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Microbiological cultural, cytologic, and immunologic observations were made on 30 calves. The eyes, nares, and bronchioalveolar region were subjected to microbiological cultural examination for mycoplasmas. Four of the examinations of 30 eyes, 15 of those of 30 nasal tissues, and 25 of those of the 30 bronchioalveolar regions from the 30 calves were positive for mycoplasmas. Mycoplasma bovis and M bovirhinis were the most prevalent species. Cytologic examinations of peripheral blood and bronchioalveolar washes did not show pathologic changes. Results of indirect hemagglutination, enzyme-linked immunosorbent assay, lymphocyte-stimulation tests on peripheral blood cells, and skin testing demonstrated only a low prevalence of immune recognition of M bovis. Infection and immune response were studied in 3 calves for 10 weeks before, and for 4 weeks after, intratracheal administration of live M bovis.

Typical mycoplasma mastitis spread to all four quarters in each of four cows after each cow was inoculated in one quarter with 70 colony-forming units of Mycoplasma bovis. Up to 50 days after inoculation, immunoglobulin (Ig) M was the predominant Ig responsible for serum indirect hemagglutination titers. After 57 days, IgG was predominant. Milk whey indirect hemagglutination titers did not distinguish between quarters resolving the infection and those that did not. Milk whey IgG and IgA concentrations increased in quarters that resolved the infection and in quarters that did not. In the initially inoculated quarters, total daily production of IgG was greater in the quarter resolving the infection than in the three quarters not resolving the infection. Total IgA production was also higher at and after the peak of infection in the quarter resolving the infection than in the quarters that did not.

Four cows were vaccinated with Mycoplasma bovis five times at two week intervals: three times subcutaneously in Freund's complete adjuvant, and two times with M. bovis alone in two of four quarters by intramammary infusion. The effect of vaccination on the immune response was evaluated in the serum and whey of the four vaccinated and control (placebo) cows experimentally challenged in two of four quarters with live M. bovis. Vaccination resulted in markedly increased M. bovis-specific, serum IgM, IgG and IgG2, but not IgA, reactivity. Challenge exposure with live M. bovis by intramammary infusion resulted in high specific serum IgM, IgG1 and IgG2 reactivity and a noticeable IgA response in both vaccinated and control cows. Whey from quarters on vaccinated cows had elevated, specific IgG1 reactivity at the time of challenge but no other differences were observed. Challenge exposure with live M. Bovis resulted in high antibody levels of all isotypes in quarters which were challenged, but highly elevated reactivities in unchallenged quarters occurred only with IgG1 and IgG2. These results indicate that vaccination elevated M. bovis-specific IgG1 but not other immunoglobulin reactivity in quarters on vaccinated cows, and that live organisms are necessary to elicit a local, specific IgA response.

The enzyme-linked immunosorbent assay (ELISA) was optimized for detection of Mycoplasma bovis-specific IgG in bovine serum. The test is rapid, reproducible, convenient, and sensitive. With this assay, the serum from naturally infected and immunized calves demonstrated the presence of antibodies early in infection and rapid increase in titers during the infection. Cross-reactivity of bovine serum with mycoplasma antigens of bovine, caprine, avian, and environmental sources was tested with this assay system. Cross-reaction was measurable in all instances, with the strongest reaction measured between M bovis and M agalactiae.

Most bovine mastitis due to mycoplasmas is initiated by passage of mycoplasmas through the teat canal into the teat and gland cisterns. Within a few days, mycoplasma numbers increase to as much as 10(6) or 10(8), and the cows react with a strong inflammatory response. Alveolar epithelium undergoes degenerative changes and exudate replaces milk secretion. The interstitium between alveoli is invaded with lymphocytes, macrophages, plasma cells and fibroblasts. The extent and duration of these changes vary greatly. In milder cases, they may be reversed within days or weeks with a return to normal or reduced milk production. Often, destruction and atrophy of alveoli are complete with extensive fibrosis throughout the udder. Milk ducts may undergo invasive and obliterative fibrosis. Cell-mediated responses are suppressed, while hypersensitivity is suspected of enhancing the adverse responses. Immunity in cows that recover is variable and of limited duration.

