BACKGROUND
the guillain-barré syndrome  is the most frequent form of acute immune-mediated neuropathy.
the miller fisher syndrome  is a variant of gbs, affecting mainly the eye muscles  <cit> .
a respiratory or gastro-intestinal infection preceding the neurological symptoms is reported by nearly two-thirds of all patients  <cit> .
the most frequently identified infectious agent is campylobacter jejuni, which is also the predominant cause of bacterial diarrhoea worldwide  <cit> .
the neuropathy is probably induced by molecular mimicry between gangliosides in nerve tissue and lipo-oligosaccharides  on the campylobacter cell surface  <cit> .
this structural resemblance leads to a cross-reactive immune response causing neurological damage.
biochemical and serological studies have revealed that many c. jejuni strains express ganglioside-like structures in their los  <cit> .
however, not all strains expressing ganglioside mimics induce gbs.
it is estimated that only  <dig> in every 1000– <dig> c. jejuni infections is followed by gbs  <cit> , which suggests that additional bacterial determinants and/or host-related factors are important as well.
many researchers have studied collections of gbs-associated and control "enteritis-only" strains in search of gbs-specific microbial features.
several reports describe an overrepresentation of specific penner  serotypes among gbs-associated strains from certain geographical areas  <cit> .
the hs: <dig> and hs: <dig> serotypes are the predominant serotypes preceding gbs in japan and south africa, respectively  <cit> .
because hs: <dig> and hs: <dig> strains represent a clonal population  <cit> , the observed overrepresentation of these serotypes does not imply that the determinant of the penner serotyping system, the capsular polysaccharide, is involved in the pathogenesis of gbs  <cit> .
in addition, this phenomenon is not seen in other regions, where gbs-associated strains are genetically heterogeneous  <cit> .
various molecular typing techniques have been used in search of gbs-specific features in c. jejuni, such as flaa-pcr-restriction fragment length polymorphism , pulsed field gel electrophoresis , randomly amplified polymorphic dna  analysis, ribotyping, amplified fragment length polymorphism  analysis and multi locus sequence typing , but none of these have identified gbs-specific markers  <cit> .
very recently, leonard et al.
also failed to detect gbs-specific features by the use of an open reading frame -specific c. jejuni dna microarray  <cit> .
however, this array was based on the genome sequence of strain nctc  <dig> and orfs that are not present in this strain will not be detected.
in addition, possible gbs-factors other than those relating to presence or absence of certain genes will not be detected using this approach.
based on the molecular mimicry hypothesis, other researchers focused on genes involved in los biosynthesis and found significant associations with gbs  <cit> .
however, these associations are not absolute and the question remains whether other gbs-specific microbial factors, either los-related or not, may exist.
comparative genomics technology facilitates genetic marker identification but not all methods may be equally suited for high-throughput marker searches.
molecular typing techniques for campylobacter strains differ in sensitivity and the overall coverage of genome regions screened.
for the detection of specific disease markers it is desirable to use a technique that screens diversity in the overall genome with a very high resolution.
mlst and flaa pcr-rflp analyze restricted parts of the genome and are not suitable for the detection of additional gbs-markers  <cit> .
pfge is based on digestion of genomic dna with a rare cutting restriction enzyme and only large insertions or deletions and mutations in the restriction sites will be detected  <cit> .
pfge patterns normally display between  <dig> and  <dig> fragments, which covers  <dig> nucleotides when a six nucleotide restriction enzyme recognition sequence is involved.
aflp analysis is considerably more sensitive than pfge for the detection of dna sequence polymorphism.
in a conventional aflp analysis, the use of two restriction enzymes and a primer pair with  <dig> or  <dig> selective nucleotides leads to a dna fingerprint pattern consisting of approximately 50– <dig> fragments per strain.
this approach physically covers in the order of 600– <dig> nucleotides of the total genome for sequence polymorphism  <cit> .
even the use of two restriction enzymes in pfge will not make up for the difference observed under a single aflp reaction.
as indicated above, dna microarrays cover the full genome but will only detect differences in the presence of known genes.
recently, we described a new high throughput aflp  approach for the identification of dna polymorphism in mycobacterium tuberculosis  <cit> .
this method has the capacity to detect mutations in more than  <dig>  nucleotides scattered throughout the genome, depending on the number of restriction enzymes and primer pairs used.
the choice of primers and restriction enzymes is crucial and selection of these requires close attention.
a wrong choice may lead to crowding of amplified fragments, caused by limiting  resolution of the gelsystem.
correct, computer-mediated comparison of aflp fingerprints may then be compromised.
however, especially the enhanced resolution makes htaflp an excellent candidate technique for the detection of potential disease-associated markers.
the main objective of the current study was to search an elaborate collection of c. jejuni stains isolated from gbs patients for genetic markers associated with bacterial neuropathogenicity.
to this aim, we developed htaflp for c. jejuni.
we analyzed six isolates of strain nctc  <dig>  the "genome" strain, obtained from different laboratories, with the aim to detect base-level polymorphism introduced by sub-culturing or storage.
in search of potential gbs-specific markers, we compared the nctc  <dig> aflp patterns with those of four gbs-associated strains.
in addition, we analyzed a collection of highly clonal gbs-associated and control strains from south africa.
potential gbs-specific htaflp markers were further identified by dna sequencing and pcr-rflp tests were developed.
these pcr-rflp tests were used to screen a larger collection of gbs-associated and control strains for confirmation of the potential gbs markers.
