Isolation, Characterization and Pathology of Citrobacter freundii Infection in Native Brazilian Catfish Pseudoplatystoma

Bacterial diseases are responsible for important economic losses in aquaculture over the world, and South American catfishes are susceptible to Gram negative bacteria causative of haemorrhagic septicemia. This study registers the biochemical characteristics, antimicrobial resistance and pathological effects of Citrobacter freundii infection in cachara Pseudoplatystoma reticulatum. Moribund juvenile fish with non-specific clinical signs were used for bacteriological and parasitological diagnosis. No parasitic infections were found in examined fish, but two isolate obtained from the kidney and encephalon was characterized by Gram negative bacilli, catalase positive and oxidase negative. Isolates were submitted to biochemical identification by a commercial API 20E. Susceptibility analysis to 15 drugs was performed by the diffusion method in Agar Muller Hinton discs. Experimental assay was also made to confirm the Kock postulate. The isolates showed biochemical profile corresponding to Citrobacter freundii. A multiresistance at 66.7% of the antibiotic molecules tested was observed. In experimental infection haemorrhagic septicaemia, severe enteritis and important lesions in kidney and gills are described.

Isolation, Characterization and Pathology of Citrobacter freundii Infection in Native Brazilian Catfish Pseudoplatystoma

Bacterial diseases are responsible for important economic losses in aquaculture over the world, and South American catfishes are susceptible to Gram negative bacteria causative of haemorrhagic septicemia. This study registers the biochemical characteristics, antimicrobial resistance and pathological effects of Citrobacter freundii infection in cachara Pseudoplatystoma reticulatum. Moribund juvenile fish with non-specific clinical signs were used for bacteriological and parasitological diagnosis. No parasitic infections were found in examined fish, but two isolate obtained from the kidney and encephalon was characterized by Gram negative bacilli, catalase positive and oxidase negative. Isolates were submitted to biochemical identification by a commercial API 20E. Susceptibility analysis to 15 drugs was performed by the diffusion method in Agar Muller Hinton discs. Experimental assay was also made to confirm the Kock postulate. The isolates showed biochemical profile corresponding to Citrobacter freundii. A multiresistance at 66.7% of the antibiotic molecules tested was observed. In experimental infection haemorrhagic septicaemia, severe enteritis and important lesions in kidney and gills are described.(AU)

Weissella sp. outbreaks in commercial rainbow trout (Oncorhynchus mykiss) farms in Brazil.

The genus Weissella contains 14 bacterial species that usually occur in nutrient-rich environments and in fermented foods and beverages. Outbreaks of hemorrhagic septicemia were reported in three commercial rainbow trout (Oncorhynchus mykiss) farms in Brazil in 2008 and 2009. Seventy-seven Gram-positive isolates were obtained from 41 diseased fish from these farms. The bacterial strains were identified as Weissella at the genus level using biochemical tests, Weissella genus-specific PCR, and 16S rRNA sequencing. To evaluate potential routes of infection, rainbow trout juveniles were experimentally infected with the pathogen. In addition, the resistance of the pathogen to five antibiotics was tested, and provisional epidemiological cut-off values were calculated using the normalized resistance interpretation (NRI). All isolates presented similar phenotypic profiles and positive reactions for Weissella genus-specific PCR. The 16S rRNA sequences of the Brazilian strains showed 100% similarity with sequences of Chinese isolates that previously were identified as the first case of Weissella sp. infection in fish. The disease was successfully reproduced in the laboratory by intraperitoneal injection, immersion, and cohabitation between diseased and healthy fish. All isolates were resistant to sulfonamide, and based on NRI analysis, one, two, and three isolates were classified as non-wild-type (NWT) for erythromycin, oxytetracycline, and norfloxacin, respectively. This is the first description of multiple cases of Weissella sp. infection in rainbow trout farms outside of China, of infectious routes for the disease, and of provisional epidemiological cut-off values for resistance of these bacteria to four antibiotics.

