Role of immunosuppression in an antibiotic stewardship intervention and its association with clinical outcomes and antibiotic use: protocol for an observational study (RISC-sepsis).

INTRODUCTION: Sepsis is characterised by a dysregulated immune response to infection, with exaggerated pro-inflammatory and anti-inflammatory responses. A predominant immunosuppressive profile affecting both innate and adaptive immune responses is associated with increased hospital-acquired infection and reduced infection-free survival. While hospital-acquired infection leads to additional antibiotic use, the role of the immunosuppressive phenotype in guiding complex decisions, such as those affecting antibiotic stewardship, is uncertain. This study is a mechanistic substudy embedded within a multicentre clinical and cost-effectiveness trial of biomarker-guided antibiotic stewardship. This mechanistic study aims to determine the effect of sepsis-associated immunosuppression on the trial outcome measures. METHODS AND ANALYSIS: RISC-sepsis is a prospective, multicentre, exploratory, observational study embedded within the ADAPT-sepsis trial. A subgroup of 180 participants with antibiotics commenced for suspected sepsis, enrolled in the ADAPT-sepsis trial, will be recruited. Blood samples will be collected on alternate days until day 7. At each time point, blood will be collected for flow cytometric analysis into cell preservation tubes. Immunophenotyping will be performed at a central testing hub by flow cytometry. The primary outcome measures are monocyte human leucocyte antigen-DR; neutrophil CD88; programmed cell death-1 on monocytes, neutrophils and T lymphocytes and the percentage of regulatory T cells. Secondary outcome measures will link to trial outcomes from the ADAPT-sepsis trial including antibiotic days; occurrence of hospital-acquired infection and length of ICU-stay and hospital-stay. ETHICS AND DISSEMINATION: Ethical approval has been granted (IRAS 209815) and RISC-sepsis is registered with the ISRCTN (86837685). Study results will be disseminated by peer-reviewed publications, presentations at scientific meetings and via patient and public participation groups and social media.

High susceptibility prevalence for F4+ and F18+Escherichia coli in Flemish pigs.

F4 and/or F18 enterotoxigenic Escherichia coli (F4+/F18+ ETEC) are responsible for diarrhea while F18+ verotoxigenic E. coli (F18+ VTEC) cause edema disease in pigs. Both infections can result in severe economic losses, which are mainly the result of the medication, growth retardation and mortality. The susceptibility of piglets to these pathogens is determined by the presence of F4 and F18 receptors (F4R and F18R). Understanding the composition of the susceptibility phenotypes of pigs is useful for animal health and breeding management. This study aimed to determine the prevalence of the F4 ETEC susceptibility phenotypes and F18+E. coli susceptibility among Flemish pig breeds by using the in vitro villous adhesion assay. In this study, seven F4 ETEC susceptibility phenotypes were found, namely A (F4abR+,acR+,adR+; 59.16%), B (F4abR+,acR+,adR-; 6.28%), C (F4abR+,acR-,adR+; 2.62%), D (F4abR-,acR-,adR+; 6.28%), E (F4abR-,acR-,adR-; 24.08%), F (F4abR+,acR-,adR-; 1.05%) and G (F4abR-,acR+,adR-; 0.52%). F4ab and F4ac E. coli showed a stronger degree of adhesion to the intestinal villi (53.40% and 52.88% strong adhesion, respectively), compared to F4ad E. coli (43.46% strong adhesion). Furthermore, the correlation between F4ac and F4ab adhesion was higher (r=0.78) than between F4ac and F4ad adhesion (r=0.41) and between F4ab and F4ad adhesion (r=0.57). For F18+E. coli susceptibility, seven out of 82 pigs were F18R negative (8.54%), but only two of these seven pigs (2.44%) were also negative for F4R. As such, the chance to identify a pig that is positive for a F4 ETEC variant or F18+E. coli is 97.56%. Therefore, significant economic losses will arise due to F4+ and/or F18+E. coli infections in the Flemish pig population due to the high susceptibility prevalence.

Maternal immunity enhances systemic recall immune responses upon oral immunization of piglets with F4 fimbriae.

