Introduction
Infectious diseases represent a major constraint on sustainable aquaculture production, driving substantial economic losses and raising significant animal welfare concerns (Hadiuzzaman et al., 2022; Vallejos-Vidal et al., 2021). In the absence of broad-spectrum prophylactic measures, the aquaculture sector has increasingly adopted non-antibiotic immunostimulants to enhance host immune competence (Vetvicka & Vetvickova, 2020). Among these, β-1,3/1,6-glucans derived from yeast cell walls (YCW) have attracted particular interest due to their capacity to act as pathogen-associated molecular patterns (PAMPs) and microbial-associated molecular patterns (MAMPs), thereby activating innate immune signalling pathways in both freshwater and marine teleost species (Rodrigues et al., 2020; Machuca et al., 2022). Upon recognition by pattern recognition receptors (PRRs) — including functional homologs of C-type lectin receptors — β-glucans trigger downstream activation of nuclear factor-��B (NF-��B) and the transcriptional upregulation of pro-inflammatory cytokines such as interleukin-1β (IL-1β) and tumour necrosis factor-α (TNF-α) (Rodrigues et al., 2020; Hadiuzzaman et al., 2022). Despite broad evidence of their immunostimulatory efficacy, comparisons between YCW products differing in yeast origin remain limited, and the influence of production method on the quality of the immune response has not been thoroughly characterised.
Materials and Methods
Immune modulatory effects were assessed using the rainbow trout (Oncorhynchus mykiss) intestinal epithelial cell line RTgutGC (passage 81), derived from a distal intestinal segment of an adult female and provided by aQuaTox-Solutions GmbH (Winterthur, Switzerland). Cells were exposed to one of four treatments: (1) exposure medium L-15/ex only (negative control; Neg. CON); (2) lipopolysaccharide (LPS) from Escherichia coli (positive control; Pos. CON); (3) a primary-grown yeast cell wall product (Primary-YCW; TechnoMos��, Biochem GmbH, Lohne, Germany); or (4) a by-product ethanol yeast cell wall preparation (Ethanol-YCW). All treatments were applied at equivalent concentrations under standardised exposure conditions. Following incubation, total RNA was extracted and reverse-transcribed, and relative gene expression of three immune markers — IL-1β, TNF-α, and the Toll-like receptor 4 (TLR4) interactor with leucine-rich repeats (TRIL) — was quantified by quantitative polymerase chain reaction (qPCR). Expression data were normalised to appropriate reference genes and analysed for statistically significant differences between treatment groups (P < 0.05).
Results and Discussion
Both YCW products elicited statistically significant upregulation of IL-1β and TNF-α relative to the negative control (P < 0.05), demonstrating immunostimulatory activity at the intestinal epithelial level. Critically, gene expression levels for both cytokines remained substantially lower than those induced by LPS, indicating that neither product provoked a hyper-inflammatory response. This profile of calibrated immune activation — sufficient to prime innate defence mechanisms without triggering deleterious inflammation — is considered functionally desirable in aquaculture settings, where excessive immune activation incurs energetic costs and compromises productive performance (Hadiuzzaman et al., 2022; Vetvicka & Vetvickova, 2020).A pronounced differential response was observed for TRIL, a TLR4 accessory molecule previously identified in teleost fish and implicated in the modulation of both LPS and viral RNA recognition (Pietretti et al., 2013). The Primary-YCW induced TRIL expression at a level statistically equivalent to LPS stimulation, whereas Ethanol-YCW produced only a modest, albeit significant, upregulation above the negative control. Because elevated TRIL expression enhances PRR sensitivity to circulating PAMPs, a higher level of TRIL induction may confer an earlier and more rapid pathogen detection capacity — an effect analogous to a broad-spectrum, unspecific innate immune priming (Pietretti et al., 2013). Importantly, this TRIL-mediated sensitisation occurred without corresponding amplification of pro-inflammatory cytokine transcription, suggesting that the tested primary-grown YCW decouples heightened pathogen surveillance from overt inflammatory cascades.These findings are consistent with and extend prior reports demonstrating that the immunostimulatory potency of YCW products is contingent on yeast origin and production method (Machuca et al., 2022; Rodrigues et al., 2020). Published data on the pathogen-binding capacity of the mannan oligosaccharide (MOS) fraction further indicate that primary-grown yeast-derived MOS binds approximately 100 % more pathogens than MOS derived from ethanol yeast (Eckart, 2024), corroborating the functional superiority of primary-grown YCW products across multiple immune-relevant parameters.
Acknowledgement
The authors are grateful to the team at aQuaTox-Solutions GmbH for providing the immune challenger assay used to generate the experimental data presented herein, and for their expert technical input and scientific discussions.
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