Infections of Digenetic Trematode Metacercariae in Wrestling Halfbeak, Dermogenys pusilla from Bangkok Metropolitan Region in Thailand

This study aimed to investigate metacercarial infections in the wrestling halfbeak, Dermogenys pusilla, collected from Bangkok metropolitan region of Thailand. A total of 4,501 fish from 78 study sites were commonly examined with muscle compression and digestion methods (only head part of fish) during September 2017 to July 2018. The overall prevalence of metacercarial infection was 86.1% (3,876/4,501 individuals), and the mean intensity was 48.9 metacercariae per fish infected. Four species, i.e., Posthodiplostomum sp., Stellantchasmus falcatus, Cyathocotylidae fam. sp., and Centrocestus formosanus, of digenetic trematode metacercariae (DTM) were detected. The prevalences were 65.8%, 52.0%, 2.1%, and 1.2%, respectively and their mean intensities were 23.1, 51.6, 1.4, and 3.2 per fish infected, respectively. The seasonal prevalences were 81.0% in winter, 87.8% in summer and 87.4% in rainy, and the mean intensities were 38.9, 46.6, and 55.2 metacercariae per fish infected, respectively. Conclusively, it was confirmed that the wrestling halfbeak play the role of second intermediate hosts of 4 species of digenetic trematodes including S. falcatus and Posthodiplostomum sp. in Bangkok metropolitan region. And then the metacercariae of C. formosanus and Cyathocotylidae fam. sp. are to be first found in the wrestling halfbeak by this study.

Prevalence of cercarial infections in freshwater snails and morphological and molecular identification and phylogenetic trends of trematodes

Objective: To investigate the prevalence of cercarial infections in freshwater snails from several water sources in Nakhon Nayok, Nonthaburi, and Pathum Thani provinces of Central Thailand, and to reconstruct a phylogenetic tree for improved understanding of the relationships in the cercarial stage. Methods: The snail specimens were collected from 34 total sampling sites and investigated for cercarial infections using the crushing method. The cercarial specimens were classified and used for the phylogenetic tree analysis using the Internal Transcribed Spacer 2 (ITS2). Results: A total of 1 921 snail specimens were classified into five families and seven species. The results showed that four snail species were identified as intermediate hosts of the larval stages of trematodes, with an overall prevalence of infection of 2.45% (47/1 921). The infected snail specimens included five groups of the cercarial type: cercariaeum cercariae, echinostome cercaria, megalurous cercaria, parapleurolophocercous cercaria, and xiphidiocercariae. This is particularly true of xiphidiocercariae, which was found to be the dominant type among cercarial infections in bithyniid snails by approximately 38.00%. With regard to molecular identification, the phylogenetic tree was reconstructed using the neighbor-joining method with 10 000 bootstraps and separated the trematodes into three clades: Echinostomatoidea, Microphalloidea and Opisthorchioidea. Conclusions: The study reveals a high prevalence of cercarial infection for each cercarial type and maturation into a definite trematode genus and delineates morphological characteristics and evolutionary trends among each larval trematode in Nakhon Nayok, Nonthaburi and Pathum Thani provinces. In addition, the ITS2 sequence data of cercariae could be used to examine classification of these species at the family level.

Prevalence of Centrocestus formosanus Metacercariae in Ornamental Fish from Chiang Mai, Thailand, with Molecular Approach Using ITS2

The prevalence of Centrocestus formosanus metacercariae was investigated in ornamental fish purchased from a pet shop in Chiang Mai, Thailand, including Carassius auratus (goldfish), Cyprinus carpio (Koi), Poecilia latipinna (Sailfin Molly), Danio rerio (Zebrafish), and Puntigrus tetrazona (Tiger barb). The parasite species was identified by the morphology of worms as well as by a molecular approach using ITS2. The results showed that 50 (33.3%) of 150 fish examined were infected with the metacercariae. The highest prevalence was found in C. auratus (83.3%), and the highest intensity was noted in C. carpio (70.8 metacercariae/fish). The most important morphological character was the presence of 32–34 circumoral spines on the oral sucker. The phylogenetic studies using the rRNA ITS2 region revealed that all the specimens of C. formosanus in this study were grouped together with C. formosanus in GenBank database. This is the first report on ornamental fish, C. carpio, P. latipinna, D. rerio, and P. tetrazona, taking the role of second intermediate hosts of C. formosanus in Thailand. Prevention and control of metacercarial infection in ornamental fish is urgently needed.

