Differential Cell Composition and Cytokine Expression Within Lymph Node Granulomas from BCG-Vaccinated and Non-vaccinated Cattle Experimentally Infected with Mycobacterium bovis.

Cattle vaccination against bovine tuberculosis (bTB) has been proposed as a supplementary method to help control the incidences of this disease. Bacillus Calmette-Guérin (BCG) is currently the only viable candidate vaccine for immunization of cattle against bTB, caused by Mycobacterium bovis (M. bovis). In an attempt to characterize the differences in the immune response following M. bovis infection between BCG-vaccinated and non-vaccinated animals, a combination of gross pathology, histopathology and immunohistochemical (IHC) analyses was used. BCG vaccination was found to significantly reduce the number of gross and microscopic lesions present within the lungs and lymph nodes. Additionally, the microscopically visible bacterial load of stages III and IV granulomas was reduced. IHC using cell surface markers revealed the number of CD68+ (macrophages), CD3+ (T lymphocytes) and WC1+ cells (γδ T cells) to be significantly reduced in lymph node granulomas of BCG-vaccinated animals, when compared to non-vaccinated animals. B lymphocytes (CD79a+) were significantly increased in BCG-vaccinated cattle for granulomas at stages II, III and IV. IHC staining for iNOS showed a higher expression in granulomas from BCG-vaccinated animals compared to non-vaccinated animals for all stages, being statistically significant in stages I and IV. TGFß expression decreased alongside the granuloma development in non-vaccinated animals, whereas BCG-vaccinated animals showed a slight increase alongside lesion progression. IHC analysis of the cytokines IFN-γ and TNF-α demonstrated significantly increased expression within the lymph node granulomas of BCG-vaccinated cattle. This is suggestive of a protective role for IFN-γ and TNF-α in response to M. bovis infection. Findings shown in this study suggest that the use of BCG vaccine can reduce the number and severity of lesions, induce a different phenotypic response and increase the local expression of key cytokines related to protection.

The effect of tuberculin testing on the development of cell-mediated immune responses during Mycobacterium bovis infection.

Protection against tuberculosis (TB) is associated with Th1-type cell-mediated immunity (CMI). Whilst the intradermal injection of partially purified derivatives of tuberculin (PPD) represents the classic test assessing the delayed type hypersensitivity (DTH) response used in both humans and cattle for diagnosing TB, it has been suggested that the test may modulate host CMI responses. To investigate the kinetics of the development of the DTH response and its subsequent effect on CMI responses, groups of 6-month old calves were inoculated intranasally with 8 x 10(4) cfu of Mycobacterium bovis, subjected to the comparative intradermal tuberculin test (TT) using bovine and avian PPD (PPD-B, PPD-A) at various time intervals post-infection, and immune responses compared. These included DTH, lymphocyte proliferation, IgG production, and synthesis of the cytokines: IFNgamma, IL-10, IL-4, IL-6, and IL-13. All animals were subjected to post-mortem examination. The kinetics of the development of the DTH response assessed in the TT was such that infected cattle could be identified as early as 3 weeks post-infection, which correlated with the detection of an antigen-specific IFNgamma response. Transient increases in plasma-derived IFNgamma as a result of TT during an established TB infection were more pronounced when blood was stimulated with PPD-A compared with PPD-B stimulation. This has the potential to mask diagnosis of infection as a result of the stronger avian-bias if the IFNgamma test is used the week following TT. Disease pathology was not affected by TT. A transient failure to a second TT was observed in 1 of 30 animals and the time (post-infection) at which the TT is administered may be of significance. In serum, IgG responses to PPD-B, which were undetectable prior to TT, were elevated after TT and were most pronounced in cattle that were TT at 6 weeks post-infection. Other cytokines were also affected by the TT; IL-4 mRNA levels increased and IL-6 mRNA levels decreased, whilst PPD-B specific IL-10 protein synthesis was enhanced. These observations may offer the potential for further diagnostic assays that could complement the TT and IFNgamma test.

Production of interleukin-12 as a self-processing 2A polypeptide.

