Aquisição e eliminação de contaminantes em tecidos de moluscos bivalves

Tese (doutorado) - Universidade Federal de Santa Catarina, Programa de Pós-Graduação em Biotecnologia e Biociências, Florianópolis, 2014O consumo de ostras pode ser responsável pela veiculação de doenças para humanos, pois estes animais podem acumular substâncias tóxicas e microrganismos patogênicos em seus tecidos. Vírus entéricos humanos podem apresentar padrões distintos de acumulação nas ostras, dificultando que sejam removidos durante a depuração. Estudos da cinética de decaimento de contaminantes microbiológicos e químicos nas ostras, durante a depuração, são escassos. O objetivo desse trabalho foi estudar a dinâmica de aquisição e eliminação de contaminantes químicos e microbiológicos, no meio ambiente e durante os processos de depuração. Esta pesquisa abrangeu duas etapas distintas, envolvendo a alocação de ostras por 14 dias em quatro locais na baía de Florianópolis-SC: dois liberados ao cultivo (RIB-Ribeirão da Ilha e SAL-Santo Antônio de Lisboa) e dois impróprios (TAP-Tapera e BUC-estuário do rio Bücheler). Na primeira etapa (capítulo I) foi medida a contaminação ambiental destes locais e na segunda (capítulo II), realizada a contaminação (diferentes níveis) das ostras, para que fossem submetidas aos testes de depuração, com diferentes tratamentos (7 dias). No capítulo I foram analisadas: ostras, água e sedimento marinhos dos locais, sendo que as amostras procedentes diretamente do laboratório fornecedor das ostras (Laboratório de Cultivo de Moluscos Marinhos/UFSC-LCMM) funcionaram como o tempo zero da contaminação (T0 dia). Foram detectados: (1) Bactérias: E. coli na água do mar (acima de 43 UFC/100 mL em TAP e BUC) e Salmonella sp. nas ostras (em BUC), por cultura bacteriana; (2) Protozoários: Cryptosporidium nas ostras (TAP) e Giardia na água do mar (BUC), pela Imunoseparação Magnética e Imunofluorescência tendo esta última sido sequenciada para a confirmação da espécie (G.duodenalis); (3) Vírus entéricos: Adenovírus Humano/HADV (nos quatro locais), Vírus da Hepatite A nas ostras (BUC), Norovírus Humano (NoV) GI nas ostras (TAP e BUC) e GII na água do mar (BUC), Poliomavírus-JC nas ostras (LCMM), todos por (RT) qPCR. Também se verificou a ausência de HAdV infecciosos nas ostras, por teste de Placa de Lise. (4) Pesticidas Organoclorados; Hidrocarbonetos Alifáticos/HAs e Policíclicos Aromáticos/HPAs; Alquibenzenos Lineares/LABs foram detectados em vários locais, por Cromatografia Gasosa, nas ostras e sedimentos. No capítulo II, somente ostras foram analisadas, antes e durante a depuração. Foram utilizadas lâmpadas UV (18 e 36 W) na descontaminação das águas de depuração e comparadas as cinéticas de decaimento dos contaminantes nos tecidos das ostras. Parte dos animais de cada local, foi artificialmente contaminada com HAdV2 e Norovírus Murino/MNV-1, funcionando como controles positivos de contaminação viral durante as depurações. Não houve eliminação dos HAdV e protozoários das ostras durante a depuração. MNV-1 foi eliminado após quatro dias de depuração. Também foram detectados HAs, HPAs e LABs, antes e após as depurações. Concluídos os testes, depuradoras com UV 36 W foram disponibilizadas em quatro restaurantes em Florianópolis e realizadas análises virais das ostras depuradas por quatro dias (43 amostras). Somente HAdV foi detectado (uma amostra) e não estava infeccioso. Laudos das análises foram entregues aos comerciantes. As contaminações detectadas ameaçam a produção de ostras em Florianópolis, sendo necessário o aumento na fiscalização nas regiões de cultivo. Embora a depuração tenha removido o MNV-1 (vírus com genoma de RNA, como a maioria dos vírus entéricos humanos veiculados pelas ostras), não eliminou outros patógenos e compostos orgânicos investigados. Além dos ensaios descritos na tese, também foram realizadas outras pesquisas (Apêndice A), referentes à bioacumulação diferencial das espécies de NoV GI e Sapovírus em ostras e inativação termal de Rotavírus em mexilhões durante o cozimento. Estas