Formalin-fixed stool improves the performance of the Kato-Katz method.

Background and Aim: Parasitic infections are a public health problem worldwide, including in Thailand. An epidemiological survey for helminthiasis based on stool examination uses the Kato-Katz method as recommended by the World Health Organization. Limitations of this method include the need for fresh stool, time requirement, and lack of quality control. The aim of this study was to enhance the efficiency of the Kato-Katz technique using formalin and glycerol solutions and to implement specimen preparation in fieldwork. Materials and Methods: For the Kato-Katz method, stool samples were divided into formalin-fixed and unfixed groups at various time points and processes. Fresh echinostome eggs were added to each stool group. Incubation with glycerol increased the clearing process. Each group was observed and photographed using a light microscope. Parasite eggs were imaged and compared using the standard Kato-Katz method. Results: Visualization of echinostome eggs from formalin-fixed stool slides was significantly better than that from unfixed stool slides (p < 0.01). Stool samples fixed for 7 days retained normal echinostome eggs morphology. Incubation with glycerol for 1 h resulted in increased Kato-Katz performance by digesting the stool content and enhancing egg observation. Moreover, the results of the Kato-Katz method using fixed and fixed stool plus glycerol for natural helminth infection showed good quality of Opisthorchis viverrini and Taenia egg visualization and normal morphology with a clear background of slides. Conclusion: Formalin-fixed stool could be more suitable than fresh stool for the Kato-Katz method.

Prevalence of Zoonotic Intestinal Parasites in Meat Goats in Southern Thailand.

Background: Intestinal parasites not only lead to poor goat health and productivity but can also affect human health. We investigated the distribution of zoonotic intestinal parasites among goats in southern Thailand to assess the human health risks. Materials and Methods: Overall, 561 fecal samples were collected randomly from goats in farms (55) in Songkhla, Satun, Pattalung, Pattani, Yala, and Narathiwat provinces between January 2020 to January 2022. Parasite eggs were detected via the standard protocol formalin ether concentration technique (FECT). Results: Samples containing one or more intestinal parasites accounted for 97.33% (543/561) of the total samples, and the percentage of zoonotic parasite was 80.04% (449/561). Among the positive samples, two types of zoonotic helminths were identified, Strongylid egg type (79.32%) and Fasciola spp. (2.14%). Strongylid egg type was highly abundant in samples from Satun (84.62%), Songkhla (82.74%), Pattalung (79.31%), and Yala (77.27%). Fasciola spp. was mostly found in samples form Pattalung (13.79%) and Songkhla (1.12%). Eimeria spp. was the mostly identified protozoa 25.67%. It was mostly identified in samples from Narathiwat (75%), Pattani (41.9%), and Songkhla (31.41%). Mixed infections accounted for 40.82% of the total with 33 patterns. It was found that Strongylid egg types occurred in 28 patterns (84.85%). Strongylid egg types-Eimeria spp.-pattern was the most commonly found. Conclusion: Based on the results, zoonotic helminth infection was found together with widespread and high diversity of intestinal parasites infection in meat goats, which affect animal health and may pose a risk to human health. Mass deworming programs with a proper anthelmintic drugs and good agricultural practices need to be encouraged by the government so that people who contact with goats should practice good hygiene behavior. Farm management should be applied by intensive goat raising with pasture circulating to reduce parasite egg contamination. Moreover, fresh fertilizers from goat faces should be avoided.

Reduction of carcinogens in fermented fish (pla-ra and pla-som) by heating.

Background and Aim: The risk factors for cholangiocarcinoma (CCA) are opisthorchiasis and the intake of a combination of nitroso compounds through the consumption of traditionally fermented fish, which is very popular in areas where liver flukes are endemic. The incidence of CCA remains high because this cultural habit of rural people has been altered. Therefore, decreasing nitrate and nitrite concentrations in fermented fish are an alternative approach to reducing the risk of CCA. Thus, this study aimed to reduce nitrate and nitrite concentrations in fermented foods by heating and investigated its effect on CCA development in a hamster model. Materials and Methods: We used Association of Official Analytical Chemists method 973.31 to measure the nitrate and nitrite concentrations in both fermented fish (pla-ra [PR]) and pickled fish (pla-som [PS]) before and after boiling for 5 and 30 min, respectively. The same samples were fed to Opisthorchis viverrini (OV)-infected or -uninfected hamsters for 3 months. Thereafter, the hamsters' liver and blood were collected for analysis. Results: The levels of nitrates and nitrites in PS and PR significantly decreased following boiling for 5 and 30 min. The OV-PR and OV-PS groups showed dramatically increased numbers of inflammatory cells, fibrosis surrounding the bile duct, and focal fibrotic areas. However, after boiling the fermented dishes for 5 and 30 min, the extent of inflammatory cell infiltration and intensity of fibrosis in these groups were decreased. Conclusion: Our findings suggest that boiling reduces nitrate and nitrite toxicity in fermented dishes, as evidenced by reduced hepatic inflammation. However, regardless of heating, kidney tissues are adversely affected when fermented meals are consumed daily.

