title: Development of an improved ESAT-6 and CFP-10 peptide-based cytokine flow cytometric assay for bovine tuberculosis.
abstract: Control of bovine tuberculosis (bTB) continues to be a problem world-wide because of difficulties in identifying infected animals at all stages of infection. The use of the IFN-γ release assays (IGRA) as an ancillary test with the tuberculin skin tests has improved the ability to identify infected animals. However, infected animals may still be missed. The objective of the present study was to evaluate a rapid flow-cytometric assay based on intracellular cytokine staining as an alternative to the in vitro IFN-γ release assay (IGRA). Antigen-specific cells producing IFN-γ were identified after a 6h stimulation with PPD-B, PPD-A and ESAT-6/CFP-10. Defined groups of animals naturally infected with Mycobacterium bovis (Mbv), animals infected with non-tuberculous mycobacteria (NTM), and uninfected control animals were analysed to evaluate the sensitivity and specificity of the optimized assay. Both antemortem and postmortem diagnostic tests were carried out to verify the status of infection. We show that IFN-γ is induced in T cells from whole blood samples from cattle infected with Mbv 6h post stimulation with PPD-B, PPD-A and ESAT-6/CFP-10, whereas non-infected animals did not respond. Four colour flow cytometric analysis demonstrated responding cells were CD45R0(+)CD69(+)CD4(+) memory T cells. Also, the response to stimulation with ESAT-6/CFP-10 can be used to distinguish between cattle infected with Mbv and cattle exposed to NTM. Although further studies are needed, the results indicate that detection of intracellular IFN-γ may represent an important alternative approach for improved method of detection of cattle secreting IFN-γ below levels of detection in culture medium.
authors.forename: Mahmoud M
authors.lastname: Elnaggar
authors.affiliation: Department of Microbiology, Faculty of Veterinary Medicine, Alexandria University, Alexandria, Egypt.
authors.forename: Mahmoud M
authors.initials: MM
authors.lastname: El-Naggar
authors.affiliation: Department of Microbiology, Faculty of Veterinary Medicine, Alexandria University, Alexandria, Egypt. Electronic address: gaber.abdellatif@alexu.edu.eg.
authors.forename: Gaber S
authors.initials: GS
authors.lastname: Abdellrazeq
authors.affiliation: Department of Transplant and Infection Immunology, Institute of Virology, Saarland University, Homburg, Germany.
authors.forename: Martina
authors.initials: M
authors.lastname: Sester
authors.affiliation: Department of Microbiology, Faculty of Veterinary Medicine, Alexandria University, Alexandria, Egypt.
authors.forename: Samy A
authors.initials: SA
authors.lastname: Khaliel
authors.affiliation: LIONEX Diagnostics & Therapeutics, Braunschweig, Germany.
authors.forename: Mahavir
authors.initials: M
authors.lastname: Singh
authors.affiliation: Department of Microbiology, Faculty of Veterinary Medicine, Alexandria University, Alexandria, Egypt.
authors.forename: Helmy A
authors.initials: HA
authors.lastname: Torky
authors.affiliation: Department of Veterinary Microbiology and Pathology, College of Veterinary Medicine, Pullman, WA, USA.
authors.forename: William C
authors.initials: WC
authors.lastname: Davis
mesh_terms: D000818:Animals; D000942:Antigens, Bacterial / Q000276:immunology*; D015703:Antigens, CD / Q000276:immunology; D000945:Antigens, Differentiation, T-Lymphocyte / Q000276:immunology; D001426:Bacterial Proteins / Q000276:immunology*; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology*; D002417:Cattle; D005434:Flow Cytometry / Q000379:methods*; D007156:Immunologic Memory; D007371:Interferon-gamma / Q000097:blood*; D059425:Interferon-gamma Release Tests / Q000379:methods*; D037181:Lectins, C-Type / Q000276:immunology; D017493:Leukocyte Common Antigens / Q000276:immunology; D008222:Lymphokines; D009163:Mycobacterium bovis / Q000276:immunology; D010455:Peptides / Q000276:immunology; D012680:Sensitivity and Specificity; D014374:Tuberculin Test; D014380:Tuberculosis, Bovine / Q000175:diagnosis*; D000099192:CD69 Antigens
chemical_list: D000942:Antigens, Bacterial; D015703:Antigens, CD; D000945:Antigens, Differentiation, T-Lymphocyte; D001426:Bacterial Proteins; D007371:Interferon-gamma; D037181:Lectins, C-Type; D017493:Leukocyte Common Antigens; D008222:Lymphokines; D010455:Peptides; D000099192:CD69 Antigens; C553218:CFP-10 protein, Mycobacterium bovis; C525005:ESAT-6 antigen, Mycobacterium leprae; C043695:lymphocyte proliferation potentiating factors
keywords: Bovine tuberculosis; Cytokine flow cytometry; ESAT-6/CFP-10; IFN-γ release assay; Mycobacterium bovis