京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22004号 / 医博第4518号 / 新制||医||1038(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 辻川 明孝, 教授 中川 一路, 教授 高橋 淳 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Orientador: Luis Guilherme de Oliveira / Resumo: Mycoplasma suis e Mycoplasma parvum se ligam fortemente aos eritrócitos e causam hemoplasmose em suínos. Os animais infectados podem ser portadores assintomáticos ou apresentar sinais inespecíficos, acometendo todas as faixas etárias. No Brasil, os estudos sobre a ocorrência e a diversidade genética associada aos hemoplasmas suínos (HS) são escassos. Portanto, este trabalho teve como objetivo detectar, quantificar e caracterizar a diversidade genética da HS em animais terminados de granjas tecnificadas no estado de Goiás, Brasil. Amostras de sangue total de 450 de 30 granjas diferentes localizadas no estado de Goiás, foram coletadas em tubos contendo ácido etilenodiaminotetracético (EDTA) e armazenadas a -80 °C. A extração de DNA seguiu o método “in house” descrito previamente. A PCR convencional (cPCR) direcionada ao gene endógeno gapdh foi realizada para evitar resultados falso-negativos nos testes seguintes. Os ensaios de PCR em tempo real quantitativa (qPCR) foram realizados a fim de detectar e quantificar DNA de Mycoplasma baseado no gene 16S rRNA para HS. Para avaliar a diversidade genética da HS, realizou-se a clonagem e sequenciamento dos genes 16S rRNA e 23S rRNA para hemoplasmas. Os resultados da qPCR mostraram uma ocorrência de HS de 68,89%, onde todas as fazendas tinham pelo menos dois animais positivos. A quantificação na qPCR variou de 8.43x10-1 a 4.69x106 µL, e 52.71% das amostras apresentaram 1x103 copies/µL. Além disso, o teste do coeficiente de Spearman reve... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Mycoplasma suis and Mycoplasma parvum are strongly bound to erythrocytes and cause hemoplasmosis in pigs. Infected animals can be asymptomatic carriers or have nonspecific signs, affecting all age groups. In Brazil, studies on the occurrence and genetic diversity associated with porcine hemoplasmas (PH) are scarce. Therefore, this work aimed to detect, quantify and characterize the genetic diversity of PH in finished animals from technified farms in the state of Goiás, Brazil. Whole blood samples from 450 animals from 30 different farms located in the state of Goiás, were collected in tubes containing ethylenediaminetetraacetic acid (EDTA) and stored at -80°C. DNA extraction followed the "in house" method previously described. Conventional PCR (cPCR) targeting the gapdh endogenous gene was performed to avoid false negative results in the following tests. Quantitative real-time PCR (qPCR) assays were performed in order to detect and quantify Mycoplasma DNA based on the 16S rRNA gene for PH. To assess the genetic diversity of PH, cloning and sequencing of the 16S rRNA and 23S rRNA genes for hemoplasmas was performed. The results of the qPCR showed a PH occurrence of 68.89%, where all farms had at least two positive animals. The quantification in the qPCR varied from 8.43x10-1 to 4.69x106 µL, and 52.71% of the samples presented 1x103 copies / µL. Furthermore, the Spearman coefficient test revealed that the occurrence of PH is inversely associated with the number of births per we... (Complete abstract click electronic access below) / Mestre

Mycoplasma synoviae (Ms) causes respiratory infection and synovitis in chickens and turkeys and is an economically important pathogen of poultry worldwide. It is critically important to characterize Ms strains, especially in countries in which poultry flocks are vaccinated with the live attenuated Ms strain MS-H. Vaccination with this vaccine may cause sero-conversion and persistence of the vaccine strain in the respiratory tract and will frequently result in positive Ms cultures and PCR results. Vaccination of flocks therefore complicates the diagnosis of Ms by the presence of detectable antibodies in the blood. Many diagnostic techniques cannot distinguish between the vaccine strain and wild type strain. Single stranded conformation polymorphism (SSCP) and real-time PCR with high melting curve (HRM) analysis can discriminate between the different Ms strains obtained from the field and also distinguish them from the