Dissertationen zum Thema „Protozoa, Pathogenic“

The purpose of the present study was to evaluate the potential usefulness of tubulin inhibitors when complexed with hydroxypropyl-beta-cyclodextrin (HP-beta-CD) against a range of protozoan parasites. This approach involved investigations into the complexation of these drugs with HP-beta-CD, and subsequent investigations of these drugs and their complexes in regard to cytotoxicity, pharmacokinetics, in vitro efficacy against Giardia, Cryptosporidium and rodent malaria (Plasmodium chabaudi), and their in vivo efficacy against Giardia and malaria. Albendazole (ABZ) is a benzimidazole carbamate with a broad anti-parasite spectrum, while the dinitroanilines trifluralin (TF) and oryzalin (OZ) have recently been found to exhibit activity against certain parasites. All three compounds are microtubule antagonists in either nematodes or weeds and have poor aqueous solubility, with the solubility of ABZ and OZ dependent on pH. Cyclodextrins (CD) have a hydrophobic cavity that allows them to form inclusion complexes with hydrophobic drugs, resulting in increased drug aqueous solubility, and often, improved drug dissolution and bioavailability. Thus the complexation of these drugs with HP-beta-CD was investigated. All three compounds exhibited type AL phase solubility diagrams with HP-beta-CD complexation, with additional increases in ABZ and OZ solubility achieved through the manipulation of temperature and pH. OZ displayed a stronger interaction with HP-beta-CD when ionised over its neutral form. However, insufficient concentrations of the TF/HP-beta-CD complex were achieved for drug efficacy studies. The cytotoxicity of the drugs and their complexes was assessed using the assay kit Cytotox 96 with human carcinoma cells. This is a colourimetric assay that measures lactate dehydrogenase release as a consequence of compromised cellular and membrane integrity. Both ABZ and OZ are cytotoxic to rapidly proliferating and differentiating cells but are not cytotoxic to cells in the stationary phase. Complexation did not affect drug cytotoxicity. In pharmacokinetic studies, complexation improved ABZ (and metabolites) bioavailability, but had no significant affect on OZ bioavailability. In vitro drug assessment studies found ABZ to be highly effective against Giardia, and effectiveagainst Cryptosporidium and malaria. OZ on the other hand exhibited no activity against Giardia, but was effective against Cryptosporidium and malaria. Complexation did not improve the antiprotozoal efficacy of either ABZ or OZ. In particular, excess HP-beta-CD decreased the antigiardial effects of ABZ, possibly due to competitive complex formation. In addition, complexation did not improve the antiprotozoal effects of ABZ in vivo. However, the cytotoxic effect of the ABZ/HP-beta-CD complex was more evident in the treatment of malaria in vivo, resulting in increased anaemia and suppression in weight gain, due to the improved bioavailability of ABZ and metabolites. HP-beta-CD alone was found to be cytotoxic at greater than 2.5%, and inhibited Giardia both in vitro and in vivo at greater than 1% and 2% respectively. This was attributed to membrane disruption caused by the dissolution and removal of membrane components. In comparison, malaria grew better in the presence of HP-beta-CD in vitro, with no detrimental effect observed at up to 8% HP-beta-CD. This was attributed to either the increased solubilization of a necessary media component, or the complexation and removal of an inhibitory compound from the cultivation medium. Therefore HP-beta-CD complexation did not improve the antiprotozoal activity of the tubulin antagonists ABZ and OZ. However, the results of the pharmacokinetic studies suggest that anthelmintic activity of ABZ, particularly against systemic infections, may be improved with oral administration of the ABZ/HP-beta-CD complex. In addition, the antiparasitic activity of HP-beta-CD alone may be promising, especially against intestinal infections. Finally, the improved in vitro cultivation of P. chabaudi in the presence of HP-beta-CD presents a promising approach to its potential long term cultivation.

Traditionally, the hygienic quality of beaches has been determined by monitoring the water for microbial indicators of fecal pollution. Beach sand, which may also be an important medium for the transmission of fecal borne pathogens, has rarely been examined. The aims of this study where to examine the prevalence of fecal indicator organisms in tidally affected beach sand and in dryer upper beach sand, relative to water; identify the potential sources of indicator organisms in beach sand; examine the prevalence of selected eukaryotic microbes at sandy beaches; and investigate the potential health risks related to beach use. Three south Florida Beaches (Ft. Lauderdale Beach, Hollywood Beach, Hobe Beach) were sampled bimonthly for a I year period. Significantly, enterococci, fecal coliform, and E. coli levels were consistently present at higher concentrations in beach sand compared to the seawater at all 3 study beaches. Levels of somatic and F-specific coliphages were also present at higher concentrations in beach sand. Microbial- source tracking analysis by carbon utilization profiling suggested that the predominate sources of enterococci in beach sand were seagulls, and transiently replicating indigenous populations. Acanthamoeba spp. was the most commonly isolated free-living naked amoeba in this study and molecular analysis revealed that 19 of the 20 beach sand clones were genotype T4, the Acanthamoeba keratitis-associated genotype. With respect to salinity, the growth characteristics of beach sand Acanthamoeba isolates were similar to Acanthamoeba isolated from corneal scrapings. Results from the beach survey indicated that beach goers may have an increased risk for acquiring contact related ailments at Hobe Beach. Accordingly, bacterial and viral fecal indicator microbes were detected at the highest frequency and greatest average concentrations from Robe Beach. Reports of enteric and respiratory related symptoms were not higher in beach goers compared to the control cohort.

Acanthamoeba causes Granulomatous Amoebic Encephalitis (GAE) and Amoebic Keratitis (AK) in humans and in its cystic form is resistant to extreme environmental conditions. Both human pathogenic water-borne viruses and free-living protozoa share the same aquatic environment. This study set out to test the ability of both Acanthamoeba and Tetrahymena to internalise and protect enteric viruses; coxsackievirus (B3, B5), poliovirus (PV) and rotavirus (RV) following co-culture. Viral uptake was assessed by infection of cultured mammalian cells, by indirect immunofluorescence (IF), and by reverse transcriptase-polymerase chain reaction (RT-PCR). The results showed that none of the free suspended viruses were internalised in Acanthamoeba or Tetrahymena. However, both coxsackievirus B3N and rotavirus Wa could be detected within Acanthamoeba by IF and confirmed by RT-PCR when the amoebae were co-cultured (fed) with virally infected mammalian cells. The co-cultured amoeba was allowed to encyst but following this procedure no viruses were detected either by cell culture or RT-PCR. In a second series of experiments, the efficacy of solar disinfection (SODIS) against viruses either alone or when co-cultured with Acanthamoeba was assessed. SODIS reduced the viral infectivity by over 3log10 after 1 h for CVB3N and over 2log10 for PV after 2 h. Repeating these experiments in the presence of riboflavin, a 6log10 reduction was observed for CVB3N after 1 h of light exposure and 6log10 after 6 h for all other viruses tested. The results suggest that Acanthamoeba does not internalise or protect viruses in suspension. However, if a virus is located with an infected mammalian cell then it may be internalised; a new potential mechanism for virus dissemination in the environment. Secondly, solar disinfection is an effective treatment method for water contaminated with viruses which is further enhanced by the addition of riboflavin. This study provides a practical example of low technology methods which could be utilised to provide safe drinking water in various circumstances.

