{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,21]],"date-time":"2026-05-21T15:17:07Z","timestamp":1779376627347,"version":"3.53.1"},"update-to":[{"DOI":"10.1371\/journal.ppat.1011992","type":"new_version","label":"New version","source":"publisher","updated":{"date-parts":[[2024,3,11]],"date-time":"2024-03-11T00:00:00Z","timestamp":1710115200000}}],"reference-count":49,"publisher":"Public Library of Science (PLoS)","issue":"2","license":[{"start":{"date-parts":[[2024,2,28]],"date-time":"2024-02-28T00:00:00Z","timestamp":1709078400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000865","name":"Bill and Melinda Gates Foundation","doi-asserted-by":"publisher","award":["OPP1117675"],"award-info":[{"award-number":["OPP1117675"]}],"id":[{"id":"10.13039\/100000865","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000865","name":"Bill and Melinda Gates Foundation","doi-asserted-by":"publisher","award":["OPP1151701"],"award-info":[{"award-number":["OPP1151701"]}],"id":[{"id":"10.13039\/100000865","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000865","name":"Bill and Melinda Gates Foundation","doi-asserted-by":"publisher","award":["Investment 44418"],"award-info":[{"award-number":["Investment 44418"]}],"id":[{"id":"10.13039\/100000865","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000865","name":"Bill and Melinda Gates Foundation","doi-asserted-by":"publisher","award":["GH VAP NG-ID20"],"award-info":[{"award-number":["GH VAP NG-ID20"]}],"id":[{"id":"10.13039\/100000865","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["www.plospathogens.org"],"crossmark-restriction":false},"short-container-title":["PLoS Pathog"],"abstract":"<jats:p>Recent advances in the <jats:italic>in vitro<\/jats:italic> cultivation of <jats:italic>Cryptosporidium parvum<\/jats:italic> using hollow fiber bioreactor technology (HFB) have permitted continuous growth of parasites that complete all life cycle stages. The method provides access to all stages of the parasite and provides a method for non-animal production of oocysts for use in clinical trials. Here we examined the effect of long-term (&gt;20 months) <jats:italic>in vitro<\/jats:italic> culture on virulence-factors, genome conservation, and <jats:italic>in vivo<\/jats:italic> pathogenicity of the host by <jats:italic>in vitro<\/jats:italic> cultured parasites. We find low-level sequence variation that is consistent with that observed in calf-passaged parasites. Further using a calf model infection, oocysts obtained from the HFB caused diarrhea of the same volume, duration and oocyst shedding intensity as <jats:italic>in vivo<\/jats:italic> passaged parasites.<\/jats:p>","DOI":"10.1371\/journal.ppat.1011992","type":"journal-article","created":{"date-parts":[[2024,2,28]],"date-time":"2024-02-28T18:35:45Z","timestamp":1709145345000},"page":"e1011992","update-policy":"https:\/\/doi.org\/10.1371\/journal.ppat.corrections_policy","source":"Crossref","is-referenced-by-count":6,"title":["Genomic and virulence analysis of in vitro cultured Cryptosporidium parvum"],"prefix":"10.1371","volume":"20","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-7493-4346","authenticated-orcid":true,"given":"Nigel","family":"Yarlett","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mary","family":"Morada","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Deborah A.","family":"Schaefer","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kevin","family":"Ackman","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Elizabeth","family":"Carranza","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rodrigo de Paula","family":"Baptista","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michael W.","family":"Riggs","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jessica