Tuanchai Srisawangwong

Epidemiology of Strongyloides stercoralis in north-east Thailand: application of the agar plate culture technique compared with the enzyme-linked immunosorbent assay.

Cross-sectional surveys of parasitic infection were performed using the agar plate culture technique (APCT) and modified formalin-ethyl acetate concentration technique (MFECT) to assess the true prevalence of Strongyloides stercoralis relative to other parasites in north-east Thailand. The enzyme-linked immunosorbent assay (ELISA) for diagnosis of S. stercoralis infection was used to estimate the seroprevalence for comparison with coproprevalence. Faecal and serum samples were collected from study participants during October-November 2000. Within the sample population of 332 rural northeast Thais from 3 communities, S. stercoralis was the most common parasitic infection (average 28.9%, range 27.7-30.3%) as determined by APCT; by MFECT the average was 5.4% (range 1.8-8.6%). Other intestinal parasites by order of prevalence were Opisthorchis viverrini (average 14.2%, range 8.6-19.4%), hookworm (average 12.3%, range 4-20.2%), Echinostoma sp. (7.5%), Giardia intestinalis (0.9%), Trichuris trichiura (0.6%), and Taenia sp., Hymenolepis nana and Entamoeba coli (all 0.3%). In an analysis of a subset of the sample population for which serum samples were available (n = 120), coproprevalence by APCT was 33.3% (range 27-53.8%) and seroprevalence was 47.5% (range 29.7-57.9%) by modified unit-based ELISA and 34.2% (range 21.6-42.1%) by conventional optical density (OD)-based ELISA. Taking APCT as the reference method for diagnosis of strongyloidiasis, the sensitivity and specificity of the OD-based ELISA were 65% and 81.3%, respectively, and of the unit-based ELISA were 77.5% and 71.3%, respectively. Our results indicate that S. stercoralis is the predominant parasite in rural north-east Thailand, and that APCT and ELISA should be used as complementary diagnostic methods for community-based parasite surveys, at least among those in high-risk groups.

Genetic variation in Opisthorchis viverrini (Trematoda: Opisthorchiidae) from northeast Thailand and Laos PDR based on random amplified polymorphic DNA analyses.

Genetic variation in Opisthorchis viverrini adults originating from different locations in northeast Thailand and Laos, People’s Democratic Republic (PDR), was examined using random amplified polymorphic DNA (RAPD) analyses. In an initial analysis, the genomic DNA of one fluke from each of ten localities was amplified using 15 random primers (10-mers); however, genetic variation among O. viverrini specimens was detected reliably for only four primers. A more detailed RAPD analysis using these four primers was conducted on ten individuals from nine localities. Considerable genetic variation was detected among O. viverrini from different geographical areas and among some individuals from the same collecting locality. Comparison of the RAPD profiles revealed that O. viverrini adults from Laos PDR were genetically distinct from those from northeast Thailand. The taxonomic significance of this finding needs to be explored in more detail. The RAPD markers established in the present study provide opportunities to examine the biology and epidemiology of O. viverrini and fish-borne trematodes within the region. Additionally, application of these markers in such studies could have important implications in relation to the prevalence of cholangiocarcinoma in different regions of Asia.

Light and electron microscopy observations of embryogenesis and egg development in the human liver fluke, Opisthorchis viverrini (Platyhelminthes, Digenea).

