Attenuated virulence of an Aeromonas salmonicida subsp. salmonicida type III secretion mutant in a rainbow trout model

Sarah E. Burr1, Dmitri Pugovkin1, Thomas Wahli2, Helmut Segner2 and Joachim Frey1

1 Institute of Veterinary Bacteriology, University of Berne, Länggass-Strasse 122, Postfach, CH-3001 Berne, Switzerland
2 Centre for Fish and Wildlife Health, Institute of Animal Pathology, University of Berne, Länggass-Strasse 122, Postfach, CH-3001 Berne, Switzerland

Correspondence
Joachim Frey
joachim.frey{at}vbi.unibe.ch


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Aeromonas salmonicida subsp. salmonicida is the causative agent of furunculosis, a severe systemic disease affecting salmonid fish. This bacterium contains a type III protein secretion system that is responsible for the secretion and translocation of the ADP-ribosylating toxin, AexT, into the cytosol of fish cells. This study showed that inactivation of the type III secretion system by marker-replacement mutagenesis of the gene ascV, which encodes an inner-membrane component of the type III secretion system, attenuated virulence in a rainbow trout model. The isogenic ascV deletion mutant was phagocytosed by peripheral blood leukocytes but the wild-type (wt) A. salmonicida subsp. salmonicida isolate was not. Histological examination of fish experimentally infected with the wt bacterium revealed extensive tissue necrosis and bacterial aggregates in all organs examined, including the heart, kidney and liver, indicating that the isolate established a systemic infection. Cumulative mortality of fish experimentally infected with the wt bacterium reached 88 %. In contrast, no mortality was observed among fish infected with the same dose of the ascV mutant, and histological examination of fish infected with this strain revealed healthy organs. The results indicate that the type III secretion system of A. salmonicida subsp. salmonicida is required to establish systemic infection.


Abbreviations: i.m., intramuscular(ly); i.p., intraperitoneal(ly); Km, kanamycin; p.i., post-infection; wt, wild-type; SPF, specific pathogen-free

The GenBank/EMBL/DDBJ accession number for the nucleotide sequence of the complete A. salmonicida subsp. salmonicida type III secretion locus in strain JF2267 is AJ616218.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Aeromonas salmonicida subsp. salmonicida, often referred to as typical A. salmonicida, is the causative agent of furunculosis, a systemic disease of salmonid fish characterized by high mortality and morbidity. Furunculosis derives its name from the boils or furuncles that develop on the skin and in the musculature of fish affected with the chronic form of the disease. In the acute form, the disease causes rapid septicaemia resulting in the formation of necrotic lesions in the skin and haemorrhages in the internal organs. Such infections are often fatal within as little as 2 or 3 days.

Because of the negative impact A. salmonicida subsp. salmonicida has on the aquaculture industry, extensive work has been done to identify virulence factors produced by this bacterium. Recently, a functional type III protein secretion system has been identified in an A. salmonicida subsp. salmonicida field isolate (Burr et al., 2002, 2003). Such secretion systems enable the movement or translocation of effector proteins, produced in the bacterial cytoplasm, into the cytosol of eukaryotic cells (see Ghosh, 2004, for a recent review). Once within the eukaryotic cytosol, effector proteins are able to disrupt the cytoskeleton or interfere with cell signalling cascades (Cornelis, 2002; Galán, 2001; Zaharik et al., 2002). As a result, type III secretion systems are important virulence factors of many Gram-negative bacteria including the pathogenic Yersinia spp., Salmonella spp., Shigella spp., enteropathogenic and enterohaemorrhagic Escherichia coli and Pseudomonas aeruginosa (Ghosh, 2004).

