Orientador: Luiz Francisco Prata / Banca: Ana Maria Centola Vidal Martins / Banca: Angela Cleusa de Fátima Banzatto de Carvalho / Resumo: Este trabalho teve o objetivo de avaliar a eficácia de uma nova ferramenta de gestão da qualidade do leite cru refrigerado, comparativamente à utilização dos testes tradicionais de controle, nas condições da Instrução Normativa 51 (IN 51). Foram utilizadas 374 amostras individuais de leite cru refrigerado (produtor) e 125 amostras de leite cru de conjunto (bocas de tanques de caminhões transportadores, referindo-se a leites desses mesmos produtores). As amostras foram submetidas a duas análises microbiológicas, sendo as individuais submetidas à prova rápida de redução em tubos - o "Tetra-Test", com o objetivo de estimar a carga microbiana do leite e a microbiota predominante, enquanto as amostras de conjunto foram submetidas à contagem padrão em placas (CPP) como método de referência. Paralelamente, buscou-se verificar os efeitos do processo de centrifugação nas características e/ou propriedades da matériaprima industrial durante a execução do processo por Ultra Alta Temperatura (UAT). Foram utilizadas 56 amostras de leite provenientes da linha de processamento, tomadas imediatamente antes e depois da etapa de centrifugação interposta no início do processo, as quais foram submetidas a ambos os testes microbiológicos ("Tetra- Test" e CPP), além da determinação da variabilidade da composição do leite e determinação do índice proteolítico da k-caseína. O "Tetra-test" se mostrou eficaz na avaliação da qualidade microbiológica do leite cru podendo ser utilizado como uma ferramenta de gestão, uma vez que seus resultados se correlacionaram proporcionalmente aos obtidos pela CPP e possibilitaram informações complementares sobre as características da microbiota dominante, oferecendo vantagens sobre os tradicionais testes de redução. Os resultados mostraram que grande... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: This study aimed to evaluate the efficacy of a new tool for quality management of refrigerated raw milk, compared to traditional control tests in accordance to the Brazilian Ministry of Agriculture, Livestock and Food Supply (MAPA) Normative Instruction No. 51. A total of 374 individual samples of refrigerated raw milk (from producers) and 125 samples of bulk raw milk (from milk transport tankers from these same producers) were studied. Samples were submitted to microbiological analyses, being individual samples submitted to rapid reduction in test tubes - the "Tetra-Test", in order to estimate the microbial load of milk and predominant microbiota, while the bulk samples were analyzed by standard plate count as a reference method. At the same time, it was investigated the effects of the centrifugation on the characteristics and/or properties of industrial raw material into the incoming of UHT process. Were analyzed 56 samples of milk from the processing line, obtained immediately before and after centrifugation performed early in the process. Both of them were subjected to microbiological tests ("Tetra-Test" and PCA). In addition it was determined the variability of milk composition and proteolytic rate of k-casein. The "Tetra-test" showed to be effective in assessing the microbiological quality of raw milk and can be used as a management tool, since its results correlated proportionally with those obtained by standard plate count and make possible to obtain more information about predominant... (Complete abstract click electronic access below) / Mestre

Milk quality continues to be a topic of intense debate in the dairy industry, medical and public health communities. Production of maximum quantities of high-quality milk is an important goal of every dairy operation. High-quality milk must contain a low number of somatic cells and low bacteria count, and must be free of human pathogens and antibiotic residues. The objective of this study was to determine the prevalence of E. coli in unpasteurized milk recovered from Middledrift and Fort Hare dairy. In this study 400 milk samples were collected from two commercial farms (Middledrift and Fort Hare) in the Eastern Cape, South Africa, 200 raw milk samples from each farm. Samples were cultured on violet red bile mug-agar (VRB-MUG Agar) and incubated at 37ºC for 24 hours and preliminary identified by Gram stain and catalase test. Isolates that were Gram negative and catalase positive were screened for a marker of E. coli uidA gene using PCR assays. Middledrift dairy farm had 50 (25%) E. coli isolated from raw milk and Fort Hare farm showed 37 (18.5%) E. coli present in the milk samples. The presence of E. coli found in the milk samples points to the fact that fecal contamination was unavoidable and traditional practices are likely to contribute to the contamination of the milk and proliferation of the microorganisms.

