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Abstract Background Colonization of large part of Europe by the Asian tiger mosquito Aedes albopictus is causing autochthonous transmission of chikungunya and dengue exotic arboviruses. While pyrethroids are recommended only to reduce/limit transmission, they are widely implemented to reduce biting nuisance and to control agricultural pests, increasing the risk of insurgence of resistance mechanisms. Worryingly, pyrethroid resistance (with mortality

Abstract Background Sand flies (Diptera: Psychodidae) are medically important vectors of human and veterinary disease-causing agents. Among these, the genus Leishmania (Kinetoplastida: Trypanosomatidae), and phleboviruses are of utmost importance. Despite such significance, updated information about sand fly fauna is missing for Balkan countries where both sand flies and autochtonous leishmaniases are historically present and recently re-emerging. Therefore, a review of historical data on sand fly species composition and distribution in the region was followed by a large-scale entomological survey in eight Balkan countries to provide a recent update on local sand fly fauna. Methods The literature search involved the period 1910–2019. The entomological survey was conducted at 1189 sampling stations in eight countries (Bulgaria, Bosnia and Herzegovina, Croatia, Kosovo, Montenegro, North Macedonia, Serbia and Slovenia), covering 49 settlements and 358 sampling sites between June and October in the years 2014 and 2016, accumulating 130 sampling days. We performed a total of 1189 trapping nights at these stations using two types of traps (light and CO2 attraction traps) in each location. Sampling was performed with a minimal duration of 6 (Montenegro) and a maximal of 47 days (Serbia) between 0–1000 m.a.s.l. Collected sand flies were morphologically identified. Results In total, 8490 sand fly specimens were collected. Morphological identification showed presence of 14 species belonging to genera Phlebotomus and Sergentomyia. Historical data were critically reviewed and updated with our recent findings. Six species were identified in Bosnia and Herzegovina (2 new records), 5 in Montenegro (2 new records), 5 in Croatia (2 new records), 9 in Bulgaria (5 new records), 11 in North Macedonia (1 new record), 10 in Serbia (no new records), 9 in Kosovo (3 new records) and 4 in Slovenia (no new records). Conclusions This study presents results of the first integrated sand fly fauna survey of such scale for the Balkan region, providing first data on sand fly populations for four countries in the study area and presenting new species records for six countries and updated species lists for all surveyed countries. Our findings demonstrate presence of proven and suspected vectors of several Leishmania species.

Abstract Background Culex pipiens is the major vector of West Nile virus in Europe, and is causing frequent outbreaks throughout the southern part of the continent. Proper empirical modelling of the population dynamics of this species can help in understanding West Nile virus epidemiology, optimizing vector surveillance and mosquito control efforts. But modelling results may differ from place to place. In this study we look at which type of models and weather variables can be consistently used across different locations. Methods Weekly mosquito trap collections from eight functional units located in France, Greece, Italy and Serbia for several years were combined. Additionally, rainfall, relative humidity and temperature were recorded. Correlations between lagged weather conditions and Cx. pipiens dynamics were analysed. Also seasonal autoregressive integrated moving-average (SARIMA) models were fitted to describe the temporal dynamics of Cx. pipiens and to check whether the weather variables could improve these models. Results Correlations were strongest between mean temperatures at short time lags, followed by relative humidity, most likely due to collinearity. Precipitation alone had weak correlations and inconsistent patterns across sites. SARIMA models could also make reasonable predictions, especially when longer time series of Cx. pipiens observations are available. Conclusions Average temperature was a consistently good predictor across sites. When only short time series (~ 4 years) are available, SARIMAs can provide better statistical descriptions of Cx. pipiens dynamics, without the need for further weather variables. This suggests that density dependence is also an important determinant of Cx. pipiens dynamics.

Abstract Background Recently, Balkan virus (BALKV, family Phenuiviridae, genus Phlebovirus) was discovered in sand flies collected in Albania and genetically characterised as a member of the Sandfly fever Naples species complex. To gain knowledge concerning the geographical area where exposure to BALKV exists, entomological surveys were conducted in 2014 and 2015, in Croatia, Bosnia and Herzegovina (BH), Kosovo, Republic of Macedonia and Serbia. Results A total of 2830 sand flies were trapped during 2014 and 2015 campaigns, and organised as 263 pools. BALKV RNA was detected in four pools from Croatia and in one pool from BH. Phylogenetic relationships were examined using sequences in the S and L RNA segments. Study of the diversity between BALKV sequences from Albania, Croatia and BH showed that Albanian sequences were the most divergent (9–11% [NP]) from the others and that Croatian and BH sequences were grouped (0.9–5.4% [NP]; 0.7–5% [L]). The sand fly infection rate of BALKV was 0.26% in BH and 0.27% in Croatia. Identification of the species content of pools using cox1 and cytb partial regions showed that the five BALKV positive pools contained Phlebotomus neglectus DNA; in four pools, P neglectus was the unique species, whereas P. tobbi DNA was also detected in one pool. Conclusions We report here (i) the first direct evidence that the Balkan virus initially described in coastal Albania has a much wider dissemination area than originally believed, (ii) two real-time RT-PCR assays that may be useful for further screening of patients presenting with fever of unknown origin that may be caused by Balkan virus infection, (iii) entomological results suggesting that Balkan virus is likely transmitted by Phlebotomus neglectus, and possibly other sand fly species of the subgenus Larroussius. So far, BALKV has been detected only in sand flies. Whether BALKV can cause disease in humans is unknown and remains to be investigated.

The epidemiology of West Nile (WNV) and Usutu virus (USUV) has changed dramatically over the past two decades. Since 1999, there have been regular reports of WNV outbreaks and the virus has expanded its area of circulation in many Southern European countries. After emerging in Italy in 1996, USUV has spread to other countries causing mortality in several bird species. In 2009, USUV seroconversion in horses was reported in Italy. Co-circulation of both viruses was detected in humans, horses and birds. The main vector of WNV and USUV in Europe is Culex pipiens, however, both viruses were found in native Culex mosquito species (Cx. modestus, Cx. perexiguus). Experimental competence to transmit the WNV was also proven for native and invasive mosquitoes of Aedes and Culex genera (Ae. albopictus, Ae. detritus, Cx. torrentium). Recently, Ae. albopictus and Ae. japonicus naturally-infected with USUV were reported. While neuroinvasive human WNV infections are well-documented, USUV infections are sporadically detected. However, there is increasing evidence of a role of USUV in human disease. Seroepidemiological studies showed that USUV circulation is more common than WNV in some endemic regions. Recent data showed that WNV strains detected in humans, horses, birds, and mosquitoes mainly belong to lineage 2. In addition to European USUV lineages, some reports indicate the presence of African USUV lineages as well. The trends in WNV/USUV range and vector expansion are likely to continue in future years. This mini-review provides an update on the epidemiology of WNV and USUV infections in Southern Europe within a multidisciplinary “One Health” context.

