Cytotoxicity and antimicrobial activity of synthetic peptides alone or in combination with conventional antimicrobials against fish pathogenic bacteria.

AIMS: This study aimed to evaluate the in vitro cytotoxicity and efficacy of synthetic host defence peptides (HDPs), alone or in combination with florfenicol (FFC), oxytetracycline (OTC) or thiamphenicol (TAP), against different pathogenic bacteria isolated from diseased fish. METHODS AND RESULTS: Solid-phase synthesis, purification and characterization of several HDPs were performed manually, using the fluorenylmethyloxycarbonyl protecting group in different resins and via high-performance liquid chromatography-mass spectrometry, respectively. The in vitro cytotoxicity and antimicrobial activity of HDPs, FFC, OTC and TAP against Nile tilapia red blood cells (RBCs) and relevant fish pathogenic bacteria (Aeromonas, Citrobacter, Edwardsiella, Streptococcus, Lactococcus and Vibrio) was determined using the haemolysis assay and broth microdilution method, respectively. The checkerboard assay was used to evaluate the synergy between the most active HDPs and other antimicrobials against the tested strains. MUC 7 12-mer, FFC, OTC and TAP were not cytotoxic to Nile tilapia RBCs, in all tested concentrations. LL-37, (p-BthTX-I)2 and Hylin-a1 were not cytotoxic at concentrations up to 78·13, 19·53 and 9·77 µg ml-1 , respectively. HDPs demonstrated potent antimicrobial activity (minimum inhibitory concentration ≤31·25 µg ml-1 ) against Aeromonas jandaei (KR-12-a5), Citrobacter freundii (Kr-12-a5; (p-BthTX-I)2 ; LL-37; and Hylin a1), Streptococcus agalactiae (Hylin a1; (p-BthTX-I)2 and LL-37), Lactococcus garviae (Hylin a1), and Vibrio fluvialis (KR-12-a5). The combinations of (p-BthTX-I)2 with TAP and LL-37 with FFC showed synergistic activity against C. freundii (fractional inhibitory concentration index of 0·25 and 0·50, respectively). CONCLUSIONS: Synthetic HDPs have the potential as a good treatment option for bacterial diseases in aquaculture. SIGNIFICANCE AND IMPACT OF THE STUDY: The in vivo effectiveness of synthetic HDPs such as KR-12-a5; LL-37; (p-BthTX-I)2 and Hylin a1 can be tested alone or in combination with conventional antimicrobials as a treatment option to reduce the use of antimicrobials in aquaculture.

In vivo phagocytosis and hematology in Astyanax altiparanae, a potential model for surrogate technology / Fagocitose in vivo e hematologia em Astyanax altiparanae, um potencial modelo para tecnologia de propagação mediada

Abstract Although the potential of surrogate propagation technology for aquaculture and conservation of Neotropical fish, the poor understanding of the host immune system may results in rejection and destruction of the donor material. Thus, it is necessary to study and to develop methods to evaluate the effects of immunosuppressive drugs employment and to evaluate the immunocompatibility between donor and receptor. Thus, the present study aimed to optimize a methodology to assess in vivo phagocytosis in Astyanax altiparanae using Saccharomyces cerevisiae and to evaluate their hematological response resultant from the inflammatory induction. To this, S. cerevisiae were labeled with Congo red and injected in the coelomic cavity of A. altiparanae at the concentration of 2.5 x 106 cells mL-1. A PBS solution and a non-injected group were kept as control. Fish blood was sampled and the phagocytic capacity and index were determined at 1, 2, 3 and 6 h post-injection (hpi). The yeast injection successfully stimulated phagocytosis, with the best result for phagocytosis assessment after 2 hpi. Moreover, it was achieved a high traceability of phagocytized and non-phagocytized yeast under optic microscopy analysis due to the Congo red labeling. The hematological profile was similar to usually observed in early infections, indicating lymphocyte migration to inflammatory site and increase in number of circulating phagocytes due to natural response to inflammatory stimulus. In conclusion, our method was efficient to assess in vivo phagocytosis in A. altiparanae and will be an important tool to evaluate the efficacy of immunosuppressive drugs in this species. Additionally, these results may serve as support for further studies in fish immunocompetence, both in laboratory and in field conditions.

