Plasmodium species including two human parasites,P. ovale and P. malariae, and theevolutionary history of SERA genes was analyzed together with 7 known species.All have one each of Group I to III cysteine-type SERA genes and varying numberof Group IV serine-type SERA genes in tandem cluster. Notably, Group IV SERAgenes were ascertained in all mammalian parasite lineages; and in two primateparasite lineages gene events such as duplication, truncation, fragmentation andgene loss occurred at high frequency in a manner that mimics the birth-and-deathevolution model. Transcription profile of individual SERA genes varied greatlyamong rodent and monkey parasites. Results support the lineage-specificevolution of the Plasmodium SERA gene family. These findingsprovide further impetus for studies that could clarify/provide proof-of-conceptthat duplications of SERA genes were associated with the parasites'expansion of host range and the evolutionary conundrums of multigene families inPlasmodium.SERA gene sequences were newly determined from 11 primate Plasmodium, is one of the most seriousinfectious diseases prevalent in the tropics. There were an estimated 243 millioncases and 863,000 malaria deaths in 2008 . The emergence ofdrug-resistant parasites has made its control more difficult than before and, thus,a better understanding of the biology of malaria parasites is required to gaininsights into new effective control measures including malaria vaccines and newantimalarial drugs. The genome of Plasmodium presents basicinformation for this understanding. One of the prominent features ofPlasmodium genomes is the presence of various unique multigenefamilies, such as the Plasmodium interspersed repeats,pirpir families are highly species-specific, suggesting evolutionof lineage-specific immune evasion mechanisms. By far the best documented ofmultigene families is the var gene family of P.falciparum, the most virulent human malaria parasite. Products ofvar genes appear on the surface of infected erythrocytes andare involved in antigenic variation to evade host immunity. Other species-specificgene families encode proteins involved in host cell invasion, e.g.rhoptry proteins and parasite surface antigens, merozoite surface protein-3 and -7.In sharp contrast to a very large number  of rRNA gene family inother eukaryotes Plasmodium has a very limited array(n = 4–7 units). Thus, Plasmodiumpossesses unique multigene family members in its genome with distinctiveevolutionary conundrums.Malaria, caused by the genus Plasmodium also has the serine repeat antigen (SERA) multigenefamily that encodes proteins with a putative papain-like cysteine protease motif. InP. falciparum, SERA5 (Pfa-SERA5), one of nine SERAs, is avaccine candidate now on phase Ib clinical trial in Uganda -SERA5 in individuals living in malariaendemic areas protect infants from clinical malaria and inhibit invitro parasite growth Plasmodiumspecies have shown that these can be categorized into Groups I to IV, according togene structure and phylogenetic relatedness P. falciparum (Pfa-SERA1 to Pfa-SERA8) is clusteredhead-to-tail on chromosome 2 between a conserved hypothetical protein (HP) gene anda putative iron-sulfur assembly protein gene (hesB) Plasmodium species examined, except for SERA3 ofP. gallinaceum, an avian parasite P.gallinaceum to 12 in P. vivax, the benign humanmalaria parasite. Outside the genus Plasmodium, no apicomplexanparasite has a SERA ortholog, except Theileria, a closely relatedprotozoan parasite of cattle, which has one SERA ortholog Plasmodium SERA multigene family, however, remains largelyunknown. Also, no study has been done for SERA multigene families of human malariaparasites, P. malariae and P. ovale; andP. vivax-related monkey malaria parasites. Since P.vivax became a human parasite by host switch from a monkey parasite, itis worth to see whether P. vivax SERA gene family underwent uniqueevolution, distinctive from closely related monkey parasites.Previous evolutionary studies of SERA genes from eight Plasmodium, we newly determined SERA genes from 11primate Plasmodium species: nine P. vivax-relatedmonkey parasites; and P. malariae and P. ovale.Together with previously reported SERA sequences, we performed evolutionary analysesof the gene family. Results obtained here show that the number of SERA genesremarkably differs among parasite lineages and the variation in mammalian parasiteswas found only in serine-type SERA gene but not in cysteine-type SERA gene. We notedthat the gene number variation occurred lineage-specifically, which was particularlyevident in human, ape and monkey parasite groups. In addition, we found thattranscription of individual SERA genes varied greatly among rodent and monkeyparasites, supporting lineage-specific evolution of the PlasmodiumSERA gene family.In this study, in our attempt to unravel the evolutionary history of the SERA genefamily of Plasmodium species analyzed in this study and theirnatural hosts are: P. falciparum, P. vivax,P. malariae andP. ovale (humans); P.reichenowi (chimpanzees); P.gonderi (African Old World monkeys);P. fragile,P. coatneyi,P. knowlesi,P. inui, P.fieldi, P.simiovale, and P.cynomolgi (Asian Old World monkeys),P. hylobati (gibbons),P. yoelii, P. berghei and P.chabaudi (rodents); and P. gallinaceum (birds)[Underline denotes newly determined SERA sequences in this study].P. vivax is closely related to Asian Old World monkeyparasites P. falciparum and P. reichenowi;and group 2 for P. malariae, P. ovale,P. vivax and the nine Old World monkey parasite speciesPlasmodium species  to 12 (P.vivax) among 18 Plasmodium species. All parasitespecies have one SERA gene from Group I, Group II and Group III (cysteine-typeSERA gene), with the exception of P. gallinaceum, which hasonly two SERA genes, one from Group I and the other from a common ancestor ofGroups II and III (Plasmodium species lies in Group IV (serine-type SERAgene). Likewise, while orthologous relationship of SERA genes in Groups I to IIIwere readily identified from sequence similarity and phylogenetic analysis gene and the iron-sulfur assembly protein gene (hesB).Except  and III . Thus, tanalysis, the relThe SERA gene family has a common exon/intron structure: four exons and threeintrons, with few exceptions. SERA genes of Group I have six exons and fiveintrons structure, except for Pfa-SERA8 and Pvi-SERA12, which lack one intron.Other exceptions are detailed in Amino acid sequence alignments reveal the consensus primary structure of SERAgenes -Xaa-(Gly/Ala)-Paa,in which Xaa is any amino acid residue and Paa is a non-polar residue except forLeu zed here .In the zed here .DegenerP. vivax and relatedmonkey malaria parasite lineage. In P. falciparum andP. reichenowi, sequences are highly conserved in alldomains of Groups I to IV, except for Group IV variable domains 1 and 2. Inrodent parasite lineage, sequences are somewhat variable in the two variabledomains of Groups I to IV, but relatively conserved in both the putativepro-enzyme and enzyme domains. In P. vivax and related monkeyparasites, putative pro-enzyme and putative enzyme domains remains relativelyconserved compared to two variable domains whose sequence variations are veryhigh. Thus, overall, although putative pro-enzyme and putative enzyme domainsseem fairly conserved, the extent of variation in variable domains 1 and 2differs greatly among parasite lineages.Amino acid sequence variations of Groups I to IV SERA genes varied greatly amongparasite lineages , with thPlasmodium species, using 392 unambiguouslyaligned amino acid sites (The maximum likelihood (ML) phylogenetic tree was constructed from 134 SERA genesrepresenting 18 id sites . The besP. falciparum and P.reichenowi, (ii) rodent Plasmodium species, (iii)P. ovale, (iv) P. malariae and (v)P. vivax and P. vivax-related monkeyparasite species  were further categorizedinto six orthologous gene groups (Clade 1 to Clade 6), except for Pgo-SERA2 andPvi-SERA7 (P. fragile) to 12(P. vivax). Orthologous relationships of the SERA genes andtheir locations are shown in P. vivax and relatedmonkey malaria parasites had at least 6 SERA genes of Group IV, followed by geneduplications and gene deletions in each lineage. Pgo-SERA2 and Pvi-SERA7 have noorthologous genes. Pgo-SERA2 was located at the earliest branching position inGroup IV SERA genes of P. vivax and related monkey malaria parasites.Group IV SERA genes of vi-SERA7 . ThenumRA genes . An orthRA genes , which iP. berghei, and monkey parasitesP. knowlesi, P. cynomolgi and P.coatneyi. The amount of each SERA gene transcript is presentedrelative to that of β-tubulin in P. berghei,Pbe-SERA3 (Group III SERA gene) was predominantly expressed followed to a lesserextent by other SERA genes except for SERA 5 that did not show detectableexpression. In three monkey parasites, the abundantly expressed genes aremembers of Group IV Clade 3: Pcy-SERA3 and Pcy-SERA5, Pco-SERA3 and Pkn-SERA2.Other SERA genes of Group IV, except Pcy-SERA1 and Pco-SERA1, were alsoexpressed to varying degrees. All Group III SERA gene expression was evident;whereas, no expression was observed for all Group I SERA genes. From above, SERAgenes were differently expressed between rodent and primate parasites, with theexception of Group I SERA genes, which were not expressed at the blood stageparasites of both P. berghei and three monkey parasites.Transcription of SERA genes was analyzed for the late trophozoite to schizontstages of the rodent parasite, PlasmodiumSERA gene family . Following divergence of mammalian malaria parasite species, alineage leading to P. falciparum and P. reichenowi(primate parasite group1) had 5– 6 SERA genes of Group IV by multiple geneduplications. In the lineage leading to three rodent parasites, a Group IV SERA genewas duplicated. In lineages of primate parasite group2 , gene duplications of Group IV SERA gene took place ineach parasite lineage resulting to 4 SERA genes in P. ovale, 7 SERAgenes in P. malariae and 2–9 SERA genes in P.vivax and related monkey parasites. Simultaneously, gene deletions aswell as pseudogenization and truncation also took place in Group IV SERA genes ofP. vivax and related monkey parasites. It is notable that geneduplication events occurred only in Group IV SERA genes in mammalian parasites andthis duplication was particularly frequent in parasite species that infect humans,apes and monkeys.This study presents an overview of the evolution of the e family .Since tPlasmodium SERAgenes is in concordance with the birth-and-death model, though traits of geneconversion are detected in a few Group IV SERA genes of limited