Development of a bovine ileal cannulation model to study the immune response and mechanisms of pathogenesis of paratuberculosis

Abstract

An ileal cannulation model was developed in conjunction with a flow cytometric assay to gain a better understanding of the mechanisms of immunopathogenesis of Johne's disease caused by Mycobacterium avium subsp. paratuberculosis. Initial studies with calves showed that M. avium subsp. paratuberculosis DNA is detectable by PCR in ileal biopsies during the first months following experimental infection. Inflammatory lesions were not detected on endoscopic evaluation up to 8 months postexperimental infection. M. avium subsp. paratuberculosis DNA was detected in multiple tissues at necropsy 8 months postinfection. Examination of the activation status of epithelial lymphocytes from the jejunum and ileum from infected and control animals at necropsy revealed that none of the major subsets of lymphocytes (NK, CD2(+), and CD2(-) gammadelta T lymphocytes, or CD4 and CD8 alphabeta T lymphocytes) expressed activation molecules CD25, CD26, CD71, ACT1, or ACT16. Subsets of CD4 and CD8 T lymphocytes from control and infected animals expressed CD26. The majority of CD4 and CD8 T lymphocytes expressed CD45R0, the memory T-lymphocyte marker. An immune response to M. avium subsp. paratuberculosis was detected by 3 months postinfection, dominated by a strong proliferative response of CD4 memory T lymphocytes. The findings indicate an immune response develops following initial exposure to M. avium subsp. paratuberculosis that controls but does not eliminate the pathogen. This persistence of M. avium subsp. paratuberculosis possibly leads to erosion and dysregulation of protective immunity at later time points postinfection. Continuous access to the ileum offers an opportunity to elucidate the cellular and molecular events leading to immune dysregulation and development of chronic inflammatory ileitis.

FIG. 1.

Pictures showing the modified cannula used in the study (A), appearance of cannula after surgery (B), endoscopic field showing no inflammation present at the time of inoculation with M. avium subsp. paratuberculosis (C), and collection of pinch biopsy (D). Other images show the status of cannula 8 months postsurgery just before necropsy. Inflammation at the site of implantation was kept at a minimum by keeping the site clean (E and F). Inspection of the internal portion of the cannula showed minimal inflammation (G).

FIG. 2.

Gating strategies used to follow the development of a CD4 T-lymphocyte response to M. avium subsp. paratuberculosis antigens following exposure to M. avium subsp. paratuberculosis through the cannula. (A to F) Lymphocytes at time zero. (G to L) lymphocytes cultured in RPMI medium alone for 6 days. (M to R) Lymphocytes cultured with PPD for 6 days. The numbers in the upper-right quadrant show the relative percentages of cells in each quadrant. The S and L gates in the plots in panels A, G, and M show the change in the relative proportions of activated proliferating cells that occurred following culture with and without antigen stimulation. The color coding of the cells in the S and L gates was used to track and distinguish resting (red, unactivated) cells from activated, proliferating (blue) cells. The color coding facilitated observing the actual change in the proportion of activated cells shown in the respective plots for NK cells (B, H, and N), γδ T cells (C, I, and O), and CD4 T cells (D, F, J, L, P, and R). Putting a third selective gate on CD4 let us enumerate the proportion of activated CD4 cells present at T₀ and after culture with and without antigen (compare plots E, K, and Q).

FIG. 3.

PCR of M. avium subsp. paratuberculosis DNA isolated from the indicated tissues at necropsy.

FIG. 4.

Comparison of the response to PPD in uninfected and experimentally infected calves. There was a significant difference (P = 0.0001) between activated CD 4 T lymphocytes in the experimentally inoculated group (animals 136, 137, and 139) compared to the negative control group (132, 142, and 143) over time. Neg, negative; Pos, positive.

FIG. 5.

Comparison of the relative proportions of naïve and memory CD4 and CD8 T lymphocytes expressing five activation molecules at the initiation of culture (time zero, 0T) and following 6 days (6D) of culture in RPMI medium alone or stimulation with PPD. M⁺, CD25⁺ memory T cells; M⁻, CD25⁻ memory T cells.

FIG. 6.

Gating strategies used to determine the relative proportion and activation status of NK (CD335), γδ T lymphocytes, and CD4 T lymphocytes in intraepithelial lymphocytes isolated from the jejunum and ileum from a calf 5 months p.i.

FIG. 7.

Comparison of the relative proportion of naïve and memory CD4 and CD8 T IEL expressing five activation molecules in the jejunum and ileum. M⁺, CD25⁺ memory T cells; M⁻, CD25⁻ memory T cells.