DETECTION OF ORNITHOBILHARZIA TURKESTANIKUM CERCARIA (TREMATODA) BY NESTED-PCR IN INTERMEDIATE HOST SNAIL, LYMNAEA GEDROSIANA

摘要

Trematodes are important in economic and public health. Ornithobilharzia turkestanicum (0. turkestanicum) is one of the important economic trematodes in domestic animals. Ornithobilharzia turkestanicum in intermediate host (Lymnaea gedrosiana ) can be detected by either exposing snails to light to induce cercarial shedding or by squeezing them between glass slides to detect parasites. The current available diagnostic methods are inefficient for identification of prepatent infections and/or after dead of snails. For the above difficulties we adapted a nested polymerase chain reaction (Nested-PCR) assay for sensitive detection of O. turkestanicum in clinical samples and its cercaria in snails. The life cycle of parasite was maintained in sheep and snails in laboratory in Razi Institute. Adult worms were isolated from sheep and DNA was extracted from worms by a procedure using DNA extraction solution developed in NIGEB. PCR and nested-PCR primers were designed based on 28s ribosomal RNA gene of O. turkestanicum and the DNA was amplified by PCR assay. PCR product was purified and cloned in pTZ57Rff and sequenced. The comparison of the obtained sequences with the GenBank using blast program was showed in NCBI Sequence viewer just 682 bp. PCR amplicon was submitted in GenBank and can be assessed using AY862391 accession number. DNA was extracted from the infected and non-infected snails 2-5 days post-infection. The infected snails could be rapidly detected with Nested-PCR. Results indicate that this assay is specific for detecting 0. turkestanicum. The high sensitivity of the test enabled identification of single infected snail even when its DNA was pooled with uninfected snails. Thus demonstrating the possibility of mass diagnosis in pools of snails, therefore, the assay has the potential for large-scale demonstration of prepatent infection prevalence in snails and offers a new diagnostic tool for evaluation of bilharziosis transmission and for control of infection as discussed.