Impact of geography and time on genetic clusters of Opisthorchis viverrini identified by microsatellite and mitochondrial DNA analysis.

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.

Infection Dynamics of Opisthorchis viverrini Metacercariae in Cyprinid Fishes from Two Endemic Areas in Thailand and Lao PDR.

The infection dynamics of Opisthorchis viverrini metacercariae was analyzed in cyprinid fish from endemic areas in Mukdahan Province, Thailand, and Khammouane Province, Lao PDR. The fish were collected during the cool-dry (November-February), hot-dry (March-May), and rainy (June-October) seasons in 2017. They were examined by the digestion method, and the infection status was statistically analyzed by study area, season, and fish size. The prevalence (no. of fish positive/no. of fish examined × 100) and metacercarial intensities (no. of metacercariae detected/no. of fish positive) of O. viverrini in both study areas depended on season, being high in the cool-dry season and varying in the hot-dry and rainy seasons. In Mukdahan Province, the average prevalence was 18.3% (range 11.0-46.7%, n = 420) and the intensity was 4.07 ± 5.86 cysts/fish (mean ± SD), whereas in Khammouane Province, the prevalence was 51.9% (range 9.1-70.6%, n = 673) and the intensity was 6.67 ± 12.88 cysts/fish. Among the cyprinid fish species examined, the infection was associated with fish body size and predominantly found in Hampala dispar (86.5%), Cyclocheilichthys armatus (73.2%), and Puntius brevis (42.7%). The distribution of O. viverrini metacercariae in fish was skewed, with most of the fish having a low worm burden with an average of four to six cysts/fish. The findings that seasonality, sampling locality, fish size, and species of fish play roles in the risk of O. viverrini infection imply that these host and environmental factors are important for the transmission dynamics and control of O. viverrini.

Culture of fecal indicator bacteria from snail intestinal tubes as a tool for assessing the risk of Opisthorchis viverrini infection in Bithynia snail habitat.

BACKGROUND: Like many trematodes of human health significance, the carcinogenic liver fluke, Opisthorchis viverrini, is spread via fecal contamination of snail habitat. Methods for assessing snail exposure to fecal waste can improve our ability to identify snail infection hotspots and potential sources of snail infections. We evaluated the feasibility of culturing fecal indicator bacteria from Bithynia snail intestinal tubes as a method for assessing snail exposure to fecal waste. Snails and water samples were collected from a site with a historically high prevalence of O. viverrini infected snails ("hotspot" site) and a site with historically no infected snails ("non-hotspot" site) on two sampling days. Snails were tested for O. viverrini and a stratified random sample of snails from each site was selected for intestinal tube removal and culture of gut contents for the fecal indicator bacteria, Escherichia coli. Water samples were tested for E. coli and nearby households were surveyed to assess sources of fecal contamination. RESULTS: At the hotspot site, 26 of 2833 Bithynia siamensis goniomphalos snails were infected with O. viverrini compared to 0 of 1421 snails at the non-hotspot site. A total of 186 snails were dissected and cultured. Escherichia coli were detected in the guts of 20% of uninfected snails, 4% of O. viverrini-positive snails and 8% of snails not examined for cercarial infection at the hotspot site. Only one of 75 snails from the non-hotspot site was positive for E. coli. Accounting for sampling weights, snails at the hotspot site were more likely to have gut E. coli than snails from the non-hotspot site. The concentration of fecal indicator bacteria in surface water was higher at the hotspot vs non-hotspot site on only the first sampling day. CONCLUSIONS: Fecal indicator bacteria can be detected in the intestinal tubes of Bithynia snails. The presence of fecal indicator bacteria in Bithynia snail guts may indicate risk of O. viverrini infection in snail populations. This method has the potential to aid in identifying locations and time windows of peak snail infection risk and may be applicable to other trematodes of human-health significance.

Cercarial emergence patterns for Opisthorchis viverrini sensu lato infecting Bithynia siamensis goniomphalos from Sakon Nakhon Province, Thailand.

UNLABELLED: Opisthorchis viverrini sensu lato is a food-borne trematode which is classified as a class 1 carcinogen, with infection potentially leading to cholangiocarcinoma. Snails of the genus Bithynia act as the first intermediate hosts and an amplifying point in the parasite life cycle. In order to investigate seasonal effect on transmission dynamics of O. viverrini in Bithynia siamensis goniomphalos, cercarial emergence and output profiles were monitored at different season. A total of 4533 snails originating from Sakon Nakhon Province, Thailand, collected during the three main seasons, were analyzed for O. viverrini s.l. INFECTION: Emergence of O. viverrini s.l. cercariae from snails was monitored daily from 06:00 to 18:00 h for seven consecutive days. The prevalence of infection in the snails was highest in the hot-dry season and declined in the rainy and cool-dry seasons. Peak cercarial emergence occurred between 08:00 and 10:00 h during the rainy and cool-dry seasons and between 10:00 and 12:00 h during the hot-dry season. The cercarial output was highest in the hot-dry season, similar to a previous study from Lao People's Democratic Republic (PDR). Average cercarial output/snail in Thailand was higher than in Lao PDR. The number of cercariae emerging from the snails was strongly related to snail size, but the relationship between prevalence of infection and snail size differed between seasons. Observed discrepancies in the emergence patterns and per capita cercarial release may reflect differences in environmental, snail, and/or parasite factors particularly biological characteristics between the cryptic species of O. viverrini s.l. and B. s. goniomphalos from Thailand and Lao PDR.