Identification and Characterization of Orientia chuto in Trombiculid Chigger Mites Collected from Wild Rodents in Kenya.

We present data that concurs with the reported geographical expansion of scrub typhus outside the "Tsutsugamushi Triangle" and addition of Orientia chuto as a second species in the Orientia genus. Wild rodents were caught in Marigat, Baringo County, Kenya, and ectoparasites, including chiggers, were recovered. Rodent and chigger species were identified by taxonomic features. DNA was extracted from the chiggers and used to amplify and/or sequence the 47-kDa high temperature transmembrane protein (TSA47), the 56-kDa type-specific antigen (TSA56), and the 16S rRNA (rrs) Orientia genes. The main rodent hosts identified were Acomys wilsoni, Crocidura sp., and Mastomys natalensis, which accounted for 59.2% of the total collection. Of these, A. wilsoni and M. natalensis harbored most of the chiggers that belonged to the Neotrombicula and Microtrombicula genera. A pool of chiggers from one of M. natalensis was positive for Orientia by TSA47 PCR, but Orientia did not amplify with the TSA56 primers. On sequencing the 850 bp of the TSA47 gene, the closest phylogenetic relative was O. chuto, with 97.65% sequence homology compared to 84.63 to 84.76% for O. tsutsugamushi 16S rRNA deep sequencing also revealed O. chuto as the closest phylogenetic relative, with 99.75% sequence homology. These results and the existing immunological and molecular reports are strongly suggestive of the existence of Orientia species in Kenya.

Variable clinical responses of a scrub typhus outbred mouse model to feeding by Orientia tsutsugamushi infected mites.

Rodents are the natural hosts for Leptotrombidium mites that transmit Orientia tsutsugamushi, the causative agent of scrub typhus, a potentially fatal febrile human disease. Utilizing mite lines that included O. tsutsugamushi infected and non-infected Leptotrombidium species we investigated the varied infection response of outbred mice (ICR) exposed to L. chiangraiensis (Lc), L. imphalum (Li) and L. deliense (Ld). Each of six mite lines (Lc1, Lc5, Li3, Li4, Li7 and Ld) was separately placed in the inner ears of ICR mice either as a single individual (individual feeding, IF) or as a group of 2-4 individuals (pool feeding, PF). The species of infected chigger feeding on mice significantly affected mortality rates of the mice, with mite lines of Lc causing higher mean (±SE) mortality (90.7 ± 3.6 %) than mite lines of Li (62.9 ± 5.6 %) or Ld (53.6 ± 5.8 %). Mouse responses which included time to death, food consumption and total mice weight change depended on mite species and their O. tsutsugamushi genotype, more than on feeding procedure (IF vs. PF) except for mite lines within the Lc. Infected mite lines of Lc were the most virulent infected mites assessed whereas the infected Ld species was the least virulent for the ICR. Mice killed by various mite lines showed enlarged spleens and produced ascites. The results of this investigation of the clinical responses of ICR mice to feeding by various infected mite lines indicated that the different species of infected mites and their O. tsutsugamushi genotype produced different clinical presentations in ICR mice, a scrub typhus mouse model which mimics the natural transmission of O. tsutsugamushi that is critical for understanding scrub typhus disease in terms of natural transmission, host-pathogen-vector interaction and vaccine development.

Retrospective Survey of Borrelia spp. From Rodents and Ticks in Thailand.

