Serum resistance as a good indicator for virulence in Aeromonas hydrophila strains isolated from diseased fish in South-East Asia

Abstract. Aeromonas hydrophila strains isolated from diseased fish in South-East Asia were studied for their virulence in naive tilapia, Oreochrommis aureus (Steindachner), and blue gourami, Trichogaster trichopterus (Pallas). Five of the most virulent strains used in this study shared a common resistance to the killing effect of naive fish serum. Other factors such as lack of autoagglutination in 0.2% acriflavine, instability after boiling, production of an S-layer, and proteases and haemolysins did not correlate well with virulence. In addition, serotyping could not group all the virulent strains. Therefore, serum resistance is considered as a good indicator for screening virulence of A. hydrophila strains isolated from diseased fish in South-East Asia.     

Insights into the virulence‐related genes of Edwardsiella tarda isolated from turbot in Europe: genetic homogeneity and evidence for vibrioferrin production

Edwardsiella tarda has long been known as a pathogen that causes severe economic losses in aquaculture industry. Insights gained on E. tarda pathogenesis may prove useful in the development of new methods for the treatment of infections as well as preventive measures against future outbreaks. In this report, we have established the correlation between the presence of virulence genes, related with three aspects typically involved in bacterial pathogenesis (chondroitinase activity, quorum sensing and siderophore‐mediated ferric uptake systems), in the genome of E. tarda strains isolated from turbot in Europe and their phenotypic traits. A total of 8 genes were tested by PCR for their presence in 73 E. tarda isolates. High homogeneity was observed in the presence/absence pattern of all the strains. Positive results in the amplification of virulence‐related genes were correlated with the detection of chondroitinase activity in agar plates, in vivo AHL production during fish infection and determination of type of siderophore produced by E. tarda. To the best of our knowledge, this is the first study carried out with European strains on potential virulence factors. Furthermore, we demonstrated for the first time that E. tarda produces the siderophore vibrioferrin.     

Isolation and identification of fish pathogen Edwardsiella tarda from mariculture in China

The causative agent was isolated from diseased turbots (Scophthalmus maximus) stricken by a high‐mortality outbreak of bacterial septicaemia occurring in a mariculture farm in Yantai, a northern coastal city of China. Seven pure isolates, namely EH‐15, EH‐103, EH‐107, EH‐202, EH‐203, EH‐305 and EH‐306, belonged to Edwardsiella tarda. The phenotypic features of the cultures were analysed extensively. Three of the isolates showed high 16S rDNA sequence similarities with E. tarda sequence (GenBank accession no. EF467289). However, unlike the E. tarda ATCC 15947, all the isolates, except EH‐15, contained a novel large plasmid sized about 23.7 kb. Furthermore, pathogenicity of the isolates was addressed by experimental challenges with fish models. The isolates exhibited strong virulence to swordtail fish with LD₅₀ ranging between 3.8 × 10³ and 3.8 × 10⁵ CFU g^?1, and EH‐202 displaying the lowest LD₅₀ value among them. Antibiotic susceptibilities of E. tarda isolates were assayed. Compared with E. tarda ATCC 15947, the isolates displayed strong resistance to chloramphenicol, and the probable dominant chloramphenicol resistance determinant was cat III. Depicting the main biological properties of turbot‐borne E. tarda strains in China, the study provided useful information for further unveiling their pathogenic mechanisms.     

Effects of dietary chitosan oligosaccharide complex with rare earth on growth performance and innate immune response of turbot,Scophthalmus maximus L.

