J Arthropod-Borne Dis, June 2019, 13(2): 224–233 E Ozan et al.: A Study on the … 224 http://jad.tums.ac.ir Published Online: June 24, 2019 Original Article A Study on the Identification of Five Arboviruses from Hematophagous Mosquitoes and Midges Captured in Some Parts of Northern Turkey Emre Ozan1; Harun Albayrak2; Semra Gumusova2; Cenk S. Bolukbas3; Mithat Kurt2; Gokmen Z. Pekmezci4; Yunus E. Beyhan5; Hamza Kadi1; Selma Kaya1; Ismail Aydin1; *Zafer Yazici2 1Veterinary Control Institut, Ministry of Food Agriculture and Livestock, Samsun, Turkey 2Department of Virology Faculty of Veterinary Medicine Ondokuz Mayıs University, Samsun, Turkey 3Department of Parasitology, Faculty of Veterinary Medicine Ondokuz Mayıs University, Samsun, Turkey 4Department of Aquatic Animal Diseases, Faculty of Veterinary Medicine, Ondokuz Mayis University, Samsun, Turkey 5Department of Parasitology, Faculty of Medicine, Yuzuncu Yil University, Van, Turkey (Received 18 Jan 2018; accepted 11 May 2019) Abstract Background: Whether zoonotic or not, arboviral infections are continuing to be a major threat to human health as well as the livestock industry all around the world. This project presented the results of the identification study on five arboviruses, including West Nile virus (WNV), Bovine ephemeral fever virus, Akabane virus, Bluetongue virus, and Epizootic hemorrhagic disease virus, in mosquitos and midges from eight provinces of the Black Sea Region. Methods: During 2011 and 2012, 3193 mosquitoes were captured around natural streams, rivers, lakes, and ponds using dry-baited miniature light-traps. Identification studies were concluded by employing molecular methods. Results: According to the morphological identification, blood-sucking mosquitoes and biting-midges belonged to Aedes (44.69%), Anopheles (28.34%), Culex (22.14%) and Culicoides (4.83%) species. Overall, 146 pools were made up of captured mosquitos and midges. None of the five viruses were directly identified by mosquitoes. Conclusion: Mosquitoes and midges have got a crucial role in the transmission of arboviruses. The risk of occurrence for the investigated arboviruses will continue depending upon many factors including the presence of these viruses in Turkey and its neighboring countries, uncontrolled livestock movements, global warming and climate changes. Keywords: Mosquito; Midge; Identification; Real-time RT-PCR; Arbovirus Introduction Viruses transmitted by arthropod vectors can either be zoonotic or non-zoonotic. Cur- rently, more than 100 species of arbovirus are categorized as zoonoses due to their transmis- sibility from arthropods to human (1). Most ar- boviruses belong to the virus families Flaviviri- dae, Togaviridae, Reoviridae as well as both Peribunyaviridae and Phenuiviridae within the order Bunyavirales, formerly known as Bun- yaviridae (1-3). Mosquitoes and ticks are the main vectors for the majority of arboviruses (1, 4). There are approximately 300 species of mosquito and 116 species of ticks known to be vectors of arboviruses (5, 6). The vast ma- jority of mosquitos are Aedes and Culex spe- cies, with 115 and 105 species respectively (6). In addition, 25 midge species, mainly Culi- coides, and Sandflies, Lasiohelea blackflies, stink-bugs, lice, mite, might have played a role in transmitting arboviruses (5). Even though certain viruses are transmitted by specific vec- tors, some arboviruses, for example, West Nile virus (WNV), can be transmitted additional- ly by ticks and other arthropods (1). Infection with certain arboviruses can re- sult in serious symptoms, including high fever, *Corresponding author: Dr Zafer Yazici, E-mail: zyazici@omu.edu.tr J Arthropod-Borne Dis, June 2019, 13(2): 224–233 E Ozan et al.: A Study on the … 225 http://jad.tums.ac.ir Published Online: June 24, 2019 haemorrhage, meningitis, encephalitis and death (1). Moreover, in animals, abortions, stillbirth, congenital anomalies arthrogryposis-hydranen- cephaly are also seen in as a result of infection by certain arboviruses, for example, Bluetongue virus (BTV), and Akabane virus (AKAV). Equally, some arboviral infections are asymp- tomatic and the patients recover from infection after a few weeks. WNV, dengue virus (DENV) and