BACKGROUND
aeromonas salmonicida, the causative agent of furunculosis in salmonid fish, is a non-motile, gram-negative bacterium, and one of the most studied bacterial pathogens of fish.
furunculosis is an important disease in wild and cultured stocks of fish with the potential for severe negative economic impact.
the canadian aquaculture industry alliance estimated the total direct and indirect costs of infectious diseases within the canadian aquaculture industry at over $ <dig> m annually, with furunculosis contributing approximately $ <dig> m annually to these losses  <cit> .
a. salmonicida is not limited to salmonids and many species of fish are affected.
several excellent reviews of a. salmonicida and furunculosis are available  <cit> .
although bergey's manual of systematic bacteriology  <cit>  recognizes five subspecies of a. salmonicida: salmonicida, achromogenes, masoucida, smithia, and pectinolytica, many laboratories currently classify a. salmonicida subsp.
salmonicida as "typical" and any isolate deviating phenotypically as "atypical".
morphological and biochemical differences are used to distinguish typical and atypical isolates.
these are pigment production, colony size and growth rate, haemolysis, and sucrose fermentation  <cit> .
a. salmonicida subsp.
salmonicida  isolates grow well on blood agar with large colonies, produce brown, diffusible pigment, are haemolytic and do not ferment sucrose.
historically, typical strains are thought to be extremely homogenous  <cit> , and therefore any deviation in any of these characteristics has been considered enough evidence to classify a strain as "atypical"  <cit> .
however, some of these parameters are subjective, and are largely based on the experience of the operator/microbiologist, possibly leading to misclassification.
molecular techniques are not yet used on a regular basis in many laboratories that type a. salmonicida.
although typical isolates are generally phenotypically homogenous some variation does exist  <cit> , particularly with regard to the production of hemolysin and degradation of casein.
antibiotic resistance patterns have shown potential as epizootiological markers in specific geographical origins.
typing schemes based on biochemical and other phenotypic methods are dependent on many factors, including plastic bacterial phenotypes and the often low consistency of inter-laboratory testing  <cit> .
genetic analysis provides a more stable basis for microbiological investigation than phenotypic methods.
several molecular techniques, including finger-printing by randomly amplified dna polymorphism   <cit> , amplified fragment length polymorphism   <cit> , pulsed-field gel electrophoresis   <cit> , restriction enzyme fragmentation patterns   <cit> , plasmid profiling  <cit> , and ribotyping  <cit> , have been used to study different strains of a. salmonicida.
while the results generally support the phenotypical evidence that typical strains are genetically homogenous and may well be clonal, and that atypical isolates are heterogeneous, there is little congruence between the techniques in establishing relationships between subspecies, strains and isolates.
austin et al.
<cit>  showed that among  <dig> isolates of atypical a. salmonicida taken from a wide host and geographical range, there was no agreement between pcr, rapd, ribotyping, or phenotypic typing methods  <cit> .
the use of genome arrays containing whole genomes or large sets of genes, either as a result of high-coverage genome sequencing or selected after suppressive subtractive hybridization have been used to study genome variability among strains of bacterial pathogens, including campylobacter jejuni  <cit> , flavobacterium psychrophilum  <cit> , listeria monocytogenes  <cit> , and burkholderia species  <cit> .
to examine the genomic diversity of virulence factors among clinical isolates of a. salmonicida and other aeromonads, we constructed a dna microarray of  <dig> selected virulence-related genes from a. salmonicida subsp.
salmonicida strain a <dig>  a <dig> is a wild-type typical virulent clinical isolate which has been sequenced and it is currently undergoing annotation.
we are undertaking a research program to examine the molecular mechanisms of interaction between a. salmonicida and atlantic salmon .
the applied goal of this program is to develop vaccines, vaccine delivery systems and other health management tools.
in order to develop vaccines we must first identify virulence-associated genes as potential vaccine candidates, the process known as "reverse vaccinology"  <cit> .
accurate typing is important in searching for vaccine candidates if there are known differences in pathogenicity of the sub-species, or if there is variation between the degree of conservation of virulence genes within each subspecies.
