Gioia Bongiorno

Host-feeding patterns of aedes albopictus (Diptera: Culicidae) in urban and rural contexts within Rome province, Italy

Knowledge of the frequency of contact between a mosquito species and its different hosts is essential to understand the role of each vector species in the transmission of diseases to humans and/or animals. However, no data are so far available on the feeding habits of Aedes albopictus in Italy or in other recently colonized temperate regions of Europe, due to difficulties in collecting blood-fed females of this diurnal and exophilic species. We analyzed Ae. albopictus host-feeding patterns in two urban and two rural sites within the area of Rome (Italy). Ae. albopictus was collected using sticky-traps and the blood-meal origin of 303 females was determined by direct dot-ELISA. The blood-fed sample, although representing only 4% of the total Ae. albopictus collected, demonstrates the useful application of sticky-trap in studying the feeding behavior of the species. The human blood index was significantly different among sites, ranging from 79-96% in urban sites to 23-55% in rural sites, where horses and bovines represented the most bitten hosts. The results obtained confirm the plastic feeding behavior shown by Ae. albopictus in its original range of distribution and highlights the high potential of this species as a vector of human pathogens in urban areas of Italy, where both humans and the mosquito itself may reach very high densities.

Seasonal Dynamics of Phlebotomine Sand Fly Species Proven Vectors of Mediterranean Leishmaniasis Caused by Leishmania infantum.

Background The recent geographical expansion of phlebotomine vectors of Leishmania infantum in the Mediterranean subregion has been attributed to ongoing climate changes. At these latitudes, the activity of sand flies is typically seasonal; because seasonal phenomena are also sensitive to general variations in climate, current phenological data sets can provide a baseline for continuing investigations on sand fly population dynamics that may impact on future scenarios of leishmaniasis transmission. With this aim, in 2011–2013 a consortium of partners from eight Mediterranean countries carried out entomological investigations in sites where L. infantum transmission was recently reported. Methods/Principal Findings A common protocol for sand fly collection included monthly captures by CDC light traps, complemented by sticky traps in most of the sites. Collections were replicated for more than one season in order to reduce the effects of local weather events. In each site, the trapping effort was left unchanged throughout the survey to legitimate inter-seasonal comparisons. Data from 99,000 collected specimens were analyzed, resulting in the description of seasonal dynamics of 56,000 sand flies belonging to L. infantum vector species throughout a wide geographical area, namely P. perniciosus (Portugal, Spain and Italy), P. ariasi (France), P. neglectus (Greece), P. tobbi (Cyprus and Turkey), P. balcanicus and P. kandelakii (Georgia). Time of sand fly appearance/disappearance in collections differed between sites, and seasonal densities showed variations in each site. Significant correlations were found between latitude/mean annual temperature of sites and i) the first month of sand fly appearance, that ranged from early April to the first half of June; ii) the type of density trend, varying from a single peak in July/August to multiple peaks increasing in magnitude from May through September. A 3-modal trend, recorded for P. tobbi in Cyprus, represents a novel finding for a L. infantum vector. Adults ended the activity starting from mid September through November, without significant correlation with latitude/mean annual temperature of sites. The period of potential exposure to L.infantum in the Mediterranean subregion, as inferred by adult densities calculated from 3 years, 37 sites and 6 competent vector species, was associated to a regular bell-shaped density curve having a wide peak center encompassing the July-September period, and falling between early May to late October for more than 99% of values. Apparently no risk for leishmaniasis transmission took place from December through March in the years considered. We found a common pattern of nocturnal females activity, whose density peaked between 11 pm and 2 am. Conclusions Despite annual variations, multiple collections performed over consecutive years provided homogeneous patterns of the potential behavior of leishmaniasis vectors in selected sites, which we propose may represent sentinel areas for future monitoring. In the investigated years, higher potential risk for L. infantum transmission in the Mediterranean was identified in the June-October period (97% relative vector density), however such risk was not equally distributed throughout the region, since density waves of adults occurred earlier and were more frequent in southern territories.

