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Streptococcus suis is a zoonotic pathogen of swine involved in arthritis, polyserositis, and meningitis. Colonization of piglets by S. suis is very common and occurs early in life. The clinical outcome of infection is influenced by the virulence of the S. suis strains and the immunity of the animals. Here, the role of innate immunity was studied in cesarean-derived colostrum-deprived piglets inoculated intranasally with either virulent S. suis strain 10 (S10) or non-virulent S. suis strain T15. Colonization of the inoculated piglets was confirmed at the end of the study by PCR and immunohistochemistry. Fever (≥40.5 °C) was more prevalent in piglets inoculated with S10 compared to T15 at 4 h after inoculation. During the 3 days of monitoring, no other major clinical signs were detected. Accordingly, only small changes in transcription of genes associated with the antibacterial innate immune response were observed at systemic sites, with S10 inducing an earlier response than T15 in blood. Local inflammatory response to the inoculation, evaluated by transcriptional analysis of selected genes in nasal swabs, was more sustained in piglets inoculated with the virulent S10, as demonstrated by transcription of inflammation-related genes, such as IL1B, IL1A, and IRF7. In contrast, most of the gene expression changes in trachea, lungs, and associated lymph nodes were observed in response to the non-virulent T15 strain. Thus, S. suis colonization in the absence of systemic infection induces an innate immune response in piglets that appears to be related to the virulence potential of the colonizing strain.

Influenza A virus infections have great impact on human health and welfare and significant resources are linked to influenza epidemics due to excess hospitalizations and lost productivity. Up to 15% of the human population is affected when Influenza spreads around the world in seasonal epidemics (WHO). Animal models are essential in understanding the mechanisms involved in human infectious disease and for the development of effective prevention and treatment strategies. It is increasingly realized that large animal models like the pig are exceptionally human like and serve as an excellent model for disease and inflammation. Pigs are fully susceptible to human influenza, and have been demonstrated to be involved in influenza evolution and ecology. Pigs share many similarities with humans regarding lung physiology and innate immune cell infiltration of the respiratory system and thus seem to be an obvious large animal model for respiratory infections. This study aimed at providing a better understanding of the involvement of circulating non-coding RNA and innate immune factors in porcine blood leukocytes during influenza virus infection. By employing the pig as a model we were able to perform highly controlled experimental infections and to study changes of symptoms, viral titer, and expression of microRNAs/mRNAs as the influenza infection progresses in time, generating information that would be difficult to obtain from human patients. The Gram-negative bacterium Actinobacillus pleuropneumoniae causes pneumonia in pigs, a disease which is associated with high morbidity and mortality, as well as impaired animal welfare. The rapidly evolving pneumonia is characterized by large areas of lung necrosis resulting from the combined effect of tissue damage caused by the bacteria, and a strong proinflammatory immune response. To obtain in-depth understanding of this infection, concurrent gene expression of host and pathogen in lung samples collected from pigs experimentally infected with A. pleuropneumoniae was studied. We applied high-throughput RT-qPCR for the simultaneous analysis of host and pathogen gene expression. This parallel analysis was done in lung tissue samples as well as in the immediate surroundings of infection loci after laser capture microdissection. Regulation of gene expression of several immune factors was observed in agreement with protein levels of these factors in lung tissue, infection status and histopathological findings.

