A proof of concept for a targeted enrichment approach to the simultaneous detection and characterization of rickettsial pathogens from clinical specimens.

Infection with either Rickettsia prowazekii or Orientia tsutsugamushi is common, yet diagnostic capabilities are limited due to the short window for positive identification. Until now, although targeted enrichment had been applied to increase sensitivity of sequencing-based detection for various microorganisms, it had not been applied to sequencing of R. prowazekii in clinical samples. Additionally, hybridization-based targeted enrichment strategies had only scarcely been applied to qPCR of any pathogens in clinical samples. Therefore, we tested a targeted enrichment technique as a proof of concept and found that it dramatically reduced the limits of detection of these organisms by both qPCR and high throughput sequencing. The enrichment methodology was first tested in contrived clinical samples with known spiked-in concentrations of R. prowazekii and O. tsutsugamushi DNA. This method was also evaluated using clinical samples, resulting in the simultaneous identification and characterization of O. tsutsugamushi directly from clinical specimens taken from sepsis patients. We demonstrated that the targeted enrichment technique is helpful by lowering the limit of detection, not only when applied to sequencing, but also when applied to qPCR, suggesting the technique could be applied more broadly to include other assays and/or microbes for which there are limited diagnostic or detection modalities.

First Molecular Identification of Rickettsia aeschlimannii and Rickettsia africae in Ticks from Ghana.

The threats from vector-borne pathogens transmitted by ticks place people (including deployed troops) at increased risk for infection, frequently contributing to undifferentiated febrile illness syndromes. Wild and domesticated animals are critical to the transmission cycle of many tick-borne diseases. Livestock can be infected by ticks, and serve as hosts to tick-borne diseases such as rickettsiosis. Thus, it is necessary to identify the tick species and determine their potential to transmit pathogens. A total of 1,493 adult ticks from three genera-Amblyomma, Hyalomma, and Rhipicephalus-were identified using available morphological keys and were pooled (n = 541) by sex and species. Rickettsia species were detected in 308 of 541 (56.9%) pools by genus-specific quantitative polymerase chain reaction assay (Rick17b). Furthermore, sequencing of the outer membrane protein A and B genes (ompA and ompB) of random samples of Rickettsia-positive samples led to the identification of Rickettsia aeschlimannii and Rickettsia africae with most R. africae DNA (80.2%) detected in pools of Amblyomma variegatum. We report the first molecular detection and identification of the rickettsial pathogens R. africae and R. aeschlimannii in ticks from Ghana. Our findings suggest there is a need to use control measures to prevent infections from occurring among human populations in endemic areas in Ghana. This study underscores the importance of determining which vector-borne pathogens are in circulation in Ghana. Further clinical and prevalence studies are needed to understand more comprehensively the clinical impact of these rickettsial pathogens contributing to human disease and morbidity in Ghana.

Correction: A time-course comparative clinical and immune response evaluation study between the human pathogenic Orientia tsutsugamushi strains: Karp and Gilliam in a rhesus macaque (Macaca mulatta) model.

[This corrects the article DOI: 10.1371/journal.pntd.0010611.].

Bartonella Species in Cambodia, Ghana, Laos, and Peru: Results from Vector and Serosurveys.

Background: Bartonella species are fastidious gram-negative vector-borne bacteria with a wide range of mammalian reservoirs. While it is understood that some species of Bartonella are human pathogens, the extent of human exposure to Bartonella species (both pathogenic and nonpathogenic) is yet to be fully understood. Materials and Methods: To this end, residual sera from participants enrolled in undifferentiated fever studies in Cambodia, Ghana, Laos, and Peru were screened for the presence of IgG antibodies against Bartonella quintana and Bartonella henselae, using the FOCUS diagnostics Dual Spot- Bartonella IgG Immunofluorescence assay. Forty-eight patients with suspected or confirmed Bartonella bacilliformis exposure or infection in Peru were screened to assess cross-reactivity of the FOCUS assay for IgG against other Bartonella species. Results: Ten of 13 patients with confirmed B. bacilliformis infection were Bartonella-specific IgG positive, and overall, 36/48 of the samples were positive. In addition, 79/206, 44/200, 101/180, and 57/100 of the samples from Peru, Laos, Cambodia, and Ghana, respectively, were Bartonella-specific IgG positive. Furthermore, ectoparasite pools from Cambodia, Laos, and Peru were tested using quantitative real-time PCR (qPCR) for the presence of Bartonella DNA. Of the sand fly pools collected in Peru, 0/196 were qPCR positive; 15/140 flea pools collected in Cambodia were qPCR positive; while 0/105 ticks, 0/22 fleas, and 0/3 louse pools collected in Laos tested positive for Bartonella DNA. Conclusion: Evidence of Bartonella in fleas from Cambodia supports the possibility that humans are exposed to Bartonella through this traditional vector. However, Bartonella species were not found in fleas, ticks, or lice from Laos, or sand flies from Peru. This could account for the lower positive serology among the population in Laos and the strictly localized nature of B. bacilliformis infections in Peru. Human exposure to the Bartonella species and Bartonella as a human pathogen warrants further investigation.

