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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.

The California Dairy Herd Improvement Association records of 29 California dairies which experienced clinical mycoplasmal mastitis between January 1975 and December 1977 were examined and compared to the records of selected control herds. A 15-fold greater risk of clinical mycoplasmal mastitis was found among large herds as compared to small herds. On average, herds with clinical mycoplasmal mastitis culled 5 % more cows than did control herds (33 % vs 28 %). No difference was found in average milk production. These findings compare closely with the findings of a previous report where infected herds were identified by the presence of pathogenic mycoplasma in bulk tank milk. The similarity of results support the use of frequent bacteriologic culture of bulk tank milk as a routine surveillance strategy for mycoplasmal mastitis in endemic areas. The similarity of results also supports the use of routine clinical diagnostic data in the study of the epidemiology of diseases of veterinary importance.

Mycoplasmas serologically different from the known Mycoplasma species were found to be important causes of mastitis in California. We describe the cultural, morphological, biological, and serological characteristics of one of these isolates, strain ST-6. In growth inhibition and immunofluorescence tests, we found that strain ST-6 differed from all 75 currently accepted Mycoplasma species and serogroups. Therefore, we regard this strain as a member of a new species for which we propose the name Mycoplasma californicum. Strain ST-6, the type strain, has been deposited in the Collaborating Center for Animal Mycoplasmas, Institute of Medical Microbiology, University of Aarhus, Denmark, under the accession number AMRC-C 1077 and in the American Type Culture Collection under the number ATCC 33461.

Summary Variations in precision of the Breed method for cell counts in milk were investigated by utilizing different working factors (WF) for the same smear and by using the same WF over a major portion of the probable cell count range. A significant inverse relationship was found between precision and the WF. With a constant WF on the other hand, the precision of the count increased very significantly as the actual cell count increased. Formulas showing the relationship between the expected high and low for any given cell count were computed via the least squares method for a WF of 20,000. Evidence was presented that a WF of 5,000 or below would be necessary when a good estimate of cell content is important.

A procedure for staining mycoplasmata colonies directly on agar blocks for examination by fluorescent microscopy is described. Areas of the agar surface appropriate for staining were demarcated by use of Lucite cylinders. Direct fluorescent-antibody staining was superior to indirect staining. The technique was very useful for determining whether cultures were mixed and for identification of mycoplasmas in either pure or mixed cultures.

Mycoplasma colonies and Mycoplasma cells in preparations from infected milk and lymph nodes were observed for their fluorescent qualities after treatment with acridine orange. Mycoplasma colonies fluoresced brilliant red or red-orange. When treated after exposure to ribonuclease, the colonies fluoresced lime-green. There was no fluorescence when both ribonucleic acid and deoxyribonucleic acid were destroyed by perchloric acid. Detection of Mycoplasma in smears of mastitic milk or smears of infected lymph nodes was not definitive because of the large amount of nonspecific ribonucleic acid-rich material present during inflammatory reactions.

The effects of three changes in the optical equipment used for Direct Microscopic Somatic Cell Count in milk (National Mastitis Council method) on performance of the method have been measured and subjected to statistical analysis of probable significance. Replicate slides of six milk samples were counted by eight technicians among the authors' laboratories. Three levels of cell concentration were represented among the samples. No consistent differences in estimated cell concentration were demonstrated in the comparisons of narrow versus wide reticle band, wide field versus Huyghenian eyepieces, or low (high dry) versus high (oil immersion) magnification. In a few instances within each comparison, individual counters produced markedly different results when the optical equipment was modified. These individual biases were large and consistent with regard to change in objective magnification. No evidence was found to preclude use of wide field eyepieces in performing the DMSCC.

Summary By means of direct microscopic observation of cells in stained smears and projection of a series of color transparencies, differences in criteria used by individuals for identification of cells to be counted in Prescott-Breed smears were discovered and described. Extensive studies revealed a unique pattern of cell distribution within smears which can materially affect the outcome of a cell count depending upon the area selected for cell counting. Among other factors, variation in size of the smear was found to have a marked affect upon resultant sample count.

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.

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.

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.

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.

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.

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.

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.