Resumo Apesar do potencial apresentado pela tecnologia de propagação mediada para a aquicultura e conservação de peixes Neotropicais, o pobre entendimento do sistema imune do hospedeiro pode resultar na rejeição e destruição do material do doador. Com isso, se fazem necessários o estudo e o desenvolvimento de métodos para análise tanto dos efeitos de drogas imunossupressoras quanto para a avaliação da imunocompatibilidade entre doadores e receptores. Logo, o presente estudo teve como objetivo aperfeiçoar um método para analisar a fagocitose in vivo em Astyanax altiparanae usando Saccharomyces cerevisiae marcado e avaliar seu perfil hematológico resultante da indução inflamatória. Para isso, S. cerevisiae foram marcados com vermelho Congo e injetados na cavidade celomática dos A. altiparanae na concentração de 2,5 x 106 células.mL-1. Peixes injetados com PBS e peixes não injetados foram mantidos como controle. Sangue foi colhido e a capacidade fagocítica e o índice fagocítico foram determinados após 1, 2, 3 e 6 horas após à injeção (hpi). A injeção de levedura estimulou a fagocitose com sucesso, com o melhor resultado atingido após 2 hpi. Ainda, foi observada uma alta rastreabilidade das leveduras fagocitadas e não fagocitadas sob microscopia óptica devido à marcação com vermelho Congo. O perfil hematológico foi similar ao observado usualmente em infecções recém-induzidas, indicando migração de linfócitos ao sítio inflamatório e aumento no número de fagócitos circulantes devido à resposta natural ao estímulo inflamatório. Como conclusão, nosso método foi eficiente para analisar a fagocitose in vivo em A. altiparanae e será uma ferramenta importante para a avaliação de eficácia de drogas imunossopressoras para esta espécie. Em adição, estes resultados podem contribuir para futuros estudos em imunocompetência em peixes, tanto em âmbito laboratorial quanto a campo.

In vivo phagocytosis and hematology in Astyanax altiparanae, a potential model for surrogate technology.

Although the potential of surrogate propagation technology for aquaculture and conservation of Neotropical fish, the poor understanding of the host immune system may results in rejection and destruction of the donor material. Thus, it is necessary to study and to develop methods to evaluate the effects of immunosuppressive drugs employment and to evaluate the immunocompatibility between donor and receptor. Thus, the present study aimed to optimize a methodology to assess in vivo phagocytosis in Astyanax altiparanae using Saccharomyces cerevisiae and to evaluate their hematological response resultant from the inflammatory induction. To this, S. cerevisiae were labeled with Congo red and injected in the coelomic cavity of A. altiparanae at the concentration of 2.5 x 106 cells mL-1. A PBS solution and a non-injected group were kept as control. Fish blood was sampled and the phagocytic capacity and index were determined at 1, 2, 3 and 6 h post-injection (hpi). The yeast injection successfully stimulated phagocytosis, with the best result for phagocytosis assessment after 2 hpi. Moreover, it was achieved a high traceability of phagocytized and non-phagocytized yeast under optic microscopy analysis due to the Congo red labeling. The hematological profile was similar to usually observed in early infections, indicating lymphocyte migration to inflammatory site and increase in number of circulating phagocytes due to natural response to inflammatory stimulus. In conclusion, our method was efficient to assess in vivo phagocytosis in A. altiparanae and will be an important tool to evaluate the efficacy of immunosuppressive drugs in this species. Additionally, these results may serve as support for further studies in fish immunocompetence, both in laboratory and in field conditions.

Effect of nutraceuticals on acanthocephalan Neoechinorhynchus buttnerae and its toxicity to the host tambaqui Colossoma macropomum.

