DEVELOPMENT OF MONOCLONAL ANTIBODY-BASED ASSAYS FOR DETECTION AND DIFFERENTIATION OF TRYPANOSOME SPECIES IN THE TSETSE FLY (GLOSSINA SPP.) A Thesis Presented to The Board of Graduate Studies, University of Ghana, Legon. Ghana. In fulfilment of the Requirements for the Degree of Doctor of Philosophy (Ph.D.) (Animal Science), By KWABENA MANTE BOSOMPEM BSc. (Hons.) Department of Animal Science, Faculty of Agriculture, University of Ghana, Legon, Accra. Ghana. March, 1993. University of Ghana http://ugspace.ug.edu.gh <^33732 University of Ghana http://ugspace.ug.edu.gh i i DECLARATION I do hereby declare that except for references to other people's investigations which have been duly acknowledged, this exercise is the result of my own original research, and that this thesis, either in whole, or in part, has not been presented for another degree elsewhere. University of Ghana http://ugspace.ug.edu.gh I l l D E D I C A T I O N To Dr. J. E. Fletcher for the help in determining my career, and to my wife Emestina and my children for their understanding of the circumstances. University of Ghana http://ugspace.ug.edu.gh iv ACKNOWLEDGEMENT It is my first duty to acknowledge with pleasure, my indebtedness to all the individuals, organisations and institutions that have contributed in various ways to the formulation, execution and submission of the work described in this thesis. I will begin by expressing my sincere thanks to the Animal Science Department, Faculty of Agriculture, University of Ghana, for making me the beneficiary of the Research Fellowship awarded by the International Laboratory for Research on Animal Diseases (ILRAD), and to my employers, Noguchi Memorial Institute for Medical Research (NMIMR), University of Ghana Legon, for granting me a study leave to undertake this study. I am also grateful to the ILRAD Administration and the Training Department for providing such excellent facilities and atmosphere under which this work was carried out. I am particularly indebted to Prof. Reginald K. G. Assoku and Dr. Vinand Nantulya, my supervisors, not only for the guidance, encouragement and stimulating discussions, but also for their unfailing courtesy and cooperation. I also wish to express my deep gratitude to Dr. Rachael Masake, for acting as my ILRAD supervisor during the absence of Dr. Nantulya. I am particularly appreciative of the way in which Dr. Nantulya continued to find time for this project, even after his departure from active service in ILRAD. I am thankful to him. I am extremely thankful to Dr. S. K. Moloo, head of the ILRAD tsetse laboratory, for the help and encouragement he offered, and all his technicians, particularly Messrs Joseph Muia, John Kabata and Clement Sabwa for their unfailing cooperation and assistance in all the experiments involving the use of laboratory-bred tsetse flies. University of Ghana http://ugspace.ug.edu.gh I gratefully acknowledge the support granted to me by the Kenya Trypanosomiasis Research Institute (KETRI) in making it possible to conduct part of this work on the Galana Ranch, and the assistance received from that institute from Dr. Elizabeth Opiyo. I am thankful to Dr. Chris Green for his assistance in trapping tsetse flies, and Mr. T. Wanyama for helping me in both trapping and dissection of tsetse at the Ranch. I owe a special debt of gratitude to Dr. Ian Gumm and Dr. Ron Kaminsky for supplying culture-derived trypanosomes for this project whilst they were in ILRAD, and Mr. Francis Chuma of ILRAD for similar assistance. Also, I remember Dr. Joe McNamara of the Tsetse Research Laboratory, Bristol, England for supplying Trypanosoma grayi antigens for this study and Dr. Andrew Peregrine of ILRAD for making this arrangement possible. The helpful advice and encouragement that was freely given by Dr. Phelix Majiwa, Dr. M. Toure and Mr. Stephen Minja all of ILRAD, is greatly appreciated. I wish to thank Dr. M. K. Shaw for technical help in immunolocalization studies; Henry Gathuo for introducing me to tissue culture and immunofluorescence techniques; Messrs John Ngatti, James Thuo, Jackson Makau, Benson Gichuki and Stephen Ngava for helping me to master the various techniques described in this work; David Elsworth and J. Mwaura for advice on preparation and presentation of illustrations, and Francis Shikhubari for his expert assistance in photography. Finally, I wish to thank Mr. Maxwell Appawu of the NMIMR, for the immeasurable help he offered during my absence from Ghana. V University of Ghana http://ugspace.ug.edu.gh VI TABLE OF CONTENTS Page No. TITLE PAGE .............................................................................................................................................. W DECLARATION ........................................................................................................................................ (ii) DEDICATION ............................................................................................................................................. 4 and O.lmM KH2PO4 , pH 7.4). The molten agarose was poured onto microscope slides and allowed to solidify. Wells were then cut into the solid gel and each was filled with approximately 10/d of reagent. Antisera were placed in a central well and test samples (culture supamatants concentrated ten-fold by ammonium sulphate precipitation) were placed in the surrounding wells. A precipitin line formed in-between a sample well and the homologous antiserum in a central well. The precipitin reaction was allowed to develop in up to 48 hr at room temperature in a wet chamber and observed by viewing the gels against light. For preservation, agarose gels were thoroughly washed with PBS, pH 7.4, followed by distilled water to remove unprecipitated proteins, dried, and stained using Coomassie Brilliant blue (Williams, 1971). University of Ghana http://ugspace.ug.edu.gh 97 4.3.10.2 Sodium dodecvl sulphate-polvacrvlamide gel electrophoresis (SDS- PAGE) Electrophoresis of trypanosome proteins was performed, using the Bio Rad Protean II cell apparatus (Bio Rad, Italy) and following the SDS-Tris- glycine discontinuous buffer system (Laemmli, 1970). 4.3.10.3 Assembly of slab gel apparatus, and preparation of resolution and stacking gels The gel casting apparatus consisted of four transparent glass plates (16cm x 20cm), four 1.5mm thick plastic spacers, and four plastic clamps. These were assembled such that the two glass plates wiped clean, using 70% ethanol, were separated by the plastic spacers along the 16cm edges and clamped together. The assembled glass plates were then secured vertically on a gel casting platform so that the lower gap between each pair of plates was sealed by a rubber gasket. Resolution acrylamide gradient gels (7.5-15%) were prepared as follows: Solution A. 7.5% resolution gel (one gel) Deionised water 7.34ml 30% (w/v) acrylamide, 0.8% (w/v) N ' -methylene bis-acrylamide 3.75ml 1.5M,pH 8 .8 ,tris-(hydroxymethyl) -aminomethane (Tris) 3.75ml 10% (w/v) sodium dodecyl sulphate (SDS) 0.3ml N,N,N'-,N'-tetramethylethylenediamine (TEMED) 0.01ml 10%(w/v) ammonium persulphate (APS) 0.04ml University of Ghana http://ugspace.ug.edu.gh 98 Solution B. 15% resolution gel (one gel) Deionised water 3.84ml 30%(w/v)acrylamide; 0.8%(w/v) bis-acrylamide 7.5ml 1.5M Tris, pH 8 . 8 3.75ml 10%(w/v) SDS 0.3ml TEMED 0.01ml 10% APS 0.04ml The acrylamide/bis-aerylamide solution and TEMED were stored in brown bottles whilst 10% APS was prepared fresh and added to Solutions A and B just before gel casting. All solutions were kept at 4°C, except 10% SDS which was kept at room temperature. A 7.5-15% resolution acrylamide gradient gel was prepared by slowly mixing solution A and B using Bio-Rad Model 385 gradient former. Each solution was swirled to mix and poured into one of the two separate chambers of the gradient mixer. Solution B, with the highest acrylamide concentration was placed in the chamber next to the outlet. The valve between the two chambers was opened and a magnetic stirrer placed in solution B started. A peristaltic pump (LKB, Sweden) set at a flow rate of 3ml/min was used to deliver the gel mixture via rubber tubing into the space between the two glass plates. The gel former apparatus was then immediately rinsed with distilled water. The poured gel was overlaid with 500-1000^1 of water- saturated butanol using a micropipette or pasteaur pipette and left for approximately 1 hr to polymerize. After the resolution acrylamide gel (separating gel) had set, the gel overlay was removed and the top of the gel rinsed with distilled water. A stacking gel (Solution C) prepared as below was then poured on top of the separating gel and a comb carefully inserted to cast the desired wells for sample application. University of Ghana http://ugspace.ug.edu.gh 99 Solution C. 3% stacking gel (one gel) Deionised water 7.5ml 30%(w/v) acrylamide; 0.8%(w/v) bis-acrylamide 1.5ml 0.5M Tris, pH 6 . 8 3.0ml 10%(w/v) SDS 0.125ml TEMED 0.01ml 10% APS 0.05ml The stacking gel was allowed 30 min to polymerize and the comb removed. The cast gel units were then assembled in a Bio-Rad Protean II cell electrophoresis apparatus. The upper electrophoretic chamber, at the cathode, was filled with 0.4 litres of running-buffer (24.8mM Tris, 191.8mM Glycine and 3.47mM SDS) and the lower chamber, at the anode, filled with 1.6 litres of running-buffer diluted with 0.5 litres of deionised water. 4.3.10.4 Preparation of samples and electrophoresis run The trypanosome crude extracts were adjusted for protein and diluted with sample buffer [150mM Tris, pH 6 .8 , 104mM SDS, 3%(v/v) mercaptoethanol, 30%(v/v) glycerol and 4%(v/v) bromophenol blue] at 2:1 sample to buffer ratio, to give a final protein concentration of approximately lmg/ml. Standard high molecular weight markers (Rainbow Markers, MW 14,300 to 200,000; Amersham International pic, Amersham, UK) were diluted 1:1 with sample buffer without bromophenol blue. All the samples and standard markers were boiled for 5 and 1 min, respectively, at 100°C in a waterbath and centrifuged at 9,900 Xg for 5 min to remove particulate matter. About 200/xg of the boiled trypanosome extracts or 5 to 10^1 of standard molecular weight markers were loaded per lane of about 0.5cm width. Electrophoresis was performed using the Bio-Rad protean II cell apparatus, cooled to 10°C with a Lauda RC20 cooler (Bremen, Germany). A constant voltage using electrophoresis power supply (EPS 500/400, Pharmacia Fine University of Ghana http://ugspace.ug.edu.gh 1 00 Chemicals) either at 50-70V/gel overnight, or at 300V/gel for 3-4 hr was applied until the bromophenol blue tracer dye migrated almost to the end of the separating gel. At the completion of the run, the assembled gel units were removed from the electrophoresis chamber and dismantled. 4.3.10.5 Staining, destaining and western immunoblot A vertical strip of the gel was cut using a surgical blade, and transferred into a plastic tray containing staining solution [0.5%(w/v) coomassie blue, 10%(v/v) acetic acid and 30 %(v/v) isopropyl] for 15 min. The stained gel was then transferred to a destaining solution containing 10%(v/v) methanol and 7%(v/v) acetic acid on a gentle rotor (Red-Rotor Model PR70, Hoeffer) with several changes of the solution until stained protein bands were clearly visible in the gel (Weber and Osborn, 1969). Separated trypanosome proteins were transferred electrophoretically from unstained gels to nitrocellulose sheets, as described by Towbin, Staechelin and Gordon (1979) and Burnette (1981). Briefly, 3mm Whatman chromatography paper (Whatman, Maidstone, England) was soaked with transfer buffer [25mM Tris, 192mM glycine, 0.1%(w/v) SDS and 20%(v/v) methanol] and placed on top of a scouring pad (Scotch-Brite, Hoefer Scientific Instruments, USA) wetted in the same buffer and supported by a stiff plastic grid. The gel was then placed on top of the chromatography paper. A sheet of nitrocellulose filter (0.45/an pore size, Schleicher and Schuell, Inc., Keen, NH, USA) trimmed to fit the gel, was briefly wetted with transfer buffer and carefully placed on top of the gel without trapping air bubbles. A second chromatography paper and scouring pad, both soaked in the same buffer, and a plastic grid were added in that order and clipped. The sandwiched gel was then fitted in a Transphor Electrophoresis Unit, Model TE50 (Hoeffer Scientific Instruments, San Francisco, USA) filled with transfer buffer. Electrophoretic transfer was run at a constant voltage (either at 10V overnight University of Ghana http://ugspace.ug.edu.gh 1 0 1 or at 70V for 3 hr) with the nitrocellulose sheet facing the cathode. The nitrocellulose was removed, cut into strips and immuno-assayed using the method described for the detection of in vitro propagated tiypanosomes by dot- ELISA (section 4.3.9.1). 4.3.10.6 Electro-elution of proteins from polyacrylamide gels Individual coomassie-stained protein bands of interest were cut out of polyacrylamide gels using a scalpel blade, and chopped into small pieces. The two screw cups of the electrophoresis concentration chambers of the Ecu- 040 electrophoresis elution apparatus (CBS Scientific Company, Inc., USA), were fitted with Spectra/Por dialysis membrane, molecular weight cut-off 3,500 (Spectrum Medical Industries, Inc., USA) and the cut gel placed in the large wells. All the chambers of the apparatus were filled with elution buffer (50mM ammonium bicarbonate, 0.97mM l,4'-Dithiothreitol and 0.1%(w/v) SDS) to volumes recommended by the manufacturer. Protein elution was performed at a constant current of 12mA/cell for 17-24 hr at room temperature. The elution buffer was then carefully replaced with dialysis buffer (lOmM NH3HCO3 and 0.02%(w/v) SDS) without disturbing the eluted proteins concentrated in the small well of the concentration chamber. Dialysis was performed using the same current for 2 hr. At the end of the run, the dialysis buffer was carefully pipetted out of the concentration chambers and the eluted proteins resuspended in a small volume of PBS, pH 7.4. These samples were immediately used to immunize mice for the production of MoAbs, or stored frozen at -20°C before use. 4.3.11 Determination of the biochemical nature of antigenic epitopes 4.3.11.1 Detection of MoAbs specific for carbohydrate epitopes The micro-plate ELISA based periodate oxidation at acid pH described by Woodward, Young and Bloodgood (1985) was used in University of Ghana http://ugspace.ug.edu.gh 102 determining whether the antigenic epitopes detected by the specific MoAbs were carbohydrate in nature. Crude trypanosome extracts were diluted in coating buffer (34.5mM NaHC03 and 15.1mM Na2C03) at dilutions previously determined by titration, and dispensed (100/tl/well) into a 96-well micro-plate (Immulon, Dynatech Laboratories, Chantilly, Virginia, USA) and blocked overnight at 4°C. The plates were rinsed once with washing buffer consisting of 0.05%(v/v) Tween 20 in PBS, pH 7.4, followed by a second rinse using 50mM sodium acetate buffer, pH 4.5. Sets of wells were then incubated with varying concentrations of periodate (0, 10, and 20mM) in sodium acetate buffer (1 0 0 /xl/'well) for 1 hr at room temperature in the dark. The plates were rinsed once with sodium acetate buffer, and incubated with 1% glycine, 100/d/well, for 30 min at room temperature, after which they were rinsed five more times with washing buffer. The wells were incubated with MoAbs of murine origin diluted appropriately, in washing buffer, 1 0 0 /d/well, for 1 hr at room temperature, and the plates washed five times with washing buffer to get rid of excess unbound antibody. To each well was then added 100/d of HRPO-conjugated goat anti-mouse antibodies diluted at 1:1000 in washing buffer. The plates were washed five times with washing buffer and incubated with substrate solution [40mM 2,2'-azino bis-(3- ethylbenz-thiazoline sulfonic acid) diammonium salt (ABTS) and 0.01%(v/v) hydrogen peroxide in 50mM citric acid buffer, pH 4.0], The reaction was allowed to proceed for 30 min and the plates read at a wavelength of 414nm using a Titertek Multiskan micro-plate ELISA reader (MCC/340, Labsystems and Flow Laboratories, Finland). 4.3.11.2 Detection of MoAbs specific for protein epitopes Monoclonal antibodies (MoAbs) with specificity for protein antigenic epitopes were detected using enzymatic digestion with proteinase-K according to the methods described by Martin, Larose, Hamel, Lagac'e and University of Ghana http://ugspace.ug.edu.gh 103 Brodeur (1988) and Lussier et al. (1989) with some modifications. Fifty micrograms of proteinase-K (Bethesda Research Laboratories, USA) diluted in PBS, pH 7.4, was added to 100/zg of fresh trypanosome crude antigens extracted in PBS by the freeze and thaw method. One hundred microgram amounts of each antigen extract were pipetted into two different eppendorf tubes. Fifty micrograms of proteinase-K in 50/xl PBS was added to one sample, whilst the other sample was diluted with 50pd of plain PBS. Both tubes were incubated at 37°C in a waterbath for 1 hr and 3/xl samples pipetted onto nitrocellulose strips in dots. The strips were assayed as described under section 4.3.9.1. 4.3.12 Micro-plate ELISA Non-competitive ELISA techniques were used for the detection of antibody, employing the double antibody sandwich method (Cheng, L.Y., 1987; Beards and Bryden, 1981) and for the detection of antigen, using the indirect-system (Sandwich-ELISA) as described by Nantulya et al. (1987) and Nantulya (1989). 4.3.12.1 Coating microtitre plates with antigen or antibody Trypanosome extracts or purified MoAbs were diluted in carbonate-bicarbonate buffer consisting of 34.5mM NaHC03 and 15.1mM Na2C0 3 , pH 9.6 (coating buffer). Flat-bottomed 96-well microtitre plates were coated with 100jul/well of 10-15/xg of crude antigen or 2.5-5.0 jug/ml of purified MoAb. The coating concentrations were determined by chequerboard titrations as described by Voller, Bidwell and Bartlett (1980). The plates were covered and the adsorption of antigen or antibody onto the polystyrene wells achieved by overnight incubation at 4 °C. University of Ghana http://ugspace.ug.edu.gh 104 4.3.13 Micro-plate ELISA procedure 4.3.13.1 Antibody detection ELISA Antibody detection ELISA was used for screening hybridoma culture fluids for the selection of trypanosome species-specific MoAbs. This method was also used for screening sera from immunized mice. In this assay, micro-ELISA plates were coated with antigens of a particular trypanosome species, and then rinsed once with washing buffer to remove excess unbound antigen. Culture fluids and positive controls (prefusion sera taken from immunized mice diluted, 1:500) and negative controls (normal mouse serum diluted 1:500) or titrated sera from immunized mice, were transferred to a micro-ELISA plate (100^1/well) and incubated for 15 min at 37°C. The micro-ELISA plates were rinsed once with washing buffer to remove excess unbound antibody, and all the wells incubated with 1 0 0 /zl/well goat anti-mouse HRPO conjugate diluted at 1:1000 for 15 min at 37°C. The plates were then washed 3 times, each by 10 min incubation with washing buffer to remove excess unbound conjugate. The presence of bound conjugate was revealed by the addition of substrate solution consisting of 40mM 2,2'- azino-bis (3- ethylbenzthiazoline-6 -sulfonic acid) (ABTS) and 0.01%(v/v) hydrogen peroxide in 50mM citric acid buffer, pH 4.0. The substrate was incubated for 30 min at room temperature. The colourless substrate solution changed to green in wells with bound enzyme conjugates. The optical densities were read at 414nm wavelength using a Titertek Multiskan micro-ELISA reader. 4.3.13.2 Antigen detection sandwich-ELISA A simplified sandwich-ELISA using MoAb-coated polystyrene micro-ELISA plates was used for antigen detection. The plates were coated with 100/i/well of 2.5-5.0 jug/ml of purified MoAbs and rinsed once with washing buffer to remove uncoated antibody. The plates were then incubated for 15 min at 37°C with 100/xl/well of trypanosome antigen extracts titrated University of Ghana http://ugspace.ug.edu.gh 105 serially (using washing buffer as diluent) to give protein concentrations of 1 0 - 0.714 /ig/ml. Antigen molecules were captured specifically by the coating MoAb. The plates were rinsed twice with washing buffer to remove uncaptured antigen. This was followed by incubation with 100/il/well horseradish peroxidase-labelled MoAb, diluted 1:500 in washing buffer at 37°C for 15 min. During this step, the conjugated MoAb reacted with the antigen that had previously been captured by the coating MoAb. The plates were washed, the substrate solution added, and the results read as described for antibody detection-ELISA (section 4.3.13.1). 4.3 .13.3 Inhibition ELISA Inhibition ELISA was used to study the relationship between trypanosome species-specific MoAbs. By this method it was possible to determine whether the binding of one antibody inhibited binding by another. Micro-ELISA plates were coated with antigens of a particular trypanosome species and rinsed once with washing buffer. Sets of wells were then incubated for 15 min at 37°C with 100/xl/well serial dilutions of different MoAbs specific for the coating trypanosome species. MoAbs with specificity to different trypanosome species (non-related MoAbs) were titrated as above and used as controls. A second control consisted of a set of wells that were not incubated with any antibody. The plates were rinsed twice to remove excess unbound antibody and then incubated as above with a uniform concentration of MoAb-HRPO conjugate of one of the specific MoAbs used earlier. The plates were washed three times (10 min/wash) to remove excess antibody-conjugate. Substrate solution was added and the optical densities read as described previously (section 4.3.13.1). The effect of the non-related MoAbs on conjugate binding, was interpreted as protein to protein interactions that were not due to specific inhibition. The conjugate activity in the control wells that were not incubated with antibody, gave the level of conjugate University of Ghana http://ugspace.ug.edu.gh 1 06 binding without interference. The results of these inhibition experiments were interpreted by two-way analysis of variance (Snedecor and Cochram, 1980). University of Ghana http://ugspace.ug.edu.gh 1 0 7 4.4.1 Selection of MoAbs from those produced previously Experiments were carried out using the nitrocellulose membrane-based dot-ELISA, the indirect immunofluorescent antibody test (IFAT) and the micro-plate ELISA assays, to re-examine the specificities of the MoAbs produced previously by Nantulya et al. (1987). Also, the sensitivity, in terms of the minimum number of procyclics or epimastigotes that could be detected by the extant MoAbs in the dot-ELISA, was studied. Following those experiments, the MoAbs listed in Table 7 were selected for further studies. The selected MoAbs reacted specifically with various trypanosome species or subspecies as shown in Table 7. The minimum number of trypanosomes that each of these MoAbs could detect, are however reported in Chapter 5. 4.4.2 Immunizing Antigens and Antibody Responses in Immunized Mice The BALB/c mice responded well to the various trypanosome antigen preparations that were used in the immunizations. In general, high serum antibody responses, with titres far beyond 1 :1 0 ,0 0 0 , were obtained against homologous trypanosome antigens, as determined by the double-antibody sandwich micro-plate ELISA. However, differences were found in the ability of the three different trypanosome antigen preparations (namely, formaldehyde-fixed whole trypanosomes, trypanosome crude-antigen extracts or purified trypanosome antigens) to induce antibody responses that were essentially species-specific. Figure 9, illustrates the mean antibody responses of mice following immunization with T. brucei procyclic crude antigen extract (BPCAE). The individual curves in this figure show that the mice produced antibodies that 4.4 Results University of Ghana http://ugspace.ug.edu.gh 108 Table 7: Reactivity of selected extant MoAbs with procyclics or epimastigotes of different trypanosome species/subspecies as determined by dot-ELISA, IFAT and micro-plate ELISA Monoclonal Antibody Isotype T. brucei * T. vivax ** T. congolense * T. simiae * TR7/47.37.16 IgM + - - - TV8/8.33.42 IgG3 - + - - C2 IgGi - - + - TC6/42.6.3 IgG, - - + - TC40/30.15.40 IgM - - + - TC39/30.25.95 IgM - - + - TC16/5.12.33 IgGi - - + + TC6/25.25.4 IgG3 - + + * procyclics. ** epimastigotes. + = antibody reacts with trypanosomes. - = antibody does not react with trypanosomes. University of Ghana http://ugspace.ug.edu.gh 1 0 9 Figure 9 Serum antibody response of BALB/c mice against antigens of different trypanosome species following immunization with T. brucei procyclic crude antigen extract. Each point represents the mean of three test readings obtained for three different mice + the standard error. O.D. = Optical density. T.b.Ag = Curve showing serum antibody response against T. brucei antigen. T.c.Ag = Curve showing serum antibody response against T. congolense antigen. T.v.Ag = Curve showing serum antibody response against T. vivax antigen. University of Ghana http://ugspace.ug.edu.gh ® oc CO S2u.o V) JQ < Q o 2.00-1 1.50 1 .0 0 t 0.50 o.oo r ---------- r -------- r 100 200 400 - A — T.b. Ag l-O H I | j |------------ T V 800 1600 3200 6400 12800 25600 51200 Reciprocal dilution — T.c. Ag —O— T.v. Ag Figure 9 University of Ghana http://ugspace.ug.edu.gh 110 cross-reacted with equal concentrations of antigens derived from three different trypanosome species (T. brucei, T. congolense and T. vivax). Even though the mean antibody titre against immunizing T. brucei antigens was the highest, the titres recorded against T. congolense or T. vivax were substantially high, with both maintaining optical densities > 0 .5 for serum dilutions of up to 1:200. On the other hand, the mean antibody response against T. brucei antigens was maintained at optical densities > 1 .0 for serum dilutions of up to 1:6,400. The mean antibody responses of other mice which were immunized with purified T. brucei procyclic antigens (PBPA), are shown in Figure 10. At a serum dilution of 1:100, the optical density of the mean response against T. brucei antigens was >1 .0 whereas, that against T. congolense or T. vivax was <0 .25 . At a serum dilution of 1:400, the mean antibody response against T. brucei antigens still gave an optical density >1.0 , whereas, that against T. congolense and T. vivax were reduced to zero. At a serum dilution of 1:12,800, the optical density of the reactivity against T. brucei antigens, remained higher than that against T. congolense or T. vivax at 1:100 serum dilution. This immunization with purified T. brucei procyclic antigens, thus demonstrates antibody responses that were essentially species-specific. The ability of purified trypanosome antigens to induce antibody responses that were essentially species-specific, was also demonstrated for T. vivax and T. simiae. In the case of T. vivax, BALB/c mice were immunized with purified T. vivax epimastigote antigens (PVEA), and screened for serum antibody response against equal concentrations of T. vivax, T. brucei, T. congolense and T. simiae antigens. The individual curves shown in Figure 11, illustrate the mean antibody responses against the different antigens. At a serum dilution of 1:400, the mean optical density of serum antibody reactivity against T. vivax antigens was > 1 .5 , whilst that against the other trypanosome antigens, was < 0 .5 . The mean serum reactivity against T. vivax antigens maintained University of Ghana http://ugspace.ug.edu.gh I l l Figure 10 Serum antibody response of BALB/c mice against antigens of different trypanosome species following immunization with purified T. brucei procyclic antigens. Each point represents the mean of three test readings obtained for three different mice + the standard error. O.D. = Optical density. T.b.Ag = Curve showing serum antibody response against T. brucei antigen. T.c.Ag =Curve showing serum antibody response against T. congolense antigen. T.v.Ag = Curve showing serum antibody response against T. vivax antigen. University of Ghana http://ugspace.ug.edu.gh 1.50n 100 200 400 “ A— T.b. Ag f t ----------- ? ----------- 1 f ------------- t “ -------$ 600 1600 3200 6400 12800 25600 51200 Reciprocal dilution — T.c. Ag - O - T.v. Ag Figure 10 University of Ghana http://ugspace.ug.edu.gh 1 1 2 Figure 11 Serum antibody response of BALB/c mice against antigens of different trypanosome species following immunization with purified T. vivax epimastigote antigens. Each point represents the mean of three test readings obtained for three different mice + the standard error. O.D. = Optical density. T.v.Ag = Curve showing serum antibody response against T. vivax antigens. T.b.Ag = Curve showing serum antibody response against T. brucei antigens. T.c.Ag = Curve showing serum antibody response against T. congolense antigens. T.s.Ag = Curve showing serum antibody response against T. simiae antigens. University of Ghana http://ugspace.ug.edu.gh O .D . (A bs or ba nc e) Reciprocal dilution —O— T.v. Ag —A— T.b. Ag — T.c. Ag — T.s. Ag Figure 11 University of Ghana http://ugspace.ug.edu.gh 113 an optical density of about 0.5 at a serum titre of 1:6,400, when reactivity against antigens of the other trypanosome species were almost nonexistent (Figure 11). Serum antibody responses of mice immunized with purified T. simiae procyclic antigens (PSPA) are shown in Figure 12. The individual curves show the mean serum antibody responses against equal amounts of T. simiae, T. brucei and T. vivax antigens. An elevated antibody response is clearly shown against T. simiae antigens as compared with the responses against antigens of the other trypanosome species. At a serum titre of 1:100, the mean optical density of the reactivity against T. simiae antigens was >1.25, whereas, that against antigens of T. brucei, T. congolense and T. vivax was each below 0.5. Furthermore, for serum dilutions >1:3,200, the reactivity on T. simiae antigens gave a mean optical density of about 0.75, whereas there were no reactions at all against antigens of the other trypanosome species, thus demonstrating specific reactivity with T. simiae. Screening for serum antibody responses in immunized mice, was also performed using the IFAT technique. Table 8 summarizes the results obtained for serum antibody responses of one of the mice immunized with formaldehyde fixed T. vivax epimastigotes (FFVE). To enable the selection of the best responder mice for cell fusion, using this method of screening, the fluorescence on test trypanosomes were graded from; negative (-); weak positive (+); to strong positive (+ + + +). Antibodies produced in this mouse reacted strongly with epimastigote antigens of East African T. vivax (EATV) as well as those of West African T. vivax (WATV), with antibody titres of up to 1:400 and 1:800 respectively (Table 8). Also the antibodies cross-reacted weakly < (+ +) with procyclic antigens of T. brucei (TB), T. congolense (TCK) and T. simiae (TS), with antibody titres ranging from 1:50 to 1:100. University of Ghana http://ugspace.ug.edu.gh 1 1 4 Figure 12 Serum antibody response of BALB/c mice against antigens of different trypanosome species following immunization with purified T. simiae procyclic antigens. Each point represents the mean of three test readings obtained for three different mice + the standard error. O.D. = Optical density. T.s.Ag = Curve showing serum antibody response against T. simiae antigens. T.c.Ag = Curve showing serum antibody response against T. congolense antigens. T.b.Ag = Curve showing serum antibody response against T. brucei antigens. T.v.Ag = Curve showing serum antibody response against T. vivax antigens. University of Ghana http://ugspace.ug.edu.gh ■O < Q o 0 .50 - 0 . 