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.

In order to develop a cheaper alternative to the conventional enzyme-linked immunosorbent assay system, application of dye molecules as labels in immunoassay was investigated in this study. This chromogenic dye-antibody conjugate could be used in colourimetric immunodetection diagnostic assays that could be used in a rural African setting. The chemistry of the interaction between twenty-six dyes of anionic, cationic and ligand dye classes with IgY and other proteins were studied for protein detection and conjugation to antibodies. Out of the twenty-six dyes studied, Direct Red 81 proved to be a good protein stain on nitrocellulose and polyacrylamide gels with comparable sensitivity to Coomassie Blue R 250. Direct Red stained proteins faster (< 5 min) than Coomassie Blue R 250 in polyacrylamide gels. Aurintricarboxylic Acid, Ethyl Red and Gallocyanine with carboxylic acid and/or hydroxyl functional groups were selected, activated with carbonyldiimidazole (CDI) to form amine reactive-imidazole intermediates and conjugated to anti-rabbit albumin IgY. Gallocyanine gave the best molar coupling ratio with IgY (76:1 dye:IgY). The dye-antibody conjugates were used to detect rabbit albumin on nitrocellulose. Aurintricarboxylic Acid-IgY and Gallocyanine-IgY detected 50 ng of rabbit albumin on nitrocellulose, which was 10 fold less sensitive than HRPO-IgY conjugate. Cross-linking of the antibodies by the dyes compromised the immunoreactivity of the Aurintricarboxylic Acid-IgY and Gallocyanine-IgY conjugates. The immunoreactivity of Ethyl Red-IgY was not compromised. Anti-rabbit albumin IgY was conjugated to derivatized dextran as an alternative immunoassay reagent and used to detect rabbit albumin on nitrocellulose by staining the polysaccharide (dextran) in the immune complex with PAS reagent. IgY-dextran complex was able to detect 25 ng of rabbit albumin on nitrocellulose, but PAS staining resulted in high background staining of the nitrocellulose membrane. Dextran-antibody conjugates may have better potential as immunodetecting reagent than dye-IgY conjugates, if a more sensitive and specific method of detecting the dextran in the Ag:Ab-dextran immune complex is developed. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2004.

The aim of this study was to develop a highly simplified, sensitive and specific malarial diagnostic test at the lowest possible cost. Initial work and optimisation of procedures was achieved with chicken antibodies by covalently attaching commercially available dye to them. Chicken antibodies were easily isolated from egg yolk and dye is cheap, easily visible and requires no equipment for identification of results. A dipstick dye-immunoassay was developed with nitrocellulose as the capture phase. The dye-immunoassay is an alternative to the traditional enzyme linked immunosorbent assay (ELISA) technique, which employs the use of an enzyme-substrate reaction. Numerous dyes were investigated and included Reactive black 5, trypan blue, Cibacron Blue, Congo red, Acid-black 2, dianix blue, dianix red, para-nitroaniline and primulin. Most of these dyes have dark colours which are essential for good contrast on nitrocellulose and in a microtitre plate. Some dyes contain amino (NH2) groups, which are targeted in a covalent linking step and attached to the lysine residues on antibody molecules or to the carbohydrate groups on antibody molecules. Attachment of dye molecules to antibodies with glutaraldehyde was the chief coupling method explored and conditions were optimized in this study. Unbound dye was removed by dialysis. Reactive black 5 is sensitive down to 50 nanograms of antigen on nitrocellulose. A second covalent coupling method was investigated by means of attaching dye to the carbohydrate moieties on the antibody. Reactive black 5 was sensitive down to 50 nanograms of antigen. The carbohydrate method appears to be more sensitive than the glutaraldehyde method at lower antibody concentrations. Primulin, a yellow dye, was similarly investigated. This dye does not have a dark colour initially, but can be diazotized to change its colour to orange or purple. It also fluoresces under ultra-violet light. This dye was sensitive down to 500 nanograms of antigen with both the glutaraldehyde and carbohydrate coupling techniques. A dye-linked immunosorbent assay (D-LlSA) protocol for direct antigen detection has been developed whereby the dye-antibody solution (dianix blue dye) acts as the primary antibody and substrate respectively. Sensitivity levels compare with traditional ELISAs. Dianix blue is sensitive down to 25 nanograms of antigen in a microtitre plate. Unique protein staining abilities of the dyes used in this study were indicated by staining IgY in electrophoretic gels. Acid-black 2 indicated better protein staining abilities than that of Coomassie brilliant blue. Evidence shows that dye was successfully covalently attached to antibodies and that antigen detection is possible by visualising the dye developed spots. Although malarial antibodies were not used, all procedures with chicken antibodies were optimised. Highly simplified, sensitive and specific diagnostic tests were developed. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2003.

