Circulating microRNAs as Biomarkers of Hepatic Fibrosis in Schistosomiasis Japonica Patients in the Philippines.

Host-derived microRNAs (miRNAs) play important regulatory roles in schistosomiasis-induced hepatic fibrosis. This study analyzed selected serum miRNAs among Filipino schistosomiasis japonica patients with ultrasound (US)-detectable hepatic fibrosis. A prospective cohort study design with convenience sampling was employed from 2017 to 2019. The study sites were eight endemic barangays in Leyte, Philippines. Eligible chronic schistosomiasis patients with varying severities of hepatic fibrosis were enrolled in the cohort and serially examined at 6, 12, and 24 months from baseline. Baseline serum miR-146a-5p, let-7a-5p, miR-150-5p, miR-122-5p, miR-93-5p, and miR200b-3p were measured using RT-qPCR. A total of 136 chronic schistosomiasis patients were included in this prospective cohort study. Approximately, 42.6% had no fibrosis, 22.8% had mild fibrosis, and 34.6% had severe fibrosis at baseline The serum levels of the antifibrotic miR-146a (p < 0.0001), miR-150 (p = 0.0058), and let-7a (p < 0.0001) were significantly lower in patients with hepatic fibrosis while the profibrotic miR-93 (p = 0.0024) was elevated. miR-146a-5p (AUC = 0.90, 95% CI [0.84, 0.96], p < 0.0001) has the most promising potential to differentiate patients with (n = 78) versus without (n = 58) hepatic fibrosis. The baseline level of serum miR-146-5p was significantly different in patients with progressive fibrosis (n = 17) compared to those who never developed fibrosis (n = 30, p < 0.01) or those who had fibrosis reversal (n = 20, p < 0.01) after 24 months. These findings demonstrate the potential utility of serum miRNAs, particularly of miR-146a, as a supplementary tool for assessing hepatic fibrosis in chronic schistosomiasis japonica patients.

Diagnostic Performance of Parasitological, Immunological, Molecular, and Ultrasonographic Tests in Diagnosing Intestinal Schistosomiasis in Fieldworkers From Endemic Municipalities in the Philippines.

Schistosomiasis remains to ha/ve a significant public health impact in the Philippines. The Kato-Katz (K-K) technique is the reference standard and most used technique for definitive diagnosis of intestinal schistosomiasis for control programs in endemic regions. However, this has a very low sensitivity when applied in areas of low endemicity and patients with light infection. Hence, this study determined the diagnostic performance of immunological, molecular, parasitological, and ultrasonographic tests in diagnosing intestinal schistosomiasis in endemic municipalities in the Philippines. We performed a community-based cross-sectional study to determine the positivity of schistosomiasis in Leyte, Philippines. The diagnostic performance of five different detection techniques: (1) three stool K-K with duplicate smears; (2) soluble egg antigen IgG ELISA; (3) urine point-of-care circulating cathodic antigen (POC-CCA) test; (4) detection of Schistosoma japonicum circulating DNA (SjcDNA) in serum and urine samples; (5) focused abdominal ultrasound (US), were also obtained in this study. Multiple stool examinations enhanced the sensitivity of K-K from 26.2% (95% CI [16.4, 38.8]) with single stool to 53.8% (95% CI [41.1, 66.1]) and 69.2% (95% CI [56.4, 80.0]) with two and three stools from consecutive days, respectively. Among the SjcDNA nucleic acid amplification test (NAAT)-based detection assays, loop-mediated isothermal amplification (LAMP) PCR using sera had the highest sensitivity at 92.3% (95% CI [82.2, 97.1]) with LAMP consistently identifying more positive cases in both serum and urine samples. This study showed that single stool K-K, which remains the only diagnostic test available in most endemic areas in the Philippines, had low sensitivity and failed to identify most patients with light infection. SjcDNA detection assay and POC-CCA urine test were more sensitive than stool microscopy in detecting schistosomiasis. On the other hand, US was less sensitive than the widely utilized K-K technique in diagnosing schistosomiasis. This study emphasizes the need to revisit the use of single stool K-K in the surveillance and case detection of schistosomiasis in endemic areas of the Philippines. The availability of advanced and more sensitive diagnostic tests will help better control, prevent, and eliminate schistosomiasis in the country.

