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VETERINARY IMMUNOLOGY

Enhancement of the Sensitivity of the Whole-Blood Gamma Interferon Assay for Diagnosis of Mycobacterium bovis Infections in Cattle

Michel Denis, D. Neil Wedlock, Allison R. McCarthy, Natalie A. Parlane, Paul J. Cockle, H. Martin Vordermeier, R. Glyn Hewinson, Bryce M. Buddle
Michel Denis
1AgResearch, Hopkirk Research Institute, Palmerston North, New Zealand
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D. Neil Wedlock
1AgResearch, Hopkirk Research Institute, Palmerston North, New Zealand
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Allison R. McCarthy
1AgResearch, Hopkirk Research Institute, Palmerston North, New Zealand
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Natalie A. Parlane
1AgResearch, Hopkirk Research Institute, Palmerston North, New Zealand
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Paul J. Cockle
2Veterinary Laboratory Agency, Weybridge, Surrey, United Kingdom
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H. Martin Vordermeier
2Veterinary Laboratory Agency, Weybridge, Surrey, United Kingdom
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R. Glyn Hewinson
2Veterinary Laboratory Agency, Weybridge, Surrey, United Kingdom
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Bryce M. Buddle
1AgResearch, Hopkirk Research Institute, Palmerston North, New Zealand
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  • For correspondence: bryce.buddle@agresearch.co.nz
DOI: 10.1128/CVI.00291-07
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  • FIG. 1.
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    FIG. 1.

    Modulation of IFN-γ production by anti-IL-10 in naturally infected cattle from Great Britain. Blood samples from 31 field reactor cattle from herds with culture-confirmed bovine tuberculosis were stimulated as described in the text for New Zealand cattle with PPD-a, PPD-b, and the ESAT-6/CFP-10 fusion protein in the absence (−) or presence (+) of MAb neutralizing bovine IL-10. Antigen-specific IFN-γ responses, determined by BOVIGAM ELISA, are shown, with medium control values subtracted (ΔIFN-γ).

  • FIG. 2.
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    FIG. 2.

    Antigen-specific IL-10 and IFN-γ production in field reactors from Great Britain. PBMC, prepared from 24 naturally infected cattle, were incubated with PPD-a and PPD-b (10 μg/ml), a cocktail of ESAT-6 and CFP10 proteins (5 μg/ml each), or staphylococcal enterotoxin B (SEB) (1 μg/ml). Supernatants were collected after 48 h of incubation and assessed for antigen-specific IL-10 (A) or IFN-γ (B) production. The results are expressed as mean antigen-specific responses plus standard errors of the mean with background (medium control) values subtracted (ΔIL-10 and ΔIFN-γ).

Tables

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  • TABLE 1.

    Culturing of blood with antigens and immunomodulators

    AntigenImmunomodulator
    PPD-a, PPD-b, or ESAT-6/CFP-10PBSa
    PPD-a, PPD-b, or ESAT-6/CFP-10Anti-IL-10 antibodyb
    PPD-a, PPD-b, or ESAT-6/CFP-10Anti-TGF-β antibodyc
    PPD-a, PPD-b, or ESAT-6/CFP-10MMLAd
    PPD-a, PPD-b, or ESAT-6/CFP-10Tryptophane
    PPD-a, PPD-b, or ESAT-6/CFP-10IL-2f
    PPD-a, PPD-b, or ESAT-6/CFP-10GM-CSFg
    PBSPBSh
    • ↵ a Positive control.

    • ↵ b Neutralizing antibodies against IL-10 (1 μg/ml) (examines the role of endogenous IL-10 in PPD-b responsiveness).

    • ↵ c Neutralizing antibodies against TGF-β (10 μg/ml) (examines the role of TGF-β in PPD-b responsiveness).

    • ↵ d 5 mg/ml (examines the role of NO in PPD-b responsiveness).

    • ↵ e 5 mg/ml (examines the role of tryptophan degradation in PPD-b responsiveness).

    • ↵ f 1 to 10 U/ml (examines whether deficiency of IL-2 is involved in PPD-b responsiveness).

    • ↵ g 10 to 100 ng/ml (determines whether GM-CSF can enhance sensitivity to PPD-b).

    • ↵ h Negative control.

  • TABLE 2.

    Adding MMLA, tryptophan, or antibodies to TGF-β does not consistently modify PPD-b-driven IFN-γ release by PBMC of cattle experimentally infected with M. bovis

    AnimalaIFN-γ release driven byb:
    PPD-bPPD-b + MMLAPPD-b + anti-TGF-βPPD-b + tryptophanPBS
    11.561.511.881.610.05
    23.183.203.073.190.06
    32.862.733.032.680.09
    41.481.491.901.410.06
    52.101.912.491.950.06
    60.400.370.510.360.05
    70.760.751.060.740.11
    83.663.593.203.580.14
    91.851.701.771.630.07
    Uninfected animal Ac0.050.060.060.050.05
    Uninfected animal Bc0.110.100.040.120.05
    • ↵ a Animals 1 to 9 were infected with 5 × 103 CFU of M. bovis by the intratracheal route, and blood was sampled 15 weeks after infection.

    • ↵ b Results are expressed in OD units for duplicate wells.

    • ↵ c Uninfected cattle used as controls.

  • TABLE 3.

