Identification of common human infectious and potentially zoonotic novel genotypes of Enterocytozoon bieneusi in cavernicolous bats in Thailand.

Enterocytozoon bieneusi is a common cause of human microsporidiosis and can infect a variety of animal hosts worldwide. In Thailand, previous studies have shown that this parasite is common in domestic animals. However, information on the prevalence and genotypes of this parasite in other synanthropic wildlife, including bats, remains limited. Several pathogens have been previously detected in bats, suggesting that bats may serve as a reservoir for this parasite. In this study, a total of 105 bat guano samples were collected from six different sites throughout Thailand. Of these, 16 from Chonburi (eastern), Ratchaburi (western), and Chiang Rai (northern) provinces tested positive for E. bieneusi, representing an overall prevalence of 15.2%. Based on ITS1 sequence analysis, 12 genotypes were identified, including two known genotypes (D and type IV) frequently detected in humans and ten novel potentially zoonotic genotypes (TBAT01-TBAT10), all belonging to zoonotic group 1. Lyle's flying fox (Pteropus lylei), commonly found in Southeast Asia, was identified as the host in one sample that was also positive for E. bieneusi. Network analysis of E. bieneusi sequences detected in this study and those previously reported in Thailand also revealed intraspecific divergence and recent population expansion, possibly due to adaptive evolution associated with host range expansion. Our data revealed, for the first time, multiple E. bieneusi genotypes of zoonotic significance circulating in Thai bats and demonstrated that bat guano fertilizer may be a vehicle for disease transmission.

Experimental transmission of Leishmania (Mundinia) parasites by biting midges (Diptera: Ceratopogonidae).

Leishmania parasites, causative agents of leishmaniasis, are currently divided into four subgenera: Leishmania, Viannia, Sauroleishmania and Mundinia. The recently established subgenus Mundinia has a wide geographical distribution and contains five species, three of which have the potential to infect and cause disease in humans. While the other Leishmania subgenera are transmitted exclusively by phlebotomine sand flies (Diptera: Psychodidae), natural vectors of Mundinia remain uncertain. This study investigates the potential of sand flies and biting midges of the genus Culicoides (Diptera: Ceratopogonidae) to transmit Leishmania parasites of the subgenus Mundinia. Sand flies (Phlebotomus argentipes, P. duboscqi and Lutzomyia migonei) and Culicoides biting midges (Culicoides sonorensis) were exposed to five Mundinia species through a chicken skin membrane and dissected at specific time intervals post bloodmeal. Potentially infected insects were also allowed to feed on ear pinnae of anaesthetized BALB/c mice and the presence of Leishmania DNA was subsequently confirmed in the mice using polymerase chain reaction analyses. In C. sonorensis, all Mundinia species tested were able to establish infection at a high rate, successfully colonize the stomodeal valve and produce a higher proportion of metacyclic forms than in sand flies. Subsequently, three parasite species, L. martiniquensis, L. orientalis and L. sp. from Ghana, were transmitted to the host mouse ear by C. sonorensis bite. In contrast, transmission experiments entirely failed with P. argentipes, although colonisation of the stomodeal valve was observed for L. orientalis and L. martiniquensis and metacyclic forms of L. orientalis were recorded. This laboratory-based transmission of Mundinia species highlights that Culicoides are potential vectors of members of this ancestral subgenus of Leishmania and we suggest further studies in endemic areas to confirm their role in the lifecycles of neglected pathogens.

In vitro susceptibility to miltefosine of amphotericin B-resistant Leishmania (Mundinia) martiniquensis.