parasites. Thebirth-and-death model also has been recently proposed for gene duplication/genedeletion of msp-7, an immune target parasite surface antigen gene Plasmodium SERA multigenefamily was, likewise, driven by the birth-and-death evolution.In general, duplicated genes undergo either (i) concerted evolution or (ii)birth-and-death evolution P. vivax and related monkey malariaparasites. The reason for this lineage-specific evolutionary event is presentlyunknown. We consider that the gene duplication/gene deletion may be associated withan expansion of host range during the radiation of macaques that took place about4–5 million years ago in Asia P. vivaxrelated monkey parasites generally infect multiple hosts. For example, P.knowlesi and P. cynomolgi have the ability to infect awide variety of macaques and humans P.vivax, which became a human parasite by host switch It is worth noting that gene duplication/gene deletion of serine-type Group IV SERAgenes occurred primarily in P. falciparum and P. berghei, many SERA genescould be disrupted without any obvious phenotypic change and appear to benon-essential. If non-functional, the birth-and-death evolution model assumes thatthese genes could have been gradually deleted or become pseudogenes. But manyPlasmodium SERA genes have sequence similarity with each othereven in different species and, moreover, transcription and/or translation can bedetected in the different family members. These observations suggest that some SERAgene family member may play a role in the parasite life cycle. Titers ofanti-Pfa-SERA5 IgG antibodies show a strong negative correlation with malariasymptom P. malariae, P.ovale and P. falciparum/P. reichenowi. Recent findingsof infections of these parasites as well as new parasite species in Great Apes indicate that these parasites show a wider hostrange than previously thought In P. falciparum and P.berghei, shed some light on the importance of SERA proteins to malariaparasites. The presence of Group I SERA gene in all 18 Plasmodiumspecies examined suggests that the gene is maintained by Plasmodiumfor its function. Disruption of P. berghei Group I SERA (Pbe-SERA5)blocked parasite development at the mosquito stage by inhibiting egress ofsporozoites from an oocyst P. berghei and three P. vivax-related monkeyparasites. Group II SERA gene is present in all Plasmodium species,except P. gallinaceum. This group is also expressed, as evidentfrom the four parasites used for expression analysis and, likewise, from studies onGroup II SERA genes of P. falciparumP.vivaxP. falciparum Pfa-SERA7 and P. bergheiPbe-SERA4 (Group II SERA genes) were nonlethal . All 17 mammalian Plasmodium species have GroupIII SERA gene; and disruption of Group III SERA genes (Pfa-SERA6 and Pbe-SERA3) hasbeen unsuccessful in both P. falciparum and P.bergheiP. vivax-related monkey parasites suggests that Group IIISERA gene is essential to all mammalian Plasmodium at the bloodstage.The present evolutionary and experimental (expression) analyses, when coupled withprevious gene disruption studies using P. falciparum and P.reichenowi, most SERA genes of Group IV are orthologous. Pfa-SERA5 hasbeen shown to be essential for parasite survival P. berghei,simultaneous disruption of the two SERA genes does not affect parasite growth P.vivax and related monkey parasites, Group IV SERA genes are furthercategorized into six clades. In Clade 5, all ten species have one SERA gene and inthe P. vivax ortholog, Pvi-SERA8, expression has been detectedP. fragile have one to three SERA genes in Clade 3. TwoP. vivax orthologs, Pvi-SERA4 and Pvi-SERA5 have been shown tobe abundantly expressed P. vivax-related monkey parasites. These suggest that SERA genes of Clade3 and 5 likely play an important role at the blood stage. The substantial expressionof SERA genes observed for other clades in the three monkey parasites as well as inP. vivaxIn contrast to Groups I to III, Group IV includes multiple SERA genes in allmammalian parasites. Contrary to clear orthologous relationship of Group I to IIISERA genes, orthologous relationship in Group IV was not clearly seen across allmammalian parasites, although relationship was identified in mammalian parasitesub-groups . ThissuPlasmodium SERA gene family. The gene family was incipientlyborn in a common ancestor of the genus Plasmodium. Geneduplications during the parasite evolution generated two types of SERA genes, thecysteine-type SERA genes (Groups I to III) and the serine-type SERA genes (GroupIV). Of note is that in mammalian malaria parasites, gene duplication occurred onlyin Group IV SERA genes, particularly frequent in primate parasites. Diversificationof duplicated SERA genes supports the birth-and-death evolution in this gene family.It is intriguing to assume that duplications of SERA genes were associated with theparasite's expansion of host range. This study thus points to unique featuresof the Plasmodium SERA gene family and reinforces the importance ofinvestigating other uncharacterized gene families of Plasmodium tofurther understand the evolutionary history and biology of this parasite.In conclusion, this study presents an overview of the evolution of thePlasmodium SERA gene family were determinedfor the following eleven parasite species: P. malariae,P. ovale, P. gonderi, P.fragile, P. coatneyi, P.knowlesi, P. inui, P. fieldi,P. simiovale, P. cynomolgi, and P.hylobati . The extension time, y(min), was set at 1 min per 1 kb. Amplified fragments were cloned into pCR BluntII TOPO vector or pCR XL TOPO vector (Invitrogen). Fragments amplified usingKOD-FX were dA-tailed with A-attachment mix (TOYOBO) before TA-cloning. DNAsequencing was conducted on a 3130 Genetyx Analyzer . SERA gene sequences reported in this study were deposited in DDBJ,with accession numbers AB576870-AB576881  trees were constructed using PROML programs in PHYLIPversion 3.69 P.berghei (ANKA) and from Japanese macaques, Macacafuscata, infected with P. coatneyi (CDC),P. knowlesi (H) or P. cynomolgi (B). Bloodwas taken several times with 4 to 12 hour intervals and parasites at the latetrophozoite stage were selected for transcription analyses.Parasitized erythrocytes were obtained from mice infected with Studies using mice were approved by Animal Care and Use Committee of GunmaUniversity and conducted in compliance with guidelines (Permit ID: 10-007). Theexperimental monkeys were second-generation offspring bred in captivity. Theinvestigators adhered to the Guidelines for the Use of Experimental Animalsauthorized by the Japanese Association for Laboratory Animal Science. Theprotocol was approved by the Committee on Ethics of Animal Experiments at DokkyoUniversity School of Medicine (Permit Number: 0536). All procedures wereperformed under anesthesia by a combination of ketamine hydrochloride  and xylaxine , and all efforts were made to minimizesuffering. The details of animal welfare/care and steps taken to amelioratesuffering were in accordance with the recommendations of the Weatherall report,“The use of non-human primates in research”.P. cynomolgi-SERA1 gene and P.coatneyi-SERA1 gene, these were excluded from further analysis.Total RNA was isolated by RNeasy Mini kit (QIAGEN) according to themanufacture's protocol. First strand cDNA was synthesized with SuperscriptIII First-strand System for RT-PCR (Invitrogen) using 20 µg of total RNA.To confirm SERA gene transcriptions, PCR amplifications were performed usingsynthesized cDNA and specific primers for each SERA gene. Since no PCR productcan be obtained for Real time quantitative PCR was performed using ABI PRISM 7900 (AppliedBiosystems), and results were analyzed with the SDS software version 2.2(Applied Biosystems). A 15 ul mixture was formulated with 7.5 ul of TaqMan GeneExpression Master Mix (Applied Biosystems), an appropriate volume of firststrand cDNA, 3 pmol of forward and reverse primers, and 0.5 pmol TaqMan probe.Sequences of primers and probes are shown in Figure S1The SERA gene family drawn to scale in17Plasmodiumspecies. The 5′-end of each gene map is set at the startcodon position of the conserved hypothetical protein gene, whosetranscriptional direction is opposite to that of SERA genes. SERA genes ofGroups I to III (cysteine-type) and those of Group IV (serine-type) areshown in green and blue, respectively. TSERA denotes truncated SERA genesshown in yellow. SERA gene fragments (putative pseudogenes) are in gray. Theregion that was not successfully sequenced in P. gonderi isindicated by a double slash [P. reichenowi SERA genesin this region are not shown due to the lack of completesequences].(EPS)Click here for additional data file.Figure S2Amino acid sequence alignments of 134Plasmodium SERAgenes. Amino acid sites used for constructing phylogenetic treesfor (DOC)Click here for additional data file.Figure S3The maximun likelihood phylogenetic tree of thePlasmodium SERA genefamily. This unrooted tree was constructed from 134 SERA genesusing 392 amino acid sites under the JTT and Γ (8 catogories) model(α = 0.96) with 500 heuristic replicates. Bootstrapvalues >50% are shown along nodes. Groups I-III and Group IV arecysteine-type and serine-type protease SERA genes, respectively.(EPS)Click here for additional data file.Figure S4Amino acid sequence alignments of Group IV Clade 6 SERA genes ofP. vivax andP. vivax-related monkeymalaria parasite species. The catalytic serine residue and otheractive site residues are shaded in green and yellow, respectively. Asterisksdenote stop codons. Putative PfSUB1 recognition motifs are shaded in blue.The long deletion in truncated SERA genes of P. coatneyi,P. knowlesi and P. fragile arehighlighted in black.(DOC)Click here for additional data file.Figure S5Sequencing strategy for the SERA gene family in 11Plasmodiumspecies. Thick bars indicate conserved protease domain of SERAgenes targeted for PCR amplification and sequencing. Primer names are givenin either ends of bars, and their sequences are listed in P.gonderi is shown by a double slash. SERA genes of Group I toIII (cysteine-type SERA gene) and those of Group IV (serine-type SERA gene)are shown in green and blue, respectively. TSERA denotes truncated SERA geneshown in yellow. SERA gene fragments are shown in gray. In this study, the Hstrain of P. knowlesi (ATCC 30158) is different from the Hstrain used for the genome sequencing (EPS)Click here for additional data file.Table S1PCR Primers used.(PDF)Click here for additional data file.Table S2SERA gene accession numbers in PlasmoDB database.(PDF)Click here for additional data file.Table S3Primers and probes used for real time PCR.(PDF)Click here for additional data file.


