Romeo Bellini

Chikungunya Virus in Aedes albopictus, Italy

Chikungunya Virus in Aedes albopictus, Italy

Population structure of Aedes albopictus (Skuse): the mosquito which is colonizing Mediterranean countries

Multilocus electrophoresis analysis has been used to study the genetic structure of 18 populations of Aedes albopictus newly introduced to Italy, in comparison with two populations in the United States, four in Japan, and four in Indonesia. Allozyme analysis revealed that 15 out of the 18 studied loci were polymorphic among the 28 populations. No significant deviations from Hardy–Weinberg equilibrium were found at polymorphic loci. High genetic affinity was observed between the Italian populations and those from the United States and Japan. The analysis of variance in allele frequencies showed that variance among subpopulations accounted for most of the total variance, suggesting that isolation of the Italian populations is not related to distance. Analysis of linkage disequilibrium using Ohta’s method shows that the variance in the frequency of allele combinations could be explained by the action of the genetic drift which accompanies the establishment of new populations. The colonization process of Ae. albopictus in Italy is following a trend similar to that previously observed in the U.S.A., probably because both infestations derive from several successive introductions, each with large numbers of individuals.

Evidence of simultaneous circulation of West Nile and Usutu viruses in mosquitoes sampled in Emilia-Romagna region (Italy) in 2009.

Background In recent years human diseases due to mosquito-borne viruses were increasingly reported in Emilia-Romagna region (Italy), from the chikungunya virus in 2007 to the West Nile virus (WNV) in 2008. An extensive entomological survey was performed in 2009 to establish the presence and distribution of mosquito arboviruses in this region, with particular reference to flaviviruses. Methodology/Principal Findings From May 6 to October 31, a total of 190,516 mosquitoes were sampled in georeferenced stations, grouped in 1,789 pools according date of collection, location, and species, and analyzed by reverse transcription polymerase chain reaction (RT-PCR) to detect the presence of RNA belong to Flavivirus genus. WNV was detected in 27 mosquito pools, producing sequences similar to those of birds and human strains obtained in 2008 outbreak, pointed out the probable virus overwintering. Isolation of WNV was achieved from one of these pools. Moreover 56 pools of mosquitoes tested positive for Usutu virus (USUV). Most PCR positive pools consisted of Culex pipiens, which also was the most analyzed mosquito species (81.4% of specimens); interestingly, USUV RNA was also found in two Aedes albopictus mosquito pools. Simultaneous circulation of WNV and USUV in the survey area was highlighted by occurrence of 8 mosquito WNV- and USUV-positive pools and by the overlaying of the viruses “hot spots”, obtained by kernel density estimation (KDE) analysis. Land use of sampled stations pointed out a higher proportion of WNV-positive Cx. pipiens pool in rural environments respect the provenience of total sampled pool, while the USUV-positive pools were uniformly captured in the different environments. Conclusions/Significance Obtained data highlighting the possible role of Cx. pipiens mosquito as the main vector for WNV and USUV in Northern Italy, and the possible involvement of Ae. albopictus mosquito in USUV cycle. The described mosquito-based surveillance could constitute the foundation for a public health alert system targeting mosquito borne arboviruses.

On the Competition Occurring Between Aedes albopictus and Culex pipiens (Diptera: Culicidae) in Italy

Abstract Aedes albopictus (Skuse) was detected for the first time in Italy in 1990. Thanks to its high biological adaptability, it spread rapidly to colonize various urban areas in central and northern Italy. The purpose of this study was to examine the ecological niche occupied by Ae. albopictus and the role of competition with Culex pipiens L., which is a widespread indigenous species, in urban areas. The larval development cycle and competition for food between the two species was also studied under laboratory conditions. The study showed that both species can colonize any type of artificial water container. Ae. albopictus mainly occupies saucers under flower pots and other, usually smaller-sized, containers, whereas Cx. pipiens tends to develop more in large tanks (100–200 liters) used in residential gardens and other larger-sized containers. It would seem that the two species interact mainly in medium-sized containers (10–50 liters) such as manholes and tires. Laboratory studies showed that, at 25°C, the food-biomass conversion coefficient is significantly higher in Ae. albopictus than in Cx. pipiens, indicating that the new established species is more efficient in transforming food into biomass. As a consequence, it has a greater capacity to exploit food resources and therefore grow more rapidly than Cx. pipiens. In larval association where food was scarce, it was noted that competition between the two species takes place and is influenced by temperature: at 25 ± 2°C, Ae. albopictus prevails but is much less likely to do so at 20°C.

