Previous studies have shown that the formulation of an active pharmaceutical ingredient (API) entrapped in the Pheroid® (Pheroid for simplification) delivery system enhances absorption of the API, suppresses its metabolism, and may contribute to an increase in the quantity of the API present at the site of action. Higher drug levels at the active site should particularly increase the effectiveness of a drug with a narrow therapeutic index and reduce the incidence of the resistance that may otherwise arise if the sub-therapeutic levels of the API are in contact with the site of interest. Two approaches were followed in this study. First, the radioactive tracer molecule 99mTechnetium methylene diphosphonate (99mTc MDP) was used. Intravenously injected 99mTc MDP is an extremely effective bone-seeking radiopharmaceutical used in the diagnosis of bone disorders such as bone metastases in patients. However, if entrapped inside a Pheroid vesicle, it will locate to that site, usually an organ, where the Pheroid vesicles may tend to accumulate. Experiments conducted with 99mTc MDP alone or with Pheroid will therefore establish how efficiently Pheroid vesicles localize and will also indicate the preferred site of localization inside a body. The process would involve the oral administration of 99mTc MDP either alone or with Pheroid, involving an animal model. It would also involve tracking localization to particular organs, blood or other sites. The second approach requires the use of chloroquine (CQ) labeled with carbon-14 (14C-CQ,) to compare absorption of the drug both with and without the Pheroid system. The intention was to compare oral absorption and bio-distribution of 14C-CQ administered either alone or entrapped in the Pheroid system. It was also possible to establish whether the Pheroid affects the biological half-lives of the CQ and residence times of CQ in the different organs of the body. Absorption of free 99mTc MDP (orally adminsistered) through the intestinal tract is negligible but it was anticipated that increased absorption will be observed when 99mTc MDP was entrapped in the Pheroid system. In the 99mTc MDP study, different routes of administration of 99mTc MDP, as well as 99mTc MDP entrapped and not entrapped in the Pheroid system, were investigated. The Sprague Dawley rat was used as animal model. Rats were divided into three groups of four rats each for the first part of the study. In the first group, only 99mTc MDP was injected intravenously in order to establish natural distribution of the 99mTc MDP. For the second group, 99mTc MDP was administered orally in order to establish whether there was any absorption through the intestinal tract. In the third group, the 99mTc MDP was entrapped in Pheroid vesicles and this formulation was administered orally in order to establish whether the Pheroid system enhanced oral absorption. The animals were sacrificed four hours after administration and organs were harvested and were counted for radioactivity to determine the percentage of injected/administrated dose in each organ. After oral administration, the Pheroid system was found to have facilitated absorption of 99mTc MDP through the intestinal tract into the blood. 99mTc MDP concentrations in the femur, although lower, were still comparable with that observed after intravenous administration of 99mTc MDP in the absence of Pheroid. Thus, overall, excellent absorption of the Pheroid entrapped 99mTc MDP through the intestinal tract was seen in contrast to little or zero absorption of the compound in the reference formulations. The half-life of the radio-labelled compound in the blood was prolonged after oral administration owing to the Pheroid. To investigate the bio-distribution of radioactive chloroquine (14C-CQ) Sprague Dawley rats were divided into two groups of four rats each. In the first group, 14C-CQ in deionised (DI) water was administered orally, and in the second group 14C-CQ entrapped in Pheroid vesicles was administered, also orally. The animals were sacrificed one, two and four hours after administration and subjected to comprehensive macroscopic inspection. All the organs were harvested and radioactivity was determined with liquid scintillation after applicable sample preparation. The Pheroid system produced much higher organ and blood concentrations of 14C-CQ and enhanced residence times within the organs and blood in comparison with that of 14C-CQ administered alone. Commercial applications of these results are possible, as a number of radiopharmaceutical products can presently be administered only intravenously. The added potential of these new Pheroid formulations could be of significance in the treatment of malaria, as chloroquine is inexpensive and widely available. Another point of interest is that the use of these formulations may enable micromolar drug concentrations to be achieved using drug dosage regimes that usually produce only nanomolar levels. However, safety aspects would have to be carefully monitored. / PhD (Pharmaceutics), North-West University, Potchefstroom Campus, 2015

