Abstract
BackgroundRed deer (Cervus elaphus) is regarded as an epidemiologically relevant host for Mycobacterium bovis (M. bovis) and closely related members of the Mycobacterium tuberculosis complex that cause animal tuberculosis (TB). The standard antemortem screening test for the detection of TB in deer is the intradermal tuberculin skin test, but the detection of interferon-gamma (IFNγ) produced by white blood cells exposed to M. bovis antigens can be used as an alternative or supplemental assay in most TB eradication/control programs. This study aims to develop an in-house sandwich ELISA for deer IFNγ, based on the cross-reactivity of the antibodies to both cervid and bovine IFNγ, and to evaluate the potential of this assay to detect M. bovis-infected red deer in response to the in vitro stimulation of whole-blood cells with bovine purified protein derivative (bPPD), p22 protein complex derived from bPPD or using the specific tuberculous mycobacterial proteins ESAT-6/CFP-10, Rv3615c and Rv3020c. The positive control stimulant used in this study was pokeweed mitogen, which resulted in a consistent induction of IFNγ in samples from red deer, thus allowing the interpretation of the assay.ResultsThe percentage of animals correctly classified by this technique as M. bovis non-infected was 100%. The detection of infected animals as positive was high and ranged widely depending upon the antigen and the cut-off value applied, as well as the time after infection. Our findings indicate that this protocol may serve as a reliable assay for the antemortem diagnosis of TB from the initial stage of M. bovis-infection, and may also be adequately sensitive.ConclusionsThe suggested optimal antigens and cut-off are bPPD, p22 and the combination of ESAT-6/CFP-10 and Rv3020c with a 0.05 Δ optical density, which yielded a up to 100% correct classification of TB positive and negatve red deer under our experimental conditions. This technique will aid in TB testing of farmed and translocated deer. Future studies should evaluate the ability of this IFNγ assay to detect specific responses under field conditions.
Highlights
Red deer (Cervus elaphus) is regarded as an epidemiologically relevant host for Mycobacterium bovis (M. bovis) and closely related members of the Mycobacterium tuberculosis complex that cause animal tuberculosis (TB)
This study was designed to develop an in-house sandwich enzyme-linked immunosorbent assays (ELISA) for the detection of cervid Interferon gamma (IFNγ) in red deer, based on the cross-reactivity of the antibodies specific to bovine IFNγ, and to evaluate the potential of this assay to detect M. bovis-infected deer in response to the in vitro stimulation of wholeblood cells with bovine purified protein derivative (bPPD), p22 protein complex derived from bPPD and Avian purified protein derivative tuberculin (PPD) (aPPD) or using specific M. bovis proteins, such as ESAT-6/CFP-10, Rv3615c or Rv3020c
The intratracheal inoculation of M. bovis in the deer resulted in gross tuberculosis lesions in every animal, which were especially prominent in lung, tracheobronchial and mediastinal lymph nodes (LNs)
Summary
Red deer (Cervus elaphus) is regarded as an epidemiologically relevant host for Mycobacterium bovis (M. bovis) and closely related members of the Mycobacterium tuberculosis complex that cause animal tuberculosis (TB). The standard antemortem screening test for the detection of TB in deer is the intradermal tuberculin skin test, but the detection of interferon-gamma (IFNγ) produced by white blood cells exposed to M. bovis antigens can be used as an alternative or supplemental assay in most TB eradication/control programs. Animal tuberculosis (TB) caused by Mycobacterium bovis (M. bovis) has been identified in a wide range of hosts, including both domestic animals and wildlife, which can become reservoirs of infection and contribute to infection maintenance [1]. The red deer (Cervus elaphus) is considered an epidemiologically important wildlife host for M. bovis and closely related members of the M. tuberculosis complex (MTC) that cause TB [1]. Pre-movement tests are currently compulsory for the translocations of live deer in Spain
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