HK1145627B - Attenuated mycoplasma gallisepticum strains - Google Patents

Description

Attenuated strain of mycoplasma gallisepticum

Technical Field

The present invention relates to the fields of microbiology and immunology. More specifically, the present invention relates to novel vaccines against bacterial pathogens.

Background

Mycoplasma (Mycoplasma) is a small prokaryote (0.2 to 0.3 μm) belonging to the class Mollicutes, all of which lack a cell wall and have a small genome. The mollicutes include at least 100 mycoplasma species. Mycoplasma are the causative agent of several diseases in human and non-human animals and plants. For example, mycoplasma gallisepticum causes serious illness in poultry. Mycoplasma gallisepticum is associated with acute respiratory diseases in chickens and turkeys and can also cause upper respiratory disease in game birds. In addition, mycoplasma gallisepticum was identified as the cause of north american cardinal conjunctivitis.

An effective strategy for preventing and treating diseases caused by mycoplasma gallisepticum infection is immunization with live strains of attenuated mycoplasma gallisepticum bacteria. In general, advantages of attenuated live vaccines include having all relevant immunogenic determinants of the pathogen facing the host's immune system in their native form, and the relatively small amount of immunizing agent required due to the ability of the pathogen to expand in the vaccinated host.

Live attenuated vaccine strains are often produced by serial passage of virulent strains several times in culture. Although live attenuated vaccine strains against mycoplasma gallisepticum have been obtained by serial passage, these strains are generally not well characterized at the molecular level. It is hypothesized that attenuated strains produced by serial passage have accumulated mutations that render the microorganism less virulent but still capable of replication. However, for attenuated strains of mycoplasma gallisepticum, the results of mutations that result in attenuation of virulence (e.g., identifying proteins with altered expression patterns in the attenuated strain) are generally unknown.

Therefore, there is a need in the art for new live attenuated mycoplasma gallisepticum bacteria that have been characterized at the proteomic level and are safe and effective in vaccine formulations.

Disclosure of Invention

The present invention is based, in part, on the surprising discovery that when used as a vaccine against avian mycoplasma gallisepticum infection, a vaccine exhibiting a vh sequence having SEQ ID NO: 1 is safe and effective. SEQ ID NO: 1 is also referred to as "MGA _062 l", with the NCBI accession number NP _ 852784.

Accordingly, the present invention relates to live attenuated mycoplasma gallisepticum bacteria that exhibit reduced expression of MGA _0621 relative to a wild-type mycoplasma gallisepticum. In certain, non-limiting, exemplary embodiments, the present invention provides live attenuated mycoplasma gallisepticum strains 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. According to certain embodiments of the invention, the live, attenuated mycoplasma gallisepticum bacteria of the present invention are characterized by reduced expression of one or more of the foregoing proteins by proteomic analysis. According to an exemplary embodiment of the present invention, the live attenuated mycoplasma gallisepticum strain is a strain that exhibits reduced expression of MGA _0621, pyruvate dehydrogenase, phosphopyruvate hydratase, 2-deoxyribose-5-phosphate aldolase, and ribosomal protein L35 relative to a wild-Type mycoplasma gallisepticum bacterium, deposited at American Type Culture Collection (ATCC), p.o.box 1549, Manassas, VA 20108, and assigned accession number PTA-8485 on day 19/6 2007. This strain is also referred to herein as "Mycoplasma gallisepticum strain MGx + 47" or "MG-P48".

The invention also provides vaccine compositions comprising live attenuated mycoplasma gallisepticum bacteria of the invention, and methods of vaccinating an animal against mycoplasma gallisepticum infection. In addition, the invention provides methods for generating and/or identifying attenuated mycoplasma gallisepticum clones. According to this aspect of the invention, the method comprises subjecting an initial population of mycoplasma gallisepticum bacteria to attenuating conditions, analysing whether expression of MGA _0621 in individual clones is reduced relative to a wild-type mycoplasma gallisepticum, and testing the clones for virulence. Mycoplasma gallisepticum clones produced according to this aspect of the invention exhibit reduced expression of MGA _0621, and may optionally exhibit reduced expression of one or more other proteins selected from the group consisting of: pyruvate dehydrogenase, phosphopyruvate hydratase, 2-deoxyribose-5-phosphate aldolase, and ribosomal protein L35. Preferably, the strain exhibiting reduced expression of at least one of the foregoing proteins also exhibits reduced virulence relative to a wild-type Mycoplasma gallisepticum bacterium.