Malaria is an infectious disease caused by various forms of the Plasmodium parasite. It is responsible for thousands of deaths yearly with 90 % of those deaths being in sub-Saharan Africa, thus making it a disease of global importance. The global burden of malaria is worsened by resistance to current treatment, a lack in funding and limited research outputs. More alternative ways of treatment must be explored and may include the co-formulation of antimalarial drug substances as well as alternative ways of drug delivery. Antifolates are drugs which interfere with an organism’s folate metabolism by inhibiting dihydropteroate synthase (DHPS) or dihydrofolate reductase (DHFR). Dapsone is a synthetic sulfone which has a mechanism of action that is very similar to that of sulphonamides. The mechanism of action is characterised by the inhibition of folic acid synthesis through the inhibition of dihydropteroate synthase (DHPS). Another antifolate drug, proguanil, is the prodrug of cycloguanil. Its mechanism involves the inhibition of dihydrofolate reductase (DHFR), thus inhibiting the malaria parasite to metabolise folates and therefore stunting its growth. Unfortunately, dapsone has a serious side-effect in people with a deficiency of the enzyme glucose-6-phosphate dehydrogenase (G6PD) causing oxidative stress on the red blood cells leading to the rupturing of these cells. The main objective of this study was to formulate and characterise TMC-TPP microparticles loaded with the effective but toxic drug combination of dapsone and proguanil and to determine if these drug-containing microparticles had in vivo efficacy against malaria. N-trimethyl chitosan chloride (TMC), a partially quaternised chitosan derivative, shows good water solubility across a wide pH range thus having mucoadhesive properties and excellent absorption enhancing effects even at neutral pH. A faster, more efficient microwave irradiation method was developed as an alternative to the conventional synthesising method of TMC. TMC with the same degree of quaternisation (DQ), ± 60 %, was obtained in a quarter of the reaction time (30 min) by using the newly developed method. The TMC synthesised with the microwave irradiation method also exhibited less degradation of the polymer structure, thus limiting the chance for the formation of any unwanted by-products (Omethylation, N,N-dimethylation and N-monomethylation). The formation of complexes by ionotropic gelation between TMC and oppositely charged macromolecules, such as tripolyphosphate (TPP), has been utilised to prepare microparticles which are a suitable drug delivery system for the dapsone-proguanil combination. Both these drugs were successfully entrapped. These particles were characterised and the in vivo efficacy against the malaria parasites was determined. The microparticles with both the drugs, separately and in combination, displayed similar or better in vivo efficacy when compared to the drugs without the TMC microparticles. An in vitro dissolution study was also performed by subjecting the dapsone and proguanil TMC formulations to 0.1N HCl dissolution medium. Samples were withdrawn after predetermined time points and the drug concentration was determined with HPLC. It was found that the TMC microparticles resulted in a sustained release profile since only 73.00 ± 1.70 % (dapsone) and 55.00 ± 1.90 % (proguanil) was released after 150 minutes. The in vivo bioavailability of the dapsone and proguanil TMC formulations was evaluated in mice by collecting blood samples at predetermined time points and analysing the samples with a sensitive and accurate LC-MS/MS method. The in vivo bioavailability of the dapsone TMC formulation relative to the normal dapsone formulation was found to be 244 % and 123 % for the proguanil TMC formulation relative to the normal proguanil formulation. These TMC-TPP microparticles formulations showed better in vivo efficacy and bioavailability when compared to the normal formulation. Together with the sustained release, these formulations may be a promising cheaper and more effective treatment against malaria. / PhD (Pharmaceutics), North-West University, Potchefstroom Campus, 2015

Malaria endemic areas not only pose a public health threat, but affects 3.3 billion people worldwide. In 2011, estimated malaria related deaths amounted to 660 000 out of 219 million reported cases, with 81% of these and 91% of malaria related mortality occurred in the African region. Those most affected were pregnant women, children under the age of five and immunocompromised individuals. Malaria is the fifth deadliest disease worldwide and accounts for the second highest death rate in Africa, following HIV/Aids. To combat this parasitic infection of antiquity, the ideal malaria pharmacotherapy would be a cost effective and easily obtainable monotherapy. The malaria parasite, however, has an intrinsic ability to develop drug resistance through various mechanisms. Widespread resistance towards antimalarial drugs has rendered traditionally used drugs therapeutically ineffective, hence accentuating the efficacy of the artemisinins as first line treatment option for uncomplicated Plasmodium falciparum (P. falciparum). A devastating reality of the challenging battle against malaria is the confirmed prolonged parasitic clearance times of the artemisinins, despite adequate drug exposure, which emphasises the urgent need for identifying and developing new, effective and safe therapies. During this study, 9-aminoacridines and artemisinin-acridine hybrids were successfully synthesised through nucleophillic substitution and their chemical structures confirmed by means of nuclear magnetic resonance spectroscopy (NMR), high resolution mass spectroscopy (HRMS) and infrared spectroscopy (IR). The hybrid compounds were synthesised through microwave