Characterisation of in vitro excretory-secretory components of the ovine intestinal nematode, Trichostrongylus vitrinus

Trichostrongylus vitrinus is one of the principal causative nematodes of ovine parasitic gastro-enteritis within Scotland and infests the proximal small intestine of the sheep. At present, control is achieved mainly by the administration of anthelmintic drugs, but with the increasing emergence of anthelmintic resistance, much research is now centred on vaccine development. Recent evidence has suggested that the excretory-secretory components (ES) from parasitic nematodes may be an important source of host-protective antigens. The overall aim of the present study was to characterise the nature and properties of T. vitrinus ES components. The initial part of the work involved the partial characterisation of two of the enzymes, acetylcholinesterase (AChE) and proteinases, excreted and secreted during the in vitro culturing of adult T. vitrinus. These enzymes were defined on the basis of their substrate specificity, molecular size, pH optima and inhibitor sensitivity. Attempts were also made to isolate cDNA fragments encoding AChE from an adult T. vitrinus cDNA pool, using the polymerase chain reaction (PCR) and degenerate oligonucleotide primers directed towards highly conserved regions of AChEs that had previously been identified by comparison of known polypeptide sequences from a number of higher eukaryotic organisms. No adult T. vitrinus cDNA fragments encoding AChE were amplified, suggesting that T. vitrinus AChE(s) is/are distinctly different to AChEs from higher eukaryotes, at least at the level of nucleic acid sequence. Subsequent research focused on the molecular characterisation of adult T. vitrinus ES. Immunoscreening of an adult T. vitrinus cDNA lambda gt11 library with antiserum raised against adult T. vitrinus ES, resulted in the isolation of ten immunopositive clones. Their inserts were sequenced and the results were analysed using computer databases. Three of the clones were identified as harbouring inserts that encoded proteins that shared significant homology to myosin heavy chain, vitellogenin and serine proteinase inhibitor (serpin) respectively. The other seven clones contained inserts that showed no significant homology to any of the sequences present in the computer databases.