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National Research Council of Canada Institute for Marine Biosciences, 1411 Oxford Street, Halifax, Nova Scotia, Canada
Correspondence
A. Dacanay
andrew.dacanay{at}cnrc-nrc.gc.ca
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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ascC mutant strain was avirulent by both intraperitoneal (i.p.) injection and immersion, did not appear to establish a clinically inapparent infection and did not confer protection from subsequent rechallenge with the parental strain. 1H NMR spectroscopy-based metabolite profiling of plasma from all fish showed significant differences in the metabolite profiles between the animals exposed to the parental strain or ascC. The experimental infection by immersion with aopO was indistinguishable from that of the parental strain, that of aexT was delayed, whilst the virulence of aopH was reduced significantly but not abolished. By i.p. injection, aexT, aopH and aopO caused an experimental disease indistinguishable from that of the parental strain. These data demonstrate that while the TTSS is absolutely essential for virulence of A. salmonicida subsp. salmonicida in Atlantic salmon, removal of individual effectors has little influence on virulence but has a significant effect on colonization. The ascC i.p. injection data also suggest that in addition to host invasion there is a second step in A. salmonicida pathogenesis that requires an active TTSS.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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-Proteobacteria group. It is the aetiological agent of furunculosis, an infectious bacteraemia of salmonid fish. Many fundamental aspects of the host–pathogen relationship between A. salmonicida subsp. salmonicida (hereafter referred to as A. salmonicida) and its salmonid hosts remain poorly understood. Many proteins and systems in A. salmonicida have been implicated in virulence including the S-layer (vapA; Trust et al., 1983
), siderophores and their receptors (fstC, fstB, hupA; Ebanks et al., 2004), superoxide dismutase (sodA, sodB; Barnes et al., 1996; Garduño et al., 1997; Dacanay et al., 2003) and extracellular toxins such as glycerophospholipid : cholesterol acetyltransferase (GCAT) and the serine protease AspA (Salte et al., 1992). Despite the presence of multiple virulence systems, until recently no single system appeared to contribute significantly to virulence, as shown by the retention of virulence by strains deficient in any given system (Ellis et al., 1988; Olivier, 1990; Vipond et al., 1998; Fernandez et al., 1998). A type III secretion system (TTSS) in A. salmonicida has been described recently (Burr et al., 2002, 2003a; Stuber et al., 2003) and appears to be the exception to this rule (Burr et al., 2003b, 2005).
The TTSSs of pathogenic Gram-negative bacteria utilize a transmembrane injection apparatus composed of integral membrane proteins and a needle-like structure to translocate a range of effector proteins from the cytosol directly into host cells. The best-characterized TTSS systems are those of the pathogenic yersiniae (Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica), which consist of at least six effectors in addition to the inner-, outer- and target cell-transmembrane pores. Secreted effectors act directly upon intracellular signalling pathways by targeting proteins such as Rho or Rac. The downstream effects include modulation of phagocytosis and inhibition of paracrine signalling (Cornelis & Wolf-Hanz, 1997; Hueck, 1998), allowing the bacteria to modulate innate and acquired immune responses. In addition to the pathogenic yersiniae, the TTSS is a virulence factor for many pathogenic bacteria including Pseudomonas aeruginosa, Shigella flexneri, Salmonella enterica serovar typhimurium, enteropathogenic Escherichia coli (reviewed by Hueck, 1998) and Aeromonas hydrophila AH-1 (Yu et al., 2004). In common with other bacteria, the A. salmonicida TTSS consists of bacterial inner- and outer-membrane secretory pores, a host-cell translocation pore and a number of effector molecules. Unlike the yersiniae, where the TTSS is carried on a single 70 kb plasmid (pYV), the various genes of the TTSS of A. salmonicida are carried both on plasmids and chromosomally (Burr et al., 2002; Stuber et al., 2003). Two laboratory-derived TTSS-deficient strains of A. salmonicida JF2267 have been described as avirulent in a rainbow trout (Oncorhynchus mykiss) challenge model. One strain was deficient in the 140 kbp plasmid that carries the TTSS system. The second was a knockout mutant strain in ascV, the orthologue of Yersinia yscV, which forms part of the inner bacterial membrane pore (Burr et al., 2002, 2005).
