BACKGROUND
streptococcus uberis is an important cause of mastitis in modern dairy herds.
it is responsible for a significant proportion of clinical and subclinical infections in both lactating and nonlactating cows  <cit> .
s. uberis is considered as an environmental mastitis pathogen in that it has a ubiquitous and widespread distribution in the cow's environment.
indeed, streptococcus uberis can be isolated from bedding and pasture, as well as intramammary and extramammary sites on the cow  <cit> .
this may contribute to the poor efficiency of recommended mastitis control measures toward this pathogen, such as teat dipping and intramammary antimicrobial therapy at the end of each lactation period.
the main route of transmission appears to be from environmental sources but recent epidemiological studies suggest that cow-to-cow transmission is likely to be occurring  <cit> .
understanding the different sources, the dynamics of spread and transmission of s. uberis is a prerequisite for the development of mastitis control programs.
in an attempt to differentiate s. uberis strains, a number of typing methods have been developed, among them restriction endonuclease fingerprinting, random amplified polymorphic dna , repetitive extragenic palindromic -pcrs and dna macrorestriction analysis by pulse-field gel electrophoresis   <cit> .
all these methods enlighted the high genomic diversity of s. uberis strains but most of them display limitations in terms of discriminatory power, standardization and reproducibility.
pfge is reliable, reproducible and highly discriminatory but its use in large epidemiological studies and routine surveillance is limited because it is technically demanding, time-consuming, expensive and complex dna patterns may be difficult to interpret, especially for large collections of isolates  <cit> .
recently, two multilocus sequence typing  systems were described for s. uberis subtyping  <cit> .
the first one, based on four housekeeping genes and two virulence genes, was evaluated with  <dig> s. uberis isolates and compared to ribotyping and rapd typing.
mlst was superior to the other techniques in terms of discriminatory power, concordance with epidemiological data, and quantitative information regarding relatedness of isolates  <cit> .
the other mlst system relies on the use of sequences from seven housekeeping gene fragments.
it was used to study s. uberis isolates from united kingdom and new zealand.
these two populations were shown to be distinct and three major clonal complexes with different geographic prevalences have been identified.
this method is suitable for the analysis of s. uberis population structure and evolutionary relationships and a database has been constructed on a central mlst web site  <cit> .
however, mlst may not be suitable for routine surveillance and local epidemiological studies because of the high cost and the necessity of access to a high-throughput dna sequencing facility.
polymorphic tandem repeat typing is a new generic technology which has been proved to be very efficient for bacterial pathogens such as b. anthracis, m. tuberculosis, p. aeruginosa, y. pestis and is compatible with the development of internet-based resources  <cit> .
it takes advantage of the release of genome sequence data for the identification of tandem repeats .
the development of an assay then requires the evaluation of tandem repeats polymorphism, i.e.
tandem repeats showing interindividual length polymorphism, on well-selected sets of isolates.
by using different polymorphic tandem repeats , it is possible to discriminate strains for a low cost with ordinary molecular biology equipment and the data can be easily exchanged and compared  <cit> .
this method, called multiple locus vntr analysis , has been recently applied to several bacterial species including staphylococcus aureus  <cit>  and escherichia coli o157:h <dig>  <cit> .
mlva can evolve with resources as fragment size, repetition number of tandem repeat can be determined by capillary electrophoresis with a dna analyzer, as well as nucleotidic sequence.
the aim of the present study was to identify polymorphic tandem repeats in s. uberis genomes that could be included in a mlva procedure able to differentiate isolates and to contribute to the comprehension of s. uberis epidemiology and ecology in dairy herds.
