BACKGROUND
enteric red mouth disease , caused by yersinia ruckeri, is one of the most important infectious diseases in rainbow trout  aquaculture in europe.
signs of a clinical infection of rainbow trout may include haemorrhages in various tissues and organs, particularly around the mouth, in the gills, muscles, peritoneum and the lower intestine.
the bacterium has been isolated from various fish species, including rainbow trout, steelhead , lake trout , cutthroat , brown  and brook trout , coho , sockeye , chinook  and atlantic salmon   <cit> .
in europe, it is endemic in many trout farms and can cause high economic losses  <cit> .
the disease most commonly affects younger rainbow trout at temperatures above 10°c.
outbreaks are often related to adverse situations or stressed carrier fish which shed the pathogen and facilitate the onset of an infection.
in infected trout populations, erm can be controlled effectively by antibiotic treatment and by the application of inactivated whole cell vaccines  <cit> , which are available commercially and are considered to be successful  <cit> .
nevertheless, outbreaks of erm are still periodically observed, especially in endemic areas.
among y. ruckeri isolates, several serological variations have been reported  <cit> .
while initially most outbreaks of erm were caused by y. ruckeri isolates of serotype o <dig> , y. ruckeri isolates of different serotypes have been more recently obtained from sick fish during outbreaks of erm in the uk, spain and the u.s.a.  <cit> .
moreover, several of the commercial erm vaccines seemed to not provide sufficient protection against infections caused by some of these isolates  <cit> .
all these ‘vaccine-tolerant’ isolates lacked motility , while isolates from previous outbreaks were all motile   <cit> .
it was hypothesised that vaccination resulted in a selective pressure that enabled the emergence of non-motile isolates that are tolerant to the commercial vaccines.
therefore, there is a high risk that these non-motile vaccine-tolerant isolates spread and cause severe outbreaks of disease in trout farms.
wheeler et al.
<cit>  found that the bt <dig> isolates in mainland europe have arisen independently from the local bt <dig> isolates.
recently, welch et al.
<cit>  indicated that the european bt <dig> isolates were not from the same clonal group.
in germany, the earliest report about bt <dig> isolates was published in  <dig>  <cit> .
recently, ‘vaccine-tolerant’ y. ruckeri were isolated from outbreaks in trout hatcheries in north west germany, in particular in the federal state of north rhine-westphalia .
in order to avoid further spreading of these ‘vaccine-tolerant’ isolates, an epidemiological survey on the occurrence and further phenotypic and molecular characterisation of y. ruckeri from trout hatcheries was performed.
in this study, interspecies differences of y. ruckeri isolates from north west germany were detected via biochemical and molecular traits to determine how related isolates from different origins are.
for this, several typing methods were compared for their ability to differentiate y. ruckeri isolates from rainbow trout and other fish species.
RESULTS
biochemical and physiological characterisation
among the variable biochemical characteristics of all  <dig> y. ruckeri isolates , gelatine hydrolysis, voges-proskauer  reaction and tween hydrolysis were the most variable biochemical reactions.
the results of vp, nitrate production, citrate utilization and gelatine hydrolysis from both, conventional biochemical analysis and testing by the api 20e system, were listed also in table  <dig>  for motility, tween 80/ <dig> hydrolysis and citrate utilization, the isolates obtained in lower saxony  from fish species other than rainbow trout were more similar to the reference strain dsm  <dig>  than the isolates obtained from rainbow trout hatcheries in nrw and ls.
only  <dig> isolates hydrolysed tween 80/ <dig>  for these isolates motility was confirmed by microscopical inspection as well as by cultivation in api m medium.
those isolates were recognized as non-motile isolates.
in particular, isolates collected from trout hatcheries in nrw were lacking flagella and motility; 92% of the isolates from this federal state were non-motile .
yersinia ruckeri
isolates from fish in aquaculture in north west germany
* api 20e test system.
l: lower saxony, h: hessen, n: north rhine-westphalia.
in the api 20e test,  <dig> isolates showed the numeric profile  <dig> , <dig>   <dig> isolates with the numeric profile  <dig> , <dig>  these profiles differed from that of the reference strain dsm  <dig>  with the profile  <dig> , <dig>  for a biochemical typing of the isolates, typing numbers from “a” to “g” were assigned to individual isolates according to their profile number .
