Lectinhistoquímica del granuloma inducido por el bacilo de Calmette Guérin en Piaractus mesopotamicus

Objective: This study was conducted to evaluate, by means of lectinhistochemistry (LHC), the expression of carbohydrates in granulomas induced by the bacillus Calmette-Guerin (BCG) in muscle tissue of Piaractus mesopotamicus after 33 days. Material and methods: Histological sections with 3 µm thick were incubated with the following lectins: WGA (Wheat germ agglutinin), DBA (Dolichos biflorus agglutinin) and HPA (Helix pomatia agglutinin), and the results were evaluated by light microscopy. Results: Acid fast bacilli were stained by Ziehl Neelsen (ZN) and strong labeled by WGA in the cytoplasm of macrophages. Labeling with DBA was intense in fibroblasts and weak in macrophages. On the other hand, HPA binding was stronger in macrophages, especially in those that were in close contact with epithelioid cells, without evidence of binding to fibroblasts. The epithelioid cells were not labeled by the used lectins, but they were identified by Hematoxilin-Eosin (HE). The lectins labeled specific type saccharides in glycoproteins, as N-acetylglucosamine present in bacilli and macrophages, as well as N-acetyl-galactosamine in macrophages. The control group showed no inflammation or lectin binding. Conclusions. This technique may be useful in identifying receptors for WGA, DBA and the HPA lectins in epithelioid granuloma induced by BCG in P. mesopotamicus.Objetivo: El presente estudio fue realizado para evaluar por medio de lectinhistoquímica (LHC), la expresión de carbohidratos en granulomas inducidos por el bacilo de Calmette-Guérin (BCG) en músculo de Piaractus mesopotamicus después de 33 días. Materiales y métodos: Cortes histológicos de 3 µm de grosor fueron incubados con las siguientes lectinas: WGA (Wheat germ aglutinin), DBA (Dolichos biflorus agglutin) y HPA (Helix pomatia agglutinin), y los resultados evaluados por medio de microscopia de luz. Resultados: Bacilos ácido resistentes fueron identificados por la tinción de Ziehl Neelsen(ZN). Se observó un marcaje intenso con WGA en el citoplasma de macrófagos. El marcaje con DBA fue intenso en fibroblastos y débil en macrófagos. Con la lectina HPA el marcaje fue intenso en macrófagos, principalmente en los que estaban en estrecho contacto con las células epitelióides, externamente se observó marcaje débil en fibroblastos. Las células epitelióides no fueron marcadas por las lectinas, pero fueron identificadas con la tinción de Hematoxilina-Eosina (HE). Las lectinas tuvieron un tipo de marcaje específico en algunos monosacáridos, como N-acetilglucosamina presente en los bacilos y en macrófagos, y N-acetilgalactosamina en macrófagos. En el grupo control no fue observada inflamación así como tampoco marcaje con las lectinas. Conclusiones. Esta técnica resultó eficiente en la identificación de receptores para las lectinas WGA, DBA y HPA en el granuloma epitelióide inducido por BCG en P. mesopotamicus.Facultad de Ciencias Veterinaria

Lectinhistoquímica del granuloma inducido por el bacilo de Calmette Guérin en Piaractus mesopotamicus

