BACKGROUND
thermophilic campylobacter species, primarily campylobacter jejuni and c. coli are the most frequently recognized cause of acute bacterial gastroenteritis in humans in the western world.
in relation to human campylobacteriosis, c. upsaliensis, c. hyointestinalis, c. lari, c. fetus and c. sputorum biovar sputorum have also been demonstrated to be implicated as gastrointestinal pathogens though these are rare  <cit> .
these campylobacter organisms have also been isolated from animals.
moreover, c. concisus, c. curvus and so on are detected in association with the oral cavity  <cit> .
alternatively, c. sputorum biovar fecalis is isolated from animals  <cit> .
a multiplex pcr assay has recently developed for the identification of c. coli, c. fetus, c. hyointestinalis subsp.
hyointestinalis, c. jejuni, c. lari and c. upsaliensis  <cit> .
thus, at this time, the genus campylobacter comprises  <dig> species  <cit>
as already shown, the genus campylobacter is, in general, indicated to carry the three copies of rrna gene operon  <cit>
in relation to bacterial 23s rrna genes, the occurrence of intervening sequences   <cit>  and the fragmentation of 23s rrna  <cit>  have been demonstrated.
in the genus campylobacter, the ε-subdivision of the proteobacteria, the occurrence of internal transcribed spacers was first described in helix  <dig> region within 23s rrna gene in two of four c. jejuni, in both c. fetus and in one of two c. upsaliensis strains, when a total of  <dig> campylobacter strains  were examined  <cit> .
in addition, three of seven c. jejuni isolates examined were found to carry fragmented 23s rrna  <cit> .
moreover, the occurrence of fragmented 23s rrna correlated with the presence of an ivs within the 23s rrna genes.
it was described that the presence of transcribed spacers is common in campylobacter spp.
<cit> .
all campylobacter isolates containing transcribed spacers in their 23s rrna gene sequences produced fragmented 23s rrnas  <cit> .
most recently, among  <dig> strains of c. coli from turkeys,  <dig> strains harbored ivss in all three 23s rrna genes, whereas the other  <dig> strains lacked ivss from at least one of the genes  <cit> .
we have already reported the absence of ivss shown in both the helix  <dig>  and  <dig>  regions within 23s rrna genes among a total of  <dig> isolates of c. lari  obtained from different sources and in several countries, by using pcr amplification, ta cloning and sequencing procedures  <cit> .
in addition, the intact 23s rrna was also identified in the c. lari isolates examined, resulting in no production of the fragmented 23s rrna  <cit> .
thus, it would be important to clarify the molecular biological entities of the occurrence and the sequence structures of ivss within the 23s rrna genes in the much more isolates of several other species than c. lari of the genus campylobacter including atypical species.
however, studies on molecular characterization and comparative analysis of ivss within the 23s rrna genes and these 23s rrna fragmentations in much more than  <dig> campylobacter isolates of c. jejuni, c. coli, c. fetus, and some other atypical campylobacter species, namely c. upsaliensis, c. hyointestinalis, c. sputorum biovar sputorum, biovar fecalis, biovar paraureolyticus, c. concisus and c. curvus have not yet been reported.
therefore, we aimed to clarify molecular characteristics of ivss within the 23s rrna gene sequences and 23s rrna fragmentations in these campylobacters other than c. lari, which has already been demonstrated not to harbor any ivss  <cit> .
in addition, the authors wished to comparatively analyze the ivss among the campylobacter organisms.
RESULTS
ivss in the helix  <dig> region
in the present study, two pcr primer pairs, f-/r-cl23h <dig>  designed to generate the helix  <dig>  and, f-/r-cl23h <dig>  the helix  <dig>  regions within the 23s rrna gene sequences with the  <dig> campylobacter isolates were employed.
when pcr was first carried out on the  <dig> isolates using the primer pair , amplicons were generated.
some of the examples are shown in fig.
<dig>  following sequencing and analysis, only the four cases, c. sputorum biovar sputorum lmg <dig> and biovar fecalis lmg <dig>  lmg <dig> and lmg <dig> isolates, were shown to carry ivss in the helix  <dig> region among these isolates of more than  <dig>  the sequence data in the helix  <dig> region from c. sputorum isolates are aligned in fig.