Leishmania (Mundinia) martiniquensis is a newly described species that causes human visceral, disseminated, and mucocutaneous leishmaniases. Amphotericin B deoxycholate (AmpB) is the first-line drug for the treatment of leishmaniasis in Thailand; however, several relapse cases of leishmaniasis caused by L. martiniquensis have been documented. In this study, in vitro susceptibility to AmpB and miltefosine (MIL) of wild-type (before treatment, LSCM1) and two AmpB-resistant L. martiniquensis strains (an in vitro-induced AmpB-resistant strain, AmpBRP2i, and a relapse strain, LSCM1-6) were determined. Results reveal that the IC50 value and resistance index against both drugs of promastigotes and intracellular amastigotes of the AmpBRP2i and LSCM1-6 strains were statistically significantly higher than those of the LSCM1 strain suggesting that cross-resistance with MIL occurred in both AmpB-resistant strains. The results of this study advocate further investigation into mechanisms that involve the complex nature of AmpB/MIL resistance in L. martiniquensis and development of effective methods for the identification of the AmpB-resistant parasites to help delivery of appropriate treatments for patients and for epidemiological surveys to survey the potential spread of drug-resistant strains.

Genetic characterization of chikungunya virus isolates from Aedes aegypti mosquitoes collected during a recent outbreak in Bangkok, Thailand.

Chikungunya virus (CHIKV) is a mosquito-borne emerging pathogen that is transmitted to humans through the bite of female Aedes mosquitoes. CHIKV infection has become a major public health concern worldwide, as it has a significant impact on the healthcare system. Since 2004, the virus has emerged in Africa and subsequently spread to countries located near the Indian Ocean, including India, and to Europe, the Americas, and Asia. In Thailand, a large CHIKV outbreak occurred during 2008-2009 and was caused by a virus originating from the east/central/south African (ECSA) CHIKV genotype. Since then, the ECSA genotype of CHIKV has continued to circulate and has caused sporadic cases in different areas in Thailand. Approximately 20,000 reported cases have been confirmed by the Bureau of Epidemiology, Ministry of Public Health, Thailand, from January 1, 2018 to July 31, 2020. However, the causes of this CHIKV re-emergence remain unclear. To obtain a better understanding of CHIKV circulation during the recent outbreak in Bangkok, Thailand, complete genome analysis of CHIKV isolates from field-caught mosquitoes collected in outbreak areas was performed. A total of 28 Ae. aegypti samples (21 females and 7 males) were collected, and individual mosquitoes were used for CHIKV detection and isolation. Eleven of 28 (39.29%) female and three of 28 (10.71%) male mosquitoes were positive for CHIKV by E1 nested RT-PCR. Four CHIKV isolates were successfully isolated from four female Ae. aegypti mosquitoes. Based on complete genome analysis, several amino acid substitutions were identified in the protein coding region. The E1:K211E and E2:V264A mutations in the background of the E1:226A mutation were observed in all four CHIKV isolates. An important observation was the presence of one amino acid substitution, leading to an E1:K245R change. This mutation was found in all four CHIKV isolates from mosquitoes in this study and in Thai patients described previously. Additionally, phylogenetic analysis indicated that the four CHIKV isolates belonged to the Indian Ocean clade of the ECSA genotype. The results obtained in this study provide detailed information on the molecular characteristics and evolution of currently circulating CHIKV strains in Thailand, which are useful for developing prevention and control strategies.

Genome sequences of chikungunya virus isolates from an outbreak in southwest Bangkok in 2018.

An outbreak of chikungunya virus (CHIKV) infection occurred in southwest Bangkok during the 2018 rainy season. The envelope glycoprotein E1 gene sequence of the infecting strain belonged to an East/Central/South African lineage with alanine at residue 226. Mutations in the predicted E1 (K211E) and E2 (V264A) proteins of CHIKV were identified in CHIKV-infected patients and in an Aedes aegypti mosquito. Analysis of the complete genome sequences showed marked differences from the strains causing previous outbreaks in Thailand in 2008-2009 and 2013 but showed similarities to strains from more recent CHIKV outbreaks in South and Southeast Asia.

Synthesis and antileishmanial activity of fluorinated rhodacyanine analogues: The 'fluorine-walk' analysis.

In a search for potent antileishmanial drug candidates, eighteen rhodacyanine analogues bearing fluorine or perfluoroalkyl substituents at various positions were synthesized. These compounds were tested for their inhibitory activities against Leishmania martiniquensis and L. orientalis. This 'fluorine-walk' analysis revealed that the introduction of fluorine atom at C-5, 6, 5', or 6' on the benzothiazole units led to significant enhancement of the activity, correlating with the less negative reduction potentials of the fluorinated analogues confirmed by the electrochemical study. On the other hand, CF3 and OCF3 groups were found to have detrimental effects, which agreed with the poor aqueous solubility predicted by the in silico ADMET analysis. In addition, some of the analogues including the difluorinated species showed exceptional potency against the promastigote and axenic amastigote stages (IC50 = 40-85 nM), with the activities surpassing both amphotericin B and miltefosine.