This work relates the arginine dihydrolase and polyamine pathways to the synthesis of nitric oxide in Trichomonas vaginalis, a microaerophilic eukaryotic protozoan which is the causative agent of the sexually transmitted disease trichomoniasis. When organisms were grown overnight in the presence of 5mM difluoromethylornithine (DFMO), an inhibitor of polyamine synthesis: a combination of plasma and hydrogenosome membrane potential decreased, electron-dense inclusions appeared in greater numbers in hydrogenosomes, and the oxygen consumption rates of hydrogenosomes increased by approximately two-fold. Upon adding exogenous sources of spermine or spermidine, various effects inflicted by the presence of DFMO were virtually reversed. Based on enzyme assays conducted, polyamine depletion by DFMO also caused changes in activities of enzymes in the arginine dihydrolase pathway. An important addition to the polyamine pathway was discovered in the body of this work: the production of nitric oxide by both Giardia intestinalis, an intestinal parasite that causes giardiasis, as well as Trichomonas vaginalis. Fluorimetric detection by confocal laser scanning microscopy and flow cytometry was performed after preincubation with the NO-specific fluorogen 4-amino-5methylamino-2'7'-difluorescein (DAF-FM). Microscopy indicated population heterogeneity with respect to NO production in freshly-harvested organisms and this was confirmed by the broad distribution of fluorescence intensities in the flow cytograms. Specific activities were determined for two nitric oxide synthases from T vaginalis, one located in the cytosol and the other in the hydrogenosome, and one nitric oxide synthase from G. intestinalis, localized to the granular fraction. Bioinformatic searches confirmed the presence of two NOS genes and contain protein motifs typically associated with NOS sequences. The N- terminal domains of both genes lack a NO synthase motif and instead contain motifs normally associated with various iron sulfur proteins (Fe-hydrogenase). Implications of NO production in the evolution, biology and pathogenicity of these important parasites are discussed.

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

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

All natural aquatic systems harbour a vast variety of microorganisms. In the aquatic microbial food web, the larger microorganisms (i.e. protozoa) feed on the smaller microorganisms (i.e. bacteria and phytoplankton). An increase in nutrient availability results in changes of the microbial food web structure, like altered community composition and blooms of toxic phytoplankton. In this thesis work I hypothesised that nutrient-rich aquatic environments, with strong protozoan predation, favour the occurrence of predation-resistant bacteria like F. tularensis, and that the microbial food web may provide a reservoir for the bacterium between outbreaks. By using a size-structured ecosystem food web model it was shown that the protozoan predation pressure on bacteria, defined as protozoan predation per bacterial biomass, increases with increasing nutrient availability in aquatic systems (estimated chlorophyll a 0.2 to 112 μg L-1). This dynamics was caused by increasing growth-rate of a relatively constant number of bacterial cells, maintaining the growth of an increasing number of protozoan cells. The results were supported by meta-analysis of field studies. Thus my results suggest that protozoa control the bacterial community by predation in nutrient-rich environments. In a field study in a natural productivity gradient (chlorophyll a 1.4 to 31 μg L-1) it was shown that intense selection pressure from protozoan predators, favours predation-resistant forms of bacteria. Thus, the abundance of predation-resistant bacteria increases with increasing nutrient availability in lakes. Furthermore, I could demonstrate that the bacterium Francisella tularensis, the causative agent of tularemia, was present in eutrophic aquatic systems in an emerging tularemia area. Isolated strains of the bacterium were found to be resistant to protozoan predation. In a microcosm study, using natural lake water, high nutrient availability in combination with high abundance of a small colourless flagellate predator favoured the occurrence of F. tularensis holarctica. In laboratory experiments F. tularensis strains were able to form biofilm at temperatures between 30-37°C, but not below 30°C. In conclusion, I have shown that the protozoan predation pressure on bacteria increases with increasing nutrient availability in aquatic systems. Predation-resistant forms of bacteria, such as F. tularensis are favoured in nutrient-rich environments. The complexity of the microbial food web and nutrient-richness of the water, influence the transmission of the pathogenic F. tularensis holarctica. However, over long periods of time, the bacterium survives in lake water but may lose its virulence. The temperature-regulated biofilm formation by F. tularensis may play a role in colonization of vectors or for colonization of hosts, rather than for survival in aquatic environments.

The aim of this thesis was to provide a picture of the trypanosomosis and drug resistance prevalence in Eastern Province of Zambia, to understand the underlying factors of drug resistance (drug use habits), to improve the diagnosis of trypanosomosis in livestock and finally, to improve the diagnosis of isometamidium resistance in T.congolense. After an introductory part where available trypanosomosis and trypanocide resistance diagnostic methods are described and discussed, the body of the thesis is divided in two main sections. In the first section are presented the results of a cross-sectional and a longitudinal epidemiological survey describing the geographical distribution of trypanosomosis cases, of resistant isolates and of cattle treated with isometamidium chloride. The results of the monitoring of unsupervised treatments of cattle with isometamidium by farmers and veterinary assistants with the Isometamidium-ELISA technique are also presented. The second section describes the development of two new diagnostic methods, the first one allowing the diagnosis of trypanosome infections with high sensitivity and specificity through semi-nested polymerase chain reaction and restriction fragment length polymorphism. This is the first report of a pan-trypanosome PCR test (a single PCR test for the diagnosis of all important pathogenic trypanosomes of cattle). The second new method that was developed allows the diagnosis of isometamidium resistant T.congolense strains by PCR-RFLP. This is the first report of a PCR based diagnostic test of trypanocide resistance in T. congolense.

Doctorat en sciences, Spécialisation biologie moléculaire
info:eu-repo/semantics/nonPublished

The presence of agrochemical residues in both urban and agricultural water bodies has become ubiquitous, often producing deleterious effects in the impacted watershed including reductions in biodiversity, alterations in species interactions, and toxicity to non-target organisms. While these effects have been studied on metazoan consumers, the consequences of agrochemical contamination on microorganisms, such as bacteria, protozoa, and viruses, are poorly understood. Agrochemicals could act directly on microorganisms, including pathogens, by either facilitating their survival or decreasing their abundance. Further, a multitude of indirect effects of agrochemicals on microorganisms are possible, whereby agrochemicals alter predation, competition, or parasitism on or available nutrient to microbes. The primary method by which agrochemicals enter water bodies is through stormwater and agricultural runoff, which can also introduce agriculturally-associated zoonotic pathogens. Presently, regulatory standards utilize fecal indicator bacteria (FIB) to predict the presence of pathogens in contaminated watersheds. However, if agrochemicals have different effects on FIB and bacterial pathogens, then these regulatory standards might be confounded by the presence of pesticide residues in impacted water bodies. Additionally, if agrochemicals promote the survival of zoonotic pathogens, then the presence of pesticide residues could potentially increase risks to human health. The studies in this dissertation investigated both the direct and indirect effects of agrochemicals on the growth and survival of FIBs ( Escherichia coli and Enterococcus faecalis), zoonotic bacterial pathogens (E. coli O157:H7, and Salmonella enterica), and two virus groups (human polyomaviruses and adenoviruses). The agrochemicals utilized in these experiments are among the most prominently used in their respective pesticide classes and included the herbicide atrazine, the insecticide malathion, the fungicide chlorothalonil and inorganic fertilizer containing phosphate and fixed nitrogen. Initially, complex mesocosms containing zooplankton, phytoplankton, leaf litter, and vertebrate and invertebrate species were used to examine net (direct and indirect) effects of agrochemicals on FIB in sediments. Subsequent studies utilized experiments in simplified microcosms to detect direct or indirect effects (i.e., predation, competition or effects on nutrient resources) on FIBs and pathogens. In complex mesocosms, atrazine and fertilizer significantly increased FIB densities in the sediment; however, because of the complexity of the mesocosms, it was not possible to determine whether these results were the product of direct or indirect agrochemical effects. Simplified microcosms, limited to predominantly direct effects, as well as in vitro growth curves, revealed no direct effects of any agrochemical treatment on either growth or survival of FIB or bacterial pathogens. When algal communities were allowed to establish, however, atrazine significantly reduced both phytoplankton and E. coli densities in the water column, but increased E. coli densities within the sediments. These effects on E. coli were indirect because they required the presence of algal species. To investigate indirect effects of predation on FIBs and E. coli O157:H7, we manipulated the presence and absence of an obligate heterotroph, Tetrahymena pyriformis, a facultative heterotroph, Ochromonas danica, and natural protozoan populations. In both laboratory and greenhouse microcosm experiments, the fungicide chlorothalonil significantly reduced all protozoan populations, which resulted in increased densities of FIBs and E. coli O157:H7 because of reduced predation. Atrazine was not found to have any significant direct effect on the densities of T. pyriformis or natural protozoans; however, atrazine did significantly reduce O. danica densities in greenhouse experiments. In laboratory experiments with O. danica, atrazine treatments resulted in decreased densities of E. coli O157:H7. Presumably, atrazine prevented or reduced photosynthesis forcing O. danica to increase its predation on E. coli thus shifting its trophic level. These studies reveal that agrochemicals can have a significant effect on microbial communities, but that these effects are often indirect and mediated through alterations of nutrient resources and predation. Atrazine application reduced FIB and pathogen densities in the water column via reduction of phytoplankton and increased predation by O. danica. These data suggest that the net effects of atrazine is deleterious to FIB survival in the water column and that application of this herbicide could result in an ecosystem service, reducing the abundance of zoonotic pathogens and lessening the risk to human health. However, elevation of FIB densities was observed in the sediments when atrazine was applied. The potential resuspension of increased sediment bacteria may negate or out-weigh the deleterious effects of atrazine on bacteria in the water column. Chlorothalonil application decreased protozoan densities, lessening the stress of predation on the bacterial targets and increasing FIB and E. coli O157:H7 densities. The use of chlorothalonil may therefore have negative implications for human health risks, as the reduction in predation seems to facilitate the survival of zoonotic waterborne pathogens. Understanding the net effects of agrochemicals is important for public health, as pesticide applications can act to either maintain or diminish potential bacterial and protozoan pathogens of humans. These studies show that indirect effects of agrochemicals on non-target microbes tend to be more prominent than direct effects and can significantly impact the fate of bacterial pathogens in aquatic environments.

