Molecular characterization of Dirofilaria spp. circulating in Portugal

Abstract

Background: Dirofilariosis 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.

Methods: Blood 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.

Results: Of 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.

Conclusions: The 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.

Keywords: Dirofilaria; Dog; Internal transcribed spacer; PCR; Portugal.

Fig. 1

Alignment of heterozygous ITS1 sequences of D. immitis from Portuguese canine samples. The haplotypes were inferred based on circulating haplotypes, considering the most parsimonious hypothesis that at least one haplotype is the same as the most common in circulation in the population. The first position on the alignment corresponds to position 604 of the first sequence, AF217800, reversed. The nucleotide codes K, R, S, and W, correspond, respectively to T/G, A/G, G/C and A/T

Fig. 2

Alignment of heterozygous ITS2 sequences of D. immitis from Portuguese canine samples. Haplotypes 7, 9, 10 and 18 include Portuguese sequences with codes LN626262-7 that are from infected mosquitoes. Haplotypes were inferred using the programme PHASE, by comparison with homozygous sequences, with 100 iterations, 100 thinning interval and 100 burn-in settings. The first position on the alignment corresponds to position 162 of the sequence, EU087699 (H18). The nucleotide codes are as standard (M, R, Y and W, correspond, respectively to A/C, A/G, C/T and A/T). Base assignment in position 11 (R) is considered uncertain (probability of 58-9%), as is for position 26 (Y) of sample 723 (50%), position 43 (Y) of sample 7, and position 51 (R) of sample 52 (50%). Haplotypes: H1: JX481279, JX866681, EU182329; H4: U182331, JN084166, JX889634, JX8896351, JX889636, JX889637, JX889638; H6: JN084168; H7: LN626265; H8: FJ263455; H9: LN626264/66; H10: LN626262; H12: FJ263458/66/67; H13: FJ263459/60/63; H14: FJ263457/64; H15: FJ263468, H16. FJ263461; H17: FJ263465; H18: EU087699, FJ263456, LN626263.1, LN626267.1; H19: EU182330. Haplotypes H2, H3, H5 and H11 result from inference