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Abstract Background Equine herpesvirus 4 (EHV-4) causes respiratory disease in horses, and the virus is considered endemic in the global equine population. However, outbreaks can occur when several horses are gathered in relation to shows, competitions, breeding units and at hospitals. In the spring year 2022, an EHV-4 outbreak occurred at the Large Animal Teaching Hospital, University of Copenhagen, Denmark. Nine horses were tested EHV-4 positive during the outbreak, which lasted approx. seven weeks. In addition, a tenth horse “Eq10” tested EHV-4 positive almost three weeks after the last of the outbreak horses tested positive. Detailed clinical registrations were obtained from all ten horses as well as their location and movement during hospitalization. Nasal swabs were obtained throughout the outbreak and tested by qPCR for EHV-4. Additionally, pre- and post-infection sera were tested for the presence of EHV-4 antibodies. Selected samples were characterized by partial and full genome sequencing. Results The most common clinical signs of the EHV-4 infected horses during this outbreak were pyrexia, nasal discharge, mandibular lymphadenopathy and increased lung sounds upon auscultation. Based on the locations of the horses, EHV-4 detection and antibody responses the most likely “patient zero” was identified as being “Eq1”. Partial genome sequencing revealed that Eq10 was infected by another wild type EHV-4 strain, suggesting that the hospital was able to eliminate the outbreak by testing and reinforcing biosecurity measures. The complete genome sequence of the outbreak strain was obtained and revealed a closer relation to Australian and Japanese EHV-4 strains rather than to other European EHV-4 strains, however, very limited sequence data are available from Europe. Conclusion The study illustrated the transmission of EHV-4 within an equine facility/hospital and provided new insights into the viral shedding, antibody responses and clinical signs related to EHV-4 infections. Finally, sequencing proved a useful tool in understanding the transmission within the hospital, and in characterizing of the outbreak strain.

Collegiality in upper-secondary school: Teacher identity in a changing school organization (1960–2010). In this article, it is argued that the teachers at the Norwegian upper secondary school had a distinct collegial practice characterised by both formal and informal aspects of collegiality, as well as close interaction between institutional and disciplinary bodies at the school. This practice enabled the teachers to be involved in their own academic and professional development, as well as in the governance of the school. With the democratisation of the school in the 1960s and 70s, and with the new ideals of governance in the 1990s, the characteristics of the teachers’ collegial culture were seen as a hindrance rather than a resource in the development of the school and the teaching profession. Drawing on unique sources from school archives, the article examines the teachers’ collegiality and how it changed from 1960 to 2010.

IntroductionInfluenza A virus (IAV) infection is a global respiratory disease, which annually leads to 3-5 million cases of severe illness, resulting in 290,000-650,000 deaths. Additionally, during the past century, four global IAV pandemics have claimed millions of human lives. The epithelial lining of the trachea plays a vital role during IAV infection, both as point of viral entry and replication as well as in the antiviral immune response. Tracheal tissue is generally inaccessible from human patients, which makes animal models crucial for the study of the tracheal host immune response.MethodIn this study, pigs were inoculated with swine- or human-adapted H1N1 IAV to gain insight into how host adaptation of IAV shapes the innate immune response during infection. In-depth multi-omics analysis (global proteomics and RNA sequencing) of the host response in upper and lower tracheal tissue was conducted, and results were validated by microfluidic qPCR. Additionally, a subset of samples was selected for histopathological examination.ResultsA classical innate antiviral immune response was induced in both upper and lower trachea after infection with either swine- or human-adapted IAV with upregulation of genes and higher abundance of proteins associated with viral infection and recognition, accompanied by a significant induction of interferon stimulated genes with corresponding higher proteins concentrations. Infection with the swine-adapted virus induced a much stronger immune response compared to infection with a human-adapted IAV strain in the lower trachea, which could be a consequence of a higher viral load and a higher degree of inflammation.DiscussionCentral components of the JAK-STAT pathway, apoptosis, pyrimidine metabolism, and the cytoskeleton were significantly altered depending on infection with swine- or human-adapted virus and might be relevant mechanisms in relation to antiviral immunity against putative zoonotic IAV. Based on our findings, we hypothesize that during host adaptation, IAV evolve to modulate important host cell elements to favor viral infectivity and replication.

