In vitro quantitation of Theileria parva sporozoites for use - TropMed ...

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Chapter 1: Quantitation of Theileria parva sporozoites: Review of literature 23 ______________________________________________________________________________________________ to feed for this process to occur. Sporozoites are about 0.75 – 1.5 µm in diameter and have a surface coat that is 20 – 25 nm thick (Shaw et al., 1991; Shaw and Young, 1994) Transmission to the bovine host occurs when the tick injects sporozoites suspended in the saliva as it feeds. Purnell and Joyner (1967) demonstrated that infected ticks feeding on blood products presented in glass capillary tubes salivated infective stages of T. parva into the blood meal in the tube. The tick's lifespan may be reduced by virtue of being infected (Watt and Walker, 2000). Similarly, reduced viability in Boophilus species heavily infected with Babesia has been observed (Ouhelli et al., 1987; De Vos et al., 1989). 1.1.2.4. Cycle of Theileria parva in the bovine host Theileria parva successively invades lymphocytes and erythrocytes. It differs in a number of respects from other apicomplexans in that it does not have a well defined apical complex (Fawcett et al., 1982b) and the secretory products of rhoptries and microspheres are ejected after internalisation (Shaw et al., 1991). The sporozoite attaches to the lymphocyte and is internalized by active endocytosis within three minutes of attachment (at 37°C) (Shaw et al., 1991). In vitro studies have shown that attachment is a random event and not energy dependent, whereas internalization is energy dependent and optimised at 37°C (Shaw et al., 1991). It begins with close approximation of the parasitic and host cell membranes most possibly by legand-receptor mechanisms. T. parva differs from its apicomplexan counterparts in that it can attach to the target cell in any orientation (Fawcett et al., 1982b). The sporozoite is then progressively internalised by becoming tightly apposed to the host membrane, in a "zippering" phenomenon described by Fawcett et al. (1982b) (Figure 2). It sheds its own surface coat (Webster et al., 1985) and then dissolves the host cell membrane to escape into the host's cytosol (Fawcett et al., 1982b) possibly as a defensive mechanism against the host cell's lysozymes. The internalised sporozoite quickly becomes associated with microtubules of the host cell, stabilises them (Shaw, 2003) and in the first few days following infection, develops into a pyriform shape that resembles Babesia piroplasms (Stagg et al., 1981). From about day five post infection, development into a multinucleated syncytium, i.e. a macroschizont, takes place. This stage transforms the host cell and the lymphoblast starts to divide exponentially in synchrony with the schizont's own divisions so that both daughters cells usually inherit parasite material (Hulliger et al., 1964; Stagg et al., 1980; Stagg et al., 1981). Rocchi et al. (2006) showed that not all originally infected cells are transformed and that even among transformed cells, there is sometimes failure for some daughter cells in receiving progeny
24 Chapter 1: Quantitation of Theileria parva sporozoites: Review of literature ______________________________________________________________________________________________ schizonts. The schizont is pulled apart on the microtubule spindle as the cells divide and sometimes one of the daughter cells is not infected or receives more schizont material than the other. This seems the most plausible explanation for the presence of uninfected cells in continuous cell lines of dividing lymphoblasts. Figure 2: Drawing illustrating the sequence of events during the entry and establishment of Theileria parva in bovine lymphocytes. Reproduced from Shaw et al. (1991). (1) Binding of sporozoite to lymphocyte surface membrane (2) a very close apposition of parasite and lymphocyte membranes forms (3) "zippering" effect of the two membranes progressively internalises the sporozoite (4) the enclosing lymphocyte membrane separates from the sporozoite as contents of microspheres and rhoptries are released (5) lymphocyte membrane is dissolved and the sporozoite escapes into the host cell's cytoplasm (6) lymphocyte microtubules surround the sporozoite. A proportion of these schizonts undergoes merogony and the merozoites are released into the general circulation upon rupture of the host cell. Merozoites infect erythrocytes in which they become piroplasms, the end stage of the cycle in the bovine host. Merogony at very low frequency may be the main mechanism by which infection is maintained in the carrier status (Maritim et al., 1989; Dolan, 1999). The mechanism bringing about lymphocyte transformation is not completely understood but several theories have been proposed. Some pieces of research implicate suppression of the lymphocyte's programmed cell death (PCD) by transcriptional factors or induction of heat shock proteins (Heussler et al., 1999; Heussler et al., 2001). This process would support the phenomenon of immortalisation. Other research pieces propose that oncogenes are switched on or the parasite inactivates pathways that prevent uncontrolled cell replication or both through transcription factors as reviewed by Chaussepied and Langsley (1996) to explain the cancerous
Page 1 and 2: Victor Mbao In vitro quantitation o
Page 3 and 4: II Front cover photograph Copyright
Page 6 and 7: V ACKNOWLEDGEMENTS This work was ma
Page 8 and 9: VII TABLE OF CONTENTS ACKNOWLEDGEME
Page 10: IX APPENDIX I .....................
Page 14 and 15: General Introduction 13 ___________
Page 16 and 17: General Introduction 15 ___________
Page 18: Chapter 1 - Quantitation of Theiler
Page 21 and 22: 20 Chapter 1: Quantitation of Theil
Page 23: 22 Chapter 1: Quantitation of Theil
Page 44 and 45: Chapter 2: Objectives 43 __________
Page 46: Chapter 3 -Comparison of two Theile
Page 49 and 50: 48 Chapter 3: Theileria parva extra
Page 58 and 59: Chapter 4: Suspension media and mul
Page 68: Chapter 5 - Snap freezing of Theile
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74 Chapter 5: Snap freezing of Thei
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Chapter 6: Storage of Theileria par
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Chapter 7: Lyophilisation of Theile
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Chapter 8: SNA and refinement of ti
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Chapter 9: General Discussion 119 _
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APPENDICES
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SUMMARY
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Curriculum Vitae
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List of Abbreviations 183 _________
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