Effects of autochthonous strains mixture on gut microbiota and metabolic profile in cobia (Rachycentron canadum).

The fish immune system is a topic or subject that offers a unique understanding of defensive system evolution in vertebrate heredity. While gut microbiota plays several roles in fish: well-being, promoting health and growth, resistance to bacterial invasion, regulation of energy absorption, and lipid metabolism. However, studies on fish gut microbiota face practical challenges due to the large number of fish varieties, fluctuating environmental conditions, and differences in feeding habits. This study was carried out to evaluate the impacts of supplemented three autochthonous strains, Bacillus sp. RCS1, Pantoea agglomerans RCS2, and Bacillus cereus RCS3 mixture diet on cobia fish (Rachycentron canadum). Also, chromatography, mass spectrometry and high throughput sequencing were combined to explore composition and metabolite profile of gut microbiota in juvenile cobia fed with supplemented diet. In the trial group, juvenile cobia received diets supplemented with 1 × 1012 CFU mL-1 autochthonous strains for ten weeks and a control diet without supplementation. Juvenile cobia receiving diets supplementation exhibited significantly improved growth than those without additives (control). Haematological indices, such as red blood cells, white blood cells, corpuscular haemoglobin concentration, mean corpuscular volume, haemoglobin, and mean corpuscular haemoglobin, were higher in the supplemented group. Similarly, digestive enzymes (trypsin, lipase, amylase, pepsin and cellulose, activities) activities were higher in supplemented diet with an indigenous isolates mixture. Serum biochemical parameters albumin, globulin, and total protein were significantly higher, while triglyceride, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and cholesterol showed no significant difference. On the other hand, glucose was significantly (P < 0.05) higher in the group without supplementation. On gene expression in the midgut, Immunoglobulin, Colony-stimulating factor receptor 1, major histocompatibility complex 1 were up-regulated by native isolates while T cell receptor beta, and Major histocompatibility complex 2 showed no significant difference. Gut bacterial composition was altered in fish receiving supplemented diet with autochthonous strains. Metabolomics also revealed that some metabolic pathways were considerably enriched in fish fed with supplemented diet; pathway analysis based on Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment revealed that differentially expressed metabolites were involved in galactose metabolism, tryptophan metabolism, carbohydrate digestion and absorption, purine metabolism, and ABC transporters. Functional analysis of bacterial community showed that differences in enriched metabolic pathways generally comprised carbohydrate and its metabolites, nucleotide and its metabolites, amino acid and its metabolites, heterocyclic compounds, and tryptamines, cholines, pigments. The current investigation results showed that autochthonous strains mixture has significantly enhanced the growth, survival, and innate and adaptive immunities of juvenile cobia.

Effects of probiotics on digestive enzymes of fish (finfish and shellfish); status and prospects: a mini review.

Digestive enzymes are found in the digestive tract of animals which assist in the breakdown of larger food molecules into more easily absorbed particles that can then be used by the body. The ability of fish to break down a diet is highly dependent on the availability of suitable digestive enzymes which mediate specific degradation pathways and on both the physical and chemical nature of food. Probiotics are known to produce helpful enzymes that aid in digestion and protect the gastrointestinal tract (GIT) of animals. When applied appropriately, probiotics improve intestinal microbial balance which also improves digestive enzyme activities, food absorption, and decrease pathogenic issues in the GIT. They work hand-in-hand with the digestive enzymes in the GIT of animals as supplements thereby improvings nutrition. This in turn leads to higher feed efficiency and growth as well as the prevention of antinutritional factors present in the ingredients, intestinal disorders, and pre-digestion. This review seeks to present summaries of the results of research findings on the application of probiotics on the activities of digestive enzymes including amylase, lipase, and protease. Further, this review points out gaps in available literature and suggests ideas that could be explored in further investigations to better understand and enhance the activities of these digestive enzymes to increase feed and nutrient utilization and the production of aquaculture species.

Mechanisms and the role of probiotic Bacillus in mitigating fish pathogens in aquaculture.

Diseases are natural components of the environment, and many have economic implications for aquaculture and fisheries. Aquaculture is a fast-growing industry with the aim to meet the high protein demand of the ever-increasing global population; however, the emergence of diseases is a major setback to the industry. Probiotics emerged as a better solution to curb the disease problem in aquaculture among many alternatives. Probiotic Bacillus has been proven to better combat a wide range of fish pathogens relative to other probiotics in aquaculture; therefore, understanding the various mechanisms used by Bacillus in combating diseases will help improve their mode of action hence yielding better results in their combat against pathogens in the aquaculture industry. Thus, an overview of the mechanisms (production of bacteriocins, suppression of virulence gene expression, competition for adhesion sites, production of lytic enzymes, production of antibiotics, immunostimulation, competition for nutrients and energy, and production of organic acids) used by Bacillus probiotics in mitigating fish pathogens ranging from Aeromonas, Vibrio, Streptococcus, Yersinia, Pseudomonas, Clostridium, Acinetobacter, Edwardsiella, Flavobacterium, white spot syndrome virus, and infectious hypodermal and hematopoietic necrosis virus proven to be mitigated by Bacillus have been provided.