y. ruckeri
patterns from different genotypic method
a
b
5
apcr-based typing methods: rep-pcr, repetitive extragenic palindromic-pcr; box-pcr, box-a1r-based repetitive extragenic palindromic-pcr; 5-pcr, polytrinucleotide 5-pcr; eric-pcr, enterobacterial repetitive intergenic consensus-pcr.
macrorestriction-based typing method, pfge macrorestriction analyis by pulsed-field gel electrophoresis.
breference strain dsm  <dig>
capi 20e profiles were as follows: a , b , c , d , e , f , g , h .
genetic characterisation
when the isolates were analysed by repetitive sequence-based pcr assays, two amplicon patterns were obtained after rep-pcr, five patterns by eric-pcr, four patterns by 5-pcr and three patterns by box-pcr .
the similarity rates in rep-pcr were clearly lower than in the other repetitive sequence-based pcrs.
in box-pcr and 5-pcr, the reference strain dsm  <dig>  exhibited the same amplicon patterns as most of the isolates .
by rep-pcr,  <dig> or  <dig> bands were seen in the size range of 200–2000 bp; by box-pcr, 18– <dig> bands were found distributed between 340–1550 bp; by 5-pcr, 11– <dig> bands were detected from 390–2500 bp; and by eric-pcr, 20– <dig> bands were present in the range from 200–2000 bp .
five isolates, which were positive in sorbitol fermentation, did not exhibit the 430 bp amplicon in eric-pcr.
only five isolates from fish species other than rainbow trout showed some differences in repetitive pcrs.
these five isolates were additionally confirmed as y. ruckeri  by 16s rdna sequencing; while the nucleotide sequence of the reference strain dsm  <dig>  was not completely identical to the sequence reported from the strain atcc  <dig>  .
conversely, all the isolates from rainbow trout showed rather uniform patterns of repetitive sequence-based pcr.
y. ruckeri
isolates constructed using the upgma method  using gel compar ii , based on fingerprints of different repetitive sequence-based pcrs: a, rep-pcr; b, box-pcr; c, 5-pcr; d, eric-pcr.
noti-directed pfge yielded four major pfge groups a-d. a total of  <dig> different pfge types  were detected among the  <dig> y. ruckeri isolates examined .
cluster analysis of these pfge types showed that the isolates from north west germany were highly uniform, whereby  <dig> isolates  belonged to pfge type c <dig> and  <dig> isolates to pfge type c <dig> .
compared with the reference strain dsm  <dig> , all the isolates were different.
the isolates from rainbow trout were clustered together .
isolates from pike, koi and brown trout  showed the greatest variation when compared to rainbow trout isolates.
all isolates previously assigned to biotype  <dig> clustered in pfge group c. isolates from this pfge group were found in the federal states of ls, hessen  and nrw .
most of the isolates were distributed in all three federal states , while isolates of pfge type c <dig> were found only in ls and nrw .
discriminatory indices of the typing methods applied
as a discriminatory index, simpson’s index of diversity was calculated for each typing method used to differentiate all y. ruckeri isolates.
discriminatory indices varied distinctly between the different typing methods.
rep-pcr had the lowest d value of  <dig> .
this means that two randomly selected isolates of this test population had a probability of  <dig> % of showing a different rep-pcr pattern.
slightly higher d values were calculated for box-pcr , 5-pcr  and eric-pcr .
highest d values were seen for biotyping via api 20e  and noti-directed pfge .
according to their biochemical and molecular characteristics as obtained by testing with api 20e, pfge and various pcr-based methods, the y. ruckeri isolates from north west germany could be subdivided into  <dig> different groups .
most of the isolates from north west germany  belonged to the group .
additional information about the origins and isolated years were shown in additional file 1: table s <dig>
discussion
since the first isolation of y. ruckeri in idaho, usa in 1950s, outbreaks of erm were frequently reported  <cit> , especially from the aquaculture industry in countries such as croatia  <cit> , france  <cit> , finland  <cit> , germany  <cit> , italy  <cit> , switzerland  <cit>  and the uk  <cit> .
the bacterium was isolated from different host species  <cit> , including several salmonid species, channel catfish, goldfish, common carp, european eel, coalfish, and perch.
typing of y. ruckeri isolates from various geographic locations or different fish species has been performed for taxonomic or epidemiological purposes and initially it was based on phenotypical characteristics including the results of biochemical and serological tests  <cit> .