Using marker-replacement mutagenesis, the type III secretion system of A. salmonicida subsp. salmonicida, has been found to be responsible for the secretion and translocation of the ADP-ribosylating toxin, AexT, into the cytosol of cultured fish cells (Burr et al., 2002, 2003). Inactivation of the secretion system has also been shown to reduce the cytoxicity of A. salmonicida subsp. salmonicida towards fish cell lines. We were therefore interested to determine the role of the type III secretion system in the virulence of A. salmonicida subsp. salmonicida towards the fish host. In the current study, we examined the virulence of a defined type III secretion mutant of A. salmonicida subsp. salmonicida using an in vivo rainbow trout model and found that this sytem is essential for virulence of the bacterium. We also investigated the presence of type III secretion genes in atypical A. salmonicida strains and other aeromonad fish isolates.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Bacterial strains, plasmids and culture conditions.
All bacterial strains and plasmids used in cloning and cellular and in vivo assays are listed in Table 1. Aeromonas spp. type strains and field isolates used in the dot blot hybridization assay are described in Table 2. The identity of field isolates was verified by sequence analysis of the 16S rRNA gene as previously described (Kuhnert et al., 2002). Escherichia coli DH5{alpha} (Sambrook et al., 1989) was used as a negative control in the dot blot hybridization assay. Strains were routinely grown on Luria–Bertani (LB) agar at 18 °C (Aeromonas spp.) or 37 °C (E. coli). When indicated, antibiotics were added to the culture media at the following final concentrations: for E. coli, ampicillin (100 µg ml–1), tetracycline (20 µg ml–1), kanamycin (50 µg ml–1); for A. salmonicida, chloramphenicol (20 µg ml–1), kanamycin (40 µg ml–1), ampicillin (100 µg ml–1).


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Table 1. Bacterial strains and plasmids used in cloning and in cellular and in vivo assays

 

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Table 2. Origin of Aeromonas spp. isolates and presence of type III secretion genes ascV and ascU as demonstrated by dot blot hybridization

ATCC, American Type Culture Collection, Manassas, VA, USA; CDC, Centers for Disease Control, Atlanta, GA, USA; CIP, Collection bactérienne de l'Institut Pasteur, Paris, France; DSM, Deutsche Sammlung von Mikroorganismen, Braunschweig, Germany; JF, Culture Collection of the Institute of Veterinary Bacteriology, University of Berne, Switzerland; NCIMB, National Culture Collection of Industrial and Marine Bacteria, Aberdeen, Scotland; NZ, Culture Collection of the National Centre for Enteropathogenic Bacteria, University of Berne, Switzerland. A superscript T denotes a type strain.

 
DNA sequencing and sequence data analysis.
Total DNA was prepared using the EZNA bacterial DNA kit (Peqlab Biotechnologie). Primer walking was carried out using the Vectorette system (Genosys) according to the manufacturer's instructions. PCR was performed using a GeneAmp 9700 thermocycler (Applied Biosystems). The reaction mixtures contained 1x PCR buffer, 170 µM each dNTP, 0·25 µM custom-synthesized forward and reverse primers (Microsynth), 2 units DNA polymerase containing proof-reading capability (Expand Long Template PCR System, Roche Diagnostics) and approximately 20 ng template DNA. Details of all primer sequences used are available upon request. PCR conditions were as follows: 3 min at 94 °C followed by 40 cycles of 30 s at 94 °C, 30 s at 58 °C and up to 4 min at 68 °C.

DNA sequencing was performed with the dRhodamine Terminator Cycle Sequencing Kit (Applied Biosystems) according to the manufacturer's protocol. Sequences were determined on both strands. Reaction products were analysed on an ABI Prism 310 genetic analyser (Applied Biosystems) and sequence alignment and editing were performed using the software Sequencher (Gene Codes Corporation). Comparison of DNA sequences and their corresponding amino acid sequences with the Swiss Prot and EMBL databases were performed using BLAST (http://www.ncbi.nlm.nih.gov/blast/). The molecular mass and theoretical isoelectric point (pI) of the type III secretion proteins were calculated using ProtParam (http://www.expasy.org/tools/protparam.html).

Marker-replacement mutagenesis.
The A. salmonicida subsp. salmonicida gene ascV was inactivated by marker-replacement mutagenesis. A 1061 bp fragment from ascV was excised using the restriction enzymes KpnI and SpeI (Roche Diagnostics) and replaced with the kanamycin (Km) cassette from pSSVI186 that had been previously excised on a 1·3 kb KpnI–SpeI fragment. The inactivated ascV and flanking genes were then cloned into the mobilizable suicide vector pSUP202sac. The resulting plasmid was transformed into E. coli S17-1 and conjugated into A. salmonicida subsp. salmonicida strain JF2267 by filter mating (Simon et al., 1983). Double crossover mutants were selected directly by growth on Tryptic Soy Agar (Difco) containing 15 % (w/v) sucrose, 40 µg kanamycin ml–1 and 20 µg chloramphenicol ml–1 at 15 °C for 7 days. Chloramphenicol served to select against the E. coli donor as A. salmonicida subsp. salmonicida strain JF2267 is resistant to this antibiotic. The absence of the wild-type (wt) ascV gene and the insertion of the Km cassette were verified by PCR. Western blot analysis was also carried out to determine whether the {Delta}ascV mutant retained the ability to express S-layer protein (Belland & Trust, 1987).

