Comparing PCR techniques against conventional cercarial shedding methods for detecting Schistosoma mansoni infection in Biomphalaria snails.

The detection of Schistosoma mansoni infection in both its intermediate (snail) and definitive (human) hosts is useful in providing information on the transmission of schistosomiasis. Three pairs of previously designed PCR primers (SM1-7, SMF/R & ND5) used for the detection of S. mansoni infection were tested. We assess the utility of each of these primer sets for detecting S. mansoni infection both in artificially exposed laboratory bred Biomphalaria glabrata, and field infected African Biomphalaria sudanica and Biomphalaria pfeifferi. Two of the three primer sets (SMF/R & ND5) detected S. mansoni infection in snails, but amplification of S. mansoni DNA with SM1-7 was unreliable. For the artificially exposed laboratory bred B. glabrata snails, SMF/R and ND5 both detected infection in more snails than the cercarial shedding method. Infection detection rates were 62.4% for ND5, 57.1% for SMF/R and 50.4% using traditional cercarial shedding methods. Both SMF/R and ND5 detected S. mansoni infection in 91% of snails observed shedding cercariae, increasing to 98.5% when low stringency PCR methods were used. When comparing each of the detection methods using a Bayesian latent class analysis model, ND5 had the highest detection sensitivity and negative predictive value (NPV), while SMF/R had the highest detection specificity and positive predictive value (PPV). In field collected Biomphalaria snails, ND5 detected S. mansoni infection in 21 of 24 snails categorised as shedding S. mansoni cercariae and 4 of 24 snails categorised as shedding non-S. mansoni cercariae, while SMF/R detected infection in 18 of 24 snails categorised as shedding S. mansoni cercariae and in 3 of 24 snails categorised as shedding non-S. mansoni cercariae. All SMF/R and ND5 PCR products were shown to be S. mansoni indicating that these field snails must have been infected with both S. mansoni and cercariae from other Schistosoma species. This indicates that the two primer sets are specific for S. mansoni and will not amplify non-S. mansoni species when used at their recommended annealing temperatures. Both the SMF/R and ND5 primers effectively detected S. mansoni infection in three Biomphalaria species and have improved detection sensitivity over cercarial shedding.

Assessing S. mansoni prevalence in Biomphalaria snails in the Gombe ecosystem of western Tanzania: the importance of DNA sequence data for clarifying species identification.

BACKGROUND: Snails are essential for the transmission and maintenance of schistosomiasis in endemic areas, as they serve as intermediate hosts for schistosome parasites. A clear understanding of the snail species present, their local distribution and infection status is therefore a prerequisite for effective control of schistosomiasis. The purpose of this study was to establish the infection status and distribution of Schistosoma mansoni in snails in the Gombe area along the shores of Lake Tanganyika in western Tanzania, using both detection of cercarial shedding and molecular approaches. METHODS: Snails were collected from streams located close to human settlements in Gombe National Park, as well as from nearby villages (Kiziba, Mtanga, Mwamgongo and Bugamba) and the largest town in the region (Kigoma). Snails were individually exposed to light to induce shedding of schistosome larvae, which were examined using a compound light microscope. Additionally, the internal transcribed spacer (ITS) region of the ribosomal RNA gene cluster was simultaneously amplified in both snails and their trematodes using a single polymerase chain reaction (PCR) and sequenced to confirm species identification. RESULTS: Snails morphologically identified as Biomphalaria pfeifferi were present in all streams except at Mtanga but their distribution was patchy in both time and space. Sequencing of PCR products indicated that not all snails were B. pfeifferi. None of the snails from Gombe or Bugamba shed schistosome larvae, while larvae were shed at all other sites. Overall, an infection prevalence of only 12% was observed in snails based on cercarial shedding. While 47% of the snails were PCR-positive for the 500 bp ITS fragment, which was predicted to indicate infection with S. mansoni, sequence data demonstrated that these bands are not species-specific and can be amplified from other trematode infections. In addition, a 1000 bp band was amplified in 14% of samples, which was identified as a trematode in the family Derogenidae. CONCLUSIONS: The results support the previous assumption that B. pfeifferi snails may be involved in transmitting schistosomiasis in the area but suggest that the community structure of both snails and trematodes may be more complicated than previously thought. This emphasises the importance of confirming species identifications using sequencing, rather than relying only on PCR-based diagnostics or cercarial shedding.

Opisthorchis viverrini infection in the snail and fish intermediate hosts in Central Vietnam.

