Our study aimed to investigate the association of survivin gene polymorphisms with the risk of HCC in Chinese han population.Survivin, one of the strongest apoptosis inhibitors, plays a critical role in the development and progression of hepatocellular carcinoma (HCC). By comparison, relatively little is known about the effect of survivin gene, including three loci of rs8073069, rs9904341 and rs1042489, were selected and genotyped by a polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP) technique. Association analysis of genotypes/alleles and haplotypes from these loci with the risk of HCC was conducted under different genetic models.A case-control study was conducted in Chinese han population consisting of 178 HCC cases and 196 cancer-free controls. Information on demographic data and related risk factors was collected for all subjects. Polymorphisms of the P > 0.05). Linkage disequilibrium (LD) analysis showed that these loci were in LD. Multivariate logistic regression indicated that with no G-C-T haplotype as reference, the haplotype of G-C-T from these loci was associated with a lower risk for HCC under the recessive model : 0.24~0.90, P = 0.023). Both HBsAg+ and the medical history of viral hepatitis type B were risk factors for HCC. However, no statistically significant haplotype-environment interaction existed.Using univariate analysis of rs8073069, rs9904341 and rs1042489 under different genetic models, no statistically significant difference was found in genotype or allele distribution of HCC cases relative to the controls (survivin gene and the risk of HCC is found in Chinese han population, but rs8073069G-rs9904341C- rs1042489T is perhaps a protective haplotype for HCC.No association between rs8073069, rs9904341 or rs1042489 in  Hepatocellular carcinoma (HCC) is the sixth most common cancer in the world and the third most common cause of cancer-related death, which represents a major and constantly rising health burden throughout the world. In addition, HCC shows great geographical variation, with a very high incidence in China, where approximately 55% of annual new cases worldwide emerge ,2. NowadSurvivin is a novel member of the inhibitor of apoptosis family of proteins (IAPs), containing a single baculovirus IAP repeat (BIR) domain . It is isurvivin gene promoter [survivin gene, such as -31G/C, -625G/C and -644C/T. -31G/C polymorphism is a common mutation in cancer cell lines leading to overexpression of survivin and the aberrant cell cycle-dependent transcription, mediated via functional disruption of binding at the CDE/CHR repressor motifs [survivin gene polymorphisms were associated with human cancers [survivin gene polymorphisms and the risk of HCC [Survivin is expressed in a cell cycle-regulated manner, with a peak in the G2/M phase of the cell cycle, while it rapidly declines in the G1 phase . This ispromoter . Severalr motifs . Several cancers . Howeverk of HCC . No simisurvivin gene polymorphisms with its expression and other cancers, we hypothesized that polymorphisms in survivin gene might modulate susceptibility to HCC. To test this hypothesis, we investigated the association between survivin gene polymorphisms and the risk of HCC in Chinese han population.Based on the key role of survivin in carcinogenesis and the association of This case-control study consisted of 178 HCC patients and 196 cancer-free controls. All subjects were unrelated han nationality living in Haimen city, Jiangsu province. HCC patients were diagnosed by doctors according to the standards established by Chinese Society of Liver Cancer (CSLC) . Control2O, 12.5 μl 2 × Taq PCR Master Mix , 1 μl 10 μmol/l each primer. The annealing temperature was 60.2°C, 64.4°C and 64.4°C for rs8073069, rs9904341 and rs1042489 respectively. PCR products were digested by the corresponding restriction enzymes  and then separated by 2.0~3.0% agarose gel electrophoresis with ethidium bromide (EB). The genotypes are shown in Figure The genomic DNA was extracted from each blood sample using RelaxGenne Blod DNA system . Each polymorphism of rs8073069, rs9904341 and rs1042489 was identified via the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. The primers for PCR, the restriction enzymes, and the fragments length after digestion are shown in Table http://ihg.gsf.de/cgi-bin/hw/hwa1.pl. Differences in the distribution of genotypes or alleles under different genetic models  within