The pathology and immunology of paratuberculosis in sheep and goats

Abstract

Mycobacterium avium subspecies paratuberculosis {Map) infects macrophages and causes severe lesions of chronic granulomatous enteritis in sheep and other ruminants (Johne's disease). In this study, ovine paratuberculosis cases were examined histologically, and the presence of two distinct forms of intestinal pathology confirmed, characterised by either multibacillary lesions which exhibited a positive correlation with the presence of a marked macrophage infiltrate (lepromatous cases), or paucibacillary lesions which showed correlation with a cellular infiltrate which was lymphocytic in nature (tuberculoid cases). The presence of acid-fast bacteria was also found to correlate with evidence of serum antibody. Genomic DNA was extracted from the ileum of infected sheep and polymerase chain reaction (PCR) performed using IS900 primers to confirm Map infection.Immunoperoxidase staining of ileum demonstrated that the lymphocyte populations differed in density and relative percentages between both histological groups. The tuberculoid group had higher densities of CD4+, CD8+ and y5TCR+ subsets, and the lepromatous group lower densities of CD4+ and CD8+ subsets, when compared with control animals. Tuberculoid cases were associated with an increase in the relative percentage of CD4+ lymphocytes, whereas lepromatous cases had an increased relative percentage of γ5TCR+ cellsFlow cytometry of lamina propria lymphocytes (LPL) isolated from the ileum of infected and control animals confirmed increased percentages of ySTCR+ cells in lepromatous cases than in controls, and higher percentages of CD8+ and y8TCR+ cells than in tuberculoid cases, which had correspondingly higher percentages of CD4+ cells. Higher percentages of ySTCR+ cells were also noted in mesenteric lymph node lymphocytes (MLNL) from lepromatous cases compared with control animals. Peripheral blood lymphocytes (PBL) of infected animals had increased percentages of B cells, and an associated increase in the percentage of MHC Class II positive cells compared with normal controls.The relationship of histological lesions to serum antibody, Map antigen-specific lymphocyte proliferation and cytokine production was investigated. LPL, MLNL, and PBL were cultured with Map PPD, and proliferative responses measured by incorporation of tritiated thymidine. Interferon (IFN)-y and interleukin (IL)-2 production were measured by ELISA and bioassay respectively. Humoral responses were measured by serum antibody ELISA. A range of immune responses was seen that corresponded to the type of histopathology present, with animals being divisible into two groups. One group was characterised by dominant cell mediated immunity (CMI), lower humoral responses and higher levels of IFN-y and IL-2, suggesting a Thl (CMI help) like response. The other group had low antigen-specific proliferation, low IFN-y and IL-2 and higher antibody levels suggesting a Th2 (B cell help) like response. These groups corresponded to the recognised tuberculoid and lepromatous types of intestinal histopathology respectively, and suggest different pathogenic mechanisms for each form of the disease.PBL from infected sheep and goats were subjected to CD4+, CD8+ and y5TCR+ subset depletion using magnetic activated cell sorting (MACS) technique. Proliferative responses to Map PPD were diminished in cell cultures which had been CD4+ T-cell depleted.RNA was extracted from the ileum of infected and control sheep, reverse transcribed to cDNA and subjected to PCR using primers for the amplification and detection of mRNA for the cytokines IFNy, IL-4, and IL-10 and for IL-2 receptor. mRNA for all cytokines was detectable in both groups of infected animals, however higher levels of IL-10 mRNA were present in lepromatous than tuberculoid tissues.The findings of this study suggest that ovine paratuberculosis is a disease with an immunological spectrum broadly comparable with that described for leprosy, with an apparent Thl like response in the tuberculoid form and a Th2 like response in the lepromatous form.