Patent Publication Number: US-2012045476-A1

Title: Live attenuated mycoplasma strains

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is a continuation application of co-pending U.S. application Ser. No. 12/207,698, filed Sep. 10, 2008, which claims priority to U.S. provisional application No. 60/993,456, filed Sep. 11, 2007. The entire disclosures are hereby incorporated by reference in their entirety. 
    
    
     BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The present invention relates to the fields of microbiology and immunology. More specifically, the invention relates to novel vaccines against bacterial pathogens. 
     2. Background Art 
     Mycoplasmas are small prokaryotic organisms (0.2 to 0.3 μm) belonging to the class Mollicutes, whose members lack a cell wall and have a small genome size. The mollicutes include at least 100 species of  Mycoplasma. Mycoplasma  species are the causative agents of several diseases in human and non-human animals as well as in plants. 
     In humans, for example,  M. pneumoniae , is a major cause of community-acquired pneumonia (non-pneumococcal bacterial pneumonia). Another human-pathogenic  Mycoplasma, M. hominis , is associated with pathological conditions in the urogenital tract of men and the upper urogenital tract of women.  M. hominis  has been implicated as a cause of nongonococcal urethritis, urethroprostatitis, vaginitis, endometritis, pelvic inflammatory disease, cervicitis, infertility, postpartum septicemia, pregnancy wastage, low birth weights and birth defects. Other human-pathogenic  Mycoplasma  species include  M. genitalium  (implicated in arthritis, chronic nongonococcal urethritis, chronic pelvic inflammatory disease, other urogenital infections, infertility and AIDS/HIV),  M. fermentans  (implicated in Arthritis, Gulf War Syndrome, Fibromyalgia, Chronic Fatigue Syndrome, Lupus, AIDS/HIV, autoimmune diseases, ALS, psoriasis and Scleroderma, Crohn&#39;s and IBS, cancer, endocrine disorders, Multiple Sclerosis and diabetes),  M. salivarium  (implicated in arthritis, TMJ disorders, eye and ear disorders and infections, gingivitis and periodontal diseases including cavities),  M. incognitus  and  M. penetrans  (implicated in AIDS/HIV, urogenital infections and diseases, and autoimmune disorders and diseases),  M. pirum  (implicated in urogenital infections and diseases, and AIDS/HIV),  M. faucium, M. lipophilum , and  M. buccale  (implicated in diseases of the gingival crevices and respiratory tract).  M. gallisepticum  and  M. synoviae  are responsible for significant disease conditions in poultry.  M. gallisepticum , for example, is associated with acute respiratory disease in chickens and turkeys and can also cause upper respiratory disease in game birds. In addition,  M. gallisepticum  has been recognized as a cause of conjunctivitis in house finches in North America. With regard to  M. synoviae , infection of poultry with this species leads to a decrease in body weight gain and loss of egg production. 
     In swine,  M. hyopneumoniae  is the etiologic agent of mycoplasmal pneumonia, causing significant economic loss in the swine industry due to reduced weight gain and poor feed efficiency. Infection of pigs with  M. hyopneumoniae  causes a chronic cough, dull hair coat, retarded growth and unthrifty appearance lasting several weeks. Characteristic lesions of purple to gray areas of consolidation, particularly in ventral apical and cardiac lobes are observed in infected animals. 
       M. bovis  is a bovine pathogen in housed or intensively reared beef and dairy cattle. The most frequently reported clinical manifestation is pneumonia of calves, which is often accompanied by arthritis, also known as pneumonia-arthritis syndrome. Its etiological role has also been associated with mastitis, otitis, and reproductive disease or disorders of cows and bulls. 
     An effective strategy for preventing and managing diseases caused by  Mycoplasma  infection is by vaccination with live, attenuated strains of  Mycoplasma  bacteria. The advantages of live attenuated vaccines, in general, include the presentation of all the relevant immunogenic determinants of an infectious agent in its natural form to the host&#39;s immune system, and the need for relatively small amounts of the immunizing agent due to the ability of the agent to multiply in the vaccinated host. 
     Live attenuated vaccine strains are often created by serially passaging a virulent strain multiple times in media. Although live attenuated vaccine strains against certain  Mycoplasma  species have been obtained by serial passaging, such strains are generally poorly characterized at the molecular level. It is assumed that attenuated strains made by serial passaging have accumulated mutations which render the microorganisms less virulent but still capable of replication. With regard to attenuated  Mycoplasma  strains, however, the consequences of the mutations that result in attenuation (e.g., the identity of proteins whose expression pattern has been altered in the attenuated strain) are usually unknown. 
     Accordingly, a need exists in the art for new live, attenuated  Mycoplasma  bacteria that have been characterized at the proteomic level and that are safe and effective in vaccine formulations. 
     BRIEF SUMMARY OF THE INVENTION 
     The present invention is directed to live, attenuated  Mycoplasma  bacteria that 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  Mycoplasma  bacterium of the same species. The live attenuated  Mycoplasma  bacteria of the invention can be of any  Mycoplasma  species. In a specific, non-limiting, exemplary embodiment, the invention provides a live, attenuated  M. gallisepticum  strain that exhibits reduced expression of pyruvate dehydrogenase, phosphopyruvate hydratase, 2-deoxyribose-5-phosphate aldolase, and ribosomal protein L35, relative to wild-type  M. gallisepticum  bacteria. According to certain embodiments of the present invention, the live, attenuated  Mycoplasma  bacteria of the invention are characterized by proteomic analysis as having reduced expression of one or more of the aforementioned proteins. 