RESULTS
detection and identification of dna sequence polymorphism in nctc  <dig> strains of diverse origin
htaflp analysis of c. jejuni was performed with one enzyme combination.
genomic dna was digested with mboi and ddei and the restricted dna was amplified by using all  <dig> possible combinations of +1/+ <dig> selective primer pairs.
this resulted in the generation of approximately  <dig> fragments per strain.
the average fragment size was  <dig> basepairs , ranging from  <dig> to  <dig> bp.
comparative htaflp analysis of the six nctc  <dig> isolates revealed  <dig> polymorphic fragments.
after excision from the gel, these fragments were amplified and the dna sequences were determined.
blast analysis of the dna sequences resulted in the identification of 13/ <dig> polymorphisms, which were spread throughout the genome .
for the other polymorphic fragments, repeated amplification and sequencing failed to generate dna sequences of sufficient quality for blast analysis.
validation of nctc  <dig> polymorphism with a pcr-rflp approach
to verify whether the htaflp-polymorphic fragments represent true dna sequence polymorphism, we analyzed the six nctc  <dig> isolates by pcr-rflp analysis.
an aflp polymorphism that is based on mutations in the restriction site will result in a polymorphic rflp pattern, whereas insertions or deletions in the aflp fragment will result in size differences between the pcr products.
based on the blast hit sequences , pcr tests for amplifying twelve marker fragments and their flanking regions were developed.
fragments of correct size were produced with all primer sets and for all isolates .
next, pcr products of the six nctc  <dig> strains were digested in separate reactions with the aflp restriction enzymes .
none of the digests showed polymorphic rflp patterns.
thus, restriction site polymorphism or insertions/deletions could not be confirmed as cause of the observed aflp polymorphisms.
nine out of twelve  digests with ddei and six of twelve  digests with mboi resulted in rflp patterns as expected based on the nctc  <dig> dna sequence.
an aflp polymorphism can also be the result of a mutation in the nucleotides complementary to the selective primer nucleotides.
because all  <dig> possible combinations of the +1/+ <dig> primer pairs were used in this htaflp, such a mutation would be expected to result in an additional polymorphism, with the same fragment length and localization on the genome, in the aflp pattern generated with a different primer pair.
we did not detect such complementary polymorphisms in the nctc  <dig> comparison.
in conclusion, the polymorphic aflp bands observed in the nctc  <dig> comparison, representing approximately  <dig> % of all bands, probably represent the low "background noise" of the htaflp technique.
comparison of nctc  <dig> with gbs-associated strains for the detection and identification of potential markers for gbs
for the detection of putative markers for the guillain-barré syndrome, we compared the nctc  <dig> isolates with strain gb <dig>  which was isolated from a gbs patient.
strain nctc  <dig> was originally isolated from a patient with gastroenteritis without neurological symptoms.
it had previously been shown that gb <dig> and nctc  <dig> are genetically closely related  <cit> .
because of this relatedness, these strains are very suitable for the detection of potential gbs-specific markers.
htaflp analysis of nctc  <dig> and gb <dig> generated  <dig> putative gbs markers.
overall,  <dig> of  <dig> markers could be successfully identified with dna sequencing and blast analysis.
a proportion of the marker fragments that were excised from the gel could not be reliably reamplified and were excluded from the analysis.
although blast searches were conducted against all dna sequences in the pubmed database, the most significant homology for all aflp dna sequences was with c. jejuni dna sequences.
to further reduce this excessive number of putative gbs markers, we analyzed three additional gbs-associated strains, not related to the nctc  <dig> and gb <dig> strains .
this reduced the number of successfully sequenced putative gbs markers to  <dig> .
three of these markers were located in the los biosynthesis gene locus.
other genes encoded a putative periplasmic protein and proteins involved in signal transduction, metabolism, transport, binding, amino acid biosynthesis, fatty acid biosynthesis and dna replication.
three genes were of unknown function.
markers  <dig> and  <dig> displayed distinct restriction site polymorphism concordant with the aflp polymorphism.
these markers contained largely overlapping dna sequences and showed a complementary pattern of presence and absence in the gbs-associated and control strains .
comparison of the dna sequences revealed that these markers were based on one dna polymorphism: the presence of an additional restriction site in the gbs strains due to a point mutation .
screening of a large strain collection for potential gbs markers by pcr-rflp analysis
after htaflp analysis of five strains to identify potential gbs markers, we developed pcr-rflp tests to screen a large collection of  <dig> gbs/mfs-associated and  <dig> control enteritis strains for the presence of these markers .
the strains used in the htaflp analysis were also included in the pcr-rflp analysis.
one randomly selected nctc  <dig> isolate was included.
based on the blast hit sequences , pcr tests for amplifying 11/ <dig> marker fragments and their flanking regions were developed .
because markers  <dig> and  <dig> represented the same dna polymorphism, they were included in one pcr test.
fragments of correct, expected size were produced with all primer sets.
for 5/ <dig> markers a pcr product was absent in a variable proportion of strains , probably due to primer site sequence heterogeneity.
for example, we have observed previously that gene cj <dig>  part of the los biosynthesis gene locus and containing marker  <dig>  shows a large degree of dna sequence heterogeneity between strains .
