{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,5]],"date-time":"2026-09-05T13:59:17Z","timestamp":1788616757051,"version":"build-2803163510"},"reference-count":67,"publisher":"American Society for Microbiology","issue":"10","license":[{"start":{"date-parts":[[2025,10,8]],"date-time":"2025-10-08T00:00:00Z","timestamp":1759881600000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"funder":[{"DOI":"10.13039\/100000199","name":"U.S. Department of Agriculture","doi-asserted-by":"publisher","award":["USDA CRIS Project 8042-32420-007-00D"],"award-info":[{"award-number":["USDA CRIS Project 8042-32420-007-00D"]}],"id":[{"id":"10.13039\/100000199","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000060","name":"National Institute of Allergy and Infectious Diseases","doi-asserted-by":"publisher","award":["R01 AI148667"],"award-info":[{"award-number":["R01 AI148667"]}],"id":[{"id":"10.13039\/100000060","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000060","name":"National Institute of Allergy and Infectious Diseases","doi-asserted-by":"publisher","award":["R01 AI148667"],"award-info":[{"award-number":["R01 AI148667"]}],"id":[{"id":"10.13039\/100000060","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["mBio"],"published-print":{"date-parts":[[2025,10,8]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:sec>\n                    <jats:title\/>\n                    <jats:p>\n                      <jats:italic toggle=\"yes\">Cryptosporidium<\/jats:italic>\n                      is a globally endemic parasite genus with over 40 recognized species. While\n                      <jats:italic toggle=\"yes\">C. hominis<\/jats:italic>\n                      and\n                      <jats:italic toggle=\"yes\">C. parvum<\/jats:italic>\n                      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\n                      <jats:italic toggle=\"yes\">Cryptosporidium<\/jats:italic>\n                      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\n                      <jats:italic toggle=\"yes\">Cryptosporidium<\/jats:italic>\n                      amplicons to a species using a custom database. This approach demonstrated sensitivity, successfully detecting and accurately identifying as little as 0.001 ng of\n                      <jats:italic toggle=\"yes\">C. parvum<\/jats:italic>\n                      DNA in a complex stool background. Notably, we differentiated mixed infections and demonstrated the ability to identify potentially novel species of\n                      <jats:italic toggle=\"yes\">Cryptosporidium<\/jats:italic>\n                      both\n                      <jats:italic toggle=\"yes\">in situ<\/jats:italic>\n                      and\n                      <jats:italic toggle=\"yes\">in vitro<\/jats:italic>\n                      . Using this method, we identified\n                      <jats:italic toggle=\"yes\">Cryptosporidium parvum<\/jats:italic>\n                      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\n                      <jats:italic toggle=\"yes\">C. hominis<\/jats:italic>\n                      . Thus, this pipeline provides a sensitive tool for\n                      <jats:italic toggle=\"yes\">Cryptosporidium<\/jats:italic>\n                      species-level identification, allowing for the detection and accurate identification of minor variants and mixed infections.\n                    <\/jats:p>\n                    <jats:sec>\n                      <jats:title>IMPORTANCE<\/jats:title>\n                      <jats:p>\n                        <jats:italic toggle=\"yes\">Cryptosporidium<\/jats:italic>\n                        is a eukaryotic parasite and a leading global cause of waterborne diarrhea, with over 40 recognized species infecting livestock, wildlife, and people. While we have effective tools for detecting\n                        <jats:italic toggle=\"yes\">Cryptosporidium<\/jats:italic>\n                        in clinical and agricultural water samples, there is still a need for a method that can efficiently identify known species as well as infections with multiple\n                        <jats:italic toggle=\"yes\">Cryptosporidium<\/jats:italic>\n                        species, which are increasingly being reported. In this study, we utilized sequencing of a specific region to develop a sensitive and accurate identification workflow for\n                        <jats:italic toggle=\"yes\">Cryptosporidium<\/jats:italic>\n                        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\n                        <jats:italic toggle=\"yes\">Cryptosporidium<\/jats:italic>\n                        species in complex clinical and agricultural samples. This has important implications for clinical diagnostics, biosurveillance, and understanding disease transmission, ultimately benefiting clinicians and produce growers.\n                      <\/jats:p>\n                    <\/jats:sec>\n                  <\/jats:sec>","DOI":"10.1128\/mbio.01109-25","type":"journal-article","created":{"date-parts":[[2025,9,9]],"date-time":"2025-09-09T13:00:44Z","timestamp":1757422844000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":2,"title":["Amplicon sequencing detects, identifies, and quantifies minority variants in mixed-species infections of\n                    <i>Cryptosporidium<\/i>\n                    parasites"],"prefix":"10.1128","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-2441-5115","authenticated-orcid":false,"given":"Randi","family":"Turner","sequence":"first","affiliation":[{"name":"Agricultural Research Service, United States Department of Agriculture (USDA)","place":["Beltsville, USA"]},{"id":[{"id":"https:\/\/ror.org\/00te3t702","id-type":"ROR","asserted-by":"publisher"}],"name":"University of Georgia","place":["Athens, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Doaa","family":"Naguib","sequence":"additional","affiliation":[{"name":"Agricultural Research Service, United States Department of Agriculture (USDA)","place":["Beltsville, USA"]},{"id":[{"id":"https:\/\/ror.org\/01k8vtd75","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Hygiene and Zoonoses, Faculty of Veterinary Medicine, Mansoura University","place":["Mansoura, Egypt"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Elora","family":"Pierce","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/0432jq872","id-type":"ROR","asserted-by":"publisher"}],"name":"Mississippi State University College of Veterinary Medicine","place":["Mississippi State, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alison","family":"Li","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/05qwgg493","id-type":"ROR","asserted-by":"publisher"}],"name":"Boston University","place":["Boston, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Matthew","family":"Valente","sequence":"additional","affiliation":[{"name":"Agricultural Research Service, United States Department of Agriculture (USDA)","place":["Beltsville, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Travis C.","family":"Glenn","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/00te3t702","id-type":"ROR","asserted-by":"publisher"}],"name":"University of Georgia","place":["Athens, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Benjamin M.","family":"Rosenthal","sequence":"additional","affiliation":[{"name":"Agricultural Research Service, United States Department of Agriculture (USDA)","place":["Beltsville, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jessica C.","family":"Kissinger","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/00te3t702","id-type":"ROR","asserted-by":"publisher"}],"name":"University of Georgia","place":["Athens, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9862-2507","authenticated-orcid":true,"given":"Asis","family":"Khan","sequence":"additional","affiliation":[{"name":"Agricultural Research Service, United States Department of Agriculture (USDA)","place":["Beltsville, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","reference":[{"key":"e_1_3_5_2_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0140-6736(13)60844-2"},{"key":"e_1_3_5_3_2","doi-asserted-by":"publisher","DOI":"10.1097\/QCO.0b013e328357e569"},{"key":"e_1_3_5_4_2","first-page":"636","article-title":"Prevalence and risk factors of Cryptosporidium species among domestic animals in rural communities in Northern South Africa","volume":"34","author":"Samie A","year":"2017","unstructured":"Samie A, Hlungwani AH, Mbati PA. 2017. 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