Drosophila pseudoobscura subgroup to reconstruct their evolutionary histories and past patterns of genic exchange. Partial genomic assemblies were generated from two subspecies of Drosophila pseudoobscura (D. ps.) and an outgroup species, D. miranda. These new assemblies were compared to available assemblies of D. ps. pseudoobscura and D. persimilis, two species with overlapping ranges in western North America. Within inverted regions, nucleotide divergence among each pair of the three species is comparable, whereas divergence between D. ps. pseudoobscura and D. persimilis in non-inverted regions is much lower and closer to levels of intraspecific variation. Using molecular markers flanking each of the major chromosomal inversions, we identify strong crossover suppression in F1 hybrids extending over 2 megabase pairs (Mbp) beyond the inversion breakpoints. These regions of crossover suppression also exhibit the high nucleotide divergence associated with inverted regions. Finally, by comparison to a geographically isolated subspecies, D. ps. bogotana, our results suggest that autosomal gene exchange between the North American species, D. ps. pseudoobscura and D. persimilis, occurred since the split of the subspecies, likely within the last 200,000 years. We conclude that chromosomal rearrangements have been vital to the ongoing persistence of these species despite recent hybridization. Our study serves as a proof-of-principle on how whole genome sequencing can be applied to formulate and test hypotheses about species formation in lesser-known non-model systems.In nature, closely related species may hybridize while still retaining their distinctive identities. Chromosomal regions that experience reduced recombination in hybrids, such as within inversions, have been hypothesized to contribute to the maintenance of species integrity. Here, we examine genomic sequences from closely related fruit fly taxa of the  Drosophila pseudoobscura species subgroup, which includes the hybridizing species pair D. pseudoobscura pseudoobscura and D. persimilis. Recent models suggest that chromosomal inversions can facilitate the persistence of hybridizing species because of their effects on recombination, whereby inverted regions would exhibit higher nucleotide divergence than non-inverted regions. Indeed, D. pseudoobscura-D. persimilis nucleotide divergence outside these inverted regions is lower than within or near inversions, resembling D. ps. pseudoobscura levels of within-species nucleotide diversity. We also observe that recombination suppression in F1 hybrids extends greater than 2 Mbp outside the inversion breakpoints. Furthermore, when genomic sequence of D. persimilis is compared to two sister subspecies—the hybridizing subspecies, D. ps. pseudoobscura, and a non-hybridizing control subspecies, D. ps. bogotana—autosomal divergence is lower in the former, demonstrating recent gene exchange. These lines of evidence support a speciation model in which the two hybridizing species persist despite the presence of recent genic introgression in collinear regions of the genome because of the reduced recombinational effects of the inversions that distinguish them.The transformation of populations into distinct species depends on whether hybridization, recombination, and subsequent gene introgression can be suppressed between diverging species. We use partial genome sequences to reconstruct this evolutionary process in the  One of the most significant empirical insights in evolutionary biology is that a large number of species naturally hybridize with close relatives Several recent studies suggested that genomic regions of low recombination may provide a means to create “islands of differentiation” between species Assembled whole genome sequences and inexpensive resequencing technologies can complement locus-specific sequencing studies and genetic mapping studies for clarifying the role of regions of low recombination in species persistence. Fruit flies from the genus Drosophila have been a major focus of many studies of nucleotide divergence between closely related species and mapping studies of traits that prevent introgression such as hybrid sterility. The recent sequencing of multiple closely related Drosophila species Drosophila pseudoobscura species subgroup is comprised of two D. pseudoobscura subspecies (D. ps. pseudoobscura and D. ps. bogotana), and two closely related species, D. persimilis and D. miranda. The D. pseudoobscura subspecies are geographically isolated , share chromosomal arrangements, and represent the earliest stages of species divergence D. persimilis and D. miranda are restricted to the west coast of North America, where they co-occur with D. ps. pseudoobscura. Both D. pseudoobscura subspecies differ from the close relative species D. persimilis by fixed (or nearly fixed) chromosomal inversion differences on three of their major chromosome arms, and F1 hybrid males from crosses between these species are sterile . In contrast, D. miranda is an outgroup species which cannot produce any fertile hybrids with D. pseudoobscura or D. persimilisD. ps. pseudoobscura-D. ps. bogotana) D. persimilis) D. miranda) is well established by DNA sequences, chromosomal inversions, and reproductive isolation D. ps. pseudoobscura and D. persimilisD. ps. bogotana, and outgroup, D. miranda).The D. ps. pseudoobscura and D. persimilis is high within and near the three chromosomal inversions Nucleotide divergence between the hybridizing species D. ps. pseudoobscura and D. persimilisD. miranda, one from a second strain of the North American subspecies, D. ps. pseudoobscura, and one from the South American subspecies, D. ps. bogotana. By providing controls for divergence in the absence of gene flow, these newly obtained genomic sequences allow for more robust analysis than previous studies. Our new results suggest that the chromosomal regions inverted between D. persimilis and D. ps. pseudoobscura arose in allopatry, and that D. pseudoobscura, D. persimilis, and D. miranda all diverged within a relatively short time frame. We also find compelling evidence for autosomal gene exchange between D. persimilis and D. ps. pseudoobscura in collinear regions since the split of D. ps. pseudoobscura and D. ps. bogotana, likely within the past 200,000 years. Overall, our analyses utilize genome sequence data in an existing framework to demonstrate the importance of chromosomal inversions in maintaining the persistence of hybridizing species and to consolidate previous tentative conclusions about divergence in this group. Further, this research serves as a model for how whole genome shotgun sequence data can be used with a reference genome sequence to address fundamental questions regarding evolutionary changes leading to the formation of species.To better understand the genealogical history of this subgroup, we use published genome sequence assemblies of D. ps. pseudoobscura, and divergence between D. ps. pseudoobscura and each of D. persimilis and D. miranda across four of the five major chromosome arms. Each datapoint within the sliding window represents the fraction of bases differentiating two genome sequences along a 500 kilobase pairs (kbp) interval, iterated every 100 kbp. Very similar plots were generated for intergenic regions or introns alone (not shown). We only scored positions for which aligned sequences were available for all four taxa  Fixed inversions on chromosomes XL, XR, and 2 distinguish D. ps. pseudoobscura and D. miranda, exhibit a different pattern. There was no consistent decline in divergence between these two species in regions near the centromeres. Furthermore, diversity within D. ps. pseudoobscura was not significantly correlated with divergence to D. miranda along any chromosome arm except chromosome 4 , suggesting that D. miranda and D. ps. pseudoobscura are not sharing many polymorphisms. According to a model where D. miranda is the outgroup, we predict that the range (maximum minus minimum) of divergences across windows should be greater for the purportedly more divergent species pair, D. miranda - D. ps. pseudoobscura, than the pair of more recently diverged species, D. persimilis - D. ps. pseudoobscura. While this prediction was met for windows along the collinear chromosome 4, we observed instead a greater range of divergences in the D. persimilis - D. ps. pseudoobscura pairing on the chromosome arms  that harbor inversions distinguishing these species . We recovered 0.25%–0.55% recombinants at markers 2.8 Mbp outside of each inversion, indicating that complete recombination suppression extends greater than 2.1 Mbp, but not more than 2.8 Mbp outside inversions. Strong crossover suppression, resulting in less than one percent recombinants, is observed relative to one marker 3.35 Mbp outside of the XR chromosomal inversion. Crossing over is largely restored at 4.55 Mbp outside inversions, with a crossover rate greater than 5% observed from one marker on chromosome 2 (see Inversions prevent gene exchange because the products of recombination are not recovered. We confirmed that recombinant products are not recovered within 2.1 megabase pairs (Mbp) of fixed inversions along chromosome XL, XR, and 2 in heterozygotes  when the inversions arose  should be more similar in sequence to North American (NA) than South American (SA) subspecies of D. pseudoobscura , indicating that divergence is lower between D. persimilis and North American D. ps. pseudoobscura than between D. persimilis and South American D. ps. bogotana.Because of hybridization between the North American taxa, a very simple expectation is that oobscura . We limiD. miranda (Dmir), than between the North American subspecies and D. miranda . Nonetheless, we can test for recent gene exchange more rigorously by specifically counting “shared-derived” base pair substitutions polarized with D. miranda. Counts of [Dmir = SA≠Dper = NA] were compared to counts of individual base pairs in which [Dmir = NA≠Dper = SA], where the latter half of the inequality denotes potential shared-derived bases. Again, we observe a slight, borderline significant excess of the first category , suggesting that D. persimilis and North American D. ps. pseudoobscura share more derived bases.The above test does not account for possible faster divergence within the South American subspecies lineage, either through increased mutation rate or more frequent fixation of slightly deleterious alleles. Testing for differences in lineage rates, we did not observe greater divergence between the South American subspecies and the non-hybridizing species, Adh) region has been reported to have introgressed recently between these species using analyses independent of divergence from the South American species, D. ps. bogotanaDmir = SA≠Dper = NA] vs. [Dmir = NA≠Dper = SA] for this region. In this region which bears Adh .Finally, introgression between species is not expected to be homogeneous outside inverted regions. The Alcohol dehydrogenase . When we polarized the bases and compared (Dmir = SA≠Dper = NA) vs. (Dmir = NA≠Dper = SA), we observed a nonsignificant difference opposite in direction to our expectation (46 vs. 62). However, there was only 27% as much sequence to analyze more than 2.5 Mbp from inversions on the X-chromosome than on the autosomes.We applied the same analyses to test for recent gene exchange along X-linked regions from both XL and XR distant from the inversion breakpoints. We observed a nonsignificant difference in number of bases categorized as [Genome