live vaccinestrains. These techniques provide effective tools for the further study of the epidemiology and spread of Ms strains in chickens in South Africa. This project was undertaken to establish whether SSCP and HRM analyses can be used effectively to discriminate between Ms field isolates and the vaccine strain. Mycoplasma synoviae DNA was extracted from samples and conventional PCR was performed using oligonucleotide primers complementary to the single-copy conserved 5’ end of the variable lipoprotein and haemagglutinin encoding gene (vlhA). Twenty six samples were separated by agarose gel electrophoresis and prepared for SSCP and real-time PCR and HRM curve analysis. Results obtained from SSCP were compared to real-time PCR/HRM. Differences obtained by SSCP and melting curve analysis between different isolates were confirmed by sequencing. Results obtained from the different techniques differentiated the strains from the vaccine strain (isolate Ms10), which had a different melting temperature to the others and a different band pattern on the SSCP gel. These results confirmed that HRM and SSCP methods can be used to detect and discriminate between Mycoplasma synoviae field isolates and the vaccine strain. / Dissertation (MSc)--University of Pretoria, 2012. / Veterinary Tropical Diseases / unrestricted

Since the first report of Mycoplasma-associated polyarthritis in farmed Nile crocodiles in 1995, the disease has spread across Zimbabwe and South Africa and has resulted in significant economic losses on infected farms. Due to poor response to antimicrobial treatment and frequent relapses, the use of an autogenous vaccine to manage disease outbreaks was evaluated. Two previous trials had been performed with a similar vaccine and the results suggested that the vaccine could be effective in alleviating disease, although the numbers of animals were limited in both. This trial aimed to evaluate an inactivated, alum-adjuvanted M. crocodyli whole-cell vaccine in a large group of yearling crocodiles under field conditions on a farm in Zimbabwe where repeated M. crocodyli outbreaks have been reported. The safety of the vaccine was assessed by administrating the vaccine intraperitoneally to a subset of crocodiles. No adverse clinical reactions were observed in any of these crocodiles. A group of two thousand two hundred crocodiles received two intramuscular vaccinations four weeks apart in the autumn of 2011, while another group of two thousand two hundred crocodiles served as unvaccinated controls. Serum was collected from a subset of the vaccinated and unvaccinated crocodiles at different time-points before and after vaccination to evaluate the humoral response to vaccination. Latex slide agglutination tests (LAT) were performed on all samples and positive samples were titrated with the latex slide agglutination test and metabolism inhibition assay. A low percentage of sera were positive with serological tests done prior to vaccination, suggesting either circulating Mycoplasma or maternal immunity. Statistically significant increase in sero-positivity was detected with LAT four weeks after primary vaccination, although the titre remained low. Six weeks after the booster vaccination the percentage seropositive vaccinated crocodiles had decreased and there were no statistically significant difference between the percentage seropositive vaccinated and unvaccinated crocodiles. A significant outbreak of Mycoplasma-like polyarthritis was encountered 6 months after vaccination, in October 2011. Both vaccinated and unvaccinated crocodiles were affected. Serum samples from different subsets of crocodiles were collected and evaluated similar to the vaccine trial. The results indicated that a similar rate of sero-positivity was present in all crocodiles, irrespective of vaccination- or disease status Sera collected during this trial was used to evaluate the performance of the latex slide agglutination assay compared to the metabolism inhibition assay (“Gold standard” assay), as the performance of the LAT had not been evaluated previously. The calculated diagnostic sensitivity was 72%, diagnostic specificity was 32%, the predictive value of the positive test was 36% while the predictive value of the negative test was 69%. This trial indicated that the autogenous, inactivated, alum-adjuvanted, whole-cell vaccine against M. crocodyli was not able to protect farmed Nile crocodiles on an infected farm against clinical Mycoplasma-associated polyarthritis. It was also found that the latex slide agglutination assay could be useful as a robust, pen-side assay to evaluate exposure to M. crocodyli, although other assays, such as PCR, bacterial culture or growth inhibition assays, has to be performed to confirm the presence of disease. / Dissertation (MSc)--University of Pretoria, 2012. / Veterinary Tropical Diseases / unrestricted

