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National audience; The goal of this study is to evaluate the immunogenicity and the specificity of 35 novel M. avium subsp paratuberculosis (Map) proteins in mice and bovine models. 23 of these 35 candidates were identified in standard Map ATCC 19698 biofilm cultures on Sauton medium by proteomics and immunoproteomics (Leroy et al, 2007). 6 were identified in in vitro dormancy models based on Sauton cultures submitted to different stress conditions (hypoxia, acidic pH, nutrient starvation or non-toxic NO) (unpublished data). Finally, 6 were identified using an in silico analysis of Map genome (Leroy et al., 2009). The 35 proteins were produced as recombinant histidine-tagged proteins in E. coli. All proteins were evaluated in BALB/c and C57BL/6 mice intravenously infected with Map ATCC 19698, M. avium subsp. avium (Maa)ATCC 15769 or M. bovis (Mb) AN5. Murine spleen cell IFN-γ production following in vitro stimulation with the proteins and antibody specific proteins were analysed. 14 and 6 of these 35 proteins were respectively evaluated in French and Belgian context. In French context, cattle from one certified Map free herd, one Silirum® vaccinated herd and three Map infected herds were tested in IFN-g release assay and ELISA (IDEXX and ID-vet) whereas in Belgian context, cattle from one Map culture-confirmed herd, five Map free herds and two M. bovis infected herds were tested. In Map infected mice, 6 of the 35 proteins induced very high IFN-γ production in spleen cell cultures. Although some proteins were recognized more strongly in Map/Maa than in M. bovis infected mice, no real species-specific antigens could be identified. In cattle, among the 14 proteins tested for IFN-γ response in French context, 8 showed significant Spearman correlation with Johnin, with particular high mean S/P ratio response. In Belgium context 3 out of the 6 tested proteins induce a Map specific IFN-γ production. This study reveals interesting candidates that could be use in the cellular and/or serology diagnosis. One of the most promising candidate is MAP0586c as it protect partially MAP infected BALB/C mice (Roupie et al., 2008) and induces significantly and specific IFN-γ production in cattle.

The goal of this study is to evaluate the immunogenicity and the specificity of 35 novel M. avium subsp paratuberculosis (Map) proteins in mice and bovine models. 23 of these 35 candidates were identified in standard Map ATCC 19698 biofilm cultures on Sauton medium by proteomics and immunoproteomics (Leroy et al, 2007). 6 were identified in in vitro dormancy models based on Sauton cultures submitted to different stress conditions (hypoxia, acidic pH, nutrient starvation or non-toxic NO) (unpublished data). Finally, 6 were identified using an in silico analysis of Map genome (Leroy et al., 2009). The 35 proteins were produced as recombinant histidine-tagged proteins in E. coli. All proteins were evaluated in BALB/c and C57BL/6 mice intravenously infected with Map ATCC 19698, M. avium subsp. avium (Maa)ATCC 15769 or M. bovis (Mb) AN5. Murine spleen cell IFN-γ production following in vitro stimulation with the proteins and antibody specific proteins were analysed. 14 and 6 of these 35 proteins were respectively evaluated in French and Belgian context. In French context, cattle from one certified Map free herd, one Silirum® vaccinated herd and three Map infected herds were tested in IFN-g release assay and ELISA (IDEXX and ID-vet) whereas in Belgian context, cattle from one Map culture-confirmed herd, five Map free herds and two M. bovis infected herds were tested. In Map infected mice, 6 of the 35 proteins induced very high IFN-γ production in spleen cell cultures. Although some proteins were recognized more strongly in Map/Maa than in M. bovis infected mice, no real species-specific antigens could be identified. In cattle, among the 14 proteins tested for IFN-γ response in French context, 8 showed significant Spearman correlation with Johnin, with particular high mean S/P ratio response. In Belgium context 3 out of the 6 tested proteins induce a Map specific IFN-γ production. This study reveals interesting candidates that could be use in the cellular and/or serology diagnosis. One of the most promising candidate is MAP0586c as it protect partially MAP infected BALB/C mice (Roupie et al., 2008) and induces significantly and specific IFN-γ production in cattle.