Morphological Characteristics and Phylogenetic Trends of Trematode Cercariae in Freshwater Snails from Nakhon Nayok Province, Thailand

The prevalence of cercarial infection in freshwater snails and their evolutionary trends were studied in Nakhon Nayok province, Thailand. A total of 2,869 individual snails were examined for parasitic infections. The results showed that 12 snail species were found to host larval stages of trematodes with an overall prevalence of 4.7%. The infected specimens included 7 types at the cercarial stage; cercariae, megalurous cercariae, echinostome cercariae, furcocercous cercariae, parapleurolophocercous cercariae, virgulate cercariae, and xiphidiocercariae. Regarding molecular identification, ITS2 sequence data of each larval trematode were analyzed, and a dendrogram was constructed using the neighbor-joining method with 10,000 replicates. The dendrogram was separated into 6 clades (order/family), including Echinostomatida/Echinostomatidae, Echinostomatida/Philophthalmidae, Opisthorchiida/Heterophyidae, Plagiorchiida/Prosthogonimidae, Plagiorchiida/Lecithodendriidae, and Strigeatida/Cyathocotylidae. These findings were used to confirm morphological characteristics and evolutionary trends of each type of cercariae discovered in Nakhon Nayok province. Furthermore, this investigation confirmed that the ITS2 data of cercariae could be used to study on phylogenetic relationships or to determine classification of this species at order and/or family level when possible.

Multiplex PCR assay for discrimination of Centrocestus caninus and Stellantchasmus falcatus

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 0.312 5 ng/μL. The lowest concentration of the DNA template of this multiplex PCR was 2.5 ng/μL. 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.

Epidemiological situation and molecular identification of cercarial stage in freshwater snails in Chao-Phraya Basin, Central Thailand

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 Lymnaeidae, 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.

Developmental and Phylogenetic Characteristics of Stellantchasmus falcatus (Trematoda: Heterophyidae) from Thailand

This study aimed to investigate the infection status, worm development, and phylogenetic characteristics of the intestinal trematode, Stellantchasmus falcatus. The metacercariae of S. falcatus were detected only in the half-beak (Dermogenus pusillus) out of the 4 fish species examined. Their prevalence was 90.0%, and the intensity of infection was 919 metacercariae on average. Worms were recovered from 33 (97.1%) of 34 chicks that were experimentally infected with 200 S. falcatus metacercariae each, and the average recovery rate was 43.0%. The body size and inner organs of S. falcatus quickly increased in the experimental chicks over days 1-2 post-infection (PI). In addition, ITS2 sequence data of this parasite were analyzed to examine the phylogenetic relationships with other trematodes using the UPGMA method. The results indicated that the ITS2 sequence data recorded from trematodes in the family Heterophyidae appeared to be monophyletic. This study concluded that D. pusillus serves as a compatible second intermediate host of S. falcatus in Thailand and that S. falcatus can develop rapidly in the experimental chicks. Data collected from this study can help to close the gap in knowledge regarding the epidemiology, biology, and phylogenetic characteristics of S. falcatus in Thailand.

Molecular phylogeny of trematodes in Family Heterophyidae based on mitochondrial cytochrome c oxidase subunit I (mCOI)

OBJECTIVE@#To analyze a phylogenetic tree for understanding the molecular systematic of trematode in Family Heterophyidae, which are highly distributed in Thailand.@*METHODS@#Based on thirteen sequences of mitochondrial cytochrome c oxidase subunit I (mCOI) gene from six genera of heterophyid trematodes, viz. Haplorchis, Stellantchasmus, Centrocestus, Metagonimus, Pygidopsis, and Haplorchoides were aligned automatically using the Clustal × 2.0 program. A phylogenetic tree was constructed by maximum likeinghood (ML) and neighbor-joining (NJ) methods, with 1 000 bootstrap using the 5.0 program.@*RESULTS@#The phylogenetic relationship from both methods was similar and separated into three groups consisting of Haplorchoides pumilio group, Haplorchoides taichui group and another heterophyid genera.@*CONCLUSIONS@#The sequence data of mtCOI can be used to investigate the phylogenetic relationships of trematodes at the genus level. Each clade of different genera of heterophyid trematodes can be separated into sister groups that correlated with the morphological characteristic, kind of secondary intermediate host and geographic distribution.

