Safety Evaluation Of Recombinant Lactobacillus Casei Expression Of Alpha Beta2 Epsilon Beta1 Toxin Of Clostridium Perfringens And Analysis Of Immune Effect

Clostridium perfringens is the major pathogenic bacterium which can cause traumatic gas gangrene, alimentary toxicosis, enteritis necroticans and enterotoxaemia. Its causative agent is sixteen secretory exotoxin of clostridium perfringens, α, β, ε and ι are main toxins. Type A, Type B, Type C, Type D and Type E are five serotypes of clostridium perfringens, according to differennt types of exotoxin.Clostridium perfringens infection is the most common Zoonosis in human being, livestock, economic animals, wild animals, its acutely and rapidly death causing huge economic losses to the development of animal husbandry in China. The disease often caused due to variety toxins attacked, a single immunization of the toxins can no t effectively resist the heterologous toxin attacks, so preparing immunogen of a variety of toxins to resist to heterologous toxin attack have practical significance to effective prevention of Clostridium perfringens diseases.In this research we used rabbit as experiment animal which was he susceptible animal,and firstly made a safety evaluation of the recombinant Lactobacillus Casei pPG-2-α-β₂-ε-β₁/L.casei 393. we did research about the recombinant Lactobacillus casei digestive environment survivability and colonization ability of the digestive tract, productivity, blood index, histopathology observation, the capacity of its released into surrounding environment after orally administration. The results showed that the recombinant Lactobacillus Casei p PG-2-α-β₂-ε-β₁/L. casei 393 could keep14.9% survival rate in gastric fluids of p H 4.5 after 5h. It also showed more than 2% survival rate in the intestinal fluid environment and a good tolerance to 2% of bile and 8% of Na Cl. The colonization ability in intestinal tracts showed that p PG-2-α-β₂-ε-β₁/L.casei 393 would begin to colonize in the gut of rabbit after day 1 4 by intragastric administration and the c FDA-SE-labeled lactobacilli percentages were 15.1%,25.3%,48.2%,29.3% in duodenum, ileum,cecum and colon, respectively. These indicated that p PG-2-α-β₂-ε-β₁/L.casei 393 was able to colonize in different sections of rabbits intestine, and it can most colonize in cecum. The rabbits were not caused to death orally fed with pPG-2-α-β₂-ε-β₁/L.casei 393, while which could improve daily intake, daily weight gain compared with the PBS group rabbits. The detection result of physical indexes of blood, the liver and kidney function test and histopathological observation did not showed bad influence to the rabbits fed with p P G-2-α-β₂-ε-β₁/L.casei 393. The PCR detection results by fodder, water, cage swab and bedding indicated that p PG-2-α-β₂-ε-β₁/L.casei 393 could not released to the surrounding environment after oral administration.To explore the application value of recombinant of Lactobacillus casei with Clostridium perfringens α-β₂-ε-β₁ toxin, this experiment used rabbit as test animal for immunology response. The rabbits of Group I were fed with p PG-2-α-β₂-ε-β₁/L.casei 393, Group II were fed with p PG-2/L. casei 393 and Group III were fed with PBS as negative control. The procedure is: orally inject 5mL×1010CFU/mL number of bacterial, then immune every 2w, three times immunity in total, with each immunity period lasting for 3 days, and immunize rabbits one time per day. A fter immunization, we measure the secreted Ig A taken from rabbits excrement and nasal cavity wash and the level of specific IgG in rabbits blood serum. The result of ELISA showed that: In rabbits excrement, nasal cavity wash, we detect highly specific s Ig A, which were significantly different compared with p PG-2 /L.casei 393 and PBS group. Meanwhile, in rabbit serum, we also detect high level of anti-serum, which means the recombinant of Lactobacillus casei can induce local mucosal immune response, as well as stimulate humoral immune response in rabbit body. Besides, we toxify the rabbits, which is after its third period of immunization, with toxin of Clostridium perfringens, and then collected each group rabbits organs for histopathological observation, so as to determine the immune function of recombinant to antibody against α-β₂-ε-β₁ toxin in rabbits body, the result also showed that the antibody against α-β₂-ε-β₁ toxin, which is expressed after transformation of recombined Lactobacillus casei, can suppress 1LD100 of the natural toxin activity produced by Clostridium perfringens.In general, the recombinant Lactobacillus Casei pPG-2-α-β₂-ε-β₁/L.casei 393 had a good property of safety and could not cause physical indexes of blood, the liver and kidney function, histopathology change and also could not release to the surroundin g environment. It can also induce local mucosal immune response, as well as stimulate humoral immune response in rabbit body by oral administration, which would provide a good protection to the animals.The study could established a good foundation for application of Clostridium perfringens α-β₂-ε-β₁-toxin oral vaccine.