Probiotic supplementation causes hematological changes and improves non-specific immunity in Brycon amazonicus

A commercialprobioticcontaining Bacillussubtilis(109 CFU g-1) was evaluated incaged matrinxã,Bryconamazonicus,by measuring hematological parametersand macrophage activity after 42 and 84 days after feeding. The product wasadded tocommercial feed using 2%soybean oil as a protectant. A randomized three-treatmentexperiment was performed using fourreplicates per treatment. The groups included: (a) control without probiotic, (b) 5 g kg-1probiotic, and (c) 10 g kg-1probiotic. Forhematological analysis,eightfishper treatmentwere used to determinetotal cell count (RBC); thrombocytes, differential, and total leukocyte count (TLC); hematocrit (Htc); hemoglobin tax; mean corpuscular volume (MCV); and mean corpuscular hemoglobin concentration (MCHC). Furthermore, plasma cortisol and glucose levels were measured in blood samples. Macrophage phagocytic activity was evaluated by injecting Saccharomyces cerevisiae(11,000 cells in a 3 mLvolume) into the coelomic cavity incubating for 8hours.Addition of probiotics to the diet of caged matrinxã altered the Htc, RBC, MCV, MCHC, TLC, lymphocyte, and eosinophil values. We observed increased cortisol and glucose levels and phagocytic activity, but no increase in the phagocytic index. We thus conclude that supplementing caged Brycon amazonicuswith probiotics improves their non-specific immunity and alters blood profiles.

Isolation of Saprolegnia aenigmatica oomycetes and protocol for experimental infection of pacu (Piaractus mesopotamicus) / Isolamento do oomiceto Saprolegnia aenigmatica e protocolo de infecção experimental em pacu (Piaractus mesopotamicus)

Successful disease treatment depends on molecular studies under indoor conditions with experimental infection protocols that facilitate understanding the disease and the drug`s efficacy. The internal transcribed spacer (ITS) region was sequenced from three isolates, which were identified as Saprolegnia aenigmatica. Subsequently, healthy fish were immunosuppressed with dexamethasone (1.2 mg kg-1) and descaled to the skin using a sharp scalpel. These individuals were isolated in individual aquariums maintained at 22°C. Individuals in one group were subcutaneously inoculated with 9,000 zoospores (DDZ treatment), a second group was exposed to oomycetes in water with three colonized baits (DDB), a third group was maintained in water without zoospores (DD), and a control group (C) consisted of healthy animals. After 48 and 96 hours, two animals from each group were euthanized for fungal reisolation. The fish from groups DD and C did not show clinical signs, and no oomycetes were isolated. The animals from the DDZ and DDB groups showed cotton-wool-like masses on the skin, and S. aenigmatica was re-isolated. Thus, for infection using zoospores or baits parasitized by S. aenigmatica, an immunosuppressor (dexamethasone) and a sharp scalpel can be used effectively to establish an experimental infection in P. mesopotamicus.

O sucesso do tratamento de uma enfermidade depende do estudo de moléculas em condições de laboratório por meio de protocolos de infecção experimental que viabilizam a compreensão da doença e da eficácia dos fármacos. Pela sequência ITS foram identificadas três cepas de Saprolegnia aenigmatica. Dessa forma, pacus sadios foram submetidos à imunossupressão com dexametasona (1,2 mg kg-1), esfoliados com auxílio de bisturi e distribuídos em aquários a 22ºC. Após este procedimento, um grupo de animais foi inoculado com 9.000 zoósporos/peixe subcutâneo (DEZ), a outro foram adicionadas três iscas colonizadas com o oomiceto na água (DEI), um terceiro grupo foi mantido sem contato com o oomiceto (DE) e um quarto grupo, de animais sadios, representaram o controle (C). Após 48 e 96h deste procedimento, foram eutanaziados animais de cada grupo para reisolamento. Os animais do grupo DE e C não apresentaram sinais clínicos e não foi reisolado o oomiceto. Porém, tanto os animais do grupo DEZ quanto como os animais do grupo DEI apresentaram micélio branco na pele e foi reisolado Saprolegnia aenigmatica. Assim, a infecção com zoósporos ou com iscas colonizadas por S. aenigmatica, com o uso de dexametasona e abrasivo epitelial são formas eficazes de infecção experimental em P. mesopotamicus.

