Persistence of protozoan parasites and Escherichia coli on plant tissue in soil

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ABSTRACT Tilling under is a method growers employ in an effort to remediate instances of widespread contamination in fields of crops, but its effectiveness with respect to specific pathogens remains poorly understood. This study had two overarching goals: first, to assess the survival dynamics of Cryptosporidium parvum and Eimeria tenella (used as a surrogate for Cyclospora cayetanensis ) oocysts, and Escherichia coli TVS355 in soil following inoculation onto plant tissue and a simulated “tilling under” process; and second, to evaluate the potential of monitoring mRNA levels over time as oocysts die off as an alternative to traditional infectivity-based methods to determine oocyst viability. Basil, cilantro, and soil without plant tissue were inoculated with C. parvum (10 6 oocysts), E. tenella (10 6 oocysts), and E. coli TVS355 (10 8 CFU) and then incorporated into 120 g of soil; samples were then incubated at 12°C or 32°C for up to 12 months. Monthly, samples were removed and assessed for organism survival, E. coli TVS355 through traditional dilution and plating, and oocyst decay through two methods: cell culture followed by qPCR, and mRNA extraction followed by RT-qPCR to measure gene expression. E. coli remained detectable in samples for 10 months, and, through infectivity methods, C. parvum and E. tenella remained infectious for 9 months. However, results from mRNA methods indicated that oocysts remained detectable for at least 12 months, with approximately 1.22 log oocysts/sample detected at that point for both C. parvum and E. tenella . Temperature impacted E. coli TVS355 and E. tenella recovery (as determined by infectivity methods) only after 7 months, when slightly more of each organism was recovered from samples incubated at 12°C. Meanwhile, mRNA methods detected significantly ( P < 0.05) fewer oocysts of both C. parvum and E. tenella in samples incubated at 32°C as compared to those incubated at 12°C throughout the experiment. IMPORTANCE Contaminated crops may be tilled under and buried in the soil, which may lead to the decay of contaminating microbes. This study looked at three microbes that can contaminate produce: Escherichia coli (a bacterium), Cryptosporidium parvum (a zoonotic protozoan pathogenic parasite), and Eimeria tenella (an avian protozoan parasite used as a stand-in for the human parasite Cyclospora cayetanensis ). Inoculated basil and cilantro were mixed in soil and stored at 12°C and 32°C for 1 year. Both protozoan parasites remained infectious for 9 months, and E. coli remained viable for 10 months. Protozoa genetic material and mRNA, as a sign of metabolic activity, were also assessed over time. The type of plant tilled under did not make a meaningful difference. Tilling contaminated crops into soil does not quickly eliminate pathogens. Some pathogens can survive for many months, meaning risks remain in fields for longer than expected.

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