An 8‐week‐feeding trial was conducted to investigate the effect of dietary chitosan oligosaccharide complex with rare earth (COS‐REE) on growth performance and innate immune response of turbot, Scophthalmus maximus L. (Initial average weight was (12.1 ± 0.1) g) as well as disease resistance against Edwardsiella tarda. Six practical diets (approximately 53.01% protein and 12.57% lipid) were formulated to contain graded levels (0, 75, 150, 300, 600 and 1200 mg kg^?1) of COS‐REE. Results of the present study showed that, compared to the control group (0 mg kg^?1), the specific growth rate (SGR) was significantly higher in fish fed the diet with 300 mg kg^?1 COS‐REE (P < 0.05), while the feed conversion ratio (FCR) significantly decreased (P < 0.05). The phagocytic index (PI) and the activity of super oxide dismutase (SOD) of serum in fish fed the diet with 300 mg kg^?1 COS‐REE was significantly higher than fish fed the control diet (P < 0.05), but no significant differences were observed in malondialdehyde (MDA) and hepatic metallothionein (MT) concentrations. After 8 weeks, fish were challenged by intraperitoneal injection with E. tarda, and COS‐REE‐treated fish demonstrated increased protection capability. These results suggested that COS‐REE could enhance growth, innate immunity and disease resistance in turbot, and the optimum dose was approximately 300 mg kg^?1.     

Effect of temperature and salinity on virulence of Edwardsiella tarda to Japanese flounder, Paralichthys olivaceus (Temminck et Schlegel)

Experiments were designed to determine the effects of temperature and salinity on the virulence of Edwardsiella tarda to Japanese flounder, Paralichthys olivaceus. In the temperature experiment, a two‐factor design was conducted to evaluate the effects of both pathogen incubation temperature and fish cultivation temperature on pathogen virulence. E. tarda was incubated at 15, 20, 25 and 30±1°C, and the fish (mean weight: 10 g) were reared at 15, 20 and 25±1°C respectively. The fish reared at different temperatures were infected with the E. tarda incubated at different temperatures. The results of a 4‐day LD₅₀ test showed that temperature significantly affected the virulence of E. tarda (P<0.01) and the interaction between the two factors was also significant (P<0.01). For fish reared at 15°C the virulence of E. tarda was the highest at 25°C of pathogen incubation, followed by 20, 15 and 30°C. When the fish rearing temperature was raised to 20 and 25°C, the virulence of E. tarda incubated at all temperatures increased. Isolation testing demonstrated results similar to those of LD₅₀. The higher rearing temperature increased the proliferation rate of the pathogen in fish. In the salinity experiment, the incubation salinity of E. tarda was at 0, 10, 20 and 30 g L^?1, respectively, and the fish with mean weight of 50 g were cultured in natural seawater of 30 g L^?1. The results of one‐way anova in 4‐day LD₅₀ test showed that incubation salinity significantly affected virulence. Virulence was lower when the salinity of the incubation medium was at 0 and 30 g L^?1, higher at 10 and 20 g L^?1. The results of isolation test were in accordance with those of LD₅₀. At 20 g L^?1E. tarda had a faster proliferation rate than that at 10 g L^?1.     

In vitro markers for virulence in Yersinia ruckeri

In this study, different traits that have been associated with bacterial virulence were studied in Yersinia ruckeri. Two isolates that had been shown to cause disease and mortality in experimentally infected rainbow trout were compared with five avirulent isolates. Both virulent isolates showed high adhesion to gill and intestinal mucus of rainbow trout, whereas the majority of non‐virulent strains demonstrated significantly lower adhesion. A decrease in adherence capability following bacterial treatment with sodium metaperiodate and proteolytic enzymes suggested the involvement of carbohydrates and proteins. All strains were able to adhere to and invade chinook salmon embryo cell line (CHSE‐214), fathead minnow epithelial cell line (FHM) and rainbow trout liver cell line (R1). One non‐virulent strain was highly adhesive and invasive in the three cell lines, whereas the virulent strains showed moderate adhesive and invasive capacity. The internalization of several isolates was inhibited by colchicine and cytochalasin‐D, suggesting that microtubules and microfilaments play a role. For all strains, intracellular survival assays showed a decrease of viable bacteria in the cells 6 h after inoculation, suggesting that Y. ruckeri is not able to multiply or survive inside cultured cells. Analysis of the susceptibility to the bactericidal effect of rainbow trout serum demonstrated that virulent Y. ruckeri strains were serum resistant, whereas non‐virulent strains were generally serum sensitive.     