Interleukin-12 (IL-12) is a heterodimeric cytokine composed of two disulfide-linked subunits (p40 and p35) encoded by separate genes. We used the apparent autocleavage property of a 2A peptide from the foot-and-mouth disease virus (FMDV) to express bovine (Bo) IL-12 as a self-processing polypeptide (p402Ap35). We demonstrate that 2A will mediate the cleavage of p402Ap35 into two separate subunits in a manner similar to that observed during the processing of the FMDV polypeptide. Furthermore, this 2A polypeptide encoded a functional heterodimer, which elicited activities associated with IL-12 in other species. We propose that this strategy of self-processing polypeptides may be used in many applications where the coordinated and stoichiometric expression of complex proteins is required.

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.

Contribution of in vivo and ex vivo studies to understanding the role of antigen-presenting cells and T cell subsets in immunity to cattle diseases.

In vivo and ex vivo studies of the immune system in relation to infectious disease that are carried out in the natural target species provide data that are relevant to understanding the biology of the immune cells and immunity to infection. This is particularly the case for diseases that show host specificity. Ex vivo studies that exploit the surgical cannulation of lymphatic ducts have allowed access to natural dendritic cells. Investigations of these cells have revealed the presence of subpopulations that differ in their ability to stimulate T cells and differ in the range of cytokines synthesized. These differences would be forecast to have major effects on the bias and type of immune response that are induced. Studies in vivo of the effect of depleting T-cell populations with monoclonal antibodies (mAbs) have shown how different T-cell populations have differing critical roles for different infectious diseases, and how they may contribute to the immune response and pathology after infection. Here the case is made for how studies in cattle have aided our understanding of immunity to several infections that can be exploited for the rational design of effective vaccination and control strategies.

MicroRNA expression profiling of PPD-B stimulated PBMC from M. bovis-challenged unvaccinated and BCG vaccinated cattle.

There is an urgent need to identify additional diagnostic biomarkers for bovine TB to complement existing read-out systems such as interferon-gamma and for predictive markers of vaccine efficacy to accelerate vaccine development. To evaluate the potential of miRNAs as such biomarkers, we have analysed their expression in bovine PPD stimulated PBMC isolated from unvaccinated and BCG vaccinated cattle before and following Mycobacterium bovis (M. bovis) infection. Using a bovine microRNA microarray, miR-155 was found to show a significant up-regulation in expression in early (week 2) and late (week 11) M. bovis post-infection samples from unvaccinated cattle, while in BCG vaccinated cattle up-regulation was observed only in late post-infection samples. No differential expression of miR-155 was observed in pre-infection samples from unvaccinated and vaccinated cattle. These observations suggest that miR-155 could be exploited as a marker distinguishing vaccinated from infected animals (DIVA). Analysis by TaqMan RT-PCR, verified the up-regulation of miR-155 in unvaccinated cattle post-infection. Significant correlation was found between the degree of pathology and miR-155 induction in the experimentally infected cattle, suggesting miR-155 is a biomarker of disease development and/or severity. Induction of miR155 expression in cattle sourced from farms with confirmed bTB that tested positive in the tuberculin skin or interferon-gamma blood test was found to be significantly higher in cattle presenting with more advanced pathology (defined by the presence of visible TB lesions) compared to infected cattle without visible pathology and thus likely to be of lower infectivity than those with more advanced disease. In conclusion, our data indicate that miR-155 has potential both as a diagnostic and prognostic biomarker that could be used to identify animals with advanced pathology and as a DIVA test read-out. Its role in the immune biology of bovine TB will also be discussed.

Effect of dose and route of immunisation on the immune response induced in cattle by heterologous Bacille Calmette-Guerin priming and recombinant adenoviral vector boosting.

BCG is used experimentally as a vaccine against tuberculosis (TB), induced by Mycobacterium bovis, in cattle (bTB). However, the efficacy of BCG is variable in humans, cattle and guinea pigs. An adenoviral vector expressing Antigen 85A (Ad5Ag85A) has enhanced protection against TB in mice when used in combination with BCG for prime-boost experiments. However, the route of immunisation affects the degree of protection seen. This work examines the immunogenicity of a new vectored vaccine (Ad5-TBF) that expresses Ag85A, Rv0287, Rv0288 and Rv0251c to explore the effects of dose of adenoviral boost and route of inoculation on immunogenicity. We found that 2×10(9) infectious units (iu) delivered intradermally conferred the most consistent and strongest responses of the different regimes tested.