Taking precautions to restrain aflatoxin M1 (AFM1) from milk is critical, particularly due to the health and economic impact AFM1 imposes. The predominant post-harvest means of reducing AFM1 in milk includes the addition of sequestering agents to feed to diminish the bioavailability of aflatoxin B1 (AFB1), the parent compound of AFM1 found in contaminated feed. Still, residual AFM1 has been found in the milk. Using sequestering agents added to raw milk, we found that activated carbon was the most effective binder to reduce AFM1 contamination. The combination of 0.75% granular activated carbon (GAC) and a flow rate of 0.4 mL/min to pump contaminated milk through a glass column were chosen as optimum conditions for the removal of AFM1. These conditions obtained a 98.4% reduction of 0.75 ng/mL AFM1 from raw milk. The treated milk was also analyzed to assess the effects of GAC on milk constituents. The results determined that GAC had no significant effect on major nutritive milk constituents: total protein, lactose, minerals, and fat. Additionally, we optimized an extraction method coupled to high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) that minimized matrix effects, lowered the levels of detection, and reduced analysis costs. The optimized extraction method was based on QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe). Results determined 5 mL milk (15°C) with 10 mL acetonitrile, 3200 g centrifugation, and 0.2 μm syringe filter were the optimum conditions for the extraction of 0.5 ng/mL AFM1 from raw milk. The method was validated according to AOAC guidelines. This study reports experimental results on AFM1 remediation from raw bovine milk. The use of GAC for the removal of AFM1 in raw milk has reduced the amount of AFM1 below the FDA action limit and European Union maximum regulatory level. This method could have a global health impact, particularly, for people in developing nations and for infants and children who are more susceptible to the adverse effects of AFM1.

Changing weather patterns, the declining social fabric of rural communities, and economic uncertainty increasingly pose challenges to Vermont communities. The socially and environmentally embedded production practices within sustainable agriculture present a potential solution to these problems. In order to make the most of the potential benefits of these practices society must maximize their adoption. This requires an understanding of both farmer adoption of these practices and consumer perceptions of the resulting food products. This thesis contributes two original articles on sustainable agriculture through the analysis of factors driving both farmer adoption and consumer perceptions of products and practices often thought of as sustainable. The first article seeks to understand farmer adoption of climate change best management practices (CCBMPs). Farmer perceptions of risk and profitability of best management practices (BMPs) are key determinants of adoption, which traditional incentive programs like the Environmental Quality Incentives Program (EQIP) attempt to address by providing financial and technical support. To ensure appropriate price points are offered through these programs, regional price structures must be based upon locally established costs. Thus, this article focuses on the economic cost of implementing and maintaining CCBMPs for twelve diverse farms in Vermont. Specifically, three CCBMPs for Vermont are examined: cover cropping, management intensive rotational grazing (MIRG), and riparian buffer strips. Results of a yearlong farmer based data collection process indicate that the average cost for cover cropping is $129.24/acre, for MIRG is $79.82/acre, and for a tree based riparian buffer strip is $807.33/acre. We conclude that existing incentive payments for cover cropping and MIRG are below costs, likely resulting in under-adoption. The second article reports on a study which seeks to understand the factors influencing Vermont consumer perceptions of raw milk safety. While this article makes no assertion regarding the sustainability of raw milk, an association is established between the motivations for raw milk consumption and sustainable agriculture support. Vermonterâ??s appear to be continuing the trend of consuming raw milk at an increasing rate despite continued declarations from local and national public health officials that raw milk is too microbiologically dangerous to justify its consumption. Thus this study was designed to increase understanding of the factors driving consumer perceptions of raw milk safety. A conceptual model was developed to establish potential factors and related questions were incorporated into the 2014 Vermonter Poll. Resulting data were analyzed using a Probit regression analysis. We conclude that observable factors have the greatest influence on perceptions of raw milk safety. Specifically, perceived health benefits, presence of children in the household, and taste all influence perceptions of raw milk safety.

