Peyer's patch-deficient mice demonstrate that Mycobacterium avium subsp. paratuberculosis translocates across the mucosal barrier via both M cells and enterocytes but has inefficient dissemination

Abstract

Mycobacterium avium subsp. paratuberculosis, the agent of Johne's disease, infects ruminant hosts by translocation through the intestinal mucosa. A number of studies have suggested that M. avium subsp. paratuberculosis interacts with M cells in the Peyer's patches of the small intestine. The invasion of the intestinal mucosa by M. avium subsp. paratuberculosis and Mycobacterium avium subsp. hominissuis, a pathogen known to interact with intestinal cells, was compared. M. avium subsp. paratuberculosis was capable of invading the mucosa, but it was significantly less efficient at dissemination than M. avium subsp. hominissuis. B-cell knockout (KO) mice, which lack Peyer's patches, were used to demonstrate that M. avium subsp. paratuberculosis enters the intestinal mucosa through enterocytes in the absence of M cells. In addition, the results indicated that M. avium subsp. paratuberculosis had equal abilities to cross the mucosa in both Peyer's patch and non-Peyer's patch segments of normal mice. M. avium subsp. paratuberculosis was also shown to interact with epithelial cells by an alpha(5)beta(1) integrin-independent pathway. Upon translocation, dendritic cells ingest M. avium subsp. paratuberculosis, but this process does not lead to efficient dissemination of the infection. In summary, M. avium subsp. paratuberculosis interacts with the intestinal mucosa by crossing both Peyer's patches and non-Peyer's patch areas but does not translocate or disseminate efficiently.

FIG. 1.

Microbial burdens in organs of infected C57BL/6 black mice. (A and B) Mice were infected as described in Materials and Methods, and microbial burdens in animals infected with M. avium subsp. paratuberculosis (MAP) (A) or M. avium subsp. hominissuis (MAH) (B) were determined as a function of time. Note that different scales are used for the y axes in panels A and B. (C, D, and E) The same data are plotted by using the same y axis scale for all graphs, highlighting the comparison of organ burdens between the two M. avium subspecies. Mice were infected with 2.4 × 10⁶ M. avium subsp. hominissuis organisms or with 3.8 × 10⁶ M. avium subsp. paratuberculosis organisms. Each bar represents the mean data collected from 20 mice. Error bars, standard errors of the means. P < 0.05 for the comparison between M. avium subsp. hominissuis and M. avium subsp. paratuberculosis at the same time point for each of the organs.

FIG. 2.

Transmission electron micrograph of a sample from a mouse orally infected with M. avium subsp. paratuberculosis. The intestine was harvested at 2 days postinfection. M. avium subsp. paratuberculosis can be observed within enterocytes (arrows). Magnification, ×10,000.

FIG. 3.

Uptake and survival of M. avium subsp. paratuberculosis in dendritic cells. (A) Uptake of M. avium subsp. paratuberculosis by bovine dendritic cells following translocation across a polarized monolayer of MDBK cells. Uptake by dendritic cells was allowed for 1 h. Bacteria obtained from 7H9 broth medium supplemented with mycobactin J were used as a control. (B) Number of M. avium subsp. paratuberculosis bacteria in dendritic cells 10 days after infection (no significant differences in growth were observed).

FIG. 4.

Mouse model of M. avium infection. Shown are predicted outcomes of oral infection of wild-type (C57BL/6J WT) (A) and B-cell-deficient (C57BL/6J B-cell KO) (B) mice with M. avium subsp. hominissuis (MAH) or M. avium subsp. paratuberculosis (MAP). Dots indicate bacillary burdens, and arrow widths are proportional to the numbers of translocated bacilli. In the model depicted, M. avium subsp. hominissuis is translocated mostly via enterocytes in WT mice.