# Patent

## Title
Vaccines for Mycoplasma bovis and methods of use

## Abstract

The invention of novel, effective vaccines against Mycoplasma bovis for use in cattle is described. These vaccines demonstrate no undesirable side effects and protect against M. bovis related disease, such as contagious mastitis, respiratory pneumonia, joint infections, keratoconjunctivitis and middle ear infections. The novel vaccines also lessen the effect M. bovis infections on milk production, weight gain and animal health. Methods of diagnosing characterizing and treating M. bovis infections as specific biotypes are also disclosed. Vaccine compositions made in accordance with the invention may be either of the attenuated or inactivated variety. Vaccines may also include antigens from other pathogens so as to provide a protective immunogenic response to diseases other than those caused by M. bovis.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a divisional of prior U.S. patent application Ser. No. 09/708,352 filed on Nov. 8, 2000 which claims benefit of U.S. Patent Application Ser. No. 60/164,286, filed on Nov. 8, 1999, the disclosures of which are incorporated herein, in their entirety.


FIELD OF THE INVENTION
This invention relates to novel vaccines for protection against Mycoplasma bovis disease in animals, compositions for the diagnosis of such infections, and methods of diagnosis and vaccination.
BACKGROUND OF THE INVENTION
Mycoplasma bovis is a pathogenic prokaryote belonging to a class of organisms that is intermediate in size between a bacteria and virus. These mycoplasmas are the smallest of the free-living microorganisms. They are characterized by the lack of a cell wall and are enveloped with only a cell membrane, which allows for varying morphological shapes and unique growth requirements.
Mycoplasmas are known to cause infectious disease in most species of animals. In bovine species, Mycoplasma bovis is an opportunistic microorganism that causes infectious disease that is of significant economic importance to the livestock industry. Mycoplasma bovis isolation in a diseased bovine can be the result of its role as either a primary or secondary causative etiological disease agent. Clinical disease and losses associated with infections caused by Mycoplasma bovis in beef and dairy cattle include: contagious mastitis, respiratory pneumonia, joint infections (arthritic conditions), keratoconjunctivitis, and middle ear infections. Even though several species of mycoplasmas have been isolated in cattle, by far the most prevalent is Mycoplasma bovis. For mastitis infections, the prevalence of M.bovis is reported to be 70% or more.
Diseases caused by mycoplasmas are often resistant to antimicrobial therapy, leaving no effective means of treatment. Consequently, the only effective control method is to cull animals from a herd. This has enormous economic implications in the dairy industry where losses are measured by the value of the culled animals as well as the impact on both milk quality and quantity due to clinical and subclinical infections. Mycoplasma infections resulting in bovine mastitis are increasing in prevalence and geographical distribution. In the United States, this higher prevalence is due to a larger and more intense cattle production industry in which herds are rapidly expanding, placing them at greater risk. Increased incidence of M. bovis infection and related infectious disease in dairy herds has been noted worldwide (Jasper, D E 1982, J. Amer. Vet. Med. Assn. 181:158-162).
Control of disease caused by mycoplasmas in swine and avian species has occurred in recent years as the result of successful vaccination programs using safe and efficacious products. The design and development of an effective commercial vaccine in the United States to control Mycoplasma bovis has not yet occurred, even though changes in cattle production methods and husbandry practices have resulted in a greater commercial need to control this agent from both an economic and food quality perspective. Although there have been numerous attempts to produce vaccine to protect against Mycoplasma bovis, the resulting experimental vaccines have been deemed unacceptable due to the lack of protection as well as unacceptable site reactions in vaccinated animals (Boothby, et al. 1986 Cornell Vet 76: 188-197; Boothby et al. 1987 Can J. Veterinary Research 51:121-125; Howard et al. 1987 Veterinary Record 121:372-376; Boothby, et al. 1988 Can J. Veterinary Research 52:355-359). Thus, there remains a need in the veterinary and animal health profession to provide a safe and effective vaccine to prevent infectious disease caused by Mycoplasma bovis with no unfavorable host animal reactions.
SUMMARY OF INVENTION
The invention disclosed herein provides safe and effective vaccines for the prevention of Mycoplasma bovis disease in cattle. Also disclosed are methods for characterizing biotypes of Mycoplasma bovis in cattle, bulk milk tanks, and barns.



DESCRIPTION OF FIGURES
FIG. 1 is an illustration of the gel electrophoretic pattern for DNA products produced by Polymerase Chain Reactions from different Mycoplasma bovis isolates. On the left side of the figure, molecular weight standards based on restriction endonuclease digests of lambda and phi phage are shown. The size of the bands in the standard digests are, from top to bottom, for lambda; n/d, n/d, n/d, 2027, 1904, 1584, 1375, 947, 831 and 564 base pairs, and for Phi X174; 1353, 1078, 872, 603, 310, 284/271, 234, 194 and 118 base pairs. The relative location of PCR-generated markers for different biotypes, designated A and B, are shown in lanes 5-12 to the right of the standards. Arrows in lanes 5 and 7 indicate the presence of three and two characteristic bands for biotypes A and B, respectively.
FIG. 2 is an illustration of the gel electrophoretic pattern for DNA products produced by Polymerase Chain Reactions from a second set of Mycoplasma bovis isolates. On the left side of the figure, molecular weight standards based on restriction endonuclease digests of lambda and phi phage are shown. The size of the bands in the standard digests are, from top to bottom, for lambda; n/d, n/d, n/d, 2027, 1904, 1584, 1375, 947, 831 and 564 base pairs, and for Phi X174; 1353, 1078, 872, 603 and 310 base pairs. The relative location of PCR-generated markers for different biotypes, designated A and C, are shown in lanes 5-11 to the right of the standards. Arrows in lanes 5 and 8 indicate the presence of the three and two characteristic bands for biotypes A and C, respectively.



DETAILED DESCRIPTION OF THE INVENTION
As used throughout the specification and in the claims, “a,” “an” or “the” can mean one or more, depending upon the context in which it is used.
In accordance with the purposes of this invention, as embodied and broadly described herein, this invention, in one aspect, provides a vaccine composition which is protective against Mycoplasma bovis disease in a bovine species, comprising one or more inactivated or attenuated Mycoplasma bovis biotype(s) and a pharmaceutically acceptable excipient. The term “inactivated,” also referred to as “killed,” means that the microorganisms are treated by any of several means known to the art so that they no longer grow or reproduce, but that the microorganisms are still capable of eliciting an immune response in the target animal. Examples of inactivating agents are: formalin, azide, freeze-thaw, sonication, heat treatment, sudden pressure drop, detergent (especially non-ionic detergents), lysozyme, phenol, proteolytic enzymes, propiolactone, Thimerosal (see U.S. Pat. No. 5,338,543 Fitzgerald, et al.), and binary ethyleneimine (see U.S. Pat. No. 5,565,205 Petersen, et al.). In a specific embodiment, the Mycoplasma bovis strains used in the vaccine are inactivated with beta-propiolactone (BPL).
Alternatively, the M. bovis biotypes used in the vaccine can be attenuated. The term “attenuated,” also referred to as “modified live,” is intended to refer to a living biotype of Mycoplasma bovis which has been attenuated (modified) by any of a number of methods known in the art including, but not limited to, multiple serial passage, temperature sensitive attenuation, mutation, or the like such that the resultant strain is relatively non-pathogenic to a bovine species. The modified live strain should be capable of limited replication in the vaccinated animal and of inducing a protective immune response which is protective against disease caused by virulent or wild-type Mycoplasma bovis. 
The term “pharmaceutically acceptable” means a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an animal along with the immunogenic material (i.e. inactivated or attenuated M. bovis biotypes) without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the vaccine in which it is contained. Examples of such pharmaceutically acceptable excipients include water and physiological saline (for further examples, see Arnon, R. (Ed.) Synthetic Vaccines 1:83-92, CRC Press, Inc., Boca Raton, Fla., 1987).
The invention disclosed herein is based in part on the discovery that Mycoplasma bovis infections in the field comprise mixtures of biotypes. The term “biotype” means a variant of a species, i.e. a strain, that can be distinguished by one or more characteristics, such as ribosomal RNA sequence variation, DNA polymorphisms, serological typing, or toxin production (see e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989; DNA cloning: A Practical Approach, Volumes I and II, Glover, D. M. ed., IRL Press Limited, Oxford, 1985; Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, N.Y. (1988)).
In another aspect of this invention, to produce an effective vaccine against Mycoplasma bovis, the vaccine must contain antigen derived from a biotype of Mycoplasma bovis. Examples of specific embodiments would include vaccines containing antigen derived from M. bovis biotypes A, B, or C. In a further specific embodiment, the vaccine comprises antigen derived from more than one M. bovis biotypes (e.g., A and B, A and C, B and C, or A, B and C). In a further specific embodiment, the vaccine comprises antigen derived from one or more M. bovis biotypes and antigen derived from another pathogen. In a further specific embodiment, the vaccine comprises inactivated or attenuated M. bovis biotype A, B or C. In a further specific embodiment, the vaccine comprises at least two inactivated or attenuated M. bovis biotypes (e.g., A and B, A and C, B and C, or A, B and C). In a further specific embodiment, the vaccine comprises at least one inactivated or attentuated M. bovis biotype with antigen derived from another pathogen. In a preferred embodiment, the vaccine comprises inactivated or attenuated M. bovis biotype A, as defined herein, and at least one other biotype of M. bovis. 
It is anticipated that additional biotypes of M. bovis may emerge and may be isolated with continued animal production. Additional biotypes can be added to the vaccine as needed. It is a matter of routine practice to sample bulk milk tanks and blood from cows to isolate Mycoplasma bovis cultures. These cultures can then be biotyped according to any of several typing techniques, as listed hereinabove. Vaccines can be formulated based on the prevalence of M. bovis biotypes present in the environment. Autogenous vaccines, i.e. those vaccines for use on the farm where the microorganisms are isolated, can be custom-designed to contain all biotypes found on the farm, but not any other biotype. Vaccines developed for use by a mass market, i.e. those vaccines produced for general use on many different farms containing pre-selected biotypes, can also be developed, marketed and used.
In another aspect, this invention provides a vaccine comprising a single, inactivated or attenuated Mycoplasma bovis biotype, a pharmaceutically acceptable excipient, and a suitable adjuvant. In a specific embodiment, the vaccine contains inactivated or attenuated M. bovis biotypes A, B or C or any mixture thereof and may further contain antigens from other pathogens.
In a preferred embodiment, the inactivated vaccines of this invention are produced from biotypes freshly isolated from infected animals or from cryopreserved biotype cultures freshly prepared from infected animals. In a preferred embodiment, the attenuated vaccines of this invention are produced from cultures of biotypes which have been treated so as to retain a limited ability to replicate within the vaccinated animal, but which does not retain the ability to infect other animals and cause mycoplasma-related disease. The preparation and use of attenuated vaccines is well-known to practitioners of ordinary skill in the art.
