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.

Expression of perforin, granzyme A and Fas ligand mRNA in caecal tissues upon Eimeria tenella infection of naïve and immune chickens.

Cytotoxic cells of the immune system may kill infected or transformed host cells via the perforin/granzyme or the Fas ligand (FasL) pathways. The purpose of this study was to determine mRNA expression of perforin, granzyme A and FasL in Eimeria tenella-infected tissues at primary infection and infection of immune chickens as an indirect measure of cytotoxic cell activity. Chickens were rendered immune by repeated E. tenella infections, which were manifested as an absence of clinical signs or pathological lesions and significantly reduced oocyst production upon challenge infection. During primary E. tenella infection, perforin, granzyme A and FasL mRNA expression in caecal tissue was significantly increased at 10 days after infection, compared to uninfected birds. In contrast, at infection of immune birds, perforin and granzyme A mRNA expression in caecal tissue was significantly increased during the early stages of E. tenella challenge infection, days 1-4, which coincided with a substantial reduction of parasite replication in these birds. These results indicate the activation of cytotoxic pathways in immune birds and support a role for cytotoxic T cells in the protection against Eimeria infections.

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.

Is there a need for improved Cryptosporidium diagnostics in Swedish calves?

Cryptosporidium parvum is a common pathogen in preweaned calves but in Sweden Cryptosporidium bovis, which is considered apathogenic, is the most common species in this age group and it has been identified in diarrhoeal samples, indicating that it could be a cause of diarrhoea. In routine diagnostic procedures, infection is determined by microscopy, which is not sufficient to differentiate these species. We investigated whether routine Cryptosporidium diagnostic procedures need improvement to include species determination. The relation of Cryptosporidium spp. and subtype with the clinical picture and other pathogens was also investigated. A total of 782 diarrhoeal calf samples were analysed and Cryptosporidium infection was diagnosed in 198 samples. Cryptosporidium parvum was identified in 178, C. bovis in six and mixed C. bovis/C. parvum in seven samples. Twenty-seven C. parvum subtypes were identified, of which 16 were newly described. Except for three herds, only one subtype per herd was identified. Cryptosporidium parvum-positive calves were younger than C. bovis-positive calves and most C. parvum infections were seen at 1-3 weeks of age. Oocyst counts were higher in C. parvum samples. Yellow faecal colour was associated with C. parvum infection. Watery faeces had no greater association with C. parvum infection, but C. parvum subtype family IIa was more common than subtype family IId in watery faecal samples. No other pathogens were detected in the six C. bovis-infected calves, indicating a pathogenic potential. Our results show that species determination does not need to be included in routine Cryptosporidium diagnostic procedures in order to estimate the clinical relevance of infection in diarrhoeal calves. The maximum age when analysis for clinical cryptosporidiosis is performed can be lowered to 6 weeks of age. However, the indicated pathogenic potential of C. bovis warrants further attention.

IgG avidity pattern in cattle after ingestion of Neospora caninum oocysts.

The avidity (functional affinity) of specific antibodies are being used to estimate duration of bovine Neospora caninum infection. Here, we report for the first time the avidity pattern in cattle orally inoculated with N. caninum oocysts. In all, 16 pregnant cows and 7 calves were administered N. caninum oocysts. In the cows, the avidity increased during the early course of infection. In all but one, the avidity was < or = 35 during the first 6 weeks after infection and no cow had an avidity value >50 until week 9. The calves were sampled either week 6 (n = 3) or week 9 (n = 9) after infection, and by then had avidities between 2 and 17. The results are in agreement with results from previous investigations of naturally infected cattle, and calves that were experimentally infected with tachyzoites. They further validate the ability of the N. caninum iscom avidity ELISA to accurately assess the duration of infection.