Borrelia is a genus of spirochetal bacteria with several species known to cause disease in humans. The distribution of Borrelia has rarely been studied in Thailand. In this study, a retrospective survey of Borrelia was conducted in ticks and wild rodents to better characterize the prevalence, diversity, and distribution of Borrelia across Thailand. Several pools of DNA from tick samples were positive for Borrelia spp. (36/258, 13.9%). Borrelia theileri/B. lonestari was found in 17 tick samples (16 pools of Haemaphysalis bandicota and 1 pool of Rhipicephalus sp.), and Borrelia yangtzensis was found in 8 tick samples (2 pools of H. bandicota and 6 pools of Ixodes granulatus). Borrelia spp. were detected at low prevalence levels in rodent tissue samples (24/2001, 1.2%), with 19 identified as B. theileri or B. lonestari and 5 identified as B. miyamotoi. Several geographic and species-specific infection trends were apparent, with Ixodes ticks infected with B. yangtzensis and Haemaphysalis and Rhipicephalus ticks infected with both B. yangtzensis and B. theileri/B. lonestari. Notably, B. yangtzensis showed a similar geographic distribution to B. miyamotoi, which was identified in new areas of Thailand in this study. The flagellin gene sequence from B. miyamotoi was more similar to European (99.3-99.9%) than Japanese (96.9-97.6%) genotypes. This study greatly expands the knowledge of Borrelia in Thailand and identified several Borrelia species for the first time. It also found several ticks and rodents infected with the pathogen that were not previously known to carry Borrelia.

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.

Assessing scrub typhus and rickettsioses transmission risks in the Chiang Rai province of northern Thailand.

BACKGROUND: Scrub typhus is an important disease in the Asia-Pacific countries with increasing relevance for public health worldwide. Entomological risk assessment for scrub typhus and rickettsial disease in Phu Chi Fah village-Chiang Rai was performed to determine areas at greatest risk for disease transmission in order to increase awareness of diseases to travelers and healthcare workers. METHODS: From 2016 to 2018, rodents and chiggers were collected from 7 sites covering residential, grassland, and forest areas to determine the prevalence of pathogen of interest. The correlation between land type and vector-host-pathogen interaction system was investigated. RESULT: High prevalence of O. tsutsugamushi-infected and Rickettsia-infected chiggers was observed especially in areas with grassland and forest ecotones. Chigger and rodent species composition were negatively associated with the level of human disturbance. Increased density of rodents was responsible for a higher abundance of chigger population and increased prevalence of O. tsutsugmaushi infection in chigger in the following year. CONCLUSION: Communities in the study areas have an increased exposure risk to scrub typhus and potentially spotted fever group rickettsiosis. Travellers to this endemic area should pay more attention pathogen risks so as to avoid vector and disease exposure. Seasonal rodenticidal activity may help migitate the risk of pathogen transmission.

Low Prevalence of Leptospira Carriage in Rodents in Leptospirosis-Endemic Northeastern Thailand.

Leptospirosis is a neglected zoonotic disease affecting mostly the world's tropical regions. The rural people of northeastern Thailand suffer from a large number of leptospirosis infections, and their abundant rice fields are optimal rodent habitats. To evaluate the contribution of diversity and carriage rate of pathogenic Leptospira in rodent reservoirs to leptospirosis incidence, we surveyed rodents, between 2011 and 2012, in four provinces in northeastern Thailand with the highest incidence rates of human leptospirosis cases. We used lipL32 real-time PCR to detect pathogenic Leptospira in rodent kidneys, partial 16S rRNA gene sequencing to classify the infecting Leptospira species, and whole 16S rDNA sequencing to classify species of isolated Leptospira. Overall prevalence of Leptospira infection was 3.6% (18/495). Among infected rodents, Bandicotaindica (14.3%), Rattusexulans (3.6%), and R. rattus (3.2%) had renal carriage. We identified two pathogenic Leptospira species: L. interrogans (n = 15) and L. borgpetersenii (n = 3). In addition, an L. wolffii (LS0914U) isolate was recovered from the urine of B. indica. Leptospira infection was more prevalent in low density rodent populations, such as B. indica. In contrast, there was a lower prevalence of Leptospira infection in high density rodent populations of R. exulans and R. rattus.

Surveillance for Scrub Typhus, Rickettsial Diseases, and Leptospirosis in US and Multinational Military Training Exercise Cobra Gold Sites in Thailand.