C.","family":"Kissinger","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2024,2,28]]},"reference":[{"issue":"9888","key":"ppat.1011992.ref001","doi-asserted-by":"crossref","first-page":"209","DOI":"10.1016\/S0140-6736(13)60844-2","article-title":"Burden and aetiology of diarrhoeal disease in infants and young children in developing countries (the Global Enteric Multicenter Study, GEMS): a prospective, case-control study.","volume":"382","author":"KL Kotloff","year":"2013","journal-title":"Lancet"},{"issue":"1","key":"ppat.1011992.ref002","doi-asserted-by":"crossref","first-page":"85","DOI":"10.1016\/S1473-3099(14)70772-8","article-title":"A review of the global burden, novel diagnostics, therapeutics, and vaccine targets for cryptosporidium","volume":"15","author":"W Checkley","year":"2015","journal-title":"Lancet Infect Dis"},{"issue":"5","key":"ppat.1011992.ref003","doi-asserted-by":"crossref","first-page":"e0004729","DOI":"10.1371\/journal.pntd.0004729","article-title":"The Burden of Cryptosporidium Diarrheal Disease among Children < 24 Months of Age in Moderate\/High Mortality Regions of Sub-Saharan Africa and South Asia, utilizing Data from the Global Enteric Multicenter Study (GEMS).","volume":"10","author":"SO Sow","year":"2016","journal-title":"PLoS Negl Trop Dis."},{"issue":"5","key":"ppat.1011992.ref004","doi-asserted-by":"crossref","first-page":"707","DOI":"10.4269\/ajtmh.1999.61.707","article-title":"Association of early childhood diarrhea and cryptosporidiosis with impaired physical fitness and cognitive function four-seven years later in a poor urban community in northeast Brazil","volume":"61","author":"DI Guerrant","year":"1999","journal-title":"Am J Trop Med Hyg"},{"issue":"9343","key":"ppat.1011992.ref005","doi-asserted-by":"crossref","first-page":"1375","DOI":"10.1016\/S0140-6736(02)11401-2","article-title":"Effect of nitazoxanide on morbidity and mortality in Zambian children with cryptosporidiosis: a randomised controlled trial","volume":"360","author":"B Amadi","year":"2002","journal-title":"Lancet"},{"issue":"9","key":"ppat.1011992.ref006","doi-asserted-by":"crossref","first-page":"1464","DOI":"10.1093\/infdis\/jis216","article-title":"Novel in vitro and in vivo models and potential new therapeutics to break the vicious cycle of Cryptosporidium infection and malnutrition","volume":"205","author":"LB Costa","year":"2012","journal-title":"J Infect Dis"},{"issue":"7561","key":"ppat.1011992.ref007","doi-asserted-by":"crossref","first-page":"477","DOI":"10.1038\/nature14651","article-title":"Genetic modification of the diarrhoeal pathogen Cryptosporidium parvum","volume":"523","author":"S Vinayak","year":"2015","journal-title":"Nature"},{"issue":"1","key":"ppat.1011992.ref008","doi-asserted-by":"crossref","first-page":"21","DOI":"10.1016\/j.ijpara.2015.07.006","article-title":"Continuous culture of Cryptosporidium parvum using hollow fiber technology","volume":"46","author":"M Morada","year":"2016","journal-title":"Int J Parasitol"},{"issue":"3","key":"ppat.1011992.ref009","doi-asserted-by":"crossref","first-page":"e00731","DOI":"10.1128\/IAI.00731-16","article-title":"Novel Bioengineered Three-Dimensional Human Intestinal Model for Long-Term Infection of Cryptosporidium parvum","volume":"85","author":"MA DeCicco RePass","year":"2017","journal-title":"Infect Immun"},{"issue":"1","key":"ppat.1011992.ref010","doi-asserted-by":"crossref","first-page":"17288","DOI":"10.1038\/s41598-017-17304-2","article-title":"Three-dimensional (3D) culture of adult murine colon as an in vitro model of cryptosporidiosis: Proof of concept.","volume":"7","author":"M Baydoun","year":"2017","journal-title":"Sci Rep."