<dig>  as shown in fig.
<dig>  identical ivs occurred in the helix  <dig> region within 23s rrna genes from the four c. sputorum isolates.
regarding the three c. sputorum biovar fecalis isolates, moreover, two different kinds of the 23s rrna genes were identified to occur with and without the ivs, respectively .
ivss in the helix  <dig> region
then, we carried out pcr amplification of the ivss, in the central region  within 23s rrna gene sequences with the  <dig> campylobacter isolates, using the primer pair f-/r-cl23h <dig>  some examples of the pcr amplicons are shown in fig.
<dig>  following sequencing and alignment analyses, in the helix  <dig> region,  <dig> c. hyointestinalis, fourteen c. sputorum biovar sputorum, biovar fecalis and paraureolyticus and  <dig> c. concisus isolates were shown not to carry any ivss.
in addition, however, regarding the other campylobacter organisms examined in the present study,  <dig> of  <dig> c. jejuni ,  <dig> of  <dig> c. coli ,  <dig> of  <dig> c. fetus ,  <dig> of  <dig> c. upsaliensis  and  <dig> of  <dig> c. curvus  isolates were shown to carry the ivss in the helix  <dig> region.
some of the sequence data of the ivss in the helix  <dig> region were aligned in fig.
<dig>  regarding the ivs sequences in the helix  <dig> region, four ivss with similar sequences occurred in the c. jejuni and c. upsaliensis species, respectively, and two also in the c. curvus isolates .
in addition, one kind of ivs with an identical sequence occurred in the c. coli and c. fetus isolates, respectively , moreover, the eight ivss in the c. jejuni and c. upsaliensis isolates showed high sequence similarities to each other , and one kind of ivs in the c. jejuni and c. coli showed an identical sequence .
four kinds of ivss in the c. upsaliensis isolates, interestingly, carried two characteristic insertion sequences of several base pairs  and twenty and several bp at the two positions .
in c. jejuni  and c. upsaliensis , two different kinds of the 23s rrna genes were identified to occur with and without the ivs in the helix  <dig> region.
moreover, multiple and heterogeneous ivss were shown in c. upsaliensis 48- <dig> and 68- <dig> isolates, respectively.
consequently, identification of the ivss within the 23s rrna genes from the  <dig> campylobacter isolates is summarized in the table  <dig>
secondary structure models of the ivss
regarding the ivss identified in the present study, within the 23s rrna gene sequences from the campylobacter isolates examined, secondary structure models were constructed with all the ivss shown in table  <dig>  fig.
<dig> and  <dig> show some examples of the secondary structure models of the ivss in helix  <dig>  and helix  <dig>  regions.
in the present models, stem and loop structures were identified in all ivss.
gel electrophoresis of purified rna
denaturing agarose gel electrophoresis profiles of purified rna from the campylobacter isolates was carried out to clarify if the primary rna transcripts of 23s rrna were fragmented in the isolates or not.
purified rna from e. coli dh5α cells, identified to lack ivss, was also employed as a reference marker .
in the purified rna fraction from the isolates of c. sputorum biovar sputorum lmg <dig> , whose 23s rrna gene was demonstrated to carry ivss in the helix  <dig>  no 23s rrna was evident in the fraction .
instead of the 23s rrna, other smaller rna fragments were identified .
regarding the c. sputorum biovar fecalis lmg <dig>  two large rrna bands consisting of an intact and a fragmented 23s rrnas, were identified to occur in the isolate .
some other examples of 23s rrnas whose genes were identified not to carry ivss in the helix  <dig> region, are also shown in the figure.
.
thus, intact 23s rrnas were identified in campylobacter isolates containing no ivss in the helix  <dig> region.
in addition, in fig.
