Skip to main content
PMC home page
Applied and Environmental Microbiology logoLink to Applied and Environmental Microbiology
. 1996 Feb;62(2):643–647. doi: 10.1128/aem.62.2.643-647.1996

Simplified method for recovery and PCR detection of Cryptosporidium DNA from bovine feces.

X Leng 1, D A Mosier 1, R D Oberst 1
PMCID: PMC167830  PMID: 8593065

Abstract

An assay to identify Cryptosporidium DNA in bovine feces has been developed emphasizing standardization of sample preparation and simplification of the DNA recovery process for PCR amplification and DNA hybridization detection. The Cryptosporidium DNA recovery-PCR detection procedure (CR-PCR) can recover DNA suitable for PCR amplification without using or generating hazardous chemicals or wastes. In comparisons with a commercial enzyme-linked immunoassay (Color Vue-Cryptosporidium; Seradyn, Indianapolis, Ind.), the CR-PCR could detect 10(3) to 10(4) times fewer purified oocysts diluted in solution (water or buffered saline) and 10(2) times fewer oocysts from diarrheic feces and showed earlier detectability from solid, nondiarrheic feces in an experimental infection. This assay may prove useful for detecting Cryptosporidium oocysts in feces and in clarifying the role of livestock in waterborne outbreaks of cryptosporidiosis.