Opisthorchis viverrini, a carcinogenic fish borne fluke, requires freshwater snails and fish as intermediate hosts. Opisthorchiasis is endemic in parts of Southeast Asia, including Central and South Vietnam. In this region the transmission by intermediate hosts has received little attention. Therefore, freshwater snails and wild fish from Bau My Tho, an opisthorchiasis endemic area in Binh Dinh Province were collected for examination of O. viverrini cercariae and metacercariae, respectively. A total of 12,000 snails belonging to six families, of which 1616 Bithynia snails representing Bithynia siamensis goniomphalos and Bithynia funiculata; as well as 754 fish representing 12 species were examined. Shedding of O. viverrini cercariae was observed only in B. s. goniomphalos and B. funiculata, at infection rates of 0.86% and 0.14%, respectively. O. viverrini infection in Bithynia spp. was significantly associated with the habitat but not with the species and the shell size of Bithynia spp. O. viverrini metacercariae were found in 10 fish species representing both Cyprinidae and non-Cyprinidae families. The prevalence of O. viverrini infection in fish was significantly associated with species. Carassius auratus, a fish species commonly eaten raw, Rasbora aurotaenia and Puntius brevis had the highest prevalence of 74.0%, 55.8% and 31.6%, respectively. Sharing of the same snail and fish intermediate host species was found for O. viverrini and a O. viverrini duck-genotype that are sympatric in the study region. This study is the first to report on the intermediate host species of O. viverrini in Central Vietnam and indicates a high risk of acquiring opistorchiasis when eating raw fish dishes.

DISCRIMINATION 28S RIBOSOMAL GENE OF TREMATODE CERCARIAE IN SNAILS FROM CHIANG MAI PROVINCE, THAILAND.

Trematode cercariae are commonly found in many freshwater gastropods. These cercariae can serve to identify the occurrence of such trematodes as Centrocestus formosanus, Haplorchis taichui, Haplorchoides sp, and Stellantchasmus falcatus, which are important parasites in Chiang Mai Province, Thailand. As the species of these cercariae cannot be identified accurately based on morphology, this study employed sequencing of a fragment of 28S ribosomal DNA and phylogenetic analysis to identify the trematode cercariae found in freshwater gastropods in Chiang Mai Province. Eight types of trematode cercariae were identified, namely, distome cercaria (grouped with Philophthalmus spp clade), echinostome cercaria (grouped with Echinostoma spp clade), furcocercous cercaria (grouped with Posthodiplostomum sp/Alaria taxideae/Hysteromorpha triloba clade), monostome cercaria (grouped with Catatropis indicus clade), parapleurolophocercous cercaria (grouped with Haplorchoides sp clade), pleurolophocercous cercaria (grouped with Centrocestusformosanus clade), transversotrema cercaria (grouped with Transversotrema spp clade), and xiphidiocercaria (grouped with Prosthodendrium spp clade). These results provide important information that can be used for identifying these parasites in epidemiological surveys.

Biomass and productivity of trematode parasites in pond ecosystems.

1. Ecologists often measure the biomass and productivity of organisms to understand the importance of populations and communities in the flow of energy through ecosystems. Despite the central role of such studies in the advancement of freshwater ecology, there has been little effort to incorporate parasites into studies of freshwater energy flow. This omission is particularly important considering the roles that parasites sometimes play in shaping community structure and ecosystem processes. 2. Using quantitative surveys and dissections of over 1600 aquatic invertebrate and amphibian hosts, we calculated the ecosystem-level biomass and productivity of trematode parasites alongside the biomass of free-living aquatic organisms in three freshwater ponds in California, USA. 3. Snails and amphibian larvae, which are both important intermediate trematode hosts, dominated the dry biomass of free-living organisms across ponds (snails = 3.2 g m(-2); amphibians = 3.1 g m(-2)). An average of 33.5% of mature snails were infected with one of six trematode taxa, amounting to a density of 13 infected snails m(-2) of pond substrate. Between 18% and 33% of the combined host and parasite biomass within each infected snail consisted of larval trematode tissue, which collectively accounted for 87% of the total trematode biomass within the three ponds. Mid-summer trematode dry biomass averaged 0.10 g m(-2), which was equal to or greater than that of the most abundant insect orders (coleoptera = 0.10 g m(-2), odonata = 0.08 g m(-2), hemiptera = 0.07 g m(-2) and ephemeroptera = 0.03 g m(-2)). 4. On average, each trematode taxon produced between 14 and 1660 free-swimming larvae (cercariae) infected snail(-1) 24 h(-1) in mid-summer. Given that infected snails release cercariae for 3-4 months a year, the pond trematode communities produced an average of 153 mg m(-2) yr(-1) of dry cercarial biomass (range = 70-220 mg m(-2) yr(-1)). 5. Our results suggest that a significant amount of energy moves through trematode parasites in freshwater pond ecosystems, and that their contributions to ecosystem energetics may exceed those of many free-living taxa known to play key roles in structuring aquatic communities.

