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Abstract Background Mosquitoes (Culicidae), as disease vectors, represent a risk for human health worldwide. Repeated introductions of alien mosquito species and the spread of invasive species have been recorded in different countries. Traditionally, identification of mosquitoes relies on morphological observation. However, morphology-based identification is associated with a number of potential disadvantages, such as the high level of specialisation of the operator and its limited applicability to damaged samples. In these cases, species identification is achieved through molecular methods based on DNA amplification. Molecular-based taxonomy has also enabled the development of techniques for the study of environmental DNA (eDNA). Previous studies indicated the 16S mitochondrial ribosomal RNA (rRNA) gene as a promising target for this application; however, 16S rRNA sequences are available for only a limited number of mosquito species. In addition, although primers for the 16S rRNA gene were designed years ago, they are based on limited numbers of mosquito sequences. Thus, the aims of this study were to: (i) design pan-mosquito 16S rRNA gene primers; (ii) using these primers, generate a 16S rRNA gene mosquito reference library (with a focus on mosquitoes present in Italy); and (iii) compare the discriminatory power of the 16S rRNA gene with two widely used molecular markers, cytochrome c oxidase subunit 1 mitochondrial gene (COI) and internal transcribed spacer 2 (ITS2). Methods A total of six mosquito genera (28 mosquito species) were included in this study: Aedes (n = 16 species), Anopheles (5 species), Coquillettidia (1 species), Culex (3 species), Culiseta (2 species) and Uranotaenia (1 species). DNA was extracted from the whole mosquito body, and more than one specimen for each species was included in the analysis. Sanger sequencing was used to generate DNA sequences that were then analysed through the Barcode of Life Data Systems (BOLD). Phylogenetic analyses were also performed. Results Novel 16S rDNA gene, COI and ITS2 sequences were generated. The 16S rRNA gene was shown to possess sufficient informativeness for the identification of mosquito species, with a discriminatory power equivalent to that of COI. Conclusions This study contributes to the generation of DNA barcode libraries, focussed on Italian mosquitoes, with a significant increase in the number of 16S rRNA gene sequences. We hope that these novel sequences will provide a resource for studies on the biodiversity, monitoring and metabarcoding of mosquitoes, including eDNA-based approaches. Graphical abstract

Migratory birds play an important role in transporting ixodid ticks and tick-borne pathogens between continents. During the Boreal spring, migratory birds reach Europe, mainly from sub-Saharan Africa or from northern African countries but not much is known about the diversity and ecology of the ticks they spread. From 2017 to 2022, in the framework of two consecutive projects focused on sampling migratory birds from Africa to Europe, a total of 27 immature Amblyomma ticks were collected from migratory birds, belonging to 8 species, captured on the Island of Ventotene, an important stop-over site in the Mediterranean Sea. In the absence of adult specimens, morphological identification was limited to assigning these ticks to the Amblyomma genus. In this study, sequencing and comparative analysis of three mitochondrial molecular markers (12S rDNA, 16S rDNA, COI) were performed to achieve taxonomic identification. Sequences obtained from Ventotene specimens matched at 100% identity with Amblyomma sparsum. In conclusion, this study documented that immature stages of this species belonging to the Amblyomma marmoreum complex reached the Pontine Islands for six consecutive years. The entry of alien tick species and their potentially transmitted pathogens deserves further study, also in light of the globally ongoing climate change.

Abstract Background Anopheles sacharovi, a member of the Anopheles maculipennis complex, was a historical malaria vector in Italy, no longer found since the last report at the end of 1960s. In September 2022, within the Surveillance Project for the residual anophelism, a single specimen of An. maculipennis sensu lato collected in Lecce municipality (Apulia region) was molecularly identified as An. sacharovi. This record led to implement a targeted entomological survey in September 2023. Methods Investigation was conducted in the areas around the first discovery, focusing on animal farms, riding stables and potential breeding sites. Adult and immature mosquitoes were collected, using active search or traps, in several natural and rural sites. Mosquitoes belonging to An. maculipennis complex were identified morphologically and molecularly by a home-made routine quantitative polymerase chain reaction (qPCR) assay, developed specifically for the rapid identification of An. labranchiae, and, when necessary, by amplification and sequencing of the ITS-2 molecular marker. Results Out of the 11 sites investigated, 6 were positive for Anopheles presence. All 20 An. maculipennis s.l. (7 adults, 10 larvae and 3 pupae) collected in the areas were identified as An. sacharovi by ITS-2 sequencing. Conclusions The discovery of An. sacharovi, considered to have disappeared from Italy for over 50 years, has a strong health relevance and impact, highlighting an increase in the receptivity of the southern areas. As imported malaria cases in European countries are reported every year, the risk of Plasmodium introduction by gametocyte carriers among travellers from endemic countries should be taken into greater consideration. Our findings allow rethinking and building new models for the prediction and expansion of introduced malaria. Furthermore, to prevent the risk of reintroduction of the disease, the need to strengthen the surveillance of residual anophelism throughout the South should be considered. Graphical Abstract

