INTRODUCTION

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Bovine tuberculosis (TB) is caused by Mycobacterium bovis, a bacterium closely related to Mycobacterium tuberculosis, the major cause of human TB. M. bovis can infect a wide range of animal species and was the cause of approximately 2,000 human deaths per annum (6%) in England and Wales in the 1930s and 1940s (12). Infected cattle can spread the bacteria by aerosol and through infected milk. However, the introduction of pasteurization of milk and the test and slaughter of tuberculous cattle has dramatically reduced the transmission from cattle to humans, and in 1995 only 32 (1%) of the 3,200 isolates from patients with TB in Great Britain (GB) were identified as M. bovis (12). However, bovine TB has severe implications for animal welfare and animal health, since it causes reduced productivity and premature death in cattle and affected farms suffer severe economic losses. Therefore, a compulsory eradication program based on tuberculin testing and slaughter of reactor animals began in GB in 1950, and by 1960 the program had been successfully implemented throughout the whole of GB. Despite these measures, the number of cases of herd breakdowns reported has been steadily rising since 1988, mainly in the southwest of England but also in the rest of England and Wales. A recent independent scientific panel set up to review the control of bovine TB in GB (18) has concluded that the development of a cattle vaccine against M. bovis holds the best long-term prospect for TB control in British herds. A sine qua non for cattle vaccination is the development of a complementary diagnostic test to differentiate between vaccinated animals and those infected with M. bovis so that test-and-slaughter-based control strategies can continue alongside vaccination.

Despite the unpredictable and widely diverging efficacies of vaccination with M. bovis BCG (2, 9, 31, 37), it is still the only realistic vaccine candidate which could be applied to the field situation in the short term. Encouraging results with BCG have been reported from New Zealand, where a significant level of protection has been observed in vaccinated cattle subsequently experimentally challenged with M. bovis (3, 4). However, vaccination with BCG compromises tuberculin purified protein derivative (PPD) specificity (2, 14, 16). Thus, the development of cattle vaccines based on BCG (or attenuated M. bovis strains) will require the identification of specific, defined antigens that allow the discrimination of M. bovis-infected animals from vaccinated animals (differential diagnosis). Such antigens are ESAT-6 and MPB64, which due to a gene deletion are not expressed in BCG Pasteur (10, 11, 26), as well as MPB70 and MPB83, which are expressed at only low levels in BCG Pasteur and are serodominant in infected cattle (15, 40). It has been demonstrated that both ESAT-6 and MPB64, when used as skin test reagents, discriminated between infected and BCG-vaccinated guinea pigs (5). In addition, ESAT-6 was found to discriminate between cattle infected with TB and cattle sensitized by environmental mycobacteria (27). An alternative approach to using recombinant proteins is the application of synthetic peptides derived from antigens such as those described above. Synthetic peptides would have the advantages of lower production costs and easier standardization and quality control and carry no risk of infection since they are fully chemically synthesized.

To assess the feasibility of developing differential diagnostic tests for a live vaccine, we tested recombinant forms of ESAT-6, MPB64, MPB70, and MPB83 alone or in combination using peripheral blood mononuclear cells (PBMC) from M. bovis-infected, BCG-vaccinated, and Mycobacterium avium-sensitized calves. In addition, a cocktail composed of synthetic peptides encompassing "promiscuous peptides" derived from ESAT-6, MPB64, MPB70, and MPB83 was also assessed. Our results provide proof of the principle that both protein-based and suitably formulated peptide-based cocktails can be employed to specifically diagnose M. bovis-infected field reactor cattle without being compromised by BCG vaccination, thus satisfying the definition of reagents for differential diagnosis.

	MATERIALS AND METHODS

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Cattle. Six- to 12-month-old calves of various breeds were obtained from herds free of bovine TB and kept in the Animal Services Unit. Four different groups of cattle were used in this study.