Seven calves seven to 30 days of age were given Mycoplasma bovis antigen by different routes. Immunization was in two phases. The first consisted of single or multiple SC, IV or oral doses of antigen for two to four weeks. The second phase consisted of multiple SC or ID injections given from the eighth to the 19th week. The experiment was terminated at 26 weeks. Antibody titers were followed by indirect hemagglutination, growth inhibition and tetrazolium reduction inhibition. Total serum protein, protein fractions and IgG and IgM concentrations were determined in serums of one calf and the distribution of indirect hemagglutination antibodies in IgG and IgM classes were determined in serums of two of the calves. Indirect hemagglutination titers of 1280 and peak titers of20,480 occurred after the first and second phases respectively. There was no relationship between total serum IgG or IgM concentrations and indirect hemagglutination titers. In one calf given M. bovis antigen in one dose SC and five weekly doses IV in phase I, indirect hemagglutination antibodies appeared in IgM within one week and IgG by four weeks, IgG antibody activity rose steadily until the 17th week but declined at the 26th week, whereas IgM activity after the initial rise dropped at the 13th week but rose even higher as a result of second phase ID injections. Another calf given six weekly IV doses of M. bovis antigen in phase I developed indirect hemagglutination antibodies in IgM peaking at four weeks then declining but with no IgG response. Activity in both IgM and IgG occurred after the second phase. Growth inhibition antibodies were found only on two occasions in one calf serum and tetrazolium reduction inhibition activity when tested never gave titres exceeding 1:32.

An enzyme-linked immunosorbent assay (ELISA) was adapted to detect Mycoplasma californicum-specific antibodies in bovine serum. Cross-reactive antibody was found in the M californicum-positive reference serum when assayed against each of 7 solid-phase antigens of heterologous mycoplasma species. Cross-reactivity was further demonstrated by inhibition of ELISA reactivity to M californicum solid-phase antigen by incubation of sera with antigen suspensions of each heterologous species. Incubation of test sera with a cross-reacting antigen mixture containing equal proportions of the 7 cross-reactive mycoplasmas was used to minimize cross-reactivity in the M californicum-specific ELISA. Specificity of antibody reactivity to M californicum, as measured by ELISA, was determined by enzyme-linked immunosorbance inhibition, in which sera were incubated with M californicum antigen suspensions before determining ELISA reactivity to M californicum solid-phase antigen. Seropositive and suspect sera (n = 55) were obtained from 3 dairies that had bacteriologically verified epizootics of M californicum mastitis. The percentage of inhibition demonstrated in enzyme-linked immunosorbance inhibition was determined for each serum. Inhibition percentages below the 15th percentile (61% inhibition) of this distribution were classified as nonspecific.

This study characterized the immune responses in four vaccinated and four control cows in response to vaccination and experimental intramammary inoculation with Mycoplasma bovis. Specific antibody responses occurred in serum and milk in response to vaccination and experimental infection. Lymphocytes from peripheral blood, but not from the mammary gland of vaccinated cows had increased responsiveness to mitogens. No lymphocytes tested were responsive to M. bovis antigen. Both vaccination and experimental infection resulted in skin test reactivity. These results imply that vaccination results in immune responses which may alter the course of experimental M. bovis mastitis, but may contribute to cellular inflammation.

Serologic and cultural observations were made in three herds with and three herds without histories of mycoplasma mastitis. Nasal swabs and sera were collected from dairy animals of various ages over an eight month peiod. The overall prevalence of Myocopalsma bovis in the nares was 34% in diseased herds and 6% in the non-diseased herds without mastitis. Mycoplasma bovis was isolated in the highest prevalence in those young animals fed infected milk. Slight serologic differences were seen in these animals. Nasal prevalence of M. bovis was low but readily detectable in non diseased herds as well as in prepartum heifers in the diseased herds with mycoplasma mastitis.

The gel electrophoresis-derived enzyme-linked immunosorbent assay (GED-ELISA) technique combines the high resolving power of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to separate complex molecules by their molecular weights, with the high sensitivity of the ELISA to detect specific antibody, Sera from 4 cows that demonstrated resistance to challenge exposure and 4 cows that were susceptible to challenge exposure with live virulent Mycoplasma bovis strain 201 were subjected to GED-ELISA to determine reactivity with M bovis antigenic components separated by SDS-PAGE. The GED-ELISA mean reactivity of sera from the 2 groups did not differ significantly (P = 0.17) when subjected to analysis of variance. Sera from both groups recognized distinct fractions of M bovis.

ELISA for use in epidemiologic field studies of bovine mastitis, were developed to measure serum antibody to Mycoplasma bovis and M. californicum. Varying levels of serological cross-reactivity to seven heterologous bovine mycoplasmal species were demonstrated in each assay. Cross-reactivity was minimized by preincubation of cattle sera with suspensions of heterologous mycoplasma antigens, prior to measuring serum antibody to solid-phase antigen. Heterologous absorption improved the immunological specificity of the assays while avoiding the need to prepare species-unique antigens. Serum antibody was measured at one serum dilution. Test results were expressed as a ratio of the reactivity of a positive and a negative reference serum. A negative reference population (n = 127) was assembled. The percentile distribution of ELISA reactivity of these 127 sera were used to establish the classification criteria for each assay. The statistical methods used, while easily applied, were found to be sensitive to outlying values in the reference population. The resulting classification criteria provided controlled or known probabilities of false-positive misclassification in the two ELISA test systems. Sera from cattle with defined exposure histories were tested and classified according to these criteria.