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Morphological changes and viability of Cryptosporidium parvum sporozoites after excystation in cell-free culture media

Published online by Cambridge University Press:  27 August 2010

M. MATSUBAYASHI ,
H. ANDO ,
I. KIMATA ,
H. NAKAGAWA ,
M. FURUYA ,
H. TANI  and
K. SASAI
M. MATSUBAYASHI
Affiliation:
Department of Food and Nutrition, Osaka Yuhigaoka Gakuen Junior College, Osaka, Osaka543-0073, Japan
H. ANDO
Affiliation:
Department of Veterinary Internal Medicine, Division of Veterinary Science, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Izumisano, Osaka598-8531, Japan
I. KIMATA
Affiliation:
Department of Parasitology, Graduate School of Medicine, Osaka City University, Osaka, Osaka545-8585, Japan
H. NAKAGAWA
Affiliation:
Department of Central Laboratory, Graduate School of Medicine, Osaka City University, Osaka, Osaka545-8585, Japan
M. FURUYA
Affiliation:
Department of Veterinary Internal Medicine, Division of Veterinary Science, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Izumisano, Osaka598-8531, Japan
H. TANI
Affiliation:
Department of Veterinary Internal Medicine, Division of Veterinary Science, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Izumisano, Osaka598-8531, Japan
K. SASAI*
Affiliation:
Department of Veterinary Internal Medicine, Division of Veterinary Science, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Izumisano, Osaka598-8531, Japan
*
*Corresponding author: Department of Veterinary Internal Medicine, Division of Veterinary Science, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Izumisano, Osaka 598-8531, Japan. Tel: +81 72 463 5385. Fax: +81 72 463 5387. E-mail: ksasai@vet.osakafu-u.ac.jp.
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Summary

Cryptosporidium parvum, belonging to the phylum Apicomplexa, is a major cause of waterborne gastroenteritis throughout the world. The sporozoites are thought to invade host enterocytes using an active process termed gliding motility. However, the biological and morphological changes within the sporozoites during this process are not fully understood. In the present study, excysted sporozoites of C. parvum were analysed ultrastructurally in vitro and their viability was evaluated using fluorescent dyes. The sporozoites excysted from oocysts changed morphologically from banana-shaped to rod-shaped and finally to a rounded shape, in culture media in 3 h. Transmission microscopy revealed that the distance between the apical end and the nucleus was markedly reduced, dense granules were present close to the rhoptry in the apical region, amylopectin granules were absent, and membranes of round sporozoites were less clear. A fluorescent assay showed that the rate of survival decreased from 89% to 56% at 0–3 h (84·3% for banana-shaped and 49·2% for rod-shaped sporozoites). Therefore, post-excysted sporozoites in vitro underwent morphological changes and a rapid loss of viability. This staining method is useful, inexpensive and provides an alternative to more costly and intensive flow cytometric assays or infectivity assays with host cells in vitro.

Keywords

Cryptosporidium parvumexcystationmorphological changessporozoitesviability
Type
Research Article
Information
Parasitology , Volume 137 , Issue 13 , November 2010 , pp. 1861 - 1866
Copyright
Copyright © Cambridge University Press 2010