Distúrbios morfológicos em leucócitos de exemplar de kinguio (Carassius auratus auratus Linnaeus, 1758) com septicemia hemorrágica bacteriana: relato de caso / Morphologic disorders in leukocytes of goldfish sample (Carassius auratus auratus Linnaeus, 1758) with bacterial hemorrhagic septicemia: case report

O presente estudo teve por objetivo relatar um caso sobre as alterações morfológicas em leucócitos de kinguio comsepticemia hemorrágica bacteriana. Um exemplar foi atendido no Laboratório de Patologia de Organismos Aquáticos doCAUNESP, Unesp de Jaboticabal, apresentando apatia, natação errática, anorexia e discreta perda de equilíbrio, buscandoconstantemente a superfície da água. Através de análise visual da superfície corpórea, foram observadas áreas de hemorragiapetequial na base das nadadeiras peitoral, pélvica e caudal, além da órbita bucal, periocular e anal e efusão celomática. Naextensão sanguínea foram observadas a presença de monócitos ativados, hiperativados, e fagócitos mononuclearesrealizando eritrofagocitose e leucofagocitose, neutrófilos com basofilia citoplasmática e com vacuolização citoplasmática,além de imunócitos e leucócitos imaturos com anisocitose e anisocariose. As alterações morfológicas descritas no presenteestudo revelaram parte da patogenia e resposta orgânica de kinguio com septicemia hemorrágica bacteriana. Dessa forma,as análises qualitativas das células sanguíneas devem ser também utilizadas como ferramenta complementar aos estudosdo estado geral da saúde de peixes em cultivo.

The morphological changes in leukocytes of goldfish with bacterial hemorrhagic septicemia were reported. One sample was served at the Laboratory of Pathology of Aquatic Organisms of CAUNESP, Unesp in Jaboticabal. The fish showed lethargy, erratic swimming and without force, slight loss of balance, trying constantly to the water surface. In the body inspection, areas of petechial hemorrhage at the base of the pectoral fins, pelvic fins and caudal fin were observed, beyond the periocular and oral orbitand anal. Blood smears presented hiperactivated monocytes, performing erythrophagocytosis and leukophagocytosis, the cytoplasm of neutrophils presented basophilia and vacuolization, beyond immunocytes, immature leukocytes with anisocytosis and anisokaryosis. The morphological changes described in this study revealed part of the pathogenesis and organic response of Kinguio with bacterial hemorrhagic septicemia. Then, the qualitative analysis of blood cells should also be used as a complementary tool to study the general health status of fish farmed

Distúrbios morfológicos em leucócitos de exemplar de kinguio (Carassius auratus auratus Linnaeus, 1758) com septicemia hemorrágica bacteriana: relato de caso / Morphologic disorders in leukocytes of goldfish sample (Carassius auratus auratus Linnaeus, 1758) with bacterial hemorrhagic septicemia: case report

O presente estudo teve por objetivo relatar um caso sobre as alterações morfológicas em leucócitos de kinguio comsepticemia hemorrágica bacteriana. Um exemplar foi atendido no Laboratório de Patologia de Organismos Aquáticos doCAUNESP, Unesp de Jaboticabal, apresentando apatia, natação errática, anorexia e discreta perda de equilíbrio, buscandoconstantemente a superfície da água. Através de análise visual da superfície corpórea, foram observadas áreas de hemorragiapetequial na base das nadadeiras peitoral, pélvica e caudal, além da órbita bucal, periocular e anal e efusão celomática. Naextensão sanguínea foram observadas a presença de monócitos ativados, hiperativados, e fagócitos mononuclearesrealizando eritrofagocitose e leucofagocitose, neutrófilos com basofilia citoplasmática e com vacuolização citoplasmática,além de imunócitos e leucócitos imaturos com anisocitose e anisocariose. As alterações morfológicas descritas no presenteestudo revelaram parte da patogenia e resposta orgânica de kinguio com septicemia hemorrágica bacteriana. Dessa forma,as análises qualitativas das células sanguíneas devem ser também utilizadas como ferramenta complementar aos estudosdo estado geral da saúde de peixes em cultivo.

