in vitro culture and isoenzyme analysis of giardia lamblia

More documents

Recommendations

Info

4.2.3 In vivo Excystation This method was based on that of Dr TE Nash & JT Conrad of the N.I.H (Bethesda, Maryland) (personal communication) and modified for local experimentation. Suspensions containing 1000 cysts/m I in 0,1 ml aliquots of distilled water were injected into each of the stomachs of 1-2 day old suckling mice in a litter (a litter consisted of at least 4 up to 9 pups). A total of 53 litters were inoculated. Depending on the availability of neonatal mice and the number of cysts in a sample, the inoculations were replicated either within the same strain of mice or between the two strains. Seven to ten days after inoculation, the infected mice were sacrificed by cervical dislocation. The proximal 8-cm of small intestine was removed and macerated with scissors in 1 ml of cold TYI-S-33 medium in a Petri dish. The mixture was transferred to a sterile 9' 0 x 10mm Kimax glass tube filled to 80% capacity with the TYI-S-33 culture medium containing antibiotics and its cap was immediately screwed on. Inoculated tubes were chilled for 30 minutes to detach trophozoites from the macerated intestines and then incubated at 35,5° C for 1 hour at a five-degree angle to allow the trophozoites to adhere to glass. Medium containing intestine fragments was filtered through a double layer of gauze to eliminate the toxic intestines. The filtrate, which contained the unattached trophozoites, was transferred to a sterile 8ml Kimax tube and the latter filled to 80% capacity with fresh TYI-S-33 medium with antibiotics. All the tubes were incubated at 35,5°C for 1 hour and supernatant medium gently decanted and replaced with an equal volume of fresh TYI-S-33 with antibiotics. All tubes were then incubated for 1 hour and the medium was changed one more time. The tubes were examined on an inverted microscope 89
for attached trophozoites and maintained at 35,5°C for at least seven days even if no trophozoites were detected on the first day. Note: Aliquots of all batches of cysts inoculated into mice were concurrently subjected to attempted excystation in vitro by the modified acid pepsin method described in 3.2.3. Where cysts numbers were sufficient, (and depending on the availability of neonatal mice when cysts were isolated) repeated inoculations of the same mouse strains and different mice strains were attempted using the same cyst batches which had been aliquoted and maintained at 4°C. 90
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 and 104: an unreliable indicator of viabilit
Page 105 and 106: propagating Giardia for subsequent
Page 107 and 108: 4.2 MATERIALS AND METHODS 4.2.1. An
Page 109: A litter comprising of 7 C57BU6 mic
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?