Identification and isolation of ORF virus from sheep in the Republic of Tuva in 2015

Orf is infectious disease of sheep and goats, characterized by lesions of mucosa of the oral cavity, skin of lips, head, mammary glands and limbs. The causal agent (virus of contagious ecthyma - Orf virus) is a member of genus Parapoxvirus, subfamily Chordopoxvirinae, family Poxviridae. Genome of the Orf virus consists of linear double stranded DNA (138 kbp) and contains 132 open reading frames. Its antigenic structure is poorly understood, and strains are not serologically identical. That is why the investigation of novel isolates of Orf virus is actual. However, we are not aware of such research in Russia possibly due to rare outbreaks recognized. We first identified Orf virus in sheep from Tuva Republic and studied biological properties of the isolate. Disease of sheep, characterized by erythema, vesicles, pustules and scabs on ears and legs has been registered in August 2015 in Tuva Republic. The scabs were sampled for isolation and identification of the causative agent. For this result three lambs were experimentally infected with the scab suspension. The mucocutaneous borders of the lips along the labial commissures were scarified and inoculum was loaded by cotton swabs. The lip vesicles appeared at days 4-5 and then formed pustules and scabs. After 2 weeks the lesions healing occurred. The suspension of scabs from lips of infected lambs was used to inoculate sheep kidney cell culture. In 7 days post inoculation the cell monolayer was harvested for second passage. However, we did not observe specific cytopathic effect in the monolayers during these passages. The skin swabs from experimentally infected lambs were examined by Real-Time PCR in accordance to the protocol developed by G. Venkatesan et al. (2014), and Orf virus DNA was detected at days 7 to 28 post infection. We also detected Orf virus DNA in scabs from the infected lambs and in lysates of the monolayers harvested after second passage. The PCR test was positive for reference strain IA82 of Orf virus unlike other viruses (nodular dermatitis vaccine strain, sheep pox strains Mongolian and B5/96 or goatpox strain QA/A-04) that confirmed specificity of the PCR system. Its analytical sensitivity to detect Orf virus genomic DNA was 1.3±0.03 lg TCD50/cm3. Thus, the pathogen which caused the disease of sheep in Tuva Republic in 2015 was isolated from experimentally infected lambs using sheep kidney cell culture and identified as Orf virus.