Scrub typhus, caused by the obligate intracellular bacterium Orientia tsutsugamushi, afflicts one million people annually. Despite being a global health threat, little is known about O. tsutsugamushi pathogenesis. Here, we demonstrate that O. tsutsugamushi modulates the ER and ER-associated processes as mechanisms of nutritional virulence and immune evasion. To obtain amino acids to fuel replication, O. tsutsugamushi simultaneously induces ER stress and the unfolded protein response (UPR) while inhibiting ER-associated degradation (ERAD) during early infection time points. During exponential growth, the bacterium releases the ER bottleneck, resulting in generation of ERAD-derived amino acids that it parasitized for replication. The O. tsutsugamushi effector, Ank4, is linked to this process, as it impedes ERAD when ectopically expressed. O. tsutsugamushi expression of ank4 peaks during the ERAD inhibition window, but is absent when the pathway is restored. These data reveal a novel mechanism of nutritional virulence, whereby an obligate intracellular pathogen coordinates the modulation of multiple ER-associated processes. Like other intracellular pathogens, O. tsutsugamushi inhibits expression of MHC-I, but it does so in a novel manner by degrading the master regulator of MHC-I, NLRC5. This impedes production of the MHC-I components, human leukocyte antigen A and Beta-2 microglobulin. The NLRC5-reduction mechanism recapitulates across diverse cell types, but the degree and duration of inhibition is cell type-specific. NLRC5 modulation and MHC-I inhibition are linked to another O. tsutsugamushi Ank, Ank5. NLRC5 is a putative interacting partner of Ank5. Moreover, NLRC5 and MHC-I levels are reduced in cells ectopically expressing Ank5. To our knowledge, these are the first examples of a pathogen modulating NLRC5 to negatively regulate MHC-I expression and of a bacterial effector interacting with NLRC5. As we learn more about the bacterium’s ability to regulate its host cell, a unifying theme has emerged: modulation of the ER and ER-associated pathways. These projects reveal two novel mechanisms of O. tsutsugamushi pathogenesis, strategies to acquire the amino acids needed for replication and to decrease MHC-I antigen presentation by the host cell. These insights help in understanding how O. tsutsugamushi and potentially other related pathogens co-opt host cell processes to cause disease.

Magister Scientiae (Biodiversity and Conservation Biology) - MSc (Biodiv and Cons Biol) / The human population in sub-Saharan Africa has been increasing due to decreases in mortality rates and increases in average human age; in turn increasing poverty and pressure placed on agriculture and agricultural production. However, livestock production in South Africa, and globally, is declining due to disease and parasite prevalence, lack of feed, poor breeding, marketing management, change in nutrition in both livestock and humans, rapid urbanization, encroachment on wildlife and unfavourable climatic conditions brought about by global change. One unintended consequence has been the emergence and spread of transboundary animal diseases and, more specifically, the resurgence and emergence of zoonotic disease. Zoonotic diseases are sicknesses transmissible from animals to humans, resulting from direct contact or environmental reservoirs. Previous studies have identified small-scale farmers as the group most prevalent to contracting zoonotic diseases, especially those working in a communal dispensation. Therefore, this study focused on the communal farming area of Steinkopf in the semi-arid Namaqualand region of South Africa. Steinkopf is one of the largest Act 9 areas, with communal land tenure and a mixed farming system, sheep and goats, on about 759 ha. Steinkopf is divided into two rainfall regions, the Succulent Karoo (winter rainfall region) and the Nama Karoo (summer rainfall region). This study aims to identify and characterise the bacterial microbial communities found in the topsoil layer and faecal matter (dung) within the winter and summer rainfall regions of Steinkopf communal rangeland using Next-generation sequencing. Further, the aim is to assess whether pathogenic bacteria are present within the rangeland and what their potential impact on the local farming community might be if present. A high-throughput sequencing technique (Next-generation sequencing) was used to amplify 16S rRNA targeting the V3-V4 hypervariable regions. The phylotypes produced were 37 phyla, 353 families and 634 genera of which the most abundant bacterial phyla were Planctomycetes, Firmicutes and Bacteroidetes and the most abundant genera were Gemmata, Akkermansia and Arthrobacter. Alpha diversity indices showed a variation in species diversity, evenness and richness between soil and dung samples, it shows a higher species richness, evenness and unique OTUs detected in summer soil samples and at natural water holes. Through these analysis soil samples were regarded as superior to dung samples within this particular environment and for this particular study. Natural water holes were identified as a safer option when compared to man-made water holes as there are natural systems in place that combat the spread and growth of harmful bacterial microbes. It was found that seasonality has a great impact on the development and growth of environmental bacterial microbiota and that the current randomness of grazing routes and migrations within the Steinkopf communal rangeland is not a detriment but instead acts as a benefits to environmental and livestock health. Furthermore, a total of three pathogenic bacteria were identified however, they occurred at relatively low abundances. It can thus be concluded that this study thoroughly describes the usefulness of using a high-throughput sequencing technique such as Next-generation sequencing when amplifying a small sample size in order to achieve a large volume of information; and that currently the Steinkopf communal rangeland is not subjected to or at risk of a potential zoonotic threat.