Renewed Risk for Epidemic Typhus Related to War and Massive Population Displacement, Ukraine.

Epidemic typhus, caused by Rickettsia prowazekii bacteria and transmitted through body lice (Pediculus humanus corporis), was a major public health threat in Eastern Europe as a consequence of World War II. In 2022, war and the resulting population displacement in Ukraine risks the return of this serious disease.

A time-course comparative clinical and immune response evaluation study between the human pathogenic Orientia tsutsugamushi strains: Karp and Gilliam in a rhesus macaque (Macaca mulatta) model.

BACKGROUND: Scrub typhus is a vector-borne febrile illness caused by Orientia tsutsugamushi transmitted by the bite of Trombiculid mites. O. tsutsugamushi has a high genetic diversity and is increasingly recognized to have a wider global distribution than previously assumed. METHODOLOGY/PRINCIPLE FINDINGS: We evaluated the clinical outcomes and host immune responses of the two most relevant human pathogenic strains of O. tsutsugamushi; Karp (n = 4) and Gilliam (n = 4) in a time-course study over 80 days post infection (dpi) in a standardized scrub typhus non-human primate rhesus macaque model. We observed distinct features in clinical progression and immune response between the two strains; Gilliam-infected macaques developed more pronounced systemic infection characterized by an earlier onset of bacteremia, lymph node enlargement, eschar lesions and higher inflammatory markers during the acute phase of infection, when compared to the Karp strain. C-reactive protein (CRP) plasma levels, interferon gamma (IFN-γ, interleukin-1 receptor antagonist (IL-1ra), IL-15 serum concentrations, CRP/IL10- and IFN-γ/IL-10 ratios correlated positively with bacterial load in blood, implying activation of the innate immune response and preferential development of a T helper-type 1 immune response. The O. tsutsugamushi-specific immune memory responses in cells isolated from skin and lymph nodes at 80 dpi were more markedly elevated in the Gilliam-infected macaques than in the Karp-infected group. The comparative cytokine response dynamics of both strains revealed significant up-regulation of IFN-γ, tumor necrosis factor (TNF), IL-15, IL-6, IL-18, regulatory IL-1ra, IL-10, IL-8 and granulocyte-colony-stimulating factor (G-CSF). These data suggest that the clinical outcomes and host immune responses to scrub typhus could be associated with counter balancing effects of pro- and anti-inflammatory cytokine-mediated responses. Currently, no data on characterized time-course comparisons of O. tsutsugamushi strains regarding measures of disease severity and immune response is available. Our study provides evidence for the strain-specificity of host responses in scrub typhus, which supports our understanding of processes at the initial inoculation site (eschar), systemic disease progression, protective and/or pathogenic host immune mechanisms and cellular immune memory function. CONCLUSIONS/SIGNIFICANCE: This study characterised an improved intradermal rhesus macaque challenge model for scrub typhus, whereby the Gilliam strain infection associated with higher disease severity in the rhesus macaque model than the previous Karp strain infection. Difficulties associated with inoculum quantitation for obligate-intracellular bacteria were overcome by using functional inoculum titrations in outbred mice. The Gilliam-based rhesus macaque model provides improved endpoint measurements and contributes towards the identification of correlates of protection for future vaccine development.

Systematic Surveillance of Rickettsial Diseases in 27 Hospitals from 26 Provinces throughout Vietnam.

