The Development Of Toxiod Vaccine And Anti-Toxin Serum Of Clostridium Perfringens Type E

Clostridium perfringens?C.perfringens?,formerly known as Clostridium welchii,is a member of the genus Clostridium,which can form spores and secrete exotoxins rapidly.According to the four major exotoxins produced?alpha,beta,epsilon and iota toxins?,it is divided into five toxin types,namely C.perfringens type A,B,C,D and E.The onset or death of animals depends on secretion of the exotoxins.C.perfringens type E mainly secretes alpha and iota toxin,which can cause dysentery in yaks and lambs,causing varying degrees of damage to the intestinal tracts of calves and lambs,affecting the absorption of nutrients by animals,and even the death of animals.In order to understand,prevent,and treat diseases caused by C.perfringens type E,the study was divided into three parts.The first part:The study of pathogenicity of C.Perfringens type E.In order to understand the pathogenicity of the exotoxins of C.perfringens type E,this part of the experiment measured the LD₅₀0 of the exotoxin on mice.And pathological changes in the intestines and liver are also tested.The exotoxins were produced through the process of resuscitating,proliferating,and secretion.The exotoxins were de-salted and concentrated by Sephadex G-25 and dialysis bags to obtain the exotoxin that were refined.At the same time,by setting up a standard curve and equation for bovine serum albumin,the concentration of exotoxin extracted in the test was measured to be 1.2 mg/mL.The exotoxin was refined twice-fold dilution,and the selected concentration gradient was used to challenge the mice by intraperitoneal injection and mortality was recorded.Result showed that the LD₅₀0 is 2.84 mg/kg through the Reed-Muench method.In order to further understand the damage of the animal's intestine and liver caused by the exotoxins of C.perfringens type E,this experiment used the mouse as an animal model and adopted the jejunum,colon and liver of the death mice.The tissues were subjected to paraffin sectioning and HE staining.By pathological section observation,the mice in the experiment showed severe damage relative to the control group.The second part:The preparation of C.perfringens type E toxiod vaccine.In order to prevent the harm caused by C.perfringens type E,this part of the experiment prepared a C.perfringens type E white-oil adjuvant toxiod vaccine and an aluminum hydroxide toxiod vaccine.First of all,the inactivation condition of the exotoxins of C.perfringens type E was at 37°C,0.3%formaldehyde,and inactivation for 24 h.Two kinds of adjuvants were added to make C.perfringens type E toxiod vaccine.According to Veterinary Biological Products Quality Standards of the People's Republic of China,the vaccine was tested for safety and sterility.The prepared white oil adjuvant and aluminium hydroxide gum toxoid vaccine were inoculated to New Zealand rabbits and the serum antibody abundance was determined by ELISA.The results showed that the white oil adjuvanted toxoid vaccine reached its highest level in the 5th week,and the antibody level was maintained for 18 weeks;the serum antibody level of New Zealand rabbits immunized with aluminum hydroxide toxoid vaccine reached the highest level in the third week,and the maintenance time was shorter.In the fifth week,the antibody titer of rabbit serum immunized with white oil adjuvant toxoid vaccine was 1:3200;the serum antibody titer of rabbits vaccinated with aluminum hydroxide toxoid vaccine was 1:1600.Further,the protective effect of the white oil adjuvant toxoid vaccine on mice was tested by the toxin challenge experiment.The results showed that the mice were completely protected by the subcutaneous injection of 0.2 mL of the white oil adjuvant toxoid vaccine at 4 weeks.This proves that the white oil adjuvant toxoid vaccine has a reliable immunoprotective effect and can be used in production.The third part:The preparation of C.perfringens type E antitoxin serum.In order to be able to treat animals suffering from C.perfringens type E disease,this part of the experiment conducted the preparation of C.perfringens type E antitoxin serum.The prepared C.perfringens type E white-oil adjuvant toxoid vaccine was inoculated subcutaneously to sheep according to a certain immune procedure,and the serum was collected aseptically.Neutralization experiments determined that this hyperimmune serum can neutralize 50MLD.In conclusion,the research in this paper can provide basis for further basic research and prevention of C.perfringens type E disease.Domestic preparation of this hyperimmune serum for the first time provides protection for the emergency treatment of C.perfringens type E infections.