     The present invention also provides vaccine compositions comprising the live, attenuated  Mycoplasma  bacteria of the invention, as well as methods of vaccinating an animal against  Mycoplasma  infection. 
     In addition, the present invention provides methods for making and/or identifying attenuated  Mycoplasma  clones. According to this aspect of the invention, the methods comprise subjecting an initial population of  Mycoplasma  bacteria to attenuating conditions, assaying individual clones for 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  Mycoplasma  bacterium of the same species, and testing the clones for virulence.  Mycoplasma  clones produced according to the methods of this aspect of the invention will preferably exhibit reduced expression of at least one of the aforementioned proteins and reduced virulence relative to a wild-type  Mycoplasma  bacterium of the same species. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWING 
         FIG. 1  is a photograph of a two-dimensional (2-D) polyacrylamide gel depicting protein spots of the attenuated  M. gallisepticum  strain MGx+47. Circled 105 spots numbered 19, 49, 74, 108, 114, 127, 147, 166, 175 and 225 correspond to proteins that are up-regulated in MGx+47 relative to wild-type strain R-980. Circled spots numbered 40, 68, 98, 99, 130, 136 and 217 correspond to proteins that are down-regulated in MGx+47 relative to wild-type strain R-980. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The present invention is directed to live, attenuated  Mycoplasma  bacteria that are suitable for use in vaccine formulations. The  Mycoplasma  bacteria of the present invention exhibit reduced expression of one or more of the following proteins: pyruvate dehydrogenase, phosphopyruvate hydratase, 2-deoxyribose-5-phosphate aldolase, and/or ribosomal protein L35, relative to the expression of these proteins in a wild-type  Mycoplasma  bacterium of the same species. 
       Mycoplasma  Species 
     The present invention is based, in part, on the surprising discovery of a new live, attenuated  Mycoplasma gallisepticum  vaccine strain that was demonstrated by proteomic analysis to have reduced levels of proteins such as pyruvate dehydrogenase, phosphopyruvate hydratase, 2-deoxyribose-5-phosphate aldolase, and ribosomal protein L35. (See Example 3 herein). The invention is exemplified by working examples using  M. gallisepticum ; however, the finding that reduced levels of pyruvate dehydrogenase, phosphopyruvate hydratase, 2-deoxyribose-5-phosphate aldolase, and ribosomal protein L35 correlates with bacterial attenuation is applicable to all species of  Mycoplasma  due to conservation of these proteins across  Mycoplasma  species. 
     For instance, homologues of the  M. gallisepticum  pyruvate dehydrogenase protein (also known as AcoA) are found in, inter alia,  M. hyopneumoniae  232 , M. hyopneumoniae  7448 , M. hyopneumoniae  J,  M. florum, Mycoplasma capricolumn  subsp.  capricolum, Mycoplasma genitalium, Mycoplasma  mobile 163K,  Mycoplasma mycoides  subsp.  mycoides  SC,  Mycoplasma penetrans, Mycoplasma pneumoniae, Mycoplasma pulmonis , and  Mycoplasma synoviae.    
     Homologues of the  M. gallisepticum  phosphopyruvate hydratase protein (also known as Eno) are found in, inter alia,  M. hyopneumoniae  232 , M. hyopneumoniae  7448 , M. hyopneumoniae  J,  M. florum, Mycoplasma capricolumn  subsp.  capricolum, Mycoplasma genitalium, Mycoplasma  mobile 163K,  Mycoplasma mycoides  subsp.  mycoides  SC,  Mycoplasma penetrans, Mycoplasma pneumoniae, Mycoplasma pulmonis, Mycoplasma synoviae , Onion yellows  phytoplasma, Ureaplasma urealyticum/parvum , and Aster yellows witches-broom  phytoplasma.    
     Homologues of the  M. gallisepticum  2-deoxyribose-5-phosphate aldolase protein (also known as DERA or DeoC) are found in, inter alia,  M. hyopneumoniae  232 , M. hyopneumoniae  7448 , M. hyopneumoniae  J,  M. florum, Mycoplasma capricolumn  subsp.  capricolum, Mycoplasma genitalium, Mycoplasma  mobile 163K,  Mycoplasma mycoides  subsp.  mycoides  SC,  Mycoplasma penetrans, Mycoplasma pneumoniae, Mycoplasma pulmonis, Mycoplasma synoviae,  and  Ureaplasma urealyticum/parvum.    
     Homologues of the  M. gallisepticum  ribosomal protein L35 protein (also known as Rpml) are found in, inter alia,  M. hyopneumoniae  232 , M. hyopneumoniae  7448 , M. hyopneumoniae  J,  M. florum, Mycoplasma genitalium, Mycoplasma pneumoniae , and  Mycoplasma pulmonis.    
     The above lists of homologues are intended to be illustrative and are not intended to be exhaustive, and it will be appreciated by those of ordinary skill in the art that additional homologues of  M. gallisepticum  AcoA, Eno, DeoC and/or Rpml exist in  Mycoplasma  species in addition to those listed above. 
     Since most  Mycoplasma  species express a version of pyruvate dehydrogenase, phosphopyruvate hydratase, 2-deoxyribose-5-phosphate aldolase and ribosomal protein L35, and since these proteins apparently serve homologous functions across species, it follows that reduced expression of these proteins is a defining characteristic of attenuated  Mycoplasma  strains as exemplified by the attenuated  M. gallisepticum  strain described in the Examples herein. 
     The attenuated  Mycoplasma  bacteria of the present invention may be of any  Mycoplasma  species. In a preferred embodiment, the attenuated bacteria are derived from animal-pathogenic  Mycoplasma  bacteria. As used herein, the term “animal-pathogenic  Mycoplasma  baceterium” means a bacterium that, in its wild-type, un-attenuated state, can infect and cause disease and/or illness in an animal. “Disease and/or illness in an animal” includes adverse physical manifestations in an animal as well as clinical signs of disease or infection indicated solely by histological, microscopic and/or molecular diagnostics. 
