Coordinate synthesis and turnover of heat shock proteins in Borrelia burgdorferi: degradation of DnaK during recovery from heat shock.

The synthesis and turnover of heat shock proteins (Hsps) by Borrelia burgdorferi, the Lyme disease spirochete, was investigated by radiolabeling of whole spirochetes and spheroplasts, comparison of one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and use of immunochemistry. The approximately 72-kDa DnaK homolog and three additional Hsps of 39, 27, and 21 kDa increased in amount by 3- to 15-fold between 2 and 6 h following temperature upshift from 28 to 39 degrees C. Temperature downshift experiments following the transfer of spirochetes from 40 to 28 degrees C showed that within 15 to 30 min, synthesis of most of the major Hsps returned to levels seen in spirochetes statically maintained at the lower temperature. Spheroplasts of B. burgdorferi produced by treatment with EDTA and lysozyme were radiolabeled, and specific Hsps were localized to either the cytoplasm or membrane fraction. Further analysis by two-dimensional electrophoresis demonstrated three constitutively expressed DnaK isoforms with pIs near 5.5. A pattern suggestive of DnaK degradation was observed following recovery from heat shock but not in spirochetes maintained entirely at a low temperature. Some of these putative degradation products were recognized by monoclonal antibodies directed against the B. burgdorferi DnaK protein. These data suggest that following a period of peak synthesis, DnaK is actively degraded as the spirochete reestablishes its metabolic thermometer. These findings provide a new interpretation of previous work suggesting that 10 to 15 B. burgdorferi polypeptides, including DnaK have a common epitope.

Molecular characterization of Pasteurella testudinis isolated from desert tortoises (Gopherus agassizii) with and without upper respiratory tract disease.

Isolates of Pasteurella testudinis recovered from clinically healthy desert tortoises (Gopherus agassizii) and tortoises with upper respiratory tract disease (URTD) were characterized in an attempt to identify strains associated with disease. Eighty-nine isolates, 52 from ill and 37 from healthy tortoises collected from Nevada (USA), June 1990 to September 1991, were genomically fingerprinted and grouped based on ribotype similarity. Twelve isolates (six from ill and six from healthy tortoises) were further characterized with regard to whole-cell protein (WCP) and outer membrane protein (OMP) composition and their ability to survive in normal tortoise plasma. The 89 isolates were initially distributed into 33 distinct ribotype groups using the restriction enzyme EcoRI; five ribotypes contained over 50% of the isolates. Only one EcoRI ribotype was comprised of multiple isolates (n = 4) exclusively recovered from tortoises with URTD. When the ten EcoRI ribotypes that contained more than one isolate per ribotype were further studied using a second restriction enzyme, EcoRV, one EcoRI/EcoRV ribotype contained five isolates recovered from URTD tortoises and none from healthy animals. The EcoRI ribotype comprised of four isolates, all from tortoises with URTD, was further separated into three distinct groups with EcoRV. All 12 isolates studied grew equally well in normal tortoise plasma, and when broth-grown WCP and OMP profiles were evaluated, no proteins were unique to isolates from URTD tortoises. Iron-regulated OMP's were produced in three isolates examined, but these OMP's apparently were not virulence-related.

Thermoregulation of protein synthesis in Borrelia burgdorferi.

Borrelia burgdorferi, the etiological agent of Lyme disease, infects humans via the bite of a tick. The microbe survives in at least two vastly different environments: an arthropod vector and a warm-blooded host. We examined protein synthesis in B. burgdorferi B31 in response to sudden heat stress, which is similar to that which occurs during the transmission from vector to host. Proteins synthesized after shifts from 28 degrees C to higher temperatures and in pulse-chase experiments were labeled with 3H-labeled amino acids for 4 h and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. The synthesis of four proteins we designated as heat stress proteins (HSPs) was increased by shifts to higher temperatures (HSP-1, 75 kilodaltons [kDa]; HSP-2, 42 kDa; HSP-3, 39 kDa; and HSP-4, 27 kDa); and the amount of one protein we designated as heat-labile protein 1 (29.5 kDa) was decreased at higher temperatures. At 37 to 40 degrees C, the major heat stress protein, HSP-1, represented 14 to 18% of the total cell protein compared with 1 to 2% of the total cell protein at 28 degrees C. HSP-1 was stable during a 4-h chase at either 40 or 28 degrees C. Demonstration of similar HSPs in low-passage, pathogenic strains of B. burgdorferi suggests that the heat stress response may be common among B. burgdorferi strains and may play a role in Lyme disease.

