Master of Agribusiness / Department of Agricultural Economics / Keith D. Harris / Food processing facilities are faced with many challenges in ensuring that the food supply is safe for consumption. Listeria monocytogenes is a food pathogen that has been linked to ready-to-eat foods, including tree nuts. Listeria monocytogenes is part of the ubiquitous microorganism genus, Listeria. The most likely cause of Listeria contamination in food is post-processing contamination. The purpose of this research is to identify and examine possible solutions a nut processing facility might employ to mitigate a food safety risk. The outcome of this research helps to establish the most financially viable method a processing facility may implement to address and mitigate an established risk given defined premise construction and constraints. The research objective is to identify a solution, implement a course of action, and establish safeguards to prevent recurrence of the issue. Factoring in facility specific variables as well as industry data and relevant analyses, the research conducted concludes with recommended actions for the facility to make, including a combination of structural design changes coupled with extensive chemical sanitation techniques.

The study was made at Lubigi sewage treatment plant in Kampala, Uganda, during February and March 2018. The aim of this master thesis was to treat faecal sludge with two different methods, urea and lime, to investigate the efficiency of the chemicals to inactivate pathogens and to estimate the feasibility and the costs of the treatment. The chemical treatments were performed on sludge of two different moisture contents. The results from the treated sludge were compared with an untreated drying bed filled at the start of the study period to use as a control. The investigated pathogens were E. coli, bacteriophages and Ascaris eggs. The total solids and volatile solids were analysed, and the pH was measured. The results of the study including a calculation of costs were used to assess the feasibility of these treatment methods at Lubigi sewage treatment plant. The results show that the treatment using lime and urea reduces the level of active pathogens in the faecal sludge. The drying process in the beds at the treatment plant also decreases the level of detected pathogens in the sludge, but not to the same extent as when adding chemicals. The E. coli in the treated sludge were under the detection limit before the study period was done. Ascaris eggs were still detected in the sludge by the final sampling occasion. Further monitoring of the treated sludge might show an additional decrease of Ascaris since the time of storage after treatment enables pathogen die-off. The bacteriophages analyses only succeeded for two sampling occasions, although a decrease of detected phages is visible in the results. Again, the time of storage is significant for pathogen reduction, which is why a decrease of bacteriophages is expected if the sludge were to be further monitored. The feasibility of the two treatment methods is mainly restricted by costs. Lime treatment is approximately seven times more expensive than urea treatment and is also required in a larger amount to treat the sludge. On the other hand, using lime to treat faecal sludge is a proven and effective method. Further studies would improve the knowledge of the sludge characteristics at Lubigi and help determine the most preferable treatment for the sludge to protect the environment and public health. For example, by focusing on one treatment method, more detailed information can be gathered, and if performing a study in a larger scale, the representativeness would increase. To make sure there is no risk of spreading pathogens to the environment, further analyses should be carried out directly before selling the sludge to farmers.

Viral sequence data are increasingly being used to estimate evolutionary and epidemiological parameters to understand the dynamics of viral diseases. This thesis focuses on developing novel and improved computational methods for high-throughput analysis of large viral sequence datasets. I have developed a novel computational pipeline, Pipelign, to detect potentially unrelated sequences from groups of viral sequences during sequence alignment. Pipelign detected a large number of unrelated and mis-annotated sequences from several viral sequence datasets collected from GenBank. I subsequently developed ANVIL, a machine learning-based recombination detection and subtyping framework for pathogen sequences. ANVIL's performance was benchmarked using two large HIV datasets collected from the Los Alamos HIV Sequence Database and the UK HIV Drug Resistance Database, as well as on simulated data. Finally, I present a computational pipeline named Phlow, for rapid phylodynamic inference of heterochronous pathogen sequence data. Phlow is implemented with specialised and published analysis tools to infer important phylodynamic parameters from large datasets. Phlow was run with three empirical viral datasets and their outputs were compared with published results. These results show that Phlow is suitable for high-throughput exploratory phylodynamic analysis of large viral datasets. When combined, these three novel computational tools offer a comprehensive system for large scale viral sequence analysis addressing three important aspects: 1) establishing accurate evolutionary history, 2) recombination detection and subtyping, and 3) inferring phylodynamic history from heterochronous sequence datasets.