In Vietnam, the public health burden of rickettsial infections continues to be underestimated due to knowledge gaps in the epidemiology of these diseases. We conducted a systematic study among 27 hospitals from 26 provinces in eight ecological regions throughout Vietnam to investigate the prevalence, distribution, and clinical characteristics of rickettsial diseases. We recruited 1834 patients in the study from April 2018 to October 2019. The findings showed that rickettsial diseases were common among undifferentiated febrile patients, with 564 (30.8%) patients positive by qPCR for scrub typhus, murine typhus or spotted fever. Scrub typhus (484, 85.8%) was the most common rickettsial disease, followed by murine typhus (67, 11.9%) and spotted fever (10, 1.8%). Rickettsial diseases were widely distributed in all regions of Vietnam and presented with nonspecific clinical manifestations.

Development of a New Genus-Specific Quantitative Real-Time PCR Assay for the Diagnosis of Scrub Typhus in South America.

Scrub typhus is a potentially severe rickettsiosis, caused by Orientia tsutsugamushi in the Asia-Pacific region. Recently, however, two distinct pathogens, "Candidatus Orientia chuto" and "Candidatus Orientia chiloensis", have been discovered in the Middle East and South America, respectively. Since the novel pathogens differ significantly from O. tsutsugamushi, many established diagnostic methods are unreliable. This work describes the development and validation of a new quantitative real-time PCR (qPCR) assay (Orien16S) for the detection of all known Orientia species. Based on a 94 bp sequence of the 16S rRNA gene (rrs), Orien16S recognized DNA samples from O. tsutsugamushi (n = 41), Ca. O. chiloensis (n = 5), and Ca. O. chuto (n = 1), but was negative for DNA preparations from closely related rickettsiae and other members of the order Rickettsiales (n = 22) as well as unrelated bacterial species (n = 11). After its implementation in Chile, the assay was verified, correctly identifying all tested eschar and buffy coat samples (n = 28) of clinical suspected cases. Furthermore, Orien16S detected Orientia DNA in trombiculid mites collected in endemic regions in southern Chile. The presented novel qPCR assay provides a useful tool for detecting Orientia and diagnosing scrub typhus from all geographical regions.

Analyses of Bloodmeal Hosts and Prevalence of Rickettsia parkeri in the Gulf Coast Tick Amblyomma maculatum (Acari: Ixodidae) From a Reconstructed Piedmont Prairie Ecosystem, North Carolina.

Host feeding patterns and the prevalence of infection with Rickettsia parkeri were determined for the primary vector, Amblyomma maculatum Koch as well as sympatric tick species A. americanum (Linnaeus) and Dermacentor variabilis (Say) collected from a reconstructed prairie in the Piedmont region of North Carolina during 2011 and 2012. The occurrence of R. parkeri among A. maculatum adults and nymphs was 36.9% (45/122) and 33.3% (2/6), respectively. Rickettsia parkeri was detected in a single male A. americanum 2.3% (1/43). A PCR-reverse line blot hybridization assay of a 12S rDNA fragment amplified from remnant larval and nymphal bloodmeals of host-seeking ticks was used to identify bloodmeal hosts. Of the tick samples tested, bloodmeal host identification was successful for 29.3% (12/41) of adult A. americanum and 39.2% (20/51) of adult D. variabilis. For A. maculatum, bloodmeal host identification was successful for 50% (61/122) of adults collected from vegetation and 100% (4/4) of nymphs removed from cotton rats (Sigmodon hispidus Say and Ord). The cotton rat was the most common bloodmeal host with 59.0% (36/61) identified for adult A. maculatum. No statistically significant association was observed, however, between bloodmeal host and pathogen prevalence for any tick species. While the cotton rat was an important bloodmeal host for A. maculatum nymphs, this vertebrate did not appear to be the primary source of R. parkeri infection for A. maculatum.

Molecular Characterization by Multilocus Sequence Typing and Diversity Analysis of Rickettsia asembonensis in Peru.