Toxoplasma gondii and Neospora caninum serological status of different canine populations from Uberlândia, Minas Gerais

Este trabalho teve como objetivo analisar a ocorrência de anticorpos contra Toxoplasma gondii e Neospora caninum em cães da cidade de Uberlândia, MG. As taxas de reatividade sorológica dos animais frente a esses parasitos foram comparadas em relação à sua origem e condições de vida. Um total de 369 amostras de soro canino foi obtido de três grupos de cães: (A) 213 pacientes do Hospital Veterinário da Universidade Federal de Uberlândia, (B) 62 pacientes de clínicas veterinárias particulares da cidade e (C) 94 cães errantes da cidade. As amostras foram analisadas para a presença de anticorpos contra os parasitos por meio de ELISA. Os resultados mostraram soropositividades de 30,3 por cento para T. gondii, 9,2 por cento para N. caninum e 5,7 por cento para ambos os parasitos. O grupo de cães errantes apresentou maior taxa de prevalência de anticorpos para os dois parasitos estudados. Pode-se concluir que as taxas de soropositividades para T. gondii e N. caninum nas populações caninas estudadas parecem ser fortemente influenciadas por suas origens e condições de vida.

Experimental reproduction of winter dysentery in lactating cows using BCV -- comparison with BCV infection in milk-fed calves.

Infection models were developed for adult cows and for young calves using the same strain of bovine coronavirus (BCV), which for the first time allows experimental reproduction of winter dysentery (WD) in seronegative lactating cows. The cattle were infected through direct contact with an experimentally inoculated calf. All experimental cattle shed faecal BCV with development of diarrhoea, being profusely watery with small amounts of blood in the most severely affected animals, including both cows and calves. The cows, in contrast to the calves, showed depressed general condition and appetite leading to a marked decrease in milk yield. Further age-associated differences were a shorter incubation period in the two youngest calves, but with milder fever and milder decrease in white blood cell counts. These findings shed light on the apparent epidemiological differences between WD and calf BCV diarrhoea suggesting that, (1) the same strains of BCV cause natural outbreaks of calf diarrhoea and WD, (2) seronegative cows are more severely affected by the infection than seronegative conventionally reared calves, and (3) unaffected general condition in diarrhoeic calves may lead to underestimation of the occurrence of calf diarrhoea in WD outbreaks. In response to infection, all cattle produced early interferon type 1 in serum and, except for one calf, in nasal secretions. A finding not previously reported is the detection of interferon type 1 responses in bovine milk. All cattle developed high IgM antibody responses and long-lasting IgA antibody responses both systemically and locally. The serum IgM antibody responses came earlier in most of the calves than in the cows. Prolonged IgM antibody responses were detected in serum and milk, while those in nasal secretions were much shorter. BCV-specific IgA was present in nasal secretions from all cattle throughout the 6 months follow-up. The IgA antibody response in serum was detected up to 17 months post-infection and the duration showed an age-related variation indicating a more prominent IgA memory in the adult cattle and in the older calves than in the younger ones. BCV-specific IgG was detected in all cattle during the experimental period of up to 22 months. In conclusion, WD was reproduced in seronegative lactating cows. The cows showed a more severe general diseases than seronegative calves infected concurrently. Very long-lasting IgA antibody responses were detected both systemically and locally.

Capture ELISA systems for the detection of bovine coronavirus-specific IgA and IgM antibodies in milk and serum.

Isotype-capture ELISAs for BCV-specific IgA and IgM were developed and tested on milk and serum samples from Swedish cattle. The capture ELISAs showed higher sensitivity than indirect ELISAs for detection of BCV-specific IgA and IgM. In the capture ELISAs the agreement between detection in milk and serum samples was 94% for IgA and 86% for IgM. The correlation between log(10) titres in milk and serum was r=0.82 (P<0.001) for IgA and 0.84 (P<0.001) for IgM. Milk seemed a better target than serum for diagnosing specific IgA at low levels. There was no variation in the isotype-specific BCV antibody titres between healthy quarters of the same udder, but subclinical mastitis was associated with higher levels of IgA antibodies and weak false IgM positive reactions in undiluted milk. Bovine IgA and IgM antibodies in milk and serum showed high stability towards freezing and thawing and storage at room temperature. The antibody responses to BCV were followed in milk and serum from six dairy cows and in serum from four calves for a period of 1 year after an outbreak of winter dysentery (WD). In this outbreak some animals became reinfected with BCV. The IgA and IgM capture ELISAs differentiated between primarily BCV infected and reinfected animals. In the primarily infected cattle, IgM antibodies were first detected in milk and serum four to nine days after the first WD symptoms observed, and were subsequently detected for at least 2-3 weeks. IgM was also detected in the reinfected cows, but mostly at lower levels and for a shorter period of time than in the primarily infected animals. In milk, however, the IgM response of the reinfected cows was detected for a longer period of time than in serum. Six months after the outbreak, IgA was still detected in both serum and milk of all six cows and also in serum of one calf. The reinfected cows showed higher and more long-lasting peak levels of IgA in milk and serum than the primarily infected cows, indicating boosting of the IgA response.