The mosquito Culex pipiens occurs in two ecotypes differing in their mating and overwintering behavior: pipiens mate in open environments and diapause, and molestus also mate in small spaces and is active throughout the year. Cx. pipiens carry Wolbachia endosymbionts of the wPip strain, but the frequency of infection differs between studied populations. Wolbachia infection affects the host reproductive success through cytoplasmic incompatibility. wPip Wolbachia is divided into five types, wPip I–V. The type of wPip carried varies among Cx. pipiens populations. In northern European locations different wPip types are found in the two ecotypes, whereas in southern locations, they often carry the same type, indicating differences in hybridization between ecotypes. In this study, Cx. pipiens specimens of both ecotypes were collected from Sweden and compared to specimens from Norway, England, Italy, and the Netherlands, as well as Cx. quinquefasciatus from Mali and Thailand. The abundance varied, but all specimens were infected by Wolbachia, while the tested specimens of other mosquito species were often uninfected. The wPip strains were determined through the sequence analysis of Wolbachia genes ank2 and pk1, showing that Cx. pipiens ecotypes in Scandinavia carry different wPip strains. The observed differences in wPip strains indicate that hybridization is not frequent and may contribute to barriers against hybridization of the ecotypes in Sweden and Norway.

The frequency of locally transmitted dengue virus (DENV) infections has increased in Europe in recent years, facilitated by the invasive mosquito species Aedes albopictus, which is well established in a large area of Europe. In Italy, the first indigenous dengue outbreak was reported in August 2020 with 11 locally acquired cases in the Veneto region (northeast Italy), caused by a DENV-1 viral strain closely related to a previously described strain circulating in Singapore and China. In this study, we evaluated the vector competence of two Italian populations of Ae. albopictus compared to an Ae. aegypti lab colony. We performed experimental infections using a DENV-1 strain that is phylogenetically close to the strain responsible for the 2020 Italian autochthonous outbreak. Our results showed that local Ae. albopictus is susceptible to infection and is able to transmit the virus, confirming the relevant risk of possible outbreaks starting from an imported case.

The role of migratory birds in the spread of ticks and tick-borne pathogens along their routes from Africa to Europe is increasingly emerging. Wild birds can host several tick species, often infected by bacteria responsible for zoonoses. The aim of the study is to assess the possible introduction of exotic ticks carried by migratory birds into Italy from Africa and to detect the presence of Rickettsia species and Coxiella burnetii they may harbor. During a two-year survey, we collected ticks from migratory birds captured during their short stop-over on Ventotene Island. Specimens were first identified by morphology or sequencing molecular targets when needed, and then tested by real-time PCR for the presence of selected pathogens. A total of 91% of the collection consisted of sub-Saharan ticks, more than 50% of which were infected by Rickettsia species belonging to the spotted fever group, mainly represented by R. aeschlimannii. In contrast, the suspected C. burnetii detected in two soft ticks were confirmed as Coxiella-like endosymbionts and not the pathogen. Although there are still gaps in the knowledge of this dispersal process, our findings confirm the role of migratory birds in the spread of ticks and tick-borne pathogens, suggesting the need for a continuous surveillance to monitor the potential emergence of new diseases in Europe.

Abstract The correct identification of mosquito vectors is often hampered by the presence of morphologically indiscernible sibling species. The Maculipennis complex is one of these groups that include both malaria vectors of primary importance and species of low/negligible epidemiological relevance, of which distribution data in Italy are outdated. Our study was aimed at providing an updated distribution of Maculipennis complex in Northern Italy through the sampling and morphological/molecular identification of specimens from five regions. The most abundant species was Anopheles messeae (2032), followed by Anopheles maculipennis s.s. (418), Anopheles atroparvus (28) and Anopheles melanoon (13). Taking advantage of ITS2 barcoding, we were able to finely characterize tested mosquitoes, classifying all the Anopheles messeae specimens as Anopheles daciae, a taxon with debated rank to which we referred as species inquirenda (sp. inq.). The distribution of species was characterized by Ecological Niche Models (ENMs), fed by recorded points of presence. ENMs provided clues on the ecological preferences of the detected species, with An. daciae sp. inq. linked to stable breeding sites and An. maculipennis s.s. more associated to ephemeral breeding sites. We demonstrate that historical Anopheles malaria vectors are still present in Northern Italy.

