Rapid delineation of closely-related filarial parasites using genetic markers in spacer rDNA

Two closely-related species of filarial parasite, Litomosoides galizai and L. sigmodontis, were characterised using a polymerase chain reaction-linked restriction fragment length polymorphism (PCR-RFLP) technique. The rDNA region spanning the first and second internal transcribed spacers as well as the 5.8S gene (ITS +) was amplified by PCR from each of the species using conserved primers to the 18S and 28S ribosomal genes, digested separately with a range of restriction endonucleases and the fragments separated by agarose gel electrophoresis. PCR-RFLP of ITS + using endonucleases Alu I, Cfo I, Dra I, Rsa I and Vsp I produced characteristic patterns for each species. No variation in RFLP patterns was detected among different DNA sample preparations or the sexes of each species. The present study demonstrates that the ITS+ provides genetic markers for the differentiation of L. galizai from L. sigmodontis and suggests that internal transcribed spacer rDNA may provide species markers for other filarioid nematodes. Such markers have implications for diagnosis and for studying the biology, pathogenesis and systematics of filarial parasites.