Application of indomethacin as a potentiator of lymphocyte blastogenesis in Brucella abortus exposed cattle.

Lymphocytes from Brucella abortus field strain infected, strain 19 vaccinated, non-exposed and field strain infected, but immunologically unresponsive cattle were incubated with B. abortus antigen and indomethacin. There were significant increases (P less than 0.005) in the blastogenic responses, as measured by [3H] thymidine uptake, in cultures with indomethacin as compared to cultures without indomethacin. Lymphocyte blastogenic responses to B. abortus antigen were potentiated by indomethacin in both B. abortus exposed and non-exposed cultures. However, potentiation of sensitized lymphocyte blastogenic responses by indomethacin was significantly greater (P less than 0.005) than that in non-exposed lymphocytes. Additionally, indomethacin significantly potentiated Brucella-induced lymphocyte blastogenic responses in lymphocytes from anergic cattle.

Levamisole potentiation of antigen specific lymphocyte blastogenic response in Brucella abortus exposed but nonresponsive cattle.

Incubation of Brucella abortus (field strain) infected and strain 19 vaccinated bovine peripheral blood lymphocytes with B. abortus antigen and levamisole caused a consistently significant increase in [3H] thymidine uptake when compared to cultures without levamisole. Levamisole did not potentiate B. abortus-induced blastogenic response of lymphocytes from non-exposed cattle. A dose response study showed that 10 micrograms/culture induced maximum potentiation of B. abortus-induced lymphocyte stimulation. Using the 10 micrograms/well concentration of levamisole, further studies were conducted to determine the net potentiation of the blastogenic responses in lymphocytes from B. abortus (field strain) infected cattle. B. abortus strain 19 vaccinated but nonresponsive and non-exposed cattle. Levamisole significantly potentiated the B. abortus-induced lymphocyte blastogenesis in lymphocytes from unresponsive cattle.

Development of a migration inhibitory factor assay under agarose of bovine mononuclear leukocytes, using an antigen of Brucella abortus.

A study was conducted to develop a migration inhibitory factor assay under agarose of bovine mononuclear leukocytes, with an antigen of Brucella abortus. Different concentrations of mononuclear leukocytes were prepared by the Ficoll-Hypaque technique from the blood of nonvaccinated calves and from calves previously vaccinated with strain 19. Concentrations of 0.5, 1, 2, 3, 4, and 5 x 10(6) leukocytes were suspended in RPMI-1640 medium and various dilutions (20, 10, 1, and 0.1 microgram) of B abortus-soluble antigen, dispensed in triplicate wells cut in 1% agarose containing minimal essential medium and 10% bovine fetal serum. These agarose plates were incubated for 4-, 8-, 12-, 16-, 20-, and 24-hour periods and then were fixed; leukocytes were stained with Wright's stain. Migration distances were measured, and statistical analyses of the data revealed a concentration of 2 x 10(6) cells/well and an antigen concentration of 10 microgram/well. An incubation period of 20 hours was optimal for the assay.

Studies on in vitro lymphocyte stimulation assay in cattle naturally infected with Brucella abortus and in cattle vaccinated with strain 19.

Cell-mediated immune responses of cattle in infected herds vaccinated with Brucella abortus, strain 19, and of nonexposed controls were studied by an in vitro lymphocyte stimulation test (LST). Brucella abortus soluble antigen was used as a specific stimulator of lymphocytes. Lymphocyte suspensions were prepared from peripheral blood of cattle by the Ficoll-diatrizoate technique. Cultures were labeled with [3H]thymidine and were assayed for [3H]thymidine incorporation into DNA by liquid scintillation spectrometry. Seroagglutination tests and cultures from Brucella were conducted simultaneously with LST. Brucella abortus soluble antigen induced significantly higher (P less than or equal to 0.005) lymphocyte stimulation responses in lymphocytes from cattle infected with B abortus than in lymphocytes from cattle vaccinated with strain 19 or from nonexposed controls. There was a significant P = agreement in results of LST and seroagglutination tests in infected cattle. Among seropositive-vaccinated cattle and controls, the LST was negative.