Abstract Background On 11 March 2020, the World Health Organisation (WHO) declared the coronavirus disease 2019 (COVID-19) outbreak to be a pandemic. As the mosquito season progressed, the understandable concern that mosquitoes could transmit the virus began to increase among the general public and public health organisations. We have investigated the vector competence of Culex pipiens and Aedes albopictus, the two most common species of vector mosquitoes in Europe, for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Due to the very unusual feeding behaviour of Ae. albopictus, we also evaluated the role of this mosquito in a potential mechanical transmission of the virus. Methods For the vector competence study, mosquitoes were allowed to take several infectious blood meals. The mosquitoes were then collected and analysed at 0, 3, 7 and 10 days post-feeding. For the mechanical transmission test, Ae. albopictus females were allowed to feed for a short time on a feeder containing infectious blood and then on a feeder containing virus-free blood. Both mosquitoes and blood were tested for viral presence. Results Culex pipiens and Ae. albopictus were found not be competent vectors for SARS-CoV-2, and Ae. albopictus was unable to mechanically transmit the virus. Conclusions This is the first study to show that the most common species of vector mosquitoes in Europe do not transmit SARS-CoV-2 and that Ae. albopictus is unable to mechanically transmit the virus from a positive host to a healthy host through host-feeding.

Abstract Background The European region achieved interruption of malaria transmission during the 1970s. Since then, malaria control programs were replaced by surveillance systems in order to prevent possible re-emergence of this disease. Sporadic cases of non-imported malaria were recorded in several European countries in the past decade and locally transmitted outbreaks of Plasmodium vivax, most probably supported by Anopheles sacharovi, have been repeatedly reported from Greece since 2009. The possibility of locally-transmitted malaria has been extensively studied in Italy where the former malaria vector An. labranchiae survived the control campaign which led to malaria elimination. In this study, we present paradigmatic cases that occurred during a 2017 unusual cluster, which caused strong concern in public opinion and were carefully investigated after the implementation of the updated malaria surveillance system. Methods For suspected locally-transmitted malaria cases, alerts to Ministry of Health (MoH) and the National Institute of Health (ISS) were mandated by the Local Health Services (LHS). Epidemiological investigations on the transmission modes and the identification of possible infection’s source were carried out by LHS, MoH and ISS. Entomological investigations were implemented locally for all suspected locally-transmitted cases that occurred in periods suitable to anopheline activity. Molecular diagnosis by nested-PCR for the five human Plasmodium species was performed to support microscopic diagnosis. In addition, genotyping of P. falciparum isolate was carried out to investigate putative sources of infection and transmission modalities. Results In 2017, a cluster of seven non-imported cases was recorded from August through October. Among them, P. ovale curtisi was responsible of one case whereas six cases were caused by P. falciparum. Two cases were proved to be nosocomial while the other five were recorded as cryptic at the end of epidemiological investigations. Conclusions The epidemiological evidence shows that the locally acquired events are sporadic, often remain unresolved and classified as cryptic ones despite investigative efforts. The “cluster” of seven non-imported cases that occurred in 2017 in different regions of Italy therefore represents a conscious alert that should lead us to maintain a constant level of surveillance in a former malaria endemic country.