assisted radiation, by covalently linking the artemisinin- and amino-functionalised acridine pharmacophores by means of a liable aminoethyl ether chain. The target compounds were screened in vitro for antimalarial activity against both the chloroquine sensitive (NF54) and chloroquine resistant (Dd2) strains of P. falciparum. Their cytotoxicities were assessed against various mammalian cells of different origins, viz. the Chinese hamster ovarian cells (CHO) from animal origin, and from human origin, hepatocellular- (HepG2), neuroblastoma- (SH-SY5Y) and cervical cancer (HeLa) cells. The synthesised hybrids exhibited antimalarial activity against both Plasmodium strains. Compound 7, featuring an ethylenediamine moiety in the linker, was the most active hybrid, with 50% inhibitory concentration (IC50) values of 2.6 nM and 35.3 nM against the NF54 and Dd2 strains, respectively. It had gametocytocidal activity against the NF54 strain, comparable to dihydroartemisinin (DHA) and artesunate (AS) and it is significantly more potent than chloroquine (CQ), whilst possessing a resistance index value of 14, indicative of a significant loss of activity against the CQ resistant strain. Contrary, the promising hybrid 10, containing a 2-methylpiperazine linker, had gametocytocidal activity, comparable to CQ and was found to be six-fold more potent than CQ against the Dd2 strain, with a resistance index (RI) value of 2, whilst it further showed highly selective action towards the parasitic cells. Compound 10 was also found to possess anticancer activity against the HeLa cell line, comparable to DHA and AS, but fivefold higher than that of CQ, with the same levels of hepatotoxicity and neurotoxicity. The artemisinin-acridine hybrids displayed superior antimalarial activity, compared to the derived 9-aminoacridines against both the Plasmodium strains. They, however, did not have the ability to overcome resistance, reduce the toxicity of acridine, nor induce synergistic activity. The hybrids, indeed displayed promising anticancer activity against HeLa cells. It is anticipated that these compounds may stand as drug candidates for further investigation in the search for new anti-cervical cancer drugs, rather than as antimalarials. / MSc (Pharmaceutical Chemistry), North-West University, Potchefstroom Campus, 2014

Malaria is endemic in 106 countries worldwide. This disease is caused by a parasite from the genus Plasmodium. Of the five species that infect humans, Plasmodium falciparum is the most virulent, with over three billion people at risk and around 660 000 deaths reported in 2011. Of these deaths, 91% were in the African region, while 86% were children under the age of five. In light of the widespread development of resistance by malaria parasites against the classic antimalarial drugs, such as chloroquine (CQ) and now the established tolerance towards the widely used artemisinins, an immense need exists for identifying and developing new and effective antiplasmodial drugs. In search for such new antimalarial drugs, three chalcone based series of compounds were prepared and investigated during this study. The first series (Chapter 3) comprised 4-aminoquinolinyl-chalcone amides, which were synthesized through amidation of carboxylic acid-functionalised chalcone with aminoquinolines, using 1,1'-carbonyldiimidazole (CDI) as coupling agent. These compounds were screened alongside CQ against the CQ sensitive (3D7) and CQ resistant (W2) strains of P. falciparum. Cytotoxicity was assessed against the WI-38 cell line. The amide, featuring the 1,6-diaminohexane linker, was found the most active of all these new novel compounds tested. It was found to be as potent as CQ against 3D7, while displaying a two-fold higher activity than CQ against the W2 strain, coupled with good selective antimalarial activity (SI = 435) towards the parasitic cells. The second series (Chapter 4) consisted of aminoferrocenyl-chalcone amides, synthesized through condensation of a chalcone with an aminoferrocenyl. These compounds were screened against the 3D7, and antifolate- and CQ resistant (FCR3) strains of P. falciparum and cytotoxicity was determined against the WI-38 line. The most active compound of this series was the amide, containing the 1,2-diaminoethane linker, which showed 130- and 42 times less potency than CQ against the 3D7 and W2 strains, respectively. The third series of antimalarials (Chapter 5) involved dihydroartemisinyl-chalcone esters, synthesized through esterification of chalcones with DHA. These compounds were screened against 3D7 and W2 strains of P. falciparum, while the cytotoxicity was determined against the WI-38 line. Those esters featuring oxygenated aryl rings were three- to four-fold more potent than current clinically used artesunate against both P. falciparum strains. They were also screened in vitro against a panel of three cancer cell lines consisting of TK-10, UACC-62 and MCF-7. Thermogravimetric analysis revealed that the targeted hybrids were all thermally more stable than DHA as a result of the presence of the chalcone moiety in their structures. This could prove beneficial to the high temperature storage conditions that prevail in most malaria endemic countries. This study resulted in a number of compounds with varying antiplasmodial activity ranges. The compounds in series 3 were overall the most active, due to the incorporation of the highly active dihydroartemisinin pharmacophore. The chalcone moiety, especially, demonstrated a large scope for future development, owing to the ease of synthesis and the relatively low costs involved. The most active compounds of the three series could serve as potential lead compounds in the future development of more effective antimalarial drugs. / PhD (Pharmaceutical Chemistry), North-West University, Potchefstroom Campus, 2014