Chikungu- nya virus (CHIKV) are the best known and globally distributed human arboviruses (1, 7). BTV, AKAV and Bovine ephemeral fever vi- rus (BEFV) are important, non-zoonotic arbo- viruses of animals. In particular, large epidem- ics of BTV-serotype 8 in EU countries includ- ing Holland, Czech Republic, Belgium, Swe- den, the UK, and Switzerland emerged between 2006–2008 and caused major economic loss- es in all of these countries (8). In Turkey, the presence of WNV, BTV, AKAV, BEFV and Epizootic hemorrhagic dis- ease virus (EHDV) has been reported follow- ing serological studies (9-12). Furthermore, a limited number of studies have been conduct- ed to identify arboviruses from invertebrate vectors (7). The spread of arboviruses is influenced by many factors, such as increasing internation- al travel, globalization of trade and climate change. Whether symptomatic or not, arbo- viral infections may cause a large social and economic burden for societies (1). The detec- tion of arboviruses in vectors is important for public health and the livestock industry and al- lows a lot of detailed information to be gathered about seasonal arbovirus circulation, helping to create time-dependent preventive measures. As the geographical bridge between Eu- rope and Asia, Turkey has a rich capacity as for natural and ecological conditions that pro- vide a suitable habitat for arthropod popula- tions, thus paving the way for the emergence of new arthropod-borne viruses. At the same time, Turkey is also at risk due to the known presence of these viruses in neighboring coun- tries such as Iran, Iraq, Syria, the Balkan and Blacksea states. The aim of this study was to identify five arboviruses from field trapped mosquitos and midges at different provinces in Northern Tur- key. Captured vectors were examined by em- ploying the real-time RT-PCR and RT-PCR. Materials and Methods Samples During summer months of 2011 and 2012, eight provinces of Northern Turkey (Fig. 1) were chosen to collect mosquito and midge samples. In the sampling period, two signifi- cant points were taken into account for the in- stallation of traps: (i) areas of sampling where mosquito and midges were particularly abun- dant, such as cowsheds and sheepfolds, near or close to lakes, ponds, dam-lakes, natural streams and rivers, were chosen in the immediate vi- cinity of least 3 residential area in each prov- ince. (ii) Mosquitos and midges were collected on days that were without rain and wind dur- ing the night between 6pm and 12pm. Mos- quitos and midges were caught in glass con- tainers containing water and then were trans- ported immediately to the laboratory under cold chain. Mosquitoes and midges were separated un- der the stereomiscroscope in order to morpho- logically identify species (13, 14). Overall, 146 pools from 3193 mosquitos and midges were made up according to each capture province and capture time, and were stored at -80 °C until use. The vast majority of them were Aedes spp. corresponding to 1427 (44.69%) mosquitoes. Furthermore, we identified the numbers of Anopheles spp., Culex spp. and Culicoides spp. as 905 (28.34%), 707 (22.14%) and 154 (4.83%), respectively (Table 1). Homogenization of mosquitoes and midges Insect pools were transferred into magnalys- er green bead tubes (Roche Germany). 1ml of J Arthropod-Borne Dis, June 2019, 13(2): 224–233 E Ozan et al.: A Study on the … 226 http://jad.tums.ac.ir Published Online: June 24, 2019 PBS (Gibco) was then added to each tube. Pools were homogenized using a 1.4mm magnalyser ceramic bead by shaking MagNa Lyser (Roche, Germany). Following homogenization, the un- soluable particulates were pelleted by centrif- ugation at 4 °C, 3000rpm for 15min. The supernatant from each pool was then transferred into a new tube and used for nu- cleic acid purification using the total RNA ex- traction kit (Qiagen, Spain) according to the manucfacturer’s instructions. RNA extractions and RT-PCR tests Positive controls of viral RNA used for PCR tests were kindly provided by University of Texas Medical Branch (Galveston, USA) for WNV, Pendik Veterinary Control Institute (Is- tanbul, Turkey) for BTV, Veterinary Diagnos- tic Technology Inc (USA) for EHDV, Veter- inary