Neutrophils provide a rapid first response to invading pathogens and orchestrate the immune response. They are able to employ potent antipathogenic mechanisms such as phagocytosis, reactive oxygen species (ROS) generation, protease release from granules, and formation of neutrophil extracellular traps (NETs). Despite this, certain pathogens have evolved mechanisms to benefit from neutrophil effector functions. Human cytomegalovirus (HCMV) is a clinically important pathogen that infects the majority of the human population. Monocytes are considered the main vehicle of HCMV dissemination throughout the body, but little research has been done on its interaction with neutrophils. The virus encodes a range of immunomodulatory proteins including an IL-8 homologue that acts as a powerful neutrophil chemoattractant. Viral conservation of a protein that recruits neutrophils to the site of HCMV infection suggests that the interaction between neutrophils and HCMV provides an overall advantage to the virus, but little evidence exists so far to suggest this is the case. Here I report that human peripheral blood neutrophils exposed to a clinical strain of HCMV display a profound survival phenotype, as assessed by morphology, phosphatidylserine exposure, cell permeability, and caspase-3/7 activity. This occurs in the absence of viral gene production. Neutrophils also upregulated their release of inflammatory cytokines in response to HCMV, with higher concentrations of IL-6, IL-8, and MIP-1α detected in the secretomes of infected neutrophils. These secretomes induced monocyte chemotaxis and increased monocyte permissivity to HCMV infection, as well as augmented survival in healthy, uninfected neutrophils. These experiments were confirmed with clean HCMV after the discovery of contaminating Mycoplasma spp. in the viral inocula of the initial experiments. Mycoplasma-HCMV coinfection induced an autophagic phenotype in neutrophils, as assessed by Western blotting and qPCR of autophagy-related components. Inhibition of autophagy using 3-MA reversed a profound survival effect. The unintended inclusion of Mycoplasma spp. further led to the serendipitous discovery of yet another pathogenic ability to overcome neutrophil immune functions: contaminating Mycoplasma spp. as well as Mycoplasma pneumoniae profoundly degraded NETs. These extracellular chromatin structures were stimulated using PMA or pyocyanin, and their release was dependent on the generation of ROS: severely ROS-deficient murine bone marrow neutrophils were unable to generate NETs. However, small amounts of ROS were sufficient for NETs generation, as neutrophils from acute respiratory distress syndrome patients, including many that had attenuated ROS-responses, were still capable of NETs generation. The NETs-degradative properties of mycoplasma were confirmed by fluorescence confocal and scanning electron microscopy, as well as spectrophotometry and agarose gel electrophoresis. This study demonstrates that two pervasive pathogens, HCMV and M. pneumoniae, both frequently found in coinfections in clinical contexts, are able to overcome neutrophil antipathogenic mechanisms to potentially enhance pathogen dissemination. These data provide not only a novel example of manipulation of an anti-viral response in a cell not productively infected, but also a novel example of pathogenic NETs degradation. These findings may have implications on our understanding of mycoplasma and HCMV pathogenesis and provide new targets for the generation of therapies.

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.