pesquisas adicionais foram realizadas durante estágio doutoral, 11 meses, no Institut Français de Recherche pour l'Exploitation de la Mer (IFREMER), na França.Abstract: The consumption of oysters may be responsible for transmission of diseases to humans because these animals can accumulate toxic substances and pathogenic microorganisms in their tissues. Human enteric viruses may exhibit distinct patterns of accumulation in oysters, making it difficult to be removed during depuration. Studies of the decay kinetics of microbiological and chemical contaminants in oysters, during depuration, are scarce. The aim of this work was to study the dynamics of acquisition and disposal of chemical and microbiological contaminants in the environment and during the process of depuration. This research comprised two stages, involving the allocation of oysters for 14 days in four locations in the Bay of Florianópolis-SC: two released for cultivation (RIB-Ribeirão da Ilha and SAL-Santo Antonio de Lisboa) and two improper (TAP-Tapera and BUC-Bücheler river estuary). In the first step (Chapter I), environmental contamination of these sites was measured and in the second (Chapter II), held contamination (different levels) of oysters, that were submitted to the depuration tests with different treatments (7 days). In the Chapter I, were analyzed: oyster, seawater and marine sediments. All samples obtained from the oyster supplier (Laboratory of Cultivation of Marine Mollusks/UFSC-LCMM) were considered time zero of contamination (T0 day). Were detected: (1) Bacteria: E. coli in seawater (above 43 CFU/100 mL in TAP and BUC) and Salmonella sp. in oysters (in BUC) by bacterial culture; (2) Protozoa: Cryptosporidium in oysters (TAP) and Giardia in seawater (BUC), by Immunomagnetic Separation and Immunofluorescence, and this was also been sequenced to confirm the species (G. duodenalis); (3) Enteric Viruses: Human Adenovirus / HAdV (in four locations), Hepatitis A virus in oysters (BUC), Human Norovirus GI (NoV GI) in oysters (TAP and BUC) and GII in seawater (BUC), Polyomavirus-JC in oysters (LCMM), all by (RT) qPCR. It was also verified the absence of infectious HAdV oysters by the Plaque Assay. (4) Organochlorine Pesticides; Aliphatic/AHs and Polycyclic Aromatic/PAHs Hydrocarbons; Linear alkylbenzenes/LABs were detected in several locations, by Gas Chromatography, in oysters and sediments. In Chapter II, only oysters were examined before and during depuration. UV lamps (18 and 36 W) were used in the decontamination of water and the kinetics of decay of contaminants was compared, in the oyster tissues. Part of the animals from each sitewas artificially contaminated with HAdV2 and Norovirus Murino/MNV-1, acting as positive controls of viral contamination during the depurations. There was no elimination of HAdV and protozoa from oysters during depuration. MNV-1 was eliminated after four days of depuration. AHs, LABs and PAHs were detected before and after the depurations. After the end of the laboratory tests, the depuration tanks with UV 36 W were allocated in four restaurants, in Florianopolis and viral analyzes of oysters depurated during four days (43 samples) were performed. Only HAdV was detected (one sample) and was not infectious. Reports of analyzes were delivered to traders. The contamination pointed hazards for oyster consumption and showed the importance for continuous surveillance in mollusks growing areas. Although depuration removed the MNV-1 (viruses with RNA genomes, such as most human enteric viruses transmitted by oysters) it did not eliminate other pathogens and organic compounds investigated. In addition to the tests described in the thesis, other research (Appendix A) related to differential bioaccumulation of NoV GI and Sapovirus species in oysters and the thermal inactivation of Rotavirus in mussels during cooking were performed. These additional studies were conducted during the doctoral stage during 11 months, at the Institut Français de Recherche pour l'Exploitation de la Mer (IFREMER), France

