RESUMEN
Objective: To develop the multiplex PCR method based on the internal transcribed spacer 2 to discriminate the intestinal trematodes, Centrocestus caninus (C. caninus), and Stellantchasmus falcatus (S. falcatus). Methods: Four species of heterophyid trematodes including C. caninus, S. falcatus, Haplorchis taichui and Haplorchoides sp. were amplified and the specific primer was designed based on the internal transcribed spacer 2 region. Two specific primers were used to validate the optimized PCR conditions:the specificity test and the sensitivity test. Results: Both of these specific primers confirmed the specificity through multiplex PCR reaction which generated both PCR products (231 and 137 bp) in the mixed DNA template of C. caninus and S. falcatus with no cross-reaction with other heterophyid trematodes. The optimum annealing temperature of both primers was 54–59 ?C. The sensitivity test used the two-fold serial dilution DNA template, which was concentrated between 10 and 0.3125 ng/mL. The lowest concentration of the DNA template of this multiplex PCR was 2.5 ng/mL. Conclusions: The technique described here proved to be a species-specific technique and was found to be a rapid method for the diagnosis of C. caninus and S. falcatus in terms of the larval and adult stages in intermediate and/or definitive hosts in the endemic area.
RESUMEN
Objective: To investigate the prevalence of cercarial trematode infection in snails and to examine the reconstruction of the phylogenetic relationship to explain the molecular system of cercarial stage trematodes to estimate the infection rate of in the definite host from the Chao-Phraya Basin. Methods: The snails were collected from 10 provinces of the Chao-Phraya Basin, Thailand by stratified sampling method. The snails were examined for cercarial infection by the crushing method. All DNA specimens were amplified with internal transcribed spacer 3 (ITS3) and ITS4 primer based on PCR technique. The sequence data were aligned and used to reconstruct the phylogenetic tree by unweighted pair-group method with arithmetic means with 10 000 bootstraps. Results: The overall rate of cercarial infection was found to be 5.90%(122/2 067). Snails in the family Thiaridae were found to be in the highest prevalence followed by Lym-naeidae, Bithyniidae, Planorbidae, Viviparidae, and Ampullariidae, respectively, while the Buccinidae family (Clea helena) did not reveal any infections. The frequently found species of cercariae were parapleurolophocercous cercariae, cercariae and megarulous cercariae. The monophyletic tree separated the snails into five groups comprised of Heterophyidae, Strigeidae, Lecithodendriidae, Philophthalmidae and Echinostomatidae using the sequence of Angiostrongylus cantonensis as an out-group. Conclusions: This study was the first to report on cercarial infection in the Chao-Phraya Basin, Thailand. This revealed that a high variety of freshwater snails were infected by cercariae stage trematodes with a high prevalence. The sequence data of ITS2 can be used to investigate the phylogenetic relationships of trematodes at the family level and in each clade of different families separated by the definitive hosts.