Patterns of intestinal schistosomiasis among mothers and young children from Lake Albert, Uganda: water contact and social networks inferred from wearable global positioning system dataloggers.

The establishment of a national control programme (NCP) in Uganda has led to routine treatment of intestinal schistosomiasis with praziquantel in the communities along Lake Albert. However, because regular water contact remains a way of life for these populations, re-infection continues to mitigate the sustainability of the chemotherapy-based programme. A six-month longitudinal study was conducted in one Lake Albert community with the aim of characterizing water contact exposure and infection among mothers and their young preschool-aged children as the latter are not yet formally included within the NCP. At baseline the cohort of 37 mothers, 36 preschool-aged children had infection prevalences of 62% and 67%, respectively, which diminished to 20% and 29%, respectively, at the 6-month post-treatment follow-up. The subjects wore global positioning system (GPS) datalogging devices over a 3-day period shortly after baseline, allowing for the estimation of time spent at the lakeshore as an exposure metric, which was found to be associated with prevalence at follow-up (OR = 2.1, P = 0.01 for both mothers and young children and odds ratio (OR) = 4.4, P = 0.01 for young children alone). A social network of interpersonal interactions was also derived from the GPS data, and the exposures were positively associated both with the number and duration of peer interaction, suggesting the importance of socio-cultural factors associated with water contact behaviour. The findings illustrate reduction in both prevalence and intensity of infection in this community after treatment as well as remarkably high rates of water contact exposure and re-infection, particularly among younger children. We believe that this should now be formally considered within NCP, which may benefit from more in-depth ethnographic exploration of factors related to water contact as this should provide new opportunities for sustaining control.

Reversing the resistance phenotype of the Biomphalaria glabrata snail host Schistosoma mansoni infection by temperature modulation.

Biomphalaria glabrata snails that display either resistant or susceptible phenotypes to the parasitic trematode, Schistosoma mansoni provide an invaluable resource towards elucidating the molecular basis of the snail-host/schistosome relationship. Previously, we showed that induction of stress genes either after heat-shock or parasite infection was a major feature distinguishing juvenile susceptible snails from their resistant counterparts. In order to examine this apparent association between heat stress and snail susceptibility, we investigated the effect of temperature modulation in the resistant snail stock, BS-90. Here, we show that, incubated for up to 4 hrs at 32°C prior to infection, these resistant snails became susceptible to infection, i.e. shedding cercariae at 5 weeks post exposure (PE) while unstressed resistant snails, as expected, remained resistant. This suggests that susceptibility to infection by this resistant snail phenotype is temperature-sensitive (ts). Additionally, resistant snails treated with the Hsp 90 specific inhibitor, geldanamycin (GA) after heat stress, were no longer susceptible to infection, retaining their resistant phenotype. Consistently, susceptible snail phenotypes treated with 100 mM GA before parasite exposure also remained uninfected. These results provide direct evidence for the induction of stress genes (heat shock proteins; Hsp 70, Hsp 90 and the reverse transcriptase [RT] domain of the nimbus non-LTR retrotransposon) in B. glabrata susceptibility to S. mansoni infection and characterize the resistant BS-90 snails as a temperature-sensitive phenotype. This study of reversing snail susceptibility phenotypes to S. mansoni provides an opportunity to directly track molecular pathway(s) that underlie the B. glabrata snail's ability to either sustain or destroy the S. mansoni parasite.

[Effect of modified crushing-cercariae escaping method on detection of infected Oncomelania snails in field].

OBJECTIVE: To evaluate the effect of a modified crushing-cercariae escaping method on the detection of infected Oncomelania snails in the field. METHODS: A snail survey was carried out in Jingzhou City in the spring of 2011, and the villages were randomly divided into several groups, the coincidence rates, detection rates and the labour cost of the modified crushing-cercariae escaping method and crushing method were compared. RESULTS: A total of 14 snail spots were surveyed, and the coincidence rate of the two methods was 100%. In the spring snail survey, 539 villages and 3 536 spots with snails were detected by the modified crushing-cercariae escaping method, and 671 villages and 11 375 spots with snails were detected by the crushing method. The detection rates of villages with infected snails of the two methods were 25.79% and 28.46%, respectively, the difference between them was not statistically significant (chi2 = 1.079 5, P > 0.05); and those of spots with infected snails were 5.57% and 3.66%, respectively, which had no significant difference between them (chi2 = 95.464 1, P < 0.01). Compared with the crushing method, the modified crushing-cercariae escaping method reduced labour cost by 87.86%, and saved 12.95 person-days per township. CONCLUSION: The modified crushing-cercaria escaping method can identify the spots with infected snails quickly and accurately with less labour cost, therefore, it is suitable for detecting environments with infected snails in batch in endemic areas.