This dissertation examined the microbial ecology of Listeria monocytogenes in three distinctly different environments: a cheese microbial community; a farm environment; and a soil microbial community. The aim of the first study was to investigate the effects of L. monocytogenes on the composition of the surface microflora on washed rind soft cheese. Two trials with washed rind cheeses that were inoculated with 100cfu cm⁻² of a L. monocytogenes six strain cocktail were conducted. The first trial had to be terminated early (day 28) as contamination of Pseudomonas spp. from the initial brine did not produce the expected characteristics of the cheese during the aging period. For the second trial, cheese samples were aged in the lab for 60 days according to the cheesemakers specifications. Surface cheese rind samples were collected from both control and inoculated cheeses every 7 days. Cheese rind samples were analyzed through the standard BAM method for enumeration of L. monocytogenes and through amplification of the V4 region of 16S rRNA and ITS regions for identification of the surface rind bacterial and fungal communities, respectively. Our data showed that Pseudomonas spp. significantly changed the composition of the microorganisms found on the surface of the rind while L. monocytogenes had little effect. In addition, although the concentration of L. monocytogenes increased to levels of 10⁶ cfu cm⁻² based on the enumeration data, the genetic data was not able to identify it in the flora due to the fact that other genera were found at much higher concentrations, which is a limitation of molecular methods used for identification of pathogens in foods. For the second study the presence and incidence of L. monocytogenes on farms that either produce raw milk cheese or supply the milk for raw milk cheese production was investigated. Five farms were visited and in total 266 samples were collected from barn, environmental, and milk sites. L. monocytogenes prevalence was found to be at 6% from all the farms tested with 10 isolates found in the barn samples, 5 from environmental sites and 1 from milking equipment. Samples were identified to the genus level through a modified BAM method and speciated though multiplex PCR. Included in the pathogenic isolates was a DUP-1042B L. monocytogenes strain that has been implicated in major outbreaks, which emphasizes the adaptability and persistence of highly pathogenic stains in food manufacturing environments. Results from this study continue to support the fact that contaminated silage can be an important reservoir of the pathogen in a dairy farm setting. From our data and field observations we identified that drinking water sources for the animals is also an important reservoir of L. monocytogenes in farm environments. More importantly this study has shown the importance of continuous monitoring of environmental sites for the presence of the pathogen, particularly in silage. Lastly manure amended soils in the northeastern U.S. were tested for the presence and survival of rifampicin resistant Escherichia coli (rE. coli), generic E. coli (gE. coli) and Listeria spp.. Both gE.coli and rE.coli samples were processed using either direct enumeration, MPN or bag enrichment methods. Samples were taken from both tilled and surface dairy solid manure-amended plots. Listeria samples were processed using a modified BAM method. Listeria presence was constant throughout the study. In contrast, rE. coli and gE. coli levels declined with time. The main conclusions of this study were that soil type, location and physical characteristics have a significant role in the survival of bacterial populations of rE. coli, gE. coli and Listeria spp. in soil. Dairy solids application does not seem to have a long term effect on the natural microbial population of soils. Tilling of soils results in increased survival of the bacterial population due to the fact that it increases soil pore size and facilitates moisture entry, which in turn has been shown to increase bacterial survival rates. Data from this research will assist in the creation of preventative measures that lead to the elimination of pathogen reservoirs. It will be further used to verify that a 120 day interval following manure application should be sufficient to ensure food safety of edible crops subsequently planted on these soils.

