BOOK NUMBER Q P 'S if c Z ..... A b 6 ...... C ' j [ !■ 3510N NO GM8330 University of Ghana http://ugspace.ug.edu.gh - 1 - Monomorphic and Pleomorphic Trypanosoma brucei rhodesiense: Biochemical, morphological and ultrastructural comparisons. A thesis submitted for the Degree of Master of Science of The University of Ghana, Legon by Tamara Aboagye-Kwarteng 1983 Department of Biochemistry University of Ghana http://ugspace.ug.edu.gh - 2 - A C K N O W L E D G E M E N T S I will like to thank the University of Ghana and the Department of Biochemistry for arranging for me. t.o do the work contained in this thesis at the London School of Hygiene and Tropical Medicine. My special thanks go to Dr. K.K. Oduro, my internal supervisor for reading the manuscript and for the helpful suggestions he made. I am supremely grateful to Dr. W.E. Ormerod, my supervisor at the London School of Hygiene and Tropical Medicine, for all the practical help and advice he gave me with this work. I will also like to thank Professor Wallace Peters, Head of the Department of Medical Protozoology at the London School of Hygiene and Tropical Medicine, for allowing me to work there in the first place. Finally a big thank you to all my friends in Room 310, Joe, Chris and Shiela for their help and patience. University of Ghana http://ugspace.ug.edu.gh - 3 - A B S T R A C T The long slender and short stumpy forms of pleomorphic T . b. rhodeslense (LSHTM 180) have been compared to each other and to the monomorphic T. b. rhodesiense Liverpool Normal (Liv). 1. The agar film technique showed that morphologically the stumpy LSHTM 180 can be differentiated from the slender LSHTM 180 and the monomorphic T. b. rhodesiense (Liv) because it contains lipid granules. 2. Starch gel and cellulose acetate electrophoresis did not show any major differences in the isoenzyme profiles of 8 out of 9 enzymes studied. However, the presence of an aconitase band in the stumpy LSHTM 180 further differentiated it from the slender LSHTM 180 and the monomorphic T. b. rhodesiense (Liv). 3. Polypeptide profiles showed the presence of a 58,000 dalton polypeptide in extracts of both the slender and monomorphic trypomastigotes which was absent in the stumpy form. 4. Like starch gel and cellulose acetate electrophore­ sis, enzyme assays gave little information as to University of Ghana http://ugspace.ug.edu.gh - 4 - differences between the 3 trypomastigotes . There was, however, a higher NADP-isocitrate dehydrogenase activity in the stumpy form than the other two. 5. Ultrastructural studies showed the stumpy form had more cristae in the mitochondrion than either the slender LSHTM 180 or the monomorphic T. b. rhodesiense (Liv). 6 . Trypomastigotes of the pleomorphic strain LSHTM 180 infected the ependymal cells of the choroid plexus, but the monomorphic T. b. rhodesiense (Liv) was unable to do this . University of Ghana http://ugspace.ug.edu.gh - 5 - C O N T E N T S Page Acknowledgements - - - - - - - - - 2 Abstract - - - - - - - - - - - - - 3 Chapter One - Introduction 1.1 General - - - - - - - - - - - - - 9 1.2 Biochemistry of Trypanosomes - - - 12 1.2.1 Energy Metabolism - - - - - - - - 12 1.2.2 Hydrolytic Enzymes - - - - - - - - 16 1.3 Characterization of Isoenzymes - - 17 1.4 Polypeptide Profiles - - - - - - - 19 1.5 The Functional Anatomy of Trypanosomes - - - - - - - - - - - 20 Chapter Two - Laboratory Materials and Methods 2 . 1 Materials - - - - - - - - - - - - 22 2.1.1 List of Materials - - - - - - - - 22 2.1.2 Buffers - - - - - - - - - - - - - 22 2. 2 Equilibration of DEAE-Cellulose - 22 2. 3 Trypanosomes - - - - - - - - - - - 23 2.3.1 Liverpool Normal (Liv) - - - - - - 23 2.3.2 LSHTM 180F - - - - - - - - - - - - 23 2. 4 Rodents - - - - - - - - - - - - - 24 2. 5 Inoculation - - - - - - - - - - - 24 2. 6 Agar Film Technique - - - - - - - 24 2. 7 Separation of Trypomastogotes from Blood Elements - - - - - - - 26 2. 8 Preparation of Lysates for Electrophoresis - - - - - - - - - 27 2. 9 Subcellular Fractionation of Trypanosome Lysates - - - - - - - 27 2.10 Protein Determination - - - - - - 30 2.11 Starch Gel Electrophoresis (SGE) - 30 2.11.1 Electrophoresis - - - - - - - - - 31 2.11.2 Staining for Enzymes - - - - - - - 31 2.12 Cellulose Acetate Electrophoresis (CAE) - - - - - - - - - - - - - - 33 2.12.1 Enzyme Development - - - - - - - - 33 2.13 SDS-Polyacrylamide Gel Electro­ phoresis (SDS-PAGE) - - - - - - - 34 2.14 Enzyme Assays - - - - - - - - - - 35 2.14.1 Calculation of Enzyme Activity - - 36 2.15 Electron Microscopy - - - - - - - 37 2.15.1 Fixation and Embedding - - - - - - 37 University of Ghana http://ugspace.ug.edu.gh - 6 - Chapter Three - Results 3.1 Agar Film Technique - - - - - - - 38 3.2 Isoenzyme Patterns on Starch Gel and Cellulose Acetate - - - - - - 38 3.3 Polypeptide Profiles from SDS-PAGE 39 3.4 Subcellular Fractionation and Assay of Enzyme Activities - - - - 39 3.5 The Course of Infection - - - - - 46 3.6 Electron Microscopy - - - - - - - 64 Chapter Four - Discussion and Conclusion 4.1 Morphology and Pleomorphism - - - 71 4.2 Isoenzyme Characterization - - - - 73 4.3 Polypeptide Profiles - - - - - - - 75 4.4 Enzyme Assay Analysis - - - - - - 76 4.5 Electron Microscopy - - - - - - - 84 4.5.1 Choroid Plexus - - - - - - - - - - 84 4.5.2 Ultrastructure - - - - - - - - - - 85 4.6 Conclusion - - - - - - - - - - - - 88 APPENDICES A. Materials and Sources - - - - - - 92 B. Buffers 95 C. Protein Determination - - - - - - 97 D. Electrophoretic Conditions for Starch Gel Electrophoresis (SGE) (Tables 3 and 4 ) - - - - - - - - - 99 E. Electrophoretic Conditions for Cellulose Acetate (Tables 5 and 6 ) 104 F. SDS-PAGE Solutions and Reagents - 106 G. Stock Enzyme Assay Mixtures (Table 7) - - - - - - - - - - - - 108 H. p-Nitrophenol Standard Curve (Figure 18) - - - - - - - - - - - m REFERENCES - - - - - - - - - - - - - - - - - - 1 1 2 L I S T O F F I G U R E S I. Aerobic Glycolysis - - - - - - - - 14 2. Anaerobic Glycolysis - - - - - - - 15 3. T. b. rhodesiense LSHTM 180F - - - 25 4A. Sub-cellular Fractionation of Trypanosome Homogenate (Opperdoes e t al., 1977 ) - 28 4B. Subcellular Fractionation of Trypanosome Homogenate - - - - - - 29 5. Diagram of an Electrophoresis Tank 32 6 . The course of infection of T. b. rhodesiense LSHTM 180 in the blood of rats 47 Page University of Ghana http://ugspace.ug.edu.gh - 7 - List of Figures (continued) 7. Subcellular Distribution of HK - - - 55 8 . " " GPI - - 5 6 9 . " " ICD - - 57 10. " " MDH - - 58 1 1 . " " ALAT - - 5 9 12. ” " G6PD - - 60 13. " " PGM - - 61 14. ” " MPI - - 62 15. " " Acid Phosphatase 63 16. The Glyoxylate Cycle - - - - - - - - 79 17. The Protein Standard Curve - - - - - 98 18. p-Nitrophenol Standard Curve 112 L I S T O F T A B L E S 1. Enzyme Activities - - - - - - - - - - 54 2. A Comparison of Selected Enzymes in T. brucei and other organisms - - - 79 3. Electrophoretic Conditions for Starch Gel Electrophoresis (SGE) - - - 99 4. Enzyme Developers for SGE - - - - - - 1 0 1 5. Electrophoretic Conditions for Cellulose Acetate - - - - - - - - - - 1 04 6 . Enzyme Developers for Cellulose Acetate Electrophoresis - - - - - - - 1 05 7. Stock Enzyme Assay Mixtures - - - - - 108 L I S T O F P L A T E S 1. T. b. rhodesiense (Liverpool Normal) - 43 Page 2. Slender Forms of T. b. rhodesiense LSHTM 180 - - - - - - - - - - - - - - 44 3. Giant Form T. b. rhodesiense LSHTM 180 44 4. Short Stumpy Form, T. b. rhodesiense LSHTM 180 - - - - - - - - - - - - - - 45 Isoenzyme Patterns 5. SGE - ACON - - - - - - - - - - - - - - 48 6. SGE - MDH - - - - - - - - - - - - - - 48 7. CAE - M E - - - - - - - - - - - - - - - 48 8 . CAE - ICD - - - - - - - - - - - - - - 49 9. SGE - GPI 49 10. SGE - PGM 49 11. CAE - G6PD - - - - - - - - - - - - - - 50 12. SGE - N H - - - - - - - - - - - - - - - 50 13. CAE - ALAT - - - - - - - - - - - - - - 50 University of Ghana http://ugspace.ug.edu.gh - 8 - 14. Polypeptide Profiles from SDS-PAGE - - 51 15. Polypeptide Profiles from SDS-PAGE - - 52 Electron Microscopy 16. T. b. rhodesiense (Liv) x 20,000 - - - 64 17. T. b. rhodesiense (Liv) x 126,000 with section of tubular mitochondrion - - - 64 18. T. b. rhodesiense (Liv) x 20,000 with double flagellum - - - - - - - - - - - 65 19. LSHTM 180 LS x 20,000, showing nucleus 66 20. LSHTM 180 LS x 126,000 with mitochon­ drion, no cristae - - - - - - - - - - 66 21. LSHTM 180 ST x 20,000 with mitochon­ drion and cristae - - - - - - - - - - 67 22. LSHTM 180 x 80,000, mitochondrion - - 67 23. - 26. Section through choroid plexus of mouse infected with T. b. rhodesiense (Liv) 72 hours after inoculation 23. x 20,000: trypanosomes and red blood cells - - - - - - - - - - - - - - - - 68 24. x 80,000: trypanosome in blood vessel 69 25. x 50,000: Golgi body and flagellum - - 69 26. x 126,000: T. b. rhodesiense (Liv) - - 69 Page Sections through choroid plexus of rats infected with LSHTM 180 24 - 27 days after infection. (4 Plates) - - 70 University of Ghana http://ugspace.ug.edu.gh - 9 - C H A P T E R O N E INTRODUCTION 1.1 General The trypanosome has been described as a destructive piece of machinery, depriving its host of essential substances while burdening it with unwanted wastes; it inflicts mechanical damage through its incessant physical acti­ vity; and it goads the host into responding to its presence and then evades the backlash (Vickerman, 1978). In two vast areas of the world, tropical Africa and South America, trypanosomes are responsible for serious sickness in man and his domestic animals. They also attack draught animals in the Middle East and tropical Asia. Because the trypanosomiases are mainly associated with rural areas in developing countries, accurate fig­ ures are uncertain but the general estimate is that 35 million people are at risk from sleeping sickness in Africa and over 10 million are infected with Chagas disease in Central and South America (WHO, 1976). Throughout Africa other forms of the disease harm domes­ University of Ghana http://ugspace.ug.edu.gh tic stock and the subsequent indirect nutrition and economy are probably more serious than actual human trypanosomiasis. For reasons related to the diseases they cause, the method of transmission and their morphology and biochem­ istry, pathogenic trypanosomes are classified into Salivarian (mainly African) and Stecorarian species. The most important Stecorarian species is T . cruz i which causes Chagas disease in man, and is transmitted by triatomid bugs. Salivarian African trypanosomes are tsetse-borne and their distribution coincides with that of their vectors, represented by flies of the purely African genus Glo s s ina. The most important tsetse-borne Salivarian species from the medical and veterinary points of view include T. b. gambiense and T. b. rhodesiense (the causative agents of "gambian" and "rhodesian" sleep- OJ\i ing sickness respectively),, T. b. brucei, T. vivax andA T. congolense, which cause nagana in cattle. Pathogenic salivarian trypanosomes also include two other species, T . evan s i and T . e q u i p e r dum, which - though not tsetse- borne - are closely related to T. b. brucei. T. evansi is transmitted directly by mechanical inoculators (Tabanid flies), and T. equiper dum, (the causative agent of dourine, a venereal disease of horses) is transmitted by sexual contact between the mammalian hosts. Due to their morphological similarity, T . evansi, T. equiperdum, - 10 - University of Ghana http://ugspace.ug.edu.gh - 1 1 - T » b. brucei, T. b. gambiense and T. b. rhodesiense have been classified together into the brucei group, j[. (Trypanozoon) brucei. T. b. rhodesiense, like