We report findings of field surveillance for disease vectors and the prevalence of Orientia tsutsugamushi, the causative agent for scrub typhus, and other Rickettsial species that cause murine typhus and spotted fever group rickettsioses, in chigger mites and small rodents; and Leptospira in rodent kidney, urine, and environmental water samples. The study sites included various Royal Thai Army military installations and other training sites, and surrounding areas where the multinational military training exercise Cobra Gold was conducted in Thailand in 2017 and 2018. The overall prevalence of O. tsutsugamushi and Rickettsia infection in chiggers was 1.3% (20/1,594) and 7.5% (119/1,594), respectively. Serum samples of the captured rodents indicated previous exposure to O. tsutsugamushi infection with a seropositive rate of 12.2%. Leptospira species were isolated from rodent kidneys and water samples collected from catchment areas as well as tap water used for hand washing. Findings from this surveillance are important in determining the potential for scrub typhus, rickettsioses, and leptospirosis risk to military and US government personnel, as well as for informing regional and combatant commanders for prevention, correct diagnosis, prompt treatment, and timely and focused implementation of vector control and personal protective measures.

Heterogeneity of Orientia tsutsugamushi genotypes in field-collected trombiculid mites from wild-caught small mammals in Thailand.

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.

Comparison of immune response against Orientia tsutsugamushi, a causative agent of scrub typhus, in 4-week-old and 10-week-old scrub typhus-infected laboratory mice using enzyme-linked immunosorbent assay technique.

Using the indirect enzyme-linked immunosorbent assay (ELISA) technique, we compared the immune response of specific antibodies (IgM and IgG) in serum samples of scrub typhus-infected and non-infected mice against Orientia sutsugamushi (a causative agent of scrub typhus). Two different age groups (4-week-old and 10-week-old) of ICR laboratory mice were infected with O. tsutsugamushi by the animal passage procedure. Serum samples were detected for scrub typhus-specific antibodies using ELISA technique. Results from determining the presence of IgM and IgG in the serum samples obtained from scrub typhus-infected mice showed that the IgG was first detected on day-13 after the initial infection in both the 4-week-old and 10-week-old mice. The IgG titer levels of both groups were not significantly different. Although the presence of IgM in the in serum samples obtained from scrub typhus-infected mice was first detected on day 13 in the 4-week-old mice and on day-12 in the 10-week-old mice, the IgM titer in the 4-week-old mice was slightly lower than in the 10-week-old mice. Clinical observations of the scrub typhus-infected mice showed that the older mice become ill on day 9 whereas the younger mice exhibited the symptoms on day 12. Considering the earlier detection and slightly higher level of specific IgM antibody, it could be interpreted that the older mice may appear to have responded against O. tsutsugamushi faster than the younger mice.

Transovarial Transmission of Co-Existing Orientia tsutsugamushi Genotypes in Laboratory-Reared Leptotrombidium imphalum.

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.

The Distribution and Diversity of Bartonella Species in Rodents and Their Ectoparasites across Thailand.

Our study highlights the surveillance of Bartonella species among rodents and their associated ectoparasites (ticks, fleas, lice, and mites) in several regions across Thailand. A total of 619 rodents and 554 pooled ectoparasites (287 mite pools, 62 flea pools, 35 louse pools, and 170 tick pools) were collected from 8 provinces within 4 regions of Thailand. Bandicota indica (279), Rattus rattus (163), and R. exulans (96) were the most prevalent species of rats collected in this study. Real-time PCR assay targeting Bartonella-specific ssrA gene was used for screening and each positive sample was confirmed by PCR using nuoG gene. The prevalence of Bartonella DNA in rodent (around 17%) was recorded in all regions. The highest prevalence of Bartonella species was found in B. savilei and R. rattus with the rate of 35.7% (5/14) and 32.5% (53/163), respectively. High prevalence of Bartonella-positive rodent was also found in B. indica (15.1%, 42/279), and R. norvegicus (12.5%, 5/40). In contrast, the prevalence of Bartonella species in ectoparasites collected from the rats varied significantly according to types of ectoparasites. A high prevalence of Bartonella DNA was found in louse pools (Polyplax spp. and Hoplopleura spp., 57.1%) and flea pools (Xenopsylla cheopis, 25.8%), while a low prevalence was found in pools of mites (Leptotrombidium spp. and Ascoschoengastia spp., 1.7%) and ticks (Haemaphysalis spp., 3.5%). Prevalence of Bartonella DNA in ectoparasites collected from Bartonella-positive rodents (19.4%) was significantly higher comparing to ectoparasites from Bartonella-negative rodents (8.7%). The phylogenetic analysis of 41 gltA sequences of 16 Bartonella isolates from rodent blood and 25 Bartonella-positive ectoparasites revealed a wide range of diversity among Bartonella species with a majority of sequences (61.0%) belonging to Bartonella elizabethae complex (11 rodents, 1 mite pool, and 5 louse pools), while the remaining sequences were identical to B. phoceensis (17.1%, 1 mite pool, 5 louse pools, and 1 tick pool), B. coopersplainensis (19.5%, 5 rodents, 1 louse pool, and 2 tick pools), and one previously unidentified Bartonella species (2.4%, 1 louse pool).