Teflubenzuron as a tool for control of trichodinids in freshwater fish: Acute toxicity and in vivo efficacy.

In this study we describe the anti-Trichodina effects of teflubenzuron (TFB) for Oreochromis niloticus and for Piaractus mesopotamicus. We also evaluated the acute toxicity, for both species, by using TFB in the concentrations of 700.0, 800.0, 900.0 and 1000.0 mg L(-1) and a control, without the drug. To assess the efficacy of TFB against Trichodina spp., we used the concentrations of 30.0 or 50.0 mg L(-1) for one hour exposure in tilapia, and the concentration of 30.0, 50.0 and 80.0 mg L(-1) for one hour and 50 mg L(-1) for two hours exposures in pacu. Teflubenzuron did not present significant toxicity in either species, with LC50;48h > 1000.0 mg L(-1). The drug effectiveness was observed against four identified Trichodina species: T. magna, T. heterodentata, T. compacta and T. centrostrigeata, with 87.9% parasite reduction with one hour exposure to 50.0 mg L(-1) TFB on O. niloticus and 96.1% with two hours exposure to 50.0 mg L(-1) TFB on P. mesopotamicus. Teflubenzuron is a drug with potential to be used in Brazilian aquaculture; it attends to important requirements, such as low toxicity and high efficacy in controlling Trichodina spp. infection in O. niloticus and P. mesopotamicus.

Hematological changes and cytogenotoxicity in the tilapia Oreochromis niloticus caused by sub-chronic exposures to mercury and selenium.

Fish bioassays are valuable tools that can be used to elucidate the toxicological potential of numerous substances that are present in the aquatic environment. In this study, we assessed the antagonistic action of selenium (Se) against the toxicity of mercury (Hg) in fish (Oreochromis niloticus). Six experimental groups with six fish each were defined as follows: (1) control, (2) mercury (HgCl(2)), (3) sodium selenite (Na(2)Se(4)O(3)), (4) sodium selenate (Na(2)Se(6)O(4)), (5) mercury + sodium selenite (HgCl(2) + Na(2)Se(4)O(3)), and (6) mercury + sodium selenate (HgCl(2) + Na(2)Se(6)O(4)). Hematological parameters [red blood cells (RBC), white blood cells (WBC), and erythroblasts (ERB)] in combination with cytogenotoxicity biomarkers [nuclear abnormalities (NAs) and micronuclei (MN)] were examined after three, seven, ten, and fourteen days. After 7 days of exposure, cytogenotoxic effects and increased erythroblasts caused by mercury, leukocytosis triggered by mercury + sodium selenite, leukopenia associated with sodium selenate, and anemia triggered by mercury + sodium selenate were observed. Positive correlations that were independent of time were observed between WBC and RBC, ERB and MN, and NA and MN. The results suggest that short-term exposure to chemical contaminants elicited changes in blood parameters and produced cytogenotoxic effects. Moreover, NAs are the primary manifestations of MN formation and should be included in a class characterized as NA only. Lastly, the staining techniques used can be applied to both hematological characterization and the measurement of cytogenotoxicity biomarkers.

Dietary administration of Bacillus subtilis on hematology and non-specific immunity of Nile tilapia Oreochromis niloticus raised at different stocking densities.