The morphological changes in leukocytes of goldfish with bacterial hemorrhagic septicemia were reported. One sample was served at the Laboratory of Pathology of Aquatic Organisms of CAUNESP, Unesp in Jaboticabal. The fish showed lethargy, erratic swimming and without force, slight loss of balance, trying constantly to the water surface. In the body inspection, areas of petechial hemorrhage at the base of the pectoral fins, pelvic fins and caudal fin were observed, beyond the periocular and oral orbitand anal. Blood smears presented hiperactivated monocytes, performing erythrophagocytosis and leukophagocytosis, the cytoplasm of neutrophils presented basophilia and vacuolization, beyond immunocytes, immature leukocytes with anisocytosis and anisokaryosis. The morphological changes described in this study revealed part of the pathogenesis and organic response of Kinguio with bacterial hemorrhagic septicemia. Then, the qualitative analysis of blood cells should also be used as a complementary tool to study the general health status of fish farmed(AU)

Evaluation of DNA vaccination of spotted sand bass (Paralabrax maculatofasciatus) with two major outer-membrane protein-encoding genes from Aeromonas veronii.

Genes encoding two major outer membrane proteins (OMPs) of the bacterial pathogen Aeromonas veronii, Omp38 and Omp48, were used to construct DNA vaccines. The protective effect of such vaccines against motile aeromonad septicaemia was evaluated in spotted sand bass (Paralabrax maculatofasciatus), an endemic species of the Mexican Northwest Pacific coast and a potential resource for the aquaculture industry. Weak protein expression, as determined by immunoblotting, was observed after transfection of eukaryotic cells with the DNA vaccines. Fish immunized with a single intramuscular injection of 20 microg of the omp38 and omp48 DNA vaccines showed slightly, but significantly elevated serum antibody levels 4 and 6 weeks after vaccination, compared to fish vaccinated with the control plasmid pcDNA3.1. Spotted sand bass vaccinated with the omp38 and omp48 DNA vaccines and challenged with A. veronii by intraperitoneal route recorded a relative percent survival (RPS) between 50 and 60%. Histopathological signs of motile aeromonad septicaemia were observed in around 40% of omp38 and omp48-vaccinated fish and 80% of pcDNA3.1-vaccinated control fish. The results indicate that P. maculatofasciatus vaccinated with a single dose of DNA plasmids encoding the major OMPs from A. veronii shows partial protection against infection and mortality by A. veronii experimental infection.

Peste suína clássica: II. Estudo sobre um vírus amostra chinesa, adaptado ao cultivo celular (amostra Porto Alegre) / Classical swine fever: II. A study of a chinese strain adapted in tissue culture (Strain Porto Alegre)

Uma amostra genuína chinesa foi adaptada em cultura de células de linhagem de rim de coelho (RK13), através de passagens sucessivas e clonagem, na temperatura de 37§ C. Na décima passagem, após a clonagem, a semente foi liofilizada e estocada a - 70§ C. A partir dessa semente, entäo denominada da Cepa Porto Alegre (CPA), foram produzidas várias suspensöes víricas com as quais preparou-se uma vacina experimental. As características "in vitro" e "in vivo" da amostra CPA foram entäo estudadas. "In vitro", a amostra de vírus cresce regularmente em células RK13, demonstrada pelo teste e imunofluorescência direta com formaçäo de grandes placas de células com citoplasma fluorescente. Entretanto a amostra CPA näo cresceu em células PK15 nas primeiras 5 passagens. "In vivo" a amostra CPA näo provocou fluorescência em tecido de suínos vacinados, mas é hipertermizante para coelhos. O vírus liofilizado resistiu bem a 27§ C, durante 5 meses e por 12 meses na temperatura entre 15 e 36§ C. Reconstituído com o respectivo diluente e mantido a 25-26§ C, manteve título infeccioso estável por 24 horas e conferiu 100//de proteçäo em vacinas até 72 horas pós diluiçäo. Esta amostra näo mostrou virulência residual para suínos imunodeprimidos e é inócua para porcas gestantes, a partir de 40 dias da cobertura. Näo possui capacidade de difusäo de suíno para suíno, e näo foi possível recuperar o vírus de órgäos de suínos, após 42 dias de vacinaçäo. A amostra CPA só pode ser recuperada do suíno, através de inoculaçäo de macerado de baço em coelhos, por via endovenosa. Os suínos vacinados e desafiados 14 dias após foram 100//protegidos, näo havendo pique febril causado pelo vírus de desafio