The production of tambaqui Colossoma macropomum has been undergoing financial losses due to parasitic infection by the acanthocephalan Neoechinorhynchus buttnerae, raising an alert for aquaculture in South America. The lack of adequate treatment and use of unlicensed chemicals encourages research for alternative solutions with minimal side effects. The objectives of this study were to evaluate the in vitro antiparasitic potential of commercial nutraceutical products (Natumix® and BioFish®) against N. buttnerae and to assess the respective in vivo toxic effects on the host tambaqui. For in vitro assays, parasitized fish were necropsied for acanthocephalans sampling. The parasites were exposed to three concentrations (0.078, 0.313 and 1.25 mg/ml) of each product, as well as controls (one without product and another with a solubilizer). For the in vivo acute toxicity test, juvenile fish (<0.1 g) were exposed to five increasing concentrations of each product. Mortality of tambaqui was recorded at 24, 48, 72 and 96 h. The estimated lethal concentration (LC) for 10, 50, 90 and 99% of fish was determined to classify the toxicity of the products on the target species. After in vitro efficacy tests, the highest concentrations (1.25 mg/ml) caused 100% mortality of the parasites in both products, but only Natumix® caused 100% mortality using the intermediate concentration (0.313 mg/ml) after 24 h. According to the acute toxicity result, the LC50 classified the nutraceutical products as slightly toxic for tambaqui. The tested products had a parasiticidal effect on N. buttnerae, and the toxicity test showed that both products have therapeutic potential when added to the diet.

In vivo phagocytosis and hematology in Astyanax altiparanae, a potential model for surrogate technology

Abstract Although the potential of surrogate propagation technology for aquaculture and conservation of Neotropical fish, the poor understanding of the host immune system may results in rejection and destruction of the donor material. Thus, it is necessary to study and to develop methods to evaluate the effects of immunosuppressive drugs employment and to evaluate the immunocompatibility between donor and receptor. Thus, the present study aimed to optimize a methodology to assess in vivo phagocytosis in Astyanax altiparanae using Saccharomyces cerevisiae and to evaluate their hematological response resultant from the inflammatory induction. To this, S. cerevisiae were labeled with Congo red and injected in the coelomic cavity of A. altiparanae at the concentration of 2.5 x 106 cells mL-1. A PBS solution and a non-injected group were kept as control. Fish blood was sampled and the phagocytic capacity and index were determined at 1, 2, 3 and 6 h post-injection (hpi). The yeast injection successfully stimulated phagocytosis, with the best result for phagocytosis assessment after 2 hpi. Moreover, it was achieved a high traceability of phagocytized and non-phagocytized yeast under optic microscopy analysis due to the Congo red labeling. The hematological profile was similar to usually observed in early infections, indicating lymphocyte migration to inflammatory site and increase in number of circulating phagocytes due to natural response to inflammatory stimulus. In conclusion, our method was efficient to assess in vivo phagocytosis in A. altiparanae and will be an important tool to evaluate the efficacy of immunosuppressive drugs in this species. Additionally, these results may serve as support for further studies in fish immunocompetence, both in laboratory and in field conditions.

Resumo Apesar do potencial apresentado pela tecnologia de propagação mediada para a aquicultura e conservação de peixes Neotropicais, o pobre entendimento do sistema imune do hospedeiro pode resultar na rejeição e destruição do material do doador. Com isso, se fazem necessários o estudo e o desenvolvimento de métodos para análise tanto dos efeitos de drogas imunossupressoras quanto para a avaliação da imunocompatibilidade entre doadores e receptores. Logo, o presente estudo teve como objetivo aperfeiçoar um método para analisar a fagocitose in vivo em Astyanax altiparanae usando Saccharomyces cerevisiae marcado e avaliar seu perfil hematológico resultante da indução inflamatória. Para isso, S. cerevisiae foram marcados com vermelho Congo e injetados na cavidade celomática dos A. altiparanae na concentração de 2,5 x 106 células.mL-1. Peixes injetados com PBS e peixes não injetados foram mantidos como controle. Sangue foi colhido e a capacidade fagocítica e o índice fagocítico foram determinados após 1, 2, 3 e 6 horas após à injeção (hpi). A injeção de levedura estimulou a fagocitose com sucesso, com o melhor resultado atingido após 2 hpi. Ainda, foi observada uma alta rastreabilidade das leveduras fagocitadas e não fagocitadas sob microscopia óptica devido à marcação com vermelho Congo. O perfil hematológico foi similar ao observado usualmente em infecções recém-induzidas, indicando migração de linfócitos ao sítio inflamatório e aumento no número de fagócitos circulantes devido à resposta natural ao estímulo inflamatório. Como conclusão, nosso método foi eficiente para analisar a fagocitose in vivo em A. altiparanae e será uma ferramenta importante para a avaliação de eficácia de drogas imunossopressoras para esta espécie. Em adição, estes resultados podem contribuir para futuros estudos em imunocompetência em peixes, tanto em âmbito laboratorial quanto a campo.