An 84-day feeding trial was conducted to evaluate the effect of the dietary administration of Bacillus subtilis on the growth performance, body composition, intestinal probiotic recovery, hematology, and non-specific immunity of Nile tilapia (Oreochromis niloticus) raised at two stocking densities. Five hundred twenty male Nile tilapias (32.63 ± 1.25 g) were distributed in 16,800-L tanks. The experimental design was completely randomized using four replications and a 2 × 2 factorial scheme with two stocking densities (18.75 fish m(-3) 62.50 fish m(-3)) and two diets (control and with probiotic). The probiotic-supplemented diet included 5 × 10(6) CFU g feed(-1). There were no significant differences (P > 0.05) in the growth performance, body composition, and levels of cortisol and glucose between the animals fed with the control diet and the animals fed with the probiotic-supplemented diet. Differences in the growth performance were observed between the fish reared at different stocking densities; in particular, the fish raised at the high stocking density exhibited reduced weight gain, feed intake, and specific growth rate compared with those raised at the low stocking density. The B. subtilis remained viable after its inclusion in the feed, storage, and passage through the stomach, which demonstrations the feasibility of using this bacteria as a probiotic. Higher values (P < 0.05) in the plasma lysozyme levels and phagocytic activity were observed in the fish that received the probiotic-supplemented diet and reared at the high stocking density, but this difference was not observed in the fish raised at the low stocking density and fed the different diets. The administration of the probiotic caused decreases in the number of erythrocytes and the hematocrit level in the fish reared at the high stocking density, but these erythrocytes showed higher values of mean corpuscular hemoglobin. The stocking density was shown to be a stressor agent that causes a lower fish growth rate. The fish fed the control diet and raised at the high stocking density had lower lysozyme levels than the fish subjected to the other treatments. The inclusion of the probiotic bacteria B. subtilis at a concentration of 5 × 10(6) CFU g feed(-1) benefited the innate immune system of Nile tilapia by decreasing the stress associated with exposure to a high stocking density, increasing the mean corpuscular hemoglobin, and improving the innate immune system (lysozyme and phagocytic activities of macrophages).

Determination of selenium toxicity to Oreochromis niloticus based on hematological parameters / Determinação da toxicidade do Selênio por meio de análises hematológicas em Oreochromis niloticus

Selenium (Se) is described as an essential micronutrient and participates in different biological functions, as the antioxidant defense systems maintenance and regulation. However, when in high concentrations, Se may cause toxic effects as well as hematological changes in fish. The aim of the present study was to determine the toxicity of selenium in the form of sodium selenate (Na2Se6+O4) in Oreochromis niloticus based on hematological parameters, after exposure to different concentrations (0.01, 0. 14 and 1.4 mg Se 6+ L-1). The erythrocytic and leukocytic series were examined over 14 days at intervals of 0, 3, 5, 7,10 and 14 days. The erythrocytic series showed significant alterations in the first 7 days, including the control group. Neutrophils and monocytes showed variations in the first 3 days at a concentration of 1.40 mgSe6+ L-1 characterizing an acute response. The total number of leukocytes was different in relation to time zero on all Se concentrations. The thrombocyte count also differed statistically from time zero and control in the first 3 days at 0.14 mgSe6+ L-1. These results indicate that different concentrations induce an acute response with diminution of total leukocytes, neutrophilia, monocytosis and thrombocytosis.

O selênio (Se) é descrito como um micronutriente essencial e participa de diversas funções biológicas. No entanto, quando em concentrações elevadas, o Se pode causar efeitos tóxicos, bem como alterações hematológicas em peixes. O objetivo do presente estudo foi determinar a toxicidade do selênio na forma de Selenato de sódio (Na2Se6+ O4) em Oreochromis niloticus com base em parâmetros hematológicos, após a exposição a diferentes concentrações (0,01, 0,14 e 1,4 mg + Se6+ L-1). A série eritrocitária e leucocitária foram examinadas por 14 dias, em intervalos de 0, 3, 5, 7,10 e 14 dias. A série eritrocítica mostrou alterações significativas nos primeiros 7 dias, incluindo o grupo controle. Neutrófilos e monócitos apresentaram variações nos primeiros 3 dias na concentração de 1,40 mgSe6+ L-1, caracterizando uma resposta aguda. O número total de leucócitos foi diferente em relação ao tempo zero em todas as concentrações de Se. A contagem de trombócitos também diferiram estatisticamente entre o tempo zero e os primeiros 3 dias a 0,14 mgSe6+ L-1. O resultados indicam que diferentes concentrações induzem a resposta aguda com diminuição dos leucócitos totais, neutrofilia, monocitose e trombocitose.