0 0 100 200 400 T.s. Ag 800 1600 3200 6400 Reciprocal dilution — ♦ — T.c Ag —A— T.b. Ag 12800 25600 51200 —O— T.v. Ag Figure 12 University of Ghana http://ugspace.ug.edu.gh 1 15 Table 8 Antibody response of a BALB/c mouse following immunization with formaldehyde fixed Trypanosoma vivax epimastigotes Serum dilution Reactivity of serum with different trypanosome species by IFAT EATV WATV TB TCK TS 1/50 + + + + + + + + + + + + 1 / 1 0 0 + + + + + + + + + 1 / 2 0 0 + + + + + - 1/400 + + + - - 1/800 - + - - EATV = East African T. vivax epimastigotes. WATV = West African T. vivax epimastigotes. TB - T. brucei procyclics. TCK = T. congolense Kilifi type procyclics. TS = T. simiae procyclics. University of Ghana http://ugspace.ug.edu.gh 116 Other mice immunized with FFVE produced serum antibodies that reacted similarly as the one described above. 4.4.3 Cell Fusions and Selection for Hybridomas Twenty eight independent cell fusions were made for the production of trypanosome species-specific MoAbs. Of those fusions, 10, 8 and 10 were made for the production of T. brucei, T. vivax and T. simiae specific MoAbs, respectively. Antibody secreting hybridomas were cloned at least twice by limiting dilution, and the class and subclass of the secreted MoAbs determined by the Ouchterlony double immunodiffusion method (section 4.3.10.1). 4.4.3.1 Anti-T. brucei MoAb secreting hybridomas The results of ten independent cell fusions made towards the production of T. brucei specific MoAbs are summarized in Table 9. Four of these fusions (TB39a, TB40a, TB40b and TB42) were carried out using spleen cells from mice immunized with formaldehyde fixed T. brucei procyclics (FFBP). From these fusions, only one hybridoma from TB39a had the desired specificity for T. brucei. Another hybridoma obtained from TB40a secreted a MoAb that reacted specifically with T. brucei antigens in the double antibody sandwich ELISA. However that MoAb cross-reacted with antigens of T. congolense and T. simiae when tested using the Western immunoblot technique (Table 9). Cell fusions (TB40c, TB40d and TB41) were undertaken using spleen cells from mice immunized with crude antigen extracts of T. brucei procyclics (BPCAE) (Table 9). None of the hybridoma cells derived from those three fusions secreted any MoAb that was T. brucei specific, despite a high number of fusion wells with antibody activity (Table 9). The highest success in deriving hybridomas that secreted T. brucei specific MoAbs was achieved from fusions TB43 and TB44, both of which were carried out using spleen cells University of Ghana http://ugspace.ug.edu.gh 1 17 Table 9: Results of cell fusions made towards the production of T. brucei specific MoAbs Fusion Origin o f spleen cells Immunogen Myeloma parent Spleen cell count for fusion Numbers o f wells showing growth of hybrids Number (%) of positive wells a Number o f wells with specific reactivity^ Number o f hybrids cross-reacting by other tests Number o f stable hybrids secreting specific antibodys TB39a BALB/c FFBP NSI/lAg4.1 3 .6 x l06 16/48 10(62%) 1 0 1 TB40a - 4 .1 x l0 6 48/48 41(85%) 1 l w 0 TB40b ' ■ 1 3.2x10® 42/48 31(74%) 0 n/a n/a TB40c " BPCAE 2 .1 x l0 6 45/48 36(80%) 0 n/a n/a TB40d ti - i 3 .2 x l0 5 48/48 47(98%) 0 n /a n /a TB41 * « • 5 .2 x l07 20/48 10(50%) 0 n/a n/a TB42 n FFBP n 8x l06 12/48 3(25%) 0 n/a n/a TB43 « PBPA X63/AG8.653 6 .9x l07 42/48 16(38%) 4 0 3 TB44 - - 9 .4 x l0 6 40/48 6(15%) 4 0 2 TB45 n n 1 7 .9 x l0 7 48/48 48(100%) 3 3w 0 a Supernatants were tested in double antibody sandwich ELISA using T. brucei antigen coated micro-ELISA plates, b Tested in double antibody sandwich ELISA using 4 different antigens (T. brucei, T. vivax, T. congolense and T. simiae). s Hybridoma cells which secreted specific antibody into culture supernatants after two months o f continuous culture in vitro. w Number o f hybridomas that secreted cross-reacting MoAbs when tested using the Western immunoblot technique. 073 not applicable. FFBP = Formaldehyde fixed T. brucei procyclics. BPCAE = T. brucei procyclic crude antigen extract. PBPA = Purified T. brucei procyclic antigens. University of Ghana http://ugspace.ug.edu.gh 118 from mice immunized with a purified T. brucei procyclic antigen preparation (PBPA). These two fusions yielded 5 T. brucei specific antibody-producing hybridomas (Table 9). One other fusion which was undertaken using spleen cells from a mouse that was immunized with PBPA produced three hybridomas that secreted MoAbs that reacted specifically with T. brucei in the micro-plate ELISA. However, those MoAbs were found to cross-react with T. congolense, T. simiae and T. vivax in the Western immunoblot assay (Figure 13). Cell clones that were isolated from T. brucei specific antibody-secreting hybridomas were found to be stable as determined by sustained MoAb secretion in continuous in vitro culture for two months. Figure 14 shows an example of the micro-plate ELISA results when used for screening 45 out of 48 wells from the T. brucei cell fusion TB43. The reactions in wells A(5&6) and F(9&10) occur on all three microtitre plates, indicating that the hybridoma cells from the corresponding tissue culture well secreted antibodies that cross-reacted with antigens from all the three species of trypanosomes. On the other hand, the reactions in wells B(l&2), C(5&6), D(5&6), E(5&6) and F(5&6) are only seen on the T. brucei coated plate, demonstrating that the hybridoma cells from the corresponding tissue culture wells were secreting antibodies that react specifically with only T. brucei antigens. 4.4.3.2 Anti-T. vivax MoAb secreting hybridomas Table 10 gives a summary of all the cell fusions performed with the aim of producing T. vivax specific MoAbs. Five of these fusions (TV30, TV31, TV32, TV33 and TV34) were made using spleen cells from mice immunized with formaldehyde fixed T. vivax epimastigotes. Of these one fusion (TV34) produced no wells with antibody activity even though two wells had hybridoma cell colonies (Table 10), and two (TV30 and TV33) produced University of Ghana http://ugspace.ug.edu.gh 1 1 9 Figure 13 Western immunoblot cross-reactivity of a MoAb secreted by a hybridoma which was produced by fusing a myeloma cell with a spleen lymphocyte from a BALB/c mouse immunized with purified T. brucei procyclic antigens. Strip (A) shows the reactivity with electrophoresed T. brucei IL2616 antigens; (B) with T. congolense K/83/IL/97/2 antigens; (C) with T. simiae KETRI 2431 antigens, and (D) with T. vivax IL1392 antigens. Notice the weak cross-reactivity of the MoAb with a 58 kDa antigen. University of Ghana http://ugspace.ug.edu.gh B C cDa 58 - Figure 13 University of Ghana http://ugspace.ug.edu.gh Figure 14 Screening for T. brucei specific MoAbs in culture supernatants from cell fusion TB43 using micro-plate ELISA. The spleen cell donor BALB/c mouse was immunized with purified T. brucei procyclic antigens. Plate I was coated with T. brucei procyclic antigen; Plate II with T. congolense procyclic antigen, and Plate III with T. vivax epimastigote antigen. Culture supernatants from fusion wells were tested in duplicates in identical wells on all three plates. Wells G(ll&12) were incubated with 1:100 dilution of normal mouse serum as negative controls, and H(ll&12) were incubated with 1:100 dilution of pre-fusion mouse serum as positive controls. Also, wells A(l&2) was used to assess non-specific conjugate binding by omitting the addition of samples. University of Ghana http://ugspace.ug.edu.gh 1 2 3 4 5 6 7 8 9 10 11 12 A r ' . r ’r ^ "S ^ B # ,< # r A A A A *S -S ^ -S C e 6 e A F 4 • > ' G 1 2 3 4 5 6 7 8 9 10 11 12 o o V \ SA o U J \ j sJ \ j J J V v V ■■ - ■' ^ J J O 'V j .J vJ K,yr. ■ _ ' s " . • ■ • -.j W ^ J :'y y- y - - ’ -' V V ^ •' V - ^ ^ J \ - W y » y ;y v v 1y iv ;V is / ,'VW 1 2 3 4 5 6 7 8 9 10 11 12 c. v ^ o o 1 I----------------- I L Plate I Plate l l P late I I I Figure 14 University of Ghana http://ugspace.ug.edu.gh 121 Table 10: Results of cell fusions made towards the production of T. vivax specific MoAbs Fusion Origin of spleen cells Immunogen Myeloma parent Spleen cell count for fusion Numbers o f wells showing growth of hybrids Number (%) o f positive wells3 Number o f wells with specific reactivity*3 Number o f hybrids cross-reacting by other testsw Number of stable hybrid, secreting specific antibodys TV30 BALB/c FFVE NSI/lAg4.1 1 .25xl07 5/48 2(40%) 0 n/a n/a TV31 ■ - 1 .65xl07 17/48 7(41%) ll 0 1 TV32 » " ■ 5 .8x l07 38/48 19(50%) 31 0 3 TV33 ' 8 .8 x l06 26/48 4(15%) 0 n/a n/a TV34 - Tl Sp2/OAG14 6 .4 x l0 5 2/48 0(0%) 0 n/a n/a TV35 " CVEAL 5x l0 6 15/48 5(33%) 0 n/a n/a TV36 * X63/AG8.653 2 .4 x l0 6 45/48 30(67%) 0 n/a n/a TV37 ■ PVEA 3 .2x l07 48/48 48(100%) 9d 0 9 a Supernatants were tested in double antibody sandwich ELISA using T. vivax antigen coated micro-ELISA plates, b Tested in double antibody sandwich ELISA using 4 different antigens (T. brucei, T. vivax, T. congolense and T. simiae). s Hybridoma cells which secreted specific antibody into culture supernatants after one month of continuous culture in vitro. I Supernatants were tested in Indirect Fluorescent Antibody Test (IFAT) using 4 different antigens (T. vivax, T. brucei, T. congolense and T. simiae). D Tested in dot enzyme immunoassay (dot-ELISA) using 4 different antigens (T. vivax, T. brucei, T. congolense and T. simiae). w Tested for cross-reactivity using the Western immunoblot technique. not applicable. FFVE = Formaldehyde fixed T. vivax epimastigotes. CVEAL = Crude T. vivax epimastigote antigen lysate. PVEA = Purified T. vivax epimastigote antigens. University of Ghana http://ugspace.ug.edu.gh 1 22 wells with antibody activity, but none of them was specific for T. vivax. The remaining two fusions (TV31 and TV32) produced four hybridomas of desired specificity for T. vivax as determined by IFAT. All the positive clones derived from those wells were stable (Table 10). None of two fusions (TV35 and TV36) made with spleen cells from mice immunized with crude T. vivax epimastigote antigen lysate (CVEAL) produced wells with specific activity against T. vivax, although both fusions had wells with antibody activity against trypanosome antigens. Cell fusion (TV37) was made using spleen cells from mouse MTV32 immunized with a purified T. vivax epimastigote antigen (see Figure 11). The products from this cell fusion were screened by dot-ELISA (Figure 15). All the 48 fusion wells showed antibody activity against the T. vivax epimastigote antigen (Figure 15). Of these, wells (1, 4, 19, 32, 33, 34, 35, 42 and 43) contained antibodies that reacted specifically with the T. vivax antigen. The cells from wells 19, 33 and 34 were selected for cloning based on the stronger reactivity of the secreted antibodies and the relatively fewer cells that were present in those wells. The hybridoma (KD37/19.3) derived from well 19 was selected for further studies. 4.4.3.3 Anti-T. simiae MoAb secreting hybridomas Of ten cell fusions carried out with the aim of producing T. simiae specific MoAbs, four were made using spleen cells from mice previously immunized with formaldehyde fixed T. simiae procyclics (FFSP). All these four fusions (TS1, TS2, TS3 and TS4) produced hybridomas which secreted antibodies to trypanosome antigens (Table 11). However, only two of those fusions (TS3 and TS4) yielded hybridomas with antibodies specific for either T. simiae or the Nannomonas subgenus. TS3 had one well with hybrids that secreted T. simiae specific antibodies. These hybrids were, however, not University of Ghana http://ugspace.ug.edu.gh 123 Figure 15 Screening for T. vivax specific MoAbs in culture supernatants from wells of cell fusion TV37 using dot-ELISA. The spleen cell donor BALB/c mouse was immunized with purified T. vivax epimastigote antigens. Culture supernatant from each of the 48 fusion wells was incubated with one strip of NC membrane which was previously "dotted" with lxlO 5 T. congolense K/83/IL/97/2 (TCK), T. brucei IL2616 (TB) and T. simiae KETRI 2431 (TS) cultured procyclics, and lxlO5 T. vivax IL1392 (TV) cultured epimastigotes. (a.) represents culture supernatants that contained antibodies that reacted specifically with T. vivax epimastigotes; (■) represents culture supernatants that contained antibodies that cross-reacted with all the four trypanosome species that were tested. University of Ghana http://ugspace.ug.edu.gh 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 Figure 15 University of Ghana http://ugspace.ug.edu.gh 124 Table 11: Results of cell fusions made towards the production of T. simiae specific MoAbs Fusion Origin of spleen cells Immunogen Myeloma parent Spleen cell count for fusion Numbers of wells showing growth o f hybrids Number (%) o f positive wellsa Number o f wells with specific reactivity Number o f hybrids cross-reacting by other testsw Number of stable hybrids secreting specific antibodys TS1 BALB/c FFSP NSI/lAg401 1 .2x l07 11/48 6(54%) 0 n/a n /a TS2 n " 6.8x10® 9/48 7(78%) 0 n/a n/a TS3 n " 2 .3 x l0 5 8/48 4(50%) l 1 0 0 TS4 7.1x10® 10/48 8(80%) I * I 0 1 TS5 n CSPAL Sp20AG14 3.8x10® 16/48 12(75%) 2D;11*D 0 1 TS6 t " 4 .4 x l0 7 48/48 47(98%) 0 n/a n/a TS7 " PSPA X63/AG8.653 7 .2 x l0 7 48/48 48(100%) 1D 0 0 TS8 " (41.7-43.6)kDa** 6 .9x l07 8/48 2(25%) 0 n/a n/a TS9 ■ 75 kDa** 8 .9x l07 13/48 6(46%) 0 n/a n/a TS10 " 107 kDa** " l . lx lO 7 18/48 10(56%) 0 n/a n/a a Supernatants were tested in double antibody sandwich ELISA using T. sim iae antigen coated micro-ELISA plates. s Hybridoma cells which secreted specific antibody into culture supernatants after one month o f continuous culture in vitro. I Supernatants were tested in Indirect Fluorescent Antibody Test (IFAT) using 4 different antigens (T. simiae, T. congolense, T. brucei and T. vivax). D Tested in dot enzyme immunoassay (dot-ELISA) using 4 different antigens (T. simiae, T. congolense, T. brucei and T. vivax). w Tested for cross-reactivity by the Western blot technique. H^ a Not applicable. FFSP = Formaldehyde fixed T. simiae procyclics. CSPAL = Crude T. simiae procyclic antigen lysate. PSP A = Purified T. simiae procyclic antigens. * Hybridoma secreting Nannomonas species-specific monoclonal antibody. ** Antigen band electro-eluted from polyacrylamide gels. University of Ghana http://ugspace.ug.edu.gh 1 25 stable as they stopped antibody secretion when maintained in continuous culture in vitro. As a result, the hybrid was lost during cloning. The fusion (TS4) had one well with specificity for the Nannomonas subgenus. This hybrid was cloned successfully. Two fusions (TS5 and TS6) were made using spleen cells from mice immunized with crude T. simiae procyclic antigen lysate (CSPAL). One of these fusions (TS5) produced one hybridoma with specific antibodies to the Nannomonas subgenus (Table 11). However, none of the hybridomas from TS6 secreted antibodies that were specific for either T. simiae or the Nannomonas subgenus. The fusions (TS8, TS9 and TS10) were made using spleen cells from mice immunized with T. simiae antigen bands of varying molecular weights that had been electro-eluted from polyacrylamide gels (Table 11, Figure 16). These bands were selected based on the following rationale. T. simiae and T. congolense procyclic extracts were electrophoresed side by side on polyacrylamide gels and the resolved bands studied for differences in molecular weight (MW). All T. simiae antigen bands that occur at MW where there were no corresponding T. congolense bands, were pin­ pointed. Reasoning that some of the T. simiae unique bands may contain antigens that define T. simiae specificity, the bands were separately electro­ eluted and used for immunizing BALB/c mice. Though each of die three fusions made with spleen cells from mice immunized with electro-eluted antigen bands produced hybridomas with antibody activity, none of them was specific for T. simiae or the Nannomonas subgenus. The fusion (TS7) which was made using spleen cells from the mouse MTS7 immunized with a purified T. simiae procyclic antigen produced the most promising results (Figure 17). Figure 17 shows the result of this cell fusion as screened by dot-ELISA. All the wells from this fusion had colonies of hybrid cells that were secreting antibodies to trypanosome antigens. Of these, 11 wells had antibodies specific to the Nannomonas subgenus, whereas 2 University of Ghana http://ugspace.ug.edu.gh 126 Figure 16 Comparison of coomassie stained polyacrylamide gel electrophoresed T. simiae (KETRI 2431) and T. congolense (K/83/IL/97/2) antigens. Lane 'C' shows the staining pattern of T. simiae antigens; Lane 'B' the staining pattern of T. congolense antigens; and lane 'A ', the molecular weight markers. T. simiae antigen bands without corresponding T. congolense bands of identical molecular weights were numbered 1 to 8 . University of Ghana http://ugspace.ug.edu.gh A i------- 1 92 .5— 69 - 46 - * 30 - +> 21 .5 - * 14 .3 - | 2 0 0 - University of Ghana http://ugspace.ug.edu.gh 1 2 7 Figure 17 Screening for T. simiae specific MoAbs in culture supernatants from wells of cell fusion TS7 using dot-ELISA. The spleen cell donor BALB/c mouse was immunized with purified T. simiae procyclic antigens. Culture supernatant from each of the 48 fusion wells was incubated with one strip of NC membrane which was previously "dotted" with lxlO5 T. congolense K/83/IL/97/2 (TCK), T. brucei IL2616 (TB) and T. simiae KETRI 2431 (TS) cultured procyclics, and lxlO5 T. vivax IL1392 (TV) cultured epimastigotes. (a ) represents culture supernatants that contained antibodies that reacted mainly with T. simiae procyclics; ( □ ) represents culture supernatants that contained antibodies that reacted specifically with trypanosomes of the Nannomonas subgenus; ( o ) represents culture supernatants that contained antibodies that cross-reacted with all the four trypanosome species that were tested. University of Ghana http://ugspace.ug.edu.gh Figure 17 University of Ghana http://ugspace.ug.edu.gh 128 wells (14 and 44) had activity confined mainly to the T. simiae antigen. The antibodies from well 14 reacted strongly with the T. simiae antigen dot and very weakly with the T. congolense antigen dot, but not at all with the dots representing T. brucei or T. vivax. Likewise, the antibodies from well 44 reacted strongly with the T. simiae antigen dot, but showed weak reactivity with both the T. congolense and the T. brucei antigen dots. It also did not react with the dot representing the T. vivax antigen. Based on this reactivity, the cells from those two wells were extensively cloned. Of the first clones originating out of well 14, only 4 out of 200 tested positive and all were specific to T. simiae when screened by dot-ELISA (Figure 18). The clones marked 1, 2 and 3 were selected for further cloning on the basis of their stronger reactivity. Screening of the re-clones revealed that only about 1% of the cells continued to secrete the T. simiae specific MoAb, suggesting that the hybridoma was unstable. It was, therefore, decided to explore the possibility of isolating some stable hybrids by re-cloning positive clones several times over. Unfortunately, after several re-cloning attempts, the trend remained unchanged. As a result, continuous culturing of these cells was not possible. However, culture supernatants obtained from the earlier cultures were concentrated by ammonium sulphate precipitation and dialysed. This fraction was tested by dot-ELISA and found to be active, and used for further characterization of the antibody and antigen. Some cells from the original well 14 as well as cells from the first and second positive clones were cryopreserved in liquid nitrogen. Two hundred first clones were derived from TS7 fusion well 44, but none of these tested positive when screened by dot-ELISA. Hence, only two hybrids (one from fusion TS3 and the other from fusion TS7) secreting antibodies with specific reactivity to T. simiae were ever produced, and none of these hybrids was stable. University of Ghana http://ugspace.ug.edu.gh 1 29 Figure 18 Dot-ELISA reactivity of MoAbs secreted by four hybridoma cell clones obtained from well 14 of cell fusion TS7. Culture supernatant from each clone was tested for antibody reactivity by incubating with a strip of NC membrane that was previously "dotted" with lxlO 5 T. congolense K/83/IL/97/2 (TCK), T. brucei IL2616 (TB) and T. simiae KETRI 2431 (TS) cultured procyclics, and lxlO5 T. vivax IL1392 (TV) cultured epimastigotes. Strips 1, 2, 3 and 4 (Group I) shows the specific reactivity of the MoAbs secreted by the four hybrid clones, with T. simiae parasites. The strips shown in Group II illustrate specific reactivity of control MoAbs in the same test: T. brucei specific MoAb (KT39a); T. vivax specific MoAb (KD32); Nannomonas specific MoAb (KN4); and T. congolense specific MoAb (TC6 ). University of Ghana http://ugspace.ug.edu.gh 1 2 3 4 TCK — TB - TS - T V - I Group I Figure University of Ghana http://ugspace.ug.edu.gh K T 3 9 a CM CO CD Group II 18 University of Ghana http://ugspace.ug.edu.gh 1 30 4.4.4 Reactivity of the New MoAbs Produced From the 28 fusions that were made, 8 additional hybridomas each of which secreted a trypanosome species or subspecies specific MoAb were derived. The specificity of each of the MoAbs was confirmed by testing for cross-reactivity using the more sensitive Enzyme-linked Immunoelectrotransfer Blot Technique (Western immunoblot) analysis. The isotypes and reactivity patterns of the 8 additional MoAbs are listed in Table 12. Three of these MoAbs [KT39a/18.17 (IgM), KT43/33.32 (IgGj) and KT43/27.32 (IgG2a)] were T. brucei specific; two [KD32/48.17 (IgGj) and KD37/19.3 (IgGj)] were T. vivax specific; one [KNS7/14.X(IgG1)] was T. simiae specific; and two [KN4/13.9(IgG3) and KN5/6.15(IgGj)] were Nannomonas subgenus-specific. 4.4.5 Characterization of MoAbs and the Antigens that they Detect The remaining sections of this Chapter record the results of the characterization studies of the MoAbs listed in (Tables 7 and 12), and the specific antigens that they detected. For purposes of convenience, abbreviated names of those MoAbs will henceforth be used in the text. Table 13 lists the full names of the MoAbs and their abbreviated forms. 4.4.6 Immunolocalization of the Species-specific Antigens bound bv the MoAbs Immunolocalization studies made by IFAT, revealed that some trypanosome species-specific antigens bound by the MoAbs were located on the surface membrane of procyclics or epimastigotes (Figures 19a, 19b and 19c) whilst others were intracytoplasmic (Figure 19d). Table 14 summarizes the results on the localization of the specific antigens bound by all the MoAbs as determined by IFAT. Three out of four T. brucei specific antigens localize to the surface membrane of T. brucei procyclics, whereas one is intracytoplasmic. Likewise, two out of three T. vivax specific antigens University of Ghana http://ugspace.ug.edu.gh 1 31 Table 12 Reactivity of the new MoAbs against procyclics or epimastigotes of different trypanosome species Monoclonal Antibody Isotype T. brucei * T. vivax ** T. congolense * T. simiae * KT39a/18.17 IgM + - - - KT43/33.32 IgGi + - KT43/27.32 I§G2a + - KD32/48.17 IgGi - + - - KD37/19.3 IgGj “ + KNS7/14.X IgGj - - + KN4/13.9 IgG3 - - + + KN5/6.15 IgGi - - + + * procyclics. ** epimastigotes. + = antibody reacts with trypanosomes. = antibody does not react with trypanosomes. University of Ghana http://ugspace.ug.edu.gh 132 Table 13 Abbreviated forms of the names of selected MoAbs Monoclonal Antibody (FULL NAME) Abbreviated name Isotype Specificity TR7/47.37.16 TR7 IgM T. brucei KT39a/18.17 KT39a IgM T. brucei KT43/33.32 KT43/33 IgGi T. brucei KT43/27.32 KT43/27 IgG2a T. brucei TV8/8.33.42 TV8 IgG3 T. vivax KD32/48.17 KD32 IgGj T. vivax KD37/19.3 KD37 IgGi T. vivax C2 C2 IgGi T. congolense TC6/42.6.3 TC6 IgGj T. congolense TC40/30.15.40 TC40 IgM T. congolense TC39/30.25.95 TC39 IgM T. congolense KNS7/14.X KNS7 IgGi T. simiae TC16/5.12.33 TC16 IgGi Nannomonas TC6/25.25.4 TC6/25 IgG3 Nannomonas KN4/13.9 KN4 IgG3 Nannomonas KN5/6.15 KN5 IgGi Nannomonas University of Ghana http://ugspace.ug.edu.gh 1 33 Figure 19a Light micrograph of Trypanosoma simiae (KETRI 2431) procyclics showing surface membrane fluorescence following incubation with KN4 and anti-mouse-FITC. Photographed at xlOO magnification. f = flagella. m - membrane. University of Ghana http://ugspace.ug.edu.gh V * * " ' m Figure 19a University of Ghana http://ugspace.ug.edu.gh 1 3 4 Figure 19b Light micrograph of Trypanosoma vivax (IL1392) epimastigotes showing surface membrane fluorescence following incubafjfp with KD32 and anti-mouse-FITC. Photographed at x 100 magnification. f = flagella. m — membrane. University of Ghana http://ugspace.ug.edu.gh Figure 19b University of Ghana http://ugspace.ug.edu.gh University of Ghana http://ugspace.ug.edu.gh 13 5 Figure 19c Light micrograph of Trypanosoma brucei (IL2616) procyclics showing surface membrane fluorescence following incubation with KT39a and anti-mouse-FITC. Photographed at xlOO magnification. f — flagella. m = membrane. University of Ghana http://ugspace.ug.edu.gh Figure University of Ghana http://ugspace.ug.edu.gh 119c University of Ghana http://ugspace.ug.edu.gh 136 Figure 19d Light micrograph of Trypanosoma brucei procyclics showing cytoplasmic staining following incubated with KT43/27 and anti- mouse-FITC. Photographed at xlOO (A) without and (B) with fluorescence. m = membrane, n - nucleus. University of Ghana http://ugspace.ug.edu.gh Figure 19d University of Ghana http://ugspace.ug.edu.gh 1 37 Table 14 Immunolocalization of the trypanosome species-specific antigens by indirect fluorescent antibody test (IFAT) Monoclonal Antibody Isotype Specificity Membrane staining Cytoplasmic staining TR7 IgM T. brucei + KT39a IgM T. brucei + KT43/33 IgGj T. brucei + KT43/27 IgG2a T. brucei + TV8 IgG3 T. vivax + KD32 IgGi T. vivax + - KD37 IgG! T. vivax + C2 IgGi T. congolense + TC6 IgGj T. congolense + TC40 IgM T. congolense + TC39 IgM T. congolense + KNS7 IgGi T. simiae - + TC16 IgGi Nannomonas + TC6/25 IgG3 Nannomonas + KN4 IgG3 Nannomonas + - KN5 IgGi Nannomonas + - + = stained. = not stained. University of Ghana http://ugspace.ug.edu.gh 1 38 are surface membrane antigens of epimastigotes, and one is intracytoplasmic. All the T. congolense specific antigens are located within the cytoplasm of procyclic organisms. The T. simiae specific antigen is also located in the cytoplasm of procyclics. Unlike the T. congolense specific MoAbs, all the Nannomonas subgenus-specific antigens are located on the surface membranes of procyclics. IFAT analysis, using live trypanosomes, showed that three T. brucei specific MoAbs (TR7, KT39a and KT43/33); four Nannomonas specific MoAbs (TC16, TC6/25, KN4 and KN5), and one T. vivax specific MoAb (KD32) bound antigens on the surface of living trypanosomes. Characterization of the specific antigens by the Western immunoblot technique revealed that some of the MoAbs could not work in this assay (Table 15). Each trypanosome species-specific MoAb was assayed on SDS-PAGE separated antigens of four different trypanosome species (T. brucei, T. vivax, T. congolense and T. simiae). Of the four T. brucei specific MoAbs, TR7 (an IgM antibody) bound three protein bands of MW between 21 and 27 kDa whilst KT43/33 (an IgGj antibody) bound multiple bands ranging between 21 and 47 kDa (Figure 20). The third T. brucei antibody, KT39a, an IgM isotype, bound a 90 kDa protein band whilst the fourth, KT43/27, an IgG2 a, did not work in this assay. The T. vivax specific MoAbs TV8 (IgG3), KD32 (IgG^ and KD37 (IgGj) did not bind any bands in electrophoresed T. vivax bloodstream form or epimastigote lysates. C2 and TC6 , both T. congolense specific and of IgGj isotype, bound protein bands within the same molecular weight range (30-40) kDa. C2 bound a doublet of protein bands at 30 and 31 kDa and another doublet at 38 and 40 kDa, whilst TC6 bound only one band at 30 kDa. The T. simiae specific MoAb, KNS7 (IgGj), did not work in the Western immunoblot assay. Also, University of Ghana http://ugspace.ug.edu.gh 139 Table 15 The molecular weights of the antigens detected by the trypanosome species-specific MoAbs as determined by Western inununoblot analysis Monoclonal Antibody Isotype Specificity Molecular weight (kDa) TR7 IgM T. brucei (21, 24, 27) KT39a IgM T. brucei (90) KT43 IgG! T. brucei (21 - 47) KT43/27 !gG2 a T. brucei — TV8 IgG3 T. vivax _ KD32 IgGi T. vivax -- KD37 IgGi T. vivax -- C2 IgGi T. congolense (30 - 40) TC6 IgGi T. congolense (30) TC40 IgM T. congolense (51, 60, 85) TC39 IgM T. congolense (21 30) KNS7 IgGi T. simiae — TC16 IgGj Nannomonas (18, 89)tck (31)ts TC6/25 IgG3 Nannomonas -- KN4 IgG3 Nannomonas -- KN5 IgGj Nannomonas -- — = antibody does not bind any antigens. TCK= T. congolense Kilifi type procyclic lysate. TS = T. simiae procyclic lysate. University of Ghana http://ugspace.ug.edu.gh 1 4 0 Figure 20 Specific reactivity of the MoAb KT43/33 in the Western immunoblot assay and the molecular weights of the antigens that it bound. Strip 'A' shows the reactivity of KT43/33 with antigens in electrophoresed T. brucei IL2616 procyclic lysate. Strips B, C and D contained respectively, electrophoresed T. congolense, T. simiae and T. vivax antigens. KT43/33 reacted specifically with T. brucei antigen peptides of molecular weights ranging between 21 and 47 kDa. University of Ghana http://ugspace.ug.edu.gh 1 41 three of the four Nannomonas specific MoAbs did not work. However, TC16, also Nannomonas specific, bound antigens in electrophoresed T. congolense and T. simiae procyclic extracts at 18, 89 kDa and 31 kDa, respectively (Table 15). 4.4.7 The Biochemical Nature of the Trypanosome Species-specific Antigenic Determinants Periodate oxidation of carbohydrate residues and proteinase-K modification of polypeptide residues were used to study the biochemical nature of the specific antigenic determinants bound by the MoAbs. Binding by three of the four T. brucei specific MoAbs was totally abrogated by treatment with proteinase-K (Table 16). This result suggests that the antigenic determinants involved are protein in nature. The fourth T. brucei specific MoAb (KT39a) was only partially affected by proteinase-K digestion, but not by sodium periodate oxidation, indicating that the antigenic determinant involved is partly protein and partly either a carbohydrate or a lipid (Table 16). Two of the three T. vivax specific MoAbs bound a proteinase-K sensitive antigenic determinant, whilst the remaining one (TV8 ) was insensitive to both periodate oxidation and proteinase-K digestion. All the four Nannomonas as well as the T. simiae specific MoAbs detected periodate sensitive antigenic determinants in glycoprotein or glycolipid antigens. In contrast, all the T. congolense specific MoAbs detected protein antigenic epitopes, as evidenced by their sensitivity to proteinase-K and insensitivity to periodate oxidation (Table 16). 