(i) M. bovis infection. Calves were infected with an M. bovis field strain from GB (AF 2122/97) by intratracheal instillation of 10⁴ CFU as described earlier (3, 4).

(ii) BCG vaccination. Calves were vaccinated with BCG Pasteur by subcutaneous injection of 10⁶ CFU into the side of the neck (3, 4), followed 8 weeks later by a booster injection with the same route and dose.

(iii) M. avium-biased cattle and other controls. Several animals with stronger responses to avian PPD (PPD-A) than to mammalian PPD (PPD-M) were included as examples of sensitization with environmental mycobacteria. Blood was also obtained from animals from herds free of TB as negative controls.

(iv) Field samples. Blood samples were obtained from cattle of mixed breeds from farms in Gloucestershire and Worcestershire, United Kingdom, that had been designated tuberculin test reactors following skin testing with the single intradermal comparative tuberculin test (SICTT). The skin tests were performed as specified previously (6a). All samples were transported by courier to our laboratory within 8 h of sampling and processed immediately upon arrival.

Antigens. ESAT-6, MPB64, MPB70, and MPB83 were expressed in Escherichia coli by using the plasmid vector pET21d and purified by Ni affinity chromatography according to standard methodologies recommended by the manufacturer (Novagen). Ag85abc complex purified from BCG culture filtrate was kindly provided by K. Huygen, Brussels, Belgium. Synthetic peptides (16 residues long and overlapping by 8 residues) were synthesized by solid-phase peptide synthesis as described earlier (36). Peptide purity and sequence fidelity were confirmed by analytical reverse-phase high-pressure liquid chromatography and by electron-spray mass spectrometry, respectively. Proteins were used in cultures singly or in a cocktail of ESAT-6, MPB64, and MPB83, at 5 to 10 µg/ml, and peptides were used at 1 to 25 µg/ml. The sequences of peptides used in the peptide cocktail are given in Table 1. PPD-M and PPD-A tuberculins were obtained from the Tuberculin Production Unit at the Veterinary Laboratories AgencyWeybridge and used in culture at 10 µg/ml.

Lymphocyte transformation assay (LTA). PBMC were isolated from heparanized blood by Histopaque-1077 (Sigma, Poole, United Kingdom) gradient centrifugation and cultured in RPMI 1640 supplemented with 5% CPSR-1 (Sigma), nonessential amino acids (Sigma), 5 × 10⁵ M 2-mercaptoethanol, 100 U of penicillin per ml, and 100 mg of streptomycin sulfate per ml. PBMC (2 × 10⁵/well in 0.2-ml aliquots) were cultured in triplicate for 6 days in flat-bottom 96-well microtiter plates in the presence of antigen and radiolabelled during the last 16 to 20 h of culture with 37 kBq of [³H]thymidine (Amersham, Amersham, United Kingdom) per well, harvested onto glass fiber filters, and counted in a beta counter. Positive responses were defined as a stimulation index (SI), i.e., radioactivity with antigen/radioactivity without antigen, of 3 together with a signal strength of 1,000 cpm (13, 35, 41).

IFN- and interleukin-2 (IL-2) assays. Whole-blood cultures were performed in 96-well plates in 0.25-ml/well aliquots by mixing heparinized blood with an equal volume of antigen-containing solution. Supernatants were harvested after 24 h of culture, and gamma interferon (IFN-) responses were determined by using the BOVIGAM enzyme-linked immunosorbent assay (ELISA) kit (CSL, Melbourne, Australia) (44). In vitro IFN- responses to PPD-M and PPD-A were interpreted as set out in the instructions accompanying the BOVIGAM ELISA kit. Results obtained with recombinant proteins, peptides, and diagnostic cocktails were deemed positive when the IFN- SI (ISI), i.e., optical density at 450 nm (OD₄₅₀) with antigen/OD₄₅₀ without antigen, was 2.0 (21, 22).

