Made available in DSpace on 2015-03-26T13:51:53Z (GMT). No. of bitstreams: 1 texto completo.pdf: 550423 bytes, checksum: 038e82245fcf06c56e234826a321a4a6 (MD5) Previous issue date: 2006-09-11 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Ninety seven bacteria from the rumen and eighty seven ones from the feces were isolated from three dairy cows, feeding ration (24% protein) and corn ensilage. The resistance of the isolates to antibiotics were evaluated by using the agar dilution procedure for the following antimicrobials: nalidixic acid (NAL), ampicillin (AMP), chloramphenicol (CHL), erythromycin (ERY), streptomycin (STR), penicillin (PEN) and tetracycline (TET). The genetic diversity of 29 isolates from the rumen and 28 isolates from the feces were evaluated by Randomly Amplified Polymorphic DNA (RAPD). Approximately fifty seven percent isolates were obtained from animal one, thirteen percent from animal two, and twenty nine percent from animal three. The percent isolates obtained from feces were 62.1; 10.3; 27.6 for animal one, two and three, respectively. Considering all rumen isolates, the percent isolates resistant to the antimicrobial under test were: NAL 100 %; AMP 59.8 %; CHL 3.1 %; ERY 21.6 %; SRT 10.3 %; PEN 97.9 %; and TET 78.3 %. The overall resistance of the isolates from the feces were: NAL 100 %; AMP 54.0 %; ERY 20.7 %; STR 2.3 %; PEN 96.5 %; and TET 32.2 %. No isolates from the rumen or feces were simultaneously resistant to all antibiotics. The rumen isolates presenting the highest number of resistance marks were simultaneously resistant to six antibiotics, whereas the feces isolates were simultaneously resistant to five antibiotics. Among the rumen isolates showing six or five marks, neither one was resistant to chloramphenicol. Most isolates showing four marks were simultaneously resistant to the nalidixic acid, ampicillin, penicillin and tetracycline, whereas most isolates showing three marks were resistant to the nalidixic acid, ampicillin and penicillin. Those data suggest the profile resistant of the isolates from the rumen to be related to the resistance pattern of the isolates from the feces, relative to nalidixic acid, ampicillin, erythromycin, penicillin and tetracycline. The values of the genetic distance for rumen isolates ranged from 58% to 100%, therefore indicating high genetic diversity among those isolates. The values of the genetic distance among the feces isolates ranged from 16% to 96%, so indicating a high genetic diversity among isolates, as being those values lower, compared to rumen isolates. / Neste trabalho, foram isoladas 97 bactérias do rúmen e 87 bactérias das fezes de três bovinos, alimentados com ração (24% proteína) e silagem de milho. A resistência dos isolados bacterianos aos antibióticos foi avaliada, por meio do método de diluição em agar, utilizando-se os seguintes antimicrobianos: ácido nalidíxico (NAL), ampicilina (AMP), cloranfenicol (CHL), eritromicina (ERY), estreptomicina (STR), penicilina (PEN) e tetraciclina (TET). A diversidade genética de 29 isolados do rúmen e 28 isolados das fezes foi avaliada, por meio da técnica de polimorfismo de DNA amplificado ao acaso (RAPD). Dentre as bactérias isoladas do rúmen, aproximadamente cinqüenta e sete por cento foram obtidas do animal 1, treze por cento obtidas do animal 2 e vinte e nove por cento foram obtidas do animal 3.Os percentuais de isolados, obtidos das fezes, foram 62,1, 10,3 e 27,6 para os animais 1, 2 e 3, respectivamente. Considerando todos os isolados do rúmen, os percentuais de isolados resistentes aos antibióticos foram: NAL 100 %, AMP 59,8 %, CHL 3,1 %, ERY 21,6 %, STR 10,3 %, PEN 97,9 % e TET 78,3 %. Para todos os isolados das fezes, os percentuais de isolados resistentes foram: NAL 100 %, AMP 54,0 %, ERY 20,7 %, STR 2,3 %, PEN 96,5 % e TET 32,2 %. Nenhum isolado do rúmen ou das fezes foi, simultaneamente, resistente aos sete antibióticos. Os isolados do rúmen, que apresentaram maior número de marcas de resistência, foram simultaneamente resistentes a seis antibióticos, enquanto os obtidos das fezes foram simultaneamente resistentes a cinco antibióticos. Entre os isolados do rúmen, que apresentaram resistência a cinco ou seis antibióticos, nenhum foi resistente ao cloranfenicol. A maioria dos isolados, que apresentaram quatro marcas, foram simultaneamente resistentes ao ácido nalidíxico, à ampicilina, à penicilina e à tetraciclina, enquanto a maioria dos isolados que apresentaram três marcas de resistência foram resistentes ao ácido nalidíxico, à ampicilina e à penicilina. Os dados obtidos indicam uma provável relação entre o perfil de resistência das bactérias do rúmen e perfil de resistência das bactérias das fezes, quanto aos antibióticos: ácido nalidíxico, ampicilina, eritromicina, penicilina e tetraciclina. Os valores de distância genética para os isolados do rúmen variaram de 58% a 100 %, indicando grande diversidade genética entre esses isolados. Os valores de distância genética entre os isolados das fezes variaram de 16 % a 96 %, indicando grande diversidade genética entre os isolados, sendo estes valores menores em comparação com os isolados do rúmen.