Depuração e tratamento térmico para redução dos níveis de patógenos em moluscos bivalves produzidos em Santa Catarina, Brasil

This review summarises the findings of studies, conducted in Santa Catarina State (SC), on the reduction of pathogens in mollusks via post-harvest treatments. Studies indicate that 48h depuration cycles can efficiently reduce the levels of Salmonella and naturally occurring Vibrio spp. in oysters, whereas viruses (hepatitis A virus and murine norovirus) require more than 48 hours to be completely inactivated. The tested depuration protocols were unable to eliminate the Protozoa Cryptosporidium and Giardia. The heat treatment protocol commonly adopted by local industries (steam cooking for 6 min at 100ºC) can eliminate Vibrio spp. from mussels and murine norovirus from oysters. Despite some limitations, adopting these post-harvest treatments is an important strategy to improve the safety of mollusk consumption in SC.Esta revisão resume os achados de estudos realizados em Santa Catarina (SC) sobre redução de patógenos em moluscos por meio de tratamentos pós-colheita. Estudos indicam que os níveis de Salmonella e Vibrio spp. de ocorrência natural podem ser eficientemente reduzidos em ostras por meio de ciclos de depuração de 48h, enquanto vírus (hepatite A e norovírus murino) requerem mais de 48 horas para serem completamente inativados. Protozoários (Cryptosporidium e Giardia) não foram eliminados pelos protocolos de depuração testados. O protocolo de tratamento térmico adotado nas indústrias locais (cozimento a vapor durante 6 min a 100ºC) é capaz de eliminar Vibrio spp. de mexilhões e norovírus murino de ostras. Apesar de algumas limitações, a adoção desses tratamentos pós-colheita é uma estratégia importante para melhorar a segurança dos moluscos em SC

Comparação entre a imuno-separação magnética, acoplada à imunofluorescência, e as técnicas de Faust et al. e de Lutz para o diagnóstico de cistos de Giardia lamblia em fezes humanas

No presente trabalho, o desempenho da técnica de Imunoseparação Magnética, acoplada à Imunofluorescência (IMS-IFA), foi comparado com aqueles das técnicas parasitológicas de FAUST et al. e de Lutz na detecção de cistos de Giardia lamblia em fezes humanas. Foram processadas 127 amostras de fezes pelas três técnicas paralelamente e a detecção de cistos foi de 27,5% para IMS-IFA e de 15,7% para as técnicas de FAUST et al. e de Lutz concomitantemente. A análise dos resultados mostrou maior sensibilidade da IMS-IFA na detecção de cistos de G. lamblia quando comparada aos métodos de FAUST et al. e Lutz. A utilização desta metodologia como procedimento de rotina proporciona o processamento de várias amostras simultaneamente, além de aumentar a recuperação de cistos de G. lamblia e reduzir o tempo de estocagem das amostras.In the present study, the performance of Immunomagnetic Separation technique, coupled with Immunofluorescence (IMS-IFA), was compared with the FAUST et al. and Lutz parasitological techniques for the detection of Giardia lamblia cysts in human feces. One hundred and twenty-seven samples were evaluated by the three techniques at the same time showing a rate of cyst detection of 27.5% by IMS-IFA and 15.7% by both Faust et al. and Lutz techniques. Data analysis showed a higher sensitivity of IMS-IFA for the detection of G. lamblia cysts in comparison with the techniques of FAUST et al. and Lutz. The use of this methodology as a routine procedure enables the processing of many samples simultaneously, in order to increase recovery rate of G. lamblia cysts and reduce the time of sample storage

Avaliação de técnicas de diagnóstico de Cryptosporidium spp. e Giardia lamblia em fezes humanas

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências da Saúde. Programa de Pós-Graduação em Farmácia.A diarréia é responsável por mais de 3 milhões de mortes ao ano no mundo e a maioria dos casos ocorre em crianças de países em desenvolvimento. A diarréia em crianças, além dos problemas relacionados à desidratação, ainda pode ocasionar má absorção dos nutrientes causando um quadro de desnutrição afetando, até mesmo, as funções cognitivas. Muitos patógenos causadores de diarréia podem ser veiculados pela água e por alimentos contaminados. Eles podem ser vírus, bactérias e parasitas. Os protozoários Cryptosporidium spp. e Giardia lamblia são importantes causadores de diarréia, principalmente em crianças. Estima-se que no Brasil a prevalência destes patógenos seja de 8% e 28,5%, respectivamente. Devido a cistos de Giardia e oocistos de Cryptosporidium serem estruturas muito pequenas, sua detecção torna-se difícil, já que as fezes naturalmente possuem muitos detritos e microrganismos que podem mascarar ou ser confundidos com estes protozoários. Para facilitar e tornar mais eficiente este diagnóstico, este trabalho teve por objetivo padronizar a Técnica de Imunoseparação Magnética (IMS) acoplada à Imunofluorescência (IFA) para o diagnóstico de Cryptosporidium spp. e Giardia lamblia em fezes humanas, além de avaliar sua eficiência quando comparada às Técnicas Clássicas descritas por Faust et al. (1939) e por Lutz (1919), ou Hoffman Pons & Janner (1934), quando utilizadas para o diagnóstico da giardíase; e por fim avaliar as prevalências de Cryptosporidium spp. e Giardia lamblia em uma creche e em ambulatórios de dois Hospitais da Grande Florianópolis-SC. Os resultados da Padronização da Técnica, utilizando-se 4 tratamentos com 3 repetições para cada, demonstraram que a IMS-IFA foi capaz de recuperar oocistos de Cryptosporidium spp. (média de 4,7%) e cistos de Giardia lamblia (média de 1,3%) em fezes humanas. Quando comparadas, as recuperações das duas espécies, não apresentaram diferenças significativas (F > 0,05). Ao se comparar o desempenho das Técnicas de IMS-IFA com a de Faust et al. e a de Lutz no diagnóstico de G. lamblia, utilizando-se 127 amostras de fezes de crianças, percebeu-se que a primeira deteve vantagem quando comparada às outras duas técnicas em conjunto, mas quando se comparou a recuperação de cistos pela primeira em relação à soma dos resultados da segunda (confirmados ou não pela Técnica de Lutz) observou-se que as diferenças não foram significativas (?2< 0,05). Avaliando-se as prevalências de Cryptosporidium spp. e Giardia lamblia encontradas em 77 crianças de uma creche e de 50 crianças de ambulatório, ambos na Grande Florianópolis, observou-se que para Cryptosporidium spp. estas foram baixas e próximas nas duas populações (1,3% e 2% respectivamente). Para Giardia lamblia, elas se comportaram de forma bastante distinta (45,5% e 2% respectivamente) e estatisticamente significativa. Também se detectou diferenças quando foram comparadas crianças menores com as maiores de 5 anos de idade dentro da mesma população (creche ou ambulatório) e na prevalência de giardíase comparando-se as faixas etárias analisadas das duas populações. A IMS-IFA mostrou-se capaz de detectar cistos de Giardia e oocistos de Cryptosporidium em fezes humanas e, devido a sua especificidade, não se fez necessária a confirmação através de outras metodologias de diagnóstico para Cryptosporidium