Hematology, micronuclei and nuclear abnormalities in fishes from São Francisco river, Minas Gerais state, Brazil / Hematologia, micronúcleos e anomalias nucleares em peixes do rio São Francisco, Estado de Minas Gerais, Brasil

This study aimed to determine the variables leukocytary, erythrocytary, frequency of micronuclei and nuclear abnormalities in peripheral erythrocytes of Prochilodus argenteus, Pimelodus maculatus and Myleus micans from São Francisco river basin, Minas Gerais State, during the summer and winter. Thrombocytes, hematocrit and leukocyte of P.argenteus series, was influenced by climatic conditions. In P. maculatus, not were significant differences for all leukocytes and thrombocytes. In M. micans, values are unprecedented in the literature, with MCV and absolute number of leukocytes found superior to other species of the family Characidae. The frequency of micronuclei and nuclear abnormalities in erythrocytes, the three species not showed seasonal differences between the sites. However, the highest values were found in summer. Furthermore, we observed a positive correlation between the increase in the percentage of micronuclei with nuclear abnormalities

O presente estudo teve como objetivo, determinar as variáveis leucocitárias, eritrocíticas, freqüência de micronúcleos e anomalias nucleares nos eritrócitos do sangue periférico de Prochilodus argenteus, Pimelodus maculatus e Myleus micans de um trecho da bacia do rio São Francisco, Minas Gerais, no verão e inverno. Trombóctios, hematócrito e a serie leucocítária de P.argenteus, foi influenciado pela sazonalidade. Em P. maculatus, não ocorreram diferenças significativas para todos os leucócitos e trombócitos. Em M. micans, os valores são inéditos na literatura, apresentando VCM e número absoluto de leucócitos superior ao encontrados para outras espécies da família Characidae. A freqüência de micronúcleos e de anomalias nucleares nos eritrócitos, nas três espécies não apresentou diferenças sazonais e entre os pontos amostrais. No entanto, os valores mais altos foram encontrados no verão. Além disso, foi possível observar correlação positiva entre o aumento da porcentagem de anomalias nucleares com micronúcleos.

Tempo de migração dos macrófagos em matrinxã, Brycon amazonicus, por meio da técnica de inoculação de leveduras Saccharomyces cerevisiae / Macrophagic migration time in matrinxã, Brycon amazonicus, through inoculation of yeast, Saccharomyces cerevisiae, technique

Na aquicultura são utilizados análises da ativação e incremento da migração de macrófagos, com intuito de verificar a capacidade imunológica inespecífica dos peixes frente a um desafio. Neste sentido, o objetivo deste estudo foi determinar o tempo de migração de monócitos/macrófagos para a cavidade peritoneal em matrinxã, Brycon amazonicus, por meio da técnica de inoculação de leveduras Saccharomyces cerevisiae, e verificar as possíveis alterações dos parâmetros hematológicos após o estímulo. Foram utilizados 30 matrinxãs com peso médio de 101,55 ± 24,50 g e comprimento médio de 19,75 ± 1,72 cm. Os tempos de inoculação utilizados foram 2, 4, 8 e 12 horas, sendo utilizados 6 animais por tempo. Após os períodos de incubação (2, 4, 8 e 12 horas), os exemplares foram anestesiados e alíquotas de sangue foram coletadas por punção do vaso caudal, para a análise: número total de células, contagem diferencial e total dos leucócitos e contagem total de trombócitos, hematócrito, taxa de hemoglobina e índices hematimétricos (VCM, HCM e CHCM). Os resultados mostram que a capacidade fagocítica do macrófago não apresentou diferenças significativas entre os tempos experimentais. Com relação ao índice fagocítico, o tempo de 2 horas representa o tempo em que os macrófagos fagocitaram maior número de leveduras com diferenças significativas em relação aos outros tempos experimentais, indicando que este tempo (2 horas) de incubação foi suficiente para a migração e ativação máxima dos macrófagos da cavidade peritoneal, da espécie estudada. Os valores do número de eritrócitos apresentaram diferenças entre os tempos de incubação. Entretanto, os valores dos outros parâmetros hematológicos não apresentaram diferenças significativas.

In aquaculture, analysis of activation and increased macrophages migration are used in order to verify the ability of the nonspecific immune fish exposed to a challenge. This study aimed to determine the time of macrophages migration in matrinxã, Brycon amazonicus, through the technique of yeast Saccharomyces cerevisiae inoculation, verifying possible changes in hematological parameters. Thirty animals with average weight of 101.55 ± 24.50 g and average length of 19.75 ± 1.72 cm were employed. The experimental inoculation periods were 2, 4, 8 and 12 hours. Thereafter, animals were anesthetized and blood was withdrawn through a caudal puncture for the determination of total erythrocytes number, differential and total leukocyte counts and total thrombocytes count, hematocrit, hemoglobin concentration and calculation of the erythrocytes index. The results for the phagocytic capacity were not significantly different between experimental periods. In the phagocytic index, the period of 2 hours presented the highest rate of phagocytized cells, indicating that 2 hours of incubation was sufficient for the macrophages migration in B. amazonicus. The number of erythrocyte was the only parameter that presented significant difference among periods.

