Study Of The Intestinal Flora Structure Of Grass Carp Infection With Aeromonas Hydrophila

Aeromonas hydrophila is the main pathogens of bacterial hemorrhagic disease for freshwater fish, widespread around the world. It can cause disease among different growth stages of the fish. The disease cycle is long and it cause high mortality. This often leads to huge economic losses in the aquaculture industry. Over the years, using antibiotics is the main way of the prevention and treatment of disease occurrence in fishery production, but it can cause a serious impact on the safety of environment and food. Currently, the research and development of probiotic for alternative antibiotics to control aquatic animal diseases becomes the focus and expectations. As an important source of probiotics development, the beneficial intestinal flora of aquatic animals has good prospects for study. This study collected grass carps suffering from varying degrees of bacterial septicemia from the breeding waters.Based on the isolation and identification of pathogen, we have studied the amount of pathogen in grass carp tissues and the intestinal microbial flora changes of grass carp, to discuss the relationship between pathogens and the species and quantity of intestinal flora.Then we made grass carps long-term infect with pathogenic bacteria to furtherly clarify the relations between the variation of the main intestinal flora of grass carp and the amount of pathogen, trying to find some probiotics which may antagonize pathogenic bacteria in the intestinal flora of grass carp. The study provided the basis for the development of disease-resistant probiotics of grass carp. Main results obtained as follows:1. The isolation and identication of the pathogens from grass carp suffered bacterial hemorrhagic disease: Collecting grass carps suffering from varying degrees of bacterial septicemia from Hanzhong, we got three bacterial strains with different forms from the grass carp suffered bacterial hemorrhagic disease, and the number of ZB-1 bacterial strain was maximum. The lethality rate of ZB-1 bacterial strain was 90% much than others in the bacterial attack test for healthy grass carp, using intraperitoneal injection method. We confirmed ZB-1 bacterial strain as the target bacterial strain. By physiological and biochemical reactions and the sequence alignment of 16 S rRNA gene, we confirmed ZB-1bacterial strain as Aeromonas hydrophila.2. The study of intestinal microbial flora changes of grass carp grass carp suffered bacterial hemorrhagic disease: Doing quantitative detection of ZB-1 bacterial strain in tissues of grass carp, we divided the nine diseased fishes into A, B, C three groups according to the low-to-high amount of bacteria in kidney. Using Illumina Mi Seq sequencing system to detect the intestinal microflora structure of the grass carps, we found that with the amount of bacteria in kidney of A, B, C three groups gradually increased, at the phylum level: the frequency of Firmicutes bacteria dropped by about 20%, the frequency of Proteobacteria bacteria dropped about 10%, the frequency of Fusobacteria bacteria presented a clear upward trend, with an average increase of about 20%; at the genus level: the frequency of Bacillus and Pseudomonas bacteria all dropped by an average proportion of about 10%, the frequency of Cetobacterium bacteria showed a clear upward trend, with an average rise of nearly 20%.3. The study of intestinal microbial flora changes of grass carp infected pathogens artificially: Making three different bacterial concentrations of pathogen and long-term soaking to infect grass carp, we got samples at 3 d, 6 d, 9 d, 12 d, 15 d. Using Illumina MiSeq sequencing system to detect the intestinal microflora structure of grass carp, we found that, at the phylum level, 3d, the frequency of Firmicutes decreased about 15% compared to the control, and then showed an upward trend with time; in terms of overall the frequency of Proteobacteria in the treatment groups, lower than the control; the frequency of Fusobacteria at 3d in the treatment groups, there is a 5%-30% increase when compared with the control and then decreased after reaching the highest. At the genus level, 3d, the frequency of Bacillus in the treatment groups reduced about 15%-20% compared with the control, then showed an upward trend with time; compared with the control, Pseudomonas and Lactococcus showed an upward trend with time, contrary Aeromonas had a frequency of 15%-20% dropped significantly; the frequency of Cetobacterium at 3d infection in the treatment groups, there a10%-30% increase when compared with the control, and then decrease after reaching the maximum.These results suggest that, when grass carp infected Aeromonas hydrophila, in the intestinal flora: the proportion of Firmicutes and Proteobacteria will decrease with the amount of pathogen infection increasing, the proportion of Fusobacteria will rise; the proportion of Bacillus will decrease with the amount of pathogen infection increasing, the proportion of Cetobacterium will rise, the ratio of Aeromonas will decline.