Differentiation of dorsal-spined elaphostrongyline larvae by polymerase chain reaction amplification of ITS-2 of rDNA

Molecular genetics was used to devise the first reliable diagnostic tool fo r differentiating morphologically indistinguishable dorsal-spined, first-st age larvae (L1's) and other stages of the nematode protostrongylid subfamil y Elaphostrongylinae. A polymerase chain reaction (PCR) assay employing spe cifically designed primers was developed to selectively amplify DNA of the ITS-2 region of the ribosomal gene. Amplification of the entire ITS-2 regio n differentiated between larvae of the genera Elaphostrongylus and Parelaph ostrongylus, based on the lengths of fragments produced. Three sets of prim ers were designed and used successfully to distinguish larvae at the specie s level. Although it was demonstrated that one primer set in a single PCR a ssay was capable of distinguishing each of the three Parelaphostrongylus sp p., a second primer set would be required for confirmation in routine diagn ostic use. Two of the three primer sets were capable of amplifying DNA from all six elaphostrongyline species and of identifying Elaphostrongylus alce s and Parelaphostrongylus odocoilei. Although two separate fragments were p roduced from each Elaphostrongylus cervi and Elaphostrongylus rangiferi, it was not possible to distinguish these two parasites from each other based on the fragment size. The use of various nematodes, hosts, and fecal contro ls demonstrated the reliability of the primers for all developmental stages including L1's, third-stage larvae, and adult worms. These primers also ha ve potential for identifying other lungworms as was shown by the amplificat ion of Umingmakstrongylus pallikuukensis, the muskox protostrongylid, and D ictyocaulus sp, from white-tailed deer. Although this assay may benefit fro m further refinement, its present design provides researchers, wildlife man agers, clinicians, and animal health regulators with a practical tool for t he control, management, and study of meningeal and tissue worms and their c lose relatives.