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.

Prevalence of Ehrlichia-, Babesia-, and Hepatozoon-infected brown dog ticks in Khon Kaen Province, Northeast Thailand.

Background and Aim: The brown dog tick, Rhipicephalus sanguineus sensu lato, is the most common tick found on domestic dogs in Southeast Asia, including Thailand. Canine tick-borne pathogens are a public health concern worldwide. Tick-borne diseases are diagnosed by identifying pathogens based on the morphological or molecular analyses of dog blood samples. However, the collection of ticks, a non-invasive procedure, is easier than drawing blood. This study aimed to demonstrate the usefulness of collecting brown dog ticks for the diagnosis of tick-borne diseases and for estimating the prevalence of tick-borne pathogens among companion dogs in Khon Kaen, Northeast Thailand. Materials and Methods: Seventy brown dog ticks from 70 companion dogs in Khon Kaen Province, Thailand, were evaluated for molecular evidence of tick-borne pathogens, including Babesia spp., Ehrlichia canis, and Hepatozoon canis. Ticks were collected from dogs at a private animal hospital based on the presence of at least one of the three inclusion criteria: fever, anorexia, or lethargy. Molecular diagnosis was performed using conventional polymerase chain reaction for the detection of pathogens. Results: Of the 70 ticks collected from 70 sick dogs, 55 (78.57%) were positive for tick-borne pathogens. The most common infection was a single infection with H. canis (65.71%) followed by Babesia spp. (31.43%) and E. canis (30.00%). Coinfection was observed in 14 ticks (20.00%), and coinfection with Babesia spp. and E. canis was the most prevalent double infection (n = 6). The prevalence of coinfection was identical for H. canis mixed with Babesia spp. and H. canis mixed with E. canis (n = 4). Conclusion: The present study showed that tick-borne pathogens are highly prevalent among companion dogs in Khon Kaen Province. Therefore, we encourage an increase in tick control or the reduction and prevention of tick-borne diseases in this region. Furthermore, this study revealed that ticks are valuable samples for the molecular detection of tick-borne pathogens.

Susceptibility patterns of Bithynia siamensis siamensis and Bithynia funiculata to Opisthorchis viverrini infection: an indication of the risk of opisthorchiasis transmission in non-endemic areas.

Among the snail species acting as hosts for medically significant trematodes, only three taxa of Bithynia are responsible for transmitting the carcinogenic liver fluke Opisthorchis viverrini to humans in different geographical areas. Although B. siamensis goniomphalos is the primary species responsible for O. viverrini transmission in endemic areas, B. siamensis siamensis and B. funiculata remain potential hosts for transmission. This study objects to determine the susceptibility of B. siamensis siamensis and B. funiculata to O. viverrini to assess the risk of O. viverrini transmission in non-endemic areas. The snails of both species were first introduced to O. viverrini eggs, after which O. viverrini infection was investigated using specific PCR primers after a period of 1, 7, 14, 28, and 56 days post-infection (dpi). Opisthorchis viverrini infection in both B. siamensis siamensis and B. funiculata was high in the early period (1 and 7 dpi) while decreasing over time. It was also shown that the odds of susceptibility to O. viverrini infection in B. siamensis siamensis were 64.5% higher relative to the odds of susceptibility in B. funiculata (P < 0.05). Results of this study provide an early insight into the Bithynia-Opisthorchis relationship and thus have great potential to assess risk and raise awareness of opisthorchiasis in non-endemic regions, especially in regions endemic for B. siamensis siamensis.

Performance of Mini Parasep® SF stool concentrator kit, Kato-Katz, and formalin-ethyl acetate concentration methods for diagnosis of opisthorchiasis in Northeast Thailand.