Les Escherichia coli producteurs de Shiga-toxines (STEC) sont des bactéries pathogènes originaires du tractus digestif des ruminants qui peuvent contaminer certains aliments. Une fois ingérés, ils peuvent provoquer des pathologies graves. Le comportement des STEC n’appartenant pas au sérotype O157:H7 dans les fromages était mal connu. Par ailleurs, en dépit d’une forte prévalence des STEC dans les fromages, cet aliment est moins impliqué dans les épidémies que la viande. C’est pourquoi nous avons étudié dans un premier temps le devenir de ces STEC au cours de la fabrication de 5 schémas technologiques de fromages différents. Dans un deuxième temps, nous avons analysé le comportement et la survie de STEC ayant survécu à la fabrication d’un fromage au cours d’une digestion artificielle. Nos résultats ont montré que certaines technologies sont favorables à la croissance des STEC. Seul un affinage de plus de 60 jours a permis une réduction du taux des STEC. Les étapes de cuisson du caillé ou de coagulation longue de certaines technologies semblent inhiber la croissance des STEC. De plus, la croissance et la survie observées pour les souches E. coli O157:H7 étaient plus faibles que pour les souches E. coli non-O157:H7. L’étude de la survie des souches STEC au cours de la digestion artificielle a montré une différence de comportement entre les 2 souches STEC testées : la souche E. coli O157:H7 a survécu à la digestion tandis que celle de sérotype O26:H11 s’est fortement développée en fin de digestion. / Shiga-toxin producing Escherichia coli (STEC) are an important cause of foodborne illness. They are originated from the digestive tract of the ruminants and can contaminate certain food. Once ingested, they can be responsible for a variety of clinical outcomes. To date, most authors have focused their work on the behavior of E. coli O157:H7 during cheese manufacturing: little is known about the behavior of non-O157:H7 STEC. Furthermore, the presence of STEC strains has been found in raw milk cheeses but dairy products remain less implicated in outbreaks than meat. Consequently, we chose to study the behavior of O157:H7 and non-O157:H7 STEC strains during the manufacturing of 5 different cheese technologies. Then, secondly, it appeared interesting to evaluate the survival of STEC isolated from cheeses during artificial digestion. Our results showed that certain technologies are favorable to the growth of the STEC. Only a long ripening step (>60 days) could then allow a reduction of the STEC rate. In contrast, the cooking step of the curd or the long coagulation of certain technologies inhibited their growth. Our results also showed that the growth and the survival of the E. coli O157:H7 strains were lower than those of the non-O157:H7 E. coli strains. The study of the survival of STEC strains during the artificial digestion highlighted an important difference of behavior for the 2 strains tested: the E. coli O157:H7 strain was able only to survive whereas the E. coli O26:H11 strain knew an important growth at the end of digestion.

A cadeia produtiva do leite pode favorecer a contaminação por Listeria monocytogenes bem como outras espécies de Listeria sp, e o leite cru pode servir de fonte de contaminação para a indústria e subprodutos. Fatores como saúde do animal, ordenhador, boas práticas de manejo e limpeza adequada de equipamentos e utensílios garantem um leite de melhor qualidade e com baixa contagem de coliformes. A refrigeração do leite é um aspecto importante implementado pela IN 51, na qual o leite cru deve permanecer a 4ºC na propriedade leiteira em tanques ou a 7ºC em latões, porém a esta temperatura pode ocorrer a proliferação de microrganismos psicrotróficos como as espécies de Listeria sp. Três Regiões A, B e C com 25 propriedades fornecedoras de leite para cada região foram caracterizadas por meio da aplicação de um questionário e avaliadas quanto à presença de Listeria monocytogenes, coliformes totais e E. coli em leite cru e avaliadas quanto a presença de Listeria sp em ambiente de ordenha. Foram avaliadas 287 amostras de leite cru, 10 Mechas de Moore e 49 amostras ambientais. As análises foram feitas de acordo com protocolo preconizado pelo FDA descrito no Bacteriological Analitycal Manual (BAM), com enriquecimento em BLEB e plaqueamento em meios Oxford e ALOA. A confirmação dos isolados foi feita em Kit Api Listeria. Das 75 fazendas estudadas, 77,3% (n=58) apresentaram condições insatisfatórias