Orientador: Alexandre Amstalden Moraes Sampaio / Banca: Atushi Sugohara / Banca: Rymer Ramiz Tullio / Resumo: O ensaio foi conduzido na FCAV/ Unesp, utilizando 16 bovinos machos não castrados da raça Canchim, com peso médio inicial de 315 kg. Os animais foram alojados em baias individuais onde receberam os seguintes tratamentos: SF - concentrado farelado com farelo de soja; SE - concentrado extrusado com farelo de soja; AF - concentrado farelado com farelo de algodão; AE - concentrado extrusado com farelo de algodão. Como volumoso ofereceu-se a silagem de milho numa relação volumoso:concentrado de 50:50 com base na matéria seca. O período experimental foi de 112 dias. Os resultados foram avaliados num delineamento inteiramente casualizado, segundo esquema fatorial 2 x 2 (fontes protéicas x tratamentos físicos). As médias de ganho de peso, ganho em área de olho de lombo e ganho em espessura de gordura de cobertura, para os tratamentos SF, SE, AF e AE foram 1,64; 1,46; 1,35 e 1,35 kg; 20,24; 18,72; 22,88 e 16,21 cm2; 0,67; 0,58; 0,50 e 0,93 mm respectivamente, não apresentando diferença (P>0,05) entre qualquer um dos tratamentos. Na avaliação da conversão alimentar e eficiência protéica a análise estatística detectou diferença apenas entre fontes protéicas (P<0,05) com médias de conversão alimentar de 4,73 e 5,31 kg MS/kg PC e eficiência protéica de 1,78 e 1,59 kg PC/kg PB, respectivamente para os tratamentos com farelo de soja e farelo de algodão. As médias de balanço de nutrientes para MS, FDN e FDA para os tratamentos SF, SE, AF e AE foram 66,2; 66,7; 64,4 e 64,7%; 47,4; 43,3; 45,8 e 38,47% e 51,3; 45,2; 49,0 e 40,0% respectivamente, não apresentando diferença (P>0,05) entre os tratamentos ...(Resumo completo, clicar no acesso eletrônico abaixo) / Abstract: - The present work was carried out in Beef Cattle Setor of FCA V/Unesp, being used 16 males bovines non castrated of the Canchim crossbreed with initial average body weight of 315 kg. The animais were housed in individual stalls where they received the following treatments: GS - grounded concentrate with soybean meal; ES - extruded concentrate with soybean meal; GC - grounded concentrate with cottonseed meal; EC - extruded concentrated with cottonseed meal. The exclusive houghage offered was the com silage in a relationship of 50:50 in the dry matter basis. The experimental period was 112 days, sub-divided in four sub-periods of 28 days, which it was evaluated the variations of the body weight, loin eye area and the backfat. For the data statistical treatment, the arrangement used was the total randomized design, in a factorial scheme 2 x 2 (two protein sources x two physical treatments), being the averages compared by the Tukey test. The averages of daily weight gain, loin eye area and backfat gains, for the treatments GS, ES, GC and EC were 1.64, 1.46, 1.35 and 1.35 kg; 20.24, 18.72, 22.88 and 16.21 cm2; 0.67, 0.58, 0.50 and 0.93 mm respectively, not presenting difference (P>0,05) among the treatments. In the evaluation of the feed conversion the statistical analysis detected differences among protein sources (P<0,05) with averages of 4.73 and 5.31 kg DM/kg BW respectively for the treatments with soybean meal and cottonseed meal. The averages of DMB, NDFB and ADFB for the treatments GS, ES, GC and EC were 66.16, 66.66, 64.43 and 64.72%; 47.36, 43.30, 45.81 and 38.47% and 51.30, 45.24, 49.03 and 39.98% respectively, not presenting difference (P>0,05) among treatments ... (Complete abstract, click eletronic address below) / Mestre

Made available in DSpace on 2014-06-11T19:30:16Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-03-03Bitstream added on 2014-06-13T18:40:15Z : No. of bitstreams: 1 ribeiro_gm_me_jabo.pdf: 848451 bytes, checksum: 7e0a8600342eb2f0539ebbb975c83975 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / O ensaio foi conduzido na FCAV/ Unesp, utilizando 16 bovinos machos não castrados da raça Canchim, com peso médio inicial de 315 kg. Os animais foram alojados em baias individuais onde receberam os seguintes tratamentos: SF - concentrado farelado com farelo de soja; SE - concentrado extrusado com farelo de soja; AF - concentrado farelado com farelo de algodão; AE - concentrado extrusado com farelo de algodão. Como volumoso ofereceu-se a silagem de milho numa relação volumoso:concentrado de 50:50 com base na matéria seca. O período experimental foi de 112 dias. Os resultados foram avaliados num delineamento inteiramente casualizado, segundo esquema fatorial 2 x 2 (fontes protéicas x tratamentos físicos). As médias de ganho de peso, ganho em área de olho de lombo e ganho em espessura de gordura de cobertura, para os tratamentos SF, SE, AF e AE foram 1,64; 1,46; 1,35 e 1,35 kg; 20,24; 18,72; 22,88 e 16,21 cm2; 0,67; 0,58; 0,50 e 0,93 mm respectivamente, não apresentando diferença (P>0,05) entre qualquer um dos tratamentos. Na avaliação da conversão alimentar e eficiência protéica a análise estatística detectou diferença apenas entre fontes protéicas (P<0,05) com médias de conversão alimentar de 4,73 e 5,31 kg MS/kg PC e eficiência protéica de 1,78 e 1,59 kg PC/kg PB, respectivamente para os tratamentos com farelo de soja e farelo de algodão. As médias de balanço de nutrientes para MS, FDN e FDA para os tratamentos SF, SE, AF e AE foram 66,2; 66,7; 64,4 e 64,7%; 47,4; 43,3; 45,8 e 38,47% e 51,3; 45,2; 49,0 e 40,0% respectivamente, não apresentando diferença (P>0,05) entre os tratamentos... / The present work was carried out in Beef Cattle Setor of FCA V/Unesp, being used 16 males bovines non castrated of the Canchim crossbreed with initial average body weight of 315 kg. The animais were housed in individual stalls where they received the following treatments: GS - grounded concentrate with soybean meal; ES - extruded concentrate with soybean meal; GC - grounded concentrate with cottonseed meal; EC - extruded concentrated with cottonseed meal. The exclusive houghage offered was the com silage in a relationship of 50:50 in the dry matter basis. The experimental period was 112 days, sub-divided in four sub-periods of 28 days, which it was evaluated the variations of the body weight, loin eye area and the backfat. For the data statistical treatment, the arrangement used was the total randomized design, in a factorial scheme 2 x 2 (two protein sources x two physical treatments), being the averages compared by the Tukey test. The averages of daily weight gain, loin eye area and backfat gains, for the treatments GS, ES, GC and EC were 1.64, 1.46, 1.35 and 1.35 kg; 20.24, 18.72, 22.88 and 16.21 cm2; 0.67, 0.58, 0.50 and 0.93 mm respectively, not presenting difference (P>0,05) among the treatments. In the evaluation of the feed conversion the statistical analysis detected differences among protein sources (P<0,05) with averages of 4.73 and 5.31 kg DM/kg BW respectively for the treatments with soybean meal and cottonseed meal. The averages of DMB, NDFB and ADFB for the treatments GS, ES, GC and EC were 66.16, 66.66, 64.43 and 64.72%; 47.36, 43.30, 45.81 and 38.47% and 51.30, 45.24, 49.03 and 39.98% respectively, not presenting difference (P>0,05) among treatments ... (Complete abstract, click eletronic address below)

