Effects of potential probiotic Bacillus velezensis K2 on growth, immunity and resistance to Vibrio harveyi infection of hybrid grouper (Epinephelus lanceolatus♂ × E. fuscoguttatus♀).

Nowadays, there is no suitable treatment for vibriosis in groupers. So an eco-efficient and environmentally friendly treatment is necessary for the grouper industry. Probiotic-feeding has been a promising strategy to control the bacterial pathogens in aquaculture. A new Bacillus velezensis strain named K2 was isolated from the intestinal tract of healthy grouper, and exhibited wide antimicrobial spectrum of against fish pathogens, including Vibrio harveyi, Vibrio alginolyticus, Aeromonas hydrophila, Aeromonas veronii, Aeromonas caviae, Enterococcus casseliflavus and Lactococcus garvieae. Moreover, results of the safety of B. velezensis K2 showed that intraperitoneal injection of K2 in healthy grouper did not cause any pathological abnormality or death, indicating this bacteria could be considered as a candidate probiotic in aquaculture. Groupers were fed with the diets containing 1 × 107 cfu/g of B. velezensis K2 for 4 weeks. Various immune parameters were examined at 1, 2, 3, and 4 weeks of post-feeding. Results showed that diets supplemented with K2 significantly increased serum acid phosphatase (ACP) activity (P < 0.05). Results of the mRNA expression of immune-related genes in the head kidney of hybrid grouper showed that the expression of lysozyme gene was significantly upregulated after 1 and 2 weeks of feeding (P < 0.05). A significant up-regulation of the expression of piscidin, IgM and MyD88 were detected at day 21, whereas the TLR3 and TLR5 showed lower expression compared to the controls during 21 days, and a significant decrease of TLR3 gene was found at day 28 (P < 0.05). After challenge with V. harveyi, the survival rate of fish administrated with the strain K2 for 28 days was signifiacantly higher than the controls without this strain (P < 0.05). These results collectively suggest that B. velezensis K2 is a potential probiotic species to improve health status and disease resistance and can be developed as a probiotic agent in grouper industry.

Immunosuppression-induced alterations in fish gut microbiota may increase the susceptibility to pathogens.

Intestinal bacteria play an important role in the health and provide a variety of beneficial effects to host. Immunosuppressant can reduce the immunity of host and increase the susceptibility to pathogens. But it is not clear whether the increased susceptibility caused by immunosuppressant is related to changes of gut microbiota. In this study, we used crucian carp administrated with dexamethasone to explore the effects of immunosuppressants on gut microbial communities and further evaluate the potential association between changes in gut microbiota and susceptibility to pathogens. The results of MANOVA based on the top 10 PCoA axis scores from unweighed/weighted UniFrac distances showed that administration of dexamethasone (P = 0.021) and the administration time (P = 0.027) had a significant impact on the gut microbial composition, regardless of pathogens infection status (P = 0.35). After administration with dexamethasone, the fish had higher abundance of Cetobacterium and lower abundance of Bacillus and Lactococcus, and the abundance of genus Bacillus, Pseudomonas and Lactococcus decreased along with prolong administration time of dexamethasone. The results may help us understand the correlation between the host susceptibility to pathogenic bacteria and gut microbial community shift, and extend our knowledge regarding the role of gut microbiota in keeping the balance between pathogenic and symbiotic bacteria.

Display of GCRV vp7 protein on the surface of Escherichia coli and its immunoprotective effects in grass carp (Ctenopharyngodon idella).

Infection with Grass carp reovirus (GCRV) is becoming unprecedentedly widespread in grass carp (Ctenopharyngodon idella) aquaculture industry, yet the management of GCRV infection still remains a challenge. Therefore, it is of importance to develop effective means against GCRV. As a delivery system of viral antigens, surface displaying of heterologous proteins on bacteria using anchoring motifs has successfully been implemented in human and veterinary vaccines research. In this study, a novel vaccine (BL21/InpN/vp7) was developed based on surface displaying a major capsid protein (vp7) of GCRV using the anchoring motif of N-terminal unique domain of ice-nucleation protein (InpN) on Escherichia coli BL21 (DE3) vaccine. Then the grass carp were immunized by surface displaying BL21/InpN/vp7 vaccine against GCRV using both intraperitoneal injection and bath immunization and their immune responses were tested. The results revealed that some non-specific immune parameters (acid phosphatase (ACP), alkaline phosphatase (AKP) and total antioxidant capacity (T-AOC)) were strongly increased in grass carp post injection inoculation (vp7 dose ranged from 10 to 20 µg). The specific antibody levels against GCRV and the transcriptional of immune-related genes (TNF-α, IL-1ß, MHCI and IgM) were also significantly enhanced in grass carp by injection inoculation (vp7 dose ranged from 5 to 20 µg). On the other hand, only the highest dose of bath vaccination significantly induced the production of specific antibody and up-regulated transcriptions of several immune-related genes (IgM and MHCI) in grass carp. The lower cumulative mortality of grass carp in vaccinated groups after GCRV challenge clearly demonstrated that surface displayed vp7 vaccine could protect fish against GCRV infection. The relative percentage survival (RPS) value in injection vaccinated group (88.89%) was much higher compared to bath group (18.89%), which was in consistent with the production of specific serum antibodies, non-specific immune response and immune related genes expression. To sum up, our results indicated the surface display of heterologous antigenic proteins on E. coli BL21 (DE3) using the anchoring motif of ice-nucleation protein may provide a promising approach to the vaccine development of aquatic animals and suggested its potential to be used as vaccine to fight against GCRV infection.

