MALACOLOGICAL INVESTIGATION OF THE FULLY OPERATIONAL NAM THEUN 2 HYDROELECTRIC DAM PROJECT IN KHAMMOUANE PROVINCE, CENTRAL LAO PDR.

We conducted a malacological investigation in four districts of the Nam Theun 2 (NT2) hydroelectric dam project area, Khammouane Province, central Lao PDR (Nakai, Gnommalath, Mahaxai and Xe Bang Fai), after the first and second years of full operation in March 2010 and November 2011 to determine health risks for humans. A total 10,863 snail specimens (10 families/23 species) from 57 sampling stations and 12,902 snail specimens (eight families/21 species) from 66 sampling stations were collected in 2010 and 2011, respectively. Neotricula aperta (gamma race), the intermediate host for Schistosoma mekongi, was found in large numbers (5,853 specimens) in 2010 in Nam Gnom (downstream) at Station 25 (Mueang Gnommalath: Gnommalath District) and in fewer numbers (170 specimens) at Station 26 (Ban Thathod: Gnommalath District). In 2011, significantly fewer numbers (434 specimens) of N. aperta were found at Station 25. No snails were found to be infected with S. mekongi; however, 3.6% and 0.45% of Bithynia (D.). s. goniomphalos specimens collected were found to be infected with Opisthorchis viverrini (human liver fluke) during 2010 and 2011, respectively. Pomacea canaliculata, the rice crop pest, the intermediate host of Angiostrongylus (Parastrongylus) cantonensis, was found in the greatest numbers during 2010 and 2011; the prevalence increased significantly from 1.3% in 2010 to 53.3% in 2011. We also found seasonal variation in snail populations in terms of abundance and diversity. The snail fauna and risk for transmission of parasitic diseases need to be monitored continuously to evaluate the long-term impact of the dam project.

Pyrosequencing for rapid molecular identification of Schistosoma japonicum and S. mekongi eggs and cercariae.

Schistosomiasis, which is caused by Schistosoma japonicum and S. mekongi, is a chronic and dangerous widespread disease affecting several countries in Asia. Differentiation between S. japonicum and S. mekongi eggs and/or cercariae via microscopic examination is difficult due to morphological similarities. It is important to identify these etiological agents isolated from animals and humans at the species or genotype level. In this study, a pyrosequencing assay designed to detect S. japonicum and S. mekongi DNA in fecal samples and infected snails was developed and evaluated as an alternative tool to diagnose schistosomiasis. New primers targeting the 18S ribosomal RNA gene were designated for specific amplification. S. japonicum and S. mekongi were identified using a 43-nucleotide pattern of the 18S ribosomal RNA gene and were differentiated using 7 nucleotides within this region. S. japonicum and S. mekongi-infected snails and fecal samples derived from infected mice and rats were differentially detected within a short period of time. The analytical sensitivity of the method enabled the identification of as little as a single cercaria artificially introduced into a pool of 10 non-infected snails and 2 eggs inoculated in 100mg of non-infected fecal sample. To evaluate the comparative efficacy of the assay, identical samples were also analyzed via microscopy and Sanger sequencing. The pyrosequencing technique was found to be superior to the microscopy method and more rapid than the Sanger sequencing method. These results suggest that the pyrosequencing assay is rapid, simple, sensitive and accurate in identifying S. japonicum and S. mekongi in intermediate hosts and fecal samples of the final host.

Molecular differentiation of Schistosoma japonicum and Schistosoma mekongi by real-time PCR with high resolution melting analysis.

Human schistosomiasis caused by Schistosoma japonicum and Schistosoma mekongi is a chronic and debilitating helminthic disease still prevalent in several countries of Asia. Due to morphological similarities of cercariae and eggs of these 2 species, microscopic differentiation is difficult. High resolution melting (HRM) real-time PCR is developed as an alternative tool for the detection and differentiation of these 2 species. A primer pair was designed for targeting the 18S ribosomal RNA gene to generate PCR products of 156 base pairs for both species. The melting points of S. japonicum and S. mekongi PCR products were 84.5±0.07℃ and 85.7±0.07℃, respectively. The method permits amplification from a single cercaria or an egg. The HRM real-time PCR is a rapid and simple tool for differentiation of S. japonicum and S. mekongi in the intermediate and final hosts.

