Transboundary and Emerging Diseases
SHORT COMMUNICATION
First Confirmation of Schmallenberg Virus in Cattle in Spain: Tissue Distribution and Pathology mez Antona2 and J. F. Garcıa Marın3 A. Balseiro1, L. J. Royo1, A. Go 1 2 3
n y Desarrollo Agroalimentario, Centro de Biotecnologıa Animal, Gijo n, Asturias, Spain SERIDA, Servicio Regional de Investigacio Guijuelo, Salamanca, Spain n, Leo n, Spain Facultad de Veterinaria, Universidad de Leo
Keywords: Schmallenberg virus; cattle; Spain Correspondence A. Balseiro. SERIDA, Servicio Regional de n y Desarrollo Agroalimentario, Investigacio Centro de Biotecnologıa Animal, 33394 n, Asturias, Spain. Tel.: +34 984502010; Gijo Fax: +34 984502012; E-mail: [email protected] Received for publication August 26, 2013
Summary Between January and June 2013, nine stillborn bovine foetuses with congenital malformations from nine cattle herds located in Salamanca (central Spain) were detected. Necropsy was performed on two calves. Pathological lesions together with molecular genetics and serological results allowed a definitive diagnosis: first confirmation of Schmallenberg virus (SBV) infection in cattle in Spain. SBV was detected in different tissues and organic fluids in both animals including blood, suggesting a possible viraemia. The umbilical cord was also positive for the presence of SBV in both animals. The former tissue provides an easy to obtain sample and might be a sample of choice when necropsy is carried out in the field.
doi:10.1111/tbed.12185
Introduction In November 2011, a previously unknown Orthobunyavirus named Schmallenberg virus (SBV) was detected in cattle in Germany, Europe (Hoffmann et al., 2012). Analysis of the SBV genome sequence showed that it belonged to the Simbu serogroup of the Bunyaviridae family, genus Orthobunyavirus (Hoffmann et al., 2012). As a midgetransmitted disease, the occurrence of SBV is closely related to the distribution of the primary vector, the Culicoides obsoletus group biting midges. To date, more than 8700 holdings have been reported with SBV cases confirmed by serological or molecular techniques from several European Member States (EFSA (European Food Safety Authority), 2013). SBV has mostly been detected in ruminant hosts, being confirmed in cattle, sheep, goat, deer, buffalo and moose (Linden et al., 2012; EFSA (European Food Safety Authority), 2013; Larska et al., 2013). Clinical signs in adult animals are either absent or non-specific, and include fever, decreased milk production and diarrhoea (Hoffmann et al., 2012; Lievaart-Peterson et al., 2012). Transplacental transmission occurs over a limited gestation range and results in the production of congenital abnormalities. © 2013 Blackwell Verlag GmbH • Transboundary and Emerging Diseases.
Infection in Spain was first reported in two newborn lambs in the Andalucıa region, Southern Spain in 2012 (EFSA (European Food Safety Authority), 2013). Afterwards, a seroprevalence of 36.8% was obtained in a study performed in five herds from the same area where those cases occurred. In 2012 and 2013, six cattle farms in Toledo and Lugo were shown to be seropositive for SBV but virus was not detected by PCR (Rojo Montejo et al., 2013). To the best of our knowledge, no further cases of SBV in domestic or wildlife species have been described in Spain since then. Material and Methods The cases Between January and June 2013, nine stillborn bovine foetuses with congenital malformations belonging to nine cattle herds located in Salamanca (central Spain) were detected. Foetuses were born at the normal time of gravidity. Pathological studies Systematic necropsies were performed on two of them, and gross lesions were recorded. Samples for histopathology were taken from the brain (cerebrum, midbrain, cerebellum 1