sequencing has recently become affordable for individual investigators, but how the resultant data can be applied to address evolutionary questions about species formation or diversification has been less clear. Here, we use partial genome sequence data to: 1) evaluate the role of chromosomal inversions in maintaining the distinction between two hybridizing Drosophila species, 2) estimate when gene exchange occurred between these species, and 3) clarify contradictory interpretations from earlier studies that attempted to address related questions. Overall, this research demonstrates how present-day patterns within genomic data can help to infer past processes involved in speciation.D. ps. pseudoobscura and D. persimilisD. ps. pseudoobscura and D. miranda were uncorrelated with nucleotide sequence differences between two strains of D. ps. pseudoobscura. This finding suggests that our comparisons to D. miranda are not hindered by introgression or extensive shared ancestral polymorphisms If two species share extensive polymorphism through introgression or incomplete lineage sorting resulting from a recent split, we predict that nucleotide sequence diversity within species should be correlated with average pairwise nucleotide differences between species. Extensive polymorphism sharing was shown previously in the case of D. ps. pseudoobscura and D. persimilis share variation far outside the fixed inversions that distinguish these species D. ps. pseudoobscura and D. persimilis is higher and more comparable to differences between two strains of D. ps. pseudoobscura in regions distant from the inversions. In contrast, divergence between D. ps. pseudoobscura and D. persimilis is comparable to that between D. ps. pseudoobscura and the non-hybridizing outgroup, D. miranda, for regions inside and just outside the chromosomal inversions that separate them.Previous DNA sequence-based studies observed that D. ps. pseudoobscura and D. persimilis largely speciated in allopatry, close in time to the split of these species from D. miranda, and recent secondary contact between the first two resulted in the dissolution of differences outside the inverted regions. In contrast, Noor et al D. ps. pseudoobscura and D. persimilis speciated under a sympatric “divergence-with-gene-flow” model. In other words, differences in divergence between inversions reveal when each inversion arose as both species evolved in sympatry.These new results can be used to formulate a hypothesis for the evolutionary history of these species and reconcile previously contradictory inferences. Machado et al D. ps. pseudoobscura and D. persimilis, followed by chromosome 2 and finally chromosome arm XR (see D. pseudoobscura and D. persimilis within each inverted region was similar to the divergence between D. ps. pseudoobscura and D. miranda (shown at single loci by D. ps. pseudoobscura divergence from D. miranda that was shown previously for divergence from D. persimilisD. miranda) from the D. pseudoobscura-D. persimilis ancestor, and these three derived forms segregated exclusively in D. persimilis. Alternatively, and arguably more parsimoniously, the three species diverged close in time, D. persimilis acquired three new inversions sometime after the split from D. pseudoobscura, and secondary contact between D. persimilis and D. pseudoobscura homogenized the noninverted regions.Our study recapitulates both sets of results and allows us to suggest a resolution. As in Noor et al m XR see . HoweverMany recent studies have analyzed DNA sequence polymorphism and divergence to identify the statistical signature of recent introgression. However, these tests did not typically identify a time frame within which introgression occurred except as variance in the time of divergence D. persimilis and D. ps. pseudoobscura. One D. pseudoobscura subspecies co-occurs and hybridizes with D. persimilis while the other subspecies lives isolated on a different continent. Hence, we can attribute differences in divergence between D. persimilis and these D. pseudoobscura subspecies to hybridization that has occurred more recently than the split of the subspecies, estimated to have been 200,000 years ago D. persimilis and the two D. pseudoobscura subspecies, suggesting recent introgression between the co-occurring taxa, but we fail to detect such evidence for introgression across comparable regions of the X-chromosome.Here, we use a comparison between subspecies to infer the timing of introgression between D. ps. pseudoobscura populations co-occurring with D. persimilis compared to those elsewhere in North America D. ps. pseudoobscura, the approach used by Kulathinal and Singh Although we detected a statistically significant signature of introgression along autosomal loci, the signature was faint, suggesting that recent gene exchange has not been extensive. DNA sequence-based studies previously identified the statistical signature of historical introgression D. ps. pseudoobscura and D. persimilis, however, we observe evidence for introgression on the autosomes while not on the X-chromosome in regions outside the inversions, suggesting that these differences may be reflective of sex-linkage per se. This observation may be consistent with a higher density of factors conferring hybrid sterility or other barriers to gene flow on the X-chromosome than on the autosomes A significant difference between sex-linked and autosomal loci in introgression has been a recurring theme in divergence population genetics In this study, we used sparse whole-genome shotgun sequences from multiple taxa to infer the evolutionary history of a species group and to identify genomic features associated with their divergence. Our system was well-leveraged in that we initially began the investigation already having an assembled and annotated full-genome sequence for two of the focal species Nonetheless, the cost of next-generation sequencing is dropping for both model and non-model systems, even between the execution of this study and its publication. Because of cost constraints, our study approached these questions using light resequencing (effectively utilizing the power of millions of markers) but producing extensive gaps and a majority of aligned positions being covered by single sequence traces. However, our approach serves as a proof-of-principle for future genomic studies on lesser developed systems. We attempted to reduce systematic biases by applying stringent filters, specific tests (including averaging across 500 kbp windows) and by employing the use of a well-assembled reference genome sequence. Future, more rigorous approaches enabled by less-expensive sequencing technologies will allow researchers greater power to infer historical evolutionary processes such as speciation and historical introgression in non-model systems.D. miranda  and the subspecies, D. ps. bogotana  were each extracted and purified using the Gentra PureGene DNA isolation kit. For D. miranda, genomic DNA was nebulized and single stranded libraries generated before being sequenced at light coverage on a single Roche/454 Life Sciences GS-FLX run at Duke University's IGSP core sequencing facility, yielding approximately 100 Mbp of sequence  as accession SRA008268. Additionally, two previously sequenced and assembled genomes, D. ps. pseudoobscura (Release 2) and D. persimilis (Release 1), were used for comparative analysis D. ps. pseudoobscura, previously sequenced Roche/454 reads (NCBI SRA accession SRA000268) from a second line  linkage groups. Individual base calls were filtered to exclude nucleotides that are: within 3 base pairs of an alignment gap, harbor low quality scores (below 10), contain greater than 30% mismatches within a 7 base pair window, are in regions of high divergence (divergence to D. persimilis is greater than 30% in a 7 base pair window). Alignments from the two previously sequenced reference genomes, Drosophila ps. pseudoobscura and D. persimilis were obtained via chain files from the UCSC Genome Browser (genome.ucsc.edu). Site-specific annotation information such as intron and codon position was extracted from D. ps. pseudoobscura Release 2.3 annotations from FlyBase (flybase.org). Chromosome arms (including ordered contigs) 2, 4, XL, and XR were used  were used in this cross and recombinants were screened among 384 progeny of F1 females backcrossed to D. pseudoobscura. The following markers were used to assay recombination rate at varying distances from the inversions – chromosome 2 inversion: DPS2019 (2.77 Mbp from inversion on telomeric side), DPS2026 (associated with inversion) and DPS2031 (2.8 Mbp from inversion on centromeric side), XL inversion: DPSX_7446z (2.84 Mbp from inversion on centromeric side), DPSX046 (associated with inversion), DPSX008 (0.4 Mbp from inversion on telomeric side), and DPSXL_3a_0.8 (2.8 Mbp from inversion on telomeric side), XR inversion: DPSXR_6_2.7 (3.35 Mbp from inversion on centromeric side), DPSX063 (associated with inversion), DPSX037nA3 (1.4 Mbp from inversion on telomeric side), DPSX037N (2.1 Mbp from inversion on telomeric side), and DPSX058 (2.8 Mbp from inversion on telomeric side). Primer sequences are available upon request.Chromosome arms XL, XR, and 2 differ by single inversions between Figure S1D. ps. pseudoobscura. Divergence estimates were then divided by diversity, the former of which was measured between D. ps. pseudoobscura and each of D. persimils (red) and D. miranda (black). The difference in overall divergence between D. miranda and D. persimilis against D. ps. pseudoobscura is found in grey.Ratio of divergence to polymorphism (Dxy/Pxy), and differences in divergence over 500 kbp windows. Inversion breakpoints for chromosomes 2, XL, and XR are shown in green. Polymorphism was estimated by comparing genomic sequences between two strains of (0.11 MB TIF)Click here for additional data file.Table S1“Short-read” sequences used in this study.(0.02 MB DOC)Click here for additional data file.Table S2D. pseudoobscura (published genome), D. pseudoobscura , D. persimilis (published genome), and D. miranda . This common set of base pairs were used in Aligned and filtered nucleotides common to four species. The total aligned sequence per chromosome with scored bases for all of the following four samples: (0.02 MB DOC)Click here for additional data file.Table S3D. ps. pseudoobscura nucleotide diversity across studies. Estimates of nucleotide diversity from D. ps. pseudoobscura from our study (focusing on intergenic regions) were compared to a previous study of nucleotide diversity in focal intergenic regions . The results show that roughly the same range of values of diversity were recovered from the two works (0.0026–0.0210 vs 0.0024–0.0179).Comparison of (0.03 MB DOC)Click here for additional data file.Table S4D. pseudoobscura across 400 kbp windows. Numbers indicate lowest and highest percent differences across windows for each chromosome arm separately, as well as the observed range (highest-lowest).Percent difference from (0.02 MB DOC)Click here for additional data file.Table S5Markers surveyed to examine recombination fractions in hybrids for various points at known physical distances from the inverted regions. For each chromosome, markers are all listed from the centromere to telomere, including the marker within the inversion. Physical distances from each marker to the nearest inversion breakpoint and the recombination fractions observed from the marker within the inversion are also indicated.(0.03 MB DOC)Click here for additional data file.


