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Early in life and particularly around weaning, piglets are susceptible to infections because of abrupt social, environmental, and dietary changes. Dietary interventions with probiotic bacteria have gained popularity because of the increased awareness of the direct link between diet and health. In this study, piglets received the probiotic strain Escherichia coli Nissle 1917 (EcN) or a control treatment perorally from day 2 after birth until 2 weeks post-weaning. To investigate spatio-temporal effects of EcN on the gut microbiota composition, intestinal epithelial gene expression and immune system, feces, digesta, blood, scraping material and mesenteric lymph node tissue were collected at different time points. In addition, oral vaccinations against Salmonella enterica serovar Typhimurium were administered on days 21 and 45 of the study to assess the immunocompetence. EcN-treated pigs showed a reduced diversity of taxa within the phylum Proteobacteria and a lower relative abundance of taxa within the genus Treponema during the pre-weaning period. Moreover, EcN induced T cell proliferation and Natural Killer cell activation in blood and enhanced IL-10 production in ex vivo stimulated mesenteric lymph node cells, the latter pointing toward a more regulatory or anti-inflammatory state of the local gut-associated immune system. These outcomes were primarily observed pre-weaning. No significant differences were observed between the treatment groups with regards to body weight, epithelial gene expression, and immune response upon vaccination. Differences observed during the post-weaning period between the treatment groups were modest. Overall, this study demonstrates that the pre-weaning period offers a ‘window of opportunity’ to modulate the porcine gut microbiota and immune system through dietary interventions such as EcN supplementation.

The zoonotic pathogen Salmonella enterica serotype Enteritidis (SE) causes severe disease in young chickens. Restriction on antibiotic use requires alternative SE control strategies such as nutritional solutions to improve the resistance of chickens. In this study, chickens were fed long-chain glucomannan (GM) or standard diet and challenged with SE at seven days of age. During 21 days post-infection (dpi), we determined numbers and responsiveness of natural killer (NK) and T cells in ileum and spleen, and SE-specific antibody titers in serum. Microbiota compositions in ileum and caeca were determined, as well as correlations of these with numbers and function of immune cells. Some of the samples in the control group had numerically higher CFUs than the GM-treated group. In addition, the relative abundance of SE based on DNA assessment was significantly lower at 21 dpi upon GM supplementation. At 3 dpi, numbers of intraepithelial NK cells were significantly higher, while activation of intraepithelial NK cells (7 dpi), numbers of intraepithelial cytotoxic CD8+ T cells (14 dpi) and SE-specific antibodies (14 dpi) were numerically higher. Furthermore, relative abundance of the commensal lactic acid bacteria (LAB) significantly increased with GM supplementation post-infection. Higher relative abundance of streptococci was associated with reduced SE in ileal and caecal contents at 21 dpi. Relative abundance of streptococci negatively correlated with SE counts and positively correlated with NK cell activation and SE-specific antibodies, which suggests involvement of the commensal LAB in NK cell responsiveness. These results indicate that GM supplementation modulates the immune system, intestinal microbiota and impacts SE infection of young chickens.

Salmonella enterica serotype Enteritidis (SE) is a zoonotic pathogen which causes foodborne diseases in humans as well as severe disease symptoms in young chickens. More insight in innate and adaptive immune responses of chickens to SE infection is needed to understand elimination of SE. Seven-day-old broiler chickens were experimentally challenged with SE and numbers and responsiveness of innate and adaptive immune cells as well as antibody titers were assessed. SE was observed in the ileum and spleen of SE-infected chickens at 7 days post-infection (dpi). At 1 dpi numbers of intraepithelial cytotoxic CD8+ T cells were significantly increased alongside numerically increased intraepithelial IL-2Rα+ and 20E5+ natural killer (NK) cells at 1 and 3 dpi. At both time points, activation of intraepithelial and splenic NK cells was significantly enhanced. At 7 dpi in the spleen, presence of macrophages and expression of activation markers on dendritic cells were significantly increased. At 21 dpi, SE-induced proliferation of splenic CD4+ and CD8+ T cells was observed and SE-specific antibodies were detected in sera of all SE-infected chickens. In conclusion, SE results in enhanced numbers and activation of innate cells and we hypothesized that in concert with subsequent specific T cell and antibody responses, reduction of SE is achieved. A better understanding of innate and adaptive immune responses important in the elimination of SE will aid in developing immune-modulation strategies, which may increase resistance to SE in young broiler chickens.