Burkholderia ubonensis is an environmental bacterium belonging to the Burkholderia cepacia complex (Bcc), a group of genetically related organisms that are associated with opportunistic but generally nonfatal infections in healthy individuals. In contrast, the near-neighbour species Burkholderia pseudomallei causes melioidosis, a disease that can be fatal in up to 95% of cases if left untreated. B. ubonensis is frequently misidentified as B. pseudomallei from soil samples using selective culturing on Ashdown’s medium, reflecting both the shared environmental niche and morphological similarities of these species. Additionally, B. ubonensis shows potential as an important biocontrol agent in B. pseudomallei-endemic regions as certain strains possess antagonistic properties towards B. pseudomallei. Current methods for characterising B. ubonensis are laborious, time-consuming and costly, and as such this bacterium remains poorly studied. The aim of our study was to develop a rapid and inexpensive real-time PCR-based assay specific for B. ubonensis. We demonstrate that a novel B. ubonensis-specific assay, Bu550, accurately differentiates B. ubonensis from B. pseudomallei and other species that grow on selective Ashdown’s agar. We anticipate that Bu550 will catalyse research on B. ubonensis by enabling rapid identification of this organism from Ashdown’s-positive colonies that are not B. pseudomallei. Burkholderia spp. comprise an ecologically diverse group containing over 70 species (http://www.bacterio.cict.fr/b/burkholderia.html), some of which are pathogenic to humans, animals or plants Burkholderia pseudomallei is the best-known member of the genus due to its ability to cause the potentially fatal disease melioidosis B. pseudomallei was recently added as a Tier 1 Select Agent in the United States, a category that includes those organisms of greatest threat to human and animal health. B. pseudomallei is commonly recovered in the environment in northern Australia (particularly the “Top End“ of the Northern Territory) and north-eastern Thailand, but has also been described from a much wider endemic region including most other countries in Southeast Asia, the Indian subcontinent, Taiwan, southern China and Hong Kong B. pseudomallei in Africa and the Americas has also been described but the extent of its distribution remains unclear Burkholderia spp. reside in ecological niches where B. pseudomallei is present, and some of these species can also cause opportunistic, albeit less serious, infections in humans. Many of these species fall into the Burkholderia cepacia complex (Bcc), which contains at least 17 Burkholderia species, including Burkholderia ubonensisThe Gram-negative Burkholderia spp. has implications for environmental studies, clinical diagnosis and biosecurity responses B. pseudomallei, where false-negative and false-positive results may have serious consequences. Species misidentification can have an economic impact, as demonstrated by false-positive calls of near-neighbour species under the BioWatch program, which was introduced in the United States in 2003 to monitor aerosol samples for the presence of Select Agent organisms in the environment B. pseudomallei from clinical samples is also a nontrivial endeavour. Most hospital laboratories use standard culture media  for culturing of clinical specimens. Morphological identification of B. pseudomallei in non-endemic areas is therefore difficult due to unfamiliarity, a lack of selective media available for identification B. pseudomallei using automated systems such as VITEK 2 B. pseudomallei is typically enriched from environmental specimens using broth selection B. pseudomallei-specific. Indeed, many Burkholderia spp. residing in the same niches as B. pseudomallei, including B. ubonensis, are morphologically similar on ASA Misidentification of Burkholderia uboniae” species was first proposed in 2000 B. ubonensis, despite being a potentially important biocontrol agent for B. pseudomalleirecA and 16S sequencing have been developed for Burkholderia spp. characterisation B. ubonensis from other Burkholderia spp. including B. pseudomallei. For example, the type III secretion system 1 (TTS1) assay B. pseudomallei, and thus cannot further identify other species that grow on ASA. Therefore, the major aim of our study was to differentiate B. ubonensis from B. pseudomallei, with a secondary aim of differentiating B. ubonensis from other members of the Bcc and non-Burkholderiaecae organisms that also grow on ASA.Since the “The Australian isolates used in our study were obtained from either private land or from Aboriginal communities. Prior to private land soil and water sampling, we obtained signed or verbal permission from land owners. Sampling permits were obtained from Northern Land Council  prior to sample collection from Aboriginal communities. As per permit conditions, we obtained further permission from the community representatives prior to sampling. No specific permits were required for collection of the Thai isolates as they were obtained from unregulated public lands. Our field collection did not involve endangered or protected species.B. pseudomallei according to the TTS1 assay recA sequencing or whole-genome sequencing (WGS) as part of this and other studies to confirm genus and, where possible, for species assignment. All isolates were subcultured for purity on chocolate agar or ASA  prior to DNA extraction. The Qiagen DNeasy kit  was used for DNA extraction as previously described 2O prior to PCR.Our laboratories have ongoing collections of isolates from soil and water samples obtained from both the Northern Territory and Thailand, comprising isolates that grow on ASA B. pseudomallei near-neighbour isolates were subjected to WGS: Burkholderia spp. MSMB175, Burkholderia spp. MSMB49, B. cenocepacia MSMB101, B. cenocepacia MSMB139, Burkholderia multivorans MSMB104, B. multivorans MSMB105, Burkholderia oklahomensis C6786, B. pseudomallei MSHR684, B. pseudomallei MSHR1079, Burkholderia thailandensis-like strain MSMB121, B. thailandensis MSMB59, B. ubonensis MSMB56, B. ubonensis MSMB106, B. ubonensis MSMB108, B. ubonensis MSMB145, B. ubonensis MSMB157, B. ubonensis MSMB166, B. ubonensis MSMB169 and B. ubonensis MSMB170. The Illumina GAIIx platform  was used to generate WGS data. An assembly of B. ubonensis MSMB170 was performed on paired-end Illumina v1.9 reads with Velvet v1.2.07 B. ubonensis-specific loci were identified by locating regions with 100% read coverage in all eight B. ubonensis strains but with <50% coverage in other Burkholderia species. Eleven candidate loci ≥5 kb were identified. One locus, Bu550, was chosen for real-time PCR assay design following confirmation of in silico specificity for B. ubonensis using Microbial Nucleotide BLAST.Nineteen B. ubonensis-specific 7 kb locus. Four putative protein products are encoded within this locus; a major facilitator superfamily transporter (GenBank ID: WP_010089641), a hypothetical protein (WP_010089640), a carbamoyltransferase (WP_010089639) and a tannase (WP_010089638). Unlabelled primers Bu550-F (5′-ATGCCGTGATCGACAACGAT) and Bu550-R (5′-ACTCCAGAAACAGTTCAGGCGT)  were used to amplify a conserved 91-bp fragment within this locus. A Black Hole Quencher (BHQ) probe  was included to increase specificity. BLAST analysis was conducted on the primers and probes to ensure assay specificity and accuracy. Real-time PCR was performed in 384-well optical plates . Each 5 µL reaction contained 0.3 µM of each primer, 0.2 µM of probe, 1X TaqMan Environmental Master Mix (Applied Biosystems) and 1 µL genomic DNA, to a total reaction volume of 5 µL. We also tested 1X TaqMan Universal Master Mix (Applied Biosystems) to determine assay robustness across different mastermixes. The 306 isolates used in this study  was developed to target a candidate is study  were tesB. pseudomallei from clinical and environmental isolates in endemic regions, and is commercially available in Australia. The isolates examined in our study contain a cross-section of isolates that grow on ASA but are TTS1-negative B. pseudomallei. Several species  possessed morphologies . Nucleotide BLAST analysis (http://blast.ncbi.nlm.nih.gov/) of 9,404 complete or draft microbial genomes confirmed specificity of this locus, with only a single significant hit occurring in B. ubonensis Bu .Comparative whole-genome analysis of 19 B. ubonensis-specific locus, encoding the hypothetical protein BuboB_03639. We chose BHQ probe technology due to its comparatively low cost compared with conventional TaqMan minor groove-binding probes (Applied Biosystems). In addition, the Bu550 assay can be multiplexed in a single PCR tube with the existing B. pseudomallei TTS1 BHQ assay B. ubonensis and B. pseudomallei isolates  probe-based real-time PCR assay isolates . In tota amplify ; Table 1Burkholderia ubonensis is now known to be an abundant environmental bacterium in northern Australia. Both regions are also endemic for B. pseudomallei, a pathogenic bacterium that causes significant morbidity and mortality, with up to 50% of infected individuals succumbing to disease B. pseudomallei from clinical and environmental specimens. However, ASA also supports the growth of other Burkholderia spp., several other Gram-negative bacteria and even some gentamicin-resistant strains of Gram-positive Staphylococcus spp. (B. pseudomallei species, we showed that B. ubonensis is the most frequently isolated in northern Australia due to its strong morphological resemblance to B. pseudomallei. Like B. pseudomallei, B. ubonensis possesses several morphotypes on ASA (B. pseudomallei morphotypes B. ubonensis from B. pseudomallei based on morphological characteristics alone.Originally identified from soil collected in Ubon Ratchathani province, Thailand, in 1989 cus spp. . Of thess on ASA , many ofB. ubonensis and B. pseudomallei based on morphology, we developed a cost-effective, rapid PCR-based method for differentiating B. ubonensis from other soil- and water-borne species that grow on ASA, particularly B. pseudomallei. Current methods of characterising Burkholderia spp. rely on dideoxy sequencing Burkholderia spp., including eight B. ubonensis isolates, to identify a locus specific for B. ubonensis. The specificity of one candidate locus, 550, was confirmed using in-depth in silico BLAST analysis. We subsequently developed a cost-effective real-time PCR assay, Bu550, for its interrogation. The performance of Bu550 was validated against a diverse collection of Burkholderiaceae and other bacterial species that grow on ASA, obtained from soil and water across the Northern Territory and Thailand. Our results indicate that Bu550 is highly specific towards B. ubonensis, with all other species we tested failing to amplify.Due to the inherent difficulty in distinguishing B. ubonensis-specific BHQ probe-based assay enables multiplex capability with the B. pseudomallei-specific assay, TTS1. B. ubonensis has demonstrated antagonistic activity towards B. pseudomallei and shows promise for biocontrol of naturally occurring B. pseudomallei, particularly in areas of high endemicity B. ubonensis affect the natural prevalence of B. pseudomallei. Our future work will involve quantifying B. ubonensis and B. pseudomallei from soils obtained in B. pseudomallei-endemic regions to determine the relative abundance of these species, thereby improving our understanding of B. pseudomallei and B. ubonensis ecology. Bu550, in combination with TTS1, will be an important and valuable tool in such ecological studies.The design of the


Brain-derived neurotrophic factor (BDNF) has established physiological roles in the development and function of the vertebrate nervous system. BDNF has also been implicated in several human malignancies, including breast cancer (BC). However, the precise biological role of BDNF and its utility as a novel biomarker have yet to be determined. The objective of this study was to determine the mRNA and protein expression of BDNF in a cohort of women with BC. Expression levels were compared with normal background tissues and evaluated against established pathological parameters and clinical outcome over a 10 year follow-up period.BC tissues (n = 127) and normal tissues (n = 33) underwent RNA extraction and reverse transcription, BDNF transcript levels were determined using real-time quantitative PCR. BDNF protein expression in mammary tissues was assessed with standard immuno-histochemical methodology. Expression levels were analyzed against tumour size, grade, nodal involvement, TNM stage, Nottingham Prognostic Index (NPI) and clinical outcome over a 10 year follow-up period.Immuno-histochemical staining revealed substantially greater BDNF expression within neoplastic cells, compared to normal mammary epithelial cells. Significantly higher mRNA transcript levels were found in the BC specimens compared to background tissues (p = 0.007). The expression of BDNF mRNA was demonstrated to increase with increasing NPI; NPI-1 vs. NPI-2 (p = 0.009). Increased BDNF transcript levels were found to be significantly associated with nodal positivity (p = 0.047). Compared to patients who remained disease free, higher BDNF expression was significantly associated with local recurrence (LR) (p = 0.0014), death from BC (p = 0.018) and poor prognosis overall (p = 0.013). After a median follow up of 10 years, higher BDNF expression levels were significantly associated with reduced overall survival (OS) . BDNF emerged as an independent prognostic variable in multivariate analysis for disease free survival (DFS) (p = 0.026) and approached significance for OS (p = 0.055).BDNF expression was found to be significantly higher in BC specimens compared to normal tissue. Higher transcript levels were significantly associated with unfavourable pathological parameters including nodal positivity and increasing NPI; and adverse clinical outcomes including LR, death from BC, poor prognosis, reduced DFS and OS. BDNF offers utility as a prognostic marker and potential for targeted therapeutic strategies. BDNF) is regulated by the presence of multiple activity dependent and tissue-specific promoters  and  and NTR henotype . TrkB hahenotype . Signallhenotype . Emerginhenotype . Studieshenotype . BDNF ishenotype . BDNF hahenotype ,46. In shenotype . It has henotype . This mahenotype . In addihenotype ,50. DespNTR, and other markers of invasiveness and metastatic competence would also be of value.Limitations of the present study include the use of background parenchyma from BC patients to provide 'normal tissue' for comparison is also contentious. Ideally, such material should be derived from patients without BC in order to avoid any 'field change' which may exist within cancer bearing tissues. Although the sample size and follow-up period were substantial, it is possible that a larger cohort, particularly with regard to subgroup analysis, may have influenced several results which approached, but failed to reach, statistical significance. In addition to the measurement of mRNA transcript levels and qualitative immuno-histochemistry, quantitative analysis of protein expression should be undertaken to ensure concordance. Correlation with associated molecules, in particular TrkB and P75BDNF expression was found to be significantly higher in BC specimens compared to normal tissues. Higher transcript levels were significantly associated with unfavourable pathological parameters including nodal positivity and increasing NPI; and adverse clinical outcomes including LR, death from BC, poor prognosis, reduced DFS and OS. In addition to the prognostic utility of BDNF, further mechanistic studies are warranted to explore the potential for therapeutic manipulation in human BC.The authors declare that they have no competing interests.NP: Literature Review, Data Interpretation, Manuscript Writing & Editing. WJ: Study Design/Concept, Laboratory Methodology, Data Acquisition/Analysis. KM: Principal Investigator, Study Design/Concept, Patient Recruitment, Data Analysis. All authors read and approved the final manuscript.