Drawings

FIG. 1 is a photograph of a two-dimensional (2-D) polyacrylamide gel showing protein spots of an attenuated strain MGx +47 of Mycoplasma gallisepticum. The circled points numbered 19, 49, 74, 108, 114, 127, 147, 166, 175 and 225 correspond to proteins that are up-regulated in MGx +47 relative to the wild type strain R-980. The circled points 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

The present invention relates to live attenuated mycoplasma gallisepticum bacteria suitable for use in vaccine formulations. Mycoplasma gallisepticum of the present invention exhibits reduced expression of the protein designated MGA _ 0621. In certain embodiments, the mycoplasma gallisepticum bacteria of the present invention further exhibit reduced expression of one or more other proteins selected from the group consisting of: pyruvate dehydrogenase, phosphopyruvate hydratase, 2-deoxyribose-5-phosphate aldolase, and ribosomal protein L35.

The NCBI accession number NP _852784 of MGA _0621, comprising the following 162 amino acid sequence: MTRTMKNKKAKKKERRFTDLSADLDEEVEKIDPEYEDFKEIKIEKNKDNQVIDKNDPFFYSESFEEARIQLIKDKKVEVKKEEEKVQETTVKNKISEAKKEEAKDVYIDSSLEIASQEPLTKGMHFYTNSRIIRKVRECAKNKGLSISRLITMILDKSIKEE (SEQ ID NO: 1).

Reduced expression of Mycoplasma gallisepticum protein

One of ordinary skill in the art will be able to determine, using routine molecular biology techniques, whether an attenuated mycoplasma gallisepticum bacterium exhibits reduced expression of one or more proteins that are normally expressed in wild-type mycoplasma gallisepticum bacterial cells. Whether an attenuated bacterium exhibits reduced expression of a particular protein (e.g., MGA _0621, pyruvate dehydrogenase, phosphopyruvate hydratase, 2-deoxyribose-5-phosphate aldolase, ribosomal protein L35, etc.) relative to a wild-type bacterium can be determined by several methods known in the art. Exemplary methods include, for example, quantitative antibody-based methods such as Western blotting, Radioimmunoassay (RIA), and enzyme-linked immunosorbent assay (ELISA), in which an antibody that detects a protein of interest (protein of interest) and is linked to the protein of interest is used. Furthermore, because messenger RNA (mRNA) levels generally reflect the amount of protein they encode, quantitative nucleic acid-based methods can also be used to determine whether expression of one or more proteins is reduced in an attenuated Mycoplasma gallisepticum bacterium. For example, quantitative reverse transcription/polymerase chain reaction (RT-PCR) methods can be used to determine the amount of mRNA corresponding to a particular protein of interest. Many nucleic acid-based quantification methods are known in the art.

The following is a non-limiting exemplary method that can be used to determine whether an attenuated mycoplasma gallisepticum bacterium exhibits reduced expression of a protein such as MGA _ 0621.

First, a population of attenuated Mycoplasma gallisepticum cells and a population of wild-type Mycoplasma gallisepticum cells are grown under substantially the same conditions in substantially the same medium. The two cell populations are then subjected to cell disruption conditions. The disrupted cells (or protein-containing portions thereof) are subjected to SDS polyacrylamide gel electrophoresis (SDS-PAGE) in parallel, followed by Western blotting using an antibody that can bind to Mycoplasma gallisepticum MGA _0621 protein (e.g., an antibody that can be obtained using standard methods well known in the art). The labeled secondary antibody is then used to provide a measurable signal proportional to the amount of protein from the cell. If an attenuated strain of Mycoplasma gallisepticum exhibits a smaller amount of signal than a wild-type Mycoplasma gallisepticum strain, it can be concluded that the expression of MGA _0621 in the attenuated strain is reduced relative to the wild-type strain. Modifications to this exemplary method, as well as alternatives thereto, will be apparent to those of ordinary skill in the art.