Schmallenberg virus in cattle in Spain
A. Balseiro et al.
and brain stem), spinal cord, liver, kidney, adrenal glands, lungs, spleen, lymph nodes, skeletal muscle, tongue, thymus, placenta and gut. They were fixed in 10% neutral buffered formalin and processed routinely to obtain haematoxylin and eosin (H&E) and Masson’s trichrome and Kl€ uber–Barrera luxol fast blue stained 4 lm sections. Molecular genetic studies Samples of different tissues (brain, spinal cord, placenta, umbilical cord, skeletal muscle and spleen) and body fluids (cerebrospinal fluid and blood) were taken for molecular analysis and immediately stored at 80°C. All samples were analysed in duplicate from two different tissue/fluid homogenates. Total RNA was extracted (TRIzol reagent; Gibco BRL, Grand Island, NY, USA) and treated with DNase I (Takara Bio Inc., Kyoto, Japan). The SBV genome was detected using the RT-qPCR protocol previously described (Bilk et al., 2012), using b-actin gen as an internal control system (Hoffmann B., personal communication). The RTqPCR was performed in a 7500 Real-Time PCR System (Applied Biosystems, Madrid, Spain). SBV presence was estimated by means of qbase+software (http://www.biogazelle.com/qbaseplus), using b-actin as the reference gene. Serological studies An indirect ELISA kit for the detection of anti-SBV antibodies in serum was used to identify seropositive cows (ID Screen Schmallenberg Virus Indirect Multispecies; Idexx, Montpellier, France). Sera from 11 cows from three suspected herds located in the same area in Salamanca were tested. Two cows were mothers of malformed calves presented in this study.
kyphosis, brachygnathia and severe hypoplasia of the central nervous system (CNS), leading to microcephaly and cerebellar and spinal cord hypoplasia. Calf Nº1 also showed hydranencephaly and lissencephaly, while calf Nº2 showed internal hydrocephalus. Atrophy and loss of skeletal muscle mass (especially in limbs and neck) and depleted fat deposits were also observed. Persistence of foramen ovale and right ventricle hypertrophy in heart was observed in calf Nº1 and palatoschisis in calf Nº2. The most important histopathological lesions were observed in the CNS and skeletal muscles (Fig. 2). Skeletal muscles and CNS malformations were more severe in calf Nº1 than in calf Nº2 where brain was hypoplasic but the basic structure was preserved. The predominant CNS lesions were cerebellar and cerebral hypoplasia, meningeal fibrosis, porencephaly, micromyelia, lack of neurons in brain and spinal cord and demyelination without inflammation. Midbrain showed dilatation of the aqueduct and lack of nervous nuclei but red nuclei (Fig. 2a). Cerebellar hypoplasia was characterized by severe reduced thickness of granular and molecular cell layers, demyelination, vacuoles and almost complete absence of Purkinje neurons (Fig. 2b, 2c). Medulla oblongata and cervical and thoracic spinal cord showed micromyelia and severely diminished diameter with severe reduction in gray matter, demyelination and lack of neurons in dorsal horns (Fig. 2d, 2e). Ventral meningeal fibrosis, especially in the median fissure, was also observed using Masson’s trichrome stain (Fig. 2d). Inflammation features and neuronal necrosis observed in other studies were not present in these animals (Herder et al., 2012). In skeletal muscle and tongue, most muscle fibres were missing and replaced by myxoid tissue, and remaining fibres showed myofibrillar hypoplasia (Fig. 2f). The remaining organs lacked significant histopathological lesions.
Results Pathology Gross lesions observed in both calves consisted of congenital malformations (Fig. 1): arthrogryposis, torticollis, scoliosis, (a)
(b)
Genetic studies The RT-qPCR results are presented in Table 1 along with the relative presence of the virus in different tissues or (c)
Fig. 1. Macroscopic features in SBV-infected stillborn bovine foetuses. (a) Aborted calf foetus infected with Schmallenberg virus (SBV) showing arthrogryposis, torticollis and scoliosis. (b) Central nervous system with severe hypoplasia, leading to microcephaly, hydranencephaly and cerebellar and spinal cord hypoplasia. (c) Hypoplasia of the central nervous system with internal hydrocephalus.
2
© 2013 Blackwell Verlag GmbH • Transboundary and Emerging Diseases.