Comparison of the immunogenicity and protection against bovine tuberculosis following immunization by BCG-priming and boosting with adenovirus or protein based vaccines.

There is a requirement for vaccines or vaccination strategies that confer better protection against TB than the current live attenuated Mycobacterium bovis Bacillus Calmette-Guerin (BCG) vaccine for use in cattle. Boosting with recombinant viral vectors expressing mycobacterial proteins, such as Ag85A, has shown a degree of promise as a strategy for improving on the protection afforded by BCG. Experiments in small animal models have indicated that broadening the immune response to include mycobacterial antigens other than Ag85A, such as Rv0288, induced by boosting with Ad5 constructs has a direct effect on the protection afforded against TB. Here, we compared the immunogenicity and protection against challenge with M. bovis afforded by boosting BCG-vaccinated cattle with a human type 5 (Ad5)-based vaccine expressing the mycobacterial antigens Ag85A (Ad5-85A); or Ag85A, Rv0251, Rv0287 and Rv0288 (Ad5-TBF); or with protein TBF emulsified in adjuvant (Adj-TBF). Boosting with TBF broaden the immune response. The kinetics of Ad5-TBF and Adj-TBF were shown to be different, with effector T cell responses from the latter developing more slowly but being more durable than those induced by Ad5-TBF. No increase in protection compared to BCG alone was afforded by Ad5-TBF or Adj-TBF by gross pathology or bacteriology. Using histopathology, as a novel parameter of protection, we show that boosting BCG vaccinated cattle with Ad5-85A induced significantly better protection than BCG alone.

Upregulation of IL-17A, CXCL9 and CXCL10 in early-stage granulomas induced by Mycobacterium bovis in cattle.

To gain further insight into the immunopathogenesis of bovine tuberculosis (bTB), the cytokine and chemokine expression of cattle experimentally infected with Mycobacterium bovis was analysed in TB granulomas, using immunohistochemistry (IHC) and laser capture microdissection (LCM) followed by qPCR. Immunohistochemistry was conducted for cell types using labelling for CD68, CD3, CD4, CD8, WC1 and CD79a and for the cytokines IFN-γ, TNF-α and TGF-ß as well as inducible form of nitric oxide synthase (iNOS). qPCR was conducted for mRNA expression of IFN-γ, TNF-α, TGF-ß, IL-17A, IL-22, IL-2, granzyme A and the chemokines CXCL9 and CXCL10. Early stages of granuloma were primarily comprised of epithelioid MΦs expressing high levels of IFN-γ and iNOS, with significantly upregulated expression of CXCL9 and CXCL10 when compared with control tissue. These chemokines displayed a trend of decreasing mRNA expression as lesion progressed, suggesting a higher level of importance during the early stages of the immune response to mycobacterial infection. IL-22 levels showed a strong trend of decrease through granuloma development, and IL-17A was shown to be upregulated, supporting its investigation as a potential biomarker of bTB. The use of LCM and qPCR may prove especially useful for the study of IL-17A as previous attempts to analyse its expression using IHC and in situ hybridization proved unsuccessful.

Duration of immunity against Mycobacterium bovis following neonatal vaccination with bacillus Calmette-Guérin Danish: significant protection against infection at 12, but not 24, months.

