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 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.
2. The method of claim 1 , wherein said attenuating conditions of (a) comprise passaging said initial population of Mycoplasma gallisepticum bacteria at least 2 times in vitro.
3. The method of claim 2 , wherein said attenuating conditions of (a) comprise passaging said initial population of Mycoplasma gallisepticum bacteria at least 5 times in vitro.
4. The method of claim 3 , wherein said attenuating conditions of (a) comprise passaging said initial population of Mycoplasma gallisepticum bacteria at least 10 times in vitro.
5. The method of claim 1 , 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.
6. The method of claim 1 , wherein said attenuating conditions of (a) comprise exposing said initial population of Mycoplasma gallisepticum to a chemical mutagen or ultra violet light.
7. The method of claim 1 , 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).
8. The method of claim 1 , wherein said individual clones of said putatively attenuated bacterial population are assayed in (b) for reduced expression of said protein by Western blot.
9. The method of claim 1 , 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.