Abstract Avian influenza in wild birds and poultry flocks constitutes a problem for animal welfare, food security and public health. In recent years there have been increasing numbers of outbreaks in Europe, with many poultry flocks culled after being infected with highly pathogenic avian influenza (HPAI). Continuous monitoring is crucial to enable timely implementation of control to prevent HPAI spread from wild birds to poultry and between poultry flocks within a country. We here utilize readily available public surveillance data and time-series models to predict HPAI detections within European countries and show a seasonal shift that happened during 2021–2022. The output is models capable of monitoring the weekly risk of HPAI outbreaks, to support decision making.

Abstract Background Recently, in-feed medicinal zinc has been phased out in pig production in the European Union. This makes updated knowledge about porcine post-weaning diarrhea (PWD) crucial. The objectives of the present study were to investigate (i) the clinical presentation of PWD in pigs housed in Danish herds that did not use medicinal zinc, specifically the prevalence of diarrhea and whether PWD was associated to clinical signs of dehydration or altered body temperature; (ii) which microorganism are associated to PWD; and iii) whether measurements of the fecal pH have a potential to be used diagnostically to differentiate between infectious etiologies in cases of PWD. Results The prevalence of diarrhea varied considerably between the outbreaks in the nine studied herds (median = 0.58, range = 0.10; 0.94). In a cross-sectional design (n = 923), diarrhea was associated with reduced rectal temperature and alkaline feces. Diarrhea was also associated with observably reduced skin elasticity, possibly indicating dehydration. In both diarrheic case pigs (n = 87) and control pigs (n = 86), the presence of Brachyspira pilosicoli, Clostridium perfringens, Cryptosporidium spp., Cystoisopora suis, enterotoxigenic Escherichia coli, Lawsonia intracellularis, porcine circovirus types 2 and 3, rotavirus A, B, C, and H, Samonella enterica spp. enterica, and Trichuris suis was described. PWD was associated with high levels of enterotoxigenic E. coli shedding (odds ratio versus no E. coli detection = 4.79 [CI 1.14; 12.62]). Diarrhea was associated with high levels of rotavirus A shedding (odds ratio versus no/low rotavirus A = 3.80 [CI 1.33; 7.97]). The association between microbiological findings in diarrheic pigs and fecal pH was negligible. Conclusions Enterotoxigenic E. coli was confirmed to be a cause of PWD; however, cases of PWD where enterotoxigenic E. coli was not detected in high levels occurred commonly, and this adds to the increasing evidence suggesting that PWD is not necessarily a result of enteric colibacillosis. Rotaviral enteritis might be a differential diagnosis of PWD. pH-measurements cannot be used to differentiate between differential diagnoses for PWD.

Abstract Background Post-weaning diarrhoea (PWD) is a multifactorial condition and the most well documented infectious cause is enterotoxigenic Escherichia coli. The objective of the study was to investigate possible associations between pathological manifestations and pathogens in pigs with and without PWD. The study was conducted as a case–control study and included a total of 173 pigs from 9 different commercial intensive indoor production herds in eastern Denmark. Results Based on clinical examination, a total of 89 piglets with PWD (cases) and 84 piglets without PWD (controls) were included. Most of the pigs (n = 105/173) presented gastric lesions, which were more frequently observed in the control group. The odds of gastric ulcers were lower among pigs with PWD compared to pigs without PWD with an odds ratio (OR) of 0.2 (0.0; 0.7). Abnormal content in the colon was associated with PWD, with an OR of 6.5 (3.2; 14.3). No apparent association was found between lesions and the various pathogens or a combination of these. The odds of neutrophilic granulocyte infiltration were lower in the jejunum among pigs with PWD (OR 0.3 [0.1; 0.6]) compared to pigs without PWD. The association between neutrophilic granulocyte infiltration in jejunum and PWD differed between the herds (P = 0.03). Furthermore, the associations between PWD and hyperleukocytosis (P = 0.04) or infiltration of eosinophilic granulocytes (P = 0.04) in ileum were also herd dependent. Histopathology revealed several lesions not relatable to PWD. Conclusion The association between lesions and specific pathogens or PWD is more complex than anticipated.