although all subspecies of a. salmonicida have been implicated in clinical cases in fishes, aeromonas salmonicida subsp.
salmonicida is more prevalent within salmonids and "typical" isolates are more often associated with disease outbreaks in salmon  <cit> .
moreover, there are indications that there are differences in the susceptibility and immune response of salmonids and other fishes to "atypical" isolates  <cit> .
therefore it is essential that any characterization or typing schemes be accurate, if health management strategies are to be based, even partly, on those schemes.
we used m-cgh to study the genetic relationships between aeromonas species, subspecies and strains based on gene conservation profiles and to examine genomic diversity.
we compared strains and isolates from selected geographic areas and host species to explore correlations between geographic or host origin and conservation or diversity of genes.
these strains were selected to provide a diverse sample of clinical and laboratory isolates from the differing geographic regions and host species infected.
our goal was to identify a set of virulence-associated genes conserved across these strains to consider for future consideration as vaccine candidates.
we hypothesized greater variability of these virulence genes between subspecies and no correlation between the "typical" and "atypical" classifications and gene variability.
we also hypothesized positive correlation between the degree of variability and the functional category of the variable genes involved.
RESULTS
comparative genomic hybridization  profiles for each of the aeromonas isolates listed in table  <dig> were obtained by competitively hybridizing labeled genomic dna  from the relevant isolate and from the a <dig> control strain to an a. salmonicida microarray comprising  <dig> putative virulence genes, selected from a draft genome sequence of the wild type strain a. salmonicida subsp.
salmonicida a <dig>
hierarchical clustering of the seventeen aeromonas isolates based on the overall variability in the m-cgh data is represented by the dendrogram in figure 1a.
as expected, the ten a. salmonicida subsp.
salmonicida strains and the control strain a <dig> clustered together.
one atypical strain  also clustered in this group.
there is very strong support  for the clustering of the eleven a. salmonicida subsp.
salmonicida strains with the atypical strain n <dig>  but much lower bootstrap values for most branches within this main cluster.
figure 1b shows hierarchical clustering of the seventeen aeromonas samples based only on "chromosomal" genes, after genes assigned to the plasmid and transposon functional categories were removed.
the ten a. salmonicida subsp.
salmonicida strains cluster with a <dig> and the atypical strain n <dig>  in each dendrogram, as expected, a. bestiarum and a. caviae showed the most divergence from the main cluster, followed by a. salmonicida subsp.
masoucida and a. salmonicida subsp.
achromogenes, consistent with the species/subspecies boundaries.
it is interesting to note that one of the a. salmonicida atypical samples  clustered with a. salmonicida subsp.
achromogenes, whereas the second atypical strain  clustered with the a. salmonicida subsp.
salmonicida a <dig> control strain.
these results suggest that neither of these should be considered a. salmonicida atypical strains.
the m-cgh data for the sixteen aeromonas samples ordered with hierarchical clustering of the genes is shown in figure  <dig>  genes were considered to be conserved if the signal differed by less than a factor of two between the tester and the control , as described by taboada et al.,  <dig>  <cit> .
the lower cutoff of - <dig>  is fairly conservative as we have shown previously that it encompasses over  <dig> % of highly conserved genes but less than 1% of missing genes  <cit> .
to highlight the variable genes, the log ratio value was set to  <dig>  for conserved genes, and genes which were conserved in all of the samples have been removed.
genes with log ratio values less than - <dig>   were considered to be divergent in sequence or absent from the tester strain, while those with log ratios values greater than + <dig>   were expected to be present in higher copy number in the tester than in the control strain.
as shown in figure 2a, clusters with the highest variability across the samples correspond to plasmid- and transposon-associated genes.
as expected, a. bestiarum and a. caviae have higher numbers of variable genes than the a. salmonicida strains.
figure 2b shows the subset of genes that are not associated with transposons or plasmids but are divergent in at least one of the fourteen a. salmonicida samples.
these divergent "chromosomal" genes show some clustering of genes coding for outer membrane proteins and flagella/pili.
the number of genes divergent or absent according to the function categories assigned in table  <dig> and by species/strain is presented in table  <dig>  the highest degree of divergence compared to a <dig> can be seen for a. caviae and a. bestiarum, followed by the type strains of a. salmonicida subspp.
