Tadpoles fed supplemented diet with probiotic bacterium isolated from the intestinal tract of bullfrog Lithobates catesbeianus: Haematology, cell activity and electron microscopy.

The aim of this study is to select and isolate autochthonous bacteria with probiotic potential for use in a supplemented diet for bullfrog tadpoles, Lithobates catesbeianus. A total of 20 strains of lactic acid bacteria were isolated. Nine out of these were used in the following in vitro assays: antagonism against pathogenic bacteria (ANT), antimicrobial activity from extracellular compounds (MIC), tolerance to bile salts (TBS), pH reduction, protease production, sensitivity to antimicrobial tetracycline, cell viability, growth rate and doubling time. Using these data was defined an ideotype (ideal strain) based on the best results. Distances were estimated with the Mahalanobis (D2) test, and the best candidates, presenting the shortest ideotype distances, were considered to be used. The best strain was found to be Lactobacillus plantarum because it presented 10.00 ± 0.50 mm of ANT against Aeromonas hydrophila, 3.99 ± 0.01 of MIC independent of pathogenic bacteria, 85.07 ± 0.01 of TBS, 4.20 ± 0.02 of final pH, 17.67 ± 1.15 of protease production, 13.50 ± 2.00 sensitivity to antimicrobial tetracycline, 9.36 ± 0.04 of cell viability, 0.20 ± 0.00 of growth rate and 3.46 ± 0.00 doubling time. Therefore this probiotic candidate was then supplemented (2.045 ± 1.07 × 107 colony forming unities. g-1) into the diets of bullfrog tadpoles for a period of 42 days. At the end of the trial, samples of blood and intestines were collected to verify the haematological alterations and the intestinal morphology using transmission and scanning electron microscopy. Tadpoles fed the supplemented diet showed successful lactic acid bacterium colonisation, an increased number of circulating thrombocytes, monocytes, eosinophil and LG-PAS+ and also an increase in the length and density of intestinal microvilli. This study shows the feasibility of using probiotics isolated from farmed bullfrogs as a supplement in the diets of tadpoles, providing a promising alternative for modulating the health of these animals.

Dairy production practices and associated risks for bovine vaccinia exposure in cattle, Brazil.

A cross-sectional serosurvey was performed to identify environmental features or practices of dairy farms associated with risk for exposure to vaccinia-like viruses in dairy cattle in Brazil. Sera from 103 cows from 18 farms in Minas Gerais state were examined for Orthopoxvirus-neutralizing antibodies. A database of 243 binary or multiple-selection categorical variables regarding the physical features and surrounding ecology of each property was obtained. Thirteen of 46 presumptive predictor variables were found to be significantly associated with Orthopoxvirus serostatus by univariate logistic regression methods. Use of teat sanitizer and having felids on the property were independently associated with virus exposure by multivariable analysis. Rodents have long been suspected of serving as maintenance reservoirs for vaccinia-like viruses in Brazil. Therefore, domestic felids are not only effective predators of small rodent pests, but also their urine can serve as a deterrent to rodent habitation in buildings such as stables and barns. These results corroborate previous evidence of the high significance of rodents in the Vaccinia virus transmission cycle, and they also raise questions regarding the common use of teat sanitizers in dairy production areas.

Effect of probiotic supplemented diet on marine shrimp survival after challenge with vibrio harveyi / Efeito do uso de dieta suplementada com o probiótico sobre a sobrevivência de camarões marinhos após o desafio com vibrio harveyi