Exploring the risk factors of avian influenza (AI) occurrence helps us to monitor and control the disease. Since late 2020, the number of avian influenza outbreaks in domestic and wild birds has increased in most European countries, including Denmark. This study was conducted to identify potential risk factors for wild birds and poultry during the epidemic in 2020/2021 in Denmark. Using Danish AI surveillance data of actively surveyed poultry and passively surveyed wild birds from June 2020 to May 2021, we calculated geographical attributes for bird locations and assessed the potential risk factors of AI detections using logistic regression analyses. 4% of actively surveyed poultry and 39% of passively surveyed wild birds were detected with AI circulating or ongoing at the time. Of these, 10 and 99% tested positive for the H5/H7 AI subtypes, respectively. Our analyses did not find any statistically significant risk factors for actively surveyed poultry within the dataset. For passively surveyed wild birds, bird species belonging to the Anseriformes order had a higher risk of being AI virus positive than five other taxonomic bird orders, and Galliformes were of higher risk than two other taxonomic bird orders. Besides, every 1 km increase in the distance to wetlands was associated with a 5.18% decrease in the risk of being AI positive (OR (odds ratio) 0.95, 95% CI 0.91, 0.99), when all other variables were kept constant. Overall, bird orders and distance to wetlands were associated with the occurrence of AI. The findings may provide targets for surveillance strategies using limited resources and assist in risk-based surveillance during epidemics.

Abstract Background Diagnosing and treatment of diseases in pigs are important to maintain animal welfare, food safety and productivity. At the same time antimicrobial resistance is increasing, and therefore, antibiotic treatment should be reserved for individuals with a bacterial infection. The aim of the study was to investigate gross and histological lesions and related pathogens in pigs that died during the nursery period in five Danish farms. In addition, high throughput, real-time qPCR monitoring of specific porcine pathogens in fecal sock and oral fluid samples were carried out to investigate the between-farm and between-batch variation in the occurrence of pathogens. Results Twenty-five batches of nursery pigs from five intensive, indoor herds were followed from weaning (approximately four weeks) to the end of nursery (seven to eight weeks post weaning). Gross and histological evaluation of 238 dead and 30 euthanized pigs showed the highest prevalence of lesions in the skin, respiratory system, gastrointestinal tract, and joints. Gross and histological diagnoses of lung and joint lesions agreed in 46.5% and 62.2% of selected pigs, respectively. Bacteriological detection of Escherichia coli, Streptococcus suis or Staphylococcus aureus infections in joints, lungs and livers was confirmed as genuine infection on immunohistochemical staining in 11 out of 70 tissue sections. The real-time qPCR analysis of pooled samples showed that most pathogens detected in feces and in oral fluid in general followed the same shedding patterns in consecutive batches within herds. Conclusions Gross assessment should be supplemented with a histopathological assessment especially when diagnosing lesions in the lungs and joints. Moreover, microbiological detection of pathogens should optimally be followed up by in situ identification to confirm causality. Furthermore, routine necropsies can reveal gastric lesions that may warrant a change in management. Real-time qPCR testing of fecal sock samples and oral fluid samples may be used to monitor the infections in the individual herd and testing one batch seems to have a good predictive value for subsequent batches within a herd. Overall, optimal diagnostic protocols will provide a more substantiated prescription of antibiotics.

