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Abstract
BackgroundDirofilariosis is a potentially zoonotic parasitic disease, mainly transmitted by mosquito vectors in many parts of the world. Data concerning the canine Dirofilaria species currently circulating in Portugal is scarce. Thereby, a large-scale study was conducted to determine the Dirofilaria spp. present in Portugal, based on a molecular approach, and also to optimize a reliable and highly sensitive species-specific polymerase chain reaction (PCR) assay that could be used for the simultaneous detection and differentiation of Dirofilaria immitis, Dirofilaria repens, and other concurrent filarial species in animal reservoirs.MethodsBlood samples were collected from three districts of Portugal (Coimbra, Santarém and Setúbal) between 2011 and 2013. Samples were tested using rapid immunomigration tests (Witness® Dirofilaria), modified Knott’s technique and acid phosphatase histochemical staining. In addition, molecular analysis was performed by amplification of the internal transcribed spacer (ITS) region using two different PCR protocols, specific for molecular screening of canine filarial species.ResultsOf the 878 dogs sampled, 8.8% (n = 77) were positive for D. immitis circulating antigen and 13.1% (n = 115) positive for microfilariae by the modified Knott’s technique. Of the 134 samples tested by acid phosphatase histochemical staining, 100 (74.6%) were positive for D. immitis. Overall, 13.7% (n = 120) were positive by PCR for D. immitis by ITS2, of which 9.3% (67/720) were also positive by ITS1. ITS2 PCR was the most sensitive and specific method, capable of detecting mixed D. immitis and A. reconditum infections. Heterozygosity, in the form of double peaks, was detected by sequencing of both ITS regions. No D. repens was detected by any of the diagnostic methods.ConclusionsThe present study confirmed D. immitis as the dominant species of the genus Dirofilaria infecting Portuguese dogs, based on sequencing of ITS1 and ITS2 PCR fragments. Additionally, ITS2 PCR was the most adequate method for diagnosis and prevalence estimation.
Highlights
Dirofilariosis is a potentially zoonotic parasitic disease, mainly transmitted by mosquito vectors in many parts of the world
The aim of the present study was to identify the Dirofilaria species currently circulating in Portuguese dogs through an optimised reliable and highly sensitive species-specific polymerase chain reaction (PCR) assay for the simultaneous detection and differentiation of D. immitis, D. repens and other concurrent filariids in animal reservoirs
Higher analytical sensitivity was observed for internal transcribed spacer 2 (ITS2)-PCR, with 12.9% of the blood samples positive for D. immitis (Table 1)
Summary
Dirofilariosis is a potentially zoonotic parasitic disease, mainly transmitted by mosquito vectors in many parts of the world. Dirofilariosis is a potentially zoonotic filarial parasitic disease, present in several parts of the world, transmitted mainly by mosquito vectors. The species Dirofilaria immitis and Dirofilaria repens (Filarioidea, Onchocercidae) are widely present in the Mediterranean basin and are the causative agents of cardiopulmonary and subcutaneous dirofilariosis, respectively. Both nematodes are transmitted by mosquito species of the family Culicidae and can infect. A. reconditum has been reported in humans [8] These filarial species release circulating microfilariae (Mf) in the blood of their definitive hosts. Molecular protocols have been developed for reliable detection and differentiation of filarial species, in particular, a speciesspecific PCR assay and multiplex PCR and restriction fragment length polymorphism (RFLP) assays for simultaneous detection of different Dirofilaria spp., either in the vector or in blood [12, 14,15,16,17,18,19,20,21]
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