Historical cases of anthrax in Sweden 1916-1961.

As in most European countries, anthrax was common in Swedish livestock during the centuries leading up to the mid-twentieth century. After 1957, the disease was regarded as practically extinct. However, in the past 7 years, three outbreaks have caused public alarm because of the risk of environmental contamination. Properly buried carcasses should present little risk of spore contamination, and instructions were in place to ensure this since the 1890s. However, as has been demonstrated in recent outbreaks, carcasses were not always adequately buried and viable spores may remain in some sites. This study was prompted by the lack of historical information to assess the geographical risk of old anthrax spores. The aim was to obtain sufficient information to map old anthrax outbreaks, to study clusters and variation between years. Historical data were retrieved from Official National and Regional Veterinary Archives. In the years 1916 to 1961, anthrax was reported from more than 3000 farms and all 24 counties in Sweden were affected. Most cases were single animals, but there were also some larger outbreaks mainly involving cattle. Anthrax in horses was mostly reported before the mid-twentieth century, and the same was seen for pigs and wildlife. A ban in 1957, on the import of bone meal for animal feed led to a drastic reduction of outbreaks. The majority of cases were reported during the summer months in animals on pasture. Historical records proved useful for the investigation of current outbreaks. If handled properly, old carcasses pose no substantial risk, but if not, they may present a risk to grazing animals in some areas. Historical information is useful for all planning of work that involves digging or relocation of soil masses. Anthrax can be regarded as one of the diseases where history is a key to present knowledge.

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.

Investigations and actions taken during 2011 due to the first finding of Echinococcus multilocularis in Sweden.

Echinococcus multilocularis is a parasite that can cause alveolar echinococcosis disease. After the first positive finding of E. multilocularis in Sweden in 2011, a consulting group with representatives from relevant authorities was summoned. In this group, all relevant information was shared, strategies for information dissemination and any actions to be taken due to the finding of E. multilocularis were discussed and decided. The present paper describes the actions taken during 2011 and the results thereof, including surveillance in animals, risk assessment for humans to become infected and recommendations given to the public. Further discussion about whether the parasite was introduced, and if so, how, as well as possible future development of the infection in animals and humans in Sweden and future actions are included.

Possible means of introduction of bluetongue virus serotype 8 (BTV-8) to Sweden in August 2008: comparison of results from two models for atmospheric transport of the Culicoides vector.

In September 2008, bluetongue virus serotype 8 (BTV-8) infection was detected for the first time in Sweden, in a dairy herd on the west coast. Two different previously published operational atmospheric dispersion models indicate that midges from infected regions in Europe are likely to have reached Sweden by atmospheric transport during an estimated infection window. Both models indicated that the likely dates for the incursion of midges were overnight on August 6 to 7 and August 14 to 15; however, the less constrained model indicated a number of additional possible dates. The distribution of infected herds detected by active surveillance coincides with the regions that were indicated by the models to have been reached by midges from regions in Denmark and Germany with infected herds. It is likely that several points of introduction of infected midges occurred, possibly on different occasions. No alternative routes for introduction of the infection to Sweden were identified, supporting the theory that BTV-8 was introduced by infected midges carried by the wind.

Infection with bluetongue virus serotype 8 in Sweden in 2008.

On September 6, 2008, bluetongue was detected for the first time in Sweden. Intensified active surveillance in cattle and vector surveillance, prompted by the situation in northern Europe, preceded the detection. A vaccination campaign and intensive surveillance activities were initiated nationally, but with a focus on the southern part of the country. Measures included both active and passive surveillance by serology and PCR in ruminants, along with vector surveillance. The investigations revealed that the infection occurred over a large area of southern Sweden during September and October 2008, despite comparatively low vector activity, an apparently low viral load and no clinical disease. Transplacental infection was detected in one case.

Emergence of porcine reproductive and respiratory syndrome in Sweden: detection, response and eradication.

Porcine reproductive and respiratory syndrome (PRRS) is characterized by reproductive failure in sows and respiratory problems in growing pigs. The disease is present in most countries throughout the world but was not diagnosed in Sweden until the summer of 2007 when it was first detected through the national PRRS surveillance program. The immediate mobilization of veterinary authorities, field veterinarians and the pig industry was a prerequisite for preventing the spread of the disease. Within 10 days seven herds were verified as infected and the measures taken included stamping out, cleaning, disinfection and a vacancy period of 3 weeks before the herds were repopulated. To evaluate the effectiveness of these measures, a national sero-surveillance was carried out during the autumn of 2007. Approximately 90% of the pig production was covered by this screening and all samples tested were negative with regard to antibodies to PRRS virus.

Recognition of the Nor98 variant of scrapie in the Swedish sheep population.

