Mycoplasma hyopneumoniae é uma bactéria de tamanho diminuto, caracterizada por um genoma pequeno, com baixo conteúdo GC. Está associada com doenças respiratórias de suínos, resultando em prejuízos produtivos e econômicos na indústria animal. A presença de sequências de DNA repetitivas, que ocorrem em grandes quantidades em células eucarióticas, vem sendo cada vez mais identificadas em genomas de procariotos, sendo também associadas a um potencial papel regulador. Uma vez que a regulação da transcrição nesses organismos ainda é pouco entendida, o objetivo do presente estudo foi realizar uma busca in silico por elementos repetitivos nas regiões intergênicas do genoma de M. hyopneumoniae linhagem 7448. Dois tipos de repetições foram selecionados para a busca inicial: tandem e palindromes. Regiões intergênicas de até 500 pb a montante do sítio de início da tradução de todas as CDSs do genoma de M. hyopneumoniae linhagem 7448 foram utilizadas para a predição. Para cada tipo de elemento dois programas computacionais independentes foram utilizados. As predições in silico resultaram em 144 repetições em tandem e 1.171 palindromes. O DNA repetitivo se encontra distribuído a montante de 86% das unidades transcricionais de M. hyopneumoniae linhagem 7448. Análises comparativas entre genomas de micoplasmas demonstraram diferentes níveis de conservação dos elementos repetitivos entre linhagens patogênicas e não-patogênicas. Linhagens patogênicas revelaram uma conservação de 59%, enquanto que a não patogênica, somente de 46%. Através de ensaios de amplificação quantitativa de DNA, foi observado diferentes níveis de expressão em genes codificantes para importantes proteínas, como glicina hidroximetiltransferase, lipoproteína, adesinas e proteína ligadora de GTP. Os genes codificantes para essas proteínas divergiam no número de repetições palindromes e tandens na sua respectiva região intergênica. Além disso, repetições encontradas em 206 genes já descritos como regulados em diferentes condições em M. hyopneumoniae linhagem 232 mostraram aproximadamente 80% de conservação em relação à linhagem M. hyopneumoniae linhagem 7448. Todos esses resultados sugerem um potencial papel regulador das repetições de DNA em tandem e palindromes em Mycoplasma. / Mycoplasma hyopneumoniae is a diminutive bacterium, characterized by a small genome with a low GC content. It is commonly associated with swine respiratory diseases, resulting in productivity and economic losses in the animal industry. Repetitive DNA, which occurs in large quantities in eukaryotic cells, has been increasingly identified in prokaryotic genomes, and has been associated with a potential regulatory function. Once transcription regulation in these organisms is still poorly understood, the aim of the current study was to perform an in silico search of repeat elements in the genomic intergenic regions of M. hyopneumoniae strain 7448. Two types of repeats were selected for initial search: Tandem and Palindromic. Intergenic regions up to 500 bp upstream from start codon of M. hyopneumoniae strain 7448 CDSs were used as input for the software’s prediction. For each type of repeat sequence, two independent software packages were used. Computational analysis results in 144 tandem repeats and 1,171 palindrome elements. The repeats were distributed in the upstream region of 86% of transcriptional units of M. hyopneumoniae strain 7448. Comparative analysis between distinct mycoplasmas, demonstrate different indices of repeat conservation among pathogenic and non-pathogenic strains. Pathogenic strains revealed 59% conservation, while non-pathogenic only 46%. Through assays of quantitative amplification of DNA, different levels of expression in genes coding important proteins have been demonstrated, as glycine hydroxymethyltransferase, lipoprotein, adhesins and GTP-binding protein. These protein coding genes differ in number of palindromes or tandem repeats in respective upstream regions. In addition, repeats found in 206 genes already described to be regulated in different grow conditions in M. hyopneumoniae strain 232 showed almost 80% of conservation in relation to M. hyopneumoniae strain 7448. All these findings, suggests a potential regulatory role of tandem and palindrome DNA repeats.

