Genomic insights and vaccine evaluation of a virulent MLST ST234 Bacillus cereus infecting Asian sea bass (Lates calcarifer).

An outbreak of disease in farmed Asian sea bass (Lates calcarifer) was investigated. A Bacillus cereus group strain, SB01, was consistently isolated from internal organs and identified by groEL PCR. Transmission electron microscopy (TEM) revealed rod-shaped cells with prominent flagella. Experimental infection demonstrated dose-dependent mortality with an LD50 of 5.6 × 106 CFU/fish and pathological signs including ascites, splenomegaly, and multi-organ necrosis. Whole-genome sequencing revealed a 5.26 Mb chromosome and a 240 kb plasmid (pSB01) encoding anthrax toxin-related genes, including protective antigen (PA), edema toxin, and a lethal factor-related metalloprotease. Multilocus sequence typing assigned SB01 to B. cereus sequence type (ST) 234. Given that PA is a well-established vaccine target in Bacillus anthracis, its presence and characteristics were examined in ST234 strains. Several amino acid substitutions were identified within PA domain 4 (PD4), corresponding to receptor-binding and major neutralizing epitope regions, compared with B. anthracis, leading to the selection of PD4 as a candidate subunit vaccine. In parallel, the prominent flagella observed by TEM prompted inclusion of flagellin as a secondary antigen. Formalin-killed cells (FKC), recombinant thioredoxin (rTrx), Trx-tagged PD4 (rPD4), and flagellin (rFla) were expressed, purified, formulated with Montanide ISA 763A, and evaluated in vaccination trials. Relative percent survival values were 0% for rTrx, 100% for rPD4 and FKC, and 75.6% for rFla. Vaccination induced early innate immune responses and elevated antigen-specific IgM levels. Overall, SB01 represents a highly virulent ST234 B. cereus strain, and both whole-cell and PD4/flagellin subunit vaccines conferred effective protection in Asian sea bass.