Dirofilaria immitis and D. repens are the two most widespread and important species of mosquito-borne nematodes, posing a significant threat to veterinary health and particularly affecting canines and felines. While D. immitis causes cardiopulmonary dirofilariasis, D. repens causes subcutaneous infections in dogs and other carnivores. Despite the extensive knowledge on these parasites, little is known about their natural vectors in Serbia. The parasite Setaria tundra, known to infect deer, has not yet been detected in Serbia but has been documented in neighboring countries. Thus, the aim of this study was to (i) further map out Dirofilaria sp. hotspots in the Vojvodina Province and detect S. tundra for the first time, (ii) detect positive mosquito species that can provide insights into how the nematodes spread in Serbia, and (iii) analyze the blood-fed female mosquitoes of species found to be infected, in order to identify the potential source of parasite infection. A total of 2902 female mosquitoes were collected across 73 locations during 2021 and 2022. Molecular biology methods, based on conventional PCR, were used to analyze non-blood-fed (2521 specimens) and blood-fed (381 specimens) female mosquitos, in order to detect filarial nematode presence and identify blood-meal sources, respectively. When the parasite genome was detected, the amplicon (cox1 gene, 650 bp fragment) was sent for Sanger sequencing, further confirming the presence of nematodes and species assignation. D. immitis was detected in three Culex pipiens mosquitoes collected in Zrenjanin (August 2021) and Glogonj and Svetozar Miletić (both in July 2021). Additionally, Setaria tundra was detected in Aedes vexans collected in Iđoš (mid-August 2021) and Aedes caspius, which was collected in Mali Iđoš (end of July 2021). This work identifies two new locations where D. immitis occurs in Vojvodina, and is the first report of S. tundra in Serbian territory. Blood-meal analysis provided insights into the preferences of mosquitoes that were positive for Dirofilaria sp. and S. tundra.

Urban areas are often populated by specific species of insects, some colorful and appealing, such as ladybugs and butterflies, and others irritating as nuisance bitters or as vectors of pathogens of public health importance. Mosquitoes in urban areas often utilize habitats adjacent to human residences, while phytophagous insect species such as stink bugs often colonize ornamental plants and utilize human-made structures including houses as overwintering shelters. This article discusses the early detection and the current distribution of two invasive mosquito species, Aedes albopictus Skuse 1894 and Ae. japonicus (Theobald 1901), in Serbia, introduced in 2009 and 2018, respectively. From the first findings until today, regular monitoring has been carried out and the establishment of both species in the newly invaded areas has been confirmed. Both species can become nuisance species, especially at high population densities, but more importantly, they are capable of transmitting a wide variety of arboviruses of public health importance. This article also discusses two invasive stink bug species Halyomorpha halys Stål 1855 and Nezara viridula Linnaeus 1758, introduced in Serbia in 2015 and 2008, respectively. These two stink bug species have also been monitored, and the establishment of their populations in the country has been confirmed. Both species have caused damage to a wide range of crops and ornamental plants and sometimes become nuisance pests in urban areas.

The pathogen transmitting Aedes albopictus mosquito is spreading rapidly in Europe, putting millions of humans and animals at risk. This species is well-established in Albania since its first detection in 1979. The sterile insect technique (SIT) is increasingly gaining momentum worldwide as a component of area-wide-integrated pest management. However, estimating how the sterile males will perform in the field and the size of target populations is crucial for better decision-making, designing and elaborating appropriate SIT pilot trials, and subsequent large-scale release strategies. A mark-release-recapture (MRR) experiment was carried out in Albania within a highly urbanized area in the city of Tirana. The radio-sterilized adults of Ae. albopictus Albania strain males were transported by plane from Centro Agricoltura Ambiente (CAA) mass-production facility (Bologna, Italy), where they were reared. In Albania, sterile males were sugar-fed, marked with fluorescent powder, and released. The aim of this study was to estimate, under field conditions, their dispersal capacity, probability of daily survival and competitiveness, and the size of the target population. In addition, two adult mosquito collection methods were also evaluated: BG-Sentinel traps baited with BG-Lure and CO2, (BGS) versus human landing catch (HLC). The overall recapture rates did not differ significantly between the two methods (2.36% and 1.57% of the total male released were recaptured respectively by BGS and HLC), suggesting a similar trapping efficiency under these conditions. Sterile males traveled a mean distance of 93.85 ± 42.58 m and dispersed up to 258 m. Moreover, they were observed living in the field up to 15 days after release with an average life expectancy of 4.26 ± 0.80 days. Whether mosquitoes were marked with green, blue, yellow, or pink, released at 3.00 p.m. or 6.00 p.m., there was no significant difference in the recapture, dispersal, and survival rates in the field. The Fried competitiveness index was estimated at 0.28. This mark-release-recapture study provided important data for better decision-making and planning before moving to pilot SIT trials in Albania. Moreover, it also showed that both BG-traps and HLC were successful in monitoring adult mosquitoes and provided similar estimations of the main entomological parameters needed.

Human and animal vector-borne diseases, particularly mosquito-borne diseases, are emerging or re-emerging worldwide. Six Aedes invasive mosquito (AIM) species were introduced to Europe since the 1970s: Aedes aegypti, Ae. albopictus, Ae. japonicus, Ae. koreicus, Ae. atropalpus and Ae. triseriatus. Here, we report the results of AIMSurv2020, the first pan-European surveillance effort for AIMs. Implemented by 42 volunteer teams from 24 countries. And presented in the form of a dataset named “AIMSurv Aedes Invasive Mosquito species harmonized surveillance in Europe. AIM-COST Action. Project ID: CA17108”. AIMSurv2020 harmonizes field surveillance methodologies for sampling different AIMs life stages, frequency and minimum length of sampling period, and data reporting. Data include minimum requirements for sample types and recommended requirements for those teams with more resources. Data are published as a Darwin Core archive in the Global Biodiversity Information Facility- Spain, comprising a core file with 19,130 records (EventID) and an occurrences file with 19,743 records (OccurrenceID). AIM species recorded in AIMSurv2020 were Ae. albopictus, Ae. japonicus and Ae. koreicus, as well as native mosquito species.

Abstract 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.