In this study we created deletion mutant strains in the genes of the outer bacterial transmembrane pore and three TTSS effector genes of A. salmonicida strain A449. In addition to conventional methods for assessing effects on virulence of the bacterium in one of its natural hosts, the Atlantic salmon (Salmo salar), we also used metabolite profiling (metabonomics) to examine the host response to infection by A. salmonicida.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. The parental strain for all knockouts was Aeromonas salmonicida strain A449 (hereafter abbreviated to A449). All A. salmonicida strains were grown in tryptic soy broth (TSB) or agar (TSA) (Difco) at 17 °C with shaking. Escherichia coli strains were grown in Luria–Bertani (LB) broth or agar at 37 °C. Antibiotics were used at the following concentrations: E. coli, 100 µg ampicillin ml–1; A. salmonicida, 50 µg ampicillin ml–1; 20 µg chloramphenicol ml–1.
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). Genomic DNA was isolated from A449 using the PureGene DNA isolation kit (Gentra Systems). Oligonucleotides were prepared by Integrated DNA Technologies. PCR was performed with either rTaq (Amersham) or Pfu (MBI Fermentas) following the manufacturers' directions.
Construction of deletion mutant strains.
All four mutant strains were created by making in-frame, unmarked deletions in the relevant gene using crossover PCR (Link et al., 1997). PCR primers used are described in Table 2. Briefly, two self-complementary PCR fragments per gene were amplified from A449 chromosomal DNA. The two PCR fragments were mixed together and amplified with the two external primers to generate a large fragment encoding the relevant gene with a large internal deletion and flanking sequences. These fragments were cloned into the pir-dependent, sucrase expressing vector pWM91, which was conjugated into A449 from E. coli BW20767. Single crossover integrants were selected by plating on TSA supplemented with ampicillin and chloramphenicol. Double crossover mutants were isolated by selection on TSA with 15 % sucrose to select against plasmid-containing colonies.
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Challenge.
For the immersion challenge there were two tanks per group with 40 fish per tank. Fish were removed from the resident tank and placed in 
40 l aerated fresh water in a large plastic container to which 106 c.f.u. ml–1 A449, ascC, aexT, aopH or aopO had been added as well as anaesthetic (15 mg tricaine methanosulphonate l–1, Syndel Laboratories) to sedate the fish during the immersion. Bacterial doses were retrospectively confirmed by direct colony counts on TSA. After 30 min, the fish were removed and replaced in the resident tanks. Control fish experienced identical handling but were exposed to PBS only. For the intraperitoneal (i.p.) injection challenge, there were two tanks per group and 30 fish per tank. Fish were anaesthetized with 50 mg TMS l–1 until laterally recumbant and injected with 105 c.f.u. per animal in 100 µl PBS of either A449, ascC, aexT, aopH or aopO. The doses were confirmed by direct colony counts on TSA. Control animals received an equal volume of PBS only.
All fish were monitored closely post-exposure. Moribund and dead fish were removed immediately; moribund animals were killed with an overdose of TMS. The posterior kidney, in accordance with standard procedures for determining A. salmonicida infections (Schotts, 1994), was sampled onto TSA supplemented with 20 µg chloramphenicol ml–1.
Rechallenge.
Animals that had survived challenge with the parental strain or ascC were rechallenged with the parental strain. For the immersion challenge, the survivors were rechallenged 85 days after the initial immersion with 106 c.f.u. A449 ml–1 by immersion as before. For the i.p. challenge, survivors were rechallenged 43 days after the initial injection with 105 c.f.u. A449 per animal in 100 µl PBS by injection as before.
Plasma sampling.
At the termination of the rechallenge, the survivors were killed. At this time blood was drawn from the caudal vein into heparinized containers (Vacutainer, Becton-Dickinson). The erythrocytes were removed by centrifugation at 3000 g, the plasma was aliquoted and stored at –20 °C.
Stress test.
