Invasion and persistence of Mycobacterium avium subsp. paratuberculosis during early stages of Johne's disease in calves

Abstract

Infection with Mycobacterium avium subsp. paratuberculosis causes Johne's disease in cattle and is a serious problem for the dairy industry worldwide. Development of models to mimic aspects of Johne's disease remains an elusive goal because of the chronic nature of the disease. In this report, we describe a surgical approach employed to characterize the very early stages of infection of calves with M. avium subsp. paratuberculosis. To our surprise, strains of M. avium subsp. paratuberculosis were able to traverse the intestinal tissues within 1 h of infection in order to colonize distant organs, such as the liver and lymph nodes. Both the ileum and the mesenteric lymph nodes were persistently infected for months following intestinal deposition of M. avium subsp. paratuberculosis despite a lack of fecal shedding of mycobacteria. During the first 9 months of infection, humoral immune responses were not detected. Nonetheless, using flow cytometric analysis, we detected a significant change in the cells participating in the inflammatory responses of infected calves compared to cells in a control animal. Additionally, the levels of cytokines detected in both the ileum and the lymph nodes indicated that there were TH1-type-associated cellular responses but not TH2-type-associated humoral responses. Finally, surgical inoculation of a wild-type strain and a mutant M. avium subsp. paratuberculosis strain (with an inactivated gcpE gene) demonstrated the ability of the model which we developed to differentiate between the wild-type strain and a mutant strain of M. avium subsp. paratuberculosis deficient in tissue colonization and invasion. Overall, novel insights into the early stages of Johne's disease were obtained, and a practical model of mycobacterial invasiveness was developed. A similar approach can be used for other enteric bacteria.

FIG. 1.

Bacterial colonization of calves infected with M. avium subsp. paratuberculosis at very early times following infection. (A) Colonization of the ileum and mesenteric lymph nodes 1 h after inoculation of 10⁹ CFU/animal for calf 1021 and inoculation of 10⁸ CFU/animal for calves 1026 and 1027. (B) Colonization of calf 1022 organs 4 days after infection with 10¹⁰ CFU M. avium subsp. paratuberculosis. The error bars indicate standard errors of the means.

FIG. 2.

Differential colonization of calf tissues during persistent infection with M. avium subsp. paratuberculosis. The colony counts in both the ileum and the mesenteric lymph nodes were determined for 9 months following surgical infection with M. avium subsp. paratuberculosis (10⁹ CFU/calf). The error bars indicate standard errors of the means. An asterisk indicates that the levels of colonization of the lymph nodes and the ileum are significantly different, as determined by Student's t test (P < 0.05).

FIG. 3.

Histopathology of calf tissues at different times following infection with M. avium subsp. paratuberculosis strain K-10. All sections were stained with hematoxylin and eosin. (A and B) Sections of a normal lymph node (A) and Peyer's patches of the ileum (B) from a control calf sacrificed 60 days after infection. (C) Small intestine with enlarged lamina propria (arrows) at 8 months postinfection. (D) Ileal Peyer's patches with large follicles and Mott cells (arrows) at 9 months postinfection. (E) Mesenteric lymph node with necrotic centers (arrow), scattered macrophages, and giant cells (arrowheads) at 9 months postinfection. (F) Liver with macrophage aggregate (arrow) at 9 months postinfection.

FIG. 4.

Induction of cell-mediated immunity in calves after intestinal challenge with M. avium subsp. paratuberculosis. (A) Two-parameter dot plot profiles of PBMC stained with three MAbs specific for CD4, CD45R0 (memory T-cell marker), and CD25. Electronic gates were placed on small nonproliferating lymphocytes (left panel, side light scatter [SSC] versus forward light scatter [FSC], lower left quadrant) and large proliferating cells (right lower quadrant) to determine the relative proportions of small and large lymphocytes in the cell preparation. An additional electronic gate was placed on CD4 T cells (middle panel, circled area in lower right quadrant) to isolate all CD4 T cells for analysis and show the relative proportion of CD4 T cells present in the cell preparation. All three gates were used to analyze the expression of CD25 on CD4 naïve and memory T cells (right panel). This gating strategy was used to analyze expression of all the activation molecules on CD4 and CD8 memory T cells. (B) Summary of fluorescence-activated cell sorting analysis of CD4 memory T cells expressing MHC class II and activation molecules at time zero and 6 days after stimulation in RPMI medium alone or with PPD of M. avium subsp. paratuberculosis. The histogram shows the relative proportions of CD4 memory T cells expressing different activation molecules after 6 days of culture. (C) Summary of fluorescence-activated cell sorting analysis of CD8 memory T cells expressing different MHC class II and activation molecules.

FIG. 5.

Cytokine gene expression in calf tissues at different times following infection with M. avium subsp. paratuberculosis. Quantitative RT-PCR was used to examine the transcription of bovine cytokines (IL-4, IFN-γ, TNF-α, and IL-12). The fold changes in transcriptional levels in infected animals were estimated by comparison with the control calf tissue at 6, 8, and 9 months postinfection. Colony counts for the same tissues were also determined (y axis on the right). (A) Transcriptional profile of cytokines in mesenteric lymph nodes. (B) Transcriptional profile of cytokines in intestinal tissues.

FIG. 6.

(A) Diagram of the gcpE operon in M. avium subsp. paratuberculosis. The position of a Tn5367 insertion present in the ΔgcpE mutant, as characterized in a previous study (30), is indicated. (B) Assessment of virulence of the ΔgcpE mutant using the intestinal calf model of paratuberculosis. The competitive index for the ΔgcpE mutant was calculated based on the levels of tissue colonization by the mutant strain and a virulent strain, M. avium subsp. paratuberculosis wild-type strain ATCC 19698.