Investigation of Salmonella infection in goats fed corn silage grown on land fertilized with sewage sludge.

A total of 36 goats were fed for 17 months with corn silage grown on land fertilized with human sewage sludge. These animals were investigated for salmonella infections. Salmonellae were not detected in cultures of fecal or silage samples. No significant agglutination titers were detected in goat sera examined. Salmonella newport C2 was isolated from the sludge used as fertilizer on the cornfields. The public health aspects of the findings are discussed as they relate to the increasing use of sewage sludge for agricultural fertilizers, as well as to the resultant effects on human food and livestock feed.

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.

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.

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.

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.

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.

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.

Cell-mediated immune responses in swine from a herd infected with Brucella suis.

Cell-mediated immune (CMI) responses in swine naturally infected with Brucella suis biotype 3, swine suckling an infected sow and Brucella-noninfected swine were studied by an in vitro lymphocyte transformation procedure. The antigen used was a soluble antigen prepared from killed cells of B suis biotype 3. Lymphocytes were prepared from peripheral swine blood by the Ficoll-Hypaque technique, suspended in RPMI-1640 medium (1.5 X 10(6) lymphocytes/ml), cultured with B suis-soluble antigen or concanconcanavalin A, and incubated for 6 days. Sixteen hours prior to termination of incubation, cultures were labeled with 1 muCi of [3H]thymidine and, after harvesting, were assayed for [3H]thymidine incorporation into DNA by liquid scintillation spectrometry. Agglutination tests were conducted on sera collected simultaneously with samples for lymphocyte-stimulation tests. The B suis-soluble antigen elicited specific stimulation in lymphocytes from infected pigs. On a group basis, there was high correlation between the amount of serum antibodies and specific lymphocyte stimulation, but on an individual animal basis, there was little correlation of the results of both systems in infected swine. There was high correlation between recovery of Brucella from the tissues of animals and the degree of CMI response. Suckling pigs from an infected sow did not develop CMI responses, as measured by our system.

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.

Specific lymphocyte stimulation in cattle naturally infected with strains of Brucella abortus and cattle vaccinated with Brucella abortus strain 19.

Cell-mediated immune responses in cattle naturally infected with strains of Brucella abortus and in cattle vaccinated with B abortus strain 19 during calfhood were studied by an in vitro lymphocyte-stimulation procedure. Lymphocytes were prepared from peripheral bovine blood by the Ficoll-diatrizoate technique, suspended in RPMI-1640 medium (1.5 X 10(6) lymphocytes/ml), cultured with B abortus-soluble antigen or phytohemagglutinin, and incubated for 6 days. Sixteen hours prior to termination of incubation, cultures were labeled with 1 muCi of [3H]thymidine (3HdT) and, after harvesting, assayed for 3HdT incorporation into DNA by liquid scintillation spectrometry. Lymphocytes from cattle with bacteriologically confirmed isolation of B abortus underwent a significantly higher lymphocyte stimulation with B abortus-soluble antigen than did cattle vaccinated with B abortus strain 19 during calfhood (P less than 0.005). Standard seroagglutination tests were conducted simultaneously with lymphocyte-stimulation tests, but there was no apparent correlation between levels of humoral antibodies and the cell-mediated immune responses as measured by in vitro specific lymphocyte stimulation.

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.

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.

In vitro stimulation of bovine peripheral blood lymphocytes: effect of short-term storage of blood prior to lymphocyte culture.

Storage of peripheral blood from Mycobacterium bovis-sensitized cattle from 1 to 48 hours at 4, 22, and 37 C was shown not to alter markedly the lymphocyte blastogenic response to M bovis-purified protein derivative. Concanavalin A-induced lymphocyte mitogenic responses were unaffected by storage of blood for 1, 24, or 48 hours at 22 C and 37 C; however, storage of blood for 48 hours at 4 C significantly lowered (P less than 0.05) mitogenic responses to concanavalin A, as compared with responses to blood kept at 22 C. Mononuclear cell recovery from stored blood at all temperatures was markedly less than that from freshly drawn blood samples. Cell recoveries were most affected by storage of blood at 4 C and 37 C.