Eriocheir sinensis larvae were stimulated with a mixture of three pathogen strains . A total of 44,767,566 Illumina clean reads corresponding to 4.52 Gb nucleotides were generated and assembled into 100,252 unigenes . 17,097 (26.09%) of 65,535 non-redundant unigenes were annotated in NCBI non-redundant protein (Nr) database. Moreover, 23,188 (35.38%) unigenes were assigned to three Gene Ontology (GO) categories, 15,071 (23.00%) to twenty-six Clusters of orthologous Groups (COG) and 8,574 (13.08%) to six Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, respectively. Numerous genes were further identified to be associated with multiple immune pathways, including Toll, immune deficiency (IMD), janus kinase (JAK)-signal transducers and activators of transcription (STAT) and mitogen-activated protein kinase (MAPK) pathways. Some of them, such as tumor necrosis factor receptor associated factor 6 (TRAF6), fibroblast growth factor (FGF), protein-tyrosine phosphatase (PTP), JNK-interacting protein 1 (JIP1), were first identified in E. sinensis. TRAF6 was even first discovered in crabs. Additionally, 49,555 single nucleotide polymorphisms (SNPs) were developed from over 13,309 unigenes. This is the first transcriptome report of whole bodies of E. sinensis larvae after immune challenge. Data generated here not only provide detail information to identify novel genes in genome reference-free E. sinensis, but also facilitate our understanding on host immunity and defense mechanism of the crab at whole transcriptome level.To study crab immunogenetics of individuals, newly hatched  Eriocheir sinensis, belonging to Grapsidae family of decapod crustaceans, is a catadromous species with a lifetime about two years. The crab has one reproductive season and dies shortly after reproduction [E. sinensis larvae normally experience several developmental processes, including five typical Zoea-stages (Zoea I-V) and a Megalopa-stage [E. sinensis under facility condition has started since 1980s [E. sinensis is easily to be artificially propagated and transported over long distance, which may make the species as a model organism in aquaculture studies. However, with development of intensive culture, various diseases like tremor disease (TD) and black gill syndrome (BGS) frequently occur and seriously threaten E. sinensis stocks. In particular, larvae of the species suffer from diseases more often than adult crabs. High mortalities of larvae can be easily caused by infection with microorganisms of Vibrio, Micrococcus and Fungus [Chinese mitten crab oduction . After bpa-stage . Culturece 1980s  and consd Fungus . It is cE. sinensis are performed and numerous immune related sequences are consequently obtained [E. sinensis, such as crustin [E. sinensis is still scattered and inadequate.Analysis of expressed sequence tags (ESTs) from cDNA library by Sanger sequencing method is proved to be useful for gene identification and expression profiling analysis. Several EST analyses of haemocytes and hepatopancreas from healthy obtained ,5,6. The crustin , antioxi crustin -10, anti crustin -13, prop crustin , serine  crustin ,16. Howede novo assembly of genome reference-free species [Litopenaeus vannamei [Musca domestica [Galleria mellonella [Apis cerana cerana [Newly-developed high-throughput sequencing technologies, such as Roche/454, Solexa/Illumina and ABI/SOLiD, furnish the opportunity to produce large numbers of sequence data in non-model organisms . They pr species . Roche 4 species -22. Howe species ,24. In r species ,25-29. Svannamei , Musca domestica , Gallerillonella  and Apisa cerana . These rE. sinensis hepatopancreas [E. sinensis larvae were challenged by a mixture of three pathogen strains . These pathogens represented three different types of major microbes that infected the crab and brought about serious diseases in aquaculture. The experimental analysis was expected to completely reveal sequence information, especially the important immune genes of challenged E. sinensis, which could be valuable to study crab immunogenetics and enhance crab resistance to various microorganisms. Besides, SNPs that were ready for marker development were identified in this study. The investigation might provide useful information for future studies on genetics and immunity of E. sinensis and other economic crustaceans.Previously, we have identified numerous immune-related genes from transcriptome of microbial challenged pancreas . It provAll animal treatments of the study were strictly carried out according to the Guide for Care and Use of Laboratory Animals by Chinese Association for Laboratory Animal Sciences (No. 2011-2).Healthy berried female mitten crabs were obtained from a farm in Panjin, China and cultured in aerated seawater at 18±1 °C. During whole period of the experiment, all crabs were fed with clam meat once daily at night. Egg incubation and larval hatching were carried out using the same method with Sui et al . In brieVibrio alginolyticus, Gram-positive bacteria Micrococcus luteus and fungi Pichia pastoris) were mixed and suspended in 0.1 mol/L PBS (pH 7.0) with the final pathogen concentration of 108 cfu·mL-1. Lots of small newly hatched larvae were too hard to be directly injected into the body. It might also influence crab responses by physical stimulation as well. To overcome these shortcomings, hundreds of zoea larvae were cultured in seawater containing 100 μL the mixture of pathogens. At 1h post-challenge, whole bodies of all the treated larvae were collected with mesh grid and pooled as one sample for RNA isolation and transcriptome analysis. They were immediately placed in liquid nitrogen until use.Three pathogen strains . RNA quality and concentration were determined by 1% agarose gel electrophoresis and a NanoDrop spectrophotometer. Polyadenylated mRNA was purified from total RNA using oligo(dT) magnetic beads and Oligotex mRNA Kits (Qiagen). They were fragmented by treating with heat and divalent cations before cDNA synthesis. The cDNA was reverse transcribed with random hexamer primers, end repaired by DNA polymerase and adapter ligated with T4 DNA ligase, according to Illumina manufacturer’s protocol.Ligated products were PCR-amplified and sequenced from both 5' and 3' ends on an Illumina HiSeq 2000 platform. Raw data of Illumina sequencing were obtained after base calling and stored in fastq format. Cleaning steps of the raw reads were as follows: (1) trimming adapter sequences; (2) removing the reads that contain ambiguous ‘N’ nucleotides over 10%; (3) filtering the reads with more than 50% bases having a quality score lower than 5. All subsequent analyses were based on the remaining clean reads.De novo assembly of full-length transcripts (sinensis) using Blast program with an E-value threshold of 1E-5.nscripts  was perfnscripts . The k-mE-value cut off: < 10-10) with public sequences in NCBI non-redundant protein (Nr), non-redundant nucleotide (Nt) databases (http://www.ncbi.nlm.nih.gov/), and UniProtKB/Swiss-Prot sequence database (http://www.ebi.ac.uk/uniprot/). Gene encoding protein domains were identified by searching against Protein Family (Pfam) database (http://pfam.janelia.org/) by means of hmmpfam program. Moreover, Gene Ontology (GO) (http://www.geneontology.org/) categorization was done with Blast2go program and WEGO software. Clusters of Orthologous Groups (COG) (http://www.ncbi.nlm.nih.gov/COG/) based analysis was then conducted to predict gene functions according to the known orthologous products. Using Enzyme Commission (EC) terms, biochemical pathway information was collected by downloading relevant maps from Kyoto Encyclopedia of Genes and Genomes (KEGG) (http://www.genome.jp/kegg/). Both COG and KEGG classifications were also performed using Blast algorithm.After being compared with NCBI EST dataset, transcriptomic dataset was analyzed using an established approach. Briefly, assembled unigenes were annotated using Blastx algorithm . Totally 46,099,408 raw reads were obtained from whole bodies of microbial challenged s larvae . After es larvae . Remainis larvae . Assembls larvae . After eE. sinensis. A total of 16,987 ESTs were downloaded from NCBI Genebank, of which 87.07%  were matched to transcriptome unigenes. However, only 9.49%  unigenes could be matched to NCBI ESTs  had an E-value from 1E-10 to 1E-50, while 10.24%  with the E-value to be zero  unigenes were annotated in Nt database and 14,481 (22.10%) in Swiss-Prot database . UnigeneAssembled non-redundant unigenes were also subjected to GO, COG and KEGG databases for blast searching. Summary statistics of them were shown in GO is an international standardized gene functional classification system to comprehensively describe characteristics of different genes and their products. In this study, 23,188 unigenes were categorized by GO analysis . Second-COG database is a database in which orthologous gene products are classified. To further evaluate the completeness of our transcriptome library and the effectiveness of the annotation process, annotation of COG were selected and 15,071 unigenes were clustered in different processes . Five laKEGG pathway-based analysis facilitated systematical study on complicated metabolic pathways and biological behaviors of functional molecules. Thousands of unigenes were consequently classified into specific pathways , among wUsing the transcriptome data as references, immune relevant genes, metabolic and signaling pathways were analyzed to gain deep insight into immune system of the crab. As shown in Drosophila melanogaster, shrimps and other relative species [Well-studied signaling pathways involved in innate immunity are Toll pathway and IMD pathway, which actively participate in anti-bacterial processes. In the study, we found many key components of the two pathways, referring to the knowledge in  species -38. Memb species .Different members of Jak-Stat pathway and MAPK pathway were detected based on reference information of KEGG mapping. Major effectors involved in Jak-Stat pathway were cytokines, cytokine-receptors (CytokineR), JAK and STAT . Their dPutative SNPs were screened following specific criteria according to base quality score, read depth and minor allele frequency . With these criteria, 49,555 putative SNPs were identified from 13,039 assembled unigenes , which wTo analyze sequence variants of immune genes, 176 candidate SNPs from 38 unigenes were found to be involved with the four mentioned immune pathways . The numDaphnia pulex is sequenced in Crustacea [E. sinensis. In this study, whole bodies of E. sinensis larvae after microbial challenge are used for the first time to analyze E. sinensis transcriptome and discover immune functional genes.Knowledge of genetic information is essential for aquaculture management and sustainable development of crustacean fisheries. However, only genome of rustacea . Lack ofE. sinensis have been performed from single organ and tissue of the crab [E. sinensis larvae and contains fuller transcriptional genes of the organism. It largely enriches transcriptional sources of mitten crab. Transcriptome data is known to be the completed RNA transcripts in a cell. Characterization of transcriptome is important to explain functional complexity of genome and to understand cell activities like growth, development, disease and immune response [Previous transcriptome studies of the crab ,33,40-43response . TherefoE. sinensis by Sanger sequencing approach [E. sinensis larvae, while up to 90.51% unigenes could not matched to NCBI ESTs of E. sinensis. It implies deep coverage of E. sinensis larval transcriptome and supplies considerable gene resources of the crab. In comparison, 60.1% NCBI ESTs from muscle, blood, hepatopancreas and other organs of L. vannamei are matched to transcriptome unigenes in L. vannamei larvae, whereas 85.8% of larvae unigenes