Enteral plasma feeding improves gut function and immunity in piglets after birth asphyxia.

INTRODUCTION: Birth asphyxia may negatively affect gut function and immunity in newborns. Conversely, immunomodulatory milk diets may protect the gut and immune system against damage caused by asphyxia. Using caesarean-derived pigs as models, we hypothesised that enteral feeding with plasma improves gut and immune functions in asphyxiated newborns. METHODS: Near-term pig fetuses (98% gestation,) were delivered by caesarean section after 8 min umbilical cord occlusion, leading to transient birth asphyxia (ASP, n = 75) and compared with non-occluded controls (CON, n = 69). Piglets were further randomised to supplementation with/without porcine plasma (plasma, PLA/vehicle, VEH), into bovine colostrum (first 24 h) or formula (until 72 h). RESULTS: Compared with CON, ASP piglets took longer to achieve stable respiration and showed reduced blood pH, weight gain and survival. Independent of asphyxia, plasma supplementation reduced gut haemorrhagic lesions, permeability and inflammatory cytokines together with improved villous morphology and brush-border enzyme activities. Asphyxia reduced blood cytokine responses to ex vivo bacterial stimulation, whereas plasma supplementation ameliorated this effect. CONCLUSION: Dietary plasma supplementation improves survival, gut functions and immunity in both normal and asphyxiated newborns. The components in plasma that mediate gut-protective effects in piglets remain to be identified, but may benefit also birth-compromised newborn infants. IMPACT: Complicated deliveries leading to birth asphyxia, may negatively affect gut, liver and immune adaptation in the first days after birth. Using a model of birth asphyxia in caesarean-derived piglets, we show that enteral feeding with maternal plasma exerts gut maturational and immunomodulatory effects in both control and asphyxiated animals in the first days of life. The mechanisms behind the gut-protective effects of plasma are unknown, but plasma components hold potential for new oral therapies for compromised newborn infants as well as piglets.

Pig Acute Phase Proteins as Non-Antibody Systemic Biomarkers of Intracellular Infections.

As an alternative to traditional serological markers, that is, antibodies, for serum-based specific diagnosis of infections, circulating non-antibody markers may be used to monitor active disease. Acute phase proteins (APPs) are a prominent class of such markers widely used for diagnosing ongoing inflammation and infection. In this chapter, basic theoretical and practical considerations on developing APP assays and using APPs as markers of ongoing infection are presented with a specific focus on intracellular infections in pigs. Examples on APP-based monitoring of infection in pigs with viruses such as porcine respiratory and reproductive syndrome virus (PRRSV), porcine endemic diarrhea virus (PEDV), and influenza A virus (IAV), as well as intracellular bacteria (Lawsonia intracellularis) and the protozoan intracellular parasites Toxoplasma gondii and Cryptosporidium parvum are presented, with an emphasis on major pig APPs C-reactive protein (CRP), haptoglobin, serum amyloid A (SAA), and pig major acute phase protein (pig-MAP). The performance of these APPs as biomarkers in a range of experimental infection studies in pigs is described as examples on their use for estimating the severity of infection, vaccine efficacy, herd health characterization, and differential diagnosis.

Novel genetically glycoengineered human dendritic cell model reveals regulatory roles of α2,6-linked sialic acids in DC activation of CD4+ T cells and response to TNFα.

Dendritic cells (DCs) are central for the initiation and regulation of appropriate immune responses. While several studies suggest important regulatory roles of sialoglycans in DC biology, our understanding is still inadequate primarily due to a lack of appropriate models. Previous approaches based on enzymatic- or metabolic-glycoengineering and primary cell isolation from genetically modified mice have limitations related to specificity, stability, and species differences. This study addresses these challenges by introducing a workflow to genetically glycoengineer the human DC precursor cell line MUTZ-3, described to differentiate and maturate into fully functional dendritic cells, using CRISPR-Cas9, thereby providing and validating the first isogenic cell model for investigating glycan alteration on human DC differentiation, maturation, and activity. By knocking out (KO) the ST6GAL1 gene, we generated isogenic cells devoid of ST6GAL1-mediated α(2,6)-linked sialylation, allowing for a comprehensive investigation into its impact on DC function. Glycan profiling using lectin binding assay and functional studies revealed that ST6GAL1 KO increased the expression of important antigen presenting and co-stimulatory surface receptors and a specifically increased activation of allogenic human CD4 + T cells. Additionally, ST6GAL1 KO induces significant changes in surface marker expression and cytokine response to TNFα-induced maturation, and it affects migration and the endocytic capacity. These results indicate that genetic glycoengineering of the isogenic MUTZ-3 cellular model offers a valuable tool to study how specific glycan structures influence human DC biology, contributing to our understanding of glycoimmunology.

