in vitro culture and isoenzyme analysis of giardia lamblia

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Parts of this work were presented at the following conferences: 1. In Vitro Culture of Giardia lamblia.1996. Kwitshana ZL, Reddy SG, Sturm AW & Jackson TFHG. Presentation at the 10 th Annual Congress of the Parasitological Association of Southern Africa. 5-6 September 1996. Waterfront, Cape Town. 2. In vitro culture and isoenzyme analysis of Giardia lamblia.1998 Kwitshana ZL, Reddy SG, Sturm A.W & Jackson TFHG. 12 th Annual Congress of the Parasitological Association of Southern Africa. 9-11 September 1998. Mont Auxources. Drakensburg. 3. Determination of viability of Giardia lamblia cysts: vital staining, in vitro and in vivo excystation. Kwitshana Z.L.; Reddy S.G.; Sturm A.W. & Jackson T.F.H.G.14 th Annual Congress of the Parasitological Association of Southern Africa. 19-23 October 1999. Augrabies Falls. Eastern Cape. Publications. In Vitro Culture of Giardia lamblia. (Abstract). 1996. Kwitshana Z.L. Jackson T.F.H.G. Journal of South African Veterinary Association 67(4): 175 In vitro culture and isoenzyme analysis of Giardia lambli8. 1998. (Abstract) Kwitshana Z.L.; Reddy S.G.; Sturm A.W. & Jackson T.F.H.G. Journal of South African Veterinary Association. 69:6 VI
ACKNOWLEDGEMENTS The author wishes to express her sincere gratitude to the following individuals for their assistance in preparation of this thesis: • Professor TFHG Jackson, (Supervisor) Regional Executive Director, S.A. Medical Research Council, Durban for supervising and affording me the opportunity to do this work. His constructive criticism, constant encouragement, expert advice, support and guidance are highly appreciated. • Professor AW Sturm, (Co-supervisor} Head of Department, Microbiology, University of Natal, Medical School, for his expert Microbiology contribution, advice and encouragement. • Mr. Selvan Reddy fQr his scientific input, patience, encouragement, support and professional criticism. • The Superintendent of King Edward Hospital for giving permission to collect stool specimens from patients at the hospital. • Professor J Coovadia Head of Paediatrics Dept. for allowing stool specimen collection from children's wards. • Or. T.E.Nash and Mr. J.T.Conrad of the Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases (NIH), Maryland, for their invaluable assistance in the establishment of the animal model and kind supply of Giardia reference strains. • The Medical Research Council and the University of Natal for providing financial support for this project. • Staff in Medical Microbiology at King Edward as well as Hlabisa hospital Laboratories for their assistance with retrieval of stool specimens. • Mr Sathyanand Suparsad for his kind assistance with isoenzyme electrophoresis. Celia Anderson, Shirley Epstein, Annalies Gumede & Welcome Mkhasibe for their assistance during stool collections and all the staff at the MRC Amoebiasis Research Unit for their moral support. • Ms. Charmine Bux & Mr James Wesley-Smith for their assistance with invertoscope and phase contrast photography respectively. • Albert Hirashen and all the technical staff at the Medical Media Services for their assistance with photography. • Staff at the MRC's Animal Facility: A. Saikoolal, Rould Cibane & Joseph Shozi for their assistance with animal care. vu
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: Declaration This study represents o
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 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
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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
Page 73 and 74:
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
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
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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
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not be infected in comparison with
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A proportionately higher percentage
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These findings strongly suggest tha
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CHAPTERS IN VITRO CULTURE OF GIARDI
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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
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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
Page 155 and 156:
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
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,
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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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in vitro culture and isoenzyme analysis of giardia lamblia

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