Made available in DSpace on 2014-12-02T11:16:51Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-02-26Bitstream added on 2014-12-02T11:21:19Z : No. of bitstreams: 1 000783735.pdf: 592510 bytes, checksum: 4c75607716aba43fa9491069b97e3bb6 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Micoplasmas são micro-organismos altamente contagiosos em bovinos, podendo causar mastite, além de pneumonias, artrite, otite e menos frequentemente abortamentos e meningites. São resistentes a maioria dos antimicrobianos utilizados e a mastite causada por este agente pode se tornar crônica e com alta contagem de células somáticas (CCS). Na maioria dos estudos sobre mastite, relata-se a alta prevalência de Streptococcus agalactiae e Staphylococcus aureus, já que estes patógenos podem ser isolados e identificados utilizando métodos microbiológicos convencionais. Em contraste, Mycoplasma bovis necessita de meios de cultura e condições especiais para isolamento. Dessa forma, acredita-se que a real prevalência de Mycoplasma bovis é, provavelmente, subestimada. Assim, o presente estudo teve por objetivo avaliar a prevalência de Mycoplasma bovis em rebanhos leiteiros do estado de São Paulo. Para tanto, o estudo foi dividido em fase de triagem, com coleta de leite de tanques e fase de coleta individual de leite dos animais. Utilizou-se cultivo em meio seletivo (SP4) e PCR para detecção do agente. O cultivo foi negativo para todas as amostras e a PCR revelou uma prevalência de 1,4% de M.bovis em tanques. Pode-se concluir com o presente estudo pela baixa prevalência de M.bovis nos rebanhos do estado de São Paulo, mostrando assim uma discreta importância deste agente como causa de mastites / FAPESP: 11/16055-3 / FAPESP: 11/21009-0

Orientador: Helio Langoni / Banca: José Carlos Figueiredo Pantoja / Banca: Márcio Garcia Ribeiro / Banca: Vera Mores Rall / Resumo: Micoplasmas são micro-organismos altamente contagiosos em bovinos, podendo causar mastite, além de pneumonias, artrite, otite e menos frequentemente abortamentos e meningites. São resistentes a maioria dos antimicrobianos utilizados e a mastite causada por este agente pode se tornar crônica e com alta contagem de células somáticas (CCS). Na maioria dos estudos sobre mastite, relata-se a alta prevalência de Streptococcus agalactiae e Staphylococcus aureus, já que estes patógenos podem ser isolados e identificados utilizando métodos microbiológicos convencionais. Em contraste, Mycoplasma bovis necessita de meios de cultura e condições especiais para isolamento. Dessa forma, acredita-se que a real prevalência de Mycoplasma bovis é, provavelmente, subestimada. Assim, o presente estudo teve por objetivo avaliar a prevalência de Mycoplasma bovis em rebanhos leiteiros do estado de São Paulo. Para tanto, o estudo foi dividido em fase de triagem, com coleta de leite de tanques e fase de coleta individual de leite dos animais. Utilizou-se cultivo em meio seletivo (SP4) e PCR para detecção do agente. O cultivo foi negativo para todas as amostras e a PCR revelou uma prevalência de 1,4% de M.bovis em tanques. Pode-se concluir com o presente estudo pela baixa prevalência de M.bovis nos rebanhos do estado de São Paulo, mostrando assim uma discreta importância deste agente como causa de mastites / Abstract: Not available / Mestre

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.

Submitted by Mario BC (mario@bc.ufrpe.br) on 2018-06-19T12:55:46Z No. of bitstreams: 1 Allison Alves de Macedo.pdf: 650201 bytes, checksum: b68962f58a5ff7ccd111367548d6a064 (MD5) / Made available in DSpace on 2018-06-19T12:55:47Z (GMT). No. of bitstreams: 1 Allison Alves de Macedo.pdf: 650201 bytes, checksum: b68962f58a5ff7ccd111367548d6a064 (MD5) Previous issue date: 2017-02-06 / This study’s objective was to conduct a Mycoplasma bovigenitalium and Ureaplasma diversum infections epidemiological investigation in cattle in the Ipanema Valley microregion, Pernambuco state. Vaginal swabs were collected from 355 reproductive age cows and submitted to polymerase chain reaction (PCR) and microbiological isolation. An investigative questionnaire was used to risk factors analysis associated with Mollicutes infection. According to PCR, 9.29% (33/355) of the samples were positive for Mycoplasma bovigenitalium and 21.69% (77/355) for Ureaplasma diversum. Coinfection was observed in 2.81% (10/355) of the samples. According to isolation, there was growth on Hayflick medium of 81.81% (27/33) from the samples classified as Mycoplasma spp. and 24.67% (19/77) growth on UB medium, being classified as Ureaplasma diversum. Risk factors associated to Mollicutes infection were considered: semi-intensive breeding system (OR = 4.6), pasture rent (OR = 3.6), reproductive