Construction Of Bivalent Streptomycin-dependent Strains For Fowl Cholera By Protoplast Fusion

With streptomycin-dependent mutants Pst(8:A) and Cst(5:A) which were mutagenized respectively from P Multocida P1059(8:A)and C4~1(5:A), drug-resistant strains Psa(Ampr,Kans) and Csk(Kanr,Amps) were induced by Ampicillin and Kanamycin. Using Psa and Csk as parental strains, their protoplasts were prepared by lysozyme-EDTA and fusion were accomplished with 40% PEG4000 as assistant solvent in the presence of DNase I. Four fusion strains (F3, F4, F7 and F 11) were obtained which were all streptomycin dependent. They possessed the resistance to the two antibiotics (Ampr,Kanr) and the serotypes of the two parental strains (8:A,5:A) Their hereditary propett~es were identical with the two parental strains, such as morphology, staining, biochemical features, etc. After a series of passages, the hereditary properties of the 4 fusion strains were found to be stable. Mice challenged. 14 days after immunization with the culture prepared from the fusion strain F3, showed the protection rates as high as 100% respectively, when challenged with 10 minimum lethal dose (MLD) in single and mixed culture of the 2 virulent strains (P1059 and C48.). It is suggested that the further work should be done in chickens, using the fusion strains as immunogens.