Immunological longitudinal study of synthetic antigen L5P response in bovine herds naturally exposed to <em>Mycobacterium avium</em> subsp <em>paratuberculosis</em>

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Cell mediated immune response to L5P in longitudinal study of heifers from naturally<em> Mycobacterium avium</em> subsp <em>paratuberculosis</em> infected herd

National audiencePeptidyl moiety of cell wall lipopentapeptide (L5P) specific of Mycobacterium avium subsp paratuberculosis (Map) is immunogenic and a target for specific humoral response in Map infected animals. A chemically synthesized L5P is able to induce specific cell mediated immune response (CMIR) in IFN-γ release assay (IGRA) in selected cows from Map infected herds comparatively to non-infected or M. bovis infected. Following these observations, the aim of this study was to evaluate if L5P was an antigen of early specific immune response and potentially a predictive tool of Map infection. 113 heifers of 6 herds were included in a two years’ longitudinal study: 71 animals from three Map culture-confirmed herds, 11 animals from a Map infected herd Silirum® vaccinated during the study and 31 animals from two certified Map free herds. The analysis of the CMIR was investigated by IGRA following whole blood stimulation with synthetic L5P or mycobacterium purified protein derivative (PPD) from M. avium (PPDa), M. bovis (PPDb), Map (PPDj) and M. phlei (PPDp). Humoral immune response was quantified by L5P-based ELISA using an internal procedure and two commercial Map kits. Moreover, bacilli excretion was estimated by isolation and culture from faecal sample. PPDs’ CMIR was more or less high depending of infected herds context, became high over 2 S/P ratio for 10/11 animals just after Silirum® vaccination and was low, less than 0.1 S/P ratio, in certified Map free herds. L5P CMIR was observed in 9 of 71 animals from Map culture-confirmed herds. These 9 animals with a L5P CMIR positive between 0.05 and 0.6 S/P ratio were from the same herd, knowing that L5P CMIR was previously detected in all included Map culture-confirmed herds. L5P CMIR was fluctuant as already described for PPD but was significantly correlated with PPDj CMIR. For 2 of the 9 animals, the L5P CMIR was predictive of the Map positive serology, whereas it was concomitant with seropositivity for 2 others and that for 5 animals was several times observed without seroconversion. And no seroconversion was observed in other herds. The continuation of this study would assess the predictive potential of L5P CMIR for paratuberculosis diagnosis

Lipopentapeptide, L5P a <em>Mycobacterium avium paratuberculosis</em> specific antigen induces cell mediated immune response: potential tool in early Johne's disease

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L5P a specific antigen suitable to detect mycobacterium avium spp. paratuberculosis infection by cell mediated immune response

International audienceContext: The livestock management needs an early diagnostic test to prevent dramatic rise of Mycobacterium avium spp paratuberculosis (Map) infection in cattle. Current serology based diagnostic tests available, detect animals in later stages when they are shedding huge amount of bacillus. Cell mediated immune (CMI) responses that can be detected by Interferon Gamma Release Assays, appear before or combined with antibody responses. Therefore, diagnostic tools based on IGRA could help to identify recently infected animals in order to prevent disease transmission. The sensitivity and specificity of these tests need to define Map-specific T-cell antigens. Map produces a specific cell-wall lipopentapeptide called L5P or LP-01. L5P is suitable to detect Map-infected animals by serodiagnostic. Moreover, L5P can be synthetized chemically at high purity, large scale, and low cost. Objectives: The purpose of this work was to assess the potential of L5P to detect Map-specific cell mediated immune responses to develop an IGRA test. Methods: A panel of 36 cows were selected from two naturally infected herds where clinical paratuberculosis had occurred. We performed 1) serology with the commercial IDEXX diagnostic test and a house-made test using L5P antigen 2) IGRA with Purified Protein Derivative avian (PPD-A) or synthetic L5P antigen 3) microbiological analyses including isolation and identification of bacillus from faeces. Results: In this study 47.2 % of cows were scored positive by the commercial IDEXX diagnostic test but only 22% developed anti-L5P antibodies. PPD-A induced CMI responses in 97 % of cows while 22.2% animals were L5P responders. Map was isolated in 25.7% of animals. We provide, for the first time, evidence that Map-specific L5P is a suitable antigen for serologic and IGRA diagnosis. Perspectives: We will now carry out a longitudinal study to investigate the potential of L5P-based IGRA to predict clinical outcome of Map-infected 18-24 months cattle

Interferon gamma response to Mycobacterium avium subsp. paratuberculosis specific lipopentapeptide antigen L5P in cattle

After Mycobacterium avium subsp. paratuberculosis (Map) infection the cell-mediated immune (CMI) response indicative of early Th1 activation may be detected using interferon-gamma release assay (IGRA). Currently, the purified protein derivatives (PPDs), i.e., the total extract of mycobacteria antigens are used to recall CMI responses against Map. This study aimed to assess the ability of the chemically synthesized Map specific cell wall lipopentapeptide L5P to induce CMI response in cows infected by Map compared to PPD. L5P and PPD elicited an IFN-γ response in 12 and 35 animals from two Map infected herds respectively, but IFN-γ was not detected in the 13 cows recruited from a non-infected herd. Levels of IFN-γ detected were higher with PPD than with L5P. There was no correlation between the IFN-γ response and the humoral response to Map or faecal culture

Immunological analysis of 35 recombinant antigens of <em>M. avium</em> subsp <em>paratuberculosis</em> in mice and cattle

National audienceThe 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