Molecular and Cellular Probes (1995) 9, 315-320 Genetic markers for closely-related parasitic nematodes R. B. Gasser 1",2 and H. Hoste 2 ~Department of Veterinary Science, The University of Melbourne, Princes Highway, Werribee, Victoria 3030, Australia and 21nstitut National de la Recherche Agronomique, Station de Pathologie Aviaire et de Parasitologie, Centre de Tours, 37380 Nouzilly, France (Received 17 May 1995, Accepted 30 May 1995) Seven species of closely-related nematode parasite (Trichostrongylus axei, T. colubriformis, T. probolurus, T. retortaeformis, T. rugatus, T. vitrinus and T. tenuis) were characterized using a polymerase chain reaction-linked restriction fragment length polymorphism technique (PCR-RFLP). The rDNA region spanning the first and second internal transcribed spacers as well as the 5.8S rDNA gene (ITS +) was amplified from isolates of each of the seven species, digested separately with six restriction endonucleases (Dra I, Hinf I, Rsa I, Vsp I, Nla III and Tsp 509 I) and the fragments separated by agarose gel electrophoresis. PCR-RFLP of ITS+ produced characteristic patterns for each Trichostrongylus species examined. No variation in RFLP patterns was observed among different isolates for species where multiple isolates were examined. The present study demonstrates that the ITS+ provides genetic markers for the species identification of closely- related parasitic nematodes, and indicates the usefulness of these markers for diagnostic purposes, and epidemiologic and molecular-systematic studies on parasites and other eukaryotic organisms. © 1995 Academic Press Limited KEYWORD$: ribosomal DNA, internal transcribed spacer, PCR-RFLP, genetic markers, parasitic nematodes, species identification, differentiation. INTRODUCTION Nematode parasites are of major animal and human health importance in many countries. The accurate identification of a nematode parasite, irrespective of life cycle stage, is central to studying its life cycle, population biology and epidemiology. The identification of adult worm stages is based on morphological characteristics, * yet, the species iden- tification of eggs, certain larval stages and female worms is not always possible using morphology. 2 Larvae produced from eggs by culturing techniques can usually be identified to the genus level under a microscope, but species recognition is sometimes unreliable and the technique of larval culture suffers from inaccuracies2 -s Recently, DNA techniques have been used to reliably identify parasite species and assess genetic diversity in parasite populations. 6-I° Ribosomal genes (rDNA) are useful targets because they are abundant in the organism, 7 making the de- velopment of highly sensitive techniques feasible. Reports describe that rDNA spacers are useful for the identification of parasites.11-17We have recently shown that the second internal transcribed rDNA spacer can provide markers to identify some species of nematodes, 18'~9but in certain genera the level of *Author to whom correspondence should be addressed. 0890-8508/95/050315 + 05 $12.00/0 315 © 1995 Academic Press Limited