The Asian tiger mosquito, Aedes albopictus, is an invasive mosquito species that is considered a potential vector of about 22 arboviruses, among which dengue, chikungunya and Zika. Here we report the first record of Aedes albopictus in the territory of the Republic of Kosovo. The first finding, in July 2020, was driven by a photo of an adult mosquito published by a citizen in social media. The subsequent field investigation in July 2020 confirmed the presence of adult mosquitoes by human landing catch and collection of eggs in ovitraps at the village Zhur. Monitoring was performed for 7 weeks with ovitraps and BG-Sentinel adult traps at 36 sampling stations from 7 localities, in the Southern part of Kosovo, on the border with Albania. Fifty-two out of 81 ovitrap samples resulted positive for the presence of Ae. albopictus. A total of 2,711 eggs are collected in 22 out 36 stations and a total of 78 adults from 4 out 4 stations with BG Sentinel trap and 15 adults with handled electric aspirator. Our finding shows that the tiger mosquito is expanding its geographical range in the Balkans, southeastern Europe.

Abstract Invasive species can encounter environments different from their source populations, which may trigger rapid adaptive changes after introduction (niche shift hypothesis). To test this hypothesis, we investigated whether postintroduction evolution is correlated with contrasting environmental conditions between the European invasive and source ranges in the Asian tiger mosquito Aedes albopictus. The comparison of environmental niches occupied in European and source population ranges revealed more than 96% overlap between invasive and source niches, supporting niche conservatism. However, we found evidence for postintroduction genetic evolution by reanalyzing a published ddRADseq genomic dataset from 90 European invasive populations using genotype–environment association (GEA) methods and generalized dissimilarity modeling (GDM). Three loci, among which a putative heat‐shock protein, exhibited significant allelic turnover along the gradient of winter precipitation that could be associated with ongoing range expansion. Wing morphometric traits weakly correlated with environmental gradients within Europe, but wing size differed between invasive and source populations located in different climatic areas. Niche similarities between source and invasive ranges might have facilitated the establishment of populations. Nonetheless, we found evidence for environmental‐induced adaptive changes after introduction. The ability to rapidly evolve observed in invasive populations (genetic shift) together with a large proportion of unfilled potential suitable areas (80%) pave the way to further spread of Ae. albopictus in Europe.

Abstract Background Culicoides (Diptera: Ceratopogonidae) is a genus of small biting midges (also known as “no-see ums”) that currently includes 1368 described species. They are proven or suspected vectors for important pathogens affecting animals such as bluetongue virus (BTV) and Schmallenberg virus (SBV). Currently little information is available on the species of Culicoides present in Serbia. Thus, the aim of this study was to examine species diversity, host preference and the presence of BTV and SBV RNA in Culicoides from the Stara Planina Nature Park in south-eastern Serbia. Results In total 19,887 individual Culicoides were collected during three nights of trapping at two farm sites and pooled into six groups (Obsoletus group, Pulicaris group, “Others” group and further each group according to the blood-feeding status to freshly engorged and non-engorged). Species identification was done on subsamples of 592 individual Culicoides specimens by morphological and molecular methods (MALDI-TOF mass spectrometry and PCR/sequencing). At least 22 Culicoides species were detected. Four animal species (cow, sheep, goat and common blackbird) as well as humans were identified as hosts of Culicoides biting midges. The screening of 8291 Culicoides specimens in 99 pools for the presence of BTV and SBV RNA by reverse-transcription quantitative PCR were negative. Conclusions The biodiversity of Culicoides species in the natural reserve Stara Planina was high with at least 22 species present. The presence of C. imicola Kieffer was not recorded in this area. Culicoides showed opportunistic feeding behaviour as determined by host preference. The absence of SBV and BTV viral RNA correlates with the absence of clinical disease in the field during the time of sampling. These data are the direct outcome of a training programme within the Institutional Partnership Project “AMSAR: Arbovirus monitoring, research and surveillance-capacity building on mosquitoes and biting midges” funded by the programme SCOPES of the Swiss National Science Foundation.

Background: Aedes albopictus is currently the most widespread invasive mosquito species in the world. It has paramount medical importance since females are efficient vectors of important viruses affecting humans. The development of alternative control strategies to complement control measures has become an imperative and involves the Sterile Insect Technique (SIT). Research to improve the productivity of mass-rearing, as well as the quality of mass-reared males is of essential importance for the success of SIT. Methods: This study compared the influence of three differently sized cages for Ae. albopictus mass-rearing on wing length, adult survival and egg production during 20 generations of colonization. Plexiglas cages of 40x40x40 cm (C1), 100 × 20 × 100 cm (C2) and 100 × 65 × 100 cm (C3) were loaded with equal adult density, and sex ratio of 1:1. An open source image processing and analysis programme (ImageJ) was used for the wing measurement and egg counting. Results: In all tested cages, we identified two periods separated by the generation showing the minimum value of each considered parameter (wing length, adult survival and egg production). The wing length and adult survival passed through the phases of initial decrease to about intermediate colonization time, and increased afterwards. Fecundity was steady during the first period and increased in the second one. Cage C1 demonstrated not only the best values for all parameters but also the smallest decrease in the initial phase. Recovering of the caged mosquitoes in the second half of the study was higher in cages C1 and C2, than in C3. Conclusions: C1 provided the least negative selection pressure on wing length, adult survival and egg production for reared Ae. albopictus. Anyhow, since maximising mosquito density by exploiting the minimum space is a priority in mosquito mass-rearing, C2 might be a better choice for better fitting the space of mass-rearing rooms.

Aedes (Stegomyia) albopictus (Skuse, 1895) is an invasive important medical and veterinary pest species. The sterile insect technique (SIT) involves the mass rearing of males, and their sterilization and release into the habitat to compete with wild males. Our research objective was to compare the effectiveness of three larval diet recipes (IAEA-BY, BCWPRL, and MIX-14) in the laboratory rearing of Ae. albopictus males to evaluate the available economical feeding alternatives. The separation of sexes was done in the pupal stage by sieving. Reared males were tested for flight capacity and longevity. The application of the BCWPRL diet resulted in a higher portion of sieved male pupae than females, but the development of males was the slowest, and the number of obtained males (pupae and adults) was lower compared to the other two diets. The adult mean survival time was the highest in males fed with MIX-14 and the lowest in males fed with IAEA-BY. Males fed by IAEA-BY also demonstrated higher initial mortality in the adult stage. The diets BCWPRL and MIX-14 are economically more convenient than IAEA-BY (2.28 and 5.30 times cheaper, respectively). The cheapest diet, MIX-14, might represent a candidate for replacing the effective but still expensive IAEA-BY larval diet, providing lower costs of sterile male production.

