Protective Effect of Nanobodies Targeting Sip Protein Against Streptococcus agalactiae Infection in Tilapia (Oreochromis niloticus)

<i>Streptococcus agalactiae</i> (GBS) has emerged as one of the most prevalent bacterial pathogens causing severe economic losses in tilapia aquaculture due to its highly contagious and lethal nature. Nanobodies (Nbs), characterized by their small molecular size, enhanced tissue penetration, high tolerance, and exceptional antigen-binding affinity, represent a promising green alternative to conventional antibiotics. In the present study, the objective was to explore the potential of specific Nbs in the treatment of tilapia GBS disease. We first screened specific Nbs targeting the surface immunogenic (Sip) protein of GBS from a naïve phage display library, and a novel nanobody Nb30 was obtained. Nb30 was expressed in <i>Escherichia coli</i> and purified using the Ni-NTA Agarose column. Indirect ELISA showed that Nb30 had a high affinity against Sip and GBS in vitro. Moreover, Nb30 significantly reduced GBS colonization in the liver, spleen, and brain of GBS-infected tilapia. The survival rate in the control groups was 53%, whereas it was increased to 86% after treatment with 100 mg/kg Nb30. Transcriptome profiling revealed that Nb30 could modulate critical biological processes, including antioxidant defense, immune regulation, amino acid/protein synthesis, and energy metabolism in the liver tissues of GBS-infection tilapia. Notably, the expression levels of antioxidant enzymes (<i>cat</i> and <i>gpx</i>) were significantly up-regulated, and the TLR/MyD88/NF-κB pathway-related genes (<i>tlr5</i>, <i>myd88</i>, <i>irak4</i>, <i>traf6</i>, <i>Rela</i>, and <i>NF-κB2</i>) were significantly down-regulated after treatment with Nb30. Collectively, these findings establish a novel therapeutic strategy for controlling GBS infection in tilapia and provide evidence supporting the application of nanobodies as sustainable alternatives to antibiotics in aquaculture disease management.