Development of a PCR- and probe-based test for the sensitive and specific detection of the dog heartworm, Dirofilaria immitis, in its mosquito intermediate host.

The mosquito-borne filarial worm, Dirofilaria immitis, causes heartworm disease in dogs. Detection of this parasite in its mosquito intermediate host currently involves dissection and microscopic examination for larval stages. Although this method is used commonly as a screening tool for epidemiological surveys, it lacks both sensitivity and specificity. In this study, a more efficient PCR- and probe-based diagnostic assay was developed. The target selected for this assay is a segment of the 16 S rRNA gene. The assay specifically detects as little as 10 pg of D. immitis genomic DNA, equivalent to DNA derived from one third stage larva (L(3)), but does not detect 100 ng (10 000-fold excess) of the purified DNA from several other filarial nematodes, including Dirofilaria striata, Dirofilaria tenuis, Dipetalonema reconditum, Wuchereria bancroftii, Brugia pahangi, B. malayi, Onchocerca volvulus or Loa loa. This assay also detects one L(3)of D. immitis, the minimal biological unit of infection, in a pool of 200 mosquito heads. This assay can serve as a highly specific and sensitive tool for efficiently screening the large numbers of mosquitoes to determine, with statistical validity the seasonal transmission pattern of D. immitis in a locality prior to designing a rational preventive medication program for that parasite.