Full Text

The Full Text of this article is available as a PDF (337.1 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Aarnaes S. L., Blanding J., Speier S., Forthal D., de la Maza L. M., Peterson E. M. Comparison of the ProSpecT and Color Vue enzyme-linked immunoassays for the detection of Cryptosporidium in stool specimens. Diagn Microbiol Infect Dis. 1994 Aug;19(4):221–225. doi: 10.1016/0732-8893(94)90035-3. [DOI] [PubMed] [Google Scholar]
  2. Anusz K. Z., Mason P. H., Riggs M. W., Perryman L. E. Detection of Cryptosporidium parvum oocysts in bovine feces by monoclonal antibody capture enzyme-linked immunosorbent assay. J Clin Microbiol. 1990 Dec;28(12):2770–2774. doi: 10.1128/jcm.28.12.2770-2774.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Arrowood M. J., Hurd M. R., Mead J. R. A new method for evaluating experimental cryptosporidial parasite loads using immunofluorescent flow cytometry. J Parasitol. 1995 Jun;81(3):404–409. [PubMed] [Google Scholar]
  4. Baxby D., Hart C. A. The incidence of cryptosporidiosis: a two-year prospective survey in a children's hospital. J Hyg (Lond) 1986 Feb;96(1):107–111. doi: 10.1017/s0022172400062598. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Berkelman R. L. Emerging infectious diseases in the United States, 1993. J Infect Dis. 1994 Aug;170(2):272–277. doi: 10.1093/infdis/170.2.272. [DOI] [PubMed] [Google Scholar]
  6. Black R. E., Lopez de Romaña G., Brown K. H., Bravo N., Bazalar O. G., Kanashiro H. C. Incidence and etiology of infantile diarrhea and major routes of transmission in Huascar, Peru. Am J Epidemiol. 1989 Apr;129(4):785–799. doi: 10.1093/oxfordjournals.aje.a115193. [DOI] [PubMed] [Google Scholar]
  7. Casemore D. P., Armstrong M., Sands R. L. Laboratory diagnosis of cryptosporidiosis. J Clin Pathol. 1985 Dec;38(12):1337–1341. doi: 10.1136/jcp.38.12.1337. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Current W. L., Reese N. C., Ernst J. V., Bailey W. S., Heyman M. B., Weinstein W. M. Human cryptosporidiosis in immunocompetent and immunodeficient persons. Studies of an outbreak and experimental transmission. N Engl J Med. 1983 May 26;308(21):1252–1257. doi: 10.1056/NEJM198305263082102. [DOI] [PubMed] [Google Scholar]
  9. DuPont H. L., Chappell C. L., Sterling C. R., Okhuysen P. C., Rose J. B., Jakubowski W. The infectivity of Cryptosporidium parvum in healthy volunteers. N Engl J Med. 1995 Mar 30;332(13):855–859. doi: 10.1056/NEJM199503303321304. [DOI] [PubMed] [Google Scholar]
  10. Farrington M., Winters S., Walker C., Miller R., Rubenstein D. Cryptosporidium antigen detection in human feces by reverse passive hemagglutination assay. J Clin Microbiol. 1994 Nov;32(11):2755–2759. doi: 10.1128/jcm.32.11.2755-2759.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Garber L. P., Salman M. D., Hurd H. S., Keefe T., Schlater J. L. Potential risk factors for Cryptosporidium infection in dairy calves. J Am Vet Med Assoc. 1994 Jul 1;205(1):86–91. [PubMed] [Google Scholar]
  12. Garcia L. S., Brewer T. C., Bruckner D. A. Fluorescence detection of Cryptosporidium oocysts in human fecal specimens by using monoclonal antibodies. J Clin Microbiol. 1987 Jan;25(1):119–121. doi: 10.1128/jcm.25.1.119-121.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Garcia L. S., Bruckner D. A., Brewer T. C., Shimizu R. Y. Techniques for the recovery and identification of Cryptosporidium oocysts from stool specimens. J Clin Microbiol. 1983 Jul;18(1):185–190. doi: 10.1128/jcm.18.1.185-190.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kehl K. S., Cicirello H., Havens P. L. Comparison of four different methods for detection of Cryptosporidium species. J Clin Microbiol. 1995 Feb;33(2):416–418. doi: 10.1128/jcm.33.2.416-418.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kuhls T. L., Greenfield R. A., Mosier D. A., Crawford D. L., Joyce W. A. Cryptosporidiosis in adult and neonatal mice with severe combined immunodeficiency. J Comp Pathol. 1992 May;106(4):399–410. doi: 10.1016/0021-9975(92)90024-o. [DOI] [PubMed] [Google Scholar]
  16. Laxer M. A., D'Nicuola M. E., Patel R. J. Detection of Cryptosporidium parvum DNA in fixed, paraffin-embedded tissue by the polymerase chain reaction. Am J Trop Med Hyg. 1992 Oct;47(4):450–455. doi: 10.4269/ajtmh.1992.47.450. [DOI] [PubMed] [Google Scholar]
  17. Laxer M. A., Timblin B. K., Patel R. J. DNA sequences for the specific detection of Cryptosporidium parvum by the polymerase chain reaction. Am J Trop Med Hyg. 1991 Dec;45(6):688–694. doi: 10.4269/ajtmh.1991.45.688. [DOI] [PubMed] [Google Scholar]
  18. LeChevallier M. W., Norton W. D., Lee R. G. Giardia and Cryptosporidium spp. in filtered drinking water supplies. Appl Environ Microbiol. 1991 Sep;57(9):2617–2621. doi: 10.1128/aem.57.9.2617-2621.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. LeChevallier M. W., Norton W. D., Siegel J. E., Abbaszadegan M. Evaluation of the immunofluorescence procedure for detection of Giardia cysts and Cryptosporidium oocysts in water. Appl Environ Microbiol. 1995 Feb;61(2):690–697. doi: 10.1128/aem.61.2.690-697.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Ma P., Soave R. Three-step stool examination for cryptosporidiosis in 10 homosexual men with protracted watery diarrhea. J Infect Dis. 1983 May;147(5):824–828. doi: 10.1093/infdis/147.5.824. [DOI] [PubMed] [Google Scholar]
