Culicoides species responsible for the transmission of Epizootic Haemorrhagic Disease virus (EHDV) serotype 8 in Italy.

Epizootic haemorrhagic disease (EHD) is a viral disease transmitted by Culicoides biting midges that affects wild and domestic ruminants. The causative agent, EHD virus (EHDV), belongs to the family Sedoreoviridae, genus Orbivirus. The virus has never been reported in Europe until October 2022, when the virus was for the first time detected in Sicily and Sardinia. After the first clinical cases, an intensive entomological field activity was carried out in five EHD affected farms located in Sardinia, with the aim of assessing the EHDV vector competence in European species of Culicoides. EHDV­8 was detected in C. imicola, C. obsoletus/scoticus, C. newsteadi, C. pulicaris ss, and C. bysta. The first 4 species have also been demonstrated to be able to transmit bluetongue virus (BTV). According to these results, it is likely that EHDV­8, sharing the same transmission patterns of BTV, can also spread to Europe.

Species of mosquitoes present in Abruzzo and Molise and their possible role as vector of Usutu and West Nile viruses.

In 2019, entomological survey on mosquitoes was carried out in Abruzzo and Molise regions in central Italy to obtain data on local mosquito fauna. Collection sites were selected based on a previous ecoregion classification of the territory.  From 2019 to 2021 virological surveillance for West Nile virus (WNV) and Usutu virus (USUV) on mosquitoes was carried out in the same regions, selecting ecoregions where virus circulation and vector presence were more likely,  all mosquitoes were collected and identified, and the female mosquitoes were sorted in 3046 pools and tested for the presence of WNV and USUV by Real-time PCR. All pools tested negative for WND, while USUV was detected in 7 pools of Aedes caspius collected in Molise region, 17 pools of Culex pipiens s.l. (2 collected in Molise, 15 in Abruzzo), and 1 pool of Culiseta longiareolata collected in Molise. These results suggests the presence of an USUV enzootic cycle, maintained by Culex pipiens s.l. and Aedes caspius in both Italian regions, as well as providing a useful picture in terms of species presence and abundance for both regions. Ecoregions proved to be a very valuable tool in determining high risk areas for vector borne diseases.

Direct and Indirect Role of Migratory Birds in Spreading CCHFV and WNV: A Multidisciplinary Study on Three Stop-Over Islands in Italy.

The annual movements of migratory birds can contribute to the spread of African ticks and tick-borne pathogens of potential public health concern across Europe. The aim of the study was to investigate their role in the possible introduction of African ticks and tick-borne pathogens into European countries during spring migration. A total of 2344 ticks were collected during three spring seasons from 1079 birds captured on three Italian stop-over islands during their northbound migration. Once identified, each tick was tested by RT-PCR for the presence of Crimean-Congo hemorrhagic fever (CCHFV), West Nile (WNV), and Usutu (USUV) viruses. Moreover, carcasses of birds found dead were collected and tested for the possible presence of WNV and USUV. Results confirmed a higher contribution of trans-Saharan migrants compared to intra-Palearctic ones and the prevalence of African tick species in the sample. CCHFV was detected for the second time in Italy in a Hyalomma rufipes, and WNV was found in two ticks of the same genus, all carried by trans-Saharan birds. WNV lineage 1 was also found in the organs of a Garden warbler. These results confirm the role of migratory birds in carrying African ticks, as well as viruses of zoonotic importance, from Africa into Europe.

The Absence of Abdominal Pigmentation in Livestock Associated Culicoides following Artificial Blood Feeding and the Epidemiological Implication for Arbovirus Surveillance.

Culicoides midges (Diptera: Ceratopogonidae), the vectors of economically important arboviruses such as bluetongue virus and African horse sickness virus, are of global importance. In the absence of transovarial transmission, the parity rate of a Culicoides population provides imperative information regarding the risk of virus dispersal. Abdominal pigmentation, which develops after blood feeding and ovipositioning, is used as an indicator of parity in Culicoides. During oral susceptibility trials over the last three decades, a persistent proportion of blood engorged females did not develop pigment after incubation. The present study, combining a number of feeding trials and different artificial feeding methods, reports on this phenomenon, as observed in various South African and Italian Culicoides species and populations. The absence of pigmentation in artificial blood-fed females was found in at least 23 Culicoides species, including important vectors such as C. imicola, C. bolitinos, C. obsoletus, and C. scoticus. Viruses were repeatedly detected in these unpigmented females after incubation. Blood meal size seems to play a role and this phenomenon could be present in the field and requires consideration, especially regarding the detection of virus in apparent "nulliparous" females and the identification of overwintering mechanisms and seasonally free vector zones.