in addition, molecular techniques, including random amplification of polymorphic dna , multilocus sequence typing  or pulsed-field gel electrophoresis  have been applied to study the genetic diversity of y. ruckeri isolates  <cit> .
in a study on isolates from various geographic origins, including chile, peru, u.s.a., uk, and mainland europe, a considerable intraspecies diversity of y. ruckeri was found on the basis of the phenotypical characteristics obtained from api 20e, lipopolysaccharide  and outer membrane profiles  <cit>  as well as by genetic analysis using mlst profiles  <cit> .
the isolates from north west germany investigated in the present study, showed a strong homogeneity of biochemical results and api 20e profiles with some variability in gelatine hydrolysis, vp reaction and tween hydrolysis.
variation in some biochemical tests for y. ruckeri has been reported in previous studies  <cit> , but this variation could not be used as a distinguishing characteristic for phenotypical traits such as serotype or biotype  <cit> .
while the majority of y. ruckeri isolates from farmed rainbow trout in peru  <cit>  or atlantic salmon in chile  <cit>  were motile, only  <dig> out of  <dig> isolates from the present study belonged to biotype  <dig> .
all other isolates could not hydrolyse tween 80/ <dig>  were lipase-negative and lacked flagella.
the api 20e profiles  <dig> , <dig>   <dig> , <dig>   <dig> , <dig> were previously obtained from y. ruckeri isolates from south-west germany  <cit> , the profiles  <dig> , <dig> and  <dig> , <dig> were already reported from spanish isolates  <cit> .
while the profiles  <dig> , <dig> and  <dig> , <dig> were also previously reported from y. ruckeri isolates,  <cit>  there was no record about an isolate with the numeric profile  <dig> , <dig>  however, 16s rrna sequencing confirmed this isolate as y. ruckeri .
in addition to the biochemical characteristics, some genetic variability was observed in isolates using several repetitive sequence-based pcr techniques including box-pcr, eric-pcr, 5-pcr, rep-pcr as well as pulsed-field gel electrophoresis.
an analysis of the isolates from north west germany by repetitive sequence based pcrs gave  <dig> to  <dig> groups, with the majority of isolates in one group.
similar observations were already reported from latin american isolates  <cit> .
eric-pcr showed the highest discriminatory power.
while in previous studies both eric-pcr and rep-pcr were considered of some value as tools to study erm epidemiology  <cit> , in our study, rep-pcr showed the lowest discriminatory power and could not differentiate the isolates very clearly.
this could depend on the different origin of the isolates: our isolates originated mainly from rainbow trout, while bastardo et al.
<cit>  studied isolates from atlantic salmon.
when y. ruckeri isolates from peruvian rainbow trout were analysed by rep-pcr, more homogeneous amplicon patterns were observed as well  <cit> .
pfge has been frequently applied for detailed molecular typing of different bacteria  <cit> , as well as for a differentiation among isolates of y. ruckeri <cit> .
in the present study, the typing results by pfge showed strong homogeneity among the isolates obtained from farmed fish species in north west germany.
more than half of the y. ruckeri isolates belonged to the two pfge types c <dig> and c <dig>  with  <dig> % of the isolates sharing the same pfge type c <dig>  these two pfge types belong to the group c pfge cluster, which comprises isolates from rainbow trout and represents the majority of the isolates.
the isolates showing pfge types a1/a <dig> or b1/b <dig> also differed from the other isolates in at least some of their repetitive sequences-based pcr patterns .
in the present study, no distinct differences were observed in pfge types from biotype  <dig> and biotype  <dig> isolates from rainbow trout.
the data presented in this study may suggest that the isolates from north west germany originated from the same ancestor and could indicate a clonal population structure.
in a previous study on the genetic diversity of y. ruckeri isolates, analysed by mlst, a large number of isolates also belonged to just two sequence types , which formed one clonal complex with  <dig> out of  <dig> recognised sts  <cit> .
however, the mlst analysis revealed that genetic recombination appeared to play a greater role than mutation for the generation and maintenance of genetic diversity within the population of y. ruckeri <cit> .
this would be contradictory to the concept of clonal expansion of a population from a common ancestor that diversified mainly by mutation rather than recombination  <cit> .
mlst results from y. ruckeri rather suggest an epidemic population structure, as it was observed for instance for vibrio parahaemolyticus <cit> .