Development and characterization of rifampicin-resistant mutants from high virulent strains of Flavobacterium columnare

Flavobacterium columnare is divided into three genetic groups or genomovars, genomovar II being highly virulent for channel catfish. A modified live vaccine is currently available to prevent columnaris disease under the licensed name Aquavac‐Col^®. The strain of F. columnare used to generate the avirulent rifampicin‐resistant mutant used in Aquavac‐Col^® belonged to genomovar I, the less virulent group towards channel catfish. In this study, we describe the generation and characterization of rifampicin‐resistant mutants from genomovar II strains. A total of 13 new mutants were obtained, and eight of them (two from each parent strain) were genetically and phenotypically characterized. Highly conserved regions within the ribosomal operons were identical between parent and mutant strains. Genetic differences between mutants and their parent strains were revealed by amplified fragment length polymorphism (AFLP). Genetic changes were distinctive among different mutants. Analysis of the lipopolysaccharide (LPS) showed that while some mutants lacked a few molecular bands of the LPS, some exhibited the same LPS profiles as their parent strains. Comparison between immunogenic proteins from mutants and parents was carried out by immunoblot analysis and further confirmed the uniqueness of individual mutants. A complete set of rifampicin‐resistant mutants with different genetic and immunogenic properties from the highly virulent genomovar II has been created. These mutants may have the potential of becoming vaccine candidates against columnaris disease.     

Dietary levamisole modulates the immune response and disease resistance of Asian catfish Clarias batrachus (Linnaeus)

In order to determine the immunomodulatory effect of dietary levamisole in Asian catfish (Clarias batrachus), fish were fed four different diets for 10 days: a formulated diet as control and the same diet supplemented with 50, 150 or 450 mg levamisole kg^?1 feed. The serum bacterial agglutination titre against Aeromonas hydrophila as a measure of specific immunity, serum haemagglutination titre, natural haemolytic complement activity (ACH₅₀), myeloperoxidase and lysozyme activities, total protein level and oxidative radical production by neutrophils as a measure of non‐specific immunity as well as disease resistance against A. hydrophila challenge to separate vaccinated and non‐vaccinated groups were evaluated at 0, 1, 2 and 3 weeks after last administration of levamisole. Levamisole supplement at the lowest level (50 mg kg^?1) significantly enhanced oxidative radical production and serum myeloperoxidase (MPO) content immediately after 10 days of feeding, which reached peak values after 3 and 2 weeks of feeding respectively. Haemolytic complement and haemagglutination titre were significantly enhanced after 3 and 1 weeks respectively. Haemolytic complement activity and MPO activities were significantly raised to 150 mg kg^?1 after 3 and 2 weeks, respectively. At the highest level of levamisole feeding (450 mg kg^?1) significant decreases in superoxide production and complement activity were measured immediately after levamisole feeding, which returned to the normal level after 1 week post‐ feeding. Fish were challenged with a virulent strain of A. hydrophila at 0, 1, 2 and 3 weeks after levamisole feeding, and the cumulative per cent survival was recorded over 10 days. Feeding levamisole at 50, 150 or 450 mg kg^?1 increased per cent survival in vaccinated fish immediately after levamisole feeding, and survival was significantly higher at 450 mg kg^?1. There was no difference in mortality patterns in non‐vaccinated fish. The results support the use of levamisole at 50 mg kg^?1 feed for 10 days as an immunostimulant in Asian catfish farming.     