The present invention includes attenuated mycoplasma gallisepticum bacteria that exhibit any degree of reduction in the expression of proteins (e.g., MGA _0621, pyruvate dehydrogenase, phosphopyruvate hydratase, 2-deoxyribose-5-phosphate aldolase, ribosomal protein L35, etc.) as compared to the expression of proteins observed in wild-type strains. In certain embodiments, the attenuated bacterium exhibits at least a 5% reduction in expression of the protein relative to a wild-type bacterium. For example, if a given amount of a wild-type strain of Mycoplasma gallisepticum exhibits 100 units of a particular protein expression, while an equal amount of an attenuated strain of a candidate Mycoplasma gallisepticum exhibits 95 units of protein expression, then it can be concluded that the attenuated strain expresses 5% less protein relative to the wild-type bacterium (other examples of calculating a "percent reduction in expression" are found elsewhere herein). In certain other embodiments, the attenuated bacterium exhibits at least a 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% reduction in expression of the protein relative to a wild-type mycoplasma gallisepticum bacterium. In other embodiments, the attenuated strain of mycoplasma gallisepticum exhibits no protein expression (i.e., 100% less expression) relative to the wild-type bacterium.

In certain exemplary embodiments of the invention, the attenuated bacterium exhibits at least 5% less expression of MGA _0621, and optionally 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.

As used herein, the "percent reduction in expression" of a particular protein exhibited by an attenuated strain of mycoplasma gallisepticum relative to a wild-type strain is calculated by the following formula: (A-B)/Ax 100; wherein a is the relative level of protein expression in a wild-type mycoplasma gallisepticum strain; and B ═ the relative level of protein expression in the attenuated strain. For illustration only, if the wild-type mycoplasma gallisepticum strain exhibits expression of 0.2500 units of protein "Y" and the attenuated strain of mycoplasma gallisepticum exhibits expression of 0.1850 units of protein "Y", it can be said that the attenuated strain exhibits a reduction in expression of protein "Y" relative to the wild-type strain of [ (0.2500-0.1850)/0.2500 × 100] ═ 26%. Table 5 in example 3 herein provides further examples of calculating the percent reduction in expression of an exemplary attenuated strain of mycoplasma gallisepticum relative to a wild-type strain of mycoplasma gallisepticum.

Vaccine composition

The invention also includes a vaccine composition comprising a live, attenuated mycoplasma gallisepticum bacterium of the invention and a pharmaceutically acceptable carrier. As used herein, the expression "live, attenuated mycoplasma gallisepticum bacteria of the present invention" includes any live, attenuated mycoplasma gallisepticum bacteria described and/or claimed elsewhere herein. The pharmaceutically acceptable carrier may be, for example, water, a stabilizer, a preservative, a culture medium or a buffer. Vaccine formulations comprising the mycoplasma gallisepticum attenuated bacteria of the present invention may be prepared in suspended form or in lyophilized form or alternatively in frozen form. If frozen, glycerol or other similar agents may be added to enhance stability upon freezing.

Method for vaccinating an animal

The invention also includes methods of vaccinating an animal against mycoplasma gallisepticum infection. The method according to this aspect of the invention comprises administering to the animal an immunologically effective amount of a vaccine composition comprising a live, attenuated mycoplasma gallisepticum bacterium of the invention. As used herein, the expression "live, attenuated mycoplasma gallisepticum bacteria of the present invention" includes any live, attenuated mycoplasma gallisepticum bacteria described and/or claimed elsewhere herein. The expression "immunologically effective amount" means the amount of vaccine composition required to elicit protective levels of antibody production in an animal following vaccination. The vaccine composition can 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. It may also be administered using a needleless delivery device. Administration may be by a combination of routes, e.g., first using a parenteral route, then using a mucosal route, etc.

