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Studies on the in vitro cultivation and development of cryptosporidium species

Hijjawi, Nawal S. (2003) Studies on the in vitro cultivation and development of cryptosporidium species. PhD thesis, Murdoch University.

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In this study, Cryptosporidium was cultured successfully for the first time with the completion and long-term maintenance of the whole life cycle. The success of this study was due to modifications to the pH throughout the period of in vitro culturing and the inhibition of host cell overgrowth by subculturing and gamma irradiation. These appear to be critical factors that may have contributed to the failure in Cryptosporidium culturing and long-term maintenance in previous studies. The addition of growth supplements N-2 and B-27 also appeared to enhance the growth of Cryptosporidium and attempts to cryopreserve in vitro-derived oocysts resulted in the maintenance of viable sporozoites within these oocysts.

The life cycle for three species of Cryprtosporidium (C. hominis, C. parvum and C. andersoni) was completed in vitro during this study from sporozoites to sporulated oocysts. Cryptosporidium hominis and C. parvum genotypes (cattle and pig) were successfully proliferated and maintained in HCT-8 cells. This is the first report of the successful culturing and maintenance of C. andersoni and C. hominis in cell culture. The culture derived oocysts of C. parvum (cattle genotype) were infective to 3-4 day old ARC/Swiss mice, however, culture derived oocysts of C. hominis failed to infect mice under the same experimental conditions supporting other in vivo results of the failure of this species to infect mice. Furthermore, oocysts of C. andersoni, C. hominis and C. parvum (pig genotype) isolated from the faeces of infected hosts failed to infect mice, a result consistent with previous results confirming the host specificity of the Cryptosporidium species and genotypes.

The completion of the life cycle for C. hominis and C. parvwn (cattle genotype) was further confirmed using TEM microscopy. All the developmental stages (sexual and asexual) were detected in cross sections prepared from HCT-8 monolayers infected with C. hominis and C. parvum (cattle genotype). The presence of sporozoites in the sections prepared after 96 hours post-infection again confirmed their release from oocysts formed in culture. Comparing the endogenous development of different Cryptosporidium species and genotypes, it was found that they all followed the same pattern of development with some minor differences. Cryptosporidium andersoni endogenous stages were larger than those of C. hominis and C. parvum (cattle genotype) and easier to monitor at lower magnifications. Cryptosporidium hominis appeared to be more adapted to grow in HCT-8 cells and completed the life cycle earlier than the C. parvum (cattle genotype). This observation was made with both light and electron microscopy.

During the present study, the modification of the culturing system facilitated the study of the complete life cycle of Cryptosporidium and allowed the description of new extracellular stages in its life cycle for the first time. The extracellular stages were first observed in culture of C. andersoni and subsequently in the faeces of cattle chronically infected with C. andersoni. The presence of this extracellular stage in the life cycle of C. andersoni was further confirmed following its isolation from the faeces of infected cattle using a laser microdissection technique. This stage was present in large numbers and some of them were seen undergoing syzgy. Extraction of DNA from the extracellular stage, followed by PCR-RFLP and sequencing of the 18S rDNA confirmed that this is a stage in the life cycle of C. andersoni.

Comparative observations with C. parvum (cattle genotype) also confirmed the presence of extracellular stages. Extracellular stages were recovered from in vitro culture after 5 days post-infection with the cattle genotype of C. parvum and from infected mice. At least two morphologically different stages (stages 1 & 2) were purified from mice after 72 hours of infection. The presence and morphological characterisation of extracellular developmental stages in the life cycle of Cryptosporidium confirms its relationship to gregarines and provides important implications for our understanding of the taxonomic and phylogenetic affinities of the genus Cryptosporidium. The growth of Cryptosporidium in cell culture now provides a means of studying its development, metabolism, and behaviour as well as testing its response to different therapeutic agents.

Item Type: Thesis (PhD)
Murdoch Affiliation: Division of Veterinary and Biomedical Sciences
Notes: Note to the author: If you would like to make your thesis openly available on Murdoch University Library's Research Repository, please contact: Thank you.
Supervisor(s): Thompson, Andrew, Ryan, Una and Meloni, Bruno
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