Vaccination of neonatal calves with Mycobacterium bovis bacillus Calmette-Guérin (BCG) induces a significant degree of protection against bovine tuberculosis, caused by infection with virulent M. bovis. In two independent experiments, we assessed the duration of the protective immunity induced in calves by neonatal vaccination with BCG Danish. Protection from disease was assessed at 12 and 24 months postvaccination in cattle challenged via the endotracheal route with M. bovis. We also assessed antigen-specific immune responses to assess their utility as correlates of protection. At 12 months postvaccination, significant reductions in lung and lymph node pathologies were observed compared to nonvaccinated M. bovis-challenged control cattle. At 24 months post-BCG vaccination, there was a reduction in lung and lymph node pathology scores and in bacterial burden. However, when comparing vaccinated and control groups, this did not reach statistical significance. Vaccination induced long-lived antigen (purified protein derivative [PPD])-specific gamma interferon (IFN-γ) release in whole-blood cultures, which remained above baseline levels for more than 20 months (approximately 90 weeks). The number of antigen-specific IFN-γ-secreting central memory T cells present at the time of M. bovis challenge was significantly higher in vaccinated than in control animals at 12 months postvaccination, but not at 24 months. Vaccination of neonatal calves with BCG Danish induced protective immune responses against bovine TB which were maintained for at least 12 months postvaccination. These studies provide data on the immunity induced by BCG vaccination in calves; the results could inform vaccination strategies for the control of bovine TB in United Kingdom cattle herds.

Identification of surrogates and correlates of protection in protective immunity against Mycobacterium bovis infection induced in neonatal calves by vaccination with M. bovis BCG Pasteur and M. bovis BCG Danish.

Vaccination of neonatal calves with Mycobacterium bovis bacillus Calmette-Guérin (BCG) induces a significant degree of protection against infection with virulent M. bovis, the causative agent of bovine tuberculosis (bTB). We compared two strains of BCG, Pasteur and Danish, in order to confirm that the current European human vaccine strain (BCG Danish) induced protective immunity in calves, and we assessed immune responses to determine correlates of protection that could assist future vaccine evaluation in cattle. Both vaccine strains induced antigen (purified protein derivate [PPD])-specific gamma interferon (IFN-γ) in whole-blood cultures. These responses were not significantly different for BCG Pasteur and BCG Danish and peaked at week 2 to 4 postvaccination. Vaccination with either BCG Danish or BCG Pasteur induced significant protection against bTB, with reductions in both lesion score and bacteriological burden evident in both groups of vaccinated calves compared with nonvaccinated control calves. Measurement of IFN-γ-expressing T lymphocytes postvaccination and postchallenge revealed both correlates and surrogates of protective efficacy. The frequency of central memory T lymphocytes present at 12 weeks postvaccination (at the time of M. bovis challenge) correlated significantly with protection. Conversely, the number of IFN-γ-expressing effector T cells present after M. bovis challenge was correlated with disease. These results demonstrate that vaccination of neonatal calves with either BCG Pasteur or BCG Danish induces protective immune responses against TB. In addition, we show that measurement of antigen-specific T lymphocyte populations may provide a reliable means for identifying protective vaccine candidates.

Cytokine expression profiles of bovine lymph nodes: effects of Mycobacterium bovis infection and bacille Calmette-Guérin vaccination.

Cytokine expression in lymph nodes from cattle inoculated intranasally with Mycobacterium bovis was compared to that of non-infected animals using real-time polymerase chain reaction. The effect of M. bovis infection, 4 months post-challenge, was to suppress the expression of anti-inflammatory cytokines interleukin (IL)-4 and IL-10 as well as the pro-inflammatory cytokines tumour necrosis factor (TNF) and IL-6. Expression of interferon (IFN)-gamma and IL-12 was maintained. Animals vaccinated with bacille Calmette-Guérin responded differently to challenge with M. bovis. In particular, no decrease in expression of IL-4 or IL-6 was observed following challenge of vaccinated animals and decreased IFN-gamma was detected. Also, vaccinated animals had higher levels of IL-4 and IL-10 transcripts compared to unvaccinated animals following challenge. These changes in cytokine expression levels led to a significant shift in the IFN-gamma/IL-4 or IFN-gamma/IL-10 ratio within the lymph node following challenge. Challenged animals generally showed a strong Th1 bias that was not seen in animals vaccinated prior to challenge. An inverse correlation between the level of pathology and bacterial load within the lymph node and the expression of IL-4, IL-10 and TNF was also observed. These results suggest that in the lymph nodes of cattle with established tuberculosis and a persisting bacterial infection, maintenance of the pro-inflammatory response in combination with a suppressed anti-inflammatory response may control the infection but contribute to host-induced tissue damage. Vaccination, which reduces the bacterial load and consequently the IFN-gamma response, may result in less suppression of anti-inflammatory cytokines.

