Intranasal Inoculation with Classical Swine Fever Virus Provided a More Consistent Experimental Disease Model Compared to Oral Inoculation.

The severity of disease resulting from classical swine fever virus (CSFV) infection is determined by several factors, including virus strain and host factors. The different outcomes of experimental studies in pigs with the same strain of CSFV emphasize the need to elucidate the influence of individual factors within experimental protocols. In this study, we investigated the outcome of disease after oral and intranasal inoculation with a moderately virulent CSFV strain in young pigs. To compare the two routes of inoculation, various infection parameters were examined during a period of two weeks. While all intranasally inoculated pigs (n = 5) were directly infected, this was only the case for two out of five pigs after oral inoculation. In addition, the intranasally inoculated pigs developed a more pronounced clinical disease and pathological lesions, as well as markedly more change in hematological and immunological parameters than the orally inoculated pigs. The wide variation among the orally inoculated pigs implied that statistical evaluation was markedly impaired, leaving this route of application less suitable for comparative studies on classical swine fever. Furthermore, our study provides additional details about the immunomodulatory effects of CSFV on the kinetics of CRP, TNF-α, and leukocyte sub-populations in pigs after infection with the CSFV strain Paderborn.

Computed Tomography as a Method for Age Determination of Carnivora and Odontocetes with Validation from Individuals with Known Age.

Traditional methods for age determination of wildlife include either slicing thin sections off or grinding a tooth, both of which are laborious and invasive. Especially when it comes to ancient and valuable museum samples of rare or extinct species, non-invasive methods are preferable. In this study, X-ray micro-computed tomography (µ-CT) was verified as an alternative non-invasive method for age determination of three species within the order of Carnivora and suborders Odontoceti. Teeth from 13 red foxes (Vulpes vulpes), 2 American mink (Neogale vison), and 2 harbor porpoises (Phocoena phocoena) of known age were studied using µ-CT. The number of visible dental growth layers in the µ-CT were highly correlated with true age for all three species (R2 = 96%, p < 0.001). In addition, the Bland-Altman plot showed high agreement between the age of individuals and visible dental layers represented in 2D slices of the 3D µ-CT images. The true age of individuals was on average 0.3 (±0.6 SD) years higher than the age interpreted by the µ-CT image, and there was a 95% agreement between the true age and the age interpreted from visible dental layers in the µ-CT.

Gastro-intestinal lesions are not relatable to diarrhoea or specific pathogens in post-weaning diarrhoea (PWD) in pigs.

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.

Immunogenicity of Bacillus Calmette-Guérin in pigs: potential as a translational model of non-specific effects of BCG.

Background: Clinical 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. Methods: In 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. Results: The 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. Discussion: BCG 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.

Lesions and pathogens found in pigs that died during the nursery period in five Danish farms.

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.

First finding of Streptococcus phocae infections in mink (Neovison vison).

Streptococcus phocae infection has been described in salmon, sea otters, and several families of pinnipeds. The pathology of the infected animals has mainly been located in the respiratory tract and reproductive system, and with indications of septicemia. In this study, we report the finding of S. phocae in diagnostic material from three unrelated cases of farmed mink. Since S. phocae initially has been described in pinnipeds, two isolates from wild harbor seals were included. All isolates originated from Denmark. To our knowledge, this is the first report of S. phocae infection in mink. The animals (three mink, two seals) were necropsied, and samples were collected for bacteriology, virology, and histopathology. Additionally, the S. phocae isolates were whole genome sequenced and compared to sequences of previously reported isolates from other host species. S. phocae was isolated from the lungs of one mink and one seal with bacteremia, and from one seal with pneumonia. The two remaining mink had dermal infections on the paws and S. phocae was isolated from the lesions. The analysis of the sequence data showed that the three mink isolates and one seal isolate were closely related. Further investigation is needed to conclude whether S. phocae is establishing as commensal in farmed mink and to uncover the infection related pathology in mink. Streptococcus phocae has been described as an emerging pathogen in other species, therefore future awareness and surveillance of this pathogen is crucial.

