Cell-mediated immune responses in cattle vaccinated with killed Brucella melitensis strain H-38 vaccine or infected with viable Brucella abortus strain 2308 organisms, or both.
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Biomedical subjects
C C Muscoplat
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Temporal cell-mediated immune responses of cattle following experimental and natural exposure to living Brucella abortus.
A study on cell-mediated immune responses in cattle with different exposure experiences to Brucella abortus was conducted by an in vitro lymphocyte stimulation assay. The purpose of this study was to determine how soon the cell-mediated immune responses would be detected following experimental exposure to B. abortus and to study the cell-mediated immune trend following experimental and natural exposure of cattle to B. abortus. The first positive cell-mediated immune responses occurred one to two weeks after experimental inoculation with living B. abortus strain 2308. The cell-mediated immune responses in these animals appeared at least one week before the appearance of of B. abortus serum agglutinating antibodies. Animals which were naturally infected with B. abortus biotypes 1 and 2 demonstrated positive cell-mediated immune responses throughout the study.
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Cell-mediated immune responses in cattle vaccinated with Brucella abortus strain 19 vaccine and nonexposed control animals of the same age.
Cell-mediated immune (CMI) responses in cattle vaccinated with Brucella abortus strain 19 vaccine during calfhood were studied by an in vitro lymphocyte stimulation assay. Cattle were grouped in six groups according to the age after vaccination, and CMI responses of these groups, as well as of individual animals, were compared. Lymphocytes were prepared from peripheral blood of these cattle by the Ficoll-diatrizoate technique. Lymphocytes were then cultured with B abortus-soluble antigen. Culture results were assayed for [3H]thymidine incorporation into DNA by liquid scintillation spectrometry. On a group basis, B abortus-soluble antigen induced lymphocyte stimulation responses in lymphocytes from all the groups, except the sixth group which contained animals that had been vaccinated the longest time (18 to 24 months before this experiment). Animals that had been vaccinated for 3 to 6 months had the highest lymphocyte stimulation response. Seroagglutination tests were conducted simultaneously with the lymphocyte stimulation test, but there was no apparent correlation between the concentrations of humoral antibodies and the CMI responses as measured by in vitro specific lymphocyte stimulation. The lymphocyte stimulation test exhibited significantly higher specificity (P less than 0.005) than the serologic tests.
Agglutination Tests↗
Cell-mediated immune responses in cattle adult-vaccinated with Brucella abortus strain 19 and in cattle infected with Brucella abortus field strain.
Cell-mediated immune responses in cattle adult-vaccinated with Brucella abortus strain 19, cattle infected with B abortus field strain, and nonexposed cattle were studied by an in vitro lumphocyte-stimulation test (LST). Lymphocytes were prepared from peripheral bovine blood by the Ficoll-diatrizoate technique, and results were assayed for [3H]thymidine incorporation into DNA by liquid scintillation spectrometry. Serotests and bacteriologic isolation attempts were conducted simultaneously with LST. Lymphocytes from cattle infected with field strains had significantly (P = 0.01) higher specific lymphocyte-stimulation inexposed controls. The LST, the serum standard-tube agglutination test (STT), the Rivanol (RIV) test, and the complement-fixation (CF) test correctly classified cattle from which field strains and strain 19 of B abortus were isolated. The LST was negative in cattle vaccinated with B abortus strain 19 (nonshedding), but the three serotests had many false-positive reactions. The CF test had the least false-positive reaction, followed by the RIV test, and the STT was the least specific. Well before the three serotests became positive, the LST was positive in samples from some cattle during the incubation period of the infection. There was little or no correlation between cell-mediated immune responses (as measured by LST) and serum antibody responses (as measured by STT, RIV test, and CF test) in vaccinated but culture-negative cattle and in some nonvaccinated cattle during the incubation period.
Agglutination Tests↗
Enumeration of T cells, B cells and monocytes in the peripheral blood of normal and lymphocytotic cattle.