Fish maintenance.
Juvenile rainbow trout (Oncorhynchus mykiss Walbaum), raised in closed systems under specific pathogen-free (SPF) conditions, were used in all in vivo assays. Prior to the onset of experiments, 10 fish were screened for bacterial, viral and parasitic pathogens according to routine diagnostic procedures. Fish, measuring 11–16 cm total length, were stocked in aerated 13 l volume PVC-tanks to a stocking densitiy of 24 kg m–3. Flowthrough of tap water was set at 0·6 l min–1. The fish were adapted to these conditions for 2 weeks before the experiments began and were fed a commercial fish diet at 1 % body weight per day.

Experimental infection.
Experimental infections were carried out with permission from the Ethical Committee for Animal Experiments, Canton Berne, Switzerland. One hundred and twenty rainbow trout were randomly distributed into tanks containing 10 fish per tank. Suspensions of the A. salmonicida subsp. salmonicida wt or {Delta}ascV mutant strains were prepared in 10 mM PBS, pH 7·4. Fish (four tanks per group) were injected intraperitoneally (i.p.) with 1x105 c.f.u. of either one of the bacterial suspensions in 50 µl PBS or with PBS only. Mortality was recorded daily or, in cases of high mortality, twice daily for 3 weeks. The temperature range during the course of the experiment was 11·8±0·5 °C. All dead fish were investigated for the presence of A. salmonicida subsp. salmonicida as were at least three surviving fish from each tank at the end of the experiment. Material was collected from the liver and kidney of each fish using sterile loops and used to inoculate Columbia sheep blood agar (BioMérieux). The plates were incubated at 18 °C for up to 5 days and the identity of A. salmonicida subsp. salmonicida isolated from dead fish was confirmed by PCR.

Histology.
Rainbow trout were injected intramuscularly (i.m.) with either the A. salmonicida subsp. salmonicida wt or {Delta}ascV mutant strains (five fish for each strain) using an infectious dose of 5x104 c.f.u. per fish. Four days following infection, live and moribund fish were sacrificed and examined for macroscopic alterations. For histological examination, muscle and organ tissues were fixed in 4 % buffered formalin, embedded in paraffin wax and sectioned according to routine histological procedures to produce 5 µm sections. Sections were then stained with haematoxylin and eosin and examined by light microscopy. At least 10 slides from each fish were examined.

Expression of green fluorescent protein.
The gfp gene, encoding green fluorescent protein, and its ribosome-binding site were excised from pBCgfp using the restriction endonucleases EcoRI and HindIII and subsequently cloned into pMMB66EH for expression. The resulting plasmid, pMMB66EH-gfp, was transformed into E. coli S17-1 and transferred to A. salmonicida subsp. salmonicida by filter-mating, as described above. Transconjugants were selected on LB agar containing ampicillin and chloramphenicol, and the presence of pMMB66EH-gfp was confirmed by PCR. Expression of gfp was induced by addition of IPTG to the culture medium at a final concentration of 1 mM.

Phagocytosis assay.
Heparinized blood, obtained from a naïve rainbow trout raised under SPF conditions, was combined with an equal volume of Eagle's minimum essential medium (EMEM, Gibco) before being layered onto a discontinuous Ficoll separating solution with a 1·077 gradient (Biochrom KG). Following centrifugation at 1200 g for 30 min, the layer at the interface, which contained peripheral blood leukocytes, was collected and washed three times with EMEM. The cells were then seeded into six-well culture plates (Falcon), containing glass cover slips coated with Matrigel (BD Biosciences) in 1 ml L-15 medium (Sigma) supplemented with 5 % fetal bovine serum and cyproxin (20 µg ml–1), to a concentration of 1x106 cells ml–1. Cells were incubated for 12 h at 15 °C to allow for attachment to the surface of the cover slips. Following incubation, the medium was replaced with 1 ml L-15 medium containing 5 % fetal bovine serum and 1 mM IPTG. The cultures were then infected with A. salmonicida subsp. salmonicida wt or {Delta}ascV mutant strains. Four hours post-infection (p.i.), the cells were fixed in 4 % buffered formalin and the preparations were examined by fluorescence microscopy. Three independent trials, each in duplicate, were performed.