Analysis of significant factors for dengue fever incidence prediction.

BACKGROUND: Many popular dengue forecasting techniques have been used by several researchers to extrapolate dengue incidence rates, including the K-H model, support vector machines (SVM), and artificial neural networks (ANN). The time series analysis methodology, particularly ARIMA and SARIMA, has been increasingly applied to the field of epidemiological research for dengue fever, dengue hemorrhagic fever, and other infectious diseases. The main drawback of these methods is that they do not consider other variables that are associated with the dependent variable. Additionally, new factors correlated to the disease are needed to enhance the prediction accuracy of the model when it is applied to areas of similar climates, where weather factors such as temperature, total rainfall, and humidity are not substantially different. Such drawbacks may consequently lower the predictive power for the outbreak. RESULTS: The predictive power of the forecasting model-assessed by Akaike's information criterion (AIC), Bayesian information criterion (BIC), and the mean absolute percentage error (MAPE)-is improved by including the new parameters for dengue outbreak prediction. This study's selected model outperforms all three other competing models with the lowest AIC, the lowest BIC, and a small MAPE value. The exclusive use of climate factors from similar locations decreases a model's prediction power. The multivariate Poisson regression, however, effectively forecasts even when climate variables are slightly different. Female mosquitoes and seasons were strongly correlated with dengue cases. Therefore, the dengue incidence trends provided by this model will assist the optimization of dengue prevention. CONCLUSIONS: The present work demonstrates the important roles of female mosquito infection rates from the previous season and climate factors (represented as seasons) in dengue outbreaks. Incorporating these two factors in the model significantly improves the predictive power of dengue hemorrhagic fever forecasting models, as confirmed by AIC, BIC, and MAPE.

Early detection of novel Leishmania species DNA in the saliva of two HIV-infected patients.

BACKGROUND: Leishmaniasis caused by two new species of Leishmania; L. siamensis and L. martiniquensis have been recently described in Thailand. The disease has mainly been documented in AIDS patients from southern Thailand. In this study, polymerase chain reaction (PCR) was used to determine HIV-Leishmania co-infection in southern Thailand. METHODS: One ml of saliva and 3 ml of EDTA blood were collected from HIV-infected patients for PCR detection of Leishmania DNA, cloning and sequencing. The positive PCR samples were then cultured on Schneider's insect medium. RESULTS: Three out of 316 saliva samples collected from HIV-infected patients were found to be positive for Leishmania DNA (0.95%). Among the positive samples, one patient was observed with disseminated cutaneous lesions and also tested positive via saliva, whole blood and buffy coat in PCR. The second case presenting with nodular lesions also gave a positive saliva test via PCR two months prior to buffy coat. This diagnosis was confirmed by microscopic examination and a culture of biopsy samples from a nodule. The last case was an asymptomatic Leishmania infection which tested PCR positive only in saliva with a consecutive sample collection conducted for three months. CONCLUSIONS: The prevalence of Leishmania infection in HIV infected patients within this study is 0.95%. Leishmania DNA was detected in saliva by PCR prior to blood and buffy coat of two HIV infected patients. Early detection of Leishmania DNA in saliva would be beneficial for the follow up of asymptomatic Leishmania infected patients, the early treatment of leishmaniasis and for surveillance survey purpose. However, full evaluation of sensitivity and specificity of this technique with a large cohort of patients is required before deployment.

Animal reservoirs and potential vectors of Leishmania siamensis in southern Thailand.