Pulmonary MicroRNA expression after heterologous challenge with swine influenza A virus (H1N2) in immunized and non-immunized pigs.

MicroRNAs (miRNAs) contribute to post-transcriptional modulation of the host response during influenza A virus (IAV) infection and may be involved in shaping disease severity. Differential disease severity was achieved in two groups of pigs by immunization of one group with a commercial swine IAV vaccine prior to heterologous IAV (H1N2) challenge of both groups. Lung tissue was harvested 1, 3, and 14 days after challenge and miRNA expression was quantified. Gene Ontology term enrichment analysis was employed to examine the functional relevance of genes potentially regulated by differentially expressed miRNAs in pigs with varying degrees of disease severity following IAV infection. Results suggested that the miRNA response associated with less severe disease may modulate host mechanisms essential for viral life cycle, e.g. transcription, translation, and protein trafficking. During more severe disease, miRNA-mediated regulation may focus on dampening virus-specific processes e.g. virion assembly and viral protein processing, and controlling host metabolism.

Effects of different immunomodulating liposome-based adjuvants and injection sites on immunogenicity in pigs.

Vaccine adjuvants, such as liposome-based cationic adjuvant formulations (CAFs), are able to boost immune responses and, by incorporation of distinct immunomodulators, steer immunity towards a desired direction in mice, non-human primates and humans, while less studied in pigs. Here we used commercial pigs to investigate polarizing adjuvant effects of CAFs with immunomodulators: C-type lectin receptor ligands trehalose-6,6'-dibehenate and monomycolyl glycerol, toll-like receptor 3 ligand Poly(I:C) or retinoic acid. Vaccines were formulated with a recombinant Chlamydia model protein antigen and administered via three injection routes. All adjuvants significantly increased antigen-specific IgG in serum, compared to non-adjuvanted antigen. Administering the vaccines through intramuscular and intraperitoneal routes induced significantly higher antigen-specific IgG and IgA serum antibodies, than the perirectal route. Although immunizations triggered cell-mediated immunity, no significant differences between adjuvants or injection sites were detected. Genes depicting T cell subtypes revealed only minor differences. Our findings suggest that specific signatures of the tested adjuvant immunomodulation do not translate well from mice to pigs in standard two-dose immunizations. This study provides new insights into immune responses to CAFs in pigs, and highlights that adjuvant development should ideally be carried out in the intended species of interest or in models with high predictive validity/translational value.

Intraarticular treatment with integrin α10ß1-selected mesenchymal stem cells affects microRNA expression in experimental post-traumatic osteoarthritis in horses.