Abstract Background After Action Reviews (AAR) with a One Health perspective were performed in Slovenia, Italy, Serbia and Greece following a severe West Nile virus (WNV) transmission season in 2018. A protocol combining traditional techniques and organizational process analysis was developed and then implemented in each country. Results In 2018, response to the unusually intense transmission season of WNV in Slovenia, Italy, Serbia and Greece took place through routine response mechanisms. None of the four countries declared a national or subnational emergency. We found a very strong consensus on the strengths identified in responding to this event. All countries indicated the availability of One Health Plans for surveillance and response; very high laboratory diagnostic capacity in the human, veterinary and entomology sectors and strong inter-sectoral collaboration with strong commitment of engaged institutions as critical in the management of the event. Finally, countries implementing One Health surveillance for WNV (in terms of early warning and early activation of prevention measures) consistently reported a positive impact on their activities, in particular when combining mosquito and bird surveillance with surveillance of cases in humans and equids. Recurring priority areas for improvement included: increasing knowledge on vector-control measures, ensuring the sustainability of vector monitoring and surveillance, and improving capacity to manage media pressure. Conclusions The AARs presented here demonstrate the benefit of cross-sectoral and cross-disciplinary approaches to preparedness for West Nile virus outbreaks in Europe. In the coming years, priorities include fostering and strengthening arrangements that: enable coordinated One Health surveillance and response during WNV transmission seasons; ensure adequate laboratory capacities; strengthen risk communication; and fund longer-term research to address the knowledge gaps identified in this study.

An amendment to this paper has been published and can be accessed via the original article.

Abstract Background Pediculus humanus, the human body louse, is widespread where overcrowding and lack of hygiene are present, in areas of the world affected by poverty, war, famine and presence of refugees. It has recently been considered re-emerging among homeless populations in developed countries. In Italy, it was last reported in 1945. Pediculus humanus is a vector of highly relevant human pathogens. Methods In October 2018, a woman found small insects on a T-shirt bought second-hand in a local street market in a village 35 km south of Rome (central Italy). Insects were identified both morphologically and by molecular analysis. Moreover, they were analyzed molecularly for the presence of Rickettsia prowazekii, Borrelia recurrentis, Bartonella quintana, Coxiella burnetii and Yersinia pestis. Results Morphological and molecular analyses of the insects identified them as 26 lice (12 females, 10 males and 4 nymphs) of the species P. humanus. Many nits were found on the T-shirt seams. DNA of the investigated pathogens was not detected in any of the lice. Conclusions The exceptionality of the described case lies both in the report of P. humanus from a country where it had not been reported since 1945, and in its finding from second-hand clothes for sale in a market, constituting a potential source of infection for people buying this type of goods. The question arises, how did adults and nits of P. humanus infest clothes for sale on a market stall in a country where it had not been reported for decades. Given that the body louse requires frequent blood meals to survive and develop, its arrival on clothes imported from abroad is highly improbable. Hence, it must be presumed that people infected with the human body louse are present in Italy. This report points out a serious regulatory problem regarding the management of second-hand clothes prior to sale and, more generally, of controls in street markets.

Abstract Background Europe and Italy were declared malaria free since the 1970s although the presence of competent vectors and the high number of yearly imported malaria cases make this disease a potential rising health issue. In September 2017, a cryptic fatal case of Plasmodium falciparum malaria in the Province of Trento, Italy, raised the concern of health authorities on the possible resurgence of this disease in the Mediterranean Basin. Methods An entomological surveillance by means of BG traps, CDC light traps and larval search was performed. Sites were chosen among urban and suburban environments (e.g. private houses, public parks, schools, cemeteries, ecotone urban/forest, farms), ranging from an altitude of 91 to 1332 m above sea level. All the mosquitoes collected were morphologically identified and about half of them (103; 49%) were confirmed with the sequencing analysis of the rRNA internal transcribed spacer 2 (ITS-2). Results In the present study 287 sites were screened for the presence of Anopheles spp. and 211 specimens were collected and identified. Hundred-eighteen individuals (56%) belonged to Anopheles plumbeus, 56 (26.5%) to Anopheles maculipennis complex, 10 (4.7%) to Anopheles claviger and 27 were identified only at genus level. This is the first record for the presence of An. plumbeus in the study area. Conclusions The presence of Anopheles spp. mosquitoes in the Province of Trento, Italy, has been updated with the occurrence of An. plumbeus. The risk of malaria endemicity in the area is to be considered very low, but urban and peri-urban habitat may act as potential breeding sites for the presence of mosquito vectors and should be constantly monitored.

