A study on partial characterisation of excretory-secretory(ES) and domatic antigens of explanatum explanatum and establishment of its phylogenetic relationship by genetic chracterisation
Loading...
Date
2005
Authors
Mallawaarachchi, R. S.
Journal Title
Journal ISSN
Volume Title
Publisher
The University of Peradeniya
Abstract
Explanatum explanatum inhabits the bile duct of ruminants (Family: Paramphistomidae) and causes paramphistomiasis. The disease is shown to affect the productive and reproductive functions of the host animal which in turn cause significant economical losses.
Diagnosis of E. explanatum infection is done by routine microscopic examination of eggs -in faeces which is not very accurate and practical enough. Serological tests, developed taking specific antigens into consideration, will provide more accurate diagnosis.
Attempts were made in this study to isolate and characterise somatic and ES antigens of E. explanatum which could be used in developing a serodiagnostic test in the future. Further, genetic characterisation was attempted in order to establish the phylogenetic relationship of E. explanatum with other trematode species.
Samples of E. explanatum were collected from the infected livers of cattle and buffaloes in the abattoir of Colombo Municipal Council. Rumen paramphistomes which were used in antigenic analysis of this study were collected at Municipal Abattoir, Kandy.
ES antigens were prepared by invitro culture of flukes in RPMI medium at 37°C, 5% CO₂ up to 5 days. Somatic antigens were prepared by grinding flukes in cold PBS. Protein concentration of ES and somatic antigen preparations were determined by bicinchoninic acid protein assay kit. Antigen preparations were further subjected to dot blot ELISA using antibovine IgG conjugate to detect contamination with host proteins. Analysis of ES and somatic antigens were carried out through sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE). Protein bands were visualised using Coomassie brilliant blue and silver staining.
ES products of E. explanatum revealed 8 additional bands in the protein profile i.e. first three bands of molecular weight between 45-66KDa, 4ᵗʰ and 5ᵗʰ band between 36-29KDa, 6ᵗʰ band of 29KDa,7ᵗʰ and 8ᵗʰ bands of molecular weight between 20-24KDa. They were not shared with ES antigens of ruminant species. These 8 bands seem to be possible candidates which could be used for development of a diagnostic tool.
SDS PAGE of somatic antigens of E. explanatum revealed 4 additional bands i.e. 1ˢᵗ band of > 66KDa, 2ⁿᵈ band of molecular weight between 45-66KDa, 3ʳᵈ band of 45KDa and 4ᵗʰ band of molecular weight between 29-36KDa with respect to that of ruminant species.
Genomic DNA of £. explanatum was extracted from individual flukes. Second internal transcribed spacer (ITS2) region of the ribosomal gene repeat was amplified using Polymerase Chain Reaction (PCR). PCR conditions were 94° C for 1 minute, 50°C for 1 minute, 72°C for 2 minutes for 30 cycles. Primers used were 3S, forward primer (5’- CGG TGG ATC ACT CGG CTC GT-3’) and A28, reverse primer (5’°-CCT GGTTAGTTT CTT TTC CTC CGC-3’), PCR products were eluted and sequenced using the same primers. Sequenced portion of ITS2 region which was 503bp in length was aligned with that of Paragonimus, Fasciola and Schistosoma spp. Phylogenic analysis was performed using Parsimony method in PAUP (Ver. 4).
Parsimony trees*revealed that £. explanatum is related more closely to Paragonimus spp than to Fasciola and Schistosoma spp.
Description
Keywords
Biochemistry and Molecular Biology , Explanatum Explanatum