The domestic pig as human-relevant large animal model to study adaptive antifungal immune responses against airborne Aspergillus fumigatus.

Pulmonary mucosal immune response is critical for preventing opportunistic Aspergillus fumigatus infections. Although fungus-specific CD4+ T cells in blood are described to reflect the actual host-pathogen interaction status, little is known about Aspergillus-specific pulmonary T-cell responses. Here, we exploit the domestic pig as human-relevant large animal model and introduce antigen-specific T-cell enrichment in pigs to address Aspergillus-specific T cells in the lung compared to peripheral blood. In healthy, environmentally Aspergillus-exposed pigs, the fungus-specific T cells are detectable in blood in similar frequencies as observed in healthy humans and exhibit a Th1 phenotype. Exposing pigs to 106 cfu/m3 conidia induces a long-lasting accumulation of Aspergillus-specific Th1 cells locally in the lung and also systemically. Temporary immunosuppression during Aspergillus-exposure showed a drastic reduction in the lung-infiltrating antifungal T-cell responses more than 2 weeks after abrogation of the suppressive treatment. This was reflected in blood, but to a much lesser extent. In conclusion, by using the human-relevant large animal model the pig, this study highlights that the blood clearly reflects the mucosal fungal-specific T-cell reactivity in environmentally exposed as well as experimentally exposed healthy pigs. But, immunosuppression significantly impacts the mucosal site in contrast to the initial systemic immune response.

A porcine animal model to mimic the restart of enteral nutrition (refeeding-model).

With the aim towards establishing an animal model of total parenteral nutrition (TPN), 12 piglets aged 9 weeks (mean body weight 21 kg) were surgically provided with central venous catheters. Six piglets were nourished parenterally with the objective to reach a 14-d period of TPN; the other six piglets served as control and were fed normally. Only one animal from each group could be monitored over the whole period. Nine piglets were euthanised on d 13 and one on d 12. No animal showed fever or signs of septicaemia during the study. The levels of Ca, Mg, Na and P in the blood were within the normal range as were those for blood glucose and plasma creatinine. Symptoms of the TPN included: transient diarrhoea, occasional appearance of faecal blood and occasional absence of defecation. A reduced small intestine length and altered mucosal morphology and function were observed. One animal showed bile stasis at the end of the study. All TPN animals showed a remarkably high level of blood urea early in the morning. The intestinal symptoms observed may resemble the human situation during TPN. However, due to the fast growth rate, pigs aged 9 weeks have higher nutrient requirements per kg body weight. Consequently, the osmolality of the nutrient solution was necessarily high. Whether the significantly higher blood urea observed in the TPN group reflected a catabolic metabolism during the starving period at night-time could not be conclusively shown. Alternatively, it could reflect a slower growth rate and a resulting quantitative excess of amino acids (AA), or could have been the consequence of a suboptimal AA composition. A permanent infusion would be favourable in order not to overcharge the capacity for glucose uptake and amino acid metabolism during the infusion.

The expression of NKG2D on porcine IEL and its possible relation to the adaptive intestinal immune system.

The gastrointestinal tract contains a multitude of components which include intraepithelial lymphocytes (IEL). IELs have been reported to express a variety of surface receptors that enable cross talk among various cell populations. The purpose of the reported investigation was to determine which IEL populations express the natural killer cell receptor NKG2D which is an activating receptor that plays a role in cytolytic responses. In a feeding experiment with piglets, IELs were isolated from jejunal tissue at three different stages post weaning. The time dependent development of different cell populations was evaluated and an elevated number of lymphocytes (CD45+) shortly after weaning was observed compared to later time points. The number of T cells (CD3), including cytotoxic T cells (CD8ß/CD16-), appeared to be particularly affected by the weaning period. Correlation analysis revealed an association between the NKG2D expression in jejunal tissue and the frequency of lymphocytes, esp. CD8ß+ cytotoxic T cells. Gene expression analysis of NKG2D were performed on several isolated IEL populations and support the hypothesis that cytotoxic T cells (CD8ß) in the porcine gut epithelium are capable of communicating with the surrounding enterocytes and inducing immune reactions via NKG2D. Unlike previous observations in porcine blood, the γδ T cells of the gut epithelium also showed expression of the stress factor binding NKG2D receptor. Subsequent analysis of the isolated IELs revealed that T cells appear to only express the receptor after isolation with an anti-CD3 mab, indicating that a previous stimulation of the TCR/CD3 complex may reinforce this signal transduction pathway.

MicroRNA Response of Primary Human Macrophages to Arcobacter Butzleri Infection.