Control Institute (Samsun, Turkey) for AKAV and Department of Virology, Faculty of Veterinary Medicine, University of Sel- cuk (Konya, Turkey) for BEFV. Detailed in- formation regarding primers and Taqman probes labelled FAM-TAMRA are given in Table 2. Viral RNAs of WNV, BTV, EHDV and AKAV was detected in 96 well plates using a Light Cycler 2.0 real-time PCR systems (Roche, Germany). The results were assessed using the LightCycler Software 4.05 (Roche, Germany). Arktik thermal cycler (Thermo Sci- entific, UK) were used for RT-PCR in order to detect BEFV RNA. The concentration and purity of RNA were used by absorbance at 260–280nm wavelenghts using a UV spectrophotemeter (Helios Gamm, Thermo Spectronic, UK). To perform a real-time RT-PCR for the detection of WNV-RNA in mosquito pools, specific primers and a probe (15) were used. The Taqman probe was labeled at the 5’ end with the reporter dye FAM and labeled at the 3’ end with quencher dye TAMRA. Briefly, the reaction mixture was made up in a total volume of 50µL containing 2µL sample RNA, 75mM Tris-HCl (25 ºC, pH 8.8), 20mM (NH4)2 SO4, 6mM MgCl2, 0.2mM of dNTP mix, 50 pmol of primers 10pmol of prob, 1µL of Tri- ton X-100, 5U RNase inhibitor, 100U Molo- ney murine leukemia virus reverse transcrip- tase and 2,5U Taq DNA polimerase (Thermo Scientific, UK) and water. The mixture was cycled as follow: 1 cycle 30min at 42 ºC for re- verse transcription, 1 cycle of 2min at 94 ºC, 40 cycles of 30sec at 94 ºC, 30sec at 55 ºC and 20sec at 72 ºC and 1 cycle of 30sec at 40 ºC. To investigate BTV-RNA in pools, the re- al-time RT-PCR method (16) was carried out using the QIAamp Viral RNA Mini Kit (Qi- agen, Spain) according to the manufacturer's instructions. RNA was eluted in 50μl nucle- ase-free water and then stored at -20 °C. For RNA sample denaturation prior to the initial RT step, heat denaturation of RNA was eval- uated by heating 6μl samples at 98 °C for 5 min followed by rapid cooling on ice. The re- action mixture contained, 20pmol of each pri- mer, 5pmol of prob, 1.25mM MgCl2, 0,4mM dNTPs, 0.8µL of enzyme mix, 4µL of 5X RT- PCR buffer, 3µL denatured RNA and water to a final volume of 20µL.The mixture was cycled in using following procedure: After 1 cycle of 30min at 50 ºC for reverse transcrip- tion, 1cyle of 15min at 95 ºC for denaturation, 45 cycles of 15sec at 95 ºC and 1min at 60 ºC. Test was evaluated at the end of 30s at 40 ºC. To investigate AKAV-RNA in pools, we used touch-down rRT-PCR with some modi- fications (17). The reaction mixture was made up in a total volume of 50µL containing 3µL sample RNA, 75mM Tris-HCl (25 ºC, pH 8.8), 20mM (NH4)2SO4, 6mM MgCl2, 0.2mM dNTP Mix, 20pmol of primers 10pmol of prob, 1µL of Triton X-100, 5U RNase inhibitor, 100U Moloney murine leukemia virus reverse tran- scriptase and 2.5U Taq DNA polymerase (Ther- mo Scientific, UK). Cycling conditions includ- ed: RNA was reverse transcribed at 42 ºC for 30min and was followed by an inactivation step at 94 ºC for 2min). After reverse-transcrip- tion and inactivation steps, 10 cycles of 15sec J Arthropod-Borne Dis, June 2019, 13(2): 224–233 E Ozan et al.: A Study on the … 227 http://jad.tums.ac.ir Published Online: June 24, 2019 at 95 ºC, 60sec at 60 ºC (decreased by 2 ºC per cycle so that it reached 50 ºC after 10 cycles) and 15min at 72 ºC were carried out. Finally, a further 35 cycles of 15sec at 95 ºC, 1min at 50 ºC and 15sec at 72 ºC and 40 ºC for 2min followed. To investigate EHDV-RNA in pools, the mixture was prepared in a total volume of 20 µL containing 3µL sample RNA, 4µL of 5X master mix, 1.25mM MgCl2, 0.5mM dNTPs, 20pmol of primers, 5 pmol of probe, 1µL of enzyme mix (Qiagen, Spain) and water. Cy- cling conditions of the reaction were as fol- lows: 1 cycle of 30min at 50 ºC for reverse transcription step, 15min at 95 ºC for inacti- vation and denaturation step, and 40 cycles of 10sec at 95 ºC, 1min at 55 ºC, 30 sec at 72 ºC and finally incubation 30sec at 40 ºC. Sev- en primer pairs and 3 probes were used to in- vestigate all of the EHD serotypes. EHDV-1 and 2 were tested using the same primer pair. The different probes were used for EHDV 3 and 4 while the same probes were using for EHDV-1, 2, 5, 6, 7 and 8 (18). BEFV-RNA