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Gene orf18, which is situated in the conjugative transposon Tn916 from the bacterial pathogen Enterococcus faecalis, encodes a putative ArdA (alleviation of restriction of DNA) protein. ArdA from Tn916 may be responsible for the apparent immunity of the transposon to DNA restriction and modification (R/M) systems and for ensuring that the transposon has a broad host range. The orf18 gene was engineered for overexpression in Escherichia coli and the recombinant ArdA protein was purified to homogeneity. Biophysical characterisation of ArdA demonstrated tight association between ArdA and the M.EcoKI. Also, ArdA was shown to efficiently inhibit restriction and modification by all four major classes of Type I R/M enzymes in vivo. Thus, ArdA can overcome the restriction barrier following conjugation and so helps to increase the spread of antibiotic resistance genes by horizontal gene transfer. The amino acid sequence of KlcA, from the incompatibility plasmid pBP136 from Bordetella pertussis, showed a high degree of similarity with the antirestriction protein ArdB from the IncN plasmid pKM101. In this study the solution structure of KlcA was solved with high-resolution NMR and its antirestriction function demonstrated. The structure of KlcA showed a rigid globular molecule with a novel fold. No antimodification function was observed for KlcA in vivo and the antirestriction function of KlcA has been successfully shown in vivo but not in vitro. Because no direct binding of KlcA to EcoKI was observed in vitro, the mechanism of the endonuclease blocking was assumed to be different from that of ArdA. Preliminary experiments including coimmunoprecipitation assays were conducted in order to elucidate the antirestriction mechanism of KlcA.

To improve understanding of the effectiveness of host resistance against Leptosphaeria spp., three aspects of effectiveness of resistance were investigated. With focus on the major Rlm-mediated resistance against L. maculans, changes in effectiveness of Rlm7-mediated resistance to prevent initiation of disease epidemics at the leaf spot stage were investigated in winter oilseed rape field experiments at five sites in the UK over the period with the cropping seasons 2009/2010 - 2013/2014. L. maculans isolates virulent against Rlm7 were identified in the UK. This may be associated with observed changes in lesion phenotypes on the Rlm7 cultivars in field conditions. However, despite increased severity of phoma leaf spotting on Rlm7 cultivars, there was no associated increase in phoma stem canker severity at the end of the cropping seasons. The effectiveness of winter oilseed rape cultivars for control of phoma stem canker (caused by L. maculans or L. biglobosa) was affected by the coexistence of the two Leptosphaeria species in oilseed rape crops. Weather conditions influenced ascospore release of both species and favoured L. biglobosa ascospore release in 2011, resulting in subsequent increased L. biglobosa phoma leaf spotting and stem canker severity. However, coexistence of Leptosphaeria spp. on oilseed rape crops was affected by the cultivar resistance against L. maculans. CE experiments showed that there were interactions between the two Leptosphaeria spp. in planta. Their coexistence on B. napus was influenced by the different host responses that they trigger during host colonisation. Effects of increased temperature on effectiveness of resistance against L. maculans and on severity of symptoms by Leptosphaeria spp. on B. napus were investigated. Increased temperature affected both Rlm4- and Rlm7-mediated resistance, when assessed by phenotypic and molecular techniques. Increased temperature was associated with increased symptom severity, for both L. maculans and L. biglobosa lesions on plants. Cultivar quantitative resistance background increased effectiveness of resistance against phoma stem canker pathogens at increased temperature and should be deployed in in strategies for adaptation to climate change to avoid increased phoma stem canker epidemics in the future.