T. b. brucei and T . b . gambiense, is mainly extracellular. The trypanosomes live in the body fluids including the blood and later the cerebrospinal fluid where they grow and divide by binary fission. Recently Abolarin et al. , ( 1982) have shown that intracellular multiple division forms of the para­ site occur in the ependymal cells of the choroid plexus. When a susceptible mammal (e.g. rat or mouse) is cyclic­ ally infected with trypanosomes by a tsetse-fly; the first trypoma'stigotes to be seen in the blood are long, thin, cylindrical in cross-section and agranular with a knou?n as free flagellum,^ the long narrow or LN forms. As the infection develops they become leaf-like, have a flat cross-section and begin to divide in the blood (the long flat oT LF form). The LN and LF forms are usually class­ ified together as the long slender or LS form. With the progress of the infection the LF trypomastigotes become short and stumpy, lose the free flagellum and acquire lipid granules. These are called short stumpy or ST forms. This phenomenon, called pleomorphism} is a variable character among the members of the brucei group. The University of Ghana http://ugspace.ug.edu.gh - 12 - most characteristic forms are the LF forms since they are always present in all the species, whereas the propor­ tions of the LN and ST forms, which appear constantly in the tsetse-borne species (T. b . brucei, T. b. gambiense, T. b. rhodesiense) fluctuate in the course of the infec­ tion and may disappear completely after prolonged mechan­ ical passages. Thus laboratory strains maintained by syringe passage eventually become monomorphic with only the LF forms present. They are also the only kind of trypanosomes in those species which are normally trans­ mitted by the mechanical method, viz. in T. evansi and T . equiperdum. Syringe-passaged strains lose their transmissibility by Glossina (tsetse-fly) and their virulence with rodents increases with time (Duke, 1934, 1935). Thus an intra- peritoneal inoculation of a syringe-passaged T . b . rhodesiense into a mouse or rat produces a fulminating infection that kills the rodent in 72-90 hours. 1.2 Biochemistry of Trypanosomes 1.2.1 Energy metabolism Trypanosomatids contain little or no endogenous energy reserves, and without an exogenous carbohydrate supply they stop respiring and become immobile within a minute (Opperdoes e t a1 , 1976). Bloodstream trypomastigotes in University of Ghana http://ugspace.ug.edu.gh - 13 - the brucei group contain some Krebs cycle enzymes, but the cycle is largely inoperative. They are therefore completely dependent on glycolysis for their energy requirements (Bowman and Flynn, 1976), and they metabolize glucose 50 times faster than the mammalian host tissues they infect (von Brand, 1951). Under aerobic conditions, these trypomastigotes catabolize glucose to pyruvate, producing 2 moles ATP per mole glucose. LDH is absent (Dixon, 1966) and NADH generated in glycolysis is reoxidized indirectly by molecular oxygen via DHAP: L-Q!- glycerophosphate dehydrogenase and L—Q!- glycerophosphate oxidase enzyme complex are involved (Grant and Sargent, 1960). (Figure 1.) Under anaerobic conditions the continual reoxidation of NADH is achieved by the operation of glycerokinase (Figure 2). Under these conditions equimolar amounts of pyruvate and glycerol are produced (Ryley; 1956; Grant and Fulton, 1957), and ATP production is halved (Brohn and Clarkson, 1978). Anaerobic conditions can be simulated by incubating trypanosomes with salicylhydroxamic acid (SHAM), an inhibitor of L-O'- glycerophosphate oxidase. Because of the alternate NADH-reoxidation pathway (Figure 2), SHAM is not lethal to trypanosomes (Opperdoes et al., 1976). A combination of SHAM and glycerol, however, has been shown to represent a potent trypanocidal agent. University of Ghana http://ugspace.ug.edu.gh - 14 - Figure 1. AEROBIC GLYCOLYSIS ATP- Glucose G - 6 - P _ADP F - 6 - P ATP- F - 1 , 6 - di P ►ADP 2PGA PEP SDP" T ? S at p^ Pyruvate 1: Hexokinase, 2: Glucose phosphate isomerase, 3: Phospho-fructokinase, 4: Aldolase, 5: Triosephosphate isomerase, 6 : Glyceraldehyde-3-phosphate dehydrogenase, 7: Phosphoglycerate kinase, 8 : Phosphoglycerate mutase, 9: Enolase, 10: Pyruvate kinase, 11 L -Oi- glycerophosphate dehydrogenase, 12 L -01- Glycerophosphate oxidase. University of Ghana http://ugspace.ug.edu.gh - 15 - Figure 2 ANAEROBIC GLYCOLYSIS IGly-3-P 10 Pyruvat e Glucose 1 1 I I F - 1 , 6 - di P 4 ; 5 ■NAD+■ -NADH- DHAP 11 L-Of- glycerophosphate 13 glycerol 1 - 1 1 are as for Figure 1 13 - Glycerokinase University of Ghana http://ugspace.ug.edu.gh - 16 - It has been shown in trypanosomes that the glycolytic enzymes involved in the first part of the glycolytic sequence, the conversion of glucose to 3-phospho glyceric acid (3PGA) are located in microbody-like organelles called glycosomes (Oduro, 1977 ; Oduro et al. , 1980; Opperdoes and Borst, 1977). Recently Oduro (personal communication) has confirmed through electron microscope studies that T . b. brucei glycosomes are microbodies. Glycosomes constitute a separate pool of glycolytic intermediates and adenine nucleotides within the cell (Visser et al. , 1981). Such compartmentation is probably responsible for the very high rate of glycolysis in the parasite. The glycosome has no parallel in the mammalian host and is therefore a logical target for chemotherapy. 1.2.2 Hydrolytic Enzymes Trypomastigotes have been shown to contain lysosomes; a bag of enzymes involved in digestion, some of which are acid proteases, leucine aminopeptidase, phosphatases, Qt-mannosidases , deoxyribonucleases and lipases (Venka­ tesan et al. , 1977 ; Steiger, 1975 ) as well as phospho- lipases (Tizard et al., 1977 ). T . bruce i trypomastigotes sequester host serum protein by pinocytosis via the flagellar pocket (Langreth and Balber, 1975). The proteins fuse with and are digested by so-called primary lysosomes. University of Ghana http://ugspace.ug.edu.gh - 17 - Venkatesan et al., (1977) have shown that the stumpy forms contain higher levels of acid phosphatase and Cathepsin D than the slender forms. This seems to explain the observation made by A. H. Fairlamb (unpub­ lished) that stumpy forms sequester host serum proteins at twice the rate of slender forms. In fact autophagy, the digestion of isolated islands of cytoplasm containing unwanted organelles - also appears to be more active in the stumpy forms (Langreth and Balber, 1975). There is also a marked difference in the localization of acid phosphatase in the two forms. In the slender forms, acid phosphatase is localized in the flagellar pocket and in discrete particles around it, apparently being trans­ ported into the pocket for purposes of digestion of par­ ticles in the pocket (Jadin and Creemers, 1970; Langreth and Balber, 1975; Venkatesan et al., 1977). In the stumpy forms, however, acid phosphatase is distributed throughout the Golgi zone and the rough endoplasmic reticulum. 1 . 3 Characterization of isoenzymes Enzyme electrophoresis has been widely used in the characterization of parasitic and non-parasitic protozoa, e.g. Entamoeba (Reeves and Bischoff, 1968; Sargeaunt et a 1. , 1978); Le i s hmania (Al-Taqi and Evans, 1978); Trypanosoma (Godfrey and Kilgour, 1976 ; Miles et al. , University of Ghana http://ugspace.ug.edu.gh - 18 - 1977 ). Godfrey and Kilgour ( 1976) differentiated T . b . gambiense from T. b. brucei and T. b. rhodesiense by its characteristic aminotransferase pattern. Miles et a l . , (1977) identified two distinct strain groups of T. cruzi circulating in independent sylvatic and domestic cycles in Brazil. Isoenzymes have been defined as multiple separable forms of enzymes occurring within the same organism and having similar catalytic activities but not necessarily similar molecular structures (Shaw, 1969). Harris and Hopkinson (1976) classified isoenzymes into three major groups. 1. Those arising from multiple genetic loci coding for functionally the same enzyme. 2. Those arising from multiple alleles at a single locus: 3 Those arising from post-translational changes - so-called secondary isoenzymes. The demonstration of structural differences among iso­ enzymes by electrophoresis depends largely on the iso­ enzymes having different electrical charges; therefore a substitution of one amino acid for another with an equal charge will not be detected. Thus two enzymes are not necessarily structurally identical because their elec­ trophoretic mobilities are the same; hence two samples are not necessarily from related organisms because they have the same electrophoretic pattern for one enzyme. However the more enzymes that are examined without any University of Ghana http://ugspace.ug.edu.gh - 19 - differences being found between the two samples, the greater the likelihood that the two S«twpl e.5 are from related organisms, and if so the closer the relation­ ship . 1.4 Polypeptide Profiles Like isoenzyme patterns, polypeptide profiles can give an idea of the relationship between two organisms. Sodium dodecyl sulphate (SDS) - polycrylamide gel electro­ phoresis (SDS - PAGE) has been used to study the proteins and RNA's of viruses (Studier; 1973; Laemmli, 1970), and the proteins of bacteria (Swindlehurst et al., 1977). It has also been used to characterize Trypanosoma, (Schizotrypanum) species from insectivorous bats (Micro- chiroptera) , (Taylor et al. , 1982), and subgenus Trypanozoon (Taylor et al. , 1983), as well as to differ­ entiate between strains of South American trypanosomes (Taylor and Williams, 1977). The method involves coating the soluble proteins of an organism with negative charges and electrophoresing them on polyacrylamide gels; "this produces characteristic poly­ peptide profiles for different organisms. University of Ghana http://ugspace.ug.edu.gh - 20 - 1 . 5 The Functional Anatomy of Trypanosomes Bloodstream trypanosomes in the brucei group have essen­ tially the same ultrastructure. Pellicular microtubules , longitudinally disposed, lie immediately beneath the plasma membrane of the flagellates body and maintain its elongate shape. The single flagellum arises from a basal body (Kinetosome) submerged in the floor of the flagellar pocket. There is a single mitochondrion which is tubular and runs the length of the trypanosome. It arises from the kinetoplast, a DNA-containing organelle. In the slender forms, internal cristae are almost absent in the mitochondrion, but as the flagellates develop through intermediate to stumpy forms the width of the mitochon­ drial canal increases and more cristae are formed (Vickerman, 1965). However, in both these forms, the mitochondrion is non-functional with respect to Krebs cycle and electron transport. In the trypomastigote and epimastigote forms in the tsetse-fly mid gut and salivary glands respectively, the mitochondrion has abundant cristae, with fully functional Krebs cycle enzymes and cytochromes (Vickerman> 1965), The nucleus has a nucleolus and, as in all animal cells, it is bounded by a double membrane punctured by pores. The outer membrane is continuous with a system of mem­ University of Ghana http://ugspace.ug.edu.gh - 21 - branous cytoplasmic tubes - the endoplasmic reticulum, many of which are studded with ribosomes. Golgi apparatus, which are involved in the packaging of cellular secretions into membrane-bound vessicles, lysosomes, are also present in the flagellates. As mentioned earlier, trypanosomes also contain glyco­ somes (microbodies) which have the unique biochemical property of containing most of the enzymes of the glycolytic pathway (Oduro, 1977). In the bloodstream trypomastigotes in the brucei group, the plasma membrane is overlaid by a surface coat of glycoprotein 12-15 nm. thick which contains the variable antigen (VA) of trypanosomes (Vickerman, 1969; Cross, 1975 ; Fruit et al. , 1977), and is responsible for antigenic variation exhibited by the flagellates (Cross, 1978; Vickerman, 1978). In this study, enzyme activity analysis (total enzyme assays and isoenzyme studies), polypeptide chain profiles and ultrastructural studies have been used to compare the slender and stumpy forms of a pleomorphic strain and a monomorphic strain of T. b. rhodesiense. This is an attempt to discover what changes, if any, take place when a slender trypomastigote becomes stumpy, and when a pleomorphic strain becomes monomorphic. University of Ghana http://ugspace.ug.edu.gh C H A P T E R T W O M A T E R I A L S A N D M E T H O D S University of Ghana http://ugspace.ug.edu.gh - 22 - LABORATORY MATERIALS AND METHODS 2 . 