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The American dog tick, Dermacentor variabilis (Say) (Acari: Ixodidae), is a vector for several human disease-causing pathogens such as tularemia, Rocky Mountain spotted fever, and the understudied spotted fever group rickettsiae (SFGR) infection caused by Rickettsia montanensis. It is important for public health planning and intervention to understand the distribution of this tick and pathogen encounter risk. Risk is often described in terms of vector distribution, but greatest risk may be concentrated where more vectors are positive for a given pathogen. When assessing species distributions, the choice of modeling framework and spatial layers used to make predictions are important. We first updated the modeled distribution of D. variabilis and R. montanensis using maximum entropy (MaxEnt), refining bioclimatic data inputs, and including soil variables. We then compared geospatial predictions from five species distribution modeling frameworks. In contrast to previous work, we additionally assessed whether the R. montanensis positive D. variabilis distribution is nested within a larger overall D. variabilis distribution, representing a fitness cost hypothesis. We found that 1) adding soil layers improved the accuracy of the MaxEnt model; 2) the predicted ‘infected niche’ was smaller than the overall predicted niche across all models; and 3) each model predicted different sizes of suitable niche, at different levels of probability. Importantly, the models were not directly comparable in output style, which could create confusion in interpretation when developing planning tools. The random forest (RF) model had the best measured validity and fit, suggesting it may be most appropriate to these data.

Scrub typhus is a potentially fatal rickettsiosis caused by Orientia species intracellular bacteria of the genus Orientia. Although considered to be restricted to the Asia Pacific region, scrub typhus has recently been discovered in southern Chile. We analyzed Orientia gene sequences of 16S rRNA (rrs) and 47-kDa (htrA) from 18 scrub typhus patients from Chile. Sequences were ≥99.7% identical among the samples for both amplified genes. Their diversity was 3.1%-3.5% for rrs and 11.2%-11.8% for htrA compared with O. tsusugamushi and 3.0% for rrs and 14.8% for htrA compared with Candidatus Orientia chuto. Phylogenetic analyses of both genes grouped the specimens from Chile in a different clade from other Orientia species. Our results indicate that Orientia isolates from Chile constitute a novel species, which, until they are cultivated and fully characterized, we propose to designate as Candidatus Orientia chiloensis, after the Chiloe Archipelago where the pathogen was identified.

Rickettsia and Leptospira spp. are under-recognized causes of acute febrile disease worldwide. Rickettsia species are often placed into the spotted fever group rickettsiae (SFGR) and typhus group rickettsiae (TGR). We explored the antibody prevalence among humans for these two groups of rickettsiae in four regions of Peru (Lima, Cusco, Puerto Maldonado, and Tumbes) and for Leptospira spp. in Puerto Maldonado and Tumbes. We also assessed risk factors for seropositivity and collected serum samples and ectoparasites from peri-domestic animals from households in sites with high human seroprevalence. In total, we tested 2,165 human sera for antibodies (IgG) against SFGR and TGR by ELISA and for antibodies against Leptospira by a microscopic agglutination test. Overall, human antibody prevalence across the four sites was 10.6% for SFGR (ranging from 6.2% to 14.0%, highest in Tumbes) and 3.3% for TGR (ranging from 2.6% to 6.4%, highest in Puerto Maldonado). Factors associated with seroreactivity against SFGR were male gender, older age, contact with backyard birds, and working in agriculture or with livestock. However, exposure to any kind of animal within the household decreased the odds ratio by half. Age was the only variable associated with higher TGR seroprevalence. The prevalence of Leptospira was 11.3% in Puerto Maldonado and 5.8% in Tumbes, with a borderline association with keeping animals in the household. We tested animal sera for Leptospira and conducted polymerase chain reaction (PCR) to detect Rickettsia species among ectoparasites collected from domestic animals in 63 households of seropositive participants and controls. We did not find any association between animal infection and human serostatus.

Endemic scrub typhus was recently detected on Chiloe Island in southern Chile. We report a series of cases, acquired over a wide geographical range in continental Chile during 2016-2018, demonstrating that this emerging rickettsial infection is also found on the mainland of South America.

Scrub typhus was the predominant rickettsial disease diagnosed among hospitalized patients with acute undifferentiated fever in northern Vietnam., Rickettsioses are endemic to Vietnam; however, only a limited number of clinical studies have been performed on these vectorborne bacteria. We conducted a prospective hospital-based study at 2 national referral hospitals in Hanoi to describe the clinical characteristics of scrub typhus and murine typhus in northern Vietnam and to assess the diagnostic applicability of quantitative real-time PCR assays to diagnose rickettsial diseases. We enrolled 302 patients with acute undifferentiated fever and clinically suspected rickettsiosis during March 2015–March 2017. We used a standardized case report form to collect clinical information and laboratory results at the time of admission and during treatment. We confirmed scrub typhus in 103 (34.1%) patients and murine typhus in 12 (3.3%) patients. These results highlight the need for increased emphasis on training for healthcare providers for earlier recognition, prevention, and treatment of rickettsial diseases in Vietnam.

The American dog tick, Dermacentor variabilis (Say), is a vector for several human disease causing pathogens such as tularemia, Rocky Mountain spotted fever, and the understudied spotted fever group rickettsiae (SFGR) infection caused by Rickettsia montanensis. It is important for public health planning and intervention to understand the distribution of this tick and pathogen encounter risk. Risk is often described in terms of vector distribution, but greatest risk may be concentrated where more vectors are positive for a given pathogen. When assessing species distributions, the choice of modeling framework and spatial layers used to make predictions are important. We first updated the modeled distribution of D. variabilis and R. montanensis using MaxEnt, refining bioclimatic data inputs, and including soils variables. We then compared geospatial predictions from five species distribution modeling (SDM) frameworks. In contrast to previous work, we additionally assessed whether the R. montanensis positive D. variabilis distribution is nested within a larger overall D. variabilis distribution, representing a fitness cost hypothesis. We found that 1) adding soils layers improved the accuracy of the MaxEnt model; 2) the predicted ‘infected niche’ was smaller than the overall predicted niche across all models; and 3) each model predicted different sizes of suitable niche, at different levels of probability. Importantly, the models were not directly comparable in output style, which could create confusion in interpretation when developing planning tools. The random forest (RF) model had the best measured validity and fit, suggesting it may be most appropriate to these data.