in pools were tested by us- ing one-step reverse transcription PCR meth- od. Specific primers with the Qiagen OneStep RT-PCR kit were used for ampflying a 809bp DNA fragment (19). The RT-PCR mixture was prepared in a total volume of 50µL containing 5µL sample RNA, 10µL of 5X mastermix, 0.2 mM of dNTP mix, 10pmol of primers, 10U RNase inhibitor and 2µL of enzyme mix and water. Amplification was conducted using ther- mal cycler according to the reaction condi- tions as follow: following reverse-transcrip- tion at 50 ºC for 30min, 1 cycle of 5min at 95 ºC, then 35 cycles of 30sec at 95 ºC, 1min at 56 ºC and 1min at 72 ºC. The amplication process was terminated after a final exten- sion of 10min at 72 ºC. 5µL of PCR products were visualized on a 1.5% agarose gel stained ethidium bromide. Results Hematophagous mosquitos and biting midges were caught using dry-baited miniature light-traps located near to natural streams, riv- ers, lakes and ponds. Following morphologi- cal identification under the stereomicroscope, 146 pools were made, representing 3193 mos- quitos and biting midges. At the end of mor- phological identification, overall proportions of hematophagous mosquitos and biting-midg- es were found to be 95.17% and 4.83%, re- spectively. Captured mosquitos were found to belong to Aedes (44.69%), Anopheles (28.34%) and Culex (22.14%) species. Midges were iden- tified as Culicoides (4.83%), The amplication results of four arbovirus including WNA, BTV, EHDV and AKAV by a real time RT-PCR test were shown in Fig. 2. Positive controls for 4 viruses worked very well and gave a good amplification earlier. A detectable fluorescence signals above the thresh- old for positive controls occurred at 12 cycles for WNV, 28 cycles for BTV, 28 cycles for EHDV and 11 cycles for AKAV. No viral RNA for these viruses could be identified by employing real-time RT-PCR in any of the insect pools. Similarly, BEFV-RNA was not detected using one-step RT-PCR in any of the mosquito pools. In all of 8 provinces, none of five viruses could be determined during the sampling period. Table 1. The numbers of captured insect species according to provinces where the traps were installed Captured Mosquitos and Midges Longitude Latitute Trapping Aedes Anopheles Culex Culicoides (°E) (°N) Provinces 35.09 42.01 Sinop 19 540 158 27 36.20 41.17 Samsun 1372 195 168 44 J Arthropod-Borne Dis, June 2019, 13(2): 224–233 E Ozan et al.: A Study on the … 228 http://jad.tums.ac.ir Published Online: June 24, 2019 36.43 40.19 Amasya 3 20 46 20 35.50 40.40 Tokat 23 136 181 35 37.53 41.00 Ordu 3 3 82 26 38.24 40.55 Giresun - 4 35 2 39.43 41.00 Trabzon 5 4 60 - 40.31 41.02 Rize 2 3 7 - Total 1427 905 707 154 Table 2. Primers and Probes sequences that employ for this research Primer/Probes Sequences(5’-3’) Position WN3 NC-F CAGACCACGCTACGGCG 10,668–10,684 WN3 NC-R CTAGGGCCGCGTGGG 10,770–10,756 WN3 NC-P TCTGCGGAGAGTGCAGTCTGCGAT 10,691–10,714 BTVrsa 291–311F GCGTTCGAAGTTTACATCAAT 291–311 BTVrsa 387–357R CAGTCATCTCTCTAGACACTCTATAATTACG 387–357 BTVuni 291–311F GCTTTTGAGGTGTACGTGAAC 291–311 BTVuni 381–357R TCTCCCTTGAAACTCTATAATTACG 381–357 BTV 341–320P CGGATCAAGTTCACTCCACGGT 341–320 BTV 346–323P TCCTCCGGATCAAGTTCACTCCAC 346–323 EHD 1/2F GCGTTGGATATATTGGACAAAGC 165–187 EHD 1/2R GCATACGAAGCATAAGCAACCTT 275–253 EHD 1/2P TCAAATCAAACGGGCGCAACTATGG 192–216 EHD 3F AGCCCTCGACATTCTGGATAAG 164–185 EHD 3R CGCGACTTTCTCCACTTTTTG 259–239 EHD 3P CAAATCAAACTGGTGCCACGATGGC 206–230 EHD 4F AAGTTGCCCTCGATATCCTAGATAAG 160–185 EHD 4R GTAAGCGACCTTTTCTACCTTTTGA 266–242 EHD 4P CAATGTCTAACCAAACAGGCGCTACAATGG 199–228 EHD 5F GCTGGATATACTCGACAAAGCAATG 167–191 EHD 5R ACGCGACCTTTTCTACTTTTTGA 262–240 EHD 6F GAGTCGCGCTGGATATACTC 160–179 EHD 6R GCATATGATGCATACGCGACCTTT 276–253 EHD 7F CAAAAGTTGCCCTTGACATTTTAG 157–180 EHD 7R CATATGCTACTTTTTCTACCTTCTGC 266–243 EHD 8F CCAAAAGTCGCCCTTGATATTT 156–177 EHD 8R CATATGCCACTTTTTCTACCTTCTGC 268–243 AK151F GCTAGAGTCTTCTTCCTCAACCAGAA 151 AK231R AAAAGTAAGATCGACACTTGGTTGTG 231 AK182P CCAAGATGGTCTTACATAAG 182 BEF346 F TATTACCCTCCTGCCGGATGCTTT 346–369 BEF1155 R AGGTCTGTATTCGCACCAAGCTCT 1155-1132 Fig. 1. A map showing the provinces targetted in htis study for the capture of mosquitoes and biting-midges during 2011 and 2012. Provinces