Effects of thymol supplementation on performance, mortality and branchial energetic metabolism in grass carp experimentally infected by Aeromonas hydrophila.

We determined whether thymol supplementation of would minimize the negative effects of Aeromonas hydrophila infection on branchial energy metabolism, weight loss and mortality in grass carp (Ctenopharyngodon idella). We found that the infected fish all died, while 62.5% of those supplemented with 100 mg/kg thymol survived. Cytosolic and mitochondrial creatine kinase (CK) activities, as well as adenylate kinase (AK) and pyruvate kinase (PK) activities were significant lower in gills of A. hydrophila-infected fish than those of the control group, and adenosine triphosphate (ATP) levels were significant lower in the infected group. Finally, branchial reactive oxygen species (ROS) were significant higher in A. hydrophila-infected fish than in the control group. Supplementation with 100 and 300 mg thymol/kg diet prevented inhibition of branchial cytosolic and mitochondrial CK activities caused by infection, and also inhibited the reduction of branchial ATP levels. Supplementation with 100, 200 and 300 mg thymol/kg prevented the inhibition of branchial AK and PK activities induced by aeromonosis. Supplementation of 100 mg thymol/kg prevented weight loss after A. hydrophila infection. These data suggest that supplementation with 100 mg thymol/kg exerts potent bactericidal properties and augments longevity. Supplementation at all concentrations of thymol prevented A. hydrophila-induced branchial bioenergetics; nevertheless, higher concentrations were associated with side-effects.

Resistência aos antimicrobianos de bactérias obtidas de carpas (Cyprinus carpio) cultivadas em sistema semi-intensivo / Antimicrobial drugs resistance of bacteria from carp (Cyprinus carpio) raised in a semi- intensive system

Objetivando determinar o perfil de sensibilidade às drogas antimicrobianas de bactérias presentes em carpas, foi utilizado o teste de sensibilidade de difusão em disco Kirb Bauer modificado. Foram avaliados 60 isolados bacterianos, analisados e encontrados os seguintes gêneros: Staphylococcus spp. (27), Streptococcus spp. (03), Aeromonas spp. (15), Proteus spp. (04), Acinetobacter spp. (05), Pseudomonas spp. (01) e Enterobacteriaceae (05). O perfil de sensibilidade observado foi de: amicacina (35%), amoxicilina (65%), apramicina (32%), ceftiofur (37%), doxicilina (32%), enrofloxacina (15%), josamicina (75%), lincomicina (37%), nitrofurantoina (60%), ácido nalidíxico (32%), novobiocina (82%), penicilina (70%) e sulfozotrim (40%). O índice de resistência múltipla às drogas antimicrobianas médio variou de 0,33 para Streptococcus spp até 0,71 para bactérias da família Enterobacteriaceae. Dessa forma, o maior perfil de resistência às drogas antimicrobianas testadas foi observado para novobiocina, enquanto que o menor foi observado para a enrofloxacina. Os isolados bacterianos obtidos de carpas apresentaram resistência múltipla às drogas testadas, sendo três isolados resistentes a todos os antimicrobianos testados.(AU)

The present work has the purpose to determine the susceptibility pattern to antimicrobial drugs of bacteria isolates from carps, using Kirb Bauer modified disk diffusion test. We analyzed 60 bacterial isolates from these groups: Staphylococcus spp. (27), Streptococcus spp. (03), Aeromonas spp. (15), Proteus spp. (04), Acinetobacter spp. (05), Pseudomonas spp. (01) and Enterobacteriaceae (05). The percentual of susceptibility was: amikacine (35%), amoxyciline (65%), apramycin (32%), ceftiofur (37%), doxycyline (32%), enrofloxacin (15%), josamycin (75%), lyncomicin (37%), nitrofurantoin (60%), nalidixic acid (32%), novobiocin (82%), penicillin (70%) and trimethoprim: sulfamethoxazole (40%). The multiple resistance ratios to antimicrobial drugs were from 0.33 to Streptococcus spp. to 0.71 Enterobacteriaceae bacteria. Thus, the highest resistance profile of the antimicrobial drugs tested was observed for novobiocin, while the lowest was observed for enrofloxacin. Bacterial isolates from carp showed multiple resistance to the drugs tested, with three isolates resistant to all tested antimicrobials.(AU)

Resistência aos antimicrobianos de bactérias obtidas de carpas (Cyprinus carpio) cultivadas em sistema semi-intensivo / Antimicrobial drugs resistance of bacteria from carp (Cyprinus carpio) raised in a semi- intensive system

