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ARS Home » Midwest Area » Bowling Green, Kentucky » Food Animal Environmental Systems Research » Research » Publications at this Location » Publication #191631


item JANAKI, L
item Cook, Kimberly - Kim
item BERK, S

Submitted to: American Society for Microbiology
Publication Type: Abstract Only
Publication Acceptance Date: 2/17/2006
Publication Date: 5/22/2006
Citation: Janaki, L., Cook, K.L., Berk, S.G. Interactions between mycobacterium avium subsp. paratuberculosis and protozoa isolated from a watering trough of a cow with johne's disease. American Society for Microbiology. CDROM

Interpretive Summary:

Technical Abstract: Introduction: Johne’s disease, caused by Mycobacterium avium subsp. paratuberculosis (Map), is a disease of cattle and other ruminants, and is economically important for beef and dairy industries. Environmental sources of the bacteria are not well understood; and no studies have considered protozoa as hosts for Map in the farm environment, particularly the water of drinking troughs of cows. The purpose of the present investigation was to obtain protozoa from watering troughs and examine their interactions with Map. Methods: Water and biofilm from a trough used by a cow with Johne’s disease was processed to enrich for protozoa. Of many species of protozoa observed, two amoebal species were isolated, and tentatively identified as a small species of Acanthamoeba and a Vanella-like species. They were cultured in bacterized cereal leaves medium to which Klebsiella was added as a food source. For experiments the amoebae (3.5 X 104 cells ml-1) were washed and suspended in a buffered saline solution amended with washed Map (ATCC 700535; 2 X 105 cells ml-1). Confocal microscopy was used to visualize fluorescent acid-fast stained Map in amoebae, and quantitative real-time PCR (QRT-PCR) was used to monitor changes in copies of the IS900. Controls were unfed amoebae. An axenically grown ciliate, Tetrahymena sp., isolated from moist soil, was also fed Map. Results: Confocal microscopy revealed large numbers of Map in the Acanthamoeba strain after 24 to 48 hours, and QRT-PCR revealed that the numbers of Map increased 3-fold, whereas the controls decreased. The Vanella-like amoeba expelled many food vacuoles containing viable Map cells, determined by the BacLight Live/Dead stain. Likewise, after only 16 h, the ciliate released vesicles with the same proportion of live to dead Map cells as were in the original Map suspension. Such vesicles may protect bacteria from harsh environmental conditions. Conclusion: These results suggest that various species of protozoa may interact differently to serve as environmental reservoirs or amplifiers of Map populations, especially in the watering trough environments of farm animals.