Exploring the second intermediate hosts and morphology of human- and cat-specific Opisthorchis viverrini-like populations.

Infection by the zoonotic fish-borne trematode, Opisthorchis viverrini, remains a crucial health issue in Thailand and neighboring countries. Recently, molecular analysis revealed two populations of putative O. viverrini: one found primarily in human hosts ("human-specific" population) and the other primarily in cats ("cat-specific" population). It is unclear how the infective stages (metacercariae) of these different populations circulate among definitive and reservoir hosts in nature. To gain an insight into this, mitochondrial cox1 and nad1 gene sequences of metacercariae from fish intermediate hosts were examined. None of 192 metacercariae from cyprinid fish in Lao PDR and Thailand had sequences typical of "cat-specific" O. viverrini, suggesting that cyprinid fish are not the main second intermediate hosts of this population. Interestingly, all 20 O. viverrini-like metacercariae from snakehead fish (Channa striata) shared 99.51-100% sequence identity with eggs from cats naturally infected in a previous study. Hence, we propose a modification of the known transmission dynamics of O. viverrini: consumption of metacercariae within snakehead fish provides another pathway for cats and (occasionally) humans to acquire infection. We also performed morphological comparisons of eggs, metacercariae, and adult flukes (raised in hamsters) of both Opisthorchis populations. The "cat-specific" population has eggs that are narrower and adults that are shorter and wider than in the human-specific population. The metacercaria of the "cat-specific" population is elliptical, while that of the "human-specific" population is oval, occasionally rounded. Our results confirmed that O. viverrini-like metacercariae from snakehead fish are the infective stages of the "cat-specific" fluke. This provides a new insight into the dissemination and transmission of each population in the second intermediate host. The identity of the cat-specific population is discussed.

Prevalence of gastrointestinal parasites in captive mammals at Khon Kaen Zoo, Thailand.

Background and Aim: Captive animals are susceptible to parasitic diseases due to the stress and confinement they experience. In addition, they can serve as reservoirs of zoonotic parasites that have the potential to infect humans. To investigate this possibility, we estimated the prevalence of gastrointestinal (GI) parasites in captive mammals at Khon Kaen Zoo, Thailand. Materials and Methods: One hundred and forty-seven individual mammals (37 primates, 43 carnivores, 62 herbivores, and 5 rodents) were examined for parasitic infections by fecal examination daily for 3 consecutive days using the formalin-ethyl acetate concentration technique (FECT) and the agar plate culture method. Results: According to FECT, the overall prevalence of GI parasites was 62.6% (92/147). Within animal groups, the numbers were as follows: 67.6% (25/37) in primates, 23.3% (10/43) in carnivores, 85.5% (53/62) in herbivores, and 80.0% (4/5) in rodents. Using the agar plate culture method, 21.43% (27/126) were positive for Strongyloides spp. and hookworm infections. The GI parasites identified belonged to three categories: protozoa (including Entamoeba histolytica species complex, Entamoeba coli, Giardia spp., coccidia, and ciliated protozoa), trematodes (minute intestinal flukes and rumen flukes), and nematodes (strongyle/hookworm, Strongyloides spp., Ascarididae, and Trichuris spp.). Conclusion: The findings of this study indicate the prevalence of several GI parasites in zoo animals with the potential for transmission to humans, given the animals' close proximity to both visitors and animal caretakers.

Influence of Water Irrigation Schemes and Seasonality on Transmission Dynamics of Opisthorchis viverrini in the Snail Intermediate Host, Bithynia siamensis goniomphalos in Rice Paddy Fields in Northeast Thailand.

Opisthorchis viverrini is a fish-borne zoonotic trematode that causes significant public health problems in Southeast Asia. Its life cycle requires Bithynia snails as the first intermediate hosts, fish, and human and/or carnivore hosts. This study assessed impacts of land use practice for rice cultivation and seasonality on the transmission dynamics of O. viverrini in Bithynia siamensis goniomphalos in rice paddy field habitats. The transmission of O. viverrini cercariae in B. s. goniomphalos was monitored at monthly intervals over a 4-year period from January 2010 to December 2013. From a total of 59,727 snails examined by standard cercarial shedding, the prevalence of O. viverrini was 0.7% (range, 0.0-4.1%). The prevalence of O. viverrini infection in B. s. goniomphalos varied with the amount of rainfall, with peaks of infection occurring in the cool-dry season, that is, after each rainy season. A shift of peak prevalence from cool-dry to hot-dry season observed in 2013 was associated with the increase in preceding water irrigation to support the production of second annual rice crop. Significant positive correlations were found between the prevalence and intensity of cercarial infection and the size of snails. Our results revealed substantial variation between years so that to have a clear understanding of the population dynamics of this complex system, studies should be conducted over an extended period (> 1 year). Results from this study highlight that water irrigation schemes in rice paddy cultivation and seasonality have a significant effect on the prevalence of O. viverrini in B. s. goniomphalos.