Objetivando determinar o perfil de sensibilidade às drogas antimicrobianas de bactérias presentes em carpas, foi utilizado o teste de sensibilidade de difusão em disco Kirb Bauer modificado. Foram avaliados 60 isolados bacterianos, analisados e encontrados os seguintes gêneros: Staphylococcus spp. (27), Streptococcus spp. (03), Aeromonas spp. (15), Proteus spp. (04), Acinetobacter spp. (05), Pseudomonas spp. (01) e Enterobacteriaceae (05). O perfil de sensibilidade observado foi de: amicacina (35%), amoxicilina (65%), apramicina (32%), ceftiofur (37%), doxicilina (32%), enrofloxacina (15%), josamicina (75%), lincomicina (37%), nitrofurantoina (60%), ácido nalidíxico (32%), novobiocina (82%), penicilina (70%) e sulfozotrim (40%). O índice de resistência múltipla às drogas antimicrobianas médio variou de 0,33 para Streptococcus spp até 0,71 para bactérias da família Enterobacteriaceae. Dessa forma, o maior perfil de resistência às drogas antimicrobianas testadas foi observado para novobiocina, enquanto que o menor foi observado para a enrofloxacina. Os isolados bacterianos obtidos de carpas apresentaram resistência múltipla às drogas testadas, sendo três isolados resistentes a todos os antimicrobianos testados.

The present work has the purpose to determine the susceptibility pattern to antimicrobial drugs of bacteria isolates from carps, using Kirb Bauer modified disk diffusion test. We analyzed 60 bacterial isolates from these groups: Staphylococcus spp. (27), Streptococcus spp. (03), Aeromonas spp. (15), Proteus spp. (04), Acinetobacter spp. (05), Pseudomonas spp. (01) and Enterobacteriaceae (05). The percentual of susceptibility was: amikacine (35%), amoxyciline (65%), apramycin (32%), ceftiofur (37%), doxycyline (32%), enrofloxacin (15%), josamycin (75%), lyncomicin (37%), nitrofurantoin (60%), nalidixic acid (32%), novobiocin (82%), penicillin (70%) and trimethoprim: sulfamethoxazole (40%). The multiple resistance ratios to antimicrobial drugs were from 0.33 to Streptococcus spp. to 0.71 Enterobacteriaceae bacteria. Thus, the highest resistance profile of the antimicrobial drugs tested was observed for novobiocin, while the lowest was observed for enrofloxacin. Bacterial isolates from carp showed multiple resistance to the drugs tested, with three isolates resistant to all tested antimicrobials.

A 425 T>C polymorphism in complement C7 association with resistance to Aeromonas hydrophila in grass carp.

Aeromonas hydrophila, a widespread bacterium in the aquatic environment, causes hemorrhagic septicemia in fish. In the last decade, the disease has caused mass mortalities and tremendous economic loss in cultured fish. The complement component C7 is a terminal component of complement that interacts in a sequence of polymerization reactions with other terminal complement components to form a membrane attack complex. The formation of the membrane attack complex creates a pore in the membranes of certain pathogen that can lead to their death. The objective of this study was to identify single nucleotide polymorphisms (SNPs) in the C7 gene and to assess their association with A. hydrophila resistance in grass carp. A resource population consisting of 186 susceptible and 191 resistant grass carp was constructed. We sequenced a total of 7826 bp of the C7 gene and identified 6 SNPs that were genotyped in the resource population. The SNP -1575 A>C was positioned in the promoter region of the gene. The SNP 425 C>T identified in the coding exon was a synonymous substitution in the fourth exon. Statistical analysis showed that SNP 425 C>T was associated with the incidence of hemorrhagic septicemia. The SNPs -1575 A>C, -688 T>C, and -266 A>C were highly linked together (r(2) > 0.85). No haplotypes generated with these 3 SNPs were associated with resistance to A. hydrophila in grass carp. These findings suggest that the 425 C>T polymorphism in C7 gene may be a significant molecular marker for resistance to A. hydrophila in grass carp.

Differential expression of interleukin-12 p35 and p40 subunits in response to Aeromonas hydrophila and Aquareovirus infection in grass carp, Ctenopharyngodon idella.