Influence of high dietary α-tocopherol intakes on specific immune response, nonspecific resistance factors and disease resistance of healthy and aflatoxin B1-induced immunocompromised Indian major carp, Labeo rohita (Hamilton)

The effect of aflatoxin treatment and/or feeding of a high level of α‐tocopherol on immune response and disease resistance was investigated in Indian major carp, Labeo rohita. Group A served as a healthy control, group B was treated with aflatoxin, group C was fed a high level of α‐tocopherol whereas group D was exposed both to aflatoxin and a high level of dietary α‐tocopherol for 60 days. Aflatoxin B₁ (AFB₁) was injected once intraperitoneally into fish on the first day of the experiment (groups B & D). High levels of DL‐α‐tocopherol (1000 mg kg^–1 feed) were provided to healthy as well as AFB₁‐treated immunocompromised fish for 60 days (groups C & D). At the end of the experiment blood samples were assayed for changes in nonspecific immunity and humoral protein levels. Disease resistance against two common bacterial pathogens viz., Aeromonas hydrophila and Edwardsiella tarda were evaluated in all groups. Significant (P < 0.05) suppression of specific immunity as measured through haemagglutination (HA) titre against sheep red blood cells (SRBCs) as well as bacterial (formalin‐killed E. tarda) agglutination titre; nonspecific resistance factors viz., globulin level, serum bactericidal and lysozyme activities, neutrophil activities, and disease resistance against two bacterial pathogens only in aflatoxin‐treated fish with respect to the control group, clearly indicated the immunosuppressive nature of aflatoxin. Feeding of a high level of α‐tocopherol to AFB₁‐treated immunocompromised fish significantly (P < 0.05) raised specific immunity, nonspecific resistance factors and disease resistance capacity when compared with aflatoxin‐exposed fish. Disease resistance and enhancement of immune status through feeding of high levels of α‐tocopherol to healthy as well as AFB₁‐treated immunocompromised fish confirmed the potential of α‐tocopherol in carp feed for prevention of disease and for combating natural/environmental immunosuppressants.     

Microbiological study results (bacteriology and electron microscopy) of diarrhea in dog whelps

Bacteriological examinations of 159 faeces and intestinal contents of dogs with diarrhoea revealed E. coli in 157 specimens. 73 of these samples contained non haemolytic strains, 18 haemolytic isolates, and 66 haemolytic, as well as non haemolytic strains. Klebsiella sp. and Staphylococcus aureus were found in 9 cases, and Salmonella sp. (group B) was isolated once only. By electron microscopy parvovirus could be detected in 19 samples. Ten were positive for coronavirus and one for rotavirus. Morphologically not finally identified coronavirus like and picornavirus like particles were found in 3 cases, respectively. A significant relationship between the occurrence of virus infections and the isolation of certain bacteria species was not found. In 45 E. coli strains virulence factors, such as the heat labile enterotoxin (LT) and the verotoxin (VT), could not be detected, but 31 of these isolates showed different haemagglutination patterns which were still present by 14 of them in the presence of mannose (mannose resistant haemagglutination, MR-HA). These MR-HA inducing E. coli isolates were present more often in parvovirus positive samples (in 6 of 10) than they could be detected in parvovirus negative ones (in 8 of 35), implicating a potential pathogenic role of these E. coli strains for the parvovirus enteritis of dogs.     

Adherence, haemagglutination and cell surface characteristics of motile aeromonads virulent for fish

Abstract. Motile Aeromonas spp. virulent for fish were studied with regard to their adhesion profile. Electron microscopy demonstrated the presence of fimbriae (pili) on Aeromonas cells regardless of virulence potential. The results show no significant correlations between ability to haemagglutinate, yeast cell co-agglutination and virulence. All strains expressed mannose-sensitive haemagglutinin activity against guinea pig erythrocytes. Using four types of red blood cells no characteristic haemagglutinin pattern related to virulence could be discerned. Expression of surface haemagglutin(s) on Aeromonas hydrophila appears to be medium dependent; strains grown in liquid media demonstrated enhanced haemagglutination activity. Both virulent and avirulent strains had in vitro epithelial cell adhesive capabilities. Cell surface characteristics measured by agglutination in acriflavine and stability after boiling indicated that most virulent strains agglutinated in the presence of acriflavine, but not all sedimented after boiling. The ability of 10 selected strains of A hydrophila to grow in normal pooled catfish serum was determined. Only 17% of the virulence variations can be explained by their sensitivity to serum.     

