Serological testing of Schmallenberg virus in Swedish wild cervids from 2012 to 2016.

BACKGROUND: Schmallenberg virus (SBV) first emerged in Europe in 2011, and in Sweden in late 2012. The virus was still circulating in parts of Europe in 2015. In recent testing, the virus has not been detected in Swedish domestic animals, indicating that it is no longer circulating in Sweden. It is not known if the virus has circulated and is still circulating in Swedish wild cervid populations and whether wildlife can act as virus reservoirs. The aim of this study was to investigate whether SBV has circulated, and is still circulating among wild cervids in Sweden. RESULTS: Ninety-two sera from moose (Alces alces, n = 22), red deer (Cervus elaphus, n = 15), fallow deer (Dama dama, n = 44), and roe deer (Capreolus capreolus, n = 11) were collected and analyzed for antibodies against SBV. The sampling occurred in the southern and middle part of Sweden during three time periods: 1) before the vector season in 2012, 2) after the vector season in 2012, and 3) after the vector season in 2015. Animals from periods 1 and 2 were of varying ages, whereas animals collected in period 3 were born after the vector season 2013. Animals from period 1 (n = 15) and 3 (n = 47) were seronegative, but, 53% (16 of 30) of animals from period 2 were seropositive, determined by SBV competitive ELISA. Samples from period 2 were additionally analyzed for SBV-neutralizing antibodies. Such antibodies were detected in 16/16 SBV-N-antibody-positive, 3/12 negative and 2/2 doubtful sera. The two tests were in accordance at SBV-neutralizing antibody titers of 1:32 or higher. CONCLUSION: Our results show that SBV circulated among wild cervids during the vector season of 2012. Three years later, no SBV-antibodies were detected in animals born after the vector season 2013. The likely absence of SBV circulation in Sweden, in contrast to other parts of Europe, might be explained by the annual occurrence of a vector-free season due to climate conditions. Interpretations are limited by the small sample-size, but the results suggest that the SBV competitive ELISA has high specificity but might have slightly lower sensitivity compared to a seroneutralization assay, when using samples from wild cervids.

Emergence of a new rhabdovirus associated with mass mortalities in eelpout (Zoarces viviparous) in the Baltic Sea.

We report the first description of a new Rhabdoviridae tentatively named eelpout rhabdovirus (EpRV genus Perhabdovirus). This virus was associated with mass mortalities in eelpout (Zoarces viviparous, Linnaeus) along the Swedish Baltic Sea coast line in 2014. Diseased fish showed signs of central nervous system infection, and brain lesions were confirmed by histology. A cytopathogenic effect was observed in cell culture, but ELISAs for the epizootic piscine viral haemorrhagic septicaemia virus (VHSV), infectious pancreas necrosis virus (IPNV), infectious haematopoietic necrosis virus (IHNV) and spring viraemia of carp virus (SVCV) were negative. Further investigations by chloroform inactivation, indirect fluorescence antibody test and electron microscopy indicated the presence of a rhabdovirus. By deep sequencing of original tissue suspension and infected cell culture supernatant, the full viral genome was assembled and we confirmed the presence of a rhabdovirus with 59.5% nucleotide similarity to the closest relative Siniperca chuatsi rhabdovirus. The full-genome sequence of this new virus, eelpout rhabdovirus (EpRV), has been deposited in GenBank under accession number KR612230. An RT-PCR based on the L-gene sequence confirmed the presence of EpRV in sick/dead eelpout, but the virus was not found in control fish. Additional investigations to characterize the pathogenicity of EpRV are planned.

Companion Animals as a Source of Viruses for Human Beings and Food Production Animals.

