Unregulated use of growth promoting antibiotics like Tetracyclines in agricultural feeds is becoming an increasing problem in antibiotic resistance. Undigested antibiotics leads to significant concentrations in livestock waste. These concentrations provide continuous selection pressure for the development of antibiotic resistance genes in the environment. Antibiotic resistance related deaths are projected to surpass cancer related deaths by 2050 making antibiotic resistance a pressing public health issue. The purpose of this study is to determine the abundance and persistence of tetracycline (tet) resistance genes in swine waste over a period of 100 days in an anaerobic digester system. Tet(A), tet(B), tet(G), tet(M), tet(O), tet(Q), and tet(W) were quantified by quantitative polymerase chain reaction after DNA extraction. Primers that target ribosomal protection proteins and efflux proteins were used. Antibiotic resistance genes decreased from day one but were found to be present throughout the study.

Fungi in the genus Trichoderma have been characterized as biocontrol agents of plant pathogens since the 1930s. The use of biologicals for disease management has increased in recent years, typically marketed as a safer alternative to chemical applications. However, biologicals often lack consistent control across varying environmental conditions. To overcome the loss in efficacy due to environmental conditions, biologicals can be combined with common fungicide seed-treatments to provide improved control. Additionally, the presence of a biological organism could slow the development of a pathogen population. Greenhouse trials were conducted to determine the baseline root colonization of three Trichoderma spp. used in conjunction with five commonly used seed treatments. In field trials, a stand-alone treatment of the Trichoderma isolates was assessed for management of Rhizoctonia root rot (caused by Rhizoctonia solani) and frogeye leaf spot (caused by Cercospora sojina). The greenhouse trial provided evidence that isolates of T. virens and T. hamatum can colonize the roots of plants in which seeds were treated with metalaxyl + prothioconazole + penflufen or metalaxyl + prothioconazole + penflufen + fluopyram. Surprisingly, in the Rhizoctonia root rot trials, the soybean seedlings treated with Trichoderma spp. had significantly reduced stand compared to the R. solani inoculated control. For the frogeye leaf spot trial, an application of T. virens conidial suspensions as a foliar treatment significantly (P ≤ 0.10) reduced frogeye leaf spot severity of soybean compared to a non-treated control. Future research is warranted to better understand the potential efficacy in additional environments and the mechanism(s) of action used by the Trichoderma isolates evaluated in these experiments.

Parasite life histories influence many aspects of infection dynamics, from the parasite infrapopulation diversity to the fitness of the parasite (the number of successfully transmitted parasites). Studies of medically important parasites, such as the parasite responsible for malaria (Plasmodium spp.), demonstrate the usefulness of investigating the life histories of parasites to better understand infection characteristics such as parasite load and probability of transmission. The gregarines are a diverse group of apicomplexan parasites that infect invertebrates, and are particularly common in insects and annelids. Given the great biodiversity and importance of their hosts, coupled with their close evolutionary relationship with important human pathogens such as Cryptosporidium spp., relatively little is known about gregarine life histories. The exemplar gregarine genus, Monocystis, is an excellent example of how a well-known gregarine parasite can have relatively little known about its life history. Specifically, the low reproductive output of Monocystis spp. and the absence of asexual replication makes the currently accepted life cycle untenable. More data are needed on the life history traits and strategies of Monocystis spp. that allow the parasite to be maintained at high prevalence and parasitemia. Here, a newly discovered species of Monocystis, infecting the invasive earthworm Amynthas agrestis, is described and investigated to determine key life history traits and strategies. First, I propose improvements to the current standard of gregarine species descriptions by standardizing nomenclature and biometrics and including molecular data. I described the newly discovered M. perplexa using the proposed improvements to gregarine species descriptions and found evidence of host species-specificity and widespread prevalence of the parasite in local earthworm populations. Such important data would not otherwise be collected with use of the current standard of gregarine species description and demonstrates the potential of implementing the suggested improvements. The life history of M. perplexa was investigated by measuring various life history traits throughout the course of infection. Key life history traits such as parasite phenology, resource allocation, and reproductive output were measured for infections at three local earthworm populations to determine if there is variation in parasite life histories within or among earthworm populations. Substantial variation in parasite life history traits and trade-offs between traits were identified. Notably, there was a trade-off between timing to parasite maturity, resource allocation, and reproductive output. Surprisingly, there was a near-complete lack of transmissible stages produced in one population, despite being maintained at high prevalence and parasitemia. To determine whether life history strategies, such as mode of transmission and asexual replication, can explain the currently untenable life cycle of M. perplexa, the presence of vertical transmission and asexual replication was revealed. Quantitative real-time PCR was used to detect and quantify minute amounts of parasite in the worm eggs and embryos and revealed a high rate of vertical transmission at all local sites. Evidence of parasite replication within the developing host embryo was found; however, no asexual replication was identified early in the host's season. Last, all worm tissues examined had high concentrations of parasite DNA, including the clitellum, the organ that produces the worm egg capsule.

