Project seeks to simplify Cyclospora sampling, filtration

January 11, 2023 - Cyclospora cayetanensis is a complex protozoan parasite, making it nearly impossible to culture in the laboratory and requiring complicated microscopy for detection in samples.

Researchers leading one of two separate CPS-funded projects plan to simplify Cyclospora detection in water samples, while those with the other project hope to validate an economical water-filtration system to significantly reduce protozoan risks.
One project seeks to use “aptamers” — or short strands of synthesized DNA — to bind to the target of interest. In this case, it’s Cyclospora.
Researchers will then use the aptamers to create a paper-based low-cost, easy-to-use water test similar to rapid COVID or pregnancy tests.
The other project is examining zero-valent iron (ZVI) sand filters. In trials, they significantly reduced pathogens, both in the initial flow and in the back-flush.
Researchers also will examine whether zero-valent iron affects pathogen viability and virulence.
Paper-based in-field water test

Lia Stanciu, Ph.D., with Purdue University, is heading efforts to find a cost-effective alternative method to detecting Cyclospora in irrigation water based on selected molecules, or DNA sequences.

Known as “aptamers,” these short strands of synthesized DNA only bind to the target of interest. In this case, the researchers focused on Cyclospora oocysts, or the transmission stage. 
Lia Stanciu, Ph.D.
Purdue University
“We were able to identify certain proteins on the cell membrane of Cyclospora that are unique only to Cyclospora and to which we can bind some DNA sequences,” Stanciu said. “We can synthesize those DNA-specific molecules. The next step would be to integrate those DNA molecules into something similar to a rapid COVID test or pregnancy test.”

Joining her as co-principal investigator is Amanda Deering, Ph.D., with Purdue, whose expertise in microbiology complements Stanciu’s chemistry and engineering background.

The researchers plan to use the aptamers to design simple low-cost microfluidic paper-based colorimetric tests for rapid in-field detection of oocysts. Once the treated paper has been exposed to the test water, workers place it in a small portable image processing box. The paper changes color in response to the presence or absence of oocysts.

While pink is negative and purple is positive, the imaging box analyzes for color subtleties and provides results. 

“It’s much more sensitive than the human eye,” Stanciu said. 

The researchers’ next step is to validate their paper-based testing system on Cyclospora samples obtained from specialized laboratories with the Food and Drug Administration.

She said this type of easy-to-use, cost-effective test will help the produce industry be more proactive in managing potential Cyclospora risks tied to irrigation water.

“If you can detect it in the water in the field, it’s going to be really helpful to the industry,” Stanciu said.
Zero-valent iron sand filters

Benjamin Rosenthal, a doctor of science with the U.S. Department of Agriculture Agricultural Research Service, is leading research into whether simple sand filters and/or zero-valent iron (ZVI) sand filters may significantly reduce Cyclospora in irrigation water.

Joining him as co-principal investigators are USDA ARS colleagues Jitender Dubey, Ph.D.; Mark Jenkins, Ph.D.; and Manan Sharma, Ph.D. Kalmia Kniel, Ph.D., with the University of Delaware, also is a co-PI.
Benjamin Rosenthal, S.D.
U.S. Department of Agriculture Agricultural Research Service
Rosenthal pointed to Kniel and Sharma’s previous work on filtration to reduce bacterial and viral pathogens in water as basis for the current project.

“We’re evaluating the basic principle that physical exclusion might be an option to reduce parasite burdens,” Rosenthal said.

As part of proof-of-concept work, the researchers will use Eimeria as a Cyclospora surrogate. A close relative of Cyclospora, the chicken pathogen Eimeria is readily available and safe for humans to work with.

In their initial laboratory trials, they inoculated water with a known quantity of Eimeria, ran it through a PVC tube filled with sand to simulate a sand filter and measured the pathogen reduction in the outflow. Rosenthal said the results were encouraging. 

But when they back-flushed their make-shift filters, they recovered about 80% of the original Eimeria population.

They repeated the experiment using a filter comprising 50% sand and 50% ZVI. They found a similar reduction in pathogens in the outflow. But when they back-flushed the ZVI medium, they detected very few Eimeria.

“We’re seeing when you have ZVI, it’s tighter holding, so I think it’s doing something physical that sand alone doesn’t do,” Rosenthal said.

To determine whether ZVI has an additional effect on the pathogen, he said they plan to swirl it in a solution of zero-valent iron and water.

“Then we can take those parasites and examine them to see if they are looking injured,” he said. “More importantly, we’ll feed them to the chickens to see if they are capable of causing infection.”

For the final test, the researchers will run Cyclospora through the ZVI filtration system to determine whether they see a population reduction similar to Eimeria.

Rosenthal said he hoped the project will provide scientific data the industry can use to make risk assessments.

“It’s certainly my hope that they might find these kinds of data helpful on practical applications as a means to reduce or manage risk,” he said. “We’re here to understand and try to address real problems for our stakeholders.”
Learn more about CPS-funded projects here.
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The Center for Produce Safety (CPS) is a 501(c)(3), U.S. tax-exempt, charitable organization focused exclusively on providing the produce industry and government with open access to the actionable information needed to continually enhance the safety of produce.
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