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.

Cattle lice are obligatory blood-sucking parasites, which is the cause of animal health problems worldwide. Recently, several studies have revealed that pathogenic bacteria could be found in cattle lice, and it can act as a potential vector for transmitting louse-borne diseases. However, the cattle lice and their pathogenic bacteria in Thailand have never been evaluated. In the present study, we aim to determine the presence of bacterial pathogens in cattle lice collected from three localities of Thailand. Total genomic DNA was extracted from 109 cattle louse samples and the Polymerase Chain Reaction (PCR) of 18S rRNA was developed to identify the cattle louse. Moreover, PCR was used for screening Bartonella spp., Acinetobacter spp., and Rickettsia spp. in cattle louse samples. The positive PCR products were cloned and sequenced. The phylogenetic tree based on the partial 18S rRNA sequences demonstrated that cattle lice species in this study are classified into two groups according to reference sequences; Haematopinus quadripertusus and Haematopinus spp. closely related to H. tuberculatus. The pathogen detection revealed that Bartonella spp. DNA of gltA and rpoB were detected in 25 of 109 samples (22.93%) both egg and adult stages, whereas Acinetobacter spp. and Rickettsia spp. were not detected in all cattle lice DNA samples. The gltA and rpoB sequences showed that the Bartonella spp. DNA was found in both H. quadripertusus and Haematopinus spp. closely related to H. tuberculatus. This study is the first report of the Bartonella spp. detected in cattle lice from Thailand. The finding obtained from this study could be used to determine whether the cattle lice can serve as a potential vector to transmit these pathogenic bacteria among cattle and may affect animal to human health.

Biting midges of the genus Culicoides have been identified as potential vectors for the transmission of species of the Leishmania subgenus Mundinia, the causative agents of autochthonous leishmaniasis in Thailand. Vector competence may be potentially affected by bacterial endosymbionts; however, there is no known correlation between these endosymbionts and Leishmania parasites in Culicoides biting midges. In this study, we aimed to explore the prevalence and association of bacterial endosymbionts and the detection of Leishmania DNA in Culicoides spp. Female midges were captured at five sampling sites in areas with autochthonous leishmaniasis in northern and southern Thailand. Culicoides species were identified using both morphological characteristics and cox1 sequencing. The presence of Wolbachia, Cardinium, and Leishmania DNA in individual midges was molecularly screened targeting the wsp and 16S rRNA genes, and the ITS1 region, respectively. All amplification products were sequenced and subjected to phylogenetic analysis. A total of 593 female midges were collected, comprising 21 species of Culicoides and one species of Culicoides (Trithecoides). The Wolbachia isolates from infected Culicoides spp. were phylogenetically classified into supergroups A, B, and F. Six Wolbachia putative strains belonged to clade Wol-b (Wol-b1 to Wol-b6), two to clade Wol-a (Wol-a1 and Wol-a2), and one to clade Wol-f (Wol-f1). Three Wolbachia strains were identified as wKerlac, wBeva_B, and wCauA. Moreover, both Cardinium groups A and C were identified. It is noteworthy that co-infections between bacterial endosymbionts and Leishmania spp. showed a significant association. To the best of our knowledge, this study provides the first evidence of Wolbachia and Cardinium in Culicoides spp. from leishmaniasis-affected areas in Thailand. Detecting bacterial endosymbionts co-occurring with Leishmania spp. in Culicoides biting midges may suggest a potential, but unconfirmed, antagonistic effect on Leishmania. This provides preliminary data that could inform the development of new vector control strategies for diseases transmitted by Culicoides spp. in Thailand.

Sand flies are potential carriers of various diseases that are transmittable to humans and animals. In this study, United States Centers for Disease Control light traps were set up in four tourist caves in the Thai provinces of Surat Thani, Nakhon Si Thammarat, Satun, and Chumphon to capture Idiophlebotomus asperulus sand flies. Over a period of three months, April to June, in 2017, a total of 181 female Idiophlebotomus asperulus sand flies were captured during nightly operations. The sand flies were dissected into 23 external and internal parts to identify their morphological characteristics. Statistical analysis was then conducted on these morphological characteristics, involving both univariate analysis (one-way analysis of variance and the Kruskal–Wallis test) and multivariate analysis (canonical discriminant analysis). Levene’s, the Kolmogorov–Smirnov, and Box’s M tests were used for the preliminary statistical screening of the data. The test results revealed significant morphological differences in the sand flies from the four provinces with regard to their antenna segments 5, palpal segments 3, pharynxes, hindlegs, femurs, and spermathecae. These morphological differences in the southern Thai Idiophlebotomus asperulus sand fly population suggest the possibility that at least three morphologically different populations are found in this region.

Bats represent over 1400 species globally, accounting for approximately one-fifth of all mammalian diversity, yet their gastrointestinal parasite communities remain understudied. Among these parasites, the genus Eimeria is one of the most commonly documented coccidian groups infecting bats. To date, more than 40 Eimeria spp. have been described from bats worldwide; however, molecular data are limited, and their evolutionary relationships with congeners infecting other vertebrate hosts remain largely unresolved. This study aims to elucidate the evolutionary connections between Eimeria parasites infecting bats and rodents, addressing a key question about shared ancestry and host-switching events across deeply divergent hosts. We investigated the genetic diversity and phylogenetic relationships of Eimeria spp. infecting Thai bats. Ninety-six genomic DNA samples extracted from bat guano, collected across six geographically distinct sites in prior research, were screened using SSU rRNA-PCR and nanopore amplicon sequencing. Host identification based on vertebrate cox1 gene sequencing revealed seven bat species (Pteropus lylei, Taphozous melanopogon, Cynopterus brachyotis, Eonycteris spelaea, Mops plicatus, Hipposideros armiger, and Pteropus vampyrus) along with the Hipposideros larvatus species complex. Haplotype network construction and phylogenetic analyses using Bayesian inference and maximum likelihood identified five putative genetic clusters of Eimeria sequences, including novel Haplogroups 1 and 2 that formed clearly distinct groups. The remaining three clusters showed close genetic affinities to known Eimeria species from bats (Eimeria sp. Bat10 and Eimeria sp. Bat31) and rodents (E. ferrisi). Notably, despite the deep evolutionary divergence between bats and rodents, Eimeria parasites infecting these hosts did not form entirely separate clades. Both phylogenies consistently revealed the polyphyletic nature of bat-derived Eimeria species, with multiple independent lineages interspersed among rodent taxa. This pattern supports hypotheses of shared ancestry or host-switching events, highlighting the complex evolutionary dynamics shaping Eimeria diversity across vertebrate hosts. The widespread distribution and genetic patterns observed in Haplogroup 1 suggest a recent population expansion potentially driven by ecological adaptability and host range dynamics. By focusing on the evolutionary relationships between bat and rodent Eimeria, this study advances our understanding of Eimeria diversity and host-parasite coevolution, emphasizing the importance of integrative molecular approaches in unravelling parasite evolutionary history across vertebrate taxa.