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Cryptosporidium cell culture infectivity assay design

Published online by Cambridge University Press:  18 March 2011

B. J. KING ,
A. R. KEEGAN ,
B. S. ROBINSON  and
P. T. MONIS
B. J. KING*
Affiliation:
Australian Water Quality Centre, SA Water Corporation, Adelaide, South Australia, Australia, 5000
A. R. KEEGAN
Affiliation:
Australian Water Quality Centre, SA Water Corporation, Adelaide, South Australia, Australia, 5000
B. S. ROBINSON
Affiliation:
Australian Water Quality Centre, SA Water Corporation, Adelaide, South Australia, Australia, 5000
P. T. MONIS
Affiliation:
Australian Water Quality Centre, SA Water Corporation, Adelaide, South Australia, Australia, 5000
*
*Corresponding author: Tel: +61 8 742 42114. E-mail: brendon.king@sawater.com.au
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Summary

Members of the genus Cryptosporidium, which cause the gastrointestinal disease cryptosporidiosis, still represent a significant cause of water-borne disease worldwide. While intensive efforts have been invested in the development of techniques for parasite culture, in vitro growth has been hampered by a number of factors including low levels of infectivity as well as delayed life-cycle development and poor synchronicity. In this study we examined factors affecting the timing of contact between excysted sporozoites and target host cells and the subsequent impact of this upon the establishment of infection. We demonstrate that excystation rate impacts upon establishment of infection and that in our standard assay format the majority of sporozoites are not close enough to the cell monolayer when they are released from the oocyst to successfully establish infection. However, this can be easily overcome by centrifugation of oocysts onto the cell monolayer, resulting in approximately 4-fold increases in sporozoite attachment and subsequent infection. We further demonstrate that excystation procedures can be tailored to control excystation rate to match the assay end purpose and that excystation rate can influence data interpretation. Finally, the addition of both a centrifugation and washing step post-sporozoite attachment may be appropriate when considering the design of in vitro culture experiments for developmental analysis and stage-specific gene expression as this appears to increase the synchronicity of early developmental stages.

Keywords

Cryptosporidiumexcystationcentrifugationmonolayer washingapical organelle discharge
Type
Research Article
Information
Parasitology , Volume 138 , Issue 6 , May 2011 , pp. 671 - 681
Copyright
Copyright © Cambridge University Press 2011

