Pathogenicity of Plesiomonas shigelloides : interactions with eukaryotic host cells in vitro

Diarrhoea is one of the leading causes of morbidity and mortality in populations in developing countries and is a significant health issue throughout the world. Despite the frequency and the severity of the diarrhoeal disease, mechanisms of pathogenesis for many of the causative agents have been poorly characterised. Although implicated in a number of intestinal and extra-intestinal infections in humans, Plesiomonas shigelloides generally has been dismissed as an enteropathogen due to the lack of clearly demonstrated virulence-associated properties such as production of cytotoxins and enterotoxins or invasive abilities. However, evidence from a number of sources has indicated that this species may be the cause of a number of clinical infections. The work described in this thesis seeks to resolve this discrepancy by investigating the pathogenic potential of P. shigelloides using in vitro cell models. The focus of this research centres on how this organism interacts with human host cells in an experimental model. Very little is known about the pathogenic potential of P. shigel/oides and its mechanisms in human infections and disease. However, disease manifestations mimic those of other related microorganisms. Chapter 2 reviews microbial pathogenesis in general, with an emphasis on understanding the mechanisms resulting from infection with bacterial pathogens and the alterations in host cell biology. In addition, this review analyses the pathogenic status of a poorly-defined enteropathogen, P. shigelloides. Key stages of pathogenicity must occur in order for a bacterial pathogen to cause disease. Such stages include bacterial adherence to host tissue, bacterial entry into host tissues (usually required), multiplication within host tissues, evasion of host defence mechanisms and the causation of damage. In this study, these key strategies in infection and disease were sought to help assess the pathogenic potential of P. shigelloides (Chapter 3). Twelve isolates of P. shigelloides, obtained from clinical cases of gastroenteritis, were used to infect monolayers of human intestinal epithelial cells in vitro. Ultrastructural analysis demonstrated that P. shigelloides was able to adhere to the microvilli at the apical surface of the epithelial cells and also to the plasma membranes of both apical and basal surfaces. Furthermore, it was demonstrated that these isolates were able to enter intestinal epithelial cells. Internalised bacteria often were confined within vacuoles surrounded by single or multiple membranes. Observation of bacteria within membranebound vacuoles suggests that uptake of P. shigelloides into intestinal epithelial cells occurs via a process morphologically comparable to phagocytosis. Bacterial cells also were observed free in the host cell cytoplasm, indicating that P. shige/loides is able to escape from the surrounding vacuolar membrane and exist within the cytosol of the host. Plesiomonas shigelloides has not only been implicated in gastrointestinal infections, but also in a range of non-intestinal infections such as cholecystitis, proctitis, septicaemia and meningitis. The mechanisms by which P. shigelloides causes these infections are not understood. Previous research was unable to ascertain the pathogenic potential of P. shigel/oides using cells of non-intestinal origin (HEp-2 cells derived from a human larynx carcinoma and Hela cells derived from a cervical carcinoma). However, with the recent findings (from this study) that P. shigelloides can adhere to and enter intestinal cells, it was hypothesised, that P. shigel/oides would be able to enter Hela and HEp-2 cells. Six clinical isolates of P. shigelloides, which previously have been shown to be invasive to intestinally derived Caco-2 cells (Chapter 3) were used to study interactions with Hela and HEp-2 cells (Chapter 4). These isolates were shown to adhere to and enter both nonintestinal host cell lines. Plesiomonas shigelloides were observed within vacuoles surrounded by single and multiple membranes, as well as free in the host cell cytosol, similar to infection by P. shigelloides