Osteoarthritis (OA) remains a major cause of lameness in horses, which leads to lost days of training and early retirement. Still, the underlying pathological processes are poorly understood. MicroRNAs (miRNAs) are small non-coding RNAs that serve as regulators of many biological processes including OA. Analysis of miRNA expression in diseased joint tissues such as cartilage and synovial membrane may help to elucidate OA pathology. Since integrin α10ß1-selected mesenchymal stem cell (integrin α10-MSC) have shown mitigating effect on equine OA we here investigated the effect of integrin α10-MSCs on miRNA expression. Cartilage and synovial membrane was harvested from the middle carpal joint of horses with experimentally induced, untreated OA, horses with experimentally induced OA treated with allogeneic adipose-derived MSCs selected for the marker integrin α10-MSCs, and from healthy control joints. miRNA expression in cartilage and synovial membrane was established by quantifying 70 pre-determined miRNAs by qPCR. Differential expression of the miRNAs was evaluated by comparing untreated OA and control, untreated OA and MSC-treated OA, and joints with high and low pathology score. A total of 60 miRNAs were successfully quantified in the cartilage samples and 55 miRNAs were quantified in the synovial membrane samples. In cartilage, miR-146a, miR-150 and miR-409 had significantly higher expression in untreated OA joints than in control joints. Expression of miR-125a-3p, miR-150, miR-200c, and miR-499-5p was significantly reduced in cartilage from MSC-treated OA joints compared to the untreated OA joints. Expression of miR-139-5p, miR-150, miR-182-5p, miR-200a, miR-378, miR-409-3p, and miR-7177b in articular cartilage reflected pathology score. Several of these miRNAs are known from research in human patients with OA and from murine OA models. Our study shows that these miRNAs are also differentially expressed in experimental equine OA, and that expression depends on OA severity. Moreover, MSC treatment, which resulted in less severe OA, also affected miRNA expression in cartilage.

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.

A Gently Processed Skim Milk-Derived Whey Protein Concentrate for Infant Formula: Effects on Gut Development and Immunity in Preterm Pigs.

SCOPE: Processing of whey protein concentrate (WPC) for infant formulas may induce protein modifications with severe consequences for preterm newborn development. The study investigates how conventional WPC and a gently processed skim milk-derived WPC (SPC) affect gut and immune development after birth. METHODS AND RESULTS: Newborn, preterm pigs used as a model of preterm infants were fed formula containing WPC, SPC, extra heat-treated SPC (HT-SPC), or stored HT-SPC (HTS-SPC) for 5 days. SPC contained no protein aggregates and more native lactoferrin, and despite higher Maillard reaction product (MRP) formation, the clinical response and most gut and immune parameters are similar to WPC pigs. SPC feeding negatively impacts intestinal MRP accumulation, mucosa, and bacterial diversity. In contrast, circulating T-cells are decreased and oxidative stress- and inflammation-related genes are upregulated in WPC pigs. Protein aggregation and MRP formation increase in HTS-SPC, leading to reduced antibacterial activity, lactase/maltase ratio, circulating neutrophils, and cytotoxic T-cells besides increased gut MRP accumulation and expression of TNFAIP3. CONCLUSION: The gently processed SPC has more native protein, but higher MRP levels than WPC, resulting in similar tolerability but subclinical adverse gut effects in preterm pigs. Additional heat treatment and storage further induce MRP formation, gut inflammation, and intestinal mucosal damage.

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.

Dietary seaweed (Saccharina latissima) supplementation in pigs induces localized immunomodulatory effects and minor gut microbiota changes during intestinal helminth infection.

Brown seaweeds have a rich bioactive content known to modulate biological processes, including the mucosal immune response and microbiota function, and may therefore have the potential to control enteric pathogens. Here, we tested if dietary seaweed (Saccharina latissima) supplementation could modulate pig gut health with a specific focus on parasitic helminth burdens, gut microbiota composition, and host immune response during a five week feeding period in pigs co-infected with the helminths Ascaris suum and Oesophagostomum dentatum. We found that inclusion of fermented S. latissima (Fer-SL) at 8% of the diet increased gut microbiota α-diversity with higher relative abundances of Firmicutes, Tenericutes, Verrucomicrobia, Spirochaetes and Elusimicrobia, and lower abundance of Prevotella copri. In the absence of helminth infection, transcription of immune-related genes in the intestine was only moderately influenced by dietary seaweed. However, Fer-SL modulated the transcriptional response to infection in a site-specific manner in the gut, with an attenuation of infection-induced gene expression in the jejunum and an amplification of gene expression in the colon. Effects on systemic immune parameters (e.g. blood lymphocyte populations) were limited, indicating the effects of Fer-SL were mainly localized to the intestinal tissues. Despite previously documented in vitro anti-parasitic activity against pig helminths, Fer-SL inclusion did not significantly affect parasite egg excretion or worm establishment. Collectively, our results show that although Fer-SL inclusion did not reduce parasite burdens, it may modify the gut environment during enteric parasite infection, which encourages continued investigations into the use of seaweeds or related products as novel tools to improve gut health.

