Beta-Glucan Effects on Vaccine Responses and Innate Immunity in Layers

The product tested is a purified yeast fraction which is highly concentrated in β-1.3/1.6 glucans (BG). β-1.3/1.6 glucans stimulate the immune system by triggering the Dectin-1 receptors located on the surface of phagocytes, stimulating a release of cytokines. Cytokines induce different immune pathways causing an immune response. When phagocytes that have previously been exposed to β-1.3/1.6 glucans, their immune responses toa pathogen is faster and stronger than innate cells that haven’t encountered BG. This innate memory improvement is called trained immunity.

The aim of this study was to evaluate the effect of supplementing laying birds from D0 with beta-glucan product for a period of 8 weeks on inducing the training immunity from antigen presenting cells (APCs) in certain tissues such as the spleen and intestine as well as evaluating the enhancement of inflammatory cytokine production by the cells from these tissues. Having better priming APC should lead to an improvement of adaptive immune response such as T and B cells responses. To confirm this hypothesis antigen-specific immunoglobulins for Salmonella, Infectious Bronchitis (IB) as well as Newcastle Disease (ND) were evaluated in test and control birds using ELISA assay and a T cell was performed.

This trial, which involved 47 Lohmann Brown layers, ran from Day 1 to 23 weeks of age. 27 birds were kept as a control (C) and 20 were given feed supplemented with 125 g/T of the betaglucan product. Blood samples were taken at 3, 6, 9, and 23 weeks old, to assess antigenspecific immunoglobulins quantification by ELISA. Tissue samples (spleen and intestine) were collected at 3, 6, and 9 weeks old, to select different types of Antigen Presenting Cells (APC). This was done by flow cytometry. TNF-alpha cytokine relative expression was also evaluated. Birds from all groups were subjected to a commercially used vaccination regime (Table 2). All vaccinations were prescribed by a veterinarian and no on farm medicinal treatments such as anti-inflammatories or anti-microbials were rendered after arrival of chicks on the farm.

At specified ages, blood was collected from assigned chickens in designated groups. Antibodies were determined by HI and ELISA methods. At sampling days, spleen and ileum were individually collected in small containers containing PBS at approximately 4 °C. Sera were also collected form each bird. Tissue samples were collected from all treatment groups on the same day. After cell isolation, APCs were quantified using a flow cytometer and gating on live CD45+ (immune cells), CD3- (T cells), Bu1- (B cells), MHC II+ (ID card specifically expressed on APC). Cells isolated were also stimulated with LPS for 24h to quantify the trained immunity ability of those cells.

In a second set of experiment, immune cells isolated from blood were stimulated using a specific antigen lysate from Newcastle disease (ND), or infectious bronchitis (ID) or Salmonella. Data analysis was performed using R (version 3.2.5) and data was collected on body weight (BW), average daily gain (ADG), daily feed intake (DFI) and feed conversion ratio (FCR). This data was analyzed using a linear regression model with treatment group as a fixed effect. Investigational groups were compared to the negative control group as a standard. Statistical significance was assessed at P < 0.05.

Immune results were analysed with graph pad prism software using a Mann-Whitney nonparametric test or a Kruskal-Wallis test (one-way ANOVA analysis) was used to compare significance between groups according to the data set. A p< 0.05 was considered statistically significant.

Flow cytometry analysis showed a numerical increase of the phagocytes in the spleen and in the intestine after 6 weeks of beta-glucan feed supplementation compared to the control group indicating that APCs in animal fed with the beta-glucan product are able to perform immunity have been significatively increased in the intestine.

The immune cells isolated from the intestine of animals fed with the beta-glucan product at 125g/t showed a higher production of TNFɑ only during the LPS challenge compared to immune cells isolated from the control group.

Flow cytometry analysis showed a numerical increase of APC in the spleen at 3 and 6 weeks old, and a significant increase of the same cells in the intestine of beta-glucan treated animals at 6 weeks (see graph 1). Analyzing the same tissues, the relative expression of TNF-alpha inflammatory cytokine, produced by immune cells, was evaluated and increased after a LPS challenge in the beta-glucan treated animals (see graph 2).

Serological analysis by ELISA showed an increase of antibodies in the beta-glucan group (see graph 3) specific to Salmonella at 9 and 23 weeks old; to Infectious Bronchitis virus (IBV) at 9 weeks, and to Newcastle Disease virus (NDV) at 23 weeks. Supplementation with the beta-glucan product from day 0 trained the immune system to react stronger to a pathogenic challenge, such as LPS, or to vaccinations with higher antibody titres. This effect is important in preparing the immune system to react better to vaccinations which are given before egg production.

Trial results show that by training the immune system, the beta-glucan product helps improve innate immunity with more Antigen Presenting Cells and more inflammatory cytokine, in response to a LPS challenge. It also helps improve adaptive immunity, creating more specific antibody titres at the beginning of egg production. Layers are then better protected against viral diseases and bacterial contamination during their egg production period.