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International audience; Despite its potential for early diagnosis of Mycobacterium avium subsp. paratuberculosis (MAP) infection, the IFN-γ release assay is not used routinely, because of low specificity of the established crude antigen preparation Johnin (PPDj). Limited data are available assessing the potential of MAP-derived protein and lipopeptide antigens to replace PPDj in assays for goats, while cattle and sheep have been studied more extensively. Furthermore, MAP infection is claimed to interfere with the diagnosis of bovine tuberculosis when other crude antigen preparations (PPDb, PPDa) are applied. In this study, the diagnostic potential of MAP-derived recombinant protein antigens, synthetic MAP lipopentapeptides and of Mycobacterium bovis-specific peptide cocktails was assessed compared to crude mycobacterial antigen preparations in experimentally infected goats. Goats were inoculated with MAP, or Mycobacterium avium subsp. hominissuis (MAH) as surrogate for environmental mycobacteria, non-exposed animals served as controls. Mycobacterium avium Complex-specific antibody and PPDj-induced IFN-γ responses were monitored in vivo. Infection status was assessed by pathomorphological findings and bacteriological tissue culture at necropsy 1 year after inoculation. The IFN-γ response to 13 recombinant protein antigens of MAP, two synthetic MAP lipopentapeptides and three recombinant peptide cocktails of Mycobacterium bovis was investigated at three defined time points after infection. At necropsy, MAP or MAH infection was confirmed in all inoculated goats, no signs of infection were found in the controls. Antibody formation was first detected 3-6 weeks post infection (wpi) in MAH-inoculated and 11-14 wpi in the MAP-inoculated goats. Maximum PPDj-induced IFN-γ levels in MAH and MAP exposed animals were recorded 3-6 and 23-26 wpi, respectively. Positive responses continued with large individual variation. Antigens Map 0210c, Map 1693c, Map 2020, Map 3651cT(it), and Map 3651c stimulated increased whole blood IFN-γ levels in several MAP-inoculated goats compared to MAH inoculated and control animals. These IFN-γ levels correlated with the intensity of the PPDj-induced responses. The two synthetic lipopentapeptides and the other MAP-derived protein antigens had no discriminatory potential. Stimulation with Mycobacterium bovis peptide cocktails ESAT6-CFP10, Rv3020c, and Rv3615c did not elicit IFN-γ production. Further work is required to investigate if test sensitivity will increase when mixtures of the MAP-derived protein antigens are applied.

Mycobacterium avium subsp. paratuberculosis is the etiologic agent of Johne's disease and is endemic to the national cattle herds of many countries. Because of the very low level of genetic heterogeneity of this organism, it is difficult to select a workable procedure for strain differentiation at a resolution sufficient to investigate epidemiological links between herds or different ruminant species and the suggested zoonotic potential of M. avium subsp. paratuberculosis for Crohn's disease. Analysis of restriction fragment length polymorphisms (RFLPs) based on the insertion element IS900 (IS900 RFLP) with four restriction enzymes and 10 markers of specific mycobacterial interspersed repetitive units (MIRUs) and variable-number tandem repeats (VNTRs) was applied to 71 bovine M. avium subsp. paratuberculosis isolates originating from 14 herds from different regions in Germany. Among these isolates, all of which belonged to the M. avium subsp. paratuberculosis type II group, 17 genotypes were detected by IS900 RFLP and consisted of a combination of seven BstEII, eight PstI, nine PvuII, and four BamHI restriction patterns. Novel RFLP types were found. The diversity of the M. avium subsp. paratuberculosis isolates inside the herds was different depending on the frequency of animal purchase. The results of typing by IS900 RFLP and MIRU-VNTR analyses were not associated. Fifteen MIRU-VNTR patterns were identified with a discriminatory index of 0.905. The most common BstEII-based IS900 RFLP type, type C1 (72%), was subdivided into 14 types by MIRU-VNTR analysis. A combination of fingerprinting and PCR-based techniques resulted in 24 M. avium subsp. paratuberculosis genotypes and achieved a discriminatory index of 0.997. By using only BstEII and PstI digestion together with typing by MIRU-VNTR analysis, a discriminatory index of 0.993 was achieved. This is high enough to support epidemiological studies on a national as well as a global scale.