Formação de biofilme por Vibrio parahaemolyticus isolados de pescados / Biofilm formation by Vibrio parahaemolyticus isolated from fish

O pescado é um alimento altamente perecível, possui pH próximo a neutralidade, elevada atividade de água e alto teor de nutrientes facilmente utilizáveis por micro-organismos. Vibrio parahaemolyticus pode ser encontrado em ambientes com salinidade entre 3% e 8% e tem pH ideal para multiplicação entre 7,8 e 8,6. É um patógeno que pode causar gastrenterite aguda pelo consumo de frutos do mar contaminados, crus ou mal cozidos. Mesmo os processos de tratamento de água como cloração, adição de antibióticos e filtros apresentam dificuldade em reduzir a contaminação por Vibrio, sendo suposto que este gênero bacteriano pode formar biofilmes em diferentes superfícies. O objetivo do trabalho foi verificar a capacidade de V. parahaemolyticus isolados de pescados formarem biofilme após estresse subletal. No decorrer de um ano, foram realizadas 12 coletas mensais de amostras de peixes capturados no estuário da Lagoa dos Patos, as quais foram analisadas quanto à presença de V. parahaemolyticus. Concomitantemente, foram coletadas assepticamente amostras de água do estuário para análise de sanilidade e pH. Os isolados de Vibrio foram analisados pela reação em cadeia da polimerase (PCR) para identificação da espécie pela presença dos genes toxR. Além dos isolados obtidos no presente trabalho, também foram estudadas 15 outras cepas de V. parahaemolyticus previamente isoladas em outros trabalhos. As cepas foram avaliadas quanto à capacidade de produção de biofilme em placas de microtitulação. A capacidade de produção de biofilme após as cepas serem submetidas a diferentes tipos de estresse subletal (42ºC, 20ºC, 4ºC e pH ácido) também foi testada. Dentre os 120 peixes analisados, foram isolados V. parahaemolyticus de quatro (3,33%) pescados, sendo Mugil platanus a única espécie de peixe na qual o micro-organismo foi encontrado. Das 19 cepas analisadas, 89,5% foram capazes de formar biofilme, o que parece indicar que essa capacidade tem um papel importante na sobrevivência do micro-organismo nos pescados. Dessas, 25% das cepas aumentaram a capacidade de formar biofilme. Com base nos resultados, conclui-se que peixes da espécie M. platanus do estuário da Lagoa dos Patos são hospedeiros de V. parahaemolyticus e que a quase totalidade das cepas são formadoras de biofilme. A exposição a condições subletais de estresse tem efeito distinto sobre as diferentes cepas, induzindo aumento na capacidade de formar biofilme em algumas. Este foi o primeiro estudo realizado com V. parahaemolyticus, para avaliar o efeito de fatores de estresse sobre a formação de biofilme.(AU)

Fish is a highly perishable food, has a neutral pH, high water activity and high content nutrient, which makes it favorable to the microorganisms multiplication. Vibrio parahaemolyticus may be found in environments with a salinity of 3% and 8% and has optimal pH for multiplication between 7.8 and 8.6. This pathogen can cause acute gastroenteritis by consumption of contaminated raw or undercooked seafood. There is difficulty in reducing Vibrio contamination during fish processing, being supposed that this bacterial genus can form biofilm on different surfaces. The aim of this study was to verify the ability of V. parahaemlyticus isolated from fish from biofilm after sublethal stress. In the course of one year, 12 monthly samples of fish caught in the Lagoa dos Patos Estuary were analyzed for the presence of V. parahaemolyticus. Concurrently, water samples from estuary were collected aseptically for salinity analysis and pH. Vibrio isolates were analyzed by polymerase chain reaction (PCR) to identification of the species by presence of the toxR gene. In addition to the isolates obtained in this study were also studied 15 other strains of V. parahaemolyticus previously isolated in other works. The strains were evaluated for biofilm production capacity in microtiter plates. The biofilm production capacity after the strains had being subjected to different types of sublethal stress (42oC, 20°C, 4°C and acid pH) was also tested. Among the 120 analyzed fish, V. parahaemolyticus were isolated from four (3.33%) fishes, and Mugil platanus was the only species in which the microorganism was found. Among the 19 strains analyzed, 89.5% were able to form biofilm, which seems to indicate that this ability has an important role in the microorganism survival in the fish. Among these strains, 25% increased the ability to form biofilm after sublethal exposure. Based on the results, we concluded that fish of the species M. platanus of the Lagoa dos Patos Estuary are hosts of V. parahaemolyticus and that almost all of these strains are forming biofilm. Exposure to sublethal stress conditions has distinct effect on different strains, inducing an increase in the ability to form biofilm in some. This was the first study about the effects of stress on the V. parahaemolyticus biofilms formation.(AU)