this leads to primer mismatches and absence of pcr products in a proportion of strains.
in 2/ <dig> pcr tests , the htaflp gbs-associated strains could be distinguished from strain nctc  <dig> through the pattern of presence and absence of pcr products.
pcr products for marker  <dig> were absent in the gbs-associated strains used in the htaflp and present in nctc  <dig>  which seemed to be in contrast with the observation that the original aflp fragment of marker  <dig> was present in the gbs strains and absent in nctc  <dig>  however, this apparent inconsistency can be explained by the fact that the primer sequences of the pcr test were based on the nctc  <dig> dna sequence.
for marker  <dig>  a pcr product was seen significantly more frequently in control enteritis strains  gbs/mfs strains versus 9/ <dig>  control enteritis strains, p =  <dig> ).
next, we subjected the pcr products to a combined digestion with the aflp restriction enzymes .
in 4/ <dig> pcr tests , the rflp types were concordant with the aflp analysis i.e.
the htaflp gbs-associated strains shared the same rflp type whereas nctc  <dig> displayed a different rflp type.
in 3/ <dig> pcr tests , the htaflp gbs-associated strains did not have identical rflp types , but these rflp types were also different from that of nctc  <dig>  this is not necessarily in contrast with the htaflp results, because different rflp types among the htaflp gbs-associated strains may be due to heterogeneity in the flanking regions of the aflp fragment.
for marker  <dig>  the rflp types of the htaflp strains were not concordant with the htaflp polymorphism: the nctc  <dig> and gb <dig> rflp types were the same.
rflp types  <dig> and  <dig> of marker  <dig>  located in gene cj1139c, were only detected in gbs/mfs-associated strains  gbs/mfs-associated strains versus 0/ <dig>  enteritis strains, p < <dig> ).
although a pcr product for marker  <dig>  located in gene cj <dig>  was absent in the majority of strains, rflp type  <dig> was more frequently found in enteritis strains .
analysis of south-african gbs-associated and control hs: <dig> strains by htaflp
in south africa, serotype hs: <dig> is over-represented among gbs-associated strains  <cit> .
previous studies have shown that hs: <dig> strains, both gbs-associated and controls, are highly clonal  <cit> .
as expected, htaflp of six gbs-associated, one mfs-associated and six control hs: <dig> strains generated very homogeneous banding patterns .
a total of forty-five aflp polymorphisms were detected, but there were no gbs-specific markers.
interestingly,  <dig> aflp polymorphisms displayed a similar pattern: fragments were present in the mfs-associated strain and two or three control enteritis strains but absent in the other strains .
these fragments were excised and dna sequences were determined.
blast analysis of five dna sequences revealed homologies with various bacterial plasmidal dna sequences .
discussion
this study describes a high-throughput aflp approach for the detection and identification of dna polymorphism and putative gbs-markers in c. jejuni.
previously, we showed that htaflp is an excellent tool for assessing the population structure and the expansion of pathogenic clones in staphylococcus aureus and for identification of genetic polymorphism in the clonal microorganism mycobacterium tuberculosis  <cit> .
the optimal enzyme and selective primer pair combinations are determined by in silico calculations using the whole genome dna sequence of the target microorganism.
the optimal number of aflp fragments to be generated depends on the aim of the study.
for the detection and identification of potential disease markers, such as gbs-specific markers in c. jejuni in the current study, it is desirable to screen the genome with high resolution.
for this, the generation of a large number of aflp fragments per strain is needed.
such high resolution aflp approach limits the number of strains that can be analyzed, but this can be overcome by the subsequent analysis of a large number of strains by pcr-rflp tests, translated from the potential markers as detected by the preceding htaflp analysis.
it is, of course, possible that a disease-specific marker is not detected by htaflp because this approach does not result in 100% genome coverage, which can only be reached with whole genome sequencing.
for c. jejuni, one enzyme combination  and  <dig> different +1/+ <dig> selective primer pair combinations physically covered approximately  <dig>  nucleotides per strain, which represents approximately 2% of the genome.
to find out whether subculturing or storage of c. jejuni strains leads to the emergence of dna polymorphism, we compared  <dig> isolates of the "genome" strain nctc  <dig> obtained from different laboratories worldwide by htaflp analysis.
the observed aflp polymorphisms, approximately  <dig> % of the fragments per strain, could not be confirmed by pcr-rflp analysis.
this indicates that the aflp polymorphisms probably represent the low background noise of the htaflp technique and that true dna polymorphism could not be detected in the six nctc  <dig> isolates.
the observed background noise equals a reproducibility of approximately  <dig> %, which is still very high when compared to other genotyping techniques  <cit> .
recently, two groups described differences in virulence properties, i.e.
the ability to colonise chickens, between different nctc  <dig> isolates that were not included in the current study  <cit> .
full transcriptional profiling revealed expression differences for several gene families in the nctc  <dig> strains.
htaflp analysis of the two nctc  <dig> isolates that were studied by carrillo et al.
