Orientia tsutsugamushi in Chiggers and Small Mammals in Laos.

Background: Scrub typhus is a leading cause of febrile illness in Laos and accounts for a high burden of disease. There have been no previous studies on the causative agent, Orientia tsutsugamushi, in vector mites ("chiggers") or their small mammal hosts in Laos. Materials and Methods: Small mammals and free-living chiggers were trapped in districts of Vientiane Province and Capital. Tissues were tested for O. tsutsugamushi by PCR and serum for IgG to O. tsutsugamushi by immunofluorescence assays (IFAs). Chiggers removed from small mammals and collected in their free-living stage using black plates were identified and tested for O. tsutsugamushi by PCR. Results: Over an 18-month period, 131 small mammals of 14 species were collected in 5 districts. Seventy-eight of 131 small mammals were infested with chiggers, but all tissues were O. tsutsugamushi PCR negative. Eighteen species of chigger were identified and 1,609 were tested by PCR. A single pool of chiggers tested O. tsutsugamushi positive. Sera from 52 small mammals were tested by IFA, with 16 testing positive. Conclusions: These are the first molecular and serological data on O. tsutsugamushi in chiggers and small mammals in Laos. Further studies are needed to better understand the key vector species and ecology of scrub typhus in areas with high disease incidence in Laos.

Orientia tsutsugamushi dynamics in vectors and hosts: ecology and risk factors for foci of scrub typhus transmission in northern Thailand.

BACKGROUND: Scrub typhus is an important neglected vector-borne zoonotic disease across the Asia-Pacific region, with an expanding known distribution. The disease ecology is poorly understood, despite the large global burden of disease. The key determinants of high-risk areas of transmission to humans are unknown. METHODS: Small mammals and chiggers were collected over an 18-month period at three sites of differing ecological profiles with high scrub typhus transmission in Chiang Rai Province, northern Thailand. Field samples were identified and tested for Orientia tsutsugamushi by real-time PCR. The rates and dynamics of infection were recorded, and positive and negative individuals were mapped over time at the scale of single villages. Ecological analyses were performed to describe the species richness, community structure and interactions between infected and uninfected species and habitats. Generalised linear modelling (GLM) was applied to examine these interactions. RESULTS: The site with the highest rates of human infection was associated with the highest number of infected chigger pools (41%), individual chiggers (16%), proportion of the known vector species Leptotrombidium deliense (71%) and chigger index (151). Chigger species diversity was lowest (Shannon diversity index H': 1.77) and rodent density appeared to be high. There were no consistent discrete foci of infection identified at any of the study sites. The small mammals Rattus tanezumi and Bandicota indica and the chiggers L. deliense and Walchia kritochaeta emerged as central nodes in the network analysis. In the GLM, the end of the dry season, and to a lesser extent the end of the wet season, was associated with O. tsutsugamushi-infected small mammals and chiggers. A clear positive association was seen between O. tsutsugamushi-positive chigger pools and the combination of O. tsutsugamushi-positive chigger pools and O. tsutsugamushi-positive small mammals with lowland habitats. CONCLUSIONS: These findings begin to reveal some of the factors that may determine high-risk foci of scrub typhus at a fine local scale. Understanding these factors may allow practical public health interventions to reduce disease risk. Further studies are needed in areas with diverse ecology.

Targeted capture and sequencing of Orientia tsutsugamushi genomes from chiggers and humans.

Scrub typhus is a febrile disease caused by Orientia tsutsugamushi, transmitted by larval stage Trombiculid mites (chiggers), whose primary hosts are small mammals. The phylogenomics of O. tsutsugamushi in chiggers, small mammals and humans remains poorly understood. To combat the limitations imposed by the low relative quantities of pathogen DNA in typical O. tsutsugamushi clinical and ecological samples, along with the technical, safety and cost limitations of cell culture, a novel probe-based target enrichment sequencing protocol was developed. The method was designed to capture variation among conserved genes and facilitate phylogenomic analysis at the scale of population samples. A whole-genome amplification step was incorporated to enhance the efficiency of sequencing by reducing duplication rates. This resulted in on-target capture rates of up to 93% for a diverse set of human, chigger, and rodent samples, with the greatest success rate in samples with real-time PCR Ct values below 35. Analysis of the best-performing samples revealed phylogeographic clustering at local, provincial and international scales. Applying the methodology to a comprehensive set of samples could yield a more complete understanding of the ecology, genomic evolution and population structure of O. tsutsugamushi and other similarly challenging organisms, with potential benefits in the development of diagnostic tests and vaccines.