in vitro culture and isoenzyme analysis of giardia lamblia

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Plates 3.3.(a) and (b) Excysted trophozoite dividing to form daughter trophozoites joined at the posterior end. Bacteria are seen in the background. (400x) 75
Twenty-four batches of cysts (71 % of 34 excystation trials) excysted successfully with this method. The overall percent excystation ranged between 2 and 41% and viability ranged between 30 and 100% by the eosin dye exclusion method. Addition of Glutathione to the induction medium did not improve the level of excystation (which remained below 40% in all but one of the excysted samples). The results of successfully excysted samples (expressed in percentages), their viabilities as determined by dye-exclusion and total counts are summarised in Table 3.1. This data was extracted from the full list of all attempted excystations in Appendix 3. The eosin exclusion method showed higher viability levels (with a mean percent viability of 75) whilst the mean excystation value was 8,9%. In most experiments, low levels of excystation were attained. Only in 3 of the 25 successful excystations were the levels of excystation above 30%, the rest were below 20%. The method described by AI Tukhi et al. (1991) (3.2.1) was attempted on seven samples that had been successfully excysted previously using the Hamilton & Jackson (1990) method. These cyst samples did not excyst using the former method. 76
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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
Page 45 and 46: impact on the physical growth of 29
Page 47 and 48: 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: Plate 3.2 (b) A completely excysted
Page 99 and 100: 3.3.3 The modified Acid Pepsin excy
Page 101 and 102: 3.4 DISCUSSION Since our source of
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
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Cryopreservation and successful ret
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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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