masoucida and achromogenes as well as one of the atypical samples .
there was much less variation between the a. salmonicida subsp.
salmonicida strains and the other a. salmonicida atypical strain .
of the a. salmonicida subsp.
salmonicida strains, n <dig>  isolated from turbot, and ss <dig> , the only isolate from a pacific salmon species , showed the highest degree of variability among chromosomal genes.
these data notwithstanding, there does not appear to be a strong correlation between the level of genomic variability and either host or geographical origin.
the m-cgh data for genes associated with mobile elements  in the sixteen aeromonas samples is shown in figure  <dig>  hierarchical clustering of the plasmid-associated genes  shows clustering of highly variable genes matching a known plasmid of ~ <dig> kb .
a subset of genes on this plasmid matches type three secretion system  genes found in other bacteria, which is unsurprising considering that ttss genes can be plasmid-borne in a. salmonicida subsp.
salmonicida  <cit> .
we observed genomic variability consistent with the lower intensity signal for some plasmid genes, including ttss genes, for several strains .
of these, the strain ss <dig>  is an avirulent, laboratory-derived strain created by treating an a. salmonicida subsp.
salmonicida strain with ethidium bromide  <cit> .
this likely resulted in the loss of at least one plasmid , which would contribute to this observation.
on the other hand, many genes on plasmid  <dig> appear to be present in higher copy number in many of the tester strains than in a <dig>  figure 3b shows the m-cgh data for genes associated with transposons.
many of these genes appear to be highly divergent, present at lower copy number, or missing in various tester strains, and many of the genes match known transposases.
the number of variable genes assigned to each functional category is shown in figure  <dig>  as well as the percent of genes in each category that were observed to be variable.
within the function categories other than transposon or plasmid, most genes that appeared to be divergent or missing in various aeromonas strains are associated with transport, surface carbohydrate biosynthesis, outer membrane, and flagella/pili proteins.
in contrast, genes associated with secretion were most likely to be present in higher copy numbers in the tester strains compared to a <dig>  in summary, the highest degree of variability was seen in aeromonas genes associated with mobile elements: the plasmids or the transposons, whereas genes within the chromosome of the bacterium displayed the lowest level of variability.
discussion
in this study, patterns of variability of the  <dig> selected virulence-associated genes of a. salmonicida subsp.
salmonicida strain a <dig> within the sixteen various test isolates of a. salmonicida and other aeromonas species were examined.
it is noteworthy that genes divergent and absent in some tester strains seem to be distributed across the entire a <dig> genome sequence and across all the predicted functional categories whereas genes shown to be at higher copy number than in the reference strain were clustered in selected loci associated with plasmids.
the number of divergent and absent genes correlated strongly with species and subspecies boundaries.
for a. bestiarum and a. caviae ~16% of the genes were divergent or missing, whereas for the achromogenes and masoucida subspecies ~8% of the genes were divergent or missing.
the range across the various a. salmonicida subsp.
salmonicida strains was between  <dig> % and 2%, again reinforcing the single clone theory for this species.
overall, for the  <dig> a. salmonicida isolates studied, approximately 19% of the genes were divergent in at least one sample.
for comparison, our previous meta-analysis of m-cgh studies of  <dig> strains of c. jejuni showed ~33% of the genes in the genome were divergent in at least one strain  <cit> .
it is likely that as we examine more strains of a. salmonicida the number of divergent genes will increase.
one interesting finding is that almost 70% of the virulence-associated genes on the dna microarray are conserved across all the aeromonas species.
because more than 80% of these highly conserved genes appear to be chromosomal, this suggests that the divergence of a. bestiarum, a. caviae, and a. salmonicida may have occurred fairly recently.
the majority of a. salmonicida subsp.
salmonicida isolates studied were isolated from atlantic salmon and gave homogenous results, i.e., there is no correlation between host or geographic origin and m-cgh patterns in subsp.
salmonicida isolates.
highest variability amongst the subsp.
salmonicida isolates was observed in an isolate  from coho salmon and in an isolate  from turbot.
