J Arthropod-Borne Dis, December 2015, 9(2): 195–203 M Bakhshesh and D Abdollahi: A Checklist of … 195 Original Article Bovine Ephemeral Fever in Iran: Diagnosis, Isolation and Molecular Characterization *Mehran Bakhshesh 1, Darab Abdollahi 2 1Animal Virology Department, Research and Diagnosis, Razi Vaccine and Serum Research Institute, Karaj, Iran 2Iranian Veterinary Organization, Tehran, Iran (Received 16 Mar 2014; accepted 28 June 2014) Abstract Background: Bovine ephemeral fever (BEFV) is an arthropod-borne disease of cattle and water buffaloes. BEFV occurs seasonally in tropical, subtropical and temperate regions of Africa, Asia and Australia. It has been known for the past decades in Iran based on clinical signs but lack of an accurate diagnosis has made the real feature of disease obscured. This is the first scientific report on isolation and identification of the agent in which molecular diagnosis of BEFV was also set up with high sensitivity and specificity. Methods: The viral agent was successfully isolated through serial passages in brain of suckling mice and cell cul- ture. In addition, the circulating virus during the autumn 2012 in Iran was molecularly characterized based on partial G gene. Results: Alignment of 3 virus sequences from different parts of Iran revealed that they are identical suggesting that the circulating viruses were most likely the same in this period. Phylogenetic analysis of the Iranian sequences with 17 sequences in the GenBank from the world showed that it is identical to the virus circulated in Turkey during the same period suggesting that the virus was circulated in a large geographic region. Conclusion: These results offer primary information about BEFV in Iran. To better understanding the epidemiology of the virus, further studies based on seroepidemiology, molecular epidemiology, entomology and meteorology to- gether with finding the model of animal transportation in the region are necessary. Keywords: Bovine ephemeral fever, Iran, Diagnosis, Isolation, Phylogenetic analysis Introduction Bovine ephemeral fever (BEFV) is a non- contagious, arthropod-borne disease of cattle and water buffaloes proposed to be trans- mitted by Culicoides biting midges and mosquitoes. BEFV has been isolated from Culicoides brevitarsis, Culicoides coarctatus, and Anopheles bancroftii, however the ar- thropod vectors are yet to be defined (Walker et al. 2012). The viral agent is classified as the type species of the genus Ephemerovirus within the Rhabdoviridae family. BEFV is bullet shaped and its genome consists of five structural protein genes; N, P, M, G and L (Walker et al. 2012). The G gene encodes glycoprotein G which is responsible for cell attachment and entry, and also contains type- specific and neutralizing antigenic sites (Cybinski et al. 1990, Walker et al. 1991). BEFV occurs seasonally in tropical, sub- tropical and temperate regions of Africa, Asia, Australia and the Middle-East and is a disabling disease with considerable eco- nomic impact due to loss of milk production, loss of condition in beef herds, infertility and abortion. The morbidity rate is high (up to 100%), however the mortality rate is gener- ally low (rarely exceeds 1%). Typical clini- cal signs include a sudden onset of fever as high as 41 °C, a sudden and severe drop in milk production, inappetence, lethargy, sali- *Corresponding author: Dr Mehran Bakhshesh, E-mail: M.Bakhshesh@rvsri.ac.ir http://jad.tums.ac.ir Published Online: March 11, 2015 J Arthropod-Borne Dis, December 2015, 9(2): 195–203 M Bakhshesh and D Abdollahi: A Checklist of … 196 vation, nasal discharge, depression, stiffness, ruminal stasis and dyspnea (Walker et al. 2012). Treatment is based on remission of clinical signs using non-steroidal anti-in- flammatory drugs and administration of cal- cium borogluconate to hypocalcemic animals. Vaccination