A. Balseiro et al.
Schmallenberg virus in cattle in Spain
(a)
(b)
(c)
(d)
(e)
(f)
Fig. 2. Microscopic features in SBV-infected stillborn bovine foetuses. (a) Midbrain showing dilatation of the aqueduct (asterisk) and lack of nervous nuclei. Disorganized and underdeveloped red nuclei (arrow) can be observed remaining in midbrain. H&E stain. (b) Severe cerebellar hypoplasia characterized by severe reduce thickness of granular and molecular cell layers. H&E stain. (c) Cerebellar hypoplasia with greatly reduced of Purkinje neurons (arrow) in molecular layer. H&E stain. (d) Spinal cervical cord with severe reduction in gray matter and lack of nervous nuclei. Ventral meningeal fibrosis (asterisk), especially in the median fissure is also observed. H&E stain. Inset: Ventral meningeal fibrosis (asterisk) is demonstrated using Mas€ber–Barrera luxol fast blue stain. Inset: A control son’s trichrome stain. (e) Demyelination in thoracic spinal cord (asterisk) is demonstrated using Klu €ber–Barrera luxol fast blue stain. (f) Skeletal muscle where most muscle fibres are missing section from an age-matched control animal stained by Klu and replaced by myxoid (star) and adipose (arrowhead) tissue. Fibres remaining show myofibrillar hypoplasia (asterisk). HE stain. Table 1. Relative presence of SBV virus in different tissues and organ fluids using RT-qPCR Calf Nº1
Calf Nº2
Sample
Homogenate 1
Homogenate 2
Homogenate 1
Homogenate 2
Spleen Brain Spinal Cord Placenta Umbilical Cord Muscle Cerebrospinal fluid Blood
1.00 4.56 29.45 1965.00 630.30 95.01 34.30 261.40
1.00 27.67 52.35 4608.00 1938.00 109.10 184.80 639.10
1.00 1.95 770.70 Non-detected 6.59 Non-detected Non-detected Non-detected
1.00 Non-detected 1.813 Non-detected Non-detected 1.06 Non-detected 0.93
Data were normalized by means of b-actin amplification. Spleen sample in each homogenate-calf couple was used as reference sample to which all values are related.
organic fluids. Spleen was used as reference sample. In calf Nº1, SBV was detected in all tested samples, but no virus genome was detected in cerebrospinal fluid or placenta in calf Nº2.
study were positive. Mother of calf Nº1 presented a strong positive result and mother of calf Nº2 presented a weak positive result. One of 11 cows from one herd was doubtful.
Serological studies
Discussion
Anti-SBV antibodies were detected in 5 of 11 cows and in 3 of 3 herds. Mothers of both calves presented in this
Pathological lesions together with molecular genetics and serological results allowed a definitive diagnosis: SBV
© 2013 Blackwell Verlag GmbH • Transboundary and Emerging Diseases.
3
Schmallenberg virus in cattle in Spain
infection in cattle in central Spain. This case confirms the infection in a species other than sheep in Spain and the detection of the virus in areas of Spain other than Andalucıa. However, one year ago, a sheep herd, located in the same area where this case occurred, suffered similar malformations in lambs (G omez Antona A., unpublished data). Although a diagnosis was not achieved in this case, the facts suggest that this disease might have been present in this area for at least one year, with very recent introduction of SBV into the cattle population in the area. SBV was detected in different tissues and organic fluids in both animals including blood, suggesting a possible viraemia. The umbilical cord was also positive for the presence of SBV in both animals. The former tissue provides an easy to obtain sample and might be a sample of choice when necropsy is carried out in the field (Bilk et al., 2012). Both animals presented the same lesions but with different grades of severity. Any foetal immune response depends upon the state of development and maturation of the foetal immune system, and intensity or time of infection (Herder et al., 2013). Some samples from calf Nº2 gave a positive PCR result in only one of the duplicates, which seems to indicate an inhomogeneous distribution of SBV in the selected tissues (Bilk et al., 2012). These results agree with the less acute lesions found in calf Nº2 and may point out a relationship between virus distribution and presence and lesion severity (Herder et al., 2013). Antibodies against SBV were also higher in mother of calf Nº1 than in mother of calf Nº2. Infection of pregnant cows by SBV often results in transmission to the foetus across the placenta, and this transmission leads to abortion, stillbirth and congenital deformities if maternal infection occurred before the foetuses became immunocompetent, that is, before the 150th day of gestation (Charles, 1994). Thus, we deduced that the new virus and its vectors actively circulated throughout the area in summer 2012. This is the first time that SBV infection has been confirmed in Spain in cattle. The mechanism of SBV emergence is unknown. Congenital malformations in neighbouring communities were not observed. Transmission of SBV into these herds might have coincided with the introduction of animals purchased from France in that time period (Larska et al., 2013). However, no animals were purchased in those three herds at least in the last ten years. This case, together with the one previously described in Andalucıa, underlines the necessity to implement a specific surveillance plan in Spain focused on midges, wildlife species and livestock. This plan will be crucial to determine the actual impact of SBV in hunting, animal breeding and human health. 4
