Presence or absence of mucin specific binding of pathogens.

Presence or absence of mucin specific binding of pathogens.</p

Effect of fluid velocity on bacterial binding to mucins.

A and B. A. salmonicida binding to Atlantic salmon skin and distal intestinal mucins increased with growing linear velocity of the surrounding liquid. A. salmonicida bound with higher avidity to skin mucins at a fluid velocity of 2 cm/s (p≤0.05; n = 7) and to distal intestinal mucins at 1.5 cm/s and 2 cm/s fluid velocity (p≤0.05 and p≤0.01, respectively; n = 7). C. A. hydrophila binding to skin mucins was reduced at 2 cm/s fluid velocity (p≤0.05; n = 7). D. A. hydrophila binding was higher at 1.5 cm/s and 2 cm/s fluid velocity compared to the static environment (p≤0.05 and p≤0.01, respectively; n = 5). Data points represent mean±SEM of biological replicates. The results were reproduced twice. Statistics: One-Way ANOVA with Dunnet´s post-hoc test (compared to 0 cm/s velocity).</p

Standard curves for calculation of adherent bacteria from luminescent signals.

Luminescence produced by A. hydrophila (A), V. harveyi (B), M. viscosa (C) and Y. ruckeri (D) using the Bac Titer-Glo™ reagent. The standard curves allow for transformation of Signal/noise ratios of luminescent signals into number of adhered bacteria/cm2 surface of the plate well using linear regression. The data points are expressed as mean±SEM and are technical replicates (due to low variation between replicates, the error bars are small, and what looks like symbols in the graph are the error bars). CFU = colony forming unit.</p

Qualitative analysis of binding specificity of pathogens to Atlantic salmon mucins.

A. Mucins were isolated by isopycnic density gradient centrifugation, and fractions were collected from the bottom of the tube. Fractions were analyzed for carbohydrate content (glycan), DNA content and density. The glycan peak at fractions 8–11 corresponds to the mucins, and for the experiments presented in Figs 3 and 4, the fractions were pooled based on the glycan peak for each sample. B.-D. Examples of pathogen binding patterns to gradient fractions. B. Low avidity, mucin-specific binding of M. viscosa to an Atlantic salmon skin sample. The binding signal is low but exceeds that of the control and follows the mucin glycan peak (fractions 9–11 in this sample). C. High avidity, mucin-specific binding of A. hydrophila to Atlantic salmon gill sample. The binding follows the mucin glycan peak (fractions 7–10), although avidity appears stronger to a low density glycoform of the main mucin peak (i.e. the binding curve is shifted slightly to the right of the main mucin peak). D. Absence of Y. ruckeri binding to a pyloric cecal sample. The binding signal to the sample is lower than to the non-mucin control and does not follow the mucin glycan peak (fractions 9–11). Binding is expressed as signal/noise luminescence. The horizontal dashed lines denote the binding signal of bacteria to the plastic well. The results of all these qualitative binding analyses are summarized in Table 1. Abbreviations: EU count = europium count; Lum = luminescence, expressed as signal/noise (S/N).</p

Quantitative analysis of pathogen binding to Atlantic salmon mucins.

Mucin containing fractions from individual fish and tissue sites were pooled according to the method shown in Fig 1A (n = 5 for each tissue). Pathogen binding to each of these 25 samples was analyzed using the Bac Titer-Glo method, and the luminescence signals were transformed to CFU/cm2 according to standard curves for each pathogen (Fig 2) to allow comparison of binding levels between pathogens. A. A. hydrophila binding to gill mucins was higher compared to the proximal intestinal mucins: p≤0.05; n = 5). B. V. harveyi bound with no distinguishable organ preference (p = n.s.). C. The level of M. viscosa binding differed between mucin groups (distal intestine vs. skin and gill: p≤0.05). D. Y. ruckeri bound to proximal and distal intestinal mucins more than to gill mucins (p≤0.01 and p≤0.05). Bars denote median ± interquartile range of biological replicates, after subtracting the background signal. The results were reproduced twice. Statistics: Kruskal-Wallis test by ranks with Dunn´s Post Hoc test to compare binding to mucins from different epithelial sites. The numerical p values on the graphs show the result of the test, without the post hoc test. Abbreviations: Pyloric = pyloric cecal mucins; Proximal = proximal intestinal mucins; Distal = distal intestinal mucins.</p

Data_Sheet_1_Improving Chicken Responses to Glycoconjugate Vaccination Against Campylobacter jejuni.pdf

Campylobacter jejuni is a common cause of diarrheal disease worldwide. Human infection typically occurs through the ingestion of contaminated poultry products. We previously demonstrated that an attenuated Escherichia coli live vaccine strain expressing the C. jejuni N-glycan on its surface reduced the Campylobacter load in more than 50% of vaccinated leghorn and broiler birds to undetectable levels (responder birds), whereas the remainder of the animals was still colonized (non-responders). To understand the underlying mechanism, we conducted three vaccination and challenge studies using 135 broiler birds and found a similar responder/non-responder effect. Subsequent genome-wide association studies (GWAS), analyses of bird sex and levels of vaccine-induced IgY responses did not correlate with the responder versus non-responder phenotype. In contrast, antibodies isolated from responder birds displayed a higher Campylobacter-opsonophagocytic activity when compared to antisera from non-responder birds. No differences in the N-glycome of the sera could be detected, although minor changes in IgY glycosylation warrant further investigation. As reported before, the composition of the microbiota, particularly levels of OTU classified as Clostridium spp., Ruminococcaceae and Lachnospiraceae are associated with the response. Transplantation of the cecal microbiota of responder birds into new birds in combination with vaccination resulted in further increases in vaccine-induced antigen-specific IgY responses when compared to birds that did not receive microbiota transplants. Our work suggests that the IgY effector function and microbiota contribute to the efficacy of the E. coli live vaccine, information that could form the basis for the development of improved vaccines targeted at the elimination of C. jejuni from poultry.</p