Live vaccine against colibacillosis

Abstract

A vaccine for the immunization of domestic fowl, such as turkeys and chickens, against E. coli infections (Colibacillosis) is disclosed which contains an effective amount of a live temperature sensitive mutant of E. coli dispersed in a physiologically acceptable, non-toxic liquid vehicle. The E. coli mutant disclosed exhibits growth at 32° C. but not at 41° C. and has a reversion frequency of less than about 1×10 −8 .

What is claimed is:  
     
         1 . A vaccine comprising an immunogenic amount of a live temperature sensitive mutant of  Escherichia coli  dispersed in a physiologically acceptable non-toxic liquid vehicle, which amount is effective to immunize a susceptible domestic fowl against colibacillosis, said mutant exhibiting growth at 32° C. but not at 41° C. and having a reversion frequency of less than about 1×10 −8 .  
     
     
         2 . The vaccine of  claim 1  wherein said temperature sensitive mutant is a mutant of  E. coli  serotype 078.  
     
     
         3 . The vaccine of  claim 1  wherein said temperature sensitive mutant is a mutant of  E. coli  serotype 01a.  
     
     
         4 . The vaccine of  claim 1  wherein said temperature sensitive mutant is a mutant of  E. coli  serotype 02a.  
     
     
         5 . The vaccine of  claim 1  wherein the immunogenic live temperature sensitive mutant colonizes the upper nasal mucosa of said domestic fowl for at least 20 days post inoculation.  
     
     
         6 . The vaccine of  claim 1  wherein said mutant has a reversion frequency of less than 1×10 −9 .  
     
     
         7 . The vaccine of  claim 1  wherein said domestic fowl is a turkey.  
     
     
         8 . The vaccine of  claim 1  wherein said domestic fowl is a chicken.  
     
     
         9 . A method to immunize a domestic fowl against colibacillosis comprising administering to said domestic fowl an effective amount of the vaccine of  claim 1 .  
     
     
         10 . The method of  claim 9  wherein the vaccine is administered by aerosol.  
     
     
         11 . The method of  claim 9  wherein the vaccine is administered orally.  
     
     
         12 . A method for obtaining a temperature sensitive mutant of  Escherichia coli  capable of colonizing the nasal mucosa of a domestic fowl, comprising the steps of: 
 (a) treating a culture of  Escherichia coli  with a mutagen and a protein synthesis inhibitor, said mutagen and protein synthesis inhibitor being employed in an amount sufficient to maximize mutation and minimize reversion frequency;    (b) selecting said culture mutants exhibiting growth at 32° C. but not at 41° C. and having a reversion frequency of less than 1×10 −8 .    
     
     
         13 . The method of  claim 12  wherein said mutagen is N-methyl-N-nitro-N-nitrosoguanidine.  
     
     
         14 . The method of  claim 12  wherein said protein synthesis inhibitor is chloramphenicol.  
     
     
         15 . The method of  claim 13  wherein said culture is treated with about 25 μg/ml chloramphenicol.  
     
     
         16 . A method for obtaining a stable temperature sensitive mutant of  Escherichia coli  capable of colonizing the nasal mucosa and enhancing immunological resistance to colibacillosis in domestic fowl, comprising the steps of: 
 (a) treating a culture of a parental strain of  E. coli  with amounts of a mutagen and protein synthesis inhibitor sufficient to produce temperature sensitive mutants having a reversion frequency of less than 1×10 −9 ;    (b) incubating a sample of said treated culture on agar plates at about 32° C. for a period of time sufficient to produce colony growth;    (c) employing replica plating to transfer colonies onto agar plates incubated at 32° C. and 41° C.;    (d) selecting mutants based on smaller colony morphology than the parental strain and inhibited growth at 41° C.;    (e) growing said selected mutants at 32° C. and 41° C. to determine mutant stability against reversion; and    (f) selecting a mutant having a reversion frequency of less than 1×10 −9 ;    
     
     
         17 . The method of  claim 16  wherein said culture is treated with about 25 μg/ml of chloramphenicol.  
     
     
         18 . The method of  claim 17  wherein said culture is treated with about 1000 μg/ml N-nitro-N-nitrosoguanidine.