4.4.8 Distribution of the Specific Antigenic Determinants fEnitones't on Antigens The antigen detection sandwich-ELISA was used to study the University of Ghana http://ugspace.ug.edu.gh Figure 20 University of Ghana http://ugspace.ug.edu.gh 142 Table 16 The nature of the antigens detected by the trypanosome species-specific MoAbs as determined by periodate and proteinase-K digestion Monoclonal Antibody Isotype Specificity Sensitivity to Nature of antigenic epitopePeriodate Proteinase-K TR7 IgM T. brucei - + P KT39a IgM T. brucei - + /- P, C?, L? KT43 IgGi T. brucei + P KT43/27 IgG2a T. brucei + P TV8 IgG3 T. vivax _ _ C?, L? KD32 IgGj T. vivax - + P KD37 IgGi T. vivax - + P C2 IgGi T. congolense - + P TC6 IgGi T. congolense + P TC40 IgM T. congolense + P TC39 IgM T. congolense + P KNS7 IgGi T. simiae + - C TC16 IgGi Nannomonas + _ C TC6/25 IgG3 Nannomonas + - C KN4 IgG3 Nannomonas + C KN5 IgGj Nannomonas + C + = sensitivity to periodate or proteinase-K. - = insensitivity to periodate or proteinase-K. + = partial sensitivity to proteinase-K. P = protein antigenic determinant. C = carbohydrate antigenic determinant. L = lipid antigenic determinant. ? = not certain. University of Ghana http://ugspace.ug.edu.gh 143 distribution of trypanosome species-specific antigenic epitopes on antigens. In these experiments, MoAbs specific to each trypanosome species or subgenus were placed into separate groups. Each antibody in a group was used to trap the antigen(s) on which its specific epitope is expressed. The trapped antigen(s) was then revealed by HRPO conjugate of each antibody in the group in separate experiments. The results obtained for the T. brucei specific MoAbs are shown in Table 17a. When the MoAb TR7 was used to capture the antigen(s) on which its specific epitope is expressed, it was possible to reveal the captured antigen(s) using the conjugates of all the T. brucei specific MoAbs, including the homologous conjugate of TR7 (Table 17a). This meant that the TR7 specific antigenic epitope is repeated on the captured antigen(s), so that when the antigen(s) was trapped by TR7 the same antigen(s) could be revealed by that MoAb's conjugate. Also, the result suggested that the antigen(s) captured by TR7 expressed all the antigenic epitopes bound by the other T. brucei specific MoAbs. Similar results were recorded when KT39a was used as capture antibody (Table 17a). The results also showed that the IgM MoAbs were better capture antibodies compared with the IgG's. This was clearly shown by the strong reactivity of the conjugate of KT43/33(IgGi) with the antigen(s) captured by KT39a(IgM), and yet the absence of reactivity when the conjugate of KT39a was used to reveal the antigen(s) captured by KT43/33 (Table 17a). Furthermore, it was evident from the pattern of reactivity that KT43/33 was the best antibody for revealing captured antigen (Table 17a). Each T. congolense MoAb captured antigen(s) that could be revealed by conjugates of all the others including that of the capture MoAb (Table 17b). This result suggested that the antigen(s) captured by these MoAbs is likely to be the same. Also, the ability of the MoAbs to capture their University of Ghana http://ugspace.ug.edu.gh 144 Table 17a Relationship between the T. brucei specific MoAbs as revealed by sandwich-ELISA Reveal antibody conjugate Capture ----------------------------------------------------------------- antibody TR7 KT39a KT43/33 KT43/27 TR7(IgM) + + + + + + + + + KT39a(IgM) + + + + + + + + KT43/33(IgG!) + - + + KT43/27(IgG2a) + + = ability of MoAb conjugate to reveal captured antigen. - = MoAb conjugate unable to reveal captured antigen. University of Ghana http://ugspace.ug.edu.gh 145 Table 17b Relationship between the T. congolense specific MoAbs as revealed by sandwich-ELISA Capture antibody Reveal antibody conjugate C2 TC6 TC40 TC39 C2 + + + + + TC6 + + + + + TC40 + + + + + + TC39 + + + + + + + + + = ability of MoAb conjugate to reveal captured antigen. University of Ghana http://ugspace.ug.edu.gh 146 respective antigens and reveal them by their own conjugates, indicated that the antigenic determinants are repeated on the antigen(s). As shown in Table 17c, the Nannomonas subgenus-specific MoAb TC6/25 captured antigen(s) that could be detected by conjugates of itself, KN4 and KN5 but not TC16. Likewise, the antigen(s) captured by TC16 could not be revealed by any of the conjugates other than that of TC16 itself. These results indicate that the antigenic epitope bound by TCI6 is on a different antigen. It is also seen from Table 17c that both KN4 and KN5 captured antigen(s) that could not be revealed by their own conjugates. This observation suggests that the epitopes bound by these MoAbs (KN4 and KN5) are not significantly repetitive on the respective antigens. The T. vivax MoAb, TV8 , captured antigen(s) that could be revealed by conjugates of each of the three T. vivax MoAbs (Table 17d), suggesting that the antigenic determinants bound by all these MoAbs are distributed on the same antigen(s). KD32 captured antigen(s) that could be revealed by its own conjugate, but weakly by conjugates of TVS or KD37, yet the same KD32 conjugate could reveal very well the antigen(s) captured by TVS. This suggests that the weak reactivity of the TV8 conjugate may be due to altered epitope accessibility due to conformational changes in the antigen, brought about by the binding to KD32. 4.4.9 Inhibition ELISA To further elucidate the relationship between the specific epitopes bound by the different MoAbs (section 4.4.8), experiments were conducted to examine the effect of the binding of one MoAb on binding by another, using micro-plate-based inhibition ELISA. The results obtained showed that the T. brucei specific MoAbs, KT43/33 and KT43/27 could not inhibit each other, showing that the two epitopes were different. However, two other T. brucei specific MoAbs University of Ghana http://ugspace.ug.edu.gh 1 47 Table 17c Relationship between the Nannomonas specific MoAbs as revealed by sandwich-ELISA Capture antibody Reveal antibody conjugate TC6/25 TCI 6 KN4 KN5 TC6/25 + - + + TC16 - + - KN4 + - - + KN5 + + + = ability of MoAb conjugate to reveal captured antigen. - = MoAb conjugate unable to reveal captured antigen. University of Ghana http://ugspace.ug.edu.gh 148 Table 17d Relationship between the T. vivax specific MoAbs as revealed by sandwich-ELISA Capture antibody Reveal antibody conjugate TV8 KD32 KD37 TV8 + + + + + KD32 + + ± KD37 + + + = ability of MoAb conjugate to reveal captured antigen. = MoAb conjugate unable to reveal captured antigen. ± = very weak reactivity. University of Ghana http://ugspace.ug.edu.gh 1 49 (KT39a and TR7) inhibited each others binding, suggesting that the two MoAbs are directed at the same epitope. Each of the four T. congolense specific MoAbs (C2, TC6 , TC39 and TC40) was able to inhibit the binding of the others. 4.4.10 Reactivity of the Various MoAbs with Different Trypanosome Stocks and Clones The aim of the experiments described here was to determine the suitability of the trypanosome species-specific MoAbs as diagnostic reagents. This assessment was based on their ability to react with trypanosomes from different geographical areas. All the trypanosome stocks and clones used in the present study, are listed, together with their places of origin, in Table 18. With the exception of KT43/27, the T. brucei specific MoAbs detected all the different developmental stages of T. brucei organisms that were tested (Table 19). The reactivity patterns indicate that KT43/27 was able to detect all the in vitro propagated T. brucei procyclic organisms. However, the same MoAb could not detect T. brucei bloodstream forms or insect forms from tsetse gut or salivary glands. Studies with the T. vivax specific MoAbs showed that TV8 and KD37 could detect all the epimastigotes and blood stream forms tested (Table 20). Thus, in addition to East and West African T. vivax, these two MoAbs detected IL3841 which originated from Colombia, South America. These MoAbs also detected T. vivax insect forms of IL3096 from tsetse mouthparts. In contrast, KD32 which was derived against epimastigotes of West African T. vivax (IL1392), was unable to detect bloodstream forms of IL1392, IL2160 and IL3841, even though the same MoAb detected bloodstream forms of University of Ghana http://ugspace.ug.edu.gh l aoie 18: Trypanosom e stocks and clones from d ifferen t geographical a rea s used in determ in ing th e range o f reactiv ity o f the trypanosom e species-specific M oAbs : ■’ , 150 Species Trypanosome stock/clone Origin T. congolense *K/83/IL/97/2 Kilifi, Kenya (K) K/82/IL/60/1 Kilifi, Kenya (K) IL3779 Nguruman, Kenya (S) CP81 Taita, Kenya (S) *ILC49 Transmara, Kenya (S) *IL13-E3 Busoga, Uganda (S) *IL2079 Serengeti, Tanzania (S) *IL1180 Serengeti, Tanzania (S) IL3900 Bobodioulasso, Burkina Faso (R) *IL3274 Banankeledaga, Burkina Faso (R) MSUS/LR/77/TSW103 Duoplay, L iberia (K) MOVS/KE/81/WG84 Matuga, Kenya (R) MBOI/NG/60/1-148 Donga Valley, N igeria (S) T. simiae KETRI 2431 Ukunda, Kenya *TS1 Ukunda, Kenya *TS4 Ukunda, Kenya IL3815 Ukunda, Kenya T. vivax IL3895 Kipini, Kenya IL2005 Teso, Uganda IL1392 Zaria, Nigeria IL2160 Zaria, Nigeria IL3096 Zaria, Nigeria *ILDat 1.9 Zaria, N igeria *IL3841 Lorica, Colombia T. b. brucei CP 2137 Nairobi, Kenya M iTat 1.2 Lugala, Uganda CP 547/R Jilib, Somalia IL2616 Serengeti, Tanzania IL375 Serengeti, Tanzania IL3579 Serengeti, Tanzania T. b. gambiense TREU 1442 Nigeria Th-17/78 E(020) Cote d 'Ivoire T. b. rhodesiense IL1984 Lugala, Uganda IL1478 Lambwe Valley, Kenya T. grayi GPAG/GM/88/BAN1 Bansang, The Gambia * = clone. K = K ilifi type. S = savannah type. R = riverine-forest type. University of Ghana http://ugspace.ug.edu.gh 1 5 1 Table 19: Reactivity of the T. brucei specific MoAbs with different stocks and clones of T. brucei as defined by dot-ELISA Monoclonal antibody IL2616 (Proc) Th-17/87 (Proc) TREU-1442 (Proc) M iT atl.2 (Proc) IL1984 (Proc) IL1478 (Proc) CP2137 (b/d) CP547/R (b/d) IL375 (i/f)G IL375 (i/f)SG IL3579 (i/f)G IL3579 (i/f)SG TR7 + + + + + + + + + + + + KT39a + + + + + + + + + + + + KT43/33 + + + + + + + + + + + + KT43/27 + + + + + - - - - - - - + = antibody reacts with trypanosomes. = antibody does not react with trypanosomes. (Proc) = procyclic forms propagated in vitro. (b/d) = bloodstream forms propagated in vivo. (i/f)G = insect forms from the gut. (i/f)SG = insect forms from the salivary glands. University of Ghana http://ugspace.ug.edu.gh 1 52 Table 20: Reactivity of the T. vivax specific MoAbs with different stocks and clones of T. vivax as defined by dot-ELISA Monoclonal antibody IL1392 (Epis) IL1392 (b/d) IL3895 (Epis) ILDatl.9 (Epis) IL2160 (b/d) IL2005 (b/d) IL3841 (b/d) IL3096 (i/f)MP TVS + + + + + + + + KD32 + - + + - + - + KD37 + + + + + + + n.t. + = antibody reacts with trypanosomes. = antibody does not react with trypanosomes. (Epis) = epimastigote forms propagated in vitro. (b/d) = bloodstream forms propagated in vivo. (i/f)MP = insect forms from the mouthparts. n.t =not tested. University of Ghana http://ugspace.ug.edu.gh 153 IL2005 (Table 20), indicating a differential expression of the epitope involved in insect stages of the parasite, and in bloodstream forms of some stocks (KD37 was not tested against the insect forms of IL3096 because the MoAb was derived late in the course of these studies). Each of the four T. congolense specific MoAbs detected all the different stocks and clones of T. congolense that were tested, regardless of the developmental stages of the organism (Table 21). The reactivity of the Nannomonas specific MoAbs is given in Table 22. The four specific MoAbs detected all the epimastigote and tsetse gut forms of T. congolense, as well as the procyclics of T. congolense and T. simiae. The reactivity of these MoAbs with bloodstream forms, however, presented a different picture. Whilst KN5 detected all the T. congolense bloodstream forms that were tested, TC6/25, TC16 and KN4 were unable to detect the bloodstream forms of IL2079, and CP81. Yet, the same MoAbs (TC6/25, TC16 and KN4) could detect bloodstream forms of IL1180, IL3779 and IL3900, suggesting that the antigenic epitopes detected by these MoAbs were not expressed in the bloodstream forms of all the different stocks of T. congolense organisms. All the specific MoAbs were screened against T. grayi procyclics. The object of this exercise was to determine whether any of the MoAbs would cross-react with T. grayi, since it also infects tsetse flies. None of the MoAbs reacted with the T. grayi parasites when tested with the dot-ELISA (Figure 21). The results of the screening of TC6/25, KN4, TC6/42, KT39a and KD32 on T. grayi, and T. congolense (savannah type, riverine/forest type and Kilifi type) are summarized in Figure 21. As it is indicated, the T. congolense MoAb (TC6 ) and Nannomonas MoAbs (TC6/25 and KN4) detected all the different types of T. congolense that were tested. University of Ghana http://ugspace.ug.edu.gh 154 Table 21: Reactivity of the T. congolense specific monoclonal antibodies with different stocks and clones of T. congolense as defined by dot-ELISA Monoclonal antibody IL/60/1 (Proc) IL/97/2 (Proc) IL2079 (Epis) IL3900 (b/d) IL2079 (b/d) ELC49 (b/d) CP81 (Epis) CP81 (b/d)* CP81 (i/f)G IL1180 (b/d) IL1180 (i/f)G EL13-E3 (i/f)G IL3274 (i/f)G IL3779 (i/f)G MOVS (Proc) MBOI (Proc) MSUS (Proc) C2 + + + + + + + + + + + + + + + + + TC39 + + + + + + + + + + + + + + + + + TC40 + + + + + + + + + + + + + + + + + TC6 + + + + + + + + + + + + + + + + + + = antibody reacts with tiypanosomes. IL/60/1 = K/82/IL/60/1. IL/97/2 = K/83/IL/97/2. (Proc) = procyclic forms propagated in vitro. (Epis) = epimastigote forms propagated in vitro. (b/d)* = bloodstream forms propagated in vitro. (b/d) = bloodstream forms propagated in vivo. (i/f)G - insect forms from the gut. MOVS = MOVS/KE/81/WG84. MBOI = MBOI/NG/60/1-148. MSUS = MSUS/LR/77/TSW103. University of Ghana http://ugspace.ug.edu.gh 1 55 Table 22: Reactivity of the Nannomonas specific MoAbs with different stocks and clones of T. congolense and T. simiae as defined by dot-ELISA Monoclonal antibody IL/97/2 (Proc) IL/60/2 (Proc) TScl (Proc) TSc4 (Proc) IL2079 (Epis) IL2079 (b/d)* CP81 (Epis) CP81 (b/d)* CP81 (i/f)G IL1180 (b/d) IL1180 (i/f)G IL3779 (b/d) IL3779 (i/f)G IL3900 (b/d) CP813 (i/f)G IL3274 (i/f)G MOVS (Proc) MBOI (Proc) MSUS (Proc) TC6/25 + + •+ + + - + - + + + + + + + + + + + TC16 + + + + + - + - + + + + + + + + + + + KN4 + + + + + - + - + + + + + + + + + + + KN5 + + + + + + + + + + + + + + + + + + + + = antibody reacts with trypanosomes. = antibody does not react with trypanosomes. IL/60/1 = K/82/IL/60/1. IL/97/2 = K/83/IL/97/2. (Proc) = procyclic forms propagated in vitro. (Epis) = epimastigote forms propagated in vitro. (b/d)* = bloodstream forms propagated in vitro. (b/d) = bloodstream forms propagated in vivo. (i/f)G = insect forms from the gut. (i/f)SG = insect forms from the salivary glands. MOVS = MOVS/KE/81/WG84. MBOI = MBOI/NG/60/1-148. MSUS = MSUS/LR/77/TSW103. University of Ghana http://ugspace.ug.edu.gh 156 Figure 21 Reactivity of some trypanosome species-specific MoAbs against lxlO4 parasites/dot of Trypanosoma grayi (TG), and T. congolense savannah type (TCST), K ilifi type (TCK7) anci 3X103 forest type (K T 1) in the dot-ELISA. The control antigens consisted of lxlO 5 parasites per dot of T. congolense K/83/IL/97/2 (TCK), T. brucei IL2616 (TB) and T. simiae KETRI 2431 (TS) cultured procyclics, and lxlO5 T. vivax IL1392 (TV) cultured epimastigotes. Strip 'C' was a conjugate control; not incubated with specific MoAb. KD32 and KT39a were respectively, T. vivax and T. brucei specific MoAbs. TC6/25 and KN4 were Nannomonas subgenus-specific, and TC6 was a T. congolense specific MoAb. Note that none of the MoAbs reacted with T. grayi. University of Ghana http://ugspace.ug.edu.gh T.G T.C ST T.C FT T.CKT TB -| TS TV - T C K - • o TC 62 5 University of Ghana http://ugspace.ug.edu.gh COo H CO 0 5 CO \— * CM CO D * Figure 21 University of Ghana http://ugspace.ug.edu.gh 1 5 7 The main aim of this study was to obtain trypanosome species-specific MoAbs that could be useful in developing a field applicable assay for detecting and differentiating trypanosome species in the vector (Glossina spp.). It had been previously shown that T. brucei, T. vivax, T. congolense and the Nannomonas subgenus, possess species-specific and/or subgenus specific antigens (Parish, Morrison and Pearson 1985, Richardson et al., 1986; Nantulya et al., 1987). The initial step, therefore, was to re-examine the specificity and sensitivity of the extant trypanosome species-specific MoAbs that were available at ILRAD, to select some for further study and then to produce additional MoAbs, if necessary, for eventual detection and differentiation of vector-borne trypanosome species. These studies were made using IFAT, micro-plate ELISA, dot-ELISA and Western immunoblot analysis, and the selection criteria were as follows: (1 ) specificity as determined in all the assay systems, (2 ) sensitivity in terms of the minimum number of insect form trypanosomes that can be detected in the dot-ELISA and (3) the ability of a MoAb to bind antigens in the Western immunoblot assay. A series of MoAbs were also produced against insect forms of T. brucei, T. vivax, T. simiae and the Nannomonas subgenus as indicated, and each of these MoAbs was screened using IFAT, micro-plate ELISA, dot-ELISA and Western immunoblot assays, and their specific reactivity with the trypanosome species against which they were derived established. Data presented in this Chapter shows that immunization of BALB/c mice with trypanosome antigens purified according to the method described by (Ijagbone et al., 1989) increased the fusion success rate as well as the chances of obtaining hybridomas that secreted trypanosome species-specific MoAbs. 4.5 Discussion University of Ghana http://ugspace.ug.edu.gh 158 Although this method of antigen purification was originally described for the purpose of improving serological diagnosis of trypanosomiasis, it has successfully been used in this study to produce T. brucei specific MoAbs (KT43/33, KT43/27), T. vivax specific MoAb (KD37) and T. simiae specific MoAb (KNS7). It has also been demonstrated in this study that the dot-ELISA procedure is suitable for screening the products of cell fusions, especially where it is desired to screen MoAb against a large number of different antigens simultaneously. This suggestion is in agreement with the findings of Hawkes, Niday and Gordon (1982), that the NC membrane-based dot-ELISA was well suited for screening of MoAbs. Two different hybridomas that secreted T. simiae specific MoAb were derived. However, both hybridomas were not stable since the ability to secrete the specific MoAb was lost when grown in continuous culture. This phenomenon has indeed been reported by Pearson, Pinder, Raelants, Kar, Lundin, Mayor-Withey and Hewett (1980), the underlying reason having been attributed to the loss of chromosome chains (Goding, 1980). Hybrid cells are unstable and tend to lose chromosomes especially during the early cell divisions following fusion (Goding, 1980). It is, therefore, possible for hybrid cells to lose one or more of the chromosomes that encode the genes for the expression of immunoglobulins. The stability of the cells increases with chromosome loss, and eventually the cells become relatively stable. Consequently, it has been established that recloning hybrid cells should produce higher cloning efficiency and increase numbers of active cells. This was, however, not the case with the T. simiae hybridomas reported here. This observation may be explained by the fact that the chances of a hybrid cell losing immunoglobulin genes never really ceases. Conditions necessary to minimise the occurrence of unstable hybrid cells include; a suitable immunization schedule; the state of health of the cells used in cell fusion; rapid identification of MoAb secreting cells; early cell cloning and the use of feeder University of Ghana http://ugspace.ug.edu.gh 1 59 cells; and the choice of FBS used in the preparation of medium (reviewed by Goding, 1980). Though these conditions were closely followed, the two T. simiae specific MoAb secreting hybridomas were not stable. This occurrence is, however, not the first, since it has been reported that some hybrids are inherently more stable than others. Hence even with intensive care and repeated cloning, some hybrid cells will lose production (Goding, 1980). The results, however, show that T. simiae specific antigens exist at least in the procyclic stage of that species, and that these antigens are sufficiently immunogenic and can be utilized in traditional cell fusion methods for the production of T. simiae specific MoAbs. This is very useful information since no T. simiae specific MoAb has been reported in the literature. The low frequency in occurrence of MoAbs that are specific for T. simiae compared with T. congolense may be attributed to fewer or less immunogenic T. simiae specific antigens. IFAT studies carried out to immunolocalize the antigens detected by the MoAbs, revealed that antigens that define T. brucei specificity were not restricted to the cell membrane. Thus, the MoAb KT43/27 was also shown to bind antigens located in the cytoplasm of T. brucei procyclics, and membrane bound T. brucei specific antigens have been reported earlier (Nantulya et al., 1987). Despite the obvious differences in location, it was not possible to determine whether the antigenic molecule bound by KT43/27 was different from that bound by any of the other T. brucei specific MoAbs. Parish, Morrison and Pearson (1985) reported the identification of an antigen specific to T. congolense using MoAbs. This MoAb, TC6/42.6.4, was shown to bind a membrane antigen that was made accessible to the antibody following treatment of bloodstream trypanosomes with acetone. In this study, it was shown using IFAT, that the four T. congolense specific MoAbs tested did not bind to live procyclic trypanosomes, unlike the Nannomonas subgenus- specific MoAbs which all bound to live procyclic trypanosome membranes. It University of Ghana http://ugspace.ug.edu.gh 160 has also^sliown using IFAT that the T. simiae specific MoAb did not bind to live T. simiae procyclics. All the MoAbs produced reacted specifically with the trypanosome species against which they were derived when tested in the IFAT, micro-plate ELISA and the dot-ELISA. However, not all of them bound their respective antigens with the Western immunoblot analysis, whether as MoAb in culture supernatants or as purified fractions. This, perhaps, was not surprising, since in SDS-PAGE analysis, antigen samples were heated to 100°C in SDS in the presence of a reducing agent, a process that could lead to the denaturation of antigem (Su and Prestwood, 1990). Also, the conformation of the antigens after they are transferred to NC membrane is not known. Moreover, it had been reported that the reactivity of immune serum made against native antigens is usually much weaker when tested against denatured antigens (Amon, 1973). Chaicumpa, Ruangkunapom, Kalambaheti, Limavongpranee, Kitikoon, Khusmith, Pungpak, Cbongsa-Nguan and Sommani (1991) had also pointed out that antisera against native proteins normally contain some clonal products which recognize the denatured antigens, allowing the Western immunoblot to function. In contrast, MoAbs against native antigens may or may not bind the denatured products. It is therefore expected that many clones of MoAb may fail to bind denatured antigens (Goding, 1983), which could explain why the T. simiae KNS7 failed to bind any antigens with the Western immunoblot assay. This inability of KNS7 to bind the specific antigens, did not allow purification of that antigen by electro-elution. As a result, it would have been necessary to resort to immunoaffinity or immuno-precipitation techniques for isolation of the antigen. However, time did not permit such a study to be conducted. The T. brucei MoAbs TR7 and KT43/33; T. congolense MoAbs (C2, TC39 and TC40); and Nannomonas MoAb TC16, reacted with multiple bands in the Western immunoblot assay. This observation is not new, since, it has University of Ghana http://ugspace.ug.edu.gh 1 6 1 been reported that certain MoAbs often show variable staining intensities or multiple band staining (Braun, Pereira, Norrid and Roizman, 1983; Mandrell and Zollinger, 1984; Steinemann, Fenner, Binz and Parish, 1984; Turner, 1983). The case of TR7 and KT39a may be explained by the findings obtained from micro-plate based antigen capture studies, which indicated that the two T. brucei specific MoAbs bound repeated determinants which possibly were located in multiple peptide bands. It has indeed been reported that the usual treatment of antigens with a reducing agent (sodium dodecyl sulphate) and heat in the Western immunoblot assay, leads to the breakdown of antigens into several peptides (Chung, 1987). Moreover, autodegradation of the antigens could produce a similar effect. Both of these processes could lead to the distribution of the antigenic epitopes detected by MoAbs on several peptides of varying molecular weights, and thus lead to multiple band staining (Chaicumpa et al. , 1991). It has also been argued that MoAbs that reveal multiple bands in the Western immunoblot assay may be binding common sequences or repeated determinants (Bers and Garfin, 1985; Chaicumpa, Thin-inta, Khusmith, Tapchaisri, Echeverria, Kalamba-heti and Chongsa-Nguan, 1988) in different polypeptide chains produced by denaturing conditions. The studies described in this Chapter also showed that each of the four T. congolense specific MoAbs captured antigen molecules that could be revealed by conjugates of all the others. These results thus suggest that the antigenic epitopes for those MoAbs were located on the same antigen molecule(s). Furthermore, inhibition ELISA studies which showed that each of those T. congolense specific MoAbs could inhibit binding by any of the others, suggested two possibilities: (1 ) that the antigenic epitope(s) recognised by all those four MoAbs was the same, or (2) that the antigenic epitopes detected by these MoAbs were not necessarily the same, instead they might be located so close to one another in a way that binding by one MoAb led to interference in the binding of another MoAb. University of Ghana http://ugspace.ug.edu.gh 1 62 Proteinase-K digestion of peptide residues and periodate oxidation of carbohydrate residues have been described and used by several workers in the characterization of the antigens detected by MoAbs (Bright, Chen, Flebbe, Lei and Morrison, 1990; Woodward et al., 1985). Investigations of the biochemical nature of the epitope specificities of the MoAbs produced had revealed that three of the T. brucei MoAbs bound protein specific antigenic determinants, since their binding was completely abrogated by proteolysis. Binding by the fourth T. brucei MoAb KT39a was, however, only partially affected by proteolysis, suggesting that the antigenic determinant was at least partly protein in nature. Binding by that same antibody was not affected by periodate oxidation of carbohydrate residues. It was, however, difficult to rule out any part played by carbohydrate. This is because according to Woodward et al. (1985), antigenic determinants affected by periodate oxidation are carbohydrates in glycoprotein or glycolipids, yet some carbohydrate residues are insensitive to periodate oxidation. It is, therefore, likely that KT39a bound a glycoprotein or a lipoprotein. The studies also showed that the four T. congolense MoAbs bound protein antigenic determinants, whilst the four Nannomonas subgenus-specific MoAbs were directed at carbohydrate antigenic determinants. The only Nannomonas specific MoAb (TC16) that bound antigens with the Western immunoblot assay, was also shown to bind an antigenic determinant which was different from the common determinant bound by the other three Nannomonas MoAbs. This meant that there were at least two different antigenic determinants that defined Nannomonas species specificity, both of which were of carbohydrate nature. Although none of the T. vivax specific MoAbs could bind antigens in the Western immunoblot assay, the results obtained using micro-plate ELISA revealed that the antigenic determinant bound by TV8 was different from that bound by KD32. This observation is supported by the finding that TV8 University of Ghana http://ugspace.ug.edu.gh 163 reacted with the bloodstream forms of the South American T. vivax IL3841, whilst KD32 could not. Also, the determinant bound by TV8 was insensitive to proteinase-K digestion whilst that bound by KD32 was sensitive to that treatment. It may be further argued that the antigenic determinants bound by TV8 was different from that bound by KD37. This is because unlike TV8 , the KD37 epitope was sensitive to proteolysis, and yet both MoAbs detected the bloodstream forms of IL3841. These findings could mean that there were at least three different antigenic determinants that express T. vivax species specificity, two of which were proteins and the other of carbohydrate or lipid nature. Studies on the reactivity of the trypanosome species-specific MoAbs with trypanosome stocks isolated from different geographical areas, have clearly shown that the reactivity of some of the MoAbs was indeed broad. The studies showed that two of the T. vivax specific MoAbs, TV8 and KD37, were capable of detecting T. vivax originating from East and West Africa, as well as from South America. All the T. congolense species-specific and Nannomonas subgenus-specific MoAbs were also shown to be capable of detecting the three different types of T. congolense (savannah, riverine-forest and Kilifi types) tested. In the application of DNA probes for the differentiation of trypanosome species, the absence of a probe that could hybridize with all the different types of T. congolense has been a major limitation (Kukla et al., 1987). It is important to mention here, though, that the recently identified Tsavo type T. congolense was not tested in this study because of failure to obtain suitable samples. Another important observation was that some MoAbs showed a stage specificity in their reactivity. Of the Nannomonas specific MoAbs, for instance, only KN5 detected all the different T. congolense bloodstream forms tested. The others could not react with the bloodstream stages of two T. congolense stocks even though they reacted with the insect stages of the same University of Ghana http://ugspace.ug.edu.gh 164 stocks. It was also shown that unlike TV8 and KD37, the T. vivax specific MoAb KD32 reacted with the vector stages but not the bloodstream forms of the parasite. These observations were not unusual since MoAbs that were specific to the procyclic stages of trypanosomes had been reported earlier (Richardson et al., 1986). A second point was that one of the T. brucei specific MoAbs, KT43/27, failed to react with some in vitro propagated T. brucei procyclics and insect forms from the gut and the salivary glands of tsetse infected with some T. brucei stocks. Moreover, KT43/27 could not react with bloodstream forms isolated from laboratory rodents (mice and rats) infected with two different T. brucei stocks. This observation suggests that the antigenic epitope bound by KT43/27 might not be expressed in the procyclic as well as other stages of some T. brucei stocks. However, it should be noted that of the four T. brucei MoAbs, only KT43/27 could bind cytoplasmic antigens; and this was the only T. brucei specific MoAb that did not bind any antigens with the Western immunoblot assay. The internal localization of the antigen detected by KT43/27 suggested that, of all the T. brucei specific MoAbs, it was the one that would most likely be affected by degradative substances such as proteases and lysozymes from the parasites during sample preparation. It is also likely that the dot-ELISA technique was not suitable for detecting the antigen targeted by KT43/27, possibly because when applied to NC membrane, the antigen could bind in such a way that the epitope detected by KT43/27 was concealed. These findings indicated that, apart from KT43/27, the MoAbs included in this study were likely to be useful in the development of MoAb- based assays for the detection and differentiation of procyclic forms of African trypanosomes, propagated in vitro as well as those in