Brief description of research area: A common problem in swine production is diarrhoea in newly weaned piglets, and huge quantities of antibiotics go to treat post-weaning diarrhoeas in piglets. The use of antibiotics can lead to the development of multi- and fully resistant bacteria, which consequently pose a great threat to human health. Therefore, sustainable alternatives for treating post-weaning diarrhoea without using antibiotics are in demand. Swine that are old (and big) enough for slaughter have during their upbringing been challenges by many different pathogens and thus have developed immunity towards these pathogens, which include pathogen-specific immunoglobulins (antibodies). We hypothesis that by harvesting natural immunoglobulins from porcine blood plasma, a waste product from swine slaughter, and feeding these immunoglobulins to the piglets this can subsequently (by passive immunisation) prevent and treat post-weaning diarrhoea.Our challenge is to find a suitable method for stabilising the immunoglobulins for oral provision in order for the immunoglobulins to pass as unharmed as possible through the digestive system and still retaining their anti-pathogenic properties.What we know: It is possible to multimerise immunoglobulins, which results in an advantage when binding to their respective antigens in comparison to the non-multimerised immunoglobulins, but too high degree of multimerisation abates immunoglobulin reactivity. Unfortunately, a preliminary study showed that multimerisation destabilises the immunglobulins. On the other hand, proteolytical resistance correlates with increased immunoglobulin concentration.What we need: To investigate the effect of increasing the concentration of multimerised immunoglobulins on proteolytical resistance.To investigate multimerised immunoglobulins’ ability in inhibiting microbial (E. coli) adhesion on relevant matrices, such intestinal villi and/or intestinal cell lines.A toxicological study on (if any) adverse side effects occurs when enteral providing immunoglobulins to piglets.

Swine influenza is a highly infectious respiratory disease in pigs caused by influenza A virus. Activation of a frontline of pattern-recognition receptors (PRRs) expressed by epithelial cells as well as immune cells of the upper respiratory tract, leads to a potent type 1 interferon (IFN) release and simultaneous proinflammatory cytokine expression. A transient induction of cytokines is required for an efficient antiviral defence; however, an over-reactive and prolonged inflammatory response may lead to excessive infiltration of immune cells, contributing to immunopathology of the infected lung. Thus, this response must be tightly regulated. Recently, microRNA (miRNA) has been proposed to play an important role in modulating and fine tuning the innate immune response in order to avoid such harmful overreactions. Little is known about the significance of miRNA regulation in the lung during acute influenza A infection. The present work aimed of providing a better understanding of the involvement of innate immune factors including miRNA in the host response to establishment and progression of influenza virus infection. Twenty pigs were challenged by aerosol containing H1N2 (A/swine/Denmark/12687/03) influenza virus. Expression of mRNA coding for cytokines, chemokines, pattern recognition receptors and other antiviral effector molecules were quantified in lung tissue at different time points after challenge (24h PI, 72h PI, and 14days PI). Likewise, microRNA in the lung tissue was quantified at the same time points. Our results demonstrate a significant regulation of several microRNAs and their targeted mRNA in the lungs of pigs during acute influenza.

Enteric infections cause major problems in most intensive animal production sectors, including poultry, pigs and cattle, leading to disease, reduced production and compromised welfare. In addition some of these infections are zoonotic, and they are to a large extent responsible for the continued massive use of antibiotics in food animals. Thus there is a pressing need for economically feasible, efficient, non-antibiotics based means for controlling the problem. Passive immunization has been known for decades as an efficient way of endowing humans or animals with short-term (weeks) immunity. To control enteric infections by passive immunization a bolus of immunoglobulin may simply be administered orally. For this to work, large amounts of active immunoglobulins are needed. To be a real alternative to antibiotics the price of the immunoglobulin product needs to be low. We combined an efficient and mild high-capacity method for extracting immunoglobulins directly from raw materials like milk, whey and blood plasma with a novel method for stabilizing activity. In a first experiment a total of 15 kg unstabilized bovine immunoglobulin was purified from whey (35.000 liters) and administered to colostrum-deprived calves (225-300 g pr calf during the first 24 hours after birth). No difference in resulting immunoglobulin serum concentration, weight gain or disease frequency were seen in this group of calves compared to a control group given full access to high-quality colostrum. The effect of orally administered bovine immunoglobulin is currently being tested in a calf herd with persistent diarrhea problems. Furthermore, it was shown in a Campylobacter challenge model in chickens that caecal and faecal counts of Campylobacter were between 0.5 and 1.0 logs lower in birds when given 200 mg avian immunoglobulins orally together with the challenge (at day 21 of age) compared to a placebo group receiving immunoglobulin with no reactivity against Campylobacter. While clearly preliminary, these results show that immunoglobulin can be produced from renewable sources at a price enabling passive immunization as a viable strategy for control of infectious diseases in the intensive animal production, with the potential to significantly reduce antibiotics consumption.