disorders nonisolated animals (OR = 3.2), and natural mating plus artificial insemination (OR = 3.5). This is the first record of Mycoplasma bovigenitalium and Ureaplasma diversum infection in semiarid region cattle herds of Pernambuco state, Brazil. From this, preventive measures directed to the identified risk factors can contribute to Mollicutes’ occurrence decrease in the cattle herds studied. / Objetivou-se, com este estudo, realizar uma investigação epidemiológica da infecção por Mycoplasma bovigenitalium e Ureaplasma diversum em bovinos na microrregião do Vale do Ipanema, estado de Pernambuco. Foram coletados suabes vaginais de 355 vacas em idade reprodutiva que foram submetidos à Reação em Cadeia da Polimerase (PCR) e isolamento microbiológico. Um questionário investigativo foi utilizado para a análise dos fatores de risco associados à infecção por Mollicutes. Pela PCR, 9,29% (33/355) amostras foram positivas para Mycoplasma bovigenitalium e 21,69% (77/355) para Ureaplasma diversum. Observou-se coinfecção em 2,81% (10/355) das amostras. A partir do isolamento microbiológico das amostras positivas na PCR, houve crescimento no meio Hayflick de 81,81% (27/33) das amostras, sendo classificadas como Mycoplasma spp. e 24,67% (19/77) cresceram no meio “UB”, sendo classificadas como U. diversum. Foram considerados fatores de risco associados à infecção por Mollicutes: sistema de criação semi-intensivo (OR=4,6); aluguel de pastagens (OR=3,6); não isolamento dos animais com distúrbios reprodutivos (OR=3,2) e realização de monta natural mais inseminação artificial (OR=3,5). Este é o primeiro registro da infecção por Mycoplasma bovigenitalium e Ureaplasma diversum em rebanhos bovinos na região semiárida do estado de Pernambuco, Brasil. A partir dos resultados obtidos, medidas de prevenção direcionadas aos fatores de riscos identificados podem contribuir com a diminuição da ocorrência de Mollicutes nos rebanhos bovinos estudados.

A partir de meados da década de 80, com a expansão da cultura do milho para além das épocas tradicionais de cultivo, quer pela prática da safrinha ou por plantios irrigados, vem ocorrendo um aumento na incidência de doenças a secundária. Dentro deste contexto, o enfezamento vermelho do milho, relatado no país primeiramente em 1970, vem ocorrendo de forma freqüente, apresentando altos índices de ocorrência, muitas vezes com comprometimento total da produção. As plantas infectadas apresentam uma sintomatologia complexa facilmente confundida com viroses. O sintoma mais característico é o avermelhamento foliar. Além do avermelhamento as plantas apresentam redução na altura, perfilhamento basal e axilar, espigas extranumerárias e colmos afinados. Essa doença é causada por um procarioto não cultivável em meio de cultura, habitante exclusivo do floema, denominado fitoplasma, veiculado de forma persistente e propagativa pela cigarrinha Dalbulus maidis. Devido às características do patógeno, a única forma de controle promissora é a utilização de variedades tolerantes/resistentes. Para eficiência na obtenção destas variedades é necessário um diagnóstico correto e conhecimento sobre a variabilidade metodologia mais eficiente tanto para diagnose correta como para investigar essa variabilidade tem sido o PCR. O PCR, utilizando oligonucleotídeos baseados no gene 16SrDNA seguido da análise de RFLP, proporciona uma identificação mente a classificação de fitoplasmas é fundamentada no perfil molecular obtido por análise de RFLP de fragmentos do gene 16SrDNA amplificados. Com base nestas considerações, o trabalho teve como objetivo caracterizar molecularmente isolados do fitoplasma associado ao enfezamento vermelho do milho, coletados em quatro regiões produtoras de milho do estado de São Paulo. A sintomatologia para cada amostra de milho foi anotada. Foram usados dois pares de oligonucleotídeos universais para fitoplasmas em duplo PCR, um par de oligonucleotídeo especificamente desenvolvido para detecção do fitoplasma do enfezamento vermelho do milho, além de oligonucleotídeos para detecção de fitoplasmas pertencentes a grupos específicos. Após amplificação e eletroforese, 29 isolados foram selecionados para a identificação através de RFLP. Fragmentos de DNA foram submetidos à digestão com diferentes enzimas de restrição com o objetivo de identificar/classificar o fitoplasma. Os padrões de bandas obtidos após a eletroforese em gel de poliacrilamida foram comparados com os padrões atuais para classificação dos fitoplasmas. Todos os isolados analisados apresentaram idênticos padrões de bandas, para cada enzima de restrição, considerada individualmente. Não houve diferenciação de acordo com a região geográfica de coleta ou de acordo com intensidade de