Worldwide, overweight/obesity and associated chronic diseases such as type 2 diabetes, have reached epidemic proportions. Statistics show that overweight/obesity and chronic disease is prevalent amongst adults and children in South Africa. In addition to chronic disease/non-communicable diseases, overweight/obesity has been shown to alter immune and sympathetic activation. There is limited information on immune function (mucosal) and sympathetic activation on children both internationally and nationally and in particular investigating these parameters using non-invasive methods such as salivary biomarkers. The aim of this thesis was to investigate the levels of salivary biomarkers of immune function and sympathetic activation in children and determine the association with overweight/obesity, cardiorespiratory fitness (CRF) and increased physical activity (PA). Methods This thesis is divided into six chapters. These include an introductory chapter (Chapter One), a review of the literature (Chapter Two) and then three chapters that are written in article format and that have each been submitted to accredited journals for publication. Chapter Three is a review article that discusses salivary biomarkers in children as they relate to exercise, PA and obesity. Chapter Four is a study that examined salivary biomarkers of mucosal immunity and sympathetic activation as predicted by age, body composition and cardiorespiratory variables in one hundred and thirty-two black South African children (age 10.05 ± 1.68y, 74 females, 58 males). Chapter Five is a study that investigated salivary biomarkers of mucosal immunity and sympathetic activation in response to 12 weeks of soccer training in thirty-four black male South African children (11 – 13y) from a youth football training academy. Chapter Six includes a summary of the research findings, conclusions and well as recommendations for future research. A review of the literature revealed that participation in regular moderate intensity PA or exercise appears to enhance mucosal immunity (increases salivary IgA (sIgA)) in preadolescent children. In contrast, poor fitness and inactivity as well as strenuous training appear to compromise the mucosal immune system thereby increasing the risk of upper respiratory tract infections (URTIs). Children reporting higher levels of body fat and with a greater BMI appear to have lower sIgA levels and a greater incidence of infections. The limited research examining salivary C-reactive protein (sCRP) suggests a strong association between poor cardio-respiratory fitness (CRF) and/or overweight/obesity and inflammatory status in children based on elevated sCRP levels. Research surrounding salivary alpha-amylase (sAA) indicates that exercise can result in a marked increase in sAA as seen by an increase sympathetic activity via increased adrenergic activity in the salivary glands. The limited research suggests exercise may also pose a high stress on young athletes as seen with an increase in sAA. Additionally it appears that BMI may be a strong predictor of stress-induced sAA increases in children. Greater hypothalamic pituitary adrenal (HPA) axis response, as seen by increases in salivary cortisol, appear to be influenced greatly by increases in obesity. Higher salivary cortisol secretions have been observed in obese versus lean children in response to exercise. School study: The outcomes of the one-way ANOVAs examining the differences by body mass index (BMI) categories showed there were significant differences in weight (F = 