laboratory. We present evidence for the continuous cultivation in three cell lines of Candidatus R. andeanae isolated from an A. maculatum tick (Portsmouth, Virginia).