Parasitology Research

, Volume 102, Issue 3, pp 381–387 | Cite as

CP2 gene as a useful viability marker for Cryptosporidium parvum

  • Soo-Ung Lee
  • Migyo Joung
  • Myoung-Hee Ahn
  • Sun Huh
  • Hyunje Song
  • Woo-Yoon Park
  • Jae-Ran Yu
Original Paper


The validity of the CP2 gene of Cryptosporidium parvum as a viability marker was evaluated using absolute quantitative real-time polymerase chain reaction (qPCR) assays. Total ribonucleic acid (RNA) was isolated from live and heat-killed C. parvum oocysts, and complementary deoxyribonucleic acid was synthesized and used as a template. The most accurate number of viable C. parvum oocysts was predicted when the CP2 gene was used as a target gene. The lower detection limit of the CP2 gene was ten oocysts, which was the most sensitive among examined target genes. With heat shock induction, only hsp70 messenger RNA (mRNA) was induced, and the predicted viable oocyst number was increased by heat shock for this marker. The CP2, hsp70, Cryptosporidium oocyst wall protein, and β-tubulin mRNAs were not detected in heat-killed oocysts, but the 18S ribosomal ribonucleic acid (rRNA) showed heat stability until 48 h after heat killing. Although the 18S rRNA demonstrated the fastest response in crossing point (CP) value among the examined primer sets in qPCR, overestimation of viable oocysts was noted in the analysis with this gene. In conclusion, the CP2 gene was identified as the most sensitive, reliable, and accurate candidate of a viability marker of C. parvum by qPCR evaluation.