The inactivated or attenuated M. bovis biotype(s) may be further processed to fractionate and/or standardize the antigenic mass. For example, specific biotypes might be isolated from samples and combined to form specific combinations of biotypes in specific ratios. Similarly, components from a specific inactivated or attentuated M. bovis biotype might be fractionated and a subset of those fractions combined with similarly fractionated components of another biotype to standardize the antigenic component of the vaccine preparation and to optimize its efficacy. In one embodiment, the antigenic components derived from a single biotype are enriched by removing non-immunogenic components from the cells of the biotype. In another embodiment, the vaccine preparations are standardized to provide a required minimum cell content per formulated dose. In a preferred embodiment, the vaccine comprising inactivated M. bovis biotype(s) is formulated to deliver at least 108 M. bovis cell equivalents of each biotype per dose. A complete vaccination of a bovine species comprises the administration of recommended doses. In a preferred embodiment, two such doses will be administered. In a further preferred embodiment, three such doses will be administered. In another preferred embodiment, the vaccine comprising attenuated M. bovis biotype(s) is formulated to deliver at least 105 M. bovis cells per biotype. It is understood by those skilled in the art that the critical value in describing a vaccination dose is the total amount of immunogen needed to elicit a protective response by the host animal to infectious disease caused by virulent or wild-type M. bovis. The number and volume of doses used can be varied and are determined by the practitioner based on costs and the need to avoid deleterious side effects in the animal caused by the administration. For example, the volume of one administration typically does not exceed 2-5 milliliters. The number of doses of inactivated vaccine needed in adult animals is typically one initial dose followed by 1-2 additional doses and annual revaccination. The number of doses of attenuated vaccine in adult animals is one initial dose followed by a booster. Subsequently, annual boosters are administered.
The vaccines of the present invention may further comprise antigenic material of other viruses and/or microorganisms known to be bovine pathogens, including, but not limited to, attenuated (modified-live) or inactivated viruses or microorganisms. Such combination vaccines provide protection against a plurality of diseases to which the bovine species are exposed, including but not limited to immunogenic compositions for Staphylococcus aureus, Pasteurella hemolytica, Pasteurella multocida, Hemophilus somnus, Bovine Respiratory Syncytial Virus, Bovine Diarrhea Virus, E. coli and Infectious Bovine Rhinotrachial Disease.
In other embodiments, the vaccine of this invention further comprises a suitable adjuvant. As used herein, an “adjuvant” is a potentiator or enhancer of the immune response. The term “suitable” is meant to include any substance which can be used in combination with the vaccine immunogen (i.e. inactivated or attenuated M. bovis biotypes or fractions thereof) to augment the immune response, without producing adverse reactions in the vaccinated animal. Effective amounts of a specific adjuvant may be readily determined so as to optimize the potentiation effect of the adjuvant on the immune response of an animal vaccinated. In a preferred embodiment, adjuvanting of the vaccines of this invention is a 2-stage process utilizing firstly a 2% aluminum hydroxide solution and secondly a mineral oil. In specific embodiments, suitable adjuvants can be chosen from the following group: mineral, vegetable or fish oil with water emulsions, incomplete Freund's adjuvant, E. coli J5, dextran sulfate, iron oxide, sodium alginate, Bacto-Adjuvant, certain synthetic polymers such as Carbopol (BF Goodrich Company, Cleveland, Ohio), poly-amino acids and co-polymers of amino acids, saponin, carrageenan, REGRESSIN (Vetrepharm, Athens, Ga.), AVRIDINE (N, N-dioctadecyl-N′,N′-bis(2-hydroxyethyl)-propanediamine), long chain polydispersed β (1,4) linked mannan polymers interspersed with O-acetylated groups (e.g. ACEMANNAN), deproteinized highly purified cell wall extracts derived from non-pathogenic strain of Mycobacterium species (e.g. EQUIMUNE, Vetrepharm Research Inc., Athens Ga.), Mannite monooleate, paraffin oil, and muramyl dipeptide.
In another aspect, this invention discloses a method for immunizing bovine animals against infectious disease caused by Mycoplasma bovis comprising administering to a bovine animal immunogenic amounts of inactivated or attentuated Mycoplasma bovis biotypes to elicit a protective immune response by the animal. Preferably, the method comprises administering at least two inactivated or attenuated Mycoplasma bovis biotypes to elicit a protective immune response by the animal. Immunization may be performed orally, intranasally, intratracheally, intramuscularly, intramammarily, subcutaneously, intravenously, or intradermally. The vaccine containing the inactivated or attenuated M. bovis biotypes can be administered by injection, by inhalation, by ingestion, or by infusion. Repeated doses of the vaccine preparations, i.e. “boosters”, are preferable at periodic time intervals to enhance the immune response initially or after a long period of time since the last dose. The time interval between vaccinations varies depending on the age and condition of the animal. For lactating and adult animals, the first vaccination is preferably given at the end of the lactation cycle (i.e. “dry-off”), followed by a “booster” dose 2-4 weeks later, and preferably followed by a second booster dose 2-4 weeks thereafter. Newborn calves are preferably vaccinated at birth, followed by booster doses every 3-5 weeks until the calves are 4-6 months old and annually thereafter. However, at risk or exposed stocker and feeder animals should be vaccinated more often, preferably no less than once every 6 months.