Estimation of the Age and Reproductive Performance of Wild-Born and Escaped Mink (Neovison vison) Caught in the Wild in Denmark.

The feral mink population in Denmark consists of two groups of animals: mink born in the wild and mink that have recently escaped from farms. The aims of this study were to: (1) estimate the reproductive performance and mortality of the Danish mink born in the wild (wild-born) and mink escaped from farms (captive-born); (2) discuss the likelihood of a self-sustaining population of wild-born mink in Denmark; and (3) model the relationship between the pulp cavity width and the age of mink. During 2018, 247 wild caught mink were sent for necropsy at the Danish National Veterinary Institute. Based on body length, 112 were determined as captive-born and 96 as wild-born. The mean litter size ± SE of wild-born females was 7.6 ± 0.9 (range: 5-11 kits) and for captive-born females 5.9 ± 0.9 (range: 1-10 kits). The relationship between age (in months) of mink and pulp cavity width was highly significant. Individuals with a pulp cavity width of >35% were younger than one year. Based on fecundity, the turnover of the mink population was estimated to be 66%, and the yearly mortality was estimated at 69%. Hence, the population is slightly declining. In conclusion, a feral reproducing mink population in Denmark persists without a continuous influx of captive-born mink from farms.

Diagnostic studies of abortion in Danish cattle 2015-2017.

BACKGROUND: Abortion is a major source of economic losses in cattle breeding. Abortion occurs due to a wide range of causes, but infections are the most frequently diagnosed. However, establishing an aetiological diagnosis remains challenging due to the large variety of bacteria, protozoa, viruses, and fungi that have been associated with abortion in cattle. Economic restraints limit the range of diagnostic methods available for routine diagnostics, and decomposition of the conceptus or lack of proper fetal and/or maternal samples further restrict the diagnostic success. In this study, we report recent diagnostic findings from bovine abortions in Denmark, a country that has a large dairy sector and is free from most infectious agents causing epizootic abortion in cattle. The aims of the study were: (i) to identify infectious causes of bovine abortion in Denmark, (ii) to categorise the diagnostic findings based on the level of diagnostic certainty, and (iii) to assess the diagnostic rate. Due to economic restraints, only a limited panel of routine diagnostic methods were available. Placentas and/or fetuses from mid- to late-term abortions and stillbirths (n = 162) were submitted to the Danish National Veterinary Institute between January 2015 and June 2017. The aborted materials were examined macroscopically, histologically, and by bacterial culture. Maternal blood samples were tested for bovine viral diarrhoea virus (BVDV) antibodies. RESULTS: The likely aetiology of the abortion was diagnosed in 52 cases, resulting in a diagnostic rate of 33%. The most common cause was protozoal infection (19%) followed by infection with Trueperella pyogenes (3%), Staphylococcus aureus (2%), and non-haemolytic Escherichia coli (2%). Lesions in fetuses with a protozoal infection were consistent with neosporosis. In many cases (38%), inflammatory changes were found in the placenta and/or fetal organs but no specific aetiology was identified. Neither infection with Brucella spp. nor maternal BVDV antibodies were detected. The majority of submitting herds (92%) were each represented by fewer than three abortion cases over the study period. CONCLUSIONS: Protozoal infection, most likely neosporosis, was the most commonly diagnosed cause of abortion and the only one associated with potential epizootic abortion events. Despite using a reduced number of diagnostic methods in comparison to other abortion studies, the diagnostic rate of this study was within the range reported in an earlier Danish study, as well as in recent international studies. The low number of submitted cases per herd and the sparse anamnestic information provided at submission hampered conclusions on the potential epizootic character of the abortion events in question.

Methods for the identification of farm escapees in feral mink (Neovison vison) populations.

