the inhibitory properties of and mode of action of plant essential oils ...

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Effect of purification methods on in vitro excystation..................................117 Effect of acidification on spontaneous excystation of C. parvum oocysts....118 3:4.2 PEO treatment experiments...........................................................................120 Control oocysts.............................................................................................120 Effect of time and temperature on the excystation of untreated C. parvum oocysts...........................................................................................................120 Effect of PEOs on the spontaneous excystation of C. parvum oocysts.........121 Determining whether PEO treatment influences maximised in vitro excystation when standard excystation triggers are used.............................121 Incubation of oocysts with PEOs / constituents............................................122 Incubation of C. parvum oocysts with PEOs and their constituents over a 24 h time course.................................................................................................123 3:5 DISCUSSION....................................................................................................127 CHAPTER 4 – Trypanosoma cruzi........................................................................137 The activity of various PEOs and their constituents, on Trypanosoma cruzi epimastigote viability in vitro.......................................................................137 4:1 ABSTRACT......................................................................................................138 4:2 INTRODUCTION............................................................................................139 4:3 MATERIALS AND METHODS.....................................................................144 4:3.1 T. cruzi in vitro culture...................................................................................144 Source and maintenance of T. cruzi epimastigotes.......................................144 T. cruzi enumeration for experiments...........................................................144 Comparison of 25°C and 37°C incubation in the MTS assay and T. cruzi titration for in vitro experiments...................................................................144 4:3.2 Experimental treatments of epimastigotes.....................................................145 Viability in ethanol........................................................................................145 PEO preparation...........................................................................................145 Incubation of T. cruzi epimastigotes with PEOs...........................................145 Incubation of T. cruzi epimastigotes with PEOs and PEO constituents over a 2 h time course..............................................................................................146 Incubation of T. cruzi epimastigotes with PEO constituents........................147 xv
4:3.3 Epimastigote culture analysis........................................................................147 T. cruzi cell count assay................................................................................147 Statistical analysis........................................................................................148 4:4 RESULTS..........................................................................................................149 Comparison of incubation at 25°C and 37°C in the MTS assay and the optimisation of T. cruzi epimastigote concentrations....................................................................149 T. cruzi epimastigote morphological alterations (TBMM assay).............................151 Effects of ethanol on T. cruzi viability......................................................................152 Incubation of T. cruzi epimastigotes with PEOs.......................................................153 Screening of PEOs........................................................................................153 Titration of PEOs..........................................................................................157 Incubation of T. cruzi epimastigotes with PEO constituents....................................161 Incubation of T. cruzi epimastigotes with PEOs over a 2 h time course..................162 4:5 DISCUSSION....................................................................................................164 CHAPTER 5 - The effect of blueberry extracts on Giardia duodenalis viability and spontaneous excystation of Cryptosporidium parvum oocysts, in vitro........172 5:1 ABSTRACT......................................................................................................173 5:2 INTRODUCTION............................................................................................174 5:3 MATERIALS AND METHODS.....................................................................178 5:3.1 Source of parasites..........................................................................................178 Source of G. duodenalis trophozoites...........................................................178 Source of oocysts...........................................................................................178 5:3.2 Experimental treatments of parasites............................................................178 Pressed blueberry extract and beverage preparation..................................178 Preparation and analysis of polyphenol and anthocyanin contents of blueberry polyphenolic-rich extract.............................................................................178 Incubation of G. duodenalis trophozoites with FB, BB and PRBE..............179 Incubation of C. parvum oocysts with FB, BB and PRBE............................179 5:3.3 Parasite enumeration and analysis................................................................180 G. duodenalis cell count assay......................................................................180 Oocyst analysis.............................................................................................180 xvi
Page 1 and 2: A thesis submitted in partial fulfi
Page 3 and 4: ABSTRACT The main aims and objectiv
Page 5 and 6: ABBREVIATION LIST 1DE: 1-dimensiona
Page 7 and 8: ml -1 : Per millilitre MgCl2: Magne
Page 9 and 10: FIGURE AND TABLE LIST CHAPTER 1 - I
Page 11 and 12: Figure 4.9 Incubation of T. cruzi e
Page 13 and 14: Life cycle.........................
Page 15: Effect of oil constituents on viabi
Page 19 and 20: 7:1 ABSTRACT.......................
