Skip to main content
ARS Home » Northeast Area » Beltsville, Maryland (BARC) » Beltsville Agricultural Research Center » Animal Parasitic Diseases Laboratory » Research » Publications at this Location » Publication #428580
Research Project: Foodborne Parasites and their Impact on Food Safety

Location: Animal Parasitic Diseases Laboratory

Title: Amplicon sequencing detects, identifies, and quantifies minority variants in mixed-species infections of Cryptosporidium parasites

Author
item TURNER, RANDI - Orise Fellow
item NAGUIB, DOAA - Orise Fellow
item PIERCE, ELORA - Mississippi State University
item LI, ALISON - Boston University
item Valente, Matthew
item GLENN, TRAVIS - University Of Georgia
item Rosenthal, Benjamin
item KISSINGER, JESSICA - University Of Georgia
item Khan, Asis

Submitted to: bioRxiv
Publication Type: Pre-print Publication
Publication Acceptance Date: 4/10/2025
Publication Date: 4/11/2025
Citation: Turner, R., Naguib, D., Pierce, E., Li, A., Valente, M., Glenn, T.C., Rosenthal, B.M., Kissinger, J.C., Khan, A. 2025. Amplicon sequencing detects, identifies, and quantifies minority variants in mixed-species infections of Cryptosporidium parasites. bioRxiv. 10(1101). Article e648298. https://doi.org/10.1101/2025.04.10.648298.
DOI: https://doi.org/10.1101/2025.04.10.648298

Interpretive Summary: Cryptosporidium is a leading cause of waterborne diarrhea, with over 40 recognized species infecting livestock, wildlife, and people. While we have effective tools for detecting Cryptosporidium in clinical and agriculture water samples, there is still a need for a method that can efficiently identify known species as well as mixed infections, which are increasingly being reported. In this study, we utilized amplicon sequencing of a single locus to develop a sensitive and accurate identification pipeline for Cryptosporidium species based on high-throughput sequencing. This method can distinguish between all 40 recognized species and accurately detect mixed infections. Our approach provides a sensitive and reliable means to identify Cryptosporidium species in complex clinical and agriculture samples. This has important implications for clinical diagnostics, biosurveillance, and understanding disease transmission, ultimately benefiting produce growers, clinicians, and regulatory agencies.

Technical Abstract: Cryptosporidium is a globally endemic parasite genus with over 40 recognized species. While C. hominis and C. parvum are responsible for most human infections, human cases involving other species have also been reported. Furthermore, there is increasing evidence of simultaneous infections with multiple species. Therefore, we devised a new means to identify various species of Cryptosporidium in mixed infections by sequencing a 431 bp amplicon of the 18S rRNA gene encompassing two variable regions. Using the DADA2 pipeline, amplicons were first identified to a genus using the SILVA 132 reference database; then Cryptosporidium amplicons to a species using a custom database. This approach demonstrated sensitivity, successfully detecting and accurately identifying as little as 0.001 ng of C. parvum DNA in a complex stool background. Notably, we differentiated mixed infections and demonstrated the ability to identify potentially novel species of Cryptosporidium both in situ and in vitro. Using this method, we identified Cryptosporidium parvum in Egyptian rabbits with three samples showing minor mixed infections. By contrast, no mixed infections were detected in Egyptian children, who were primarily infected with C. hominis. Thus, this pipeline provides a sensitive tool for Cryptosporidium species-level identification, allowing for the detection and accurate identification of minor variants and mixed infections.