Sixty-six days after exposure by immersion and 14 days after the cessation of mortality, half of the surviving animals exposed to the parental strain, ascC and PBS-negative control groups were assessed for clinically inapparent infections by application of a stress test (adapted from Specker et al., 1994). Briefly, 100 mg cortisol (hydrocortisone, Sigma-Aldrich) in a vegetable oil/vegetable fat emulsion was administered by i.p. injection. This was followed by an increase in water temperature from 14 °C to 18 °C over 2 h, which was maintained for the remainder of the experiment. The remaining animals were left as unstressed controls. All moribund animals were processed as before. The stress test ceased after 10 days, at which time all surviving animals were killed with an overdose of TMS.
Statistics.
Statistical differences in cumulative morbidity between groups were assessed by the G-test (a modified 
2 test). Three indices were used to compare morbidity rates between groups: (a) survival curves were directly compared using the Mantel–Haenszel test; a P value <0.05 indicated the curves were significantly different; (b) calculation of hazard ratios, the ratio of deaths in the test group compared to the positive control group; and (c) calculation of the median survival time, the time in days to 50 % morbidity in each group. All tests except the G-test, which was calculated manually, were calculated using GraphPad Prism 3.0 (GraphPad Software).
1H NMR spectral acquisition.
Plasma samples from the rechallenge survivors were thawed. A 100 µl aliquot of each sample was mixed with 50 µl D2O for analysis in a 2.5 mm outer diameter (o.d.) tapered Wilmad 520-1A NMR tube. Proton nuclear magnetic resonance (1H NMR) spectra were acquired at 4 °C on a Bruker Avance-DRX 500 MHz spectrometer operating at 500.13 MHz using a 5 mm Bruker triple-axis gradient, triple-band inverse (TBI) probe. Three types of 1D 1H spectra with different pulse sequences were acquired for each sample, as for previous studies with salmon plasma (Solanky et al., 2005). The sequences were: presaturation (PS), WATERGATE (WG) and Carr–Purcell–Meiboom–Gill (CPMG). Conditions for data acquisition, processing and analysis were also as described by Solanky et al. (2005), apart from the use of 2.5 mm o.d. NMR tubes allowing spectra from smaller volumes of plasma (100 µl) to be acquired.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Three TTSS effectors were also identified. One, AexT, was chromosomally located, had 100 % amino acid identity to a previously characterized translocated ADP-ribosylating cytotoxin from A. salmonicida strain JF2267 and had similarity to exoT of P. aeruginosa (Braun et al., 2002, 2003b). The other two, located on plasmid pAsa5, were termed aop (Aeromonas outer protein) H and aopO, with similarity to Yersinia yopH and yopO/ypkA, respectively (Table 3). Neither aopH nor aopO are located within a cluster of genes encoding the TTSS apparatus, which is also located on pAsa5 (Table 3).
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Immersion challenge
Immersion with 106 c.f.u. ml–1 A. salmonicida strain A449 or the isogenic mutant strains aexT, aopH and aopO caused an experimental infection in Atlantic salmon that started at 7 days after immerson for aopO, 14 days for aopH and A449 and 15 days for aexT. Morbidity ceased 60 days after challenge (data not shown). A449 or the appropriate isogenic mutant strain was isolated from the posterior kidney of all morbid and dead animals. Cumulative morbidity, hazard ratio and median survival time data for the immersion challenge are reported in Table 4. There was no A. salmonicida-related morbidity in groups exposed to ascC or PBS.
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aopO and the parental strain. The median survival time for animals exposed to aopO was 29 days, 2 days longer than for the parental strain. The hazard ratio was 0.99, indicating that the morbidity rates between the two strains were essentially the same. Similarly, there were no significant differences in cumulative morbidity between the parental strain and aexT (47.5 %; G-test, P=0.0559) and the survival curve was not significantly different from that of the parental strain (Mantel–Haenszel test; P=0.14). However, the median survival time was 39 days, 12 days longer than that for the parental strain and the hazard ratio was 1.37, indicating that the morbidity rate for the parental strain was 1.4 times that of aexT. There was significantly lower morbidity with aopH (35.0 %; G-test, P=0.0008) compared to the parental strain. The survival curve was also significantly different (Mantel–Haenszel; P=0.02). This difference was reflected in both a median survival time of 49 days, 22 days longer than the parental strain, and a hazard ratio of 1.68, showing that the morbidity rate for the parental strain was 1.68 times greater than for aopH.