},{"issue":"1","key":"ppat.1011992.ref011","doi-asserted-by":"crossref","first-page":"135","DOI":"10.1016\/j.chom.2019.05.006","article-title":"A Genetically Tractable, Natural Mouse Model of Cryptosporidiosis Offers Insights into Host Protective Immunity.","volume":"26","author":"A Sateriale","year":"2019","journal-title":"Cell Host Microbe"},{"issue":"7658","key":"ppat.1011992.ref012","doi-asserted-by":"crossref","first-page":"376","DOI":"10.1038\/nature22337","article-title":"K inhibitor is a drug candidate for cryptosporidiosis","volume":"546","author":"UH Manjunatha","year":"2017","journal-title":"Nature"},{"issue":"5","key":"ppat.1011992.ref013","doi-asserted-by":"crossref","first-page":"413","DOI":"10.1016\/j.ijpara.2020.01.006","article-title":"Bumped kinase inhibitors as therapy for apicomplexan parasitic diseases: lessons learned","volume":"50","author":"R Choi","year":"2020","journal-title":"Int J Parasitol"},{"issue":"5","key":"ppat.1011992.ref014","doi-asserted-by":"crossref","first-page":"1275","DOI":"10.1021\/acsinfecdis.1c00076","article-title":"Repurposing infectious disease hits as anti-cryptosporidium leads.","volume":"7","author":"MA Hulverson","year":"2021","journal-title":"ACS Infect Dis"},{"issue":"1","key":"ppat.1011992.ref015","doi-asserted-by":"crossref","first-page":"e0156021","DOI":"10.1128\/AAC.01560-21","article-title":"Pharmacokinetics and pharmacodynamics of clofazimine for the treatment of cryptosporidiosis.","volume":"66","author":"CX Zhang","year":"2022","journal-title":"Antimicrob Ag Chemother"},{"key":"ppat.1011992.ref016","doi-asserted-by":"crossref","first-page":"e2947","DOI":"10.21769\/BioProtoc.2947","article-title":"Long-term in vitro Culture of Cryptosporidium parvum.","volume":"8","author":"N Yarlett","year":"2018","journal-title":"Bio Protoc"},{"issue":"3","key":"ppat.1011992.ref017","doi-asserted-by":"crossref","first-page":"187","DOI":"10.3354\/dao051187","article-title":"Comparison of in vitro-cultured and wild-type Perkinsus marinus. I. Pathogen virulence","volume":"51","author":"SE Ford","year":"2002","journal-title":"Dis Aquat Organ"},{"issue":"11","key":"ppat.1011992.ref018","doi-asserted-by":"crossref","first-page":"914","DOI":"10.1016\/j.pt.2020.08.008","article-title":"From Circulation to Cultivation: Plasmodium in vivo versus in vitro","volume":"36","author":"AC Brown","year":"2020","journal-title":"Trends Parasitol"},{"issue":"12","key":"ppat.1011992.ref019","doi-asserted-by":"crossref","first-page":"441","DOI":"10.1111\/pim.12056","article-title":"Virulence loss and amastigote transformation failure determine host cell responses to Leishmania mexicana","volume":"35","author":"KS Ali","year":"2013","journal-title":"Parasite Immunol"},{"issue":"1","key":"ppat.1011992.ref020","doi-asserted-by":"crossref","first-page":"e1469","DOI":"10.1371\/journal.pntd.0001469","article-title":"Impact of continuous axenic cultivation in Leishmania infantum virulence.","volume":"6","author":"D Moreira","year":"2012","journal-title":"PLoS Negl Trop Dis"},{"key":"ppat.1011992.ref021","doi-asserted-by":"crossref","first-page":"253","DOI":"10.1007\/978-1-4939-9748-0_15","article-title":"Calf Clinical Model of Cryptosporidiosis for Efficacy Evaluation of Therapeutics.","volume":"2052","author":"MW Riggs","year":"2020","journal-title":"Methods Mol Biol"},{"issue":"6","key":"ppat.1011992.ref022","doi-asserted-by":"crossref","first-page":"1445","DOI":"10.1080\/21645515.2018.1498436","article-title":"The piglet acute diarrhea model for evaluating efficacy of treatment and control of cryptosporidiosis.","volume":"15","author":"S Lee","year":"2019","journal-title":"Human Vaccines Immunotherapeutics"},{"key":"ppat.1011992.ref023","volume-title":"Guide for the care and use of laboratory animals","author":"National Research Council","year":"2011","edition":"8"},{"key":"ppat.1011992.ref024","volume-title":"Guide for the care and use of agricultural animals in research and teaching","author":"Federation of Animal Science Societies","year":"2020","edition":"3"},{"issue":"1","key":"ppat.1011992.ref025","doi-asserted-by":"crossref","first-page":"203","DOI":"10.1101\/gr.275325.121","article-title":"Long-read assembly and comparative evidence-based reanalysis of Cryptosporidium genome