This study aimed to enhance our understanding of the agreement between two sampling methods for the detection of bovine respiratory disease (BRD) pathogens in calves using high-throughput real-time qPCR (ht-RT-qPCR). In total, 233 paired nasal swab (NS) and non-endoscopic bronchoalveolar lavage (nBAL) samples were collected from 152 calves from 12 Danish cattle herds. In 202 of the observations, the calves were examined using a standardized clinical protocol. Samples were tested for three viruses (bovine respiratory syncytial virus, bovine corona virus, and influenza D virus) and six bacteria (Histophilus somni, Mannheimia haemolytica, Mycoplasma bovis, Mycoplasma species, Pasteurella multocida, and Truepurella pyogenes). The results showed age-related differences in disease and pathogen occurrence, with the highest detection rates in calves aged 35 days or older. Poor to moderate agreement was found between the NS and nBAL results. The presence of Mannheimia haemolytica in both NS and nBAL in younger calves and in nBAL in older calves was associated with clinical BRD. There was a potential link between BRD and influenza D virus in older calves, although it was only found in one herd in a small sample size. Overall, NS was a relatively poor predictor of pathogens in the lower respiratory tract. The present study confirms the complexity of pathogen detection in BRD, with marked influences of age and the sampling method on pathogen detection and disease associations.

BackgroundClinical and immunological studies in humans show that the live attenuated Bacillus Calmette-Guérin (BCG) vaccine has beneficial non-specific effects, increasing resistance against diseases other than tuberculosis. The underlying mechanisms are currently being explored. The pig exhibits considerable physiological similarity to humans in anatomy and physiology, suggesting that similar responses to BCG could be expected. Studies of the non-specific effects of BCG in pigs are scarce. We investigated the feasibility of using pigs as a large animal model to investigate the non-specific immunological effects of BCG.MethodsIn a series of experiments, we randomized newborn or young piglets from conventional farms to receiving BCG or placebo and investigated the persistence of live BCG bacteria in various tissues, the immunogenicity of BCG in ex vivo blood and in vitro stimulation assays, and the acute phase protein and clinical responses to heterologous infectious challenge with influenza A virus or Actinobacillus pleuropneumoniae.ResultsThe BCG vaccine was generally well tolerated. In contrast to humans, no skin reaction in the form of abscesses, ulcers, or scars was observed. Live BCG was recovered from draining lymph nodes in 2/13 animals 20 weeks after vaccination. Specific in vitro responses of IFN-γ to antigen-specific re-stimulation with mycobacterial antigen were increased but not TNF-responses to TLR2 or TLR4 agonists. A few genes were differentially expressed in blood after vaccination, including the antiviral genes RIG-I and CSF1, although the effect disappeared after correction for multiple testing. Clinical symptoms after heterologous bacterial or viral respiratory infections did not differ, nor did virus copies in nasopharyngeal samples after the challenge. However, the acute phase protein response was significantly reduced in BCG-vaccinated animals after influenza challenge but not after A. pleuropneumoniae challenge.DiscussionBCG was safe in pigs, inducing specific immunological responses, but our model did not corroborate the innate immunological responsiveness to BCG seen in humans. The dose of BCG or the bacterial and viral challenges may have been sub-optimal. Even so, the acute phase protein response to influenza infection was significantly reduced in BCG-vaccinated animals.

Abstract Background Along with an expanding global swine production, the commercial housing and management of swine herds, provide an optimal environment for constant circulation of swine influenza virus (swIAV), thereby challenging farmers and veterinarian in determining optimal control measures. The aim of this study was to investigate the role of gilts in the swIAV transmission dynamics, and to evaluate the impact of different control measures such as quarantine and gilt vaccination. Methods The study was conducted as a cross-sectional study in ten Danish sow herds, including five swIAV vaccinated and five unvaccinated herds. Blood- and nasal swab samples of gilts, first parity sows and their piglets were collected at different stages in the production system (quarantine in/out, mating, gestation and farrowing) and analyzed for the presence of swIAV and swIAV antibodies. Associations between the detection of swIAV, seroprevalence, antibody levels, sow and gilt vaccination strategy and quarantine biosecurity were thereafter investigated to identify possible risk factors for swIAV introductions and persistence within the herds. Results Nine of the ten herds of the study had swIAV circulation and swIAV was detected in the quarantine, mating- and farrowing unit. The prevalence of seropositive gilts and first parity sows was significantly higher in the vaccinated herds, but swIAV was still present in nasal swabs from both gilts, first parity sows and piglets in these herds. Quarantine gilt vaccination and all-in/all-out management resulted in a significant reduction of swIAV positive gilts at the end of the quarantine period. Conclusion The results underline that herd vaccination and/or quarantine facilities are crucial to avoid swIAV introductions into sow herds.