1 Materials 2.1.1 List of Materials 2 .1 . 2 See Appendix A. Buf f er s The quantities of material used to make up buffers are given in Appendix B. a) For trypanosome separation and mainte­ nance: PSG - phosphate-buffered saline glucose pH 8.0. b) For cell fractionation: TSE - Tris- sucrose EDTA pH 7.8. 2 . 2 Equlibriation of DEAE-Cellulose (DE-52) pH adjusted to 8.0 with 5% H„P0,3 4 The dry DE-52 was suspended in PSG and the The material was then washed a minimum of 6 times to remove all fines. The pH and conductivity of the slurry were checked and the material stored at -20°C until required. University of Ghana http://ugspace.ug.edu.gh - 23 - 2.3 Trypanosomes Two strains of T. b. rhodesiense were used: 2.3.1 Liverpool Normal (Liv). This strain was isolated from man in 1923 (Yorke et al. , 1929). It was maintained in mice by syringe passage every 2-3 days until it was frozen in 1960. It produces a very high parasitemia in rats and mice and usually kills the rodent host 72 hours after inoculation. The infection is completely monomorphic - i.e. produces only one type of parasite, the LF. The stabilate was obtained from the LSH & TM cryobank at Winches Farm Field Station. 2.3.2 LSHTM 180F. This strain was isolated from man (Dumelo) in Botswana by Ormerod in 1960 (Apted et al. , 1963). Since then it has been passaged through rats and mice and frozen a number of times (Fig. 3). The strain is pleomorphic, and therefore produces a gradation between blood forms of the parasite in the course of infection. The first trypomastigotes seen in the blood about 30 hours after infection are long, slender, cylindrical and agranular (long, narrow or LN forms). As the infection progresses, these gradually become flattened (long flat or LF forms) and finally short, stumpy with increasing number of lipid granules (short stumpy or ST forms). University of Ghana http://ugspace.ug.edu.gh - 24 - The course of the Infection is shown in Fig. 6 . In the present state of passage of the LSHTM 180 strain, the rodent host usually dies 3 - 4 weeks after inoculation. 2.4 Rodents Adult male and female albino Wistar rats weighing 150­ 2 0 0g were used for the passage and harvesting of trypano- s ones. was checked by counting the number of motile trypanosomes in a drop of tail blood under a X40 objective of the light microscope 72 - 96 hours after infection. For harvesting trypanosomes 10 - 15 rats were each infected with 0.5ml of heavily infected blood from the donor. 2 . 6 Agar Film Technique The method is that of Ormerod et al. , ( 1963 ). It allows the simultaneous counting of trypanosomes and their lipid granules, so enabling one to differentiate between the LF University of Ghana http://ugspace.ug.edu.gh - 25 - Figure 3. T. b. rhodesiense LSHTM 180F Man.(ttumelo): Mababe, Ngamiland, Botswana Rat 6 March 19 60 EATRO 180 1 April 1960 LSHTM 180A 26. Oct. 1968 Y t LSHTM 180 B1A 18,Anril 1975 ¥ Mouse 14 May 19 7 6 LSHTM 180E 6 Match 1978Mane VMouse 7 March 1978 LSHTM 180B 5 July 1970 Mous e LSHTM 180D 11 July 1976 Rat LSHTM 180F 15 March 1978 Syringe Passage : Frozen in liquid nitrogen University of Ghana http://ugspace.ug.edu.gh - 26 - and ST trypomastigotes. The technique involves making agar slides from 1.2% agar in 0.85% NaCl. A "pinhead” of blood from the infected animal is placed on the slide and sandwiched between a coverslip and the agar film. The slides were left on the bench for about a minute to allow the trypomastigotes to move to the phalanx of the blood film on agar. From a recognizable point on the phalanx, the trypomastigotes were counted per agar field using X100 oil-immersion phase contrast objective on a Zeiss microscope. Counting was continued until the starting point was reached and the number of trypomastigotes with and without granules per field was recorded. 2.7 Separation of Trypamastigotes from Blood Elements When the desired stage of parasitaemia was obtained, the rats were bled under anaesthesia by cardiac puncture using heparin as anticoagulant. The blood was diluted 1:3 with cold PSG and kept on ice. The trypanosomes were separated from blood elements by the method of Lanham and Godfrey (1970). The infected blood was passed through equilibrated DE-52 in a Buchner funnel with a sintered glass base, and the trypanosomes eluted with cold PSG. The column eluates were collected on ice and centrifuged at 1500g for 20 minutes at 4°C. University of Ghana http://ugspace.ug.edu.gh - 27 - The supernatant was discarded and the packed trypanosomes were washed twice by resuspending them in ice-cold PSG before centrifuging again at 1500g for 20 minutes. 2.8 Preparation of Lysates for Electrophoresis The washed trypanosome pellet was resuspended in an equal volume of ■'cocktail" containing 2mM each of E-amino-n- caproic acid, dithiothreitol and EDTA in water and mixed thoroughly. The resultant mixture was freeze-thawed in liquid nitrogen 3 times and then centrifuged at 10,000g for 30 minutes at 4“C. The pellet was discarded and 20ul aliquots of the supernatant dropped into liquid nitrogen to form beads which were stored in liquid nitrogen until required. 2.9 Subcellular Fractionation of Tryapanosome Lysates Subcellular fractionation was carried out to provide material to study the distribution of certain enzymes in trypanosomes. Washed trypanosome pellet was suspended in twice the volume of cold TSE buffer pH 7.8 and homogen­ ized on ice in a Potter-Elvejham homogenizer with a teflon piston for 5 minutes at 300 revolutions/minute. After checking for at least 90% broken cells under a X40 objective, the homgenate obtained was divided into two and treated as shown in Figures 4A. and 4B. University of Ghana http://ugspace.ug.edu.gh - 28 - Figure 4A. Subcellular fractionation of trypanosome homogenate This is the procedure described by Opperdoes et al., (1977). Trypanosome Homogenate s lOOOg, 10 minutes x 2 I1 KS 71 -►di s card I 139 ,000g T 3 1 hour Suspended in TSE buffer and beaded 1 (Supernatant) 139 KS 1 139 KP (Pellet) JBeaded K.r ISuspended in TSE buffer and beaded. As well as 14.5 KP, 139 KP and 139 KS , a sample of 1 KS was also beaded. All the beads were stored in liquid nitrogen until required. 1. KS refers to the resultant supernatant after centri­ fugation at a certain speed, e.g. 1 KS is the super- tant after centrifugation at lOOOg. 2. KP is the pellet after centrifugation, e.g. 1KP being the pellet from lOOOg centrifugation. 3. TSE is the Tris-Sucrose - EDTA buffer pH 7.8. University of Ghana http://ugspace.ug.edu.gh - 29 - Figure 4B. Subcellular fractionation of trypanosome homogenate Trypanosome homogenate lOOOg, 10 minutes x 2 fdiscard 1 KP (pellet) 1 KS (supernatant) 3 , 400g , 10 minutes x 2 f3.4 KS (supernatant) 42 ,250g, 10 minutes x 2 3.4 KP (pellet) Suspended in TSE and beaded (pellet) 42 KP * 1. 42 KS (supernatant) 105 , OOOg , 1 hour Suspended in TSE and beaded (supernatant) 105 KS 1beaded 105 KP (pellet) i Suspended in TSE and beaded. All the beads were stored in liquid nitrogen. University of Ghana http://ugspace.ug.edu.gh - 30 - 2.10 Protein Determination Protein was determined according to the method of Lowry e t al. , (1951). Detail of reagents and standard curve are shown in Appendix C. 2.11 Starch Gel Electrophoresis (SGE) Electrophoresis was carried out on thin starch gel essentially as described by Smith, (1976) using the modifications and conditions described by Miles et al. , (1980). The buffers and electrophoretic conditions are as shown in Table 3, Appendix D The enzymes analyzed were: acid phosphatase (E.C:3.1.23, ACP), aconitase (E.C.42.1.3, ACON), esterase (E .C .3.1.1.1, ES), glucose phosphate isomerase (E .C.5.3.1.9 , GPI) , malate dehydrogenase (E.C . 1 .1.1■37 , MDH), mannosephosphate isomerase (E.C . 5 . 3 .1 . 8 , MPI), purine nucleoside hydrolase (E.C . 3 . 2 . 2 . 1 , NH) , phosphoglucomutase (E .C.2.7 . 5 . 1 , PGM), alanine aminotransferase (E .C .2 . 6 . 1 . 2 , ALAT), aspartate aminotransferase (E .C .C . 2 . 6 . 1 . 1 , ASAT), malate dehydrogenase (oxaloacetate decarboxylating) (NADP+) (E .C.1.1.1•40 , ME), isocitrate dehydrogenase (NADP+) (E .C .1.1•1.42, ICD), glucose-6-phosphate dehydrogenase (E.C.1.1.1.49 , G6PD). University of Ghana http://ugspace.ug.edu.gh - 31 - 2.11.1 Electrophoresis Thin layer of starch gel plates measuring 0.1 x 14.0 x 321.5 cm were made with 11.2% solution of hydrolyzed ("Table 3, Appendix t>) potato starch in the appropriate buffer^. When the gels had solidified and cooled, slots were made in them with a slot template and samples applied to the slots on pieces of boiled cotton thread (Anchor 6-stranded embroidery cotton) cut to size. The tanks for electrophoresis were set up as shown in Figure 5. Electrophoresis was carried out at a constant voltage with variable current, the plates being held at 8 °C for the duration of the run by means of a cooling plate. The electrophoretic conditions for each enzyme are shown in Table 3, Appendix D. 2.11.2 Staining for Enzymes After electrophoresis, zones of enzyme activity were located by applying an appropriate developer solution either as an agar overlay or on filter paper (see Table 4, Appendix D). Agar overlays were used for developers with formazan (MTT) dye-linked reaction. Fluorescent developers were usually applied on filter paper, and the reactions monitored under UV light (360 nm) . The developer plates were incubated at 37°C until fully developed as judged by eye, from 10 minutes for ALAT and GPI to 2 hours for ACON. Initially control plates were University of Ghana http://ugspace.ug.edu.gh - 32 - Figure 5. Diagram of an electrophoresis tank a : bridge buffer b: electrode buffer c : cooling plate d: wick e: Melinex sheet f: gel plate g: cover plate h : cover plate University of Ghana http://ugspace.ug.edu.gh - 33 - set up for each enzyme, for which an essential ingredient like a specific enzyme substrate was left out of the developer. This was to ensure that the enzyme activity produced was due to the enzyme in question. 2.12 Cellulose Acetate Electrophoresis (CAE) CAE was used for the enzymes which gave poor results with thin layer SGE, e.g. ICD, G6PD, ME. Electrophoresis was carried out essentially as described by Lanham et al. , (1981). The basic Helena system (Beaumont, Texas) was used, with Titan III cellulose acetate plates (94 x 76mm, Helena Cat. No. 3024). The tank buffers and electro­ phoretic conditions are shown in Table 5, Appendix E. The cellulose acetate plates were presoaked in the appropriate buffer as per the manufacturer's instruc­ tions, and samples loaded wih the 8-sample CPK applica­ tion system (Cat. No. 7092). Electrophoresis was carried out as directed by the manufacturer with a Titan Power supply providing a constant voltage with a variable current. 