Attenuated mycoplasma gallisepticum strains

Abstract

The present invention provides live, attenuated Mycoplasma gallisepticum bacteria that exhibit reduced expression of a protein identified as MGA_0621. In certain embodiments, the attenuated bacteria may additionally exhibit reduced expression of one or more proteins selected from the group consisting of pyruvate dehydrogenase, phosphopyruvate hydratase, 2-deoxyribose-5-phosphate aldolase, and ribosomal protein L35, relative to a wild-type M. gallisepticum bacterium. Also provided are vaccines and vaccination methods involving the use of the live, attenuated M. gallisepticum bacteria, and methods for making live attenuated M. gallisepticum bacteria. An exemplary live, attenuated strain of M. gallisepticum is provided, designated MGx+47, which was shown by proteomics analysis to exhibit significantly reduced expression of MGA_0621, and was shown to be safe and effective when administered as a vaccine against M. gallisepticum infection in chickens.

1 . A live, attenuated  Mycoplasma gallisepticum  bacterium that exhibits reduced expression of a protein having the amino acid sequence of SEQ ID NO:1, relative to a wild-type  Mycoplasma gallisepticum.    
     
     
         2 . The bacterium of  claim 1 , wherein said bacterium exhibits at least 25% less expression of said protein relative to a wild-type  Mycoplasma gallisepticum.    
     
     
         3 . The bacterium of  claim 2 , wherein said bacterium exhibits at least 50% less expression of said protein relative to a wild-type  Mycoplasma gallisepticum.    
     
     
         4 . The bacterium of  claim 3 , wherein said bacterium exhibits at least 75% less expression of said protein relative to a wild-type  Mycoplasma gallisepticum.    
     
     
         5 . The bacterium of  claim 1 , wherein said bacterium further exhibits reduced expression of pyruvate dehydrogenase relative to a wild-type  Mycoplasma gallisepticum.    
     
     
         6 . The bacterium of  claim 1 , wherein said bacterium further exhibits reduced expression of phosphopyruvate hydratase relative to a wild-type  Mycoplasma gallisepticum.    
     
     
         7 . The bacterium of  claim 1 , wherein said bacterium further exhibits reduced expression of 2-deoxyribose-5-phosphate aldolase relative to a wild-type  Mycoplasma gallisepticum.    
     
     
         8 . The bacterium of  claim 1 , wherein said bacterium further exhibits reduced expression of ribosomal protein L35 relative to a wild-type  Mycoplasma gallisepticum.    
     
     
         9 . The bacterium of  claim 1 , wherein said bacterium further exhibits reduced expression of pyruvate dehydrogenase, phosphopyruvate hydratase, 2-deoxyribose-5-phosphate aldolase, and ribosomal protein L35 relative to a wild-type  Mycoplasma gallisepticum.    
     
     
         10 . A vaccine composition comprising:
 (a) a live, attenuated  Mycoplasma gallisepticum  bacterium that exhibits reduced expression of a protein having the amino acid sequence of SEQ ID NO:1, relative to a wild-type  Mycoplasma gallisepticum ; and   (b) a pharmaceutically acceptable carrier.   
     
     
         11 . The vaccine composition of  claim 10 , wherein said bacterium exhibits at least 25% less expression of said protein relative to a wild-type  Mycoplasma gallisepticum.    
     
     
         12 . The vaccine composition of  claim 11 , wherein said bacterium exhibits at least 50% less expression of said protein relative to a wild-type  Mycoplasma gallisepticum.    
     
     
         13 . The vaccine composition of  claim 12 , wherein said bacterium exhibits at least 75% less expression of said protein relative to a wild-type  Mycoplasma gallisepticum.    
     
     
         14 . The vaccine composition of  claim 10 , wherein said bacterium further exhibits reduced expression of pyruvate dehydrogenase relative to a wild-type  Mycoplasma gallisepticum.    
     
     
         15 . The vaccine composition of  claim 10 , wherein said bacterium further exhibits reduced expression of phosphopyruvate hydratase relative to a wild-type  Mycoplasma gallisepticum.    
     
     
         16 . The vaccine composition of  claim 10 , wherein said bacterium further exhibits reduced expression of 2-deoxyribose-5-phosphate aldolase relative to a wild-type  Mycoplasma gallisepticum.    
     
     
         17 . The vaccine composition of  claim 10 , wherein said bacterium further exhibits reduced expression of ribosomal protein L35 relative to a wild-type  Mycoplasma gallisepticum.    
     
     
         18 . The vaccine composition of  claim 10 , wherein said bacterium further exhibits reduced expression of pyruvate dehydrogenase, phosphopyruvate hydratase, 2-deoxyribose-5-phosphate aldolase, and ribosomal protein L35 relative to a wild-type  Mycoplasma gallisepticum.    
     
