Delayed-type skin hypersensitivity and in vitro lymphocyte immunostimulation responses of swine following inoculation with Mycobacterium avium cell walls and a mycobacterial immunopotentiating glycolipid

Miniature swine (n = 5 per group) were inoculated intradermally with mineral oil-in-water emulsions containing either 150 μg of mycobacterial immunopotentiating glyco-lipid P3 (EP3), 150 μg of lyophilized Mycobacterium avium (serotype 8) cell walls (E-MaCW), or 150 μg P3 and 150 μg M. avium cell walls (EP3-MaCW). Swine vaccinated with E-MaCW and EP3-MaCW developed antigen-sensitive lymphocytes detectable with delayed-type hypersensitivity (DTH) skin tests and lymphocyte transformation assay. Swine injected with EP3 were not sensitized. In general EP3-MaCW evoked a more pronounced in vivo DTH tuberculin skin test and in vitro lymphocyte transformation responses than E-MaCW. Time-course studies indicated a more persistent response in swine injected with EP3-MaCW than in those given E-MaCW. Commercial type Yorkshire swine (n = 5) inoculated intradermally with EP3-MaCW developed cell-mediated immune (CMI) responses to avian tuberculin detectable in vivo with delayed-type skin hypersensitivity and in vitro with lymphocyte immunostimulation responses.     

Cell-mediated and humoral immune responses of cattle to Brucella abortus, Mycobacterium bovis, and tetanus toxoid: evaluation of immunization and assay techniques.

A concentration of 2.5 X 10(-5) M 2-mercaptoethanol (2-ME) added to the medium in lymphocyte blastogenesis assays increased both the uptake of [3H]thymidine in unstimulated lymphocyte cultures and the probability of detecting antigen-sensitized cattle. The use of 2-ME did not cause lymphocytes from unsensitized cattle to react positively in blastogenesis assays. A crude brucella lysate prepared from Brucella abortus strain 19 was compared with a well-characterized brucella protein allergen prepared from B melitensis and was found to be equally suitable for use in blastogenesis assays. Cell-mediated immunity was produced most effectively in 4-month-old calves by tetanus toxoid, then by Mycobacterium bovis, and least effectively by B abortus.     

The effect of repeated tuberculin skin testing of cattle on immune responses and disease following experimental infection with Mycobacterium bovis

The comparative intradermal skin test, in which a delayed type hypersensitivity (DTH) response to purified protein derivative of tuberculin (PPD) from Mycobacterium bovis and M. avium is assessed and compared, may be used repeatedly on non-infected animals on farms where bovine tuberculosis (TB) has occurred. A skin test is known to affect subsequent skin tests in infected animals. The reported study was to determine whether repeated skin testing prior to infection with M. bovis might affect the development of the comparative skin test and IFNgamma response subsequent to exposure to virulent M. bovis. The comparative intradermal skin test was applied to one group of six calves five times at 8-week intervals. These and six control calves were subsequently inoculated intratracheally with a dose of M. bovis that produced mild disease. The development of the DTH reaction, IFNgamma, IL-10 and proliferative responses were compared in the two groups of animals. No differences in IFNgamma, IL-10 and proliferative responses were seen between the two groups of calves prior to challenge. After infection with M. bovis no differences in the development of the DTH and IFNgamma responses to PPD were noted as a consequence of the repeated skin testing prior to challenge. No differences between the groups were evident when ESAT-6 was used as antigen and IFNgamma was assayed, although two animals that responded to PPD did not respond with ESAT-6. However, there did appear to be subtle effects of repeated skin testing on the immune response post-challenge that did not affect the diagnostic tests. After challenge control animals showed greater proliferative responses than animals given repeated skin tests prior to challenge, indicating that the procedure did have consequences for immune responses following infection. In both groups a marked reduction in the intensity of the skin test and in the number of animals that would be recognized as reactors was evident when animals were tested 15 weeks post-infection compared to their responses 8 weeks earlier that could have consequences for diagnosis of TB. An antibody response was not evident as a result of repeat skin testing prior to infection but was seen in both groups of calves following skin testing performed 7 weeks after infection.     