Doctor of Philosophy / Biology / Michael A. Herman / Nematodes play an important role in various habitats where numerous factors serve to shape their communities. One such factor is the potentially pathogenic nematode-prey interaction. This project is focused on the elucidation of the genes that the bacterivorous nematode Caenorhabditis elegans employs to respond to the emerging nosocomial bacterial pathogen Stenotrophomonas maltophilia. A virulent S. maltophilia strain JCMS requires the action of several C. elegans conserved innate immune pathways that serve to protect the nematode from other pathogenic bacteria. However, insulin-like DAF-2/16 signaling pathway mutants that are typically pathogen resistant are susceptible to JCMS, and several DAF-2/16 regulated genes are not significantly differentially expressed between JCMS and avirulent E. coli OP50. We have determined the complete set of mRNA transcripts under different bacterial treatments to identify genes that might explain this JCMS specific DAF-2/16 pathway evasion. The identified set included 438 differentially expressed transcripts among pairwise comparisons of wild-type nematodes fed OP50, JCMS or avirulent S. maltophilia K279a. Candidate genes were nominated from this list of differentially expressed genes using a probabilistic functional connection model. Six of seven genes that were highly connected within a gene network generated from this model showed a significant effect on nematode survival by mutation. Of these genes, C48B4.1, mpk-2, cpr-4, clec-67 and lys-6 are needed for combating JCMS, while dod-22 was solely involved in K279a response. Only dod-22 had a documented role in innate immunity, which merits our approach in the identification of gene candidates. To a lesser extent, we have also focused on the identification of virulence factors and the mode of action employed by S. maltophilia. JCMS virulence requires rpfF, xps and involves living bacteria that accumulate in the intestinal lumen. Additionally, the bacterial secretion encoding genes cs, p773, p1176, pi1y1 and xdi are involved in JCMS evasion of daf-2. In summary, we have discovered a novel host-pathogen interaction between C. elegans and S. maltophilia JCMS, revealed genes that are involved in each partner of the interaction, and established a new animal model for the study of S. maltophilia mode of action.

abstract: Studies of ancient pathogens are moving beyond simple confirmatory analysis of diseased bone; bioarchaeologists and ancient geneticists are posing nuanced questions and utilizing novel methods capable of confronting the debates surrounding pathogen origins and evolution, and the relationships between humans and disease in the past. This dissertation examines two ancient human diseases through molecular and bioarchaeological lines of evidence, relying on techniques in paleogenetics and phylogenetics to detect, isolate, sequence and analyze ancient and modern pathogen DNA within an evolutionary framework. Specifically this research addresses outstanding issues regarding a) the evolution, origin and phylogenetic placement of the pathogen causing skeletal tuberculosis in New World prior to European contact, and b) the phylogeny and origins of the parasite causing the human leishmaniasis disease complex. An additional chapter presents a review of the major technological and theoretical advances in ancient pathogen genomics to frame the contributions of this work within a rapidly developing field. This overview emphasizes that understanding the evolution of human disease is critical to contextualizing relationships between humans and pathogens, and the epidemiological shifts observed both in the past and in the present era of (re)emerging infectious diseases. These questions continue to be at the forefront of not only pathogen research, but also bioarchaeological and paleopathological scholarship. / Dissertation/Thesis / Doctoral Dissertation Anthropology 2014