The withdrawal of water sources concluded the reuse of treated wastewaters, especially for non-potable purposes. Agricultural use of the reclaimed wastewaters is one of the reuse options. However health considerations of the reuse of reclaimed wastewaters for public related purposes are underestimated, since wastewaters contain a variety of microbial pathogens, which may be transmitted to workers and consumers through the crops irrigated. Of these, parasitic eggs have a special place, as they are capable of surviving in the soil for months or even years, depending on environmental conditions. There is insufficient accumulated information on the health related criteria for the reuse of treated wastewaters in Turkey. The aim of this study was therefore to determine the helminthic eggs in raw sewage and in effluents of ASKi municipal wastewater treatment plant in Ankara. The study involved examining to decide whether these organisms exist in the wastewaters at all, and if so in what concentrations. Modified Bailenger&rsquo
s method, which published in the &ldquo
WHO Laboratory Manual of Parasitological and Bacteriological Techniques&rdquo
and &ldquo
U.S.EPA ICR Microbial Laboratory Manual&rdquo
were used in developing the specific methods used in this study.

Our hypothesis proposed that Trichomonas vaginalis would induce apoptosis in host cells as part of its pathogenic assault. This hypothesis was tested in an established co-culture system; previous research demonstrated that the parasite destroyed McCoy cell monolayers. The TUNEL assay was used to identify cells undergoing apoptotic DNA fragmentation as a result of co-incubation with T. vaginalis. Early experiments suggested that apoptosis may have been occurring in the McCoy monolayers, but this was later disproved with the help of a sandwich ELISA to detect DNA/histone fragments that characteristically result from apoptosis. Instead, the cells that had originally been identified as apoptotic were found to be adherent trichomonads. While McCoy cell apoptosis in response to co-culture with T. vaginalis was not observed, T. vaginalis did, over time, cause detachment of almost all monolayer cells, and also caused irreparable damage in a portion of these cells. Induction of McCoy monolayer detachment was determined to be contact-independent, given that trichomonad culture supernatants had a similar effect. (Abstract shortened by UMI.)

Fecal indicator bacteria (FIB) such as Escherichia coli and enterococci are used to assess microbiological water quality in recreational waters worldwide. FIB are used with the assumption that their presence correlates with that of fecal-associated pathogens in recreational waters. In aquatic habitats, several factors can interfere with the predictive relationship between FIB and pathogens including extended survival of FIB in secondary habitats such as sediment, vegetation and sand. Furthermore, many biotic (e.g. predation from bacterivorous protozoa and competition from indigenous bacteria) and abiotic factors (e.g. temperature, salinity, ultraviolet (UV) light irradiation, and nutrient availability) can influence the fate of FIB and pathogens associated with gastrointestinal tracts of animals (enteric pathogens) in secondary habitats. The relative importance of these factors is not well characterized, thus limiting our knowledge on the efficacy of FIB as indicators of fecal contamination and microbial pathogens in water. The studies presented in this dissertation investigated the influence of biotic (predation from bacterivorous protozoa and competition from indigenous bacteria) and abiotic factors (e.g. nutrient availability) on the survival of FIB (E. coli and Enterococcus faecalis) and pathogens (E. coli O157 and Salmonella enterica) in aquatic habitats. Water and sediment samples were collected from a fresh water river source (Hillsborough River, Tampa, FL) and used to prepare a series of outdoor mesocosm experiments. In each experiment, biota treatments were varied to include various combinations of predation and competition, both or neither. Manipulation of biota treatments involved disinfection of water and baking of sediments to remove indigenous microbiota, or addition of cycloheximide or kanamycin to diminish the effect of predation from natural protozoa or competition from indigenous bacteria respectively. Bacterial levels in all experiments were monitored over a five day period. In the mesocosms investigating the effect of predation and competition on FIB (E. coli and Ent. faecalis) and a pathogen (E. coli O157:H7), predation had a detrimental effect on the survival of the FIB and pathogen in the water column but only influenced the survival of the FIB in the sediment. Unlike predation, competition from indigenous bacteria influenced the survival of E. coli but not Ent. faecalis in both water and sediment. The second set of mesocosms investigated the effect of predation on two motile and non-motile enteric bacteria types (E. coli O157 and S. enterica), each with a motile and non-motile counterpart. An allochthonous predation source (Tetrahymena pyriformis) was added into the mesocosms to supply a consistent level of predation. Motility had a significant positive effect on the survival of S. enterica in the water and sediment but had negative significant effect for E. coli O157 in sediment only. Motility also played a more important role in the sediment compared to predation while predation played a more important role in the water column for both bacteria types. The third study compared the relative effects of predation, competition and nutrients on the survival of E. coli. Natural waters (not amended with nutrients) served as a baseline condition to which organic nutrients were added in two increments. Significant interactions among predation, nutrients and competition (all possible combinations) were observed. Interactions between predation and nutrients as well as competition and predation also accounted for the greatest effects (10% and 8% respectively). The interaction between predation and competition was particularly pronounced at the highest nutrient level. These studies reveal that predation, competition and nutrients are all important factors in the survival of FIB and enteric bacteria in water and sediment, and provide new observations on the relative magnitude of these effects. I show that survival characteristics of FIB and enteric bacteria in secondary habitats can vary depending on bacteria type (FIB or pathogen), location (water or sediment), prey characteristics (motile or non-motile) and specific environmental stressor present (predation, competition or nutrients). The findings of this dissertation provide new insights on the ecology of FIB and enteric bacteria in secondary habitats and underscore the importance of biotic and abiotic factors as determinants of the fate of FIB and enteric bacteria in secondary habitats.

Trichomonas vaginalis carries out unique mode of carbohydrate decarboxylation by an intracellular compartment, hydrogenosome. It was suggested that enzymes exclusively associated with hydrogenosome were responsible for activating metronidazole. This provoked researchers to target hydrogensosomal enzymes such as pyruvate: ferredoxin oxioreductase and ferredoxin, to treat trichomoniasis caused by T. vaginalis. Recent studies have shown alternative pathways responsible for activating metronidazole without the involvement of hydrogenosomal enzymes. This pathway requires the participation of thioredoxin and thioredoxin reductase. These enzymes are essential for T.vaginalis' survival as it is responsible for maintaining cell's redox system, as well as many other cellular processes. Targeting thioredoxin and thioredoxin reductase could be a novel drug target to treat metronidazole-resistant T. vaginalis. In this study, recombinant T. vaginalis TrxR and T. vaginalis Trx were produced to test its activity against known TrxR inhibitor, auranofin, using DTNB assay. Cell lysates of metronidazole susceptible and metronidazole resistant lines were also tested to see its activity against auranofin. Auranofin derivatives that were effective on E. histolytica recombinant TrxR were also tested to compare their effects on T. vaginalis recombinant TrxR. The results showed that auranofin effectively inhibits recombinant TvTrxR. Aurnofin derivatives were also shown to have different effects on inhibiting the activity of recombinant TvTrxR. It is known that auranofin also targets mammalian TrxR. With the degree of different inhibitions observed between auranofin derivatives, it opened the possibility of developing an auranofin derivative that can specifically targets thioredoxin reductase of parasitic protozoan, T. vaginalis.

