Francisella tularensis is an intracellular pathogen that causes tularemia in humans and the public health importance of this bacterium has been well documented in recent history. Francisella philomiragia, a distant relative of F. tularensis, is thought to constitute an environmental lineage along with Francisella novicida. Nevertheless, both F. philomiragia and F. novicida have been associated with human disease, primarily in immune-compromised individuals. To understand the genetic relationships and evolutionary contexts among different lineages within the genus Francisella, the genome of Francisella spp. strain TX07-7308 was sequenced and compared to the genomes of F. philomiragia strains ATCC 25017 and 25015, F. novicida strain U112, and F. tularensis strain Schu S4.Francisella , several strain-specific genes were identified. Strains TX07-7308 and ATCC 25017 contained an operon with six open reading frames encoding proteins related to enzymes involved in thiamine biosynthesis that was absent in F. novicida strain U112 and F. tularensis strain Schu S4. Strain ATCC 25017 contained an operon putatively involved in lactose metabolism that was absent in strain TX07-7308, F. novicida strain U112, and F. tularensis strain Schu S4. In contrast, strain TX07-7308 contained an operon putatively involved in glucuronate metabolism that was absent in the genomes of strain ATCC 25017, F. novicida strain U112, and F. tularensis strain Schu S4. The polymorphic nature of polysaccharide biosynthesis/modification gene clusters among different Francisella strains was also evident from genome analyses.The size of strain ATCC 25017 chromosome was 2,045,775 bp and contained 1,983 protein-coding genes. The size of strain TX07-7308 chromosome was 2,035,931 bp and contained 1,980 protein-coding genes. Pairwise BLAST comparisons indicated that strains TX07-7308 and ATCC 25017 contained 1,700 protein coding genes in common. NUCmer analyses revealed that the chromosomes of strains TX07-7308 and ATCC 25017 were mostly collinear except for a few gaps, translocations, and/or inversions. Using the genome sequence data and comparative analyses with other members of the genus environmental lineages within the genus Francisella. The inability to acquire new genes coupled with the loss of ancestral traits and the consequent reductive evolution may be a cause for, as well as an effect of, niche selection of F. tularensis. Sequencing and comparison of the genomes of more isolates are required to obtain further insights into the ecology and evolution of different species within the genus Francisella.From genome comparisons, it appeared that genes encoding novel functions have contributed to the metabolic enrichment of the  Pasteurella in many features was isolated in 1959 from a moribund muskrat found on the shoreline of a marsh on the Bear River Migratory Bird Refuge in northern Utah[Yersinia philomiragia (ATCC strain 25015T). Additional isolations  of bacteria similar to the muskrat pathogen were made in 1960 from surface water samples collected in marshy areas within few miles of the Bear River Research Station, Utah[T, 25016, 25017, and 25018 were biochemically and genetically different from members of the genus Yersinia[Yersinia philomiragia were reported from clinical cases[Francisella philomiragia  in 1989[A small gram-negative bacterium that resembled members of the genus al cases. These h in 1989.F. philomiragia are from human cases associated with saltwater exposure and the first environmental isolates were from a marshy area that forms part of the Great Salt Lake waterway[F. philomiragia is halotolerant and grows in media containing 6% NaCl, whereas Francisella tularensis does not[F. philomiragia is less fastidious than F. tularensis and does not require cysteine for growth[F. philomiragia is considered an opportunistic pathogen and is most commonly isolated from patients with chronic granulomatous disease or those who experienced near-drowning in saltwater[F. tularensis is a highly infectious zoonotic agent and tularemia in healthy individuals exposed to contaminated freshwater sources has been reported[F. tularensis is generally not associated with saltwater exposure and is not known to cause near-drowning-associated pneumonia[F. philomiragia in the clinical laboratory have been sporadic and only four human cases have been reported in the last two decades[F. philomiragia has also been reported in a North Carolina dog that lived in a coastal town[Most of the clinical isolates of waterway,4. Not sdoes not. Furtheraltwater,4. In coneumonia. Isolati decades-11. A catal town.Francisella spp. from environmental samples using cysteine heart agar with 9% chocolatized sheep blood have yielded three strains  from seawater collected in the Galveston Bay area, Texas[sdhA genes from these strains with those of other Francisella spp.  have indicated that strain TX07-7308 is more similar to F. philomiragia than to F. tularensis[Francisellaceae has clustered strain TX07-7308 with an uncultured bacterium clone SSW64Au obtained from the Salton Sea in California[F. philomiragia has been detected in samples obtained from a brackish-water pond in Martha’s Vineyard, Massachusetts[F. philomiragia and Francisella noatunensis, an emerging fish pathogen associated with marine environments, has also been established[Recent attempts at direct isolation of a, Texas. Compariularensis. A phylolifornia. In addichusetts. Furtherablished.Francisella novicida is thought to constitute an environmental lineage along with F. philomiragia, and the genome of F. novicida strain U112 has been sequenced and compared to the genome sequences of F. tularensis strains pathogenic to humans[F. novicida-like strains Fx1 and 3523, which are clinical isolates from the USA and Australia, respectively, have also been sequenced and compared to the genome sequence of strain U112[F. philomiragia, the complete genome of strain ATCC 25017  and a draft genome of strain ATCC 25015T  have been sequenced. Comparative genomic analyses of these strains with F. novicida and F. tularensis isolates have revealed major differences in metabolic competency and putative pathogenic characteristics[F. noatunensis isolate GM2212 and its comparison to F. philomiragia have been reported[Francisella strain TX07-7308 and to identify the genetic differences between this strain and F. philomiragia strain ATCC 25017 using a comparative genomics approach. Since genetic relationships and evolutionary contexts could be better understood by whole-genome analyses of conserved operons, it was envisaged to include the genomes of different Francisella species and strains in the comparisons, when feasible.o humans. The genain U112. In vieweristics,20. In aBacterial cultivation and chromosomal DNA extraction were performed at the Centers for Disease Control and Prevention, Fort Collins, using standard procedures,22. Genoe.g., remnants of IS elements, integration sites), and the similarity of proteins encoded by genes at a locus of interest (>90% identity at the predicted protein level for orthologs).Multiple genome comparisons were performed using the ‘progressive alignment’ option available in the program MAUVE version 2.3.0. Default scoring and parameters were used for generating the alignment. A synteny plot was generated using the program NUCmer. The program uses exact matching, clustering, and alignment extension strategies to create a dot plot based on the number of identical alignments between two genomes. Prophage regions (PRs) were identified using Prophinder (http://aclame.ulb.ac.be/Tools/Prophinder/), an algorithm that combines similarity searches, statistical detection of phage-gene enriched regions, and genomic context for prophage prediction. Insertion sequences (ISs) were identified by whole genome BLAST analysis of strains TX07-7308 and ATCC 25017 using the IS finder (http://www-is.biotoul.fr/). Gene acquisition and loss among the different strains were determined by comparing gene order, orientation of genes (forward/reverse), GC content of genes (% above or below whole genome average), features of intergenic regions  was used for comparing the genomes and identification of shared and unique protein encoding genes. The phylogenetic profiling tool within IMG was used to find single gene homologs. Three 3-way comparisons were performed using different genome orders, and the lowest number was used to represent the core genes in the venn diagram. Pairwise comparisons were performed bidirectionally for each pair of genomes and the lowest number was used in the diagram. Strain-specific genes were obtained by subtracting the larger number of common genes from the total number of genes in the genome. The specific parameters used for the analysis were: E-value: 1e-05; minimum percent identity: 30%; algorithm: by present/absent homologs; minimum taxon percent with homologs: 100%.sdhA) gene sequences from high quality, finished Francisella genomes were included in these comparisons.Multiple sequence alignments for phylogenetic analyses of strains TX07-7308 and ATCC 25017 were performed using the program MUSCLE available at the website http://www.phylogeny.fr/,28. The F. novicida strain U112 . Strain TX07-7308 contained yet another genomic island  that was also absent in strain ATCC 25017, but was partially present in F. novicida strain U112 . Strain ATCC 25017 contained a genomic island  that was absent in strain TX07-7308 and F. novicida strain U112. Furthermore, strain TX07-7308 lacked plasmids, but strain ATCC 25017 contained a single plasmid[The chromosome of strain ATCC 25017 was 9,844 bp larger than that of strain TX07-7308. Although the chromosomes of strains ATCC 25017, ATCC 25015, and TX07-7308 had minor differences in size, their average GC content and the percentage of sequence that encodes proteins were similar . Strain ATCC 25017 chromosome encoded 196 protein-coding genes with no homologs in strains TX07-7308 and U112. Strain TX07-7308 chromosome encoded 249 protein-coding genes with no homologs in strains U112 and ATCC 25017. Strain U112 encoded 211 protein-coding genes with no homologs in strains TX07-7308 and ATCC 25017 . Notably, the chromosomes of strains ATCC 25017 and TX07-7308 contained an unlinked 119 bp 5S rRNA sequence  that was also present in other members of the genus Francisella . Furthermore, the positions of the three rRNA operons were not conserved in the chromosomes of strains ATCC 25017 and TX07-7308, but they were relatively conserved among the chromosomes of F. novicida and F. tularensis strains.Bacterial rRNA operons generally contain the 16S, 23S, and 5S rRNA genes linked together by one or more tRNA genes located in the internal transcribed spacer. ChromosFrancisella contain 37–39 tRNA genes (~1.8% of the genome), representing all 20 amino acids. Although some redundancy of tRNA genes was apparent among all Francisella genomes , they each contained a single copy of tRNAAsn, tRNAAsp, tRNACys, tRNAGln, tRNAGlu, tRNAHis, tRNAPhe, tRNAPro, tRNATrp, and tRNATyr genes. In all of the publicly available Francisella genomes, genes encoding tRNAIle and tRNAAla were never present outside of the rRNA operons. This is generally true of most other bacteria and the occurrence of genes encoding tRNAIle and tRNAAla genes within the rRNA operons has also been noted in Escherichia coli K12 and Rhodopseudomonas palustris[Francisella genomes contained genes encoding putative aminoacyl tRNA synthetases specific for each of the 20 amino acids. They also contained a single gene encoding a putative peptidyl-tRNA hydrolase .A positive correlation between the abundance of tRNA and rRNA genes among bacterial genomes has been observed. Typicalpalustris,34. FurtF. tularensis genomes and these IS elements are thought to be generally stable among different isolates despite their diverse geographical origins[F. tularensis strains Schu S4 and OSU18, which contained at least six