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Despite advancements in diagnosis and control, Aeromonas infections are considered the leading cause of economic aquaculture loss. In this study, to enhance DNA vaccine efficacy against Aeromonas infections, a fused DNA fragment (1504 bp) of the OmpAI gene from Aeromonas veronii (A. veronii) combined with the C5–I gene from the common carp was generated with splicing by overlapping PCR (SOE-PCR) and expressed in Lactobacillus casei strain CC16. Protein C5–I served as a molecular adjuvant for the antigen OmpAI. Two types of fusion antigens were developed (anchored and secretory). Generally, anchored-type antigens are more effective in inducing immune responses in fish than secretory antigens. Western blot analysis showed that the bands of both antigens were present at 58 kDa. After oral immunization, both DNA vaccines enhanced the serum levels of AKP, ACP, SOD and LZM in immunized carp; the genes IL-10, IL-1β, TNF-α, and IFN-γ in the heart, liver, spleen, head kidney, and intestinal tract were upregulated; and a stronger phagocytic response was triggered in immunized fish. In addition, common carp administered the fused antigens were more protected from Aeromonas challenge (60–73.3% protection). Recombinant Lactobacillus bacteria expressing the fused protein showed a greater propensity for colonization in the intestinal tract in immunized fish than in controls. Here, we provide a promising approach to improve DNA vaccine immunogenicity for protecting common carp from A. veronii infections.

Aeromonas veronii is an emerging aquatic pathogen causing hemorrhagic septicemia in humans and animals. Probiotic is an effective strategy for controlling enteric infections through reducing intestinal colonization by pathogens. Here we report that the consumption of Bacillus velezensis regulated the intestinal innate immune response and decreased the degree of intestinal inflammation damage caused by the A. veronii in Crucian carp. In this study, we isolated four strains of B. velezensis, named C-11, S-22, L-17 and S-14 from apparently healthy Crucian carp, which exerted a broad-spectrum antimicrobial activity inhibiting both Gram-positive and Gram-negative bacteria especially the fish pathogens. B. velezensis isolates showed typical Bacillus characteristics by endospore staining, physiological and biochemical test, enzyme activity analysis (amylase, protease, and lipase), and molecular identification. Here, Bacillus-containing dietary was orally administrated to Crucian carp for 8 weeks before A. veronii challenge. Immunological parameters and the expression of immune-related genes were measured at 2, 4, 6, 8, and 10 weeks post-administration. The results showed that B. velezensis was found to promote the increase in the phagocytic activities of peripheral blood leukocytes (PBLs) and head kidney leukocytes (HKLs), as well as the increase in interleukin 1β (IL-1β), IL-10 and tumor necrosis factor α (TNF-α) concentration of serum. Lysozyme levels (113.76 U/mL), ACP activity (25.32 U/mL), AKP activity (130.08 U/mL), and SOD activity (240.63 U/mL) were maximum (P < 0.05) in the B. velezensis C-11 treated group at 8 week. Our results showed that Crucian carp fed with the diet containing B. velezensis C-11 and S-22 developed a strong immune response with significantly higher (P < 0.05) levels of IgM in samples of serum, mucus of skin and intestine compared to B. velezensis L-17 and S-14 groups. Moreover, B. velezensis spores appeared to show no toxicity and damage in fish, which could inhabit the gut of Crucian carp. B. velezensis restrained up-regulation of pro-inflammation cytokines (IL-1β, IFN-γ, and TNF-α) mRNA levels in the intestine and head kidney at final stage of administration, and the expression of IL-10 was increased throughout the 10-week trial. A. veronii infection increased the population of inflammatory cells in the intestinal villi in the controls. In contrast, numerous goblet cells and few inflammatory cells infiltrated the mucosa in the B. velezensis groups after challenge with A. veronii. Compared with A. veronii group, B. velezensis could safeguard the integrity of intestinal villi. The highest post-challenge survival rate (75.0%) was recorded in B. velezensis C-11 group. The present data suggest that probiotic B. velezensis act as a potential gut-targeted therapy regimens to protecting fish from pathogenic bacteria infection.IMPORTANCEIn this work, four Bacillus velezensis strains isolated from apparently healthy Crucian carp, which exhibited a broad-spectrum antibacterial activity especially the fish pathogens. Administration of B. velezensis induced the enhancement of the intestinal innate immune response through reducing intestinal colonization by pathogens. The isolation and characterization would help better understand probiotic can be recognized as an alternative of antimicrobial drugs protecting human and animal health.