Genome size estimation of liver fluke Opisthorchis viverrini by real-time polymerase chain reaction based method.

The human liver fluke, Opisthorchis viverrini, has been categorized as a class one carcinogenic organism according to its strong association with cholangiocarcinoma, bile duct cancer which has high incidence in the northeast of Thailand. The lack of genome database of this parasite limited the studies aimed to understand the basic molecular biology of this carcinogenic liver fluke. The determination of the genome size is an initial step prior to the full genome sequencing. In this study, we applied an absolute quantitative real-time polymerase chain reaction for this aspect. Our results indicated the genome size of O. viverrini is 75.95 Mb or C value 0.083. The information of O. viverrini genome size is useful for estimation of sequence coverage and the cost of the parasite's whole genome sequencing using next-generation sequencing technologies.

Molecular detection of Schistosoma japonicum in infected snails and mouse faeces using a real-time PCR assay with FRET hybridisation probes.

A real-time polymerase chain reaction (PCR) assay with fluorescence resonance energy transfer (FRET) hybridisation probes combined with melting curve analysis was developed to detect Schistosoma japonicum in experimentally infected snails and in faecal samples of infected mice. This procedure is based on melting curve analysis of a hybrid between an amplicon from the S. japonicum internal transcribed spacer region 2 sequence, which is a 192-bp S. japonicum-specific sequence, and fluorophore-labelled specific probes. Real-time FRET PCR could detect as little as a single cercaria artificially introduced into a pool of 10 non-infected snails and a single egg inoculated in 100 mg of non-infected mouse faeces. All S. japonicum-infected snails and all faecal samples from infected mice were positive. Non-infected snails, non-infected mouse faeces and genomic DNA from other parasites were negative. This assay is rapid and has potential for epidemiological S. japonicum surveys in snails, intermediate hosts and faecal samples of final hosts.

Molecular detection of Schistosoma japonicum in infected snails and mouse faeces using a real-time PCR assay with FRET hybridisation probes

A real-time polymerase chain reaction (PCR) assay with fluorescence resonance energy transfer (FRET) hybridisation probes combined with melting curve analysis was developed to detect Schistosoma japonicum in experimentally infected snails and in faecal samples of infected mice. This procedure is based on melting curve analysis of a hybrid between an amplicon from the S. japonicum internal transcribed spacer region 2 sequence, which is a 192-bp S. japonicum-specific sequence, and fluorophore-labelled specific probes. Real-time FRET PCR could detect as little as a single cercaria artificially introduced into a pool of 10 non-infected snails and a single egg inoculated in 100 mg of non-infected mouse faeces. All S. japonicum-infected snails and all faecal samples from infected mice were positive. Non-infected snails, non-infected mouse faeces and genomic DNA from other parasites were negative. This assay is rapid and has potential for epidemiological S. japonicum surveys in snails, intermediate hosts and faecal samples of final hosts.

Molecular evidence of Opisthorchis viverrini in infected bithyniid snails in the Lao People's Democratic Republic by specific hybridization probe-based real-time fluorescence resonance energy transfer PCR method.

Naturally occurring bithyniid snails, Bithynia siamensis goniomphalos (Prosobranchia: Bithyniidae), and their intermediate hosts were sampled from Khammouane Province, the Lao People's Democratic Republic, and the prevalence of the carcinogenic human liver fluke, Opisthorchis viverrini, was examined. The presence of O. viverrini cercariae in snails was examined by cercarial shedding test and then confirmed by specific hybridization probe-based real-time fluorescence resonance energy transfer (FRET) PCR method. The real-time FRET PCR method is based on a fluorescence melting curve analysis of a hybrid between an amplicon produced from the pOV-A6 specific sequence (Genbank accession no. S80278), a 162-bp repeated sequence specific to O. viverrini, and specific fluorophore-labeled probes. Mean melting temperature of O. viverrini DNA from the cercariae and each of two positive snails by shedding test was 66.3 ± 0.1. The O. viverrini infection rate in snails was 2.47% (2/81) by cercarial shedding test but was 8.52% (4/47) by real-time FRET PCR method. The real-time FRET PCR method is rapid and effective in examining a large number of snail samples simultaneously. Validation using molecular evidence from this procedure provides another tool for surveying the prevalence of O. viverrini-infected snails in Southeast Asian countries.