The effect of a Lactobacillus plantarum-supplemented diet on shrimp growth, digestive tract bacterial microbiota, survival, and some hemato-immunological parameters after an experimental challenge with Vibrio harveyi was studied. No difference (P>0.05) was observed in shrimp survival (80±6 percent) and final weight (6.63±0.56g) after 60 days feeding trial. Total bacteria count and Vibrio spp. count in the digestive tract were not diferent (P>0.05) until day 40, but they were lower (P<0.05) in the probiotic-supplemented group on day 60. Total lactic bacteria in the shrimp digestive tract was higher after day 20 in the probiotic-supplemented group. Ten hours after V. harveyi challenge, survival of the probiotic-supplemented group (65.7±2.9 percent) was higher (P>0.05) than the control group (39.9±4.4 percent). Bacterial counts in hemolymph and hepatopancreas was lower (P<0.05) in the probiotic-supplemented group than in the control group after V. harveyi challenge. Total hemocyte count and serum agglutination activity were higher (P>0.05) in the probiotic-supplemented group after challenge with V. harveyi. Probiotic-supplemented diet modifies shrimp digestive tract bacterial microbiota, increasing resistance to V. harveyi infection.

Avaliou-se o efeito de uma dieta suplementada com Lactobacillus plantarum na engorda de Litopenaeus vannamei sobre a microbiota bacteriana do trato digestivo, a sobrevivência e alguns parâmetros hematoimunológicos após desafio com Vibrio harveyi. Não foi observada diferença significativa na sobrevivência (80±6 por cento) e no peso final dos camarões (6,63±0,56g) durante a engorda (60 dias). A população de bactérias totais e Vibrio spp. no intestino não diferiu até o 40º dia de cultivo, mas foi inferior nos camarões alimentados com dieta suplementada com probiótico no 60º dia de cultivo. A população de bactérias lácticas foi superior no intestino de camarões alimentados com dieta suplementada com probióticos após o 20º dia de cultivo. Dez horas após o desafio, a sobrevivência dos camarões alimentados com dieta suplementada com probiótico foi superior (65,7±2,9 por cento) a dos camarões do controle (39,9±4,4 por cento). Camarões alimentados com dieta suplementada com probióticos e desafiados com V. harveyi apresentaram menor contagem de bactérias na hemolinfa e hepatopâncreas. A contagem total de hemócitos e a atividade aglutinante da hemolinfa foram superiores nos camarões inoculados com V. harveyi alimentados com dieta suplementada com probióticos. A suplementação da dieta com probióticos modifica a microbiota bacteriana intestinal dos camarões aumentando sua resistência ao desafio com V. harveyi.

Isolation and experimental infection with Vibrio alginolyticus in the sea horse, Hippocampus reidi Ginsburg, 1933 (Osteichthyes: Syngnathidae) in Brazil.

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 thiosulphate citrate bilesalt sucrose agar broth. The strains were identified by API 20E kit with 99.1% 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 x 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% 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.

Haematological changes in Nile tilapia experimentally infected with Enterococcus sp.

This study evaluated the haematological changes in Nile tilapia experimentally infected with 1 x 10(3) and 1 x 10(6) colony-forming units (CFU)/mL of Enterococcus sp. in the swim bladder. The experiment consisted of four treatments in triplicates: non-injected fish (NI); fish injected with 1 mL of sterile saline solution 0.65% (SAL); fish injected with 1 x 10(3) and 1 x 10(6) CFU/mL of Enterococcus diluted in 1 mL sterile saline. Twenty-four hours after injection, the fish were anesthetized and the blood collected. The haematological tests included red blood cell (RBC) and white blood cell (WBC) counts, hematocrit, number of total thrombocytes, and differential counting of WBC. Fish injected with 1 x 10(6) CFU/mL of Enterococcus showed a higher number of thrombocytes than the other treatments. White blood cell and lymphocyte numbers increased significantly in fish injected with 1 x 10(6) CFU/mL of Enterococcus when compared to non-injected control. There was significant increase in the number of neutrophils in saline injected fish and reduced number of monocytes after injections with 1 x 10(6) CFU/mL of Enterococcus. Hematocrit increased in fish injected with 1 x 10(3) and 1 x 10(6) CFU/mL of Enterococcus.