the vector (Glossina spp.). In addition, some of the newly derived MoAbs could be useful in studies aimed at diagnosing trypanosomiasis in the vertebrate host. The usefulness of the MoAbs as diagnostic reagents in detecting and differentiating University of Ghana http://ugspace.ug.edu.gh 165 between culture derived vector stage trypanosome species, would be the subject of investigation in the next Chapter, as a prelude to the diagnosis of infections in the vector. University of Ghana http://ugspace.ug.edu.gh CHAPTER 5 166 DIFFERENTIATION BETWEEN IN VITRO PROPAGATED INSECT-STAGE TRYPANOSOME SPECIES USING DOT-ELISA University of Ghana http://ugspace.ug.edu.gh 167 A sensitive and specific nitrocellulose (NC) membrane-based dot- ELISA, utilizing a panel of monoclonal antibodies (MoAbs), was developed for differentiation between in vitro derived procyclic forms of Trypanosoma brucei, T. congolense and T. simiae, and epimastigotes of T. vivax. Trypanosomes were applied onto NC membrane in dots and probed with unlabelled trypanosome species-specific MoAb. Bound MoAb was revealed by enzyme labelled anti-mouse IgG and precipitable chromogenic substrate. The assay detected the afore-mentioned trypanosome species in both single and artificially mixed preparations. Six T. brucei, four T. vivax, seven T. congolense and three T. simiae procyclic stocks and clones from different geographical areas were tested and identified using the specific MoAbs in the dot-ELISA which had a specificity greater than 99.9%. Some of the T. brucei, T. congolense and Nannomonas specific MoAbs could detect as low as 10 trypanosomes per dot, whilst one T. vivax MoAb was able to detect a minimum of 100 trypanosomes per dot in mono-species preparations. A concentration of lxlO4 trypanosomes//xl/dot was eventually determined as ideal for testing in the dot-ELISA. Antigen dots made from the different trypanosome species, and stored at 4°C under desiccated conditions did not show any loss in activity in up to 90 days. However, when stored under similar conditions at room temperature (17-26°C), the T. congolense specific antigen remained unaffected up to 60 days, and then showed decreased activity when tested on day 90. The ability of the dot-ELISA to distinguish between the various stocks and clones of trypanosomes that were used, and the ability to identify the constituent species in mixed trypanosome preparations, indicated that this test might prove useful as a laboratory tool for the determination of the identity of in vitro derived procyclic trypanosomes. Also, the dot-ELISA 5.1 Summary University of Ghana http://ugspace.ug.edu.gh 168 developed could be a useful first step in the development of a field applicable MoAb-based assay for diagnosis of trypanosome infections in the tsetse fly. University of Ghana http://ugspace.ug.edu.gh 169 In the studies described in this Chapter, the trypanosome species and subgenus-specific MoAbs derived against in vitro propagated procyclics of T. brucei, T. congolense and T. simiae, and epimastigotes of T. vivax, were employed in the development and standardization of a simple, sensitive and specific NC membrane-based dot-ELISA for the differentiation of readily available in vitro cultivated forms of those trypanosome species. It was intended to ultimately apply the assay to the diagnosis of trypanosome infections in infected tsetse flies. The feasibility of this approach stems from the finding that procyclic tsetse midgut forms and culture forms of the African trypanosomes express similar antigens (Richardson et al., 1986; Pearson, Moloo and Jenni, 1987). The NC membrane-based dot-ELISA was selected for this application for two reasons. Firstly, it offered the best opportunity for the development of a diagnostic test that is simple and easy to perform, in addition to being both sensitive and specific. Secondly, such an assay could be easily modified for field diagnosis of trypanosome infections in the tsetse fly (Glossina spp.). Furthermore, the introduction of a simple, specific and sensitive assay capable of detecting and differentiating between in vitro propagated trypanosome species, would facilitate other studies. For example, the search for a solution to the trypanosomiasis problem has necessitated extensive studies into the biology, biochemistry, response to chemotherapy, antigenic constitution, as well as characterization of the causative organisms. To facilitate these studies, techniques have been developed for in vitro culture of various developmental stages of the parasite. Thus the procyclic, epimastigote, metacyclic and the bloodstream forms of trypanosomes can be propagated in large numbers in artificial cultures in vitro (Hirumi, Doyle and Hirumi, 1977; Ross, Gray, Taylor and Luckins, 1985; Zweygarth, Gumm, 5.2 Introduction University of Ghana http://ugspace.ug.edu.gh 1 70 Gray, Cheruiyot, Webster and Kaminsky, 1989; Hirumi, Nelson and Hirumi, 1983; Baltz, Baltz, Giroud and Crocket, 1985; Bran and Schonenberger, 1979). This has made it possible to conduct trypanosome drug sensitivity analysis in vitro (Kaminsky and Zweygarth, 1989; Kaminsky, Chuma and Zweygarth, 1989; Ross and Taylor, 1990). Moreover, in vitro culturing of trypanosomes has made possible, the isolation of the parasites from various organs of infected tsetse flies into artificial cultures (Cunningham, 1977; Trager, 1959; Gumm, 1991; Gray, Cunningham, Gardiner, Taylor and Luckins, 1981). This unlimited opportunity to grow freshly isolated trypanosomes in culture, and the ability to cryopreserve samples of such organisms, calls for the availability of simple reliable techniques for ascertaining the species of trypanosomes present in in vitro cultures. Two diagnostic techniques, DNA hybridization analysis and isoenzyme characterization, are currently used for this purpose (Gashumba, Gibson and Opiyo, 1986; Kukla et al., 1987). These methods are, however, not simple enough and cannot be performed in most laboratories. The ability of a simple MoAb-based dot-ELISA to detect and differentiate between culture derived procyclic trypanosomes, is reported. University of Ghana http://ugspace.ug.edu.gh 171 5.3.1 In vitro cultivation of trypanosomes 5.3.1.1 Cultivation of procyclic forms Procyclic trypanosomes were cultivated in culture using complete- (SM) medium filtered through 0.2 to 0.45/xm membrane bottle filter (Costar). Cryopreserved T. brucei, T. congolense and T. simiae procyclic trypanosomes were resuscitated into culture medium and the cultures initiated as described earlier (section 3.2.1). The trypanosomes were allowed to multiply until the trypanosome density reached approximately lx l0 7 /ml. The T. brucei IL2616 procyclics were well adapted to culture. Maintenance of this trypanosome stock was achieved by removal of all but 1ml of the lx l 0 7/ml trypanosome suspension from a culture flask and replacement with fresh medium in quantities of up to 20 times the residual volume. However, the T. congolense clone K/83/IL/97/2 and T. simiae clones TS1 and TS4 procyclics were poorly adapted to culture. These were maintained by removal of half of the lx l0 7/ml procyclic suspension and replacement with an equal volume of new medium usually thrice weekly. The trypanosomes were grown in 15-20ml volumes in 75cm2 flasks (Costar; Falcon). The cultures were gassed whenever the flasks were opened. Flasks were closed tightly soon after gassing and incubated at 27°C. Old flasks that had been used for maintaining over six passages were replaced with new ones. 5.3.1.2 Cultivation of epimastigote forms In vitro propagation of T. vivax epimastigotes, East African (EA) stock IL2337 and West African (WA) stock IL1392 were achieved by transformation of freshly isolated bloodstream forms into epimastigotes at 27°C in Iscove's modified Dulbecco's minimum essential medium (M- DMEM,; Flow Laboratories, Irvine, Scotland, UK) that had transferrin, 5.3 Materials and methods University of Ghana http://ugspace.ug.edu.gh 172 soybean lecithin, and bovine serum albumin incorporated in it (Iscove and Melchers, 1978). Foetal bovine serum (FBS), purchased from Hyclone Laboratories Inc., was heat inactivated at 56°C for 30 min, and used at 20%(v/v) final concentration. This medium was modified again by the addition of 0.3% (w/v) sodium bicarbonate and adjusting the pH to 7.0 (It will henceforth be referred to as "complete-(M-DMEM) medium"). Mice were infected by intraperitoneal injection of lxlO 5 trypanosomes in 0.5ml of PSG, pH 7.4. Blood taken from the tail veins of infected mice was examined microscopically, at x400 magnification, for trypanosomes as described by Herbert and Lumsden (1976). Mice with peak parasitaemia of more than lxlO9 trypanosomes/ml were killed by terminal anaesthesia with diethy 1-ether and immediately sterilized by immersion in 70% ethanol. Infected blood was drawn aseptically by cardiac puncture, using a 22 gauge hypodermic needle, into heparinized syringes containing 5IU heparin/ml. Five microlitre volumes of infected blood were slowly deposited by pipette at the bottom, close to the edges of three of the wells in a six well plate (Costar, USA) containing 1ml of complete-(M-DMEM) medium each. The culture plate was then incubated at 27°C for 90 min. During this incubation, bloodstream form trypanosomes migrated from the infected blood into the medium. Five hundred microlitres of medium, containing the trypanosomes, were pipetted away from the deposited blood from each of the three wells and transferred to the three remaining wells containing 1ml of complete-(M-DMEM) medium each. The three new wells were incubated for another 90 min, after which 1ml volumes of medium, containing trypanosomes, were pipetted from each well away from the site of deposition and pooled (3ml) into a 25cm2 culture flask. The flask was capped tightly and incubated at 27°C. No attempt was made to change or add medium to the culture for at least five days, and even then only if an increase in trypanosome numbers and a decrease in pH were observed. Thereafter, up to 50% of the University of Ghana http://ugspace.ug.edu.gh 173 medium was changed over 2 to 3 days. Subculture of the EA IL2337 and WA IL1392 were made when colonies of epimastigote forms covered at least 75% of the plastic surface (Gumm, 1991). Epimastigote colonies were scraped off, using disposable cell scrapers (Costar). Half of the medium containing epimastigotes was transferred from one flask to a new one, and an equal volume of fresh medium added. 5.3.1.3 Transformation of bloodstream form trypanosomes into procyclics Transformation of T. brucei and T. congolense bloodstream trypomastigotes into procyclics was initiated at 27°C in complete-(SM) medium. Infected parasitaemic mice were killed by terminal anaesthesia as usual and immediately dipped into 70% ethanol. Infected blood was drawn aseptically into a heparinised syringe by cardiac puncture as previously described. The blood was washed two times in complete-(SM) medium by centrifugation at 400 Xg for 10 min. The pelleted blood cells and trypanosomes were resuspended in complete-(SM) medium to a final concentration of Ix l0 6 - lx l0 7 red bloodcells/ml and 4ml volumes pipetted into 25cm2 sterile culture flasks (Costar). The flasks were gassed with 5% CO2 in air for 10-20 seconds, tightly closed and incubated at 27°C. Each flask was examined daily, using an inverted microscope (Nikon, 46212, Japan) under x200 magnification. When the trypanosome density reached approximately lx l 0 7 /ml, half of the medium was removed and replaced by an equal volume of fresh medium. This process was repeated until all blood cells were eliminated from the culture. The cultures were then expanded by two fold volume increases and transferred to larger (75cm2) culture flasks (Costar). University of Ghana http://ugspace.ug.edu.gh 174 5.3.2 Sample Preparation for Dot-ELISA 5.3.2.1 Preparation of trypanosomes for dot-ELISA Procyclic forms of T. brucei and T. congolense, as well as epimastigote forms of T. vivax were propagated in culture as described under sections 5,3.1.1 and 5.3.1.2. Trypanosomes were harvested from in vitro cultures and washed 2 times in PBS, pH 7.4, PSG, pH 8.0 or normal saline, by centrifugation at 1000 Xg for 5 min each. The trypanosome pellets were resuspended in small volumes of the appropriate buffer and counted, using an Improved Neubauer counting chamber. To allow for the proper estimation of the minimum number of trypanosomes detected, the trypanosome suspensions were adjusted to lxlO 8 trypanosomes/ml and ten-fold serial dilutions made down to lxlOJ trypanosomes/ml. 1 /xl samples were then pipetted from those tubes and placed in dots onto strips of NC membrane, pore size 0.45/mi. Trypanosome numbers ranging between lxlO5 trypanosomes/dot and 0 trypanosomes/dot were thus obtained. Each sample was dotted onto several strips so that strips with the same samples could be tested against different trypanosome species- specific MoAbs. Also, procyclic T. congolense (savannah, riverine/forest and Kilifi types) and T. grayi, each suspended in normal saline and dotted onto NC filters (lxlO4 or 7xl03) per dot, were prepared and donated by Dr. J. McNamara of the Tsetse Research Laboratory, Bristol, England. The ability of the MoAbs to identify mixed trypanosome populations was also studied. Trypanosome mixtures, each consisting of equal numbers of two different species from the group (T. brucei, T. congolense, T. simiae and T. vivax), were made in all possible combinations. Each mixed sample was titrated in PBS (pH 7.4) to give lxlO5, lxlO4, lxlO3, lxlO2, lxlO 1 and 0 trypanosomes per microlitre, and 1/xl volumes dotted onto NC membrane strips. Trypanosomes were also lysed by suspension in distilled water, and the lysed suspensions titrated to determine the effect of lysis on University of Ghana http://ugspace.ug.edu.gh 17 5 specificity and sensitivity of the MoAbs. The antigen "dotted" NC membrane strips were left to dry at room temperature (17-26°C) for 15 min before they were assayed. The suitability of some other membrane supports, namely, 0.45/jm pore size hybridization transfer membranes, Hybond-C (Cat. RPN.203C, Lot. 18425), Hybond-N (Cat. RPN.303N, Lot.20849) both from Amersham, and 0.45/xm pore size immuno-affinity membrane (Pall Immunodyne Lot. 141743; Pall Bio Support Division, East Hills, NY, USA), was also investigated. 5.3.3 Dot-ELISA Procedure 5.3.3.1 Detection of in vitro propagated trypanosomes by dot-ELISA In vitro derived trypanosomes were suspended in buffer and applied in dots onto NC membrane filters, and tested as described previously (Chapter 4, section 4.3.9). 5.3.3.2 Titration of specific MoAbs and enzyme conjugated antibody Trypanosome species-specific MoAbs were tested in the dot-ELISA to determine the optimal working dilution and time of incubation. MoAb in culture supernatants were tested in four-fold dilutions, starting with a dilution of 1:2. Ammonium sulphate concentrated MoAb fractions were tested in two­ fold dilutions (starting with 1:5 dilution), and affinity purified MoAb fractions were tested in two-fold dilutions, starting from 1:250. All MoAb dilutions were also tested for optimum time of incubation, namely 30 min, Ihr, and 3hr. Enzyme-conjugated antibody was also tested in two-fold dilutions from 1:250 to 1:2,000. The dilution and time of incubation at which an antibody reacted specifically and gave the highest intensity of colour development were selected and tested against various conjugate incubation times (15 min, 30 min and lhr) for optimisation. University of Ghana http://ugspace.ug.edu.gh 1 76 5.4.1 Optimal working dilution and incubation period for MoAbs and enzvme-coniugate Investigations into the influence of period of incubation on the reactivity of the specific MoAbs, indicated that a 3hr incubation period was suitable for all the MoAbs. In contrast, different MoAb dilutions were determined to be ideal, depending on the source of MoAb and the purification method used (Table 23). Figure 22 shows the results obtained when purified fractions of KT43/33 and KN4 were tested to determine the optimal working dilutions. Both MoAbs reacted specifically at all the dilutions tested. The intensity of the positive colour reactions remained virtually unchanged from 1:250 to 1:1,000 and decreased at 1:2,000. A working dilution of 1:1,000 was selected for both MoAbs. One hour incubation of enzyme-conjugate was found to give better reactions when compared to 30 min or 15 min incubations (Figure 23). 5.4.2 Specificity of the dot-ELISA in identifying trypanosomes in mono­ species preparations The ability of a panel of trypanosome species-specific MoAbs to differentiate between in vitro derived procyclics of T. brucei, T. congolense and T. simiae and epimastigotes of T. vivax, was investigated with the dot- ELISA. Figure 24 summarizes the reactivity of two of the T. brucei specific MoAbs (TR7 and KT39a) in this assay. It was observed that both TR7 and KT39a reacted with the dot containing T. brucei antigen, and neither of them reacted with the antigen dots representing T. congolense, T. simiae or T. vivax. At 1:5 dilution, the culture supernatant containing KT39a MoAb, clearly left negative impressions on the T. congolense and T. simiae dots, thereby 5.4 Results University of Ghana http://ugspace.ug.edu.gh 1 77 Table 23 Working dilution of the specific MoAbs used in the dot-ELISA Monoclonal Antibody Isotype Specificity Source of antibody Dot-ELISA titre TR7/47.37.16 IgM T. brucei A.S. ppt 1 : 2 0 KT39a/18.17 IgM T. brucei C.S. 1:50 KT43/33.32 IgGj T. brucei Purified 1 :1 , 0 0 0 KT43/27.32 IgG2 a T. brucei Purified 1 :1 , 0 0 0 TV8/8.33.42 Ig°3 T. vivax Purified 1 :1 , 0 0 0 KD32/48.17 IgGi T. vivax Purified 1 :1 , 0 0 0 KD37/11.1 IgGi T. vivax A.S. ppt 1:50 C2 IgGi T. congolense Purified 1:500 TC6/42.6.3 IgGj T. congolense A.S. ppt 1 : 2 0 TC40/30.15.40 IgM T. congolense Purified 1 :2 , 0 0 0 TC39/30.25.95 IgM T. congolense Purified 1 :2 , 0 0 0 KNS7/14.X IgGi T. simiae A.S. ppt 1 : 2 0 TC16/5.12.33 IgGj Nannomonas Purified 1 :1 , 0 0 0 TC6/25.25.4 IgG3 Nannomonas Purified 1 :1 , 0 0 0 KN4/13.9 IgG3 Nannomonas Purified 1 :1 , 0 0 0 KN5/6.15 IgGi Nannomonas A.S. ppt 1 : 2 0 A.S. ppt= x20 concentration of culture supernatant by ammonium sulphate precipitation. C.S. = culture supernatant. Purified = purified MoAb fraction. University of Ghana http://ugspace.ug.edu.gh 1 7 8 Figure 22 Titration of purified fractions of KT43/33 (Tb ruce i species-specific) and KN4 (Nannomonas subgenus-specific) MoAbs to determine the optimal dilution of antibody for use in the dot-ELISA. Each strip of NC membrane was applied with varying concentrations of trypanosomes per dot (Tryps/dot) of T. congolense K/83/IL/97/2 (TCK), T. brucei IL2616 (TB) and T. simiae KETRI 2431 (TS) cultured procyclics. MoAbs were tested at several dilutions as shown. University of Ghana http://ugspace.ug.edu.gh CO CO — , CO 5r • • , • i U « • — o o o o o W O l f l O O CM 10 N- O O ±1 - “ T- CM z * i r • • • • • • * ! ♦ i • * l Tryps/dot 1x10® 1 x 1 0 4 1x10 1x10" 3 O O O O O^ o ^ o o MoAb. CM 10 Is- O O ... . ^ ^ ^ r . dilution Figure 22 University of Ghana http://ugspace.ug.edu.gh Figure 23 Titration of goat anti-mouse horseradish peroxidase conjugate to determine optimal incubation time for reactions in the dot-ELISA. Each strip of NC membrane was applied with varying concentrations of trypanosomes per dot (Tryps/dot) of T. congolense K/83/IL/97/2 (TCK), T. brucei IL2616 (TB) and T. simiae KETRI 2431 (TS) cultured procyclics. Strip 'C represents a conjugate control, that was not incubated with specific MoAb. University of Ghana http://ugspace.ug.edu.gh — Tryps/dot 1 x 1 0 5 1x104 1x103 1x102 1x101 0 University of Ghana http://ugspace.ug.edu.gh 180 Figure 24 An illustration of the specific reactivity of two Trypanosoma brucei specific MoAbs (TR7 and KT39a) in the dot-ELISA. Each strip shown was applied with various concentrations of trypanosomes per dot (Tryps/dot) of either T. vivax IL1392 cultured epimastigotes (TV) or procyclics of T. congolense K/83/IL/97/2 (TCK), T. brucei IL2616 (TB) and T. simiae KETRI 2331 (TS). University of Ghana http://ugspace.ug.edu.gh KT39a MoAb ^ ^ dilution University of Ghana http://ugspace.ug.edu.gh 181 illustrating clear T. brucei specificity (Figure 24). Figures 25, 26 and 27 illustrate respectively, the specificity of two T. vivax specific MoAbs (TV8 and KD32), four T. congolense specific MoAbs (C2, TC6 , TC40 and TC39), and four Nannomonas subgenus-specific MoAbs (TC16, TC6/25, KN4 and KN5). These specific reactions remained unaltered even at trypanosome concentrations of lxlO5 trypanosomes/dot in 1 pci volumes. The reactivity pattern of the entire panel of MoAbs, as determined by dot-ELISA, is shown in (Table 24). 5.4.3 Specificity of the dot-ELISA in identification of the constituent trypanosome species in artificially mixed preparations Mixtures consisting of two different trypanosome species each, were made in all possible combinations, using T. brucei, T. vivax, T. congolense and T. simiae organisms. The mixed trypanosome suspensions were doted onto NC membrane and tested to determine the ability of the various MoAbs to differentiate the constituent species. Figure 28 illustrates the ability of the T. brucei specific MoAb (KT39a) to detect T. brucei in brucei/congolense and brucei/simiae mixtures. In the same experiment, KT39a did not react with the congolense/simiae mixture. Also, Figure 28 shows the specific detection of T. congolense by C2, TC6 , TC40 and TC39 in congolense/brucei and congolense/simiae mixtures. The absence of a reaction on the brucei/simiae dot, clearly showed that those MoAbs were indeed T. congolense specific. TVS and KD32, both T. vivax specific, had been shown not to react with antigen mixtures of brucei/congolense, brucei/simiae and congolense/simiae (Figure 29). Yet, the Nannomonas specific MoAbs (TC16, TC6/25, KN4 and KN5) are shown to detect their target species (T. congolense and T. simiae) in those mixtures. University of Ghana http://ugspace.ug.edu.gh 182 Figure 25 Reactivity of Trypanosoma vivax specific MoAbs (TV8 and KD32) in the dot- ELISA. Each strip shown was applied with various concentrations of trypanosomes per dot (Tryps/dot) of either T. vivax IL1392 cultured epimastigotes (TV) or procyclics of T. congolense K/83/IL/97/2 (TCK), T. brucei IL2616 (TB) and T. simiae KETRI 2431 (TS). University of Ghana http://ugspace.ug.edu.gh 0 0 > CM CO o * — ■ Tryps/dot TV 1x105 1x104 1x103 1 x 1 0 2 1x101 1x10° 00 > TCK TB TS Figure 25 K D 32 University of Ghana http://ugspace.ug.edu.gh 183 Figure 26 Reactivity of Trypanosoma congolense specific MoAbs (C2, TC6 , TC40 and TC39) in the dot-ELISA. Each strip shown was applied with various concentrations of trypanosomes per dot (Tryps/dot) of either T. vivax IL1392 cultured epimastigotes (TV) or procyclics of T. congolense K/83/IL/97/2 (TCK), T. brucei IL2616 (TB) and T. simiae KETRI 2431 (TS). University of Ghana http://ugspace.ug.edu.gh CM o O CD CO CO o o o I— H- I— CD CM Q O I- L_ Tryps/dot TS 5 ^ 1 x 1 0 Figure 26 TC 40 University of Ghana http://ugspace.ug.edu.gh 1 8 4 Figure 27 Reactivity of Nannomonas subgenus-specific MoAbs (TC16, TC6/25, KN4 and KN5) in the dot-ELISA. Each strip shown was applied with various concentrations of trypanosomes per dot (Tryps/dot) of either T. vivax IL1392 cultured epimastigotes (TV) or procyclics of T. congolense K/83/EL/97/2 (TCK), T. brucei IL2616 (TB) and T. simiae KETRI 2431 (TS). University of Ghana http://ugspace.ug.edu.gh - TV Tryps/dot y B ^ T S 1x105 1x104 1 x 103 1 x 1 0 2 1 x 1 0 1 1x10 Figure 27 University of Ghana http://ugspace.ug.edu.gh 1 85 Table 24 The specificity of the MoAbs as determined by their reactivity with procyclics and epimastigotes of different trypanosome species in the dot-ELISA Monoclonal Antibody Isotype T. brucei * T. vivax ** T. congolense * T. simiae * TR7/47.37.16 IgM + KT39a/18.17 IgM + KT43/33.32 IgGi + - - KT43/27.32 IgG2 a + - - TV8/8.33.42 IgG3 - + - - KD32/48.17 IgGi + - KD37/11.1 IgGj - + - C2 IgGi - + TC6/42.6.3 IgGi - - + TC40/30.15.40 IgM - + TC39/30.25.95 IgM + KNS7/14.X IgGi - - + TC16/5.12.33 IgGi + + TC6/25.25.4 IgG3 + + KN4/13.9 IgG3 - + + KN5/6.15 IgGi - + + * procyclics. ** epimastigotes. + = antibody reacts with trypanosomes. = antibody does not react with trypanosomes. University of Ghana http://ugspace.ug.edu.gh Figure 28 Reactivity of T. brucei species-specific MoAbs (TR7 and KT39a) and T. congolense specific MoAbs (C2, TC6 , TC40 and TC39) with mixtures of cultured trypanosome procyclics in the dot-ELISA. Each NC membrane strip was applied with varying concentrations of trypanosome mixtures per dot (Tryps/dot/spp.). The trypanosome species used were: T. brucei IL2616 (TB), T. congolense K/83/IL/97/2 (TCK) and T. simiae KETRI 2431 cultured procyclics. ( a ) indicates NC membrane strips which were "dotted" with trypanosome antigens prepared by suspending whole organisms in deionised water, ( a ) indicates NC membrane strips which were "dotted" with whole trypanosomes suspended in phosphate buffered saline (PBS) pH 7.4. University of Ghana http://ugspace.ug.edu.gh K QC 1- TB TCK — TB TS - TCK TS — K T3 9a University of Ghana http://ugspace.ug.edu.gh 9 01 O O) ^ COo oI— I— 1 i 1 T ryps/dot/spp. r p i j - - r a 5x104 5x103 5x102 5x101 5 0 A A A A A A A A Figure 28 University of Ghana http://ugspace.ug.edu.gh 1 8 7 Figure 29 Reactivity of T. vivax specific MoAbs (TV8 and KD32) and Nannomonas subgenus-specific MoAbs (TC16, TC6/25, KN4 and KN5) with mixtures of cultured trypanosome procyclics in the dot-ELISA. Each NC membrane strip was applied with varying concentrations of trypanosome mixtures per dot (Tryps/dot/spp.). The trypanosome species used were: T. brucei IL2616 (TB), T. congolense K/83/IL/97/2 (TCK) and T. simiae KETRI 2431 cultured , procyclics. ( a ) indicates NC membrane strips which were "dotted" with trypanosome antigens prepared by suspending whole organisms in deionised water, (a ) indicates NC membrane strips which were "dotted" with whole trypanosomes suspended in phosphate buffered saline (PBS) pH 7.4. University of Ghana http://ugspace.ug.edu.gh Figure 29 University of Ghana http://ugspace.ug.edu.gh 1 Tryps/dot/spp. 5x104 5x103 5x102 5x101 5 0 University of Ghana http://ugspace.ug.edu.gh 188 Suspending mixed trypanosomes in PBS and dotting them as whole organisms or lysing the mixed organisms in deionised water before applying the samples in dots onto NC membrane, gave similar results (Figure 28, 29). On the whole, it was found that each of the MoAbs could detect it's target trypanosome species, irrespective of which other species were present (Table 25). Also, the MoAbs were tested against trypanosome stocks or clones from different geographical areas and shown to detect specifically all the different isolates (Table 26). 5.4.4 The suitability of ascites as a source of MoAb for the dot-ELISA The use of ascitic fluid as a source of specific MoAb for the dot-ELISA presented some problems. Most ascites fractions cross-reacted extensively with all the different trypanosome species when used at dilutions lower than 1:100. At higher dilutions, specific reactions were usually obtained, but this was normally at the expense of sensitivity. Figure 30 shows the results obtained from the titration of ascites containing the T. congolense specific MoAb TC39. The trend clearly illustrates the decreasing sensitivity as specific reactivity on the T. congolense antigen dot (TCK) was being achieved. This loss in sensitivity was unreasonably high when compared to MoAb purified from culture supernatants, and remained unchanged even when ascites was purified. It was, therefore, decided to discontinue the use of ascites as a source of specific MoAb for the dot-ELISA in favour of direct use of culture supernatants or purified fractions thereof. 5.4.5 Cross-reactivitv in the dot-ELISA Cross-reactivity due to factors such as the source of MoAb and concentrations of MoAb or conjugate or antigen, was encountered in the dot- ELISA, prior to standardization of the assay. High concentrations of purified MoAb or HRPO-conjugated antibody, increased the non-specific reactivity, University of Ghana http://ugspace.ug.edu.gh 1 8 9 Table 25: Ability of the specific MoAbs to identify trypanosome species in artificial mixtures of cultured insect stages of the parasites Reactivity of MoAbs with trypanosome mixtures tested T. brucei T. brucei T. brucei T. congolenseT. congolense T. simiae Monoclonal & & & & & & Antibody Isotype Specificity T. congolense T. simiae T. vivax T. simiae T. vivax T. vivax TR7/47.37.16 IgM T. brucei + + + ~ ~ KT39a/18.17 IgM T. brucei + + + - - - KT43/33.32 IgGi T. brucei + + + - - - KT43/27.32 !SG2 a T. brucei + + + - - - TV8/8.33.42 IgG3 T. vivax - + - + + KD32/48.17 IgGi T. vivax - - + - + + KD37/11.1 IgGx T. vivax - - + - + + C2 IgGi T. congolense + - - + + _ TC6/42.6.3 IgGi T. congolense + - - + + - TC40/30.15.40 IgM T. congolense + - - + + - TC39/30.25.95 IgM T. congolense + - - + + - KNS7/14.X IgGi T. simiae - + - + - + TC16/5.12.33 IgGj Nannomonas + + _ + + + TC6/25.25.4 IgG3 Nannomonas + + - + + + KN4/13.9 IgG3 Nannomonas + + - + + + KN5/6.15 IgGi Nannomonas + + - + + + + = antibody reacts with trypanosomes. - = antibody does not react with trypanosomes. University of Ghana http://ugspace.ug.edu.gh 1 90 Table 26: Reactivity of the specific MoAbs with different stocks and clones of T. brucei, T. vivax, T. congolense and T. simiae as defined by dot-ELISA Reactivity of the monoclonal antibodies with uncoated procyclic or epimastigote trypanosomes of: T. brucei T. vivax T. congolense T. simiae Monoclonal IL2616 Th-17/87TREU-1442MiTatl .2 IL1984 IL1478 IL1392 IL3895 ILDatl .9 CP2331 IL/60/1 IL/97/2 1L2079 CP81 MOVS MBOI MSUS TS1 TS4 KETRI243 antibody (Proc) (Proc) (Proc) (Proc) (Proc) (Proc) (Epis) (Epis) (Epis) (Epis) (Proc) (Proc) (Proc) (Proc) (Proc) (Proc) (Proc) (Proc) (Proc) (Proc) TR7 + + + + + + _ _ . - _ _ _ _ _ _ _ KT43/33 + + + + + + - - - - - - - - - - - - - - KT39a + + + + + + - - - - - - - - - - - - - - KT43/27 + + + + + + - - - - - - - - - - - - - - TV8 - - - - - - + + + + - - - - - - - - - - KD32 - - - - - - + + + + - - - - - - - - - - KD37 - - - - - - + + + + - - - - - - - - - - C2 - ■ - - - - ■ - - + + + + + + + - - - TC6 - - - - - - - - - - + + + + + + + - - - TC40 - - - - - - - - - - + + + + + + + - - - TC39 - - - - - - - - - - + + + + + + + - - - KNS7 - - - - - - - KNS7 - - - - - - - - - - - - TC16 - - - - - - - - - - + + + + + + + + + TC6/25 - - - - - - - - - - + + + + + + + + + + KN4 - - - - - - - - - - + + + + + + + + + + n- m- KN5 - - - - - - - - - - + + + + + + + + + + + = antibody reacts with trypanosomes. = antibody does not react with trypanosomes. EL/60/1 = K/82/IL/60/1 EL/97/2 = K/83/IL/97/2 MSUS = MSUS/LR/77/TSW103 (Proc) = procyclic forms propagated in vitro (Epis) = epimastigote forms propagated in vitro MOVS = MOVS/KE/81/WG84 MBOI = MBQI/NG/60/1-148 University of Ghana http://ugspace.ug.edu.gh 1 9 1 Figure 30 Reactivity of ascitic fluid containing the T. congolense species-specific MoAb (TC39) with T. congolense K/83/IL/97/2 (TCK), T. brucei IL2616 (TB) and T. simiae KETRI 2431 cultured procyclics. Trypanosomes were titrated tenfold from lxlO5 to 0 trypanosomes per dot (Tryps/dot). Note the decreasing sensitivity as the ascites was diluted further in order to achieve specificity. University of Ghana http://ugspace.ug.edu.gh TC39 TCK TB TS o Q ‘ © o © o Oj O - I ® CM 00 CM " t 00 ^ " CO CD CM Tryps/do t 1x105 1 x 1 0 1x10" 1x10* 1x10 1x10 1 Ascites dilution Figure 30 University of Ghana http://ugspace.ug.edu.gh 1 92 even though culture supernatants, or higher dilutions of the same MoAbs fractions, reacted specifically. Similarly, it was found that excess antigen ( > l x l 0 8 trypanosomes/dot//K.l) induced non-specific reactions, although the observed reaction intensities were generally low. All cross-reactions were, however, abolished following standardization of the assay. 