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® 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′-CCGTCGAATACATGTCATCC-3′; PTP2, 5′-TCGTCAAGACGATGATTCCC-3′). 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, 250μM deoxynucleotide triphosphates, 0.5μM of each primer, and 1 U of Taq DNA polymerase (Promega, Madison, WI). Cycling parameters included denaturation at 94°C for 3 minutes, followed by 40 cycles of denaturation at 94°C for 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® Gel Extraction Kit (Qiagen) according to the manufacturer’s instructions. Extracted DNA was cloned with pCR® 4-TOPO® plasmid (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Sequencing of the plasmid was performed at the Integrated Biotechnology Laboratories at the University of Georgia with T3 and T7 primers and a 3100 Genetic Analyzer (Applied Biosystems Inc, Foster City, CA).
Nucleotide sequence analysis of the 241–base pair PCR product revealed 98% identity to a P tenuis second internal transcribed spacer sequence in the GenBank® database (accession No. AF504029). The consensus nucleotide sequences of the nematodes have been deposited in GenBank® under the accession No. GU122925. To our knowledge, this is the first confirmation of P tenuis infection in a horse based on PCR and nucleotide sequencing techniques.