RESULTS
the genome sequence of s.uberis strain 0140j  was explored with the tandem repeats finder software using the advanced version with default parameters.
in these conditions, twenty-seven tandem repeats  with a maximal unit length of  <dig> bp were detected around the chromosome.
two tandem repeats displaying a period size smaller than  <dig> bp were not considered in this study, although one of them is comprised in tr <dig>  this choice was motivated by the relative ease with which allelic differences can be resolved by agarose gel electrophoresis.
among the  <dig> trs studied, the lowest value of nucleotide sequence identity between individual repeat units was 72% and copy numbers of repeat units in s. uberis 0140j genome varied from  <dig>  to  <dig>  .
twelve trs had a repeat unit length with multiples of  <dig> and all of them, except tr <dig>  were embedded in predicted open reading frames  according to the current preliminary gene prediction and s. uberis blast server.
trs  <dig> and  <dig> were partially contained in orfs and displayed the start and stop codons, respectively, while tr <dig> contained a putative small orf.
other trs were localized in non-coding regions of the genome.
a set of  <dig> epidemiologically unrelated s. uberis isolates, i.e.
collected at different times in herds from diverse french areas, were used to evaluate the size polymorphism of the trs by pcr with specific primers.
overlapping or adjoining trs, namely trs  <dig> and  <dig>   <dig> and  <dig>   <dig> and  <dig>  and  <dig> and  <dig> were co-amplified with a single set of primers .
a total of  <dig> trs were found to be polymorphic as diverse size amplicons were obtained but two of them  were amplified for a minority of isolates.
these trs were not investigated further.
the seven polymorphic trs for which amplicons were obtained for all isolates were used to type the entire set of strains.
these polymorphic trs are the followings: trs  <dig>  11+ <dig>   <dig>   <dig>   <dig>   <dig> and  <dig>  with the exception of tr <dig> which may be part of an orf , all polymorphic trs were localized in intergenic regions.
among the monomorphic trs, tr6+ <dig> and tr  <dig> were not amplified in all isolates.
the inability to amplify tr6+ <dig>  tr <dig>  tr16+ <dig> or tr <dig> for some strains may be due to a local genetic variability in the area annealing with primers or to the absence of the corresponding sequences in their genomes.
mlva typing of the entire set of  <dig> s. uberis isolates was performed with the seven selected trs.
the set of alleles obtained with these trs is presented in figure  <dig>  a total of  <dig> mlva types were obtained with  <dig> types among the  <dig> strains isolated from the milk of cows belonging to our experimental herd, and  <dig> types for the  <dig> epidemiologically unrelated strains, i.e.
collected in different herds and areas .
with the exception of the s. parauberis strain ncdo  <dig>  all the isolates used in the study were defined as s. uberis according to pcr amplification results using primers targeting species-specific parts of the 16s rrna gene as previously described  <cit> .
this confirmed that the occurence of s. parauberis as causative agent of bovine mastitis appears to be rare  <cit> .
the s. parauberis strain ncdo  <dig> was the sole strain for which no amplification occurred with most of the trs .
this result is probably the consequence of some genetic variability and it would be interesting to determine if this variability is specific to the ncdo  <dig> strain or if it represents a general feature of s. parauberis genotype.
concerning other major streptococcus species implicated in bovine mastitis, i.e.streptococcus agalactiae and streptococcus dysgalactiae, none of the  <dig> polymorphic trs were amplified from the genomic dna preparations of the  <dig> strains that we tested .
moreover, no significant alignments of the  <dig> trs sequences with available s. agalactiae genomic sequences were found when a blast analysis was performed.
the mlva system described in this study displayed a satisfactory discriminatory power as it was able to distinguish most of the s. uberis isolates collected in different herds or from the cows belonging to our experimental herd .
these results are in agreement with previous studies that showed, using diverse typing methods, the high genetic variability of s. uberis strains  <cit> .
the  <dig> isolates collected from  <dig> of the cows in our experimental herd were divided into  <dig> mlva types with  <dig> types  represented by  <dig> isolates collected from the milk of different cows.
strains isolated simultaneously from different quarters of individual cows harboured different mlva types.
these observations are congruent with previous reports stating that the environment rather than infected mammary glands was the likely source of s. uberis infections, even if cow to cow transmission could occur  <cit> .
overall, the  <dig> s. uberis strains used in this study were classified in  <dig> mlva types among them  <dig>  comprised  <dig> strains that were isolated in different herds and years.
even if some s. uberis strains isolated in different herds and countries have already been demonstrated to belong to the same type using methods such as pfge  <cit>  or mlst  <cit> , the strains belonging to mlva types  <dig> and  <dig> may be distinguished with another typing method or additional trs.