Motivated by the One Health paradigm, we found the expected changes in temperature and UV radiation (UVR) to be a common trigger for enhancing the risk that viruses, vectors, and diseases pose to human and animal health. We compared data from the mosquito field collections and medical studies with regional climate model projections to examine the impact of climate change on the spreading of one malaria vector, the circulation of West Nile virus (WNV), and the incidence of melanoma. We analysed data obtained from ten selected years of standardised mosquito vector sampling with 219 unique location-year combinations, and 10 years of melanoma incidence. Trends in the observed data were compared to the climatic variables obtained by the coupled regional Eta Belgrade University and Princeton Ocean Model for the period 1961-2015 using the A1B scenario, and the expected changes up to 2030 were presented. Spreading and relative abundance of Anopheles hyrcanus was positively correlated with the trend of the mean annual temperature. We anticipated a nearly twofold increase in the number of invaded sites up to 2030. The frequency of WNV detections in Culex pipiens was significantly correlated to overwintering temperature averages and seasonal relative humidity at the sampling sites. Regression model projects a twofold increase in the incidence of WNV positive Cx. pipiens for a rise of 0.5°C in overwintering TOctober-April temperatures. The projected increase of 56% in the number of days with Tmax ≥ 30°C (Hot Days-HD) and UVR doses (up to 1.2%) corresponds to an increasing trend in melanoma incidence. Simulations of the Pannonian countries climate anticipate warmer and drier conditions with possible dominance of temperature and number of HD over other ecological factors. These signal the importance of monitoring the changes to the preparedness of mitigating the risk of vector-borne diseases and melanoma.

The Asian bush mosquito, Aedes japonicus japonicus (Theobald, 1901), a potential vector of several pathogens, has recently established in North America and Central Europe. In 2013, it was found on the Slovenian-Croatian border, and during the following years, it emerged in more and more counties of northwestern Croatia. Surveillance of Ae. j. japonicus and other invasive mosquito species was subsequently extended both spatially and temporally in Croatia and neighbouring Bosnia and Herzegovina and Serbia. Mosquito collections were conducted in 2017 and 2018, based on adult trapping through dry ice-baited CDC traps and BG-Lure-baited BG-Sentinel traps, larval sampling through dippers and nets, and ovitrapping. Aedes j. japonicus specimens from collected samples were subjected to population genetic analysis by comparing microsatellite signatures and nad4 DNA sequences between sampled locations and with data previously obtained from more western European distribution areas. Aedes j. japonicus immature stages were found at 19 sites in Croatia, two sites in Bosnia and Herzegovina and one site in Serbia. In Croatia, four new counties were found colonised, two in the east and two in the south of the previously known distribution area. A spread of 250 km could thus be documented within five years. The findings in Bosnia and Herzegovina and Serbia represent the first records of Ae. j. japonicus in these countries. Genetic analysis suggests at least two introduction events into the surveyed area. Among the locations analysed, Orahovica can be considered a genetic border. The individuals collected west of this point were found to be similar to samples previously collected in the border regions of Southeast Germany/Austria and Austria/Slovenia, while the specimens from more eastern Croatian localities, together with those from Bosnia and Herzegovina and Serbia, were genetically different and could not be assigned to a probable origin. Thus, introduction from Central Europe, possibly by vehicular traffic, into the study area is likely, but other origins, transportation routes and modes of entry appear to contribute. Further dispersal of Ae. j. japonicus to other parts of southeastern Europe is anticipated.

Genetic based mosquito control methods have been gaining ground in recent years for their potential to achieve effective suppression or replacement of vector populations without hampering environments or causing any public health risk. These methods require the mass rearing of the target species in large facilities sized to produce millions of sterile males, as already well established for a number of insects of agricultural importance. Assessing the performance of released males in Sterile Insect Technique (SIT) control programs is of the utmost importance for the success of the operation. Besides the negative effects of mass rearing and sterilization, the handling of sterilized insects and shipment to distant areas may also negatively impact the quality of sterilized males. The aim of the current study was to design and executive quality control (QC) tests for sterilized Aedes albopictus (Asian tiger mosquito) males delivered by air shipment from the mass production facility located in Italy to Greece and Montenegro field release sites. Mass reared mosquito strains were based on biological materials received from Italy, Greece and Montenegro. Tests conducted at the mass rearing facility before transportation revealed a rather high residual female contamination following mechanical sex separation (approximately 1.5% females, regardless of the mosquito strain). Irradiated males of all three mosquito strains induced high levels of sterility to females. Shipment lasting approximately 24 h resulted in approximately 15% mortality, while when shipment lasted nearly two days this increased to almost 40%. The flight ability of sterilized males following one day transportation time was satisfactory (over 60%). The response of sterile males to food and water starvation was comparable and slightly lower than that of wild non-transported males. Longevity of sterile males was shorter than that of wild counterparts and it seems it was not affected by mating to wild females. Both mating propensity and mating competitiveness for wild virgin females was higher for the wild, control males compared to the sterile, transported ones. Overall, the performance of sterile male Ae. albopictus delivered from the mass rearing facility of Italy to Greece in approximately 24 h was satisfactory. Transportation lasting two days or longer incurred detrimental effects on males, which called into question the outcome of the SIT release programs. In conclusion, our results demonstrate the need of quality control procedures, especially when sterile male production facilities are not near to the releasing point. Transportation could be a serious drawback for the implementation of Sterile Insect Releases and, consequently, it is important to establish an efficient and fast transportation of sterilized males in advance.

Abstract Dirofilaria repens is a nematode affecting domestic and wild canids, transmitted by several species of mosquitoes. It usually causes a non-pathogenic subcutaneous infection in dogs and is the principal agent of human dirofilariosis in the Old World. In the last decades, D. repens has increased in prevalence in areas where it has already been reported and its distribution range has expanded into new areas of Europe, representing a paradigmatic example of an emergent pathogen. Despite its emergence and zoonotic impact, D. repens has received less attention by scientists compared to Dirofilaria immitis. In this review we report the recent advances of D. repens infection in dogs and humans, and transmission by vectors, and discuss possible factors that influence the spread and increase of this zoonotic parasite in Europe. There is evidence that D. repens has spread faster than D. immitis from the endemic areas of southern Europe to northern Europe. Climate change affecting mosquito vectors and the facilitation of pet travel seem to have contributed to this expansion; however, in the authors’ opinion, the major factor is likely the rate of undiagnosed dogs continuing to perpetuate the life-cycle of D. repens. Many infected dogs remain undetected due to the subclinical nature of the disease, the lack of rapid and reliable diagnostic tools and the poor knowledge and still low awareness of D. repens in non-endemic areas. Improved diagnostic tools are warranted to bring D. repens diagnosis to the state of D. immitis diagnosis, as well as improved screening of imported dogs and promotion of preventative measures among veterinarians and dog owners. For vector-borne diseases involving pets, veterinarians play a significant role in prevention and should be more aware of their responsibility in reducing the impact of the zoonotic agents. In addition, they should enhance multisectorial collaboration with medical entomologists and the public health experts, under the concept and the actions of One Health-One Medicine.