Field Evaluation of Recombinant Antigen ELISA in Detecting Zoonotic Schistosome Infection Among Water Buffaloes in Endemic Municipalities in the Philippines.

In this study, we investigated the use of recombinant antigens thioredoxin peroxidase-1 (rSjTPx-1) and tandem repeat rSj1TR in evaluating the antibody positivity rates of Schistosoma japonicum infection among water buffaloes from four endemic areas in the Philippines, two municipalities with high endemicity (Calatrava, Negros Occidental and Catarman, Northern Samar) and two municipalities nearing elimination with no cases of human schistosomiasis (Talibon and Trinidad, Bohol). These recombinant antigen ELISA assays were compared with other diagnostic tests including SEA-ELISA, FECT, and fecal-based PCR. Results showed that rSj1TR-ELISA has the highest agreement with PCR in all study areas. Furthermore, significant positivity rates among water buffaloes were seen in Talibon and Trinidad, indicating that water buffaloes are maintaining the schistosome parasites in transmission areas even in the absence of human infection. Hence, serological assay using a more sensitive and specific rSj1TR-ELISA can be used for animal surveillance to prevent emergence and re-emergence of human schistosomiasis.

Analyses of the expression, immunohistochemical properties and serodiagnostic potential of Schistosoma japonicum peroxiredoxin-4.

BACKGROUND: Schistosoma japonicum, which inhabits the mesenteric vein of the mammalian hosts for about 20 to 30 years, is subjected to the oxidative stresses from the host defense mechanism during their intra-mammalian stages. To counteract this host immune attack, the parasite utilizes their antioxidant system for survival inside the host. Peroxiredoxins (Prxs), thiol-specific antioxidant proteins, play an essential role for protecting the parasite against oxidative stress by reducing hydrogen peroxide to water. Only three types of 2-Cys Prxs have been previously characterized in S. japonicum whereas a fourth Prx has been identified for Schistosoma mansoni as Prx-4. A sequence coding homologous to this gene in the S. japonicum database was identified, characterized and expressed as recombinant SjPrx-4 protein (rSjPrx-4). Furthermore, rSjPrx-4 was evaluated in this study for its diagnostic potentials in detecting S. japonicum infection in humans. RESULTS: The gene found in the parasite genome contained 2 active-site cysteines with conserved sequences in the predicted amino acid (AA) sequence and showed 75% identity with that of the previously characterized Prx (TPx-1) of S. japonicum. The gene was expressed in different stages of schistosome life-cycle with highest transcription level in the adult male. The gene was cloned into a plasmid vector and then transfected into Escherichia coli for expression of rSjPrx-4. Anti-rSjPrx-4 mouse sera recognized native SjPrx-4 in egg and adult worm lysate by western blotting. The result of a mixed function oxidation assay in which rSjPrx-4 prevented the nicking of DNA from hydroxyl radicals confirmed its antioxidant activity. Subsequently, immunolocalization analysis showed the localization of SjPrx-4 inside the egg, on the tegument and in the parenchyma of the adult worm. Enzyme-linked immunosorbent assay results showed that rSjPrx-4 has 83.3% sensitivity and 87.8% specificity. Its diagnostic potential was further evaluated in combination with recombinant SjTPx-1 protein, yielding an improved sensitivity and specificity of 90% and 92.7%, respectively. CONCLUSIONS: These results suggest that SjPrx-4 plays a role as an antioxidant dealing with oxidative stresses of S. japonicum, and its diagnostic potential improved by coupling it with SjTPx-1 is a proof for developing a serological test with better diagnostic performance for human schistosomiasis.

Oncomelania hupensis quadrasi: Snail intermediate host of Schistosoma japonicum in the Philippines.