F4 enterotoxigenic Escherichia coli (ETEC) cause diarrhoea and mortality in piglets leading to severe economic losses. Oral immunization of piglets with F4 fimbriae induces a protective intestinal immune response evidenced by an F4-specific serum and intestinal IgA response. However, successful oral immunization of pigs with F4 fimbriae in the presence of maternal immunity has not been demonstrated yet. In the present study we aimed to evaluate the effect of maternal immunity on the induction of a systemic immune response upon oral immunization of piglets. Whereas F4-specific IgG and IgA could be induced by oral immunization of pigs without maternal antibodies and by intramuscular immunization of pigs with maternal antibodies, no such response was seen in the orally immunized animals with maternal antibodies. Since maternal antibodies can mask an antibody response, we also looked by ELIspot assays for circulating F4-specific antibody secreting cells (ASCs). Enumerating the F4-specific ASCs within the circulating peripheral blood mononuclear cells, and the number of F4-specific IgA ASCs within the circulating IgA(+) B-cells revealed an F4-specific immune response in the orally immunized animals with maternal antibodies. Interestingly, results suggest a more robust IgA booster response by oral immunization of pigs with than without maternal antibodies. These results demonstrate that oral immunization of piglets with F4-specific maternal antibodies is feasible and that these maternal antibodies seem to enhance the secondary systemic immune response. Furthermore, our ELIspot assay on enriched IgA(+) B-cells could be used as a screening procedure to optimize mucosal immunization protocols in pigs with maternal immunity.

Variation in 12 porcine genes involved in the carbohydrate moiety assembly of glycosphingolipids does not account for differential binding of F4 Escherichia coli and their fimbriae.

BACKGROUND: Glycosphingolipids (GSLs) are important membrane components composed of a carbohydrate structure attached to a hydrophobic ceramide. They can serve as specific membrane receptors for microbes and microbial products, such as F4 Escherichia coli (F4 ETEC) and isolated F4 fimbriae. The aim of this study was to investigate the hypothesis that variation in genes involved in the assembly of the F4 binding carbohydrate moiety of GSLs (i.e. ARSA, B4GALT6, GAL3ST1, GALC, GBA, GLA, GLB1, GLB1L, NEU1, NEU2, UGCG, UGT8) could account for differential binding of F4 ETEC and their fimbriae. RESULTS: RT-PCR could not reveal any differential expression of the 12 genes in the jejunum of F4 receptor-positive (F4R(+)) and F4 receptor-negative (F4R(-)) pigs. Sequencing the complete open reading frame of the 11 expressed genes (NEU2 was not expressed) identified 72 mutations. Although some of them might have a structural effect, none of them could be associated with a F4R phenotype. CONCLUSION: We conclude that no regulatory or structural variation in any of the investigated genes is responsible for the genetic susceptibility of pigs towards F4 ETEC.

Refined candidate region for F4ab/ac enterotoxigenic Escherichia coli susceptibility situated proximal to MUC13 in pigs.

F4 enterotoxigenic Escherichia coli (F4 ETEC) are an important cause of diarrhea in neonatal and newly-weaned pigs. Based on the predicted differential O-glycosylation patterns of the 2 MUC13 variants (MUC13A and MUC13B) in F4ac ETEC susceptible and F4ac ETEC resistant pigs, the MUC13 gene was recently proposed as the causal gene for F4ac ETEC susceptibility. Because the absence of MUC13 on Western blot from brush border membrane vesicles of F4ab/acR+ pigs and the absence of F4ac attachment to immunoprecipitated MUC13 could not support this hypothesis, a new GWAS study was performed using 52 non-adhesive and 68 strong adhesive pigs for F4ab/ac ETEC originating from 5 Belgian farms. A refined candidate region (chr13: 144,810,100-144,993,222) for F4ab/ac ETEC susceptibility was identified with MUC13 adjacent to the distal part of the region. This candidate region lacks annotated genes and contains a sequence gap based on the sequence of the porcine GenomeBuild 10.2. We hypothesize that a porcine orphan gene or trans-acting element present in the identified candidate region has an effect on the glycosylation of F4 binding proteins and therefore determines the F4ab/ac ETEC susceptibility in pigs.

Effect of fatty acid composition of the sow diet on the innate and adaptive immunity of the piglets after weaning.

This study investigated whether the immunocompetence of piglets at weaning was modulated by including different sources of n-3 polyunsaturated fatty acids (PUFA) in the maternal diet. From day 73 of gestation until weaning at 4 weeks, 32 pregnant sows were fed a palm oil-based diet (control group) or a diet including 1% linseed oil (C18:3n-3), 1% echium oil (C18:3n-3, C18:4n-3, C18:3n-6) or 1% fish oil (C20:5n-3, C22:6n-3). It was hypothesized that each diet would differently affect immune function through effects such as specific eicosanoid production. Piglets were fed a conventional diet without added n-3 PUFA from weaning until day 35 post-weaning. At weaning and 21 days post-weaning, four piglets per litter were immunized with bovine thyroglobulin. Blood samples were taken from weaning until day 35 post-weaning to determine thyroglobulin-specific antibodies, serum amyloid A (SAA) concentration and fatty acid composition. The fatty acid composition of the maternal diets was reflected in the plasma and red blood cells of the weaned piglets. The onset of the thyroglobulin-specific IgM response differed between dietary groups, with a delay in response for piglets from sows fed the fish oil diet. No significant dietary effects were observed on the thyroglobulin-specific IgG and IgA titres or on SAA concentrations in the piglet serum. Including n-3 PUFA in the maternal diet at the concentrations used in the present study had no major effects on the adaptive and innate immunity of the piglets after weaning.