     Animal-pathogenic  Mycoplasma  bacteria include human- and non-human-pathogenic  Mycoplasma  bacteria. Human-pathogenic  Mycoplasma  bacteria include, but are not limited to, e.g., bacteria of the  Mycoplasma  species  M. genitalium, M. fermentans, M. salivarium, M. hominis, M. pneumonia, M. incognitus, M. penetrans, M. pirum, M. faucium, M. lipophilum , and  M. buccale . Non-human-pathogenic  Mycoplasma  bacteria include, e.g., avian-, porcine-, ovine-, bovine-, caprine- or canine-pathogenic  Mycoplasma  bacteria. Avian-pathogenic  Mycoplasma  bacteria include, but are not limited to, e.g., bacteria of the  Mycoplasma  species  M. cloacale, M. gaffinarum, M. gallisepticum, M. gallopavonis, M. glycophilum, M. iners, M. iowae, M. lipofaciens, M. meleagridis , and  M. synoviae . Porcine-pathogenic  Mycoplasma  bacteria include, but are not limited to, e.g., bacteria of the  Mycoplasma  species  M. flocculare, M. hyopneumoniae, M. hyorhinis , and  M. hyosynoviae . Ovine-, bovine-, caprine- or canine-pathogenic  Mycoplasma  bacteria include, but are not limited to, e.g., bacteria of the  Mycoplasma  species  M. capricolumn  subsp.  capricolum, M. capricolumn  subsp.  capripneumoniae, M. mycoides  subsp.  mycoides  LC,  M. mycoides  subsp.  capri, M. bovis, M. bovoculi, M. canis, M. californicum , and  M. dispar.    
     Reduced Expression of  Mycoplasma  Proteins 
     A person of ordinary skill in the art will be able to determine, using routine molecular biological techniques, whether an attenuated  Mycoplasma  bacterium exhibits reduced expression of one or more proteins that are normally expressed in wild-type  Mycoplasma  bacterial cells. Determining whether an attenuated bacterium exhibits reduced expression of a particular protein (e.g., pyruvate dehydrogenase, phosphopyruvate hydratase, 2-deoxyribose-5-phosphate aldolase, ribosomal protein L35, etc.), relative to a wild-type bacterium, can be accomplished by several methods known in the art. Exemplary methods include, e.g., quantitative antibody-based methods such as Western blotting, radioimmunoassays (RIAs), and enzyme-linked immunosorbant assays (ELISAs), in which an antibody is used which detects and binds to the protein of interest. In addition, since messenger RNA (mRNA) levels generally reflect the quantity of the protein encoded therefrom, quantitative nucleic acid-based methods may also be used to determine whether an attenuated  Mycoplasma  bacterium exhibits reduced expression of one or more proteins. For example, quantitative reverse-transcriptse/polymerase chain reaction (RT-PCR) methods may be used to measure the quantity of mRNA corresponding to a particular protein of interest. Numerous quantitative nucleic acid-based methods are well known in the art. 
     The following is a non-limiting, exemplary method that can be used for determining whether an attenuated  Mycoplasma  bacterium exhibits reduced expression of, e.g., phosphopyruvate hydratase. For purposes of this illustrative method, it will be assumed that the  Mycoplasma  bacterium is of the species  M. gallisepticum , however, it will be appreciated by persons of ordinary skill in the art that this exemplary method can be applied equally to all species of  Mycoplasma  and can be used to assess the relative expression of any  Mycoplasma  protein. 
     First, a population of attenuated  M. gallisepticum  cells and a population of wild-type  M. gallisepticum  cells are grown under substantially identical conditions in substantially the same culture medium. Next, the two populations of cells are subjected to cell-disrupting conditions. The disrupted cells (or the protein-containing fractions thereof) are subjected, in parallel, to SDS polyacrylamide gel electrophoresis (SDS-PAGE) and then to Western blotting using an antibody which binds to the  M. gallisepticum  phosphopyruvate hydratase protein (such antibodies can be obtained using standard methods that are well known in the art). A labeled secondary antibody is then applied in order to provide a measurable signal that is proportional to the amount of the protein derived from the cells. If the amount of signal exhibited by the attenuated  M. gallisepticum  strain is less than the amount of signal exhibited by the wild-type  M. gallisepticum  strain, then it can be concluded that the attenuated strain exhibits reduced expression of phosphopyruvate hydratase relative to the wild-type strain. Variations on this exemplary method, as well as alternatives thereto, will be immediately evident to persons of ordinary skill in the art. 
     The present invention includes attenuated  Mycoplasma  bacteria that exhibit any degree of reduction in expression of a protein (e.g., pyruvate dehydrogenase, phosphopyruvate hydratase, 2-deoxyribose-5-phosphate aldolase, ribosomal protein L35, etc.) compared to the expression of that protein observed in a wild-type strain. In certain embodiments, the attenuated bacterium exhibits at least about 5% less expression of the protein relative to a wild-type bacterium. As an example, if a given quantity of a wild-type  Mycoplasma  strain exhibit 100 units of expression of a particular protein and the same quantity of a candidate attenuated  Mycoplasma  strain of the same species exhibits 95 units of expression of the protein, then it is concluded that the attenuated strain exhibits 5% less expression of the protein relative to the wild-type bacterium (additional examples for calculating “percent less expression” are set forth elsewhere herein). In certain other embodiments, the attenuated bacterium exhibits at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% less expression of the protein relative to a wild-type  Mycoplasma  bacterium. In yet other embodiments, the attenuated  Mycoplasma  strain exhibits no expression (i.e., 100% less expression) of the protein relative to a wild-type  Mycoplasma  bacterium. 
     In certain exemplary embodiments of the present invention, the attenuated bacteria exhibit at least 5% less 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  Mycoplasma  bacterium of the same species. 
