Bacillus thuringiensis subsp. israelensis (B.t.i.) produces a plasmid encoded parasporal crystalline protein which is larvacidal to mosquitoes carrying parasites for malaria and other infectious diseases. The purpose of this study was to construct a library of random fragments from the nine plasmids of wild type B.t.i. strain 402. The library was to be utilized in order to clone a 135kDa mosquitocidal polypeptide carried on a 108 kb B.t.i. plasmid.The library construction involved isolation of plasmid DNA by equilibrium density centrifugation, generation of random fragments of the nine plasmids by a partial Sau3A restriction digest, and ligation of these fragments into XbaI-BamHI restricted Lambda GEM-11 vector. Escherichia coli strain LE392 was infected by the packaged recombinant lambda and over 1000plaques were pooled to comprise the library. In order to verify construction of the library, both plaque screens of the library and Southern Analysis of restricted clones subjected to agarose gel electrophoresis were performed with labeled probes. The labeled probes were included: 1) radioactive end-labeled oligonucleotides constructed from published sequences of the B.t.i. 135 kDa toxic protein, 2) radioactive end-labeled random fragments from all nine plasmids of B.t.i., 3) radiolabeled entire plasmids of all nine plasmids of B.t.i., and 4) dioxigenin-labeled oligonucleotides. No homology between the lambda library digested DNA and the B.t.i. plasmid was observed. The results suggested that no lambda library of B.t.i. was constructed and, therefore, a lambda clone encoding the 135 kDa mosquitocidal polypeptide was not isolated. / Department of Biology

Thesis (M.Sc.(Med.))--University of the Witwatersrand, Faculty of Health Sciences, 2013.

A Thesis submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg for the Degree of Doctor of Philosophy / This thesis describes the characterisation of southern African isolates of Plasmodium falciparum according to isoenzyme type, antigen variants and drug sensitivity. Nineteen southern African isolates and a Gambian reference isolate were cultured in vitro in gassed tissue culture flasks. Polyacrylamide gel electrophoresis with use of 5 enzymes revealed little variation amongst the isolates and the frequencies of enzyme forms were similar to those of isolates from other parts of the world. Antigenic diversity was demonstrated using a panel of 9 monoclonal antibodies in the indirect fluorescent antibody test. The antigenic composition of 70% of isolates was markedly different to any obtained in other geographical areas. Both characterisation techniques revealed a mixture of parasite types in some isolates. However I the characteristics of most of these heterogeneous isolates were not stable with time in culture. Most isolates proved to be resistant to chloroquine in a 48-hour growth inhibition test. A wide range in Pyrimethamine susceptibilities was detected although most isolates exhibited a low level of resistance to this drug. In the radioisotope uptake assay, the halofantrine IC50 values obtained were comparable with those of West African isolates but were higher than the TC50s of south-East Asian isolates. All of the isolates resistant to chloroquine in the 48-hour test were resistant in the radioisotope assay. However, 1 isolate shown to be sensitive to chloroquine in the first test was found to be resistant in the 3Hhypoxanthine incorporation method. This was assumed to be the result of selection of resistant clones. Southern African isolates were shown to be fully susceptible to mefloquine, but had reduced susceptibility to quinine and sulphadoxine/ pyrimethaInine. Most isolates were sensitive to amodiaquine. A field study of 31 KwaZulu isolates of P. falciparum obtained in 1987 and 1988 showed 29 to be resistant to chloroquine in the 24-hour microtechnique, with the majority being highly resistant. Chloroquine resistance was confirmed in this area in a field study carried out in 1989 but in 1990 all 4 isolates tested were sensitive to the drug. In 1989, 12 out of 13 KwaZulu isolates were resistant to amodiaquine and most isolates showed reduced susceptibility to quinine and sulphadoxine/ pyrimethamine. / Andrew Chakane 2018

Typescript, etc. / Digitized by Kansas Correctional Industries

Thesis (M. Sc.(Microbiology)) -- University of the North, 1987 / Refer to the document