O objetivo deste projeto foi entender a organização genômica e o conteúdo de genes de dois fitopatógenos relacionados geneticamente, mas que infectam diferentes hospedeiros: Leifsonia xyli subsp. xyli (Lxx), um patógeno de cana-de-açúcar; e Leifonia xyli subsp. cynodontis (Lxc), um patógeno de gramíneas do gênero Cynodon. Os resultados do seqüenciamento parcial do genoma de Lxc são descritos, incluindo as seqüências comuns ao genoma de Lxx e os fragmentos de organização distinta e genes específicos de Lxc. Para alcançar o objetivo, bibliotecas genômicas do genoma de Lxc foram construídas. A estratégia revelou seqüências específicas, algumas provavelmente adquiridas por transferência horizontal, e regiões não sintênicas do genoma de Lxc, quando comparadas com Lxx. Regiões específicas somaram 311.353 pb e foram anotadas. Devido a associação de elementos genéticos móveis com reorganização cromossômica e transferência horizontal de genes, um estudo detalhado dos transposons do tipo IS, presentes em ambos os genomas, foi realizado. A análise revelou um número variável de elementos para cada genoma atuando na diversificação dos mesmos. / This study aimed to understand genome organization and gene content of two closely related plant pathogens that infect different hosts: Leifsonia xyli subsp. xyli (Lxx), a sugarcane pathogen; and Leifonia xyli subsp. cynodontis (Lxc), a Cynodon grass pathogen. We describe the results of a partial genome sequence of Lxc, assessing the similarity to Lxx completely sequenced genome, describing differences in genome organization and uncovering genes specific to Lxc genome. To accomplish the objective, genomic libraries were constructed. The strategy uncovered specific sequences, some probably acquired by horizontal transfer and non-syntenic regions of Lxc genome compared to Lxx. Specific regions of Lxc genome accounted for 311,353 bp and were annotated. Because mobile genetic elements are often associated with rearrangements and horizontal gene transfer, a detailed study of all insertion sequence (IS) elements presented in both genomes were realized. The analysis revealed a variable number of transposable elements acting upon genomic diversity.

CoordenaÃÃo de AperfeÃoamento de Pessoal de NÃvel Superior / O cajueiro (Anacardium occidentale L.) à uma planta nativa do Brasil com grande valor comercial. Isso contribui com a geraÃÃo de milhares de empregos diretos e indiretos, especialmente na RegiÃo Nordeste, em Ãpoca de estiagem. Programas de melhoramento genÃtico vem selecionando cultivares de cajueiro melhores adaptados ao ambiente semiÃrido a fim de colocÃ-lo em um mercado cada vez mais competitivo. Entre os tipos de proteÃnas de sementes vÃrias tÃm funÃÃo de reserva, estrutural ou metabÃlica. AlÃm disso, as plantas sÃo dispostas de uma variedade de mecanismos de defesa contra o ataque de patÃgenos. Uma das respostas de defesa mais estudada diz respeito à expressÃo de proteÃnas relacionadas com a patogÃnese nas quais se inclui o grupo das quitinases. A enzima quitinase (EC 3.2.1.14) hidrolisa o polÃmero de quitina para a N-acetil glucosamina por qualquer uma das endo ou exo clivagens da ligaÃÃo β (1-4). As respostas moleculares nos perfis das plantas a nÃvel transcricional tÃm demonstrado serem cruciais para o estabelecimento de um conjunto de mecanismos de defesa contra patÃgenos invasores. Usando ferramentas de bioinformÃtica foram identificados, no transcriptoma de cajueiro CCP076, dez contigs apresentando alto grau de semelhanÃa com quitinases, endoquitinases, e quitinases-like das famÃlias GH18 e GH 19 de Ricinus communis, Cicer arietinum, Mangifera indica, Citrus sinensis, Euonymus europaeus, Vitis vinifera, Aegilops tauschii e Hevea brasiliensis. Os ensaios enzimÃticos das proteÃnas da castanha confirmaram a presenÃa quitinases em seu proteoma. Ainda revelaram uma atividade catalÃtica Ãtima em pH 5. Um perfil de proteÃnas com sitio de ligaÃÃo à quitina tambÃm foi encontrado. Dessa forma, à demonstrado um grande potencial biotecnolÃgico nas quitinases provenientes da castanha de cajueiro CCP76. / The cashew (Anacardium occidentale L.) is a plant native to Brazil with high market value. This contributes to the generation of thousands of direct and indirect jobs, especially in the Northeast, in the dry season. Breeding program has been selecting the best cashew cultivars adapted to semi-arid environment in order to put it in an increasingly competitive market. Among the types of seed proteins have several reservation function, structural or metabolic. Furthermore, plants are arranged in an array of defense mechanisms against pathogen attack. One of the most studied defense response with respect to the expression of pathogenesis related proteins which are included in the group of chitinases. The enzyme chitinase (EC 3.2.1.14) hydrolyse the polymer chitin to N-acetyl glucosamine by either endo or exo cleavage of β connection (1-4). The molecular profiles of responses in plants transcriptional level have been shown to be crucial for the establishment of a set of defense mechanisms against invading pathogens. Using bioinformatics tools were identified in the transcriptome cashew CCP076 ten contigs having high degree of similarity with chitinases, endoquitinases and chitinase-like the GH18 family and GH 19 of Ricinus communis, Cicer arietinum, Mangifera indica, Citrus sinensis, Euonymus europaeus , Vitis vinifera, Aegilops tauschii and Hevea brasiliensis. The enzyme assays of chestnut chitinase protein confirmed the presence in their proteome. Also revealed a great catalytic activity at pH 5. A protein profile with chitin-binding site was also found. Of these, it is demonstrated great potential in biotechnological chitinase from CCP76 cashew nuts.

