Submitted to: PLoS One
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: November 20, 2010
Publication Date: December 20, 2010
Citation: Ravva, S.V., Sarreal, C.Z., Mandrell, R.E. 2010. Identification of protozoa in dairy lagon Wwsterwater that consume Escherichia coli O157:H7 preferentially. PLoS One. 5:12 e15671. Interpretive Summary: Outbreaks of foodborne illnesses caused by pathogenic bacteria are increasingly being linked to fresh produce. Animal manure is a potential pathogen reservoir, and the close proximity of dairy operations and croplands in California cannot be ignored. We have worked on developing improved detection methods for pathogens, and on understanding how manure management systems affect the chemical environment on dairies. Our long-term goals are to develop integrated manure management systems that reduce pathogen numbers on dairies, thereby reducing chances for food contamination in the field. Data on reduction of a foodborne pathogen, Escherichia coli O157:H7, by protozoa native to wastewater from dairy lagoons are presented.
Technical Abstract: Escherichia coli O157:H7 (EcO157), an agent of life threatening hemolytic-uremic syndrome, resides in ruminants and released in feces at numbers as high as 10 million cells per gram. EcO157 could survive in manure for as long as 21 months, but we observed a rapid disappearance of an outbreak strain of EcO157 with a D-value (days for 90% decline) of 0.5 day in wastewater from dairy lagoons. Although chemical, environmental and biological factors may be responsible for this decrease, we observed an 11-fold increase in native protozoa when wastewater was re-inoculated with 2 x 107 cells of EcO157 per ml. These protozoa engulfed the green fluorescent protein labeled EcO157 within 2 hours after inoculation, but expelled vacuoles filled with live EcO157 cells within 3 days into surrounding wastewater, whereas other protozoa retained the EcO157-filled vacuoles for 7 days. EcO157 was not detected by confocal microscopy either inside or outside protozoa after 7 days. Mixed cultures of protozoa enriched from wastewater consumed EcO157 preferentially as compared to native aerobic bacteria, but failed to eliminate them when EcO157 cells declined to 104 per ml. We isolated three protozoa from mixed cultures and typed them by 18S sequencing as Vorticella microstoma, Platyophyra sp. and Colpoda aspera. While all three protozoa internalized EcO157, only Platyophyra and Colpoda acted as predators. Similar to mixed cultures, these protozoa failed to eliminate EcO157 from PBS containing no other supplemental nutrients or prey. However, spiking PBS with cereal grass medium as nutrients induced predation of EcO157 by Platyophyra sp. after 3 days or enhanced predation by Colpoda after 5 days. Therefore, attempts to enrich protozoa to decrease EcO157 from dairy lagoons, may correspond to an increase in protozoa similar to Vorticella and possibly facilitate transport of bacterial pathogens to food crops grown in proximity.