Human seroepidemiology of Rickettsia and Orientia species in Chile - A cross-sectional study in five regions.

In recent years, the spectrum and epidemiology of human rickettsioses has become an emerging topic in Chile. This survey aimed to assess the seroprevalence of spotted fever group rickettsiae (SFGR), typhus group rickettsiae (TGR), and scrub typhus group orientiae (STGO) in northern, central, and southern Chile. We performed a cross-sectional study of healthy adults in rural and urban settings of five regions. Participants were chosen by double stratified random sampling in urban and by convenience in rural locations (n = 1302). Serum specimens were analyzed for group-specific IgG antibodies against SFGR, TGR, and STGO by enzyme-linked immunosorbent assays (ELISAs). Overall seroprevalences to SFGR, TGR, and STGO were 5.3 %, 1.2 %, and 0.4 %, respectively. Prevalences showed geographical differences. Statistical analyses revealed an association of older age with seropositivity to SFGR and to TGR and of rural setting and male gender with seropositivity to SFGR. The study indicates that SFGR, TGR, and STGO are endemic in Chile. The very low STGO seroprevalence might indicate an insufficient sensitivity of serological tests using Asian O. tsutsugamushi strains as ELISA antigens for the detection of antibodies against Chilean Orientia species.

Serological Evidence of Rickettsia, Orientia, and Coxiella in Domestic Animals from Bhutan: Preliminary Findings.

There is no information on rickettsial diseases in domestic animals in Bhutan. This study provides preliminary serological data on exposure of domestic animals to Rickettsia, Orientia, and Coxiella. Animal sera were collected opportunistically from Bhutan and tested in the Australian Rickettsial Reference Laboratory for IgG antibodies against spotted fever group (SFG) and typhus group (TG) Rickettsia, scrub typhus group (STG), and Q fever (QF). Of the 294 animals tested, 136 (46%) showed serological evidence of past exposure to one or more rickettsiae: 106 (36%), 62 (21%), 45 (15%), and 11 (4%) being positive against SFG Rickettsia, Orientia, TG Rickettsia, and Coxiella, respectively. Dogs appeared to exhibit the highest seropositivity against SFG (55%) and TG Rickettsia (45%), horses against STG (91%), while goats were mostly positive for Coxiella (9%). Dogs also appeared to have high risk of being exposed to SFG Rickettsia (odd ratios [OR] 5.71, 95% confidence interval [CI] 3.02-10.80, p < 0.001), TG Rickettsia (OR 48.74, 95% CI 11.29-210.32, p < 0.001), and STG (OR 6.80, 95% CI 3.32-13.95, p < 0.001), but not against QF (OR 1.95, 95% CI 0.42-8.95, p = 0.390). Differences in seropositivity rates between animal species may have been significant for SFG, TG, and STG, but not for QF. The differences in the seropositivity rates of the four infections between districts appeared to be significant for TG and STG, but not for SFG and QF. The seropositivity rates of domestic animals to the four rickettsial infections were consistent with similar studies on the human population in the same areas and appear to demonstrate a high prevalence of exposure to rickettsiae in Bhutan. These preliminary findings constitute baseline data for Bhutan. The findings of this study call for an increased human-livestock sector collaboration in rickettsial diseases research aimed at developing diagnostic and therapeutic guidelines and formulating preventive and control measures through a One Health approach.

Infection Density Dynamics and Phylogeny of Wolbachia Associated with Coconut Hispine Beetle, Brontispa longissima (Gestro) (Coleoptera: Chrysomelidae), by Multilocus Sequence Type (MLST) Genotyping.

The intracellular bacterium Wolbachia pipientis is widespread in arthropods. Recently, possibilities of novel Wolbachia-mediated hosts, their distribution, and natural rate have been anticipated, and the coconut leaf beetle Brontispa longissima (Gestro) (Coleoptera: Chrysomelidae), which has garnered attention as a serious pest of palms, was subjected to this interrogation. By adopting Wolbachia surface protein (wsp) and multilocus sequence type (MLST) genotypic systems, we determined the Wolbachia infection density within host developmental stages, body parts, and tissues, and the results revealed that all the tested samples of B. longissima were infected with the same Wolbachia strain (wLog), suggesting complete vertical transmission. The MLST profile elucidated two new alleles (ftsZ-234 and coxA-266) that define a new sequence type (ST-483), which indicates the particular genotypic association of B. longissima and Wolbachia. The quantitative real-time PCR analysis revealed a higher infection density in the eggs and adult stage, followed by the abdomen and reproductive tissues, respectively. However, no significant differences were observed in the infection density between sexes. Moreover, the wsp and concatenated MLST alignment analysis of this study with other known Wolbachia-mediated arthropods revealed similar clustering with distinct monophyletic supergroup B. This is the first comprehensive report on the prevalence, infection dynamics, and phylogeny of the Wolbachia endosymbiont in B. longissima, which demonstrated that Wolbachia is ubiquitous across all developmental stages and distributed in the entire body of B. longissima. Understanding the Wolbachia infection dynamics would provide useful insight to build a framework for future investigations, understand its impacts on host physiology, and exploit it as a potential biocontrol agent.