sequences reveal expanded transporter repertoire and duplication of entire chromosome ends including subtelomeric regions","volume":"32","author":"RP Baptista","year":"2022","journal-title":"Genome Res"},{"key":"ppat.1011992.ref026","doi-asserted-by":"crossref","first-page":"D419","DOI":"10.1093\/nar\/gkj078","article-title":"CryptoDB: a Cryptosporidium bioinformatics resource update","volume":"34","author":"M Heiges","year":"2006","journal-title":"Nucleic Acids Res"},{"key":"ppat.1011992.ref027","first-page":"1754","article-title":"Fast and accurate short read alignment with Burrows-Wheeler transform","volume":"25","author":"H Li","year":"2009","journal-title":"Bioinformatics (Oxford, England)."},{"key":"ppat.1011992.ref028","author":"Broad Institute"},{"key":"ppat.1011992.ref029","doi-asserted-by":"crossref","first-page":"1297","DOI":"10.1101\/gr.107524.110","article-title":"The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data","volume":"20","author":"A McKenna","year":"2010","journal-title":"Genome Res"},{"issue":"2","key":"ppat.1011992.ref030","first-page":"80","article-title":"A program for annotating and predicting the effects of single nucleotide polymorphisms","volume":"6","author":"P Cingolani","year":"2012","journal-title":"SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118"},{"key":"ppat.1011992.ref031","doi-asserted-by":"crossref","first-page":"35","DOI":"10.3389\/fgene.2012.00035","article-title":"Using Drosophila melanogaster as a Model for Genotoxic Chemical Mutational Studies with a New Program, SnpSift.","volume":"3","author":"P Cingolani","year":"2012","journal-title":"Front Genet"},{"issue":"6","key":"ppat.1011992.ref032","first-page":"841","article-title":"BEDTools: a flexible suite of utilities for comparing genomic features","volume":"26","author":"AR Quinlan","year":"2010","journal-title":"Bioinformatics (Oxford, England)"},{"key":"ppat.1011992.ref033","author":"JC Oliveros","journal-title":"Venny: An interactive tool for comparing lists with Venn\u2019s diagrams. (2007\u20132015"},{"issue":"5","key":"ppat.1011992.ref034","doi-asserted-by":"crossref","first-page":"455","DOI":"10.1089\/cmb.2012.0021","article-title":"SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing","volume":"19","author":"A Bankevich","year":"2012","journal-title":"J Computational Biol"},{"issue":"1","key":"ppat.1011992.ref035","doi-asserted-by":"crossref","first-page":"224","DOI":"10.1186\/s13059-019-1829-6","article-title":"RaGOO: fast and accurate reference-guided scaffolding of draft genomes","volume":"20","author":"M Alonge","year":"2019","journal-title":"Genome Biol"},{"issue":"12","key":"ppat.1011992.ref036","doi-asserted-by":"crossref","first-page":"1639","DOI":"10.1093\/bioinformatics\/btaa1016","article-title":"Liftoff: accurate mapping of gene annotations","volume":"37","author":"A Shumate","year":"2020","journal-title":"Bioinformatics"},{"key":"ppat.1011992.ref037","author":"J. Dainat","journal-title":"AGAT: Another Gff Analysis Toolkit to handle annotations in any GTF\/GFF format"},{"issue":"4","key":"ppat.1011992.ref038","doi-asserted-by":"crossref","first-page":"772","DOI":"10.1093\/molbev\/mst010","article-title":"MAFFT multiple sequence alignment software version 7: improvements in performance and usability","volume":"30","author":"K Katoh","year":"2013","journal-title":"Mol Biol Evol"},{"issue":"9","key":"ppat.1011992.ref039","doi-asserted-by":"crossref","first-page":"1236","DOI":"10.1093\/bioinformatics\/btu031","article-title":"InterProScan 5: genome-scale protein function classification","volume":"30","author":"P Jones","year":"2014","journal-title":"Bioinformatics"},{"issue":"12","key":"ppat.1011992.ref040","doi-asserted-by":"crossref","first-page":"1856","DOI":"10.1093\/infdis\/jiw488","article-title":"Novel bumped kinase Inhibitors are safe and effective therapeutics in the calf clinical model for