Vaccination with LiESP/QA-21 (CaniLeish ® ) reduces the intensity of infection in Phlebotomus perniciosus fed on Leishmania infantum infected dogs—A preliminary xenodiagnosis study

Ten Beagle dogs at different stages of Leishmania infantum infection, among which 6 had received a full course of LiESP/QA-21 (CaniLeish(®); Virbac) vaccination, were exposed to the bites of reared Phlebotomus perniciosus to assesses their infectiousness potential. This was found to be negligible/nil in 2 seronegative dogs with subpatent infection. Among the 8 dogs with active infection (=positive serology, bone-marrow qualitative PCR and lymph node culture), 2/5 vaccinated (40.0%) and 2/3 nonvaccinated dogs (66.7%) were infectious to the sand flies (p=0.5). However significantly fewer of the sand flies which fed on the vaccinated dogs were infected when compared to those which fed on the control dogs (10/82 compared to 30/49) (chi-squared test, p<0.0001; mixed binomial model with the dog identity included as a random effect, p=0.03). Furthermore, there was a significant difference in the proportion of sand flies with >500 parasites in their gut (i.e. a higher risk for subsequent transmission): 3.7% for vaccinated dogs compared with 28.6% for nonvaccinated dogs (Fishers exact test, p<0.0001; binomial mixed model, p=0.006). Although preliminary, these results suggest value in further investigations on L. infantum transmissibility parameters in LiESP/QA-21 vaccinated dogs.

First Surveys to Investigate the Presence of Canine Leishmaniasis and Its Phlebotomine Vectors in Hungary

Hungary is regarded as free of leishmaniasis because only a few imported cases have been reported. However, southern Hungary has a sub-Mediterranean climate, and so it was included in the EU FP6 EDEN project, which aimed to map the northern limits of canine leishmaniasis (CanL) in Europe. The numbers of traveling and imported dogs have increased in the last decade, raising concerns about the introduction of CanL caused by Leishmania infantum. Serum samples were collected from 725 dogs (22 localities, 6 counties) that had never traveled to endemic countries, as well as from other potential reservoir hosts (185 red foxes and 13 golden jackals). All sera were tested by the indirect fluorescent antibody test, but they were sero-negative using the OIE cut-off of 1:80 serum dilution except for those of two dogs resident since birth in southern Hungary. These had not received a blood transfusion, but the mode of transmission is unclear because no sandfly vectors were caught locally. From 2006 to 2009, phlebotomine sandflies were sampled in the summer months at 47 localities of 8 counties. They were trapped with castor-oil-impregnated sticky-paper, light, and CO(2)-baited traps. Small numbers of two vectors of Leishmania infantum were found. Phlebotomus neglectus occurred in three villages near to Croatia and one in north Hungary at latitude 47 °N, and Phlebotomus perfiliewi perfiliewi was trapped at two sites in a southeastern county close to the sites where it was first found in 1931-1932. Our report provides baseline data for future investigations into the northward spread of CanL into Hungary, which we conclude has yet to occur.

Viral Isolates of a Novel Putative Phlebovirus in the Marche Region of Italy

Thirty pools from 900 (540 females and 360 males) Phlebotomus perfiliewi sandflies collected during the summer of 2012 in the Fermo area (Marche Region, central Italy) were tested for the presence of Phleboviruses. A nested polymerase chain reaction was performed using degenerated primers amplifying a fragment of the polymerase gene (large segment) and a fragment of the nucleoprotein gene (small segment) of the genus Phlebovirus. One pool was positive for Toscana virus, as expected from results of studies in the area, and six pools were positive for a putative novel Phlebovirus. Virus isolation in Vero cells was performed. Minimum field infection rates/1,000 insects processed for the novel and Toscana viruses were 6.7 and 1.0, respectively. Phylogenetic analysis of the novel Phlebovirus, tentatively named Fermo virus, placed it in the Sandfly Fever Naples virus serocomplex.