    Adding IL-2 or GM-CSF enhances IFN-γ release in response to antigens but also stimulates IFN-γ release without antigens in experimentally infected and uninfected cattle

    AnimalaIFN-γ release driven byb:
    PPD-bPPD-b + GM-CSFPPD-b + IL-2PPD-aPPD-a + GMCSFPPD-a + IL-2IL-2 onlyGMCSF onlyPBS
    11.562.992.551.013.082.420.752.740.05
    23.182.973.312.213.013.150.483.100.06
    32.863.173.290.513.352.120.683.150.09
    41.483.413.370.423.472.270.793.330.06
    52.103.563.340.702.832.651.883.340.06
    60.403.451.510.183.381.240.662.590.05
    70.763.542.360.483.282.150.923.250.11
    83.663.413.772.172.593.410.072.600.14
    91.853.793.580.593.342.671.253.340.07
    Uninfected animal Ac0.052.280.130.062.450.150.142.450.05
    Uninfected animal Bc0.110.042.000.160.041.831.500.040.05
    • ↵ a Animals 1 to 9 were infected with 5 × 103 CFU of M. bovis by the intratracheal route, and blood was sampled 15 weeks after infection.

    • ↵ b Results are expressed in OD units for duplicate wells.

    • ↵ c Uninfected cattle used as controls.

  • TABLE 4.

    Neutralizing antibodies against IL-10 enhance PPD-b- and PPD-a-driven IFN-γ release, and do not induce IFN-γ release without antigen in experimentally infected cattle

    AnimalaIFN-γ release driven byb:
    PPD-bPPD-b + anti-IL-10PPD-aPPD-a + anti-IL-10Anti-IL-10 onlyPBS
    11.562.981.012.300.060.05
    23.182.962.213.120.060.06
    32.863.270.513.100.070.09
    41.483.290.423.530.060.06
    52.103.480.703.470.070.06
    60.401.700.181.690.060.05
    70.763.430.483.560.120.11
    83.663.652.173.540.130.14
    91.853.690.593.610.070.07
    Uninfected animal Ac0.050.060.060.070.060.05
    Uninfected animal Bc0.110.0410.160.040.040.05
    • ↵ a Animals 1 to 9 were infected with 5 × 103 CFU of M. bovis by the intratracheal route, and blood was sampled 15 weeks after infection.

    • ↵ b Results are expressed in OD units for duplicate wells.

    • ↵ c Uninfected cattle used as controls.

  • TABLE 5.

    Adding a neutralizing antibody against IL-10 enhances the PPD-b-driven IFN-γ release in some naturally infected cattle

    AnimalaIFN-γ release driven byb:
    PPD-bPPD-b + anti-IL-10PBS
    10.290.410.06
    22.583.520.07
    33.813.850.11
    40.842.270.08
    53.283.370.07
    63.153.680.09
    • ↵ a Animals 1 to 6 had typical tuberculosis lesions at necropsy.

    • ↵ b Results are expressed in OD units for duplicate wells. Insufficient blood was obtained to test for responsiveness to PPD-a.

  • TABLE 6.

    Adding a neutralizing antibody against IL-10 enhances the ESAT-6/CFP-10-driven IFN-γ release in most experimentally infected cattle

    AnimalaIFN-γ release driven byb:
    PBSAnti-IL-10 onlyESAT-6/CFP-10ESAT-6/CFP-10 + anti-IL-10
    10.060.060.460.73
    20.060.062.103.02
    30.070.071.081.87
    40.060.060.461.89
    50.060.070.832.29
    60.080.060.140.59
    70.120.120.401.00
    80.050.132.053.20
    90.080.070.743.16
    Uninfected animal Ac0.060.060.060.06
    Uninfected animal Bc0.080.040.070.05
    • ↵ a Animals 1 to 9 were infected with 5 × 103 CFU of M. bovis by the intratracheal route, and blood was sampled 15 weeks after infection.

    • ↵ b Results are expressed in OD units for duplicate wells.

    • ↵ c Uninfected cattle used as controls.

  • TABLE 7.

    Adding a neutralizing antibody against IL-10 in “false-positive” cattle does not induce IFN-γ release with or without antigens

    AnimalaIFN-γ release driven byb:
    PBSPBS + anti-IL-10 (no antigen)ESAT-6/CFP-10ESAT-6/CFP-10 + anti-IL-10
    10.1740.0910.1660.122
    20.0590.0950.0480.046
    30.0710.0730.0690.07
    40.0410.0810.0530.038
    50.0350.0790.0320.031
    60.0430.0850.0350.041
    70.0460.0890.0470.06
    80.1120.0920.01260.117
    90.0820.0870.0850.093
    100.0440.0390.0440.086
    • ↵ a Representative data from 10 cattle that were false positive in the tuberculin skin test. The IFN-γ response of animal 10 was positive for ESAT-6/CFP-10 + anti-IL-10 compared to PBS (ESAT-6/CFP-10 + anti-IL-10 OD − PBS OD > 0.040).

    • ↵ b Results are expressed in OD units for duplicate wells.

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Enhancement of the Sensitivity of the Whole-Blood Gamma Interferon Assay for Diagnosis of Mycobacterium bovis Infections in Cattle
Michel Denis, D. Neil Wedlock, Allison R. McCarthy, Natalie A. Parlane, Paul J. Cockle, H. Martin Vordermeier, R. Glyn Hewinson, Bryce M. Buddle
Clinical and Vaccine Immunology Nov 2007, 14 (11) 1483-1489; DOI: 10.1128/CVI.00291-07

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Enhancement of the Sensitivity of the Whole-Blood Gamma Interferon Assay for Diagnosis of Mycobacterium bovis Infections in Cattle
Michel Denis, D. Neil Wedlock, Allison R. McCarthy, Natalie A. Parlane, Paul J. Cockle, H. Martin Vordermeier, R. Glyn Hewinson, Bryce M. Buddle
Clinical and Vaccine Immunology Nov 2007, 14 (11) 1483-1489; DOI: 10.1128/CVI.00291-07
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KEYWORDS

Interferon-gamma
Interleukin-10
Mycobacterium bovis
Tuberculosis, Bovine

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