2.12.1 Enzyme Development Enzyme developer solutions were applied either as agar underlays or on filter paper. Agar underlays, which were used with MTT dye-linked reactions were made up and University of Ghana http://ugspace.ug.edu.gh - 34 - allowed to set before the end of electrophoresis. After the run, the cellulose acetate plate was placed face down on the underlay, excluding all air bubbles, and incubated at 37°C until the zones of enzyme activity were fully developed as judged by the eye. Fluorescent developers were applied on filter paper. The plate was sandwiched together with the developer-soaked filter paper between two rectangular glass plates pressed tightly together with strong bulldog clips, and incubated at 37°C. The development of zones of enzyme activity was monitored under UV light (360 nm). The enzyme developers are shown in Table 6 of Appendix E. 2.13 SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) SDS-PAGE was carried out in a discontinuous buffer system (Laemmli, 1970), using an LKB2001 Vertical Electrophor­ esis unit with an LKB 2197 constant wattage power supply. Subcellular trypanosome fractions were separated on a 12% polyacrylamide gel with a 3% stacking gel. (See Appendix F.) Samples (containing about 50ug protein) were dis­ solved in an equal volume of SDS-sample buffer (Appendix F), boiled for 2 minutes and applied to the gel. Elec­ trophoresis was carried out at 40mA for 5 hours or until the bromophenol blue tracking dye reached the bottom of the gel. Gels were stained overnight in Coomassie blue University of Ghana http://ugspace.ug.edu.gh - 35 - (0.025% Coomassie blue in 40.5ml methanol: 52 ml H^O: 0.7 mis glacial acetic acid and destained over a 24-hour period in a solution consisting of 405 ml ethanol, 250 ml H^O and 70 ml glacial acetic acid. Gels were dried by sandwiching them between a polythene sheet and wet 3MM filter paper, clamped tightly to a flat rigid support with bulldog clips and left overnight at 37°C. 2.14 Enzyme Assays The assay procedures used for all enzymes except acid phosphatase (E .C .3.1 . 2 . 3 , ACP) were based on the oxida­ tion or reduction of pyridine nucleotide co-enzymes. Changes in optical density at 340nm were followed at 25°C in a Unicam SP1700 UV spectrophometer coupled to a Unicam AR25 Linear Recorder. All assays were carried out in a final volume of 1.0ml using a 1cm light path. Acid phos­ phatase activity was measured by following the release of p-nitrophenol from p-nitrophenyl phosphate at 430 nm in the same spectrophotometer. Equal volumes (0.5ml) of sub-cellular fractionation sample and 0.025M p-nitrophen­ yl phosphate in 0.1M Acetate buffer pH 5.0 were incubated at 38°C for 30 minutes. The reaction was stopped by the addition of 2.0 ml 5% trichloroacetic acid. The reaction tubes were then centrifuged at 500g for 15 minutes to obtain a clear supernatant. Liberated p-nitrophenol was estimated at 430 nm after the addition of 1ml of 2M University of Ghana http://ugspace.ug.edu.gh - 36 - NH^OH (pH 13.5) to 1 ml of TCA supernatant. A standard curve was prepared with a known range of p-nitrophenol concentration which gave a linear curve (Figure 18, Appendix H). Details of all enzyme assay mixtures are given in Appendix G. 2.14.1 Calculation of Enzyme Activity a) Acid Phosphatase The amount of p-nitrophenol released, in moles, was determined from a p-nitrophenol standared curve, Figure 18 (Appendix H). The units of activity are mole/min/mg protein. b) Other Enzymes Enzyme activities were calculated from the rate of production or utilization of reduced pyridine nucleotide using an extinction coefficient at 340 nm of 6 . 2 2 x 1 0 ̂ cm /mole. Specific activity = V DE ------------------------ x — mole/min/mg protein E x d x v x c (protein Dt where V = total assay volume = 1.00ml E = extinction coefficient of NADH and NADPH at 340nm 3 2= 6 . 2 2 x 10 cm /mole, d = light path = 1cm DE = change in extinction v = sample volume = 0.01ml Dt = reaction time c = protein concentration of sample in mg/ml. University of Ghana http://ugspace.ug.edu.gh - 37 - 2.15 Electron Microscopy Electron microscopy was used to: 1 . compare the ultrastructure of the different trypomastigotes, pure samples of which were obtained by the method of Lanham and Godfrey (1970) described on page 26. 2 . study the choroid plexuses of rats infected with the two strains for evidence of intracellular trypomas­ tigotes. To this end, choroid plexuses were dissec­ ted from brains of rats which had been infected with either T. b. rhodesiense (Liv) or LSHTM 180 3 days or 3 - 4 weeks respectively after infection. 2.15.1 Fixation and Embedding Material (choroid plexus or trypomastigotes) was immedi­ ately placed in 3% glutaraldehyde with cacodylate buffer at pH 7.4 and left for 8 hours, washed overnight in the same buffer, and post-fixed with 1 % osmium tetraoxide (OsO^) for 2 hours. The material was then dehydrated in a graded series of 30%, 60%, 90% and 100% methanol, each for 5 minutes duration. The blocks were stained with 1% phosphotungstic acid and embedded in 'Spurr' (Spurr, 1969). Sections were cut with a Cambridge Huxley Ultra­ Microtome and observed with a Zeiss Em9 electron microscope. University of Ghana http://ugspace.ug.edu.gh C H A P T E R T H R E E R E S U L T S University of Ghana http://ugspace.ug.edu.gh - 38 - 3.1 Agar Film Technique The agar film technique shows the morphological differ­ ences between the slender and stumpy forms of LSHTM 180 (Plates 2 - 4). Plate 2 shows that the slender trypo- mastigote forms of LSHTM 180 are agranular and have longer flagella than the stumpy forms (Plate 4) which have lipid granules (arrowed dark spots, Plate 4). The technique was also used to study the course of the infection of LSHTM 180 in rats. Figure 6 shows that the infection is synchronous, producing alternate peaks of slender and stumpy forms at 72 hours, and 144 hours respectively• 3.2 Isoenzyme Patterns on Starch Gel and Cellulose Acetate The three trypomastigote forms, LSHTM 180 slender and stumpy and T. b. rhodesiense (Liv) had identical isoenzyme patterns for 8 of the 9 enzymes analysed (Plates 6 - 13). The stumpy form of LSHTM 180 showed a single electrophoretic band on starch gel for aconitase (ACON), which was absent in both the slender LSHTM 180 and the monomorphic T. b. rhodesiense (Liv) (Plate 5). University of Ghana http://ugspace.ug.edu.gh - 39 - 3.3 Polypeptide Profiles from SDS-PAGE The polypeptide profiles of subcellular fractions from the three trypomastigotes are shown in Plates 14 and 15. Clear differences exist: 1 . between the three trypomastigote forms for each subcellular fraction, e.g. profiles 1 - 3 on each plate; 2 . between the subcellular fractions for each trypo­ mastigote; e.g. between profile 1, the lOOOg super­ natant (IKS) and profile 4, the 42,000g pellet (42KP) of slender LSHTM 180 (Plate 14). The polypeptide molecular weight range where the greatest differences are found is between 60,000 daltons (60K) and 40,000 daltons (40K). A 58,000 daltons (58K) molecular weight polypeptide is consistently absent from all fractions of the stumpy LSHTM 180, but is present in some fractions of both slender LSHTM 180 and T. b. rhodesiense (Liv), most prominently in the IKS profiles (Plate 14). 3.4 Subcellular Fractionation and Assay of Enzyme Activities Trypanosome homogenates were fractionated by differential centrifugation to provide large granule (14.5KP), small granule (139KP) and final supernatant (139KS) fractions for all enzyme assays except acid phosphatase. The University of Ghana http://ugspace.ug.edu.gh - 40 - results obtained by assaying these fractions are shown in Table 1 and Figures 7 - 1 5 . Figure 7 shows that there is about twice as much hexokinase (HK) in the slender and stumpy LSHTM 180 as there is in T. b. rhodesiense (Liv). LSHTM 180 LS and ST have identical distribution of HK, with about 80% of the activity in the 14.5KP fraction, 18% in the 139KP fraction and 2% in the final supernatant (139KS). As with HK, the slender and stumpy LSHTM 180 has similar distribution of glucose phosphate isomerase (GPI) (Figure 8 ). About 60% and 30% of the activity was found in the 14.5KP and 139KS fractions respectively, and the remain­ der in the 139KP fraction. T. b. rhodesiense (Liv) had a different distribution - 61% of GPI activity was in the 139KS fraction with 24% and 15% in the 139KP and 14.5KP fractions. All the isocitrate dehydrogenase (ICD) activity of the stumpy LSHTM 180 and the monomorphic T. b. rhodesiense (Liv) was found in the 139KS fraction. In the slender LSHTM 180, about 65% of ICD activity was in the 14.5KP fraction and the rest in the 139KS fraction. The ICD activity of the stumpy LSHTM 180 was 2i times and 6 times higher than in the slender LSHTM 180 and monomorphic T . b. rhodesiense (Liv) respectively - Figure 9. The distribution of malate dehydrogenase (MDH), the other Krebs cycle enzyme studied, was identical in all three trypomastigotes with less than 10% of the activity in the University of Ghana http://ugspace.ug.edu.gh - 41 - 14.5KP fraction, about 20% in the 139KP fraction and over 70% in the final supernatant (139KS) - Figure 10. The distribution pattern of alanine amino transferase (ALAT) shown in Figure 11 is similar to that of MDH. The distribution of glucose-6-phosphate dehydrogenase (G6PD) is shown in Figure 12. The slender and stumpy forms of LSHTM 180 showed about equal G6PD activity which was about 3 times the activity in T. b. rhodesiense (Liv). The three trypomastigotes showed different distribution patterns. The % distribution in the 14.5KP, 139KP, and 139KS are as follows: LSHTM 180. slender - 34%: 21%: 55%, stumpy LSHTM 180 - 63%: 39%: 8%, T. b. rhodesiense (Liv) 38%: 54%: 8%. Figure 13 shows that all the phosphoglucomutase (PGM) activity was located in the final supernatant (139KS) in all three trypomastigotes, with stumpy. LSHTM 180 showing twice as much activity as LSHTM 180 LS and three times as much activity as T. b. rhodesiense (Liv). Mannose phosphate isomerase (MPI) activity was distribu­ ted between the 14.5 KP and 139KS fractions (Figure 14). In T. b. rhodesiense (Liv), MPI was evenly distributed between the two fractions. About 70% of MPI activity in LSHTM 180, LS was located in the 139KS fraction and 30% in the 14.5KP fraction. The reverse was true for the stumpy LSHTM 180. Acid phosphotase (ACP) activity was determined in sub- University of Ghana http://ugspace.ug.edu.gh - 42 - cellular fractions from the differential centrifugation scheme shown in Figure 4B. The distribution of ACP activity is shown in Figure 15. The long slender LSHTM 180 and the monomorphic T. b. rhodesiense (Liv) had identical distribution patterns with about 30% of the activity in the 42KP fraction, 60% in the 105 KP fraction and less that 10% in the final supernatant (105KS) The stumpy LSHTM 180 showed 65% activity in 42KP, 35% in 105KP and 5% in 105KS fractions. University of Ghana http://ugspace.ug.edu.gh - 43 - Agar Film T e chnique Plate 1 T . b . rhodesien s e (Liverpool Normal) on agar film. Judging by the eye the sizes of these trypomastigotes are about the same as the short stumpy form of LSHTM 180 (Plate 4) , but unlike them the Liverpool Normal Strain (Liv) is devoid of lipid granules. University of Ghana http://ugspace.ug.edu.gh University of Ghana http://ugspace.ug.edu.gh - 44 - P 1* a t e 2 T he s l e n d e r f o rm s ( LN a n d LF) of T. b. r h o d e s i ­ e n s e L S H T M 