Molecular Approach for the Diagnosis of Blood and Skin Canine Filarioids

Abstract

The zoonotic <i>Onchocerca lupi</i> and tick-transmitted filarioids of the genus <i>Cercopithifilaria</i> remain less well known due to the difficulties in accessing to skin samples as target tissues. Here, we proposed a molecular approach reliying on multiplex qPCR assays that allow the rapid identification of filarioids from canine blood, skin, and tick samples. This includes two newly developed duplex qPCR tests, the first one targeting filarial and <i>C. grassii</i> DNA (CanFil-<i>C. grassii</i>). and the second qPCR assay designed for the detection of <i>Cercopithifilaria bainae</i> and <i>Cercopithifilaria</i> sp. II DNAs (<i>C. bainae</i>-C.spII). The third one is a triplex TaqMan <i>cox 1</i> assay targeting DNA of blood microfilariae (e.g., <i>Dirofilaria immitis, Dirofilaria repens</i> and <i>Acanthocheilonema reconditum</i>). The novel duplex qPCRs developed were validated in silico and by screening of known DNA collection. The qPCR assays were also used for screening the blood and tick samples of 72 dogs from Algeria. This allowed the identification of canine filariasis infection with 100% of specificity and 89.47% and 100% of sensitivity from naturally infected blood and tick samples, respectively. The prevalences of 26.39% for <i>D. immitis</i> and 5.56% for both <i>D. repens</i> and <i>A. reconditum</i> were reported in blood and tick samples. <i>Cercopithifilaria</i> DNAs were detected only in tick samples, with a prevalence of 4.17% and 5.56% for <i>C. bainae</i> and <i>Cercopithifilaria</i> sp. II, respectively. Co-infections were diagnosed in 6.94% and 13.89% of blood and tick samples, respectively. Whereas all samples were negative for <i>C. grassii</i> DNA. The use of engorged ticks instead of blood and skin samples could be an easier option for the surveillance of all canine filarioids herein investigated. The multiplex qPCR assays herein validated were shown to be useful in the detection of filarial co-infections by overcoming sequencing of positive samples.