Essential oils to control ichthyophthiriasis in pacu, Piaractus mesopotamicus (Holmberg): special emphasis on treatment with Melaleuca alternifolia.

In vitro effect of the Melaleuca alternifolia, Lavandula angustifolia and Mentha piperita essential oils (EOs) against Ichthyophthirius multifiliis and in vivo effect of M. alternifolia for treating ichthyophthiriasis in one of the most important South American fish, Piaractus mesopotamicus (Holmberg), were evaluated. The in vitro test consisted of three EOs, each at concentrations of 57 µL L(-1) , 114 µL L (-1) , 227 µL L(-1) and 455 µL L (-1) , which were assessed once an hour for 4 h in microtitre plates (96 wells). The in vitro results demonstrated that all tested EOs showed a cytotoxic effect against I. multifiliis compared to control groups (P < 0.05). The in vivo treatment for white spot disease was performed in a bath for 2 h day(-1) for 5 days using the M. alternifolia EO (50 µL L (-1) ). In this study, 53.33% of the fish severely infected by I. multifiliis survived after the treatment with M. alternifolia (50 µL L (-1) ) and the parasitological analysis has shown an efficacy of nearly 100% in the skin and gills, while all the fish in the control group died. Furthermore, the potential positive effect of M. alternifolia EO against two emergent opportunistic bacteria in South America Edwardsiella tarda and Citrobacter freundii was discussed.

Pharmacokinetic study of enrofloxacin in Nile tilapia (Oreochromis niloticus) after a single oral administration in medicated feed.

The objective of this study was to evaluate the disposition kinetics of enrofloxacin (ENR) in the plasma and its distribution in the muscle tissue of Nile tilapia (Oreochromis niloticus) after a single oral dose of 10 mg/kg body weight via medicated feed. The fish were kept at a temperature between 28 and 30 °C. The collection period was between 30 min and 120 h after administration of the drug. The samples were analyzed by high-performance liquid chromatography with a fluorescence detector (HPLC-FLD). The ENR was slowly absorbed and eliminated from the plasma (Cmax = 1.24 ± 0.37 µg/mL; Tmax = 8 h; T1/2Ke  = 19.36 h). ENR was efficiently distributed in the muscle tissue and reached maximum values (2.17 ± 0.74 µg/g) after 8 h. Its metabolite, ciprofloxacin (CIP), was detected and quantified in the plasma (0.004 ± 0.005 µg/mL) and muscle (0.01 ± 0.011 µg/g) for up to 48 h. After oral administration, the mean concentration of ENR in the plasma was well above the minimum inhibitory concentrations (MIC50 ) for most bacteria already isolated from fish except for Streptococcus spp. This way the dose used in this study allowed for concentrations in the blood to treat the diseases of tilapia.

Phytotherapy as an alternative for treating fish disease.

Intensification of livestock rearing often promotes an increase in inappropriate practices that disregard care for the environment, animal health, and workers' health. Intensive fish farming systems are often associated with higher stocking density and massive use of artificial feed. Currently, outbreaks of parasitic, bacterial, and fungal diseases act as major limiting factors for fish farming, meaning that producers have to make use of massive amounts of antibiotics, disinfectants, and pesticides in order to control mortality and avoid huge economic losses. Because of adverse effects on the aquatic environment, terrestrial organisms, and human health (both fish handlers and consumers), this therapy has been criticized. Use of herbal medicines within animal production has shown promise, in that it is natural and biodegradable and has antimicrobial activity against various pathogens, including those relating to fish. Recently, researchers have reported promising effects from many herbal medicines for treating parasitic diseases caused by protozoa and metazoa, and broad activity against bacteria and fungi. This review addresses the current issues regarding indiscriminate use of chemicals and antibiotics in aquaculture and discusses the main findings and methodologies of the latest research on herbal medicines to stimulate and accelerate research in this field, especially in developing countries.