Characterization based on the 56-Kda type-specific antigen gene of Orientia tsutsugamushi genotypes isolated from Leptotrombidium mites and the rodent host post-infection.

Abstract. Characterization of the 56-kDa type-specific antigen (TSA) genes of Orientia tsutsugamushi (OT) from three naturally infected, laboratory-reared mite colonies comprising three species (Leptotrombidium deliense [Ld], Leptotrombidium imphalum [Li], and Leptotrombidium chiangraiensis [Lc]) has revealed the presence of single and coexisting OT genotypes found in individual chiggers. The Karp genotype was found in all of the chiggers examined, whereas Gilliam and UT302 genotypes were only observed in combination with the Karp genotype. From analysis of these OT genotypes after transmission from chiggers to mice it was determined that with the Lc and Li mites, the OT genotype composition in the rodent spleens post-infection had not changed and therefore resembled that observed in the feeding chiggers. However, only the Karp genotype was found in rodents after feeding by Ld chiggers carrying Karp and Gilliam genotypes. The current findings reveal a complex association among the host, pathogen, and vector.

Surveys of rodent-borne disease in Thailand with a focus on scrub typhus assessment.

The epidemiology of many rodent-borne diseases in South-East Asia remains ill-defined. Scrub typhus and lep-tospirosis are common and medically significant, while other zoonotic diseases, such as spotted fever group Rickettsiae have been identified, but their overall medical significance is unknown. Rodent surveillance was conducted from June 2002 to July 2004 in 18 provinces from Thailand. Traps were set up for one to three nights. Blood and serum samples and animal tissue samples (liver, spleen, kidney and urinary bladder) were collected. Chiggermites, ticks and fleas were removed from captured rodents. A total of 4536 wild-caught rodents from 27 species were captured over two years of animal trapping. Rattus rattus was the dominant species, followed by Rattus exulans and Bandicota indica. Almost 43 000 ectoparasites were removed from the captured animals. Approximately 98% of the ectoparasites were chigger-mites, of which 46% belonged to the genus Leptotrombidium (scrub typhus vector). Other genera included Schoengastia and Blankaartia. Tick and flea specimens together comprised less than 1% of the sample. Among the five species of ticks collected, Haemaphysalis bandicota was the predominant species caught, followed by Ixodes granulatus other Haemaphysalis spp., Rhipicephalus spp. and Dermacentor spp. Only two species of fleas were collected and Xenopsylla cheopis (rat flea) was the predominant species. Using both commercial diagnostic kits and in-house molecular assays, animal tissue samples were examined and screened for zoonotic diseases. Seven zoonotic diseases were detected: scrub typhus, leptospirosis, murine typhus, tick typhus, bartonella, babesiosis and trypanosomiasis. Most samples were positive for scrub typhus. Other zoonotic diseases still under investigation include borrelosis, ehrlichiosis, the plague, and other rickettsial diseases. Using geographic information systems, global positioning systems and remote sensing technology, epidemiological and environmental data were combined to assess the relative risk in different biotopes within highly endemic areas of scrub typhus in Thailand.