In another embodiment of the methods of this invention, the multiple M. bovis biotypes comprising the vaccine can be delivered in separate administrations to the animal. For example, the vaccine comprising inactivated M. bovis biotypes A and B can be delivered by separately administering an immunogenic amount of biotype A in one injection and an immunogenic amount of biotype B in another injection. In a further embodiment, each separately administered biotype can be administered as a combination vaccination, comprising antigenic material of other viruses and/or microorganisms known to be bovine pathogens.
The term “immunogenic amount” means an amount of an immunogen, i.e. the inactivated or attenuated M. bovis biotype(s) or a portion thereof, which is sufficient to induce an immune response in a vaccinated bovine species and which protects the animal against disease caused by wild-type or virulent M. bovis infections upon exposure thereto or which has a commercially beneficial effect that lessens the effect of M. bovis on milk production, weight gain or animal health. In a preferred embodiment, bovine animals are immunized by administering at least approximately 108 M. bovis cell equivalents of each inactivated biotype in the vaccine. In a specific embodiment, animals are immunized by administering at least approximately 108 M. bovis biotype A cell equivalents and approximately 108 M. bovis biotype B cell equivalents, which have been inactivated, in at least two injections. In another specific embodiment, bovine animals are immunized by administering at least approximately 108 M. bovis biotype A cell equivalents, 108 M. bovis biotype B cell equivalents and approximately 108 M. bovis biotype C cell equivalents, which have been inactivated, in at least two injections.
In another preferred embodiment, bovine animals are immunized by administering at least approximately 105 M. bovis cells of each attenuated biotype in the vaccine. In a specific embodiment, bovine animals are immunized by administering at least approximately 105 M. bovis biotype A attenuated cells and at least approximately 105 M. bovis biotype B attenuated cells. In another specific embodiment, bovine animals are immunized by administering at least approximately 105 M. bovis biotype A cells, 105 M. bovis biotype B cells, and 105 M. bovis biotype C cells.
In another aspect, this invention discloses a method for producing a Mycoplasma bovis vaccine comprising contacting at least two live Mycoplasma bovis biotypes with an inactivating material and incorporating the inactivated Mycoplasma bovis biotypes into a pharmaceutically acceptable excipient with a suitable adjuvant to produce a Mycoplasma bovis vaccine. In a preferred method, selected Mycoplasma bovis biotypes are grown separately as pure cultures, free of contamination by viruses, bacteria or any other microbial agent, including other biotypes of M. bovis, to the desired cell equivalents, inactivated as described herein, and then combined in equal amounts with a pharmaceutically acceptable excipient to produce a Mycoplasma bovis vaccine. Alternatively, the biotypes can be grown together as a mixed culture to the desired cell equivalents, inactivated and then, optionally, combined with a pharmaceutically acceptable excipient and a suitable adjuvant to produce a Mycoplasma bovis vaccine.
In a further embodiment of the hereinabove disclosed method of producing a Mycoplasma bovis vaccine, the inactivated or attenuated Mycoplasma bovis biotypes are mixed with a suitable adjuvant. In a preferred method, the suitable adjuvant is an aluminum hydroxide-oil emulsion.
Selected M. bovis biotypes may be used as the basis for diagnostic tools to detect the presence of M. bovis. In one aspect of this invention, samples from cattle would be tested for the presence of antibodies specific for M. bovis by contacting the samples with M. bovis cells or antigens derived from M. bovis. Examples of technologies that could be adapted to such a method include, but are not limited to, RIA, ELISA and immunoblot. Examples of specific embodiments would include antigens derived from one or more M. bovis biotypes (e.g., A, B, C, A and B, B and C, A and C, or A, B and C). In a preferred embodiment, antigen from each of the M. bovis biotypes A, B and C would be utilized to test for the presence of antibodies specific for each of the M. bovis biotypes, thus allowing an autogenous vaccine to be administered. In another embodiment, antibodies raised against M. bovis biotypes or antigens derived from selected biotypes would be used to test for the presence of M. bovis biotypes A, B and C. Examples of specific embodiments would include antibodies reactive to antigens derived from one or more M. bovis biotypes (e.g., A, B, C, A and B, B and C, A and C, or A, B and C). In another embodiment, antigens derived from different biotypes would be utilized to test for the presence of antibodies specific against antigens from a given biotype.
In a further embodiment, the present invention provides an isolated Mycoplasma bovis biotype A, Mycoplasma bovis biotype B, Mycoplasma bovis biotype C, or any combination thereof.
Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.
EXAMPLES
Example 1
Characterization and Typing of Field Isolates
Strains were collected from infected animals or milk tanks. Single colonies were cultured, and each culture was analyzed for cytotoxicity and for the presence of specific DNA markers by PCR fingerprinting.
PCR fingerprinting: Arbitrarily-chosen primers were selected; Primer 1 and Primer 2 below (I representing deoxyinosine and A, C, T and G representing the four naturally-occurring bases of DNA):









