Epigenetic reprogramming around IFN1 and IFNy2 promoters in rainbow trout cells inoculated with infectious pancreatic necrosis virus (IPNV).

Infectious pancreatic necrosis virus (IPNV) has proven to effectively evade the host antiviral responses. This study clarifies whether the modulation of the antiviral immune response exerted by IPNV involves epigenetic mechanisms. An in-silico characterization of the rainbow trout IFN1 and IFNγ2 promoters was performed, identifying the islands or sequences rich in CpG dinucleotides and the putative transcription factor binding sites (TBS) for both gene promoters. RTS11 cells (rainbow trout monocyte/macrophage) were infected with IPNV, and the course of viral infection was followed up to 48 h post infection (hpi). Infected cells showed increased IFN1 and IFNγ2 transcriptional expression at 6 and 24 hpi, respectively. IPNV infection caused increases and decreases in global IFNγ2 promoter methylation at 6 and 24 hpi, respectively. The CpG dinucleotides at positions -392 and + 38 of this promoter were the most sensitive to methylation changes. The IFN1 promoter remained fully unmethylated during the course of the infection, similar to the control. The changes in the methylation pattern observed for the IFNγ2 promoter were coincident with the changes in DNA methyltransferase (DNMT) expression levels, increasing at 6 hpi and decreasing below basal level at 24 hpi. Similarly, the H4 histones associated with the IFN1 and IFNγ2 promoters were hyperacetylated at 6 hpi, subsequently decreasing their acetylation below basal levels at 24 hpi, in both promoters. Coincidentally with the above, overexpression of histone acetyltransferase (HAT) was observed at 6 hpi and of histone deacetylase (HDAC) at 24 hpi, with return to baseline of HAT. These results suggest that IPNV would epigenetically modulate the expression of IFN1 by changing acetylation levels of the histones H4 associated with its promoter. Also, the modulation of the expression of IFNy2 would be by switching methylation/demethylation levels of its promoter, in addition to changes in acetylation levels of histones H4 associated with this promoter. This study is the first to demonstrate the effect of epigenetic reprogramming after IPNV infection in salmonid cells, demonstrating that promoter methylation/demethylation level and changes in the histone code associated with promoters may play a role in the modulation of the immune response induced by the virus.

High doses of Francisella noatunensis induces an immune response in Eleginops maclovinus.

Francisella noatunensis subsp. noatunensis, the etiological agent of Francisellosis, affects a large number of farmed species such as Salmo salar. This species coexists with several native species in the same ecosystem, including Eleginops maclovinus. Our objective was to evaluate the susceptibility, presence of clinical symptoms, and the ability of Eleginops maclovinus to respond to Francisella infection. For this, healthy individuals were inoculated with 1.5 × 101, 1.5 × 105, and 1.5 × 1010 bact/µL of Francisella by intraperitoneal injection, subsequently the fish were sampled on days 1, 3, 7, 14, 21, and 28 post injection (dpi). At the end of the experiment, no mortality, nor internal and external clinical signs were observed, although in the high dose anaemia was detected. Additionally, bacteria were detected in all three doses, however there was replication at day 28 only in the liver in the high dose. Analysis of gene expression by qPCR showed that the spleen generated an immune response against infection from day 1 dpi, however at day 7 dpi most of the genes suffered repressed expression; observing over expression of the genes C3, NLRC3, NLRC5, MHCI, IgM. In contrast, expression in the anterior kidney did not vary significantly during the challenge. IgM quantification showed the production of antibodies in the medium and high doses. This study provides new knowledge about Francisella infection and the long-lasting and specific immune response generated by Eleginops maclovinus. It also demonstrates its susceptibility to Francisellosis where there is a difference in the immune response according to the tissue.

Effect of Flavobacterium psychrophilum on the neuroendocrine response of rainbow trout (Oncorhynchus mykiss) in a time course experiment.