7b, some of the denaturing agarose gel electrophoresis profiles of purified rna from the campylobacter isolates, whose helix  <dig> regions were examined, are shown.
no 23s rrna and fragmented other smaller rna fragments were evident in the some purified rna fractions, and intact 23s rrnas were evident in other rna fractions.
in relation to the 16s rrna molecules from the four isolates of c. sputorum biovar sputorum lmg <dig> , biovar fecalis lmg <dig>  and lmg <dig>  and c. curvus lmg <dig> , surprisingly, slightly shorter rnas than the 16s were identified in these isolates, instead of the 16s rrna species.
discussion
we have already shown no ivss, in the helix  <dig> regions within the 23s rrna genes among a total of  <dig> isolates of c. lari [n =  <dig> un c. lari; n =  <dig> uptc  <cit> .
consequently, in  <dig> isolates of  <dig> campylobacter isolates of the nine species  examined, the absence of ivss was identified in helix  <dig> region within 23s rrna genes.
moreover, until now, no ivss have been identified in the helix  <dig> region within 23s rrna genes, from more than  <dig> campylobacter isolates of the  <dig> species  by other research groups  <cit> .
thus, ivs is extremely rare in the helix  <dig> region within the 23s rrna genes from the campylobacter organisms.
therefore, this is the first scientifically significant report of ivss in the helix  <dig> from c. sputorum biovar sputorum and biovar fecalis among campylobacter organisms.
in addition, no ivss have been identified to occur in the helix  <dig> from c. sputorum strains   <cit> .
regarding the 23s rrna, however, fragments smaller than intact 23s rrna were visible on the gel for c. sputorum biovar bubulus and fecalis strains by using a northern blot hybridization analysis  <cit> .
in relation to the ivss in the helix  <dig> from the c. jejuni and c. coli isolates, a total of  <dig> isolates  have already been examined  <cit> .
in the two major and typical c. jejuni and c. coli species of campylobacter, ivss occur in helix  <dig> at high percent degree   <cit> .
in the present study, the occurrence of ivss with the two typical campylobacter species, were shown in helix  <dig> region at a high similar percentage , as shown in table  <dig>  in addition, ivss have already been shown to occur in the helix  <dig> region for only a few other campylobacter species, than the typical c. jejuni and c. coli , three ivss being identified to occur in c. fetus and in c. upsaliensis  <cit> .
at present, we identified the majority  of isolates from the three campylobacter species of c. fetus, c. upsaliensis and c. curvus to carry ivss in helix  <dig> within 23s rrna genes.
however, in a total of  <dig> isolates of the three campylobacter species of c. hyointestinalis , c. sputorum  and c. concisus , no ivss were identified in helix  <dig> region, as shown in table  <dig>  these are also scientifically significant observations.
thus, in conclusion, no ivss were identified in  <dig> isolates of three campylobacter species  both in the  <dig> and  <dig> helix regions within the 23s rrna genes.
overall, in the present study, two different kinds of the 23s rrna genes with and without the ivss occurred in the seven campylobacter isolates  .
in addition, in the present study, electrophoretic profiles of the purified rna from campylobacter organisms were examined.
in the purified rna fractions of some isolates from c. sputorum and c. curvus, slightly shorter 16s rrna were identified to occur , as described above.
these may be partially due to occurrence of ivs within the 16s rrna genes from these isolates and fragmentation of the primary 16s rrna transcripts among these isolates.
however, we have not clarified the nature of the 16s rrna genes from these isolates, yet.
therefore, sequencing and alignment analyses of the complete 16s rrna genes from these isolates are needed to identify the nature of the rrna from these two campylobacter species.
research to examine this is now in progress.
CONCLUSIONS
consequently, in  <dig> isolates of  <dig> campylobacter isolates of the nine species  examined, the absence of ivss was identified in helix  <dig> region within 23s rrna genes.
thus, ivs is extremely rare in the helix  <dig> region within the 23s rrna genes from the campylobacter organisms.
the occurrence of ivss with the two typical campylobacter species, were shown in helix  <dig> region at a high percentage .
we also identified the majority  of isolates from the three campylobacter species of c. fetus, c. upsaliensis and c. curvus to carry ivss in helix  <dig>  however, in a total of  <dig> isolates of the three species of c. hyointestinalis , c. sputorum  and c. concisus , no ivss were identified in the region.
thus, in conclusion, no ivss were identified in  <dig> isolates of three campylobacter species  both in the  <dig> and  <dig> helix regions.
in addition, intact 23s rrnas were identified in the purified rna fractions in campylobacter isolates containing no ivss, and no 23s rrna and fragmented other smaller rna fragments were evident in the isolates containing ivss.