<cit>  failed to identify polymorphisms responsible for the difference in virulence properties .
it has to be emphasized that by htaflp still only a random proportion of the genome is screened.
dna sequence variation  leading to biological differences may therefore not be detected.
in search of gbs-specific markers, we first compared the nctc  <dig> patterns with the aflp patterns of the genetically related gbs-associated strain gb <dig>  however, although nctc  <dig> was originally isolated from the faeces of a patient with gastroenteritis, we cannot exclude that nctc  <dig> can induce gbs if a patient with the right host susceptibility factors becomes infected with this strain.
the only substantial but probably very important difference between nctc  <dig> and gb <dig> that has been found so far, is that the los biosynthesis gene locus strongly diverges between these strains, probably as result of a horizontal exchange event  <cit> .
comparison of nctc  <dig> with gb <dig> led to the detection of more than two hundred possible gbs markers, which was substantially higher than the expected background noise of  <dig> %, underscoring the phylogenetic relevance of the polymorphisms.
the number of possible gbs markers was reduced to  <dig> after adding three additional gbs-associated strains.
for  <dig> markers, the location on the genome could be identified after dna sequence analysis.
a relatively large proportion of potential gbs markers  was located in the los biosynthesis gene locus, whereas this locus only comprises 1% of the c. jejuni genome .
although this may represent a true pathogenic association with gbs, it is also possible that htaflp preferentially picked up the los locus because it is a highly polymorphic region.
however, analysis of a larger c. jejuni strain collection by pcr-rflp analysis showed that the three los-specific markers were indeed associated with gbs .
these findings are concordant with the proposed pathogenic mechanism of gbs and with previous reports that certain genes involved in los biosynthesis or specific nucleotide sequences within these genes occur more frequently in gbs-associated c. jejuni strains  <cit> .
there are several possible explanations for the fact that we did not find molecular markers that are 100% specific for the guillain-barré syndrome.
first, it is possible that truly gbs-specific features do not exist in c. jejuni.
there is a broad variability in the severity and spectrum of clinical symptoms in gbs patients  <cit> .
different ganglioside mimicking structures and anti-ganglioside antibody specificities have been associated with certain clinical presentations  <cit> , and therefore, c. jejuni markers may be associated with a subset of various disease entities.
because of this heterogeneity and the presumed importance of host-related factors, the existence of features in c. jejuni that are specific for gbs may be questionable.
second, a certain combination of multiple c. jejuni genes may be required for the induction of gbs.
detection of such combinations of markers  is extremely labour-intensive and cannot be achieved with the current approach.
finally, it is possible that htaflp failed to detect gbs-specific markers because htaflp does not accomplish 100% genome coverage.
one of the three additional gbs-associated strains mentioned above was from a collection of south-african hs: <dig> strains.
in south africa, the hs: <dig> serotype is over-represented among gbs-associated strains  <cit> .
a certain feature of these strains may be responsible for their capacity to trigger gbs.
hs: <dig> strains were found to be indistinguishable by previous genotyping studies, indicating that hs: <dig> strains form a genetically stable clone  <cit> .
it is important to note that the enteritis-only hs: <dig> strains may have the same gbs-inducing capacity as the gbs-associated strains, because host-related factors are also crucial for developing gbs.
we analyzed both gbs-associated and control enteritis-only hs: <dig> strains, as well as an mfs-associated isolate by htaflp.
although we did not detect gbs-specific bands, we found that the mfs-associated isolate and half of the enteritis-only strains contained several additional fragments that appeared to be linked.
dna sequences of these fragments showed homologies with plasmidal dna sequences, indicating that a subset of the hs: <dig> strains contained a plasmid.
to our knowledge, the south african hs: <dig> strains we used in this study have never been analyzed for the presence of plasmids.
whether the presence of a plasmid is of importance for the virulence or neuropathogenic potential of hs: <dig> strains currently remains unknown, but seems unlikely based on the distribution of plasmidal dna in the tested strains.
CONCLUSIONS
previous searches for c. jejuni markers for gbs-invoking potential were unsuccessful when performed with general genotyping techniques.
some studies that focussed at specific loci or sometimes even specific genes found potential, though not absolute, gbs markers within the los biosynthesis genes  <cit> .
we have used a method, htaflp, that detects sequence polymorphisms in a wide, non-gene dependent scale.
theoretically approximately 2% of the total genome is covered by this approach.
however, we still conclude that bacterial gbs markers are not absolute, limited in number and located in the los biosynthesis gene locus.
this corroborates the current consensus that los mimicry with human gangliosides may be the prime etiologic determinant of gbs.
in addition to bacterial factors, host-related factors probably play an important role in the pathogenesis of gbs as well.
furthermore, our results demonstrate that htaflp, with its high reproducibility and resolution, is an adequate technique for the detection and subsequent identification of putative disease and epidemiological markers.
analysis of a limited number of strains in great detail by htaflp and subsequent screening of a large collection of strains with simple pcr-rflp tests combines high sensitivity with the possibility to screen large groups of strains.
this allows for the identification of regions of genomic instability or variability.
finally, htaflp does not require complete genome sequences and it is not influenced by the presence of sequences not present in the genome strain.
as such, htaflp is the second best option, after complete sequencing of the genome of multiple strains, for the unbiased detection of genome polymorphisms associated with pathogenicity or other features of bacterial isolates from diverse clinical and environmental origin.