Strains of the well-studied plant pathogen Pseudomonas syringae show large differences in their ability to colonize plants epiphytically and to inflict damage to hosts. Additionally, P. syringae can infect some sap-sucking insects and at least one P. syringae strain is highly virulent to insects, causing death to most individuals within as few as 4 days and growing to high population densities within insect hosts. The likelihood of agricultural pest insects coming into contact with transient populations of P. syringae while feeding on plants is high, yet the ecological implications of these interactions are currently not well understood as virulence has not been tested across a wide range of strains. To investigate virulence differences across strains we exposed the sweet potato whitefly, Bemisia tabaci, and the pea aphid, Acyrthosiphon pisum, both of which are cosmopolitan agricultural pests, to 12 P. syringae strains. We used oral inoculations with bacteria suspended in artificial diet in order to assay virulence while controlling for other variables such as differences in epiphytic growth ability. Generally, patterns of pathogenicity remain consistent across the two species of hemipteran insects, with bacterial strains from phylogroup II, or genomospecies 1, causing the highest rate of mortality with up to 86% of individuals dead after 72 h post infection. The rate of mortality is highly variable across strains, some significantly different from negative control treatments and others showing no discernable difference. Interestingly, one of the most pathogenic strains to both aphids and whiteflies (Cit7) is thought to be nonpathogenic on plants. We also found Cit7 to establish the highest epiphytic population after 48 h on fava beans. Between the nine P. syringae strains tested for epiphytic ability there is also much variation, but epiphytic ability was positively correlated with pathogenicity to insects, suggesting that the two traits may be linked and that strains likely to be found on plants may often be entomopathogenic. Our study highlights that there may be a use for epiphytic bacteria in the biological control of insect crop pests. It also suggests that interactions with epiphytic bacteria could be evolutionary and ecological drivers for hemipteran insects.

This PhD examined the epidemiology of two potentially devastating plant pathogens, Phytophthora ramorum and P. kernoviae, at Benmore Botanic Garden in Argyll & Bute and Brodick Castle Garden in Arran. Spore traps, river baiting, bait plants and soil sampling were used to both confirm the presence of, and measure the amount of inoculum in the environment in order to quantify the relationship between inoculum levels and disease development. P. ramorum was detected in spore traps at high levels under a sporulating host throughout the year at Benmore. Also, findings at sites where infected hosts had been removed before the study led to the conclusion that the low level spore traps detect inoculum from soil splash. Rhododendron and Vaccinium bait plants were also infected with P. ramorum via soil splash at sites within Benmore where there was no sporulating host present. P. kernoviae was detected in spore traps at Brodick throughout the year but only where there was a sporulating host overhead. P. kernoviae infected bait plants only where an infected host is overhead. Water baiting confirmed the presence of P. ramorum in two streams at Benmore but P. kernoviae was not detected using this method despite the large-scale P. kernoviae infection at Brodick. Inoculum continued to be detected in soil in areas of both gardens where infected hosts had been removed two years previously, confirming that both of these pathogens can survive in soil for a considerable period. A number of statistical models were produced to describe conditions required for P. ramorum sporulation and infection. Sporulation occurred during warmer and wetter conditions and infection of bait plants occurred in wet conditions and where an infected host is in close proximity. A statistical model was also used to produce a P. ramorum risk map, uniquely at the garden scale, to enable garden managers at Benmore to visualise the areas most at risk within their garden. The findings of this study have clear management implications for the control of disease establishment and spread within the garden setting.