of Caco-2 cells. Comparisons of the number of bacteria adhered to and present intracellularly within Hela, HEp-2 and Caco-2 cells revealed a preference of P. shigelloides for Caco-2 cells. This study conclusively showed for the first time that P. shigelloides is able to enter HEp-2 and Hela cells, demonstrating the potential ability to cause an infection and/or disease of extra-intestinal sites in humans. Further high resolution ultrastructural analysis of the mechanisms involved in P. shigelloides adherence to intestinal epithelial cells (Chapter 5) revealed numerous prominent surface features which appeared to be involved in the binding of P. shige/loides to host cells. These surface structures varied in morphology from small bumps across the bacterial cell surface to much longer filaments. Evidence that flagella might play a role in bacterial adherence also was found. The hypothesis that filamentous appendages are morphologically expressed when in contact with host cells also was tested. Observations of bacteria free in the host cell cytosol suggests that P. shigelloides is able to lyse free from the initial vacuolar compartment. The vacuoles containing P. shigel/oides within host cells have not been characterised and the point at which P. shigelloides escapes from the surrounding vacuolar compartment has not been determined. A cytochemical detection assay for acid phosphatase, an enzymatic marker for lysosomes, was used to analyse the co-localisation of bacteria-containing vacuoles and acid phosphatase activity (Chapter 6). Acid phosphatase activity was not detected in these bacteria-containing vacuoles. However, the surface of many intracellular and extracellular bacteria demonstrated high levels of acid phosphatase activity, leading to the proposal of a new virulence factor for P. shigelloides. For many pathogens, the efficiency with which they adhere to and enter host cells is dependant upon the bacterial phase of growth. Such dependency reflects the timing of expression of particular virulence factors important for bacterial pathogenesis. In previous studies (Chapter 3 to Chapter 6), an overnight culture of P. shigelloides was used to investigate a number of interactions, however, it was unknown whether this allowed expression of bacterial factors to permit efficient P. shigelloides attachment and entry into human cells. In this study (Chapter 7), a number of clinical and environmental P. shigelloides isolates were investigated to determine whether adherence and entry into host cells in vitro was more efficient during exponential-phase or stationary-phase bacterial growth. An increase in the number of adherent and intracellular bacteria was demonstrated when bacteria were inoculated into host cell cultures in exponential phase cultures. This was demonstrated clearly for 3 out of 4 isolates examined. In addition, an increase in the morphological expression of filamentous appendages, a suggested virulence factor for P. shigel/oides, was observed for bacteria in exponential growth phase. These observations suggest that virulence determinants for P. shigel/oides may be more efficiently expressed when bacteria are in exponential growth phase. This study demonstrated also, for the first time, that environmental water isolates of P. shigelloides were able to adhere to and enter human intestinal cells in vitro. These isolates were seen to enter Caco-2 host cells through a process comparable to the clinical isolates examined. These findings support the hypothesis of a water transmission route for P. shigelloides infections. The results presented in this thesis contribute significantly to our understanding of the pathogenic mechanisms involved in P. shigelloides infections and disease. Several of the factors involved in P. shigelloides pathogenesis have homologues in other pathogens of the human intestine, namely Vibrio, Aeromonas, Salmonella, Shigella species and diarrhoeaassociated strains of Escherichia coli. This study emphasises the relevance of research into Plesiomonas as a means of furthering our understanding of bacterial virulence in general. As well it provides tantalising clues on normal and pathogenic host cell mechanisms.

