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Objective: To explore genetic variations of Hypoderaeum conoideum collected from domestic ducks from 12 different localities in Thailand and Lao PDR, as well as their phylogenetic relationship with American and European isolates. Methods: The nucleotide sequences of their nuclear ribosomal DNA (ITS), mitochondrial cytochrome c oxidase subunit 1 (CO1), and NADH dehydrogenase subunit 1 (ND1) were used to analyze genetic diversity indices. Results: We found relatively high levels of nucleotide polymorphism in ND1 (4.02%), whereas moderate and low levels were observed in CO1 (2.11%) and ITS (0.96%), respectively. Based on these polymorphisms, the 20 ND1, 12 CO1, and 18 ITS haplotypes were classified, and several common haplotypes were observed in all samples. At least three major lineages, namely American, European and Asian lineages, have been classified by phylogenetic analyses based on ND1 sequences. Conclusions: Our report demonstrates that the ND1 gene is the most suitable genetic marker to explore genetic variation and phylogenetic relationship of Hypoderaeum conoideum. However, a combination of all loci for ND1, CO1 and ITS would be of great value toward further genetic investigation of this endemic worldwide parasite. Thus, comprehensive molecular genetic analyses of Hypoderaeum conoideum from its worldwide distribution is needed to further understanding of the evolutionary and systematic relationships of this parasite. [ABSTRACT FROM AUTHOR]

Background: Few studies have been conducted on the presence of Salmonella in the rodents that inhabit the wet markets that play an important role in daily life in Southeast Asia. The results of studies of rodents as carriers of Salmonella vary greatly, ranging from an absence of Salmonella to high prevalences. Previous studies investigated habitats such as farms and urban and wild areas where there is less rodent-human interaction than in wet markets. Consequently, the potential role of rodents as reservoirs and transmitters of Salmonella in wet markets is of great interest. Methods: Rodents were trapped in eight traditional wet markets in Thailand and identified to species level. Subsequently, they were screened for Salmonella and isolates were serotyped. Results: A total of 110 rats (Rattus norvegicus and Rattus exulans) were examined. Overall, the prevalence of Salmonella in rats was 49.10%, but varied between 0% and 73.3% among markets. Three serovars were identified: Salmonella Typhimurium (30%), S. Weltevreden (12.7%), and S. 4,[5],12:i:- (6.4%). Conclusions: Our results show that rodents in wet markets are a potential reservoir of Salmonella due to the close contact they have with humans and food. The three isolated serovars, of which serovar S. 4,[5],12:i:- is reported for the first time in rodents, are among the 10 commonest serovars isolated from humans in Thailand. Thus, more attention should be paid to rodents as potential reservoirs of Salmonella. [ABSTRACT FROM AUTHOR]

Fifty-six samples ofNeotricula aperta-like snails were collected from six locations in Cambodia. Their mitochondrial cytochrome c oxidase subunit 1 (cox1) sequences were examined using haplotype network and neighbor-joining (NJ) tree analysis. Twenty-seven haplotypes (H1-H27) were observed and were divided into two different groups/lineages. Of 27, 17 haplotypes (H11-H27) were clustered with the reference samples of the γ-raceN. aperta. The remaining 10 haplotypes (H1-H10) were clustered in a separate group/lineage, differing from the reference samples of the α-, β-, and γ-raceN. aperta, suggesting a new lineage belonging the genusNeotricula. Our results show that both the γ-race and a new lineage were sympatrically present approximately 60 km upstream of the Mekong River near the Kratie port, Cambodia. Further morphological and molecular studies are required to confirm the taxonomic status of this new, unidentified lineage. [ABSTRACT FROM AUTHOR]

Background: Paragonimiasis is a foodborne parasitic infection caused by lung flukes of the genus Paragonimus. Several species of Paragonimus are endemic in Japan: P. westermani (diploid and triploid) P. miyazakii, P. ohirai and P. iloktsuenensis. The taxonomic status and genetic variability of these lung flukes remains poorly understood. Methods: The second intron of domain 1 of the taurocyamine kinase gene (TKD1int2) region was used to explore genetic variation and differentiation of diploid and triploid P. westermani, as well as P. miyazakii, P. ohirai and P. iloktsuenensis originating from Japan. Results:We found high levels of intraspecific variation in P. westermani, but only low levels of variation within the other species studied. Haplotype network and phylogenetic tree analyses demonstrated the sister-group relationship of P. ohirai and P. iloktsuenensis and the phylogenetically distant relationship of P. westermani with the other species. All individuals except for triploid P. westermani were homozygous. Each triploid contained at least one allele similar to that seen in most diploids from Chiba and one allele resembling that seen in diploids from Oita. One triploid contained three different sequences. Conclusions: Our findings suggested that the TKD1int2 region is a suitable marker for use in studying the genetic variation and phylogenetics of Paragonimus species, as well as providing clues to the origins of triploidy in P. westermani. [ABSTRACT FROM AUTHOR]