Submitted in partial fulfillment for the Degree of Master of Applied Sciences in Biotechnology, Durban University of Technology, Durban, South Africa, 2017. / The study aims to assess and compare the concentration of microbial contaminants, their sources and distribution in surface water and sediment, and to determine the impact of seasonal variations and corresponding risks of faecal contamination using conventional and molecular methods. Historical data analysis was conducted using E. coli values from the eThekwini Water and Sanitation (EWS) department for 66 months (2009-2014). E. coli and Enterococci were analysed in surface water and sediment samples using the mFC/ spread plate and Colilert-18 (IDEXX) methods. The impact of seasonal variations was assessed using E. coli and Enterococci data collected during rainfall and no rainfall events, using an auto-sampler and sediment trap in parallel. Conventional standard membrane filtration methods using mFC agar, Slanetz & Bartley/ Bile Esculin and Brilliance E. coli selective agar were compared to the enzymatic Colilert-18 and Enterolert (IDEXX) test methods along the Isipingo and Palmiet Rivers. In addition, comparison of the analytical performance of droplet digital PCR (ddPCR) and qPCR for the detection of Salmonella targeting ttr gene in river sediment samples collected from the four sites of the Palmiet River in Durban, South Africa was done. In order to assess the public health risk associated with exposure of men, women and children to microbial pathogens in polluted surface water during recreational activities, the QMRA tool was employed in relation to the risk exposure to pathogenic E. coli, Campylobacter, Salmonella and Shigella. Also, the risk associated with crop irrigation (on farmers) as well as the consumption of crops irrigated with surface water from the Isipingo river was determined. Analysis of the historical data gave a baseline of the two rivers of interest, thus helps understand the current situation of the rivers enabling researchers to pick up potential gaps. In this study after the analysis of the historical data it was evident that at the Palmiet river, microbial analysis must be conducted around the QRI settlements which is a major pollution source. Also, from this study it was found that sampling points situated close to wastewater treatment plants, pump stations or informal settlements were of major concern, thus were considered for the study. It was found that sediment exhibited higher microbial concentrations than surface water, which was observed in both rivers. Also, rainfall had a significant impact on microbial variability. Higher microbial concentrations (indicator organisms) were observed in surface water after a heavy rainfall as appose to when there was no rainfall. This was due to contamination that is washed off into the river and sediment resuspension. Methodology comparison revealed that Colilert-18 and Brilliance E. coli were more selective compared to mFC agar. Brilliance E. coli /Coliform agar was comparable with Colilert-18 IDEXX, which was also observed with Slanetz & Bartley and Enterolert IDEXX. However, when mFC agar was compared with Colilert-18 IDEXX, significant difference was observed. In comparison of two Molecular methods, ddPCR were found to be fully amenable for the quantification of Salmonella and offer robust, accurate, high-throughput, affordable and more sensitive quantitation than qPCR in complex environmental samples like sediments. Quantitative Microbial Risk Assessment (QMRA) relating to recreational and occupational exposure showed that children were at the highest risk of getting infected. Also, it was observed that the probability of infection upon exposure to surface water from the Isipingo and Palmiet rivers was significantly high, hence exceeded the WHO guidelines values. Risk assessment on crops revealed that pathogenic bacteria may pose a risk to the consumer, however, a 9-log reduction may be achieved according to the WHO multi-barrier approach which involves proper washing and proper cooking of the crop before ingestion. Overall the sampling points that had the highest pollution level and constantly exceeded the WHO and DWAF guidelines at the Isipingo river were the points situated and named “Next to the WWTP”, and “Downstream of QRI” at the Palmiet River. / M

La tuberculose bovine est une maladie infectieuse contagieuse causée par Mycobacterium bovis. Cette maladie touche les bovins et de nombreuses espèces de mammifères domestiques et sauvages, ainsi que l’homme. La circulation de la bactérie dans des systèmes multi-hôtes variés favorise l’entretien de la maladie et la contamination des bovins vivant à proximité des animaux sauvages infectés. En marge de la transmission directe de M. bovis par voie respiratoire, la transmission indirecte aux bovins, liée à l’inhalation ou à l’ingestion de matrices environnementales contaminées par un animal infecté excréteur, est suspectée dans plusieurs régions du monde. L’existence de réservoirs environnementaux où le bacille M. bovis est capable de persister, pourrait donc être un facteur important de la réémergence puis du maintien de la maladie dans les systèmes multi-hôtes.En Côte d’Or, département fortement touché par la tuberculose bovine depuis 2004, la transmission indirecte de la bactérie entre la faune sauvage infectée et les bovins est suspectée dans plusieurs élevages. Pour évaluer la présence et la survie de cette bactérie dans l’environnement, nous avons analysé un grand nombre d’échantillons prélevés dans des zones partagées par les bovins et/ou la faune sauvage infectés dans le but de déterminer la distribution environnementale de M. bovis. Pour ce faire, nous avons développé