The role of microRNAs (miRNAs) in infectious diseases is becoming more and more apparent, and the use of miRNAs as a diagnostic tool and their therapeutic application has become the major focus of investigation. The aim of this study was to identify miRNAs involved in the immune signaling of macrophages in response to Arcobacter (A.) butzleri infection, an emerging foodborne pathogen causing gastroenteritis. Therefore, primary human macrophages were isolated and infected, and miRNA expression was studied by means of RNAseq. Analysis of the data revealed the expression of several miRNAs, which were previously associated with bacterial infections such as miR-155, miR-125, and miR-212. They were shown to play a key role in Toll-like receptor signaling where they act as fine-tuners to establish a balanced immune response. In addition, miRNAs which have yet not been identified during bacterial infections such as miR-3613, miR-2116, miR-671, miR-30d, and miR-629 were differentially regulated in A. butzleri-infected cells. Targets of these miRNAs accumulated in pathways such as apoptosis and endocytosis - processes that might be involved in A. butzleri pathogenesis. Our study contributes new findings about the interaction of A. butzleri with human innate immune cells helping to understand underlying regulatory mechanisms in macrophages during infection.

Probiotic Treatment Decreases the Number of CD14-Expressing Cells in Porcine Milk Which Correlates with Several Intestinal Immune Parameters in the Piglets.

Modulating the mucosal immune system of neonates by probiotic treatment of their mothers is a promising approach which can only be investigated through the use of animal models. Here, we used sows and their piglets to investigate the impact of a bacterial treatment on the sow's milk and on the neonate piglet intestinal immune system. In previous experiments, feed supplementation of sows with the probiotic Enterococcus faecium NCIMB 10415 during pregnancy and lactation had been shown to affect intestinal microbiota and cytokine expression of the offspring during the suckling and weaning periods. We therefore investigated the composition of the milk from treated sows in comparison to samples from a control group. In treated sows, the amount of lactose increased, and the somatic cell numbers were reduced. In all milk samples, the percentage of cells expressing membranous CD14 (mCD14) was greater than the fractions of immune cells, indicating expression of mCD14 on mammary epithelial cells. However, in the milk of E. faecium-treated sows, mCD14(+) cells were reduced. Furthermore, the number of CD14(+) milk cells was positively correlated with the percentages of B cells and activated T cells in the ileal MLN of the piglets. This study provides evidence for the expression of mCD14 by the porcine mammary epithelium, and suggests an immunological effect of mCD14(+) milk cells on the piglets' intestinal immune system. Our study further suggests that mCD14(+) mammary epithelial cell populations can be modulated by probiotic feed supplementation of the sow.

Impact of a probiotic Bacillus cereus strain on the jejunal epithelial barrier and on the NKG2D expressing immune cells during the weaning phase of piglets.

In a feeding experiment, the probiotic Bacillus cereus var. Toyoi was fed to sows and piglets in order to test whether it influences the stress response of enterocytes, thereby causing intestinal immune activation, possibly accompanied by an impairment of the epithelial integrity. The impact of B. cereus on the piglets' intestinal enterocytes (EC) and on the communicating intraepithelial lymphocytes (IEL) was investigated during the weaning phase where significant immunological changes might be expected. The expression of the stress-induced MHC class I-related molecule 2 (MIC2) and the UL16 binding protein (ULBP) was measured in jejunal EC and the frequencies of the main present IEL populations in the jejunum were monitored. To find out which of the IEL populations can be activated by the stress-induced molecules the sorted IEL were tested for the expression of the activating natural killer receptor 2D (NKG2D). The piglets fed with B. cereus showed an impaired intestinal barrier function shortly after weaning. However, a significant impact on the expression of stress-induced molecules was not observed. The mRNA expression of NKG2D was confirmed in intraepithelial CD5+ γδ T cells. The ratio of IEL (CD45+) to EC was lower in the B. cereus treated group, which could be explained by lower frequencies of CD8αß+ T cells in the jejunal epithelium (p ≤ 0.005 for ages 32 and 34). Although a consistently increased expression of stress-induced MHC class I-related molecules was not found, this study suggests a negative impact of B. cereus on the intestinal barrier function and supports immune-modulating properties of the probiotic feed supplement.

Colostrum and milk selenium, antioxidative capacity and immune status of dairy cows fed sodium selenite or selenium yeast.

Dietary selenium (Se) can be supplemented from organic or inorganic sources and this may affect Se metabolism and functional outcome such as antioxidative status and immune functions in dairy cows. A feeding trial was performed with 16 Holstein-Friesian dairy cows fed with a total mixed ration (0.18 mg Se/kg dry matter (DM)) either without Se supplement (Control, n = 5), or with Se from sodium selenite (Group SeS, n = 5) or Se yeast (Group SeY, n = 6). In Groups SeS and SeY, the Se supplementation amounted to an additional intake of 4 mg Se and 6 mg Se/d during gestation and lactation, respectively. The effect of both Se sources was characterised by milk Se and antioxidant levels, and the phenotyping and functional assessment of phagocytic activity of milk immune cells. Se yeast has been found to increase (p ≤ 0.001) the milk Se and antioxidant levels markedly compared to the control group. The experimental treatment did not affect the immune parameters of the cows. Lymphocyte subpopulations and phagocytosis activity of neutrophilic granulocytes were affected neither by the Se intake nor by the two different dietary supplements. It can be concluded that sodium selenite and Se yeast differ considerably in their effects on antioxidant status in dairy cows. However, the basal dietary Se concentration of 0.18 mg/kg DM seemed to be high enough for the measured immune variables.

