Bacillus subtilis-597 induces changes in lung pathology and inflammation during influenza A virus infection in pigs.

In recent years, it has become apparent that imbalances in the gastrointestinal system can impact organs beyond the intestine such as the lungs. Given the established ability of probiotics to modulate the immune system by interacting with gastrointestinal cells, our research aimed to investigate whether administering the probiotic strain Bacillus subtilis-597 could mitigate the outcome of influenza virus infection in pigs. Pigs were fed a diet either with or without the probiotic strain B. subtilis-597 for 14 days before being intranasally inoculated with a swine influenza A H1N2 strain (1 C.2 lineage). Throughout the study, we collected fecal samples, blood samples, and nasal swabs to examine viral shedding and immune gene expression. After seven days of infection, the pigs were euthanized, and lung and ileum tissues were collected for gene expression analysis and pathological examination. Our findings indicate that the administration of B. subtilis-597 exhibit potential in reducing lung lesions, possibly attributable to a general suppression of the immune system as indicated by reduced C-reactive protein (CRP) levels in serum, decreased expression of interferon-stimulated genes (ISGs), and localized reduction of the inflammatory marker serum amyloid A (SAA) in ileum tissue. Notably, the immune-modulatory effects of B. subtilis-597 appeared to be unrelated to the gastrointestinal microbiota, as the composition remained unaltered by both the influenza infection and the administration of B. subtilis-597.

Experimental infection of pigs and ferrets with "pre-pandemic," human-adapted, and swine-adapted variants of the H1N1pdm09 influenza A virus reveals significant differences in viral dynamics and pathological manifestations.

Influenza A viruses are RNA viruses that cause epidemics in humans and are enzootic in the pig population globally. In 2009, pig-to-human transmission of a reassortant H1N1 virus (H1N1pdm09) caused the first influenza pandemic of the 21st century. This study investigated the infection dynamics, pathogenesis, and lesions in pigs and ferrets inoculated with natural isolates of swine-adapted, human-adapted, and "pre-pandemic" H1N1pdm09 viruses. Additionally, the direct-contact and aerosol transmission properties of the three H1N1pdm09 isolates were assessed in ferrets. In pigs, inoculated ferrets, and ferrets infected by direct contact with inoculated ferrets, the pre-pandemic H1N1pdm09 virus induced an intermediary viral load, caused the most severe lesions, and had the highest clinical impact. The swine-adapted H1N1pdm09 virus induced the highest viral load, caused intermediary lesions, and had the least clinical impact in pigs. The human-adapted H1N1pdm09 virus induced the highest viral load, caused the mildest lesions, and had the least clinical impact in ferrets infected by direct contact. The discrepancy between viral load and clinical impact presumably reflects the importance of viral host adaptation. Interestingly, the swine-adapted H1N1pdm09 virus was transmitted by aerosols to two-thirds of the ferrets. Further work is needed to assess the risk of human-to-human aerosol transmission of swine-adapted H1N1pdm09 viruses.

Investigation of the SARS-CoV-2 post-vaccination antibody response in Canadian farmed mink.

Currently, SARS-CoV-2 have been detected in farmed mink in 13 different countries. Due to the high susceptibility and transmissibility among mink, great concerns of mink serving as a reservoir to generate novel variants with unknown virulence and antigenic properties arose. These concerns have consequently resulted in entire mink productions being culled and banned. This study investigates the post-vaccination antibody response in the Canadian farmed mink vaccinated with a commercial Index spike protein-based vaccine, approved for use in cats, and compares the antibody response to that observed post infection in Danish farmed mink. Blood samples were obtained from 50 mink at the Canadian Centre for Fur Animal Research (CCFAR), Dalhousie University (Truro, Canada). The sera were initially analyzed for antibodies by enzyme-linked immunosorbent assay (ELISA), and selected sera was subsequently tested in a virus neutralization tests. The levels of neutralizing antibodies were evaluated for an ancestral D614G strain and a recent circulating SARS-CoV-2 variant of concern (Omicron BA.4). The results revealed that the vaccine induced a strong antibody response in mink by reaching antibody titer levels of up to 1:12800 in the ELISA. Moreover, high levels of neutralizing antibodies were obtained, and despite the great level of genetic differences between the ancestral and Omicron BA.4 strains, the vaccinated mink showed high levels of cross-reacting neutralizing antibodies. Interestingly, the antibody levels towards SARS-CoV-2 in the Canadian vaccinated mink were significantly higher than observed in recently SARS-CoV-2 infected Danish mink and equal to anamnestic responses following re-infection. In conclusion, the vaccine used in the Canadian farmed mink was able to induce a strong and broad-reacting antibody response in mink, which could limit the spread of SARS-CoV-2 in farmed mink and thereby reduce the risk of mink serving as a SARS-CoV-2 reservoir for human infections.