Oncomelania hupensis quadrasi is the snail intermediate host of Schistosoma japonicum in the Philippines. It was discovered by Dr. Marcos Tubangui in 1932 more than two decades after the discovery of the disease in the country in 1906. This review, the first for O. h. quadrasi, presents past and present works on the taxonomy, biology, ecology, control, possible paleogeographic origin of the snail intermediate host and future in research, control and surveillance of the snail. Extensive references are made of other subspecies of O. hupensis such as the subspecies in China for which majority of the advances has been accomplished. Contrasting views on whether the snail is to be considered an independent species of Oncomelania or as one of several subspecies of Oncomelania hupensis are presented. Snail control methods such as chemical methods using synthetic and botanical molluscicides, environmental manipulation and biological control are reviewed. Use of technologies such as Remote Sensing, Geographical Information System and landscape genetics is stressed for snail surveillance. Control and prevention efforts in the Philippines have consistently focused on mass drug administration which has proved inadequate in elimination of the disease. An integrated approach that includes snail control, environmental sanitation and health education has been proposed. Population movement such as migration for employment and economic opportunities and ecotourism and global climate change resulting in heavy rains and flooding challenge the gains of control and elimination efforts. Concern for possible migration of snails to non-endemic areas is expressed given the various changes both natural and mostly man-made favoring habitat expansion.

Development of an effective alternative model for in vivo hypnozoite-induced relapse infection: A Japanese macaque (Macaca fuscata) model experimentally infected with Plasmodium cynomolgi.

In the present study, we demonstrate that the Japanese macaque (Macaca fuscata) can be used as an effective alternative in vivo model for investigating hypnozoite-induced relapsing infection caused by Plasmodium cynomolgi B strain, and that this model is comparable to the rhesus macaque model. Two female Japanese macaques (JM-1 and JM-2; aged 5 years; weighing about 4.0 kg) were used for the experiment. To produce sporozoites in mosquitoes, blood infected with P. cynomolgi B strain was collected from the donor monkey JM-1 and fed to approximately 200 mosquitoes using the standard artificial membrane feeding method. The isolated sporozoites (2 × 105) were intravenously inoculated into the JM-2 monkey, and the blood stage of the parasite was detected on day 8 after the infection. Chloroquine sulfate (CQ) was intramuscularly administered at a dosage of 6.0 mg/kg into the JM-2 monkey for 6 consecutive days from day 12 onward, after which the parasites disappeared from the peripheral blood. The first relapse occurred on day 26, which was treated again with CQ. Then, the second relapse occurred on day 44, which was cured by CQ treatment followed by the administration of primaquine phosphate (PQ) at a dosage of 1.0 mg/kg/day for 15 days. The JM-2 monkey was observed until 69 days after PQ administration, and there was no relapse during the entire follow-up period. We propose that the Japanese macaque model could contribute not only to drug screening for anti-hypnozoite activity, but could also be used as a powerful tool for investigating hypnozoite biology.

Detection of Schistosoma japonicum and Oncomelania hupensis quadrasi environmental DNA and its potential utility to schistosomiasis japonica surveillance in the Philippines.

In recent years, the prevalence and infection intensity of Schistosoma japonicum in endemic areas of the Philippines have significantly decreased due to yearly population-based treatment strategies, yet transmission rates remain high and uninterrupted. An important indicator of active disease transmission is the presence of Schistosoma japonicum and its snail intermediate host Oncomelania hupensis quadrasi in freshwater habitats. In this study, we sought to apply a species-specific real-time PCR (qPCR) assay for the detection of S. japonicum and O. hupensis quadrasi in freshwater samples using environmental DNA approach that can complement the commonly utilized malacological survey in determining potential transmission foci in order to have a more effective snail surveillance strategy for schistosomiasis japonica in endemic areas. The newly developed assay was specific to S. japonicum and O. hupensis quadrasi with no amplification detected against non-target trematode Fasciola spp. and snails such as Lymnaea spp., Pomacea canaliculata, and Melanoides spp. that typically co-exist in the same environment. The assay effectiveness was determined using 19 environmental water samples collected from Northern Samar (N = 5 sites), Leyte (N = 11 sites) and Compostela Valley (N = 3 sites) and compared to malacological survey for determining O. hupensis quadrasi snail colonies and snail crushing to visualize S. japonicum cercariae. TaqMan qPCR targeting a short fragment of the cytochrome c oxidase subunit 1 (cox1) gene was positive for S. japonicum in 9 sites, for O. hupensis quadrasi in 9 sites, and for both S. japonicum and O. hupensis quadrasi in 5 sampling sites. Moreover, it was able to detect O. hupensis quadrasi in 3 out of 12 sites found negative and 6 out of 7 sites found positive through malacological survey, and in 4 of the 5 snail sites positive for snails with cercariae. Overall, this method can complement malacological surveys for monitoring of schistosomes in endemic areas of the Philippines, especially those with high risk of human infection.