Preliminary genetic evidence of two different populations of Opisthorchis viverrini in Lao PDR.

Opisthorchis viverrini is a major public health concern in Southeast Asia. Various reports have suggested that this parasite may represent a species complex, with genetic structure in the region perhaps being dictated by geographical factors and different species of intermediate hosts. We used four microsatellite loci to analyze O. viverrini adult worms originating from six species of cyprinid fish in Thailand and Lao PDR. Two distinct O. viverrini populations were observed. In Ban Phai, Thailand, only one subgroup occurred, hosted by two different fish species. Both subgroups occurred in fish from That Luang, Lao PDR, but were represented to very different degrees among the fish hosts there. Our data suggest that, although geographical separation is more important than fish host specificity in influencing genetic structure, it is possible that two species of Opisthorchis, with little interbreeding, are present near Vientiane in Lao PDR.

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.

EVALUATION OF A COMMERCIAL STOOL CONCENTRATOR KIT COMPARED TO DIRECT SMEAR AND FORMALIN-ETHYL ACETATE CONCENTRATION METHODS FOR DIAGNOSIS OF PARASITIC INFECTION WITH SPECIAL REFERENCE TO OPISTHORCHIS VIVERRINI SENSU LATO IN THAILAND.

Opisthorchiasis and cholangiocarcinoma (CCA) are major public health problems in Thailand and countries in the lower Mekong Subregion. Elimination of opisthorchiasis will be an important step toward the prevention, control and reduction of CCA. In order to achieve this goal, a sensitive and robust diagnostic method is required to identify people with current Opisthorchis viverrini sensu lato infection as the parasite is a group 1 carcinogen believed to be an etiology of CCA. To date, sensitive parasitological methods, such as formalin-ethyl acetate concentration technique (FECT) is preferred, but it is not practical in a remote primary care setting. In this study, we evaluated the diagnostic accuracy of a commercial stool concentrator kit with that of a direct simple smear method and a modified FECT. In diagnosing parasite infection and opisthorchiasis, the commercial kit had greater sensitivity (43.8-58.5%) than direct smear method (12.5-31.7%), but was less sensitive than FECT (73.2-75%). In a separate sample population, similar results were obtained when comparing the diagnostic accuracy of the commercial kit and FECT. However, the commercial kit was more effective in a field setting than FECT, and had better accuracy than direct smear method, which suggests that the kit could have potential utility in epidemiological studies and control programs of opisthorchiasis, as well as other parasitic infections. The design of the self-contained one-tube kit plus its long storage time after sample preparation provides a considerable advantage over other methods, such as direct or Kato thick smear method, under similar field conditions.

A cross-sectional study on the potential transmission of the carcinogenic liver fluke Opisthorchis viverrini and other fishborne zoonotic trematodes by aquaculture fish.

Throughout Southeast Asia and China, eating raw and or partially cooked cyprinid fish causes liver (hepatobiliary) disease and cancer (cholangiocarcinoma) due to fishborne zoonotic trematodes (FZT), in particular Clonorchis sinensis and Opisthorchis viverrini. The primary source of transmission is by native fish, but aquaculture fish are also reported to have high infective potential. Here, a cross-sectional survey of FZT in fish farms was conducted in an endemic area in Khon Kaen Province, Thailand. By using conventional and polymerase chain reaction (PCR) methods, we detected O. viverrini and FZT metacercariae (Centrocestus formosanus and Haplorchis taichui) in two popular fish species, Barbonymus gonionotus (silver barb) and Cirrhinus mrigala (mrigal), from aquaculture farms. Both species were infected in five of six farms examined by PCR but not by conventional methods, yet the prevalence of FZT metacercariae in aquaculture fish was high (46.9%). In addition to O. viverrini (17.1%), the native fish Cyclocheilichthys armatus and Hampala dispar had a prevalence of FZT of 81.4%, which included 5.7% for C. formosanus and 17.1% for H. taichui by conventional method. To our knowledge, this is the first discovery of O. viverrini in aquaculture fish in Thailand. More comprehensive studies are required to determine if human-induced disease transmission coupled with natural transmission cycle occurs throughout the aquaculture industry in the region. This has significant impact on food quality and safety, and provides the basis for the development of an effective strategy for the prevention and control of foodborne diseases.

Population genetic structuring in Opisthorchis viverrini over various spatial scales in Thailand and Lao PDR.

