Genetic markers for the identification and characterization of Opisthorchis viverrini, a medically important food borne trematode in Southeast Asia

Abstract

The liver fluke, Opisthorchis viverrini, is one of the major food borne trematodes in Southeast Asia, where infection causes hepatobiliary disease and subsequent development of cholangiocarcinoma. In Thailand, O. viverrini is most prevalent in the northeast where there is marked regional variation in the rate of infection in humans at provincial, district and village levels. To date, the roles of genetic variation of O. viverrini on this observed variability in infection, transmission and associated disease are not known. We have applied multilocus enzyme electrophoresis (MEE), specifically allozyme electrophoresis, to isolates of O. viverrini from Thailand and Laos to establish genetic markers to examine its systematics and population structure. Forty-six enzymes commonly found useful for genetic characterisation in parasitic helminths were screened, and of these, 33 enzymes gave sufficient staining and resolution to act as potential genetic markers. Sixteen enzymes were monomorphic and 17 enzymes were polymorphic in the pools of worms examined. Whether they are indicative of different enzyme loci, heterozygosity or unique genotypes within the pools of worms examined remains to be determined. Preliminary investigations examining five individual worms at enzyme loci where pools of worms showed multiple bands have confirmed the diagnostic value of the enzyme loci established as well as providing evidence of potential population sub structuring and heterozygosity. For the first time, we have established at least 17 enzymes that provide the basis to undertake comprehensive genetic analyses of the systematics and population structure of O. viverrini, a medically important food borne trematode in Southeast Asia.

Genetic relationships within the Opisthorchis viverrini species complex with specific analysis of O. viverrini from Savannakhet, Lao PDR by multilocus enzyme electrophoresis

“…Comparison of the RAPD profiles revealed that O. viverrini from Lao PDR was genetically distinct from those collected in Thailand ). This finding was supported by allozyme electrophoresis analyses using 32 enzyme loci as genetic markers to examine systematics and population structure of O. viverrini (Saijuntha et al 2006a;b;Saijuntha et al 2007). O. viverrini from 15 different localities in Thailand and Lao PDR were analyzed with 32 enzyme loci.…”

Evidence of a species complex within the food-borne trematode Opisthorchis viverrini and possible co-evolution with their first intermediate hosts

“…Based on our previous MEE studies to define a sufficient number of enzyme loci to genetically characterise O. viverrini (Saijuntha et al, 2006a,b) 28 enzymes encoding a presumptive 32 loci were used herein for the genetic characterization of O. viverrini . This number of enzyme loci is considerably more than that employed in MEE studies on other species of food-borne trematodes (Agatsuma et al, 1986; 1994; Park et al, 2002) and provides the opportunity to assess the significance of genetic variation detected among geographical localities, and to define a sufficient number of independent genetic markers for biological and epidemiological studies.…”

Analysis of the population genetics of Opisthorchis viverrini sensu lato in the Nam Ngum River wetland, Lao PDR, by multilocus enzyme electrophoresis

“…Five polymorphic enzymes, ENOL, FDP, PGAM, PGM and TPI, all of which are known to be polymorphic in O. viverrini from Thailand (Saijuntha et al 2006;Saijuntha et al 2007), were examined in samples from populations of the O. viverrini species from Lao PDR.…”

“…Supernatants were removed directly by loading pen from each well and loaded onto the cellulose acetate gel (Cellogel; Milan) as the support medium. Each gel was stained histochemically for a specific enzyme as described for the five enzymes that were previously shown to be polymorphic (Saijuntha et al 2006;Saijuntha et al 2007). The polymorphic enzymes used for the population genetic study in Thailand were used for in the present study for O. viverrini from Lao PDR, namely: enolase (ENOL, EC 4.2.1.11), fructose-1,6-diphosphatase (FDP, 3.1.3.11), phosphoglycerate mutase (PGAM, 2.7.5.3), phosphoglucomutase (PGM, EC 2.7.5.1), and triose phosphate isomerase (TPI, EC 5.3.1.1).…”