Case Report: Acute Amebic Colitis Triggered by Colonoscopy: Exacerbation of Asymptomatic Chronic Infection with Entamoeba histolytica Accompanied by Dysbiosis.

Recent data show that the gut microbiome plays a role in determining the clinical outcome of Entamoeba histolytica infection. We report the case of a patient who developed recurrent acute amebic colitis (second episode of acute colitis) after colonoscopy. Genotyping of E. histolytica revealed that she developed a second episode of acute amebic colitis with the same genotype as that of the first episode, indicating chronic infection had persisted asymptomatically for > 10 months between the first and second episodes. Analysis of the gut microbiome, in addition to the clinical findings, suggested that dysbiosis at colonoscopy induced the change in the clinical form of E. histolytica infection from asymptomatic chronic infection to symptomatic colitis.

Analysis of Environmental DNA and Edaphic Factors for the Detection of the Snail Intermediate Host Oncomelania hupensis quadrasi.

BACKGROUND: The perpetuation of schistosomiasis japonica in the Philippines depends to a major extent on the persistence of its intermediate host Oncomelania hupensis quadrasi, an amphibious snail. While the malacological survey remains the method of choice in determining the contamination of the environment as evidenced by snails infected with schistosome larval stages, an emerging technology known as environmental DNA (eDNA) detection provides an alternative method. Previous reports showed that O. hupensis quadrasi eDNA could be detected in water, but no reports have been made on its detection in soil. METHODS: This study, thus focused on the detection of O. hupensis quadrasi eDNA from soil samples collected from two selected schistosomiasis-endemic barangays in Gonzaga, Cagayan Valley using conventional and TaqMan-quantitative (qPCR) PCRs. RESULTS: The results show that qPCR could better detect O. hupensis quadrasi eDNA in soil than the conventional method. In determining the possible distribution range of the snail, basic edaphic factors were measured and correlated with the presence of eDNA. The eDNA detection probability increases as the pH, phosphorous, zinc, copper, and potassium content increases, possibly indicating the conditions in the environment that favor the presence of the snails. A map was generated to show the probable extent of the distribution of the snails away from the body of the freshwater. CONCLUSION: The information generated from this study could be used to determine snail habitats that could be possible hotspots of transmission and should, therefore, be targeted for snail control or be fenced off from human and animal contact or from the contamination of feces by being a dumping site for domestic wastes.

Detection of canine Schistosoma japonicum infection using recombinant thioredoxin peroxidase-1 and tandem repeat proteins.

Humans and dogs live very close together and share various pathogens causing zoonotic parasitoses like schistosomiasis. A previous population genetics study done for schistosomes in the Philippines suggested that there is a high transmission level of Schistosoma japonicum among humans and dogs proving that the latter are important reservoirs for this zoonotic parasite. A more sensitive and specific test detecting schistosome infection in dogs will therefore strengthen the zoonotic surveillance, which might help in the possible elimination of this ancient disease. In this study, recombinant thioredoxin peroxidase-1 (SjTPx-1) and tandem repeat proteins (Sj1TR, Sj2TR, Sj4TR, Sj7TR) previously tested on human and water buffalo samples were used to assess its diagnostic applicability to dogs. Fifty-nine dog serum and stool samples were collected in the schistosomiasis-endemic municipalities of Calatrava, Negros Occidental and Catarman, Northern Samar in the Philippines and examined using the ELISA as compared to microscopy and fecal sample-based PCR. Samples positive for Babesia gibsoni and Dirofilaria immitis were also used to check for cross-reaction. Results showed that SjTPx-1 (80% sensitivity, 92.3% specificity) and Sj7TR (73.3% sensitivity, 92.3% specificity) have good potentials for diagnosing S. japonicum infection in dogs. These diagnostic antigens will therefore improve the surveillance in the transmission of the parasites from dogs to humans.

Schistosoma japonicum cathepsin B as potential diagnostic antigen for Asian zoonotic schistosomiasis.