BACKGROUND: Control and elimination of the liver fluke (Opisthorchis viverrini) is a primary preventive strategy against cholangiocarcinoma in Southeast Asia. A sensitive parasitological diagnostic method is required to facilitate a surveillance and control program. In this study, we evaluated the performance of Mini Parasep® SF stool concentrator kit (stool kit) compared with Kato-Katz (KK) and the quantitative formalin-ethyl acetate concentration technique (FECT) for detection of O. viverrini and co-endemic parasitic infections. METHODS: A cross-sectional survey for parasitic infection in residents aged > 15 years in a community in Kalasin province, Northeast Thailand, was conducted in 2018. Fecal samples were collected and screened by KK method, and a subset of samples was further examined by the stool kit and FECT methods. The results were analyzed for prevalence of parasitic infections in addition to the diagnostic performance of the methods for qualitative and quantitative detection of helminthiases. RESULTS: The initial survey of parasitic infection determined by the KK method (n = 567) showed the prevalence of O. viverrini was 32.63%, followed by Taenia 2.65%, echinostomes 1.76%, hookworms 1.41%, Trichuris trichiura 0.53% and Strongyloides stercoralis 0.53%. Within a subset of samples tested with multiple diagnostics (n = 150), the detection rates of O. viverrini by the stool kit, FECT and KK methods were 27.3%, 30.7% and 28.7%, respectively. The diagnostic sensitivity for opisthorchiasis was similar for FECT (75.5%), KK(66.0%) and the stool kit (67.3%). For other parasitic infections, FECT and stool kit methods performed better than KK, particularly in detecting minute intestinal flukes (MIF), S. stercoralis and coinfections. When measuring the intensity of O. viverrini infection (fecal egg counts), the stool kit results showed a significant positive correlation with KK and FECT (P < 0.05). CONCLUSIONS: As the stool kit is simple to use and shows a comparable performance to FECT, it may serve as an alternative method of fecal examination for screening of helminthiasis including opisthorchiasis.

Application of Urine and Copro Antigen Assays after Primary Infection and Drug Treatment in an Experimental Opisthorchiasis Animal Model.

Infection by Opisthorchis viverrini causes significant health problems, including cholangiocarcinoma (CCA); thus control and elimination of this trematode is an important strategy for the reduction of CCA. Currently, urine and copro antigen detection is more sensitive than parasitological examination of the feces for the diagnosis of opisthorchiasis. Given limitations in human studies, we used an animal model to quantify the parasite antigen profiles in urine and feces in O. viverrine-infected hamsters, and postdrug treatment. The positive detections of O. viverrini antigen began from week 1 in urine and week 2 in feces after infection until week 28 of the study. The recoveries of O. viverrini worms were detected starting from week 1 and eggs of O. viverrini were detected in feces from week 3 after infection and remained detectable throughout the study period. There was a significant positive correlation of urine and copro antigen levels with the number of fecal egg counts (P < 0.01) and worm recovery (P < 0.01). In the drug-treatment experiment, treatment of infected hamsters with praziquantel significantly reduced worm burden, fecal egg output, and antigen in urine and feces compared with the untreated controls (P < 0.001). At 4 weeks posttreatment, the egg and worm reduction rates were 100% and 95.5%, respectively. The positive antigen detections in urine and feces corresponded with partial worm clearance from praziquantel treatment. This study demonstrated a direct link of urine and copro antigen tests with worms infecting the liver thereby reaffirming the reliability of urine and copro antigen assay in opisthorchiasis diagnosis.

Population dynamics and diversity of trematode infections in Bithynia siamensis goniomphalos in an irrigated area in northeast Thailand.

Several trematodes including Opisthorchis viverrini utilize Bithynia siamensis goniomphalos as a snail intermediate host in their life cycles. In order to capture a comprehensive range of host­parasite interactions and their transmission dynamic patterns, B. s. goniomphalos were sampled monthly over 4 consecutive years in an irrigated paddy-field habitat in northeast Thailand. Using a standard cercarial shedding method, a high diversity of trematodes (17 types) was recovered. Virgulate xiphidiocercariae were the most prevalent (7.84%) followed by O. viverrini (0.71%). In addition to seasonal and environmental factors, the quantity of irrigation water for rice cultivation correlated with transmission dynamics of trematodes in B. s. goniomphalos. The peak prevalence of all trematode infections combined in the snails shifted from the cool-dry season in 2010­2012 to the hot-dry season in 2013 associated with an increasing quantity of water irrigation. A low frequency of mixed trematode infections was found, indicating that the emergence of virgulate cercariae, but not of O. viverrini, was negatively impacted by the presence of other trematodes in the same snail. Taken together, the observed results suggest that interactions between host and parasite, and hence transmission dynamics, depend on specific characteristics of the parasite and environmental factors including irrigated water for rice cultivation.

