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Use of Markers to Determine Cryptosporidium Genotypes for Epidemiology Tracking and Detection

  • Anson V. KoehlerEmail author
  • Jan ŠlapetaEmail author
Protocol
  • 339 Downloads
Part of the Methods in Molecular Biology book series (MIMB, volume 2052)

Abstract

Polymerase chain reaction (PCR) enables amplification of specific DNA fragments for the detection and tracking of Cryptosporidium spp. Newly obtained DNA are compared to an ever-growing database of Cryptosporidium sequences with uncertain or outdated metadata in primary public repositories (i.e., EMBL/DDBJ/GenBank). Here, we describe standard operating procedures to obtain DNA sequences from Cryptosporidium spp. marker genes. Small-subunit ribosomal RNA gene, large-subunit ribosomal RNA gene, and glycoprotein 60 are amplified using conventional PCR. Amplified and sequenced genes are compared to a reference library of up-to-date curated gene sequences to identify Cryptosporidium species and variants.

Keywords

PCR DNA Genotyping LSU SSU gp60 Cryptosporidium 

References

  1. 1.
    Checkley W, White AC Jr, Jaganath D, Arrowood MJ, Chalmers RM, Chen XM, Fayer R, Griffiths JK, Guerrant RL, Hedstrom L, Huston CD, Kotloff KL, Kang G, Mead JR, Miller M, Petri WA Jr, Priest JW, Roos DS, Striepen B, Thompson RC, Ward HD, Van Voorhis WA, Xiao L, Zhu G, Houpt ER (2015) A review of the global burden, novel diagnostics, therapeutics, and vaccine targets for Cryptosporidium. Lancet Infect Dis 15(1):85–94.  https://doi.org/10.1016/S1473-3099(14)70772-8CrossRefPubMedGoogle Scholar
  2. 2.
    Zahedi A, Paparini A, Jian F, Robertson I, Ryan U (2016) Public health significance of zoonotic Cryptosporidium species in wildlife: critical insights into better drinking water management. Int J Parasitol Parasit Wildl 5(1):88–109.  https://doi.org/10.1016/j.ijppaw.2015.12.001CrossRefGoogle Scholar
  3. 3.
    Ryan U, Zahedi A, Paparini A (2016) Cryptosporidium in humans and animals-a one health approach to prophylaxis. Parasite Immunol 38(9):535–547.  https://doi.org/10.1111/pim.12350CrossRefPubMedGoogle Scholar
  4. 4.
    Šlapeta J (2013) Cryptosporidiosis and Cryptosporidium species in animals and humans: a thirty colour rainbow? Int J Parasitol 43(12–13):957–970.  https://doi.org/10.1016/j.ijpara.2013.07.005CrossRefPubMedGoogle Scholar
  5. 5.
    Šlapeta J (2018) DNA barcoding of Cryptosporidium. Parasitology 145(5):574–584.  https://doi.org/10.1017/S0031182017001809CrossRefPubMedGoogle Scholar
  6. 6.
    Xiao L, Morgan UM, Limor J, Escalante A, Arrowood M, Shulaw W, Thompson RC, Fayer R, Lal AA (1999) Genetic diversity within Cryptosporidium parvum and related Cryptosporidium species. Appl Environ Microbiol 65(8):3386–3391CrossRefGoogle Scholar
  7. 7.
    Koehler AV, Korhonen PK, Hall RS, Young ND, Wang T, Haydon SR, Gasser RB (2017) Use of a bioinformatic-assisted primer design strategy to establish a new nested PCR-based method for Cryptosporidium. Parasit Vectors 10(1):509.  https://doi.org/10.1186/s13071-017-2462-4CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Alves M, Xiao L, Sulaiman I, Lal AA, Matos O, Antunes F (2003) Subgenotype analysis of Cryptosporidium isolates from humans, cattle, and zoo ruminants in Portugal. J Clin Microbiol 41(6):2744–2747CrossRefGoogle Scholar
  9. 9.
    Power ML, Cheung-Kwok-Sang C, Slade M, Williamson S (2009) Cryptosporidium fayeri: diversity within the GP60 locus of isolates from different marsupial hosts. Exp Parasitol 121(3):219–223.  https://doi.org/10.1016/j.exppara.2008.10.016CrossRefPubMedGoogle Scholar
  10. 10.
    Stensvold CR, Beser J, Axen C, Lebbad M (2014) High applicability of a novel method for gp60-based subtyping of Cryptosporidium meleagridis. J Clin Microbiol 52(7):2311–2319.  https://doi.org/10.1128/JCM.00598-14CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Li N, Xiao L, Alderisio K, Elwin K, Cebelinski E, Chalmers R, Santin M, Fayer R, Kvac M, Ryan U, Sak B, Stanko M, Guo Y, Wang L, Zhang L, Cai J, Roellig D, Feng Y (2014) Subtyping Cryptosporidium ubiquitum, a zoonotic pathogen emerging in humans. Emerg Infect Dis 20(2):217–224.  https://doi.org/10.3201/eid2002.121797CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Stensvold CR, Elwin K, Winiecka-Krusnell J, Chalmers RM, Xiao L, Lebbad M (2015) Development and application of a gp60-based typing assay for Cryptosporidium viatorum. J Clin Microbiol 53(6):1891–1897.  https://doi.org/10.1128/JCM.00313-15CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Guo Y, Cebelinski E, Matusevich C, Alderisio KA, Lebbad M, McEvoy J, Roellig DM, Yang C, Feng Y, Xiao L (2015) Subtyping novel zoonotic pathogen Cryptosporidium chipmunk genotype I. J Clin Microbiol 53(5):1648–1654.  https://doi.org/10.1128/JCM.03436-14CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Yan W, Alderisio K, Roellig DM, Elwin K, Chalmers RM, Yang F, Wang Y, Feng Y, Xiao L (2017) Subtype analysis of zoonotic pathogen Cryptosporidium skunk genotype. Infect Genet Evol 55:20–25.  https://doi.org/10.1016/j.meegid.2017.08.023CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Xiao L, Bern C, Limor J, Sulaiman I, Roberts J, Checkley W, Cabrera L, Gilman RH, Lal AA (2001) Identification of 5 types of Cryptosporidium parasites in children in Lima, Peru. J Infect Dis 183(3):492–497.  https://doi.org/10.1086/318090CrossRefPubMedGoogle Scholar
  16. 16.
    Kumar S, Stecher G, Tamura K (2016) MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33(7):1870–1874.  https://doi.org/10.1093/molbev/msw054CrossRefPubMedGoogle Scholar
  17. 17.
    Ryan U, Xiao L, Read C, Zhou L, Lal AA, Pavlasek I (2003) Identification of novel Cryptosporidium genotypes from the Czech Republic. Appl Environ Microbiol 69(7):4302–4307CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Department of Veterinary Biosciences, Melbourne Veterinary School, Faculty of Veterinary and Agricultural SciencesThe University of MelbourneParkvilleAustralia
  2. 2.Sydney School of Veterinary Science, Faculty of ScienceThe University of SydneySydneyAustralia

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