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

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Chapter 7: Lyophilisation of Theileria parva stabilates 97 _______________________________________________________________________________ Katete Stabilates were reconstituted by adding 0.8 ml of sterile water to each vial. After centrifugations and separations of supernates, they were distributed in 50 µl aliquots in microtitration-plate wells, 40 wells per cryopreservative type. An equal volume of PBMC suspension containing 3*10 5 cells was then added to each well and the plates incubated for 10 days. For control, cryopreserved (un-lyophilised) stabilate was assessed in the same in vitro sessions. 7.2.4.2. In vivo assessment Lyophilised stabilate CA was appraised by injecting 20 ml of reconstituted material into a Friesian heifer 10 ml subcutaneously below the right ln. parotideus superficialis and 10 ml intravenously into the jugular vein. 7.2.5. Vital staining Vital staining was performed using Ethidium monoazide (EMA, 3-amino-8-azido-5-ethyl-6-phenylphenanthridium bromide: C 21 H 18 N 5 Br) (Sigma-Aldrich ® , Germany). EMA penetrates damaged cell membranes (dead cells) to covalently bind with genomic DNA and prevents such bound DNA from being amplified by PCR. Thus, presence of a PCR product means the membranes are intact and therefore from viable cells (Nogva et al., 2003; Rudi et al., 2005). Samples of lyophilised and nonlyophilised stabilates (CA) were EMA stained to check viability by PCR. Briefly, EMA was added to reconstituted and purified stabilate at a final concentration of 20 µg/ml in 1.5 ml microtubes. The mixtures were placed on ice and photo-activated by placing the tubes for 30 min under laboratory fluorescent lighting. The tubes were then centrifuged at 14,000 g for one minute, the pellet washed once in 400 µl PBS buffer and DNA extraction performed by the method of Boom et al. (1990; 1999). DNA extracts were then subjected to PCR and products resolved by electrophoresis through a 2% (w/v) Agarose gel. 7.3. Results 7.3.1. In vitro and in vivo infectivity assessment Cultures of PBMC incubated with various T. parva lyophilised sporozoite materials were negative. The steer that had been injected with lyophilised stabilate did not show any clinical or parasitological reactions. PBMC incubated with control stabilates (cryopreserved) became infected and demonstrated schizonts.
98 Chapter 7: Lyophilisation of Theileria parva stabilates ______________________________________________________________________________________________ 7.3.2. Vital staining DNA extracts from both the lyophilised and cryopreserved T. parva stabilates were amplified as shown by PCR bands on Agarose gel (Figure 18). L L C C + _ L C + _ L L C C + _ Figure 18: PCR bands of extracts of EMA treated (L) lyophilised and (C) cryopreserved Theileria parva stabilates with (+) positive and (-) negative controls. 7.4. Discussion The various attempts at lyophilisation of T. parva stabilates and other infective materials were unsuccessful. It is evident that the deleterious stage is that of drying and/or resuscitation and not the freezing stage. The freezing stage is unequivocally less damaging as evidenced by the ability for the cryopreserved un-lyophilised stabilates to infect PBMC in vitro in this work and the largely successful cryopreservation of immunizing stabilates in the field. Vital staining with EMA showed that the integrity of the sporozoite wall is well preserved in spite of loss of viability. This is an encouraging finding and may be indicative of a denaturing process limited to functional proteins. Marcotty et al. (2003) demonstrated a successful lyophilisation of T. parva stabilate. However, attempts to repeat this were not successful (Marcotty, personal communication, 2004). They reported that, even in the successful event, the majority of sporozoites could have been killed by the process putting the surviving proportion at less than 1% (Marcotty et al., 2003). It is speculated that the stabilate used in their work contained a very high concentration of viable sporozoites consequently raising the number surviving lyophilisation Possible reasons for failure include choice of lyoprotectants. Carpenter and Crowe (1988), suggest that the ability for a sugar to stabilise a protein during drying may depend on how it binds to the protein during the process. It may be that the sugars used in our studies did not bind sufficiently well to functional proteins. This would allow unfolding of the proteins during dehydration. Another
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Victor Mbao In vitro quantitation o
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II Front cover photograph Copyright
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V ACKNOWLEDGEMENTS This work was ma
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VII TABLE OF CONTENTS ACKNOWLEDGEME
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IX APPENDIX I .....................
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General Introduction 13 ___________
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General Introduction 15 ___________
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Chapter 1 - Quantitation of Theiler
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20 Chapter 1: Quantitation of Theil
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Chapter 2: Objectives 43 __________
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Chapter 3 -Comparison of two Theile
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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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