Intraperitoneal challenge
Intraperitoneal (i.p.) injection with 105 c.f.u. per animal of either A449 or the isogenic mutant strains aexT, aopH or aopO caused an experimental infection that started for all strains 3 days after injection and ceased after 21 days (data not shown). A449 or the appropriate isogenic mutant strain was isolated from the posterior kidney of all morbid and dead animals. Cumulative morbidity, hazard ratio and median survival for the i.p. challenge are shown in Table 4
. Again there was no A. salmonicida-related morbidity in groups exposed to ascC or PBS.
There was high morbidity in the group injected with the parental strain (76.3 % cumulative morbidity). There were no significant differences in morbidity (G-test; P>0.05) between the parental strain and any of the TTSS effector mutant strains: aexT (71.0 % cumulative morbidity), aopH (67.2 %) and aopO (84.9 %). The survival curves were not significantly different (Mantel–Haenszel test; P>0.05). The hazard ratio was 0.9 for all three mutant strains when compared to A449 and the median survival time was equal at 5 days post-challenge.
Stress test
Clinically inapparent (covert) A. salmonicida infection levels were assessed by a stress test: the application of the twin stressors of an increase in water temperature from 14 °C to 18 °C and injection of 100 mg cortisol per animal (Specker et al., 1994). In a parallel experiment with the immersion challenge, Atlantic salmon were exposed by immersion to A449, PBS or ascC and stress tested 66 days after the initial exposure. Ten days after the stress test started there was no A. salmonicida-related morbidity in either the PBS- or ascC-exposed groups. There was 85 % A. salmonicida-related morbidity in the A449 group and the median survival was 7 days.
Rechallenge
We tested whether prior exposure to the avirulent ascC strain conferred protection from subsequent challenge with the virulent parental strain. Survivors from both the i.p. and immersion challenges were rechallenged with A449; data from these rechallenges are shown in Table 5. When rechallenged with A449 by immersion 85 days after an immersion exposure to A449, ascC or PBS, the latter two groups experienced high morbidity; PBS (57.5 % cumulative mortality) and ascC (57.9 %). Median survival was 15 days for both groups. Morbidity in the group initially exposed to A449 (4.2 % cumulative morbidity) was significantly lower than in either the PBS or ascC group (G-test; P<0.0001).
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ascC by injection showed high morbidity upon rechallenge with the parental strain. Animals initially exposed to the parental strain showed some protection from rechallenge, as cumulative morbidity was significantly reduced compared to that of the PBS group (28.6 %; G-test; P=0.005).
Principal components analysis (PCA) of 1H NMR spectra of plasma
Metabolite profiles from plasma samples drawn from survivors of the immersion and rechallenge were compared by 1H NMR and PCA. Samples were (I) exposure to PBS/re-exposure to PBS (naïve controls, n=29); (II) immersion challenge with A449/survivors rechallenged by immersion with A449 (n=18); (III) exposure to PBS/rechallenge by immersion with A449 (n=9); (IV) exposure by immersion to ascC/rechallenge by immersion with A449 (n=8) and (V) exposure by immersion to ascC only (n=21). PS, WG and CPMG spectra were recorded, giving three independent datasets, which were analysed separately by PCA (Aries et al., 1991; Eriksson et al., 1999). The spectral data of the naïve control group (PBS/PBS; group I) were compared with the spectra from each of the four bacteria-exposed groups (II–V) by analyses of scores plots (Fig. 1a–f). Fig. 1(a–d) shows the scores plots for PS spectra from groups II, III, IV and V compared to group I. The scores plots show clearly that the metabolite profiles of plasma from the A449/A449 group (group II) clustered together, occupying a distinct region separate from that of the PBS/PBS group (Fig. 1a), showing that the metabolic response to infection of the A449/A449 group was significantly different from that of naïve controls. The spectral profiles of groups III, IV and V clustered with those of the naïve group (Fig. 1b–d), indicating there was no difference in the metabolite profiles for these groups. Similar data were obtained after analysis of the WG and CPMG spectra; scores plots showing the comparison of the A449/A449 and PBS/PBS groups are shown in Fig. 1(e, f).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), the roles performed by individual effectors or the TTSS itself are not necessarily conserved (Hueck, 1998). To investigate the contribution of the TTSS in the virulence of A. salmonicida, deletion mutations were created in the outer-membrane pore gene, ascC, and three effectors aexT, aopH and aopO. Atlantic salmon, one of the natural hosts of this bacterium, were exposed to these strains by either immersion or i.p. injection. Animals that survived the initial infection were later assessed for a clinically inapparent (covert) infection or for protection from a subsequent challenge with the parental strain.