categories of IS elements (ISFtu1-ISFtu6). Whole genome BLASTP analyses showed that strains ATCC 25017 and TX07-7308 each contained a single copy of the ISFtu2 sequence , but they lacked ISFtu1 and ISFtu3-ISFtu6 sequences , are the hallmark of  origins,35. Pairquences . FurtherFrancisella piscicida strain GM2212 , it was absent in all other publicly available Francisella genomes, including strain ATCC 25015.In strain TX07-7308, the locus adjacent to F7308_1638 encoded an integral membrane protein , a phosphoserine phosphatase , a lignostilbene-alpha, beta-dioxygenase , and a beta carotene dioxygenase . Interestingly, the homologs of these ORFs in strain ATCC 25017 (Fphi_0135-0138) were associated with Fphi_0139, which encoded a putative integrase (314 aa). Strain ATCC 25017 contained 41 full-length and 5 truncated orthologs of Fphi_0139 . WhereasFrancisella genomes, except strain ATCC 25015. In strain ATCC 25017, this transposable element was associated with a gene encoding a putative chitinase  whose ortholog was present in strain ATCC 25015, but not in strain TX07-7308 and all other publicly available Francisella genomes. Strain TX07-7308 mutator family transposase was associated with genes encoding a putative chitinase  and a two-component response regulator  whose orthologs were present in strain ATCC 25015, but not in strain ATCC 25017 and all other publicly available Francisella genomes. Furthermore, strain ATCC 25017 contained a locus , an integrase , a Tn3 family transposase , a transposase-like protein , and an invertase/recombinase/resolvase-like protein . These genes were absent in all other publicly available complete Francisella genomes, including strain TX07-7308. Several genes encoding putative phage integrases were also identified in the genomes of strains ATCC 25017 and TX07-7308  and F. novicida strain U112 using transmission electron microscopy[F. tularensis and comparisons of available Francisella genomes have revealed the heterogeneity of genes putatively involved in the assembly of type IV pili[F. novicida and F. tularensis strains (data not shown), the biochemical and functional similarity of type IV pili among these bacteria cannot be ruled out. Nevertheless, the role of type IV pili in the survival of F. philomiragia in the environment needs to be carefully studied, especially in light of recent reports of the ability of this bacterium to form biofilms and interact with the aquatic protist Acanthamoeba castellanii[Several species of pathogenic gram-negative bacteria contain type IV pili, also known as fimbriae, which facilitate bacterial adhesion to host cells, biofilm formation, and twitching motility. Structucroscopy,38. Rece IV pili,40. Strastellanii. oppABCDF operon[oppD and oppF occur as separate ORFs in Type A strains of F. tularensis, but a 960 bp internal deletion has caused the disruption of oppD and oppF coding sequences in Type B strains of F. tularensis[F. philomiragia strain ATCC 25017 and F. novicida strain U112 contained an opp locus . Within this locus, FTN_1590 encoded a putative full-length OppD protein (322 aa), but Fphi_1027 was annotated as a pseudogene because of a frame-shift mutation , which are ATP-binding cassette transporters of oligopeptides. The impF operon,43. It hularensis. Genome Francisella pathogenicity island (FPI) and is found in a single copy in F. novicida strain U112 (FTN_1309-1325), but is duplicated in the genomes of F. tularensis[F. novicida strain U112, was truncated in strain TX07-7308 (F7308_1011) and homologs of FTN_1318-FTN_1320  were absent in strains ATCC 25017 and TX07-7308.A cluster of 17–19 genes has been proposed to constitute the ularensis. Genome F. tularensis strain Schu S4 and F. novicida strain U112 has been described previously[fur, Fphi_0928/F7308_0528) and a cluster of genes putatively involved in the biosynthesis of a polycarboxylate siderophore (Fphi_0922-0926 and F7308_0522-0526). An ortholog of strain Schu S4 fupA (FTT_0918), whose product is required for efficient utilization of siderophore-bound iron[fur, and fupA in almost all Francisella genomes suggests that these functions are essential for their survival in the environment and/or host.The biosynthesis of a polycarboxylate siderophore in eviously,46. Straund iron, was alsF. novicida-like strains Fx1 and 3523[Some bacteria have evolved mechanisms for the metabolism of uronic acids and uronates using the Entner-Doudoroff pathway. In thisand 3523.E. coli UxuA (ECs5281), KdgK (ECs4406), KdgA (ECs2560), and UxaC (ECs3974), respectively . These enzymes catalyze the dehydration of D-mannonate to KDG, phosphorylation of KDG, cleavage of KDPG to pyruvate and G3P, and the conversion of D-glucuronate to D-fructuronate, respectively[Bacillus stearothermophilus T-6, which has been predicted to metabolize GlcUA akin to E. coli and Bacillus sutbtilis[The predicted mannonate dehydratase, 2-keto-3-deoxygluconokinase, KDPG aldolase, and glucuronate isomerase proteins from strain TX07-7308 had 57%, 37%, 36%, and 28% identities to ectively. The Glcsutbtilis.Francisella. However, strain TX07-7308 inositol oxygenase had 37% identity to Mus musculus myo-inositol oxygenase , which catalyzes the conversion of myo-inositol to GlcUA[Myo-inositol and its derivatives are ubiquitous among eukaryotes and archaea, but their synthesis and metabolism is believed to be less common among bacteria[F. tularensis genomes sequenced to date had ORFs encoding proteins putatively involved in the transport and/or metabolism of myo-inositol, most of them had a suhB homolog . A suhB homolog was also found in the genomes of strains TX07-7308 and ATCC 25017 (F7308_0980 and Fphi_1342). This evolutionarily conserved gene encoded inositol-1-monophosphatase, which hydrolyzes myo-inositol-1-phosphate to yield free myo-inositol[Francisella can convert myo-inositol-1-phosphate to free myo-inositol. However, only some strains may be able to utilize myo-inositol to synthesize GlcUA, which is then metabolized using the Entner-Doudoroff pathway.The ORFs encoding a putative inositol oxygenase in strains TX07-7308 and ATCC 25015 had no bacterial homologs in the public databases outside of the genus bacteria. Althouginositol,53. Thuslac operon, which contains the structural genes encoding proteins that facilitate lactose metabolism, is found in a variety of bacteria. Lactose is imported into the cell as a free sugar by means of a permease and the enzyme β-galactosidase hydrolyzes this disaccharide into galactose and glucose[lac operons were also identified in F. novicida-like strains Fx1 and 3523[Carnobacterium maltaromaticum[Bacillus subtilis[E. coli. Strain ATCC 25017 LacY had 25% identity to the putative oligogalacturonide transporter  of E. coli CFT073 and 22% identity to the putative sugar transporter  of Bacillus cereus. However, genes encoding the galactoside O-acetyltransferase (lacA) and the regulatory protein (lacI) were not found in strains ATCC 25015 and ATCC 25017. In strain ATCC 25017, an ORF (Fphi_0306) encoding a putative transposase/integrase-like protein was found near the lac operon. Furthermore, several F. tularensis genomes  contained a truncated/vestigial lacZ, but lacked a lacY (data not shown).The  glucose. Genome and 3523. Strain romaticum and 26%  subtilis, but waslac operon of E. coli consists of three ORFs (lacZYA), which are regulated by the product of the adjacent lacI repressor gene, bacteria containing only lacZY or lacZ have been identified[E. coli lac operon is uncertain[lac genes near integrative and conjugative elements and the identification of E. coli-like lac operons in some gram-positive bacteria suggest their lateral mobility[F. philomiragia strain ATCC 25017 and F. tularensis strains may have acquired the lacZY operon by transposon-mediated horizontal transfer. Nevertheless, detailed phylogenetic analyses are required to establish the evolutionary origin of the Francisella lac operon. The loss of the lacZY operon in most F. tularensis strains may be due to niche selection or through genetic drift, and a similar mechanism for some members of Enterobacteriaceae has been proposed[Francisella that have retained an ancestral copy of the lac operon. The lacZ gene identified in this report could be useful in studies involving pathogenic F. tularensis strains that require a native reporter protein.Although the classic entified-59. Whilmobility-62. From1) is involved in several microbial metabolic functions[de novo or acquire it from their niche[Thiamine pyrophosphate  encoding proteins related to enzymes involved in thiamine biosynthesis in several prokaryotes  is found within plasmid pEA29 of the plant pathogen Erwinia amylovora strain Ea88[Ralstonia eutropha H16 also contains a thiCOSGE locus that is proposed to be involved in de novo synthesis of thiamine[R. etli (AAC45972) and Ra. eutropha (H16_A0235), whereas strain ATCC 25017 ThiF was 34% identical to ThiF of E. amylovora . Furthermore, ATCC 25017 ThiO and ThiS were 30% identical to ThiO (H16_A0236) and ThiS (H16_A0237) of Ra. eutropha, respectively . Putative thiazole synthase ThiG of strain ATCC 25017 was ~51% identical to ThiG of R. etli  and Ra. eutropha . Strains ATCC 25017 and TX07-7308 contained an operon with six ORFs . Homologs of Fphi_0087 or F7308_1159 were found in several bacteria  and plants . It has been proposed that these bifunctional enzymes are involved in the synthesis of HMP-PP as well as the condensation of HMP-PP and HET-P to produce thiamine monophosphate[′ untranslated regions of operons involved in thiamine biosynthesis and transport have been shown to contain a regulatory element called THI-box sequence[thiC in strains TX07-7308 and ATCC 25017. This suggests a thiamine-dependent regulation of thiC in strains ATCC 25017 and TX07-7308, as in other bacteria that have THI-box sequences upstream of TBS genes[In strains ATCC 25017 and TX07-7308, Fphi_0087 and F7308_1159, respectively, appeared to encode a putative fused protein containing hydroxy-phosphomethylpyrimidine kinase and thiamine-phosphate pyrophosphorylase domains. In some bacteria, these functions are encoded by two different ORFs . Strain ATCC 25017 TPK was 27% identical to Bacillus subtilis TPK , which catalyzes the direct conversion of thiamine to thiamine pyrophosphate[Francisella are capable of salvage of dephosphorylated thiamine intermediates, but some strains  can synthesize thiamine de novo, when thiamine intermediates are not available in their environments.In bacteria that lack a TBS pathway, thiamine kinases may facilitate the salvage of dephosphorylated thiamine intermediates from the environment or growth medium. A gene hosphate. Thus it2 (riboflavin) is the precursor of coenzymes flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), which are cofactors for several biochemical reactions[de novo using one molecule of GTP and two molecules of ribulose 5-phosphate as substrates[Vitamin Beactions. Most babstrates. Strainsbstrates,76. The bstrates. Strainsde novo using pimelic acid as a precursor, some others have evolved mechanisms for importing this essential cofactor from their natural environments[bioABFCD operon of E. coli, the bioA and bioBFCD genes are divergently transcribed and encode the enzymes catalyzing the biosynthesis of biotin[bioABFCD operon of E. coli . The occurrence of these genes/loci in almost all Francisella genomes suggests that these functions are essential for survival in their natural environments. However, the genomes of F. tularensis strains conspicuously lacked genes for the biosynthesis of thiamine. It is possible that these strains lost the ancestral TBS genes as a consequence of host adaptation and reductive evolution. The TBS genes identified in this study could be useful in rescuing the thiamine auxotrphy of F. tularensis strains and other bacteria in the laboratory. Furthermore, it was evident from comparative genome analyses that strains ATCC 25017 and TX07-7308 as well