<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.

<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.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2016-08-25T13:37:26Z No. of bitstreams: 2 Dissertação - Nathânia Dábilla Alves Silva - 2016.pdf: 1926312 bytes, checksum: 08f15d1c8a44d1ed6118f432a6917f2d (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-08-25T13:38:53Z (GMT) No. of bitstreams: 2 Dissertação - Nathânia Dábilla Alves Silva - 2016.pdf: 1926312 bytes, checksum: 08f15d1c8a44d1ed6118f432a6917f2d (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-08-25T13:38:53Z (GMT). No. of bitstreams: 2 Dissertação - Nathânia Dábilla Alves Silva - 2016.pdf: 1926312 bytes, checksum: 08f15d1c8a44d1ed6118f432a6917f2d (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-04-13 / The norovirus (NoVs) are important viral causative agents of acute gastroenteritis (AGE), affecting individuals of all ages in distinct parts of the word; however, the highest morbi-mortality rates occur mainly in children under five years of age and the elderly. The aim of this study was to to screening NoV by Polymerase Chain Reaction Post Reverse Transcription (RT-qPCR) and real-time (RT-qPCR) in fecal and nasal swab of children up to six years of age, with and without AGE symptoms. Samples were obtained at the Materno Infantil Hospital, from May/2014 to May/2015. Secretor status of children was also determined by enzyme immunoassay and genotyping (FUT2 gene) from the sediment of nasal swab epithelial cells. A global positivity index of 17% (37/219) for NoV in feces, and from these positive children, 48.6% (18/37) had AGE symptoms. Mean viral load in fecal samples was 2.59 x 1010 CG/g from symptomatic and 1.37 x 109 CG/g in asymptomatic. A higher positivity rate (70%) was observed GII NoV, compared to GI NoV (30%) and a higher positivity in children up to 24 month old (67.5%), although not statistically significant. As for the secretor status of children positive for NoV in fecal samples, 94.6% positive secretory status. The NoV were detected in practically every month of the study, and no particular pattern of circulation in relation to dry or rainy seasons was observed. Most children positive for NoV (70%) had the record they have received at least the first dose of the vaccine against Rotavirus, being the highest viral load detected among vaccinated children. The NoV RNA was detected in 8.7% of nasal swab samples of the children and of these, 58% had AGE symptoms. The mean viral load in swab samples from symptomatic children was 2.10 x 108 and in the asymptomatic children was 2.41 x 107 CG/mL. A high NoV genotype variability was found in the study (GI.2, GI.3, GI.5, GII.3, GII.4 and GII.6), with a predominance of GII.4 (28.6%), with this being the first report of NoV GI.5 in Brazil. The data obtained in this study reveal a high frequency, viral load, and genetic variability of NoVs among children attended in a hospital of Goiânia, Goiás. The results are important for a better understanding of NoV epidemiology in nosocomial environment, and may constitute useful information on the advent of the development of an effective vaccine. The viral load in nasal swab samples is a novel data that may contribute for the elucidation of a possible alternative rout of NoV transmission. / Os norovírus (NoVs) são importantes agentes virais causadores de gastroenterite aguda (GEA), atingindo indivíduos de todas as idades em diversas partes do mundo, porém os maiores índices de morbimortalidade ocorrem principalmente nas crianças menores de cinco anos e idosos. O objetivo do presente estudo foi realizar a pesquisa de NoV, através da Reação em Cadeia pela Polimerase Pós-Transcrição Reversa (RT-PCR) e por tempo real (RT-qPCR) em amostras fecais e swab nasal de crianças com até seis anos de idade, com e sem sintomas de GEA. As amostras foram coletadas no Hospital Materno Infantil, entre maio/2014 e maio/2015. Procedeu-se ainda à determinação do status secretor das crianças, por Ensaio Imunoenzimático e genotipagem (gene FUT2), em sedimento de células epiteliais do swab nasal. Foi observado um índice de positividade global de 17% (37/219) para NoV nas fezes, sendo que destas crianças positivas, 48,6% (18/37) apresentavam sintomas de GEA. A carga viral média nas amostras de fezes de crianças com sintomas foi 2,59 x 1010 CG/g e 1,37 x 109 CG/g nas assintomáticas. Observou-se maior positividade para NoV GII (70%) quando comparado ao GI (30%) e maior positividade nas crianças de até 24 meses de idade (67,5%), entretanto este dado não foi estatisticamente significativo. Quanto ao status secretor das crianças positivas para NoV nas fezes, 94,6% status secretor positivo. Os NoV foram detectados em praticamente todos meses do estudo, não sendo observado padrão de circulação definido em relação às estações seca e chuvosa. A maioria das crianças positivas para NoV (70%) tinham o registro de terem recebido pelo menos a primeira dose da vacina contra Rotavírus, sendo a carga viral mais elevada detectada entre crianças vacinadas. O RNA de NoV foi ainda detectado em 8,7% (19/219) das amostras de swab nasal das crianças e destas, 58% apresentavam sintomas de GEA. A carga viral média nas amostras de swab das crianças sintomáticas foi 2,10 x 108 CG/mL e nas assintomáticas 2,41 x 107 CG/mL. Foi observada elevada variabilidade de genótipos de NoV no estudo (GI.2, GI.3, GI.5, GII.3, GII.4 e GII.6), com maior predominância de GII.4 (28,6%), sendo este o primeiro relato de NoV GI.5 no Brasil. Os dados obtidos neste estudo revelam elevada frequência, carga viral e variabilidade genética de NoVs entre crianças atendidas em um hospital de Goiânia, Goiás. Os resultados são importantes para o melhor entendimento da epidemiologia dos NoVs em ambiente nosocomial, e poderão ser uteis como informação no advento do desenvolvimento de uma vacina eficaz. A determinação da carga viral de NoV em xii amostras de swab nasal é um dado novo, podendo este contribuir para a elucidação de uma possível rota alternativa de transmissão dos NoV.