indeed, s. uberis strains harbouring the same alleles in a mlst study appeared to be different using pvuii ribotyping and some strains belonging to the same pvuii ribotype were considered different using mlst  <cit> .
the mlva system described here has already a high discriminatory power but, considering the variability of s. uberis genotype, it would be interesting to look for additional polymorphic trs to complete this set.
among the  <dig> trs used, tr11+ <dig> and tr <dig> were the most efficient with a simpsons diversity index of  <dig>  and  <dig> , respectively.
sequencing of amplicons corresponding to tr11+ <dig> showed that tr <dig> and tr <dig> are both polymorphic and that some strains may harbour only one of them.
indeed, the sequence analysis of one representative of allele  <dig> revealed that tr <dig> was not contained in the genome of the corresponding strain.
tr  <dig>  tr <dig> and tr <dig> exhibited a simpsons diversity index value of  <dig> ,  <dig>  and  <dig> , respectively.
tr <dig>  and tr <dig>  were the less discriminatory.
indeed, tr <dig> displayed only  <dig> alleles with our strains and tr <dig> allowed us to define  <dig> alleles but most of the strains possessed the allelic forms  <dig> or  <dig>
CONCLUSIONS
this investigation validates the usefulness of the mlva typing method for bovine s. uberis strains and a first set of markers.
the proposed mlva system can be evaluated with collections of strains from diverse geographical origins, completed by additional polymorphic tandem repeats and compared with other typing methods.
in contrast to pfge or mlst, the mlva method is easy and fast to perform, low cost and could constitute the method of choice for short-term epidemiological studies when population structure and evolutionary relationships are not a concern.
investigations at the herd level such as analysis of outbreaks, strains implicated in persistent infections or demonstrating different antibiotic treatment susceptibilities, should be facilitated and allow the development of adapted herd management measures.

 streptococcus uberis is an important cause of mastitis in modern dairy herds. it is responsible for a significant proportion of clinical and subclinical infections in both lactating and nonlactating cows. s. uberis is considered as an environmental mastitis pathogen in that it has a ubiquitous and widespread distribution in the cow's environment. indeed, streptococcus uberis can be isolated from bedding and pasture, as well as intramammary and extramammary sites on the cow. this may contribute to the poor efficiency of recommended mastitis control measures toward this pathogen, such as teat dipping and intramammary antimicrobial therapy at the end of each lactation period. the main route of transmission appears to be from environmental sources but recent epidemiological studies suggest that cow-to-cow transmission is likely to be occurring. understanding the different sources, the dynamics of spread and transmission of s. uberis is a prerequisite for the development of mastitis control programs. in an attempt to differentiate s. uberis strains, a number of typing methods have been developed, among them restriction endonuclease fingerprinting, random amplified polymorphic dna, repetitive extragenic palindromic -pcrs and dna macrorestriction analysis by pulse-field gel electrophoresis. all these methods enlighted the high genomic diversity of s. uberis strains but most of them display limitations in terms of discriminatory power, standardization and reproducibility. pfge is reliable, reproducible and highly discriminatory but its use in large epidemiological studies and routine surveillance is limited because it is technically demanding, time-consuming, expensive and complex dna patterns may be difficult to interpret, especially for large collections of isolates. recently, two multilocus sequence typing systems were described for s. uberis subtyping. the first one, based on four housekeeping genes and two virulence genes, was evaluated with s. uberis isolates and compared to ribotyping and rapd typing. mlst was superior to the other techniques in terms of discriminatory power, concordance with epidemiological data, and quantitative information regarding relatedness of isolates. the other mlst system relies on the use of sequences from seven housekeeping gene fragments. it was used to study s. uberis isolates from united kingdom and new zealand. these two populations were shown to be distinct and three major clonal complexes with different geographic prevalences have been identified. this method is suitable for the analysis of s. uberis population structure and evolutionary relationships and a database has been constructed on a central mlst web site. however, mlst may not be suitable for routine surveillance and local epidemiological studies because of the high cost and the necessity of access to a high-throughput dna sequencing facility. polymorphic tandem repeat typing is a new generic technology which has been proved to be very efficient for bacterial pathogens such as b. anthracis, m. tuberculosis, p. aeruginosa, y. pestis and is compatible with the development of internet-based resources. it takes advantage of the