180. The LN are the f i r s t b l o o d f o r m s w h i c h a r e s e e n f o l l o w e d by the LF c i r c u l a t i n g a b o u t 30 h o u r s a f t e r i n o c u l a t i o n . L i k e the L i v . S t r a i n , t h e s e t r y p o m a s t i g o t e s a r e a g r a n u l a r P l a t e 3 G i a n t f o r m of T. b. r h o d e s i e n s e L S H T M 180. It is a b i g g e r v e r s i o n of the s l e n d e r f o r ® , and is in fact d i v i d i n g . University of Ghana http://ugspace.ug.edu.gh PLATE 2 6 o o ° r ' j Q & O r P LAT E 3 V J r v * . . . . * 1 . © University of Ghana http://ugspace.ug.edu.gh - 45 - P l a t e 4 T. b. r h o d e s i e n s e L S H T M 180, s h o r t s t u m p y form. T h e s e t r y p o m a s t i g o t e s c o n t a i n g r a n u l e s w h i c h h a v e b e e n s h o w n to be l i p o i d ( O r m e r o d a n d P a g e , 1 9 6 7 ) . T h i s f o r m f i rs t a p p e a r s in the b l o o d a b o u t 96 h o u r s a f t e r i n o c u l a t i o n , t h e i r n u m b e r s i n c r e a s i n g u n t i l 144 h o u r s a f t e r i n o c u l a t i o n w h e n t h e y a r e the o n l y b l o o d f o r m s s e e n c i r c u l a t i n g . T he a r r o w e d t r y p o m a s t i g o t e has at l e a s t two f l a g e l l a a n d a p p e a r s e i t h e r to be d i v i d i n g by m u l t i p l e f i s s i o n or to be a r r e s t e d at t h a t s t a g e . University of Ghana http://ugspace.ug.edu.gh University of Ghana http://ugspace.ug.edu.gh - 46 - 3.5 The C o u r s e of I n f e c t i o n T h e c o u r s e of i n f e c t i o n of L S H T M 180 w i t h p a r t i c u l a r r e f e r e n c e to c y t o p l a s m i c i n c l u s i o n s was s t u d i e d in d e t a i l b y O r m e r o d ( 19 6 3 ) a n d by O r m e r o d and V e n k a t e s s a n (19 7 1 ) As s h o w n in F i g u r e 6 , the i n f e c t i o n p r o d u c e d is s u f f i ­ c i e n t l y s y n c h r o n o u s to a l l o w p u r e p o p u l a t i o n s of l ong s l e n d e r ( a g r a n u l a r ) a n d s h o r t s t u m p y ( g r a n u l a r ) f o r m s to be i s o l a t e d at 72 h o u r s and 144 h o u r s r e s p e c t i v e l y . T h i s s y n c h r o n y , h o w e v e r , o c c u r s o n l y at the b e g i n n i n g of the i n f e c t i o n . A f t e r the f i r s t r e m i s s i o n w h e n v e r y few p a r a s i t e s are s e e n in the b l o o d ( a b o u t 168 h o u r s ) , m i x e d p o p u l a t i o n s c o - e x i s t at a l l t i m e s . As the i n f e c t i o n p r o g r e s s e s , t h e r e are r e p e a t e d r i s e s and f a l l s in b l oo d t r y p o m a s t i g o t e l e v e l s u n t i l the r o d e n t h o s t d i e s 3 - 4 w e e k s a f t e r i n o c u l a t i o n . T h e L i v e r p o o l N o r m a l S t r a i n p r o d u c e s a f u l m i n a t i n g i n f e c t i o n in the b l o o d w i t h o u t r e m i s s i o n , k i l l i n g the r o d e n t h o s t a b o u t 72 h o u r s a f t e r i n o c u l a t i o n . University of Ghana http://ugspace.ug.edu.gh P A R A S I T A I I M I A (N o of T r y p a n o s o m e s / f i e l d ) - 47 - Figure 6. The course of infection of T. b. rhodesiense LSHTM 180 in the blood of rats HOURS OF INFECTION University of Ghana http://ugspace.ug.edu.gh - 48 - I s o e n z y m e P a t t e r n s on S t a r ch G e l and C e l l u l o s e A c e t a t e P l a t e 5 ACONITASE (ACOMJ - STARCH GEL ELECTROPHORESIS (SGE) 0 . 1 M T r i s - O . W 2 8 M C i t r i c a c i d pH 7.5; 2 0 v / c m ; 180 m i n u t e s . 1. T. b. r h o d e s i e n s e (Liv) 2 . L S H T M 180, ST 3. L S H T M 180, LS P l a t e 6 MALATE DEHYDROGENASE (MDH) - SGE 0 . 1 M Na HPO - 0 . 0 1 4 M c i t r i c a c i d , pH 7.0; 2 0 v / c m ;2 4 9 0 m i njit es S a m p l e s 1 - 3 as for P l a t e 5. P l a t e 7 MALIC ENZYME (ME) - CELLULOSE ACETATE ELECTROPHORESIS (CAE) 0 . 0 0 6 M Na HPO. - 0 . 0 1 4 M N a H PO , pH 8.0; 2 0 0v; L 4 2 4 50 m i n u t e s 1- T . b . r h o d e s i e n s e ( L iv) 10 KS* 2. L S H T M 180, ST 10 KS 3. L S H T M 180, LS 10 KS 4. T. b. r h o d e s i e n s e ( L iv) 139 K.P 5 . L S H T M 1 8 0 , ST 1 3 9 .KP 6 . L S H T M 1 8 0 , LS 1 3 9 K P * The s u p e r n a t a n t from a 1 0 , 0 0 0 g c e n t r i f u g a t i o n of a t r y p a n o s o m e h o m o g e n a t e - University of Ghana http://ugspace.ug.edu.gh University of Ghana http://ugspace.ug.edu.gh Plate 8 ISOCITRATE DEHYDROGENASE (ICD) - CAE C o n d i t i o n s the s a m e as ME a b o v e . S a m p l e s 1 - 6 , as for P l a t e 7. P l a t e 9 GLUCOSE PHOSPHATE ISOMERASE (GPI) - SGE 0 . 1 6 2 . N a ^ H P O ^ - 0 .038 N a H PO^ , pH 7.4; 2 0 v / c m ; 135 1. T. b. r h o d e s i e n s e (Liv) 2. L S H T M 180, LS 3. L S H T M 180, ST Plate 10 PHOSPHOGLUCOMUTASE (PGM) - SGE - 49 - m i n u t e s . 0 . 1 M T r i s - 0 . 1 M m a l e i c a c i d - 0 . 0 1 M E D T A - 0 . 0 1 M M g C l 2 pll 7.4; 1 3 . 5 v / c m ; 180 m i n u t e s . S a m p l e s 1 - 3 as for P l a t e 9. University of Ghana http://ugspace.ug.edu.gh PLATES 8 - 1 0 1 2 3 4 5 6 4 a « T " 2 3 10 University of Ghana http://ugspace.ug.edu.gh - 50 - GLUCOSE- 6 -PHOSPHATE DEHYDROGENASE (G6 PD) - CAE 0 . 0 5 . T r i s - 0 . 0 5 M m a l e i r a c i d - 0 . 0 0 5 M E D T A - 0 . 0 0 5 M M g C 1 2 pH 8 . 6 ; 13Qv; 55 m i n u t e s . 1. T. b. r h o d e s i e n s e ( L i v ) 1 4 . 5 K P 2. L S H T M 180, ST 1 4 . 5 K P 3. L S H T M 180, ST 1 4 . 5 K P 4. T. b. r h o d e s i e n s e ( L i v ) 1 3 9 K P 5. L S H T M 180, LS 1 3 9K P 6 . L S H T M 180, ST 1 3 9 K P P l a t e 1 2 NUCLEOSIDE HYDROLASE (NH) - SGE 0 . 0 4 M T ris - 0 . 4 4 M b o r i c a c i d pH 6.5; 2 0 v / c m ; 180 m i n u t e s . S a m p e s 1 - 3 are for P l a t e s 9 a n d 10. Plate 13 ALANINE AMINOTRANSFERASE (ALAT) - CAE 0 . 6 6 M T ris - 0 . 0 B 3 M c i t r i c a c i d pH 8 . 6 ; 200v; 25 m i n u t e s . Plate 11 S a m p l e s 1 - 6 are as for p l a t e 7. University of Ghana http://ugspace.ug.edu.gh PLATES 11 - 13 13 University of Ghana http://ugspace.ug.edu.gh - 51 - P o l y p e p t i d e P r o f l l s s f r o m SDS*~PAGE Plate 14 M* M a r k e r 1 . L S H T M 180, LS IKS 2 . L S H T M 180, ST IKS 3 . T. b. r h o d e s i e n s e ( L i v ) IKS 4 . L S H T M 180, LS 4 2 KP 5 . L S H T M 180, ST 4 2 K P 6 . T. b . r h o d e s i e n s e ( L i v ) 4 2 KP 7 . L S H T M 180, LS 1 0 5 K P 8 . L S H T M 180, ST 1 0 5 K P 9 . T. b. r h o d e s i e n s e ( L i v ) 1 0 5 K P of the f o l l o w i n g p r o t e i n s : M o l e c u l a r w e i g h t in d a l t o n s 68 ,000 58 ,000 41 ,000 1 7 . 0 0 0 * The m a r k e r M was m a d e up B o v i n e S e r u m A l b u m i n (BSA) A l d o l a s e Myoglfi'aia University of Ghana http://ugspace.ug.edu.gh University of Ghana http://ugspace.ug.edu.gh 52 - P l a t e 1 5 M H a r k e r 1 . L S H T M 1 80, LS 3 . 4K.P 2 . L S H T M 1 80, ST 3 . 4 KP 3 . T. b . r h o d e s i e n s e ( L i v ) 3 . 4KP 4 . L S H T M 1 8 0 , LS 1 4 . 5 K P 5 . L S H T M 1 8 0 , ST 1 4.5 KP 6 . T. b . r h o d e s i e n s e ( L i v ) 1 4 . 5KP 7 . L S H T M 1 8 0 , LS 1 3 9K S 8 . L S H T M 1 80, ST 1 3 9 K S 9 . T. b. r h o d e s i e n s e ( L i v ) 1 3 9 K S University of Ghana http://ugspace.ug.edu.gh -A TE 15 University of Ghana http://ugspace.ug.edu.gh - 53 - E N Z Y M E A S S A Y S T h e e n z y m e a c t i v i t i e s m e a s u r e d in the s u b c e l l u l a r f r a c t i o n s of the t r y p a n o s o m e s a r e s h o w n in T a b l e 1, and F i g u r e s 7 to 15 s h o w the s u b c e l l u l a r d i s t r i b u t i o n of the e n z y m e . University of Ghana http://ugspace.ug.edu.gh Table 1. Enzyme Activities (nmole/min/mg protein)* ENZYME FRACTION Alanine 14.5KP aminotransferase 139KP E.C.2.6.12; ALAT I39KS Glucose phos- 14.5KP phate isomenase 139KP E.C.5.3.1.9; GPI 139KS Glucose-6- 14.5KP phosphate dehydrogenase 139KP E.C.1.1.1.49; G6PD 139KS Malate 14.5KP dehydrogenase 139KP E.C.1.1.1.37; MDH 139KS Mannose phosphate 14.5KP isomerase 139KP E.C.5.3.1.8; MPI 139K.S Hexoklnase 14.5KP E.C.2.7.1.1; HK 139KP 139KS Isocitrate 14.5KP dehydrogenase 139KP E.C.1.1.1.42; ICD 139KS Phosphogluco 14.5KP mutase 139KP E.C.2.7.5.1; PGM 139KS Acid 14.5K.P phosphotase 139KP E.C.3.1.2.3; ACP 139KS LSHTM 180 LSHTM 180 T. b. (LS) (ST) rhodesiei 147.0 126.0 54.0 335.0 160.0 287.0 1195.0 879.0 1320.0 1847.0 1660.0 348.0 287.0 130.0 575.0 874.0 697.0 1484.0 21.0 49.0 9.0 13.0 23.0 13.0 28.0 6.05 2.06 42.0 34.0 41.0 144.0 77.0 182.0 539.0 273.0 619.0 13.0 19.0 5.0 40.0 7.0 5.0 1946.0 1469.0 587.0 447.0 309.0 435.0 53.0 5.4 7.4 3.5 - - 1.7 12.0 2.1 7.0 15.0 4.1 10.6 24.3 15.0 18.3 11.0 26.0 0.7 1.4 3.7 * All enzyme activities are in nmole/min/mg protein, except acid phosphate which is in mole/min/mg protein. University of Ghana http://ugspace.ug.edu.gh - 55 - Fig u r e 7, S u b c e l l u l a r d i s t r i b u t i o n of HK m T» b. rhodesiense, long slender T. b. rhodesiense, short stumpy T. b. rhodesiense, Liverpool normal 14.5KP 139 KP 139 KS 1 2 3 1 2 3 1 2 3 University of Ghana http://ugspace.ug.edu.gh - 56 - Fig u r e 8. S u b c e l l u l a r d i s t r i b u t i o n of GPI T. b. rhodesiense, long slender T. b. rhodesiense, short stumpy T . b . rhode s i ens e , Liverpool normal 1 = 14 . 5KP 2 = 139 KP 3 = 139 KS 1 2 3 1 2 3 1 2 3 University of Ghana http://ugspace.ug.edu.gh - 57 - Figu r e 9. S u b c e l l u l a r d i s t r i b u t i o n of ICD T. b. rhodesiense, long slender T. b. rhodesiense, short stumpy T. b. rhodesiense, Liverpool normal 15. 0 c QJ 4-J ou. C l CJJ QJ O 1 0 .0 - 5. 0 U QJCLLT> 14.5 KP 139 KP 139 KS 1 2 3 1 2 3 1 2 3 University of Ghana http://ugspace.ug.edu.gh - 58 - Figure 10. S u b c e l l u l a r d i s t r i b u t i o n of MDH W T. b. rhodesiense, long slender T. b. rhodesiense, short stumpy T. b. rhodesiense, Liverpool normal 1 = 14.5KP 2 = 1 3 9 KP 3 = 139 KS +-> o -I- s_ > CL •r— cn U E - r - CD U .— QJ O Q- E CO CL 1 2 3 1 2 3 1 2 3 University of Ghana http://ugspace.ug.edu.gh - 59 - Fig ur e 11. S u b c e l l u l a r d i s t r i b u t i o n of ALA T m T. b. rhodesiense, long slender T. b. rhodesiense, short stumpy T. b. rhodesiense, Liverpool normal 1500 14.5 KP 13 9 KP 139 KS ai o 1000 ■ XJ > 4-1o u QJ 500 . 1 2 3 1 2 3 1 2 3 University of Ghana http://ugspace.ug.edu.gh - 60 - F i g u re 12. S u b c e l l u l a r d i s t r i b u t i o n of G6FD m T. b. rhodesiense, long slender T. b. rhodesiense, short stumpy T. b. rhodesiense, Liverpool normal 14.5 KP 139 KP 139 KS University of Ghana http://ugspace.ug.edu.gh - 61 - F i g u re 13. S u b c e l l u l a r d i s t r i b u t i o n of P GM T. b. rhodesiense, long slender T. b. rhodesiense, short stumpy T. b» rhodesiense, Liverpool normal 15 . 0 - +jo SwQ. C •i—£\aj oE Sc •i— >»4J *r—> U IT3 U CD a = 14.5 KP b = 139 KP c = 139 KS University of Ghana http://ugspace.ug.edu.gh 3 # 5 E l e c t r o n M i c r o s c o p y P l a t e 16 E l e c t r o n m i c r o g r a p h (x 2 0 , 0 0 0 ) of (Liv). N = n u c l e u s . P l a t e 17 - 64 - b. r h o d e s i e n s e E l e c t r o n m i c r o g r a p h (x 1 2 6 . 