are not matched to NCBI ESTs [L. vannamei and greatly enrich transcriptional data of important economic crustaceans. Additionally, Pfam searching of protein homologous domain/motif shows many genes without blast hits in any species. It will be helpful to explore new sequences and study on them, such as molecular characterization, sequence structure analysis, expression pattern analysis and biological activity test.In detail, many ESTs are obtained from tissues like testis, haemocyte and hepatopancreas of approach ,5,40 andCBI ESTs . Our resMicrococcus luteus, Gram-negative bacteria Vibrio alginolyticus and fungi Pichia pastoris, can help to acquire abundant information of immune genes. A considerable amount of genes relating to Toll, IMD, JAK-STAT and MAPK pathways are fully and systematically characterized in our study. Most genes are also detected in infected Fenneropenaeus chinensis pleopod and E. sinensis hepatopancreas [The research shows that infection with various microbes, including Gram-positive bacteria pancreas ,33. ThosE. sinensis [E. sinensis larvae. Moreover, the molecule is recently characterized to be a new STAT3 interactor and negatively regulate activation of JAK-STAT signaling pathway [de novo sequencing and resequencing) of the genome offers another strategy to find candidate functional genes [Comparing with previous transcriptome data of sinensis , severalsinensis . It is asinensis ,46. As e pathway . All theal genes ,49. WithE. sinensis. Alternatively, it may be because of the complicated gene background of Eriocheir species or other crustaceans and their limited sequence information. Besides, there might be some differences on gene sequences of the crab and other animal species. Considering these, it is quite common that many unigenes cannot be matched. With more crab genetic information being studied and high-throughput sequencing data being applied, sequences obtained in the study will be further annotated and characterized.In this analysis, although high data output is produced, only a few unigenes have blast hits in public databases. Sequences that are not definitively annotated possibly represent genes of unknown functions in G. mellonella [Bactrocera dorsalis [Spodoptera exigua [Potential genes and pathways are also annotated in other larval transcriptome researches of arthropod species, including llonella , Bactrocdorsalis  and Spoda exigua . The patE. sinensis larval transcriptome sequences. In the analysis, SNP types of A/G and C/T are quite common and SNP densities vary among different genes. It may be partly due to the relative functional importance of individual genes and the effects of selection [E. sinensis.Another important application of high-throughput sequencing technology is to identify genetic variants. It has been well established and demonstrated using Illumina technique to detect SNP mutations -54. Our election . DiscoveMany SNPs are also derived in other crustacean species with EST sequences through traditional sequencing method ,56. Bothde novo assembly technique, we have derived a dataset from whole bodies of E. sinensis larvae after microbial challenge. This data comprised 44,767,566 clean reads and 100,252 assembled unigenes. Enormous functional genes are detected to be related with multiple immune pathways, including Toll, IMD, JAK-STAT and MAPK pathways. Some important genes, including TRAF6, FGF, PTP and JIP1, are identified in E. sinensis for the first time. Particularly, TRAF6 is even first found of existence in crabs. 49,555 putative SNPs are also identified from the transcriptome data, which are useful to marker assisted selection of new strains in E. sinensis. Collectively, this is the first transcriptome report of microbial challenged E. sinensis larvae and it will provide valuable data to research immune mechanism and molecular biological of the crab.High-throughput sequencing technology offers a powerful approach to analyze gene expression and SNP markers in genome reference-free organisms. Using Illumina platform and Table S1E. sinensis larvae.Putative immune genes involved in Toll pathway of (DOC)Click here for additional data file.Table S2E. sinensis larvae.Putative immune genes involved in IMD pathway of (DOC)Click here for additional data file.Table S3E. sinensis larvae.Putative immune genes involved in JAK-STAT pathway of (DOC)Click here for additional data file.Table S4E. sinensis larvae.Putative immune genes involved in MAPK pathway of (DOC)Click here for additional data file.


NLRP3 gene have not been investigated so far.Central nervous system (CNS) involvement is common in cryopyrin-associated periodic syndromes (CAPS), especially in children. Neurological manifestations of the low-penetrance cryopyrin mutations V198M and Q703K encoded by exon 3 of the To determine the frequency of the V198M and Q703K substitutions in adult patients with possible inflammatory CNS disease and at least two symptoms compatible with CAPS and to describe the clinical phenotype of mutation-positive patients.94 unrelated, consecutive patients with possible inflammatory CNS disease and at least two symptoms compatible CAPS were prospectively screened for the V198M and Q703K mutations. In addition, the clinical, laboratory, and MRI features of mutation carriers were assessed.NLRP3 gene . CAPS-associated systemic symptoms consisted of recurrent inflammation of the eyes, arthralgias, myalgias, urticarial rash, abdominal pain, and severe fatigue. CNS manifestation included optic nerve inflammation and/or atrophy, cranial nerve palsy, migraine, recurrent meningitis, and sensoneurinal hypacusis. Eight patients (53%) fulfilled the diagnostic criteria for multiple sclerosis (MS) according to the McDonald criteria. Brain magnetic resonance imaging (MRI) showed abnormalities in all but one patient.15 patients  were identified to carry one of the two low-penetrance mutations in exon 3 of the So far, the V198M and Q703K mutations have been only rarely described in association with MS or CNS inflammation. We observed a a surprisingly high frequency of these two low-penetrance mutations in the cohort studied, leading to a heterogeneous pattern of CNS manifestations in affected patients. Thus, molecular genetic testing should be considered in patients with an unusual CNS inflammation and/or MS, who report additional symptoms compatible with CAPS.T. Kümpfel Grant / Research Support from: TK has received grant support by Novartis Pharma, E. Schuh: None declared, R. Hohlfeld: None declared, P. Lohse: None declared


Corynebacterium ulcerans can cause a diphtheria-like illness, especially when the bacterium is lysogenized with a tox gene-carrying bacteriophage that produces diphtheria toxin. Acquisition of toxigenicity upon phage lysogenization is a common feature of C. ulcerans and C. diphtheriae. However, because of a lack of C. ulcerans genome information, a detailed comparison of prophages has not been possible between these two clinically important and closely related bacterial species.C. ulcerans 0102 isolated in Japan. The genomic sequence showed a striking similarity with that of Corynebacterium pseudotuberculosis and, to a lesser extent, with that of C. diphtheriae. The 0102 genome contained three distinct prophages. One of these, ΦCULC0102-I, was a tox-positive prophage containing genes in the same structural order as for tox-positive C. diphtheriae prophages. However, the primary structures of the individual genes involved in the phage machinery showed little homology between the two counterparts.We determined the whole genome sequence of the toxigenic tox-positive prophage in this strain of C. ulcerans has a distinct origin from that of C. diphtheriae NCTC 13129.Taken together, these results suggest that the  Corynebacterium ulcerans is increasing in clinical importance in developed countries and is now regarded as “diphtheria” in Europe [C. ulcerans occurs in a wide range of hosts, including cats, dogs, pigs, cows, and whales [C. diphtheriae, whose host species is thought to be limited to humans [tox-encoding bacteriophages, they become toxigenic and are able to produce the potent diphtheria toxin [tox, situated inside prophages lysogenized in the bacterial genome of C. diphtheriae[C. diphtheriae and C. ulcerans[C. ulcerans tox gene is also encoded in a genome region surrounded by phage attachment (att) sites conserved between the two species [C. ulcerans tox genes were published by Sing et al. They showed some diversity in the genetic sequence among C. ulcerans strains, in contrast to the highly conserved C. diphtheriae tox gene [A diphtheria-like infectious disease caused by n Europe ,2. Infecd whales -9. The fd whales . This iso humans . Neverthia toxin ,10. Thisphtheriae. The prophtheriae. Some ty ulcerans-16. Furt species ,16. The tox gene ,18.C. diphtheriae strain NCTC13129 was reported [C. diphtheriae strains [tox-positive prophage flanked by the att regions was confirmed and supported the findings of previous reports [C. ulcerans has not yet been reported. In the present study, we determined the nucleotide sequence of the toxigenic C. ulcerans isolate 0102 genome, obtained in 2001 from the pharyngeal pseudomembrane of a 52-year-old woman presenting with a sore throat and fever. This was the first toxigenic C. ulcerans infection reported in Japan. This patient had been living with nearly 20 cats before the onset of illness [tox-positive prophage because of its unexpectedly novel structure.In 2003, the nucleotide sequence of the whole genome of reported . The seqreported , uncommo reports . Despite illness . Details illness . Our anaC. ulcerans 0102, obtained short reads were assembled into five contigs by de novo assembly. Each gap was filled by direct PCR and sequencing. A circular chromosome sequence of C. ulcerans 0102 represents 2,579,188 bp, with a G + C content of 53.4%   and a nitric oxide reductase (nor) gene, respectively, instead of a prophage  and BR-AD22  [C. ulcerans 0102 shares many common features with the two previously reported strains, including 12 virulence factors. Strain 0102 is distinctive with respect to the features of prophages integrated in its genome. It possesses a unique tox-positive prophage, ΦCULC0102-I, in its chromosome  gene [C. ulcerans 0102 prophage differs from the corresponding prophage in C. diphtheriae. Although the integrase and tox gene sequences of ΦCULC0102-I showed high similarity to those of the corynephage encoding tox in C. diphtheriae NCTC 13129, the major phage machinery genes in ΦCULC0102-I are distinct from those in other corynephages in C. diphtheriae  . C. ulcetC) gene   carry tox pseudogenes that are relatively similar to the tox genes of C. ulcerans  appears to be a ‘hotspot’ for the acquisition of ΦCULC0102-I-like prophages by homologous integrase.In a recent report, whole genome sequence analysis of non-toxigenic  BR-AD22 , the β-cC. ulcerans 809 and BR-AD22 contain possible virulence factors, such as corynebacterial protease (CP40), phospholipase D (Pld), neuraminidase (NanH), venom serine protease (Vsp1), trypsin-like serine protease (TspA), Rpf interacting protein (RpfI), cell wall-associated hydrolase (CwlH), and five surface-anchored proteins (SpaB–F) [spaABCsrtA gene cluster, is considered to play a crucial role in adhesion of C. diphtheriae[spaA) was absent in C. ulcerans 0102, a feature consistent with previous findings in C. ulcerans 809 and BR-AD2 [C. ulcerans strains, can contribute to host-cell adhesion in the absence of SpaA [C. ulcerans[The whole genome sequences of (SpaB–F) . The Spaphtheriae. The gend BR-AD2 . As SpaB of SpaA , this ma ulcerans.C. ulcerans 809 strain was isolated from a patient with a rapid fatal pulmonary infection. The 809 strain-unique virulence factor  is located adjacent to the truncated integrase (CULC809_00176) and corresponds to the integrase