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Detection and Differentiation of Cryptosporidium Oocysts in Water by PCR-RFLP

  • Protocol
Public Health Microbiology

Part of the book series: Methods in Molecular Biology ((MIMB,volume 268))

Abstract

Consumption of contaminated water has been implicated as a major source of Cryptosporidium infection in various outbreak investigations and case control studies. Surveys conducted in various regions of the United States demonstrated the presence of Cryptosporidium oocysts in 67–100% of wastewaters, 24–100% of surface waters, and 17–26.8% of drinking waters (1–4). The identity and human infective potential of these waterborne oocysts are not known, although it is likely that not all oocysts are from human-infecting Cryptosporidium species. Likewise, the source of the oocyst contamination is also not fully clear. Farm animals and human sewage discharge are generally considered to be the major sources of surface water contamination with C. parvum (5). Because Cryptosporidium infection is common in wildlife, it is conceivable that wildlife can also be a source for Cryptosporidium oocysts in waters (4). The presence of host-adapted Cryptosporidium spp. and genotypes makes it possible to develop molecular tools to assess the human infection potential and source of Cryptosporidium oocysts in water.

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References

  1. LeChevallier, M. W., Norton, W. D., and Lee, R. G. (1991) Giardia and Cryptosporidium spp. in filtered drinking water supplies. Appl. Environ. Microbiol. 57, 2617–2621.

    PubMed  CAS  Google Scholar 

  2. LeChevallier, M. W., Norton, W. D., and Lee, R. G. (1991) Occurrence of Giardia and Cryptosporidium spp. in surface water supplies. Appl. Environ. Microbiol. 57, 2610–2616.

    PubMed  CAS  Google Scholar 

  3. Madore, M. S., Rose, J. B., Gerba, C. P., Arrowood, M. J., and Sterling, C. R. (1987) Occurrence of Cryptosporidium oocysts in sewage effluents and selected surface waters. J. Parasitol. 73, 702–705.

    Article  PubMed  CAS  Google Scholar 

  4. Rose, J. B. (1997) Environmental ecology of Cryptosporidium and public health implications. Annu. Rev. Public Health 18, 135–161.

    Article  PubMed  CAS  Google Scholar 

  5. Meinhardt, P. L., Casemore, D. P., and Miller, K. B. (1996) Epidemiologic aspects of human cryptosporidiosis and the role of waterborne transmission. Epidemiol. Rev. 18, 118–136.

    PubMed  CAS  Google Scholar 

  6. Xiao, L., Bern, C., Limor, J., et al. (2001) Identification of 5 types of Cryptosporidium parasites in children in Lima, Peru. J. Infect. Dis. 183, 492–497.

    Article  PubMed  CAS  Google Scholar 

  7. Chrisp, C. E. and LeGendre, M. (1994) Similarities and differences between DNA of Cryptosporidium parvum and C. wrairi detected by the polymerase chain reaction. Folia Parasitol. 41, 97–100.

    PubMed  CAS  Google Scholar 

  8. Johnson, D. W., Pieniazek, N. J., Griffin, D. W., Misener, L., and Rose, J. B. (1995) Development of a PCR protocol for sensitive detection of Cryptosporidium oocysts in water samples. Appl. Environ. Microbiol. 61, 3849–3855.

    PubMed  CAS  Google Scholar 

  9. Laxer, M. A., Timblin, B. K., and Patel, R. J. (1991) DNA sequences for the specific detection of Cryptosporidium parvum by the polymerase chain reaction. Am. J. Trop. Med. Hyg. 45, 688–694.

    PubMed  CAS  Google Scholar 

  10. Webster, K. A., Pow, J. D., Giles, M., Catchpole, J., and Woodward, M. J. (1993) Detection of Cryptosporidium parvum using a specific polymerase chain reaction. Vet. Parasitol. 50, 35–44.

