Antigen-specific B-cell unresponsiveness induced by chronic Mycobacterium avium subsp. paratuberculosis infection of cattle

Abstract

Mycobacterium avium subsp. paratuberculosis infection of cattle results in a chronic granulomatous enteritis. Clinical disease (i.e., cachexia, diarrhea, and high fecal bacterial counts) is preceded by a lengthy subclinical stage of disease. The immunologic mechanisms associated with the progression of infected cattle from subclinical to clinical disease are unclear. In this study, a cell proliferation assay was used in combination with flow cytometry to compare peripheral blood lymphocyte responses of cattle with subclinical paratuberculosis to responses of cattle with clinical paratuberculosis. B cells from cattle with subclinical disease proliferated vigorously upon stimulation with M. avium subsp. paratuberculosis antigen, with up to 12.4% of the total B cells responding. However, B cells from cattle with clinical disease did not proliferate upon antigen stimulation despite good proliferation in response to concanavalin A stimulation. In addition, these animals had high percentages of peripheral blood B cells. B cells from noninfected animals did not proliferate upon M. avium subsp. paratuberculosis antigen stimulation. Thus, it appears that B-cell proliferation is a sensitive indicator of subclinical Johne's disease. Furthermore, the immunologic mechanisms responsible for the antigen-specific unresponsiveness of peripheral blood B cells may be significant in the eventual progression from subclinical to clinical Johne's disease in cattle.

FIG. 1

Comparison of two assays for antigen-specific proliferation of PBMC isolated from noninfected (controls) and M. avium subsp. paratuberculosis-infected cattle (identified by number below the bars). The hatched bars represent the number of cells proliferating/5,000 PBMC in response to 10 μg of antigen (whole-cell sonicate of M. avium subsp. paratuberculosis)/ml as measured by the PKH2 assay (see Materials and Methods). The solid bars represent the SI of PBMC in response to antigen as measured by [³H]thymidine uptake (see Materials and Methods).

FIG. 2

Antigen-specific B-cell proliferation of PBMC isolated from noninfected (controls) and M. avium subsp. paratuberculosis-infected cattle (identified by number below the bars). The hatched bars represent the percentages of PBMC that are B cells as detected by flow cytometry. The solid bars represent the number of B cells proliferating/5,000 PBMC in response to 10 μg of antigen (whole-cell sonicate of M. avium subsp. paratuberculosis)/ml as measured by the PKH2 assay (see Materials and Methods).

FIG. 3

Flow cytometric analysis of PKH2 staining of B cells from cultures of PBMC from noninfected and M. avium subsp. paratuberculosis-infected cattle. The PBMC were stained with PKH2 prior to culture, cultured with or without antigen (whole-cell sonicate), and harvested for flow cytometric analysis after 5 days of incubation. The cells were examined for expression of BAQ155A (a cell surface marker of bovine B cells) and PKH2 staining. Cells from subclinical animals had a lower intensity of PKH2 staining in antigen-stimulated cultures (D) compared to nonstimulated cultures (C), whereas animals with clinical disease (E and F) and noninfected animals (A and B) had similar PKH2 staining patterns in nonstimulated and antigen-stimulated cultures. The decreased intensity of PKH2 staining (as detected in antigen-stimulated cultures from subclinical animals) indicates cell division, since daughter cells have reduced PKH2 staining within their membranes compared to that of their parent generation. Animals with clinical disease (E and F) had higher percentages of B cells compared to noninfected animals (A and B) and animals with subclinical disease (C and D). PE, phycoerythrin.

FIG. 4

Antigen-specific T-cell proliferation of PBMC isolated from control (noninfected) and M. avium subsp. paratuberculosis-infected cattle (identified by number below the bars). The hatched bars represent the percentages of PBMC that are T cells (CD3⁺) as detected by flow cytometry. The solid bars represent the number of T cells proliferating/5,000 PBMC in response to 10 μg of antigen (whole-cell sonicate of M. avium subsp. paratuberculosis)/ml as measured by the PKH2 assay (see Materials and Methods).

FIG. 5

IFN-γ production of whole-blood cultures from control (noninfected) and M. avium subsp. paratuberculosis-infected cattle (identified by number below the bars). The open bars represent optical density readings from nonstimulated cultures, the hatched bars represent those from M. avium PPD-stimulated cultures, and the solid bars represent those from antigen (10 μg of whole-cell sonicate of M. avium subsp. paratuberculosis/ml)-stimulated cultures as measured by a commercially available ELISA kit. Optical density (OD) readings of >0.1 for antigen-stimulated cultures compared to nonstimulated cultures were considered positive (the accepted method used by diagnostic laboratories for this kit).