Widespread use of tuberculosis vaccines in domestic animals depends on the development of diagnostic tests which can readily differentiate between vaccinated and tuberculosis-infected individuals. The present work used a whole-blood IFN-γ assay to detect immune responses to different mycobacterial antigens in BCG-vaccinated and M. bovis-infected cattle. Since vaccination of cattle with BCG induced little protection in this study, immune responses to selected mycobacterial antigens could be measured in BCG-vaccinated animals which subsequently developed an infection with M. bovis.
The majority of the vaccinated calves produced moderate responses to PPDB and PPDA in the IFN-γ assay but very weak responses to the recombinant mycobacterial antigens. Sixteen weeks after challenge with M. bovis, there were marked differences in IFN-γ responses to recombinant mycobacterial antigens in the nonvaccinated and BCG-vaccinated groups of animals that were culture positive. In the nonvaccinated group, an infection with M. bovis induced strong IFN-γ responses to ESAT-6 (mean, 52.2 ODI units) which were comparable to those for PPDB, while IFN-γ responses to MPB59, MPB64, and MPB70 were weak to moderate (means ranged from 7.8 to 18.8 ODI units). The responses to all of these mycobacterial antigens were significantly greater for the culture-positive animals than for culture-negative animals from the same group. In contrast, in the BCG-vaccinated group ESAT-6 was the only recombinant mycobacterial antigen for which the IFN-γ responses were significantly greater for the culture-positive animals than for the culture-negative animals. This interesting finding indicates that a reagent based on ESAT-6 may discriminate active disease from exposure to M. bovis and no infection.
The disparity in responses to the different mycobacterial antigens between culture-positive and culture-negative animals from the two groups could be related to the stage of the M. bovis
infection. Pollock and Andersen (20
) recently showed that cattle in the early stages of experimental infection were characterized by strong IFN-γ responses directed predominantly against the low-molecular-mass ESAT-6. Cattle in later stages of experimental infection (16 weeks postinfection) exhibited a broader recognition of antigens of various molecular masses. Cattle with field cases of bovine tuberculosis preferentially recognized low-molecular-mass antigens, which is characteristic of animals in the early stages of infection. There is a suggestion from the present study that the M. bovis
infections in the BCG-vaccinated animals were at an early stage, since two of the M. bovis
-infected animals which had been vaccinated had no macroscopic tuberculous lesions and one of these had microscopic tuberculous lesions. Secondly, in a previous study (4
), the IFN-γ responses to PDDB in M. bovis
-infected cattle which had previously been vaccinated with BCG peaked 2 months after corresponding responses observed in M. bovis
-infected animals which were not vaccinated.
It is unlikely that the pulmonary isolates from M. bovis
-infected animals were BCG, since no M. bovis
isolates were recovered from pulmonary lymph nodes of BCG-vaccinated cattle which had not been challenged. Furthermore, in three vaccination-challenge cattle studies (4
) in which M. bovis
isolates were typed by molecular techniques, all of the pulmonary isolates corresponded to the challenge strain and not to BCG. No tuberculous lesions were seen in any of the studies in which BCG-vaccinated animals had not been challenged.
The reason that BCG induced no or little protection in the present trial is not clearly understood. However, some of the calves had transient reactions to PPDA in the IFN-γ assay prior to vaccination, suggesting exposure to environmental mycobacteria (data not shown). Prior exposure to environmental mycobacteria has been proposed as an explanation for the failure of BCG to induce protection in a number of human tuberculosis vaccine trials (2
). In two earlier cattle trials where animals had no sensitization to PPDA prior to BCG vaccination, significantly fewer BCG-vaccinated animals than nonvaccinated animals developed tuberculous lesions following challenge (4
). In these two trials, in which BCG vaccination induced protection, the mean IFN-γ responses to PPDB following initial vaccination were stronger than in the present trial and in one other trial where there was no protection (6
). However, for individual animals, there has been no correlation between the strength of the IFN-γ response to PPDB postvaccination and subsequent protection.
Although IFN-γ responses to the recombinant mycobacterial antigens in the present study were very weak following vaccination with BCG, the responses of the BCG-vaccinated animals were still significantly stronger than those of the nonvaccinated animals. The result for MPB59 could be expected since the gene for MPB59 is found in BCG strains and in one study 78% of human BCG vaccinees recognized MPB59 at the cellular level (23
). In addition, T-cell responses to MPB59 in cattle could be stimulated by environmental mycobacteria as well as by M. bovis
). In contrast, the genes for MPB64 and ESAT-6 are not present in BCG Pasteur (12
), and MPB70 is expressed only at low levels by this strain of BCG (10
). The most likely explanation is that there is a low level of cross-reactivity between these antigens and those from BCG Pasteur, since it did not appear that the BCG-vaccinated animals nonspecifically responded to other antigens. There have been reports of cross-reactivity between mycobacterial antigens. MPT64 has been shown to have regions of some sequence similarity to the antigen 85 family members (24
). A second possibility is that since the recombinant antigens were expressed in E. coli
, very low concentrations of E. coli
antigens or lipopolysaccharide may be present in the antigen preparations, and these may cross-react with antigens from BCG. However, it is important to note that the IFN-γ responses to the recombinant antigens in the BCG-vaccinated animals were very weak.
IFN-γ responses to the pooled recombinant mycobacterial antigen preparation were very similar to the strongest response to an individual mycobacterial antigen. Following BCG vaccination, the responses to the pooled antigens were similar to those to MPB59, and in the M. bovis-infected animals, they were similar to the responses to ESAT-6. Hence, the response to the pooled antigen preparation was not additive, and use of a selected pool of mycobacterial antigens may be helpful in the diagnosis of field cases of bovine tuberculosis in which all of the infected animals may not respond to a single mycobacterial antigen.
Based on the Office International des Epizooties standard, the skin test was effective in identifying all of the M. bovis culture-positive cattle; however, five of the seven BCG-vaccinated cattle which were culture negative also showed positive responses. In contrast, when a positive cutoff ODI of ≥11 was used for the ESAT-6 IFN-γ response, all of the M. bovis culture-positive animals (n = 10) were correctly identified and the responses of all of the culture-negative animals (n = 14) were below the positive cutoff. Larger numbers of naturally infected and BCG-vaccinated animals need to be tested to confirm these results.
In conclusion, the strong correlation between the IFN-γ responses to ESAT-6 and those to PPDB in M. bovis
-infected and noninfected cattle confirmed previous findings (20
) that ESAT-6 would be a very useful reagent for specific diagnosis of bovine tuberculosis. IFN-γ responses to ESAT-6 were very weak in calves following vaccination with BCG, contrasting with consistently strong IFN-γ responses to ESAT-6 in M. bovis
-infected animals for both nonvaccinated and BCG-vaccinated groups. These findings indicate that ESAT-6 should be a suitable antigen for use in diagnostic tests for differentiating between BCG-vaccinated and M. bovis