Thesis (Ph.D.)--University of Delaware, 2006. / Principal faculty advisor: Adam G. Marsh, College of Marine and Earth Studies. Includes bibliographical references.

Techniques for the large-scale isolation of Cryptosporidium oocysts and sporozoites, obtained from the feces of experimentally infected Holstein calves, were developed employing discontinuous sucrose gradients and isopycnic Percoll gradients. The three step oocyst recovery method utilized two sequential discontinuous sucrose gradients followed by one Percoll gradient. Recovered oocysts were essentially free of debris and bacteria and represented 34% of the original oocyst suspension. Sporozoites were recovered from excystation mixtures on a single Percoll gradient. Sixty-three percent of the original sporozoites were recovered with 2.2% contamination by intact oocysts and virtually no oocyst walls. Eight anti-oocyst hybridomas were derived from oocyst-immunized mice: five from BALB/c mice and three from RBF/Dn mice. The monoclonal antibody (Mab) OW3 reacted specifically with C. parvum oocysts in immunofluorescent assays (IFA) and was shown to be superior to conventional stains for detecting oocysts in fecal smears from infected individuals. Sixteen anti-sporozoite hybridomas were derived from sporozoite-immunized BALB/c mice. The Mabs appeared to react with cell surface and cytoplasmic antigens by IFA. Two anti-sporozoite Mabs (C8C5, C6B6) reacted with a 20 kDa sporozoite antigen in western blots while the Mab C4A1 reacted with multiple antigens in western blots. These three Mabs (C8C5, C6B6, C4A1) were examined for potential modulation of cryptosporidial infections in vivo by oral Mab administration to oocyst-inoculated neonatal mice. The role for colostrum and breast milk in controlling cryptosporidial infections was examined by immunizing mouse dams and experimentally infecting their neonatal offspring. Colostrum and Mab-treated neonatal mice were sacrificed four days post infection. No difference in infection rates was observed among the treatment groups. Suckling mice treated daily with orally administered mixtures of Mabs (purified or ascitic fluid) showed significantly reduced parasite loads compared to control mice at four days post infection. In vitro cultivation of C. parvum was successful through asexual stages in human fetal lung, bovine turbinate and murine L929 cells. Parasite numbers that developed in the cell cultures varied from infection run to infection run.

Thesis (Ph. D.)--University of Texas at El Paso, 2007. / Title from title screen. Vita. CD-ROM. Includes bibliographical references. Also available online.

No description available.

Thesis submitted to the Division of Veterinary and Biomedical Sciences. Bibliography: leaves 237-283.

Thesis (Ph.D.)--Murdoch University, 2002. / Thesis submitted to the Division of Veterinary and Biomedical Sciences. Bibliography: leaves 237-283.

M.Sc. / In this research project, a variety of sites and ecosystems were studied. These ranged from tropical coral reefs (north-eastern Coast of Australia), to warm temperate intertidal pools (South Coast of South Africa, SA) and sub-tropical estuaries (East Coast of SA). The overall aims of the thesis were to examine the haematophagous gnathiid ectoparasites and blood protozoans of some host teleosts found in these systems, and to some extent to investigate the role gnathiids might play as vectors of the protozoans. Gnathiid research in Australia on the Great Barrier Reef (GBR) focuses mainly on gnathiid ecology and not taxonomy. This is not the case in SA, where gnathiid taxonomy is researched more regularly and gnathiid ecology has received little attention. In this thesis, two new gnathiid species, Gnathia aureamaculosa and Gnathia sp. B, were described from a number of teleost fishes of the GBR and several morphological features, including live colouration patterns, were highlighted as useful in future gnathiid identification and discrimination. The feeding ecology of Gnathia africana from SA was also examined and this feeding study was based on similar work done on coral reef gnathiids in Australia. Gnathiids of the GBR are mainly nocturnal due to cleaner fish predation during the day, whereas G. africana was found to have a preference for dawn/early morning/midday feeding on an intertidal teleost, Clinus superciliosus. Gnathia africana‟s behaviour/feeding patterns, especially of its different juvenile stages, are therefore determined by time of day, and likely by locality and predation by other organisms, such as fishes, though probably not by cleaner fish. Gnathiid feeding behaviours/patterns are thus, it seems, determined by environmental and biological factors, and these vary according to the type of ecosystem studied. Several gnathiids and fish blood protozoan species are known from the South Coast of SA, but the East Coast has remained largely unexplored. Sampling along the East Coast yielded the first records of haemogregarines from the blood of fishes in this region, in particular new hosts and locality records probably for both probable Haemogregarina bigemina and a Haemogregarina quadrigemina – like haemogregarine. However, both haemogregarines displayed unusual features compared with the original species descriptions, in size, development patterns, or effect on host cells. Limited data suggested that juveniles of Gnathia pilosus were possible haematophagous vectors of these haemogregarines, but further studies are required to confirm this.

