Evaluation of three alum-precipitated Aeromonas hydrophila vaccines administered to Labeo rohita, Cirrhinus mrigala and Ctenopharyngodon idella: immunokinetics, immersion challenge and histopathology / Avaliação de três vacinas de Aeromonas hydrophila precipitadas com alúmen administradas a Labeo rohita, Cirrhinus mrigala e Ctenopharyngodon idella: imunocinética, desafio de imersão e histopatologia

Abstract Aeromonas hydrophila is a cause of infectious disease outbreaks in carp species cultured in South Asian countries including Pakistan. This bacterium has gained resistance to a wide range of antibiotics and robust preventive measures are necessary to control its spread. No prior use of fish vaccines has been reported in Pakistan. The present study aims to develop and evaluate inactivated vaccines against local strain of A. hydrophila in Pakistan with alum-precipitate as adjuvant. The immunogenic potential of vaccine was evaluated in two Indian major carps (Rohu: Labeo rohita, Mori: Cirrhinus mrigala) and a Chinese carp (Grass carp: Ctenopharyngodon idella). Fish were vaccinated intraperitoneally followed by a challenge through immersion. Fish with an average age of 4-5 months were randomly distributed in three vaccinated groups with three vaccine concentrations of 108, 109 and 1010 colony forming unit (CFU)/ml and a control group. Fixed dose of 0.1ml was applied to each fish on 1st day and a booster dose at 15 days post-vaccination (DPV). Blood samples were collected on 14, 28, 35, 48 and 60 DPV to determine antibody titers in blood serum using compliment fixation test (CFT). Fish were challenged at 60 DPV with infectious A. hydrophila with 108 CFU/ml through immersion. Significantly higher levels of antibody titers were observed from 28 DPV in all vaccinated groups as compared to those in the control group. In challenge experiment the average RPS (relative percent survivability) was 71% for groups vaccinated with 109 and 1010 CFU/ml and 86% for 108 CFU/ml. Vaccine with 108 CFU/ml induced highest immune response followed by 109 and 1010 CFU/ml. The immune response of L. rohita and C. idella was better than that of C. mrigala. In general, normal histopathology was observed in different organs of vaccinated fish whereas minor deteriorative changes were found in fish vaccinated with higher concentrations of the vaccine.

Resumo Aeromonas hydrophila é uma causa de surtos de doenças infecciosas em espécies de carpas cultivadas em países do sul da Ásia, incluindo o Paquistão. Essa bactéria ganhou resistência a uma ampla gama de antibióticos, e medidas preventivas robustas são necessárias para controlar sua disseminação. Nenhum uso anterior de vacinas para peixes foi relatado no Paquistão. O presente estudo tem como objetivo desenvolver e avaliar vacinas inativadas contra cepa local de A. hydrophila no Paquistão com precipitado de alúmen como adjuvante. O potencial imunogênico da vacina foi avaliado em duas carpas principais indianas (Rohu: Labeo rohita, Mori: Cirrhinus mrigala) e uma carpa chinesa (Grass Carp: Ctenopharyngodon idella). Os peixes foram vacinados por via intraperitoneal, seguido de um desafio por imersão. Peixes com idade média de 4-5 meses foram distribuídos aleatoriamente em três grupos vacinados com três concentrações de vacina de 108, 109 e 1010 unidades formadoras de colônias (UFC) / ml e um grupo de controle. Foi aplicada dose fixa de 0,1ml em cada peixe no 1º dia e dose de reforço 15 dias pós-vacinação (DPV). Amostras de sangue foram coletadas em 14, 28, 35, 48 e 60 DPV para determinar os títulos de anticorpos no soro sanguíneo usando o teste de fixação de elogio (CFT). Os peixes foram desafiados a 60 DPV com infecciosa A. hydrophila com 108 CFU / ml por imersão. Níveis significativamente mais elevados de títulos de anticorpos foram observados em 28 DPV em todos os grupos vacinados, em comparação com aqueles no grupo de controle. Na experiência de desafio, o RPS médio (sobrevivência percentual relativa) foi de 71% para os grupos vacinados com 109 e 1010 CFU / ml e 86% para 108 CFU / ml. A vacina com 108 UFC / ml induziu a maior resposta imune seguida por 109 e 1010 UFC / ml. A resposta imune de L. rohita e C. idella foi melhor do que a de C. mrigala. Em geral, histopatologia normal foi observada em diferentes órgãos de peixes vacinados, enquanto pequenas alterações deteriorantes foram encontradas no grupo de controle e nos peixes vacinados com concentrações mais altas da vacina.