A. Balseiro et al.
Acknowledgements This paper was partially funded by a Grant from INIARTA2011-00010-00-00 (FEDER-Cofinantiated). We would like to thank G. Belver and S.Morales for their kind help and support. We also thank Dr. Kevin Dalton for critically reviewing the manuscript. A. Balseiro is a recipient of a ‘Contrato de Investigaci on para Doctores’ from INIA (Spain). References Bilk, S., C. Schulze, M. Fischer, M. Beer, A. Hlinak, and B. Hoffmann, 2012: Organ distribution of Schmallenberg virus RNA in malformed newborns. Vet. Microbiol. 159, 236–238. Charles, J. A., 1994: Akabane virus. Vet. Clin. North Am. Food Anim. Pract. 10, 525–546. EFSA (European Food Safety Authority), 2013. Schmallenberg virus: Analysis of the epidemiological data and assessment of impact. EFSA J., EN-429. Herder, V., P. Wohlsein, M. Peters, F. Hansmann, and W. Baumg€artner, 2012: Salient lesions in domestic ruminants infected with the emerging so-called Schmallenberg virus in Germany. Vet. Pathol. 49, 588–591. Herder, V., F. Hansmann, P. Wohlsein, M. Peters, M. Varela, M. Palmarini, and W. Baumg€artner, 2013: Immunophenotyping of inflammatory cells associated with Schmallenberg virus infection of the central nervous system of ruminants. PLoS One 8, e62939. Hoffmann, B., M. Scheuch, D. H€ oper, R. Jungblut, M. Holsteg, H. Schirrmeier, M. Eschbaumer, K. V. Goller, K. Wernike, M. Fischer, A. Breithaupt, T. C. Mettenleiter, and M. Beer, 2012: Novel Orthobunyavirus in cattle, Europe, 2011. Emerg. Infect. Dis. 18, 469–472. Larska, M., M. P. Polak, M. Grochowska, L. Lechowski, J. S. Zwiazzek, and J. F. Zmudzinski, 2013: First report of Schmallenberg virus infection in cattle and midges in Poland. Transbound Emerg. Dis. 60, 97–101. Lievaart-Peterson, K., S. J. M. Luttikholt, R. van den Brom, and P. Vellema, 2012: Schmallenberg virus infection in small ruminants – First review of the situation and prospects in Northern Europe. Small Rum. Res. 106, 71–76. Linden, A., D. Desmecht, R. Volpe, M. Wirtgen, F. Grefoire, J. Pirson, J. Paternostre, D. Kleijnen, H. Schirrmeier, M. Beer, and M. M. Garigliany, 2012: Epizzotic spread of Schmallenberg virus among wild cervids, Belgium, Fall 2011. Emerg. Infect. Dis. 18, 2006–2008. Rojo Montejo, S., J. M. San Miguel Ayanz, J. J. Vizuete Barrios, O. Garcıa Fari~ na, V. Navarro Lozano, E. Collantes Fernandez, G. Ad uriz, and L. M. Ortega Mora, 2013. Descripci on de varios casos clınicos asociados a la infecci on por el virus de la enfermedad de Schmallenberg en ganado bovino en Espa~ na. In: Proceedings of XVIII Congreso Internacional ANEMBE de Medicina Bovina. 24–26th April 2013, pp. 196–198, ANEMBE, Lleida, Spain.
