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

  • Lihua Xiao
  • Altaf A. Lal
  • Jianlin Jiang
Part of the Methods in Molecular Biology book series (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, 2, 3, 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.

Keywords

Cryptosporidium Oocyst Cryptosporidium Infection Polymerase Chain Reaction Inhibitor Cryptosporidium Species Polymerase Chain Reaction Tube 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 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.PubMedGoogle Scholar
  2. 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.PubMedGoogle Scholar
  3. 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.PubMedCrossRefGoogle Scholar
  4. 4.
    Rose, J. B. (1997) Environmental ecology of Cryptosporidium and public health implications. Annu. Rev. Public Health 18, 135–161.PubMedCrossRefGoogle Scholar
  5. 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.PubMedGoogle Scholar
  6. 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.PubMedCrossRefGoogle Scholar
  7. 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.PubMedGoogle Scholar
  8. 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.PubMedGoogle Scholar
  9. 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.PubMedGoogle Scholar
  10. 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.PubMedCrossRefGoogle Scholar
  11. 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.PubMedCrossRefGoogle Scholar
  12. 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.PubMedCrossRefGoogle Scholar
  13. 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.PubMedGoogle Scholar
  14. 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.PubMedCrossRefGoogle Scholar
  15. 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.PubMedCrossRefGoogle Scholar
  16. 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.PubMedGoogle Scholar
  17. 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.PubMedGoogle Scholar
  18. 18.
    Sulaiman, I. M., Xiao, L. H., and Lal, A. A. (1999) Evaluation of Cryptosporidium parvum genotyping techniques. Appl. Environ. Microbiol. 65, 4431–4435.PubMedGoogle Scholar
  19. 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.PubMedCrossRefGoogle Scholar
  20. 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.PubMedCrossRefGoogle Scholar
  21. 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.PubMedCrossRefGoogle Scholar
  22. 22.
    Fayer, R., Morgan, U., and Upton, S. J. (2000) Epidemiology of Cryptosporidium: transmission, detection and identification. Int. J. Parasitol. 30, 1305–1322.PubMedCrossRefGoogle Scholar
  23. 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.PubMedGoogle Scholar
  24. 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.PubMedGoogle Scholar
  25. 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.PubMedCrossRefGoogle Scholar
  26. 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.PubMedCrossRefGoogle Scholar
  27. 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.PubMedCrossRefGoogle Scholar
  28. 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.CrossRefGoogle Scholar
  29. 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.PubMedGoogle Scholar
  30. 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.CrossRefGoogle Scholar
  31. 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.PubMedGoogle Scholar
  32. 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.PubMedCrossRefGoogle Scholar
  33. 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.PubMedGoogle Scholar
  34. 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.PubMedGoogle Scholar
  35. 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.PubMedGoogle Scholar
  36. 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.PubMedGoogle Scholar
  37. 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.PubMedCrossRefGoogle Scholar
  38. 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.PubMedGoogle Scholar
  39. 39.
    Toze, S. (1999) PCR and the detection of microbial pathogens in water and wastewater. Water Res. 33, 3545–3556.CrossRefGoogle Scholar
  40. 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.CrossRefGoogle Scholar
  41. 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.PubMedCrossRefGoogle Scholar
  42. 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.PubMedCrossRefGoogle Scholar
  43. 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.PubMedCrossRefGoogle Scholar
  44. 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.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc.Totowa, NJ 2004

Authors and Affiliations

  • Lihua Xiao
    • 1
  • Altaf A. Lal
    • 1
  • Jianlin Jiang
    • 1
  1. 1.Division of Parasitic Diseases, Centers for Disease Control and Prevention, Public Health ServiceUnited States Deparment of Health and Human ServicesAtlanta

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