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Clinical and Diagnostic Laboratory Immunology, September 1999, p. 675-682, Vol. 6, No. 5
1071-412X/99/$04.00+0
Development of Diagnostic Reagents To Differentiate
between Mycobacterium bovis BCG Vaccination and M. bovis Infection in Cattle
H. M.
Vordermeier,1,*
P. C.
Cockle,1
A.
Whelan,1
S.
Rhodes,1
N.
Palmer,2
D.
Bakker,3 and
R.
G.
Hewinson1
TB Research Group,1
and TB Diagnostic Unit,2 Bacteriology
Department, Veterinary Laboratories Agency
Weybridge, New Haw,
Addlestone, KT15 3NB, United Kingdom, and Department of Small
Ruminants Health, Animal Health Service, Boxtel, The
Netherlands3
Received 13 January 1999/Accepted 20 May 1999
 |
ABSTRACT |
In Great Britain a recent independent scientific review for the
government has concluded that the development of a cattle vaccine
against Mycobacterium bovis holds the best long-term
prospect for tuberculosis control in British herds. A sine qua non for 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. In order to assess the
feasibility of developing a differential diagnostic test for a live
vaccine, we chose M. bovis BCG Pasteur as a model system.
Recombinant forms of antigens which are expressed in M. bovis but not, or only at low levels, in BCG Pasteur (ESAT-6,
MPB64, MPB70, and MPB83) were produced. These reagents were tested
either alone or in combination by using peripheral blood mononuclear
cells from M. bovis-infected, BCG-vaccinated, and
Mycobacterium avium-sensitized calves. All four antigens
induced in vitro proliferation and gamma interferon responses only in
M. bovis-infected animals. A cocktail composed of ESAT-6,
MPB64, and MPB83 identified infected animals but not those vaccinated
with BCG. In addition, promiscuous T-cell epitopes of ESAT-6, MPB64,
and MPB83 were formulated into a peptide cocktail. In T-cell assays
with this peptide cocktail, infected animals were identified with
frequencies similar to those obtained in assays with the protein
cocktail, while BCG-vaccinated or M. avium-sensitized animals did not respond. In summary, our results suggest that peptide
and protein cocktails can be designed to discriminate between M. bovis infection and BCG vaccination.
| |
INTRODUCTION |
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 |
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 × 10
5 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).
IL-2 production in the same supernatants was determined by the ability
to sustain proliferation of lymphoblasts generated
by stimulation with
concanavalin A (ConA) (
6). Briefly, ConA-induced
lymphoblasts (5 µg of ConA/ml) were cocultured with the test
supernatants
for 24 h and then labelled for 24 h with 37 kBq
of [
3H]thymidine/well, harvested onto glass fiber
filters, and counted
in a beta counter. A standard of recombinant IL-2
(provided by
R. Collins, Institute for Animal Health, Compton, United
Kingdom)
was included in each
assay.
| |
RESULTS |
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.
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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.
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In contrast, PBMC from BCG-vaccinated or from PPD-A-biased responders
did not recognize these antigens, thus confirming that
ESAT-6, MPB64,
MPB70, and MPB83 might be suitable antigens to
differentiate between
M. bovis infection and BCG vaccination.
BCG-vaccinated
animals reacted with Ag85 complex antigens, while
M. avium
reactor cattle did not (Fig.
1). Similarly, PBMC from
field reactor
cattle produced IFN-
in response to ESAT-6, MPB64,
and MPB83, while
BCG-vaccinated or PPD-A-biased reactor cattle
did not (data not
shown).
To define responder frequencies for ESAT-6, MPB64, and MPB83, a cohort
of 18 field reactors was tested with these antigens.
Proliferative
responses were induced in 66, 33, and 50% of the
animals, respectively
(Table
2). These results confirm that
only
a subset of animals will respond to any given antigen, as
described
earlier (
7,
8). In this context, although ESAT-6
was the
dominant antigen, MPB83 and MPB64 were recognized by some
animals
that did not recognize ESAT-6 (Table
2).
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TABLE 2.
Immune responses against recombinant mycobacterial
proteins in field animals that were positive by the single
comparative tuberculin testa
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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.
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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.
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Both cocktails were further tested with PBMC from calves experimentally
infected with
M. bovis (2 animals), SICTT-positive
field
reactors (3 animals), animals vaccinated with BCG (7 animals),
or
PPD-A-biased responders (13 animals). Proliferative immune
responses
were induced in all field reactors and experimentally
infected calves
tested in this experiment. In contrast, only one
of the BCG-vaccinated
animals and 1 of the 13 PPD-A-biased responders
or tuberculin-negative
cattle had a weak reaction to the protein
cocktail. Furthermore, none
of the vaccinated or
M. avium-sensitized,
SICTT-negative
animals reacted with the peptide cocktail (Fig.
3), whereas 6 of the 7 BCG-vaccinated
cattle responded strongly
to both bovine and avian tuberculin. Similar
response patterns
were observed when antigen-induced IFN-
was
measured in culture
supernatants (data not shown).
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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.
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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).
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TABLE 3.
Proliferative responses against diagnostic protein and
peptide cocktails in field animals that were positive by the single
comparative tuberculin testa
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TABLE 4.
IFN- responses against diagnostic protein and peptide
cocktails in field animals that were positive by the single comparative
tuberculin testa
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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.
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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.
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| |
DISCUSSION |
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.
| |
ACKNOWLEDGMENTS |
We acknowledge the valuable assistance of the Veterinary Field
Service Staff of the Ministry of Agriculture Fisheries and Food,
particularly in the Gloucester and Worcester Animal Health Offices, in
the collection of blood samples from field reactor cattle for use in
this study. We also express our appreciation for the staff of the
Animal Services Unit at Veterinary Laboratories AgencyWeybridge.
The work performed was jointly funded by the Ministry of Agriculture
Fisheries and Food, Great Britain, and EU contract AIR CT 92-0697.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: TB Research
Group, Bacteriology Department, Veterinary Laboratories
AgencyWeybridge, New Haw, Addlestone, KT15 3NB, United Kingdom.
Phone: 44 1932 357 884. Fax: 44 1932 357 684. E-mail:
mvordermeier.vla{at}gtnet.gov.uk.
| |
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Abstract
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