Background: Acute respiratory infections (ARIs) represent an important cause of morbidity and mortality in children, remaining a major public health concern, especially affecting children under 5 years old from low-income countries. Unfortunately, information regarding their epidemiology is still limited in Peru. Methods: A secondary data analysis was performed from a previous cross-sectional study conducted in children with a probable diagnosis of Pertussis from January 2010 to July 2012. All samples were analyzed via Polymerase Chain Reaction (PCR) for the following etiologies: Influenza-A, Influenza-B, RSV-A, RSV-B, Adenovirus, Parainfluenza 1 virus, Parainfluenza 2 virus, Parainfluenza 3 virus, Mycoplasma pneumoniae and Chlamydia pneumoniae. Results: A total of 288 patients were included. The most common pathogen isolated was Adenovirus (49%), followed by Bordetella pertussis (41%) from our previous investigation, the most prevelant microorganisms were Mycoplasma pneumonia (26%) and Influenza-B (19.8%). Coinfections were reported in 58% of samples and the most common association was found between B. pertussis and Adenovirus (12.2%). Conclusions: There was a high prevalence of Adenovirus, Mycoplasma pneumoniae and other etiologies in patients with a probable diagnosis of pertussis. Despite the presence of persistent cough lasting at least two weeks and other clinical characteristics highly suspicious of pertussis, secondary etiologies should be considered in children under 5 years-old in order to give a proper treatment. / Revisión por pares

Antimicrobials are commonly used to treat mycoplasmosis in animals. In spite of this and the fact that antimicrobial resistance has been recorded for this group of bacteria there are no universally accepted in vitro means of testing for this resistance, nor is resistance testing for mycoplasmas a routine in most veterinary laboratories. So prior to testing for resistance to a number of mycoplasmas isolated from animals in South Africa it was necessary to compare different tests including broth and agar microdilution tests to find out which one would perform best. Using the field strains M. bovis, M. crocodyli, M. felis, M. gallisepticum and M. synoviae, and the reference strains M. gallisepticum 56USDA, M. gallisepticum VaxSafe MG vaccine strain, M. mycoides T1/44 vaccine strain, and M. mycoides Ygoat (11706) broth- and agar-microdilution minimum inhibitory concentration (MIC)tests were performed using either modified Hayflicks or Mycoplasma synoviae media. Two different metabolism indicator systems were compared in the broth microdilution test (BrMIC) namely sugar fermentation (glucose or pyruvate) with phenol red (SFS) and evidence of reduction with resazurin (AlamurBlue®). It was also tested whether amoxicillin and clavulanic acid (ACA) could be used in the tests to reduce problems associated with contamination. Statistical analyses of the tests (repeatability and linear association) indicated that the BrMIC with SFS was the most reproducible method (pooled standard deviation = 0.14). The antimicrobial ACA was found to not to affect the MIC values (R2= 0.976 to 0.996). Furthermore one hundred forty two field strains including 93 M. bovis, 5 M. synoviae, 21 M. gallisepticum, 13 M. bovirhinis, 8 M. crocodyli and 6 M. felis were tested using the BrMIC+SFS with ACA method. Generally the mycoplasmas originating from poultry were resistant to commonly used antimicrobials and had higher MIC50 and MIC90 values than isolates originating from cattle, crocodiles and cats. It was found that most of the mycoplasmas were susceptible to doxycycline (tetracycline) and enrofloxacin with the exception of M. gallisepticum where 17.9% of strains