Characterization of the second internal transcribed spacer (ITS-2)rDNA region among the Elaphostrongylinae (Nematoda:Protostrongylidae)

Abstract

Four species of elaphostrongyline nematodes are known to infect cervids in North America. One species, Parelaphostrongylus tenuis, can cause neurologic disease (parelaphostrongylosis) in cervid hosts other than white tailed deer. Another, Elaphostrongylus rangiferi, can cause cerebrospinal elaphostrongylosis (CSE) in young, heavily infected caribou. The remaining species, P. andersoni and P. odocoilei do not cause neurologic disease but can cause verminuous pneumonia. Moreover, two European species, E. cervi and E. alces, are capable of infecting North American cervid populations if they enter with imported game animals, such as red deer. Differentiation of these species is problematic as they all produce first stage larvae (LI) that are morphologically indistinguishable. This is a major concern for wildlife biologists who attempt to identify and limit the spread of pathogenic nematodes in North America. This study improves upon existing methods of extraction and amplification of protostrongylid DNA by addressing the difficulties of obtaining DNA data from preserved as well as single nematodes, both adult and larval. A modified commercial kit extraction and purification procedure (QIAamp, Qiagen, Valencia, California) was developed and PCR parameters, such as cycling temperatures and times, were optimised to address these difficulties. Second internal transcribed spacer (TTS-2) rDNA sequence data was obtained for all six elaphostrongyline species as well as an unidentified nematode from bighorn sheep in Washington. Elaphostrongylus cervi and E. rangiferi are both 585 base pairs (bp) long, E. alces is 575 bp, P. tenuis is 554 bp, P. andersoni is 545 bp, and P. odocoilei is 561 bp long.