La microflore des raisins est importante d’un point de vue technologique car elle conditionne en partie la qualité du vin. Or, la diversité des flores fongiques présentes sur baies de raisin ainsi que leur potentiel de contamination du produit final ne sont pas encore pleinement connus. Dans ce cadre, la caractérisation des flores fongiques cultivables présentes sur baies de raisin a été réalisée par PCR ITS-RFLP. 41 espèces de moisissures différentes sur les 43 étudiées appartenant à 11 genres différents ont été caractérisées de façon fiable. Seules les espèces Penicillium thomii et Penicillium glabrum ont présenté le même profil. Ainsi 96.3% des souches étudiées ont été caractérisées avec au maximum 4 enzymes de restriction et 41.5% des souches ont pu l’être avec seulement 2 enzymes de restriction. Ces résultats ont permis d’enrichir les bases de données, moyennement pourvues en séquences ITS caractéristiques de genres ou d’espèces de moisissures présentes sur baies de raisin. De plus, une étude exhaustive des moisissures présentes sur baies de raisin en Bourgogne a permis, par PCR ITS-RFLP, d’identifier 199 souches au niveau de l’espèce et ce quelque soit le genre. Penicillium spinulosum est l’espèce majoritaire isolée pour le millésime 2008 en Bourgogne. Parallèlement, la quantification de Botrytis cinerea, choisi comme micro-organisme modèle, a été réalisée par qPCR. La technique qPCR décrite dans ce travail présente (i) une bonne sensibilté avec une limite de détection de 6.4 pg d’ADN correspondant à 540 spores, (ii) l’originalité de travailler en échantillons naturellement contaminés et la fiabilité d’utiliser un standard interne. L’évaluation de l’efficacité de différentes stratégies de traitements anti-Botrytis a confirmé l’importance de la prophylaxie (effeuillage) dans la lutte contre Botrytis cinerea. / Microbial population of grapes is important from a technological point of view because it determines the quality of wine. But few studies have focused on fungal populations of grapes. A better knowledge of the fungal diversity on grapes, particularly as concerns species responsible for wine defects, may help efforts to control their development.We report the development of a PCR ITS-RFLP method as a fast and easy technique for identifying species of fungal genera present on grapes. By this methode, 41 different fungal species among 43 studied species belonging to 11 genera were characterized at the species level. Only P. thomii remained indistinguishable from P. glabrum. Using this PCR-ITS-RFLP, 96.3% strains tested could be differentiated to the species level with only four enzymes and 41.5% only with two enzymes. Moreover this work has contributed to the enriching of the database of fungal ITS sequences.Thus 199 isolated strain were on grapes in Burgundy vineyard were chacacterized at species level indepdantly of the genus by this method. P. spinolusum was the most frequently isolated species of Penicillium in Burgundy for 2008 vintage. Paralelly, the quantification of Botrytis cinerea, used as model, was developped by qPCR. The assay contained an internal amplification control to compensate for variations in DNA extraction and the various compounds from grapes, had high efficiency and the limit of detection was estimated to be 6.3 pg DNA (corresponding to 540 spores). This method was applied to assess the effects of various treatment strategies against Botrytis in the vineyard and demonstrates the importance of the prophylactic method.

