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.

Metagenomic Approach to Characterizing Disease Epidemiology in a Disease-Endemic Environment in Northern Thailand.

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.

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.

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.

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.

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.

Prevalence and diversity of Bartonella in rodents of northern Thailand: a comparison with Bartonella in rodents from southern China.

We report results of the first study to investigate the distribution and diversity of Bartonella in rodents from Thailand. Whole blood from 195 rodents, representing six species, was tested for the presence of Bartonella species using standard culture techniques. Isolates were obtained from 17 (8.7%) of the samples, and 14 of those isolates represented distinct strains, based upon partial sequencing of the citrate synthase (gltA) gene. Phylogenetic analysis of the isolates and other Bartonella species indicated that five unique isolates from Bandicota indica form a cluster that may represent a new Bartonella species. Two additional isolates from B. indica clustered together, and were nearly identical to an isolate from Apodemus draco collected in southern China. Importantly, a number of the isolates from Thailand rodents are closely related to B. grahamii and B. elizabethae, species which have been associated with human illness.

Occurrence of Orientia tsutsugamushi in small mammals from Thailand.

Extensive sampling of small mammals was conducted in eight provinces of Thailand between September 9, 1992 and April 29, 2001. A total of 3,498 specimens representing 22 species were collected. Eighty-eight percent (3,089 of 3,498) of the animals were collected from a region in Chiangrai Province, which is commonly recognized as endemic for human scrub typhus. Blood and tissue samples from each animal were tested for the presence of Orientia tsutsugamushi, the etiologic agent of scrub typhus. The predominant species collected were Rattus rattus (53%, n = 1,863), R. losea (18%, n = 638), Bandicota indica (16%, n = 564), and R. exulans (4%, n = 146). Orientia tsutsugamushi was detected in 10 of the 22 species of mammals that included R. bukit (25% infected, 1 of 4), R. rattus (23%, 419 of 1,855), R. argentiventer (22%, 5 of 23), R. berdmorei (22%, 2 of 9), R. losea (13%, 82 of 638), B. indica (9%, 52 of 564), R. koratensis (8%, 1 of 12), B. savilei (3%, 1 of 30), R. exulans (1%, 2 of 146), and Tupaia glis (2%, 1 of 49). Infected animals were found in Chiangrai (18% infected, 563 of 3,084), Bangkok (11%, 1 of 9), Sukothai (3%, 1 of 30), and Nonthaburi (1%, 1 of 69) Provinces. The implications towards scrub typhus maintenance and transmission are discussed.

Development of an in vitro method for the evaluation of candidate repellents against Leptotrombidium (Acari: Trombiculidae) chiggers.

We developed a rapid and economical in vitro procedure with which to evaluate the efficacy of candidate repellents against chiggers. The procedure requires only 5 min and a small number of chiggers to obtain a valid estimate of the median effective dose. We used this procedure to evaluate the repellent activity of 11 compounds against the chigger, Leptotrombidium imphalum Vercammen-Grandjean and Langston. Median effective doses were determined for 10 of the 11 compounds. DM-165-2 (N,N-diethyl-3-flurobenzamide) was the only compound that was significantly more effective than deet.

Efficiency of Leptotrombidium chiggers (Acari: Trombiculidae) at transmitting Orientia tsutsugamushi to laboratory mice.

Thirteen different laboratory colonies of Leptotrombidim chiggers [L. chiangraiensis Tanskul & Linthicum, L. deliense Walch and L. imphalum (Vercammen-Grandjean &Langston)] were evaluated for their ability to transmit Orientia tsutsugamushi (Hyashi) to mice. Of 4,372 transmission attempts using individual chiggers from all 13 colonies, 75% (n = 3,275) successfully infected mice. Transmission rates for the individual chigger colonies ranged from 7 to 80%. Increasing the number of chiggers that fed on a given mouse generally increased transmission rates. Transmission of O. tsutsugamushi to mice by different generations (F1-F11) of certain chigger colonies was stable; however, transmission rates varied greatly in other colonies. Transmission rates (both vertical and horizontal) of several L. changraiensis colonies and the L. deliense colony were the highest, suggesting that these colonies may be useful for the development of a chigger-challenge model that can be used to evaluate the efficacy of candidate scrub typhus vaccines or therapeutic agents in laboratory mice.

Antibodies to leptospirosis in rodents from Thailand using a modified human diagnostic assay.

The number of reported cases of Leptospirosis in Thailand has grown since 1996. Identification of major reservoirs and endemic areas is essential in surveillance of Leptospira species in Thailand. To assist in the effort of surveillance, a dipstick assay for detecting Leptospira antibodies in mammals was adapted from a human diagnostic assay and tested in a field trial in Thailand. Antibodies to Leptospira were detected in 18 of 60 wild rodents. Four of 9 culture positive rodents were positive by the dipstick assay. The proportion of sera positive for antibodies by dipstick was correlated with positive culture outcome using McNemar test for correlated proportions (0.83, P> 0.05). The dipstick assay was effective in detecting antibodies to Leptospira in mammals and may be useful in resource poor areas or under circumstances where the microagglutination test (MAT) is not practical.