Conflict in the Intracellular Lives of Endosymbionts and Viruses: A Mechanistic Look at Wolbachia-Mediated Pathogen-blocking.

At the forefront of vector control efforts are strategies that leverage host-microbe associations to reduce vectorial capacity. The most promising of these efforts employs Wolbachia, a maternally transmitted endosymbiotic bacterium naturally found in 40% of insects. Wolbachia can spread through a population of insects while simultaneously inhibiting the replication of viruses within its host. Despite successes in using Wolbachia-transfected mosquitoes to limit dengue, Zika, and chikungunya transmission, the mechanisms behind pathogen-blocking have not been fully characterized. Firstly, we discuss how Wolbachia and viruses both require specific host-derived structures, compounds, and processes to initiate and maintain infection. There is significant overlap in these requirements, and infection with either microbe often manifests as cellular stress, which may be a key component of Wolbachia's anti-viral effect. Secondly, we discuss the current understanding of pathogen-blocking through this lens of cellular stress and develop a comprehensive view of how the lives of Wolbachia and viruses are fundamentally in conflict with each other. A thorough understanding of the genetic and cellular determinants of pathogen-blocking will significantly enhance the ability of vector control programs to deploy and maintain effective Wolbachia-mediated control measures.

Tick- and fly-borne bacteria in ungulates: the prevalence of Anaplasma phagocytophilum, haemoplasmas and rickettsiae in water buffalo and deer species in Central Europe, Hungary.

BACKGROUND: Hunting constitutes an important industry in Europe. However, data on the prevalence of vector-borne bacteria in large game animal species are lacking from several countries. Blood or spleen samples (239 and 270, respectively) were taken from red, fallow and roe deer, as well as from water buffaloes, mouflons and wild boars in Hungary, followed by DNA extraction and molecular analyses for Anaplasma phagocytophilum, haemoplasmas and rickettsiae. RESULTS: Based on blood samples, the prevalence rate of A. phagocytophilum infection was significantly higher in red deer (97.9%) than in fallow deer (72.7%) and roe deer (60%), and in all these compared to mouflons (6.3%). In addition, 39.2% of the spleen samples from wild boars were PCR positive for A. phagocytophilum, but none of the buffalos. Based on blood samples, the prevalence rates of both Mycoplasma wenyonii (Mw) and 'Candidatus M. haemobos' (CMh) infections were significantly higher in buffaloes (Mw: 91.2%; CMh: 73.3%) than in red deer (Mw: 64.6%; CMh: 45.8%), and in both of them compared to fallow deer (Mw: 30.3%; CMh: 9.1%) and roe deer (Mw: 20%; CMh: 1.5%). The prevalence of Mw and CMh infection significantly correlated with the body sizes of these hosts. Furthermore, Mw was significantly more prevalent than CMh in buffaloes, red and roe deer. Mycoplasma ovis was detected in mouflons, M. suis in wild boars, R. helvetica in one fallow deer and one mouflon, and an unidentified Rickettsia sp. in a fallow deer. CONCLUSIONS: Forest-dwelling game animal species were found to be important carriers of A. phagocytophilum. In contrast, animals grazing grassland (i.e. buffaloes) were less likely to get infected with this Ixodes ricinus-borne pathogen. Water buffaloes, deer species, mouflons and wild boars harbored haemoplasmas that may affect domestic ungulates. Evaluated animals with larger body size had significantly higher prevalence of infection with haemoplasmas compared to smaller deer species. The above host species rarely carried rickettsiae.