Background: Because the eggs of Paragonimus, Echinostoma and Fasciola are very similar in size and shape, it is difficult to distinguish and accurately identify species by the morphology of their eggs, which is a standard diagnostic method. Methods: In this study, a novel assay combining a real-time fluorescence resonance energy transfer PCR and melting curve analysis using one set of primers and fluorophore-labelled hybridization probes specific for the 28S rDNA region was developed for the molecular detection of Paragonimus heterotremus, Echinostoma malayanum and Fasciola gigantica eggs. Results: This assay could detect and distinguish P. heterotremus, E. malayanum and F. gigantica DNA with the distinct melting temperature (Tm) values of 57.99+0.08, 62.12+0.15 and 74.10+0.18, respectively. The assay can also be used to detect and distinguish DNA from P. bangkokensis, P. harinasutai, P. machorchis, E. revolutum, Hypodereum conoideum and F. hepatica, which have different Tmvalues. The sensitivity of this assay enabled the detection of one egg of P. heterotremus, E. malayanum or F. gigantica per 100 mg of faeces. In addition, the specificity testing showed no fluorescence signal for other parasites. Conclusions: Due to the sensitivity and specificity of our assay in detecting P. heterotremus, E. malayanum and F. gigantica, our method could be used to accurately diagnose these three medically important parasitic groups and has potential implications for molecular epidemiological investigations of human and/or animal infections. [ABSTRACT FROM AUTHOR]

The giant water bug, Lethocerus indicus (Lepeletier and Serville, 1825), is one of the most important edible insects in Thailand. This study investigated the genetic diversity of L. indicus from natural resources in northeast Thailand by using mitochondrial DNA sequences. Ninety adults of L. indicus were collected from 12 different localities by light trap and hand-net methods. Nucleotide and haplotype diversity of cytochrome c oxidase subunit 1 (CO1) sequences were relatively high. The 43 CO1 haplotypes (Li1–Li43) with 26 unique haplotypes were classified and used to generate a star-like haplotype network. The most common haplotypes were Li6 and Li2 shared between 19 and 15 sequences, respectively from nine different localities. A phylogenetic tree clearly separated L. indicus from the other species and closely clustered with a sibling species, L. patruelis (Stål, 1854). However, the comprehensive genetic variation of L. indicus covering broad range of areas needs to be further explored. [ABSTRACT FROM AUTHOR]

Cholangiocarcinoma (CCA) is an aggressive malignancy arising from the damaged epithelial cells of the biliary tract. Previous studies have reported that the multi-potent mesenchymal stem cells (MSCs) activate a series of tumor signaling pathways by releasing several cytokines to influence tumor cell development. However, the roles and mechanisms of human chorion-derived MSCs (CH-MSCs) in cholangiocarcinoma progression have not been fully addressed. This present study aims to examine the effects of conditioned media derived from CH-MSCs (CH-CM) on CCA cell lines and investigate the respective underlying mechanism of action. For this purpose, MSCs were isolated from chorion tissue, and three cholangiocarcinoma cell lines, namely KKU100, KKU213A, and KKU213B, were used. MTT assay, annexin V/PI analysis, and JC-1 staining were used to assess the effects of CH-CM on proliferation and apoptosis of CCA cells, respectively. Moreover, the effect of CH-CM on caspase-dependent apoptotic pathways was also evaluated. The western blotting assay was also used for measuring the expression of JAK2/STAT3 signaling pathway-associated proteins. The results showed that CH-CM suppressed proliferation and promoted apoptosis of CCA cell lines. CH-CM treatment-induced loss of mitochondrial membrane potential (∆Ψm) in CCA cell lines. The factors presented in the CH-CM also inhibited JAK2/STAT3 signaling, reduced the expression of BCL-2, and increased BAX expression in CCA cells. In conclusion, our study suggests that the CH-CM has a potent anti-cancer effect on cholangiocarcinoma cells and thus provides opportunities for use in alternative cell therapy or in combination with a conventional chemotherapeutic drug to increase the efficiency of CCA treatment. [ABSTRACT FROM AUTHOR]