The rickettsioses of the “Far East” or Asia–Australia–Pacific region include but are not limited to endemic typhus, scrub typhus, and more recently, tick typhus or spotted fever. These diseases embody the diversity of rickettsial disease worldwide and allow us to interconnect the various contributions to this special issue of Tropical Medicine and Infectious Disease. The impact of rickettsial diseases—particularly of scrub typhus—was substantial during the wars and “police actions” of the last 80 years. However, the post-World War II arrival of effective antibiotics reduced their impact, when recognized and adequately treated (chloramphenicol and tetracyclines). Presently, however, scrub typhus appears to be emerging and spreading into regions not previously reported. Better diagnostics, or higher population mobility, change in antimicrobial policies, even global warming, have been proposed as possible culprits of this phenomenon. Further, sporadic reports of possible antibiotic resistance have received the attention of clinicians and epidemiologists, raising interest in developing and testing novel diagnostics to facilitate medical diagnosis. We present a brief history of rickettsial diseases, their relative importance within the region, focusing on the so-called “tsutsugamushi triangle”, the past and present impact of these diseases within the region, and indicate how historically, these often-confused diseases were ingeniously distinguished from each another. Moreover, we will discuss the importance of DNA-sequencing efforts for Orientia tsutsugamushi, obtained from patient blood, vector chiggers, and rodent reservoirs, particularly for the dominant 56-kD type-specific antigen gene (tsa56), and whole-genome sequences, which are increasing our knowledge of the diversity of this unique agent. We explore and discuss the potential of sequencing and other effective tools to geographically trace rickettsial disease agents, and develop control strategies to better mitigate the rickettsioses.

Recently, an intradermal inoculation of the rhesus macaque model of scrub typhus has been characterized at our institution. The current project was to establish a rhesus macaque model of scrub typhus using the naturally infected chigger challenge method that faithfully mimics the natural route of pathogen transmission to fully understand the host-pathogen-vector interactions influencing pathogen transmission. Unlike the needle-based inoculation route, Orientia tsutsugamushi-infected chiggers introduce both pathogen and chigger saliva into the host epidermis at the bite site. However, information on the interaction or influence of chigger saliva on pathogenesis and immunity of host has been limited, consequently hindering vaccine development and transmission-blocking studies. To characterize chigger inoculated O. tsutsugamushi in rhesus macaques, we determined the minimum chigger attachment time required to efficiently transmit O. tsutsugamushi to the immunocompetent hosts and preliminary assessed clinical parameters, course of bacterial infection, and host’s immunological response to identifying potential factors influencing pathogen infection. Chigger infestation on hosts resulted in: (i) Rapid transmission of O. tsutsugamushi within 1 h and (ii) antigen-specific type I and II T-cell responses were markedly increased during the acute phase of infection, suggesting that both systems play critical roles in response to the pathogen control during the primary infection. In summary, we demonstrate that O. tsutsugamushi infection in rhesus macaques via chigger challenge recapitulates the time of disease onset and bacteremia observed in scrub typhus patients. Levels of proinflammatory cytokines and chemokines were positively correlated with bacteremia.

Encounters with ticks harboring pathogenic agents have demonstrated increasing public health implications. Tick surveillance in the Republic of Korea (ROK) is essential for determining tick distributions and the potential regions where tick-borne pathogens may be found. Extensive tick collections (tick drags and tick flagging) were previously performed by Force Health Protection & Preventive Medicine (FHP&PM), Medical Activity-Korea (MEDDAC-K)/65th Medical Brigade (MED BDE) personnel, in collaboration with the Public Health Activity-Korea in the ROK. A total of 144,131 ticks were collected from 2,019 locations during 2004 to 2016. The associated location data (GPS coordinates) for each of the collection sites were incorporated into distribution maps using ArcGIS and combined with environmental data in the Maxent ecological niche modeling program (n = 733 geographical unique locations from 1,429 presence records/collection locations) to produce estimates of tick distributions for each species. The predominant tick species found and modeled were, in order of prevalence: Haemaphysalis longicornis, H. flava, Ixodes nipponensis, H. phasiana, I. turdus, Amblyomma testudinarium, H. japonica, and I. persulcatus. Haemaphysalis longicornis, H. flava, and I. nipponensis were the most widely distributed and most commonly collected species of ticks. The maps and models of suitable habitat regions produced in this study provide a better understanding of where there are potential risks of encountering a particular tick species, and which, as demonstrated herein with rickettsiae, can be used to study tick-pathogen dynamics of diseases. Knowledge of the distribution of ticks is important in the ROK because of the presence of tick-borne diseases, such as severe fever with thrombocytopenia syndrome, tick-borne encephalitis, rickettsioses, and borrelioses.

Scrub typhus, a neglected infectious disease caused by the obligate intracellular bacterium Orientia tsutsugamushi, is a major cause of fever across the Asia Pacific region with more than a billion people at risk. Treatment with antibiotics such as doxycycline or chloramphenicol is effective for the majority of patients. In the 1990s, reports from northern Thailand raised a troubling observation; some scrub typhus patients responded poorly to doxycycline, which investigators attributed to doxycycline resistance. Despite the controversial nature of these reports, independent verification was neglected, with subsequent studies speculating on the role of doxycycline resistance in contributing to failure of treatment or prophylaxis. In this review, we have outlined the evidence for drug-resistant Orientia tsutsugamushi, assessed the evidence for doxycycline resistance, and highlight more recent findings unsupportive of doxycycline resistance. We conclude that doxycycline resistance is a misconception, with treatment outcome likely to be determined by other bacterial, host, and pharmacological factors., Scrub typhus is a major cause of fever across the Asia Pacific region. Resistance to doxycycline, the main antibiotic used for treatment, was described in the 1990s. Assessment of historical evidence and recent reports suggest this finding was a misconception.

Background Scrub typhus is an emerging vector-borne zoonosis, caused by Orientia spp. and transmitted by larvae of trombiculid mites, called chiggers. It mainly occurs within a region of the Asia-Pacific called the tsutsugamushi triangle, where rodents are known as the most relevant hosts for the trombiculid vector. However, the reservoir(s) and vector(s) of the scrub typhus outside Asia-Pacific are unknown. The disease has recently been discovered on and is considered endemic for Chiloé Island in southern Chile. The aim of the present work was to detect and determine the prevalence of chiggers on different rodent species captured in probable sites for the transmission of orientiae responsible for scrub typhus on Chiloé Island in southern Chile and to molecularly examine collected chiggers for the presence of Orientia DNA. Methodology/Principal findings During the austral summer 2018, rodents were live-trapped in six sites and examined for chigger infestation. All study sites were rural areas on Chiloé Island, previously identified as probable localities where human cases acquired the scrub typhus. During a total of 4,713 trap-nights, 244 rodents of seven species were captured: the most abundant was Abrothrix olivacea. Chiggers were detected on all seven rodent species with a 55% prevalence rate. Chiggers showed low host specificity and varied according to site specific host abundance. Three genera of trombiculids were identified. Herpetacarus was the most abundant genus (93%), prevalent in five of the six sites. Infestation rates showed site specific differences, which were statistically significant using a GLM model with binomial errors. Molecular analyses proved that 21 of 133 (15.8%) mite pools were positive for Orientia species, all of them belonged to the genus Herpetacarus. Conclusions/Significance This study firstly reports the presence of different rodent-associated chigger mites positive for Orientia sp., in a region endemic for scrub typhus in southern Chile. Herpetacarus and two other genera of mites were found with high infestation rates of rodents in sites previously identified as probable exposure of scrub typhus cases. A substantial percentage of mite pools were positive for Orientia DNA, suggesting that chigger mites serve as vectors and reservoirs of this emerging zoonosis in South America., Author summary Scrub typhus is a chigger-transmitted zoonotic infection caused by Orientia species, which is endemic to the tsutsugamushi triangle in Asia-Pacific region. Recently, a focus of scrub typhus in South America has been confirmed on Chiloé Island in southern Chile. However, the vectors of scrub typhus in this region remain unknown. We undertook a survey to study the presence of chiggers on different rodent species in areas identified as probable sites of exposure to scrub typhus on Chiloé Island. The study showed that 55% of rodents were infested by trombiculids. Three mite genera were identified, of which Herpetacarus was the most abundant. Chiggers showed low host specificity, but spatial differences. Using molecular techniques, the trombiculid mites were found to be infected with Orientia species. These findings suggest that chigger mites play a role in the life cycle and transmission of this emerging infectious disease in Chile.

