| Tilapia is one of the most cultured aquaculture species second to carps with regards to total global aquaculture production.It is an important source of protein for people in most developed and developing countries.The occurrence of diseases in tilapia aquaculture as a result of high stocking densities to meet the high demand for fish is a major blow to the aquaculture industry.The use of probiotics to combat diseases as well as to improve immunity and water quality is well elucidated in aquaculture.However most probiotics used in aquaculture are mostly from non-fish sources as a result,they are relatively ineffective in eliciting the desired effects in aquatic animals.Host-associated probiotics,“bacteria originally isolated from the rearing water or the GI tract of the host to improve growth and health of the host”are therefore,considered as the future of aquaculture.Among the many microbial candidates,probiotic Bacillus has sporulation capacity that makes them survive harsh environmental conditions（heat-tolerance and longer shelf-life）,are non-pathogenic and non-toxic（though not all）when fed to fish.They can produce a wide range of antimicrobial substances making them more suitable candidates compared to other probiotics.This study is therefore aimed at isolating and characterizing Bacillus species from the digestive tract of freshwater fish,Oreochromis niloticus based on their morphological,biochemical,and evolutionary relationships using in vitro methods.Further,in vivo assessment of the probiotic potentials of the isolates in O.niloticus was carried out through dietary administration taking into consideration their individual effects as well as their consortia effects.The effects of the isolates on growth,digestive and antioxidant enzymes capacity,serum and mucus immunity,gene expression as well as their influence on the microbiota and intestinal morphology of O.niloticus were evaluated.Also,the ability of the isolated Bacillus species to protect O.niloticus against Streptococcus agalactiae and Aeromonas hydrophila infection was assessed.Three Bacillus strains identified as Bacillus velezensis TPS3N（MK130897）,Bacillus subtilis TPS4（MK130899）and Bacillus amyloliquefaciens TPS17（MK130898）were successfully isolated and characterized from the gut of O.niloticus.B.velezensis TPS3N and B.amyloliquefaciens TPS17 wereα-haemolytic while B.subtilis TPS4 wasγ-haemolytic.In vitro studies revealed that,the three isolates had higher viability ability after exposure to high temperatures（80 oC,90 oC and 100 oC）and were resistant to low p H（1）and low bile salt concentration（0.5%）as well as had high cell surface hydrophobicity and auto-aggregation.The three isolates were compatible with one another 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 B.subtilis TPS4）and furazolidone（except B.amyloliquefaciens TPS17）.The antimicrobial assessment showed that among the three isolates,B.velezensis TPS3N and B.amyloliquefaciens TPS17 exhibited good antimicrobial activity against the three fish pathogens（Streptococcus agalactiae,Aeromonas hydrophila,Vibrio harveyi）tested,while B.subtilis TPS4 was effective against S.agalactiae only.Four weeks feeding trial was conducted to assess the in vivo probiotic potentials of the three host-associated Bacillus species in O.niloticus.The fish were divided into five treatment groups and fed with diets containing no bacteria denoted as group CT（control）,B.velezensis TPS3N denoted as group V,B.subtilis TPS4 denoted as group S,B.amyloliquefaciens TPS17denoted as group A and a 5th group which comprise the combination of the three Bacillus species at a ratio 1:1:1 denoted as group CB.At the end of the feeding trial,enhanced growth performance in terms of final weight（FW）,weight gain（WG）,specific growth rate（SGR）as well as significantly lower feed conversion ratio（FCR）were recorded in the Bacillus treatment groups with the CB group having the best performance.Improved immune/biochemical（nitric oxide（NO）,immunoglobulin M（Ig M）,lysozyme（LZM）,acid phosphatase（ACP）,and alkaline phosphatase（AKP））indexes,antioxidant enzymes（superoxide dismutase（SOD）and catalase（CAT））,and digestive enzymes（lipase and trypsin）were also recorded in the treatment groups relative to the CT group.Significantly lower GOT（glutamate oxalate transaminase）and MDA（malondialdehyde）activities were also observed in the V,S,A,and CB groups than the CT group.Growth-related genes GHR-1 and IGF-1 were upregulated significantly in both liver and head kidney（HK）of fish fed Bacillus supplemented diets.Likewise,the CB group induced significant upregulation of TNF-α,TLR-2,Ig M（except in the liver）,and C-LYZ in both liver and HK.A subsequent challenge test with S.agalactiae resulted in cumulative mortalities（%）of 60.0%,33.33%,26.67%,20.0%,and 13.33%in the CT,V,S,A,and CB treatment groups respectively.A further study to evaluate the effects of the 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 carried out.The fish were divided into five treatment groups and fed with diets containing no bacteria denoted as CT group,Bacillus velezensis TPS3N denoted as group V,Bacillus subtilis TPS4denoted 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（Ig M）（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 was 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 CT group.Dietary probiotic supplementation also influenced the intestinal microflora composition of Nile tilapia.In general,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 harmful bacteria viz Staphylococcus and Aeromonas were reduced in the Bacillus supplemented groups in comparison to the CT group.A challenge test with A.hydrophila resulted in lower mortalities（%）in the Bacillus treated groups thus86.67%,50.00%,43.33%,63.33%,and 30.00%for Nile tilapia fed Control,V,S,A,and CB diets respectively.In conclusion,three Bacillus species were isolated from the gut of Nile tilapia and in vitro studies suggested that the three isolates could be considered as potential probiotics in tilapia aquaculture.In vivo studies through dietary supplementation confirmed that the three Bacillus species singularly or in combination could improve Nile tilapia’s growth,digestive capacity,serum and mucous immune titres,intestinal health and microbiota as well as protect Nile tilapia against S.agalactiae and A.hydrophila infection.These results put together,suggests that B.velezensis TPS3N,B.subtilis TPS4,and B.amyloliquefaciens TPS17 could be considered as potential probiotics in tilapia aquaculture.However,a combination of the three isolates may yield the best results. |
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