Seasonal pattern of questing ticks and prevalence of pathogenic Rickettsia and Anaplasmataceae in Khao Yai national park, Thailand.

BACKGROUND: Tick-borne diseases (TBD) are considered neglected diseases in Thailand with disease burden likely underestimated. To assess risk for emerging TBD in Thailand, the seasonality of questing tick and pathogen prevalence were studied in Khao Yai National Park, a top tourist destination. METHODS: During 2019, questing ticks around tourist attractions were systematically collected bimonthly and analyzed for Rickettsia and Anaplasmataceae bacterial species by polymerase chain reaction and DNA sequencing. RESULTS: Larvae and nymphs of questing ticks peaked in Khao Yai National Park during the late rainy-winter season, though no specific trends were observed in adult ticks. Winter (November to February) was the highest risk for human tick-bites due to higher numbers of both ticks and visitors. Of the total 5916 ticks analyzed (651 pools), Anaplasma phagocytophilum, Neoehrlichia mikurensis, Ehrlichia ewingii, and Ehrlichia chaffeensis were detected at low rates (≤0.05%). There was a higher prevalence of human rickettsioses (0.2-7%) in ticks surveyed with Rickettsia tamurae, Rickettsia raoultii, and Rickettsia montana the major species. Amblyomma ticks had the highest prevalence of Rickettsia (85%, 35/44 Amblyomma adults), in which only R. tamurae and R. raoultii were found in Amblyomma with mixed species infections common. We report the first detection of R. africae-like and N. mikurensis in Ixodes granulatus adults in Thailand, suggesting I. granulatus as a potential vector for these pathogens. CONCLUSION: This study demonstrated the risk of emerging TBD in Thailand and underscores the need for tick-bite prevention among tourists in Thailand.

Applying next generation sequencing to detect tick-pathogens in Dermacentor nuttalli, Ixodes persulcatus, and Hyalomma asiaticum collected from Mongolia.

Ticks and tick-borne diseases represent major threats to the public health of the Mongolian population, of which an estimated 26% live a traditional nomadic pastoralist lifestyle that puts them at increased risk for exposure. Ticks were collected by dragging and removal from livestock in Khentii, Selenge, Tuv, and Umnugovi aimags (provinces) during March-May 2020. Using next-generation sequencing (NGS) with confirmatory PCR and DNA sequencing, we sought to characterize the microbial species present in Dermacentor nuttalli (n = 98), Hyalomma asiaticum (n = 38), and Ixodes persulcatus (n = 72) tick pools. Rickettsia spp. were detected in 90.4% of tick pools, with Khentii, Selenge, and Tuv tick pools all having 100% pool positivity. Coxiella spp. were detected at an overall pool positivity rate of 60%, while Francisella spp. were detected in 20% of pools and Borrelia spp. detected in 13% of pools. Additional confirmatory testing for Rickettsia-positive pools demonstrated Rickettsia raoultii (n = 105), Candidatus Rickettsia tarasevichiae (n = 65) and R. slovaca/R. sibirica (n = 2), as well as the first report of Candidatus Rickettsia jingxinensis (n = 1) in Mongolia. For Coxiella spp. reads, most samples were identified as a Coxiella endosymbiont (n = 117), although Coxiella burnetii was detected in eight pools collected in Umnugovi. Borrelia species that were identified include Borrelia burgdorferi sensu lato (n = 3), B. garinii (n = 2), B. miyamotoi (n = 16), and B. afzelii (n = 3). All Francisella spp. reads were identified as Francisella endosymbiont species. Our findings emphasize the utility of NGS to provide baseline data across multiple tick-borne pathogen groups, which in turn can be used to inform health policy, determine regions for expanded surveillance, and guide risk mitigation strategies.

Borrelia miyamotoi a neglected tick-borne relapsing fever spirochete in Thailand.

