Polymerase Chain Reaction-Based Method for the Detection of Canine Heartworm (Filarioidea: Onchocercidae) in Mosquitoes (Diptera: Culicidae) and Vertebrate Hosts

Abstract

A polymerase chain reaction (PCR)–based test was developed to aid in identification of Dirofilaria sp. and for use in surveys for infected vectors of Dirofilaria immitis (Leidy). A set of PCR primers was designed based on the DNA sequence of a D. immitis surface antigen gene. The predicted product was a 378 base-pair DNA fragment. The target fragment was amplified from free D. immitis larvae (both L3 and microfilariae), individual infected mosquitoes, pools of 30 mosquitoes (including a single infected mosquito), infected mosquitoes stored desiccated at room temperature for 7 mo, and whole blood from a dog infected with D. immitis. These primers did not amplify a homologous fragment from a mermithid or from 4 other species of filarioid nematodes (including 1 other Dirofilaria species). Third-stage larvae from field-collected mosquitoes also were tested. Field-collected L₃, identified tentatively as D. immitis, were confirmed by PCR. There was no amplification using the PCR test for L₃ identified tentatively as Dirofilaria sp. (possibly D. tenuis Chandler). These data provide a strong indication of the specificity of these primers. The potential utility of this technique for detecting the presence of D. immitis in field populations of mosquitoes is discussed.