is considered as the only effec- tive approach to control the disease. Live- attenuated, inactivated, subunit and recombi- nant vaccines have been applied experimen- tally and commercially while live vaccines generally induce more-prolonged immunity (Radostitis et al. 2007). Because of the short course of viremia in affected animals and proper shipping require- ments of blood samples to the laboratory, de- tection and isolation of BEFV is not usually successful. Bovine ephemeral fever has been reported for the past decades in Iran and some efforts have been taken to isolate the virus (Hazrati et al. 1975). According to clin- ical observations, the economic losses due to the virus seem to be significantly high, how- ever due to the lack of an accurate laboratory diagnosis the real feature of the disease re- mained unknown. The aim of this study was isolation and adaptation of the viral agent on cell culture, and molecular characterization of the circu- lating virus in Iran during the autumn 2012 while accurate diagnosis of BEFV was also set up. The results are useful for any epide- miological study and are prerequisites for fur- ther research to manufacture a potent vaccine against the virus in Iran. Materials and Methods Sampling Blood samples with anticoagulant were taken from clinically suspected animals dur- ing febrile phase and shipped on ice to the Animal Virology Department, Research and Diagnosis of Razi Vaccine and Serum Re- search Institute. Viral RNA isolation and PCR amplification Viral RNA was extracted from whole blood, cell culture and tissues using RNTX™ solu- tion (Cinagen), according to manufacturer's protocol. The purified RNA was subjected to reverse transcription (RT). Reverse transcription (RT) was carried out using RevertAid™ First Strand cDNA synthesis Kit (Fermentas) as follows: 6 μl RNA and 1 μl Oligo (dT) primer with 5 μl DEPC-treated water heated at 65 °C for 10 minutes and cooled on ice. Then, 4 μl 5X Reaction buffer, 1 μl Ribolock™ Rnase in- hibitor (20u/μl), 2 μl of 10 mM dNTP Mix, 1 μl RevertAid™ M-MulV Reverse Tran- scriptase (200 u/μl) were added to the solu- tion to a final volume of 20 μl. The resulting cDNA was amplified by polymerase chain reaction (PCR) using AccuPower® PCR PreMix (BIONEER), in which 2 μl of cDNA was mixed with 10 pmol of each forward: 5'-ATGTTCAAGGTCCTC ATAATTACC-3' (nt 1–24) and reverse: 5'- AATGATCAAAGAACCTATCATCAC-3' (nt 1874–1871) (Wang et al. 2001) and dionized water to a final volume of 20 μl to amplify full length of G gene. Thermal cy- cling condition was initial denaturation at 95 °C for 5 min then 35 cycles of 95 °C for 40 s, 52 °C for 40 s, 72 °C for 2 min and 30 s and final extension of 72 °C for 10 min. The PCR product (1μl) was diluted (1/100) and 1 μl of the dilution was subjected for Nested-PCR using AccuPower® PCR PreMix (BIONEER) as described above. Nested-PCR amplified an 809 bp PCR product (nt 346–1155) using forward: 5'- TATTACCCTCCTGCCGGAT GTTT-3' and reverse: 5'- AGGTCTGTATT CGCACCAAGCTCT-3' primers (Aziz-Boaron et al. 2012). Thermal cycling program was carried out as initial denaturation at 95 °C for 4 min then 35 cycles of 95 °C for 30 s, 56 °C for 30 s, 72 °C for 40 s and final ex- tension of 72 °C for 5 min. All PCR products were run on agarose gel and stained with http://jad.tums.ac.ir Published Online: March 11, 2015 J Arthropod-Borne Dis, December 2015, 9(2): 195–203 M Bakhshesh and D Abdollahi: A Checklist of … 197 ethidium bromide and amplicons were ob- served in all positive samples. In order to minimize the risk of nucleo- tide miscorporation for phylogenetic analy- sis of the 809 bp amplicon, additional PCR reaction was carried out on cDNAs using Pfu enzyme with the same thermal cycling condition. The PCR reaction mixture (50 μl total volume) comprised 5 μl 10× Pfu buffer, 2 μl dNTP mix (5mM), 1 μl (10 