The animal to which the attenuated strain of mycoplasma gallisepticum is administered is preferably a bird, e.g., a chicken or turkey. When the animal is a bird, the vaccine formulation of the present invention may be administered such that the formulation immediately or eventually contacts the respiratory mucosa of the bird. Thus, the vaccine formulation may be administered to the bird, e.g., intranasally, orally, and/or intraocularly. Vaccine compositions for avian administration may be formulated as described above and/or in a form suitable for administration by spraying, including by nebulizer (for intranasal administration) or in drinking water (for oral administration).

The vaccine composition of the present invention administered by spraying or nebuliser may be formulated by incorporating live attenuated mycoplasma gallisepticum bacteria into small liquid particles. The primary particle size of the particles may be between about 10 μm to about 100 μm. These particles can be produced by, for example, conventional spray equipment and spray generators, including commercially available spray generators for backpack sprays, hatching chamber sprays, and sanitizing sprays.

Method for producing attenuated clones of mycoplasma gallisepticum

In another aspect of the invention, the invention provides methods for identifying and/or generating attenuated Mycoplasma gallisepticum clones. The method according to this aspect of the invention comprises subjecting an initial population of mycoplasma gallisepticum bacteria to attenuating conditions, thereby producing a putative attenuated population. Next, individual clones of putative attenuated colonies were analyzed to determine whether expression of MGA _0621 was reduced relative to wild-type Mycoplasma gallisepticum. Clones identified as having reduced expression of MGA _0621 were then tested for virulence. Clones that showed reduced expression of MGA _0621 and reduced virulence relative to wild-type mycoplasma gallisepticum bacteria were identified as mycoplasma gallisepticum attenuated clones.

According to this aspect of the invention, the "initial population of Mycoplasma gallisepticum bacteria" may be any number of Mycoplasma gallisepticum bacteria. In certain embodiments the bacterium is a wild-type bacterium. Alternatively, the bacterium may comprise one or more mutations. Preferably, however, the bacteria in the initial population are allogeneic or substantially allogeneic; that is, the bacteria are preferably all derived from a single parent mycoplasma gallisepticum bacterial cell and/or a cell having the same or substantially the same genotypic and/or phenotypic characteristics.

As used herein, the term "attenuating conditions" means any condition or combination of conditions that may introduce one or more genetic alterations (e.g., nucleotide mutations) into the genome of a mycoplasma gallisepticum bacterium. Exemplary, non-limiting attenuating conditions include, for example, passaging the bacteria in culture, transforming the bacteria with a genome-insertable genetic element, such as a transposon (e.g., a transposon that can be randomly inserted into the mycoplasma gallisepticum genome), and exposing the bacteria to one or more mutagens (e.g., chemical mutagens or ultraviolet light, etc.). When the cell is attenuated by in vitro passaging, the cell may be passaged any number of times, for example, 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 passaging.

After the application of attenuating conditions, the initial population of mycoplasma gallisepticum cells is referred to herein as a putative attenuated population. Individual clones of putative attenuated colonies may be obtained by standard microbiological techniques including, for example, serial dilution of the cells and plating of individual cells on a suitable medium. Once individual clones are obtained, individual clones of putative attenuated populations can be analyzed for reduced expression of MGA _0621 and/or one or more other specific proteins. Methods for determining whether an attenuated mycoplasma gallisepticum bacterium exhibits reduced expression of one or more proteins that are normally expressed in a wild-type mycoplasma gallisepticum bacterial cell are described herein. Exemplary methods include, for example, RT-PCR based methods, Western blotting, and the like.