release of genome sequence data for the identification of tandem repeats. the development of an assay then requires the evaluation of tandem repeats polymorphism, i.e. tandem repeats showing interindividual length polymorphism, on well-selected sets of isolates. by using different polymorphic tandem repeats, it is possible to discriminate strains for a low cost with ordinary molecular biology equipment and the data can be easily exchanged and compared. this method, called multiple locus vntr analysis, has been recently applied to several bacterial species including staphylococcus aureus and escherichia coli o157:h. mlva can evolve with resources as fragment size, repetition number of tandem repeat can be determined by capillary electrophoresis with a dna analyzer, as well as nucleotidic sequence. the aim of the present study was to identify polymorphic tandem repeats in s. uberis genomes that could be included in a mlva procedure able to differentiate isolates and to contribute to the comprehension of s. uberis epidemiology and ecology in dairy herds.  the genome sequence of s.uberis strain 0140j was explored with the tandem repeats finder software using the advanced version with default parameters. in these conditions, twenty-seven tandem repeats with a maximal unit length of bp were detected around the chromosome. two tandem repeats displaying a period size smaller than bp were not considered in this study, although one of them is comprised in tr this choice was motivated by the relative ease with which allelic differences can be resolved by agarose gel electrophoresis. among the trs studied, the lowest value of nucleotide sequence identity between individual repeat units was 72% and copy numbers of repeat units in s. uberis 0140j genome varied from to. twelve trs had a repeat unit length with multiples of and all of them, except tr were embedded in predicted open reading frames according to the current preliminary gene prediction and s. uberis blast server. trs and were partially contained in orfs and displayed the start and stop codons, respectively, while tr contained a putative small orf. other trs were localized in non-coding regions of the genome. a set of epidemiologically unrelated s. uberis isolates, i.e. collected at different times in herds from diverse french areas, were used to evaluate the size polymorphism of the trs by pcr with specific primers. overlapping or adjoining trs, namely trs and and and and and were co-amplified with a single set of primers. a total of trs were found to be polymorphic as diverse size amplicons were obtained but two of them were amplified for a minority of isolates. these trs were not investigated further. the seven polymorphic trs for which amplicons were obtained for all isolates were used to type the entire set of strains. these polymorphic trs are the followings: trs 11+ and with the exception of tr which may be part of an orf, all polymorphic trs were localized in intergenic regions. among the monomorphic trs, tr6+ and tr were not amplified in all isolates. the inability to amplify tr6+ tr tr16+ or tr for some strains may be due to a local genetic variability in the area annealing with primers or to the absence of the corresponding sequences in their genomes. mlva typing of the entire set of s. uberis isolates was performed with the seven selected trs. the set of alleles obtained with these trs is presented in figure a total of mlva types were obtained with types among the strains isolated from the milk of cows belonging to our experimental herd, and types for the epidemiologically unrelated strains, i.e. collected in different herds and areas. with the exception of the s. parauberis strain ncdo all the isolates used in the study were defined as s. uberis according to pcr amplification results using primers targeting species-specific parts of the 16s rrna gene as previously described. this confirmed that the occurence of s. parauberis as causative agent of bovine mastitis appears to be rare. the s. parauberis strain ncdo was the sole strain for which no amplification occurred with most of the trs. this result is probably the consequence of some genetic variability and it would be interesting to determine if this variability is specific to the ncdo strain or if it represents a general feature of s. parauberis genotype. concerning other major streptococcus species implicated in bovine mastitis, i.e.streptococcus agalactiae and streptococcus dysgalactiae, none of the polymorphic trs were amplified from the genomic dna preparations of the strains that we tested. moreover, no significant alignments of the trs sequences with available s. agalactiae genomic sequences were found when a blast analysis was performed. the mlva system described in this study displayed a satisfactory discriminatory power as it was able to distinguish most of the s. uberis isolates collected in different herds or from the cows belonging to our experimental herd. these results are in agreement with previous studies that showed, using diverse typing methods, the high genetic variability of s. uberis strains. the isolates collected from of the cows in our experimental herd were divided into mlva types with types represented by isolates collected