 thermophilic campylobacter species, primarily campylobacter jejuni and c. coli are the most frequently recognized cause of acute bacterial gastroenteritis in humans in the western world. in relation to human campylobacteriosis, c. upsaliensis, c. hyointestinalis, c. lari, c. fetus and c. sputorum biovar sputorum have also been demonstrated to be implicated as gastrointestinal pathogens though these are rare. these campylobacter organisms have also been isolated from animals. moreover, c. concisus, c. curvus and so on are detected in association with the oral cavity. alternatively, c. sputorum biovar fecalis is isolated from animals. a multiplex pcr assay has recently developed for the identification of c. coli, c. fetus, c. hyointestinalis subsp. hyointestinalis, c. jejuni, c. lari and c. upsaliensis. thus, at this time, the genus campylobacter comprises species as already shown, the genus campylobacter is, in general, indicated to carry the three copies of rrna gene operon in relation to bacterial 23s rrna genes, the occurrence of intervening sequences and the fragmentation of 23s rrna have been demonstrated. in the genus campylobacter, the ε-subdivision of the proteobacteria, the occurrence of internal transcribed spacers was first described in helix region within 23s rrna gene in two of four c. jejuni, in both c. fetus and in one of two c. upsaliensis strains, when a total of campylobacter strains were examined. in addition, three of seven c. jejuni isolates examined were found to carry fragmented 23s rrna. moreover, the occurrence of fragmented 23s rrna correlated with the presence of an ivs within the 23s rrna genes. it was described that the presence of transcribed spacers is common in campylobacter spp.. all campylobacter isolates containing transcribed spacers in their 23s rrna gene sequences produced fragmented 23s rrnas. most recently, among strains of c. coli from turkeys, strains harbored ivss in all three 23s rrna genes, whereas the other strains lacked ivss from at least one of the genes. we have already reported the absence of ivss shown in both the helix and regions within 23s rrna genes among a total of isolates of c. lari obtained from different sources and in several countries, by using pcr amplification, ta cloning and sequencing procedures. in addition, the intact 23s rrna was also identified in the c. lari isolates examined, resulting in no production of the fragmented 23s rrna. thus, it would be important to clarify the molecular biological entities of the occurrence and the sequence structures of ivss within the 23s rrna genes in the much more isolates of several other species than c. lari of the genus campylobacter including atypical species. however, studies on molecular characterization and comparative analysis of ivss within the 23s rrna genes and these 23s rrna fragmentations in much more than campylobacter isolates of c. jejuni, c. coli, c. fetus, and some other atypical campylobacter species, namely c. upsaliensis, c. hyointestinalis, c. sputorum biovar sputorum, biovar fecalis, biovar paraureolyticus, c. concisus and c. curvus have not yet been reported. therefore, we aimed to clarify molecular characteristics of ivss within the 23s rrna gene sequences and 23s rrna fragmentations in these campylobacters other than c. lari, which has already been demonstrated not to harbor any ivss. in addition, the authors wished to comparatively analyze the ivss among the campylobacter organisms.  ivss in the helix region in the present study, two pcr primer pairs, f-/r-cl23h designed to generate the helix and, f-/r-cl23h the helix regions within the 23s rrna gene sequences with the campylobacter isolates were employed. when pcr was first carried out on the isolates using the primer pair, amplicons were generated. some of the examples are shown in fig. following sequencing and analysis, only the four cases, c. sputorum biovar sputorum lmg and biovar fecalis lmg lmg and lmg isolates, were shown to carry ivss in the helix region among these isolates of more than the sequence data in the helix region from c. sputorum isolates are aligned in fig. as shown in fig. identical ivs occurred in the helix region within 23s rrna genes from the four c. sputorum isolates. regarding the three c. sputorum biovar fecalis isolates, moreover, two different kinds of the 23s rrna genes were identified to occur with and without the ivs, respectively. ivss in the helix region then, we carried out pcr amplification of the ivss, in the central region within 23s rrna gene sequences with the campylobacter isolates, using the primer pair f-/r-cl23h some examples of the pcr amplicons are shown in fig. following sequencing and alignment analyses, in the helix region, c. hyointestinalis, fourteen c. sputorum biovar sputorum, biovar fecalis and paraureolyticus and c. concisus isolates were shown not to carry any ivss. in addition, however, regarding the other campylobacter organisms examined in the present