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

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Chapter 7: Lyophilisation of Theileria parva stabilates 93 _______________________________________________________________________________________________ "Now faith is being sure of what we hope for and certain of what we do not see" Hebrews 11:1 7. CHAPTER 7 7.1. Introduction East Coast fever (ECF), a disease of cattle caused by Theileria parva is of major economic concern due to direct losses incurred through its control and mortality (Mukhebi et al., 1992). Among the control measures targeted at East Coast fever is immunisation by the "Infection and Treatment" (I & T) method. However, the method has a few constraints. Since the immunity generated is strain specific, it calls for isolation and judicial use of different T. parva stocks for specific localities. Secondly, it relies on a costly cold chain both for storage and delivery. T. parva stabilates have to be kept in liquid nitrogen to preserve parasite viability. Simplification of this cold chain by assessing viability of stored stabilates on ice (Musisi et al., 1996b; Marcotty et al., 2001) and attempts to freeze-dry the stabilate (Marcotty et al., 2003) has been explored. Delivery of stabilate on ice is currently the method of choice in the eastern province of Zambia. Successful resuscitation of freeze-dried sporozoites was demonstrated by Marcotty et al. (2003). However, studies to repeat this success or optimize it were not conclusive. As this is a potentially less expensive strategy for storage and delivery of immunising stabilates, we conducted further T. parva stabilates lyophilisation experiments using two lyoprotectants and several lyophilisation protocols. 7.2. Materials and Methods 7.2.1. Parasite material and cryoprotectants Two stocks of T. parva, Katete and Chitongo, were used. The Chitongo and Katete stocks were isolated from Namwala and Katete districts of the southern and eastern provinces of Zambia, respectively in the early 1980's, (Berkvens et al.,1988; Geysen et al., 1999). Parasite materials were suspended in RPMI 1640 (25 mM HEPES, Life Technologies # 52400-025) medium supplemented with BSA (Acros organics # 240401000 and #240400100) at 3.5% (w/v) and antibiotics. Two lyoprotectants were used: Sucrose (Sigma #S1888-500g) or Trehalose (Sigma # T-0167) at molar concentrations of 0.3 M and 0.1 M, respectively.
94 Chapter 7: Lyophilisation of Theileria parva stabilates ______________________________________________________________________________________________ A first batch of Katete stabilate (AV) was prepared at a concentration of 10 ticks/ml. It was dispensed in 1 ml aliquots in 5 ml penicillin glass bottles giving a depth of about 5 mm. The second batch of Katete stabilate (KA) was similarly prepared from a separate batch of ticks. It was aliquoted into 5 ml and 25 ml penicillin bottles, 1 and 3 ml to a bottle, respectively. The 25 ml bottles were placed on their sides and frozen in that position to give a shallower depth of stabilate material of about 4 mm but larger surface area. The T. parva Chitongo parasite material was prepared as: the normal stabilate (CA), sediment portion (CA-sed), whole infected salivary glands (CA-acini) and whole ticks (CA-tick). The sediment was collected at the interface of the supernate and debris after centrifugation of the ground up tick material. It was presumed this layer would contain a higher concentration of sporozoites than the supernate. Glands dissected from 50 female ticks were stored as 1 ml sucrose aliquots, five pairs to a vial. Fifty whole ticks, five to a vial, were aliquoted in 1 ml sucrose medium. Whole ticks and glands were used on the assumption that the tissue would provide extra protection for the sporozoites during drying. 7.2.2. Freezing protocol The Katete stabilate was used in one set of experiments and the Chitongo material in another. Both had been pre-frozen at production to -80°C by placing the vials in polystyrene boxes that were then put in an ultra freezer. This is the first step in cryopreserving stabilates for field use (OIE, 2005). 7.2.3. Drying protocols 7.2.3.1. Theileria parva Chitongo stock Chitongo parasite material (CA) was lyophilised using a Labconco ® FREEZE DRYER 5 (Labconco Corp, Kansas City). Vials were weighed before and after drying. Materials were dried with several variations of a protocol as illustrated in Table 9. Table 9: Lyophilisation protocols for Theileria parva infected material Material Shelf temp (°C) P c (hPa) Time CA -35 0.02 11 CA -20 0.02 11 CA-sed -30 0.02 10 CA-sed -18 0.01 2 CA-tick -36 0.01 19 CA-acini -10 0.02 2
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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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APPENDICES
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SUMMARY
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Curriculum Vitae
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List of Abbreviations 183 _________
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