Parasitological diagnosis in food fish produced in a fish farm, in the Zona da Mata, Minas Gerais, southeastern Brazil / Diagnóstico parasitológico em peixes para consumo produzidos em uma psicultura, na Zona da Mata, Minas Gerais, região sudeste do Brasil

Abstract This study identified the parasitic species in juvenile freshwater finfishes during the fattening stage, from a fish farm located in the Zona da Mata (MG), southeastern Brazil, and revealed both macro and microscopical lesions in fish gills. A total of 172 juvenile fishes of different species (Oreochromis niloticus, Ictalurus punctatus, Ctenopharyngodon idella, Cyprinus carpio, Astyanax bimaculatus and Brycon amazonicus) were transported to a laboratory in São Paulo city. The fish were sedated and then euthanized for parasitological analysis. All fish were infected by at least one parasite species. Ten different species of parasites were identified: Apiosoma sp., Epistylis sp., Ichthyobodo sp., trichodinids, Piscinoodinium pillulare, Ichthyophthirius multifiliis, Tetrahymena sp., monogeneans, Centrocestus formosanus metacercariae, and Dermocystidium sp. The best management practices and lack of sanitary control were also discussed.

Resumo Este trabalho identificou espécies parasitas em peixes de produção juvenis de água doce, durante a fase de engorda, oriundos de uma piscicultura da Zona da Mata (MG), na região sudeste do Brasil, além das lesões de brânquias, causadas tanto macro quanto microscopicamente. Um total de 172 peixes juvenis de diferentes espécies (Oreochromis niloticus, Ictalurus punctatus, Ctenopharyngodon idella, Cyprinus carpio, Astyanax bimaculatus e Brycon amazonicus) foram transportados para um laboratório na cidade de São Paulo. Os peixes foram anestesiados e eutanasiados para análise parasitológica. Todos os peixes estavam acometidos por pelo menos uma espécie de parasito. Dez diferentes espécies de parasitos foram identificadas: Apiosoma sp., Epistylis sp., Ichthyobodo sp., tricodinídeos, Piscinoodinium pillulare, Ichthyophthirius multifiliis, Tetrahymena sp., monogeneas, metacercárias de Centrocestus formosanus e Dermocystidium sp. As boas práticas de manejo e inadequado controle sanitário também foram discutidos.

High concentration of levamisole in the diet of Colossoma macropomum (Pisces: Serrasalmidae) is effective for controlling monogenean parasites / Alta concentração de levamisol na dieta de Colossoma macropomum (Pisces: Serrasalmidae) é efetiva para controlar parasitos monogenea

Abstract This study investigated the effects of diets supplemented with levamisole on monogeneans on the gills of Colossoma macropomum. Fish were fed with diets containing levamisole at concentrations of 0, 300, 600, 900 and 1200 mg kg-1 for 24, 96 and 240 h and the infection by Anacanthorus spatulatus, Notozothecium janauachensis and Mymarothecium boegeri were evaluated. None of the levamisole concentrations caused either mortality or behavioral alterations in fishes during 240 h of feeding. After 24 h of feeding with 1200 mg kg-1 of levamisole, the abundance of N. janauachensis decreased in comparison with treatments of 0, 300, 600 and 900 mg kg-1, as did the abundance of M. boegeri after 240 h of feeding with 1200 mg kg-1 of levamisole. The efficacy of 900 mg kg-1 of levamisole was only 55.7% after 96 h of feeding, but it was 84.6% after 240 h of feeding with 1200 mg kg-1. Our results show that 1200 mg kg-1 of levamisole for 10 days has good anthelmintic efficacy against monogeneans of C. macropomum. Since monogeneans elicit some of the worst problems in C. macropomum, this study has provided evidence of an effective control method that may be used in fish farms.