Edwardsiellosis in farmed turbot,Scophthalmus maximus (L.), associated with an unusual variant of Edwardsiella tarda: a clinical,aetiological and histopathological study

During 2005 and 2010, a survey of edwardsiellosis on eight turbot, Scophthalmus maximus (L.), farms was conducted in China. This report presents the detailed results of the study on this disease. Diseased turbot displayed two distinct types of gross signs: black discoloration of the dorsal skin on the posterior portion of the body; and red cutaneous foci on the ventral side. Internally, the most pronounced clinical signs in all fish examined were enlarged kidneys. The causal agent of the disease was finally proved to be one species of bacterium that was identified as Edwardsiella tarda by physiological and biochemical tests, API 32E and 16S ribosomal RNA sequence analysis. It is noteworthy that unlike the commonly described E. tarda strains, the isolates in this study were non‐motile strains without flagella. A histopathological study revealed that E. tarda infection was systemic in turbot and that kidney showed the most significant pathological changes, including acute focal necrosis, an influx of macrophages and formation of granuloma. The most common histopathological characteristics of this disease are the proliferation of macrophage in various organs and formation of granuloma. In addition, this article also gave background information on the disease and presented the results of virulence tests with the E. tarda strain identified in this study.     

Intra- and interspecific phenotypic characteristics of fish-pathogenic Edwardsiella ictaluri and E. tarda

Intra‐ and interspecific characteristics of fish‐pathogenic Edwardsiella ictaluri, and E. tarda were determined by numerical analysis of gel electrophoresed protein profiles, fatty acid methyl esters (FAMEs) and immunoblotting. The 18 E. ictaluri isolates revealed a high degree of homogeneity (70% similarity or higher) in their protein profiles and 95% similarity in their FAME, while the nine E. tarda isolates revealed 30% similarity in their protein profiles and 95% similarity in their FAME. Immunoblots probed for antigenic epitopes with goat antiserum produced against E. ictaluri and E. tarda, respectively, revealed that E. ictaluri were more homogeneous compared with the E. tarda isolates. Overall, there was a considerable degree of relatedness between the two species. Our findings suggest that phenotypically E. ictaluri represents a clonal bacterial population structure compared with the less monomorphic E. tarda.     

Mortality and pathology of hybrid catfish,Clarias macrocephalus (Günther) × Clarias gariepinus (Burchell), associated with Edwardsiella ictaluri infection in southern Thailand

Enteric septicaemia of catfish (ESC) caused by Edwardsiella ictaluri is becoming an increasing problem in aquaculture and has been reported worldwide in a variety of fish species. This study reports ESC in hybrid catfish, Clarias macrocephalus (Günther) × Clarias gariepinus (Burchell), cultured in southern Thailand. The bacteria were identified as E. ictaluri by conventional and rapid identification systems, as well as by genetic and phylogenetic characterization. Analysis of 16S rRNA indicated 100% homology to the 16S rRNA sequence of several E. ictaluri strains in GenBank. Plasmid profiles demonstrated 4.0‐ and 5.6‐kb plasmids, compared with the 4.8‐ and 5.6‐kb plasmids in the US isolates, and representative genes of three of the four known pathogenicity islands of US isolates were present. Serologically, lipopolysaccharide (LPS) purified from the Thai isolates was not recognized by a monoclonal antibody against the LPS of US isolates. Fish experimentally infected with E. ictaluri showed 23–100% mortality within 14 days with a 168‐h LD₅₀ of 6.92 × 10⁷ CFU mL^?1 by immersion and a 96‐h LD₅₀ of 1.58 × 10⁶ CFU fish^?1 by intraperitoneal injection. Examination of tissue sections obtained from both naturally and experimentally infected fish indicated that infection of hybrid catfish with E. ictaluri produced lesions in several organs including liver, kidney, spleen, heart and brain. Histopathology findings included cellular necrosis, focal haemorrhage, infiltration of lymphocytes and multifocal granulomatous inflammation in the infected organs.     