     As used herein, the “percent less expression” of a particular protein exhibited by an attenuated  Mycoplasma  strain relative to a wild-type strain is calculated by the following formula: (A−B)/A×100; wherein A=the relative level of expression of the protein in a wild-type  Mycoplasma  strain; and B=the relative level of expression of the protein in the attenuated strain. Solely for the purpose of illustration, if a wild-type  Mycoplasma  strain exhibited 0.2500 units of expression of protein “Y”, and an attenuated strain of  Mycoplasma  exhibited 0.1850 units of expression of protein “Y” then the attenuated strain is said to exhibit [(0.2500−0.1850)/0.2500×100]=26% less expression of protein “Y” relative to the wild-type strain. Table 5 in Example 3 herein provides additional illustrative examples of percent less expression calculated for an exemplary attenuated strain of  M. gallisepticum  relative to a wild-type  M. gallisepticum  strain. 
     Vaccine Compositions 
     The present invention also includes vaccine compositions comprising a live, attenuated  Mycoplasma  bacterium of the invention and a pharmaceutically acceptable carrier. As used herein, the expression “live, attenuated  Mycoplasma  bacterium of the invention” encompasses any live, attenuated  Mycoplasma  bacterium that is described and/or claimed elsewhere herein. The pharmaceutically acceptable carrier can be, e.g., water, a stabilizer, a preservative, culture medium, or a buffer. Vaccine formulations comprising the attenuated  Mycoplasma  bacteria of the invention can be prepared in the form of a suspension or in a lyophilized form or, alternatively, in a frozen form. If frozen, glycerol or other similar agents may be added to enhance stability when frozen. 
     Methods of Vaccinating an Animal 
     The present invention also includes methods of vaccinating an animal against  Mycoplasma  infection. The methods according to this aspect of the invention comprise administering to an animal an immunologically-effective amount of a vaccine composition comprising a live, attenuated  Mycoplasma  bacterium of the invention. As used herein, the expression “live, attenuated  Mycoplasma  bacterium of the invention” encompasses any live, attenuated  Mycoplasma  bacterium that is described and/or claimed elsewhere herein. The expression “immunologically-effective amount” means that amount of vaccine composition required to invoke the production of protective levels of antibodies in an animal upon vaccination. The vaccine composition may be administered to the animal in any manner known in the art including oral, intranasal, mucosal, topical, transdermal, and parenteral (e.g., intravenous, intraperitoneal, intradermal, subcutaneous or intramuscular) routes. Administration can also be achieved using needle-free delivery devices. Administration can be achieved using a combination of routes, e.g., first administration using a parental route and subsequent administration using a mucosal route, etc. 
     In embodiments of the invention wherein the live, attenuated  Mycoplasma  bacterium is an avian-pathogenic  Mycoplasma  bacterium, e.g., an  M. gallisepticum  bacterium, the animal to which the attenuated bacterium is administered is preferably a bird, e.g., a chicken or a turkey. Where the animal is a bird, the vaccine formulations of the invention may be administered such that the formulations are immediately or eventually brought into contact with the bird&#39;s respiratory mucosal membranes. Thus, the vaccine formulations may be administered to birds, e.g., intranasally, orally, and/or intraocularly. The vaccine compositions for avian administration may be formulated as described above and/or in a form suitable for administration by spray, including aerosol (for intranasal administration) or in drinking water (for oral administration). 
     Vaccine compositions of the present invention that are administered by spray or aerosol can be formulated by incorporating the live, attenuated  Mycoplasma  bacteria into small liquid particles. The particles can have an initial droplet size of between about 10 μm to about 100 μm. Such particles can be generated by, e.g., conventional spray apparatus and aerosol generators, including commercially available spray generators for knapsack spray, hatchery spray and atomist spray. 
     Methods for Making Attenuated Mycoplasma Clones 
     In another aspect of the present invention, the invention provides methods for identifying and/or making attenuated  Mycoplasma  clones. The methods according to this aspect of the invention comprise subjecting an initial population of  Mycoplasma  bacteria to attenuating conditions, thereby producing a putatively attenuated bacterial population. Next, individual clones of the putatively attenuated bacterial population are assayed for reduced expression of one or more proteins selected from the group consisting of pyruvate dehydrogenase, phosphopyruvate hydratase, 2-deoxyribose-5-phosphate aldolase, ribosomal protein L35, relative to a wild-type  Mycoplasma  bacterium of the same species. The clones that are identified as having reduced expression of one or more of the above-mentioned proteins are then tested for virulence. Clones that exhibit both reduced expression of one or more of the above-mentioned proteins and reduced virulence relative to a wild-type  Mycoplasma  bacterium of the same species are identified as attenuated  Mycoplasma  clones. 
     According to this aspect of the invention, the “initial population of  Mycoplasma  bacteria” can be any quantity of  Mycoplasma  bacteria. The bacteria, in certain embodiments are wild-type bacteria. Alternatively, the bacteria may contain one or more mutations. Preferably, however, the bacteria in the initial population are clonally identical or substantially clonally identical; that is, the bacteria preferably are all derived from a single parental  Mycoplasma  bacterial cell and/or have identical or substantially identical genotypic and/or phenotypic characteristics. 