pmol) of each primer, 5 μl cDNA, 3.2 μl MgSo4 (25 mM) and 0.8 μl Pfu polymerase (2.5 u/μl, Fermentas). Virus isolation BEFV was successfully grown and adapted on cell culture by inoculating the virus intracerebrally into suckling mice pri- marily (Van der Westhuizen 1967). Using this approach, viral isolation was achieved as explained briefly. The blood samples were centrifuged at 2000 ×g for 5 min. Buffy coats of blood in anticoagulant were washed with phosphate-buffered saline (PBS). The buffy coats were resuspended in PBS and were frozen and thawed 3 times and 20 μl was inoculated into suckling mice' brains. The suckling mice were examined carefully every day for clinical signs. After 6 days, their brain were extracted and were grinded with PBS, the homogenates were frozen and thawed 3 times, centrifuged at 2000 × g for 5 min and the supernatant was inoculated into suckling mice's brains again until the 3rd pas- sages when clinical signs were observed. The controlled mice were injected with 20 μl PBS repeatedly in all 3 passages. Viral RNA extraction and RT-PCR were performed on all inoculated and controlled mice. The ho- mogenates from the 3rd passage were used for inoculation of cell culture. Confluent Green Monkey Kidney (Vero) cell monolayers in 25 cm2 flask were inocu- lated with 0.5 ml of homogenate from the 3rd passages of virus in brains of suckling mice and incubated at 37 °C for 1 h, then cell maintenance medium containing: DMEM (Gibco), Glucose, Calf serum (5%), NaHCO3, Penecillin and Streptomycin, was added into the monolayers. The cultures were main- tained at 37 °C for 8 days and observed every 24 h intervals. The flasks were frozen- thawed 3 times, they were centrifuged and the supernatant were inoculated into new monolayers. A monolayer flask was also con- sidered as control which was inoculated with PBS in serial passages. Viral RNA extrac- tion and RT-PCR were also carried out on all inoculated and controlled monolayers. Sequence and phylogenetic analysis The 809 PCR products were purified by High pure PCR product purification kit (Roche) and directly sequenced in both ori- entations with PCR primers mentioned above. The obtained sequences were assem- bled by Genious (4.8.3) software. The partial G gene sequences from Miandoab (36° N, 46° E), Shahriar (35° N, 51° E) and Mamasani (30° N, 51° E) located in North-West, center and South of Iran, respectively, were aligned using Clustal W program (Thampson et al. 1994). An Iranian sequence named SH (Shahriar) was deposited in GenBank with Accession number of KF 14868. This se- quence was then aligned with 17 sequences from Australia, Taiwan, Japan, China, Egypt, Israel and Turkey (Table 1). The phylogenetic tree was constructed by the maximum like- lihood method (Tamura et al. 2011) using Kimura's two-parameter formula (Kimura 1980). The reliability of the branching was evaluated by the bootstrap test with 100 replicates (Felsenstein 1985). Results Polymerase chain reaction (PCR) All PCR reactions were successfully car- ried out. The first reaction amplified an 1871 bp product spanning the whole G gene and the nested reaction amplified an 809 bp product. http://jad.tums.ac.ir Published Online: March 11, 2015 J Arthropod-Borne Dis, December 2015, 9(2): 195–203 M Bakhshesh and D Abdollahi: A Checklist of … 198 Phylogenetic analysis of the partial G gene sequences Using Clustal W program, the 3 sequenc- es obtained from different parts of Iran in- cluding Shahriar, Miandoab and Mamasani were absolutely identical as depicted in Fig. 1. Additionally, alignment of Iranian se- quence with 17 sequences from the world revealed that the Iranian sequence is 99.9 % identical with the Turkish isolate TR- ETLIK-2-BEF which was isolated in 2012. According to the phylogenetic tree, all se- quences grouped into three distinct lineages including