<i>Mycoplasma bovis</i> is generally considered the causative pathogen associated with Chronic Pneumonia and Polyarthritis Syndrome (CPPS) in feedlot cattle. However, <i>M. bovis</i> virulence may vary between strains as it is also isolated from asympytomatic cattle. The following study aims to determine the prevalence of <i>M. bovis</i> in the respiratory tract of western Canadian cattle using two sampling methods and at two time points following feedlot entry. Three study groups were sampled. In the first group nasal swabs (NS) and bronchoalveolar lavages (BAL) were taken from 36 clincally healthy cattle at the University of Saskatchewan feedlot at both 14 and 90 days on feed (DOF). In a second experiment, NS were taken from 56 animals upon arrival at a commercial feedlot and one week to three months later upon treatment for respiratory disease. Lung and joint tissue swabs were collected at necropsy from a third group of 19 animals with CPPS clinical pathology originating in 10 different western Canadian feedlots. All samples were selectively cultured for <i>Mycoplasma</i> spp. DNA was extracted from isolated putative <i>Mycoplasma</i> colonies and amplified with universal 16S rRNA gene primers for identification. Amplified Fragment Length Polymorphism (AFLP) was used to genetically differentiate <i>M. bovis</i> positive isolates. More <i>M. bovis</i> was isolated from NS than BAL and <i>M. bovis</i> prevalence increased with DOF in the feedlot in both the University of Saskatchewan and commercial feedlot trials. Three genetically distinct clusters (A, B, and C) were isolated from the necropsy group. Two of these clusters were primarily associated with isolates collected from feedlot cattle and one strain was exclusively found in CPPS-associated mortalities. No significance difference in the prevalence of <i>M. bovis</i> strains was observed between different days on feed or sampling methods. It was concluded that either the difference in disease state is a host dependent outcome, due to a multi-factorial disease complex, or the AFLP assay was not sensitive enough to differentiate strains based on virulence.

Los micoplasmas son bacterias sin pared celular que pertenecen a la clase Mollicutes. Estos microorganismos se caracterizan por su pequeño tamaño y por tener un genoma reducido con un bajo porcentaje de C+G. Muchas especies de mycoplasma actúan como parásitos y patógenos de un amplio rango de huéspedes, causando enfermedades comunes en humanos. Por ejemplo, Mycoplasma genitalium, objeto de esta tesis, es el causante de la uretritis no gonocócica. La secuenciación del genoma de algunos micoplasmas ha favorecido el conocimiento de la fisiología y genética de estos microorganismos, pero algunos mecanismos como la regulación de la expresión génica y la división celular siguen siendo aún un misterio por desvelar. M. genitalium, con tan sólo 525 genes, es considerado un modelo de célula mínima. Posee el genoma más pequeño de entre todas las bacterias que se pueden cultivar axénicamente, siendo así un candidato ideal para el estudio de los procesos biológicos con el mínimo número de genes implicados. El trabajo presentado en esta tesis se divide en tres capítulos independientes. En el primero se estudia la regulación de la expresión génica en M. genitalium y M. pneumoniae. El trabajo con M. pneumoniae fue llevado a cabo durante una estancia en el laboratorio del Prof. J. Stülke (Göttingen, Alemania). Este primer trabajo dio lugar a un artículo publicado en la revista Microbiology. La obtención de un mutante por transposición de M. genitalium que deleccionaba el gen ftsZ, descrito como esencial para la división celular en la mayor parte de bacterias, derivó en el segundo capítulo de esta tesis. En este segundo trabajo se investiga la división celular en micoplasma en ausencia de este gen. Este estudio ha sido publicado recientemente en la revista Molecular Microbiology. Por último, con el objetivo de profundizar en el tema de la división celular en micoplasma, en el tercer capítulo se analiza la funcionalidad del gen mraZ, que junto con mraW, mg223 y ftsZ conforma el operón de división celular de M. genitalium. / Mycoplasmas are the smallest and simplest free-living microorganisms. They are members of the Mollicutes class which is characterized by the absence of cell wall and by having genomes with a low G+C content. Despite this apparent simplicity, some mycoplasma species are parasites and pathogens of a wide range of hosts. For instance, Mycoplasma genitalium, which is the object of this thesis, is the agent of non-gonococcal and non-chlamydial urethritis. Genome sequencing has advanced the knowledge concerning the physiology and genetics of these microorganisms, but some mechanisms such as the regulation of gene expression and the cell division remain unclear. With only 525 genes M. genitalium is considered a model of minimal cell, since it has the smallest genome of any microorganism that can be grown in a pure or axenic culture. It is considered a perfect candidate to study biological processes with the minimal set of genes. This thesis is divided into three independent chapters. In the first one we study the regulation of gene expression in M. genitalium and M. pneumoniae. This work was published in 2007 in the journal "Microbiology". A minitransposon insertion was found in the ftsZ gene of M. genitalium, indicating that this gene was not essential for cell division in this microorganism. In the second chapter of this thesis we investigate the cell division process in M. genitalium in the absence of ftsZ. This work was recently published in the journal "Molecular Microbiology". Finally, to gain insight into the cell division process in mycoplasma, in the third chapter we analyze the function of the mraZ gene, which together with mraW, mg223 and ftsZ comprise the cell division operon of M. genitalium.