A window into the human immune system: comprehensive characterization of the complexity of antibody complementary-determining regions in functional antibodies.

The human immune system uses antibodies to neutralize foreign antigens. They are composed of heavy and light chains, both with constant and variable regions. The variable region has six hypervariable loops, also known as complementary-determining regions (CDRs) that determine antibody diversity and antigen specificity. Knowledge of their significance, and certain residues present in these areas, is vital for antibody therapeutics development. This study includes an analysis of more than 11,000 human antibody sequences from the International Immunogenetics information system (IMGT). The analysis included parameters such as length distribution, overall amino acid diversity, amino acid frequency per CDR and residue position within antibody chains. Overall, our findings confirm existing knowledge, such as CDRH3's high length diversity and amino acid variability, increased aromatic residue usage, particularly tyrosine, charged and polar residues like aspartic acid, serine, and the flexible residue glycine. Specific residue positions within each CDR influence these occurrences, implying a unique amino acid type distribution pattern. We compared amino acid type usage in CDRs and non-CDR regions, both in globular and transmembrane proteins, which revealed distinguishing features, such as increased frequency of tyrosine, serine, aspartic acid, and arginine. These findings should prove useful for future optimization, improvement of affinity, synthetic antibody library design, or the creation of antibodies de-novo in silico.

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.

Agaricus subrufescens fermented rye affects the development of intestinal microbiota, local intestinal and innate immunity in suckling-to-nursery pigs.

BACKGROUND: Agaricus subrufescens is considered as one of the most important culinary-medicinal mushrooms around the world. It has been widely suggested to be used for the development of functional food ingredients to promote human health ascribed to the various properties (e.g., anti-inflammatory, antioxidant, and immunomodulatory activities). In this context, the interest in A. subrufescens based feed ingredients as alternatives for antibiotics has also been fuelled during an era of reduced/banned antibiotics use. This study aimed to investigate the effects of a fermented feed additive -rye overgrown with mycelium (ROM) of A. subrufescens-on pig intestinal microbiota, mucosal gene expression and local and systemic immunity during early life. Piglets received ROM or a tap water placebo (Ctrl) perorally every other day from day 2 after birth until 2 weeks post-weaning. Eight animals per treatment were euthanized and dissected on days 27, 44 and 70. RESULTS: The results showed ROM piglets had a lower inter-individual variation of faecal microbiota composition before weaning and a lower relative abundance of proteobacterial genera in jejunum (Undibacterium and Solobacterium) and caecum (Intestinibacter and Succinivibrionaceae_UCG_001) on day 70, as compared to Ctrl piglets. ROM supplementation also influenced gut mucosal gene expression in both ileum and caecum on day 44. In ileum, ROM pigs showed increased expression of TJP1/ZO1 but decreased expression of CLDN3, CLDN5 and MUC2 than Ctrl pigs. Genes involved in TLR signalling (e.g., TICAM2, IRAK4 and LY96) were more expressed but MYD88 and TOLLIP were less expressed in ROM pigs than Ctrl animals. NOS2 and HIF1A involved in redox signalling were either decreased or increased in ROM pigs, respectively. In caecum, differentially expressed genes between two groups were mainly shown as increased expression (e.g., MUC2, PDGFRB, TOLLIP, TNFAIP3 and MYD88) in ROM pigs. Moreover, ROM animals showed higher NK cell activation in blood and enhanced IL-10 production in ex vivo stimulated MLN cells before weaning. CONCLUSIONS: Collectively, these results suggest that ROM supplementation in early life modulates gut microbiota and (local) immune system development. Consequently, ROM supplementation may contribute to improving health of pigs during the weaning transition period and reducing antibiotics use.

Decreased water temperature enhance Piscine orthoreovirus genotype 3 replication and severe heart pathology in experimentally infected rainbow trout.

