The Growth of Eimeria tenella: Characterization and Application of Quantitative Methods to Assess Sporozoite Invasion and Endogenous Development in Cell Culture

In vitro development of the complete life cycle of Eimeria species has been achieved in primary cultures of avian epithelial cells with low efficiency. The use of immortalized cell lines simplifies procedures but only allows partial development through one round of parasite invasion and intracellular replication. We have assessed the suitability of Madin-Darby Bovine Kidney (MDBK) cells to support qualitative and quantitative studies on sporozoite invasion and intracellular development of Eimeria tenella. Analysis of parasite ultrastructure by transmission electron microscopy and serial block face—scanning electron microscopy proved the suitability of the system to generate good quality schizonts and first-generation merozoites. Parasite protein expression profiles elucidated by mass spectrometry corroborated previous findings occurring during the development of the parasite such as the presence of alternative types of surface antigen at different stages and increased abundance of proteins from secretory organelles during invasion and endogenous development. Quantitative PCR (qPCR) allowed the tracking of development by detecting DNA division, whereas reverse transcription qPCR of sporozoite- and merozoite-specific genes could detect early changes before cell division and after merozoite formation, respectively. These results correlated with the analysis of development using ImageJ semi-automated image analysis of fluorescent parasites, demonstrating the suitability and reproducibility of the MDBK culture system. This systems also allowed the evaluation of the effects on invasion and development when sporozoites were pre-incubated with anticoccidial drugs, showing similar effects to those reported before. We have described through this study a series of methods and assays for the further application of this in vitro culture model to more complex studies of Eimeria including basic research on parasite cell biology and host-parasite interactions and for screening anticoccidial drugs

Innate responses and biomarkers of resistance to Eimeria infection in the chicken