A previous population genetics study of Opisthorchis viverrini from a locality in an endemic area in Thailand found little genetic variation over time and second intermediate fish host species. Since a similar comparative analysis is not available for Lao PDR, we conducted a study of O. viverrini from different endemic foci in Vientiane Province, Lao PDR, based on spatial, temporal and fish host species. A total of 620 adult O. viverrini originating from the Nam Ngum River wetland were analysed at five previously defined polymorphic enzyme loci. Of these worms, 252 were from six different localities (spatial samples), 162 worms from different years (temporal samples) and 206 worms from four different cyprinid fish species. Significant heterozygote deficiency was found in most O. viverrini populations with levels of genetic differentiation ranging between F 0.0000 and 0.0197 suggesting that gene flow occurred at a variable rate. The role of temporal factors and fish host species had little influence on the level of genetic differentiation. As for O. viverrini from Thailand, these findings indicate that self-fertilization and/or a clonal distribution of O. viverrini occurs in Lao PDR. Unlike the results for O. viverrini from Thailand, spatial population substructuring may be the underlying population processes for O. viverrini in Lao PDR. These findings indicate that geographical variation may contribute to the transmission dynamics of the parasite with implications for parasite control. However, other host factors, such as snail intermediate hosts and mammal reservoir hosts, as well as human beings, may also play significant roles. [ABSTRACT FROM AUTHOR]

• Cryptic genetic lineages of Simulium chumpornense were evaluated. • Morphology and molecular data indicated the lineages are different species. • ITS2 sequences accurately identified cryptic species of S. chumpornense. • Two Leucocytozoon and one Trypanosoma species were found in S. khelangense. Species of the Simulium varicorne group in Thailand have veterinary significance as vectors of haemosporidian parasites. Accurate identification is, therefore, critical to the study of vectors and parasites. We used morphology and molecular markers to investigate cryptic genetic lineages in samples identified as Simulium chumpornense Takaoka & Kuvangkadilok, 2000. We also tested the efficiency of the nuclear internal transcribed spacer 2 (ITS2) marker for the identification of species in this group. Morphological examinations revealed that S. chumpornense lineage A is most similar to S. khelangense Takaoka, Srisuka & Saeung, 2022, with minor morphological differences. They are also genetically similar based on mitochondrial cytochrome c oxidase I (COI) sequences. Geographically, the sampling site where paratypes of S. khelangense were originally collected is <50 km from where S. chumpornense lineage A was collected. We concluded that cryptic lineage A of S. chumpornense is actually S. khelangense. COI sequences could not differentiate S. kuvangkadilokae Pramual and Tangkawanit, 2008 from S. chumpornense and S. khelangense. In contrast, ITS2 sequences provided perfect accuracy in the identification of these species. Molecular analyses of the blood protozoa Leucocytozoon and Trypanosoma demonstrated that S. khelangense carries L. shoutedeni, Leucocytozoon sp., and Trypanosoma avium. The Leucocytozoon sp. in S. khelangense differs genetically from that in S. asakoae Takaoka & Davies, 1995, signaling the possibility of vector-parasite specificity. [ABSTRACT FROM AUTHOR]

Cyanobacteria are distributed worldwide, and many new cyanobacterial species are discovered in tropical region. The Nostoc-like genus Amazonocrinis has been separated from the genus Nostoc based on polyphasic methods. However, species diversity within this genus remains poorly understood systematically because only one species (Amazonocrinis nigriterrae) has been described. In this study, two novel strains (NUACC02 and NUACC03) were isolated from moist rice field soil in Thailand. These two strains were characterized using a polyphasic approach, based on morphology, 16S rRNA phylogenetic analysis, internal transcribed spacer secondary structure and ecology. Phylogenetic analyses based on 16S rRNA gene sequences confirmed that the two novel strains formed a monophyletic clade related to the genus Amazonocrinis and were distant from the type species A. nigriterrae. The 16S rRNA gene sequence similarity (<98.1%) between novel strains and all other closely related taxa including the Amazonocrinis members exceeded the cutoff for species delimitation in bacteriology, reinforcing the presence of a new Amazonocrinis species. Furthermore, the novel strains possessed unique phenotypic characteristics such as the presence of the sheath, necridia-like cells, larger cell dimension and akinete cell arrangement in long-chains and the singularity of D1-D1', Box-B, V2, and V3 secondary structures that distinguished them from other Amazonocrinis members. Considering all the results, we described our two strains as Amazonocrinis thailandica sp. nov. in accordance with the International Code of Nomenclature for Algae, Fungi and Plants. [ABSTRACT FROM AUTHOR]

