Cryptosporidiosis is an infection caused by Cryptosporidium; a protozoan parasite that infects the gastrointestinal tract. The infection is of major public health concern in both developed and developing countries. Faecal samples were collected from 160 in-patient adults, with complaint of diarrhoea, admitted at Victoria hospital in Alice, Nkonkobe Municipality. Twenty apparently healthy subjects were included as controls. All diarrhoea positive patients were interviewed to record socio-demographic information, water supply and animal contact. Initial screening was carried out by microscopy and ELISA to detect positive Cryptosporidium. Genomic DNA was extracted from microscopically positive samples and a PCR reaction was perform to amplify the (18S) SSUrRNA gene for further identification and epidemiology of Cryptosporidium. Data were analysed using Pearson‘s χ2 and Fisher‘s exact test to assess the univariate association between Cryptosporidium infection and the possible risk factors. Of the 180 subjects screened for cryptosporidial infection, Cryptosporidium antigen was detected in 122 giving an overall prevalence of 67.8 percent. In HIV-positive diarrhoea patients, prevalence increased with ages; between 31-43 (mean age 36.5 yr) and 70-82 (mean age 75.8 yr) had a higher prevalence (100 percent) of the antigen than 18-30 (mean age 23.2 yr) and 83-95 (mean age 88.8 yr) (50.0 percent) in HIV-positive diarrhoea patients (P > 0.05). In HIV-negative diarrhoea patients, prevalence was highest in the 18-30 (mean age 23.2 yr) (87.5 percent) and least (35.7 percent) in those aged 83-95 (mean age 88.8 yr) (P > 0.05). Cryptosporidium antigen was higher in females than in males. Of 115 females (mean age 46.7yr) who participated in the study, antigen was detected in 90 (78.2 percent) against 32 (71.1 percent) of 45 males (mean age 42.6yr). None of the 20 apparently healthy control subjects was found to be infected with Cryptosporidium. Cryptosporidium was detected in 27 HIV-positive and 97 HIV-negative diarrhoea patients by any one of the techniques. Antigen detection by ELISA 14 showed the highest positivity 96 (76.8 percent) in HIV- negative and 26 (74.3 percent) in HIV- positive diarrhoea patients. PCR detected eighty-nine (71.2 percent) cases in HIV-negative and 23 (65.7 percent) in HIV-positive patients with diarrhoea. Only 13 (37.1 percent) HIV-positive and 34 (27.2 percent) HIV-negative diarrhoea patients were found positive for Cryptosporidium by modified ZN. No significant difference was observed in sensitivity of antigen detection by ELISA and PCR (96.9 percent) in HIV-negative diarrhoea patients, respectively. Specificity of the staining technique was 88.9 percent in HIV-positive and 96.6 percent in HIV-negative diarrhoea patients. No significant difference was found in specificity of antigen detection by ELISA and PCR in HIV-positive and HIV-negative diarrhoea patients, respectively. Positive predictive value of ZN staining in both HIV-positive and HIV-negative diarrhoea patients (92.3 and 96.9 percent) was statistically higher than ELISA and PCR. No significant difference was observed in negative predictive value of ZN technique for detection of Cryptosporidium between HIV-positive and HIV- negative diarrhoea patients. Differences found in prevalence rates due to water source, suggest that the high infection rates of specific groups are associated with their exposure to the contaminated water supply. The results indicate that Cryptosporidium infection is highly prevalent in adult faecal specimens in the Nkonkobe Municipality, an indication of active infection that is likely to emerge as major human pathogen in this location due to socioeconomic changes which favour transmission. However, sequencing analysis is required to differentiate between Cryptosporidium genotypes in the various outbreaks

MSc (Biochemistry) / Department of Biochemistry / Malaria is caused by protozoa of the genus Plasmodium. Malaria accounts for approximately more than half a million deaths yearly. Of the five species of Plasmodium, P. falciparum accounts for the most deadly form of the disease. P. falciparum survives under various physiological conditions during its life cycle. The parasite employs its molecular chaperones machinery particularly heat shock proteins (Hsps) to protect its protein constituents during physiological stress. Hsps are conserved molecules that constitute a major part of the cell’s molecular chaperone system. P. falciparum Hsps play an important cyto-protective role guaranteeing that the malarial parasite survives under the severe conditions that prevail in the host environment. PFF1010c is a type IV P. falciparum heat shock protein 40. PFF1010c is predicted to be expressed only at the gametocyte stage of the malarial parasite’s life cycle. The aim of the current study was to investigate the expression PFF1010c by parasites and the gametocyte stage as well as characterize the structure-function features of the protein. PFF1010c was successfully expressed in E. coli cells. Despite successful expression of the protein, its purification proved problematic. The attempt to purify PFF1010c was carried out under both native and denaturing conditions. Far Western blot analysis to investigate direct interaction between PFF1010c and PfHsp70-1 was conducted and no interaction was observed. Malarial parasites were harvested at different stages and total protein was isolated. The expression of PFF1010c was confirmed to occur at the gametocyte stage of the parasite’s development using Western blot analysis. This study confirmed that PFF1010c is only expressed at the gametocyte stage of the malarial parasite. Furthermore, PFF1010c was not expressed at the asexual stage. Possible interactors of PFF1010c were predicted by STRING, a bioinformatics based tool. The expression of PfHsp90, PfHop and PfHsp70-1 at the gametocyte stage was investigated and confirmed by Western blot analyses.