Coccidiosis is an intestinal disease caused by the protozoan parasite Eimeria, of which E. tenella and E. maxima are a common cause of disease in the poultry industry, causing weight gain loss, decreased feed efficiency and mortality in poultry. Coccidiosis is usually controlled by the application of anti-coccidial drugs or by vaccination, but drug resistance in Eimeria has been reported and vaccines require the passage of live Eimeria oocysts through the birds and are therefore expensive and difficult to produce. Alternative solutions are to develop subunit vaccines and to breed chickens for resistance to Eimeria by identifying resistance biomarkers, both of which require characterisation of the chicken immune response to Eimeria. To characterise immune responses to Eimeria, this study aimed to investigate the response of antigen presenting cells (APC) to Eimeria and determine which chicken pathogen recognition receptors (PRRs) recognise Eimeria vaccine candidates using in vitro techniques. The role of T helper (Th) 17 cells during E. maxima and E. tenella infection was also investigated in vivo through infection of a commercial broiler line. This study also aimed to identify biomarkers of Eimeria resistance by characterising the immune response to E. maxima and E. tenella in chicken lines which exhibit differential resistance and susceptibility to both these Eimeria spp. The development of chicken bone marrow-derived macrophage (BMM) and dendritic cell (BMDC) cultures provides an opportunity to study the responses of host-derived APC to Eimeria antigens and potential vaccine candidates in vitro. Here, both BMM and BMDC responded to an E. tenella oocyst crude lysate by upregulating mRNA expression of proinflammatory mediators (IL1B, IL6 and NOS2), BMM appeared more regulatory in nature (upregulated IL10 mRNA expression) and BMDC appeared more Th1-promoting (upregulated IFNG mRNA expression). Immune mapped protein 1 (IMP1) and apical membrane antigen 1 (AMA1) are two Eimeria vaccine candidates that have been shown to elicit protective immunity to Eimeria. In response to vaccine candidates IMP1 and AMA1, BMM responded in an inflammatory fashion through increased expression of IL6 and NOS2 mRNA. These results indicate that chicken macrophages and dendritic cells can recognise Eimeria and Eimeria vaccine candidates and facilitate inflammation though production of proinflammatory cytokines, but also have roles in promoting Th1 responses and in immune regulation. In order to trigger innate immune responses, pathogen associated molecular patterns (PAMPs) must be recognised by host PRRs, present on the surface of APC. Currently it is not known which Eimeria PAMPs are detected by which chicken PRRs. Use of a reporter gene assay identified that recombinant IMP1 and AMA1 are recognised by Toll-like receptor (TLR)1LB/2A heterodimers however further investigation is needed to determine other Eimeria PAMPs that are recognised by other chicken PRRs. Th1 responses are known to be important for the resolution of Eimeria infection however Th17 responses during Eimeria infection are less well characterised. Thought to contribute to immunopathology during Eimeria infection, Th17 responses represent a potential target in improving the outcome of Eimeria infection in chickens. Surprisingly, RT-qPCR analysis revealed no changes in the mRNA expression of Th17-associated cytokines in the gut of E. maxima- or E. tenella-infected Ross 308 broilers with the exception of IL21, indicating that IL-21 is acting in another capacity than as a Th17 effector during Eimeria infection. IL-21 is a highly pluripotent cytokine and further study would be required to characterise the role of IL-21 during Eimeria infection. In order to breed chickens for resistance, biomarkers of Eimeria resistance must first be identified. Line 15I and C.B12 chickens display inverse resistance and susceptibility to E. maxima and E. tenella. To identify biomarkers of resistance to E. maxima and E. tenella, the immune response of these lines to both Eimeria spp. was phenotyped. A higher increase in serum IL-10 was observed in E. maxima-infected susceptible line 15I than line C.B12 supporting a previous report that IL-10 is involved in susceptibility to E. maxima. RT-qPCR analysis revealed earlier increases of IFNG, IL10 and IL21 mRNA in the gut of resistant line C.B12 birds following E. maxima infection, indicating that a prompt immune response is a factor in resistance to E. maxima. No biomarkers of resistance to primary E. tenella infection were identified and further interrogation of the immune responses of these lines is required, particularly in response to secondary E. tenella infection. The results of this study have furthered our understanding of the role of APCs during Eimeria infection and following vaccination with IMP1 and AMA1 and support IMP1 and AMA1 as suitable vaccine candidates. IL-21 was identified as an important cytokine during Eimeria infection and further study is required to assess if IL-21 is beneficial or damaging to clearance of the parasite and to evaluate its potential as a therapeutic target. This study also confirmed previous findings that IL- 10 is involved in susceptibility to Eimeria and identified that a rapid response is important for resistance to E. maxima, providing a basis for further study to identify biomarkers of Eimeria resistance

Investigation of the anti-toxoplasma activity of arprinocid and the application of proteomics to the analysis of drug-resistance