Exogenous enzyme complex prevents intestinal soybean meal-induced enteritis in Mugil liza (Valenciennes, 1836) juvenile

ABSTRACT Four soybean meal-based diets containing increasing levels of an enzyme complex (E50, E100, E150 and E200 at 50, 100, 150 and 200 g ton-1, respectively) and one soybean meal-based diet without the enzyme complex (E0) were fed in triplicate to M. liza juveniles in a semi-static flow system with 20 fish per tank for 75 days. There were no differences between the treatments for animal performance parameters, but fish fed the enzyme complex treatment exhibited significantly (P<0.05) higher values of calcium bone retention compared with control fish. Although there was no relationship between bacterial counts in different sections of the gastrointestinal tract or enzyme levels, filamentous bacteria were increased in E50 compared with E150. All of the treatments resulted in higher bacterial counts in the stomach than in intestinal segments. Histological screening showed serious to moderate infiltration of inflammatory cells, modification in villus morphology and necrosis in some cases in fish fed the E0 diet. In addition, fish from the E0 treatment exhibited significantly (P<0.05) lower lipid deposition in the peritoneal cavity. Therefore, the use of low levels of exogenous enzyme is recommended in diets for M. liza when soybean meal is used as the main source of protein.

Isolation and experimental infection with Vibrio alginolyticus in the sea horse, Hippocampus reidi Ginsburg, 1933 (Osteichthyes: Syngnathidae) in Brazil / Isolamento e infecção experimental de Vibrio alginolyticus em cavalo-marinho, Hippocampus reidi Ginsburg, 1933 (Osteichthyes: Syngnathidae) no Brasil

The aim of this study was to evaluate the pathogenicity of Vibrio alginolyticus isolated from an outbreak of sea horse Hippocampus reidi reared in the State of Santa Catarina, Brazil, by experimental infection. Sea horses with necrosis on the mouth epithelium were collected from aquaria at the Aquaculture Department, UFSC and the bacterium isolated from the mouth, liver, heart and blood in tiosulphate citrate bilesalt sucrose agar broth. The strains were identified by API 20E kit with 99.1 percent probability as Vibrio alginolyticus. Twelve adult sea horses (9.63 ± 2.42 g and 15.12 ± 0.87 cm) were distributed in six aquaria of 10 L capacity with aerated sea water. Fish from three aquaria were submitted to an immersion bath in a solution containing 1.0 × 10(7) CFU of V. alginolyticus/mL for 15 minutes. Fish from the other three aquaria received the same procedure without bacteria. Twenty four hours after this challenge, 100 percent mortality was observed in the animals infected with V. alginolyticus. No mortality was observed in non-infected fish. Hyperplasia, displacement and fusion of secondary lamellae of the gills; leukocyte infiltration and necrotic foci in the kidney; hyperplasia, sinusoidal deformation and necrotic foci in the liver were observed in histopathological analysis. The V. alginolyticus isolated in this study was pathogenic to H. reidi and constitutes an important sanitary problem to its culture.