Selon le dernier rapport de l’EFSA-ECDC (EFSA Journal 2014), les parasites se classent en 8ème position des agents étiologiques impliqués dans les épidémies d’origine alimentaire reportées en 2012 en Europe. Par ailleurs, un récent rapport de l’OMS et la FAO (2014) classe Toxoplasma gondii en première position des parasites protozoaires à considérer dans le domaine alimentaire, suivi de Cryptosporidium spp et Giardia duodenalis. Les oocystes de T. gondii et Cryptosporidium spp et les kystes de G. duodenalis sont des formes très résistantes excrétées en très grande quantité dans les selles des individus malades. Lorsqu’ils se retrouvent dans l’environnement, ils peuvent y persister longtemps et contaminer certaines matrices alimentaires (végétaux et mollusques) lors de leur production primaire. A l’heure actuelle, en l’absence de méthodes d’analyse standardisées dans les aliments pour ces 3 parasites, peu de données de prévalence sont disponibles dans la littérature et les épidémies d’origine alimentaire restent négligées. Pour combler ce manque, une norme pour la détection/quantification des kystes de G. duodenalis et des oocystes de Cryptosporidium spp. dans les végétaux à feuilles vertes et fruits rouges à baies par microscopie à fluorescence est en cours de rédaction (ISO 18744). Des approches moléculaires, plus compatibles avec l’analyse de routine ont été développées par ACTALIA et l’équipe PROTAL pour détecter les 3 parasites simultanément sur des matrices végétales (Protofood, ANR-09-ALIA-009). Cependant, quelque soit la méthode de détection utilisée, elle met en évidence les parasites vivants et morts. Or, seul un parasite viable pourra être infectieux et donc potentiellement provoquer une maladie. A l’heure actuelle, les modèles in vivo constituent la méthode de choix pour évaluer l’infectiosité de manière précise, sensible et quantitative. Ils sont en revanche coûteux, lourds à mettre en œuvre et présentent un délai de réponse de plusieurs jours voire semaines qui n’est pas compatible avec les attentes des professionnels de l’agroalimentaire. L’objectif de la thèse est de développer des méthodes moléculaires pour caractériser la viabilité des 3 protozoaires dans des matrices alimentaires et disposer d’un outil permettant l’évaluation du risque lié à la détection de ces dangers dans les aliments. Ces méthodes seront comparées à celles qui permettent de mesurer l’infectiosité. Elles seront ensuite mises en œuvre pour évaluer leur potentiel pour déterminer l’efficacité d’inactivation de traitements technologiques sur des matrices alimentaires. / In the latest report from EFSA-ECDC (EFSA Journal 2014), parasites are ranked in the 8th position of the etiological agents involved in foodborne outbreaks reported in Europe in 2012. Moreover, in a recent report from the WHO and FAO (2014), Toxoplasma gondii is designated as the first protozoan parasite to be considered in the food domain, followed by Cryptosporidium spp. and Giardia duodenalis. Oocysts of T. gondii and Cryptosporidium spp., and cysts of G. duodenalis are excreted in big quantity by infected hosts and are particularly resistant. Consequently they can be found in the environment during long period and contaminate food matrices (vegetables and molluscs) during primary production. For now, since there are no standard methods to detect these 3 parasites in food samples, only few occurrence data are available and foodborne outbreaks remain neglected. To fill this gap, an ISO standard which describes a method for the detection and quantification of Cryptosporidium and Giardia in green leafy vegetables and red berries fruit by fluorescence microscopy is being draft (ISO 18744). Molecular approaches which are more suitable for routine analyses were developed by ACTALIA and PROTAL to simultaneously detect the 3 parasites in vegetable matrices (Protofood, ANR-09-ALIA-009). Nevertheless, whatever the used detection method, it highlights alive and dead parasites. But solely a living parasite can be infectious and induce pathology. For the moment, animal models are the favorite method to quantitatively evaluate infectivity with accuracy and sensitivity. However they are costly, heavy to implement and display a long time-to-result (from days to weeks) which does not fit with the agro-industrial needs. The objective of the thesis is to develop molecular methods to characterize the viability of the three protozoa in food matrices in order to have a tool allowing risk assessment in food safety. These methods will be compared to infectivity measurement methods. Then they will be implemented to evaluate their potential to determine the efficiency of technological treatments to inactivate protozoa in food matrices.