Previous studies have shown that the formulation of an active pharmaceutical ingredient (API) entrapped in the Pheroid® (Pheroid for simplification) delivery system enhances absorption of the API, suppresses its metabolism, and may contribute to an increase in the quantity of the API present at the site of action. Higher drug levels at the active site should particularly increase the effectiveness of a drug with a narrow therapeutic index and reduce the incidence of the resistance that may otherwise arise if the sub-therapeutic levels of the API are in contact with the site of interest. Two approaches were followed in this study. First, the radioactive tracer molecule 99mTechnetium methylene diphosphonate (99mTc MDP) was used. Intravenously injected 99mTc MDP is an extremely effective bone-seeking radiopharmaceutical used in the diagnosis of bone disorders such as bone metastases in patients. However, if entrapped inside a Pheroid vesicle, it will locate to that site, usually an organ, where the Pheroid vesicles may tend to accumulate. Experiments conducted with 99mTc MDP alone or with Pheroid will therefore establish how efficiently Pheroid vesicles localize and will also indicate the preferred site of localization inside a body. The process would involve the oral administration of 99mTc MDP either alone or with Pheroid, involving an animal model. It would also involve tracking localization to particular organs, blood or other sites. The second approach requires the use of chloroquine (CQ) labeled with carbon-14 (14C-CQ,) to compare absorption of the drug both with and without the Pheroid system. The intention was to compare oral absorption and bio-distribution of 14C-CQ administered either alone or entrapped in the Pheroid system. It was also possible to establish whether the Pheroid affects the biological half-lives of the CQ and residence times of CQ in the different organs of the body. Absorption of free 99mTc MDP (orally adminsistered) through the intestinal tract is negligible but it was anticipated that increased absorption will be observed when 99mTc MDP was entrapped in the Pheroid system. In the 99mTc MDP study, different routes of administration of 99mTc MDP, as well as 99mTc MDP entrapped and not entrapped in the Pheroid system, were investigated. The Sprague Dawley rat was used as animal model. Rats were divided into three groups of four rats each for the first part of the study. In the first group, only 99mTc MDP was injected intravenously in order to establish natural distribution of the 99mTc MDP. For the second group, 99mTc MDP was administered orally in order to establish whether there was any absorption through the intestinal tract. In the third group, the 99mTc MDP was entrapped in Pheroid vesicles and this formulation was administered orally in order to establish whether the Pheroid system enhanced oral absorption. The animals were sacrificed four hours after administration and organs were harvested and were counted for radioactivity to determine the percentage of injected/administrated dose in each organ. After oral administration, the Pheroid system was found to have facilitated absorption of 99mTc MDP through the intestinal tract into the blood. 99mTc MDP concentrations in the femur, although lower, were still comparable with that observed after intravenous administration of 99mTc MDP in the absence of Pheroid. Thus, overall, excellent absorption of the Pheroid entrapped 99mTc MDP through the intestinal tract was seen in contrast to little or zero absorption of the compound in the reference formulations. The half-life of the radio-labelled compound in the blood was prolonged after oral administration owing to the Pheroid. To investigate the bio-distribution of radioactive chloroquine (14C-CQ) Sprague Dawley rats were divided into two groups of four rats each. In the first group, 14C-CQ in deionised (DI) water was administered orally, and in the second group 14C-CQ entrapped in Pheroid vesicles was administered, also orally. The animals were sacrificed one, two and four hours after administration and subjected to comprehensive macroscopic inspection. All the organs were harvested and radioactivity was determined with liquid scintillation after applicable sample preparation. The Pheroid system produced much higher organ and blood concentrations of 14C-CQ and enhanced residence times within the organs and blood in comparison with that of 14C-CQ administered alone. Commercial applications of these results are possible, as a number of radiopharmaceutical products can presently be administered only intravenously. The added potential of these new Pheroid formulations could be of significance in the treatment of malaria, as chloroquine is inexpensive and widely available. Another point of interest is that the use of these formulations may enable micromolar drug concentrations to be achieved using drug dosage regimes that usually produce only nanomolar levels. However, safety aspects would have to be carefully monitored. / PhD (Pharmaceutics), North-West University, Potchefstroom Campus, 2015