ou modifié des systèmes de détection moléculaire adaptés aux matrices environnementales complexes. Nous avons également évalué l’impact de la température et des propriétés physico-chimiques de deux sols sur la survie de M. bovis, ainsi que le rôle de la mésofaune du sol (lombrics en particulier) dans la dissémination de la bactérie à partir de matière organique contaminée. L’étude environnementale a mis plus particulièrement en évidence la contamination de deux biotopes: les zones humides des pâtures et les sols de terriers de blaireaux. De plus, les études expérimentales ont montré que M. bovis pouvait survivre plusieurs mois dans le sol à 4°C et que les lombrics pouvaient disséminer la bactérie dans le sol, voire jouer un rôle potentiel de vecteur pour les animaux qui les consomment. Ces résultats apportent de nouvelles connaissances sur la persistance et la circulation de M. bovis dans l’environnement en Côte d’Or et permettront de proposer des améliorations aux mesures de biosécurité déjà existantes dans les élevages bovins. / Bovine tuberculosis is an infectious disease caused by Mycobacterium bovis. This disease affects cattle, and many species of domestic and wild mammals, and humans. The circulation of the bacteria in various multi-host systems promotes the maintenance of the disease and the contamination of cattle in the vicinity. Beside direct transmission of the bacteria through the respiratory route, indirect transmission, through inhalation or ingestion of environmental matrices contaminated by an infected animal excretory, is suspected in several countries. Environmental contamination with M. bovis appears to be a crucial factor in the persistence of the infection in multi-host systems.In Côte d'Or, a French department affected by bovine tuberculosis since 2004, the indirect transmission of the bacteria from infected wildlife to cattle is suspected in several cases. To assess this type of transmission of the bacillus, we evaluated the environmental contamination with M. bovis on a large number of samples taken in areas shared by cattle and / or wildlife infected. For this purpose, we developed or modified molecular detection systems adapted for environmental complex matrices. We also assessed the impact of physicochemical properties of both soil and temperature on survival of M. bovis and the role of earthworms in the spread of the bacteria from contaminated organic material. The environmental study showed the contamination of two media in particular: wetlands pastures and soil badger setts. Moreover, experimental studies have shown that M. bovis can survive in soil for several months at 4 ° C and the worms could spread the bacteria in the soil, or even play a potential role for vector animals that consume them. These results will propose improvements to existing biosecurity measures on cattle farms and provide new knowledge about the persistence and circulation of M. bovis in the environment in Côte d'Or.

INTRODUÇÃO: A microbiota intestinal tem sido alvo de diversos estudos moleculares, principalmente através da introdução de plataformas de sequenciamento de nova geração, devido à sua importância e amplo relacionamento com o hospedeiro humano. Entretanto, o armazenamento de amostras fecais antes da extração do DNA é crítico ao caracterizar a composição da microbiota intestinal. Com base nesses dados, o presente estudo buscou compreender os efeitos de diferentes métodos de armazenamento de amostras fecais para caracterizar a microbiota intestinal através do sequenciamento da nova geração, bem como estabelecer um método alternativo de conservação do material genético bacteriano nessas amostras, utilizando guanidina. MÉTODO: Foram coletadas amostras de fezes de 10 voluntários saudáveis. Cada amostra foi dividida em cinco alíquotas, uma alíquota extraída imediatamente após a coleta (fresca) e duas alíquotas submetidas ao congelamento, à temperaturas de -20°C e -80°C e extraídas após 48 horas. As outras duas alíquotas restantes foram armazenadas em guanidina à temperatura ambiente e a 4°C e extraídas após 48 horas. Para observar a presença de alterações na microbiota intestinal, durante um período de armazenamento maior das amostras de fezes, três amostras foram armazenadas em guanidina à temperatura ambiente e a 4ºC e extraídas após o período de 60 dias. A região hipervariável v4 do gene 16S rRNA bacteriano foi amplificada por PCR. Os amplicons gerados foram sequenciados utilizando a plataforma Ion PGM Torrent e os dados analisados utilizando o software QIIME. A determinação da significância estatística foi realizada utilizando-se o teste não-paramétrico de Kruskal-Wallis. RESULTADOS: Não foram encontradas diferenças significativas em nenhum dos níveis taxonômicos (filo, classe, família, ordem e gênero) entre amostras frescas analisadas e os métodos de armazenamento testados. As análises de coordenadas principais (PCoA) mostraram que as amostras se agruparam de acordo com os indivíduos analisados, tendo as amostras referentes a cada indivíduo agrupado-se com maior proximidade do que com outras amostras do mesmo grupo de armazenamento. CONCLUSÃO: Nossos dados sugerem que o congelamento e o uso de guanidina para armazenamento de amostras de fezes, para a caracterização da microbiota intestinal, podem efetivamente preservar o material genético bacteriano nessas amostras ao longo de um período de 48 horas para amostras submetidas ao congelamento e durante 60 dias para amostras armazenadas em guanidina / INTRODUCTION: The gut microbiota has been the target of several molecular studies, mainly through the introduction of next generation sequencing platforms, due to its importance and wide relationship with the human host. However, the storage of fecal samples prior to DNA extraction is critical when characterizing the composition of the intestinal microbiota. Based on these facts, the present study aimed to understand the effects of different methods of storage of fecal samples to characterize the intestinal microbiota by next generation sequence, as well as establishing an alternative conservation method of the bacterial genetic material in these samples using guanidine. METHODS: Stool samples from 10 healthy volunteers were collected. Each collected sample was divided into five aliquots, one aliquot extracted immediately after collection (fresh) and two aliquots subjected to freezing at -20°C and -80°C temperatures and extracted after 48 hours. The others