Contagious Bovine Pleuropneumonia (CBPP), caused by Mycoplasma mycoides mycoides small colony (MmmSC), is a devastating respiratory disease of cattle in Africa, Asia and the Middle East. Little investigation has been done on molecular disease pathogenesis and host response beyond soluble cytokine detection. This study developed and characterized models for three strains of MmmSC of varying severity. Strains used were Gladysdale, Ondangwa and Shawawa. Samples of bronchoalveolar lavage fluid, bronchial biopsy, nasal epithelial cells and blood were obtained prior to and at weekly time points post-infection. Microarray analysis of RNA extracted from samples revealed host cellular pathways and genes important in the pathogenesis of CBPP, including multiple immune system and inflammatory response pathways. A number of pathways whose influence on disease pathogenesis was not immediately clear were also activated, including pathways involved in amino acid synthesis, fat metabolism, and endocrine hormone responses. Microarray results were confirmed with real-time polymerase chain reaction (RT-PCR) of selected genes. Comparative RT-PCR analysis of selected genes between the three strains of MmmSC revealed genes possibly responsible for differential strain virulence, including interleukins 1B, 6, 8, and 18 and the gene nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor, alpha (NFKBIA). A similar analysis of selected genes between survivors and nonsurvivors of the virulent Gladysdale strain of MmmSC suggested genes involved in survival, including interleukin 8, calmodulin 2 (CALM2), and NFKBIA. Avenues of additional study were identified.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2002. / Härtill 4 uppsatser.

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2016-09-05T18:15:16Z No. of bitstreams: 1 texto completo.pdf: 693325 bytes, checksum: 2afbc494cbadf4704a75769d027d64d1 (MD5) / Made available in DSpace on 2016-09-05T18:15:16Z (GMT). No. of bitstreams: 1 texto completo.pdf: 693325 bytes, checksum: 2afbc494cbadf4704a75769d027d64d1 (MD5) Previous issue date: 2015-02-24 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Actinobacillus pleuropneumoniae é o agente etiológico da pleuropneumonia suína, uma severa enfermidade que acomete suínos de todas as idades, gerando perdas econômicas significativas para a suinocultura mundial. Embora os 15 sorotipos conhecidos dessa bactéria possam causar a doença, existem diferenças marcantes de virulência entre eles. A virulência de A. pleuropneumoniae é multifatorial e está relacionada à composição e estrutura de polissacarídeos da cápsula, LPS, e toxinas da família RTX, além desses fatores, a aderência em forma de biofilme e a resistência a agentes antimicrobianos podem ser determinantes para virulência. Este trabalho estabeleceu um modelo de infecção alternativo para o estudo de A. pleuropneumoniae, utilizando larvas de Galleria mellonella e, posteriormente esse modelo foi usado para investigar a virulência de isolados clínicos de A. pleuropneumoniae sorotipo 8. Os mesmos isolados foram avaliados quanto ao potencial de formação de biofilme e resistência a antimicrobianos comumente empregados em campo. A partir dessas informações, isolados clínicos com diferenças significativas na virulência, no potencial de formação de biofilme e no perfil de resistência foram selecionados para o sequenciamento genômico. Os resultados mostraram que o modelo de infecção A. pleuropneumoniae – G. mellonella é capaz de diferenciar níveis de virulência de isolados clínicos de mesmo sorotipo, além de permitir a avaliação da eficiência de agentes antimicrobianos contra este patógeno. O modelo também mostrou eficiência para diferenciar virulência entre linhagens selvagem e mutante da mesma bactéria. Uma análise de correlação entre os dados de virulência, formação de biofilme e resistência a antimicrobianos permitiu que seis isolados fossem selecionados para o sequenciamento. Com a montagem e anotação foi possível verificar que os genomas de A. pleuropneumoniae sorotipo 8 apresentam tamanho de 2,2 ± 0,004 Mpb, com o conteúdo GC de 40,33% ± 0,263 e regiões codificadoras com uma média de tamanho de 817,3 ± 6,8 pb. As regiões codificadoras correspondem a 89,05% ± 0,13 do genoma, das quais a maior parte foi anotada como genes funcionais, o que permitirá a realização de estudos comparativos. Estes genomas apresentam em média 79,5 ± 24,05 genes exclusivos, revelando a alta variabilidade genética dessa espécie, que pode estar relacionada com a variação da virulência entre os isolados estudados. / Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia, a severe disease that affects pigs of all ages, causing significant economic losses to the swine industry worldwide. Although the 15 serotypes of this bacterium are known to cause the disease, there are marked differences in virulence between them. A. pleuropneumoniae virulence is multifactorial and involves capsular polysaccharides, LPS, and toxins of the RTX family. In addition to these factors, the adhesion in biofilm form and resistance to antimicrobial agents may be determinant for virulence. This work has established an alternative infection model for the study of A. pleuropneumoniae, using larvae of Galleria mellonella and this model was subsequently used to investigate the virulence of clinical isolates of A. pleuropneumoniae serotype 8. The same isolates were evaluated for biofilm formation potential and resistance to antimicrobials commonly used in the field. From this information, clinical isolates with significant differences in virulence, biofilm formation potential and resistance profile were selected for genomic sequencing. Results show that the A. pleuropneumoniae - G. mellonella infection model is capable of differentiating levels of virulence of clinical isolates of the same serotype. Furthermore, it can be used to evaluate the effectiveness of antimicrobial agents against this pathogen. The model also showed efficiency to differentiate the virulence between wild and mutant strains of the same bacteria. A correlation analysis between the virulence data, biofilm formation and antibiotic resistance allowed six isolates to be selected for genome sequencing. With the assembly and annotation, we found that the genomes of A. pleuropneumoniae serotype 8 present size of 2.2 ± 0.004 Mpb, with GC content of 40.33% ± 0.263 and coding regions with an average size of 817.3 ± 6.8 bp. The coding regions correspond to 89.05 ± 0.13% of the genome, most of which was recorded as functional genes, enabling the comparative studies with these genomes. These genomes have 79.5 ± 24.05 exclusive proteins, revealing the high genetic variability of the species, which may be related to the variation in virulence between these isolates.