that indicated with numbers are 1: Sinop, 2: Samsun 3: Ordu, 4: Giresun, 5: Trabzon, 6: Rize, 7: Amasya, 8: Tokat Table 1. Continued … J Arthropod-Borne Dis, June 2019, 13(2): 224–233 E Ozan et al.: A Study on the … 229 http://jad.tums.ac.ir Published Online: June 24, 2019 Fig. 2. The amplication results of four arbovirus including WNA, BTV, EHDV and AKAV by a real time RT-PCR test. Curves indicated the real time PCR plots of positive controls. A:WNV, B:BTV, C: EHDV, and D:AKAV. A detectable fluorescence signals above the threshold for positive controls of four viruses occurred at 12 cycles for WNV, 28 cycles for BTV, 28 cycles for EHDV and 11 cycles for AKAV.As it seen in all figures, no positive pools for 4 viruses could be determined Discussion Whether zoonotic or not, arboviral infec- tions are continuing to be a major threat to hu- man health as well as the livestock industry all around the world (1, 20). In the last decade, many outbreaks in humans and animals caused by arboviruses have been reported. Identifi- cation of arboviruses in vectors can provide us much information to help develop control strategies against them. Historically, there are only a few studies on arboviruses in vectors in Turkey, limited to the Flaviviridae and To- gaviridae (7, 21). In one such study, 15.9% of collected mosquitos were reported as WNV positive (7). Our study was planned for five animal arboviruses, including WNV, BTV, EHDV, AKAV and BEFV to identify from their vectors. With the exception of WNV, all of the viruses are non-zootic. This study can be defined as the first compherensive identi- fication study of mosquitoes and midges caught in the Middle and East Black Sea Re- gion in Turkey. During 2011–2012, 3190 mosquitoes and midges were collected using light traps in 8 provinces of the Central and East Blacksea re- gion of Northern Anatolia, Turkey. According to the morphological identification results, the vast of majority of the overall vector population was mosquito (95.17%), comprising 44.69% Aedes, 28.34% Anopheles and 22.14% Culex, species known as vector for WNV, BEFV (22, 23). Culicoides midges, known as the important biological vector of BTV (8), EHDV (11) and AKAV (24), represented 4.83% of catches. J Arthropod-Borne Dis, June 2019, 13(2): 224–233 E Ozan et al.: A Study on the … 230 http://jad.tums.ac.ir Published Online: June 24, 2019 In Turkey, serologically the presence of WNV for the first time was reported in differ- ent animal species including horses (13.5%), dogs (37.7%), cattle (4%) and sheep (1%) (9). The first identification of WNV from haema- tophagous mosquitoes in Eastern Thrace ter- ritory of Turkey was carried out (7). In the previous years, a few identification studies on WNV had been reported in the Black Sea region and no WNV could be identified from insects and blood samples of various animal species such as horses and wild birds (25, 26). Moreover, the serological diagnosis of WNV could also not be possible in various animal species with the exception of goats that sero- positivity rate was reported as 2.85% (27, 28). In our comprehensive study, although 80% of the captured insects were a specific vector for WNV, the identification of WNV was not pos- sible using real-time RT-PCR. The lack of pres- ence of WNV may be due to the absence of certain vector specific determinats of emer- gence, such as the seasonal activity, climate change, ecological and environmental condi- tions (7, 9). BTV and EHDV both belong to the fam- ily Reoviriadae, and both viruses are trans- mitted between ruminant hosts by Culicoides biting midges. During the past decade, BTV outbreaks have occurred in the western part of Turkey (8, 29). An EHDV outbreak was also reported in Western Anatolia in 2007 (11). However, infections caused by these viruses in Northern Anatolia had not been seen be- fore. On the other hand, there are serological data regarding both viruses showing the his- torical presence of these viruses. Seroposi- tivity for EHDV had been reported in cattle as 1.33%, with evidence of BTV seropositive animals, including 11% for cattle, 3% for sheep and 4% for goat (25, 27–28, 30). In our study, no BTV-RNA and EHDV-RNA could be iden- tified by using real-time