Companion animals comprise a wide variety of species, including dogs, cats, horses, ferrets, guinea pigs, reptiles, birds and ornamental fish, as well as food production animal species, such as domestic pigs, kept as companion animals. Despite their prominent place in human society, little is known about the role of companion animals as sources of viruses for people and food production animals. Therefore, we reviewed the literature for accounts of infections of companion animals by zoonotic viruses and viruses of food production animals, and prioritized these viruses in terms of human health and economic importance. In total, 138 virus species reportedly capable of infecting companion animals were of concern for human and food production animal health: 59 of these viruses were infectious for human beings, 135 were infectious for food production mammals and birds, and 22 were infectious for food production fishes. Viruses of highest concern for human health included hantaviruses, Tahyna virus, rabies virus, West Nile virus, tick-borne encephalitis virus, Crimean-Congo haemorrhagic fever virus, Aichi virus, European bat lyssavirus, hepatitis E virus, cowpox virus, G5 rotavirus, influenza A virus and lymphocytic choriomeningitis virus. Viruses of highest concern for food production mammals and birds included bluetongue virus, African swine fever virus, foot-and-mouth disease virus, lumpy skin disease virus, Rift Valley fever virus, porcine circovirus, classical swine fever virus, equine herpesvirus 9, peste des petits ruminants virus and equine infectious anaemia virus. Viruses of highest concern for food production fishes included cyprinid herpesvirus 3 (koi herpesvirus), viral haemorrhagic septicaemia virus and infectious pancreatic necrosis virus. Of particular concern as sources of zoonotic or food production animal viruses were domestic carnivores, rodents and food production animals kept as companion animals. The current list of viruses provides an objective basis for more in-depth analysis of the risk of companion animals as sources of viruses for human and food production animal health.

Tick-borne encephalitis.

Tick-borne encephalitis (TBE), a zoonotic arbovirosis caused by tick-borne encephalitis virus (TBEV), is an increasing public health concern. Infections result in neurological symptoms in humans and the virus has rapidly expanded to new geographical areas. Three subtypes are currently present in different parts of Europe and Asia. The virus is transmitted by ticks, mainly Ixodes spp., between small mammals such as rodents, which serve as virus amplifying hosts. Humans are infected sporadically, either by a tick bite or by ingestion of infected milk or milk products. Other mammals (e.g. ruminants) can also be infected, but most of the time do not show clinical signs. In contrast to rodents, other wild and domestic mammals probably play only a very small direct role in maintaining TBEV in an area, but they might play an important role as hosts in sustaining a large tick population. Therefore, the virus prevalence and the occurrence of TBE can be influenced by several environmental, genetic and behavioural factors associated with the virus, the vectors or the hosts, and understanding these factors is essential for implementation of effective control measures. This article reviews virus characteristics and the epidemiological and clinical aspects of TBEV infections and examines pathogenesis, diagnostic approaches and control measures.

Schmallenberg Virus beyond Latitude 65°N.

Extensive and rapid spread of Schmallenberg virus (SBV) in Sweden was detected by consecutive serological bulk milk surveys conducted before and after the vector season of 2012. Whereas <0.2% of cattle herds tested positive in a first survey in spring 2012, SBV-specific antibodies were detected in almost 75% of 723 bulk milk samples randomly collected all over the country 6 months later, beyond the 65th northern latitude, and with an observed spatial distribution suggesting multiple introductions of the virus. Circulation of virus was later confirmed by the detection of SBV in malformed lambs and calves starting from November 2012 and January 2013, respectively. These observations suggest SBV circulation starting from July 2012, with a peak in transmission between August and October. A local heterogeneity of within-herd seroprevalence was found, indicating that SBV-naïve animals remain also in highly infected areas enabling the re-emergence of the infection in the coming vector season.

Avian influenza A(H10N7) virus involvement in mass mortality of harbour seals (Phoca vitulina) in Sweden, March through October 2014.

We provide the first scientific report of influenza A virus involvement in a mass mortality event among harbour seals (Phoca vitulina) off the west coast of Sweden. Avian influenza A (H10N7) virus was detected in the lungs of two affected animals. This subtype has not been reported in seals to date, nor has influenza A-associated mortality been reported in seals in Europe. Circulation of avian influenza viruses in mammals may have implications for public health.

Limited interlaboratory comparison of Schmallenberg virus antibody detection in serum samples.

