in vitro culture and isoenzyme analysis of giardia lamblia

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Table 3.3 Summary results of the 3-excystation methods. At least two excystation attempts were made on each sample. AI Tukhi et al Acid Induction* Acid Pepsint 34 34 24 71% 103 43 42% *Hamilton & Jackson, 1990 tBingham & Meyer, 1979 Summary of Excystation Hamilton/Jackson (Bingham/Meyer) AI Tukhi • Total excystment experiments • Successful excystments Fig.3.1 A graphical representation of the summary of in vitro excystation using 3 methods. The total number of trials as well as the number of successful attempts are indicated for each method. It is evident that that the modified Acid Induction method of Hamilton & Jackson resulted in higher levels of successful excystation (overall 71 %) than the Acid Pepsin method (overall 42%). 79
3.4 DISCUSSION Since our source of material for culture initiation was faecally-derived cysts, progress of this work was largely dependent on successful excystation of trophozoites. Several excystment procedures were therefore explored in search of optimal results. It has been acknowledged that none of the available excystment methods work all the time (Nash, 1988). Furthermore, Schaefer (1990) reported large variations in excystation results (2-80%) in his review of available techniques and he concluded that, " Giardia duodenalis do not excyst routinely at high levels with available methods". There are also other investigators of the same opinion (Gordts et al., 1985, Mayrhofer et al., 1992). Similarly, in the current study, varying results were obtained using different excystment methods. In the classic in vitro excystation work of Bingham and Meyer (1979), it was shown that hydrochloric acid alone was capable of inducing excystation and that salts and digestive enzymes did not significantly alter the level of excystation. In our hands, however, 68 replicated attempts at inducing excystation of cysts obtained from both symptomatic and asymptomatic donors by use of Hel only and subsequently incubating in the culture medium (TYI-S-33) as described by AI Tukhi et al. (1991) were unsuccessful. To exclude the possibility of variation in ability of cysts to excyst in vitro, the method was repeated (with no success) on seven samples which had been previously excysted with success using the method of Hamilton and Jackson, (1990). Therefore failure to successfully repeat the method described by AI-Tukhi et al. (1991) could not be explained in terms of an inherent inability of cysts to excyst in vitro. When salts and trypsin were incorporated into the induction medium (modified 80
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IN VITRO CULTURE AND ISOENZYME ANAL
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(j)edicated to my {ate parents :No
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the positive specimens by sucrose g
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Declaration This study represents o
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ACKNOWLEDGEMENTS The author wishes
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Table of Contents Page CHAPTER 1 -
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4.2.1. Animal Sourcing and Care ...
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APPENDIX 8 List of enzyme systems u
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List of Tables Page Table 1.1 Commo
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List of Plates Page Plate 2.1 An eo
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ABBREVIATIONS AND UNITS OF MEASUREM
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agi/is in 1882. In 1914, Alexeieff
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unique about Giardia in humans, whi
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To-date, use of the different speci
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Differentiation into the cyst stage
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1.3 Transmission 1.3.1 Human hosts
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Owen, 1984) 1.3. 2 The Zoonosis of
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1.4 Distribution The morbidity and
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children by Schutte et al. in 1981
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of (a) the enormous absorptive capa
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Ig M (100% of patients), IgG (70%),
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chymotrypsin) between different iso
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impact on the physical growth of 29
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Figure 1.3. Transmission Electron m
Page 49 and 50: among children between the ages of
Page 51 and 52: where immunocompromised mice have p
Page 53 and 54: up to 4 distinct genotypes within e
Page 55 and 56: in initiation of infection (excysta
Page 57 and 58: In order to address these and many