Borrelia miyamotoi is a relapsing fever spirochete that shares the same vector as Lyme disease causing Borrelia. This epidemiological study of B. miyamotoi was conducted in rodent reservoirs, tick vectors and human populations simultaneously. A total of 640 rodents and 43 ticks were collected from Phop Phra district, Tak province, Thailand. The prevalence rate for all Borrelia species was 2.3% and for B. miyamotoi was 1.1% in the rodent population, while the prevalence rate was quite high in ticks collected from rodents with an infection rate of 14.5% (95% CI: 6.3-27.6%). Borrelia miyamotoi was detected in Ixodes granulatus collected from Mus caroli and Berylmys bowersi, and was also detected in several rodent species (Bandicota indica, Mus spp., and Leopoldamys sabanus) that live in a cultivated land, increasing the risk of human exposure. Phylogenetic analysis revealed that the B. miyamotoi isolates detected in rodents and I. granulatus ticks in this study were similar to isolates detected in European countries. Further investigation was conducted to determine the serological reactivity to B. miyamotoi in human samples received from Phop Phra hospital, Tak province and in rodents captured from Phop Phra district using an in-house, direct enzyme-linked immunosorbent assay (ELISA) assay with B. miyamotoi recombinant glycerophosphodiester-phosphodiesterase (rGlpQ) protein as coated antigen. The results showed that 17.9% (15/84) of human patients and 9.0% (41/456) of captured rodents had serological reactivity to B. miyamotoi rGlpQ protein in the study area. While a low level of IgG antibody titers (100-200) was observed in the majority of seroreactive samples, higher titers (400-1,600) were also detected in both humans and rodents. This study provides the first evidence of B. miyamotoi exposure in human and rodent populations in Thailand and the possible roles of local rodent species and Ixodes granulatus tick in its enzootic transmission cycle in nature.

Metagenomic profiles of Dermacentor tick pathogens from across Mongolia, using next generation sequencing.

Tick-borne diseases are a major public health concern in Mongolia. Nomadic pastoralists, which make up ~ 26% of Mongolia's population, are at an increased risk of both tick bite exposure and economic loss associated with clinical disease in herds. This study sought to further characterize tick-borne pathogens present in Dermacentor ticks (n = 1,773) sampled in 2019 from 15 of Mongolia's 21 aimags (provinces). The ticks were morphologically identified and sorted into 377 pools which were then screened using Next-Generation Sequencing paired with confirmatory PCR and DNA sequence analysis. Rickettsia spp. were detected in 88.33% of pools, while Anaplasma spp. and Bartonella spp. were detected in 3.18 and 0.79% of pools, respectively. Khentii had the highest infection rate for Rickettsia spp. (76.61%; CI: 34.65-94.79%), while Arkhangai had the highest infection rate for Anaplasma spp. (7.79%; CI:4.04-13.72%). The exclusive detection of Anaplasma spp. in tick pools collected from livestock supports previous work in this area that suggests livestock play a significant role in disease maintenance. The detection of Anaplasma, Bartonella, and Rickettsia demonstrates a heightened risk for infection throughout Mongolia, with this study, to our knowledge, documenting the first detection of Bartonella melophagi in ticks collected in Mongolia. Further research deploying NGS methods is needed to characterize tick-borne pathogens in other endemic tick species found in Mongolia, including Hyalomma asiaticum and Ixodes persulcatus.

The Bacterial Community in Questing Ticks From Khao Yai National Park in Thailand.