Objective: This study was conducted to evaluate, by means of lectinhistochemistry (LHC), the expression of carbohydrates in granulomas induced by the bacillus Calmette-Guerin (BCG) in muscle tissue of Piaractus mesopotamicus after 33 days. Material and methods: Histological sections with 3 µm thick were incubated with the following lectins: WGA (Wheat germ agglutinin), DBA (Dolichos biflorus agglutinin) and HPA (Helix pomatia agglutinin), and the results were evaluated by light microscopy. Results: Acid fast bacilli were stained by Ziehl Neelsen (ZN) and strong labeled by WGA in the cytoplasm of macrophages. Labeling with DBA was intense in fibroblasts and weak in macrophages. On the other hand, HPA binding was stronger in macrophages, especially in those that were in close contact with epithelioid cells, without evidence of binding to fibroblasts. The epithelioid cells were not labeled by the used lectins, but they were identified by Hematoxilin-Eosin (HE). The lectins labeled specific type saccharides in glycoproteins, as N-acetylglucosamine present in bacilli and macrophages, as well as N-acetyl-galactosamine in macrophages. The control group showed no inflammation or lectin binding. Conclusions. This technique may be useful in identifying receptors for WGA, DBA and the HPA lectins in epithelioid granuloma induced by BCG in P. mesopotamicus.Objetivo: El presente estudio fue realizado para evaluar por medio de lectinhistoquímica (LHC), la expresión de carbohidratos en granulomas inducidos por el bacilo de Calmette-Guérin (BCG) en músculo de Piaractus mesopotamicus después de 33 días. Materiales y métodos: Cortes histológicos de 3 µm de grosor fueron incubados con las siguientes lectinas: WGA (Wheat germ aglutinin), DBA (Dolichos biflorus agglutin) y HPA (Helix pomatia agglutinin), y los resultados evaluados por medio de microscopia de luz. Resultados: Bacilos ácido resistentes fueron identificados por la tinción de Ziehl Neelsen(ZN). Se observó un marcaje intenso con WGA en el citoplasma de macrófagos. El marcaje con DBA fue intenso en fibroblastos y débil en macrófagos. Con la lectina HPA el marcaje fue intenso en macrófagos, principalmente en los que estaban en estrecho contacto con las células epitelióides, externamente se observó marcaje débil en fibroblastos. Las células epitelióides no fueron marcadas por las lectinas, pero fueron identificadas con la tinción de Hematoxilina-Eosina (HE). Las lectinas tuvieron un tipo de marcaje específico en algunos monosacáridos, como N-acetilglucosamina presente en los bacilos y en macrófagos, y N-acetilgalactosamina en macrófagos. En el grupo control no fue observada inflamación así como tampoco marcaje con las lectinas. Conclusiones. Esta técnica resultó eficiente en la identificación de receptores para las lectinas WGA, DBA y HPA en el granuloma epitelióide inducido por BCG en P. mesopotamicus.Facultad de Ciencias Veterinaria

Acute Toxicity and Determination of the Active Constituents of Aqueous Extract of Uncaria tomentosa

Uncaria tomentosa is a medicinal plant used in folk medicine by Amazon tribes. In this study the constituents of aqueous extract of U. tomentosa bark were quantified by chromatographic technique and its lethal concentration 50 (48 h) in Hyphessobrycon eques was determined. The chromatography showed high levels of oxindole alkaloids, quinovic acid glycosides, and low molecular weight polyphenols. The CL50 48 h was 1816 mg/L. Fish showed behavior changes at concentrations above 2000 mg/L, accompanied by a significant decrease of dissolved oxygen. At the highest concentration 100% mortality was observed attributed to oxygen reduction by the amount of oxindole alkaloids, polyphenols accumulation of the extract in the gills, and the interaction of these compounds with dopamine. In conclusion, the aqueous extract of U. tomentosa did not alter the chemical components and it was shown that U. tomentosa has low toxicity to H. eques; therefore, it can be used safely in this species