Page 21 and 22: CHAPTER 1 Introduction 1
Page 23 and 24: ecome resistant to both chloroquine
Page 25 and 26: Leptomonas colosoma, Crithidia fasc
Page 27 and 28: viability of a population) determin
Page 29 and 30: changes in phospholipid composition
Page 31 and 32: killed by garlic (Zenner et al. 200
Page 33 and 34: Table 1.2 Plant oils that demonstra
Page 35 and 36: arouse, until the stomach was clear
Page 37 and 38: Dermatological reactions Many plant
Page 39 and 40: 2002). With industrialised nations,
Page 41 and 42: elongation and joining of large cyt
Page 43 and 44: the ER is the formation and folding
Page 45 and 46: of the cysts through the stomach ex
Page 47 and 48: Figure 1.5 Life-cycle of G. duodena
Page 49 and 50: Cryptosporidium is an apicomplexan
Page 51 and 52: Table 1.4 - Continued Species of Cr
Page 53 and 54: Upon excystation, sporozoites attac
Page 55 and 56: MacKenzie et al. 1994b). However, a
Page 57 and 58: Figure 1.7 Life-cycle of C. parvum.
Page 59 and 60: identified by the relative position
Page 61 and 62: Amastigote These replicative stages
Page 63 and 64: Life Cycle The sylvatic cycle start
Page 65 and 66: Acute phase An intense and localise
Page 67 and 68:
inflammatory reactions induced by t
Page 69 and 70:
1:8 Research Hypothesis Little scie
Page 71 and 72:
able to identify them molecularly a
Page 73 and 74:
CHAPTER 2 Giardia duodenalis The in
Page 75 and 76:
2:2 INTRODUCTION The protozoan para
Page 77 and 78:
example, the understanding of antig
Page 79 and 80:
Jassim & Naji 2003) and more recent
Page 81 and 82:
2:3 MATERIALS AND METHODS 2.3.1 Par
Page 83 and 84:
concentration equivalent to 0.02% v
Page 85 and 86:
whole oil respectively. Elemi oil c
Page 87 and 88:
Plus Protein standard; unstained; 1
Page 89 and 90:
2:4 RESULTS Ethanol and DMSO titrat
Page 91 and 92:
Figure 2.1 In vitro viability of G.
Page 93 and 94:
and elemi was 0.005% although > 80%
Page 95 and 96:
Trypan Stained / Non-viable Trophoz
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(10.90%). Whilst both the main cons
Page 99 and 100:
Incubation of G. duodenalis trophoz
Page 101 and 102:
Trophozoite protein profiling by SD
Page 103 and 104:
used in this protein analysis exper
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MIC of 0.005%. This phenomenon was
Page 107 and 108:
Effect of elemi oil on viability as
Page 109 and 110:
0.05). This effect though, was less
Page 111 and 112:
kDa 50- 37- 50- 37- 50- 37- MW G HK
Page 113 and 114:
2:5 DISCUSSION These experiments we
Page 115 and 116:
Using microscopic evaluation of par
Page 117 and 118:
cell death may have been initiated,
Page 119 and 120:
Using this information, investigati
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flavanones, anthocyanidins and isof
Page 123 and 124:
protein was also abolished in elemi
Page 125 and 126:
CHAPTER 3 Cryptosporidium parvum In
Page 127 and 128:
3:2 INTRODUCTION The enteric parasi
Page 129 and 130:
disease in extraintestinal location
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constituents and speed of activity.
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(empty), partially excysted and int
Page 135 and 136:
and incubated for a further 1, 4 or
Page 137 and 138:
3:4 RESULTS 3:4.1 Acidification exp
Page 139 and 140:
When compared with MD, the IOWA iso
Page 141 and 142:
with incubation at 5ºC (-0.5 ± 1.
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constituent in palmarosa oil which
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Spontaneous Excystation Rate (%) 10
Page 147 and 148:
3:5 DISCUSSION Interventions in the
Page 149 and 150:
When these acid treated oocysts wer
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PEOs augment the effect of temperat
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spontaneous excystation within 4 h.
Page 155 and 156:
Trans-anethole is used as a flavour
Page 157 and 158:
CHAPTER 4 Trypanosoma cruzi The act
Page 159 and 160:
4:2 INTRODUCTION The disease caused
Page 161 and 162:
It is clear that alternative therap
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analyse the activity of 12 differen
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µl of parasites. One 96 well plate
Page 167 and 168:
Incubation of T. cruzi epimastigote
Page 169 and 170:
4:4 RESULTS Comparison of incubatio
Page 171 and 172:
MTS Absorbance (492nm) 1 0.9 0.8 0.