Severe Human Case of Zoonotic Infection with Swine-Origin Influenza A Virus, Denmark, 2021.

During routine surveillance at the National Influenza Center, Denmark, we detected a zoonotic swine influenza A virus in a patient who became severely ill. We describe the clinical picture and the genetic characterization of this variant virus, which is distinct from another variant found previously in Denmark.

The role of gilts in transmission dynamics of swine influenza virus and impacts of vaccination strategies and quarantine management.

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.

Molecular Characterization of Highly Pathogenic Avian Influenza Viruses H5N6 Detected in Denmark in 2018-2019.

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.

Co-circulation of multiple influenza A reassortants in swine harboring genes from seasonal human and swine influenza viruses.

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.

Infection Dynamics of Swine Influenza Virus in a Danish Pig Herd Reveals Recurrent Infections with Different Variants of the H1N2 Swine Influenza A Virus Subtype.

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.

A recombination between two Type 1 Porcine Reproductive and Respiratory Syndrome Virus (PRRSV-1) vaccine strains has caused severe outbreaks in Danish pigs.

Porcine reproductive and respiratory syndrome virus (PRRSV) is prevalent in Danish swine herds. In July 2019, PRRSV-1 was detected in a PRRSV-negative boar station and subsequently spread to more than 38 herds that had received semen from the boar station. Full genome sequencing revealed a sequence of 15.098 nucleotides. Phylogenetic analyses showed that the strain was a recombination between the Amervac strain (Unistrain PRRS vaccine; Hipra) and the 96V198 strain (Suvaxyn PRRS; Zoetis AH). The major parent was the 96V198 strain that spanned ORFs 1-2 and part of ORF 3 and the minor parent was the Amervac strain, which constituted the remaining part of the genome. The virus seems to be highly transmissible and has caused severe disease in infected herds despite a high level of genetic identity to the attenuated parent strains. The source of infection was presumable a neighbouring farm situated 5.8 km from the boar station.

Substantial Antigenic Drift in the Hemagglutinin Protein of Swine Influenza A Viruses.

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.

Acute Influenza A virus outbreak in an enzootic infected sow herd: Impact on viral dynamics, genetic and antigenic variability and effect of maternally derived antibodies and 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.

Limited impact of influenza A virus vaccination of piglets in an enzootic infected sow herd.

Recent studies have questioned the effect of maternal derived antibodies (MDAs) to protect piglets against infection with influenza A virus (IAV). The lack of protection against IAV infections provided by MDAs has encouraged alternative vaccination strategies targeting young piglets in an attempt to stimulate an early antibody response. There is a lack of studies documenting the efficacy of piglet vaccination. In the present study, we monitored a group of vaccinated and non-vaccinated piglets in a Danish sow herd that initiated piglet vaccination with » dose of an inactivated swine influenza vaccine at the time of castration (day 3-4). A total of 160 piglets from 11 sows were included and either vaccinated with 0.5 mL inactivated swine influenza vaccine or sham-vaccinated. From week 0 until week 6, all included piglets were clinically examined and nasal swapped once per week and weighed at weeks 0, 3 and 6. Blood samples were collected from sows at week 0 and from piglets at week 3. Vaccination of piglets had limited effect on clinical signs, body weight, antibody development and viral shedding, within the first 6 weeks of life. At least 50% of all pigs of each treatment group tested positive for IAV at week 2, and very early onset of IAV shedding was observed. In total, 18 pigs were IAV positive in nasal swabs for more than one consecutive sampling time indicating prolonged shedding and 14 pigs were IAV positive with negative samplings in between indicating re-infection with the same IAV strain.

Longitudinal field studies reveal early infection and persistence of influenza A virus in piglets despite the presence of maternally derived antibodies.

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.