Mechnism Study Of The Risk Associated With Anti-infection Use Of Lactobacillus Spp.

Probiotics has been studied and applied as alternative of anbiotics for a few decades in aquatic animals. Commercial probiotics has been applied extensive in aquaculture practices. Adhesive ability was considered as an important characterization of lactobacillus. However, there has been nopublished, systematic comparison of probiotic effects of adhesive strains and non-adhesive strains on aquatic animal hosts in vivo. In addition, effects of ceasing supplement lactobacilluson fish anti-infection of pathogenic bacteria were few considered. This dissertation was aimed to investigate the effects of higher-adhesiveandless-adhesive lactobacillus on tilapia. Then an effective method for rapid evaluation of immersion challenge with G- pathogenic bacteria was established and applied to study the change of fish after ceasing dietary lactobacillus supplementation. We found that tilapia may be more sensitive to pathogen in some case and the pathogenetic mechanism was analyzed. At last, effects of sodium butyrate, a probiotic component of lactobacillus on intestinal damage caused by oxidized oil of common carp was assessed. Primary results were as follows: During a 5-week feeding trial the fish were fed a diet containing 0 to 109 cells/g feed of the two Lactobacillus strains, higher-adhesive L. brevis JCM 1170(HALB) or less-adhesive L. acidophilus JCM 1132(LALB). Samples of intestine, kidney, and spleen were taken at the start and at 10, 20, and 35 days for analysis of stress tolerance and cytokine gene mRNA levels and to assess the diversity of adhesive gut bacterial communities. A 14-day immersion challenge with Aeromonas hydrophila NJ-1 was also performed following the feeding trial. The results showed no significant differences in survival rate, weight gain, or feed conversion in the different dietary treatments. The adhesive gut bacterial communities were strikingly altered in the fish fed either HALB or LALB, but the response was more rapid and substantial with the adhesive strain. The two strains induced similar changes in the patterns(upregulation or downregulation) of intestinal, splenic or kidney cytokine expression, but they differed in the degree of response for these genes. Changes in intestinal HSP70 expression levels coincided with changes in the similarity coefficient of the adhesive gut bacterial communities between the probiotic treatments. The highest dose of the HALB appeared to protect against the toxic effects of immersion in A. hydrophila(P < 0.05). In conclusion, the degree to which lactobacillus strains adhere to the gut may be a favorable criterion in selecting probiotic strain for aquaculture. Tilapia fed different diets, basal(CK), FOS(0.1%), L. brevis JCM 1170(108 cell/g), L. brevis JCM 1170(108 cell/g) and FOS(0.1%), L. plantarum JCM1149(108 cell/g), L. plantarum JCM1149(108 cell/g) and FOS(0.1%) for 5 weeks. A 21-day immersion challenge with Aeromonas hydrophila NJ-1 was also performed following the feeding trial. After infected for 24 h, intestine samples were collected for measurement of alkaline phosphatase(IAP) activity and number of adhesive A. hydrophila NJ-1. Results showed that co-relative between relative level of protection(RLP) and IAP was significantly in short term(3, 4 d). And linear and quadratic regression between RLP and IAP was significantly at 21 d. IAP activities were consistent with the number of adhesive A. hydrophila NJ-1. These results indicated that IAP can be used as a rapid evaluation mehod for immersion challenge with G- pathogenic bacteria in aquaculture experiment. In another trial, tilapia fed basal diet(CK), L. brevis JCM 1170(108 cell/g) and FOS(0.1%)(1170 treatment), L. plantarum JCM1149(108 cell/g) and FOS(0.1%)(1149 treatment) for 2 weeks. At the end of the 2nd week(S0 day)and after ceasing dietary lactobacillus supplementation for 3 days(S3 day), fish were immersion challenged with A. hydrophila NJ-1 for 24 h. Results showed that IAP activities were lower in 1149 and 1170 treatments at S0 day when compared to control group. However, IAP activities of treatment 1149 were significantly higher at S3 days but not in treatment 1170. These suggested that continuously applied JCM1149 and JCM1170 can prevent A.hydrophila NJ-1 adhere to fish intestine. After stopping dietary lactobacillus, fish from JCM1149 treatment was more sensitive to A. hydrophila NJ-1. At the end of 2 weeks feeding with lactobacillus supplementation(S0 day) and after ceasing dietary lactobacillus supplementation for 3 days(S3) days, the numbers of JCM1149 and JCM1149 adhered on intestine were counted through pure culture on MRS agar plate. The number of JCM1170 and JCM1149 was reduce, the reduce degree of JCM1170 was not significantly while that of JCM1149 was extremely significantly. The intestinal adhesive bacterial community, bacterial inhibitation ability of intestinal mucin, intestinal metabolome and immune response of fish before or after ceasing dietary lactobacillus supplementation for 3 d were analyzed. We found that after ceasing dietary lactobacillus supplementation for 3 d, gut dysmicrobialism and gut barrier dysfuction were present. In addition, intestinal metabolite was obviously affected after ceasing dietary lactobacillus supplementation for 3 days. Therefore, during the reconstruction period of host intestinal bacteria after ceasing dietary lactobacillus supplementation, the host was more sensitive to pathogenic bacteria, which was related to the gut dysmicrobialism and gut barrier dysfuction and metabolic disorders. Butyric acid and butyrate are important effective component of LAB. We investigated the effects of different dietary sustained-release microencapsulated sodium butyrate(MSB)(0: non supplement, 1.5, 3.0 h) for control or oxidised soybean oil(oxi-oil) diets on fish production, intestinal mucosa condition, immunity and bacteria in juvenile common carp(Cyprinus carpio). Dietary MSB considerably increased the weight gain(WG) and reduced the feed conversion ratio(FCR) within the control or oxi-oil groups despite no statistical significance. Fish gut mucosa was damaged in the oxi-oil group without MSB, but normal appearance was present when fish were fed MSB1.5 and MSB3.0 in the oxi-oil group. Microvilli density was increased in fish fed the MSB1.5 and MSB3.0 treatments in the oxi-oil group(P < 0.001), and microvilli density was affected by different pre-fed diets in the mid-gut(MG)(P < 0.001) while it was affected by different sustained-release time of MSB in the distal gut(DG)(P = 0.003). Interaction between pre-fed diet and dietary sustained-release time of MSB was significant for the relative expression levels of gut HSP70, pro-inflammatory cytokines(IL-1? and TNF-?), and anti-inflammatory cytokines(TGF-?) genes within each segment besides HSP70 in the DG and IL-1? in the fore gut(FG). However, modulation of gut adhesive bacteria within each segment investigated was not obvious when common carp were fed diets with MSB, as similarity coefficients of > 0.79 were observed. These results indicated that butyrate as a probiotic component could be used as dietary supplement to repair or prevent damage to the intestine of carp fed oxidised oil, and butyrate did not interference gut bacterial communities.