In this study, the diagnostic value of Schistosoma japonicum cathepsin B (SjCatB) was evaluated as an antigen for the early detection of S. japonicum infection. SjCatB is a key protease used by the cercaria to penetrate the intact skin of the host for transdermal infection. The early exposure of the host's immune system to this enzyme may elicit early production of antibodies against this molecule. Therefore, the recombinant SjCatB (rSjCatB) was expressed in Escherichia coli with N-terminal 6xHis-tag. rSjCatB was tested for its performance as a diagnostic antigen using indirect enzyme-linked immunosorbent assay (ELISA) with sera from experimentally infected mice collected at > 8 weeks post-infection. Showing 100% sensitivity and 95.0% specificity in the ELISA, rSjCatB was then evaluated with sera from experimentally infected mice collected at 1-7 weeks post-infection to determine how early the antibodies can be detected. Results showed that as early as 6 weeks post-infection, 2 of the 3 infected mice were found to be positive with the antibodies against SjCatB. Furthermore, the potential of the recombinant antigen in detecting human schistosomiasis was evaluated with archived serum samples collected from individuals who had been diagnosed with S. japonicum infection by stool examination. Results showed 86.7% sensitivity and 96.7% specificity suggesting its high diagnostic potential for human schistosomiasis. In addition, SjCatB showed minimal cross-reaction with the sera collected from patients with other parasitic diseases. In conclusion, the results of this study suggest that SjCatB will be useful in the development of a sensitive and specific early detection test for S. japonicum infection.

Occurrence of Bovine Cysticercosis in Two Regions of the State of Tocantins-Brazil and the Importance of Pathogen Identification.

Bovine cysticercosis, caused by Taenia saginata metacestodes, is the cause of significant economic losses to the meat production chain by condemnation and downgrading of infected carcasses. It is also a public health issue causing human taeniasis. This study evaluated the occurrence of bovine cysticercosis at the meat inspection procedures in slaughterhouses of south and north regions of the Tocantins State in Brazil. Specimens identified as cysts of T. saginata were collected and analyzed by molecular (PCR) and histopathological techniques. The cysts were collected from March to December of 2010 in slaughterhouses located in the cities of Alvorada (South) and Araguaína (North). The frequency of cystic lesions during the study was 0.033% (53/164,091) with 69.81% of calcified lesions and 30.9% of live cysts at meat inspection. From 14 samples submitted to molecular analysis, 28.57% (4/14) were positive for T. saginata. The histopathological analysis of the non-T. saginata samples showed lesions suggestive of granuloma and hydatid disease. The results indicated that the identification of the etiological agent is difficult by macroscopic inspection, emphasizing the need to associate specific diagnostic methods at meat inspection in abattoirs. In addition, species-specific PCR would be an effective tool for diagnosis, monitoring, and identifying cysticercosis, assisting the conventional tests.

Usefulness of environmental DNA for detecting Schistosoma mansoni occurrence sites in Madagascar.

OBJECTIVES: Schistosomiasis is an important disease in Madagascar, and several studies on the disease have focused on the occurrence of the parasite in humans. However, the range of the pathogen in the environment and its impact on human infection is difficult to predict. An environmental DNA (eDNA) detection system for Schistosoma mansoni was developed to improve schistosomiasis eco-epidemiology studies. METHODS: Primers and probes were designed and tested in experimental biotopes. The field study was conducted in Maevatanana District of Madagascar. Seven water sources with human use were sampled, with a total of 21 water samples collected. Snails were collected, and patients were examined by ultrasound to determine the occurrence of schistosomiasis in the study area. RESULTS: One water source with active transmission was identified through the detection of S. mansoni eDNA in the water and the intermediate host Biomphalaria pfeifferi collected from the same water source. People with clinical schistosomiasis were found in the area, reinforcing the findings. CONCLUSIONS: The application of eDNA in eco-epidemiology enables the determination of hot spots and safe spots in endemic areas, constituting an alternative ecological tool for follow-up and monitoring of control programs for schistosomiasis, and contributing information on water safety for improving the standard of living of the people in endemic areas.

Utilization of real time PCR for the assessment of egg burden in the organs of Schistosoma japonicum experimentally infected mice.

