scholarly journals Differentiation between Mycobacterium bovis BCG-Vaccinated and M. bovis-Infected Cattle by Using Recombinant Mycobacterial Antigens

1999 ◽  
Vol 6 (1) ◽  
pp. 1-5 ◽  
Author(s):  
Bryce M. Buddle ◽  
Natalie A. Parlane ◽  
Denise L. Keen ◽  
Frank E. Aldwell ◽  
John M. Pollock ◽  
...  
Keyword(s):  
Mycobacterium Bovis ◽  
Bovine Tuberculosis ◽  
Secreted Proteins ◽  
Infected Cattle ◽  
Recombinant Antigens ◽  
Purified Protein Derivative ◽  
Ifn Γ ◽  
Mycobacterial Antigens ◽  
Culture Positive ◽  
Culture Negative

ABSTRACT Tuberculosis continues to be a worldwide problem for both humans and animals. The development of tests to differentiate between infection with Mycobacterium tuberculosis orMycobacterium bovis and vaccination with M. bovis BCG could greatly assist in the diagnosis of early infection as well as enhance the use of tuberculosis vaccines on a wider scale. Recombinant forms of four major secreted proteins ofM. bovis—MPB59, MPB64, MPB70, and ESAT-6—were tested in a whole-blood gamma interferon (IFN-γ) assay for differentiation between cattle vaccinated with BCG and those experimentally infected with M. bovis. BCG vaccination induced minimal protection in the present study, with similar numbers of animals infected withM. bovis in BCG-vaccinated and nonvaccinated groups. Following vaccination with BCG, the animals produced moderate IFN-γ responses to bovine purified protein derivative (PPDB) but very weak responses to the recombinant antigens. Cattle from both the BCG-vaccinated and nonvaccinated groups which were M. bovisculture positive following challenge produced IFN-γ responses to PPDB and ESAT-6 which were significantly stronger than those observed in the corresponding M. bovis culture-negative animals. IFN-γ responses to MPB59, MPB64, and MPB70 were significantly weaker, and these antigens could not discriminate between vaccinated animals which develop disease and the culture-negative animals. The results of the study indicate that of the four antigens tested in the IFN-γ assay, only ESAT-6 would be suitable for differentiating BCG-vaccinated animals from those infected with bovine tuberculosis.

scholarly journals Evaluation of Gamma Interferon (IFN-γ)-Induced Protein 10 Responses for Detection of Cattle Infected with Mycobacterium bovis: Comparisons to IFN-γ Responses

2012 ◽  
Vol 19 (3) ◽  
pp. 346-351 ◽  
Author(s):  
W. R. Waters ◽  
T. C. Thacker ◽  
B. J. Nonnecke ◽  
M. V. Palmer ◽  
I. Schiller ◽  
...  
Keyword(s):  
Mycobacterium Bovis ◽  
Similar Pattern ◽  
Gamma Interferon ◽  
Content Type ◽  
Purified Protein Derivative ◽  
Testing Protocol ◽  
Archived Samples ◽  
Ifn Γ ◽  
Highly Correlated ◽  
Mycobacterial Antigens

ABSTRACTGamma interferon (IFN-γ)-induced protein 10 (IP-10) has recently shown promise as a diagnostic biomarker ofMycobacterium tuberculosisinfection of humans. The aim of the current study was to compare IP-10 and IFN-γ responses uponMycobacterium bovisinfection in cattle by using archived samples from two aerosol inoculation studies. In the first study (104CFUM. bovisby aerosol,n= 7),M. bovispurified protein derivative (PPDb)-specific IP-10 and IFN-γ gene expression was detected as early as 29 days after challenge. PPDb-specific IP-10 and IFN-γ mRNA responses followed a similar pattern of expression over the course of this study and were highly correlated (r= 0.87). In the second study (105CFUM. bovisby aerosol,n= 5), IP-10 and IFN-γ (protein) responses to mycobacterial antigens were compared following challenge. IFN-γ responses to mycobacterial antigens were detected at 29 days after challenge and were sustained during the remainder of the study. IFN-γ responses to mycobacterial antigens exceeded corresponding responses in nonstimulated cultures. IP-10 responses to mycobacterial antigens exceeded preinfection responses at 7, 29, and 63 days after challenge. In contrast to IFN-γ responses, IP-10 responses to mycobacterial antigens generally did not exceed the respective responses in nonstimulated cultures. IP-10 responses to medium alone and to mycobacterial antigens followed a similar pattern of response. Correlations between IP-10 and IFN-γ (protein) responses were modest (r≈ 0.50 to 0.65). Taken together, these findings do not support the use of IP-10 protein as a biomarker for bovine tuberculosis using the current testing protocol and reagents; however, mRNA-based assays may be considered for further analysis.