two remaining aliquots were stored in guanidine at room temperature and at 4°C and extracted after 48 hours. In order to observe the presence of alterations in the intestinal microbiota, during a longer storage period of the stool samples, three samples were stored in guanidine at room temperature and at 4°C and extracted after 60 day period. The v4 hypervariable region of bacterial and archeal 16S rRNA gene were amplified by PCR. The generated amplicons were sequenced using Ion PGM Torrent platform and the data analyzed using the software QIIME. Determination of statistical significance was performed using non-parametric Kruskal-Wallis test. RESULTS: No significant differences were found in any of the taxonomic levels (phylum, class, family, order and genus) between analyzed fresh samples and the others different storage methods. The principal coordinates analysis (PCoA) unweighted showed that the samples clustered based on the host each sample originated from, rather than by storage group. CONCLUSION: Our data suggest that both freezing and the use of guanidine to store stool samples for gut microbiota characterization can effectively preserve the bacterial genetic material in these samples over a 48 hours period for samples subjected to freezing and for up to 60 days for samples stored in guanidine

INTRODUÇÃO: A microbiota intestinal tem sido alvo de diversos estudos moleculares, principalmente através da introdução de plataformas de sequenciamento de nova geração, devido à sua importância e amplo relacionamento com o hospedeiro humano. Entretanto, o armazenamento de amostras fecais antes da extração do DNA é crítico ao caracterizar a composição da microbiota intestinal. Com base nesses dados, o presente estudo buscou compreender os efeitos de diferentes métodos de armazenamento de amostras fecais para caracterizar a microbiota intestinal através do sequenciamento da nova geração, bem como estabelecer um método alternativo de conservação do material genético bacteriano nessas amostras, utilizando guanidina. MÉTODO: Foram coletadas amostras de fezes de 10 voluntários saudáveis. Cada amostra foi dividida em cinco alíquotas, uma alíquota extraída imediatamente após a coleta (fresca) e duas alíquotas submetidas ao congelamento, à temperaturas de -20°C e -80°C e extraídas após 48 horas. As outras duas alíquotas restantes foram armazenadas em guanidina à temperatura ambiente e a 4°C e extraídas após 48 horas. Para observar a presença de alterações na microbiota intestinal, durante um período de armazenamento maior das amostras de fezes, três amostras foram armazenadas em guanidina à temperatura ambiente e a 4ºC e extraídas após o período de 60 dias. A região hipervariável v4 do gene 16S rRNA bacteriano foi amplificada por PCR. Os amplicons gerados foram sequenciados utilizando a plataforma Ion PGM Torrent e os dados analisados utilizando o software QIIME. A determinação da significância estatística foi realizada utilizando-se o teste não-paramétrico de Kruskal-Wallis. RESULTADOS: Não foram encontradas diferenças significativas em nenhum dos níveis taxonômicos (filo, classe, família, ordem e gênero) entre amostras frescas analisadas e os métodos de armazenamento testados. As análises de coordenadas principais (PCoA) mostraram que as amostras se agruparam de acordo com os indivíduos analisados, tendo as amostras referentes a cada indivíduo agrupado-se com maior proximidade do que com outras amostras do mesmo grupo de armazenamento. CONCLUSÃO: Nossos dados sugerem que o congelamento e o uso de guanidina para armazenamento de amostras de fezes, para a caracterização da microbiota intestinal, podem efetivamente preservar o material genético bacteriano nessas amostras ao longo de um período de 48 horas para amostras submetidas ao congelamento e durante 60 dias para amostras armazenadas em guanidina / INTRODUCTION: The gut microbiota has been the target of several molecular studies, mainly through the introduction of next generation sequencing platforms, due to its importance and wide relationship with the human host. However, the storage of fecal samples prior to DNA extraction is critical when characterizing the composition of the intestinal microbiota. Based on these facts, the present study aimed to understand the effects of different methods of storage of fecal samples to characterize the intestinal microbiota by next generation sequence, as well as establishing an alternative conservation method of the bacterial genetic material in these samples using guanidine. METHODS: Stool samples from 10 healthy volunteers were collected. Each collected sample was divided into five aliquots, one aliquot extracted immediately after collection (fresh) and two aliquots subjected to freezing at -20°C and -80°C temperatures and extracted after 48 hours. The others two remaining aliquots were stored in guanidine at room temperature and at 4°C and extracted after 48 hours. In order to observe the presence of alterations in the intestinal microbiota, during a longer storage period of the stool samples, three samples were stored in guanidine at room temperature and at 4°C and extracted after 60 day period. The v4 hypervariable region of bacterial and archeal 16S rRNA gene were amplified by PCR. The generated amplicons were sequenced using Ion PGM Torrent platform and the data analyzed using the software QIIME. Determination of statistical significance was performed using non-parametric Kruskal-Wallis test. RESULTS: No significant differences were found in any of the taxonomic levels (phylum, class, family, order and genus) between analyzed fresh samples and the others different storage methods. The principal coordinates analysis (PCoA) unweighted showed that the samples clustered based on the host each sample originated from, rather than by storage group. CONCLUSION: Our data suggest that both freezing and the use of guanidine to store stool samples for gut microbiota characterization can effectively preserve the bacterial genetic material in these samples over a 48 hours period for samples subjected to freezing and for up to 60 days for samples stored in guanidine