Haematological changes in Nile tilapia experimentally infected with Enterococcus sp[period] / Alterações hematológicas em tilápia do Nilo infectada experimentalmente com Enterococcus sp[ponto final]

This study evaluated the haematological changes in Nile tilapia experimentally infected with 1 x 10³ and 1 x 10(6) colony-forming units (CFU)/mL of Enterococcus sp. in the swim bladder. The experiment consisted of four treatments in triplicates: non-injected fish (NI); fish injected with 1 mL of sterile saline solution 0.65 percent (SAL); fish injected with 1 x 10³ and 1 x 10(6) CFU/mL of Enterococcus diluted in 1 mL sterile saline. Twenty-four hours after injection, the fish were anesthetized and the blood collected. The haematological tests included red blood cell (RBC) and white blood cell (WBC) counts, hematocrit, number of total thrombocytes, and differential counting of WBC. Fish injected with 1 x 10(6) CFU/mL of Enterococcus showed a higher number of thrombocytes than the other treatments. White blood cell and lymphocyte numbers increased significantly in fish injected with 1 x 10(6) CFU/mL of Enterococcus when compared to non-injected control. There was significant increase in the number of neutrophils in saline injected fish and reduced number of monocytes after injections with 1 x 10(6) CFU/mL of Enterococcus. Hematocrit increased in fish injected with 1 x 10³ and 1 x 10(6) CFU/mL of Enterococcus.

Este estudo avaliou alterações hematológicas em tilápia do Nilo infectada experimentalmente com 1 x 10³ e 1 x 10(6) unidades formadoras de colônia (CFU)/mL de Enterococcus sp. na bexiga natatória. O experimento consistiu de quatro tratamentos com três repetições cada: peixes não injetados (NI); peixes injetados com 1 mL de solução salina a 0,65 por cento esterilizada (SAL); peixes injetados com 1 x 10³ e 1 x 10(6) CFU/mL de Enterococcus diluída em 1 mL de solução salina esterilizada. Vinte e quatro horas após as injeções, os peixes foram anestesiados com benzocaína e o sangue coletado. Os testes hematológicos incluíram as contagens totais de eritrócitos (RBC), de leucócitos (WBC) e de trombócitos, o percentual do hematócrito e a contagem diferencial de leucócitos. Peixes injetados com 1 x 10(6) CFU/mL de Enterococcus mostraram maior número de trombócitos no sangue do que os dos outros tratamentos. O número de leucócitos totais e o número de linfócitos foram significativamente mais altos após injeção de 1 x 10(6) CFU/mL de Enterococcus, quando comparados ao controle não injetado. Houve aumento no número de neutrófilos nos injetados com salina e redução no número de monócitos nos injetados com 1 x 10(6) CFU/mL de Enterococcus. O hematócrito aumentou nos animais injetados com 1 x 10³ e 1 x 10(6) CFU/mL de Enterococcus.

Characterization and experimental infection of Flexibacter maritimus (Wakabayashi et al. 1986) in hatcheries of post-larvae of Litopenaeus vannamei Boone, 1931.

A preliminary study to characterize filamentous bacteria, whose presence is related to high mortality of Litopenaeus vannamei larvae cultured in Santa Catarina State, Brazil, is reported. The extract of infected larvae was diluted in different concentrations, cultured in marine agar (Difco, Marine Agar 2216) and incubated at 30 degrees C for 48 hours. The biochemical characterization included hydrolytic reactions of starch, gelatin and tyrosine, growth in TCBS agar, growth in 0 and 37 per thousand salinity, pigment production in tyrosine agar, production of H2S, nitrate reduction, congo red reaction, oxidase and catalase. The isolated bacteria belong to the species Flexibacter maritimus, Gram-negative bacilli of 0.4-0.5 microm width and 15 microm length. Experiments were carried out on pathogenicity of F. maritimus in post-larvae of L. vannamei. Survival and symptoms in L. vannamei post-larvae 24 hours after inoculation with F. maritimus and its growth in marine agar were evaluated. Mortality was detected around 92,5% as well as symptoms like melanized lesions in several parts of body, discolouration of gills, bad formation of appendages and of the last abdominal segment, low motility and feeding reduction. The experimental infection results suggested that isolated bacteria of the genus Flexibacter are pathogenic to the shrimp Litopenaeus vannamei post-larvae.

