Development Of Inactivated Vaccines Against Two Bacterial Pathogens Of Chinemys Reevesiis And Preliminary Study On Cell Culture Of Tilapia

In 2018,the total number of turtle breeding in the country was 47676 tons,an increase of 4.10%over 2017;the national freshwater cultured fish species was 25.44million tons,of which tilapia cultured production was 1.62 million tons,accounting for6.4%.In recent years,the intensification of the aquaculture industry has increased,and epidemics,especially bacterial diseases,have become one of the main problems restricting the development of the industry.Klebsiella Pneumoniae and Edwardsiella tarda are common bacterial pathogens in turtles.Tilapia’s Edwardsiellosis and Streptococcus agalactiae diseases are two major outbreaks.The above three bacteria are all zoonotic bacteria,which not only affect the sustainable development of the breeding industry,but also pose a threat to human life and health.The development of turtle vaccine is relatively lagging behind.The vaccines for Klebsiella pneumoniae and Edwardsiella tarda are still in the blank.Although Streptococcus agalactiae and Edwardsiella tarda have been studied by many scholars,there are relatively few studies at the cellular level.therefore,this article carried out the following work:1. S27 and S28 pathogenic bacteria isolated from the diseased tortoise（Chinemys reevesii）were identified by purification,growth characterization,biochemical experiments,and molecular biology experiments,and the LD₅₀was determined respectively,and 23 drug pairs were selected.Isolate the strains for drug sensitivity experiments.The results showed that S27 and S28 were identified as Klebsiella pneumoniae and Edwardsiella tarda.The LD₅₀for turtles was 3×10⁷CFU/m L and2.4×10⁶CFU/m L,respectively.In the drug susceptibility test,Klebsiella pneumoniae was only highly sensitive to cefepime,moderately sensitive to aztreonam,tetracycline,insensitive to 20 antibiotics such as florfenicol,amoxicillin,and extremely resistant.Edwardsiella tarda is highly sensitive to cefepime,aztreonam,enrofloxacin,florfenicol,etc.moderately sensitive to neomycin,azithromycin,chloramphenicol,etc.,not to amoxicillin,cefalexin sensitive.2. Explore the production conditions of inactivated vaccines of Klebsiella pneumoniae and Edwardsiella tarda,using the best inactivation conditions to inactivate.After the safety test was passed,70 turtles were immunized for the first time by intraperitoneal injection,and 40 of them were immunized a second time after 14 days.During the 42 days of the experiment,5 turtles were selected randomly every 7 days to collect blood from the jugular sinus,and the serum was separated to determine the lysozyme activity,C3 complement activity and serum antibody titer in the serum.On the 14th day after the first immunization and the second immunization,20 mice were challenged with the live bacteria concentration of 5LD₅₀to obtain mortality and calculate the immune protection rate.The results showed that the best preparation conditions for inactivated seedlings were 0.03%formaldehyde concentration,30℃,and48 hours inactivation.The lysozyme activity reached its peak on the 35th day,and was significantly higher than that of the control group.The Klebsiella pneumoniae experimental group had the highest C3 complement expression on 28d,and the Klebsiella pneumoniae group had increased antibody titers on the 14th day after two immunizations,which were 4log2 and 6log2,but the maintenance time was shorter and the decline rate was fast,The immune protection rate was 28%and 50%;the C3complement of the Edwardsiella tarda experimental group reached its peak on the 35th day,and the titer of the antibody on the 14th day of the first immunization of the Edwardsiella tarda was 6log2.It increased to 10log2 on the 42nd day,and there was no downward trend yet.The immune protection rates were 60%and 73%.3. Using tissue block adherence method,choose M199 medium containing 2% double antibody and 20%FBS,can successfully culture the primary cells of turtle heart,liver and spleen,the heart and spleen begin to migrate out of cells on 7d,liver tissue The cells began to migrate out around 10 days,and the heart,liver,and spleen cells reached more than 80%of the bottom area of the culture flask on the 35th,40d,and 50d monolayers.The heart cells are mostly triangular,the liver cells are mostly polygonal,and the spleen cells are mainly elongated and are fibroblasts.The heart and liver cells were passed to the sixth generation.Due to the large size,flat and multilateral growth,and slow growth of the dominant cells,the spleen cells fell off under normal growth conditions,and all the cells passed to the eighth generation.4. Establish tilapia brain cell line by normal temperature trypsin digestion method,screen the optimal growth conditions,count the survival rate of the cells by cryopreservation and recovery,and use the Streptococcus agalactiae and Edwardsiella tarda for the 30th generation cells Stimulate by time to explore the regulation of immune factors of tilapia brain cells stimulated by Gram-positive bacteria and Gram-negative bacteria.The results showed that the single-cell suspension of tilapia brain cells after pancreatin digestion began to adhere to the cells 7 days after inoculation,and the bottom of the bottle was over 30 days to be passaged.It has been passed to 40generations,and the 5th generation cells are frozen.After 2 months,the cell recovery survival rate is about 75%,the optimal medium for cell growth is M99 medium,the optimal growth temperature is 28℃,the optimal serum concentration is 15%,both bacteria inhibit My D88 expression,first After inhibiting the expression of NF-κB,the expression of NF-κB is induced,and the expression of IL-1?and TNF-αis induced.Among them,the gram-negative bacterium slows Edwardsella tarda to induce IL-1?more than the gram-positive bacterium Streptococcus agalactiae.,And the induction of TNF-ɑnisin is significantly stronger than that of Edwardsiella tarda.