title: Development of a Gene Expression Assay for the Diagnosis of Mycobacterium bovis Infection in African Lions (Panthera leo).
abstract: Mycobacterium bovis infection, the cause of bovine tuberculosis (BTB), is endemic in wildlife in the Kruger National Park (KNP), South Africa. In lions, a high infection prevalence and BTB mortalities have been documented in the KNP; however, the ecological consequences of this disease are currently unknown. Sensitive assays for the detection of this infection in this species are therefore required. Blood from M. bovis-exposed, M. bovis-unexposed, M. tuberculosis-exposed and M. bovis-infected lions was incubated in QuantiFERON®-TB Gold (QFT) tubes containing either saline or ESAT-6/CFP-10 peptides. Using qPCR, selected reference genes were evaluated for expression stability in these samples and selected target genes were evaluated as markers of antigen-dependent immune activation. The abundance of monokine induced by gamma interferon (MIG/CXCL9) mRNA, measured in relation to that of YWHAZ, was used as a marker of ESAT-6/CFP-10 sensitization. The gene expression assay results were compared between lion groups, and lenient and stringent diagnostic cut-off values were calculated. This CXCL9 gene expression assay combines a highly specific stimulation platform with a sensitive diagnostic marker that allows for discrimination between M. bovis-infected and M. bovis-uninfected lions.
authors.affiliation: DST-NRF Centre of Excellence for Biomedical Tuberculosis Research/SAMRC Centre for TB Research/Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Tygerberg, South Africa.
authors.forename: T T
authors.identifier: http://orcid.org/0000-0002-9631-9133
authors.initials: TT
authors.lastname: Olivier
authors.affiliation: DST-NRF Centre of Excellence for Biomedical Tuberculosis Research/SAMRC Centre for TB Research/Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Tygerberg, South Africa.
authors.forename: I M
authors.initials: IM
authors.lastname: Viljoen
authors.affiliation: Veterinary Wildlife Services, Kruger National Park, Skukuza, South Africa.
authors.forename: J
authors.initials: J
authors.lastname: Hofmeyr
authors.affiliation: DST-NRF Centre of Excellence for Biomedical Tuberculosis Research/SAMRC Centre for TB Research/Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Tygerberg, South Africa.
authors.forename: G A
authors.initials: GA
authors.lastname: Hausler
authors.affiliation: DST-NRF Centre of Excellence for Biomedical Tuberculosis Research/SAMRC Centre for TB Research/Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Tygerberg, South Africa.
authors.forename: W J
authors.initials: WJ
authors.lastname: Goosen
authors.affiliation: Department of Paraclinical Sciences, Faculty of Veterinary Science, University of Pretoria, Pretoria, South Africa.|National Zoological Gardens of South Africa, Pretoria, South Africa.
authors.forename: A S W
authors.initials: ASW
authors.lastname: Tordiffe
authors.affiliation: Veterinary Wildlife Services, Kruger National Park, Skukuza, South Africa.
authors.forename: P
authors.initials: P
authors.lastname: Buss
authors.affiliation: DST-NRF Centre of Excellence for Biomedical Tuberculosis Research/SAMRC Centre for TB Research/Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Tygerberg, South Africa.
authors.forename: A G
authors.initials: AG
authors.lastname: Loxton
authors.affiliation: DST-NRF Centre of Excellence for Biomedical Tuberculosis Research/SAMRC Centre for TB Research/Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Tygerberg, South Africa.
authors.forename: R M
authors.initials: RM
authors.lastname: Warren
authors.affiliation: DST-NRF Centre of Excellence for Biomedical Tuberculosis Research/SAMRC Centre for TB Research/Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Tygerberg, South Africa.
authors.forename: M A
authors.initials: MA
authors.lastname: Miller
authors.affiliation: DST-NRF Centre of Excellence for Biomedical Tuberculosis Research/SAMRC Centre for TB Research/Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Tygerberg, South Africa.
authors.forename: P D
authors.initials: PD
authors.lastname: van Helden
authors.affiliation: DST-NRF Centre of Excellence for Biomedical Tuberculosis Research/SAMRC Centre for TB Research/Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Tygerberg, South Africa.
authors.forename: S D C
authors.identifier: http://orcid.org/0000-0002-9033-9686
authors.initials: SDC
authors.lastname: Parsons
mesh_terms: D000818:Animals; D008045:Lions / Q000382:microbiology*; D009163:Mycobacterium bovis / Q000235:genetics* / Q000302:isolation & purification; D015995:Prevalence; D013019:South Africa / Q000453:epidemiology; D014376:Tuberculosis / Q000175:diagnosis / Q000453:epidemiology / Q000382:microbiology / Q000662:veterinary*
keywords: 
  Mycobacterium bovis
  ; 
  Panthera leo
  ; African lion; diagnosis; tuberculosis; wildlife