Abstract Background Identification of vectors is of prime importance in the field of medical entomology for both operational and research purposes. An external quality assessment of mosquito identification capacities was carried out within the MediLabSecure Network, which is composed of laboratories located in 19 countries close to the European Union around the Mediterranean and Black seas. Methods A set of blind samples consisting of 7 or 8 adult mosquitoes and 4 larvae was given to each participant laboratory. In all, 138 adult mosquitoes and 76 larvae of different species were distributed for genus and species identification. Results All identifications were exclusively morphology based. Overall, 81% of identifications were correct at the genus level, 64% at the species level. The results were highly varied among the 19 participating laboratories. The levels of correct identifications were: 100% (three laboratories), 90–95% (four laboratories), 50–75% (six laboratories) and < 50% (six laboratories). Conclusions This evaluation showed the need to maintain efforts in capacity building and quality control in the field of medical entomology and, more specifically, in the morphological identification of the Culicidae.

Abstract Background The prime significance of species belonging to the genus Culicoides Latreille, 1809 (Diptera: Ceratopogonidae) is their ability to transmit viruses such as bluetongue virus (BTV) to wild and domestic ruminants. Prior to 1998, BTV was considered exotic in Europe, but according to recent history of its outbreaks, it has become endemic in southern and eastern European countries circulating beyond its expected historical limits, into the Balkan region. The wind-borne long-distance dispersal of Culicoides spp. over water bodies and local spreading between farms emphasize the necessity of filling in the information gaps regarding vector species distribution. In most Balkan countries, data on Culicoides fauna and species distribution are lacking, or information is old and scarce. Results During this study, 8586 specimens belonging to 41 species were collected. We present the first faunistic data on Culicoides species in the former Yugoslav Republic of Macedonia (FYROM), Kosovo, Montenegro and Serbia. For other countries (Bosnia and Herzegovina, Bulgaria and Croatia), all historical records were compiled for the first time and then expanded with our findings to various extents. In all countries, confirmed or suspected BTV vector species belonging to the subgenera Avaritia and Culicoides were collected. The total number of species sampled during our field collections was 20 in Bosnia and Herzegovina (15 new records), 10 in Bulgaria (2 new records), 10 in Croatia (5 new records), 13 in FYROM, 9 in Kosovo, 15 in Montenegro, and 28 in Serbia. Of these, 14 species were registered for the first time in this part of the Balkans. Conclusions This paper provides the first data about Culicoides fauna in FYROM, Kosovo, Montenegro and Serbia, as well as new records and an update on the checklists for Bosnia and Herzegovina, Bulgaria and Croatia. These findings provide preliminary insights into the routes of BTV introduction and spreading within the Balkans, and present a valuable contribution to further research related to Culicoides-borne diseases in Europe.

The results of the Serbian national integrated West Nile virus (WNV) surveillance program conducted in 2018 and funded by the Serbian Veterinary Directorate are presented. The WNV surveillance program encompassed the entire territory of Serbia and was conducted by the veterinary service in collaboration with entomologists and ornithologists. The objective of the program was early detection of WNV circulation in the environment and timely reporting to the public health service and local authorities to increase clinical and mosquito control preparedness. The program was based on the detection of WNV presence in wild birds (natural hosts) and mosquitoes (virus vectors) and on serological testing of sentinel horses (WNV-specific IgM antibodies). The season 2018 was confirmed to be the season of the most intensive WNV circulation with the highest number and severity of human cases in Serbia ever reported. The most intense WNV circulation was observed in the northern and central parts of Serbia including Vojvodina Province, the Belgrade City area, and surrounding districts, where most positive samples were detected among sentinel animals, mosquitoes and wild birds. The majority of human cases were preceded by the detection of WNV circulation during the surveillance. The WNV surveillance program in 2018 showed satisfactory results in the capacity to indicate the spatial distribution of the risk for humans and sensitivity to early detection of WNV circulation in the environment.

Abstract Background Leishmaniasis in Serbia was an endemic disease, and is considered to be eradicated for more than 40 years. In the past decade sporadic cases of canine leishmaniasis started to emerge for the first time in Vojvodina Province (previously non-endemic region of Serbia). Reports of introduced, and later on autochthonous cases of leishmaniasis alerted the possibility of disease emergence. The aim of this study was to bridge more than a half a century wide gap in entomological surveillance of sand fly vectors in Vojvodina, as well as to verify the presence of the vector species that could support Leishmania spp. circulation. Results During the period 2013–2015, a total of 136 sand flies were collected from 48 of 80 surveyed locations. Four sand fly species of the genus Phlebotomus were detected: P. papatasi, P. perfiliewi, P. mascittii and P. neglectus. Detection of P. mascittii represents the first record of this species for the sand fly fauna in Vojvodina and in Serbia. All female specimens (n = 80) were tested for Leishmania spp. DNA, and three blood-fed P. papatasi specimens were positive (4%). One positive DNA sample was successfully amplified by ITS1 nPCR. The RFLP analysis of the resulting 350 bp fragment showed a typical pattern of L. infantum, and the ITS1 partial sequence blasted in GenBank confirmed 100% identity with L. infantum and L. donovani complex sequences. This result represents the first record of both Leishmania spp. and L. infantum DNA from sand flies in Vojvodina, and in Serbia. Conclusions Presence of autochthonous canine leishmaniasis cases, records of Phlebotomus (Larroussius) species proven vectors of L. infantum (P. perfiliewi and P. neglectus) and detection of L. infantum DNA from wild caught (non-competent) vectors, prove that L. infantum is present in Vojvodina and indicates a probable circulation in the region.