Les moyens mis en oeuvre chez les animaux domestiques pour lutter contre les maladies dépendent de leur vitesse de propagation, de leur virulence et surtout de leur capacité à passer la barrière d'espèce. Certaines maladies entraînent des pertes économiques importantes chez l'animal(des centaines de milliers de vaches ont été abattues pendant l'épidémie de BSE). Bien plus, dans le cas des zoonoses, maladies transmissibles à l'homme, il convient d'appliquer des mesures de lutte extrêmement onéreuses s'organisant dans les élevages mais aussi à l'échelle des populations. Plusieurs stratégies de prévention et/ou de traitement des maladies infectieuses sont possibles: les traitements chimiques, la vaccination, la désinfection des bâtiments d'élevage et la conduite des troupeaux, le contrôle sanitaire et la traçabilité des produits alimentaires,l'abattage partiel ou total des troupeaux infectés. Ces mesures peuvent être très onéreuses et parfois peu efficaces. Ces maladies peuvent être mono ou multifactorielles. Quand une maladie est en partie régulée par une prédisposition génétique, il est possible de choisir des reproducteurs les plus résistants et diminuer ainsi l'incidence de la maladie. Si cette prédisposition est monogénique, ou pour le moins sous l'influence d'un gène à effet majeur, comme par exemple dans la tremblante ovine,la sélection assistée par gène peut être une stratégie complémentaire aux stratégies déjà existantes. Mais cette sélection ne peut se faire indépendamment de celle des caractères traditionnels de production qui ont permis d'améliorer par exemple la quantité de lait en races laitières ou le nombre et la croissance des animaux en races bouchères. La question qui se pose alors est d'arriver le plus rapidement possible à une incidence quasi nulle de la maladie en créant une population essentiellement porteuse des allèles de résistance au gène sélectionné tout en évitant de perdre le gain génétique acquis sur les caractères traditionnels de production. L'objectif de cette thèse a été de répondre à cette question en proposant une approche paramétrique permettant de modéliser et d'optimiser la stratégie de sélection. Le paramétrage du modèle permet de décrire de manière détaillée les caractéristiques du/des gène(s) de résistance sélectionné(s) et celles du schéma de sélection sur les caractères de production, souvent complexes chez les animaux domestiques. Les sorties du modèle sont l'évolution des distributions des caractères de production (moyennes, variabilités) et des fréquences des allèles de résistance / sensibilité. Afin de prendre en compte le processus de sélection, une modélisation dynamique a été utilisée. Des approches déterministe et stochastique ont été développées et ont été optimisées à l'aide d'un algorithme génétique. La stratégie optimale difficile à promouvoir sur le terrain a été alors considérée comme référence pour la mise en place de stratégies de sélection acceptables et efficaces. Il est possible ainsi d'évaluer différents scénarios concernant par exemple l'organisation du génotypage (pour le gène de résistance) dans la population sélectionnée, le choix des conjoints selon leur génotype, ainsi que la méthode de diffusion des reproducteurs vers les élevages de production. La méthodologie développée lors de ce travail a été appliquée au cas de la tremblante du mouton, un exemple possible parmi d'autres maladies. Différentes stratégies proches de l'optimale ont été combinée à un modèle épidémiologique afin d'analyser l'impact de ces dernières sur l'évolution de la maladie. / The resources to be used in domestic animals to fight against the diseases depend on their diffusion speed, their virulence and especially their ability to pass the species barrier. Some diseases cause significant economic losses in animals (hundreds of thousands of cows were culled during the BSE epidemic). Moreover, in the zoonotic case, diseases transmissible to humans, measures to be implemented are generally extremely expensive both at the farm and population levels. Several strategies for the prevention and / or treatment of infectious diseases are possible : chemical treatments, vaccination, disinfection of livestock buildings and herd management, health monitoring and traceability of food products, or partial or full slaughtering of herds . These measures can be very costly and sometimes ineffective. These diseases can be mono or multifactorial. In the case of genetic diseases (most often single gene such as sheep scrapie), gene-assisted selection can be a complementary strategy to existing strategies. But this selection can not be done independently of the selection of the traditional production traits such as the quantity of milk in dairy breeds or the number and the growth of animals in beef breeds. The question that arises is how to reach as quickly as possible an almost zero incidence of the disease by creating a population displaying a high proportion of the resistant allele at the gene of interest while minimizing the loss of genetic progress on traditional production traits. The objective of this thesis was to answer this question by proposing a parametric approach to model and optimize the selection strategy. The parametrization of the model allowed to describe in detail the characteristics of the gene (s) selected for resistance (s) and those of the breeding scheme on production traits, often complex in domestic animals. The model outputs were the evolution of distributions of production traits (means, variability) and allele frequencies of resistance / sensitivity. To take into account the selection process, a dynamic model was used. Deterministic and stochastic approaches have been developed and have been optimized using a genetic algorithm. The optimal strategy may be difficult to apply. The optimal strategy may be taken as a reference for the development of selection strategies. It is thus possible to evaluate different scenarios such as the genotyping organization (for the resistance gene) in the selected population, the choice of the mating according to their genotype, the method of dissemination of reproducers to the production farms. The methodology developed in this work has been applied to the scrapie case, a possible example among other diseases. Different strategies close to optimal have been combined with an epidemiological model to analyze their impact of the spread of the disease.