however, more isolates from pacific salmon should be analyzed before drawing definite conclusions on the variation between isolates from atlantic and pacific salmon.
the relatively low number of divergent genes in most of the a. salmonicida subsp.
salmonicida strains supports the findings of garcia et al.
who identified a single clone of a. salmonicida subsp.
salmonicida as responsible for most outbreaks of disease worldwide  <cit> .
differentiation of typical and atypical strains of a. salmonicida is important economically if it can be shown that there are significant differences in the degree of variation in the genes associated with virulence and pathogenicity, and which affect the immune response of the host.
if this is the case, then accurate typing methods and differentiation between typical and atypical strains and subspecies are required for diagnostic methods and for vaccine development.
lund et al.
have shown variation in the protective ability of some commercial furunculosis vaccines  against atypical strains infecting farmed spotted wolffish  <cit>  and halibut .
in this context, m-cgh provides an objective means to type clinical isolates based upon the presence, absence or divergence of a large number of genes.
phenotypical variation amongst subsp.
salmonicida isolates is rare but has been described  <cit> .
the increased resolution of m-cgh compared to phenotypic typing can be illustrated on consideration of strain n <dig> which was considered an atypical strain on phenotypical grounds.
however, both m-cgh and subsequent pulsed-field gel electrophoresis  suggest that this strain is very closely related to, and should perhaps more accurately be considered a typical strain.
similarly, atypical strain n <dig>  may be more accurately placed within the subspecies achromogenes based upon m-cgh results.
further comparative studies will be required to establish the level of strain resolution capable by m-cgh for aeromonads.
however, in the absence of complete genome sequence data for each aeromonas strain used, m-cgh, which in this study uses over  <dig>  separate genetic markers, represents a comprehensive, high- resolution methodology for comparing genome information.
recent work by our group used m-cgh to examine the quantitative relationship between the log ratio and probe/target identity, and these analytical processes were used in the study  <cit> .
it is preferable that vaccine candidates be strongly conserved among strains that cause disease.
the m-cgh data from these experiments will provide a list of conserved genes, sorted by function category, which can be further analyzed to generate these candidates.
for example, there are bioinformatics programs available to predict sub-cellular location  <cit>  to select candidates which may be exposed to the host immune system, or to predict adhesins  <cit> .
these downstream analyses, in combination with m-cgh analysis will reduce the number of genes required to be cloned in order to test the immune response of their encoded proteins.
these experiments are currently underway.
it must be noted that m-cgh does not reveal information about gene expression or the specific role of targeted genes in pathogenicity or host immune responses.
however, it is a powerful tool in reverse vaccinology and m-cgh is a sensitive and comprehensive technique that can determine genomic variation between pathogen strains, and it also can be used as a screening tool for target identification, and/or a typing method.
CONCLUSIONS
m-cgh is a powerful first screen for vaccine target identification, and is the basis of reverse vaccinology  <cit> , as it facilitates identification of conserved and duplicated genes associated with virulent strains.
this technique may also prove useful in strain typing and epidemiological studies.
comparative analysis of genomic data of a. salmonicida and related organisms reveals candidates common to all clinical isolates or those strains shown to be associated with virulence and disease.
our work on knock-out mutants of a <dig> virulence genes, combined with proteomic analysis of selected isolates confirm the results of our m-cgh analyses and will enable us to select specific antigens for vaccine development .
the findings reported here support our initial hypotheses that variability exists between subspecies, that there is no correlation between 'typical' and 'atypical' classifications and gene variability.
we also found that there is a positive correlation between the degree of variability and the functional category of the genes involved.
future work will include the use of microarray transcript profiling experiments to further focus the choice of target genes.
this will allow us to develop an ongoing, dynamic list of vaccine candidates to be used in live challenges for virulence and protection assays and then in further vaccine development.