Dot blot hybridization.
Digoxigenin-11-dUTP (DIG)-labelled probes directed against type III secretion genes ascV and ascU of A. salmonicida subsp. salmonicida strain JF2267 were prepared by PCR using the following primer pairs: AslcrD-L (5'-GCCCGTTTTGCCTATCAA-3') and AslcrD-R (5'-GCGCCGATATCGGTACCC-3') for ascV, and AscU-5' (5'-CGATGAGCGGCGAGAAAAC-3') and AscU-3' (5'-AGCGTGCAGCCCTACCACTG-3') for ascU. PCR conditions were 3 min at 94 °C followed by 35 cycles of 30 s at 94 °C, 30 s at 58 °C and 30 s at 72 °C. Following PCR amplification, the probes were purified using the High Pure PCR Product Purification Kit (Roche) according to the manufacturer's instructions.

Total DNA was prepared using the EZNA bacterial DNA kit (Peqlab Biotechnologie) and denatured in 0·4 M NaOH, 10 mM EDTA. Denatured DNA was spotted onto positively charged nylon membranes (Roche) and fixed by baking at 80 °C under vacuum. Hybridization was performed overnight at 65 °C and membranes were washed under low-stringency conditions (Ausubel et al., 1999). Hybridization reactions were detected using anti-DIG antibodies and the chemiluminescent reagent CPD-Star (Roche) according to the manufacturer's protocol. DNA from A. salmonicida subsp. salmonicida strain JF2267 and E. coli DH5{alpha} served as positive and negative controls respectively.


   RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Identification and sequence analysis of type III secretion genes in A. salmonicida subsp. salmonicida
To date, three genetic loci encoding type III secretion genes have been identified in A. salmonicida subsp. salmonicida strain JF2267; these are virA, virB and virT (Burr et al., 2002, 2003). In an effort to complete the sequencing of this type III secretion system, primer walking was carried out downstream of the previously sequenced type III secretion genes. As a result, a further 15 ORFs displaying homology to known type III secretion genes were identified (Fig. 1). Downstream of the aopD gene, we identified homologues of the exsC, exsB, exsA and exsD genes of Pseudomonas aeruginosa followed by homologues of the Yersinia enterocolitica and P. aeruginosa type III secretion genes yscB–L and pscB–L respectively. The yscL/pscL homologue in A. salmonicida subsp. salmonicida, ascL, appears to be the last gene of this type III secretion system as directly downstream of this gene we have identified a partial ORF displaying sequence similarity to the gene ruvC. [Homologues of ruvC are found in many eubacteria and encode RuvC, a nuclease that resolves Holliday junction intermediates during homologous recombination (Sharples et al., 1999).]. Relevant characteristics of the predicted A. salmonicida subsp. salmonicida type III secretion proteins based on the ORFs we have identified are shown in Table 3.



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Fig. 1. Genetic map of type III secretion genes found in A. salmonicida subsp. salmonicida strain JF2267. ORFs are indicated by grey arrows; grey boxes represent partial ORFs. Potential promoter sequences, represented by black arrowheads, have been identified upstream of genes exsC, exsA and exsD. Cut sites for the restriction endonucleases BamHI and PstI are indicated.