Brucella species, Brucella microti, was determined. B. microti is a member of the genus Brucella within the Alphaproteobacteria, which consists of medically important highly pathogenic facultative intracellular bacteria. In contrast to all other Brucella species, B. microti is a fast growing and biochemically very active microorganism with a phenotype more similar to that of Ochrobactrum, a facultative human pathogen. The atypical phenotype of B. microti prompted us to look for genomic differences compared to other Brucella species and to look for similarities with Ochrobactrum.Using a combination of pyrosequencing and conventional Sanger sequencing, the complete genome sequence of the recently described novel B. microti is almost identical to that of Brucella suis 1330 with an overall sequence identity of 99.84% in aligned regions. The most significant structural difference between the two genomes is a bacteriophage-related 11,742 base pairs insert only present in B. microti. However, this insert is unlikely to have any phenotypical consequence. Only four protein coding genes are shared between B. microti and Ochrobactrum anthropi but impaired in other sequenced Brucella. The most noticeable difference between B. microti and other Brucella species was found in the sequence of the 23S ribosomal RNA gene. This unusual variation could have pleiotropic effects and explain the fast growth of B. microti.The genome is composed of two circular chromosomes of 2,117,050 and 1,220,319 base pairs. Unexpectedly, we found that the genome sequence of B. microti is highly similar to that of known Brucella species, and is remotely related to the one of O. anthropi. How the few differences in gene content between B. microti and B. suis 1330 could result in vastly different phenotypes remains to be elucidated. This unexpected finding will complicate the task of identifying virulence determinants in the Brucella genus. The genome sequence of B. microti will serve as a model for differential expression analysis and complementation studies. Our results also raise some concerns about the importance given to phenotypical traits in the definition of bacterial species.Contrary to expectations from the phenotypic analysis, the genome sequence of  Brucella comprises important mammal and human pathogens. Low infectious doses  in the Czech Republic  catalyzes the following reaction: ATP + N-acetyl-D-glucosamine = ADP + N-acetyl-D-glucosamine 6-phosphate.Also involved in glutamate and other amino-acids metabolisms, the B. suis 1330 (BRA0722). This enzyme [EC:1.5.1.12] catalyzes the reaction: (S)-1-pyrroline-5-carboxylate + NAD(P)+ + 2 H2O = L-glutamate +NAD(P)H + H+ and is involved in glutamate, arginine and proline metabolism.The proline/dehydrogenase/delta-1-pyrroline-5-carboxylate dehydrogenase (BMI_II715) is a pseudogene in B. microti than in its counterparts in other Brucella. A small conductance mechanosensitive ion channel protein encoding gene (BMI_I1045) has frameshifts in B. suis 1330 (pseudogene BR1042), B. ovis and B. canis and is intact in B. microti and O. anthropi. A second small conductance mechanosensitive ion channel protein encoding gene (BMI_I1334) is impaired in B. suis 1330 (pseudogene BR1322), B. canis and B. ovisWe also observed a number of changes in genes encoding membrane proteins. The outer membrane protein encoding gene BMI_I75 exhibits large differences in size, being larger in Mesorhizobium sp. BNC1 than to its homologues in all Brucella and Ochrobactrum.An outer membrane autotransporter, encoded by BMI_II170, is paradoxically most similar to the protein Meso_3532 in B. microti membrane protein Bme3, encoded by BMI_II422, corresponds to pseudogene BRA0425 in B. suis 1330. Genes at this locus have been reported to be involved in polysaccharide synthesis  synthesis genes) apparently entered the Brucella genomes through this mechanism, the B. microti island appears devoid of functional genes. Paradoxically, the most striking genome structure difference between B. microti and other Brucella is probably of no phenotypic consequence.The specific 12 kbp genomic island of probable phage origin is of obvious interest for identification purpose, and has already been used for the recovery of  samples . Blast  153 bp long and the 3' fragment 2.6 kbp long. The IVS is composed of palindrome sequences and repeated motifs forming stable secondary structures (stem-loop)  Figure  38]. Th. ThB. mifunction ,40. The Brucella , howeverlmonella  indicateBrucella that have been characterized include the Type IV secretion system  [B. suis 1330 [NC_004310 (large) and NC_004311 ] [B. abortus 9–941 [NC_006932 (large) and NC_006933 ] [B. melitensis biovar Abortus 2308 [NC_007618 (large) and NC_007624 ], B. ovis [NC_009505 (large) and NC_009504] [B. suis ATCC 23445 [NC_010169 (large) and NC_010167 ], B. canis ATCC 23365 [NC_010103 (large) and NC_010104 ] and Brucella abortus S19 [NC_010742 (large) and NC_010740 ]. In addition we used the genomic sequence of Ochrobactrum anthropi ATCC 49188 . Dotplots of the genome sequences were performed using programs from the MUMMER package [The genomic sequences reported in this article were compared to the available genomic sequences of ] , B. suis] , B. suis package .B. suis 1330 and B. microti were performed using software from the LAGAN Toolkit [B. microti. The alignment files are available as Additional Files Alignment of chromosomes of  Toolkit . An in-hB. suis 1330 and B. microti, a large use was made of the full alignment of sequences that is presented in Additional Files Brucella. Insertion sequences that were not present in the genome of B. suis 1330 were identified with the help of the Biotoul IS-Finder [Due to the close similarity between the genomes of S-Finder ,57.B. microti or B. suis 1330 were identified as follows. First, the most similar and intact version of the homologous ORF was selected among one of the available Brucella genomes. This sequence was then used to query (using tblastn [-50) the remaining Brucella and Ochrobactrum genomes to detect the eventual absence or disruption of the homologous gene . 170 cases of premature terminations or frameshifts were found and are listed in Additional file B. microti and intact in at least one other Brucella, while 120 correspond to genes presumably functional in B. microti and altered in its closest relative B. suis 1330.Candidate pseudogenes either in  tblastn , no filtB. microti chromosomes were grouped with those of 8 other Brucella and O. anthropi in a file containing 33,053 proteins. A blastp [-5) of this set against itself yielded a table of blast results which was used to cluster proteins using a Markov chain clustering algorithm [Brucella and in O. anthropi. The 1,486 Brucella core proteins were first aligned individually using MUSCLE (v3.7)[The proteins of the A blastp  search (lgorithm . We seleLE (v3.7). Poorly LE (v3.7) using thLE (v3.7). The resLE (v3.7). The defLE (v3.7) and MEGALE (v3.7).B. microti and O. anthropi but impaired in all other Brucella were identified. These genes are a priori the most likely to contribute to the Ochrobactrum-like phenotypic traits of B. microti. The corresponding protein sequences were searched against the nr database with the blastp program  using the Phylogeny.fr platform [Only 4 genes common to platform . The homThe authors declare that they have no competing interests.HCS designed the project, performed the experiments and wrote the manuscript. SA, ML and JMC analyzed the data and wrote the manuscript.B. microti and B. suis 1330.Supplementary File 1: Whole genome alignment with superimposed annotation of the large chromosomes of  Letter x in the consensus line denotes an indel or a point mutation. In protein coding regions, the reading frame is indicated.Click here for fileB. microti and B. suis 1330.Supplementary File 2: Whole genome alignment with superimposed annotation of the small chromosomes of  Letter x in the consensus line denotes an indel or a point mutation. In protein coding regions, the reading frame is indicated.Click here for fileB. microti and B. suis 1330.Supplementary Table 1: List of indels between the genomes of  The coordinates in B. microti and B. suis are given as well as the localization and the putative effect of the indels on gene products.Click here for fileB. microti as confirmed by the genome sequence.Supplementary Table 2: Distinctive VNTR typing of  For each tandem repeat we listed the primer pair, the theoretical length of the amplimer in B. suis 1330, B. melitensis 16M, B. abortus 9–941 and B. microti CCM 4915, and the location of the theoretical PCR product in the genome of B. microti.Click here for fileO. anthropi and other Brucella studied in this work.Supplementary Figure 2: Alignment of the 23S ribosomal RNA gene sequences in  In addition, we included the sequences of B. ceti and Brucella inopinata B01. Abbreviations: oan, O. anthropi; bcs, B. canis; bmt, B. suis ATCC 23445; bms, B. suis 1330; bmb, B. abortus 9–941; bmf, B. melitensis biovar abortus 2308; bmc, B. abortus S19; bce, B. ceti; bme, B. melitensis 16M; bov, B. ovis; bmi, B. microti and B01, Brucella inopinata B01.Click here for fileB. microti genome sequence, showing that Brucella inopinata sp. nov. strain B01 diverged earlier than the other Brucella studied in this work.Supplementary Figure 1: Phylogenetic representation of the alignment of the regions corresponding to the first 10,000 nucleotides of  The sequence of B. ceti is also included. B. ceti and Brucella inopinata B01 sequences were obtained from the PATRIC web site [web site .Click here for fileB. microti and B. suis 1330 .Supplementary Table 3: Extended list of orthologous genes exhibiting annotation differences between  For each gene, we give its identification in B. microti and B. suis 1330 as well as its status in other Brucella. Abbreviations include: * for internal stop, a number indicates multiple stops, fs for frameshift, + for an intact sequence, Mult. Diffs for multiple difference, Mult. Fs for multiple frameshifts, NF for not found.Click here for fileB. microti and O. anthropi and impaired in the other Brucella.Supplementary Figure 4: Phylogenetic tree for the 4 genes conserved in  The trees were built using the Phylogeny.fr Web Server [Brucella; B) BMI_I1566, aspartyl/asparaginyl beta-hydroxylase; C) BMI_I1599, extracellular solute-binding protein belonging to an ABC-type transport system involved probably in dipeptide transport and D) BMI_II978, MarR family transcriptional regulator.b Server  using deClick here for fileO. anthropi and interrupted in Brucella.Supplementary Figure 3: Genomic representation of the region around the cluster of flagella assembly genes that is contiguous in  Intact genes are represented as black arrows, pseudogenes as red arrows.Click here for file


Staphylococcus aureus and S. epidermidis biofilms differ in structure, growth and regulation, and thus the high-throughput method of evaluating biofilm susceptibility that has been published for S. epidermidis cannot be applied to S. aureus without first evaluating the assay's reproducibility and reliability with S. aureus biofilms.Staphylococcus aureus biofilms were treated with eleven approved antibiotics, lysostaphin, or Conflikt®, exposed to the oxidation reduction indicator Alamar blue, and reduction relative to untreated controls was determined visually and spectrophotometrically. The minimum biofilm inhibitory concentration (MBIC) was defined as ≤ 50% Alamar blue reduction and a purple/blue well 60 min after the addition of Alamar blue. Because all of the approved antibiotics had MBICs >128 μg/ml (most >2048 μg/ml), lysostaphin and Conflikt®, with relatively low MBICs, were used to correlate Alamar blue reduction with 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction and viable counts (CFU/ml) for S. aureus ATCC 29213 and three clinical isolates. Alamar blue's stability and lack of toxicity allowed CFU/ml to be determined from the same wells as Alamar blue absorbances.®, Alamar blue reduction had excellent correlation with XTT reduction (r = 0.93-0.99) and with CFU/ml (r = 0.92-0.98). For one of the clinical isolates, the results were moderately correlated for Conflikt®  and had excellent correlation for lysostaphin .Overall, Alamar blue reduction had excellent correlation with XTT reduction and with CFU/ml. For ATCC 29213 and two clinical isolates treated with lysostaphin or ConfliktS. aureus biofilms. The described method provides researchers with a simple, nontoxic, relatively inexpensive, high throughput measure of viability after drug treatment. A standardized biofilm Alamar blue assay should greatly increase the rate of discovery of S. aureus biofilm specific agents.A reliable, reproducible method for evaluating biofilm susceptibility was successfully applied to  Staphylococcus aureus and S. epidermidis are the most common infectious agents associated with foreign device infections . The stus study  used a vStaphylococcus biofilm specific agents.The benefits of AB over other methods of biofilm susceptibility testing are numerous and include simplicity, relative cost, compatibility with high throughput, lack of toxicity and importantly, AB measures viability, not simply mass. The major benefit of AB over XTT/menadione is its lack of toxicity (menadione has human toxicity), and the obvious benefit of AB over CFU/ml determination is its simplicity. The AB biofilm assay can be performed in any lab with a spectrophotometer, and is an excellent choice for high throughput labs. A standardized biofilm AB assay should greatly increase the rate of discovery of The authors declare that they have no competing interests.RKP designed the studies, performed the statistical analyses, and wrote the manuscript. CAW performed the studies and participated in data analyses. GRP arranged financial support and critically evaluated the manuscript. All authors read and approved the final manuscript.