In this study, we used a metagenomic approach to analyze bacterial communities from diverse populations (humans, animals, and vectors) to investigate the role of these microorganisms as causative agents of disease in human and animal populations. Wild rodents and ectoparasites were collected from 2014 to 2018 in Nan province, Thailand where scrub typhus is highly endemic. Samples from undifferentiated febrile illness (UFI) patients were obtained from a local hospital. A total of 200 UFI patient samples were obtained and 309 rodents and 420 pools of ectoparasites were collected from rodents (n = 285) and domestic animals (n = 135). The bacterial 16S rRNA gene was amplified and sequenced with the Illumina. Real-time PCR and Sanger sequencing were used to confirm the next-generation sequencing (NGS) results and to characterize pathogen species. Several pathogens were detected by NGS in all populations studied and the most common pathogens identified included Bartonella spp., Rickettsia spp., Leptospira spp., and Orientia tsutsugamushi. Interestingly, Anaplasma spp. was detected in patient, rodent and tick populations, although they were not previously known to cause human disease from this region. Candidatus Neoehrlichia, Neorickettsia spp., Borrelia spp., and Ehrlichia spp. were detected in rodents and their associated ectoparasites. The same O. tsutsugamushi genotypes were shared among UFI patients, rodents, and chiggers in a single district indicating that the chiggers found on rodents were also likely responsible for transmitting to people. Serological testing using immunofluorescence assays in UFI samples showed high prevalence (IgM/IgG) of Rickettsia and Orientia pathogens, most notably among samples collected during September-November. Additionally, a higher number of seropositive samples belonged to patients in the working age population (20-60 years old). The results presented in this study demonstrate that the increased risk of human infection or exposure to chiggers and their associated pathogen (O. tsutsugamushi) resulted in part from two important factors; working age group and seasons for rice cultivation and harvesting. Evidence of pathogen exposure was shown to occur as there was seropositivity (IgG) in UFI patients for bartonellosis as well as for anaplasmosis. Using a metagenomic approach, this study demonstrated the circulation and transmission of several pathogens in the environment, some of which are known causative agents of illness in human populations.

Background Resurgence of scrub typhus was reported in Northeast India in 2010 after a gap of 67 years since World War II. However, the presence of other rickettsial infections remained unknown from this region. A seroepidemiological investigation was undertaken in the scrub typhus affected areas from 2013-2015 in Assam, Arunachal Pradesh and Nagaland to assess the exposure to other rickettsial diseases besides scrub typhus. Methods Samples were collected from people residing in scrub typhus reporting areas. Serology was performed by an indirect ELISA for the three rickettsial agents' viz., scrub typhus group orientiae (STGO), spotted fever group rickettsiae (SFGR) and typhus group rickettsiae (TGR). A sample with total net absorbance ≥1.000 was considered as positive. An entomological survey was also carried out in the affected areas. Results Overall, 1265 human blood samples were collected, of which 30.8% (n=390), 13.8% (175) and 4.2% (53) had antibodies against STGO, SFGR and TGR respectively. Presence of antibodies against more than one of the rickettsial groups was also detected. Among the arthropods collected, chiggers of Leptotrombidium deleinse, fleas belonging to Ctenocephalides felis and Pulex irritans, ticks belonging to Rhipicephalus microplus, Haemaphysalis spp. were predominant. Candidatus Rickettsia senegalensis was detected in C. felis. Conclusions Our findings confirm wide circulation of rickettsial infections and their probable vectors in the northeast region of India.Accession numbers: KU163367, KU163368, KU499847, KU499848.

To increase knowledge of undifferentiated fevers in Kenya, we tested paired serum samples from febrile children in western Kenya for antibodies against pathogens increasingly recognized to cause febrile illness in Africa. Of patients assessed, 8.9%, 22.4%, 1.1%, and 3.6% had enhanced seroreactivity to Coxiella burnetii, spotted fever group rickettsiae, typhus group rickettsiae, and scrub typhus group orientiae, respectively.

Rickettsia montanensis has long been considered a nonpathogenic member of the spotted fever group rickettsiae. However, the infection potential of R. montanensis is being revisited in light of its recent association with a case of human infection in the United States and the possibility that additional cases may have been misdiagnosed as Rocky Mountain spotted fever. To this end, DNA was extracted from American dog ticks (Dermacentor variabilis) removed from Department of Defense (DoD) personnel and their dependents at DoD medical treatment facilities (MTFs) during 2002-2012 (n = 4792). These 4792 samples were analyzed for the presence of R. montanensis (n = 36; 2.84%) and all vector DNA was confirmed to be of D. variabilis origin using a novel Dermacentor genus-specific quantitative real-time polymerase chain reaction procedure, Derm, and a novel Dermacentor species multilocus sequence typing assay. To assess the risk of R. montanensis infection, the positive and negative samples were geographically mapped utilizing MTF site locations. Tick localities were imported into a geographical information systems (GIS) program, ArcGIS, for mapping and analysis. The ecological niche modeling (ENM) program, Maxent, was used to estimate the probability of tick presence in eastern United States using locations of both R. montanensis-positive and -negative ticks, climate, and elevation data. The ENM for R. montanensis-positive D. variabilis estimated high probabilities of the positive ticks occurring in two main areas, including the northern Midwest and mid-Atlantic portions of the northeastern regions of United States, whereas the R. montanensis-negative D. variabilis tick model showed a wider estimated range. The results suggest that R. montanensis-positive and -negative D. variabilis have different ranges where humans may be at risk and are influenced by similar and different factors.

