Accidental infection of veterinary personnel with Mycobacterium tuberculosis at necropsy: a case study.

Mycobacterium tuberculosis is the main cause of human tuberculosis. Infection in companion animals is mainly acquired from close contact to a diseased human patient and hence rarely diagnosed in countries with low tuberculosis incidence rates. Therefore the general awareness of the disease might be low. Here we report the potential risk of infection for veterinary personnel with M. tuberculosis during the clinical and pathological examination of a dog with unexpected disseminated tuberculosis. The dog had presented with symptoms of a central nervous system disease; rapid deterioration prevented a complete clinical workup, however. Post-mortem examination revealed systemic mycobacteriosis, and M. tuberculosis was identified by PCR amplification of DNA extracts from paraffin-embedded tissue sections and spoligotyping. Contact investigations among the owners and veterinary personnel using an IFN-γ release assay indicated that the index dog did not infect humans during its lifetime. Serological and IFN-γ release assay results of one of two cats in direct contact with the index dog, however, suggested that transmission of M. tuberculosis might have occurred. Importantly, all three pathologists performing the necropsy on the dog tested positive. Accidental infection was most likely due to inhalation of M. tuberculosis containing aerosols created by using an electric saw to open the brain cavity. As a consequence routine necropsy procedures have been adapted and a disease surveillance program, including tuberculosis, has been initiated. Our results highlight the importance of disease awareness and timely diagnosis of zoonotic infectious agents in optimizing work safety for veterinary personnel.

Isoniazid treatment of Mycobacterium bovis in cattle as a model for human tuberculosis.

Cattle infected with Mycobacterium bovis spoligotype 9 were treated with Isoniazid (INH) from three to 14 weeks post infection, rested for fourweeks to allow INH depletion and then challenged with M. bovis spoligotype 35. Post mortem examination (PME) 35 weeks after the initial infection showed partial protection against infectious challenge following INH-attenuated infection compared with the spoligotype 35 challenge controls. Antigen-specific IFN-gamma responses decreased over time with INH therapy, following a similar pattern to that observed in the treatment of M. tuberculosis infection in humans. Following cessation of therapy, specific IFN-gamma responses increased more strongly in those calves that were visibly lesioned at PME. IFN-gamma responses were also used to identify two antigens, TB10.4 and Acr2, that induced anamnestic responses in INH-treated, re-challenged calves, suggesting a role for both antigens in protective immunity. Specific IL-10 responses were observed in all calves following treatment with INH suggesting a role for IL-10 in the resolution of infection.

1,25-dihydroxyvitamin D3 and development of tuberculosis in cattle.

This report describes the presence and activity of 1,25-dihydroxyvitamin D3 (1,25-D3) in experimental bovine tuberculosis. Animals that went on to develop tuberculous lesions exhibited a rapid transient increase in serum 1,25-D3 within the first 2 weeks following infection with Mycobacterium bovis. 1,25-D3-positive mononuclear cells were later identified in all tuberculous granulomas by immunohistochemical staining of postmortem lymph node tissue. These results suggest a role for 1,25-D3 both at the onset of infection and in the development of the granuloma in these infected animals. Using a monoclonal antibody to the vitamin D receptor (VDR) as a VDR agonist, we confirmed that activation of the vitamin D pathway profoundly depresses antigen-specific, but not mitogenic, bovine peripheral blood T-cell responses (proliferation and gamma interferon production). Investigation of the mechanism of this suppression showed that the VDR antibody modified the expression of CD80 by accessory cells, such that a significant positive correlation between T-cell proliferation and accessory cell CD80 emerged.

Antigen recognition and immunomodulation by gamma delta T cells in bovine tuberculosis.

This report describes the in vitro proliferative responses of peripheral blood gammadelta T cells to defined mycobacterial protein Ags and the immunomodulatory effect of gammadelta T cells in cattle infected with Mycobacterium bovis. gammadelta T cell responses were specific to M. bovis infection because they were detected in cattle either experimentally or naturally infected with M. bovis, but were not present in uninfected controls. Proliferating gammadelta T cell cultures produced enhanced levels of IFN-gamma and TGF-beta, but not IL-2 in response to the more immunodominant mycobacterial AGS: Depletion of gammadelta T cells from PBMC resulted in an increased Ag-specific proliferation in half the animals tested, indicating a suppressive effect of gammadelta T cells upon other (alphabeta) T cell responses. Because gammadelta T cells constitute a major T cell population in the peripheral blood of cattle, the activities of gammadelta T cells described in this report could make a significant contribution to the immune response in bovine tuberculosis.

Distinct response kinetics of gamma interferon and interleukin-4 in bovine tuberculosis.