 

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Table 3. Characteristics of predicted A. salmonicida subsp. salmonicida type III secretion proteins and their percentage amino acid identity and similarity to homologues of other bacteria

 
A functional type III secretion system in A. salmonicida subsp. salmonicida prevents phagocytosis by peripheral blood leukocytes
To further study the role of the type III secretion system in A. salmonicida subsp. salmonicida virulence, we examined the interaction between A. salmonicida subsp. salmonicida and phagocytic cells. Two A. salmonicida subsp. salmonicida strains were chosen: a wt isolate, strain JF2267, and an isogenic mutant of strain JF2267 containing a mutation in the gene encoding AscV, an inner-membrane component of the type III secretion channel. Both A. salmonicida subsp. salmonicida strains were engineered to express green fluorescent protein and then used to infect peripheral blood leukocytes isolated from rainbow trout. Four hours p.i., the cells were fixed and examined using fluorescence microscopy. The wt A. salmonicida subsp. salmonicida strain, JF2267, was consistently observed outside the leukocytes, either free in the culture medium or adhered to the cell surface (Fig. 2A). In contrast, the {Delta}ascV mutant was most often found within the leukocytes, indicating that this mutant was phagocytosed (Fig. 2B). These results demonstrate that a functional type III secretion system enables A. salmonicida subsp. salmonicida to avoid uptake by phagocytic cells. A similar role has been assigned to the type III secretion system recently identified in Aeromonas hydrophila AH-1 (Yu et al., 2004).



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Fig. 2. Interaction between peripheral blood leukocytes and A. salmonicida subsp. salmonicida. Fluorescence micrographs of primary leukocytes taken 4 h following inoculation with A. salmonicida subsp. salmonicida wt strain (A) and the isogenic {Delta}ascV mutant (B). Bars, 10 µm.

 
The A. salmonicida subsp. salmonicida type III secretion mutant has attenuated virulence in rainbow tout
Virulence of the wt A. salmonicida subsp. salmonicida isolate, strain JF2267, was compared to that of the isogenic {Delta}ascV mutant in vivo using a rainbow trout model. The results (Fig. 3) showed that in fish injected i.p. with 1x105 c.f.u. of the wt strain, mortality was first seen at 4 days p.i. The dead fish presented with macroscopic alterations typical of acute furunculosis including internal haemorrhages and a swollen vent. Within 5 days p.i., mortality occurred in each of the four tanks containing fish that had been injected with this strain. Cumulative mortality increased to 85±13 % by day 9 p.i. and reached 88±13 % by day 16 (Fig. 3). In contrast, there was no mortality in fish infected with the {Delta}ascV mutant or in any of the control fish injected with PBS only. All dead fish from each of the four tanks containing fish injected with the wt strain were investigated for the presence of A. salmonicida subsp. salmonicida. The bacterium could be re-isolated from the liver and kidneys of each of these fish. In contrast, no bacteria could be isolated from the internal organs of fish infected with the {Delta}ascV mutant.



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Fig. 3. Cumulative mortality of rainbow trout injected i.p. with A. salmonicida subsp. salmonicida wt ({circ}) or {Delta}ascV mutant ({bullet}), or with PBS (control, {square}). Four replicates, each containing 10 fish per tank, were carried out for every bacterial strain tested. Bar lines indicate standard deviation between replicates.

 
Attenuation of the A. salmonicida subsp. salmonicida {Delta}ascV mutant correlates with histological findings
To further characterize the virulence of both the wt and {Delta}ascV A. salmonicida subsp. salmonicida strains, experimental infection of rainbow trout was carried out by i.m. injection with 5x104 c.f.u. per fish. Within 4 days p.i., all fish injected with the wt isolate, strain JF2267, were dead or moribund. Macroscopic examination of these fish revealed an enlarged spleen and dilated intestines. Petechiae were observed in the gills and muscle tissue. Extensive tissue necrosis and liquefaction were also seen in the muscle tissue surrounding the injection site (data not shown).

Histological examination was carried out on the internal organs and muscle tissue of the infected fish. In fish that were injected with the wt isolate, strain JF2267, extensive tissue damage and large aggregates of bacteria could be seen in all organs examined, including the kidney, liver and heart (Fig. 4A, C and E), indicating that this strain established a systemic infection. Tissue from the posterior kidney revealed focal to coalescing necrosis of the interstitial tissue and, to a lesser extent, necrosis of single glomeruli and tubuli (Fig. 4A). In the liver, sinusoids were distended and partially filled with granular, eosinophilic material (oedema fluid) (Fig. 4C). Many cells with pycnotic nuclei were observed randomly distributed throughout the liver tissue (Fig. 4C). The lymphoid tissue of the spleen was severely depleted and foci of necrosis could be seen. The mucosa of the intestine was multifocally or generally necrotic. The heart muscle displayed a massive separation of muscle fibres due to oedema (Fig. 4E) and focal to coalescing myolysis could be seen in the body muscle tissue. In contrast, none of the fish that were infected with the {Delta}ascV A. salmonicida subsp. salmonicida mutant displayed signs of disease at 4 days p.i. Histological examination revealed no pathological changes in any organ (Fig. 4B, D and F) and no bacterial cells were seen in the internal organs or muscle tissue surrounding the initial injection site. Furthermore, we were unable to re-isolate the bacterium from the internal organs of these fish. These findings indicate that the type III secretion system of A. salmonicida subsp. salmonicida is required by the bacterium to establish a systemic infection.