Il y a désormais un consensus autour de l’idée que nous devons réduire l’usage des pesticides (Plan Ecophyto). Il est donc nécessaire de développer des systèmes de cultures réduisants l’utilisation des pesticides au profit d’un système de production dans lequel on stimule les mécanismes de défense «naturels» des plantes. Dans ce contexte, on voit aujourd’hui se développer de nombreuses solutions d’origines chimique ou biologique à base de stimulateurs de défenses des plantes (SDP) mais dont l’efficacité est souvent jugée aléatoire. Dans ce travail, notre choix a porté sur l’utilisation des rayonnements UV-C pour stimuler les défenses de la laitue et de la tomate contre Botrytis cinerea. L’idée d’exploiter les rayonnements UV-C, comme stimulateurs des défenses des plantes à intérêt alimentaire en culture, n’a jamais été testée. Nos résultats démontrent clairement un effet stimulateur des défenses des plantes par des doses faibles et répétées d’UV-C (inférieur à 1,70 kJ/m2). Cette stimulation est associée au renforcement de la paroi cellulaire, à l’augmentation des composés phénoliques et à l’augmentation de l’activité de certaines enzymes antioxydantes. Lorsque le traitement par les rayonnements UV-C est combiné à l’agent biologique Bacillus subtilis aucun effet synergique ou même additif n’a été observé par rapport à un traitement simple UV-C ou B. subtillis. / There is now a consensus around the idea that we must reduce theuse of pesticides (Plan Ecophyto). It is therefore necessary to develop cropsystems that have less need to be protected by pesticides in favor of aproduction system in which the "natural" defense mechanisms arestimulated. In this context, many solutions of chemical or biological originbased on stimulation of plant defense are now being developed but theireffectiveness is often judged to be random. In this work, we chose to useUV-C radiation to stimulate the defenses of lettuce and tomato againstBotrytis cinerea. The idea of exploiting radiation UV-C, as a stimulant ofplant food-borne defenses in culture, has never been tested. Our resultsclearly demonstrate a stimulatory effect of plant defenses by low andrepeated doses of UV-C (less than 1.70 kJ/m2). This stimulation isassociated with a reinforcement of the cell wall, an increase in the phenoliccompounds and the activity of certain antioxidant enzymes. When treatmentwith UV-C radiation is combined with a biological agent Bacillus subtilis, nosynergistic or even additive effect has been observed compared with asimple UV-C or B. subtillis treatment. / Se ha establecido en el mundo entero la importancia sobre el controlde uno de los principales organismos fitopatológicos como lo es Botrytiscinérea, el cual demanda en la actualidad a los productores aplicarcontroles cónsonos con la protección del medio ambiente. Por consiguiente,se evaluó el uso de diferentes dosis de luz ultravioleta sola o encombinación con un agente de biocontrol (Basillus subtilis), con la finalidadde estimular las defensas naturales de dos especies vegetales (lechuga ytomate). Al respecto, los resultados establecieron que dosis menores de1,70 kJ/m2 de UV-C logran disminuir la sensibilidad de las plantas, sinafectar significativamente el desenvolvimiento del aparato fotosintético deambas especies. Así como, se observó un efecto antagónico del biocontrolsobre la UV-C cuando se combinan.