Weissella confusa N17 Derived from Loach (Misgurnus anguillicaudatus) Exhibits Promising for Further Applications in Loach Aquaculture.

The application of probiotics, in aquaculture, is becoming increasingly widespread and have had positive application effects. However, reports of loach-derived probiotics are quite limited. In this study, two representative strains of lactic acid bacteria with excellent traits, namely, Weissella confusa N17 and Lactobacillus saniviri N19, were screened from the intestine of healthy loaches. W. confusa N17 and L. saniviri N19 could inhibit different common various pathogenic bacteria, especially Aeromonas spp., and were sensitive to the most common antibiotics. The survival rate of the two strains exceeded 50% after 4 h of incubation in 10% loach bile. Moreover, the two strains showed significant tolerance to trypsin. Their autoaggregation capacity and hydrophobicity were greater than 30%. In addition, the aggregation ability of both strains was higher than 30% for both A. veronii TH0426 and A. hydrophila TPS. The two strains had a high biofilm-forming ability and strong adhesion to epithelioma papulosum cyprini (EPC) cells. Scanning electron microscopy results showed that the culture supernatants of the two strains had a significantly destructive effect on A. veronii TH0426 and A. hydrophila TPS. Overall, the traits of W. confusa N17 were better than those of L. saniviri N19. Genome sequencing and analysis demonstrated a lack of virulence factor-related or drug resistance-related genes in genome N17. The diet supplemented with the W. confusa N17 strain significantly improved the resistance of loaches to A. veronii infection, and the protection rate reached 57.1%. Therefore, W. confusa N17 exhibits promising for further applications in loach aquaculture.

The effects of dietary supplementation of ginseng stem and leaf saponins on the antioxidant capacity, immune response, and disease resistance of crucian carp, Carassius auratus.

This is the first study to explore the positive effects of ginseng stem and leaf saponins (GSLS) on antioxidant capability, immunity, and disease resistance of crucian carp. Seven hundred fifty crucian carps (initial body weight: 25 ± 0.15 g (mean ± SE)) were randomly allocated into five groups with three replicates each; five diets supplemented with the final concentration of 0, 1, 2, 4, and 8 g/kg GSLS were fed to crucian carp for 5 weeks. The results demonstrated that, at a concentration of 8 g/kg, the contents of IgM, C4, SOD, GSH-Px, and the activity of AKP in serum of crucian carp gradually increased at 7, 14, 21, 28, and 35 days, and the expression of immune-relative cytokine genes (TNF-α, IL-10, IFN-γ) in the liver, spleen, and the intestinal tract also had a significant up-regulation (P < 0.05), and which were significant difference compared with control (P < 0.05). The above results demonstrated that dietary GSLS showed enhancement effects on the antioxidant and anti-inflammatory capability, and innate immune response of crucian carp. The feed of 8 g/kg GSLS for 1 week could improve the survival rate 44% more than the control group when crucian carp infected Aeromonas hydrophila (A. hydrophila). In conclusion, the addition of GSLS at a concentration of 8 g/kg in the diet improve immune-related enzyme activity better, immune-relative cytokine expression, and disease resistance.

Cadaverine reverse transporter (CadB protein) contributes to the virulence of Aeromonas veronii TH0426.

Aeromonas veronii is one of the main pathogens causing sepsis and ulcer syndrome in freshwater fish. Analysis of the results of epidemiological investigations in recent years has revealed that the virulence of A. veronii and its tolerance to drugs have been increasing year by year. Currently, most of the research on A. veronii focuses on its isolation, identification, and drug susceptibility, whereas research on its virulence factors and pathogenesis mechanisms is relatively rare. In this study, we identified and obtained the highly expressed TH0426 cadaverine reverse transporter (CadB) of A. veronii. We used efficient suicide plasmid-mediated homologous recombination to delete the cadB gene in TH0426 and constructed a cadB deletion strain. The LD50 of ΔcadB was 93.2 times higher than that of TH0426 in zebrafish, the toxicity of ΔcadB was 9.5 times less than that of TH0426 in EPC cells, and the biofilm formation ability of ΔcadB was 5.6-fold greater than that of TH0426. In addition, motility detection results indicated that ΔcadB had lost its swimming ability. The results of flagellar staining and TEM demonstrated that ΔcadB shed the flagella. In summary, the virulence and adhesion of A. veronii TH0426 were significantly decreased by the deletion of cadB, which might provide a theoretical basis for research into A. veronii virulence factors.

Oral immunization with recombinant Lactobacillus casei expressing flaB confers protection against Aeromonas veronii challenge in common carp, Cyprinus carpio.

Aeromonas veronii is an important type of gram-negative pathogen of human-livestock-aquatic animal and causes great economic losses in the aquaculture industry. Vaccination is an effective method of defence against A. veronii. There are many factors that restrict the use of vaccination, and the development of new oral vaccines is urgently needed. The selection of suitable antigens is of great significance for the development of aquaculture vaccines. Bacterial flagellin can specifically bind to TLR5 and induce the release of cytokines from the organism, which could be used in the development of vaccines. In this study, we constructed two recombinant Lactobacillus casei (L. casei) (surface-displayed or secretory) expressing the flaB of A. veronii and evaluated the effect of immune responses in common carp. The flaB gene (900 bp) of A. veronii was subcloned into the L. casei expression plasmids pPG-1 (surface-displayed) and pPG-2 (secretory). Western blot and immunofluorescence assays confirmed the expression of the recombinant flaB protein. Common carp immunized with Lc-pPG-1-flaB and Lc-pPG-2-flaB via oral administration route exhibited induction of antibody expression and innate immune responses. The results indicated that Lc-pPG-1-flaB and Lc-pPG-2-flaB can induce high levels of IgM, ACP, AKP, LZM and SOD activity in organisms, and Lc-pPG-1-flaB can induce even higher levels. The recombinant L. casei may effectively induce humoral immunity and increase the serum immunological index. Furthermore, leukocytes phagocytosis percentage and index of the recombinant L. casei were enhanced. The results of qRT-PCR showed that recombinant L. casei can significantly increase the expression of IL-10, IL-ß, IFN-γ and TNF-α in the tissues of immunized common carp, compared with control groups. Viable recombinant L. casei strains, which were delivered directly survived throughout the intestinal tract. Common carp that received Lc-pPG-1-flaB (66.7%) and Lc-pPG-2-flaB (53.3%) exhibited higher survival rates than the controls after challenge with the pathogen A. veronii. Our work indicated that Lc-pPG-1-flaB and Lc-pPG-2-flaB had beneficial effects on immune response and enhanced the disease resistance of common carp against A. veronii infection. The combination of flaB delivery and the Lactic acid bacteria (LAB) approach may be a promising method for the development of oral vaccines for treating A. veronii. In future research, we will focus on the colonization ability of LAB in the intestines and on the impact of these bacteria on intestinal flora.