Proceedings of the 10th International Colloquium on Paratuberculosis

#142 Identification <strong>of</strong> new antigens <strong>of</strong> Mycobacterium avium subsp. paratuberculosis by
cross-immunoproteomic approach
Sophie Viart, Virginie Roupie, Marc Govaerts, Kris Huygen, Ruddy Wattiez
University <strong>of</strong> Mons-Hainaut, Belgium; WIV-ex-Pasteur Institute Brussels, Belgium; Veterinary and Agrochemical
Research Center Uccle, Belgium
We have identified in a previous study, combining proteomic and reverse genomic approaches, twenty-five new
Mycobacterium avium subsp. paratuberculosis (Map) antigenic proteins (1). From <strong>the</strong> ten first proteins studied,
a cocktail <strong>of</strong> three has been selected using a panel <strong>of</strong> 69 sera (21 positive / 48 negative) from cattle categorised
by faecal culture, Pourquier and ID-Vet seroassays. Based on this panel, this cocktail yielded a more
efficient diagnostic ELISA assay than <strong>the</strong> most widely used European paratuberculosis commercial seroassay
(Pourquier test). However, when expanding performance assessment to a larger serum reference panel (81
positive / 50 negative), responses <strong>of</strong> positive reference sera appeared unpredictable and <strong>the</strong> sensitivity <strong>of</strong> <strong>the</strong>
cocktail dropped significantly from 94.74% to 19.67%.
The aim <strong>of</strong> this study was <strong>the</strong>refore to identify, by cross-immunoproteomic approach, new specific
antigenic targets which would complement <strong>the</strong> cocktail, and increase its sensitivity. In this context, 5 Pourquier
and faecal culture positive sera, that failed to react with <strong>the</strong> antigenic cocktail, were used to probe 2-D Western
blots <strong>of</strong> <strong>the</strong> secretome and cellular protein extract <strong>of</strong> Map. As expected, all failed to detect any 3 <strong>of</strong> <strong>the</strong>
cocktail proteins. Yet, 7 antigenic proteins complementary to <strong>the</strong> original cocktail were detected, identified and
characterized.
In parallel, we have grown MAP in different culture conditions (acid pH, hypoxia, presence <strong>of</strong> nontoxic
concentrations <strong>of</strong> NO and nutrient starvation) presumed to mimic in vivo latency conditions. Antigens present in
<strong>the</strong>se culture filtrates and extracts may have a potential for early diagnosis and Johne’s disease control. Interestingly,
among <strong>the</strong> candidates previously identified by cross-immunoproteomic approach, few appeared be
over-expressed in latency conditions.
With <strong>the</strong> prospect <strong>of</strong> serological mass screening for paratuberculosis control, this study indicates <strong>the</strong>
necessity to use a cocktail <strong>of</strong> complementary antigens. Moreover, <strong>the</strong> development <strong>of</strong> latency models opens
perspectives for <strong>the</strong> discovery <strong>of</strong> novel early diagnosis antigens.
Reference: 1. Leroy, B., et al., Proteomics, 2007. 7: 1164-76.
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