de produção de leite, higienização de equipamentos e infra-estrutura; 20% (n=15) em condições regulares e apenas 2,7% (n=2) em condições satisfatórias. Quanto à enumeração de coliformes totais, nas regiões A, B e C, as amostras de leite apresentaram 86% (n=85), 75% (n=71) e 72% (n=66) de contagens acima de 103 NMP/mL, respectivamente e E. coli esteve presente em 66% (região A) , 66% (região B) e 49% (região C) das amostras. Das amostras de leite cru em relação à presença de L. monocytogenes todas foram negativas. Das mechas de Moore todas foram negativas para Listeria sp e as amostras do ambiente de ordenha foram positivas em uma propriedade da região C em dois pontos, sendo eles: ralo e chão de ordenha representando 4,44% das amostras ambientais, sendo os isolados pertencentes à espécie Listeria innocua. Nas duas cepas foram feitas a análise por sorotipagem pela Fio Cruz/RJ e o resultado foi de sorotipo 6a e 6b. No ralo de ordenha foram testadas duas colônias sendo encontrados dois sorotipos diferentes 6a e 6b indicando a presença de dois sorotipos em uma mesma amostragem e no piso da sala de ordenha as duas colônias testadas foram do sorotipo 6a. Embora não tenha sido detectada a presença de L. monocytogenes, a presença de L. innocua nas amostras pode indicar presença presumida de L. monocytogenes visto que as mesmas apresentam características fisiológicas semelhantes e podem ocorrer no mesmo ambiente. Quanto à produção do leite, apesar da vigência da IN 51, as regiões estudadas ainda encontram-se, em geral, em condições precárias e são necessárias muitas mudanças para se obter um leite de melhor qualidade. / The production chain of milk may promote contamination by Listeria monocytogenes and other species of Listeria sp and raw milk can serve as a source of contamination for the industry and byproducts. Factors such as animal health, milker, good management practices and proper cleaning of equipment and utensils provide a better quality milk with low coliform counts. The cooling of milk is an important aspect introduced by the IN 51, in which the raw milk should remain at 4 °C in dairy farms in tanks or 7 °C in cans, but at this temperature can occur proliferation of psychrotrophic microorganisms, such as species of Listeria sp. Three regions A, B and C with 25 farms which supply milk for each region were characterized by applying a questionnaire and evaluated for the presence of Listeria monocytogenes, coliforms and E. coli in raw milk and for the presence of Listeria sp in the milking environment. We evaluated 287 samples of raw milk, Moore 10 wicks and 49 environmental samples. Analyses were made according to a protocol recommended by the FDA as described in the Bacteriological Analitycal Manual (BAM), with enrichment and plating media Bleb Oxford and ALOA. Confirmation of isolates was done in Kit Api Listeria. Of the 75 farms studied, 77.3% (n = 58) showed unsatisfactory conditions of milk production, hygiene equipment and infrastructure, 20% (n = 15) on a regular basis and only 2.7% (n = 2 ) in satisfactory condition. As for the enumeration of coliforms, in regions A, B and C, milk samples showed 86% (n = 85), 75% (n = 71) and 72% (n = 66) scores above 103 MPN / mL, respectively, and E. coli was present in 66% (region A), 66% (region B) and 49% (region C) of the samples. All the raw milk samples were negative for the presence of L. monocytogenes. The fuses Moore were all negative for Listeria sp, and the environmental samples were positive for milking on a farm in the region C in two points, namely: the floor drain and milking, which represent 4.44% of environmental samples, and the isolates belong to the species Listeria innocua. In the two strains the analysis were made by serotyping, executed by Fio Cruz / RJ, and the results were serotype 6a and 6b. In the drain of milking two colonies were tested, founding two different serotypes, 6a and 6b, indicating the presence of two serotypes in the same sampling and floor in the parlor. The two colonies tested were of serotype 6a. Although not detected the presence of L. monocytogenes, the presence of L. innocua in samples may indicate presumed presence of L. monocytogenes since they have similar physiological characteristics and may occur in the same environment. For milk production, despite the validity of the IN 51, the regions studied are still generally in poor condition and many changes are necessary to achieve a better quality milk.