Abstract Background Porcine post-weaning diarrhea (PWD) has reemerged as an important topic in pig production, as common control strategies based on prophylactic use of antimicrobials and zinc oxide have been deemed unsustainable. The objectives of this study were to estimate the cumulative incidence of porcine post-weaning diarrhea with different etiologies in production systems weaning without zinc oxide and prophylactic antimicrobials, to assess risk factors for post-weaning diarrhea, and to estimate the impact of post-weaning diarrhea on growth rate. A cohort study was conducted at two commercial indoor producers weaning without medicinal zinc oxide and prophylactic antimicrobials. Results Piglets were included at birth (n = 300) and 272 survived until weaning. After insertion to the nursery units, the piglets were clinically examined every day for 14 days, and rectal swabs were collected and analyzed for enterotoxigenic Escherichia coli (ETEC) and rotavirus A. The cumulative incidences of PWD the first 14 days after insertion to the nursery units were 41.8% (CI 33.6, 50.4) and 51.1% (CI 42.3, 60.0) at the two producers, respectively. We found a low incidence of cases associated to ETEC, and detected a substantial proportion of cases associated to rotavirus. We observed a biphasic pattern in the assumed etiology with rotavirus occurring first, and then a shift towards cases associated to ETEC/non-ETEC hemolytic E. coli. Being offspring of older sows was a protective factor for the development of PWD (Hazard ratio = 0.88 [CI 0.78, 0.99] per unit increase in parity of the dam). Low birth weight reduced the post-weaning growth rate (− 5.2 g/day [CI − 7.5, − 2.9] per 100 g decrease in birthweight) and increased the hazard of developing PWD (Hazard ratio for birthweight below 1100 g: 2.30 [CI 1.41–3.74]). The combined effect of having diarrhea for 2 days or more and receiving antimicrobial treatment was associated with an increased average daily weight gain. Conclusions This study suggests novel insights regarding pathogen dynamics and risk factors for PWD in productions not using prophylactic antimicrobials and medicinal zinc. The findings may have important implications for both antimicrobial usage and prevention strategies.

Major geographical transmission of porcine reproductive and respiratory syndrome virus (PRRSV) occurs via semen when a boar stud is infected. This happened in Denmark in 2019, providing an opportunity to compare previous experimental PRRSV boar studies with natural PRRSV-1 infection in boars. The aim of this study was to investigate the association between the presence of PRRSV RNA in serum, semen, testicles, and epididymis of boars naturally infected with PRRSV and to describe the histological lesions in the testes and epididymis combined with direct visualisation of PRRSV-infected cells by immunohistochemical staining (IHC). The exact timing of infection of each boar was not determined, but based on serology the boars were divided into two groups: acute and late infections. All boars included were sampled the same day. In this study, 35 boars and 10 healthy boars from another PRRSV-negative boar stud were included as histological controls. PRRSV RNA was found most often in serum (51%) and least frequently in semen (22%) and was more often detected in the reproductive tract in the acute phase of infection (p < 0.0001; RR: 2.58). Mononuclear cells and multinuclear giant cells were present in the adluminal compartment of the testis and epididymis in PRRSV-infected boars, but not in control boars (p < 0.05), which supports the hypothesis that macrophages are involved in the venereal spread of the virus.

Since the influenza pandemic in 2009, there has been an increased focus on swine influenza A virus (swIAV) surveillance. This paper describes the results of the surveillance of swIAV in Danish swine from 2011 to 2018. In total, 3800 submissions were received with a steady increase in swIAV-positive submissions, reaching 56% in 2018. Full-genome sequences were obtained from 129 swIAV-positive samples. Altogether, 17 different circulating genotypes were identified including six novel reassortants harboring human seasonal IAV gene segments. The phylogenetic analysis revealed substantial genetic drift and also evidence of positive selection occurring mainly in antigenic sites of the hemagglutinin protein and confirmed the presence of a swine divergent cluster among the H1pdm09Nx (clade 1A.3.3.2) viruses. The results provide essential data for the control of swIAV in pigs and emphasize the importance of contemporary surveillance for discovering novel swIAV strains posing a potential threat to the human population.