Serum bactericidal activity as indicator of innate immunity in pacu Piaractus mesopotamicus (Holmberg, 1887) / Atividade bactericida do soro como indicador de imunidade inata de pacu Piaractus mesopotamicus (Holmberg, 1887)

The immune system of teleost fish has mechanisms responsible for the defense against bacteria through protective proteins in several tissues. The protein action can be evaluated by serum bactericidal activity and this is an important tool to analyze the immune system. Pacu, Piaractus mesopotamicus, is one of the most important fish in national aquaculture. However there is a lack of studies on its immune responses. In order to standardize and assess the accuracy of the serum bactericidal activity assay, fish were briefly challenged with Aeromonas hydrophila and sampled one week after the challenge. The bacterial infection increased the concentration of protective proteins, resulting in a decrease of colony-forming unit values expressed as well as an enhanced serum bactericidal activity. The protocol showed a reliable assay, appropriate to determine the serum bactericidal activity of pacu in the present experimental conditions.

O sistema imune de peixes teleósteos tem mecanismos responsáveis pela defesa contra bactérias e atua através de proteínas presentes em diversos tecidos. A ação destas proteínas pode ser avaliada pela atividade bactericida do soro, sendo esta uma importante ferramenta para analisar o sistema imune. O pacu, Piaractus mesopotamicus, é um peixe nativo muito importante para aquicultura nacional, entretanto há pouco conhecimento sobre o funcionamento de seu sistema imune. Assim foi realizado experimento para padronizar e avaliar a eficiência do ensaio de atividade bactericida. Resumidamente, peixes foram desafiados por Aeromonas hydrophila e amostradas uma semana após o desafio. A infecção bacteriana promoveu um aumento na concentração de proteínas protetoras, resultando em diminuição dos valores de unidades formadoras de colônias ou expressos também como aumento da atividade bactericida do soro. O protocolo se mostrou confiável, sendo apropriado para determinar a atividade bactericida do soro de pacu nas condições experimentais.

Paratrichodina africana (Ciliophora): a pathogenic gill parasite in farmed Nile tilapia.

Trichodinids are ciliated protozoa that are widely known as one of the main groups of fish parasites. The genus Trichodina presents the greatest species diversity. However, records of Paratrichodina species are scarce, and little is known about their pathogenicity in hosts. The present study provides new records of Paratrichodina africana Kazubski and El-Tantawy (1986) in Nile tilapia from South America and descriptions of pathological changes and seasonality. A total of 304 farmed fish were examined. From gill scraping, parasites were identified using Klein's nitrate impregnation method. Gill samples were fixed for histopathological analysis. Small trichodinid found in this study have a prominent blade apophysis and narrow central part and blade shape that corresponds to the characteristics of P. africana Kazubski and El-Tantawy (1986). Gill lesions were proportional to parasite intensity, in which the gill tissue was compromised in heavy infestation. Proliferative disturbances were found, including epithelial hyperplasia, desquamation, and mononuclear and eosinophilic infiltrate that culminated in necrosis. We did not observe a seasonality effect on the occurrence of P. africana. This ciliated protozoan causes compromised respiratory capacity that leads to severe gill lesions and currently is an important pathogen that afflicts intensive tilapia cultures in Brazil.

First record of Chilodonella hexasticha (Ciliophora: Chilodonellidae) in Brazilian cultured fish: a morphological and pathological assessment.

Chilodonelids are small ciliated protozoans found worldwide and can be dangerous in culture conditions. This study presents morphometric data on the ciliate Chilodonella that is found in cultured Nile tilapia (Oreochromis niloticus), native bait fish tuvira (Gymnotus aff. inaequilabiatus) and native pacu (Piaractus mesopotamicus) and includes a histopathological assessment of the changes that occur in the pacu. For parasitic diagnosis, skin and gill samples were scraped onto slides, dried at room temperature, stained with Giemsa or impregnated with silver nitrate, and the measurements were obtained from photomicrographs. In the diseased pacu, the first gill arch was collected and fixed in a 10% buffered formalin solution for histopathological analysis. Parasite specimens from the different collection sites were identified morphologically as C. hexasticha Kiernik (1909). Diseased fish exhibited depigmentation, skin ulceration, scale loss, excessive mucus production and gill lesions. Histopathological analysis of pacu gills displayed epithelial proliferation with mononuclear inflammatory infiltrate, hemorrhages, and scattering necrosis. In Brazilian-farmed fish this is the first record of C. hexasticha, which has great pathogenic potential in cultured freshwater species. In addition, two new hosts are presented.