Hematological parameters in Nile Tilápia, Oreochromis niloticus exposed to sub-letal concentrations of mercury

Mercury toxicity in tilapia, Oreochromis niloticus, (Linnaeus, 1758) was investigated by the hematological parameters after long-term (14 days) exposure to various Hg concentrations (0.02, 0.002, 0.0002mg/L Hg). Test groups were set up with three replicates for each concentration, plus the control group. Blood samples were collected from six individuals for each concentration at 0, 3, 7, 10 and 14 days of exposure. The hematological parameters analyzed were: total red blood cell count (RBC), hemoglobin concentration (Hb), hematocrit (Ht), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), total white blood cell count (WBC) and differential leukocyte counts and total thrombocyte count (Tr). There were no significant differences among the mean hematological values at the different Hg concentrations indicating that Hg at the concentrations studied was not toxic to tilapia.

A toxicidade do mercúrio foi avaliada em tilápia, Oreochromis niloticus (Linnaues, 1758) através da análise dos parâmetros hematológicos após exposição a diferentes concentrações sub-letais, durante um período de 14 dias. O bioensaio foi conduzido no laboratório de toxicologia do Instituto de Pesca, SP. Foram utilizados alevinos (12.44 ± 0.84 cm, e 27.13 ± 4.67 g) e aquários com capacidade para 50 litros e preenchidos com água declorada e mais a quantidade de solução de mercúrio (HgCl2) correspondendo as seguintes concentrações: 0,02; 0,002; 0.0002 mg.L-1 Hg. Foram utilizadas 3 repetições de cada concentração e grupo controle. Amostras de sangue foram coletadas de seis animais de cada concentração nos tempos 0, 3, 7, 10 e 14 dias de exposição. Foram avaliados: a contagem de eritrócitos (RBC), concentração de hemoglobina (Hb), hematócrito (Ht), volume corpuscular médio (VCM), hemoglobina corpuscular média (HCM) e concentração de hemoglobina corpuscular média (CHCM), trombócitos totais (Tr), contagem diferencial e total de leucócitos (Lc). Os resultados demonstram que as concentrações de Hg testadas, não alteraram significativamente os parâmetros hematológicos, permitindo concluir que a quantidade de Hg na água não foram suficientes para afetar o quadro hematológico de Oreochromis niloticus.

Effects of an experimental challenge with Mycobacterium marinum on the blood parameters of Nile tilapia, Oreochromis niloticus (Linnaeus, 1757)

Cem exemplares adultos de tilápia, Oreochromis niloticus, foram inoculadas intraperitonealmente com 108 UFC de Mycobacterium marinum e outras 100 foram mantidas sem inoculação. Inicialmente, o sangue de 6 exemplares não inoculados foi colhido e considerado como "tempo zero". Do grupo dos inoculados foi retirado sangue de 6 animais por colheita, 1, 3, 7, 14, 21, 28, 35, 42, 49, 56 e 84 dias após inoculação. Os parâmetros hematológicos: hematócrito (Hct), taxa de hemoglobina (Hgb), contagem de eritrócitos (RBC), foram determinados e os índices hematológicos (VCM, HCM e CHCM), calculados. Foram determinados também o número de trombócitos e o número total e diferencial de leucócitos. Não ocorreram alterações nos valores de Hct, Hgb, HCM e CHCM. Entretanto houve diminuição dos valores de RBC e VCM. Os valores médios de RBC e Hct dos animais no "tempo zero" foram maiores e os de VCM e HCM, menores que os dos animais inoculados. Ocorreu neutrofilia e, após 3 dias de inoculação, ocorreram linfocitose e neutropenia nos animais. No 49º. dia após inoculação bacteriana, ocorreu monocitose.