Cryptosporidium spp., Giardia lamblia and Entamoeba histolytica are the most frequently identified enteric protozoa in water-borne disease outbreaks. Many PCR assays, with satisfactory results in terms of sensitivities and specificities, have been developed for detection of the aforementioned three protozoa in faecal specimens but the majority of these assays have a limited usage in the clinical laboratories due to being more costly and more time-consuming than the conventional diagnostic methods. Based on published oligonucleotide primers, three individual uniplex PCR assays were developed, properly optimised and subsequently combined into a conventional multiplex PCR format to screen for the three protozoa in the same stool specimen. The multiplex PCR assay was clinically validated with 185 control and 212 randomly selected stool samples. The assay was optimised with DNA directly retrieved from stool samples using a modified QIAamp® DNA Stool Mini Kit (Qiagen) DNA extraction protocol subsequent amplification using single-round well-controlled PCR protocol. Like the individual PCRs, the multiplex PCR assay detected genomic DNA from control isolates matching 12, 12 and four copies of the Cryptosporidium, G. lamblia and E. histolytica genomes, respectively. Similarly, ~100 (oo)cysts per 200μl stool were successfully identified by the multiplex and the matching uniplex-PCR assays as detection limits. The diagnostic sensitivity, specificity, negative predictive value and positive predictive value of the multiplex and the individual PCR assays were comparable and equal to 97 %, 100 %, 95 % and 100 %, respectively. Furthermore, by nominating three nested PCRs as 'gold standards', the multiplex PCR demonstrated specificity and sensitivity exceeding that achieved by the combined copro-antigen immunoassay adopted for Cryptosporidium/Giardia diagnosis at the Clinical Microbiology laboratory, Leicester Royal Infirmary, University Hospital of Leicester. In conclusion, the newly developed multiplex PCR was demonstrated to be a simple, cost-effective, adequately sensitive and highly specific assay. An assay with this broad-spectrum format has a great potential to be adopted as a routine test in diagnostic laboratories especially those in resource-poor countries where parasitic protozoal infections are endemic.

Orientador: Bruno Coraucci Filho
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia Civil
Made available in DSpace on 2018-08-03T18:54:05Z (GMT). No. of bitstreams: 1 Pires_MartaSivieroGuilherme_D.pdf: 5702842 bytes, checksum: 77ad92c5acda8dafac231e6107994972 (MD5) Previous issue date: 2003
Resumo: o lodo de esgoto é um resíduo gerado no final do processo de tratamento de esgotos. É produzido em grandes quantidades e há necessidade de promover sua disposição de maneira adequada. A disposição do lodo no solo é uma alternativa que combina reuso e reciclagem de constituintes orgânicos e minerais, pois o lodo contém matéria orgânica e elementos como N e P, que são importantes para o desenvolvimento das plantas. No entanto, este lodo pode estar contaminado por patógenos, como os protozoários e helmintos, ou por metais pesados, causando problemas em relação à disposição. O objetivo desta pesquisa foi avaliar a presença destes patógenos no solo e no líquido percolado que recebeu aplicação de lodo em 3 dosagens diferentes: 2,5 ; 5,0 e 7,5 TDS/ha, com pH natural e 5,0 TDS/ha pH corrigido, além do grupo controle. Também foram testadas duas formas de desinfecção do lodo: calagem (20, 30 e 50%) e desinfecção natural utilizando a luz solar, sendo que neste experimento, além de helmintos e protozoários, foram feitas análises de coliformes totais e E. colí. Os resultados obtidos para aplicação de lodo no solo mostram que os patógenos concentram-se na camada superficial do solo (0-20cm), e quanto maior a dose de lodo aplicada maior a concentração destes organismos. No líquido percolado não foram detectados patógenos. Os testes de calagem indicam que a 50% os patógenos são eliminados em 15 dias, e o experimento de desinfecção natural também demonstra que este método pode ser utilizado, com eliminação total da E. colítambém em periodo de 15 dias
Abstract: Sewage sludge is a residue trom wastewater treatment process that has been produced in large scale and must be disposed appropriatelly. Land disposal of sludge is an altemative that arrange reuse and recicling ot organic and mineral constituints like nitrogen (N) and phosporus (P). However, sludge can be contamined with pathogens, helminth and protozoan, or heavy metal, causing problems with disposal. The objective was to evaluate the pathogens in soil and inflitrated liquid in sai', that received sewage sludge application in three differents dosagens: 2,5; 5,0 e 7,5 TDS/ha in natural pH and 5,OTDS/ha in corriged pH. There were tested two forms os sludge disinfection: chemical stabilization with lime a (20, 30 and 50%) and natural solar disinfection. In the last was checkin total coliforms and E. colí. The research to land appplication in soU shows that the pathogens are concentring on superficial layer at soil (D-20cm) and the concentration increases in accordance with elevation dosage. In the infilirated liquid has not been detected pathogens. The chemical stabilization with lime (50%) shows that pathogens are eliminated within 15 days. Solar disinfection proved an alternative efficient, with destruction of E coli within 15 days too
Doutorado
Saneamento e Ambiente
Doutor em Engenharia Civil

Foodborne illnesses are a significant public health challenge in the United States, with an estimated 9.4 million illnesses annually attributed to the consumption of contaminated food, of which 59% are estimated to be caused by viruses, 39% by bacteria and 2% by parasites. Timely detection and identification of the pathogens causing foodborne outbreaks is vital for the implementation of outbreak control strategies, allowing public health officials to prevent additional illnesses and maintain confidence in the food supply. Public health laboratories employ a variety of traditional and molecular testing techniques to identify foodborne outbreak etiologic agents. One technology is the Luminex XMap® microsphere system, which is also marketed as the Bio-Plex™ 200. This platform has a multiplexing capability with the potential to simultaneously detect up to 100 targets in one reaction. The studies described here show that the combination of two Bio-Plex assays with real-time virus assays and one extraction method provides a flexible foodborne outbreak screening algorithm that potentially identifies an outbreak-associated pathogen on the first day of specimen submission and aids in focusing confirmatory laboratory testing. In these studies, two microsphere-based assays were designed for use on the Bio-Plex 200 system as screening assays for the detection of four enteric protozoa (Giardia intestinalis, Cyclospora cayetanensis, Cryptosporidium parvum, Entamoeba histolytica) and six virulence determinants of shiga toxin-producing Escherichia coli (STEC), enterotoxigenic Escherichia coli (ETEC), enteroinvasive Escherichia coli (EIEC) and Shigella spp. Precision and limits of detections were established for both assays. The sensitivity and specificity of the protozoan assay as compared to reference methods ranged from 81.25% to 100% for most targets, while sensitivity for the E. histolytica target was 42.86%. Sensitivity and specificity for the bacterial assay was 100% as compared to reference methods. However, cross-reactivity of the protozoan assay E. histolytica target with E. dispar and of the bacterial assay uidA target with enteropathogenic E. coli strains was noted. Additionally, real-time detection of norovirus and rotavirus nucleic acids extracted with the QIAamp DNA Stool Mini Kit was statistically comparable to detection when extracted with the Ambion® MagMAX™-96 Viral RNA Isolation Kit combined with the KingFisher® Magnetic Particle Processor.