Correction to: The tree that hides the forest: cryptic diversity and phylogenetic relationships in the Palaearctic vector Obsoletus/Scoticus complex (Diptera: Ceratopogonidae) at the European level.

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

Species composition and relative abundance of the genus Culicoides (Diptera: Ceratopogonidae) in Romania.

BACKGROUND: Culicoides biting midges are vectors involved in the biological transmission cycle of important animal diseases such as bluetongue and African horse sickness. In Romania, the first outbreaks of bluetongue were reported in 2014, leading to increased activities within the existing entomological surveillance network. The main goals of the surveillance activities were the establishment of the vector free period in relation to animal trade and the identification of Culicoides species involved in the transmission of the pathogen. This study was conducted on the composition and relative abundance of the species belonging to the genus Culicoides (Diptera: Ceratopogonidae) in certain regions of Romania and provided the opportunity to update the existing checklist of Culicoides species of this country. METHODS: The study was conducted in 33 of the 42 administrative units (counties), including a total of 659 catches, in 102 locations. The collections were carried out with UV blacklight suction traps (OVI type). The collected insects were preserved in 70% ethanol. Morphological insect identification was carried out using a stereomicroscope, according to established identification keys. In ten localities the relative abundance of the cryptic species of the Obsoletus complex was determined by multiplex PCR assay based on the ITS2 segment. The identification of the Culicoides chiopterus (Meigen) species by morphological examination was confirmed by PCR assay based on the ITS1 segment. RESULTS: Eleven species were identified using morphological and PCR tools. The rest of the individuals were separated into five taxa. The species of the Obsoletus complex (grouping Culicoides obsoletus (Meigen) and Culicoides scoticus Downes & Kettle) were the most abundant, accounting for 59% of the total number of captured Culicoides spp. Three of the identified species are mentioned, according to our knowledge, for the first time in Romania: Culicoides newsteadi Austen, Culicoides flavipulicaris Dzhafarov and Culicoides bysta Sarvasová, Kocisová, Candolfi & Mathieu. CONCLUSIONS: Our study demonstrates that the Culicoides species most commonly cited as being involved in the transmission of arboviruses in Europe (i.e. bluetongue and Schmallenberg viruses) make up a high proportion of adult Culicoides trapped in Romania.

The tree that hides the forest: cryptic diversity and phylogenetic relationships in the Palaearctic vector Obsoletus/Scoticus Complex (Diptera: Ceratopogonidae) at the European level.

BACKGROUND: Culicoides obsoletus is an abundant and widely distributed Holarctic biting midge species, involved in the transmission of bluetongue virus (BTV) and Schmallenberg virus (SBV) to wild and domestic ruminants. Females of this vector species are often reported jointly with two morphologically very close species, C. scoticus and C. montanus, forming the Obsoletus/Scoticus Complex. Recently, cryptic diversity within C. obsoletus was reported in geographically distant sites. Clear delineation of species and characterization of genetic variability is mandatory to revise their taxonomic status and assess the vector role of each taxonomic entity. Our objectives were to characterize and map the cryptic diversity within the Obsoletus/Scoticus Complex. METHODS: Portion of the cox1 mitochondrial gene of 3763 individuals belonging to the Obsoletus/Scoticus Complex was sequenced. Populations from 20 countries along a Palaearctic Mediterranean transect covering Scandinavia to Canary islands (North to South) and Canary islands to Turkey (West to East) were included. Genetic diversity based on cox1 barcoding was supported by 16S rDNA mitochondrial gene sequences and a gene coding for ribosomal 28S rDNA. Species delimitation using a multi-marker methodology was used to revise the current taxonomic scheme of the Obsoletus/Scoticus Complex. RESULTS: Our analysis showed the existence of three phylogenetic clades (C. obsoletus clade O2, C. obsoletus clade dark and one not yet named and identified) within C. obsoletus. These analyses also revealed two intra-specific clades within C. scoticus and raised questions about the taxonomic status of C. montanus. CONCLUSIONS: To our knowledge, our study provides the first genetic characterization of the Obsoletus/Scoticus Complex on a large geographical scale and allows a revision of the current taxonomic classification for an important group of vector species of livestock viruses in the Palaearctic region.