The aim of this study was to examine the effects of Flavobacterium psychrophilum, a pathogen that is economically important in the aquaculture sector, on the neuroendocrine response of Oncorhynchus mykiss during a time course experiment with sampling at 0.5, 1, 2, 6, 10, and 30 days post injection (dpi). In the brain, serotonin (5HT) content increased in the infected group at all the measured time points, a similar pattern was observed for 5-hydroxyindole-3-acetic acid (5HIAA). Infected fish presented an increase in brain dopamine levels on day 0.5 and 1 dpi. A non-significant variation in noradrenaline levels was observed on all treatment days. Foregut 5-HT and 5-HIAA content in the infected group presented the highest 5-HT concentrations with 248.6 and 983.5 ng/g tissue at 0.5 dpi respectively. Midgut 5-HT and 5-HIAA levels presented the highest 5-HT concentrations, 486.9 ng/g tissue and 1006.4 ng/g tissue respectively, at the beginning of the experiment (0.5 dpi). 5-HT levels in the hindgut presented the highest concentrations with 233.9 ng/g tissue at 0.5 dpi, while 5-HIAA presented the highest concentrations, 690.5 ng/g tissue, at the same time point. After injection with F. psychrophilum the neuroendocrine response in rainbow trout was tissue dependent. Brain levels of 5HT and 5HIIA indicate that the neuroendocrine response increased together with dopamine following intramuscular infection. These increases are in line with reports from other authors, indicating an early response of catecholamines as neurotransmitters to stressful stimulus. In addition the intestinal response was also increased, implying that there could be a possible relationship between the serotonergic system at the intestinal level and the immune system.

First identification and characterization of Streptococcus iniae obtained from tilapia (Oreochromis aureus) farmed in Mexico.

This is the first study to isolate, identify and characterize Streptococcus iniae as the causative disease agent in two tilapia (Oreochromis aureus) populations. The populations were geographically isolated, of distinct origins, and did not share water sources. Affected fish showed various external (e.g., exophthalmia and cachexia, among others) and internal (e.g., granulomatous septicaemia and interstitial nephritis, among others) signs. All internal organ samples produced pure cultures, two of which (one from each farm, termed S-1 and S-2) were subjected to biochemical, PCR and 16S rRNA sequencing (99.5% similarity) analyses, confirming S. iniae identification. The two isolates presented genetic homogeneity regardless of technique (i.e., RAPD, REP-PCR and ERIC-PCR analyses). Pathogenic potentials were assessed through intraperitoneal injection challenges in rainbow trout (Oncorhynchus mykiss) and zebrafish (Danio rerio). Rainbow trout mortalities were respectively 40% and 70% at 104 and 106  CFU per fish with the S-1 isolate, while 100% mortality rates were recorded in zebrafish at 102 and 104  CFU per fish with the S-2 isolate. The obtained data clearly indicate a relationship between intensified aquaculture activities in Mexico and new disease appearances. Future studies should establish clinical significances for the tilapia industry.

Iron acquisition and siderophore production in the fish pathogen Renibacterium salmoninarum.

Renibacterium salmoninarum is the causative agent of bacterial kidney disease, which significantly affects salmonid farming worldwide. Despite this impact, there is scarce data on its iron uptake ability, a factor of pathogenesis. This study investigated the iron acquisition mechanisms of R. salmoninarum and its capacity to uptake iron from different sources. Thirty-two Chilean isolates and the DSM20767T type strain grew in the presence of 2,2'-Dipyridyl at varying concentrations (250-330 µm), and all isolates positively reacted on chrome azurol S agar. Subsequently, inocula of four Chilean isolates and the type strain were prepared with or without 200 µm of 2,2'-Dipyridyl for uptake assays. Assay results revealed differences between the isolates in terms of iron acquisition. While a prior iron-limited environment was, for most isolates, not required to activate the uptake of iron (II) sulphate, ammonium iron (III) citrate or iron (III) chloride at higher concentrations (100 µm), it did facilitate growth at lower iron concentrations (10 µm and 1 µm). An exception was the H-2 isolate, which only grew with 100 µm of iron sulphide. In turn, 100 µm of haemin was toxic when isolates were grown in normal KDM-2. In silico R. salmoninarumATCC 33209T genome analysis detected various genes coding iron uptake-related proteins. This is the first study indicating two iron acquisition systems in R. salmoninarum: one involving siderophores and another involving haem group utilization. These data represent a first step towards fully elucidating this virulence factor in the pathogenic R. salmoninarum.

Effectiveness of egg yolk immunoglobulin against the intracellular salmonid pathogen Piscirickettsia salmonis.