The grass carp (Ctenopharyngodon idella) aquaculture industry in Asia is prone to bacterial and viral hemorrhagic diseases. Effective adjuvants for vaccine formulation are the need of the hour for control of these diseases and long-term sustainability of grass carp farming. In this study, the involvement of interleukin-12 (IL-12) from grass carp (gcIL­12) in anti-bacterial and anti-viral immune responses was demonstrated via expression profiles of gcIL-12 subunits in immune tissues of the fish, following infection by Aeromonas hydrophila and Aquareovirus. Additionally, cDNA of the gcIL-12 subunits, p35 and p40 was cloned and characterized. We found that most of the structurally and functionally important features of vertebrate orthologues were conserved in gcIL-12 subunits, p35 and p40, with some features specific to grass carp. High levels of gcIL-12 p35 expression in the brain and gills suggest that IL-12 plays an important role in neural and immune systems. High expression levels in the heart, blood, and immune-related tissues suggest an important role in circulation and the immune system as well. Infections by both, A. hydrophila and Aquareovirus stimulated the mRNA expression of gcIL-12 subunits, p35 and p40 in most immune tissues. Significant upregulation or downregulation of gcIL-12 subunits, p35 and p40 by bacterial and viral infection confirms their potential role in anti-bacterial and anti-viral immune responses in fish.

Molecular characterization, expression, and immunological response analysis of the TWEAK and APRIL genes in grass carp, Ctenopharyngodon idella.

TWEAK and APRIL are important members of the TNF superfamily, which play a crucial role in several diseases. Here, we describe the identification of grass carp (Ctenopharyngodon idella) homologs of TWEAK and APRIL (designated gcTWEAK and gcAPRIL, respectively) and their response to Aeromonas hydrophila and Aquareovirus infection. The gcTWEAK cDNA sequence contains 2273 bases with an open reading frame of 753 bases encoding 250-amino acid residues. The gcTWEAK protein contains a predicted transmembrane domain, a putative furin protease cleavage site, 3 conserved cysteine residues, and a typical TNF homology domain. The gcAPRIL cDNA sequence contains 1408 bases with an open reading frame of 747 bases encoding 248-amino acid residues. The gcAPRIL protein contains a predicted transmembrane domain, a putative furin protease cleavage site, 2 conserved cysteine residues, and a typical TNF homology domain corresponding to other, known APRIL homologs. Reverse transcription-polymerase chain reaction analysis shows that both gcTWEAK and gcAPRIL transcripts are predominantly expressed in the skin, spleen, and head kidney, and they are significantly upregulated in most immune tissues by A. hydrophila and Aquareovirus infections. Our results demonstrate that liver is the most responsive tissue against bacterial infection, whereas gill is the most responsive tissue against viral infection. The association of increased gcTWEAK and gcAPRIL expression after bacterial and viral infections suggests that they play a potentially important role in the immune system of fish.

Investigations in associated protozoa-bacterial infections of cyprinids from a fish farm situated on the Jijia river in N-E of Romania / Investigações sobre infecções protozoárias-bacterianas associadas a ciprinídeos da exploração piscícola no rio Jijia, situado no NE da Romênia

In autumn 2011 in cyprinid farms located in Iasi on the Jijia river, several infections with bacterial strains and macroscopical external cysts on the skin were diagnosedwhich developed as a result of the stress induced by biotic and abiotic factors. On the examination of the cyst contents the presence of numerous spores was observed, mostly of the Dermocystidium sp genusThe samples were taken from the common carp (Cyprinus carpio) and crucian carp (Carassius auratus gibelio) species from the fish farm as well as from the Jijia River. 35 fish were examined, all of them showing cysts, fragmentation of their dorsal fin and congestion of the gills. Histological examination of the skin showed a field of multiple dermal cysts with round light eosinophilic formations (14-16µm) containing a central refractable body similar to that reported for Dermocystidium sp. Gills samples were taken from the affected areas for the SEM examination with the purpose of evaluating not only aspects of normal morphology, but also aspects of some modifications of the affected areas as well as the presence of the etiologically incriminated bacteria Pseudomonas fluorescens. The isolates were identified through phenotypic methods. All the strains that showed mobility and oxidase-positivity were tested using API 20 NE strip. Consequently, they were taxonomically grouped into the species Pseudomonas fluorescens. The scanning electron microscope (SEM) was used for the first time in the characterization of the bacterial lesions produced by Pseudomonas strains on Cyprinus carpio and Carassius auratus gibelio gills. The diagnosis of septicemia with conditional pathogen species of Pseudomonas fluorescens was correlated with the results of the physico-chemical investigations of water and the data concerning the breeding conditions of the investigated livestock.(AU)

