in vitro culture and isoenzyme analysis of giardia lamblia

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Plate 7.5. Malic enzyme Lane1 . Uninoculated TVI. Lane 2. SA305. Lanes 3-4. E. histo/ytica group ii and xi Lanes 5-11. H7, WB, SAS, 7, 18, 24 and 29. Similar Giardia enzymes (arrowhead) migrated slower than those of the Entamoeba controls (arrow). Entamoeba histo/ytica demonstrated typical HK activity. Marked heterogeneity was observed in Giardia isolates in which 3 isolates (WB, H7 and SAS) showed double bands of different mobilities for each isolate and 3 isolates displayed indistinct single bands of at least 2 different mobilities (i) (SA7, SA24 and SA305); (ii) (SA18 and SA29). The isoenzymes of all Giardia isolates migrated farther than those of E. histo/ytica (Plate 7.S). 147
Plate 7.6. Hexokinase Lane 1. Uninoculated TVI. Lane 2. E. histolytica control. Lanes 3-11. WB, H?, SA6, SA?, SA18, SA24, SA29 SA305a and SA305b, SA305c. 305a =54 x subcultured; 306b =111 subcultures, SA305c=216 subcultures. Consistent bands in all 3 lanes. Distinct double bands(3 different patterns) were consistently exhibited by 3 Giardia isolates WB, H? and SA6 (arrow-heads), indistinct single bands were seen in isolates SA18, 24 and 29 (big arrows) while very poor band resolution was seen for isolates SA? and 305 (small arrows). All 3 isolates with single bands had a different pattern. Shorter migration for E. histolytica resulted from the shorter run time and Giardia enzymes migrated farther. InG6PD 4 electrophoretic patterns were observed; in the fist pattern, WB isolate migrated farther than the rest of the isolates, in the second, SA6SA 18, SA24 and SA305 showed similar migration while in the third, H? and SA? migrated slowest. One isolate (SA29) showed double banding in the fourth pattern. Intense bands were observed in all but one isolate (SA305), seen in Plate?? 148
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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
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among children between the ages of
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where immunocompromised mice have p
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up to 4 distinct genotypes within e
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in initiation of infection (excysta
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In order to address these and many
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multiple stool examinations in 4 of
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2.2 MATERIALS AND METHODS 2.2.1 Spe
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Table 2.1. Semi-quantitative assess
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2.2.3.2 The modified Zinc sulphate
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Preliminary work with five samples
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% Viability = unstained cysts/total
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Table 2.3. Prevalence of Giardia in
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Table 2. 4. Monthly summary of stoo
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spp (59%). Other parasites less fre
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Observations • A large proportion
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Plate 2.1. An eosin-stained prepara
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2.4 DISCUSSION In terms of yield, i
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examination of wet preparations. Th
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ecognised (on unstained preparation
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cultivation in vitro (Kasprzak & Ma
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3.2 MATERIALS AND METHODS Three exc
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3.2.3 The modified Acid Pepsin meth
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• After 15 minutes incubation: Ma
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Plate 3.2 (b) A completely excysted
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Twenty-four batches of cysts (71 %
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3.3.3 The modified Acid Pepsin excy
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3.4 DISCUSSION Since our source of
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an unreliable indicator of viabilit
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propagating Giardia for subsequent
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4.2 MATERIALS AND METHODS 4.2.1. An
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A litter comprising of 7 C57BU6 mic
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for attached trophozoites and maint
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days pi. However, accurate quantifi
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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
Page 151 and 152: 6.3 RESULTS Trophozoites have been
Page 153 and 154: Table 6.1. A longevity record of sa
Page 155 and 156: Diamond (1995) stated that an impor
Page 157 and 158: into two categories. One group cont
Page 159 and 160: technique to the first local (South
Page 161 and 162: for Giardia Iysates. These included
Page 163 and 164: 4. To exclude the presence of bacte
Page 165 and 166: Plate 7.1 Banding pattern of 8 seri
Page 167: PGM activity was displayed by Giard
Page 171 and 172: Plate 7.8. Phosphogluconate dehydro
Page 173 and 174: stably expressed between different
Page 175 and 176: techniques, which would allow diffe
Page 177 and 178: Belosevic M, Faubert GM, MacLean JD
Page 179 and 180: Farthing MJG, Mata L, Urrutia JJ, K
Page 181 and 182: Heidelberg pp.397-398. Hiatt RA, Ma
Page 183 and 184: patients with X-linked agammaglobul
Page 185 and 186: Press, Calgary. pp 57-58. Nash TE,
Page 187 and 188: of Entamoeba histolytica in a group
Page 189 and 190: Wolfe M. S.1979. Giardiasis. Sympos
Page 191 and 192: APPENDICES APPENDIX 1 Information t
Page 193 and 194: Unayo imvume yokunqaba ukungenela l
Page 195 and 196: Appendix 3 Excystation and culture
Page 197 and 198: Appendix 4 Results of excystation a
Page 199 and 200: Appendix 5 Results of in vitro (aci
Page 201 and 202: Key to Appendix 5 *Replicated inocu
Page 203 and 204: Appendix 7 Preparation of culture m
Page 205 and 206: Appendix 8 List of enzyme systems u
Page 207 and 208: Appendix 9 Appendix (9a) Preparatio
Page 209: Appendix 10. Quantitative descripti
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