Development of a multiplex qPCR-based approach for the diagnosis of Dirofilaria immitis, D. repens and Acanthocheilonema reconditum

Younes Laidoudi,El Hadji Amadou Niang,Oleg Mediannikov,Florence Fenollar,Marie Varloud,Bernard Davoust

doi:10.1186/s13071-020-04185-0

Abstract

BackgroundDirofilaria immitis, D. repens and Acanthocheilonema reconditum are the main causative agents of zoonotic canine filariosis.MethodsWe developed a combined multiplex approach for filaria and Wolbachia detection using the 28S-based pan-filarial and 16S-based pan-Wolbachia qPCRs, respectively, involving a fast typing method of positive samples using triplex qPCR targeting A. reconditum, D. immitis and D. repens, and a duplex qPCR targeting Wolbachia of D. immitis and D. repens. The approach was complemented by a duplex qPCR for the differential diagnosis of heartworms (D. immitis and Angiostrongylus vasorum) and pan-filarial cox1 and pan-Wolbachia ftsZ PCRs to identify other filarial parasites and their Wolbachia, respectively. A total of 168 canine blood and sera samples were used to validate the approach. Spearmanʼs correlation was used to assess the association between filarial species and the strain of Wolbachia. Positive samples for both the heartworm antigen-test after heating sera and at least one DNA-positive for D. immitis and its Wolbachia were considered true positive for heartworm infection. Indeed, the presence of D. repens DNA or that of its Wolbachia as well as A. reconditum DNA indicates true positive infections.ResultsThe detection limit for Wolbachia and filariae qPCRs ranged from 5 × 10−1 to 1.5 × 10−4 mf/ml of blood. When tested on clinical samples, 29.2% (49/168) tested positive for filariae or Wolbachia DNA. Filarial species and Wolbachia genotypes were identified by the combined multiplex approach from all positive samples. Each species of Dirofilaria was significantly associated with a specific genotype of Wolbachia. Compared to the true positives, the approach showed excellent agreement (k = 0.98–1). Unlike D. immitis DNA, no A. vasorum DNA was detected by the duplex qPCR. The immunochromatographic test for heartworm antigen showed a substantial (k = 0.6) and a weak (k = 0.15) agreements before and after thermal pre-treatment of sera, respectively.ConclusionsThe proposed approach is a reliable tool for the exploration and diagnosis of occult and non-occult canine filariosis. The current diagnosis of heartworm disease based on antigen detection should always be confirmed by qPCR essays. Sera heat pre-treatment is not effective and strongly discouraged.

Highlights

Dirofilaria immitis, D. repens and Acanthocheilonema reconditum are the main causative agents of zoonotic canine filariosis
Another qPCR system for Wolbachia targeting the 16S gene has been proposed as a complementary diagnosis from human blood of the lymphatic filariosis caused by Wuchereria bancrofti [42]
The molecular approach developed represents an improvement in the diagnosis of canine filariosis

Summary

Introduction

Dirofilaria immitis, D. repens and Acanthocheilonema reconditum are the main causative agents of zoonotic canine filariosis. Acanthocheilonema reconditum, A. dracunculoides, Cercopithifilaria grassii, Brugia ceylonensis, B. patei, B. malayi, B. pahangi, Onchocerca lupi and Thelazia callipaeda [1,2,3,4] These arthropod-borne filarioids produce blood, cutaneous or mucous microfilariae, where they are available to arthropod vectors [5]. The most common and medically important species affecting dogs are D. immitis, D. repens and A. reconditum [6]. Acanthocheilonema reconditum is an occasional zoonotic agent that affects the subcutaneous tissue and the perirenal fat [9, 10] causing a common but clinically less important infection in dogs [11]

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