     As used herein, the term “attenuating conditions” means any condition or combination of conditions which has/have the potential for introducing one or more genetic changes (e.g., nucleotide mutations) into the genome of a  Mycoplasma  bacterium. Exemplary, non-limiting, attenuating conditions include, e.g., passaging bacteria in culture, transforming bacteria with a genome-insertable genetic element such as a transposon (e.g., a transposon that randomly inserts into the  Mycoplasma  genome), exposing bacteria to one or more mutagens (e.g., chemical mutagens or ultraviolet light), etc. When bacterial cells are attenuated by passaging in vitro, the cells may be passaged any number of times, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or more times in vitro. 
     The initial population of  Mycoplasma  cells, after being subjected to attenuating conditions, are referred to herein as a putatively attenuated bacterial population. Individual clones of the putatively attenuated bacterial population can be obtained by standard microbiological techniques including, e.g., serially diluting the cells and plating out individual cells on appropriate media. Once obtained, the individual clones of the putatively attenuated bacterial population are assayed for reduced expression of one or more specified proteins. Methods for determining whether an attenuated  Mycoplasma  bacterium exhibits reduced expression of one or more proteins that are normally expressed in wild-type  Mycoplasma  bacterial cells are described elsewhere herein. Exemplary methods include, e.g., RT-PCR-based methods, Western blot, etc. 
     Individual clones that are identified as having reduced expression of one or more proteins (e.g., pyruvate dehydrogenase, phosphopyruvate hydratase, 2-deoxyribose-5-phosphate aldolase, ribosomal protein L35) can be tested for virulence by administration of the clones to an animal that is susceptible to infection by the wild-type (unattenuated) version of the bacterium. As used herein, “an animal that is susceptible to infection by a wild-type  Mycoplasma  bacterium” is an animal that shows at least one clinical symptom after being challenged with a wild-type  Mycoplasma  bacterium. Such symptoms are known to persons of ordinary skill in the art. For example, in the case of a putatively attenuated  M. gallisepticum  strain that exhibits reduced expression of, e.g., pyruvate dehydrogenase, the strain can be administered to, e.g., turkeys or chickens (which are normally susceptible to infection by wild-type  M. gallisepticum ). Clinical symptoms of  M. gallispeticum  infection of poultry animals include, e.g., acute respiratory symptoms, pericarditis, perihepatitis, air sacculitis, trachea thickening, reduced weight gain, deciliation, abnormal goblet cells, capillary distension, increased numbers of lymphocytes, plasma cells and/or heterophils, and in some cases reduced egg production. Thus, if the putatively attenuated  M. gallisepticum  strain, when administered to a chicken or turkey, results in fewer and/or less severe symptoms as compared to a turkey or chicken that has been infected with a wild-type  M. gallisepticum  strain, then the putatively attenuated  M. gallisepticum  strain is deemed to have “reduced virulence.” Any degree of reduction in symptoms will identify the putatively attenuated strain as having reduced virulence. In certain embodiments, the putatively attenuated strain will be avirulent. 
     According to the present invention, a  Mycoplasma  clone that exhibits 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, and that exhibits reduced virulence relative to a wild-type  Mycoplasma  bacterium of the same species is an attenuated  Mycoplasma  clone. 
     The following examples are illustrative, but not limiting, of the method and compositions of the present invention. Other suitable modifications and adaptations of the variety of conditions and parameters normally encountered in molecular biology and chemistry which are obvious to those skilled in the art in view of the present disclosure are within the spirit and scope of the invention. 
     EXAMPLES 
     Example 1 
     Generation of a Live, Attenuated  M. gallisepticum  Strain 
     A new live, attenuated  Mycoplasma gallisepticum  strain was generated by passaging a wild-type  M. galliespticum  strain R980 multiple times in vitro. In particular, 0.1 mL seed material of wild-type  M. gallisepticum  strain R-980 was inoculated into 20 mL of modified Frey&#39;s medium (Frey et al.,  Am. J. Vet. Res.  29:2163-2171 (1968) (also referred to herein as “MG culture medium”). The wild-type cells were grown until media color changed to bright yellow. The bright yellow cultures were subsequently used to re-inoculate fresh MG culture media as described above. The culture was passaged a total of 47 times in this manner. The resulting strain was tested for attenuation by vaccinating groups of birds followed by challenge using the wild-type  M. gallisepticum . All the birds were necropsized two weeks post-challenge and mycoplasma related pathologies were observed. High passage strain (x+47) provided protection against the clinical signs associated with  Mycoplasma gallisepticum  infection. This attenuated  M. gallisepticum  strain designated MGx+47 (also referred to as “MG-P48”) was deposited with the American Type Culture Collection, P.O. Box 1549, Manassas, Va. 20108, on Jun. 19, 2007 and was assigned accession number PTA-8485. 
     Example 2 
     Safety and Efficacy Evaluation of a Live, Attenuated  M. gallisepticum Vaccine in Chickens    
     In this Example, the safety and efficacy of the new  M. gallisepticum  vaccine strain MGx+47 obtained in Example 1 was assessed in chickens. 
     Seventy one SPF white leghorn chickens were divided into seven groups as follows: 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Study Design 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Group 
                 # Chickens 
                 Vaccinated 
                 Challenged 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 1 
                 11 
                 No 
                 Yes 
               