  1. Belosevic M, Guy RA, Taghi-Kilani R, Neumann NF, Gyürék LL, Liyanage LRJ, Millard PJ, Finch GR (1997) Nucleic acid stains as indicators of Cryptosporidium parvum oocyst viability. Int J Parasitol 27:787–798PubMedCrossRefGoogle Scholar
  2. Cacciò S, La Rosa G, Pozio E (1997) The beta-tubulin gene of Cryptosporidium parvum. Mol Biochem Parasitol 89:307–311PubMedCrossRefGoogle Scholar
  3. Cai X, Woods KM, Upton SJ, Zhu G (2005) Application of quantitative real-time reverse transcription-PCR in assessing drug efficacy against the intracellular pathogen Cryptosporidium parvum in vitro. Antimicrob Agents Chemother 49:4437–4442PubMedCrossRefGoogle Scholar
  4. Casemore DP, Armstrong M, Sands RL (1985) Laboratory diagnosis of cryptosporidiosis. J Clin Pathol 8:1337–1341CrossRefGoogle Scholar
  5. Chappell CL, Okhuysen PC, Sterling CR, DuPont HL (1996) Cryptosporidium parvum: intensity of infection and oocyst excretion patterns in healthy volunteers. J Infect Dis 73:232–236Google Scholar
  6. Di Giovanni GD, LeChevallier MW (2005) Quantitative PCR assessment of Cryptosporidium parvum cell culture infection. Appl Environ Microbiol 71:1495–1500PubMedCrossRefGoogle Scholar
  7. DuPont HL, Chappell CL, Sterling CR, Okhuysen PC, Rose JB, Jakubowski W (1995) The infectivity of Cryptosporidium parvum in healthy volunteers. N Engl J Med 332:855–859PubMedCrossRefGoogle Scholar
  8. Fontaine M, Guillot E (2003) Study of 18S rRNA and rRNA stability by real-time RT-PCR in heat-inactivated Cryptosporidium parvum oocysts. FEMS Microbiol Lett 226:237–243PubMedCrossRefGoogle Scholar
  9. Guy RA, Payment P, Krull UJ, Horgen PA (2003) Real-time PCR for quantification of Giardia and Cryptosporidium in environmental water samples and sewage. Appl Environ Microbiol 69:5178–5185PubMedCrossRefGoogle Scholar
  10. Hallier-Soulier S, Guillot E (2003) An immunomagnetic separation-reverse transcription polymerase chain reaction (IMS-RT-PCR) test for sensitive and rapid detection of viable waterborne Cryptosporidium parvum. Environ Microbiol 5:592–598PubMedCrossRefGoogle Scholar
  11. Higgins JA, Fayer R, Trout JM, Xiao L, Lal AA, Kerby S, Jenkins MC (2001) Real-time PCR for the detection of Cryptosporidium parvum. J Microbiol Methods 47:323–337PubMedCrossRefGoogle Scholar
  12. Jenkins MB, Anguish LJ, Bowman DD, Walker MJ, Ghiorse WC (1997) Assessment of a dye permeability assay for determination of inactivation rates of Cryptosporidium parvum oocysts. Appl Environ Microbiol 63:3844–3850PubMedGoogle Scholar
  13. Jenkins MC, Trout J, Abrahamsen MS, Lancto CA, Higgins J, Fayer R (2000) Estimating viability of Cryptosporidium parvum oocysts using reverse transcriptase–polymerase chain reaction (RT-PCR) directed at mRNA encoding amyloglucosidase. J Microbiol Methods 43:97–106PubMedCrossRefGoogle Scholar
  14. Khramtsov NV, Tilley M, Blunt DS, Montelone BA, Upton SJ (1995) Cloning and analysis of a Cryptosporidium parvum gene encoding a protein with homology to cytoplasmic form Hsp70. J Eukaryot Microbiol 42:416–422PubMedCrossRefGoogle Scholar
  15. Korich DG, Mead JR, Madore MS, Sinclair NA, Sterling CR (1990) Effects of ozone, chlorine dioxide, chlorine, and chloramine on Cryptosporidium parvum oocyst viability. Appl Environ Microbiol 56:1423–1428PubMedGoogle Scholar
  16. Le Blancq SM, Khramtsov NV, Zamani F, Upton SJ, Wu TW (1997) Ribosomal RNA gene organization in Cryptosporidium parvum. Mol Biochem Parasitol 90:463–478PubMedCrossRefGoogle Scholar
  17. Lindquist S (1986) The heat-shock response. Annu Rev Biochem 55:1151–1191PubMedCrossRefGoogle Scholar
  18. Lindquist S, Petersen R (1990) Selective translation and degradation of heat-shock messenger RNAs in Drosophila. Enzyme 44:147–166Google Scholar
  19. Mac Kenzie WR, Hoxie NJ, Proctor ME, Gradus MS, Blair KA, Peterson DE, Kazmierczak JJ, Addiss DG, Fox KR, Rose JB, Davis JP (1994) A massive outbreak in Milwaukee of Cryptosporidium infection transmitted through the public water supply. N Engl J Med 331:161–167PubMedCrossRefGoogle Scholar
  20. MacDonald LM, Sargent K, Armson A, Thompson RC, Reynoldson JA (2002) The development of a real-time quantitative-PCR method for characterization of a Cryptosporidium parvum in vitro culturing system and assessment of drug efficacy. Mol Biochem Parasitol 121:279–282PubMedCrossRefGoogle Scholar