0 0 0 ) of T. b. r h o d e s i e n s e (Liv) s h o w i n g a s e c t i o n of the t u b u l a r m i t o c h o n d r i o n (M) w i t h a d o u b l e m e m b r a n e . University of Ghana http://ugspace.ug.edu.gh PLATE 16 University of Ghana http://ugspace.ug.edu.gh - 65 - P l a t e 18 E l e c t r o n m i c r o g r a p h (x 2 0 , 0 0 0 ) of T. b. r h o d e s l e n s e (Llv) w i t h a d o u b l e f l a g e l l u m ( n a r r o w e d , p r o b a b l y d i v i d i n g . University of Ghana http://ugspace.ug.edu.gh University of Ghana http://ugspace.ug.edu.gh - 66 - E l e c t r o n m i c r o g r a p h (x 2 0 , 0 0 0 ) of L S H T M 180, LS s h o w i n g the n u c l e u s (N). P l a t e 20 E l e c t r o n m i c r o g r a p h (x 1 2 6 , 0 0 0 ) of L S H T M 180, LS s h o w i n g a s e c t i o n of the l o n g t u b u l a r m i t o c h o n d r i o n (M) b o u n d e d by a d o u b l e m e m b r a n e but d e v o i d (f d r i s t a e . P l « l:e 19 University of Ghana http://ugspace.ug.edu.gh University of Ghana http://ugspace.ug.edu.gh - 67 - P l a t e 2 1 x 2 0 , 0 0 0 e l e c t r o n m i c r o g r a p h of L S H T M 180, ST, s h o w i n g the n u c l e u s (N) a n d n u c l e o l u s (Nu) a n d the m i t o c h o n d r i o n (M) w i t h c r i s t a e ( a r r o w e d ) . P l a t e 22 A c l o s e - u p (x 80 ,000) of the m i t o c h o n d r i o n of L S H T M 180, ST. ( C r i s t a e a r r o w e d . ) University of Ghana http://ugspace.ug.edu.gh University of Ghana http://ugspace.ug.edu.gh Plate 23 E l e c t r o n m i c r o g r a p h (x 2 0 , 0 0 0 ) of a s e c t i o n t h r o u g h t h e c h o r o i d p l e x u s of a m o u s e i n f e c t e d w i t h T . b . r h o d e s i e n s e ( L i v ) j u s t b e f o r e d e a t h , ( i . e . a b o u t 72 h o u r s a f t e r i n o c u l a t i o n ) , s h o w i n g t r y p a n o s o m e s (T) a n d red b l o o d c e l l s ( r . b . c . ) in a b l o o d v e s s e l . - 68 - University of Ghana http://ugspace.ug.edu.gh University of Ghana http://ugspace.ug.edu.gh E l e c t r o n m i c r o g r a p h (x 8 0 , 0 0 0 ) of a s e c t i o n t h r o u g h the c h o r o i d p l e x u s of a m o u s e i n f e c t e d w i t h T. b. r h o d e s i e n s e ( L i v ) , 72 h o u r s a f t e r i n f e c t i o n , s h o w i n g a t r y p a n o s o m e in a b l o o d v e s s e l . N = n u c l e u s . P l a t e 25 T. b. r h o d e s i e n s e ( L i v ) in a c h o r o i d p l e x u s v e s s e l of a m o u s e s h o w i n g G o l g i b o d y (Gb) a n d f l a g e l l u m (f) (x 50 ,000) . Plate 26 - 69 - Plate 24 T. b. r h o d e s i e n s e ( L i v ) in a c h o r o i d p l e x u s b l o o d v e s s e l of a m o u s e (x 1 2 6 , 0 0 0 ) . f = f l a g e l l u m . University of Ghana http://ugspace.ug.edu.gh University of Ghana http://ugspace.ug.edu.gh University of Ghana http://ugspace.ug.edu.gh - 70 - E l e c t r o n m i c r o g r a p h s of s e c t i o n s t h r o u g h the c h o r o i d p l e x u s of r a t s i n f e c t e d w i t h L S H T M 180 24 - 27 dajfls a f t e r i n f e c t i o n ( A b o l a r i n e t a l . , 1 982 ). 1. T r y p o m a s t i g o t e (T) in an i n t a c t e p e n d y m a l c e l l of c h o r o i d p l e x u s . 2. M u l t i p l e - d i v i s i o n f o r m (md) in i n t a c t e p e n d y m a l c e l l (N = n u c l e u s of e p e n d y m a l c e l l ) f r o m rats t r e a t e d w i t h S H A M a n d g l y c e r o l . F l a g e l l a r p r o f i l e s a r r o w e d . 3. M u l t i p l e d i v i s i o n f o r m ( md ) a n d t r y p o m a s t i g o t e (T) in d e g e n e r a t i n g e p e n d y m a l c e l l (N = n u c l e u s of e p e n d y m a l c e l l ) . L i m i t i n g m e m b r a n e s of a d j o i n i n g c e l l s a r r o w e d . Plate 27 4. L i b e r a t i o n of t r y p o m a s t i g o t e s a f t e r d e s t r u c t i o n of h o s t e p e n d y m a l c e ll. University of Ghana http://ugspace.ug.edu.gh PL AT E 27 University of Ghana http://ugspace.ug.edu.gh C H A P T E R F O U R D I S C U S S I O N A N D C O N C L U S I O N University of Ghana http://ugspace.ug.edu.gh - 71 - D I S C U S S I O N 4.1 Morphology and Pleomorphism For many years the pleomorphism of trypanosomes has been studied by drawing individuals by camera lucida and by measuring and recording lengths and other parameters of the drawings (Bruce et al. , 1912, Fairbairn and Culwick, 1949). By this method trypomastigotes were divided into "slender", "intermediate" and "stumpy', but the parameters show so much variability that drawing and measuring sufficient numbers becomes a tedious operation. Wijers (1959) further elaborated this method by classifying the trypomastigotes which did not conform to any of the above types either as "long-intermediate" or "short-intermediate", but in the final percentages of categories of "slender", "intermediate" and "stumpy" half the long-intermediates were counted as slender and the other half as intermediate, similarly the short- intermediates were divided among intermediate and stumpy forms. While this method has been useful in relieving much of the tedium of drawing and measuring, it tends to "create" an intermediate form which some workers have considered to be independent and not just a transition from slender to stumpy. The agar film technique developed by Ormerod (1958). which was used in this study, showed that under phase University of Ghana http://ugspace.ug.edu.gh - 72 - contrast microscopy the first trypanosomes to appear in the blood had no cytoplasmic granules, but as the infection developed, granules appeared and increased in number up to the time of the remission when very few trypanosomes are seen circulating in the blood. The use of granules as a parameter for the study or pleomorphism has three clear advantages: 1. it is a less tedious process; 2 . it gives an absolute definition of a long thin try- panosome as one without cytoplasmic granules; 3. a quantitative assessment of intermediate and stumpy forms can be made according to the number of granules. Measurement of the overall volume of the trypanosome and of the volumes of cytoplasmic organelles such as the mitochondrion can be obtained from electron microscopic data; this method has also been used to study pleomorph­ ism (Hecker et al. , 1972 ; Hecker, 1980). However, each method produces different types of result and further work is required to study the correlation between the methods• Monomorphic infection of T» b. rhodesiense (Liv) is so acute that it kills the rodent host at the first peak of parasitemia, and there is no time for the development of more than one form of the parasite, the agranular trypo- University of Ghana http://ugspace.ug.edu.gh - 73 - mastigote. The infection of LSHTM 180 in rats is synchronous. This behaviour is not unique either to LSHTM 180 or to the strains obtained in Botswana (Ormerod, 1963); it occurs to some extent in other recently isolated strains of T. brucei, but it is most marked in strains isolated from man in this region. In the absence of proper separation methods of these two forms, LSHTM 180 was particularly useful in obtaining pure populations of both slender and stumpy forms for biochemical and ultrastructural studies. 4 . 2 Isoenzyme Characterization LSHTM 180, LS and ST, and T. b. rhodesiense (Liv) all had identical isoenzyme patterns for eight of the nine enzymes examined; only an aconitase (ACON) band was unique to LSHTM 180 ST. The similar isoenzyme patterns for eight enzymes suggests great similarity between the trypomastigotes examined. This means that as far as isoenzymes are concerned, there Is little change when a slender trypomastigote becomes stumpy or when a pleomor­ phic strain becomes monomorphic. It also suggests that both LSHTM 180 and T. b. rhodesiense (Liv) belong to the same zymodeme. Records about where T. b. rhodesiense (Liv) was isolated have become rather confused. It is known that it was isolated in 1923 and was used by Yorke and co-workers for many years at the Liverpool School of University of Ghana http://ugspace.ug.edu.gh - 74 - T r o p i c a l M e d i c i n e ( Y o r k e et a l . , 1 9 2 9 ) . It p r e s u n a b ^ o r i g i n a t e d f r o m the L u a n g w a v a l l e y , Z a m b i a , w h e r e Y o r k e a n d o t h e r s h a d i n v e s t i g a t e d an o u t b r e a k of “R h o d e s i a n " s l e e p i n g s i c k n e s s c i r c a 1912; at a n y r a t e , a c c o r d i n g to G i b s o n ( 1 9 7 9 ) , in t he e a r l y 1 9 2 0 ' s T. r h o d e s i e n s e w a s k n o w n o n l y f r o m C e n t r a l E a s t A f r i c a . G e o g r a p h i c a l l y , the L u a n g w a v a l l e y is c l o s e e n o u g h to M a b a b e , B o t s w a n a , w h e r e L S H T M 180 w a s i s o l a t e d , for t h i s c l o s e s i m i l a r i t y in t h e i r i s o e n z y m e p a t t e r n s to be a c c e p t a b l e . T h e a b s e n c e of a c o n i t a s e b a n d s in L S H T M 180, LS a n d T . b. r h o d e s i e n s e ( L iv) w a s n ot d ue to t he u s e of d i l u t e s a m p l e s b e c a u s e the s a m e r e s u l t w a s o b t a i n e d w i t h m o r e c o n c e n t r a t e d l y s a t e s . A c o n i t a s e ( A C O N ) , l i k e m a l a t e d e h y d r o g e n a s e ( M D H ) a n d i s o c i t r a t e d e h y d r o g e n a s e ( I C D ) , is a K r e b s c y c l e e n z y m e ; w h i l e the c y c l e is not o p e r a ­ t i o n a l in b l o o d s t r e a m t r y p o m a s t i g o t e s in th e b r u c e i g r o u p , s o m e of its e n z y m e s h a v e b e e n s h o w n to be p r e s e n t ( B o w m a n a n d F l y n n , 1 9 7 6 ) , for e x a m p l e M D H a n d I C D w e r e p r e s e n t in a l l l y s a t e s ; but the p r e s e n c e of A C O N in o n l y the s t u m p y L S H T M 180 s u g g e s t s that: 1. m o r e K r e b s c y c l e e n z y m e s m a y be f o r m e d or a c t i v a t e d in the c h a n g e f r o m s l e n d e r to s t u m p y ; 2. the a c t i v i t y of som e K r e b s c y c l e e n z y m e s m a y be lost w h e n a p l e o m o r p h i c s t r a i n b e c o m e s m o n o m o r p h i c . It is a l s o s i g n i f i c a n t to n o t e tha t b o t h the s l e n d e r f o r m of L S H T M 180 an d T. b. r h o d e s i e n s e ( L iv) l a c k l i p i d University of Ghana http://ugspace.ug.edu.gh - 75 - g r a n u l e s ; th e a b s e n c e of A C O N in b o t h t h e s e f o r m s s u g g e s t s an e v e n g r e a t e r s i m i l a r i t y b e t w e e n them. 4 . 3 P o l y p e p t i d e P r o f i l e s C h a n g e s in t he g e n e t i c c o d e a s s o c i a t e d w i t h the c o n v e r s i o n of a s l e n d e r t r y p o m a s t i g o t e to a s t u m p y or a p l e o m o r p h i c to a m o n o m o r p h i c s t r a i n w o u l d be d e m o n s t r a t e d by c h a n g e s in t he p r o t e i n s p r o d u c e d by t he o r g a n i s m . T h e s e p r o t e i n s n e e d n o t n e c e s s a r i l y be e n z y m e s . P o l y ­ p e p t i d e p r o f i l e s on SD S - p o l y a c r y l a m i d e g e l s a l l o w the s t u d y of p r o t e i n s , b o t h e n z y m i c a nd n o n - e n z y m i c . T he m e t h o d i n v o l v e s b r e a k i n g up the p r o t e i n s i n t o s m a l l e r p o l y p e p t i d e s , c o a t i n g t h e m w i t h n e g a t i v e c h a r g e s (SDS) a n d s e p a r a t i n g t h e m a c c o r d i n g to t h e i r m o l e c u l a r w e i g h t s . P l a t e s 14 a n d 15 s h o w the p o l y p e p t i d e p r o f i l e s of d i f ­ f e r e n t f r a c t i o n s of the t h r e e t r y p o m a s t i g o t e s . Th e p r o f i l e s f o r d i f f e r e n t f r a c t i o n s of the s a m e s a m p l e of t r y p o m a s t i g o t e s (e.g. 3 . 4 K P a nd 1 4 . 