Characterization of Echinostoma cinetorchis endoribonuclease, RNase H.

Echinostoma cinetorchis is an oriental intestinal fluke causing significant pathological damage to the small intestine. The aim of this study was to determine a full-length cDNA sequence of E. cinetorchis endoribonuclease (RNase H; EcRNH) and to elucidate its molecular biological characters. EcRNH consisted of 308 amino acids and showed low similarity to endoribonucleases of other parasites (<40%). EcRNH had an active site centered on a putative DDEED motif instead of DEDD conserved in other species. A recombinant EcRNH produced as a soluble form in Escherichia coli showed enzymatic activity to cleave the 3'-O-P bond of RNA in a DNA-RNA duplex, producing 3'-hydroxyl and 5'-phosphate. These findings may contribute to develop antisense oligonucleotides which could damage echinostomes and other flukes.

Allozyme analysis of the temporal populations of Echinostoma revolutum collected from domestic ducks in Khon Kaen Province, Thailand.

Four temporal populations of Echinostoma revolutum (ER1, ER2, ER3, ER4) were collected from domestic ducks in Khon Kaen Province, Thailand during February-October 2008. The ER1, ER2, ER3 and ER4 were collected in February, April, June and October, respectively. The 12 enzymes encoding 15 loci were examined. Two loci were found in each of 3 enzymes, namely glucose-6-phosphate dehydrogenase (G6PD), malic enzyme (ME) and peptidase valine-leucine (PEPA). Of these, three loci, namely, G6pd-1, Me-1 and PepA-2, were polymorphic. Genotypes were assigned for the specific allelic profiles detected at these three polymorphic loci. Twenty-eight genotypes were observed in the 4 temporal populations of E. revolutum. Three genotypes, Er22, Er23 and Er25, were found in all populations. The Er6 genotype occurred had the highest frequency of all the populations. These 28 genotypes were clustered into 3 groups with genetic differences of 2-12% among the loci. A cluster of genotypes (Er1, Er3, Er9 and Er12) showed the greatest genetic difference among the genotypes (12% difference). These results show intraspecific variation exists in E. revolutum populations in domestic ducks from Khon Kaen Province, Thailand.

A comparison of Echinostoma caproni and Echinostoma trivolvis (Trematoda: Echinostomatidae) adults using isoelectrofocusing.

An isoenzymatic analysis using thin-layer agarose gel isoelectrofocusing on laboratory strains of Echinostoma trivolvis and Echinostoma caproni adults showed characteristic monomorphic phenotypes for phosphoglucomutase and glucose phosphate isomerase. The fixed allelic variation observed between these 2 taxa is consistent with their current classification as distinct species.

Electrophoretic analysis of proteases in Echinostoma Caproni and Echinostoma trivolvis.

Gelatin substrate sodium dodecyl sulfate polyacrylamide gel electrophoresis was used to analyze proteases in 14 day-old adults of Echinostoma caproni and Echinostoma trivolvis. At pH 8.0, E. caproni adults showed 2 protease bands at 36 kDa and 58 kDa, whereas E. trivolvis adults showed 6 bands at 39, 64, 77, 96, 120, and 168 kDa. Each species also showed distinct protease banding patterns in their excretory/secretory (E/S) products. The E. caproni E/S proteases were at 36 and 58 kDa, whereas those of E. trivolvis were at 120 and 168 kDa. Further characterization of E. caproni adult proteases revealed 2 bands (58 and 66 kDa) with optimal activity at pH 3.0-4.5 and 3 bands (38, 61, and 96 kDa) that were most active at pH 7.0-8.0. Four low molecular weight bands (19, 21, 25, and 30 kDa) appeared when E. caproni worm extracts were incubated in the presence of CaCl2 at pH 8.0 but were inhibited with ethylenediaminetetraacetic acid and 1,10-phenanthroline. Echinostoma caproni protease bands at 58 and 38 kDa in the whole worm samples and the E/S products and the 36-kDa band in the whole worm samples were inhibited with phenylmethylsulfonyl fluoride. By showing protease differences in addition to recent work on nucleotide differences, this study helps distinguish these 2 related allopatric species of 37-collar-spined Echinostoma.

