However, these are not ideal antigens for immunodiagnosis because of the phase and antigenic variation seen in Vsps and the similarities between Hsp60 and P48 and their orthologs in closely related species such asMycoplasma agalactiae. enzyme-linked immunosorbent assays (ELISAs). The IgM MKT 077 ELISA detectedM. bovis-specific IgM antibody 2 weeks after contamination with 97.1% sensitivity and experienced a specificity of MKT 077 63.3%, while the IgG ELISA detectedM. bovis-specific IgG 3 weeks after contamination with 92.86% sensitivity and experienced a specificity of 98.7%, demonstrating that this IgG ELISA has potential for use as a sensitive and specific assay for detecting infection in cattle. == INTRODUCTION == Mycoplasma bovisis the most frequently isolated etiological agent in naturally occurring outbreaks of bovine respiratory disease (BRD) in veal calves (1), a chronic disease characterized by pneumonia and arthritis.M. bovisis also responsible for outbreaks of keratoconjunctivitis, mastitis, meningitis, otitis media, and genital tract disease (25). The significance of respiratory tract contamination withM. bovishas become progressively apparent in recent years because of greater recognition of the role ofM. bovisin pneumonia in many parts of the world (6) and due to the increasing resistance of this bacterium to antimicrobial drugs (7). In the absence of an effective vaccine or antimicrobial therapy, an alternative strategy to controlM. bovisinfection in cattle is usually to separate infected from uninfected cattle (4). Currently,M. bovisdiagnostic methods, including culture (8), PCR assays (9), and antigen capture enzyme-linked immunosorbent assays (ELISAs) (10,11), have not completely fulfilled the need for a rapid diagnostic test to detect contamination, and there is a clear need for a sensitive and specific serological assay based on a species-specific immunologically reactive antigen. MKT 077 Recently published genome sequences of severalM. bovisstrains (12,13) have contributed to the identification of novel antigenic proteins. Most of the effort thus far has been concentrated on identifying and characterizing the variable surface proteins (Vsps) (14). Apart from the Vsps, only a few antigenic proteins ofM. bovishave been recognized and characterized, including Hsp60 and P48 (15,16). However, these are not ideal antigens for immunodiagnosis because of the phase and antigenic variance seen in Vsps and the similarities between Hsp60 and P48 and their orthologs in closely related species such asMycoplasma agalactiae. Therefore, there remains a need to identify a betterM. bovisantigen DDR1 for serological diagnosis. The aim of this study was to identify an immunogenic protein by Western blotting usingM. bovis-specific calf sera and characterize its function, and then to develop an indirect ELISA by using this protein and assess the sensitivity of the ELISA using a panel of sera collected from experimentally infected calves. == MATERIALS AND METHODS == == Bacterial strains and plasmids. == Mycoplasma bovisstrain 3683, originally isolated from a joint lesion in a calf in Queensland, Australia, was produced at 37C for 17 h in mycoplasma culture medium. The pGEX-4T-1 plasmid was utilized for cloning different regions ofmilA, andEscherichia coliJM109 cells were used to express the recombinant glutathioneS-transferase (GST)-mycoplasma immunogenic lipase A (MilA) proteins. == Serum samples from experimentally MKT 077 infected calves. (i) Experiment 1. == Sixteen 1-month-old Friesian-cross calves were weighed and allocated into one of two groups. Group 1 consisted of 4 animals uncovered only to mycoplasma culture medium, while group 2 consisted of 12 calves exposed to an overnight culture ofM. bovisstrain 3683 twice on days 1 and 3, with each calf receiving an estimated dose of 105.2color-changing units (CCU), using an aerosol exposure method explained previously (17). The calves in groups 1 and 2 were held in individual facilities. Blood samples were taken from all the calves on days 0, 10, 17, and 24 after contamination. All calves were euthanized and necropsied at day 24 and examined for lesions as explained previously (17). == (ii) Experiment 2. == Thirty-five 1-month-old Friesian-cross calves were stratified according to their weight and then randomly allocated into 1 of 2 groups. Group 1, the uninfected group, consisted of 5 calves exposed to an aerosol of mycoplasma culture medium, and group 2 consisted of 30 calves exposed to an aerosol ofM. bovisstrain 3683. Contamination and sampling were performed as explained for experiment 1. All calves in the two experiments were tested for.