© 2013 Blackwell Verlag GmbH • Transboundary and Emerging Diseases.
SHORT COMMUNICATION
First Confirmation of Schmallenberg Virus in Cattle in Spain: Tissue Distribution and Pathology mez Antona2 and J. F. Garcıa Marın3 A. Balseiro1, L. J. Royo1, A. Go 1 2 3
n y Desarrollo Agroalimentario, Centro de Biotecnologıa Animal, Gijo n, Asturias, Spain SERIDA, Servicio Regional de Investigacio Guijuelo, Salamanca, Spain n, Leo n, Spain Facultad de Veterinaria, Universidad de Leo
Keywords: Schmallenberg virus; cattle; Spain Correspondence A. Balseiro. SERIDA, Servicio Regional de n y Desarrollo Agroalimentario, Investigacio Centro de Biotecnologıa Animal, 33394 n, Asturias, Spain. Tel.: +34 984502010; Gijo Fax: +34 984502012; E-mail: [email protected] Received for publication August 26, 2013
Summary Between January and June 2013, nine stillborn bovine foetuses with congenital malformations from nine cattle herds located in Salamanca (central Spain) were detected. Necropsy was performed on two calves. Pathological lesions together with molecular genetics and serological results allowed a definitive diagnosis: first confirmation of Schmallenberg virus (SBV) infection in cattle in Spain. SBV was detected in different tissues and organic fluids in both animals including blood, suggesting a possible viraemia. The umbilical cord was also positive for the presence of SBV in both animals. The former tissue provides an easy to obtain sample and might be a sample of choice when necropsy is carried out in the field.
doi:10.1111/tbed.12185
Introduction In November 2011, a previously unknown Orthobunyavirus named Schmallenberg virus (SBV) was detected in cattle in Germany, Europe (Hoffmann et al., 2012). Analysis of the SBV genome sequence showed that it belonged to the Simbu serogroup of the Bunyaviridae family, genus Orthobunyavirus (Hoffmann et al., 2012). As a midgetransmitted disease, the occurrence of SBV is closely related to the distribution of the primary vector, the Culicoides obsoletus group biting midges. To date, more than 8700 holdings have been reported with SBV cases confirmed by serological or molecular techniques from several European Member States (EFSA (European Food Safety Authority), 2013). SBV has mostly been detected in ruminant hosts, being confirmed in cattle, sheep, goat, deer, buffalo and moose (Linden et al., 2012; EFSA (European Food Safety Authority), 2013; Larska et al., 2013). Clinical signs in adult animals are either absent or non-specific, and include fever, decreased milk production and diarrhoea (Hoffmann et al., 2012; Lievaart-Peterson et al., 2012). Transplacental transmission occurs over a limited gestation range and results in the production of congenital abnormalities. © 2013 Blackwell Verlag GmbH • Transboundary and Emerging Diseases.
Infection in Spain was first reported in two newborn lambs in the Andalucıa region, Southern Spain in 2012 (EFSA (European Food Safety Authority), 2013). Afterwards, a seroprevalence of 36.8% was obtained in a study performed in five herds from the same area where those cases occurred. In 2012 and 2013, six cattle farms in Toledo and Lugo were shown to be seropositive for SBV but virus was not detected by PCR (Rojo Montejo et al., 2013). To the best of our knowledge, no further cases of SBV in domestic or wildlife species have been described in Spain since then. Material and Methods The cases Between January and June 2013, nine stillborn bovine foetuses with congenital malformations belonging to nine cattle herds located in Salamanca (central Spain) were detected. Foetuses were born at the normal time of gravidity. Pathological studies Systematic necropsies were performed on two of them, and gross lesions were recorded. Samples for histopathology were taken from the brain (cerebrum, midbrain, cerebellum 1