No outono de 2011, em fazendas de ciprinídeos localizadas em Iasi, no rio Jijia, diversas infecções bacterianas e cistos externos macroscópicos na pele se desenvolveram como resultado do estresse induzido por fatores bióticos e abióticos. No exame do conteúdo dos cistos, a presença de diversos esporos foi observada, a maioria do gênero Dermocystidium sp. As amostras foram colhidas das seguintes espécies: carpa comum (Cyprinus carpio) e carpa cruciana (Carassius auratus gibelio) de fazenda piscícola, além do rio Jijia. Assim sendo, 35 peixes foram examinados, todos demonstrando cistos, fragmentação da barbatana dorsal e congestão das guelras. O exame histológico da pele mostrou um campo de múltiplos cistos dérmicos com formações circulares claras eosinofílicas (14-16µm) contendo corpo central refratado similar ao relatado para Dermocystidium sp. Amostras de guelras foram retiradas das áreas afetadas para exame MEV, com o propósito de se avaliar não apenas os aspectos da morfologia normal, mas também os aspectos de algumas modificações das áreas afetadas, além da presença da bactéria etiologicamente incriminada: Pseudomonas fluorescens. Os isolados foram identificados por meio de métodos fenotípicos. Todas as amostras que mostraram mobilidade e positividade-oxidase foram testadas usando-se fita API 20 NE. Consequentemente, estas foram taxonomicamente agrupadas na espécie Pseudomonas fluorescens. O microscópio eletrônico de varredura (MEV) foi usado pela primeira vez na caracterização de lesões bacterianas produzidas por Pseudomonas nas guelras de Cyprinus carpio e Carassius auratus gibelio. O diagnóstico de septicemia com espécies condicionais de patogênico de Pseudomonas fluorescens foi correlacionado com os resultados das investigações físico-químicas da água e de dados sobre as condições de reprodução dos animais investigados.(AU)

Investigations in associated protozoa-bacterial infections of cyprinids from a fish farm situated on the Jijia river in N-E of Romania / Investigações sobre infecções protozoárias-bacterianas associadas a ciprinídeos da exploração piscícola no rio Jijia, situado no NE da Romênia

In autumn 2011 in cyprinid farms located in Iasi on the Jijia river, several infections with bacterial strains and macroscopical external cysts on the skin were diagnosedwhich developed as a result of the stress induced by biotic and abiotic factors. On the examination of the cyst contents the presence of numerous spores was observed, mostly of the Dermocystidium sp genusThe samples were taken from the common carp (Cyprinus carpio) and crucian carp (Carassius auratus gibelio) species from the fish farm as well as from the Jijia River. 35 fish were examined, all of them showing cysts, fragmentation of their dorsal fin and congestion of the gills. Histological examination of the skin showed a field of multiple dermal cysts with round light eosinophilic formations (14-16µm) containing a central refractable body similar to that reported for Dermocystidium sp. Gills samples were taken from the affected areas for the SEM examination with the purpose of evaluating not only aspects of normal morphology, but also aspects of some modifications of the affected areas as well as the presence of the etiologically incriminated bacteria Pseudomonas fluorescens. The isolates were identified through phenotypic methods. All the strains that showed mobility and oxidase-positivity were tested using API 20 NE strip. Consequently, they were taxonomically grouped into the species Pseudomonas fluorescens. The scanning electron microscope (SEM) was used for the first time in the characterization of the bacterial lesions produced by Pseudomonas strains on Cyprinus carpio and Carassius auratus gibelio gills. The diagnosis of septicemia with conditional pathogen species of Pseudomonas fluorescens was correlated with the results of the physico-chemical investigations of water and the data concerning the breeding conditions of the investigated livestock...

No outono de 2011, em fazendas de ciprinídeos localizadas em Iasi, no rio Jijia, diversas infecções bacterianas e cistos externos macroscópicos na pele se desenvolveram como resultado do estresse induzido por fatores bióticos e abióticos. No exame do conteúdo dos cistos, a presença de diversos esporos foi observada, a maioria do gênero Dermocystidium sp. As amostras foram colhidas das seguintes espécies: carpa comum (Cyprinus carpio) e carpa cruciana (Carassius auratus gibelio) de fazenda piscícola, além do rio Jijia. Assim sendo, 35 peixes foram examinados, todos demonstrando cistos, fragmentação da barbatana dorsal e congestão das guelras. O exame histológico da pele mostrou um campo de múltiplos cistos dérmicos com formações circulares claras eosinofílicas (14-16µm) contendo corpo central refratado similar ao relatado para Dermocystidium sp. Amostras de guelras foram retiradas das áreas afetadas para exame MEV, com o propósito de se avaliar não apenas os aspectos da morfologia normal, mas também os aspectos de algumas modificações das áreas afetadas, além da presença da bactéria etiologicamente incriminada: Pseudomonas fluorescens. Os isolados foram identificados por meio de métodos fenotípicos. Todas as amostras que mostraram mobilidade e positividade-oxidase foram testadas usando-se fita API 20 NE. Consequentemente, estas foram taxonomicamente agrupadas na espécie Pseudomonas fluorescens. O microscópio eletrônico de varredura (MEV) foi usado pela primeira vez na caracterização de lesões bacterianas produzidas por Pseudomonas nas guelras de Cyprinus carpio e Carassius auratus gibelio. O diagnóstico de septicemia com espécies condicionais de patogênico de Pseudomonas fluorescens foi correlacionado com os resultados das investigações físico-químicas da água e de dados sobre as condições de reprodução dos animais investigados...

