Genetics, immunity and DNA fingerprinting in the identification of protective antigens of Eimeria maxima


Damer P. Blake, Adrian L. Smith and Martin W. Shirley.
Institute for Animal Health, Compton, Berkshire, RG20 7NN, UK.
Email: damer.blake@bbsrc.ac.uk

Many protozoan genomes are the subject of extensive sequencing projects, but the identification of genes that encode antigens capable of stimulating a protective immune response remains a demanding task. The rational identification of genuinely immunoprotective gene products encoded by unsequenced genomes is even more daunting. The majority of screens for vaccine candidates protective against protozoan infection have been largely empirical, based upon the induction of an immune response rather than protection. However, consideration of the genetics of immune escape by different strains of Eimeria maxima has raised the possibility of the targeted identification of a gene (or genes) under immune selection. Central to our strategy is the concept that the inheritance of genetic markers is influenced by their proximity within the genome to loci under deleterious selection. Initially multiple independent hybrid populations were created through the mating of two strains of E. maxima that each induce a lethal strain-specific protective immune response in the host and show a differential response to the anticoccidial drug robenidine. Escape by a sub-population within the progeny of this cross from a double-barrier consisting of immune and chemotherapeutic selection lethal to either parent proved that loci encoding molecules stimulating strain-specific protective immunity or resistance to robenidine segregate independently. Comparison of parasite replication in the presence of the selective double-barrier, either individual component of the barrier or no barrier, implicates a highly restricted number of key protective-antigen-encoding loci. The selection of recombinant parasites through the double-barrier imposed genetic-bottleneck was accompanied by the elimination of ~3% of the polymorphic DNA markers that defined the parent strain used to immunise the host. Hybridisation studies with Southern-blotted digested and undigested karyotypes resolved by PFGE and a BAC library derived from the parent strain under immune selection have revealed two clusters of these DNA markers correlated with selection that cover independent regions of ~220 Kb (13 markers) and ~400 Kb (8 markers) within the genome. Our identification of polymorphic DNA markers that associate with immune-mediated killing and are physically co-localised within the genome support our strategy to identify loci under selection in Eimeria spp. and should be applicable to other loci under selection in other Apicomplexa including Plasmodium spp.