Snails and trematode infection after Indian Ocean tsunami in Phang-Nga Province, southern Thailand.

The tsunami and non-tsunami affected areas of Takua Pa District, Phang-Nga Province were investigated for fresh- and brackish-water snails that transmit human parasitic diseases during 2006 and 2007. Among 46 snail species found, 17 species of 8 families were freshwater snails, 28 species of another 7 families were brackish-water snails, and 1 species was a land snail. Of these species, 11 freshwater snails, 4 brackish-water snails and 1 land snail were of medical importance. The fresh-water snails were Pomacea canaliculata, Pila angelica, P. gracilis, P. polita, Filopaludina (S.) martensi, F. (F.) s. polygramma, Melanoides tuberculata, Indoplanorbis exuxtus, Radix rubiginosa, Helicorbis umbilicalis, Gyraulus convexiusculus. Four brackish-water snails were Cerithidea cingulata, C. djadjarensis, C. alata, Sermyla riqueti and Achatina fulica was the land snail. I. exutus, M. tuberculata and F. (F.) s. polygramma harbored Xiphidio, Microcercus, Furocercus, Echinostome cercariae, and cercaria without eyespots or tail with hair. Three species of brackish-water snails, Cerithidia cingulata, C. djadjariensis, and C. alata presented with 6 types of trematode cercariae and rediae. Knowledge of medically important snails and their parasitic diseases, and prevention were given to Takua Pa people by poster, pamphlets and broadcasting through community radio.

Intertidal snail-trematode communities on the southern Thailand before and after the South Asia tsunami.

Intertidal snail-trematode communities in southern Thailand were examined before and after the South Asia tsunami. Infection rates and species diversity of cercaria in the host snail Cerithidea in tidal zones did not change significantly from one year before to one month after the tsunami. However, the host snails C. quadrata, C. alata and C. obtusa disappeared from greatly damaged sites. It is important to follow up on the intertidal snail-trematode community recovery process after destruction of the intertidal ecosystem.

Freshwater mollusks at designated areas in eleven provinces of Thailand according to the water resource development projects.

The study was conducted at 75 collecting loci in 15 districts of 11 provinces in Thailand during 1999-2004. A total of 12,079 live mollusks were collected, 11,874 were snails and 205 were clams. The snails were comprised of 39 species and classified into 9 families: Ampullariidae, Bithyniidae, Buccinidae, Potamiopsidae, Stenothyridae, Thiaridae, Viviparidae, Planorbidae and Lymnaeidae. The clams were comprised of 14 species classified into 2 families: Amblemidae and Corbiculidae. Fifteen species were medically important snails: Pomacea canaliculata, Pila ampullacea, P. pesmei, P. polita, Bithynia (Digoniostoma) funiculata, B. (D.) siamensis goniomphalos, B. (D.) s. siamensis, Filopaludina (Siamopaludina) martensi martensi, F. (Filopaludina) sumatrensis polygramma, Melanoides tuberculata, Tarebia granifera, Helicorbis umbilicalis, Gyraulus convexiusculus, Indoplanorbis exustus and Radix rubiginosa. Of these 3 snail species harbored trematode cercariae. I. exustus harbored Echinostoma malayanum, Xiphidio and Schistosoma spindale, and R. rubiginosa and B. (D.) siamensis goniomphalos harbored Xiphidio and intestinal flukes, respectively.

Malacological survey in Phang-Nga Province, southern Thailand, pre- and post-Indian Ocean tsunami.

Three malacological surveys were conducted in the Takua Pa District of Phang-Nga Province, southern Thailand, before and after the Indian Ocean Tsunami disaster. Twenty-nine species of fresh- and brackish-water snails were found, in which 10 species of freshwater snails were present, including live Pila polita; 8 species were of medical importance. Two brackish-water snails, Nerita articulata and Littorinopsis scabra, were absent after the tsunami disaster, while brackish-water Cerithidea cingulata and C. djadjariensis harbored 9 types of trematode cercariae.

Freshwater mollusks of medical importance in Kalasin Province, northeast Thailand.

