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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.

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.

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.

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.

Escherichia coli, Enterobacter aerogenes, and Klebsiella pneumoniae were responsible for 63, 10, and 11 percent, respectively, of 158 coliform organisms recovered from mastitis cases in 8 California herds. The severity and nature of the coliform mastitis problem were found to vary greatly among herds but were characteristic for each of 4 herds studied in detail.

Mastitis due to mycoplasma has been reported sporadically from several countries in Europe, Australia, Israel, New Zealand, and many states in the United States. Most reported occurrences are due to Mycoplasma bovis, but 5 other mycoplasmas are reported to cause mastitis. A total of 5 species of mycoplasma have been isolated from cows with mastitis in California. Therapy trials with M bovis infections have not been encouraging. Recovery is slow, and shedding of mycoplasma into milk of apparently recovered cows for an indefinite period is common.

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.

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.

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.

Mycoplasmas isolated from bovine mastitis in California were classified into five distinct species. These included Mycoplasma bovis, M. bovigenitalium, M. alkalescens, M. canadenfe, and an unidentified strain, ST-6. Strains frequently recovered from the nose of young calves proved to be M. arginini, M. bovirhinis was recovered from the respiratory tract but was not a common finding.

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%.

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.

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.

Calves were given (subcutaneous inoculations) viable Mycoplasma bovis with and without Freund's complete adjuvant (FCA). In the calves given M bovis with FCA, immediate and delayed cutaneous reactivity to formolized M bovis and increases in serum indirect hemagglutination (IHA) titers were pronounced. None of the calves showed evidence of an anamnestic response to a 2nd inoculation of M bovis. Treatment of the test serums from all calves with 2-mercaptoethanol (2-ME) revealed the presence of increased immunoglobulin M-IHA activities throughout the 70-day postinoculation (PI) period. Strong cell-mediated responses, as shown by the lymphocyte-activation test, were not observed until PI day 44. Lymphocytes from calves given M bovis with FCA gave the greatest response to M bovis antigens. However, the lymphocyte activation response to M bovis rapidly decreased after day 44 PI. The in vitro lymphocyte responses to phytohemagglutinin (PHA), as well as the control rates of lymphocyte incorporation of tritiated thymidine, appeared to be depressed as a result of the M bovis inoculations. The aqueous "supernatant" of M bovis was generally suppressive to in vitro lymphocyte blastogenesis. Mycoplasma bovis appeared to affect the immune responsiveness of the host directly by either stimulation of suppressor cell function or transiently depressing T-lymphocyte activities. The addition of FCA with M bovis had an enhancing effect on humoral and cell-mediated responses probably by increasing macrophage presentation of antigen to T-lymphocytes. The delayed development of cell-mediated immune responses may assist in the explanation of why some hosts are unsuccessful in mounting an effective immune response against M bovis.

Seven teat dip and sanitizer products were tested in vitro and in vivo for mycoplasmacidal activity against Mycoplasma agalactiae subsp. bovis (M. bovimastitidis). Most, but not all products tested appeared to kill the mycoplasma at satisfactory dilutions. These mycoplasma survived longer on teat skin during humid, rainy weather than during warm, dry weather. Acholeplasma laidlawii was frequently found on normal teat skin.

Seven species of mycoplasma plus one or more unknown species were found to cause bovine mastitis in California. Both the frequency of cases and number of species of mycoplasma in samples received at the laboratory have increased from 1976 to 1978. By survey, nearly 4% of samples of bulk tank milk from dairy farms were found to contain mycoplasma of potential pathogenic significance. Acholeplasma laidlawii was frequently isolated from samples both from cows and from farm bulk tanks during wet, rainy weather in the spring of 1978, apparently as contaminants only. The prevalence of positive bulk tank milk samples in an area appeared to parallel the prevalence of clinical mycoplasmal mastitis problem herds.

Mycoplasma alkalescens was isolated from 6 of 7 synovial fluid samples taken by arthrocentesis from 3-week- to 4-month-old Holstein-Friesian calves with severe arthritis (tibiotarsal or carpal joints). Approximately 30 of 215 calves in the herd were affected. In one 6-week-old calf, M alkalescens was isolated from the liver, right tibiotarsal joint, right and left popliteal lymph nodes, and an exposed umbilical artery. Intraarticular inoculations of broth cultures of M alkalescens initially induced a febrile response and then severe fibrinopurulent arthritis. Intravenous inoculation of M alkalescens induced only a febrile response. The natural disease may have been a complication of umbilical exposure to M alkalescens, causing omphaloarteritis and subsequent arthritis. Before and during the arthritis problem, the umbilicus of newborn calves was dipped in an organic iodine product with 10% glycerin, marketed as a postmilking teat dip. After the cause of the arthritis was determined, the umbilicus of each newborn calf was treated with 7% tincture of iodine and no new cases of arthritis occurred.

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.

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.

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.

Rabbits were given 3 serologically-related trains of mycoplasma isolated from mastitic bovine udders. Growth inhibiting antibodies were not found. High-titered sera from each rabbit immunized with each strain developed at least 4 precipitin lines to each organism and at least one line of identity was demonstrated between the organisms. Significant indirect hemagglutination titers were found in all rabbits in 1 week, with titers reaching 1.0 X 10(5) to 3.3 X 10(5) in 4 to 6 weeks but despite continued intravenous antigenic stimulation, titers quickly fell to low levels thereafter.

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.

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.

Coliform organisms were isolated from bedding, udders of cows exhibiting acute or chronic mastitis, and milk of cows tested for udder pathogens. The bactericidal activity of standard bovine serum was tested on 362 isolates, of which 2.4% were killed at a serum dilution of less than 1:5; 2.7% were killed at a dilution of 1.5; 3.0% were killed at a dilution of 1:12.5; and 4.4% were sensitive to a dilution of 1:25. Organisms isolated from mastitic udders were not killed by serum. Sensitivity to agglutination in 1:500 solution of acriflavine was tested on 157 isolates, and only 54 had any degree of agglutination. Of these 54, only 4 organisms had any susceptibility to killing by serum. Results suggest that most organisms in the environment of the cow are resistant to serum, few serum-sensitive coliforms cause mastitis, and agglutination in acriflavine (indicating organisms in the rough phase) does not correlate with sensitibity to serum.

A dairy herd having a high incidence of coliform mastitis was observed by means of pre- and post-milking samples and teat apex culture. Infected quarters were the major source of teat apex contamination with coliform bacteria and therefore constituted a risk to unifected quarters and to other cows. Coliform populations on the apex from environmental sources appeared to be transitory.

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.

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.