Aliivibrio salmonicida is the causative agent of cold-water vibriosis in marine aquaculture. The Gram-negative bacterium causes tissue degradation, hemolysis and sepsis in vivo.The fish pathogen A. salmonicida has a six partite architecture with two chromosomes and four plasmids. Sequence analysis revealed a highly fragmented genome structure caused by the insertion of an extensive number of insertion sequence (IS) elements. The IS elements can be related to important evolutionary events such as gene acquisition, gene loss and chromosomal rearrangements. New A. salmonicida functional capabilities that may have been aquired through horizontal DNA transfer include genes involved in iron-acquisition, and protein secretion and play potential roles in pathogenicity. On the other hand, the degeneration of 370 genes and consequent loss of specific functions suggest that A. salmonicida has a reduced metabolic and physiological capacity in comparison to related Vibrionaceae species.In total, 4 286 protein coding sequences were identified, and the 4.6 Mb genome of A. salmonicida unable to grow on the polymer form of chitin. These, and other losses could restrict the variety of carrier organisms A. salmonicida can attach to, and associate with. Gene acquisition and gene loss may be related to the emergence of A. salmonicida as a fish pathogen.Most prominent is the loss of several genes involved in the utilisation of the polysaccharide chitin. In particular, the disruption of three extracellular chitinases responsible for enzymatic breakdown of chitin makes  Aliivibrio salmonicida  is a facultative pathogen of fish responsible for causing cold-water vibriosis (CV) in farmed Atlantic salmon , sea farmed rainbow trout (Oncorhynchus mykiss) and captive Atlantic cod (Gadus morhua) .The sequence was annotated using Artemis software . InitialComparison of the genome sequences was facilitated by using the Artemis Comparison Tool (ACT) , which eIn order to determine if duplicated genes originated from the plasmids or from the chromosomes and to predict the order in which the plasmids were acquired we performed a CAI and a CBI analysis: CAI and a CBI analysis: CAI used the Highly expressed genes  as the reference; CBI used the codon usage of all the genes in the genome and measured the adaptation of each gene to that. The CAI was done via EMBOSS cai and the CBI was done via EMBOSS codcmp .Amplification of genes from other isolates was performed by PCR using Platinum Pfx DNA Polymerase  according to the protocol supplied by the manufacturer. PCR amplification products were analyzed in 0.8% agarose gels stained with ethidium bromide.A. salmonicida, A. wodanis and V. splendidus were grown in LB medium containing 2.5% NaCl, diluted in A. salmonicida minimal medium 2SO4, 3.9 μM FeSO4, 2.5% NaCl, 0.81 mM MgSO4, 2 mM Valin, 0.5 mM Isoleucin, 0.5 mM Cystein, 0.5 mM Methionin, 40 mM Glutamate]) and transferred to Vsmm agar supplemented with 10 mg/ml α-chitin (Sigma-Aldrich) or N-acetyl-α-D-glucosamine . Plates were incubated from 2 to 7 days at 12°C  and 22°C (V. splendidus) and growth evaluated by visual examination.Isolates of IS: insertion sequence; CV: cold-water vibriosis; chr: chromosome; CDS: protein coding sequence; CAI: Codon Adaptation Index; CBI: Codon Bias Index; CPS: capsular polysaccharides; LPS: lipopolysaccharide; (GlcNAc): N-acetyl-D-glucosamine; CBP: chitin binding protein; QS: quorum sensing; HGT: horizontal gene transfer; GI: genomic island.EH: study conception, data analysis, research design, manuscript writing. MSL: research design, data collection, manuscript production. MTGH: research design, manuscript production. KS: data collection. SP: research design, manuscript production. NB: data collection. CC: data collection. DH: data collection. HN: data collection. MAQ: data collection. SS: data collection. ST: data collection. JP: study conception, manuscript production. NPW: study conception, manuscript production. NRT: research design, study conception, manuscript writing.A. salmonicida LFI1238 plasmids; B: Putative duplicated regions of the plasmids in comparison to chromosome I.A: Schematic circular diagrams of  A: Appropriate categories are shown as pairs of concentric circles representing both coding strands. Key to the chromosomal circular diagrams (outside to inside): scale (in kb), annotated CDSs, unique CDSs compared to the other Vibrionaceae species (red), orthologues shared with the other Vibrionaceae species (green), IS element transposases (purple), % G+C content, G+C deviation . Colour coding for CDSs (according to predicted function): dark blue, pathogenicity/adaptation; black, energy metabolism; red, information transfer; dark green, surface associated; cyan, degradation of large molecules; magenta, degradation of small molecules; yellow, central/intermediary metabolism; pale green, unknown; pale blue, regulators; orange, conserved hypothetical; brown, pseudogenes; pink, phage + IS elements; grey, miscellaneous. B: CDSs are represented as blocked arrows showing the direction of transcription. Identity at nucleotide level is indicated in grey boxes.Click here for fileA. salmonicida genome.Types and distribution of IS elements encoded in the  The data provided shows the distribution of the different types of IS elements identified in the six replicons.Click here for fileCodon Adaptation Index (CAI) of genes plotted against Codon Bias Index (CBI). Colour coding for the genes are: grey, chromosomal genes; light blue, highly expressed genes ; red, pVSAL840; dark green, pVSAL320; yellow, pVSAL54; light green, pVSAL43; black triangles, duplicated genes.Click here for fileA. salmonicida and A. fischeri.Linear DNA comparison between the chromosomes of  The grey bars represent the forward and reverse strands, and red and blue lines between the genomes indicate regions with similarity and inversions, respectively. Black boxes represent IS elements in A. salmonicida.Click here for fileA. salmonicida inserted phage ϕ VS4 and comparison with the related phage K139.Genomic organisation of the  The red lines between the phages indicate regions with amino acid similarity. CDSs are represented as blocked arrows showing the direction of transcription, with colour codes according to their functional annotation. The length of the arrows approximately reflects the sizes of the CDSs. The G+C content and G+C average was analysed using Artemis with a window size of 500 nt.Click here for fileA. salmonicida.CDSs harboured on putative genomic islands and phages in  The data in this table is additional to the data in Table Click here for fileA. salmonicida LFI1238; B: Agarose gel electrophoresis of PCR products confirming the presence of the duplication in all tested A. salmonicida isolates.A: The two duplicated regions in  The first gene of each duplicate and the flanking genes are indicated as grey boxes . B) Agarose gel electrophoresis of duplication products VSAL_I0408-I0407 (PCR product 1) and VSAL_I0264-I0265 (PCR product 2), and the corresponding regions of different A. salmonicida isolates derived by PCR from genomic DNA. Primers are designed from LFI1238 and are indicated by arrows . The A. salmonicida isolates were obtained from various outbreaks of cold water vibriosis from salmon and cod. The isolate numbers are indicated above each lane. S, 100 bp DNA ladder.Click here for fileA. salmonicida genes involved in key steps of the chitinolytic cascade, together with orthologues from other sequenced Vibrionaceae.Functional and putative non-functional  The data provided in this table lists all identified A. salmonicida CDSs that are known to be involved in the chitinolytic cascade in other Vibrionaceae. Disrupted CDSs and pseudogenes are labelled in the table.Click here for fileVibrionaceae isolates on α-chitin and GlcNAc.Growth of different  The data provided shows that the A. salmonicida isolates investigated in this study are not able to utilise α-chitin or GlcNAc.Click here for fileA. salmonicida with potential roles in pathogenicity.Predicted CDSs of  The data in this table gives a comprehensive list of all CDSs with possible functions that may be associated with mediating cold-water vibriosis in fish.Click here for fileA. salmonicida genome.Functional distribution of putative inactivated genes in the  The data provided in this table includes all CDSs disrupted or truncated by IS elements and CDSs containing translational frameshifts of premature stop codons.Click here for file


Trichinella papuae.In 2006, the Thailand Ministry of Public Health studied 28 patients from a village in northern Thailand. All had myalgia, edema, fever, and gastrointestinal symptoms; most had eaten wild boar. A muscle biopsy specimen from a patient showed nonencapsulated larvae with a cytochrome oxidase I gene sequence of  Trichinella spiralis .Trichinella spp. was collected. A muscle biopsy of 1 case-patient was performed.For laboratory investigation, samples were collected from case-patients and controls for complete blood counts and serologic testing. In the hospital, laboratory results, including complete blood counts and creatine phosphokinase (CPK), were reviewed and blood for detection of antibodies to T. zimbabwensis and T. papuae in GenBank . Primers for amplification were Tri-COIF (forward: 5′-GTTTATAT(C/T)(C/T)TAGTACTA CC-3′) and Tri-COIR (reverse: 5′-GC(G/A)TTTGATAGTCT(A/G)ACTCC-3′). DNA alignment analyses were conducted by using ClustalW version 1.83 , and nucleotide substitutions were identified by using BioEdit version 7.0.1  . The partial cytochrome oxidase subunit I (COI) region was amplified by primers, which were designed from partial COI sequences of mitochondrial DNA from Trichinella spp. and a sample of fermented barking deer meat for larvae examination. No uncooked wild boar meat was available for laboratory analysis. All larvae and human serum specimens were sent to the Department of Helminthology, Mahidol University, Thailand. Immunoblot tests for human trichinellosis were conducted by using the 109-kDa diagnostic band   had an illness that met the case definition (4 were suspected cases). Of the 28 case-patients , 18 were male. The attack rate in the village was 10.3% (28/273); there were no deaths. The outbreak occurred during May 24–June 26, 2006, and the epidemic curve was characteristic of a common point source . All casThe environmental study found that villagers were avid hunters of deer, boar, and other local game; they also raised domesticated pigs . Villagers shared domesticated pig meat from various sties every 2–3 weeks. On May 20, a successful wild boar hunt resulted in distribution of wild boar meat to villagers. On May 21, domesticated pig meat was shared among villagers. A successful hunt for barking deer also obtained meat that was prepared in various styles, including cooked, raw, and fermented, and was distributed to villagers during April and May.Trichinella spp. Among 28 blood samples from case-patients, all had eosinophilia  and 21 had leukocytosis, . All 3 hospitalized case-patients had elevated CPK levels . One domesticated pig was randomly selected from each of 8 pigsties and tested by ELISA for antibodies to Trichinella spp.; all pig samples were negative. A human gastrocnemius muscle biopsy specimen from a hospitalized case-patient was positive for nonencapsulated Trichinella spp. larvae . Eighty-two blood samples were collected during the investigation; 32 (39%) had antibodies to . larvae , which pTrichinella spp. and were included in the analytic study. Of the 54 villagers who did not meet the descriptive case definition, 46 had negative serologic test results and were included as controls. Eating uncooked wild boar from the hunt showed the highest risk for illness   had antibodies to .0–30.1) . Associa.0–30.1) . AlthougTrichinella spp. These findings indicate that the geographic range of T. papuae is greater than previously thought.Eating undercooked wild boar meat was strongly implicated as the source of this trichinellosis outbreak. The villagers were instructed about the importance of thoroughly cooking meat potentially contaminated with