Vector Competence of Italian Populations of Culicoides for Some Bluetongue Virus Strains Responsible for Recent Northern African and European Outbreaks.

The distribution of Bluetongue virus (BTV) in Europe can be represented by two distinct and interconnected epidemiological systems (episystems), each characterized by different ecological characteristics and vector species. This study investigated the vector competence of Italian populations of Culicoides imicola and Culicoides obsoletus/scoticus to some representative BTV strains after artificial oral infection. The BTV strains were selected according to their ability to spread to one or both episystems and included BTV-4 ITA, responsible of the recent Italian and French BTV-4 outbreaks; the BTV-2 strain which caused the first BTV incursion in Italy, Corsica, and Balearic Islands; BTV-4 MOR, responsible for the epidemic in Morocco; and BTV-8, the strain which spread through Europe between 2006 and 2008. Blood-soaked cotton pledgets and Hemotek membrane feeder using Parafilm® membrane were used to artificially feed midges. For each population/strain, recovery rates (positive/tested heads) were evaluated using serogroup- and serotype-specific RT-PCR. The trial demonstrated that, except for the Abruzzo population of C. obsoletus/C. scoticus, which was refractory to BTV-4 MOR infection, all the investigated Culicoides populations are susceptible to the selected BTV strains and that, if prompt vaccination programs and restriction measures had not been implemented, BTV-2 and BTV-4 MOR could have spread all over Europe.

Defining ecological regions in Italy based on a multivariate clustering approach: A first step towards a targeted vector borne disease surveillance.

Ecoregionalization is the process by which a territory is classified in similar areas according to specific environmental and climatic factors. The climate and the environment strongly influence the presence and distribution of vectors responsible for significant human and animal diseases worldwide. In this paper, we developed a map of the eco-climatic regions of Italy adopting a data-driven spatial clustering approach using recent and detailed spatial data on climatic and environmental factors. We selected seven variables, relevant for a broad set of human and animal vector-borne diseases (VBDs): standard deviation of altitude, mean daytime land surface temperature, mean amplitude and peak timing of the annual cycle of land surface temperature, mean and amplitude of the annual cycle of greenness value, and daily mean amount of rainfall. Principal Component Analysis followed by multivariate geographic clustering using the k-medoids technique were used to group the pixels with similar characteristics into different ecoregions, and at different spatial resolutions (250 m, 1 km and 2 km). We showed that the spatial structure of ecoregions is generally maintained at different spatial resolutions and we compared the resulting ecoregion maps with two datasets related to Bluetongue vectors and West Nile Disease (WND) outbreaks in Italy. The known characteristics of Culicoides imicola habitat were well captured by 2/22 specific ecoregions (at 250 m resolution). Culicoides obsoletus/scoticus occupy all sampled ecoregions, according to its known widespread distribution across the peninsula. WND outbreak locations strongly cluster in 4/22 ecoregions, dominated by human influenced landscape, with intense cultivations and complex irrigation network. This approach could be a supportive tool in case of VBDs, defining pixel-based areas that are conducive environment for VBD spread, indicating where surveillance and prevention measures could be prioritized in Italy. Also, ecoregions suitable to specific VBDs vectors could inform entomological surveillance strategies.

Mosquito species involved in the circulation of West Nile and Usutu viruses in Italy.

Usutu (USUV) and West Nile (WNV) are mosquito-borne Flavivirus emerged in Italy in 1996 and 1998, respectively, and reappeared 10 years later. The aim of this work is to review the Italian mosquito species found positive for WNV and USUV between 2008 and 2014. Moreover, the role of mosquitoes in promoting the overwintering of these viruses is discussed, as a result of the mosquito collections performed in Molise region between September 2010 and April 2011. Overall 99,000 mosquitoes were collected: 337 and 457 mosquito pools tested positive by real time reverse transcriptase polymerase chain reaction (real time RT-PCR) for WNV and USUV, respectively. West Nile virus was detected in pools of Culex pipiens s.l. (329), Ochlerotatus caspius (4), Culex modestus (2), and Culex spp. (2). Positive USUV pools were from Cx. pipiens s.l. (435), Aedes albopictus (12), Oc. caspius (5), Culex spp. (2), Anopheles maculipennis s.l. (1), Culiseta annulata (1), and Ochlerotatus detritus (1). In Molise region, 1,694 mosquitoes were collected, and USUV was identi ed in Cx. pipiens s.l., Cs. annulata, and Oc. detritus pools. This paper shows that Cx. pipiens s.l. is the mosquito species most involved in the WNV and USUV circulation in Italy, although other species would also support the spread of both the viruses during Winter.