AIMS: To produce and characterize egg yolk immunoglobulin (IgY) against the fish intracellular pathogen Piscirickettsia salmonis as well as to evaluate the antibacterial activity of IgY in vitro and the availability in the serum of fish immunized orally. METHODS AND RESULTS: Specific IgY was produced by immunizing hens with P. salmonis proteins. The IgY was obtained from egg yolks using the ammonium sulphate precipitation method and it was characterized by SDS-PAGE, Western-blot and ELISA, demonstrating that anti-P. salmonis IgY strongly reacted specifically against P. salmonis proteins. In an in vitro neutralization assay, IgY inhibited the growth of P. salmonis in liquid medium at concentrations ranging from 128 to 256 µg ml(-1) in a dose-dependent manner. Interestingly, IgY against P. salmonis also generates a strong protective effect on the infection of P. salmonis in salmon head kidney-1 cells. In addition, the bacteriostatic function of IgY appears to result possibly from agglutination by the interaction of IgY with surface components of the pathogen. Finally, to confirm this IgY as an alternative for salmonid treatment, Atlantic salmon (Salmo salar) specimens were orally inoculated with IgY. The analysis of the sera demonstrates that IgY was effectively transported by fish intestine and that this immunoglobulins maintains its properties and recognizes several proteins of P. salmonis up to 12 h after inoculation of IgY against P. salmonis. CONCLUSIONS: Specific IgY effectively inhibited the growth of P. salmonis and this immunoglobulin can be released in the Atlantic salmon sera when administered orally to fish. SIGNIFICANCE AND IMPACT OF THE STUDY: We propose that this specific IgY against this fastidious micro-organism could be a useful strategy for the treatment of piscirickettsiosis.

Bacterial lipopolysaccharide induces rainbow trout myotube atrophy via Akt/FoxO1/Atrogin-1 signaling pathway.

Lipopolysaccharide (LPS) is considered as a powerful inducer of muscle atrophy in higher vertebrates due to skeletal muscle cell recognition of the endotoxin and a consequent activation of catabolic signaling pathways. In contrast, there is no evidence of LPS directly inducing skeletal muscle atrophy in lower vertebrates, such as fish. For years it has been assumed that fish are resistant to LPS, mainly due to differences in the key features of toll-like receptor (TLR) signaling pathways when compared with mammals. In this study, we report that the stimulation of cultured rainbow trout (Oncorhynchus mykiss) myotubes with LPS (100 ng/ml) resulted in a transient decrease in the pAkt/Akt ratio, a subsequent reduction in the pFoxO1/FoxO1 ratio, and a significant increase in atrogin-1 transcript expression. Preincubation with polymyxin B, an LPS-neutralizing agent, and 740 Y-P, an agonist of p85-PI3K, blocked the effects of LPS. Additionally, LPS treatment induced an increase in protein ubiquitination and a reduction in myotube diameter, both of which are associated with muscular atrophy that is not observed under polymyxin B and 740 Y-P pretreatments. Finally, rainbow trout myotubes expressed the genes tlr1, tlr3, tlr5m, tlr8a1, tlr8a2, tlr9, and tlr22, with significantly increased expressions of tlr5m and tlr9 under LPS stimulation. These results indicate that LPS is an inducer of fish skeletal muscle atrophy and suggest that TLR5M and TLR9 may play important roles in detecting LPS, which supports for the first time the hypothesis that LPS is a direct inducer of skeletal muscle atrophy in teleost species.

Cell-surface properties of Vibrio ordalii strains isolated from Atlantic salmon Salmo salar in Chilean farms.

