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Background   Dirofilaria immitis ,  D. repens  and  Acanthocheilonema reconditum  are the main causative agents of zoonotic canine filariosis.    Methods  We 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  cox 1 and pan- Wolbachia fts Z 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.    Results  The 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.    Conclusions  The 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.

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