cryptosporidiosis","volume":"214","author":"DA Schaefer","year":"2016","journal-title":"J Infect Dis"},{"key":"ppat.1011992.ref041","doi-asserted-by":"crossref","first-page":"41","DOI":"10.1016\/j.vetpar.2012.02.014","article-title":"Antibody fusions reduce onset of experimental Cryptosporidium parvum infection in calves","volume":"188","author":"M. Imboden","year":"2012","journal-title":"Vet Parasitol"},{"issue":"1","key":"ppat.1011992.ref042","doi-asserted-by":"crossref","first-page":"159","DOI":"10.1086\/345876","article-title":"A new method for culturing Plasmodium falciparum shows replication at the highest erythrocyte densities","volume":"187","author":"T Li","year":"2003","journal-title":"J Infect Dis"},{"issue":"4","key":"ppat.1011992.ref043","first-page":"761","article-title":"Optimizing the culture of Plasmodium falciparum in hollow fiber bioreactors","volume":"41","author":"P Preechapornkul","year":"2010","journal-title":"Southeast Asian J Trop Med Public Health"},{"issue":"7940","key":"ppat.1011992.ref044","doi-asserted-by":"crossref","first-page":"534","DOI":"10.1038\/s41586-022-05466-7","article-title":"In vitro production of infectious Plasmodium falciparum sporozoites","volume":"612","author":"AG Eappen","year":"2022","journal-title":"Nature"},{"issue":"7","key":"ppat.1011992.ref045","doi-asserted-by":"crossref","first-page":"814","DOI":"10.1038\/s41564-018-0177-8","article-title":"Modelling Cryptosporidium infection in human small intestine and lung organoids","volume":"3","author":"I Heo","year":"2018","journal-title":"Nat Microbiol"},{"key":"ppat.1011992.ref046","doi-asserted-by":"crossref","first-page":"124","DOI":"10.1016\/j.mib.2020.10.002","article-title":"Intestinal organoid\/enteroid-based models for Cryptosporidium","volume":"58","author":"S Bhalchandra","year":"2020","journal-title":"Curr Opin Microbiol"},{"issue":"7","key":"ppat.1011992.ref047","first-page":"2288","article-title":"P-Glycoprotein-mediated efflux reduces the in vivo efficacy of a therapeutic targeting the gastrointestinal parasite Cryptosporidium","volume":"220","author":"SLM Arnold","year":"2019","journal-title":"J Infect Dis"},{"key":"ppat.1011992.ref048","doi-asserted-by":"crossref","first-page":"335","DOI":"10.1007\/978-1-4939-9748-0_19","article-title":"In vitro Culture of Cryptosporidium parvum Using Hollow Fiber Bioreactor: Applications for Simultaneous Pharmacokinetic and Pharmacodynamic Evaluation of Test Compounds.","volume":"2052","author":"N Yarlett","year":"2020","journal-title":"Methods Mol Biol"},{"issue":"Suppl 1","key":"ppat.1011992.ref049","first-page":"S40","article-title":"Molecular Characterization of the Cryptosporidium parvum IOWA Isolate Kept in Different Laboratories","volume":"53","author":"VA Cama","year":"2006","journal-title":"J Eukaryot Microbiol"}],"updated-by":[{"DOI":"10.1371\/journal.ppat.1011992","type":"new_version","label":"New version","source":"publisher","updated":{"date-parts":[[2024,3,11]],"date-time":"2024-03-11T00:00:00Z","timestamp":1710115200000}}],"container-title":["PLOS Pathogens"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.ppat.1011992","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,3,11]],"date-time":"2024-03-11T17:46:53Z","timestamp":1710179213000},"score":1,"resource":{"primary":{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.ppat.1011992"}},"subtitle":[],"editor":[{"given":"Ana Maria","family":"Cevallos","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"editor"}]}],"short-title":[],"issued":{"date-parts":[[2024,2,28]]},"references-count":49,"journal-issue":{"issue":"2","published-online":{"date-parts":[[2024,2,28]]}},"URL":"https:\/\/doi.org\/10.1371\/journal.ppat.1011992","relation":{"new_version":[{"id-type":"doi","id":"10.1371\/journal.ppat.1011992","asserted-by":"object"}]},"ISSN":["1553-7374"],"issn-type":[{"value":"1553-7374","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,2,28]]}}}