of ΦCULC0102-I. It appears that virulence factors have been acquired as a cassette gene in the ΦCULC0102-I-like prophage. It is intriguing to note that the 0102 strain does not carry the 809 strain-unique virulence factors , but instead carries the tox gene on ΦCULC0102-I, which resulted in a diphtheria-like illness in a 52-year-old woman.The C. ulcerans are generally obtained from a diverse range of animals, including humans. Isolation of a human pathogen C. diphtheriae from animals has been reported previously, although it is rare [tox gene might be frequently transmitted through common prophages with the aid of the highly homologous regions among Corynebacterium spp., including C. diphtheriae and C. ulcerans isolated from animal sources.Isolates of  is rare . The toxC. ulcerans is an emerging pathogen that can be transmitted from animals to humans [C. diphtheriae, the tox gene [tox-positive C. ulcerans prophage with no significant sequence homology to those in C. diphtheriae. This suggests distinct origins of the prophages and thus may also explain the difference in the primary structures of their tox genes. The tox-positive bacteriophages may increase the dissemination risk of toxigenic C. ulcerans isolates, therefore, C. ulcerans isolates from both human and animal sources should be investigated further to determine the level of variation.Toxigenic o humans . In the tox gene  is encodThis research was not carried out on humans. No experimental research on animals was carried out.C. ulcerans isolate 0102 was obtained in 2001 as a human clinical isolate [The toxigenic  isolate ,23.Genomic DNA was isolated by conventional methods, using phenol extraction and ethanol precipitation from heat-killed bacterial cells propagated in brain-heart infusion liquid medium.C. ulcerans 0102 were prepared using a genomic DNA Sample Prep Kit . DNA clusters were generated on a slide using a Cluster Generation Kit (ver. 4) on an Illumina Cluster Station (Illumina), according to the manufacturer’s instructions. Sequencing runs for 80-mer short reads were performed using an Illumina Genome Analyzer IIx (GA IIx) and TruSeq SBS kit v5. Fluorescent images were analyzed using the Illumina base-calling pipeline RTA2.6/SCS2.8 to obtain FASTQ-formatted sequence data.DNA libraries of the ~600 bp insert length of The 80-mer reads were assembled  using ABySS-pe v1.2.0 [easyrun Perl-command [SfiI  and electrophoresed in a CHEF DRII apparatus  at 11°C with a pulse time of 5–20 s for the first 20 h and 1–5 s for the following 18 h.To validate the genome sequence, 40–mer short reads were re-aligned with the sequence using Maq software (ver. 0.7.1) and the -command . Read al-command . PFGE an-command . Bacteri-command , embeddehttp://www.ncbi.nlm.nih.gov/genomes/static/pipeline.html). Several of the suggested errors were revised manually. Pseudogenes that were identified by PGAAP were checked using the read-mapping correction described above. Genomic information, such as nucleic acid variations and circular representation, was analyzed using IMC-GE software . A BLASTN homology search [C. pseudotuberculosis FRC41 (accession no. NC_014329), C. ulcerans 0102, and C. diphtheriae NCTC 13129 (accession no. NC_002935). Aligned images of the homologous regions were visualized with the ACT program [Gene prediction from the complete sequence was performed using the NCBI Prokaryotic Genomes Automatic Annotation Pipeline  software was used to display the generated tree.Phylogenetic analyses of all nucleotide sequences were conducted using the neighbor-joining method with 1,000-times bootstrapping in ClustalW2 . FigTreeC. ulcerans 0102 strain has been deposited in the DNA Data Bank of Japan .The complete chromosome sequence for the The authors declare that they have no competing interests.TS and FT carried out the genome sequencing studies, participated in the sequence alignment and drafted the manuscript. TKo carried out maintenance, quality control and propagation of the bacterial strain for genome sequencing. AY and TKe participated in the design of the study. MT and KS conceived of and participated in coordination of the study, respectively. MK and MI coordinated the study, and drafted and finalized the manuscript. All authors read and approved the final manuscript.C. ulcerans 0102 genome. From the outside inward, the outer circle 1 indicates the size in base pairs (Mb). The red bars on Circle 2 show prophage region. Circles 3 and 4 show the positions of CDS transcribed in clockwise and anticlockwise directions, respectively. The dark blue bars on circle 5 indicate ribosomal DNA loci. Circle 6 shows a plot of G + C content (in a 20 kb window). Circle 7 shows a plot of GC skew .Circular representation of the Click here for fileC. ulcerans 0102 with four restriction enzyme digestions.PFGE analysis of Click here for filerpoB gene region among Corynebacterium isolates with 1,000-fold bootstrapping. Scale bar indicates number of substitutions per site. The number at each branch node represents the bootstrapping value. GenBank accession nos. given in parentheses.Jukes-Cantor-derived phylogenetic tree based on the partial Click here for fileC. ulcerans 0102 and C. diphtheriae NCTC 13129. The red characters show regions annotated as tRNAArg.Alignment of the nucleotide sequences of attachment site common regions among Click here for fileCorynebacterium spp. using the Neighbor-joining method with 1,000-fold bootstrapping. Scale bar indicates number of substitutions per site. The number at each branch node represents the bootstrapping value. GenBank accession nos. given in parentheses.Phylogenetic tree based on the tox genes among toxgenic and nontoxigenic Click here for file


Columba livia f. domestica) reported in Germany and the United States. It is caused by the apicomplexan parasite Sarcocystis calchasi which is transmitted by Accipter hawks. In contrast to other members of the Apicomplexa such as Toxoplasma and Plasmodium, the knowledge about the pathophysiology and host manipulation of Sarcocystis is scarce and almost nothing is known about PPE. Here we show by mRNA expression profiling a significant down-modulation of the interleukin (IL)-12/IL-18/interferon (IFN)-γ axis in the brains of experimentally infected pigeons during the schizogonic phase of disease. Concomitantly, no cellular immune response was observed in histopathology while immunohistochemistry and nested PCR detected S. calchasi. In contrast, in the late central-nervous phase, IFN-γ and tumor necrosis factor (TNF) α-related cytokines were significantly up-modulated, which correlated with a prominent MHC-II protein expression in areas of mononuclear cell infiltration and necrosis. The mononuclear cell fraction was mainly composed of T-lymphocytes, fewer macrophages and B-lymphocytes. Surprisingly, the severity and composition of the immune cell response appears unrelated to the infectious dose, although the severity and onset of the central nervous signs clearly was dose-dependent. We identified no or only very few tissue cysts by immunohistochemistry in pigeons with severe encephalitis of which one pigeon repeatedly remained negative by PCR despite severe lesions. Taken together, these observations may suggest an immune evasion strategy of S. calchasi during the early phase and a delayed-type hypersensitivity reaction as cause of the extensive cerebral lesions during the late neurological phase of disease.Pigeon protozoal encephalitis (PPE) is an emerging central-nervous disease of domestic pigeons ( Sarcocystis calchasi is an apicomplexan parasite and the causative agent of pigeon protozoal encephalitis (PPE), an emerging neurological disease of the domestic pigeon (Columba livia f. domestica) . No . No 5]. S. calchasi merozoites, schizonts and tissue cysts containing bradyzoites (data not shown). A positive labelling for S. calchasi was observed in the cerebrum of pigeons of group A and B. Four of eight pigeons of group A had rare schizonts in the neuropil . Three of seven pigeons of group B had few, randomly distributed tissue cysts located in areas of the neuropil not associated with pathological lesions or inflammatory cell reactions . All reference animals were negative for S. calchasi by immunohistochemistry.Immunohistochemical testing revealed that the anti-merozoite antibody is capable of labelling l Figure . Mean scAreas with cerebral lesions including necrosis and gliosis as well as areas with mononuclear cell infiltration had strong MHC-II labelling in all pigeons of group B Figure . Most moS. calchasi in the brains of all pigeons of group A and all but one (pigeon no. 14) of group B  positive horses [Sarcocystis spp. in general may exhibit an immune evasion strategy that disrupts IFN-γ signaling. Since the Th1-biased immune response is of major importance in clearing infection with T. gondii and N. caninum [T. gondii[T. gondii[S. calchasi infection. This temporary immune suppression may, similar to T. gondii[S. calchasi to infect host cells and to replicate in the absence of a protective immune response.Compared to closely related members of the Apicomplexa such as  in mice . IFN-γ i in mice . While Itibodies . Furthertibodies , while atibodies . There ie horses . Isolatee horses  and the e horses . Togethe caninum ,31, a baT. gondii,18. FurtT. gondii. IL-15 aT. gondii. InteresT. gondii, facilitIn contrast to the first phase, the second neurological phase of disease is associated with a massive mononuclear cell infiltration and characterized by a markedly up-modulated IFN-γ expression. Notably, IL-12 and IL-18 as important inducers of IFN-γ were not up-modulated. IFN-γ is the key cytokine for the activation of mononuclear cells. This correlates well with the prominence of T-cells, the granulomatous character of the lesions and prominent MHC-II signaling in the pigeon brains. Chicken IFN-γ increases the expression of class II MHC on antigen presenting cells and directly activates macrophages and natural killer cells which can excrete further inflammatory effector molecules such as TNF-α that may destroy tissue. -36. SincS. calchasi depict neurological signs such as incoordination about eight weeks after infection when tissue cysts mainly located in skeletal muscles contain infectious bradyzoites [Sarcocystis spp. (i.e. S. neurona) depict neurological signs associated with encephalitis only during the schizogonic phase which does not allow transmission to a final host [Microtus arvalis) and lemmings (Dicrostonyx richardsoni) infected by birds of prey-transmitted Sarcocystis cernae and Sarcocystis rauschorum, respectively, have been suggested to enhance parasite transmission due to increased predation rates [S. calchasi may be regarded as a parasite’s adaptation to enhance its fitness or simply as side effect due to a delayed-type hypersensitivity reaction requires further investigation to meet the manipulation hypothesis [Pigeons infected with dyzoites . This alnal host ,9,37,38.on rates ,40. Howepothesis .S. calchasi development is down-modulated in the intermediate host. The absence of a strong host’s cellular immune response in the pigeon may facilitate parasite evasion during acute disease and subsequent formation of tissue cysts. The results of this study further suggest that during the late central nervous phase of PPE a T-cell mediated delayed-type hypersensitivity reaction may cause the cerebral lesions.In conclusion, the observations of this study suggest that the Th-1 immune response during the schizogonic phase of the The authors declare that they have no competing interests.Conceived and designed the experiments: PO, AM, ML, AG. Performed the experiments: PO, AM. Analyzed the data: PO, AM, RK, BK. Contributed reagents/ materials/ analysis tools: PO, ML, BK, AG. Wrote the paper: PO. All authors have read and approved the final manuscript.