Eggs of most species digenean flukes hatch in the external environment to liberate larvae that seek and penetrate a snail intermediate host. Those of the human liver flukes, Opisthorchis viverrini, hatch within the gastrointestinal canal of their snail hosts. While adult parasites are primarily responsible for the pathology in cases of human opisthorchiasis, their eggs also contribute by inducing granulomata and in serving as nidi for gallstone formation. In view of the peculiar biology of O. viverrini eggs and their contribution to pathology, we investigated embryogenesis in this species by light and transmission electron microscopy. Egg development was traced from earliest stages of coalescence in the ootype until full embryonation in the distal region of the uterus. Fully mature eggs were generally impermeable to resin and could not be examined by conventional electron microscopy methods. However, the use of high-pressure freezing and freeze-substitution fixation of previously fixed eggs enabled the internal structure of mature eggs, particularly the subshell envelopes, to be elucidated. Fertilization occurs in the ootype, and the large zygote is seen therein with a single spermatozoon wrapped around its plasma membrane. As the zygote begins to divide, the spent vitellocytes are pushed to the periphery of the eggs, where they progressively degrade. The early eggshell is formed in the ootype by coalescing eggshell precursor material released by approximately six vitelline cells. The early eggs have a thinner eggshell and are larger than, but lack the characteristic shape of, mature eggs. Characteristic shell ornamentation, the “muskmelon” appearance of eggs, appears after eggshell polymerization in the ootype. Pores are not present in the shell of O. viverrini eggs. The inner and outer envelopes are poorly formed in this species, with the outer envelope evident beneath the eggshell at the opercular pole of the mature egg. The miracidium has a conical anterior end that lacks the distinctive lamellar appearance of the terebratorium of other digeneans, such as the schistosomes. The miracidium is richly glandular, containing an apical gland in the anterior end, large cephalic gland, and posterior secretory glands. Each gland contains a secretory product with different structure. The paucity of vitelline cells associating with eggs, the reduced size of eggs, and reduced complexity of the extraembryonic envelopes are interpreted as adaptations to the peculiar hatching biology of the miracidia.

Turmeric reduces inflammatory cells in hamster opisthorchiasis

The curcumin compound from turmeric is effective in the treatment of many inflammatory diseases. The aim of our present study was to evaluate the efficacy of turmeric on reducing the histopathological changes of hamster opisthorchiasis. Hamsters were infected with Opisthorchis viverrini and then administered turmeric. Using light microscopic observation, liver function tests for alanine transaminase (ALT), alkaline phosphatase, and direct bilirubin were investigated. The resulting histopathological changes show that turmeric has anti-inflammatory properties—during both N-nitrosodimethylamine administration and O. viverrini infection—by reducing the aggregation of inflammatory cells surrounding the hepatic bile ducts, which correlates with a decreased serum ALT level. The decrease in direct bilirubin levels in the hamsters treated with turmeric suggests that turmeric may enhance biliary contraction. The present study found that turmeric clearly reduces the inflammatory cells in hamster opisthorchiasis at an early stage. This finding may be connected with a reduction in the risk factors of cholangiocarcinoma development.

Involvement of c-Ski Oncoprotein in Carcinogenesis of Cholangiocacinoma Induced by Opisthorchis viverrini and N-nitrosodimethylamine

Opisthorchiasis is the major public health problem in the endemic areas of Thailand and Laos because Opisthorchis viverrini infection causes serious hepatobiliary diseases including CCA. The molecular mechanism of the CCA carcinogenesis induced by the infection remains obscure. To reveal the potential genes and signaling pathways to involve in the carcinogenesis, the present study investigated the expression of c-Ski, an oncogene, and two TGF-β signaling pathway relative genes, TGF-β and Smad4, during the development of CCA induced by O. viverrini infection in hamster model, and in human opisthorchiasis associated CCA. The results showed that the expression of c-Ski gene was greatly up-regulated during the carcinogenesis of CCA in hamster model. The overexpression of c-Ski was confirmed by immunohistological staining result which showed the increased expression of c-Ski protein in cytoplasm of the epithelial lining of hepatic bile ducts. Moreover, the immunohistological staining of the specimens of human opisthorchiasis associated CCA revealed the up-regulated expression of c-Ski and Smad4 proteins in the cytoplasm of the epithelial lining of hepatic bile ducts and stomal fibrosis respectively. The expression of TGF-β and Smad4 were up-regulated, which expression kinetics was time-dependent of CCA development. These results suggest that c-Ski is likely involved in the carcinogenesis of CCA induced by O. viverrini infection through regulating TGF-β signaling pathway.