The co-evolution of Orientia tsutsugamushi and its vector/host Leptotrombidium mites is important for this bacterium to survive and exist in its environment. The data in this study demonstrated that O. tsutsugamushi has adapted to take advantage of the parasitic nature of the host's larval stage and thus increase its chance of transmission to a vertebrate host and potentially to other vector mites by increasing its density at the time of transmission. Our data demonstrated that during the larval stage the density of O. tsutsugamushi was at its highest level compared to other life stages (Kruskal-Wallis, p < 0.0001). We further revealed that the different O. tsutsugamushi 56-kDa type-specific antigen (TSA) genotypes within the mite were maintained and preserved during transovarial transmission from the Leptotrombidium imphalum, lines Li-3 and Li-5. No sequence difference of 56-kDa TSA gene (variable domain I-III, 765 bp) was observed between the UT302-like genotype found in mothers and their offspring (100% identity). However, one or two nonsynonymous mutations in the 56-kDa TSA gene were observed in the Karp-like genotypes found in the F1 offspring with a percent difference ranging from 0.13 to 0.26 for nucleotide sequences and from 0.39 to 0.78 for amino acid sequences. Additionally, the composition of co-existing O. tsutsugamushi genotypes was maintained in L. imphalum lines through transsovarial and transstadial transmission processes; however, the proportion of these genotypes in each stage varied (larva, nymph, adult). These results show some of the key characteristics of O. tsutsugamushi maintenance within and transmission among its vector/host L. imphalum.

To expand the documentation of rickettsioses in Indonesia, we conducted an ectoparasite and small mammal investigation involving four major islands: Java, Sumatra, Sulawesi, and Kalimantan. Coastal and highland regions on each island surveyed were chosen to represent different ecologies in Indonesia. Indication of the presence of Rickettsia spp. was evident in all areas sampled. Typhus group rickettsiae-specific antibodies had significantly higher prevalence among small mammals captured in Java compared to the other islands surveyed (78% in coastal and 50% in highland regions) and the prevalence of spotted fever group rickettsiae-specific antibodies was significantly higher in Kalimantan than the other islands investigated. Hosts and vectors were restricted by Rickettsia spp. but not by coastal or highland regions. Our findings expand the range in which rickettsial pathogens have been documented within the Indonesian archipelago and point to a significant risk to human health.

We assessed the diagnostic accuracy of two immunochromatographic tests (ICTs), the Access Bio CareStart Scrub Typhus test (Somerset, NJ) (IgM), and the SD BIOLINE Tsutsugamushi test (Kyonggi-do, Republic of Korea) (IgG, IgM, or IgA) compared with indirect immunofluorescence assay (IFA) and real-time PCR results as ref- erence tests using 86 paired acute and convalescent specimens from febrile patients. The sensitivity and specificity of the CareStart test were 23.3% (95% confidence interval (CI): 11.8-38.6) and 81.4% (95% CI: 66.6-91.6), respectively, for acute specimens and 32.6% (95% CI: 19.1-48.5) and 79.1% (95% CI: 64.0-90.0), respectively, for convalescent specimens. For the SD BIOLINE test, sensitivity and specificity were 20.9% (95% CI: 10.0-36.0) and 74.4% (95% CI: 58.8-86.5), respectively, for acute specimens and 76.7% (95% CI: 61.4-88.2) and 76.7% (95% CI: 61.4-88.2), respec- tively, for convalescent specimens. The poor sensitivity obtained for both ICTs during this study when performed on acute specimens highlights the difficulties in prompt diagnosis of scrub typhus.

Using a large, passive, febrile surveillance program in Iquitos, Peru, we retrospectively tested human blood specimens for scrub typhus group orientiae by ELISA, immunofluorescence assay, and PCR. Of 1,124 participants, 60 (5.3%) were seropositive, and 1 showed evidence of recent active infection. Our serologic data indicate that scrub typhus is present in the Peruvian Amazon.

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.

To explore the risk of antimicrobial resistant (AMR) non-typhoidal Salmonella during asymptomatic infection passage between pet dogs and human caregivers in Khon Kaen, Thailand, one hundred forty paired fecal samples (n = 280) were obtained from companion dogs and their human caregivers, interviewed from 140 households during 2019&ndash, 2020. The purified Salmonella isolates were serotype-identified and tested for antimicrobial resistance against ampicillin, ciprofloxacin, chloramphenicol, nalidixic acid, streptomycin, sulfamethoxazole&ndash, trimethoprim, and tetracycline. The homologous Salmonella isolate pairs (suggesting Salmonella infections may have been due to passage between each one of the pair, or derived from the same source) were subsequently characterized by serotype screening, pulsed field gel electrophoresis (PFGE), and Synchrotron Fourier transform infrared spectroscopy (SR-FTIR). The Salmonella prevalence observed in dogs, 12.86% (18/140), was not significantly different from that observed in humans, 17.86% (25/140) using McNemar&rsquo, s test. The AMR patterns (the patterns among the isolates of pet dogs and caregivers) and the serotypes (thirteen serotypes with 18 isolates from pet dogs plus thirteen serotypes with 25 isolates from humans) between pet dogs and humans were not significantly different using Pearson&rsquo, s chi-squared test. The homologous Salmonella isolates from the Salmonella-present households was 5.13% (2/39). This study demonstrated that the hypothesis that there is a high risk of Salmonella infection passage between dogs and humans with close contact in Khon Kaen is doubtful. Only 5.13% of homologous Salmonella isolates from Salmonella-present households were found in Khon Kaen, Thailand, although the prevalence of Salmonella-positive samples, serotypes, and antimicrobial resistance patterns were quite similar among the two populations.

Scrub typhus and its etiological agents, Orientia species, have been around for a very long time. Historical reference to the rickettsial disease scrub typhus was first described in China (313 AD) by Hong Ge in a clinical manual (Zhouhofang) and in Japan (1810 AD) when Hakuju Hashimoto described tsutsuga, a noxious harmful disease in the Niigata prefecture. Other clinicians and scientists in Indonesia, Philippines, Taiwan, Australia, Vietnam, Malaysia, and India reported on diseases most likely to have been scrub typhus in the early 1900s. All of these initial reports about scrub typhus were from an area later designated as the Tsutsugamushi Triangle—an area encompassing Pakistan to the northwest, Japan to the northeast and northern Australia to the south. It was not until the 21st century that endemic scrub typhus occurring outside of the Tsutsugamushi Triangle was considered acceptable. This report describes the early history of scrub typhus, its distribution in and outside the Tsutsugamushi Triangle, and current knowledge of the causative agents, Orientia species.

Orientia tsutsugamushi, formerly Rickettsia tsutsugamushi, is an obligate intracellular pathogen that causes scrub typhus, an underdiagnosed acute febrile disease with high morbidity. Scrub typhus is transmitted by the larval stage (chigger) of Leptotrombidium mites and is irregularly distributed across endemic regions of Asia, Australia and islands of the western Pacific Ocean. Previous work to understand population genetics in O. tsutsugamushi has been based on sub-genomic sampling methods and whole-genome characterization of two genomes. In this study, we compared 40 genomes from geographically dispersed areas and confirmed patterns of extensive homologous recombination likely driven by transposons, conjugative elements and repetitive sequences. High rates of lateral gene transfer (LGT) among O. tsutsugamushi genomes appear to have effectively eliminated a detectable clonal frame, but not our ability to infer evolutionary relationships and phylogeographical clustering. Pan-genomic comparisons using 31 082 high-quality bacterial genomes from 253 species suggests that genomic duplication in O. tsutsugamushi is almost unparalleled. Unlike other highly recombinant species where the uptake of exogenous DNA largely drives genomic diversity, the pan-genome of O. tsutsugamushi is driven by duplication and divergence. Extensive gene innovation by duplication is most commonly attributed to plants and animals and, in contrast with LGT, is thought to be only a minor evolutionary mechanism for bacteria. The near unprecedented evolutionary characteristics of O. tsutsugamushi, coupled with extensive intra-specific LGT, expand our present understanding of rapid bacterial evolutionary adaptive mechanisms.

