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

doi:10.1128/CVI.00291-07

DOI: 10.1128/CVI.00291-07

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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).
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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).

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TABLE 1.

Culturing of blood with antigens and immunomodulators

Antigen	Immunomodulator
PPD-a, PPD-b, or ESAT-6/CFP-10	PBS^a
PPD-a, PPD-b, or ESAT-6/CFP-10	Anti-IL-10 antibody^b
PPD-a, PPD-b, or ESAT-6/CFP-10	Anti-TGF-β antibody^c
PPD-a, PPD-b, or ESAT-6/CFP-10	MMLA^d
PPD-a, PPD-b, or ESAT-6/CFP-10	Tryptophan^e
PPD-a, PPD-b, or ESAT-6/CFP-10	IL-2^f
PPD-a, PPD-b, or ESAT-6/CFP-10	GM-CSF^g
PBS	PBS^h

↵ 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

Animal^a	IFN-γ release driven by^b:
Animal^a	PPD-b	PPD-b + MMLA	PPD-b + anti-TGF-β	PPD-b + tryptophan	PBS
1	1.56	1.51	1.88	1.61	0.05
2	3.18	3.20	3.07	3.19	0.06
3	2.86	2.73	3.03	2.68	0.09
4	1.48	1.49	1.90	1.41	0.06
5	2.10	1.91	2.49	1.95	0.06
6	0.40	0.37	0.51	0.36	0.05
7	0.76	0.75	1.06	0.74	0.11
8	3.66	3.59	3.20	3.58	0.14
9	1.85	1.70	1.77	1.63	0.07
Uninfected animal A^c	0.05	0.06	0.06	0.05	0.05
Uninfected animal B^c	0.11	0.10	0.04	0.12	0.05

↵ 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

Animal^a	IFN-γ release driven by^b:
Animal^a	PPD-b	PPD-b + GM-CSF	PPD-b + IL-2	PPD-a	PPD-a + GMCSF	PPD-a + IL-2	IL-2 only	GMCSF only	PBS
1	1.56	2.99	2.55	1.01	3.08	2.42	0.75	2.74	0.05
2	3.18	2.97	3.31	2.21	3.01	3.15	0.48	3.10	0.06
3	2.86	3.17	3.29	0.51	3.35	2.12	0.68	3.15	0.09
4	1.48	3.41	3.37	0.42	3.47	2.27	0.79	3.33	0.06
5	2.10	3.56	3.34	0.70	2.83	2.65	1.88	3.34	0.06
6	0.40	3.45	1.51	0.18	3.38	1.24	0.66	2.59	0.05
7	0.76	3.54	2.36	0.48	3.28	2.15	0.92	3.25	0.11
8	3.66	3.41	3.77	2.17	2.59	3.41	0.07	2.60	0.14
9	1.85	3.79	3.58	0.59	3.34	2.67	1.25	3.34	0.07
Uninfected animal A^c	0.05	2.28	0.13	0.06	2.45	0.15	0.14	2.45	0.05
Uninfected animal B^c	0.11	0.04	2.00	0.16	0.04	1.83	1.50	0.04	0.05

↵ 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

Animal^a	IFN-γ release driven by^b:
Animal^a	PPD-b	PPD-b + anti-IL-10	PPD-a	PPD-a + anti-IL-10	Anti-IL-10 only	PBS
1	1.56	2.98	1.01	2.30	0.06	0.05
2	3.18	2.96	2.21	3.12	0.06	0.06
3	2.86	3.27	0.51	3.10	0.07	0.09
4	1.48	3.29	0.42	3.53	0.06	0.06
5	2.10	3.48	0.70	3.47	0.07	0.06
6	0.40	1.70	0.18	1.69	0.06	0.05
7	0.76	3.43	0.48	3.56	0.12	0.11
8	3.66	3.65	2.17	3.54	0.13	0.14
9	1.85	3.69	0.59	3.61	0.07	0.07
Uninfected animal A^c	0.05	0.06	0.06	0.07	0.06	0.05
Uninfected animal B^c	0.11	0.041	0.16	0.04	0.04	0.05

↵ 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
Animal^a IFN-γ release driven by^b:
PPD-b PPD-b + anti-IL-10 PBS
1 0.29 0.41 0.06
2 2.58 3.52 0.07
3 3.81 3.85 0.11
4 0.84 2.27 0.08
5 3.28 3.37 0.07
6 3.15 3.68 0.09
↵ 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

Animal^a	IFN-γ release driven by^b:
Animal^a	PBS	Anti-IL-10 only	ESAT-6/CFP-10	ESAT-6/CFP-10 + anti-IL-10
1	0.06	0.06	0.46	0.73
2	0.06	0.06	2.10	3.02
3	0.07	0.07	1.08	1.87
4	0.06	0.06	0.46	1.89
5	0.06	0.07	0.83	2.29
6	0.08	0.06	0.14	0.59
7	0.12	0.12	0.40	1.00
8	0.05	0.13	2.05	3.20
9	0.08	0.07	0.74	3.16
Uninfected animal A^c	0.06	0.06	0.06	0.06
Uninfected animal B^c	0.08	0.04	0.07	0.05

↵ 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

Animal^a	IFN-γ release driven by^b:
Animal^a	PBS	PBS + anti-IL-10 (no antigen)	ESAT-6/CFP-10	ESAT-6/CFP-10 + anti-IL-10
1	0.174	0.091	0.166	0.122
2	0.059	0.095	0.048	0.046
3	0.071	0.073	0.069	0.07
4	0.041	0.081	0.053	0.038
5	0.035	0.079	0.032	0.031
6	0.043	0.085	0.035	0.041
7	0.046	0.089	0.047	0.06
8	0.112	0.092	0.0126	0.117
9	0.082	0.087	0.085	0.093
10	0.044	0.039	0.044	0.086

↵ 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.