were resistant to both. Resistance to tiamulin (100%) and tylosin (20 to 64%) was high for the poultry mycoplasmas. Most field isolates tested were resistant to erythromycin, nalidixic acid, florfenicol, norfloxacin, neomycin, sulphamethoxazole, trimethoprim and sulphamethoxazole/ trimethoprim combination, mostly resistant to norfloxacin and florfenicol. It is concluded that BrMIC+SFS with ACA method is a reproducible method that reduces any problems with bacterial contamination. As observed with the poultry strains, it is quite clear that antimicrobial resistance is developing to commonly used antimicrobials such as tylosin, the related pleuromutilins, fluoroquinolones and tetracyclines. In species where antimicrobial therapy is applied routinely such as poultry and possibly feedlot cattle, it is recommended that MIC testing is done prior to any therapeutic interventions. / Dissertation (MSc)--University of Pretoria, 2010. / Veterinary Tropical Diseases / unrestricted

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Le « porcine respiratory disease complex » (PRDC) est un trouble multifactoriel dû à une infection simultanée ou séquentielle de divers micro-organismes pouvant intensifier ou prolonger les signes cliniques des porcs. On retrouve dans ce complexe Mycoplasma hyopneumoniae, un des agents initiateurs du PRDC et agent primaire de la pneumonie enzootique (EP) chez les porcs. Streptococcus suis est l’un des agents pathogènes secondaires du PRDC, c’est également un agent pathogène important induisant principalement des méningites, des septicémies et la mort subite des porcelets post-sevrés. Mycoplasma hyorhinis est également l’un des agents pathogènes secondaires du PRDC, et va induire des inflammations sérofibrineuses chez les porcelets. Comme ces trois pathogènes sont retrouvés au sein du PRDC et au niveau des voies respiratoires supérieures des porcs, il pourrait exister un effet positif des mycoplasmes sur la pathogénèse de S. suis. C’est pourquoi différentes expériences in vitro ont été réalisées avec les cellules épithéliales porcines (NPTr), les macrophages alvéolaires porcins (PAMs) et les cellules dendritiques porcines (BM-DCs) qui ont été pré-infectés par les mycoplasmes puis infectés avec S. suis. Il a été observé que la cytotoxicité et l’inflammation des cellules porcines ont été significativement augmentées lorsqu’elles ont été pré-infectées par les mycoplasmes puis infectées par S. suis. Cependant, la pré-infection des cellules n’a pas joué de rôle sur l’adhésion et l’invasion de S. suis, sur la phagocytose et la survie intracellulaire de la bactérie. Cette étude semble montrer que la pré-infection des mycoplasmes pourraient induire un contexte inflammatoire favorisant la pathogenèse de S. suis. / Porcine respiratory disease complex (PRDC) is a multifactorial disorder due to simultaneous or sequential infection with various microorganisms that can intensify or prolong clinical signs in pigs. Included in this complex is Mycoplasma hyopneumoniae, one of the initiating agents of PRDC and the primary agent of enzootic pneumonia (EP) in pigs. Streptococcus suis is one of the secondary pathogens of PRDC and is also an important pathogen, mainly causing meningitis, septicemia, and sudden death in post-weaned piglets. Mycoplasma hyorhinis is also one of the secondary pathogens of PRDC and will also induce serofibrinous inflammation in piglets. As all three pathogens are found in the PRDC and in the upper respiratory tract of pigs there may be a positive effect of mycoplasma on the pathogenesis of S. suis. Therefore, different in vitro experiments were performed with newborn pig tracheal cells (NPTr), primary porcine alveolar macrophages (PAMs) and porcine bone-marrow-derived dendritic cells (BM-DCs) that were pre-infected with mycoplasma and then infected with S. suis. It was observed that cytotoxicity and inflammation of pig cells were significantly increased when they were pre-infected with mycoplasma and then infected with S. suis. However, pre-infection of the cells did not play a role in the adhesion and invasion of S. suis and in the phagocytosis and intracellular survival of the bacteria. This study suggests that pre-infection with mycoplasma may induce an inflammatory context favoring the pathogenesis of S. suis.