Utilidad del diagnóstico molecular precoz de fiebre Q y rickettsiosis en pacientes con fiebre de duración intermedia / Usefulness of the early molecular diagnosis of Q fever and rickettsial diseases in patients with fever of intermediate duration

La mayor parte de los casos de fiebre de duración intermedia (FDI) en España corresponden a enfermedades infecciosas (principalmente fiebre Q y rickettsiosis). En la práctica clínica el diagnóstico causal de estas entidades se basa en el inmunodiagnóstico, con una escasa utilidad en fases precoces. Por ello, el objetivo de este trabajo fue la evaluación de la utilidad de técnicas moleculares en el diagnóstico precoz de fiebre Q y rickettsiosis en pacientes con FDI. Se estudió mediante PCR la presencia de material genético de Coxiella burnetii y Rickettsia spp. en muestras sanguíneas de 271 pacientes con FDI. La especificidad de ambas técnicas es elevada, permitiendo el diagnóstico en casos no diagnosticados mediante detección de anticuerpos específicos. Estos datos sugieren que el empleo de técnicas moleculares, con una adecuada selección de la muestra de estudio y el empleo de cebadores adecuados, es un elemento útil en el diagnóstico precoz de las principales causas de FDI, principalmente si la serología es negativa o no es concluyente (AU)

Most cases of fever of intermediate duration (FDI) in Spain are associated with infectious diseases (mainly Q fever and rickettsia infections). In clinical practice, the causal diagnosis of these entities is based on immunodiagnostic techniques, which are of little help in the early stages. Therefore, the aim of this study was to evaluate the usefulness of molecular techniques for the early diagnosis of Q fever and rickettsia diseases in patients with FDI. A PCR method was used to detect the presence of genetic material of Coxiella burnetii and Rickettsia spp. in blood specimens from 271 patients with FDI. The specificity of both techniques is high, allowing diagnosis in cases undiagnosed by specific antibodies detection. These data suggest that the use of molecular techniques, with proper selection of the study specimen, and using appropriate primers is a useful tool in the early diagnosis of the main causes of FDI, especially if serology is negative or inconclusive (AU)

Molecular detection of Anaplasma platys, Anaplasma phagocytophilum and Wolbachia sp. but not Ehrlichia canis in Croatian dogs.

The bacteria Anaplasma platys, Anaplasma phagocytophilum and Ehrlichia canis are tick-borne agents that cause canine vector-borne disease. The prevalence of these pathogens in South Eastern Europe is unknown with the exception of an isolated case of A. platys detected in a dog imported into Germany from Croatia. To gain a better insight into their presence and prevalence, PCR-based screening for these bacterial pathogens was performed on domesticated dogs from different regions of Croatia. Blood samples from 1080 apparently healthy dogs from coastal and continental parts of Croatia as well as tissue samples collected from 63 deceased dogs with a history of anaemia and thrombocytopenia were collected for molecular screening by an Anaplasmataceae-specific 16S rRNA conventional PCR. Positive samples were confirmed using a second Anaplasmataceae-specific PCR assay with the PCR product sequenced for the purpose of bacterial species identification. All sequenced isolates were georeferenced and a kernel intensity estimator was used to identify clusters of greater case intensity. 42/1080 (3.8%; CI 2.7-5.0) of the healthy dogs were PCR positive for bacteria in the Anaplasmataceae. Sequencing of the 16S rRNA gene amplified from these positive samples revealed the presence of A. platys in 2.5% (CI 1.6-3.4%, 27 dogs), A. phagocytophilum in 0.3% (CI 0-0.6%, 3 dogs) and a Wolbachia endosymbiont in 1.1% (CI 0.4-1.6%, 12 dogs) of dogs screened in this study. Necropsied dogs were free from infection. Notably, no evidence of E. canis infection was found in any animal. This survey represents a rare molecular study of Anaplasmataceae in dogs in South Eastern Europe, confirming the presence of A. platys and A. phagocytophilum but not E. canis. The absence of E. canis was surprising given it has been described in all other Mediterranean countries surveyed and raises questions over the regional vector capacity of the Rhipicephalus sanguineus tick.

The spread of Wolbachia through mosquito populations.