scholarly journals Cellular and Cytokine Responses in the Granulomas of Asymptomatic Cattle naturally infected with Mycobacterium bovis in Ethiopia

2020 ◽  
Author(s):  
Begna Tulu ◽  
Henny M Martineau ◽  
Aboma Zewude ◽  
Fekadu Desta ◽  
David A Jolliffe ◽  
...  
Keyword(s):  
T Cells ◽  
Mycobacterium Bovis ◽  
Addis Ababa ◽  
Natural Infection ◽  
Stage Iv ◽  
Stage I ◽  
Tnf Α ◽  
Ifn Γ ◽  
Culture Positive ◽  
Culture Negative

ABSTRACTCellular (CD3+ T cell and CD68+ macrophages), cytokines (IFN-γ+ and TNF-α+) and effector molecule (iNOS+) responses were evaluated in the lymph nodes and tissue of cattle naturally infected with Mycobacterium bovis. Detailed post mortem and immunonohistochemical examinations of lesions were performed on 16 cows positive for single intradermal cervical comparative tuberculin test (SICCTT) and identified from dairy farms located around the Addis Ababa City. The severity of the gross lesion was significantly higher (p=0.003) in M. bovis culture positive (n=12) cows than in culture negative (n=4). Immunohistochemical techniques showed that the mean percentage labelling of CD3+ T cells decreased as the stage of granuloma increased from stage I to stage IV in culture positive cows (p<0.001). On the other hand, the proportional fraction of CD68+ macrophages and the concentrations of IFN-γ+, TNF-α+ and iNOS+ increased significantly from stage I to stage IV (p< 0.001) in culture positive cows. At the early stage of the granuloma, the culture negative cows showed significantly higher mean proportions of CD68+ macrophages (p=0.03) as well as the concentrations of iNOS+ (p=0.007) compared to culture positive cows. Similarly, at advanced granuloma stages, culture negative cows demonstrated significantly higher mean proportions of CD3+ T cells (p< 0.001) compared to culture positive cows. Thus, the present study demonstrated that following natural infection of cows with M. bovis, as the stage of granuloma increases from stage I to stage IV, the proportion of CD3+ cells decreases while the immunolabeling fraction of CD68+ macrophages, IFN-γ+, TNF-α and iNOS+ increases.

scholarly journals Specificity of the Tuberculin Skin Test Is Modified by Use of a Protein Cocktail Containing ESAT-6 and CFP-10 in Cattle Naturally Infected with Mycobacterium bovis

2012 ◽  
Vol 19 (5) ◽  
pp. 797-803 ◽  
Author(s):  
S. Flores-Villalva ◽  
F. Suárez-Güemes ◽  
C. Espitia ◽  
A. O. Whelan ◽  
M. Vordermeier ◽  
...  
Keyword(s):  
Skin Test ◽  
Mycobacterium Bovis ◽  
Bovine Tuberculosis ◽  
Infected Cattle ◽  
Test Format ◽  
Mycobacterial Species ◽  
Purified Protein Derivative ◽  
Skin Response ◽  
Low Prevalence ◽  
Tuberculosis Prevalence