5.4.6 Sensitivity of the dot-ELISA Application of the dot-ELISA in identification of trypanosomes in mono-species cultures revealed that, some MoAb fractions of TR7, KT39a, TC40, TC6/25 and KN4 were capable of detecting a minimum of 10 trypanosomes per dot, but one of the T. vivax specific MoAbs (KD37) detected a minimum of 100 organism per dot. Table 27 summarizes the results of the sensitivity in terms of the minimum number of trypanosomes detectable in mono-species culture preparations by the dot-ELISA, using the panel of MoAbs prepared. The minimum number of trypanosomes that could be detected in the dot-ELISA increased by a factor of 50, when the assay was applied for the specific identification of trypanosomes in artificially mixed preparations. Thus, a MoAb fraction that detected a minimum of 100 trypanosomes per dot in the mono-species preparations, now would detect a minimum of 100 x 50 (5,000) trypanosomes per dot in the mixed preparations. Table 28 summarizes the sensitivity in terms of the minimum number of trypanosomes that could be detected by the various MoAbs in the dot-ELISA when applied for the differentiation of artificially mixed trypanosomes. This drop in sensitivity could be due to competition between antigens of the mixed trypanosome species for the binding sites on the NC membrane. Analysis of the data on sensitivity in terms of the minimum number of trypanosomes that could be detected in mono-species trypanosome cultures, University of Ghana http://ugspace.ug.edu.gh 193 Table 27: Sensitivity in terms of the minimum number of trypanosomes detected in mono-species preparations by the dot-ELISA Specific monoclonal antibodies raiaMLc number detected TR7 T. brucei KT39a KT43/33 KT43/27 T. vivax TV8 KD32 KD37 C2 T. congolense TC6 TC40 TC39 T. simiae KNS7 TC16 Nannomonas TC6/25 KN4 KN5 1 0 5 + + + + + + + + + + + + + + + + 1 0 4 + + + + + + + + + + + + + + + + 1 0 3 + + + - + + - + + + + - + + + + 1 0 2 + + - - + - + + + + - - + + - 1 0 1 + + - - - - - - + _ _ _ + + _ 1 0 ° - - - - - - - - - - - - - - - 0 - - - - - - - - - - - - - - + = visible reactions observed with that number of trypanosomes in a dot. - = visible reaction not observed with that number of trypanosomes in a dot. University of Ghana http://ugspace.ug.edu.gh 194 Table 28: Sensitivity in terms of the minimum number of trypanosomes detected in mixed trypanosome preparations by the dot-ELISA Parasite number detected Specific monoclonal antibodies TR7 T. brucei KT39a KT43/33 KT43/27 TV8 T. vivax KD32 KD37 C2 T. congolense TC6 TC40 TC39 T. simiae KNS7 TC16 Nannomonas TC6/25 KN4 KN5 5xl04 5xl03 5xl02 5X101 5 + + + + + + + ' 1 1 + + + + + + + + + + + + + + + + + + + i i i + + 1 0 - - - - - - - - - - - - - - - - + = visible reactions observed with that number of trypanosomes in a dot. - = visible reaction not observed with that number of trypanosomes in a dot. University of Ghana http://ugspace.ug.edu.gh 195 revealed that this sensitivity varied between different assays as well as between different fractions of the same MoAb. Thus, using the most active fractions, some of the MoAbs could detect as low as 10 trypanosomes per dot. However, the repeatability of the assay was better when higher trypanosome numbers were used. It was found that all the MoAbs could detect 5xl03 trypanosomes/spp./dot, and this was 1 0 0 % reproducible, compared with 60% reproducibility obtained with 1 0 organisms/dot. Based on these results, and the intensity of the positive colour reactions obtained, lxlO4 trypanosomes/dot was considered to be the most suitable parasite number for use in identifying trypanosome cultures with the dot- ELISA. 5.4.7 Stability of dotted antigens NC membrane strips with trypanosomes deposited as dots were stored under desiccated conditions at 4°C, and at room temperature (17-26°C), and tested for antigenic reactivity at intervals of 30 days using the panel of MoAbs. No significant loss in assay reactivity was observed for periods of up to 60 days, regardless of whether the strips were blocked with 5% skimmed-milk diluted in TBS before storage or not. However, after 90 days of storage at room temperature, the T. congolense specific MoAbs showed reduced reactivity, with a loss in sensitivity from lxlO2 to lxlO4 trypanosomes/dot, irrespective of whether the strips were pre-blocked or not. Again, no change in reactivity was observed in strips stored at 4°C for up to 90 days. 5.4.8 Use of different immuno-affinitv membranes The effects of different brands of immuno-affinity membranes (NC membrane, Hybond-C, Hybond-N and Pall Immunodyne) on the dot-ELISA reactions, were also investigated. No differences in either specificity or sensitivity were observed with regard to the NC membrane, Hybond-C and University of Ghana http://ugspace.ug.edu.gh 196 Hybond-N. The Pall Immunodyne membrane, however, gave less satisfactory results when compared with the other three membranes. There was loss of sensitivity of the order of xlO magnitude, from lxlO3 to lxlO4 trypanosomes/dot. This drop in sensitivity was attributed to a tendency for samples to diffuse into the Pall Immunodyne membrane, leading to lack of clearly defined positive staining reaction zones. University of Ghana http://ugspace.ug.edu.gh 19 7 In order to develop a MoAb-based assay for the differentiation of in vitro propagated insect stage trypanosome species, emphasis was placed on the utilization of an existing technique that offered the best opportunity to develop an assay which would be simple and easy to perform, in addition to being both sensitive and specific. It was anticipated that such an assay could easily be modified for use in the field in the diagnosis of trypanosome infections in infected tsetse flies (Glossina spp.). Of the existing techniques namely, dot-ELISA, micro-plate ELISA and radio-immunoassay, the assay that could best be developed for purposes of this study, was the NC membrane-based dot-ELISA. The dot-ELISA is a highly versatile, solid-phase immunoassay useful for both antibody or antigen detection. The technique is rapid, easy to perform and interpret, reagent conservative, cost effective and field portable (Pappas, 1988a). The dot-ELISA described in this Chapter was successfully applied to the identification of in vitro derived vector stages of T. brucei, T. congolense, T. simiae and T. vivax. The assay could correctly identify the trypanosome species involved in mono-species preparations as well as in artificial mixtures of trypanosome species. This assay was also able to identify the various stocks and clones of the different trypanosome species isolated from different geographical areas. This broad application of the dot-ELISA is a distinct advantage over the DNA hybridization technique which at present employs probes that are intra-species specific (Gardiner, 1989) and, therefore, are known to fail to identify new genotypes (McNamara et al., 1991; Majiwa etal., 1993). Some of the MoAbs detected as low as 10 trypanosomes/dot with the dot-ELISA. This high sensitivity, in terms of the minimum number of trypanosomes detected, was not surprising, since it had been reported that NC 5.5 Discussion University of Ghana http://ugspace.ug.edu.gh 198 membrane avidly binds a wide variety of parasite antigen preparations (Pappas et al., 1983; Pappas et al., 1986; Zimmerman, Nelson and Clark, 1985; Boctor et al., 1987; Whelen, Richardson and Wikel, 1986). Furthermore, unlike. plate-ELISA in which adsorption of soluble antigens to well surface had been reported to vary significantly (Burt, Carter, and Kricka, 1979; Kricka, Carter, Burt, Kennedy, Holder, Holliday, Telford and Wisdom, 1980), with the dot-ELISA technique the entire antigen applied is immobilized on the NC membrane, which has a large surface area as a result of its porosity, and thus increases the sensitivity of the assay (McFarlane, Tolley, Major, McFarlane and Williams, 1983; Kumar, Band, Samantaray, Dang and Talwar, 1985). However, in this work, it was found that the smaller the number of trypanosomes dotted onto NC membrane (ie,, at least 100 or less) the less reproducible was the assay results. This reduced reproducibility is believed to be due to small variations in the test conditions which may affect MoAb binding; some of these conditions were temperature, pH, concentrations of buffers and washing. This finding therefore suggested that the dot-ELISA would not be suitable, especially under field conditions if such low numbers of trypanosomes were to be detected. However, the finding that at least 1,000 or more trypanosomes/dot could produce 1 0 0 % reproducible results, indicated that at higher antigen concentrations, this dot-ELISA method could be robust and suitable for use in the field. Using the dot-ELISA method, Pappas and colleagues (1983) had shown that as little as 2.5xl04 parasites/dot gave sensitive, specific and reproducible results. In this study, lxlO4 trypanosomes/dot, applied in 1 fi\ volumes, was established to give satisfactory results, and this was therefore recommended for use in the dot-ELISA for subsequent studies. The application of antigen in small volumes was shown to give better results, since the intensity of the colour that developed was more dependent on the density, than on the amount of antigen in a dot (Towbin and Gordon, 1984), Volumes University of Ghana http://ugspace.ug.edu.gh 19 9 as small as 0.1 /xl/dot (Hawkes, Niday and Gordon, 1982) and 1 /xl/dot (Pappas, Hajkowski and Hockmeyer, 1983) had been used previously. In this dot-ELISA method, the use of whole ascites or immunoglobulin fractions was found unsuitable, as they cross-reacted extensively. Ascitic fluids contain antibodies secreted by hybridoma cells, as well as some of the animal's own immunoglobulins. Consequently, immunoglobulins, purified from this source are not monospecific (Boeye, 1986). Hence, depending on the type of work, the contaminating antibodies in ascites may be more or less a nuisance. Pappas, Hajkowski and Hockmeyer (1984) attributed the decreased reactivity of antigen in dotted samples stored on NC membrane to oxidation of adsorbed antigen by nitro groups present in the NC membrane matrix. This phenomenon is likely to be, in part, the cause of the deterioration of the T. congolense specific antigen when dotted samples were stored at room temperature (17-26°C) for more than 60 days. Another likely cause of decreased reactivity was epitope sensitivity to temperature variations. It has been reported that other types of membrane, for example cellulose membrane (Londner, Rosen, Sintov and Spira, 1987) and opaque, white plastics (Lin and Halbert, 1986) could be used in dot-ELISA. In this study, it was found that some brands of membrane supports were more suitable than others in the dot- ELISA. Positive results obtained with the dot-ELISA method were easily observed visually as brown dots on white NC filter paper, and the assay did not require a high level of technical expertise to perform or interpret. The potential of this procedure for diagnosis of trypanosome infections in the vector (Glossina spp.) of the African trypanosomes was noted, and this was indeed the subject of investigation in the studies described in the next chapter. University of Ghana http://ugspace.ug.edu.gh CHAPTER 6 2 0 0 DETECTION AND DIFFERENTIATION BETWEEN TRYPANOSOMES IN EXPERIMENTALLY-INFECTED TSETSE FLIES (GLOSSINA SPP'.) USING DOT-ELISA University of Ghana http://ugspace.ug.edu.gh 2 0 1 A modification of the NC membrane-based dot-ELISA developed in Chapter 5, was successfully used to detect and differentiate between T. brucei, T. congolense and T. simiae procyclics in the midguts of experimentally infected tsetse flies. The modification of the assay consisted of (a) the lysis of T. congolense or T. simiae in NC membrane applied sample dots using Triton X-114, and (b) a hydrogen peroxide destaining step, in which stains made on NC membrane strips by applied sample dots were removed. The afore-mentioned trypanosome species were specifically detected and differentiated without any cross-reactivity. In these assays, T. brucei and T. congolense parasites were detected directly using MoAbs specific to each of them, whereas T. simiae parasites were detected by exclusion using a T. congolense specific and Nannomonas subgenus-specific MoAb. The sensitivity of the assay was 90.5% in detecting T. brucei infections, 85.4% in detecting T. congolense infections and 94.4% in detecting T. simiae infections. The sample preparation from the gut of each tsetse fly could be replicated in 15 different dots, allowing some samples to be stored for testing at a later date. Sample dots stored at room temperature (19-26°C) under desiccated conditions did not show any loss in activity in 90 days. However, after seven days of storage, a ring-pattem reaction appeared on most sample dots that were tested with the T. brucei specific MoAb, irrespective of whether T. brucei antigens were present or not. These ring reactions, however, did not interfere with the correct interpretation of the assay results. Substitution of the PBS or PSG sample buffers used in the original dot-ELISA described in Chapter 5 for Na2EDTA buffer, led to the detection of T. brucei parasites in the salivary glands of infected tsetse flies using a T. brucei specific MoAb. This dot ELISA had a specificity greater than 99.9% and a sensitivity of 90%. Also, a dot-ELISA employing T. vivax 6.1 Summary University of Ghana http://ugspace.ug.edu.gh 2 02 and T. congolense specific MoAbs and utilizing the biotin-streptavidin reaction amplification technique, was successfully used to detect T. vivax and T. congolense in the mouthparts of infected tsetse flies. The specificity of the assays were as good as for detecting T. brucei in infected tsetse salivary glands, but the sensitivity was lower, 43.8% in detecting T. vivax and 55.6% in the case of T. congolense. The successful modification and application of the dot-ELISA in detecting and differentiating between trypanosome species in the midguts, salivary glands and mouthparts of experimentally infected tsetse flies, is the best indication that the assays developed may be capable of specific identification of trypanosome species in naturally infected Glossina species. University of Ghana http://ugspace.ug.edu.gh 203 Earlier studies of the life-cycle of trypanosomes in the vector established that each trypanosome subgenus characteristically develops in a particular organ of the tsetse fly (Lloyd and Johnson, 1924). As a result, the standard method for diagnosis of trypanosome infections in tsetse, has been by dissection and microscopy. By this method, infections in the gut and salivary glands have been deemed to be due to the Trypanozoon subgenus, whereas, infections in the midgut and proboscis have been assigned to the Nannomonas subgenus. Infections confined to the midgut are classified as immature Nannomonas or Trypanozoon, whilst those confined to the proboscis have been ascribed to the Duttonella subgenus. It is, however, not possible by these criteria to differentiate between the recognised species within the various subgenera, since all species within a subgenus have identical cycles of development in the vector (Hoare, 1972). The differential diagnosis is further complicated when mixed infections occur in the vector (Godfrey, 1966). Besides, Trypanosoma grayi and T. suis are known to reside in the midgut and salivary glands of infected tsetse, respectively (Hoare, 1972), and their presence, therefore, could lead to a misdiagnosis. Currently, a recombinant DNA-based technique, first applied to trypanosome identification by Kukla et al. (1987), provides the best known alternative for the detection of, and differentiation between trypanosome species in the Glossina species. This technique has subsequently been used for the identification of Nannomonas species (Gibson et al., 1988), differentiation between T. congolense and T. simiae (Majiwa and Webster, 1987; McNamara, Dukes, Snow and Gibson, 1989; Majiwa and Otieno, 1990; McNamara and Snow, 1991) and identification of T. brucei and T. vivax (Kukla et al., 1987; Dicken and Gibson, 1989) in infected tsetse flies. However, this method also has some disadvantages. The most important disadvantage is that the existing 6.2 Introduction University of Ghana http://ugspace.ug.edu.gh 204 DNA probes employed in the technique are intra-species specific (Majiwa et al., 1993). As a result, the probes have shown that there are five types of T. congolense (Kilifi type, West African riverine-forest type, Savannah type, Godfrey type and Tsavo type) recognised by five different probes. The most recent discovery, the Tsavo type, was established following failure of the four previously existing T. congolense probes to hybridize to a new Nannomonas isolate from Tsavo, Kenya. It is also quite likely that other T. congolense populations exist which would not react with any of the five probes developed, so that a negative result would not necessarily indicate that a given Nannomonas trypanosome population does not belong to the T. congolense species. Another, disadvantage is that the technique is not simple enough, and therefore cannot be performed in most laboratories. The development of a test which is simple, rapid, sensitive and specific and applicable under both laboratory and field conditions is, therefore, highly desirable. In the studies described in the previous chapter, a nitrocellulose (NC) membrane based dot-ELISA that utilizes trypanosome species-specific MoAbs was developed for identification and differentiation between in vitro derived trypanosome species. In the study reported here, the dot-ELISA thus developed, was successfully modified and used to detect and differentiate between T. brucei, T. congolense and T. simiae procyclics in the midgut, T. brucei in the salivary glands, and T. congolense and T. vivax in the mouthparts of experimentally infected Glossina species. University of Ghana http://ugspace.ug.edu.gh 2 0 5 6.3.1 Experimental animals 6.3.1.1 Goats Adult male castrated goats (crossbreeds between East African Masai and Galla), aged 8-10 months and weighing between 20 and 25Kg, were used in this study. They were purchased from farms in the Kumanchu location, Laikipia district of Kenya, an area known to be free from tsetse flies and trypanosomiasis. The goats were quarantined in fly-proof housing for one month, after being dipped and treated with long-acting tetracycline, coccidiostats and anthelmintics on arrival. Prior to being used, they were confirmed to be uninfected with trypanosomes, using the thick, thin and wet blood films as well as by the darkground/phase contrast buffy coat technique described by Murray, Murray and McIntyre (1977). 6.3.1.2 Pigs Male and female, 6 -month old, Large-white pigs were bred at the Veterinary Research Laboratory, Kabete, Kenya, an area known to be free from tsetse and trypanosomiasis. Those which were purchased for this study, came from herds kept in the area. The animals were kept in fly-proof quarters and screened for trypanosomiasis, using the haematocrit centrifugation technique as well as the thick, thin and wet blood film microscopy method. 6.3.2 Tsetse flies 6.3.2.1 Laboratory bred tsetse flies The tsetse flies used came from the ILRAD laboratory-reared Glossina morsitans centralis which had previously been obtained from the East African Trypanosomiasis Research Organisation (EATRO), Tororo, Uganda in 6.3 Materials and methods University of Ghana http://ugspace.ug.edu.gh 206 1979 (Moloo, Kutuza, Bakakimpa, Kamunya, Desai and Pereira, 1985). This colony was first initiated in 1969 at EATRO, with adults which had emerged from pupae collected in the field at Singida, mainland Tanzania (Moloo and Kutuza, 1969). The Glossina pallidipes flies used had originated from Nguiuman and Shimba Hills in Kenya, and were also part of the ILRAD tsetse colony collection. 6.3.3 Infection of goats, pigs and tsetse flies 6.3.3.1 Infection of goats Seven goats were each infected with one of the following 7 trypanosome stocks or clones: T. brucei stock IL375; T. vivax stocks IL3096 and IL2337; T. congolense stock IL3779 and clones, IL1180, IL3274, and IL13-E3. Each goat was infected by the intramuscular route with about lxlO 7 trypanosomes, diluted in 3ml of phosphate-buffered saline-glucose (PSG), pH 8.0 (Lanham and Godfrey, 1970). To monitor parasitaemia, each goat was bled daily from a marginal ear vein, and the blood examined for the presence of trypanosomes, using the wet blood film phase contrast microscopy at x400 magnification or the microhaematocrit/dark ground technique (Murray et al. , 1977). 6.3.3.2 Infection of pigs Three pigs were each infected subcutaneously in the neck region with either T. simiae stock CP11(IL3879) or IL3815. To monitor parasitaemia, peripheral blood was drawn daily from either an ear or tail vein and examined for trypanosomes as described for goats above. University of Ghana http://ugspace.ug.edu.gh 207 6.3.3.3 Infection, maintenance and identification o f infected tsetse flies T. brucei, T. vivax and T. congolense parasites were transmitted to G. m. centralis flies from infected goats and T. simiae to G. pallidipes from infected pigs, by fly feeding. Five days after infections had become patent in goats, and 1 day afterwards in pigs, teneral tsetse were allowed to feed on the shaven flanks of an infected goat for a period of 30 days in the case of T. brucei and 25 days for T. vivax and T. congolense. Thereafter, the flies were starved for two days and those with mature infections identified by the warm-slide probe method of Burtt (1946). With regard to T. simiae, teneral tsetse were fed once only on infected pigs, after which the flies were maintained by feeding on rabbits for 25 days and starved for 2 days prior to probing. All tsetse flies confirmed to be infected by the extrusion of metacyclics were maintained by feeding on rabbits. 6.3.4 Preparation of samples for dot-ELISA 6.3.4.1 Dissection of tsetse and extraction of midgut tissue Tsetse flies were killed by crushing the thorax with gentle pressure exerted with a finger or by anaesthesia using chloroform, and the wings and legs pulled off. Several ways of dissection and preparation of the midgut samples were examined: 6.3.4.1.1 The fly was placed on a microscope slide under a dissecting microscope (Wild M5A binocular; Wild, Heerbrugg, Switzerland) at xl20 magnification and about 30/d of PBS (pH 7.4) or PSG (pH 8.0) added. The abdomen of the fly was then tom open at the ventral surface, using a pair of forceps and a dissecting pin. The gut was pulled into the buffer on the slide and the midgut cut out and covered with a coverslip and examined for trypanosomes using a compound microscope (Leitz larlux; Leitz Wetzlar, Germany) at University of Ghana http://ugspace.ug.edu.gh 208 x320 magnification. The examined tissues were then transferred with forceps into an Eppendorf tube containing 50/d of the appropriate buffer. 6.3.4.1.2 Alternatively, the tsetse gut was dissected out onto a slide as above, and cut below the proventriculus and above the rectum. The whole gut, including the foregut, midgut and hindgut, was examined microscopically for the presence of trypanosomes and then transferred into 50/d of either PBS or PSG. 6.3.4.1.3 In the last method tried, the distal quarter of the abdomen of tsetse was excised with a pair of dissecting scissors and discarded (Kukla et al., 1987). The whole abdominal contents were then squeezed out by applying firm pressure in a rolling motion, from the anterior portion toward the posterior end of the abdomen. The protruding gut was then tom apart with forceps and transferred into 50/d of buffer. 6.3.4.2 Preparation and application of tsetse midgut suspensions onto NC membrane Tsetse gut tissue dissected out as indicated variously above, and suspended in either PBS or PSG, were treated again in three different ways before applying sample dots onto NC membrane strips for testing: (1) Suspended tsetse gut samples were agitated to release trypanosomes into the buffer, by tapping the base of the tube gently with a finger. The samples were then allowed to stand for up to 1 hr, and agitated once more before pipetting out 3 /d samples onto NC membrane strips in dots. (2) The gut suspensions were mixed gently by pipetting the fluid up and down a few times, using a 50/d micropipette, and 3/d samples dotted soon after. (3) Trypanosomes in suspended gut tissues were released by physical University of Ghana http://ugspace.ug.edu.gh 2 0 9 maceration, using the tip of a 50/u.l micropipette, together with vigorous pipetting up and down about 10 times, before 3/xl sample volumes were applied in dots onto NC membrane strips for testing. As controls, lxlO5 T. brucei, T. congolense and T. simiae procyclics, and epimastigotes of T, vivax obtained from in vitro cultures were separately applied in dots onto each NC membrane strip. 6.3.4.3 Preparation of touch blots Tsetse abdomen was slit open as above (section 6 .3.4.1.3). The abdomen was squeezed several times and the protruding gut touch- blotted by pressing gently onto NC membrane. Alternatively, the protruding gut was cut with a scalpel blade before touch-blotting. 6.3.4.4 Preparation of dot-blots using lvsed trypanosomes Attempts were made to lyse gut forms of T. congolense and T. simiae so as to expose internal trypanosome species-specific antigens for uptimum reactivity in the dot-ELISA. 6.3.4.4.1 Lvsis of T. consolense or T. simiae in infected tsetse gut samples in suspension Several detergents, namely, Nonidet P-4Q (NP-40), Saponin, Sodium dodecyl sulphate (SDS), all from (Sigma, England); and Triton X-114 (Fluka Chemie AG, Switzerland), were tested in experiments intended to lyse T. congolense or T. simiae procyclic midgut forms in infected tsetse gut samples. Each of the detergents was used to prepare a "lysis-buffer" which consisted of 0.05, 0,1, 0.2, 0.3, 0.4 or 0.5% detergent, and lOjug/ml Leupeptin and E-64 in either PBS or PSG. Fifty microlitre aliquots of each concentration of lysis-buffer were pipetted into Eppendorf tubes, and infected tsetse midguts dissected out and suspended in them. Trypanosomes were University of Ghana http://ugspace.ug.edu.gh 2 1 0 released into the sample lysis buffers by gently pipetting the fluid up and down a few times, using a 50/xl pipette. The samples were allowed to stand for 30 min, and mixed once again before 3/d volumes were pipetted out and applied onto NC membrane strips in dots. 6.3.4.4.2 Lvsis of T. comolense or T. simiae gut forms in sample dotted NC membrane strips NC membrane strips were "dotted" with experimentally-infected T. congolense or T. simiae tsetse gut samples, as previously described. The sample dotted strips were then incubated in different concentrations of detergent solutions for varying time periods (either 30 min, lhr or 2hr, at room temperature of 19-25°C) without shaking. The detergents used were either: NP-40, Saponin, SDS or Triton X-114. Each was tested at six different concentrations (0.05, 0.1, 0.2, 0.3, 0.4, or 0.5%), diluted in blocking solution which consisted of 5% (w/v) skimmed milk in Tris-buffered saline (TBS)(50mM Tris and 150mM NaCl, pH 8.0). 6.3.4.5 Preparation of tsetse salivary glands for dot-ELISA Tsetse salivary glands were dissected out in one of two ways: 6.3.4.5.1 Each fly was placed on a microscope slide, ventral surface up, and 30/d of PSG added onto the head region. The thorax was then pinned down with a dissecting needle close to the base of one wing, and the head grasped with fine forceps and pulled gently, but steadily, away from the thorax in a straight line, under a dissection microscope at xl20 magnification. The glands were pulled out whilst immersed in the buffer until they were out of the thorax. When a gland broke, it was seized with forceps and pulled out. 6.3.4.5.2 A fly was placed on a slide as above, and 30/d of buffer added to the abdominal area. The abdomen was then torn open at the anterior University of Ghana http://ugspace.ug.edu.gh 2 1 1 end close to the thorax and the elastic tissue pulled backwards towards the posterior end. The exposed gut was pulled back and a drop of PSG added into the cavity. The tissues at the anterior most part of the exposed abdominal cavity were grasped with a pair of fine forceps and pulled into the buffer on the slide. The two salivary glands were easily located by this method. The isolated salivary glands were transferred, using forceps, into lOjul of 5mM Na2EDTA buffer. The suspended glands were pipetted up and down several times, using a 1 0 /d micro-pipette, before 3/zl volumes were pipetted onto NC membrane strips in dots. 6.3.4.6 Preparation of tsetse proboscides for dot-ELISA Several ways of dissection and preparation of tsetse mouthparts were explored: 6.3.4.6.1 The proboscis of a tsetse fly was cut a third of the way down from the thecal bulb, with a pair of scissors. The thorax was then squeezed gently to expel a drop of fluid through the cut proboscis and gently touch-blotted onto a demarcated spot on NC membrane. 6.3.4.6.2 The head of a tsetse fly was placed on a microscope slide, ventral surface up. The posterior base of the thecal bulb, close to the head, was pressed gently but firmly with a dissecting needle placed almost parallel to the slide surface, and pulled away from the head. The labrum and hypopharynx were then examined under a light microscope (Leitz labrlux; Leitz Wetzlar, Germany) at x320 magnification, for the presence of trypanosomes. The separated proboscis was then transferred into about 1 0 /xl of distilled water, under a dissection microscope at xl80 magnification, and the labrum, labium and hypopharynx separated, using two dissecting University of Ghana http://ugspace.ug.edu.gh 2 12 needles, before they were transferred into about 1 0 /xl of distilled water in an Eppendorf tube. The sample was left to stand for about lhr before pipetting up and down about ten times using a 5/A micropipette, and five microlitres transferred onto a spot on NC membrane. The sample was air dried at room temperature and the remaining 5/xl pipetted onto the same spot. 6.3.4.6.3 Tsetse proboscis was separated from the head and dissected as described above, in about 7/xl of distilled water, PBS or PSG or 5mM Na2EDTA buffer, in the wells of a teflon coated multitest immunofluorescence slide. The dissected labrum and hypopharynx were then cut into pieces in the wells with a small knife, and the suspension transferred from the well directly onto NC membrane in a dot. 6.3.4.6.4 The sample preparation, as previously described (section 6 .3.4.6 .3) was repeated using silicon coated multitest slides. 6.3.4.6.5 The proboscis was separated from the head of tsetse and cut at the base of the thecal bulb. The mouth parts were then transferred into an Eppendorf tube containing about 7^1 of distilled water, PBS, PSG, or 5mM Na2EDTA buffer. The sample was left to stand for at least 1 hr, after which the proboscis was broken up into small pieces with the tip of a micropipette. The sample was pipetted up and down about 5 times and the whole volume transferred onto a NC membrane in a dot. 6.3.5 Estimation of trypanosome numbers in midgut suspensions Tsetse midgut suspensions were prepared and mixed as described earlier, and large particulate tissue matter removed and discarded. A small volume was then pipetted out of each sample preparation and dropped onto a microscope slide, and covered with coverslip. University of Ghana http://ugspace.ug.edu.gh 213 The number of trypanosomes per millilitre in each sample was then estimated using the "rapid matching method" of Herbert and Lumsden (1976). 