In many regions of the world, mosquito-borne viruses pose a growing threat to human health. As an alternative to traditional control measures, the bacterial symbiont Wolbachia has been transferred from Drosophila into the mosquito Aedes aegypti, where it can block the transmission of dengue and Zika viruses. A recent paper has reported large-scale releases of Wolbachia-infected Ae. aegypti in the city of Cairns, Australia. Wolbachia, which is maternally transmitted, invaded and spread through the populations due to a sperm-egg incompatibility called cytoplasmic incompatibility. Over a period of 2 years, a wave of Wolbachia infection slowly spread out from 2 release sites, demonstrating that it will be possible to deploy this strategy in large urban areas. In line with theoretical predictions, Wolbachia infection at a third, smaller release site collapsed due to the immigration of Wolbachia-free mosquitoes from surrounding areas. This remarkable field experiment has both validated theoretical models of Wolbachia population dynamics and demonstrated that this is a viable strategy to modify mosquito populations.

Detection of 'Candidatus Neoehrlichia mikurensis' and other Anaplasmataceae and Rickettsiaceae in Canidae in Switzerland and Mediterranean countries.

INTRODUCTION: 'Candidatus Neoehrlichia mikurensis' is an emerging tick-borne zoonotic agent that primarily affects immunocompromised human patients. Dogs and foxes are frequently exposed to ticks, and both species are in close proximity to humans. This is the first study to systematically investigate the occurrence of 'Candidatus Neoehrlichia mikurensis' in Canidae in Europa. We analyzed 1'739 blood samples from dogs in Switzerland, Italy, Spain and Portugal and 162 blood samples from free-ranging red foxes (Vulpes vulpes) in Switzerland. All samples were tested using a previously described multiplex real-time PCR for the Anaplasmataceae family, the 'Candidatus Neoehrlichia' genus and the 'Candidatus Neoehrlichia mikurensis' species. All Anaplasmataceae positive samples were subsequently tested using specific real-time PCRs for Anaplasma phagocytophilum, Anaplasma platys, Ehrlichia canis and Rickettsia helvetica. Among the tested animals, one dog from Zurich tested positive for 'Candidatus Neoehrlichia mikurensis'. The 12-year old West Highland white terrier had been splenectomized 3 months prior to the blood collection and presented with polyuria/polydipsia. Fanconi syndrome was diagnosed based on glucosuria with normoglycemia and hyperaminoaciduria. A. platys and E. canis were detected in 14/249 dogs from Sicily and Portugal; two of the dogs were coinfected with both agents. Four Swiss foxes tested positive for A. phagocytophilium. R. helvetica was detected for the first time in a red fox. In conclusion, 'Candidatus Neoehrlichia mikurensis' infection should be considered in sick dogs, particularly when immunocompromised. The pathogen seems not to be widespread in Canidae in the investigated countries. Conversely, other Anaplasmataceae were more readily detected in dogs and foxes.

INTRODUCTION: 'Candidatus Neoehrlichia mikurensis' est un agent de zoonose transmis par les tiques qui gagne en importance et concerne principalement les patients immunosupprimés. Les chiens comme les renards sont souvent concernés par des morsures de tiques et vivent en contact étroit avec les êtres humains. Dans le présent travail, nous étudions pour la première fois systématiquement la présence de 'Candidatus Neoehrlichia mikurensis' chez les canidés en Europe. Les échantillons sanguins analysés provenaient de 1'739 chiens de Suisse, d'Italie, d'Espagne et du Portugal ainsi que de 162 renards (Vulpes vulpes) de Suisse. Tous les échantillons ont été examinés avec un test de PCR multiplex en temps réel déjà publié quant à la présence d'agents de la famille des Anaplasmataceae, du genre 'Candidatus Neoehrlichia' et de l'espèce 'Candidatus Neoehrlichia mikurensis'. Les échantillons positifs aux Anaplasmataceae ont ensuite été testés avec un test PCR en temps réel spécifique quant à Anaplasma phagocytophilum, Anaplasma platys, Ehrlichia canis und Rickettsia helvetica. Parmi les échantillons examinés se trouvait celui d'un chien de Zürich qui était infecté par 'Candidatus Neoehrlichia mikurensis'. Ce West Highland White Terrier de 12 ans avait été présenté pour polyurie/polydipsie; il avait été splénectomisé trois mois avant la prise de l'échantillon. Au vu d'une glycosurie et d'une hyperaminoacidurie accompagnées d'une glycémie normale, on a posé le diagnostic de syndrome de Fanconi. A. platys et E. canis ont été mis en évidence chez 14/249 chiens provenant de Sicile et du Portugal; deux chiens étaient infectés par les deux agents pathogènes. Quatre renards suisses étaient positifs à A. phagocytophilium et R. helvetica a été trouvé pour la première fois chez un renard. En résumé, on peut dire qu'une infection à 'Candidatus Neoehrlichia mikurensis' chez un chien malade doit être prise en considération comme diagnostic différentiel, particulièrement chez les anomaux immunosupprimés. Toutefois cet agent n'est pas très répandu chez les canidés des pays examinés, contrairement aux autres Anaplasmataceae spp. qui ont été trouvées plus souvent chez les chiens et les renards.