ABSTRACTThe mycobacterial immunodominant ESAT-6 and CFP-10 antigens are strongly recognizable in tuberculosis-infected cattle, and they do not elicit a response in cattle without infection. In addition, they are absent in most environmental mycobacterial species, and therefore, their use can be an alternative to purified protein derivative (PPD) tuberculin in the development of a more specific skin diagnostic test in cattle. The aim of the current study was to assess the potential of an ESAT-6 and CFP-10 (E6-C10) protein cocktail in a skin test format in naturally tuberculosis-infected and paratuberculosis-infected cattle. We also included MPB83 as a third component in one of the protein cocktail preparations. The protein cocktail was tested at different dose concentrations (5, 10, and 15 μg per protein). The best skin response to the E6-C10 protein cocktail was obtained with 10 μg. Subsequently, this concentration was tested in 2 herds with high and low bovine tuberculosis prevalence, the latter with paratuberculosis coinfection. Our data show that the E6-C10 cocktail allows identification of an important proportion of animals that PPDB is not able to recognize, especially in low-prevalence herds. The protein cocktail did not induce reactions in tuberculosis-free cattle or in paratuberculosis-infected cattle. Addition of MPB83 to the protein cocktail did not make any difference in the skin reaction.

scholarly journals Cellular and Cytokine Responses in the Granulomas of Asymptomatic Cattle Naturally Infected with Mycobacterium bovis in Ethiopia

2020 ◽  
Vol 88 (12) ◽  
Author(s):  
Begna Tulu ◽  
Henny M. Martineau ◽  
Aboma Zewude ◽  
Fekadu Desta ◽  
David A. Jolliffe ◽  
...  
Keyword(s):  
T Cells ◽  
Mycobacterium Bovis ◽  
Natural Infection ◽  
Stage Iv ◽  
Stage I ◽  
Content Type ◽  
Tnf Α ◽  
Ifn Γ ◽  
Culture Positive ◽  
Culture Negative

ABSTRACT Cell (CD3+ T cell and CD68+ macrophages), cytokine (interferon gamma-positive [IFN-γ+] and tumor necrosis factor alpha-positive [TNF-α+]), and effector molecule (inducible nitric oxide synthase-positive [iNOS+]) responses were evaluated in the lymph nodes and tissues of cattle naturally infected with Mycobacterium bovis. Detailed postmortem and immunohistochemical examinations of lesions were performed on 16 cows that were positive by the single intradermal cervical comparative tuberculin (SICCT) test and that were identified from dairy farms located around the city of Addis Ababa, Ethiopia. The severity of the gross lesion was significantly higher (P = 0.003) in M. bovis culture-positive cows (n = 12) than in culture-negative cows (n = 4). Immunohistochemical techniques showed that in culture-positive cows, the mean immunolabeling fraction of CD3+ T cells decreased as the stage of granuloma increased from stage I to stage IV (P < 0.001). In contrast, the CD68+ macrophage, IFN-γ+, TNF-α+, and iNOS+ immunolabeling fractions increased from stage I to stage IV (P < 0.001). In the early stages, culture-negative cows showed a significantly higher fraction of CD68+ macrophage (P = 0.03) and iNOS+ (P = 0.007) immunolabeling fractions than culture-positive cows. Similarly, at advanced granuloma stages, culture-negative cows demonstrated significantly higher mean proportions of CD3+ T cells (P < 0.001) than culture-positive cows. Thus, this study demonstrates that, following natural infection of cows with M. bovis, as the stage of granuloma increases from stage I to stage IV, the immunolabeling fraction of CD3+ cells decreases, while the CD68+ macrophage, IFN-γ+, TNF-α+, and iNOS+ immunolabeling fractions increases.

scholarly journals Assessment of Mycobacterium tuberculosis OmpATb as a Novel Antigen for the Diagnosis of Bovine Tuberculosis

2009 ◽  
Vol 16 (9) ◽  
pp. 1314-1321 ◽  
Author(s):  
Irene Schiller ◽  
H. Martin Vordermeier ◽  
W. Ray Waters ◽  
Mitchell Palmer ◽  
Tyler Thacker ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Bovine Tuberculosis ◽  
Diagnostic Tests ◽  
Protein A ◽  
Infected Cattle ◽  
Purified Protein Derivative ◽  
Specificity And Sensitivity ◽  
Combined Use ◽  
Release Assay ◽  
Ifn Γ