Abstract A longitudinal study was performed in three Danish farrow to grower (30 kilos) herds over a 4-month period to investigate the dynamics and clinical impacts of influenza A virus (IAV) infections. In each herd, four batches consisting of four sows each with five ear-tagged piglets were included. Nasal swabs and/or blood were sampled from the sows and/or the piglets prior to farrowing and at weeks 1, 3, and 5 and at the end of the nursery period. Clinical examinations were performed at each sampling time. The sows and piglets were tested for IAV and IAV antibodies in nasal swabs and blood samples, respectively. The results revealed three enzootically infected herds, where the majority of the pigs were infected during the first 5 weeks after birth. Infected piglets of only 3 days of age were detected in the farrowing unit, where the sows were also shedding virus. In all herds, low to moderate numbers of infected pigs (ranging from 3.6 to 20.7%) were found to be virus positive in nasal swabs at two consecutive sampling times. Furthermore, clinical signs of respiratory disease were associated with IAV detection. The findings of this study documented that IAV can persist in herds and that piglets as young as 3 days can be infected despite the presence of maternally derived antibodies.

Bovine respiratory disease (BRD) results from interactions between pathogens, environmental stressors, and host factors. Obtaining a diagnosis of the causal pathogens is challenging but the use of high-throughput real-time PCR (rtPCR) may help target preventive and therapeutic interventions. The aim of this study was to improve the interpretation of rtPCR results by analysing their associations with clinical observations. The objective was to develop and illustrate a field-data driven statistical method to guide the selection of relevant quantification cycle cut-off values for pathogens associated with BRD for the high-throughput rtPCR system “Fluidigm BioMark HD” based on nasal swabs from calves. We used data from 36 herds enrolled in a Danish field study where 340 calves within pre-determined age-groups were subject to clinical examination and nasal swabs up to four times. The samples were analysed with the rtPCR system. Each of the 1,025 observation units were classified as sick with BRD or healthy, based on clinical scores. The optimal rtPCR results to predict BRD were investigated for Pasteurella multocida, Mycoplasma bovis, Histophilus somni, Mannheimia haemolytica, and Trueperella pyogenes by interpreting scatterplots and results of mixed effects logistic regression models. The clinically relevant rtPCR cut-off suggested for P. multocida and M. bovis was ≤ 21.3. For H. somni it was ≤ 17.4, while no cut-off could be determined for M. haemolytica and T. pyogenes. The demonstrated approach can provide objective support in the choice of clinically relevant cut-offs. However, for robust performance of the regression model sufficient amounts of suitable data are required.

Abstract Background Many factors can influence the occurrence of neonatal diarrhoea in piglets. Currently, well-known pathogens such as enterotoxigenic Escherichia coli and Clostridium perfringens type C appear to play a minor role in development of disease. Other infectious pathogens may be involved. In this study, we aimed to investigate the presence of selected infectious pathogens in neonatal piglets with clinical and pathological signs of enteric disease. The association between rotavirus A, Enterococcus hirae, Clostridium difficile and Clostridium perfringens type A/C and diarrhoea was investigated in a case control study on piglet level. The possible role of E. coli virulence factors was investigated in a multistep-procedure using herd-pools of E.coli isolates to screen for their presence. Results Rotavirus A was detected more often in cases (25%) than in controls (6%) (P

In recent years, there has been a considerable increase in the use of Modified Live PRRSV Vaccines (MLV) for mass vaccination in Denmark. The potential risks and negative impact of this strategy have been sparsely studied. The aim of this study was to investigate the impact of quarterly sow mass vaccination in two Danish sow herds. The study was performed as an observational prospective cohort of 120 sows in each of two commercial breeding herds in a paired design. Blood samples were taken from sows and oral fluid samples from nursery pigs (four to ten weeks old) before and after vaccination. The presence of PRRSV-1 RNA was measured by real time quantitative reverse transcription-polymerase chain reaction (RT-qPCR), and the level of PRRSV-1 specific antibodies was measured by two different serological assays. PRRS virus was not detected in the sow herds two days before and two weeks after vaccination, but the vaccine strain virus was detected in the nursery pigs. The prevalence of sows without antibodies towards PRRSV-1 went from 6–15% before vaccination to 1–4% after vaccination depending on the serological assay used, despite the fact that they had previously been repeatedly vaccinated. Four sows tested negative for antibodies in both assays after vaccination.