 the guillain-barré syndrome is the most frequent form of acute immune-mediated neuropathy. the miller fisher syndrome is a variant of gbs, affecting mainly the eye muscles. a respiratory or gastro-intestinal infection preceding the neurological symptoms is reported by nearly two-thirds of all patients. the most frequently identified infectious agent is campylobacter jejuni, which is also the predominant cause of bacterial diarrhoea worldwide. the neuropathy is probably induced by molecular mimicry between gangliosides in nerve tissue and lipo-oligosaccharides on the campylobacter cell surface. this structural resemblance leads to a cross-reactive immune response causing neurological damage. biochemical and serological studies have revealed that many c. jejuni strains express ganglioside-like structures in their los. however, not all strains expressing ganglioside mimics induce gbs. it is estimated that only in every 1000– c. jejuni infections is followed by gbs, which suggests that additional bacterial determinants and/or host-related factors are important as well. many researchers have studied collections of gbs-associated and control "enteritis-only" strains in search of gbs-specific microbial features. several reports describe an overrepresentation of specific penner serotypes among gbs-associated strains from certain geographical areas. the hs: and hs: serotypes are the predominant serotypes preceding gbs in japan and south africa, respectively. because hs: and hs: strains represent a clonal population, the observed overrepresentation of these serotypes does not imply that the determinant of the penner serotyping system, the capsular polysaccharide, is involved in the pathogenesis of gbs. in addition, this phenomenon is not seen in other regions, where gbs-associated strains are genetically heterogeneous. various molecular typing techniques have been used in search of gbs-specific features in c. jejuni, such as flaa-pcr-restriction fragment length polymorphism, pulsed field gel electrophoresis, randomly amplified polymorphic dna analysis, ribotyping, amplified fragment length polymorphism analysis and multi locus sequence typing, but none of these have identified gbs-specific markers. very recently, leonard et al. also failed to detect gbs-specific features by the use of an open reading frame -specific c. jejuni dna microarray. however, this array was based on the genome sequence of strain nctc and orfs that are not present in this strain will not be detected. in addition, possible gbs-factors other than those relating to presence or absence of certain genes will not be detected using this approach. based on the molecular mimicry hypothesis, other researchers focused on genes involved in los biosynthesis and found significant associations with gbs. however, these associations are not absolute and the question remains whether other gbs-specific microbial factors, either los-related or not, may exist. comparative genomics technology facilitates genetic marker identification but not all methods may be equally suited for high-throughput marker searches. molecular typing techniques for campylobacter strains differ in sensitivity and the overall coverage of genome regions screened. for the detection of specific disease markers it is desirable to use a technique that screens diversity in the overall genome with a very high resolution. mlst and flaa pcr-rflp analyze restricted parts of the genome and are not suitable for the detection of additional gbs-markers. pfge is based on digestion of genomic dna with a rare cutting restriction enzyme and only large insertions or deletions and mutations in the restriction sites will be detected. pfge patterns normally display between and fragments, which covers nucleotides when a six nucleotide restriction enzyme recognition sequence is involved. aflp analysis is considerably more sensitive than pfge for the detection of dna sequence polymorphism. in a conventional aflp analysis, the use of two restriction enzymes and a primer pair with or selective nucleotides leads to a dna fingerprint pattern consisting of approximately 50– fragments per strain. this approach physically covers in the order of 600– nucleotides of the total genome for sequence polymorphism. even the use of two restriction enzymes in pfge will not make up for the difference observed under a single aflp reaction. as indicated above, dna microarrays cover the full genome but will only detect differences in the presence of known genes. recently, we described a new high throughput aflp approach for the identification of dna polymorphism in mycobacterium tuberculosis. this method has the capacity to detect mutations in more than nucleotides scattered throughout the genome, depending on the number of restriction enzymes and primer pairs used. the choice of primers and restriction enzymes is crucial and selection of these requires close attention. a wrong choice may lead to crowding of amplified fragments, caused by limiting resolution of the gelsystem. correct, computer-mediated comparison of aflp fingerprints may then be compromised. however, especially the enhanced resolution makes htaflp an excellent candidate technique for the detection of potential disease-associated markers. the main objective of the current study was to search an elaborate collection of c. jejuni stains isolated from gbs patients for genetic markers associated with bacterial neuropathogenicity. to this aim, we developed htaflp for c. jejuni. we analyzed six isolates of strain nctc the "genome" strain, obtained from different laboratories, with the aim to detect base-level polymorphism introduced by sub-culturing or storage. in search of potential gbs-specific markers, we compared the nctc aflp patterns with those of four gbs-associated strains. in addition, we analyzed a collection of highly clonal gbs-associated and control strains from south africa. potential gbs-specific htaflp markers were further identified by dna sequencing and pcr-rflp tests were developed. these pcr-rflp tests were used to screen a larger collection of gbs-associated and control strains for confirmation of the potential gbs markers.  