In vitro Assessment of the Probiotic Characteristics of Three Bacillus Species from the Gut of Nile Tilapia, Oreochromis niloticus.

Probiotics used in aquaculture are mostly from non-fish sources, as a result ineffective in eliciting the desired effects in aquatic animals. In this study, three Bacillus species were isolated from the digestive tract of freshwater fish Oreochromis niloticus and characterised based on their morphological, biochemical and evolutionary relationships. Their probiotic potentials were evaluated based on their ability to tolerate low pH, bile salt concentration, high temperatures, adhesion ability (auto-aggregation and hydrophobicity), haemolytic activity and antimicrobial activity including biosafety assay. Three Bacillus strains identified as Bacillus velezensis TPS3N (MK130897), Bacillus subtilis TPS4 (MK130899) and Bacillus amyloliquefaciens TPS17 (MK130898) were designated as TPS3N, TPS4 and TPS17, respectively. TPS3N and TPS17 were α-haemolytic, while TPS4 was γ-haemolytic. The three isolates had higher viability ability after exposure to higher temperatures (80 °C, 90 °C and 100 °C) and were resistant to low pH (1) and bile salt concentration (0.5%) as well as high cell surface hydrophobicity and auto-aggregation. The three isolates were compatible with one another and thus can be used in consortia. These strains were susceptible to gentamicin, cephalexin, ampicillin, ceftriaxone, kanamycin, amikacin, penicillin, cefoperazone, chloramphenicol, erythromycin, tetracycline, doxycycline, ciprofloxacin, clindamycin (except TPS4) and furazolidone (except TPS17). The antimicrobial assessment showed that among the three isolates, TPS3N and TPS17 exhibited good antimicrobial activity against the three fish pathogens (Streptococcus agalactiae, Aeromonas hydrophila, Vibrio harveyi), while TPS4 was effective against Streptococcus agalactiae only. The results of this work suggest that Bacillus strains TPS3N, TPS4 and TPS17 could be considered as potential probiotics in tilapia aquaculture.

Effects of three host-associated Bacillus species on mucosal immunity and gut health of Nile tilapia, Oreochromis niloticus and its resistance against Aeromonas hydrophila infection.

Skin and intestinal mucosa lymphoid tissues are known to be the fish's first line of defence since they serve as the first point of contact for pathogens. Only few studies have investigated the influence of host-associated Bacillus on mucosal immunity. In this study, the effects of three host-associated Bacillus species on mucosal immunity, intestinal morphology, intestinal digestive enzymes activity, intestinal microbiome and resistance of Nile tilapia against Aeromonas hydrophila infection was evaluated. The fish were divided into five treatment groups and fed with diets containing no bacteria denoted as Control, Bacillus velezensis TPS3N denoted as group V, Bacillus subtilis TPS4 denoted as group S, Bacillus amyloliquefaciens TPS17 denoted as group A and a 5th group containing the three Bacillus species at a ratio 1:1:1 denoted as group CB. At the end of the feeding trial, significant enhancement of both skin mucus and intestinal immune titres were recorded in terms of nitric oxide (NO) (except in the mucus of V and S groups), immunoglobulin M (IgM) (except in the intestine of group V), lysozyme (LZM), and alkaline phosphatase (AKP) in all fish fed the Bacillus supplemented groups relative to the untreated group. Intestinal antioxidant enzymes (catalase (CAT) (except in the intestine of group S) and superoxide dismutase (SOD)) capacity of Nile tilapia were higher in the Bacillus groups. Intestinal lipase activity was elevated in the Bacillus supplemented groups. The intestinal morphological parameters (villus height, villus width, goblet cells count (except in group S and A), and intestinal muscle thickness) were significantly enhanced in the Bacillus supplemented groups relative to the Control group. Dietary probiotic supplementation also influenced the intestinal microflora composition of Nile tilapia. Proteobacteria recorded the highest abundance followed by Firmicutes, Fusobacteria, and Bacteroidetes at the phylum level in this study. At the genus level, the abundance of pathogenic bacteria viz Staphylococcus and Aeromonas were reduced in the Bacillus supplemented groups in comparison to the Control group. A challenge test with A. hydrophila resulted in lower mortalities (%) in the Bacillus treated groups thus 86.67%, 50.00%, 43.33%, 63.33%, and 30.00% for Nile tilapia fed Control, V, S, A, and CB diets respectively. In conclusion, the inclusion of B. velezensis TPS3N, B. subtilis TPS4, and B. amyloliquefaciens TPS17 in the diet of Nile tilapia singularly or in combination, could enhance the mucosal immunity, intestinal health, and resistance of Nile tilapia against A. hydrophila infection.

Traditional Chinese Medicine Enhances Growth, Immune Response, and Resistance to Streptococcus agalactiae in Nile Tilapia.