Genetic diversity and population structure of Haemonchus contortus in goats from Thailand.

Haemonchus contortus is one of the most economically important parasitic nematodes affecting small ruminant livestock worldwide. This study was conducted to elucidate the genetic diversity and population structure of this nematode in Thailand based on mitochondrial DNA markers, the nicotinamide dehydrogenase subunit 4 (nad4) and the cytochrome c oxidase subunit 1 (cox1) genes. One hundred and thirty-six adult worms were obtained from 86 abomasa of slaughtered goats from 13 different localities in 5 regions of Thailand. Identification to the genus Haemonchus was done using morphology. DNA sequences of the nuclear ribosomal second internal transcribed spacer (ITS2) identified each specimen to species: three fixed nucleotide (SNP) differences distinguished H. contortus from H. placei. Genetic analysis defined 118 and 122 unique haplotypes in partial sequences of nad4 (alignment length 723 bp) and cox1 (645 bp) genes, respectively. Nucleotide diversities were 0.031 and 0.043 for nad4 and cox1 genes, respectively. Low genetic differentiation was observed among H. contortus samples from various provinces in Thailand. This is the first study on the genetic diversity and population structure of H. contortus of goats in Thailand. This study has provided insights into the transmission dynamics of this parasitic nematode, information which is essential for farm management and parasite control.

First Molecular Detection of Babesia gibsoni in Stray Dogs from Thailand.

In southern Thailand, the increasingly growing population of stray dogs is a concern to public health and environmental safety because of the lack of medical attention and control. More importantly, these animals are considered reservoirs for many zoonotic pathogens. The objective of this study was to molecularly detect canine vector-borne pathogens, and to perform genetic characterization of Babesia gibsoni present in stray dogs from southern Thailand. Blood samples were collected from 174 stray dogs in two provinces (Songkhla and Narathiwat) in southern Thailand. PCR analyses were executed using specific primers based on the Babesia spp. 18S rRNA gene, Babesia gibsoni Internal transcribed spacer 1 (ITS1) region, Ehrlichia canis citrate synthase (gltA) gene, Hepatozoon spp. 18S rRNA gene and Anaplasma platys heat shock protein (groEL) gene. The most common canine vector-borne pathogen found infecting stray dogs in this study was Hepatozoon canis (24.7%) followed by A. platys (14.9%), Babesia vogeli (8.0%), B. gibsoni (6.3%), and E. canis (1.72%). Concurrent infection with more than one pathogen occurred in 72 cases. Phylogenetic analysis based on the ITS1 region and 18S rRNA gene revealed that the B. gibsoni isolates from this study shared a large proportion of their identities with each other and with other reported B. gibsoni genotypes from Asia. This study highlights the molecular detection of B. gibsoni in dogs in Thailand for the first time and presents the genetic characterization by sequencing the ITS1 region and 18S rRNA gene of B. gibsoni from Thailand. Follow-up studies are needed to elucidate the origin, distribution, and vectors of B. gibsoni parasites circulating in dogs in Thailand, as well as to determine to what extent dogs are important reservoir hosts for zoonotic canine vector-borne disease infection in the studied area.

The first molecular identification of benzimidazole resistance in Haemonchus contortus from goats in Thailand.