detection and identification of dna sequence polymorphism in nctc strains of diverse origin htaflp analysis of c. jejuni was performed with one enzyme combination. genomic dna was digested with mboi and ddei and the restricted dna was amplified by using all possible combinations of +1/+ selective primer pairs. this resulted in the generation of approximately fragments per strain. the average fragment size was basepairs, ranging from to bp. comparative htaflp analysis of the six nctc isolates revealed polymorphic fragments. after excision from the gel, these fragments were amplified and the dna sequences were determined. blast analysis of the dna sequences resulted in the identification of 13/ polymorphisms, which were spread throughout the genome. for the other polymorphic fragments, repeated amplification and sequencing failed to generate dna sequences of sufficient quality for blast analysis. validation of nctc polymorphism with a pcr-rflp approach to verify whether the htaflp-polymorphic fragments represent true dna sequence polymorphism, we analyzed the six nctc isolates by pcr-rflp analysis. an aflp polymorphism that is based on mutations in the restriction site will result in a polymorphic rflp pattern, whereas insertions or deletions in the aflp fragment will result in size differences between the pcr products. based on the blast hit sequences, pcr tests for amplifying twelve marker fragments and their flanking regions were developed. fragments of correct size were produced with all primer sets and for all isolates. next, pcr products of the six nctc strains were digested in separate reactions with the aflp restriction enzymes. none of the digests showed polymorphic rflp patterns. thus, restriction site polymorphism or insertions/deletions could not be confirmed as cause of the observed aflp polymorphisms. nine out of twelve digests with ddei and six of twelve digests with mboi resulted in rflp patterns as expected based on the nctc dna sequence. an aflp polymorphism can also be the result of a mutation in the nucleotides complementary to the selective primer nucleotides. because all possible combinations of the +1/+ primer pairs were used in this htaflp, such a mutation would be expected to result in an additional polymorphism, with the same fragment length and localization on the genome, in the aflp pattern generated with a different primer pair. we did not detect such complementary polymorphisms in the nctc comparison. in conclusion, the polymorphic aflp bands observed in the nctc comparison, representing approximately % of all bands, probably represent the low "background noise" of the htaflp technique. comparison of nctc with gbs-associated strains for the detection and identification of potential markers for gbs for the detection of putative markers for the guillain-barré syndrome, we compared the nctc isolates with strain gb which was isolated from a gbs patient. strain nctc was originally isolated from a patient with gastroenteritis without neurological symptoms. it had previously been shown that gb and nctc are genetically closely related. because of this relatedness, these strains are very suitable for the detection of potential gbs-specific markers. htaflp analysis of nctc and gb generated putative gbs markers. overall, of markers could be successfully identified with dna sequencing and blast analysis. a proportion of the marker fragments that were excised from the gel could not be reliably reamplified and were excluded from the analysis. although blast searches were conducted against all dna sequences in the pubmed database, the most significant homology for all aflp dna sequences was with c. jejuni dna sequences. to further reduce this excessive number of putative gbs markers, we analyzed three additional gbs-associated strains, not related to the nctc and gb strains. this reduced the number of successfully sequenced putative gbs markers to. three of these markers were located in the los biosynthesis gene locus. other genes encoded a putative periplasmic protein and proteins involved in signal transduction, metabolism, transport, binding, amino acid biosynthesis, fatty acid biosynthesis and dna replication. three genes were of unknown function. markers and displayed distinct restriction site polymorphism concordant with the aflp polymorphism. these markers contained largely overlapping dna sequences and showed a complementary pattern of presence and absence in the gbs-associated and control strains. comparison of the dna sequences revealed that these markers were based on one dna polymorphism: the presence of an additional restriction site in the gbs strains due to a point mutation. screening of a large strain collection for potential gbs markers by pcr-rflp analysis after htaflp analysis of five strains to identify potential gbs markers, we developed pcr-rflp tests to screen a large collection of gbs/mfs-associated and control enteritis strains for the presence of these markers. the strains used in the htaflp analysis were also included in the pcr-rflp analysis. one randomly selected nctc isolate was included. based on the blast hit sequences, pcr tests for amplifying 11/ marker fragments and their flanking regions were developed. because markers and represented the same dna polymorphism, they were included in one pcr test. fragments of correct, expected size were produced with all primer sets. for 5/ markers a pcr product was absent in a variable proportion of strains, probably due to primer site sequence heterogeneity. for example, we have observed previously that gene cj part of the los biosynthesis gene locus and containing marker shows a large degree of dna sequence heterogeneity between strains. this leads to primer mismatches and absence of pcr products in a proportion of strains. in 2/ pcr tests, the htaflp gbs-associated strains could be distinguished from strain nctc through the pattern of presence and absence of pcr products. pcr products for marker were absent in the gbs-associated strains used in the htaflp and present in nctc which seemed to be in contrast with the observation that the original aflp fragment of marker was present in the gbs strains and absent in nctc however, this apparent inconsistency can be explained by the fact that the primer sequences of the pcr test were based on the nctc dna sequence. for marker a pcr product was seen significantly more frequently in control enteritis strains gbs/mfs strains versus 9/ control enteritis strains, p = ). next, we subjected the pcr products to a combined digestion with the aflp restriction enzymes. in 4/ pcr tests, the rflp types were concordant with the aflp analysis i.e. the htaflp gbs-associated strains shared the same rflp type whereas nctc displayed a different rflp type. in 3/ pcr tests, the htaflp gbs-associated strains did not have identical rflp types, but these rflp types were also different from that of nctc this is not necessarily in contrast with the htaflp results, because different rflp types among the htaflp gbs-associated strains may be due to heterogeneity in the flanking regions of the aflp fragment. for marker the rflp types of the htaflp strains were not concordant with the htaflp polymorphism: the nctc and gb rflp types were the same. rflp types and of marker located