Giardia peramelis

Hillman, Alison, Ash, Amanda, Elliot, Aileen, Lymbery, Alan, Perez, Catherine & Thompson, R. C. Andrew, 2016, Confirmation of a unique species of Giardia, parasitic in the quenda (Isoodon obesulus), International Journal for Parasitology: Parasites and Wildlife 5 (1), pp. 110-115 : 111

2.2. G. peramelis morphological description-trophozoites

Wet mounts were prepared from the small intestinal mucosa of two quenda carcasses, which were positive for G. peramelis on faecal testing (and were not positive for any other species of Giardia ), and were considered sufficiently fresh, with minimal mucosal autolysis, for detection of trophozoites. The first third of the small intestine was gently scraped and the scrapings mounted on a microscope slide. The slides were examined for trophozoites using an Olympus BX50 microscope. A sample of mucosal scrapings from each quenda was also used to seed flasks containing Giardia media (section 2.2, below), and cultures were monitored regularly for the appearance of trophozoites.

Excystation of G. peramelis cysts was attempted three times, using faecal samples from three quenda that were positive for G. peramelis by immunofluorescence microscopy and PCR (and not positive for any other species of Giardia ).

To purify G. peramelis cysts, 1 g of fresh faeces containing G. peramelis was homogenised in 1X PBS, passed through layers of gauze, and centrifuged at 0.6 G. Two wash steps were carried out, where the supernatant was removed, the pellet was resuspended in 1X PBS, and the sample was centrifuged at 0.6 G. Two sucrose samples were made-one to a specific gravity of 1.18, and another made up to 0.8 M. The 0.8 M solution was layered on top of the SG 1.18 solution, and the purified sample was layered on top. The sample was centrifuged at 0.2 G, and cysts were collected at the water/sucrose interphase. 1X PBS was added to this isolation and it was centrifuged at 0.6 G, with the supernatant removed subsequently. The cysts were resuspended in a final volume of 1 ml 1X PBS, and examined under the microscope.

For excystment, cysts were resuspended in 10 mL of acidified Hanks Balanced Salt Solution (HBSS) (30 g /L biosate peptone (BD, Annapolis, USA), 10 g /L glucose (Sigma ‾ Aldrich, St. Louis, USA), 2 g / L sodium chloride (Chem-Supply, Adelaide, Australia), 2 g /L cysteine (Sigma ‾ Aldrich, St. Louis, USA), 1 g /L K 2 HPO 4 (Chem-Supply, Adelaide, Australia), 0.6 g /L KH 2 PO 4 (Merck, Melbourne, Australia), 0.01 g /L ferric ammonium citrate (Sigma ‾ Aldrich, St. Louis, USA), 0.2 g /L ascorbic acid (Sigma ‾ Aldrich, St. Louis, USA), 0.5 g /L bovine bile (Sigma ‾ Aldrich, St. Louis, USA), 100 ml/L newborn calf serum (SAFC Biosciences, Lenexa, USA) and 10 ml/L penicillin/streptomycin (Sigma ‾ Aldrich, St. Louis, USA), pH 2) and incubated at 37 ǫC for 30 min. Cysts were then centrifuged at 0.2 g and washed twice in HBSS (pH7.2), and finally resuspended in HBSS (pH 7.2) medium and incubated at 37 ǫC. Cysts were monitored daily for excystment, for one week.