title: First molecular identification of mosquito vectors of Dirofilaria immitis in continental Portugal.
abstract: 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.
authors.affiliation: Medical Parasitology Unit, Medical Parasitology & Microbiology Unit-UPMM, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Rua da Junqueira 100, 1349-008, Lisboa, Portugal. cferreira@ihmt.unl.pt.
authors.forename: Cátia Alexandra Costa
authors.initials: CA
authors.lastname: Ferreira
authors.affiliation: Medical Parasitology Unit, Medical Parasitology & Microbiology Unit-UPMM, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Rua da Junqueira 100, 1349-008, Lisboa, Portugal. veronica_mixao@hotmail.com.
authors.forename: Verónica
authors.initials: V
authors.lastname: de Pinho Mixão
authors.affiliation: Medical Parasitology Unit, Medical Parasitology & Microbiology Unit-UPMM, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Rua da Junqueira 100, 1349-008, Lisboa, Portugal. tenovo@ihmt.unl.pt.
authors.forename: Maria Teresa Lourenço Marques
authors.initials: MT
authors.lastname: Novo
authors.affiliation: Medical Parasitology Unit, Medical Parasitology & Microbiology Unit-UPMM, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Rua da Junqueira 100, 1349-008, Lisboa, Portugal. mcalado@ihmt.unl.pt.
authors.forename: Maria Manuela Palmeiro
authors.initials: MM
authors.lastname: Calado
authors.affiliation: International Public Health and Biostatistics Unit, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Rua da Junqueira 100, 1349-008, Lisboa, Portugal. luziag@ihmt.unl.pt.|Centro de Estatística e Aplicações da Universidade de Lisboa, Faculdade de Ciências da Universidade de Lisboa, Bloco C6 - Piso 4, Campo Grande, 1749-016, Lisboa, Portugal. luziag@ihmt.unl.pt.
authors.forename: Luzia Augusta Pires
authors.initials: LA
authors.lastname: Gonçalves
authors.affiliation: Medical Parasitology Unit, Medical Parasitology & Microbiology Unit-UPMM, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Rua da Junqueira 100, 1349-008, Lisboa, Portugal. silvanabelo@ihmt.unl.pt.
authors.forename: Silvana Maria Duarte
authors.initials: SM
authors.lastname: Belo
authors.affiliation: Medical Parasitology Unit, Medical Parasitology & Microbiology Unit-UPMM, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Rua da Junqueira 100, 1349-008, Lisboa, Portugal. palmeida@ihmt.unl.pt.|Zoonosis Research Unit, Faculty of Health Sciences, University of Pretoria, Pretoria, South Africa. palmeida@ihmt.unl.pt.
authors.forename: António Paulo Gouveia
authors.initials: AP
authors.lastname: de Almeida
mesh_terms: D000330:Aedes / Q000145:classification / Q000469:parasitology; D000818:Animals; D000852:Anopheles / Q000145:classification / Q000469:parasitology; D003465:Culex / Q000145:classification / Q000469:parasitology; D009033:Culicidae / Q000145:classification* / Q000469:parasitology; D018509:DNA, Helminth / Q000235:genetics; D004183:Dirofilaria immitis / Q000235:genetics / Q000302:isolation & purification*; D004184:Dirofilariasis / Q000453:epidemiology* / Q000469:parasitology / Q000635:transmission*; D004285:Dogs; D005260:Female; D007303:Insect Vectors / Q000145:classification*; D007814:Larva; D016133:Polymerase Chain Reaction / Q000662:veterinary; D011174:Portugal / Q000453:epidemiology; D012621:Seasons
chemical_list: D018509:DNA, Helminth