Preparation And Protective Effect Evaluation Of Clostridium Perfringens Type G Toxoid Vaccine

Clostridium perfringens is a common pathogenic microorganism in nature.It not only exists widely in the external environment such as soil,feces and sewage,but also in the intestinal tract of animals.Under certain conditions,Clostridium perfringens can proliferate rapidly in animal intestinal tract of animals,cause Clostridium perfringens disease in livestock and poultry.According to the toxin of Clostridium perfringens,it can be divided into seven types:A,B,C,D,E,F and G.Studies in recent years have shown that Clostridium perfringens type G plays an important role in the occurrence of chicken necrotizing enteritis（NE）.From New Year’s day of 2020,with the comprehensive prohibition of antibiotics as feed additives in China,the incidence rate of chicken necrotizing enteritis caused by Clostridium perfringens type G increased gradually,which has brought great harm to poultry breeding industry in China.Therefore,the purpose of this study was to develop a safe and efficient Clostridium perfringens toxoid vaccine to prevent and control necrotizing enteritis in chickens.Firstly,the optimal culture time of Clostridium perfringens type G isolated and preserved in the laboratory in the toxin producing medium was explored,and the optimal culture time was determined to be 8 h.The bacterial solution after toxin production was centrifuged at high speed at 4°C,and the supernatant was filtered to remove the bacteria to obtain the natural exotoxin.Then the pathogenicity of exotoxins of two G-type strains to mice was tested,and the 7a9-2 strain with strong virulence was selected as the candidate strain for vaccine preparation.Finally,the natural toxin filtered and sterilized was inactivated with formaldehyde.The optimum inactivation conditions were determined as 37℃,0.4%formaldehyde and 72 hours.The water phase（inactivated exotoxin）and oil phase（white oil adjuvant）are mixed in a volume ratio of 1:2 to prepare a G-type Clostridium perfringens toxoid vaccine.Then the physical characteristics,safety and sterility of the prepared vaccine were tested.In this study,160 one-day-old Sanhuang chickens were randomly divided into four groups A,B,C and D,with 40 chickens in each group.The group A was the Primary immunization control group and group B was the Primary immunization group;Group C was the secondary immunization control group,and group D was the secondary immunization group.As this bacterium is a kind of conditional pathogen,coccidia and Clostridium perfringens were used in the challenge experiment of this experiment.Group B was immunized with 0.5ml of Clostridium perfringens type G toxoid vaccine on day 7.Groups A and B took 1ml chicken coccidia oocysts（22000/ml）orally at per chicken on day 25,and infected with Clostridium perfringens type G（1×10~8CFU/ml,2 ml/chicken）on days29,30,31,and 32,once in the morning and once in the evening.Group D was immunized with0.5ml of Clostridium perfringens type G toxoid vaccine at the on day 7 and 1ml of Clostridium perfringens type G toxoid vaccine at the on day 28.Groups C and D took 1ml chicken coccidia oocysts（22000/ml）orally per chicken on day 46,and infected with Clostridium perfringens type G（1×10~8CFU/ml,3 ml/chicken）on days 50,51,52,and 53,once in the morning and once in the evening.During the whole experimental process,they were fed with full-price pelleted feed.The feeding cycle of group A and B was 42 days,and that of group C and D was 63 days.Chicken feed intake was recorded every day,the body weight was weighed every week,and finally the production performance was counted.Serum was collected weekly for antibody testing.On days 2 and 4 after the completion of virus attack,6 chickens in each group were randomly selected for autopsy,the pathological changes were observed and the pathological score was counted.The final experimental results are as follows:1.Intestinal lesions score:At the 34-day and 36-day autopsy of the group A and group B,it was found that the intestinal mucosal lesions were more serious in group A than in group B.The incidence rate in group A was 58.3%,and the incidence rate in group B was 16.7%.At the 55-day and 57-day autopsy of the group C and group D,it was found that the intestinal mucosal lesions were more serious in group C than in group D.The incidence rate in group C was 33.4%,and incidence rate of group D was 8.3%.Through the above analysis,it is found that vaccine immunization can significantly reduce the degree of intestinal lesions.2.Growth performance:Through comparison,it was found that the body weight of group A was significantly lower than that of group B（P<0.05）,and the weight of group C was extremely significantly lower than that of group D（P<0.01）.In average daily weight gain,group B was increased by 16.3%compared with group A,and there was a extremely significant difference between the two groups（P<0.01）;group D was increased by 9.2%compared with group C,and there was a significant difference between the two groups（P<0.05）.In terms of average daily feed intake,there was no significant difference between group A and group B,and between group C and group D（P>0.05）;In terms of feed conversion ratio,group B was extremely significantly higher than group A（P<0.01）,group D was significantly higher than group C（P<0.05）,but there was no significant difference between group A and group C,and between group B and group D（P>0.05）.Through the above analysis,it is found that the vaccine immunization can effectively slow down the decline of animal production performance.3.Detection of antibodies against Clostridium perfringens type G:Firstly,the exotoxin of the G-type strain was used as the antigen,and the optimal antigen coating concentration of indirect ELISA was explored,and finally the optimal coating concentration was determined to be 4μg/ml.Other conditions such as sealing conditions,sample reaction time and enzyme-labeled secondary antibody reaction time refer to the conditions explored by Xiaojia Lin,and color development conditions refer to the instruction manual of TMB single-component color developing solution.Finally,an indirect ELISA method was established to detect the rise and decline of antibodies in the serum of experimental chickens.After the serum to be tested was diluted 100 times,the indirect ELISA results showed that the antibody level increased rapidly in the first three weeks after primary immunization,and the antibody level reached 6.34×log₂,after challenge,the antibody titer reached the highest level at the 4th week,up to 6.83×log₂,but the antibody level decreased in the fifth week.After the second immunization,the antibody titer has been rising slowly for 5 weeks,and also upward trend after the challenge,up to 8.34×log₂.Through the above analysis,it was found that the antibody level was significantly increased after secondary immunization.To sum up,the vaccine of Clostridium perfringens toxoid prepared in this experiment can not only make chickens obtain higher anti-G exotoxin antibody level,but also significantly reduce the intestinal lesions after Clostridium perfringens infection,slow down the decline of its production performance,and have a good immune protection against chicken necrotizing enteritis.