Development of a Reverse Transcriptase-Polymerase Chain Reaction-Based Assay for the Detection and Differentiation of Canine and Phocine Distemper Viruses
James B. Stanton1; Steven Poet2, DVM, PhD; Salvatore Frasca Jr.3, DVM, PhD, DACVP; J. Lawrence Dunn4, VMD; Thomas P. Lipscomb5, DVM, DACVP; Seamus Kennedy6; Pierre-Yves Daoust7, DVM, PhD, DACVP; Corrie C. Brown1, DVM, PhD, DACVP
1Department of Pathology, University of Georgia, Athens, GA, USA; 2Department of Medical Microbiology and Parasitology, University of Georgia, Athens, GA, USA; 3Department of Pathobiology, University of Connecticut, Storrs, CT, USA; 4Mystic Aquarium, Mystic, CT, USA; 5Department of Veterinary Pathology, Armed Forces Institute of Pathology, Washington, DC, USA; 6Veterinary Sciences Division, Belfast, UK; 7Department of Pathology and Microbiology, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, PE, Canada
Canine distemper virus (CDV) and phocine distemper virus (PDV) are closely related members of the genus Morbillivirus.1 Both viruses will infect and cause disease in phocids.1,2 Diagnostic differentiation by immunologic means is difficult due to antigenic similarity. Virus isolation would be ideal, but it is complicated by the difficulty in handling live animals, the poor sensitivity when sampling deceased animals, and the difficulty in preserving field-collected specimens. An alternative technique that complements postmortem diagnosis is the use of nucleic acid-based assays, which can utilize sequence analysis to differentiate the two viruses. We developed a semi-nested reverse transcriptase-polymerase chain reaction (RT-PCR) technique and compared its ability to detect CDV and PDV with that of immunohistochemistry (IHC). The procedure was tested first on canine tissue and was shown to consistently amplify a CDV nucleotide sequence from tissue with histopathologic lesions consistent with CDV infection. One case of a CDV infected Caspian seal (Phoca caspica) also yielded a positive RT-PCR result. We also tested three phocine cases (Phoca groenlandica, Cystophora cristata, and an unidentified third species) which were independently confirmed to be infected with PDV; all were positive by our semi-nested RT-PCR technique as well. Sequencing of the amplified product revealed that the two viruses could be distinguished, thus confirming the presence of either CDV or PDV. These data demonstrate the utility of this semi-nested RT-PCR protocol for the accurate diagnosis of these two Morbillivirus viruses and as a complement to other diagnostic procedures.
1. Blixenkrone-Møller M. Biological properties of phocine distemper virus and canine distemper virus. APMIS Suppl. 1993;36:1–51.
2. Mamaev LV, Denikina NN, Belikov SI, Volchkov VE, Visser IK, Fleming M, Kai C, Harder TC, Liess V, Osterhaus AD, Barrett T. Characterisation of morbilliviruses isolated from Lake Baikal seals (Phoca sibirica). Vet Microbiol. 1995;44:251–259.