In Denmark, American mink (Neovison vison) have been bred for their fur since the mid-1920s. Mink escaping from farms may supply the feral population. Often, it is of biological and management interest to separate the population of feral mink (i.e. mink caught in the wild) in two groups: 1) mink born on farms i.e., escapees, and 2) mink born in the wild. In this study, two methods were used for separating feral mink into the two groups: a) Comparison of body length of farmed mink and feral mink, and b) Presence of a biomarker (tetracycline: an oral antibiotic used on mink farms). A total of 367 wild caught mink (from the mainland of Denmark and the island of Bornholm), and 147 mink from farms, collected during the period 2014-2018, were used for the analysis of body length. For the testing of tetracycline (TC) as a biomarker, 78 mink from farms where there was knowledge about TC treatment (with or without) were examined for fluorescent markings in the canine teeth. Results from both univariate analyses and Gaussian mixture model analysis demonstrated clear divisions between the mean body length (mean ± S.E., range) of farmed males (52.1 cm ± 0.4, 48-68) and farmed females (mean 44.0 ± 0.2, 40-50), and between farmed mink and wild caught mink. Mixture analysis identified two groups within each sex of the wild caught mink, one assigned to farmed mink (born in captivity) and another group of smaller mink suspected of being born in the wild. On Bornholm, the mean (±SD, range) length of males born in the wild was 43.7cm (± 0.3, 36-57) and for females 37.5cm (± 0.3, 32-45). The mean length (±SD, range) of males born in the wild in the mainland of Denmark was 42.5cm (± 2.3, 36-46) and for females 36.1cm (± 1.0, 34-37). Among the feral mink from mainland Denmark, 28.4% of males and 21.6% of females were identified as escapees, while 0% of the males and 1% of the females were identified as escapees among the wild caught mink on Bornholm. Eight percent of mink from farms using tetracycline were false negatives, while no false positives were found among mink from farms not using TC. TC fluorescence was found in five of 217 mink caught in the wild equivalent to 22% escapees in mainland Denmark. No TC markings were found in mink caught in the wild on Bornholm. In conclusion, both methods a) the body length of mink, and b) fluorescent biomarkers in canine teeth are considered as useful tools to identifing mink that have escaped from farms.

The Effect of Inactivated Mycobacterium Paratuberculosis Vaccine on the Response to a Heterologous Bacterial Challenge in Pigs.

Background: Vaccines may have non-specific effects, affecting resistance to heterologous pathogens. Veterinary vaccines have seldom been investigated for their non-specific effects. However, recent observational studies suggest that an inactivated paratuberculosis vaccine reduced all-cause mortality in goats and cattle. Aim: We tested if vaccination with a killed mycobacterial vaccine may have heterologous effects in swine (Sus domesticus), specifically on the pathogenic and clinical effects of a heterologous challenge with Actinobacillus pleuropneumoniae in young pigs. Methods: Newborn piglets were randomized to vaccination s.c. with the inactivated paratuberculosis vaccine Gudair (Zoetis Inc.) (n = 17) or no vaccine (n = 16). At 4-5 weeks after vaccination, all piglets were challenged intra-nasally with a high (Gudair: n = 8; control: n = 8) or a low (Gudair: n = 9; control: n = 8) dose of the gram-negative bacterium A. pleuropneumoniae causing acute porcine pleuropneumonia. The effect and severity of pathogen challenge was evaluated by measuring acute phase proteins C-reactive protein, haptoglobin and Porcine α1-acid glycoprotein, and by gross pathology 1 day post challenge. Specific and non-specific in vitro cytokine responses to vaccination were evaluated in whole blood before bacterial challenge. Results: The vaccine was immunogenic in the pigs as evidenced by increased IFN-γ responses to purified protein derivative of Mycobacterium paratuberculosis. However, Gudair vaccine did not affect IL-6 responses. The gross pathology of the lungs as well as the acute phase protein responses after the high A. pleuropneumoniae dose challenge was slightly increased in the vaccinated animals compared with controls, whereas this was not seen in the animals receiving the low-dose bacterial challenge. Conclusion: The inactivated paratuberculosis vaccine exacerbated the pathological and inflammatory effects of an experimental A. pleuropneumoniae infection in young pigs.