Identification of antibiotic resistance cassettes in class 1 integrons in Aeromonas spp. strains isolated from fresh fish (Cyprinus carpio L.).

Forty-six Aeromonas spp. strains were isolated from fresh fish and investigated for their antimicrobial susceptibility, detection of Class 1 integrons by PCR, and arrangement of gene cassettes. Selected isolates were further characterized by enterobacterial repetitive intergenic consensus-PCR. Twenty isolates were found to carry Class 1 integrons. Amplification of the variable regions of the integrons revealed diverse bands ranging in size from 150 to 1,958 pb. Sequence analysis of the variable regions revealed the presence of several gene cassettes, such as adenylyl transferases (aadA2 and aadA5), dihydrofolate reductases (dfrA17 and dfrA1), chloramphenicol acetyl transferase (catB3), ß-lactamase (oxa2), lincosamide nucleotidil transferase (linF), aminoglycoside-modifying enzyme (apha15), and oxacillinase (bla OXA-10). Two open reading frames with an unknown function were identified as orfC and orfD. The aadA2 cassette was the most common integron found in this study. Interestingly, five integrons were detected in the plasmids that might be involved in the transfer of resistance genes to other bacteria. This is a first report of cassette encoding for lincosamides (linF) resistance in Aeromonas spp. Implications on the incidence of integrons in isolates of Aeromonas spp. from fresh fish for human consumption, and its possible consequences to human health are discussed.

Biologic responses of bacteria communities living at the mucus secretion of common carp (Cyprinus carpio) after exposure to the carbon nanomaterial fullerene (C60).

Bacteria communities living in mucus secretions of common carp Cyprinus carpio (Cyprinidae) were exposed to the organic nanomaterial fullerene (C(60)) to evaluate its potential bactericidal effects. End points analyzed were viability, growth, reactive oxygen species (ROS) concentration, and total antioxidant competence against peroxyl radicals. Viability was not affected (p > 0.05), whereas growth was arrested (p < 0.05) after 3 hours of exposure to the three concentration of C(60) assayed (0.1, 1, and 10 mg/L). Levels of RO measured at different C(60) concentration showed that some colonies were reactive (significant dose-response relation, p < 0.05) to C(60), whereas others were not. The nonreactive colonies to C(60) presented higher antioxidant competence to peroxyl radicals compared with the reactive colonies (p < 0.05). The strains isolated and identified by polymerase chain reaction (PCR) products of 16S rRNA showed a predominance of Aeromonas genus between all the isolated Gram-negative bacteria. Thus, the present results indicate that C(60) affects bacterial communities that live in mucus secretions of common carp.

In vivo transfer of plasmid pRAS1 between Aeromonas salmonicida and Aeromonas hydrophila in artificially infected Cyprinus carpio L.

This study investigated the possible in vivo transfer of plasmid pRAS1 between Aeromonas salmonicida and A. hydrophila inhabiting two different organs of Cyprinus carpio L. To distinguish transconjugants from naturally occurring antibiotic resistant bacteria, twelve luminescent transposon-tagged A. hydrophila strains using mini Tn5luxCDABEKm2 transposon were generated. In conjugal transfer experiments, fish were conditioned with the donor bacteria and subsequently immersed in water containing the recipient strain. Bacteria were recovered from gills and intestines and isolated by growth on selective plates. Transconjugants were identified by their resistance to the pRAS1 encoded antimicrobials and by light emission. In vivo transfer frequencies ranged between 10(-3) and 10(-6) and were somewhat lower in intestines, compared to gills. Transfer frequencies were also smaller relative to those obtained in vitro. The minimal amount of donor and recipient bacteria needed to yield detectable transconjugants in vivo was 1 x 10(4) CFU mL(-1). Implications of this plasmid transfer in natural settings and its possible consequences to human health are discussed.