Experimental infection of a bovine model with human isolates of Mycobacterium avium subsp. paratuberculosis

Abstract

Mycobacterium avium subsp. paratuberculosis (Map), the etiologic agent of Johne's disease (JD) in ruminants, has been implicated in the pathogenesis of Crohn's disease (CD) in humans. We developed a bovine ileal cannulation model to facilitate comparison of the immune response to Map and the mechanisms of pathogenesis in cattle and humans. Initial studies showed a T cannula could be maintained for up to a year in calves without inducing inflammation or adversely affecting intestinal function. Map introduced through the cannula established a persistent low level of infection without inflammation. Infection elicited an immune response to Map antigens detectable by flow cytometry. Further studies now show the cannulation model can be used with cows during the later stage of infection, affording access to the target tissue at all stages of infection. The studies also revealed no difference in infectivity or immunogenicity of isolates of Map obtained from cattle or humans with CD. Comparison of the immune response to Map during the early and late stages of infection using PCR, flow cytometry and QRT-PCR, showed the immune response early in the disease process is dominated by CD4 T cells. A CD8 response is delayed but comparable at later stages of infection. Genes for pro-inflammatory cytokines IFN-γ and the recently identified genes encoding IL-17 and IL-22 are up regulated in infected animals. These findings reveal that both human and bovine isolates of Map can establish infection and induce similar immune responses in a bovine model. They also reveal the cytokine responses elicited in cattle are similar to those implicated in CD pathogenesis.

Figure 1

Representative dot plots of CD4 T cells labeled with anti-CD4, -CD45R0, and -CD25, showing the gating strategy used to electronically isolate activated proliferating cells for analysis, (SSC vs FSC). Panel A, electronic gates were used to identify and color code resting (red) and activated proliferating (blue) cells. Panel B, an additional gate was used to isolate cell subsets for analysis (FSC vs CD4). Panel C, gated CD4 cells showing relative proportion of naïve and CD25^- and CD25⁺ memory CD4 T cells. Panel D, gated CD4 cells showing the proportion of naïve and memory T cells expressing CD25. Panels E and F, additional gates were placed on resting and activated cells to show the relative proportion of resting and activated naïve and memory CD4 cells expressing CD25. Note that all activated CD4 cells expressed CD25. For presentation of the data, the proportion of activated cells present in preparations of PBMC cultured in RPMI alone (as shown in panel C) were subtracted from the proportion of activated cells present in preparations of PBMC cultured in jPPD or SAg.

Figure 2

Three endoscopic pictures of cannulated ileums. Picture A is of a normal ileum from the control negative calf 132. Picture B is from calf 129 (experimentally inoculated) with little to no changes in the ileal mucosa, and picture C shows the corrugated thickened appearance of the ileal mucosa from a naturally infected cow 1181 with clinical Johne's disease.

Figure 3

Comparison of the expression of CD25 on activated CD4 and CD8 memory cells. The first bar shows pre-inoculation samples that were taken from all of the calves. The second bar shows the mean of the control negative calf sampled over 3 time points and bars 3-5 show the means of the inoculated calves at 4, 5 and 11 months PI. Bar 6 shows expression of CD25 on cells from clinical cows was similar to the expression on cells from experimentally inoculated calves. Asterisks indicate statistical significance difference P < .05 compared to pre inoculation results. Results from clinical cows were not significantly different from results from calves 11 months PI.

Figure 4

Cytokine gene expression from ileocecal lymph nodes from clinically (naturally) infected cows (CI) n = 3 and experimentally inoculated calves (EI) n = 2 compared to negative controls (NC) n = 3. Control negative values were set at 0. IFN-γ and IL-22 are statistically significant with a P > .05.