Despite several reports worldwide documenting the presence of Rickettsia asembonensis in samples derived from ectoparasites, animals and more recently humans, genomic information of these specimens remains scarce, and when available, is usually limited to small genomic fragments of limited value. We generated complete sequences for two conserved (17-kDa antigen gene and gltA) and three variable (sca4, ompB and ompA) genes in five R. asembonensis DNA samples detected in cat and dog fleas in Peru. Complete gene sequences were used to conduct multi-locus sequence typing and phylogenetic analyses to assess diversity and infer relationships among strains and other reference sequences. The 17-kDa antigen gene was highly conserved across Rickettsia species. Of the variable genes ompB was the most variable, but this diversity was not captured through phylogenetics alone even when efforts were made to maximize potential diversity in terms of flea species, animal host and location. Through a combination of de novo and reference-based genome assembly we identified a 75 bp insertion in ompA that encodes a 25 aa repetitive motif found in other Rickettsia species, but not present in the original prototype strain from Kenya. R. asembonensis has only recently been shown to be a bona-fide human pathogen. As such, and compounded by a lack of available genomic information, it remains understudied. Our work directly addresses the lack of genomic information available worldwide for the study of these novel Rickettsia species and specifically contributes to our understanding of the diversity and molecular epidemiology of R. asembonensis in Peru.

Building Scientific Capability and Reducing Biological Threats: The Effect of Three Cooperative Bio-Research Programs in Kazakhstan.

Cooperative research programs aimed at reducing biological threats have increased scientific capabilities and capacities in Kazakhstan. The German Federal Foreign Office's German Biosecurity Programme, the United Kingdom's International Biological Security Programme and the United States Defense Threat Reduction Agency's Biological Threat Reduction Program provide funding for partner countries, like Kazakhstan. The mutual goals of the programs are to reduce biological threats and enhance global health security. Our investigation examined these cooperative research programs, summarizing major impacts they have made, as well as common successes and challenges. By mapping various projects across the three programs, research networks are highlighted which demonstrate best communication practices to share results and reinforce conclusions. Our team performed a survey to collect results from Kazakhstani partner scientists on their experiences that help gain insights into enhancing day-to-day approaches to conducting cooperative scientific research. This analysis will serve as a basis for a capability maturity model as used in industry, and in addition builds synergy for future collaborations that will be essential for quality and sustainment.

Establishment of a Rhesus Macaque Model for Scrub Typhus Transmission: Pilot Study to Evaluate the Minimal Orientia tsutsugamushi Transmission Time by Leptotrombidium chiangraiensis Chiggers.

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.

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

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

Carios kelleyi (Acari: Ixodida: Argasidae) Infected With Rickettsial Agents Documented Infesting Housing in Kansas, United States.

During September-December 2018, 25 live ticks were collected on-post at Fort Leavenworth, Kansas, in a home with a history of bat occupancy. Nine ticks were sent to the Army Public Health Center Tick-Borne Disease Laboratory and were identified as Carios kelleyi (Cooley and Kohls, 1941), a species that seldom bites humans but that may search for other sources of blood meals, including humans, when bats are removed from human dwellings. The ticks were tested for numerous agents of human disease. Rickettsia lusitaniae was identified by multilocus sequence typing to be present in two ticks, marking the first detection of this Rickettsia agent in the United States and in this species of tick. Two other Rickettsia spp. were also detected, including an endosymbiont previously associated with C. kelleyi and a possible novel Rickettsia species. The potential roles of C. kelleyi and bats in peridomestic Rickettsia transmission cycles warrant further investigation.

International Rickettsia Disease Surveillance: An Example of Cooperative Research to Increase Laboratory Capability and Capacity for Risk Assessment of Rickettsial Outbreaks Worldwide.

Cooperative research that addresses infectious disease surveillance and outbreak investigations relies heavily on availability and effective use of appropriate diagnostic tools, including serological and molecular assays, as exemplified by the current COVID-19 pandemic. In this paper, we stress the importance of using these assays to support collaborative epidemiological studies to assess risk of rickettsial disease outbreaks among international partner countries. Workforce development, mentorship, and training are important components in building laboratory capability and capacity to assess risk of and mitigate emerging disease outbreaks. International partnerships that fund cooperative research through mentoring and on-the-job training are successful examples for enhancing infectious disease surveillance. Cooperative research studies between the Naval Medical Research Center's Rickettsial Diseases Research Program (RDRP) and 17 institutes from nine countries among five continents were conducted to address the presence of and the risk for endemic rickettsial diseases. To establish serological and molecular assays in the collaborative institutes, initial training and continued material, and technical support were provided by RDRP. The laboratory methods used in the research studies to detect and identify the rickettsial infections included (1) group-specific IgM and IgG serological assays and (2) molecular assays. Twenty-six cooperative research projects performed between 2008 and 2020 enhanced the capability and capacity of 17 research institutes to estimate risk of rickettsial diseases. These international collaborative studies have led to the recognition and/or confirmation of rickettsial diseases within each of the partner countries. In addition, with the identification of specific pathogen and non-pathogen Rickettsia species, a more accurate risk assessment could be made in surveillance studies using environmental samples. The discoveries from these projects reinforced international cooperation benefiting not only the partner countries but also the scientific community at large through presentations (n = 40) at international scientific meetings and peer-reviewed publications (n = 18). The cooperative research studies conducted in multiple international institutes led to the incorporation of new SOPs and trainings for laboratory procedures; biosafety, biosurety, and biosecurity methods; performance of rickettsia-specific assays; and the identification of known and unknown rickettsial agents through the introduction of new serologic and molecular assays that complemented traditional microbiology methods.