the epidemiology of yersinia ruckeri, which started as a geographically isolated disease and quickly became disseminated  <cit> , also was considered to support the epidemic model  <cit> .
pulsed-field gel electrophoretic analysis of y. ruckeri isolates resulted in a discriminatory index  of  <dig> , whereas repetitive sequence-based pcr approaches yielded low d values between  <dig>  and  <dig> .
as the d value describes the probability that two randomly selected isolates show different profiles by the method applied, methods with low d values are considered not to be sufficiently discriminative for the detection of differences among y. ruckeri isolates.
in a previous analysis, “medium to high” d values ranging between  <dig> – <dig>  were obtained by the api 20e, lipopolysaccharide and outer membrane protein analysis  <cit> , while eric-and rep-pcr were valuable in combination with other techniques.
similar results were reported from y. ruckeri isolated in turkey  <cit> .
the combination of several typing methods, as performed by bastardo et al.
<cit>  showed that isolates with similar characteristics were associated with certain fish species and/or predominated in some geographical areas.
in the present study, the combination of six typing methods, including api 20e, pfge and four different repetitive sequence pcr techniques facilitated the identification of  <dig> different groups.
with this set of methods, most of the isolates from rainbow trout  could be differentiated from the isolates obtained from pike, koi and brown trout.
biotype  <dig> isolates were only detected in rainbow trout in this study.
the genomic variation of y. ruckeri isolates provides information for comparing their distribution and the relationships among individual isolates from different regions in north west germany.
CONCLUSIONS
most of the isolates from north west germany belonged to biotype  <dig>  the combination of different typing methods could well differentiate y. ruckeri isolates.
the results of this study showed the diversity of isolates present in north west germany.
moreover, these data enable the identification and in-depth characterisation of pathogenic y. ruckeri isolates.
as a consequence of this detailed characterisation, these data can be used to examine the distribution of these isolates in the field and may be used as a basis for preventive disease monitoring plans.

 enteric red mouth disease, caused by yersinia ruckeri, is one of the most important infectious diseases in rainbow trout aquaculture in europe. signs of a clinical infection of rainbow trout may include haemorrhages in various tissues and organs, particularly around the mouth, in the gills, muscles, peritoneum and the lower intestine. the bacterium has been isolated from various fish species, including rainbow trout, steelhead, lake trout, cutthroat, brown and brook trout, coho, sockeye, chinook and atlantic salmon. in europe, it is endemic in many trout farms and can cause high economic losses. the disease most commonly affects younger rainbow trout at temperatures above 10°c. outbreaks are often related to adverse situations or stressed carrier fish which shed the pathogen and facilitate the onset of an infection. in infected trout populations, erm can be controlled effectively by antibiotic treatment and by the application of inactivated whole cell vaccines, which are available commercially and are considered to be successful. nevertheless, outbreaks of erm are still periodically observed, especially in endemic areas. among y. ruckeri isolates, several serological variations have been reported. while initially most outbreaks of erm were caused by y. ruckeri isolates of serotype o, y. ruckeri isolates of different serotypes have been more recently obtained from sick fish during outbreaks of erm in the uk, spain and the u.s.a.. moreover, several of the commercial erm vaccines seemed to not provide sufficient protection against infections caused by some of these isolates. all these ‘vaccine-tolerant’ isolates lacked motility, while isolates from previous outbreaks were all motile. it was hypothesised that vaccination resulted in a selective pressure that enabled the emergence of non-motile isolates that are tolerant to the commercial vaccines. therefore, there is a high risk that these non-motile vaccine-tolerant isolates spread and cause severe outbreaks of disease in trout farms. wheeler et al. found that the bt isolates in mainland europe have arisen independently from the local bt isolates. recently, welch et al. indicated that the european bt isolates were not from the same clonal group. in germany, the earliest report about bt isolates was published in. recently, ‘vaccine-tolerant’ y. ruckeri were isolated from outbreaks in trout hatcheries in north west germany, in particular in the federal state of north rhine-westphalia. in order to avoid further spreading of these ‘vaccine-tolerant’ isolates, an epidemiological survey on the occurrence and further phenotypic and molecular characterisation of y. ruckeri from trout hatcheries was performed. in this study, interspecies differences of y. ruckeri isolates from north west germany were detected via biochemical and molecular traits to determine how related isolates from different origins are. for this, several typing methods were compared for their ability to differentiate y. ruckeri isolates from rainbow trout and other fish species.  