First molecular identification of mosquito vectors of Dirofilaria immitis in continental Portugal
Ferreira, Catia Alexandra Costa; Mixao, Veronica de Pinho; Novo, Maria Teresa Lourenco Marques; Calado, Maria Manuela Palmeiro; Goncalves, Luzia Augusta Pires; Belo, Silvia Maria Duarte; De Almeida, Antonio Paulo Gouveia
BACKGROUND : Canine dirofilariasis due to Dirofilaria immitis is known to be endemic in continental Portugal.
However, information about the transmitting mosquito species is still scarce, with only Culex theileri identified to
date, albeit with L1-2, through dissection. This study was carried out to investigate the potential vectors of Dirofilaria
spp. in continental Portugal.
METHODS : Mosquitoes were collected in three distinct seasons (Summer, Autumn and Spring), 2011–2013, in three
districts. CDC traps and indoor resting collections were carried out in the vicinity of kennels. Mosquitoes were kept
under controlled conditions for 7 days to allow the development of larval stages of Dirofilaria spp.. DNA extraction
was performed separately for both head+thorax and abdomen in order to differentiate infective and infected
specimens, respectively, in pools, grouped according to the species and collection site (1–40 specimen parts/pool),
and examined by PCR using pan-filarial specific primers. Mosquito densities were compared using non-parametric
tests. Dirofilaria development units (DDU) were estimated.
RESULTS : In total, 9156 female mosquitoes, from 11 different species, were captured. Mosquito densities varied
among the 3 districts, according to capture method, and were generally higher in the second year of collections.
From 5866 specimens screened by PCR, 23 head+thorax and 41 abdomens pools, corresponding to 54 mosquitoes
were found positive for D. immitis DNA. These belonged to 5 species: Culex (Cux) theileri (estimated rate of infection
(ERI)=0.71%), Cx. (Cux) pipiens f. pipiens and f. molestus (ERI=0.5%), Anopheles (Ano) maculipennis s.l. (ERI=3.12%),
including An. (Ano) atroparvus, Aedes (Och) caspius (ERI=3.73%) and Ae. (Och) detritus s.l. (ERI=4.39%). All but Cx.
pipiens, had at least one infective specimen. No D. repens infected specimens were found. Infection rates were:
3.21% in Coimbra, 1.22% in Setúbal and 0.54% in Santarém. DDU were at least 117/year in the study period.
CONCLUSIONS : Culex theileri, Cx. pipiens, An. maculipennis s.l. An. atroparvus, Ae.caspius and Ae. detritus s.l. were
identified as potential vectors of D. immitis in three districts of Portugal, from Spring to Autumn, in 5 of the 6
collection dates in 2011–2013. Implications for transmission, in the context of climate changes, and need for
prophylactic measures, are discussed.