Vibrio ordalii is the causative agent of atypical vibriosis and has the potential to cause severe losses in salmonid aquaculture, but the factors determining its virulence have not yet been elucidated. In this work, cell-surface-related properties of the isolates responsible for outbreaks in Atlantic salmon were investigated. We also briefly examined whether pathogenicity against fish varied for V. ordalii strains with differing cell-surface properties. Hydrocarbon adhesions indicated the hydrophobic character of V. ordalii, although only 4 of 18 isolates induced haemagglutination in Atlantic salmon erythrocytes. A minority of the studied isolates (6 of 18) and the type strain ATCC 33509T produced low-grade biofilm formation on polyethylene surface after 2 h post-inoculation (hpi), but no strains were slime producers. Interestingly, V. ordalii isolates showed wide differences in hydrophobicity. Therefore, we chose 3 V. ordalii isolates (Vo-LM-03, Vo-LM-18 and Vo-LM-16) as representative of each hydrophobicity group (strongly hydrophobic, relatively hydrophobic and quasi-hydrophilic, respectively) and ATCC 33509T was used in the pathogenicity studies. All tested V. ordalii strains except the type strain resisted the killing activity of Atlantic salmon mucus and serum, and could proliferate in these components. Moreover, all V. ordalii isolates adhered to SHK-1 cells, causing damage to fish cell membrane permeability after 16 hpi. Virulence testing using rainbow trout revealed that isolate Vo-LM-18 was more virulent than isolates Vo-LM-03 and Vo-LM-16, indicating some relationship between haemagglutination and virulence, but not with hydrophobicity.

PCR protocol for detection of Vibrio ordalii by amplification of the vohB (hemolysin) gene.

Vibrio ordalii is the causative agent of atypical vibriosis and has the potential to cause severe losses in salmonid aquaculture. To prevent and control outbreaks, a rapid, reproducible, sensitive, and effective diagnostic method is needed. We evaluated a new conventional polymerase chain reaction (PCR) and real-time PCR (qPCR) protocol using a primer set (VohB_Fw-VohB_Rv) designed to amplify a 112 bp fragment flanking the vohB gene (coding for hemolysin production), against 24 V. ordalii strains isolated from different fish species, the V. ordalii type strain, and 42 representative related and unrelated bacterial species. The primer set was species-specific, recognizing all V. ordalii strains evaluated, with no cross-reaction with the other bacterial species. A sensitivity of 103 copies of the vohB gene was obtained with a standard curve. When the VohB_Fw-VohB_Rv qPCR protocol was applied to Atlantic salmon seeded tissues (kidney, liver, spleen, and muscle), the detection limit ranged from 5.27 × 102 to 4.13 × 103 V. ordalii CFU ml-1, i.e. 62 to 145 copies of the vohB gene, using the previously calculated standard curve. The conventional PCR also detected V. ordalii, but the total reaction time was 1 h longer. When the qPCR protocol was applied to naturally infected cage-cultured Atlantic salmon samples, 5 of 8 fish tested positive for V. ordalii, but only one of them was diagnosed as positive by direct cultivation on agar. We conclude that the PCR protocol evaluated is fast, specific, and sensitive enough to detect V. ordalii in infected tissues and is an important tool for secure diagnosis of atypical vibriosis, and is therefore helpful for the control of the disease through the prompt detection within fish populations.

Flavobacterium chilense sp. nov. and Flavobacterium araucananum sp. nov., isolated from farmed salmonid fish.

Three Gram-staining-negative non-endospore-forming strains were isolated from farmed fish in Chile: one (LM-09-Fp(T)) from a rainbow trout (Oncorhynchus mykiss) and the others (LM-19-Fp(T) and LM-20-Fp) from two Atlantic salmon (Salmo salar). Phylogenetic analyses based on 16S rRNA gene sequences indicated that all three isolates belonged to the genus Flavobacterium. In these analyses, strain LM-09-Fp(T) appeared most closely related to the type strains of Flavobacterium chungangense (98.5 % sequence similarity), Flavobacterium glaciei (98.2 %), Flavobacterium aquidurense (97.6 %), Flavobacterium saccharophilum (97.6 %) and Flavobacterium hercynium (97.6 %). The 16S rRNA gene sequences of strains LM-19-Fp(T) and LM-20-Fp were found to be identical and most similar to the corresponding sequences of the type strains of Flavobacterium aquidurense (98.6 %), Flavobacterium frigidimaris (98.5 %), Flavobacterium hercynium (97.9 %), Flavobacterium saccharophilum (97.7 %) and Flavobacterium pectinovorum (97.7 %). For each of the three novel strains, menaquinone (MK-6) was the predominant respiratory quinone and the major compounds in the polar lipid profile were phosphatidylethanolamine, an unidentified aminolipid, phosphatidylserine and two or three unknown lipids. The fatty acid profile of each strain, which comprised major amounts of iso-C(15:0), C(15:0) and summed feature 3 (C(16:1)ω7c and/or iso-C(15:0) 2-OH) as well as smaller amounts of various hydroxylated fatty acids (e.g. iso-C(16:0) 3-OH, iso-C(17:0) 3-OH, C(16:0) 3-OH and C(15:0) 3-OH), indicated that each belonged to the genus Flavobacterium. Based on their physiological and biochemical characteristics and the results of DNA-DNA hybridizations, which showed relatively low levels of relatedness between the novel strains and the most closely related Flavobacterium species, strain LM-09-Fp(T) ( = LMG 26360(T) = CCM 7940(T)) represents a novel species within the genus Flavobacterium, for which the name Flavobacterium chilense sp. nov. is proposed, and strains LM-19-Fp(T) ( = LMG 26359(T) = CCM 7939(T)) and LM-20-Fp ( = LMG 26331) represent a second novel species within the same genus, for which the name Flavobacterium araucananum sp. nov. is proposed.

