in vitro culture and isoenzyme analysis of giardia lamblia

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The mouse inoculation method using Giardia from humans is favoured by renowned Giardia authorities like Nash. This method proved to be the most successful for obtaining trophozoites from cysts in his laboratory with a consequent 70% success rate ofaxenisation of Giardia (Nash, 1988). Although in vitro excystation was successfully accomplished (Chapter 3) by various methods, axenic cultures (Chapter5) could never be established with trophozoites from this source. An alternative method for sourcing trophozoites was needed. Or T.E. Nash and J. Conrad of the National Institutes for Health (N.I.H) Laboratory for Parasitic Diseases in Bethesda in U.S.A were consulted. They recommended the in vivo model and assisted with establishing this method in our laboratory in Durban. The current chapter describes the adaptation of the method to our local environment and the results obtained. 85
4.2 MATERIALS AND METHODS 4.2.1. Animal Sourcing and Care C57BU6 inbred mice were recommended by Nash and Conrad (personal communication) and these (eight weeks old) were obtained from the Biomedical Resource Centre at the University of Durban Westville. In addition, an indigenous species, readily available in our laboratory, Praomys (Mastomys) coucha (outbred) was used. The mice were housed in cages cushioned with sawdust and the temperature maintained at 25°C. A standard pellet diet and fresh water were supplied to the animals ad lib. Soiled sawdust was replaced daily. 4.2.2 Preliminary Experiments: To ensure that the animals were well suited for the in vivo experiments, some pilot studies were necessary to: ~ eliminate any endogenous infections ~ *establish appropriate cyst dosages with which to effectively inoculate the mice ~ *establish the appropriate time to retrieve the trophozoites from infected mice in order to ensure that maximum numbers of organisms are obtained. *Although these have been published previously elsewhere, this type of work was being done for the first time in SA and in our laboratory, furthermore the Mastomys strain used here differs from those used by the other investigators. 4.2.2.1 Exclusion of Endogenous Giardia Infections During the acclimatisation period, mouse faecal samples were collected on wetted absorbent cotton-wool pads daily from each of the cages and were screened for the presence of Giardia cysts using the formal-ether technique (2.2.2.1) for ten days. The mice were deemed free of parasite infection if all faecal screens were 86 -
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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
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
Page 105: propagating Giardia for subsequent
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
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
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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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in vitro culture and isoenzyme analysis of giardia lamblia

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