Use of the Sterile Insect Technique Against Aedes albopictus in Italy: First Results of a Pilot Trial

In Europe, the mosquito Aedes albopictus (Skuse) is widespread in Italy, Albania and most probably in neighbouring Montenegro. Recent introductions have also been reported in France, Spain and southern Switzerland. In Italy, the species is currently recognized as the most noxious mosquito, thus requiring the implementation of intensive control programmes. Ae. albopictus is also a potential vector of human diseases, which has raised the issue of whether eradication campaigns are called for. This species is particularly suitable for application of the sterile insect technique (SIT) because of its urban-related distribution, recent introduction, low active dispersal potential, low population density which may be maintained by conventional control measures, and ease of mass-rearing. In 1999, a programme was initiated that focused on the application of the SIT against Ae. albopictus. A pilot rearing facility, targeted at the production of up to 20 000 male pupae per week has been established. Blood feeding is performed with a thermostatically controlled device using defibrinated bovine blood, egg hatching is stimulated with a nutrient broth culture, and egg counts conducted automatically. Larval density and larval diet are still being investigated in order to improve productivity, the separation of males is currently conducted at the pupal stage using calibrated metal sieves, and irradiation studies are performed at the 60Co plant Calliope, Italian National Agency for New Technologies, Energy and the Environment in Rome. During the summer of 2004, eight weekly sterile male pupal releases were organized in Rimini to evaluate sterile male performances in the field against a natural population. A significant difference was observed in the release area compared with the control area when the effects on egg fertility and egg density were cumulated. It is planned to continue the programme on a larger scale to improve rearing efficiency and obtain a preliminary benefit/cost evaluation.

Mosquito, bird and human surveillance of West Nile and Usutu viruses in Emilia-Romagna Region (Italy) in 2010.

Background In 2008, after the first West Nile virus (WNV) detection in the Emilia-Romagna region, a surveillance system, including mosquito- and bird-based surveillance, was established to evaluate the virus presence. Surveillance was improved in following years by extending the monitoring to larger areas and increasing the numbers of mosquitoes and birds tested. Methodology/Principal Findings A network of mosquito traps, evenly distributed and regularly activated, was set up within the surveyed area. A total of 438,558 mosquitoes, grouped in 3,111 pools and 1,276 birds (1,130 actively sampled and 146 from passive surveillance), were tested by biomolecular analysis. The survey detected WNV in 3 Culex pipiens pools while Usutu virus (USUV) was found in 89 Cx. pipiens pools and in 2 Aedes albopictus pools. Two birds were WNV-positive and 12 were USUV-positive. Furthermore, 30 human cases of acute meningoencephalitis, possibly caused by WNV or USUV, were evaluated for both viruses and 1,053 blood bags were tested for WNV, without any positive result. Conclusions/Significance Despite not finding symptomatic human WNV infections during 2010, the persistence of the virus, probably due to overwintering, was confirmed through viral circulation in mosquitoes and birds, as well as for USUV. In 2010, circulation of the two viruses was lower and more delayed than in 2009, but this decrease was not explained by the relative abundance of Cx. pipiens mosquito, which was greater in 2010. The USUV detection in mosquito species confirms the role of Cx. pipiens as the main vector and the possible involvement of Ae. albopictus in the virus cycle. The effects of meteorological conditions on the presence of USUV-positive mosquito pools were considered finding an association with drought conditions and a wide temperature range. The output produced by the surveillance system demonstrated its usefulness and reliability in terms of planning public health policies.

Characterization of a New Aedes albopictus (Diptera: Culicidae)-Wolbachia pipientis (Rickettsiales: Rickettsiaceae) Symbiotic Association Generated by Artificial Transfer of the wPip Strain from Culex pipiens (Diptera: Culicidae)

ABSTRACT Wolbachia is a maternally inherited endosymbiont inducing various effects in insects and other invertebrate hosts that facilitate the invasion of naïve host populations. One of the effects is a form of sterility known as cytoplasmic incompatibility (CI) through which females are effectively sterilized when they mate with males harboring a different Wolbachia strain. The repeated mass release of cytoplasmically incompatible males can be a tool to suppress insect populations. Here, we attempt to infect an Aedes albopictus (Skuse) (Diptera: Culicidae) strain, artificially deprived of the natural Wolbachia infection, with a new Wolbachia strain from Culex pipiens (L.) (Diptera: Culicidae). Further experiments were designed to study the effects of the new infection on Ae. albopictus fitness and evaluate key parameters that affect infection dynamics, including CI level and maternal inheritance. Using embryonic microinjection, the new Wolbachia strain was successfully established in Ae. albopictus. Crosses demonstrated a pattern of bidirectional CI between naturally infected and transinfected individuals. Specifically, egg hatch was essentially absent (i.e., CI was very high) in all crosses between the transinfected males and females with a different infection status. Furthermore, naturally infected Ae. albopictus males were incompatible with the transinfected females. Maternal inheritance was close to 100%. Moreover, the new infection did not affect immature and adult survivorship, but it significantly reduced female fecundity and egg hatch rate. The results are discussed in relation to the potential use of the new Ae. albopictus—Wolbachia symbiotic association as a suitable system for the study and development of CI-based strategies for suppressing populations of this important pest and disease vector.

