Continuous culture of Cryptosporidium parvum using hollow fiber technology

Morada, Mary; Lee, Sangun; Gunther-Cummins, Leslie; Weiss, Louis M.; Widmer, Giovanni; Tzipori, Saul; Yarlett, Nigel

doi:10.1016/j.ijpara.2015.07.006

Cited by 109 publications

(111 citation statements)

References 34 publications

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“…More recently, Morada et al developed a culture system employing a hollow fiber technology with HCT-8 cells surrounding hollow fibers (36). This system allowed the in vitro production of C. parvum oocysts for 6 months but required specialized equipment to actively pump in nutrients and remove waste products.…”

Section: Discussionmentioning

confidence: 99%

Novel Bioengineered Three-Dimensional Human Intestinal Model for Long-Term Infection of Cryptosporidium parvum

et al. 2017

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Cryptosporidium spp. are apicomplexan parasites of global importance that cause human diarrheal disease. In vitro culture models that may be used to study this parasite and that have physiological relevance to in vivo infection remain suboptimal. Thus, the pathogenesis of cryptosporidiosis remains poorly characterized, and interventions for the disease are limited. In this study, we evaluated the potential of a novel bioengineered three-dimensional (3D) human intestinal tissue model (which we developed previously) to support long-term infection by Cryptosporidium parvum. Infection was assessed by immunofluorescence assays and confocal and scanning electron microscopy and quantified by quantitative reverse transcription-PCR. We found that C. parvum infected and developed in this tissue model for at least 17 days, the extent of the study time used in the present study. Contents from infected scaffolds could be transferred to fresh scaffolds to establish new infections for at least three rounds. Asexual and sexual stages and the formation of new oocysts were observed during the course of infection. Additionally, we observed ablation, blunting, or distortion of microvilli in infected epithelial cells. Ultimately, a 3D model system capable of supporting continuous Cryptosporidium infection will be a useful tool for the study of hostparasite interactions, identification of putative drug targets, screening of potential interventions, and propagation of genetically modified parasites. KEYWORDS 3D model, Cryptosporidium, in vitro culture, intestinal epithelial cells C ryptosporidium spp. are apicomplexan parasites of global importance that cause diarrheal disease in humans and animals worldwide (1, 2). Though immunocompetent individuals experience self-limiting or asymptomatic infection, immunocompromised hosts, such as untreated patients with HIV infection or AIDS (3) and malnourished children (1, 2) in resource-limited settings, may experience severe diarrhea, wasting, and death. In a recent case-control study, Cryptosporidium was one of four pathogens responsible for moderate to severe diarrhea in children under the age of 5 years and was associated with an increased risk of death in children from 1 to 2 years of age in seven countries in sub-Saharan Africa and South Asia (4). The only FDA-approved drug for the treatment of cryptosporidiosis is nitazoxanide, a drug with broad antiparasitic properties (5). However, this drug is not effective in immunocompromised patients (6) and has not been widely tested in malnourished children in resource-limited countries. Though significant improvements in water purification methods have occurred (7) since the outbreak of waterborne cryptosporidiosis in Milwaukee, WI, in 1993 (8), industrialized countries are seeing increased numbers of cases, largely due to recreational water outbreaks (9).Unlike many other apicomplexan parasites which often require more than one host to complete their life cycles (10), Cryptosporidium completes its entire life cycle in a

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Section: Discussionmentioning

confidence: 99%

Novel Bioengineered Three-Dimensional Human Intestinal Model for Long-Term Infection of Cryptosporidium parvum

et al. 2017

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“…It would be of great interest to determine whether such amino acid substitutions have an effect on the biology of the Cryptosporidium parasite. Recent breakthroughs in the ability to genetically manipulate (31) or continuously culture (32) Cryptosporidium should provide a platform for more direct functional studies on both reference and clinical Cryptosporidium species samples.…”

Section: Discussionmentioning

confidence: 99%

Genomic Variation in IbA10G2 and Other Patient-Derived Cryptosporidium hominis Subtypes

et al. 2017

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In order to improve genotyping and epidemiological analysis of Cryptosporidium spp., genomic data need to be generated directly from a broad range of clinical specimens. Utilizing a robust method that we developed for the purification and generation of amplified target DNA, we present its application for the successful isolation and whole-genome sequencing of 14 different Cryptosporidium hominis patient specimens. Six isolates of subtype IbA10G2 were analyzed together with a single representative each of 8 other subtypes: IaA20R3, IaA23R3, IbA9G3, IbA13G3, IdA14, IeA11G3T3, IfA12G1, and IkA18G1. Parasite burden was measured over a range of more than 2 orders of magnitude for all samples, while the genomes were sequenced to mean depths of between 17ϫ and 490ϫ coverage. Sequence homologybased functional annotation identified several genes of interest, including the gene encoding Cryptosporidium oocyst wall protein 9 (COWP9), which presented a predicted loss-of-function mutation in all the sequence subtypes, except for that seen with IbA10G2, which has a sequence identical to the Cryptosporidium parvum reference Iowa II sequence. Furthermore, phylogenetic analysis showed that all the IbA10G2 genomes form a monophyletic clade in the C. hominis tree as expected and yet display some heterogeneity within the IbA10G2 subtype. The current report validates the aforementioned method for isolating and sequencing Cryptosporidium directly from clinical stool samples. In addition, the analysis demonstrates the potential in mining data generated from sequencing multiple whole genomes of Cryptosporidium from human fecal samples, while alluding to the potential for a higher degree of genotyping within Cryptosporidium epidemiology.

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“…Until recently (6,7), the inability to propagate Cryptosporidium in vitro and the lack of a system for genetic manipulation have hindered the discovery and validation of new molecular targets. Still, many studies, including our own, have demonstrated the importance of mucin-like glycoproteins and lectins in mediating Cryptosporidium infection in vitro and in vivo (8,9).…”

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confidence: 99%

The Cryptosporidium parvum C-Type Lectin CpClec Mediates Infection of Intestinal Epithelial Cells via Interactions with Sulfated Proteoglycans

Ludington

Ward

2016

Infect Immun

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Continuous culture of Cryptosporidium parvum using hollow fiber technology

Cited by 109 publications

References 34 publications

Novel Bioengineered Three-Dimensional Human Intestinal Model for Long-Term Infection of Cryptosporidium parvum

Novel Bioengineered Three-Dimensional Human Intestinal Model for Long-Term Infection of Cryptosporidium parvum

Genomic Variation in IbA10G2 and Other Patient-Derived Cryptosporidium hominis Subtypes

The Cryptosporidium parvum C-Type Lectin CpClec Mediates Infection of Intestinal Epithelial Cells via Interactions with Sulfated Proteoglycans

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