Immunobinding assay for detection of Mycoplasma bovis in milk.

An immunobinding dot-blot assay (IBA) was developed for the detection of mycoplasma in milk. The test was highly species specific when monoclonal antibody preparations were employed in the assay system. Reactions were obtained with all mycoplasma species tested when polyclonal antisera preparations were used. Preincubation for 48-72 hours was necessary with milk samples containing only a few mycoplasma. Time from sample receipt to diagnosis in most positive samples could be reduced from several days by culture to a few hours by the IBA, thus enabling control procedures to be quickly initiated.

Clinical mastitis in two California dairy herds participating in contagious mastitis control programs.

The occurrence of clinical mastitis in two large California dairy herds over a 3-yr period is described. Herds had been participating for 15 or 22 yr in mastitis control programs against Streptococcus agalactiae and Staphylococcus aureus, had low bulk tank SCC, and had maintained good standards of hygiene and husbandry, but clinical mastitis remained a serious problem. A total of 1654 clinical mastitis cases were detected; the annual incidence in each herd was 49%. Coliform bacteria and environmental streptococci were etiological agents in 60% of the total clinical mastitis cases; coliforms produced 1.6 times more clinical mastitis than environmental streptococci. A higher susceptibility to clinical mastitis, primarily from coliform bacteria and environmental streptococci, was found in the first months of lactation. Clinical mastitis incidence peaked for cows in lactations 4 and 5 and was lowest during the first lactation. Highest incidence of clinical mastitis due to coliform bacteria and environmental streptococci at each dairy occurred during the rainy season (late fall and winter).

Prevention of clinical coliform mastitis in dairy cows by a mutant Escherichia coli vaccine.

A prospective cohort study was undertaken in two commercial California dairies. The treatment group, 246 cows, received three doses of a whole cell bacterin of J5 Escherichia coli (mutant of E. coli O111:B4) plus Freund's incomplete adjuvant vaccine (two in the dry period and one after calving) while 240 unvaccinated cows served as controls. Thirty-five cases of clinical coliform mastitis were diagnosed, six in vaccinated cows and 29 in unvaccinated cows. Bacteria isolated from the clinical cases included 15 E. coli five Klebsiella pneumoniae, three K. oxytoca, three K. ozaenae, five Enterobacter aerogenes, three Serratia marcescens and one Serratia spp. Four control cows were culled, three of them because of chronic coliform mastitis and one because of postcoliform infection agalactia. Incidence rate of clinical gram-negative mastitis was 2.57% in vaccinated cows and 12.77% in unvaccinated cows. The estimated risk ratio, the measure of risk of having clinical gram-negative mastitis for vaccinated cows to unvaccinated cows, was 0.20 (p less than 0.005), indicating a strong relationship between vaccination and lack of clinical gram-negative mastitis. The results of this trial indicate that the administration of the E. coli J5 vaccine is protective against natural challenge to gram-negative bacteria, and reduces the incidence of clinical gram-negative mastitis in dairy cows during the first three months of lactation.

Prevalence of udder infections and mastitis in 50 California dairy herds.

The California mastitis test (CMT) and bacteriologic culture were performed on samples of bulk-tank milk and cow-composite milk (n = 23,138 cows) from 50 California dairies, 19 of the 50 with known mastitis problems. Thirty-eight (76.0%) bulk-tank milk samples and 12,334 (53.3%) cows were positive by results of the CMT. Potential mastitis agents were isolated from 5,085 (22%) cows. Staphylococcus aureus was isolated from all 50 herds, Streptococcus agalactiae was isolated from 47 herds, and Mycoplasma sp was isolated from 24 herds. For cow-composite milk samples, the prevalences were 9.3% for Str agalactiae, 9.1% for S aureus, 0.9% for Mycoplasma sp, 1.2% for coliform bacteria, 0.9% for other streptococci, 0.8% for coagulase-negative staphylococci, and 1.3% for other organisms. The relative sensitivity and the relative specificity of the CMT performed on cow-composite milk samples were 83.4% and 55.2%, respectively, and the predictive value of positive CMT results was 34.2%.

Immune responses to Mycoplasma bovis vaccination and experimental infection in the bovine mammary gland.

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.

Detection of bovine serum antibody specific to Mycoplasma bovis and Mycoplasma californicum by enzyme-linked immunosorbent assay (ELISA).

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.

Pathogenesis of bovine mycoplasma mastitis.

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.

Enzyme-linked immunosorbent assay for detection of Mycoplasma californicum-specific antibody in bovine serum: optimization of assay determinants and control of serologic cross-reactions.

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.

Experimental intramammary inoculation with Mycoplasma bovis in vaccinated and unvaccinated cows: effect on local and systemic antibody response.

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.

Relationship between mastitis pathogen numbers in bulk tank milk and bovine udder infections in California dairy herds.