Arboviral Survey of Mosquitoes in Two Northern Italian Regions in 2007 and 2008

Recently, Italy-particularly the Emilia-Romagna region-was the location of consecutive outbreaks of human diseases caused by the arboviruses chikungunya virus and West Nile virus. The two outbreaks, spread by different species of mosquitoes, were not related, but pointed out the lack of an arboviral surveillance program in this region. Beginning in 2007 entomological surveillance was initiated in the Emilia-Romagna region, and in 2008 the program was improved and extended at Lombardia region. Using CO(2)-baited traps, 65,292 mosquitoes were collected; pooled by date of collection, location, and species; macerated manually; and tested by reverse transcription (RT)-polymerase chain reaction for the presence of alphaviruses, orthobunyaviruses, and flaviviruses. Amplicons were sequenced and employed for identification of viral RNA by basic local alignment search tool search in GenBank. Results of these assays showed (1) the presence of West Nile virus in two pools of Culex pipiens mosquitoes, (2) the presence of RNA of two orthobunyaviruses, Tahyna virus in a pool of Ochlerotatus caspius mosquitoes and Batai virus in a pool of Anopheles maculipennis mosquitoes, and (3) the presence of flavivirus RNAs in pools of Oc. caspius, Aedes albopictus, and Aedes vexans mosquitoes; the sequences of these amplicons were most closely related to flaviviruses that have been detected only in mosquitoes and had no recognized vertebrate host (Aedes flavivirus, Culex flavivirus, and Kamiti River virus).

Pilot Field Trials with Aedes albopictus Irradiated Sterile Males in Italian Urban Areas

ABSTRACT The pilot field studies here presented are part of a long-term research program aimed to develop a cost-effective sterile insect technique (SIT) methodology to suppress Aedes albopictus (Skuse) populations. Aedes albopictus is a mosquito species mainly developing in man-made containers and with an island-like urban and suburban distribution. These two features make the application of the sterile insect technique a possible control strategy. Five trials have been performed in three small towns from 2005 to 2009 (Emilia-Romagna region, northern Italy). Reared male pupae, sexed by a sieving technique allowing the recovery of ≈26–29% of males, were exposed to gamma rays and immediately released in the field. Adult population density was estimated based on a weekly monitoring of egg density in the ovitraps, whereas induced sterility was estimated by measuring the hatching percentage of weekly collected eggs in SIT and control areas. Results showed that sterile males released at the rate of 896–1,590 males/ha/wk induced a significant sterility level in the local population. In addition, when the sterility level achieved values in the range of 70–80%, a similar reduction also was found for the egg density in the ovitraps. We could estimate that the minimum egg sterility value of 81% should be maintained to obtain suppression of the local population. Immigration of mated females was not a main issue in the small villages where trials have been run.

Detection of Usutu Virus Within a West Nile Virus Surveillance Program in Northern Italy

Usutu virus (USUV) is a mosquito-borne flavivirus belonging to the Japanese encephalitis serocomplex, recently related to neurological disease in immunosuppressed patients. In the same area of Northern Italy where USUV human cases occurred in 2009, a regional West Nile virus (WNV) surveillance program based on mosquito monitoring and wild birds screening has been implemented since 2008. Mosquito pools and wild birds were tested using three different polymerase chain reactions (Flavivirus, WNV, and USUV). During summer 2009, 56 pools (54 consisting of Culex pipiens and 2 of Aedes albopictus) and 27 pools (Cx. pipiens) out of 1789 mosquito pools were, respectively, USUV and WNV positive. Moreover, out of 1218 wild birds tested, 44 were WNV positive, whereas only 11 birds were USUV positive by polymerase chain reaction. Data collected during 2009 prove a cocirculation of USUV and WNV in Northern Italy, but these two viruses show different incidence values in both mosquitoes and birds, suggesting involvement of different animals (other bird species or mammals) in their natural cycles. The cocirculation of WNV and USUV poses a new potential threat to human health in this area. The extent of WNV surveillance to other Flaviviruses will require new diagnostic procedures able to process a large number of samples in a limited period of time and highlights the importance of developing more specific serological tests that could be used in field.