Abstract not available

Diseases in natural ecosystems are often assumed to be less severe than those observed in domestic cropping systems due to the extensive biodiversity exhibited in wild vegetation communities. In Australia, it is this natural biodiversity that is now under threat from Phytophthora cinnamomi. The soilborne Oomycete causes severe decline of native vegetation communities in south-western Victoria, Australia, disrupting the ecological balance of native forest and heathland communities. While the effect of disease caused by P. cinnamomi on native vegetation communities in Victoria has been extensively investigated, little work has focused on the Anglesea healthlands in south-western Victoria. Nothing is known about the population structure of P. cinnamomi at Anglesea. This project was divided into two main components to investigate fundamental issues affecting the management of P. cinnamomi in the Anglesea heathlands. The first component examined the phenotypic characteristics of P. cinnamomi isolates sampled from the population at Anglesea, and compared these with isolates from other regions in Victoria, and also from Western Australia. The second component of the project investigated the effect of the fungicide phosphonate on the host response following infection by P. cinnamomi. Following soil sampling in the Anglesea heathlands, a collection of P, cinnamomi isolates was established. Morphological and physiological traits of each isolate were examined. All isolates were found to be of the A2 mating type. Variation was demonstrated among isolates in the following characteristics: radial growth rate on various nutrient media, sporangial production, and sporangial dimensions. Oogonial dimensions did not differ significantly between isolates. Morphological and physiological variation was rarely dependant on isolate origin. To examine the genetic diversity among isolates and to determine whether phenotypic variation observed was genetically based, Random Amplified Polymorphic DNA (RAPD) analyses were conducted. No significant variation was observed among isolates based on an analysis of molecular variance (AMQVA). The results are discussed in relation to population biology, and the effect of genetic variation on population structure and population dynamics. X australis, an arborescent monocotyledon indigenous to Australia, is highly susceptible to infection by P. cinnamomi. It forms an important component of the heathland vegetation community, providing habitat for native flora and fauna, A cell suspension culture system was developed to investigate the effect of the fungicide phosphonate on the host-pathogen interaction between X. australis and P. cinnamomi. This allowed the interaction between the host and the pathogen to be examined at a cellular level. Subsequently, histological studies using X. australis seedlings were undertaken to support the cellular study. Observations in the cell culture system correlated well with those in the plant. The anatomical structure of X australis roots was examined to assist in the interpretation of results of histopathological studies. The infection of single cells and roots of X. australis, and the effect of phosphonate on the interaction are described. Phosphonate application prior to inoculation with P. cinnamomi reduced the infection of cells in culture and of cells in planta. In particular, phosphonate was found to stimulate the production of phenolic material in roots of X australis seedlings and in cells in suspension cultures. In phosphonate-treated roots of X australis seedlings, the deposition of electron dense material, possibly lignin or cellulose, was observed following infection with P. cinnamomi. It is proposed that this is a significant consequence of the stimulation of plant defence pathways by the fungicide. Results of the study are discussed in terms of the implications of the findings on management of the Anglesea heathlands in Victoria, taking into account variation in pathogen morphology, pathogenicity and genotype. The mode of action of phosphonate in the plant is discussed in relation to plant physiology and biochemistry.

