Molecular Approach for the Diagnosis of Blood and Skin Canine Filarioids.

Younes Laidoudi,Samia Bedjaoui,Bernard Davoust,Hacène Medkour,Idir Bitam,Oleg Mediannikov,Domenico Otranto,Abdeslam Mekroud,Maria Stefania Latrofa

doi:10.3390/microorganisms8111671

Abstract

The zoonotic Onchocerca lupi and tick-transmitted filarioids of the genus Cercopithifilaria 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 C. grassii DNA (CanFil-C. grassii). and the second qPCR assay designed for the detection of Cercopithifilaria bainae and Cercopithifilaria sp. II DNAs (C. bainae-C.spII). The third one is a triplex TaqMan cox 1 assay targeting DNA of blood microfilariae (e.g., Dirofilaria immitis, Dirofilaria repens and Acanthocheilonema reconditum). 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 D. immitis and 5.56% for both D. repens and A. reconditum were reported in blood and tick samples. Cercopithifilaria DNAs were detected only in tick samples, with a prevalence of 4.17% and 5.56% for C. bainae and Cercopithifilaria 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 C. grassii 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.

Highlights

Canine filarioses are a group of diseases caused by arthropod-borne filarioids (Spirurida: Onchocercidae) belonging to the genera Dirofilaria, Acanthocheilonema, Cercopithifilaria, Brugia, and Onchocerca [1,2,3]
Microfilariae in the subcutaneous tissues such as Cercopithifilaria spp. and the zoonotic O. lupi are less diagnosed or completely non-diagnosed in comparison to those circulating in blood such as D. immitis, D. repens, A. reconditum and Acanthocheilonema dracunculoides, where they are routinely diagnosed by several assays such as morphological identification, molecular and serological tests [5]
No DNA amplification was obtained from both free-filarial tick and skin samples as well as from the panel of negative controls summarized in Supplementary Table S1

Summary

Introduction

Canine filarioses are a group of diseases caused by arthropod-borne filarioids (Spirurida: Onchocercidae) belonging to the genera Dirofilaria, Acanthocheilonema, Cercopithifilaria, Brugia, and Onchocerca [1,2,3]. In addition to their veterinary importance, many of them are zoonotic. The diagnosis of Cercopithifilaria spp. and O. lupi is based mainly on microscopic examination of dog skin snip sediments and the identification of adults embedded in cutaneous/ocular nodules [3] This method is quite invasive since it requires a skin biopsy, representing a major limitation to this diagnosis technique in the clinical routine [20]. Molecular techniques have recently been standardized for the detection of O. lupi DNA [3]

Methods

Results

Discussion

Conclusion