STABILITY OF HEMATOLOGICAL VARIABLES IN Colossoma macropomum BLOOD STORED WITH DIFFERENT ANTICOAGULANTS

Hemograma é uma ferramenta de diagnóstico imprescindível na medicina humana e veterinárias. O recomendável é que as análises hematológicas ocorram pouco tempo após coleta do sangue. No entanto, há situações nas quais as amostras são coletadas em locais distantes dos laboratórios, fazendo-se necessário o armazenamento. Assim, o presente trabalho avaliou a viabilidade das amostras de sangue coletado com ácido etilenodiamino tetra-acético tripotássico – EDTA k3 10% (1.3mg mL-1/EDTAa), EDTA k3 10% (2.0 mg mL-1/EDTAb), heparina de sódio (15 UI/ml) e citrato trissódico (3%) em tambaqui, Colossoma macropomum, comparando os valores do hematócrito e hemoglobina armazenada em temperatura de refrigeração (4ºC). As amostras de sangue foram obtidas através da venopunção caudal e determinado o hematócrito (Ht) e a hemoglobina (Hb), durante 8 dias consecutivos com as análises realizadas em intervalo de 24 horas. Os resultados foram analisados através teste Tukey (5%). Não houve diferença estatística (p>0.05) entre os tempos nos parâmetros hematológicos. Entretanto, observou-se hemólise crescente ao longo do tempo em todas as amostras conservadas com citrato, iniciando horas pós coleta (HPC); no grupo EDTAa verificou-se hemólise em 100% das amostras em 192 HPC. Nas amostras com heparina verificou-se coágulo em 25%, 75%, 87,5% e 100% nos tempos de 2, 3, 4 e 6 dias de armazenagem respectivamente. Conclui-se que o anticoagulante citrato trissódico (3%) e heparina de sódio (15 UI/ml) não são recomendáveis para análises hematológicas em C. macropomum. O EDTA K3 10% na quantidade de 2.0 mg mL-1 foi a melhor opção testada nesse estudo, por apresentar estabilidade das amostras sob refrigeração, evitando hemólise e coagulação.                               PALAVRAS-CHAVE: Hematócrito, Tambaqui, Hemoglobin, Coagulação, Hemólise.El recuento sanguíneo es una herramienta de diagnóstico esencial para la medicina humana y veterinaria. Lo más recomendado es que los análisis hematológicos se realicen poco después de la extracción de sangre. Sin embargo, hay situaciones en las que las muestras se recolectan en lugares alejados de los laboratorios, lo que hace necesario el almacenamiento. Por lo tanto, el presente estudio evaluó la viabilidad de las muestras de sangre recolectadas con ácido tripotásico de etilendiamina tetraacético - EDTA k3 10% (1.3mg mL-1 / EDTAa), EDTA k3 10% (2.0 mg mL-1 / EDTAb), heparina sodio (15 UI / ml) y citrato trisódico (3%) de tambaquis, Colossoma macropomum, comparando los valores de hematocrito y hemoglobina almacenados a temperatura refrigerada (4ºC). Se obtuvieron muestras de sangre mediante venopunción caudal y se determinaron hematocrito (Ht) y hemoglobina (Hb) durante 8 días consecutivos con análisis realizados a intervalos de 24 horas. Los resultados se analizaron utilizando la prueba de Tukey (5%). No hubo diferencia estadística (p> 0.05) entre los tiempos en los parámetros hematológicos. Sin embargo, se observó un aumento de la hemólisis con el tiempo en todas las muestras conservadas con citrato, horas de inicio después de la recolección (HPC); En el grupo EDTAa, se encontró hemólisis en el 100% de las muestras a 192 HPC. En las muestras con heparina hubo un coágulo en 25%, 75%, 87.5% y 100% en los períodos de 2, 3, 4 y 6 días de almacenamiento, respectivamente. Se concluye que el citrato trisódico anticoagulante (3%) y la heparina de sodio (15 UI / ml) no se recomiendan para el análisis hematológico en C. macropomum, ya que EDTA K3 10% en la cantidad de 2.0 mg mL-1, el mejor opción probada en este estudio, por apresentar muestras de estabilidad bajo refrigeración, evitando la hemólisis y la coagulación. PALABRAS CLAVES: Hematocrito, Tambaqui, Hemoglobina, Coagulación, Hemólisis. The blood count is an essential diagnostic tool for human and veterinary medicine. The most recommended is that hematological analyzes should take place shortly after blood collection. However, there are situations in which samples are collected in places far from the laboratories, making storage necessary. Thus, the present work evaluated the viability of blood samples collected with tripotassic ethylenediamine tetraacetic acid - EDTA k3 10% (1.3mg mL-1 / EDTAa), EDTA k3 10% (2.0 mg mL-1 / EDTAb), heparin sodium (15 IU / ml) and trisodium citrate (3%) of tambaquis, Colossoma macropomum, comparing the values of hematocrit and hemoglobin stored at refrigerated temperature (4ºC). Blood samples were obtained through caudal venipuncture and hematocrit (Ht) and hemoglobin (Hb) were determined for 8 consecutive days with analyzes performed at 24-hour intervals. The results were analyzed using the Tukey test (5%). There was no statistical difference (p> 0.05) between times in hematological parameters. However, increasing hemolysis was observed over time in all samples preserved with citrate, starting hours post collection (HPC); in the EDTAa group, hemolysis was found in 100% of the samples at 192 HPC. In the samples with heparin there was a clot in 25%, 75%, 87.5% and 100% in the periods of 2, 3, 4 and 6 days of storage respectively. It is concluded that the anticoagulant trisodium citrate (3%) and sodium heparin (15 IU / ml) are not recommended for hematological analysis in C. macropomum, since EDTA K3 10% in the amount of 2.0 mg mL-1, the best option tested in this study, for presenting samples stability under refrigeration, avoiding hemolysis and coagulation.KEYWORDS: Hematocrit, Tambaqui, Hemoglobin, Coagulation, Hemolysis