6.3.6 Monoclonal antibodies The panel of trypanosome species, and subgenus-specific MoAbs used in this study were produced and selected as described in Chapter 4. 6.3.7 Dot-ELISA procedure 6.3.7.1 Detection of T. brucei in midguts of infected tsetse flies bv a modified dot-ELISA I All incubations and washings in this assay were performed at room temperature on a gentle rocker. Each tsetse sample was applied in dots onto several strips so that every sample could be assayed for reactivity with various trypanosome species-specific MoAbs. The strips were first destained by incubating for 1 hr in a "destaining solution" containing 5%(v/v) H2O2 in blocking solution. They were then washed three times, 10 min each, with TBS pH 8.0, and then incubated for 3 hr with specific MoAbs, diluted in a blocking solution. This was followed by two washes (10 min/wash) with the same buffer. The strips were then incubated for 1 hr with goat anti-mouse immunoglobulins labelled with horseradish peroxidase (HRPO) (Sigma, USA) and diluted in a blocking solution. They were washed two times, after which they were immersed for three minutes in a substrate solution containing 0.15%(v/v) hydrogen peroxide (H2 0 2) and 0.05%(w/v) 3,3'-diaminobenzidine (DAB) in phosphate-Na2EDTA buffer (lOmM NaH2P04, lOmM Na2HP0 4 and lOmM Na2 EDTA). The strips were then rinsed two times in deionised water and the substrate reaction stopped as described earlier (Chapter 4 , section 4.3.8.1). The results were read visually. Positive reactions appeared as brown dots, whereas negative results remained colourless. A control University of Ghana http://ugspace.ug.edu.gh 2 14 section on each strip was applied with lxlO5 trypanosomes/dot of T. brucei, T. congolense and T. simiae. These were used to assess the performance of the specific MoAbs. The conjugate control strips dotted with all the test samples were assayed omitting the step in which specific MoAb was added. Any reactions on these trips therefore gave a measure of non-specific background reactivity. 6.3.7.2 Detection of T. coneolense and T. simiae in the midguts of infected tsetse flies bv a modified dot-ELISA II NC membrane strips applied with sample dots were first incubated for I hr with 0.1%(v/v) Triton X-114 dissolved in blocking solution, and washed three times (10 min/wash) with Tris-buffered saline pH 8.0. The Triton X-114 was used to lyse whole trypanosomes in the dotted samples so as to expose internal trypanosome antigens. The rest of the procedure was the same as described previously for the detection of T. brucei in infected tsetse midgut. 6.3.7.3 Detection of T. brucei in the salivary glands of infected tsetse flies bv dot-ELISA Salivary gland samples were prepared as described earlier and dotted onto NC membrane strips in triplicates. All the steps in this assay were performed at room temperature. Also, all incubations and washings were done on a gentle rocker. The strips were first blocked by incubation in blocking solution for 1 hr. The blocking solution was discarded and the strips further incubated for 3 hr with trypanosome species-specific MoAbs diluted in blocking solution. They were then washed three times (10 min/wash) with TBS and incubated for 1 hr with HRPO-conjugated goat anti-mouse immunoglobulins diluted in blocking solution. They were washed as above University of Ghana http://ugspace.ug.edu.gh 2 15 and then incubated with substrate solution for 3 min. The substrate reaction was stopped and the results read as before. 6.3.7.4 Detection of T. vivax and T. consolense in the mouthparts of infected tsetse flies bv dot-ELISA Each tsetse proboscide sample was placed onto a NC membrane strip in a single dot. The sample dotted strips were assayed either following the procedure described earlier for the detection of T. brucei in the salivary glands or following the modified procedure briefly described here under: Briefly, after washing off excess monoclonal antibody, the strips were incubated for 1 hr with biotinylated sheep anti-mouse immunoglobulins, diluted in Tris- buffered saline pH 8.0 and containing l%(w/v) bovine serum albumin. The strips were washed three times as usual to remove excess unbound biotinylated antibody and then incubated for 1 hr with streptavidin-conjugated horseradish peroxidase (diluted in blocking solution). This was followed by washing as described above, before incubating with the substrate. As usual, a control section was incorporated into the system, consisting of four dots containing, respectively, lxlO5 trypanosomes/dot of T. vivax, T. congolense, T. brucei and T. simiae. (There were no controls with the conjugate since tsetse proboscide samples were not replicated). University of Ghana http://ugspace.ug.edu.gh 2 16 6.4.1 Detection of trypanosomes in the midgut of experimentally infected tsetse flies, using the standardized dot-ELISA developed for differentiating between in vitro derived insect stage trypanosomes Gut samples prepared from laboratory-reared tsetse flies and applied in dots onto white NC membrane, were found to stain the membrane with varying coloration and intensity. The stains were predominantly either, reddish, reddish-brown, brown or blackish-brown to black, and occasionally greenish to almost colourless, depending on the stage of digestion in the fly. The strip labelled "P" in Figure 31 shows the stains made on NC membrane by "dotted" tsetse gut samples from five T. congolense infected flies, five uninfected flies, and in vitro derived procyclic T. congolense, T. brucei and T. simiae. Two important observations were evident. Firstly, midgut samples from both infected and uninfected tsetse flies stained the membrane. Secondly, sample dots consisting of in vitro derived trypanosomes did not stain the membrane. Figure 31 shows an example of the results of direct application of the dot-ELISA developed and standardized in Chapter 5 when applied for the detection of T. congolense in the midgut of experimentally infected G. m. centralis. The Nannomonas subgenus-specific MoAb (TC6/25) and the T. congolense specific MoAb (TC6 ) reacted with samples from the T. congolense infected flies as expected (Figure 31). However, these MoAbs also reacted with samples from all the uninfected flies. The reactions with the control trypanosome dots on the strips incubated with these two MoAbs, showed cross-reactivity with other trypanosome species to which they were known not to react (Chapter 5). Furthermore, both the T. brucei (KT39a) and T. vivax (KD32) specific MoAbs, reacted with samples from all the T. congolense 6.4 Results University of Ghana http://ugspace.ug.edu.gh 2 1 7 infected as well as all the uninfected flies. These false reactions were also seen on samples that were tested as conjugate controls (without incubation with specific MoAbs), suggesting that the reactions were not the result of non specific binding of the MoAbs. Comparison of the staining on the preserved NC membrane strip P with those on the tested strips, revealed that the staining intensity of the sample from uninfected fly UF5 was amplified by the dot- ELISA (Figure 31). 6.4.1.1 Removal of high background activity from the dot-ELISA The high background encountered was assumed to be due to two causes: (1) persistent staining of the NC membrane due to the physical coloration of the test samples, and (2 ) non-specific reactivity due to interference by haem. To remove the background, investigations were conducted into several alternatives. These were: (1) testing only flies with very low amount of undigested blood meal (2 ) changing the handling of samples prior to dotting, such as using the touch blot technique (3) changing the enzyme substrate chromogen system so that positive reactions are distinguishable from the background (4) reducing the amount of biological debris in the tsetse gut samples, such as by partial isolation of trypanosomes (5) removing the background without destroying the diagnostic antigens such as by a destaining process, and (6 ) developing an alternative assay in which background originating from tsetse gut samples present no problems. The findings on each of these investigations are presented serially here under: 6.4.1.1.1 Testing flies with very low amounts of undigested blood msal The suitability of the option to test only flies with m in im a l amounts of residual blood meal clearly depended on the proportion of flies that could be tested using that selection criterion. To determine this proportion in University of Ghana http://ugspace.ug.edu.gh 218 Figure 31 Dot-ELISA of midgut samples from five T. congolense (IL1180) infected tsetse flies (IF1 to IF5) and five uninfected control flies (UF1 to UF5), The sample from each fly was dotted across in a row, one replicate dot on each strip. A control sector at the lower section of each strip was applied with 1x10s trypanosomes per dot of in vitro propagated T. congolense K/83/IL/97/2 (TCK), T. brucei IL2616 (TB) and T. simiae KETRI 2431 (TS). The antigen "dotted" strips were tested using Nannomonas subgenus-specific MoAb (TC6/25), a T. brucei species-specific MoAb (KT39a), a T. congolense specific MoAb (TC6 ) and a T. vivax specific MoAb (KD32). Strip 'C' was a conjugate control, and strip 'P ' was preserved without processing through the dot-ELISA. Note the sample stains shown on strip 'P '. University of Ghana http://ugspace.ug.edu.gh CM COO) CO CM/VSCD o i - h * O I- Q M c P IF1 • • --- • • • IF2 o 9 Q © 0 # IF3 # • * • • • IF4 • • • 0 0 • IF5 • • • • • • UF1 0 0 o • • « UF2 • ® • * • UF3 o © 0 ---- • • U F 4 U F 5 r P o • » • • • • ■«* TCK - § 'I j TB - TS - • 1... Figure 31 University of Ghana http://ugspace.ug.edu.gh 2 1 9 the case of laboratory reared tsetse flies, an experiment was first conducted using newly emerged and 8 -week old G. m. centralis. Figure 32a shows the staining pattern obtained from midgut samples prepared from newly emerged male and female G. m. centralis from 1 hr after feeding (day 0) through day 1, 2, 3 to 4 after feeding. Non-specific staining was observed with samples obtained up to 4 days post-feeding. Samples prepared and dotted on day 0, 3 and 4 after feeding presented less staining problems, compared with the samples prepared and dotted on days 1 and 2 after feeding. Analysis of the results obtained from the experiments conducted showed that <10% of male, and <5% of female flies could be tested 1 hr after feeding. The flies that were most suitable for testing were those that did not fully engorge. None of the flies from either sex could be tested on day 1 and 2 after feeding, though the female flies presented more intense staining problems. By day 3 and 4 after feeding, about 10% of the flies from either sex could be tested, and there appeared to be no differences in the intensity of staining with reference to sex. The pattern of staining from dotted gut samples of the eight week old G. m. centralis flies is shown in Figure 32b. It was evident that gut samples from male flies stained NC membrane less intensely when compared with samples from female flies. Whilst about 10% of male flies could be tested 1 hr after feeding and 35% could be tested on day 3 or 4 after feeding, less than 2% of female flies could be tested on day 3 and 4 post-feeding combined. 6.4.1.1.2 Use of different sample application techniques Efforts were made to reduce non-specific staining by reducing the amount of undigested material that was applied onto NC membrane for testing. Investigations were conducted into the suitability of use of the tsetse abdominal touch blot technique previously described by Kukla et al. (1987). University of Ghana http://ugspace.ug.edu.gh 2 2 0 Figure 32a, b The staining pattern obtained on NC membrane following application of midgut samples prepared from male (0 ") and female (9) G. morsitans centralis from 1 hr after feeding (day 0) through days 1 to 4 after feeding, (a) staining pattern from young flies that have fed only once; (b) staining pattern from 8 - week old adult flies. Midgut samples were prepared from four randomly selected flies from each of the two sex groups on days 0, 1, 2, 3 and 4 after feeding, and applied in dots onto NC membrane. The strips were tested in the dot-ELISA without incubating with specific MoAbs, but with conjugate and substrate, so as to determine non-specific background. University of Ghana http://ugspace.ug.edu.gh .• m • Midgut contents of l * _ • • v. randomly selected1 — ----- ----- — ----- tsetse flies • *1 m t— ---- ----1 • o ! • 1 1 /s after feeding ^ 0 1 2 3 _1j l 0 1 2 3 4 i Figure 32a Midgut contents of randomly selected tsetse fSes 9 a • 0 ■» o • o O • ♦ ♦ • • • • • • Days after feeding • 9 Figure 32b University of Ghana http://ugspace.ug.edu.gh 2 2 1 The results showed that touch blotted samples left very little or no stains on NC membrane. However, the sensitivity of the dot-ELISA was so much lowered that less than 5% of T. brucei midgut infections could be detected. 6.4.1.1.3 Use of different enzymes and chromogenic substrates It was thought likely that the false reactions could be the result of oxidation of the chromogenic substrate [3,3'-diaminobenzidine (DAB)] by H2 0 2 through catalysis by haem which acted as a peroxidase (Saunders, Holmes-Siedle and Staak, 1964). To abolish those non-specific reactions, the HRPO enzyme used in the dot-ELISA was replaced with glucose oxidase which generates H2 0 2 by its action on glucose (Decker, 1977), thereby permitting the omission of H2 0 2 in the substrate solution. The presence of haem without glucose oxidase, therefore, would result in no false reactions since H2 0 2 would be absent. Figure 33 summarizes the results of a comparison of HRPO-conjugate and glucose oxidase-conjugated antibodies in the detection of T. congolense (IL1180) in the midguts of experimentally infected Glossina. The results showed that the glucose oxidase-conjugate was not able to eliminate the background. It was also found that the glucose oxidase-conjugate gave a higher assay background compared to HRPO. The chromogenic substrate 4-Chloro-l-Naphthol (4C1N) which normally gives a blue reaction, was compared with the brown reaction given by DAB so as to determine whether it was possible to distinguish positive reactions from the background by colour. The results obtained showed that neither the blue reaction of 4C1N nor the brown reaction of DAB could allow clear distinction of positive reactions from the background in the dot-ELISA. 6.4.1.1.4 Removal of background activity bv destaining A destaining step involving the use of hydrogen peroxide (H2 0 2) was introduced into the dot-ELISA and investigated to establish University of Ghana http://ugspace.ug.edu.gh 2 2 2 Figure 33 Comparison of HRPO-conjugated and glucose oxidase-conjugated antibodies in the detection of T. congolense (IL1180) in the midguts of experimentally- infected G. morsitans centralis using dot-ELISA. Each strip was "dotted" with samples from ten infected flies (IF1 to IF10) and three uninfected control flies (UF1 to UF3). The strips marked '1' were tested using HRPO-conjugate, and those marked '2' with glucose oxidase-conjugate. The MoAbs used were: TC6 , T. congolense specific; TC6/25, Nannomonas subgenus-specific; TR7 andKT39a, T. brucei species-specific; and KD32, T. vivax specific. Strip 'P' was preserved without processing through the dot-ELISA. University of Ghana http://ugspace.ug.edu.gh CO 1 I——--- 1 IF 1 e • 6 IF2 IF 3 o ~ ♦ & — i IF 4 I ET E • o Q 0 I F IF6 Q % O — IF7 0 m © © IF # O & ; o IF f o IF 10 UF1 • 0 • ® - • • UF3 UF3 — — — H 1 2 1 2 T C 6/ 25 University of Ghana http://ugspace.ug.edu.gh hr GCm i (0 CO CM CO Q Figure 33 University of Ghana http://ugspace.ug.edu.gh 22 3 whether it could remove the non-specific stains. Figure 34 shows the results of an experiment in which the stains made by gut samples from T. brucei infected flies as well as uninfected flies were removed by the destaining method. Strip P which was not subjected to any testing, shows the stains made by the original dotted samples. Strip C was run through the already established dot-ELISA as conjugate control (without incubation with specific MoAbs). This strip showed the non-specific reactions and stains described earlier. The third strip Cm was run through a modified dot-ELISA that included an H2O2 destaining step, also as a conjugate control. The removal of all the non-specific stains in the sample dots on strip Cm are clearly shown. It is important to mention, however, that the H2O2 treatment at this dilution appeared to be harsh, considering the manner in which the sample dots foamed, and pieces of debris broke loose and floated. Two important questions, therefore, arose from this experiment. These were: (1) the effect of H2O2 on the diagnostic antigens detected by the MoAbs, and (2) the stability of those antigens on the NC membrane during treatment with H2O2 . These questions were subsequently addressed in the following experiments. 6.4.1.2 The effect of hydrogen peroxide on the trypanosome species- specific antigens detected bv the MoAbs Figure 35 shows the effect of a 1 hr incubation of varying concentrations of H2 0 2 on the T. brucei specific antigenic epitope detected by KT43/27. The reactions on the control strip (incubated with 0% H2 0 2) and those on strips incubated with 0.5-30% H2 0 2 showed that both the specificity and sensitivity of the MoAb were unaffected by this treatment. In a similar experiment illustrated in Figure 36, the Nannomonas subgenus-specific MoAb KN4 was shown to react specifically with H2 0 2 treated T. congolense and T. simiae parasites without any noticeable changes in sensitivity. University of Ghana http://ugspace.ug.edu.gh 2 2 4 Figure 34 Removal of non-specific stains from NC membrane strips "dotted" with G. morsitans centralis midgut samples from five T. brucei infected flies (IF1 to IF5) and five uninfected control flies (UF1 to UF5), using H2O2 in the dot- ELISA. The sample from each fly was dotted across in a row, one replicate dot on each strip. 'P ' was a preserved strip that was not subjected to any testing. It shows the original stains made on NC membrane by the applied gut samples. 'C ' was a strip processed through the dot-ELISA as conjugate control, without incubation with specific MoAb, and strip 'Cm ' was processed through a modified dot-ELISA that included an H2O2 destaining step. University of Ghana http://ugspace.ug.edu.gh P c cm IF 1 • • IF2 IF3 O o IF4 IF5 o o UF1 ■ UF2 I UF3 ' UF4 UF5 Figure 34 University of Ghana http://ugspace.ug.edu.gh 225 Figure 35 The effect of H2 0 2 on the T. brucei species-specific antigenic epitope detected by KT43/27. In vitro derived T. congolense K/83/IL/97/2 (TCK), T. brucei IL2616 (TB) and T. simiae KETRI 2431 (TS) were applied onto NC membrane strips starting from lxlO 5 trypanosomes per dot to I trypanosome per dot for each species. University of Ghana http://ugspace.ug.edu.gh TCK— TB — TS - n Tryps /do t 1 x 10 ' 1 x 10 * 1x1CT 1 x 1 0 ' 1 x 101 1 x 10 I o i O r i n N i o W ^ i n i f l i o i c s a i o i o i o o i n o 6 OJ CO T t lO t- i - CM co Figure 41 University of Ghana http://ugspace.ug.edu.gh 2 3 7 reacted very weakly with the infected samples. This inability to strongly detect clearly, the T. congolense IL1180 midgut infections was found to be the case for all the T. congolense specific MoAbs. On the contrary, each of the Nannomonas subgenus-specific MoAbs was able to detect those infections with strong positive reactions. 6.4.3.1 Improved detection of T. consolense in the midguts of experimentally infected tsetse flies using the modified dot- ELISA Attempts were made to improve the accessibility of the T, congolense species-specific internal antigens (Chapter 4) for reactivity in the dot-ELISA, by lysis of the trypanosomes in gut suspensions before applying samples onto NC membrane for testing. The results, however, showed an abolition of the weak reactivity of the T. congolense specific MoAbs, and a decreased reactivity of the Nannomonas subgenus-specific MoAbs. It was assumed that the decreased binding of the Nannomonas specific MoAbs, could be due to increased competition for binding to NC membrane between the trypanosome species-specific antigens and the mass of debris released by midgut contents. Figure 42 shows a light micrograph of T. congolense midgut procyclic forms together with the characteristic mass of biological debris usually present in the tsetse gut samples. To circumvent this problem, it was decided to first dot the gut samples onto NC membrane, before lysing the bound trypanosomes. Preliminary investigations showed that each of the detergents used (NP-40, Saponin, SDS and Triton X-114), was capable of enhancing the reactivity of the T. congolense specific MoAbs. Triton X-114 was, however, selected due to its superior ability to give stronger reactions. Figure 43 shows the results of an experiment in which T. congolense (IL1180) in the midguts of University of Ghana http://ugspace.ug.edu.gh 238 Figure 42 Light micrograph of a trypanosome infected tsetse midgut sample showing biological debris and T. congolense parasites (t). University of Ghana http://ugspace.ug.edu.gh University of Ghana http://ugspace.ug.edu.gh University of Ghana http://ugspace.ug.edu.gh 2 3 9 Figure 43 Detection of T. congolense IL1180 in the midguts of experimentally infected G. morsitans centralis using a modified dot-ELISA. Each of the NC membrane strips was "dotted" with midgut sample preparations from, five infected flies (IF1 to IF5), and five uninfected control flies (UF1 to UF5). A control sector at the lower section of each strip was applied with lxlO5 trypanosomes per dot of in vitro propagated T. congolense K/83/IL/97/2 (TCK), T. brucei IL2616 (TB) and T. simiae KETRI 2431 (TS). The antigen "dotted" strips were tested using Nannomonas subgenus-specific MoAb (KN4), T. congolense specific MoAb (TC6) and T. brucei species-specific MoAb (KT39a). Strip 'C' was tested as a conjugate control. University of Ghana http://ugspace.ug.edu.gh •'t z CO O s- CC 0> CO £ IF 1 IF2 IF3 IF4 IF5 UF 1 UF2 UF3 UF4 UF5 TCK — TB — TS — © Figure 43 University of Ghana http://ugspace.ug.edu.gh 240 experimentally infected G. m. centralis were detected following lysis of trypanosomes in sample bound NC membrane strips using 0.1% Triton X-114. Both the Nannomonas subgenus-specific MoAb (KN4) and the T. congolense specific MoAb (TC6) elicited stronger reactions and detected clearly, 4 out of the five infected flies. Samples from all the uninfected flies tested negative, and the reactions on the control trypanosome dots showed that both MoAbs reacted specifically. The T. brucei specific MoAb KT39a reacted with the T. brucei control parasite dot and detected none of the flies tested, thus, demonstrating the specificity of the test. 6.4.4 Detection of T. simiae in the midguts of experimentally infected tsetse flies using the modified dot-ELISA The dot-ELISA described for detection of T. congolense in infected tsetse guts was used for the detection of T. simiae in the same organ. Figure 44 summarizes the results of an experiment in which T. simiae parasites were detected and differentiated from T. congolense using the modified dot- ELISA. The preserved strip P, which was not run through the assay, shows the original staining of NC membrane by the gut samples. The conjugate control strip C, which was not tested with specific MoAb shows no reactions, confirming that the non-specific stains were removed. As seen on the strip tested with the T. brucei specific MoAb (KT39a), none of the tested tsetse samples reacted with this MoAb even though the specific reactivity of the antibody was clearly illustrated by its reaction with the T. brucei control. Both the Nannomonas and T. congolense specific MoAbs (KN4 and TC6) reacted specifically in the assay as shown by their reactivity with the control T. congolense and T. simiae parasite dots. Yet it was clearly shown that, whereas KN4 detected all the five infected flies, TC6 did not, thus indicating that the infections were due to T. simiae. T. simiae infections were successfully University of Ghana http://ugspace.ug.edu.gh 2 4 1 Figure 44 Detection of T. simiae CP11 in the midguts of experimentally infected G. morsitans centralis using a modified dot-ELISA. Each of the NC membrane strips was "dotted" with midgut sample preparations from, five infected flies (IF1 to IF5), and five uninfected control flies (UF1 to UF5). A control sector at the lower section of each strip was applied with lxlO5 trypanosomes per dot of in vitro propagated T. congolense K/83/IL/97/2 (TCK), T. brucei IL2616 (TB) and T. simiae KETRI 2431 (TS). The antigen applied strips were tested using Nannomonas subgenus-specific MoAb (KN4), T. congolense specific MoAb (TC6) and T. brucei species-specific MoAb (KT39a). Strip 'C' was tested as a conjugate control, and strip 'P ' was preserved without processing through the dot-ELISA. Note the successful removal of sample stains shown on strip 'P '. University of Ghana http://ugspace.ug.edu.gh Figure 44 University of Ghana http://ugspace.ug.edu.gh 24 2 detected in males and females of both experimentally infected G. m. centralis and G. pallidipes. 6.4.5 Selected method of dissecting out tsetse gut tissue, choice of sample buffer, and method of gut sample preparation Dot-ELISA assays performed using gut tissue dissected out as described in the materials and methods, indicated that, infecting trypanosome species in tsetse gut could be detected using any of the gut sections. In the preparation of tsetse gut for testing, no significant differences were found between the use of PBS or PSG as sample buffers. The release of trypanosomes from suspended tsetse gut tissue by mixing through gentle pipetting and application of samples onto NC membrane was found to be the best method. Using this method, positive reactions were strong and easily seen. The release of trypanosomes by complete disruption of gut tissue gave the strongest positive reactions. However, the method also gave high background on negative sample dots, thereby making interpretation of the results of the dot-ELISA difficult. 6.4.6 MoAbs selected for use in the modified dot-ELISA Of the four T. brucei specific MoAbs tested in the modified dot- ELISA, KT43/27 (IgG2 a) was found to be unsuitable, since it could not detect T. brucei infections in the midguts of experimentally infected Glossina. The other three MoAbs [TR7 (IgM), KT43/33 (IgGj) and KT39a (IgM)] were able to detect those infections. KT39a was selected for routine use in the detection of T. brucei in infected tsetse guts on the basis of its ability to strongly react with this parasite species in that organ Four T. congolense specific MoAbs [TC6 (IgGj), C2 (IgGj), TC39 (IgM) and TC40 (IgM)] were tested in the modified dot-ELISA for the detection of T. congolense in the midguts of infected tsetse flies. TC6, TC39 University of Ghana http://ugspace.ug.edu.gh 243 and TC40 reacted more strongly with infected samples compared to C2. Though TC6, TC39 and TC40 did equally well, TC6 was selected for use in the modified dot-ELISA. From the four Nannomonas subgenus-specific MoAbs tested, the two IgG3 MoAbs (KN4 and TC6/25) reacted more strongly with positive samples compared to the two IgGj (TC16 and KN5). Both KN4 and TC6/25 were found to be suitable for the identification of T. congolense and T. simiae infections in the midguts of infected Glossina. 6.4.7 Specificity of the modified dot-ELISA As reported under section 6.4.2.1 and 6.4.2.2, a ring-pattem reaction occurred round some uninfected samples when tested in the modified dot-ELISA using the T. brucei specific MoAb KT39a. Since the ring reactions could be differentiated from specific reactions, they did not lead to difficulty in the interpretation of the results. Hence, none of the 315 uninfected tsetse flies that were tested using the T. brucei specific MoAb KT39a gave false positive reactions in the modified dot-ELISA. Of one hundred and ten (110) uninfected flies tested using the Nannomonas subgenus-specific MoAb (KN4) and the T. congolense specific MoAb (TC6), none reacted positively in the dot-ELISA. 6.4.8 Sensitivity of the modified dot-ELISA The ability of the modified dot-ELISA to detect trypanosomes in the midguts of experimentally infected tsetse flies was 91.6% (Table 29). Further breakdown of this sensitivity into the ability to detect T. brucei, T. congolense or T. simiae are also provided in the same table. Of 95 T. brucei, 130 T. congolense and 90 T. simiae infected flies tested, 86, 111 and 85 were detected, giving sensitivities of 90.5, 85.5 and 94.4% respectively. University of Ghana http://ugspace.ug.edu.gh 244 Table 29 Detection of trypanosomes in the midguts of experimentally infected Glossina by a modified dot-ELISA Trypanosome species Number of infected flies tested Number of flies positive % positive (sensitivity) T. brucei 95 86 90.5 T. congolense 130 111 85.4 T. simiae 90 85 94.4 Total 315 282 91.6 The number of flies detected by both T. congolense and Nannomonas specific MoAbs used in the assays. University of Ghana http://ugspace.ug.edu.gh 245 In the case of the detection of T. congolense, two different MoAbs (one Nannomonas and one T. congolense specific) were used. Confirmed T. congolense infections were those that were detected by both the Nannomonas and the T. congolense specific MoAbs. As shown in Table 29, 111 out of 130 T. congolense infected flies were detected by both the Nannomonas MoAb and the T. congolense specific MoAbs, giving a sensitivity of 85.4%. However, as shown in Table 30, more of the known T. congolense infections were detected by the Nannomonas MoAb (117) compared to those detected by the T. congolense specific MoAb (111). Thus, 6 out of 130 known T. congolense infected flies were detected by the Nannomonas but not the T. congolense specific MoAb. Also, as shown in Table 30, one out of the 111 T. congolense infections detected by the T. congolense specific MoAb, was not detected by the Nannomonas specific MoAb. Investigations into the sensitivity of the modified dot-ELISA in terms of the number of trypanosomes required in test samples before they could be detected positive, revealed that, as low as 5x10s trypanosomes/ml or 1.5xl03 trypanosomes/dot could be detected in gut samples. 