Hepatitis E Virus in Manure and Its Removal by Psychrophilic anaerobic Biodigestion in Intensive Production Farms, Santa Catarina, Brazil, 2018–2019

Hepatitis E virus (HEV) is an important enteric agent that can circulate in swine; it is excreted in manure, and of zoonotic interest. The present study investigated, by RT-qPCR, the circulation of HEV in swine manure from different types of pig farms (maternity, nursery, and grow-finish farms) in Santa Catarina State, the major pig production area of Brazil, and also evaluated the HEV removal efficiency of psychrophilic anaerobic biodigesters (PABs). While HEV was consistently detected in manure from grow-finish pig farms (&gt;4 log HEV genome copies (GC) L&minus;1), the virus was not detected in manure from maternity and nursery farms. These findings suggest a potential high biosafety status during primary-swine production, with a subsequent contamination in grow-finish production. The anaerobic biodigestion process reduced more than 2 log10 HEV GC in the processed swine manure. However, the virus concentration in final effluent remained high, with an average value of 3.85 log10 HEV GC L&minus;1. Consequently, our results demonstrate that PABs can be a robust tool for effective inactivation of HEV, while reinforcing the need for sanitary surveillance and legislation of swine manure-derived biofertilizers, to avoid the spread of zoonotic enteric pathogens such as HEV

Presence of enteric viruses, bioaccumulation and stability in Anomalocardia brasiliana clams (Gmelin, 1791)

Bivalve mollusks are filter feeders and may accumulate human pathogens in their tissues. Many studies demonstrated human diseases associated with bivalve consumption, especially oysters. Anomalocardia brasiliana clams are distributed along the Brazilian coastal area and are an exotic ingredient for some typical dishes in Brazil. Even though there are several reports describing the contamination of oysters and mussels with human pathogens, there is a lack of studies reporting contamination of A. brasiliana with human pathogens. An evaluation of natural microbiological contamination in A. brasiliana samples over a period of 18 months (November 2014 to April 2016) showed that the bacteria indices were in accordance with Brazilian regulations (E. coli < 230 MPN and Salmonella sp. absent in 25 g of meat). However, the enteric viruses evaluated were detected throughout the analysis period, with the highest result for the hepatitis A virus (HAV); followed by Rotavirus-A (RVA); Human Adenovirus (HAdV) and Norovirus GI (NoV GI). The bioaccumulation of enteric viruses by A. brasiliana during a period of 24 h was performed using NoV GI and GII, HAV, RVA and HAdV as models. Interestingly the mollusk demonstrated different uptake behaviors in relation to these viruses throughout the time period. NoV GI was the most adsorbed virus after 24 h. HAV concentration was < 1% at 3 h, but it increased to < 10% at 8 h, remaining unchanged until 12 h, and decreasing to < 3% at 24 h; HAdV reached its highest concentration at 12 h, being released by the animals and lowering to < 3% at 24 h. RVA bioaccumulation was unstable over time, reaching its highest values after 24 h (< 5%); NoV GII bioaccumulation remained < 1%. Thermal inactivation of HAdV-2 in A. brasiliana was also evaluated. After the usual gentle cooking procedure using different times (0, 1, 1.5, 3 and 5 mins), viral infectivity was evaluated using ICC-et-RT-qPCR. The temperature inside the DT remained < 80 °C over time and after 5 min of cooking the HAdV reached a decay of 90% (1 log10). The results showed a real warn to the consumers that can be exposed to infectious human viruses if they eat these clams improperly cooked. HAV was the most detected virus in these animals, which may lead to outbreaks. A. brasiliana exhibited distinct behavior in NoV GI bioaccumulation and persistence, pointing to the need for further studies about the cellular ligands used by these viruses to become attached to these clams