    Article  PubMed  CAS  Google Scholar 

  11. Awad-el-Kariem, F. M., Warhurst, D. C., and McDonald, V. (1994) Detection and species identification of Cryptosporidium oocysts using a system based on PCR and endonuclease restriction. Parasitology 109, 19–22.

    Article  PubMed  CAS  Google Scholar 

  12. Jellison, K. L., Hemond, H. F., and Schauer, D. B. (2002) Sources and species of Cryptosporidium oocysts in the Wachusett reservoir watershed. Appl. Environ. Microbiol. 68, 569–575.

    Article  PubMed  CAS  Google Scholar 

  13. Kimbell, L. M., Miller, D. L., Chavez, W., and Altman, N. (1999) Molecular analysis of the 18S rRNA gene of Cryptosporidium serpentis in a wild-caught corn snake (Elaphe guttata guttata) and a five-species restriction fragment length polymorphism-based assay that can additionally discern C. parvum from C. wrairi. Appl. Environ. Microbiol. 65, 5345–5349.

    PubMed  CAS  Google Scholar 

  14. Leng, X., Mosier, D. A., and Oberst, R. D. (1996) Differentiation of Cryptosporidium parvum, C. muris, and C. baileyi by PCR-RFLP analysis of the 18S rRNA gene. Vet. Parasitol. 62, 1–7.

    Article  PubMed  CAS  Google Scholar 

  15. Sturbaum, G. D., Reed, C., Hoover, P. J., Jost, B. H., Marshall, M. M., and Sterling, C. R. (2001) Species-specific, nested PCR-restriction fragment length polymorphism detection of single Cryptosporidium parvum oocysts. Appl. Environ. Microbiol. 67, 2665–2668.

    Article  PubMed  CAS  Google Scholar 

  16. Xiao, L. H., Escalante, L., Yang, C. F., et al. (1999) Phylogenetic analysis of Cryptosporidium parasites based on the small-subunit rRNA gene locus. Appl. Environ. Microbiol. 65, 1578–1583.

    PubMed  CAS  Google Scholar 

  17. Xiao, L. H., Morgan, U. M., Limor, J., et al. (1999) Genetic diversity within Cryptosporidium parvum and related Cryptosporidium species. Appl. Environ. Microbiol. 65, 3386–3391.

    PubMed  CAS  Google Scholar 

  18. Sulaiman, I. M., Xiao, L. H., and Lal, A. A. (1999) Evaluation of Cryptosporidium parvum genotyping techniques. Appl. Environ. Microbiol. 65, 4431–4435.

    PubMed  CAS  Google Scholar 

  19. Morgan, U. M., Deplazes, P., Forbes, D. A., Spano, F., Hertzberg, H., Sargent, K. D., Elliot, A., and Thompson, R. C. A. (1999) Sequence and PCR-RFLP analysis of the internal transcribed spacers of the rDNA repeat unit in isolates of Cryptosporidium from different hosts. Parasitology 118, 49–58.

    Article  PubMed  CAS  Google Scholar 

  20. Morgan, U. M., Monis, P. T., Fayer, R., Deplazes, P., and Thompson, R. C. A. (1999) Phylogenetic relationships among isolates of Cryptosporidium: evidence for several new species. J. Parasitol. 85, 1126–1133.

    Article  PubMed  CAS  Google Scholar 

  21. Sulaiman, I. M., Morgan, U. M., Thompson, R. C., Lal, A. A., and Xiao, L. (2000) Phylogenetic relationships of Cryptosporidium parasites based on the 70-kilodalton heat shock protein (HSP70) gene. Appl. Environ. Microbiol. 66, 2385–2391.

    Article  PubMed  CAS  Google Scholar 

  22. Fayer, R., Morgan, U., and Upton, S. J. (2000) Epidemiology of Cryptosporidium: transmission, detection and identification. Int. J. Parasitol. 30, 1305–1322.

    Article  PubMed  CAS  Google Scholar 

  23. Morgan, U., Weber, R., Xiao, L., et al. (2000) Molecular characterization of Cryptosporidium isolates obtained from human immunodeficiency virus-infected individuals living in Switzerland, Kenya, and the United States. J. Clin. Microbiol. 38, 1180–1183.

