As condições climáticas presentes nas regiões produtoras de trigo, do Brasil e dos principais países do qual o produto é importado, favorecem o aparecimento de doenças importantes desta cultura, dentre elas a fusariose, causada principalmente pelo fungo Fusarium graminearum Schwabe. Além dos danos diretos causados pela doença, os grãos infectados podem ser tóxicos para o homem e animais devido à presença de micotoxinas especialmente o desoxinivalenol (DON). Um total de 100 amostras de trigo, sendo 50 de trigo nacional (provenientes do Estado de São Paulo, Paraná e Rio grande do Sul) e 50 de trigo importado (Argentina e Paraguai) foram coletadas de empresas que normalmente comercializam ou processam trigo durante o período de maio a dezembro de 2005. Foram avaliados o percentual de freqüência de fungos, especialmente Fusarium graminearum, a contaminação com DON, percentual de grãos giberelados e realizadas correlações entre os parâmetros avaliados. Os resultados indicaram que freqüência média de Fusarium graminearum e F. spp. foram baixas (&#8804;2,6 e &#8804;3,6%, respectivamente), porém foi maior no trigo nacional do que no trigo importado. Do total de amostras avaliadas 94% do trigo nacional e 88% do trigo importado apresentaram-se contaminadas com DON em níveis médios de 332 µg.kg-1 (nacional) e 90 µg.kg-1 (importado). Existiu correlação positiva e significativa entre contaminação com DON e percentual de grãos giberelados (r = 0,83; p<0,0001), freqüência de Fusarium graminearum (r = 0,92; p<0,0001) e freqüência de Fusarium spp. (r = 0,86; p<0,0001). / The climatic conditions present in wheat producing areas from Brazil and main countries from which the product is imported favor the occurrence of important diseases in this crop, among them the Fusarium Head Blight or scab. It is mainly caused by the fungus Fusarium graminearum Besides, direct damages caused by this disease, the infected kernels may be toxic for humans and animals due to presence of mycotoxins (e.g deoxynivalenol). A total of 100 wheat samples, being 50 from national production (São Paulo, Paraná and Rio Grande Do Sul states) and 50 from imported one (Argentina and Paraguay), were collected during the period of May to December 2005 from companies that normally commercialize or process wheat. Frequency (%) of fungi occurrence, specially Fusarium graminearum and Fusarium spp., DON contamination and Fusarium damaged kernels (%) were evaluated. Correlations between the evaluated parameters were carried out. Frequency of Fusarium graminearum and Fusarium spp. were low (&#8804;2.6 and &#8804;3.6%, respectively), however it was higher in Brazilian wheat when compared with imported wheat. Ninety-four percent of national wheat samples and 88% of the imported samples were DON contaminated (mean levels, 332 µg.kg-1 and 90 µg.kg-1, respectively). The occurrence of DON was highly correlated with percentage of Fusarium damaged kernels, (r = 0,83; p<0.0001), percentual frequency of Fusarium graminearum (r = 0,92; p<0,0001) and percentual frequency of Fusarium spp. (r = 0,86; p<0,0001).

Bacillus cereus is a ubiquitous organism and a potential foodborne pathogen that can cause two types of gastrointestinal diseases: emesis and diarrhea. The emetic syndrome is caused by a heat and acid stable peptide toxin that is pre-formed in food, while the diarrheal syndrome is associated to two 3-protein, heat labile enterotoxin complexes that are formed in the intestine after ingestion of the organism. There are many reports on the isolation and characterization of Bacillus cereus from various foods, however there are no studies on the levels, toxigenicity and physical characteristics of B. cereus isolated from U.S. retail spices. A huge part of spices sold in the U.S. are imported from developing nations. Developing nations lack hygienic practices during processing and packaging of spices, due to which there is a high chance of imported spices being contaminated with B. cereus. Therefore, the main objective of this thesis work was to characterize B. cereus spores from U.S. retail spices. Levels of aerobic spores and B. cereus spores were determined. B. cereus spores were further analyzed for their enterotoxigenic ability, growth characteristics and physical spore characteristics. In the 247 spice samples analyzed 77 were found to contain B. cereus, while 11 were positive for B. thuringiensis. Eighty four of the 88 spices tested possessed either one of the enterotoxin genes. None of the isolates tested positive for the emetic toxin (ces) gene. Seventy five of the B. cereus isolates grew at 12 °C, although only two isolates grew well at 9 °C. Seven selected diarrheal B. cereus spore strains had D95-values ranging from 0.64-3.53 min while the two emetic strains had D95-values of 7.04 min and 6.64 min. B. cereus grew well in pre-cooked rice. After 48 h, counts of 1.26 X 107 and 3.8 X 107 B. cereus/ 10 g were obtained in pre-cooked rice maintained at 17 °C and 20 °C respectively. At 12 °C, counts did not reach 104 CFU/ 10g even after 48 h of incubation. The aerobic mesophilic bacterial population and B. cereus population of 0.1% crushed pepper in pre-cooked rice over a period of 48h at temperature 20 °C and 17 °C were also analyzed. Counts of B. cereus in pepper rice samples reached a maximum of 1600 MPN/ 10 g and 1100 MPN/ 10 g at 20 °C and 17 °C respectively while the aerobic mesophilic counts per 10 g were 2.4 X 108 and 4.4 X 106 at these temperatures. The low B. cereus counts and high aerobic mesophilic population indicates competition of nutrients in cooked rice by background flora other than B. cereus. The physical spore characteristics of five B. cereus and 3 B. thuringiensis strains were studied using transmission electron microscopy (TEM). Tubular, whip-like appendages were present in four B. cereus and two B. thuringiensis, while all seven isolates possessed exosporia.