Whitening Race: Essays In Social And Cultural Criticism In Australia

Aileen Moreton-Robinson has brought together scholars from a range of disciplines: philosophy, cultural and gender studies, education, social work, sociology and literary studies. All engage critically with the location of the social and discursive construction of whiteness.

Implementation and evaluation of a new model of nursing practice in radiation oncology

Patients undergoing radiation therapy for cancer face a series of challenges that require support from a multidisciplinary team which includes radiation oncology nurses. However, the specific contribution of nursing, and the models of care that best support the delivery of nursing interventions in the radiotherapy setting, is not well described. In this case study, the Interaction Model of Client Health Behaviour and the associated principles of person-centred care were incorporated into a new model of care that was implemented in one radiation oncology setting in Brisbane, Australia. The new model of care was operationalised through a Primary Nursing/Collaborative Practice framework. To evaluate the impact of the new model for patients and health professionals, multiple sources of data were collected from patients and clinical staff prior to, during, and 18 months following introduction of the practice redesign. One cohort of patients and clinical staff completed surveys incorporating measures of key outcomes immediately prior to implementation of the model, while a second cohort of patients and clinical staff completed these same surveys 18 months following introduction of the model. In-depth interviews were also conducted with nursing, medical and allied health staff throughout the implementation phase to obtain a more comprehensive account of the processes and outcomes associated with implementing such a model. From the patients’ perspectives, this study demonstrated that, although adverse effects of radiotherapy continue to affect patient well-being, patients continue to be satisfied with nursing care in this specialty, and that they generally reported high levels of functioning despite undergoing a curative course of radiotherapy. From the health professionals’ perspective, there was evidence of attitudinal change by nursing staff within the radiotherapy department which reflected a greater understanding and appreciation of a more person-centred approach to care. Importantly, this case study has also confirmed that a range of factors need to be considered when redesigning nursing practice in the radiotherapy setting, as the challenges associated with changing traditional practices, ensuring multidisciplinary approaches to care, and resourcing a new model were experienced. The findings from this study suggest that the move from a relatively functional approach to a person-centred approach in the radiotherapy setting has contributed to some improvements in the provision of individualised and coordinated patient care. However, this study has also highlighted that primary nursing may be limited in its approach as a framework for patient care unless it is supported by a whole team approach, an appropriate supportive governance model, and sufficient resourcing. Introducing such a model thus requires effective education, preparation and ongoing support for the whole team. The challenges of providing care in the context of complex interdisciplinary relationships have been highlighted by this study. Aspects of this study may assist in planning further nursing interventions for patients undergoing radiotherapy for cancer, and continue to enhance the contribution of the radiation oncology nurse to improved patient outcomes.

Multiple indicators of ambient and personal ultraviolet radiation exposure and risk of non-Hodgkin lymphoma (United States)

Recent epidemiologic studies have suggested that ultraviolet radiation (UV) may protect against non-Hodgkin lymphoma (NHL), but few, if any, have assessed multiple indicators of ambient and personal UV exposure. Using the US Radiologic Technologists study, we examined the association between NHL and self-reported time outdoors in summer, as well as average year-round and seasonal ambient exposures based on satellite estimates for different age periods, and sun susceptibility in participants who had responded to two questionnaires (1994–1998, 2003–2005) and who were cancer-free as of the earlier questionnaire. Using unconditional logistic regression, we estimated the odds ratio (OR) and 95% confidence intervals for 64,103 participants with 137 NHL cases. Self-reported time outdoors in summer was unrelated to risk. Lower risk was somewhat related to higher average year-round and winter ambient exposure for the period closest in time, and prior to, diagnosis (ages 20–39). Relative to 1.0 for the lowest quartile of average year-round ambient UV, the estimated OR for successively higher quartiles was 0.68 (0.42–1.10); 0.82 (0.52–1.29); and 0.64 (0.40–1.03), p-trend = 0.06), for this age period. The lower NHL risk associated with higher year-round average and winter ambient UV provides modest additional support for a protective relationship between UV and NHL.

Characterisation of the tritrophic interactions between tobacco yellow dwarf virus, its vector and host-plants