groups were estimated by univariate statistical analyses (chi-square test) on SPSS 16.0. Pair-wise linkage disequilibrium (LD) between SNPs was calculated by Haploview 4.1. In addition, haplotypes analysis was carried out by multivariate logistic regression analysis on Hapstat 3.0. The odds ratios (ORs) and 95% confidence intervals (CIs) were presented for association analysis. For all hypothesis tests, a two-tailed significance level of P < 0.05 was considered statistically significant.Genotypes were tested for Hardy-Weinberg equilibrium by an online soft P = 0.343 and 0.703 respectively). Besides, all subjects were local ethnic han population in Haimen, which indicated that both groups were comparable in demographic characteristics. With regard to the risk factors, Table A total of 374 subjects were recruited in our study, including 178 HCC cases and 196 controls. Their general characteristics are summarized in Table χ2 = 2.239, 2.820 and 2.675 respectively; for controls, χ2 = 0.143, 0.078 and 1.451 respectively; all P values were higher than 0.05), illustrating that our subjects presented the source population well. We compared the genotype or allele frequencies of every polymorphism within groups under the dominant, recessive, additive and multiple genetic models respectively. As shown in Table The genotype and allele frequencies of rs8073069, rs9904341, rs1042489 in both HCC cases and controls are presented in Table 2 r= 0.288, D' = 0.916, D' 95%CI (0.830~0.960); rs8073069 and rs1042489 were also in strong LD, with the 2 r= 0.425, D' = 0.945, D' 95%CI (0.880~0.980); rs9904341 and rs1042489 were in weak LD, with the 2 r= 0.026, D' = 0.189, D' 95%CI (0.070~0.290). These results stated clearly the three identified survivin gene polymorphisms were in LD.LD analysis showed that rs8073069 and rs9904341 were in strong LD, with the AIC). With no G-C-T haplotype as reference, it was found that the haplotype of G-C-T from these loci was associated with a lower risk for HCC under the recessive model , whereas none of other significant haplotypes were found in this model. It also indicated that HBsAg+ and the medical history of viral hepatitis type B were risk factors for HCC, with the OR being 27.03 (P < 0.001) and 55.16 (P < 0.001) respectively. However, no statistically significant haplotype-environment interaction was detected under different genetic models, as shown in Table With the help of multivariate logistic regression, factors like drinking, HBsAg+, the medical history of viral hepatitis type B and different haplotypes constructed by rs8073069-rs9904341-rs1042489 were included into models. As shown in Table Suvivin gene, located in 17q25, containing three exons and four introns, can encode an apoptosis inhibitor consisting of 142 amino acids. Nowadays, several SNPs have been identified in this gene [survivin gene polymorphisms with the risk of HCC in Chinese han population for the first time. Our results suggested that none of rs8073069, rs9904341 or rs1042489 polymorphisms in survivin gene correlated with the susceptibility to HCC. The polymorphism of rs1042489 is in the 3' untranslated region of survivin gene, studies about this polymorphism haven't been reported yet. Our result showed that rs1042489 C/T polymorphism didn't correlate with the risk of HCC, indicating this polymorphism probably had nothing to do with the stability of survivin mRNA or its translational efficiency [his gene . The presurvivin gene. There were two studies on the relationship between this polymorphism and cancers, whereas no similar studies have been conducted on HCC. Yang et al. [OR of CC genotype being 2.404 in contrast to GG genotype. Yang also described different survivin expression levels between subgroups with different rs8073069 G/C variants in ESCC patients. His study suggested that rs8073069 G/C polymorphism was associated with the susceptibility to ESCC, perhaps by influencing survivin expression. However, Jang et al. [The locus of rs8073069 -625 G/C) is located in the promoter region of  G/C is lg et al.  discoversurvivin gene promoter. Several studies on the association of this polymorphism with cancers have been carried out. However their results were inconsistent. Jang et al. [The locus of rs9904341 -31 C/G) is located in g et al.  discover C/G is lg et al. ,32,33. Ig et al.  demonstrOR = 0.46, 95%CI: 0.24~0.90). This suggested G-C-T haplotype was perhaps a