Influenza A virus (IAV) is a highly contagious pathogen in pigs. Swine IAV (swIAV) infection causes respiratory disease and is thereby a challenge for animal health, animal welfare and the production economy. In Europe, the most widespread strategy for controlling swIAV is implementation of sow vaccination programs, to secure delivery of protective maternally derived antibodies (MDAs) to the newborn piglets. In this study we report a unique case, where a persistently swIAV (A/sw/Denmark/P5U4/2016(H1N1)) infected herd experienced an acute outbreak with a new swIAV subtype (A/sw/Denmark/HB4280U1/2017(H1N2)) and subsequently decided to implement a mass sow vaccination program. Clinical registrations, nasal swabs and blood samples were collected from four different batches of pigs before and after vaccination. Virus isolation, sequencing of the virus strain and hemagglutinin inhibition (HI) tests were performed on samples collected before and during the outbreak and after implementation of mass sow vaccination. After implementation of the sow mass vaccination, the time of infection was delayed and the viral load significantly decreased. An increased number of pigs, however, tested positive at two consecutive sampling times indicating prolonged shedding. In addition, a significantly smaller proportion of the 10-12 weeks old pigs were seropositive by the end of the study, indicating an impaired induction of antibodies against swIAV in the presence of MDAs. Sequencing of the herd strains revealed major differences in the hemagglutinin gene of the strain isolated before- and during the acute outbreak despite that, the two strains belonged to the same HA lineage. The HI tests confirmed a limited degree of cross-reaction between the two strains. Furthermore, the sequencing results of the hemagglutinin gene obtained before and after implementation of mass sow vaccination revealed an increased substitution rate and an increase in positively selected sites in the globular head of the hemagglutinin after vaccination.

Beginning in late 2017, highly pathogenic avian influenza (HPAI) H5N6 viruses caused outbreaks in wild birds and poultry in several European countries. H5N6 viruses were detected in 43 wild birds found dead throughout Denmark. Most of the Danish virus-positive dead birds were found in the period from February to April 2018. However, unlike the rest of Europe, sporadic HPAI H5N6-positive dead wild birds were detected in Denmark in July, August, September, and December 2018, with the last positive bird being found in January 2019. HPAI viruses were not detected in active surveillance of apparently healthy wild birds. In this study, we use full genome sequencing and phylogenetic analysis to investigate the wild bird HPAI H5N6 viruses found in Denmark. The Danish viruses were found to be closely related to those of contemporary HPAI H5N6 viruses detected in Europe. Their sequences formed two clusters indicating that at least two or more introductions of H5N6 into Denmark occurred. Notably, all viruses detected in the latter half of 2018 and in 2019 grouped into the same cluster. The H5N6 viruses appeared to have been maintained undetected in the autumn 2018.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes severe respiratory distress and reproductive failure in swine. Modified live virus (MLV) vaccines provide the highest degree of protection and are most often the preferred choice. While somewhat protective, the use of MLVs is accompanied by multiple safety issues, why safer alternatives are urgently needed. Here, we describe the generation of virus replicon particles (VRPs) based on a classical swine fever virus genome incapable of producing infectious progeny and designed to express conserved PRRSV-2 cytotoxic T-cell epitopes. Eighteen pigs matched with the epitopes by their swine leucocyte antigen-profiles were vaccinated (N = 11, test group) or sham-vaccinated (N = 7, control group) with the VRPs and subsequently challenged with PRRSV-2. The responses to vaccination and challenge were monitored using serological, immunological, and virological analyses. Challenge virus load in serum did not differ significantly between the groups, whereas the virus load in the caudal part of the lung was significantly lower in the test group compared to the control group. The number of peptide-induced interferon-γ secreting cells after challenge was higher and more frequent in the test group than in the control group. Together, our results provide indications of a shapeable PRRSV-specific cell-mediated immune response that may inspire future development of effective PRRSV vaccines.

