Leukocyte-derived chemotaxin-2 (LECT2) is a multifunctional immunoregulator that plays several pivotal roles in the host\'s defense against pathogens. This study aimed to elucidate the specific functions and mechanisms of LECT2 (CaLECT2) in the northern snakehead (Channa argus) during infections with pathogens such as Nocardia seriolae (N. seriolae). We identified CaLECT2 in the northern snakehead, demonstrating its participation in the immune response to N. seriolae infection. CaLECT2 contains an open reading frame (ORF) of 459 bp, encoding a peptide of 152 amino acids featuring a conserved peptidase M23 domain. The CaLECT2 protein shares 62%-84 % identities with proteins from various other fish species. Transcriptional expression analysis revealed that CaLECT2 was constitutively expressed in all examined tissues, with the highest expression observed in the liver. Following intraperitoneal infection with N. seriolae, CaLECT2 transcription increased in the spleen, trunk kidney, and liver. In vivo challenge experiments showed that injecting recombinant CaLECT2 (rCaLECT2) could protect the snakehead against N. seriolae infection by reducing bacterial load, enhancing serum antibacterial activity and antioxidant capacity, and minimizing tissue damage. Moreover, in vitro analysis indicated that rCaLECT2 significantly enhanced the migration, respiratory burst, and microbicidal activity of the head kidney-derived phagocytes. These findings provide new insights into the role of LECT2 in the antibacterial immunity of fish.

Persistent nocardiosis has prompted exploration of the effectiveness of heterologous approaches to prevent severe infections. We have previously reported the efficacy of a nucleic acid vaccine in protecting groupers from highly virulent Nocardia seriolae infections. Ongoing research has involved the supplementation of recombinant cholesterol oxidase (rCho) proteins through immunization with a DNA vaccine to enhance the protective capacity of orange-spotted groupers. Recombinant rCho protein exhibited a maturity and biological structure comparable to that expressed in N. seriolae, as confirmed by Western blot immunodetection assays. The immune responses observed in vaccinated groupers were significantly higher than those observed in single-type homologous vaccinations, DNA or recombinant proteins alone (pcD:Cho and rCho/rCho), especially cell-mediated immune and mucosal immune responses. Moreover, the reduction in N. seriolae occurrence in internal organs, such as the head, kidney, and spleen, was consistent with the vaccine\'s efficacy, which increased from approximately 71.4 % to an undetermined higher percentage through heterologous vaccination strategies of 85.7 %. This study underscores the potential of Cho as a novel vaccine candidate and a heterologous approach for combating chronic infections such as nocardiosis.

The complement system is pivotal in innate immune defense, with Complement 1qb (C1qb) playing a key role in recognizing immune complexes and initiating the classical pathway. In this research, we cloned the full-length cDNA of silver pomfret (Pampus argenteus) c1qb and demonstrated its role in mediating defense responses against Nocardia seriolae (N. seriolae) infection, which notably causes significant economic losses in the aquaculture industry. Our investigation revealed that N. seriolae infection led to tissue damage in fish bodies, as observed in tissue sections. Subsequent analysis of differential genes (DEGs) in the transcriptome highlighted genes linked to apoptosis and inflammation. Through experiments involving overexpression and interference of c1qb in vitro, we confirmed that c1qb could suppress N. seriolae-induced apoptosis and inflammation. Moreover, overexpression of c1qb hindered N. seriolae invasion, and the purified and replicated C1qb protein displayed antimicrobial properties. Additionally, our study unveiled that overexpression of c1qb might stimulate the expression of membrane attack complexes (MAC), potentially enhancing opsonization and antibacterial effects. In conclusion, our findings offer valuable insights into the immune antibacterial mechanisms of c1qb and contribute to the development of strategies for controlling N. seriolae.

Nocardia seriolae is the primary pathogen causing nocardiosis in various fish species, leads to significant economic losses in the aquaculture industry. In this study, 10 bacterial strains isolated from Micropterus salmoides and Channa argus infected with nocardiosis, were identified as N. seriolae by physiological and biochemical identification, as well as 16S rDNA sequencing. Moreover, the key virulence-related genes such as ESX-1, T7SS-2, T7SS-3, EspG1, sodC, sod2 and ESAT6 were all positive, and showing high homology among different strains. Pathogenicity testing revealed mortality rates ranging from 70 to 100%, accompanied by the presence of white nodules in the viscera of deceased fish. The drug sensitivity test demonstrated that LY21811, the most lethal strain, exhibited high sensitivity to nine types of antibiotics, including azithromycin, doxycycline, florfenicol and compound sulfamethoxazole, yet showed complete resistance to β-lactam antibiotics. Additionally, the tannic acid also demonstrated potent inhibitory effects against LY21811, with a minimum inhibitory concentration of 0.0625 mg/mL. These results showed that N. seriolae originated from M. salmoides and C. argus in Zhejiang Province were highly conserved, demonstrating a high homogeneity in genetic characteristics, pathogenicity and antimicrobial susceptibilities. These results provide a foundation for further research on the pathogenic characteristics and disease prevention of N. seriolae infections.