Within the framework of the active surveillance for transmissible spongiform encephalopathies in sheep in Sweden, 4 cases of the atypical form of scrapie, Nor98, were identified during 2003. Nor98 is a recently recognized and poorly understood variant of scrapie, first described in Norway. The cases were positive by the rapid test (enzyme-linked immunosorbent assay). Immunohistochemical staining showed diffuse thin-granular staining of the cerebellar cortex. Western immunoblotting analysis of specimens of brain stem and cerebellum showed a light band of approximately 12 kDa. Typical scrapie was ruled out based on the confirmatory testing. The affected ewes were from 4 different flocks. They were between 7 and 9 years old. Two were of the ARQ/ARQ genotype, 1 ARR/ARQ, and 1 ARR/AHQ. Two ewes had shown ataxia, and the other 2 had no clinical signs. Whole-flock slaughter was applied, and testing of the flock mates did not reveal additional cases. Nor98 differs from typical scrapie in its epidemiology, frequency of genotypes of sheep affected, clinical signs, microscopic lesions, distribution of scrapie prion protein in the brain, and characteristics of the immunostaining and immunoblotting profiles.

Causes of stillbirth and time of death in Swedish Holstein calves examined post mortem.

This study was initiated due to the observation of increasing and rather high levels of stillbirths, especially in first-calving Swedish Holstein cows (10.3%, 2002). Seventy-six Swedish Holstein calves born to heifers at 41 different farms were post mortem examined in order to investigate possible reasons for stillbirth and at what time in relation to full-term gestation they had occurred. The definition of a stillborn calf was dead at birth or within 24 h after birth after at least 260 days of gestation. Eight calves were considered as having died already in uterus. Slightly less than half of the examined calves (46.1%) were classified as having died due to a difficult calving. Four calves (5.3%) had different kinds of malformations (heart defects, enlarged thymus, urine bladder defect). Approximately one third of the calves (31.6%) were clinically normal at full-term with no signs of malformation and born with no indication of difficulties at parturition or any other reason that could explain the stillbirth. The numbers of male and female calves were rather equally distributed within the groups. A wide variation in post mortem weights was seen in all groups, although a number of the calves in the group of clinically normal calves with unexplained reason of death were rather small and compared with e.g. those calves categorised as having died due to a difficult calving, their average birth weight was 6 kg lower (39.9 +/- 1.7 kg vs. 45.9 +/- 1.5 kg, p < or = 0.01). It was concluded that the cause of stillbirth with a non-infectious aetiology is likely to be multifactorial and difficult calving may explain only about half of the stillbirths. As much as one third of the calves seemed clinically normal with no obvious reason for death. This is a target group of calves that warrants a more thorough investigation in further studies.

Detection of IgM responses to bovine respiratory syncytial virus by indirect ELISA following experimental infection and reinfection of calves: abolition of false positive and false negative results by pre-treatment of sera with protein-G agarose.

The IgM responses in three panels of sera generated by infection and reinfection of calves with bovine respiratory syncytial virus (BRSV) were measured by indirect ELISA (I-ELISA). The effect of depleting serum IgG by pre-treatment with protein G agarose (PGA) was evaluated. Following primary infection a weak IgM response was detected in the untreated sera of 3 out of 4 calves with maternally derived antibody (MDA). Both the magnitude and duration of the specific IgM responses in these calves were increased by pre-treatment with PGA. In addition, the fourth infected calf tested gave a single positive IgM result following PGA treatment. Transient or persistent IgM responses which were abolished by pre-treatment of sera with PGA were detected in 4/8 calves following reinfection. These were considered to be false positive results, consistent with the influence of IgM rheumatoid factor (IgM-RF). One of these calves and two additional calves showed transient increases in IgM which were resistant to PGA treatment. These were considered to represent specific IgM responses to reinfection. The results indicate the ability of PGA treatment to eliminate both false positive and false negative results and emphasise the necessity for controlling the influence of IgM-RF in IgM-specific indirect ELISAs.

Evaluation of an IgM-specific indirect enzyme-linked immunosorbent assay for serodiagnosis of bovine respiratory syncytial virus infection: influence of IgM rheumatoid factor on test results with field sera.

