title: The elephant interferon gamma assay: a contribution to diagnosis of tuberculosis in elephants.
abstract: Mycobacterium tuberculosis (M. tb) has been shown to be the main causative agent of tuberculosis in elephants worldwide. M. tb may be transmitted from infected humans to other species including elephants and vice versa, in case of prolonged intensive contact. An accurate diagnostic approach covering all phases of the infection in elephants is required. As M. tb is an intracellular pathogen and cell-mediated immune (CMI) responses are elicited early after infection, the skin test is the CMI assay of choice in humans and cattle. However, this test is not applicable in elephants. The interferon gamma (IFN-γ) assay is considered a good alternative for the skin test in general, validated for use in cattle and humans. This study was aimed at development of an IFN-γ assay applicable for diagnosis of tuberculosis in elephants. Recombinant elephant IFN-γ (rEpIFN-γ) produced in eukaryotic cells was used to immunize mice and generate the monoclonal antibodies. Hybridomas were screened for IFN-γ-specific monoclonal antibody production and subcloned, and antibodies were isotyped and affinity purified. Western blot confirmed recognition of the rEpIFN-γ. The optimal combination of capture and detection antibodies selected was able to detect rEpIFN-γ in concentrations as low as 1 pg/ml. The assay was shown to be able to detect the native elephant IFN-γ, elicited in positive-control cultures (pokeweed mitogen (PWM), phorbol myristate acetate plus ionomycin (PMA/I)) of both Asian and African elephant whole-blood cultures (WBC). Preliminary data were generated using WBC from non-infected elephants, a M. tb infection-suspected elephant and a culture-confirmed M. tb-infected elephant. The latter showed measurable production of IFN-γ after stimulation with ESAT6/CFP10 PPDB and PPDA in concentration ranges as elicited in WBC by Mycobacterium tuberculosis complex (MTBC)-specific antigens in other species. Hence, the IFN-γ assay presented potential as a diagnostic tool for the detection of elephant tuberculosis. Validation of the assay will require its application in large populations of non-infected and infected elephants.
authors.affiliation: National Elephant Institute, Hangchart, Lampang, Thailand; Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, the Netherlands.
authors.forename: T
authors.initials: T
authors.lastname: Angkawanish
authors.forename: D
authors.initials: D
authors.lastname: Morar
authors.forename: P
authors.initials: P
authors.lastname: van Kooten
authors.forename: I
authors.initials: I
authors.lastname: Bontekoning
authors.forename: J
authors.initials: J
authors.lastname: Schreuder
authors.forename: M
authors.initials: M
authors.lastname: Maas
authors.forename: W
authors.initials: W
authors.lastname: Wajjwalku
authors.forename: A
authors.initials: A
authors.lastname: Sirimalaisuwan
authors.forename: A
authors.initials: A
authors.lastname: Michel
authors.forename: E
authors.initials: E
authors.lastname: Tijhaar
authors.forename: V
authors.initials: V
authors.lastname: Rutten
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002417:Cattle; D004606:Elephants / Q000097:blood / Q000276:immunology / Q000382:microbiology*; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D007371:Interferon-gamma / Q000097:blood / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000276:immunology / Q000302:isolation & purification*; D014376:Tuberculosis / Q000175:diagnosis* / Q000276:immunology / Q000662:veterinary*
chemical_list: D000911:Antibodies, Monoclonal; D007371:Interferon-gamma
keywords: elephant; interferon gamma; tuberculosis