in gene cj1139c, were only detected in gbs/mfs-associated strains gbs/mfs-associated strains versus 0/ enteritis strains, p < ). although a pcr product for marker located in gene cj was absent in the majority of strains, rflp type was more frequently found in enteritis strains. analysis of south-african gbs-associated and control hs: strains by htaflp in south africa, serotype hs: is over-represented among gbs-associated strains. previous studies have shown that hs: strains, both gbs-associated and controls, are highly clonal. as expected, htaflp of six gbs-associated, one mfs-associated and six control hs: strains generated very homogeneous banding patterns. a total of forty-five aflp polymorphisms were detected, but there were no gbs-specific markers. interestingly, aflp polymorphisms displayed a similar pattern: fragments were present in the mfs-associated strain and two or three control enteritis strains but absent in the other strains. these fragments were excised and dna sequences were determined. blast analysis of five dna sequences revealed homologies with various bacterial plasmidal dna sequences. discussion this study describes a high-throughput aflp approach for the detection and identification of dna polymorphism and putative gbs-markers in c. jejuni. previously, we showed that htaflp is an excellent tool for assessing the population structure and the expansion of pathogenic clones in staphylococcus aureus and for identification of genetic polymorphism in the clonal microorganism mycobacterium tuberculosis. the optimal enzyme and selective primer pair combinations are determined by in silico calculations using the whole genome dna sequence of the target microorganism. the optimal number of aflp fragments to be generated depends on the aim of the study. for the detection and identification of potential disease markers, such as gbs-specific markers in c. jejuni in the current study, it is desirable to screen the genome with high resolution. for this, the generation of a large number of aflp fragments per strain is needed. such high resolution aflp approach limits the number of strains that can be analyzed, but this can be overcome by the subsequent analysis of a large number of strains by pcr-rflp tests, translated from the potential markers as detected by the preceding htaflp analysis. it is, of course, possible that a disease-specific marker is not detected by htaflp because this approach does not result in 100% genome coverage, which can only be reached with whole genome sequencing. for c. jejuni, one enzyme combination and different +1/+ selective primer pair combinations physically covered approximately nucleotides per strain, which represents approximately 2% of the genome. to find out whether subculturing or storage of c. jejuni strains leads to the emergence of dna polymorphism, we compared isolates of the "genome" strain nctc obtained from different laboratories worldwide by htaflp analysis. the observed aflp polymorphisms, approximately % of the fragments per strain, could not be confirmed by pcr-rflp analysis. this indicates that the aflp polymorphisms probably represent the low background noise of the htaflp technique and that true dna polymorphism could not be detected in the six nctc isolates. the observed background noise equals a reproducibility of approximately %, which is still very high when compared to other genotyping techniques. recently, two groups described differences in virulence properties, i.e. the ability to colonise chickens, between different nctc isolates that were not included in the current study. full transcriptional profiling revealed expression differences for several gene families in the nctc strains. htaflp analysis of the two nctc isolates that were studied by carrillo et al. failed to identify polymorphisms responsible for the difference in virulence properties. it has to be emphasized that by htaflp still only a random proportion of the genome is screened. dna sequence variation leading to biological differences may therefore not be detected. in search of gbs-specific markers, we first compared the nctc patterns with the aflp patterns of the genetically related gbs-associated strain gb however, although nctc was originally isolated from the faeces of a patient with gastroenteritis, we cannot exclude that nctc can induce gbs if a patient with the right host susceptibility factors becomes infected with this strain. the only substantial but probably very important difference between nctc and gb that has been found so far, is that the los biosynthesis gene locus strongly diverges between these strains, probably as result of a horizontal exchange event. comparison of nctc with gb led to the detection of more than two hundred possible gbs markers, which was substantially higher than the expected background noise of %, underscoring the phylogenetic relevance of the polymorphisms. the number of possible gbs markers was reduced to after adding three additional gbs-associated strains. for markers, the location on the genome could be identified after dna sequence analysis. a relatively large proportion of potential gbs markers was located in the los biosynthesis gene locus, whereas this locus only comprises 1% of the c. jejuni genome. although this may represent a true pathogenic association with gbs, it is also possible that htaflp preferentially picked up the los locus because it is a highly polymorphic region. however, analysis of a larger c. jejuni strain collection by pcr-rflp analysis showed that the three los-specific markers were indeed associated with gbs. these findings are concordant with the proposed pathogenic mechanism of gbs and with previous reports that certain genes involved in los biosynthesis or specific nucleotide sequences within these genes occur more frequently in gbs-associated c. jejuni strains. there are several possible explanations for the fact that we did not find molecular markers that are 100% specific for the guillain-barré syndrome. first, it is possible that truly gbs-specific features do not exist in c. jejuni. there is a broad variability in the severity and spectrum of clinical symptoms in gbs patients. different ganglioside mimicking structures and anti-ganglioside antibody specificities have been associated with certain clinical presentations, and therefore, c. jejuni markers may be associated with a subset of various disease entities. because of this heterogeneity and the presumed importance of host-related factors, the existence of features in c. jejuni that are specific for gbs may be questionable. second, a certain combination of multiple c. jejuni genes may be required for the induction of gbs. detection of such combinations of markers is extremely