sintomas apresentados. Todos os isolados foram identificados como pertencentes ao grupo I e subgrupo B da classificação molecular atualmente adotada para estes microorganismos. / Since the middle 80s, an increase in year round cropping of maize resulted in a spread of secondary diseases in the crop’s major production areas. In this context, maize busy stunt, firstly appointed in Brazil in 1970, is occurring more frequently, often with total damage of production. Infected plants show a complex symptomatology, easily confounded with virus-caused diseases. The most characteristic symptom is leaf reddening. Besides the reddening diseased plants show stunting, often developing tillering. This disease is caused by a phytoplasma, a wall- less prokaryote, uncultivable, phloem inhabitant. This pathogen is transmitted by the leafhopper Dalbulus maidis, a in persistent and propagative manner. Due to the pathogen’s characteristics, the best control measure is the use of tolerant/resistant plants. For efficiency in breeding, accurate procedures of detection and an investigation of the pathogen’s genetic variability are necessary. The more accurate manner is using PCR. PCR, using 16SrDNA based primers pairs and followed by RFLP analysis, offers a safe identification of the pathogen. Today the phytoplasma classification is based in molecular patterns obtained by RFLP analysis of amplified 16SrDNA gene fragments. This work’s objective was the molecular characterization of maize bushy stunt phytoplasma strains collected in four corn production areas in São Paulo state, Brazil. The simptomatology to every maize sample was saved. Two primer pairs in nested PCR and a specific primer pair developed to MBS detection were used, besides group specific phytoplasma primers pairs. After amplification and electrophoresis, 29 samples were selected. These selected samples were digested with different restriction enzymes to identify/classify the phytoplasma. The fragment’s sizes obtained by electrophoresis through 4,5% polyacrilamide gel were compared with the reference’s classification patterns. All analyzed samples showed identical fragment-size patterns, for each restriction enzyme considered individually. There was no difference between these samples according to geographic collect region or according to symptoms. All strains were identified as belonging to group I and subgroup B of molecular classification.

Mycoplasma genitalium (M. genitalium) är en liten bakterie utan cellvägg som kan smitta sexuellt och efter en veckas inkubationstid orsaka genitala infektioner. Bakterien är svårodlad, men med PCR-teknik kan DNA-sekvens, specifik för M. genitalium, detekteras i prover från uretra/vagina. M. genitalium-infektionen ger ofta inga symptom. Symptom som sveda, klåda, blödningsrubbningar och flytningar kan dock ses vid M. genitalium-infektion, men dessa symptom liknar C. trachomatis-infektion, en annan vanlig sexuellt överförbar infektion. M. genitalium-bakterien har påvisats i prov från patienter med uretrit och cervicit men även i uppåtstigande infektioner, som benämns med det engelska namnet Pelvic Inflammatory Disease (PID). Obehandlade i tid, kan dessa uppåtstigande infektioner orsaka infertilitet, ektopisk graviditet och kroniska buksmärtor. Detta litteraturarbete har som syfte att redovisa hur vanligt M. genitalium- infektion är bland unga kvinnor. I några av studierna undersöks prevalensen av infektionen bland olika riskgrupper med eller utan symptom. Andra studier undersöker prevalensen av M. genitalium-infektion bland kvinnor som har diagnostiken cervicit och PID. I en studie påvisades en prevalens av M. genitalium-infektion på 2,1 % bland unga kvinnor som rekryterats inom primärvården. En betydligt högre prevalens på 22 % för samma infektion fann man hos riskgrupper för sexuellt överförbara infektioner (STI). I samma studie var 54 % av kvinnorna med M. genitalium-infektion smittade med även andra sexuellt överförbara patogener.  Arbetet undersöker också om behandlingen med doxycyklin, ett vanligt tetracyklin som används vid behandling av C. trachomatis-infektion, även fungerar för att behandla M. genitalium-infektionen. I två av studierna användes 1 g azitromycin (en makrolid) som singeldos för eradikering av M. genitalium. Detta gav en eradikeringsfrekvens mellan 85 - 91 %, vilket är betydligt högre än för doxycyklin på17-37 %. Azitromycinbehandling med 500 mg dag 1 och 250 mg dag 2 till 5 gav en eradikeringsfrekvens på 96 %. En retrospektiv studie visade att kvinnor diagnostiserade med PID och positiva för M. genitalium – infektion hade fått dåliga eradikerings- och behandlingsresultat med en standardbehandling som bestod av cefoxitin och doxycyklin. Dessa kvinnor drabbades senare i livet av infertilitet, kronisk smärta och återfall av PID. Utifrån de studier som användes i arbetet framkom att M. genitalium-infektion år 2010-2013 har en prevalens på mellan 2 till 4 % bland unga kvinnor mellan 17och 27 år och att det finns behov av screening och kontroll av infektionen. Incidens av M. genitalium-infektion ligger på ungefär 1 % per 100 kvinnoår. Det behövs nya behandlingsstrategier som undviker doxycyklin och cefoxitin, vilka har en dålig eller helt overksam effekt för eradikering av M. genitalium-bakterien.  Screening för både M. genitalium- och C. trachomatis – infektioner behövs för att välja ett antibiotikum som båda är känsliga för.

