Molecular characterization of Dirofilaria spp. circulating in Portugal

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.

Exposure of humans to the zoonotic nematode Dirofilaria immitis in Northern Portugal

“…Human pulmonary dirofilariosis is an emerging zoonotic disease caused by Dirofilaria immitis . Humans are accidentally infected in endemic areas, where dogs act as reservoirs of the parasite and the climate conditions favour the proliferation of the mosquito vectors [1]. D. immitis is considered a public health concern because of its zoonotic potential; in humans, the pre-adult parasite stages form coin-shaped cysts in the branches of the pulmonary arteries, which are generally asymptomatic.…”

First Molecular Detection of Dirofilaria immitis and D. repens in Dogs from Kyrgyzstan

“…Third one is mosquito vectors are not common in the sampling region due to the climatic conditions. Prevalence of D. immitis was determined as 21.3% in France [38], 14.5% in Tunisia [29], 13.7% in Portugal [10], 8.0% in Mexico [40], 2.3-4.02% in Iran [4,24], 1.5-3.7% in Turkey [3,13,37] and 0.0% in Cape Verde [20] with PCR. Environmental factors particularly climate and ecologic features influence mosquito population and this can be related to prevalence of dirofilariosis.…”

“…Dirofilaria immitis, a pathogenic filarial nematode in dogs, other canids and humans [19], cause cardiopulmonary problems (heartworm disease) in many geographic regions of Asia [14,22,27,37,41,43], Europe [9,10,12,30], Africa [29,39], Australia [8] and America [40].…”

Sero-prevalence of Dirofilaria repens Infection in Dogs by Indirect-ELISA using Microfiarial and Adult Worm Antigen

“…Many researchers across world were reported seroprevalence of D. immitis infection in dogs using commercial ELISA test kits in the range of 1.3 -42.8 per cent (Alves et al, 1999;Svobodova et al, 2005;Simsek et al, 2008;Ben Mahdi and Mohamed, 2009;Rapti and Rehbein, 2010;Ionica et al, 2014;Girdan et al, 2015;Diakou et al, 2016;Ferreira et al, 2017 andAtas et al, 2018). Whereas, Borthakur et al, (2015) reported 18.03 per cent seroprevalence of D. immitis infection in dogs from north-eastern states of India by using commercial ELISA test kit.…”