Wolbachia often acts as a subtle parasite that manipulates insect reproduction, resulting potentially in reproductive isolation between host populations. Whilst distinct Wolbachia strains are documented in a group of evolutionarily closely related mosquitoes known as the Culex pipiens complex, their impact on mosquito population genetics remains unclear. To this aim, we developed a PCR-RFLP test that discriminates the five known Wolbachia groups found in this host complex. We further examined the Wolbachia genetic diversity, the variability in the coinherited host mitochondria and their partitioning among members of the Cx. pipiens complex, in order to assess the impact of Wolbachia on host population structure.The maternally inherited bacterium Wolbachia and mitochondrial haplotypes indicating a stable co-transmission in mosquito populations. Despite evidence that members of the Cx. pipiens complex are genetically distinct on the basis of nuclear DNA, the association of Wolbachia and mtDNA with members of the Cx. pipiens complex were limited. The Wolbachia wPip-I group, by far the most common, was associated with divergent Cx. pipiens members, including Cx. quinquefasciatus, Cx. pipiens pipiens form pipiens and Cx. pipiens pipiens form molestus. Four other wPip groups were also found in mosquito populations and all were shared between diverse Cx. pipiens members.There was a strong association between wPip infections, and their allied mitochondria, are associated with regular transfers between Cx. pipiens members rather than specific host associations. Overall, this is suggestive of a recent and likely ongoing cytoplasmic introgression through hybridization events across the Cx. pipiens complex.This data overall supports the hypothesis that  Wolbachia are extremely widespread in insects[Wolbachia is typically maternally inherited through the egg cytoplasm and has evolved a variety of interactions with its hosts, exerting subtle effects such as manipulation of host reproduction or protection against natural enemies[Wolbachia to spread rapidly through insect populations. These effects are also of ecological and evolutionary importance to the particular host species that is infected, potentially inducing reproductive isolation or driving changes in sexuality[Symbiotic associations with the intracellular bacterium  insects-3. Wolba enemies-6. Each exuality-6.Culex pipiens complex have long been recognized to exhibit a great variability of effects associated with Wolbachia. In this host, Wolbachia, known as wPip, is associated with cytoplasmic incompatibility (CI), a sperm-egg incompatibility between infected males and uninfected females, so that infected females have a reproductive advantage[wPip strains[Wolbachia multilocus strain typing (MLST)[Wolbachia surface protein gene wsp present no variation among these wPip strains, showing that they delineate a recent and monophyletic clade into the B Wolbachia supergroup[ank2 and pk1 genes encoding proteins with ankyrin (ANK) motifs, revealed the existence of more than 100 genetically distinct wPip strains[wPip-I to wPip-V groups[wPip strains are mutually incompatible but also that some others, although genetically distinct, are fully compatible[wPip has also emerged as a conditional mutualist that protects Cx. pipiens against mortality induced by the avian malaria parasite Plasmodium relictum[wPip is an important associate of most mosquitoes in the Cx. pipiens complex potentially driving its evolution.Mosquitoes of the dvantage-9. Apart strains. Five hog (MLST) and the pergroup. The rec strains,14, belompatible,14. Mean relictum. OverallwPip infections interact with the genetic structure within the Cx. pipiens complex remains still unclear. Mosquitoes in this complex have a global distribution in all temperate and tropical regions, with a recent history of association with human migration[Cx. pipiens complex have controversial taxonomic statuses  and, even though they are genetically distinct, they remain difficult to separate morphologically. Four species, or subspecies according to the authors, are frequently recognized as members of the complex: Cx. pipiens, Cx. quinquefasciatus, Cx. australicus, and Cx. globocoxitus, as well as two subspecies: Cx. pipiens pipiens in Europe and North and South Africa, and Cx. p. pallens, in Asia. In addition, two sympatric forms, pipiens and molestus, are also encountered in Cx. p. pipiens in the Northern hemisphere. Cx. quinquefasciatus, commonly known as the southern house mosquito, exists all across the tropics and the lower latitudes of temperate regions. Cx. australicus and Cx. globocoxitus are restricted to Australia and are poorly known. Although the exact taxonomic status of the Cx. pipiens members remains controversial, their close evolutionary association has been repeatedly supported by genetic analyses and the relative abundance of hybrids in areas where distributions overlap[wPip was never detected in Cx. australicus and Cx. globocoxitus, infection frequency was near or at fixation in almost all populations of both Cx. pipiens and Cx. quinquefasciatus[wPip groups were found associated within given infected members of the Cx. pipiens complex[Cx. pipiens and Cx. quinquefasciatus laboratory lines have been examined, making this result difficult to interpret. This issue is of special importance since Wolbachia may either produce reproductive isolation between host populations infected with different Wolbachia strains, or reinforce an existing divergence by selecting for pre-mating isolation mechanisms[Cx. pipiens complex is thus a relevant system to test such a hypothesis.How igration,17. The  overlap-22. Remafasciatus,23-25. W complex. Howeverchanisms,5. The pwPip infections in natural populations of the four main members of the Cx. pipiens complex , spanning 118 natural populations and 64 laboratory lines. With this aim, we characterized each wPip individual infection using two to five Wolbachia markers and identified Cx. pipiens members using diagnostic nuclear markers, including microsatellites for a subsample of populations. Since Wolbachia and host mitochondria are co-transmitted in egg cytoplasm and therefore are in linkage disequilibrium, we also examined the mitochondrial (mtDNA) diversity through the sequencing of one to three mtDNA markers. Using this approach, we thus attempted to infer the contribution of Wolbachia in shaping the genetic diversity within the Cx. pipiens complex.Here we have approached this issue by undertaking an extensive screening for the presence, the diversity and the partitioning of Cx. pipiens complex: Cx. p. pipiens form pipiens, Cx. p. pipiens form molestus, Cx. p. pallens as well as Cx. quinquefasciatus, hereinafter respectively referred to as pipiens, molestus, pallens and quinquefasciatus  using a series of specific PCR-RFLP (restriction fragment length polymorphism) assays based on two ANK Wolbachia markers, ank2 and pk1 : a (one RFLP fragment: 313 bp), b , c , d  and e (415 bp). TaqI digestion of the pk1 PCR products allowed discrimination of four specific wPip alleles : a/e , b , c  and d . The pk1 a and e alleles were next distinguished using a digestion of the pk1 PCR products with PstI: a  and e . For a subsample of specimens, three additional Wolbachia markers were sequenced: the DNA mismatch repair protein gene MutL, the putative secreted protein gene GP15  and the regulatory protein gene RepA  of these five Wolbachia genes were present in one single copy in the wPip(Pel) genome while the fifth (pk1) is present in three identical copies  chromosome, were thus examined. None of these genes was amplified from Wolbachia-free Cx. pipiens lines, which confirmed their Wolbachia origin.The DNA was extracted from individual mosquitoes using a CetylTrimethylAmmonium Bromide (CTAB) or phenoCx. pipiens mtDNA haplotypes were determined through the sequencing of an 852 bp fragment from the cytochrome b (cytb) gene  and 5   gene  implemented in GENEPOP[The significance of non-random associations between  GENEPOP.Wolbachia in 1935 specimens from 118 natural populations and 64 isofemale laboratory lines  for ank2 and pk1 : alleles at these loci are not randomly associated showing that they are stably co-transmitted within the wPip chromosome. Hence, the ank2 and pk1 allelic profiles are congruent and allowed unambiguously attributing the wPip group to 1836 (94.9%) of 1935 specimens  from samples with null alleles at either MutL, GP15 or RepA loci indicate satisfactory DNA template quality in these cases. Phylogenetic tree using ank2, pk1, MutL, GP15 and RepA concatenated sequences (5624 bp) showed that almost all new wPip haplotypes fall into one of the five wPip groups  remaining not assigned.We further used the wPip-I group, 326 (16.9%) by wPip-II, 414 (21.4%) by wPip-III, 261 (13.5%) by wPip-IV, 108 (5.6%) by wPip-V and 3 (0.1%) by undetermined wPip groups  in electropherograms.Taking into account PCR-RFLP and phylogenetic grouping, we thus found that 823 specimens (42.5%) were infected by the oup, 326 6.9% by wwPip showed an important spatial variation over the distribution area of the Cx. pipiens complex . Thus, oversampling does not seem to be a likely explanation for the great wPip diversity found in Europe.The diversity of  Figure A, but a  Figure B. Given wPip group was detected per population: 88 (74.6%) of the 118 natural populations harboured only one wPip group while, in the remaining 30 (25.4%), two to three wPip groups per population were observed. Twenty-two of the 30 populations were located in Europe and half (18 of 30) harboured Cx. pipiens individuals infected either by wPip-II or wPip-III. Aside from the wPip-II/wPip-III mixed populations found in Western Europe, at least five other geographic contact zones between wPip groups exist: in the North (wPip-II/wPip-IV) and in the South of Italy (wPip-I/wPip-IV), in North Africa (wPip-I/wPip-IV), in South America (wPip-I/wPip-III) and Eastern Asia (wPip-I/wPip-V)  were obtained  with a clear pattern of cytb haplotype specificity to wPip groups  in a suwPip groups, very little incongruence were observed. For instance, although the mtDNA-1 group contains 65 wPip-I-infected specimens, seven specimens infected with wPip-V were also found in this group (Table wPip-I-infected specimen was found in the mtDNA-4 group (otherwise associated to 20 wPip-IV-infected specimens), one wPip-II-infected specimen in the mtDNA-3 group (associated to 44 wPip-III-infected specimens), and 3 wPip-III-infected specimens in the mtDNA-2 group (associated to 39 wPip-II-infected specimens). To improve our understanding of this incongruence, two additional mtDNA genes (ND2 and ND5) were sequenced and phylogenetic analyses were conducted using cytb, ND2 and ND5 concatenated genes  described above. The resulting phylogenetic tree does not group the seven incongruent specimens infected by wPip-V within the extended mtDNA-1 group, and they can not be assigned to a specific mtDNA group. These results suggest that the primary wPip-mtDNA incongruence observed in these specimens is likely due to low polymorphism in the mitochondrial gene studied here , pipiens , molestus , and pallens  as well as a few hybrids  , as shown with quinquefasciatus that appeared mainly infected with the wPip-I group , pipiens with wPip-II (55 of 118) and pallens with wPip-V (19 of 20) . Overall, the association wPip/Culex pipiens taxa is thus far from exclusive since no wPip group was unique to a particular Cx. pipiens member: each Cx. pipiens taxa harbours two to five different wPip groups. For instance, pipiens harbours infections of all five wPip groups and quinquefasciatus of three wPip groups .To investigate the association of us n = 20, 49.1%, ens taxa,30 with wPip infections among Cx. pipiens members could be based on potential artefacts from molecular typing: for some samples, taxa identification was done through the characterization of two nuclear markers, ace-2 and CQ11, an approach that could fail to detect hybrid individuals. To address