We report a rare case of spontaneous pneumomediastinum due to perforation of sigmoid cancer in a patient suffering from Vogt-Koyanagi-Harada syndrome and temporal arteritis, two rare diseases. This patient, who generally receives corticosteroid and methotrexate therapy, was admitted to hospital with vague abdominal and left flank pain, urinary disorders and low grade fever one day prior to admission. Initial evaluation including X-ray and laboratory tests was normal. Several hours later a repeat chest X-ray showed pneumomediastinum. Chest and abdominal Computed Tomgraphy were performed because of worsening abdominal pain, and revealed a perforated sigma due to carcinoma. Pneumomediastinum (PM) is uncommon, and spontaneous PM is rare. It occurs when air leaks from any part of the lung or airways into the mediastinum. One cause is increased pressure within the lungs or airways that ruptures the air sacs or airways, allowing air to escape into surrounding structures. Such pressure can be caused by excessive coughing, sneezing, vomiting or repeated Valsalva maneuvers . PM may also occur following perforation of the trachea, rapid ascents in altitude, or use of a breathing machine . It also can occur in association with pneumothorax or other diseases . Pneumom3, hemoglobin 12.4 g/dl, and platelets 227000/mm3. Blood glucose 160 mg/dl, sodium, potassium and electrolytes were normal. Kidney and liver function tests including amylase were all in the normal range. Urine test was normal. Chest and abdominal X-ray at admission showed normal findings. Chest X-ray was repeated several hours later because of the appearance of mild chest pain and dyspnea, and revealed an initial small PM (th day post-operative with the diagnosis of perforated cancer of sigma.A 60-year-old Arab woman with a history of Vogt-Koyanagi-Harada (VKH) syndrome is an uveo encephalomeningitis and temporal arteritis for several years was admitted to our department for evaluation of left flank pain and vague abdominal discomfort, low grade fever, and mild urinary disorders. The patient had been treated with corticosteroids (prednisone 20 mg/day), methotrexate 15 mg/weekly and folic acid 5 mg/day for her rare chronic diseases. On admission, the patient appeared well, complained of mild, constant pain in left flank and diffuse abdomen, without nausea, vomiting or diarrhea. She had no symptoms of dyspnea or chest pain; stools were normal. Physical examination revealed good performance status and no respiratory distress. Body temperature was 37.8Cº, blood pressure 130/65 mmHg, heart rate 108/min, and respiratory rate 15/min. Cardiac examination revealed no gallop, murmurs, or friction rub. Lungs were clear. The abdomen was soft, with mild tenderness on deep palpation especially in the left lower quadrant, but without signs of peritoneal irritation. Bowel sounds were present. Laboratory results upon admission revealed leukocyte count 18000/mmsmall PM . On the small PM ,3. The pSPM is a rare condition. A 2001 review by Chalumeau et al. summarized the available literature . Based o


Leishmania transmission as it was shown to increase parasite virulence. On the other hand, immunity to salivary components impedes the establishment of infection. Therefore, it is most desirable to gain a deeper insight into the composition of saliva in sand fly species which serve as vectors of various forms of leishmaniases. In the present work, we focused on Phlebotomus (Adlerius) arabicus, which was recently shown to transmit Leishmania tropica, the causative agent of cutaneous leishmaniasis in Israel.Sand fly saliva plays an important role in blood feeding and P. arabicus females was constructed and transcripts were sequenced and analyzed. The most abundant protein families identified were SP15-like proteins, ParSP25-like proteins, D7-related proteins, yellow-related proteins, PpSP32-like proteins, antigen 5-related proteins, and 34 kDa-like proteins. Sequences coding for apyrases, hyaluronidase and other putative secreted enzymes were also represented, including endonuclease, phospholipase, pyrophosphatase, amylase and trehalase. Mass spectrometry analysis confirmed the presence of 20 proteins predicted to be secreted in the salivary proteome. Humoral response of mice bitten by P. arabicus to salivary antigens was assessed and many salivary proteins were determined to be antigenic.A cDNA library from salivary glands of P. arabicus salivary glands is the first description of salivary proteins of a sand fly in the subgenus Adlerius. Proteomic analysis of P. arabicus salivary glands produced the most comprehensive account in a single sand fly species to date. Detailed information and phylogenetic relationships of the salivary proteins are provided, expanding the knowledge base of molecules that are likely important factors of sand fly-host and sand fly-Leishmania interactions. Enzymatic and immunological investigations further demonstrate the value of functional transcriptomics in advancing biological and epidemiological research that can impact leishmaniasis.This transcriptomic analysis of  Leishmania parasites, the causative agents of leishmaniasis. During the feeding process sand flies inject saliva into the site of the bite to facilitate successful acquisition of a blood meal [Leishmania while feeding; thus, the parasite is always introduced to the host as a mixture with sand fly saliva. Sand fly saliva facilitates the transmission of Leishmania parasites to mammalian hosts; at the same time, immune response to salivary components was shown to partially protect the host from Leishmania infection [Phlebotomus papatasi, the vector of Leishmania major, and Lutzomyia longipalpis, the vector of L. infantum, have been extensively studied. In addition, cDNA libraries from several other sand fly species were characterized and include other sand flies that vector L. major (P. duboscqi), L. infantum (P. ariasi and P. perniciosus) and L. donovani (P. argentipes).Phlebotomine sand flies are the arthropod vectors of ood meal . An infenfection . TherefoPhlebotomus (Adlerius) arabicus is distributed in certain parts of East Africa and the Middle East. In Ethiopia, P. arabicus females infected with uncharacterized Leishmania sp. were reported [P. arabicus is the proven vector of cutaneous leishmaniasis caused by L. tropica [L. tropica is found in a vast discontinuous area reaching from the south-western Mediterranean to Turkey, north-western India and Sub-Saharan Africa [L. tropica was recently shown to occur as an anthropozoonosis as well [P. arabicus is a permissive vector, meaning it is susceptible to development of more than one species of Leishmania, including L. major and L. infantum [reported , and in  tropica ,5. Cutann Africa . Long su as well ,5. Laborinfantum .P. (Adlerius) arabicus were studied by cDNA sequencing, electrophoretic and proteomic methods. This is the first study of the repertoire of salivary molecules of a vector of L. tropica and it is the first report of the composition of salivary proteins in the subgenus Adlerius.In the present study, salivary gland transcripts and proteins of Phlebotomus arabicus females dissected one day after emergence. From this cDNA library, 1152 random transcripts were selected and sequenced, resulting in 985 high quality sequences. Sequences were clustered together based on sequence homology and produced 107 clusters and 288 sequences were assessed as singletons (only one sequence). The term cluster may refer to either singletons or multiple homologous sequences. Similar to other sand flies studied so far, the most abundant transcripts were those coding for putative secretory proteins and resulted in 74 clusters with an average number of 7.65 sequences per cluster. Predicted proteins containing retention signals for endoplasmic reticulum and/or transmembrane domains were not treated as putatively secreted. An example of such proteins is the translocon-associated protein complex, δ subunit , which has homologs previously designated as 16 kDa or 16.1 kDa salivary protein in P. ariasi or L. longipalpis, respectively.A cDNA library was constructed from salivary glands of L. infantum vectors P. (Larroussius) ariasi and P. (L.) perniciosus. The expected values of these matches were highly significant at values lower than E-60. To a lesser extent, similarity to sequences of salivary proteins of P. (Euphlebotomus) argentipes, the vector of L. donovani in India, was also observed. These findings are fully in concert with the close evolutionary relationship of Larroussius and Adlerius subgenera reported by Aransay et al. [Members of 21 different families were found among putative secretory proteins. BLAST comparison of translated nucleotide sequences with the NR protein database showed that overall, high similarity was observed namely with salivary proteins of y et al. .P. papatasi brought strong evidence that SP-15 is a multicopy gene [P. arabicus salivary gland cDNA library may reflect gene duplication events or allelic variation.Some of the protein families contained multiple members. The observed variability among individual protein family members might be explained by intraspecific polymorphism, as the tested sample was heterogeneous . Nevertheless, analysis of genetic variation of SP15 salivary protein in opy gene . While iw) and isoelectric point (pI) of the mature protein, and the GenBank accession number of the nucleotide coding sequence. The table also includes information on the presence of individual proteins in P. arabicus salivary proteome, as confirmed by Edman degradation and/or mass spectrometry. A more detailed description of the putative secreted proteins follows, starting with proteins encoded by the most abundant transcripts:Full-length sequences were obtained for most clusters coding for putatively secreted proteins. Only sequences containing a signal peptide and a polyA tail in the coding cDNA were considered full-length. Table P. papatasi SP15 conferred partial protection against L. major infection [P. arabicus salivary gland cDNA library and clustered into three separate groups . The amino acid sequences of mature proteins coded by P. arabicus transcripts share 22.5% amino acid identity and 23.3% amino acid similarity. When SP15-like proteins from other sand flies were added to the analysis, only the six cysteines and three other amino acids were conserved in the amino acid sequence of mature proteins , corresponding to the fact that most Phlebotomus spp. sand fly salivary proteins have very high predicted pI values.Thus far, SP15-like proteins have only been reported in sand flies and their function remains unknown. It was suggested that SP15-like proteins were derived from an ancestral odorant-binding protein and were closely related to short D7 proteins . Immuniznfection . Transcrs Figure , reflecty Figure . The preP. ariasi 27 kDa salivary protein  and P. perniciosus 29 kDa salivary protein  were found in the P. arabicus salivary gland cDNA library. There are no other homologs of these proteins in accessible databases, no conserved domains were found in the translated sequences, and no function has been assigned to these proteins. However, in P. arabicus cDNA library they represent the second most abundant protein family. Transcripts coding for ParSP25-like proteins occurred in long  and short forms , with very little variability among individual clusters. The mature proteins coded by these transcripts have a predicted Mw of 27 kDa and 25 kDa, respectively, and are composed of 90.5% identical amino acids and 0.9% similar amino acids. The predicted pI of the proteins is acidic (average pI = 5.03), unlike most sand fly salivary proteins described thus far.Six clusters coding for proteins related to L. longipalpis [P. arabicus salivary gland cDNA library . Predicted mature proteins have Mw of 26–28 kDa and an average basic pI of 9.24. Two of the seven clusters have potential N-glycosylation sites, as predicted by NetNGlyc server. The protein sequences of mature proteins were 20.3% identical and 15.5% similar . The predicted Mw of the protein is 42.9 kDa with a pI of 8.4. No N-glycosylation sites were predicted in the protein sequence by amino acid submission to the NetNGlyc server.Yellow-related proteins are common in insects; in bloodsucking Diptera, yellow-related proteins were described from mosquitoes and sand flies. In proteins  and, accproteins , it mighoperties . Sand floperties -26. In