A Population Growth Trend Analysis for Neotricula aperta,the Snail Intermediate Host of Schistosoma mekongi,after Construction of the Pak-Mun Dam

Background

The Pak-Mun dam is a controversial hydro-power project on the Mun River in Northeast Thailand. The dam is sited in a habitat of the freshwater snail Neotricula aperta, which is the intermediate host for the parasitic blood-fluke Schistosoma mekongi causing Mekong schistosomiasis in humans in Cambodia and Laos. Few data are available which can be used to assess the effects of water resource development on N. aperta. The aim of this study was to obtain data and to analyze the possible impact of the dam on N. aperta population growth.

Methodology/Principal Findings

Estimated population densities were recorded for an N. aperta population in the Mun River 27 km upstream of Pak-Mun, from 1990 to 2011. The Pak-Mul dam began to operate in 1994. Population growth was modeled using a linear mixed model expression of a modified Gompertz stochastic state-space exponential growth model. The N. aperta population was found to be quite stable, with the estimated growth parameter not significantly different from zero. Nevertheless, some marked changes in snail population density were observed which were coincident with changes in dam operation policy.

Conclusions/Significance

The study found that there has been no marked increase in N. aperta population growth following operation of the Pak-Mun dam. The analysis did indicate a large and statistically significant increase in population density immediately after the dam came into operation; however, this increase was not persistent. The study has provided the first vital baseline data on N. aperta population behavior near to the Pak-Mun dam and suggests that the operation policy of the dam may have an impact on snail population density. Nevertheless, additional studies are required for other N. aperta populations in the Mun River and for an extended time series, to confirm or refine the findings of this work.     

Sex chromosome differences among the three races (alpha, beta, gamma) of the snail intermediate host of Schistosoma mekongi, Neotricula aperta

Chromosome analyses were performed on three races (alpha, beta, gamma) of Neotricula aperta which were previously reported to show variation in the chromosome number and pairing in meiosis. We used an air-drying method, which was more reliable for chromosome preparations from fresh animal tissues than a squash method. Each of the races had the same number of chromosomes, 2n=33 for males, and 2n=34 for females. The sex determination system was XO type (male: 32+X, female: 32+XX). The sex chromosome of each race was distinct in its morphology, but not in its length. Karyotyping revealed that the X-chromosome of the alpha race was metacentric (M), whereas it was acrocentric (A) in the beta and the gamma races. An association between the X-chromosome and a small autosome in meiosis suggested that a neo-Y chromosome probably lies in the terminal region of the small autosome.     

The pig as a host for Schistosoma mekongi in Laos.

A survey of helminths in domestic pigs was conducted in Khong District, Laos, to elucidate if these domestic animals could act as definitive hosts for Schistosoma mekongi and to obtain a general overview of their helminthological infection status. Fecal samples were collected from 98 pigs. Twelve pigs (12.2%) were found to excrete S. mekongi eggs. Infection was confirmed by detection of S. mekongi eggs in tissues of liver, rectum, and cecum of 2 pigs. A total of 75.8% of the pigs was infected with 1 or more helminth species. This study showed that pigs may act as a definitive host for S. mekongi.     

The evaluation of control measures against Schistosoma mekongi in Cambodia by a mathematical model

We constructed a mathematical model for the transmission of Schistosoma mekongi in Cambodia. The simulation of the model will be instrumental in planning schistosomiasis control measures. The model includes two definitive hosts, humans and dogs, as animal reservoirs. Dogs are recognized to play an important role in schistosomiasis transmission in Cambodia. For the purpose of dealing with age-specific prevalence and intensity of infection, the human population was classified into eight age categories in the model. To describe the seasonal fluctuation of the intermediate host population of S. mekongi, the "Post-Spate Survival" hypothesis was adopted for the population dynamics of Neotricula aperta present in the Mekong River. We carried out simulations to evaluate the effect of universal treatment (UT) and targeted mass treatment (TT) with praziquantel on the reduction in prevalence of S. mekongi. The simulations indicated that biyearly UT for 8 years or yearly TT for 5 years after three courses of yearly UT could reduce the prevalence to below 5% when a UT or TT coverage of 85% of inhabitants was achieved. The simulation suggested that the suppression of S. mekongi in Cambodia would be possible by UT or TT with a high coverage rate.     