Salmonellosis is a common infection in poultry, which results in huge economic losses in the poultry industry. At the same time, Salmonella infections are a threat to public health, since contaminated poultry products can lead to zoonotic infections. Antibiotics as feed additives have proven to be an effective prophylactic option to control Salmonella infections, but due to resistance issues in humans and animals, the use of antimicrobials in food animals has been banned in Europe. Hence, there is an urgent need to look for alternative strategies that can protect poultry against Salmonella infections. One such alternative could be to strengthen the innate immune system in young chickens in order to prevent early life infections. This can be achieved by administration of immune modulating molecules that target innate immune cells, for example via feed, or by in-ovo applications. We aimed to review the innate immune system in the chicken intestine; the main site of Salmonella entrance, and its responsiveness to Salmonella infection. Identifying the most important players in the innate immune response in the intestine is a first step in designing targeted approaches for immune modulation.

Introduction: Before release, vaccine batches are assessed for quality to evaluate whether they meet the product specifications. Vaccine batch tests, in particular of inactivated and toxoid vaccines, still largely rely on in vivo methods. Improved vaccine production processes, ethical concerns, and suboptimal performance of some in vivo tests have led to the development of in vitro alternatives. Areas covered: This review describes the scientific constraints that need to be overcome for replacement of in vivo batch tests, as well as potential solutions. Topics include the critical quality attributes of vaccines that require testing, the use of cell-based assays to mimic aspects of in vivo vaccine-induced immune responses, how difficulties with testing adjuvanted vaccines in vitro can be overcome, the use of altered batches to validate new in vitro test methods, and how cooperation between different stakeholders is key to moving the transition forward. Expert opinion: For safety testing, many in vitro alternatives are already available or at an advanced level of development. For potency testing, in vitro alternatives largely comprise immunochemical methods that assess several, but not all critical vaccine properties. One-to-one replacement by in vitro alternatives is not always possible and a combination of methods may be required.

Inactivated poultry vaccines are subject to routine potency testing for batch release, requiring large numbers of animals. The replacement of in vivo tests for cell-based alternatives can be facilitated by the identification of biomarkers for vaccine-induced immune responses. In this study, chicken bone marrow-derived dendritic cells were stimulated with an inactivated vaccine for infectious bronchitis virus and Newcastle disease virus, as well as inactivated infectious bronchitis virus only, and lipopolysaccharides as positive control, or left unstimulated for comparison with the stimulated samples. Next, the cells were lysed and subjected to proteomic analysis. Stimulation with the vaccine resulted in 66 differentially expressed proteins associated with mRNA translation, immune responses, lipid metabolism and the proteasome. For the eight most significantly upregulated proteins, mRNA expression levels were assessed. Markers that showed increased expression at both mRNA and protein levels included PLIN2 and PSMB1. Stimulation with infectious bronchitis virus only resulted in 25 differentially expressed proteins, which were mostly proteins containing Src homology 2 domains. Stimulation with lipopolysaccharides resulted in 118 differentially expressed proteins associated with dendritic cell maturation and antimicrobial activity. This study provides leads to a better understanding of the activation of dendritic cells by an inactivated poultry vaccine, and identified PLIN2 and PSMB1 as potential biomarkers for cell-based potency testing.

This dataset contains data collected during an in vivo experiment with piglets as part of the PhD Thesis Projects of Hugo de Vries and Mirelle Geervliet (first authors of the manuscript).

Piglets are susceptible to infections in early life and around weaning due to rapid environmental and dietary changes. A compelling target to improve pig health in early life is diet, as it constitutes a pivotal determinant of gut microbial colonization and maturation of the host’s immune system. In the present study, we investigated how supplementation of yeast-derived β-glucans affects the gut microbiota and immune function pre-and post-weaning, and how these complex systems develop over time. From day two after birth until two weeks after weaning, piglets received yeast-derived β-glucans or a control treatment orally and were subsequently vaccinated against Salmonella Typhimurium. Faeces, digesta, blood, and tissue samples were collected to study gut microbiota composition and immune function. Overall, yeast-derived β-glucans did not affect the vaccination response, and only modest effects on faecal microbiota composition and immune parameters were observed, primarily before weaning. This study demonstrates that the pre-weaning period offers a ‘window of opportunity’ to alter the gut microbiota and immune system through diet. However, the observed changes were modest, and any long-lasting effects of yeast-derived β-glucans remain to be elucidated.