Piscine orthoreovirus genotype 3 (PRV-3) was first discovered in Denmark in 2017 in relation to disease outbreaks in rainbow trout (Oncorhynchus mykiss). While the virus appears to be widespread in farmed rainbow trout, disease outbreaks associated with detection of PRV-3 have only occurred in recirculating aquaculture systems, and has predominantly been observed during the winter months. To explore the possible effects of water temperature on PRV-3 infection in rainbow trout, an in vivo cohabitation trial was conducted at 5, 12, and 18°C. For each water temperature, a control tank containing mock-injected shedder fish and a tank with PRV-3 exposed fish were included. Samples were collected from all experimental groups every 2nd week post challenge (WPC) up until trial termination at 12 WPC. PRV-3 RNA load measured in heart tissue of cohabitants peaked at 6 WPC for animals maintained at 12 and 18°C, while it reached its peak at 12 WPC in fish maintained at 5°C. In addition to the time shift, significantly more virus was detected at the peak in fish maintained at 5°C compared to 12 and 18°C. In shedders, fish at 12 and 18°C cleared the infection considerably faster than the fish at 5°C: while shedders at 18 and 12°C had cleared most of the virus at 4 and 6 WPC, respectively, high virus load persisted in the shedders at 5°C until 12 WPC. Furthermore, a significant reduction in the hematocrit levels was observed in the cohabitants at 12°C in correlation with the peak in viremia at 6 WPC; no changes in hematocrit was observed at 18°C, while a non-significant reduction (due to large individual variation) trend was observed at cohabitants held at 5°C. Importantly, isg15 expression was positively correlated with PRV-3 virus load in all PRV-3 exposed groups. Immune gene expression analysis showed a distinct gene profile in PRV-3 exposed fish maintained at 5°C compared to 12 and 18°C. The immune markers mostly differentially expressed in the group at 5°C were important antiviral genes including rigi, ifit5 and rsad2 (viperin). In conclusion, these data show that low water temperature allow for significantly higher PRV-3 replication in rainbow trout, and a tendency for more severe heart pathology development in PRV-3 injected fish. Increased viral replication was mirrored by increased expression of important antiviral genes. Despite no mortality being observed in the experimental trial, the data comply with field observations of clinical disease outbreaks during winter and cold months.

Influence of vitamin D metabolites on vitamin D status, immunity and gut health of piglets.

Immediately post-weaning, piglets are prone to gastrointestinal infectious diseases. The active metabolite of vitamin D 1,25-dihydroxyvitamin D has direct impact on immune cell function and responses. Thus, a low vitamin D status may compromise the immune responses during infectious diseases. The aim of this study was to examine the effect of supplementation of different forms of vitamin D (25-OH-D3 and vitamin D3) to suckling piglets' vitamin D status at weaning. In addition, to determine whether the vitamin D status could affect the immune development in piglets and their robustness against E. coli challenge. Genetically E. coli F4 susceptible litters of piglets were divided into two treatment groups: group 1 (n = 16) provided milk formula supplemented with vitamin D3 (CON), and group 2 (n = 16) provided milk formula supplemented with 25-OH-D3 (TREAT). Piglets were offered the experimental milk formulas from day 3 after farrowing until weaning (at day 28 of age). A commercial weaner diet with high protein content were provided to induce weaning stress. Milk formulas, sow and weaner diets as well as plasma and milk samples obtained from sows (n = 8) were analysed for vitamin D metabolites. Vitamin D status in piglets was investigated by collection of plasma samples on day 3, 15, 28 and 35 of age. Eight piglets randomly selected from each dietary group (in total 16 pigs) were inoculated with E. coli F4 O149 on day 2 and 3 post-weaning. Blood samples collected on day 2 and 9 post-weaning (pre- and post E. coli inoculation, respectively) were analysed for haematological and immunological parameters including immunoglobulins, antibodies specific to E. coli O149 K88, cytokines and C-reactive protein. In addition, intestinal samples were obtained one week after E. coli inoculation to study the influence of infection and vitamin D status on immune responses at different sites of the intestine. This was accomplished by gene expression of various cytokines and tight junction proteins. In general, vitamin D status of the piglets were low. However, piglets provided TREAT during the suckling period had increased vitamin D status at weaning compared to piglets provided CON. Vitamin D was used during activation of the immune system as pigs inoculated with E. coli had lower plasma concentrations of 25-OH-D3 than non-inoculated pigs possibly due to mobilising of vitamin D in the liver. Hence, increased vitamin D status at weaning might improve piglets' resistance to E. coli infection.