protective genetic factor for HCC in Chinese han population. Nonetheless, it requires further studies to confirm. Both HBsAg+ and the medical history of viral hepatitis type B were risk factors for HCC, whereas no statistically significant haplotype-environment interaction existed.Haplotype analysis can obtain more information than single SNP and thus elevates the statistical power by making an assay of several SNPs within the same gene simultaneously . LD analsurvivin gene with HCC has been reported. Since polymorphisms often vary among different ethnic groups, additional and lager sample size studies are required to validate the association of survivin gene polymorphisms with HCC in diverse ethnic populations. However, our study was considered credible, suggesting survivin gene polymorphisms  didn't correlate with HCC, at least in Chinese han population.To date, only one similar study on the correlation of survivin gene polymorphisms with the risk of HCC in Chinese han population. No association between rs8073069, rs9904341 or rs1042489 polymorphisms in survivin gene and the risk of HCC is found in the present study, but rs8073069G-rs9904341C-rs1042489T is perhaps a protective haplotype for HCC in Chinese han population. Further studies will be needed to see whether survivin gene polymorphism has a role in HCC in other geographical region.In conclusion, this is the first report regarding the association of the The authors declare that they have no competing interests.The study's chief researcher WM was responsible for identifying the research question, designing the study, obtaining ethics approval and overseeing the study. WL and FJ were in charge of the study fields, collecting subjects' data. YL and JW majored in the laboratorial work, analyzing the data, writing the manuscript. All authors were responsible for drafting the manuscript, read and approved the final version.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2350/13/1/prepub


We present data on the anti-HIV activity and toxicity using a broad armamentarium of L.vaginalis, L. iners, L. jensenii, L. gasseri, L. crispatus, A vaginae, G. vaginalis) was measured for the lead molecules UAMC00838 and UAMC01009. Dapivirine (TMC120) was used as a bench mark throughout the study.In a primary screen, the anti-HIV activity against the laboratory strain Ba-L and against a primary subtype C isolate was determined in the TZM-bl cell line. Cellular toxicity on TZM-bl cells was evaluated using WST-1. Subsequently, a selection of 17 compounds was further evaluated for anti-HIV activity in different primary cells, including peripheral blood mononuclear cells, dendritic cells and CD4+ T lymphocytes. In addition, the activity against NNRTI-resistantviruses  was tested. The toxicity profile was further investigated using blood cells and epithelial cells originating from the female genital tract (FGT) and in a dual chamber assay modeling the FGT and underlying mucosae. Finally, toxicity towards vaginal flora . Similar nM activities were found in primary cells for a selection of 17 compounds. Interestingly, these compounds retained fairly good potency  against a resistant strain carrying the NNRTI-resistance mutations V106A or Y181C. However, potency was diminished  when tested against a mutant virus carrying L100I/K103N. Compounds UAMC00838 and UAMC01009 were identified as lead molecules based on their activity/toxicity profile and chemical structure.vaginalis and A vaginae was inhibited at the highest compound concentration. Compound UAMC01009 showed additional low level toxicity against L. iners and high level toxicity against L. crispatus.These novel compounds showed similar or better toxicity profiles as the bench mark molecule dapivirine, in TZM-bl cells as well as in FGT epithelial cells and in a dual chamber system modeling the FGT. Finally, while no toxicity against vaginal lactobacilli was observed up to concentrations approx. 80,000 times above the EC50 of compound UAMC00838, the growth of G. vaginalis and A vaginae, which are associated with bacterial vaginosis, a risk factor for HIV acquisition. Ongoing studies on solubility and formulation will reveal their potential as intravaginal/-rectal microbicides.We present data of highly active NNRTIs with a favorable toxicity profiles compared to the bench mark NNRTI dapivirine. Compound UAMC00838 does not affect the normal vaginal flora, but could inhibit G.