Primer 1:




5′ III ICG ICG ICA TCI GGC 3′;
[SEQ ID NO: 1]


and


 


Primer 2:


5′ ICG ICT TAT CIG GCC TAC 3′;
[SEQ ID NO: 2]






Mycoplasma bovis DNA was isolated and amplified, using these primers, in a polymerase chain reaction (PCR) as follows: The initial cycling step was for 120 seconds at 94° C. Denaturation was for 30 seconds at 94° C., followed by annealing for 90 seconds at 40° C., extension for 120 seconds at 72° C., with a final extension for 240 seconds at 72° C. A total of 35 cycles of amplification were used.

The resulting DNA products of the PCR were separated by non-denaturing 1.5% agarose gel electrophoresis and were visualized by staining with ethidium bromide and illuminating the gel with a UV light source. Comparison of the resultant patterns, characteristic for a given biotype, with molecular weight standards such as the EcoRI/HindIII digest of lambda phage or the HaeIII digest of phiX174 phage, electrophoresed alongside the PCR products, allows consistent and reproducible biotyping of M. bovis strains. Examples of biotyping results using this method are provided below.



















Biotype



Strain ID
Culture #
% Cytotoxicity
Profile





















BA2580
1
95
A




2
0
A



BA2491
1
82
A




2
100
A



498
1
100
A




3
91
A



4082
1
100
B




2
91
B




3
83
B



Tank 2-18
1
90
A




2
100
A




3
100
A



Tank 2-19
1
100
A




2
20
A




3
100
A



L-56291
1
100
A




2
100
A




3
86
A



L-477
1
84
C




2
76
C




3
90
C



L-53219
1
66
A




2
100
A




3
100
A










Both cytotoxic (i.e. ≧40% cytotoxic) and noncytotoxic strains are pathogenic. While the majority of isolates are homogeneously cytotoxic, a few isolates, e.g. BA2580 and Tank 2-19, are a mixture of non-cytotoxic and cytotoxic colonies. Following extensive passage in culture, all strains become noncytotoxic, while passage through calves accentuates the initial phenotype, whether non-cytotoxic or cytotoxic. The PCR fingerprints for three M. bovis biotypes are illustrated in FIGS. 1 and 2 alongside the standards formed by the EcoRI/HindIII restriction endonuclease digest of lambda phage and the HaeIII restriction endonuclease digest of phi phage. The sizes of the resultant standard fragments, in base pairs, are listed in the description of FIGS. 1 and 2. A blank and a positive control for the PCR fingerprinting reactions are included in lanes 3 and 4, respectively, for both FIGS. 1 and 2. Further description of PCR fingerprinting can be found in Artiushin et al. Int. J. Syst. Bacteriol. 46: 324-328 (1996), Fan et al. Avian Dis. 39: 729-735 (1995) and elsewhere. Other methods of biotyping mycoplasma, or other microorganisms, are well-known to the art and may also be used in the practice of the invention (see e.g., Sambrook et al., Molecular Cloning. A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989; DNA cloning: A Practical Approach, Volumes I and II, Glover, D. M. ed., IRL Press Limited, Oxford, 1985; Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, N.Y. (1988)).
Example 2
Preparation of an Inactivated Vaccine Against Biotypes of Mycoplasma 
A Mycoplasma bovis vaccine is prepared by inactivating a selected biotype of Mycoplasma bovis and combining this preparation with an adjuvant.
A. Selection of M. bovis Biotypes
Isolates of M. bovis were obtained from samples of infected milk. These isolates were then cultured using standard techniques, such as those described by Knudtson et al. Vet. Microbiol. 11: 79-91 (1986).
Selected isolates were further expanded and characterized by biotype. Cultures of isolates representative of the characteristic biotypes, as determined by PCR fingerprinting, were selected and stocks of these biotypes were preserved by combining them with a gelatin protein hydrolysate stabilizing solution and subjecting the product to cryopreservation. Pure biotype cultures were used to inoculate a controlled fermentation of the microorganism for use in producing vaccine. PCR fingerprinting of these cultures for vaccine production confirmed their purity as single biotypes. Subsequent testing of the cryo-preserved stock(s) was performed in a USDA licensed facility according to Title 9 Code of Federal Regulations to validate purity and identity of the culture(s). Identity was determined to be Mycoplasma bovis by two independent laboratories when tested by indirect immunofluorescence with specific antisera to the following species:

    
    
        Mycoplasma bovis 
        Mycoplasma califoricum 
        Mycoplasma alkalescens 
        Mycoplasma canadense 
        Mycoplasma bovigenitalium 
        Mycoplasma bovirhinis 
        Mycoplasma arginnii 
        Acholeplasma laidlawii 

B. Propagation of the Pure, Isolated Biotypes

    
    


Selected strains, or biotypes, identified as being pure, were propagated in a defined media and further processed to make vaccines.
Mycoplasma bovis biotypes can be propagated in a variety of different general purpose, growth-promoting, defined media that are known to those knowledgable in the art, including, but not limited to, Hayflick Media, Adler Media, and Gourlay Media. In a preferred embodiment of this invention, the propagation medium is:

    
    
        Yeast Extract: 5 grams per liter
        Proteose Peptone: 2 grams per liter
        Mixed substrate
        
            Peptone, such as
            Enhancetone: 20 grams per liter
        
        
        Dextrose: 2 grams per liter
        Sodium Chloride: 5 grams per liter
        Sodium Phosphate: 2.5 grams per liter
        Glycerol: 1 gram per liter
        Nutrient
        
            Horse Serum: 50 ml per liter
        
        
        1% NAD/Cysteine: 20 ml per liter
        Water: to volume of 1000 ml
    
    