Background Scrub typhus is a neglected vector-borne zoonosis causing life-threatening illnesses, endemic in the Asian-Pacific region and, as recently discovered, in southern Chile. Scrub typhus is rarely reported in travelers, most probably due to the lack of clinical experience and diagnostic tests in non-endemic countries. We report the first case of imported scrub typhus in South America. Case presentation A 62-year-old tourist from South Korea presented severely ill with fever, rash, and eschar in Santiago, Chile. Laboratory exams showed thrombocytopenia and elevated inflammation parameters, hepatic enzymes, and LDH. With the clinical suspicion of scrub typhus, empirical treatment with doxycycline was initiated and the patient recovered rapidly and without complications. The diagnosis was confirmed by IgM serology and by real-time PCR, which demonstrated infection with Orientia tsutsugamushi (Kawasaki clade). Conclusions Only due to the emerging clinical experience with endemic South American scrub typhus and the recent implementation of appropriate diagnostic techniques in Chile, were we able to firstly identify and adequately manage a severe case of imported scrub typhus in South America. Physicians attending febrile travelers need to be aware of this rickettsiosis, since it requires prompt treatment with doxycycline to avoid complications.

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.

Although flea-borne rickettsiosis is endemic in Los Angeles County, outbreaks are rare. In the spring of 2015 three human cases of flea-borne rickettsiosis among residents of a mobile home community (MHC) prompted an investigation. Fleas were ubiquitous in common areas due to presence of flea-infested opossums and overabundant outdoor cats and dogs. The MHC was summarily abated in June 2015, and within five months, flea control and removal of animals significantly reduced the flea population. Two additional epidemiologically-linked human cases of flea-borne rickettsiosis detected at the MHC were suspected to have occurred before control efforts began. Molecular testing of 106 individual and 85 pooled cat fleas, blood and ear tissue samples from three opossums and thirteen feral cats using PCR amplification and DNA sequencing detected rickettsial DNA in 18.8% of the fleas. Seventeen percent of these cat fleas tested positive for R. felis-specific DNA compared to under two (, Author summary Outbreaks of flea-borne rickettsiosis are rare despite the endemic status in Los Angeles County. In the spring of 2015 three human cases of flea-borne rickettsiosis among residents of a mobile home community (MHC) prompted an investigation. Fleas were found in all common areas at the MHC due to presence of flea-infested opossums and overabundant outdoor cats and dogs. The MHC was summarily abated in June 2015, and within five months, flea control and removal of animals significantly reduced the flea population. Two additional epidemiologically-linked human cases detected at the MHC were considered to have occurred before control efforts began. Molecular testing of cat fleas, immunological testing of opossums and feral cats collected at the site indicated active transmission of flea-borne rickettsiosis. This study represents the first flea-borne rickettsial outbreak that summary abatement approach was used to reduce its intensity.

Trombiculid mites are the vectors of scrub typhus, with infected larval mites (chiggers) transmitting the causative agent, Orientia tsutsugamushi, during feeding. Co-existence of multiple O. tsutsugamushi strains within infected mites has previously been reported in naturally infected, laboratory-reared mite lines using molecular methods to characterize the 56-kDa type-specific antigen (TSA) gene. In the current study, more advanced next-generation sequencing technology was used to reveal the heterogeneity of O. tsutsugamushi genotypes in field-collected trombiculid mites from rodents and small mammals in scrub typhus-endemic areas of Thailand. Twenty-eight trombiculid mites collected from 10 small mammals were positive for O. tsutsugamushi, corresponding to a prevalence rate of 0.7% within the mite population. Twenty-four of the infected mites were Leptotrombidium spp., indicating that this genus is the main vector for O. tsutsugamushi transmission in Thailand. In addition, O. tsutsugamushi was detected in the mite genera Ascoschoengastia, Blankaartia, Gahrliepia, and Lorillatum. Of the 10 infested small animal hosts, six had 2–10 infected mites feeding at the time of collection. Deep sequencing was used to characterize mixed infections (two to three O. tsutsugamushi genotypes within an individual mite), and 5 of the 28 infected mites (17.9%) contained mixed infections. Additionally, 56-kDa TSA gene sequence analysis revealed identical bacterial genotypes among co-feeding mites with single or mixed infections. These results suggest that co-feeding transmission may occur during the feeding process, and could explain the occurrence of mixed infections in individual mites, as well as the recovery of multiple infected mites from the same host. This study also revealed highly diverse within-host O. tsutsugamushi genotypes. The occurrence of multiple O. tsutsugamushi genotypes within individual mites has important implications, and could provide a mechanism for pathogen evolution/diversification in the mite vector., Author summary Scrub typhus is a leading cause of undifferentiated febrile illness, putting 1 billion people at risk of infection. Trombiculid mites are the major vectors of the causative agent of scrub typhus, Orientia tsutsugamushi (OT), which is transferred through the bite of an infected mite. Previously, we reported the co-existence of multiple OT strains within laboratory-reared mites. Here, we extended our investigation into wild trombiculid mite populations collected from areas of Thailand where scrub typhus is endemic. More advanced next-generation sequencing (NGS) technology was used to examine the presence and abundance of mixed infection in individual mites based on sequence analysis of the 56-kDa type-specific antigen gene. NGS data revealed heterogeneity of OT genotypes in field-collected mites, and mixed infection with two to three OT genotypes was consistently observed (prevalence rate = 17.9%). Moreover, the identical OT genotype detected among co-feeding mites with single or mixed infections suggesting that co-feeding transmission may occur during the feeding process. This result could explain the occurrence of mixed infections in individual mites, as well as the recovery of multiple infected mites from the same host.

Investigation of a scrub typhus outbreak in Thailand during September 2013 found that 9.1% of Thai soldiers and 11.1% of residents living in areas surrounding training sites had antibodies against the causative agent, Orientia tsutsugamushi. Sequence analysis of O. tsutsugamushi from rodents and chiggers identified 7 genogroups and 3 genotypes.