Ltrometry . In the P. papatasi, have only been found in sand flies and their function is unknown. In P. perniciosus they possess a collagen-related internal sequence [P. arabicus, however, these proteins bear no significant similarity to collagen; this feature is shared with PpSP32-like proteins of P. papatasi, P. ariasi or P. argentipes. Similarly to other protein families analyzed, the phylogenetic position of P. arabicus PpSP32-like proteins is close to that of P. ariasi and P. perniciosus homologs , the mature proteins being 88.1% identical  and very basic (pI>11.5) amino acids  [Culicoides sequences. Close relationship of P. arabicus Ag5-related protein to P. perniciosus and P. ariasi was observed, much in the same way as in other salivary protein families .Antigen 5 (Ag5) protein is present in vespid venom  and relaJ439532) ,28,30. MJ439532) , this sas Figure . The preCimex apyrase family [P. arabicus cDNA library (98.4% identity) . The predicted average pI of P. arabicus apyrases is 8.85 and the predicted Mw is 35.3 kDa.Apyrases are widespread in saliva of bloodsucking insects. The antihemostatic effects of saliva are, for a great part, due to apyrase anti-platelet activity . Sand fle family . Three vP. arabicus cDNA library . Similar sequences were reported from P. ariasi, P. perniciosus, P. argentipes, and L. longipalpis salivary glands. Cluster PabSP49 encodes an endonuclease domain, which is typical for DNA/RNA non-specific endonucleases. Since all residues composing the active site, the substrate binding site and the Mg2+ binding site are conserved in this cluster; we suggest that PabSP49 might possess endonuclease activity. The predicted pI of the mature protein is 9.45 and the predicted Mw of the mature protein is 40.5 kDa. Possible roles for a salivary endonuclease include reducing the viscosity of the blood pool during feeding and liberating nucleosides. Exogenous nucleosides, primarily adenosine, can exhibit regulatory effects on blood clotting, immune and inflammatory responses, and Leishmania pathogenesis [A cluster encoding a putative endonuclease was identified in the ogenesis .P. papatasi and P. duboscqi salivary glands do not contain hyaluronidase transcripts. Nonetheless, the enzyme activity was detected in salivary gland samples from these species [P. arabicus, hyaluronidase activity was also observed. As revealed by zymography, the apparent molecular weight of the P. arabicus hyaluronidase holoenzyme is approximately 110 kDa , a phospholipase A2 (PLA2) domain is present, containing all conserved residues of both catalytic and metal-binding sites of PLA2. In hymenopteran venoms, PLA2 represents a major allergen. In the salivary glands of blood-feeding insects, sequences coding for PLA2-like proteins were reported only from sand flies of the subgenus Larroussius [P. arabicus specifically for PLA2 activity and did not detect any positive reaction. Cluster 52 contains an exceptionally long 5' untranslated region (5' UTR) compared to other clusters from this cDNA library coding for secreted proteins. The 5' UTR in this cluster is more than 500 nucleotides long. Thus, the regulation of expression of this transcript might be different from other transcripts reported herein.In the amino acid translation of sequence cluster 52 , amylase , an enzyme involved in digestion of dietary starch [PabSP315, GenBank accession number EZ000625). Previously, sequences coding for pyrophosphatase-like proteins were reported in P. argentipes and P. duboscqi sand flies [P. arabicus homolog reported herein, contain a conserved phosphodiesterase domain, typical for enzymes cleaving phosphodiester and phosphosulphate bonds in NAD, deoxynucleotides and nucleotide sugars [L. longipalpis salivary glands and midguts as well as P. papatasi midguts [L. longipalpis and P. papatasi salivary gland samples [P. arabicus salivary glands. The putative trehalase enzyme from P. arabicus salivary glands might either be an intrinsic component of insect metabolism, or might be related to sugar feeding and digestion. Trehalose is the main energy source in insect hemolymph in general. Trehalases are involved in its hydrolysis, yielding glucose molecules which are then readily available to various cells of the insect body [P. papatasi [Other sequences coding for other putative enzymes could not be obtained as full-length clones. These included a pyrophosphatase-like protein . These proteins are seemingly sand fly-specific; apart from 5 sand fly species ; no other related proteins from any organism were reported. Another family of putative sand fly-specific proteins contain homologs of P. arabicus 46 kDa salivary protein . Transcripts coding for such proteins were reported from P. perniciosus, P. ariasi, P. duboscqi and L. longipalpis. Homologs of P. arabicus 2.7 kDa peptide  were only found in P. ariasi and P. perniciosus [Larroussius [PabSP75 and PabSP97; GenBank accession numbers FJ474087 and FJ474088, respectively) were found in P. arabicus; homologous molecules were identified in P. ariasi [PabSP63 and PabSP64; GenBank accession numbers FJ474085 and FJ474086, respectively) are homologous to molecules identified in P. argentipes [There were a number of transcripts with no homology to known enzymes or structural proteins; however, eight of these transcripts encode potentially secreted proteins with high homology to other sand fly salivary molecules. niciosus . Our fin. ariasi . AdditioP. arabicus cDNA library show no similarity with known sand fly sequences. Cluster 107  is homologous to Ae. aegypti putative salivary secreted mucin 3, as well as the IgE binding protein icarapin from honeybee venom [Anopheles gambiae (XP_001230739) and Aedes aegypti (XP_001650286) were also predicted to contain multiple O-glycosylation sites. These proteins might be involved in hypersensitivity to bites of blood-feeding insects. The second cluster 126 , encodes a homolog of conserved hypothetical proteins of culicine as well as anopheline mosquitoes. The predicted molecular weight of the mature protein from P. arabicus is 17.2 kDa and the predicted pI is 5.34. No N- or O-glycosylation sites were predicted in cluster 126 protein and nothing is known about these hypothetical proteins.Two sequence clusters coding for putatively secreted proteins in the ee venom . The preP. arabicus salivary gland samples separated by SDS-PAGE were subjected to Edman degradation and mass spectrometry. Edman degradation resulted in the identification of 7 different N-terminal sequences. These were representative of two 14 kDa proteins , yellow-related protein , and endonuclease . An N-terminal sequence common to all six variants of salivary proteins similar to ParSP25 was also detected by Edman degradation , as well as N-terminal sequences common to apyrases  and to D7-related proteins A and C . From the data obtained by Edman degradation analysis it could not be concluded which variants of polymorphic salivary proteins were present in the proteome.For the proteome analysis, P. arabicus salivary proteome. By this method, 19 putative secreted proteins were identified in the proteome , yellow-related protein , two 34 kDa salivary proteins , all three apyrase-like proteins , two PpSP32-like proteins , antigen 5-related protein , four 25 kDa salivary proteins similar to ParSP25 , three D7-related proteins , and two PpSP15-like proteins . In addition, one high-molecular weight protein (>70 kDa) analyzed by mass spectrometry revealed no similarity to predicted P. arabicus secreted salivary proteins. We assume this protein represents a component of salivary gland wall rather than a secreted protein present in the saliva. Accordingly, in P. duboscqi female salivary glands, we previously detected multiple protein bands running at molecular weight protein >70 kDa which were specifically present in the wall of salivary glands [Mass spectrometry was used for a more detailed analysis of e Figure . These py glands .P. arabicus salivary gland samples . A number of paralogous transcripts were identified, such as those in the SP15 and D7 families. The presence of duplicate gene copies has been observed in other blood feeding arthropods [P. arabicus that were characterized in the transcriptome. In addition, one protein was identified by mass spectrometry that did not match any of the characterized transcripts. This is the most comprehensive description of sand fly salivary proteome to date and also demonstrates that the transcriptome represents >95% of the most abundant proteins present in the salivary gland.Some of the most abundant molecules identified in the transcriptome that have a predicted signal secretion peptide include a 14 kDa protein (PabSP2), a D7-related protein (PabSP20), a yellow-related protein (PabSP26), an Antigen 5-related protein (PabSP4) and 25 kDa and 27 kDa proteins similar to thropods ,44 and cP. arabicus salivary gland transcriptome four sequences were identified as encoding a putative hyaluronidase. Hyaluronidase is an enzyme that has been identified in a number of phlebotomine salivary glands including Lutzomyia longipalpis, P. Phlebotomus papatasi, P. Phlebotomus duboscqi, P. Paraphlebotomus sergenti and P. Adlerius halepensis [In the analysis of the lepensis . The zymP. arabicus demonstrates the abundance and diversity of antigenic molecules in the saliva. Future work may focus on the use of functional transcriptomics  to use these P. arabicus salivary proteins to evaluate the role of these molecules in the epidemiology of leishmaniasis.Having described the repertoire of saliva molecules opens more doors in the research of vector-host and vector-parasite interactions, pharmacology and insect biochemistry. The antigenicity of sand fly saliva is one important aspect of the vector-host interaction. Evaluating the humoral response of mice bitten by P. arabicus (Israel) was reared in the insectary of Charles University in Prague in standard conditions as described by Benkova and Volf [P. arabicus females were dissected and stored in Tris buffer .The colony of and Volf . For mRN® III Gold Packaging Extract (Stratagene) was used for packaging the phage particles. The libraries were plated by infecting log-phase XL-1 blue Escherichia coli (Clontech). Several plaques from each plate were selected and a PCR with vector primers flanking the inserted cDNA was performed. The presence of recombinants was checked by visualisation the PCR products on 1.1% agarose gel with ethidium bromide.Salivary gland mRNA was isolated from 30 pairs of glands using Micro-FastTrack mRNA isolation kit (Invitrogen). PCR-based cDNA library was made following the manufacturer's instructions for SMART™ cDNA library Construction Kit (BD Clontech) with some modifications described by Chmelar et al. . The cDNPlaques were randomly selected from the plated libraries and transferred to 96-well polypropylene plate containing 75 μl of water per well. The PCR reaction amplifying randomly selected cDNAs was performed using FastStart PCR Master mix (Roche), 3 μl of the phage sample as a template and primers described elsewhere . AmplifiDetailed description of the bioinformatic treatment of the data can be found elsewhere ,46. BrieProtein sequences of the members of identified protein families were compared with related sequences of other sand fly species obtained from GenBank. Sequences were aligned using ClustalW version 1.4  running P. arabicus females were homogenized by 5 freeze-thaw cycles. Samples were reduced using sample buffer with 2-mercaptoethanol, and electrophoretically separated in 12% polyacrylamide SDS gel. Gels were stained for total proteins with Coomassie G-250  or for glycoproteins with Pro-Q Emerald 300 glycoprotein stain (Invitrogen). Mass spectrometric analysis was performed with individual bands cut from the Coomassie-stained gel. The individual bands were placed in microtubes and covered with 100 μl 50 mM ammonium bicarbonate (ABC) buffer in 50% acetonitrile (ACN) with 50 mM dithiothreitol (DTT). The samples were subjected to sonication in an ultrasonic bath for 5 minutes. After 15 minutes the supernatant was discarded and the gel was covered with 100 μl of 50 mM ABC/50% ACN with 50 mM iodoacetamide and sonicated for 5 minutes. After 25 minutes, the supernatant was discarded and exchanged for 100 μl 