Cryptosporidium

Davidson, Rebecca K., Amundsen, Hanne, Lie, Nora Oftenes, Luyckx, Katrien, Robertson, Lucy J., Verocai, Guilherme G., Kutz, Susan J. & Ytrehus, BjØrnar, 2014, Sentinels in a climatic outpost: Endoparasites in the introduced muskox (Ovibos moschatus wardi) population of Dovrefjell, Norway, International Journal for Parasitology: Parasites and Wildlife 3 (2), pp. 154-160 : 156

2.4. Analysis of species/genotype for Cryptosporidium and Giardia

Samples were selected for genotyping on the basis of microscopy results; one of the Giardia samples scored ++, and for all samples selected some parasites were nucleated (included DAPI in the nuclei).

The centrifuge pellets of these samples (retained refrigerated) were re-suspended in 1.5 mL water, and transferred to a microcentrifuge tube. The Cryptosporidium oocysts and/or Giardia cysts were isolated by a modified immunomagnetic separation procedure, as previously published ( Robertson et al., 2006), using 15 µL of beads coated with the relevant monoclonal antibody (GC-Combo, Invitrogen). The isolated parasites were then re-suspended in 100 µL Tris–EDTA buffer and placed for one hour in a heat block set at 100 ° C for Cryptosporidium oocysts and 90 ° C for Giardia . DNA was then isolated using a QIAmp DNA mini-kit (QIAGEN GmbH, Germany) following the manufacturer’s protocol.

For Giardia samples, PCR was run at both the glutamate dehydrogenase (gdh) gene (approximately 460 bp) and the ss-giardin gene (approximately 515 bp). For Cryptosporidium isolates, PCR was targeted at the SSU rRNA gene (approximately 800 bp). Published primers and protocols were used with slight modifications ( Xiao et al., 1999, 2nd PCR only of nested protocol; Read et al., 2004; Lalle et al., 2004). PCR amplification products from positive samples were purified (High Pure PCR product purification kit, Roche Applied Science) according to the manufacturer’s protocol, and sequenced on both strands at a commercial facility (Eurofins MWG Operon, Germany). Chromatograms and sequences were examined using Chromas and DNA Baser. Consensus sequences were constructed and compared using BioEdit (http://www.mbio. ncsu.edu/BioEdit/page2.html).

Sequence searches were conducted using BLAST (http://blast. ncbi.nlm.nih.gov/Blast.cgi) and the sequences obtained in this study compared with sequences published in GenBank.