  21. Miller R. A., Bronsdon M. A., Morton W. R. Experimental cryptosporidiosis in a primate model. J Infect Dis. 1990 Feb;161(2):312–315. doi: 10.1093/infdis/161.2.312. [DOI] [PubMed] [Google Scholar]
  22. Müller H. M., Ranucci L., Pozio E., Crisanti A. A method for collecting large quantities of Cryptosporidium parasites. Parasitol Today. 1993 Jul;9(7):261–263. doi: 10.1016/0169-4758(93)90072-n. [DOI] [PubMed] [Google Scholar]
  23. Newman R. D., Jaeger K. L., Wuhib T., Lima A. A., Guerrant R. L., Sears C. L. Evaluation of an antigen capture enzyme-linked immunosorbent assay for detection of Cryptosporidium oocysts. J Clin Microbiol. 1993 Aug;31(8):2080–2084. doi: 10.1128/jcm.31.8.2080-2084.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Nieminski E. C., Schaefer F. W., 3rd, Ongerth J. E. Comparison of two methods for detection of Giardia cysts and Cryptosporidium oocysts in water. Appl Environ Microbiol. 1995 May;61(5):1714–1719. doi: 10.1128/aem.61.5.1714-1719.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Parisi M. T., Tierno P. M., Jr Evaluation of new rapid commercial enzyme immunoassay for detection of Cryptosporidium oocysts in untreated stool specimens. J Clin Microbiol. 1995 Jul;33(7):1963–1965. doi: 10.1128/jcm.33.7.1963-1965.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Petersen C. Cryptosporidiosis in patients infected with the human immunodeficiency virus. Clin Infect Dis. 1992 Dec;15(6):903–909. doi: 10.1093/clind/15.6.903. [DOI] [PubMed] [Google Scholar]
  27. Riggs M. W., Perryman L. E. Infectivity and neutralization of Cryptosporidium parvum sporozoites. Infect Immun. 1987 Sep;55(9):2081–2087. doi: 10.1128/iai.55.9.2081-2087.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Rosenblatt J. E., Sloan L. M. Evaluation of an enzyme-linked immunosorbent assay for detection of Cryptosporidium spp. in stool specimens. J Clin Microbiol. 1993 Jun;31(6):1468–1471. doi: 10.1128/jcm.31.6.1468-1471.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Rusnak J., Hadfield T. L., Rhodes M. M., Gaines J. K. Detection of Cryptosporidium oocysts in human fecal specimens by an indirect immunofluorescence assay with monoclonal antibodies. J Clin Microbiol. 1989 May;27(5):1135–1136. doi: 10.1128/jcm.27.5.1135-1136.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Siddons C. A., Chapman P. A., Rush B. A. Evaluation of an enzyme immunoassay kit for detecting cryptosporidium in faeces and environmental samples. J Clin Pathol. 1992 Jun;45(6):479–482. doi: 10.1136/jcp.45.6.479. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Smith H. V., Patterson W. J., Hardie R., Greene L. A., Benton C., Tulloch W., Gilmour R. A., Girdwood R. W., Sharp J. C., Forbes G. I. An outbreak of waterborne cryptosporidiosis caused by post-treatment contamination. Epidemiol Infect. 1989 Dec;103(3):703–715. doi: 10.1017/s0950268800031101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Stehr-Green J. K., McCaig L., Remsen H. M., Rains C. S., Fox M., Juranek D. D. Shedding of oocysts in immunocompetent individuals infected with Cryptosporidium. Am J Trop Med Hyg. 1987 Mar;36(2):338–342. doi: 10.4269/ajtmh.1987.36.338. [DOI] [PubMed] [Google Scholar]
  33. Sterling C. R., Arrowood M. J. Detection of Cryptosporidium sp. infections using a direct immunofluorescent assay. Pediatr Infect Dis. 1986 Jan-Feb;5(1 Suppl):S139–S142. doi: 10.1097/00006454-198601001-00022. [DOI] [PubMed] [Google Scholar]
  34. Ungar B. L. Enzyme-linked immunoassay for detection of Cryptosporidium antigens in fecal specimens. J Clin Microbiol. 1990 Nov;28(11):2491–2495. doi: 10.1128/jcm.28.11.2491-2495.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Weber R., Bryan R. T., Bishop H. S., Wahlquist S. P., Sullivan J. J., Juranek D. D. Threshold of detection of Cryptosporidium oocysts in human stool specimens: evidence for low sensitivity of current diagnostic methods. J Clin Microbiol. 1991 Jul;29(7):1323–1327. doi: 10.1128/jcm.29.7.1323-1327.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Webster K. A., Pow J. D., Giles M., Catchpole J., Woodward M. J. Detection of Cryptosporidium parvum using a specific polymerase chain reaction. Vet Parasitol. 1993 Oct;50(1-2):35–44. doi: 10.1016/0304-4017(93)90005-8. [DOI] [PubMed] [Google Scholar]
  37. Xiao L., Herd R. P. Quantitation of Giardia cysts and Cryptosporidium oocysts in fecal samples by direct immunofluorescence assay. J Clin Microbiol. 1993 Nov;31(11):2944–2946. doi: 10.1128/jcm.31.11.2944-2946.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Applied and Environmental Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES