Between 2007 and 2012, a variety of disease outbreaks most often characterized by skin disorders were observed among different species of freshwater fish in Poland. In most cases, the clinical signs included focally necrotized gills, necrotic skin lesions or ulcers. Internally, haemorrhages, oedematous kidney and abnormal spleen enlargement were generally noted. The disorders were accompanied by increased mortality. Most of the problems concerned cultured common carp Cyprinus carpio L. and rainbow trout Oncorhynchus mykiss (Walbaum). Fish have been examined from a number of these farms, and additionally, the wild and ornamental fish with similar clinical signs of diseases were also tested. Bacteria were isolated consistently from lesions and internal organs. They had characteristic orange-pigmented colonies which grew in pure culture or constituted 55-95% of total bacterial flora. One hundred and eighteen isolates were collected and biochemically identified as Shewanella putrefaciens group, and this was confirmed by sequencing. Challenge tests confirmed the pathogenicity of these bacteria. This is the first report characterizing and describing S. putrefaciens as a pathogen of different species of freshwater fish in Europe.

The aim of this study was to characterise Acinetobacter sp. isolated from fish. Eight isolates obtained from diseased rainbow trout and common carp cultured in Poland were analysed. The isolates were identified using API 20 NE system as Acinetobacter sp. Afterwards, they were identified by sequencing 16S rDNA gene fragment. The bacteria were identified as A. johnsonii (two isolates), A. lwoffii (two isolates), A. junii/johnsonii (one isolate), A. calcoaceticus (one isolate), and Acinetobacter sp. (two isolates). The drug resistance of isolates was examined. The majority of the isolates were resistant to ampicilin, amoxicillin, and cephalothin and all demonstrated sensitivity to fluoroquinolones, except of one isolate. Two isolates were selected for the experimental infection of trout and carp to confirm their pathogenicity. Experimentally infected fish showed disease symptoms similar to those observed in fish naturally infected with these bacteria. This is the first report concerning pathogenicity of A. johnsonii for rainbow trout and A. lwoffii for common carp. These bacteria were regarded as emerging opportunistic pathogens of fish farmed in Poland. Acinetobacter strains are commonly known as microorganisms transmitting the antibiotic resistance genes. Therefore, they might have a great impact on the resistance transfer in aquaculture.

Aeromonas isolates were collected from cultured fish, characterized phenotypically and identified to species using 16S rDNA. The pathogenicity of all isolates was assayed on the basis of haemolytic and proteolytic activity and challenge tests were performed for isolates from healthy fish. A total of 131 Aeromonas isolates were obtained and identified as follows: A. hydrophila (13), A. bestiarum (23), A. salmonicida (motile biogroup) (19), A. caviae (2), A. sobria (18), A. veronii bt. sobria (42), A. jandaei (1), A. encheleia (11) and A. allosaccharophila (2). All isolates of A. hydrophila and A. bestiarum and most isolates of A. salmonicida and A. veronii were classified as pathogenic. Aeromonas hydrophila was isolated only from diseased trout except for one isolate obtained from carp fry. The other potentially pathogenic Aeromonas species were present in diseased as well as healthy fish. The pathogenicity of isolates from healthy fish was correlated with their enzymatic activity and was also tested by challenge experiments. The dominant pathogenic species were A. veronii bt. sobria, A. bestiarum and A. salmonicida in common carp and A. hydrophila in rainbow trout.

Aims: To evaluate the relationship between the genomospecies, phenotypic profile and pathogenicity for carp of 37 motile Aeromonas strains. Methods and Results: Aeromonas strains were identified to genomospecies level by the 16S rDNA restriction fragment length polymorphism (RFLP) method and characterized phenotypically by the API 20E and API Zym systems and by conventional tube or plate methods. 16S rDNA RFLP analysis showed that the strains belonged to five species, Aeromonas bestiarum (5), Aerom. salmonicida (13), Aerom. veronii (11), Aerom. sobria (6) and Aerom. encheleia (2). Most strains of Aerom. bestiarum (80%) and Aerom. salmonicida (85%) could be separated by growth at 4 and 42°C, autoagglutination after boiling, reaction for lipase (C14) and naphthol-AS-BIphosphohydrolase. All strains of Aerom. veronii corresponded to Aerom. veronii biotype sobria and could be separated from Aerom. sobria by citrate utilization, growth at 37 and 42°C, amygdalin and cellobiose fermentation. All strains of Aerom. bestiarum and most strains of Aerom. salmonicida (76AE9%) and Aerom. veronii (63AE6%) were pathogenic for carp. Conclusions: The biochemical identification of carp Aeromonas strains is not entirely clear. Some association between Aeromonas species, phenotypic profile and specific disease signs was observed. Significance and Impact of the Study: The results will be useful for ichthyopathology laboratories in the diagnosis of motile aeromonad septicaemia in carp.

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An attempt was made to delineate the relationship between ofAeromonasspecies and/or serogroups and specific disease symptoms in common carpCyprinus carpioL. and rainbow troutOncorhynchus mykissWalbaum. The adhesion ofAeromonasstrains to various tissues in relation to disease spectrum was also tested. All strains ofA. hydrophilacaused skin ulcers as well as septicaemia in both carp and trout while the other strains were able to cause only skin ulcers or some specific internal lesions with or without septicaemia depending on which species and/or serogroup they represented. Disease symptoms depended also on fish species. It was found that adhesion intensity ofAeromonasstrains tested was significantly higher to tissues, which were susceptible to infection with these strains. The results indicate that adhesion to various cells of the fish organism is principal marker to detect virulentAeromonasstrains. The findings presented in this study may be helpful in the appraisal of aeromonads disease risk and kind of the infection in particular fish farms by epizootiological studies or/and during routine fish examinations. They will also be useful to improve and facilitate diagnosis of bacterial fish disease.

Chemical analyses and mass spectrometry were used to study the structure of the lipopolysaccharide (LPS) isolated from Aeromonas bestiarum strain K296, serotype O18. ESI-MS revealed that the most abundant A. bestiarum LPS glycoforms have a hexa-acylated or tetra-acylated lipid A with conserved architecture of the backbone, consisting of a 1,4′-bisphosphorylated β-(1→6)-linked d-GlcN disaccharide with an AraN residue as a non-stoichiometric substituent and a core oligosaccharide composed of Kdo1Hep6Hex1HexN1P1. 1D and 2D NMR spectroscopy revealed that the O-specific polysaccharide (OPS) of A. bestiarum K296 consists of a branched tetrasaccharide repeating unit containing two 6-deoxy-l-talose (6dTalp), one Manp and one GalpNAc residues; thus, it is similar to that of the OPS of A. hydrophila AH-3 (serotype O34) in both the sugar composition and the glycosylation pattern. Moreover, 3-substituted 6dTalp was 2-O-acetylated and additional O-acetyl groups were identified at O-2 and O-4 (or O-3) positions of the terminal 6dTalp. Western blots with polyclonal rabbit sera showed that serotypes O18 and O34 share some epitopes in the LPS. The very weak reaction of the anti-O34 serum with the O-deacylated LPS of A. bestiarum K296 might have been due to the different O-acetylation pattern of the terminal 6dTalp. The latter suggestion was further confirmed by NMR.

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