Longitudinal study on the detection of canine Leishmania infections by conjunctival swab analysis and correlation with entomological parameters

A longitudinal study was carried out on kennelled stray dogs in a canine leishmaniasis (CanL) endemic area, to evaluate early and late diagnostic performance of a non-invasive conjunctival swab (CS) nested (n)-PCR analysis for Leishmania detection in 2 cohorts of dogs, respectively. (A) Sixty-five IFAT- and CS n-PCR-negative dogs exposed to, and followed up once or twice a month during a full sand fly season (July-November 2008). In parallel, a sand fly survey was performed on site using standard sticky traps set twice a month, for a cumulative surface of 63 m(2). (B) Seventeen IFAT- and CS n-PCR-negative dogs found positive in July 2008 at the peripheral blood buffy-coat (BC) n-PCR. These dogs were examined again by BC n-PCR in September and November 2008, and before the subsequent transmission season (May 2009) along with CS n-PCR and IFAT. None of the cohort (A) dogs converted to positive CS n-PCR during the transmission season. Although approximately 2500 phlebotomine specimens were collected with peaks of 100-147 specimens/m(2) sticky trap, the cumulative density of the only proven CanL vector in the area (Phlebotomus perniciosus) was found to be very low (0.5/m(2)). All cohort (B) dogs remained substantially seronegative; BC n-PCR showed an intermittent positive trend during the period surveyed, resulting in 82% conversions to negative by the end of the study, in contrast with 71% conversions to positive at the CS n-PCR analysis. In conclusion, while CS n-PCR was not found effective for the early detection of Leishmania contacts in dogs exposed to a low pressure of vectorial transmission, this assay showed to slowly convert to positive in a high rate of dogs, in the absence of seroconversion. CS n-PCR technique can be a suitable marker for assessing Leishmania exposure in dogs as a non-invasive alternative to current serological and molecular tools.

Aspects of the bionomics of Phlebotomus sergenti sandflies from an endemic area of anthroponotic cutaneous leishmaniasis in Aleppo Governorate, Syria.

Aspects of the bionomics of phlebotomine sandflies (Diptera: Psychodidae) were studied from June to November 2005 in three foci of anthroponotic cutaneous leishmaniasis (ACL) in Aleppo Governorate, Syria, where the agent Leishmania tropica (Wright) (Kinetoplastida: Trypanosomatidae) is transmitted by Phlebotomus sergenti Parrot. Syria has been designated by the World Health Organization as one of four countries in the Old World where cutaneous leishmaniasis is hyperendemic, but little is known about the biology of local vector populations. Standard collections by sticky traps showed two peaks in density, in June and late August. In total, 1840 sandflies were caught, comprising five species: Phlebotomus papatasi (Scopoli) (68.0%); P. sergenti (25.4%); Sergentomyia minuta (Rondani) (6.4%); Phlebotomus tobbi Adler & Theodor (0.1%), and Phlebotomus mascittii canaaniticus Adler & Theodor (0.1%). Similar numbers of P. sergenti were caught indoors (246 specimens) and outdoors (222), whereas P. papatasi was significantly more abundant indoors (1096 specimens) than outdoors (156) (χ2 = 241, P < 0.01). In total, 212 blood‐fed females were tested for host blood determination, of which 176 (83.0%) reacted with anti‐species reagent. Results from 20 P. sergenti suggest that this species is an opportunistic feeder, imbibing human, ovine, avian, bovine and feline blood, although more bloodmeals were taken from humans and cattle than expected in relation to the relative proportions of potential hosts present (the forage ratio, FR). The bionomics of P. sergenti are discussed in relation to the inefficacy of control campaigns based on indoor spraying with residual insecticides that have been implemented by the Syrian Ministry of Health to control the epidemics of ACL in the Aleppo Governorate.