Scrub typhus is a mites-borne rickettsiosis caused by the obligate intracellular Gram-negative bacterium Orientia tsutsugamushi. The disease is potentially life threatening and is prevalent in tropical Asia, islands of the western Pacific Ocean and northern Australia where an estimated one million cases occur annually. Orientia tsutsugamushi is transmitted by the bite of larval mites in the genus Leptotrombidium. In the present study, the composition of the microbiome in larvae, deutonymphs and adult males and females from laboratory colonies of L. imphalum that were infected as well as uninfected with O. tsutsugamushi were investigated by high-throughput sequencing of the bacterial 16S rRNA gene. Notably, the bacterial microbiomes of infected adult females were dominated by sequences of O. tsutsugamushi and an unidentified species of Amoebophilaceae, which together comprised 98.2% of bacterial sequences. To improve the taxonomic resolution of the Amoebophilaceae OTU a nearly full length sequence of the 16S rRNA gene was amplified, cloned, and Sanger sequenced. Infected female mites had 89 to 92% nucleotide identity with the Amoebophilaceae family, indicating that the bacterium was likely to be a species of a novel genus. The species composition of bacterial communities varied between mite life stages regardless of their infection status. Uninfected adults exhibited greater species diversity than adults infected with O. tsutsugamushi. In the infected colony, the rate of filial infection with Orientia was less than 100%. Larval and male mites that were PCR-negative for Orientia contained low numbers of sequences of Amoebophilaceae (0.01 and 0.06%, respectively) in their taxonomic profiles, suggesting that a mutualistic relationship exists between the novel species of Amoebophilaceae and O. tsutsugamushi. Our study findings provide the basis for further research to determine the influence of the novel Amoebophilaceae species on the bacterial microbiome and on vector susceptibility to and transovarial transmission of O. tsutsugamushi.

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.

Background Scrub typhus is an important endemic disease in tropical Asia caused by Orientia tsutsugamushi for which no effective broadly protective vaccine is available. The successful evaluation of vaccine candidates requires well-characterized animal models and a better understanding of the immune response against O. tsutsugamushi. While many animal species have been used to study host immunity and vaccine responses in scrub typhus, only limited data exists in non-human primate (NHP) models. Methodology/Principle findings In this study we evaluated a NHP scrub typhus disease model based on intradermal inoculation of O. tsutsugamushi Karp strain in rhesus macaques (n = 7). After an intradermal inoculation with 106 murine LD50 of O. tsutsugamushi at the anterior thigh (n = 4) or mock inoculum (n = 3), a series of time course investigations involving hematological, biochemical, molecular and immunological assays were performed, until day 28, when tissues were collected for pathology and immunohistochemistry. In all NHPs with O. tsutsugamushi inoculation, but not with mock inoculation, the development of a classic eschar with central necrosis, regional lymphadenopathy, and elevation of body temperature was observed on days 7–21 post inoculation (pi); bacteremia was detected by qPCR on days 6–18 pi; and alteration of liver enzyme function and increase of white blood cells on day 14 pi. Immune assays demonstrated raised serum levels of soluble cell adhesion molecules, anti-O. tsutsugamushi-specific antibody responses (IgM and IgG) and pathogen-specific cell-mediated immune responses in inoculated macaques. The qPCR assays detected O. tsutsugamushi in eschar, spleen, draining and non-draining lymph nodes, and immuno-double staining demonstrated intracellular O. tsutsugamushi in antigen presenting cells of eschars and lymph nodes. Conclusions/Significance These data show the potential of using rhesus macaques as a scrub typhus model, for evaluation of correlates of protection in both natural and vaccine induced immunity, and support the evaluation of future vaccine candidates against scrub typhus., Author summary Scrub typhus is a febrile illness caused by bacteria that invade and live within cells of the immune and blood vessel systems. Small earth-bound mites can bite humans and transmit these bacteria into the skin. Scrub typhus is treatable with antibiotics, but currently there is no scrub typhus vaccine available. Unfortunately if humans get scrub typhus, the immune response is usually weak and short-lived, especially against different strains, and affected individuals can get ill again within a year. This is a problem in areas where the infection is very common and a vaccine could be an effective approach to protect susceptible humans against scrub typhus. In this study, we characterized the immune response and disease features of scrub typhus in rhesus macaques by inoculating the bacteria directly into the skin–similar to the mite bite in nature–previously this had only been done in cynomolgus macaques (Macaca fascicularis). We found that the scrub typhus symptoms and immune responses of rhesus macaques resemble more closely the human responses than those of cynomolgus macaques. Studying the immune response in rhesus macaques will help us to understand how humans react against different bacterial proteins, to identify new markers of protection and to find the strongest vaccine candidates. This will then help us develop new and better vaccines (and also diagnostics) against scrub typhus in the future.

Scrub typhus is an acute, and sometimes fatal, human febrile illness, typically successfully treated using chloramphenicol or one of the tetracyclines. Over the past several years, descriptions of strains of Orientia tsutsugamushi with reduced susceptibility to antibiotics have appeared. Because case-fatality ratios approached 50% during the pre-antibiotic era, antibiotic-resistant scrub typhus is concerning. Herein, we review the data on resistant scrub typhus, describe how the theoretical existence of such resistance is affected by interpretation of treatment outcomes, and propose a plan to further identify whether true drug resistance is present and how to deal with drug resistance if it has evolved. Limited resistance is not unambiguous, if present, and antibiotic resistance in scrub typhus is not a dichotomous trait. Rather, evidence of resistance shows a continuous gradation of increasing resistance. The availability of genomes from isolates of O. tsutsugamushi allows the search for loci that might contribute to antibiotic resistance. At least eighteen such loci occur in all genomes of O. tsutsugamushi examined. One gene (gyrA) occurs as a quinolone-resistant form in the genome of all isolates of O. tsutsugamushi. At least 13 other genes that are present in some members of the genus Rickettsia do not occur within O. tsutsugamushi. Even though reports of scrub typhus not responding appropriately to chloramphenicol or a tetracycline treatment have been in the literature for approximately 23 years, the existence and importance of antibiotic-resistant scrub typhus remains uncertain.

Three novel isolates of the genus Bartonella were recovered from the blood of two patients enrolled in a clinical trial for the treatment of chronic stage Bartonella bacilliformis infection (verruga peruana) in Caraz, Ancash, Peru. The isolates were initially characterized by sequencing a fragment of the gltA gene, and found to be disparate from B. bacilliformis. The isolates were further characterized using phenotypic and genotypic methods, and found to be genetically identical to each other for the genes assessed, but distinct from any known species of the genus Bartonella, including the closest relative B. bacilliformis. Other characteristics of the isolates, including their morphology, microscopic and biochemical properties, and growth patterns, were consistent with members of the genus Bartonella. Based on these results, we conclude that these three isolates are members of a novel species of the genus Bartonella for which we propose the name Bartonella ancashensis sp. nov. (type strain 20.00T = ATCC BAA-2694T = DSM 29364T).