as most strains of F. tularensis lacked the vitamin B12 biosynthetic pathway. Thus it appears that most extant members of the genus Francisella are vitamin B12 auxotrphs and may require its supplementation for in vitro growth.Most of the publicly available Francisella spp. has several unique features and has been demonstrated to undergo antigenic variation[F. tularensis strains are well characterized and their LPS is implicated in pathogenesis[F. philomiragia has been proposed to contain lipooligosaccharide and the lipid A of strain ATCC 25015 has been shown to be rich in shorter fatty acid chains in comparison to F. tularensis[wbt gene cluster of F. tularensis strain Schu S4  is involved in LPS biosynthesis and contained 15 ORFs[F. novicida strain U112  contained only 12 ORFs[F. novicida and F. tularensis have been shown to be structurally and immunologically distinct, due in part to the differences in wbt genes involved in their biosynthesis[The lipopolysaccharide (LPS) of ariation. The bioogenesis,88. F. pularensis-91. The  15 ORFs. In cont 12 ORFs. The LPSynthesis,93.wbt gene clusters of F. tularensis strain Schu S4 and F. novicida strain U112.wbt gene clusters of these bacteria. Although strains Schu S4 and U112 contained manB and manC ORFs encoding proteins putatively involved in mannose modification adjacent to the wbt gene cluster, strains ATCC 25017 and TX07-7308 contained only a manB ORF. Among the other genes found in the wbt gene cluster of F. tularensis strain Schu S4, only FTT_1450c, FTT_1451c, and FTT_1462c-1464c had homologs among strains ATCC 25017 and TX07-7308. The wbt gene cluster of strain ATCC 25017 contained 7 genes  that had no homologs in strains TX07-7308, Schu S4, and U112. Similalry, the wbt gene cluster of strain TX07-7308 contained 20 genes that had no homologs in strains ATCC 25017, Schu S4, and U112 . In contrast, the wbt gene cluster of strain Schu S4 contained four genes that had no homologs in strains ATCC 25017, TX07-7308, and U112 (FTT_1452c-1454c and FTT_1458c), whereas the wbt gene cluster of strain U112 contained six genes that had no homologs in strains ATCC 25017, TX07-7308, and Schu S4 .Comparative genomic analyses revealed that strains ATCC 25017 and TX07-7308 contained a cluster of 28  and 42  ORFs, respectively, that were related to the wbt gene clusters of strains ATCC 25017 and TX07-7308 contained groups of two , three (Fphi_1262-1264 and F7308_0869-0871), and five (Fphi_1265-1269 and F7308_0876-0880) contiguous orthologous ORFs that had no homologs in F. tularensis strain Schu S4 and F. novicida strain U112. They also contained three non-contiguous ORFs  within their wbt gene clusters that had no homologs in strains Schu S4 and U112. The wbt gene clusters of strains Schu S4 and U112 contained three contiguous orthologous ORFs (FTT_1459c-1461c and FTN_1425-1427) that had no homologs in strains ATCC 25017 and TX07-7308. Furthermore, the wbt gene cluster of strain U112 contained a copy of ISFtu3 transposase (233 aa) that appeared to have truncated the ORF encoding a putative dTDP-D-glucose 4,6-dehydratase  and the wbt gene cluster of strain Schu S4 contained ISFtu1 transposases (126 aa each). However, the wbt gene clusters of strains ATCC 25017 and TX07-7308 were devoid of transposase genes within or adjacent to them , was also identified in F. novicida-like strains Fx1 and 3523[psl cluster, strain TX07-7308 contained only two (F7308_1111 and F7308_1118) strain-specific ORFs within this cluster. Furthermore, strain Schu S4 contained 3 strain-specific ORFs (FTT_0794-0796) within the psl cluster and strain U112 contained only one (FTN_1216) strain-specific ORF within this cluster. These strain-specific ORFs were flanked by a single orthologous ORF encoding a putative HAD family hydrolase (FTT_0800/Fphi_1481/F7308_1119/FTN_1211) and a sugar transferase (FTT_0790/Fphi_1468/F7308_1110/FTN_1220). Based on gene content and organization, it may be surmised that the psl gene cluster was involved in LPS and/or exopolysaccharide (EPS) biosynthesis. Since the psl gene cluster of strain ATCC 25017 contained more genes compared to strains TX07-7308, Schu S4, and U112, it is possible that the LPS/EPS of this strain is more complex.Biosynthesis of polysaccharides requires several glycosyltransferases (GTs), which catalyze the transfer of sugars from an activated donor to an acceptor molecule and are usually specific for the glycosidic linkages created. The genand 3523. WhereasarsRBC) wherein the conversion of arsenate to arsenite is accomplished by a reductase (product of arsC), arsenite is transported out of the cell by a membrane-bound efflux pump (product of arsB), and arsR encodes an arsenic-resistance regulatory protein. Plasmid or transposon-mediated horizontal transfer of genes that confer arsenic resistance has been well documented[Arsenic is an environmental pollutant and some microorganisms have evolved mechanisms of resistance to this cytotoxic agent. Arsenic exists in two oxidation states, arsenite (AsIII) and arsenate (AsV), in biological systems. In mostcumented.arsB, 27 aa), an arsenate reductase , a sulfate permease , a hypothetical protein , and a mechanosensitive ion channel family protein . This locus was adjacent to the one that encoded several integrative/transposable elements . Strain ATCC 25017 contained yet another locus encoding putative arsenical resistance proteins  and Fphi_1817  had 97% identities to F7308_1298 (342 aa) and F7308_1299 (110 aa), respectively. Strain TX07-7308 contained yet another locus encoding putative arsenical resistance proteins  and F7308_0309 , respectively.Strain ATCC 25017 contained a locus encoding a truncated arsenite/antimonite exporter . Although a gene encoding a putative IS4 family transposase (247 aa) was found adjacent to the arsRB locus of strain U112, no such gene was found near the orthologous arsRB loci of strains ATCC 25017 and TX07-7308. Furthermore, at the predicted protein level, strain ATCC 25017 ArsB (Fphi_1818) was 61% identical to the arsenite efflux transporter of Bacillus subtilis  and ArsR (Fphi_1817) was 37% identical to ArsR repressor of B. subtilis . Although BSU25790 and BSU25810 have been shown to be involved in arsenate and arsenite resistance in B. subtilis[Francisella strains. In addition, adjacent to Fphi_1817-1818, strain ATCC 25017 contained a gene encoding a putative protein  that was 42% identical to the small multidrug resistance antiporter (EmrE) of E. coli. Interestingly, none of the F. tularensis genomes sequenced to date contained homologs of arsRB loci or emrE. The occurrence of an ortholog of emrE in strains U112  and TX07-7308  suggests that arsRB and emrE loci are ancestral and evolutionarily conserved among some environmental lineages of the genus Francisella.An arsenical resistance locus was also found in  subtilis, biochemFrancisella, the following genetic traits are obvious. Whereas strain TX07-7308 lacked the lac operon, strain ATCC 25017 lacked genes for GlcUA utilization. However, strains ATCC 25017 and TX07-7308 contained genes for thiamine biosynthesis. Although Type A and Type B strains of F. tularensis had lost loci for GlcUA utilization and thiamine biosynthesis, they do contain a vestigial lacZ gene.Based on whole genome comparisons of different strains of F. novicida-like strain Fx1 contained the lac operon in addition to genes for GlcUA utilization, F. novicida-like strain 3523 contained the lac operon along with GlcUA utilization and thiamine biosynthesis genes[F. novicida strain U112 lacked genes/loci for GlcUA utilization, thiamine biosynthesis, and lactose utilization. However, strain U112 contained an arsRB locus, which was absent in strains 3523 and Fx1[F. novicida, had retained an arsRB locus, but had lost loci for lactose and GlcUA utilization as well as genes for thiamine biosynthesis because of niche selection. Similarly, extant strains of F. tularensis appeared to have lost the arsRB locus, loci for lactose and GlcUA utilization, in addition to genes for thiamine biosynthesis as a consequence of host adaptation and reductive evolution. Based on comparative genome analyses, it may be concluded that the genes/loci encoding putative phenotypes such as arsenite resistance, lactose utilization, biosynthesis of various vitamins/cofactors, type IV pili, oligopeptide ABC transporter system, and iron metabolism are more ancient within the genus Francisella. FigureFrancisella strain TX07-7308, F. philomiragia strains ATCC 25015 and ATCC 25017, F. novicida-like strains 3523 and Fx1, F. novicida strain U112, and F. tularensis strain Schu S4. While is genes. Notably and Fx1, but wasF. philomiragia is more ancient than F. tularensis[F. philomiragia from F. tularensis[Francisella genomes have indicated that the average nucleotide identity between F. tularensis and F. philomiragia ranges between 80.6% and 81.2%[F. novicida is ancient to F. tularensis and that both acquisition and loss of genes have occurred during the evolution of different species within the genus Francisella[F. novicida represents an intermediate environmental and/or pathogenic lineage between F. philomiragia and F. tularensis. Phylogenetic analyses based on full-length 16S rRNA and sdhA genes support these observations Click here for file Table S2. Comparison of the wbt gene clusters of four different strains within the genus Francisella. This table contains data related to the comparison of the wbt gene clusters of strains TX07-7308, ATCC 25017, U112, and Schu S4.Click here for file Table S3. Comparison of the psl gene clusters of four different strains within the genus Francisella. This table contains data related to the comparison of the psl gene clusters of strains TX07-7308, ATCC 25017, U112, and Schu S4. (PDF 99 kb)Click here for file


Mycoplasma suis-induced infectious anemia is associated with hemorrhagic diathesis, and coagulation dysfunction. However, intravasal coagulation and subsequent consumption coagulopathy can only partly explain the sequence of events leading to hemorrhagic diathesis manifesting as cyanosis, petechial bleeding, and ecchymosis, and to disseminated coagulation. The involvement of endothelial activation and damage in M. suis-associated pathogenesis was investigated using light and electron microscopy, immunohistochemistry, and cell sorting. M. suis interacted directly with endothelial cells in vitro and in vivo. Endothelial activation, widespread endothelial damage, and adherence of red blood cells to the endothelium were evident in M. suis-infected pigs. These alterations of the endothelium were accompanied by hemorrhage, intravascular coagulation, vascular occlusion, and massive morphological changes within the parenchyma. M. suis biofilm-like microcolonies formed on the surface of endothelial cells, and may represent a putative persistence mechanism of M. suis. In vitro analysis demonstrated that M. suis interacted with the endothelial cytoskeletal protein actin, and induced actin condensation and activation of endothelial cells, as determined by the up-regulation of ICAM, PECAM, E-selectin, and P-selectin. These findings demonstrate an additional cell tropism of HM for endothelial cells and suggest that M. suis interferes with the protective function of the endothelium, resulting in hemorrhagic diathesis.Hemotrophic mycoplasmas (HM) are highly specialized red blood cell parasites that cause infectious anemia in a variety of mammals, including humans. To date, no in vitro cultivation systems for HM have been available, resulting in relatively little information about the pathogenesis of HM infection. In pigs,  Mycoplasma suis, M. haemofelis and M. ovis have been isolated from diseased humans[Candidatus M. haemohominis, was recently reported in a human subject and was associated with clinical symptoms of pyrexia and hemolytic anemia[Hemotrophic mycoplasmas (HM) are global etiological agents of infectious anemia in a variety of animals. HM