this potential bias we refined our analysis by restricting our analyses to a subsample of 113 specimens from 12 populations  that were typed with microsatellite loci. Although these mosquito populations were originally typed using 7–12 microsatellite loci, the loci used were however different depending on what Cx. pipiens taxa and studies they were for. Further analyses were thus conducted using four microsatellite loci  indicating that most individuals are not admixed and come from a cluster  and also confirmed that this association is not exclusive since at least four wPip groups are shared by different Cx. pipiens members  for Cx. pipiens members was assayed in 166 individuals , but not exclusive: although mtDNA-1 group was common in quinquefasciatus  and mtDNA-2 group was common in pipiens (35 of 67), no mtDNA group was specific to a particular Cx. pipiens member . However, we did not observe this pattern: similar wPip groups and mtDNA haplotypes are found in different taxa and no cytoplasm type is specific to a given Cx. pipiens taxa. We rather showed that the cytoplasmic diversity tends to be homogenized across the four members of the Cx. pipiens complex we examined. Collectively, the data suggest a recent, and possibly still ongoing, cytoplasmic exchange between Cx. pipiens taxa.Because of vertical transmission, one should expect that persistence of Cx. pipiens taxa share similar cytoplasms. The first process is the relatively recent emergence of some Cx. pipiens taxa. The pipiens and molestus forms are very closely related and several scenarios place their divergence at 10,000 years ago[wPip groups [Cx. quinquefasciatus. This taxon has emerged long before the pipiens-molestus divergence[wPip infection in European populations. It is thus likely that wPip has secondarily spread from Cx. pipiens to Cx. quinquefasciatus following cytoplasmic introgression since recurring hybridization occurs where their distribution overlaps[wPip groups in Cx. p. pallens: this latter taxa is thought to be the result of unidirectional hybridization between Cx. p. pipiens females and Cx. quinquefasciatus males[Cx. p. pipiens and Cx. p. pallens share two wPip groups. Cytoplasmic introgression is likely still underway in the Cx. pipiens complex: hybrids between pipiens and molestus forms have been documented in North America and in southern Europe[Cx. quinquefasciatus and Cx. p. pallens in Asia[wPip infections may commonly flow from either side into the hybrid zone.Two non-exclusive processes can explain why different ears ago, which iars ago;). Assumiression). This paus males, a patte in Asia, and wPiWolbachia impacts the Cx. pipiens diversity in different ways. The wPip distribution presented here showed a well-structured picture, and underlines a critical example of cytoplasmic introgression through sibling taxa. This situation is actually similar to adaptive introgression of beneficial alleles. Previous studies examining loci involved in insecticide resistance concluded that resistant alleles are undergoing frequent expansion across the complex through hybridization between Cx. pipiens members[Cx. pipiens neutral loci, however, show that recombination may break the connection between selected and neutral loci very quickly, maintaining the genetic differentiation between taxa and allowing the inter-taxa expansion of selected genes[wPip infections seem to operate in a similar manner as insecticide resistance genes in the Cx. pipiens members, suggesting that introgression has a crucial role in the dispersal of wPip infections. Of note, however, in the case of Wolbachia, infection spread may have a negative impact on population dynamics of mosquito hosts since it could drive deleterious mitotypes to fixation through cytoplasmic hitchhiking. It remains to be seen what role, if any, CI between incompatible wPip strains play in divergence between different geographic populations within the Cx. pipiens complex.It is now clear that  members,47. Surved genes. HybridiThe authors declare that they have no competing interests.Conceived and designed the experiments: ED, CMA, MW and OD. Performed the experiments: ED, CMA, OD, P Makoundou, SU, DMF, EVS. Analyzed the data: ED, P Milesi and OD. Wrote the paper: ED, MW and OD. All authors read and approved the final manuscript.Culex pipiens populations. LL, laboratory lines; ND, not determined. Table S2. List of primers and gene features. Table S3. Nucleotide polymorphism in the cytb, ND2 and ND5 mitochondrial genes. Only polymorphic sites are represented and a dash indicates similarity with the top sequence. Position expressed in nucleotides bases on the complete mitochondrial sequence of Pel Culex pipiens line . Table S4. Pair-wise FST values among mosquito populations . *, significant FST values after Bonferroni correction. Figure S1. Identification of ank2 and pk1 allelic profiles. (A) HinfI digestion of the ank2 PCR products allowed discrimination of five alleles (a to e): a (one RFLP fragment: 313 bp), b , c , d  and e (415 bp). (B) TaqI digestion of the pk1 PCR products allowed discrimination of four specific wPip alleles : a/e , b , c  and d . (C) PstI digestion of the pk1 PCR products allowed discrimination alleles a  and e . Figure S2.mtDNA phylogeny constructed using Maximum likelihood method based on concatenated sequences of cytb, ND2 and ND5 genes. mtDNA haplotypes originally described by Atyame et al.(2011b) are marked by full circles. Triangles show the seven specimens presenting incongruences between wPip infection and mtDNA haplotypes. Numbers on branches indicate percentage bootstrap support for major branches (500 replicates). The scale bar indicates the number of substitutions.Detailed results of the screen of Click here for file


Facklamia hominis is a rare causative organism of infective endocarditis (IE). Only few cases of infection due to F. hominis have been reported in the literature. We describe a case of IE due to Gram-positive, alpha-haemolytic, catalase-negative coccus F. hominis in an adult patient with rheumatic mitral stenosis. Isolated mitral stenosis is an uncommon valve lesion predisposing to IE. The following paper is being presented to emphasize the possibility of IE due to F. hominis, and laboratories need to be alert of the potential significance in appropriate clinical setting. Streptococci, Staphylococci, Enterococci, and fastidious Gram-negative coccobacilli. In their absence, unusual organisms should be considered. Facklamia hominis is one such rare organism [Facklamia species are Gram-positive, alpha-haemolytic, catalase negative cocci, which resemble Streptococcus viridians on 5% sheep blood agar. Only few cases of infection due to F. hominis have been reported in the literature. We present a case of IE due F. hominis in an adult patient with rheumatic mitral stenosis. Isolated mitral stenosis is an uncommon valve lesion predisposing to IE. The following paper is being presented to emphasize the possibility of IE due to F. hominis, and laboratories need to be alert of the potential significance in appropriate clinical setting.Infective endocarditis (IE) is one of the major complications in individuals with structural heart disease. The majority of cases of IE are caused by organism . FacklamR FA bottles (Biomerieux). These bottles were incubated in the Bact/ALERT microbial detection system. Pending culture reports, empirical intravenous treatment with crystalline penicillin  and gentamicin (1 mg/kg IV every 8 hr) was initiated. Results routine blood tests were: haemoglobin 12.2 g/dL, white cell count 16.2 × 109/L (neutrophils: 81%), platelets 226 × 109/L, and erythrocyte sedimentation rate (ESR) 102 mm/1 h. Renal parameters, liver function tests, blood sugars, urine routine, and chest X-ray were normal. Serology for HIV was negative. ECG showed a normal sinus rhythm. Two-dimensional transthoracic echocardiography revealed mobile vegetation attached to the anterior mitral leaflet on the left atrial side . The isolate was susceptible to penicillin, amoxicillin, cefotaxime, ceftazidime, ceftriaxone, gentamicin, ciprofloxacin, levofloxain, tetracycline, rifampicin, and vancomycin by the Kirby Bauer method on Muller Hinton Agar with 5% sheep blood, as per Clinical and Laboratory Standards Institute. The patient continued to be febrile ten days after initiation of treatment with crystalline penicillin and gentamicin. There were no complications of IE. Antibiotic treatment was changed from crystalline penicillin to ceftriaxone . Intravenous treatment was continued for a period of six weeks (6 weeks of ceftriaxone and 4 weeks of gentamicin). The patient's general condition improved, fever subsided, and ESR reduced to 30 mm/1 h at the end of treatment. Repeat blood culture after completion of antibiotic course yielded no growth.A 35-year-old male with rheumatic heart disease was admitted to our hospital for evaluation of fever of 3-weeks duration. He had been empirically treated for enteric fever by a primary care physician. The patient had received oral ciprofloxacin for 1 week prior to admission. He was on regular benzathine penicillin prophylaxis and had undergone balloon mitral valvotomy 15 years earlier. The temperature at presentation was 101°F. The patient was hemodynamically stable. Cardiovascular system examination revealed a middiastolic murmur at the apex. The rest of the systemic examination was unremarkable. Three separate sets of blood cultures were obtained for possible IE. Blood cultures were performed using BacT/ALERTial side . Additiocubation . The isoStreptococcus viridans and Staphylococcus aureus are the common causative agents. However, unusual organisms like F. hominis cannot be ignored when isolated from multiple blood cultures. IE due to F. hominis is a rare entity. Todate, only one case of F. hominis endocaritis has been published in the literature [The microbiology of native valve IE is relatively predictable. terature  and as sFacklamia species are gram positive cocci resembling Streptococcus viridans on 5% sheep blood agar. Since the first description of the genus Facklamia in 1997 [Facklamia have been isolated. The species of the genus Facklamia described include F. hominis, F. ignava, F. sourekii, F. languida, and F. tabaciasalis [F. tabaciasalis, the other Facklamia species have been isolated from human clinical specimens. Clinical source of Facklamia species include blood cultures, abscess, bone, cerebrospinal fluid, gall bladder, urine, placenta, and vaginal swab [Facklamia species is the female genital tract [Facklamia is challenging because of varied susceptibility and resistance patterns. The duration of treatment in IE is unclear. The earlier reported case of F. hominis IE had a fatal myocardial infarction 2 days following initiation of intravenous antibiotics [ in 1997 , severalciasalis . With thnal swab –5. The nal tract . TherefoR system, as in our report, saves time and points towards diagnosis of IE in suspected individuals. The use of fully automated VitekR 2 system for accurate identification of organism to species level, compared to traditional biochemical tests, is highlighted. In addition, antibiotic susceptibility is possible, which guides the treating physician for initiation of pertinent antibiotics.A positive blood culture is a major criterion for the diagnosis of IE. Hence, early detection of bacteraemia using automated BacT/ALERTAn additional interesting finding in this report is the cardiac lesion predisposing to infective endocarditis. This patient had an isolated rheumatic mitral stenosis, which is an uncommon valve lesion predisposing to IE. The estimated risk of IE in patients with mitral stenosis is 0.17 per 1000 patient years . IE is mF. hominis should be considered in the microbiological spectrum causing infective endocarditis. Isolates of Facklamia species from blood cultures are frequently discarded as contaminants. This paper highlights the potential significance of such isolates in the setting of infective endocarditis.To conclude, rare organisms like