  21. Neumann NF, Gyurek LL, Gammie L, Finch GR, Belosevic M (2000) Comparison of animal infectivity and nucleic acid staining for assessment of Cryptosporidium parvum viability in water. Appl Environ Microbiol 66:406–412PubMedGoogle Scholar
  22. Nichols GL (2000) Food-borne protozoa. Br Med Bull 56:209–235PubMedCrossRefGoogle Scholar
  23. Nichols RA, Campbell BM, Smith HV (2006) Molecular fingerprinting of Cryptosporidium oocysts isolated during water monitoring. Appl Environ Microbiol 72:5428–5435PubMedCrossRefGoogle Scholar
  24. O’Donoghue PJ (1995) Cryptosporidium and cryptosporidiosis in man and animals. Int J Parasitol 25:139–195PubMedCrossRefGoogle Scholar
  25. O’Hara SP, Yu JR, Lin JJ (2004) A novel Cryptosporidium parvum antigen, CP2, preferentially associates with membranous structures. Parasitol Res 92:317–327PubMedCrossRefGoogle Scholar
  26. Okhuysen PC, Chappell CL, Crabb JH, Sterling CR, DuPont HL (1999) Virulence of three distinct Cryptosporidium parvum isolates for healthy adults. J Infect Dis 180:1275–1281PubMedCrossRefGoogle Scholar
  27. Petry F, Robinson HA, McDonald V (1995) Murine infection model for maintenance and amplification of Cryptosporidium parvum oocysts. J Clin Microbiol 33:1922–1924PubMedGoogle Scholar
  28. Robertson LJ, Campbell AT, Smith HV (1993) In vitro excystation of Cryptosporidium parvum. Parasitology 106:13–19PubMedCrossRefGoogle Scholar
  29. Rochelle PA, Ferguson DM, Handojo TJ, De Leon R, Stewart MH, Wolfe RL (1997) An assay combining cell culture with reverse transcriptase PCR to detect and determine the infectivity of waterborne Cryptosporidium parvum. Appl Environ Microbiol 63:2029–2037PubMedGoogle Scholar
  30. Rochelle PA, Marshall MM, Mead JR, Johnson AM, Korich DG, Rosen JS, De Leon R (2002) Comparison of in vitro cell culture and a mouse assay for measuring infectivity of Cryptosporidium parvum. Appl Environ Microbiol 68:3809–3817PubMedCrossRefGoogle Scholar
  31. Shin GA, Linden KG, Arrowood MJ, Sobsey MD (2001) Low-pressure UV inactivation and DNA repair potential of Cryptosporidium parvum oocysts. Appl Environ Microbiol 67:3029–3032PubMedCrossRefGoogle Scholar
  32. Spano F, Puri C, Ranucci L, Putignani L, Crisanti A (1997a) Cloning of the entire COWP gene of Cryptosporidium parvum and ultrastructural localization of the protein during sexual parasite development. Parasitology 114:427–437PubMedCrossRefGoogle Scholar
  33. Spano F, Putignani L, McLauchlin J, Casemore DP, Crisanti A (1997b) PCR-RFLP analysis of the Cryptosporidium oocyst wall protein (COWP) gene discriminates between C. wrairi and C. parvum, and between C. parvum isolates of human and animal origin. FEMS Microbiol Lett 150:209–217PubMedCrossRefGoogle Scholar
  34. Stinear T, Matusan A, Hines K, Sandery M (1996) Detection of a single viable Cryptosporidium parvum oocyst in environmental water concentrated by reverse transcription PCR. Appl Environ Microbiol 62:3385–3390PubMedGoogle Scholar
  35. Tanriverdi S, Tanyeli A, Başlamişli F, Köksal F, Kilinç Y, Feng X, Batzer G, Tzipori S, Widmer G (2002) Detection and genotyping of oocysts of Cryptosporidium parvum by real-time PCR and melting curve analysis. J Clin Microbiol 40:3237–3244PubMedCrossRefGoogle Scholar
  36. Widmer G, Orbacz EA, Tzipori S (1999) β-Tubulin mRNA as a marker of Cryptosporidium parvum oocyst viability. Appl Environ Microbiol 65:1584–1588PubMedGoogle Scholar
  37. Yang S, Healey MC (1993) The immunosuppressive effects of dexamethasone administered in drinking water to C57BL/6N mice infected with Cryptosporidium parvum. J Parasitol 79:626–630PubMedCrossRefGoogle Scholar
  38. Yu JR, O’Hara SP, Lin JL, Dailey ME, Cain G, Lin JL (2002) A common oocyst surface antigen of Cryptosporidium recognized by monoclonal antibodies. Parisitol Res 88:412–420CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • Soo-Ung Lee
    • 1
  • Migyo Joung
    • 1
  • Myoung-Hee Ahn
    • 2
  • Sun Huh
    • 3
  • Hyunje Song
    • 4
  • Woo-Yoon Park
    • 5
  • Jae-Ran Yu
    • 1
  1. 1.Department of Environmental and Tropical MedicineKonkuk University School of MedicineChungjuRepublic of Korea
  2. 2.Department of Parasitology, College of MedicineHanyang UniversitySeoulRepublic of Korea
  3. 3.Department of Parasitology, College of MedicineHallym UniversityChuncheonRepublic of Korea
  4. 4.Departmetnt of PathologyGwangju Health CollegeGwangjuRepublic of Korea
  5. 5.Department of Radiation Oncology, College of MedicineChungbuk National UniversityCheongjuKorea

Personalised recommendations