Genomic Approach to Identification of
            <i>Mycobacterium bovis</i>
            Diagnostic Antigens in Cattle

Aagaard, Claus; Govaerts, Marc; Okkels, Limei Meng; Andersen, Peter; Pollock, J.M.

doi:10.1128/jcm.41.8.3719-3728.2003

Cited by 50 publications

(45 citation statements)

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“…The diagnostic antigen selected should ideally accommodate the possible application of the M. bovis bacillus CalmetteGuérin (BCG) vaccine in the future (9). Genetic comparison of M. bovis and a number of BCG strains has revealed several large genomic regions in BCG strains that have been lost during the attenuation process (5,18); and because of their theoretical lack of interference with a BCG vaccine, the proteins encoded by these regions have received growing attention as diagnostic antigens, especially antigens from region of difference (RD) RD1 (1,10,19,26,30). All studies have clearly identified the two RD1 antigens ESAT6 and CFP10 as being the most immune dominant of those tested with experimentally infected cattle.…”

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Optimizing Antigen Cocktails for Detection ofMycobacterium bovisin Herds with Different Prevalences of Bovine Tuberculosis: ESAT6-CFP10 Mixture Shows Optimal Sensitivity and Specificity

et al. 2006

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Bovine tuberculosis is a major problem in many countries; hence, new and better diagnostic tools are urgently needed. In this work, we have tested ESAT6, CFP10, PE13, PE5, MPB70, TB10.4, and TB27.4 for their potentials as diagnostic markers in field animals from Northern Ireland, Mexico, and Argentina, regions with low, medium, and high prevalences of bovine tuberculosis, respectively. At all three sites, ESAT6 and CFP10 were superior diagnostic antigens, while their combination performed even better at the two sites where the combination was tested, providing the best coverage for the detection of diseased populations. The high sensitivity in the skin test reactor groups, combined with the high specificity in the tuberculosis-free groups, indicated that a diagnosis could correctly be made for 85% of the infected animals, based on their responses to these two antigens. Furthermore, TB10.4, PE13, and PE5 have the potential to supplement ESAT6 and CFP10 in a future five-component diagnostic cocktail.

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Optimizing Antigen Cocktails for Detection ofMycobacterium bovisin Herds with Different Prevalences of Bovine Tuberculosis: ESAT6-CFP10 Mixture Shows Optimal Sensitivity and Specificity

et al. 2006

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“…A kit based on whole-blood production of IFN-␥ detected by enzyme-linked immunosorbent assay (ELISA) is available and has the advantage of relative simplicity, but being based on PPD, it does not ameliorate the poor specificity of skin testing (6). In addition, this test requires 10 ml of blood, a large amount if one considers its application in pediatric practice.We were interested to determine, by the use of a sensitive IFN-␥ ELISA, whether a form of the whole blood stimulation assay that is used by many groups to assay antigen specific responses to RD1 encoded antigens (1,3,7,9,16) could match the performance of the ELISPOT assay. Ethical permission for this study was provided by the Harrow local research ethics committee (Harrow LREC 1646).…”

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confidence: 99%

“…We were interested to determine, by the use of a sensitive IFN-␥ ELISA, whether a form of the whole blood stimulation assay that is used by many groups to assay antigen specific responses to RD1 encoded antigens (1,3,7,9,16) could match the performance of the ELISPOT assay. Ethical permission for this study was provided by the Harrow local research ethics committee (Harrow LREC 1646).…”

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Gamma Interferon-Based Immunodiagnosis of Tuberculosis: Comparison between Whole-Blood and Enzyme-Linked Immunospot Methods

et al. 2004

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The gamma interferon secretion levels of 52 adults whose T cells had been stimulated with the Mycobacterium tuberculosis antigens CFP-10 and ESAT-6 were measured by enzyme-linked immunospot methods and a whole-blood-based assay. The test results correlated well (r ‫؍‬ 0.689, P < 0.0001). Its simple format and use of only small volumes of blood make the whole-blood assay suitable for pediatric practice and field trials.Mycobacterium tuberculosis is a significant threat to global health (11). The skin test reagent used to aid diagnosis of both active and latent tuberculosis, purified protein derivative (PPD), is impaired in specificity and sensitivity (22), and the skin reaction may cause discomfort. Recent interest in the field of immunodiagnosis has therefore concentrated on antigens encoded by the RD1 genomic segment of M. tuberculosis, which is deleted from all BCG strains (5,15,19). In addition, the idea of replacing the tuberculin skin test (TST) by a method that assays the in vitro production of gamma interferon (IFN-␥) in response to M. tuberculosis antigens has gained popularity (2, 6, 9, 17). The most promising approach combines the recognized high sensitivity of ELISPOT analysis with the use of peptides covering the sequence of the RD1-encoded ESAT-6 molecule into a diagnostic test of greater sensitivity and specificity than the TST (4, 13, 18, 21). The enzyme-linked immunospot (ELISPOT) technique requires separation of cells from blood, the use of multiple wells of an expensive precoated plate, and an automated ELISPOT counter. Some have therefore wondered whether and how these advances could be applied to medically underserved environments (3). A kit based on whole-blood production of IFN-␥ detected by enzyme-linked immunosorbent assay (ELISA) is available and has the advantage of relative simplicity, but being based on PPD, it does not ameliorate the poor specificity of skin testing (6). In addition, this test requires 10 ml of blood, a large amount if one considers its application in pediatric practice.We were interested to determine, by the use of a sensitive IFN-␥ ELISA, whether a form of the whole blood stimulation assay that is used by many groups to assay antigen specific responses to RD1 encoded antigens (1,3,7,9,16) could match the performance of the ELISPOT assay. Ethical permission for this study was provided by the Harrow local research ethics committee (Harrow LREC 1646). To perform the whole-blood assay, heparinized blood was diluted 1:10 in RPMI and 180 l of the diluted blood was stimulated with 2.5 g/ml ESAT-6 prepared as described previously (20) or 5 g/ml CFP-10 (encoded by the gene adjacent to ESAT-6, obtained commercially from Lionex, Braunschweig, Germany) or no stimulus in duplicate on a 96-well plate. Diluted blood was cultured at 37°C in a CO 2 incubator for 72 h, at which point the supernatants were aspirated for determination of IFN-␥ level by ELISA (Antibody pair from BD Pharmingen catalogue no. 554548 and 554550). This ELISA has a working sensitivity of Ͻ10 pg/ml. Initially...