A research report submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in partial fulfilment of the requirements for the degree of Master of Science in Biology and Control of African Disease Vectors. Johannesburg, February 2012 / In Tanzania both Anopheles funestus and An. gambiae s.l. play a role as major vectors of malaria. Different species exist in the An. funestus group and the An. gambiae complex and play different roles in disease transmission. For malaria vector control programmes knowledge of vector species and their behaviour is key. A recent report on increased exophagy of An. funestus in southern rural Tanzania as a response to increased use of insecticide treated bed nets raised the question of whether there was misidentification of species and/or behavioural insecticide resistance playing a part. The present study used molecular tools to identify the species and determine human biting rates indoors and outdoors along with development and field evaluation of a new tool for sampling malaria vectors which is more effective than human landing catches. The results showed that the majority (96.2%) of the An. funestus group that were collected were An. funestus s.s. by PCR assay. Also, the exophagic proportion (45.9%) of An. funestus was lower than the endophagic proportion (54.1%), similar to other places in Africa, although in this study the difference was insignificant when untreated bed nets and treated bed nets were used. In addition, there was significant outdoor biting activity early in the evening that could lead to the malaria transmission cycle being unaffected by ITNs. The new trap, “Sticky Bucket Trap”, caught considerably fewer mosquitoes (14.2%) than that caught by human landing catches (85.8%), with statistical significance of P>0.05. These results imply that the sticky bucket trap is not a suitable substitute for human landing catches and some modifications are needed to make it more effective. Whereas indoor and outdoor proportions insignificant difference in feeding preference imply that both indoor and outdoor interventions should be used to control this vector.

In order to employ the Sterile Insect Technique (SIT), biologically fit mosquitoes able to compete with their wild counterparts, suitable field sites for mass release of sterilized male mosquitoes, and appropriate methods of rearing genetic sex-separation (GSS) mosquito strains are required. The life history traits and biological fitness of four laboratory-reared, southern African Anopheles arabiensis strains were investigated. Despite being colonized at different times, the four strains demonstrated comparable levels of biological fitness. Three sites in the Kruger National Park were assessed as possible SIT field sites. Mosquito collections were conducted at each site during three summer months. Anopheles arabiensis was predominant at Malahlapanga during each collection period, establishing Malahlapanga as the most appropriate site for SIT field trials. A standard larval diet was shown to be appropriate for rearing An. arabiensis GSS. This work formed the laboratory basis for the evaluation of a SIT strategy for South Africa.

Malaria is transmitted by the mature, blood feeding portion of mosquito vector populations. Malaria vector control programs based on indoor residual spraying (IRS) of insecticides are designed to target resting adult Anopheles mosquitoes before or after they have blood fed. When a female mosquito acquires a blood meal, she could also ingest harmful xenobiotics that are present in the blood. During the resting period after feeding, many processes are initiated in order to assist in the digestion and assimilation of the blood. Ultimately, this enables the mosquito to absorb those amino acids needed for the biosynthesis of yolk proteins, which are essential for subsequent egg maturation. Since the regulation of xenobiotic (including insecticides) detoxification enzyme systems is likely to be altered in response to the ingestion of blood, this study aimed to investigate the effect of a blood meal on insecticide tolerance in insecticide resistant and susceptible southern African strains of the major malaria vector Anopheles funestus. Through the use of CDC bottle bioassays it was demonstrated that blood fed An. funestus carrying a pyrethroid resistant phenotype are even more tolerant of pyrethroid intoxication than their unfed counterparts. Using another major malaria vector, An. gambiae, microarray analysis revealed that a general increase in delta class glutathione-s-transferase (GST) expression occured in response to a blood meal. One gene, GSTD3, was over-expressed in both blood fed An. gambiae and An. funestus. Although this gene could not be validated with real time quantitative PCR, it serves as a viable target for future investigations. Since the pyrethroid resistant phenotype of southern African An. funestus has been linked to the over-expression of the duplicate copy gene CYP6P9, the expression levels of both copies of this gene were investigated. CYP6P9 and its copy, CYP6P13, showed a small but significant increase in expression in response to a blood meal. The increased expression of these major effect genes in response to blood feeding may be responsible for the increase in insecticide tolerance seen in the bottle bioassays. In an effort to repeat these experiments on wild caught An. funestus, field material was collected from Karonga in northern Malawi. Specimens were morphologically identified as members of the An. funestus group. However, attempts to molecularly identify them to species level failed. Through the use of ITS2 and D3 sequence analysis, cytogenetics and cross mating studies it was possible to conclude that these wild caught specimens were a new species. They have been provisionally named An. funestus-like.

Anopheles funestus is a major vector of malaria in the southern African region. Insecticide resistance to pyrethroid and carbamate insecticides has been recorded in populations of this species in South Africa and Mozambique. This study aimed to determine the relationship between pyrethroid resistance and gene expression of two closely related genes, CYP6P9 and CYP6P13, by age and sex in a resistant strain An. funestus from southern Africa, FUMOZ-R. The insecticide susceptibility assays showed that percentage survival in both FUMOZ-R sexes significantly decreased as age increased. The mRNA expressions of CYP6P9 and CYP6P13 were higher in FUMOZ-R relative to the insecticide susceptible strain (FANG). The expression of permethrin resistance varies with age in An. funestus FUMOZ-R. The results indicate that other genes may also be involved in insecticide resistance. In addition to this, the expression profile of other metabolic genes involved in insecticide resistance was also investigated. A microarray based approach was used to identify genes differentially expressed in FUMOZ-R and FANG. As the full set of detoxification genes in An. funestus are unknown, this study investigated the utility of the An. gambiae detox chip to screen for differentially expressed detoxification genes in An. funestus. After optimization of the hybridisation conditions, over 90% of the probes showed a positive signal. Only three genes were significantly (P<0.001) differentially expressed in the females, CYP6P9, COI and CYP6M7. The same genes were also significantly differentially expressed in the adult males, together with an additional 21 transcripts. The third part of this study investigated the gene expression in the first instar, fourth instar and 3-day old adults in FUMOZ-R using the An. gambiae detox chip. The variation in metabolic enzyme gene transcription at the different developmental stages in An. funestus are not known. The identification of differentially transcribed genes at the different life stages provides some insight into the role and function of these genes. A large number of cytochrome P450s (monooxygenases), esterases, glutathione S-transferases (GSTs) and other additional genes were differentially expressed in all life stages. This study provided vital information regarding genes potentially involved in pyrethroid resistant and is the first to provide metabolic or detoxifying transcription gene information in An. funestus.

A thesis submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg In fulfilment of the requirements for the Degree of Master of Science June, 30, 2015 / The world suffers under a serious threat of malaria with about 584 000 deaths reported each year and most of these fatalities being children under five years of age. Malaria is caused by the protozoan parasite of the genus Plasmodium. Five different malaria species infect humans and cause disease: P. vivax, P. malariae, P. ovale, P. knowlesi and the cause of most malaria deaths, P. falciparum. The main reason for this disturbing situation is the emergence of drug resistance which reduces the effectiveness of most antimalarials. Hence, there is an urgent need for new drugs that will possibly be effective against both wild type and mutant strains of Plasmodium species. Pyrimethamine, a dihydrofolate reductase (DHFR) inhibitor, has been used most widely as an antimalarial antifolate drug for the treatment of malaria. However, rapid development of parasite resistance to this drug occurred because of its rigidity. Parasitic resistance to antimalarial antifolates arises from single mutations at various amino acid residues surrounding the PfDHFR active site. In this project, we aimed to design and synthesise a novel series of flexible pyrimidine analogues of a dihydrotriazine hit compound prepared in a previous study. These compounds were designed to target folate metabolism in the malaria parasite. The initial series of compounds prepared in this project were synthesised over 5 steps in an overall yield of 10%. The flexible pyrimidine analogues were screened for antimalarial activity in an in vitro P. falciparum screen on the Gambian FCR-3 strain (chloroquine and cycloguanil resistant strain) with dihydroartemisinin, methotrexate and quinine as controls. 5-(3-(3,5-Dichlorophenoxy)propyl)-6-phenylpyrimidine-2,4-diamine displayed the best antimalarial activity (IC50 = 0.09 μM) of the compounds in this series. Surprisingly; this was the only compound prepared in this series that proved to be as effective as our original hit dihydrotriazine (IC50 ~50 nM). In the second generation of compounds prepared in this study, we used a multicomponent coupling approach to synthesise three flexible pyrimidines bearing a non-aromatic side chain at the 6-position of the pyrimidine ring. For comparison, two analogues bearing a phenyl group at the 6-position of the pyrimidine ring were also prepared. Once again; only one compound of this series [5-((4-chlorophenethylamino)methyl)-6-cyclopropylpyrimidine-2,4-diamine, (IC50 = 0.03 μM)] showed activity comparable with our original hit compound. iv Finally, ten substituted pyrimidines bearing a flexible side chain at the 6-position of the pyrimidine ring, were prepared. These compounds are structurally similar to P65, [6-methyl-5-(3-(2,4,5-trichlorophenoxy)propoxy)pyrimidine-2,4-diamine] an analogue of a potent antifolate, WR99210, found to have good oral bioavailability in rats. Once again, the antimalarial activity of the compounds prepared was assessed in an in vitro P. falciparum screen on the Gambian FCR-3 strain. The most promising compound of this series was 6-(3-(3,4-dichlorophenoxy)propoxy)pyrimidine-2,4-diamine, which exhibited antimalarial activity in the low micromolar range (IC50 = 4.46 μM).