Arcanobacterium phocae infection in mink (Neovison vison), seals (Phoca vitulina, Halichoerus grypus) and otters (Lutra lutra).

BACKGROUND: Infectious skin disorders are not uncommon in mink. Such disorders are important as they have a negative impact on animal health and welfare as well as on the quality and value of the fur. This study presents the isolation of Arcanobacterium phocae from mink with severe skin lesions and other pathological conditions, and from wild seals and otters. RESULTS: In 2015, A. phocae was isolated for the first time in Denmark from outbreaks of dermatitis in mink farms. The outbreaks affected at least 12 farms. Originating from these 12 farms, 23 animals cultured positive for A. phocae. The main clinical findings were necrotizing pododermatitis or dermatitis located to other body sites, such as the lumbar and cervical regions. A. phocae could be isolated from skin lesions and in nine animals also from liver, spleen and lung, indicating a systemic spread. The bacterium was also, for the first time in Denmark, detected in dead seals (n = 9) (lungs, throat or wounds) and otters (n = 2) (throat and foot). CONCLUSIONS: An infectious skin disorder in mink associated with A. phocae has started to occur in Danish farmed mink. The origin of the infection has not been identified and it is still not clear what the pathogenesis or the port of entry for A. phocae infections are.

Progression of experimental chronic Aleutian mink disease virus infection.

BACKGROUND: Aleutian mink disease virus (AMDV) is found world-wide and has a major impact on mink health and welfare by decreasing reproduction and fur quality. In the majority of mink, the infection is subclinical and the diagnosis must be confirmed by serology or polymerase chain reaction (PCR). Increased knowledge based on a systematically description of clinical signs, pathology and histopathology might be a tool to reduce the risk of infection from subclinically infected mink to AMDV free herds. The aim of this study was to give a histopathological description of the progression of a chronic experimental infection with a currently circulating Danish strain of AMDV, Saeby/DEN/799.1/05. These results were compared with the pathogenesis of previously published AMDV stains. RESULTS: This experimental AMDV infection resulted in only decreased appetite and soft or discolored feces, primarily within the first 8 weeks after AMDV inoculation. Gross pathology revealed few and inconsistent findings mainly associated with the liver, spleen and kidneys. The majority of the AMDV inoculated wild type mink (n = 41) developed various histopathological changes consistent with AMDV infection in one or more organs: infiltrations of mononuclear cells in liver, kidney and brain, reduced density of lymphocytes and increased numbers of plasma cells in lymph nodes and spleen. Natural infection, as occurred in the sentinel sapphire mink (four of six mink), progressed similar to the experimentally inoculated mink. CONCLUSIONS: Experimental AMDV inoculation mainly resulted in subclinical infection with unspecific clinical signs and gross pathology, and more consistent histopathology appearing at any time after AMDV inoculation during the 24 weeks of observation. Thus, the observed histopathology substantiates AMDV infection and no correlation to time of inoculation was found. This confirms that diagnosing AMDV infection requires serology and/or PCR and the Saeby/DEN/799.1/05 AMDV strain results in histopathology consistent with other AMDV strains.

An outbreak of bovine meningoencephalomyelitis with identification of Halicephalobus gingivalis.