The in vitro efficacy and mechanism of action of two purine analogues, arprinocid and its in vivo metabolite arprinocid-1-N-oxide, were investigated against T. gondii tachyzoites using two contrasting approaches. Firstly, a global proteomics approach was undertaken for the analysis of proteins expressed in the tachyzoite stage of T. gondii, as a preliminary to analysing differences between arprinocid-1-N-oxide-resistant and - sensitive parasites. Secondly, a biochemical approach to investigate purine transporters of T. gondii as possible conduits or targets for arprinocid and arprinocid-1-N-oxide. Initial work using 3H-uracil growth uptake to measure the growth of tachyzoites enabled the measurement of the basic anti-parasitic properties of the compounds. The IC50 values for arprinocid and arprinocid-1-N-oxide were 22.4 +/- 5.0 muM and 0.061 +/- 0.028 muM, respectively. Both compounds were specific to T. gondii at therapeutic concentrations and acted irreversibly within a short period of time. High-resolution two-dimensional electrophoresis (2-DE) using the pH ranges 4-7 and 6- 11 reproducibly separated over 1,000 polypeptides, whilst further separations using narrow range gels suggested that at least 3,000-4,000 polypeptides should be resolvable by 2-DE using multiple single pH unit gels. Peptide mass fingerprint (PMF) data acquired by MALDI-time-of flight mass spectrometry, enabled unambiguous protein identifications to be made where full gene sequence information was available. However, interpretation of the T. gondii EST database using PMF data was less reliable. In contrast, peptide fragmentation data, acquired by MALDI-post-source decay mass spectrometry, proved a successful strategy for the putative identification of proteins using the T. gondii EST database. Moreover, peptide fragmentation data permitted the identification of T. gondii proteins based on peptide homology to known proteins from other organisms. The data demonstrated that proteomic analyses are now viable for T. gondii and other protozoa for which there are good EST databases, even in the absence of complete genome sequence. Work presented in this thesis demonstrated the usefulness of proteomics for the investigation of strain variation, protein changes as a consequence of genetic manipulation, and protein expression differences between arprinocid-l-N-oxide-sensitive and -resistant T. gondii lines. Detailed analysis of these drug-sensitive and -resistant mutants indicated that they reproducibly differed in only one protein, although it is suspected that further analysis using narrow range IPG strips may yield more differences. Unfortunately, this differentially expressed protein remains unidentified probably because of the incompatibility of silver-staining with mass spectrometry analysis. Although proteomics is a powerful collection of tools for the investigation of biological questions, currently it is in an early development phase. In contrast, classical biochemical approaches, such as the oil-stop technique for measurements of purine transport, are more standardised. Characterisation of purine transport in T. gondii resulted in the identification of a high affinity transporter for hypoxanthine (TgHTl; Km = 0.91 +/- 0.19 muM) and low affinity transporters for inosine (TglTl; 656 +/- 259 muM) and adenosine (TgATl; Km = 105 +/- 22 muM). No saturable transport of [3H]-adenine was observed. The discovery of a hypoxanthine transporter with a 100-fold higher affinity for substrate than the purine nucleoside transporters indicates that TgATl may not be the main carrier responsible for purine salvage in this organism. Both arprinocid and arprinocid-l-N-oxide inhibited TgATl with high affinity (K = 3.3 +/- 1.1 muM and Ki = 10.4 +/- 3.4 muM, respectively), suggesting that these drugs may be substrates for, or inhibitors of, this transporter. Although their exact mechanisms of action remain to be elucidated, neither drug acts on T. gondii by interfering with the hypoxanthine-xanthine- guanine-phosphoribosyltransferase (HXGPRT)-mediated purine salvage

Nutritional strategies to control clostridium perfringens in gastrointestinal tract of broiler chickens

A series of experiments were conducted to examine the effect of chemical composition of the diet on intestinal Clostridium perfringens populations and necrotic enteritis (NE) in broiler chickens. In the first experiment, birds were fed high concentrations of dietary protein (fish meal or soy protein concentrate) and soluble fiber (guar gum). Clinical NE was not observed, however, there was a high level of C. perfringen colonization especially in guar gum fed birds. The next set of experiments examined the effect of various levels of DL-Met or MHA-FA on C. perfringen and other intestinal microbes. These experiments demonstrated a significant reduction (P < 0.05) in C. perfringen growth with methionine supplementation in ileum and cecum. The results suggest that both DL-Met and MHA-FA may reduce intestinal populations of C. perfringen in broiler chickens when used in high concentrations. The next three experiments were conducted to examine the effect of dietary glycine levels on gut C. perfringen populations, &#945;-toxin production and NE lesion scores. Majority of birds showed clinical signs of disease with 4.16-8.33% mortality. There was a direct correlation between intestinal C. perfringen populations, NE lesions scores and mortality with dietary glycine level. However, due to the use of gelatin as the dietary source of glycine in these experiments, the diets also contained high proline levels which confounded our results. The last study was conducted to establish a direct causative relationship between dietary glycine concentration and C. perfringen growth and/or NE in broiler chickens using encapsulated amino acids. Birds fed diets containing high levels of encapsulated glycine had higher NE lesion scores than those fed encapsulated proline or no encapsulated amino acids, thus demonstrating a direct effect of glycine on intestinal C. perfringen growth. It is concluded that amino acid composition of dietary protein is an important determinant of intestinal microbial growth, particularly C. perfringen, and could affect incidence of NE in broiler chickens

