The partial purification of clostridium perfringens toxins and production of Quaderaly toxoid vaccine.

number: 
356
إنجليزية
Degree: 
Imprint: 
Biotechnology
Author: 
Ali Abdul Rhman Al-Sheikhly
Supervisor: 
Dr. Ala H. AL-Omran
Dr. Faddeel Abaas Al-Jasim
year: 
1999
Abstract:

Three types of Clostridium perfringens were adopted in this study. The type A was used to produced alpha toxin, type C was used to produced beta and enterotoxin, while the type D was used to produce epsilon toxin. The virulance of Egyptian and Borose Wellcome strains (8237, 3180 and 8346 of types A, C and D) was compared before and after reactivation in guinea pigs. The minimum lethal dose (MLD) per ml in mice was observed with Borose Wellcome strains after reactivation in guinea pigs to give 50, 1000 and 3000 MLD/ml of types A, C and D respectively. In addition to the MLD measurement, the erythemal activity determination was depend in order to measured the enterotoxin units per ml, also lecithinase activity of alpha toxin was adopted to study the stability of the toxin. The beta, entero and epsilon toxins were precipitated with ammonium sulphate, while the DEAE-Sepharose was used to purify alpha and epsilon toxin. Further purification of alpha and beta was performed by Sephadex G-100. The purified alpha toxin followed by gel filtration gave shorter death time (30 minutes) in mice at fraction no. 79, while the gel fractionation of beta toxin obtained shorter death time (2 minutes) in mice at fraction no. 26. The ion-exchange of epsilon fraction gave 4000 MLD/ml in mice, while the enterotoxin precipitated with 80% and 15% saturated ammonium sulphate caused 1.05 cm in diameter as erythemal reaction (equivalent to 26 U/ml) in the skin of guinea pig. Detoxification of purified toxins achieved by 0.6% formaline (40% concentration) at 6.8-7.0 pH and 37°C within 3 days. These toxoids was adopted to produce antitoxin in rabbits. All the toxoids and antitoxins obtained was subjected to the standardization methods described in the British veterinary codex (1970). The total combining power of beta and epsilon toxoid was 16 and 4 unit/ml, while the potency of prepared anti beta and anti epsilon toxins was 64 and 321.U/ml. The potency of prepared anti alpha toxin revealed power of neutralization of 10 MLD/ml alpha toxin prepared in this study, while the potency of antienterotoxin was 10 LU/ml when examined to neutralized the erythemal activity of enterotoxin in the skin of guinea pig. Equal concentration of alpha, beta, entero and epsilon toxoid was mixed and 1, 0.5,0.25, 0.125 mg/ml of the quderaly toxoids preparation was used to inject subceutaneosly in 15 sheep purchased from AL-Dabooni farm in the south east of Baghdad. The higher optical density (OD) (0.187) was observed in animals sera had been injected with 1 mg/ml, while the higher OD was observed with anti enterotoxin followed by anti beta, anti alpha and anti epsilon toxins appeared in the polyvalent antitoxin of animals injected with 0.125 mg/ml