Toxoplasma gondii, Neospora caninum et Plasmodium falciparum sont des parasites protozoaires intracellulaires obligatoires, respectivement responsables de la toxoplasmose, de la néosporose et du paludisme. Les différents traitements mis en œuvre reposent sur une association médicamenteuse. Cependant, des échecs thérapeutiques et des résistances aux traitements ont été décrits. Notre travail a porté sur l’identification de molécules actives isolées par Chomatographie de Partage Centrifuge (CPC) à partir d’extraits d’écorces d’Anogeissus leiocarpus, un arbre d’Afrique de l’ouest connu pour son activité antipaludique, et de dix arbres de la région Champagne-Ardenne. Nous nous sommes penchés, dans un premier temps, sur l’activité antiparasitaire des fractions obtenues à partir d’extrait d’écorce d’A. leiocarpus. La trachélospérogénine E et l’extrait global sans tanin se sont révélés actifs, notamment en inhibant l’invasion des cellules hôtes par T. gondii. Cet extrait a également préservé la survie des souris atteintes de toxoplasmose chronique. Les mêmes composés naturels ont eu un effet contre N. caninum et P. falciparum. Dans une seconde partie, 30 extraits d’écorces de dix arbres de la région Champagne-Ardenne ont été testés sur T. gondii et N. caninum. Les composés responsables de l’activité antiparasitaire présents chez Alnus glutinosa semblent être la bétuline et ses dérivés. Dans la dernière partie, nous nous sommes intéressés à l’activité de 400 molécules de synthèse de la Pathogen Box. Huit d’entre elles ont eu un effet significatif contre T. gondii, dont trois avec une sélectivité importante. Des expérimentations sont toutefois à réaliser pour N. caninum
Toxoplasma gondii, Neospora caninum and Plasmodium falciparum are mandatory intracellular protozoan parasites and are responsible for toxoplasmosis, neosporosis and malaria, respectively. The different treatments used are based on drug combination. However therapeutic failures and drug resistances have been described. Our work focused on the identification of active compounds isolated by Centrifugal Partition Chromatography (CPC) from crude barks extracts from Anogeissus leiocarpus, a West African tree known for its antimalarial activity, and ten trees from the Champagne-Ardenne region. First we studied the activity of the fractions obtained from the crude bark extract from A. leiocarpus. Trachelosperogenin E and the global extract without tannin showed a good activity by inhibiting host cell invasion by T. gondii. The latter was able to preserve mice survival toward chronic toxoplasmosis. These extracts were also active on N. caninum and P. falciparum. In a second part 30 crude barks extracts from ten trees located in the Champagne-Ardenne region were screened on T. gondii and N. caninum. Compounds responsible for the antiparasitic activity found in Alnus glutinosa were especially betulin and its derivatives. In the last part of this study we focused on the antiparasitic activity of 400 synthetic molecules from the Pathogen Box. Eight out of them were significantly efficient against T. gondii, among which three showed an important selectivity. Further experiments must be completed in the case of N. caninum

Etude d'inhibiteurs de voies metaboliques du trypanosome. Synthese et action d'inhibiteurs possibles d'enzymes de replication. Etude d'enzymes de la glycolyse, cible cle dans le trypanosome, celui-ci utilisant le glucose comme seule source d'energie. Puis caracterisation d'une proteine qui presente les proprietes de recepteur vis-a-vis d'une nouvelle famille de trypanocides derives du diphenyl methane

Mise au point d'un modele in vitro pour l'etude du cycle hepatique d'especes plasmodiales pathogenes pour l'homme utilisant des cultures d'hepatocytes humains. Evaluation des facteurs interferant avec le developpement du schizonte hepatique. Application du modele a la prediction de l'efficacite de la reponse immune anti-proteine circumsporozoite (pv): evaluation de l'effet biologique d'anticorps specifiques des sequences repetes de pc, d'anticorps induits chez l'homme par immunisation par des sporozoites, et de cytokines. Etude de la composition antigenique du schizonte hepatique in vitro

Inventaire de la faune amoebienne de differents biotopes (bacs de climatiseurs, un bassin naturel d'eau pluviale, differents niveaux d'un effluent d'une source hyperthermale et bacs de cultures industrielles d'une algue marine unicellulaire) 68 souches d'amibes ont ete isolees. Le pouvoir pathogene de deux genres, acanthamoeba et naegleria a ete teste sur des souris blanches. Certains facteurs abiotiques dont la temperature et la salinite, influencent la repartition des genres et especes dans les differents sites de prelevement