Schmallenberg virus in cattle in Spain
A. Balseiro et al.
and brain stem), spinal cord, liver, kidney, adrenal glands, lungs, spleen, lymph nodes, skeletal muscle, tongue, thymus, placenta and gut. They were fixed in 10% neutral buffered formalin and processed routinely to obtain haematoxylin and eosin (H&E) and Masson’s trichrome and Kl€ uber–Barrera luxol fast blue stained 4 lm sections. Molecular genetic studies Samples of different tissues (brain, spinal cord, placenta, umbilical cord, skeletal muscle and spleen) and body fluids (cerebrospinal fluid and blood) were taken for molecular analysis and immediately stored at 80°C. All samples were analysed in duplicate from two different tissue/fluid homogenates. Total RNA was extracted (TRIzol reagent; Gibco BRL, Grand Island, NY, USA) and treated with DNase I (Takara Bio Inc., Kyoto, Japan). The SBV genome was detected using the RT-qPCR protocol previously described (Bilk et al., 2012), using b-actin gen as an internal control system (Hoffmann B., personal communication). The RTqPCR was performed in a 7500 Real-Time PCR System (Applied Biosystems, Madrid, Spain). SBV presence was estimated by means of qbase+software (http://www.biogazelle.com/qbaseplus), using b-actin as the reference gene. Serological studies An indirect ELISA kit for the detection of anti-SBV antibodies in serum was used to identify seropositive cows (ID Screen Schmallenberg Virus Indirect Multispecies; Idexx, Montpellier, France). Sera from 11 cows from three suspected herds located in the same area in Salamanca were tested. Two cows were mothers of malformed calves presented in this study.
kyphosis, brachygnathia and severe hypoplasia of the central nervous system (CNS), leading to microcephaly and cerebellar and spinal cord hypoplasia. Calf Nº1 also showed hydranencephaly and lissencephaly, while calf Nº2 showed internal hydrocephalus. Atrophy and loss of skeletal muscle mass (especially in limbs and neck) and depleted fat deposits were also observed. Persistence of foramen ovale and right ventricle hypertrophy in heart was observed in calf Nº1 and palatoschisis in calf Nº2. The most important histopathological lesions were observed in the CNS and skeletal muscles (Fig. 2). Skeletal muscles and CNS malformations were more severe in calf Nº1 than in calf Nº2 where brain was hypoplasic but the basic structure was preserved. The predominant CNS lesions were cerebellar and cerebral hypoplasia, meningeal fibrosis, porencephaly, micromyelia, lack of neurons in brain and spinal cord and demyelination without inflammation. Midbrain showed dilatation of the aqueduct and lack of nervous nuclei but red nuclei (Fig. 2a). Cerebellar hypoplasia was characterized by severe reduced thickness of granular and molecular cell layers, demyelination, vacuoles and almost complete absence of Purkinje neurons (Fig. 2b, 2c). Medulla oblongata and cervical and thoracic spinal cord showed micromyelia and severely diminished diameter with severe reduction in gray matter, demyelination and lack of neurons in dorsal horns (Fig. 2d, 2e). Ventral meningeal fibrosis, especially in the median fissure, was also observed using Masson’s trichrome stain (Fig. 2d). Inflammation features and neuronal necrosis observed in other studies were not present in these animals (Herder et al., 2012). In skeletal muscle and tongue, most muscle fibres were missing and replaced by myxoid tissue, and remaining fibres showed myofibrillar hypoplasia (Fig. 2f). The remaining organs lacked significant histopathological lesions.
Results Pathology Gross lesions observed in both calves consisted of congenital malformations (Fig. 1): arthrogryposis, torticollis, scoliosis, (a)
(b)
Genetic studies The RT-qPCR results are presented in Table 1 along with the relative presence of the virus in different tissues or (c)
Fig. 1. Macroscopic features in SBV-infected stillborn bovine foetuses. (a) Aborted calf foetus infected with Schmallenberg virus (SBV) showing arthrogryposis, torticollis and scoliosis. (b) Central nervous system with severe hypoplasia, leading to microcephaly, hydranencephaly and cerebellar and spinal cord hypoplasia. (c) Hypoplasia of the central nervous system with internal hydrocephalus.
2
© 2013 Blackwell Verlag GmbH • Transboundary and Emerging Diseases.