Trypanosoma brucei is the causative agent of Human African Trypanosomiasis (HAT), also known as African sleeping sickness. T. brucei is unique in several ways that distinguish this organism from other eukaryotes. One of the unique features of T. brucei is the organism’s mitochondrial DNA, which is organized in a complex structure called kinetoplast DNA (kDNA). Since kDNA is unique to the kinetoplastids, kDNA may serve as a good drug target against T. brucei. Previews studies have shown that kDNA has 4 different family A mitochondrial DNA polymerases. Three of these mitochondrial DNA polymerases (POLIB, POLIC, and POLID) are essential components of kDNA synthesis and replication. POLID and POLIC dynamically localize throughout the cell cycle. POLID is found dispersed in the matrix before the kDNA has undergone replication and is re-localized at the antipodal sites when the kDNA is dividing. POLIC is found in the kinetoflagellar zone (KFZ) at low concentrations when the kDNA is not replicating and relocalizes to the antipodal sites when dividing. Based on the dynamic localization of these two DNA polymerases, we hypothesize that POLIB undergoes dynamic localization at some point during the cell cycle stage. Here, a POLIB/PTP single expressor cell line was analyzed by immunofluorescence microscopy in an unsynchronized population. We characterized the localization pattern of POLIB-PTP at different cell cycle stages and found different localization patterns throughout cell cycle. Cells at 1N1K (the majority of cell in an unsynchronized population) have single foci, but at 1N1Kdiv two different patterns are mainly observed, diffuse and segregated. When the kDNAs are separated POLIB-PTP is again seen as a distinct foci in each kDNA. By doing TdT labeling and a quantitative analysis, we found that at early stages of minicircles replication POLIB-PTP start relocalizing to the kDNA disk with a diffuse pattern being the main. By the time the minicircles are being reattached in the disk (late TdT), POLIB is seen in the disk as a bilobe shape.

Mycobacteria, a genus within Actinobacteria Phylum, are well known for two pathogens that cause human diseases: leprosy and tuberculosis. Other than the obligate human mycobacteria, there is a group of bacteria that are present in the environment and occasionally cause diseases in immunocompromised persons: the non-tuberculosis mycobacteria (NTM). Mycobacterium kansasii, which was first discovered in the Kansas state, is the main etiologic agent responsible for lung infections caused by NTM and raises attention because of its co-infection with human immunodeficiency virus (HIV). Five subspecies of M. kansasii (Type I-V) were described and only M. kansasii Type I is pathogenic to humans. M. kansasii is a Gram-positive bacteria that has a unique cell wall and secretion system, which is essential for its pathogenicity. We undertook a comparative genomics and transcriptomic approach to identify components of M. kansasii Type I pathogenicity. Our previous study showed that espA (ESX-1 essential protein) operon, a major component of the secretion system, is exclusively present in M. kansasii Type I. The purpose of this study was to test the functional role of the espA operon in pathogenicity and identify other components that may also be involved in pathogenicity. This study provides a new molecular diagnostic method for M. kansasii Type I infection using PCR (Polymerase Chain Reaction) technique to target the espAoperon. With detailed manual curation of the comparative genomics datasets, we found several genes exclusively present in M. kansasii Type I including ppsA/ppsC and whiB6, that we believe are involved, or have an effect on ESX-mediated secretion system. We have also highlighted, in our study, the differences in genetic components coding for the cell membrane composition between the five subspecies of M. kansasii. These results shed light on genetic components that are responsible for pathogenicity determinants in Type I M. kansasii and may help to design better control measures and rapid diagnostic tools for monitoring these group of pathogens.

