Development and evaluation of an interferon gamma assay for the diagnosis of tuberculosis in red deer experimentally infected with Mycobacterium bovis
Abstract Background Red deer (Cervus elaphus) is regarded as an epidemiologically relevant host for Mycobacterium bovis (M. bovis) and closely related members of the Mycobacterium tuberculosis complex that cause animal tuberculosis (TB). The standard antemortem screening test for the detection of TB...
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doaj-019df86a76d44776a6ed97670233aba62020-11-24T22:18:06ZengBMCBMC Veterinary Research1746-61482017-11-0113111110.1186/s12917-017-1262-6Development and evaluation of an interferon gamma assay for the diagnosis of tuberculosis in red deer experimentally infected with Mycobacterium bovisMaría Ángeles Risalde0Jobin Thomas1Iker Sevilla2Miriam Serrano3Jose Antonio Ortíz4Joseba Garrido5Mercedes Domínguez6Lucas Domínguez7Christian Gortázar8Jose Francisco Ruíz-Fons9SaBio (Health and Biotechnology), Instituto de Investigación en Recursos Cinegéticos IREC (CSIC-UCLM)SaBio (Health and Biotechnology), Instituto de Investigación en Recursos Cinegéticos IREC (CSIC-UCLM)NEIKER-Tecnalia, Animal Health DepartmentNEIKER-Tecnalia, Animal Health DepartmentMedianilla Red Deer GeneticsNEIKER-Tecnalia, Animal Health DepartmentServicio de Inmunología, Centro Nacional de Microbiología, Instituto de Salud Carlos IIIVISAVET Health Surveillance Centre. Complutense University of MadridSaBio (Health and Biotechnology), Instituto de Investigación en Recursos Cinegéticos IREC (CSIC-UCLM)SaBio (Health and Biotechnology), Instituto de Investigación en Recursos Cinegéticos IREC (CSIC-UCLM)Abstract Background Red deer (Cervus elaphus) is regarded as an epidemiologically relevant host for Mycobacterium bovis (M. bovis) and closely related members of the Mycobacterium tuberculosis complex that cause animal tuberculosis (TB). The standard antemortem screening test for the detection of TB in deer is the intradermal tuberculin skin test, but the detection of interferon-gamma (IFNγ) produced by white blood cells exposed to M. bovis antigens can be used as an alternative or supplemental assay in most TB eradication/control programs. This study aims to develop an in-house sandwich ELISA for deer IFNγ, based on the cross-reactivity of the antibodies to both cervid and bovine IFNγ, and to evaluate the potential of this assay to detect M. bovis-infected red deer in response to the in vitro stimulation of whole-blood cells with bovine purified protein derivative (bPPD), p22 protein complex derived from bPPD or using the specific tuberculous mycobacterial proteins ESAT-6/CFP-10, Rv3615c and Rv3020c. The positive control stimulant used in this study was pokeweed mitogen, which resulted in a consistent induction of IFNγ in samples from red deer, thus allowing the interpretation of the assay. Results The percentage of animals correctly classified by this technique as M. bovis non-infected was 100%. The detection of infected animals as positive was high and ranged widely depending upon the antigen and the cut-off value applied, as well as the time after infection. Our findings indicate that this protocol may serve as a reliable assay for the antemortem diagnosis of TB from the initial stage of M. bovis-infection, and may also be adequately sensitive. Conclusions The suggested optimal antigens and cut-off are bPPD, p22 and the combination of ESAT-6/CFP-10 and Rv3020c with a 0.05 Δ optical density, which yielded a up to 100% correct classification of TB positive and negatve red deer under our experimental conditions. This technique will aid in TB testing of farmed and translocated deer. Future studies should evaluate the ability of this IFNγ assay to detect specific responses under field conditions.http://link.springer.com/article/10.1186/s12917-017-1262-6Animal tuberculosisCellular immune responseFarmed Cervus elaphusIn vivo testMycobacterium tuberculosis complex |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
María Ángeles Risalde Jobin Thomas Iker Sevilla Miriam Serrano Jose Antonio Ortíz Joseba Garrido Mercedes Domínguez Lucas Domínguez Christian Gortázar Jose Francisco Ruíz-Fons |