Ticks are known vectors for a variety of pathogens including bacteria, viruses, fungi, and parasites. In this study, bacterial communities were investigated in active life stages of three tick genera (Haemaphysalis, Dermacentor, and Amblyomma) collected from Khao Yai National Park in Thailand. Four hundred and thirty-three questing ticks were selected for pathogen detection individually using real-time PCR assays, and 58 of these were subjected to further metagenomics analysis. A total of 62 ticks were found to be infected with pathogenic bacteria, for a 14.3% prevalence rate, with Amblyomma spp. exhibiting the highest infection rate (20.5%), followed by Haemaphysalis spp. (14.5%) and Dermacentor spp. (8.6%). Rickettsia spp. were the most prevalent bacteria (7.9%) found, followed by Ehrlichia spp. (3.2%), and Anaplasma spp. and Borrelia spp. each with a similar prevalence of 1.6%. Co-infection between pathogenic bacteria was only detected in three Haemaphysalis females, and all co-infections were between Rickettsia spp. and Anaplasmataceae (Ehrlichia spp. or Anaplasma spp.), accounting for 4.6% of infected ticks or 0.7% of all examined questing ticks. The prevalence of the Coxiella-like endosymbiont was also investigated. Of ticks tested, 65.8% were positive for the Coxiella-like endosymbiont, with the highest infection rate in nymphs (86.7%), followed by females (83.4%). Among tick genera, Haemaphysalis exhibited the highest prevalence of infection with the Coxiella-like endosymbiont. Ticks harboring the Coxiella-like endosymbiont were more likely to be infected with Ehrlichia spp. or Rickettsia spp. than those without, with statistical significance for Ehrlichia spp. infection in particular (p-values = 0.003 and 0.917 for Ehrlichia spp. and Rickettsia spp., respectively). Profiling the bacterial community in ticks using metagenomics revealed distinct, predominant bacterial taxa in tick genera. Alpha and beta diversities analyses showed that the bacterial community diversity and composition in Haemaphysalis spp. was significantly different from Amblyomma spp. However, when examining bacterial diversity among tick life stages (larva, nymph, and adult) in Haemaphysalis spp., no significant difference among life stages was detected. These results provide valuable information on the bacterial community composition and co-infection rates in questing ticks in Thailand, with implications for animal and human health.

Anopheles bionomics in a malaria endemic area of southern Thailand.

BACKGROUND: Ivermectin mass drug administration (MDA) could accelerate malaria elimination in the Greater Mekong Subregion. This study was performed to characterize the bionomics of Anopheles in Surat Thani province, Thailand. METHODS: Mosquitoes were collected via human landing collections between February and October 2019. Anopheles mosquitoes were morphologically identified to species. Primary Anopheles malaria vectors were dissected to assess parity status, and a subset were evaluated for molecular identification and Plasmodium detection. RESULTS: A total of 17,348 mosquitoes were collected during the study period; of these, 5777 were Anopheles mosquitoes. Morphological studies identified 15 Anopheles species, of which the most abundant were Anopheles minimus (s.l.) (87.16%, n = 5035), An. dirus s.l. (7.05%, n = 407) and An. barbirostris s.l. (2.86%, n = 165). Molecular identification confirmed that of the An. minimus s.l. mosquitoes collected, 99.80% were An. minimus (s.s.) (n = 484) and 0.2% were An. aconitus (n = 1), of the An. dirus (s.l.) collected, 100% were An. baimaii (n = 348), and of the An. maculatus (s.l.) collected, 93.62% were An. maculatus (s.s.) (n = 44) and 6.38% were An. sawadwongporni (n = 3). No Anopheles mosquito tested was Plasmodium positive (0/879). An average of 11.46 Anopheles were captured per collector per night. There were differences between species in hour of collection (Kruskal-Wallis H-test: χ2 = 80.89, P < 0.0001, n = 5666), with more An. barbirostris (s.l.) and An. maculatus (s.l.) caught earlier compared to An. minimus (s.l.) (P = 0.0001 and P < 0.0001, respectively) and An. dirus (s.l.) (P = 0.0082 and P < 0.001, respectively). The proportion of parous An. minimus (s.l.) captured by hour increased throughout the night (Wald Chi-square: χ2 = 17.31, P = 0.000, odds ratio = 1.0535, 95% confidence interval 1.0279-1.0796, n = 3400). Overall, An. minimus (s.l.) parity was 67.68% (2375/3509) with an intra-cluster correlation of 0.0378. A power calculation determined that an An. minimus (s.l.) parity reduction treatment effect size = 34%, with four clusters per treatment arm and a minimum of 300 mosquitoes dissected per cluster, at an α = 0.05, will provide 82% power to detect a significant difference following ivermectin MDA. CONCLUSIONS: The study area in Surat Thani province is an ideal location to evaluate the impact of ivermectin MDA on An. minimus parity.