Screening And Evalution Of Potential Probiotics Against Disease In Litopenaenaeus Vannamei

Part 1: The screening and application of probiotics in aquatic animalsWith the rapid development of large-scale and intensive culture, the diseases occur frequently. In recent years, people attempt to use probiotics to improve the farming ecological environment and restrain pathogenic microorganisms, thereby reducing the occurrence of diseases. In this part, the definition and biological function of probiotics are summarized; it also introduces the approach of srains screening and identification. In effects evaluation of probiotics, we put the emphasis on the following four aspects: promoting the growth of shrimp, improving water quality, strengthening the immunity and inhibiting pathogenic microorganism. Finally, based on aspects above, we discuss the main problems and the development prospects of the screening of the disease-resist microorganisms. Part2: The screening and identification of probiotics against vibrios that resulted in AHPNDA total of 244 bacterial strains were isolated from the gut of shrimps and screened for antagonistic activity against Vibrio parahaemolyticus, which results in acute hepatopancreatic necrosis disease(AHPND). Two of the isolated strains(m8, a22) displayed antagonistic activity against AHPND-pathogenic Vibrioparahaemolyticus(VPAHPND), which opens the way to their potential use as probiotics in aquaculture.(Zone diameters were 9.33±0.61 mm for m8 and 4.33±0.11 mm for a22). The putative probiotic strains m8 and a22 were identified as Pseudoalteromonas spp. based on their morphological properties、biochemical properties and 16 S r RNA gene sequencing.The present study confirmed the potential of probionts, under both in vivo and vitro conditions, which in turn can be used for controlling AHPND. Part3:The effects of disease resistantance and immunocompetence of two potential probiotics to LitopenaeusvannameiThe two probiotic strains(m8, a22) were then added to the feed of Litopenaenaeus vannamei. The shrimps were fed four different diets: Group A diet with m8 dosed at 106 CFU/m L; Group B diet with a22 dosed at 106 CFU/m L; Group C mixed diet comprised of strains a22 and m8 at a ratio of 1:1, dosed at107 CFU/m L; Group D control diet with no probiotics. Each diet was randomly fed to quadruplicate groups of 20 shrimps. Bacteriological analysis indicated that additional probiotics in the diets would not affect the total heterotrophic bacterialnumbers. The counts in the intestinal tracts of the two treated groups(A and B) were significantly lower than in the control group(D).After a 28-day feeding trial, the shrimps were challenged with VPAHPND. The cumulative mortalities of shrimps fed with m8 or a22 only were 30.0%±0.18 and 68.3%±0.03, respectively, significantly lower than the control group, which had a cumulative mortality of 95.0 % ±0.15. The q PCR assay results demonstrated that the quantities of virulence plasmids in the shrimp gills ranged from 8.43±3.10×101 to 6.63±0.83×103 copies per μg DNA. Part4:Effects of Bacillus cereus PC465 as probiotics adding to the rearing water on disease resistance and immune gene expression of Litopenaeus vannameiThis part evaluated the effects of three different doses of bacillus cereus as probiotics adding to the rearing water on bacteria numbers, water quality and the disease resistant ability of Litopenaeus vannamei in comparison to untreated control group. The experimental results show that the addition of the probiotics can reduce the number of bacteria in the intestinal tract of Litopenaeus vannamei, which was significantly different compared to the control group, it also reduce the number of Vibrio spp. in culture water(P<0.05). However, no significant difference in the level of ammonia nitrogen and nitrite were recorded in this study. Four weeks later, shrimp were challenged with white spot syndrome virus(WSSV), statistical analysis show the cumulative mortality of shrimps of the group H(106CFU/m L)and group M(105CFU/m L) were 64﹪and74﹪,significantly decreased than the control group of 100﹪mortality(P<0.05. Subsequently the quantitative PCR was also employed to determine the m RNA level of three immune related genes, statistical analysis revealed Lipopolysaccharideaβ-1,3-glucan-binding protein(LGBP),beta-1,3-glucan-binding protein-Lipoprotein(βGBP-HDL), Heat shock proteins(Hsp70)show a significantly up-regulated after the infection of WSSV. The above results demonstrated that probiotics can stimulate the expression of immune related genes, which can in turn improve the disease resistant ability.