Algae “super blooms” are a commonly encountered environmental issue in fresh and brackish water that occurs due to the buildup of cyanobacteria. Many of the commonly encountered cyanobacteria such as Mycrocystis aeruginosa (M. aeruginosa) produce potent cyanotoxins (microcystins) that pose serious health threats and even death to local wild life and humans. Microcystin contaminated fresh-water that empties into the ocean has been shown to lethally affect marine life in the area of contamination. Human consumption of tainted sea life and other routes of mycrocystin exposure can lead to serious liver damage and even death. Thus, a method was developed for forensic postmortem analysis of microcystins RR, LR and YR by Two-Dimensional (2D) Liquid Chromatography (LC) - tandem Mass Spectrometry (MS/MS). A final 2D LC-MS/MS method was selected from 6x6 automated method development experiments. Each microcystin were subjected to a total of 36 methods, which were completed over an 18hr period. The extraction process was performed using a reverse-phase sorbent (Oasis HLB, Waters Corporation, Milford, MA) with a 3cc solid phase extraction (SPE) barrel using sequential elution. From acetonitrile (ACN) and methanol (MeOH) stock solutions, 10 μL of the internal standard (IS) Nodularin was added to the final extract. The concept of sequential micro extraction was designed to capture the retention behaviour of the target analyte in response to various extraction parameters (sorbent strength, elution polarity, and solubility). Therefore, optimized conditions were selected to excise the region of interest during extraction. The elution solvents chosen for the microcystins were acetonitrile, methanol and acetone with 10 % sequential increments. Since microcystins exhibit a zwitterionic structure, three sets of elution solutions were created to evaluate their elution profile (pH 3, pH 7, pH 10). When the elution profile for low pH and high pH are compared, microcystin RR was eluted over the 40% to 70% methanol fractions under low pH conditions with a slight shift towards higher organic % (50%-70% fractions) under high pH. This elution behaviour suggests that the basic moieties of the structure demonstrate a stronger retention for the stationary phase. Microcystin LR and YR however, eluted at a higher organic solvent percentage under low pH conditions and at a lower organic solvent percentage under high pH conditions, indicates that the acidic moieties of the structures have stronger retention. The urine sample gave recovery values for all three microcystins in the 80-90% range, as to be expected with type of complexity associated with biological samples. The sequential extraction protocol produced an extraction method that delivered a clean extract after a 30 min workflow using a single and optimized 2D LC-MS/MS method. The total analytical run time was set at 10 minutes.

Made available in DSpace on 2014-07-29T15:07:32Z (GMT). No. of bitstreams: 1 Dissertacao Marilia Cristina Sola.pdf: 489950 bytes, checksum: 4d267a18376c43b47c56d28c43d655a5 (MD5) Previous issue date: 2011-02-28 / Brucellosis is a chronic infectious disease caused by bacterium of the genus Brucella, which affects humans and different species of animals. Despite the implementation of programs aimed at the disease controlling and eradicating, brucelosis is endemic in many countries, especially in developing ones, resulting in significant economic losses and serious implications for animal and public health, due to its zoonotic character. The disease can be transmitted by direct or indirect contact with infected animals, fetal membranes, and also transmitted to humans by contaminated animal products, especially milk and dairy products that have not undergone thermal processing, by raw meat and the handling of carcasses and visceras at the slaughterhouse. The National Programme for Control and Eradication of Brucellosis (PNCEBT), established in the country in 2001, determines the diagnosis of animals in order to establish both the distribution and characterization of the agent. In this context and seeking for a rapid, safe and precise diagnosis of this disease in cattle, we aimed to detect Brucella