DEPARTMENT OF VETERINARY DISEADSE BIOLOGY, SECTION FOR PARASITOLOGY

FACULTY OF LIFE SCIENCES

UNIVERSITY OF COPENHAGEN

A MOLECULAR METHOD TO DIFFERENTIATE BETWEEN THREE GENOTYPES OF TRICHURIS RECOVERED FROM HUMANS AND PIGS
Al-Jubury,A.;Nissen,S.;Olsen,A.;Christensen,H.;Thamsborg,S.;Nejsum,P.
Danish Centre for Experimental Parasitology, Department of Veterinary Disease Biology, Faculty of Life Sciences, University of Copenhagen, Denmark

Introduction
The whipworms Trichuris trichura and T.suis in human and pigs respectively are believed to be closely related but different species. No reliable morphological character has been identified which can discriminate worms from the two hosts. Molecular method PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) have been developed, based on recent sequensis analysis has suggested 3 major ITS-2 genotypes are present in trichuris obtained from human and pigs, it could give reliable method to identify worms genotypes.

Trichuris male worm

Photo by sofie Nissen.Uganda 2008

Method
Trichuris Trichura, T.suis worms were collected from different humans and pigs in various countries. DNA extraction have been done for most of these worms samples by using Qiagen kit.
Trichuri s femail worm

PCR reaction was conducted on 29 T. Trichura and 59 T.suis DNA ,using 2 primers forward primer was used based on the 5.8S of T.suis and conserved primer from literature. PCR products were digested by BSEL I enzyme ( PCR-RFLP). After digestion , the product examend on Gel electrophoresis (1.5% Agarose)

Photo by stefan Andersen

Results
The distribution of the three genotypes in woems from pigs were 2,0 and 5 respectivley and 26,2and 1 in human. The result suggest that Trichuris in human and pigs belong to two different population (p<0.001). We have seen evidence of some few cases of heterozygote worms.

Table 1: Shows the distribution of (88) Trichuris samples according to genotypes.

samples Type 1 heterozygote Type 2 Total Human Pig Total 26 2 28 2 2 1 57 58 29 59 88

Discussion
The present study confirm the credibility of T.trichura and T.suis are two different species. It became possible to distinguish between these two spp. By using PCR-RLFP. The present method can make several samples at one time , so it is time consuming. The result shows an evidence for a few cases of cross-infection and the existence of heterozygote worms , suggesting cases of hybridization or retention of ancestral polymorphism.

Human-derived worms

Pig-derived worms

Fig: Gel showing 10,9 human and pigs respectively using PCR-RFLP. The different between 3 genotype are clear to distinguish accoring to the marker.( 1:type one genotype. 2: type two genotype. 3: Hetrozygotes genotype.

Danish Centre for Experimental Parasitology

FAO Collaborating Centre on Helminthology - WHO/FAO Collaborating Centre for Parasitic Zoonoses