Bluetongue virus detection: a safer reverse-transcriptase polymerase chain reaction for prediction of viremia in sheep.

A reversible target capture viral RNA extraction procedure was combined with a reverse-transcriptase nested polymerase chain reaction (PCR) to develop a capture PCR assay providing a rapid and safe prediction method for circulating bluetongue virus in infected ruminants. This new assay was compared with virus isolation and a recently developed antigen-capture enzyme-linked immunosorbent assay (ELISA) for the detection of bluetongue virus. Eight Warhill crossbred sheep were inoculated subcutaneously with bluetongue virus serotype 10, and blood samples were taken sequentially over a period of 28 days. The capture PCR detected the peak of viremia, as determined by virus isolation and antigen-capture ELISA, from day 5 to day 14 after challenge. The results indicate that the rapid-capture bluetongue virus PCR provides a rapid indicator of samples in which virus can be isolated. In addition, this capture bluetongue virus PCR procedure does not require a lengthy phenol extraction or the use of the highly toxic methyl mercury hydroxide denaturant.

Bluetongue virus seropositivity in sheep flocks in North West Frontier Province, Pakistan.

The objectives of this study were to describe the prevalence and distribution of serum antibodies to Bluetongue virus (BTV) in a sample of 38 sheep flocks in northern areas of the North West Frontier Province of Pakistan and to identify demographic and productivity variables that are associated with BTV seropositivity. Blood samples were taken from a random sample of ewes in each flock in April 1995. The owners of the flocks were interviewed regarding some demographic, husbandry and productivity variables of the flocks on the day of blood sampling. A competitive enzyme-linked immunosorbent assay was conducted to test the serum samples for BTV group-specific antibodies. BTV seropositive reactions were obtained in 184 (48.4%) out of 380 tested sera, and in 89.5% (34/38) of the flocks. In the 34 seropositive flocks, the prevalences ranged from 12.5 to 100% (median = 47). A multivariable logistic analysis was carried out to study the influence of demographic and productivity variables on the BTV serological status of the sheep flocks. Abortion risk in the previous lambing season was mildly associated with the serological status of the flock (adjusted odds ratio = 1.16, P = 0.07). For the seropositive flocks, a linear multiple regression showed that distance travelled by the flock during transhuman movement was significantly associated with percent seropositivity (partial regression coefficient (+/- SE) = -0.091 +/- 0.045).