In accordance with the reports of others (Burr et al., 2002, 2005; Yu et al., 2004), the outer-membrane pore of the secretion apparatus was clearly required for A. salmonicida virulence. The ascC strain caused no morbidity when administered either i.p. or by immersion (Table 4). This was likely due to the inability of this strain to release effectors, since AscC is critical for secretory apparatus assembly and yscC knockouts completely block TTSS secretion in Y. enterocolitica (Koster et al., 1997) and Y. pestis (Plano & Straley, 1995). The avirulence of ascC may reflect the inability of this strain to resist phagocytosis; secretion-apparatus-deficient mutants are reported to be more readily phagocytosed than their respective parental strains (Burr et al., 2005; Yu et al., 2004).
The precise site of infection during the clinically inapparent infection state remains unknown but most workers recognize that it is due to the colonization of an outer surface of the fish by A. salmonicida (Hiney et al., 1997). The stress test apparently showed ascC was not capable of establishing such an infection. Whereas this should be prima facie evidence for a significant role for the TTSS of A. salmonicida in colonization, the inability of ascC to cause clinical disease even by direct injection suggests that it would have been equally unable to cause disease during the stress test. Therefore as overt disease is the ‘positive’ outcome for the stress test, these data cannot be used as evidence that the TTSS is required for host colonization.
Prior exposure to ascC by either immersion or injection did not confer protection from rechallenge. In an attempt to understand why, we assayed plasma from the rechallenge survivors. Analysis of specific anti-A. salmonicida immunoglobulin by ELISA was inconclusive (data not shown). Metabonomic analysis was also performed on these plasma samples. Metabonomics uses PCA of 1H NMR spectra to qualitatively and quantitatively compare metabolites in biofluids from, in this case, control and challenged individuals. The data, when presented as scores plots (Fig. 1a–f), where each spectrum is represented by a single point (for more details see Eriksson et al., 1999; Solanky et al., 2005), show that similar spectra cluster together whilst dissimilar spectra do not. We have shown recently that metabonomic analysis of plasma can discriminate between infected and uninfected Atlantic salmon following exposure to A. salmonicida (Solanky et al., 2005). The use of three different spectroscopic conditions provided three independent datasets for each sample. PS and WG use different means to suppress the H2O signal whilst CPMG suppresses broad signals from high-molecular-mass compounds in order to highlight otherwise superimposed sharp signals from low-molecular-mass metabolites. The plasma metabolite profiles correlated with a protective immune response. The A449/A449 group showed clear protection during the rechallenge and its metabolic response clustered distinctly from the naïve group (PBS/PBS). The metabolic responses of the other challenged groups (groups III, IV and V) were not distinguishable from the naïve group nor were any of these groups protected from rechallenge. Thus neither the rechallenge nor metabonomic data shows evidence of acquired immunity in Atlantic salmon in response to exposure to ascC.
This is believed to be the first study that has investigated the in vivo behaviour of A. salmonicida TTSS effectors by conducting animal challenges with mutants deleted in three effector genes. A. salmonicida TTSS effectors are poorly characterized compared with those of other systems; only the functionality of AexT has been studied in any detail (Braun et al., 2002, 2003b, 2005). The in vivo behaviour of the three effector mutant strains mimicked the behaviour of a suite of effector mutant strains created in Y. enterocolitica O : 8 (Trülzsch et al., 2004). Both yopO and yopE mutant strains of Y. enterocolitica O : 8 were capable of colonizing mice and causing an overt disease following oral challenge; yopO behaved as wild-type and was lethal whereas yopE was able to colonize mice but did not cause an overt disease and was eventually cleared. Virulence was abolished in the yopH mutant strain. Similar findings are reported for Y. pseudotuberculosis TTSS effectors (Logsdon & Mecsas, 2003). In this study both aexT and aopO behaved as the parental strain; there was only a subtle reduction in the virulence of aexT. Whereas virulence was significantly reduced in aopH, it was not abolished.