Eight veterinary institutes in seven different countries in Europe participated in a limited interlaboratory comparison trial to evaluate laboratory performances of Schmallenberg virus (SBV) antibody detection in serum. Seven different sheep sera and three different cattle sera were circulated, and all participating institutes were asked to test these sera using SBV antibody detection assay(s) in place in their laboratories. All laboratories within the trial performed a virus neutralisation test (VNT) as well as one or two ELISAs on all samples, and swiftly detected SBV antibodies using these assays. VNT was more sensitive in detecting SBV antibodies than several of the used ELISA assays. Based on the test results, one cattle and one sheep SBV antibody-positive serum were selected to serve as reference sera, which now can be supplied to other laboratories on request.

Incursions of foot-and-mouth disease virus into Europe between 1985 and 2006.

Foot-and-mouth disease (FMD) is one of the biggest threats to animal health in European countries. In the last 22 years (1985-2006), FMD has occurred 37 times in 14 European countries. Serotype O was most frequently involved in these outbreaks followed by A, C and Asia 1. Sometimes, epidemics were very limited and at other times, they were the cause of devastating economic losses. In most cases (22/37), the origin of the outbreaks could not be determined. For some of these outbreaks, however, routes of introduction and spread were identified through epidemiological inquiries. Moreover, in some cases, the origin of the virus was also traced by phylogenetic analysis of the partial or complete sequences of VP1 genes. Lessons learned from the outbreaks are still useful as most of the same risk factors persist. However, efforts made by FMD-free countries to help those where the disease is endemic are a valuable strategy for the reduction of the global risk. The present and the future potential sources of FMD infection need to be identified to best focus European efforts.

Epidemiological patterns of foot-and-mouth disease worldwide.

Foot-and-Mouth Disease (FMD) is a clinical syndrome in animals due to FMD virus that exists in seven serotypes, whereby recovery from one sero-type does not confer immunity against the other six. So when considering intervention strategies in endemic settings, it is important to take account of the characteristics of the different serotypes in different ecological systems. FMD serotypes are not uniformly distributed in the regions of the world where the disease still occurs. For example, the cumulative incidence of FMD serotypes show that six of the seven serotypes of FMD (O, A, C, SAT-1, SAT-2, SAT-3) have occurred in Africa, while Asia contends with four sero-types (O, A, C, Asia-1), and South America with only three (O, A, C). Periodically there have been incursions of Types SAT-1 and SAT-2 from Africa into the Middle East. This paper describes the global dynamics for the seven sero-types and attempts to define FMD epidemiological clusters in the different regions of the world. These have been described on a continent by continent basis. The review has reaffirmed that the movement of infected animals is the most important factor in the spread of FMD within the endemically infected regions. It also shows that the eco-system based approach for defining the epidemiological patterns of FMD in endemic, which was originally described in South America, can apply readily to other parts of the world. It is proposed that any coordinated regional or global strategy for FMD control should be based on a sound epidemiological assessment of the incidence and distribution of FMD, identifying risk sources as either primary or secondary endemic eco-systems.

Evaluation of laboratory tests for SAT serotypes of foot-and-mouth disease virus with specimens collected from convalescent cattle in Zimbabwe.

During a field study in Zimbabwe, clinical specimens were collected from 403 cattle in six herds, in which the history of foot-and-mouth disease (FMD) vaccination and infection appeared to be known with some certainty. Five herds had reported outbreaks of disease one to five months previously but clinical FMD had not been observed in the sixth herd. A trivalent vaccine (South African Territories [SAT] types 1, 2 and 3) had been used in some of the herds at various times either before and/or after the recent outbreaks of FMD. The primary aim of this study was to evaluate the performance of serological tests for the detection of SAT-type FMD virus infection, particularly elisas for antibodies to non-structural proteins (NSPs) of FMD virus and solid phase competition ELISAS (SPCEs) for serotypes SAT1 and SAT2. Secondary aims were to examine NSP seroconversion rates in cattle that had been exposed to infection and to compare virus detection rates by virus isolation and real-time reverse transcriptase-PCR (rtRT-PCR) tests on both oesophagopharyngeal fluids and nasopharyngeal brush swabbings. In addition, the hooves of sampled animals were examined for growth arrest lines as clinical evidence of FMD convalescence. Laboratory tests provided evidence of FMD virus infection in all six herds; SAT2 viruses were isolated from oesophagopharyngeal fluids collected from two herds in northern Zimbabwe, and SAT1 viruses were isolated from three herds in southern Zimbabwe. Optimised rtRT-PCR was more sensitive than virus isolation at detecting FMD virus persistence and when the results of the two methods were combined for oesophagopharyngeal fluids, between 12 and 35 per cent of the cattle sampled in the convalescent herds were deemed to be carriers. In contrast, nasopharyngeal swabs yielded only two virus-positive specimens. The overall seroprevalence in the five affected herds varied with the different NSPS from 56 per cent to 75 per cent, compared with 81 per cent and 91 per cent by homologous SPCE and virus neutralisation tests respectively. However, if serological test results were considered only for the cattle in which persistent infection with FMD virus had been demonstrated, 70 to 90 per cent scored seropositive in the different NSPs.