Brachyspira pilosicoli causes enteric disease in avian, porcine and human hosts, amongst others. To date, the only available genome sequence of B. pilosicoli is that of strain 95/1000, a porcine isolate. In the first intra-species genome comparison within the Brachyspira genus, we report the whole genome sequence of B. pilosicoli B2904, an avian isolate, the incomplete genome sequence of B. pilosicoli WesB, a human isolate, and the comparisons with B. pilosicoli 95/1000. We also draw on incomplete genome sequences from three other Brachyspira species. Finally we report the first application of the high-throughput Biolog phenotype screening tool on the B. pilosicoli strains for detailed comparisons between genotype and phenotype.The anaerobic spirochaete B. pilosicoli strains, although the genomes of B2904 and WesB were larger than that of 95/1000 . Genome rearrangements were observed which correlated largely with the positions of mobile genetic elements. Through comparison of the B2904 and WesB genomes with the 95/1000 genome, features that we propose are non-essential due to their absence from 95/1000 include a peptidase, glycine reductase complex components and transposases. Novel bacteriophages were detected in the newly-sequenced genomes, which appeared to have involvement in intra- and inter-species horizontal gene transfer. Phenotypic differences predicted from genome analysis, such as the lack of genes for glucuronate catabolism in 95/1000, were confirmed by phenotyping.Feature and sequence genome comparisons revealed a high degree of similarity between the three B. pilosicoli genome sequences has allowed us to demonstrate the substantial genomic variation that exists between these strains, and provides an insight into genetic events that are shaping the species. In addition, phenotype screening allowed determination of how genotypic differences translated to phenotype. Further application of such comparisons will improve understanding of the metabolic capabilities of Brachyspira species.The availability of multiple  Brachyspira is the sole genus of the family Brachyspiraceae within the order Spirochaetales, which belongs to the spirochaete phylum [Brachyspira are Gram-negative, loosely coiled, aerotolerant anaerobes that colonise the lower gastrointestinal (GI) tract of mammals and birds, but vary in pathogenicity. There are seven Brachyspira species that are currently officially recognised: B. aalborgi, a potential human pathogen [B. hyodysenteriae[B. alvinipulli[B. intermedia[B. pilosicoli[B. innocens[B. murdochii, which is of uncertain pathogenic potential [B. canis” [B. pulli” [B. suanatina” [Brachyspira as pathogenic, intermediate pathogenic or non-pathogenic is subject to debate.Spirochaetes represent a monophyletic lineage and a major branch in eubacterial evolution; e phylum . Brachyspathogen ; the porsenteriae; the avivinipulli and B. intermedia; the aviilosicoli; non-pat innocens and B. motential . In addi. canis” , “B. pul. pulli”  and “B. anatina”  amongst B. pilosicoli is the only species considered to be a pathogen of birds, pigs and humans. The species is quite diverse, and it seems unlikely that there are barriers to cross-species and zoonotic transmission [B. pilosicoli is an aetiological agent of colitis and occasional spirochaetaemia in humans [B. pilosicoli is commonly found in humans living in densely populated areas with poor hygienic conditions [B. pilosicoli infections are highly prevalent in intensively farmed swine and poultry, inducing inflammation in the colon and caeca, diarrhoea and reducing growth and productivity [B. hyodysenteriae, B. pilosicoli has a chemoattraction to mucin that facilitates penetration of the mucus and association with the underlying intestinal epithelial surface [B. pilosicoli infection, and shared only by B. aalborgi, is the ability to insert one cell end into the luminal surface of enterocytes in the large intestine, forming a pit-like structure, with arrays of such attached spirochaetes giving the appearance of a “false brush-border” [B. pilosicoli also occurs in Caco-2 cells in vitro, resulting in apoptosis, actin rearrangement and elevated interleukin expression [smission . B. pilon humans , and a cn humans . It may n humans . B. pilonditions -17, and nditions . B. pilouctivity . Chemota surface ,20. The  surface . An unus-border” ,23. Thispression .Brachyspira have undergone to colonise the lower GI tract, and for the pathogenic species to induce disease. Brachyspira whole genome sequences have only recently been made available for the following species: B. hyodysenteriae[B. intermedia[B. murdochii[B. pilosicoli[B. pilosicoli