ALTERAÇÕES MORFOLÓGICAS DE LEUCÓCITOS E TROMBÓCITOS SANGUÍNEOS EM Piaractus mesopotamicus DURANTE A SEPSE

Sepsis is defined as organ dysfunction with high mortality. During this process, the hematological system plays a critical role. Thus, the work aimed to analyze the morphological changes in the leukogram and thrombogram by the indirect method in the course of sepsis in Piaractus mesopotamicus. For this, 98 pacus were used, divided into two groups, one received 0.5 ml of sterile 0.65% sodium chloride solution (control) and the other received the same volume containing the bacterial inoculum (1.8 x 108 CFU/ml – challenge). To assess such changes in the evolution of sepsis, blood samples were collected from fish 1, 3, 6 and 9 hours after inoculation and in the control group (n = 10). One aliquot of the blood was used for blood culture and another for determining the number of leukocytes and thrombotics by the indirect method and the evaluation of morphological changes in blood extensions. Aeromonosis was confirmed by positive blood culture in all samples from the challenged groups. The leukogram analysis showed a significant increase (p <0.05) of leukocytes in the later times, 9 and 6 HPI in relation to the control group. Differential analysis showed leukopenia (p <0.05; 3 HPI) and consumption of thrombocytes. It was found that with the increase of the infection the septicemic pacus develops a picture of granulocytosis and monocytosis (p <0.05). It was concluded that inoculation with A. hydrophila induced sepsis with positive blood culture with leukocytosis in the challenged pacus and caused an intense change in the leukocyte and thrombotic morphology.KEYWORDS: Hemoculture, Leukogram, Morphology, Aeromonas hydrophila, Teleosts.La sepsis se define como disfunción orgánica con alta mortalidad. Durante este proceso, el sistema hematológico juega un papel crítico. Por lo tanto, el trabajo tuvo como objetivo analizar los cambios morfológicos en el leucograma y el trombograma por el método indirecto en el curso de la sepsis en Piaractus mesopotamicus. Para esto, se utilizaron 98 pacus, divididos en dos grupos, uno recibió 0,5 ml de solución estéril de cloruro de sodio al 0,65% (control) y el otro recibió el mismo volumen que contenía el inóculo bacteriano (1,8 x 108 UFC / ml - desafío). Para evaluar dichos cambios en la evolución de la sepsis, se recogieron muestras de sangre de peces 1, 3, 6 y 9 horas después de la inoculación y en el grupo de control (n = 10). Una parte alícuota de la sangre se usó para el hemocultivo y otra para determinar el número de leucocitos y trombóticos mediante el método indirecto y la evaluación de los cambios morfológicos en las extensiones de sangre. La aeromonosis se confirmó por hemocultivo positivo en todas las muestras de los grupos desafiados. El análisis de leucogramas mostró un aumento significativo (p <0.05) de leucocitos en los últimos tiempos, 9 y 6 HPI en relación con el grupo de control. El análisis diferencial mostró leucopenia (p <0.05; 3 HPI) y consumo de trombocitos. Se encontró que con el aumento de la infección, el pacus septicémico desarrolla una imagen de granulocitosis y monocitosis (p <0.05). Se concluyó que la inoculación con A. hydrophila indujo sepsis con hemocultivo positivo con leucocitosis