Eco-climatic indicators for three Culicoides species of the Obsoletus complex in Italy.

Bluetongue, Schmallenberg and African horse sickness viruses are transmitted by vectors belonging to the genus Culicoides (Diptera: Ceratopogonidae). Among this genus, species of the Obsoletus complex play a crucial role in Europe. In Italy the complex includes three species: Culicoides obsoletus sensu strictu, Culicoides scoticus and Culicoides montanus. These three sibling species were identified in 80 collection sites across Italy through a multiplex PCR test. Sixty­four sites were classified with a predominant species through a Bayesian approach. Environmental, topographic and climatic factors together with landscape metrics were investigated in each species group using the non­parametric Mann­Whitney test. Culicoides obsoletus s.s. resulted the most widely distributed species across the peninsula (51 sites). This species was collected in many eco­climatic conditions, at a wide range of temperature and altitudes, mainly in anthropogenic landscape. Conversely C. scoticus collection sites were dominated by natural vegetated areas or forest, at medium higher altitudes, preferably in a wilder and more pristine environment (predominant species in 8 sites). Culicoides montanus was a rarer species, statistically predominant in only 3 sites located in Southern Italy, characterized by temperatures higher than the other species. These results, together with other on­going researches on the vector competence of the species belonging to the Obsoletus complex, and with investigation of larval and breeding habitats, could greatly improve epidemiological knowledge of possible spreading Culicoides­borne viruses.

Analysis of climatic factors involved in the BTV-1 incursion in Central Italy in 2014.

In 2012, six years after the previous epidemic, Bluetongue virus serotype 1 (BTV-1) re-emerged in Sardinia causing a limited number of outbreaks. Due to impossibility of implementing a vaccination campaign, the BTV-1 then spread all over the island in 2013 with about 7,000 outbreaks and, in September 2013, the virus reached Central Italy, with a limited number of outbreaks located along the Tyrrhenian coast. The surveillance system in place in Italy detected viral circulation during the following winter, when a few seroconversions were notified. Starting from mid July 2014, a huge number of outbreaks were reported and the disease spread toward inland territories, affecting Umbria, Abruzzo and Marche. In 2014, BTV-1 affected areas where Culicoides species belonging to the Obsoletus and Pulicaris complexes were identified as main vectors. The analysis of temperature and rainfall in Central Italy revealed a significant warmer winter (2013-2014) and a cooler and rainy summer season (2014). These climatic aspects might have certainly favored the overwintering of the virus in local vector or host populations in the Tyrrhenian coast, and, then, the spread of the virus to the rest of Central Italy. However, the heavy circulation of BTV-1 and the severity of clinical outbreaks recorded leave a number of 'open questions' that are currently under investigations.

The 'Culicoides obsoletus group' in Italy: relative abundance, geographic range, and role as vector for Bluetongue virus.

As Bluetongue virus (BTV) spread in Italy following its first incursion in 2000, it soon became apparent that, besides Culicoides imicola, additional species of the subgenus Avaritia were involved as vectors, namely one or more of the species that belong to the so-called 'Culicoides obsoletus group', which comprises C. dewulfi, C. chiopterus, C. obsoletus sensu stricto, C. scoticus and C. montanus; the three last named species are considered generally as forming the Obsoletus complex. This study presents the findings made over the last decade and more, within the Italian entomological surveillance program for Bluetongue. It describes the integrated morphological and molecular approach used to identify the species of the 'C. obsoletus group', maps in detail their relative abundances and geographic ranges in Italy, clarifies the hitherto unknown comparative seasonal abundances of C. obsoletus s.s. and C. scoticus in a site in Central Italy, and provides further details on the potential vector status of five species of the 'C. obsoletus group', with emphasis on C. obsoletus s.s., C. scoticus and C. montanus. Unlike the situation in Northern Europe, Culicoides dewulfi and C. chiopterus are uncommon to rare in Italy. In contrast, the Obsoletus complex occurs abundantly throughout Italy, with C. obsoletus s.s. being the most prevalent and ecologically adaptive of the three species making up the complex. A longitudinal study conducted at a site in Central Italy revealed that: (i) species of the Obsoletus complex prefer horses to sheep; (ii) their parity rates range from 10% (March) to 56% (November); (iii) throughout the year C. scoticus is consistently more abundant than C. obsoletus s.s.; (iv) abundances in both, C. obsoletus s.s. and C. scoticus, peak in May-June, with the peak of the latter species being more evident. Bluetongue virus was first isolated from wild caught midges of the Obsoletus complex in 2002. Thereafter, pools of selected parous midges collected across Italy, and during multiple outbreaks of BT, have been found consistently PCR-positive for the virus. More recently, viral RNA has been detected in field specimens of C. dewulfi, C. obsoletus s.s., C. scoticus and C. montanus.

Epizootic haemorrhagic disease in Italy: vector competence of indigenous Culicoides species and spatial multicriteria evaluation of vulnerability.

Epizootic haemorrhagic disease (EHD) is an infectious non-contagious viral disease transmitted by Culicoides, which affects wild and domestic ruminants. The disease has never been reported in Europe, however recently outbreaks of EHD occurred in the Mediterranean Basin. Consequently, the risk that Epizootic haemorrhagic disease virus (EHDV) might spread in Italy cannot be ignored. The aim of this study was to evaluate the risk of EHDV transmission in Italy, in case of introduction, through indigenous potential vectors. In Italy, the most spread and abundant Culicoides species associated to livestock are Culicoides imicola and the members of the Obsoletus complex. Culicoides imicola is a competent vector of EHDV, whereas the vector status of the Obsoletus complex has not been assessed yet. Thus, its oral susceptibility to EHDV was here preliminary evaluated. To evaluate the risk of EHDV transmission a geographical information system-based Multi-Criteria Evaluation approach was adopted. Distribution of vector species and host density were used as predictors of potential suitable areas for EHDV transmission, in case of introduction in Italy. This study demonstrates that the whole peninsula is suitable for the disease, given the distribution and abundance of hosts and the competence of possible indigenous vectors.

Vector species of Culicoides midges implicated in the 2012­2014 Bluetongue epidemics in Italy.

In 2012, serotypes 1 and 4 of bluetongue virus (BTV) entered and co­circulated in Sardinia. The following year, BTV­1 spread all over Sardinia and invaded Sicily and the Italian Tyrrenian coast. In 2014, this strain spread extensively in mainland Italy, causing severe outbreaks. In late 2014, BTV­4 was detected in Southern Italy (Apulia region). This study reports the detection of BTV in species of Culicoides (Diptera: Ceratopogonidae) collected in Italy during the epidemics between 2012 and 2014. A total of 2,925 pools (83,102 midges), sorted from 651 collections made on 339 affected farms of 12 Italian regions, were tested for the presence of BTV by real time polymerase chain reaction (RT­PCR). The study clearly shows that Culicoides imicola and Obsoletus complex have played a crucial role in the bluetongue (BT) epidemics in Italy in 2012­2014. Nevertheless, it also shows that other species may have played a role in transmitting BTV during these outbreaks. Culicoides dewulfi and at least 3 species of the Pulicaris complex, namely Culicoides pulicaris, Culicoides newsteadi and Culicoides punctatus, were found positive to BTV. Serotype 1 was detected in all species tested, whereas the BTV­4 was detected in the Obsoletus complex, C. imicola, and C. newsteadi.

Orbivirus detection from Culicoides collected on African horse sickness outbreaks in Namibia.

African horse sickness (AHS), a non-contagious infectious disease caused by a RNA virus in the Orbivirus genus within the Reoviridae family affecting all equids, is endemic in sub-Saharan Africa. The virus is transmitted by some species of biting midges in the genus Culicoides (Diptera: Ceratopogonidae). In April 2011, 8 Culicoides collections were performed in 6 districts of 4 regions of the Republic of Namibia (Africa), all within a 400 km radious from the capital Windhoek. Six farms - Khomas (Windhoek and Steinhausen), Erongo (Karibib and Omaruru), Otjozondjupa (Okahandja), and Omaheke (Gobabis) involved in the AHS outbreaks, were sampled. Overall 194,211 Culicoides were collected and identified. Culicoides imicola was largely the most abundant species at all farms (99.4%). A total of 18,687 parous and gravid Culicoides females were assayed for AHS virus (AHSV) by real time RT-PCR. Of the 248 assayed pools, 227 consisted of C. imicola, 13 of Culicoides pycnostictus and 5 of Schultzei complex. Only 1 pool each of Culicoides nivosus, Culicoides leucostictus, and Culicoides tropicalis was assayed. Of the 248 pools examined by real time RT-PCR, 81 tested positive for AHSV, all consisting of C. imicola collected at Omaruru, resulting in a field vector infection rate of 0.91%. No viable AHSV could be isolated from 88 of the tested pools (n = 1,463). However, bluetongue virus (BTV) serotype-1 and 10 were isolated from 3 of these pools, each consisting of 100 C. imicola collected at Windhoek. The present study confirms the relative low infection prevalence in field collected Culicoides and the strict relationship between the high abundance of C. imicola and outbreaks of AHSV.

The emergence of Schmallenberg virus across Culicoides communities and ecosystems in Europe.

Schmallenberg virus (SBV), a novel arboviral pathogen, has emerged and spread across Europe since 2011 inflicting congenital deformities in the offspring of infected adult ruminants. Several species of Culicoides biting midges (Diptera: Ceratopogonidae) have been implicated in the transmission of SBV through studies conducted in northern Europe. In this study Culicoides from SBV outbreak areas of mainland France and Italy (Sardinia) were screened for viral RNA. The role of both C. obsoletus and the Obsoletus complex (C. obsoletus and C. scoticus) in transmission of SBV were confirmed in France and SBV was also discovered in a pool of C. nubeculosus for the first time, implicating this species as a potential vector. While collections in Sardinia were dominated by C. imicola, only relatively small quantities of SBV RNA were detected in pools of this species and conclusive evidence of its potential role in transmission is required. In addition to these field-based studies, infection rates in colony-derived individuals of C. nubeculosus and field-collected C. scoticus are also examined in the laboratory. Rates of infection in C. nubeculosus were low, confirming previous studies, while preliminary examination of C. scoticus demonstrated that while this species can replicate SBV to a potentially transmissible level, further work is required to fully define comparative competence between species in the region. Finally, the oral competence for SBV of two abundant and widespread mosquito vector species in the laboratory is assessed. Neither Aedes albopictus nor Culex pipiens were demonstrated to replicate SBV to transmissible levels and appear unlikely to play a major role in transmission. Other vector competence data produced from studies across Europe to date is then comprehensively reviewed and compared with that generated previously for bluetongue virus.

First cases of Schmallenberg virus in Italy: surveillance strategies.

Following the first report of Schmallenberg virus (SBV) in the brain of a dystocic goat foetus in 2012 in Northern Italy, immediate response actions were adopted to avoid the virus circulation. The brain tested positive by 2 different one-step real-time RT-PCR protocols; these results were also confirmed by partial sequencing of the viral genome. At that time this was the first detection of the new Orthobunyavirus genus within the Bunyaviridae family in Italy. An epidemiological investigation in the involved farm was carried out in collaboration with the CESME - National Reference Centre for the study and verification of Foreign Animal Diseases (Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise 'G. Caporale', Italy). Epidemiological information on the flock was provided and analysed, whole blood and serum samples were also collected from all animals in the farm for both virological and serological investigations. All blood samples tested negative for SBV, whereas serological positive results were obtained by virus-neutralization (VN). Epidemiological investigations indicated the possible virus circulation in the area. The subsequent surveillance actions were mainly based on the standardization and reenforcement of passive surveillance protocols, a risk-based serological surveillance programme through VN and an entomological surveillance programme in the involved geographical areas were also put in place. Eventually SBV local circulation was confirmed by real time RT-PCR in 6 Culicoides pools, collected between September and November 2011 in 3 farms in the surroundings of the area of SBV outbreak.