6.4.9 Cross-reactivity in the modified dot-ELISA Some results did raise questions about cross-reactivity. Figure 45 illustrates one such case. The three MoAbs, TC6/25 (Nannomonas specific), TC6 (T. congolense specific) and KT39a (T. brucei specific) used in this experiment were shown to react specifically with the control trypanosome dots. As shown on the strip tested with the Nannomonas specific antibody (TC6/25), four of the five infected flies were clearly positive, suggesting that those flies were infected with T. congolense or T. simiae. Three of these four flies (IF2, IF3 and IF4) were negative on the strips tested with the T. congolense specific MoAb (TC6) as well as the T. brucei specific University of Ghana http://ugspace.ug.edu.gh 2 46 Table 30 Detection of T. congolense in the midguts of experimentally infected Glossina by a modified dot-ELISA Number of flies positive when tested using: Percentage of flies detected as T. congolense T. simiae Trypanosome species Number of infected flies Nannomonas species- specific MoAbs T. congolense specific MoAbs T. congolense 130 117 111 85.4 4.6** One of the flies was detected by the T. congolense, but not the Nannomonas specific MoAb. T. congolense infections that could be mistakenly attributed to T. simiae. University of Ghana http://ugspace.ug.edu.gh 2 4 7 Figure 45 Detection of T. simiae CP813 in the midguts of experimentally infected G. morsitans centralis using a modified dot-ELISA. Each of the NC membrane strips was "dotted" with midgut samples from five T. simiae infected flies (IF1 to IF5) and five uninfected control flies (UF1 to UF5), as well as cultured control trypanosomes consisting of lxlO5 parasites per dot of T. congolense K/83/IL/97/2 (TCK), T. brucei IL2616 (TB) and T. simiae KETRI 2431 (TS). The MoAbs used were: Nannomonas subgenus- specific (TC6/25); T. congolense specific (TC6); and T. brucei species- specific (KT39a). Strip 'C ' was processed through the modified dot-ELISA as a conjugate control. University of Ghana http://ugspace.ug.edu.gh Figure 45 University of Ghana http://ugspace.ug.edu.gh 248 MoAb (KT39a), when compared with the control strip C, indicating by exclusion that those flies were infected with T. simiae. However, the sample from one fly IF5, appeared to be positive when tested with TC6 as well as KT39a, thus suggesting cross-reactivity. The results also showed that the sample from fly IF5 gave a reaction when tested on the conjugate control strip C which was not incubated with any specific MoAb, suggesting that the reactions seen on the sample dots from IF5 were non-specific. Further analysis of the results revealed that, whilst the intensity of the reactions on the sample dots from IF5 were uniform on the conjugate control, and T. congolense and T. brucei incubated strips, it was far more intense on the strip tested with the Nannomonas subgenus-specific MoAb. This indicated that despite a general background given by that sample, the Nannomonas subgenus- specific MoAb was the only one that reacted with it. 6.4.10 Stability of dotted samples Sample "dotted" NC membrane strips were stored under desiccated conditions at room temperature (19-26°C). Some of the strips were retrieved and tested for antigen potency at various time intervals with selected trypanosome species-specific MoAbs (KT39a, TC6, KN4 and TC6/25), in the modified dot-ELISA. No significant loss in assay reactivity was observed in up to 90 days of storage for any of the tested MoAbs. However, after 7 days of storage, the frequency of occurrence, as well as the intensity of the non­ specific ring reaction increased on strips tested with the T. brucei specific MoAb (KT39a). 6.4.11 Detection of T. brucei in the salivary glands of experimentally infected tsetse flies Attempts to use the dot-ELISA developed and standardized for the differentiation of in vitro derived trypanosomes, in detecting T. brucei in University of Ghana http://ugspace.ug.edu.gh 2 4 9 the salivary glands of infected tsetse flies were not successful. None of the four T. brucei specific MoAbs (KT39a, TR7, KT43/33 and KT43/27) was able to detect T. brucei (IL375) in microscopically confirmed G. m. centralis infected salivary glands, even though each of the MoAbs reacted specifically with T. brucei control parasite dots. The most likely causes of this failure to detect T. brucei in infected salivary glands, were thought to be: (1) interference due to factors such as enzymes, and (2) inability of the sample buffers (PBS and PSG) to release infecting trypanosomes from the salivary glands and promote their binding onto NC membrane in a way that they can be detected. Addition of protease inhibitors (Leupeptin and E-64) to PBS, PSG or plain distiled water, did not solve the problem. Further experimentation revealed that 5mM Na2EDTA was a suitable sample buffer for the assay. Figure 46 shows the results of one experiment in which using 5mM Na2EDTA as sample buffer, the dot-ELISA was used to detect T. brucei in the salivary glands of infected tsetse flies. The two T. brucei specific MoAbs (KT39a and KT43/33) reacted specifically with the T. brucei control parasite dots, and the conjugate control strip C showed no background. The T. brucei specific MoAb (KT39a) which was selected for diagnosing T. brucei in infected tsetse gut was able to detect 3 of the 5 infected salivary glands. In comparison, KT43/33 clearly detected all the five T. brucei infected salivary glands. As shown in Figure 46, the reactivity of KT39a on the control T. brucei parasite dot was always far stronger than that of KT43/33. The superior ability of KT43/33 to detect T. brucei salivary gland infections was confirmed in all other experiments. University of Ghana http://ugspace.ug.edu.gh 2 50 Figure 46 Detection of T. brucei IL375 in the salivary glands of experimentally infected G. morsitans centralis using dot-ELISA. Each of the NC membrane strips was "dotted" with salivary gland samples from five infected flies (IF1 to IF5) and five uninfected control flies (UF1 to UF5). A control sector at the lower section of each strip was applied with lxlO5 trypanosomes per dot of in vitro propagated T. congolense K/83/IL/97/2 (TCK), T. brucei IL2616 (TB) and T. simiae KETRI2431 (TS). The MoAbs used were: KT43/33 and KT39a, both T. brucei species-specific. Strip 'C ' was processed as a conjugate control. University of Ghana http://ugspace.ug.edu.gh Figure University of Ghana http://ugspace.ug.edu.gh 2 5 1 6.4.12 Specificity and sensitivity of the dot-ELISA used in detecting T. brucei in experimentally infected tsetse salivary glands During the development and standardization of the assay, sample dots made from some uninfected salivary glands reacted positively in the dot-ELISA. Whenever they occurred, such false positive reactions were found on all dots made from the same sample, irrespective of whether they were incubated with specific MoAb or not. The presence of such reactions on strips tested as conjugate control (without reaction with specific MoAb), indicated that the reactions were indeed not specific (background). Investigations into the cause of this background revealed that it was due to contamination of salivary gland samples with pigments originating from the gut, through spillage of gut contents during dissection. Following this finding, dissection of the salivary glands were always completed before dissection of the gut in the same fly. As a result, none of 70 uninfected salivary glands tested using the standardized dot-ELISA were positive. Of 70 T. brucei infected salivary glands that were tested, 63 reacted positively (Table 31), giving a sensitivity of 90%. 6.4.13 Detection of T. coneolense and T. vivax in the proboscides of experimentally infected tsetse flies Several methods of tsetse proboscide sample preparations were tested using dot-ELISA. The most simplified method investigated was the excision of the proboscis about a third way from the tip using a pair of scissors and subsequent dotting of fluid expelled by squeezing the thorax of the fly. Samples obtained from T. congolense or T. vivax infected tsetse proboscides using this method were, however, not successfully diagnosed using the dot- ELISA. In an attempt to reveal very weak, inconspicuous reactions that may be present, the biotin/streptavidin reaction amplification steps were incorporated into the dot-ELISA procedure. Nevertheless, none of the University of Ghana http://ugspace.ug.edu.gh 252 Table 31 Detection of trypanosomes in the salivary glands of experimentally infected Glossina by dot-ELISA Trypanosome Number of Number of flies % positive species infected flies positive by test (sensitivity) T. brucei 70 63 90 University of Ghana http://ugspace.ug.edu.gh 253 microscopically confirmed T. congolense or T. vivax infected tsetse proboscides tested, using the method, was positive. Another procedure involving the suspension of dissected tsetse mouthparts in distiled water which was expected to lyse infecting trypanosomes and release their constituent antigens, was tried. However, this method of tsetse proboscide sample preparation also failed to give positive results using known infected mouthparts. In order to ensure the release of trypanosomes from infected proboscides, tsetse mouthparts were dissected in distiled water, PBS, PSG or 5mM Na2EDTA in the wells of multitest immunofluorescence slides, and the labrum and hypopharynx cut into pieces using a small knife, and the sample fluids transferred from the wells directly onto NC membrane strips in dots. This method of sample preparation, permitted the detection of about 20% of T. congolense infected tsetse proboscides using the biotin/streptavidin amplified dot-ELISA. The use of distiled water did not give any positive results even though microscopically confirmed infected proboscides were tested. Microscopic examination of the sample remnants in multitest slide wells in which mouthparts were dissected revealed the presence of some trypanosomes, showing that not all the infecting organisms were transferred onto NC membrane for testing. Following that finding, further experiments were conducted using silicon coated multitest slides. The results were, however, not better than those obtained earlier using the uncoated slides. In order to maximise the number of trypanosomes transferred onto NC membrane for testing, each tsetse proboscis including the thecal bulb was separated from the insects head and tested. In this experiment., all the infected as well as the uninfected proboscides tested positive, irrespective of which sample buffer was used (data not shown). The loss of specificity was in part attributable to higher concentrations of biological debris originating from the massive thecal bulb. To reduce the amount of biological debris in the test University of Ghana http://ugspace.ug.edu.gh 25 4 samples without drastically reducing the number of trypanosomes available for testing, the thecal bulb was cut at a position leaving about a third of it still attached to the mouthparts, and the two thirds discarded. The mouthparts together with the third of the thecal bulb were transferred into Eppendorf tubes and disrupted with the tip of a pipette. The samples were then transferred onto NC membrane in dots and tested using the amplified dot-ELISA. The results of this experiment revealed that PBS, PSG or 5mM Na2EDTA could be used to detect T. congolense (IL1180) infections in the mouthparts of infected G. m. centralis. Using this method of sample preparation, T. vivax (IL3096) was successfully detected in the mouthparts of experimentally infected G. m. centralis (Figure 47a). Both strips were tested using the T. vivax specific MoAb KD32. The reactivity of the MoAb with only the T. vivax (TV) control antigen showed that it reacted specifically in the test. Whilst none of the uninfected proboscides was positive, the infected fly proboscides were all positive on each of the two strips. Figure 47b shows the results obtained for another experiment in which two T. congolense specific MoAbs (TC6 and TC39) were used to detect that parasite in tsetse mouthparts. Both MoAbs were shown to react specifically with the T. congolense control antigen dots (Figure 47b). Furthermore, each of the MoAbs detected clearly, four out of five infected proboscides, and none of the uninfected samples. 6.4.14 Specificity and sensitivity of the dot-ELISA used in detecting T. consolense and T. vivax in experimentally infected tsetse proboscides Using the standardized dot-ELISA, a total of 45 and 64 uninfected tsetse proboscides were tested using the T. congolense specific MoAb (TC6) and the T. vivax specific MoAb (KD32), respectively. None of University of Ghana http://ugspace.ug.edu.gh Figure 47a, b Detection of T. vivax (A) and T. congolense (B) in the mouthparts of experimentally infected G. morsitans centralis using dot-ELISA. Each infected or uninfected tsetse mouthpart was processed and placed onto NC membrane in a single dot for testing. KD32 was a T. vivax specific MoAb, whilst TC6 and TC39 were both T. congolense specific. University of Ghana http://ugspace.ug.edu.gh CM CO O * Two infected mouthparts Two uninfected mouthparts TV - TCK — TB - TS - Figure 47a CM CO Q * Five infected mouthparts Five uninfected mouthparts TCK TB - TS - Figure 47b University of Ghana http://ugspace.ug.edu.gh 2 5 6 these uninfected proboscides tested positive in the dot-ELISA, giving a specificity of 100%. The overall sensitivity of the assay was, however, lower than 50% (Table 32). Thus, of 45 T. congolense infected tsetse proboscides that were tested, 25 reacted positively, giving a sensitivity of 55.6%, whilst 28(43.8%) of the 64 T. vivax infected proboscides were detected. University of Ghana http://ugspace.ug.edu.gh 2 5 7 Table 32 Detection of trypanosomes in tbe mouthparts of experimentally infected Glossina by dot-ELISA Trypanosome Number of Number of flies % positive species infected flies positive by test (sensitivity) T. congolense 45 25 55.6 T. vivax 64 28 43.8 University of Ghana http://ugspace.ug.edu.gh 2 58 The p r im a ry a im of this particular part of the study, as indicated before, was to apply the dot-ELISA developed and standardized for the differentiation of in vitro derived insect stage trypanosomes, to the detection of T. brucei, T. congolense and T. simiae in the gut, T. brucei in salivary glands, and T. congolense and T. vivax in the proboscides of experimentally infected tsetse flies. Unlike trypanosome suspensions from in vitro cultures, which did not stain NC membrane upon dotting, tsetse gut suspensions were found to stain the membrane when applied for testing. The staining colours ranged from red through brown to black, with occasional greenish or colourless shades, depending on the state of digestion of the blood meal in the fly. These stains affected the specificity of the dot-ELISA when used for the detection and differentiation of trypanosome species in infected tsetse guts. Also, false positive reactions were encountered even in uninfected control sample dots processed with substrate and chromogen alone, ie., without incubation with MoAb or anti-MoAb HRPO-conjugate. This observation suggested that there were peroxidase-like substances in the sample dots. Such substances could utilise H202 and cause the oxidation of the chromogenic substrate (DAB), and thereby elicit positive reactions. This indeed appeared to be the case. According to Bursell (1970), haematin is split off from haemoglobin in the gut of tsetse during the early stages of digestion, but it is not absorbed from the gut. It means therefore that haematin is likely to be present in much of the digestive residue found in the tsetse gut sections that were dissected out and processed for testing. Moreover, Saunders and co­ workers (1964) reported that some haem-proteins could utilize either oxygen or hydrogen peroxide (H20 2) as a substrate and carry out oxidation, hydroxylation or oxygenation. Williams (1974) called these haem-proteins, 6.5 Discussion University of Ghana http://ugspace.ug.edu.gh 2 59 peroxidases, in accordance with earlier practice, but also indicated that they could as well be oxidases or oxygenases. Thus, the high background activity encountered with the dot-ELISA, was attributable to: (1) staining of NC membrane by pigmented gut samples, and (2) false reactivity resulting from oxidation of DAB by haem. It was reasoned, therefore, that one method of eliminating the non-specific reactions was to change the enzyme/substrate system to one that did not require the addition of free H20 2 to the solution. To achieve this, the HRPO enzyme was substituted for glucose oxidase, which made it possible to use a substrate solution in which H2O2 was substituted for glucose. This change of enzyme, however, did not substantially reduce the non-specific staining. Moreover, the use of glucose oxidase-conjugated antibodies, introduced an additional problem of higher "assay background" which made it more difficult to detect weak positive reactions. A similar contribution to high assay background has been reported for alkaline phosphotase-conjugated antibodies (Pappas, 1988b). Consequently, the use of glucose oxidase- conjugated antibodies in the dot-ELISA was abandoned. Another possibility was to degrade the haem-proteins in the test samples through a chemical process, such as oxidation, prior to analysis of the samples with the dot-ELISA. The most suitable of the oxidizing agents tried was H20 2. H20 2 is a strong oxidising agent. Earlier research by Lemberg (cited by Jackson, 1974) had shown that the first step in haemoglobin catabolism is an oxidative degradation of the molecule, which leads to separation of the haem from globin and the loss of iron. Treatment of haematin with excess H20 2 might lead to a similar degradation of the haem- protein, with accompanying loss of colour. This discovery that H20 2 could be used to decolorize pigmented samples obtained from the guts of laboratory reared tsetse flies, without affecting the integrity of trypanosome species- University of Ghana http://ugspace.ug.edu.gh 260 specific diagnostic antigens, suggests that: (1) the modified dot-ELISA that utilized this innovation, may do even better when it came to detecting and differentiating between trypanosome species in naturally infected Glossina; since such flies are known to digest their blood meals faster (Langley, 1967b) and will, therefore, present less staining problems; (2) H2O2 destaining may be potentially useful in the development of colorimetric assays for the detection of parasites in pigmented samples. Tellez-Giron, Ramos, Dufour, Alvarez and Montante (1987) reported the occurrence of a non-specific ring-pattem reaction in a dot-ELISA that they used for detecting Cysticercus cellulosae antigens in cerebrospinal fluid. There was, however, no explanation offered for the occurrence of the so-called "ring phenomenon". In this study, a similar non-specific "ring- pattem" reaction occurred on some sample dots, despite the use of H2O2 . These ring reactions did not occur on samples tested as conjugate controls, thus showing that specific MoAb played a role in their occurrence. Of the four T, brucei specific MoAbs tested with the dot-ELISA, only one (KT39a) induced the ring reactions, although not all samples tested with that particular MoAb showed the phenomenon. Despite the need to elucidate the mechanisms responsible for the ring formation, it has been shown in this work that the presence of the rings did not hamper interpretation of the results of the modified dot-ELISA. The inability to enhance the reactivity of the T. congolense specific MoAbs by lysing the infecting organisms in suspension, was attributable to two causes. Firstly, it is known that some detergents such as NP-40 could remove protein from NC membranes (Lin and Kasamatsu, 1983), suggesting that their presence in the sample suspensions could reduce the binding efficiency of the antigens. Secondly, lysing the trypanosomes, together with the mass of biological debris present in the gut suspensions, could enormously increase the competition for binding between released University of Ghana http://ugspace.ug.edu.gh 2 6 1 trypanosome antigens and other substances. The method of applying T. congolense infected gut suspensions onto NC membrane before lysing the parasites in the bound sample, circumvented the above listed problems and led to successful identification of these infections with the modified dot-ELISA. The success of this procedure could be explained by the earlier finding that the T. congolense specific MoAbs detected antigens that were not exposed on the surface of intact trypanosomes (Chapter 4). Parasite detection and identification methods intended for field use need to be simple, in addition to being specific and sensitive. Methods that reduce time spent on sample preparation are, therefore, desirable. In this study, tsetse gut samples collected by simply cutting away the distal end of the insect's abdomen and squeezing out its contents for testing in the dot-ELISA, gave results that were comparable, in terms of detection and identification of parasites, to those following the more time consuming methods of tsetse midgut dissection. The suitability of this simplified method of tsetse gut tissue extraction for the dot-ELISA, was indicated by the finding that none of the trypanosome species-specific MoAbs cross-reacted with T. grayi, which is known to reside mainly in the hindgut of tsetse flies infected with the species (Hoare, 1972). The tsetse gut sample preparation methods, employed in this dot-ELISA, afford the opportunity of analysing multiple dots of the same sample, using different species and/or subgenus-specific MoAbs. This has made it possible, for instance, to use T, congolense specific MoAbs and Nannomonas subgenus-specific MoAbs to specifically detect T. congolense infections in tsetse gut, or detect T. simiae infections in this organ in the same fly, by exclusion of T. congolense. Also, with the present dot-ELISA, it should be possible to tell whether a tsetse fly was infected with T. grayi in the gut, by excluding the presence of T. congolense, T. simiae, and T. brucei. University of Ghana http://ugspace.ug.edu.gh 2 62 Theoretically, it would be expected that all T. congolense infections should be detected by T. congolense specific MoAbs and by the Nannomonas subgenus-specific MoAbs. However, it was shown in this study that T. congolense infections could be detected by Nannomonas specific MoAbs without being detected by T. congolense specific MoAbs. It is, however, important to note that the frequency of occurrence of this discordant reaction was low (<5%) as only 6 out of 117 T. congolense infected flies were detected by the Nannomonas but not by the T. congolense specific MoAb. This inability of the T. congolense specific MoAb to detect all the T. congolense tsetse gut infections revealed by the Nannomonas MoAb, could be attributed to differences in sensitivity between the two groups of MoAbs. Another observation made in this study was that one T. congolense tsetse gut infection out of 110 (<1%) was detected by the T. congolense specific MoAbs without being detected by Nannomonas specific MoAbs in the same experiment. Two possible explanations could be offered for this unusual reactivity: Firstly the occurrence of 1 out of 110 is statistically insignificant and could be simply due to variations in test conditions, such as the amount of residual Triton X-114 or H2O2 that remained on a test strip following washings. Secondly, it might be the result of the destruction of the Nannomonas subgenus-specific antigen by factors such as enzymes in tsetse gut which nevertheless did not affect the T. congolense species-specific antigen. The likelihood of the second possibility was supported by earlier findings, that the T. congolense specific antigenic epitope(s) were of protein nature, whilst the Nannomonas specific antigenic epitope(s) were of carbohydrate nature (Chapter 4). The opportunity to test each tsetse gut sample at least 15 times using the dot-ELISA, made the technique even more suitable for the purpose of detecting and differentiating between trypanosome species in infected tsetse gut. This is because, five main species of trypanosomes (T. brucei, T. University of Ghana http://ugspace.ug.edu.gh 263 congolense, T. simiae, T. suis and T. grayi) are known to infect the gut of the Glossina species. This ability to replicate tsetse gut originated test samples, therefore, offered two additional advantages. The first was the possibility of detecting mixed infections in tsetse by employing the different trypanosome species and subgenus-specific MoAbs. The second advantage was the opportunity to utilize undigested tsetse blood meal in unused gut samples, in the identification of tsetse host, using methods such as described with the micro-plate ELISA developed by Rurangirwa, Minja, Musoke, Nantulya, Grootenhuis and Moloo (1986). In this study, a total of 315 tsetse flies experimentally infected with T. brucei, T. congolense or T simiae in the guts, were tested using the modified dot-ELISA. The sensitivity of the assays was high, as 90.5% of the tsetse infected with T brucei, 85.4% of those infected with T. congolense and 94.4% of those infected with T. simiae were correctly identified. This gave an overall sensitivity of 91.6% for detecting and differentiating between trypanosome species in the guts of experimentally infected tsetse flies. The specificity of these assays was greater than 99.9%. Decreased reactivity of the T. congolense specific antigen was recorded when culture-derived vector stage trypanosomes were applied and stored on NC membrane at room temperature (17-26°C) for more than 60 days (Chapter 5). However, no significant loss in reactivity was observed in up to 90 days of storing trypanosome infected tsetse gut samples under similar conditions. This stability of the antigens, introduces a degree of flexibility in this test, since the collected samples need not be analysed at once. The T. brucei specific MoAb (KT39a) which was used for detecting this parasite in infected tsetse gut, was not used for the detection of the parasite in the salivary glands of the vector. This was because, another T. brucei specific MoAb (KT43/33) performed better and was, therefore, selected. This difference in the performance of the two MoAbs was explained University of Ghana http://ugspace.ug.edu.gh 2 64 as follows. It was found in Chapter 4 of this thesis that, KT39a bound to an antigenic epitope which was only partially sensitive to proteinase-K and which was located on a 90 kDa antigen peptide. On the other hand, KT43/33 bound to an antigenic epitope which was completely susceptible to proteinase-K and which was located on an antigen which localized in a series of peptide bands ranging between 21 and 47 kDa. These findings suggested that the T. brucei species-specific antigens detected by the two MoAbs (KT39a and KT43/33), were indeed different. It is, therefore, possible that the T. brucei specific antigens detected by the two MoAbs are expressed in different quantities in the different life cycle stages of the parasite. If this were the case, then the antigen detected by KT39a was better expressed in the procyclic stages of the parasites which predominate in the vector's midgut, whilst the antigen detected by KT43/33 was better expressed in the epimastigote and metacyclic stages in the vector's salivary glands. Another possible cause of the differences in the performance of the two MoAbs, was differential susceptibility of the specific antigens to degradative enzymes that may be present in the tsetse salivary glands. As shown in the case of the identification of trypanosomes in the guts of infected tsetse flies, the sensitivity of the dot-ELISA for the detection of T. brucei in infected salivary glands was high (90%). The specificity of the assay was greater than 99.9%. Furthermore, the opportunity to test each pair of tsetse salivary glands at least three times, indicated that if the need arose, and MoAbs were made against T. suis which can also infect the vector's salivary glands, then this parasite could also be tested for alongside T. brucei in the dot-ELISA. It has also been shown in this work that both T. congolense and T. vivax could be detected in the proboscides of infected tsetse flies. However, the sensitivity of the dot-ELISA in detecting these two trypanosome species in the target organ was low (55.6% for T. congolense and 43.8% for University of Ghana http://ugspace.ug.edu.gh 2 6 5 T. vivax). This low detection rate of trypanosomes in the proboscis of Glossina, might be related to three factors: (1) the relative numbers of T. congolense or T. vivax parasites present in the proboscis at a certain point in their life cycles in the vector; (2) difficulties in releasing trypanosomes from the proboscis as a result of anatomical peculiarities of that organ, and (3) differences in sensitivity of the trypanosome species-specific MoAbs employed in the study. The inability to test each tsetse proboscis more than once, is clearly a drawback. One way to circumvent this limitation is to randomly place suspected tsetse proboscides into two groups, and to test one group with T. vivax specific MoAb and the other with T. congolense specific MoAb. From this study, it could be concluded that the dot-ELISA is potentially a practical method for the diagnosis of trypanosome infections in tsetse flies. However, in order to determine the full potential of this technique, there was the need to investigate its applicability in the field. This was done, and the results obtained were recorded in the next chapter. University of Ghana http://ugspace.ug.edu.gh CHAPTER 7 266 FIELD EVALUATION OF A DOT-ELISA DEVELOPED FOR THE DETECTION AND DIFFERENTIATION OF TRYPANOSOME SPECIES IN INFECTED TSETSE FLIES (GLOSSINA SPP.) University of Ghana http://ugspace.ug.edu.gh 2 6 7 A rapid, visually read, dot-ELISA developed for the detection and differentiation of trypanosome species in tsetse flies (Glossina spp.), was field tested alongside the standard fly dissection method on a ranch in south eastern Kenya. Of a total of 104 G. pallidipes dissected, two were found to be infected with trypanosomes in their midguts. By the dissection method the infecting trypanosome species could not be identified, as both flies were free from salivary gland infections. However, using the dot-ELISA, the two flies were shown to be infected with T. congolense in their midguts. The midguts of an additional 6(5.8%) of the 104 G. pallidipes tested positive for T. congolense in the dot-ELISA, even though no trypanosomes were seen on dissection. The infection rate for this fly species as determined using the dot- ELISA, therefore, was 7.7% for T. congolense in midgut infections compared to 1.9% identified by fly dissection. The salivary glands and mouthparts of the 6 additional tsetse flies identified by dot-ELISA, were all negative as determined by the two techniques. None of 390 G. longipennis flies dissected and examined for trypanosomes in the midgut, salivary glands and mouthparts was shown, by this method, to be infected. Using the dot-ELISA, however, 17(4.4%) of the flies tested positive for T. congolense in the midguts, whilst the salivary glands and mouthparts of the same flies were negative. Thus, the dot-ELISA appears to be more sensitive than the fly dissection method under field conditions. Moreover, the dot-ELISA was performed in the field without electricity. It was simple to perform, and was not affected by high ambient temperatures (22-32°C), or by contamination of reactants with dust. 7.1 Summary University of Ghana http://ugspace.ug.edu.gh 2 68 The tsetse dissection method, first introduced by Lloyd and Johnson (1924), is the method used in routine epidemiological surveys to determine trypanosome infection rates in Glossina. This is still the case even though a recombinant DNA technique was introduced by Kukla and colleagues (1987), for the diagnosis of trypanosome infections in the tsetse fly. The reason for this is partly because, the present DNA probes cannot recognise all the intra-species variants of targeted trypanosome species. Secondly, the DNA technique is not simple enough to be performed in most laboratories. In the studies recorded in this Chapter, the dot-ELISA developed for differentiating between in vitro propagated trypanosome species (Chapter 5), and which was successfully modified for detecting and differentiating between infecting trypanosome species in the mouthparts, salivary glands or midguts of experimentally-infected Glossina species (Chapter 6), was evaluated in the field for the diagnosis of natural trypanosome infections in the vector. It is shown here, that the dot-ELISA developed hereto, is capable of detecting and identifying infecting trypanosome species in naturally infected tsetse flies when the assay was performed under field conditions. 