A aflatoxina M1 (AFM1) é um metabólito hidroxilado da aflatoxina B1 (AFB1), e é detectada no leite, após a ingestão de alimentos contaminados com a AFB1. Foram avaliadas durante o período de dez meses (julho/2009 a abril/2010): a ocorrência de AFM1 em leite bovino cru, produzido em 45 fazendas de três mesorregiões produtoras de leite do Estado de São Paulo (Araçatuba, Bauru e Vale do Paraíba); a variação da contaminação entre as mesorregiões produtoras; e a correlação da concentração de AFM1 com parâmetros de qualidade (contagem bacteriana total e contagem de células somáticas) e composição (teores de gordura, proteína, lactose e sólidos totais). A análise de AFM1 foi realizada empregando-se coluna de imunoafinidade para purificação associada à cromatografia líquida de alta eficiência, com detector de fluorescência. Os valores do limite de detecção e de quantificação foram 0,003 µg L-1 e 0,01 µg L-1, respectivamente. A recuperação média da metodologia avaliada com amostras artificialmente contaminada em três concentrações (0,04, 0,1 e 0,2 µg L-1) foi 83%. A AFM1 foi detectada em 210 (49%) das 429 amostras analisadas numa faixa de concentração de traços a 0,617 µg L-1 com média de 0,017 µg L-1 e mediana igual a não detectada (LD<0,003 µg L-1). A concentração média de AFM1 das amostras da mesorregião de Bauru (0,031 µg L-1) foi estatisticamente maior do que da mesorregião de Araçatuba (0,015 µg L-1) e do Vale do Paraíba (traços). Apenas 3 amostras (0,7%) encontraram-se acima do limite máximo permitido no Brasil (0,5 µg L-1) e 28 amostras (6,5%) apresentaram contaminação acima do limite da Comunidade Européia (0,05 µg L-1). A média dos valores de contagem de células somáticas e de contagem bacteriana total foi de 497 103 células mL-1 e 515 103 UFC mL-1, com faixa de 72 a 1411 103 células mL-1 e 17 a 5014 103 UFC mL-1, respectivamente. Os valores médios dos parâmetros de composição do leite foram: gordura 3,59%, proteína 3,15%, lactose 4,50% e sólidos totais 12,22%. Não foi observada correlação significativa entre a ocorrência de AFM1 e os parâmetros de qualidade e de composição do leite. / The AFM1 is a hydroxylated metabolite of aflatoxin B1 (AFB1), and is detected in milk, after ingestion of food/feed contaminated with B1. During the period of ten months (July/2009 to April/2010), it was evaluated: the occurrence of aflatoxin M1 (AFM1) in raw cow milk produced in 45 farms of three mesoregions of São Paulo State (Araçatuba, Bauru and Vale do Paraíba); the variability of contamination among mesoregions; and the correlation of AFM1 contamination with quality parameters (total bacterial count and somatic cell count) and composition (fat, protein, lactose and total solid contents) of raw milk. The AFM1 analysis were performed using an immunoaffinity column for clean up, and high performance liquid chromatography, with fluorescence detection. The detection and quantification limits were 0.003 µg L-1 and 0.01 µg L-1, respectively. The mean recovery of the methodology obtained with spiked aflatoxin-free milk samples at three concentrations (0.04, 0.1 and 0.2 µg L-1) was 83%. The AFM1 was detected in 210 (49%) of 429 samples ranged from traces to 0.617 µg L-1 with a mean level of 0.017 µg L-1 and median not detected (LOD <0.003 µg L-1). The mean AFM1 concentration of samples from Bauru mesoregion (0.031 µg L-1) was statistically higher than Araçatuba (0.015 µg L-1) and Vale do Paraíba (traces) mesorregions. Only 3 samples (0.7%) were above the maximum limit allowed in Brazil (0.5 µg L-1) and 28 samples (6.5%) were above the limit of the European Community (0.05 µg L-1). The mean values of somatic cell count and total bacterial count was 497 103 cells mL-1 and 515 103 CFU mL-1 ranged from 72-1411 103 cells mL-1 and from 17-5014 103 CFU mL-1, respectively. The mean values of the parameters of milk composition were: fat 3.59%, protein 3.15%, lactose 4.50% and total solids 12.22%. There was no significant correlation among the occurrence of aflatoxin M1 and the quality and composition parameters of milk.