This study shows that gamma interferon (IFN-gamma) and interleukin-4 (IL-4) cytokine responses are produced by peripheral blood cells in cattle infected with Mycobacterium bovis. The different kinetics of the IFN-gamma and IL-4 responses to bovine tuberculin and to ESAT-6 following experimental intratracheal infection with M. bovis are described. An early increase in IFN-gamma was observed that was maintained throughout the period studied. In contrast, the IL-4 response was delayed and confined to a peak of activity lasting 6 to 8 weeks. Interestingly, an experimental challenge of cattle with a lower dose of M. bovis which did not result in the development of lesions, positive DTH skin test, or substantial IFN-gamma responses nevertheless generated strong specific IL-4 responses. Investigation of naturally infected M. bovis field reactors showed increased IFN-gamma and IL-4 responses compared to uninfected cattle and that both of these cytokines were equally able to differentiate infected from uninfected animals. The magnitude of the M. bovis-induced IL-4 responses were found to be similar to the antigen-specific IL-4 responses of cattle infected with the parasitic nematode Onchocerca ochengi, further supporting the presence of this type 2 cytokine in bovine tuberculosis.

Antigen specificity in experimental bovine tuberculosis.

This report describes the kinetics of T-cell responses to a panel of mycobacterial antigens (PPD-M, PPD-A, ESAT-6, Ag85, 38kD, MPB64, MPB70, MPB83, hsp16.1, hsp65, and hsp70) following experimental infection of cattle with Mycobacterium bovis. Increased antigen-specific lymphocyte proliferation, gamma interferon, and interleukin-2 responses were observed in all calves following infection. Positive lymphocyte proliferation and cytokine responses to PPD-M and ESAT-6 were observed throughout the infection period studied. In contrast, responses to all other antigens were more variable and were not constantly present, suggesting that antigen cocktails rather than individual antigens should be used for immunodiagnosis. The detection of cytokine responses in the absence of lymphocyte proliferation, particularly during the early stages of infection, suggests a role for antigen-specific cytokine readout systems in the early identification of M. bovis infection in cattle.

Bovine tuberculosis: immune responses in the peripheral blood and at the site of active disease.

This report describes a comparison of immune responses in the peripheral blood and at the site of active disease in cattle 20 weeks after experimental infection with Mycobacterium bovis. Lymphocyte proliferation, and the production of interferon-gamma (IFN-gamma) and interleukin (IL)-2 were measured in response to tuberculin and a number of mycobacterial antigens, including ESAT-6, MPB64, MPB70, MPB83, hsp 16.1, hsp 65, hsp 70 and the 38 000 MW lipoprotein antigen. The level of transforming growth factor-beta (TGF-beta) was measured following stimulation of cells with tuberculin. Our results suggest little difference in the responses of peripheral blood and lymph node cells to most of the antigens used. However, tuberculin purified protein derivative (PPD) and ESAT-6 elicited stronger responses in the peripheral blood compared with lymph node cells. Investigation of the responding T-cell subpopulations in the peripheral blood showed that both CD4+ and, to a lesser extent, gammadelta T-cell receptor-positive (TCR+) T cells contributed to these responses. This is the first report to compare peripheral and local immune responses in bovine tuberculosis. Unlike cases of human tuberculosis where immune activity at the site of disease and anergy in the peripheral blood have been reported, our results suggest that for bovine tuberculosis immune responses occurring in the peripheral blood reflect those at the site of disease.

Differential cytokine responses of CD4+ and CD8+ T cells in response to bovine viral diarrhoea virus in cattle.

Virus-specific T cell responses were measured in cattle seropositive for bovine viral diarrhoea virus (BVDV) and compared with those from BVDV-seronegative animals. CD4+ and CD8+ T cells were purified and co-cultured in vitro with autologous, BVDV-infected monocytes over a time-course to assess the kinetics of the proliferative response. Supernatants from parallel T cell cultures were harvested and the presence of the cytokines interleukin (IL)-2, IL-4 and interferon-gamma (IFN-gamma) were measured by ELISA (IFN-gamma) or by a bioassay (IL-2 and IL-4). CD4+ and CD8+ T cells from all seropositive, but not seronegative, cattle proliferated specifically in response to BVDV-infected monocytes. Measurement of cytokines in the supernatants from proliferating T cell cultures showed that the CD4+ T cell response was type 2-like, with extremely high levels of B cell growth factor and IL-4 activity together with comparatively low levels of IL-2 activity and IFN-gamma protein. The CD8+ T cell response, although more variable, appeared to be type 1-like, with increased IL-2 and IFN-gamma but no IL-4 or B cell stimulatory activity.