ABSTRACT In the search for better tools to control bovine tuberculosis, the development of diagnostic tests with improved specificity and sensitivity has a high priority. We chose to search for novel immunodiagnostic reagents. In this study, Rv0899 (outer membrane protein A of Mycobacterium tuberculosis [OmpATb]) was evaluated as a stimulation antigen in a gamma interferon (IFN-γ) release assay to diagnose bovine tuberculosis. OmpATb induced IFN-γ responses in cattle experimentally infected with M. bovis as early and as persistently as ESAT-6 and CFP-10, the current lead diagnostic antigens. In naturally infected cattle, OmpATb stimulated IFN-γ production in 22 of 26 animals (85%). Importantly, OmpATb detected a portion of M. bovis-infected cattle which did not respond to ESAT-6 and CFP-10 (five of six cattle). The combined diagnostic sensitivity of OmpATb, ESAT-6, and CFP-10 for a preselected group consisting of naturally infected cattle with an overrepresentation of ESAT-6/CFP-10 nonresponders was 96% (25 of 26 animals). The specificity of OmpATb for uninfected cattle was 100% (27 cattle were tested; 12 of them gave false-positive results with tuberculins). In summary, our results indicate that OmpATb has the potential to enhance the sensitivity of previously described diagnostic tests based on ESAT-6 and CFP-10 and that the combined use of OmpATb, ESAT-6, CFP-10, and other proteins may achieve at least equal sensitivity to that obtained with purified protein derivative, but at a higher specificity. Further studies evaluating the diagnostic performance of OmpATb in combination with other proteins are ongoing.

scholarly journals Flow Cytometric Detection of Gamma Interferon Can Effectively Discriminate Mycobacterium bovis BCG-Vaccinated Cattle from M. bovis-Infected Cattle

2006 ◽  
Vol 13 (12) ◽  
pp. 1343-1348 ◽  
Author(s):  
P. Sopp ◽  
C. J. Howard ◽  
J. C. Hope
Keyword(s):  
Mycobacterium Bovis ◽  
Bovine Tuberculosis ◽  
Human Health Risk ◽  
Flow Cytometric Analysis ◽  
Gamma Interferon ◽  
Accurate Diagnosis ◽  
Economic Losses ◽  
Infected Cattle ◽  
Flow Cytometric ◽  
Ifn Γ

ABSTRACT Mycobacterium bovis is the causative agent of bovine tuberculosis, a disease that is increasing in incidence in United Kingdom cattle herds. In addition to increasing economic losses, the rise in bovine tuberculosis poses a human health risk. There is an urgent requirement for effective strategies for disease eradication; this will likely involve vaccination in conjunction with current test and slaughter policies. A policy involving vaccination would require an accurate diagnosis of M. bovis-infected animals and the potential to distinguish these animals from vaccinates. Currently used diagnostic tests, the skin test and gamma interferon (IFN-γ) blood test, have a sensitivity of up to 95%. A further complication is that M. bovis BCG-vaccinated animals are also scored positive by these tests. We tested the hypothesis that the quantification of IFN-γ-producing lymphocytes by flow cytometric analysis of intracellular IFN-γ expression would provide a more accurate discrimination of M. bovis-infected animals from BCG vaccinates. Significant numbers of IFN-γ-expressing CD4+ T cells were detected following culture of heparinized blood from M. bovis-infected animals, but not from BCG vaccinates, with purified protein derived from M. bovis (PPD-B) or live mycobacteria. Only 1 of 17 BCG-vaccinated animals had a significant number of CD4+ T lymphocytes expressing IFN-γ, compared with 21/22 M. bovis-infected animals. This assay could allow an accurate diagnosis of M. bovis and allow the discrimination of BCG-vaccinated cattle from infected cattle.

scholarly journals Screening of Highly Expressed Mycobacterial Genes Identifies Rv3615c as a Useful Differential Diagnostic Antigen for the Mycobacterium tuberculosis Complex

