Viability of Giardia intestinalis cysts : assessing viability under environmental conditions : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University, Palmerston North, New Zealand

Abstract

Much work has been put into the detection and monitoring of Giardia, but once found, it is not easy to tell whether the cysts are viable and thus infective. There are fluorescently labelled monoclonal antibody kits which can be used to identify Giardia, but are the Giardia cysts viable? Excystation has been the main method used to determine the viability of cysts. This is quite unreliable as varying excystation conditions seem to be required for different strains of cysts. Using samples of fresh cysts, certain batches consistently measured 80-95% viable, while others resulted in viability measurements of 0-10%. The cysts themselves displayed the normal morphology of viable cysts. The assumption that partially excysted trophozoites as well as completely excysted trophozoites are viable may also lead to over-estimation of viable cyst numbers. Another commonly used method for estimating the viability of Giardia is staining with vital dyes, in particular the combination of fluorescein diacetate (FDA) and propidium iodide (PI). These also gave unexpected results where none of the cysts in a fresh sample stained with FDA, which usually stains viable cysts. An alternative dye, 4',6-diamidino-2-phenylindole (DAPI) was used in the place of FDA. The combination of DAPI and PI showed viabilities of 85.7% for cyst samples. This correlated well with 88% viability using excystation. Using the DAPI/PI combination, the viability of G. intestinalis cysts over time was monitored under different temperature conditions, and in sea water. Temperature was quite significant in the viability of the cysts – cysts stored at 4°C remained viable for 62 days, while those stored at 25°C were non-viable after 5 days. Sea-water had an immediately lethal effect on the G. intestinalis cysts, with all cysts non-viable after 45 minutes. Giardia intestinalis trophozoites can be cultured in the laboratory. By the addition of bile to the growth media, it is possible to transform these into cysts. Over the course of four days in encystation media, a large proportion of the trophozoites in the culture were converted into cysts, 3.5 X 10⁵ cysts/ml from an initial trophozoite concentration of 7.2 X 10⁵ organisms/ml. However, the cysts generated from the strains of G. intestinalis used were completely non-viable, compared with viability rates for fresh in vivo cysts of 80-95%. A population of hamsters was found to be carrying a Giardia which seemed different to recognised species. An analysis was carried out by PCR and sequencing of sections of the ribosomal DNA of this Giardia. Through this it was found to be closely related to Giardia muris, but perhaps not as closely related as to be a species of G. muris, possibly a sub-species. The rDNA analysis used may be very useful in typing other strains and species of Giardia