The rosette-forming capacity of bovine peripheral blood lymphocytes (PBL) was determined with dextran and 2-aminoethylisothiouronium bromide (AET)-treated sheep erythrocytes (SRBC). Both dextran and AET-enhanced rosette formation; however, AET-treated SRBC detected a larger percentage of rosette-forming cells and thus was used in this study. The specificity of rosette formation by bovine thymus-derived (T) lymphocytes was shown by (1) demonstration of rosettes and surface-membrane immunoglobulins sIg) on different cells in PBL and nylon-wool fractionated lymphocyte populations and (2) rosette formation by a large percentage (83--90%) of thymocytes from three bovine foetuses and two 14-month-old heifers. A procedure was also developed to identify bovine monocytes by latex phagocytosis and 10--30% latex-ingesting cells were detected in PBL preparations isolated by Ficoll-Hypaque flotation. The frequency of sIg-bearing latex-ingesting, and sIg-bearing latex non-ingesting cells in bovine peripheral blood was also determined. These procedures were utilized to determine the distribution of T and bone-marrow derived (B) lymphocytes in peripheral blood of normal and lymphocytotic cattle. PBL from twenty normal cattle contained approximately 63% T and 11% B (sIg+ latex non-ingesting) lymphocytes. In peripheral blood of three cattle with persistent lymphocytosis, a prodromal stage of bovine leukaemia, the percentage of B cells was elevated approximately to 59% whereas T lymphocytes decreased to 35%, thus providing additional evidence that persistent lymphocytosis is a B-cell disease.
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Ontogeny of T cells, B cells and monocytes in the bovine foetus.
The ontogeny of lymphocyte subpopulations and monocytes was studied in developing bovine foetuses. Fifty-four bovine foetuses ranging in age from 3 to 9 months of gestation were collected and examined for the presence of thymus-derived (T lymphocytes), bone marrow-derived (B lymphocytes) and phagocytic (macrophage) cells from thymus, spleen and peripheral blood. Detection of the cells expressing T lymphocyte surface markers was done using a modified sheep red blood cell rosette assay. Lymphocytes bearing surface immunoglobulin (sIg) were detected by staining with a fluoresceinated rabbit antibody prepared against bovine IgG. The procedure used for detecting monocytes was a latex bead phagocytic assay. In the thymus, the percentage of T lymphocytes was relatively constant throughout gestation at approximately 60-70%, with B lymphocytes remaining at about 1%, whereas the phagocytic cells showed an increase from 1% at 3 months to approximately 8% at term. The spleen showed an increasing percentage of T lymphocytes, from 11% at 3 months to over 40% at term, whereas the percentage of B lymphocytes remained consistent at about 2-3% from 3 months until term. The percentage of monocytes increases from 1% at 3 months to over 20% at 9 months. Peripheral blood T lymphocytes increased steadily from approximately 1% at 3 months to 45% at term. B lymphocyte levels remained low, about 1%, throughout gestation, whereas monocytes increased from 1% at 3 months to 5% at 9 months.
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B-lymphocyte differentiation, using pokeweed mitogen stimulation: in vitro studies in leukemic and normal cattle.
B-lymphocytes from 7 normal cattle and from 3 persistent lymphocytotic, 6 lymphosarcomatosus, and 3 bovine leukemia virus-infected animals with diverse defects in humoral immunity were examined for their capacity to undergo terminal differentiation in vitro. Pokeweek mitogen-stimulated cells from normal and leukemic cattle were stimulated to synthesize and secrete immunoglobulins, and these were detected by radial immunodiffusion. Lymphocytes from normal cattle synthesized more IgM than those from persistent lymphocytotic-, lymphosarcomatous-, and bovine leukemia virus-infected cattle. Leukemic cattle have a higher percentage of cells containing intracytoplasmic immunoglobulins; most cells are believed to contain IgG. The serum IgM in normal cattle is significantly (P less than 0.05) higher than that in leukemic cattle. The results indicate that there is an abnormality either in synthesis or secretion of IgM or in abnormal catabolism of Ig from leukemic B-lymphocytes.
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Cryopreservation of bovine mononuclear leukocytes.
A technique of cryopreservation of bovine mononuclear leukocytes for use in lymphocyte stimulation tests and cell identification studies has been developed. Dimethyl sulfoxide was used as the cryopreservation agent. Cells were frozen to --40 C at a controlled rate of --1 C/minute and then to --80 C at a rate of --4 C/minute, and were subsequently stored in liquid nitrogen (--109 C). The cells were than recovered by rapid thawing in a 37-C water bath, diluted with tissue culture media, washed, and used for lymphocyte stimulation and cell identification tests. The magnitude of the lymphocyte blastogenic responses of frozen and thawed mononuclear leukocytes was similar to that seen with freshly collected cells. Additionally, percentages of T cells, B cells, and monocytes were similar between frozen and thawed cells and freshly collected cells.
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Suppression of specific lymphocyte blastogenesis after intravenous tuberculin injection in Mycobacterium bovis-sensitized cattle.