Tobacco yellow dwarf virus (TbYDV, family Geminiviridae, genus Mastrevirus) is an economically important pathogen causing summer death and yellow dwarf disease in bean (Phaseolus vulgaris L.) and tobacco (Nicotiana tabacum L.), respectively. Prior to the commencement of this project, little was known about the epidemiology of TbYDV, its vector and host-plant range. As a result, disease control strategies have been restricted to regular poorly timed insecticide applications which are largely ineffective, environmentally hazardous and expensive. In an effort to address this problem, this PhD project was carried out in order to better understand the epidemiology of TbYDV, to identify its host-plant and vectors as well as to characterise the population dynamics and feeding physiology of the main insect vector and other possible vectors. The host-plants and possible leafhopper vectors of TbYDV were assessed over three consecutive growing seasons at seven field sites in the Ovens Valley, Northeastern Victoria, in commercial tobacco and bean growing properties. Leafhoppers and plants were collected and tested for the presence of TbYDV by PCR. Using sweep nets, twenty-three leafhopper species were identified at the seven sites with Orosius orientalis the predominant leafhopper. Of the 23 leafhopper species screened for TbYDV, only Orosius orientalis and Anzygina zealandica tested positive. Forty-two different plant species were also identified at the seven sites and tested. Of these, TbYDV was only detected in four dicotyledonous species, Amaranthus retroflexus, Phaseolus vulgaris, Nicotiana tabacum and Raphanus raphanistrum. Using a quadrat survey, the temporal distribution and diversity of vegetation at four of the field sites was monitored in order to assess the presence of, and changes in, potential host-plants for the leafhopper vector(s) and the virus. These surveys showed that plant composition and the climatic conditions at each site were the major influences on vector numbers, virus presence and the subsequent occurrence of tobacco yellow dwarf and bean summer death diseases. Forty-two plant species were identified from all sites and it was found that sites with the lowest incidence of disease had the highest proportion of monocotyledonous plants that are non hosts for both vector and the virus. In contrast, the sites with the highest disease incidence had more host-plant species for both vector and virus, and experienced higher temperatures and less rainfall. It is likely that these climatic conditions forced the leafhopper to move into the irrigated commercial tobacco and bean crop resulting in disease. In an attempt to understand leafhopper species diversity and abundance, in and around the field borders of commercially grown tobacco crops, leafhoppers were collected from four field sites using three different sampling techniques, namely pan trap, sticky trap and sweep net. Over 51000 leafhopper samples were collected, which comprised 57 species from 11 subfamilies and 19 tribes. Twentythree leafhopper species were recorded for the first time in Victoria in addition to several economically important pest species of crops other than tobacco and bean. The highest number and greatest diversity of leafhoppers were collected in yellow pan traps follow by sticky trap and sweep nets. Orosius orientalis was found to be the most abundant leafhopper collected from all sites with greatest numbers of this leafhopper also caught using the yellow pan trap. Using the three sampling methods mentioned above, the seasonal distribution and population dynamics of O. orientalis was studied at four field sites over three successive growing seasons. The population dynamics of the leafhopper was characterised by trimodal peaks of activity, occurring in the spring and summer months. Although O. orientalis was present in large numbers early in the growing season (September-October), TbYDV was only detected in these leafhoppers between late November and the end of January. The peak in the detection of TbYDV in O. orientalis correlated with the observation of disease symptoms in tobacco and bean and was also associated with warmer temperatures and lower rainfall. To understand the feeding requirements of Orosius orientalis and to enable screening of potential control agents, a chemically-defined artificial diet (designated PT-07) and feeding system was developed. This novel diet formulation allowed survival for O. orientalis for up to 46 days including complete development from first instar through to adulthood. The effect of three selected plant derived proteins, cowpea trypsin inhibitor (CpTi), Galanthus nivalis agglutinin (GNA) and wheat germ agglutinin (WGA), on leafhopper survival and development was assessed. Both GNA and WGA were shown to reduce leafhopper survival and development significantly when incorporated at a 0.1% (w/v) concentration. In contrast, CpTi at the same concentration did not exhibit significant antimetabolic properties. Based on these results, GNA and WGA are potentially useful antimetabolic agents for expression in genetically modified crops to improve the management of O. orientalis, TbYDV and the other pathogens it vectors. Finally, an electrical penetration graph (EPG) was used to study the feeding behaviour of O. orientalis to provide insights into TbYDV acquisition and transmission. Waveforms representing different feeding activity were acquired by EPG from adult O. orientalis feeding on two plant species, Phaseolus vulgaris and Nicotiana tabacum and a simple sucrose-based artificial diet. Five waveforms (designated O1-O5) were observed when O. orientalis fed on P. vulgaris, while only four (O1-O4) and three (O1-O3) waveforms were observed during feeding on N. tabacum and the artificial diet, respectively. The mean duration of each waveform and the waveform type differed markedly depending on the food source. This is the first detailed study on the tritrophic interactions between TbYDV, its leafhopper vector, O. orientalis, and host-plants. The results of this research have provided important fundamental information which can be used to develop more effective control strategies not only for O. orientalis, but also for TbYDV and other pathogens vectored by the leafhopper.