Herbal supplements are suitable for improving fish health and combating diseases in fish culture. However, the mechanism of action of many herbal mixtures remains unclear. This study was conducted to evaluate the effects of traditional Chinese medicine (TCM; a mix of Astragalus membranaceus, Angelica sinensis, and Crataegus hupehensis at a ratio of 1:1:1 on a weight basis) on growth, immune response, and disease resistance in Nile tilapia Oreochromis niloticus. Experimental fish (mean ± SE weight = 57 ± 1 g) were divided into two groups: a control group and a TCM (10 g/kg) group. After 4 weeks of the experimental trial, a significant increase in weight gain and specific growth rate and a lower feed conversion ratio were observed in fish fed a TCM-supplemented diet compared with control fish. Similarly, the immune response of Nile Tilapia in the TCM group showed enhanced lysozyme, superoxide dismutase, catalase, and immunoglobulin levels compared with the control fish. In comparison with the control, fish fed TCM showed significant up-regulation of ß-defensin, lysozyme, heat shock protein 70, superoxide dismutase, and catalase genes in the intestine and head-kidney tissues. After a Streptococcus agalactiae challenge, survival of Nile Tilapia in the TCM group was 70% compared with 35% in the control. These results indicate that the TCM mixture in this study can elevate the immune response and disease resistance of Nile Tilapia.

A review on the application of Bacillus as probiotics in aquaculture.

Probiotics use in aquaculture has gained attention as microbial candidates to maintain the health and the well-being of many aquaculture animals. Among the many microbial candidates, probiotic Bacillus has sporulation capacity that makes them survive harsh environmental conditions, are non-pathogenic and non-toxic when fed to fish, and can produce antimicrobial substances making them more suitable candidates compared to other probiotics. In this review, we discussed the necessity of using the probiotic Bacillus in sustainable aquaculture as a good alternative to improve feed utilization, stress response, immune response and disease resistance, maintenance of tissue integrity, and as well improvement of water quality for sustainable aquaculture. Therefore the findings of current researches about the effects of Bacillus application to improve the culture of aquatic animals for future research and development of Bacillus application in aquaculture have been summarised.

Effects of a commercial probiotic BS containing Bacillus subtilis and Bacillus licheniformis on growth, immune response and disease resistance in Nile tilapia, Oreochromis niloticus.

The present study evaluated a commercial probiotic designated as BS (a mix of B. subtilis and B. licheniformis) to ascertain its efficacy and the dose necessary to improve growth, immune response, and disease resistance in tilapia, Oreochromis niloticus. Fish (53.01 ±â€¯1.0 g) were fed with a basal diet supplemented with 0 g kg−1 (CT), 3 g kg−1(BS3), 5 g kg−1 (BS5), 7 g kg−1 (BS7), and 10 g kg−1 (BS10) [corrected] of the probiotic BS for 4 weeks. At the end of the feeding trial, the weight gain, specific growth rate, and feed conversion ration were enhanced in all probiotic BS enriched groups but with better (P < 0.05) improvement in the BS10 group. The lysozyme, protease, anti-protease, superoxide dismutase activities, and immunoglobulin M level were significantly (P < 0.05) highest in the BS10 group in both serum and skin mucus. Enhanced (P > 0.05) catalse activity in all treated groups in the serum and myeloperoxidase activity in the B10 group in both serum and skin mucus were observed. The expression of C-lysozyme, heat shock protein 70, ß-defensin, transforming growth factor beta, and small body size decapentaplegic homolog 3, genes in the mid-intestines and the head-kidney were up-regulated in all treated groups with the BS10 group provoking the highest up-regulation (P < 0.05). After challenge with Streptococcus agalactiae, cumulative mortality was 80 %, 47.5 %, 42.8 %, 30 %, and 20 % [corrected] for fish fed with CT, BS3, BS5, BS7, BS10 groups respectively. In conclusion, probiotic BS application at 10 g kg−1(BS10) [corrected] can be considered to improve growth and immunological status in tilapia farming.

NK-lysin from Oreochromis niloticus improves antimicrobial defence against bacterial pathogens.

NK-lysin, an effector of cytotoxic T cells and natural killer cells, is a potent antimicrobial peptide widely distributed in mammals. Homologues of NK-lysin have been discovered in several teleost species, but only several of their natural functions was recorded so far. Here we identified an NK-lysin from Nile tilapia (Oreochromis niloticus), On-NKL, and analysed its expression model and biological effects on pathogen infection. The open reading frame of On-NKL sequence spans 432 bp, codes for 143 amino acids and shares 27%-62% overall sequence identities with NK-lysin of other species. The deduced mature peptide of On-NKL possesses a saposin B domain and six well-conserved cysteine residues that essential for antimicrobial activity by forming three intrachain disulphide bonds. The results of qRT-PCR showed that On-NKL expression was observed in multiple tissues and head kidney leucocytes and nonspecific cytotoxic cells (NCCs) and is most abundant in gills. After bacterial challenge, On-NKL expression significantly varied in different tissues and NCCs. Following bacterial infection, On-NKL-overexpressing fish featured significantly lower pathogen loads in tissues than control fish. On-NKL-overexpressing fish also exhibited 33.3% relative percent survival compare with control groups. Findings suggested that On-NKL could be the potential effector of NCCs and act as immune-related gene that enhances antimicrobial defence.