Virulence can be tested by administering individual clones identified as having reduced expression of MGA _0621 to an animal susceptible to infection by wild-type (non-attenuated) bacteria. As used herein, an "animal susceptible to infection by a wild-type mycoplasma gallisepticum bacterium" is an animal that develops at least one clinical symptom following infection with a wild-type mycoplasma gallisepticum bacterium. These symptoms are known to those of ordinary skill in the art. For example, for a putative attenuated strain of mycoplasma gallisepticum that exhibits reduced expression of, for example, MGA _0621, the strain may be administered to, for example, turkeys or chickens (which are typically susceptible to infection with wild-type mycoplasma gallisepticum). Clinical symptoms of avian mycoplasma gallisepticum infection include, for example, acute respiratory symptoms, pericarditis, perihepatitis, airsacculitis, airway thickening, slow weight gain, disappearance of lint, goblet cell abnormalities, telangiectasia, increased lymphocytes, plasma cells, and/or xenophiles, and sometimes decreased egg production. Thus, a putative attenuated strain of mycoplasma gallisepticum is considered "less virulent" if it is administered to a chicken or turkey that produces fewer and/or less severe symptoms than a turkey or chicken infected with a wild-type mycoplasma gallisepticum strain. Any degree of symptom reduction will identify the putative attenuated strain as having reduced virulence. In certain embodiments, the putative attenuated strain is not toxic.

According to the present invention, a mycoplasma gallisepticum clone that exhibits reduced expression of MGA _0621 (and/or one or more other specific proteins) and reduced virulence relative to a wild-type mycoplasma gallisepticum bacterium is a mycoplasma gallisepticum attenuated clone. An exemplary live attenuated Mycoplasma gallisepticum clone of the present invention is the strain designated MGx +47, which exhibits reduced expression of MGA _0621 (with reduced expression of pyruvate dehydrogenase, phosphopyruvate hydratase, 2-deoxyribose-5-phosphate aldolase, and ribosomal protein L35). MGx +47 was deposited at the American Type culture Collection (American Type culture Collection, P.O. Box 1549, Manassas, VA 20108) on 19.6.2007 with the designation of accession number PTA-8485.

The following examples are intended to illustrate, but not limit, the methods and compositions of the present invention. Appropriate modifications and variations to various conditions and parameters, which are conventional in molecular biology and chemistry, will be apparent to those skilled in the art in light of this disclosure and are intended to be within the spirit and scope of the invention.

Examples

Example 1

Production of live attenuated Mycoplasma gallisepticum strains

A new live attenuated strain of Mycoplasma gallisepticum was generated by passaging the wild-type Mycoplasma gallisepticum strain R980 multiple times in vitro. Specifically, 0.1mL of seed material of wild-Type Mycoplasma gallisepticum strain R-980 was inoculated into 20mL of modified free medium (free et al, am. J. Vet. Res.29: 2163-, box 1549, Manassas, VA 20108), and designates accession number PTA-8485.

Example 2

Safety and efficacy assessment of live attenuated mycoplasma gallisepticum vaccines in chickens

In this example, the new mycoplasma gallisepticum vaccine strain MGx +47 obtained in example 1 was evaluated for safety and efficacy in chickens.

Seventy-one SPF white leghorn chickens were divided into seven groups as follows:

table 1: design of experiments

Group of	Number of chickens	Whether or not to vaccinate	Whether or not to infect
				1	11	Whether or not	Is that
2	10	Is that	Whether or not
				3	11	Is that	Is that
4a	10	Is that	Whether or not
				4b	11	Is that	Whether or not
4c	9	Is that	Whether or not
				5	9	Whether or not	Whether or not

At four weeks of age, attenuated strain MGx +47 was used as a coarse spray at 3.72X 10⁷CCU/mL/bird vaccinated chickens of groups 2, 3, 4a, 4b and 4 c. Seven weeks old, 0.5mL of Mycoplasma gallisepticum strain R at 7.74X 10⁵CCU/mL infected chickens in groups 1 and 3 through the trachea. At nine weeks of age, the chickens of groups 1, 2, 3 and 5 were dissected, and the chickens of groups 4a, 4b and 4c were dissected at days 7, 14 and 21 post-vaccination (DPV), respectively. Average weight gain, pericarditis, perihepatitis, air sacculitis and tracheitis were evaluated in chickens. The results are shown in Table 2.

