Preliminary Results of Schmallenberg virus Serosurveillance in Romania

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1 Preliminary Results of Schmallenberg virus Serosurveillance in Romania DOINA DANES 1, STELIAN BARAITAREANU 1, MARIA RODICA GURAU 1, MARIUS DAN 1, ALIN BARTOIU 1, HORATIU MOLDOVAN 2, MIHAI DANES 3 1 University of Agronomical Sciences and Veterinary Medicine of Bucharest, Faculty of Veterinary Medicine Campus, Splaiul Independentei 105, 5 th District, Bucharest, ROMANIA 2 MSD, Intervet Romania SRL, Sos. Bucuresti-Ploiesti, nr.1a, 1 th District, Bucharest, ROMANIA 3 University of Agronomical Sciences and Veterinary Medicine of Bucharest, Agronomic Campus, Marasti 59, 1 th District, Bucharest, ROMANIA doruvet@yahoo.com Abstract: Schmallenberg virus (SBV), the newest Orthobunyavirus occurred at the end of the 2011 in northwest Germany and The Netherlands. SBV spread rapidly in almost all Europe in less than three years after its first report. The objective of study was the serosurveillance of SBV / Simbu group viruses in cattle and sheep herds in southeastern Romania, as preliminary study of SBV circulation throughout Romania. Serological test of samples collected from September do December 2013 were 22.65% positive in cattle and 5.67% positive in sheep. Evaluation of Schmallenberg virus / Simbu group viruses by ELISA Ab test Kit could be a useful tool in active surveillance and assessment of virus circulation in Romania. Even if the ELISA for antibody detection do not provide a definite diagnosis, the advantages of this technique recommend it to be use in testing a large number of animals. These data recommend the implementation of the surveillance program to establish the herds status for SBV in Romania, as keystone for the Romanian veterinary policy decision, as actor in the European Union monitoring program. Key-Words: arbovirus, Orthobunyavirus, ruminant reproductive pathology, arboviral diseases 1 Introduction Schmallenberg virus (SBV) is a new arbovirus (family Bunyaviridae, genus Orthobunyavirus) that emerged in Europe in 2011 affecting cattle, sheep and goat from several countries [5, 15]. In less than three years after first reporting, SBV has spread rapidly in almost all Europe [7]. Late in 2011, an unknown disease in cattle was registered in Germany and the Netherlands. Clinical features of the new diseases were fever, decreased milk production, and diarrhea. Rapidly as never before, using metagenomic analysis, identified a novel Orthobunyavirus, isolated further from blood of affected animals. Surveillance was initiated to test malformed newborn animals in the affected region [12]. From DNA and RNA isolated from plasma, samples libraries were generated. Seven Orthobunyavirus sequences were detected in the library prepared from pooled RNA. Further studies on sequences M and L segments enabled to locate the new virus in Simbu group. Up to the origin of the first positive sample, the new Bunyavirus was named Schmallenberg virus (SBV). SBV was reported in Germany (2011), The Netherlands (2011), Belgium (2011), United Kingdom (2012), France (2012), Luxembourg (2012), Italy (2012), Spain (2012), Denmark (2012), Switzerland (2012), Austria (2012), Poland (2012), Sweden (2012), Finland (2012), Ireland (2012), Norway (2012), Czech Republic (2012), Estonia (2013) and Slovenia (2013) [6, 7], and new reports are publishing. The members of the family Bunyaviridae are widely distributed; representants of Simbu group are spread mainly through biting midges as Culicoides spp. and mosquitoes. Akabane, Aino, and Shamonda viruses are the members of the Simbu group, well known as pathogens of ruminants, but none have been detected in Europe [16]. Abortion and stillbirths with foetal abnormalities, mainly in sheep but in cattle and goats too, were identified in the Netherlands, Germany and Belgium from December In England the disease and the SBV has been found in Central and Southern part, in lambs and calves, suggesting the introduction of the SBV by wind-blown insect originating in Europe, during the active midge season, opening the way to previously unaffected areas [3]. Doceul et al. (2013) reported outbreaks of Schmallenberg virus in cattle, sheep and goats, and not in horses, dogs or humans [7]. Now is already know that are sensitive to this virus sheep and could become ill, cattle and goats and it s also ISBN:

