in vitro culture and isoenzyme analysis of giardia lamblia

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7.4 DISCUSSION As Giardia lamblia infections may have an asymptomatic course or they may produce acute or chronic diarrhoea, a mechanism of establishing if the different clinical outcome of these infections could be due to genetic strain differences would be useful. Isoenzyme analysis has proved to be an invaluable biochemical tool in differentiating pathogenic and non-pathogenic strains of Entamoeba histolytica (Jackson et al., 1982; Sargeaunt et al., 1982). Genetic variation in Giardia has peen shown by isoenzyme analysis (Cedillo-Rivera et al., 1989; Isaac Renton et al., 1993) however none of the investigations have demonstrated genetic variations that are correlated to pathogenecity. In the present study, it was not possible to characterise and group strains from symptomatic and asymptomatic subjects owing to limited numbers of isolates. Five isolates were obtained from "presumably" healthy children and 1 from a patient with diarrhoea. However, in the latter concomitant infection with Salmonella spp was present; therefore, symptomatology could not be unequivocally assigned to giardiasis. Furthermore, there was marked heterogeneity among the few isolates that were analysed by the enzyme systems described in the current work. An exhaustive genetic interpretation of the banding patterns of these isolates was therefore not made. Nonetheless, some polymorphism was demonstrated in this limited study. This suggests that the different isolates analysed in the present study posses enzyme variants, and therefore heterogeneous. In this study, it is shown that serial cultivation of trophozoites does not alter the isoenzymes as shown by consistent banding patterns of serially cultivated isolates. This could indicate that the genes encoding the respective enzymes are 151
stably expressed between different subcultures; or that the culture conditions induce expression of stable genotypes. Therefore isoenzyme analysis of cultured trophozoites seems to be a reliable method for characterisation of different strains. The findings reported in this work are comparable to results obtained by other investigators. For example, in ME, a homogenous pattern of single bands were exhibited by all isolates analysed in this study and Cedillo-Rivera et al., (1989) reported strong homogeneity of electrophoretic patterns (all comprising of a single band) for ME when they analysed 19 strains isolated from symptomatic and asymptomatic patients in Mexico. However, other investigators reported different isoenzyme patterns when they analysed strains obtained from (i) widelydistributed geographic locations and (ii) both human and animal strains. Proctor et al., (1989) found single ME bands with 3 different patterns when they analysed 32 G. duodena/is isolates from humans and animals of various geographic locations. Similar findings (2 different patterns of Single bands) were reported by Moss et al., (1992) for 11 G.lamblia strains from various geographic locations; Isaac-Renton et al., 1993, reported similar findings for this enzyme. Meloni et al., (1988) obtained single and multiple banding of various patterns for 30 isolates from humans and felines with worldwide distribution. From these studies it would appear therefore that the ME homogeneity is restricted to human isolates from a defined locality. In the present study, no bands were obtained in the GOT enzyme system despite replicated attempts. Similarly, Proctor et al., 1989 reported unsatisfactory results 152
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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
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not be infected in comparison with
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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 and 168: PGM activity was displayed by Giard
Page 169 and 170: Plate 7.6. Hexokinase Lane 1. Unino
Page 171: Plate 7.8. Phosphogluconate dehydro
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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