No description available.

<i>Actinobacillus pleuropneumoniae</i> is the etiological agent of contagious swine pleuropneumonia, an economically important disease of the swine industry worldwide. Improved control of this disease requires enhanced understanding of the factors contributing to pathogenesis. The objectives of this study were to investigate the immune response and virulence properties of the 110-kilodalton (110-KDa) hemolysins [hemolysin I (HlyI) and hemolysin II (HlyII)] of <i>A. pleuropneumoniae</i>. Several monoclonal antibodies (MAb) to the hemolysins were developed. An IgGl. MAb (8C2) specific for HlyII, as determined by immunoblotting, was cross-linked to Protein A-Sepharose, and HlyII was purified from serotypes 1 and 5 by immunoaffinity chromatography. An indirect enzyme-linked immunosorbent assay (ELISA) using MAb 8C2, or affinitypurified rabbit IgG to both hemolysins, was developed for detection of swine antibody to one or both hemolysins, respectively. In comparison with the complement fixation test, the ELISA was highly sensitive and specific, and was able to identify animals infected with or exposed to most, if not all, serotypes of <i>A. pleuropneumoniae</i>. Several nonhemolytic mutants of <i>A. pleuropneumoniae</i> serotype 5 were isolated following electroporation of the parent with an hemolysin gene whose open-reading-frame was disrupted with a kanamycin resistance gene. One mutant was characterized for phenotypic and pathogenic properties. Biochemical profiles, growth rate, capsule content, and lipopolysaccharide and whole cell protein electrophoretic profiles of the parent and one of the mutants were similar. The nonhemolytic mutant lacked both HlyI and HlyII proteins in culture supernatant and in whole cell lysates as determined by immunoblot analysis; extracellular and intracellular hemolytic and cytotoxic activity was also absent. The mutant was avirulent in mice and pigs at doses greater than 10 times the lethal dose of the parent. Unlike the parent, the nonhemolytic mutant failed to confer protection against lethal challenge in mice following immunization. Thus, one or both hemolysins are essential for virulence and immunoprotection in <i>A. pleuropneumoniae</i> serotype 5. / Ph. D.