83.64, df = 2, 129, P < 0.0001), BMI (F = 193.36, df = 2, 129, P < 0.0001), waist-to-hip ratio (F = 193.36, df = 2, 129, P < 0.0001), body fat percentage (F = 336.98, df = 2, 129, P = 0.0001), SBP (F = 5.72, df = 2, 129, P = 0.0042), DBP (F = 291.76, df = 2, 129, P < 0.0001), VO2max (F = 521.00, df = 2, 129, P < 0.0001), sAA concentration (F = 17.05, df = 2, 129, P < 0.0001), sAA secretion rate (F = 15.15, df = 2, 129, P < 0.0001), sIgA concentration (F = 11.30, df = 2, 129, P < 0.0001), and sIgA secretion rate (F = 8.08, df = 2, 129, P = 0.0005), between children of different BMI categories. According to the CDC-BMI-for-age standards, the participants were grouped into the following CDC-BMI-for-age categories: normal weight (< 85th percentile), overweight (≥ 85th percentile to < 95th percentile), and obese (≥ 95th percentile) (Ogden and Flegal, 2010). Tukey’s post hoc analyses revealed that obese children had significantly (P < 0.01) higher weight, BMI, body fat percentage, DBP, SBP, sAA concentration and secretion rate, compared to overweight and normal weight children, as well as a significantly lower aerobic capacity (VO2max) than both normal (P < 0.001) weight and overweight (P < 0.05) children. In addition, sIgA concentration and secretion rate were significantly lower between normal weight and obese children (P < 0.01). Multiple linear regression revealed that BMI, DBP and VO2max predicted sAA. BMI (P = 0.04) and DBP (P = 0.04) were found to be independent predictors of sAA concentration. Age and BMI category predicted sIgA secretion rate. BMI category (P = 0.0006) was found to be an independent predictor of sIgA secretion rate. Soccer study: Significant differences after 12 weeks of soccer specific training were found to be significant between pre vs. post for BMI (P =0.034), waist-to-hip ratio (P = 0.046), age (P < 0.0001), height (P < 0.0001), body fat % (P < 0.0001) and LMM (P < 0.0001). Decreases in BMI, waist-to-hip ratio, body fat % and LMM were found while age and height increased throughout the 12 weeks. Significant differences were also found between sIgA secretion rate pre vs. post training (P =0.025) as increases in these values pre to post were observed. Conclusions The results from the studies on the school children and soccer players suggested that mucosal immune function and sympathetic activation appear to be affected by body composition, CRF and chronic exercise training. The main findings for the school study revealed that BMI, DBP and VO2 predict sAA and that age and BMI category predict sIgA. This study also found that obesity (based on BMI) has a major role to play and that obese children have elevated sAA, lowered sIgA, and poor CRF. The finding of an increase in sIgA secretion rate in the soccer study suggested that a structured 12 week exercise programme can elevate mucosal immune function in youth soccer players. The underlying mechanism responsible may be an exercise-induced increase in the transport of sIgA across the mucosal epithelium and/or enhanced production of IgA in the mucosa via mediating cytokines. The literature review demonstrated that PA and overweight/obesity may have an impact on salivary biomarkers of mucosal immunity and sympathetic activation in children, however further research with regards to optimal intensity, duration and modality need to be assessed in the pre-pubescent population.Physical activity, obesity, immunity, neuro-endocrine, children, salivary biomarkers, sympathetic activation. / Thesis (Ph.D.)-University of KwaZulu-Natal, Durban, 2012.