Australian, Middle- East and East Asian. The Iranian 2012/SH with the Turkish 2012/ TR-ETLIK-2-BEF and TRCP3 were placed with East Asian in one cluster (Fig. 2). Virus isolation From the third passage on suckling mice, clinical signs including paralysis, stiffness in hind legs and death were observed on fourth day after inoculation, while no clinical sign and death were observed in the control mice which were inoculated with PBS (Fig. 3). Moreover, the monolayer Vero cells showed CPE (Cytopathic effect) including rounding of cells, chromatin condensation followed by detachment from flasks on fifth day after in- oculation from the second passage (Fig. 4). The presence of BEFV genome was con- firmed in brains of suckling mice and cells inoculated with the virus by RT-PCR while the control mice and cells were negative. Table 1. Bovine ephemeral fever virus sequences used for the phylogenetic analysis Accession numberYear of isolationCountryIsolates AF2345331968AUSTRALIABB7921 AF0583231998AUSTRALIACS1619 AF0583221984AUSTRALIACS1647 AB4620281966JAPANYHL AB4620442004JAPANON-04-1 AY9352391984TAIWAN1984/TW/TN1 AY8181942004TAIWANTN-2004-124 GQ2294522008TURKEYTRCP77 KC6094262012TURKEYTR-ET/IK-2 KC4703102012TURKEY2012/TR/CP3 KC3371182012TURKEY2012/TR/SKR GQ2277452005EGYPTEGY-2005 JN6460902008ISRAELIL-2008 JN8336352012ISRAELISR10/3 JX5666401976CHINAJB76H JX1746612011CHINASHANDONG JX5646392002CHINAJT02L KF1486852012IRANSH http://jad.tums.ac.ir Published Online: March 11, 2015 J Arthropod-Borne Dis, December 2015, 9(2): 195–203 M Bakhshesh and D Abdollahi: A Checklist of … 199 ..|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| .. 390 400 410 420 430 440 450 460 470 Mamasani TTATAAGA TCCGATCCCA TAATGATTCT TTTTAACCAT TCCCCCTCTT GTTGATGTTC TCCAATCTAT ACTGCAATCC TTTGAAACTC GA Miandoab TTATAAGA TCCGATCCCA TAATGATTCT TTTTAACCAT TCCCCCTCTT GTTGATGTTC TCCAATCTAT ACTGCAATCC TTTGAAACTC GA Shahriar TTATAAGA TCCGATCCCA TAATGATTCT TTTTAACCAT TCCCCCTCTT GTTGATGTTC TCCAATCTAT ACTGCAATCC TTTGAAACTC GA ..|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| .. 480 490 500 510 520 530 540 550 560 Mamasani CCTGAATC TGGTAGGGAC AGACAAAGTT TTTCTTGCCA ACTTGTTTGC TCAAATAAAT ATGCATAATC TCTTCCTGGT CTTGTGGGAG CT Miandoab CCTGAATC TGGTAGGGAC AGACAAAGTT TTTCTTGCCA ACTTGTTTGC TCAAATAAAT ATGCATAATC TCTTCCTGGT CTTGTGGGAG CT Shahriar CCTGAATC TGGTAGGGAC AGACAAAGTT TTTCTTGCCA ACTTGTTTGC TCAAATAAAT ATGCATAATC TCTTCCTGGT CTTGTGGGAG CT ..|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| .. 570 580 590 600 610 620 630 640 650 Mamasani AAGTAAGA CATATCCAAG GTATTTATAT TTTCTTTATT AAGTATCTTG CTAATGGTAT TTAAACATCT TTCATGTTCT AATTCTGCCT TA Miandoab AAGTAAGA CATATCCAAG GTATTTATAT TTTCTTTATT AAGTATCTTG CTAATGGTAT TTAAACATCT TTCATGTTCT AATTCTGCCT TA Shahriar AAGTAAGA CATATCCAAG GTATTTATAT TTTCTTTATT AAGTATCTTG CTAATGGTAT TTAAACATCT TTCATGTTCT AATTCTGCCT TA ..|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| .. 660 670 680 690 700 710 720 730 740 Mamasani ATATCTAA TTCTTCAAAT TCTGTTCTAT CTGTGTGCAT CCTAAAACCC GGTTTTTTAC CCTTACATCT TTCAATTTTA AAATTTTGGA AA Miandoab ATATCTAA TTCTTCAAAT TCTGTTCTAT CTGTGTGCAT CCTAAAACCC GGTTTTTTAC CCTTACATCT TTCAATTTTA AAATTTTGGA AA Shahriar ATATCTAA TTCTTCAAAT TCTGTTCTAT CTGTGTGCAT CCTAAAACCC GGTTTTTTAC CCTTACATCT TTCAATTTTA AAATTTTGGA AA ..|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| .. 750 760 770 780 790 800 810 820 830 Mamasani TCAGATTC AGTTTGGTTT TCTATACTCC ACCATTCTCC ATCCTCAAAA ATGATGCCAT TTCTTCCACA AAATGTAGAC AGACATCCCT TA Miandoab TCAGATTC AGTTTGGTTT TCTATACTCC ACCATTCTCC ATCCTCAAAA ATGATGCCAT TTCTTCCACA AAATGTAGAC AGACATCCCT TA Shahriar TCAGATTC AGTTTGGTTT TCTATACTCC ACCATTCTCC ATCCTCAAAA ATGATGCCAT TTCTTCCACA AAATGTAGAC AGACATCCCT TA ..|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| .. 