2 recommended to be aware to exotic or wild ruminants and cervid families [1]. In 2012, rise up the first data about dogs susceptibility, from a dog breeding kennel in northwestern France (Orne), observed in a litter of 5 puppies: at birth they express neurologic signs and stunted growth and four of the puppies, died at 5 6 weeks of age. The female dogs tested for SBV sowed positive results by virus neutralization against the virus of the kennel, proving the exposure. This is not clear if this was un isolate event or if they are many cases undiagnosed or under investigated. Few other reports on different Orthobunyavirus infections in dogs are available, as serological survey carried out in US [9] and Mexico [2]. In pigs SBV infections induce seroconversion but are ineffective in terms of virus replication and excretion, suggesting that pigs do not play in the SBV epidemiology [14]. The clinical signs vary up to the age of animal at exposure time; adult cows express diarrhoea, fever, and reduction in milk yield, followed by a full and rapid recovery several days later. In adult seeps clinical signs did not reported [12]. In all species of domestic ruminants, in newborns and foetuses, the disease, is associate with arthrogryposis, twisted neck or spine, a domed appearance to the skull, short lower jaw and brain deformities and severe damage to the spinal cord [8, 10]. Some animals seem normal at birth but show nervous signs such as blindness, ataxia, recumbency, inability to suck and even convulsions. The foetal deformities vary depending on the age of pregnancy at the time of infection; typical malformations are neuromusculoskeletal disorder (ankylosis, arthrogryposis, severe torticollis, brachygnathia inferior, kyphosis, lordosis scoliosis) and neurological disorders (behavioral abnormalities, amaurosis, ataxia,) [1, 11]. Sheep and goats seem to have clinical and sessional features much mildly than cattle [7]. In small ruminants were described abortions and birth of malformed lambs and kids [7, 18]. Because several of related viruses from Bunyaviruses can affect humans, one of the main concern at the occurrence of this pathogen was the human risk. The human pathogens belonging to the group due this ability to a gene sequence missing in Schmallenberg virus. Tis is in accord to the lack of any of human cases in any country where SBV was isolated. The conclusion of risk assessment performed by the European Centre for Disease Prevention and Control (ECDC), the Robert Koch Institute (RKI), Germany, and the National Institute of Public Health and the Environment (RIVM), Netherlands, vas that it is very unlikely that SBV poses a risk to humans and that Schmallenberg virus is very unlikely to cause illness in people [1]. Short time after virus identification, indirect ELISA tests for Schmallenberg antibody were designed [e.g. IDEXX Schmallenberg Ab test Kit (Switzerland), ID-VET ID Screen Schmallenberg virus Indirect (France)] [4, 17]. Assays shown sensitive value in detection of antibodies in foetal or procolostral sera and diagnose SBV in newborn calves and lambs [4]. Sensitivity of those test appears to be even higher than RT-qPCR, Van Maanen et al. (2012) in a study concerning anti- SBV antibodies in foetal blood samples from calves and lambs obtained 74% ELISA positive results and 27% RT-qPCR positive results [19]. Also, ELISA tests for anti-sbv antibodies detection in milk have been developed [13] and, to control the epidemic, it is available on the market a commercial vaccine. 