BACKGROUND AND OBJECTIVES: Streptococcus agalactiae (Group B Streptococcus, GBS) is the leading cause of neonatal infections and deaths in human. It can also cause infections in pregnant women and non-pregnant adults. Penicillin and ampicillin are antibiotics of choice for the treatment of GBS infections. Erythromycin and clindamycin are used as alternative therapy in penicillin allergic patients, however resistance to these agents has been increasingly observed. This present study was undertaken to determine the colonization rate of GBS, susceptibility profile and the mechanism of antibiotic resistance in pregnant women and their babies at Dr. George Mukhari Academic Hospital in Pretoria. METHODS: Rectal and vaginal swabs were collected from pregnant women; ear and umbilical swabs from newborns over an 11 month period. Samples were cultured on selective media (CNA agar and Todd-Hewitt broth) and GBS positively identified using morphological and biochemical tests including Gram staining, hemolytic activity, catalase test, bile esculin, CAMP test and Latex agglutination test. The susceptibility testing was done using the Kirby-Bauer and E-test methods. The D-test method was used to determine the inducible clindamycin resistance. Multiplex PCR with were used to detect different genes coding for resistance. RESULTS: Out of the 413 patients evaluated, 128 (30.9%) were positive with GBS. All isolates were sensitive to penicillin and ampicillin. Erythromycin and clindamycin resistance was 21.1% and 17.2% respectively; of which 69% harbouring constitutive MLBB, 17.4% inducible MLSB. The alteration of ribosomal target encoded by ermB genes was the commonest mechanism of resistance observed in 55% of isolates, 38% of isolates had both ermB and linB genes and efflux pump mediated by mefA genes was detected in one of isolates. Conclusion: This study reaffirms the appropriateness of penicillin as the antibiotic of choice for treating GBS infection. However it raises the challenges of resistance to the macrolides and lincosamides. More GBS treatment options for penicillin allergic patients need to be researched. / Health Studies / M.Sc. (Life Sciences (Microbiology))

BACKGROUND AND OBJECTIVES: Streptococcus agalactiae (Group B Streptococcus, GBS) is the leading cause of neonatal infections and deaths in human. It can also cause infections in pregnant women and non-pregnant adults. Penicillin and ampicillin are antibiotics of choice for the treatment of GBS infections. Erythromycin and clindamycin are used as alternative therapy in penicillin allergic patients, however resistance to these agents has been increasingly observed. This present study was undertaken to determine the colonization rate of GBS, susceptibility profile and the mechanism of antibiotic resistance in pregnant women and their babies at Dr. George Mukhari Academic Hospital in Pretoria. METHODS: Rectal and vaginal swabs were collected from pregnant women; ear and umbilical swabs from newborns over an 11 month period. Samples were cultured on selective media (CNA agar and Todd-Hewitt broth) and GBS positively identified using morphological and biochemical tests including Gram staining, hemolytic activity, catalase test, bile esculin, CAMP test and Latex agglutination test. The susceptibility testing was done using the Kirby-Bauer and E-test methods. The D-test method was used to determine the inducible clindamycin resistance. Multiplex PCR with were used to detect different genes coding for resistance. RESULTS: Out of the 413 patients evaluated, 128 (30.9%) were positive with GBS. All isolates were sensitive to penicillin and ampicillin. Erythromycin and clindamycin resistance was 21.1% and 17.2% respectively; of which 69% harbouring constitutive MLBB, 17.4% inducible MLSB. The alteration of ribosomal target encoded by ermB genes was the commonest mechanism of resistance observed in 55% of isolates, 38% of isolates had both ermB and linB genes and efflux pump mediated by mefA genes was detected in one of isolates. Conclusion: This study reaffirms the appropriateness of penicillin as the antibiotic of choice for treating GBS infection. However it raises the challenges of resistance to the macrolides and lincosamides. More GBS treatment options for penicillin allergic patients need to be researched. / Health Studies / M. Sc. (Life Sciences (Microbiology))