Broth medium for the successful culture of the fish pathogen Piscirickettsia salmonis .

Piscirickettsiosis or salmonid rickettsial septicaemia (SRS) caused by Piscirickettsia salmonis constitutes one of the main problems in farmed salmonid and marine fishes. Since the first reports of the disease, it has been successfully isolated and maintained in eukaryotic cell--culture systems, but these systems are time-consuming, the media are costly, and eliminating heavily contaminated host cell debris is difficult. In this report, we describe a marine-based broth supplemented with L-cysteine, named AUSTRAL-SRS broth, that facilitates superior growth of P. salmonis strains. Strains reached an optical density of approximately 1.8 when absorbance was measured at 600 nm after 6 d incubation at 18°C. Several passages (n = 6) did not alter the culture kinetics. We report for the first time the purification of DNA, lipopolysaccharide (LPS) and whole membrane protein obtained from P. salmonis grown in this liquid medium, and thus provide a suitable platform to simplify the preparation of P. salmonis cells for genetic and serological studies. Moreover, the results of the cytopathic effect test showed that P. salmonis grown in AUSTRAL-SRS broth maintained their virulence properties, inducing apoptosis after 3 d. This makes the medium a good candidate for the successful growth of P. salmonis and an excellent basis for the development of low cost vaccines.

Infectivity study of Streptococcus phocae to seven fish and mammalian cell lines by confocal microscopy.

Streptococcus phocae is a beta-haemolytic bacterium that causes systemic infections in Atlantic salmon, Salmo salar L., cultured in southern Chile and also in seals. In this study, the host-pathogen interaction between S. phocae and seven types of cell lines (fish and mammalian) was examined using an indirect fluorescent antibody and confocal microscopy (CM). Chinook salmon embryo (CHSE-214), epithelioma papulosum cyprini (EPC), salmon head kidney (SHK-1) and Atlantic salmon kidney were used as the fish cell lines, while human cervix epithelial adenocarcinoma (HeLa), African green monkey kidney fibroblast (Cos-7) and mouse leukaemic monocyte macrophage (Raw 264.7) were included as mammalian cell lines. Streptococcus phocae type strain ATCC 51973(T) and isolates LM-08-Sp and P23 were selected as representatives from the salmon and seal host, respectively. For the CM examination, monolayers seeded on round coverslips were studied at 2- and 20-h post-inoculation (pi). The results showed that there is no common infectivity pattern between the three S. phocae strains at 2-h pi and the cell lines tested, regardless of the source of isolation (seal or salmon). All S. phocae strains could internalize and were found inside the fish and mammalian cell cytoplasm after 20-h pi. Regardless of the cells studied (fish or mammal) and incubation (2 and 20 h), S. phocae was never observed inside the nuclei. Seal and salmon isolates showed the highest number of bacteria entering into the primate cell lines (HeLa and Cos-7) from 2-h pi, while ATCC 51973(T) was not found outside or inside the HeLa and Cos-7 cells.

Multiplex PCR for the detection of Piscirickettsia salmonis, Vibrio anguillarum, Aeromonas salmonicida and Streptococcus phocae in Chilean marine farms.