Leishmania siamensis is newly described as the causative pathogen of autochthonous leishmaniasis in Thailand. Potential vectors and animal reservoirs of L. siamensis are not thoroughly studied. An environmental survey was carried out in the affected area in two provinces in southern Thailand: Songkhla and Nakhon Si Thammarat. Ninety-nine villagers, 378 sandflies, and potential animal reservoirs were examined. Leishmania DNA amplicon was identified in two species of female sandflies, Sergentomyia (Neophlebotomus) and Sergentomyia (Parrotomyia) barraudi. The DNA amplicon was also identified in black rats (Rattus rattus). A phylogenetic tree of confirmed patients, sandflies and black rats fell into a single clade and separate from other Leishmania species. This study showed the potential involvement of R. rattus and Sergentomyia (Neophlebotomus and Parrotomyia) sandflies in transmission of L. siamensis.

Wolbachia supergroups A and B in natural populations of medically important filth flies (diptera: muscidae, calliphoridae, and sarcophagidae) in Thailand.

Filth flies, belonging to suborder Brachycera (Family; Muscidae, Calliphoridae and Sarcophagidae), are a major cause of nuisance and able to transmit pathogens to humans and animals. These insects are distributed worldwide and their populations are increasing especially in sub-tropical and tropical areas. One strategy for controlling insects employs Wolbachia, which is a group of maternally inherited intracellular bacteria, found in many insect species. The bacteria can cause reproductive abnormalities in their hosts, such as cytoplasmic incompatibility, feminization, parthenogenesis, and male lethality. In this study we determined Wolbachia endosymbionts in natural population of medically important flies (42 females and 9 males) from several geographic regions of Thailand. Wolbachia supergroups A or B were detected in 7 of female flies using PCR specific for wsp. Sequence analysis of wsp showed variations between and within the Wolbachia supergroup. Phylogenetics demonstrated that wsp is able to diverge between Wolbachia supergroups A and B. These data should be useful in future Wolbachia-based programs of fly control.

Nanopore-Based Surveillance of Leishmania Parasites in Culicoides Latrielle (Diptera: Ceratopogonidae) Caught from the Affected Community and Tham Phra Cave in Chiang Rai Province, the Endemic Area of Leishmaniasis in Northern Thailand.

In this research, we elucidated the species composition of Culicoides biting midges, infection prevalence, and genetic diversity of Leishmania parasites circulating in the affected community in Chiang Rai Province, being the most endemic area in Northern Thailand. A total of 146 parous and gravid females, belonging to at least twelve Culicoides species in five subgenera and one species group, were trapped from three collection sites with an overall Leishmania prevalence of 26.7% (39/146). Leishmania was detected, using ITS1-PCR, in C. mahasarakamense (15), C. guttifer (11), C. (Trithecoides) spp. (8), C. jacobsoni (2), C. oxystoma (2), and C. orientalis (1). The evidence of Leishmania infection in these last five species represents new records in Northern Thailand. Given a high infection rate in cavernicolous specimens, this indicates an increased risk of parasite exposure when visiting the cave. Using the nanopore amplicon sequencing, L. martiniquensis was ubiquitously identified in all positives, and more than half of these were also co-infected with L. orientalis. The genetic diversity analysis revealed 13 and 17 unique haplotypes for L. martiniquensis and L. orientalis, respectively. Higher haplotype diversity and relatively low nucleotide diversity were observed in both parasite populations, suggesting recent population divergence. Neutrality tests (Tajima's D and Fu and Li's D) showed to be significantly negative, indicating rapid population growth or a selective sweep. Moreover, dominant haplotypes of both Leishmania species were 100% identical to those in all leishmaniasis patients previously reported from Northern Thailand, strongly supporting the imperative role of Culicoides spp. in disease transmission. Essentially, this research provides the first entomological surveillance data representing the sympatric existence, transmission dynamics, and genetic complexity of two autochthonous Leishmania (Mundinia) parasites in several Culicoides species in the endemic area of Northern Thailand. This would contribute to a more complete understanding of the epidemiology of vector infection and facilitate the development of vector control programs to effectively reduce the transmission of this neglected tropical disease in endemic areas of Northern Thailand.

Comparative analysis of midgut bacterial communities in Chikungunya virus-infected and non-infected Aedes aegypti Thai laboratory strain mosquitoes.