Les gastroentérites aiguës affectent chaque année entre un quart et la moitié des personnes dans le Monde. Elles sont causes de morbidité, de mortalité et de coûts de santé importants. Leur transmission directe ou indirecte via l’eau, les aliments, l’air ou les surfaces inertes dépend de leur étiologie (virale, bactérienne ou parasitaire) et du contexte local. Bogotá et sa région présentent plusieurs spécificités : des eaux usées rejetées en rivière souvent sans ou après seulement un traitement primaire, la mise en décharge des papiers toilettes, couches et protections souillés par les excréments, et une consommation de fruits et légumes faible et limitée à des produits bon marché irrigués par des eaux pouvant être contaminées fécalement. Notre thèse visait à évaluer les flux de certains pathogènes entériques de l’Homme dans l’environnement à proximité de Bogotá et à essayer de relier ces flux à la santé de la population.La thèse a associé trois contributions. Premièrement, une méthode de culture du norovirus humain a été mise au point en utilisant des villosités intestinales isolées de souris comme modèle cellulaire présentant toute la diversité des cellules épithéliales intestinales. Plusieurs concentrations en trypsine ont été testées pour activer les norovirus ; la méthode a été appliquée à des échantillons fécaux et environnementaux. Deuxièmement, les contaminations en E. coli et en pathogènes entériques de l’Homme ont été suivies dans des eaux (lixiviat de décharge, eau de ruissellement, rivière, eau d’irrigation, eau potable), des légumes-feuilles mangés crus (blettes) et l'air (au-dessus d’une décharge, en zone rurale, en zone urbaine) dans la région de Bogotá. Troisièmement, l’impact des contextes socioéconomiques et des pratiques individuelles (alimentation, hygiène et santé) sur les cas de gastroentérites aiguës a été testé à partir d’enquêtes réalisées dans un district de Bogotá et analysées par divers outils (analyse en composante principale, modélisation …).Nous avons montré que les villosités intestinales isolées de souris permettent l'infection et la réplication du norovirus humain. Le virus doit être activé avec de la trypsine et a un cycle réplicatif moyen de 10 h. Les villosités sont efficaces pour obtenir un matériel biologique abondant et sont idéales pour étudier l'activité biologique du norovirus ou générer des anticorps. Elles ont permis de voir des norovirus non détectés par méthode moléculaire dans certains excréments ou échantillons environnementaux ; les échantillons positifs par méthode moléculaire ou en immunodot-blot contenaient quasiment tous des norovirus infectieux. Au niveau régional, les rejets d'eaux usées dans les rivières Bogotá et Balsillas et dans le marais Tres Esquinas contaminent le réseau d'irrigation de La Ramada au nord-ouest de Bogotá en E. coli et potentiellement en pathogènes entériques de l’Homme. Les blettes récoltées dans cette zone étaient fortement contaminées, en contraste d’autres zones de culture. Leur contamination évoluait de leur production à leur achat dans les commerces de proximité, les lavages pouvant être contaminants ou décontaminants, les manipulations sur l’étal des marchands étant contaminantes. L’air était souvent contaminé par E. coli et par Shigella spp., sans pouvoir attribuer à la décharge Doña Juana un rôle particulier. La présence de Shigella spp. était observée parallèlement dans plus de la moitié des selles des personnes diarrhéiques. Les enquêtes réalisées ont montré que la fréquence annuelle des gastroentérites aiguës diminuait avec l’accroissement de l’âge des personnes ; elle semblait plus faible dans les foyers avec personnes âgées, peut-être en lien avec des pratiques plus strictes en matière d’hygiène, alimentaire notamment
Acute gastroenteritis affect between a quarter and a half of people in the World each year. They are responsible for significant morbidity, mortality and healthcare costs. Their direct or indirect transmissions via water, food, air or inert surfaces depend on their aetiology (viral, bacterial or parasitic) and the local context. Bogotá and its region have several specificities: wastewater are often discharged into rivers without or after primary treatment only, the deposit in landfill of toilet papers and diapers soiled by excrement, and the low consumption of fruits and vegetables largely restricted to a handful of relatively cheap products that may be irrigated by surface freshwaters heavily contaminated with faeces. Our PhD aimed to assess the fluxes of some human enteric pathogens in the region of Bogotá and to try to relate these fluxes to the population health. The PhD combined three contributions. First, a method for culturing the human norovirus has been developed using isolated mouse intestinal villi as a cell model exhibiting the full diversity of intestinal epithelial cells. Several concentrations of trypsin were tested to activate noroviruses; the method was applied to faecal and environmental samples. Second, contamination with E. coli and some human enteric pathogens was monitored in water (landfill leachate, runoff water, river, irrigation water, drinking water), leafy vegetables eaten raw (chards) and air (above a landfill, in rural areas, in urban areas) in the Bogotá region. Third, the impact of socioeconomic contexts and individual practices (food, hygiene and health) on cases of acute gastroenteritis was assessed from surveys carried out in one district of Bogotá and analysed by various tools (principal component analysis, modelling …). We have shown that mouse isolated intestinal villi allow the infection and replication of human norovirus. The virus has to be activated with trypsin and has an average replicative cycle of 10 h. Villi are efficient in obtaining abundant biological material and are ideal for studying the biological activity of norovirus or for generating antibodies. They made it possible to see infectious noroviruses not detected by molecular method in several faeces and environmental samples; almost all samples positive by molecular method or immunodot-blot contain infectious noroviruses. At the regional level, the discharges of wastewater in the Bogotá and Balsillas rivers and in Tres Esquinas march contaminate the irrigation network of La Ramada area in the northwest of Bogotá with E. coli and potentially human enteric pathogens. Chards harvested in this area were heavily contaminated, in contrast to other growing areas. Their contamination evolved from their production to their purchase in nearby stores, washings increasing or decreasing their contamination, and handling on the merchant's stalls increasing contamination. The air was often contaminated with E. coli and Shigella spp.; it was not possible to detect a particular contribution of the Doña Juana landfill in pathogen aerosolization. The presence of Shigella spp. was observed in parallel in more than half of the stools of people with diarrhoea. Surveys have shown that the annual frequency of acute gastroenteritis decreases with increasing age; it seemed less common in households with elderly people, possibly due to stricter food hygiene practices. A transmission model of acute gastroenteritis distinguishing contamination from outside the households and contaminations between people in the same households did not show significant differences between neighbourhoods. Used to simulate numerical experiments, it suggests working on much higher numbers of surveys
La gastroenteritis aguda afecta entre una cuarta parte y la mitad de las personas en el mundo cada año. Son responsables de importantes costos de morbilidad, mortalidad y asistencia sanitaria. Sus transmisiones directas o indirectas a través del agua, alimentos, aire o superficies inertes dependen de su etiología (viral, bacteriana o parasitaria) y del contexto local. Bogotá y su región aledaña tienen varias especificidades: las aguas residuales a menudo se vierten a los ríos sin o solo después de un tratamiento primario, el depósito de papel higiénico y pañales sucios con excrementos son dispuestos generalmente en un relleno sanitario, y el bajo consumo de frutas y verduras restringido en gran medida a un puñado de productos relativamente baratos pueden ser irrigados por aguas dulces superficiales muy contaminadas con excrementos. Nuestra tesis doctoral tuvo como objetivo evaluar los flujos de algunos patógenos entéricos humanos en la región de Bogotá y tratar de relacionar estos flujos con la salud de la población. El doctorado combinó tres contribuciones. En primer lugar, se desarrolló un método para cultivar el norovirus humano utilizando vellosidades intestinales aisladas de ratón como modelo celular que exhibe la diversidad completa de células epiteliales intestinales. Se probaron varias concentraciones de tripsina para activar norovirus; el método se aplicó a muestras fecales y ambientales. En segundo lugar, se evidenció la contaminación de E. coli y patógenos entéricos humanos en el agua (lixiviados de vertedero, agua de escorrentía, río, agua de riego, agua potable), vegetales de hoja que se comen crudos (acelgas) y aire (sobre un vertedero sanitario, así como en áreas rurales y urbanas) en la región de Bogotá. En tercer lugar, se evaluó el impacto de los contextos socioeconómicos y las prácticas individuales (alimentación, higiene y salud) frente a los casos de gastroenteritis aguda a partir de encuestas realizadas en una localidad de Bogotá y analizadas mediante diversas herramientas (análisis de componentes principales, modelización…). Con este doctorado, hemos demostrado que las vellosidades intestinales aisladas de ratón permiten la infección y la replicación del norovirus humano. El virus debe activarse con tripsina y tiene un ciclo replicativo promedio de 10 h. Las vellosidades son eficaces para obtener abundante material biológico y son ideales para estudiar la actividad biológica de los norovirus o para generar anticuerpos. Ellas permitieron ver norovirus infecciosos no detectados por método molecular en varias heces y muestras ambientales; casi todas las muestras positivas por método molecular o inmunodot-blot contienían norovirus infecciosos. A nivel regional, los vertidos de aguas residuales en los ríos Bogotá y Balsillas y en el humedal Tres Esquinas contaminan la red de riego La Ramada en el noroeste de Bogotá con E. coli y potencialmete con patógenos entéricos humanos. Las acelgas recolectadas en esta área resultaron muy contaminadas, a diferencia de otras áreas de cultivo. Su contaminación evolucionó desde la producción hasta su compra en las tiendas cercanas, los lavados aumentaron o disminuyeron su contaminación y la manipulación en los puestos de comercio aumentaron la contaminación. El aire a menudo estaba contaminado con E. coli y Shigella spp., sin poder atribuir al relleno sanitario Doña Juana un rol particular. A su vez la presencia de Shigella spp. se observó en paralelo en más de la mitad de las deposiciones de personas con diarrea. Las encuestas demostraron que la frecuencia anual de gastroenteritis aguda disminuye respecto al aumento en edad; parecía menos común en hogares con personas mayores, posiblemente debido a prácticas de higiene alimentaria más estrictas. Un modelo de transmisión de gastroenteritis aguda que distinguió la contaminación fuera de los hogares y las contaminaciones entre personas dentro de los mismos hogares no mostró diferencias significativas entre vecindarios

Plusieurs évolutions sont constatées dans la recherche en biologie. Tout d’abord, les études menées reposent souvent sur des approches expérimentales quantitatives. L’analyse et l’interprétation des résultats requièrent l’utilisation de l’informatique et des statistiques. Également, en complément des études centrées sur des objets biologiques isolés, les technologies expérimentales haut débit permettent l’étude des systèmes (caractérisation des composants du système ainsi que des interactions entre ces composants). De très grandes quantités de données sont disponibles dans les bases de données publiques, librement réutilisables pour de nouvelles problématiques. Enfin, les données utiles pour les recherches en biologie sont très hétérogènes (données numériques, de textes, images, séquences biologiques, etc.) et conservées sur des supports d’information également très hétérogènes (papiers ou numériques). Ainsi « l’analyse de données » s’est petit à petit imposée comme une problématique de recherche à part entière et en seulement une dizaine d’années, le domaine de la « Bioinformatique » s’est en conséquence totalement réinventé. Disposer d’une grande quantité de données pour répondre à un questionnement biologique n’est souvent pas le défi principal. La vraie difficulté est la capacité des chercheurs à convertir les données en information, puis en connaissance. Dans ce contexte, plusieurs problématiques de recherche en biologie ont été abordées lors de cette thèse. La première concerne l’étude de l’homéostasie du fer chez la levure pathogène Candida glabrata. La seconde concerne l’étude systématique des modifications post-traductionnelles des protéines chez la levure pathogène Candida albicans. Pour ces deux projets, des données « omiques » ont été exploitées : transcriptomiques et protéomiques. Des outils bioinformatiques et des outils d’analyses ont été implémentés en parallèle conduisant à l’émergence de nouvelles hypothèses de recherche en biologie. Une attention particulière et constante a aussi été portée sur les problématiques de reproductibilité et de partage des résultats avec la communauté scientifique
Biological research is changing. First, studies are often based on quantitative experimental approaches. The analysis and the interpretation of the obtained results thus need computer science and statistics. Also, together with studies focused on isolated biological objects, high throughput experimental technologies allow to capture the functioning of biological systems (identification of components as well as the interactions between them). Very large amounts of data are also available in public databases, freely reusable to solve new open questions. Finally, the data in biological research are heterogeneous (digital data, texts, images, biological sequences, etc.) and stored on multiple supports (paper or digital). Thus, "data analysis" has gradually emerged as a key research issue, and in only ten years, the field of "Bioinformatics" has been significantly changed. Having a large amount of data to answer a biological question is often not the main challenge. The real challenge is the ability of researchers to convert the data into information and then into knowledge. In this context, several biological research projects were addressed in this thesis. The first concerns the study of iron homeostasis in the pathogenic yeast Candida glabrata. The second concerns the systematic investigation of post-translational modifications of proteins in the pathogenic yeast Candida albicans. In these two projects, omics data were used: transcriptomics and proteomics. Appropriate bioinformatics and analysis tools were developed, leading to the emergence of new research hypotheses. Particular and constant attention has also been paid to the question of data reproducibility and sharing of results with the scientific community

