Beneficial effects of indigenous Bacillus spp. on growth, antioxidants, immunity and disease resistance of Rhynchocypris lagowskii.

This study aimed to investigate the effect of Bacillus aryabhattai (LSG3-7) and Bacillus mojavensis (LSG3-8) on growth performance, antioxidant capacity, and immune response in Rhynchocypris lagowskii (Dybowski, 1869), at the trial and challenge periods. A 630 healthy fish (10.76 ± 0.05) were randomly divided into six groups: control group (D1) was fed the basal diet, D2 and D3 were supplemented with LSG 3-7 and LSG3-8 (1 × 108 CFU/g) for both of them, whereas D4 was supplemented with a mixture of both bacteria (0.5 × 108 CFU/g each), and D5 was supplemented with LSG3-7 0.75 × 108 CFU/g + LSG3-8 0.25 × 108 CFU/g, and D6 supplemented with LSG3-7 0.25 × 108 CFU/g + LSG3-8 0.75 × 108 CFU/g. After the trial, Aeromonas hydrophila was used in a challenge test for 14 days. Treatments showed significant differences (p < 0.05) in growth performance and antioxidant capacity (CAT, CuZn-SOD, GPX) in the liver and intestine compared to the control. The antioxidant-related genes CAT, CuZn-SOD, GPX, and Nrf2 in the liver and intestine showed upregulation compared with the control group. Serum IgM, LZM, C3, C4, and AKP showed a favorable superiority (p < 0.05) in treatments (D2 - D6) at the trial and challenge test compared to controls. In parallel, immune-related genes (IgM, NF-κB, TLR-1, TLR-2, and MyD88) showed an up-regulated level (p < 0.05) in treatments (D2 - D6) compared to the control. In addition, pro-inflammatory cytokines (IL-1, TNF-α) showed a downregulated level in treatments (D2 - D6). After the challenge test, the immune-related genes in the liver and muscle showed an up-regulated level in treatments compared to the controls. The survival rate showed a significant increase (p < 0.05) in the treatment groups (D2 - D6) compared to the control. Overall, individuals and the bacterial mixture of B. aryabhattai and B. mojavensis could improve the growth performance, antioxidant capacity, immune capacity, and survival rate of R. lagowskii and prevent side effects of A. hydrophila. However, B. mojavensis showed a slight improvement compared to B. aryabhattai without a significant difference between them.

Characterization of Bacillus spp. isolated from the intestines of Rhynchocypris lagowskii as a potential probiotic and their effects on fish pathogens.

Probiotics sourced from fish intestinal microbiota have a merit over other bacterial sources due to colonization ability and effective time. This study aimed to evaluate the bacilli isolated from the Rhynchocypris lagowskii intestines and their validity as a probiotic. Three isolates were selected (LSG 2-5, LSG 3-7, and LSG 3-8) and defined by morphological and 16S rRNA analysis as Bacillus velezensis, Bacillus aryabhattai, and Bacillus mojavensis, respectively. Results showed the strain tolerant abilities to gastrointestinal fluid, bile salt, pH, and temperature expotures. Additionally, all bacterial strains showed anti-pathogenic activity against at least four strains out of six tested pathogen strains (Staphylococcus aureus, Aeromonas hydrophila, Escherichia coli, Aeromonas veronii, Edwardsiella, and Aeromonas sobria). The bacterial strains also showed a high percentage of co-aggregation activity, more than 70%, with Aer. hydrophile, Staph. epidermidis, and Klebsiella aerogenes. At the same time, the results of competition, rejection, and substitution activity with Aer. hydrophila and Aer. veronii indicated the ability of the isolated strains to reduce the adhesion of pathogens to mucin. All strains showed safety properties, non-hemolytic, and sensitivity characteristics for most of tested antibiotics. In vivo test after injecting these strains into fish at various concentrations showed no side effects in the internal or external organs of fish compared to controls, proving that this is safe for these fish. Furthermore, the three strains produced lipase, amylase, and protease enzymes. The strains also showed bile salt hydrolase activity and biofilm formation, allowing them to tolerate stressful conditions. Conclusion: Based on these strains characteristics and features, they could be considered a promising candidate probiotic and can be used as an anti-pathogenic, especially in aquaculture.

Effect of Bacillus amyloliquefaciens LSG2-8 on the intestinal barrier function of Amur minnow (Rhynchocypris lagowskii).

Probiotics are widely used in aquaculture. This article aims to study the effect of Bacillus amyloliquefaciens LSG2-8 on the intestinal barrier function of Rhynchocypris lagowskii. B. amyloliquefaciens LSG2-8 were added to R. lagowskii basal diets (CK) as additives at four concentrations: 1.0 × 106 (D-6), 1.0 × 107 (D-7), 1.0 × 108 (D-8) and 1.0 × 109 (D-9) CFU g-1 by dry weight of basal diet. After a 56-day feeding experiment, the activities of intestinal digestive enzymes and immunity-related enzymes of R. lagowskii on group D-6, D-7, D-8 and D-9 diet were significantly higher than the control (P < 0.05). In molecular experiments, the authors found that the levels of TGF-ß mRNA, IL-10 mRNA, ZO-1 mRNA and claudin-3 mRNA in group D-8 R. lagowskii were significantly higher (P < 0.05) than those of the control and other groups. Furthermore, the levels of IL-1ß and IL-8 mRNA of R. lagowskii on group D-6, D-7, D-8 and D-9 diet were significantly lower than those of the control (P < 0.05). In addition, the authors found that B. amyloliquefaciens LSG2-8 can regulate the intestinal flora balance and improve the intestinal structure of R. lagowskii. In conclusion, B. amyloliquefaciens LSG2-8 can improve the intestinal barrier function of R. lagowskii and can be used as a feed additive in aquaculture.