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

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The role of the mosquito in a dengue human infection model

Recent efforts to combat the growing global threat of dengue disease, including deployment of phase IIb vaccine trials, has continued to be hindered by uncertainty surrounding equitable immune responses of serotypes, relative viral fitness of vaccine vs naturally occurring strains, and the importance of altered immune environments due to natural delivery routes. Human infection models can significantly improve our understanding of the importance of certain phenotypic characteristics of viral strains, and inform strain selection and trial design. With human models, we can further assess the importance of the natural delivery route of DENV and/or the accompanying mosquito salivary milieu. Accordingly, we discuss the use of mosquitoes in such a human infection model with DENV, identify important considerations, and make preliminary recommendations for deployment of such a mosquito improved DENV human infection model (miDHIM). © The Author 2014

Entomological Surveillance for Zika and Dengue Virus in Aedes Mosquitoes: Implications for Vector Control in Thailand

Entomological surveillance for arthropod-borne viruses is vital for monitoring vector-borne diseases and informing vector control programs. In this study, we conducted entomological surveillance in Zika virus endemic areas. In Thailand, it is standard protocol to perform mosquito control within 24 h of a reported dengue case. Aedes females were collected within 72 h of case reports from villages with recent Zika–human cases in Kamphaeng Phet Province, Thailand in 2017 and 2018. Mosquitoes were bisected into head-thorax and abdomen and then screened for Zika (ZIKV) and dengue (DENV) viruses using real-time RT-PCR. ZIKV RNA was detected in three samples from two female Ae. aegypti (1.4%). A partial envelope sequence analysis revealed that the ZIKV sequences were the Asian lineage identical to sequences from ZIKV-infected cases reported in Thailand during 2016 and 2017. Dengue virus-1 (DENV-1) and dengue virus-4 (DENV-4) were found in four Ae. aegypti females (2.8%), and partial capsid sequences were nearly identical with DENV-1 and DENV-4 from Thai human cases reported in 2017. Findings in the current study demonstrate the importance of entomological surveillance programs to public health mosquito-borne disease prevention measures and control

Heterogeneity of <i>Orientia tsutsugamushi</i> genotypes in field-collected trombiculid mites from wild-caught small mammals in Thailand

<div><p>Trombiculid mites are the vectors of scrub typhus, with infected larval mites (chiggers) transmitting the causative agent, <i>Orientia tsutsugamushi</i>, during feeding. Co-existence of multiple <i>O</i>. <i>tsutsugamushi</i> 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. <i>tsutsugamushi</i> 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 <i>O</i>. <i>tsutsugamushi</i>, corresponding to a prevalence rate of 0.7% within the mite population. Twenty-four of the infected mites were <i>Leptotrombidium</i> spp., indicating that this genus is the main vector for <i>O</i>. <i>tsutsugamushi</i> transmission in Thailand. In addition, <i>O</i>. <i>tsutsugamushi</i> was detected in the mite genera <i>Ascoschoengastia</i>, <i>Blankaartia</i>, <i>Gahrliepia</i>, and <i>Lorillatum</i>. 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 <i>O</i>. <i>tsutsugamushi</i> 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 <i>O</i>. <i>tsutsugamushi</i> genotypes. The occurrence of multiple <i>O</i>. <i>tsutsugamushi</i> genotypes within individual mites has important implications, and could provide a mechanism for pathogen evolution/diversification in the mite vector.</p></div

Bacterial microbiome of the chigger mite Leptotrombidium imphalum varies by life stage and infection with the scrub typhus pathogen Orientia tsutsugamushi.

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

Chigger infestation rates and population diversity of mites recovered from small mammals, Thailand.

<p>Chigger infestation rates and population diversity of mites recovered from small mammals, Thailand.</p

Abundance and diversity of field-collected trombiculid mites on wild-caught rodents and small mammals.

<p>(A) Abundance of chigger(s) from three dominant small mammal species. (B) Diversity of chigger(s) collected from animal(s).</p

Genotyping of <i>Orientia tsutsugamushi</i> from individual mites by next-generation sequencing.

<p>Genotyping of <i>Orientia tsutsugamushi</i> from individual mites by next-generation sequencing.</p