Giardia intestinalis : aerobic metabolism and physiology of in vitro growth : a thesis presented in partial fulfillment of the requirements for the degree of Master of Science in microbiology at Massey University, Palmerston North, New Zealand

Abstract

G. intestinalis; the causative agent of giardiasis, parasitises a number of vertebrates including man; and has a worldwide distribution. Although giardiasis is now a widely recognised public health concern; little is known of its aetiological agent. This is primarily due to the fact that a protocol for the routine axenic cultivation of this intestinal parasite was not available until 1976. With the advent of in vitro cultivation, an increasing number of reports have outlined the in vitro growth requirements of G. intestinalis; however; the physiology and unique metabolism of this protozoan still require further clarification. Utilising two strains of G. intestinalis (Bris/83/HEPU/106 and Hast/87/MUGU/68), the influences of envirornnental factors such as pH and temperature on axenic culture growth were investigated. Variations in both temperature and pH were shown to effect the in vitro growth rate of the two strains examined. Growth of Bris/83/HEFU/106 was markedly impaired at non-optimal temperature; (optimal growth of Bris/83/HEPU/106 and Hast/87/MUGU/68 occurred at 37°c); while growth of Hast/87/MUGU/68 continued, at a reduced rate, over a wider, non-optimal temperature range (30-40°c). Both strains exhibited marked pH optima for culture growth (pH 6. 75-7.50) with a rapid decline in culture growth rates outside these pH levels. Clonal growth of G. intestinalis trophozoites in semi-solid agarose has been utilised in the past as an assay of trophozoite viability in vitro. The suitability of such an assay for use during this study was investigated for both Bris/83/HEPU/106 and Hast/87/MUGU/68. Over the range of agarose concentrations examined, the colony fonning efficiency (CFE) of both strains was extremely variable. While Hast/87jMUGU/68 was better adapted to growth in agarose medium, with CFE of up to 60% recorded; these rates of clonal growth were often not reproducible, as the growth of trophozoite colonies remained inconsistent despite duplication of all assays. The thiol reducing agent L-cysteine, has been reported to be a specific growth requirement of G. intestinalis in vitro. The correlation between reducing conditions and the growth and attachment of Bris/83/HEPU/106 and Hast/87jMUGU/68 in culture, was investigated as trophozoites were exposed to a range of L-cysteine concentrations in TYl-S-33 growth medium. Enhanced growth of experimental cultures was directly related to increases in L­ cysteine concentration and corresponding decreases in the 0-R Potential of growth medium. Culture growth occurred at a maximal rate where the concentration of L-cysteine in growth medium exceeded 0.15% w/v. All cultures failed to grow in the absence of L­ cysteine. Trophozoite attachment in culture was most rapid during the 30-90 minutes following culture establishment. Under elevated L-cysteine concentrations (0.15-0.25% w/v) this attachment reached maximal levels (85-95%). In the absence of L-cysteine, attachment of trophozoites in culture continued, but at a markedly reduced rate. The oxygen sensitivity of G. intestinalis trophozoites was investigated in TYl-S-33 utilising a protocol developed during this study, where the exposure of trophozoites to dissolved oxygen was directly controlled through adjustment of oxygen flow into growth medium. Bris/83/HEPU/106 and Hast/87jMUGU/68 trophozoites displayed a similar degree of oxygen sensitivity at 37°c. A slow decline in culture viability was recorded upon exposure of trophozoites to 4.0- 6.0 ppm dissolved oxygen in growth medium. At 8.0 ppm; exponential killing of trophozoites was preceded by a 'lag phase' of 3-4 hours duration. In contrast; the killing of cultures commenced almost immediately after exposure of trophozoites to 12.0 ppm dissolved oxygen. At temperatures below 37°c (20°c and 3o0 C) , Bris/83/HEPU/106 exhibited a reduced sensitivity to elevated dissolved oxygen levels in TYl-S-33, as both the T1/2 of killing, and the lag phases preceding this killing were extended. The basis for the observed 'temperature-dependant' oxygen sensitivity of G. intestinalis is not known. Oxygen consumption by G. intestinalis has recently been reported by several workers; however; there is still very little known of the metabolic role of 'active respiration' in this 'aerotolerant anaerobe'. Consumption of oxygen by Hast/87jMUGU/68 in PBS was demonstrated. using a Model 97-08 Oxygen Electrode. Dissolved oxygen was removed from PBS by trophozoites at a rate of 3.2-5.3 10- 9 ppm/cell/hr. This oxygen consumption was inhibited. up to 50% by the flavoantagonist, Quinacrine dihydrochloride, at concentrations of 250-1000 µg/ml in PBS solution. Tbe concentrations of Quinacrine which were inhibitory to oxygen consumption by trophozoites over a 5 hour period were well in excess of the Quinacrine MLC (Minimum Lethal Concentration)