Specific antibody responses to Mycobacterium bovis in infected cattle analysed with six mycobacterial antigens in enzyme-linked immunosorbent assays

A total of 23 (15.3 per cent) of 150 cattle infected with Mycobacterium bovis and which had never been tuberculin tested showed specific antibody responses to M bovis. Their sera may be important keys to the identification of unique M bovis antigens for use in specific serodiagnostic tests. Assessment of specific and non-specific responses was done by screening sera in six indirect anti-IgG enzyme-linked immunosorbent assays using whole cell sonicates of M bovis and five members of the Mycobacterium avium-intracellulare-scrofulaceum complex as respective antigens. Sera from 16 infected cattle that had been tuberculin tested positive and nine uninfected cattle (never tuberculin tested) were also assayed for specific and non-specific responses. Three other findings emerged. First, 43 of the 150 infected animals (28.7 per cent) showed no antibody responses to any of the mycobacterial antigens used. Secondly, the cattle showing the highest antibody levels were associated with the greatest cross reactivity. Lastly, the results indicated that tuberculin injections may increase antibody responses to shared, rather than specific, M bovis antigens in infected cattle.     

Experimental Mycobacterium bovis infection of cattle: effect of dose of M. bovis and pregnancy on immune responses and distribution of lesions

Groups of 18-month-old cattle were inoculated intratracheally with 5 x 10(5) colony forming units (high dose) or 500 colony forming units (low dose) of Mycobacterium bovis to determine an appropriate dose to induce lesions similar to those seen in the natural disease. An additional group of 21-28 weeks pregnant cattle were inoculated with the high dose of M. bovis to determine if pregnancy increased the susceptibility of cattle to M. bovis infection. By 23-24 weeks after challenge, the high dose of M. bovis had induced extensive lung lesions, and tuberculous lesions were observed in the lymph nodes of the head, neck, and thoracic and abdominal cavities. In contrast, the low dose of M. bovis induced predominantly small lesions (< 1 cm diameter) which were localised to the lungs and pulmonary lymph nodes. The lesions induced by the low dose were similar to those seen in the natural disease in cattle. The majority of the high dose group cattle produced strong antibody responses to M. bovis culture filtrate, while only one low dose animal produced a detectable response. All of the M. bovis-inoculated cattle produced strong cellular immune responses to bovine PPD (skin test and interferon-gamma responses). Pregnancy did not appear to affect the susceptibility to M. bovis infection, and immune responses of the cattle in this group at the end of the study were similar to those in the high dose non-pregnant group. However, from the first test after calving, the interferon-gamma responses of peripheral blood cultures to bovine PPD were low compared with the responses prior to calving.     

Experimental Mycobacterium bovis infection of cattle: Effect of dose of M. bovis and pregnancy on immune responses and distribution of lesions

Groups of l8-month-old cattle were inoculated intratracheally with 5 X 10⁵ colony forming units (high dose) or 500 colony forming units (low dose) of Mycobacterium bovis to determine an appropriate dose to induce lesions similar to those seen in the natural disease. An additional group of 21–28 weeks pregnant cattle were inoculated with the high dose of M. bovis to determine if pregnancy increased the susceptibility of cattle to M. bovis infection. By 23–24 weeks after challenge, the high dose of M. bovis had induced extensive lung lesions, and tuberculous lesions were observed in the lymph nodes of the head, neck, and thoracic and abdominal cavities. In contrast, the low dose of M. bovis induced predominantly small lesions (< 1 cm diameter) which were localised to the lungs and pulmonary lymph nodes. The lesions induced by the low dose were similar to those seen in the natural disease in cattle. The majority of the high dose group cattle produced strong antibody responses to M. bovis culture filtrate, while only one low dose animal produced a detectable response. All of the M. bovis-inoculated cattle produced strong cellular immune responses to bovine PPD (skin test and interferon-γ responses). Pregnancy did not appear to affect the susceptibility to M. bovis infection, and immune responses of the cattle in this group at the end of the study were similar to those in the high dose non-pregnant group. However, from the first test after calving, the interferon-y responses of peripheral blood cultures to bovine PPD were low compared with the responses prior to calving.     