are small erythrocytic parasites that adhere to and invade red blood cells (RBCs)-3. To dad humans-6. Additc anemia.M. suis infection  manifests as hemolytic anemia and hemorrhagic diathesis accompanied by immune modulation and coagulation dysfunction due to intravasal coagulation and subsequent consumption coagulopathy[M. suis infection, e.g. cyanosis, petechial bleeding, and ecchymosis. One possibility is that the endothelial cells (ECs) of the vasculature are involved in the pathogenesis of IAP. Electron microscopic studies of RBCs from experimentally-infected pigs revealed massive morphological changes as well as strong aggregation of parasitized and non-parasitized cells. Blood clotting in these animals was attributed to the production of cold and warm autoreactive IgM and IgG antibodies directed against the RBC surface. In addition an acute phase response of the immune system enhanced the formation of blood clots[In pigs, acute ulopathy-13. Howeod clots,13,14.M. mycoides subsp. mycoides, M. gallisepticum, M. neurolyticum, Bartonella bacilliformis, Rickettsia conorii and Bacillus anthracis, have been described that target and damage the endothelial barrier, or interact closely with EC, triggering inflammatory responses and coagulation processes. In this manner, they are able to interfere with the protective function of ECs through a variety of mechanisms, including invasion and toxin induced endothelial damage, that latter of which could induce hemorrhagic diathesis[To date, several pathogenic bacteria, including iathesis-20.M. suis. The goal of the current study was to elucidate potential interactions between M. suis and ECs both in vivo and in vitro. Our hypothesis is that the interaction of M. suis with ECs, via either adhesion and/or activation, leads to an activated EC phenotype, thereby mediating damage to the vascular endothelium and activation of pro-inflammatory and inflammatory cascades. These direct and indirect interactions induce a systemic immune response resulting in endothelial pathophysiology. We demonstrated that M. suis closely interacts with ECs in vivo and in vitro, resulting in endothelial activation and destruction. M. suis infection led to endothelial damage, the induction of pro-inflammatory and inflammatory cascades, and immunopathology.Based on current evidence, RBCs serve as the exclusive target and host cell of M. suis infection was performed as described previously[M. suis negative health status was confirmed by a quantitative PCR[n = 10) underwent splenectomy[M. suis-containing blood from an experimentally infected pig, as described previously  and served as a negative control. Splenectomy induces an acute clinical manifestation of M. suis infection in the used pig model[M. suis-negative pigs were monitored accordingly.Experimental eviously-23 in active PCR and ELIStive PCR. Afterwaenectomy. Six pigig model,26. Pigs2.Primary porcine aortic endothelial cells  were used for the in vitro adhesion assays. The immortalized porcine aortic endothelial cell line PEDSV.15 was used for in vitro activation. PAECs w2 in 1.5 mL of DMEM in 12-well tissue culture plates.PEDSV.15 cells  were maintained in DMEM supplemented with 10% FCS, 1 mM sodium pyruvate, 1 mM L-glutamine, non-essential amino acids (1×), and 20 mM HEPES . PEDSV.1M. suis was purified from the plasma of infected pigs as described previously, with slight modifications[g to sediment the erythrocytes. Plasma was removed and subjected to centrifugation at 20 000 × g for 1 h at room temperature (RT) . The resulting pellet was washed twice and then resuspended in phosphate buffered saline . M. suis was quantified by quantitative LightCycler (LC) PCR analysis[ications,23. Brieanalysis. As a neM. suis-infected and control pigs to examine the effect of M. suis on the ECs. Blood vessels and parenchyma were immediately fixed in 4% phosphate-buffered formaldehyde (FA) for 24 h. Other tissues  were immediately fixed in methanol/glacial acid (2:1 for 24 h) or in Bouin’s fluid, and then embedded in paraffin according to standard procedures.Blood vessels  and parenchyma (heart and liver) were collected from euthanized FA-fixed blood vessels and parenchyma were post-fixed in 2.5% phosphate-buffered glutaraldehyde (GA) for 24 h and then stored in 0.1 M cacodylate buffer until further processing. Strips 2 cm in length (long axis) of the fixed blood vessels were cut in half for macroscopic documentation under a Leica Z16 APO light microscope . After microscopy, tissues were post-fixed for 1 h at RT in 1% osmium tetroxide  in 0.1 M cacodylate buffer, dehydrated through a graded ethanol series, and then subjected to critical point drying . Finally, the samples were sputter-coated with 12 nm of platinum using the BAL-TEC MED 020 coating system, mounted on an aluminum stub, and then analyzed on a Zeiss Supra 50 VP  scanning electron microscope.M. suis-specific rabbit polyclonal antibody directed against α-enolase (1:200 in PBS)[For immunohistochemistry, the fixed and paraffin embedded tissues were cut into sections (5 μm thick) and mounted on Superfrost® glass slides. After deparaffinization and antigen retrieval in a microwave oven in 10 mM sodium citrate buffer at pH 6.0 (3 × 5 min at 700 W), sections were subjected to immunohistochemistry using an  in PBS). BrieflyFormaldehyde-fixed blood vessels and parenchyma were cut into 1–2 mm segments and post-fixed in 2.5% phosphate buffered GA solution for 24 h. Samples were washed in 0.05 M cacodylate buffer and stored at 4°C until further processing. Following post fixation in 2% osmium tetroxide in cacodylate buffer, sections were washed once in 0.05 M cacodylate buffer. Samples were dehydrated in increasing concentrations of ethanol (70–100%) and then infiltrated by increasing concentrations (33 and 50%) of Epon 812 (Fluka Chemie) in ethanol (100%) for 1 h each. After incubation in a mixture of 75% Epon and 25% ethanol overnight, samples were infiltrated with fresh pure Epon for 2–3 h. Specimens were embedded in fresh Epon in flat silicone rubber molds and hardened for 12 h at 60°C. After preparation of ultrathin sections using an Ultracut E microtome , sections were placed on copper grids , contrasted consecutively with 4% uranyl acetate (Fluka Chemie) and lead citrate as described previously (Reynold 1963), and then analyzed with a Philips CM100 transmission electron microscope equipped with a Gatan Orius CCD camera .M. suis surface protein, MSG1 (10 μg/mL). Sections were then washed three times for 20 min each with PBS and then incubated with 1.2 nm colloidal gold particles conjugated to goat anti-mouse IgG (1:1000)[FA-fixed blood vessels were cut into 5–8 mm segments, equilibrated in 30% sucrose and then embedded in tissue-Tek  at −40°C. Horizontal segments (40 μm thick) were sectioned using a Kryostat HYRAX C 60  to view the subcellular matrix underneath the endothelium. Sections were permeabilized with 0.2% Triton X-100  for 1 h at RT and then incubated for 24 h with a monoclonal antibody against the M. suis  or the negative control purification preparation (100 μL/well) for different time periods (5 min to 5 days). After washing twice with PBS, cells were fixed in 2.5% phosphate buffered FA for 2 h at 4°C. After washing again with PBS, PAECs were permeabilized with 0.2% Triton X-100 (Sigma-Aldrich) for 2 min at RT. Cells were washed with PBS and non-reacted aldehydes were blocked with 0.1 M glycine  in PBS for 20 min. Non-specific binding of antibodies was reduced by incubation of samples in blocking buffer (3% BSA in PBS) for 30 min. Adhesion of M. suis to PAECs was visualized by staining with rabbit anti-HspA1 serum (1:100)[PAECs on cover slips were incubated with purified  (1:100) for 1 h M. suis, PEDSV.15 cells at 60% confluence were incubated in duplicate with purified M. suis  or the negative control purification preparation. Cells were harvested and fixed in 2.5% FA after 2 h and 4 h for the analysis of E (CD62E)- and P (CD62P)-selectin expression, respectively, and after 24 h, for the analysis of intercellular adhesion molecule 1  and platelet endothelial cell adhesion molecule 1  expression[To analyze the activation of endothelial cells by pression-34. Thespression,36. FITCP value ≤ 0.05 was considered statistically significant.SPSS statistical software was used to test differences in the expression of cell surface markers between groups. Differences were calculated using the unpaired Wilcoxon test and a M. suis-infected pigs revealed marked alterations of the luminal surface of the abdominal aortic vessel, which appeared rough and covered in reddish spots. An additional file shows this in more detail  revealed massive alterations in tissue structure, particularly in the vascular system  of the aortic vessel of e Figurea and2b.M. suis aggregates of approximately 5–50 μm in diameter attached to the surface of the vessel walls. These micro-colonies were characterized by round or elongated cross-linked cells of 200–400 nm in diameter embedded in a three-dimensional granular matrix  arranged in parallel and with no attached blood cells or bacteria were observed in non-infected pigs . Microvilli formation was evident on the ECs of the abdominal aorta of s Figurea. In soms Figureb. M. suin Figurec, d. M. e Figuree.M. suis-induced morphological changes, endothelial morphology and intravascular coagulation in the organ capillaries of the liver and heart parenchyma were analyzed by TEM.To further examine M. suis-infected pigs were irregular in shape with localized swelling and finger-like pseudopodia  of the liver of s Figured, e. In s Figuref. Liver s Figured, e and s Figuref had a nM. suis with ECs in vitro, PAECs were infected with purified M. suis and then analyzed by confocal laser scanning microscopy following immunohistochemical staining to differentiate the cytoskeleton, cell nucleus, and M. suis. M. suis cells were detected as aggregates on the surface of PAECs as early as 90 min post-infection. Actin condensation was observed at the sites of bacterial cell attachment , i.e. ICAM-1 (CD54), PECAM-1 (CD31), P-selectin (CD62P) and E-selectin (CD62E) was used as activation marker. The CAMs expression levels (number of CAMs expressing PEDSV.15 cells) were determined using FACS analysis and CAM-specific monoclonal antibodies. PEDSV.15 cells were responsive for activation as shown by the induction of CAMs upregulation after TNF-α and LPS stimulation. TNF-α induces a 2.3 fold more CD54, a 2.0-fold more E-selectin (CD62E), a 15.8-fold more P-selectin CD62P, and 2.1-fold PECAM-1 (CD31) expression when compared with the negative control (cell culture medium). LPS induces a 17.5 fold more CD54, a 6.9-fold more E-selectin (CD62E), a 14-fold more P-selectin CD62P, and 2.1-fold PECAM-1 (CD31) expression when compared with the negative control (cell culture medium).To assess whether the presence of M. suis (P ≤ 0.05). As shown in Figure6M. suis-stimulated PEDSV-15 showed a significant increase in CAM cell-surface expression compared with that in the negative control preparation M. suis-incubated PEDSV-15 expressed on average 1.9-fold more ICAM-1 , 2.6-fold more E-selectin , 7.5-fold more P-selectin  and 1.9-fold more PECAM-1  compared with negative control preparation.Overall, there was a significant increase in CAMs expressing PEDSV.15 cells upon infection with M. suis infection is accompanied by coagulopathy, vascular thrombosis, and hemorrhaging of the skin and organs; symptoms that are indicative of the involvement of vascular ECs in the pathogenesis of acute M. suis infection[M. suis pathogenesis has received little attention to date because consumption coagulopathy is considered to be the primary cause of hemorrhagic diathesis[M. suis and ECs, and the resulting endothelial disruption and damage to the vasculature.Hemolytic anemia as a result of acute nfection,11,12. HM. suis-infected pigs. Blood vessels exhibited detached ECs over a considerable portion