50 mM ABC/50% ACN with 50 mM DTT and sonicated for 5 minutes to remove any excess iodoacetamide. The supernatant was discarded and samples were sonicated for 5 minutes in 100 μl of HPLC water. The water was discarded and samples were sonicated for another 5 minutes in 100 μl of ACN. ACN was discarded and microtubes with samples were left open for a couple of minutes to allow the rest of ACN to evaporate. Five ng of trypsin (Promega) in 10 μl of 50 mM ABC were added to the gel. Samples were incubated at 37°C overnight. Trifluoroacetic acid (TFA) and ACN were added to reach final concentration 1% TFA, 30% ACN. Samples were sonicated for 10 minutes and 0.5 μl drop was transferred onto MALDI target and let to dry. Dried droplets were covered with 0.5 μl drop of alpha-cyano-hydroxycinnamic acid solution (2 mg/ml in 80% ACN) and let to dry. Samples were measured using a 4800 Plus MALDI TOF/TOF analyzer (Applied Biosystems/MDS Sciex) equipped with a Nd:YAG laser .Salivary glands from 5-day-old Peak lists from the MS spectra were generated by 4000 Series Explorer V 3.5.3 (Applied Biosystems/MDS Sciex) without smoothing, peaks with local signal to noise ratio greater than 5 were picked and searched by local Mascot v. 2.1 (Matrix Science) against a database of proteins sequences derived from cDNA library. Database search criteria were as follows – enzyme: trypsin, taxonomy: none, fixed modification: carbamidomethylation, variable modification: methionine oxidation, peptide mass tolerance: 120 ppm, one missed cleavage allowed. Only hits that were scored as significant (p < 0.0001) are included.Phlebotomus arabicus salivary glands were electrophoretically separated on 1 mm thick 4–20% NuPAGE Novex Bis-Tris gels using MES SDS buffer (Invitrogen). A sample containing 30 glands was reduced with NuPAGE Sample Reducing Agent (Invitrogen) and run in parallel with non-reduced samples (50 glands) on the same gel. Wet blotting on a PVDF membrane was performed using XCell II™ Blot Module (Invitrogen). SeeBlue® Pre-Stained Standards (Invitrogen) were used to estimate molecular weight (Mw) of separated proteins and assess transfer efficiency. The membrane was stained with 0.025% Coomassie blue without acetic acid. Stained bands were cut and subjected to Edman degradation using a Procise 494cLC sequencer (Applied Biosystems). cDNA sequences corresponding to obtained N-terminal amino acid sequences of salivary proteins were identified using an in-house search program [P. arabicus cDNA sequencing project with the amino acid sequences.For the Edman degradation analysis,  program . This prP. arabicus females were tested for the activities of hyaluronidase and phospholipase A2. Salivary glands were dissected in Tris buffer  and stored at -20°C. Before use, the glands were mechanically disrupted, samples were centrifuged at 12000 g for 5 minutes, and the supernatant was used in the assays.Salivary gland samples from 5-day-old The zymographic hyaluronidase assay was performed as described by Volfova et al. . Briefly® Phospholipase A2 Assay Kit (Invitrogen). The manufacturer's instructions were followed. Samples containing 0.5, 1, 2, 5, 10 and 20 salivary glands in 50 μl were assayed; as a positive control, PLA2 from honey bee venom supplied with the kit was used. The assay was performed at 26°C, and fluorescence emission at 515 nm was detected after an incubation step of 12 minutes (Tecan Infinite 200).The phospholipase A2 activity was tested in the same salivary gland samples using the EnzChekP. arabicus sand flies. In biweekly intervals, the mice were anaesthetized with ketamine (1.5 mg/10 g body weight) and xylazin (0.15 mg/10 g body weight) and exposed to sand fly females. Each time, an average of 60 females fed on each mouse (SE = 14.9). Ten days after the sixth exposure the mice were bled and obtained sera were kept at -20°C.Experiments on mice were done in accordance with Czech Act No. 246/1992 and approved by IACUC of the Faculty of Science, Charles University in Prague. Female BALB/c mice, 8 weeks old  were used for the exposure to Phlebotomus arabicus salivary gland proteins were disrupted by three freeze-thaw cycles in liquid nitrogen and separated by SDS-PAGE on 12% polyacrylamide gel, 0.75 mm thick using Mini-Protean III apparatus (BioRad). As a reducing agent, 2.5% 2-mercaptoethanol was used in sample buffer. Biotinylated low range standards (BioRad) were run on the same gel. Separated proteins were electro-transferred onto a nitrocellulose membrane by iBlot™ Gel Transfer Device (Invitrogen), and the membrane was cut into strips corresponding to sample load of 5 salivary glands per strip. These strips carrying salivary gland samples were either stained with amidoblack  or blocked with 5% BSA in phosphate-buffered saline, pH 7.4, with 0.05% Tween-20 (PBS-Tw) overnight. The sample-carrying strips were incubated for 1 hour with pre-immune or immune sera of BALB/c mice exposed to P. arabicus (diluted 1:250 in PBS-Tw), and then with horseradish peroxidase-conjugated swine anti-mouse antibody  for 45 minutes. Streptavidin-conjugated peroxidase  was used for the biotinylated standards. The colour reaction was developed using H2O2 and diaminobenzidine in PBS. Washes between individual steps were done with PBS-Tw.JH participated in the cDNA library construction and annotation, sequence alignment and phylogenetic analysis, immunization of mice by sand flies and the detection of humoral response to sand fly saliva, and drafting the manuscript. JM sequenced all cDNA clones selected from the library. VV dissected the salivary glands and carried out the enzymatic assays. IR participated in sample preparation for Edman degradation, which was carried out by MG. PV and JGV conceived the study, participated in its design and coordination and revised the manuscript. RCJ carried out the bioinformatic analysis of transcript sequences, participated in coordination of the study and drafting the manuscript. All authors have read and approved the final manuscript.


Gut bifidobacteria are believed to influence immune-related diseases. The objective of this study was to assess the possible relationships between the gut bifidobacteria composition and coeliac disease (CD) in children.Bifidobacterium genus and species were analyzed by real-time PCR.A total of 48 faecal samples  and 33 duodenal biopsy specimens of CD patients  were analysed. Samples  from a control age-matched group of children were also included for comparative purposes. Gut Bifidobacterium and B. longum species in faeces and duodenal biopsies than controls, and these differences were particularly remarkable between active CD patients and controls. B. catenulatum prevalence was higher in biopsies of controls than in those of active and non-active CD patients, whereas B. dentium prevalence was higher in faeces of non-active CD patients than in controls. Correlations between levels of Bifidobacterium and B. longum species in faecal and biopsy samples were detected in both CD patients and controls.Active and non-active CD patients showed lower numbers of total Bifidobacterium and B. longum populations were associated with both active and non-active CD when compared to controls. These bacterial groups could constitute novel targets for adjuvant dietary therapies although the confirmation of this hypothesis would require further investigations.Reductions in total  Coeliac disease (CD) is a chronic inflammatory disorder of the small intestine that presents in genetically predisposed individuals following gluten consumption. Gluten removal from the diet is currently the only treatment available. This disease often presents in early childhood with small intestinal villous atrophy and signs of malabsorption . RecentlBifidobacterium genus constitutes an important bacterial group in the human gut, where this is thought to be essential to maintain health via beneficial metabolic, trophic, and protective functions [Bifidobacterium is the predominant intestinal bacterial genus during the first year of life, particularly in full-term breastfed infants, although becomes quantitatively less important in adult's microbiota [Bifidobacterium species composition have been related to the development of inflammatory diseases such as allergy, irritable bowel syndrome (IBS), inflammatory bowel diseases (IBD) and colorectal cancer compared to healthy controls [Bifidobacterium species and strains that in turn were related to different disease risks. Strains of B. adolescentis have been shown to be more proinfammatory or had non-effect on immunity, while strains of the species B. bifidum and B. longum were shown to have immunoregulatory properties [Bifidobacterium strains colonizing the human gut could contribute to regulate disturbances in the balance of T-helper 1 (Th1)/Th2 lymphocyte responses to exogenous antigens related to either allergic diseases (characterized by a Th2-phenotype polarization) or Crohn and CD (characterized by a Th1 phenotype polarization). As a consequence, Bifidobacterium species have been regarded as particularly attractive targets for dietary intervention within the gut ecosystem to maintain intestinal homeostasis and host health.unctions ,7. Bifidcrobiota ,9. Qualicontrols -12. In aoperties -15. In tBifidobacterium species composition of duodenal biopsies and faecal samples from CD patients (with active and non-active disease) and age-matched controls by the use of quantitative real-time PCR technique to elucidate their possible role in this disorder.The objective of this study was to assess the Three groups of children were included in this study: (1) active CD patients on a normal gluten-containing diet; (2) non-active CD patients after following a gluten-free diet for at least 2 years; and (3) control children without known gluten intolerance. Biopsy specimens of the control group were obtained from children who were investigated for weight loss, growth retardation or functional intestinal disorders of non-coeliac origin, confirmed by showing a normal villous structure after diagnosis by biopsy examination.The following faecal samples and duodenal biopsy specimens from these group of subjects were included in the analyses: 30 faecal and 25 biopsy samples from active CD patients; 18 faecal and 8 biopsy samples from non-active CD patients and 30 faecal and 8 biopsy samples from control children.None of the children included in the study was treated with antibiotics for at least 1 month before the sampling time and they were recommended not consuming probiotic-containing products for at least 15 days prior the sampling time to limit the detection of food-related bifidobacteria without delaying too much the diagnosis procedure. The adherence to this dietary recommendation was checked at the sampling time and children that did not comply with this recommendation were not included in the study. The study protocol was approved by the local committee on ethical practice from CSIC and Hospitals taking part in the study. Children were enrolled in the study after written informed consent was obtained from their parents.Samples were collected from every subject in sterile plastic recipients, frozen at -20°C immediately and kept at -80°C until further processing. Duodenal biopsy specimens were obtained by capsule and endoscopy after a 12-h fasting period. Faeces (1 g) and duodenal biopsy samples (10–15 mg) were weighted, diluted 1:10 (w/v) in PBS (pH 7.2) and homogenized by thorough agitation in a vortex. Aliquots of these dilutions were used for DNA extraction. DNA from both type of samples (faeces and biopsies) and from pure cultures of the different bacterial strains used as reference were extracted by using the QIAamp DNA stool Mini kit  following the manufacturer's instructions.