Malaria is an infectious disease caused by various forms of the Plasmodium parasite. It is responsible for thousands of deaths yearly with 90 % of those deaths being in sub-Saharan Africa, thus making it a disease of global importance. The global burden of malaria is worsened by resistance to current treatment, a lack in funding and limited research outputs. More alternative ways of treatment must be explored and may include the co-formulation of antimalarial drug substances as well as alternative ways of drug delivery. Antifolates are drugs which interfere with an organism’s folate metabolism by inhibiting dihydropteroate synthase (DHPS) or dihydrofolate reductase (DHFR). Dapsone is a synthetic sulfone which has a mechanism of action that is very similar to that of sulphonamides. The mechanism of action is characterised by the inhibition of folic acid synthesis through the inhibition of dihydropteroate synthase (DHPS). Another antifolate drug, proguanil, is the prodrug of cycloguanil. Its mechanism involves the inhibition of dihydrofolate reductase (DHFR), thus inhibiting the malaria parasite to metabolise folates and therefore stunting its growth. Unfortunately, dapsone has a serious side-effect in people with a deficiency of the enzyme glucose-6-phosphate dehydrogenase (G6PD) causing oxidative stress on the red blood cells leading to the rupturing of these cells. The main objective of this study was to formulate and characterise TMC-TPP microparticles loaded with the effective but toxic drug combination of dapsone and proguanil and to determine if these drug-containing microparticles had in vivo efficacy against malaria. N-trimethyl chitosan chloride (TMC), a partially quaternised chitosan derivative, shows good water solubility across a wide pH range thus having mucoadhesive properties and excellent absorption enhancing effects even at neutral pH. A faster, more efficient microwave irradiation method was developed as an alternative to the conventional synthesising method of TMC. TMC with the same degree of quaternisation (DQ), ± 60 %, was obtained in a quarter of the reaction time (30 min) by using the newly developed method. The TMC synthesised with the microwave irradiation method also exhibited less degradation of the polymer structure, thus limiting the chance for the formation of any unwanted by-products (Omethylation, N,N-dimethylation and N-monomethylation). The formation of complexes by ionotropic gelation between TMC and oppositely charged macromolecules, such as tripolyphosphate (TPP), has been utilised to prepare microparticles which are a suitable drug delivery system for the dapsone-proguanil combination. Both these drugs were successfully entrapped. These particles were characterised and the in vivo efficacy against the malaria parasites was determined. The microparticles with both the drugs, separately and in combination, displayed similar or better in vivo efficacy when compared to the drugs without the TMC microparticles. An in vitro dissolution study was also performed by subjecting the dapsone and proguanil TMC formulations to 0.1N HCl dissolution medium. Samples were withdrawn after predetermined time points and the drug concentration was determined with HPLC. It was found that the TMC microparticles resulted in a sustained release profile since only 73.00 ± 1.70 % (dapsone) and 55.00 ± 1.90 % (proguanil) was released after 150 minutes. The in vivo bioavailability of the dapsone and proguanil TMC formulations was evaluated in mice by collecting blood samples at predetermined time points and analysing the samples with a sensitive and accurate LC-MS/MS method. The in vivo bioavailability of the dapsone TMC formulation relative to the normal dapsone formulation was found to be 244 % and 123 % for the proguanil TMC formulation relative to the normal proguanil formulation. These TMC-TPP microparticles formulations showed better in vivo efficacy and bioavailability when compared to the normal formulation. Together with the sustained release, these formulations may be a promising cheaper and more effective treatment against malaria. / PhD (Pharmaceutics), North-West University, Potchefstroom Campus, 2015

This thesis investigates antigenic variation and its evolution in Plasmodium falciparum, the cause of the most deadly form of human malaria. Antigenic variation is a strategy for evading immunity by switching between antigenic variants during infection. In P. falciparum, such variable antigens confer different binding phenotypes that may affect parasite survival and have also been linked to pathology. Here, a new statistical method is described for determining the switching patterns that underlie antigenic variation. This method is then applied to experimental data to yield a full description of an antigenic switching network in P. falciparum. In light of the findings, theoretical modelling is used to show how immune selection and binding phenotypes may have contributed to the evolution of antigenic repertoire structure, expression order and virulence. Related models are also used to investigate parasite population diversity, providing possible explanations for observations reported here and elsewhere, with implications for vaccine design. Together, these chapters advance understanding of P. falciparum immune evasion and how it relates to pathology. This work further reinforces the role of host immunity in shaping pathogen population diversity at multiple levels.