O presente trabalho foi planejado com o objetivo de estudar os atributos do teste tuberculínico e da abreugrafia em confronto com o diagnóstico bacteriológico da tuberculose. A população de estudo foi constituída por 15.056 pessoas com 15 e mais anos de idade, matriculadas no Centro de Saúde de Ribeirão Preto durante 6 meses consecutivos em 1973. Constatou-se que o valor predictivo do teste tuberculínico negativo foi de 99,98 por cento e o valor predictivo positivo da abreugrafia foi de 14,45 por cento ; a realização de abreugrafia apenas em reatores fortes elevaria o seu valor predictivo positivo para 18,96 por cento melhorando consideravelmente a eficácia desse instrumento sem prejuízo da sua sensibilidade. Esses resultados permitem recomendar o emprêgo do teste tuberculínico em Saúde Pública visando a exclusão de tuberculose doença bem corno para a triagem de adultos para exame abreugráfico do tórax. / This study was designed to evaluate the tuberculin test and roentgenphotography attributes in comparison with the bacteriological diagnosis of tuberculosis. The study population consisted of 15.056 persons aged 15 or above, registered at the Health Center of Ribeirão Preto, Brazil, during 6 consecutive months in 1973. It was observed that the predictive value of negative tuberculin test was of 99,98 per cent and the predictive value of abnormal roentgenphotography was of 14,45 per cent ; when the late was taken only of \"strong reactors\" its predictive positive value raised to 18,96 per cent , what increased its efficacy without impairing sensibility. These findings permit to recommend the use of the tuberculin test as a Public Health routine procedure to exclude tuberculosis disease in adults as well as a screening procedure for roentgenphotography examination.

Os queijos artesanais, elaborados com leite cru, são apreciados por suas características sensoriais peculiares. Com objetivo de assegurar a inocuidade destes produtos, a legislação brasileira estabeleceu padrões microbiológicos sanitários para estes alimentos. Microorganismos probióticos são conhecidos por sua capacidade de modular favoravelmente a microbiota do hospedeiro por meio da produção de substâncias antimicrobianas, em especial, os ácidos orgânicos e bacteriocinas. Assim, o presente estudo buscou avaliar a utilização de micro-organismos probióticos como alternativa para melhorar a qualidade microbiológica de queijos artesanais usando o Queijo Artesanal da Serra da Mantiqueira como modelo. Para tanto, foi incorporada à microbiota do leite uma preparação composta de 16 cepas de micro-organismos probióticos, obtendo-se uma concentração final de 3x106 UFC/ml de leite. A estratégia adotada consistiu em comparar os queijos inoculados com micro-organismos probióticos em relação aos queijos não inoculados (controle), durante o período de maturação de 45 dias, em três estações do ano (inverno, verão e outono). As amostras foram coletadas nos dias 1, 15, 30 e 45 para a determinação dos parâmetros: umidade, pH, concentrações de carboidratos, ácidos orgânicos e análises microbiológicas. Avaliou-se também a composição da microbiota dos queijos pela técnica do pirossequenciamento dos genes do rRNA 16S. Os resultados permitiram verificar que os gêneros predominantes na microbiota dos queijos foram Streptococcus, Lactobacillus e Lactococcus , em diferentes níveis nas três estações do ano. Foi possível observar que a incorporação das culturas probióticas resultou em diferentes efeitos nas estações avaliadas no que se refere às concentrações de carboidratos e ácido cítrico, bem como de ácidos orgânicos ao final dos 45 dias de maturação, possivelmente devido à variabilidade da microbiota verificada nas diferentes estações. No tocante às características