Development of a Multiplex Real-Time PCR to Disambiguate Culicoides sonorensis within Culicoides variipennis Complex, the Proven Vector of Bluetongue and Epizootic Hemorrhagic Disease Viruses in North America

Abstract

Species delimitation of Culicoides complex species can be challenging. Among species within the Culicoides variipennis complex, C. sonorensis is considered the primary vector of bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV) in North America. Morphological identification of C. sonorensis within the C. variipennis complex is laborious, time-consuming, and requires entomology expertise. Therefore, in this study we developed and validated a multiplex real-time PCR for rapid detection and differentiation of C. sonorensis from the two other main cryptic species (C. variipennis and C. occidentalis) within the C. variipennis complex. The assay targets the EF1α gene and has a built-in internal control targeting 18 S. The specificity and the sensitivity of the multiplex real-time PCR were evaluated using morphologically identified reference and field-collected specimens. The multiplex PCR was 100% specific when nucleic acid extracted from C. variipennis, sonorensis, and occidentalis specimens was tested. When nucleic acid extracted from pools of midges was tested, the multiplex PCR was able to detect all three Culicoides species with comparable sensitivity. The multiplex assay, however, failed to detect eight morphologically identified C. sonorensis specimens collected from Alberta in 2014. The EF1α gene sequences of these specimens formed a distinct phylogenetic cluster, amongst those from C. variipennis, sonorensis, and occidentalis, suggesting that they belong to a different species. We hypothesize that those specimens might be C. albertensis, the only other species remaining in the C. variipennis complex with known geographical distribution in North America. We believe that this highly sensitive and specific multiplex real-time PCR assay could be an effective tool for rapid detection and differentiation of C. sonorensis, the known vector of BTV and EHDV, in trap collections in future vector surveillance programs.

Keywords: Culicoides sonorensis; Culicoides variipennis complex; bluetongue; epizootic hemorrhagic disease; real-time PCR.

Figure 1

Forward and reverse primers are shown in green and red, respectively. Probe target sites are shown in orange with black arrows identifying unique single nucleotide polymorphisms for C. sonorensis (PP529686), C. variipennis (PP529717), or C. occidentalis (PP529684). Consensus nucleotides in the sequence alignment are indicated by an asterisk.

Figure 2

Phylogenetic tree and multiple-sequence alignment analysis for species of the C. variipennis complex using EF1α sequence data. Sequences derived from specimens collected for this study are shown with the addition of squares to branch tips. The topology shows Bayesian inference trees using Geneious Prime 2021.2.2. Only pairwise identity values above 70% are shown. Different species are identified by different colors.

Figure 3

Distribution of a subset of Culicoides species within the C. variipennis complex across Canada from 2014–2016, including the direct comparison of changes in species between 2014–2015 and 2023 in British Columbia.