En aquesta Tesis, s'ha estudiat per una banda, 1) la dinàmica de infecció de Mycoplasma hyopneumoniae (M. hyopneumoniae) a nivell de granja i la seva relació amb els diferents sistemes de producció i 2) la correlació entre la detecció d'aquest patogen a diferents nivells del tracte respiratori (cavitat nasal, tonsil.la, i bronqui) amb la seroconversió, i presència de lesions macroscópiques i microscópiques suggestives de la Pneumònia Enzoòtica (PE) a nivell de casos clínics i nivell d'animals de camp. Del estudi de la dinàmica de infecció en vam concloure que característiques inherents de cada granja tenien un efecte molt important en el patró de circulació de M.hyopneumoniae. Fins i tot dins d'una mateixa granja, es podien trobar dinàmiques de infecció diferents entre diferents lots d'animals, repercutint en diferents graus i moments d'aparició de les lesions. A més a més, el tipus de producció utilitzat (1-2 llocs versus 3 llocs) també influenciava la dinàmica de infecció del patogen. Els resultats obtinguts en les granges en 3 llocs, clarament donen suport a la característica tardana aparició del complex respiratori porcí en els sistemes de producció en múltiples fases. De fet, la colonització en transició era menor i més tardana en els sistemes en 3 llocs comparats amb en 1-2 llocs. A més a més, en els sistemes de producció en múltiples fases, s'observava un increment molt brusc de la proporció d'animals infectats associat a brot respiratoris tardans.Malgrat que la infecció per M.hyopneumoniae es va detectar en totes les granges, aquest patogen no estava sempre involucrat en els problemes respiratoris observats. De fet, es van observar garnges infectades clínica i subclínicament per M.hyopneumoniae Aquest fet pot ser important en el moment de prendre decisions sobre les estratègies de prevenció i control de malalties. Els seroperfils no van resultar ser discriminatius per determinar el paper del agent en els problemes respiratoris observats. Els bacterioperfils van ser més útils per determinar el paper del patogen, ja que l'aparició de la simptomatologia clínica estava estretament lligada a un increment de la proporció d'animals infectats (basat en la detecció de M.hyopneumoniae en hisops nasals). Tot i no ser discriminatiu, la proporció d'animals seropositius va ser major també en les granges afectades clínicament que en les subclinicament. La seroconversió en front a aquest agent ha estat descrita com lenta, retardada i molt variable. Ens els estudis de camp presentats en aquesta Tesis, la seroconversió també va ser variable. Sembla ser, que la seroconversió estava més relacionada amb la detecció de M.hyopneumoniae en els hisops bronquials que en els nasal o tonsil.lars, i a la vegada amb el percentatge d'animals amb els hisops bronquials positius.Tal i com s'esperava, l'hisop bronquial va ser el millor en predir la presencia de lesions macroscópiques i microscópiques de PE coincidint amb els treball prèviament publicats. Sorprenentment, la proporció relativa de detecció de M.hyopneumoniae en hisops nasals va ser menor en l'estudi de camp que en l'estudi on les mostres de diagnòstic. La major proporció d'hisops positius en els animals de diagnòstic es podria explicar per: un increment de l'excreció de M. hyopneumoniae degut a situacions estressants (tal com el transport) o el increment de la multiplicació de les bactèries després d'unes hores de la mort de l'animal. Els resultats d'aquesta Tesis, han demostrat que M. hyopneumoniae es pot detectar en tonsil.la, on no hi ha cèl·lules epitelials. Malgrat el mostreig d'hisops tonsil.lar en animals vius es possible però molt laboriós, pot esdevenir un lloc de mostreig més adequat per monitorejar la infecció per M.hyopneumoniae a nivell de granja. Per altre banda, el resultats obtinguts, demostren que quan l'hisop bronchial és positiu, la probabilitat de tenir lesions macroscópiqes i microscópiques compatibles amb PE és alta. Tanmateix, la presencia d'aquestes lesions no sempre implica la detecció de M.hyopneumoniae en bronqui, ja que poden ser causades per altres patògens com pot ser SIV. / The research presented in this Thesis assessed 1) the dynamics of infection of M. hyopneumoniae at a herd level and its relationship with production systems and 2) its detection in three respiratory levels (nasal cavities, tonsil and bronchi), taking into account the presence of seroconversion and macroscopic and microscopic lung lesions suggestive of Enzootic Pneumonia (EP)The studies on infection dynamics showed that the factor with greatest effect on M. hyopneumoniae circulation pattern was the farm. Even within a farm, significant differences in the dynamics of infection between different batches were observed, which were also related with different degrees and timings of M. hyopneumoniae -related lesions appearance.Moreover, the type of production system (1/2-site versus 3-site herds) was another factor that influenced M.hyopneumoniae infection dynamics. Results of our field trial in regards M.hyopneumoniae, involving 12 Spanish herds clearly support the characteristic late appearance of Porcine Respiratory Disease Complex (PRDC) in multisite production systems. M. hyopneumoniae colonization in nurseries was lower and occurred later in time in 3-site versus in 1/2-site production systems. Moreover, an abrupt increase in the proportion of infected animals, associated with late respiratory outbreaks was seen in the multisite production systems. Although M. hyopneumoniae infection was detected in all farms, it was not always involved in the respiratory disease. An important contribution of this work is that, although seroconversion and M. hyopneumoniae infection were detected in all herds, clinical and subclinical infections were described. This is important for decision making on the control strategies to implement at the farm. Serumprofiles were not discriminative to determine the involvement of M. hyopneumoniae in a clinical outbreak. Bacteriumprofiles were more useful in determining M. hyopneumoniae involvement in respiratory disease, since an increase of the proportion of infected pigs (based on detection of M. hyopneumoniae in nasal swabs) was clearly related with appearance of the clinical outbreak. Although not discriminative, the proportion of seropositive pigs was also greater in clinically than in subclinically infected herds. Dynamics of serocoversion against M. hyopneumoniae was another issue addressed in both, transversal and longitudinal field trials. Several authors have described the time of seroconversion under field conditions as slow, delayed and vaery variable. In field studies presented here time of seroconversion was also variable. Seroconversion was more related to detection of M. hyopneumoniae in bronchial swabs rather than in nasal or tonsillar swabs, and to the detection of a higher percentage of pigs with a positive nPCR bronchial swab. As expected, bronchial swabs were the best predictors of presence of microscopic and gross EP lesions coinciding with previously published studies. However, to sample bronchial swabs from live animals is not possible. Surprisingly, the relative proportion of nasal detection was lower in the field study than in the study using pigs received at the diagnostic laboratory. An increase of M.hyopneumoniae shedding in nasal cavities due to stressful situations (such as transport) or due to increase multiplication of bacteria after the animal's death have been proposed as potential explanations for a larger proportion nPCR positive nasal samples obtained in diagnostic samples.Results of this study demonstrated that detection of M.hyopneumoniae in tonsil where no ciliated epithelial cells exists, is possible. Although tonsillar sampling in live animals is more laborious than nasal samplings, it is possibly more adequate to monitor M. hyopneumoniae detection at a farm level. Results obtained in this Thesis and from previous works, showed that when bronchial swabs are positive the probability to show EP-compatible microscopic lesions in the animal is high. However, presence of EP-compatible lesions does not always imply M. hyopneumoniae detection in bronchi, and that may be caused by other pathogens such as SIV.