biochemical and physiological characterisation among the variable biochemical characteristics of all y. ruckeri isolates, gelatine hydrolysis, voges-proskauer reaction and tween hydrolysis were the most variable biochemical reactions. the results of vp, nitrate production, citrate utilization and gelatine hydrolysis from both, conventional biochemical analysis and testing by the api 20e system, were listed also in table  for motility, tween 80/ hydrolysis and citrate utilization, the isolates obtained in lower saxony from fish species other than rainbow trout were more similar to the reference strain dsm than the isolates obtained from rainbow trout hatcheries in nrw and ls. only isolates hydrolysed tween 80/ for these isolates motility was confirmed by microscopical inspection as well as by cultivation in api m medium. those isolates were recognized as non-motile isolates. in particular, isolates collected from trout hatcheries in nrw were lacking flagella and motility; 92% of the isolates from this federal state were non-motile. yersinia ruckeri isolates from fish in aquaculture in north west germany * api 20e test system. l: lower saxony, h: hessen, n: north rhine-westphalia. in the api 20e test, isolates showed the numeric profile, isolates with the numeric profile, these profiles differed from that of the reference strain dsm with the profile, for a biochemical typing of the isolates, typing numbers from “a” to “g” were assigned to individual isolates according to their profile number. y. ruckeri patterns from different genotypic method a b 5 apcr-based typing methods: rep-pcr, repetitive extragenic palindromic-pcr; box-pcr, box-a1r-based repetitive extragenic palindromic-pcr; 5-pcr, polytrinucleotide 5-pcr; eric-pcr, enterobacterial repetitive intergenic consensus-pcr. macrorestriction-based typing method, pfge macrorestriction analyis by pulsed-field gel electrophoresis. breference strain dsm capi 20e profiles were as follows: a, b, c, d, e, f, g, h. genetic characterisation when the isolates were analysed by repetitive sequence-based pcr assays, two amplicon patterns were obtained after rep-pcr, five patterns by eric-pcr, four patterns by 5-pcr and three patterns by box-pcr. the similarity rates in rep-pcr were clearly lower than in the other repetitive sequence-based pcrs. in box-pcr and 5-pcr, the reference strain dsm exhibited the same amplicon patterns as most of the isolates. by rep-pcr, or bands were seen in the size range of 200–2000 bp; by box-pcr, 18– bands were found distributed between 340–1550 bp; by 5-pcr, 11– bands were detected from 390–2500 bp; and by eric-pcr, 20– bands were present in the range from 200–2000 bp. five isolates, which were positive in sorbitol fermentation, did not exhibit the 430 bp amplicon in eric-pcr. only five isolates from fish species other than rainbow trout showed some differences in repetitive pcrs. these five isolates were additionally confirmed as y. ruckeri by 16s rdna sequencing; while the nucleotide sequence of the reference strain dsm was not completely identical to the sequence reported from the strain atcc. conversely, all the isolates from rainbow trout showed rather uniform patterns of repetitive sequence-based pcr. y. ruckeri isolates constructed using the upgma method using gel compar ii, based on fingerprints of different repetitive sequence-based pcrs: a, rep-pcr; b, box-pcr; c, 5-pcr; d, eric-pcr. noti-directed pfge yielded four major pfge groups a-d. a total of different pfge types were detected among the y. ruckeri isolates examined. cluster analysis of these pfge types showed that the isolates from north west germany were highly uniform, whereby isolates belonged to pfge type c and isolates to pfge type c. compared with the reference strain dsm, all the isolates were different. the isolates from rainbow trout were clustered together. isolates from pike, koi and brown trout showed the greatest variation when compared to rainbow trout isolates. all isolates previously assigned to biotype clustered in pfge group c. isolates from this pfge group were found in the federal states of ls, hessen and nrw. most of the isolates were distributed in all three federal states, while isolates of pfge type c were found only in ls and nrw. discriminatory indices of the typing methods applied as a discriminatory index, simpson’s index of diversity was calculated for each typing method used to differentiate all y. ruckeri isolates. discriminatory indices varied distinctly between the different typing methods. rep-pcr had the lowest d value of. this means that two randomly selected isolates of this test population had a probability of % of showing a different rep-pcr pattern. slightly higher d values were calculated for box-pcr, 5-pcr and eric-pcr. highest d values were seen for biotyping via api 20e and noti-directed pfge. according to their biochemical and molecular characteristics as obtained by testing with api 20e, pfge and various pcr-based methods, the y. ruckeri isolates from north west germany could be subdivided into different groups. most of the isolates from