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.

Atrazine levels in the Jaboticabal water stream (São Paulo State, Brazil) and its toxicological effects on the pacu fish Piaractus mesopotamicus.

The aim of this study was to determine the environmental concentration of atrazine (ATZ) in five streams located in the north of São Paulo state (Brazil) and evaluate its toxicological impact on young specimens of the pacu fish Piaractus mesopotamicus. Samples of water were collected on three occasions between 2010 and 2011, corresponding to periods signifying the beginning, middle, and end of rain season. ATZ levels were estimated by a high-performance liquid chromatography coupled with a mass spectrometry (HPLC-MS/MS) triple quadrupole. Later, the quotient of environmental risk (QR) was determined based on the medium lethal concentration (LC50 48 h), non-observable effect concentration (NOEC), and the estimated environmental concentration (EEC) of ATZ detected in the environment. Histological changes in gills and liver were also studied, along with the brain activity of the enzyme acetylcholinesterase (AChE). The highest concentration of ATZ measured was 10.4 µg L(-1). The ATZ LC50 (48 h) for young P. mesopotamicus was 24.46 mg L(-1) and the QR was classified as "safe". Although the QR indicated that ATZ could be safe for the tested species, it caused many histological alterations in the liver and gills of the exposed specimens, and an increase in the AChE levels.

Histopathological biomarkers in pacu (Piaractus mesopotamicus) infected with aeromonas hydrophila and treated with antibiotics.

Pharmacochemicals usage in fish farming disease treatment can cause morphological and functional changes in absorption capacity, metabolism and excretion organs. The aim of this research was to evaluate the histopathological biomarkers in the gills, liver, kidney and skin of pacu (P. mesopotamicus), which have been infected with A. hydrophila and treated with oxytetracycline (OTC) and florfenicol (FFC). Fish were exposed to 2.4×10(7) mL(-1) of A. hydrophila bacteria experimental infection and after 24h exposed to FFC treatment for ten days and OTC for seven days. OTC was not effective in the A. hydrophila control in pacu in up to 170.0 mg kg(-1) concentration. Nevertheless, FFC was 100% effective with 10.0 mg kg(-1) concentration. After the treatment, skin, gills, liver and kidney samples were collected and processed for histopathological analysis. A. hydrophila caused lamellar fusion, sub epithelial edema, mucous hypertrophy and hyperplasia, lining, pillar and chloride cells. Hepatocytes hypertrophy was observed on liver, as a result of the antibiotics metabolism and bacteria cell wall. The histopathological biomarkers show the effects of the presence of the A. hydrophila. The use of the antibiotic florfenicol decreases the bacterial action effectiveness on tissues evaluated. Thus, the histopathological biomarkers show the A. hydrophila effect and the antibiotics treatment. The skin and liver are exposure biomarkers for both.

Efeitos tóxicos de surfactantes fitossanitários para o peixe mato grosso (Hyphessobrycon eques) / Toxic effects of phytosanitary surfactants for jewel tetra (Hyphessobrycon eques)

Os surfactantes são moléculas anfipáticas que reduzem a tensão superficial da água e fazem parte dos componentes inertes das formulações de produtos fitossanitários. Assim, os objetivos deste trabalho foram: estimar a concentração letal (CL(I)(50;96h)); classificar e avaliar a qualidade da água durante os testes de toxicidade aguda dos surfactantes: Agral®, Aterbane® BR, Ag-Bem®, Energic®, Fixade® e Gotafix® para o peixe mato grosso (Hyphessobrycon eques); e os sinais de intoxicação nos animais. Para tanto, os peixes foram aclimatados por dez dias em sala de bioensaio. Os animais foram expostos aos surfactantes em DIC com três repetições. A CL(I)(50;96h) do surfactante Agral® foi 3,29 mg L-1; do Aterbane® BR, 8,21 mg L-1; do Energic®, 2,34 mg L-1; do Gotafix®, 4,37 mg L-1; do Fixade®, 3,38 mg L-1 e do Ag-Bem®, 34,95 mg L-1. As variáveis de qualidade da água não foram alteradas. Os peixes apresentaram aumento do batimento opercular após a exposição; 4 e 24h, perda da capacidade de arfagem; 48h, batimento opercular lento e 72 e 96h, recuperação. Os surfactantes Energic®, Agral®, Gotafix®, Aterbane® BR e Fixade® podem ser classificados como moderadamente tóxicos e o Ag-Bem® como pouco tóxico para H. eques e este organismo apresenta sinais de intoxicação semelhantes para todos os surfactantes.

Surfactants are amphipatic molecules that reduce the surface tension of water and make up the inert components of pesticide formulations. Thus, the objectives of this study were: to estimate the lethal concentration (LC(I)50;96h); classify and evaluate water quality during testing of the following surfactants: Agral®, Aterbane® BR, Ag-bem®, Energic®, Fixade® and Gotafix® for jewel tetra (Hyphessobrycon eques); and the signs of intoxication in the animals. For this, the fish were acclimated for ten days in the bioassay room. The animals were exposed to the surfactants in an entirely randomized design with three replications. The LC(I)(50;96h) of surfactant Agral® was 3.29 mg L-1; Aterbane® BR 8.21 mg L- 1; Energic® 2.34 mg L-1; Gotafix® 4.37 mg L- 1; Fixade® 3.38 mg L-1; and Ag-bem® 34.95 mg L-1. The variables of water quality were unchanged. The fish showed an increase in the opercular beating after exposure; 4 and 24 hours, loss of gasping ability; 48 hours, slow opercular beating; and 72 and 96 hours later, recovery. The surfactants Energic®, Agral®, Gotafix®, Aterbane® BR and Fixade® can be classified as moderately toxics, and Ag-Bem® as slightly toxic for H. eques; this organism shows similar intoxication signs for all surfactants.