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Fig. 4. Histological sections of rainbow trout organs. Haematoxylin and eosin stained tissue sections of the kidney (A, B), liver (C, D) and heart (E, F) from fish injected with A. salmonicida subsp. salmonicida wt strain (A, C, E) and {Delta}ascV mutant (B, D, F). Large aggregates of bacteria can be seen in the organs of fish infected with the wt strain (black arrows). In the kidney (A), extended necrosis of interstitial tissue is evident (black arrowheads). Necrosis of single tubuli (white arrowhead) and alteration of glomeruli (white arrow) can also be seen (A). In the liver (C), there are randomly distributed cells with pycnotic nuclei (black arrowheads) and enlarged sinusoid spaces (white arrowheads). In the heart (E), spaces between muscle fibres are widened (black arrowhead). Bars, 50 µm.

 
Identification of type III secretion genes in atypical A. salmonicida strains and other aeromonad fish isolates
The results obtained from the experimental infections clearly indicate that type III protein secretion is required for virulence of A. salmonicida subsp. salmonicida strain JF2267. Therefore, we were interested to ascertain if atypical A. salmonicida isolates, which are increasingly being associated with disease outbreaks in both wild and cultivated fish (Gudmundsdóttir, 1998), also possess genes involved in type III protein secretion. Ten atypical A. salmonicida isolates, including all atypical type strains, were screened for two well-conserved type III secretion genes, ascV and ascU (Burr et al., 2002, 2003), by dot blot hybridization. We also included in the assay a number of other Aeromonas species isolated from fish and from water environments. In accordance with the results of Stuber et al. (2003), we considered that a positive signal for ascV during dot blot hybridization indicated the presence of a type III secretion gene cluster. The second gene probe, hybridizing to the gene ascU, was used to confirm this result. The results, shown in Table 2, indicate that these genes are present in 9 of the 10 atypical A. salmonicida strains (strains not belonging to the subspecies salmonicida) examined. Therefore, type III secretion genes are present in atypical A. salmonicida isolates from a variety of fish species. Although the atypical A. salmonicida display a wide range of virulence characteristics and are able to cause disease in both salmonid and non-salmonid hosts, the results suggest that type III protein secretion may also play a role in the virulence of these isolates.

Finally, type III secretion system genes were also found in fish isolates of Aeromonas encheleia, Aeromonas eucrenophila, Aeromonas hydrophila, Aeromonas media, Aeromonas sobria and Aeromonas veronii (Table 2). This is believed to be the first time such genes have been found in A. enceleia, A. eucrenophila and A. media, and in A. veronii isolates of non-human origin. In addition to finding ascV and ascU in such Aeromonas species isolated from fish, we also detected these genes in four isolates from water sources.

Conclusion
The type III secretion system of A. salmonicida subsp. salmonicida has been shown to be an important virulence factor. It enables the bacterium to avoid uptake by peripheral blood leukocytes, induce alterations in the internal organs and muscle tissue of the fish host and establish systemic infection. Furthermore, our findings suggest that type III secretion systems may also be important in the virulence of atypical A. salmonicida and other aeromonad fish pathogens.


   ACKNOWLEDGEMENTS
 
We are grateful to Brian Austin, Antonella Demarta, Bjarnheidur Gudmundsdóttir and William Kay for the gift of strains, to Bernd Köllner for providing us with antibodies and to Shelia MacIntyre for providing us with plasmid pSUP202sac. We also thank Heike Schmidt for her expertise with the histological analysis. This work was supported by the Swiss National Science Foundation, grant no. SNF Nr 3100A0-101595.


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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
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Received 28 January 2005; revised 2 March 2005; accepted 7 March 2005.



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