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References

REFERENCES

Abe, N., Kimata, I. and Iseki, M. (2002). Identification of genotypes of Cryptosporidium parvum isolates from a patient and a dog in Japan. Journal of Veterinary Medical Science 64, 165–168.CrossRefGoogle Scholar
Brown, S. M., McDonald, V., Denton, H. and Coombs, G. H. (1996). The use of a new viability assay to determine the susceptibility of Cryptosporidium and Eimeria sporozoites to respiratory inhibitors and extremes of pH. FEMS Microbiology Letters 142, 203–208.CrossRefGoogle ScholarPubMed
Campbell, A. T., Robertson, L. J. and Smith, H. V. (1992). Viability of Cryptosporidium parvum oocysts: correlation of in vitro excystation with inclusion or exclusion of fluorogenic vital dyes. Applied and Environmental Microbiology 58, 3488–3493.CrossRefGoogle ScholarPubMed
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
Clark, D. P. (1999). New insights into human cryptosporidiosis. Clinical Microbiology Reviews 12, 554–563.CrossRefGoogle ScholarPubMed
Fayer, R., Speer, C. A. and Dubey, J. P. (1997). General biology of Cryptosporidium. In Cryptosporidium and Cryptosporidiosis (ed. Fayer, R.), pp. 1–41. CRC Press, Boca Raton, Florida, USA.Google Scholar
Fricker, C. R. and Crabb, J. H. (1998). Water-borne cryptosporidiosis: detection methods and treatment options. Advances in Parasitology 40, 241–278.CrossRefGoogle ScholarPubMed
Girouard, D., Gallant, J., Akiyoshi, D. E., Nunnari, J. and Tzipori, S. (2006). Failure to propagate Cryptosporidium spp. in cell-free culture. Journal of Parasitology 92, 399–400.CrossRefGoogle ScholarPubMed
Harris, J. R., Adrian, M. and Petry, F. (2003). Structure of the Cryptosporidium parvum microneme: a metabolically and osmotically labile apicomplexan organelle. Micron 34, 65–78.CrossRefGoogle ScholarPubMed
Henriquez, F. L., Richards, T. A., Roberts, F., McLeod, R. and Roberts, C. W. (2005). The unusual mitochondrial compartment of Cryptosporidium parvum. Trends in Parasitology 21, 68–74.CrossRefGoogle ScholarPubMed
Hijjawi, N. S., Meloni, B. P., Ng'anzo, M., Ryan, U. M., Olson, M. E., Cox, P. T., Monis, P. T. and Thompson, R. C. (2004). Complete development of Cryptosporidium parvum in host cell-free culture. International Journal for Parasitology 34, 769–777.CrossRefGoogle ScholarPubMed
Juranek, D. D. (1997). Cryptosporidium and water: a public health handbook. Clinical Laboratory Science 10, 272.Google Scholar
Karanis, P., Kimura, A., Nagasawa, H., Igarashi, I. and Suzuki, N. (2008). Observations on Cryptosporidium life cycle stages during excystation. Journal of Parasitology 94, 298–300.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
McDonald, V., McCrossan, M. V. and Petry, F. (1995). Localization of parasite antigens in Cryptosporidium parvum-infected epithelial cells using monoclonal antibodies. Parasitology 110, 259–268.CrossRefGoogle ScholarPubMed
Matsubayashi, M., Kimata, I., Iseki, M., Lillehoj, H. S., Matsuda, H., Nakanishi, T., Tani, H., Sasai, K. and Baba, E. (2005). Cross-reactivities with Cryptosporidium spp. by chicken monoclonal antibodies that recognize avian Eimeria spp. Veterinary Parasitology 128, 47–57.CrossRefGoogle ScholarPubMed
Matsubayashi, M., Takase, H., Kimata, I., Nakagawa, H., Tani, H., Sasai, K. and Baba, E. (2008). Electron microscopic observation of cytoskeletal frame structures and detection of tubulin on the apical region of Cryptosporidium parvum sporozoites. Parasitology 135, 295–301.CrossRefGoogle ScholarPubMed
Mercier, C., Adjogble, K. D., Däubener, W. and Delauw, M. F. (2005). Dense granules: are they key organelles to help understand the parasitophorous vacuole of all apicomplexa parasites? International Journal for Parasitology 35, 829–849.CrossRefGoogle ScholarPubMed
O'Donoghue, P. J. (1995). Cryptosporidium and cryptosporidiosis in man and animals. International Journal for Parasitology 25, 139–195.CrossRefGoogle Scholar
Petry, F., Kneib, I. and Harris, J. R. (2009). Morphology and in vitro infectivity of sporozoites of Cryptosporidium parvum. Journal of Parasitology 95, 1243–1246.CrossRefGoogle ScholarPubMed
Reduker, D. W., Speer, C. A. and Blixt, J. A. (1985). Ultrastructure of Cryptosporidium parvum oocysts and excysting sporozoites as revealed by high resolution scanning electron microscopy. Journal of Protozoology 32, 708–711.CrossRefGoogle ScholarPubMed
Schmidt, J. and Kuhlenschmidt, M. S. (2008). Microbial adhesion of Cryptosporidium parvum: identification of a colostrum-derived inhibitory lipid. Molecular and Biochemical Parasitology 162, 32–39.CrossRefGoogle ScholarPubMed
Slifko, T. R., Friedman, 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., Nichols, R. A. and Grimason, A. M. (2005). Cryptosporidium excystation and invasion: getting to the guts of the matter. Trends in Parasitology 21, 133–142.CrossRefGoogle Scholar
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
Woodmansee, D. B., Powell, E. C., Pohlenz, J. F. and Moon, H. W. (1987). Factors affecting motility and morphology of Cryptosporidium sporozoites in vitro. Journal of Protozoology 34, 295–297.CrossRefGoogle ScholarPubMed