Thesis (MSc (Veterinary Science))--University of Pretoria, 2004. / Includes bibliographical references.

Thesis (MSc (Veterinary Science))--University of Pretoria, 2005. / Includes bibliographical references.

The protozoan parasites Trypanosoma congolense and T. vivax cause trypanosomosis in cattle. The major lysosomal cysteine proteinase of T. congolense, congopain, may contribute to pathogenesis of the disease, and antibody-mediated inhibition of this enzyme may contribute to mechanisms of trypanotolerance. Oligopeptidase B, a trypanosomal serine peptidase, is also a potential virulence factor in African trypanosomes because it is released into the host circulation by dead or dying parasites, where it retains catalytic activity due to the enzyme's insensitivity to serum protease inhibitors. The vaccine potential of the catalytic domain of congopain, C2, and oligopeptidase B complexed with 0'2-macroglobulin (0'2M) was evaluated by producing antibodies in rabbits. Inhibition of congopain and oligopeptidase B activity by these antibodies was assessed. The oligopeptidase B open reading frame from T. congolense and T. vivax was cloned and expressed in Escherichia coli, from which active recombinant enzymes were purified. These recombinant enzymes exhibited trypsin-like specificity for peptide substrates, cleaving on the carboxy side of basic amino acid residues such as arginine and lysine. Enzymes were found to be optimally active between pH 8 and 10, optimally stable at pH 6, and showed activation by reducing agents and sensitivity to ionic strength. The enzymes showed typical oligopeptidase B-like inhibitor profiles, except that they were not inhibited by thiol sensitive inhibitors such as iodoacetamide and Nethylmaleimide. High yields of bovine and rabbit 0'2M were isolated by a three-step procedure of fractionation by PEG 6000, and zinc chelate and Sephacryl S-300 HR chromatography. Congopain, its catalytic domain C2, papain and cathepsin L all cleaved the bait region of bovine 0'2M and became trapped inside the 0'2M molecule, where their activity against large molecular weight substrates was inhibited. C2 could thus be complexed with 0'2M directly or used to form C2-0'2M-oligopeptidaseB complexes for immunisation purposes. iv The catalytic domain of congopain, C2, was used to immunise rabbits either without adjuvant, as a water-in-oil emulsion with Freund's adjuvant, or in a complex with either bovine or rabbit U2M. Freund's adjuvant elicited the highest anti-C2 antibody response. However, the greatest inhibition, 65%, of C2 activity against Z-Phe-Arg-AMC was obtained with antibodies produced by rabbits receiving C2-U2Mcomplexes. In a second study, C2 and oligopeptidase B were used to immunise rabbits , either in alum, or complexed to bovine U2M. Anti-C2 antibody levels were highest in rabbits immunised with the free proteins in alum, whereas anti-oligopeptidase B antibody levels were comparable for each adjuvant system. Anti-oligopeptidase antibodies produced with alum gave 100% inhibition of oligopeptidase B activity. In contrast, antibodies produced against C2-u2M-oligopeptidase B complexes had little effect on oligopeptidase B activity. However, these antibodies inhibited 55% of C2 activity. Alum was a slightly less efficient adjuvant for C2 and 50% inhibition of C2 activity was observed. It appeared that immunisation of rabbits with C2 complexed to U2M resulted in the production of antibodies that were better able to neutralise the proteolytic activity of C2 and congopain in vitro than that with conventional adjuvants . The immunisation of C2 complexed to bovine u2-macroglobulin therefore has the potential to neutralise parasite congopain in vivo, and may contribute to an anti-disease vaccine against African trypanosomosis. Complexation of oligopeptidase B to u2M offers no benefit, since antibodies produced against this complex are not able to inhibit the activity of oligopeptidase B. Immunisation with oligopeptidase B in alum is sufficient to produce efficient enzyme-inhibiting antibodies in the context of an anti-disease vaccine against African trypanosomosis. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2006.