Schistosoma japonicum, causing zoonotic intestinal schistosomiasis, is found in China, the Philippines and parts of Indonesia. Severe disease manifestations are basically due to the deposition of eggs in some vital organs such as the liver, spleen and brain. Traditionally, histopathological microscopic examination of the egg burden was used to evaluate the intensity of infection in the affected organs. However, this technique is laborious, time-consuming and requires trained personnel. In this study, real time PCR targeting the mitochondrial NADH dehydrogenase I gene was used to compare with microscopic examination of tissue sections in evaluating the egg burdens in different affected organs. Livers, spleens and brains of the S. japonicum infected mice after 8 and 18 weeks post-infection (p.i) were harvested and examined. Results showed that there were statistically significant correlations between the egg burden evaluated by tissue section examination, and the Ct values of the real time PCR of livers with heavy egg burden at 8 (r = -0.81) and 18 (r = -0.80) weeks p.i. Furthermore, a correlation (r = -0.56) between the egg burden assessed by the microscopic examination and Ct value of the real time PCR of spleens with moderate egg burden after 18 weeks p.i and not 8 weeks p.i was also observed. Brains with low egg burden showed no schistosome eggs in the microscopic examination, however one sample tested positive by real time PCR. These results suggested that real time PCR is useful in evaluating schistosome egg burden in the organs of the experimentally infected mice model that will give further insights into the pathology of schistosomiasis.

Evaluation of Schistosoma japonicum thioredoxin peroxidase-1 as a potential circulating antigen target for the diagnosis of Asian schistosomiasis.

Asian schistosomiasis caused by Schistosoma japonicum is a serious zoonotic disease endemic in China, the Philippines and parts of Indonesia. Mass drug administration in endemic areas resulted to decline in disease severity and intensity. The low intensity of infection limits the use of current parasitological methods for schistosomiasis diagnosis. Detection of parasite circulating antigens might provide more informative result as it may indicate the true status of infection. In this study, S. japonicum thioredoxin peroxidase-1 (SjTPx-1) a 22 kDa secreted antioxidant enzyme expressed throughout the life stages of the parasite was evaluated for its potential use as a biomarker for schistosomiasis japonica infection. Rabbit polyclonal antibody and mouse monoclonal antibodies (mAbs) were raised against the recombinant SjTPx-1 (rSjTPx-1). The antibodies produced against the recombinant antigen was confirmed to detect the native SjTPx-1 in crude adult worm lysate. Likewise, the specific binding of mAbs to parasite TPx-1 and not to mammalian peroxiredoxin-1 orthologues was also confirmed. The double antibody sandwich ELISA developed in this study was able to detect at least 1 ng/ml of rSjTPx-1. In addition, this method was able to detect the antigen from all serum samples of experimentally infected rabbit and mice. The diagnostic potential of SjTPx-1 in human clinical samples was also evaluated, in which 4 out of 10 stool-confirmed serum samples had detectable levels of the antigen. The results suggest that SjTPx-1 can be a potential biomarker for Asian zoonotic schistosomiasis.

Geographic strain differentiation of Schistosoma japonicum in the Philippines using microsatellite markers.

BACKGROUND: Microsatellites have been found to be useful in determining genetic diversities of various medically-important parasites which can be used as basis for an effective disease management and control program. In Asia and Africa, the identification of different geographical strains of Schistosoma japonicum, S. haematobium and S. mansoni as determined through microsatellites could pave the way for a better understanding of the transmission epidemiology of the parasite. Thus, the present study aims to apply microsatellite markers in analyzing the populations of S. japonicum from different endemic areas in the Philippines for possible strain differentiation. METHODOLOGY/ PRINCIPAL FINDINGS: Experimental mice were infected using the cercariae of S. japonicum collected from infected Oncomelania hupensis quadrasi snails in seven endemic municipalities. Adult worms were harvested from infected mice after 45 days of infection and their DNA analyzed against ten previously characterized microsatellite loci. High genetic diversity was observed in areas with high endemicity. The degree of genetic differentiation of the parasite population between endemic areas varies. Geographical separation was considered as one of the factors accounting for the observed difference between populations. Two subgroups have been observed in one of the study sites, suggesting that co-infection with several genotypes of the parasite might be present in the population. Clustering analysis showed no particular spatial structuring between parasite populations from different endemic areas. This result could possibly suggest varying degrees of effects of the ongoing control programs and the existing gene flow in the populations, which might be attributed to migration and active movement of infected hosts from one endemic area to another. CONCLUSIONS/ SIGNIFICANCE: Based on the results of the study, it is reasonable to conclude that genetic diversity could be one possible criterion to assess the infection status in highly endemic areas. Genetic surveillance using microsatellites is therefore important to predict the ongoing gene flow and degree of genetic diversity, which indirectly reflects the success of the control program in schistosomiasis-endemic areas.

