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

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Xiao et al. 1999), the Cryptosporidium oocyst wall protein (COWP) locus (Homan et al. 1999; Xiao et al. 2000b), thrombospondin-related adhesive protein 1 (TRAP-C1; (Spano et al. 1998)) and β-tubulin (Caccio et al. 1999; Rochelle et al. 1999; Sulaiman, Xiao and Lal 1999; Widmer et al. 1998). Table 1.4 Characteristics of recognized Cryptosporidium species. Species of Major Host Oocyst Site of Infection Reference Cryptosporidium Dimensions (μm) C. andersoni Cattle 5.5 x 7.4 Abomasum (Lindsay et al. 2000) C. baileyi Poultry 4.6 x 6.2 Bursa of Fabricus, (Current, Upton and cloaca and trachea Haynes 1986) C. bovis Cattle 4.7-5.3 x 4.2-4.8 Small intestine (Barker & Carbonell 1974) C. canis Dogs 4.95 x 4.71 Small intestine (Fayer et al. 2001) C. fayeri Red kangaroo 4.5-5.1 × 3.8-5.0 Intestine (Ryan, Power and (Macropus rufus) Xiao 2008) C. felis Cats 4.5 x 5.0 Small intestine (Asahi et al. 1991; Iseki 1979) C. galli Finches, 8.25 x 6.3 Proventriculus (Ryan et al. 2003; Chickens Pavlasek 2001 & 1999) C. hominis Humans 4.5 x 5.5 Small intestine (Morgan-Ryan et al. 2002) C. macropodum Grey kangaroo Not known Not known (Power & Ryan (Macropus giganteus) 2008) C. meleagridis Turkeys 4.5-4.0 x 4.6-5.2 Small intestine (Slavin 1955) C. molnari Fish 4.7 x 4.5 Small intestine and stomach 30 (Fayer 2008; Alvarez-Pellitero & Sitjà-Bobadilla 2002) C. muris Rodents 5.6 x 7.4 Stomach (Tyzzer 1907) C. parvum Man and other mammals 4.5 x 5.5 Small intestine (Tyzzer 1912) C. ryanae Cattle (Bos 2.94-4.41 x Not known (Fayer, Santin and (previously taurus) 2.94-3.68 Trout 2008; Santin Cryptosporidium deer-like genotype) et al. 2004) C. scophthalmi Fish (Turbot) 3.7-5.0 x 3.0- Intestine, very (Alvarez-Pellitero et 4.7 seldom in the stomach al. 2004) C. serpentis Snakes, Reptiles 5.6-6.6 x 4.8-5.6 Stomach, gastric glands (Levine 1980) C. suis Pigs 4.9-4.4 x 4.0-4.3 Small and large intestine (Ryan et al. 2004)
Table 1.4 – Continued Species of Cryptosporidium C. varanii (previously C. saurophilum Major Host Oocyst Dimensions (μm) Lizards 4.2-5.2 x 4.4-5.6 31 Site of Infection Reference Stomach and small intestine. (Pavlasek & Ryan 2008; Koudela & Modry 1998) C. wrairi Guinea pigs 4.9-5.0 x 4.8-5.6 Small intestine (Vetterling & Jervis 1971; Vetterling, Takeuchi and Madden 1971) At least 8 species (C. hominis, C. parvum, C. meleagridis, C. felis, C. canis, C. suis, C. andersoni and C. muris) and 5 genotypes Cryptosporidium (chipmunk, cervine [2 of 3 genotypes], monkey, and skunk) can infect humans, but it is C. parvum and C. hominis which are responsible for the majority of human instances of cryptosporidiosis (Feltus et al. 2006; Leoni et al. 2006; Nichols, Campbell and Smith 2006; Caccio et al. 2005; Ryan et al. 2004; Xiao et al. 2004). Table adapted from Smith (2008) and Smith et al. (2007). Oocyst wall structure The oocysts of Cryptosporidium are extremely resistant to environmental pressures (Nichols, Paton and Smith 2004; Robertson, Campbell and Smith 1992) and disinfectants (Angus et al. 1982; Blewett 1989; Campbell et al. 1982; Korich et al. 1990; Li et al. 2004; Pavlasek 1984; Peeters et al. 1989). The oocyst wall is the interface between the host, the oocyst and the environment and is composed of 3 main layers: outer, central and inner layers and measured to be 40 – 50 nm thick (Harris & Petry 1999; Nanduri et al. 1999). Coating the oocyst wall is a carbohydrate-rich glycocalyx, 20 – 30 nm thick and is antigenic (Nanduri et al. 1999). The outer layer of the oocyst wall is composed of acidic glycoproteins and lipids between 5 and 10 nm thick which is variably electron dense (Harris & Petry 1999; Robertson, Campbell and Smith 1993a). A 5 nm lipid-rich central layer separates the outer and inner layers of the oocyst wall. Finally there is an inner layer which is 30 nm thick and composed of 2 further layers: a 10 nm electron dense outer-inner layer and a 20 nm less electron dense inner-inner layer. With the 10 nm thick outer-inner layer a typical fibrillar array can be seen in negatively stained sections as disordered filamentous material (Smith & Ronald 2002). A protease sensitive, 20 nm thick,
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 and 16: Effect of oil constituents on viabi
Page 17 and 18: 4:3.3 Epimastigote culture analysis
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: Cryptosporidium is an apicomplexan
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
Page 97 and 98: (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
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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
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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
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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
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disease in extraintestinal location
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constituents and speed of activity.
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(empty), partially excysted and int
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and incubated for a further 1, 4 or
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3:4 RESULTS 3:4.1 Acidification exp
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When compared with MD, the IOWA iso
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with incubation at 5ºC (-0.5 ± 1.
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constituent in palmarosa oil which
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Spontaneous Excystation Rate (%) 10
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3:5 DISCUSSION Interventions in the
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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.
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Trans-anethole is used as a flavour
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CHAPTER 4 Trypanosoma cruzi The act
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4:2 INTRODUCTION The disease caused
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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
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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
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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
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using the MTS assay, where a lack o
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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
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37ºC over a 24 h period, in the ab
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against Cryptosporidium with any su
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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
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5:5 DISCUSSION Here it is shown tha
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spontaneous excystation observed (F
Page 211 and 212:
certain concentration is exceeded (
Page 213 and 214:
6:1 ABSTRACT Current conventional t
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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
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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
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Family: Lamiaceae, Pogostemon cabli
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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
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