Peripheral immune responses in pregnant cattle following Neospora caninum infection.

Experimental infection of cattle with Neospora caninum in early gestation causes foetal death, but the foetus survives infection in late gestation. An immunological mechanism of abortion has been suggested; therefore changes in the maternal immune response during pregnancy could account for these differences. We have investigated the peripheral immune responses of pregnant cattle following an intravenous inoculation with 10(7) N. caninum tachyzoites in early and late gestation. Percentages of CD2+ and CD4+ T-cells in peripheral blood mononuclear cells (PBMC) increased 1-2 weeks after infection in both early (day 70) and late (day 210) gestation, and percentages of CD8+ T-cells increased 1-2 weeks after infection at day 70. Interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) mRNA expression in PBMC increased 1-2 weeks after infection at day 210 and IL-4 increased 1-2 weeks after infection at day 70. Immunomagnetic isolation of CD4+ cells from PBMC showed that they were a major source of IL-4 and IFN-gamma, and expression of both cytokines increased in CD4+ cells after infection in early and late gestation. These results suggest that CD4+ cells proliferate and express IL-4 and IFN-gamma in response to N. caninum irrespective of the stage of gestation when infection occurs.

Bovine respiratory syncytial virus lacking the virokinin or with a mutation in furin cleavage site RA(R/K)R109 induces less pulmonary inflammation without impeding the induction of protective immunity in calves.

The BRSV fusion (F) protein is cleaved at two furin consensus sequence sites, resulting in the generation of disulphide-linked F1 and F2 subunits and the release of an intervening peptide of 27 amino acids (pep27), which is converted into a biologically active tachykinin (virokinin). The role of the virokinin and the importance of one of the furin cleavage sites, FCS-2 [RA(R/K)R109], in the pathogenesis of BRSV infection and in the subsequent development of immunity was studied in gnotobiotic calves infected with a recombinant BRSV (rBRSV) lacking pep27 (rBRSVdelta p27) or with rBRSV108/109, which contains two amino acid substitutions in FCS-2 (RANN109). Although replication of the mutant viruses and the parental wild-type (WT) rBRSV in the lungs was similar, the extent of gross and microscopic lesions induced by the mutant viruses was less than that induced by WT rBRSV. Furthermore, the numbers of eosinophils in the lungs of calves infected with the mutant viruses were significantly less than that in calves infected with WT virus. These observations suggest a role for the virokinin in the pathogenesis of BRSV infection. Following mucosal immunization with rBRSVdelta p27, the levels of BRSV-specific serum antibodies were similar to those induced by WT virus. In contrast, the level of neutralizing antibodies induced by rBRSV108/109 was 10-fold lower than that induced by WT virus. Nevertheless, resistance to BRSV challenge induced by the mutant and WT viruses was similar, suggesting that neither pep27 nor FCS-2 plays a major role in the induction of protective immunity.

Dynamics of virus infections involved in the bovine respiratory disease complex in Swedish dairy herds.

