in vitro culture and isoenzyme analysis of giardia lamblia

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pathogenesis. Malabsorption of electrolytes, solutes and water in the upper small intestine appears to be the primary mechanism of diarrhoea production in giardiasis (Buret, 1994). As pathogenesis depends on successful establishment of infection, adherence has been implicated as an indirect contributing factor. Trypsin-activated lectins have been documented to play a role in parasite adherence to the mucosa (Lev et al., 1986; Adam, 1991; Nash et al., 1991). Inge et al., 1988 showed that the parasite lectins may be required for the selective establishment of Giardia infection in the jejunal epithelia rather than colonic cells (a predilection for the host's upper small intestine). Vast reduction of the villus to crypt ratio (Fig.1.3) has been revealed by transmission electron microscopy in animal giardiasis (Roberts-Thompson et al. 1976). Disruption of the brush border in the small intestine, crypt epithelial hyperplasia and mononuclear inflammatory cell infiltration have been documented. The latter is proposed to contribute both to the disease process as well as in parasite eradication (Farthing, 1989; Owen et al. 1979). Sometimes the parasites reach the gall bladder and bile ducts and cause jaundice. 25
Figure 1.3. Transmission Electron micrograph of the small intestine in murine giardiasis (right) compared to the normal jejunum of an uninfected control animal (left) (Roberts-Thompson, et al., 1976). (x 160) (Courtesy of A.Mahmoud) Smith et al., (1982) tested four strains of Giardia from humans for toxin production in rabbits and found that these organisms did not produce toxins. To date, no reports showing evidence for toxin production have been published. However, it was suggested that Giardia excretory-secretory products may play a role in the pathogenesis of giardiasis, either as a trigger for the diffuse brush border injury, or via direct cytopathogenetic effects during close contacts between host cells and trophozoites, and this needs to be investigated (Buret, 1994). Lysosome-like vacuoles that line the surface of trophozoites have been identified (Feely et al., 1984). These organelles have been shown to release numerous hydrolases, including a thiol-dependant proteinase (Parenti, 1989). A possibility of secretion of the proteinase has been indicated, although there is only limited evidence in support of this. It was further suggested that under selected conditions, the proteinase might be released into the extracellular milieu and play a role in the pathogenesis of diarrhoeal disease (Parenti, 1989). The association of giardiasis and allergic reactions have been documented. 26
Page 1 and 2: IN VITRO CULTURE AND ISOENZYME ANAL
Page 3 and 4: (j)edicated to my {ate parents :No
Page 5 and 6: the positive specimens by sucrose g
Page 7 and 8: Declaration This study represents o
Page 9 and 10: ACKNOWLEDGEMENTS The author wishes
Page 11 and 12: Table of Contents Page CHAPTER 1 -
Page 13 and 14: 4.2.1. Animal Sourcing and Care ...
Page 15 and 16: APPENDIX 8 List of enzyme systems u
Page 17 and 18: List of Tables Page Table 1.1 Commo
Page 19 and 20: List of Plates Page Plate 2.1 An eo
Page 21 and 22: ABBREVIATIONS AND UNITS OF MEASUREM
Page 23 and 24: agi/is in 1882. In 1914, Alexeieff
Page 25 and 26: unique about Giardia in humans, whi
Page 27 and 28: To-date, use of the different speci
Page 29 and 30: Differentiation into the cyst stage
Page 31 and 32: 1.3 Transmission 1.3.1 Human hosts
Page 33 and 34: Owen, 1984) 1.3. 2 The Zoonosis of
Page 35 and 36: 1.4 Distribution The morbidity and
Page 37 and 38: children by Schutte et al. in 1981
Page 39 and 40: of (a) the enormous absorptive capa
Page 41 and 42: Ig M (100% of patients), IgG (70%),
Page 43 and 44: chymotrypsin) between different iso
Page 45: impact on the physical growth of 29
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 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
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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
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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
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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
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Table 5.1 Outlines a relative semi-
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dislodged by chilling the culture t
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Table 5.2. Sensitivity to Ciproflox
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Table 5.3. Summary results of in vi
Page 139 and 140:
Once established in culture for a l
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Plates 5.3 and 5.4 Confluent growth
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difficult to produce viable culture
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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
Page 161 and 162:
for Giardia Iysates. These included
Page 163 and 164:
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
Page 177 and 178:
Belosevic M, Faubert GM, MacLean JD
Page 179 and 180:
Farthing MJG, Mata L, Urrutia JJ, K
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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,
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of Entamoeba histolytica in a group
Page 189 and 190:
Wolfe M. S.1979. Giardiasis. Sympos
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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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