of the endothelium. In some ultrathin sections, EC were completely missing and the subcellular matrix was exposed. This likely represents the mechanism by which M. suis gained access to the sub-endothelial matrix, where it was detected by TEM. However, the extent of endothelial denudation made the microscopic detection of intracellular M. suis in ECs unfeasible. An analogy can be made with certain Bartonella species, which are also undetectable intracellularly in ECs, even though pathogenesis induced by these species was shown to involve ECs[SEM and TEM analyses clearly demonstrated marked abnormalities in the endothelium of blood vessels in olve ECs. Additioolve ECs,39, and M. suis-infected animals, which is also characteristic of an activated phenotype, and could serve to increase the frequency of adhesion of M. suis-infected RBCs, as observed in micro vessels from patients infected with P. falciparum[M. suis-infected RBCs to the endothelium might be further enhanced by M. suis-induced RBC changes, some of which could trigger a variety of pathologies such as vasoocclusion, endothelial activation, and initiation of the coagulation cascade[P. falciparum infections[The formation of microvilli-like membranes on ECs was observed in alciparum. The inc cascade. The extfections.M. suis-induced endothelial injury. This fibrin network acts as a provisional matrix during inflammation and wound healing[Numerous arterial thrombi, composed of leukocytes, RBCs, platelets and a network of fibrin fibers, were attached to ECs next to sites of  healing. Fibrin  healing,44. Acti healing. Further healing,47.M. suis in direct contact with ECs and attached to the endothelium is an entirely new finding in HM research. To date, HM has been reported to attach and invade a unique host cell, the RBC[M. suis was attached to ECs either as a single cell or as microcolonies ranging in diameter from 30–50 μm. This direct interaction of M. suis with ECs could trigger the EC destruction and exposure of the subendothelial matrix observed in the M. suis-infected pigs. This would be consistent with other pathogenic Mycoplasma species, such as the large colony strains of M. mycoides subsp. mycoides, which are able to adhere to and destroy caprine ECs, thereby exposing subendothelial collagen and causing vasculitis and coagulation disturbances[The detection of  the RBC-3. In thurbances,20.M. suis biofilm-like structures we observed were homogenous aggregates of cross-linked 200–400 nm M. suis cells that formed a three-dimensional network. Occasionally, microcolonies appeared more compact and the bacteria were embedded in a granular matrix. M. suis microcolonies attached to the vascular wall and resembling biofilms have been described for other mycoplasmas[M. suis cells probably originated from a budding-like replication mechanism, similar to that proposed for HM, as well as for other mycoplasmas, and they resembled propagating M. suis cells on RBC[M. suis is capable of propagating on ECs.The oplasmas. Within s on RBC,49,50. TM. suis cells on the surface of RBCs and attached to the vascular wall. M. suis adhere to host RBCs via fibrils[M. suis cells were observed only on the surface of RBCs attached to the endothelium Further studies are needed to better characterize the composition and function of M. suis fibrils[One of the more novel and more interesting findings of this study was the formation of small fibrils interconnecting several  fibrils,51. In t fibrils.M. suis and ECs. A considerable number of M. suis cells were attached to the PAEC surface, and actin condensation was evident at the attachment sites. Indications for an interaction between M. suis and porcine actin have been found in a previous study since M. suis-induced autoreactive IgG antibodies also bind to actin[M. suis interacts with cytoskeletal actin of endothelial cells leading to cytoskeletal rearrangements in the EC. Various invasive bacteria, such as Yersinia enterocolitica and Listeria monocytogenes, trigger rearrangement of the actin cytoskeleton to facilitate their uptake into eukaryotic cells[M. suis is internalized by a similar mechanism remains to be clarified in further studies. Analysis of the M. suis genome revealed that, similar to other Mycoplasma species, M. suis lack any of the genes required for de novo nucleotide biosynthesis[M. suis in terms of acquiring a source of nucleotides for proliferation. Despite demonstrating a clear and close interaction between M. suis and ECs, definitive proof of the intracellular localization of M. suis in ECs in vivo could not be demonstrated due to the extensive cytopathological effects of M. suis (data not shown). Damaged ECs most likely detached from the endothelium and, thus, were not available for microscopic analysis of intracellular M. suis. Additional studies of sections from infected blood vessels at an early phase of infection may provide the necessary evidence to support this hypothesis.In vitro analysis using porcine endothelial cells (PAEC and PEDSV.15 cells) provided further insight into the mechanism of interaction between to actin,53. In tic cells,55. Whetynthesis. Thus, eM. suis was demonstrated in vitro by the significant increase of ICAM-1, PECAM-1, E-selectin and P-selectin expressing cells, all of which are important markers of EC activation. Endothelial E-selectin and P-selectin are up-regulated by inflammation and mediate leukocyte capture and rolling on the endothelium[M. suis. This could explain the relatively smaller but significant difference between M. suis and negative control in case of E-selectin and PECAM-1. ICAM-1 is involved in leukocyte rolling and arrest on endothelial cells[M. suis infection results in activation of EC.The activation of ECs by othelium. The up-othelium, both ofal cells, as wellal cells. Recentlal cells. PECAM-1al cells,62, wheral cells. In summM. suis-infected pigs with subsequent occlusion of capillaries leading to ischemia (interruption of the blood supply). This would be consistent with the fact that some cases of acute M. suis infection in pigs result in death within a few days[Another of the more intriguing findings of the current study was the observation of cardiac muscle damage with disorganized and damaged and/or destroyed cardiac muscles cells. These effects could be explained by RBC aggregation in the blood vessels of few days.M. suis to form biofilm-like microcolonies on the endothelium, which may protect the organism from antimicrobial agents and host immune factors, may contribute to the persistence of HM infections.In conclusion, we report several novel findings of infection with HM leading to widespread endothelial damage, RBC adhesion to the endothelium, and vascular occlusion. These vascular alterations lead to the development of hemorrhage and organ failure. To our knowledge, this is the first demonstration that HM interacts with host cells other than RBCs. In addition, the ability of RBC: Red blood cells; Bc: Bile canaliculi; BM: Bacterial microcolony; Co: Collagen; EC: Endothelial cells; ER: Endoplasmic reticulum; F: Small fibrils; Fi: Fibrinogen fibers; IAP: Infectious anemia in pigs; ICAM 1: Intercellular adhesion molecule 1; L: Lumen; Leu: Leukocytes; LC: Lightcycler; Mi: Mitochondria; MV: Microvilli; N: Nucleus; PAEC: Porcine aortic endothelial cells; PECAM-1: Platelet endothelial cell adhesion molecule 1; Ps: Pseudopodia; PV: Plasma lemma vesicle; RM: Tunica media; SEL: Subendothelial layer; SP: Silver precipitates; V: Intracellular vacuoles; VC: Vacuolar cell; VW: Vessel wall.The authors declare that they have no competing interests.Conceived and designed the experiments: LEH, UZ, KH; performed animal experiments: LEH, KH, KMF; performed microscopic experiments: AS, KG, WMA, JMM; performed in vitro analyses and data analysis: AS, MKJS, KMF, KG; wrote the manuscript: AS, KH, LEH. All authors read and approved the manuscript.M. suis infection results in perforation of the blood vessels. Light microscopic images of the luminal surface of the aortic vessel. A. Aortic vessel of an infected pig showing RBCs attached to ECs. Scale bar = 5 mm; B. Aortic vessel of an infected control pig showing a smooth endothelial surface. Scale bar = 5 mm. Abbreviations: CT, connective tissue; I, injured endothelium with bleeding into the tissue; L, lumen; and VW, vessel wall.Click here for fileM. suis infection causes endothelial cell activation. Scanning electron micrograph of an aortic vessel from an M. suis-infected pig. A. Aortic vessel from an infected pig showing RBCs attached to ECs. ECs (white arrows) are characterized by extensive microvilli (MV) formation. Image also shows the exposed subendothelial layer (SEL) due to detachment of ECs. Scale bar = 10 μm. Abbreviations: EC, endothelial cell; MV, microvilli; RBC, red blood cell; and SEL, subendothelial layer.Click here for file


Chlamydia pneumoniae is a widespread pathogen causing upper and lower respiratory tract infections in addition to a range of other diseases in humans and animals. Previous whole genome analyses have focused on four essentially clonal (> 99% identity) C. pneumoniae human genomes , providing relatively little insight into strain diversity and evolution of this species.C. pneumoniae koala genome and four C. pneumoniae human genomes to identify species-specific genes, and more importantly, to gain an insight into the genetic diversity and evolution of the species. We selected genes dispersed throughout the chromosome, representing genes that were specific to C. pneumoniae, genes with a demonstrated role in chlamydial biology and/or pathogenicity (n = 49), genes encoding nucleotide salvage or amino acid biosynthesis proteins (n = 6), and extrachromosomal elements (9 plasmid and 2 bacteriophage genes).We performed individual gene-by-gene comparisons of the recently sequenced C. pneumoniae isolates from both human and animal origin. Such characterisation is necessary for an improved understanding of disease transmission and intervention.We have identified strain-specific differences and targets for detection of  Chlamydiaceae are obligate intracellular pathogens that undergo a unique biphasic developmental cycle involving the inter-conversion between the extracellular infectious elementary body and the intracellular, replicative reticulate body. Chlamydia 'Chlamydophila' pneumoniae is probably one of the most successful chlamydial species, having established a niche in a range of warm-blooded (homoeothermic) and cold-blooded (poikilothermic) hosts, including humans, horses, marsupials, frogs and reptiles ,,C. pneumrovars A , respectC. pneumoniae, AR39 is the only human genome to have an extrachromosomal bacteriophage (4524 nt single-stranded DNA) [C. psittaci phage), which is present in the four C. pneumoniae human genomes. This suggests earlier integration of the phage genome in the C. pneumoniae genome . The second koala LPCoLN phage remnant was a 445 nt ORF, termed CPK_ORF00730, which appears to be 'intact', sharing approximately 77% similarity (nt 342/445) to the human AR39 phage. The koala LPCoLN phage remnants are positioned between hypothetical genes in the genome and there appear to be no further remnants of the phage within this region of the genome. A comparison of the C. pneumoniae koala LPCoLN phage with other chlamydial species revealed approximately 77-79% sequence identity to partial-length sequences of the C. psittaci Chp2 phage, C. pecorum phage 3, C. caviae phiCPG1 phage and C. abortus Chp4 phage.The bacteriophage is another strain-specific extrachromosomal element reported in chlamydial species. In ded DNA) , whereasC. pneumoniae koala LPCoLN genome and previously published C. pneumoniae human genomes . The koala LPCoLN genome is larger than all four C. pneumoniae human genomes by 10-12 kbp. We combined BLAST search methods and motif analysis for use in elucidating the relationship between gene function and evolution. Even though these techniques