Enterobacter asburiae E7, a Novel Potential Probiotic, Enhances Resistance to Aeromonas veronii Infection via Stimulating the Immune Response in Common Carp (Cyprinus carpio).

Probiotics are an alternative strategy for antibiotics, but most probiotics are Gram-positive bacteria suitable for terrestrial animals. Therefore, it is imperative to develop dedicated probiotics for the common carp industry to be ecologically efficient and environmentally friendly. A novel Enterobacter asburiae named E7 was isolated from the intestine of healthy common carp and displayed an extensive antibacterial spectrum against Aeromonas hydrophila, A. veronii, A. caviae, A. media, A. jandaei, A. enteropelogenes, A. schubertii, A. salmonicida, Pseudomonas aeruginosa, Ps. putida, Plesiomonas shigelloides, and Shewanella. E7 was nonpathogenic to the host and susceptible to the majority of antibiotics used in human clinical practice. E7 could grow between 10 and 45°C and between pH 4 and 7 and was extremely resistant to 4% (wt/vol) bile salts. Diets were supplemented with 1 × 107 CFU/g E. asburiae E7 for 28 days. No significant difference in the growth of fish was observed. Expression of immune-related genes IL-10, IL-8, and lysozyme in common carp kidney was significantly upregulated at weeks 1, 2, and 4 (P < 0.01). A significant upregulation of IL-1ß, IFN, and TNF-α expression was observed after week 4 (P < 0.01). There was a significant increase in mRNA expression of TGF-ß at week 3 (P < 0.01). Following challenge by Aeromonas veronii, the survival rate (91.05%) was significantly higher than observed in the controls (54%; P < 0.01). Collectively, E. asburiae E7 is a promising new Gram-negative probiotic that can enhance health and bacterial resistance of aquatic animals and could thus be developed as an exclusive aquatic probiotic. IMPORTANCE In the present study, we evaluated for the first time the efficiency of Enterobacter asburiae as a prospective probiotic for aquaculture applications. The E7 strain showed extensive resistance to Aeromonas, no pathogenicity to the host, and stronger environmental tolerance. We observed that the resistance of common carp to A. veronii was enhanced by feeding a diet containing 1 × 107 CFU/g E. asburiae E7 for 28 days, but growth was not improved. Strain E7 can act as an immunostimulant to induce the upregulation of some innate cellular and humoral immune responses, resulting in enhanced resistance to A. veronii. Hence, the continuous activation of immune cells can be maintained by adding suitable fresh probiotics to the diet. E7 has the potential to act as a probiotic agent for green, sustainable aquaculture and aquatic product safety.

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.

Evaluation on the antiviral activity of genipin against white spot syndrome virus in crayfish.

White spot syndrome virus (WSSV) is a serious epidemic pathogen of crustaceans and cause severe economic losses to aquaculture. However, no commercial drugs presently available to control WSSV infection. Genipin (GN) is a bioactive compound extracted from the fruit of Gardenia jasminoides and exhibits potential antiviral activity. In the study, the antiviral activity of GN against WSSV was investigated in crayfish Procambarus clarkii and in shrimp Litopenaeus vannamei. In vitro antiviral test showed that GN could inhibit WSSV replication in crayfish and in shrimp, and the highest inhibition on WSSV was over 99% when treatment with 50 mg/kg of GN for 24 h. In vivo antiviral test proved that GN could be used to treat and prevent WSSV infection. GN could also effectively protect crayfish from WSSV infection by reducing the mortality rate of WSSV-infected crayfish. Moreover, GN attenuated the WSSV-induced oxidative stress and inflammatory by upregulation the expression of antioxidant-related genes and downregulation the expression of inflammatory-related genes, respectively. Mechanically, GN inhibited WSSV replication at least via decreasing STAT (signal transducer and activator of transcription) gene expression to block WSSV immediate-early gene ie1 transcription. Additionally, the inhibition of BI-1 (Bax inhibitor-1) gene expression also played an important role in the suppression of WSSV infection. In conclusion, GN represented a potential therapeutic and preventive agent to block WSSV infection.