Cryptosporidium: Identification and Genetic Typing

Jan Šlapeta1

1 Sydney School of Veterinary Science and School of Life and Environmental Sciences, Faculty of Science, University of Sydney, New South Wales
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 20B.1
DOI:  10.1002/cpmc.24
Online Posting Date:  February, 2017
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Abstract

Cryptosporidium spp. are obligate protozoan parasites of the gastrointestinal tract of vertebrates, including humans. In the majority of human cases, the diarrheal disease cryptosporidiosis is caused by either the human‐adapted species Cryptosporidium hominis or the zoonotic Cryptosporidium parvum ‘bovine genotype’ (also known as Cryptosporidium pestis). The infectious stage, environmentally resilient Cryptosporidium oocysts, are shed by the infected host. Cryptosporidium parasites are transmitted by the fecal‐oral route and are one of the major water‐borne pathogens. The cryptic nature of the microscopic Cryptosporidium oocysts coupled with the existence of several host‐adapted and zoonotic species requires molecular tools to identify Cryptosporidium spp. in either fecal or environmental samples. This unit describes methods for Cryptosporidium identification and typing using genotyping based on nuclear loci. We also provide a protocol for morphological confirmation of Cryptosporidium oocysts based on antibody labeling of the Cryptosporidium oocyst wall and a protocol for purification of oocysts from fecal material. © 2017 by John Wiley & Sons, Inc.

Keywords: Cryptosporidium; genotyping; identification; oocyst; PCR

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: PCR‐Based and Molecular Phylogenetic Identification of Cryptosporidium at the 18S rRNA Gene and Cryptosporidium Oocyst Wall Protein 1
  • Alternate Protocol 1: Alternate Nested PCR Amplification of the Cryptosporidium 18S rRNA Gene
  • Support Protocol 1: Cryptosporidium Oocyst Isolation and Purification
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: PCR‐Based and Molecular Phylogenetic Identification of Cryptosporidium at the 18S rRNA Gene and Cryptosporidium Oocyst Wall Protein 1

  Materials
  • Fecal samples (5 g) from young dairy calf (<2 weeks old)
  • MeriFluor Cryptosporidium/Giardia kit (Meridian Bioscience)
  • PowerFecal DNA Isolation Kit (Mo‐Bio), FastDNA SPIN Kit for Feces (MP Biomedicals), or ZR Fecal DNA MiniPrep (Zymo Research)
  • Tris⋅Cl, pH 8.5 ( appendix 2A)
  • MyTaq Red Mix (BioLine) or GoTaq Green Master Mix (Promega)
  • Amplification primers (10 pmol/μl or 10 μM each; Table 20.1.1)
  • Sterile double‐distilled water (PCR‐grade)
  • PCR purification kit (such as the QIAquick PCR Purification Kit; Qiagen)
  • Fluorescent microscope equipped with a filter system for fluorescein isothiocyanate (FITC) with the following parameters: excitation wavelength, 490 to 500 nm; barrier filter: 510 to 530 nm
  • Set of repeat pipettors (0.5 to 2 μl, 2 to 20 μl, 20 to 200 μl) with respective sterile filtered pipet tips
  • Fast Prep‐24 (MP Biomedicals) or benchtop vortex with the Vortex Adapter for Vortex‐Genie 2 (Mo‐Bio, cat. no. 13000‐V1‐24)
  • NanoDrop ND‐1000 spectrophotometer (Thermo Fisher Scientific)
  • Thermal cycler (Veriti 96‐well Thermal Cycler, Applied Biosystems; T100 Thermal Cycler, BioRad)
  • 200‐μl thin‐walled PCR tubes (Invitrogen)
  • DNA sequencing facility (also see Ausubel et al., , Chapter 7)
  • Additional reagents and equipment for agarose gel electrophoresis (Voytas, ) and DNA sequencing (Ausubel et al., , Chapter 7)
Table 0.0.1   MaterialsAmplification Primers for Small Subunit rRNA gene (18S rDNA) and Cryptosporidium Oocyst Wall Protein 1 (COWP1) Markers

Target marker gene Name (5′>3′ sequence) a Product size Reference
18S rDNA F2 (TTC TAG AGC TAA TAC ATG CG) ∼1,350 bp Xiao et al. ( )
18S rDNA RX (CCC ATT TCC TTC GAA ACA GGA) b ∼1,350 bp Xiao et al. ( )
18S rDNA F3N (GGA AGG GTT GTA TTT ATT AGA TAA AG) ∼850 bp Xiao et al. ( )
18S rDNA R3N (AAG GAG TAA GGA ACA ACC TCC A) ∼850 bp Xiao et al. ( )
COWP1 Cry‐9 (GGA CTG AAA TAC AGG CAT TAT CTT G) ∼550 bp Spano et al. ( )
COWP1 Cry‐15 (GTA GAT AAT GGA AGA GAT TGT) ∼550 bp Spano et al. ( )

 aSee Figure .
 bNote that RX primer is an improved version of the original R2 primer (Xiao et al., ).

Alternate Protocol 1: Alternate Nested PCR Amplification of the Cryptosporidium 18S rRNA Gene

  Additional Materials (also see protocol 1Basic Protocol)
  • Amplification primers (10 pmol/μl or 10 μM each, Table 20.1.2)
Table 0.0.2   Additional Materials (also see protocol 1Basic Protocol)Amplification Primers for Small Subunit rRNA Gene (18S rDNA) Marker

Target marker gene Name (5′>3′ sequence) aa Product size Reference
18S rDNA 18SiCF2 (GAC ATA TCA TTC AAG TTT CTG ACC) ∼800 bp Ryan et al.,
18S rDNA 18SiCR2 (CTG AAG GAG TAA GGA ACA ACC) ∼800 bp Ryan et al.,
18S rDNA 18SiCF1 (CCT ATC AGC TTT AGA CGG TAG G) ∼600 bp Ryan et al.,
18S rDNA 18SiCR1 (TCT AAG AAT TTC ACC TCT GAC TG) ∼600 bp Ryan et al.,

 aSee Figure .

Support Protocol 1: Cryptosporidium Oocyst Isolation and Purification

  Materials
  • Fecal material
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Saturated NaCl (see recipe)
  • 2% potassium dichromate (see recipe; optional)
  • 10‐ and 50‐ml centrifuge tubes
  • Falcon cell strainers (100 µm)
  • Bench‐top refrigerated centrifuge with a swing‐out rotor for 50‐ml tubes (Eppendorf 5810R, swinging‐bucket rotor for 50‐ml tubes)
  • Vacuum pump (i.e., faucet vacuum pump)
  • Disposable, sterile 1 ml Pasteur pipet
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Figures

Videos

Literature Cited

Literature Cited
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