Currently, no vaccine has been developed to protect humans from naturally acquired heterologous Orientia tsutsugamushi infections. To enhance the validity of vaccine candidates, we are developing a murine chigger challenge model with the O. tsutsugamushi Lc-1-infected Leptotrombidium chiangraiensis Line-1. To this end, an intraperitoneal (i.p.) murine challenge model using an O. tsutsugamushi Lc-1 isolate was developed for eventual validation of the chigger challenge model. We have determined that the murine lethal dose that kills 50% of the challenged mice (MuLD50) of a liver/spleen homogenate developed from O. tsutsugamushi Lc-1-infected ICR Swiss mice to be 10(-6.9). Employing different inoculum doses of this homogenate, the bacterial load using quantitative real-time PCR (qPCR) was determined to range from 60 to 1.6 × 10(5) genome equivalent copies (GEC)/μL of liver and 33.4 to 2.2 × 10(5) GEC/μL of spleen tissue. The clinical outcomes relative to homogenate dose levels followed a dose-dependent pattern. The successful development and characterization of the O. tsutsugamushi Lc-1 i.p. challenge model will assist in the development and validation of a mouse chigger challenge scrub typhus model.

Ticks are the most significant vectors of infectious diseases in the United States, inspiring many researchers to study aspects of their biology, ecology, and their effects on public health. However, regional differences in tick abundance and pathogen infection prevalence result in the inability to assume results from one area are relevant in another. Current local information on tick ranges, infection rates, and human cases is needed to assess tick-borne disease risk in any given region. The Mid-Atlantic Tick Summit III brought together over 100 area experts and researchers to share regional updates on ticks and their associated pathogens. We report some meeting highlights here. Regional meetings foster cross-disciplinary collaborations that benefit the community, and open novel lines of inquiry so that tick-bite risk can be reduced and tick-borne diseases can be treated effectively.

ABSTRACT 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.

We assessed serum samples from 1,000 US Marines deployed to Afghanistan during 2001–2010 to find evidence of 4 rickettsial pathogens. Analysis of predeployment and postdeployment samples showed that 3.4% and 0.5% of the Marines seroconverted for the causative agents of Q fever and spotted fever group rickettsiosis, respectively.

Scrub typhus is a febrile infection caused by the obligate intracellular bacterium Orientia tsutsugamushi, which causes significant morbidity and mortality across the Asia-Pacific region. The control of this vector-borne disease is challenging due to humans being dead-end hosts, vertical maintenance of the pathogen in the vector itself, and a potentially large rodent reservoir of unclear significance, coupled with a lack of accurate diagnostic tests. Development of an effective vaccine is highly desirable. This however requires better characterization of the natural immune response of this neglected but important disease. Here we implement a novel IFN-γ ELISpot assay as a tool for studying O. tsutsugamushi induced cellular immune responses in an experimental scrub typhus rhesus macaque model and human populations. Whole cell antigen for O. tsutsugamushi (OT-WCA) was prepared by heat inactivation of Karp-strain bacteria. Rhesus macaques were infected intradermally with O. tsutsugamushi. Freshly isolated peripheral blood mononuclear cells (PBMC) from infected (n = 10) and uninfected animals (n = 5) were stimulated with OT-WCA, and IFN-γ secreting cells quantitated by ELISpot assay at five time points over 28 days. PBMC were then assayed from people in a scrub typhus-endemic region of Thailand (n = 105) and responses compared to those from a partially exposed population in a non-endemic region (n = 14), and to a naïve population in UK (n = 12). Mean results at Day 0 prior to O. tsutsugamushi infection were 12 (95% CI 0–25) and 15 (2–27) spot-forming cells (SFC)/106 PBMC for infected and control macaques respectively. Strong O. tsutsugamushi-specific IFN-γ responses were seen post infection, with ELISpot responses 20-fold higher than baseline at Day 7 (mean 235, 95% CI 200–270 SFC/106 PBMC), 105-fold higher at Day 14 (mean 1261, 95% CI 1,097–1,425 SFC/106 PBMC), 125-fold higher at Day 21 (mean 1,498, 95% CI 1,496–1,500 SFC/106 PBMC) and 118-fold higher at Day 28 (mean 1,416, 95% CI 1,306–1,527 SFC/106 PBMC). No significant change was found in the control group at any time point compared to baseline. Humans from a scrub typhus endemic region of Thailand had mean responses of 189 (95% CI 88–290) SFC/106 PBMC compared to mean responses of 40 (95% CI 9–71) SFC/106 PBMC in people from a non-endemic region and 3 (95% CI 0–7) SFC/106 PBMC in naïve controls. In summary, this highly sensitive assay will enable field immunogenicity studies and further characterization of the host response to O. tsutsugamushi, and provides a link between human and animal models to accelerate vaccine development., Author summary Scrub typhus is a disease caused by bacteria that invade cells in our immune system and blood vessels. It is transmitted by mites and is treatable with antibiotics. Unfortunately diagnosis is difficult and requires techniques that are not easily accessible everywhere. Currently, there is no scrub typhus vaccine available. In order to improve diagnostics and vaccine development in future, we need to better understand our immune response against these bacteria. In this study, we developed a test where these bacteria were killed and prepared by a new purification method to stimulate the immune cells in our blood -not antibodies. We evaluated this test in hospitalized patients with scrub typhus disease and also in non-human primates to study the responses over time. The test proved to be very accurate and useful to study natural immune responses, and we found differences in responses in areas where scrub typhus is common, compared to areas where it is not common. This test will allow us to investigate the immune response to scrub typhus more in-depth in the future, and will support the development of better diagnostic tests and vaccines against scrub typhus.

International audience; Background: Rickettsiae are obligate intracellular bacteria responsible for many febrile syndromes around the world,including in sub-Saharan Africa. Vectors of these pathogens include ticks, lice, mites and fleas. In order to assessexposure to flea-associated Rickettsia species in Madagascar, human and small mammal samples from an urbanand a rural area, and their associated fleas were tested.Results: Anti-typhus group (TGR)- and anti-spotted fever group rickettsiae (SFGR)-specific IgG were detected in24 (39%) and 21 (34%) of 62 human serum samples, respectively, using indirect ELISAs, with six individuals seropositivefor both. Only two (2%) Rattus rattus out of 86 small mammals presented antibodies against TGR. Out of 117fleas collected from small mammals, Rickettsia typhi, a TGR, was detected in 26 Xenopsylla cheopis (24%) collected fromrodents of an urban area (n = 107), while two of these urban X. cheopis (2%) were positive for Rickettsia felis, a SFGR. R.felis DNA was also detected in eight (31%) out of 26 Pulex irritans fleas.Conclusions: The general population in Madagascar are exposed to rickettsiae, and two flea-associated Rickettsiapathogens, R. typhi and R. felis, are present near or in homes. Although our results are from a single district, theydemonstrate that rickettsiae should be considered as potential agents of undifferentiated fever in Madagascar.

The genus Bartonella contains >40 species, and an increasing number of these Bartonella species are being implicated in human disease. One such pathogen is Bartonella ancashensis, which was isolated in blood samples from 2 patients living in Caraz, Peru, during a clinical trial of treatment for bartonellosis. Three B. ancashensis strains were analyzed by using whole-genome restriction mapping and high-throughput pyrosequencing. Genome-wide comparative analysis of Bartonella species showed that B. ancashensis has features seen in modern and ancient lineages of Bartonella species and is more related to B. bacilliformis. The divergence between B. ancashensis and B. bacilliformis is much greater than what is seen between known Bartonella genetic lineages. In addition, B. ancashensis contains type IV secretion system proteins, which are not present in B. bacilliformis. Whole-genome analysis indicates that B. ancashensis might represent a distinct Bartonella lineage phylogenetically related to B. bacilliformis.