Molecular detection of Leishmania infantum, filariae and Wolbachia spp. in dogs from southern Portugal.

BACKGROUND: Leishmaniosis caused by the protozoan Leishmania infantum and dirofilariosis caused by the nematodes Dirofilaria immitis or Dirofilaria repens are vector-borne zoonoses widely present in the Mediterranean basin. In addition, some studies reported that the endosymbiont Wolbachia spp. play a role in the biology and pathogenesis of filarial parasites. The aim of this work was to evaluate the frequency of mono- and co-infections by L. infantum, filariae and Wolbachia spp. and their association with clinical signs in dogs from the south of Portugal. Leishmanial, filarial and Wolbachia spp. DNA were evaluated by specific real-time polymerase chain reaction (qPCR) assays in blood samples from 230 dogs. FINDINGS: One hundred and thirty-nine (60.4 %) dogs were qPCR-positive for L. infantum and 26 (11.3 %) for filariae (24 for D. immitis only, one D. immitis and for Acanthocheilonema dracunculoides and another one for Acanthocheilonema reconditum only). Wolbachia spp. DNA was amplified from 16 (64.0 %) out of the 25 D. immitis-positive dogs. Nineteen (8.3 %) dogs were co-infected with L. infantum and D. immitis, including the one (0.4 %) A. drancunculoides-positive animal. In dogs without clinical signs consistent with leishmaniosis and/or dirofilariosis, L. infantum prevalence was 69 %, whereas in those dogs with at least one clinical manifestation compatible with any of the two parasitoses prevalence was 42.7 %. Leishmania prevalence was significantly higher in apparently healthy mongrels (77.2 %) and pets (76.9 %) than in defined-breed dogs (including crosses; 58.8 %) and in dogs with an aptitude other than pet (i.e. farm, guard, hunting, shepherd or stray), respectively, whereas in those dogs with at least one clinical sign, the detection of L. infantum DNA was higher in males (53.3 %) and in those dogs not receiving insect repellents (52.8 %). CONCLUSIONS: The molecular detection of canine vector-borne disease (CVBD) agents, some of which are zoonotic, reinforces the need to implement efficient prophylactic measures, such as insect repellents and macrocyclic lactones (including compliance to administration), in the geographical areas where these agents are distributed, with the view to prevent infection and disease among mammalian hosts including humans.

Molecular detection of bacteria in the families Rickettsiaceae and Anaplasmataceae in northern crested caracaras (Caracara cheriway).

Bacterial pathogens of the families Anaplasmataceae and Rickettsiaceae are often spread to humans or other animals from bites from infected arthropod hosts. Recently, an increasing number of studies have implicated migratory birds in the circulation of these pathogens through the spread of arthropod vectors. However, few studies have examined the potential for resident bird populations to serve as reservoirs for these zoonoses. In this study, we used nested PCRs of the GroESL and 17 kDa genes to screen for Anaplasmataceae and Rickettsiaceae, respectively, in a resident population of the northern crested caracara (Caracara cheriway) from Florida (n=55). Additionally, a small number (n=6) of captive individuals from Texas were included. We identified one individual (1.64%) positive for Rickettsia felis and one (1.64%) positive for Ehrlichia chaffeensis; both these individuals were from Florida. Presence of these pathogens demonstrates that these birds are potential hosts; however, the low prevalence of infections suggests that these populations likely do not function as an ecological reservoir.

Establishment of a Wolbachia Superinfection in Aedes aegypti Mosquitoes as a Potential Approach for Future Resistance Management.