Le processus d'épileptogenèse, qui conduit à la production de décharges électro-encéphalographiques spontanées caractéristiques de la maladie épileptique, implique des mécanismes inconnus pour la plupart et probablement divers. Des études récentes soulignent le rôle potentiel de l'inflammation cérébrale dans les mécanismes précoces de l'épileptogenèse. Le syndrome d'épilepsie de la face mésiale du lobe temporal (EMLT) est souvent associé à une perte neuronale unilatérale et sélective dans l'hippocampe, suggérant que cette structure est particulièrement sensible. Notre travail a donc été consacré à l'étude de l'épileptogenèse faisant suite à l'injection intrahippocampique de toxiques chimiques capables d'entraîner des modifications tissulaires et cellulaires locales chez la souris C57BL/6. Le rôle potentiel de la neuro-inflammation a été plus particulièrement recherché. Dans notre premier modèle, un déséquilibre cholinergique est induit par l'injection de soman, un puissant inhibiteur des cholinestérases. Il est capable d'induire un processus d'épileptogenèse sans état de mal initial, associé à un déficit de la réponse émotionnelle conditionnée contextuelle mais en l'absence de remaniements tissulaires majeurs (neurodégénérescence, œdème et neuro-inflammation). En revanche, dans le modèle d'EMLT obtenu par l'injection de kaïnate, une forte activation microgliale est détectée précocement dans les zones de neurodégénérescence. Une astrogliose est également observée. Grâce à la technique quantitative de transcription inverse suivie de polymérisation en chaîne (RT-qPCR), nous avons également pu mettre en évidence que certains médiateurs moléculaires de l'inflammation sont également liés dans le temps et dans l'espace (particulièrement la transcription d'IL-1β) aux événements neurodégénératifs. Pour s'assurer de la fiabilité des données analytiques de RT-qPCR dans les régions cérébrales touchées par ces modifications, le choix des gènes de référence doit faire l'objet d'études spécifiques. Nous avons pu montrer qu'un ensemble de cinq gènes (Hprt1, Ppia, Tbp, Actb et Arbp) avait une forte stabilité dans la structure hippocampique dans ce modèle murin d'EMLT et pouvait être utilisé pour normaliser les données brutes de RT-qPCR dans ce modèle. Nos résultats indiquent que, chez la souris, des altérations neurochimiques sont capables d'initier l'épileptogenèse même en l'absence de lésions tissulaires notables et qu'une réponse neuro-inflammatoire massive n'est pas une condition sine qua non. Déterminer le caractère bénéfique ou délétère de la neuro-inflammation reste un enjeu majeur pour la découverte de nouvelles thérapeutiques anti-épileptogènes.

Celiac disease (CD) is a common heritable immune related disorder where chronic inflammationof the small intestine is induced by the ingestion of gluten. The immune response leads to theinflammation and flattening of intestinal mucosa due to the damaged villi and thus results indefects in the absorption of nutrients. This defect can affect any organ or body system and exposeto the risk of lifelong complications such as cancer, autoimmune diseases and other complexdiseases. Now a day, celiac disease is becoming one of the well-studied models of complexdisorders.The PI3K- FOX signaling pathway is activated by many regulators and growth factors and playsa key role in cell cycle. Two components of this pathway, INSR and FOX, play crucial roles indiverse aspects of embryogenesis from the initial tissue genesis up to organ formation. INSR andFOX take part in development, differentiation, proliferation, apoptosis and stress resistance aswell as metabolism. SNP´s could affect the expression of neighboring genes. These SNP´s areshown to be as eQTLs, genomic loci that regulate the expression of genes. The aim of this studywas to detect and quantitate the expression of INSR and certain FOX genes in celiac disease.Quantitative real time PCR (QPCR) was used to analyze the expression of INSR, FOXO1,FOXO4 and FOXD3 genes in 38 celiac cases and 50 control samples. Three reference genesACTB, EPCAM and PGK1 were tested for their expression stability and their average was used inthe normalization procedure. Gene expression results were analyzed using the ΔCt method. Theexpression of INSR, FOXO1, FOXO4 and FOXD3 were described as their fold change in CDcompared to normal non-celiac mucosa. Our results indicated that FOXO4 and INSR wereexpressed less by 0.60 fold and FOXO1 was expressed less by 0.23 fold in CD samples. Theresults are preliminary and further studies will be needed to confirm if these findings are a resultof the intestinal inflammation in CD or if these genes are partly driving the disease itself.

Staphylococcus aureus belongs to the normal flora. Many healthy people are colonized by the bacterium mainly in the nose but also on the skin and on other mucous membranes without showing symptoms. After damage to the skin, the bacterium can enter the wound and cause infections. Methicillin-resistant S. aureus (MRSA) is resistant to b-lactam antibiotics such as penicillin and methicillin. The gene that gives resistance characteristic of MRSA is the mecA-gene. MRSA strains are spread in both hospitals and in the community, and it is important to identify these bacteria with rapid and sensitive methods. In this study, Taq Man RT-qPCR was compared with SYBR Green RT-qPCR (LightCycler480, Roche) to explore which method had the best sensitivity with the least working hours. In addition, Bullet for automated DNA extraction and CAS 1200 ™ for automated pipetting of the samples were evaluated. Twelve patient isolates and 232 patient samples for MRSA screening were included in the study. The results showed that the primers were of major importance for the outcome of the amplification. It was also shown that the Ct-values were clearly lower when the Bullet, CAS 1200 ™ and LightCycler480 were combined compared with manual DNA extraction, manual pipetting and the Rotor-Gene 6000. In future, the former method will be used by the laboratory when screening patient samples for MRSA.