Detection of Enteric Viruses and Core Microbiome Analysis in Artisanal Colonial Salami-Type Dry-Fermented Sausages from Santa Catarina, Brazil

Microbial fermentation plays an important role in the manufacturing of artisanal sausages and can have major effects on product quality and safety. We used metagenomics and culture-dependent methods to study the presence of Hepatitis E virus (HEV) and Rotavirus-A (RV-A), and fungal and bacterial communities, in artisanal Colonial salami-type dry-fermented sausages in Santa Catarina state, Brazil. Lactic acid bacteria (LAB) and yeast dominated the microbiome. Latilactobacillus sakei and Debaryomyces hansenii were ubiquitous and the most abundant species. The DNA of some foodborne pathogens was found in very low concentrations although viable cells of most of these species were undetectable by cultivation methods. The characteristics of the raw material and hygiene of the artisanal sausage manufacturing process resulted in high loads of beneficial microorganisms and the absence of HEV and RV-A viruses as determined by RT-qPCR assays. In conclusion, high LAB load in sausages was more relevant to preventing pathogen growth than the ripening time and/or physicochemical characteristics. However, the presence of Clostridium spp. and other pathogens in some samples must be taken into account for the development of future preservation methods; appropriate LAB starter cultures and health surveillance are required in the production process to prevent foodborne outbreaks

Microbiological and physicochemical analysis of the coastal waters of southern Brazil

The aim of this study was to assess the impact of sewage discharge on coastal waters by evaluating the influence of physicochemical parameters on the presence of enteric microorganisms in seawater samples collected from 11 beaches in Florianopolis, Santa Catarina, Brazil, over a one-year period (August 2009 to July 2010). Samples were assessed for the presence of human adenoviruses (HAdV), polyomavirus (JCPyV), hepatitis A virus (HAV), and noroviruses (HuNoV GI and GII). Escherichia coli and physicochemical parameters (salinity, temperature, pH and dissolved oxygen) were also evaluated. From the 132 samples analyzed, 55% were positive for HAdV, 51.5% for HAV, 7.5% for HuNoV GI, 4.5% for HuNoV GII, and 3% for JCPyV. E. coli levels ranged from 8 to 1325 CFU/100 mL at all sites. The overall results highlight the problem of sewage discharge into coastal waters and confirm that there is no correlation between viral presence and bacterial contamination

Genomic Surveillance of SARS-CoV-2 in Healthcare Workers: A Critical Sentinel Group for Monitoring the SARS-CoV-2 Variant Shift

SARS-CoV-2 genome surveillance is important for monitoring risk groups and health workers as well as data on new cases and mortality rate due to COVID-19. We characterized the circulation of SARS-CoV-2 variants from May 2021 to April 2022 in the state of Santa Catarina, southern Brazil, and evaluated the similarity between variants present in the population and healthcare workers (HCW). A total of 5291 sequenced genomes demonstrated the circulation of 55 strains and four variants of concern (Alpha, Delta, Gamma and Omicron—sublineages BA.1 and BA.2). The number of cases was relatively low in May 2021, but the number of deaths was higher with the Gamma variant. There was a significant increase in both numbers between December 2021 and February 2022, peaking in mid-January 2022, when the Omicron variant dominated. After May 2021, two distinct variant groups (Delta and Omicron) were observed, equally distributed among the five Santa Catarina mesoregions. Moreover, from November 2021 to February 2022, similar variant profiles between HCW and the general population were observed, and a quicker shift from Delta to Omicron in HCW than in the general population. This demonstrates the importance of HCW as a sentinel group for monitoring disease trends in the general population