The objective of this study was to characterize the mitochondrial genomes of the species within the genus Ceratocystis and investigate the evolutionary process of the ribosomal RNA cistron found within these fungi. Ceratocystis incorporates a number of pathogenic species affecting a variety of hosts, making the study of these fungi economically significant. The fortuitous identification of a Ceratocystis species, C. manginecans, which contained two different internally transcribed spacer sequence types within the ribosomal rRNA cistron, enabled a study of concerted evolution in this fungus. Using this non-model organism we were able to show empirical evidence for unequal crossing over and gene conversion as the ultimate forces acting on this gene region dictating a concerted evolutionary effect. We suggest that this process is true for all eukaryotes. Using the knowledge drawn from previously characterized and annotated mitochondrial genomes of other eukaryotes, the genomes of three Ceratocystis species, namely Ceratocystis fimbriata, Ceratocystis albifundus and Ceratocystis moniliformis were fully assembled and annotated for comparative analysis. This comparative study addressed the genome size, gene content, tRNA presence as well as intron types and their homing endonucleases found among these three mitochondrial genomes. An interspecies characterization was then undertaken using the mitochondrial genomes of six different C. albifundus isolates from different geographical locations in Africa. Genetic variation and similarities among these isolates supports the previous hypothesis that the origin of this fungus is Southern Africa. It is hoped that the research presented in this thesis will contribute to the improved understanding of the mitochondrial genomes in Ceratocystis species. / Thesis (PhD)--University of Pretoria, 2013. / gm2013 / Genetics / Unrestricted

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

Please read the abstract in the section, 00front, of this document / Dissertation (MSc)--University of Pretoria, 2001. / Microbiology and Plant Pathology / MSc / unrestricted

No description available.

Candida albicans is the most common human fungal pathogen. The relationship between C. albicans and Enterobacter bacteria have yet to be explored. The hypothesis of this study is that C. albicans and both E. aerogenes and E. cloacae have a positive relationship and work together to infect the host. In this study, the physical cell-to-cell interaction, molecular components of said interaction, and the impact of the interaction on a live organism were explored. Results indicate that Enterobacter adheres to C. albicans and inhibits growth with unidentified secreted molecules. Als1p has potential involvement in the attachment of E. cloacae to C. albicans. Out of 480 E. cloacae mutants, 6 showed reductions in attachment to C. albicans. The presence of C. albicans in C. elegans may lead to less Enterobacter colonization. Future work involving this interaction should strive to identify the Enterobacter secreted molecules and genes necessary for their production.

Effective treatment of tuberculosis requires at least six months of combination therapy involving four antibiotics. Alterations in the physiological state of Mycobacterium tuberculosis during infection may reduce drug efficacy and prolong treatment, but these adaptations are incompletely defined. To investigate the mechanisms limiting antibiotic efficacy, I performed a comprehensive genetic study to identify M. tuberculosis genes and pathways important for bacterial survival during antibiotic treatment in vivo. First, I identified mutants in the glycerol kinase enzyme, GlpK, that promote survival under combination therapy. Similar glycerol catabolic mutants are enriched in extensively drug-resistant clinical isolates, indicating that these mutations may promote survival and the development of resistance in humans. A majority of these mutations are frameshifts within a homopolymeric region of the glpK gene, leading to the hypothesis that M. tuberculosis may reversibly produce drug-tolerant phenotypes through genetic variation introduced at homopolymer sites as a strategy for survival during antibiotic treatment. Second, I identified bacterial mutants with altered susceptibility to individual first-line anti-mycobacterial drugs. Many of these mutations did not have obvious effects in vitro, demonstrating that a wide variety of natural genetic variants can influence drug efficacy in vivo without altering standard drug-susceptibility tests. A number of these genes are enriched in drug-resistant clinical isolates, indicating that these genetic variants influence treatment outcome. Together, these data suggest new targets for improving therapy, as well as mechanisms of genetic adaptations that can reduce antibiotic efficacy and contribute to the evolution of resistance.