Actinobacillus pleuropneumoniae (Ap) is a gram-negative bacterium that causes porcine pleuropneumonia, which is a widespread, highly contagious and often fatal respiratory disease in swine. A total of 44 pigs were experimentally inoculated with Ap serotype 2 or 6 and samples of liver and tracheobronchial lung lymph nodes were collected 6, 12, 24 and 48 hours after experimental inoculation, as well as from six non-inoculated control pigs. Transcriptional profiles of the liver samples have been generated by preparation of 12-plexed mRNA-Seq libraries followed by sequencing on an Illumina GAIIx (51+7 cycles) obtaining more than 200 million tag sequences. The 12-plexed mRNA-Seq libraries of the lung lymph node samples have presently (April 2010) been prepared and are to be sequenced. The PCR amplicons of the liver libraries were quantified using both a fluorometer and a qPCR assay, including the use of a sequence-titrated, in-house control library. The libraries were diluted to 6 pM based on the qPCR assay, except for a single library set which was duplicated and diluted based on the fluorometer measurements as well. Analysis of the obtained sequences revealed that the qPCR based quantifications reduced the cluster density variability as compared to fluorometer based quantifications. Furthermore, it was found that the fluorometer based measurements tended to deviate for dilute as well as for more concentrated libraries. Following the sequencing of the lung lymph node samples analyses are to be conducted to study the time and serotype dependent transcriptional response to Ap infection.

Lawsonia intracellularis is the cause of proliferative enteropathy, an economically important enteric disease in pigs that causes weight loss and failure to thrive. The disease is controllable with antibiotics and an attenuated live oral vaccine (Enterisol®) is used for prophylaxis. Still these interventions have not resulted in eradication of the bacteria, which is abundantly present in most pig herds in many countries, including Denmark. In the experimental study we present here, weaned pigs received the oral L. intracellularis vaccine or a virulent field strain (Re-I pigs). The latter resulting in subclinical disease. Both groups were treated with antibiotics from day 21 to 26 and challenged at day 49 with the virulent strain. A control group (CC) only received challenge. We here report on clinical outcome, L. intracellularis infection of the intestines and immunological responses. While Re-I pigs had a serological response to L. intracellularis from day 18, no specific IgG or IgA was measurable in serum from vaccinated animals until around day 55, i.e. after challenge. Specific secretory IgA measured in faeces was at a low level and short-lasting (day 67-70) in both groups. Excretion of bacteria was measured in faeces by qPCR after primary inoculation (Re-I). After challenge Re-I pigs did not excrete bacteria, but vaccinated animals had a high excretion at a level comparable to the CC group. Acute phase response measured in serum as an indication of the general disease status of the animals was high in both Vac and Re-I groups after primary inoculation/vaccination compared to the CC group. After challenge, however Vac and CC levels were high and Re-I levels low, indicating that Re-I pigs were protected from disease whereas vaccinated pigs were not. These results show that the vaccination does not confer protective immunity comparable to that of a virulent strain.