Cultures were expanded and inoculated into media at a concentration of 107-108 cfu/milliliter. Cultures were grown at a temperature between 30 and 41° C., under normal atmospheric oxygen pressure, with the percentage of CO2 in the environment kept between 0% and 10%. Incubation times ranged from 8 hours to 72 hours. The endpoint of incubation is determined by the time at which the cultures reach stationary phase, as measured by standard microbiological methods.
Standard microbiological methods are used to determine immunogen mass, e.g. a direct plate count procedure or a spectophotometric optical density method based upon light absorbance of the Mycoplasma bovis cultured cell mass.
C. Inactivation of Mycoplasma bovis 
Beta-propiolactone (BPL) is prepared as a 10% solution (v/v) in chilled water. The chilled solution is slowly added to the M. bovis culture(s) with constant stirring, thereby allowing hydrolysis. This BPL solution is added in the amount of 10 milliliters per liter of M. bovis culture(s). The pH of the BPL-M. bovis suspension is maintained between 6.5 and 7.8, by adding sodium hydroxide as needed. The suspension is warmed to room temperature and continuously agitated for 24 hours. The cells are concentrated by centrifugation at 8,000 g or by ultrafiltration.
D. Adjuvanting and Formulation of Vaccine
Adjuvanting and final formulation of bulk concentrated inactivated M. bovis cultures were done concurrently as described in following protocol:
1) Determine the final volume batch quantity desired based upon 2 milliliters per dose. Quantity of each ingredient to be added is then calculated as described in steps 2 through 5.
2) Dispense an amount of inactivated M. bovis cell concentrate necessary to contain a protective dose quantity sufficient for the total number of doses being formulated, based on the cell counts determined in the live culture.
3) Dilute the inactivated M. bovis cell concentrate with 0.85% saline solution sufficient to bring the batch to the final desired volume (following addition of adjuvant components)
4) Adjust pH to 6.0 to 6.5 using a 10 normal hydrochloric acid solution.
5) Add an amount of 2% aluminum hydroxide solution to yield a final formulated concentration of 8% to 16%; incubate for 24 hours.
6) Using 10 N sodium hydroxide solution, readjust the pH to 7.2 to 7.4
7) Emulsify mineral oil adjuvant with the diluted aluminum hydroxide-absorbed inactivated M. bovis cells with an amount sufficient to yield 4% to 12% in the final formulation.
Example 3
Preparation of Vaccine Against M. bovis Biotypes A, B and C.
A Mycoplasma bovis vaccine was prepared containing antigen from 3 biotypes; A, B and C. The process for preparation of vaccine from line was the same as described for Example 2 above. Immunogenic components from biotypes A, B and C were combined after inactivation of selected quantities of pure cultures of each biotype.
Example 4
Preparation of Vaccine Against M. bovis Biotypes A, B, C and M. alkalescens. 
Five lung and ear isolations were obtained from necropsied calves. Using indirect immunofluorescence, the isolates were identified by Biomune as:
S99-0052—M. bovis—Lung
S99-0052—M. bovis—Ear
S99-0053—M. bovis—Lung
S99-0053—M. alkalescens—Lung.
Cultures were passaged 4× in Hayflicks modified liquid media with characterization and preparation of pure cultures.
Samples of cultures from isolates S99-0052 and S99-0053 were further characterized and were determined to be pure M. bovis and M. alkalescens by an independent laboratory. Identity of isolate S99-0053 as M. alkascens was confirmed by further testing.
Two groups of isolates were further characterized. Cytotoxicity cell culture bioassays and PCR fingerprinting were performed. These assays confirmed the identification of the cultures, to be used for vaccine production, to be pure M. bovis and M. alkalescens, 
From the isolated Mycoplasma, a vaccine containing antigens from M. bovis biotypes A, B, C and antigens from M. alkalescens was prepared using the protocol described earlier.
Example 5
Field Trial of Vaccine Against M. bovis Biotype A
Efficacy of an inactivated vaccine of this invention specific for M. bovis biotype A was determined under field conditions at a site with an endemic mycoplasma mastitis infection in the herd. An active field challenge was confirmed, based on a historical review of cull cow losses due to M. bovis, farm site M. bovis environmental isolation records, cultural isolation of M. bovis from clinical mastitis cases in the non-vaccinated animals, and isolation of M. bovis from dairy bulk tanks. Laboratory tests confirmed the identity of these isolations.
The dosage and regime protocol for field vaccinations were as follows:
Administration: 2 milliliter dose of an oil emulsion adjuvanted M. bovis vaccine; subcutaneous injection in neck region
Regime: 3 doses of vaccine
For lactating cows:

    
    
        1st Vaccination at Dry Off (End of Lactation Cycle)
        2nd Vaccination 2 to 3 Weeks Following 1st injection
        3rd Vaccination 2 to 3 Weeks Following 2nd injection
    
    


For heifers:

    
    
        The 3 doses are spaced 24 weeks apart prior to calving. Preferably, the last dose is administered at least 10 days prior to calving and the start of the lactation cycle.
    
    


Comparative results were used to measure efficacy of the vaccine. Samples taken from all animals presenting with clinical mastitis were cultured by an independent laboratory to monitor the absence or presence of Mycoplasma bovis infection of the mammary gland. Field evaluations were made by comparing clinical incidence of mastitis caused by Mycoplasma bovis following herd vaccination to the base line herd incidence prior to vaccination. Results were as follows:
Pre Vaccination Base Line Incidence:
155 confirmed positive clinical Mycoplasma bovis infections
Post Vaccination Herd Incidence:
1st year following vaccination:

    
    
        24 confirmed positive clinical Mycoplasma bovis infections
    
    


2nd year following vaccination:

    
    
        1 confirmed positive clinical Mycoplasma bovis infection
    
    


No injection reactions were observed. No inflammatory udder reactions were observed.
Animals were also evaluated for serological response using serum collected from individual animals prior to and following the 2nd vaccination. A direct ELISA was performed, with the following results for selected animals:














O.D. values










Animal ID:
Pre-vaccination
Post-vaccination












82651
.093
.313


82759
.189
.693


61043
.135
.273


3219
.198
.586


83550
.495
1.733


9296
.289
1.553









An immune response is indicated when the post-vaccination values exceed the pre-vaccination values by at least 2 fold.
Example 6
Field Trial of Vaccine Against M. bovis Biotypes A, B and C
In the 3rd calendar year of the trial described in Example 5, 200 replacement cows were introduced into the herd; 100 at the same site (Site 1) as for Example 5 and 100 into a second related site located in the same geographical area (Site 2). Neither subset of replacement cows were quarantined prior to being introduced to their respective groups. Within 2 months, serious problems with Mycoplasma mastitis were reported at both Sites 1 and 2 by personnel at each site.
Testing of all cows at both sites, approximately 4,000 animals altogether, revealed the presence of 22 animals infected with M. bovis. Initial screening of all animals was accomplished by culturing pooled milk samples (16 cows/sample). When pooled samples were identified as positive for M. bovis by culturing milk, all animals in positive groups were tested individually. Three independent studies confirmed isolation of “bovis species” and identification of the three different biotypes (A, B and C) of M. bovis was made by PCR fingerprinting. The PCR fingerprinting was carried-out as described above in Example 1.
A vaccine was prepared using antigen from 3 biotypes of M. bovis (A, B and C) as described in Example 3 above and was used to vaccinate cattle at both Site 1 and Site 2 according to the regime described in Example 5. Vaccinations began in mid-September, 1999. The incidence of Mycoplasma mastitis was monitored by independent laboratory testing for the presence of Mycloplasma in any animal determined by farm personnel to have mastitis.
Following vaccination of a significant portion of the herd at Site 1 and Site 2, the incidence of mycoplasma was greatly reduced. From Jan. 1, 2000 to Jul. 18, 2000, there were only 10 animals reported positive for Mycoplasma bovis at each site. This reduction in the incidence of Mycoplasma positive mastitis cows was regarded as a significant reduction by the operators of Sites 1 and 2. A breakdown of the incidence during Calendar year 2000 is as follows:














Site 1.
Site 2




















Jan
1
2



Feb
1
1



March
—
3



April
3
1



May
1
2



June
1
—



July
3
—










Example 7
Field Trial of Vaccine Against M. bovis Biotypes A, B, C and M. alkalescens. 
A vaccine prepared according to Example 4 comprising antigen from M. bovis biotypes A, B, C and M. alkalescens was used to vaccinate calves at a large 17,000 head-calf raising facility. It has been determined by the site's operators that Mycoplasma is a major respiratory problem. Sample bleedings and serological evaluation prior to initiation of the trial indicated that approximately 50% of calves received are serologically negative as determined by direct ELISA bioassay.
Calves selected for the trial were bled and identified with numbered tags on Oct. 19, 1999. Serum was immediately collected. Each of the calves was given the normal treatment regime for newborn cattle arriving at the site (colostrum, etc.). In addition, the calves were vaccinated three times with 2 mL of the vaccine prepared in Example 4. Vaccine was administered approximately every 7 days for the first 3 weeks. On Nov. 22, 1999, serum samples were taken from the 36 calves remaining of the original 50. On Dec. 21, 1999, serum samples were taken from 35 of the 36 calves remaining of the original 50 (one calf could not be located).
Response to vaccination was monitored using the ELISA bioassay used earlier to determine the serological status of calves at the site prior to initiating the trial. A representative random sample of calves (16) which had been serologically negative at day zero was monitored at day zero, post 2nd vaccination and post 3rd vaccination. A 7-fold increase in the immunological response over their pre-vaccinated status was realized in the animals following the vaccination procedure. This is believed to be a significant response and the vaccine's efficacy was confirmed by the fact that the vaccinated animals performed well as measured by days to market and rate of gain, both important indicators of a calf's health and well-being.
Example 8
Field Trial of Vaccine Against M. bovis Biotype B.
Biotype B was isolated from a 1200 cow Jersey dairy herd experiencing Mycoplasma mastitis infections in the herd. A vaccine against Biotype B was prepared as described in Example 2 and used in a manner consistent with the vaccination regime previously described. Following the initiation of the vaccination regime for the herd in February, 2000, a veterinarian monitored the herd for the incidence of M. bovis. The dairy reported in September 2000 that there were no confirmed cases of Mycoplasma in vaccinated animals, despite the continued challenge from the presence of confirmed, infected nonvaccinated animals. As of September, approximately 50% of the herd had been vaccinated. No unfavorable reactions resulting from the vaccine's use have been reported.
Example 9
Field Trial of Vaccine Against M. bovis Biotype C.
Biotype C was isolated from a cultured isolate derived from a Holstein dairy herd of approximately 400 animals. This herd had been experiencing Mycoplasma mastitis infections and had been experiencing positive bulk milk tanks for the year prior to May, 1999. In March, 1999, thirteen cows had been identified as positive for Mycoplasma bovis infection. A vaccine specific for biotype C was made as described in Example 2 and was used in a manner consistent with the vaccination regime previously described. The owner and herd health veterinarian monitored performance of the vaccine. There have been no reported clinical mastitis events in vaccinated animals. No unfavorable reactions in animals receiving the product have been reported.

## Claims

1. A method of immunizing bovine animals against mastitis comprising administering to bovine animals at least one inactivated or attenuated Mycoplasma bovis biotype, whereby the clinical incidence of mastitis in the bovine animals is reduced such that the number or percentage of bovine animals that show clinical Mycoplasma bovis infection is less after such administering than before such administering.

2. The method of claim 1 comprising administering at least one inactivated Mycoplasma bovis biotype to at least about 50% of the herd.

3. The method of claim 1 where the inactivated or attenuated Mycoplasma bovis biotype is administered together with an adjuvant.

4. The method of claim 3 where the adjuvant is an aluminum hydroxide-oil emulsion; a mineral, vegetable, or fish oil-water emulsion; a water-oil-water emulsion; incomplete Freund's adjuvant; E. coli J5; dextran sulfate; iron oxide; sodium alginate; Bacto-Adjuvant; a synthetic polymer; Carbopol; a poly-amino acid; a co-polymer of amino acids; saponin; carrageenan; N,N-dioctadecyl-N′-N′-bis(2-hydroxyethyl) propanediamine; a long chain polydispersed β(1,4) linked mannan polymer interspersed with O-acetylated groups; deproteinized cell wall extracts from a non-pathogenic strain of Mycobacterium; mannite monooleate; paraffin oil; or muramyl dipeptide.