Namao virus (NV) was associated with mortality in lake sturgeon Acipenser fulvescens reared as part of a conservation stocking program for this endangered species in Manitoba, Canada. The virus itself was large, doubly encapsidated and icosahedral-shaped. Phylogenetic analyses using the major capsid protein showed that NV and other epitheliotropic sturgeon nucleo-cytoplasmic large DNA viruses shared a common evolutionary past and formed a distinct evolutionary lineage within Megavirales. Three PCR tests were developed and their analytical performance was validated for detection of these viruses. Testing of wild sturgeon revealed that NV is endemic in the Nelson River water basin in Manitoba. Bath exposure resulted in transmission of NV to healthy sturgeon. The gills appeared to be the initial site of infection with virus persisting in the head skin tissue for up to 62 days. The molecular tests will be useful tools for disease management in sturgeon conservation stocking programs. / October 2015

This dissertation includes molecular and phytochemical investigations of the harmful, bloom-forming alga, Prymnesium parvum, including analysis of known polyketide metabolites as a function of salinity and growth. Initially, the development of molecular and phytochemical tools was necessary for the detection and quantification of P. parvum and its associated toxins. Suites of oligonucleotides and molecular beacons were designed for conventional and quantitative multiplex PCR to amplify four species- and gene-specific products simultaneously that were used for the detection and quantitation of P. parvum. This built-in redundancy provided increased confidence in reactions with the positive confirmation of four discrete products. Techniques were also developed for the chemical enrichment of toxins produced by P. parvum. Until now, isolation of “prymnesins” has never been reproduced. Polyketide prymnesins possess unique spectral properties that were used to generate an LC-MS fingerprint that comprised 13 ion species. Preliminary investigations using chemifluorimetric methods were also capable of detecting prymnesins in the pico- and nano-molar range. Environmental samples were tested as an independent assessment of these methods. Lastly, the roles of polyketide prymnesins were analyzed with respect to total hemolytic activity (HA) as a function of culture age and salinity. Variation in HA of supernatants was statistically significant relative to both variables (p << 0.05). Salinity was inversely related to HA wherein cultures growing in 5-25 psu were 150-200% more hemolytic. Total HA was inversely related to culture age during the first three weeks, but positively related to it during the next three weeks. Interestingly, no hemolysis was detected in fractions containing prymnesins from culture supernatants and the majority of hemolysins remained in the aqueous phase. Prymnesins extracted from cells varied significantly over the 6-week observation period (p << 0.05); HA was positively correlated during the first half and inversely related during the last half of the study. Salinity was directly related to HA from cell extracts, but these effects were not significantly different until the last three weeks. These investigations suggest that polyketide prymnesins are present at much lower quantities than previously believed, and they may not be the key compounds associated with hemolysis due to P. parvum. / text

Molecular biology has become an integral part of the diagnosis of infectious diseases. Recently, quantitative real-time PCR (QPCR) methods (often in the form of so-called TaqMan® systems) have been developed for the diagnosis of a wide range of infectious diseases; these techniques found valuable clinical application in the diagnosis and evaluation of progress and therapeutic success of viral diseases. The use of QPCR as a tool for diagnostic virological and viral research laboratories has greatly increased in recent years. It often replaces conventional PCR and amplicon detection systems which are more complex and laborious, with a higher risk of amplicon carry-over contamination. The new QPCR methods presented here utilize broadly targeted primers and probes for rational and sensitive detection and quantification of variable RNA viruses. They take advantage of the dual properties, both RNA and DNA dependent DNA polymerase activities, of the rTth thermostable polymerase, and thermolabile UNG with dUTP to protect against inadvertent contamination of samples with amplimers. In paper one, a novel QPCR approach to detect and quantify human enteroviral (EV) RNA in patients with neurological disorders such as aseptic meningitis is presented. In the second paper, the development of a novel serological technique, quantitative PCR enhanced immunoassay (QPIA), for serodiagnosis of EV infection, is described. In paper three the subject is the development of a touch-down QPCR (TD-QPCR) for detection and preliminary genogrouping of norovirus (NV), a group of Caliciviruses. In paper four a rational, broadly targeted, system for detection of diverse influenza viruses, yet being able to discriminate between influenza A, B and C, is designed and evaluated. In the last paper, another rational broadly targeted system, for detection of corona viruses in humans and animals, is described. The technologies described in this collection of papers have common features. They are a platform for further development of diagnostic tools for screening and detection of viruses in known viral diseases, maybe also for discovering new viruses.