have several limitations [C. pneumoniae evolution, and (iii) suggested targets for detection and differentiation of C. pneumoniae isolates from both human and animal origins.In this work, we applied computational analyses to explore the genome content and genetic diversity among the recently sequenced itations , our comC. pneumoniae koala genome has the full-length version. These length polymorphisms suggest that the presumed functional changes are brought about by adaptation to a specialised niche, where the ancestral gene function may no longer be required. Therefore, it is important to understand how these genetic differences may influence differences in pathogenicity and fitness in the host. Our data supports the findings of Rattei et al. [et al. [The presence of unique insertions/deletions is evidence of evolution in action. For the majority of these genes, the i et al.  and the  [et al.  in that C. pneumoniae. One hundred and twenty-three of these represented hypothetical genes with no significant similarity to genes in other organisms present in the database. Further analysis of these hypothetical genes  may reveal undiscovered biovars or subspecies in C. pneumoniae.Our analysis revealed a total of 140 genes that were specific to C. penumoniae. This suggests that the pmp gene family is subjected to high selective pressure , correlating with a relatively faster evolutionary rate for these antigens. Taken together, the polymorphism of pmp sequences in C. pneumoniae from humans and animals is dually consistent with the divergent evolution of the pmp genes under host-specific selection while maintaining the capacity to adapt to specific niches or immune responses in the two different hosts.The Pmp family is characterised by an unusual degree of sequence polymorphism, including mutations and large indels across all species -47 and spmp protein family, the strict conservation of T3S effector genes was initially surprising given the effectors' normal tendency for divergence. While the differences observed between other regions of the genomes are consistent with evolutionary changes [C. pneumoniae that are conserved in human and animal strains, such as inclusion and intracellular development. Overall, these results support a critical role for T3S less in the virulence than in the developmental biology of these organisms. Such a role has been proposed in the context of the contact-dependent T3S-mediated hypothesis of chlamydial development proposed earlier [In light of the variation seen between other families of genes and their orthologs in the human isolates, namely the  changes , the rel earlier ,49.C. trachomatis MACPF by Taylor et al. [Toxoplasma spp. have shown that ablation of the MACPF (termed TgPLP1) resulted in a reduction in virulence (in mice), whereby TgPLP1 deficient parasites were unable to exit normally and were entrapped within host cells, due to the inability to permeabilise the parasitophorous vacuole membrane [Drosophila torso-like protein involved in embryonic development [Photorhabdus luminescens which binds to the surface of insect cells [Chlamydiaceae.Orthologs of the MACPF were identified in several chlamydial species. The first biological characterisation of the r et al.  has revemembrane . If the membrane , a Drosoelopment  and Plu-ct cells . FurtherC. pneumoniae is the only chlamydial species thus far to have a udk gene encoding uridine kinase. The udk gene is a pyrimidine ribonucleoside kinase that phosphorylates uridine and cytidine into uridine or cytidine monophosphate (UMP/CMP) [Prevotella bryantii genome encodes a putative uracil DNA glycosylase and uridine kinase, likely to be involved in the removal of misincorporated uracil from DNA and its subsequent re-use [C. pneumoniae is the only species carrying the udk ortholog. This implies that an alternative gene product is involved in UMP production in the other chlamydial species. The C. muridarum genome includes a CDS (upp) encoding a uracil phosphoribosyltransferase [C. pneumoniae is unusual in having a very broad host range and therefore the fact that it is the only chlamydial species to have retained the udk gene could reflect this broad host capacity.UMP/CMP) , and is t re-use . All chlnsferase  that mayde novo synthesis, and to a degree, of salvage [pyrE should be retained. The final step in the pathway is via pyrF which is absent from the chlamydial genome, suggesting that they are unable to convert orotate to UMP. The presence in all chlamydial genomes of orthologs encoding the three downstream enzymes involved in UMP to CTP conversion and the earlier demonstration of CTP synthetase activity in these organisms [guaB, guaA and add have been selectively maintained in several chlamydial species including C. pneumoniae AR39, CWL029, TW183 and J138, C. felis Fe/C-56, C. caviae GPIC and C. muridarum Nigg. C. abortus has a guaB pseudogene, whereas the C. pneumoniae LPCoLN and C. trachomatis serovar A/HAR, B/Jali20/OT, D/UQ-3/CX, L2b/UCH-1/proctititis, L2/434/Bu and Candidatus Protochlamydia amoebophila UWE25 genomes lack all three genes [guaBA-add from C. pneumoniae koala LPCoLN and other chlamydial species suggest that these three enzymes required for inter-conversion of GMP, IMP and AMP must be acquired by other means or are clearly not essential for species survival.Most bacteria can salvage or synthesise their own purines and pyrimidines. By contrast, chlamydiae and rickettsiae (another obligate intracellular bacterium) are incapable of  salvage ,57. Giverganisms ,59 confiee genes ,35,36,60tyrP (tryptophan tyrosine permease) gene have been suggested to reflect vascular tropism and pathogenicity among C. pneumoniae human isolates with multiple copies associated with respiratory infection and single copy more frequently associated with vascular tropism [C. pneumoniae genomes also revealed variations in the tyrP (tryptophan tyrosine permease) copy number that are, however, inconsistent with the hypothesis by Gieffers et al. [Chlamydia pneumoniae TW183 and comparison with other Chlamydia strains based on whole genome sequence analysis, submitted) have a single tyrP copy while two other respiratory isolates (CWL029 and AR39) [tyrP.Copy number variations of the  tropism . A compas et al. . Among ts et al. ,30, as wnd AR39) ,26 carryC. pneumoniae koala and C. pneumoniae human. Extrachromosomal plasmids have been identified in six of the nine chlamydial species. As the plasmid is not common to C. pneumoniae, it is not known why koala LPCoLN has a plasmid.The loss and fragmentation of pre-existing genes during evolution is one of the primary distinguishing features between C. pneumoniae-specific hypothetical proteins with no predicted function were identified in the comparisons and may be worth further investigating for their potential role in host tropism, pathogenicity and niche adaptation  genes that could potentially differentiate isolates from human and animal origins, for example, length polymorphic genes including the membrane attack complex perforin and the hypothetical protein CPK_ORF00679, (iii) genes for the identification of a C. pneumoniae plasmid.While proteins with predicted or known biologic function are favoured candidate gene targets, many C. pneumoniae and their hosts, and assists in the identification of functional differences that may determine pathogenicity and virulence differences between the strains. Previously, whole genome comparisons have focused on four practically clonal C. pneumoniae human genomes, which make the identification of target genes for strain differentiation quite challenging. In this study we have made use of the recently sequenced koala LPCoLN genome, which is the largest and most unique C. pneumoniae genome sequenced thus far, in order to select target genes that represented traits of strain-specific determinants. Further investigation of these genes in other host species may provide additional clues as to what enables this pathogen to: (i) present varied clinical pathologies, (ii) occupy multiple niches, and (iii) establish an infection in cold and warm-blooded hosts. Moreover, these genes may become targets for improved diagnosis and therapeutic strategies.The study of whole genome sequences provides important clues to the natural history of C. pneumoniae AR39 (GenBank accession number AE002161), CWL029 (GenBank accession number AE001363) TW183 (GenBank accession number AE009440), J138 (GenBank accession number BA000008) and LPCoLN (GenBank accession CP001713) were used in this study.The complete genomic sequences of C. pneumoniae human genome and other organisms using an E-value cutoff of 1 × 10-4 with manual curation. This approach enabled us to identify regions of high SNP accumulation and to select target genes for comparison. These selected genes were grouped into five categories by their putative, predicted or hypothetical functions. The categories included: (1) C. pneumoniae-specific genes ; (2) genes with a prior demonstrated role in chlamydial biology and pathogenicity (n = 49); (3) genes encoding nucleotide salvage or amino acid biosynthesis proteins (n = 6); (4) extrachromosomal elements, including a plasmid (n = 9) and bacteriophage-related genes (n = 2) ;  genes wiGene alignments were performed using the Clustal W program  to identify polymorphisms and indels (insertions/deletions) . ConservA phylogenetic tree was constructed by Neighbor-Joining, tie breaking = systematic, distance corrected by the Poisson method with gaps distributed proportionally and 1,000 bootstrap replicates.CDS: Coding Sequence; Indels: Insertions/Deletions; ORF: Open Reading Frame; SNP: Single Nucleotide Polymorphism.CMM prepared the manuscript and performed comparative analyses including evaluations. KMH contributed with text, interpretation and analysis for the manuscript. PMB aided in data interpretation and critically revised the manuscript. GSAM interpretation and analysis of the genome. JAC contributed with text, interpretation and analysis for the manuscript. PT participated in the design and drafting of the manuscript, aided in data interpretation and critically revised the manuscript. All authors read and approved the final manuscript.Categories of targets for investigation. (1) genes with a prior demonstrated role in chlamydial biology and pathogenicity (n = 49); (2) genes encoding nucleotide salvage or amino acid biosynthesis proteins (n = 6); (3) extrachromosomal elements, including a plasmid (n = 9) and bacteriophage-related genes (n = 2).Click here for fileC. pneumoniae-specific genes. A list of C. pneumoniae genes that have no significant similarity to other chlamydial species or organisms. E-value cutoff of 1 × 10-4 with manual curation.Click here for fileC. pneumoniae polymorphic membrane proteins (Pmps)Comparative analysis of the . A comparison of the 21 Pmps revealed a high degree of sequence polymorphism and indels between the koala LPCoLN and human AR39, CWL029 and TW183 isolates. The TW183 and J138 isolates were well-conserved and therefore, TW183 also represents J138 in this figure. Arrows indicate the direction of transcription: green arrows show typical pmp characteristics, red arrows represent pseudogenes (numbers below arrows indicate stop codon position), and white arrows represent proteins with no relation to pmps (note the same orientation as pmpG10). Dashed lines indicate truncated products.Click here for fileC. pneumoniaePolymorphic outer membrane protein features of .Click here for fileC. pneumoniaePolymorphic outer membrane protein SNP position analysis of .Click here for fileT3S ortholog comparisons.Click here for fileChlamydia MACPF. A BLAST alignment of the C. pneumoniae MACPF protein. From top to bottom: C. pneumoniae LPCoLN, C. pneumoniae J138, C. pneumoniae CWL029, C. felis FE/C-56, C. trachomatis A/HAR-13, C. trachomatis 6276, C. trachomatis D/UW-3/CX, C. trachomatis 70, C. trachomatis 434/Bu, C. muridarum Nigg, C. pneumoniae