labour-intensive and cannot be achieved with the current approach. finally, it is possible that htaflp failed to detect gbs-specific markers because htaflp does not accomplish 100% genome coverage. one of the three additional gbs-associated strains mentioned above was from a collection of south-african hs: strains. in south africa, the hs: serotype is over-represented among gbs-associated strains. a certain feature of these strains may be responsible for their capacity to trigger gbs. hs: strains were found to be indistinguishable by previous genotyping studies, indicating that hs: strains form a genetically stable clone. it is important to note that the enteritis-only hs: strains may have the same gbs-inducing capacity as the gbs-associated strains, because host-related factors are also crucial for developing gbs. we analyzed both gbs-associated and control enteritis-only hs: strains, as well as an mfs-associated isolate by htaflp. although we did not detect gbs-specific bands, we found that the mfs-associated isolate and half of the enteritis-only strains contained several additional fragments that appeared to be linked. dna sequences of these fragments showed homologies with plasmidal dna sequences, indicating that a subset of the hs: strains contained a plasmid. to our knowledge, the south african hs: strains we used in this study have never been analyzed for the presence of plasmids. whether the presence of a plasmid is of importance for the virulence or neuropathogenic potential of hs: strains currently remains unknown, but seems unlikely based on the distribution of plasmidal dna in the tested strains.  previous searches for c. jejuni markers for gbs-invoking potential were unsuccessful when performed with general genotyping techniques. some studies that focussed at specific loci or sometimes even specific genes found potential, though not absolute, gbs markers within the los biosynthesis genes. we have used a method, htaflp, that detects sequence polymorphisms in a wide, non-gene dependent scale. theoretically approximately 2% of the total genome is covered by this approach. however, we still conclude that bacterial gbs markers are not absolute, limited in number and located in the los biosynthesis gene locus. this corroborates the current consensus that los mimicry with human gangliosides may be the prime etiologic determinant of gbs. in addition to bacterial factors, host-related factors probably play an important role in the pathogenesis of gbs as well. furthermore, our results demonstrate that htaflp, with its high reproducibility and resolution, is an adequate technique for the detection and subsequent identification of putative disease and epidemiological markers. analysis of a limited number of strains in great detail by htaflp and subsequent screening of a large collection of strains with simple pcr-rflp tests combines high sensitivity with the possibility to screen large groups of strains. this allows for the identification of regions of genomic instability or variability. finally, htaflp does not require complete genome sequences and it is not influenced by the presence of sequences not present in the genome strain. as such, htaflp is the second best option, after complete sequencing of the genome of multiple strains, for the unbiased detection of genome polymorphisms associated with pathogenicity or other features of bacterial isolates from diverse clinical and environmental origin. 
this study shows that bacterial gbs markers are limited in number and located in the los biosynthesis genes, which corroborates the current consensus that los mimicry may be the prime etiologic determinant of gbs. furthermore, our results demonstrate that htaflp, with its high reproducibility and resolution, is an effective technique for the detection and subsequent identification of putative bacterial disease markers. comparison of nctc with gbs-associated strains revealed potentially gbs-specific markers, of which were identified by dna sequencing. htaflp analysis of highly clonal south african gbs/mfs-associated and enteritis control strains did not reveal gbs-specific markers. gbs and mfs are probably induced by molecular mimicry between human gangliosides and bacterial lipo-oligosaccharides. campylobacter jejuni is the predominant cause of antecedent infection in post-infectious neuropathies such as the guillain-barré and miller fisher syndromes. this study describes a new c. jejuni-specific high-throughput aflp approach for detection and identification of dna polymorphism, in general, and of putative gbs/mfs-markers, in particular. the dna polymorphisms could not be confirmed by pcr-rflp analysis, suggesting a baseline level of % aflp artefacts. we compared different isolates of the "genome strain" nctc obtained from different laboratories. htaflp analysis generated approximately markers per stain, of which were polymorphic. a collection of gbs/mfs-associated and enteritis control strains was analyzed with pcr-rflp tests based on of these markers.
 campylobacter jejuni is the predominant cause of antecedent infection in post-infectious neuropathies such as the guillain-barré and miller fisher syndromes. gbs and mfs are probably induced by molecular mimicry between human gangliosides and bacterial lipo-oligosaccharides. this study describes a new c. jejuni-specific high-throughput aflp approach for detection and identification of dna polymorphism, in general, and of putative gbs/mfs-markers, in particular.  we compared different isolates of the "genome strain" nctc obtained from different laboratories. htaflp analysis generated approximately markers per stain, of which were polymorphic. the dna polymorphisms could not be confirmed by pcr-rflp analysis, suggesting a baseline level of % aflp artefacts. comparison of nctc with gbs-associated strains revealed potentially gbs-specific markers, of which were identified by dna sequencing. a collection of gbs/mfs-associated and enteritis control strains was analyzed with pcr-rflp tests based on of these markers. we identified markers, located in the los biosynthesis genes cj cj and cj1139c, that were significantly associated with gbs. htaflp analysis of highly clonal south african gbs/mfs-associated and enteritis control strains did not reveal gbs-specific markers.  this study shows that bacterial gbs markers are limited in number and located in the los biosynthesis genes, which corroborates the current consensus that los mimicry may be the prime etiologic determinant of gbs. furthermore, our results demonstrate that htaflp, with its high reproducibility and resolution, is an effective technique for the detection and subsequent identification of putative bacterial disease markers. 