Influence of the nature of the antigen on the boosting of responses to mycobacteria in M. bovis-BCG vaccinated cattle.

Mycobacterium bovis is the causative agent of bovine tuberculosis (TB) and of a proportion of human TB. Protection against TB requires Th1 responses and worsening of disease is associated with Th2 responses. To help clarify the nature of the response to mycobacteria, the responses from M. bovis-BCG vaccinated cattle boosted with live mycobacteria (BCG), bacterial soluble antigens (PPD) or PBS were evaluated. The results indicated that macrophages may be the major cell population ingesting and presenting mycobacteria in BCG boosted animals, while B-cells seem able to ingest and present PPD to T-cells in PPD boosted animals.

Exposure to Mycobacterium avium induces low-level protection from Mycobacterium bovis infection but compromises diagnosis of disease in cattle.

We assessed the effect of exposure to Mycobacterium avium on the development of immune responses and the pathogenesis of disease observed following Mycobacterium bovis challenge. A degree of protection against M. bovis was observed in calves which were pre-exposed to M. avium as assessed by the extent of lesions and bacterial load compared to the M. bovis alone group. The immune response following M. bovis challenge in cattle previously inoculated with M. avium was biased towards antigens (PPD) present in M. avium, whereas the response following M. bovis alone was biased towards antigens present in M. bovis, indicating an imprinting of memory to avian antigens on T lymphocytes. A consequence of the memory to M. avium antigens was failure to diagnose M. bovis infection by the skin test or the IFN(gamma) assay in some of the animals which had lesions of tuberculosis at necropsy. The use of M. bovis specific antigens ESAT-6 and CFP-10 increased IFN(gamma) test specificity in animals previously exposed to M. avium but the responses to these antigens were lower than those observed in animals exposed to M. bovis alone. The implication is that responses to M. avium, although providing some immunity, may mask diagnosis of M. bovis infection, even when specific antigens are employed, potentially contributing to disease transmission in the field.

Vaccination of neonatal calves with Mycobacterium bovis BCG induces protection against intranasal challenge with virulent M. bovis.

Vaccination of neonates with Mycobacterium bovis bacillus Calmette-Guerin (BCG) may be a strategy that overcomes reduced vaccine efficacy associated with exposure to environmental mycobacteria in humans and cattle. Preliminary comparisons indicated that 2-week-old calves produced an immune response to vaccination at least as intense as that observed in adults. Subsequently, five gnotobiotic hysterotomy derived calves aged 1 day were inoculated with BCG and 3 months later were challenged intranasally with virulent M. bovis. The number of tissues with lesions and the pathological extent of these lesions was reduced significantly in vaccinates. Furthermore, lesions were evident in the lung or associated chest lymph nodes of four of five controls but none of five vaccinates. BCG vaccination reduced significantly the level of bacterial colonization. However, lesions in the head associated lymph nodes were observed in three of five BCG-vaccinated cattle. Levels of interferon gamma (IFN-gamma) detected by enzyme-linked immunosorbent assay (ELISA) or enzyme-linked immunospot (ELISPOT) in individual vaccinated animals at challenge did not correlate with subsequent resistance and in general immune responses post-challenge were lower in vaccinated calves. Low IL-10 responses were evident but IL-4 was not detected. Responses to ESAT-6 and/or CFP-10 were evident in four of four control calves that had lesions. Two of the BCG vaccinates with lesions did not produce a response to ESAT-6 and CFP-10, indicating that these antigens did not distinguish vaccinated immune animals from vaccinated animals with lesions. Overall, vaccination of neonatal calves with BCG induced significant protection against disease and has potential as a strategy for the reduction of the incidence of bovine tuberculosis.

Effect of late administration of anti-TNF alpha antibodies on a Salmonella infection in the mouse model.

