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The Delayed type hypersensitivity skin test (DTH) and interferon-gamma assay are used for the diagnosis of bovine tuberculosis (TBB). The specificity of these diagnoses, however, is compromised because both are based on the response against purified protein derivative of Mycobacterium bovis (PPD-B). In this study, we assessed the potential of two cocktails containing M. bovis recombinant proteins: cocktail 1 (C1): ESAT-6, CFP-10 and MPB83 and cocktail 2 (C2): ESAT-6, CFP-10, MPB83, HspX, TB10.3, and MPB70. C1, C2, and PPD-B showed similar response by DTH in M. bovis-sensitized guinea pigs. Importantly, C1 induced a lower response than PPD-B in M. avium-sensitized guinea pigs. In cattle, C1 displayed better performance than PPD-B and C2; indeed, C1 showed the least detection of animals either vaccinated or Map-infected. To optimize the composition of the cocktails, we obtained protein fractions from PPD-B and tested their immunogenicity in experimentally M. bovis-infected cattle. In one highly reactive fraction, seven proteins were identified. The inclusion of FixB in C1 enhanced the recognition of M. bovis-infected cattle without compromising specificity. Our data provide a promising basis for the future development of a cocktail for TBB detection without interference by the presence of sensitized or infected animals with other mycobacteria.

Delayed-type hypersensitivity represents high levels of protein Ag-specific adaptive immunity induced by mycobacterial infection, and can be monitored in the Ag-challenged skin. Besides protein Ags, recent evidence has suggested that a substantial immunity directed against glycolipid Ags is also elicited in response to mycobacterial infection, but skin hypersensitivity to this class of Ags has not been fully assessed. To address this issue directly, glycolipid-specific skin reactions were evaluated in guinea pigs infected with Mycobacterium avium complex (MAC). Significant skin induration was observed in MAC-infected, but not mock-infected, guinea pigs, following intradermal administration of a mixture of MAC-derived glycolipids. Surprisingly, this glycolipid-specific skin response involved up-regulated expression of IL-5 mRNA in situ and marked local infiltration of eosinophils. Challenge experiments with individual glycolipid components detected an outstanding capability for trehalose dimycolate (TDM), but not a structurally related glycolipid, glucose monomycolate, to elicit the skin response. T lymphocytes derived from the spleen of MAC-infected, but not uninfected, guinea pigs specifically responded to TDM in vitro by up-regulating IL-5 transcription, and this response was not blocked by Abs that reacted to the known guinea pig group 1 CD1 proteins. Finally, the eosinophilic skin hypersensitivity to TDM was also elicited in guinea pigs vaccinated with bacillus Calmette-Guerin, which contrasted sharply with the classical delayed-type hypersensitivity response to the purified protein derivative. Therefore, the TDM-elicited eosinophilic response defines a new form of hypersensitivity in mycobacterial infection, which may account for local infiltration of eosinophils often observed at the site of 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.

In order to develop a model of Mycobacterium bovis infection with pathogenetical relevance, a modified version of the Henderson apparatus was used to deliver infectious aerosols directly to the snouts of guinea pigs. Aerosols generated from 10(6), 10(7), 10(8)CFU/ml M. bovis suspensions established disease in every animal, with estimated retained doses of 10, 100, 1000 CFU, respectively. For comparison, other guinea pigs were inoculated with 100 CFU M. bovis intramuscularly (i.m.). Pathology and bacterial colonisation of lungs and spleen varied according to the dose and route of inoculation. Animals inoculated i.m. gave a significant cutaneous tuberculin hypersensitivity reaction earlier after testing than those infected aerogenically. A serological response to M. bovis antigens was detected in all infected animals. Intensity of antigen recognition was dose-dependent and although the range of antigens recognised varied between animals, a 25 kDa antigen present in the cell fraction was serodominant. Thus, a reproducible guinea pig model has been defined that may be suitable for virulence, vaccination, and immunological studies.