Chikungunya virus (CHIKV) poses a significant global health threat, re-emerging as a mosquito-transmitted pathogen that caused high fever, rash, and severe arthralgia. In Thailand, a notable CHIKV outbreak in 2019-2020 affected approximately 20,000 cases across 60 provinces, underscoring the need for effective mosquito control protocols. Previous studies have highlighted the role of midgut bacteria in the interaction between mosquito vectors and pathogen infections, demonstrating their ability to protect the insect from invading pathogens. However, research on the midgut bacteria of Aedes (Ae.) aegypti, the primary vector for CHIKV in Thailand remains limited. This study aims to characterize the bacterial communities in laboratory strains of Ae. aegypti, both infected and non-infected with CHIKV. Female mosquitoes from a laboratory strain of Ae. aegypti were exposed to a CHIKV-infected blood meal through membrane feeding, while the control group received a non-infected blood meal. At 7 days post-infection (dpi), mosquito midguts were dissected for 16S rRNA gene sequencing to identify midgut bacteria, and CHIKV presence was confirmed by E1-nested RT-PCR using mosquito carcasses. The study aimed to compare the bacterial communities between CHIKV-infected and non-infected groups. The analysis included 12 midgut bacterial samples, divided into three groups: CHIKV-infected (exposed and infected), non-infected (exposed but not infected), and non-exposed (negative control). Alpha diversity indices and Bray-Curtis dissimilarity matrix revealed significant differences in bacterial profiles among the three groups. The infected group exhibited an increased abundance of bacteria genus Gluconobacter, while Asaia was prevalent in both non-infected and negative control groups. Chryseobacterium was prominent in the negative control group. These findings highlight potential alterations in the distribution and abundance of gut microbiomes in response to CHIKV infection status. This study provides valuable insights into the dynamic relationship between midgut bacteria and CHIKV, underscoring the potential for alterations in bacterial composition depending on infection status. Understanding the relationships between mosquitoes and their microbiota holds promise for developing new methods and tools to enhance existing strategies for disease prevention and control. This research advances our understanding of the circulating bacterial composition, opening possibilities for new approaches in combating mosquito-borne diseases.

Multilocus characterization and phylogenetic analysis of Leishmania siamensis isolated from autochthonous visceral leishmaniasis cases, southern Thailand.

BACKGROUND: Visceral leishmaniasis (VL) caused by Leishmania siamensis is an emerging disease continuously reported in six southern provinces of Thailand. To date, the phylogenetic relationships among Leishmania isolates from Thai patients and other Leishmania species are still unclear and the taxonomic diversity needs to be established. In this study, the phylogenetic inference trees were constructed based on four genetic loci (i.e., SSU-rRNA, ITS1, hsp70, and cyt b), using DNA sequences obtained from autochthonous VL patients from southern Thailand and reference sequences of reported Leishmania isolates from other studies deposited in GenBank. RESULTS: Phylogenetic analyses of hsp70 and cyt b loci supported a clade comprised of L. siamensis isolates, which is independent to the other members in the genus Leishmania. In combination with genetic distance analysis, sequence polymorphisms were observed among L. siamensis isolates and two different lineages could be differentiated, lineages PG and TR. Phylogenetic analysis of the cyt b gene further showed that L. siamensis lineage TR is closely related to L. enrietti, a parasite of guinea pigs. CONCLUSION: The finding of this study sheds further light on the relationships of L. siamensis, both in intra- and inter-species aspects. This information would be useful for further in-depth studies on the biological properties of this important parasite.

Comparative field efficacy of newly developed formulations of larvicides against Aedes aegypti (L.) (Diptera: Culicidae).