 aeromonas salmonicida, the causative agent of furunculosis in salmonid fish, is a non-motile, gram-negative bacterium, and one of the most studied bacterial pathogens of fish. furunculosis is an important disease in wild and cultured stocks of fish with the potential for severe negative economic impact. the canadian aquaculture industry alliance estimated the total direct and indirect costs of infectious diseases within the canadian aquaculture industry at over $ m annually, with furunculosis contributing approximately $ m annually to these losses. a. salmonicida is not limited to salmonids and many species of fish are affected. several excellent reviews of a. salmonicida and furunculosis are available. although bergey's manual of systematic bacteriology recognizes five subspecies of a. salmonicida: salmonicida, achromogenes, masoucida, smithia, and pectinolytica, many laboratories currently classify a. salmonicida subsp. salmonicida as "typical" and any isolate deviating phenotypically as "atypical". morphological and biochemical differences are used to distinguish typical and atypical isolates. these are pigment production, colony size and growth rate, haemolysis, and sucrose fermentation. a. salmonicida subsp. salmonicida isolates grow well on blood agar with large colonies, produce brown, diffusible pigment, are haemolytic and do not ferment sucrose. historically, typical strains are thought to be extremely homogenous, and therefore any deviation in any of these characteristics has been considered enough evidence to classify a strain as "atypical". however, some of these parameters are subjective, and are largely based on the experience of the operator/microbiologist, possibly leading to misclassification. molecular techniques are not yet used on a regular basis in many laboratories that type a. salmonicida. although typical isolates are generally phenotypically homogenous some variation does exist, particularly with regard to the production of hemolysin and degradation of casein. antibiotic resistance patterns have shown potential as epizootiological markers in specific geographical origins. typing schemes based on biochemical and other phenotypic methods are dependent on many factors, including plastic bacterial phenotypes and the often low consistency of inter-laboratory testing. genetic analysis provides a more stable basis for microbiological investigation than phenotypic methods. several molecular techniques, including finger-printing by randomly amplified dna polymorphism, amplified fragment length polymorphism, pulsed-field gel electrophoresis, restriction enzyme fragmentation patterns, plasmid profiling, and ribotyping, have been used to study different strains of a. salmonicida. while the results generally support the phenotypical evidence that typical strains are genetically homogenous and may well be clonal, and that atypical isolates are heterogeneous, there is little congruence between the techniques in establishing relationships between subspecies, strains and isolates. austin et al. showed that among isolates of atypical a. salmonicida taken from a wide host and geographical range, there was no agreement between pcr, rapd, ribotyping, or phenotypic typing methods. the use of genome arrays containing whole genomes or large sets of genes, either as a result of high-coverage genome sequencing or selected after suppressive subtractive hybridization have been used to study genome variability among strains of bacterial pathogens, including campylobacter jejuni, flavobacterium psychrophilum, listeria monocytogenes, and burkholderia species. to examine the genomic diversity of virulence factors among clinical isolates of a. salmonicida and other aeromonads, we constructed a dna microarray of selected virulence-related genes from a. salmonicida subsp. salmonicida strain a a is a wild-type typical virulent clinical isolate which has been sequenced and it is currently undergoing annotation. we are undertaking a research program to examine the molecular mechanisms of interaction between a. salmonicida and atlantic salmon. the applied goal of this program is to develop vaccines, vaccine delivery systems and other health management tools. in order to develop vaccines we must first identify virulence-associated genes as potential vaccine candidates, the process known as "reverse vaccinology". accurate typing is important in searching for vaccine candidates if there are known differences in pathogenicity of the sub-species, or if there is variation between the degree of conservation of virulence genes within each subspecies. although all subspecies of a. salmonicida have been implicated in clinical cases in fishes, aeromonas salmonicida subsp. salmonicida is more prevalent within salmonids and "typical" isolates are more often associated with disease outbreaks in salmon. moreover, there are indications that there are differences in the susceptibility and immune response of salmonids and other fishes to "atypical" isolates. therefore it is essential that any characterization or typing schemes be accurate, if health management strategies are to be based, even partly, on those schemes. we used m-cgh to study the genetic relationships between aeromonas species, subspecies and strains based on gene conservation profiles and to examine genomic diversity. we compared strains and isolates from selected geographic areas and host species to explore correlations between geographic or host origin and conservation or diversity of genes. these strains were selected to provide a diverse sample of clinical and laboratory isolates from the differing geographic regions and host species infected. our goal was to identify a set of virulence-associated genes conserved across these strains to consider for future consideration as vaccine candidates. we hypothesized greater variability of these virulence genes between subspecies and no correlation between the "typical" and "atypical" classifications and gene variability. we also hypothesized positive correlation between the degree of variability and the functional category of the variable genes involved.  