Studies in mammals, including chickens, have shown that the development of the immune system is affected by interactions with intestinal microbiota. Early life microbial colonization may affect the development of innate and adaptive immunity and may contribute to lasting effects on health and resilience of broiler chickens. We inoculated broiler chickens with adult-derived-microbiota (AM) to investigate their effects on intestinal microbiota composition and natural killer (NK) cells, amongst other immune cells. We hypothesized that AM inoculation directly upon hatch (day 0) would induce an alteration in microbiota composition shortly after hatch, and subsequently affect (subsets of) intestinal NK cells and their activation. Microbiota composition of caecal and ileal content of chickens of 1, 3, 7, 14, 21, and 35 days of age was assessed by sequencing of 16S ribosomal RNA gene amplicons. In parallel, subsets and activation of intestinal NK cells were analyzed by flow cytometry. In caecal content of 1- and 3-day-old AM chickens, a higher alpha-diversity (Faith's phylogenetic diversity) was observed compared to control chickens, whereas ileal microbiota were unaffected. Regarding beta-diversity, caecal microbiota profiles could be clustered into three distinct community types. Cluster A represented caecal microbiota of 1-day-old AM chickens and 1- and 3-day-old control chickens. Cluster B included microbiota of seven of eight 3- and 7-day-old AM and 7-day-old control chickens, and cluster C comprised microbiota of all chickens of 14-days and older, independent of inoculation. In 3-day-old AM chickens an increase in the percentages of intestinal IL-2Rα+NK cells and activated NK cells was observed compared to control chickens of the same age. In addition, an increase in relative numbers of intestinal cytotoxic CD8αα+T cells was observed in 14- and 21-day-old AM chickens. Taken together, these results indicate that early exposure to AM shapes and

In early life and around weaning, pigs are at risk of developing infectious diseases which compromise animal welfare and have major economic consequences for the pig industry. A promising strategy to enhance resistance against infectious diseases is immunomodulation by feed additives. To assess the immune stimulating potential of feed additives in vitro, bone marrow-derived dendritic cells were used. These cells play a central role in the innate and adaptive immune system and are the first cells encountered by antigens that pass the epithelial barrier. Two different feed additives were tested on dendritic cells cultured from fresh and cryopreserved bone marrow cells; a widely used commercial feed additive based on yeast-derived β-glucans and the gram-negative probiotic strain E. coli Nissle 1917. E. coli Nissle 1917, but not β-glucans, induced a dose-dependent upregulation of the cell maturation marker CD80/86, whereas both feed additives induced a dose-dependent production of pro- and anti-inflammatory cytokines, including TNFα, IL-1β, IL-6 and IL-10. Furthermore, E. coli Nissle 1917 consistently induced higher levels of cytokine production than β-glucans. These immunomodulatory responses could be assessed by fresh as well as cryopreserved in vitro cultured porcine bone marrow-derived dendritic cells. Taken together, these results demonstrate that both β-glucans and E. coli Nissle 1917 are able to enhance dendritic cell maturation, but in a differential manner. A more mature dendritic cell phenotype could contribute to a more efficient response to infections. Moreover, both fresh and cryopreserved bone marrow-derived dendritic cells can be used as in vitro pre-screening tools which enable an evidence based prediction of the potential immune stimulating effects of different feed additives.

Intramammary infections (IMI) with Staphylococcus aureus are a common cause of bovine mastitis and can result in both clinical (CM) or subclinical mastitis (SCM). Although bacterial isolates of S. aureus differ in their virulence potential it is largely unclear which bacterial virulence factors are responsible for increased clinical severity. We performed a genome wide association study and used a generalized linear mixed model to investigate the correlation between gene carriage, lineage and clinical outcome of IMI in a collection of S. aureus isolates from cattle with CM (n = 125) and SCM (n = 151) from 11 European countries. An additional aim was to describe the genetic variation of bovine S. aureus in Europa. The dominant lineages in our collection were clonal complex (CC) 151 (81/276, 29.3%), CC97 (54/276, 19.6%), CC479 (32/276, 11.6%) and CC398 (19/276, 6.9%). Virulence and antimicrobial resistance (AMR) gene carriage was highly associated with CC. Among a selection of nine virulence and AMR genes, CC151, CC479 and CC133 carried more virulence genes than other CCs, and CC398 was associated with AMR gene carriage. Whereas CC151, CC97 were widespread in Europe, CC479, CC398 and CC8 were only found in specific countries. Compared to CC151, CC479 was associated with CM rather than SCM (OR 3.62; 95% CI 1.38–9.50) and the other CCs were not. Multiple genes were associated with CM, but due to the clustering within CC of carriage of these genes, it was not possible to differentiate between the effect of gene carriage and CC on clinical outcome of IMI. Nevertheless, this study demonstrates that characterization of S. aureus CC and virulence genes helps to predict the likelihood of the occurrence of CM following S. aureus IMI and highlights the potential benefit of diagnostics tools to identify S. aureus CC during bovine mastitis.