| Probiotics is a kind of microbes that could promote host animal health.Their functions includes:promoting growth,improveing the efficiency of feed conversion,antagonism of pathogenic bacteria,improving the aquatic animal gut microbiota,enhancing immune system of aquatic animals and improving the water environment,and so on.Probiotics can be used as animal feed additive as well as potential alternative to antibiotics.At present,most probiotics for aquatic animals derived from terrestrial animals,there are fewer probiotics from aquatic animals.Therefore,it is important to screen harmless and effective aquatic animal probioticsimportant for the sustainable development of healthy aquatic animal culture1.Isolation and identification of the pathogenic bacteria of T.ovatusTo determine the pathogeny for the mass death in the golden pompano T.ovatus in Hainan in Aug 2016,China,the virulence test showed that HAINUV01 had the strongest virulence to the pompano,and the lethal dose(LD50)of 14 days was 1.75×102 CFU/g fish by intraperitoneal(i.p.)injection.Neighbor-joining phylogenetic analysis based on 16S rRNA together with four other commonly used housekeeping genes-the mreB,pyrH,recA and rpoA genes-showed that HAINUV01 had 99.2%similarity to V.ponticus CECT 5869T ?V.ponticus CECT 5870.It could be identity to V ponticus.Of the 36 antibiotics used for the susceptibility test of the pompano,19 antibiotics had no effects on the growth of HAINUV01,while only 12 antibiotics can inhibit the growth of HAINUV01.These results suggest that V.ponticus is a pathogen that can potentially lead to the mass death of T.ovatus.2.Screening and identification of probiotics and the evaluation of probiotic characteristicsThis study separately use 2216E and Marine Broth Medium agar medium isolated and cultured strains(a total of 434 strains)from T.ovatus gut,including 310 strains have no hemolysis.No hemolysis strain for producing enzyme activity and antagonist activity experiment,.In this paper,10 strains with excellent enzyme activity and antagonistic activity were selected to text the tolerance and adhesion abilities of gut.The result show that the strain A97 was selected for potential probiotics to identify.The activity of protease was Dh/Dc=3.201,the activity of amylase was Dh/Dc=1.505),the antagonist activity to V.ponticus,V.harvey,V.alginolyticus and S.agalactiae was Di/Dc=2.373,2.593,1.643 and 2.051It is showed that the strain A97 was B.pumilus.The strain A97 was safety to T.ovatus.The antibiotic susceptibility test revealed that the strain A97 was highly resistant to Streptomycin and other 10 antibiotic,moderate resistant to Ciprofloxacin and other 5 antibiotic,but sensitive to Ofloxacin and other 18 antibiotic.3.Effect of B.pumilus A97 on the growth performance,natural immunity and disease resistance of T.ovatus.B.pumilus A97,B.megaterium A20 and L.plantarum 1036 mixture with fodder(the bacteria was 108 CFU/g),continuous feeding for 56 days.Measuring body weight and length,specific growth rate(SGR),feed coefficient(FC)and feed conversion ratio(FCR).In addition significant differences(P<0.05)were recorded of the A97 group and A20 group in weight gaining,SGR,FC and FCR.Immu-serological results showed that no significant differences(P>0.05)were recorded in malondialdehyde(MDA)and total antioxidant capacity(T-AOC)between the A97 group and the control.The A97 group was significant enhanced(P<0.05)of Serum lysozyme activity(LZM)and total protein level(TP)compared to control,but it was significant reduced(P<0.05)of acid phosphatase(ACP),alkaline phosphatase(AKP),catalase(CAT),superoxide dismutase(SOD)and glutathione(GSH)compared to control.The survival rate of the T.ovatus subjected to V.ponticus within 7 day were significantly different(P<0.05)between the A97 group and the control group.The qPCR results show that in the head kidney,the A97 group can significantly increase the amount expression of TLR9 gene(P<0.05).In the intestinal tissue,the A97 group can significantly enhanced the expression of TLR8 gene(P<0.05),but the quantity expression of TLR9 gene is not obvious(P>0.05).In other tissue,the A97 group can not significantly enhanced the expression of TLR8 gene and TLR9 gene(P>0.05)... |
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