The dynamics of bovine respiratory syncytial virus (BRSV), bovine parainfluenza virus 3 (PIV-3), bovine corona virus (BCoV) and bovine viral diarrhoea virus (BVDV) infections were studied in 118 dairy herds in south western Sweden. By using serology on paired samples from three approximately 7 vs. approximately 15-month-old calves per herd, the propagation of infections was investigated over about a 1-year period. The results implied that at least 74% of calves had experienced one or more of the monitored infections at the age of approximately 7 months (Sample 1, Spring); 30%, 48%, 34% and 8% were seropositive to BRSV, PIV-3, BCoV and BVDV, respectively. Seroconversions to BRSV, PIV-3, BCoV and BVDV occurred in 26%, 38%, 50% and 3% of seronegative animals and 63% had antibodies against two or more infections at approximately 15 months (Sample 2). In total, 90-97% of animals that were seropositive in Sample 1 remained positive in Sample 2. A significant association was found between BVDV and BCoV (P = 0.01). Moreover, a significantly higher proportion of herds in which no calves had a recorded history of respiratory disease (n = 15) were classified as negative to all four infections monitored when compared to herds in which disease was observed (P = 0.0002). This study showed a high infection burden in young animals and effective spread of BRSV, PIV-3 and BCoV in one area of Sweden. BVDV infections were restricted to a few herds, reflecting the effect of a voluntary control program against BVDV in Sweden.

[Retrospective study of foot and mouth disease in West Africa from 1970 to 2003]. / Etude rétrospective de la fièvre aphteuse en Afrique de l'Ouest de 1970 à 2003.

A retrospective study of foot and mouth disease in seven West African countries was conducted for the period 1970 to 2003. The study included three cattle-exporting Sahel countries (Burkina-Faso, Mali and Niger) and four cattle-importing coastal countries (Benin, Côte d'lvoire, Ghana and Togo). Foot and mouth disease has been enzootic in these countries since 1990/1991. Four of the seven serotypes are regularly notified (O, A, SAT 1 and SAT 2). In the seven countries as a whole, 198 biological samples from identified foot and mouth disease outbreaks confirmed the involvement of the following serotypes: O (62 outbreaks); A (32 outbreaks); SAT 1 (18 outbreaks); SAT 2 (86 outbreaks). This result, which is largely underestimated, clearly demonstrates the seriousness of foot and mouth disease in West Africa, whose livestock production system characterised by continual uncontrolled animal movements facilitates the spread of the disease. Unlike in Southern Africa, for foot and mouth disease to be controlled in West Africa it is necessary immediately to introduce a regional strategy involving all countries which takes into account the real situation in the field: transhumance, nomadism and live-animal imports by coastal countries.

Selection of foot and mouth disease vaccine strains--a review.

The choice of the most appropriate strains of foot and mouth disease (FMD) virus vaccines to use in FMD control programmes and to store in vaccine antigen reserves is based on the matching of representative field isolates from outbreaks around the world to available vaccine strains. However, those involved in FMD control at a national level do not always give this work a high priority, while in countries without effective control of FMD there is little incentive to collect samples or to overcome the constraints on submission to international reference laboratories. In the short term, specific initiatives for targeted collection can provide samples on a periodic basis, but a long-term solution requires the development of FMD control measures. This must be underpinned by the strengthening of local Veterinary Services and laboratories, and by demand-driven provision of sufficient amounts of high-quality vaccine. Difficulties may be increased by commercial constraints on disclosure of the strains used for vaccine production and on the supply of reagents needed for matching tests. Vaccine matching tests are mainly based on in vitro methods - such as virus neutralisation, enzyme-linked immunosorbent assay with polyclonal antibodies and complement fixation - and are performed in a relatively small number of laboratories around the world. In addition to the difficulties of gathering representative field and vaccine strains, neither the reagents nor the methods used for vaccine matching are fully harmonised. Consequently, there is no strict equivalence in the results obtained. Alternative approaches using monoclonal antibody panels and/or viral capsid gene sequencing are being developed and could complement the currently employed serological tests. However, there is limited in vivo cross-protection information, more of which is essential for future validation of the vaccine matching methods. In response to the funding and leadership deficit for vaccine strain selection, a network of World Organisation for Animal Health (OIE) and Food and Agriculture Organization FMD reference laboratories has been established; this gives these laboratories the potential to strengthen the coordination of their work and reporting and thereby improve recommendations on vaccine strain selection.

Persistent infection of B lymphocytes by bovine respiratory syncytial virus.