An IgG avidity ELISA to discriminate between recent and chronic Neospora caninum infection.

The avidity of IgG antibodies directed to Neospora caninum was measured using an IgG avidity enzyme-linked immunosorbent assay (ELISA) employing N. caninum proteins incorporated into immunostimulating complexes as antigen. In this ELISA, low-affinity antibodies were eluted by adding an incubation step with urea after the serum incubation. The antibody titers obtained with and without incubation with urea were then used to calculate the IgG avidity values. Analysis of sequential sera collected from experimentally infected calves revealed that the avidity increased during the course of infection. Three weeks after infection, the IgG avidity was 9-18%, and 24 weeks later it had increased to 58-76%. Cattle naturally infected for more than 6 months all had avidities >50%. The results in this study, however preliminary, indicate that the IgG avidity ELISA can be used to discriminate between recent and chronic N. caninum infections and may therefore be a valuable complement to IgG assays in epidemiologic studies of N. caninum infection in cattle.

Detection of antibodies to Neospora caninum in bovine postabortion blood samples from Hungary.

In order to obtain data on the occurrence of the protozoan parasite Neospora caninum as a potential factor in the aetiology of reproduction problems in cattle, 97 postabortion sera were examined by ISCOM ELISA and IFAT for the presence of antibodies in N. caninum. The results showed 10% and 9% seropositivity by the ELISA and IFAT, respectively. In 2 of the 10 seropositive cases no other fetopathogenic agents (such as Chlamydia sp., Leptospira spp. or bovine viral diarrhoea virus) were detected. These data confirm the presence of N. caninum in cattle in Hungary.

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.

Bovine leukaemia virus: rapid detection of proviral DNA by nested PCR in blood and organs of experimentally infected calves.

The early stage of bovine leukaemia virus (BLV) infection was studied in experimentally infected calves in order to assess the diagnostic applicability of a double polymerase chain reaction (PCR). In addition, the kinetics of infection and virus distribution were evaluated. To simulate the natural route of virus transmission, the calves were infected by transferring two different infectious doses of whole blood from a BLV infected cow. The establishment of infection was determined by the double PCR and syncytia formation assay and by indirect serological methods including indirect ELISA, gp51/p24 ELISA, agar gel immunodiffusion (AGID) and Western blotting. BLV antibodies were first detected in ELISA on post infection (p.i.) day 26. Close agreement was found between the results of the various indirect methods. BLV infection was first detected in peripheral blood lymphocytes (PBL) by the PCR on p.i. day 7. No animal became seropositive to BLV prior to direct detection of BLV infection by the PCR. At slaughter, urine and saliva specimens as well as various organs were collected from the calves and tested by the double PCR. Several of the organs yielded positive results: e.g. spleen, uterus, liver, kidney, abomasum, and lymph nodes. Nine out of eleven spleen suspensions were positive by the PCR, including the spleen from one calf, which otherwise remained negative in all tests throughout the experiment. This phenomenon indicates that an animal may be infected without detectable levels of BLV proviral DNA in PBLs and without circulating antibodies, further emphasizing the diagnostic importance of the PCR. The findings indicate that the PCR is the most rapid method for the early detection of BLV infection in cattle and a valuable tool for studying the tropism of the virus.

Pedal cycling fatalities in northern Sweden.