A Chemical Genetics Approach To Understand The Regulation Of Cryptosporidium Sexual Differentiation

Cryptosporidium species are eukaryotic intracellular parasites belonging to the phylum Apicomplexa. C. hominis and C. parvum cause diarrhea in humans which is self-limiting in immunocompetent adults but can have severe consequences in immunocompromised individuals and malnourished children. In developing countries, Cryptosporidium is one of the leading causes of moderate to severe diarrhea in children under five years of age. There is an urgent need for novel therapeutics against this parasite as the current treatment option is inadequate to treat the most vulnerable population to Cryptosporidium infection. Better understandings of the biology of Cryptosporidium would greatly enhance our capability to design effective control measures. The parasite initially multiplies through several rounds of asexual replication within the intestinal epithelial cells before differentiating into sexual forms. Here we have tested the ReFRAME library, a set of ~12,000 biologically active compounds, for activities against the asexual and sexual forms of C. parvum separately. We identified and validated compounds that inhibit and/or induce the growth of either or both life cycle stages. Compounds that inhibit both stages are potentially good drug candidates. Other compounds have promising tool-like properties that can be utilized to probe different aspects of C. parvum sexual differentiation. Several inhibitors of host cell oxidative phosphorylation and purine nucleotide biosynthesis disproportionately inhibited the sexual differentiation of C. parvum, highlighting the necessity of these processes in facilitating C. parvum sexual differentiation. All the apicomplexan parasites differentiate into distinct forms in the course of their life cycle and each of the differentiation steps is associated with large scale changes in gene expression. To identify such stage specific genes and pathways associated with C. parvum sexual differentiation, we performed mRNA-seq of host cells infected with C. parvum in the presence of nine differentiation inhibitors. Ribosomal proteins were the most significantly enriched group of genes that were upregulated with multiple differentiation inhibitors, which suggested that the downregulation of these genes is associated with C. parvum sexual differentiation. Comparison of our results with a publicly available stage-specific mRNA-seq dataset of C. parvum validated this conclusion and analysis of transcriptomic datasets from other apicomplexan parasites revealed that this is a common mechanism of regulating life cycle stage differentiation in these parasites. In addition, identification of significantly enriched DNA motifs at promoters of dysregulated genes coupled with the expression pattern of several apicomplexan AP2 transcription factors strongly suggested that they play a critical role in regulating C. parvum sexual differentiation