Anopheles algeriensis Theobald, 1903, considered a competent vector of Plasmodium parasites, is a mosquito species widely distributed in the Mediterranean area but rare in Northern and Central Europe. The disappearance of its suitable breeding sites in Italy is having a detrimental effect on the occurrence of this species once common along the Southern coasts and on the islands. Recently, molecular investigations have renewed interest in this species, highlighting a genetic heterogeneity among European populations. In this study, An. algeriensis populations from Italy, Germany, Romania, and Sweden were analyzed by molecular typing of the intergenic transcribed spacer 2 (ITS2). The mitochondrial cytochrome c oxidase subunit I (COI) was also analyzed from specimens collected in Southern Italy. With the aim of investigating the population structure of this species, the obtained data were compared to all publicly available ITS2 and COI sequences of An. algeriensis, adding specimens from Spain and Portugal. The analyses of both markers indicate a split between Iberian populations (Spain for ITS2 and Spain/Portugal for COI) and those from the rest of Europe, revealing two cryptic species. The analysis of the COI barcode revealed a third clade representing a cryptic species present in Danube Delta (Romania). The high levels of genetic divergence among the clades of An. algeriensis indicate that this taxon represents a species complex, potentially harboring several distinct cryptic species.

In 2017, a chikungunya outbreak in central Italy later evolved into a secondary cluster in southern Italy, providing evidence of disease emergence in new areas. Officials have taken action to raise awareness among clinicians and the general population, increase timely case detection, reduce mosquito breeding sites, and promote mosquito bite prevention.

We detected Crimean-Congo hemorrhagic fever virus in a Hyalomma rufipes nymph collected from a whinchat (Saxicola rubetra) on the island of Ventotene in April 2017. Partial genome sequences suggest the virus originated in Africa. Detection of the genome of this virus in Italy confirms its potential dispersion through migratory birds.

Malaria still represents a potential public health issue in Italy, and the presence of former Anopheles vectors and cases imported annually merit continuous surveillance. In areas no longer endemic, the concurrent presence of gametocyte carriers and competent vectors makes re-emergence of local transmission possible, as recently reported in Greece. In October 2017, due to the occurrence of four suspected introduced malaria cases in the province of Taranto (Apulia region), entomological investigations were performed to verify the involvement of local anopheline species. In 2019–2020 entomological surveys were extended to other areas historically prone to malaria between the provinces of Taranto and Matera and the province of Foggia (Gargano Promontory). Resting mosquitoes were collected in animal shelters and human dwellings, larvae were sampled in natural and artificial breeding sites, and specimens were both morphologically and molecularly identified. A total of 2228 mosquitoes were collected, 54.3% of which were anophelines. In all the investigated areas, Anopheles labranchiae was the most widespread species, while Anopheles algeriensis was predominant at the Gargano sites, and Anopheles superpictus and Anopheles plumbeus were recorded in the province of Matera. Our findings showed a potentially high receptivity in the surveyed areas, where the abundance of the two former malaria vectors, An. labranchiae and An. superpictus, is related to environmental and climatic parameters and to anthropic activities.

Abstract Background Aedes albopictus, a known worldwide vector of several mosquito-borne disease pathogens including dengue, chikungunya and Zika viruses, was introduced into Europe in the late 1970s through global trade. First recorded in northern Italy in 1990, this mosquito species has rapidly spread throughout the country, where it was responsible for an outbreak of chikungunya in 2007 that affected more than 200 people. As part of the VectorNet project, which is aimed at improving preparedness and responsiveness for animal and human vector-borne diseases in Europe, a mosquito targeted study was carried out on the three southernmost Italian islands. The objective was to verify the current European southern distribution limits of Ae. albopictus and the potential occurrence of other invasive mosquito species, in the light of the introduction of high risk for vector-borne disease pathogens into Europe via migration flows. Results In the summer 2015, six surveys for container-breeding mosquitoes were carried out by setting up a network of oviposition traps and BG Sentinel traps in selected areas on the islands of Pantelleria, Lampedusa and Linosa. Aedes albopictus was found on all three islands under investigation. The consequences on public health with regard to the presence of this mosquito vector and the migrant people entering the country from Africa and the Middle East are also discussed here. Conclusions The detection of the Asian tiger mosquito on these islands, which represent the last European strip of land facing Africa, has important implications for public health policy and should prompt the national authorities to implement tailored surveillance activities and reinforce plans for preparedness strategies in such contexts.