BACKGROUND AND AIM: Haemonchus contortus is one of the major trichostrongyloid nematodes affecting small ruminant production worldwide, especially in tropical and subtropical regions. Adult H. contortus suck the blood from the host abomasum leading to anemia and often death in heavily infected animals. The mainstay of parasitic control is an anthelmintic drug, but long-term drug use may cause drug resistance. The aim of this study was to examine benzimidazole resistance in H. contortus of goats from different regions in Thailand by detecting the frequency of the F200Y polymorphism in the ß-tubulin isotype 1 gene. MATERIALS AND METHODS: A total of 121 H. contortus adults were obtained from 31 naturally infected out of 37 slaughtered goats from city abattoirs in five regions of Thailand. The frequency of the F200Y polymorphism in the ß-tubulin isotype 1 gene was detected following the allele-specific polymerase chain reaction protocol. RESULTS: The overall genotype frequencies in Thailand were homozygous resistant (RR: 24%), heterozygous (SR: 44.6%), and homozygous susceptible (SS: 31.4%). The allele frequencies were resistant allele (R: 46%) and susceptible allele (S: 54%). The R allele frequency and the RR genotype varied from 30% to 65% and 0% to 43.9%, respectively. The frequency of R alleles was significantly higher in the southern region (0.65) as compared to northern (0.30, p=0.001), western (0.38, p=0.04), and central regions (0.30, p=0.03). The RR genotype was also significantly higher in the southern region (43.9%) versus the northern (0 %, p=0.001), western (11.8%, p=0.012), and central regions (17.4%, p=0.001). CONCLUSION: This is the first study of the detection of single-nucleotide polymorphisms in codon 200 of the ß-tubulin isotype 1 gene of H. contortus from goats in Thailand. These findings are essential and imply that an integrated approach is needed for issues such as drug treatment, farm management, prevention, and control strategies. This is of interest to farmers, veterinarians, and the department of livestock.

Towards Evidence-based Control of Opisthorchis viverrini.

Transmission of the carcinogenic liver fluke Opisthorchis viverrini is ongoing across Southeast Asia. Endemic countries within the region are in different stages of achieving control. However, evidence on which interventions are the most effective for reducing parasite transmission, and the resulting liver cancer, is currently lacking. Quantitative modelling can be used to evaluate different control measures against O. viverrini and assist the design of clinical trials. In this article we evaluate the epidemiological parameters that underpin models of O. viverrini and the data necessary for their estimation, with the aim of developing evidence-based strategies for parasite control at a national or regional level.

Efficacy of Dipterocarpus alatus oil combination with Rhinacanthus nasutus leaf and Garcinia mangostana pericarps against canine demodicosis.

BACKGROUND AND AIM: Canine demodicosis is a skin disease that is a major global health problem in dogs. Ivermectin is a drug of choice for treatment, but it may cause toxicity in dogs carrying multidrug resistance mutation-1 gene mutations. Hence, alternative herbal medicines are used instead of the drug, such as Dipterocarpus alatus oil (YN oil), Rhinacanthus nasutus leaf (WC), and Garcinia mangostana pericarps (MG) extracts. This study aimed to determine the efficacy of D. alatus oil, R. nasutus leaf, and G. mangostana pericarp extracts on canine demodicosis in vivo. MATERIALS AND METHODS: Twenty-five mixed-breed dogs with localized demodicosis were examined. Dogs were diagnosed with demodicosis through deep skin scraping and screened with the inclusion criteria. Five dogs of each group were treated in five treatment groups (ivermectin, YN oil, YN oil+WC, YN oil+MG, and YN oil+WC+MG) for 1 month. The individual dogs were clinically evaluated, and the dermatological lesions were monitored daily for 60 days. RESULTS: Dermatological lesion improvement was predominantly observed in the group of dogs treated with YN oil+WC. This was evidenced by the disappearance of the hyperpigmentation and lichenification on day 28 post-treatment and alopecia on day 56 post-treatment. Moreover, no allergic or clinical signs were observed during treatment. CONCLUSION: YN oil+WC is an alternative herbal medicine that could be used for the treatment of localized canine demodicosis.

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.

Phylogenetic relationships within the Opisthorchis viverrini species complex with specific analysis of O. viverrini sensu lato from Sakon Nakhon, Thailand by mitochondrial and nuclear DNA sequencing.

The liver fluke Opisthorchis viverrini sensu lato causes serious public-health problems in Northeast Thailand and Southeast Asian countries. A hypothesis has been proposed that O. viverrini represents a species complex with varying levels of genetic differentiation in Thailand and Lao PDR. This study aimed to clarify whether O. viverrini populations can be genetically divided into separate taxa. We collected O. viverrini s.l. from eight different locations in Lao PDR and Thailand. The results of nad1, cox1, CF-int6, Pm-int9, ITS2 and 28S rDNA sequence analysis revealed that sub-structuring occurred between the eight populations. We found that O. viverrini s.l. from Sakon Nakhon (SK), Thailand, shows significant genetic differentiation (P < .05) from all other isolates from different localities in Thailand and Lao PDR. This was supported by haplotype and phylogenetic tree analyses in which the SK isolate was separated from all other isolates. This suggests that O. viverrini s.l. from SK is a cryptic species. The data, however, also confirm the association between genetic groups of O. viverrini s.l. and specific wetland systems, and raise important questions regarding the epidemiological significance of these genetic differences.