Page 173 and 174:
Page 175 and 176:
A B Absorbance (492nm) Trypan Stain
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Titration of PEOs As all the PEOs a
Page 179 and 180:
0.02% 0.01% 0.005% 0.0025% 0.00125%
Page 181 and 182:
Page 183 and 184:
Trypan Stained / Non-viable Epimast
Page 185 and 186:
published concerning the trypanocid
Page 187 and 188:
using the MTS assay, where a lack o
Page 189 and 190:
With myrtle, 2 constituents working
Page 191 and 192:
mixed with inert carrier oils such
Page 193 and 194:
5:1 ABSTRACT The protozoan parasite
Page 195 and 196:
37ºC over a 24 h period, in the ab
Page 197 and 198:
against Cryptosporidium with any su
Page 199 and 200:
polyphenol content of PRBE was 920
Page 201 and 202:
5:4 RESULTS Polyphenolic content of
Page 203 and 204:
Figure 5.1 A Viable and dead G. duo
Page 205 and 206:
Effect of FB, BB and PRBE on the sp
Page 207 and 208:
5:5 DISCUSSION Here it is shown tha
Page 209 and 210:
spontaneous excystation observed (F
Page 211 and 212:
certain concentration is exceeded (
Page 213 and 214:
6:1 ABSTRACT Current conventional t
Page 215 and 216:
Flavonoids and phenolic acids form
Page 217 and 218:
efore any dilutions in TYI-S-33 cou
Page 219 and 220:
6:4 RESULTS Incubation of G. duoden
Page 221 and 222:
Trypan Stained / Non-viable Trophoz
Page 223 and 224:
6:5 DISCUSSION It was demonstrated
Page 225 and 226:
vitro, is not due to the degradatio
Page 227 and 228:
CHAPTER 7 The examination of latrin
Page 229 and 230:
7:2 INTRODUCTION The examination of
Page 231 and 232:
7:3 MATERIALS AND METHODS 7:3.1 Sou
Page 233 and 234:
Figure 7.2 Excavation of Soutra Ais
Page 235 and 236:
7:3.2 Sample preparations Formol et
Page 237 and 238:
magnet. The tubes were rocked throu
Page 239 and 240:
Using the methods of Smith et al. (
Page 241 and 242:
incubated at 55°C for 3 h in a wat
Page 243 and 244:
RNA Cryptosporidium gene to determi
Page 245 and 246:
concentration recommended in publis
Page 247 and 248:
7:4 RESULTS Microscopic examination
Page 249 and 250:
Figure 7.6 Fluorescence staining of
Page 251 and 252:
These slurries were subjected to IM
Page 253 and 254:
Figure 7.10 PCR analysis for Crypto
Page 255 and 256:
Pike 1967 & 1968; Jones 1982; Aspoc
Page 257 and 258:
the digestion enzymes DraI and AseI
Page 259 and 260:
CONCLUSIONS This series of experime
Page 261 and 262:
distortion, blebbing and increased
Page 263 and 264:
trophozoites and Trypanosoma epimas
Page 265 and 266:
Reduviid bug is a means of entry fo
Page 267 and 268:
anticryptosporidial activity may be
Page 269 and 270:
Historically, many PEOs and extract
Page 271 and 272:
REFERENCES Abdo, K.M., Cunningham,
Page 273 and 274:
Anthony, J-P., Fyfe, L. Smith, H.V.
Page 275 and 276:
Best, M., Sattar, S.A., Springthorp
Page 277 and 278:
in the chronic phase of experimenta
Page 279 and 280:
entidade morbida do homem. Memorias
Page 281 and 282:
Current, W.L., Upton, S.J. and Hayn
Page 283 and 284:
Elliot, B.C., Wisnewski, A.V., John
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Ferreira, L.F., Britto, C., Cardoso
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Gillin, F.D., Reiner, D.S. and Bouc
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Hashmey, R., Smith, N.H., Cron, S.,
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Hunter, P.R. and Syed, Q. 2001. Com
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Kim, H.C. & Healey, J.M. 2001. Effe
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Le Bourhis, B. 1968. Preliminary re
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ornidazole. Transactions of the Roy
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Moo-Puc, R.E., Mena-Rejon, G.J., Qu
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length polymorphism assay. Applied
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Pecevski, J., Savkovic, D., Radivoj
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Reduker, D.W. & Speer, C.A. 1985. F
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Rothhammer, F., Allison, M., Nuñez
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Sgambatti de Andrade, A.L., Zicker,
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Sosa Estani, S., Segura, E.L., Ruiz
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Tyler, K.M. and Engman, D.M. 2001.
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Wang, M., Kikuzaki, H., Lin, C.C.,
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Yasunaka, K., Abe, F., Nagayama, A.
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Family: Burseraceae, Commiphora myr
Page 321 and 322:
Family: Lamiaceae, Pogostemon cabli
Page 323 and 324:
Family: Rutaceae, Amyris balsamifer
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Family: Ericaceae, Vaccinium vitis-
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Family: Rosaceae, Rubus fruticosus
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Family: Rosaceae, Sorbus aucuparia
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Appendix 5. SDS-Polyacrylamide gel
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5. When the glass plates are in pla
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Appendix 6: Incubation of G. duoden
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Appendix 8. Agarose gel electrophor
Page 359:
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