Evidence of a species complex within the food-borne trematode Opisthorchis viverrini and possible co-evolution with their first intermediate hosts

The food-borne trematodes, Opisthorchis viverrini, O. felineus and Clonorchis sinensis, have long been recognized as the cause of major human health problems, with an estimated 40 million infected persons. Of the three species of liver fluke, only O. viverrini is classified as a type 1 carcinogen because of its role as an initiator of chronic inflammation and the subsequent development of cholangiocarcinoma. At present, there are no techniques for the early diagnosis of cholangiocarcinoma and it is fatal for most patients. There is considerable variation in parasite prevalence and disease presentation in different geographical areas, the latter of which may be associated with genetic differences among parasites. In the present study, multilocus enzyme electrophoresis was used to provide a comprehensive genetic characterization of O. viverrini from different geographical localities in Thailand and the Peoples' Democratic Republic of Laos. Parasites from different localities were compared genetically at 32 enzyme loci. The results of the genetic analyses are sufficient to reject the null hypothesis that O. viverrini represents a single species. Therefore, O. viverrini consists of at least two genetically distinct, yet morphologically similar (i.e. cryptic) species. Moreover, there was also separation of the different populations of snails (i.e. the first intermediate hosts) into two distinct genetic groups that corresponded with the delineation of O. viverrini into two species. This suggests that there may be a history of co-evolution in this host-parasite lineage. Additionally, five distinct genetic groups of parasites were detected, each of which occurred within a different and independent river wetland system. Our findings have major implications for the implementation of effective control and surveillance programs targeted to these medically important food-borne parasites.     

A novel carbohydrate antigen expression during development of Opisthorchis viverrini- associated cholangiocarcinoma in golden hamster: a potential marker for early diagnosis

Poor prognosis of cholangiocarcinoma (CCA) is primarily due to delayed diagnosis because of the lack of appropriate tumor marker(s) to detect cancer development at an early stage. We have recently established a S121 monoclonal antibody (mAb) which recognizes an unidentified glycan epitope on MUC5AC, designated as CCA-associated carbohydrate antigen (CCA-CA). This antigen is expressed in human CCA cells but not in normal biliary epithelia. Detection of CCA-CA effectively distinguished CCA patients' sera from normal control sera with high specificity and sensitivity. In the present study, we examined a time profile of the expression of CCA-CA by immunohistochemical methods in the liver tissues of Opisthorchis viverrini (Ov)-associated CCA in a hamster model. Hamsters were divided into four groups; non-treated, Ov infected, NDMA (N-nitrosodimethamine) treated and Ov + NDMA treated groups, and animals from each group were euthanized at 1, 3 and 6 months post-treatment. CCA-CA was not detected in normal biliary cells of non-treated hamsters throughout the course of experiment. CCA-CA became detectable in the cytoplasm and apical surface of biliary cells of the NDMA and Ov + NDMA groups at early stage (1 month) of tumor development and increased with tumor progression. In contrast, CCA-CA was detected as nuclear staining at the 1 month post Ov infection and declined thereafter. These results suggest the possibility of CCA-CA as an early marker for CCA.     

Protospirura siamensis n. sp. (Nematoda: Spiruridae) from rodents in Thailand

A large number (828) of rodents belonging to nine genera (Bandicota, Berylmys, Chiropodomys, Hapalomys, Leopoldamys, Maxomys, Mus, Niviventer and Rattus) were trapped in four Thai provinces between 2008 and 2010. A new species of Protospirura (Nematoda: Spiruridae) was identified and described. Protospirura siamensis n. sp. was found in 10 rodent species from the four Thai provinces surveyed. The new species can be distinguished from known species of the genus by the arrangement of the papillae on the male and the morphology and length of the spicules. This is the first species of Protospirura to be described from South-East Asia.     

Pallisentis rexus from the Chiang Mai Basin, Thailand: ultrastructural studies on egg envelope development and the mechanism of egg expansion

Pallisentis rexus Wongkham & Whitfield, 1999 (Eoacanthocephala: Quadrigyridae) infects the freshwater snakehead fish, Channa striata, in the Chiang Mai Basin, Thailand. All stages of egg development within the body cavity of the female parasite were observed, using transmission electron microscopy. Changes in mature eggs after contact with water were also investigated. The mature egg has five egg envelopes separated from each other by four gaps. The fertilization membrane, which is formed first, is pushed centrifugally by other, subsequently formed, envelopes and gaps, which produces a final total shell thickness of 8-36 microm around the acanthor. The disappearance of the outermost layer and the unpleating of an adjacent inner layer causes the expansion of eggs on contact with water. The volume of an expanded egg is approximately 27 times that of an unexpanded one, but the density of eggs is reduced from a value greater than water to one almost equal to water. This is believed to aid the dispersion of eggs.     

Molecular genetic diversity of <Emphasis Type="Italic">Gongylonema neoplasticum</Emphasis> (Fibiger &amp; Ditlevsen, 1914) (Spirurida: Gongylonematidae) from rodents in Southeast Asia

