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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.