Influence of African Swine Fever Virus on Host Gene Transcription within Peripheral Blood Mononuclear Cells from Infected Pigs.

African swine fever virus (ASFV) has become a global threat to the pig production industry and has caused enormous economic losses in many countries in recent years. Peripheral blood mononuclear cells (PBMCs) from pigs infected with ASFV not only express ASFV genes (almost 200 in number) but have altered patterns of host gene expression as well. Both up- and down-regulation of host cell gene expression can be followed using RNAseq on poly(A)+ mRNAs harvested from the PBMCs of pigs collected at different times post-infection. Consistent with the time course of changes in viral gene expression, only few and limited changes in host gene expression were detected at 3 days post-infection (dpi), but by 6 dpi, marked changes in the expression of over 1300 host genes were apparent. This was co-incident with the major increase in viral gene expression. The majority of the changes in host gene expression were up-regulation, but many down-regulated genes were also identified. The patterns of changes in gene expression within the PBMCs detected by RNAseq were similar in each of the four infected pigs. Furthermore, changes in the expression of about twenty selected host genes, known to be important in host defence and inflammatory responses, were confirmed using high-throughput microfluidic qPCR assays.

Immobilization-Free Binding and Affinity Characterization of Higher Order Bispecific Antibody Complexes Using Size-Based Microfluidics.

Simultaneous targeting of different antigens by bispecific antibodies (bsAbs) is permitting synergistic binding functionalities with high therapeutic potential, but is also rendering their analysis challenging. We introduce flow-induced dispersion analysis (FIDA) for the in-depth characterization of bsAbs with diverse molecular architectures and valencies under near-native conditions without potentially obstructive surface immobilization. Individual equilibrium dissociation constants are determined in solution, even in higher-order complexes with both antigens involved, hereby allowing the analysis of binding cooperativity and elucidation of a potential interference between the interactions. We further illustrate bispecific binding functionality as incremental increases in complex sizes when the bsAbs are exposed to one or two antigens. The possibility for comprehensive binding analysis with low material consumption and high matrix tolerability irrespective of molecular format and with little optimization renders FIDA a versatile tool for format selection and characterization of complex bi/multispecific protein therapeutics throughout the drug development and biomanufacturing pipeline.

Outer Membrane Vesicles of Actinobacillus pleuropneumoniae Exert Immunomodulatory Effects on Porcine Alveolar Macrophages.

Outer membrane vesicles (OMVs) are spontaneously released by Gram-negative bacteria, including Actinobacillus pleuropneumoniae, which causes contagious pleuropneumonia in pigs and leads to considerable economic losses in the swine industry worldwide. A. pleuropneumoniae OMVs have previously been demonstrated to contain Apx toxins and proteases, as well as antigenic proteins. Nevertheless, comprehensive characterizations of their contents and interactions with host immune cells have not been made. Understanding the protein compositions and immunomodulating ability of A. pleuropneumoniae OMVs could help illuminate their biological functions and facilitate the development of OMV-based applications. In the current investigation, we comprehensively characterized the proteome of native A. pleuropneumoniae OMVs. Moreover, we qualitatively and quantitatively compared the OMV proteomes of a wild-type strain and three mutant strains, in which relevant genes were disrupted to increase OMV production and/or produce OMVs devoid of superantigen PalA. Furthermore, the interaction between A. pleuropneumoniae OMVs and porcine alveolar macrophages was also characterized. Our results indicate that native OMVs spontaneously released by A. pleuropneumoniae MIDG2331 appeared to dampen the innate immune responses by porcine alveolar macrophages stimulated by either inactivated or live parent cells. The findings suggest that OMVs may play a role in manipulating the porcine defense during the initial phases of the A. pleuropneumoniae infection. IMPORTANCE Owing to their built-in adjuvanticity and antigenicity, bacterial outer membrane vesicles (OMVs) are gaining increasing attention as potential vaccines for both human and animal use. OMVs released by Actinobacillus pleuropneumoniae, an important respiratory pathogen in pigs, have also been investigated for vaccine development. Our previous studies have shown that A. pleuropneumoniae secretes OMVs containing multiple immunogenic proteins. However, immunization of pigs with these vesicles was not able to relieve the pig lung lesions induced by the challenge with A. pleuropneumoniae, implying the elusive roles that A. pleuropneumoniae OMVs play in host-pathogen interaction. Here, we showed that A. pleuropneumoniae secretes OMVs whose yield and protein content can be altered by the deletion of the nlpI and palA genes. Furthermore, we demonstrate that A. pleuropneumoniae OMVs dampen the immune responses in porcine alveolar macrophages stimulated by A. pleuropneumoniae cells, suggesting a novel mechanism that A. pleuropneumoniae might use to evade host defense.