L'âge au sevrage est un trait d'histoire de vie qui affecte le succès reproductif des femelles. Sa détermination à partir d'observations de la tétée est limitée en raison de l'allaitement de confort ou de nuit. Le suivi de l'alimentation des nourrissons, à partir des isotopes stables de carbone et d'azote fécaux (δ13C, δ15N %N) est une alternative précise et non invasive aux méthodes comportementales. Les âges de sevrage chez le colobe magistrat (Colobus vellerosus) à BFMS, Ghana, ont été déterminés en utilisant les δ13C et δ15N fécaux, et ceux-ci ont été comparés aux évaluations comportementales du sevrage. Les différences d'âge au sevrage entre trois groupes de colobes différents ont également été comparées. Des échantillons fécaux ont été collectés auprès de 8 dyades de mères (N = 88 fèces) et de leurs enfants (N = 98 fèces). Les échantillons ont été homogénéisés et analysés dans un spectromètre de masse à rapport isotopique et un analyseur élémentaire. L'âge moyen du sevrage chez tous les nourrissons ayant utilisé des isotopes stables fécaux était de 15,75 mois, ce qui était supérieur à l'âge moyen du sevrage déterminé à partir des observations de l'allaitement (14,6 mois). Deux nourrissons ont été sevrés avant le début de la collecte des données fécales, deux avaient un âge isotopique au sevrage similaire à leur âge de sevrage comportemental, et deux avaient un âge isotopique au sevrage supérieur à leur âge comportemental. Deux nourrissons dont on a déterminé qu'ils n'étaient pas encore sevrés d'après les évaluations isotopiques n'ont pas été observés en train de téter et ont montré des différences δ15N nourrisson-mère alternativement plus grandes et plus petites entre 6 et 9 mois. Cela peut indiquer un processus de sevrage cyclique, les nourrissons devenant plus ou moins dépendants du lait au cours de la période de 4 mois. Il semblait y avoir des différences dans les âges moyens de sevrage isotopique entre les groupes. Mon étude a montré que les isotopes stables fécaux peuvent être utilisés avec succès pour surveiller le développement nutritionnel des nourrissons et les différences de niveau trophique entre le nourrisson et la mère chez les singes colobes arboricoles. / Age at weaning is a life-history trait that affects the reproductive success of females. Its determination from observations of suckling is limited due to comfort and night nursing. To monitor infant diets, fecal stable carbon, and nitrogen isotopes (δ13C, δ15N %N) provide an accurate and non-invasive alternative to behavioral methods. Weaning ages in ursine colobus (Colobus vellerosus) at BFMS, Ghana was determined using fecal δ13C and δ15N, and these were compared to behavioral weaning assessments. I also compared differences in weaning ages between three different colobus groups. Fecal samples were collected from 8 dyads of mothers (N = 88 feces) and their infants (N = 98 feces). The samples were homogenized and analyzed in an isotope ratio mass spectrometer and elemental analyzer. The mean weaning age among all infants using fecal stable isotopes was 15.75 months, which was older than the mean weaning age determined from observations of nursing (14.6 months). Two infants were weaned before fecal data collection began, two had an isotopic age at weaning similar to their behavioral weaning age, and two had an isotopic age at weaning that was older than their behavioral age. Two infants who were determined to be not yet weaned from isotopic assessments were not observed to nurse and showed alternately larger and smaller δ15N infant-mother differences between 6 and 9 months. This may indicate a cyclic weaning process, with infants becoming more or less dependent on milk over the 4-month period. There appeared to be differences in the average isotopic weaning ages between groups. My study showed that fecal stable isotopes can be successfully used to monitor infant nutritional development and infant-mother trophic level differences in arboreal colobus monkeys.

In off-grid holiday homes, alternative toilet solutions are needed. There is a wide range of dry toilet systems, where urine-diverting systems and incineration toilets are common solutions. Urine-diverting dry toilets require that users need to manage generated fecal fractions. This is often done by private composting or through municipal latrine bucket pick-ups. In this project, fecal management for holiday homes in Sweden is examined from three perspectives by (1) studying the biological phenomenon with composting and how a compost should be managed to generate rich humus, whilst minimizing greenhouse gas emissions associated with the act of composting, (2) examine current user experiences associated with latrine compost management, and (3) map out current latrine management systems, including laws and regulations. The goal was to develop a user-friendly concept for fecal management for urine-diverting toilets, based on this research. The project was carried out in collaboration with Harvest Moon, a company focused on the development of innovative and refined dry toilet systems. The project was initiated with a literature review, the examination of current fecal management systems, and interviews with composting experts and researchers. The background research showed that there is no such thing as perfect compost management since it depends on what end goals the users have. Research also showed that frequently turning the pile, increases ammonia (NH3) emissions, but reduces methane (CH4) formation. Furthermore, biochar can be added as a bulking agent to aerate the compost mass, and aid the hygienization process of such a mass, since it binds e.g., hormones. Regarding composting methods, a static passively aerated compost is not the fastest process but has the least compost mass reduction, which is desired when using it as a soil enhancer. In addition, it requires the least management. This method was therefore chosen for further development. Furthermore, since temperature and moisture are easily measured with sensors, it was deemed interesting to implement such sensors in a final concept, to alleviate management for the user. To assure that the final concept would reflect user needs and wishes, a phase of user studies was then initiated. The studies showed that users generally see latrine compost more as something to take care of, rather than as a resource, and therefore have no interest in using composted humus. The research also showed that because many municipalities require 2-year storage of the material in the composter, users experienced scheduling issues, which ultimately led to an inefficien composting system. The third perspective that was investigated, was the management systems of today and how they are regulated by laws. Each municipality has its own requirements on how latrine composts should be managed. These requirements are based on the Environmental Code, as well as Naturvårdsverket’s recommendation for the implementation of the law. Apart from the compilation of these regulations, this investigation showed that pyrolysis, as well as the centralization of hygienized feces, could be future alternatives to latrine composting and latrine pickup. But due to the short Time-to-Market, and the project's limited time scope, product development towards system innovation was deemed unrealistic within this project.  