• Opisthorchis viverrini life stages were sampled from 20 localities in Thailand and Lao PDR. • Microsatellite and mitochondrial sequence data indicate one widespread genetic cluster. • A second genetic cluster was found only at Phang Khon, Sakon Nakhon Province (SPk), Thailand. • The SPk cluster was largely replaced by the widespread cluster over time. • Metacercariae are genetically diverse and can represent the population. Infection by the small liver fluke, Opisthorchis viverrini , causes serious public health problems, including cholangiocarcinoma, in Thailand and southeastern Asian countries. Previous studies have reported that O. viverrini represents a species complex with varying levels of genetic differentiation in Thailand and Lao PDR. In this study, we re-examined population genetic structure and genetic diversity of O. viverrini using extensive samples of the parasite collected over 15 years from 12 geographical localities in Thailand and eight localities in Lao PDR. Parasite life-cycle stages of 721 individuals of O. viverrini (91 cercariae, 230 metacercariae and 400 adult worms) were genotyped using 12 microsatellite loci. Metacercariae exhibited genetic diversity comparable with that of experimentally raised adults: metacercariae can therefore be used to represent O. viverrini populations without the need for laboratory definitive hosts. Data obtained from larval as well as adult worms identified two distinct genetic clusters of O. viverrini. Sequences of a portion of the mitochondrial cox1 gene strongly supported the existence of these two clusters. One, the widespread cluster, was found at all sampled sites. The second cluster occurred only in Phang Khon District, Sakon Nakhon Province (SPk), within the Songkram River wetland in Thailand. A striking feature of our data relates to the temporal dynamics of the SPk cluster, which was largely replaced by representatives of the widespread cluster over time. If the SPk cluster is excluded, no marked genetic differences were seen among O. viverrini populations from Thailand and Lao PDR. The underlying causes of the observed population structure and population dynamics of O. viverrini are not known. [ABSTRACT FROM AUTHOR]

Three taxa of Bithynia snails, Bithynia funiculata, B. siamensis siamensis and B. siamensis goniomphalos, are currently recognised in Thailand. They serve as first intermediate hosts for the liver fluke Opisthorchis viverrini sensu lato. This study aims to characterise the arginine kinase gene intron regions (AKInt) for further use as molecular markers to explore the genetic variation and differentiation of Bithynia snails in Thailand. Four intron regions belonging to the AK gene, namely AK intron 1 (AKInt1) to AK intron 4 (AKInt4) were characterised for B. funiculata, B. s. siamensis and B. s. goniomphalos. Of these, AKInt1 showed a high level of genetic variability and was the most appropriate region for reliably examining the genetic variation of Bithynia species/subspecies and discriminating between them. Two different forms of AKInt1, namely AKInt1-long containing a long fragment (∼341–351 bp) and a short fragment (∼213–229 bp), and AKInt1-short containing only a short fragment, were observed. Insertions/deletions (indels) were commonly found within AKInt1 leading to observed heterozygosity. This study revealed that nucleotide polymorphism was high in the long fragment, which was observed in some specimens. The short fragment was common in all samples and can potentially be used to discriminate between Bithynia taxa. [ABSTRACT FROM AUTHOR]

Background: The genus Onchocerca Diesing, 1841 includes species of medical importance, such as O. volvulus (Leuckart, 1893), which causes river blindness in the tropics. Recently, zoonotic onchocercosis has been reported in humans worldwide. In Japan, O. dewittei japonica Uni, Bain & Takaoka, 2001 from wild boars is a causative agent for this zoonosis. Many filarioid nematodes are infected with Wolbachia endosymbionts which exhibit various evolutionary relationships with their hosts. While investigating the filarial fauna of Borneo, we discovered an undescribed Onchocerca species in the bearded pig Sus barbatus Müller (Cetartiodactyla: Suidae). Methods: We isolated Onchocerca specimens from bearded pigs and examined their morphology. For comparative material, we collected fresh specimens of O. d. dewittei Bain, Ramachandran, Petter & Mak, 1977 from banded pigs (S. scrofa vittatus Boie) in Peninsular Malaysia. Partial sequences of three different genes (two mitochondrial genes, cox1 and 12S rRNA, and one nuclear ITS region) of these filarioids were analysed. By multi-locus sequence analyses based on six genes (16S rDNA, ftsZ, dnaA, coxA, fbpA and gatB) of Wolbachia, we determined the supergroups in the specimens from bearded pigs and those of O. d. dewittei. Results: Onchocerca borneensis Uni, Mat Udin & Takaoka n. sp. is described on the basis of morphological characteristics and its genetic divergence from congeners. Molecular characteristics of the new species revealed its close evolutionary relationship with O. d. dewittei. Calculated p-distance for the cox1 gene sequences between O. borneensis n. sp. and O. d. dewittei was 5.9%, while that between O. d. dewittei and O. d. japonica was 7.6%. No intraspecific genetic variation was found for the new species. Wolbachia strains identified in the new species and O. d. dewittei belonged to supergroup C and are closely related. Conclusions: Our molecular analyses of filarioids from Asian suids indicate that the new species is sister to O. d. dewittei. On the basis of its morphological and molecular characteristics, we propose to elevate O. d. japonica to species level as O. japonica Uni, Bain & Takaoka, 2001. Coevolutionary relationships exist between the Wolbachia strains and their filarial hosts in Borneo and Peninsular Malaysia. [ABSTRACT FROM AUTHOR]