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References

REFERENCES

Arrowood, M. J. (2002). In vitro cultivation of Cryptosporidium species. Clinical Microbiology Reviews 15, 390–400.CrossRefGoogle ScholarPubMed
Beaudeau, P., De Valk, H., Vaillant, V., Mannschott, C., Tillier, C., Mouly, D. and Ledrans, M. (2008). Lessons learned from ten investigations of waterborne gastroenteritis outbreaks, France, 1998–2006. Journal of Water and Health 6, 491–503.CrossRefGoogle ScholarPubMed
Borowski, H., Clode, P. L. and Thompson, R. C. (2008). Active invasion and/or encapsulation? A reappraisal of host-cell parasitism by Cryptosporidium. Trends in Parasitology 24, 509–516.CrossRefGoogle ScholarPubMed
Borowski, H., Thompson, R. C. A., Armstrong, T. and Clode, P. L. (2010). Morphological characterization of Cryptosporidium parvum life-cycle stages in an in vitro model system. Parasitology 137, 13–26.CrossRefGoogle Scholar
Chen, X. M., O'Hara, S. P., Huang, B. Q., Nelson, J. B., Lin, J. J., Zhu, G., Ward, H. D. and Larusso, N. F. (2004). Apical organelle discharge by Cryptosporidium parvum is temperature, cytoskeleton, and intracellular calcium dependent and required for host cell invasion. Infection and Immunity 72, 6806–6816.CrossRefGoogle ScholarPubMed
Connelly, S. J., Wolyniak, E. A., Williamson, C. E. and Jellison, K. L. (2007). Artificial UV-B and solar radiation reduce in vitro infectivity of the human pathogen Cryptosporidium parvum. Environmental Science & Technology 41, 7101–7106.CrossRefGoogle ScholarPubMed
Feng, H. P., Nie, W. J., Sheoran, A., Zhang, Q. S. and Tzipori, S. (2006). Bile acids enhance invasiveness of Cryptosporidium spp. into cultured cells. Infection and Immunity 74, 3342–3346.Google ScholarPubMed
Hijjawi, N. S., Meloni, B. P., Morgan, U. M. and Thompson, R. C. A. (2001). Complete development and long-term maintenance of Cryptosporidium parvum human and cattle genotypes in cell culture. International Journal for Parasitology 31, 1048–1055.CrossRefGoogle ScholarPubMed
Huang, B. Q., Chen, X. M. and Larusso, N. F. (2004). Cryptosporidium parvum attachment to and internalization by human biliary epithelia in vitro: A morphologic study. Journal of Parasitology 90, 212–221.CrossRefGoogle ScholarPubMed
Ives, R. L., Kamarainen, A. M., John, D. E. and Rose, J. B. (2007). Use of cell culture to assess Cryptosporidium parvum survival rates in natural groundwaters and surface waters. Applied and Environmental Microbiology 73, 5968–5970.CrossRefGoogle ScholarPubMed
Keegan, A. R., Fanok, S., Monis, P. T. and Saint, C. P. (2003). Cell culture-Taqman PCR assay for evaluation of Cryptosporidium parvum disinfection. Applied and Environmental Microbiology 69, 2505–2511.CrossRefGoogle ScholarPubMed
Kim, S. B. and Corapcioglu, M. Y. (2002). Vertical transport of Cryptosporidium parvum oocysts through sediments. Environmental Technology 23, 1435–1446.CrossRefGoogle ScholarPubMed
King, B. J., Hoefel, D., Lim, S. P., Robinson, B. S. and Monis, P. T. (2009). Flow cytometric assessment of distinct physiological stages within Cryptosporidium parvum sporozoites post-excystation. Parasitology 136, 953–966.CrossRefGoogle ScholarPubMed
King, B. J., Keegan, A. R., Monis, P. T. and Saint, C. P. (2005). Environmental temperature controls Cryptosporidium oocyst metabolic rate and associated retention of infectivity. Applied and Environmental Microbiology 71, 3848–3857.CrossRefGoogle ScholarPubMed
Lalancette, C., Di Giovanni, G. D. and Prevost, M. (2010). Improved risk analysis by dual direct detection of total and infectious Cryptosporidium oocysts on cell culture in combination with immunofluorescence assay. Applied and Environmental Microbiology 76, 566–577.CrossRefGoogle ScholarPubMed
LeChevallier, M. W., Di Giovanni, G. D., Clancy, J. L., Bukhari, Z., Bukhari, S., Rosen, J. S., Sobrinho, J. and Frey, M. M. (2003). Comparison of method 1623 and cell culture-PCR for detection of Cryptosporidium spp. in source waters. Applied and Environmental Microbiology 69, 971–979.CrossRefGoogle ScholarPubMed
Li, L. J. and Haas, C. N. (2004). Inactivation of Cryptosporidium parvum with ozone in treated drinking water. Journal of Water Supply Research and Technology-Aqua 53, 287–297.CrossRefGoogle Scholar
Mason, B. W., Chalmers, R. M., Carnicer-Pont, D. and Casemore, D. P. (2010). A Cryptosporidium hominis outbreak in North-West Wales associated with low oocyst counts in treated drinking water. Journal of Water and Health 8, 299–310.CrossRefGoogle ScholarPubMed
Matsubayashi, M., Ando, H., Kimata, I., Nakagawa, H., Furuya, M., Tani, H. and Sasai, K. (2010). Morphological changes and viability of Cryptosporidium parvum sporozoites after excystation in cell-free culture media. Parasitology 137, 1861–1866.Google ScholarPubMed
Quintero-Betancourt, W., Gennaccaro, A. L., Scott, T. M. and Rose, J. B. (2003). Assessment of methods for detection of infectious Cryptosporidium oocysts and Giardia cysts in reclaimed effluents. Applied and Environmental Microbiology 69, 5380–5388.CrossRefGoogle ScholarPubMed
Rochelle, P. A., Ferguson, D. M., Johnson, A. M. and De Leon, R. (2001). Quantitation of Cryptosporidium parvum infection in cell culture using a colorimetric in situ hybridization assay. Journal of Eukaryotic Microbiology 48, 565–574.CrossRefGoogle ScholarPubMed
Schets, F. M., Engels, G. B., During, A. and Husman, A. A. D. (2005). Detection of infectious Cryptosporidium oocysts by cell culture immunofluorescence assay: Applicability to environmental samples. Applied and Environmental Microbiology 71, 6793–6798.CrossRefGoogle ScholarPubMed
Searcy, K. E., Packman, A. I., Atwill, E. R. and Harter, T. (2005). Association of Cryptosporidium parvum with suspended particles: Impact on oocyst sedimentation. Applied and Environmental Microbiology 71, 1072–1078.CrossRefGoogle ScholarPubMed
Slifko, T. R., Freidman, D., Rose, J. B. and Jakubowski, W. (1997). An in vitro method for detecting infectious Cryptosporidium oocysts with cell culture. Applied and Environmental Microbiology 63, 3669–3675.CrossRefGoogle Scholar
Smith, H. V., Robertson, L. J. and Ongerth, J. E. (1995). Cryptosporidiosis and giardiasis: The impact of waterborne transmission. Journal of Water Supply Research and Technology-Aqua 44, 258–274.Google Scholar
Upton, S. J., Tilley, M., Nesterenko, M. V. and Brillhart, D. B. (1994). A simple and reliable method of producing in-vitro infections of Cryptosporidium-parvum (Apicomplexa). FEMS Microbiology Letters 118, 45–49.CrossRefGoogle ScholarPubMed
Vesey, G., Griffiths, K. R., Gauci, M. R., Deere, D., Williams, K. L. and Veal, D. A. (1997). Simple and rapid measurement of Cryptosporidium excystation using flow cytometry. International Journal for Parasitology 27, 1353–1359.CrossRefGoogle ScholarPubMed
Weir, S. C., Pokorny, N. J., Carreno, R. A., Trevors, J. T. and Lee, H. (2001). Improving the rate of infectivity of Cryptosporidium parvum oocysts in cell culture using centrifugation. Journal of Parasitology 87, 1502–1504.CrossRefGoogle ScholarPubMed
Wetzel, D. M., Schmidt, J., Kuhlenschmidt, M. S., Dubey, J. P. and Sibley, L. D. (2005). Gliding motility leads to active cellular invasion by Cryptosporidium parvum sporozoites. Infection and Immunity 73, 5379–5387.CrossRefGoogle ScholarPubMed
Wheeler, C., Vugia, D. J., Thomas, G., Beach, M. J., Carnes, S., Maier, T., Gorman, J., Xiao, L., Arrowood, M. J., Gilliss, D. and Werner, S. B. (2007). Outbreak of cryptosporidiosis at a California waterpark: employee and patron roles and the long road towards prevention. Epidemiology and Infection 135, 302–310.CrossRefGoogle Scholar
Widmer, G., Klein, P. and Bonilla, R. (2007). Adaptation of Cryptosporidium oocysts to different excystation conditions. Parasitology 134, 1583–1588.CrossRefGoogle ScholarPubMed