Proceedings of the 10th International Colloquium on Paratuberculosis

#212 Paratuberculosis: novel antigenic targets for early diagnosis
Kathy Dernivoix, Virginie Roupie, Sophie Viart, Morgan Collin, Ruddy Wattiez, Kris Huygen, Jean-Jacques
Letesson, Marc MCJ Govaerts
Department <strong>of</strong> Bacterial Diseases, Veterinary and Agrochemical Research Center, Belgium; Laboratory <strong>of</strong>
Mycobacterial Immunology, WIV-ex-Pasteur Institute, Belgium; Department <strong>of</strong> Proteomic and Protein Biochemistry,
University <strong>of</strong> Mons-Hainaut, Belgium; Laboratory <strong>of</strong> Immunology-Microbiology, University <strong>of</strong> Namur, Belgium
Johne’s disease caused by Mycobacterium avium subsp. paratuberculosis (Map) affects domestic and wildlife
ruminants worldwide but <strong>the</strong>re are currently nei<strong>the</strong>r efficient treatment nor vaccine, and diagnostic tests require
improvement in terms <strong>of</strong> sensitivity and specificity. Recently, combining advanced proteome and reverse
genomic approaches, we identified twenty-five new antigenic targets <strong>of</strong> Map. Five <strong>of</strong> <strong>the</strong>m have already shown
promise in ELISA for serological screening (Leroy et al., 2007). In this project, we focused on IFN-γ release
assay because this test has <strong>the</strong> advantage over serology to allow for early detection <strong>of</strong> infection at subclinical
stages. We fur<strong>the</strong>r evaluated <strong>the</strong> diagnostic potential <strong>of</strong> five antigens with this test. The coding sequences
<strong>of</strong> MAP3547c, specifically detected by sera from cattle naturally infected with Map in 2-D Western blot, and
<strong>of</strong> four reverse genomic targets, Ag 5, Ag6, Ag7 and Ag8, were cloned into <strong>the</strong> pQE-80L expression vector
(Qiagen) and purified from transformed Top-10F’ E. coli cells as His-tagged recombinant proteins. They were
screened for specificity on 32 cattle originating from 2 culture-confirmed herds infected with M. bovis and for
sensitivity on 102 cattle aged 3 to 68 months from a culture-confirmed Map-infected herd. Heparinized whole
blood samples were stimulated within 8 hours <strong>of</strong> collection with each antigen at 5 µg/ml along with controls,
and incubated for 20 hours with 5% CO2 at 37°C. IFN-γ release was measured in culture supernatants using a
bovine IFN-γ sandwich ELISA (Invitrogen). Applying <strong>the</strong> manufacturer’s cut<strong>of</strong>f for positivity, preliminary results
indicate that single antigen sensitivity ranges from 0 to 97% and specificity from 91% to 100% (95% CI). The
potential to combine single antigens to improve diagnostic performance, and cut-<strong>of</strong>f optimization, are fur<strong>the</strong>r
discussed.
#233 Use <strong>of</strong> Phage Amplification Assay to rapidly enumerate viable MAP and o<strong>the</strong>r
Mycobacteria
George Botsaris, Emma Stanley, Ca<strong>the</strong>rine Rees
University <strong>of</strong> Nottingham, United Kingdom; University College London, United Kingdom
The FASTPlaqueTB assay is a commercial diagnostic test for <strong>the</strong> rapid detection <strong>of</strong> viable Mycobacterium
tuberculosis from human sputum samples. The end-point <strong>of</strong> <strong>the</strong> assay is <strong>the</strong> development <strong>of</strong> plaques following
successful infection <strong>of</strong> a viable target cell by a bacteriophage. The phage used in this assay is D29 which
has a broad host range in <strong>the</strong> Mycobacterium genus. The objective <strong>of</strong> this work was to investigate whe<strong>the</strong>r this
rapid detection method could be used to enumerate numbers <strong>of</strong> viable MAP cells when working with laboratory
cultures. Mycobacterium cells grown on agar slopes were resuspended in Media Plus to provide an inoculum.
Samples were processed through <strong>the</strong> phage assay using a modified protocol that allowed <strong>the</strong> number <strong>of</strong>
plaques to be determined at a range <strong>of</strong> dilutions <strong>of</strong> <strong>the</strong> samples being tested. The results showed a direct correlation
between viable count and plaque number. The method was fur<strong>the</strong>r used to rapidly monitor heat inactivation
<strong>of</strong> MAP cells and to determine <strong>the</strong> MIC <strong>of</strong> <strong>the</strong> antibiotic rifamycin. Using <strong>the</strong> modified phage assay data
was gained in 24 h demonstrating that <strong>the</strong> assay can successfully be used to monitor cell number and replace
culture techniques for some physiological studies <strong>of</strong> this slow growing organism.
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