Thesis (Ph.D.)--McMaster University, 2005. / Supervisor: Dr. Christian Baron. Includes bibliographical references (leaves 94-101).

The growth of bacteriophage HK75 is inhibited by specific mutations in the zinc binding domain of the host RNA polymerase beta prime subunit. It shares this rare property with bacteriophage HK022 and other phages that use RNA mediated antitermination to promote early gene expression. Recent genomic analysis of HK75 and HK022 has confirmed the relatedness of these two phages and place HK75 in the lambdoid family of bacteriophages. Lambdoid phages are temperate and can adopt a lytic or lysogenic lifestyle upon infection of a suitable host. However, HK75 only forms clear plaques and thus appears to be defective in its ability to form lysogens. Based on published analyses of other lambdoid phages, a clear plaque phenotype is commonly due to a mutation in one of 5 phage genes: cI, cII, cIII, int, xis or the phage repressor DNA binding sites. To determine which mutation is responsible for the clear plaque phenotype of HK75, we cloned the cI and cIII genes and assayed their activities. The HK75 cI gene clone prevented super-infection by HK75. This result demonstrated repressor functionality and thus the clear plaque phenotype cannot be due to a mutation in the HK75 cI gene. Several amino acid differences were noted between the HK022 and HK75 CIII proteins. To determine if the clear plaque phenotype was due to mutations in the HK75 cIII gene, we cloned it into an expression vector. Only under conditions of cIII gene overexpression were lysogens of HK75 recovered. The phage CIII protein normally protects CII from proteolysis. Stabilization of CII by mutations in specific host proteases has been shown to suppress a clear plaque phenotype caused by mutations in the cIII gene. When HK75 was plated on a protease deficient strain of E. coli, turbid plaques were formed and lysogens were recovered. These results support the idea that the clear plaque phenotype of HK75 is due to a defect in the expression of the phage cIII gene.

Ball State University LibrariesLibrary services and resources for knowledge buildingMasters ThesesThere is no abstract available for this thesis.

In some areas the effect of pathogenicity of microorganisms upon the European corn borer have been explored. The microorganisms studied were: Nosema, pyraustae, Nosema necatrix, and Bacillus thuringlensis var, thuringiensie. Most studies have been limited to either laboratory or field work. In this thesis, a comparative study of mortality, percent infection, and tune intervals in which infection occurs have been explored, in the field and the laboratory studies.It is concluded: (1) field recovered borers at different day intervals showed interaction between the experimental microorganisms and time intervals (2) that the microorganisms used in the field, especially N. necatrix, may be used as effective microbial control agents against Ostrinia nubilalis.Ball State UniversityMuncie, IN 47306

The six major drainage basins of Indiana were sampled for Aeromonas hydrophila. Daring the summer of 1979 a statewide mean of 53 colony forming units (W u) per ml with a range of 0-383 was obtained using membrane filter techniques and Rimler-Shotts differential media. Density values were correlated with the following physico-chemical water quality parameters: temperature, dissolved oxygen, and conductivity. Temperature showed the highest significant correlation when regressed with Gru, accounting for 25% of the variation during the statewide survey. Four sites on the White River, Delaware County, Indiana, were sampled biweekly, in the same manner as the statewide surrey, for cne year to determine seasonal variatic± in A:. hydrophila densities. The mean varied from a low of 2 CFC ml-1 in the winter months , a summer high in July of 205 CFU ml-1. No significant correlations could be found between CFU and water quality parameters during summer months. Antibiotic sensitivities were performed on 112 environmental isolates. All were resistant to ampicillin and carbenicillin and all but seven were resistant to cephalothin.Ball State UniversityMuncie, IN 47306