spellingShingle |
María Ángeles Risalde Jobin Thomas Iker Sevilla Miriam Serrano Jose Antonio Ortíz Joseba Garrido Mercedes Domínguez Lucas Domínguez Christian Gortázar Jose Francisco Ruíz-Fons Development and evaluation of an interferon gamma assay for the diagnosis of tuberculosis in red deer experimentally infected with Mycobacterium bovis BMC Veterinary Research Animal tuberculosis Cellular immune response Farmed Cervus elaphus In vivo test Mycobacterium tuberculosis complex |
author_facet |
María Ángeles Risalde Jobin Thomas Iker Sevilla Miriam Serrano Jose Antonio Ortíz Joseba Garrido Mercedes Domínguez Lucas Domínguez Christian Gortázar Jose Francisco Ruíz-Fons |
author_sort |
María Ángeles Risalde |
title |
Development and evaluation of an interferon gamma assay for the diagnosis of tuberculosis in red deer experimentally infected with Mycobacterium bovis |
title_short |
Development and evaluation of an interferon gamma assay for the diagnosis of tuberculosis in red deer experimentally infected with Mycobacterium bovis |
title_full |
Development and evaluation of an interferon gamma assay for the diagnosis of tuberculosis in red deer experimentally infected with Mycobacterium bovis |
title_fullStr |
Development and evaluation of an interferon gamma assay for the diagnosis of tuberculosis in red deer experimentally infected with Mycobacterium bovis |
title_full_unstemmed |
Development and evaluation of an interferon gamma assay for the diagnosis of tuberculosis in red deer experimentally infected with Mycobacterium bovis |
title_sort |
development and evaluation of an interferon gamma assay for the diagnosis of tuberculosis in red deer experimentally infected with mycobacterium bovis |
publisher |
BMC |
series |
BMC Veterinary Research |
issn |
1746-6148 |
publishDate |
2017-11-01 |
description |
Abstract Background Red deer (Cervus elaphus) is regarded as an epidemiologically relevant host for Mycobacterium bovis (M. bovis) and closely related members of the Mycobacterium tuberculosis complex that cause animal tuberculosis (TB). The standard antemortem screening test for the detection of TB in deer is the intradermal tuberculin skin test, but the detection of interferon-gamma (IFNγ) produced by white blood cells exposed to M. bovis antigens can be used as an alternative or supplemental assay in most TB eradication/control programs. This study aims to develop an in-house sandwich ELISA for deer IFNγ, based on the cross-reactivity of the antibodies to both cervid and bovine IFNγ, and to evaluate the potential of this assay to detect M. bovis-infected red deer in response to the in vitro stimulation of whole-blood cells with bovine purified protein derivative (bPPD), p22 protein complex derived from bPPD or using the specific tuberculous mycobacterial proteins ESAT-6/CFP-10, Rv3615c and Rv3020c. The positive control stimulant used in this study was pokeweed mitogen, which resulted in a consistent induction of IFNγ in samples from red deer, thus allowing the interpretation of the assay. Results The percentage of animals correctly classified by this technique as M. bovis non-infected was 100%. The detection of infected animals as positive was high and ranged widely depending upon the antigen and the cut-off value applied, as well as the time after infection. Our findings indicate that this protocol may serve as a reliable assay for the antemortem diagnosis of TB from the initial stage of M. bovis-infection, and may also be adequately sensitive. Conclusions The suggested optimal antigens and cut-off are bPPD, p22 and the combination of ESAT-6/CFP-10 and Rv3020c with a 0.05 Δ optical density, which yielded a up to 100% correct classification of TB positive and negatve red deer under our experimental conditions. This technique will aid in TB testing of farmed and translocated deer. Future studies should evaluate the ability of this IFNγ assay to detect specific responses under field conditions. |
topic |
Animal tuberculosis Cellular immune response Farmed Cervus elaphus In vivo test Mycobacterium tuberculosis complex |
url |
http://link.springer.com/article/10.1186/s12917-017-1262-6 |
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