Epidemiology of cercarial stage of trematodes in freshwater snails from Chiang Mai province, Thailand

<p><b>OBJECTIVE</b>To investigate the epidemiological situation of cercarial trematodes infection in freshwater snails from different water resources in Chiang Mai province, Thailand.</p><p><b>METHODS</b>The snail specimens were collected from 13 districts of Chiang Mai province during April 2008 to February 2012. The prevalence of cercarial infection in snails was investigated using the crushing method. The drawing was done with the help of a camera lucida for the morphological study.</p><p><b>RESULTS</b>A total of 2 479 snail individuals were collected and classified into 7 families, 11 genera, and 14 species, Among them, 8 snails species were found to be infected with an overall prevalence of 17.27% (428/2 479), which infected with nine groups of cercariae; gymnocephalous cercaria, strigea cercaria, megalurous cercaria, monostome cercaria, parapleurolophocercous cercaria (Haplorchis cercaria), pleurolophocercous cercaria, furcocercous cercaria (Transversotrema cercaria), xiphidiocercaria, and virgulate cercaria. The parapleurolophocercous cercaria was found to be the dominant type among the cercarial infection in the snails (64.25%).</p><p><b>CONCLUSIONS</b>The various species of snails found in the research location act as the intermediate hosts for the high prevalence of parasitic infection of many species of mammals. This work will provide new information on both the distribution and first intermediate host of trematodes.</p>

Epidemiology of cercarial stage of trematodes in freshwater snails from Chiang Mai province, Thailand

Objective: To investigate the epidemiological situation of cercarial trematodes infection in freshwater snails from different water resources in Chiang Mai province, Thailand. Methods:The snail specimens were collected from 13 districts of Chiang Mai province during April 2008 to February 2012. The prevalence of cercarial infection in snails was investigated using the crushing method. The drawing was done with the help of a camera lucida for the morphological study. Results:A total of 2 479 snail individuals were collected and classified into 7 families, 11 genera, and 14 species, Among them, 8 snails species were found to be infected with an overall prevalence of 17.27% (428/2 479), which infected with nine groups of cercariae;gymnocephalous cercaria, strigea cercaria, megalurous cercaria, monostome cercaria, parapleurolophocercous cercaria (Haplorchis cercaria), pleurolophocercous cercaria, furcocercous cercaria (Transversotrema cercaria), xiphidiocercaria, and virgulate cercaria. The parapleurolophocercous cercaria was found to be the dominant type among the cercarial infection in the snails (64.25%). Conclusions:The various species of snails found in the research location act as the intermediate hosts for the high prevalence of parasitic infection of many species of mammals. This work will provide new information on both the distribution and first intermediate host of trematodes.

Prevalence of Haplorchis taichui in Field-Collected Snails: A Molecular Approach

The prevalence of the cercarial stage of an intestinal trematode, Haplorchis taichui, in thiarid snails (Gastropoda: Thiaridae) was investigated using light microscope and species-specific PCR procedures. A total of 988 snails were collected from Mae Taeng district, Chiang Mai province, northern Thailand, which comprised of 3 species; Melanoides tuberculata, Tarebia granifera, and Thiara scabra. The overall prevalence of pleurolophocercous cercariae was 21.7% as determined by the morphology. For genetic detection of H. taichui infection in snails, 2 primers Hapt_F (5'-GGCCAACGCAATCGTCATCC-3') and Hapt_R (5'-GCGTCGGGTTTCAGACATGG-3'), were used. The genomic DNA of H. taichui, which was used as a positive control, gave an amplification of the 256 bp fragment. The overall prevalence of H. taichui from specific PCR was 9.7%. The proportion of H. taichui among the pleurolophocercous cercariae in this study was 44.9%.