Aedes aegypti (L.) is known as vector of dengue and chikungunya fever. Larvicides are used to control this vector. We evaluated the efficacy of newly developed formulations of larvicides to control Ae. aegypti under field conditions for 24 weeks post single application. Mosdop P and Mosdop TB containing diflubenzuron (2% and 40 mg/tablet, respectively) as the active ingredient, were applied at a dosage of 0.1 mg a.i./1 and Mosquit TB10, Mosquit TB100 and Temecal containing temephos (1%, 10% and 1%, respectively) as the active ingredient were applied at a dosage of 1 mg active ingredent (a.i.) to 200 liter water storage jars. Two water regimens were used in the jars: in one regimen the jar was kept full of water all the time and in the other regimen a full jar had half the volume removed and refilled weekly. The larvicidal efficacy was reported as the level of inhibition of emergence (IE%) calculated based on the pupal skins in the jars versus the original number of larvae added. Mosdop P, Mosdop TB, Mosquit TB10, Mosquit TB100 and Temecal showed complete larvicidal efficacy (100% IE) in the constantly full jars for 16, 17, 14, 20 and 13 weeks posttreatment, respectively; in the jars where half the volum of water was replaced weekly, the larvicides had complete larvicidal efficacy (100% IE) for 19, 20, 17, 24 and 15 weeks post-treatment, respectively. The five larvicide regimens evaluated in this study are effective for controlling Ae. aegypti larvae.

Enhancement of VGG16 model with multi-view and spatial dropout for classification of mosquito vectors.

Mosquitoes transmit pathogens that can cause numerous significant infectious diseases in humans and animals such as malaria, dengue fever, chikungunya fever, and encephalitis. Although the VGG16 model is not one of the most advanced CNN networks, it is reported that a fine-tuned VGG16 model achieves accuracy over 90% when applied to the classification of mosquitoes. The present study sets out to improve the accuracy and robustness of the VGG16 network by incorporating spatial dropout layers to regularize the network and by modifying its structure to incorporate multi-view inputs. Herein, four models are implemented: (A) early-combined, (B) middle-combined, (C) late-combined, and (D) ensemble model. Moreover, a structure for combining Models (A), (B), (C), and (D), known as the classifier, is developed. Two image datasets, including a reference dataset of mosquitoes in South Korea and a newly generated dataset of mosquitoes in Thailand, are used to evaluate our models. Regards the reference dataset, the average accuracy of ten runs improved from 83.26% to 99.77%, while the standard deviation decreased from 2.60% to 0.12%. When tested on the new dataset, the classifier's accuracy was also over 99% with a standard deviation of less than 2%. This indicates that the algorithm achieves high accuracy with low variation and is independent of a particular dataset. To evaluate the robustness of the classifier, it was applied to a small dataset consisting of mosquito images captured under various conditions. Its accuracy dropped to 86.14%, but after retraining with the small dataset, it regained its previous level of precision. This demonstrates that the classifier is resilient to variation in the dataset and can be retrained to adapt to the variation. The classifier and the new mosquito dataset could be utilized to develop an application for efficient and rapid entomological surveillance for the prevention and control of mosquito-borne diseases.

Genetic diversity and phylogenetic analyses of Asian lineage Zika virus whole genome sequences derived from Culex quinquefasciatus mosquitoes and urine of patients during the 2020 epidemic in Thailand.

Zika virus (ZIKV), a mosquito-borne flavivirus, has been continually emerging and re-emerging since 2010, with sporadic cases reported annually in Thailand, peaking at over 1000 confirmed positive cases in 2016. Leveraging high-throughput sequencing technologies, specifically whole genome sequencing (WGS), has facilitated rapid pathogen genome sequencing. In this study, we used multiplex amplicon sequencing on the Illumina Miseq instrument to describe ZIKV WGS. Six ZIKV WGS were derived from three samples of field-caught Culex quinquefasciatus mosquitoes (two males and one female) and three urine samples collected from patients in three different provinces of Thailand. Additionally, successful isolation of a ZIKV isolate occurred from a female Cx. quinquefasciatus. The WGS analysis revealed a correlation between the 2020 outbreak and the acquisition of five amino acid changes in the Asian lineage ZIKV strains from Thailand (2006), Cambodia (2010 and 2019), and the Philippines (2012). These changes, including C-T106A, prM-V1A, E-V473M, NS1-A188V, and NS5-M872V, were identified in all seven WGS, previously linked to significantly higher mortality rates. Furthermore, phylogenetic analysis indicated that the seven ZIKV sequences belonged to the Asian lineage. Notably, the genomic region of the E gene showed the highest nucleotide diversity (0.7-1.3%). This data holds significance in informing the development of molecular tools that enhance our understanding of virus patterns and evolution. Moreover, it may identify targets for improved methods to prevent and control future ZIKV outbreaks.