               
                   
                 2 
                 10 
                 Yes 
                 No 
               
               
                   
                 3 
                 11 
                 Yes 
                 Yes 
               
               
                   
                 4a 
                 10 
                 Yes 
                 No 
               
               
                   
                 4b 
                 11 
                 Yes 
                 No 
               
               
                   
                 4c 
                 9 
                 Yes 
                 No 
               
               
                   
                 5 
                 9 
                 No 
                 No 
               
               
                   
                   
               
            
           
         
       
     
     The chickens in groups 2, 3, 4a, 4b and 4c were vaccinated with attenuated strain MGx+47 at 3.62×10 7  CCU/mL/bird, administered by coarse spray at 4 weeks of age. The chickens in groups 1 and 3 were challenged intratracheally (IT) at 7 weeks of age with 0.5 mL of  Mycoplasma gallisepticum  strain R at 7.74×10 5  CCU/mL. Necropsy was performed on the chickens of groups 1, 2, 3 and 5 at 9 weeks of age, and necropsy was performed on the chickens of groups 4a, 4b and 4c at 7, 14 and 21 days post vaccination (DPV), respectively. The chickens were assessed for average weight gain, pericarditis, perihepatitis, airsacculitis, and tracheitis. The results are summarized in Table 2. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Safety and Efficacy Summary 
               
               
                 Vaccination = 3.62 × 10 7  CFU/mL/bird 
               
               
                 Challenge = 0.5 mL at 7.74 × 10 5  CFU/mL 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                 Average 
                   
                   
                   
                 Airsacculitis 
                   
               
               
                   
                   
                   
                 Weight Gain 
                   
                   
                   
                 Score (average 
                 Trachea 
               
               
                 Group 
                 Vaccinated 
                 Challenged 
                 (kg/day) 
                 Pericarditis 
                 Perihepatitis 
                 Airsacculitis 
                 of positives) 
                 (Histology) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 1 
                 No 
                 Yes 
                 0.016 
                 0/11 
                 0/11 
                 9/11 
                 3.56 
                 severe 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 tracheitis 
               
               
                 2 
                 Yes 
                 No 
                 0.018 
                 0/10 
                 0/10 
                 0/10 
                 0 
                 normal 
               
               
                 3 
                 Yes 
                 Yes 
                 0.017 
                 0/11 
                 0/11 
                 2/11 
                 2.5 
                 mixed 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 tracheitis 
               
               
                 4a 
                 Yes 
                 No 
                 0.016 
                 0/9 
                 0/9 
                 0/9 
                 0 
                 normal 
               
               
                 4b 
                 Yes 
                 No 
                 0.017 
                 0/11 
                 0/11 
                 0/11 
                 0 
                 normal 
               
               
                 4c 
                 Yes 
                 No 
                 0.017 
                 0/10 
                 0/10 
                 0/10 
                 0 
                 normal 
               
               
                 5 
                 No 
                 No 
                 0.015 
                 0/9 
                 0/9 
                 0/9 
                 0 
                 normal 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Safety Table: Histology Report of Formalin-Fixed Chicken Tracheas 
               
               
                 from Individual Vaccinated/Unchallenged Chickens (Group 4a, 4b and 4c) 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 Time 
                   
                   
                 Goblet 
                 Capillary 
                 LC/ 
                   
                 Thickness 
               
               
                 Point 
                 Chicken 
                 Cilia 
                 Cells/M 
                 Distension 
                 PC 
                 PMNs 
                 (microns) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 7 
                 1 
                 N 
                 − 
                 − 
                 − 
                 − 
                 30 
               
               
                 DPV 
                 2 
                 N 
                 − 
                 − 
                 − 
                 − 
                 30 
               
               
                   
                 3 
                 N 
                 − 
                 − 
                 − 
                 − 
                 30 
               
               
                   
                 4 
                 N 
                 − 
                 − 
                 + 
                 − 
                 30 
               
               
                   
                 5 
                 N 
                 − 
                 − 
                 − 
                 − 
                 30 
               
               
                   
                 6 
                 N 
                 − 
                 − 
                 + 
                 − 
                 30 
               
               
                   
                 7 
                 N 
                 − 
                 − 
                 + 
                 − 
                 30 
               
               
                   
                 8 
                 N 
                 − 
                 − 
                 − 
                 − 
                 30 
               
               
                   
                 9 
                 N 
                 + 
                 − 
                 − 
                 − 
                 30 
               
               
                 14 
                 1 
                 N 
                 − 
                 − 
                 − 
                 − 
                 50 
               
               
                 DPV 
                 2 
                 N 
                 + 
                 − 
                 − 
                 − 
                 50 
               
               
                   
                 3 
                 N 
                 − 
                 − 
                 + 
                 − 
                 50 
               
               
                   
                 4 
                 N 
                 − 
                 − 
                 − 
                 − 
                 50 
               
               
                   
                 5 
                 N 
                 − 
                 − 
                 − 
                 − 
                 50 
               
               
                   
                 6 
                 N 
                 − 
                 − 
                 − 
                 − 
                 50 
               
               
                   
                 7 
                 N 
                 − 
                 − 
                 − 
                 − 
                 50 
               
               
                   
                 8 
                 N 
                 − 
                 − 
                 − 
                 − 
                 50 
               
               
                   
                 9 
                 N 
                 − 
                 − 
                 + 
                 − 
                 50 
               
               
                   
                 10 
                 N 
                 − 
                 − 
                 − 
                 − 
                 50 
               
               
                   
                 11 
                 N 
                 − 
                 − 
                 + 
                 − 
                 50 
               
               
                 21 
                 1 
                 N 
                 − 
                 − 
                 − 
                 − 
                 50 
               