	RESULTS

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Recognition of recombinant mycobacterial antigens. Recombinant forms of the M. bovis antigens ESAT-6, MPB64, MPB70, and MPB83 were expressed in E. coli and purified as described in Materials and Methods. These recombinant antigens were then tested for their ability to induce T-cell proliferation and IFN- secretion in PBMC from field reactor cattle (SICTT positive), M. bovis-infected animals, BCG-vaccinated cattle, or cattle naturally sensitized with environmental mycobacteria (defined by stronger PPD-A responses than PPD-M responses). Native Ag85 complex antigens purified from BCG culture filtrate were included as a control because they are strongly expressed in both M. bovis and BCG (39). The results of a representative experiment are shown in Fig. 1. Strong proliferative responses were observed in PBMC isolated from M. bovis field reactors stimulated with ESAT-6, MPB64, and MPB83, as well as in those cultures stimulated with the Ag85 complex antigens (shown for one representative field reactor in Fig. 1). Stimulation with MPB70 gave rise to weaker yet still significant responses.

View larger version (17K): [in this window] [in a new window]   FIG. 1.   Recognition of mycobacterial proteins. PBMC isolated from one SICTT-positive field reactor (M. bovis), three BCG-vaccinated cattle (BCG), and one animal with PPD-A-biased responses (PPD-A biased) were incubated with recombinant mycobacterial proteins and avian and mammalian tuberculin at 10 µg/ml for 6 days. Results are expressed as means ± standard errors.

Definition of diagnostic cocktails composed of either recombinant proteins or promiscuous synthetic peptides of ESAT-6, MPB64, and MPB83. We next investigated whether diagnostic cocktails of pooled recombinant proteins or synthetic peptides representing their immunodominant epitopes would give rise to elevated responder frequencies as well as increased signal strength. A protein cocktail composed of ESAT-6, MPB64, and MPB83 was tested in parallel with a peptide cocktail containing seven promiscuous peptides derived from ESAT-6, MPB64, MPB70, and MPB83 (Table 1). These peptides were identified in a separate investigation which showed that they were recognized "promiscuously" by T cells from more than 50% of the M. bovis-infected cattle tested (data not shown and reference 36a). First, these diagnostic cocktails were tested in a small number of field reactors. The PBMC responses induced by cocktails were compared with those induced by their individual components. Results of a representative experiment which demonstrated that both protein and peptide cocktails induced stronger proliferative responses than any of their constituents alone are shown in Fig. 2. In addition, cocktails also compensated for the lack of recognition of individual protein or peptide constituents.

View larger version (24K): [in this window] [in a new window]   FIG. 2.   Proliferative responses induced by protein (A) and peptide (B) cocktails. PBMC isolated from a SICTT-positive reactor were incubated with recombinant ESAT-6, MPB64, or MPB83 individually (10 µg/ml) or a pool of all three (5 µg/ml each). PBMC from the same reactor animal were incubated with peptides p1 to p4 individually (10 µg/ml) or a pool of all four peptides (10 µg/ml each). Results are expressed as means ± standard errors.

View larger version (27K): [in this window] [in a new window]   FIG. 3.   Proliferative responses induced by protein and peptide cocktails. PBMC isolated from 3 SICTT-positive field reactor cattle (SICTT+), 2 cattle experimentally infected with M. bovis (M. bovis exp.), 7 BCG-vaccinated cattle (BCG), and 13 cattle with either PPD-A-biased responses or negative SICTT responses (PPD-A/uninfected) were incubated with either the protein cocktail (5 µg/ml each) or the peptide cocktail described in Table 1 (10 µg/ml each). Results are expressed as mean proliferative responses ± standard errors.