Hematology and productive performance of nile tilapia (Oreochromis niloticus) naturally infected with Flavobacterium columnare

Columnaris disease is one of the main causes of mortality in tilapia rearing and is responsible for large economic losses worldwide. Hematology is a tool that makes it possible to study organisms' physiological responses to pathogens. It may assist in making diagnoses and prognoses on diseases in fish populations. The hematological variables of nile tilapia were studied in specimens with a clinical diagnosis of columnaris disease and in specimens that were disease-free. The total erythrocyte count, hemoglobin rate, hematocrit percentage, mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular volume (MCV), organic defense blood cell percentages (leukocytes and thrombocytes) and hepatosomatic and splenosomatic index were determined. The results showed that there were changes in the erythrocytic series and in organic defense blood cells, in the fish infected with the bacterium, with reductions in erythrocytic variables and significant increases in the numbers of circulating lymphocytes and neutrophils.

Hematology and Productive Performance of Nile Tilapia (Oreochromis niloticus) Naturally Infected With Flavobacterium columnare.

Columnaris disease is one of the main causes of mortality in tilapia rearing and is responsible for large economic losses worldwide. Hematology is a tool that makes it possible to study organisms' physiological responses to pathogens. It may assist in making diagnoses and prognoses on diseases in fish populations. The hematological variables of nile tilapia were studied in specimens with a clinical diagnosis of columnaris disease and in specimens that were disease-free. The total erythrocyte count, hemoglobin rate, hematocrit percentage, mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular volume (MCV), organic defense blood cell percentages (leukocytes and thrombocytes) and hepatosomatic and splenosomatic index were determined. The results showed that there were changes in the erythrocytic series and in organic defense blood cells, in the fish infected with the bacterium, with reductions in erythrocytic variables and significant increases in the numbers of circulating lymphocytes and neutrophils.

Isolamento e resistência a antimicrobianos de cepas de Streptococcus spp. provenientes de rãs-touro (Lithobates catesbeianus) / Isolation and antimicrobial resistance of Streptococcus spp. strains from bullfrog (Lithobates catesbeiana)

Twelve bullfrogs were selected from two commercial frog farms and clinically diagnosed as attacked by Streptococcus disease. Sixty samples were collected, and Streptococcus spp. was isolated from all bullfrog, being 12 (100%) from the encephalus, seven from the kidneys (58.3%), three from the liver (25%), two from the spleen (16.6%), and one from the ascitic liquid (8.3%). Streptococcus "-hemolytic were isolated from all the 60 samples, which were sensible to chloramphenicol (100%), gentamycin (100%), vancomycin (96%), cefotaxime (96%), and cefoxitine (92%).

Isolation and characterization of Flavobacterium columnare (Bernardet et al. 2002) from four tropical fish species in Brazil.

Flavobacterium columnare is the causative agent of columnaris disease in freshwater fish, implicated in skin and gill disease, often causing high mortality. The aim of this study was the isolation and characterization of Flavobacterium columnare in tropical fish in Brazil. Piracanjuba (Brycon orbignyanus), pacu (Piaractus mesopotamicus), tambaqui (Colossoma macropomum) and cascudo (Hypostomus plecostomus) were examined for external lesions showing signs of colunmaris disease such as greyish white spots, especially on the head, dorsal part and caudal fin of the fish. The sampling comprised 50 samples representing four different fish species selected for study. Samples for culture were obtained by skin and kidney scrapes with a sterile cotton swabs of columnaris disease fish and streaked onto Carlson and Pacha (1968) artificial culture medium (broth and solid) which were used for isolation. The strains in the liquid medium were Gram negative, long, filamentous, exhibited flexing movements (gliding motility), contained a large number of long slender bacteria and gathered into columns'. Strains on the agar produced yellow-pale colonies, rather small, flat that had rhizoid edges. A total of four Flavobacterium columnare were isolated: 01 Brycon orbignyanus strain, 01 Piaractus mesopotamicus strain, 01 Colossoma macropomum strain, and 01 Hypostomus plecostomus strain. Biochemical characterization, with its absorption of Congo red dye, production of flexirubin-type pigments, H2S production and reduction of nitrates proved that the isolate could be classified as Flavobacterium columnare.