Molecular Differentiation of Schistosoma japonicum and Schistosoma mekongi by Real-Time PCR with High Resolution Melting Analysis

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

Schistosoma mekongi and Schistosoma japonicum: Differences in the distribution of eggs in the viscera of mice

The difference in the distribution of Schistosoma eggs in the viscera has not been clearly elucidated in the two closely related species Schistosoma japonicum and Schistosoma mekongi. In this study, we quantitatively compared the distribution of eggs in mice infected with the two species. In S. mekongi-infected mice, 56.6% to 69.4% of total eggs were found in the distal small intestine 9 to 15 weeks after infection, while in S. japonicum-infected mice, 48.8% to 71.8% of eggs were found in the proximal small intestine during the same period. There were significantly more eggs in the liver in mice infected with S. japonicum than in those infected with S. mekongi. The number of adult worms recovered did not differ between the two species during the study period. The total number of eggs laid in the tissues also did not differ between the two species at 12 to 15 weeks postinfection, but in the earlier period the total number of eggs was significantly fewer in S. mekongi-infected than in S. japonicum-infected mice, suggesting the delayed maturation of the former compared with the latter. These results clearly show that S. japonicum and S. mekongi exhibit different oviposition behavior in their hosts.     

Metabolomics reveal alterations in arachidonic acid metabolism in Schistosoma mekongi after exposure to praziquantel

BackgroundMekong schistosomiasis is a parasitic disease caused by the blood-dwelling fluke Schistosoma mekongi. This disease contributes to human morbidity and mortality in the Mekong region, posing a public health threat to people in the area. Currently, praziquantel (PZQ) is the drug of choice for the treatment of Mekong schistosomiasis. However, the molecular mechanisms of PZQ action remain unclear, and Schistosoma PZQ resistance has been reported occasionally. Through this research, we aimed to use a metabolomic approach to identify the potentially altered metabolic pathways in S. mekongi associated with PZQ treatment.Methodology/Principal findingsAdult stage S. mekongi were treated with 0, 20, 40, or 100 μg/mL PZQ in vitro. After an hour of exposure to PZQ, schistosome metabolites were extracted and studied with mass spectrometry. The metabolomic data for the treatment groups were analyzed with the XCMS online platform and compared with data for the no treatment group. After low, medium (IC₅₀), and high doses of PZQ, we found changes in 1,007 metabolites, of which phosphatidylserine and anandamide were the major differential metabolites by multivariate and pairwise analysis. In the pathway analysis, arachidonic acid metabolism was found to be altered following PZQ treatment, indicating that this pathway may be affected by the drug and potentially considered as a novel target for anti-schistosomiasis drug development.Conclusions/SignificanceOur findings suggest that arachidonic acid metabolism is a possible target in the parasiticidal effects of PZQ against S. mekongi. Identifying potential targets of the effective drug PZQ provides an interesting viewpoint for the discovery and development of new agents that could enhance the prevention and treatment of schistosomiasis.     

Schistosoma mekongi: a prominent neutrophil chemotactic activity of egg antigen with reference to that of Schistosoma japonicum

Schistosoma mekongi causes granulomatous lesions around eggs deposited in the liver with neutrophil-rich inflammatory reactions in the early stage of the egg laying. To define the aspects of the typical pathogenesis of S. mekongi infection, we determined the difference between soluble egg antigen (SEA) from S. mekongi and S. japonicum with a focus on chemotactic factors for neutrophils or eosinophils. Mean volume and protein amount of S. mekongi eggs was 71 and 58% of those of Schistosoma japonicum eggs, respectively. Neutrophil chemotactic activity of S. mekongi SEA was about two times higher than that of S. japonicum. In contrast, eosinophil chemotactic activity of S. mekongi SEA was about half of that of S. japonicum SEA. Molecular analysis revealed that S. mekongi SEA contains higher molecular-weight components with a lower level of glycosylation, and this is likely to be related to the intense neutrophil chemotactic activity in comparison with S. japonicum SEA. The prominent chemotactic reactivity for neutrophils is likely to be involved in the typical pathogenesis of mekongi schistosomiasis.     

The status and conservation of Galliformes in Cambodia, Laos and Vietnam

Cambodia, Laos and Vietnam form a diverse and important region for the order Galliformes with at least 21 species, comprising 10 pheasants, seven partridges, three quails and one francolin. Several are endemic to the region and several are considered globally threatened. Information was collated for each species on the Indochinese range, habitat use and conservation status. This information was used to determine gross patterns in conservation requirements, prioritised partly through the global importance of the Indochinese population. The galliform species at most immediate risk in Indochina is probably Green Peafowl, through heavy hunting exacerbated by increasing human access to former wildernesses. The endemic Edwards’s Pheasant also merits specific conservation attention. Other forest species occur widely throughout surviving habitat, which remains landscape-scale despite serious losses in recent decades. Non-forest species, notably three quails (none endemic) remain very poorly known and threat levels cannot be assessed. For galliform conservation in Indochina by far the most important action is turning the existing declared protected areas networks into functional reality conserving a multitude of landscapes, each of hundreds of square kilometres, across the region. Past assessments of threat level to Indochinese forest Galliformes have been alarmist: many species are persisting in heavily-hunted fragments of the 10–50 sq. km size class, where many other forest vertebrates have been extirpated.     