RT-PCR. When scru- tinized, these results could depend upon two main causes. First, historically neither disease has been clinically evident in this region un- til recently and, secondly, the proportion of Cu- licoides species was far fewer than other insects captured during sampling periods. Furthermore, livestock movements inside Turkey may con- found some interpretations of serological data. Culicoides is also the main vector for AKAV. Despite a serological prevalence of 22% in our study area (27), in neither mos- quito nor midge pools could be not detected AKAV-RNA by real-time PCR. This result can be interperented similarly to BTV and EHDV. In previous years, numerous BEFV out- breaks were reported in Turkey, primarily in South and Sout-East Anatolia. The climate of these regions is suitable for BEFV vectors. In addition, there are common borders with Syr- ia and Iraq where the virus has been previously reported. The latest outbreak began in the same regions in 2012 and reached a case fatality rate (31, 32). In the Central and Eastern Blacksea region of Turkey, antibodies against BEFV were shown to be 13.5% (27). Despite the serolog- ical indications, clinical BEFV has not been re- ported. In the current study, we attempted to identify BEFV from captured insects using RT- PCR. At the end of the experiments, no BEFV-RNA could be found in all pools. Ac- cording to our standpoint, there are a few un- suitable climatic conditions and vector ecol- ogy may represent important factors in de- termining whether or not infection occurs. No viral RNAs of five arboviruses could be found in pools made up of captured vari- ous mosquitos and midges during this project. Infections caused by these five viruses were not observed clinically in the Central and East Blacksea region where the project was car- ried out. However, in the light of serological data obtained from previous studies, five vi- ruses are nevertheless considered to circulate between animals and prospective vectors. Fur- thermore it is not ruled out that mosquitos and midges cannot be found in some locations of targeting regions or were never infected with five viruses. J Arthropod-Borne Dis, June 2019, 13(2): 224–233 E Ozan et al.: A Study on the … 231 http://jad.tums.ac.ir Published Online: June 24, 2019 Reservoir, vector, and climate are the most important aspects for the epidemiology of ar- boviruses. The climatic condition of a region, such as amount of rainfall, wind status and in- secticide application are particularly important factors in vector biology. In the Black Sea re- gion, yearly averages of temperature, humid- ity and rain are reported as 13.0 ºC (4.2–22.1 ºC), 71%, 842.6mm3, respectively. When com- pared, these values are lower than the West- ern Anatolia region of Turkey where the dis- ease is seen frequently, reportedly 16.3 ºC (6.4– 26.8 ºC), 63%, 2, 725.9mm3 respectively. These data are important factors affecting vector abun- dance. Hence the existence of Culicoides spe- cies in the Black Sea region was far less than the western and southern part of Turkey (14). Conclusion Mosquitoes and midges have got a crucial role in the transmission of arboviruses. The risk of occurrence for the investigated arbovirus- es will continue depending upon many factors including (i) the presence of these viruses in Turkey and its neighbouring countries (ii) un- controlled livestock movements (iii) global warming and climate changes. A large scale project, inclusive of both people and animals, should be planned. In addition, effective ar- boviral disease surveillance systems that can detect outbreaks of emerging zoonotic and non- zoonotic infection should be developed. Acknowledements This research was supported by General Directorate of Agricultural Research and Pol- icies, Republic of Turkey, Ministry of Food, Agriculture and Livestock, Project Nr: TA- GEM/HS/11/01/02/184, and carried out by col- laborating with Virology Laboratory of Sam- sun Veterinary Control Institute and Virology and Parasitology Departments of School of Vet- erinary Medicine, Ondokuz Mayis University. The authors declare that there is no con- flict of interests. References 1. 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