in vitro culture and isoenzyme analysis of giardia lamblia

More documents

Recommendations

Info

CHAPTER 4 IN VIVO EXCYSTATION 4.1 INTRODUCTION Animal models of giardiasis using mice and G.muris have been used for various studies since the 1920's. Hegner (1927) reported excystation of cysts from human faeces in laboratory rats. Similar experiments by Filice (1952) illustrated that Giardia cysts from humans can excyst and establish infections in the rodent small intestine. Roberts-Thompson et al., (1976) described a novel animal model of murine giardiasis which was later adopted by many workers. They induced reproducible infections by intra-oesophageal inoculation of G.muris cysts into Swiss albino mice and investigated the effects of varying doses of inoculated cysts (100,1000 and 10000 cysts). Twenty-two of 24 mice receiving inoculations of 100 cysts became infected while all mice receiving 1000 or 10 000 cysts were infected. Maximal cyst output did not differ significantly in the three groups, however the mice inoculated with higher dosages excreted maximum numbers of cysts earlier than those receiving the lower dose. A similar pattern of trophozoite counts was observed; maximum counts occurred on days 5,7, and 14 post inoculation with 10 000, 1000, and 100 cysts respectively. Hill and co-workers (1983) pioneered the use of suckling mice for infection with the Giardia sourced from humans. They inoculated the CF-1 strain of mice with axenically cultured G.lamblia trophozoites to assess the susceptibility of mice to infection. All of the 65 mice that were challenged became infected. Subsequently, Nash et al. (1985) first reported the use of neonatal mice as a means of 83
propagating Giardia for subsequent in vitro isolation. They axenised five strains using this technique. Recently Mayrhofer and colleagues (1992) also used the suckling mice model for primary isolation of Giardia from faecal specimens and for the subsequent growth of the organisms to obtain sufficient material for genetic analysis. Their work revealed that natural infections can be comprised of mixed genotypes. Isolation and purification can lead to selection of distinct genotypes by both in vivo and in vitro techniques. Gerbils have also been used as excystation hosts in a study to characterise isolates retrieved from humans and animals in a waterborne outbreak of giardiasis (Isaac-Renton et al., 1993). In another study, gerbils were inoculated with Giardia lamblia cysts and infections were effected (8elosevic et al., 1983). In this model it was established that the pattern of cyst release and the number of trophozoites in the intestines of orally and duodenally inoculated gerbils were similar. Visvesvara et al. (1988) stated that of all the various animals used as hosts for G./amblia, consistently good results have been obtained only with suckling mice and gerbils. These workers studied the infectivity patterns of Giardia cysts obtained from 10 subjects (7 symptomatic and 3 asymptomatic) in gerbils. A strikingly variable infection pattern was reported. One of the ten human isolates (obtained from an asymptomatic person) tested for infectivity in gerbils infected all 12 animals inoculated. Of the nine other isolates inoculated into gerbils, 4 isolates (2 from asymptomatic carriers and 2 from symptomatic patients) failed to establish infections in the animals while cysts from the other five patients produced infections in 11 to 75% of the animals. These results clearly indicated differences between human isolates of Giardia in their innate ability to infect gerbils. 84
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 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
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: an unreliable indicator of viabilit
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 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 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
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
show all

in vitro culture and isoenzyme analysis of giardia lamblia

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?