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.

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.

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.

Fish stocks as phosphorus sources or sinks: Influenced by nutritional and metabolic variations, not solely by dietary content and stoichiometry.

The study provides a descriptive understanding of when fish (Cyprinus carpio model) are the source or sink of phosphorus. Dissolved reactive phosphorus (DRP; PO4-P) losses (51.1 ± 5.9 % of intake-P) increase at excess of bioavailable P (>0.83 g 100 g-1 dry matter, DM fed) or when food (digestible) N:P mass ratio (≤4.4:1) approaches organismal storage threshold (~4:1). This is known, however, even at a sub-threshold food P content (0.57 g 100 g-1 DM) and food N:P mass ratio (7.3:1), DRP losses (57.8 ± 4.5 % of intake-P) may be extraordinary if two indispensable amino acids are biologically insufficient (lysine ≤1.43 g, methionine ≤0.39 g 100 g-1 DM fed). Given that methionine and lysine are sufficient, DRP losses cease (≈0 %) and even some P from water is absorbed, given there is support from non-protein energy (NPE). Insufficient NPE (<180 kcal 100 g-1 DM fed) may drive DRP losses (81.6 ± 4.3 % of intake-P) beyond expected levels (46-59 % of intake-P) at a given food P content (0.91 g 100 g-1 DM). Natural food seldom fulfills low P, high lysine + methionine, and high NPE contents simultaneously, thus keeping fish in a perpetual P recycling for algae (scaleless carp > scaly carp). Such P recycling ceases only during basal metabolism. During feeding state, the richness of lysine + methionine bound N and lipid + carbohydrate bound C in the food base may enhance the fishes' threshold of P storage. P storage can be diminished when they are insufficient. We show that for fish, the decision of P recycling or not recycling (for algae) may change based on the supply of specific fractions of N or C from the food web or metabolic variations (basal metabolism, presence of scales). NOVELTY STATEMENT: The ecological stoichiometry theory is better connected to fish nutritional bioenergetics for better understanding and biomanipulation of eutrophication processes.

In vitro Assessment of the Safety and Potential Probiotic Characteristics of Three Bacillus Strains Isolated From the Intestine of Hybrid Grouper (Epinephelus fuscoguttatus♀ × Epinephelus lanceolatus♂).

Probiotics serving as an alternative to the criticized antibiotics mainly focus on improving animal's growth and health. After realizing the dangers posed by diseases that have led to lots of economic losses, aquaculture scientists have sought the usage of probiotics. However, most probiotics are ineffective in eliciting aquatic animals' preferred effects, since they are from non-fish sources. Again, there are even a few marine aquatic probiotics. Given this, a study was conducted to investigate the probiotic potential of the bacteria species isolated from the digestive tract of hybrid grouper (Epinephelus fuscoguttatus♀ × Epinephelus lanceolatus♂). Based on the morphological, biochemical, 16S rRNA sequencing analysis and evolutionary relationships, the isolated species were identified as Bacillus tequilensis GPSAK2 (MW548630), Bacillus velezensis GPSAK4 (MW548635), and Bacillus subtilis GPSAK9 (MW548634), which were designated as GPSAK2, GPSAK4, and GPSAK9 strains, respectively. Their probiotic potentials including their ability to tolerate high bile salt concentration, low pH, high temperatures, adhesion ability (auto-aggregation and cell-surface hydrophobicity), antimicrobial activity and biosafety test, compatibility test, hemolytic activity, and antibiotic susceptibility test were evaluated. While GPSAK2 and GPSAK9 strains were γ-hemolytic, that of GPSAK4 was α-hemolytic. All the isolates were resistant to low pH (1) and higher bile salt concentration (0.5%), showed higher viability ability after higher temperature exposure (80, 90, and 100°C), as well as higher cell-surface percentage hydrophobicity and auto-aggregation. All isolates exhibited positive compatibility with each other, signifying their ability to be used as multispecies. The three strains were susceptible to ampicillin (except GPSAK9, which was resistant), penicillin, kanamycin, ceftriaxone, chloramphenicol, erythromycin, clindamycin, furazolidone (except GPSAK2 and GPSAK9, which were moderately susceptible and resistant, respectively), polymyxin B, vancomycin (except GPSAK9, which was resistant), sulfamethoxazole (except GPSAK9, which was moderately susceptible), amikacin, minocycline, ofloxacin, norfloxacin, doxycycline, neomycin, gentamicin, tetracycline, carbenicillin, midecamycin (except GPSAK9, which was moderately susceptible), ciprofloxacin, piperacillin, and cefoperazone. All isolates demonstrated good antimicrobial activity against four pathogens, viz. Streptococcus agalactiae, Streptococcus iniae, Vibrio harveyi, and Vibrio alginolyticus. The results collectively suggest that Bacillus strains GPSAK2, GPSAK4, and GPSAK9 could serve as potential probiotic candidates that can be used to improve the growth and health status of aquatic animals, especially grouper.

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.