A. Balseiro et al.
Schmallenberg virus in cattle in Spain
(a)
(b)
(c)
(d)
(e)
(f)
Fig. 2. Microscopic features in SBV-infected stillborn bovine foetuses. (a) Midbrain showing dilatation of the aqueduct (asterisk) and lack of nervous nuclei. Disorganized and underdeveloped red nuclei (arrow) can be observed remaining in midbrain. H&E stain. (b) Severe cerebellar hypoplasia characterized by severe reduce thickness of granular and molecular cell layers. H&E stain. (c) Cerebellar hypoplasia with greatly reduced of Purkinje neurons (arrow) in molecular layer. H&E stain. (d) Spinal cervical cord with severe reduction in gray matter and lack of nervous nuclei. Ventral meningeal fibrosis (asterisk), especially in the median fissure is also observed. H&E stain. Inset: Ventral meningeal fibrosis (asterisk) is demonstrated using Mas€ber–Barrera luxol fast blue stain. Inset: A control son’s trichrome stain. (e) Demyelination in thoracic spinal cord (asterisk) is demonstrated using Klu €ber–Barrera luxol fast blue stain. (f) Skeletal muscle where most muscle fibres are missing section from an age-matched control animal stained by Klu and replaced by myxoid (star) and adipose (arrowhead) tissue. Fibres remaining show myofibrillar hypoplasia (asterisk). HE stain. Table 1. Relative presence of SBV virus in different tissues and organ fluids using RT-qPCR Calf Nº1
Calf Nº2
Sample
Homogenate 1
Homogenate 2
Homogenate 1
Homogenate 2
Spleen Brain Spinal Cord Placenta Umbilical Cord Muscle Cerebrospinal fluid Blood
1.00 4.56 29.45 1965.00 630.30 95.01 34.30 261.40
1.00 27.67 52.35 4608.00 1938.00 109.10 184.80 639.10
1.00 1.95 770.70 Non-detected 6.59 Non-detected Non-detected Non-detected
1.00 Non-detected 1.813 Non-detected Non-detected 1.06 Non-detected 0.93
Data were normalized by means of b-actin amplification. Spleen sample in each homogenate-calf couple was used as reference sample to which all values are related.
organic fluids. Spleen was used as reference sample. In calf Nº1, SBV was detected in all tested samples, but no virus genome was detected in cerebrospinal fluid or placenta in calf Nº2.
study were positive. Mother of calf Nº1 presented a strong positive result and mother of calf Nº2 presented a weak positive result. One of 11 cows from one herd was doubtful.
Serological studies
Discussion
Anti-SBV antibodies were detected in 5 of 11 cows and in 3 of 3 herds. Mothers of both calves presented in this
Pathological lesions together with molecular genetics and serological results allowed a definitive diagnosis: SBV
© 2013 Blackwell Verlag GmbH • Transboundary and Emerging Diseases.
3
Schmallenberg virus in cattle in Spain
infection in cattle in central Spain. This case confirms the infection in a species other than sheep in Spain and the detection of the virus in areas of Spain other than Andalucıa. However, one year ago, a sheep herd, located in the same area where this case occurred, suffered similar malformations in lambs (G omez Antona A., unpublished data). Although a diagnosis was not achieved in this case, the facts suggest that this disease might have been present in this area for at least one year, with very recent introduction of SBV into the cattle population in the area. SBV was detected in different tissues and organic fluids in both animals including blood, suggesting a possible viraemia. The umbilical cord was also positive for the presence of SBV in both animals. The former tissue provides an easy to obtain sample and might be a sample of choice when necropsy is carried out in the field (Bilk et al., 2012). Both animals presented the same lesions but with different grades of severity. Any foetal immune response depends upon the state of development and maturation of the foetal immune system, and intensity or time of infection (Herder et al., 2013). Some samples from calf Nº2 gave a positive PCR result in only one of the duplicates, which seems to indicate an inhomogeneous distribution of SBV in the selected tissues (Bilk et al., 2012). These results agree with the less acute lesions found in calf Nº2 and may point out a relationship between virus distribution and presence and lesion severity (Herder et al., 2013). Antibodies against SBV were also higher in mother of calf Nº1 than in mother of calf Nº2. Infection of pregnant cows by SBV often results in transmission to the foetus across the placenta, and this transmission leads to abortion, stillbirth and congenital deformities if maternal infection occurred before the foetuses became immunocompetent, that is, before the 150th day of gestation (Charles, 1994). Thus, we deduced that the new virus and its vectors actively circulated throughout the area in summer 2012. This is the first time that SBV infection has been confirmed in Spain in cattle. The mechanism of SBV emergence is unknown. Congenital malformations in neighbouring communities were not observed. Transmission of SBV into these herds might have coincided with the introduction of animals purchased from France in that time period (Larska et al., 2013). However, no animals were purchased in those three herds at least in the last ten years. This case, together with the one previously described in Andalucıa, underlines the necessity to implement a specific surveillance plan in Spain focused on midges, wildlife species and livestock. This plan will be crucial to determine the actual impact of SBV in hunting, animal breeding and human health. 4