5. The method of claim 1 where the inactivated or attenuated Mycoplasma bovis biotype is administered together with a pharmaceutically acceptable excipient.

6. The method of claim 1 where the inactivated or attenuated Mycoplasma bovis biotype is administered orally, intranasally, intratracheally, intramuscularly, intramammarily, subcutaneously, intravenously, or intradermally.

7. The method of claim 1 where the inactivated or attenuated Mycoplasma bovis biotype is administered by injection, inhalation, ingestion, or infusion.

8. The method of claim 1 where the Mycoplasma bovis biotype has been inactivated.

9. The method of claim 8 where the Mycoplasma bovis biotype has been inactivated by treatment with: formalin, azide, freeze-thawing, sonication, heat, sudden pressure drop, detergent, lysozyme, phenol, proteolytic enzymes, β-propiolactone, Thimerosal, or binary ethyleneimine.

10. The method of claim 9 where the Mycoplasma bovis biotype has been inactivated by treatment with β-propiolactone.

11. The method of claim 1 where at least two inactivated Mycoplasma bovis biotypes are administered.

12. The method of claim 11 where the at least two inactivated Mycoplasma bovis biotypes are selected from the group consisting of Biotype A, Biotype B, and Biotype C.

13. The method of claim 11 where at least 108 cell equivalents of each Mycoplasma bovis biotype are administered.

14. The method of claim 11 where approximately 108 cell equivalents of each Mycoplasma bovis biotype are administered.

15. The method of claim 11 where at least approximately 105 cell equivalents of each Mycoplasma bovis biotype are administered.

16. The method of claim 11 where approximately 105 cell equivalents of each Mycoplasma bovis biotype are administered.

17. The method of claim 11 where the at least two inactivated Mycoplasma bovis biotypes are administered separately.

18. The method of claim 1 where at least two inactivated Mycoplasma bovis biotypes and an antigen derived from another pathogen are administered.

19. The method of claim 18 where the antigen from another pathogen is from an attenuated or inactivated virus.

20. The method of claim 18 where the antigen from another pathogen is selected from the group consisting of antigens from Staphylococcus aureus, Pasteurella hemolytica, Pasteurella multocida, Hemophilus somnus, Bovine Respiratory Syncytial Virus, E. coli, and the organism causing Infectious Bovine Rhinotrachial Disease.

21. The method of claim 11 where the at least two inactivated Mycoplasma bovis biotypes are genetically different as determined by an analysis of DNA or RNA from the biotypes.

22. The method of claim 21 where the analysis is PCR fingerprinting, analysis of ribosomal RNA, or analysis of DNA polymorphisms.

23. The method of claim 22 where the analysis is by PCR fingerprinting.

24. The method of claim 23 where the PCR fingerprinting uses arbitrarily chosen primers.

25. The method of claim 23 where the PCR fingerprinting uses as primers 5′ NNN NCG NCG NCA TCN GGC 3′ (SEQ ID NO:1) and 5′ NCG NCT TAT CNG GCC TAC 3′ (SEQ ID NO:2).

26. The method of claim 11 where the at least two Mycoplasma bovis biotypes are administered in a specific ratio.

27. The method of claim 11 where the at least two Mycoplasma bovis biotypes are grown separately as pure cultures, inactivated, and combined together in equal amounts before being administered to the animal.

28. A method for immunizing bovine animals against mastitis comprising administering to bovine animals an antigenic component from at least one inactivated or attenuated Mycoplasma bovis biotype, whereby the clinical incidence of mastitis in the bovine animals is reduced such that the number or percentage of bovine animals that show clinical Mycoplasma bovis infection is less after such administering than before such administering.

29. The method of claim 28 where antigenic components from at least two Mycoplasma bovis biotypes are administered.

30. The method of claim 1 where the administering results in greater milk production, greater weight gain, or less clinical disease in the bovine animal.

31. A method of immunizing bovine animals against mastitis comprising:
(a) testing samples from bovine animals for the presence of Mycoplasma bovis biotypes, thereby identifying specific Mycoplasma bovis biotypes in the samples;
(b) preparing a vaccine by inactivating at least 105 cell equivalents of at least one of the Mycoplasma bovis biotypes identified in step (a); and
(c) administering to the bovine animals the vaccine of step (b), whereby the bovine animals are immunized so that the clinical incidence of mastitis in the bovine animals is reduced such that the number or percentage of bovine animals that show clinical Mycoplasma bovis infection is less after such administering than before such administering.

32. The method of claim 31 where the sample is milk.

33. The method of claim 31 where step (a) comprises genetic analysis of DNA or RNA from the Mycoplasma bovis biotypes.

34. The method of claim 33 where the genetic analysis is PCR fingerprinting, analysis of ribosomal RNA, or analysis of DNA polymorphisms.

35. The method of claim 34 where the genetic analysis is PCR fingerprinting.

36. The method of claim 1 whereby the administering does not cause unfavorable reactions.

37. The method of claim 11 whereby the administering does not cause unfavorable reactions.

38. The method of claim 8 whereby the at least one inactivated Mycoplasma bovis biotype has not been inactivated with formalin.

39. The method of claim 11 whereby the at least two inactivated Mycoplasma bovis biotypes have not been inactivated with formalin.