north west germany belonged to the group. additional information about the origins and isolated years were shown in additional file 1: table s discussion since the first isolation of y. ruckeri in idaho, usa in 1950s, outbreaks of erm were frequently reported, especially from the aquaculture industry in countries such as croatia, france, finland, germany, italy, switzerland and the uk. the bacterium was isolated from different host species, including several salmonid species, channel catfish, goldfish, common carp, european eel, coalfish, and perch. typing of y. ruckeri isolates from various geographic locations or different fish species has been performed for taxonomic or epidemiological purposes and initially it was based on phenotypical characteristics including the results of biochemical and serological tests. in addition, molecular techniques, including random amplification of polymorphic dna, multilocus sequence typing or pulsed-field gel electrophoresis have been applied to study the genetic diversity of y. ruckeri isolates. in a study on isolates from various geographic origins, including chile, peru, u.s.a., uk, and mainland europe, a considerable intraspecies diversity of y. ruckeri was found on the basis of the phenotypical characteristics obtained from api 20e, lipopolysaccharide and outer membrane profiles as well as by genetic analysis using mlst profiles. the isolates from north west germany investigated in the present study, showed a strong homogeneity of biochemical results and api 20e profiles with some variability in gelatine hydrolysis, vp reaction and tween hydrolysis. variation in some biochemical tests for y. ruckeri has been reported in previous studies, but this variation could not be used as a distinguishing characteristic for phenotypical traits such as serotype or biotype. while the majority of y. ruckeri isolates from farmed rainbow trout in peru or atlantic salmon in chile were motile, only out of isolates from the present study belonged to biotype. all other isolates could not hydrolyse tween 80/ were lipase-negative and lacked flagella. the api 20e profiles,,, were previously obtained from y. ruckeri isolates from south-west germany, the profiles, and, were already reported from spanish isolates. while the profiles, and, were also previously reported from y. ruckeri isolates, there was no record about an isolate with the numeric profile, however, 16s rrna sequencing confirmed this isolate as y. ruckeri. in addition to the biochemical characteristics, some genetic variability was observed in isolates using several repetitive sequence-based pcr techniques including box-pcr, eric-pcr, 5-pcr, rep-pcr as well as pulsed-field gel electrophoresis. an analysis of the isolates from north west germany by repetitive sequence based pcrs gave to groups, with the majority of isolates in one group. similar observations were already reported from latin american isolates. eric-pcr showed the highest discriminatory power. while in previous studies both eric-pcr and rep-pcr were considered of some value as tools to study erm epidemiology, in our study, rep-pcr showed the lowest discriminatory power and could not differentiate the isolates very clearly. this could depend on the different origin of the isolates: our isolates originated mainly from rainbow trout, while bastardo et al. studied isolates from atlantic salmon. when y. ruckeri isolates from peruvian rainbow trout were analysed by rep-pcr, more homogeneous amplicon patterns were observed as well. pfge has been frequently applied for detailed molecular typing of different bacteria, as well as for a differentiation among isolates of y. ruckeri. in the present study, the typing results by pfge showed strong homogeneity among the isolates obtained from farmed fish species in north west germany. more than half of the y. ruckeri isolates belonged to the two pfge types c and c with % of the isolates sharing the same pfge type c these two pfge types belong to the group c pfge cluster, which comprises isolates from rainbow trout and represents the majority of the isolates. the isolates showing pfge types a1/a or b1/b also differed from the other isolates in at least some of their repetitive sequences-based pcr patterns. in the present study, no distinct differences were observed in pfge types from biotype and biotype isolates from rainbow trout. the data presented in this study may suggest that the isolates from north west germany originated from the same ancestor and could indicate a clonal population structure. in a previous study on the genetic diversity of y. ruckeri isolates, analysed by mlst, a large number of isolates also belonged to just two sequence types, which formed one clonal complex with out of recognised sts. however, the mlst analysis revealed that genetic recombination appeared to play a greater role than mutation for the generation and maintenance of genetic diversity within the population of y. ruckeri. this would be contradictory to the concept of clonal expansion of a population from a common ancestor that diversified mainly by mutation rather than recombination. mlst results from y. ruckeri rather suggest an epidemic population structure, as it was observed for instance for vibrio parahaemolyticus. the