BACKGROUND:Aedes mosquitoes are vectors of arboviral diseases of great relevance for public health. The recent outbreaks of dengue, Zika, chikungunya and the rapid worldwide spreading of Aedes albopictus emphasize the need for improvement of vector surveillance and control. Host antibody response to mosquito salivary antigens is emerging as a relevant additional tool to directly assess vector-host contact, monitor efficacy of control interventions and evaluate risk of arboviral transmission. METHODOLOGY/PRINCIPAL FINDINGS:Groups of four BALB/c mice were immunized by exposure to bites of either Aedes albopictus or Aedes aegypti. The 34k2 salivary proteins from Ae. albopictus (al34k2) and Ae. aegypti (ae34k2) were expressed in recombinant form and Ae. albopictus salivary peptides were designed through B-cell epitopes prediction software. IgG responses to salivary gland extracts, peptides, al34k2 and ae34k2 were measured in exposed mice. Both al34k2 and ae34k2, with some individual and antigen-specific variation, elicited a clearly detectable antibody response in immunized mice. Remarkably, the two orthologous proteins showed very low level of immune cross-reactivity, suggesting they may eventually be developed as species-specific markers of host exposure. The al34k2 immunogenicity and the limited immune cross-reactivity to ae34k2 were confirmed in a single human donor hyperimmune to Ae. albopictus saliva. CONCLUSIONS/SIGNIFICANCE:Our study shows that exposure to bites of Ae. albopictus or Ae. aegypti evokes in mice species-specific IgG responses to al34k2 or ae34k2, respectively. Deeper understanding of duration of antibody response and validation in natural conditions of human exposure to Aedes mosquitoes are certainly needed. However, our findings point to the al34k2 salivary protein as a promising potential candidate for the development of immunoassays to evaluate human exposure to Ae. albopictus. This would be a step forward in the establishment of a serological toolbox for the simultaneous assessment of human exposure to Aedes vectors and the pathogens they transmit.

Bruchidius raddianae (Anton & Delobel 2003), a species of seed beetle spread in Northern Africa, was detected for the first time in Europe in some localities of the Southern Iberian Peninsula in 2007. In Spain this coleopteran lives on the shrubs of the South African acacia tree, Vachellia karroo (Hayne) Banfi & Galasso, currently present in the Southern Iberian Peninsula. From seeds of V. karroo collected in October 2015, in Lampedusa island, Italy, where this plant is widely spread, 45 specimens of this coleopteran emerged. This observation represents the first record of B. raddianae in Italy and the second one for Europe.

Chikungunya virus (CHIKV) is an emerging arbovirus, belonging to the Togaviridae family, Alphavirus genus, transmitted by Aedes spp. mosquitoes. Since 2007, two different CHIKV strains (E1-226A and E1-226V) have been responsible for outbreaks in European countries, including Italy, sustained by Ae. albopictus mosquitoes.In this study, we assessed the susceptibility to the CHIKV E1-226V, strain responsible for the Italian 2007 outbreak, of eight Ae. albopictus populations collected in Northern, Central, Southern, and Island Italy, by experimental infections. Vector competence was evaluated by estimating infection, dissemination, and transmission rates (IR, DR, TR), through detection of the virus in the bodies, legs plus wings, and saliva, respectively. Additionally, vertical transmission was evaluated by the detection of the virus in the offspring. The results of our study demonstrated that the Italian populations of Ae. albopictus tested were susceptible to CHIKV infection, and can disseminate the virus outside the midgut barrier with high values of IR and DR. Viral infectious RNA was detected in the saliva of three populations from Central, Southern, and Island Italy, also tested for TR and population transmission rate (PTR) values. No progeny of the first and second gonotrophic cycle were positive for CHIKV.This study strongly confirms the role of Ae. albopictus as a potential CHIKV vector in Italy. This may represent a threat, especially considering both the high density of this species, which is widespread throughout the country, and the increasing number of cases of imported arboviruses.

BACKGROUND: Ticks are vectors and important reservoirs for microbial agents that cause disease in humans and animals. Among these pathogens, the members of Rickettsia species play an important role in public health. AIM AND METHODS: One hundred twenty-nine ticks belonging to four tick species (Ixodes ricinus,Rhipicephalus turanicus,Dermacentor marginatus, and Haemaphysalis punctata) were collected at different sites of the Insugherata Natural Reserve, localized in the urban area of Rome, Italy. Questing ticks were tested by PCR for Rickettsia spp., amplifying partial gene of ompA. RESULTS: Forty-six ticks were found to be infected with Rickettsia species. Five SFG rickettsiae were identified: three human pathogens Rickettsia conorii, Rickettsia massiliae and Rickettsia aeschlimannii, and two putative new strains Rickettsia sp. strain RM1 and Rickettsia sp. strain RM2. The phylogenetic analysis of partial gene sequences of ompA, gltA, and 17-kd antigen showed that they clustered with several rickettsiae with unidentified pathogenicity. However, Rickettsia sp. strain RM1 and Rickettsia sp. strain RM2 clustered in a statistically supported clade with R. massiliae, and R. monacensis, respectively. CONCLUSION: Our findings suggest that Rickettsia species other than R. conorii are implicated in human disease in Italy.