Influenza A virus (IAV) in swine, so-called swine influenza A virus (swIAV), causes respiratory illness in pigs around the globe. In Danish pig herds, a H1N2 subtype named H1N2dk is one of the main circulating swIAV. In this cohort study, the infection dynamic of swIAV was evaluated in a Danish pig herd by sampling and PCR testing of pigs from two weeks of age until slaughter at 22 weeks of age. In addition, next generation sequencing (NGS) was used to identify and characterize the complete genome of swIAV circulating in the herd, and to examine the antigenic variability in the antigenic sites of the virus hemagglutinin (HA) and neuraminidase (NA) proteins. Overall, 76.6% of the pigs became PCR positive for swIAV during the study, with the highest prevalence at four weeks of age. Detailed analysis of the virus sequences obtained showed that the majority of mutations occurred at antigenic sites in the HA and NA proteins of the virus. At least two different H1N2 variants were found to be circulating in the herd; one H1N2 variant was circulating at the sow and nursery sites, while another H1N2 variant was circulating at the finisher site. Furthermore, it was demonstrated that individual pigs had recurrent swIAV infections with the two different H1N2 variants, but re-infection with the same H1N2 variant was also observed. Better understandings of the epidemiology, genetic and antigenic diversity of swIAV may help to design better health interventions for the prevention and control of swIAV infections in the herds.

The degree of antigenic drift in swine influenza A viruses (swIAV) has historically been regarded as minimal compared to that of human influenza A virus strains. However, as surveillance activities on swIAV have increased, more isolates have been characterized, revealing a high level of genetic and antigenic differences even within the same swIAV lineage. The objective of this study was to investigate the level of genetic drift in one enzootically infected swine herd over one year. Nasal swabs were collected monthly from sows (n = 4) and piglets (n = 40) in the farrowing unit, and from weaners (n = 20) in the nursery. Virus from 1−4 animals were sequenced per month. Analyses of the sequences revealed that the hemagglutinin (HA) gene was the main target for genetic drift with a substitution rate of 7.6 × 10−3 substitutions/site/year and evidence of positive selection. The majority of the mutations occurred in the globular head of the HA protein and in antigenic sites. The phylogenetic tree of the HA sequences displayed a pectinate typology, where only a single lineage persists and forms the ancestor for subsequent lineages. This was most likely caused by repeated selection of a single immune-escape variant, which subsequently became the founder of the next wave of infections.

A major outbreak of canine distemper virus (CDV) in Danish farmed mink (Neovison vison) started in the late summer period of 2012. At the same time, a high number of diseased and dead wildlife species such as foxes, raccoon dogs, and ferrets were observed. To track the origin of the outbreak virus full-length sequencing of the receptor binding surface protein hemagglutinin (H) was performed on 26 CDV's collected from mink and 10 CDV's collected from wildlife species. Subsequent phylogenetic analyses showed that the virus circulating in the mink farms and wildlife were highly identical with an identity at the nucleotide level of 99.45% to 100%. The sequences could be grouped by single nucleotide polymorphisms according to geographical distribution of mink farms and wildlife. The signaling lymphocytic activation molecule (SLAM) receptor binding region in most viruses from both mink and wildlife contained G at position 530 and Y at position 549; however, three mink viruses had an Y549H substitution. The outbreak viruses clustered phylogenetically in the European lineage and were highly identical to wildlife viruses from Germany and Hungary (99.29% - 99.62%). The study furthermore revealed that fleas (Ceratophyllus sciurorum) contained CDV and that vertical transmission of CDV occurred in a wild ferret. The study provides evidence that wildlife species, such as foxes, play an important role in the transmission of CDV to farmed mink and that the virus may be maintained in the wild animal reservoir between outbreaks.