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.

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.

Detection of Mycoplasma gallisepticum by direct immunofluorescence using a species-specific monoclonal antibody.

A monoclonal antibody against Mycoplasma gallisepticum (MG) (strain S6) was prepared in mice and identified as isotype IgG1 by standard procedures. Although it did react at high titers (1:100,000) in the enzyme-linked immunosorbent assay (the original method for its identification), it failed to react in the agglutination, hemagglutination-inhibition, and growth-inhibition tests. When conjugated to fluorescein isothiocyanate, the monoclonal antibody reacted with the homologous and eight "atypical" strains of MG but not with M. meleagridis or M. synoviae in the direct fluorescent-antibody test. This reagent may be useful for detecting field infections involving atypical strains of MG.

An enzyme immunoassay to measure canine circulating fibronectin.

A competitive enzyme immunoassay has been used to detect and quantitate fibronectin in canine plasma. In this test, purified fibronectin, bound to microtiter plates, competes with plasma fibronectin for the conjugated antibody, rabbit-anticanine, fibronectin-horseradish peroxidase. The assay could detect fibronectin in purified standards from 58 ng/ml to 580 microgram/ml. The range of 1-100 microgram/ml was linear for plasma samples diluted 1:10, allowing samples with fibronectin concentrations from 10-1000 microgram/ml to be easily measured by this method. The mean normal fibronectin concentration of 132 dogs, by this method, was determined to be 320 +/- 74 microgram/ml.

Measurement of canine plasma fibronectin by rocket immunoelectrophoresis.

Plasma fibronectin concentrations of 148 normal canine samples were measured by rocket immunoelectrophoresis. Electrophoresis was accomplished, using 2.0% rabbit anticanine fibronectin by volume in 0.7% agarose in buffer. Films were electrophoresed 18 hours in barbital buffer, 7.5 mA/film. The mean fibronectin concentration for normal citrated dog plasma was 290 micrograms/ml +/- 50 micrograms/ml.

Changes of complement values in calves during the first month of life.

Hemolytic complement activity and the 3rd component of complement (C3) concentrations were measured in the blood sera of 8 dams before, at, and after parturition, and in the sera of their calves before and after feeding colostrum and at fixed intervals up to 1 month of life. The mean hemolytic titer in the dams, as measured by incubating guinea pig RBC sensitized with bovine natural antibodies in serially diluted serum, was slightly less than 200 and was not influenced by parturition and onset of lactation. The titers in the sera of the calves immediately after birth ranged from 63 to 149 with a mean of 99. One day later, values in all calves had dropped markedly to a mean of 39. During the following month, the titers increased and reached the precolostral levels after about 4 weeks; however, these titers were still far below the titers measured in adult cows. A similar pattern was seen in the C3 concentration. The mean value at birth was 28% of the values measured in adult cows. Values decreased to 18% one day later and increased during the following month to 43% of the adult C3 concentration.

Prevalence of mycoplasmas and immune responses to Mycoplasma bovis in feedlot calves.

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.

Characterization of antigens from mycoplasmas of animal origin.

Alcholeplasma laidlawii, Mycoplasma gallisepticum, M mycoides subsp mycoides, M agalactiae, M bovirhinis, mycoplasmal strain ST-6, and culture medium were compared with M bovis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, enzyme-linked immunosorbent assay (ELISA), and gel electrophoresis-derived ELISA. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated there were areas of homology and areas of heterology among the species tested. Sera from rabbits hyperimmunized with the mycoplasma organisms and noninoculated culture medium demonstrated ELISA reactivity with M bovis antigens immobilized on polystyrene. Absorption of the serum from a rabbit hyperimmunized with M bovis reduced 65.9% of its reactivity with culture medium, 29.7% to 32.7% of its reactivity with the heterologous species, and 21.1% of its reactivity with the homologous species. Gel electrophoresis-derived ELISA performed on immobilized M bovis antigens separated by molecular weight, using sera from rabbits hyperimmunized with the mycoplasmal species under study and noninoculated culture medium revealed antigenic components which are shared among species or with the culture medium and several components which may be unique to M bovis.

Gel electrophoresis-derived enzyme-linked immunosorbent assay of serum from cows resistant to and cows susceptible to challenge exposure with Mycoplasma bovis.

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.

Detection of Mycoplasma bovis specific IgG in bovine serum by enzyme-linked immunosorbent assay.

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.