B2904, isolated from a chicken exhibiting clinical symptoms of AIS in the UK, and the partial genome sequence of B. pilosicoli WesB, isolated from an Australian Aboriginal child with diarrhoea. Experimentally, the latter strain has been shown to colonise and cause disease in pigs [B. pilosicoli 95/1000, isolated from a pig with PIS in Australia, and which has been confirmed to be virulent in experimental infection studies in pigs [The paucity of genomic information and absence of tools for genetic manipulation are responsible, at least partly, for the lack of knowledge regarding the adaptations that senteriae, B. intentermedia, B. murdmurdochii and B. pilosicoli. The fou in pigs . Althoug in pigs . The gen in pigs . We empl in pigs  to assesB. pilosicoli B2904 was isolated from a chicken displaying clinical symptoms of AIS in the UK; WesB was isolated from an Australian child with diarrhoea [2 and 10% CO2 in N2) at 37°C or 42°C for 5 days, for phenotypic studies. For genomic DNA extraction, strains were grown in pre-reduced anaerobic broth [iarrhoea  and 95/1iarrhoea . The striarrhoea  in an anic broth  at 37°C B. pilosicoli B2904 and WesB genomes were sequenced on a Roche 454 FLX platform, using a standard preparation for a 3 Kb and 8 Kb library, respectively.Cetyltrimethylammonium bromide (CTAB) extraction was used to purify high molecular weight genomic DNA . The B. de novo assembly of the sequence reads into contiguous sequences was generated using Newbler Assembler software. The reads were assembled into one scaffold of 173 contigs with an average coverage of × 20. Remaining gaps were closed by PCR walking between unlinked, contiguous sequences [For the B2904 genome, a equences , followeFor the WesB genome, sequence data were initially assembled with Short Oligonucleotide Alignment Program (SOAP)  and subsB. pilosicoli B2904 (accession number: CP003490 Project ID: 80999) and partial nucleotide sequence and annotation of B. pilosicoli WesB B2904  have been deposited in GenBank. Scaffold sequences for unpublished genomes B. alvinipulli C1T and B. intermedia HB60 can be accessed from the authors via e-mail request. The draft genome scaffolds for B. aalborgii are available at the MetaHit website (http://www.sanger.ac.uk/resources/downloads/bacteria/metahit/).The complete nucleotide sequence and annotation of B. pilosicoli B2904 and partial B. pilosicoli WesB genomes was as previously described for B. hyodysenteriae WA1 [B. pilosicoli 95/1000 [Sequence and protein analysis and annotation (including rRNA and tRNA prediction) for the complete riae WA1  and B. p 95/1000  unless oBrachyspira strains that have undergone genome sequencing, and three that are currently within unpublished genome sequencing projects being undertaken by the authors was calculated and constructed from the concatenation of 7 gene nucleotide sequences  [I).The Multi Locus Sequence Typing (MLST) dendrogram of six hi, alp) . These chi, alp)  and the hi, alp) . The conThe open source utility ‘Freckle’ was used for sequence dot plotting (code.google.com/p/freckle/). Gene prediction and gene and protein sequence extraction was achieved using prodigal 2.50 .Brachyspira genome annotation methodologies for comparative purposes [Initial coding region annotation was completed with an in-house updated compilation of the annotation pipeline AutoFACT 3.4 . Resultipurposes ,26,28. Fhttp://www.micans.org/mcl/). The granularity of the output cluster was set with an inflation value of 2.5.Protein reciprocal blast similarity searches with a threshold maximum expected value 1e-20 were conducted with BlastlineMCL, which is an implementation of the Markov clustering algorithm (MCL) for graphs  were attached per PM panel, which were then placed into 4 oz Whirl-Pak® Long-Term Sample Retention Bags (Nasco) with the open end heat-sealed.Substrate utilisation was measured via the reduction of a tetrazolium dye (clear yellow) to formazan (purple), indicative of cellular respiration at 37°C. Experiments were also run at 42°C, using bacteria cultured at this temperature. Formazan formation was monitored at 15 min intervals for 120 h in OmniLog apparatus. Kinetic data were analyzed with OmniLog-PM software. Each experiment was performed at least twice per strain. It was noted that although tetrazolium dye reduction is indicative of cellular respiration, it can occur independent of cell growth ,30.Blank PM1 and PM2 controls were run, whereby IF-0 was added in place of the bacterial cell suspension, to assess for abiotic reactions that occur in the anaerobic atmosphere across the 120 h monitoring period. The following compounds were