from the milk of different cows. strains isolated simultaneously from different quarters of individual cows harboured different mlva types. these observations are congruent with previous reports stating that the environment rather than infected mammary glands was the likely source of s. uberis infections, even if cow to cow transmission could occur. overall, the s. uberis strains used in this study were classified in mlva types among them comprised strains that were isolated in different herds and years. even if some s. uberis strains isolated in different herds and countries have already been demonstrated to belong to the same type using methods such as pfge or mlst, the strains belonging to mlva types and may be distinguished with another typing method or additional trs. indeed, s. uberis strains harbouring the same alleles in a mlst study appeared to be different using pvuii ribotyping and some strains belonging to the same pvuii ribotype were considered different using mlst. the mlva system described here has already a high discriminatory power but, considering the variability of s. uberis genotype, it would be interesting to look for additional polymorphic trs to complete this set. among the trs used, tr11+ and tr were the most efficient with a simpsons diversity index of and, respectively. sequencing of amplicons corresponding to tr11+ showed that tr and tr are both polymorphic and that some strains may harbour only one of them. indeed, the sequence analysis of one representative of allele revealed that tr was not contained in the genome of the corresponding strain. tr tr and tr exhibited a simpsons diversity index value of, and, respectively. tr and tr were the less discriminatory. indeed, tr displayed only alleles with our strains and tr allowed us to define alleles but most of the strains possessed the allelic forms or  this investigation validates the usefulness of the mlva typing method for bovine s. uberis strains and a first set of markers. the proposed mlva system can be evaluated with collections of strains from diverse geographical origins, completed by additional polymorphic tandem repeats and compared with other typing methods. in contrast to pfge or mlst, the mlva method is easy and fast to perform, low cost and could constitute the method of choice for short-term epidemiological studies when population structure and evolutionary relationships are not a concern. investigations at the herd level such as analysis of outbreaks, strains implicated in persistent infections or demonstrating different antibiotic treatment susceptibilities, should be facilitated and allow the development of adapted herd management measures. 
streptococcus uberis is a common cause of bovine mastitis and recommended control measures, based on improved milking practice, teat dipping and antibiotic treatment at drying-off, are poorly efficient against this environmental pathogen. collected in different herds and areas. the mlva method can be applied to s. uberis genotyping and constitutes an interesting complement to existing typing methods. a simple and efficient typing method would be helpful in identifying s.uberis sources, virulent strains and cow to cow transmission. the potential of mlva for s. uberis mastitis isolates genotyping was investigated. twenty-five tandem repeats were identified and amplified by pcr with dna samples from s. uberis strains. a set of seven trs were found to be polymorphic and used for mlva typing of s. uberis isolates. this method, which is easy to perform, low cost and can be used in routine, could facilitate investigations of the epidemiology of s. uberis mastitis in dairy cows. a total of mlva types were obtained with types among strains isolated from the milk of mastitic cows belonging to our experimental herd, and types for epidemiologically unrelated strains, i.e. the genomic sequence of streptococcus uberis was analyzed for potential variable number tandem repeats.
 streptococcus uberis is a common cause of bovine mastitis and recommended control measures, based on improved milking practice, teat dipping and antibiotic treatment at drying-off, are poorly efficient against this environmental pathogen. a simple and efficient typing method would be helpful in identifying s.uberis sources, virulent strains and cow to cow transmission. the potential of mlva for s. uberis mastitis isolates genotyping was investigated.  the genomic sequence of streptococcus uberis was analyzed for potential variable number tandem repeats. twenty-five tandem repeats were identified and amplified by pcr with dna samples from s. uberis strains. a set of seven trs were found to be polymorphic and used for mlva typing of s. uberis isolates. a total of mlva types were obtained with types among strains isolated from the milk of mastitic cows belonging to our experimental herd, and types for epidemiologically unrelated strains, i.e. collected in different herds and areas.  the mlva method can be applied to s. uberis genotyping and constitutes an interesting complement to existing typing methods. this method, which is easy to perform, low cost and can be used in routine, could facilitate investigations of the epidemiology of s. uberis mastitis in dairy cows. 