The acute phase response is playing an important role, aiming to restore the healthy state after tissue injury, inflammation and infection. The biological function of this response and its interplay with other parts of innate defense reactions remain somewhat elusive. Expression of acute phase proteins (APP) outside the liver is increasingly recognized, still little is known of extra-hepatic production of APP in pigs. 14-18 h after experimental infection with Actinobacillus pleuropneumoniae, causing acute pleuropneumonia in pigs, we studied local APP gene expression changes in different locations of the infected lung (necrotic areas, areas bordering on necrotic areas, and from visually unaffected areas). Expression differences was also studied in the liver and in peripheral lymphoid tissue (tracheobronchial lymph nodes, spleen, tonsils) of infected (n=10) and non-infected (n=5) pigs using reverse transcription quantitative real-time PCR (RT-qPCR). SAA, CRP and IL-6 were further measured in serum samples before and after the infection using ELISA. Expression of ALB, A1AG, APOA1, CRP, Hp, PigMAP, SAA, TF, and TTR were found in all tissues investigated. SAA, Hp, LTF, LBP, TRF, SpA, SpD, IL-1α and IL-6 were found to be differentially expressed in all three areas of the infected lung compared to lung tissue from control pigs. Expression differences were general highest in necrotic areas. We found good correlation between ELISA and RT-qPCR data the in serum and whole blood respectively. mRNA coding for CRP, Hp, IL-6, and SAA were found to be significantly up regulated in infected pigs in whole blood and tracheobronchial lymph nodes. In the spleen we found mRNA coding for APOA1, FIB, Hp, PigMAP, SAA, TF, and IL-6 to be differentially expressed. In the tonsils IL-6 and SAA were also found differentially expressed between infected and control animals. We demonstrated that acute pleuropneumonia caused by A. pleuropneumoniae leads to a rapid disseminated local intra-lung APP response, also in apparently unaffected areas of the infected lung. Further extrahepatic expression of several acute-phase proteins was found 14-18h after experimental infection with A. pleuropneumoniae. This firmly establishes that expression of APPs is widely disseminated, involving changes in the expression of APPs at a dynamic scale comparable to the hepatic response. These results suggest that many different cell-types in the organism are involved in production of APP and further supports that extrahepatic APP might be important players of the innate defence system.

MicroRNAs (miRNAs) are a highly evolutionarily conserved group of small non-coding RNA molecules, which regulate the activity of other genes at the post-transcriptional level. Recently it has become evident that miRNA plays an important role in modulating and fine tuning of the innate and adaptive immune responses. Still, little is known about the impact of miRNAs in the development and pathogenesis of lung infections. Expression of miRNA, known to be induced by bacterial (i.e., LPS) ligands and thus supposed to play a role in the regulation of antimicrobial defence, were studied in lung tissue from pigs experimentally infected with Actinobacillus pleuropneumoniae serotype 2 and 6. Circulating miRNAs were studied in blood from pigs infected with A. pleuropneumoniae serotype 2 using real time-qPCR (RT-qPCR). Expression profiles of miRNA in blood of seven animals before and after infection, where also studied using miRCURY™ LNA arrays (Exiqon, Denmark). Piglets were inoculated by dripping 1ml bacterial suspension, into each nostril during inhalation. Each time group is a different set of 4-6 pigs. Most of the inoculated pigs revealed characteristic, well demarcated, lung lesions. No pathological changes were seen in lungs from control animals. All AP infected animals had a significantly higher level of mRNA coding for the acute-phase protein SAA-2 in the liver compared to the control group. Whole Blood samples were collected in PAXgene Blood RNA Tubes (PrenalytiX) before (control) and after infection of piglets (6 h., 12 h., 24 h. and 48 h.). Total RNA was extracted from blood samples using PAXgene™ Blood RNA kit (Qiagen/ PrenalytiX). The quantity of extracted total RNA was determined using a Nanodrop ND-1000 and the quality of extracted RNA was estimated by on-chip electrophoresis (Nanochip 6000) on an Agilent 2100 Bioanalyzer, a RNA integrity number (RIN) was assigned to each sample. Expression levels of selected miRNA were further studied in lung tissue collected at two time points (6 h. and 24 h.) after A. pleuropneumoniae serotype 2 and 6 infection. 600 ng total RNA from blood samples before and after infection were labelled with Hy5™ and Hy3™ fluorescent label, respectively, using the miRCURY™ LNA Array power labelling kit (Exiqon, Denmark). Sample were mixed pair-wise and hybridized to miRCURY™ LNA array version 11.0 (Exiqon, Denmark), which contains capture probes targeting all miRNAs for human, mouse and rat. The miRCURY™ LNA array microarray slides were scanned, and image analysis was carried out using the ImaGene 8.0 software (BioDiscovery, Inc., USA). A two-tailed T-test calculated between infected and control identified 10 of 1263 miRNA to be differentially expressed (p-values lower than 0.05). MicroRNA expression in lung tissue over time in response to the two different serotypes were very similar. miR-223 was found to be highly up regulated, followed by miR-146a and to a lesser degree miR-21 in lung tissue of the AP serotype 2 infected animals. MiR-233 was also found to be up regulated in blood based on both microarray and RT-qPCR, mir-233 is a negative regulator of neutrophil proliferation and activation and might act to limit the potentially harmful consequences of the accumulation of infiltrating neutrophils in AP infected lungs. More data of microRNA expression in blood of pigs infected with A. pleuropneumonia serotype 2 will be presented.

