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Use of Hoechst 33342 staining to detect apoptotic changes in bovine mononuclear phagocytes infected with
1Department of Bacteriology, College of Agriculture and Life Sciences, University of Wisconsin-Madison, Madison, WI 53706, USA.
Abstract:
Mycobacterium avium subsp. paratuberculosis is an intracellular pathogen of macrophages that causes a chronic enteritis (Johne's disease) in ruminants. The purpose of this study was to determine whether M. avium subsp. paratuberculosis infection causes apoptosis in bovine monocytes. Using Hoechst 33342 staining, we observed increased numbers of apoptotic monocytes within 6 h of infection with M. avium subsp. paratuberculosis, and these numbers increased further at 24 and 48 h. This effect appeared to require viable bacilli, because monocytes infected with heat-killed M. avium subsp. paratuberculosis did not exhibit a significant increase in apoptosis. Preincubation of monocytes with bovine growth hormone prior to infection with M. avium subsp. paratuberculosis did not significantly alter the number of apoptotic cells.
Insights
Mycobacterium avium subsp. paratuberculosis infection induces apoptosis in bovine monocytes, a key step in Johne's disease. This effect requires live bacteria and is not influenced by bovine growth hormone.
Area of Science:
- Veterinary immunology
- Bacterial pathogenesis
- Cell biology
Background:
- Mycobacterium avium subsp. paratuberculosis (MAP) is an intracellular pathogen responsible for Johne's disease in ruminants.
- MAP infects macrophages, leading to chronic enteritis.
- Understanding MAP's interaction with host cells is crucial for disease management.
Purpose of the Study:
- To investigate if MAP infection induces apoptosis in bovine monocytes.
- To explore the role of viable MAP bacilli in this process.
- To assess the impact of bovine growth hormone on MAP-induced apoptosis.
Main Methods:
- Bovine monocytes were infected with live or heat-killed MAP.
- Apoptosis was quantified using Hoechst 33342 staining at 6, 24, and 48 hours post-infection.
- Monocytes were preincubated with bovine growth hormone before MAP infection.
Main Results:
- A significant increase in apoptotic bovine monocytes was observed starting at 6 hours post-infection with live MAP.
- Apoptosis levels continued to rise at 24 and 48 hours.
- Heat-killed MAP did not induce significant apoptosis.
- Bovine growth hormone did not alter the rate of apoptosis.
Conclusions:
- MAP infection triggers apoptosis in bovine monocytes.
- Viable MAP bacilli are necessary for inducing monocyte apoptosis.
- Bovine growth hormone does not modulate MAP-induced monocyte apoptosis.