Spatial and temporal genetic variation of Echinostoma revolutum (Trematoda: Echinostomatidae) from Thailand and the Lao PDR.

A total of 314 individual Echinostoma revolutum were collected at different locations and times from domestic ducks from Khon Kaen Province, Thailand and Vientiane Province, the Lao People's Democratic Republic (PDR). Genetic variation of these parasites was analyzed using multilocus enzyme electrophoresis at three polymorphic loci namely, glucose-6-phosphate dehydrogenase (G6pd), malic enzyme (Me) and peptidase valine-leucine (PepA). High levels of genetic variability were found within and between populations. Significant heterozygote deficiencies compared with the predictions under Hardy-Weinberg equilibrium were detected in populations from Thailand and the Lao PDR for all loci except G6pd-1. Significant genetic differentiation was observed between spatially separated populations from Thailand and the Lao PDR. This as also true for some samples collected at different times in Thailand. The variability found may be consistent with a Wahlund effect, genetic drift and/or other factors such as the population structure of snail hosts. Our data provide further insight into the process of genetic divergence within and among geographically and temporally isolated populations of E. revolutum, and potentially other medically important echinostomes in Southeast Asia.

Echinostoma caproni: identification of enolase in excretory/secretory products, molecular cloning, and functional expression.

In order to investigate molecules that could be involved in host-trematode relationships, we have analysed the excretory/secretory products (ESP) of Echinostoma caproni following a proteomic approach. Actin, Gluthathione S-transferase (GST) and enolase have been identified in the ESP. Enolase, observed to be one of the most abundant proteins, was further characterized. The molecular cloning and in vitro expression in Escherichia coli of E. caproni enolase allowed us to determine that the protein contains 431 amino acids and a theoretical MW of 46272 Da. E. caproni enolase shows high homology to other trematode enolases. The recombinant protein binds specifically to human plasminogen in vitro, as observed for the native protein, confirming its properties as a host-interacting molecule.

5'-Nucleotidase activity and substrate affinity in digenetic trematodes.

5'-nucleotidases of eight species of digenetic trematodes were studied using five different substrates. All species showed the following preferential order of substrate affinity; AMP greater than CMP greater than GMP greater than TMP greater than UMP. It was observed that different species occupying similar habitats possessed closely related levels of enzyme activities. The function of 5'-nucleotidases in trematodes is also suggested.

The genetic relationships between Echinostoma caproni, E. paraensei, and E. trivolvis as determined by electrophoresis.

Adults of Echinostoma caproni, E. paraensei, and E. trivolvis were processed for starchgel electrophoresis. Ten enzyme systems representing 12 structural loci were examined using three different buffer systems. E. paraensei and E. caproni were found to be genetically inbred as indicated by the lack of heterozygosity in individual worms. All three taxa showed fixed differences indicating they are distinct species. Fixed differences were found between E. paraensei and E. caproni in six enzyme systems, between E. paraensei and E. trivolvis in five enzyme systems, and between E. trivolvis and E. caproni in five enzyme systems. Phenic relationships among the three species showed E. caproni was genetically more similar to E. trivolvis than to E. paraensei.

[Chemicotaxonomic study of the genus Echinostoma: comparison of a strain isolated in the Cameroon (E. sp.) and 2 African species (E. caproni and E. togoensis)]. / Etude chimiotaxonomique du genre Echinostoma: comparaison d'une souche isolée du Cameroun (E. sp.) à deux espèces africaines (E. caproni et E. togoensis).

A comparative enzymatic study is carried out on adults of three species of the genus Echinostoma (E. caproni, E. togoensis, E. sp.). Amongst 23 enzymes tested by starch gel electrophoresis, Pgm and Pgi are characterised by different zymogrammes in each species. Agarose and polyacrylamide isoelectrophoresis focusing define isoenzyme genetic structures: Pgm is coded by three loci, Pgi is coded by one locus. Pgm polymorphism allows us to extend this work to other experimental or natural stocks.

Parasites in Bulinus senegalensis (Mollusca: Planorbidae) and their detection.