More than a dozen Gongylonema spp. (Spirurida: Spiruroidea: Gongylonematidae) have been described from a variety of rodent hosts worldwide. Gongylonema neoplasticum (Fibiger & Ditlevsen, 1914), which dwells in the gastric mucosa of rats such as Rattus norvegicus (Berkenhout) and Rattus rattus (Linnaeus), is currently regarded as a cosmopolitan nematode in accordance with global dispersion of its definitive hosts beyond Asia. To facilitate the reliable specific differentiation of local rodent Gongylonema spp. from the cosmopolitan congener, the genetic characterisation of G. neoplasticum from Asian Rattus spp. in the original endemic area should be considered since the morphological identification of Gongylonema spp. is often difficult due to variations of critical phenotypical characters, e.g. spicule lengths and numbers of caudal papillae. In the present study, morphologically identified G. neoplasticum from 114 rats of seven species from Southeast Asia were selected from archived survey materials from almost 4,500 rodents: Thailand (58 rats), Cambodia (52 rats), Laos (three rats) and Philippines (one rat). In addition, several specimens from four rats in Indonesia were used in the study. Nucleotide sequences of the ribosomal RNA gene (rDNA) (5,649 bp) and the cytochrome c oxidase subunit 1 gene (cox1) (818 bp) were characterised. The rDNA showed little nucleotide variation, including the internal transcribed spacer (ITS) regions. The cox1 showed 24 haplotypes, with up to 15 (1.83%) nucleotide substitutions regardless of parasite origin. Considering that Rattus spp. have been shown to originate from the southern region of Asia and G. neoplasticum is their endogenous parasite, it is reasonable to propose that the present study covers a wide spectrum of the genetic diversity of G. neoplasticum, useful for both the molecular genetic speculation of the species and the molecular genetic differentiation of other local rodent Gongylonema spp. from the cosmopolitan congener.     

Improving the attachment and proliferation of umbilical cord mesenchymal stem cells on modified polystyrene by nitrogen-containing plasma

Wharton’s jelly mesenchymal stem cells (WJMSCs) are important alternative source of pluripotent cells for several therapeutic purposes. Understanding of adhesion properties of such cells is necessary to regulate the attachment, growth and proliferation on targeted culture surfaces. BCP-K1, a line of WJMSCs, and polystyrene (PS) culture dishes were used as membrane samples. A 13.56 MHz inductively coupled discharge plasma reactor with a mixture of N-containing gas and noble gas was used. This was expected to introduce the more hydrophilic groups on PS surface and enhance the cell adhesion. The plasma-treated PS dishes with the mixed gas of N₂ + He at 50 W and NH₃ + He at 100 W were reactive towards BCP-K1. Cellular adhesion and proliferation was significantly twice as efficient on the treated surfaces than on PS dishes. BCP-K1 also secreted more focal adhesion kinase to adhere and proliferate when cultured on N₂-treated PS dishes than on the NH₃-treated PS dishes. Stable stemness markers were detected, including CD105, CD9 and SSEA-4, expressed on BCP-K1 growing on the modified PS dish surfaces, during 7 days of culturing. The presence of –NH₂ groups on the PS dish surface were revealed by X-ray photoelectron spectroscopy and Fourier transform infrared spectroscopy. A large amount of oxygen- and nitrogen-containing functional groups, up to 9.0 %, were introduced by NH₃ plasma and N₂ plasma. The functional groups introduced on to the PS surfaces were clearly the key factors which enhanced WJMSCs attachment and stemness stability.     

Effects of albendazole on Gnathostoma spinigerum in mice.

Mice were infected with 5 advanced third-stage larvae of Gnathostoma spinigerum and, beginning on the 28th day postinfection, were treated orally with albendazole. In the first experiment, infected mice each received albendazole once a day (30, 60, or 90 mg/kg/day) for 21 consecutive days. In the second experiment, they received albendazole twice a day (30 and 30, 60 and 60, or 90 and 90 mg/kg/day) for the same length of time. All mice were killed 28 days after cessation of treatment and the carcasses were examined for parasites. With both regimens, the administration of albendazole significantly reduced the number of larvae. However, a complete larvicidal effect was obtained only with albendazole at the dosage of 90 mg/kg twice daily.     

Hybrid liposomes inhibit the growth of Cholangiocarcinoma by induction of cell cycle arrest in G1 phase

Specific accumulation and cell cycle arrest were observed in human cholangiocarcinoma cells by hybrid liposomes composed of 90 mol % l-α-dimyristoylphosphatidylcholine (DMPC) and 10 mol % polyoxyethylene(21)dodecyl ether (C₁₂(EO)₂₁) without affecting normal cholangiocytes.     

Field evaluation of the gut microbiome composition of pre-school and school-aged children in Tha Song Yang,Thailand, following oral MDA for STH infections

Soil-transmitted helminths, such as roundworms (Ascaris lumbricoides), whipworms (Trichuris trichiura) and hookworms (Necator americanus and Ancylostoma spp.), are gastrointestinal parasites that occur predominantly in low- to middle-income countries worldwide and disproportionally impact children. Depending on the STH species, health status of the host and infection intensity, direct impacts of these parasites include malnutrition, anaemia, diarrhoea and physical and cognitive stunting. The indirect consequences of these infections are less well understood. Specifically, gastrointestinal infections may exert acute or chronic impacts on the natural gut microfauna, leading to increased risk of post-infectious gastrointestinal disorders, and reduced gut and overall health through immunomodulating mechanisms. To date a small number of preliminary studies have assessed the impact of helminths on the gut microbiome, but these studies are conflicting. Here, we assessed STH burden in 273 pre-school and school-aged children in Tha Song Yang district, Tak province, Thailand receiving annual oral mebendazole treatment. Ascaris lumbricoides (107/273) and Trichuris trichiura (100/273) were the most prevalent species and often occurred as co-infections (66/273). Ancylostoma ceylanicum was detected in a small number of children as well (n = 3). All of these infections were of low intensity (<4,999 or 999 eggs per gram for Ascaris and Trichuris respectively). Using this information, we characterised the baseline gut microbiome profile and investigated acute STH-induced alterations, comparing infected with uninfected children at the time of sampling. We found no difference between these groups in bacterial alpha-diversity, but did observe differences in beta-diversity and specific differentially abundant OTUs, including increased Akkermansia muciniphila and Bacteroides coprophilus, and reduced Bifidobacterium adolescentis, each of which have been previously implicated in STH-associated changes in the gut microfauna.