en pacus desafiado y causa un cambio intenso en los leucocitos y la morfología trombóticaPALABRAS CLAVES Hemocultivo, Leucograma, Morfología, Aeromonas hydrophila, Teleósteos.A sepse é definida como disfunção orgânica com alta mortalidade. Durante esse processo o sistema hematológico desempenha um papel crítico. Assim, o trabalho objetivou analisar as alterações morfológicas no leucograma e no trombograma pelo método indireto no curso da sepse em Piaractus mesopotamicus. Para tanto, foram utilizados 98 pacus, divididos em dois grupos, um deles recebeu 0,5 ml de solução de cloreto de sódio esterilizada a 0,65% (controle) e outro recebeu o mesmo volume contendo o inoculo bacteriano (18 x 108 CFU/ml – desafio). Para avaliar tais alterações na evolução da sepse foram realizadas coletas de sangue dos peixes 1, 3, 6 e 9 horas após inoculação e no grupo controle (n=10). Uma alíquota do sangue foi destina a hemocultura e outra na determinação do número de leucócitos e de trombóticos pelo método indireto e a avaliação das alterações morfológicas nas extensões sanguíneas. A aeromonose foi confirmada pela hemocultura positiva em todas as amostras dos grupos desafiados. A análise do leucograma demonstrou aumento significativa (p<0,05) dos leucócitos nos tempos mais tardios, 9 e 6 HPI em relação ao grupo controle. Da análise diferencial observou-se leucopenia (p<0,05; 3 HPI) e consumo dos trombócitos. Verificou-se que com o aumento da infecção os pacus septicêmicos desenvolvem um quadro de granulocitose e monocitose (p<0,05). Concluiu-se que a inoculação com A. hydrophila induziu a sepse com hemocultura positiva com leucocitose nos pacus desafiados e causa intensa alteração na morfologia dos leucócitos e trombóticos.PALAVRAS-CHAVE: Hemocultura, Leucograma, Morfologia, Aeromonas hydrophila, Teleósteos

Lectinhistochemical staining of granuloma induced by bacillus Calmette-Guerin in Piaractus mesopotamicus

ABSTRACT Objetive. This study was conducted to evaluate, by means of lectinhistochemistry (LHC), the expression of carbohydrates in granulomas induced by the bacillus Calmette-Guerin (BCG) in muscle tissue of Piaractus mesopotamicus after 33 days. Material and methods. Histological sections with 3 μm thick were incubated with the following lectins :WGA (Wheat germ agglutinin), DBA (Dolichos biflorus agglutinin) and HPA (Helix pomatia agglutinin), and the results were evaluated by light microscopy. Results. Acid fast bacilli were stained by Ziehl Neelsen (ZN) and strong labeled by WGA in the cytoplasm of macrophages. Labeling with DBA was intense in fibroblasts and weak in macrophages. On the other hand, HPA binding was stronger in macrophages, especially in those that were in close contact with epithelioid cells, without evidence of binding to fibroblasts. The epithelioid cells were not labeled by the used lectins, but they were identified by Hematoxilin-Eosin (HE). The lectins labeled specific type saccharides in glycoproteins, as N-acetylglucosamine present in bacilli and macrophages, as well as N-acetyl-galactosamine in macrophages. The control group showed no inflammation or lectin binding. Conclusions. This technique may be useful in identifying receptors for WGA, DBA and the HPA lectins in epithelioid granuloma induced by BCG in P. mesopotamicu