omitted from analysis due to the nature of the abiotic reactions that occurred in wells containing these compounds, under the conditions of the study: D-arabinose and L-arabinose, dihydroxyacetone, D-glucosamine, 5-keto-D-gluconate, L-lyxose, palatinose, D-ribose, 2-deoxy-D-ribose, sorbate, D-tagatose and D-xylose.Brachyspira strains highlighted the close relationship between the three B. pilosicoli strains, with B. aalborgi being distinct, but most closely related to B. pilosicoli and distantly related to B. hyodysenteriae  [B. intermedia phenotype should be assigned to this species [A dendrogram based on the MLST data for nine e Figure , concordfindings ,28. The s (PFGE) , and a ps (PFGE) . It has  species .B. pilosicoli genomes are compared in Table B. pilosicoli genomes were very similar to each other (27.44% to 27.9%), and to that of other chromosomes in the genus, which range from 27.1% to 27.9% [B. pilosicoli B2904 consisted of a 2,765,477 bp circular chromosome . The relatively small size of the B. pilosicoli genomes is most likely due to them being members of a more specialised species that has undergone a high degree of reductive genome evolution. If this is the case, then B. pilosicoli is likely to be an older pathogen than other Brachyspira species such as B. hyodysenteriae[Brachyspira species, only B. pilosicoli and B. aalborgi show long-term intimate associations with the surface of enterocytes, into which they interdigitate one of their cell ends. In addition to their small genomes, the sequenced B. pilosicoli strains lacked plasmids, whereas the genomes of the other fully sequenced Brachyspira species have included plasmids [The general genome features of the three sequenced to 27.9% . The comsenteriae. Such a senteriae and consplasmids .The disparity between the number of open reading frames (ORF) and genome size between the B2904 and WesB strains and the high number of non-significant percentage identity (PID) and coverage ORFs in the WesB genome may be an artefact of the incomplete nature of this genome, which is the largest of the three strains. In 95/1000, 44.8% of ORFs were assigned a KEGG Orthology (KO), whereas only 40.5% and 43.6% of ORFs were assigned in B2904 and WesB, respectively. A lower proportion of ORFs were matched in COG database for B2904 and WesB compared to 95/1000.B. pilosicoli strains harboured the same number of transfer RNA (tRNA), rRNA and transfer-messenger (tmRNA) genes (Table rrf (5S) and rrl (23S) genes were co-located in all three B. pilosicoli genomes, with the rrs (16S) gene located approximately 645 Kb, 679 Kb and 773 Kb from the other rRNA genes in the 95/1000, B2904 and WesB genomes, respectively. This rRNA gene organisation has been considered a distinguishing feature of Brachyspira species [Brachyspira have been detected in the spirochaete Borrelia burgdorferi[rrs gene and rrfrrl cluster, which are either side of the oriC, was the tmRNA  gene and nine of the total 34 tRNAs that were otherwise dispersed throughout the genome  found in 95/1000 and most in B2904  and WesB (wesB2037 – wesB2051) genomes.Despite the significant chromosomal rearrangements, genome alignments showed that the majority of genome sequence was shared between the three strains, with the larger B2904 and WesB genomes possessing the greatest proportion of unique sequences Figure . Furtherchia coli, was ideB. pilosicoli strains using the COG database showed that the general distribution of features into categories was similar for the three strains , amino acid (E) and nucleotide (F) transport and metabolism categories, with the larger WesB genome containing considerably more features than the B2904 and 95/1000 genomes. In addition, compared to other Brachyspira species the B. pilosicoli strains had a reduced number of features associated with inorganic ion transport and metabolism (P) [Functional classifications assigned to each of the protein-coding genes of the three ns Table , and thilism (P) ,28.B. pilosicoli strains contained 2,132 conserved genes, and these contribute to defining the B. pilosicoli pan-genome  were identified for B. hyodysenteriae WA1, B. pilosicoli 95/1000 and B. murdochii 56-150T[B. pilosicoli WesB harboured the greatest number of unique genes, with 10.0% of its genes being absent from the other genomes; B2904 had a similar proportion (9.5%), whereas 95/1000 had considerably fewer (4.9%). B. pilosicoli B2904 and WesB shared the greatest proportion of genes (~8.9%) while B2904 shared a greater percentage of its genes with 95/1000 (1.4%) than with WesB (0.7%).The three e Figure ; this rei 56-150T. B. piloB. hyodysenteriae WA1, B. intermedia