The role of domestic dogs in the transmission of zoonotic helminthes in a rural area of Mekong river basin.

Dogs have been bred since ancient times for companionship, hunting, protection, shepherding and other human activities. Some canine helminth parasites can cause significant clinical diseases in humans as Opisthorchis viverrini causing cholangiocarcinoma in Southeast Asian Countries. In this study, socio-cultural questionnaire, canine parasitological analysis, necropsy, parasite molecular confirmation and dog roaming data were evaluated in Savannakhet, Lao-PDR, a typical Mekong Basin area. Dog owners comprised 48.8% of the studied population, with 61.2% owning one dog, 25.1% 2 dogs, 8.5% 3 dogs and 1.8% owning more than 4 dogs. Data from GPS logger attached to dogs showed they walked from 1.4 to 13.3 km per day, covering an area of 3356.38m2 average, with a routine of accessing water sources. Thirteen zoonotic helminth species were observed. Causative agents of visceral and cutaneous larva migrans occurred in 44.1% and 70% of the samples respectively. Spirometra erinaceieuropaei was detected in 44.1% of samples. Importantly, O. viverrini was found in 8.8% of samples. Besides the known importance of dogs in the transmission of Ancylostoma spp., Toxocara spp. and S. erinaceieuropaei, the observed roaming pattern of dogs confirmed it as an important host perpetuating O. viverrini in endemic areas; their routine access to waterbodies may spread O. viverrini eggs in a favorable environment for the fluke development, facilitating the infection of fishes, and consequently infecting humans living in the same ecosystem. Therefore, parasitic NTDs control programs in humans should be done in parallel with parasite control in animals, especially dogs, in the Mekong River basin area.

First Record of Paramphistomes Fischoederius cobboldi and Paramphistomum epiclitum Detected in Bovine Rumen from a Local Market of Savannakhet Province, Lao PDR.

In the present study, we report on the occurrence of paramphistomes, Fischoederius cobboldi and Paramphistomum epiclitum, in Lao PDR with the basis of molecular data. Parasite materials were collected from bovines bred in Ban Lahanam area, Savannakhet Province, Lao PDR at Lahanam public market. Morphological observations indicated 2 different species of paramphistomes. The mitochondrial gene cox1 of the specimens was successfully amplified by PCR and DNA sequencing was carried out for diagnosis of 11 specimens. Pairwise alignment of cox1 sequences were performed and confirmed F. cobboldi and P. epiclitum infecting bovines in Laos. Although there were many limiting points, as the small number of worm samples, and the restricted access of the animal host materials, we confirmed for the first time that 2 species of paramphistomes, F. cobboldi and P. epiclitum, are distributed in Lao PDR. More studies are needed to confirm the paramphistome species present in Savannakhet and its hosts to clear the natural history of these parasites of ruminants in the region and measure the impact of this parasite infection in the life and health of the local people.

Molecular characterization of sympatrically distributed Neotricula aperta-like snails in the Mekong River, Kratie, Cambodia.

Fifty-six samples of Neotricula aperta-like snails were collected from six locations in Cambodia. Their mitochondrial cytochrome c oxidase subunit 1 (cox1) sequences were examined using haplotype network and neighbor-joining (NJ) tree analysis. Twenty-seven haplotypes (H1-H27) were observed and were divided into two different groups/lineages. Of 27, 17 haplotypes (H11-H27) were clustered with the reference samples of the γ-race N. aperta. The remaining 10 haplotypes (H1-H10) were clustered in a separate group/lineage, differing from the reference samples of the α-, ß-, and γ-race N. aperta, suggesting a new lineage belonging the genus Neotricula. Our results show that both the γ-race and a new lineage were sympatrically present approximately 60 km upstream of the Mekong River near the Kratie port, Cambodia. Further morphological and molecular studies are required to confirm the taxonomic status of this new, unidentified lineage.