Dietary Supplementation with Chlorella vulgaris Confers Protection against Streptococcus agalactiae in Nile Tilapia: An NMR-Based Metabolomics Approach

Abstract

Background: Disease outbreaks, particularly those caused by Streptococcus agalactiae, represent a significant challenge in Nile tilapia (Oreochromis niloticus) aquaculture. While Chlorella vulgaris is recognized as a promising immunostimulant due to its rich nutrient profile, the molecular mechanisms underlying its protective effects during a live bacterial challenge require further elucidation. Objective: This study aimed to evaluate the efficacy of a prophylactic diet incorporated with C. vulgaris in enhancing the survival of Nile tilapia challenged with Streptococcus agalactiae and to characterize the associated metabolic signatures using an NMR-based metabolomics approach. Methodology: Juvenile Nile tilapia were fed a commercial diet supplemented with 125 mg/kg body weight of C. vulgaris biomass for 21 days. Each group of fish receiving treatment and the control group consist of six (6) fish and replicated three (3) times and one-way ANOVA was used for statistical analysis. Following the feeding period, the fish were subjected to a bacterial challenge via the immersion method using a 1x10⁸ CFU/mL concentration of S. agalactiae (exceeding the calculated LC₅₀ of 9x10⁷ CFU/mL). Spleen samples were collected on day 29 for ¹H NMR spectroscopy and multivariate data analysis (MVDA), including PCA and OPLS-DA, to identify differentiating biomarkers between control and treated groups. Data were expressed as Mean ± Standard Error of the Mean (SEM). Survival rates were analyzed using the Kaplan-Meier method and compared via the Log-Rank test. For metabolomics data, normalized spectral intensities were subjected to one-way ANOVA followed by Tukey's HSD post-hoc test (q < 0.05). MVDA models (PCA and OPLS-DA) were validated using 7-fold cross-validation (Q2) and a 100-fold permutation test. All univariate statistical analyses were performed using Software, SPSS Version 17.0 (SPSS Inc., Chicago, IL, USA), and multivariate modeling was conducted using Software, SIMCA Umetrics 14.1 (Sartorius Stedim Biotech, Sweden) and an online database, MetaboAnalyst 6.0. Results: The C. vulgaris treatment significantly improved survival rates, with the treated challenged group (CVC) exhibiting a 90% survival rate compared to only 13.3% in the control challenged group (CFC). Metabolomics analysis identified 21 metabolites in the spleen, with significant up-regulation of key biomarkers in C. vulgaris-fed fish, including palmitic acid (11.55-fold), riboflavin (6.51-fold), arginine (2.62-fold), and alanine (2.12-fold). Conversely, S. agalactiae challenge in control fish led to a severe down-regulation of essential metabolites, most notably linoleic acid (33.28-fold reduction) and stearic acid (18.46-fold reduction). This drastic metabolic depletion was effectively mitigated in fish previously fed with C. vulgaris, where metabolite levels remained stable despite the bacterial challenge. Conclusion: Prophylactic supplementation with C. vulgaris at 125 mg/kg bw effectively primes the innate immune system of Nile tilapia, providing robust protection against S. agalactiae infection. The sustained levels of vital amino acids and fatty acids in treated fish suggest that C. vulgaris maintains metabolic homeostasis and immune cell viability, offering a sustainable alternative to traditional disease management in aquaculture.