Biomedical subjects
Publications and source records attributed to D E Jasper.
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.
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.
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.
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 of >20,480 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.
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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.
Transfer of bacteria between teat cups, between teats of the same cow, and between teats of different cows was investigated by tracer bacteria applied to teat skin and by injection of tracer bacteria or colored fluids into the short milk tube. In spite of the unidirectional flow of milk and air with the single chamber teat cup, no advantage over a conventional system in transfer of tracer bacteria from cup to cup, teat to teat, and cow to cow showed.
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.
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.
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.
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.
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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.
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