840 850 860 870 880 890 900 910 920 Mamasani TTTACATG GACTAACCCG ATATCTGGAG CCTCCCATAA TCTCTCTGTC TCATTTAATT CACTCTTAAA GGATTTAACT GTAATGCATT CC Miandoab TTTACATG GACTAACCCG ATATCTGGAG CCTCCCATAA TCTCTCTGTC TCATTTAATT CACTCTTAAA GGATTTAACT GTAATGCATT CC Shahriar TTTACATG GACTAACCCG ATATCTGGAG CCTCCCATAA TCTCTCTGTC TCATTTAATT CACTCTTAAA GGATTTAACT GTAATGCATT CC ..|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| .. 930 940 950 960 970 980 990 1000 1010 Mamasani CAATGTTT GCTATTACAG TGTTCACTTA TTTCTTTGAC CCTCACATCT GGTATCCATG TCCCAGTTAT ATCTTTAAGA GGACATCCTT TT Miandoab CAATGTTT GCTATTACAG TGTTCACTTA TTTCTTTGAC CCTCACATCT GGTATCCATG TCCCAGTTAT ATCTTTAAGA GGACATCCTT TT Shahriar CAATGTTT GCTATTACAG TGTTCACTTA TTTCTTTGAC CCTCACATCT GGTATCCATG TCCCAGTTAT ATCTTTAAGA GGACATCCTT TT ..|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| .. 1020 1030 1040 1050 1060 1070 1080 1090 1100 Mamasani GTCTTACT GTCATTAATA GTACAAGGAG TTAGAAAACG AGAATCATAA ATCAAATTAT CATAAGGATT CAAAAAAGGC TTGTGTTGTA TT Miandoab GTCTTACT GTCATTAATA GTACAAGGAG TTAGAAAACG AGAATCATAA ATCAAATTAT CATAAGGATT CAAAAAAGGC TTGTGTTGTA TT Shahriar GTCTTACT GTCATTAATA GTACAAGGAG TTAGAAAACG AGAATCATAA ATCAAATTAT CATAAGGATT CAAAAAAGGC TTGTGTTGTA TT ..|....| ....|....| ....|....| ....|....| .. 1110 1120 1130 1140 Mamasani AAGACATA AAATTCTATT TCTTGATTCA TTTCAGTATT CC Miandoab AAGACATA AAATTCTATT TCTTGATTCA TTTCAGTATT CC Shahriar AAGACATA AAATTCTATT TCTTGATTCA TTTCAGTATT CC Fig. 1. Alignment of partial G gene nucleotides (at residues 383–1142) of three bovine ephemeral fever isolates collected from different parts of Iran during autumn 2012 http://jad.tums.ac.ir Published Online: March 11, 2015 J Arthropod-Borne Dis, December 2015, 9(2): 195–203 M Bakhshesh and D Abdollahi: A Checklist of … 200 Fig. 2. Phylogenetic tree of partial G gene sequences of the Iranian Shahriar isolate with 17 isolates from Turkey, Australia, Taiwan, Egypt, Israel, China and Japan during the years 1966-2012. The scale represents 1% sequence divergence Fig. 3. Suckling mice inoculated with bovine ephem- eral fever presented paralysis, stiffness and death while control mouse marked with blue dot on the head (left) showed no clinical signs Fig. 4. A) Cytopathic effect in Vero cell inoculated with bovine ephemeral fever. B) Control Vero cell line showed no Cytopathic effect http://jad.tums.ac.ir Published Online: March 11, 2015 J Arthropod-Borne Dis, December 2015, 9(2): 195–203 M Bakhshesh and D Abdollahi: A Checklist of … 201 Discussion Bovine ephemeral fever is one of the most important infectious diseases of live- stock in Iran. The disease occurs usually in southern regions but sometimes observed as epidemic in other parts. Differential diagno- sis of BEF based on clinical observation is sometimes difficult and complicated. There- fore, tentative diagnosis is essential and nec- essary to implement urgent and proper prophy- lactic action, evaluation of the extent of the disease and further epidemiological studies. This study represents an accurate and relia- ble diagnosis using molecular approach for detection of the widely spread viral agent in Iran. In future, application of a real time PCR as introduced by Stram et al. (2005) will def- initely increase the sensitivity of BEFV di- agnosis and will also help to reveal the real extent of the virus. In this research, scientific procedure for the isolation of BEFV on cell culture was also successfully set up in the country. Com- plete characterization of the isolated viruses will certainly raise our knowledge about the epidemiology of the virus in the Middle-East and the world. These viruses can also be ap- plied for manufacturing an appropriate do- mestic vaccine against the virus and are also efficient for seroepidemiologic studies using viral neutralization (VN) test. As previously reported, the sequences from the world are clustered into three distinct lineages (Kato et al. 2009, Aziz-Boaro et al. 2012, Lin and Qiu 2012, Zheng and Qiu 