Resumo Este estudo investigou os efeitos de dietas suplementadas com levamisol na infecção por monogeneas nas brânquias de Colossoma macropomum. Os peixes foram alimentados com dietas contendo 0, 300, 600, 900 e 1200 mg kg-1 de levamisol por 24, 96 e 240 h e os níveis de infecção por Anacanthorus spatulatus, Notozothecium janauachensis e Mymarothecium boegeri, foram avaliados. Nenhuma das concentrações de levamisol causou mortalidade ou alterações comportamentais nos peixes durante 240 h de alimentação. Após 24 h de alimentação com 1.200 mg kg-1 de levamisol, a abundância de N. janauachensis diminuiu quando comparada aos tratamentos com 0, 300, 600 e 900 mg kg-1, bem como a abundância de M. boegeri após 240 h de alimentação com 1200 mg kg-1 de levamisol. A eficácia de 900 mg kg-1 de levamisol foi somente de 55,7% após 96 h de alimentação, mas foi de 84,6% após 240 h de alimentação com 1200 mg kg-1. Os resultados mostram que 1200 mg kg-1 de levamisol durante 10 dias, tem uma boa eficácia antihelmíntica contra monogeneas de C. macropomum. Como monogeneas provocam alguns dos piores problemas em C. macropomum, este estudo forneceu evidências de um método de controle eficaz que pode ser usado em pisciculturas.

Exogenous enzyme complex prevents intestinal soybean meal-induced enteritis in Mugil liza (Valenciennes, 1836) juvenile

ABSTRACT Four soybean meal-based diets containing increasing levels of an enzyme complex (E50, E100, E150 and E200 at 50, 100, 150 and 200 g ton-1, respectively) and one soybean meal-based diet without the enzyme complex (E0) were fed in triplicate to M. liza juveniles in a semi-static flow system with 20 fish per tank for 75 days. There were no differences between the treatments for animal performance parameters, but fish fed the enzyme complex treatment exhibited significantly (P<0.05) higher values of calcium bone retention compared with control fish. Although there was no relationship between bacterial counts in different sections of the gastrointestinal tract or enzyme levels, filamentous bacteria were increased in E50 compared with E150. All of the treatments resulted in higher bacterial counts in the stomach than in intestinal segments. Histological screening showed serious to moderate infiltration of inflammatory cells, modification in villus morphology and necrosis in some cases in fish fed the E0 diet. In addition, fish from the E0 treatment exhibited significantly (P<0.05) lower lipid deposition in the peritoneal cavity. Therefore, the use of low levels of exogenous enzyme is recommended in diets for M. liza when soybean meal is used as the main source of protein.

Protozoan infections in farmed fish from Brazil: diagnosis and pathogenesis / Infecções por protozoários em peixes cultivados no Brasil: diagnóstico e patogênese

The Phylum Protozoa brings together several organisms evolutionarily different that may act as ecto or endoparasites of fishes over the world being responsible for diseases, which, in turn, may lead to economical and social impacts in different countries. Apart from the recent advances for the diagnosis of fish diseases in Brazil, little is known on the protozoan parasites and their relationship with environment and host. This revision presents the most important protozoan parasites found in farmed fish from Brazil, not only with emphasis on its diagnosis, biology, transmission and host-parasite relationship, but also on some information that may be useful to researchers in determining the correct diagnosis in fish farms.