Geographic distribution and modeling of ticks in the Republic of Korea and the application of tick models towards understanding the distribution of associated pathogenic agents.

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.

Detection of Rickettsia lusitaniae Among Ornithodoros sawaii Soft Ticks Collected From Japanese Murrelet Seabird Nest Material From Gugul Island, Republic of Korea.

In a follow-up to the investigations of soft ticks identified from seabird nest soil and litter collected from coastal islands of the Republic of Korea (ROK), Ornithodoros sawaii and Ornithodoros capensis were assessed for the presence and identification of rickettsiae. Ticks collected from samples of 50-100 g of nest litter and soil from seabird nests were identified individually by morphological techniques, and species confirmed by sequencing of the mt-rrs gene. Subsequently, tick DNA preparations were screened for the presence of rickettsiae using a genus-specific nested PCR (nPCR) assay targeting the 17 kDa antigen gene. The amplicons from the 17 kDa assay and two additional nPCR assays targeting the gltA and ompB gene fragments were sequenced and used to identify the rickettsiae. A total of 134 soft ticks belonging to two species, O. sawaii Kitaoka & Suzuki 1973 (n = 125) and O. capensis Neumann 1901 (n = 9), were collected. Rickettsia lusitaniae DNA was detected and identified among O. sawaii ticks (n = 11, 8.8%) collected from nest litter and soil of the Japanese murrelet (Synthliboramphus wumizusume Temminck 1836) at Gugul Island along the western coastal area of the ROK. This study confirmed for the first time the presence of R. lusitaniae associated with O. sawaii collected from migratory seabird nests in the ROK.

Molecular Characterization of Bartonella Species Discovered in Ectoparasites Collected from Domestic Animals, Cuzco, Peru.

Rickettsiae and bartonellae are Gram-negative bacteria that can cause zoonotic and human diseases and are vectored by hematophagous arthropods. In the Americas, rickettsioses and bartonelloses have reemerged as significant public health threats. Bartonella species have been identified as causing zoonotic infections responsible for a variety of clinical syndromes in humans and animals. The aim of this study was to investigate the distribution, prevalence, and molecular heterogeneity of Rickettsia spp. and Bartonella spp. among ectoparasites collected from domestic animals in 14 farming communities in the Andes Mountains of Cuzco, Peru. A total of 222 domestic animals representing 8 different species (sheep, donkeys, goats, cattle, pigs, llamas, guinea pigs, and horses) were sampled. Nine species of ectoparasites (n = 1,697) collected from 122 animals were identified resulting in 1,657 chewing lice, 39 ticks, and 1 flea. DNA was individually extracted from a random sample of 600 (35.4%) considering variability of ectoparasite species, hosts, and sample location elevation. All 600 samples were negative for rickettsial DNA by a genus-specific molecular assay. A subset of 173 (28.8%) samples were selected based on variability of arthropods species, host, and location for Bartonella testing. Ninety-one (52.6%) of these samples including Melophagus ovinus (90/110) and Bovicola bovis (1/7) were positive for Bartonella by a genus-specific molecular assay. Five Bartonella genes of seven DNA samples from M. ovinus were analyzed by the multilocus sequence typing for characterization. We identified five identical Bartonella melophagi specimens and two specimens with Bartonella species related to B. melophagi from the seven M. ovinus. The Bartonella agents detected were widely distributed and frequent in multiple studied locations.

Pathogen Carriage by Peri-Domestic Fleas in Western Kenya.

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