The holy grail of structural biology is to study a protein in situ, and this goal has been fast approaching since the resolution revolution and the achievement of atomic resolution. A cell's interior is not a dilute environment, and proteins have evolved to fold and function as needed in that environment; as such, an investigation of a cellular component should ideally include the full complexity of the cellular environment. Imaging whole cells in three dimensions using electron cryotomography is the best method to accomplish this goal, but it comes with a limitation on sample thickness and produces noisy data unamenable to direct analysis. This thesis establishes a novel workflow to systematically analyse whole-cell electron cryotomography data in three dimensions and to find and identify instances of protein complexes in the data to set up a determination of their structure and identity for success. Mycoplasma pneumoniae is a very small parasitic bacterium with fewer than 700 protein-coding genes, is thin enough and small enough to be imaged in large quantities by electron cryotomography, and can grow directly on the grids used for imaging, making it ideal for exploratory studies in structural proteomics. As part of the workflow, a methodology for training deep-learning-based particle-picking models is established. As a proof of principle, a dataset of whole-cell Mycoplasma pneumoniae tomograms is used with this workflow to characterize a novel membrane-associated complex observed in the data. Ultimately, 25431 such particles are picked from 353 tomograms and refined to a density map with a resolution of 11 Å. Making good use of orthogonal datasets to filter search space and verify results, structures were predicted for candidate proteins and checked for suitable fit in the density map. In the end, with this approach, nine proteins were found to be part of the complex, which appears to be associated with chaperone activity and interact with translocon machinery. Visual proteomics refers to the ultimate potential of in situ electron cryotomography: the comprehensive interpretation of tomograms. The workflow presented here is demonstrated to help in reaching that potential. / Der heilige Gral der Strukturbiologie ist die Untersuchung eines Proteins in situ, und dieses Ziel ist seit der Auflösungsrevolution und dem Erreichen der atomaren Auflösung in greifbare Nähe gerückt. Das Innere einer Zelle ist keine verdünnte Umgebung, und Proteine haben sich so entwickelt, dass sie sich falten und so funktionieren, wie es in dieser Umgebung erforderlich ist; daher sollte die Untersuchung einer zellulären Komponente idealerweise die gesamte Komplexität der zellulären Umgebung umfassen. Die Abbildung ganzer Zellen in drei Dimensionen mit Hilfe der Elektronenkryotomographie ist die beste Methode, um dieses Ziel zu erreichen, aber sie ist mit einer Beschränkung der Probendicke verbunden und erzeugt verrauschte Daten, die sich nicht für eine direkte Analyse eignen. In dieser Dissertation wird ein neuartiger Workflow zur systematischen dreidimensionalen Analyse von Ganzzell-Elektronenkryotomographiedaten und zur Auffindung und Identifizierung von Proteinkomplexen in diesen Daten entwickelt, um eine erfolgreiche Bestimmung ihrer Struktur und Identität zu ermöglichen. Mycoplasma pneumoniae ist ein sehr kleines parasitäres Bakterium mit weniger als 700 proteinkodierenden Genen. Es ist dünn und klein genug, um in grossen Mengen durch Elektronenkryotomographie abgebildet zu werden, und kann direkt auf den für die Abbildung verwendeten Gittern wachsen, was es ideal für Sondierungsstudien in der strukturellen Proteomik macht. Als Teil des Workflows wird eine Methodik für das Training von Deep-Learning-basierten Partikelpicken-Modellen entwickelt. Als Proof-of-Principle wird ein Dataset von Ganzzell-Tomogrammen von Mycoplasma pneumoniae mit diesem Workflow verwendet, um einen neuartigen membranassoziierten Komplex zu charakterisieren, der in den Daten beobachtet wurde. Insgesamt wurden 25431 solcher Partikel aus 353 Tomogrammen gepickt und zu einer Dichtekarte mit einer Auflösung von 11 Å verfeinert. Unter Verwendung orthogonaler Datensätze zur Filterung des Suchraums und zur Überprüfung der Ergebnisse wurden Strukturen für Protein-Kandidaten vorhergesagt und auf ihre Eignung für die Dichtekarte überprüft. Letztendlich wurden mit diesem Ansatz neun Proteine als Bestandteile des Komplexes gefunden, der offenbar mit der Chaperonaktivität in Verbindung steht und mit der Translocon-Maschinerie interagiert. Das ultimative Potenzial der In-situ-Elektronenkryotomographie – die umfassende Interpretation von Tomogrammen – wird als visuelle Proteomik bezeichnet. Der hier vorgestellte Workflow soll dabei helfen, dieses Potenzial auszuschöpfen.