Wolbachia pipientis is an endosymbiotic bacterium estimated to chronically infect between 40-75% of all arthropod species. Aedes aegypti, the principle mosquito vector of dengue virus (DENV), is not a natural host of Wolbachia. The transinfection of Wolbachia strains such as wAlbB, wMel and wMelPop-CLA into Ae. aegypti has been shown to significantly reduce the vector competence of this mosquito for a range of human pathogens in the laboratory. This has led to wMel-transinfected Ae. aegypti currently being released in five countries to evaluate its effectiveness to control dengue disease in human populations. Here we describe the generation of a superinfected Ae. aegypti mosquito line simultaneously infected with two avirulent Wolbachia strains, wMel and wAlbB. The line carries a high overall Wolbachia density and tissue localisation of the individual strains is very similar to each respective single infected parental line. The superinfected line induces unidirectional cytoplasmic incompatibility (CI) when crossed to each single infected parental line, suggesting that the superinfection would have the capacity to replace either of the single constituent infections already present in a mosquito population. No significant differences in fitness parameters were observed between the superinfected line and the parental lines under the experimental conditions tested. Finally, the superinfected line blocks DENV replication more efficiently than the single wMel strain when challenged with blood meals from viremic dengue patients. These results suggest that the deployment of superinfections could be used to replace single infections and may represent an effective strategy to help manage potential resistance by DENV to field deployments of single infected strains.

Development and Validation of an Improved PCR Method Using the 23S-5S Intergenic Spacer for Detection of Rickettsiae in Dermacentor variabilis Ticks and Tissue Samples from Humans and Laboratory Animals.

A novel nested PCR assay was developed to detectRickettsiaspp. in ticks and tissue samples from humans and laboratory animals. Primers were designed for the nested run to amplify a variable region of the 23S-5S intergenic spacer (IGS) ofRickettsiaspp. The newly designed primers were evaluated using genomic DNA from 11Rickettsiaspecies belonging to the spotted fever, typhus, and ancestral groups and, in parallel, compared to otherRickettsia-specific PCR targets (ompA,gltA, and the 17-kDa protein gene). The new 23S-5S IGS nested PCR assay amplified all 11Rickettsiaspp., but the assays employing other PCR targets did not. The novel nested assay was sensitive enough to detect one copy of a cloned 23S-5S IGS fragment from "CandidatusRickettsia amblyommii." Subsequently, the detection efficiency of the 23S-5S IGS nested assay was compared to those of the other three assays using genomic DNA extracted from 40 adultDermacentor variabilisticks. The nested 23S-5S IGS assay detectedRickettsiaDNA in 45% of the ticks, while the amplification rates of the other three assays ranged between 5 and 20%. The novel PCR assay was validated using clinical samples from humans and laboratory animals that were known to be infected with pathogenic species ofRickettsia The nested 23S-5S IGS PCR assay was coupled with reverse line blot hybridization with species-specific probes for high-throughput detection and simultaneous identification of the species ofRickettsiain the ticks. "CandidatusRickettsia amblyommii,"R. montanensis,R. felis, andR. belliiwere frequently identified species, along with some potentially novelRickettsiastrains that were closely related toR. belliiandR. conorii.

National Surveillance of Spotted Fever Group Rickettsioses in the United States, 2008-2012.

Spotted fever group (SFG) rickettsioses are notifiable conditions in the United States caused by the highly pathogenic Rickettsia rickettsii and less pathogenic rickettsial species such as Rickettsia parkeri and Rickettsia sp. 364D. Surveillance data from 2008 to 2012 for SFG rickettsioses are summarized. Incidence increased from 1.7 cases per million person-years (PY) in 2000 to 14.3 cases per million PY in 2012. During 2008-2012, cases of SFG rickettsiosis were more frequently reported among males, persons of white race, and non-Hispanic ethnicity. Overall, case fatality rate (CFR) was low (0.4%), however, risk of death was significantly higher for American Indian/Alaska Natives (relative risk [RR] = 5.4) and Asian/Pacific Islanders (RR = 5.7) compared with persons of white race. Children aged < 10 years continue to experience the highest CFR (1.6%). Higher incidence of SFG rickettsioses and decreased CFR likely result from increased reporting of tick-borne disease including those caused by less pathogenic species. Recently, fewer cases have been confirmed using species-specific laboratory methods (such as cell culture and DNA detection using polymerase chain reaction [PCR] assays), causing a clouded epidemiological picture. Use of PCR and improved documentation of clinical signs, such as eschars, will better differentiate risk factors, incidence, and clinical outcomes of specific rickettsioses in the future.

The role of CD8 T lymphocytes in rickettsial infections.

