in vitro culture and isoenzyme analysis of giardia lamblia

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SA24 , SA29 and SA305. Table 5.4. Culture results using in vivo excysted trophozoites as inoculum. Thirty percent of the culture initiates were lost through contamination, while 40% failed to establish cultures. ExcystationlT otal inoculations Contaminated No contamination but died out *No contaminationsurvived 20/53 (38%) 6/20 (30%) 8/20(40%) 6/20 (30%) *6 isolates axenised (30% success). 5.4 Observations Behaviour of trophozoites in vitro For fresh isolates, during the initial period of culture, the trophozoites adhered to the bottom of the culture tube mostly as individual cells without signs of division (Plate 5.1). This inert period varied with different isolates (from 10 to 15days) and could be as long as twenty days in some instances after which they showed signs of division (Plate 5.2). However, as the organisms became adapted to the culture environment, this period became shorter. When the trophozoites are continuously passaged they can be seen dividing by day three after subculture. A confluent monolayer of cells was seen by day 18 in some isolates (Plates 5.3 and 5.4) However one isolate took 32 days to start dividing; the concentration of fetal bovine serum was increased from 10 to 15% on day 24 and confluent growth was reached after 55 days. Interestingly, some trophozoites continually swam around, never adhering to the surface of the tube. 117
Once established in culture for a long time, the trophozoites became highly resilient. Some trophozoites survived de&pite long periods without medium replacement. Two tubes of the reference strain (W8) were kept in the 35,5°C incubator for 10 and 45 days respectively without changing the medium and later took to culture. The local strains have also survived up to 96 hours without medium changes. In these cases, although many were rounded up and detached from the glass surface, plenty were still adhering and they continued to grow well after replenishment with fresh medium. 118
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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
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 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: Table 5.3. Summary results of in vi
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 and 168: PGM activity was displayed by Giard
Page 169 and 170: Plate 7.6. Hexokinase Lane 1. Unino
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
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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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