    PubMed  CAS  Google Scholar 

  24. Pedraza-Diaz, S., Amar, C., Iversen, A. M., Stanley, P. J., and McLauchlin, J. (2001) Unusual Cryptosporidium species recovered from human faeces: first description of Cryptosporidium felis and Cryptosporidium ‘dog type’ from patients in England. J. Med. Microbiol. 50, 293–296.

    PubMed  CAS  Google Scholar 

  25. Pedraza-Diaz, S., Amar, C., and McLauchlin, J. (2000) The identification and characterisation of an unusual genotype of Cryptosporidium from human faeces as Cryptosporidium meleagridis. FEMS Microbiol. Lett. 189, 189–194.

    Article  PubMed  CAS  Google Scholar 

  26. Pieniazek, N. J., Bornay-Llinares, F. J., Slemenda, S. B., et al. (1999) New Cryptosporidium genotypes in HIV-infected persons. Emerg. Infect. Dis. 5, 444–449.

    Article  PubMed  CAS  Google Scholar 

  27. Chung, E., Aldom, J. E., Carreno, R. A., et al. (1999) PCR-based quantitation of Cryptosporidium parvum in municipal water samples. J. Microbiol. Methods 38, 119–130.

    Article  PubMed  CAS  Google Scholar 

  28. Chung, E., Aldom, J. E., Chagla, A. H., et al. (1998) Detection of Cryptosporidium parvum oocysts in municipal water samples by the polymerase chain reaction. J. Microbiol. Methods 33, 171–180.

    Article  CAS  Google Scholar 

  29. Di Giovanni, G. D., Hashemi, F. H., Shaw, N. J., Abrams, F. A., LeChevallier, M. W., and Abbaszadegan, M. (1999) Detection of infectious Cryptosporidium parvum oocysts in surface and filter backwash water samples by immunomagnetic separation and integrated cell culture-PCR. Appl. Environ. Microbiol. 65, 3427–3432.

    PubMed  Google Scholar 

  30. Gibbons, C. L., Rigi, F. M., and Awad-El-Kariem, F. M. (1998) Detection of Cryptosporidium parvum and C. muris oocysts in spiked backwash water using three PCR-based protocols. Protist 149, 127–134.

    Article  Google Scholar 

  31. Kaucner, C. and Stinear, T. (1998) Sensitive and rapid detection of viable Giardia cysts and Cryptosporidium parvum oocysts in large-volume water samples with wound fiberglass cartridge filters and reverse transcription-PCR. Appl. Environ. Microbiol. 64, 1743–1749.

    PubMed  CAS  Google Scholar 

  32. Kostrzynska, M., Sankey, M., Haack, E., et al. (1999) Three sample preparation protocols for polymerase chain reaction based detection of Cryptosporidium parvum in environmental samples. J. Microbiol. Methods 35, 65–71.

    Article  PubMed  CAS  Google Scholar 

  33. Mayer, C. L. and Palmer, C. J. (1996) Evaluation of PCR, nested PCR, and fluorescent antibodies for detection of Giardia and Cryptosporidium species in wastewater. Appl. Environ. Microbiol. 62, 2081–2085.

    PubMed  CAS  Google Scholar 

  34. Rochelle, P. A., De Leon, R., Johnson, A., Stewart, M. H., and Wolfe, R. L. (1999) Evaluation of immunomagnetic separation for recovery of infectious Cryptosporidium parvum oocysts from environmental samples. Appl. Environ. Microbiol. 65, 841–845.

    PubMed  CAS  Google Scholar 

  35. Rochelle, P. A., De Leon, R., Stewart, M. H., and Wolfe, R. L. (1997) Comparison of primers and optimization of PCR conditions for detection of Cryptosporidium parvum and Giardia lamblia in water. Appl. Environ. Microbiol. 63, 106–114.

