Molecular Confirmation of Parelaphostrongylus tenuis Infection in a Horse With Verminous Encephalitis

Abstract

>> Editor: Advances in molecular diagnostic technology have enabled us to identify and confirm an etiologic agent that was not possible when we published a case report of verminous encephalitis in a colt in Veterinary Pathology. 2 Although we presumed that the cause of the encephalitis was Parelaphostrongylus tenuis—a meningeal helminth that commonly parasitizes white-tailed deer (Odocoileus virginianus)—we could identify only the parasites as members of the family Protostrongylidae. We recently applied a polymerase chain reaction–(PCR) based approach to the DNA isolated from archival paraffin blocks. Using genus-specific primers and sequence analysis, we confirmed the infection specifically as P tenuis. Thin sections of formalin-fixed, paraffin-embedded brain tissue containing protostrongylid larvae were processed for DNA extraction with a QIAamp 1 DNA Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. The Parelaphostrongylus spp–specific primers used for PCR were described previously (PTP1, 5 0 -CCGTCGAATACATGT CATCC-3 0 ; PTP2, 5 0 -TCGTCAAGACGATGATTCCC-3 0 ). 1 Specificity of these primers for Parelaphostrongylus spp was confirmed by BLAST comparison with nonredundant sequences in the BLASTn database (National Center for Biotechnology Information). These primers amplify a 248–base pair product covering a portion of the second internal transcribed spacer of the ribosomal RNA gene in Parelaphostrongylus spp. The PCR reaction mixture included 10mM Tris-HCl (pH 9.0), 50mM KCl, 0.1% Triton X-100, 1.5mM MgCl2, 250mM deoxynucleotide triphosphates, 0.5mM of each primer, and 1 U of Taq DNA polymerase (Promega, Madison, WI). Cycling parameters included denaturation at 94 � Cf or 3 minutes, followed by 40 cycles of denaturation at 94 � Cf or 1 minute, annealing at 60 � C for 1 minute, and extension at 72 � C for 1 minute. Reaction products were examined by electrophoresis in a 1% agarose gel and were subsequently purified with a QIAquick 1 Gel Extraction Kit (Qiagen) according to