Ammonia exposure alters the expression of immune-related and antioxidant enzymes-related genes and the gut microbial community of crucian carp (Carassius auratus).

Chronic exposure of ammonia in fish can affect the activities of antioxidant enzymes but few studies investigate the influence of ammonia exposure on the expression of immune-related and antioxidant enzymes-related genes. Also, there is no study demonstrates the effect of ammonia exposure on gut microbial community of fish. In this study, crucian carp (Carassius auratus) were exposed to the ammonia concentrations, 0 (control), 10 mg L-1 (low) or 50 mg L-1 (high) for consecutive 30 days at 25 ± 1 °C temperature, respectively, and after that, the fish from all exposure groups were maintained in control conditions for another 15 days. The results showed that low concentration ammonia increased the expression of immune-related genes and antioxidant enzymes-related genes, but high concentration ammonia inhibited the expression of immune-related genes and antioxidant enzymes-related genes. After a 15-day treatment without ammonia, the expression of antioxidant enzymes-related genes and immune-related genes showed no significant changes compared with control. The results of high-throughput sequencing showed that gut microbial communities were significantly differentiated following ammonia exposure. The abundance of Bacteroides and Cetobacterium (two kinds of potential probiotics) increased while fish exposed to 10 mg L-1 ammonia. The Flavobacterium (a potential fish pathogen) showed increasing trends when the exposure dose reached 50 mg L-1, while the Bacteroides and Cetobacterium showed almost no abundance. The results also revealed that ammonia exposure concentration or time can alter the intestinal microbial community. In conclusion, ammonia exposure could induce the immune response in crucian carp, and alter the gut microbial community. The results may help us understand the correlations of gut microbial community shift and ammonia exposure and extend our knowledge to comprehend the effects of environmental factors on intestinal microbial community.

Protective immunity of grass carp induced by DNA vaccine encoding capsid protein gene (vp7) of grass carp reovirus using bacterial ghost as delivery vehicles.

Grass carp reovirus (GCRV) is one of the most pathogenic aquareovirus and can cause lethal hemorrhagic disease in grass carp (Ctenopharyngodon idella). However, management of GCRV infection remains a challenge. Therefore, it is necessary to find effective means for the control of its infection. The uses of bacterial ghost (BG, non-living bacteria) as carriers for DNA delivery have received considerable attentions in veterinary and human vaccines studies. Nevertheless, there is still no report about intramuscular administration of bacterial ghost-based DNA vaccines in fish. In the current study, a novel vaccine based on Escherichia coli DH5α bacterial ghost (DH5α-BG), delivering a major capsid protein gene (vp7) of grass carp reovirus encoded DNA vaccine was developed to enhance the efficacy of a vp7 DNA vaccine against GCRV in grass carp. The grass carp was injected intramuscularly by different treatments -i) naked pcDNA-vp7 (containing plasmid 1, 2.5 and 5 µg, respectively), ii) DH5α-BG/pcDNA-vp7 (containing plasmid 1, 2.5 and 5 µg, respectively) and iii) naked pcDNA, DH5α-BG or phosphate buffered saline. The immune responses and disease resistance of grass carp were assessed in different groups, and results indicated that the antibody levels, serum total antioxidant capacity (T-AOC), superoxide dismutase (SOD) activity, acid phosphatase (ACP) activity and alkaline phosphatase (AKP) activity and immune-related genes were significantly enhanced in fish immunized with DH5α-BG/pcDNA-vp7 vaccine (DNA dose ranged from 2.5 to 5 µg). In addition, the relative percentage survival were significantly enhanced in fish immunized with DH5α-BG/pcDNA-vp7 vaccine and the relative percentage survival reached to 90% in DH5α-BG/pcDNA-vp7 group than that of naked pcDNA-vp7 (42.22%) at the highest DNA dose (5 µg) after 14 days of post infection. Moreover, the level of pcDNA-vp7 plasmid was higher in DH5α-BG/pcDNA-vp7 groups than naked pcDNA-vp7 groups in muscle and kidneys tissues after 21 days. Overall, those results suggested that DH5α bacterial ghost based DNA vaccine might be used as a promising vaccine for aquatic animals to fight against GCRV infection.

Preliminary study on the relationship between dexamethasone and pathogen susceptibility on crucian carp (Carassius auratus).

Dexamethasone, a known immunosuppressant, can inhibit the immune response and increase the amount of pathogen in body, but the role of dexamethasone affecting susceptibility of crucian carp (Carassius auratus) to pathogen is unclear. The effects of dexamethasone on susceptibility of crucian carp to Aeromonas hydrophila were investigated in this study. The fish were divided into four groups randomly and injected intraperitoneally by dexamethasone for 0 day (group D), 3 days (group C), 6 days (group B), and 9 days (group A), respectively. The serum lysozyme activity was significantly declined in group A, B and C. Relative immune gene expression such as il-1ß, cxcl-8, tnfα and crp in kidney were down-regulation compared to group D. After that crucian carp were infected with A. hydrophila, crucian carp treated by dexamethasone had higher mortality (group A 95%, group B 76%, group C 31%) when compared to group D (4% mortality); the amount of pathogen in was significantly increased (P < 0.05) in liver, kidney and spleen of fish in group A-C compared to group D. These results implicated that higher susceptibility caused by dexamethasone may be induced by the decrease of lysozyme activity and the down-regulation of some immune genes.