A snail survey was performed in six districts around irrigation areas of Lampao Dam, in Kalasin Province. The survey caught a total of 5,479 live snails and classed them into five families, 12 genera and 15 species, of which 7 species are suspected of transmitting human parasitic diseases. The seven species were Pila polita, Pomacea canaliculata, Filopaludina (S.) m. martensi, Bithynia (Digoniostoma) siamensis goniomphalos, Melanoides tuberculata, Radix rubiginosa, and Indoplanorbis exustus. Of these, B. (D.) s. goniomphalos and I. exustus were found to harbor emergent cercariae. Only B. (D.) s. goniomphalos hosted several types of cercariae--Opisthorchis viverrini, unidentified species of intestinal flukes, echinostomes, xyphidio and furcocercous cercariae. Indoplanorbis exustus shed only echinostome cercariae. B. (D.) s. goniomphalos showed a rather high natural infection rate with O. viverrini, 1.3% in Yang Talat district, and 0.61% in Kamalasai district, in Kalasin Province.

Brackish-water mollusks of Surat Thani Province, southern Thailand.

Brackish-water mollusks inhabiting the mangrove areas along the Gulf of Thailand of Surat Thani Province were investigated for distribution, abundance and natural infections. Nine families and 32 species of brackish-water snails were recovered from 14 sampling stations. Species belonging to the genus Ceritidea of the family Potamididae were mainly examined and Cerithidea (Cerithideopsilla) Cingulata, C. (C.) djadjariensis, and C. (Cerithidea) charbonnieri were naturally infected with 2 types of trematode cercariae, and one which was undetermined. C. (C.) cingulata had the highest infection rate (38.5%). Viewing two snail communities, the first community on the mainland and the second on Samui Island in Surat Thani Province, 28 brackish-water mollusk species were present on the mainland, 15 species were evident on Samui Island, and 11 snail species were common to both the mainland and Samui Island. Measurement of community similarity based on species presence revealed an index of similarity of 0.51. Concerning land use by the local people in the station areas investigated, brackish-water snails in Surat Thani Province are facing habitat degradation by human use.

Emergence of Opisthorchis viverrini cercariae from naturally infected Bithynia (Digoniostoma) siamensis goniomphalos.

Under natural conditions, the emergence of Opisthorchis viverrini cercariae from naturally infected Bithynia (Digoniostoma) siamensis goniomphalos showed diurnal periodicity, peaking between 8:00-10:00 AM. The cercariae did not emerge during darkness, but low-intensity light could induce a release. Cercariae shedded from each field infected B.(D.) s. goniomphalos was recorded daily. The maximum output from one snail was 1,728 cercariae in a day. The total cercarial output from all five infected snails was 56,555 and the maximum of total cercariae shed from one snail was 27,692. The field-infected B. (D.) s. goniomphalos could survive for 70 days after the snails were collected.

The health belief model and factors relating to potential use of a vaccine for shigellosis in Kaeng Koi district, Saraburi province, Thailand.

Shigellosis is an important cause of morbidity and mortality throughout the world. Approximately, 1.1 million deaths occur a year due to this disease, making it the fourth leading cause of mortality worldwide. This paper explores local interest in and potential use of a vaccine for shigellosis in Thailand where Shigella poses an important public-health concern. Data for this study were collected during June-November 2002 from 522 subjects surveyed using a sociobehavioural questionnaire in Kaeng Koi district in central Thailand. The community demand and likely use of a vaccine were examined in relation to the Health Belief Model, which provides analytical constructs for investigating the multiple issues of local readiness to accept and access a new vaccine. As the key outcome variable, most respondents showed interest in receiving a vaccine against dysentery which they thought would provide useful protection against the disease. However, there was only a moderate number who perceived dysentery as serious and themselves as susceptible to it, although it was perceived to cause some burden to and additional expense for families. Most people identified a number of groups who were thought to be especially vulnerable to dysentery, such as the elderly, pre-school, and school-age children, and poor labourers. Other outcomes of the study included the identification of acceptable and convenient sites for its delivery, such as government health clinics and private clinics, and respected sources for information about the vaccine, such as health clinic personnel and community health volunteers. This information suggests that components of the Health Belief Model may be useful in identifying community acceptance of a vaccine and the means of introducing it. This health information is important for planning and implementing vaccine programmes.