Halicephalobus gingivalis is an opportunistic parasite which is known to cause fatal meningoencephalomyelitis primarily in equines but sporadically also in humans. In April 2014, laboratory examination of the head of a young dairy calf, euthanized due to severe central nervous system symptoms, revealed the presence of granulomatous to necrotizing encephalitis and myriads of nematodes in the brain lesion. Morphologically the parasites were identified as H. gingivalis. The diagnosis was confirmed by molecular analysis of the large subunit (LSU) rRNA and the small subunit (SSU) rRNA genes, revealing genetic variations of 0.5-4.4% and 0.7-8.6%, respectively, between the H. gingivalis isolated from the Danish calf and published isolates, collected worldwide from free-living and parasitic stages of the nematode. Clinical symptoms and histological changes indicated infection with H. gingivalis from another three calves in the herd. This is the first scientific publication of H. gingivalis induced meningoencephalomyelitis in ruminants. As ante mortem diagnosis is a major challenge, the infection may easily remain undiagnosed in cattle.

Associations between biosecurity and outbreaks of canine distemper on Danish mink farms in 2012-2013.

BACKGROUND: During 8 months from July 2012 to February 2013, a major outbreak of canine distemper involving 64 mink farms occurred on the Danish peninsula of Jutland. The canine distemper outbreak was associated with exposure of farmed mink to infected wild carnivores and could represent a deficit in biosecurity on the mink farms. The aim of this study was to investigate the extent and association of specific biosecurity measures with the outbreak. The study was carried out in an epidemiological case-control design. The case group consisted of the 61 farms, which had a confirmed outbreak of canine distemper from July 2012 to February 2013. The control group included 54 farms without an outbreak of canine distemper in 2012 or 2013, selected as the closest geographical neighbour to a case farm. RESULTS: The results showed that significantly more control than case farms had vaccinated their mink against canine distemper virus. Mortality was only assessed on the case farms, and there was a non-significantly lower mortality on vaccinated farms than on the non-vaccinated farms. Furthermore, the proportion of farms with observations of wild red foxes (Vulpes vulpes) inside the farm enclosures were larger for case farms, indicating that the control farms had a better biosecurity or were not equally exposed to canine distemper virus. Generally, all farms had very few specific precautions at the gate entrance in respect to human visitors as well as animals. The use of biosecurity measures was very variable in both case and control farms. Not using plastic boot covers, presence of dogs and cats, presence of demarcated area for changing clothes when entering and leaving the farm area and presence of hand washing facilities significantly lowered the odds of the farm having a canine distemper virus outbreak. CONCLUSIONS: The results of the study indicate that consistent use of correct vaccination strategies, implementation of biosecurity measures and limiting human and animal access to the mink farm can be important factors in reducing the risk for canine distemper outbreaks.

Repeated examination of natural sapovirus infections in pig litters raised under experimental conditions.

BACKGROUND: Porcine sapovirus, belonging to the family Caliciviridae, is an enteric virus that is widespread in the swine industry worldwide. A total of 14 sapovirus genogroups have been suggested and the most commonly found genogroup in swine is genogroup III (GIII). The goal of the present experiment was to examine the presence of sapovirus in 51 naturally infected pigs at two different time points. The pigs were kept under experimental conditions after weaning. Previous studies on sapovirus have primarily been of a cross sectional nature, typically prevalence studies performed on farms and abattoirs. In the present study, faecal samples, collected from each pig at 5½ weeks and 15-18 weeks of age, were analysed for sapovirus by reverse transciptase polymerase chain reaction and positive findings were genotyped by sequencing. RESULTS: At 5½ weeks of age, sapovirus was detected in the majority of the pigs. Sequencing revealed four different strains in the 5½ week olds-belonging to genogroups GIII and GVII. Ten to 13 weeks later, the virus was no longer detectable from stools of infected pigs. However, at this time point 13 pigs were infected with another GIII sapovirus strain not previously detected in the pigs studied. This GIII strain was only found in pigs that, in the initial samples, were virus-negative or positive for GVII. CONCLUSIONS: At 5 weeks of age 74 % of the pigs were infected with sapovirus. At 15-18 weeks of age all pigs had cleared their initial infection, but a new sapovirus GIII strain was detected in 25 % of the pigs. None of the pigs initially infected with the first GIII strain were reinfected with this new GIII strain, which may indicate the presence of a genogroup-specific immunity.