Local and systemic immune responses in pigs intramuscularly injected with an inactivated Mycoplasma hyopneumoniae vaccine.

The immune response induced by intramuscular administration of a commercial inactivated Mycoplasma hyopneumonie whole-cell vaccine (Suvaxyn(®)MH One) was investigated in conventional M. hyopneumoniae-free pigs. The animals were assigned randomly to two groups: non-vaccinated and vaccinated. Pigs in the vaccinated group were injected intramuscularly with the vaccine at 7 days of age, whereas non-vaccinated pigs received physiological saline solution (PBS). Pigs were euthanized and necropsied at 30, 36 and 58 days of age. Blood, bronchoalveolar lavage (BAL) fluid, spleen, lung and bronchial lymph nodes (BLN) were collected. Serum and BAL fluid were tested for the presence of antibodies by ELISA. Monomorphonuclear cells from the peripheral blood and tissues were isolated to quantify the T cell subsets by flow cytometry, and cytokine production by ELIspot and ELISA. Antibodies against M. hyopneumoniae were detected in serum of most vaccinated pigs at 30 days of age. M. hyopneumoniae specific IgG, IgM and IgA were detected in BAL fluid from vaccinated animals, but not from control animals. Significantly higher numbers of IL-12 secreting cells were observed in the lung at day 58 in the vaccinated than in the non-vaccinated group (p<0.05). The number of IL-10 secreting cells from BLN was also higher in the vaccinated group at day 58 (p<0.05). After restimulation in vitro, lymphocytes from BLN and lungs secreted significantly higher levels of IL-12 in the vaccinated group at day 58. These results show that the vaccine induced both systemic and mucosal cellular and humoral immune responses.

ETEC vaccination in pigs.

Enterotoxigenic Escherichia coli (ETEC) remain an important cause of neonatal and post-weaning diarrhoea in pigs. In general, neonatal infections can be prevented effectively by passive colostral and lactogenic immunity obtained by vaccination of the sow. In this respect, several maternal vaccines are on the market. These are applied mainly parenterally in the pregnant sow. However at weaning, lactogenic protection disappears. Strains involved in post-weaning diarrhoea mostly express F4 or F18 fimbriae. These fimbriae are important virulence factors since they allow the bacteria to bind to specific receptors on small intestinal enterocytes, resulting in colonization and subsequently the secretion of enterotoxins causing diarrhoea. Consequently, an active mucosal immunity, in which the local production of F4- and/or F18-specific sIgA plays an important role, is required to protect pigs against post-weaning diarrhoea. This review aims to give an overview of the immunization strategies applied in the pig model to prevent post-weaning diarrhoea caused by F4- and/or F18- positive ETEC in pigs. These include the use of oral live and subunit vaccines, encapsulation strategies and parenteral immunization.

Influence of polymer hydrolysis on adjuvant effect of Gantrez®AN nanoparticles: implications for oral vaccination.

The adjuvant effect of methylvinylether-co-maleic anhydride (Gantrez®AN) nanoparticles was investigated during oral vaccination of mice with F4 adhesins of F4-positive Escherichia coli. To differentiate whether the adjuvant effect originated from a nanoparticle effect or a polymer effect, 20 µg F4 was administered as slightly crosslinked F4-containing nanoparticles (g(F4)(0.01)) or as F4 mixed with slightly crosslinked pure nanoparticles (F4+g(0.01)). The F4-specific immune response was reduced using F4-containing nanoparticles due to complete shielding of F4, whereas oral administration of F4+g(0.01) increased the level of F4-specific antibody-secreting cells (ASC) in the spleen. When repeating the vaccination study after 6months using freshly prepared nanoparticles, the adjuvant effect of F4+g(0.01) was lost due to an altered polymer reactivity caused by partial hydrolysis of anhydride groups of Gantrez®AN. Combining F4 with nanoparticles stabilised with a higher crosslinker amount during nanoparticle synthesis (F4+g(0.22)) could overcome the effect of partial polymer hydrolysis, as higher levels of ASC were detected. Hence, an in-depth characterisation of the Gantrez®AN polymer is required as stability issues can alter its biological effect during oral vaccination.

Adjuvant effect of Gantrez®AN nanoparticles during oral vaccination of piglets against F4+enterotoxigenic Escherichia coli.