Foi avaliado por meio de infecção experimental a patogenicidade de Vibrio alginolyticus isolado de um surto de enfermidade em cavalo-marinho Hippocampus reidi cultivado no Estado de Santa Catarina, Brasil. Os peixes com necroses no epitélio bucal foram coletados em aquários do Departamento de Aquicultura, UFSC e as bactérias isoladas da boca, fígado, coração e sangue em meio Agar tiossulfato citrato bile sacarose. Os isolados foram identificados pelo kit API 20E como Vibrio alginolyticus com 99,1 por cento de probabilidade. Doze peixes adultos (9,63 ± 2,42 g e 15,12 ± 0,87 cm) foram distribuídos em seis aquários de 10 L com água marinha e aeração. Peixes de três aquários foram submetidos a um banho de imersão por 15 minutos em uma solução contendo 1,0 × 10(7) UFC de V. alginolyticus/mL. Nos outros três aquários realizou-se o mesmo procedimento sem a bactéria. Vinte e quatro horas após o desafio, 100 por cento de mortalidade foi observada nos animais infectados com V. alginolyticus. Não houve mortalidade nos peixes não infectados. Nas análises histopatológicas, foi observado hiperplasia, deslocamento do epitélio e fusão das lamelas secundárias das brânquias; infiltração de leucócitos e necrose no rim; hiperplasia, deformação sinusoidal e necrose no fígado nos animais desafiados com V. alginolyticus. O V. alginolyticus isolado neste estudo foi patogênico para H. reidi, constituindo-se de um importante problema para seu cultivo.

Effects of bacterial infestation caused by human wastes on the skin structures of Mugil platanus Günther, 1880 (Mugilidae) / Efeitos da infestação de bactérias provocada por resíduos humanos na pele de Mugil platanus Günther, 1880 (Mugilidae)

Manifestation of infectious pathologies in fishes usually increases in environments where organic wastes are disposed. Specimens of Mugil platanus Günther, 1880 and water samples collected at three points of the Tramandaí river were analyzed during a one year period. The macroscopic observation revealed ulcerations in the caudal peduncle area covered with a mass of amorphous and whitened tissues. Histopathologic analysis showed the presence of negative gram bacteria, probably responsible for alterations of the normal structure of the epidermic tissues. Non-parametric statistical analysis for ammonia concentration showed a significant variation among the three collected spots as well as in the multiple comparison between two spots. In this study, we describe cutaneous lesions observed in Mugil platanus specimens and tested their correlation with environmental ammonia concentration.

A manifestação de patologias infecciosas em populações de peixes costuma ser incrementada em ambientes onde são lançados despejos orgânicos. Exemplares da espécie Mugil platanus Günther, 1880 coletados em três pontos do rio Tramandaí foram utilizados para análise das alterações, e amostras de água foram tomadas durante o período de um ano para verificação da presença de bactérias. A observação macroscópica revelou ulcerações na região do pedúnculo caudal revestidas por uma massa de tecido amorfo e esbranquiçado. A análise histopatológica mostrou a presença de bactérias gram negativas, que provavelmente foram as responsáveis pela alteração da estrutura normal dos tecidos de revestimento do corpo. O resultado da análise estatística não paramétrica para a concentração de amônia mostrou variação significativa entre os três pontos amostrados bem como nas comparações múltiplas entre os pontos dois a dois. Este trabalho descreve as lesões cutâneas observadas em Mugil platanus e testa sua correlação com a concentração de amônia.

Partial purification and anti-leukemic activity of L-asparaginase enzyme of the actinomycete strain LA-29 isolated from the estuarine fish, Mugil cephalus (Linn.).

The actinomycete strain LA-29 isolated from the gut contents of the fish, Mugil cephalus of the Vellar estuary showed excellent L-asparaginase activity The enzyme was purified 18-fold and the final recovery of protein was 1.9%, which exhibited an activity of 13.57 IU/mg protein. The partially purified L-asparaginase inhibited the growth of leukemia cells in male wistar rats. Average survival period of the rats was more in an optimum enzyme dose of 100 units and the survival period was less when the dosages were increased and at the same time the enzyme became less effective when the dosages were decreased. Higher survival of 17.2 days was recorded when 100 units of the enzyme was given in three intermittent doses (50/25/25 units) at the interval of 24 hr. Analysis of cell components of the strain LA-29 has revealed the wall type-I which is the characteristic of the genus Streptomyces. Further the morphological, physiological and biochemical features along with the micromorphological results obtained for the strain LA-29 were compared with that of the Streptomyces species found in Bergey's Manual of Determinative Bacteriology and the strain LA-29 has been tentatively identified as Streptomyces canus.