5 K P for T . b . r h o d e s i e n s e [ L i v ] ) a r e d i f f e r e n t ; thi s is to be e x p e c t e d b e c a u s e e a c h f r a c t i o n c o n t a i n s d i f f e r e n t o r g a n e l l e s a n d t h e r e f o r e d i f f e r e n t p r o t e i n s . T he l O O O g s u p e r n a t a n t f r a c t i o n ( I K S , p l a t e 14) s h o w s the p r e s e n c e of a 5 8 , 0 0 0 d a l t o n (58K.) m o l e c u l a r w e i g h t p o l y p e p t i d e in b o t h the s l e n d e r L S H T M 180 a n d t he m o n o m o r p h i c T. b. r h o d e s i e n s e ( L i v ) , w h i c h is a b s e n t in t he s t u m p y L S H T M 180. In fact t h i s p o l y p e p t i d e is c o n s i s t e n t l y a b s e n t f r o m a l l t he University of Ghana http://ugspace.ug.edu.gh - 76 - f r a c t i o n s of the s t u m p y f o rm. S i n c e the p r e p a r a t i o n of a l l t h e f r a c t i o n s of the t h r e e t r y p o m a s t i g o t e s w a s d o n e u n d e r s i m i l a r c o n d i t i o n s , al l t he g e l s r u n t o g e t h e r and t h e w h o l e r e p e a t e d f o u r t i m e s w i t h c o n s i s t e n t l y t he s a m e r e s u l t s , t he a b s e n c e of the 5 8 K p o l y p e p t i d e s e e m s to be a n e s t a b l i s h e d r e s u l t an d n ot d ue to a t e c h n i c a l e r r o r . T a y l o r et a l . , ( 1982) h a v e s u g g e s t e d t h a t t h i s p o l y p e p ­ t i d e c o u l d r e p r e s e n t the s u r f a c e c o a t g l y c o p r o t e i n s , b e c a u s e it s t a i n e d p o s i t i v e l y w i t h p e r i o d i c a c i d - S c h i f f . H o w e v e r , t h i s w o u l d n e e d to be c o n f i r m e d for the t r y p o n o s o m e s u n d e r s t u d y h e r e . If the 5 8 K p o l y p e p t i d e is i n d e e d t h e s u r f a c e c o a t g l y c o p r o t e i n , t h e n it p r o v i d e s a n o t h e r p i e c e of e v i d e n c e for th e g r e a t e r s i m i l a r i t y b e t w e e n the s l e n d e r L S H T M 180 a n d t he m o n o m o r p h i c T . b . r h o d e s i e n s e ( L i v ) a l r e a d y s u g g e s t e d on the p r e v i o u s p a ge. S e c o n d l y ^ m o n o c l o n a l a n t i b o d i e s c o u l d be m a d e a g a i n s t it, a n d it c o u l d t h e r e f o r e s e r v e as a m o r e s p e c i f i c t o o l for d i f f e r e n t i a t i n g b e t w e e n s l e n d e r a n d s t u m p y t r y p o m a s t i ­ g o t e s of a p l e o m o r p h i c s t r a i n . 4 . 4 E n z y m e A s s a y A n a l y s i s V a r i o u s w o r k e r s h a v e s t u d i e d the d i s t r i b u t i o n of e n z y m e s in t r y p a n o s o m e s by s u b c e l l u l a r f r a c t i o n a t i o n ( O d u r o . 1 977 , O d u r o et a l . , 198 0 , O p p e r d o e s et a l . , 1977 ). D i f f e r e n t i a l c e n t r i f u g a t i o n a n d s u b s e q u e n t i s o p y c n i c s u e r o s e - g r a d i e n t c e n t r i f u g a t i o n h a s b e e n u s e d to i s o l a t e University of Ghana http://ugspace.ug.edu.gh c o n t a i n i n g s o m e g l y c o l y t i c p a t h w a y e n z y m e s w h i c h is u n i q u e to b l o o d s t r e a m t r y p o m a s t i g o t e s ( O d u r o , 1 9 7 7 , O d u r o et a l . , 198 0 , O p p e r d o e s et a l . , 1977 ). In t h i s s t u d y , d i f f e r e n t i a l c e n t r i f u g a t i o n h a s b e e n u s e d to c o m p a r e the s u b c e l l u l a r d i s t r i b u t i o n of s e l e c t e d e n z y m e s . T he r e s u l t s s h o w t h a t m o s t of the h e x o k i n a s e (HK) a c t i v i t y is l o c a t e d in t h e 1 4 , 5 0 0 g ( 1 4 . 5 K P ) a n d 1 3 9 , 0 0 0 g ( 1 3 9 K P ) p e l l e t s , w h i c h is in a g r e e m e n t w i t h t h o s e o b s e r v a t i o n s m a d e by O d u r o ( 1977 , 1 9 8 0 ) . H K is the f i r s t e n z y m e in t h e g l y c o l y t i c p a t h w a y a n d h a s b e e n s h o w n to be l o c a t e d in t h e g l y c o s o m e . S i n c e g l y c o s o m e s f o r m a p e l l e t at 1 4 , 5 0 0 g ( O d u r o , 1 9 7 7 , O d u r o et a l ., 1 9 80, O p p e r d o e s et a l . , 1 9 7 7 ) , it c a n be c o n c l u d e d t hat the 1 4 . 5 K P f r a c t i o n in w h i c h m o s t of the H K a c t i v i t y is l o c a t e d a l s o c o n t a i n s g l y c o s o m e s , w h i c h h a v e b e e n c o n f i r m e d by O d u r o ( p e r s o n a l c o m m u n i c a t i o n ) to be m i c r o b o d i e s . T h e d i s t r i b u t i o n of g l u c o s e - p h o s p h a t e i s o m e r a s e ( G PI) in T. b. r h o d e s i e n s e ( L i v ) d i f f e r s f r o m t h o s e in L S H T M 180 s l e n d e r a n d s t u m p y f o r m s , w h e r e m o s t of the G P I a c t i v i t y is l o c a t e d in t he 1 4 . 5 K P ( g l y c o s o m a l ) f r a c t i o n . T h i s d i f f e r e n c e m a y be d ue to l e a k a g e of a less t i g h t l y b o u n d e n z y m e d u r i n g t he p r e p a r a t i o n of the T. b. r h o d e s i e n s e ( L i v ) f r a c t i o n s . T h e d i s t r i b u t i o n of A L A T in al l t h r e e t r y p o m a s t i g o t e s is University of Ghana http://ugspace.ug.edu.gh - 78 - i d e n t i c a l , m o s t of the a c t i v i t y b e i n g l o c a t e d in the 1 3 9 , 0 0 0 s u p e r n a t a n t ( 1 3 9 K S ) , the s o l u b l e f r a c t i o n . T h i s r e s u l t c o n f i r m s the o b s e r v a t i o n s m a d e by V i s s e r a nd O p p e r d o e s ( 1 9 8 0 ) t h a t A L A T is a s o l u b l e e n z y m e . A L A T a n d o t h e r t r a n s a m i n a s e s s u c h as A S A T c a t a l y s e t he i n t e r ­ c o n v e r s i o n of K r e b s c y c l e i n t e r m e d i a t e s a nd a m i n o a c i d s . T h e d i s t r i b u t i o n of NADH— l i n k e d M D H is s i m i l a r to t h a t of A L A T , m o s t of it b e i n g l o c a t e d in t he s o l u b l e p a r t of the c e l l ( 1 3 9 K S ) . T h e p r e s e n c e of h i g h l e v e l s of M D H , a K r e b s c y c l e e n z y m e , w h i c h c a t a l y s e s the r e v e r s i b l e o x i d a ­ t i o n of m a l a t e to o x a l o a c e t a t e r e m a i n s u n e x p l a i n e d in v i e w of t he c y c l e b e i n g n o n - f u n c t i o n a l in t h e s e b l o o d s t r e a m t r y p o m a s t i g o t e s . T h e o p e r a t i o n of a g l y o x y l a t e c y c l e ( F i g u r e 16), a m o d i f i e d f o r m of t he K r e b s c y c l e w h i c h t a k e s p l a c e in m o s t p l a n t s a n d m i c r o o r g a n i s m s c o u l d e x p l a i n t h e p r e s e n c e of M D H in t h e s e t r y p o m a s t i g o t e s . H o w e v e r ; as T a b l e 2 o v e r l e a f s h o w s , the l e v e l s of m a l a t e s y n t h a s e a n d i s o c i t r a t e l y a s e a r e p r o b a b l y too l o w to m a k e t he c y c l e o p e r a t i v e . M D H s e e m s to be p r e s e n t to g e n e r a t e p r e c u r s o r s f or a n a b o l i c p a t h w a y s , n a m e l y o x a l o ­ a c e t a t e for t h e s y n t h e s i s of the a m i n o a c i d s a s p a r t a t e a n d a s p a r a g i n e . T he s p e c i f i c a c t i v i t y of M D H in p r o - c y c l i c t r y p o m a s t i g o t e s of T. b r u c e i is 1 6 0 0 n m o l e / m i n / m g p r o t e i n ( O p p e r d o e s et a l ., 1 9 8 1 ) . T h i s is 3 to 5 t i m e s as m u c h as the a c t i v i t y o b s e r v e d in t he b l o o d s t r e a m t r y p o m a s t i g o t e s , a n d is p r o b a b l y d ue to the f a c t that University of Ghana http://ugspace.ug.edu.gh - 79 - Fi g u r e 16. The G l y o x y l a t e Cycle Table 2. (Opperdoes et a l ., 1977) A comparison of selected enzymes in T. brucei and other organisms -------------------|------------------- j------------ | | Specific activity (nmole/min/mg protein) | 1 T. brucei I Enzyme | Cell-free I Large granule Other | I extract (139KS) |14.5KP fraction organisms | I Isocitrate lyase 1 | 0 .2 1 1 1 1 23a | Imalate synthase 1 1 1 1 | 1 1 X3oo |NAIH— linked ICD 1 1 1 1 I 0 .1 1 2.lc , 19d | a) Tetrahymena pyriformis cell-free extract. b) Yeast cell-free extract. c) Crithidia luciliae cell—free extract d) Rat liver mitochondrion. University of Ghana http://ugspace.ug.edu.gh - 80 - t h e s e p r o c y c l i c f o r m s f o u n d in t he i n s e c t v e c t o r h a v e a f u l l y f u n c t i o n a l K r e b s c y c l e . L S H T M 180 ST ha s 1 \ t i m e s to 5 t i m e s as m u c h I C D as the s l e n d e r f o r m of L S H T M 180 a n d T. b. r h o d e s i e n s e ( L i v ) . L i k e M D H , in the a b s e n c e of a f u n c t i o n a l K r e b s c y c l e , I CD m a y a l s o be i n v o l v e d in g e n e r a t i n g a n a b o l i c p r e c u r s o r s s i n c e it c a t a l y s e s the o x i d a t i o n of i s o c i t r a t e to 0( - k e t o g l u t a r a t e w h i c h s e r v e s as a p r e c u r s o r of a m i n o a c i d s in m a n y t r a n s a m i n a t i o n s . M o s t m i c r o o r g a n i s m s a nd t i s s u e s of h i g h e r a n i m a l s c o n t a i n 2 t y p e s of ICD, one t y p e u s e s N A D + as e l e c t r o n a c c e p t e r a n d the o t h e r N A D P + . B o t h NADH— l i n k e d a n d NADPH— l i n k e d I C D s o c c u r in the m i t o ­ c h o n d r i o n of a n i m a l t i s s u e s , but the f o r m e r is f o u n d o n l y in the m i t o c h o n d r i o n , w h e r e a s the l a t t e r is f o u n d b o t h in t h e m i t o c h o n d r i o n a n d c y t o s o l . T he I CD a c t i v i t y d e t e r m i n e d in t h i s s t u d y l o c a t e d m a i n l y in the c y t o s o l w a s N A D P - l i n k e d a n d w a s f o u n d to be 20 - 100 t i m e s the v a l u e g i v e n fo r T. b r u c e i b l o o d s t r e a m t r y p o m a s t i g o t e s by O p p e r d o e s et a l . , ( 1 9 7 7 ) . G 6 PD c a t a l y z e s the c o n v e r s i o n of g l u c o s e - 6 - p h o s p h a t e ( G 6 P) to 6- p h o s p h o g l u c o n a t e (6 PG) in the p h o s p h o g l u c o n a t e p a t h w a y . O ne of the f u n c t i o n s of t his p a t h w a y is to c o n v e r t h e x o s e s i n t o p e n t o s e s , p a r t i c u l a r l y D - r i b o s e 5- p h o s p h a t e , r e q u i r e d in the s y n t h e s i s of n u c l e i c a c i d s . In a n i m a l c e l l s the r e a c t i o n s of the p h o s p h o g l u c o n a t e p a t h w a y t a k e p l a c e in the c y t o s o l . M o s t of the G 6 PD University of Ghana http://ugspace.ug.edu.gh - 81 - a c t i v i t y in t h e s t u m p y f o r m of L S H T M 180 a n d T . b . r h o d e s i e n s e ( L i v ) a p p e a r s to be l o c a t e d in the p a r t i c u ­ l a t e f r a c t i o n s , 1 4 . 