Arthropod-borne obligately intracellular bacteria pose a difficult challenge to the immune system. The genera Rickettsia, Orientia, Ehrlichia, and Anaplasma evolved mechanisms of immune evasion, and each interacts differently with the immune system. The roles of CD8 T cells include protective immunity and immunopathology. In Rickettsia infections, CD8 T cells are protective mediated in part by cytotoxicity toward infected cells. In contrast, TNF-α overproduction by CD8 T cells is pathogenic in lethal ehrlichiosis by induction of apoptosis/necrosis in hepatocytes. Yet, CD8 T cells, along with CD4 T cells and antibodies, also contribute to protective immunity in ehrlichial infections. In granulocytic anaplasmosis, CD8 T cells impact pathogen control modestly but could contribute to immunopathology by virtue of their dysfunction. While preliminary evidence indicates that CD8 T cells are important in protection against Orientia tsutsugamushi, mechanistic studies have been neglected. Valid animal models will enable experiments to elucidate protective and pathologic immune mechanisms. The public health need for vaccines against these agents of human disease, most clearly O. tsutsugamushi, and the veterinary diseases, canine monocytotropic ehrlichiosis (Ehrlichia canis), heartwater (Ehrlichia ruminantium), and bovine anaplasmosis (A. marginale), requires detailed immunity and immunopathology investigations, including the roles of CD8 T lymphocytes.

Genomic diversification in strains of Rickettsia felis Isolated from different arthropods.

Rickettsia felis (Alphaproteobacteria: Rickettsiales) is the causative agent of an emerging flea-borne rickettsiosis with worldwide occurrence. Originally described from the cat flea, Ctenocephalides felis, recent reports have identified R. felis from other flea species, as well as other insects and ticks. This diverse host range for R. felis may indicate an underlying genetic variability associated with host-specific strains. Accordingly, to determine a potential genetic basis for host specialization, we sequenced the genome of R. felis str. LSU-Lb, which is an obligate mutualist of the parthenogenic booklouse Liposcelis bostrychophila (Insecta: Psocoptera). We also sequenced the genome of R. felis str. LSU, the second genome sequence for cat flea-associated strains (cf. R. felis str. URRWXCal2), which are presumably facultative parasites of fleas. Phylogenomics analysis revealed R. felis str. LSU-Lb diverged from the flea-associated strains. Unexpectedly, R. felis str. LSU was found to be divergent from R. felis str. URRWXCal2, despite sharing similar hosts. Although all three R. felis genomes contain the pRF plasmid, R. felis str. LSU-Lb carries an additional unique plasmid, pLbaR (plasmid of L. bostrychophila associated Rickettsia), nearly half of which encodes a unique 23-gene integrative conjugative element. Remarkably, pLbaR also encodes a repeats-in-toxin-like type I secretion system and associated toxin, heretofore unknown from other Rickettsiales genomes, which likely originated from lateral gene transfer with another obligate intracellular parasite of arthropods, Cardinium (Bacteroidetes). Collectively, our study reveals unexpected genomic diversity across three R. felis strains and identifies several diversifying factors that differentiate facultative parasites of fleas from obligate mutualists of booklice.

'Candidatus Rickettsia asemboensis' and Wolbachia spp. in Ctenocephalides felis and Pulex irritans fleas removed from dogs in Ecuador.

BACKGROUND: Flea-borne infections are distributed worldwide. Up to date there are no reports about microorganisms associated to fleas in Ecuador. METHODS: Seventy-one Pulex irritans and 8 Ctenocephalides felis fleas were removed from dogs in two Ecuadorian areas (Pastaza and Chimborazo Provinces) in December 2012. DNA extracts were tested by polymerase chain reaction (PCR) assays targeting universal 16S rRNA, as well as screened for the presence of Rickettsia spp. (gltA, htrA, ompB, sca4 and ompA genes) and Bartonella spp. (rpoB, gltA and ITS genes). RESULTS: Our results showed the presence of 'Candidatus Rickettsia asemboensis' (highly similar to R. felis) in C. felis and Wolbachia spp. endosimbionts in P. irritans collected from animals in Ecuador. No fleas were found to be positive for any Bartonella species or Yersinia pestis. CONCLUSIONS: Clinicians should be aware of the potential risk of this new Candidatus Rickettsia sp. and keep in mind other flea-borne infections since these flea species frequently bite humans.

Human spotted fever group rickettsioses are underappreciated in southern Taiwan, particularly for the species closely-related to Rickettsia felis.

