Lung lesions in bovine fetuses aborted by Brucella abortus.

Considering the poor facilities available for microbiological diagnosis in some countries where Brucella abortus is a frequent cause of bovine abortion, a study was conducted to determine if isolation of B. abortus from an aborted bovine fetus could be predicted from a detailed histological study of the formalized lung. Thirty-nine samples of B. abortus positive and 20 negative fetal samples were examined for the presence of 14 different pulmonary lesions. Differences in the frequency of observed lesions between the positive and negative groups, were determined by odds ratios and chi square statistic. The confidence of the prediction was calculated by means of the logistic computer model. The frequency of eight lung lesions was found to be significantly (p less than 0.05) different between the groups; nevertheless, these lesions were not specific enough to be able to incriminate B. abortus as the cause of abortion.     

Opsonin-dependent stimulation of bovine neutrophil oxidative metabolism by Brucella abortus

Nonopsonized Brucella abortus and bacteria treated with fresh antiserum, heat-inactivated antiserum, or normal bovine serum were evaluated for their ability to stimulate production of superoxide anion and myeloperoxidase-mediated iodination by neutrophils from cattle. Brucella abortus opsonized with fresh antiserum or heat-inactivated antiserum stimulated production of approximately 3 nmol of O2-/10(6) neutrophils/30 min. Similarly treated bacteria also stimulated the binding of approximately 4.3 nmol of NaI/10(7) neutrophils/h to protein. Significant (P less than 0.05) production of O2- and iodination activity by neutrophils were not stimulated by nonopsonized bacteria or organisms treated with normal bovine serum. Seemingly, B abortus stimulated oxidative metabolism in bovine neutrophils; however, the stimulation was dependent on the presence of bacterial associated opsonins.     

Isolation of Brucella abortus and Brucella abortus, strain 19, from cattle.

Tissues from 104 cows in herd were examined for brucellae. Brucella abortus, strain 19, was isolated from 22 cows, a field strain of B abortus, biotype 1, was isolated from 9 cows, and both strains were isolated from 2 cows.     

Enhanced intraphagocytic killing of Brucella abortus in bovine mononuclear cells by liposomes-containing gentamicin

In vitro intraphagocytic killing of Brucella abortus in bovine mononuclear leukocytes was enhanced by cationic, anionic, and neutral multilamellar liposomes-containing gentamicin. Free gentamicin not entrapped in liposomes. and liposomes without antibiotic did not enhance intraphagocytic killing of B. abortus in bovine phagocytes. In vivo killing of B. abortus in guinea pigs was also enhanced by liposomes-containing gentamicin when compared to free gentamicin. Liposomes-containing alpha tocopherol acetate failed to enhance in vivo killing of B. abortus.     

Kinetics of in vitro bovine lymphocyte immunostimulation with a Brucella abortus antigen.

A Brucella abortus-soluble antigen was investigated, using in vitro assay of lymphocyte immunostimulation, to determine which concentration of this antigen and which period of incubation of the lymphocyte cultures would induce maximum specific lymphocyte immunostimulation as an additional method for further study of B abortus infection in cattle. Soluble antigen was prepared from autoclaved cells of B abortus strain 1119-3. Peripheral blood lymphocytes were obtained from cattle infected with B abortus and from healthy control cattle not infected with B abortus. The lymphocytes were prepared by the Ficoll-Hypaque density gradient technique, suspended in RPMI 1640 medium (1.5 X 10(6)/ml), cultured with several dilutions of soluble antigen, and incubated. Prior to termination of incubation, cultures were labeled with 1 muCi of [3H]thymidine and, after harvesting, assayed for [3H]thymidine incorporation in DNA by a liquid scintillation spectrometer. Maximum specific immunostimulation of lymphocytes from B abortus-infected cattle was induced in this assay system with 6 days' incubation and 22 microgram of protein/ml/1.5 X 10(6) lymphocytes, using protein content to express concentration of soluble antigen in this system.     

Characterization of an atypical biotype of Brucella abortus.