Göttingen minipigs are a useful model for diseases having an inflammatory component, and the associated use of acute-phase proteins (APP) as biomarkers of inflammation warrants establishment of their reference ranges. The objective of this study was to establish reference values for selected APP in Göttingen minipigs and to investigate the effects of age, sex, and various stimuli on these ranges. Serum concentrations of C-reactive protein (CRP), serum amyloid A (SAA), haptoglobin, pig major acute-phase protein (PMAP), albumin, and porcine α-1 acid glycoprotein (PAGP) were evaluated in 4 age groups (6, 16, 24 and 40-48 wk) of male and female Göttingen minipigs. In addition, minipigs were tested under 2 housing conditions, after acute LPS challenge, and after diet-induced obesity with and without mild diabetes. Changing the pigs to a new environment induced significant increases in CRP, PMAP, haptoglobin and PAGP and a decrease in albumin. An acute LPS stimulus increased CRP, PMAP, haptoglobin, and SAA; PAGP was unchanged and albumin decreased. Obese pigs with and without diabetes showed increases in CRP and PAGP, albumin decreased, and haptoglobin and SAA were unchanged. PMAP was increased only in obese pigs without diabetes. In conclusion, reference values for CRP, PMAP, haptoglobin, SAA, PAGP and albumin were established for male and female Göttingen minipigs of different ages. These APP were influenced by age and sex, underlining the importance of considering these factors when designing and interpreting studies including aspects of inflammation. In addition, an APP response was verified after both acute and chronic stimuli.

A major concern amongst the Danish mink farmers is the incidence of the syndrome pre-weaning diarrhea. The syndrome causes major management issues and decreases the welfare of the mink and increases mortality in the pre-weaning period. The etiology of the syndrome is considered multifactorial as a specific cause is not fully established or understood. Adding to an increased risk of developing pre-weaning diarrhea is the fact that the mink kits are born with very low levels of circulating immunoglobulins. Rapid achievement of high levels of immunoglobulins in the bloodstream is essential for the kits early immunity and thus their resistance against pathogenic agents found in the environment. This study describes a sandwich ELISA for quantification of the concentration of total immunoglobulin G in mink blood. The ELISA was validated with serum samples from females (n=8) and their kits (litters of 4-12). Preliminary results show that the IgG concentration among kits from the same litter was similar, while litter to litter variation was high.

Vaccination against the intracellular porcine enteric pathogen Lawsonia intracellularis remains a challenge as the commercially available vaccine does not provide full protection. In an experimental challenge study, the Enterisol® Ileitis attenuated live vaccine against Lawsonia intracellularis did not induce measurable primary humoral or cell-mediated immune responses, nor was it able to reduce faecal shedding of bacteria from eight vaccinated pigs compared to seven age-matched naïve challenge-control pigs. Vaccinated pigs did, however, respond to vaccination with an acute phase protein response in serum comparable to that of ten primary infected (and later re-inoculated) pigs, and post mortem immunohistochemical examination of tissue samples revealed less L. intracellularis in vaccinated pigs compared to challenge-control pigs.In more detailed studies of the immune response after challenge, the vaccinated pigs did not show any immediate evidence of primed (faster or stronger) IgG or CMI response compared to naïve pigs. L. intracellularis-specific CMI responses 18-33 dpi were further characterized by flow cytometry for intracellular IFN-γ and cell proliferation (CFSE). Phenotypes of IFN-γ producing cells in the vaccinated pigs showed profiles primarily of CD8+(CD4neg) and CD4+CD8+ double positive lymphocytes. Similar profiles of IFN-γ producing cells were found in re-inoculated immune pigs, which experienced a boost in CMI responses. Cellular proliferation was identified in nearly all vaccinated pigs with mainly CD4+(CD8neg) and CD4+CD8+ double positive cells, whereas the immune re-inoculated pigs also included response in CD8high(CD4neg) cells. These different profiles of responsive cellular phenotypes may influence the observed differences in protection between vaccinated and re-inoculated pigs.