2008 ◽  
Vol 76 (9) ◽  
pp. 3932-3939 ◽  
Author(s):  
Ben Sidders ◽  
Chris Pirson ◽  
Philip J. Hogarth ◽  
R. Glyn Hewinson ◽  
Neil G. Stoker ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Significant Proportion ◽  
Growth Conditions ◽  
Control Measures ◽  
Human Populations ◽  
Infected Cattle ◽  
Ifn Γ ◽  
Mycobacterial Antigens ◽  
And Control ◽  
Differential Antigens

ABSTRACT Tuberculous infections caused by mycobacteria, especially tuberculosis of humans and cattle, are important both clinically and economically. Human populations can be vaccinated with Mycobacterium bovis bacille Calmette-Guérin (BCG), and control measures for cattle involving vaccination are now being actively considered. However, diagnostic tests based on tuberculin cannot distinguish between genuine infection and vaccination with BCG. Therefore, identification of differential diagnostic antigens capable of making this distinction is required, and until now sequence-based approaches have been predominant. Here we explored the link between antigenicity and mRNA expression level, as well as the possibility that we may be able to detect differential antigens by analyzing quantified global transcriptional profiles. We generated a list of 14 candidate antigens that are highly expressed in Mycobacterium tuberculosis and M. bovis under a variety of growth conditions. These candidates were screened in M. bovis-infected and naïve cattle for the ability to stimulate a gamma interferon (IFN-γ) response. We identified one antigen, Rv3615c, which stimulated IFN-γ responses in a significant proportion of M. bovis-infected cattle (11 of 30 cattle [37%] [P < 0.01]) but not in naïve or BCG-vaccinated animals. Importantly, the same antigen stimulated IFN-γ responses in a significant proportion of infected cattle that did not respond to the well-characterized mycobacterial antigens ESAT-6 and CFP-10. Therefore, use of the Rv3615c epitope in combination with previously described differential tests based on ESAT-6 and CFP-10 has the potential to significantly increase diagnostic sensitivity without reducing specificity in BCG-vaccinated populations.

scholarly journals Improved Skin Test for Differential Diagnosis of Bovine Tuberculosis by the Addition of Rv3020c-Derived Peptides

2012 ◽  
Vol 19 (4) ◽  
pp. 620-622 ◽  
Author(s):  
Gareth J. Jones ◽  
Adam Whelan ◽  
Derek Clifford ◽  
Mick Coad ◽  
H. Martin Vordermeier
Keyword(s):  
Differential Diagnosis ◽  
Skin Test ◽  
Mycobacterium Bovis ◽  
Bovine Tuberculosis ◽  
Johne's Disease ◽  
Diagnostic Sensitivity ◽  
Bacillus Calmette Guerin ◽  
Content Type ◽  
The Face ◽  
Mycobacterial Antigens

ABSTRACTA peptide cocktail derived from the mycobacterial antigens ESAT-6, CFP-10, and Rv3615c allowed differentiation betweenMycobacterium bovis-infected and M. bovis bacillus Calmette-Guérin (BCG)-vaccinated cattle when used as a skin test reagent for a “DIVA” test (i.e., a test capable ofdifferentiatinginfected and uninfectedvaccinatedanimals). Addition of the antigen Rv3020c improves the diagnostic sensitivity without compromising specificity in the face of BCG or Johne's disease vaccination.

scholarly journals Effects of Serial Skin Testing with Purified Protein Derivative on the Level and Quality of Antibodies to Complex and Defined Antigens in Mycobacterium bovis-Infected Cattle

2015 ◽  
Vol 22 (6) ◽  
pp. 641-649 ◽  
Author(s):  
W. Ray Waters ◽  
Mitchell V. Palmer ◽  
Molly R. Stafne ◽  
Kristin E. Bass ◽  
Mayara F. Maggioli ◽  
...  
Keyword(s):  
Mycobacterium Bovis ◽  
Specific Antibody ◽  
Skin Testing ◽  
Antibody Responses ◽  
Proteinase K ◽  
Skin Tests ◽  
Infected Cattle ◽  
Content Type ◽  
Purified Protein Derivative ◽  
In Series