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“…We applied a high-throughput immunological screening system based on pools of chemically synthesized peptides representing the complete amino acid sequences of target proteins. In the first set of experiments we assessed the immunogenicity of the 28 potential antigens encoded in the RD1, RD2, and RD14 gene regions that are deleted from the genome of BCG Pasteur (Brosch et al, 2007;Garnier et al, 2003) Other immunogenic antigens identified that could be useful for either diagnosis or as vaccine antigens include proteins belonging to the important ESAT-6 protein family (Pallen, 2002), such as Rv3019c or Rv0288 (Aagaard et al, 2003;Cockle et al, 2006;Vordermeier et al, 2003).…”

Section: Comparative Genomicsmentioning

confidence: 99%

Mycobacterium bovis antigens for the differential diagnosis of vaccinated and infected cattle

Vordermeier

Gordon

Hewinson

2011

Veterinary Microbiology

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The urgency for new and improved cattle vaccines and diagnostic reagents for Bovine tuberculosis (TB) has made their development a research priority in Great Britain (GB). Significant progress has been made to develop specific antigens that allow the differentiation of BCG vaccinated and M. bovis infected cattle (DIVA test). This has been greatly facilitated by the completion of the genome sequences of M. tuberculosis, M. bovis and BCG Pasteur and the subsequent application of comparative genome and transcriptome analysis to define DIVA antigens that complemented the prototype DIVA antigens ESAT-6 and CFP-10 by increasing their test sensitivity. Finally, we present an update of our current approaches in this area. Keywords: DIVA, BCG, Mycobacterium tuberculosis, Mycobacterium bovis, differential diagnosis, antigen reagentsPage 3 of 26 A c c e p t e d M a n u s c r i p t 3 Development of cattle TB vaccinesOver the last two decades the (tuberculin) test and slaughter strategy failed to prevent a dramatic rise in the incidence of TB in cattle in England and Wales (http://www.defra.gov.uk/animalh/tb/stats/county.htm). The urgency for new and improved cattle vaccines and diagnostic reagents has been acknowledged by the United Kingdom government.(http://www.defra.gov.uk/animalh/tb/vaccination/index.htm).However, the short-term development of improved cattle TB vaccines faces two major challenges: The identification of vaccine strategies that enhance the efficacy of M. bovis bacille Calmette Guérin (BCG), and the development of a diagnostic test that can be used alongside vaccination to differentiate vaccinated and infected cattle (DIVA test).The significant progress in developing TB vaccines for cattle has been reviewed in a number of recent articles Buddle et al., 2006; Hogarth et al., 2006; Hope and Vordermeier, 2005; Vordermeier et al., 2006a; Vordermeier and Hewinson, 2006). Briefly, results in cattle have shown that the most effective vaccination strategy against bovine TB is based on priming the immune system with BCG followed by boosting with subunit vaccines containing protective antigens that are present in BCG (heterologous prime-boost strategy), DNA (Skinner et al., 2003;, protein , or viral subunit vaccines (Vordermeier et al., 2006b; Vordermeier et al., 2004;Vordermeier et al., 2009). Nevertheless, all these strategies resulted in at least a proportion of animals becoming tuberculin-test positive (both in tuberculin skin and IFN-! tests). Thus, the development of Differential diagnosis of infected from vaccinated individuals (DIVA).Bovine tuberculosis in cattle is viewed as a spectral disease with cellmediated immune responses developing early post-infection and serum responses becoming patent at later and advanced stages of disease Pollock et al., 2001;Ritacco et al., 1991). Therefore, diagnostic assays like tuberculin skin testing or in vitro cytokine detection assays as the generally suffered from poor sensitivity. However, it should be noted that recent advances in this technology using b...

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Genomic Approach to Identification of Mycobacterium bovis Diagnostic Antigens in Cattle

Cited by 50 publications

References 44 publications

Optimizing Antigen Cocktails for Detection ofMycobacterium bovisin Herds with Different Prevalences of Bovine Tuberculosis: ESAT6-CFP10 Mixture Shows Optimal Sensitivity and Specificity

Optimizing Antigen Cocktails for Detection ofMycobacterium bovisin Herds with Different Prevalences of Bovine Tuberculosis: ESAT6-CFP10 Mixture Shows Optimal Sensitivity and Specificity

Gamma Interferon-Based Immunodiagnosis of Tuberculosis: Comparison between Whole-Blood and Enzyme-Linked Immunospot Methods

Mycobacterium bovis antigens for the differential diagnosis of vaccinated and infected cattle

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