Intravenous injection of 1.0 ml of old tuberculin (OT) in 4 calves previously sensitized to Mycobacterium bovis resulted in suppression of specific lymphocyte blastogenic responses to purified protein in vitro. Suppression of in vitro blastogenic responses occurred within 24 hours after injection of OT and persisted for up to 96 hours. Lymphocytes from these calves, cultured with concanavalin A, did not show any suppression of the lymphocyte blastogenic response in vitro. Suppression was temporary and in all calves was preceded by rapid febrile responses. Rectal temperature responses ranged from 40.1 to 41.6 C as early as 6 hours after tuberculin injections. Three control animals, M bovis-sensitized but not given OT, remained lymphocyte stimulation-positive throughout the study and served as controls for laboratory fluctuations.
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Potentiation of lymphocyte mitogenic responses to concanavalin A by antigen-activated peripheral blood monocytes.
Purified protein derivative (PPD)-stimulated monocytes derived from Mycobacterium bovis-sensitized cattle significantly potentiated lymphocyte mitogenic responses to concanavalin A (conA), as measured by incorporation of [3H] thymidine into cellular DNA. Monocytes were cultured for 24 hours in the presence of PPD, washed thoroughly, and mixed with purified lymphocytes; various doses of conA were added to these cultures, and the cultures were incubated for 4 days and assayed for DNA synthesis. The lymphocyte mitogenic responses to suboptimal, buy not optimal, doses of conA were significantly enhanced by the presence of PPD-activated monocytes from M bovis-sensitized cattle. Treatment nonsensitized cattle with PPD did not result in any significant enhancement of conA-induced lymphocyte mitogenic responses at any dose of conA tested.
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Induction of blastogenesis in bovine peripheral blood lymphocytes by oxidation with sodium periodate.
Suitable treatment and culture conditions are defined for the induction of blast transformation in bovine peripheral blood lymphocytes by oxidation with sodium metaperiodate (NaIO4). Stimulation with NaIO4 required slight modification of techniques used routinely for activation of lymphocytes in vitro with lectins and antigens. Gradient-separated mononuclear leukocytes responded with maximal [3H]TdR incorporation after oxidation with 0.50 to 1.0 mM NaIO4 for 30 minutes at 25 C. Oxidized cells cultured at 1 to 2 X 10(6)/ml responded better than cells cultured at any other concentration, when compared with untreated cells. Blastogenesis in response to oxidation reached its maximum rate within 48 hours of treatment, after which it declined rapidly. Partial removal of glass wool-adherent cells reduced periodate-triggered blastogenesis by 95%, but did not significantly affect activation with phytohemagglutinin, concanavalin A, pokeweed mitogen, or purified protein derivative. Reintroduction of macrophages restored responses to their precolumn level. Oxidation with NaIO4 provided a simple, rapid means of inducing blastogenesis in bovine lymphocytes. Manipulation of the well-defined triggering conditions may help to explain the mechanisms involved in lymphocyte activation.
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[Responses to lymphocyte immunostimulation in Mycobacterium infections in cattle].
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Animals↗
Enhancement of lymphocyte blastogenic and delayed hypersensitivity skin responses by indomethacin.
Incubation of Mycobacterium bovis-sensitized bovine peripheral blood lymphocytes with concanavalin A or M. bovis purified protein derivative and indomethacin caused a consistent, statistically significant increase in [3H] thymidine uptake as compared to cultures without indomethacin. The kinetics of the response showed that indomethacin must be added to lymphocyte cultures within hours after mitogen or antigen addition for enhancement of [3H]thymidine uptake to occur. Lymphocyte blastogenic responses to purified protein derivative were enhanced in both normal and M. bovis-sensitized lymphocyte cultures. However, enhancement of sensitized lymphocyte responses was significantly (P less than 0.01) greater than that in normal animals. Additionally, indomethacin treatment of M. bovis-sensitized guinea pigs singnificantly augmented delayed skin reactions to tuberculin. Delayed hypersensitive skin reactions were only enhanced when indomethacin was administered simultaneously with tuberculin.
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Brucella antigen preparations for in vitro lymphocyte immunostimulation assays in bovine brucellosis.
Three Brucella antigen preparations, Brucella abortus soluble antigen, B. abortus strain 45/20 enriched protein antigen, and B. melitensis enriched protein antigen, were compared in terms of their ability to induce specific in vitro lymphocyte immunostimulation responses. Lymphocytes were prepared from peripheral blood of cattle with different exposure experiences to B. abortus organisms. Lymphocytes were processed by the Ficoll-diatrizoate technique, and results were assayed for [3H]tymidine incorporation into DNA by liquid scintillation spectrometry. The three Brucella antigen preparations were compared both at the optimal concentrations of protein and on an equal-dry-weight basis. The results were evaluated in terms of specific lymphocyte immunostimulation responses induced by each preparation and the degree of correlation with infection. B. abortus soluble antigen-induced lymphocyte immunostimulation response correlated best with infection status followed by B. abortus 45/20 and B. melitensis enriched protein antigens. The implications of these findings are discussed and a hypothesis is proposed.