Tumor necrosis factor (TNF) signalling via the TNF receptor 1 (TNF-R1) is required for host resistance to mycobacterial infection. The role of TNF-R2 in anti-mycobacterial immunity is not known. Therefore, we compared TNF-R1 and TNF-R2 knockout (KO) mice infected with Mycobacterium bovis BCG (10(7) CFU, i.v.). While the bacterial burden of TNF-R1-deficient mice was significantly increased and the mice succumbed to infection between 4 and 5 weeks, TNF-R2 KO mice were less sensitive, and only 3 of 10 mice died within 12 weeks. Wild-type (WT) mice were resistant to BCG infection. The inability to clear the infection of TNF-R1 KO mice was associated with a reduced delayed-type hypersensitivity (DTH) response to purified protein derivative and severe impairment in forming granulomas with reduced macrophage recruitment and activation, and diminished expression of adhesion molecules. By contrast, TNF-R2 KO mice developed normal DTH response and mature mycobactericidal granulomas as the WT mice. Therefore, anti-mycobacterial immunity is largely dependent on TNF signalling via the TNF-R1, while activation of TNF-R2 plays a minor role., (Copyright 2000 S. Karger AG, Basel)

Neutralization of TNF or disruption of TNF-R1 leads to fatal Mycobacterium bovis BCG infection. Here we used TNF-LT-alpha-deficient mice to test whether a complete disruption of TNF and LT-alpha reduces further host resistance to BCG infection. The bacterial burden especially in the lungs of TNF-LT-alpha-deficient mice was significantly increased and the mice succumbed to infection between 8 and 10 weeks. In the absence of TNF-LT-alpha the granulomatous response was severely impaired and delayed. The cells in the granulomas of TNF-LT-alpha-deficient mice expressed low levels of MHC class II and ICAM-1. They contained a few T cells and F4/80-positive macrophages expressing little iNOS and acid phosphatase activity. By contrast, the lethal action of endotoxin was dramatically reduced in BCG-infected TNF-LT-alpha-deficient mice. In summary, in the absence of TNF-LT-alpha the recruitment and activation of mononuclear cells in response to BCG infection were significantly delayed and reduced resulting in immature granulomas allowing uncontrolled fatal infection., (Copyright 2000 Academic Press.)

Recombinant MPB64 (rMPB64), a mycobacterial antigen, was obtained from an Escherichia coli clone transformed with a recombinant expression vector, pMAL64c. The rMPB64 was examined for the activity to elicit delayed-type hypersensitivity (DTH) in guinea pigs injected with liver Mycobacterium tuberculosis H37Rv or live M. bovis BCG Tokyo. It was found that rMPB64 has the same reactivity as native MPB64 (nMPB64) or MPT64 (nMPT64) and the potency to elicit DTH was 13.4 times higher than that of PPD. Because MPB64 is secreted only by living M. tuberculosis and some strains of BCG, it is possible to use this antigen for the diagnosis of tuberculosis.

Research on delayed type hypersensitivity (DTH) to facultative intracellular bacteria in guinea pigs focused especially on intracutaneous DTH reactions. However, phenomena of erythematous and necrotic skin reactions were only evaluated qualitatively, dose-response relationships not investigated, differences between early and late reactions overlooked and the development of unspecific skin necrosis ignored. In this work, these aspects were investigated by characterisation of DTH-mediated reactions against mycobacterial antigens in the skin of guinea pigs. Intracutaneous injection of mycobacterial antigen into the skin of immunised and non-immunised guinea pigs induced an erythematous reaction which reached its peak after 24 h. On days two and three after challenge, the centre of the erythema blanched and necrotised. During the next days, skin necrosis increased and reached a peak on days six and seven. In immunised guinea pigs, DTH led to a specific enhancement of erythematous and necrotising skin reactions which also appeared in non-immunised guinea pigs. Thus, an early erythematous and a late necrotising DTH-reaction were identified by their morphology and time course. Both types of DTH-mediated allergy revealed dose-dependency from the intracutaneously injected antigen doses. Erythematous and necrotising DTH reactions against viable and killed mycobacteria showed a similar time course and morphology. DTH reactivity which was induced by immunisation with Mycobacterium tuberculosis showed cross-reactivity with antigens of Mycobacterium bovis.