CWL029, C. abortus S26/3, C. felis Fe/C-56, Alcanivorax sp. DG881, Saccolglossus kowaleski, Theileria parva strain Muguga....Click here for fileChlamydia has lost several steps in the pyrimidine biosynthesis pathway. All chlamydial genomes sequenced thus far, have lost the initial steps involved in pyrimidine biosynthesis. C. pneumoniae (Cpn), C. abortus (Cab), C. caviae (Cav) and C. felis (Cfe) contain a pyrE gene encoding an orotate phosphoribosyltransferase, while C. muridarum (Cmu) and C. trachomatis (Ctr) lack this gene. The next step in the pathway is via pyrF, which is absent from all chlamydial genomes. Interestingly, all six genomes have maintained the last three steps for the conversion of UMP into CTP. Adapted from Koonin and Galperin [carA, carbamoyl-phosphate synthase, small subunit; carB, carbamoyl-phosphate synthase, large subunit; pyrB, aspartate carbamoyltransferase; pyrC, dihydroorotase; pyrD, dihydroorotate dehydrogenase; pyrE, orotate phosphoribosyltransferase; pyrF, orotidine 5-phosphate decarboxylase; pyrH, uridylate kinase; ndk, nucleoside diphosphate kinase; pyrG, CTP synthase. Red boxes indicate gene loss, yellow boxes indicate the presence of a gene.Galperin . Gene naClick here for fileSequence comparison of the chlamydial plasmid. Multiple sequence alignment of the predicted amino acid sequence from C. pneumoniae koala LPCoLN (pCpnKo), C. pneumoniae horse N16 (pCpnE1), C. psittaci avian N352 (pCpA1), C. felis feline Fe/C-56 (pCfe1), C. caviae guinea pig GPIC (pCpGP1), C. muridarum mouse Nigg (pMoPn) and C. trachomatis human serovars A (pCTA), B (pJALI), E (pSW2) and L1 (pLVG440). The sequences are well-conserved across species, indicating some degree of ancestry among them. The C. pneumoniae plasmid shared a close relationship with C. psittaci, C. caviae and C. felis, while C. muridarum and C. trachomatis were highly conserved. Predicted functions include plasmid replication (ORF1 and ORF2), double-stranded DNA unwinding (ORF3), chlamydial pathogenesis (ORF5) and regulation of partitioning and copy number (ORF7 and ORF8) [nd ORF8) ,67. The Click here for filePlasmid similarity scores (%). Plasmid similarity scores based on multiple sequence alignment.Click here for fileC. pneumoniae target genesList of . Suggested target genes for detection, strain differentiation and plasmid identification in C. pneumoniae. See also reference [eference .Click here for file


AbstractLamprosomatinae, Cachiporra extremaglobosa Chamorro & Konstantinov, is described from Brazil. A new tribe, Cachiporrini, is proposed. The first phylogenetic analysis of Lamprosomatinae based on adult morphological caharacters is conducted. Comparisons are made among lamprosomatine tribes and genera. A key to tribes is provided.A new genus and species of  Cryptocephalinae addenda drawers PageBreakof the Coleoptera collection at the National Museum of Natural History (NMNH), Smithsonian Institution. Confirmation of this taxon as new was facilitated by the comprehensive chrysomelid collection at NMNH, by the relatively small number of higher-level taxa in Lamprosomatinae, and by the comprehensive studies by ; Neochlamysini Monrós, 1958 (2 genera); Sphaerocharini Clavareau, 1913 (1 genus); and Lamprosomatini Larcordaire, 1848 (10 genera)   not placing it in its own subfamily. Including Sphaerocharini in Lamprosomatinae is now the prevailing classification  . Sphaeroubfamily , as a tromatinae  or as a halinae) . Monrós fication . Monrós Cryptocephalinae, including Chlamisini, sister taxon to lamprosomatines (PageBreakare rarely iridescent), their abdominal ventrites are all the same width medially with sternal longitudinal muscles within ventrite 4 ; femalePageBreakCachiporra could not be placed into any previously recognized lamprosomatine tribe. Clavate antennae are common in many camptosomata  , which is much wider than preceding antennomeres . This kind of club is very uncommon in Chrysomelidae; it is known in only a few flea beetle (Alticini) genera commonly living in relatively dense substrate, such as leaf litter or moss cushions   , however-Lacayo)  and a feLamprosomatinae was undertaken to find taxonomic position for Chachiporra within the subfamily. Designation of a new tribe was considered appropriate and justified to accommodate this new genus from Brazil. Current higher-level classification is based on Lamprosomatinae is here inferred based on cladistic principles.A phylogenetic analysis of Exema Lacordaire and Melittochlamys Monrós (Chlamisini) were selected as outgroup to determine character polarity. All examined material is from the National Museum of Natural History (NMNH), Smithsonian Institution, Washington, DC.This study includes 12 of 14 lamprosomatine genera in 4 tribes  and 3. EPageBreakPageBreakGeneral morphological terminology follows The data matrix was created using MacClade 4.08  and analCachiporrini. Both Bremer and bootstrap values  strongly support the monophyly of Lamprosomatinae and Lamprosomatini. The placement of Sphaerocharis and the monophyly of Neochlamysini remains uncorroborated in the strict consensus. Majority-rule consensus tree retains a monophyletic Neochlamysini with Sphaerocharini as the sister taxon to Lamprosomantini . Midcranial and frontal sutures absent. Top of frons and bottom of vertex with oval impression. Orbital area swollen and situated above eye level. Internal margin of eye entire, not notched. Distance between eyes 2.64 times as large as transverse diameter of eye. Labrum with three setae on each side placed symmetrically on anterior margin. Antennae extremely capitate with last 4 antennomeres tightly fused to form sphere , insertePronotum apically shallowly sinusoidal, basally sinuate, medially extending posterad beyond elytral base. Each pronotal puncture with short seta. Sides swollen and bent ventrally so that lateral border not visible from above. Lateral border situated close to posterior margin of prosternum leaving hypomera extremely thin and limiting prosternum to small triangle. Intercoxal prosternal process in ventral view slightly longer than wide (length to width ratio 1.26), sides constricted above middle, anterior margin evenly concave with obtuse denticle in middle, posterior margin straight. Intercoxal prosternal process in lateral view nearly straight in middle, abruptly bending posterodorsally. Procoxal cavity open posteriorly.Scutellum triangular, equilateral, small . Elytra Anterior process of metasternum rectangular with straight anterior margin . PosteriPageBreakdivided into two parts: poorly sclerotized median and strongly sclerotized lateral. AA better developed than CuA. RA 3+4 absent. Cu and A form only one complete cell.fully developed . Radial PageBreak times longer than preceding, without stridularoty file (device) on distal border. Pygidium exposed.First visible abdominal ventrite as long  as rest of abdomen. Second visible ventrite narrowest, half as narrow medially as either third and fourth. Third and fourth of equal length . Last abwithout dorsal or ventral sclerites with chitinpolster dorsally and ventrally; long lateral fold sclerotized and bent upward and with small denticles .Femora dorsoventrally flat with anterior and posterior sides nearly parallel to each other. Tibiae slightly curved in ventral view gradually widening apically. All tibial apices with small spur, but without long setae and excavation. Tarsal claws relatively small, simple, and free.. Parapro“Cachiporra” is the Spanish word for “billy” club (capitulum). “Cachiporra” alludes to the club-shaped antenna of this lamprosomatine. Feminine.Brazil, Rio Grande do Norte, Natal.PageBreakChamorro & Konstantinov sp. n.urn:lsid:zoobank.org:act:464C4E20-8CAB-40C4-A072-27D057015464Brazil, Rio Grande do Norte, Natal.Length 1.81-1.85 mm. Color black, with bluish and bronzish tint. Head with frons and vertex shagreened, covered by sparse, sharply defined punctures.PageBreakPageBreakPronotum strongly shagreened, evenly covered with sharply impressed small punctures, each bearing a single, small seta. Diameter of punctures four to ten times smaller than distance between them.Elytral surface strongly shagreened, with numerous wrinkles, some of which short and placed diagonally, some exceptionally long and stretched from base of elytron to and beyond middle. Punctures with tendency to form rows.Female genitalia. Median side of the lateral sclerotization of tergite IX strongly oblique. Stylus attached slightly anteriorly from apex. Receptacle of spermatheca slightly longer than pump, slightly S-shaped with small bump near base and apically. Apex of spermathecal pump bulbous, wider than receptacle and base of pump.Cachiporra extremaglobosa Chamorro & Konstantinov, des. 2010 (NMNH).Holotype, female: 1) Brazil RG Norte, Papari: III. 1952. M. Alvarengo; 2) F. Monrós Collection, 1959; 3) Holotype Cachiporra extremaglobosa Chamorro & Konstantinov, des. 2010 (NMNH).Paratype, female: 1) Brazil Natal, R.G. Norte, 24.IX. 1951. M. Alvarengo; 2) F. Monrós Collection, 1959; 3) Paratype PageBreakPageBreakNamed for the extremely globular antennal club (capitulum). The epithet is treated as a noun in apposition.Cachiporrini include the following: antenomeres 7–11 fused into a tight capitulum (2:0); explanation of lateral margin of pronotum situated ventrally essentially covered from above by expansion of upper side of pronotum laterally (5:1); pronotal and elytral punctures with setae (15:0); wings with RA 3+4 absent (18:1); absence of sclerotized rim of kotpresse (23:1); and stylus of female genitalia longer than wide.Unique features of Cachiporra is superficially most similar in color, size, wing venation, and overall body shape to Oomorphus. The pronotum of Cachiporra is considerably different from that of Oomorphus and all other lamprosomatines in having the sides swollen and bent ventrally so that the lateral border is not visible from above. The lateral border is therefore located very close to the posterior margin of the prosternum, leaving hypomera extremely thin and limiting the intercoxal prosternal process to the shape of a small triangle. In Oomorphus the sides of the pronotum are not bent ventrally and the hypomera and the prosternum itself occupy most of the ventral side of the lateral part of prothorax. Furthermore, dorsally the pronotum in Cachiporra is basally sinuate and medially extending posterad beyond elytral base , entire eyes, completely exposed pygidium, and free and simple claws.Lamprosomatinae  (Synapomorphies of omatinae  include r states –31. Sider states  supportsmization . The tramization  is a shasomatini  under sler 11:0) ; a uniqu