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In vitro stimulation of bovine peripheral blood lymphocytes: comparison of round- and flat-bottom microtiter plates for detection of tuberculin hypersensitivity.
Lymphocytes from Mycobacterium bovis-sensitized and normal cattle were cultured in round- and/or flat-bottom microtiter plates and stimulated with M. bovis purified protein derivative (PPD) tuberculin. Blastogenic responses of lymphocytes from M. bovis-sensitized cattle to PPD cultured in round-bottom plates were significantly greater than those of lymphocytes cultured in flat-bottom microtiter plates. Normal lymphocytes of nonsensitized cattle were not stimulated by PPD in either round- or flat-bottom microtiter plates. Kinetics of lymphocyte responses in round-bottom plates are presented.
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Whole-blood lymphocyte stimulation assay for measurement of cell-mediated immune responses in bovine brucellosis.
A study was conducted to develop an in vitro whole-blood lymphocyte stimulation assay for measurement of cell-mediated immune response in bovine brucellosis. A soluble antigen (BASA) prepared from killed cells of Brucella abortus 1119-3 was used. Cattle infected with B. abortus field strains, B. abortus 19 calfhood- and adult-vaccinated cattle, and nonexposed cattle were tested. Blood was diluted 10-fold in RPMI-1640 medium (without added serum) and cultured with BASA (at a concentration of 2.2 microgram per culture) at varying times of incubation. Results were assayed for [3H]thymidine incorporation into deoxyribonucleic acid. A 6-day period was found to be optimal for incubating blood cultures to achieve maximum specific lymphocyte stimulation. Serological tests and bacteriological isolation attempts were conducted simultaneously with lymphocyte stimulation tests, and there was a significant correlation between cell-mediated immune response and bacteriological findings. There was a significant correlation between cell-mediated immune response and the level of serum antibodies on a group basis, but there was little correlation between the two systems on individual infected animals. Among vaccinated animals there was little or no correlation between cell-mediated immune and humoral responses. The whole-blood assay was found to be simple, fast, sensitive, and reproducible.
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Comparison of sensitivity and specificity of purified lymphocyte and whole-blood in vitro lymphocyte stimulation assays in detection of Brucella abortus infection in cattle.
A study was conducted to compare the sensitivity and specificity of purified lymphocyte and whole-blood in vitro lymphocyte stimulation assays in detection of Brucella abortus infection in cattle. Cattle used were infected with B. abortus field strains or strain 19. Peripheral blood was collected, and lymphocytes for the technique. The blood for the whole-blood lymphocyte stimulation assay was diluted 10-fold with RPMI 1640 medium (without additional serum supplement) and cultured. The two tests were run simultaneously, and B. abortus soluble antigen or concanavalin A was added to the cultures. The cultures were incubated for 6 days and assayed for [3H] thymidine incorporation into their DNA. Generally, cultures of the purified lymphocyte stimulation assay had higher counts per minute than those of the whole-blood lymphocyte stimulation assay, but the stimulation ratios for the two tests were comparable. The two assays were comparable in terms of their sensitivity and specificity as applied to detection of brucella infection in cattle.
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Utilization of a specific in vitro lymphocyte immunostimulation assay as an aid in detection of brucella-infected cattle not detected by serological tests.
Studies using the in vitro lymphocyte stimulation test (LST) were conducted with cattle in a dairy herd with a high percentage of reactors to several serological tests for brucellosis. Lymphocytes were prepared from peripheral bovine blood by the Ficoll-diatrizoate technique. Lymphocytes were cultured using microtitration culture plates. Brucella abortus soluble antigen, at a concentration of 4.4 microgram/culture, was added to the appropriate wells of microtitration culture plates and incubated for 6 days. The lymphocyte stimulation responses were measured by assaying for [3H]thymidine incorporation into DNA. Seroagglutination tests were conducted simultaneously with the LST, and tissues were collected after slaughter of the cattle for bacteriological culture to isolate B. abortus. All 21 animals studied were serologically negative for anti-brucella antibodies. Two of the 21 animals were classified as infected with Brucella by the LST, and B. abortus biotype 1 was isolated from tissues of these same two animals. The LST exhibited significant sensitivity and specificity in this study, and more observations of this nature might strengthen the application of this assay as an aid in the diagnosis of brucellosis.
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