2012, Walker et al. 2012). In the present study, Australian lineage is also a completely dis- tinct lineage, Israeli and some Turkish iso- lates are grouped into a separate lineage and the East Asian isolates together with Iranian, Egyptian and the other Turkish sequences construct the third lineage. This geographic distinction explained to be resulted from cir- culation of the virus in a vast distance by vectors and winds in Australia and East Asia (Murray 1970, Ogawa 1992, Shirakawa et al. 1994, Finlaison et al. 2010). However, the circulation of BEFV in the Middle East has been proposed to occur by both winds and animal transport, this hypothesis was sup- ported by close genetic relationship between some Middle East (Egyptian-2005) and East Asian sequences, and supportive documents indicating animal transport from China to Jordan in 2004 (Aziz-Baron et al. 2012). The Iranian 2012/SH was placed with the Turk- ish 2012/TR-ETLIK isolate in one branch (99.9% nucleotide identity), this branch with Chinese 2011/SHANDONG and Turkish 2012/ TRCP3 constructed a sublineage within the East Asian Lineage, the high similarity of some Turkish and the Iranian sequences with that of Chinese again provide supportive evidence for the possible two routes of transmission in the Middle East. Alignment of partial G gene sequence from 3 different regions (North-West, Centre and South) of Iran during the autumn of 2012 revealed that they are 100 % identical implying that the circulating virus was most likely the same in this period. However, this finding is not consistent with the sequences submitted in the GenBank from Turkey in the same period which are even separated into different lineages. These divergent se- quences may also support the role of animal transport beside the wind and insect in transmission of the virus in the region. Tur- key faced a severe BEF in 2012 with mor- tality rate of 15–20 % (Tonbak et al. 2012), this significant increase in mortality with acute form of BEF was also reported in China in 2011 (Zheng and Qiu 2012), but it is not clear whether the highly similar (96.4% nucleotide identity) Turkish (2012/TR-ETLIK) and Chinese (2011/SHANDONG) isolates or environmental factors (Walker 2013) are re- sponsible for the change in virulence in these two far regions. It is also remain to be eluci- http://jad.tums.ac.ir Published Online: March 11, 2015 J Arthropod-Borne Dis, December 2015, 9(2): 195–203 M Bakhshesh and D Abdollahi: A Checklist of … 202 dated how a number of Turkish viruses were co-circulated in a specific period and wheth- er this co-circulation affected the feature of the disease. Conclusion The geographic situation of Iran which is located between the East Asian and Middle- East countries places the country at high risk of BEFV transmission by both winds and animal transport. Characterization of more vi- ruses together with analysis of meteorologi- cal and entomological data and finding the model of animal transport in years will help in evaluating the risk of the virus transmis- sion in Iran and the Middle-East. In particu- lar, further research is essentially required to define the vector species and their ecology in Iran. Due to the high risk of BEFV transmis- sion and its consequences in the region, im- plementation of a specific surveillance system and large scale vaccination are recommended. Acknowledgements The author gratefully thanks to Razi Vac- cine and Serum Research Institute for finan- cial support of this study. The authors de- clare that there is no conflict of interests. References Aziz-Boaron O, Klausner Z, Hasoksuz M, Shenkar J, Gafni O, Gelman B, David D, Klement E (2012) Circulation of bovine ephemeral fever in the Middle East-Strong evidence for transmission by winds and animal transport. Vet Microbiol. 158: 300–307. 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