Antigen-induced production of interferon-γ in samples of peripheral lymph nodes from sheep experimentally inoculated with Mycobacterium avium subsp. paratuberculosis

Abstract

The production of interferon-γ (IFN-γ) in response to Johnin purified protein derivate was measured in samples of the prescapular lymph node (PLN) from 10 sheep, aged 2 years, and nine sheep, aged 1 year that had been inoculated orally with Mycobacterium avium subsp. paratuberculosis within their first month of life. Ten non-inoculated sheep, aged 1 year, constituted the negative control group. The results obtained in the PLN IFN-γ assay were compared with those derived from serological tests: a complement fixation test (CFT), agar gel diffusion test (AGID) and enzyme-linked immunosorbent assay (ELISA), as well as an IFN-γ test on samples of blood. Among the 19 inoculated sheep, 16 gave positive reactions in the PLN IFN-γ assay on samples incubated overnight, and 18 tested positive when the assay was applied to PLN samples incubated for 48 h. In comparison, three, four and seven inoculated sheep gave positive reactions in the ELISA, CFT and in the blood IFN-γ assay on samples incubated overnight, respectively. The AGID and IFN-γ assay on blood samples incubated for 48 h detected eight inoculated animals. Twelve inoculated sheep, that tested positive in the PLN IFN-γ assay were clinically normal, gave negative results in an IS900-based polymerase chain reaction (PCR) assay on samples of ileum and ileocaecal lymph node and had no histological evidence of paratuberculosis, but tested positive on more than two occasions in sequential serological testing before necropsy. None of the 10 non-inoculated sheep tested positive in the AGID, CFT, ELISA, blood IFN-γ assay on samples incubated overnight and for 48 h or the PLN IFN-γ assay on samples incubated overnight, but one gave a positive result in the PLN IFN-γ assay on samples stimulated for 48 h. It is likely that the positive reactions obtained by the PLN IFN-γ assay in the 12 inoculated sheep that tested negative in the PCR assay and histopathological examination represents immunological evidence of latent infection or previous exposure to M. paratuberculosis rather than active infection.