Characterization and experimental infection of Flexibacter maritimus (Wakabayashi et al. 1986) in hatcheries of post-larvae of Litopenaeus vannamei Boone, 1931

A preliminary study to characterize filamentous bacteria, whose presence is related to high mortality of Litopenaeus vannamei larvae cultured in Santa Catarina State, Brazil, is reported. The extract of infected larvae was diluted in different concentrations, cultured in marine agar (DifcoTM, Marine Agar 2216) and incubated at 30 °C for 48 hours. The biochemical characterization included hydrolytic reactions of starch, gelatin and tyrosine, growth in TCBS agar, growth in 0 and 37‰ salinity, pigment production in tyrosine agar, production of H2S, nitrate reduction, congo red reaction, oxidase and catalase. The isolated bacteria belong to the species Flexibacter maritimus, Gram-negative bacilli of 0.4-0.5 µm width and 15 µm length. Experiments were carried out on pathogenicity of F. maritimus in post-larvae of L. vannamei. Survival and symptoms in L. vannamei post-larvae 24 hours after inoculation with F. maritimus and its growth in marine agar were evaluated. Mortality was detected around 92,5 percent as well as symptoms like melanized lesions in several parts of body, discolouration of gills, bad formation of appendages and of the last abdominal segment, low motility and feeding reduction. The experimental infection results suggested that isolated bacteria of the genus Flexibacter are pathogenic to the shrimp Litopenaeus vannamei post-larvae.

Neste trabalho, relata-se um estudo preliminar para caracterizar bactérias filamentosas associadas a elevadas mortalidades de larvas e pós-larvas cultivadas no Estado de Santa Catarina, Brasil. Um extrato de larvas infectadas foi diluído em diferentes concentrações, cultivado em Agar Marine (DifcoTM, Marine Agar 2216) e incubado a 30° C por 48 horas. A caracterização bioquímica inclui reações de amido, gelatina e tirosesine, crescimento em Agar TCBS,crescimento a 0 e 37‰ de salinidade, produção de pigmentos em Agar Tirosine, produção de H2S, redução de nitrato, reação vermelho congo, oxidase e catalase. A bactéria isolada apresentou características de Flexibacter maritimus, um bacilo Gram negativo de 0,4-0,5 µm de largura e 15 µm de comprimento. Os experimentos foram realizados sobre a patogenicidade de F. Maritimus, em pós-larvas de L. vannamei. Foram avaliados a sobrevivência e os sintomas das pós-larvas 24 horas após a inoculação com F. Maritimus, além do seu crescimento em Agar Marine. Foram encontradas mortalidades de 92,5 por cento e sintomas próprios da enfermidade, tais como lesões melanizadas em várias partes do corpo, descoloração das brânquias e diminuição da motilidade e da alimentação. Com base nos resultados da infecção experimental, pode se concluir que a bactéria isolada do gênero Flexibacter é patogênica para pós-larvas de L. vannamei.

The water quality of the Cértima River basin (Central Portugal).

The aim of this study was to evaluate the water quality of the Cértima River basin (Central Portugal). For that purpose, surface water samples were collected in March, May and July 2003, at 10 selected sampling sites, and were analysed for physicochemical parameters, namely temperature, conductivity, pH, total suspended solids, dissolved oxygen, biochemical oxygen demand (BOD(5)), Kjeldahl nitrogen and total phosphorus. Results revealed an acceptable water quality during the spring season. Maxima of 64 mg dm(-3) for BOD(5), 39 mg dm(-3) for Kjeldahl nitrogen, and 5.2 mg dm(-3) for total phosphorus, were recorded during summer, indicating a significant degradation of the water quality in a river stretch located downstream of the town of Mealhada. These values, which did not comply with the objectives of minimum quality for surface waters prescribed by the Portuguese legislation, were related to domestic wastewater discharges and runoff waters from a cattle farm. Besides their effects on the middle stretch of the river, these pollution sources were the most likely cause of the high nutrient load in downstream waters, and thus may have a major impact on the trophic status of Pateira de Fermentelos, a sensitive wetland area located in the lower Cértima basin.