Insights from these three perspectives created a framework for the concept development phase, which was finalized with building a full-scale functional prototype. During detailed design, the concept was further developed in CAD. The final concept presented in this project is a modular, user-friendly latrine compost that can be adapted to follow different municipal regulations. It has an inner mesh that aerates the compost mass to reduce methane gas formation. The mesh is constructed with hexagonal perforated acid-proof steel. The composter has a push latch mechanism on the lid together with two gas struts, which makes it easy to open since the user only needs to push the lid once, for it to open. Temperature and moisture sensors make it easier for the user to manage their compost correctly, and a front door allows for ergonomic emptying of the finished compost humus. Future development to reduce production costs, simplify the construction, continue the CAD model development, find suitable sensors, develop product instructions as well as perform user tests with the physical prototype should be further investigated. / I fritidshus som saknar kommunalt avlopp behövs alternativa toalettlösningar. Det finns en rad olika torra toalettsystem, där urinsorterande och förbränningstoaletter är vanliga lösningar. Urinsorterande torrtoaletter kräver att användaren själv tar hand om genererade fekalier. Detta görs genom antingen privat latrinkompostering eller kommunal hämtning av latrin. I detta projekt undersöks fekaliehantering för fritidshus i Sverige från tre perspektiv genom att (1) studera biologiska fenomen i en kompost och hur en kompost ska hanteras för att få en rik humus, samt minimera växthusgasutsläpp som bildas vid just kompostering, (2) undersöka användarupplevelsen vid hantering av latrinkomposter idag, samt (3) kartlägga nuvarande system för latrinhantering, inklusive rådande lagar och förordningar. Målet med projektet var att utveckla ett användarvänligt koncept för fekaliehantering från urinsorterande toaletter, baserat på denna forskning. Projektet genomfördes i samarbete med Harvest Moon, ett företag som arbetar med att utveckla innovativa och estetiskt tilltalande torrtoalettsystem. Projektet inleddes med att studera litteratur, undersöka befintliga fekaliehanteringssystem, samt att intervjua komposteringsexperter och forskare inom området. Efter denna bakgrundsforskning kunde slutsatsen dras att det inte finns en perfekt komposthantering, då det beror på de mål som användaren har. Forskningen visade också att ju mer man vände kompostmassan, desto mer ökade utsläppen av ammoniak (NH3), dock minskade bildningen av metan (CH4). En annan insikt var att biokol kan tillsättas för att lufta, samt hjälpa till att hygienisera kompostmassan, då det binder till sig till exempel hormonrester. Gällande komposteringsmetoder så är en statisk passivt luftad kompost inte den snabbaste processen, men den leder till minst kompostreduktionen, vilket är bra om målet är att använda det komposterade materialet som jordförbättrare. Dessutom kräver det den minsta hanteringen av användaren. Denna metod valdes därför för vidareutveckling av slutkoncept. Slutligen, eftersom både temperatur och fukt lätt går att mäta med sensorer, så ansågs det intressant att implementera dessa typer av sensorer i ett slutkoncept i och med att det skulle kunna underlätta hanteringsprocessen för användaren. För att säkerställa att det slutliga konceptet skulle återspegla vad användarna behöver och önskar, inleddes sedan en fas av användarstudier. Användarstudierna visade att användare i allmänhet ser latrinkompost mer som något de måste ta hand om, än som en resurs, och har därmed inget intresse av att ta vara på det materialet som har komposterats. Studierna visade också att på grund av att många kommuner kräver två års lagring av materialet i fekaliekomposten, upplevde användare svårigheter med att få till en bra rutin kring hanteringen, vilket bidrog till att hela latrinhanteringssystemet fungerade sämre. Det tredje perspektivet som undersöktes inom projektet var hur systemen för latrinhantering ser ut idag, samt hur och av vilka lagar de regleras. Varje kommun har egna krav på hur en latrinkompost ska hanteras. Dessa krav är baserade på Miljöbalken, liksom Naturvårdsverkets rekommendationer gällande den praktiska implementeringen av Miljöbalken. Utöver sammanställningen av dessa regelverk visade studien att pyrolys, alternativt centralisering av hygieniserade fekalier, skulle kunna vara framtida alternativ till kompostering eller kommunal hämtning av latrin. Men på grund av den korta Time-to-Market för denna produkt, samt projektets begränsade tidsram ansågs produktutveckling mot systeminnovation vara orealistisk inom detta projekt. Insikter från dessa tre perspektiv bildade ett ramverk för konceptutvecklingsfasen, som avslutades med byggandet av en fullskalig funktionsprototyp. Under detaljutvecklingen fortsattes utvecklingen av konceptet i CAD. Det slutgiltiga konceptet som presenteras i projektet är en modulär, användarvänlig latrinkompost som går att anpassa till att följa olika kommunala regler. Den har en inre struktur som luftar kompostmaterialet för att minska bildandet av metangas. Strukturen består av hexagonalt perforerat syrafast stål. Komposten har en push latch mekanism på locket tillsammans med två gasfjädrar, vilket gör det enkelt att öppna eftersom användaren endast behöver trycka locket för att det ska öppnas. Temperatur- och fuktsensorer gör det lättare för användaren att hantera sin kompost korrekt, och en främre dörr möjliggör ergonomisk tömning av färdigt kompostmaterial. Framtida utveckling för att minska produktionskostnaderna, förenkla konstruktionen, vidareutveckla CAD-modellen, hitta lämpliga sensorer, utveckla användarinstruktioner, samt genomförandet av användartester med funktionsprototypen bör vidare undersökas.