comparative genomic hybridization profiles for each of the aeromonas isolates listed in table were obtained by competitively hybridizing labeled genomic dna from the relevant isolate and from the a control strain to an a. salmonicida microarray comprising putative virulence genes, selected from a draft genome sequence of the wild type strain a. salmonicida subsp. salmonicida a hierarchical clustering of the seventeen aeromonas isolates based on the overall variability in the m-cgh data is represented by the dendrogram in figure 1a. as expected, the ten a. salmonicida subsp. salmonicida strains and the control strain a clustered together. one atypical strain also clustered in this group. there is very strong support for the clustering of the eleven a. salmonicida subsp. salmonicida strains with the atypical strain n but much lower bootstrap values for most branches within this main cluster. figure 1b shows hierarchical clustering of the seventeen aeromonas samples based only on "chromosomal" genes, after genes assigned to the plasmid and transposon functional categories were removed. the ten a. salmonicida subsp. salmonicida strains cluster with a and the atypical strain n in each dendrogram, as expected, a. bestiarum and a. caviae showed the most divergence from the main cluster, followed by a. salmonicida subsp. masoucida and a. salmonicida subsp. achromogenes, consistent with the species/subspecies boundaries. it is interesting to note that one of the a. salmonicida atypical samples clustered with a. salmonicida subsp. achromogenes, whereas the second atypical strain clustered with the a. salmonicida subsp. salmonicida a control strain. these results suggest that neither of these should be considered a. salmonicida atypical strains. the m-cgh data for the sixteen aeromonas samples ordered with hierarchical clustering of the genes is shown in figure genes were considered to be conserved if the signal differed by less than a factor of two between the tester and the control, as described by taboada et al.,. the lower cutoff of - is fairly conservative as we have shown previously that it encompasses over % of highly conserved genes but less than 1% of missing genes. to highlight the variable genes, the log ratio value was set to for conserved genes, and genes which were conserved in all of the samples have been removed. genes with log ratio values less than - were considered to be divergent in sequence or absent from the tester strain, while those with log ratios values greater than + were expected to be present in higher copy number in the tester than in the control strain. as shown in figure 2a, clusters with the highest variability across the samples correspond to plasmid- and transposon-associated genes. as expected, a. bestiarum and a. caviae have higher numbers of variable genes than the a. salmonicida strains. figure 2b shows the subset of genes that are not associated with transposons or plasmids but are divergent in at least one of the fourteen a. salmonicida samples. these divergent "chromosomal" genes show some clustering of genes coding for outer membrane proteins and flagella/pili. the number of genes divergent or absent according to the function categories assigned in table and by species/strain is presented in table the highest degree of divergence compared to a can be seen for a. caviae and a. bestiarum, followed by the type strains of a. salmonicida subspp. masoucida and achromogenes as well as one of the atypical samples. there was much less variation between the a. salmonicida subsp. salmonicida strains and the other a. salmonicida atypical strain. of the a. salmonicida subsp. salmonicida strains, n isolated from turbot, and ss, the only isolate from a pacific salmon species, showed the highest degree of variability among chromosomal genes. these data notwithstanding, there does not appear to be a strong correlation between the level of genomic variability and either host or geographical origin. the m-cgh data for genes associated with mobile elements in the sixteen aeromonas samples is shown in figure hierarchical clustering of the plasmid-associated genes shows clustering of highly variable genes matching a known plasmid of ~ kb. a subset of genes on this plasmid matches type three secretion system genes found in other bacteria, which is unsurprising considering that ttss genes can be plasmid-borne in a. salmonicida subsp. salmonicida. we observed genomic variability consistent with the lower intensity signal for some plasmid genes, including ttss genes, for several strains. of these, the strain ss is an avirulent, laboratory-derived strain created by treating an a. salmonicida subsp. salmonicida strain with ethidium bromide. this likely resulted