Large animal models are essential in understanding the mechanisms involved in human infectious disease. To study the expression of host and bacterial genes involved in defense and survival mechanisms, we analyzed lung tissue from pigs experimentally infected with the Gram-negative bacterium A. pleuropneumoniae. All steps including RNA extraction and high-throughput real-time qPCR were carried out simultaneously for the two organisms. By applying this dual-organism approach, we obtained unique insights into the host-pathogen interaction at the site of infection. Differential expression of host genes involved in innate immune responses towards Gram-negative infections, including pattern recognition receptors and cytokines concurrent with expression of bacterial genes involved in lipopolysaccharide biosynthesis and adhesion was demonstrated. We also studied the gene expression in blood leukocytes after experimental H1N2 virus infection of pigs, and found the regulation of several swine encoded miRNAs and cytokines to mimic key findings from influenza studies in human patients. By employing the pig as a model we were able to perform highly controlled experimental infections and to study changes of symptoms, viral titer, and expression of microRNAs/mRNAs as the influenza infection progressed in time, generating information that would be difficult to obtain from human patients.

MicroRNAs are small non-coding RNA molecules (18-23 nt), that regulate the activity of other genes at the post-transcriptional level. Recently it has become evident that microRNA plays an important role in modulating and fine tuning innate and adaptive immune responses. Still, little is known about the impact of microRNAs in the development and pathogenesis of lung infections. Expression of microRNA known to be induced by bacterial (i.e., LPS) ligands and thus supposed to play a role in the regulation of antimicrobial defence, were studied in lung tissue and in blood from pigs experimentally infected with Actinobacillus pleuropneumoniae (AP). Expression differences of mRNA and microRNA were quantified at different time points (6h, 12h, 24h, 48h PI) using reverse transcription quantitative real-time PCR (Rotor-Gene and Fluidigm). Expression profiles of miRNA in blood of seven animals were further studied using miRCURY™ LNA arrays (Exiqon, Denmark). All AP infected animals had significantly higher levels of mRNA coding for inflammatory mediators as IL-6 and IL-8 as well as the acute phase protein SAA, in the lung compared to the control group. MiR-223 was found to be highly up regulated, followed by miR-146a and to a lesser degree miR- 155 in lung tissue of the AP infected animals. MiR-233 was also found to be up regulated in blood based on both microarray and real-time PCR. Mir-233 has been found to be a negative regulator of neutrophil proliferation and activation, and might act to limit the potentially harmful consequences of the accumulation of infiltrating neutrophils in AP infected lungs.

