Progressive bovine paratuberculosis is associated with local loss of CD4(+) T cells, increased frequency of gamma delta T cells, and related changes in T-cell function

Abstract

Bovine paratuberculosis is caused by the infection of young calves with Mycobacterium avium subsp. paratuberculosis, resulting in a chronic granulomatous infection of predominantly the ileum. After an incubation period of 2 to 5 years, the disease becomes progressive in some of the chronically infected, but asymptomatic cows. This results in a protein-losing enteropathy that will ultimately be fatal. A loss of cell-mediated immune responses in symptomatic animals has been described, but no information is available concerning immune reactivity in the intestine. We sought to investigate putative disease status-associated lymphocyte subset distributions and antigen-specific functional characteristics of mononuclear cells isolated from blood, gut-associated lymphoid tissue, and the intestinal walls of 22 cows in different stages of disease and in control animals. The results demonstrated a significant decrease in CD4(+) T-cell frequency and a significant increase in TcR1-N12(+) gamma delta T-cell frequency in ileum lamina propria lymphocytes of symptomatic animals compared to the asymptomatic shedders. Immunohistology revealed that there was also an absolute decrease in the number of CD4(+) T cells in sections of the lesional ileum. Our findings also indicated that both peripheral and intestinal cell-mediated responses are decreased in symptomatic animals compared to asymptomatic animals. We conclude that the decrease in cell-mediated responses is likely related to a loss of antigen-specific CD4(+) T cells, which is most prominent in the lesional ileum from symptomatic animals, thus contributing to the progressive nature of bovine paratuberculosis.

FIG. 1.

Distribution of lymphocyte subsets related to anatomical location. Representative data on the frequency (percent positive-staining cells in flow cytometry plus the standard error [SE], n = 5 animals [group: control]) of the different lymphocyte subsets (CD4⁺ T cells, CD8⁺ T cells, TcR1-N12⁺ γδ T cells, and WC1⁺ γδ T cells) in mononuclear cell populations isolated from blood (PBMC), the mesenteric lymph nodes (LN), the lamina propria (LPL), and the epithelium (IEL). WC1⁺ γδ T cells are a subset of the TcR1-N12⁺ γδ T cells, most γδ T cells in PBMC are WC1⁺ in contrast to those in mesenteric lymph nodes, LPL, and IEL. CD4⁺ T cells predominate in PBMC and mesenteric lymph node populations, but their frequency is very low in IEL.

FIG. 2.

CD4⁺ αβ T-cell numbers in LPL in ileum sections as determined by FACS analysis. The frequency of CD4⁺ T cells (plus the SE) in LPL populations isolated from the ileum of cows from the control (n = 5), vaccinated (n = 5), shedder (n = 6), and clinical (n = 6) groups. Letters above columns indicate the results of statistical analysis. Columns that do not share letters are significantly different (P < 0.05).

FIG. 3.

Numbers of CD4⁺ αβ T cells in LPL ileum sections. The number of CD4⁺ αβ T cells per mm² of lamina proprium (plus the SE), as determined by immunohistochemistry, is shown for sections of ileum derived from cows from the control (n = 5), shedder (n = 6), and clinical (n = 6) groups. Letters above columns indicate the result of the statistical analysis. Columns that do not share letters are significantly different (P < 0.05).

FIG. 4.

Lymphoproliferative responses of PBMC and MLNC. Proliferative responses of mononuclear lymphocyte populations isolated from blood (PBMC), the ileocecal lymph node (ileum), and a jejunal lymph node (jejunum). Responses to recombinant M. avium subsp. paratuberculosis Hsp70 (A), PPD-P (B), and whole bacteria (M. avium subsp. paratuberculosis strain 316F) (C) and the result of stimulation with the mitogen ConA (D) are illustrated. Responses, expressed as the SI (S.I.) plus the SE on a logarithmic scale, are depicted for animals from the control, vaccinated, shedder, and clinical groups. Different letters indicate statistically significant differences (P < 0.05) between the different groups, within an anatomical location.

FIG. 5.

Lymphoproliferative responses of LPL. Lymphoproliferative responses from vaccinated animals (□, n = 2), shedders (▪, n = 2), and animals with clinical signs (▧, n = 2) are expressed as the mean SI plus the SE from triplicate measurements. The responses of LPL isolated from the ileum and the jejunum to M. avium subsp. paratuberculosis Hsp70 and bovine Hsp70 are shown in panels A and C, respectively. The responses of LPL isolated from the ileum and the jejunum to M. avium subsp. paratuberculosis PPD-P and whole bacteria (M. avium subsp. paratuberculosis strain 316F [M.a.p. 316F]) are shown in panels B and D, respectively.

FIG. 6.

Photomicrographs of ileum and jejunum sections of cows with clinical signs of paratuberculosis. Photomicrographs of intestinal sections were stained according to Ziehl-Neelsen for detection of acid-fast bacteria. The panels show representative sections of the ileum (A and B) and the jejunum (C and D) of two cows with clinical signs of paratuberculosis. Panels A and C are from sections taken from the same animal; panels B and D are likewise from sections taken from the same animal. Bars, 100 μm.