Patterns of hemolysis produced by staphylococci isolated from milk were investigated on media prepared with bovine, sheep, horse, and rabbit erythrocytes under a variety of cultural circumstances. Hemolytic patterns were sharper and easier to interpret on erythrocyte agar plates when incubated at 37 degrees C in an atmosphere containing 30% CO(2.) Alpha-hemolysin production was greatly enhanced in this environment and was not detected at times when cultures were grown in air only. This was also true of deltahemolysin but to a lesser extent. Very satisfactory identification of alpha, beta- and delta-hemolysins was obtained by streaking BEA plates with the unknown strains perpendicularly to a strain producing beta-hemolysin and incubating in 30% CO(2.) This procedure avoided use of erythrocytes of different species of origin and use of staphylococcal alpha-antitoxin. Prior action of the beta-hemolysin on bovine cells was found to completely inhibit hemolysis from alpha-hemolysin but the reverse was not true. A strain inhibiting beta-hemolysis in air failed to exhibit its antihemolytic properties for 24 hours when incubated in 30% CO(2.) Patterns of hemolysis within areas subject to multiple hemolysins were found to reflect the nature and relative strength of contributing hemolysins or antihemolysins and could be modified by the time sequence of exposure to certain hemolysins or antihemolysins.

Observations were made on the value of certain selective media for isolation and identification of staphylococci from milk obtained from individual quarters and farm bulk tanks Aerobic fermentation of mannitol salt was unreliable for identification of coagulase positive strains. Coagulase positive strains tended to clump when grown in tryptose serum broth. Tellurite glycine agar plates were not entirely reliable for the selection of coagulase positive strains. Fibrinogen-tellurite-glycine plates were very useful for the isolation of staphylococci from milk in the presence of other microorganisms and for the simultaneous identification of coagulase positive strains. Plate coagulase tests utilizing fibrinogen tellurite glycine agar corresponded almost perfectly with conventional tube tests. Fibrinolytic strains were not uncommon and were found also among beta hemolysin producing coagulase positive strains.

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.

The Direct Microscopic Somatic Cell Count can be used advantageously in control of abnormal milk to perform simultaneously screening and confirmation of milk samples. Confidence limits computed according to the individual microscope Strip Factor are used to interpret the count made on a single strip of one of the two milk films. The sample is assigned on the basis of the interpreted count to one of three categories: (a) less than the legal cell concentration maximum ( P

Summary Milk smears were prepared and the weights of milk delivered were determined following use of the syringe and use of the 0.01 ml loop. Large and significant increases in the volume of milk delivered resulted when the angle of the loop with the milk was 20° rather than 90° or when the flat surface of the loop was brought out of the milk in the manner of a dipper. Slow withdrawal of the loop resulted in loss of milk and delivery of small volumes. Delivery of 0.01 ml of milk required that the loop be held in a vertical position and withdrawn quickly, but not with forced rapidity. The platinum loop, calibrated to deliver 0.01 ml of milk, has been used extensively for counting leukocytes in milk (1, 3, 5). Some limitations have been encountered, particularly the tendency for the loop to pick up an excessive volume of cell-laden fat from the suface of poorly mixed milk (1, 6). With well-mixed milk, leukocyte counts from smears made with the loop using carefully standardized techniques were found not to differ significantly from those made with the syringe1 (6). Standard procedure calls for the loop to be withdrawn vertically from the milk (1, 3). It has been observed, however, that workers from different milk quality control laboratories may withdraw the loop in differing ways so that the volumes of milk removed may vary substantially (2). During the course of these studies, it also became evident that the speed with which the loop was withdrawn influenced the volume of milk delivered. When the milk container is level, the loop is easily withdrawn vertically at an angle of about 90° to the surface of the milk, or it may be withdrawn at angles considerably smaller. When the milk container is tipped, it becomes convenient to withdraw the loop at an angle of about 20°. With the container tipped, the loop may also be used as a “dipper” by bringing it out with the broad surface almost horizontal with that of the milk. Tendencies toward use of each of these procedures have been noted in general laboratory practice. Because leukocyte counts have become a vital part of many milk quality control programs, any causes of error inherent in use of the loop should be understood. The following observations were made in order to evaluate the nature and magnitude of differences in volume of milk delivered by the loop as a consequence of variations in the manner of withdrawal of the loop from the milk.