Bovine respiratory syncytial virus (BRSV) is a major cause of respiratory disease in young cattle. Here we demonstrate BRSV persistence at low levels in tracheobronchial and mediastinal lymph nodes up to 71 days after the experimental infection of calves. Positive results were obtained on viral genomic RNA and messenger RNA coding for the nucleoprotein, glycoprotein (G), and fusion protein (F). G and F proteins were also detected in the pulmonary lymph nodes by immunohistochemistry. Double-staining experiments revealed that viral antigen was present in B-lymphocytes. Coculture experiments with the lymph node cells showed that the virus was still able to infect permissive target cells, even though no cytopathic effect was recorded. In vitro studies indicate that BRSV was still able to replicate in bovine B-lymphocyte cell lines 6 months after infection. These results may also be relevant to the understanding not only of the epidemiology and the peculiarities of the immune response of BRSV infections but also of human respiratory syncytial virus infections.

Evolution of bovine respiratory syncytial virus.

Until now, the analysis of the genetic diversity of bovine respiratory syncytial virus (BRSV) has been based on small numbers of field isolates. In this report, we determined the nucleotide and deduced amino acid sequences of regions of the nucleoprotein (N protein), fusion protein (F protein), and glycoprotein (G protein) of 54 European and North American isolates and compared them with the sequences of 33 isolates of BRSV obtained from the databases, together with those of 2 human respiratory syncytial viruses and 1 ovine respiratory syncytial virus. A clustering of BRSV sequences according to geographical origin was observed. We also set out to show that a continuous evolution of the sequences of the N, G, and F proteins of BRSV has been occurring in isolates since 1967 in countries where vaccination was widely used. The exertion of a strong positive selective pressure on the mucin-like region of the G protein and on particular sites of the N and F proteins is also demonstrated. Furthermore, mutations which are located in the conserved central hydrophobic part of the ectodomain of the G protein and which result in the loss of four Cys residues and in the suppression of two disulfide bridges and an alpha helix critical to the three-dimensional structure of the G protein have been detected in some recent French BRSV isolates. This conserved central region, which is immunodominant in BRSV G protein, thus has been modified in recent isolates. This work demonstrates that the evolution of BRSV should be taken into account in the rational development of future vaccines.

Evaluation of a nested reverse transcription-PCR assay based on the nucleoprotein gene for diagnosis of spontaneous and experimental bovine respiratory syncytial virus infections.

The first nested reverse transcription (RT)-PCR based on the nucleoprotein gene (n RT-PCR-N) of the bovine respiratory syncytial virus (BRSV) has been developed and optimized for the detection of BRSV in bronchoalveolar lavage fluid cells of calves. This test is characterized by a low threshold of detection (0.17 PFU/ml), which is 506 times lower than that obtained by an enzyme immunosorbent assay (EIA) test (RSV TESTPACK ABBOTT). During an experimental infection of 17 immunocompetent calves less than 3 months old, BRSV RNA could be detected up to 13 days after the onset of symptoms whereas isolation in cell culture was possible only up to 5 days. Compiling results obtained by conventional techniques (serology, antigen detection, and culture isolation) for 132 field samples collected from calves with acute respiratory signs revealed that n RT-PCR-N showed the highest diagnostic sensitivity and very good specificity. This n RT-PCR-N with its long period of detection during BRSV infection thus provides a valuable tool for diagnostic and epidemiological purposes.

Flow cytometric analysis of bovine CD4 and CD8 lymphocytes: influence of blood sampling and processing methods.

Technical information to facilitate bovine blood treatment for optimum lymphocyte flow cytometry analysis is reported. Murine monoclonal antibodies CC8 and CC63 were used to identify phenotypes corresponding to bovine CD4 T cells and CD8 T cells. Blood samples collected in acid citrate dextrose (ACD) enhanced leucocyte subpopulation separation compared with ethylenediamine tetra-acetic acid, heparin and sodium citrate. To preserve bovine blood before immunophenotyping, samples collected in ACD may be kept at 22 degrees C or at 4 degrees C and should be analysed within 32 hours. For isolation of white blood cells, whole blood lysis was faster and gave the same results as Ficoll gradient separation 1.077 and Ficoll gradient separation 1.083. After immunophenotyping, blood could be stored at 4 degrees C if fixed with paraformaldehyde within seven days. Owing to diurnal variations, blood should be collected at a standard time of the day.