Abstract Background West Nile virus (WNV) is a vector-borne disease responsible for causing epidemics in many areas of the world. The virus is maintained in nature by an enzootic bird-mosquito-bird cycle and occasionally transmitted to other hosts, such as equines and humans. Culex species, in particular the ubiquitous species Culex pipiens is thought to play a major vector role both in enzootic and epizootic maintenance and transmission of WNV. Introduced in Europe in recent years, since 2008 WNV has been stably circulating mainly in the Northeastern regions of Italy, although sporadic equine and/or human cases, as well as WNV infected Cx. pipiens pools, have been recorded in other Italian areas. The scope of our study was to evaluate the potential competence of some Italian populations of Cx. pipiens to transmit WNV and to assess their ability for vertical transmission of the virus. For this purpose four Italian populations, from different areas, were experimentally infected. Methods After the infectious blood meal, fed females were monitored for 32 days to determine the length of viral extrinsic incubation period. WNV titre of infected mosquitoes was evaluated both by quantitative Real Time PCR and viral titration by Plaque Forming Units/ml (PFU/mL) in VERO cells. Potential Infection, Dissemination, Transmission rates (IR, DR, TR) were assessed by detection of the virus in body, legs plus wings and saliva of the fed females, respectively. Results All tested populations were susceptible to the WNV infection. The viral presence in legs and wings demonstrated the ability of WNV to disseminate in the mosquitoes. Viral RNA was detected in the saliva of tested populations. No significant differences in TR values were observed among the four studied populations. The offspring of the Cx. pipiens infected females were WNV negative. Conclusions Our study addressed an important issue in the knowledge on the complex WNV-vector relationships in Italy, indicating that all Italian Cx. pipiens populations tested exhibited vector competence for WNV. Further studies should be performed in order to better clarify the role of other factors (vector density, climatic conditions, reservoir presence etc.) in order to predict where and when WNV outbreaks could occur.

Abstract Background Understanding wildlife disease ecology is becoming an urgent need due to the continuous emergence and spread of several wildlife zoonotic diseases. West Nile Virus (WNV) is the most widespread arthropod-borne virus in the world, and in recent decades there has been an increase both in geographic range, and in the frequency of symptomatic infections in humans and wildlife. The principal vector for WNV in Europe is the common house Culex pipiens mosquito, which feeds on a wide variety of vertebrate host species. Variation in mosquito feeding preference has been described as one of the most influential parameters driving intensity and timing of WNV infection in the United States, but feeding preferences for this species have been little studied in Europe. Methods Here, we estimated feeding preference for wild Cx. pipiens in northern Italy, using molecular analysis to identify the origin of blood meals, and avian census to control host abundance variations. Additionally, we used host bird odour extracts to test experimentally mosquito preferences in the absence of environmental variations. Results For the first time, we demonstrate a clear feeding preference for the common blackbird (Turdus merula), both for wild collected specimens and in the lab, suggesting a potential important role for this species in the WNV epidemiology in Europe. A seasonal decrease in abundance of blackbirds is associated with increased feeding on Eurasian magpies (Pica pica), and this may be linked to seasonal emergence of WNV in humans. Feeding preferences on blackbirds are more marked in rural areas, while preference for magpies is higher in peridomestic areas. Other species, such as the house sparrow (Passer domesticus) appear to be selected by mosquitoes opportunistically in relation to its abundance. Conclusions Our findings provide new insights into the ecology of Cx. pipiens in Europe and may give useful indications in terms of implementing targeted WNV surveillance plans. However, a clearer understanding of spatio-temporal variations of Cx. pipiens feeding preferences, and targeted studies on reservoir competence for WNV for these species are therefore now urgently needed as this is essential to describe disease dynamics and quantify virus transmission risk.