epidemiology of yersinia ruckeri, which started as a geographically isolated disease and quickly became disseminated, also was considered to support the epidemic model. pulsed-field gel electrophoretic analysis of y. ruckeri isolates resulted in a discriminatory index of, whereas repetitive sequence-based pcr approaches yielded low d values between and. as the d value describes the probability that two randomly selected isolates show different profiles by the method applied, methods with low d values are considered not to be sufficiently discriminative for the detection of differences among y. ruckeri isolates. in a previous analysis, “medium to high” d values ranging between – were obtained by the api 20e, lipopolysaccharide and outer membrane protein analysis, while eric-and rep-pcr were valuable in combination with other techniques. similar results were reported from y. ruckeri isolated in turkey. the combination of several typing methods, as performed by bastardo et al. showed that isolates with similar characteristics were associated with certain fish species and/or predominated in some geographical areas. in the present study, the combination of six typing methods, including api 20e, pfge and four different repetitive sequence pcr techniques facilitated the identification of different groups. with this set of methods, most of the isolates from rainbow trout could be differentiated from the isolates obtained from pike, koi and brown trout. biotype isolates were only detected in rainbow trout in this study. the genomic variation of y. ruckeri isolates provides information for comparing their distribution and the relationships among individual isolates from different regions in north west germany.  most of the isolates from north west germany belonged to biotype the combination of different typing methods could well differentiate y. ruckeri isolates. the results of this study showed the diversity of isolates present in north west germany. moreover, these data enable the identification and in-depth characterisation of pathogenic y. ruckeri isolates. as a consequence of this detailed characterisation, these data can be used to examine the distribution of these isolates in the field and may be used as a basis for preventive disease monitoring plans. 
all isolates were confirmed as y. ruckeri by the api 20e system. isolates from the field study clustered together as pfge type c. according to the results of api 20e, repetitive sequence-based pcrs and pfge, these isolates could be subdivided into different groups. the isolates originated mainly from rainbow trout and were characterised by biochemical profiling, 16s rdna sequencing, repetitive sequence-based pcrs, including 5-pcr, box-pcr, eric-pcr and rep-pcr, and pulsed-field gel electrophoresis. five isolates were additionally confirmed as y. ruckeri by y. ruckeri-specific pcr and 16s rdna sequencing. enteric redmouth disease, caused by yersinia ruckeri, is one of the most important infectious diseases in rainbow trout aquaculture in europe. the aim of this study was to analyse the variation of biochemical and molecular characteristics of y. ruckeri isolates collected in north west germany as a basis for strain differentiation. the detailed molecular and phenotypic characterisation scheme developed in this study could be used to help trace the dissemination of y. ruckeri isolates, and thus may represent part of improved disease monitoring plans in the future.
 enteric redmouth disease, caused by yersinia ruckeri, is one of the most important infectious diseases in rainbow trout aquaculture in europe. more recently, non-motile vaccine resistant isolates appear to have evolved and are causing disease problems throughout europe, including germany. the aim of this study was to analyse the variation of biochemical and molecular characteristics of y. ruckeri isolates collected in north west germany as a basis for strain differentiation. the isolates originated mainly from rainbow trout and were characterised by biochemical profiling, 16s rdna sequencing, repetitive sequence-based pcrs, including 5-pcr, box-pcr, eric-pcr and rep-pcr, and pulsed-field gel electrophoresis.  in total, isolates were characterised, including isolates collected during a field study in north west germany. all isolates were confirmed as y. ruckeri by the api 20e system. five isolates were additionally confirmed as y. ruckeri by y. ruckeri-specific pcr and 16s rdna sequencing. only isolates hydrolyzed tween 80/ sixty-six isolates were non-motile. two different patterns were obtained by rep-pcr, five patterns by eric-pcr, four patterns by 5-pcr and three patterns by box-pcr. noti-directed pfge resulted in patterns that differed from each other by 25– fragments. isolates from the field study clustered together as pfge type c. according to the results of api 20e, repetitive sequence-based pcrs and pfge, these isolates could be subdivided into different groups.  the detailed molecular and phenotypic characterisation scheme developed in this study could be used to help trace the dissemination of y. ruckeri isolates, and thus may represent part of improved disease monitoring plans in the future. enteric red mouth diseaserep-pcreric-pcrbox-pcrpfgenon-motile strains 