O filo Protozoa reúne diversos organismos evolutivamente distintos que podem atuar como ecto ou endoparasitos de peixes em todo o mundo, sendo responsáveis por doenças as quais, por sua vez, podem ocasionar impactos econômico e social nos diferentes países. Apesar dos recentes avanços no campo de diagnóstico de doenças em peixes no Brasil, ainda pouco se conhece sobre a fauna de protozoários parasitos de peixes e suas relações com o ambiente e hospedeiro. Esta revisão apresenta os mais importantes protozoários parasitos encontrados em peixes cultivados no Brasil, não apenas com ênfase no seu diagnóstico, biologia, transmissão e relação hospedeiro-parasito, mas também algumas informações que podem ser úteis para pesquisadores para o correto diagnóstico em pisciculturas.

Proposed method for agglutinating antibody titer analysis and its use as indicator of acquired immunity in pacu, Piaractus mesopotamicus / Aglutinante de anticorpos como indicador de imunidade adquirida de pacu Piaractus mesopotamicus

Antibody can be assessed by agglutinating antibody titer which is a quantitative measure of circulating antibodies in serum from fish previously immunized. The antibody evaluation has been performed with different fish species, and is considered a reliable method that can be applied to confirm several hypothesis regarding acquired immunity, even in conjunction with precise methods to describe immune mechanisms. In order to provide appropriate analytical methods for future studies on the specific immune system of native fish, the present study standardized on assay to measure the serum agglutinating antibody titer produced after immunization with inactivated A. hydrophila and levamisole administration in pacu. It was possible to determine the agglutinating antibodies titer in a satisfactorily way in pacu immunized with inactive A. hydrophila, and the highest titers were observed on fish fed with levamisole.

Os anticorpos podem ser avaliados pelo título aglutinante de anticorpos, que é uma medida quantitativa de anticorpos no soro de peixe previamente imunizados. A determinação do título de anticorpos foi realizada com diversas espécies de peixes e é considerado um método confiável que pode ser aplicado para confirmar diversas hipóteses que envolvam o sistema adquirido de defesa, mesmo em conjunto com métodos precisos, para descrever mecanismos imunes. A fim de prover métodos analíticos adequados para futuros estudos sobre o sistema imune específico de peixes nativos, o presente estudo aperfeiçoou o ensaio para avaliar o título aglutinante de anticorpos em soro de pacu imunizados com A. hydrophila e alimentados com levamisol. Foi possível determinar o título aglutinante de anticorpos de forma satisfatória, em pacus imunizados com A. hydrophila inativa, e os maiores títulos foram observados em peixes alimentados com levamisol.

Nanoparticles and microparticles of polymers and polysaccharides to administer fish vaccines

Aquaculture has become an important economic sector worldwide, but is faced with an ongoing threat from infectious diseases. Vaccination plays a critical role in protecting commercially raised fish from bacterial, viral and parasitic diseases. However, the production of effective vaccines is limited by the scarcity of knowledge about the immune system of fish. Improving vaccines implies using antigens, adjuvants and employing methods of administration that are more effective and less harmful to the fish. In this context, in recent year there have studies of methods of encapsulating antigens in matrices of different types to apply in fish vaccines. This work reviews the new methods to improve fish vaccines by encapsulating them in polymers and polysaccharides.

Vaccines for finfish aquaculture: what do we need to know to make them work?

Aquaculture still faces serious economic impacts due to the loss of animals to disease. A conservative estimate of 5 percent losses due to disease means that the finfish aquaculture industry loses over $1 billion annually on a global scale. One proven way to prevent costly disease outbreaks is to vaccinate fish against common or known pathogens. Current vaccination schemes still result in losses, however, and this may be due in part to vaccine design. Vaccines are currently designed using state of the art knowledge of immune responses, which is based primarily on mammalian studies. Just how applicable is this information to fish immunity and vaccine design, however? This review discusses what is currently known for teleost fish about two key processes that drive immune response: antigen presentation and cytokine regulation. In both cases many of the genes known to be involved have been identified; in the case of cytokines recent genome projects have added to the total rapidly in recent years. Most functional studies to date in these areas have focused on gene expression and mRNA levels, due to a lack of available antibodies that are required for studies at the protein level. These studies are confounded by the fact that in many cases the teleost equivalents of single copy mammalian genes are duplicated and are regulated in very different ways. This suggests that vaccines designed around mammalian immunological principles will not be as efficient as they could be. Future research goals for fish immunologists should be to develop the antibodies required for protein level functional studies in order to provide the true understanding of fish immunity that is required for the design of finfish aquaculture vaccines that are truly effective.