2 Problem Formulation The objective of study was the survey of SBV / Simbu group viruses in cattle and sheep herds in southeastern Romania by antibody detection, as preliminary study of SBV circulation in Romanian herds. 2.1 Materials Farms location, animals and biological samples Survey was conducted in cattle and sheep farms located in following counties: Bacau, Vrancea, Galati, Braila, Buzau, Tulcea, Constanta, Buzau, Ialomita, Calarasi, Prahova, Ilfov, Giurgiu, and Mehedinti. Collection of serum samples from 415 bovine serum and 229 ovine, from southwestern of Romania, were from September do December Method of Blood Sampling Blood samples collected from the jugular vein has been done in accordance with the Directive 2010/63/EU of the European Parliament and of the Council. Three milliliters of blood has been collected in sterile conditions by using Vacutainer blood collection tubes (Becton Dickinson, USA). The samples were refrigerated at 4 8 C before serum extraction in the same day, and serum frozen at 20 C until serologic testing. ISBN:

3 2.2 Indirect enzyme-linked immunosorbent assay (ELISA) for Schmallenberg antibody testing In accordance with the manufacturer s instructions of Schmallenberg Ab test Kit (IDEXX, Switzerland), serum samples and controls were diluted 1:10 and dispensed in volume of 100 µl in appropriate wells of the microtiter plate. After gently shaking the microtiter plates were covered and incubated for 60 minutes at C. Afterwards, microtiter wells were washed 3 times with 300 µl wash solution, 100 µl conjugate was dispensed into each well, covered and incubated for 60 minutes at C. Afterwards, microtiter wells were washed 3 times with 300 µl wash solution, 100 µl TMB substrate N.12 into each well, covered and incubated for 10 minutes at C. Color reaction was stopped by adding 100 µl Stop solution N.3 into each well. Results were read with a photometer at a wavelength of 450 nm, and the optical density (OD) of the samples were analyzed in relation to the negative and the positive controls with the formula presented in fig.1. Table 2 Sheep with antibodies for Schmallenberg virus or other Simbu serogroup viruses in southeastern Romania* Serum samples County Positive Total No. % Bacau Vrancea Galati Braila Buzau Tulcea Constanta Ialomita Calarasi Prahova Dambovita Ilfov Giurgiu Mehedinti TOTAL *IDEXX Schmallenberg Ab test Kit (Switzerland) Fig.1 Calculation formula of sample per positive proportion in percent. Values used in interpretation of results: negative if S/P% is <30%, suspect if S/P% 30% and 40%, and positive if S/P% 30%. 3 Problem Solution Serological result in table 1 for cattle and in table 2 for sheep. Table 1 Cattle with antibodies for Schmallenberg virus / Simbu group viruses in south-eastern Romania* Serum samples County Positive Total No. % Bacau Vrancea Galati Braila Buzau Tulcea Constanta Ialomita Calarasi Prahova Dambovita Ilfov Giurgiu Mehedinti TOTAL *IDEXX Schmallenberg Ab test Kit (Switzerland) Fig.2 Romanian counties with serological suspicion of Schmallenberg virus / Simbu group viruses in cattle and sheep herds in serum samples from collected September do December Dark red color corresponds to the maximum prevalence in positive counties. Green color marks the county in which none of the samples were positive. Indirect ELISA revealed presence of specific SBV / Simbu group virus antibodies in cattle and sheep (Table1, 2). 4 Conclusion Serological results of samples collected from September do December 2013 were 22.65% positive in cattle and 5.67% positive in sheep. To establish undoubtedly the SBV Romanian herd status, furter study in order to isolate the Evaluation of Schmallenberg virus / Simbu group viruses by ELISA Ab test Kit could be the solution ISBN:

4 in active surveillance and quantification of virus circulation in Romania. Even if the ELISA method not provides a definite diagnosis, advantages of this technique recommend its use in testing a large number of animals. The positive results are not related with data proving reproduction disorders or pathology in newborn. These data seem to be in accord with a DEFRA notice. Otherwise, these results strongly suggest to extend the survey and to investigate other possible way of transmission, out of vectors and human activities and also, to carry out studies for the isolation of SBV or for its genom. These data suggest development of active surveillance programs of cattle, goat and sheep for SBV in Romania, as part of European Union program of surveillance. References: [1] AHVLA, Schmallenberg virus. Page last modified: 24 May Available at: [2] Blitvich, B.J., Saiyasombat, R., Da Rosa, A.T., Tesh, R.B., Calisher, C.H., Garcia-Rejon, J.E., Orthobunyaviruses, a common cause of infection of livestock in the Yucatan Peninsula of Mexico. American Journal of Tropical Medicine and Hygiene, Vol.87, No.6, 2012, pp [3] Bradshaw, B., Evidence that exposure to Schmallenberg virus has been quite widespread in Southern and South-eastern counties of Ireland during Veterinary Laboratory Service. Sally Gaynor, National Disease Control Centre, Department of Agriculture, Food and the Marine (DAFM), Available at: imalhealthwelfare/diseasecontrols/schmallenbergvir us/schmallenbergvirussummary17jan13.pdf [4] Bréard E, Lara E, Comtet L, Viarouge C, Doceul V, Desprat A, Vitour D, Pozzi N, Cay AB, De Regge N, Pourquier P, Schirrmeier H, Hoffmann B, Beer M, Sailleau C, Zientara S: Validation of a commercially available indirect ELISA using a nucleocapside recombinant protein for detection of Schmallenberg virus antibodies. PLoS One, 2013, 8:e [5] Conraths, F., Kämer, D., Teske, K., Hoffmann, B., Mettenleiter, T.C., Beer, M., Reemerging Schmallenberg virus infections, Germany. Emerging Infectious Diseases, Vol.19, No.3, 2013, pp [6] De Regge, N., Madder, M., Deblauwe, I., Losson, B., Fassotte, C., Demeulemeester, J., Smeets, F., Tomme, M., Cay, A.B., Schmallenberg Virus Circulation in Culicoides in Belgium in 2012: Field Validation of a Real Time RT-PCR Approach to Assess Virus Replication and Dissemination in Midges. PLoS One, Vol.9, 2014, e [7] Doceu, D., Lara, E., Sailleau, C., Belbis, G., Richardson, J., Bréard, E., Viarouge, C., Dominguez, M., Hendrikx, P., Calavas, D., Desprat, A., Languille, J., Comtet, L., Pourquier, P., Eléouët, J.F., Delmas, B, Marianneau, P., Vitour, D., Zientara, S., Epidemiology, molecular virology and diagnostics of Schmallenberg virus, an emerging orthobunyavirus in Europe. Veterinary Research, Vol.44, No.1, 2013, 44/1/31. Available at: [8] Garigliany, M.M., Hoffmann, B., Dive, M., Sartelet, A., Bayrou, C., Cassart, D., Beer, M., Desmecht, D., Schmallenberg virus in calf born at term with porencephaly, Belgium. Emerging Infectious Diseases, Vol.18, No.6, 2012, pp [9] Godsey, M.S.Jr, Amoo, F., Yuill, T.M., Defoliart, G.R., California serogroup virus infections in Wisconsin domestic animals, American Journal of Tropical Medicine and Hygiene, Vol. 39, No.5, 1988, pp [10] Hahn, K., Habierski, A., Herder, V., Wohlsein, P., Peters, M., Hansmann, F., Baumgartner, W., Schmallenberg virus in central nervous system of ruminants. Emerging Infectious Diseases, Vol. 19, no.1, 2012, pp [11] Herder, V., Wohlsein, P., Peters, M., Hansmann, F., Baumgartner, W., Salient lesions in domestic ruminants infected with the emerging socalled Schmallenberg virus in Germany. Veterinary Pathology, Vol.49, No.4, 2012, pp [12] Hoffmann, B., Scheuch, M., Hoper, D., Jungblut, R., Holsteg, M., Schirrmeier, H., Eschbaumer, M., Goller, K.V., Wernike, K., Fischer, M., Breithaupt, A., Mettenleiter, T.C., Beer, M., Novel orthobunyavirus in Cattle, Europe, Emerging Infectious Diseases, Vol.18, No.3, 2011, pp [13] Humphries, D., Burr, P., Schmallenberg virus milk antibody ELISA. Veterinary Records, Vol.171, No.20, 2012, pp [14] Poskin, A., Van Campec, W., Mostinc, L., Cayb, B., De Reggeb, N. Experimental Schmallenberg virus infection of pigs. Veterinary Microbiology, Vol.170, No. 3 4, 2014, pp ISBN:

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