Khon Kaen Province in northeast Thailand is known as a hot spot for opisthorchiasis in Southeast Asia. Preliminary allozyme and mitochondrial DNA haplotype data from within one endemic district in this Province (Ban Phai), indicated substantial genetic variability within Opisthorchis viverrini. Here, we used microsatellite DNA analyses to examine the genetic diversity and population structure of O. viverrini from four geographically close localities in Khon Kaen Province. Genotyping based on 12 microsatellite loci yielded a mean number of alleles per locus that ranged from 2.83 to 3.7 with an expected heterozygosity in Hardy-Weinberg equilibrium of 0.44-0.56. Assessment of population structure by pairwise F(ST) analysis showed inter-population differentiation (P<0.05) which indicates population substructuring between these localities. Unique alleles were found in three of four localities with the highest number observed per locality being three. Our results highlight the existence of genetic diversity and population substructuring in O. viverrini over a small spatial scale which is similar to that found at a larger scale. This provides the basis for the investigation of the role of parasite genetic diversity and differentiation in transmission dynamics and control of O. viverrini.

The systematics and population genetics of Opisthorchis viverrini sensu lato: implications in parasite epidemiology and bile duct cancer.

Together with host and environmental factors, the systematics and population genetic variation of Opisthorchis viverrini may contribute to recorded local and regional differences in epidemiology and host morbidity in opisthorchiasis and cholangiocarcinoma (CCA). In this review, we address recent findings that O. viverrini comprises a species complex with varying degrees of population genetic variation which are associated with specific river wetland systems within Thailand as well as the Lao PDR. Having an accurate understanding of systematics is a prerequisite for a meaningful assessment of the population structure of each species within the O. viverrini complex in nature, as well as a better understanding of the magnitude of genetic variation that occurs within different species of hosts in its life cycle. Whether specific genotypes are related to habitat type(s) and/or specific intermediate host species are discussed based on current available data. Most importantly, we focus on whether there is a correlation between incidence of CCA and genotype(s) of O. viverrini. This will provide a solid basis for further comprehensive investigations of the role of genetic variation within each species of O. viverrini sensu lato in human epidemiology and genotype related morbidity as well as co-evolution of parasites with primary and secondary intermediate species of host.

Microsatellite loci in the carcinogenic liver fluke, Opisthorchis viverrini and their application as population genetic markers.

Opisthorchis viverrini is a carcinogenic foodborne trematode endemic in Southeast Asia especially in Thailand and the Lao People's Democratic Republic. Opisthorchiasis causes hepatobiliary diseases and cholangiocarcinoma (bile duct cancer). Currently there is substantial evidence on genetic variation of O. viverrini but the information on population genetic structure is lacking. Because microsatellite DNA of this parasite is not available, we for the first time isolated and utilized microsatellite DNA as genetic markers to examine genetic diversity and the population structure of O. viverrini. Partial genomic DNA libraries were constructed by conventional and enrichment methods which yielded microsatellite-containing clones of 0.18-0.25% and 16.84%, respectively. Within 41 microsatellite loci isolated 36.59% were perfect, 60.98% were interrupted and 2.44% were compound microsatellites. The CA repetitions were the most frequent, followed by GT and CAT. Primers specific to the flanking regions of 12 microsatellite loci were developed to genotype 150 O. viverrini individuals from geographical localities in Thailand and Lao PDR. Allele numbers per locus ranged from 2 to 15, with the mean expected heterozygosity of 0.03-0.66. Analyses of O. viverrini from 5 localities revealed a high level of genetic diversity and had significant deviation from Hardy-Weinberg equilibrium. Significant heterozygote deficiency as well as heterozygote excess was detected across all localities indicating the possibility of selfing (inbreeding) as a predominant reproductive mode. Significant genetic differentiation (F(ST)) was also detected between worms from different localities with varying levels of genetic heterogeneity. We discuss our results in terms of what these novel microsatellite markers reveal about the epidemiology and transmission dynamics of this medically important parasite, both in terms of the current study and their potential for future comprehensive population genetic studies O. viverrini sensu lato in Southeast Asia.

Biological variation within Opisthorchis viverrini sensu lato in Thailand and Lao PDR.

Previous molecular genetic analyses indicate that Opisthorchis viverrini is a complex of at least 2 cryptic species in Thailand and the Lao People's Democratic Republic (PDR). Opisthorchis viverrini sensu lato (s.l.) populations can be divided into 6 genetic groups, which correlate with 5 different river wetlands systems. The present study examines the infectivity, growth, fecundity, and body size of O. viverrini s.l. isolates from these systems. Worm recovery was lowest in the Songkhram River, which differed significantly from all other wetland systems. In addition, the Chi River and Nam Ngum River populations also differed significantly. A similar pattern was found for fecundity. These results support the genetic/molecular data indicating a subdivision of O. viverrini populations on the basis of watershed and that propose the existence of cryptic species in Thailand and the Lao PDR.