F. R. Holbrook

Genetic variation in populations of Culicoides variipennis complex in the six New England states, U.S.A.

Abstract. We investigated the identity and distribution of members of the Culicoides variipennis complex in the six New England states of the U.S.A., a region where bluetongue transmission has not been detected. Analyses of seven polymorphic isozyme‐encoding loci showed that only C.v.variipennis, not considered to be a vector of the bluetongue viruses, was present. The populations of C.v.variipennis were significantly more hetero‐zygous than C.v.sonorensis and Cv.occidentalis populations from similar studies in the state of California. Estimates of genetic diversity among populations of C.v.variipennis in New England were similar to C.v.sonorensis in the state of Colorado, but were significantly more genetically divergent than California populations of Cv.occidentalis. The impact of these findings on the status of New England as a possible bluetongue‐free region for the purpose of international trade in ruminant livestock and their germplasm is discussed.

Specificity of molecular hybridization techniques for the detection of bluetongue virus serotypes in Culicoides variipennis

Direct blot hybridization (DBH) and sandwich hybridization (SH) were evaluated for their ability to detect bluetongue virus (BTV) RNA in the biting midge Culicoides variipennis (Coquillett). Probes were derived from the L3 RNA segment of BTV, serotype 17. RNA of the five BTV serotypes occurring in the USA (BTV-2, BTV-10, BTV-11, BTV-13, and BTV-17) was extracted from pools of varying numbers of infected and uninfected biting midges and assayed by direct blot and sandwich hybridization tests. Direct blot hybridization using an RNA transcript probe or cDNA probe was a fast, efficient and sensitive technique, detecting as few as one midge infected with any BTV serotype in a pool of 50 or 100. Sandwich hybridization was able to detect the homologous serotype, BTV-17, in pools containing a single infected midge in a total of 50 or 100. However, detection of the heterologous serotypes, BTV-10, BTV-11, and BTV-13, was limited to pools containing 5 or more infected midges in a total of 50, and BTV-2 was undetectable by SH. Hybridization techniques provide an alternative to the conventional detection methods of inoculation of cell culture or embryonated chicken eggs for detection of BTV.