YopE and YopO share several intracellular targets, none of which are shared by YopH (Aepfelbacher, 2004). If the target ranges of the A. salmonicida effectors are similar, then the differing effects on virulence of the TTSS effector mutant strains may be explained. The absent/subtle effects on virulence in aexT and aopO in contrast to a more obvious effect on virulence in aopH suggest that AopO and AexT either share intracellular targets or target the same process(es); thus in aexT and aopO the presence of one effector complemented the absence of the other. Further study is required to confidently ascribe definitive targets to A. salmonicida TTSS effectors.
Unlike deletion of secretory apparatus genes, deletion of individual TTSS effector genes in A449 lessened, but did not abolish, virulence. This may be due to the presence of other effectors in A449. A. salmonicida strain JF2267 is reported to carry a fourth effector, AopP/J, not present in A449 and neither strain appears to carry YopT or YopM orthologues (Burr et al., 2002, 2003a; M. Reith, unpublished data). Even though the full genomic sequence of A449 is available, the presence of other effectors in this strain cannot be discounted.
Previous reports on the effects of the TTSS on aeromonad virulence in two species of fish assessed the virulence of TTSS-deficient mutant strains by injection (Burr et al., 2002, 2005; Yu et al., 2004). As the TTSS mediates host invasion in some species of bacteria, injection may not accurately assess the role of the TTSS in virulence. In this study bacterial virulence was assessed as the ability of the bacteria to cause disease when administered by either immersion or i.p. injection.
Attempts to quantify invasion of A449 and its isogenic TTSS mutant strains by serial sampling of tissues from apparently healthy animals after immersion were unsuccessful (data not shown). However, the challenge data suggest strongly that the TTSS of A. salmonicida is required for host invasion. By immersion, a route of administration that requires host invasion to establish an overt disease state (Cardella & Eimers, 1990; Nordmo & Ramsted, 1997), an inactive secretory complex completely abolished virulence. The level of overt disease caused by aopH was significantly reduced and aexT was also attenuated. Full virulence was restored to these effector mutants when they were administered by injection, a route that does not require invasion. The apparent inability of the ascC mutant to invade would explain the absence of involvement of the host's acquired immune system in the response to this strain.
Unlike the effector mutant strains, virulence was not restored to ascC by i.p. injection, suggesting that there is a second step in the pathogenesis of furunculosis that requires an active TTSS after host invasion. This is likely to be either TTSS-mediated cytotoxicity (Burr et al., 2003b), which in A. salmonicida has been shown to be contact mediated (Olivier et al., 1992), or a secondary invasive step such as macrophage residence as suggested by Garduño et al. (2000).
Very little is known on the portal of entry for A. salmonicida in natural infections. Work on the covert infection state has shown that prior to invasion and progression to an overt disease state A. salmonicida resides on an as-yet-unidentified exterior structure of the fish (Hiney et al., 1997). Furthermore, the pathognomic clinical sign for furunculosis is a focal dermomyonecrotic lesion (furuncle) that arises from dermally, rather than more deeply located A. salmonicida microcolonies (Bernoth, 1997; Roberts & Rodger, 2001). The requirement of the TTSS of A. salmonicida for invasion and possibly colonization of exterior surfaces prior to invasion is consistent with this.
This study has also revealed that A. salmonicida pathogenesis is a more complex process than it initially appears. Further investigation of both the bacterial virulence factors and host immune responses is required to better understand this disease.
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ACKNOWLEDGEMENTS |
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Received 13 December 2005;
revised 6 March 2006;
accepted 7 March 2006.
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)with group II (A449/A449, a) (
); with group III (PBS/A449, b) (
); with group IV (
); and with group V (
). Group II forms a distinct cluster from group I, whereas groups III, IV and IV form clusters that are indistinguishable from group I. Panels (e) and (f) show similar clustering results for group II versus group I, obtained with WG and CPMG spectral data respectively. Each symbol represents a single individual. All panels: abscissa, PC1; ordinate, PC2.