Effects of praziquantel and artesunate on the tegument of adult Schistosoma mekongi harboured in mice

The effects of praziquantel and artesunate on the tegument of adult Schistosoma mekongi harboured in mice were compared using scanning electron microscopy (SEM). Forty-two mice infected with S. mekongi for 49 days were treated intragastrically with either 300 mg/kg praziquantel or 300 mg/kg artesunate. Mice were sacrificed 1 or 3 days post-treatment. Worms were collected by perfusion and examined by SEM. One to 3 days after administration of artesunate, the tegument of S. mekongi showed severe swelling, vacuolization, fusion of the tegumental ridges and loss or shortening of the spines on the trabeculae, collapse and peeling. Praziquantel induced similar tegumental alterations as those observed after administration of artesunate, but they were less severe. Three days post-treatment, there was evidence of recovery only in the case of praziquantel. The results of our study suggest that artesunate is more effective than praziquantel in causing tegumental damage in adult S. mekongi, and provides a basis for subsequent clinical trials.     

Schistosoma mekongi: the in vitro effect of praziquantel and artesunate on the adult fluke

The efficacy and tolerance of 80 microg/ml praziquantel (PZQ) and 40 microg/ml artesunate (ATS) against adult stage Schistosoma mekongi in vitro were investigated after 3, 6, 12, and 24h incubation by monitoring worm motility and compared tegumental changes using scanning electron microscopy (SEM). Thirty mice were infected with S. mekongi cercaria for 49 days. Adult worms were collected by perfusion method and prepared for in vitro study. Contraction and decreased motor activity were observed after as little as 3h incubation with PZQ and ATS. Some of the worms were immobile 12h after exposure, and died within 24h. The tegument of S. mekongi showed severe swelling, vacuolization and disruption, fusion of the tegumental ridges, collapse and peeling. After 12-24h incubation, PZQ induced similar but they less severe, tegumental changes to those observed after exposure to ATS. The direct observation of the fluke motility and SEM study suggest that ATS is more effective than PZQ in causing tegumental damage in adult S. mekongi, and provides a basis for subsequent clinical trials.     

Genetic differentiation of Macrognathus siamensis within the Mekong River between Laos and Cambodia

This study investigated the genetic variation in the Mastacembelidae fish Macrognathus siamensis by sequencing 1047 bp of the mitochondrial ND2 gene of 48 fish samples collected in the Mekong River. They were divided into 15 mtDNA haplotypes. The haplotype network and Bayesian tree revealed striking genetic differences between the Laos and Cambodian samples. The high pairwise F statistic values (0.93–0.97) also confirmed this genetic differentiation. Analysis of the molecular clock indicated that these two samples separated approximately four million years ago. Most likely, the genetic variation was influenced by immigration and isolation events as well as by environmental factors. Furthermore, the present study results could be valuable in identifying the conservation and resource management units of this species.     

Efficacy of Praziquantel against Schistosoma mekongi and Opisthorchis viverrini: A Randomized, Single-Blinded Dose-Comparison Trial

Background

Schistosomiasis and opisthorchiasis are of public health importance in Southeast Asia. Praziquantel (PZQ) is the drug of choice for morbidity control but few dose comparisons have been made.

Methodology

Ninety-three schoolchildren were enrolled in an area of Lao PDR where Schistosoma mekongi and Opisthorchis viverrini coexist for a PZQ dose-comparison trial. Prevalence and intensity of infections were determined by a rigorous diagnostic effort (3 stool specimens, each examined with triplicate Kato-Katz) before and 28–30 days after treatment. Ninety children with full baseline data were randomized to receive PZQ: the 40 mg/kg standard single dose (n = 45) or a 75 mg/kg total dose (50 mg/kg+25 mg/kg, 4 hours apart; n = 45). Adverse events were assessed at 3 and 24 hours posttreatment.

Principal Findings

Baseline infection prevalence of S. mekongi and O. viverrini were 87.8% and 98.9%, respectively. S. mekongi cure rates were 75.0% (95% confidence interval (CI): 56.6–88.5%) and 80.8% (95% CI: 60.6–93.4%) for 40 mg/kg and 75 mg/kg PZQ, respectively (P = 0.60). O. viverrini cure rates were significantly different at 71.4% (95% CI: 53.4–84.4%) and 96.6% (95% CI: not defined), respectively (P = 0.009). Egg reduction rates (ERRs) against O. viverrini were very high for both doses (>99%), but slightly lower for S. mekongi at 40 mg/kg (96.4% vs. 98.1%) and not influenced by increasing diagnostic effort. O. viverrini cure rates would have been overestimated and no statistical difference between doses found if efficacy was based on a minimum sampling effort (single Kato-Katz before and after treatment). Adverse events were common (96%), mainly mild with no significant differences between the two treatment groups.

Conclusions/Significance

Cure rate from the 75 mg/kg PZQ dose was more efficacious than 40 mg/kg against O. viverrini but not against S. mekongi infections, while ERRs were similar for both doses.

Trial Registration

Controlled-Trials.com ISRCTN57714676