Detection of sheep poxvirus in skin biopsy samples by a multiplex polymerase chain reaction

The development of a multiplex polymerase chain reaction (PCR) method with amplification of capripoxvirus in a single-step procedure from skin biopsies using three primer pairs, two specific for capripoxvirus and one specific for α-tubulin is described. A sensitive multiplex PCR was achieved by optimization of parameters such as the primer concentrations, magnesium and dNTPs concentrations. False negative results that sometimes arise due to inhibitors of DNA amplification may be avoided by the inclusion in the assay of α-tubulin primers. The results reported on 42 skin biopsies from sheep suspected to have poxvirus infection, indicated that the assay could monitor simultaneously DNA extraction from skin biopsy samples and allow improved detection of capripoxvirus within 24 h of specimen receipt in the laboratory

Sheep poxvirus identification from clinical specimens by PCR, cell culture, immunofluorescence and agar gel immunoprecipitation assay

Some 40 clinical specimens of skin lesions from sheep pox suspected cases were investigated by four different diagnostic assays: PCR, virus isolation in lamb testis cell cultures, direct immunofluorescent assay (DIFA) and antigen detecting agar gel immune precipitation test (AGIPT). All the specimens were positive by PCR and virus isolation, 29 were positive by DIFA and 16 by AGIPT. Using virus isolation on cell cultures as the gold standard, the PCR sensitivity was 100%, while that of DIFA and AGIPT was 73% and 40%, respectively. Skin samples with orf lesions or normal skin biopsies were PCR-negative. Cross-reactions with orf virus were observed in three samples only in the AGIPT assay. The PCR described combines high specificity and sensitivity with speed. PCR was therefore shown to be the method of choice for sheep poxvirus diagnosis directly from clinical specimens

Sheep poxvirus identification by PCR in cell cultures

A simple, rapid and specific diagnostic polymerase chain reaction (PCR) method was developed for sheep poxvirus identification. The primers used were from the sequenced genomes of the capripox viruses KS-1 and InS-1. Six different sheep pox isolates were tested against two orf (parapox) and three animal herpesviruses as controls. Material from uninfected cell cultures was also used as control. The sensitivity of the PCR was approximately equivalent with each of the two primers and for the six sheep pox isolates. All the negative control virus DNAs were negative and differed clearly from those of the sheep pox strains

Caprine PRNP polymorphisms at codons 171, 211, 222 and 240 in a Greek herd and their association with classical scrapie

The association between PRNP variation and scrapie incidence was investigated in a highly affected Greek goat herd. Four mutations were identified at codons 171Q/R, 211R/Q, 222Q/K and 240P/S. Lysine at codon 222 was found to be associated with the protection from natural scrapie (P=0.0111). Glutamine at codon 211 was observed in eight animals, all of them being scrapie-negative, indicating a possible protective role of this polymorphism although statistical analysis failed to support it (P=0.1074). A positive association (P=0.0457) between scrapie-affected goats and the wild-type Q(171)R(211)Q(222)S(240) allele is presented for the first time. In addition, a novel R(171)RQS allele, which is identical to the A(136)R(154)R(171) allele that has been associated with resistance to classical scrapie in sheep, was observed in low frequency. Resistant alleles that include K(222) and Q(211) are absent or rare in sheep and can provide the basis for the development of a feasible breeding programme for scrapie eradication in goats