® Green PCR Master Mix , 1 μl of each of the specific primers at a concentration of 0.25 μM, and 1 μl of template DNA. The fluorescent products were detected at the last step of each cycle. A melting curve analysis was made after amplification to distinguish the targeted PCR product from the non-targeted PCR product. Bacterial concentration from each sample was calculated by comparing the Ct values obtained from standard curves. A standard curve was made from serial dilutions of DNA isolated from each pure culture of the different reference strains. A linear relationship was observed between the cell number and CT values (r2 = 0.99–0.97) when the DNA was isolated from cultures containing between 102 and 109 log cells/ml, as determined by epifluorescence microscopy counts using DAPI. The following reference strains were used to construct the corresponding standard curves: Bifidobacterium longum subsp. longum CECT 4503, B. longum subsp. infantis CECT 4553, B. bifidum LMG 11041, B. breve LMG 11042, B. pseudocatenulatum CECT 5776, B. animalis subsp. lactis DSMZ 10140, B. adolescentis LMG 11037, and B. dentium CECT 687. The strains were obtained from the Spanish Collection of Type Cultures (CECT), the German Collection of Microorganisms and Cell Cultures (DSMZ) and the Belgian Coordinated Collections of Microorganisms .Quantitative real time PCR was used to characterize the faecal microbiota by using group and species-specific primers described previously ,17. BrieU test. The possible correlation between variables was studied by applying the Spearman rank correlation coefficient and significance was established at 0.5%. The chi-square test was used to establish differences in species prevalence between the studied groups of children. A P < 0.050 was considered statistically significant. Bonferroni adjustment test was also applied to correct the significance for a multiple test comparisons among three groups (active and non-active CD patients and control groups), which has the advantage of reducing type I error and the disadvantage of increasing type II error.Statistical analyses were done using the SPSS 11.0 software . Due to non-normal distribution, microbial data are expressed as medians with interquartile ranges (IQR). Comparisons among data of more than two groups of children were done by applying the Kruskal-Wallis test and comparisons between data of two groups of children were done by applying the Mann-Whitney Clinical characteristics of the children groups included in the study are shown in Table B. longum was one of the most frequently detected species in biopsy samples followed by B. breve, B. bifidum, B. catenulatum and B. lactis . B. catenulatum group was detected more frequently in controls than in active CD (P = 0.050) and non-active CD patients (P = 0.038). In addition, B. lactis group was detected more frequently in active CD patients (P = 0.003) and in controls (P = 0.012) than in non-active CD patients. B. dentium was found in active CD and non-active CD patients but not in controls. The prevalence of B. lactis group was also significantly different in active CD patients and controls as compared to that of non-active CD patients by applying the Bonferroni adjustment, but this was not the case for the rest of bacterial groups.is Table . CurrentB. longum, followed by B. breve, B. lactis, B. bifidum and B. catenulatum, whereas B. longum subsp. infantis and B. dentium were less prevalent.The composition of biopsy-associated bifidobacteria assessed by qPCR is shown in Table Bifidobacterium (P = 0.040), B. longum (P = 0.017), B. breve (P = 0.018) and B. bifidum (P = 0.022). No differences were found for the other analysed species. Comparisons of bifidobacterial levels between groups by using the Mann-Whitney U test allowed the detection of significantly higher levels of total Bifidobacterium in controls than in active CD patients (P = 0.009), although no significant differences were found between non-active CD patients and controls (P = 0.461). B. longum levels were also significantly higher in controls than in active CD patients (P = 0.004) and slightly higher than in non-active CD patients although not significantly (P = 0.368). Total Bifidobacterium and B. longum group levels were also significantly different between active CD and control children by applying the Bonferroni adjustment. B. breve levels were significantly lower in non-active CD patients than in active CD patients (P = 0.020) and also slightly lower (P = 0.100) than in control children as it was the case for faeces. A similar trend was found for B. bifidum but none of the differences reached statistical significance. B. lactis levels were higher in active CD patients than in controls (P = 0.033), while this species was not detected in non-active CD patients.Significant differences were detected by using the Kruskal-Wallis test among active and non-active CD patient and control groups for total Bifidobacterium group and species detected by PCR  compared to the total number of samples tested in the study are shown in Table B. longum, B. bifidum and B. catenulatum groups were detected in all faecal samples, whereas the other Bifidobacterium species analysed were not detected in every sample . B. adolescentis was detected more frequently in non-active CD patients than in active CD patients (P = 0.016) and controls (P = 0.045). B. dentium was significantly more prevalent in non-active CD patients than in controls (P = 0.040), and the same trend was detected between active CD patients and controls but the differences were not statistically significant. B. adolescentis prevalence was also significantly different between non-active CD patients and active CD patients (P = 0.016) by applying the Bonferroni adjustment test. No significant differences were found for the other Bifidobacterium groups or species.The number of positive samples for le Table . B. brevBifidobacterium groups present in faecal samples were B. longum, B. catenulatum group and B. bifidum. Significant differences were obtained by using the Kruskal-Wallis test among active and non-active CD patient and control groups for total Bifidobacterium (P = 0.002), B. longum (P < 0.001), B. breve (P < 0.001), B. bifidum (P = 0.030) and B. adolescentis (P = 0.020). No differences were found for the other studied species. The comparison of bifidobacterial levels between groups by using the Mann-Whitney U test allowed the detection of significant differences in several cases. Total Bifidobacterium levels were significantly higher in control samples than in those of active CD (P = 0.014) and non-active CD patients (P = 0.002). No differences were found between active and non-active CD patients (P = 0.183). B. longum levels were significantly higher in controls than in active CD (P = 0.038) and non-active CD patients (P < 0.001); moreover, B. longum levels were significantly higher in non-active CD patients than in active CD patients (P = 0.030). Most of these differences were also statistically significant by applying the Bonferroni adjustment at P < 0.017 (Table B. breve levels were significantly higher in active CD (P = 0.001) and control children (P < 0.001) than in non-active CD patients, which showed the lowest counts of this species, but differences between active CD patients and controls (P = 0.860) were not found by either Mann Whitney or Bonferroni adjustment test. Similarly, B. bifidum levels were higher in active CD patients (P = 0.030) and controls (P = 0.050) than in non-active CD patients, whereas significant differences were not found between active CD and control children (P = 0.577). B. adolescentis levels were significantly higher (P = 0.050) in active CD patients than in controls while differences were not found neither between active and non-active CD patients nor between non-active CD patients and controls. However, differences in B. bifidum and B. adolescentis were not statistically significant when applying the Bonferroni adjustment test. No differences were found for any other Bifidobacterium species analysed between the three groups of children under study  of bifidobacteria than duodenal biopsies samples for every analyzed group, as anticipated , non-active CD patients  and controls . Similarly, correlations between B. longum levels in faecal and biopsy samples were significant in active CD patients , non-active CD patients  and controls . In active CD patients, correlations were also found for B. breve , B. bifidum , B. adolescentis , B. catenulatum  and B. lactis , whereas no correlations were found for B. angulatum, B. longum subsp.infantis and B. dentium. In controls, significant correlations between bifidobacterial levels in faeces and biopsy samples were also found for B. catenulatum group  and B. longum subsp.infantis  and in non-active CD patients for B. bifidum  and B. catenulatum group .Faecal samples showed higher numbers  particularly in faeces; however, the gluten-free diet could also influence the levels of this species since differences were found between active and non-active CD patients. Duodenal B. longum levels seemed to be partially restored after the gluten-free diet, following the same trend as that detected for total Bifidobacterium levels.The most predominant bifidobacterial groups detected in both, biopsies and faeces, were crobiota ,12. The Bifidobacterium have been shown to polarized Th2/Th1 responses in a specific manner, thereby modulating unbalanced cytokine production characteristic of either Th2-type  or Th1-type diseases (e.g. Crohn disease and CD) and overall inflammation [B. adolescentis and B. catenulatum group could favour Th2-biased immune responses characteristic of allergy inflammation [B. longum mainly related to their ability to stimulate regulatory cytokine production (e.g. IL-10) [B. longum detected in control samples (biopsies and faeces) compared to those found in active and non-active CD patient samples suggest that this bifidobacterial group could exert a protective role in CD. Otherwise, changes in the intestinal environment of CD patients, such as the mucus layer composition, could secondarily lead to changes in gut bacterial populations. In this context, lower levels of B. longum have also been reported in IBD and colorectal cancer patients [Bifidobacteria have been demonstrated to have a species and strain-specific influence on immunity ,15,24. Sammation ,25. It hammation . In cont. IL-10) . In thispatients .B. breve and B. bifidum numbers were particularly reduced in non-active CD patients when compared with active CD-patients and controls in both biopsy specimens, and especially in faecal samples, indicating that these reductions could be due to the gluten-free diet rather than to the disease.B. adolescentis were detected in slightly higher numbers in faecal samples of active CD patients than in controls and its prevalence was also higher in faeces of non-active CD patients than in those of controls. However, this loosely association between B. adolescentis and CD was neither confirmed in biopsies nor in a preliminary study carried out previously in a few faecal samples of active CD patients and controls by PCR-DGGE [PCR-DGGE .Bifidobacterium and the species B. longum in faecal and biopsy samples, which were the bacterial groups most clearly related to CD. Therefore, faecal alterations of Bifidobacterium and B. longum levels reflect those occurring in the duodenum. These could be used as indexes of CD in faeces without the use of invasive biopsy techniques, although further studies should be carried out in other population groups to confirm such hypothesis.In general, this study confirms that the dominant bifidobacterial species detected in faeces represented those found in duodenal biopsies although in different quantities Fig. , supportBifidobacterium populations. The microbiota of CD patients is characterised by reductions in total Bifidobacterium and B. longum numbers. These microbial deviations are not completely restored after treatment with a gluten-free diet. Thus, the results suggest that total and specific Bifidobacterium species could be possible protective factors for CD. Therefore, the administration of specific probiotics and prebiotics to increase their intestinal levels could constitute a possible adjuvant therapeutic strategy for this disorder. Confirmation of such hypothesis would require further investigations.Active and non-active CD is associated with changes in number, composition and prevalence of The author's responsibilities were as follows: YS. made the microbiological study concept and design, MCC acquired microbiology data and made the statistical analyses. MC, CR-K and ED acquired clinical data. All authors participated in preparation of the manuscript and approved the final version. None of the authors has conflict of interests.