spp by real time PCR in suspect lesions detected during routine inspection in slaughterhouses under Federal Inspection in the state of Goias, Brazil. Such lesions were related to cervical bursitis, testicular, lung and liver lesions and also in mandibular, retropharyngeal, esophageal, intercostal, apical, mediastinal, tracheobronchial, inguinal, ischiatic, popliteal and mesenteric lymphnodes. In the sampling procedure, cellulose cards were used for storage of biological material (card FTA ® Elute) by impregnating the material in the fibrous matrix of the card. Laboratory analyses were performed on 47 samples from animal tissue and fluids, collected from carcasses and visceras at 40 bovines suspected of brucellosis. Samples were processed at the Laboratório de Biologia Molecular from the Centro de Pesquisa em Alimentos, Escola de Veterinária e Zootecnia from Universidade Federal de Goiás (LBM / CPA / EVZ / UFG). Brucella spp was detected in 42.5% of the bovines with suspect lesions and in 38.3% of samples. It was verified that the use of real-time PCR associated with the FTA® Elute method is an important diagnostic tool in the process of trial and disposition of carcasses and visceras of slaughtered animals and it gives flexibility and efficiency for the diagnosis of diseases, helping the Federal Inspection Service in fulfilling its mission of providing safe food to consumers / A brucelose é uma enfermidade infectocontagiosa de caráter crônico, causada por bactérias do gênero Brucella, que acomete o homem e diferentes espécies animais. Apesar da implementação de programas que visam o controle e a erradicação da enfermidade, apresenta-se endêmica em muitos países, principalmente aqueles em desenvolvimento, resultando em prejuízos econômicos significativos aos sistemas de produção e sérias implicações em saúde animal e pública, visto seu caráter zoonótico. A doença pode ser transmitida pelo contato direto ou indireto com animais infectados e anexos fetais e, ainda, veiculada ao homem pela ingestão de produtos de origem animal contaminados, principalmente leite e seus derivados que não passaram por processamento térmico. Pode ser veiculada também por meio de carnes cruas e pela própria manipulação de carcaças e vísceras durante o abate sanitário. O Programa Nacional de Controle e Erradicação da Brucelose e Tuberculose (PNCEBT) instituído no país em 2001 determina como uma das medidas sanitárias, o diagnóstico dos animais, a fim de estabelecer a ocorrência, distribuição e caracterização do agente. Neste contexto, visando um diagnóstico rápido, seguro e preciso desta enfermidade em bovinos, objetivou-se detectar o DNA de Brucella spp por meio da técnica de PCR em Tempo Real em lesões sugestivas identificadas durante a inspeção sanitária de rotina em matadouros-frigoríficos sob Inspeção Federal no estado de Goiás. Tais lesões abrangeram a bursite cervical, lesões testiculares, pulmonares, hepáticas e de linfonodos mandibular, retrofaríngeo, esofageano, intercostal, apical, mediastínico, traqueobrônquico, inguinal, isquiático, poplíteo e mesentérico. Para tanto, no procedimento de colheita de amostras, foram utilizados cartões de celulose destinados ao armazenamento de material biológico (cartão FTA® Elute), por impregnação do material na matriz fibrosa do cartão. As análises laboratoriais foram efetuadas em 47 amostras provenientes de fluidos e tecido animal, colhidas em carcaças e vísceras de 40 bovinos com suspeita de brucelose e desenvolvidas no Laboratório de Biologia Molecular do Centro de Pesquisa em Alimentos da Escola de Veterinária e Zootecnia da Universidade Federal de Goiás (LBM/CPA/EVZ/UFG). Detectou-se o DNA de Brucella spp em 42,5% dos bovinos que apresentaram lesões sugestivas e em 38,3% das amostras totais. Verificou-se que o emprego da técnica de PCR em Tempo Real associada ao método FTA® Elute, é uma ferramenta de diagnóstico importante no processo de julgamento e destino de carcaças e vísceras dos animais de abate, tendo em vista que confere agilidade e eficiência no diagnóstico das enfermidades, auxiliando o Serviço de Inspeção Federal no cumprimento de sua missão, ou seja, de colocar à disposição do consumidor, alimentos seguros.