A commercially available indirect enzyme-linked immunosorbent assay for measuring bovine respiratory syncytial virus (BRSV)-specific IgG was adapted to measure virus-specific IgM. Using this assay, the development of rapid IgM responses in experimentally infected calves was observed 7-9 days postinfection, with peak absorbance values ranging from 1.698 to 2.873. When absorbance values were expressed as a percentage of a positive reference serum, a positive/negative threshold of 22% was determined by testing serum samples from 59 healthy 3-5-month-old calves. Acute and convalescent serum samples collected from 151 calves during 38 outbreaks of respiratory disease were tested, and 130 sera were positive. To determine the number of false-positive results due to the presence of IgM rheumatoid factor, a method for depleting serum IgG by pretreatment of sera with a suspension of protein-G-agarose was developed. All sera that initially tested IgM positive were retested following depletion of serum IgG. False-positive IgM reactions were detected in 23 sera (17.7%). Specific IgM responses were confirmed in 107 sera from 84 calves. Evidence of BRSV infection was detected in 34 of 38 outbreaks. In contrast, seroconversion was detected in 69 calves from 24 outbreaks, confirming the diagnostic potential of the IgM assay. Overall correlation between IgM and seroconversion results was 74.2%. Intra- and interassay reproducibility were 12.50% and 17.48%, respectively (mean coefficients of variation).

An experimental study of a concurrent primary infection with bovine respiratory syncytial virus (BRSV) and bovine viral diarrhoea virus (BVDV) in calves.

Experimental infections with bovine respiratory syncytial virus (BRSV) and bovine viral diarrhoea virus (BVDV) were performed to study the effect of concurrent BRSV and BVDV infections. Twelve seronegative calves, in 3 groups, were inoculated on a single occasion with pure BRSV (group A), BRSV and noncytopathogenic BVDV (group B) or mock infected (group C). Mild respiratory symptoms were recorded 4 to 5 days post inoculation (dpi) in group A and group B calves. One calf in group A was severely affected and required medical treatment. In group B, fever (40.7-41.4 degrees C) was prominent 7 to 8 dpi. Only calves in group B were BVDV positive in purified lymphocytes at 2 to 14 dpi and showed increased serum interferon levels, with a peak at 4 dpi, indicating BVDV to be responsible for inducing the rise. BRSV was detected in lung lavage fluids up to 7 dpi for group A calves, compared to 11 dpi for group B and calves in this group also seroconverted later displaying lower BRSV titers. The time lag before an antibody response and the titers recorded in group B, indicated that the duration of BVDV infection in lymphocytes negatively influenced the capacity to mount a BRSV antibody response.

The immunostimulating complex (ISCOM) is an efficient mucosal delivery system for respiratory syncytial virus (RSV) envelope antigens inducing high local and systemic antibody responses.

ISCOM is an efficient mucosal delivery system for RSV envelope proteins as measured by antibody responses in respiratory tract secretions and in sera of mice following two intranasal (i.n.) administrations. Intranasally administered RSV ISCOMs induced high levels of IgA antibodies both in the upper respiratory tract and in the lungs. In the lungs, a prominent and long-lasting IgA response was recorded, which still persisted 22 weeks after the second i.n. immunization when the experiment ended. Subcutaneous (s.c.) immunization only induced low IgA titres in the upper respiratory tract and no measurable response to RSV was found in the lungs. Differences were also noticed in serum between the i.n. and s.c. modes of immunization. ISCOMs given intranasally induced earlier, higher and longer lasting IgM and IgG1 serum anti-RSV antibody responses than those induced by the s.c. mode of administration. A low serum IgE response was only detectable at 2 weeks after i.n. immunization with ISCOMs and after s.c. immunization with an inactivated virus, but no IgE response was detectable after s.c. injection of ISCOMs. The serum IgA response was more pronounced following s.c. injection of inactivated virus than after i.n. application of ISCOMs, and a clear-cut booster effect was obtained with a second immunization. Virtually no serum IgA response was detected after the s.c. administration of ISCOMs. In conclusion, the high immune responses induced by RSV ISCOMs in the respiratory tract and serum after i.n. administration indicate prominent mucosal delivery and adjuvant properties of the ISCOMs, warranting further studies.

Genetic and antigenic analysis of the G attachment protein of bovine respiratory syncytial virus strains.

Antigenic and genetic studies of bovine respiratory syncytial virus (BRSV) were made on isolates obtained from three continents over 27 years. Antigenic variation between eight isolates was initially determined using protein G-specific monoclonal antibodies. Four distinct reaction patterns were observed, two of which corresponded to the previously established subgroups A and AB. A third pattern was produced by five Scandinavian strains and a fourth was observed from a single Dutch isolate. The genetic diversity of 27 strains of BRSV was investigated by comparative nucleotide sequence analysis of a 731 nucleotide fragment in the G protein gene. Nine of the BRSV strains were analysed by direct sequencing of RT-PCR amplicons whereas sequences of 18 BRSV and three human respiratory syncytial virus (HRSV) strains were obtained from GenBank. The analysis revealed similarities of 88-100% among BRSV strains and 38-41% between BRSV and HRSV. A phylogenetic tree created for BRSV revealed two main branches, one of which divided into five further lineages, each representing a geographic cluster. A correlation was evident between the positions of some strains in the phylogenetic tree and their antigenic pattern. For HRSV strains, a genetic similarity of only 62% allowed the distinction of two antigenic subgroups, A and B, a pattern which was not seen for BRSV. This study showed that genetic analysis was an accurate method for discriminating BRSV strains and that these viruses should be regarded as a single genetic and antigenic group, within which variants can be distinguished.