The effect of late administration of anti-TNF alpha antibodies on the course of a Salmonella infection in mice was evaluated. Administration of anti-TNF alpha antiserum as late as day 5 after challenge enhanced a sublethal primary infection with the virulent Salmonella typhimurium C5 in innately resistant (Ityr) A/J mice by preventing the suppression of exponential bacterial growth in the reticuloendothelial system (RES) (plateau phase). When the anti-TNF alpha treatment was started well after the establishment of the plateau (day 7) a prompt relapse of the infection occurred, with the rapid resurgence of bacterial growth in the reticuloendothelial system leading to the death of the animals. In contrast, late administration of the antiserum did not affect the clearance from the tissues of an avirulent temperature-sensitive mutant of S. typhimurium C5 (C5TS). Innately susceptible (Itys) BALB/c mice immunized with the SL3261 aroA live vaccine acquire solid long-lasting protection from oral challenge with the virulent C5 strain, suppressing growth of the challenge in the RES. Administration of anti-TNF alpha antibodies on day 8 of a secondary oral infection with strain C5 abrogated vaccine-induced protection, with a progressive increase of bacterial numbers in the RES leading to the death of the animals. The results indicate that TNF alpha is constantly required for the control of virulent salmonellae in the RES, both in a sublethal primary infection in innately resistant mice and also in a secondary infection in innately susceptible mice immunized with a live vaccine. TNF alpha may not be essential for bacterial clearance of avirulent organisms from the tissues.

Adoptive transfer of immunity to oral challenge with virulent salmonellae in innately susceptible BALB/c mice requires both immune serum and T cells.

The mechanisms of immunity to salmonellae conferred by immunization with live vaccines were studied by adoptive transfer using the mouse-virulent strain Salmonella typhimurium C5 and innately susceptible BALB/c (ltys) mice. This organism cannot establish a sublethal infection in naive BALB/c mice. Animals immunized 2 to 3 months earlier with the S. typhimurium SL3261 aroA live vaccine were used as donors of serum, spleen cells, and mesenteric lymph node cells for naive recipients which were challenged orally with the virulent C5 strain. Simultaneous transfer of both immune serum and immune cells was necessary for protection. Simultaneously depleting the donors of CD4+ and CD8+ T cells by administration of antisera in vivo prior to cell harvesting showed that T cells were necessary for protection. The results demonstrate that both antibody and T cells are required for recall of immunity to oral challenge with virulent salmonellae in innately susceptible mice and suggest that the ability to elicit opsonizing antibody in addition to cell-mediated immunity is important for optimal protection induced by salmonella vaccines.

Role of T cells, TNF alpha and IFN gamma in recall of immunity to oral challenge with virulent salmonellae in mice vaccinated with live attenuated aro- Salmonella vaccines.

The SL3261 Salmonella typhimurium aroA live vaccine strain confers solid protection against oral challenge with virulent salmonellae, immunity persisting long after the vaccine has been cleared from the tissues. BALB/c mice immunized with SL3261 and later subjected to in vivo depletion of both CD4+ and CD8+ T cells had impaired recall of immunity to oral challenge with the virulent S. typhimurium C5, with increased mortality and higher bacterial loads in the reticuloendothelial system (RES). Selective depletion of CD4+ cells alone significantly impaired resistance both 8 and 14 weeks after vaccination as determined by estimation of bacterial numbers in organ homogenates. Depletion of CD8+ cells alone had less effect on immunity when performed at 8 weeks than at 14 weeks after immunization. Administration of anti-IFN gamma or anti-TNF alpha antibodies also impaired recall of immunity, exacerbating a secondary infection in vaccinated mice. Challenge of T cell-depleted immune mice with virulent salmonellae caused hepatosplenomegaly with minute grossly visible focal lesions, and a marked increase in the number and severity of necrotic foci in spleen, liver and lymph nodes. A widespread mononuclear cell infiltrate was present. The histopathology in anti-IFN gamma-treated mice was qualitatively similar to that seen in T-cell depleted mice. In contrast, in the anti-TNF alpha-treated mice splenomegaly was much less than in T cell-depleted mice. Granulomas were absent, no mononuclear infiltration was observed and there was severe necrosis; the lesions appeared similar to or worse than those seen in naïve mice. Surprisingly, IFN gamma was detectable in sera of both controls and T cell-depleted mice on day 8 of the secondary infection, as well as in sera of anti-TNF alpha-treated mice on day 6 of infection. The results indicate that T cells, IFN gamma and TNF alpha are all important in the specific recall of immunity to virulent salmonellae conferred by immunization with live vaccines, with the effect of T cell and IFN gamma depletion (marked macrophage infiltration) being qualitatively very different from that of TNF alpha neutralization (no mononuclear infiltrate or granuloma formation).