Persistence of dengue genome in a remotely infected patient.

Background: Dengue virus infection is an intriguing illness. It is traditionally thought of as a self-limited and nonpersistent disease. Objectives: We report a case with persistent dengue virus genome detectable in hematopoietic cells of a person with remote infection. Methods: A patient with multiple myeloma in remission was prepared for peripheral blood stem cell (PBSC) transplantation. Plasma and G-CSF-stimulated, mobilized PBSCs were collected. Dengue-specific reverse transcription polymerase chain reaction (RT-PCR) was performed in both pre- and post-stimulated blood specimens. Anti-dengue antibodies by ELISA and by neutralization assay were measured before and after the stem cell mobilization. Results: The viral genome was detected only in the PBSC of the post-G-CSF-stimulated specimens. Anti-dengue antibodies were negative and positive, by ELISA and neutralization assays, respectively, both before and after stem cell mobilization. Conclusion: Our findings reveal a persistent infection. Whether and how this strain may interact with subsequent serotype(s) remains to be elucidated.

Species diversity of phlebotomine sand flies and sympatric occurrence of Leishmania (Mundinia) martiniquensis, Leishmania (Leishmania) donovani complex, and Trypanosoma spp. in the visceral leishmaniasis focus of southern Thailand.

Autochthonous leishmaniasis in Thailand has recently been a public health concern due to an increasing number of new clinical cases. Most indigenous cases were diagnosed with Leishmania (Mundinia) martiniquensis, and Leishmania (Mundinia) orientalis. However, some doubts regarding vector misidentification have arisen and need to be elucidated. Accordingly, we aimed to assess the species composition of sand flies and determine the molecular prevalence of trypanosomatids in the transmission area of leishmaniasis in southern Thailand. In the present study, a total of 569 sand flies were caught from the vicinity of a visceral leishmaniasis patient's house in Na Thawi District, Songkhla Province. Of these, 229 parous and gravid females consisted of Sergentomyia khawi, Se. barraudi, Phlebotomus stantoni, Grassomyia indica, and Se. hivernus, accounting for 31.4%, 30.6%, 29.7%, 7.9%, and 0.4%, respectively. However, Se. gemmea, which has previously been proposed as the most abundant species and putative vector of visceral leishmaniasis, was not found in the present study. Based on ITS1-PCR and sequence analysis, two specimens of Gr. indica and Ph. stantoni showed positive amplification of L. martiniquensis and L. donovani complex, respectively, the first one being presumed indigenous and the second one being not. Anuran Trypanosoma was also molecularly detected using SSU rRNA-PCR and ubiquitously found in 16 specimens of four dominant sand fly species except for Se. hivernus. The obtained sequences could be phylogenetically categorized into the two major amphibian clades (An04/Frog1 and An01+An02/Frog2). The existence of the monophyletic subgroup and distinct lineage suggests them as novel Trypanosoma species. The TCS network analysis of these anuran Trypanosoma sequences also revealed high haplotype diversity (Hd = 0.925 ± 0.050), but low nucleotide diversity (π = 0.019 ± 0.009). Furthermore, the living anuran trypanosomes were microscopically demonstrated in a single specimen of Gr. indica, supporting the vectorial capacity. Importantly, our data confirmed the scarcity of Se. gemmea and also uncovered, for the first time, the co-circulation of L. martiniquensis, L. donovani complex, and suspected novel anuran Trypanosoma spp. in phlebotomine sand files, implicating their potential role as vectors of trypanosomatid parasites. Therefore, the novel data from this study would greatly facilitate the comprehension of the complexity of trypanosomatid transmission and the establishment of prevention and control measures for this neglected disease more effectively.

Description of potential vectors of zoonotic filarial nematodes, Brugia pahangi, Setaria digitata, and Setaria labiatopapillosa in Thai mosquitoes.