Table 2: security and availability profile

Vaccination ═ 3.62 × 10⁷CCU/mL/bird

Infection is 7.74X 10⁵CCU/mL，0.5mL

Group of

Whether or not to vaccinate

Whether or not to infect

Average weight gain (kg/day)

Pericarditis

Perihepatitis of liver

Cystitis of the air

Air sac inflammation score (positive mean)

Trachea (histology)

1

Whether or not

Is that

0.016

0/11

0/11

9/11

3.56

Severe gas

Inflammation of the canal

2

Is that

Whether or not

0.018

0/10

0/10

0/10

0

Is normal

3

Is that

Is that

0.017

0/11

0/11

2/11

2.5

Mixed tracheitis

4a

Is that

Whether or not

0.016

0/9

0/9

0/9

0

Is normal

4b

Is that

Whether or not

0.017

0/11

0/11

0/11

0

Is normal

4c

Is that

Whether or not

0.017

0/10

0/10

0/10

0

Is normal

5

Whether or not

Whether or not

0.015

0/9

0/9

0/9

0

Is normal

Table 3: safety watch: histological report of formalin-fixed chicken tracheas from each vaccinated/uninfected chicken (groups 4a, 4b and 4 c)

Table 4: table of effectiveness: histological report of formalin-fixed chicken tracheas from each chicken

Key points of the safety and effectiveness tables (tables 3 and 4):

● all "vaccinated" birds were sprayed coarsely with vaccine strain MGx +47 at 3.62X 10⁷CCU/mL/bird vaccination;

● all "infested" birds were infected Intratracheally (IT) with 0.5mL of Mycoplasma gallisepticum strain at 7.74X 105CCU/mL

● time points (Table 3: in safety Table) are the days after inoculation when the chickens were examined and are expressed as days after inoculation (DPV).

● cilia: "N" is normal cilia; "-", is dropped;

● goblet cells/M ("-normal goblet cells;" + "-mucus on the surface of the respiratory tract);

● telangiectasia ("-", no dilatation or inflammation; "+", moderate telangiectasia or inflammation; "+", severe telangiectasia or inflammation);

● LC/PC ═ lymphocytes and plasma cells ("-" ═ none; "+" -few; "+ + + + +" -many);

● PMN ═ xenotropic cells ("-" ═ none; "+" ═ few; "+ + + + +" -many);

histological analysis of group 2 chickens (vaccinated but not infected) was substantially similar to group 5 chickens (unvaccinated, not infected), demonstrating the safety of the newly-produced MGx +47 vaccine strain. (see, e.g., Table 2 above).

With respect to effectiveness, group 3 chickens (vaccinated and infested) exhibited a significant reduction in bursitis compared to group 1 chickens (not vaccinated, infested). (see, e.g., tables 2 and 4). Furthermore, as shown in table 4, group 3 chickens exhibited fewer histological signs of mycoplasma gallisepticum infection with cilia, goblet cells, telangiectasia, lymphocytes and plasma cells (LC/PC), xenophile cells, and tracheal thickness. (see Table 4).

Thus, this example demonstrates MGx +47 as a safe, effective live attenuated mycoplasma gallisepticum vaccine strain.

Example 3

Proteomics identification of MGx +47 vaccine strains

In order to more accurately identify MGx +47 vaccine strains at the molecular level (see examples 1 and 2), proteomic analysis of the strains was performed.

In this example, total protein was isolated from wild-type Mycoplasma gallisepticum strain R-980 and the newly identified vaccine strain MGx + 47. The proteins of each strain were resolved by two-dimensional polyacrylamide gel electrophoresis, followed by computer analysis of gel imaging (see FIG. 1). Protein spots differentially expressed in the vaccine strains were identified. Protein spots that were not expressed or were significantly reduced in the vaccine strain compared to the wild type strain were excised from the gel.