Trypanosomosis is a parasitic disease in man, domestic and wild animals and is of major economic importance in many parts of the world, particularly in Sub-Saharan Africa. Trypanosoma congolense, T vivax and T brucei brucei are the major pathogenic trypanosomes infecting cattle in sub-Saharan Africa. The parasite itself is not directly responsible for the disease, but rather causes illness through the release of pathogenic factors. One of the major pathogenic factors released by trypanosomes is proteinases. Trypanotolerant cattle produce antibodies against a trypanosomal proteinase, congopain, that inhibit congopain activity. Congopain thus has vaccine potential. This study describes the mapping of immunogenic epitopes of congopain to identify peptide regions of the protein that induce enzyme inhibitory antibodies for inclusion in a trypanosome vaccine. This vaccine approach targets the disease, rather than the parasite by focusing on a pathogenic factor. These peptides also have potential for use in diagnostic assays. Peptides from the catalytic domain of a trypanosomal cysteine proteinase, congopain, were selected using an epitope prediction program. Peptides selected were from the two forms of congopain called CP1 and CP2. Antibodies against peptide-carrier conjugates were produced in chickens. The antibodies recognised native congopain, recombinant CP2 and the recombinant catalytic domain (C2). This suggests that the peptides selected have promise for use in vaccines. The peptides were also used to determine whether they are natural immunogenic epitopes of CP2 and thus have potential for use in diagnostic assays. Antibodies in the sera from T. congolense infected cattle recognised all the peptides in an ELISA. Antibodies in the sera from C2-immunised, non-infected cattle recognised most of the peptides in an ELISA. In order to distinguish between T. congolense and T vivax infection, two different peptides from the C-terminal extensions of CP2 and vivapain were used in ELISA tests with sera from infected cattle. Although anti-peptide antibodies produced against the two C-terminal extension peptides were specific for their respective peptides, thereby indicating the discriminatory power of the peptides selected, there was cross-reactivity by the sera from T. congolense and T. vivax infected cattle. Optimal antibody binding peptide sequences of these two peptides need to be identified by testing modified sequences of these two peptides to improve the sensitivity of this assay. In addition to attempting to define the epitopes of congopain, preliminary studies to increase the immunogenicity of congopain were also undertaken. Alpha 2-macroglobulin is a natural host inhibitor of proteinases. Inhibition occurs by entrapment of an active proteinase within the alpha 2-macroglobulin cage. In addition, it has been demonstrated that antigen complexed with alpha 2-macroglobulin becomes more immunogenic, resulting in enhanced antigenic presentation of an entrapped antigen. This study reports the interaction between congopain and alpha 2-macroglobulin. The preliminary results of this study showing congopain-alpha 2-macroglobulin interaction could be used to explore the possibility of increasing the immunogenicity of congopain and congopain epitopes by complexing these to alpha 2-macroglobulin. Congopain epitopes complexed with alpha 2-macroglobulin could be used to form a peptide-based vaccine. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2003.

Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: In Uganda, cattle are an important reservoir of Trypanosoma brucei rhodesiense, a parasite that causes human African trypanosomiasis or sleeping sickness. We developed mathematical models to examine the transmission of T. b. rhodesiense by tsetse vector species, Glossina fuscipes fuscipes in a host population that consists of humans, domestic and wild mammals, and reptiles. The models were developed and analysed based on the situation in Tororo district in Southeastern Uganda, where sleeping sickness is endemic and which has a cattle and human population of 40, 000 and 500, 000, respectively. Assuming populations of cattle and humans only, the impact of mass chemoprophylaxis and vector control through insecticide-treated cattle (ITC) is evaluated. Keeping 12% or 82% of the cattle population on insecticides that have an insecticidal killing effect of 100% at all times or trypanocides that have 100% efficacy, respectively, can lead to the control of T. b. rhodesiense in both humans and cattle. Optimal control of T. b. rhodesiense is shown to be achieved through ITC alone or a combination of chemoprophylaxis and ITC, the former being the cheapest control strategy. Allowing for the waning effect of insecticides and including wildhosts, T. b. rhodesiense control can be achieved by keeping 21% or 27% of the cattle population on insecticides through whole-body or restricted application, respectively. Restricting the treatment of insecticides to adult cattle only would require 24% or 33% of the adult cattle population to be kept on insecticides through whole-body or restricted application, respectively, to control T. b. rhodesiense. A cost-effectiveness and benefit-cost analysis of using ITC to control T. b. rhodesiense show that restricted application of insecticides is a cheaper and more beneficial strategy compared to whole-body treatment. The results of the study show that the restricted application of insecticides on cattle provides a cheap, safe and farmer-based strategy for controlling tsetse and trypanosomiasis. / AFRIKAANSE OPSOMMING: In Uganda is beeste ’n belangrike reservoir van Trypanosoma brucei rhodesiense, ’n parasiet wat tripanosomiase of slaapsiekte in mense veroorsaak. Ons het wiskundige modelle ontwikkel wat die oordrag van T. b. Rhodesiense deur tesetse vektor spesies, Glossina fuscipes fuscipes in ’n draer populasie wat bestaan uit mense, mak en wilde diere en reptiele, ondersoek. Die modelle was ontwikkel en geanaliseer gebaseer op die oordrag situasie in die Tororo distrik in Suidoostelike Uganda, ’n gebied waar slaapsiekte endemies is en wat ’n populasie van 40, 000 beeste en 500, 000 mense het. Die impak van massa chemoprofilakse en vektor beheer deur insekdoder-behandelde beeste is gevalueer onder die aanname van bees en mens populasies alleenlik. Beheer oor T. b. Rhodesiense in beide mense en beeste kan verkry word deur of 12% van die bees populasie te behandel met ’n insekdoder wat 100% effektief is ten alle tye of 82% van die bees populasie te behandel met tripanosiedes wat 100% effektief is. Daar is aangetoon dat optimale beheer van T. b. Rhodesiense bereik kan word deur die gebruik van insekdoders alleenlik of ’n kombinasie van insekdoders en chemoprofilakse, hoewel eersgenoemde die goedkoopste strategie is. Wanneer die kwynende effek van insekdoders asook wilde diere as draers in ag geneem word, kan T. b. Rhodesiense beheer verkry word deur 21% van beeste se hele liggaam met insekdoders te behandel of 27% gedeeltelik te behandel. As slegs volwasse beeste met insekdoders behandel word, moet 24% se hele liggaam of 33% gedeeltelik behandel word vir beheer van T. b. Rhodesiense. ’n Koste-effektiwiteit en voordeel-koste analise van insekdoders as beheermaatstaf vir T. b. Rhodesiense toon aan dat gedeeltelike behandeling van die bees se liggaam die goedkoper en meer voordelige strategie is in vergelyking met behandeling van die hele liggaam. Die resultate van die studie wys dat gedeeltelike behandeling van beeste met insekdoders ’n goedkoop, veilige en landbouer-gebaseerde strategie is om tsetse en tripanosomiase te beheer.

The aim of this thesis was to provide a picture of the trypanosomosis and drug resistance prevalence in Eastern Province of Zambia, to understand the underlying factors of drug resistance (drug use habits), to improve the diagnosis of trypanosomosis in livestock and finally, to improve the diagnosis of isometamidium resistance in T.congolense. After an introductory part where available trypanosomosis and trypanocide resistance diagnostic methods are described and discussed, the body of the thesis is divided in two main sections. In the first section are presented the results of a cross-sectional and a longitudinal epidemiological survey describing the geographical distribution of trypanosomosis cases, of resistant isolates and of cattle treated with isometamidium chloride. The results of the monitoring of unsupervised treatments of cattle with isometamidium by farmers and veterinary assistants with the Isometamidium-ELISA technique are also presented. The second section describes the development of two new diagnostic methods, the first one allowing the diagnosis of trypanosome infections with high sensitivity and specificity through semi-nested polymerase chain reaction and restriction fragment length polymorphism. This is the first report of a pan-trypanosome PCR test (a single PCR test for the diagnosis of all important pathogenic trypanosomes of cattle). The second new method that was developed allows the diagnosis of isometamidium resistant T.congolense strains by PCR-RFLP. This is the first report of a PCR based diagnostic test of trypanocide resistance in T. congolense.<p> / Doctorat en sciences, Spécialisation biologie moléculaire / info:eu-repo/semantics/nonPublished