Master of Agribusiness / Department of Agricultural Economics / Vincent R. Amanor-Boadu / Mycoplasma; it is not a household name for many Americans or people around the world, but for those in the livestock industry, it has been a major concern. Mycoplasma, a member of the class Mollicutes, has had and continues to have a major impact on the cattle, swine and poultry industry, causing conditions such as arthritis, otitis media, reduced growth rate and reduced egg production (Journal of Veterinary Internal Medicine 2011) (Okwara 2016). This class of bacteria is unlike other classes, as defined by the lack of a cell wall, and is considered by many to be the smallest self-replicating prokaryote (Jack Maniloff 1992). Due to its small size, it can reside within cells and even pass through some of the currently used sterilizing filters in the biological/pharmaceutical industry today (Pall Corporation n.d.). This creates a risk for Mycoplasma contamination for those facilities/research centers that use materials of animal origin, as Mycoplasma organisms have historically been a common contaminate of cell lines and laboratory cultures, affecting roughly 15-35% of cell cultures (Cara N. Wilder 2015). An added concern is the difficulty in treatment of infected animals once an infection is established. The Mollicutes class has been considered innately resistant to the antibiotic penicillin and other cephalosporins due to the lack of the cell wall (Jack Maniloff 1992). Due to the clinical significance and risk factors surrounding the Mollicutes class, it is a current regulatory requirement to test materials of animal origin for the presence or absence of Mycoplasma. The specific criteria for the presence or absence of Mycoplasma test is dependent upon the country in which the product is intended to be sold. For the purposes of this study, the required method and products will be for those intended for sale domestically in the United States, or countries accepting US methodologies. To test a material or product for the presence or absence of Mycoplasma according to the current USDA code of federal regulations (CFR), the method is not a rapid procedure or a simple traditional broth inoculation. The domestic method is a minimum 24 day test that requires complex broth and agar media for Mycoplasma recovery. The complex media requirement is due to the fact that Mycoplasma organisms have stringent nutritional requirements due to their simplified cell structure/genome, which often require materials of animal origin, such as serums for lipid supply/metabolism (Jack Maniloff 1992). The 24 day Mycoplasma test requires an initial inoculation into the aforementioned broth and agar media and then 4 subsequent subcultures from the broth media onto the agar media at specified time intervals. All of the broth and agar media plates are incubated at specific atmospheric conditions and temperature for the duration of the test. The initial inoculation and subcultures are all examined by a trained Microbiologist at specific time intervals to search for evidence of viable Mycoplasma growth. The examination by a trained Microbiologist/technician is a vital step as Mycoplasmas do not produce turbidity in media, such as in traditional bacterial growth, nor are they visible by traditional light microscopy (Farzaneh 2011). If a Mycoplasma contamination is found, a biological/pharmaceutical company can pay huge sums of money to investigate the cause of the contamination, initiate corrective action, decontaminate the facility and destroy impacted batches. As evidenced by the above description, Mycoplasma testing places a large burden on a biological/pharmaceutical production facility or even research institutions. The complex media and labor cost for the 24 day test is extensive, which must be repeated for each batch of new material received or produced. The cost skyrockets if any contamination event occurs or even appears to occur, as investigation and decontamination add cost due to delay of release or possible destruction.