5 K P a n d 1 3 9 K P . In the s l e n d e r f o r m of L S H T M 180, t he G 6 PD a c t i v i t y is f a i r l y e v e n l y d i v i d e d b e t w e e n t he p a r t i c u l a t e f r a c t i o n s an d the c y t o s o l . M o r e w o r k n e e d s to be d o n e to d e t e r m i n e if t h i s p a t h w a y is f u l l y f u n c t i o n a l , a n d its i m p o r t a n c e to t he t r y p a n o - s o m e s . In t he c e l l s of h i g h e r a n i m a l s , P G M c a t a l y z e s the c o n v e r s i o n of g l u c o s e - l - p h o s p h a t e ( G - l - P ) t h e end p r o d u c t of g l y c o g e n a n d s t a r c h p h o s p h o r y l a s e r e a c t i o n s , to G - 6 -P, w h i c h c a n t h e n e n t e r the g l y c o l y t i c s e q u e n c e . S i n c e b l o o d s t r e a m t r y p o m a s t i g o t e s are not k n o w n to c o n t a i n a n y e n d o g e n o u s c a r b o h y d r a t e s t o r e s (e.g. g l y c o ­ g e n ) , the f u n c t i o n of P G M in t h e s e p a r a s i t e s is o b s c u r e . If it is f u n c t i o n a l in v i v o , t h e n it c o u l d be u s e d to c o n v e r t G - l - P a b s o r b e d f r o m the h o s t b l o o d to G - 6 -P. P G M is l o c a t e d in th e c y t o s o l of al l the t r y p o m a s t i g o t e s , its a c t i v i t y in t he s t u m p y f o r m of L S H T M 180 is 2 to 3 t i m e s h i g h e r t h a n in the o t h e r two t r y p o m a s t i g o t e s . M a n n o s e - 6- p h o s p a t e , M - 6 -P, l i k e G - l - P c a n e n t e r the g l y c o l y t i c s e q u e n c e a f t e r c o n v e r s i o n i n t o one of the g l y c o l y t i c i n t e r m e d i a t e s . It is c o n v e r t e d to f r u c t o s e - 6 - p h o s p a t e ( F - 6-P) by m a n n o s e p h o s p h a t e i s o m e r a s e ( M P I ) . The p r e s e n c e of e n z y m e s l i k e P G M a n d M P I s u g g e s t s t hat t h e s e b l o o d s t r e a m t r y p o m a s t i g o t e s c a n u se m o n o s a c c h a r i d e s University of Ghana http://ugspace.ug.edu.gh - 82 - o t h e r t h a n g l u c o s e as a s o u r c e of e n e r g y . T h e s u b c e l l u l a r f r a c t i o n a t i o n s c h e m e s h o w n in F i g u r e 4B p r o v i d e d t he m a t e r i a l s f or A C P a n a l y s i s . T he s c h e m e is t h a t d e s c r i b e d by V e n k a t e s a n et a l ., ( 1 9 7 7 ) , w h o l o c a t e d A C P a c t i v i t y in t he 4 2 K P a n d the 1 0 5 K P p e l l e t s w h i c h t h e y t e r m e d ' h e a v y l y s o s o m a l 1 a n d ' l i g h t l y s o s o m a l ' r e s p e c ­ t i v e l y ; t h e y a l s o s t u d i e d the in v i v o l o c a l i z a t i o n of A C P in s l e n d e r a n d s t u m p y t r y p o m a s t i g o t e s . C y t o c h e m i c a l e v i d e n c e s h o w s t h a t A C P in t he s l e n d e r f o r m s is l o c a l i z e d in t he f l a g e l l a r p o c k e t a n d in d i s c r e t e p a r t i c l e s a r o u n d it, c o n f o r m i n g to th e s u g g e s t i o n of J a d i n a n d C r e e m e r s ( 1 9 7 0 ) a n d L a n g r e t h a n d B a l b e r ( 1 9 7 5 ) t h a t it is t r a n s ­ p o r t e d i n t o t h e f l a g e l l a r p o c k e t fo r p u r p o s e s of d i g e s ­ t i on. In t he s t u m p y t r y p o m a s t i g o t e s V e n k a t e s a n et a l . , ( 1977 ) f o u n d A C P a c t i v i t y in t he G o l g i z o n e a n d r o u g h e n d o p l a s m i c r e t i c u l u m ( R E R ) , a n d s u g g e s t t h a t t h i s is due to a d i f f e r e n t m e c h a n i s m of i n t r a c e l l u l a r d i g e s t i o n in t h e s t u m p y f o r m s . S t e i g e r et a l . , ( 1 9 8 0 ) h a v e a l s o r e p o r t e d th e l o c a l i z a t i o n of A C P in b o t h t he f l a g e l l a r p o c k e t a n d e n d o p l a s m i c r e t i c u l u m of a p l e o m o r p h i c T . b r u c e i s t r a i n . W i t h o u t s u p p o r t i n g e v i d e n c e f r o m e n z y m e c y t o l o g y , the o n l y c o n c l u s i o n s t h a t c a n be d r a w n f r o m A C P a n a l y s i s in thi s s t u d y are: 1. In a l l t h r e e t y p o m a s t i g o t e s , m o s t of A C P a c t i v i t y is l o c a l i z e d in the ' h e a v y l y s o s o m a l ' ( 4 2 K P ) a n d ' l ight University of Ghana http://ugspace.ug.edu.gh - 83 - l y s o s o m a l ' ( 1 0 5 K P ) f r a c t i o n s , as o b s e r v e d by V e n k a t e s a n et a l . . ( 1977 ). 2. T h e s l e n d e r f o r m of L S H T M 180 a n d the m o n o m o r p h i c T. b. r h o d e s i e n s e (Liv) h a v e s i m i l a r A C P d i s t r i b u t i o n w h i c h is d i f f e r e n t f r o m t h a t of the s t u m p y L S H T M 180. T h i s is f u r t h e r e v i d e n c e of the r e s e m b l a n c e b e t w e e n t h e s l e n d e r a n d m o n o m o r p h i c t r y p o m a s t i g o t e s . V e n k a t e s a n et a l . , ( 1977 ) o b s e r v e d t h a t t he c h a n g e in A C P l o c a l i z a t i o n f r o m the f l a g e l l a r p o c k e t in t h e s l e n d e r t r y p o m a s t i g o t e s to t he R E R a n d G o l g i c o m p l e x in the s t u m p y f o r m w a s a c c o m p a n i e d by an i n c r e a s e in the u p t a k e of l i p i d s as w e l l as a 3- 4 f o l d i n c r e a s e in A C P a c t i v i t y in t he s t u m p y f o r m s . F r o m t h e s e o b s e r v a t i o n s , t h e y s u g g e s t e d t h a t t he i n c r e a s e d l i p i d a b s o r p t i o n t r i g g e r e d o f f an i n c r e a s e in A C P a c t i v i t y in the R E R a n d G o l g i c o m p l e x , l e a d i n g to t h e a u t o l y s i s a n d r e m o v a l of the s t u m p y f o r m s f r o m the b l o o d ( F i g u r e 6 , r e m i s s i o n at 144 h o u r s ) . T h e r e s u l t s o b t a i n e d f r o m A C P a c t i v i t y s t u d i e s ( F i g u r e 11) do n o t s u p p o r t h is h y p o t h e s i s . W h i l e the s t u m p y f o r m of L S H T M 180 s h o w s a h i g h e r A C P a c t i v i t y t h a n t h e s l e n d e r , the d i f f e r e n c e is n o t n e a r l y as l a r g e as o b s e r v e d by V e n k a t e s a n et a l . , ( 1977 ); m o r e o v e r t he c e l l s of the m o n o m o r p h i c T. b. r h o d e s i e n s e ( L i v ) w h i c h s h o w the s a m e l e v e l of A CP a c t i v i t y as the s t u m p y a re p r o b a b l y too y o u n g to h a v e s t a r t e d a u t o l y s i s at 72 h o u r s . But s i n c e t h e p r o c e s s of c e l l a u t o l y s i s w o u l d not d e p e n d University of Ghana http://ugspace.ug.edu.gh - 84 - s o l e l y on A C P a c t i v i t y , no f i r m c o n c l u s i o n s c a n be m a d e u n t i l a t h o r o u g h s t u d y of m o r e h y d r o l y t i c e n z y m e s is m a d e . 4 . 5 E l e c t r o n M i c r o s c o p y 4 . 5 . 1 C h o r o i d P l e x u s T r y p a n o s o m e s in t he b r u c e i g r o u p a r e m a i n l y i n t r a v a s c u - lar, b ut in t he l a t t e r s t a g e s of a p l e o m o r p h i c i n f e c t i o n i n r a t s a n d m i c e , t h e y h a v e b e e n s e e n to be o u t s i d e the b l o o d v e s s e l s a n d to a c c u m u l a t e in t he p e r i v a s c u l a r s p a c e s in t h e c h o r o i d p l e x u s ( V a n M a r c k et a l ., 1 9 8 1 ) . T h e c h o r o i d p l e x u s is of s p e c i a l i n t e r e s t b o t h b e c a u s e l a r g e n u m b e r s of t r y p o m a s t i g o t e s a c c u m u l a t e in its p e r i ­ v a s c u l a r s p a c e s a n d b e c a u s e it f o r m s p a r t of the b l o o d - b r a i n b a r r i e r . O n c e in t h e p e r i v a s c u l a r s p a c e s , th e t r y p o m a s t i g o t e s c a n e n t e r t h e e p e n d y m a l c e l l s l i n i n g the c h o r o i d p l e x u s . U n t i l r e c e n t l y , t h e s e t r y p a n o s o m e s w e r e b e l i e v e d to be e n t i r e l y e x t r a - c e l l u l a r , b ut A b o l a r i n et a l ., ( 1 9 8 2 ) h a v e s h o w n w i t h p l a t e 27.1 t h a t i n t r a c e l l u l a r f o r m s do o c c u r ; a l b e i t i n f r e q u e n t l y . B l o o d - s t r e a m t r y p o m a s t i g o t e s d i v i d e by b i n a r y f i s s i o n , but p l a t e s 2 7 . 2 a n d 2 7 . 3 s h o w t hat the i n t r a c e l l u l a r f o r m s d i v i d e by m u l t i p l e d i v i s i o n , a nd are l i b e r a t e d i n t o the p e r i v a s c u l a r s p a c e s a f t e r the d e s t r u c t i o n of the h o s t e p e n d y m a l c e ll. O n c e in the h o s t University of Ghana http://ugspace.ug.edu.gh - 85 - e p e n d y m a l c e l l , t h e s e i n t r a c e l l u l a r f o r m s a r e s a f e f r o m t h e t r y p a n o c i d a l a c t i o n of d r u g s l i k e s u r a m i n w h i c h c a n ­ n o t c r o s s t he b l o o d - b r a i n b a r r i e r ; a n d f r o m th e h o s t ' s i m m u n e r e s p o n s e . T h e y a r e t h e r e f o r e t h o u g h t to be r e ­ s p o n s i b l e f o r t he t e n d e n c y to r e l a p s e a f t e r a p p a r e n t s u c c e s s f u l t r e a t m e n t , a n d s p o n t a n e o u s r e m i s s i o n s of A f r i c a n s l e e p i n g s i c k n e s s . E l e c t r o n m i c r o g r a p h s of th e c h o r o i d p l e x u s of r a t s w i t h t h e m o n o m o r p h i c T. b. r h o d e s i e n s e ( L i v ) ( P l a t e s 23 - 26) s h o w t h a t u n l i k e t he p l e o m o r p h i c s t r a i n t h e s e t r y p o m a s ­ t i g o t e s do n o t l e a v e th e b l o o d v e s s e l s . M o r e w o r k n e e d s to be d o n e to e s t a b l i s h w h y t h e s e m o n o m o r p h i c t r y p o m a s t i ­ g o t e s a r e e x c l u s i v e l y i n t r a v a s c u l a r . 1. Is it b e c a u s e t he i n f e c t i o n d o e s n ot go on l o n g e n o u g h f or t h i s to h a p p e n ? 2. Or is it o ne of the c h a n g e s w h i c h o c c u r s w h e n a p l e o m o r p h i c s t r a i n b e c o m e s m o n o m o r p h i c ? 4 . 5 . 2 U l t r a s t r u c t u r e T h e u l t r a s t r u c t u r a l i n f o r m a t i o n o b t a i n e d f r o m e l e c t r o n m i c r o s c o p y s h o w s t h a t s o m e c h a n g e s o c c u r w h e n a s l e n d e r t r y p o m a s t i g o t e b e c o m e s s t u m p y . T he m i t o c h o n d r i o n of the s l e n d e r L S H T M 180 h as a d o u b l e m e m b r a n e , but l a c k s c r i s t a e ; b ut t h a t of the s t u m p y L S H T M 180 h a s d e v e l o p e d s o m e c r i s t a e . T he f o r m of t he m i t o c h o n d r i o n in t r y p o — University of Ghana http://ugspace.ug.edu.gh - 86 - m a s t l g o t e s of the b r u c e i g r o u p v a r i e s s t r i k i n g l y f r o m one s t a g e of the l i f e c y c l e to a n o t h e r . T he s l e n d e r t r y p o ­ m a s t i g o t e s h a v e f e w or no c r i s t a e , but as t h e y are c o n v e r t e d i n t o the s t u m p y f o rm, m o r e c r i s t a e d e v e l o p . In t he t r y p o m a s t i g o t e f o r m s of the t s e t s e f ly m i d g u t an d p r o v e n t r i c u l u s , the m i t o c h o n d r i o n h a s a b u n d a n t c r i s t a e . T h e e p i m a s t i g o t e f o r m s of the s a l i v a r y g l a n d s of the fly m a i n t a i n t h i s e l a b o r a t e m i t o c h o n d r i a l m o r p h o l o g y b ut in t h e n e x t s t a g e the t r y p o m a s t i g o t e m e t a c y c l i c f o r m s h a v e m i t o c h o n d r i a w i t h r e d u c e d c r i s t a e , r a t h e r l i k e the b l o o d s t r e a m f o r m s ( V i c k e r m a n , 1 9 6 2 , 1 9 6 6 ) . B l o o d s t r e a m t r y p o m a s t i g o t e s m e t a b o l i z e g l u c o s e i n c o m p l e t e l y to p y r u v a t e due , it is g e n e r a l l y s u p p o s e d , to t he a b s e n c e of a f u n c t i o n a l K r e b s c y c l e in t he u n d e r d e v e l o p e d m i t o ­ c h o n d r i o n . T h i s is a r a t h e r w a s t e f u l u s e of g l u c o s e , so to p r o v i d e s u f f i c i e n t e n e r g y , the t r y p a n o s o m e a b s o r b s m a s s i v e a m o u n t s of g l u c o s e f r o m the h o s t ' s b l o o d , w h i c h a l s o s u p p l i e s the o x y g e n r e q u i r e d to s a t i s f y the d e m a n d s of the g l y c e r o p h o s p h a t e o x i d a s e s y s t e m ( F u l t o n a nd S p o o n e r , 195 9 ; G r a n t et a l . , 1961; D a n f o r t h , 196 7 ). The l i m i t e d q u a n t i t i e s of g l u c o s e a n d o x y g e n in the s t a g n a n t b l o o d in t he fly d e m a n d a m o r e e f f i c i e n t u s e of t h e s e r e s o u r c e s . To thi s e nd the t r y p o m a s t i g o t e s d e v e l o p a f u l l y f u n c t i o n a l m i t o c h o n d r i o n , w i t h K r e b s c y c l e e n z y m e s a n d c y t o c h r o m e s , w h i c h is r e g i s t e r e d by the a p p e a r a n c e of c r i s t a e . V i c k e r m a n ( 1 9 6 5 , 1970) ha s s u g g e s t e d f r o m u l t r a s t r u c t u r a l University of Ghana http://ugspace.ug.edu.gh - 87 - a n d e n z y m e s t u d i e s t hat t he s t u m p y t r y p o m a s t i g o t e h as an a d v a n t a g e in a c t i n g as the i n f e c t i v e f o r m f or t he f ly b e c a u s e it r e p r e s e n t s an i n t e r m e d i a t e s t a g e in the d e v e l o p m e n t of the m i t o c h o n d r i o n b e t w e e n the ' i n a c t i v e ' m i t o c h o n d r i o n of the s l e n d e r f o r m a n d the h i g h l y a c t i v e o n e of t he i n s e c t f o rm. T h e r e s u l t s o b t