Concurrent host-pathogen gene expression in the lungs of pigs challenged with Actinobacillus pleuropneumoniae.

BACKGROUND: Actinobacillus pleuropneumoniae causes pleuropneumonia in pigs, a disease which is associated with high morbidity and mortality, as well as impaired animal welfare. To obtain in-depth understanding of this infection, the interplay between virulence factors of the pathogen and defense mechanisms of the porcine host needs to be elucidated. However, research has traditionally focused on either bacteriology or immunology; an unbiased picture of the transcriptional responses can be obtained by investigating both organisms in the same biological sample. RESULTS: Host and pathogen responses in pigs experimentally infected with A. pleuropneumoniae were analyzed by high-throughput RT-qPCR. This approach allowed concurrent analysis of selected genes encoding proteins known or hypothesized to be important in the acute phase of this infection. The expression of 17 bacterial and 31 porcine genes was quantified in lung samples obtained within the first 48 hours of infection. This provided novel insight into the early time course of bacterial genes involved in synthesis of pathogen-associated molecular patterns (lipopolysaccharide, peptidoglycan, lipoprotein) and genes involved in pattern recognition (TLR4, CD14, MD2, LBP, MYD88) in response to A. pleuropneumoniae. Significant up-regulation of proinflammatory cytokines such as IL1B, IL6, and IL8 was observed, correlating with protein levels, infection status and histopathological findings. Host genes encoding proteins involved in iron metabolism, as well as bacterial genes encoding exotoxins, proteins involved in adhesion, and iron acquisition were found to be differentially expressed according to disease progression. By applying laser capture microdissection, porcine expression of selected genes could be confirmed in the immediate surroundings of the invading pathogen. CONCLUSIONS: Microbial pathogenesis is the product of interactions between host and pathogen. Our results demonstrate the applicability of high-throughput RT-qPCR for the elucidation of dual-organism gene expression analysis during infection. We showed differential expression of 12 bacterial and 24 porcine genes during infection and significant correlation of porcine and bacterial gene expression. This is the first study investigating the concurrent transcriptional response of both bacteria and host at the site of infection during porcine respiratory infection.

New reassortant and enzootic European swine influenza viruses transmit efficiently through direct contact in the ferret model.

The reverse zoonotic events that introduced the 2009 pandemic influenza virus into pigs have drastically increased the diversity of swine influenza viruses in Europe. The pandemic potential of these novel reassortments is still unclear, necessitating enhanced surveillance of European pigs with additional focus on risk assessment of these new viruses. In this study, four European swine influenza viruses were assessed for their zoonotic potential. Two of the four viruses were enzootic viruses of subtype H1N2 (with avian-like H1) and H3N2, and two were new reassortants, one with avian-like H1 and human-like N2 and one with 2009 pandemic H1 and swine-like N2. All viruses replicated to high titres in nasal wash and nasal turbinate samples from inoculated ferrets and transmitted efficiently by direct contact. Only the H3N2 virus transmitted to naïve ferrets via the airborne route. Growth kinetics using a differentiated human bronchial epithelial cell line showed that all four viruses were able to replicate to high titres. Further, the viruses revealed preferential binding to the 2,6-α-silalylated glycans and investigation of the antiviral susceptibility of the viruses revealed that all were sensitive to neuraminidase inhibitors. These findings suggested that these viruses have the potential to infect humans and further underline the need for continued surveillance as well as biological characterization of new influenza A viruses.

Baylisascaris procyonis in wild raccoons (Procyon lotor) in Denmark.