7.2 Introduction University of Ghana http://ugspace.ug.edu.gh 269 7.3.1 The dot-ELISA kit 7.3.1.1 Nitrocellulose CNC) membrane template Lines were drawn on NC membrane sheets to form a grid consisting of square and rectangular shaped areas as shown in Figure 48. The columns representing the outlines of demarcated strips were numbered, and in vitro propagated procyclic T. congolense (TCK), T. brucei (TB) and T. simiae (TS), and epimastigotes of T. vivax (TV) were applied, lxlO5 trypanosomes/dot onto each of the demarcated NC membrane strips as shown in Figure 48, to provide the controls. The NC membrane sheets were sealed in polythene bags and transported to the field for use. 7.3.1.2 Materials 7.3 Materials and methods 1. Two Bio-Rad slot incubation trays 2. One 500ml plastic measuring cylinder 3. Two 500ml plastic beakers 4. One rubber pipette aid 5. 10ml plastic pipettes 6. 0.5 to 10/d adjustable pipette and pipette tips 7. 1.5ml Eppendorf tubes 8. Multitest IF A slides. 7.3.1.3 Chemicals, reagents and buffers Chemicals, reagents and buffers, enough for screening at least 1,000 tsetse flies, were transported to the field. These were: University of Ghana http://ugspace.ug.edu.gh 2 7 0 Figure 48 A nitrocellulose membrane grid showing demarcated spaces for sample application, TCK = lxlO5 trypanosomes/dot of T. congolense Kilifi type culture procyclics. TB = lxlO5 trypanosomes/dot of T. b. brucei culture procyclics. TS = lxlO5 trypanosomes/dot of T. simiae culture procyclics. TV = lxlO5 trypanosomes/dot of T. vivax culture epimastigotes. University of Ghana http://ugspace.ug.edu.gh Figure 48 < CO 03 O Tv * * I * o >^1 - I - o o o o - o o o o I\3 o o o o CO o o o o -1^ o o o o 01 o o o o CD o o o o -J o o o o 00 o o o o CD o o o o o o o o o o o o o N> o o o o CO o o o o ■f* o o o o cn o o o o O) o o o o ~sl o o o o 00 ro 03-o3 University of Ghana http://ugspace.ug.edu.gh 2 7 1 1. 5Qmg weights of 3-3'diaminobenzidine (DAB) in 1.5ml Eppendorf tubes, each enough for assaying samples from 100 tsetse flies 2. 5g weights of skimmed milk in sealed polythene bags, each enough for assaying samples from 50 tsetse flies 3. 1ml of concentrated hydrochloric acid 4. 50ml of 30% hydrogen peroxide (H20 2) 5. Aliquots of trypanosome species or subgenus-specific MoAbs and goat anti-mouse horseradish peroxidase (HRPO)-conjugated antibodies kept on ice in thermos flasks. 6. 100ml of phosphate buffered saline, pH 7.4 7. 100 ml 5mM Na2EDTA buffer 8. 1.5 litres of xlO concentrated Tris buffered saline, pH 8. 9. 2ml of Triton X-114 10. 10 litres of deionised water 11. 200ml of Phosphate/Na2EDTA buffer 7.3.2 The dissecting kit 7.3.2.1 Equipment 1. Dissection microscope 2. Compound microscope 7.3.2.2 Other materials 1. Microscope slides and cover slips 2. Kit of dissecting instruments 3. Phosphate buffered saline, pH 7.4, listed under 7.3.1.3 University of Ghana http://ugspace.ug.edu.gh 272 7.3.3 Study area and trapping of tsetse flies 7.3.3.1 Study area Field evaluation of the dot-ELISA was conducted at the Galana Ranch which covers an area of approximately 6000 km2 in the coastal hinterland of Kenya. The mean altitude is 270 m, with an average annual rainfall of 550 mm (Wilson, Gatuta, Njogu, Mgutu and Alushula, 1986). The vegetation consists of riverine thickets along the Galana river, and thick coastal bush in the east which gives way to grasslands and scattered thickets in the west. Four species of tsetse have been identified on this ranch, inhabiting different ecological zones that make up to 35% of the area of the Ranch. The tsetse species were: Glossina longipennis in the drier savannah areas, away from the river; G. austeni, restricted to the river-bed; G. brevipalpis, in forested areas in the east; and G. pallidipes in thickets along the river and in the east. 7.3.3.2 Trapping of wild tsetse flies Male and female G. longipennis and G. pallidipes flies were trapped from their natural habitats using the F4 and biconical traps, respectively. The F4 traps (Figure 49) were set up in the late afternoon at about 4.00 pm in the grassland areas infested by G. longipennis and emptied the following day at about 9 am. Biconical traps (Figure 50) were also set up around 4.00 pm, in the G. pallidipes infested areas, in the east of the Ranch, and emptied the following day at about 4.00 pm. 7.3.4 Experimental design Tsetse flies were killed by anaesthesia using chloroform, and sorted into groups according to species. The flies were also separated into tenerals and non-tenerals. The teneral flies were discarded. University of Ghana http://ugspace.ug.edu.gh 273 Figure 49. F4 trap used for catching G. longipennis in the savannah areas at the Galana Ranch. University of Ghana http://ugspace.ug.edu.gh 2 7 4 Figure 50. Biconical trap used for catching G. pallidipes in the thickets in the west of the Galana Ranch. University of Ghana http://ugspace.ug.edu.gh 2 75 As shown in Figure 51 for G. pallidipes, each species of tsetse was first separated on the basis of sex. The flies in either sex (male and female) were subsequently sorted randomly, each into two groups; M l and M2 for the males and; FI and F2 for the females (Figure 51). The males in group M l were then added to the females in group FI to form batch 1 tsetse flies whose mouthparts were dissected and examined microscopically, and tested with a T. vivax specific MoAb (KD32/48.17) using the dot-ELISA described in Chapter 6. The mouthparts of the second batch of flies consisting of flies from M2 and F2, were also dissected, but tested with a T. congolense specific MoAb (TC6/42.6.3) in the dot-ELISA. The midgut and salivary glands of all the flies were also dissected and examined microscopically for trypanosomes and also tested using the dot- ELISA as described in Chapter 6. 7.3.5 Dot-ELISA procedure Tsetse proboscide, midgut and salivary gland samples were prepared and applied onto the NC membrane template (Figure 48) following the procedure described in Chapter 6, and the NC membrane cut out into strips. The dot-ELISA procedures were also as previously described (Chapter 6, section 6.3.7) except that the shaking of the slot incubation trays was accomplished manually. University of Ghana http://ugspace.ug.edu.gh Figure 51 Sorting G pallidipes into two batches, for testing for infecting trypanosomes in the mouthparts. Flies in 'Batch 1' were tested with a T. congolense specific MoAb, whereas flies in 'Batch 2' were tested with a T. vivax specific MoAb. University of Ghana http://ugspace.ug.edu.gh G. pa/I/c/fpes Males Females Males(M1) Males(M2) Females(FI) Females(F2) 1 * ' l ! ' s M1 + F1 = Batch 1 M2 + F2= Batch 2 University of Ghana http://ugspace.ug.edu.gh 2 7 7 7 .4 . 1 Detection of trypanosome infections in tsetse flies using the dissection method The mouthparts, salivary glands and midguts of 104 G. pallidipes (44 males and 60 females), and 390 G. longipennis (154 males and 236 females) were dissected and examined for trypanosomes. Two of the G. pallidipes flies (1.9%), both of them females, were found to be infected with trypanosomes in the midguts (Table 33), and no trypanosomes were seen in the mouthparts or salivary glands of the same flies. Trypanosomes were not detected in any of the G. longipennis. 7.4.2 Detection of trypanosome species in tsetse flies using the dot- ELISA The mouthparts, salivary glands and midguts of all the dissected tsetse flies were also tested using the dot-ELISA. Of the 104 G. pallidipes that were tested, 8(7.7%) were positive for T. congolense in the midgut (Table 33). These included the two flies which were determined by the dissection method to be infected with trypanosomes in their midguts. Gut samples from all the 8 flies reacted positively with the T. congolense specific MoAb as well as with the Nannomonas subgenus-specific MoAb (Table 34). No T. vivax or T. brucei antigens were detected in any of the organs that were tested. Out of the 390 G. longipennis tested using the dot-ELISA, 17(4.3%) were positive for trypanosome infections in the midguts (Table 33). Nine of those 17 midguts gave positive reactions with T. congolense and the Nannomonas specific MoAbs (Table 35), whilst the remaining 8 midguts tested positive with only the T. congolense specific MoAb (Table 35). 74 Results University of Ghana http://ugspace.ug.edu.gh 2 78 Table 33 Detection of trypanosome infections in the midguts of G. pallidipes and G. longipennis using the dissection and dot-ELISA techniques Tsetse species Number tested Dissection Number (% positive) Dot-ELISA Number (% positive) G. pallidipes 104 2(1.9%)* 8(7.7%) G. longipennis 390 0 17(4.3%) Total 494 2(0.4%) 25(5.1%) Infections detected only in the midgut by the fly dissection method. University of Ghana http://ugspace.ug.edu.gh 2 7 9 Table 34 Detection and differentiation of trypanosome species in the midguts of G. pallidipes using the dot-ELISA Reactivity of tsetse midguts with different Tsetse flies trypanosome species-specific MoAbs detected b y -------------------------------------------------------------------------- assay T. congolense T. brucei Nannomonas T. vivax 1 + + - 2 + + - 3* + + - 4 + + 5* + - + 6 + + 7 + + 8 + + * Tsetse flies with infected midguts as determined by the dissection method. University of Ghana http://ugspace.ug.edu.gh 2 80 Table 35 Detection and differentiation of trypanosome species in the midguts of G. longipennis using the dot-ELISA Reactivity of tsetse midguts with different Tsetse flies trypanosome species-specific MoAbs detected by ------------------------------------------------------------------------ assay T. congolense T. brucei Nannomonas T. vivax 1 + + 2 + - + - 3 + + - 4 + + - 5 + + - 6 + + - 7 + + - 8 + + - 9 + - + - 10 + - 11 + - - - 12 + - 13 + - 14 + - 15 + - 16 + - 17 + - University of Ghana http://ugspace.ug.edu.gh 2 8 1 7.4.3 Problems encountered, and special observations made during tl performance of the dot-ELISA under field conditions In the laboratory, all incubations in the dot-ELISA were made slot trays under continuous shaking on an electrical powered gentle rocke Under field conditions, the unavailability of electrical power necessitate improvisation. Consequently, shaking of the slot trays during the varioi incubation steps in the first dot-ELISA performed in the field w; accomplished manually by tilting the trays in a rocking motion continuous] for 1-2 min, and then allowing the trays to stand for 15 min before repeatiri the shaking. The reactivity of the specific MoAbs on the control trypanoson: dots included in the test were, however, weaker than those obtained in tl laboratory using the electric rocker. In order to eliminate the effect c interrupted shaking on the reaction intensities, an improvised technique we tested. The improvisation consisted of suspending the incubation trays on rope that was tied to the branch of a tree, and swinging to emulate the simp] pendulum motion (Figure 52). In addition, the trays were rocked manually a before. This procedure increased the intensity of the reactions in the doi ELISA. University of Ghana http://ugspace.ug.edu.gh 2 8 2 Figure 52. Field improvisation of a rocker during incubations. Note the suspended tray. University of Ghana http://ugspace.ug.edu.gh 283 The aim of the studies conducted in this Chapter was to evaluate the applicability of the dot-ELISA in terms of its ability to detect and identify trypanosome species involved in natural infections in the vector, when the assay was performed under field conditions. The field conditions under which the dot-ELISA was evaluated, constituted a realistic situation likely to be encountered in other tsetse infested areas. The NC membrane strips, applied with control trypanosomes, and the reagents and buffers proved to be stable over the period of one week that they were used in the field. The presence of strong winds, coupled with the abundance of sandy dust, brought about contamination of reagent solutions with dust and plant debris during the performance of the assays. However, this contamination, together with the high ambient temperatures (22 to 31°C) prevailing during the assays, did not adversely affect the results. This indicated that the dot-ELISA was robust as far as conditions such as temperature and dust were concerned. In this study, only 2 midgut infections were detected in a total of 494 tsetse flies that were screened using the dissection method, on the Galana Ranch. However, in an earlier study conducted on the same ranch by Wilson, Gatula, Njogu, Mgutu and Alushula (1986), T. vivax, T. congolense and T. brucei infection types were all detected in tsetse flies, using the dissection method, with T. vivax infections being the most common. The most likely explanation for these differences in observations made in the present study and that of Wilson et al, (1986), is that, the tsetse and trypanosomiasis control programme being implemented on the ranch (Opiyo, Njogu and Omuse, 1990) may have altered the status of trypanosome infections and transmission. This field study revealed that the trypanosome infection rate in G. pallidipes was higher than that in G. longipennis, regardless of the technique 7 .5 Discussion University of Ghana http://ugspace.ug.edu.gh 284 used. This finding was in agreement with an earlier report which showed that G. pallidipes was the major vector of trypanosomiasis on the Galana Ranch (Opiyo, Dolan, Njogu, Sayer and Mgutu, 1987). The identity of the trypanosome species involved in the two midgut infections which were detected in G. pallidipes could not be determined using the dissection method, since there were no accompanying infections in the mouthparts or salivary glands of the two flies. Using the dot-ELISA, however, it was possible to identify T. congolense as the parasite species involved, since the parasites reacted with both the Nannomonas and the T. congolense specific MoAbs. Furthermore, it was possible to ascertain the absence of T. brucei in those infections. Unfortunately, at the time that this study was conducted, the T. simiae specific MoAb reported in Chapter 4 had not been derived, and was not, therefore, included in the evaluation. It was also found that the dot-ELISA detected T. congolense antigens in the midguts of some parasitologically negative flies. This was a new observation that could be attributed to three possible causes, namely: (1) correct identification of infected tsetse by the dot-ELISA as a result of differences in sensitivity between the two tests, (2) false detection of uninfected tsetse due to reactivity of MoAbs with circulating T. congolense antigens present in the blood meal ingested from infected hosts, and (3) false detection of uninfected tsetse due to reactivity of MoAbs with trypanosome antigens originating from ingested whole bloodstream form trypanosomes that were unable to establish an active infection. However, this observation would appear to indicate that the dot-ELISA had a higher sensitivity compared with the tsetse dissection method. A statistical comparison of the dissection technique with the dot-ELISA using Chi-square analysis, indicated that the two tests were significantly different in their ability to detect trypanosomes in tsetse midgut, at P< 0.05. This possibility of diagnosing trypanosome infections in the midguts of tsetse flies, even though no infecting parasites are seen using University of Ghana http://ugspace.ug.edu.gh 2 8 5 the dissection method, has not been indicated or discounted in the use of the recombinant DNA technique. This is simply because, in all the studies that the recombinant DNA technique was tried in the field, only flies shown by the dissection method to be infected with trypanosomes, were tested. It was also interesting to note that gut samples from some of the parasitologically negative tsetse flies could react with the T. congolense specific MoAb without reacting with the Nannomonas subgenus-specific MoAb in the dot-ELISA. Three likely explanations could be offered for this unusual reactivity. Firstly, it could be due to differences in the levels of biochemical substances such as enzymes which are able to affect the integrity of the epitope on the Nannomonas subgenus-specific antigen, without affecting the T. congolense specific antigen. If this were true, it would mean that the species of tsetse may be an important factor, since the observation was associated with G. longipennis, but not G. pallidipes. Secondly, it could be that the G. longipennis were infected with different T. congolense variants, some of which may not be expressing the Nannomonas subgenus-specific antigen detected by the MoAb. This possibility is however unlikely, since the Nannomonas specific MoAb was able to react with T. congolense organisms isolated from different geographical areas (Chapter 4). Thirdly, the T. congolense specific MoAb could be cross-reacting with unidentified antigens present in the tsetse gut. However, this possibility was also thought to be unlikely, since in extensive studies with experimental tsetse flies (Chapter 6), no cross-reactivity was observed. The results obtained from this limited evaluation of the dot-ELISA clearly showed that the technique was a practical alternative to the dissection method which is currently employed in the diagnosis of trypanosome infections in Glossina species. This is especially so, considering the fact that each of the MoAbs used had been shown to react with various trypanosome isolates from different geographical areas. Besides, the materials required can easily be University of Ghana http://ugspace.ug.edu.gh 2 8 6 transported to field locations, and the assay could be performed without the need for electricity. The test is rapid, simple to perform, and will be inexpensive in screening large numbers of tsetse flies at a time. Moreover, it is specific and detects more trypanosome infections in field caught tsetse flies in comparison with the dissection method. These are strong indications that the dot-ELISA could contribute greatly to studies aimed at further elucidating the role played by the tsetse fly in the epidemiology of the African trypanosomiases. University of Ghana http://ugspace.ug.edu.gh CHAPTER 8 287 GENERAL DISCUSSION AND CONCLUSIONS University of Ghana http://ugspace.ug.edu.gh 288 Since the discovery that tsetse flies were the main vectors of the African trypanosomiases, attempts have been made to reduce the disease prevalence by vector control and by the use of trypanocidal drugs. These methods have, however, been expensive and relatively unsuccessful. As a result, vast areas of land are still infested with tsetse flies, while areas previously cleared of the fly are prone to reinfestation (MacLennan, 1981). This, together with the emergence of drug resistant strains of trypanosomes (Kupper and Wolters, 1983; Pinder and Authie, 1984), reveals the true extent of the threat posed by trypanosomiasis. Estimation of the trypanosomiasis risk or challenge to domestic animals or humans, requires the determination of several factors, including tsetse relative density, the proportion of blood meals taken from target hosts, and trypanosome infection rates in tsetse (Lloyd and Johnson, 1924; Challier and Laveisiere, 1973). Assessment of these parameters is currently important, as sites are investigated for trypanosomiasis risk in relation to productivity of trypanotolerant breeds (Leak et al., 1988). The diagnosis of trypanosome infections in the tsetse fly, at present, is by dissection. This method, however, can only be used to identify the parasites up to the subgeneric level (Hoare, 1972; Stephen, 1986; McNamara and Snow, 1991). It is, therefore, essential to develop a more accurate method that is capable of detecting and differentiating between trypanosome species in infected tsetse flies. In this thesis, the suitability of a MoAb-based approach was investigated. Several species of parasitic protozoa (including Plasmodia spp., Leishmania spp., T. cruzi and Trypanosoma spp.) possess species and/or subgenus-specific antigens (Santoro, Cochrane, Nussenzweig, Nardin, Nussensweig, Gwardz and Ferreira, 1983; Flint, Schechter, Chapman and Miles, 1984; McMahon-Pratt, Bennet and David, 1982; Parish, Morrison and University of Ghana http://ugspace.ug.edu.gh 289 Pearson, 1986; Nantulya et al., 1987). In the case of the Trypanosoma, this had been shown by the generation of MoAbs that are specific to the T. congolense species, and the Nannomonas, Trypanozoon and Duttonella subgenera. However, no MoAbs specific to T. simiae or any of the subspecies that constitute the Trypanozoon subgenus had been produced. In studies described in Chapter 4, a MoAb specific to T. simiae (KNS7/14.X) was produced using spleen cells from a BALB/c mouse that had been immunized with purified T. simiae antigens obtained using the purification procedure described by Ijagbone et al., (1989). The production of a T. simiae specific MoAb has shown for the first time that both trypanosome species within the Nannomonas subgenus (T. congolense and T. simiae), possess immunogenic species-specific antigens that could be used to differentiate between them. The potential usefulness of the trypanosome species-specific MoAbs as diagnostic reagents was demonstrated through their reactivity with vector stages of the parasites that had been isolated from different geographical areas (Chapter 4). In those experiments, it was recorded that the majority of the MoAbs could also detect the bloodstream forms of the target parasite species, indicating that some of the MoAbs may also be useful in the diagnosis of trypanosomiasis in the mammalian host. A similar observation was made by Nantulya and co-workers (1987) for MoAbs that they produced against membrane antigens of procyclic trypanosomes, and which were later utilized in developing diagnostic assays for detecting circulating bloodstream trypanosome antigens in both infected humans and animals (Nantulya and Lindqvist, 1989; Nantulya, 1989; Nantulya, Doua and Molisho, 1992). Since it had previously been established that procyclic tsetse midgut forms and culture forms of the African trypanosomes express similar antigens (Richardson et a l , 1986; Pearson, Moloo and Jenni, 1987), efforts were first initiated to establish a simple, sensitive and specific MoAb-based assay that could detect and differentiate between culture derived T. brucei, T. congolense University of Ghana http://ugspace.ug.edu.gh 290 and T. simiae procyclics, and T. vivax epimastigotes. The NC membrane- based dot-ELISA was the preferred choice. The reason for this was that, this technique had been shown to be simple, sensitive, specific and field portable (Pappas, 1988a). Ensuing experiments led to the development and standardization of a simple, sensitive and specific NC membrane-based dot- ELISA (Chapter 5) that utilized trypanosome species-specific MoAbs in the detection and differentiation of in vitro propagated forms of the aforementioned trypanosome species. In these experiments, the dot-ELISA correctly identified the trypanosome species in both single and artificially mixed trypanosome populations. This finding showed that the trypanosome species present in artificial cultures could be ascertained using the dot-ELISA. This was considered important since culture derived trypanosomes were becoming increasingly utilized in trypanosome research (Ross and Taylor, 1990). Hence, there was the need for simple reliable techniques that could be used in identification and confirmation of the propagated trypanosome species. The finding also indicated that the assay might be suitable for detecting and differentiating between trypanosome species in infected tsetse flies. Genotypic diversity had been recorded among members of the Nannomonas subgenus, especially the T. congolense species (Majiwa, Masake, Nantulya, Hamers and Matthyssens, 1985; Majiwa, Hamers, van Meirvenne and Matthyssens, 1986). As a result of this diversity, efforts aimed at producing a DNA probe that could hybridize to all the intra-species variants of T. congolense, have been unsuccessful. As reported in Chapter 4, the T. congolense specific and Nannomonas subgenus-specific MoAbs reacted with all the T. congolense isolates including the Savannah, Kilifi and riverine-forest types that were tested. This result showed that despite the reported genotypic differences, these T congolense genotypes were closely related antigenically, thus University of Ghana http://ugspace.ug.edu.gh 2 9 1 suggesting that the MoAb approach to diagnosis of trypanosome infections in Glossina may have an advantage over the recombinant DNA technique. The gut of an infected tsetse fly contains trypanosomes as well as digestive residue originating from ingested blood meal. The digestive residue is normally pigmented, mostly by haematin which is split off the haemoglobin molecule early in the digestion of tsetse blood meal, but which is not absorbed (Bursell, 1970). Efforts to test tsetse gut samples for the presence of infecting trypanosomes using the NC membrane-based dot-ELISA (Chapter 5) encountered non-specific background. This background was attributable to two likely causes (Chapter 6); namely, (1) staining of NC membrane by pigmented gut samples, and (2) false reactivity resulting from the oxidation of the chromogenic substrate (DAB) by haem. The non-specific stains were removed using hydrogen peroxide (H2O2 ) as a destaining agent in a modified dot-ELISA, without any significant effect on the integrity of the trypanosome species-specific diagnostic antigens (Chapter 6). This ability of H20 2 to decolorize NC membrane applied pigmented gut samples was attributed to its (H20 2's) oxidative degradation of haem (Jackson, 1974), and of haematin (Chapter 6). It should be of interest to further investigate the ability of H20 2 to decolorize NC membrane applied infected gut samples as well as faecal material obtained from other haematophagous arthropod vectors, such as the Triatomine bugs that transmit T. cruzi. This could lead to further development of this H20 2 innovation and allow maximum utilization of colorimetric assays, which are generally simple and can be read visually (without the aid of sophisticated equipment). The modified dot-ELISA was shown to be capable of identifying T. brucei, T. congolense and T. simiae organisms in the guts of experimentally infected tsetse flies. The assay had an overall sensitivity greater than 90 percent and a specificity greater than 99.9 percent. This successful detection of trypanosome species in the guts of laboratory-colonized tsetse flies, University of Ghana http://ugspace.ug.edu.gh 292 suggested that the modified dot-ELISA would be even more readily applied in the detection of trypanosome species in wild tsetse flies which are known to digest their blood meals faster (Langley, 1967a). Two main trypanosome species (T. brucei and T. suis) are known to infect the salivary glands of the Glossina species (Hoare, 1972; Mulligan, 1970). In this study, a T. brucei species-specific MoAb was employed in the dot-ELISA for the identification of T. brucei in experimentally infected tsetse salivary glands (Chapter 6). However, the assay could not detect the parasites in the target organ when PBS or PSG were used as sample buffer, even though these buffers were the preferred choice for the preparation of tsetse gut samples for testing with the dot-ELISA (Chapter 6). This observation was explained as below: Tsetse saliva is known to contain several enzymes and inhibitors including an antithrombin (Parker and Mant, 1979) and fibrinolytic proteases (Endege et al., 1989). These enzymes and inhibitors were believed to affect trypanosome antigens in salivary glands during sample preparation, leading to interference with the dot-ELISA. Investigations conducted into the use of alternative sample buffers showed that 5mM Na2EDTA was suitable for treating tsetse salivary glands prior to testing with the dot-ELISA (Chapter 6). The successful use of Na2EDTA for this purpose, was explained as follows. Na2EDTA is an enzyme inhibitor. Its use in the sample buffer may, therefore, have been necessary to inactivate salivary gland enzymes that affected the T. brucei diagnostic antigen or interfered with the reaction between specific MoAb and the target antigens. Trypanosoma suis had been found on only three separate occasions since its discovery (Stephen, 1986). As a result, suitable antigens of this parasite could not be obtained for immunization and production of specific MoAbs to this species. However, the opportunity to replicate test samples from tsetse salivary glands for testing with the dot-ELISA, showed that should the need arise and specific MoAbs made against T. suis, this parasite could University of Ghana http://ugspace.ug.edu.gh 293 also be tested alongside T. brucei using the dot-ELISA. The sensitivity of the dot-ELISA in detecting T. brucei parasites in the salivary glands of tsetse was high (90%), and the specificity was greater that 99.9%, with no cross­ reactivity recorded. The overall sensitivity of the dot-ELISA in detecting trypanosomes in the mouthparts of experimentally infected Glossina was low, less than 50% (Chapter 6). This low detection rate was believed to be due to the relatively low number of trypanosomes present in infected proboscides as well as to difficulties in releasing trypanosomes from the proboscis as a result of anatomical peculiarities of this organ. The specificity of the assay was, however, high (greater than 99.9%). Natural trypanosome infection rates in Glossina determined by dissection are usually low, less than 10% as reported by Jordan (1974). Evaluation of the dot-ELISA in the field, revealed similarly low trypanosome infection rates (Chapter 7). In this field study, two tsetse flies that were identified by dissection as infected with trypanosomes in the midguts, were also positive by the dot-ELISA (Chapter 7). Using the dissection technique, such infections were attributable to immature T. brucei or T. congolense or T. simiae (Lloyd and Johnson, 1924). However, using the dot-ELISA, it was possible to tell that those two flies were infected with T. congolense (Chapter The sensitivity of the dissection method had been shown to be below 100% by many investigators. For example, Ward and Bell (1971) performed experiments with the animal sub-inoculation method of revealing mature trypanosome infections in the vector, and reported that feeding T brucei infected tsetse flies individually on mice gave transmission rates that were about five times higher than those revealed by salivary gland dissections. Also, Moloo and Kutuza, (1974), used the animal sub-inoculation method to incriminate G swynnertoni as the vector of sleeping sickness in an area in University of Ghana http://ugspace.ug.edu.gh 294 Tanzania, where attempts to find the vector of the disease using the dissection method had failed. In a field evaluation (Chapter 7), the dot-ELISA detected T congolense antigens in the guts of tsetse flies that had been assumed to be uninfected as determined using the dissection technique. This high detection rate by the dot-ELISA, could be attributed to superior sensitivity of the technique as compared with the dissection method, thus suggesting that the dot-ELISA could be a better choice for revealing the true extent of the vectoral capacity of the Glossina species. In conclusion, this study provides useful information on the suitability of MoAbs as diagnostic reagents for detecting and differentiating between the vector stages of the African trypanosomes. It also provides evidence that a MoAb-based dot-ELISA could be employed as a practical alternative to the dissection technique which is currently used for diagnosis of trypanosome infections in Glossina species. University of Ghana http://ugspace.ug.edu.gh 2 95 REFERENCES Agu, E.W. (1984). 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