     
         19 . A method of vaccinating an animal against  Mycoplasma gallisepticum  infection, said method comprising administering to an animal an immunologically-effective amount of a vaccine composition, said vaccine composition comprising a live, attenuated  Mycoplasma gallisepticum  bacterium having reduced expression of a protein having the amino acid sequence of SEQ ID NO:1, relative to a wild-type  Mycoplasma gallisepticum.    
     
     
         20 . The method of  claim 19 , wherein said bacterium exhibits at least 25% less expression of said protein relative to a wild-type  Mycoplasma gallisepticum.    
     
     
         21 . The method of  claim 20 , wherein said bacterium exhibits at least 50% less expression of said protein relative to a wild-type  Mycoplasma gallisepticum.    
     
     
         22 . The method of  claim 21 , wherein said bacterium exhibits at least 75% less expression of said protein relative to a wild-type  Mycoplasma gallisepticum.    
     
     
         23 . The method of  claim 19 , wherein said bacterium further exhibits reduced expression of pyruvate dehydrogenase relative to a wild-type  Mycoplasma gallisepticum.    
     
     
         24 . The method of  claim 19 , wherein said bacterium further exhibits reduced expression of phosphopyruvate hydratase relative to a wild-type  Mycoplasma gallisepticum.    
     
     
         25 . The method of  claim 19 , wherein said bacterium further exhibits reduced expression of 2-deoxyribose-5-phosphate aldolase relative to a wild-type  Mycoplasma gallisepticum.    
     
     
         26 . The method of  claim 19 , wherein said bacterium further exhibits reduced expression of ribosomal protein L35 relative to a wild-type  Mycoplasma gallisepticum.    
     
     
         27 . The method of  claim 19 , wherein said bacterium further exhibits reduced expression of pyruvate dehydrogenase, phosphopyruvate hydratase, 2-deoxyribose-5-phosphate aldolase, and ribosomal protein L35 relative to a wild-type  Mycoplasma gallisepticum.    
     
     
         28 . A method for identifying attenuated  Mycoplasma gallisepticum  clones, said method comprising:
 (a) subjecting an initial population of  Mycoplasma gallisepticum  bacteria to attenuating conditions, thereby producing a putatively attenuated bacterial population; and   (b) assaying individual clones of said putatively attenuated bacterial population for reduced expression of a protein having the amino acid sequence of SEQ ID NO:1, relative to a wild-type  Mycoplasma gallisepticum ; and   (c) testing clones identified in (b) as having reduced expression of said protein for virulence;   wherein a  Mycoplasma gallisepticum  clone that exhibits reduced expression of said protein and reduced virulence relative to a wild-type  Mycoplasma gallisepticum  is an attenuated  Mycoplasma gallisepticum  clone.   
     
     
         29 . The method of  claim 28 , wherein said attenuating conditions of (a) comprise passaging said initial population of  Mycoplasma gallisepticum  bacteria at least 2 times in vitro. 
     
     
         30 . The method of  claim 29 , wherein said attenuating conditions of (a) comprise passaging said initial population of  Mycoplasma gallisepticum  bacteria at least 5 times in vitro. 
     
     
         31 . The method of  claim 30 , wherein said attenuating conditions of (a) comprise passaging said initial population of  Mycoplasma gallisepticum  bacteria at least 10 times in vitro. 
     
     
         32 . The method of  claim 28 , wherein said attenuating conditions of (a) comprise transforming said initial population of  Mycoplasma gallisepticum  with a transposon which randomly inserts into the  Mycoplasma gallisepticum  genome. 
     
     
         33 . The method of  claim 28 , wherein said attenuating conditions of (a) comprise exposing said initial population of  Mycoplasma gallisepticum  to a chemical mutagen or ultra violet light. 
     
     
         34 . The method of  claim 28 , wherein said individual clones of said putatively attenuated bacterial population are assayed in (b) for reduced expression of said protein by reverse transcriptase-polymerase chain reaction (RT-PCR). 
     
     
         35 . The method of  claim 28 , wherein said individual clones of said putatively attenuated bacterial population are assayed in (b) for reduced expression of said protein by Western blot. 
     
     
         36 . The method of  claim 28 , wherein said clones identified in (b) are tested for virulence in (c) by administering one or more of said clones to an animal that is susceptible to  Mycoplasma gallisepticum  infection and comparing the clinical symptoms observed in said animals after being administered said one or more clones to the clinical symptoms of control animals that are not administered said clones. 
     
     
         37 . The live, attenuated  Mycoplasma gallisepticum  bacterium deposited at the ATCC under accession number PTA-8485. 
     
     
         38 . The method of  claim 19 , wherein said vaccine composition is administered to said animal by direct injection, spray administration or drinking water administration.