For vector-borne parasites such as malaria, how within- and between-host processes interact to shape transmission is poorly understood. In the host, malaria parasites replicate asexually but for transmission via mosquitoes to occur, specialized sexual stages (gametocytes) must be produced. Once inside the mosquito vector, gametocytes immediately differentiate into male and female gametes, and motile male gametes must swim through the hostile environment of the bloodmeal to find and fertilise female gametes. Despite the central role that gametocytes play in disease transmission, explanations of why parasites adjust gametocyte production in response to in-host factors remain controversial. Furthermore, surprisingly little is known about the mating behaviour of malaria parasites once inside the mosquito. Developing drugs and/or vaccines that prevent transmission by disrupting sexual stages are major goals of biomedicine, but understanding variation in gametocyte investment and male gamete behaviour is key to the success of any intervention. First, I propose that the evolutionary theory developed to explain variation in reproductive effort in multicellular organisms provides a framework to understand gametocyte investment strategies in malaria parasites. I then demonstrate that parasites appear to change their reproductive strategies in response to environmental cues and in a manner consistent with our predictions. Next, I show how digital holographic microscopy can be used to characterise the morphology and motility of male gametes. I then provide evidence for non-random movement of male gametes and that gamete interactions with red blood cells appear to hinder mating success in a bloodmeal. Finally, I discuss the variation in gametocyte differentiation and fertilisation success when exposed to a number of factors implicated in gametocyte activation. The data presented here provides important information on the basic biology of malaria parasite reproductive stages and demonstrates considerable variation in parasite traits and behaviours in response environmental changes; both in the host and in the mosquito vector.

Malaria is a leading cause of death in some developing countries. The malaria parasite has been around for over a century, and has coevolved with humans. Coming up with an effective vaccine for P. falciparum will save millions of lives and reduce the morbidity and mortality of malaria globally. Understanding the role of exported parasite proteins i.e PfEMP1 a virulence factor and major cause of malarial pathogenesis, has been of great interest to vaccine researchers in the last decade. The focus of this review is to provide a literature review on PfEMP1s, their interaction with the human immune system, and their role in helping P. falciparum parasite to evade the immune system. This review will primarily focus on the intra-erythrocytic stage, which is the stage that results in the symptoms of malaria. A review is necessary to understand the antigenic variation of PfEMP1s, and how PfEMP1s challenge the different arms of the immune response, both the innate and adaptive. This review is unique in touching on the major parts of the immune system's interaction with the PfEMP1 antigen. Furthermore, the review explores the discussion of future research and therapeutic opportunities based on our knowledge of PfEMP1 antigens.

Thesis (MSc)--University of Stellenbosch, 2005. / ENGLISH ABSTRACT: The African penguin, which occurs along the south-eastern and south-western shores of South-Africa and Namibia, has experienced a severe reduction in population numbers due to guano and egg collection in the first half of the 19th century, and oil pollution in the second half of the 19th century as a result of oil tankers rounding the Cape of Good Hope. The population would have been reduced by a further 19% had it not been for the rehabilitation of penguins at the South African National Council for the Conservation of Coastal Birds (SANCCOB) facility. Although this has been very successful, mortalities as a result of avian malaria infection have considerably reduced the efficiency of rehabilitation. In an effort to assess the role of immunity against malaria in combating the disease, an enzyme-linked immunosorbent assay (ELISA) for the detection of antibody levels to avian malaria was developed. The ELISA was used to detect antibody levels to avian malaria of penguins on entry and during rehabilitation from October 2001 to January 2003. The aim of this study was to continue the determination of antibody levels to avian malaria of penguins entering the SANCCOB facility, in order to allow an evaluation of the antibody levels to avian malaria for two full calendar years. This investigation was combined with a polymerase chain reaction (PCR)-based method, capable of detecting any Plasmodium species in penguin serum. These two methods were also used to investigate avian malaria in several breeding colonies in order to assess the role avian malaria may play in the survival of the African penguin in the wild. Results indicated that the ability of penguins to produce anti-Plasmodium antibodies was not influenced by oiling and that infection with malaria was not due to recrudescence but rather due to infection via mosquitoes. This indicated a possible role of the SANCCOB facility in exposing the penguins to avian malaria. However a large number of penguins arrived at the facility previously infected with malaria, indicating that malaria was present in the breeding colonies. Investigations in the breeding colonies revealed extremely high avian malaria prevalence even though no sick birds or mortalities were observed. This raised the question whether different types of malaria are responsible for infection in the SANCCOB facility and breeding colonies. / AFRIKAANSE OPSOMMING: Die Afrika Pikkewyn kom langs die suid-oostelike en suid-westelike kus van Suid Afrika en Namibië voor. In die afgelope eeu het hierdie spesie ‘n geweldige afname in populasie getalle ondervind. Dit was hoofsaaklik die gevolg van die versameling van guano en pikkewyneiers in die eerste helfte van die 19de eeu en oliebesoedeling in die tweede helfde van die 19de eeu. Die “South African Foundation for Conservation of Coastal Birds” (SANCCOB) is ‘n seevoëlreddings- en rehabilitasiesentrum vir siek, beseerde en ge-oliede pikkewyne. Dit word geskat dat die Afrika Pikkewyn populasie met ‘n verdere 19% sou afgeneem het as dit nie vir die rehabilitasie by die SANCCOB sentrum was nie. Hierdie sentrum het egter aansienlike vrektes in die somer as gevolg van voëlmalaria, wat sodoende die effektiwiteit van die rehabilitasie verlaag. In ‘n poging om die rol van immuniteit teen malaria te bepaal is ‘n “enzyme-linked immunosorbent assay” (ELISA) ontwikkel vir die bepaling van antiliggaam vlakke teen malaria. Hierdie ELISA is gebruik vir die bepaling van die anti-Plasmodium antiliggaam vlakke van die pikkewyne by aankoms en ten tye van rehabilitasie by SANCCOB vanaf Oktober 2001 to Januarie 2003. Die doel van hierdie studie was eerstens om hierdie ELISA bepalings voort te sit om sodoende antiliggaam vlakke teen malaria oor twee kalender jare te kan evalueer. Hierdie ondersoek was gekombineer met ‘n polimerase ketting reaksie (PCR) metode, wat enige Plasmodium spesie in pikkewynserum sou kon opspoor. Hierdie twee metodes is ook gebruik vir ondersoeke in sommige broeikolonies, met die doel om te bepaal watter rol voëlmalaria in die oorlewing van die Afrika pikkewyn in die natuur speel. Resultate het getoon dat olie nie die vermoë van die pikkewyn beïnvloed om anti- Plasmodium antiliggame te vervaardig nie en dat malaria infeksie hoofsaaklik deur muskiete veroosaak word en nie deur heruitbraak van ‘n bestaande infeksie nie. Dit dui egter daarop dat pikkewyne blootgestel word aan voëlmalaria by die SANCCOB sentrum. Daar is ook gevind dat ‘n groot aantal pikkewyne met malaria infeksies by die sentrum opgedaag het wat dui op die voorkoms van malaria in die broeikolonies. Ondersoeke in die broeikolonies het ‘n besonder hoë voorkoms van malaria onthul. Geen vrektes of siek pikkewyne is in die broeikolonies waargeneem nie, wat moontlik kan beteken dat pikkewyne by SANCCOB met ‘n ander tipe malaria geïnfekteer word as in die broeikolonies.