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Cryptosporidium is a major cause of diarrhoeal disease and mortality in young children in resource-poor countries, for which no vaccines or adequate therapeutic options are available. Infection in humans is primarily caused by two species: C. hominis and C. parvum. Despite C. hominis being the dominant species infecting humans in most countries, very little is known about its growth characteristics and life cycle in vitro, given that the majority of our knowledge of the in vitro development of Cryptosporidium has been based on C. parvum. In the present study, the growth and development of two C. parvum isolates (subtypes Iowa-IIaA17G2R1 and IIaA18G3R1) and one C. hominis isolate (subtype IdA15G1) in HCT-8 cells were examined and compared at 24 h and 48 h using morphological data acquired with scanning electron microscopy. Our data indicated no significant differences in the proportion of meronts or merozoites between species or subtypes at either time-point. Sexual development was observed at the 48-h time-point across both species through observations of both microgamonts and macrogamonts, with a higher frequency of macrogamont observations in C. hominis (IdA15G1) cultures at 48-h post-infection compared to both C. parvum subtypes. This corresponded to differences in the proportion of trophozoites observed at the same time point. No differences in proportion of microgamonts were observed between the three subtypes, which were rarely observed across all cultures. In summary, our data indicate that asexual development of C. hominis is similar to that of C. parvum, while sexual development is accelerated in C. hominis. This study provides new insights into differences in the in vitro growth characteristics of C. hominis when compared to C. parvum, which will facilitate our understanding of the sexual development of both species., (© 2023. The Author(s).)

Cryptosporidium is a parasite responsible for widespread disease in livestock and humans. Recent phylogenetic reclassification of Cryptosporidium from a coccidian to a gregarine dictates an urgent need to reconsider the biology and behavior of this parasite. Overwhelming data now confirm that, like its close relatives, Cryptosporidium is a facultatively epicellular apicomplexan that is able to multiply in a host cell-free environment. We complement the latest phylogenetic and taxonomic proposals with advances in our understanding of Cryptosporidium's biology, with particular focus on in vitro studies that have characterized the development of Cryptosporidium stages in the absence of host cells. Opportunities to revisit in vivo infections are discussed and questions about the Cryptosporidium host cell-free life cycle that remain unanswered highlighted., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background: Aquatic biofilms often serve as environmental reservoirs for microorganisms and provide them with a nutrient-rich growth environment under harsh conditions. With regard to Cryptosporidium, biofilms can serve as environmental reservoirs for oocysts, but may also support the growth of additional Cryptosporidium stages., Results: Here we used confocal laser scanning microscopy, scanning electron microscopy (SEM), and flow cytometry to identify and describe various Cryptosporidium developmental stages present within aquatic biofilm systems, and to directly compare these to stages produced in cell culture. We also show that Cryptosporidium has the ability to form a parasitophorous vacuole independently, in a host-free biofilm environment, potentially allowing them to complete an extracellular life cycle. Correlative data from confocal and SEM imaging of the same cells confirmed that the observed developmental stages (including trophozoites, meronts, and merozoites) were Cryptosporidium. These microscopy observations were further supported by flow cytometric analyses, where excysted oocyst populations were detected in 1, 3 and 6 day-old Cryptosporidium-exposed biofilms, but not in biofilm-free controls., Conclusions: These observations not only highlight the risk that aquatic biofilms pose in regards to Cryptosporidium outbreaks from water distribution systems, but further indicate that even simple biofilms are able to stimulate oocyst excystation and support the extracellular multiplication and development of Cryptosporidium within aquatic environments.

Host-cell invasion by Cryptosporidium is a complex process that requires many different factors derived from both the parasite and the host cell. However, the exact natures of the processes have yet to be resolved. Here, research on different components of the invasion process is put in context, and the sequence of events and pathways associated with the establishment of Cryptosporidium in its unique niche is clarified. In addition, initial parasite-host contact, host-cell invasion and host-cell responses are described. The roles of parasite and host-cell-derived components in the invasion process are examined, as is the question of whether Cryptosporidium actively invades cells and to what extent host-cell responses are involved.