               
                 DPV 
                 2 
                 N 
                 − 
                 − 
                 ++ 
                 − 
                 110 
               
               
                   
                 3 
                 N 
                 − 
                 − 
                 − 
                 − 
                 50 
               
               
                   
                 4 
                 N 
                 − 
                 − 
                 − 
                 − 
                 50 
               
               
                   
                 5 
                 N 
                 − 
                 − 
                 − 
                 − 
                 50 
               
               
                   
                 6 
                 N 
                 − 
                 − 
                 + 
                 − 
                 50 
               
               
                   
                 7 
                 N 
                 − 
                 − 
                 − 
                 − 
                 50 
               
               
                   
                 8 
                 N 
                 − 
                 − 
                 − 
                 − 
                 50 
               
               
                   
                 9 
                 N 
                 − 
                 − 
                 − 
                 − 
                 50 
               
               
                   
                 10 
                 N 
                 − 
                 − 
                 − 
                 − 
                 50 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Efficacy Table: Histology Report of Formalin-Fixed Chicken Tracheas 
               
               
                 from Individual Chickens 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                 Gob- 
                   
                   
                   
                   
               
               
                   
                   
                   
                 let 
               
               
                   
                   
                   
                 Cells/ 
                 Capillary 
                 LC/ 
                   
                 Thickness 
               
               
                 Group 
                 Chicken 
                 Cilia 
                 M 
                 Distension 
                 PC 
                 PMNs 
                 (microns) 
               
               
                   
               
            
           
           
               
               
            
               
                 1 
                 Not Vaccinated; Challenged 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                 1 
                 − 
                 + 
                 ++ 
                 ++++ 
                 ++ 
                 410 
               
               
                   
                 2 
                 +/− 
                 − 
                 − 
                 + 
                 − 
                 90 
               
               
                   
                 3 
                 N 
                 + 
                 − 
                 − 
                 − 
                 50 
               
               
                   
                 4 
                 − 
                 − 
                 ++++ 
                 ++++ 
                 − 
                 420 
               
               
                   
                 5 
                 N 
                 + 
                 + 
                 + 
                 − 
                 60 
               
               
                   
                 6 
                 − 
                 + 
                 ++++ 
                 ++++ 
                 +++ 
                 400 
               
               
                   
                 7 
                 − 
                 − 
                 ++++ 
                 ++++ 
                 − 
                 440 
               
               
                   
                 8 
                 − 
                 − 
                 ++++ 
                 ++++ 
                 ++++ 
                 280 
               
               
                   
                 9 
                 − 
                 + 
                 − 
                 − 
                 − 
                 40 
               
               
                   
                 10 
                 − 
                 − 
                 ++++ 
                 ++++ 
                 − 
                 260 
               
               
                   
                 11 
                 − 
                 + 
                 ++++ 
                 ++++ 
                 +++ 
                 450 
               
            
           
           
               
               
            
               
                 3 
                 Vaccinated and Challenged 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                 1 
                 − 
                 − 
                 ++ 
                 ++++ 
                 − 
                 380 
               
               
                   
                 2 
                 N 
                 − 
                 + 
                 + 
                 − 
                 40 
               
               
                   
                 3 
                 N 
                 − 
                 + 
                 + 
                 − 
                 50 
               
               
                   
                 4 
                 − 
                 − 
                 + 
                 +++ 
                 ++ 
                 220 
               
               
                   
                 5 
                 N 
                 − 
                 + 
                 + 
                 − 
                 60 
               
               
                   
                 6 
                 N 
                 − 
                 + 
                 + 
                 − 
                 60 
               
               
                   
                 7 
                 N 
                 − 
                 − 
                 − 
                 − 
                 50 
               
               
                   
                 8 
                 N 
                 − 
                 − 
                 − 
                 − 
                 50 
               
               
                   
                 9 
                 N 
                 − 
                 + 
                 + 
                 − 
                 50 
               
               
                   
                 10 
                 +/− 
                 − 
                 + 
                 ++ 
                 − 
                 140 
               
            
           
           
               
               
            
               
                 5 
                 Not Vaccinated; Not Challenged 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                 1 
                 N 
                 − 
                 − 
                 + 
                 − 
                 50 
               
               
                   
                 2 
                 N 
                 − 
                 − 
                 + 
                 − 
                 50 
               
               
                   
                 3 
                 N 
                 − 
                 − 
                 − 
                 − 
                 50 
               
               
                   
                 4 
                 N 
                 − 
                 − 
                 + 
                 − 
                 50 
               
               
                   
                 5 
                 N 
                 − 
                 − 
                 − 
                 − 
                 50 
               
               
                   
                 6 
                 N 
                 − 
                 − 
                 + 
                 − 
                 50 
               
               
                   
                 7 
                 N 
                 − 
                 − 
                 − 
                 − 
                 50 
               
               
                   
                 8 
                 N 
                 − 
                 − 
                 + 
                 − 
                 50 
               
               
                   
                 9 
                 N 
                 − 
                 − 
                 − 
                 − 
                 50 
               
               
                   
               
            
           
         
       
     
     Key to Safety and Efficacy Tables (Tables 3 and 4): 
     
         
         