Centrocestus formosanus: surface morphology of metacercaria, adult and egg

The surface morphology of metacercariae isolated from Puntius spp., adult worms from infected hamsters and eggs of Centrocestus formosanus (Digenea: Heterophyidae) were studied using scanning electron microscopy. It was found that the surfaces of the metacercariae and adult worms were closely similar in appearance. The oral sucker was surrounded by a circumoral expansion with two rows of 32 spines (16 spines each). The ventral sucker, with six large nonciliated papillae on the lip, and the genital opening were located mid-ventrally. The excretory pore was terminal. The body surface was covered with pectinate scale-like spines of varying sizes. The scales in the middle area were larger than those in the anterior and posterior parts of the body. Sensory papillae, mostly uniciliated, were present in greater abundance at the anterior region of both stages. However, morphological variations were observed among the adults depending on the age of the worms. At 4 weeks, the oral sucker and circumoral expansion were cobblestone-like in structure and the grooves of spines split compared with their smooth surfaces at 10 days. In addition, the ventral sucker became depressed and its large papillae on the lip disappeared at 10 weeks post infection. Multiciliated papillae were also present in 10-week old worms. Each egg of this parasite possessed a prominent operculum and a latticed design on the egg shell.

Concomitant and protective immunity in mice exposed to repeated infections with Echinostoma malayanum

Concomitant immunity and its consequence against infection play roles in regulating worm burdens in helminthiasis. Under natural conditions, this immunity is generated by exposure to repeated low dose or trickle infection. In this study, concomitant immunity was induced in mice exposed repeatedly to infection with Echinostoma malayanum and its protective effect on a challenge infection evaluated. A profile of worm burden from exposure to 10 metacercariae/mouse/week rose rapidly during the first 2 weeks reaching a plateau from week 3 to 8 post infection. Based on a cumulative dose of infection, worm recoveries were around 75% in the first 2 weeks, dropped to 50% at week 3 and 19% at week 8. After week 2, adult worm burden was constant and no juvenile worms were found after week 3 of the experiment. To examine the effect of resistance against reinfection, mice in the experimental group were primarily infected with 10 metacercariae/week for 5 weeks, treated with praziquantel and were challenged with 75 metacercariae/animal. The number of worms recovered from the experimental groups was significantly lower than that from naïve control groups beginning from 24 h to 28 days post challenge. The worms in the experimental group showed growth retardation and the proportion of adult worms was lower than that in the control animals especially during the first 3 weeks of the experiment. Parasite fecundity was also suppressed compared with that in the control group. The selective effects of protective immunity on establishment, growth, and fecundity of challenged worms affected the population dynamics of E. malayanum which is a similar phenomenon to concomitant immunity in schistosomiasis.

Egg-Hatching Mechanism of Human Liver Fluke, Opisthorchis viverrini: A Role For Leucine Aminopeptidases From the Snail Host, Bithynia siamensis goniomphalos

Abstract The human liver fluke Opisthorchis viverrini (Platyhelminthes, Trematoda, Digenea) uses snails of the genus Bithynia as first intermediate host. Peculiarly among trematodes, the eggs of O. viverrini hatch within the digestive tract of its snail host. It remains uncertain whether hatching in this species is mediated through mechanical fracture of the eggshell or by digestion with specific digestive enzymes. This study aimed to characterize enzymes with specific inhibitors and factors involved in the hatching activity of O. viverrini eggs. For measuring egg hatching in vivo, 50 O. viverrini mature eggs were fed to individual Bithynia siamensis goniomphalos snails at various temperature conditions for 24 hr. Ex vivo, mature eggs were incubated with crude snail extract and commercial leucine aminopeptidase (LAP). Egg-hatching of O. viverrini was temperature dependent, with optimal hatching occurring at 24–28 C, with a peak of hatching of 93.54% in vivo and 30.55% ex vivo occurring at these temperatures. Ex vivo hatching rates increased to 45.87% under anaerobic conditions at 28 C. Some 22.70% and 16.21% of heat-killed eggs also hatched within the snail digestive tract and snail extract, respectively, indicating that host molecules are involved in the hatching response. Most eggs hatch in the anterior regions of the digestive tract. Hatching was completely inhibited in the presence of bestatin, an inhibitor of LAP, but not in the presence of phosphatase inhibitors. Bestatin inhibition of hatching was reversible. Finally, egg hatching could be induced by addition of a porcine LAP. The results indicate that this digenean utilizes both LAP of the snail host and movement of miracidia for hatching.