Interferon-γ Response of Mycobacterium avium subsp. paratuberculosis Infected Goats to Recombinant and Synthetic Mycobacterial Antigens

Abstract

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.

Peripheral IFN-ɣ Production after Blood Stimulation with Different Mycobacterial Antigens in Goats Vaccinated against Paratuberculosis

“…According to these results, in an infection with M. bovis in calves that had previously been vaccinated against paratuberculosis, but not infected with Map [ 8 ], non-specific responses were obtained in those vaccinated animals when the samples were stimulated with the same antigens used in our study. As already indicated, its specificity in the diagnosis of tuberculosis in animals infected with M. bovis or M. caprae has already been verified in different studies using both the IGRA test and the IDTB [ 6 , 8 , 14 , 15 , 16 , 17 , 18 , 41 , 42 , 43 ].…”

“…In recent years, some work has been carried out aiming to search for new and more specific antigens that would allow the differentiation of animals vaccinated against paratuberculosis from animals infected with tuberculosis [ 6 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 39 , 40 ]. Among them, the new specific antigens of M. bovis ESAT-6, CFP-10, and recombinant proteins (Rv3615c, RV peptide pool) stand out, which are part of the peptide cocktails (PC-EC and PC-HP) used in this study.…”

“…In a recent study in calves and goats [ 14 , 15 ], peptides of tuberculosis ESAT6, CFP10, and RV3615c have been shown to be more specific than bovine PPD and johnin in experimental infected, but unvaccinated, animals against Map. Identical results to ours were obtained in a vaccination experiment against paratuberculosis in goats that were later infected with M. caprae [ 6 ], where it was found that, at 3.5–4 mpv, the stimulation of peripheral blood with these antigens derived from M. bovis did not give any type of reaction, while when the infection by M. caprae occurred, there was a specific reaction against them.…”

“…These studies have been performed in some experimental kids or calves infected with M. bovis [ 6 , 8 , 12 , 13 , 14 ] and Map [ 15 ], but they have been barely assessed in relation to paratuberculosis vaccination, only being investigated in a limited number of goats or cattle [ 6 , 13 , 14 , 15 , 16 , 17 ] under experimental conditions rather than in field or experimental studies with Map-infected or vaccinated animals for longer time and at different age range.…”

Predicting Antigenic Peptides from Rocio Virus NS1 Protein for Immunodiagnostic Testing Using Immunoinformatics and Molecular Dynamics Simulation

“…However, to our knowledge, no diagnostic tests exist for ROCV. Synthetic peptides have recently emerged as novel targets for efficient serological diagnosis of infectious viral, bacterial, and parasitic diseases [ 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 ]. Such molecules could be applied in rapid diagnostic tests or other immunodiagnostic platforms such as ELISA to diagnose acute ROCV infection, offering lower production costs, higher specificity and reproducibility with no variation between batches, and larger-scale production compared to diagnosis based on whole antigens [ 30 ].…”