Recent systematic studies of Opisthorchis viverrini based on multilocus enzyme electrophoresis (MEE) have shown that there are at least five genetic groups and possibly two cryptic species occurring in Thailand and Lao PDR each associated with a specific wetland system. A study based on mitochondrial DNA (mtDNA) gene analyses of an O. viverrini population from Savannakhet (SV, Lao PDR) clustered with several isolates from Thailand and Lao PDR although they originated from different river wetland systems. We used MEE to re-examine whether O. viverrini from SV was similar genetically to isolates from Thailand and Lao PDR. The allelic profiles of O. viverrini from SV and five different wetlands representing defined genetic groups of O. viverrini were recorded at 24 enzyme loci as opposed to only two loci of mtDNA. Contrary to previous studies, O. viverrini from SV was found to have fixed genetic differences at six to eight of the 24 loci examined (24.50-35.42%). Allelic data indicated that O. viverrini from SV differed from isolates in the Nam Ngum River wetland in Lao PDR (29.33% fixed genetic differences) and clustered with O. viverrini from Nakhon Phanom and Sakon Nakhon within the Songkram River wetland in Thailand but had fixed genetic differences from these at 24.5% of loci examined. Our data confirm the association between genetic groups of O. viverrini and specific wetland systems, and raise important questions regarding the significance of the genetic differences and relationships of O. viverrini from these wetlands. [ABSTRACT FROM AUTHOR]

• COI sequence was used to identify species and sub-species of Bithynia. • Bithynia siamensis siamensis and B. s. goniomphalos were found in central Thailand. • Bithynia s. siamensis, B. s. goniomphalos and B. funiculata were found in northern Thailand. • Genetic structure of B. s. goniomphalos was related to catchment systems. • Bithynia s. siamensis and B. s. goniomphalos should be classified as " B. siamensis sensu lato" The freshwater snails, Bithynia are the first intermediate hosts of the liver fluke, Opisthorchis viverrini , the causative agent of cholangiocarcinoma (CCA) in Southeast Asia. In Thailand, there are three traditionally recognized taxa of Bithynia: Bithynia funiculata; B. siamensis siamensis; B. s. goniomphalos. This study examines the geographical distribution and genetic structure of Bithynia species from five previously reported water catchments and six new catchments in Thailand. Of these, three new catchments Kok, Wang, and Nan are from the north and the remaining three new catchments are Phetchaburi, Prachuap Khiri Khan Coast, Mae Klong from the west of Thailand. We sampled 291 Bithynia snails from 52 localities in 11 catchment systems in the northern, western and central regions of Thailand. Mitochondrial cytochrome c oxidase subunit 1 (COI) and 16S ribosomal DNA (16S rDNA) sequences were used to examine genetic diversity of Bithynia snails which revealed 200 and 27 haplotypes of COI and 16S rDNA, respectively. However, as 16S rDNA is a conserved gene, it is not suitable to distinguish Bithynia at the species and sub-species levels in our study. The phylogenetic tree and haplotype network analyses included sequences of COI from GenBank. B. funiculata was found only in the north of Thailand and the genetic structure did not differ among populations. Genetic differentiation (Φ ST) analyses showed that B. s. goniomphalos contained three distinct lineages. Lineage I contained B. s. goniomphalos from the vast majority of catchment systems in Thailand and Lao PDR. Lineage II contained all B. s. goniomphalos from the Prachin Buri and Bang Pakong catchment systems in eastern and central Thailand, including samples from all catchment systems in Cambodia. While lineage III contained B. s. goniomphalos from the Songkram and Nam Kam catchment systems in Thailand and the Nam Ngum and Huai Som Pak catchment systems in Lao PDR. Furthermore, results showed that all samples of B. s. siamensis were classified into one lineage and placed phylogenetically between B. s. goniomphalos lineages I and II. Thus, the taxonomic status of B. s. goniomphalos and B. s. siamensis requires reassessment, and they should be reclassified as belonging to the species complex " Bithynia siamensis sensu lato". [Display omitted] [ABSTRACT FROM AUTHOR]