Background: Neospora caninum is a parasite that causes disease, largely in cattle and dogs. It is a disease of significant interest within New Zealand due to its association with bovine abortion. The economic impact of bovine abortion justifies the development of a bovine vaccine against N. caninum. Aim: To develop and optimise diagnostic procedures for the detection of Neospora from a variety of blood and tissue samples and to isolate a New Zealand strain of Neospora caninum. Methods: A local strain of Toxoplasma gondii and an imported Neospora caninum strain, Nc-Liverpool, were used to optimise tachyzoite growing conditions in bovine endothelial (BE) cells and Vero host cell cultures. A serum study using 112 tissue culture flasks was performed to determine whether foetal bovine serum or horse serum supplemented media provided the optimal growing conditions for Nc-Liverpool tachyzoites. Nc-Liverpool tachyzoites were also used to determine the optimal growth period between passage, and harvest for cryopreservation and cryopreservation conditions. Percoll gradients were also tested using Nc-Liverpool tachyzoites. A known Neospora positive canine sample and murine tissues infected with Toxoplasma, were used during the development of the immunohistochemical diagnostic technique. Antibody concentrations and incubation temperatures were tested to reduce cross-reactivity and increase specific stain intensity. Immunohistochemistry was performed on sections of all tissue samples used for N. caninum isolation and experimentally infected murine tissue. Several PCR techniques were developed, the final PCR used being a combination of the different techniques, which produced a 250kb band. PCR-3 used the NF6/GA1 primer combination for Neospora detection and TF6/GA1 for Toxoplasma detection, additional Mg2+ and an annealing temperature of 55°C were required. Whole tissue was processed via DNA elution whereas cell culture and Percoll purified tachyzoites were used following crude lysis techniques. All bovine and canine tissues used for parasite isolation as well as all experimentally infected mouse tissues were tested for N. caninum using PCR. An immunoblot technique was developed for the detection of N. caninum antibodies in murine blood samples. Lysed Nc-Liverpool tachyzoites were used as antigen with varied results. The primary and secondary antibodies were commercially available and used at concentrations of 1:1,000 and 1:25,000 respectively. BALB/c and CF1 mice were experimentally infected with Toxoplasma gondii and Nc-Liverpool. Forty female BALB/c and 40 female CF1 mice were used in 2 studies to determine the optimal Nc-Liverpool inoculation dose and immunosuppression requirements. Mice were immunosuppressed with 2.5mg of methylprednisolone acetate (MPA) and Nc-Liverpool inoculation ranged from 1.3x106 to 5x103 tachyzoites. Upon death, the brain and blood was harvested from the mouse carcases. Attempts were made to isolate a New Zealand strain of N. caninum from bovine and canine central nervous system (CNS) tissue, and to maintain the parasites in cell culture and by small animal passage, in order to attenuate the parasite strain for use as a live large animal vaccine. Twenty one bovine tissue samples were used for N. caninum isolation attempts, 18 of which were positive for Neospora antibodies using a commercial IFAT. Isolation tissues were purified using a 30% Percoll gradient and inoculated onto 8 cell culture flasks and into 8 immunosuppressed mice (BALB/c and CF1). Results: Nc-Liverpool tachyzoites were found to be viable when grown at 37°C in antibiotic-MEM supplemented with either FBS or ES and grew optimally in FBS despite Neospora antibodies being detected using an IFAT. Passaging cultures at approx. 4 day intervals resulted in the greatest parasite growth. However, cryopreserved parasites should be harvested 2 days post inoculation (PI) for optimal viability. Viable parasites could be isolated using a 30% Percoll gradient and centrifuged at 2,700 x g (3,400 rpm) in a bucket centrifuge for 10 minutes. Tissue cysts could be detected using immunohistochemistry but some degree of cross reaction remained despite optimisation. Cysts were not found in tissues used for isolation attempts or in mouse brains following inoculation with Nc-Liverpool, however cysts were commonly found in mice experimentally infected with T. gondii tachyzoites. PCR-3 was successfully used to detect N. caninum and T. gondii infected tissue and tachyzoites from tissue culture. PCR-3 could detect N. caninum DNA in the brain tissue of 9/24 mice experimentally infected with Nc-Liverpool, even though most mice were culled within 1 week. Although production of N. caninum antigen was only moderately successful, N. caninum antibody detection in mouse blood using one specific antigen batch was reliable and specific. The immunoblot could only detect N. caninum antibody approximately 14 days PI, but was sensitive enough to detect 100% of mice experimentally infected with Nc-Liverpool tachyzoites. PCR-3 strongly correlated with the immunoblot results from 14 days PI. BALB/c mice were found to be far more sensitive to Nc-Liverpool than CF1 mice and developed severe disease at concentrations of approximately 1x106 Nc-Liverpool tachyzoites. Neither BALB/c nor CF1 mice developed peritoneal exudate, irrespective of the parasite inoculation concentration. Despite Neospora DNA being present in the brains of experimentally infected mice, re-isolation and continuous parasite passage from the brains could not be achieved. No mice experimentally infected with either Nc-Liverpool or isolation attempts were found to have brain cysts when tested using immunohistochemistry. Only 1 mouse inoculated with bovine isolation material was found to have a Neospora positive PCR. Through the detection of DNA, antigens and antibodies, parasites were determined to have been present in 10 of the 18 IFAT positive bovine isolation samples, indicating that 55% of calves born to seropositive dams were infected with N. caninum. However, despite numerous attempts to isolate Neospora parasites from naturally infected canine and bovine tissue and culturing using the optimised Nc-Liverpool technique, maintenance of a live culture of a New Zealand strain of N. caninum could not be established. Conclusions: Findings from this study could be used to assist in the maintenance of Neospora caninum and Toxoplasma gondii parasite strains and for detection or diagnosis of these parasites in host tissues.