Development of a whole blood lymphocyte stimulation assay for detecting hypersensitivity to PPD in cattle experimentally infected with Mycobacterium bovis

A whole blood lymphocyte stimulation assay utilizing the uptake of tritiated thymidine was developed for the detection of Mycobacterium bovis sensitivity in cattle. Results on eight M. bovis infected animals (six to ten weeks after infection) and eight control animals show that satisfactory lymphocyte stimulation can be obtained using heparinized whole blood diluted 10-fold in tissue culture medium and cultured with purified protein derivative (PPD) for three to seven days. Infected animals exhibited significantly greater stimulation when cultured with PPD than did control animals.     

Systemic and local immune responses of gnotobiotic calves to respiratory infection with Mycoplasma bovis

Gnotobiotic calves were inoculated by the intratracheal route with Mycoplasma bovis and the specific antibody response in sera and tracheo-bronchial washings examined by radioimmunoassay. In sera an IgM response which reached a peak two weeks post infection was followed by IgG1 and IgG2 antibody responses. Low levels of IgA antibody were detected in sera three and four weeks after infection. The predominant antibody in tracheo-bronchial washings 2 weeks after infection was IgA. Four weeks after infection IgG1 antibody predominated, but IgG2 and IgA antibodies were also present. Cells containing Ig were present in the cellular accumulations around the necrotic zones produced by M. bovis in the lung parenchyma two and four weeks after infection. IgG1 containing cells predominated in these cellular infiltrates. IgG2 producing cells were the next in frequency. It is concluded that the lung lesion caused by M. bovis is partly due to the host's immune response, presumably contributing to the control of the infection, and that the cells infiltrating the lung are a major source of the local and systemic IgG antibody that is detected after infection. IgA staining cells were observed in the submucosa of tissues from nasal cavity and trachea. These cells are probably the source of IgA in tracheo-bronchial washings and sera since IgA-producing cells were not a predominant component of the lesion in the lung parenchyma.     

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.     

Kinetics of detection of blastogenic responses of neonatal calves inoculated in utero with tetanus toxoid, killed Mycobacterium bovis, and killed Brucella abortus.

Nine pregnant cows were laparotomized and their fetuses were immunized with tetanus toxoid, killed Brucella abortus, and killed Mycobacterium bovis. Blastogenesis assays and total leukocyte and differential counts were done when the calves were 1, 2, 3, 7, 14, 21, 28, and 60 days of age. Initial blastogenesis responses to antigens, phytohemagglutinin, and concanavalin A were not positive as frequently as were the responses obtained when the calves were 2 to 3 weeks of age. The probability of obtaining a positive response to an antigen was positively correlated with the magnitude of the response, as determined by delayed-type hypersensitivity skin reactions. Leukocyte and differential WBC counts in immunized calves were similar to those of unimmunized calves. The mean leukocyte count for the immunized calves remained near 16,000 cells/mm3; blood obtained in the first few days after birth contained a greater number of neutrophils than lymphocytes, whereas lymphocyte-to-neutrophil ratio gradually approached those of adult cattle, in which lymphocytes predominate.     

Immune responses of cattle following vaccination with living and non-living Babesia bovis antigens

Twenty-four yearling Hereford (Bos taurus) cattle were vaccinated against Babesia bovis using either live parasites or non-living antigens obtained from the supernatant of in vitro cultures. A single dose of live parasites was given subcutaneously, while the non-living supernatant antigen (NLSA) was combined with saponin and 2 doses given, 2 weeks apart. Following vaccination with live parasites, serum antibodies remained at high levels for 6 months, but the lymphocyte transformation response was low and lasted only 10-18 days. In contrast, NLSA vaccination was followed, after 21-28 days, by a peak of serum antibodies which then slowly declined. The lymphocyte transformation response in these animals was much higher and persisted for 6 months. Following heterologous challenge all unvaccinated cattle had severe reactions and required treatment to prevent death. Cattle vaccinated with live parasites had mild reactions with only 1 of the 12 requiring treatment. Cattle vaccinated with NLSA were only partially protected and 6 of the 12 required treatment.     