study, of c. jejuni, of c. coli, of c. fetus, of c. upsaliensis and of c. curvus isolates were shown to carry the ivss in the helix region. some of the sequence data of the ivss in the helix region were aligned in fig. regarding the ivs sequences in the helix region, four ivss with similar sequences occurred in the c. jejuni and c. upsaliensis species, respectively, and two also in the c. curvus isolates. in addition, one kind of ivs with an identical sequence occurred in the c. coli and c. fetus isolates, respectively, moreover, the eight ivss in the c. jejuni and c. upsaliensis isolates showed high sequence similarities to each other, and one kind of ivs in the c. jejuni and c. coli showed an identical sequence. four kinds of ivss in the c. upsaliensis isolates, interestingly, carried two characteristic insertion sequences of several base pairs and twenty and several bp at the two positions. in c. jejuni and c. upsaliensis, two different kinds of the 23s rrna genes were identified to occur with and without the ivs in the helix region. moreover, multiple and heterogeneous ivss were shown in c. upsaliensis 48- and 68- isolates, respectively. consequently, identification of the ivss within the 23s rrna genes from the campylobacter isolates is summarized in the table secondary structure models of the ivss regarding the ivss identified in the present study, within the 23s rrna gene sequences from the campylobacter isolates examined, secondary structure models were constructed with all the ivss shown in table fig. and show some examples of the secondary structure models of the ivss in helix and helix regions. in the present models, stem and loop structures were identified in all ivss. gel electrophoresis of purified rna denaturing agarose gel electrophoresis profiles of purified rna from the campylobacter isolates was carried out to clarify if the primary rna transcripts of 23s rrna were fragmented in the isolates or not. purified rna from e. coli dh5α cells, identified to lack ivss, was also employed as a reference marker. in the purified rna fraction from the isolates of c. sputorum biovar sputorum lmg, whose 23s rrna gene was demonstrated to carry ivss in the helix no 23s rrna was evident in the fraction. instead of the 23s rrna, other smaller rna fragments were identified. regarding the c. sputorum biovar fecalis lmg two large rrna bands consisting of an intact and a fragmented 23s rrnas, were identified to occur in the isolate. some other examples of 23s rrnas whose genes were identified not to carry ivss in the helix region, are also shown in the figure.. thus, intact 23s rrnas were identified in campylobacter isolates containing no ivss in the helix region. in addition, in fig. 7b, some of the denaturing agarose gel electrophoresis profiles of purified rna from the campylobacter isolates, whose helix regions were examined, are shown. no 23s rrna and fragmented other smaller rna fragments were evident in the some purified rna fractions, and intact 23s rrnas were evident in other rna fractions. in relation to the 16s rrna molecules from the four isolates of c. sputorum biovar sputorum lmg, biovar fecalis lmg and lmg and c. curvus lmg, surprisingly, slightly shorter rnas than the 16s were identified in these isolates, instead of the 16s rrna species. discussion we have already shown no ivss, in the helix regions within the 23s rrna genes among a total of isolates of c. lari [n = un c. lari; n = uptc. consequently, in isolates of campylobacter isolates of the nine species examined, the absence of ivss was identified in helix region within 23s rrna genes. moreover, until now, no ivss have been identified in the helix region within 23s rrna genes, from more than campylobacter isolates of the species by other research groups. thus, ivs is extremely rare in the helix region within the 23s rrna genes from the campylobacter organisms. therefore, this is the first scientifically significant report of ivss in the helix from c. sputorum biovar sputorum and biovar fecalis among campylobacter organisms. in addition, no ivss have been identified to occur in the helix from c. sputorum strains. regarding the 23s rrna, however, fragments smaller than intact 23s rrna were visible on the gel for c. sputorum biovar bubulus and fecalis strains by using a northern blot hybridization analysis. in relation to the ivss in the helix from the c. jejuni and c. coli isolates, a total of isolates have already been examined. in the two major and typical c. jejuni and c. coli species of campylobacter, ivss occur in helix at high percent degree. in the present study, the occurrence of ivss with the two typical campylobacter species, were shown in helix region at a high similar percentage, as shown in table in addition, ivss have already been shown to occur in the helix region for only a few other campylobacter species, than the typical c. jejuni and c. coli, three ivss being identified to occur in c. fetus and in c. upsaliensis. at