Assessment of the diagnostic cocktails in field reactors. Having demonstrated that both the protein and peptide diagnostic cocktails described above were not recognized by PMBC from BCG-vaccinated cattle, we next set out to quantify their ability to identify infected animals by testing a cohort of 35 SICTT-positive field animals. The blood samples were obtained within 7 to 28 days after skin testing from six farms in Gloucestershire and Worcestershire, transported to our laboratory within 6 to 8 h, and processed immediately upon arrival. Parameters tested were PBMC LTA responses and whole-blood IFN- production. The results of the LTAs can be summarized as follows (Table 3): 29 (82.8%) of the 35 SICTT-positive field reactors were identified on the basis of stronger responses to PPD-M than to PPD-A. Encouragingly, T cells from 74.2% (27 of 35) of these cattle recognized the protein cocktail, while T cells from 71.4% (25 of 33) reacted with the peptide cocktail, thus achieving sensitivity levels only slightly inferior to that for PPD. When IFN- production was measured in culture supernatants of whole-blood cultures and the criteria set out in the BOVIGAM ELISA kit were used to score for positivity, 82.8% of the SICTT-positive field reactors were identified, whereas the protein cocktail identified 68.6% (24 of 35) and the peptide cocktail identified 55.2% (16 of 29) of these cattle (Table 4).

View this table: [in this window] [in a new window]   TABLE 4.   IFN- responses against diagnostic protein and peptide cocktails in field animals that were positive by the single comparative tuberculin testa

IL-2 as a surrogate for proliferation. Since LTAs are labor-intensive and take 5 to 6 days to complete, a surrogate marker for proliferation amenable to an ELISA format and to whole-blood cultures would be highly desirable. We tested whether determination of IL-2 levels in culture supernatants from 24-h whole-blood cultures could serve as such a surrogate assay. IL-2 production was measured in culture supernatants obtained from cultures from 9 field reactors and from 10 control animals from farms with no recent history of TB. The results of this pilot experiment were compared to those obtained with PBMC LTAs and suggest that IL-2 production could indeed serve as a surrogate for proliferation (Fig. 4). Neither the protein nor the peptide cocktail induced IL-2 production in control animals, whereas IL-2 production was induced in cultures from field reactors whose lymphocytes recognized both cocktails in LTAs. Interestingly, IL-2 levels were comparable to those in the control group in supernatants from two animals whose PBMC did not proliferate following stimulation with either the peptide or the protein cocktail.

View larger version (15K): [in this window] [in a new window]   FIG. 4.   IL-2 production as a surrogate marker for proliferation. Whole-blood cultures in the presence of either the protein or the peptide cocktail were performed with samples obtained from 9 SICTT-positive field reactors and from 10 negative control animals from farms with no history of TB. Supernatants were collected after 24 h, and IL-2 production was determined. Filled circles, samples from cattle recognizing the cocktails in LTAs; filled squares, samples from cattle not responding to cocktails in LTAs.

	DISCUSSION

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The development of diagnostic reagents capable of differentiating between infection and vaccination is a prerequisite for the development of a vaccine against TB in cattle so that existing test-and-slaughter control strategies can continue alongside vaccination. Such differential diagnosis would also be of benefit to human vaccination regimes so that individuals who, despite vaccination, contract TB and require chemotherapy can be identified. BCG vaccination has been shown to compromise tuberculin skin testing in cattle (and in humans) and in vitro assays which utilize tuberculin, including the IFN--based ELISA kit (42-44).

The general trend in the diagnosis of bovine TB in cattle points to a preference for blood-based tests since, in contrast to the tuberculin skin test, they require only a single farm visit, and it has been suggested that a blood-based test could be used in conjunction with the SICTT to reduce the time between consecutive tests in animals with inconclusive results (1). Such blood-based assays are also being developed for other species, including humans (20, 32, 33).