Cell mediated and humoral immune responses of white-tailed deer experimentally infected with Mycobacterium bovis

The objective of this study was to improve the understanding of immune responses of whitetailed deer (Odocoileus virginianus) infected with Mycobacterium bovis. Ten mature, female, white-tailed deer were inoculated by intratonsilar instillation of 2 x 10(3)or 2 x 10(5)colony-forming units of M. bovis. Lymphocyte proliferation and humoral response to M. bovis PPD and the M. bovis protein, MPB70 were measured. Deer were tested for exposure to M. bovis by the comparative cervical skin test. Biopsy specimens of skin test sites were examined microscopically and immunohistochemically. The comparative cervical skin test correctly identified all M. bovis -inoculated deer as exposed to M. bovis. Lymphocyte proliferative responses to MPB70 were more consistent than responses to M. bovisPPD in M. bovis -inoculated deer. Antibody responses were more prominent in deer with disseminated disease than in deer with localised disease. The cellular components of delayed-type hypersensitivity reactions at skin test sites were similar to tuberculin reactions in other species. T lymphocytes of the gamma/delta phenotype were seen in increased numbers in M. bovisPPD injection sites.     

Intravenous johnin and tuberculin tests in cattle vaccinated with Mycobacterium paratuberculosis cells and subsequently inoculated with Mycobacterium bovis.

Calves at 30 days of age were vaccinated with a killed whole-cell Mycobacterium paratuberculosis vaccine. Four months later, these calves were inoculated with Mycobacterium bovis. The intravenous tuberculin and johnin tests were applied both before and after inoculation. The results of the hematologic investigation had extremes at both high and low values and were too unsuitable for statistical analysis. The intravenous tuberculin test is considered unsuitable for diagnosis of bovine tuberculosis in cattle vaccinated against paratuberculosis.     

Immune responses associated with progression and control of infection in calves experimentally challenged with Mycobacterium avium subsp. paratuberculosis

Cells expressing CD4, CD8, major histocompatibility complex (MHC) Class II, and macrophage biomarkers in lungs of chickens were quantified by measuring total area of antigen expressed using imageJ, a software program developed at the National Institutes of Health and available at no cost. The procedures reported here were rapid, and reproducible. Total area of antigen expressed had positive correlation with manual counts of cells expressing CD4 and CD8 biomarkers after inoculation with serotype 1 Marek's disease virus (MDV) vaccines. Visual inspection and overlays prepared from outlines of cells counted by imageJ confirmed agreement between antigen expression and area measured. Total area measured was not dependent on time of image acquisition from randomly selected fields from the same slides. Total area values were not computer specific, but acquisition of the original images required standardization of microscope used and camera setup. All steps in the process from sample collection through sectioning, staining, and image acquisition must be standardized as much as possible. Chickens infected with a very virulent+ (vv(+)) isolate of MDV (648A) had increased CD4, CD8, MHC Class II, and macrophage biomarker expression compared to noninfected control chickens at 10 days post infection, but variable responses depending on the specific biomarker measured at 3 and 5 days post infection. The procedure described here is faster and more reproducible than manual counting in cases (CD4 and CD8) where the number of positive cells is low enough for manual counts. Manual counting is not possible with MHC Class II and macrophage antigens nor when CD4(+) cells are present in large numbers following proliferation to tumors, thus subjective systems are used for scoring in these conditions. Using imageJ as described eliminates the need for subjective and less reproducible methods for measuring expression of these antigens.     

Cell-mediated immune responses to sporozoites and macroschizonts of Theileria annulata.

The nature of cell-mediated immune (CMI) responses was studied in cross-bred bovine calves, immunised by attenuated and allogeneic macroschizonts of Theileria annulata. The CMI responses were also investigated in calves, destined to survive or die of tropical theileriosis (Theileria annulata) induced by a virulent dose of sporozoites or macroschizont-infected lymphoblasts. Calves suffering fatal theileriosis showed poor CMI response. Microcytotoxicity assay revealed an enhanced population of specific cytotoxic cells amongst the peripheral blood lymphocytes (PBL) of calves resolving the infection successfully. The E rosette assay showed proliferation of T cells and the assay for macrophage migration inhibition factor (MIF) demonstrated antigen sensitised cells in the PBL. Calves, immunised by allogeneic and attenuated macroschizont-infected lymphoblasts or those recovering from virulent macroschizont-induced infection, showed protective CMI responses with patterns similar to those appearing after non-fatal sporozoite infection.