           
             All “vaccinated” birds were vaccinated by coarse spray with vaccine strain MGx+47 at 3.62×10 7  CCU/mL/bird; 
             All “challenged” birds were challenged intratracheally (IT) with 0.5 mL of  Mycoplasma gallisepticum  strain Rat 7.74×10 5  CCU/mL 
             Time Point (in Table 3: Safety Table)=number of days after vaccination when the chickens were examined, expressed as # days post vaccination (DPV). 
             Cilia: “N”=normal cilia; “−”=deciliation; 
             Goblet Cells/M (“−”=normal goblet cells; “+”=mucus lying on the respiratory surface); 
             Capillary Distension (“−”=no distension or inflammation; “+”=moderate capillary distension or inflammation; “++”=severe capillary distension or inflammation); 
             LC/PC=Lymphocytes and Plasma cells (“−”=none; “+”=few; “++++”=numerous); 
             PMNs=Heterophils (“−”=none; “+”=few; “++++”=numerous); 
           
         
       
    
     The histology analysis of the group 2 chickens (vaccinated but not challenged) was substantially similar to that of the group 5 chickens (unvaccinated, unchallenged), demonstrating the safety of the newly generated MGx+47 vaccine strain. (See, e.g., Table 2 above). 
     With regard to efficacy, the group 3 chickens (vaccinated and challenged) showed significantly reduced airsacculitis compared to the group 1 chickens (unvaccinated and challenged). (See, e.g., Tables 2 and 4). In addition, as illustrated in Table 4, the group 3 chickens exhibited fewer histological signs of  M. gallisepticum  infection with regard to cillia, goblet cells, capillary distension, lymphocytes and plasma cells (LC/PC), heterophils (PMNs) and trachea thickness. (See Table 4). 
     Thus, this Example demonstrates that MGx+47 is a safe and effective live, attenuated  M. gallisepticum  vaccine strain. 
     Example 3 
     Proteomic Characterization of MGx+47 Vaccine Strain 
     In an effort to more precisely define the MGx+47 vaccine strain (see Examples 1 and 2) at the molecular level, a proteomic analysis of this strain was undertaken. 
     In this Example, total protein was isolated from the wild-type  M. gallisepticum  strain R-980 and from the newly identified vaccine strain MGx+47. Proteins from each strain were resolved by 2-dimensional polyacrylamide gel electrophoresis followed by computerized analysis of the gel images. (See  FIG. 1 ). Protein spots were identified that were differentially expressed in the vaccine strain. Protein spots that were absent, or were expressed at significantly reduced levels, in the vaccine strain compared to the wild-type strain were excised from the gel. 
     Five spots were identified that were expressed at significantly lower levels in the MGx+47 vaccine strain as compared to the wild-type  M. gallisepticum . Each of these protein spots were excised from the gel and enzmatically digested. Followed by peptide mass fingerprinting using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). The mass spectra identified for each protein spot was compared to a peptide mass database to identify the proteins and the corresponding genes that encodes them. The results of this analysis are summarized in the Table below: 
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Summary of Proteomic Analysis of MGx + 47 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                   
                 Level of 
                   
                 Percent 
               
               
                   
                   
                   
                 expression 
                 Level of 
                 decrease 
               
               
                   
                   
                   
                 in wild- 
                 expression in 
                 in 
               
               
                 Gene 
                 Product 
                 Function 
                 type MG 
                 MGx + 47 
                 expression 
               
               
                   
               
               
                 acoA 
                 Pyruvate 
                 Required for energy 
                 0.1872 
                 0.0858 
                 54.2% 
               
               
                   
                 dehydrogenase 
                 production and 
                   
                   
                   
               
               
                   
                   
                 conversion (Kreb&#39;s 
                   
                   
                   
               
               
                   
                   
                 Cycle) 
                   
                   
                   
               
               
                 eno 
                 Phospho- 
                 Catalyzes the formation 
                 0.0683 
                 0.0173 
                 74.7% 
               
               
                   
                 pyruvate 
                 of phosphoenol-pyruvate 
                   
                   
                   
               
               
                   
                 hydratase 
                   
                   
                   
                   
               
               
                 deoC 
                 2-deoxyribose-5- 
                 Required for nucleotide 
                 0.0525 
                 0.0309 
                 41.1% 
               
               
                   
                 phosphate 
                 metabolism 
                   
                   
                   
               
               
                   
                 aldolase 
                   
                   
                   
                   
               
               
                 rpml 
                 Ribosomal 
                 Translaction, ribosomal 
                 0.1171 
                 0.0259 
                 77.9% 
               
               
                   
                 protein L35 
                 structure and biogenesis 
                   
                   
                   
               
               
                 MGA_0621 
                 Hypothetical 
                 Unknown 
                 0.4534 
                 0.0835 
                 81.6% 
               
               
                   
                 protein 
               
               
                   
               
            
           
         
       
     
     The decrease in expression of the gene products can also be expressed in terms of “fold decrease in expression.” For example, in Table 5, strain MGx+47 can be said to exhibit 2.2, 3.9, 1.7, 4.5 and 5.4 fold decreased expression of acoA, eno, deoC, rpml, and MGA — 0621, respectively, relative to wild-type MG. 
     As indicated in Table 5, five gene products were identified that had significantly reduced expression in the live, attenuated MGx+47 vaccine strain as compared to the wild-type R-980 strain: AcoA, Eno, DeoC, Rmpl, and MGA — 0621 (a hypothetical protein identified under NCBI accession number NP — 852784). Importantly, three of these genes (acoA, eno and deoC) encode proteins involved in metabolic/energy generation pathways. In addition, homologues of AcoA, Eno, DeoC, and Rpml are found in most species of  Mycoplasma , strongly suggesting that down-regulation of one or more of these gene products may be a general strategy for attenuating  Mycoplasma.    
     Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, this invention is not limited to the particular embodiments disclosed, but is intended to cover all changes and modifications that are within the spirit and scope of the invention as defined by the appended claims. 
     All publications and patents mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patents are herein incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.