The pathology of canine cerebral babesiosis was examined at the gross, histological and ultrastructural levels. Gross lesions could be categorised as either global or regional. Congestive brain swelling , diffuse cerebral congestion and diffuse cerebral pallor were classified as global lesions. Multifocal haemorrhage and malacia were classified as regional lesions. Oedema was inconsistently present and could be either focal or diffuse. The majority of histological changes were observed in both cerebral babesiosis and control cases. Regional lesions were unique to cerebral babesiosis and had specific histological features. Highly localised endothelial injury was the primary lesion. Early lesions were multifocal and strictly associated with the microvasculature. Intermediate lesions, with perivascular haemorrhage and neutrophil infiltration, were suggestive of reperfusion injury. Advanced lesions were locally extensive and similar in appearance to haemorrhagic infarction. It is likely that the pathogenesis of regional lesions is by a process of microvascular infarction, as venous thrombosis could not be demonstrated. Ultrastructural evidence for adherent contact between erythrocytes and capillary endothelium was demonstrated. Endothelial cell necrosis occurred early in the development of lesions, before neuronal and glial injury. It is postulated that endothelial injury is the primary event in the development of regional lesions and secondary lesions develop as a consequence of microvascular infarction. / Die patologie van die serebrale vorm van bosluiskoors in honde is ondersoek. Die letsels is makroskopies, histologies en elektronmikroskopies beskryf. Letsels kon makroskopies in twee groepe verdeel word: Globale letsels en gelokaliseerde letsels. Kongestiewe brein swelling, diffuse serebrale kongestie en serebrale anemie kom voor as globale letsels in serebrale babesiose. Multifokale bloeding en nekrose kom voor as gelokaliseerde letsels. Edeem was nie konsekwent teenwoordig nie, en was algemeen of verspreid. Die meeste algemene histologiese veranderinge was in beide serebrale en kontrole gevalle teenwoordig. Gelokaliseerde letsels waarin spesifieke hisotpatologiese veranderinge voorgekom het, was kenmerkend van serebrale babesiose. Die primere letsel is hoogs gelokaliseerde beskadiging van endoteelselle. Beskadiging van die kapillere bloedvate ontstaan vroeg in die ontwikkeling van letsels. Verdere ontwikkeling van die letsel word gekenmerk deur peri-vaskulere bloeding en neutrofiel infiltrasie wat aanduidend is van reperfusie beskadiging. Volontwikkelde letsels is plaaslik-ekstensief en het die voorkoms van hemoragiese infarkte Dit is waarskynlik dat mikrovaskulere infarksie 'n rol speel in die patogenese van die letsels, aangesien veneuse trombose nie ontstaan nie. Noue kontak tussen rooibloedselle en kapillere endoteel is elektronmikroskopies bevestig. Endoteelselnekrose ontstaan voordat tekens van beskadiging geidentifiseer kan word in neurone of gliaselle. Dit blyk dat kapillere endoteelselbeskadiging die primere letsel by die ontstaan van gelokaliseerde lese Is is, en dat sekondere lesels ontwikkel as gevolg van mikrovaskulere infarksie. / Dissertation (MSc)--University of Pretoria, 2000. / Paraclinical Sciences / Unrestricted

Beca JAE-Predoctoral CISC; Proyecto LARECO CTM2011-25929

Giardia lamblia, a worldwide cause of diarrhoea, must differentiate into environmentally resistant cysts for dissemination and completion of its life cycle. Although G. lamblia is an early diverging eukaryote, encystation involves many complex cellular changes including formation of the cyst wall that contains at least two cyst wall proteins, cyst wall proteins 1 and 2. Cwp genes are transcribed only during encystation. In this study, we examine the regulatory elements for the encystation-specific gene cwp2. The 64 bp immediately upstream of the cwp2 open reading frame (-64 to -1 relative to ATG) was shown to be sufficient for the encystation-specific expression of luciferase. To determine which region(s) within this 64 bp contributed to encystation-specific expression in vivo, a series of deletions were cloned into a Giardia luciferase expression vector and their ability to control encystation-specific expression of luciferase was assessed. Deletion of elements in the -64 to -23 region of the cwp2 promoter significantly increased expression of luciferase in vegetative trophozoites, suggesting that this area contains a negative cis-acting element. Deletions of elements from -23 to -10 led to decreased expression in encysting cells, suggesting that this region may contain positive cis-acting elements. When the A/T-rich initiator was deleted but the cis-acting elements (-64 to -10) were retained, encystation-specific expression of luciferase was maintained but an aberrant transcriptional start site was utilised. These results indicate that Giardia has developed a classic repressor mechanism(s) that allows tight, encystation-specific control by the cwp2 promoter.

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Seasonal effects on protozoan colonization of polyurethane foam (PF) units were investigated in.an oligotrophic lake, Mountain Lake, Virginia. PF units were placed into the lake's pelagic water to simulate barren habitat islands suitable for colonization by protozoa. The results were interpreted with respect to the MacArthur-Wilson equilibrium theory of island biogeography. Results showed seasonal changes can cause a substantial amount of instability in colonization curves. Within a season, changes in the lake's planktonic species pool were more prominent during the decay of the thermocline than during Fall or Spring overturn and Summer stratification. For different seasons, the relationship between changes in the lake's planktonic species pool and species accrual on PF units was positive, for total species and for ciliate species. For different seasons, the relationship between changes in colonization rate and species accrual was consistent with MacArthur-Wilson theory. In contrast, for different seasons, the relationship between changes in equilibrium number of species and species accrual was inconsistent with MacArthur-Wilson theory, and this places in question the interseasonal predictive value of colonization curves. For some situations where the MacArthur-Wilson equation for insular colonization did not describe adequately the colonization a modified equation was developed. The modified equation was used to account roughly for species which may rapidly colonize and persist in PF unit communities and described adequately more total species colonization curves than the unmodified equation. Moreover, the modified equation was a useful tool for the interpretation of PF unit colonization by protozoa. / Master of Science

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