Brucella abortus strains were isolated from bovine tissue and milk samples from seven Ontario herds. The isolates were characterized by colonial morphology, requirement of CO2 for growth, lysis by Tbilisi phage, biochemical tests and agglutination in monospecific sera. They resembled B. abortus biotype 2 (on the basis of sensitivity to thionin and basic fuchsin) and biotype 4 (on the basis of agglutination with anti-Brucella "M" but not anti-Brucella "A" absorbed sera). Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of these isolates and B. abortus biotypes 1, 2 and 4 showed similar profiles. Immunoblots with anti-A and anti-M absorbed sera showed different antigenic regions reacting with the specific sera and also confirmed that the atypical B. abortus isolates were serologically similar to biotype 4.     

The half-life of bovine (incomplete) antibodies to Brucella abortus.

The half-life of bovine immunoglobulins was established in a group of 60 calves suckled on non-infected dams immunised with antigens to Brucella abortus. The half-life of the "incomplete antibody" as determined by the anti-bovine globulin test was 20.9 days. Up to 45 days, the mean half-life of antibodies in the calves was 17.1 days; from 46 to 99 days it was 21.1 days and for periods exceeding 100 days it was 24.4 days. In 39 calves born of and suckled on infected dams the mean half-life of antibodies was 22.4 days. Up to 100 days the figure was 19.5 days; over 100 days it was 22.8 days. There was no significant difference in the mean half-life of antibody between calves from infected and non-infected dams. In both cases the antibodies appear to be catabolised at a progressively slower rate.     

Resistance of preimplantation bovine embryos to infection with Brucella abortus

Preimplantation bovine embryos were exposed in vitro to Brucella abortus to determine if the bacteria would adhere to zona pellucida (ZP)-intact embryos or adhere to or infect ZP-free embryos. Brucella abortus was not isolated from ZP-intact or ZP-free groups of embryos after 10 sequential antibiotic-free washings. Brucella abortus was isolated from all groups containing ZP-defective embryos after the exposure period and washing. Detrimental effects on healthy in vitro development of embryos were not observed.     

Interaction of bovine chorioallantoic membrane explants with three strains of Brucella abortus.

Chorioallantoic membrane (CAM) explants were used to determine the in vitro growth and cytotoxic potential of 3 strains of Brucella abortus. Bovine CAM explants were inoculated with 2 x 10(7) colony-forming units of the pathogenic strain 2308, attenuated strain 19, or the rough strain RB51 of B abortus. After inoculation, the explants were harvested and examined at 2 or 4 hours, 12 or 14 hours, and 24 or 26 hours of incubation. Bacterial growth associated with each explant was determined by counting colony-forming units. The degree of cellular damage in each explant associated either with bacterial growth or bacterial toxins was evaluated by morphometric analysis after trypan blue staining. Significant differences were not detected in the numbers of bacteria of any strain of B abortus in the CAM explants at comparable time intervals. The rate of growth of the bacteria in CAM explants was higher between 2 and 12 hours after inoculation than between 12 and 24 hours after inoculation. Cytotoxic effects associated with strain 2308 were significantly (P less than 0.05) greater than that caused by other strains. Cytotoxic effects associated with strain 19 and rough strain RB51 were similar, and both were significantly (P less than 0.05) greater than the phosphate buffer solution control. Chorioallantoic membrane explants inoculated with a filtrate of heat-killed strain 2308 induced minimal cellular damage, compared with that caused by the viable bacteria. These results indicated that the number of B abortus in trophoblasts was independent of the degree of cellular damage.     

Characterization of Brucella abortus strain 19 isolated from human and bovine tissues and fluids

One hundred isolates of Brucella abortus, which were recovered from bovine and human tissues or fluids, were identified as strain 19 by conventional bacteriologic methods. Each isolate was examined using a Warburg respirometer to determine oxidative rates on substrates of D- and L-alanine, L-glutamic acid, d(+)-galactose, D-ribose, and i-erythritol. These results were compared with those of repository (seed) cultures of strain 19 used for making antigens and vaccines. Except on the substrate of i-erythritol, each of the 100 isolates oxidized these substrates with rates different from the repository cultures and indistinguishable from those of field strains of B abortus. Thus, oxidatively, i-erythritol was the only substrate useful to help distinguish between strain 19 and virulent strains of B abortus biotype 1.     