Nocardia seriolae pathogen causes chronic granulomatous disease, reportedly affecting over 40 species of marine and freshwater cultured fish. Hence, research is required to address and eliminate this significant threat to the aquaculture industry. In this respect, a reliable and reproducible infection model needs to be established to better understand the biology of this pathogen and its interactions with the host during infection, as well as to develop new vaccines or other effective treatment methods. In this study, we examined the pathogenicity of the pathogen and the immune response of snakehead (Channa argus) juvenile to N. seriolae using a range of methods and analyses, including pathogen isolation and identification, histopathology, Kaplan-Meier survival curve analysis, and determination of the median lethal dose (LD50) and cytokine expression. We have preliminarily established a N. seriolae - C. argus model. According to our morphological and phylogenetic analysis data, the isolated strain was identified as N. seriolae and named NSE01. Eighteen days post-infection of healthy juvenile C. argus with N. seriolae NSE01, the mortality rate in all four experimental groups (intraperitoneally injected with 1 × 105 CFU/mL - 1 × 108 CFU/mL of bacterial suspension) (n = 120) was 100 %. The LD50 of N. seriolae NSE01 for juvenile C. argus was determined to be 1.13 × 106 CFU/fish. Infected juvenile C. argus had significant pathological changes, including visceral tissue swelling, hemorrhage, and the presence of numerous nodules of varying sizes in multiple tissues. Further histopathological examination revealed typical systemic granuloma formation. Additionally, following infection with N. seriolae NSE01, the gene expression of important cytokines, such as Toll-like receptor genes TLR2, TLR13, interleukin-1 receptor genes IL1R1, IL1R2, and interferon regulatory factor IRF2 were significantly upregulated in different tissues, indicating their potential involvement in the host immune response and regulation against N. seriolae. In conclusion, juvenile C. argus can serve as a suitable model for N. seriolae infection. The establishment of this animal model will facilitate the study of the pathogenesis of nocardiosis and the development of vaccines.

In this study, we present the first cloning and identification of perforin (MsPRF1) in largemouth bass (Micropterus salmoides). The full-length cDNA of MsPRF1 spans 1572 base pairs, encoding a 58.88 kDa protein consisting of 523 amino acids. Notably, the protein contains MACPF and C2 structural domains. To evaluate the expression levels of MsPRF1 in various healthy largemouth bass tissues, real-time quantitative PCR was employed, revealing the highest expression in the liver and gut. After the largemouth bass were infected by Nocardia seriolae, the mRNA levels of MsPRF1 generally increased within 48 h. Remarkably, the recombinant protein MsPRF1 exhibits inhibitory effects against both Gram-negative and Gram-positive bacteria. Additionally, the largemouth bass showed a higher survival rate in the N. seriolae challenge following the intraperitoneal injection of rMsPRF1, with observed reductions in the tissue bacterial loads. Moreover, rMsPRF1 demonstrated a significant impact on the phagocytic and bactericidal activities of largemouth bass MO/MΦ cells, concurrently upregulating the expression of pro-inflammatory factors. These results demonstrate that MsPRF1 has a potential role in the immune response of largemouth bass against N. seriolae infection.

Nocardia seriolae adversely impacts a diverse range of fish species, exhibiting significant pathogenic characteristics that substantially impede the progress of aquaculture. N. seriolae infects in fish has a long incubation period, and clinical symptoms are not obvious in the early stages. There is presently no viable and eco-friendly approach to combat the spread of the disease. According to reports, N. seriolae primarily targets macrophages in tissues after infecting fish and can proliferate massively, leading to the death of fish. Interferon-gamma (IFN-γ) is a crucial molecule that regulates macrophage activation, but little is known about its role in the N. seriolae prevention.
IFN-γ was first defined as largemouth bass (Micropterus salmoides, MsIFN-γ), which has a highly conserved IFN-γ characteristic sequence through homology analysis. The recombinant proteins (rMsIFN-γ) were obtained in Escherichia coli (E. coli) strain BL21 (DE3). The inflammatory response-inducing ability of rMsIFN-γ was assessed in vitro using monocytes/macrophages. Meanwhile, the protective effect of MsIFN-γ in vivo was evaluated by N. seriolae infection largemouth bass model.
In the inflammatory response of the monocytes/macrophages activated by rMsIFN-γ, various cytokines were significantly increased. Interestingly, interleukin 1β (IL-1β) and tumor necrosis factor alpha (TNF-a) increased by 183- and 12-fold, respectively, after rMsIFN-γ stimulation. rMsIFN-γ improved survival by 42.1% compared with the control. The bacterial load in the liver, spleen and head kidney significantly decreased. rMsIFN-γ was also shown to better induce increased expression of IL-1β, TNF-α, hepcidin-1(Hep-1), major histocompatibility complex I (MHCI), and MHC II in head kidney, spleen and liver. The histopathological examination demonstrated the transformation of granuloma status from an early necrotic foci to fibrosis in the infection period. Unexpectedly, the development of granulomas was successfully slowed in the rMsIFN-γ group.
This work paves the way for further research into IFN-γ of largemouth bass and identifies a potential therapeutic target for the prevention of N. seriolae.