The aim of the study was to elucidate the crash and injury mechanisms in bicycle fatalities in the northern half of Sweden. All available autopsy protocols, hospital records and police reports were scrutinized. In 11 years, 146 bicyclists were fatally injured. The majority of the victims were males (66%) and the median age was 60 years. Most of the crashes (81%) occurred from May through October, during weekdays (84%), and during daylight (86%). Almost all victims (88%) died in a motor vehicle collision, in 21% with a truck. None was wearing a helmet. Poor hearing and cerebral arterosclerosis were probable risk factors among the elderly. Of the victims tested, 10% were under the influence of alcohol, half of whom were involved in single-bicycle crashes. In an additional five cases, the motor vehicle driver was impaired by alcohol. All injuries were due to blunt trauma and 69% of the victims died from head injuries. In 91% of all cases, there was an Abbreviated Injury Scale (AIS) score of the head region of > or = 3. The results indicate that separation of bicyclists from motor vehicle traffic by separate cycling tracks and protection of the head by a helmet would be beneficial.

Toxoplasmosis and border disease in 54 Swedish sheep flocks. Seroprevalence and incidence during one gestation period.

Serum samples from 704 animals from 54 Swedish sheep flocks were analysed by ELISA twice during 1 breeding season for antibodies to Toxoplasma gondii and border disease virus (BDV). An ELISA, originally developed for the detection of antibodies to bovine viral diarrhoea virus (BVDV) in cattle, was assessed on sheep sera and the results were compared with those obtained in a virus neutralization test. The correlation between the 2 assays proved good. Before breeding, 132 (19%) sheep in 42 flocks had antibodies to T. gondii and 7 (1%) sheep in 5 flocks were seropositive to BDV. During the observation period 4 sheep seroconverted to T. gondii and 13 to BDV, giving an incidence rate of 0.7% and 1.9% respectively. No clinical signs due to the infections were observed. In 5 flocks the frequency of barrenness, abortion or stillbirths exceeded 5%, 5% and 8%, respectively, but there was no evidence that this was attributable to the agents studied. The proportion of BDV-positive flocks was significantly higher among flocks that had been in contact with cattle than among those that had not.

Evaluation of an indirect ELISA for the detection of antibodies to bovine leukaemia virus in milk and serum.

An indirect enzyme-linked immunosorbent assay (ELISA) was developed to detect antibodies to BLV in milk and serum (Juntti et al., 1989). The conjugate consists of a monoclonal anti-bovine IgG1 and IgG2 labelled with horseradish peroxidase (HRP). The indirect ELISA was calibrated with EEC reference serum E 4. Standard serum E 4 was scored positive when diluted 8192 times in negative milk and between 12,800 and 25,600 times in negative serum. The sensitivity and specificity of the indirect ELISA relative to the agar gel immunodiffusion test (AGID) were 100% and 99.8%, respectively. ELISA results for milk and sera from 614 dairy cows agreed to 100%. The absorbance value in bulk milk could be used to roughly predict the rate of BLV infection among lactating cows in a herd. An infection rate of 4 to 5% in a herd could be detected in the ELISA. Results were applied in a nation-wide screening of more than 24,000 bulk-milk samples, and the subsequent introduction of an eradication programme for BLV. The aim is to eliminate the infection from Swedish herds in 5 to 10 years.

Pedal cycling accidents--mechanisms and consequences. A study from northern Sweden.

During one year, 447 persons attended the University Hospital of Umeå (Sweden) because of bicycling accidents. The incidence was highest in children, falling with advancing age. The most common accident was falling off a bicycle on an uneven or slippery road. Collisions and objects interfering with the rear or front wheel were also common causes. A high percentage of the injuries involved the head, and one-third of these were major injuries. Almost one-fifth of the injured received in-patient care (average 6 days) and a similar number were paid sickness benefit (average 26.5 days). Costs for treatment and benefit were estimated as approx. 2200 Swedish kronor (SEK) per injured person (1 USD = 4:30 SEK, 1979, and 1984 = 8:20 SEK).