Serogroup, fimbriae và các độc tố được sở hữu bởi các chủng ứng cử viên Các đặc tính độc lực (OK-kháng nguyên serogroup, fimbriae và các độc tố) trong ba chủng được lựa chọn để sản xuất vắc xin đã được xác nhận lợn và Đơn vị nghiên cứu độc lập (Phru), Victoria Sở Công nghiệp tiểu học (Bảng 1). Những dòng này sẽ được lưu giữ như là đóng băng các mẫu vật khô trong ba phòng thí nghiệm riêng biệt (NIVR, UQ và Phru) | APPENDIX ONE Characterization of vaccine candidate strains for E. coli vaccine 1 1. Serogroup fimbriae and enterotoxins possessed by the candidate strains The virulence characteristics OK-antigen serogroup fimbriae and enterotoxins of the three strains selected for vaccine production were independently confirmed by The Pig Health and Research Unit PHRU Victorian Department of Primary Industry Table 1 . These strains have been stored as freeze dried specimens in three separate laboratories NIVR UQ and PHRU . Table 1 E. coli strains used for the preparation of vaccine Designation of E. coli vaccine strains Virulence Characteristics O-serogroup Fimbriae Enterotoxin s NVP613 CARD-VN1 O8 5F- STa STb LT NVP1402 CARD-VN2 O149 K91 F4 STa STb LT NVP1372 CARD-VN3 O64 F5 STa Negative for all five recognized fimbriae associated with porcine enterotoxigenic E. coli F4 F5 F6 F18 and F41 . May therefore possess a novel fimbrial antigen. 2. Attempts to produce diagnostic antisera at NIVR to rapidly and specifically identify the E. coli O8 5F- strains ie strains with NVP613 genotype A Crude whole cell method Step 1 Preparation of antigen and vaccination of rabbits Method - Grow isolate NVP613 5F- at 37oC for 6-8 hours on shaker. - Inactivate with formalin v v overnight. - Plate onto bacteriological agar to confirm inactivation. - Wash in PBS and centrifuge at 3000 rpm for 5-10 mins x3 . - Protein determination using the Lowry protein assay an optic density of determined at 280 nm contained an estimated protein concentration of 1 mg ml . - Vaccination schedule 2 rabbits were injected intravenously at 4-5 day intervals with and 1 mg of antigen respectively. - The rabbits were bled 6-7 days after last injection and the serum harvested by centrifugation. Step 2 Preparation of antigen for cross-absorption Note E. coli grown at 18-20oC will not produce fimbriae. Therefore to prepare sera that is specific for the possible new fimbrial type the antisera from rabbits is