La peste des petits ruminants (PPR) et la Pleuropneumonie Contagieuse Caprine (PPCC) causées respectivement par un Morbillivirus (Virus de la Peste des Petits Ruminants) et un mycoplasme (Mycoplasma capricolum subsp. Capripneumoniae) sont deux maladies respiratoires très contagieuses des petits ruminants. La PPR est présente en Afrique, en Asie, au Moyen Orient, et depuis peu en Europe. Sur le continent africain, notamment en Afrique de l’Ouest, elle est en expansion et représente un facteur majeur d’insécurité alimentaire pour la population agricole. La PPCC identifiée au Niger en 1995 n’est que suspectée au Mali sur la base de résultats sérologiques.La PPR est un modèle pour l’étude des maladies transfrontalières car sa diffusion est très étroitement liée aux mouvements régionaux d’animaux vivants. La compréhension de cette diffusion est une condition essentielle à la mise en place de mesures de contrôle efficaces (vaccination, contrôle aux frontières etc.).La thèse a pour ambition de clarifier la situation épidémiologique de la PPR et de la PPCC au Mali, notamment pour savoir si ces deux maladies coexistent, afin d’en évaluer le risque pour les filières de production de caprins et de proposer des stratégies de contrôle adaptées. Nous n’avons pas réussi à mettre en évidence la présence de la PPCC au Mali. Pour la PPR, l’objectif de la thèse est de caractériser la diversité génétique de souches collectées en Afrique de l’Ouest et plus particulièrement au Mali en utilisant en première instance le gène partiel de la nucléoprotéine du virus. Nous avons ensuite estimé la diversité et le taux d’évolution du PPRV dans la région à partir de séquences génomiques complètes. Notre étude a montré qu’au Mali ainsi que dans les autres pays de l’Afrique de l’Ouest, trois lignées génétiques du PPRV circulent dont l’une d’elles, la lignée II est dominante dans la région et est caractérisée par une grande diversité génétique transfrontalière. Cette étude démontre également une progression de la lignée IV dans l’Afrique de l’Ouest et la persistance au Mali et au Niger de la lignée I (au moins jusqu’en 2001). Ces résultats reflètent par rapport aux données précédentes connues de la répartition des lignées de PPRV, une intensification des mouvements du bétail dus à l’échange et au commerce de ces animaux, flux qui n’est pas contrôlé entre tous les pays de l’ouest africain. Au Mali, il n’existe aucun moyen de contrôle, de traçabilité et d’identification animale. L’utilisation de la diversité génétique comme marqueur épidémiologique serait un moyen d’améliorer notre connaissance de la diffusion de la PPR et de là son contrôle, plus particulièrement dans les pays d’Afrique de l’Ouest. / Peste des petits ruminants (PPR) and Contagious caprine pleuropneumonia (CCPP) caused respectively by a Morbillivirus and a mycoplasma (Mycoplasma capricolum subsp. Capripneumoniae) are two highly contagious respiratory diseases of small ruminants. PPR is present in Africa, Asia, Middle East, and has just entered Europe. On the African continent, particularly in West Africa, it is emerging and is a major factor of food insecurity for low-income farmers. CCPP, identified in Niger in 1995, is only suspected in Mali on the basis of serological results.PPR is a model for the study of transboundary diseases because its diffusion is closely linked to regional movements of livestock. Understanding this diffusion is an essential condition for the implementation of effective control measures (vaccination, border control, etc.).The aims of our study is to clarify the epidemiological situation of PPR and the CCPP in Mali, including whether these two diseases coexist in order to assess the risk for goat production chains and propose appropriate control strategies.We did not succeed in confirming the presence of the CCPP in Mali. PPR has already been identified in Mali. The aim of our study for PPR is to characterize the genetic diversity and therefore the different lineages that circulate in Mali and, more generally, in the West African sub region by using at first the partial gene of Nucleoprotein of PPRV. We then estimated more accurately the diversity and rate of evolution of the virus in the region from PPRV genomic sequences. Our studies showed that three lineages of PPRV are circulating in Mali and West Africa. The lineage II is dominating and is characterized with a wide genetic diversity and extensive transboundary circulation. We also demonstrate the progression of lineage IV in West Africa and the persistence of lineage I in Mali and Niger (at least until 2001). These results reflect the large flow of uncontrolled livestock trade between all West African countries. In Mali, there is no means of control, traceability and animal identification. The use of genetic diversity as an epidemiological marker is an effective means of controlling the spread of PPR in these West African countries.

Advances in molecular methods for analyzing DNA, RNA and proteins in humans as well as in other animals, plants, fungi, bacteria or viruses have greatly increased the resolution with which we can study life’s complexity and dynamics on earth. While genomic, transcriptomic and proteomic laboratory tools for molecular diagnosis of disease are rapidly becoming more comprehensive, the access to such advanced yet often expensive and centralized procedures is limited. There is a great need for rapid and comprehensive diagnostic methods in low-resource settings or contexts where a person can not or will not go to a hospital or medical laboratory, yet where a clinical analysis is urgent. In this thesis, results from development and characterization of novel technologies for DNA and protein microarray analysis are presented. Emphasis is on methods that could provide rapid, cost-effective and portable analysis with convenient readout and retained diagnostic accuracy. The first study presents a magnetic bead-based approach for DNA microarray analysis for a rapid visual detection of single nucleotide polymorphisms. In the second work, magnetic beads were used as detection reagents for rapid differential detection of presence of pestiviral family members using a DNA oligonucleotide microarray with read-out by means of a tabletop scanner or a digital camera. In paper three, autoimmune responses from human sera were detected on a protein autoantigen microarray, again by means of magnetic bead analysis. Here, special emphasis was made in comprehensively comparing the performance of the magnetic bead detection to common fluorescence-based detection. In the fourth study, an immunochromatographic lateral flow protein microarray assay is presented for application in the classification of contagious pleuropneumonia from bovine serum samples. The analysis could be performed within 10 minutes using a table top scanner, and the performance of the assay was shown to be comparable to that of a cocktail ELISA. In the fifth paper, the lateral flow microarray framework is investigated in further detail by means of experiments and numerical simulation. It was found that downstream effects play an important role, and the results further suggest that the downstream binding profiles may find use in simple affinity evaluation. / QC 20100713