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Diagnostic biomarkers in murine Cryptosporidiosis: dose- and age-related infection

Journal of Parasitic Diseases volume 41, pages 831–836 (2017)Cite this article

Abstract

Increasing prevalence of Cryptosporidium raises the importance to explore different aspects of its infection. In the absence of reproducible in vitro culturing, animal model is the only experimental method to study Cryptosporidium. Our study evaluated Cryptosporidium infection using coproscopy, copro-antigen and copro-DNA for early detection of murine cryptosporidiosis. Hundred and forty albino mice (neonates and adult) were divided into two groups, control group received sterile PBS solution, and infected groups were inoculated with molecularly characterized Cryptosporidium parvum oocysts and further subdivided into three subgroups for infectious dose response detection. Mice fecal samples were collected every 4 h on the first day and then daily and examined for fecal oocysts, copro-antigen and copro-DNA. Four mice from each subgroup were killed at 12, 24 and 48 h post-infection (P-I), and their intestines were examined for cryptosporidial mucosal DNA. Cryptosporidium copro-antigen and copro-DNA were detected 4 and 8 h P-I in infected neonatal and adult mice, respectively, and intestinal mucosal DNA was detected after 12 h in both. Microscopy was able to detect oocysts 48 h P-I. Inoculated C. parvum oocysts were recovered in feces of infected mice without genotypic changes. Neonate mice showed higher susceptibility for cryptosporidial infection than adults without statistical differences for the given infectious doses. Both copro-immunoassay and copro-nPCR assays can early detect Cryptosporidium infection; however, nPCR was able to identify Cryptosporidium species, making nPCR a reliable biomarker for early detection in murine model.

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References

  • Abdel-Wahab A, Abdel-Maogood S (2011) Identification of Cryptosporidium species infecting camels (Camelus dromedarius) in Egypt. J Am Sci 7(2):609–612

    Google Scholar 

  • Bialek R, Binder N, Dietz K, Joachim A, Knobloch J, Zelck UE (2002) Comparison of fluorescence, antigen and PCR assays to detect Cryptosporidium parvumin faecal specimens. Diagn Microbiol Infect Dis 43:283–288

    CAS  Article  PubMed  Google Scholar 

  • Chapman PA, Rush BA, McLauchlin J (1990) An enzyme immunoassay for detecting Cryptosporidium in faecal and environmental samples. J Med Microbiol 32(4):233–237

    CAS  Article  PubMed  Google Scholar 

  • Connelly JT, Nugen SR, Borejsza-Wysocki W, Durst RA, Montagna AJ (2008) Human pathogenic Cryptosporidium species bioanalytical detection method with single oocyst detection capability. Anal Bioanal Chem 36:450–457

    Google Scholar 

  • Current WL, Garcia LS (1991) Cryptosporidiosis. Clin Microbiol Rev 4:325–358

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Current WL, Snyder DB (1988) Development of and serologic evaluation of acquired immunity to Crypto-sporidium baileyi by broiler chickens. Poult Sci 67:720–729

    CAS  Article  PubMed  Google Scholar 

  • Enriquez FJ, Sterling CR (1991) Cryptosporidium infections in inbred strains of mice. J Protozool 38:100S–102S

    CAS  PubMed  Google Scholar 

  • Gaafar MR (2007) Effect of solar disinfection on viability of intestinal protozoa in drinking water. J Egypt Soc Parasitol 37:65–86

    PubMed  Google Scholar 

  • Garcia LS (2007) Diagnostic medical parasitology, 5th edn. ASM Press, Washington, DC

    Google Scholar 

  • Harp JC, Woodmanse DB, Moon HW (1990) Resistance of calves to Cryptosporidium parvum: effects of age and previous exposure. Infect Immun 58:2237–2240

    CAS  PubMed  PubMed Central  Google Scholar 

  • Harp JA, Chen W, Harmsen AG (1992) Resistance of severe combined immunodeficient mice to infection with Cryptosporidium parvum: the importance of intestinal microflora. Infect Immun 60:3509–3512

    CAS  PubMed  PubMed Central  Google Scholar 

  • Heyworth MF (1990) Immunological response to Cryptosporidium species. Gut 31:1423–1424

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Hijjawi N (2010) Cryptosporidium: new developments in cell culture. Exp Parasitol 124(1):54–60

    Article  PubMed  Google Scholar 

  • Jacyna MR, Parkin J, Goldin R, Baron JH (1990) Protracted enteric cryptosporidial infection in selective immunoglobulin A and saccharomyces opsonin deficiencies. Gut 31:714–716

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Khalifa AM, El Temsahy MM, Abou El Naga IF (2001) Effect of ozone on the viability of some protozoa in drinking water. J Egypt Soc Parasitol 31:603–616

    CAS  PubMed  Google Scholar 

  • Lumb R, Swift J, James C, Papanaoum K, Mukh-erjee T (1993) Identification of the microsporidian parasite, Enterocytozoon bieneusi in faecal samples and intestinal biopsies from an AIDS patient. Int J Parasitol 23:793–801

    CAS  Article  PubMed  Google Scholar 

  • OIE Terestrial Manual (2008). Chapter 2.9.4: Cryptosporidiosis. http://www.oie.int/Eng/normes/mmanual/2008/pdf/2.09.04_CRYPTO.pdf

  • Mauzy MJ, Enomoto S, Lancto CA, Abrahamsen MS, Rutherford MS (2012) The Cryptosporidium parvum transcriptome during in vitro development. PLoS ONE 7(3):e31715

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Mirza-Qavami SM, Sadraei J (2011) An examination of Cryptosporidium parvum infection in neonate BALB/c mice and rats. Jundishapur J Microbiol 4(3):185–190

