Towards the Creation of a Transgenic Possum Parasite

Abstract

The brushtail possum. Trichosurus vulpecula, is New Zealand's most serious vertebrate pest; possums destroy native flora and fauna and are vectors of bovine Tb. Conventional control is considered to be unsustainable and, in the long term, biological control is seen as the only solution to reducing possum numbers. The aim of this project is to contribute to the development of a self-disseminating vector that will spread a control molecule throughout the possum population reducing fecundity or increasing mortality. The possum-specific parasite Parastrongyloides trichosuri has considerable potential a-s such a vector. A protein from P. trichosuri specifically, was found to be antigenic in possums. The antibodies to this protein were purified from positive possum serum and used to detect the antigen on the surface of infective larvae but not in the excretory/secretory products of either larvae or adults. The protein was isolated from crude infective larvae and found to show homology to the heat-shock 70 family of proteins. Genomic DNA was extracted, an oligonucleotide probe made and a genomic library screened for the Hsp70 gene. Several positive clones were found and DNA isolated and sequenced from one such clone. Five kilo bases of unambiguous sequence was obtained in which was an open reading frame of 2 kb. Theoretical translation of this gave a protein of 64 amino acids with 80% homology to the Hsp70A protein of C. elegans. The region upstream of the ATG initiator codon was amplified and 1.3 kb of the putative promoter region was cloned into a vector containing the gfp:lacZ reporter genes. This construct was microinjected, first into C. elegans to demonstrate promoter function, and then into both tree-living and parasitic adults of P. trichosuri. Reporter gene expression was shown in the progeny of microinjected parasitic adults. RNA was made from infective P. trichosuri larvae, reverse transcribed and the coding sequence for the PtHsp70 protein cloned into an expression vector and expressed in E. coli, The recombinant protein pattern had a similar pattern of trypsin digestion products as the native protein, as shown by MALDI-TOF mass spectrometry, but it was immunologically distinct from the native protein. The culmination of this project was the generation of a transgenic P trichosuri, the first vertebrate endoparasitic nematode to be heritably transformed. This is a necessary step in the development of a self-disseminating vector to be used in the biocontrol of possums.