Filariasis is classified as a vector-borne zoonotic disease caused by several filarial nematodes. The disease is widely distributed in tropical and subtropical regions. Understanding the relationship between mosquito vectors, filarial parasites, and vertebrate hosts is therefore essential for determining the probability of disease transmission and, correspondingly, developing effective strategies for prevention and control of diseases. In this study, we aimed to investigate the infection of zoonotic filarial nematodes in field-caught mosquitoes, observe the potential vectors of filaria parasites in Thailand using a molecular-based survey, conduct a study of host-parasite relationship, and propose possible coevolution of the parasites and their hosts. Mosquitoes were collected around cattle farms in Bangkok, Nakhon Si Thammarat, Ratchaburi, and Lampang provinces from May to December 2021 using a CDC Backpack aspirator for 20-30 minutes in each area (intra-, peri-, and wild environment). All mosquitoes were identified and morphologically dissected to demonstrate the live larvae of the filarial nematode. Furthermore, all samples were tested for filarial infections using PCR and sequencing. A total of 1,273 adult female mosquitoes consisted of five species: 37.78% Culex quinquefasciatus, 22.47% Armigeres subalbatus, 4.71% Cx. tritaeniorhynchus, 19.72% Anopheles peditaeniatus, and 15.32% An. dirus. Larvae of Brugia pahangi and Setaria labiatopapillosa were found in Ar. subalbatus and An. dirus mosquitoes, respectively. All mosquito samples were processed by PCR of ITS1 and COXI genes for filaria nematode species identification. Both genes showed that B. pahangi was found in four mosquitoes of Ar. subalbatus from Nakhon Si Thammarat, S. digitata was detected in three samples of An. peditaeniatus from Lampang, and S. labiatopapillosa was detected in one of An. dirus from Ratchaburi. However, filarial nematodes were not found in all Culex species. This study infers that this is the first data regarding the circulation of Setaria parasites in Anopheles spp. from Thailand. The phylogenetic trees of the hosts and parasites are congruent. Moreover, the data could be used to develop more effective prevention and control strategies for zoonotic filarial nematodes before they spread in Thailand.

Natural infection with Leishmania (Mundinia) martiniquensis supports Culicoides peregrinus (Diptera: Ceratopogonidae) as a potential vector of leishmaniasis and characterization of a Crithidia sp. isolated from the midges.

The prevalence of autochthonous leishmaniasis in Thailand is increasing but the natural vectors that are responsible for transmission remain unknown. Experimental in vivo infections in Culicoides spp. with Leishmania (Mundinia) martiniquensis and Leishmania (Mundinia) orientalis, the major causative pathogens in Thailand, have demonstrated that biting midges can act as competent vectors. Therefore, the isolation and detection of Leishmania and other trypanosomatids were performed in biting midges collected at a field site in an endemic area of leishmaniasis in Tha Ruea and a mixed farm of chickens, goats, and cattle in Khuan Phang, Nakhon Si Thammarat province, southern Thailand. Results showed that Culicoides peregrinus was the abundant species (>84%) found in both locations and only cow blood DNA was detected in engorged females. Microscopic examination revealed various forms of Leishmania promastigotes in the foregut of several C. peregrinus in the absence of bloodmeal remnants, indicating established infections. Molecular identification using ITS1 and 3'UTR HSP70 type I markers showed that the Leishmania parasites found in the midges were L. martiniquensis. The infection rate of L. martiniquensis in the collected flies was 2% in Tha Ruea and 6% in Khuan Phang, but no L. orientalis DNA or parasites were found. Additionally, organisms from two different clades of Crithidia, both possibly new species, were identified using SSU rRNA and gGAPDH genes. Choanomastigotes and promastigotes of both Crithidia spp. were observed in the hindgut of the dissected C. peregrinus. Interestingly, midges infected with both L. martiniquensis and Crithidia were found. Moreover, four strains of Crithidia from one of the clades were successfully isolated into culture. These parasites could grow at 37°C in the culture and infect BALB/c mice macrophages but no multiplication was observed, suggesting they are thermotolerant monoxenous trypanosomatids similar to Cr. thermophila. These findings provide the first evidence of natural infection of L. martiniquensis in C. peregrinus supporting it as a potential vector of L. martiniquensis.