The efficacy of bacteriological procedures for the isolation of Brucella abortus from abattoir material

A process of emulsifying and centrifuging abattoir specimens before plating out is described. Brucella abortus was isolated more successfully by this process than by conventional methods, especially in low grade infections. The 5 different media used were equally effective in our attempts at isolation, but growth did not necessarily occur on all 5 plates. In dairy cows, specimens from supra-mammary lymph nodes, udder and iliac lymph nodes accounted for a high percentage of positive isolations.     

Brucella abortus-specific immunoglobulin isotypes in serum and vaginal mucus from heifers vaccinated with Brucella abortus salt-extractable proteins and challenge exposed with virulent Brucella organisms

Serum and vaginal Brucella-specific immunoglobulin isotypes (IgG1, IgG2, IgM, and IgA), obtained from 62 crossbred beef heifers vaccinated with Brucella abortus salt-extractable proteins and subsequently challenge exposed with B abortus S2308, were studied. Brucella-specific IgG antibodies and Brucella-specific immunoglobulin isotypes were quantitated by a fluorometric immunoassay. Serum and vaginal immunoglobulin responses were evaluated as a method of distinguishing infected from noninfected heifers. Rivanol precipitation, complement-fixation, buffered-antigen brucellosis tests and an ELISA were performed on sera. For immunoglobulin isotypes, vaccinated heifers had mean antibody responses higher than baseline mean antibody responses for at least 31 weeks after vaccination. After challenge exposure, significant differences (P greater than 0.05) were not detected between mean antibody responses of vaccinated and nonvaccinated heifers. Vaginal Brucella-specific antibody responses did not correlate with protection from disease. Vaginal Brucella-specific IgM was detected only at the time of abortion. Vaginal IgA appeared specific for identification of virulent B abortus infection. All serotests appeared adequate in distinguishing baseline titers from titers of heifers that had aborted and were considered bacteriologic culture-positive. Results of serotests neither consistently distinguished vaccinates from challenge-exposed cattle nor distinguished heifers that were challenge exposed, had aborted, and were considered bacteriologic culture-positive adequately from heifers that were challenge-exposed, had not aborted, and were considered bacteriologic culture-negative. Brucella-specific IgA appeared to be the most effective in distinguishing vaccinated heifers from challenge- exposed heifers and heifers that were challenge exposed and had aborted, from heifers that were challenge exposed and had not aborted. Brucella-specific serum IgA was detected up to 13 weeks after abortion.     

Comparison of the 2-mercaptoethanol and dithiothreitol tests for determining Brucella immunoglobulin G agglutinating antibody in bovine serum.

The dithiothreitol test was evaluated as a substitute for the 2-mercaptoethanol test for determining Brucella immunoglobulin G agglutinating antibody in bovine serum. The tests were compared on 207 card-positive sera that showed a standard tube-agglutination titer of incomplete 1:50 or higher. The tests agreed within one dilution with 182 of the 207 sera tested for an 87.9% rate of agreement. When titers were not the same, those obtained with the dithiothreitol test were more frequently lower than higher than those obtained with the 2-mercaptoethanol test. Sixteen sera that showed a titer with the 2-mercaptoethanol test were negative with the dithiothreitol test and two that showed a titer with the dithiothreitol test were negative with the 2-mercaptoethanol test. Results suggest that the dithiothreitol test is not a reliable substitute for the 2-mercaptoethanol test to detect immunoglobulin G agglutinating antibody in bovine serum.     

The bovine immune response to Brucella abortus. III. Preparation of antisera against a Brucella component precipitated by sera of some infected cattle.

Two methods are described for the partial purification of a high molecular weight, heat-resistant component (CO1) of sonicates of smooth and rough Brucella abortus which is precipitated by sera of some infected cattle. Method 1, a combination of gel filtration chromatography and polyacrylamide gel electrophoresis, was used to prepare CO1 from sonicates of a smooth field strain of B. abortus. Method 2, a combination of gel filtration chromatography and heat treatment, was used to obtain CO1, from sonicates of rough B. abortus strain 45/20. Rabbit antisera produced against CO1 prepared by either method contained only CO1 precipitins but were negative in standard agglutination and complement fixation tests conducted with whole cell antigens. Evidence is presented that CO1 is identical to Brucella antigen A2, and it is proposed that in future the designation A2 be employed.     