Severe respiratory disease in dairy cows caused by infection with bovine respiratory syncytial virus.

Outbreaks of severe respiratory disease caused by bovine respiratory syncytial virus (BRSV) were recorded in dairy herds throughout Sweden in 1988 and subsequently. The virus was demonstrated in nasopharyngeal swab material from animals in the acute stage of the disease by culture, the polymerase chain reaction (PCR) and by immunofluorescence. Serological data from the herds investigated showed that the cows had seroconverted to BRSV rather than to bovine coronavirus, bovine viral diarrhoea virus or parainfluenza-3 virus. It was predominantly dairy herds in isolated areas that contracted a severe primary BRSV infection, often after the purchase of new animals. A nationwide survey for BRSV antibodies in bulk milk samples showed the highest prevalence, of 84 to 89 per cent, in the southernmost regions of Sweden and the lowest prevalence, of 41 to 51 per cent, in the north of the country. The prevalence of BRSV was highest in areas with the highest populations of cattle.

Evaluation and application of an indirect ELISA for the detection of antibodies to bovine respiratory syncytial virus in milk, bulk milk, and serum.

An indirect enzyme-linked immunosorbent assay (ELISA) was developed at the National Veterinary Institute (NVI), Uppsala, to detect antibodies to bovine respiratory syncytial virus (BRSV) in serum and milk. For the evaluation of the NVI ELISA, field sera collected from cattle in England and Sweden were tested in parallel with an ELISA in use at the Central Veterinary Laboratory (CVL), Weybridge. The tests showed 96% agreement. The sensitivity and specificity of the NVI ELISA relative to the CVL ELISA were 94% and 100%, respectively. There was evidence that the difference in sensitivity between the 2 tests was due to the detection of both IgG and IgM class antibodies by the CVL ELISA, whereas the NVI ELISA was designed specifically to detect IgG1. Milk and serum samples from individual cows were tested by the NVI ELISA for presence of antibodies to BRSV. There was a good correlation between the ability to detect antibodies in serum and the ability to detect them in milk, although the antibody titer was generally lower in milk than in serum. Bulk milk samples were collected from farms with severe clinical symptoms of respiratory distress and from farms with no history of respiratory disease. There was a clear distinction between antibody levels in diseased and healthy herds. The NVI ELISA is a rapid and reliable test for detecting antibodies to BRSV in milk, bulk milk, and serum samples.

Development of nested PCR assays for detection of bovine respiratory syncytial virus in clinical samples.

Two nested PCR assays were developed for the detection of bovine respiratory syncytial virus (BRSV). Primers were selected from the gene encoding the F fusion protein (PCR-F) and the gene encoding the G attachment protein (PCR-G). Biotinylated oligonucleotide probes, termed F and G, were selected for the hybridization of the respective PCR products. The sensitivities of the PCR-F and PCR-G assays were similar, both detecting 0.1 tissue culture infective dose of the virus. The PCR-F assay amplified all bovine strains and one human strain (RS32) tested. No cross-reactions were observed with nine heterologous respiratory viruses. PCR-F products of bovine and human RSV strains were discriminated by using endonuclease restriction enzyme ScaI, which specifically cleaved, products of BRSV. Oligonucleotide probe F was also specific for products of BRSV. The PCR-G assay detected all bovine strains and none of the human strains tested. A faint electrophoretic band was also observed with products of Sendai virus. However, probe G did not hybridize with this product, only with products of BRSV. Nasal swabs collected from cattle with no symptoms and cattle in the acute stage of respiratory disease were analyzed for BRSV by the immunofluorescence (IF) method and by the PCR-F and PCR-G assays. The virus was detected by the PCR assays in 31 of 35 (89%) samples tested. Only 23 samples (66%) were positive by the IF method, and these samples were also positive by both the PCR-F and PCR-G assays. The 31 samples detected as positive by PCR originated from cattle presenting clinical signs of acute respiratory disease; the four PCR-negative samples originated from clinically asymptomatic neighboring cattle. All sampled animals subsequently seroconverted and became reactive to BRSV. Thus, the detection of BRSV by PCR correlated with clinical observations and was considerably more sensitive (66 versus 89%) than IF. These results indicate that both nested PCR assays provide rapid and sensitive means for the detection of BRSV infection in cattle. Considering its higher specificity, the PCR-F assay can be recommended as the method of choice in the analysis of clinical specimens of BRSV.