in the loss of at least one plasmid, which would contribute to this observation. on the other hand, many genes on plasmid appear to be present in higher copy number in many of the tester strains than in a figure 3b shows the m-cgh data for genes associated with transposons. many of these genes appear to be highly divergent, present at lower copy number, or missing in various tester strains, and many of the genes match known transposases. the number of variable genes assigned to each functional category is shown in figure as well as the percent of genes in each category that were observed to be variable. within the function categories other than transposon or plasmid, most genes that appeared to be divergent or missing in various aeromonas strains are associated with transport, surface carbohydrate biosynthesis, outer membrane, and flagella/pili proteins. in contrast, genes associated with secretion were most likely to be present in higher copy numbers in the tester strains compared to a in summary, the highest degree of variability was seen in aeromonas genes associated with mobile elements: the plasmids or the transposons, whereas genes within the chromosome of the bacterium displayed the lowest level of variability. discussion in this study, patterns of variability of the selected virulence-associated genes of a. salmonicida subsp. salmonicida strain a within the sixteen various test isolates of a. salmonicida and other aeromonas species were examined. it is noteworthy that genes divergent and absent in some tester strains seem to be distributed across the entire a genome sequence and across all the predicted functional categories whereas genes shown to be at higher copy number than in the reference strain were clustered in selected loci associated with plasmids. the number of divergent and absent genes correlated strongly with species and subspecies boundaries. for a. bestiarum and a. caviae ~16% of the genes were divergent or missing, whereas for the achromogenes and masoucida subspecies ~8% of the genes were divergent or missing. the range across the various a. salmonicida subsp. salmonicida strains was between % and 2%, again reinforcing the single clone theory for this species. overall, for the a. salmonicida isolates studied, approximately 19% of the genes were divergent in at least one sample. for comparison, our previous meta-analysis of m-cgh studies of strains of c. jejuni showed ~33% of the genes in the genome were divergent in at least one strain. it is likely that as we examine more strains of a. salmonicida the number of divergent genes will increase. one interesting finding is that almost 70% of the virulence-associated genes on the dna microarray are conserved across all the aeromonas species. because more than 80% of these highly conserved genes appear to be chromosomal, this suggests that the divergence of a. bestiarum, a. caviae, and a. salmonicida may have occurred fairly recently. the majority of a. salmonicida subsp. salmonicida isolates studied were isolated from atlantic salmon and gave homogenous results, i.e., there is no correlation between host or geographic origin and m-cgh patterns in subsp. salmonicida isolates. highest variability amongst the subsp. salmonicida isolates was observed in an isolate from coho salmon and in an isolate from turbot. however, more isolates from pacific salmon should be analyzed before drawing definite conclusions on the variation between isolates from atlantic and pacific salmon. the relatively low number of divergent genes in most of the a. salmonicida subsp. salmonicida strains supports the findings of garcia et al. who identified a single clone of a. salmonicida subsp. salmonicida as responsible for most outbreaks of disease worldwide. differentiation of typical and atypical strains of a. salmonicida is important economically if it can be shown that there are significant differences in the degree of variation in the genes associated with virulence and pathogenicity, and which affect the immune response of the host. if this is the case, then accurate typing methods and differentiation between typical and atypical strains and subspecies are required for diagnostic methods and for vaccine development. lund et al. have shown variation in the protective ability of some commercial furunculosis vaccines against atypical strains infecting farmed spotted wolffish and halibut. in this context, m-cgh provides an objective means to type clinical isolates based upon the presence, absence or divergence of a large number of genes. phenotypical variation amongst subsp. salmonicida isolates is rare but has been described. the increased resolution of m-cgh compared to phenotypic typing can be illustrated on consideration of strain n which was considered an atypical strain on phenotypical grounds. however, both m-cgh and subsequent pulsed-field gel electrophoresis suggest that this strain is very closely related to, and should perhaps more accurately be considered a typical strain. similarly, atypical strain n may be more accurately placed within the subspecies achromogenes based upon m-cgh results. further comparative studies will be required to establish the level of strain resolution capable by m-cgh for aeromonads. however, in the absence of complete genome sequence data for each aeromonas strain used, m-cgh, which in this study uses over separate genetic markers, represents a comprehensive, high- resolution