<i>Mycoplasma bovis</i> is generally considered the causative pathogen associated with Chronic Pneumonia and Polyarthritis Syndrome (CPPS) in feedlot cattle. However, <i>M. bovis</i> virulence may vary between strains as it is also isolated from asympytomatic cattle. The following study aims to determine the prevalence of <i>M. bovis</i> in the respiratory tract of western Canadian cattle using two sampling methods and at two time points following feedlot entry. Three study groups were sampled. In the first group nasal swabs (NS) and bronchoalveolar lavages (BAL) were taken from 36 clincally healthy cattle at the University of Saskatchewan feedlot at both 14 and 90 days on feed (DOF). In a second experiment, NS were taken from 56 animals upon arrival at a commercial feedlot and one week to three months later upon treatment for respiratory disease. Lung and joint tissue swabs were collected at necropsy from a third group of 19 animals with CPPS clinical pathology originating in 10 different western Canadian feedlots. All samples were selectively cultured for <i>Mycoplasma</i> spp. DNA was extracted from isolated putative <i>Mycoplasma</i> colonies and amplified with universal 16S rRNA gene primers for identification. Amplified Fragment Length Polymorphism (AFLP) was used to genetically differentiate <i>M. bovis</i> positive isolates. More <i>M. bovis</i> was isolated from NS than BAL and <i>M. bovis</i> prevalence increased with DOF in the feedlot in both the University of Saskatchewan and commercial feedlot trials. Three genetically distinct clusters (A, B, and C) were isolated from the necropsy group. Two of these clusters were primarily associated with isolates collected from feedlot cattle and one strain was exclusively found in CPPS-associated mortalities. No significance difference in the prevalence of <i>M. bovis</i> strains was observed between different days on feed or sampling methods. It was concluded that either the difference in disease state is a host dependent outcome, due to a multi-factorial disease complex, or the AFLP assay was not sensitive enough to differentiate strains based on virulence.

Mycoplasma hyopneumoniae causes swine enzootic pneumonia (SEP) and leads to economic loss worldwide. The mechanism of pathogenesis is still not clear. Since this pathogen remains extracellulary after infection, the surface proteins on M. hyopneumoniae should play very important roles in adhering and affecting tracheal mucosal cells. Therefore, the potential of using the surface proteins as the basic to develop molecular vaccine is currently being investigated. The recombinant clone expressing the 42 kDa protein was isolated from the

Thesis (Ph.D.)--University of Alabama at Birmingham, 2009. / Title from PDF title page (viewed on Feb. 2, 2010). Includes bibliographical references (p. 64-72).

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 45-55).

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

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