Sep 15, 2020

Better test, water filtration for Cyclospora sought in research

Kali Kniel is navigating the relatively uncharted waters of developing a more accurate test for Cyclospora cayetanensis, an emerging pathogenic protozoan parasite possibly found in agricultural water supplies.

“Cyclospora is kind of an enigma – we still don’t understand the whole biological process and how it interacts with food products,” Kniel, Ph.D. and a professor of microbial food safety at the University of Delaware, said in a news release.

As part of her project, titled “Analysis of the presence of Cyclospora in waters of the Mid-Atlantic States and evaluation of removal and inactivation by filtration,” she also is looking into using zero-valent iron (ZVI), a byproduct of the steel industry. If the material proves an effective way to neutralize and remove bacterial, viral and chemical contaminants, it could offer a cost-effective way to reduce pathogen risks in agricultural water sources.

Kniel said filling in the knowledge gaps is crucial to the produce industry, as foodborne outbreaks tied to C. cayetanensis have increased significantly during the past five years.

Although a few researchers have looked at the prevalence of C. cayetanensis in other watersheds, she said very little is known about how widespread the pathogen is within the Delmarva – Delaware, Maryland and Virginia – region.

“I think this is going to be very interesting to see if we can find Cyclospora in surface water in the urban areas and within the rural areas as well as in samples of reclaimed water, which we know is very high in E. coli,” Kniel said. “This area has quite a bit of ag and also is a very urban area only a couple hundred miles between New York City and Washington, D.C.”

To that end, she is working to develop a testing protocol that can more accurately confirm the presence of C. cayetanensis in water samples than current assays, which can have potential false positives caused by other related protozoa.

“The first finding of Cyclospora in waters of the Mid-Atlantic States could be a bit unnerving,” Kniel said. “So, the first time we report that finding, we want to be certain. There’s some chance of a false-positive with a PCR (polymerase chain reaction) assay. We’re using advanced PCR, including two different primers and gene sequencing. That will really help us to confirm it’s there. We don’t want to alert the food safety community and produce growers until we’re really certain.”

She is working closely with the CONSERVE Center of Excellence, based in the University of Maryland School of Public Health and funded by the U.S. Department of Agriculture. Led by Amy Sapkota, Ph.D., the center is tasked with developing safe, alternative irrigation strategies that will sustain food production. As part of Kniel’s project, they collected 72 water samples over about 18 months and identified about one third as presumptive positive for Cyclospora using an older testing method.

Like many other activities this year, Kniel’s research has been slowed by the coronavirus pandemic, which significantly limited laboratory activities for a number of months. “We’ve done screening using our controls and testing the actual samples,” she said in late August. “I hope to send them off for gene sequencing in another week. I had hoped to have all of this done by now.”

Kniel also is working with Manan Sharma, Ph.D. and a research microbiologist with the USDA Agricultural Research, who is further developing ZVI and optimizing prototype filtration ZVI-sand systems for small-scale diversified farms. The system could theoretically remove protozoan oocytes – rather large, thick-bodied structures that sporulate once inside the host.  “(Oocytes) are so large that we should be able to successfully filter and remove them,” Kniel said “It will be interesting to  see if we can get some sort of inactivation by a biochemical event when the iron oxidizes with water.”

Graduate student Alyssa Kelly is working to set up the filters and film a video about the work. The benefit of ZVI is it can be easily incorporated into existing irrigation practices.

“Long term, this type of filtration could be beneficial in that it should improve the microbiological quality of water and help in compliance with water quality standards,” Kniel said. “There are several companies that could provide ZVI for incorporation into a designed filtration system.”

Of course, she said, any filtration system would be in addition to and would not be a replacement for good agricultural practices and sound cold-chain management.