Post-weaning diarrhoea (PWD) is a common condition in intensive swine production, resulting in reduced welfare of weaners and economic losses for the farmer as a result of illness, death, and treatment costs. It is also one of the main causes of antibiotics- and zinc use in the pig production industry. We aim at developing a sustainable product for protection against PWD based on natural antibodies (immunoglobulins) derived directly from inexpensive raw materials. The availability of such an inexpensive and highly active immunogloulin product would allow swine producers to reduce expenses and minimize the on antibiotics and zinc usage. Swine immunoglobulins were isolated directly from slaughterhouse swine plasma-waste by expanded bed chromatography. It was shown that the isolated Immunoglobulin fraction bound enterotoxigenic Escherichia coli (ETEC) and Salmonella ssp. and inhibited their adhesion to porcine epithelial cells in vitro. As the immunoglobulin fraction is intended for oral use as a feed supplement, we also tested the safety of feeding 4 grams of natural immunoglobulins to 4-5 week old weaner piglets for 14 days and observed no adverse effects. In an experimental model of E. coli F4+ induced PWD, we observed that piglets given IgG as a feed supplement cleared the E coli infection significantly faster than control weaner piglets not receiving an immunoglobulin feed supplement. Furthermore, deep sequencing of the ileal microbiota showed a significantly lowered colonization of the family Enterobactericea in immunoglobulin fed piglets as compared to the control group. Thus pig slaughterhouse plasma is indicated as a potential source resource of antibodies for the control of PWD.

This study aimed at providing a better understanding of the involvement of innate immune factors including microRNA (miRNA) in the local and systemic host response to influenza virus infection. Twenty pigs were challenged by influenza A virus subtype H1N2. Expression of miRNA, mRNA and proteins were quantified at different time points after challenge (24h, 72h, and 14days post infection (pi)). Gene expression was quantified using 48.48 Dynamic Arrays (Fluidigm Corporation, CA, USA) combining 48 samples with 48 primer sets for 2304 individual and simultaneous qPCR reactions. Several groups of genes were significantly regulated according to time point and infection status: Pattern recognition receptors (TLR2, TLR3, TLR7, RIG1, MDA5), IFN and IFN induced genes (IFNB, IFNG, IRF7, STAT1, ISG15 and OASL), cytokines (IL1B, IL1RN, IL6, IL7, IL10, IL12A, TNF, CCL2, CCL3 and CXCL10), and several acute phase proteins. Likewise, the following miRNAs were differentially expressed in one or more time groups compared to the control pigs: miR-15a, miR-21, miR-146, miR-206, miR-223 and miR-451. At day one pi lung tissue protein levels of IL-6, IL-12 and IFN-α were significantly increased compared to the control group, and haptoglobin and C-reactive protein were at significantly increased at day three pi. MiRNA are small non coding RNA molecules, that regulate gene expression in a wide range of organisms. Cellular miRNAs might be involved in influenza infection, both by targeting immune related host transcripts but also by targeting viral gene products. Our results suggest that in addition to a wide range of immune factors, miRNAs are involved in fine tuning of an efficient innate immune response to influenza infection.

IntroductionSubstantial morbidity and mortality is caused by secondary bacterial infections occurring in individuals after influenza A virus (IAV) infection. Results from studies in mice suggest that this may in part be due to a lack of responsiveness to Toll-like receptor (TLR) ligands in the post-IAV infected individual. Using the pig as an animal model, we have identified microRNAs (miRNAs) that are differentially expressed in lung tissue two weeks after challenge compared to uninfected controls, i.e. well after the infection has cleared. The role for differential expression of miRNA at this late time point remains unclear. We therefore seek to examine the potential involvement of miRNAs in the translational regulation of TLRs and associated proteins, thus contributing to the lowered responsiveness to bacterial TLR ligands at this late time point, making the individual vulnerable to secondary infections.Methods and outcomePigs were experimentally challenged with a Danish reassortant IAV strain (A/sw/Denmark/12687/03(H1N2)). Lung tissue was harvested 14 days after challenge, as well as from uninfected control animals. Using RNAseq and high-throughput RT-qPCR, we quantified the expression of relevant miRNAs (e.g. miR-335 and miR-146a-5p) and mRNA levels of relevant miRNA targets. Transcriptional analysis at the site of infection reveals a set of miRNAs to be regulated one week after the pigs had cleared the IAV infection (i.e. two weeks after challenge). This set included miRNAs experimentally validated or in silico predicted to bind to and regulate transcripts of TLRs and relevant co-factors and transcription factors (online tools). The antiviral immune response elicited by IAV infection thus includes late miRNA regulation, which in turn may be at the expense of host responsiveness to bacterial TLR ligands.