Feeding of the probiotic bacterium Enterococcus faecium NCIMB 10415 differentially affects shedding of enteric viruses in pigs.

Effects of probiotic bacteria on viral infections have been described previously. Here, two groups of sows and their piglets were fed with or without feed supplementation of the probiotic bacterium Enterococcus faecium NCIMB 10415. Shedding of enteric viruses naturally occurring in these pigs was analyzed by quantitative real-time RT-PCR. No differences between the groups were recorded for hepatitis E virus, encephalomyocarditis virus and norovirus. In contrast, astrovirus was exclusively detected in the non-supplemented control group. Rotavirus was shedded later and with lower amounts in the probiotic piglet group (p < 0.05); rotavirus-shedding piglets gained less weight than non-infected animals (p < 0.05). Serum titres of anti-rotavirus IgA and IgG antibodies were higher in piglets from the control group, whereas no difference was detected between sow groups. Phenotype analysis of immune cell antigens revealed significant differences of the CD4 and CD8ß (p < 0.05) as well as CD8α and CD25 (p < 0.1) T cell populations of the probiotic supplemented group compared to the non-supplemented control group. In addition, differences were evident for CD21/MHCII-positive (p < 0.05) and IgM-positive (p < 0.1) B cell populations. The results indicate that probiotic bacteria could have effects on virus shedding in naturally infected pigs, which depend on the virus type. These effects seem to be caused by immunological changes; however, the distinct mechanism of action remains to be elucidated.

Studies on the effect of an Enterococcus faecium probiotic on T cell populations in peripheral blood and intestinal epithelium and on the susceptibility to Salmonella during a challenge infection with Salmonella Typhimurium in piglets.

Although Enterococcus faecium is used as a probiotic feed supplement in animal production, feeding of the bacterium to piglets resulted in a more severe infection with Salmonella Typhimurium DT104 during a challenge experiment. To enlighten the mode of action by which E. faecium affected the piglets' health, we investigated the influence of the probiotic bacterium on the development of intestinal and circulating immune cells during a challenge experiment with S. Typhimurium DT104. To minimise varying impacts of the maternal immunity on the course of infection, only piglets were implemented that descended from Salmonella-free sows. In addition, the potency of purified blood and intraepithelial immune cells to control the growth of Salmonella was tested in vitro. In animals treated with E. faecium, a reduction of intraepithelial CD8alphabeta T cells, reduced circulating CD8alphabeta T cells and a less efficient control of intracellular Salmonella growth, mediated by peripheral blood mononuclear cells, were observed.

Influence of a probiotic strain of Enterococcus faecium on Salmonella enterica serovar Typhimurium DT104 infection in a porcine animal infection model.

The beneficial effects of probiotic Enterococcus spp. in different hosts, such as mice and humans, have previously been reported in several studies. However, studies of large domestic animals, as well as challenge studies with pathogenic microorganisms, are very rare. Here, we investigated the influence of oral treatment of pigs with the probiotic bacterium Enterococcus faecium NCIMB 10415 on Salmonella enterica serovar Typhimurium DT104 infections in weaning piglets. Clinical symptoms, fecal excretion, the organ distribution of Salmonella, and the humoral immune response (immunoglobulin G [IgG], IgM, and IgA levels) in serum were examined. A pool of 89 piglets was randomly divided into probiotic and control groups. The probiotic group received a feed supplement containing E. faecium starting on day 14 postpartum prior to challenge with Salmonella serovar Typhimurium DT104 at 28 days postpartum. After challenge with Salmonella serovar Typhimurium DT104, piglets in both groups showed no severe clinical signs of salmonellosis. However, fecal excretion and colonization of Salmonella in organs were significantly greater in piglets fed E. faecium. Likewise, the humoral immune response against Salmonella (serum IgM and IgA levels) was significantly greater in the probiotic group animals than in control animals. The results of this study suggest that E. faecium NCIMB 10415 treatment enhanced the course of infection in weaning piglets challenged with Salmonella serovar Typhimurium DT104. However, the probiotic treatment also appeared to result in greater production of specific antibodies against Salmonella serovar Typhimurium DT104.

Influence of an Enterococcus faecium probiotic on the development of Peyer's patches B cells in piglets.

To study the interactions between a probiotic bacterium and the host's immune system, we undertook a feeding trial with Enterococcus faecium SF68 (NCIMB 10415). Starting at an age of 1 day, piglets received a daily oral dose of the probiotic bacteria. Immune cells were isolated from the blood and the distal continuous Peyer's patch (PP) of the piglets. While the percentage of B cells in the distal continuous PP was not influenced by the probiotic treatment, an elevated expression of CD1 on Peyer's patch B cells was observed after probiotic treatment. Furthermore, the fraction of CD4-CD8+ cells was decreased in this organ. In blood lymphocyte fractions of the probiotic-treated piglets, the proportion of CD16-positive cells was also diminished, whereas the portion of γδ T cells and CD4-positive T cells increased. The data indicate that early administration of the probiotic Enterococcus faecium can modulate the composition of blood lymphocyte populations in piglets.