Ecological Niche Modelling of Potential West Nile Virus Vector Mosquito Species and Their Geographical Association with Equine Epizootics in Italy

In Italy, West Nile virus (WNV) equine outbreaks have occurred annually since 2008. Characterizing WNV vector habitat requirements allows for the identification of areas at risk of viral amplification and transmission. Maxent-based ecological niche models were developed using literature records of 13 potential WNV Italian vector mosquito species to predict their habitat suitability range and to investigate possible geographical associations with WNV equine outbreak occurrence in Italy from 2008 to 2010. The contribution of different environmental variables to the niche models was also assessed. Suitable habitats for Culex pipiens, Aedes albopictus, and Anopheles maculipennis were widely distributed; Culex modestus, Ochlerotatus geniculatus, Ochlerotatus caspius, Coquillettidia richiardii, Aedes vexans, and Anopheles plumbeus were concentrated in north-central Italy; Aedes cinereus, Culex theileri, Ochlerotatus dorsalis, and Culiseta longiareolata were restricted to coastal/southern areas. Elevation, temperature, and precipitation variables showed the highest predictive power. Host population and landscape variables provided minor contributions. WNV equine outbreaks had a significantly higher probability to occur in habitats suitable for Cx. modestus and Cx. pipiens, providing circumstantial evidence that the potential distribution of these two species coincides geographically with the observed distribution of the disease in equines.

Ecological Distribution and CQ11 Genetic Structure of Culex pipiens Complex (Diptera: Culicidae) in Italy.

Mosquitoes in the Culex pipiens complex are considered to be involved in the transmission of a range of pathogens, including West Nile virus (WNV). Although its taxonomic status is still debated, the complex includes species, both globally distributed or with a more limited distribution, morphologically similar and characterised by different physiological and behavioural traits, which affect their ability as vectors. In many European countries, Cx. pipiens and its sibling species Culex torrentium occur in sympatry, exhibiting similar bionomic and morphological characters, but only Cx. pipiens appears to play a vector role in WNV transmission. This species consists of two biotypes, pipiens and molestus, which can interbreed when in sympatry, and their hybrids can act as WNV-bridge vectors, due to intermediate ecological features. Considering the yearly WNV outbreaks since 2008 and given the morphological difficulties in recognising species and biotypes, our aim was to molecularly identify and characterised Cx. pipiens and Cx. torrentium in Italy, using recently developed molecular assays. Culex torrentium was not detected; as in other European countries, the pipiens and molestus biotypes were widely found in sympatry with hybrids in most environments. The UPGMA cluster analysis applied to CQ11 genotypic frequencies mainly revealed two groups of Cx. pipiens populations that differed in ecological features. The high propensity of the molestus biotype to exist in hypogean environments, where the habitat’s physical characteristics hinder and preclude the gene flow, was shown. These results confirmed the CQ11 assay as a reliable diagnostic method, consistent with the ecological and physiological aspects of the populations analysed. Since the assessment of the actual role of three biotypes in the WNV circulation remains a crucial point to be elucidated, this extensive molecular screening of Cx. pipiens populations can provide new insights into the ecology of the species and may give useful indications to plan and implement WNV surveillance activities in Italy.

Isolation of a Wickerhamomyces anomalus yeast strain from the sandfly Phlebotomus perniciosus, displaying the killer phenotype

The yeast Wickerhamomyces anomalus has been studied for its wide biotechnological potential, mainly for applications in the food industry. Different strains of W. anomalus have been isolated from diverse habitats and recently from insects, including mosquitoes of medical importance. This paper reports the isolation and phylogenetic characterization of W. anomalus from laboratory‐reared adults and larvae of Phlebotomus perniciosus (Diptera: Psychodidae), a main phlebotomine vector of human and canine leishmaniasis. Of 65 yeast strains isolated from P. perniciosus, 15 strains were identified as W. anomalus; one of these was tested for the killer phenotype and demonstrated inhibitory activity against four yeast sensitive strains, as reported for mosquito‐isolated strains. The association between P. perniciosus and W. anomalus deserves further investigation in order to explore the possibility that this yeast may exert inhibitory/killing activity against Leishmania spp.