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Background <p><i>Streptococcus agalactiae</i> (GBS) causes severe tilapia streptococcosis with heavy aquaculture losses; existing vaccines have administration or efficacy limitations. This study used CRISPR-Cas9 to construct recombinant <i>Escherichia coli</i> DH5α-ORF4-GFP (targeting GBS <i>scpB</i> gene ORF4 fragment), optimized tilapia immersion immunization doses/frequencies, and evaluated the vaccine’s protective efficacy, biosafety and regulatory effects via multi-dimensional assays.</p> Results <p>The optimal regimen was single immersion at 1.5 × 10<sup>4</sup> CFU/mL, with a maximum RPS of 73.13% and stable 65.79% in validation. Immunized tilapia showed elevated immune indices (161.40% higher platelets) and numerical increases in globulin, normal liver/kidney function, and improved oxidative stress resistance with no tissue damage. The vaccine did not alter intestinal microbial richness but modulated community structure, enriching beneficial taxa such as Alphaproteobacteria, suggesting a potential interaction between vaccination and gut microbiota that may contribute to enhanced host defense.</p> Conclusions <p>In conclusion, this study successfully developed an effective and safe genetically engineered vaccine against GBS in tilapia. The precise CRISPR-Cas9-mediated construction strategy and confirmed immune protective effect provide a novel technical approach for controlling this disease in aquaculture and offer important references for the development of related genetically engineered vaccines.</p>

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CRISPR-Cas9-mediated construction of a Streptococcus agalactiae vaccine for tilapia and evaluation of its protective efficacy

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年6月1日

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  • Mianlong Huang,
  • Xiufang Li,
  • Taian Pan,
  • Donghai Wu,
  • Gonghe Li,
  • Wende Wu

摘要

Background

Streptococcus agalactiae (GBS) causes severe tilapia streptococcosis with heavy aquaculture losses; existing vaccines have administration or efficacy limitations. This study used CRISPR-Cas9 to construct recombinant Escherichia coli DH5α-ORF4-GFP (targeting GBS scpB gene ORF4 fragment), optimized tilapia immersion immunization doses/frequencies, and evaluated the vaccine’s protective efficacy, biosafety and regulatory effects via multi-dimensional assays.

Results

The optimal regimen was single immersion at 1.5 × 104 CFU/mL, with a maximum RPS of 73.13% and stable 65.79% in validation. Immunized tilapia showed elevated immune indices (161.40% higher platelets) and numerical increases in globulin, normal liver/kidney function, and improved oxidative stress resistance with no tissue damage. The vaccine did not alter intestinal microbial richness but modulated community structure, enriching beneficial taxa such as Alphaproteobacteria, suggesting a potential interaction between vaccination and gut microbiota that may contribute to enhanced host defense.

Conclusions

In conclusion, this study successfully developed an effective and safe genetically engineered vaccine against GBS in tilapia. The precise CRISPR-Cas9-mediated construction strategy and confirmed immune protective effect provide a novel technical approach for controlling this disease in aquaculture and offer important references for the development of related genetically engineered vaccines.