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.

Comparison of infectivity, metacercarial burden and host mortality induced by Opisthorchis viverrini sensu lato cercariae from Lao PDR compared with Thailand in cyprinid fish, Barbonymus gonionotus.

BACKGROUND: Previous studies indicate that Opisthorchis viverrini sensu lato (s.l.) is a species complex with strong associations to geographical regions, i.e., specific wetlands. The present study was conducted to compare the infectivity, establishment and metacercarial burden and survival of the fish host following exposure to O. viverrini s.l. cercariae isolates from two different river wetlands. METHODS: Experimental infections were performed by exposing fish (silver barb, Barbonymus gonionotus) individually to 0 (control) and 100 O. viverrini s.l. cercariae originating from the Songkram River wetland in Thailand and the Nam Ngum River wetland, Lao PDR. Metacercarial burden and fish survivals were monitored after infection. RESULTS: Fish exposed to O. viverrini s.l. cercariae from the Nam Ngum River wetland had significantly greater metacercarial burden and more active motility at 28 and 35 days post infection than those from the Songkram River wetland. The mortality of fish infected with O. viverrini s.l. from the Nam Ngum River wetland was greater than that from the Songkram River wetland (log-rank test, p<0.01). CONCLUSIONS: The findings in this study provided additional evidence supporting the importance of cryptic species of O. viverrini s.l. and it has implications for parasite transmission dynamics, life cycle success and disease ecology.

Seasonal transmission of Opisthorchis viverrini sensu lato and a lecithodendriid trematode species in Bithynia siamensis goniomphalos snails in northeast Thailand.

Seasonal changes play roles in the transmission success of fish-borne zoonotic trematodes (FZT). This study examined the seasonal transmission patterns of Opisthorchis viverrini sensu lato (s.l.) and a virgulate cercaria (family Lecithodendriidae) in the snail intermediate host, Bithynia siamensis goniomphalos in northeast Thailand. Snail samples were collected monthly during the rainy, cool, and hot seasons during 2012-2013 to determine the prevalence and intensity of larval trematode infections. The prevalence of O. viverrini s.l. varied significantly with season, being 0.31%, 1.05%, and 0.37% in the rainy, cool, and hot seasons, respectively (P < 0.05). Similarly, the prevalence of virgulate cercariae was 3.11%, 6.80%, and 1.64% in the rainy, cool, and hot seasons, respectively (P < 0.05). The intensity of larval trematode infections also varied between seasons and peaked in the hot season (P < 0.05) in both species. The snails infected with O. viverrini s.l. were significantly smaller (P < 0.05) and those infected with virgulate cercariae were significantly larger (P < 0.05) than uninfected snails. Seasonal variation and the different sizes of B. s. goniomphalos parasitized by O. viverrini s.l. and virgulate trematodes indicate complex host-parasite interactions with important implications for the epidemiology of O. viverrini s.l.

Analysis of the population genetics of Opisthorchis viverrini sensu lato in the Nam Ngum River wetland, Lao PDR, by multilocus enzyme electrophoresis.

A previous population genetics study of Opisthorchis viverrini from a locality in an endemic area in Thailand found little genetic variation over time and second intermediate fish host species. Since a similar comparative analysis is not available for Lao PDR, we conducted a study of O. viverrini from different endemic foci in Vientiane Province, Lao PDR, based on spatial, temporal and fish host species. A total of 620 adult O. viverrini originating from the Nam Ngum River wetland were analysed at five previously defined polymorphic enzyme loci. Of these worms, 252 were from six different localities (spatial samples), 162 worms from different years (temporal samples) and 206 worms from four different cyprinid fish species. Significant heterozygote deficiency was found in most O. viverrini populations with levels of genetic differentiation ranging between F ST 0.0000 and 0.0197 suggesting that gene flow occurred at a variable rate. The role of temporal factors and fish host species had little influence on the level of genetic differentiation. As for O. viverrini from Thailand, these findings indicate that self-fertilization and/or a clonal distribution of O. viverrini occurs in Lao PDR. Unlike the results for O. viverrini from Thailand, spatial population substructuring may be the underlying population processes for O. viverrini in Lao PDR. These findings indicate that geographical variation may contribute to the transmission dynamics of the parasite with implications for parasite control. However, other host factors, such as snail intermediate hosts and mammal reservoir hosts, as well as human beings, may also play significant roles.