Isolation and characterization of Flavobacterium columnare (Bernardet et al. 2002) from four tropical fish species in Brazil / Isolamento e caracterização de Flavobacterium columnare de quatro espécies de peixes tropicais no Brasil

Flavobacterium columnare is the causative agent of columnaris disease in freshwater fish, implicated in skin and gill disease, often causing high mortality. The aim of this study was the isolation and characterization of Flavobacterium columnare in tropical fish in Brazil. Piracanjuba (Brycon orbignyanus), pacu (Piaractus mesopotamicus), tambaqui (Colossoma macropomum) and cascudo (Hypostomus plecostomus) were examined for external lesions showing signs of colunmaris disease such as greyish white spots, especially on the head, dorsal part and caudal fin of the fish. The sampling comprised 50 samples representing four different fish species selected for study. Samples for culture were obtained by skin and kidney scrapes with a sterile cotton swabs of columnaris disease fish and streaked onto Carlson and Pacha (1968) artificial culture medium (broth and solid) which were used for isolation. The strains in the liquid medium were Gram negative, long, filamentous, exhibited flexing movements (gliding motility), contained a large number of long slender bacteria and gathered into ‘columns'. Strains on the agar produced yellow-pale colonies, rather small, flat that had rhizoid edges. A total of four Flavobacterium columnare were isolated: 01 Brycon orbignyanus strain, 01 Piaractus mesopotamicus strain, 01 Colossoma macropomum strain, and 01 Hypostomus plecostomus strain. Biochemical characterization, with its absorption of Congo red dye, production of flexirubin-type pigments, H2S production and reduction of nitrates proved that the isolate could be classified as Flavobacterium columnare.

Flavobacterium columnare é o agente etiológico da columnariose em peixes de água doce, ocasionando enfermidade na pele e nas brânquias, provocando freqüentemente um grande número de mortalidade. O objetivo deste estudo foi o isolamento e a caracterização de Flavobacterium columnare em peixes tropicais no Brasil. Piracanjuba (Brycon orbignyanus), pacu (Piaractus mesopotamicus), tambaqui (Colossoma macropomum) e cascudo (Hypostomus plecostomus) foram examinados externamente com relação a sinais característicos de columnariose, como manchas acinzentadas na cabeça, região dorsal e pedúnculo caudal dos peixes. A amostragem compreendeu a coleta de 50 exemplares de peixes, representando as quatro diferentes espécies escolhidas para este estudo. Amostras para o isolamento foram obtidas através de raspado com swab estéril das lesões e do rim dos peixes clinicamente diagnosticados como acometidos por columnarios e imediatamente semeados em meios de culturas artificiais (líquido e sólido) próprios para o estudo de Flavobacterium segundo Carlson e Pacha (1968). No meio líquido, houve o desenvolvimento de microrganismos que observados em gota pendente apresentaram a forma de bacilos finos, longos, móveis por deslizamento. Através da coloração de Gram, apresentaram morfologia de bacilos finos, Gram negativos, agrupados em colunas. Em meio sólido, as colônias eram pequenas, cinza-amareladas, com borda em forma de raiz. No total, foram obtidos quatro isolamentos: 01 cepa de Brycon orbignyanus; 01 cepa de Piaractus mesopotamicus; 01 cepa de Colossoma macropomum; e 01 cepa de Hypostomus plecostomus. A caracterização bioquímica das amostras, como absorção do vermelho Congo, produção de flexirrubina, produção de H2S e redução do nitrato, sugere que os isolamentos poderiam ser classificados como Flavobacterium columnare.