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

Toxoplasma gondii is a ubiquitous coccidian protozoan that can infect a wide range of warm-blooded animals, including humans. For immunocompetent humans it is a well-adapted parasite that usually causes asymptomatic infection. However, in congenitally infected infants and immunocompromised patients it can cause a serious life threatening disease. The aim of this study was to determine the levels of anti-toxoplasma antibodies among residents of the South Backa District, Serbia. During the period from January 2014 to December 2018, sera from 11,288 persons from South Backa District were tested on the presence of IgM and IgG antibodies against Toxoplasma gondii, using ELISA test (Euroimmun, Lübeck, Germany). The testing was performed on the automatic device Euroimmun Analyzer I-2P. The avidity of IgG WNV antibodies was determined for IgG positive sera using commercial avidity test (Euroimmun, Lübeck, Germany). In total, out of 11,288 patients who were tested for toxoplasma antibodies, the results were positive for 2,513 (22.26%). In 2014 seropositivity for Toxoplasma gondii was 25.78% (464/1800), in 2015 it was 23.30% (400/1717), in 2016 it amounted 20.99% (474/2258), in 2017 it was 21.47% (529/2464) and in 2018 seropositivity was 20.96% (639/3049). Seropositivity of 26.53% (390/1470) was found in males and it amounted 21.62% (2123/9818) in females. Possible recent infection within the last 12 months was found in 1.70% (192/11288) patients. Possible acute infection or false - positive IgM result was detected in 1.31% (148/11288) patients. Past infection was found in 2173/11288 (19.25%) patients. Equivocal results were found in 1.48% of samples (167/11288). The lowest frequency of anti-toxoplasma antibodies was detected in pre-school children 13.07% (97/742) and the highest in persons older than 65 years 60.18% (65/108). Comparing the results of the research done from 2014 to 2018 with the data from 1989, a significant decline of seroprevalence in general population and women of generative age was found.

Among various arthropod-borne viruses (arboviruses), the flaviviruses stand out with regard to their number, geographic distribution and importance in both human and veterinary medicine. West Nile virus is flavivirus, present endemic in many European countries as well as in Serbia where it circulates in horses, birds, humans and mosquitoes. Usutu virus (USUV) is flavivirus morphologically, antigenically, genetically and ecologically very similar to WNV, which circulates in neighbouring countries (Hungary, Croatia, Austria). The USUV is maintained in transmissible cycle between birds and mosquitoes mainly from the genus Culex. Mammals (humans, horses, rodents) can also be infected. Humans and other mammals are “dead end” hosts. Virus is isolated from numerous bird species. The USUV infections are asymptomatic in wild African birds, while for European birds, the virus is very virulent causing necrotizing focal encephalitis, degenerative myocarditis and fatal encephalitis. It is assumed that the virus was introduced into Europe by the migratory birds that have been infected by living or passing through endemic areas in Africa. First human cases were recorded in Italy in 2009. The genome of USUV was detected in cerebrospinal fluid of woman suffering from B-cell lymphoma with meningoencephalitis and in plasma of a female, who was subjected to a liver transplantation and subsequently developed fever, headache, and fulminant hepatitis which progressed to coma. In Austria, USUV infections were confirmed in people with a skin rash of unknown aetiology using plaque reduction neutralization test. The circulation of USUV has been proven in humans in many European countries by serological studies (Germany, Italy, and Croatia). Serological study performed in 2015 revealed that USUV is present in inhabitants of South Bačka District of Vojvodina, Serbia. Serum samples were tested using commercial ELISA IgG test for USUV and IgG antibodies against USUV were detected in 5% (4/88) of patients. The molecular investigation included 216 pools of mosquitoes collected in the period from June to September, in the South Bačka District. The USUV genome was detected in two mosquito pools (2/216). In human samples tested by RT PCR, USUV genome was not found.

BackgroundThe Mediterranean Basin is historically a hotspot for trade, transport, and migration. As a result, countries surrounding the Mediterranean Sea share common public health threats. Among them are vector-borne diseases, and in particular, mosquito-borne viral diseases are prime candidates as (re)emerging diseases and are likely to spread across the area. Improving preparedness and response capacities to these threats at the regional level is therefore a major issue. The implementation of entomological surveillance is, in particular, of utmost importance. Guidance in designing entomological surveillance systems is critical, and these systems may pursue different specific objectives depending on the disease. The purpose of the proposed review is to draw up guidelines for designing effective and sustainable entomological surveillance systems in order to improve preparedness and response. However, we make it clear that there is no universal surveillance system, so the thinking behind harmonisation is to define evidence-based standards in order to promote best practises, identify the most appropriate surveillance activities, and optimise the use of resources. Such guidance is aimed at policymakers and diverse stakeholders and is intended to be used as a framework for the implementation of entomological surveillance programmes. It will also be useful to collaborate and share information with health professionals involved in other areas of disease surveillance. Medical entomologists and vector control professionals will be able to refer to this report to advocate for tailored entomological surveillance strategies. The main threats targeted in this review are the vectors of dengue virus, chikungunya virus, Zika virus, West Nile virus, and Rift Valley fever virus. The vectors of all these arboviruses are mosquitoes.MethodsCurrent knowledge on vector surveillance in the Mediterranean area is reviewed. The analysis was carried out by a collaboration of the medical entomology experts in the region, all of whom belong to the MediLabSecure network, which is currently funded by the European Union and represents an international effort encompassing 19 countries in the Mediterranean and Black Sea region.FindingsRobust surveillance systems are required to address the globalisation of emerging arboviruses. The prevention and management of mosquito-borne viral diseases must be addressed in the prism of a One Health strategy that includes entomological surveillance as an integral part of the policy. Entomological surveillance systems should be designed according to the entomological and epidemiological context and must have well-defined objectives in order to effect a tailored and graduated response. We therefore rely on different scenarios according to different entomological and epidemiological contexts and set out detailed objectives of surveillance. The development of multidisciplinary networks involving both academics and public authorities will provide resources to address these health challenges by promoting good practises in surveillance (identification of surveillance aims, design of surveillance systems, data collection, dissemination of surveillance results, evaluation of surveillance activities) and through the sharing of effective knowledge and information. These networks will also contribute to capacity building and stronger collaborations between sectors at both the local and regional levels. Finally, concrete guidance is offered on the vector of the main arbovirus based on the current situation in the area.

The aim of this study is to summarize the data on the occurrence, presence and prevalence of some zoonotic flaviviruses, which have been actively circulating in the territory of the Republic of Serbia during past decade. The genus Flavivirus, family Flaviviridae,encompasses vector-transmitted viruses with zoonotic potential. The genus is comprised of more than 70 viruses transmitted to humans by mosquitoes and ticks. Some of those are associated only with human diseases (such as yellow fever and dengue fever), whereas others can cause diseases in both humans and animals. Animals are major reservoirs and primary hosts for the latter group. The virus transmission to other animal species or humans occurs via diverse vectors – mostly mosquitoes and ticks. A range of these virus species is widely distributed worldwide, especially in tropical and sub-tropical climatic zones. Some representatives of these species have only recently been identified at the territory of Republic of Serbia. This paper gives an evidence on the presence and distribution of West-Nile virus, tick-borne encephalitis (TBE) virus and Usutu virus that were conducted within the study in Serbia. The research pointed out the presence of recently detected West-Nile virus and Usutu virus as well as the current circulation of tick-borne encephalitis virus, for which only limited serological evidence on the presence of virus-specific antibodies in humans was available so far. In the aspect of public health, the full understanding of the importance of identification and prevalence of different flaviviruses still require further comprehensive entomologicalacarological, seroepidemiological, clinical and virological research.

Aedes albopictus, an invasive mosquito species, is currently well established in many European countries, posing high risks to human health. A preliminary trial using repetitive releases of irradiated sterile male mosquitoes was designed, implemented and evaluated for the first time in Greece. The main scope of this trial was to investigate the efficacy of sterile insect technique (SIT) on wild population egg hatch rate in Greece using mass-produced sterile male mosquitoes from another country (Italy). The study was conducted in Vravrona area, close to Athens International Airport (Attica Region). The number of eggs laid in ovitraps was similar in all intervention and control plots. However, a significant reduction in egg hatch rate was recorded in the SIT plot in comparison with both control plots starting two weeks after the first release. This trial validates the logistics (transportation, releases handling and monitoring) as a major step towards implementing efficient, environmentally safe control approaches as an additional tool against the invasive Aedes species in Greece and more widely in Europe.

West Nile virus (WNV) is arbovirus distributed all around the world. In humans, 80% of infection cases are asymptomatic. In 20% of infected people, a febrile self-limiting illness is reported. WNV has the potential for fatal neuroinvasive disease. In 1% of cases, the infection may result in neuroinvasive disease with permanent neurological consequences or death outcome. Neurological forms may vary presenting with encephalitis, meningitis, meningoencephalitis or acute flaccid paralysis. Outbreaks with neurological forms of WNV infection were recorded in different areas of Greece, Italy, Romania, Hungary and Serbia. During the period from 2013 to 2016, 114 samples of cerebrospinal fluid and 107 serum samples were taken from 114 patients suspected of WNV neuroinvasive disease (WNND). The presence of specific anti-WNV IgM and IgG antibodies in cerebrospinal fluid (CSF) and sera samples were tested by WNV IgM and IgG ELISA (Euroimmun, Germany). In addition, 48 samples of CSF or/and serum of people with suspected WNV infection were examined by commercial molecular tests - real time RT-PCR (WNV Real-TM, Sacace biotechnologies, Italy). The IgM antibodies against WNV were present in 25.4% (29/114) of CSF samples, and in 31.8% (34/107) of serum samples tested from 114 patients suspected of WNND. The IgG antibodies against WNV were detected in 3.5% (4/114) of CSF samples, and in 11.2% (12/107) of serum samples. The WNV RNA was detected by real time RT-PCR test in 7 out of 48 (14.6%) CSF or/and serum samples. In this study, detection of IgM antibodies in CSF is more frequent than detection of WNV RNA in CSF or serum samples. WNV RNA detection in CSF is confirmatory diagnostic test but has limited utility in the diagnosis of WNV neuroinvasive disease due to low viremia level at the time of clinical presentation of the disease. The limitations in the use of ELISA IgM test are linked to cross - reactivity among flaviviruses and long persistence of IgM antibodies in the serum and CSF.

Studies conducted during the past few years have confirmed active West Nile virus (WNV) circulation in Serbia. Based on these studies and the epidemiological situation, the Veterinary Directorate of the Ministry of Agriculture and Environmental Protection launched national WNV surveillance programmes in 2014 and 2015. The programmes encompassed the territory of Serbia and were conducted by the veterinary service in collaboration with entomologists and ornithologists. The objective of the programmes was early detection of WNV and timely reporting to the public health service and local authorities to increase both clinical and mosquito control preparedness. The WNV surveillance programmes were based on direct and indirect surveillance of the presence of WNV by the serological testing of initially seronegative sentinel horses and chickens as well as through viral detection in pooled mosquito and wild bird samples. The most intense WNV circulation was observed in all seven districts of Vojvodina Province (northern Serbia) and Belgrade City, where most of the positive samples were detected among sentinel animals, mosquitoes and wild birds. The West Nile virus surveillance programmes in 2014 and 2015 showed satisfactory results in their capacity to indicate the spatial distribution of the risk for humans and their sensitivity to early detect viral circulation at the enzootic level. Most of the human cases were preceded by the detection of WNV circulation as part of the surveillance programmes. According to the existing data, it can be reasonably assumed that WNV infection, now an endemic infection in Serbia, will continue to present a significant problem for the veterinary service and public health.

Abstract To enable a better understanding of the overwhelming alterations in the invasive mosquito species (IMS), methodical insight into the population and environmental factors that govern the IMS and pathogen adaptations are essential. There are numerous ways of estimating mosquito populations, and usually these describe developmental and life-history parameters. The key population parameters that should be considered during the surveillance of invasive mosquito species are: (1) population size and dynamics during the season, (2) longevity, (3) biting behaviour, and (4) dispersal capacity. Knowledge of these parameters coupled with vector competence may help to determine the vectorial capacity of IMS and basic disease reproduction number (R0) to support mosquito borne disease (MBD) risk assessment. Similarly, environmental factors include availability and type of larval breeding containers, climate change, environmental change, human population density, increased human travel and goods transport, changes in living, agricultural and farming habits (e.g. land use), and reduction of resources in the life cycle of mosquitoes by interventions (e.g. source reduction of aquatic habitats). Human population distributions, urbanisation, and human population movement are the key behavioural factors in most IMS-transmitted diseases. Anthropogenic issues are related to the global spread of MBD such as the introduction, reintroduction, circulation of IMS and increased exposure to humans from infected mosquito bites. This review addresses the population and environmental factors underlying the growing changes in IMS populations in Europe and confers the parameters selected by criteria of their applicability. In addition, overview of the commonly used and newly developed tools for their monitoring is provided.