Doenças de peixes: profilaxia, diagnóstico e tratamento / Illnesses of fish: Prophylaxis, diagnosis and treatment

Publicada pela primeira vez em 1998, esta obra tem como objetivo divulgar, aos profissionais da piscicultura, informações básicas, e de certa maneira com caráter aplicado, sobre as doenças mais importantes que podem comprometer os peixes de água doce quando criados em cativeiro. Esta segunda edição tem algumas modificações em relação à anterior, embora mantenha a mesma organi-zação geral. A cultura de peixes marinhos é cada vez mais importante e, conquanto seja ainda incipiente no Brasil, terá sem dúvida, em médio prazo, uma relevância considerável. Assim, além de outras, foram também incluídas doenças que se manifestam nas pisciculturas marinhas, algumas de grande importância pelo impacto econômico que podem ter nessas explorações. Por outro lado, foram ampliadas as ilustrações fotográficas e incluídas inovações

Métodos de estudo e técnicas laboratoriais em parasitologia de peixes / Methods of study and laboratorial techniques in parasitology of fish

Quando se estudam os parasitas de peixes pode-se ter em mente vários objetivos a serem atingidos, destacando-se aqueles que compreendem os estudos sistemáticos, ecológicos, profiláticos, histopatológicos, ciclos de vida, análise das relações parasitas-hospedeiros, entre outros. Para o seu desenvolvimento, normalmente, são utilizados numerosos métodos e técnicas cujo conhecimento e correta aplicação são fundamentais para que se possam atingir os objetivos inicialmente propostos. Esses instrumentos encontram-se descritos em numerosas publicações dispersas, cuja obtenção nem sempre é uma tarefa fácil. Uma publicação que reunisse os métodos de estudo e técnicas laboratoriais utilizados em parasitologia de preixes poderia ser de grande utilidade para quem se inicia ou já trabalha nesta área da parasitologia

Application of hazard analysis critical control point (HACCP) as a possible control measure for Opisthorchis viverrini infection in cultured carp (Puntius gonionotus).

Opisthorchiasis due to Opisthorchis viverrini and transmitted through infected freshwater cyprinoid fish (carps) affects more than 8 million people in Thailand, People's Democratic Republic of Lao, and Vietnam. The Hazard Analysis Critical Control Point (HACCP)-concept has been recommended by FAO and WHO to be included in programs to control foodborne trematode infections (FBT). HACCP is a multifactorial approach to control food hazards through surveillance of diseases, foods, and operations and education. This study describes the first attempt to apply HACCP to the prevention and control of Opisthorchis viverrini in pond culture carp (Puntius gonionotus). The experiment was designed and carried out by a multidisciplinary "HACCP team" including experts in the field of public health, parasitology, epidemiology, aquaculture, fisheries extension and fish inspection. The investigation was performed in two fish ponds in the District of Sun Pa Tong, Chiang Mai, Thailand. In the experimental pond, fish was cultured according to HACCP principles and compared with the control pond, which followed conventional aquaculture practices. Water supply to the pond, fish fry, fish feed and pond conditions during the growing period were identified as critical control points (CCPs). Hazards were identified and analyzed, as well as control measures, critical limits, monitoring procedures, corrective actions, and record keeping developed for each one of the above CCPs. Complete pond preparation, particularly aiming to eliminate contamination of pond water with O. viverrini eggs, fish infected with parasite meacercariae and the first intermediate host (Bithynia spp), was conducted. After the pond was filled with water, O. viverrini metacercaria-free fry were released into the pond. The preliminary results obtained indicate that HACCP-based principles applied to carp pond culture could be used as a strategy to prevent and control O. viverrini. Further studies should be undertaken aiming to confirm these preliminary results.