PCR diagnosis of dicrocoelium dendriticum infection in mollusc and ant intermediate hosts

Abstract

Dicrocoeliosis, caused by Dicrocoelium dendriticum, is an important hepatic trematodosis which affects a wide range of mammals, mainly ruminants, in Spain and many other countries. To apply sueeessful control programmes against dicroceliosis, prior study of its epidemiology is needed. This requires specific and early diagnosis in mammals, as well as in molluscs and ants, the first and second intermediate hosts, respectively. The aim of this study was to develop an analytical method based on PCR (Polymerase chain reaction) to detect the infection by D. dendriticum in their intermediate hosts. A PCR based on the diversity of nucleotide sequences in mitochondrial DNA among species was carried out. Since no mitochondrial sequenee of D. dendriticum was available, firstly we identified conserved regions of different parasite species with phylogenetic similarity to design general primers which anneal with D. dendriticum DNA. So ten primers,and several combinations, which flanked the large and small rRNA subunits , the NADH dehydrogenase gen and different cytochrome C oxidase subunits were tested. The pair of general primers, which partly flanked the cytochrome C oxidase I and the large rRNA subunits, amplified a band from adult D. dendriticum samples. Once this band was sequenced, internal specific primers were designed fo detect the infection in the intermediate hosts. Using PCR the primers amplified DNA obtained from: D. dendriticum adults, different species of land molluscs experimentally and naturally infected with D. dendriticum and infected ants collected in tetania (in León province, Spain). The PCR products, observed in agarose gel, permitted the detection of D. dentricum sporocysts in mollusc hepatopancreas as well as metacercariae and brainworm in ant abdomen and head, respectively. The specificity of the PCR was also established after testing the primers with other different parasites such as Fasciola hepatica, Calicophoron daubneyi and Brachylaimidae.