A. Balseiro et al.
Acknowledgements This paper was partially funded by a Grant from INIARTA2011-00010-00-00 (FEDER-Cofinantiated). We would like to thank G. Belver and S.Morales for their kind help and support. We also thank Dr. Kevin Dalton for critically reviewing the manuscript. A. Balseiro is a recipient of a ‘Contrato de Investigaci on para Doctores’ from INIA (Spain). References Bilk, S., C. Schulze, M. Fischer, M. Beer, A. Hlinak, and B. Hoffmann, 2012: Organ distribution of Schmallenberg virus RNA in malformed newborns. Vet. Microbiol. 159, 236–238. Charles, J. A., 1994: Akabane virus. Vet. Clin. North Am. Food Anim. Pract. 10, 525–546. EFSA (European Food Safety Authority), 2013. Schmallenberg virus: Analysis of the epidemiological data and assessment of impact. EFSA J., EN-429. Herder, V., P. Wohlsein, M. Peters, F. Hansmann, and W. Baumg€artner, 2012: Salient lesions in domestic ruminants infected with the emerging so-called Schmallenberg virus in Germany. Vet. Pathol. 49, 588–591. Herder, V., F. Hansmann, P. Wohlsein, M. Peters, M. Varela, M. Palmarini, and W. Baumg€artner, 2013: Immunophenotyping of inflammatory cells associated with Schmallenberg virus infection of the central nervous system of ruminants. PLoS One 8, e62939. Hoffmann, B., M. Scheuch, D. H€ oper, R. Jungblut, M. Holsteg, H. Schirrmeier, M. Eschbaumer, K. V. Goller, K. Wernike, M. Fischer, A. Breithaupt, T. C. Mettenleiter, and M. Beer, 2012: Novel Orthobunyavirus in cattle, Europe, 2011. Emerg. Infect. Dis. 18, 469–472. Larska, M., M. P. Polak, M. Grochowska, L. Lechowski, J. S. Zwiazzek, and J. F. Zmudzinski, 2013: First report of Schmallenberg virus infection in cattle and midges in Poland. Transbound Emerg. Dis. 60, 97–101. Lievaart-Peterson, K., S. J. M. Luttikholt, R. van den Brom, and P. Vellema, 2012: Schmallenberg virus infection in small ruminants – First review of the situation and prospects in Northern Europe. Small Rum. Res. 106, 71–76. Linden, A., D. Desmecht, R. Volpe, M. Wirtgen, F. Grefoire, J. Pirson, J. Paternostre, D. Kleijnen, H. Schirrmeier, M. Beer, and M. M. Garigliany, 2012: Epizzotic spread of Schmallenberg virus among wild cervids, Belgium, Fall 2011. Emerg. Infect. Dis. 18, 2006–2008. Rojo Montejo, S., J. M. San Miguel Ayanz, J. J. Vizuete Barrios, O. Garcıa Fari~ na, V. Navarro Lozano, E. Collantes Fernandez, G. Ad uriz, and L. M. Ortega Mora, 2013. Descripci on de varios casos clınicos asociados a la infecci on por el virus de la enfermedad de Schmallenberg en ganado bovino en Espa~ na. In: Proceedings of XVIII Congreso Internacional ANEMBE de Medicina Bovina. 24–26th April 2013, pp. 196–198, ANEMBE, Lleida, Spain.
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