University of Technology Sydney. Faculty of Science.
𝘕𝘦𝘰𝘴𝘱𝘰𝘳𝘢 𝘤𝘢𝘯𝘪𝘯𝘶𝘮 is a cyst-forming apicomplexan parasite, responsible for economic and reproductive losses to cattle industries worldwide, and represents a serious neurological disease in canines. Although discovered over three decades ago, progress towards treatment and control strategies against neosporosis, remains stagnant. Currently, common practices to combat the disease include passivity, or expensive culling of seropositive dams. However, vaccination represents a cost-effective and efficacious option, especially using live, attenuated isolates. Members of the Apicomplexa consist of populations that vary enormously in their disease-causing potential, where 𝘪𝘯 𝘷𝘪𝘷𝘰 experiments have demonstrated pathogenic variability between 𝘕. 𝘤𝘢𝘯𝘪𝘯𝘶𝘮 isolates. The underlying question therefore, is what is the genetic basis of virulence within the species, and consequently, how can such information be exploited in vaccine development? Thus far, conventional techniques have been employed to study the intraspecies genetic diversity associated with 𝘕. 𝘤𝘢𝘯𝘪𝘯𝘶𝘮, generally involving PCR-based approaches targeting repetitive elements. However, a direct causal relationship between such diversity and important parasite phenotypes such as virulence, is yet to be established. Alternatively, burgeoning next generation sequencing (NGS) technologies and 𝘪𝘯-𝘴𝘪𝘭𝘪𝘤𝘰 tools have provided new opportunities to perform genome-wide scans in such organisms. Hence the objective of this body of work was to compare the genomes and transcriptomes of two distinct 𝘕. 𝘤𝘢𝘯𝘪𝘯𝘶𝘮 isolates, using NGS data and bioinformatics workflows, to identify sequence variants in coding and non-coding DNA. Annotation of variable regions would reveal potential virulence markers distinguishing isolates of this species. Challenges accompanying such research include the lack of optimisation and standardisation of NGS analysis tools for non-model organisms such as pathogenic Protozoa. This is compounded by the dubious accuracy of the 𝘕. 𝘤𝘢𝘯𝘪𝘯𝘶𝘮 reference genome, as well as the disturbingly large number of proteins described as ‘hypothetical’ or ‘uncharacterised’. This body of research represents a thesis by compilation, consisting of four publications, and one chapter under review. Each chapter represents an independent study, which collectively address the research objective and current gaps in the literature. The results present polymorphic “hotspots” in concentrated windows of the 𝘕. 𝘤𝘢𝘯𝘪𝘯𝘶𝘮 genome, where there is a correlation between hypervariable regions within protein-coding genes, and non-coding regions. Furthermore, an 𝘪𝘯-𝘴𝘪𝘭𝘪𝘤𝘰 pipeline is developed to annotate uncharacterised proteins, subsequently identifying a subset of proteins potentially implicated in crucial parasite mechanisms, conducive to 𝘕. 𝘤𝘢𝘯𝘪𝘯𝘶𝘮’s success. It is trusted that this thesis contributes vital knowledge pertaining to 𝘕. 𝘤𝘢𝘯𝘪𝘯𝘶𝘮 intraspecies diversity, aiding in the quest to develop a vaccine against neosporosis.

博士
國防醫學院
生命科學研究所
90
英文摘要 Iron is a key modulator of gene expression in Trichomonas vaginalis. In this study, iron induced gene regulation of T. vaginalis adhesin protein（AP65-1）was investigated. A transient DNA transfection system was established and for the analysis of ap65-1 promoter activity. The region -109/-56 was found to be critical for iron-induced transcription and -121/-102、-52/-39 and initiator element were found to be important for basal transcription. Nuclear proteins binding sites in these regions were identified by electrophoretic mobility shift assay, was shown to be -95TAACGATAT-87 and T-rich sequence in the distal promoter region. According to these results, DNA transient transfection system is usfeul to further study basal and iron-induced transcriptional regulation in T. vaginalis.

博士
國防醫學院
生命科學研究所
87
Giardia lamblia is an enteric protozoan parasite causing infectious diarrheal diseases worldwide. It also represents an early-diverging eukaryote and exhibits an unusual and fascinating biology. To understand the transcriptional regulation in this primitive organism, I have characterized the promoter region of the ras-related nuclear protein (ran) gene of G. lamblia. In addition, two molecular tools, a stable DNA transfection system and an inducible gene expression system, have also been developed for studies of gene functions in G. lamblia. I started the study by developing a stable DNA transfection system in G. lamblia. Plasmid pRANneo was constructed by placing a neomycin resistant gene under the control of the ran promoter. After transfection with this plasmid by electroporation, stable transfectants were established under G418 selection. The stable transfectants were found to contain episomal pRANneo molecules in high copy numbers. A luciferase gene directed by the glutamate dehydrogenase (gdh) promoter was inserted into pRANneo to generate another construct, pRANneo/GDHluc. Results showed that this plasmid was maintained episomally and the NEO and luciferase proteins were both highly expressed in the stable transfectants. The plasmid copy numbers and luciferase expression levels were both increased with increasing concentrations of G418. This system will be a useful tool for studying the specific effects of candidate genes in G. lamblia. I then proceeded to characterize the G. lamblia ran gene promoter. The 5''- and 3''-untranslated regions (UTRs) of the ran gene were found to confer significant activity when linked to a luciferase reporter gene and transiently transfected into G. lamblia. Deletion and mutation analyses revealed that an AT-rich region spanning -51/-20 of the ran 5''-UTR gene was required for promoter activity. This 32-bp element was also sufficient for promoter function when placed upstream of a promoterless luciferase gene, and low levels of expression remained when it was dissected into 10-bp smaller fragments. Electrophoretic mobility shift assays showed that the 32-bp element bound specifically to nuclear proteins in single-stranded but not double-stranded configurations. Results from primer extension studies revealed that the 32-bp element was able to determine the transcription start sites. These data suggest that the 32-bp AT-rich region plays a major role in the transcriptional regulation of the ran gene in G. lamblia. In order to improve the stable DNA transfection system, I have developed an inducible gene expression system by adapting the bacterial tet repressor-operator system into G. lamblia. One, two, or three tet operators were inserted into the 32-bp ran promoter context at various positions to drive the expression of luciferase reporter gene. Different G. lamblia promoters were also tested to drive the expression of the tet repressor gene. These regulatory elements were incorporated into the pRANneo stable transfection vector and the stable transfectants of the resulting constructs were established. By assaying the luciferase activity in the presence and absence of the inducer tetracycline, it was found that the best inducibility was mediated by the construct containing two tet operators downstream of the ran promoter and the tet repressor gene directed by the a-giardin promoter. The induction of luciferase expression was tetracycline dose- and time-dependent. The optimal induction ratio, ~50, was achieved after 10 hr upon addition of 10 mg/ml tetracycline. This system could be an improved tool for studies in G. lamblia since it allows adjustable expression of a target gene and thereby avoids the potential toxic effects of overexpression. These studies may form a basis for future investigation of the transcriptional mechanisms and provide valuable tools for molecular studies in G. lamblia.

Indiana University-Purdue University Indianapolis (IUPUI)
Toxoplasma gondii, a single-celled eukaryotic pathogen, has infected one-third of the world’s population and is the causative agent of toxoplasmosis. The disease primarily affects immunocompromised individuals such as AIDS, cancer, and transplant patients. The parasites can infect any nucleated cell in warm-blooded vertebrates, but because they preferentially target CNS, heart, and ocular tissue, manifestations of infection often include encephalitis, myocarditis, and a host of neurological and ocular disorders. Toxoplasma can also be transmitted congenitally by a mother who becomes infected for the first time during pregnancy, which may result in spontaneous abortion or birth defects in the child. Unfortunately, the therapy currently available for treating toxoplasmosis exhibits serious side effects and can cause severe allergic reactions. Therefore, there is a desperate need to identify novel drug targets for developing more effective, less toxic treatments. The regulation of proteins via lysine acetylation, a reversible post-translational modification, has previously been validated as a promising avenue for drug development. Lysine acetyltransferases (KATs) are responsible for the acetylation of hundreds of proteins throughout prokaryotic and eukaryotic cells. In Toxoplasma, we identified a KAT that exhibits homology to Elongator protein 3 (TgElp3), the catalytic component of a transcriptional elongation complex. TgElp3 contains the highly conserved radical S-adenosylmethionine and KAT domains but also possesses a unique C-terminal transmembrane domain (TMD). Interestingly, we found that the TMD anchors TgElp3 in the outer mitochondrial membrane (OMM) such that the catalytic domains are oriented towards the cytosol. Our results uncovered the first tail-anchored mitochondrial KAT reported for any species to date. We also discovered a shortened form of Elp3 present in mouse mitochondria, suggesting that Elp3 functions beyond transcriptional elongation across eukaryotes. Furthermore, we established that TgElp3 is essential for parasite viability and that its OMM localization is important for its function, highlighting its value as a potential target for future drug development.