Polymerase chain reaction for laboratory diagnosis of orf virus infections

Abstract

BACKGROUND: The orf virus of sheep and goats is one of several zoonotic parapoxviruses. In the ovine/caprine host it causes contagious ecthyma (contagious pustular dermatitis, scabby mouth), but in humans it normally causes solitary or clustered orf lesions, typically on hands, arms or face. In addition to disease in the animals, the virus can be quite a nuisance as an occupational hazard in farmers and butchers. Clinical diagnosis is often possible, but laboratory diagnosis is sometimes necessary. For virus isolation, primary ovine or bovine cells, not routinely present, are needed. Serological methods exist, but electron microscopy is the most commonly used method. OBJECTIVES: To develop a reliable method for the laboratory diagnosis of orf zoonoses, without virus culture and without access to an electron microscope. STUDY DESIGN: A suitable primer pair was designed for orf polymerase chain reaction (PCR), using the Oligo software and sequence information from GenBank. Orf positive controls and specimens were kindly provided by several public health centers. Molluscum contagiosum specimens were provided by a dermatologist. HSV-1, HSV-2 and VZV positive swab specimens came from our routine diagnostic service. Asymptomatic skin specimens were obtained from sheep heads from the abattoir, and swab specimens from the heads of asymptomatic sheep. Selected amplified orf PCR positive specimens were sequenced to ensure the authenticity of the PCR products. Orf positive specimens were sent to another laboratory for electron microscopy. RESULTS AND CONCLUSIONS: A robust PCR was developed, with very small inter-run variation. All specificity demands were met, and the sensitivity seems to be good or excellent. All negative specificity controls from cell cultures and non-orf viruses were negative. Twenty-two (95.7%) of 23 scab or swab specimens with suspected orf etiology were orf PCR positive. Five of eight skin specimens from sheep heads from the abattoir were positive, and all 11 swab specimens from asymptomatic sheep were negative. Electron microscopy demonstrated orf-like particles in orf-PCR positive specimens. This PCR seems to be suitable as a diagnostic test for orf in humans, but asymptomatic virus shedding in sheep or goats may complicate veterinary applications of the assay.