methodology for comparing genome information. recent work by our group used m-cgh to examine the quantitative relationship between the log ratio and probe/target identity, and these analytical processes were used in the study. it is preferable that vaccine candidates be strongly conserved among strains that cause disease. the m-cgh data from these experiments will provide a list of conserved genes, sorted by function category, which can be further analyzed to generate these candidates. for example, there are bioinformatics programs available to predict sub-cellular location to select candidates which may be exposed to the host immune system, or to predict adhesins. these downstream analyses, in combination with m-cgh analysis will reduce the number of genes required to be cloned in order to test the immune response of their encoded proteins. these experiments are currently underway. it must be noted that m-cgh does not reveal information about gene expression or the specific role of targeted genes in pathogenicity or host immune responses. however, it is a powerful tool in reverse vaccinology and m-cgh is a sensitive and comprehensive technique that can determine genomic variation between pathogen strains, and it also can be used as a screening tool for target identification, and/or a typing method.  m-cgh is a powerful first screen for vaccine target identification, and is the basis of reverse vaccinology, as it facilitates identification of conserved and duplicated genes associated with virulent strains. this technique may also prove useful in strain typing and epidemiological studies. comparative analysis of genomic data of a. salmonicida and related organisms reveals candidates common to all clinical isolates or those strains shown to be associated with virulence and disease. our work on knock-out mutants of a virulence genes, combined with proteomic analysis of selected isolates confirm the results of our m-cgh analyses and will enable us to select specific antigens for vaccine development. the findings reported here support our initial hypotheses that variability exists between subspecies, that there is no correlation between'typical' and'atypical' classifications and gene variability. we also found that there is a positive correlation between the degree of variability and the functional category of the genes involved. future work will include the use of microarray transcript profiling experiments to further focus the choice of target genes. this will allow us to develop an ongoing, dynamic list of vaccine candidates to be used in live challenges for virulence and protection assays and then in further vaccine development. 
as part of a larger research program to identify virulence genes and candidates for vaccine development, a dna microarray was constructed using a subset of genes from the draft genome sequence of a. salmonicida subsp. we used microarray-based comparative genomic hybridizations to compare selected a. salmonicida sub-species and other aeromonas species from different hosts and geographic locations. aeromonas salmonicida has been isolated from numerous fish species and shows wide variation in virulence and pathogenicity. we have used the m-cgh technique to identify subsets of conserved genes from amongst this set of a. salmonicida virulence genes for further investigation as potential vaccine candidates. salmonicida strain a the microarray included genes encoding known virulence-associated factors in a. salmonicida and homologs of virulence genes of other pathogens. results showed variable carriage of virulence-associated genes and generally increased variation in gene content across sub-species and species boundaries. unlike other bacterial characterization methods that use a small number of gene or dna-based functions, m-cgh examines thousands of genes and/or whole genomes and thus is a more comprehensive analytical tool for veterinary or even human health research.
 aeromonas salmonicida has been isolated from numerous fish species and shows wide variation in virulence and pathogenicity. as part of a larger research program to identify virulence genes and candidates for vaccine development, a dna microarray was constructed using a subset of genes from the draft genome sequence of a. salmonicida subsp. salmonicida strain a the microarray included genes encoding known virulence-associated factors in a. salmonicida and homologs of virulence genes of other pathogens. we used microarray-based comparative genomic hybridizations to compare selected a. salmonicida sub-species and other aeromonas species from different hosts and geographic locations.  results showed variable carriage of virulence-associated genes and generally increased variation in gene content across sub-species and species boundaries. the greatest variation was observed among genes associated with plasmids and transposons. there was little correlation between geographic region and degree of variation for all isolates tested.  we have used the m-cgh technique to identify subsets of conserved genes from amongst this set of a. salmonicida virulence genes for further investigation as potential vaccine candidates. unlike other bacterial characterization methods that use a small number of gene or dna-based functions, m-cgh examines thousands of genes and/or whole genomes and thus is a more comprehensive analytical tool for veterinary or even human health research. 