Broad Bean Mottle Virus: Identification, Serology, Host Range, and Occurrence on Faba Bean (Vicia Faba) in West Asia and Norm Africa

One of the faba bean viruses found in West Asia and North Africa was identified as broad bean mottle virus (BBMV) by host reactions, particle morphology and size, serology, and granular, often vesiculated cytoplasmic inclusions. Detailed research on four isolates, one each from Morocco, Tunisia, Sudan and Syria, provided new information on the virus. The isolates, though indistinguishable in ELISA or gel-diffusion tests, differed slightly in host range and symptoms. Twenty-one species (12 legumes and 9 non-legumes) out of 27 tested were systemically infected, and 14 of these by all four isolates. Infection in several species was symptomless, but major legumes such as chickpea, lentil and especially pea, suffered severely from infection. All 23 genotypes of faba bean, 2 of chickpea, 4 of lentil, 11 out of 21 of Phaseolus bean, and 16 out of 17 of pea were systemically sensitive to the virus. Twelve plant species were found to be new potential hosts and cucumber a new local-lesion test plant of the virus. BBMV particles occurred in faba bean plants in very high concentrations and seed transmission in this species (1.37%) was confirmed. An isolate from Syria was purified and two antisera were produced, one of which was used in ELISA to detect BBMV in faba bean field samples. Two hundred and three out of the 789 samples with symptoms suggestive of virus infection collected in 1985, 1986 and 1987, were found infected with BBMV: 4 out of 70 (4/70) tested samples from Egypt, 0/44 from Lebanon, 1/15 from Morocco, 46/254 from Sudan, 72/269 from Syria and 80/137 from Tunisia. This is the first report on its occurrence in Egypt, Syria and Tunisia. The virus is a potential threat to crop improvement in the region.

Host use and crop impacts of Oribius Marshall species (Coleoptera: Curculionidae) in Eastern Highlands Province, Papua New Guinea

Oribius species are small flightless weevils endemic to the island of New Guinea and far northern Cape York, Australia. The adults feed externally on leaves, developing fruit and green bark, but their impact as pests and general host use patterns are poorly known. Working in Eastern Highlands Province, Papua New Guinea, we carried out structured host use surveys, farmer surveys, shade-house growth trials, and on-farm and on-station impact trials to: (i) estimate the host range of the local Oribius species; (ii) understand adult daily activity patterns; (iii) elucidate feeding habits of the soil dwelling larvae; and (iv) quantify the impacts of adult feeding damage. Oribius inimicus and O. destructor accounted for nearly all the Oribius species encountered locally: of these two O. inimicus was the most abundant. Weevils were collected from 31 of 33 plants surveyed in the Aiyura Valley and a combination of farmer interviews and literature records provided evidence for the beetles being pestiferous on 43 crops currently or previously grown in the Highlands. Adult weevils had a distinct diurnal pattern of being in the upper plant canopy early in the morning and, to a lesser extent, again late in the afternoon. For the remainder of the day beetles resided within the canopy, or possibly off the plant. Movement of adults between plants appeared frequent. Pot trials confirmed the larvae are root feeders. Quantified impact studies showed that the weevils are damaging to a range of vegetable and orchard crops (broccoli, capsicum, celery, French bean, Irish potato, lettuce, orange and strawberry), causing average yield losses of around 30-40%, but up to 100% on citrus. Oribius weevils pose a significant and apparently growing problem for Highland’s agriculture.