Delayed (footpad) hypersensitivity and Arthus reactivity using protein-rich antigens and LPS in mice immunized with live attenuated aroA Salmonella vaccines.

Footpad reactions to protein-rich salmonella extracts and lipopolysaccharide (LPS) were studied in BALB/c mice 2 and 8 months after immunization with the Salmonella typhimurium SL3261 aroA live vaccine. T-cell depletion in vivo and adoptive serum transfer showed that protein-rich antigens induced T-cell dependent delayed hypersensitivity reactions, whereas LPS only elicited Arthus reactions. The footpad reactions to crude protein extracts were not always T-cell mediated, but depended on the nature and the dose of the antigen. Selective depletion of CD4+ T cells alone had a greater effect than depletion of CD8+ T cells alone, but neither was as marked as simultaneous depletion of both CD4+ and CD8+ T cells, which abolished the delayed type hypersensitivity (DTH) response. Crude protein-rich extracts subjected to alkaline hydrolysis (which removes some ester-linked fatty acids and causes disaggregation of LPS resulting in decreased toxicity while conserving O-specificity) still gave positive T-cell dependent reactions, but with reduced T-cell independent reactivity. Purified phenol-water LPS (2.5 micrograms) produced Arthus reactivity which could be confused with DTH. LPS induced positive reactions which still occurred in T-cell depleted mice and were transferable by immune serum. Arthus reactions did not occur when using alkali-treated LPS, which showed reduced complement fixation in vitro when using serum from immunized mice. The results indicate that footpad testing using salmonella antigens containing LPS elicit DTH but can also produce toxic reactions, some of which are T-cell independent and not necessarily a true measure of DTH. Arthus reactivity to LPS can be confused with DTH. Alkaline hydrolysis of the antigens can eliminate non-specific reactogenicity while retaining the ability of the (protein-rich) antigen to elicit a true T-cell dependent footpad response, which requires the participation of both CD4+ and CD8+ T cells.

Investigation of the role of CD8+ T cells in bovine tuberculosis in vivo.

Mycobacterium bovis is the causative agent of bovine tuberculosis (TB), and it has the potential to induce disease in humans. CD8(+) T cells (CD8 cells) have been shown to respond to mycobacterial antigens in humans, cattle, and mice. In mice, CD8 cells have been shown to play a role in protection against mycobacterial infection. To determine the role of CD8 cells in bovine TB in vivo, two groups of calves were infected with the virulent M. bovis strain AF2122/97. After infection, one group was injected with a CD8 cell-depleting monoclonal antibody (MAb), and the other group was injected with an isotype control MAb. Immune responses to mycobacterial antigens were measured weekly in vitro. After 8 weeks, the animals were killed, and postmortem examinations were carried out. In vitro proliferation responses were similar in both calf groups, but in vitro gamma interferon (IFN-gamma) production in 24-h whole-blood cultures was significantly higher in control cattle than in CD8 cell-depleted calves. Postmortem examination showed that calves in both groups had developed comparable TB lesions in the lower respiratory tract and associated lymph nodes. Head lymph node lesion scores, on the other hand, were higher in control calves than in CD8 cell-depleted calves. Furthermore, there was significant correlation between the level of IFN-gamma and the head lymph node lesion score. These experiments indicate that CD8 cells play a role in the immune response to M. bovis in cattle by contributing to the IFN-gamma response. However, CD8 cells may also play a deleterious role by contributing to the immunopathology of bovine TB.