Extracellular excystation and development of Cryptosporidium: tracing the fate of oocysts within Pseudomonas aquatic biofilm systems.

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.

Labeling surface epitopes to identify Cryptosporidium life stages using a scanning electron microscopy-based immunogold approach.

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.