BACKGROUND: Despite increased identification of spotted fever group rickettsioses (SFGR) in animals and arthropods, human SFGR are poorly characterized in Taiwan. METHODS: Patients with suspected Q fever, scrub typhus, murine typhus, leptospirosis, and dengue fever from April 2004 to December 2009 were retrospectively investigated for SFGR antibodies (Abs). Sera were screened for Rickettsia rickettsii Abs by indirect immunofluorescence antibody assay (IFA), and those with positive results were further examined for Abs against R. rickettsii, R. typhi, R. felis, R. conorii, and R. japonica using micro-immunofluorescence (MIF) tests. Polymerase chain reaction (PCR) for detection of SFGR DNA was applied in those indicated acute infections. Case geographic distribution was made by the geographic information system software. RESULTS: A total of 413 cases with paired serum, including 90 cases of Q fever, 47 cases of scrub typhus, 12 cases of murine typhus, 6 cases of leptospirosis, 3 cases of dengue fever, and 255 cases of unknown febrile diseases were investigated. Using IFA tests, a total of 49 cases with 47 (11.4%) and 4 (1.0%) cases had sera potentially positive for R. rickettsii IgG and IgM, respectively. In the 49 cases screened from IFA, MIF tests revealed that there were 5 cases of acute infections (3 possible R. felis and 2 undetermined SFGR) and 13 cases of past infections (3 possible R. felis and 10 undetermined SFGR). None of the 5 cases of acute infection had detectable SFGR DNA in the blood specimen by PCR. Possible acute infection of R. felis was identified in both one case of Q fever and scrub typhus. The geographic distribution of SFGR cases is similar with that of scrub typhus. CONCLUSIONS: Human SFGR exist and are neglected diseases in southern Taiwan, particularly for the species closely-related to R. felis.

Rickettsiaceae and Anaplasmataceae infections in Ixodes ricinus ticks from urban and natural forested areas of Poland.

BACKGROUND: Ixodes ricinus is a major vector for a range of microbial pathogens and the most prevalent and widely distributed tick species on the European continent, occurring in both natural and urban habitats. Nevertheless, little is known about the relative density of ticks in these two ecologically distinct habitats and the diversity of tick-borne pathogens that they carry. METHODS: We compared densities of questing I. ricinus nymphs and adults in urban and natural habitats in Central and Northeastern Poland, assessed the prevalence and rate of co-infection with A. phagocytophilum, Rickettsia, Ehrlichia and 'Ca. Neoehrlichia spp.' in ticks, and compared the diversity of tick-borne pathogens using molecular assays (PCR). RESULTS: Of the 1325 adults and nymphs, 6.2% were infected with at least one pathogen, with 4.4%, 1.7% and less than 0.5% being positive for the DNA of Rickettsia spp., A. phagocytophilum, Ehrlichia spp. and Ca. N. mikurensis, respectively. Although tick abundance was higher in natural habitats, the prevalence of the majority of pathogens was higher in urban forested areas. CONCLUSION: We conclude that: (i) zoonotic genetic variants of A. phagocytophilum are widely distributed in the Polish tick population, (ii) although the diversity of tick borne pathogens was higher in natural habitats, zoonotic species/strains were detected only in urban forests, (iii) and we provide the first description of Ca. N. mikurensis infections in ticks in Poland.

First case series of emerging Rickettsial neonatal sepsis identified by polymerase chain reaction-based deoxyribonucleic acid sequencing.

PURPOSE: To detect and identify the aetiological agent in the peripheral blood from the cases of neonatal sepsis. MATERIALS AND METHODS: Four neonates from geographically different regions of South India presented with signs of neonatal sepsis and all the routine clinical and laboratory investigations were performed. Blood culture by Bac T Alert 3D was negative. To establish the aetiology, polymerase chain reaction (PCR) for eubacterial genome and subsequent amplification with Gram positive and Gram negative primers were performed followed by deoxyribonucleic acid (DNA) sequencing. RESULTS: PCR for the detection of eubacterial genome was positive in all the four neonates and further amplification with designed Gram positive and Gram negative primers revealed the presence of Gram negative bacteria. The amplicons were identified as Orientia tsutsugamushi in three neonates and Coxiella burnetti in the other neonate. Multalin analysis was done to further characterise the strain variation among the three strains. CONCLUSION: PCR-based DNA sequencing is a rapid and reliable diagnostic tool to identify the aetiological agents of neonatal sepsis. This is the first case series of emerging Rickettsial neonatal sepsis in India .