Salmonella enterica are an ongoing public health problem especially in Sub-Saharan Africa. Essentially typhoid fever is associated with high mortality particularly because of the increasing prevalence of multidrug-resistant strains. Thus, a rapid blood-culture based bacterial species diagnosis including an immediate sub-differentiation of the various serovars is mandatory. At present, MALDI-TOF based intact cell mass spectrometry (ICMS) advances to a widely used routine identification tool for bacteria and fungi. In this study, we investigated the appropriateness of ICMS to identify pathogenic bacteria derived from Sub-Saharan Africa and tested the potential of this technology to discriminate S. enterica subsp. enterica serovar Typhi (S. Typhi) from other serovars. Among blood culture isolates obtained from a study population suffering from febrile illness in Ghana, no major misidentifications were observed for the species identification process, but serovars of Salmonella enterica could not be distinguished using the commercially available Biotyper database. However, a detailed analysis of the mass spectra revealed several serovar-specific biomarker ions, allowing the discrimination of S. Typhi from others. In conclusion, ICMS is able to identify isolates from a sub-Saharan context and may facilitate the rapid discrimination of the clinically and epidemiologically important serovar S. Typhi and other non-S. Typhi serovars in future implementations.Systemic infections caused by  Fever is a leading cause for hospital admission in Sub-Saharan Africa. Often malaria is thought to be the underlying disease; however bacterial blood stream infections (BSI) contribute to a significant proportion of febrile illness Staphylococcus aureus and Escherichia coli continue to be the most common causative agents of true BSIs in developed countries Salmonella enterica, are the main cause of BSI in African countries S. enterica serovar Typhi (S. Typhi) and non-typhoid Salmonella are frequently isolated from blood cultures in African countries S. Typhi, is estimated to annually cause about 21 million cases and approximately 217.000 deaths Whereas S. enterica relies on bacterial culture using different selective media S. enterica strains produce hydrogen sulfide with exception of S. Paratyphi A and some S. Typhi strains, they can generally be distinguished from other Enterobacteriaceae using thiosulfate containing agar (10). However, some species like Citrobacter freundii are also able to produce hydrogen sulfide and differentiation between S. enterica and C. freundii can therefore be challenging. Consequently, various other chromogenic media have been developed to discriminate between C. freundii and S. entericaS. enterica the White-Kauffmann-Le Minor classification scheme The laboratory diagnosis of S. Enteritidis, S. Typhimurium/4, 5, 12:i:-, S. Virchow, S. Infantis, S. Hadar, S. Choleraesuis, S. Heidelberg, and S. Gallinarum. However, the clinically most important serovar S. Typhi was not included in those particular analyses Currently, intact cell mass spectrometry (ICMS) advances to a widely used routine identification tool for bacteria and fungi S. enterica reference strains to investigate the suitability of ICMS to (i) identify pathogenic bacteria derived from sub-Saharan Africa (spectrum databases were generated with isolates originating mainly in the Western World) and (ii) test the potential of this technology to discriminate S. Typhi from other serovars.In this study, we used blood culture isolates taken from a study population suffering from febrile illness in Ghana Salmonella S. Typhi subtyping was done with the Vi phage typing scheme Isolates used for our analyses were taken from a previous epidemiologic study done in three independent locations in Ghana Salmonella enterica subsp. enterica isolates of 12 different serovars  as well as other species present in the blood cultures  at 37°C under safety conditions as required, prepared in duplicate for ICMS by smear preparation and overlaid with HCCA matrix, both under a safety cabinet and after drying transported to the MALDI device. ICMS was done by standard procedures recommended for the BioTyper 3.0 system . For analysis, 600 spectra from 2–20 kDa were gathered in 100-shots steps. Results with score values >2.000 were considered correct. Analyses for isolates not yielding a significant score were repeated once by smear preparation and in the case of 22  isolates subsequently by formic acid-acetonitrile extraction. All ICMS identification experiments were done in a blinded form. In 15 cases 16 S rDNA sequencing was used as a tie-breaker for discordant or unclear results.Salmonella (S. Typhi (10), S. Paratyphi (3), S. enterica subsp. Arizonae (2), and one spectrum each of S. Bongori, S. Anatum, S. Choleraesuis, S. enterica subspec. diarizonae, S. Dublin, S. Enteritidis, S. Gallinarum, S. Hadar, S. enterica subspec. houtenae, S. enterica subspec. indica, S. enterica subspec. salamae, S. Stanley, S. Typhimurium, and one untyped). For the global identification procedure all of these were counted as “Salmonella enterica spp.”. Phyloproteomic analyses were done using Flexanalysis and PCA algorithms implemented into the BioTyper 3.0 software . Spectra were pre-processed by baseline subtraction and smoothing, for PCA-based hierarchical clustering distance measurement was set to ‘correlation’; the linkage algorithm to ‘average’.At the time of investigation, the Biotyper 3.0 and SR databases together contained 29 spectra from the genus S. enterica control strains were re-typed using ICMS (Shigella flexneri isolate. Discrimination of S. enterica spp. from other Enterobacteriaceae including Citrobacter spp. (as well as other genera) was 100%.A total of 225 blood culture isolates plus 44 ing ICMS . No majoS. Typhi from other Salmonella serovars, score values for all Salmonella isolates were obtained for all S. enterica spp. spectra contained in the database and a “delta mean score”  calculated  clustered into several well-separated groups  were of phage type D1 (data not shown). In an overlay of spectra from the four major clusters at least three major and several smaller peaks can be identified, which separate S. Typhi isolates from other serovars  is a more subtle process. In our study this was demonstrated by the inability of the system to discriminate S. enterica subspecies even using smear spectra. This finding should be of special interest in areas where enteric bacteria, particularly Salmonella enterica, are highly prevalent as causative agent of BSI and other severe infections and together with new enrichment technologies In conclusion, our study demonstrates that (i) ICMS-based species identification is applicable to isolates from sub-Saharan Africa and (ii) that it is possible to discriminate clinically important subtypes, such as the serovars inside the


Borrelia burgdorferi-specific antibody production for the definite diagnosis of LNB. However, about 20% of early stage infections present without an elevated antibody index. Moreover, intrathecal B. burgdorferi specific antibody synthesis may persist long after successful therapy of LNB. Recently published data indicate that CXCL13 seems to be a promising diagnostic tool for early stage LNB. In addition, CXCL13 might be suitable for treatment monitoring.Current guidelines regarding Lyme neuroborreliosis [LNB] require the presence of intrathecal Borrelia garinii OspA-type 7 could be amplified from the first CSF sample. The monitoring of CXCL13 in all CSF samples documented a fast decrease from 5000 pg/ml to 450 pg/ml after appropriate antibiotic treatment.We report on a 39-year-old male patient from southern Germany, who has been suffering from subfebrile body temperatures and meningeal headache for six weeks. On the second day after hospital admission he developed peripheral palsy of the VII. cranial nerve. Cerebrospinal fluid (CSF) analysis showed granulocytic pleocytosis, elevated total protein and blood-CSF barrier dysfunction. Differential diagnostics for granulocytic pleocytosis were unremarkable. Only a second lumbar puncture, on day 6 after admission, revealed a lymphocytic pleocytosis. Serologic testing pointed to clear intrathecal Borrelia specific IgG antibody production. Interestingly, no anti-OspC antibodies were detectable. DNA of the rare CXCL13 is a novel biomarker with high sensitivity and specificity for acute LNB. Our data show, that CXCL13 might be helpful in unclear cases and support the presumption that it might be a valuable tool for treatment monitoring. Anti-OspC antibody negativity is a rare observation, given the need of OspC for infection of the human hosts. Most likely this is due to a lack of sensitivity of OspC immunoblots that are unable to detect rare OspC variants. B. burgdorferi sensu stricto, B. afzelii, B. garinii, B. spielmanii, and B. bavariensis – may cause human diseases. The most frequent disseminated form of the disease is early Lyme-neuroborreliosis [LNB]. Current international guidelines, antineutrophil cytoplasmic antibodies [ANCA] and urinanalysis were normal. Magnetic resonance imaging [MRI] of the brain with administration of contrast material revealed no signs of inflammation, malignancies or vascular complications. The patient received an eight day therapy of steroids - 70 mg prednisolone per day – resulting in a transient improvement of his condition. However, 10 days before admission to this hospital his headache worsened again.On admission to our hospital the temperature was 38.5°C, stable vital signs, blood pressure 140/75 mmHg, heart rate 70 beats per minute, oxygen saturation 97% while breathing ambient air. The weight was 79.5 kg. The skin was warm and dry. The abdomen was soft, normal bowel sounds, without distention, tenderness or masses. Neurological examination revealed positive meningeal signs, the remainder of the examination was normal. Echocardiography points to a mild pericardial effusion, while the electrocardiogram was normal - apart from nonspecific ST-segment changes. Standard laboratory tests were repeated, without significant findings . On the second hospital day, the patient developed a right sided peripheral palsy of the VII. cranial nerve. An emergency MRI of the brain was performed, but did not show any significant pathological intracranial findings. Cerebrospinal fluid [CSF] analysis showed 280 cells, predominantly granulocytes [70%] and lymphocytes [20%], lactate and CSF total-protein levels were elevated, microscopic examination and gram staining revealed no bacteria in CSF [TableThe nuclear acid testing [PCR] for HSV1, HSV2 and VZV in CSF was negative, there was no detection of enterovirus nuclear acid in the patient plasma, no clinical and laboratory signs of CNS HIV or Treponema pallidum infection were observed. CMV and EBV serology showed positive IgG titers, whereas IgM titers were negative. Additional screening for Coxiella burnetii, Brucellosis and Tuberculosis  remained negative. Borrelia serology was not done regarding predominant granulocytes in the CSF. To rule out neurosarcoidosis or systemic sarcoidosis a total body Computed Tomography [CT] was performed, but without any significant findings, in particular neopterin, soluble IL-2 receptor [sIL-2r] and angiotensin converting enzyme [ACE] were not suggestive for sarcoidosis.The follow-up lumbar puncture  activation. CXCL13 r manner. There a. garinii, whereasDRL test,13. Howeated LNB.B. burgdorferi sensu stricto strain B31, B. afzelii strain PKo, B. bavariensis strain PBi, B. garinii type strain 20047] did not show positive results, too for the establishment of the infection and dissemination of the pathogen in humans. The comlts, too. In earl present. Less throtype 7. Thereforotype 7,18. Whetaffected. It remaIn conclusion, the presented case demonstrates the efficient use of CXCL13 in diagnosis and follow-up of LNB, even with initial misleading findings. Possible pitfalls in problematic cases, due to uncommon serological findings or lack of species differentiation, underscore the need for an interdisciplinary collaboration between neurologists, infectious disease consultants and microbiologists.Written informed consent was obtained from the patient for publication of this Case report and any accompanying figures, tables and images. A copy of the written consent is available for review by the Series Editor of this journal.CXCL13: Chemokine C-X-C motif ligand 13; TLR-2: Toll-like receptor 2; LNB: Lyme neuroborreliosis; AI: Antibody index; ELISA: Enzyme linked immuno sorbent assay; Osp: Outer surface protein; VlsE: Variable major protein-like sequence expressed; VDRL Test: Veneral disease research laboratory test; CNS: Central nervous system; CSF: Cerebrospinal fluid; PCR: Polymerase chain reaction; IL-2: Interleukin 2.The authors declare that they have no competing interests.JPB and JH have made substantial contributions to the conception and drafting of the case report. VF and CK carried out CXCL13 and all molecular and genetic studies. SM and JPB were substantially involved in the clinical decision making and interpretation of laboratory data. WVK revised critically the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/12/344/prepub