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 of 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.

1 . A vaccine which is protective against  Mycoplasma bovis  clinical disease in a bovine species comprising at least one inactivated or attenuated  Mycoplasma bovis  biotype, an adjuvant, and a pharmaceutically acceptable excipient, wherein: 
 the adjuvant does not include saponin,    the clinical disease includes respiratory pneumonia, and    the vaccine does not cause unfavorable reactions.    
     
     
         2 . The vaccine of  claim 1 , wherein the  Mycoplasma bovis  biotype is inactivated.  
     
     
         3 . The vaccine of  claim 1 , wherein the  Mycoplasma bovis  biotype is attenuated.  
     
     
         4 . The vaccine of  claim 1 , wherein at least one of the inactivated or attenuated  Mycoplasma bovis  biotypes is selected from the group consisting of biotype A, biotype B, and biotype C.  
     
     
         5 . A vaccine which is protective against  Mycoplasma bovis  clinical disease in a bovine species comprising at least two inactivated or attenuated  Mycoplasma bovis  biotypes and a pharmaceutically acceptable excipient, wherein the vaccine does not cause unfavorable reactions.  
     
     
         6 . The vaccine of  claim 5 , further comprising a suitable adjuvant.  
     
     
         7 . The vaccine of  claim 6 , wherein the  Mycoplasma bovis  biotypes are inactivated.  
     
     
         8 . The vaccine of  claim 7 , wherein the inactivated  Mycoplasma bovis  biotypes are genetically different as determined by an analysis of DNA or RNA from the biotypes.  
     
     
         9 . The vaccine of  claim 10 , wherein the analysis is by PCR fingerprinting, analysis of ribosomal RNA, or analysis of DNA polymorphisms.  
     
     
         10 . The vaccine of  claim 9 , wherein the analysis is by PCR fingerprinting.  
     
     
         11 . The vaccine of  claim 10 , wherein the PCR fingerprinting uses arbitrarily chosen primers.  
     
     
         12 . The vaccine of  claim 11 , wherein 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).  
     
     
         13 . The vaccine of  claim 8 , wherein the at least two  Mycoplasma bovis  biotypes have been identified as being genetically different biotypes by a process comprising: 
 (a) isolating DNA from the biotypes;    (b) amplifying the DNA by PCR;    (c) separating the amplified DNA by gel electrophoresis; and    (d) comparing the resulting patterns from the gel electrophoresis to identify the different biotypes.    
     
     
         14 . The vaccine of  claim 5 , wherein the  Mycoplasma bovis  biotypes are attenuated.  
     
     
         15 . The vaccine of  claim 5 , wherein the  Mycoplasma bovis  biotypes are selected from the group consisting of biotype A, biotype B, and biotype C.  
     
     
         16 . A vaccine which is protective against  Mycoplasma bovis  mastitis in a bovine species comprising at least one inactivated or attenuated  Mycoplasma bovis  biotype and a pharmaceutically acceptable excipient, wherein the vaccine does not cause unfavorable reactions.  
     
     
         17 . The vaccine of  claim 16 , where the vaccine is protective against  Mycoplasma bovis  mastitis in a bovine species following systemic administration.  
     
     
         18 . The vaccine of  claim 16 , comprising at least two inactivated  Mycoplasma bovis  biotypes.  
     
     
         19 . The vaccine of  claim 18 , wherein the vaccine includes at least one inactivated  Mycoplasma bovis  biotype selected from the group consisting of biotype A, biotype B, and biotype C.  
     
     
         20 . The vaccine of  claim 18 , wherein the inactivated  Mycoplasma bovis  biotypes are genetically different as determined by an analysis of DNA or RNA from the biotypes.  
     
     
         21 . The vaccine of  claim 20 , wherein the analysis is by PCR fingerprinting, analysis of ribosomal RNA, or analysis of DNA polymorphisms.  
     
     
         22 . The vaccine of  claim 21 , wherein the analysis is by PCR fingerprinting.  
     
     
         23 . The vaccine of  claim 22 , wherein the PCR fingerprinting uses arbitrarily chosen primers.  
     
     
         24 . The vaccine of  claim 23 , wherein 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).  
     
     
         25 . The vaccine of  claim 20 , wherein the  Mycoplasma bovis  biotypes have been identified as being genetically different biotypes by a process comprising: 
 (a) isolating DNA from the biotypes;    (b) amplifying the DNA by PCR;    (c) separating the amplified DNA by gel electrophoresis; and    (d) comparing the resulting patterns from the gel electrophoresis to identify the different biotypes.    
     
     
         26 . The vaccine of  claim 16 , wherein, when the vaccine is administered to a plurality of cows in a herd of cows, the incidence of mastitis caused by  Mycoplasma bovis  in the herd before administering is greater than the incidence of mastitis caused by  Mycoplasma bovis  in the herd after administering.  
     
     
         27 . The vaccine of  claim 26 , wherein the vaccine is administered to at least about 50% of the herd.  
     
     
         28 . The vaccine of  claim 27 , where the vaccine is administered together with an adjuvant.  
     
     
         29 . The vaccine of  claim 1 , wherein 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; REGRESSIN®; 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.  
     
     
         30 . The vaccine of  claim 2 , wherein 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.  
     
     
         31 . A vaccine which is protective against  Mycoplasma bovis  clinical disease in a bovine species comprising at least one  Mycoplasma bovis  biotype that has been inactivated by treatment with β-propiolactone.  
     
     
         32 . The vaccine of  claim 31 , wherein the clinical disease is  Mycoplasma bovis  mastitis.  
     
     
         33 . The vaccine of  claim 32 , comprising at least two  Mycoplasma bovis  biotypes that have been inactivated by treatment with β-propiolactone.  
     
     
         34 . The vaccine of  claim 33 , wherein the at least two inactivated  Mycoplasma bovis  biotypes are genetically different as determined by an analysis of DNA or RNA from the biotypes.  
     
     
         35 . The vaccine of  claim 34 , wherein the analysis is by PCR fingerprinting, analysis of ribosomal RNA, or analysis of DNA polymorphisms.  
     
     
         36 . The vaccine of  claim 35 , wherein the analysis is by PCR fingerprinting.  
     
     
         37 . The vaccine of  claim 36 , wherein the PCR fingerprinting uses arbitrarily chosen primers.  
     
     
         38 . The vaccine of  claim 37 , wherein 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).  
     
     
         39 . The vaccine of  claim 34 , wherein the at least two  Mycoplasma bovis  biotypes have been identified as being genetically different biotypes by a process comprising: 
 (a) isolating DNA from the biotypes;    (b) amplifying the DNA by PCR;    (c) separating the amplified DNA by gel electrophoresis; and    (d) comparing the resulting patterns from the gel electrophoresis to identify the different biotypes.