Difference in genes between a high virulence strain G4 and a low virulence strain G18 of Flavobacterium columnare by using suppression subtractive hybridization

Flavobacterium columnare is the causative agent of columnaris disease. Different genetic groups of F. columnare show to some extent different degrees of virulence. To identify genetic differences between the high virulence strain G₄ and the low virulence strain G₁₈ of F. columnare, suppression subtractive hybridization was used. A total of 46 genes were identified from the virulent strain G₄, 35 of which showed some degree of homology with known proteins and can be classified into 11 categories: DNA replication or recombination proteins, inorganic ion transport proteins, outer membrane proteins, enterotoxin, binding proteins, YD repeat proteins, transposase, chaperon, signal transduction‐related proteins, regulatory proteins, metabolism‐related proteins. Several putative virulence factors identified in other bacteria could also be identified in the virulent strain G₄, such as ferrous iron transport protein, TonB‐dependent receptor, transposases, as well as ABC transporter permease protein. The flanking region of a putative transposase ISFclI was analysed, and a putative Rhs element was located at the downstream of the putative transposase. The analysis of isfclI gene in 24 strains of F. columnare isolated in China revealed that 11 strains have isfclI, and all the strains from Zhaoqing, Anhui and Qingjiang have isfclI.     

Effect of Sanitizers on Planktonic Edwardsiella tarda Isolated from Asian Stinging Catfish Heteropneustes fossilis (Bloch 1794)

Incidence of Edwardsiella tarda in Asian stinging catfish, Heteropneustes fossilis (Bloch), and its sensitivity to antibiotics and sanitizers was studied. Five out of 10 representative lots of H. fossilis were positive for E. tarda. The minimum bactericidal concentration (MBC) of sanitizers (hydrogen peroxide, glutaraldehyde, benzalkonium chloride, sodium hypochlorite, and iodophor) on planktonic E. tarda varied with strains, suspending media (distilled water, physiological saline, pond water, and tryptic soy broth), and combination of factors. The time taken by the sanitizers to achieve 5-log reductions varied greatly between 1 min and 180 min at X and 2X MBCs of sanitizers. Benzalkonium chloride, glutaraldehyde, and sodium hypochlorite were effective in reducing the planktonic E. tarda at concentrations ranging from 3.13 ppm for glutaraldehyde to 25 ppm for sodium hypochlorite. Edwardsiella tarda strains were highly sensitive to ciprofloxacin and exhibited varying degrees of resistance to other antibiotics.     

Occurrence of multiple antibiotic resistance and genotypic characterization in Edwardsiella tarda isolated from cage‐cultured hybrid red tilapia (Oreochromis sp.) in the Ping River,Northern Thailand

Edwardsiella tarda is a pathogen that causes edwardsiellosis in aquatic animals. The emergence of multiple antibiotic‐resistant strains makes antibiotic treatment difficult. This study aimed to investigate the antibiotic susceptibility patterns and the genotypic characterization of E. tarda isolated from cage‐cultured red tilapia in Thailand. A total of 30 isolates were identified as E. tarda using biochemical and molecular analysis. The disc diffusion method for testing antibiotic susceptibility showed all the isolates were resistant to colistin sulphate and oxolinic acid. High levels of resistance to amoxicillin, ampicillin, ceftazidime, oxytetracycline and sulphamethoxazole/trimethoprim were observed as well. The multiple antibiotic resistance index ranged from 0.25 to 0.92, indicating that these isolates had been exposed to high risk sources of contamination where antibiotics were commonly used. All the isolates carried the bla_TEM gene based on polymerase chain reaction (PCR). The tetA and sul3 genes were detected in 90% (27/30) and 26.7% (8/30) of the isolates respectively. Nine different genetic groups of isolates were obtained using enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC‐PCR). A correlation between genetic types and multiple antibiotic‐resistant patterns was found. These results highlight the potential risks of multiple antibiotic‐resistant isolates for humans and the environment.     