microbiológicas, observou-se que, durante o período de maturação, 95% dos queijos confeccionados nas diferentes condições apresentaram padrões de qualidade em conformidade com o estabelecido na legislação, no que se refere às concentrações de coliformes termotolerantes, estafilococos coagulase positivos, Salmonella sp e Listeria sp independentemente da incorporação das culturas probióticas. Entretanto, a adição das culturas probióticas reduziu a carga microbiana de gêneros da família Enterobacter iaceae , em especial Enterobacter , Citrobacter , Klebsiella , Kluyvera e Obesumbacterium durante processo de maturação. Especificamente, o emprego das culturas probióticas resultou em redução do percentual total de Enterobacter iaceae na microbiota dos queijos de 0,41% para 0,17% no verão, e de 12,42% para 6,71% no outono. Neste contexto, a adição de culturas probióticas pode constituir alternativa viável para melhoria da qualidade microbiológica de queijos elaborados com leite cru por meio do incremento do potencial antagônico da microbiota sobre espécies de micro-organismos potencialmente patogênicos, toxigênicos e deteriorantes, além de agregar valor ao produto final. / Artisanal cheeses, made with raw milk, are appreciated for their sensorial profiles, which differentiates them from industrialized cheeses. In order to ensure safety to consumers, Brazilian legislation established microbiological standards for these products. Probiotics are well known for their ability to favorably modulate the host\'s microbiota by producing antimicrobial compounds, mainly organic acids and bacteriocins. Therefore, this study aimed to evaluate the use of probiotic microorganisms as an alternative to improve the microbiological quality of artisanal cheeses through inhibition of contaminating microorganisms, using Mantiqueira Highlands Artisanal Cheese as a model. To do so, a preparation consisting of 16 probiotic strains was added to the milk, at a final concentration of 3x106 CFU/ml. Additionally, the effects of this practice on pH, carbohydrates and organic acids concentrations over a 45 day ripening period were evaluated. Analyses of cheese bacterial community composition were also undertaken by means of 16S rRNA gene pyrosequencing. The strategy consisted of comparing cheeses made with probiotic cultures with a control group, to which no probiotic culture was added. Over a 45 day ripening period, in three seasons (winter, summer and autumn), samples were collected at days 1, 15, 30 and 45 in order to conduct pH, moisture, carbohydrate, organic acids, microbiological and pyrosequencing analyses. The results showed that the addition of probiotic cultures resulted in distinct effects in the different seasons regarding carbohydrate and organic acids concentrations, possibly due to variability in bacterial community composition. The results also demonstrated that 95% of cheeses were deemed acceptable according to Brazilian law regarding coliforms, positive coagulase staphylococci, Salmonella sp. and Listeria sp, regardless of adding probiotics. However, the incorporation of probiotic microorganisms resulted in reduced populations of genera belonging to Enterobacteriaceae family, especially Enterobacter, Citrobacter, Klebsiella, Kluyvera and Obesumbacterium, throughout the whole ripening process. Specifically, the addition of probiotic cultures reduced the total percentage of Entercobacteriaceae from 0,41% to 0,17% in the summer, and from 12,42% to 6,71% in the fall. In this context, the use of probiotic microorganisms may represent a viable alternative for improving microbiological quality of raw milk cheeses by means of incrementing the antagonistic potential of cheese microbiota towards potentially pathogenic and toxigenic microorganisms.