Scrub typhus, a chigger-borne febrile illness, occurs primarily in countries of the Asia-Pacific rim and islands of the Western Pacific. The etiologic agent is the obligate intracellular rickettsial bacterium Orientia tsutsugamushi. Research on O. tsutsugamushi has relied on the availability of several prototype strains, which were isolated from human cases of scrub typhus in the 1940s and 1950s. We review the history of the three original, and most important, prototype strains, Gilliam, Karp and Kato, including information on their isolation, their culture history, their clinical characteristics, their importance within the research literature on scrub typhus, and recent advances in elucidating their molecular genomics. The importance of these strains to the research and development of clinical tools related to scrub typhus is also considered. Finally, we examine whether the strains have been genetically stable since their isolation, and whether prototype strains maintained in separate laboratories are identical, based on pairwise comparisons of several sequences from four genes. By using genetic information archived in international DNA databases, we show that the prototype strains used by different laboratories are essentially identical, and that the strains have retained their genetic integrity at least since the 1950s. The three original prototype strains should remain a standard by which new diagnostic procedures are measured. Given their fundamental position in any comparative studies, they are likely to endure as a critical part of present and future research on scrub typhus and Orientia.

During a scrub typhus outbreak investigation in Thailand, 4 isolates of O. tsutsugamushi were obtained and established in culture. Phylogenetic analysis based on the 56-kDa type-specific antigen gene demonstrated that the isolates fell into 4 genetic clusters, 3 of which had been previously reported and 1 that represents a new genotype.

We conducted serological surveys for Coxiella burnetii in archived sera from patients that visited a rural clinic in western Kenya from 2007 to 2008 and in cattle, sheep, and goats from the same area in 2009. We also conducted serological and polymerase chain reaction-based surveillance for the pathogen in 2009–2010, in human patients with acute lower respiratory illness, in ruminants following parturition, and in ticks collected from ruminants and domestic dogs. Antibodies against C. burnetii were detected in 30.9% (N = 246) of archived patient sera and in 28.3% (N = 463) of cattle, 32.0% (N = 378) of goats, and 18.2% (N = 159) of sheep surveyed. Four of 135 (3%) patients with acute lower respiratory illness showed seroconversion to C. burnetii. The pathogen was detected by polymerase chain reaction in specimens collected from three of six small ruminants that gave birth within the preceding 24 hours, and in five of 10 pools (50%) of Haemaphysalis leachi ticks collected from domestic dogs.

In July 2006, an Australian tourist returning from Dubai, in the United Arab Emirates (UAE), developed acute scrub typhus. Her signs and symptoms included fever, myalgia, headache, rash, and eschar. Orientia tsutsugamushi serology demonstrated a 4-fold rise in antibody titers in paired serum collections (1:512 to 1:8,192), with the sera reacting strongest against the Gilliam strain antigen. An Orientia species was isolated by the in vitro culture of the patient's acute blood taken prior to antibiotic treatment. The gene sequencing of the 16S rRNA gene ( rrs ), partial 56-kDa gene, and the full open reading frame 47-kDa gene was performed, and comparisons of this new Orientia sp. isolate to previously characterized strains demonstrated significant sequence diversity. The closest homology to the rrs sequence of the new Orientia sp. isolate was with three strains of O. tsutsugamushi (Ikeda, Kato, and Karp), with a nucleotide sequence similarity of 98.5%. The closest homology to the 47-kDa gene sequence was with O. tsutsugamushi strain Gilliam, with a nucleotide similarity of 82.3%, while the closest homology to the 56-kDa gene sequence was with O. tsutsugamushi strain TA686, with a nucleotide similarity of 53.1%. The molecular divergence and geographically unique origin lead us to believe that this organism should be considered a novel species. Therefore, we have proposed the name “ Orientia chuto ,” and the prototype strain of this species is strain Dubai, named after the location in which the patient was infected.

Using a large, passive, clinic-based surveillance program in Iquitos, Peru, we characterized the prevalence of rickettsial infections among undifferentiated febrile cases and obtained evidence of pathogen transmission in potential domestic reservoir contacts and their ectoparasites. Blood specimens from humans and animals were assayed for spotted fever group rickettsiae (SFGR) and typhus group rickettsiae (TGR) by ELISA and/or PCR; ectoparasites were screened by PCR. Logistic regression was used to determine associations between patient history, demographic characteristics of participants and symptoms, clinical findings and outcome of rickettsial infection. Of the 2,054 enrolled participants, almost 2% showed evidence of seroconversion or a 4-fold rise in antibody titers specific for rickettsiae between acute and convalescent blood samples. Of 190 fleas (Ctenocephalides felis) and 60 ticks (Rhipicephalus sanguineus) tested, 185 (97.4%) and 3 (5%), respectively, were positive for Rickettsia spp. Candidatus Rickettsia asemboensis was identified in 100% and 33% of the fleas and ticks tested, respectively. Collectively, our serologic data indicates that human pathogenic SFGR are present in the Peruvian Amazon and pose a significant risk of infection to individuals exposed to wild, domestic and peri-domestic animals and their ectoparasites., Author Summary Rickettsial infection remains relatively unexplored in South America compared to other regions of the world. For most regions of Peru (including the Amazon Basin), nothing more than broad serological characterization is available about circulating rickettsiae. Even less is known about the animal reservoirs and insect vectors involved in disease transmission. With this study we aimed to better characterize the circulating species of Rickettsia in humans in the Amazon Basin, as well as investigate their domestic animal reservoir and arthropod vectors. Out of 2054 fever patients enrolled we identified 38 individuals with serologic evidence for acute rickettsial infection. Their homes were visited in order to draw blood samples and collect ectoparasites from their domestic animals. Serology and molecular methods were used to test the animal blood samples as well as the ectoparasites. The information collected contributes to the understanding of the transmission dynamics of rickettsial diseases in Iquitos and leads to a better understanding of the exposure risk to rickettsial infection and it will guide approaches for prevention.

Raw milk was collected from cows in western Kazakhstan in winter 2014-2015. Samples were defatted and frozen at -20C, then tested as follows. For tick-borne encephalitis virus, 65 samples were tested using the VectorBest TBEV antigen capture kit, with 9% positive. For Coxiella burnetii, 50 samples were assayed using a species-specific qPCR assay and all were negative, though positive controls were consistently positive. For Brucella spp., PCR, ELISA and FPA testing is ongoing, with some positive results. These data suggest that consumption of raw cow's milk in western Kazakhstan is a risk factor for tick-borne encephalitis and brucellosis. The risk for Q fever appears to be small during winter, but may be present at other times of the year.