The purpose of this study was to characterize the association between anti-prothrombin antibodies and the lupus anticoagulant (LA) in order to elucidate the antigenic site of the LA. Plasma from 8 patients with the LA had evidence of anti-prothrombin antibodies on prothrombin crossed immunoelectrophoresis as characterized by material moving slower in the first dimension of electrophoresis than normal prothrombin, i.e., a trailing shoulder. Four of 5 LA patients with a prolonged prothrombin time demonstrated the most pronounced evidence of anti-prothrombin antibodies. All patients were shown to have an essentially normal level of prothrombin antigen. Using an enzyme-linked immunosorbent assay (ELISA), six of 8 LA patients tested positive for anticardiolipin antibodies (aCL) of the IgG isotype while 7 of 8 LA patients tested positive for antiphosphatidylserine antibodies (aPS) of the IgG isotype.An anti-human Factor II (prothrombin) ELISA was developed to confirm the presence of anti-Factor II (aFII) activity in LA patients. Seven of 8 LA patients were positive for aFII activity. A strong parallel existed between the presence of aPS activity, anti-human Factor II activity, and the LA, i.e., 7 of 8 LA plasmas were aPS (+)/aFII (+). An antibovine Factor II ELISA was developed to determine if the aFII activity associated with LA patients is speciesspecific. Three of 5 LA patients positive for anti-human Factor II activity were also shown to be positive for antibovine Factor II activity. Antibodies with specificity for human prothrombin were purified from LA plasmas using a prothrombin affinity column. Three of 8 LA patient eluates were shown to be positive for aPS (IgG) while none were positive for aCL (IgG or IgM) or human aFII activity. Affinity-purified eluates were assayed for LA activity using the dilute Russell viper venom time (dRVVT). None of the LA patient eluates were shown to prolong the dRVVT when present with normal plasma in concentrations up to 100 micrograms/mL. / Department of Chemistry

Human African Trypanosomiasis (HAT), also known as sleeping sickness is one of the many life threatening tropical diseases posing a serious risk to livelihoods in Africa. The disease is restricted to the rural poor across sub–Saharan Africa, where tsetse flies that transmit the disease, are endemic. Sleeping sickness is known to be caused by protozoan parasites of the genus Trypanosoma brucei, with the two sub-species: T. b. gambiense and T. b. rhodesiense, responsible for causing infection in humans. The disease develops in two stages, firstly, the infection is found in the blood and secondly, when the parasites cross the blood-brain barrier entering the nervous system. To date, no vaccines have been developed, however, there is a range of drugs and treatments available which depend on the type of infection (T. b. gambiense or T. b. rhodesiense) as well as disease stage. The trypanosome parasites have a two-host life cycle i.e. in the mammalian host as well as the tsetse fly vector. Throughout the cycle, the parasite undergoes changes, one of them being the acquisition of a variable surface glycoprotein (VSG) coat prior to entry into the human host bloodstream. Once in the host, the infection progresses and through a phenomenon known as antigenic variation, the parasite expresses a different VSG coat periodically, enabling the parasites to constantly evade the host’s immune response, facilitating their survival. The VSG genes coding for the proteins are activated by an intricate process involving the encoding of a gene which is kept silent, until activated in one of several expression sites. Despite the constant switching of VSG surface coats, there are VSG forms that are predominantly expressed in T. b. gambiense namely VSGs LiTat 1.3, LiTat 1.5 and LiTat 1.6 which are used in diagnostic tests, as antigens to detect antibodies in infected sera of HAT patients. The acquisition of these VSG antigens is, however, of high risk to staff handling the parasites, and so the first part of the study was aimed at cloning, recombinantly expressing and purifying the two VSGs known to be recognised by all gambiense HAT patients: LiTat 1.3 and LiTat 1.5, for possible use as alternative antigens in diagnostic tests. The genes encoding both VSGs, LiTat 1.3 and LiTat 1.5, were first amplified from either genomic or complementary DNA (gDNA or cDNA), cloned into a pTZ57R/T-vector and sub-cloned into pGEX or pET expression vectors prior to recombinant expression in E. coli BL21 DE3 and purification by Ni-affinity chromatography. Amplification and subsequent cloning yielded the expected 1.4 kb and 1.5 kb for the LiTat 1.3 and LiTat 1.5 genes respectively. Recombinant expression in E. coli was only successful with the constructs cloned from cDNA, i.e. the pGEX4T-1-cLiTat 1.3 and pET-28a-cLiTat 1.3 clones. Purification of the 63 kDa cLiTat 