{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,23]],"date-time":"2026-10-23T20:27:26Z","timestamp":1792787246669,"version":"4.5.0"},"reference-count":61,"publisher":"American Society for Microbiology","issue":"8","license":[{"start":{"date-parts":[[2025,8,5]],"date-time":"2025-08-05T00:00:00Z","timestamp":1754352000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2025,8,5]],"date-time":"2025-08-05T00:00:00Z","timestamp":1754352000000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"funder":[{"DOI":"10.13039\/100000060","name":"National Institute of Allergy and Infectious Diseases","doi-asserted-by":"publisher","award":["RO1AI143951"],"award-info":[{"award-number":["RO1AI143951"]}],"id":[{"id":"10.13039\/100000060","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Vermont Immunology and Infectious Disease Training Grant","award":["2T32AI055402-16A1"],"award-info":[{"award-number":["2T32AI055402-16A1"]}]},{"name":"National Institute of general","award":["P20GM125498"],"award-info":[{"award-number":["P20GM125498"]}]}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["Microbiol Spectr"],"published-print":{"date-parts":[[2025,8,5]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:sec>\n                    <jats:title\/>\n                    <jats:p>\n                      Many advances have been made recently in our understanding of\n                      <jats:italic toggle=\"yes\">Cryptosporidium<\/jats:italic>\n                      \u2019s asexual cycle and sexual differentiation. However, the process of fertilization, which is required for transmission of infectious oocysts, is not well understood. Typical cancer cell-based culture only allows robust exploration of asexual cycle and sexual differentiation of\n                      <jats:italic toggle=\"yes\">Cryptosporidium<\/jats:italic>\n                      . To facilitate exploration of sexual reproduction in\n                      <jats:italic toggle=\"yes\">Cryptosporidium parvum,<\/jats:italic>\n                      we developed an organoid-based culture system that supports\n                      <jats:italic toggle=\"yes\">Cryptosporidium\u2019s<\/jats:italic>\n                      full life cycle and a novel fertilization reporter. Organoid-derived monolayers (ODMs) supported fertilization and oocyst production and maintained the infection for up to 3 weeks. ODM-derived oocysts were infectious\n                      <jats:italic toggle=\"yes\">in vivo<\/jats:italic>\n                      . Fertilization was confirmed by successfully mating two strains of\n                      <jats:italic toggle=\"yes\">C. parvum<\/jats:italic>\n                      and with a novel fertilization switch reporter. The fertilization switch reporter utilizes a DiCre system in which cre fragments are expressed under the control of sexual stage promoters, resulting in a rapamycin-inducible switch in fluorescent protein expression from mCherry to mNeonGreen after fertilization that is spatially and temporally controlled. This results in mCherry-positive parasites in the first generation and offspring that express mNeonGreen.\n                      <jats:italic toggle=\"yes\">In vivo<\/jats:italic>\n                      validation of the fertilization switch reporter demonstrated the precision and efficiency of the fertilization switch reporter and confirmed excision of the mCherry gene sequence only after rapamycin treatment. The start of a second generation of parasites was also shown in the ODMs and rarely in HCT8s. Use of this reporter in ODMs can help investigate the\n                      <jats:italic toggle=\"yes\">Cryptosporidium<\/jats:italic>\n                      life cycle post-sexual differentiation in a physiologically relevant\n                      <jats:italic toggle=\"yes\">in vitro<\/jats:italic>\n                      system.\n                    <\/jats:p>\n                    <jats:sec>\n                      <jats:title>IMPORTANCE<\/jats:title>\n                      <jats:p>\n                        Organoid-derived monolayers enable the study of previously inaccessible aspects of\n                        <jats:italic toggle=\"yes\">Cryptosporidium<\/jats:italic>\n                        \u2019s biology. This system overcomes the disadvantages of previous organoid-based methods for\n                        <jats:italic toggle=\"yes\">Cryptosporidium<\/jats:italic>\n                        culture. It is faster and simpler than previously described systems, uses defined differentiation media to increase reproducibility, enables real-time observation, supports parasite fertilization and oocyst production, and provides a physiologically relevant tissue culture system to facilitate cell biology studies. The organoid-derived monolayer system could facilitate the study of host-pathogen interactions,\n                        <jats:italic toggle=\"yes\">Cryptosporidium<\/jats:italic>\n                        -host specificity, or innate or cellular immune responses to\n                        <jats:italic toggle=\"yes\">Cryptosporidium<\/jats:italic>\n                        infection stimulated in the intestinal epithelium. The fertilization switch reporter could be used to test factors or drugs that may have potential to interfere with\n                        <jats:italic toggle=\"yes\">Cryptosporidium\u2019s<\/jats:italic>\n                        sexual reproduction. Organoid-based cell cultures in combination with the fertilization switch reporter could increase our understanding of sexual reproduction in\n                        <jats:italic toggle=\"yes\">Cryptosporidium,<\/jats:italic>\n                        leading to vital information for the development of sexual reproduction inhibitors or vaccines that could shorten disease duration and prevent transmission.\n                      <\/jats:p>\n                    <\/jats:sec>\n                  <\/jats:sec>","DOI":"10.1128\/spectrum.00502-25","type":"journal-article","created":{"date-parts":[[2025,6,25]],"date-time":"2025-06-25T09:02:23Z","timestamp":1750842143000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":5,"title":["Organoid-based\n                    <i>in vitro<\/i>\n                    system and reporter for the study of\n                    <i>Cryptosporidium parvum<\/i>\n                    sexual reproduction"],"prefix":"10.1128","volume":"13","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-6538-3714","authenticated-orcid":true,"given":"Bethany R.","family":"Korwin-Mihavics","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/0155zta11","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Medicine, University of Vermont","place":["Burlington, USA"]},{"name":"Microbiology and Molecular Genetics, University of Vermont","place":["Burlington, USA"]},{"id":[{"id":"https:\/\/ror.org\/0155zta11","id-type":"ROR","asserted-by":"publisher"}],"name":"Cellular, Molecular, and Biomedical Sciences, University of Vermont","place":["Burlington, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Emmett A.","family":"Dews","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/0155zta11","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Medicine, University of Vermont","place":["Burlington, 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