In this study, the adjuvanticity of methylvinylether-co-maleic anhydride (Gantrez(®)AN) nanoparticles (NP) was investigated in an oral immunisation experiment of pigs against F4+enterotoxigenic Escherichia coli (F4+ETEC). In addition, Wheat Germ Agglutinin (WGA)-coating of the nanoparticles was tested for enterocyte-targeting. Pigs were either vaccinated with F4 fimbriae, F4 encapsulated in Gantrez(®)AN NP, F4 encapsulated in Gantrez(®)AN NP coated with WGA or F4 fimbriae mixed with empty Gantrez(®)AN NP. Only vaccination with the combination of F4 mixed with empty Gantrez(®)AN NP improved protection against F4+ETEC infection. In addition, vaccination with this formulation also resulted in an F4-specific serum antibody response prior to F4+ETEC challenge. Encapsulation of F4 in Gantrez(®)AN NP only raised the serum antibody response after F4+ETEC challenge compared to soluble F4, but did not improve protection, whereas WGA-coating almost completely abolished the serum antibody response. These data indicate that nanoparticle effects after F4 encapsulation were of lesser importance for the adjuvant effect of Gantrez(®)AN NP, contrarily to the reactivity of the Gantrez(®)AN polymer used to prepare the nanoparticles.

Influence of reaction medium during synthesis of Gantrez AN 119 nanoparticles for oral vaccination.

Two synthesis methods of poly(methyl vinyl ether-co-maleic anhydride) (Gantrez AN 119) nanoparticles (NP) (used for oral vaccination) were compared. Wheat germ agglutinin (WGA) was used as ligand to enhance the bioadhesive properties of NP and beta-galactosidase as antigen. The first method encapsulated beta-galactosidase in NP by co-precipitation in an acetone/water mixture containing 44% acetone. In the second method, antigen addition occurred in 100% acetone. To improve stability, NP were crosslinked with 1,3-diaminopropane. The stability of WGA-conjugated NP with encapsulated antigen diminished at lower pH and when decreasing the amount of crosslinker. The binding type between WGA and polymer depended on the synthesis method: predominantly ionic bonds were formed using the 44% acetone method, whereas synthesis via the 100% acetone method resulted in covalent bonds. The biological activity of the WGA coating, evaluated via a pig gastric mucin binding test, was lower in NP prepared via the 100% acetone method. No release of native antigen was detected after hydrolysis of NP, due to the covalent antigen binding during antigen encapsulation and the high reactivity of the polymer. Moreover, the mucosal irritation capacity was evaluated upon nanoparticle hydrolysis using a slug mucosal irritation assay. Herein, hydrolysed NP of the 44% acetone method were classified as mild irritative.

Molecular cloning and characterization of the porcine prostaglandin transporter (SLCO2A1): evaluation of its role in F4 mediated neonatal diarrhoea.

BACKGROUND: Because prostaglandins are involved in many (patho)physiological processes, SLCO2A1 was already characterized in several species in an attempt to unravel specific processes/deficiencies. Here, we describe the molecular cloning and characterization of the porcine ortholog in order to evaluate its possible involvement in F4 enterotoxigenic E. coli mediated neonatal diarrhoea, based on a positional candidate gene approach study. RESULTS: Porcine SLCO2A1 is organized in 14 exons, containing an open reading frame of 1935 bp, encoding a 12-transmembrane organic anion cell surface transporter of 644 aa. The -388 to -5 upstream region comprises a (CpG)48 island containing a number of conserved promoter elements, including a TATA box. A potential alternative promoter region was found in the conserved -973 to -700 upstream region. No consensus polyadenylation signal was discovered in the 3' UTR. Repeat sequences were found in 15% of all the non coding sequences.As expected for a multifunctional protein, a wide tissue distribution was observed. mRNA expression was found in the adrenal gland, bladder, caecum, colon (centripetal coil/centrifugal coil), diaphragm, duodenum, gallbladder, heart, ileum, jejunum, kidney, liver, longissimus dorsi muscle, lung, lymph node, mesenterium, rectum, spleen, stomach, tongue and ureter, but not in the aorta, oesophagus and pancreas.The promoter region and the exons (including the splice sites) of SLCO2A1 were resequenced in 5 F4ab/ac receptor positive and 5 F4ab/ac receptor negative pigs. Two silent and 2 missense (both S --> L at position 360 and 633) mutations were found, but none was associated with the F4ab/ac receptor phenotype. In addition, no phenotype associated differential mRNA expression or alternative/abberant splicing/polyadenylation was found in the jejunum. CONCLUSION: The molecular cloning and characterization of porcine SLCO2A1 not only contributes to the already existing knowledge about the transporter in general, but enables studies on porcine prostaglandin related processes/deficiencies as patient and/or model. Here we examined its possible involvement as receptor in F4 enterotoxigenic E. coli mediated neonatal diarrhoea. Because no phenotype associated differences could be found in the gene sequence nor in its jejunal transcription profile of F4ab/ac receptor positive/negative pigs, SLCO2A1 can most likely be excluded as receptor for F4 bacteria.