Five points were identified whose expression levels in the MGx +47 vaccine strain were significantly reduced compared to the wild-type mycoplasma gallisepticum. Each protein spot was excised from the gel and digested with enzyme. Peptide mass fingerprinting was then performed using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). The mass spectra identified for each protein spot were compared to a database of peptide masses to identify the proteins and the corresponding genes encoding them. The results of this analysis are summarized in the following table:

table 5: MGx +47 summary of proteomics analysis results

Gene	Product of	Function(s)	Expression level in wild type MG	Expression level in MGx +47	Percent reduction of expression
						acoA	Pyruvate dehydrogenase	Energy production and conversion (Kreb cycle)	0.1872	0.0858	54.2％
eno	Phosphopyruvate hydratase	Catalyzing the formation of phosphoenolpyruvate	0.0683	0.0173	74.7％
						deoC	2-deoxyribose-5-phosphate aldolase	Required for nucleotide metabolism	0.0525	0.0309	41.1％
rpml	Ribosomal protein L35	Translation, ribosomal structure and biological origin	0.1171	0.0259	77.9％
						MGA_0621	Hypothetical proteins	Is unknown	0.4534	0.0835	81.6％

The reduction in expression of the gene product is also expressed as "fold reduction in expression". For example, in table 5, it can be said that the strain MGx +47 exhibited 2.2, 3.9, 1.7, 4.5 and 5.4-fold reduced expression of acoA, eno, deoC, rpml and MGA _0621, respectively, relative to wild-type MG.

As shown in table 5, five gene products were identified in the attenuated MGx +47 live vaccine strain that were significantly reduced in expression compared to the wild-type R-980 strain: AcoA, Eno, DeoC, Rmpl and MGA _0621(NCBI accession No. NP _852784, identified as a putative protein). The most reduced expression was observed with MGA _ 0621. Thus, mutations or growth conditions that result in reduced expression of MGA _0621 may result in attenuated mycoplasma gallisepticum toxicity. Thus, down-regulation of MGA _0621 may be an effective strategy to generate attenuated strains of mycoplasma gallisepticum.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is not intended to be limited to the specific embodiments disclosed, but rather, to cover all changes and modifications that come within the spirit and scope of the invention as defined by the appended claims.

All documents 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 documents and patents are incorporated by reference herein to the same extent as if each document or patent application was specifically and individually indicated to be incorporated by reference.

Sequence listing

<110> Huishi Limited liability company

Mach-Kuma

Mohmander, a. kaen

<120> attenuated strain of Mycoplasma gallisepticum

<130>AM102888

<150>60/993447

<151>2007-09-11

<160>1

<170>PatentIn version 3.4

<210>1

<211>162

<212>PRT

<213> Mycoplasma gallisepticum

<400>1

Met Thr Arg Thr Met Lys Asn Lys Lys Ala Lys Lys Lys Glu Arg Arg

1 5 10 15

Phe Thr Asp Leu Ser Ala Asp Leu Asp Glu Glu Val Glu Lys Ile Asp

20 25 30

Pro Glu Tyr Glu Asp Phe Lys Glu Ile Lys Ile Glu Lys Asn Lys Asp

35 40 45

Asn Gln Val Ile Asp Lys Asn Asp Pro Phe Phe Tyr Ser Glu Ser Phe

50 55 60

Glu Glu Ala Arg Ile Gln Leu Ile Lys Asp Lys Lys Val Glu Val Lys

65 70 75 80

Lys Glu Glu Glu Lys Val Gln Glu Thr Thr Val Lys Asn Lys Ile Ser

85 90 95

Glu Ala Lys Lys Glu Glu Ala Lys Asp Val TyrIle Asp Ser Ser Leu

100 105 110

Glu Ile Ala Ser Gln Glu Pro Leu Thr Lys Gly Met His Phe Tyr Thr

115 120 125

Asn Ser Arg Ile Ile Arg Lys Val Arg Glu Cys Ala Lys Asn Lys Gly

130 135 140

Leu Ser Ile Ser Arg Leu Ile Thr Met Ile Leu Asp Lys Ser Ile Lys

145 150 155 160

Glu Glu

Claims (2)

1. A live, attenuated mycoplasma gallisepticum bacterium deposited at the ATCC under accession number PTA-8485.

2. A vaccine composition comprising the live, attenuated mycoplasma gallisepticum bacterium of claim 1, further comprising a pharmaceutically acceptable carrier.