Développement de nouveaux produits antibactériens issus de la forêt québécoise

L’augmentation de la résistance de certaines bactéries pathogènes aux traitements antibiotiques conventionnels et l’émergence des bactéries multi-résistantes a progressé à un rythme effréné au cours de la dernière décennie. Cette augmentation vient nous rappeler que la lutte contre les maladies infectieuses est loin d’être terminée. Les bactéries sont des organismes qui possèdent une très grande faculté d’adaptation et qui ont une croissance et une reproduction extrêmement rapide, et malheureusement les résistances bactériennes apparaissent plus rapidement que nous développons de nouveaux antibiotiques. Selon l’organisme mondial de la santé, la résistance des bactéries aux antibiotiques pourrait devenir la plus grande cause de mortalité sur la planète d’ici 2050. Trouver de nouvelles stratégies pour contrer le développement de résistances est un enjeu majeur pour la santé publique et pour la communauté scientifique. Les bactéries multi-résistantes émergent non seulement dans les hôpitaux, mais sont également souvent présentes dans le milieu communautaire. L’utilisation excessive et inadéquate des antibiotiques et leur ajout systématique dans la nourriture des animaux d’élevage contribuent à l’émergence rapide et à la propagation de ces résistances. Pour lutter efficacement, il faut d’une part diminuer le niveau de résistance chez les bactéries et d’autre part, trouver de nouveaux composés antibiotiques susceptibles d’être efficaces contre ces nouvelles bactéries multi-résistantes. Présentement les antibiotiques disponibles sur le marché proviennent exclusivement de différents composés isolés chez des champignons ou des bactéries, ou sont le fruit de la chimie de synthèse. Bien que les plantes possèdent une panoplie de composés et de méthodes de défense antibactérienne qui pourraient passer par de nouveaux mécanismes d’action et de nouvelles cibles, encore très peu d’entre elles ont été étudiées en profondeur. L’objectif principal de cette thèse était d’identifier et de développer des extraits et des composés issus de plantes de la forêt boréale possédant des propriétés antibactériennes. Deux types d’extraits de plantes de la forêt boréale pourraient posséder des propriétés antibactériennes très intéressantes pour la lutte aux bactéries résistantes: les huiles essentielles et les résines. Pour ce projet de recherche, afin de pouvoir tester adéquatement ces deux types d’extraits, un nouveau test antibactérien adapté aux composés et matrices hydrophobes a été développé. Grâce à ce test, l’activité de la résine de sapin baumier (Abies balsamea) a été confirmée in vitro contre Staphylococcus aureus et deux souches de S. aureus résistantes à la méticilline (SARM). Il a été observé que cette activité était en grande partie apportée par la présence de sept différents acides résiniques dans l’extrait. Cet extrait serait très intéressant pour le traitement cutané des infections et pour des développements futurs. Par la suite, trois huiles essentielles (Monarda didyma, Tanacetum vulgare et Tussilago farfara) ont été analysées et comparées afin de sélectionner la plus prometteuse pour remplacer les antibiotiques en nutrition chez les poulets d’élevage. Leurs compositions chimiques et leurs activités antibactériennes, anti-inflammatoires, antioxydantes et cytotoxiques ont été déterminées. L’huile essentielle de monarde didyma (Monarda didyma L.) s’est révélée très prometteuse, notamment en raison de l’activité antibactérienne contre Escherichia coli et Clostridium perfringens. Dans les tests in vivo chez les souris, une hausse significative du poids a été obtenue lors de l’ajout de l’huile essentielle dans la diète. Pour les tests in vivo directement chez les poulets d’élevage, une hausse significative du poids a également été obtenue chez ceux traités avec l’huile essentielle. Des tests supplémentaires restent à effectuer pour une utilisation plus intensive de l’huile essentielle dans la diète des poulets d’élevage, mais les résultats sont très prometteurs. Finalement la balsacone C, un composé issu de la résine des bourgeons de peuplier baumier (Populus balsamifera), a démontré des résultats antibactériens contre une banque de trente-cinq SARM. Le composé semble sélectif envers les bactéries gram positif et possède un faible risque d’apparition de résistances. Pour déterminer des éléments du mécanisme d’action l’intégrité membranaire de bactéries traitées avec la balsacone C a été testé grâce à deux fluorochromes. Une perte de l’intégrité membranaire a été observée après un court temps d’incubation en présence du composé. Par la suite, l’effet sur les membranes a été confirmé grâce à la mesure du relâchement de protéines et d’acides nucléiques dans le milieu, et par microscopie électronique à balayage (MEB). Les résultats de ce chapitre sont très importants et pourraient mener au développement d’un nouvel antibiotique issu du métabolite secondaire des végétaux. Pour conclure, bien qu’il reste encore beaucoup de recherche à faire, ce projet de thèse a permis de tester et sélectionner des extraits de plantes possédant des activités antibactériennes. Ces extraits pourraient être utilisés à plusieurs niveaux, notamment pour réduire l’incidence des bactéries résistantes dans l’élevage et dans la communauté en général, et pour traiter ces bactéries multi-résistantes lors d’infections. Les résultats présentés pourraient ouvrir des avenues très prometteuses pour ces différents extraits provenant de la forêt boréale québécoise