Page 59 and 60: multiple stool examinations in 4 of
Page 61 and 62: 2.2 MATERIALS AND METHODS 2.2.1 Spe
Page 63 and 64: Table 2.1. Semi-quantitative assess
Page 65 and 66: 2.2.3.2 The modified Zinc sulphate
Page 67 and 68: Preliminary work with five samples
Page 69 and 70: % Viability = unstained cysts/total
Page 71 and 72: Table 2.3. Prevalence of Giardia in
Page 73 and 74: Table 2. 4. Monthly summary of stoo
Page 75 and 76: spp (59%). Other parasites less fre
Page 77 and 78: Observations • A large proportion
Page 79 and 80: Plate 2.1. An eosin-stained prepara
Page 81 and 82: 2.4 DISCUSSION In terms of yield, i
Page 83 and 84: examination of wet preparations. Th
Page 85 and 86: ecognised (on unstained preparation
Page 87 and 88: cultivation in vitro (Kasprzak & Ma
Page 89 and 90: 3.2 MATERIALS AND METHODS Three exc
Page 91 and 92: 3.2.3 The modified Acid Pepsin meth
Page 93 and 94: • After 15 minutes incubation: Ma
Page 95 and 96: Plate 3.2 (b) A completely excysted
Page 97 and 98: Twenty-four batches of cysts (71 %
Page 99: 3.3.3 The modified Acid Pepsin excy
Page 103 and 104: an unreliable indicator of viabilit
Page 105 and 106: propagating Giardia for subsequent
Page 107 and 108: 4.2 MATERIALS AND METHODS 4.2.1. An
Page 109 and 110: A litter comprising of 7 C57BU6 mic
Page 111 and 112: for attached trophozoites and maint
Page 113 and 114: days pi. However, accurate quantifi
Page 115 and 116: Table 4.4. Summary of the inoculati
Page 117 and 118: not be infected in comparison with
Page 119 and 120: A proportionately higher percentage
Page 121 and 122: These findings strongly suggest tha
Page 123 and 124: CHAPTERS IN VITRO CULTURE OF GIARDI
Page 125 and 126: Diamond et al.'s (1978) medium. He
Page 127 and 128: Majewska, 1985) United States (Nash
Page 129 and 130: were evaluated. 5,2,1,2 Biosate Bio
Page 131 and 132: Table 5.1 Outlines a relative semi-
Page 133 and 134: dislodged by chilling the culture t
Page 135 and 136: Table 5.2. Sensitivity to Ciproflox
Page 137 and 138: Table 5.3. Summary results of in vi
Page 139 and 140: Once established in culture for a l
Page 141 and 142: Plates 5.3 and 5.4 Confluent growth
Page 143 and 144: difficult to produce viable culture
Page 145 and 146: dominance of certain genotypes unde
Page 147 and 148: Cryopreservation and successful ret
Page 149 and 150: adjusted to 5°C per minute from am
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6.3 RESULTS Trophozoites have been
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Table 6.1. A longevity record of sa
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Diamond (1995) stated that an impor
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into two categories. One group cont
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technique to the first local (South
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for Giardia Iysates. These included
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4. To exclude the presence of bacte
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Plate 7.1 Banding pattern of 8 seri
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PGM activity was displayed by Giard
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Plate 7.6. Hexokinase Lane 1. Unino
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Plate 7.8. Phosphogluconate dehydro
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stably expressed between different
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techniques, which would allow diffe
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Belosevic M, Faubert GM, MacLean JD
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Farthing MJG, Mata L, Urrutia JJ, K
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Heidelberg pp.397-398. Hiatt RA, Ma
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patients with X-linked agammaglobul
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Press, Calgary. pp 57-58. Nash TE,
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of Entamoeba histolytica in a group
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Wolfe M. S.1979. Giardiasis. Sympos
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APPENDICES APPENDIX 1 Information t
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Unayo imvume yokunqaba ukungenela l
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Appendix 3 Excystation and culture
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Appendix 4 Results of excystation a
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Appendix 5 Results of in vitro (aci
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Key to Appendix 5 *Replicated inocu
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Appendix 7 Preparation of culture m
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Appendix 8 List of enzyme systems u
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Appendix 9 Appendix (9a) Preparatio
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Appendix 10. Quantitative descripti
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