Cholestasis alters brain lipid and bile acid composition and compromises motor function in neonatal piglets.

Infants with neonatal cholestasis are prone to neurodevelopmental deficits, however, the underlying pathogenesis is unclear. Lipid malabsorption and accumulation of potentially neurotoxic molecules in the blood such as bile acids are important yet relatively unexplored pathways. Here, we developed a translational piglet model to understand how the molecular bile acid and lipid composition of the brain is affected by this disease and relates to motor function. Piglets (8-days old) had bile duct ligation or sham surgery and were fed a formula diet for 3 weeks. Alongside sensory-motor deficits observed in bile duct-ligated animals, we found a shift toward a more hydrophilic and conjugated bile acid profile in the brain. Additionally, comprehensive lipidomics of the cerebellum revealed a decrease in total lipids including phosphatidylinositols and phosphatidylserines and increases in lysophospholipid species. This was paralleled by elevated cerebellar expression of genes related to inflammation and tissue damage albeit without significant impact on the brain transcriptome. This study offers new insights into the developing brain's molecular response to neonatal cholestasis indicating that bile acids and lipids may contribute in mediating motor deficits.

Innate antiviral responses in porcine nasal mucosal explants inoculated with influenza A virus are comparable with responses in respiratory tissues after viral infection.

Nasal mucosal explant (NEs) cultured at an air-liquid interface mimics in vivo conditions more accurately than monolayer cultures of respiratory cell linesor primary cells cultured in flat-bottom microtiter wells. NEs might be relevant for studies of host-pathogen interactions and antiviral immune responses after infection with respiratory viruses, including influenza and corona viruses. Pigs are natural hosts for swine influenza A virus (IAV) but are also susceptible to IAV from humans, emphasizing the relevance of porcine NEs in the study of IAV infection. Therefore, we performed fundamental characterization and study of innate antiviral responses in porcine NEs using microfluidic high-throughput quantitative real-time PCR (qPCR) to generate expression profiles of host genes involved in inflammation, apoptosis, and antiviral immune responses in mock inoculated and IAV infected porcine NEs. Handling and culturing of the explants ex vivo had a significant impact on gene expression compared to freshly harvested tissue. Upregulation (2-43 fold) of genes involved in inflammation, including IL1A and IL6, and apoptosis, including FAS and CASP3, and downregulation of genes involved in viral recognition (MDA5 (IFIH1)), interferon response (IFNA), and response to virus (OAS1, IFIT1, MX1) was observed. However, by comparing time-matched mock and virus infected NEs, transcription of viral pattern recognition receptors (RIG-I (DDX58), MDA5 (IFIH1), TLR3) and type I and III interferons (IFNB1, IL28B (IFNL3)) were upregulated 2-16 fold in IAV-infected NEs. Furthermore, several interferon-stimulated genes including MX1, MX2, OAS, OASL, CXCL10, and ISG15 was observed to increase 2-26 fold in response to IAV inoculation. NE expression levels of key genes involved in antiviral responses including IL28B (IFNL3), CXCL10, and OASL was highly comparable to expression levels found in respiratory tissues including nasal mucosa and lung after infection of pigs with the same influenza virus isolate.