Enhancement of non‐specific immune responses and disease resistance on oral administration of Nyctanthes arbortristis seed extract in Oreochromis mossambicus (Peters)

The immunomodulatory effect of the seed extract of an Indian medicinal plant Nyctanthes arbortristis L. (NAT) on non‐specific immune responses and disease resistance of tilapia, Oreochromis mossambicus was investigated. Chloroform extract was administered orally as a feed supplement at doses of 0.01%, 0.1% or 1% for 3 weeks. Non‐specific immune parameters such as serum lysozyme and alternate complement haemolytic (ACH₅₀) activities, and cellular reactive oxygen species (ROS), reactive nitrogen intermediate (RNI) and myeloperoxidase production and disease resistance against live virulent Aeromonas hydrophila were investigated after 1, 2 or 3 weeks of feeding with chloroform extract‐supplemented diet. The results of this study indicated that feeding tilapia for 2 weeks with selected doses of chloroform extract of NAT seeds significantly enhanced serum lysozyme, alternate complement activities and cellular ROS, RNI and MPO production. It was evident from the disease resistance test that feed supplemented with NAT seed extract at 0.1% or 1% level significantly reduced the mortality of O. mossambicus and a 3‐week feeding with 0.1% extract‐supplemented diet appears to be the optimal regimen for maximal disease resistance. Thus, the study indicates the scope of using the NAT extract as an immunoprophylactic in finfish aquaculture.     

Surface properties of Streptococcus phocae strains isolated from diseased Atlantic salmon, Salmo salar L

Streptococcus phocae is an emerging pathogen for Chilean Atlantic salmon, Salmo salar, but the factors determining its virulence are not yet elucidated. In this work, cell surface–related properties such as hydrophobicity and haemagglutination, adhesion to mucus and cell lines, capsule detection, survival and biofilm formation in skin mucus and serum resistance of the isolates responsible for outbreaks in Atlantic salmon and seals were examined. Adhesion to hydrocarbons and the results of salt aggregation tests indicated most of the S. phocae were strongly hydrophobic. All isolates exhibited a similar ability to attach to the Chinook salmon embryo (CHSE) cells line, but were not able to enter CHSE cells. Haemagglutination was not detected. Our data clearly indicate that S. phocae can resist the killing activity of mucus and serum and proliferate in them, which could be associated with the presence of a capsular layer around the cells. Pathogenicity studies using seal and fish isolates demonstrated mortality or pathological signs in fish injected only with the Atlantic salmon isolate. No mortalities or histopathological alterations were observed in fish injected with extracellular products.     

Photobacterium damselae subsp. damselae, an emerging pathogen in Danish rainbow trout, Oncorhynchus mykiss (Walbaum), mariculture

A selection of 16 field isolates of Photobacterium damselae from marine rainbow trout farms in Denmark was subjected to phenotypic and genotypic characterization and pathogenicity to fish. All isolates belonged to the subspecies damselae , being positive for haemolysis, motility and urease. There were considerable differences in haemolytic properties, some isolates presenting a broad zone of haemolysis and others only a narrow zone. Pulsed-field gel electrophoresis revealed a high diversity indicating that P. damselae subsp. damselae is an opportunistic, not clonal pathogen in Danish marine rainbow trout. Virulence of the strains to rainbow trout was highly variable with LD₅₀ values ranging from 3.9 × 10³ to 1.5 × 10⁸ cfu at 20 °C. The virulence was significantly higher at 20 °C than at 13 °C. The strains with the strongest haemolytic properties were the most virulent suggesting a strong involvement of haemolysin in the pathogenesis. The pathological changes were consistent with a bacterial septicaemia and the haemorrhages were more pronounced than for most other bacterial infections.