    Google Scholar 

  • Moon HW, Woodmansee DB, Harp JA, Able S, Ungar BLP (1988) Lacteal immunity to enteric cryptosporidiosis in mice: immune dams do not protect their suckling pups. Infect Immun 56:649–653

    CAS  PubMed  PubMed Central  Google Scholar 

  • Morgan UM, Pallant L, Dwyer BW, Forbes DA, Rich G, Thompson RC (1998) Comparison of PCR and microscopy for detection of Cryptosporidium parvum in human faecal specimens: clinical trial. J Clin Microbiol 36:995–998

    CAS  PubMed  PubMed Central  Google Scholar 

  • Oyibo WA, Okangba CC, Obi RK, Nwanebu FC, Ojuromi T (2011) Diagnosis of intestinal Cryptosporidiosis in Africa: prospects and challenges. J Appl Biosci 40:2659–2667

    Google Scholar 

  • Pedraza-Díaz S, Amar C, Nichols GL, McLauchlin J (2001) Nested polymerase chain reaction for amplification of the Cryptosporidium Oocyst wall protein gene. Emerg Infect Dis 7(1):49–56

    Article  PubMed  PubMed Central  Google Scholar 

  • Smith HV (2008) Diagnostics. In: Fayer R, Xiao L (eds) Cryptosporidium and cryptosporidiosis, 2nd edn., 1075 Boca RatonCRC Press and IWA Publishing, Boca Raton, FL, pp 173–208

    Google Scholar 

  • Smith HV, Corcoran GD (2004) New drugs and treatment for cryptosporidiosis. Curr Opin Infect Dis 17:557–564

    CAS  Article  PubMed  Google Scholar 

  • Spano F, Putignani L, McLauchlin J, Casemore D, Crisanti A (1997) 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(2):209–217

    CAS  Article  PubMed  Google Scholar 

  • Tarazona R, Lally NC, Carmona MD, Blewett DA (1997) Characterization of secretory IgA responses in mice infected with Cryptosporidium parvum. Int J Parasitol 27:417–423

    CAS  Article  PubMed  Google Scholar 

  • Tarazona R, Blewett DA, Carmona MD (1998) Cryptosporidium parvum infection in experimentally infected mice: infection dynamics and effect of immunosuppression. Folia Parasitol 45:101–107

    CAS  PubMed  Google Scholar 

  • Tzipori S (1998) Cryptosporidiosis laboratory investigations and chemotherapy. Adv Parasitol 40:24–187

    Google Scholar 

  • Tzipori S, Angus KW, Campbell I, Gray EW (1980) Cryptosporidium: evidence for a single-species genus. Infect Immun 30:884–886

    CAS  PubMed  PubMed Central  Google Scholar 

  • Ungar BL (1990) Enzyme-linked immunoassay for detection of Cryptosporidium antigens in fecal specimens. J Clin Microbiol 28(11):2491–2495

    CAS  PubMed  PubMed Central  Google Scholar 

  • Uppal B, Singh O, Chadha S, Jha AK (2014) A comparison of nested PCR assay with conventional techniques for diagnosis of intestinal cryptosporidiosis in AIDS cases from Northern India. J Parasitol Res 706105:6

    Google Scholar 

  • Van Der Heijden PJ, Bianchi ATJ, Stok W, Bokhout BA (1988) Background (spontaneous) immunoglobulin production in the murine small intestine as a function of age. Immunology 65:243–248

    PubMed  PubMed Central  Google Scholar 

  • Vohra P, Sharma M, Chaudary U (2012) A comprehensive review of diagnostic techniques for detection of Cryptosporidium parvum in stool samples. J Pharm 2(5):15–26

    Google Scholar 

  • Weber RRT, Bryan HS, Bishop SP, Wahlquist JJ, Sullivan DD (1991) Threshold of detection of Cryptosporidium oocysts in human stool specimens: evidence for low sensitivity of current diagnostic methods. J Clin Microbiol 29:1323–1327

    CAS  PubMed  PubMed Central  Google Scholar 

  • Yu JR, Lee SU (2007) Time gap between oocyst shedding and antibody responses in mice infected with Cryptosporidium parvum. Korean J Parasitol 45(3):225–228

    Article  PubMed  PubMed Central  Google Scholar 

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Authors and Affiliations

  1. Medical Parasitology Department, Kasr Al-Ainy School of Medicine, Cairo University, Cairo, 1156, Elmanial, Egypt

    Hebat-Allah S. Yousof, Mona M. Khater & Ayman A. El-Badry

  2. Department of Medical Parasitology, Theodor Bilharz Research Institute, Imbaba, Giza, Egypt

    Shaimaa H. El-Sayed

Authors
  1. Hebat-Allah S. Yousof
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  2. Mona M. Khater
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  3. Shaimaa H. El-Sayed
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  4. Ayman A. El-Badry
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Correspondence to Shaimaa H. El-Sayed.

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All manuscript authors contributed to every activity of it; idea of paper, study design, collection of materials, methodology, writing the paper and revising it.

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Yousof, HA.S., Khater, M.M., El-Sayed, S.H. et al. Diagnostic biomarkers in murine Cryptosporidiosis: dose- and age-related infection. J Parasit Dis 41, 831–836 (2017). https://doi.org/10.1007/s12639-017-0898-2

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  • DOI: https://doi.org/10.1007/s12639-017-0898-2

Keywords

  • Cryptosporidium
  • Diagnostic biomarker
  • Murine
  • Copro-DNA
  • Nested PCR
  • Fecal immunoassay