Silvia Ciocchetta

Molecular xenomonitoring of Dirofilaria immitis and Dirofilaria repens in mosquitoes from north-eastern Italy by real-time PCR coupled with melting curve analysis

BackgroundDirofilaria immitis and Dirofilaria repens are transmitted by bloodsucking culicid mosquitoes belonging to Culex, Aedes, Ochlerotatus, Anopheles and Mansonia genera.The detection of filarioids in mosquitoes for assessing distribution of vectors and/or of pathogens in a given area (also known as “xenomonitoring”), when based on individual dissection of wild-caught female mosquitoes is time consuming and hardly applicable in large epidemiological surveys.Our study aimed to evaluate the recently developed duplex real-time PCR for screening large number of culicids and to assess their positivity for D. immitis and D. repens in an area where both species are endemic.MethodsA duplex real-time PCR was used to detect and differentiate D. immitis and D. repens in mosquitoes collected in six provinces of the Veneto region using 43 carbon dioxide-baited traps under the frame of an entomological surveillance program to monitor the vectors of West Nile disease. From early May till October 2010, unfed female mosquitoes (n = 40,892) were captured in 20 selected sites.ResultsMosquitoes identified as Culex pipiens, Ochlerotatus caspius, Aedes vexans and Culex modestus were grouped into 995 pools according to species, day and site of collection (from minimum of 1 to maximum of 57). Out of 955 pools, 23 (2.41 %) scored positive for Dirofilaria spp. of which, 21 (2.2 %) for D. immitis and two (0.21 %) for D. repens. An overall Estimated Rate of Infection (ERI) of 0.06 % was recorded, being higher in Och. caspius and Ae. vexans (i.e., 0.18 % and 0.14 %, respectively). At least one mosquito pool was positive for Dirofilaria spp. in each province with the highest ERI recorded in Vicenza and Padova provinces (i.e., 0.42% and 0.16 %, respectively). Mosquitoes collected in all provinces were positive for D. immitis whereas, only two (i.e., Padova and Rovigo) provinces scored positive for D. repens. All mosquito species, except for Cx. modestus, were positive for D. immitis, whereas D. repens was only found in Cx. pipiens.ConclusionsThe results suggest that both Dirofilaria species are endemic and may occur in sympatry in the examined area. The molecular approach herein used represents a powerful tool for surveillance programs of D. immitis and D. repens in the culicid vectors towards a better understanding of the epidemiology of the infections they cause and their seasonal transmission patterns.

Current distribution of the invasive mosquito species, Aedes koreicus (Hulecoeteomyia koreica) in northern Italy

BackgroundThe invasive species Aedes (Finlaya) koreicus was first identified in north-eastern Italy in 2011, during the ongoing surveillance activity of Aedes albopictus. Following this finding, a more intensive monitoring was carried out to assess the distribution of the species and to collect biological data. Herein, we report the new records obtained by four years of surveillance.FindingsThe presence of Ae. koreicus was checked using ovitraps, adults traps and by larval collections in all possible breeding sites from May 2011 to July 2015. The monitoring started in the site of the first detection (Province of Belluno) and was then extended in the neighbouring Provinces belonging to four Regions. Aedes koreicus was found in 73 municipalities out of 155 monitored (47.1 %), including 23 municipalities (14.8 %) previously not infested. The area of first detection of Ae. koreicus (Province of Belluno) was also the most infested (68 %). However the mosquito has also been found to the west (Province of Trento) and to the south and south-west (Provinces of Vicenza and Treviso) of the initially infested area.ConclusionsThe spread of Ae. koreicus is directed towards south and west from the original infested area, likely due to the dense road connections and the habitat suitability of the new areas. According to these records, northern Italy has a high probability to be invaded by Ae. koreicus in the next decade. These data can be useful to validate predictive models of potential distribution and dispersal of this species in Italy or in Europe.

Laboratory evidence on vector competence of the invasive mosquito Aedes koreicus [Hulecoeteomyia koreica] for Dirofilaria immitis

Aedes (Finlaya) koreicus is an exotic invasive mosquito detected for the first time in Italy in 2011. Little information on its vector competence for Dirofilaria immitis are available. The area where this species is now established (Veneto Region, north-eastern Italy) is endemic for dirofilariosis. In this study, Ae. koreicus specimens were experimentally infected with D. immitis to evaluate the development of filarial larval stages in different body districts. Aedes koreicus were reared under laboratory standard condition (temperature: 25 ± 1°C; relative humidity: 65 ± 5%; light-dark: 16-8 h). A test group (T) (n = 54 mosquitoes) and a control group (C) (n = 29 mosquitoes) were fed by an artificial feeding system (Hemotek™) using uninfected (in C) and naturally infected (in T) dog blood (3000 microfilariae/ml). Mosquitoes naturally dead and specimens killed at 1, 13, 16, 22 and 28 days post infection (dpi) were dissected; head, thorax and abdomen were examined separately. Five specimens (3 from T and 2 from C) were selected for histology. In addition, molecular confirmation by real time PCR for Dirofilariae were performed. Each larval stage was documented by pictures and videos. The experiment lasted 28 days. A total of 46 mosquitoes fed in T (85%) and 24 mosquitoes in C (83%) groups. In T, 11 mosquitoes were killed and 32 were recovered dead. The mosquito mortality rate in T was 52% during the first nine days, significantly higher compared to C (8%) (p < 0.01). In total, 31 mosquitoes (67%) were infected. The average of microfilariae, L1 (sausage stage) and L3 was 14.67, 8.56 and 3.15, respectively. Second stage larvae were observed only once (8 specimens on 13 dpi). First stage larvae were first observed on 3 dpi whereas L3 on 8 dpi. The latter were found in salivary glands and proboscis starting on 16 till 28 dpi. Aedes koreicus seems to be a suitable intermediate host for D. immitis. Despite the low initial number of microfilariae, the infective L3 stage was observed in all body districts including the proboscis. An high mosquito mortality occurred during the first days, nevertheless one third of them survived and became infective. This results show that Ae. koreicus may be involved in the natural cycle of D. immitis, increasing the risk of exposure for dogs and humans.

Seasonal and Daily Activity Patterns of Mosquito (Diptera: Culicidae) Vectors of Pathogens in Northeastern Italy

ABSTRACT The seasonal and daily activity of mosquito vectors of pathogens affecting animals and humans were studied in northeastern Italy at a site within the Po River Delta Park. A CDC-CO2 trap and a gravid trap were operated at 2-h intervals for 24 h every 15 d from May to October 2010. Overall, 5,788 mosquitoes comprising six species were collected, namely Culex pipiens L. (75.1% of total), Aedes caspius (Pallas) (15.2%), Aedes vexans (Meigen) (6.9%), Anopheles maculipennis s.l. Meigen (2.6%), Culiseta annulata (Schrank) (0.2%), and Culex modestus Ficalbi (<0.1%). The relative abundance of these species increased from May until the beginning of July and then decreased, disappearing at the beginning of October. The diel host-seeking patterns and oviposition site-seeking patterns were species specific and were differentially affected by the ecological variables recorded at the day and hour of mosquito collection or two weeks before collection. Knowledge of the seasonal and daily host-seeking patterns of mosquitoes highlights the time periods of the day and the seasons of potential exposure for animals and humans to mosquito-borne pathogens, therefore delineating the best time for the application of preventive measures. Furthermore, knowledge of the oviposition site-seeking activity of the mosquitoes optimizes the capture of gravid females, thereby enhancing the likelihood of detecting pathogens.

Laboratory colonization of the European invasive mosquito Aedes ( Finlaya ) koreicus

BackgroundAedes (Finlaya) koreicus (Edwards) is a mosquito that has recently entered Europe from Asia. This species is considered a potential threat to newly colonized territories, but little is known about its capacity to transmit pathogens or ability to compete with native mosquito species. The establishment of a laboratory colony is a necessary first step for further laboratory studies on the biology, ecology and vector competence of Ae. koreicus.ResultsA self-mating colony was established at QIMR Berghofer Medical Research Institute (Brisbane, Australia) from eggs of the F1 progeny of individuals collected as free-living larvae in northeastern Italy (Belluno province). Mosquitoes are currently maintained on both defibrinated sheep blood provided via an artificial membrane system and human blood from volunteers. Larvae are maintained in rain water and fed with Tetramin® fish food (©2015 Spectrum Brands - Pet, Home and Garden Division, Tetra-Fish). Morphometric measurements related to body size were taken and a fecundity index, based on wing length, was calculated. An in vivo technique for differentiating male and female pupae has been optimized. Our findings provide the basis for further studies on the ecology and physiology of Ae. koreicus.ConclusionWe describe the establishment of an Ae. koreicus colony in the laboratory and identify critical requirements for the maintenance of this mosquito species under artificial conditions. The laboratory colony will facilitate studies investigating the vector potential of this species for human pathogens.