Aquatic fishes are threatened by the strong pathogenic bacterium Nocardia seriolae, which challenges the current prevention and treatment approaches. This study introduces luminogens with aggregation-induced emission (AIE) as an innovative and non-antibiotic therapy for N. seriolae. Specifically, the AIE photosensitizer, TTCPy-3 is employed against N. seriolae. We evaluated the antibacterial activity of TTCPy-3 and investigated the killing mechanism against N. seriolae, emphasizing its ability to aggregate within the bacterium and produce reactive oxygen species (ROS). TTCPy-3 could effectively aggregate in N. seriolae, generate ROS, and perform real-time imaging of the bacteria. A bactericidal efficiency of 100% was observed while concentrations exceeding 4 μM in the presence of white light irradiation for 10 min. In vivo, evaluation on zebrafish (Danio rerio) confirmed the superior therapeutic efficacy induced by TTCPy-3 to fight against N. seriolae infections. TTCPy-3 offers a promising strategy for treating nocardiosis of fish, paving the way for alternative treatments beyond traditional antibiotics and potentially addressing antibiotic resistance.

IL-8 and IL-10 are crucial inflammatory cytokines that participate in defending host cells against infections. To demonstrate the function of the two interleukin genes in largemouth bass (Micropterus salmoides), we initially cloned and identified the cDNA sequences of il-8 and il-10 in largemouth bass, referred to as Msil-8 and Msil-10, respectively. The open reading frame (ORF) of Msil-8 was 324 bp in length, encoding 107 amino acids, while the ORF of Msil-10 consisted of 726 bp and encoded 241 amino acids. Furthermore, the functional domains of the SCY domain in MsIL-8 and the IL-10 family signature motif in MsIL-10 were highly conserved across vertebrates. Additionally, both MsIL-8 and MsIL-10 showed close relationships with M. dolomieu. Constitutive expression of Msil-8 and Msil-10 was observed in various tissues, with the highest level found in the head kidney. Subsequently, largemouth bass were infected with Nocardia seriolae via intraperitoneal injection to gain a further understanding of the function of these two genes. Bacterial loads were initially detected in the foregut, followed by the midgut, hindgut, and liver. The mRNA expression of Msil-8 was significantly down-regulated after infection, especially at 2 days post-infection (DPI), with a similar expression to Msil-10. In contrast, the expression of Msil-8 and Msil-10 was significantly upregulated in the foregut at 14 DPI. Taken together, these results reveal that the function of IL-8 and IL-10 was likely hindered by N. seriolae, which promoted bacterial proliferation and intercellular diffusion.

Nocardia seriolae has been identified as the causative agent of fish nocardiosis, resulting in serious economic losses in aquaculture. With an aim to screen potential candidates for vaccine development against N. seriolae, the in vivo-induced genes of N. seriolae in hybrid snakehead (Channa maculate ♀ × Channa argus ♂) model were profiled via in vivo-induced antigen technology (IVIAT) in the present study, and 6 in vivo-induced genes were identified as follows: IS701 family transposase (is701), membrane protein insertase YidC (yidC), ergothioneine biosynthesis glutamate-cysteine ligase (egtA), molybdopterin respectively-dependent oxidoreductase (mol), phosphoketolase family protein (Ppl), hypothetical protein 6747 (hp6747). Additionally, the yidC was inserted into eukaryotic expression vector pcDNA3.1-myc-his-A to construct a DNA vaccine named as pcDNA-YidC to evaluate immunoprotection in hybrid snakehead after artificial challenge with N. serioale. Results showed that the transcription of yidC was detected in spleen, trunk kidney, muscle and liver in vaccinated fish, suggesting that this antigenic gene can be recombinantly expressed in fish. Meanwhile, indexes of humoral immunity were evaluated in the vaccinated fish through assessing specific-antibody IgM and serum enzyme activities, including lysozyme (LZM), superoxide dismutase (SOD), acid phosphatase (ACP) and alkaline phosphatase (AKP). Quantitative real-time PCR analysis indicated that pcDNA-YidC DNA vaccine could notably enhance the expression of immune-related genes (CD4、CD8α、MHCIIα、TNFα、IL-1β and MHCIα) in 4 tissues (spleen, trunk kidney, muscle and liver) of the vaccinated fish. Finally, an immuno-protection with a relative survival rate of 65.71 % was displayed in vaccinated fish in comparison to the control groups. Taken together, these results indicate that pcDNA-YidC DNA vaccine could boost strong immune responses in hybrid snakehead and show preferably protective efficacy against N. seriolae, indicating that IVIAT is a helpful strategy to screen the highly immunogenic antigens for vaccine development against fish nocardiosis.