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.

Studies on the morphology of cercariae obtained from freshwater snails at Erawan Waterfall, Erawan National Park, Thailand.

The morphology of cercariae of freshwater snails from Erawan Waterfall, Erawan National Park, Kanchanaburi Province was studied between December 2002 and August 2003. The snail samples were collected by handpicking using a counts per unit of time sampling method. The cercariae, larva stage of a trematode, were investigated using the shedding method where they were categorized into three groups and six species. The first group, Pleurolophocercous cercariae, consists of Haplorchis pumillo (C1) and Stictodora tridactyla (C3). The second group, Furcocercous cercariae, consisted of Mesostephanus appendicalatus (C2), Transversotrema laruei (C6) and Cardicola alseae(C4). The third group, Xiphidio cercariae, has only one species which is Loxogenoides bicolor (C5). Out of 1163 snails, only 62 were found to be infected by cercariae, equivalent to a 5.33% infection rate. The infections grouped by species of the cercariae are as follows: C, 22 (1.9%), C, 29 (2.5%), C2 1 (0.1%), C6 1 (0.1%), C4 6 (0.5%) and C5 3 (0.3%). The freshwater snail samples consist of four species. From a total of 1163 samples, there are 687 Melanoides jugicostis, 91 Tarebia granifera, 296 Thiara scabra and 89 Melanoides tuberculata. Infections were found in 45 (6.5%), 6 (6.6%), 1 (0.3%) and 10 (11.2%), respectively.

Trematode infections of the freshwater snail family Thiaridae in the Khek River, Thailand.

The freshwater snail family Thiaridae was studied at five different locations: water sources for the Khek River, Thailand. Snail samples were collected by hand using counts per unit of time sampling method between December 2004 and October 2005. The physico-chemical quality of the water changed with the seasons and affected the sampling areas during both the dry season and the flood season. A total of 9,568 snail samples comprised of 14 species were found. These were 284 Tarebia granifera, 24 Melanoides tuberculata, 86 Thiara scabra, 3,295 Paracrostoma pseudosulcospira pseudosulcospira, 736 P. paludiformis paludiformis, 3,266 P. paludiformis dubiosa, 117 P. morrisoni, 304 Brotia (Brotia) binodosa binodosa, 1,250 B. (Brotia) microsculpta, 146 B. (Senckenbergia) wykoffi, 1 B. (Brotia) pagodula, 5 B. (Brotia) binodosa spiralis, 5 B. (Brotia) insolita and 49 B. (Brotia) manningi. The cercariae were investigated using shedding and crushing methods where they were categorized into two types and five species. The first type, Parapleurolophocercous cercariae, were comprised of Haplorchis pumilio Looss, 1899 and Centrocestus formosanus Nishigori, 1924. The second type, Xiphidiocercariae were comprised of Acanthatrium hitaense Koga, 1953, Loxogenoides bicolor Kaw, 1945 and Haematoloechus similis Looss, 1899. The cercarial infection rates in the above 5 species were 0.1% (5:9,568), 0.2% (15:9,568), 0.3% (24:9,568), 0.4% (37:9,568) and 0.1% (5:9,568), respectively. Five species of snails were susceptible to trematode infections. They were T. granifera, M. tuberculata, T. scabra, P. paludiformis paludiformis and B. (Senckenbergia) wykoffi; infections were found in 26.1% (74:284), 33.3% (8:24), 1.2% (1:86), 0.3% (2:736) and 0.7% (1:146), respectively.

Cercarial infection in Paludomus petrosus, freshwater snail in Pa La-U Waterfall.

Paludomus petrosus, the freshwater snails found in Pa La-U Waterfall, were examined for cercarial infection of trematodes. The snails were collected every other month from April, 2001 to February, 2002. Collections were taken from two sampling stations. The counts per unit of time' method was used for collection of the snails. The density of snails was highest in June 2001, and the highest of parasite infection rate was in February 2002. Four types of cercariae were found in the snails: Xiphidiocercariae, Amphistome, Furcocercous cercariae type I, and Furcocercous cercariae type II. Xiphidiocercariae were found in April 2001 to February 2002. Amphistome, Furcocercous cercariae type I and Furcocercous cercariae type II were found in February 2002.

Localization of antigenic molecules of adult Fasciola gigantica using monoclonal antibodies against the parasite tegumental antigen.

In this study, monoclonal antibodies were developed from the partially purified surface tegumental antigens of F. gigantica. Nine MoAbs: 2G11, 1G2, 1B12, 2G2, 2G5, 3C6, 3G2, 3G3 and 3F6 were used for anatomical localization of adult F. gigantica. The reaction was demonstrated by Avidin-Biotin method. The results revealed that among the sections stained with non-immune sera and control group, there were no reaction products on either the tegument or the cecal epithelium. The only brownish areas were the vitelline glands. In the sections stained with immune sera, brownish reaction products appeared on the surface membrane, the spine membrane, the cecal lumen and its epithelial cells. The experiment sections of nine monoclonal antibodies revealed that the reaction occurred mainly on the tegument of the adult worm which covered its surface and spine.

Production of monoclonal antibodies against partially purified surface tegument antigens of Fasciola gigantica.

Monoclonal antibodies (MAbs) were produced by the in vitro fusion of Balb/C mice spleen cells immunized with partially purified surface tegument antigens of Fasciola gigantica. The surface membrane and tegument antigens were purified by using gel filtration chromatography. SDS-PAGE performed on the processed proteins demonstrated that the proteins had molecular weights of 20 to 97 kDa. In this study, fifteen monoclonal antibody clones were selected from the hybridoma clones, namely: 1B7, 1B11, 1B12, 1C9, 1D4, 1G2, 1H7, 2B6, 2C3, 2C9, 2D11, 2F11, 2G2, 2G5, and 2G11. They were evaluated by immunoblot assay and were differentiated into two groups. In the first group were found 60 and 38 kDa proteins; in the second group were found 66, 60, and 38 kDa proteins. All were found to secrete IgM, kappa light-chain antibodies. These MAbs were tested for their cross-reactivity with other trematodes commonly found to infect cattle and man. All of these MAbs showed some degree of cross-reactivity with other trematode species.