ABSTRACTSeveral serological tests designed to detect antibodies to immunodominantMycobacterium bovisantigens have recently emerged as ancillary tests for the detection of bovine tuberculosis in cattle, particularly when used after the injection of purified protein derivative (PPD) for skin testing, which significantly boostsM. bovis-specific antibody responses. The present findings demonstrate the onset and duration of boosted antibody responses after the injection ofM. bovisPPD for the caudal fold test (CFT) andMycobacterium aviumandM. bovisPPDs for the comparative cervical test (CCT), administered in series in cattle experimentally infected withM. bovis. While skin tests boosted the responses to certain antigens (i.e., MPB83 and MPB70), they did not affect the responses to other antigens (e.g., ESAT-6, CFP10, MPB59, and MPB64). Administration of the CCT 105 days after the CFT resulted in an even greater secondary boost in antibody responses to MPB83 and MPB70 and to a proteinase K-digested whole-cell sonicate (WCS-PK) ofM. bovis. Both IgM and IgG contributed to the initial boost in the MPB83/MPB70-specific antibody response after the CFT. The secondary boost after the CCT was primarily due to increased IgG levels. Also, the avidity of antibodies to MPB83 and MPB70 increased after the CCT inM. bovis-infected cattle. The avidity of antibodies to the WCS-PK antigens increased in the interval between the CFT and the CCT but did not increase further after the CCT. Together, these findings demonstrate that the administration of PPDs for skin tests results in additive enhancement (i.e., when the CFT and CCT are performed in series), both qualitative and quantitative, of MPB83/MPB70-specific antibody responses.

scholarly journals Recognition of Mycobacterial Antigens Delivered by Genetically Detoxified Bordetella pertussis Adenylate Cyclase by T Cells from Cattle with Bovine Tuberculosis

2004 ◽  
Vol 72 (11) ◽  
pp. 6255-6261 ◽  
Author(s):  
H. Martin Vordermeier ◽  
Marcela Simsova ◽  
Katalin A. Wilkinson ◽  
Robert J. Wilkinson ◽  
R. Glyn Hewinson ◽  
...  
Keyword(s):  
T Cells ◽  
Adenylate Cyclase ◽  
Fusion Protein ◽  
Bordetella Pertussis ◽  
Bovine Tuberculosis ◽  
Economic Problem ◽  
T Cell Recognition ◽  
Ifn Γ ◽  
Mycobacterial Antigens ◽  
Immunodiagnostic Tests

ABSTRACT The exponential increase in the incidence of tuberculosis in cattle over the last two decades in the British national herd constitutes a significant economic problem. Therefore, research efforts are under way to develop cattle tuberculosis vaccines and specific diagnostic reagents to allow the distinction of vaccinated from infected animals. Mycobacterial antigens like ESAT-6 and CFP10 allow this distinction. This study investigates whether fusion protein of ESAT-6 or CFP10 with genetically detoxified Bordetella pertussis adenylate cyclase (CyaA) are recognized by Mycobacterium bovis-infected cattle more effectively than conventional recombinant proteins are, thus enhancing sensitivity or reducing the amount of antigens required. By measuring the frequencies of gamma interferon (IFN-γ)-producing cells, we were able to show that the presentation of CFP10 as a CyaA fusion protein enhanced the molecular efficiency of its recognition 20-fold, while the recognition of ESAT-6 was not improved by CyaA delivery. Furthermore, in the whole-blood IFN-γ test currently used in the field, the delivery of CFP10 and ESAT-6 by fusion to CyaA increased the amount of IFN-γ produced and hence the proportion of infected animals responding to CFP10. The improved T-cell recognition of CyaA336/CFP10 was found to be dependent upon interaction with CD11b. In addition, presentation of CyaA336/CFP10 to CD4+ T cells was chloroquine sensitive, and CFP10 delivery by CyaA resulted in its accelerated presentation to T cells. In conclusion, the use of CyaA fusion proteins with ESAT-6 and CFP10 has the potential to improve the sensitivity of immunodiagnostic tests detecting bovine tuberculosis in cattle.

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