It is increasingly realized that large animal models like the pig are exceptionally human like and serve as an excellent model for disease and inflammation. Pigs are fully susceptible to human influenza, share many similarities with humans regarding lung physiology and innate immune cell infiltration of the respiratory system.This study aimed at providing a better understanding of the involvement of innate immune factors and non-coding RNA in blood leukocytes during influenza A virus infection. By using the pig as a model we were able to perform highly controlled experimental infections and study early clinical signs of disease, viral titer, and transcriptional response of coding and non-coding RNA. This was completed during the first two weeks after experimental viral infection, generating information that would be difficult to obtain from human patients. Expression of a wide range of immune factors including several genes known to be centrally involved in the viral defence was quantified by high throughput qPCR (BioMark, Fluidigm). Likewise, miRNAs were quantified using the BioMark (Fluidigm) as well as by MiRCURY LNATM (Exiqon). During the first 24 hours of infection we found the expression of several antiviral genes, including interferon and interferon-related genes, to mimic key findings from human studies. Finally, several circulating miRNAs isolated from blood leukocytes was found to hold great potential as biomarkers for progression of viral lung infection. These results further consolidate the pig as a valuable model for influenza A virus infection.

In preterm infants, the serious gastrointestinal disease, necrotizing enterocolitis (NEC), is caused by the combined effect of abnormal bacterial colonization, enteral feeding, and prematurity, in-cluding immaturity of the immune system. In a well-established preterm pig model of NEC, the effect of diet on disease development has been studied thoroughly; however, the inflammatory response during NEC needs to be characterized to improve and promote the use of this model as a model for human disease. We investigated how expression of genes related to immune function and gut maturation in distal small intestinal tissue was affected by NEC development in a number of experimental diet groups. Preterm pigs delivered by Cesarean section received total parenteral nutrition for 2 d followed by enteral nutrition for an additional 2 d: bovine colostrum (n = 6), infant formula (FORM, n = 13), FORM for 6 h followed by bovine colostrum (n = 14), spray dried bovine colostrum (n = 8) or pasteurized, spray dried bovine colostrum (n = 9). At euthanasia, the gastroin-testinal tract (stomach to colon) was evaluated for NEC lesions using a severity score ranging from 1–6 (6 being severe NEC). Pigs with a severity score of minimum three in any gastrointestinal re-gion was regarded as a case of NEC. High throughput qPCR was used to investigate the gene ex-pression of 48 genes in intestinal tissue. Across all enteral diet groups, a relatively higher expres-sion of IL-6, IL-8, IL1-RA and CCL3 was seen in pigs suffering from NEC compared with healthy pigs. In conclusion, the expression of four genes coding for proteins involved in inflammation was increased in pigs suffering from NEC compared to healthy pigs irrespectively of enteral diet group, which points to inflammation as being an important component of NEC. Further studies will ad-dress the relationship of inflammation related gene expression and the development of NEC in order to elucidate cause-effect relationships leading to NEC.

Aim: To test whether melatonin reduces oxidative and inflammatory biomarkers in a closed-chest porcine model of acute myocardial infarction. Materials and Methods: Twenty pigs were randomized to receive a total dosage of 200 mg (0.4 mg/ml) of melatonin, or placebo immediately prior to reperfusion of a coronary artery balloon occlusion in a randomized, observer-blinded, placebo-controlled trial. We assessed high-sensitivity troponin T (hs-TnT), malondialdehyde and interleukin-1b, -6 and -10 at baseline, 30 min and 1, 2, 3 and 4 h after the start of reperfusion. Results: Seventeen pigs completed the trial. There was an increase in hs-TnT, but no significant difference between the melatonin-treated and placebo-treated groups. There were no significant differences in development of any of the circulating plasma markers between the two groups. Conclusion: Melatonin treatment did not result in reduction of inflammatory or oxidative stress markers after experimental myocardial infarction compared to placebo.