Chikungunya virus is an Arbo virus belonging to the family Togaviridae. In urban areas, antropophilic Aedes aegypti and Aedes albopictus mosquitoes are vectors for virus transmission to human population. Chikungunya virus has attracted the professional and scientifi c public attention in 2013 causing a massive outbreak on the American continent. In Europe, autochthonous transmissions of Chikungunya virus infections have been recorded in Italy in 2007 as well as in France in 2010 and 2014. Usutu virus is a RNA virus from the family Flaviviridae. Th e virus circulates in a transmission cycle between wild birds and Culex mosquitoes. The virus has been detected in numerous bird species across Europe. Manifestations recorded in humans include meningoencephalitis and skin rash. First human cases in Europe were recorded in immunocompromised individuals in Italy in 2009. Spirochete Borrelia burgdorferi sensu lato is transmitted to humans by ticks and causes Lyme disease, a multisystemic disease with dermatological, neurological, cardiological or articular manifestations. Ninety three persons interviewed about risk factors for vector-borne infections were examined. The examination was performed using commercial ELISA IgG for Chikungunya and Usutu virus and ELISA IgM and IgG test for Borrelia burgdorferi in line with manufacturer’s instructions (Euroimmun, Germany). Out of 93 examined individuals Usutu virus specific IgG antibodies were identified in 7.5% (7/93) persons. Th e results of ELISA IgG test for Chikungunya virus were negative in the majority of tested samples, whereas 7.5% (7/93) of samples revealed borderline result. In 9.7% (9/93) participants, antibodies against Borrelia burgdorferi were detected only by ELISA IgM test. Recent infection with Borrelia burgdorferi was confi rmed in 2.15% (2/93) individuals, whereas IgG antibodies against Borrelia burgdorferi were detected in only one participant. Th e obtained results indicated that Usutu virus is active in the territory of South Bačka District contrary to Chikungunya virus as well as that Borrelia burgdorferi is an important pathogen in the investigated region.

Aedes albopictus has become established in many parts of Europe since its introduction at the end of the 20th century. It can vector a range of arboviruses, of which Chikungunya and Dengue are most significant for Europe. An analysis of the expected climate change and the related shift in Köppen zones for Montenegro and impact on the establishment of Ae. albopictus was conducted. Outputs of a mechanistic Aedes albopictus model were validated by 2245 presence/absence records collected from 237 different sites between 2001 and 2014. Finally, model-based sampling was designed and performed at 48 sites in 2015, in a previously unexplored northern part of Montenegro, and results were validated. The Eta Belgrade University (EBU)-Princeton Ocean Model (POM) regional climate model was used with the A2 emissions scenario for the 2001⁻2030 and 2071⁻2100 integration periods. The results point to a significant increase in suitability for the mosquito and a vertical shift to higher altitudes by the end of the century. The model showed excellent results with the area under the receiver operating characteristic curve (AUC) of 0.94. This study provides a tool for prioritizing surveillance efforts (model-based surveillance), especially when resources are limited. This is the first published analysis of Climate Change that incorporates observations from the national synoptic grid and the subsequent impact on Ae. albopictus in Montenegro.

Serological and virological examination of the presence of human and animal infection caused by West Nile Virus (WNV) as well as the presence of the virus in vectors, which has been conducted during the past few years, confi rmed an active virus circulation in the territory of the Republic of Serbia. Based on the obtained results and anticipated intense circulation of WNV, which poses substantial risks for both public and animal health in Serbia, and having in mind its crucial role in the protection of public health, Veterinary Directorate of the Ministry of Agriculture and Environmental Protection infront the Veterinary Service launched and funded the national WNV monitoring program starting from April 2014. The Program encompassed the entire territory of the Republic of Serbia and was conducted by scientific and specialized veterinary institutes and fi eld veterinary service in close collaboration with qualifi ed entomologists and ornithologists. The principal objective of the monitoring – surveillance program is early detection of WNV in monitored regions, timely reporting of the virus presence and activation of human health service institutions and local authorities aimed at establishing the control measures - eradication of mosquitoes, informing the local community and taking all relevant preventive measures for human health protection. Th e surveillance program of the WNV occurrence and spread is based on direct and indirect surveillance of WNV in natural environment. Indirect surveillance encompasses serological testing of seronegative sentinel horses and poultry for the presence of WNV infection, and it is performing continuously and periodically during the most intensive mosquito activity (May – September). The number of sentinel animals that should be tested was defi ned at the district level, according to the rate of anticipated risk of WNV infection. Direct surveillance was performed through periodical and continuous testing of pooled mosquitoes samples collected at two-week intervals during peak mosquito season (May – September) and samples of wild birds (tissues of dead birds and throat swabs of captured live susceptible bird species). The number of samples was stipulated according to the anticipated risk rate in particular regions.

The aim of this study is development of a model for monitoring and control of vector-transmitted diseases, which manifest increasing tendency during the last few decades. Th e main infection reservoirs are domestic and wild animals as well as various birds, and the disease is transmitted to humans indirectly - by vectors. Recently detected epidemics of Dengue virus, Chikungunya and West Nile virus in countries where these diseases are not common strongly indicate the expansion of infections transmitted by mosquitoes and other vectors, and are the consequence of climatic changes, international trade and travelling. Currently, there are neither vaccines nor specific antiviral therapy for these infections, while the efforts put on vector control did not halt the rapid increase and global spread of the disease. Serological studies and molecular investigation on humans, mosquitoes, horses and birds have suggested the activity of flaviviruses in Serbia. The obtained information on fl avivirus infections in our region are of use in modelling the control and vector monitoring and the prevention of these infections in humans.

BACKGROUND: Among the arthropod-borne nematodes infesting dogs, Onchocerca lupi (Spirurida: Onchocercidae) is of increasing zoonotic concern, with new human cases of infection diagnosed in Turkey, Tunisia, Iran and the USA. Knowledge of the biology of this nematode is meagre. This study aimed at assessing the distribution and periodicity of O. lupi microfilariae from different body regions in naturally infested dogs. METHODOLOGY/PRINCIPAL FINDINGS: Skin samples were collected from six dogs infested with O. lupi but without apparent clinical signs. Two skin samples were collected from 18 anatomical regions of dog 1 at necropsy. In addition, single skin biopsies were performed from the forehead, inter-scapular and lumbar regions of dogs 2-6, in the morning, afternoon, and at night. Two aliquots of the sediment of each sample were microscopically observed, microfilariae counted and morphologically and molecularly identified. Most of the 1,667 microfilariae retrieved from dog 1 were in the right ear (59.6%), nose (26.5%), left ear (6.7%), forehead (3.0%), and inter-scapular (2.9%) regions. In dogs 2-6, the overall mean number of microfilariae was larger on the head (n = 122.8), followed by the inter-scapular (n = 119.0) and lumbar (n = 12.8) regions. The overall mean number of microfilariae was larger in the afternoon (153.4), followed by night (75.4) and morning (25.8). CONCLUSIONS: Onchocerca lupi microfilariae were more common in the head (i.e., ears and nose) than in the remaining part of the dog's body, indicating they tend to aggregate in specific body regions, which are the best sites to collect skin samples for diagnostic purposes. The periodicity pattern of microfilariae of O. lupi and their concentration in specific body regions is most likely a result of the co-evolution with their as-yet-unknown vector. The detection of skin microfilariae in asymptomatic animals, suggests the potential role of these animals as carriers and reservoirs of O. lupi.