Bovine respiratory disease complex (BRD) consists of a largely single clinical entity of bronchopneumonia that is usually associated with the assembly of large numbers of especially weaner cattle into a feedlot environment. It has a multifactorial aetiology and develops as a result of complex interactions between environmental factors, host or animal factors and pathogens. It is often difficult to determine the exact role played by the various pathogens involved in an individual outbreak of disease. None of the many organisms isolated will on their own, reliably reproduce the natural disease in experimental animals. Observations from research studies and clinical experience have indicated that the presence of mycoplasmas increases the severity of respiratory disease. However, the role of Mycoplasma spp. in BRD complex as a primary or secondary pathogen remains controversial. The various stresses associated with the feedlot causes a breakdown of the defense mechanisms that normally hold the nasal infections in check, resulting in a rapid proliferation of virulent Mannheimia haemolytica serotype A1 in particular and the spread to the lower respiratory tract. The various viruses and mycoplasmas have however been shown to have the same effect as stress on the Pasteurella populations of the nasal mucosa. More than 10 species of Mycoplasma have been isolated from the bovine respiratory tract, but not all are pathogenic. They are able to act as a stress-causing agent, leading to a decreased host defense mechanism by altering the immune responsiveness or by causing tissue damage and thereby allowing bacteria to invade and colonise the lung and so causing a severe pneumonia. M. bovis and M. dispar are the more important lung isolates, with M. bovis being the most invasive and destructive and has been shown to increase the severity of calf pneumonias. M. bovis has been isolated from bovine pneumonias, arthritis, mastitis, tendosynovitis, genitalia, keratoconjunctivitis and is considered to be the primary pathogen in endemic pneumonia in younger calves. According to the literature, mycoplasmas are isolated from 25% to 80% of pneumonic lungs in feedlot cattle and the aim of the study was to identify the isolation rates in South African feedlots over a period of 2000 to 2004. To achieve this, 446 transtracheal aspirates (TTA’s) were collected from more than 25 feedlots around South Africa, except for the western Cape. Collection criteria included: pulled for respiratory disease; febrile (≥40ºC); depressed; anorexia and/or lack of rumen fill; nasal discharge or failure to clean muzzle; cough; increased respiratory rate >40 and most importantly, no prior treatment. Samples were also collected from 31 ‘healthy’ animals as controls. Samples collected were used for Mycoplasma isolations, as well as the aerobic bacteria to establish an antibiogram profile of bacteria commonly isolated in cattle feedlots. Mycoplasma spp. were isolated from 52.8% of samples taken from sick animals, with 67 out of 201 isolates (33.3%) being identified as M. bovis. According to the literature, M. bovis, M. haemolytica or P. multocida are isolated from bronchial lavage fluids from healthy calves in only a few cases, with estimates being put at 5 – 10% levels for Mycoplasma. Isolation rates of Mycoplasma spp. from healthy animals in this study was 22.7%, which was considerably higher than anticipated and could possibly be due to problems with the definition of a healthy animal. Although the number of samples from healthy animals was relatively small in this study, it was possible to show that there was a statistically significant association between Mycoplasma isolation and respiratory disease, p = 0,001 and with an odds ratio (OR) of 3,75 in cattle from those feedlots included in the study and thereby proving the hypothesis put forward. / Dissertation (MMedVet (Bovine Medicine))--University of Pretoria, 2007. / Production Animal Studies / MMedVet / Unrestricted