An evaluation of a biphasic medium for the isolation of Brucella abortus from bovine tissues

The isolation of virulent Brucella abortus from specimens taken from cattle was studied using culture in biphasic medium in which solid and liquid media were contained in the same flask and on solid medium in Petri plates. A total of 8638 specimens from 441 cattle, reactors to one or more of several serological tests or to an allergic test, were used. The specimens consisted of lymph nodes, udder, spleen, uterine caruncles, cotyledons, foetal tissues, foetal stomach contents and mammary secretions. Following maceration each specimen was inoculated into 2 flasks of biphasic medium one with 1 ml (Flask A) and the other with 2 ml (Flask B), and on to solid medium in one Petri plate. The biphasic medium and solid medium were shown to have equal ability to support the growth of B. abortus. The increased rate of detection in the biphasic medium was due to the increased volume of the inoculum used. Brucella were isolated from 1151 specimens. By direct plating on the solid medium 685 specimens were positive whereas Flask A positive for 961 specimens and Flask B for 1031 specimens. The success of the biphasic medium can be judged by the increase in the number of infected animals detected. Of the 150 infected cattle, both techniques identified 126 animals in common, 4 were identified only by the plate technique and 20 only by the biphasic technique. Culture on both solid medium and in biphasic medium is recommended for optimum results.     

Recombinant bovine interleukin 2 enhances immunity and protection induced by Brucella abortus vaccines in cattle

Augmentation of immunization of cattle Brucella abortus S19 or a B. abortus soluble protein extract (SPEBA) vaccine through administration of recombinant bovine IL 2 (rBoIL 2) was evaluated. Seventy-five heifers were divided among 6 groups that were treated with the following: Group 1, no treatment; Group 2, rBoIL 2 (1microg/kg) on day 0; Group 3, SPEBA (2 mg) on day 0 and week 9; Group 4, SPEBA + rBoIL 2 on day 0, SPEBA on week 9; Group 5, S19 (10(7) CFU) on day 0 and week 9; Group 6, S19 + rBoIL 2 on day 0, S19 only on week 9. Approximately, 6 months after vaccination, cattle were bred by natural service, and at mid-gestation pregnant cattle were challenged intraconjunctivally with 9.1 x 10(5) CFU of virulent B. abortus S2308. Pre- and post-challenge antibody responses were measured by an enzyme-linked immunosorbent assay, a particle concentration fluorescence assay, and the card test. Lymphoproliferation (LP) responses to gamma-irradiated B. abortus and SPEBA antigens were measured in peripheral blood mononuclear cells. After vaccination, antibody responses to B. abortus elevated rapidly in SPEBA- and S19-vaccinates with and without rBoIL 2, however, these responses were significantly (P < 0.05) higher in vaccinates which also received rBoIL 2. Antibody levels for all vaccinated groups had returned to those of negative control groups by the challenge date with the exception of the SPEBA/rBoIL 2 group. In general, LP responses were higher in vaccinated or rBoIL 2-treated cattle than for unvaccinated controls. Challenge of 48 pregnant heifers resulted in abortions in 4/9 of Group 1, 0/9 of Group 2, 4/8 of Group 3, 2/9 of Group 4, 1/7 of Group 5, and 0/6 of Group 6 cattle. Treatment with rBoIL 2 alone (Group 2) provided significant (P < 0.05) protection from infection, abortions and induction of sero-positive status compared to untreated (Group 1) cattle. Co-administration of rBoIL 2 with S19 resulted in significant (P < 0.05) augmentation in onset, duration and magnitude of LP responses to B. abortus antigens following challenge. Characterization of the cytokine response of bovine monocyte-derived macrophages by real-time polymerase chain reaction indicated that in vitro stimulation of these cells with rBoIL 2 resulted in a profound up-regulation of genes encoding tumor necrosis factor-alpha, IL 12p40, and interferon-gamma reflecting activation of the cells. Overall, rBoIL 2-treatment was associated with fewer infections, sero-conversions and a significant (P = 0.02) level of protection against abortion as compared to vaccination alone or no treatment.