The nematode Baylisascaris procyonis, which may cause severe clinical disease in humans and animals, is emerging in Europe after its introduction with raccoons (Procyon lotor) from North America. B. procyonis has a broad spectrum of paratenic hosts, including rodents, birds, wild carnivores and primates, which are severely affected by the migrating larval stages of the parasite. We report here the recovery of B. procyonis from two out of 18 examined wild raccoons in Denmark. The parasites were identified based on morphology and their identity was confirmed by partial sequencing of the 18S rRNA gene. Follow-up telephone interviews of staffs in nine zoos housing captive raccoons and veterinarians supervising these zoos showed that knowledge of B. procyonis and its zoonotic potential were sparse. Eggs of B. procyonis were detected in two raccoons kept in one of three zoos that submitted fecal samples following the telephone interviews. Continuous monitoring and increased awareness are needed to reduce further spread of the parasite and to limit the public health risks associated with baylisascariasis.

Genetically distinct isolates of Spirocerca sp. from a naturally infected red fox (Vulpes vulpes) from Denmark.

Spirocerca lupi causes formation of nodules that may transform into sarcoma in the walls of aorta, esophagus and stomach of infected canids. In February 2013, post mortem examination of a red fox (Vulpes vulpes) hunted in Denmark revealed the presence of several nodules containing adult worms of Spirocerca sp. in the stomach and the omentum. The nodules largely consisted of fibrous tissue with infiltration of mononuclear cells, neutrophilic granulocytes and macrophages with hemosiderin deposition. Parasitological examination by three copromicroscopic methods, sedimentation, flotation with saturated sugar-salt solution, and sieving failed to detect eggs of Spirocerca sp. in feces collected from the colon. This is the first report of spirocercosis in Denmark, and may have been caused by a recent introduction by migrating paratenic or definitive host. Analysis of two overlapping partial sequences of the cox1 gene, from individual worms, revealed distinct genetic variation (7-9%) between the Danish worms and isolates of S. lupi from Europe, Asia and Africa. This was confirmed by phylogenetic analysis that clearly separated the Danish worms from other isolates of S. lupi. The distinct genetic differences of the current worms compared to other isolates of S. lupi may suggest the presence of a cryptic species within Spirocerca.

A polyvalent influenza A DNA vaccine induces heterologous immunity and protects pigs against pandemic A(H1N1)pdm09 virus infection.

The composition of current influenza protein vaccines has to be reconsidered every season to match the circulating influenza viruses, continuously changing antigenicity. Thus, influenza vaccines inducing a broad cross-reactive immune response would be a great advantage for protection against both seasonal and emerging influenza viruses. We have developed an alternative influenza vaccine based on DNA expressing selected influenza proteins of pandemic and seasonal origin. In the current study, we investigated the protection of a polyvalent influenza DNA vaccine approach in pigs. We immunised pigs intradermally with a combination of influenza DNA vaccine components based on the pandemic 1918 H1N1 (M and NP genes), pandemic 2009 H1N1pdm09 (HA and NA genes) and seasonal 2005 H3N2 genes (HA and NA genes) and investigated the protection against infection with virus both homologous and heterologous to the DNA vaccine components. We found that pigs challenged with a virus homologous to the HA and NA DNA vaccine components were well protected from infection. In addition, heterologous challenge virus was cleared rapidly compared to the unvaccinated control pigs. Immunisation by electroporation induced HI antibodies >40 HAU/ml seven days after second vaccination. Heterologous virus challenge as long as ten weeks after last immunisation was able to trigger a vaccine antibody HI response 26 times higher than in the control pigs. The H3N2 DNA vaccine HA and NA genes also triggered an effective vaccine response with protective antibody titres towards heterologous H3N2 virus. The described influenza DNA vaccine is able to induce broadly protective immune responses even in a larger animal, like the pig, against both heterologous and homologous virus challenges despite relatively low HI titres after vaccination. The ability of this DNA vaccine to limit virus shedding may have an impact on virus spread among pigs which could possibly extend to humans as well, thereby diminishing the risk for epidemics and pandemics to evolve.