Influence of glyceryl guaiacolate ether on anesthetics in tilapia compared to benzocaine and eugenol

ABSTRACT Objective. The study aimed to investigate the effectiveness of glyceryl guaiacolate ether (GGE) and compare the times of induction, recovery, hematological changes, total protein and glycaemia among anesthetics in Nile tilapia, Oreochromis niloticus. Materials and methods. A total of 60 tilapia distributed in 3 aquariums (N=20) were used, which formed the group benzocaine (100 mg/L), eugenol (50 mg/L) and guaiacol glyceryl ether (9.000 mg/L). After the induction of anesthesia fish blood samples were collected to determine the complete hemogram and glycemia. Then the animals were placed in aquariums with running water for assessing the anesthesia recovery. Results. It was verified that GGE showed longer induction and recovery times as well a significant increase (p0.05). An increase in the number of monocytes in the group treated with benzocaine (p <0.05) was observed in the analysis of the hematological parameters with no difference between groups for other variables. Conclusions. Eugenol and benzocaine allow rapid induction and recovery in Nile tilapia, without evidence of stress during handling and GGE showed high induction and recovery times, being inadequate for anesthetic use in Nile tilapia

Myxosporidiosis in intensively-reared Piaractus mesopotamicus: Histopathological diagnosis by means of Ziehl-Neelsen staining

Amostras de diferentes órgãos de Piaractus mesopotamicus mantidos em criação intensiva foram coletadas e processadas mediante as técnicas histológicas usuais para obtenção de cortes que foram corados com hematoxilina-eosina e pelo método de Ziehl-Neelsen. Ao exame em microscopia de luz foi possível identificar mixosporídeos dos gêneros Henneguya sp. e Myxobolus sp. em brânquia e rim de P. mesopotamicus respectivamente. Plasmódios com esporos imaturos de Henneguya sp. foram localizados ao longo das lamelas secundárias e mensurados (comprimento total 30,45±4,84µm e largura 3,52±0,33µm) e no rim esporos de Myxobolus sp. (comprimento total 8,94±0,82µm e largura 5,59±0,39µm). Na análise histopatológica das brânquias observaram-se plasmódios contendo esporos de Henneguya sp., com localização intralamelar e intravascular, em diferentes estágios de desenvolvimento. No rim identificaram-se esporos de Myxobolus sp., na região peritubular e no interstício e glomérulo, circundados por melanomacrófagos. em poucos casos foi registrada hemorragia focal. O uso da coloração de Ziehl-Neelsen permitiu identificar particularidades dos esporos, facilitou sua biometria e classificação em comparação com a hematoxilina-eosina, demonstrando sua utilidade no diagnóstico histopatológico da referida parasitose.Samples of different organs from intensively-reared Piaractus mesopotamicus were collected and processed using routine histological techniques in order to produce thin sections for staining with hematoxylin-eosin and with the Ziehl-Neelsen method. Through examination under an optical microscope, myxosporidians of the genera Henneguya sp. and Myxobolus sp. were identified, respectivelyin the gills and kidneys of P. mesopotamicus. Plasmodia with immature spores of Henneguya sp. were located along the secondary lamellae, with total length of 30.45±4.84µm and width of 3.52±0.33µm. Spores of Myxobolus sp. were located in the kidneys, with total length of 8.94±0.82µm and width of 5.59±0.39µm. Histopathological analysis of the gills showed plasmodia containing spores of Henneguya sp., at intralamellar and intravascular localities, at different stages of development. Spores of Myxobolus sp. were identified in the kidneys, in the peritubular region and in the interstices and glomerulus, surrounded by melanomacrophages. Focal hemorrhage was recorded in a few cases. Ziehl-Neelsen staining allowed to identify particular features of the spores and facilitated biometry and enabled classification in comparison with hematoxylin-eosin, thus demonstrating its usefulness for histopathological diagnosis of the parasitosis.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP