The ability of Mycobacterium avium subsp. paratuberculosis to enter bovine epithelial cells is influenced by preexposure to a hyperosmolar environment and intracellular passage in bovine mammary epithelial cells

Abstract

Mycobacterium avium subsp. paratuberculosis is the cause of Johne's disease in cattle and other ruminants. M. avium subsp. paratuberculosis infection of the bovine host is not well understood; however, it is assumed that crossing the bovine intestinal mucosa is important in order for M. avium subsp. paratuberculosis to establish infection. To examine the ability of M. avium subsp. paratuberculosis to infect bovine epithelial cells in vitro, Madin-Darby bovine kidney (MDBK) epithelial cells were exposed to M. avium subsp. paratuberculosis. It was observed that bacteria can establish infection and replicate within MDBK cells. M. avium subsp. paratuberculosis also has been reported to infect mammary tissue and milk, and we showed that M. avium subsp. paratuberculosis infects bovine mammary epithelial cells (MAC-T cell line). Using polarized MAC-T cell monolayers, it was also determined that M. avium subsp. paratuberculosis crosses apical and basolateral surfaces with approximately the same degree of efficiency. Because M. avium subsp. paratuberculosis can be delivered to the naïve host by milk, it was investigated whether incubation of M. avium subsp. paratuberculosis with milk has an effect on invasion of MDBK cells. M. avium subsp. paratuberculosis exposed to milk entered epithelial cells with greater efficiency than M. avium subsp. paratuberculosis exposed to broth medium or water (P < 0.01). Growth of M. avium subsp. paratuberculosis within MAC-T cells also resulted in augmented ability to subsequently infect bovine MDBK cells (P < 0.001). Microarray analysis of intracellular M. avium subsp. paratuberculosis RNA indicates the increased transcription of genes which might be associated with an invasive phenotype.

FIG. 1.

Invasion of bovine epithelial cells (MDBK) by M. avium subsp. paratuberculosis. The percentage of invasion was defined as the fraction of inoculated bacteria that became internalized after the incubation period. Values represent the means of three experiments ± standard errors of the means (SEM). *, P < 0.05 compared with the percent invasion at 2 h.

FIG. 2.

(A) Invasion of bovine mammary epithelial cells (MAC-T) by M. avium subsp. paratuberculosis. The percent invasion was defined as the fraction of inoculated bacteria that became internalized after the incubation period. Values represent the means of three experiments ± SEM. *, P ≤ 0.05 compared with the percent invasion at 2 h. (B) Representative transmission electron micrograph of mammary epithelial cells (MAC-T) after 24 h of infection by M. avium subsp. paratuberculosis bacteria (arrows). Bacterial cells can be seen within vacuoles. Magnification, ×10,000.

FIG. 3.

(A) Ability of M. avium subsp. paratuberculosis to invade MDBK epithelial cells following exposure to three different environments: milk, 7H9 broth, or water for 24 h at 37°C. Values are the means of three experiments ± SEM. *, P < 0.01 compared with 7H9 broth or water. (B) Invasion of bovine epithelial cells (MDBK) by M. avium subsp. paratuberculosis. Prior to invasion, M. avium subsp. paratuberculosis bacteria were incubated in the presence of milk (column A), casein (column B), casein plus 0.9% NaCl (column C), serum protein plus lactose (column D), or lactose (4.8% solution) (column E) for 24 h at 37°C. Values represent the means of three experiments ± SEM. *, P < 0.05 for difference between the invasion percent for milk (column A) and casein (column B) casein plus NaCl (column C).

FIG. 4.

Invasion of bovine epithelial cells (MDBK) by M. avium subsp. paratuberculosis after passage in MAC-T cells. M. avium subsp. paratuberculosis bacteria were incubated in MAC-T cells for 1 or 4 days and then used to infect MDBK epithelial cells. M. avium subsp. paratuberculosis incubated in medium was used as a control. Values represent the means of three experiments ± SEM. *, P < 0.001 compared with the invasion percent for the control.