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CP2 gene as a useful viability marker for Cryptosporidium parvum

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Abstract

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.

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References

  • 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 27:787–798

    Article  PubMed  CAS  Google Scholar 

  • Cacciò S, La Rosa G, Pozio E (1997) The beta-tubulin gene of Cryptosporidium parvum. Mol Biochem Parasitol 89:307–311

    Article  PubMed  Google Scholar 

  • 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 49:4437–4442

    Article  PubMed  CAS  Google Scholar 

  • Casemore DP, Armstrong M, Sands RL (1985) Laboratory diagnosis of cryptosporidiosis. J Clin Pathol 8:1337–1341

    Article  Google Scholar 

  • Chappell CL, Okhuysen PC, Sterling CR, DuPont HL (1996) Cryptosporidium parvum: intensity of infection and oocyst excretion patterns in healthy volunteers. J Infect Dis 73:232–236

    Google Scholar 

  • Di Giovanni GD, LeChevallier MW (2005) Quantitative PCR assessment of Cryptosporidium parvum cell culture infection. Appl Environ Microbiol 71:1495–1500

    Article  PubMed  CAS  Google Scholar 

  • 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 332:855–859

    Article  PubMed  CAS  Google Scholar 

  • 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 226:237–243

    Article  PubMed  CAS  Google Scholar 

  • 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 69:5178–5185

    Article  PubMed  CAS  Google Scholar 

  • 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 5:592–598

    Article  PubMed  CAS  Google Scholar 

  • 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 47:323–337

    Article  PubMed  CAS  Google Scholar 

  • 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 63:3844–3850

    PubMed  CAS  Google Scholar 

  • 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 43:97–106

    Article  PubMed  CAS  Google Scholar 

  • 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 42:416–422

    Article  PubMed  CAS  Google Scholar 

  • 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 56:1423–1428

    PubMed  CAS  Google Scholar 

  • Le Blancq SM, Khramtsov NV, Zamani F, Upton SJ, Wu TW (1997) Ribosomal RNA gene organization in Cryptosporidium parvum. Mol Biochem Parasitol 90:463–478

    Article  PubMed  CAS  Google Scholar 

  • Lindquist S (1986) The heat-shock response. Annu Rev Biochem 55:1151–1191

    Article  PubMed  CAS  Google Scholar 

  • Lindquist S, Petersen R (1990) Selective translation and degradation of heat-shock messenger RNAs in Drosophila. Enzyme 44:147–166

    Google Scholar 

  • 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 331:161–167

    Article  PubMed  CAS  Google Scholar 

  • 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 121:279–282

    Article  PubMed  CAS  Google Scholar 

  • 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 66:406–412

    PubMed  CAS  Google Scholar 

  • Nichols GL (2000) Food-borne protozoa. Br Med Bull 56:209–235

    Article  PubMed  CAS  Google Scholar 

  • Nichols RA, Campbell BM, Smith HV (2006) Molecular fingerprinting of Cryptosporidium oocysts isolated during water monitoring. Appl Environ Microbiol 72:5428–5435

    Article  PubMed  CAS  Google Scholar 

  • O’Donoghue PJ (1995) Cryptosporidium and cryptosporidiosis in man and animals. Int J Parasitol 25:139–195

    Article  PubMed  CAS  Google Scholar 

  • O’Hara SP, Yu JR, Lin JJ (2004) A novel Cryptosporidium parvum antigen, CP2, preferentially associates with membranous structures. Parasitol Res 92:317–327

    Article  PubMed  Google Scholar 

  • Okhuysen PC, Chappell CL, Crabb JH, Sterling CR, DuPont HL (1999) Virulence of three distinct Cryptosporidium parvum isolates for healthy adults. J Infect Dis 180:1275–1281

    Article  PubMed  CAS  Google Scholar 

  • Petry F, Robinson HA, McDonald V (1995) Murine infection model for maintenance and amplification of Cryptosporidium parvum oocysts. J Clin Microbiol 33:1922–1924

    PubMed  CAS  Google Scholar 

  • Robertson LJ, Campbell AT, Smith HV (1993) In vitro excystation of Cryptosporidium parvum. Parasitology 106:13–19

    Article  PubMed  Google Scholar 

  • 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 63:2029–2037

    PubMed  CAS  Google Scholar 

  • 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 68:3809–3817

    Article  PubMed  CAS  Google Scholar 

  • Shin GA, Linden KG, Arrowood MJ, Sobsey MD (2001) Low-pressure UV inactivation and DNA repair potential of Cryptosporidium parvum oocysts. Appl Environ Microbiol 67:3029–3032

    Article  PubMed  CAS  Google Scholar 

  • 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 114:427–437

    Article  PubMed  CAS  Google Scholar 

  • 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 150:209–217

    Article  PubMed  CAS  Google Scholar 

  • 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 62:3385–3390

    PubMed  CAS  Google Scholar 

  • 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 40:3237–3244

    Article  PubMed  CAS  Google Scholar 

  • Widmer G, Orbacz EA, Tzipori S (1999) β-Tubulin mRNA as a marker of Cryptosporidium parvum oocyst viability. Appl Environ Microbiol 65:1584–1588

    PubMed  CAS  Google Scholar 

  • Yang S, Healey MC (1993) The immunosuppressive effects of dexamethasone administered in drinking water to C57BL/6N mice infected with Cryptosporidium parvum. J Parasitol 79:626–630

    Article  PubMed  CAS  Google Scholar 

  • 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 88:412–420

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This research was supported by the program of Basic Atomic Energy Research Institute (BAERI), which is a part of the Nuclear R & D Programs funded by the Ministry of Science and Technology (MOST) of Korea in 2006. This study complies with the current laws of the Republic of Korea in which the experiments were performed.

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Authors and Affiliations

  1. Department of Environmental and Tropical Medicine, Konkuk University School of Medicine, Chungju, 380-701, Republic of Korea

    Soo-Ung Lee, Migyo Joung & Jae-Ran Yu

  2. Department of Parasitology, College of Medicine, Hanyang University, Seoul, 133-791, Republic of Korea

    Myoung-Hee Ahn

  3. Department of Parasitology, College of Medicine, Hallym University, Chuncheon, 200-702, Republic of Korea

    Sun Huh

  4. Departmetnt of Pathology, Gwangju Health College, Gwangju, 506-701, Republic of Korea

    Hyunje Song

  5. Department of Radiation Oncology, College of Medicine, Chungbuk National University, Cheongju, 361-763, Korea

    Woo-Yoon Park

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  1. Soo-Ung Lee
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  2. Migyo Joung
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  3. Myoung-Hee Ahn
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  4. Sun Huh
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  7. Jae-Ran Yu
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Correspondence to Jae-Ran Yu.

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Lee, SU., Joung, M., Ahn, MH. et al. CP2 gene as a useful viability marker for Cryptosporidium parvum . Parasitol Res 102, 381–387 (2008). https://doi.org/10.1007/s00436-007-0772-8

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  • DOI: https://doi.org/10.1007/s00436-007-0772-8

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