Protozoan infections such as Malaria, Leishmaniasis, Toxoplasmosis, Chaga’s disease and African trypanosomiasis caused by the Plasmodium, Leishmania, Toxoplasma and Trypanosoma genuses respectively; inflict a huge economic, health and social impact in endemic regions particularly tropical and sub-tropical regions. The combined infections are estimated at over a billion annually and approximately 1.1 million deaths annually. The global burden of the protozoan infections is worsened by the increased drug resistance, toxicity and the relatively high cost of treatment and prophylaxis. Therefore there has been a high demand for new drugs and drug targets that play a role in parasite virulence. Cysteine proteases have been validated as viable drug targets due to their role in the infectivity stage of the parasites within the human host. There is a variety of cysteine proteases hence they are subdivided into families and in this study we focus on the clan CA, papain family C1 proteases. The current inhibitors for the protozoan cysteine proteases lack selectivity and specificity which contributes to drug toxicity. Therefore there is a need to identify the differences and similarities between the host, vector and protozoan proteases. This study uses a variety of bioinformatics tools to assess these differences and similarities. The Plasmodium cysteine protease FP-2 is the most characterized protease hence it was used as a reference to all the other proteases and its homologs were retrieved, aligned and the evolutionary relationships established. The homologs were also analysed for common motifs and the physicochemical properties determined which were validated using the Kruskal-Wallis test. These analyses revealed that the host and vector cathepsins share similar properties while the parasite cathepsins differ. At sub-site level sub-site 2 showed greater variations suggesting diverse ligand specificity within the proteases, a revelation that is vital in the design of antiprotozoan inhibitors.

Giardia lamblia, an enteric protozoan parasite, infects a large number of individuals worldwide. In South Africa prevalences ranging between 4 and 63% are documented, however, the impact of giardiasis is underreseached in this country. Giardia infections vary from asymptomatic carriage or a self-limiting acute symptomatic illness to chronic, debilitating malabsorption syndrome. The factors responsible for development of symptomatic versus asymptomatic infection are poorly understood. It is believed by some that host factors determine the clinical outcome of infection. On the other hand, the possibility of the existence of pathogenic and non-pathogenic strains (a situation akin to Entamoeba spp.) remains to be explored. One requirement for investigation of the potential contribution of strain differences to pathogenecity of infection is establishment of laboratory cultures of different strains isolated from symptomatic and asymptomatic patients. The present study was undertaken to develop and modify existing methods for: (i) establishment of laboratory cultures of Giardia trophozoites from excystation of faecal cysts, (ii) long-term maintenance and cryopreservation of the cultures and (iii) preliminary characterisation methodology. One thousand and twenty-three stool specimens were collected from day care centres, hospital wards and Hlabisa hospital laboratory. A further 6246 were retrieved from the Microbiology Laboratory at King Edward VIII Hospital and screened by direct wet preparation. Giardia was detected by light microscopy following formol-ether concentration (127 of 1023 samples) or direct examination of wet preparations (78 of 6246 samples). Cysts were purified from the positive specimens by sucrose gradient separation. Viability was assessed by a dye-exclusion method (eosin). Three in vitro excystation techniques were employed in an attempt to obtain trophozoites for initiation and establishment of viable cultures thereof. Culture conditions were optimised using two reference strains of Giardia, WB & H7 (obtained from the National Institutes of Health, USA). The percentage excystation ranged between 0-42% with all the in vitro methods of excystment. Excysted trophozoites remained viable in TYI-S-33 culture medium for periods ranging between 12-72 hours or up to 9 days, and gradually died, hence viable trophozoite cultures could not be established. Some culture initiates (overall 65%) were lost through overwhelming bacterial and!or fungal contaminants. An animal model was subsequently set up in which C57BL/6 and Praomys (Mastomys) coucha mice were used for in vivo excystation experiments. 1-3 day old suckling mice were intragastrically injected with 10,5 -cysts/ ml in 0,1 ml distilled water. Trophozoites were retrieved from the stomachs of infected mice 7-10 days after inoculation and cultivated in TYI-S-33 medium. Six local isolates were axenised using the in vivo excystation method. They have been maintained for more than 15 months in culture after stabilates and Iysates of confluent growths had been cryopreserved in Liquid Nitrogen. Successful (100%) retrieval of the cryopreserved cultures has been achieved. Seven isoenzyme electrophoresis systems have been set up and optimised. Reproducible results were obtained in six of the enzymes. Some differences in banding patterns of the enzymes were demonstrated. / Thesis (M.Med.Sc.)-University of Natal, Durban, 1999.

Thesis (Ph. D.)--University of Texas at El Paso, 2009. / Title from title screen. Vita. CD-ROM. Includes bibliographical references. Also available online.