Background: Aquatic biofilms often serve as environmental reservoirs for microorganisms and provide them with a nutrient-rich growth environment under harsh conditions. With regard to Cryptosporidium, biofilms can serve as environmental reservoirs for oocysts, but may also support the growth of additional Cryptosporidium stages. Results: Here we used confocal laser scanning microscopy, scanning electron microscopy (SEM), and flow cytometry to identify and describe various Cryptosporidium developmental stages present within aquatic biofilm systems, and to directly compare these to stages produced in cell culture. We also show that Cryptosporidium has the ability to form a parasitophorous vacuole independently, in a host-free biofilm environment, potentially allowing them to complete an extracellular life cycle. Correlative data from confocal and SEM imaging of the same cells confirmed that the observed developmental stages (including trophozoites, meronts, and merozoites) were Cryptosporidium. These microscopy observations were further supported by flow cytometric analyses, where excysted oocyst populations were detected in 1, 3 and 6 day-old Cryptosporidium-exposed biofilms, but not in biofilm-free controls. Conclusions: These observations not only highlight the risk that aquatic biofilms pose in regards to Cryptosporidium outbreaks from water distribution systems, but further indicate that even simple biofilms are able to stimulate oocyst excystation and support the extracellular multiplication and development of Cryptosporidium within aquatic environments. [ABSTRACT FROM AUTHOR]

Background: In natural aquatic environments biofilms are known to act as environmental reservoirs for Cryptosporidium parvum oocysts. However, the fate of these oocysts within biofilms has yet to be determined. Methods: This study aimed to identify if biofilms have the ability to support the multiplication of Cryptosporidium by measuring the change in parasite number over time using quantitative polymerase chain reaction (qPCR) and detecting the possible extracellular developmental stages using a combination of confocal microscopy and immunolabelling techniques. Pseudomonas aeruginosa biofilm flow cell systems were established and C. parvum oocysts were constantly supplied over a six day period. Results: A significant (P < 0.001) increase in Cryptosporidium was detected as the biofilm matured, with the total number of C. parvum multiplying 2-3 fold during this period. With this, various Cryptosporidium developmental stages (sporozoites, trophozoites, type I and II meronts) were identified from the biofilm. Conclusion: This is the first study demonstrating that biofilms not only serve as an environmental reservoir for oocysts, but are also capable of supporting the multiplication of Cryptosporidium over time in an aquatic environment [ABSTRACT FROM AUTHOR]

Abstract: The Apicomplexan parasite Cryptosporidium parvum is responsible for the widespread disease cryptosporidiosis, in both humans and livestock. The nature of C. parvum infection is far from understood and many questions remain in regard to host–parasite interactions, limiting successful treatment of the disease. To definitively identify a range of C. parvum stages in cell culture and to begin to investigate host cell interactions in some of the lesser known life stages, we have utilized a combined scanning electron microscopy and immunolabeling approach, correlating high resolution microstructural information with definitive immunogold labeling of Cryptosporidium stages. Several life cycle stages, including oocysts, merozoites I, trophozoites, gamonts and microgametocytes, were successfully immunolabeled in an in vitro model system. Developing oocysts were clearly immunolabeled, but this did not persist once excystation had occurred. Immunolabeling visualized on the host cell surface adjacent to invasive merozoites is likely to be indicative of receptor shedding, with merozoites also initiating host responses that manifested as abnormal microvilli on the host cell surface. Small sub-micron stages such as microgametocytes, which were impossible to identify as single entities without immunolabeling, were readily visualized and observed to attach to host cells via novel membranous projections. Epicellular parasites also expressed Cryptosporidium-derived epitopes within their encapsulating membrane. These data have allowed us to confidently identify a variety of C. parvum stages in cell culture at high resolution. With this, we provide new insight into C. parvum - host cell interactions and highlight future opportunities for investigating and targeting receptor-mediated interactions between Cryptosporidium life cycle stages and host cells. [Copyright &y& Elsevier]