PCR detection of Dirofilaria immitis in Aedes aegypti and Culex pipiens from urban temperate Argentina

Darío Vezzani,María Mesplet,María F Fontanarrosa,Diego F Eiras,Leonhard Schnittger

doi:10.1007/s00436-010-2142-1

Abstract

Dirofilariasis, a mosquito-borne disease of dogs caused by the nematode Dirofilaria immitis (Leidy; Spirurida: Onchocercidae), has now become a growing zoonotic concern. Based on direct microscopical observation, Aedes aegypti (L.) and Culex pipiens L. (Diptera: Culicidae) have been previously incriminated as potential vectors of D. immitis in urban temperate Argentina. In this study, an effort was made to provide evidence for this assumption by screening of mosquitoes for D. immitis infection using a polymerase chain reaction (PCR) assay. PCR primers were developed to specifically amplify the D. immitis-16S rRNA gene and to reliably detect 100th of the genomic equivalent (10 pg) of the infective third-stage larvae in mosquito pools of up to 30 individuals. Collection of mosquitoes was performed between September 2007 and April 2008 in premises known to be inhabited by D. immitis-infected dogs in Greater Buenos Aires. The final collection comprised 453 specimens belonging to 11 mosquito species of the genera Aedes, Culex, Ochlerotatus, and Psorophora. PCR assays were performed on 82 pools (n ≤ 20) of heads and abdomens separately, as this allows differentiating infective and non-infective stages of the parasite, respectively. Identification of the non-infective stage of D. immitis in A. aegypti and C. pipiens provided additional strong support of transmission of the parasite by these species. To our knowledge, this was the first PCR screening for D. immitis-infected mosquitoes in South America.

Highlights

The dog heartworm, Dirofilaria immitis (Leidy; Spirurida: Onchocercidae), is considered a veterinary concern in most warm regions of the word
Since the first report of a mosquito infected with D. immitis in 1901, more than 60 mosquito species have been incriminated as potential vectors (Ludlam et al 1970)
Search in the GenBank database for genes that might be suitable targets for molecular diagnostics of D. immitis resulted in the identification of a ribosomal 16S rRNA partial gene (Watts et al 1999)

Summary

Introduction

The dog heartworm, Dirofilaria immitis (Leidy; Spirurida: Onchocercidae), is considered a veterinary concern in most warm regions of the word. Since the first report of a mosquito infected with D. immitis in 1901, more than 60 mosquito species have been incriminated as potential vectors (Ludlam et al 1970). The identification of the filarial larval stages in the mosquito is based on morphological criteria by microscopical diagnostics, which is considered to be laborious, of low sensitivity, and can lead to misdiagnosis (Nuchprayoon et al 2005; Plichart et al 2006; Scoles and Kambhampati 1995; Watts et al 1999). The development of molecular methods to identify filarial larvae has allowed a more accurate identification of their mosquito vectors during the last decade (McCall et al 2008)

Methods

Results

Conclusion