Liqun Xia

Background: Fish nocardiosis caused by Nocardia seriolae is a severe bacterial disease in aquaculture, causing significant economic losses. The effectiveness of antibiotics in dealing with nocardiosis induced by N. seriolae is not satisfactory. NsICL is a potential virulence factor in N. seriolae based on its crucial role in the glyoxylate cycle, its identity as a secreted protein, and its established role in virulence and intracellular survival in homologous pathogens such as Mycobacterium tuberculosis . so, the NS-ΔICL deletion strain was constructed to investigate its function. Methods: The NsICL gene was knocked out via homologous recombination to generate the mutant strain NS-ΔICL. Comparative analysis of morphology, growth, and virulence was performed between the mutant and wild-type strains. The NS-ΔICL strain was used to immunize hybrid snakehead ( Channa maculata ♀ × Channa argus ♂), with immune responses evaluated through non-specific immune parameters, specific antibodies, and expression levels of immune-related genes. Protective efficacy was assessed by challenge tests. Results: NS-ΔICL showed reduced virulence (LD ₅₀ = 2.60 × 10 ⁵ cfu/fish) compared to the wild-type strain (LD ₅₀ = 4.32 × 10 ⁴ cfu/fish). Immunized fish exhibited significantly higher levels of non-specific immune factors (AKP, ACP, POD, LZM) and specific IgM antibodies. Immune-related genes ( MHCI α, CD4, IL-8 ) were upregulated in vaccinated fish. Relative percentage survival (RPS) was 77.03% in vaccinated fish after challenge tests, which indicating strong protective efficacy. Conclusion: The NS-ΔICL deletion strain was successfully constructed in this study. It can not only induce humoral and cellular immunity in fish but also effectively protect fish against N. seriolae infection. These results provide a foundation for the development of a live attenuated vaccine for treating fish nocardiosis.

Nocardia seriolae is the primary cause of fish nocardiosis, and it is urgent to develop an effective vaccine for controlling N. seriolae infection in aquaculture. In our previous study, two potential antigenic proteins, glutamate‐cysteine ligase EgtA (EgtA) and hypothetical protein 6747 (hp6747), were determined via the in vivo–induced antigen technique (IVIAT). A DNA vaccine was aimed to be developed using these two in vivo–induced antigens as candidates for ligation with the pcDNA3.1‐myc‐his‐A plasmid, and their immunological effects were evaluated in hybrid snakehead ( Channa maculata ♀ × Channa argus ♂). Multiple epitopes of the EgtA and hp6747 proteins from N. seriolae were predicted on both T cells and B cells, and the transcription of the EgtA and hp6747 genes was found in the spleen, trunk kidney, muscle and liver in the fish after immunisation with pcDNA‐EgtA and pcDNA‐hp6747 DNA vaccines, respectively. Notably, the serum specific antibody (IgM) titres, as well as LYZ, ACP, AKP and SOD activities, were elevated in the vaccinated fish. Meanwhile, quantitative real‐time PCR analysis demonstrated that the expression levels of several immune‐related genes ( TNFα , CD4 , CD8α , IL‐1β , MHCIIα and MHCIα ) were significantly elevated across the aforementioned tissues in the vaccinated fish. Furthermore, they could provide the relative percent survival (RPS) at 61.04% and 43.73% against artificial challenge with N. seriolae in the fish after immunisation with pcDNA‐EgtA and pcDNA‐hp6747 DNA vaccines, respectively. Taken together, using these two in vivo–induced antigens of EgtA and hp6747 for vaccine development was the ideal strategy for controlling fish nocardiosis in aquaculture.

In previous studies, natural killer lysin (NK-lysin) emerged as a crucial antimicrobial peptide (AMP) discharged by NK cells and CTLs. The sequence of NK-lysin was cloned and discovered in some fishes, but its function remains unclear. In our study, we obtained a copy of NK-lysin from the spleen of the healthy clownfish (Amphiprion ocellaris; AoNK-lysin) through cloning and proceeded to investigate its potential functions and activities. The findings showed that the AoNK-lysin gene’s open reading frame (ORF) had a length of 465 base pairs (bp) and encoded 154 amino acids (aa), which included a saposin B domain and six cysteine residues that are highly conserved, forming three intrachain disulfide bonds to carry out antimicrobial activity. The AoNK-lysin gene was widely present in different tissues, with the skin showing the highest expression, followed by the eye, intestine, and muscle. Additionally, the expression of AoNK-lysin was significantly upregulated in the immune organs (spleen, gill, intestine, and head kidney) of A. ocellaris after being challenged by Singapore group iridovirus (SGIV). Furthermore, a 399 base pair cDNA sequence that encodes the fully developed peptide of AoNK-lysin was successfully inserted into a secretion plasmid called pPIC9K. Subsequently, a significant amount of the recombinant AoNK-lysin protein was efficiently manufactured using the Pichia pastoris expression system. The antibacterial test demonstrated that the AoNK-lysin protein significantly suppressed the growth of various pathogens, particularly Streptococcus agalactiae, Streptococcus iniae, Salmonella typhi, Shigella sonnei, Pseudomonas aeruginosa, and Aeromonas caviae. The minimal inhibitory concentration (MIC) was found to be 7.81 μg/mL. Further analysis of antiviral assays showed all the viral mRNA of SGIV to be significantly reduced after AoNK-lysin protein stimuli in FHM cells. Collectively, these discoveries indicate that AoNK-lysin exhibits features of both direct pathogen-killing abilities and inhibited virus replication.