Abstract: The present invention provides a bacterin comprising at least two virulent Mycoplasma bovis isolates of differing biotypes, in an amount sufficient to immunize a calf or cow against infection by Mycoplasma bovis, an adjuvant, and a pharmaceutically acceptable carrier. Further provided is a method of producing the bacterin of the present invention. Finally, the present invention also provides a method of immunizing a calf or cow against Mycoplasma bovis infection, especially infection associated with BRD, comprising administering to the calf or cow a dose of a bacterin made according to the present invention in an amount effective to elicit a protective response in the animal, as evidenced by a decrease in the mortality and morbidity associated with natural infection.