1.3His protein following solubilising and refolding did not yield pure protein and there were also signs of protein degradation. For comparison, expression was also carried out in P. pastoris and similar to the bacterial system, expression was only successful with the LiTat 1.3-SUMO construct yielding a 62.7 kDa protein. Purification of LiTat 1.3SUMO also surpassed that of cLiTat 1.3His with no degradation. The diagnostic tests based on VSGs LiTat 1.3 and LiTat 1.5 as antigens operate by binding with antibodies in infected sera, to confirm infection. These antibody detection tests have their limitations, hence an alternative would be antigen detection tests, which use antibodies to detect the respective antigens in infected sera. The second part of the study therefore involved antibody production, where chickens were immunised with the native VSGs LiTat 1.3, LiTat 1.5 as well as recombinant RhoTat 1.2 (a VSG expressed in T. evansi). Antibody production was analysed by ELISA and characterised by western blotting, prior to immunolabelling of T. b. brucei Lister 427 parasites. The chicken IgY showed a response to the immunogens, and were able to detect their respective proteins in the western blot. Interestingly, the anti-nLiTat 1.3, anti-nLiTat 1.5 and anti-rRhoTat 1.2 antibodies were able to detect their respective VSGs on the T. b. brucei trypanosome parasites in the immunofluorescence assay, thus demonstrating cross reactivity. As the antibodies showed specificity, they could potentially detect antigens in infected sera of HAT patients in an antigen detection based test. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2013.

Accurate diagnosis of the presence of an infectious organism is very important for therapeutic interventions and consequently the recovery of the individual. There is a need for identifying new diagnostic antigens for the serological diagnosis of trypanosomiasis, a disease of humans and animals in Africa caused by protozoa belonging to the genus Trypanosoma. Invariant surface glycoproteins (ISGs) are present in most strains of the parasite and have the potential to replace the variable surface glycoproteins as diagnostic antigens. In order to avoid the challenges of in vivo culturing of bloodstream form (BSF) trypanosomes in laboratory animals, ISG65 and ISG75, the two most common ISGs were heterologously expressed in Escherichia coli and Pichia pastoris expression systems. The extracellular domains of ISG65 and ISG75 of both T. b. brucei and T. b. gambiense were amplified by PCR from genomic DNA using appropriate primers to give inserts of 1121 bp and 1342 bp sizes. These were sub-cloned into the pGEX-4T1 and pET28a expression vectors. Chemically competent E. coli BL21 (DE3) were transformed using the resultant plasmids and the transformed E. coli cells were used for heterologous protein expression. The expressed proteins were purified by three phase partitioning (TPP), nickel or glutathione affinity and molecular exclusion chromatography and analysed by reducing SDS-PAGE. The glycosylation status of ISG65 and ISG75 expressed in the M5 strain of P. pastoris, which has an engineered N-glycosylation pathway that produces glycosylated proteins similar to what is obtained in trypanosomes, was determined. The enzymatic action of Endoglycosidase H resulted in a shift in the electrophoretic migration of ISG65 but not ISG75 on SDS-PAGE, confirming N-glycosylation. Anti-ISG65 and anti-ISG75 antibodies were produced in chickens and affinity purified using the respective recombinant proteins immobilised on affinity matrices. The antibodies recognised native ISG65 and ISG75 respectively in western blots of lysates of T. b. brucei parasites cultured in vitro. Similar recognition of the native ISGs by the anti-recombinant ISG antibodies was also obtained using immunofluorescence microscopy of fixed T. b. brucei parasites. The results obtained demonstrate the potential of application of the recombinant ISG65 and ISG75 and their respective antibodies in the diagnosis of African trypanosomiasis. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2013.

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Bibliography: leaves 184-218. / xx, 218, [135] leaves, [15] leaves of plates : ill. (chiefly col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / The research in this thesis describes the development and use of assays to detect cellular immune responses in ducks with application to duck hepatitis B virus (DHBV) infections. This animal model is used to provide an additional area of research which complements the study of hepadnaviruses. The introduction contains an outline of the significance of hepadnavirus research, including hepatitis B virus (HBV) epidemiology, structure, replication and clinical manifestations of the diseases caused by the virus. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1997