    PubMed  CAS  Google Scholar 

  36. Rochelle, P. A., Ferguson, D. M., Handojo, T. J., De Leon, R., Stewart, M. H., and Wolfe, R. L. (1997) An assay combining cell culture with reverse transcriptase PCR to detect and determine the infectivity of waterborne Cryptosporidium parvum. Appl. Environ. Microbiol. 63, 2029–2037.

    PubMed  CAS  Google Scholar 

  37. Sluter, S. D., Tzipori, S., and Widmer, G. (1997) Parameters affecting polymerase chain reaction detection of waterborne Cryptosporidium parvum oocysts. Appl. Microbiol. Biotechnol. 48, 325–330.

    Article  PubMed  CAS  Google Scholar 

  38. Stinear, T., Matusan, A., Hines, K., and Sandery, M. (1996) Detection of a single viable Cryptosporidium parvum oocyst in environmental water concentrates by reverse transcription-PCR. Appl. Environ. Microbiol. 62, 3385–3390.

    PubMed  CAS  Google Scholar 

  39. Toze, S. (1999) PCR and the detection of microbial pathogens in water and wastewater. Water Res. 33, 3545–3556.

    Article  CAS  Google Scholar 

  40. Wiedenmann, A., Kruger, P., and Botzenhart, K. (1998) PCR detection of Cryptosporidium parvum in environmental samples—a review of published protocols and current developments. J. Indust. Microbiol. Biotechnol. 21, 150–166.

    Article  CAS  Google Scholar 

  41. Xiao, L., Alderisio, K., Limor, J., Royer, M., and Lal, A. A. (2000) Identification of species and sources of Cryptosporidium oocysts in storm waters with a small-subunit rRNA-based diagnostic and genotyping tool. Appl. Environ. Microbiol. 66, 5492–5498.

    Article  PubMed  CAS  Google Scholar 

  42. Xiao, L., Singh, A., Limor, J., Graczyk, T. K., Gradus, S., and Lal, A. (2001) Molecular characterization of Cryptosporidium oocysts in samples of raw surface water and wastewater. Appl. Environ. Microbiol. 67, 1097–1101.

    Article  PubMed  CAS  Google Scholar 

  43. Lowery, C. J., Moore, J. E., Millar, B. C., et al. (2001) Occurrence and molecular genotyping of Cryptosporidium spp. in surface waters in Northern Ireland. J. Appl. Microbiol. 91, 774–779.

    Article  PubMed  CAS  Google Scholar 

  44. Ono, K., Tsuji, H., Rai, S. K., et al. (2001) Contamination of River Water by Cryptosporidium parvum oocysts in Western Japan. Appl. Environ. Microbiol. 67, 3832–3836.

    Article  PubMed  CAS  Google Scholar 

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Author information

Authors and Affiliations

  1. Division of Parasitic Diseases, Centers for Disease Control and Prevention, Public Health Service, United States Deparment of Health and Human Services, Atlanta, GA

    Lihua Xiao, Altaf A. Lal & Jianlin Jiang

Authors
  1. Lihua Xiao
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  2. Altaf A. Lal
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  3. Jianlin Jiang
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Editors and Affiliations

  1. Planta Piloto de Procesos Industriales Microbiológicos, (PROIMI)-CONICET, San Miguel de Tucumán, Argentina

    John F. T. Spencer  & Alicia L. Ragout de Spencer  & 

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© 2004 Humana Press Inc.Totowa, NJ

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Xiao, L., Lal, A.A., Jiang, J. (2004). Detection and Differentiation of Cryptosporidium Oocysts in Water by PCR-RFLP. In: Spencer, J.F.T., Ragout de Spencer, A.L. (eds) Public Health Microbiology. Methods in Molecular Biology, vol 268. Humana Press. https://doi.org/10.1385/1-59259-766-1:163

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  • DOI: https://doi.org/10.1385/1-59259-766-1:163

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-117-2

  • Online ISBN: 978-1-59259-766-6

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