Para obter resultados rápidos e confiáveis que permitam o monitoramento da segurança microbiológica de alimentos, seja pela indústria ou pelos órgãos de fiscalização, diversos métodos alternativos têm sido desenvolvidos para a detecção e quantificação de Salmonella. Os propósitos do estudo foram avaliar a viabilidade de emprego do QIAamp® DNA Stool Mini Kit para extração e purificação de DNA de Salmonella; validar ensaios baseados em PCR-tempo real (PCR-RT) para quantificar o DNA de Salmonella empregando ttr ou tuf e desenvolver um ensaio para quantificar Salmonella baseado na transcriptase reversa-PCR-tempo real (RT-PCR-RT). Para avaliação do QIAamp® DNA Stool Mini Kit empregaram-se fezes coletadas diretamente do reto de animais infectados ou não, sendo estas últimas artificialmente contaminadas e submetidas à extração segundo protocolo do fabricante. As amostras de DNA isoladas foram quantificadas empregando um ensaio Salmonella-específico PCR-RT utilizando como alvo o lócus ttr. O mesmo ensaio foi utilizado para células de Salmonella provenientes de meio de cultura. O ensaio PCR-RT baseado no alvo tuf foi validado empregando-se primeiramente cepas de diferentes sorotipos de Salmonella e de outras Enterobacteriaceae. A seguir sua eficiência foi avaliada para alimentos-modelo (ave e suíno) artificialmente contaminadas com elevada (&#8776; 6 log UFC/mL) e baixa (&#8776; 2 log UFC/mL) população de Salmonella Typhimurium DT 104. A validação do método quantitativo de Salmonella por RT-PCR-RT foi realizada primeiramente com células em meio de cultura e posteriormente nos mesmos alimentos-modelo utilizados para PCR-RT. Em ambos os métodos, alíquotas dos alimentos-modelo foram mantidas a 20 ºC e a 8 ºC, sendo examinadas em diferentes tempos pós-inoculação. Como controle empregou-se a enumeração de microrganismos mesófilos totais e de Salmonella por técnicas convencionais. A taxa de recuperação de Salmonella em fezes suínas artificialmente inoculadas, após tratamento com QIAamp® DNA Stool Kit, variou entre 25% a 50%, dependendo da quantidade inicial de células. Empregando o DNA extraído e submetendo-o à PCR-RT para o ttr obteve-se limite de detecção de 2,8 log UFC eq/g de fezes; método que foi menos sensível que o convencional. A quantificação de Salmonella por PCR-RT empregando tuf apresentou limite de detecção menor que 1 log UFC eq. Os resultados obtidos com este método, empregando-se células em meio de cultura ou alimentos-modelo, foram, de maneira geral, ligeiramente inferiores aos do método convencional. A eficiência de amplificação para PCR-RT e tuf foi de 94%. O método RT-PCR-RT apresentou limite de detecção semelhante ao obtido com o ttr (2 log UFC eq) e sua eficiência de amplificação foi de 100%. Observou-se que tuf é expresso na fase logarítmica de multiplicação bacteriana, o que o torna um bom indicador da viabilidade de Salmonella. / In order to get fast and trustworthy results that allow monitoring the microbiological food safety either by industries or governmental agencies, diverse alternative methods have been developed for Salmonella detection and quantification. The purposes of this study were to evaluate the viability of the use of QIAamp® DNA Stool Mini Kit for Salmonella DNA extraction and purification; to validate assays based on real time-PCR (PCR-RT) to quantify Salmonella DNA by using ttr or tuf, and to develop an assay to quantify Salmonella based on reverse transcriptase- PCR-real time (RT-PCR-RT). For QIAamp® DNA Stool Mini Kit evaluation feces taken directly from the rectum of infected or health animals were used, with the former being artificially contaminated. Samples were submitted to DNA extraction, according to manufacturers protocol. The isolated DNA were quantified using a Salmonella-specific PCR-RT targeting the ttr locus. The same assay was used for Salmonella cells originated from culture medium. The PCR-RT assay with tuf as target was first validated employing different Salmonella serovars and other Enterobacteriaceae strains. After, its efficiency was evaluated on food-models (chicken and swine) spiked with high (&#8776; 6 log CFU/mL) and low (&#8776; 2 log CFU/mL) Salmonella Typhimurium DT 104 populations. The validation of the quantitative RT-PCR-RT method was first conducted with cells grown in culture medium, and then in the same food-model used for PCR-RT. For both methods aliquots of foodmodels were maintained at 20 ºC and 8 ºC being evaluated at different incubation times. Enumeration of total mesophilic microorganisms and Salmonella based on conventional methods were used as controls. The DNA recovery rate in swine feces artificially inoculated, after QIAamp® DNA Stool Mini Kit treatment, was between 25% to 50% depending the initial amount of cells. Using the extracted DNA and submitting it to PCR-RT for ttr a detection level of 2,8 CFU eq/g of feces was obtained. This method showed lower sensitivity than the conventional. Salmonella quantification by PCR-RT employing tuf showed a detection level lower than 1 log CFU eq. The results obtained with this method and cells suspended in culture medium or in food-model systems were, in general slightly lower that those obtained with the conventional method. The efficiency of amplification for PCR-RT tuf was 94%. Detection limit of RT-PCR-RT was similar to that of ttr (2 log CFU eq) and efficiency of amplification was 100%. tuf was expressed in logarithmic phase of bacteria growth curve showing that it is a good viability indicator for Salmonella.