Introduction. This study represents a preliminary inquiry on tick fauna composition carried out in Maremma, Central Italy, where very few data are available. Materials and methods. The study area was selected in the limestone hills surrounding Tarquinia town, on the base of suitable elements directly affecting the tick occurrence and the possible circulation of tick-borne pathogens, such as wild cattle rearing, kind of vegetation and human activities. Since a proper understanding of tick ecology is critical in predicting the risk of tick-borne pathogen transmission in a given area, a series of surveys was planned in such area in 2011. Results. One hundred fifty-four ticks belonging to 4 genera and 6 species, namely Ixodes ricinus (n. = 109; 70%), Rhipicephalus bursa (n. = 18; 12%), Rhipicephalus turanicus (n. = 14; 9%), Rhipicephalus sanguineus (n. = 6; 4%), Hyalomma marginatum (n. = 4; 3%) and Dermacentor marginatus (n. = 3; 2%) were identified and reported for the area. Discussion. The results of this acarological research represent a significant contribution to the knowledge of the tick fauna of rural areas in Northern Lazio Region, as first step toward a future molecular investigation on pathogen circulation.

[b]introduction.[/b] Limited information is available about the presence of tick-borne pathogens in urban parks in Italy. To fill this gap, ticks were collected in a public park in Rome over a 1-year period and screened by molecular methods for tick-borne pathogens. [b]results and conclusion[/b]. The most abundant tick species were Rhipicephalus turanicus and Ixodes ricinus. The predominant pathogens detected were Borrelia. burgdorferi sensu lato (36%), Rickettsia spp. (36%), and Coxiella burnetii (22%). Among less frequently detected pathogens, Babesia microti was detected for the first time in Italy, with a prevalence of 4%. Neither Bartonella spp. nor Francisella tularensis were detected. With regard to co-infections, the most frequent double and triple infections involved Rickettsia spp., B. burgdorferi sl., and C. burnetii.. A positive correlation was detected between pathogens and I. ricinus. Further studies are needed in order to assess risk associated with tick-borne pathogens in urban areas.

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.

Nel presente lavoro vengono riportate le informazioni essenziali su tassonomia e biologia nonché sugli aspetti ecologici degli adulti dei Culicidi italiani. Attualmente la fauna culicidica italiana comprende 64 specie appartenenti a 2 sottofamiglie e 8 generi. Alla sottofamiglia Anophelinae appartiene soltanto il genere Anopheles, presente con 16 specie raggruppate in due sottogeneri. Alla sottofamiglia Culicinae appartengono i rimanenti 7 generi: Aedes con 6 specie raggruppate in 3 sottogeneri, Coquillettidia con 2 specie, Ochlerotatus con 20 specie raggruppate in 3 sottogeneri, Culex con 12 specie raggruppate in 4 sottogeneri, Culiseta con 6 specie raggruppate in 3 sottogeneri, Orthopodomyia e Uranotaenia con una specie ognuna. In questo contesto vengono fornite le chiavi di identificazione specifica per le zanzare adulte, in italiano e in inglese. Le chiavi sono corredate da un’ampia iconografia (figure 1-75). Alle chiavi fa seguito la diagnosi morfologica dell’adulto di ogni specie con note sulla relativa biologia e distribuzione. Per ulteriori approfondimenti viene riportata la bibliografia completa sulle zanzare della fauna italiana dal 1960 ed i precedenti lavori più autorevoli.

The early transcribed membrane proteins ETRAMPs belong to a family of small, transmembrane molecules unique to Plasmodium parasite, which share a signal peptide followed by a short lysine-rich stretch, a transmembrane domain and a variable, highly charged C-terminal region. ETRAMPs are usually expressed in a stage-specific manner. In the blood stages they localize to the parasitophorous vacuole membrane and, in described cases, to vesicle-like structures exported to the host erythrocyte cytosol. Two family members of the rodent parasite Plasmodium berghei, uis3 and uis4, localize to secretory organelles of sporozoites and to the parasitophorous membrane vacuole of the liver stages. By the use of specific antibodies and the generation of transgenic lines, we showed that the P. berghei ETRAMP family member SEP2 is abundantly expressed in gametocytes as well as in mosquito and liver stages. In intracellular parasite stages, SEP2 is routed to the parasitophorous vacuole membrane while, in invasive ookinete and sporozoite stages, it localizes to the parasite surface. To date SEP2 is the only ETRAMP protein detected throughout the parasite life cycle. Furthermore, SEP2 is also released during gliding motility of salivary gland sporozoites. A limited number of proteins are known to be involved in this key function and the best characterized, the CSP and TRAP, are both promising transmission-blocking candidates. Our results suggest that ETRAMP members may be viewed as new potential candidates for malaria control.