Samples of bulk tank milk and cow-composite milk from 23,138 dairy cows from 50 California dairies were examined by use of microbiologic procedures. The number of colonies of mastitis pathogens isolated per milliliter of bulk tank milk (used as a predictor of the percentage of infected cows in the herd) was evaluated, using simple regression analysis and Spearman's rank correlation. Correlations between the pathogens and the percentage of cows in each herd shedding the pathogens were found for Streptococcus agalactiae (r = 0.71) and mycoplasma (r = 0.59), but were considerably lower for other pathogens. When greater than or equal to 4,000 colonies of Streptococcus agalactiae were found per milliliter of bulk tank milk, at least 7% of the cows in the herd was shedding this organism. However, a pattern was not found between the number of mycoplasma colonies per milliliter of bulk tank milk and the percentage of infected cows in the herd.

Detecting Mycoplasma bovis in milk by enzyme-linked immunosorbent assay, using monoclonal antibodies.

Enzyme-linked immunosorbent assay, using monoclonal antibodies, was used to detect Mycoplasma bovis in milk samples from a dairy experiencing an epizootic of mastitis. This method was specific (100%) for M bovis. Broth enrichment increased the sensitivity from 65% to 86%, compared with standard culture methods.

Experimental intramammary inoculation with Mycoplasma bovis in vaccinated and unvaccinated cows: effect on the mycoplasmal infection and cellular inflammatory response.

The effect of vaccination on mycoplasmal infection and the cellular inflammatory response was evaluated in 4 vaccinated and 4 control cows experimentally challenged in 2 of 4 quarters with live Mycoplasma bovis. In unchallenged quarters during the first three weeks after experimental challenge exposure, 6 of 8 quarters on control cows, and 7 of 8 quarters on vaccinated cows became infected with low numbers (10(2)-10(4) cfu/ml) of M bovis. During the same period all challenge-infused quarters on both control and vaccinated animals became infected with high numbers (10(9) cfu/ml) of M bovis. Thereafter, all quarters on vaccinated cows became culture-negative for M bovis, while 2 of 8 unchallenged quarters, and 4 of 8 challenged quarters on 3 of 4 control cows remained infected. A cellular inflammatory response as measured by the California Mastitis Test accompanied the experimental infection in proportion to the infection level except in challenged quarters on vaccinated cows after the first three weeks post challenge in which the cellular inflammatory response remained high despite the advent of negative M bovis culture results. This study indicates that the course of experimental M bovis mastitis can be affected by vaccination, and that vaccination results in an adverse cellular inflammatory response in challenged quarters.

Experimental intramammary inoculation with Mycoplasma bovis in vaccinated and unvaccinated cows: effect on milk production and milk quality.

The effect of vaccination on milk production was evaluated in vaccinated and control cows experimentally challenged in two of four quarters with live Mycoplasma bovis. During the first three weeks after experimental challenge, six of eight unchallenged quarters on vaccinated cows and seven of eight unchallenged quarters on control cows became infected. Most of these quarters secreted normal milk, with negative California Mastitis Test scores and maintained normal milk production throughout most of the study (although some quarters on control cows remained infected). All challenged quarters became infected, had strong California Mastitis Test reactions, and had a drastic (greater than 85%) loss in milk production. Thereafter, four of eight challenged quarters on control cows remained infected, had mostly positive California Mastitis Test scores, produced mostly normal-appearing milk, and recovered some productive capabilities. By the end of the study no M. bovis could be recovered from challenged quarters on vaccinated cows and the milk appeared mostly normal. The California Mastitis Test scores on these quarters, however, remained elevated and milk production remained very low.

Efficacy of the API Staph-Ident system for identification of Staphylococcus species from milk.

A total of 524 staphylococcal isolates from bovine milk were identified, using the API Staph-Ident system and conventional biochemical methods. The API Staph-Ident system correctly identified 192 of 201 (95.5%) Staphylococcus aureus isolates, but was correct on only 23 of 323 (7.1%) non-S aureus isolates.

Relationships among the results of coagulase, staphylococcal toxin, and thermonuclease tests on staphylococci from cow milk.

Production of staphylococcal alpha- or alpha-beta-toxins correlated well with production of coagulase or thermonuclease (or both) in 203 Staphylococcus aureus isolates from milk and should be reliable indicators of S. aureus in the absence of Staphylococcus intermedius. Failures to produce toxin, tube coagulase, or thermonuclease occurred in only 1 to 2% of S. aureus. Evidence of beta- or alpha-beta-toxins was not found among 321 other staphylococci isolated from milk. A few coagulase- or thermonuclease-positive isolates not producing beta- or alpha-beta-toxins were found among the Staphylococcus hyicus isolates.

Acridine orange staining for diagnosis of Mycoplasma bovis infection in cow milk.

Mycoplasma organisms were readily recognized in samples of milk or udder secretions from cows with clinical Mycoplasma bovis mastitis when these samples were stained with 0.01% acridine orange at pH 3.0. Samples could be stored at -4 degrees C for several days or subjected to repeated freezing and thawing without loss of staining or fluorescence properties. Use of this procedure in diagnostic laboratories on suspect samples from cows with clinical mastitis could hasten inauguration of control measures against this highly contagious disease by several days; however, definitive diagnosis still requires standard culture methods.