ESAT-6 and MPB64, both constituents of the diagnostic cocktails used in this study, have been tested previously in cattle. ESAT-6 has been shown to induce IFN- production in 85 to 90% of the field reactors tested in Northern Ireland (27, 28). Since the gene encoding ESAT-6 is not present in BCG or in most environmental mycobacteria, ESAT-6 is an obvious candidate antigen for differential diagnosis. This study confirms that ESAT-6 induces T-cell responses in the majority of field reactors tested, albeit with a lower frequency, 66%. This lower figure could be due to a difference in the genetic makeup of the two study populations or to differences in the levels of expression of ESAT-6 in some M. bovis strains in GB. A recent study (21) has defined the responder frequency for MPB64 as 50%, which is higher than the 33% observed in our study population. These differences in responder frequencies between different study populations highlight the necessity to evaluate the frequencies of responders to specific antigens in the target population. To our knowledge, frequencies of responders to MPB83 among cattle have not been determined before. We were encouraged that 50% of the field reactors tested recognized this protein, and it was therefore incorporated into our diagnostic cocktails.

The data presented in this study provides proof of the principle that differential diagnosis discriminating between vaccination and infection can be achieved. Our study has also shown that only a subset of infected animals will respond to a single antigen, reinforcing previous observations (7, 8) and demonstrating that it is unlikely that single antigens will be able to identify the vast majority of infected animals. We have also demonstrated that stronger responses can be induced with reagent pools than with their individual components. Recently, protein pools composed of mycobacterial antigens which provide improved specificity over tuberculin in a guinea pig model have been defined (23). A further advantage in using diagnostic antigen cocktails is that it reduces the possibility of selecting "escape mutants", i.e., strains of tubercle bacilli lacking the diagnostic antigen. Such natural mutants have been described. For example, strains of M. tuberculosis which do not express the 19-kDa lipoprotein have been identified (19).

The use of synthetic peptides rather than recombinant antigens as diagnostic reagents has the added advantages of lower production costs, easier standardization, and lack of any risk of infection from the recombinant strain. However, due to the genetic diversity of the major histocompatibility complex (MHC), the use of peptides has in the past been deemed impractical, since it was believed that a large pool of epitopes would be required to achieve wide population coverage. Fortunately, it has been recognized over recent years that a significant proportion of MHC class II-restricted peptides are recognized by T cells in the context of multiple MHC alleles. This has been demonstrated to be particularly so in the recognition of mycobacterial antigens by murine, human (reviewed in reference 34), and bovine CD4⁺ T cells (29, 30). Moreover, rationally designed peptide cocktails have been shown to induce responses in PBMC from human TB patients and BCG-vaccinated donors with high sensitivity (17). The results presented in this study further confirm these results, since we were able to detect more than 70% of reactor cattle with a small pool of seven peptides.

The responder frequencies obtained for field samples by LTAs with the protein and peptide cocktails were comparable to those obtained with tuberculin (74.2 and 71.4% versus 82.8%, respectively). The same responder frequency was observed when responses to PPD-M were compared with those to PPD-A by using IFN- as a readout system. This sensitivity is within the range observed in different studies (between 76 and 98%) during the evaluation of the IFN- assay (38, 43, 44). The sensitivity of the IFN- assay, indicated by the results observed for the defined protein and peptide cocktails, was significantly lower than that indicated by results obtained in LTAs with the same antigens (68.6 and 55%, respectively). This could reflect the greater standardization of culture conditions for LTA, particularly with respect to cell numbers and culture conditions.

LTAs are highly labor-intensive, depend on specialized skills and equipment, and require several days for the results to become available. Thus, surrogate readout systems of proliferation amenable to whole-blood-based ELISAs are desirable. Determination of production of soluble IL-2 receptors has been used recently with good results (24, 25). In this context, our results demonstrating that the levels of IL-2 in 24-h whole-blood culture supernatants are in concordance with the results observed for the LTAs are highly encouraging.

In conclusion, we have demonstrated that diagnostic cocktails based on either recombinant protein or peptides derived from antigens expressed by M. bovis but not by the vaccine strain, in this case BCG Pasteur, can distinguish between vaccinated and infected individuals. We predict that the addition of other such antigens to the cocktails will increase their sensitivity further without compromising specificity. The development of such protein and peptide pools is now under investigation in our laboratory.