present, we identified the majority of isolates from the three campylobacter species of c. fetus, c. upsaliensis and c. curvus to carry ivss in helix within 23s rrna genes. however, in a total of isolates of the three campylobacter species of c. hyointestinalis, c. sputorum and c. concisus, no ivss were identified in helix region, as shown in table these are also scientifically significant observations. thus, in conclusion, no ivss were identified in isolates of three campylobacter species both in the and helix regions within the 23s rrna genes. overall, in the present study, two different kinds of the 23s rrna genes with and without the ivss occurred in the seven campylobacter isolates. in addition, in the present study, electrophoretic profiles of the purified rna from campylobacter organisms were examined. in the purified rna fractions of some isolates from c. sputorum and c. curvus, slightly shorter 16s rrna were identified to occur, as described above. these may be partially due to occurrence of ivs within the 16s rrna genes from these isolates and fragmentation of the primary 16s rrna transcripts among these isolates. however, we have not clarified the nature of the 16s rrna genes from these isolates, yet. therefore, sequencing and alignment analyses of the complete 16s rrna genes from these isolates are needed to identify the nature of the rrna from these two campylobacter species. research to examine this is now in progress.  consequently, in isolates of campylobacter isolates of the nine species examined, the absence of ivss was identified in helix region within 23s rrna genes. thus, ivs is extremely rare in the helix region within the 23s rrna genes from the campylobacter organisms. the occurrence of ivss with the two typical campylobacter species, were shown in helix region at a high percentage. we also identified the majority of isolates from the three campylobacter species of c. fetus, c. upsaliensis and c. curvus to carry ivss in helix however, in a total of isolates of the three species of c. hyointestinalis, c. sputorum and c. concisus, no ivss were identified in the region. thus, in conclusion, no ivss were identified in isolates of three campylobacter species both in the and helix regions. in addition, intact 23s rrnas were identified in the purified rna fractions in campylobacter isolates containing no ivss, and no 23s rrna and fragmented other smaller rna fragments were evident in the isolates containing ivss. 
in the purified rna fractions from the isolates which carried the 16s or 23s rrna genes with the ivss, no 16s or 23s rrna was evident, respectively. secondary structure models were also constructed with all the ivss identified in the present study. identification and characterization of intervening sequences within 23s rrna genes from campylobacter organisms including atypical campylobacters were carried out using two pcr primer pairs, designed to generate helix and regions. c. sputorum biovar fecalis lmg and lmg interestingly, carried two different kinds of the 23s rrna genes with and without the ivs, respectively. in c. jejuni and c. upsaliensis isolates, two different kinds of the 23s rrna genes were also identified to occur with and without ivss in the helix region, respectively. only c. sputorum biovar sputorum lmg and fecalis lmg lmg and lmg of a total of campylobacter isolates were shown to carry ivss in helix region. in the helix region, all the c. hyointestinalis, c. sputorum and c. concisus isolates were shown not to carry any ivss. however, the of c. jejuni isolates, of c. coli, of c. fetus, of c. upsaliensis and of c. curvus were shown to carry ivss.
 identification and characterization of intervening sequences within 23s rrna genes from campylobacter organisms including atypical campylobacters were carried out using two pcr primer pairs, designed to generate helix and regions.  only c. sputorum biovar sputorum lmg and fecalis lmg lmg and lmg of a total of campylobacter isolates were shown to carry ivss in helix region. c. sputorum biovar fecalis lmg and lmg interestingly, carried two different kinds of the 23s rrna genes with and without the ivs, respectively. consequently, in a total of isolates of including c. lari isolates examined previously, the absence of ivss was identified in the helix region. in the helix region, all the c. hyointestinalis, c. sputorum and c. concisus isolates were shown not to carry any ivss. however, the of c. jejuni isolates, of c. coli, of c. fetus, of c. upsaliensis and of c. curvus were shown to carry ivss. in c. jejuni and c. upsaliensis isolates, two different kinds of the 23s rrna genes were also identified to occur with and without ivss in the helix region, respectively.  secondary structure models were also constructed with all the ivss identified in the present study. in the purified rna fractions from the isolates which carried the 16s or 23s rrna genes with the ivss, no 16s or 23s rrna was evident, respectively. 