A multiplex (m-)PCR-based protocol was designed for the simultaneous detection of the main marine bacterial pathogens in Chilean salmon farms: Streptococcus phocae, Aeromonas salmonicida, Vibrio anguillarum and Piscirickettsia salmonis. Each of the 4 oligonucleotide primer pairs exclusively amplified the target gene of the specific bacterial pathogen. The detection limit of the m-PCR using purified total bacterial DNA was 50 pg microl(-1) for V anguillarum, 500 fg microl(-1) for P. salmonis, and 5 pg microl(-1) for S. phocae and A. salmonicida. This corresponded to average limits in the m-PCR sensitivity of 3.69 x 10(5) CFU ml(-1) of V anguillarum, 1.26 x 10(4) CFU m(-1) of S. phocae, and 5.33 x 10(4) CFU ml(-1) of A. salmonicida, while the detection limits for the spiked fish tissues, regardless of the sample (spleen, kidney, liver or muscle) were 2.64 +/- 0.54 x 10(7) CFU g(-1) for V. anguillarum, 9.03 +/- 1.84 x 10(5) CFU g(-1) for S. phocae, 3.8 +/- 0.78 x 10(3) CFU mg(-1) for A. salmonicida and 100 P. salmonis cells. However, high amounts of DNA from 3 bacterial species had a reduction of -1 log-unit on the amplification sensitivity of S. phocae or A. salmonicida when these were present in lower concentration in the multiplex reaction. The assay described in this study is a rapid, sensitive and efficient tool to detect the presence of S. phocae, A. salmonicida, V. anguillarum and P. salmonis simultaneously from pure cultures and tissues from clinically diseased fish. Therefore, it may be a useful alternative to culture-based methods for the diagnosis of infections in fish obtained from Chilean salmon farms.

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.

TaqMan real-time RT-PCR detection of infectious salmon anaemia virus (ISAV) from formalin-fixed paraffin-embedded Atlantic salmon Salmo salar tissues.

The objective of this study was to evaluate the application of a TaqMan real-time reverse transcriptase PCR (RT-PCR) assay for the detection of infectious salmon anaemia virus (ISAV) in formalin-fixed paraffin-embedded (FFPE) fish tissues from Atlantic salmon Salmo salar with and without clinical signs of infection, and to compare it with histological and immunohistochemical (IHC) techniques. Sixteen fish samples obtained in 2007 and 2008 from 4 different farms in Chile were examined. The real-time RT-PCR allowed the detection of ISAV in FFPE samples from 9 of 16 fish, regardless of the organs analyzed, whereas 4 of the real-time RT-PCR negative fish were positive as indicated by histological examination and 3 of the real-time RT-PCR positive fish were negative as indicated by immunohistochemistry evaluation. The presence of ISAV in RT-PCR positive samples was confirmed by amplicon sequencing. This work constitutes the first report on the use of real-time RT-PCR for the detection of ISAV in FFPE sections. The assay is very useful for the examination of archival wax-embedded tissues, and allows for both prospective and retrospective evaluation of tissue samples for the presence of ISAV. However, the method only confirms the presence of the pathogen and should be used in combination with histopathology, which is a more precise tool. The combination of both techniques would be invaluable for confirmatory diagnosis of infectious salmon anaemia (ISA), which is essential for solving salmon farm problems.

First description of atypical furunculosis in freshwater farmed Atlantic salmon, Salmo salar L., in Chile.

We report the first isolation, identification and characterization of a group of Chilean strains of atypical Aeromonas salmonicida isolated from freshwater farmed Atlantic salmon, Salmo salar. Affected fish showed superficial ulcers and pale liver with or without petechial haemorrhages. Outbreaks of the disease occurred in two farms in the south of Chile about 2200 km apart. Five strains were isolated in pure culture and identified by serological assays and immunofluorescence tests as belonging to Aeromonas salmonicida. Although the bacterial isolates were phenotypically homogeneous, minor differences with the reference strain A. salmonicida subsp. salmonicida ATCC 33658 were noted. Three specific primer sets and partial 16S rRNA gene sequencing allowed the identification of the Chilean isolates as atypical A. salmonicida, with A. salmonicida subsp. achromogenes and A. salmonicida subsp. masoucida as their closest relatives (100% sequence similarity). Molecular typing indicated that the atypical isolates belong to two genetic groups that were associated with the geographical origin.