Isoelectric focusing studies on enzyme variation between populations of the snail Bulinus senegalensis revealed that parasitic infections in the snails contributed additional bands of enzyme activity, particularly in the glucose phosphate isomerase (GPI) and malate dehydrogenase (MDH) systems. The patterns due to the parasite enzymes were, in most cases, clearly distinct from those of the host and different from each other. Parasites encountered included Schistosoma haematobium, S. bovis, Paramphistomum microbothrium, another amphistome probably belonging to the group which infect amphibians, Echinostoma revolutum, another echinostome (probably Echinoparyphium sp.), strigeids, xiphidiocercariae (these were resolved into 3 distinct types by the enzyme data) and ciliate protozoa. The 7 host populations which were examined showed marked differences in both the prevalence and variety of their parasitic infections and these variations were tentatively related to environmental differences in their respective habitats and to the nature of human contact patterns. Seasonal changes in the parasite fauna were also noted and some of the implications of the parasite load on the host population are briefly mentioned.

Histochemical and electrophoretic studies on phosphatases of some Indian trematodes.

The isoenzymes of acid and alkaline phosphatases and their histochemical localization were studied by polyacrylamide disc gel electrophoresis in four species of trematodes: Gigantocotyle explanatum from the liver and Gastrothylax crumenifer from the rumen of water buffalo (Bubalus bubalis) and Echinostoma malayanum and Fasciolopsis buski from the small intestine of the pig (Sus scrofa). Both acid and alkaline phosphatases were present in the tegument, gastrodermis, suckers, testes, ovary, eggs, vitellaria and uterus but alkaline phosphatase activity was demonstrated only in the parenchyma and excretory ducts. Polyacrylamide gel electrophoresis revealed two to four isoenzymes for both acid and alkaline phosphatase.

Isoenzyme analysis of Echinostoma liei: comparison and hybridization with other African species.

Isoenzyme analysis was carried out on the laboratory strain of Echinostoma liei. The results were compared with those from a preliminary study on Echinostoma caproni, Echinostoma togoensis, and Echinostoma sp. (A. Voltz, J. Richard, B. Pesson, and J. Jourdane, 1986, Annales de Parasitologie Humaine et Comparée, 61, 617-623). Isoelectrofocusing showed characteristic phenotypes for phosphoglucomutase (PGM, EC 5.4.2.2) and glucosephosphate isomerase (GPI, EC 5.3.1.9). The four experimental strains were monomorphic. Their genotypes were defined. Isoenzyme analysis of F1-hybrids and their F2 descendants indicated the subunit structure of both isoenzymes and showed that they were encoded by independent genes. Finally, it also suggested that the four strains corresponded to variants of the same species.

A genetic comparison between natural and laboratory strains of Echinostoma (Trematoda) by isoenzymatic analysis.

A comparative isoenzyme study between a natural population of Echinostoma caproni and 2 strains of E. caproni and E. togoensis, maintained for a long time in the laboratory led to the following conclusions. (1) The natural E. caproni population showed polymorphism of associated PGM and GPI genotypes. Some zymograms were identical to those of experimental strains. Allelic frequencies show that the natural population is panmictic according to the Hardy-Weinberg equilibrium. (2) The laboratory strains were monomorphic, and interbreeding produced double-heterozygous F1 progeny, identical to those of the natural population.

Esterases in natural populations of normal and Echinostoma revolutum-infected Helisoma trivolvis (Gastropoda).

Esterases of the digestive gland-gonad (DGG) complex of individual snails from a wild population of Helisoma trivolvis infected with the trematode Echinostoma revolutum were analyzed by vertical slab PAGE and compared to similar DGG homogenates of uninfected conspecifics from the same population. Our analysis indicated that: 1. Four classes of esterases, some atypical, could be resolved using diagnostic inhibitors. 2. Uninfected snails demonstrated polymorphism for two of these four esterase groups, including cholinesterases (CHE), in the 34 individual DGGs analyzed. 3. The rarer of the two ChE phenotypes in the uninfected sample (29.4%) was present in 100% of the 17 infected snails examined. However, no changes in esterase zymograms of infected DGGs due to the parasite were noted. 4. The possibility that the 'rare' ChE phenotype is somehow related to host susceptibility to Echinostoma revolutum is discussed in view of similar apparent linkages in other snail-trematode systems.