PWS/AT, B. pilosicoli 95/1000 and B. murdochii 56-150T have previously undergone comparative analysis [B. pilosicoli B2904 and WesB can now be added to these comparisons, giving the first opportunity for a Brachyspira intra-species genome comparison. A protein blastmatrix comparison was performed on the four previously sequenced genomes, the two newly-sequenced B. pilosicoli genomes and the draft genome scaffolds of B. aalborgi 513T, B. alvinipulli C1T and B. intermedia HB60 (unpublished) (Table B. pilosicoli strains shared the greatest proportion of proteins (54.9-68.4%). Of B. pilosicoli strains, B2904 had the greatest proportion of protein repeats relating to paralogs (2.7%), despite not having the largest genome. Overall, the non-pathogenic B. murdochii had the greatest proportion of protein repeats (5.3%), perhaps relating to its large genome. High proportions of shared proteins highlighted the close relationships of B. hyodysenteriae with B. intermedia (>46.7%) and B. murdochii (33.7%)  and it has the largest genome. The greatest number of clusters shared only between two strains was with B. intermedia PWS/AT and B. hyodysenteriae WA1 (n = 61), consistent with the close relationship of these species , and WesB also shared the greatest number of clusters with a non-B. pilosicoli strain, having 36 clusters in common with B. intermedia and 16 with B. murdochii. The B. pilosicoli strains collectively shared the most clusters with B. murdochii 56-150T (n = 58), and fewest with B. hyodysenteriae WA1 (n = 4), as noted previously [B. pilosicoli strains shared 173 clusters, whereas the B. pilosicoli strains shared 110 clusters, reflecting gene loss and genome reduction.A protein Markov clustering analysis of the six published eviously . Non-B. B. pilosicoli genomes  [B. pilosicoli and may contribute to host cell adherence. Other B. pilosicoli-specific clusters were for an ankyrin repeat protein , a β-lactamase , two peptidases   and phage proteins  .Of 110 protein clusters present only in the volution , 13 clus00_2023) ,28 were 00_2023) -64. ClusB. pilosicoli strains, B2904 and WesB shared most unique clusters  [Of the B. pilosicoli 95/1000 harboured the fewest and WesB the most unique features  and rfbB , but rfbC was noted only in B2904 (n = 1) and WesB (n = 2) . Genes inferred to be involved in the biosynthesis of 3,5-dideoxyhexose, an O-antigen component of lipopolysaccharide (LPS) [rfbC gene(s) in B2904 and WesB; both strains contained rfbF  and rfbG , but rfbH was present only in WesB (wesB_0129). The absence of such genes in the pathogenic strain 95/1000 suggests that they have a limited impact on virulence.An almonella,66, was  plasmid . Althougde (LPS) , were foB. pilosicoli strains possessed fewer chemotaxis genes than B. hyodysenteriae and B. murdochii .End-on attachment of the spirochaete to the luminal surface of the lower intestinal tract epithelia is characteristic of nisation ,68, and nisation . The samnisation  and the nisation , OmpA prirulence , and Tiaux pumps -74. The ux pumps . Gene dudenticola. Genes egulation ,78, in aalmonella. Genes eogenesis , TonB-deirulence , and PTSd E. coli,83. Gened E. coli, a ferroathogens ,86, and irulence . In the  pallidum was note pallidum. Genes f pallidum, with nosagB , sagC  and sagD , involved in β-haemolysis and virulence in streptococci [Salmonella Typhimurium [The complement of haemolysis-related genes was identical between the three strains. Compared to previous analysis, other genes were detected including a haemolysin, previously undetected in 95/1000 (BP951000_1925) and three streptolysin genes, BatI (Bacteroides aerotolerance) operon [nox genes and consisted of batB , batC , batD  and batE . The batA gene was in a distinct locus in the three strains .Genes related to oxidative stress were shared by the three strains. A partial ) operon  was noteB. pilosicoli strains, which may be involved in host cell interactions through their ability to bind host chromatin as in Orientia[B. pilosicoli had consistently fewer of these genes than B. hyodysenteriae[There was little difference in the number of genes encoding ankyrin-like proteins between the  Orientia. B. pilosenteriae.B. pilosicoli genomes are detailed in Additional file Clostridium carboxidivorans , consistent with the finding that Brachyspira share a high degree of gene similarity with Clostridium[C. carboxidivorans, B. hyodysenteriae, B. intermedia, B. pilosicoli and B. murdochii, suggesting that the GTA had involvement in intra- and inter-species gene transfer. The bacteriophage that was identified in B. pilosicoli 95/1000 (pP1), and in B. murdochii 56-150T  [B. pilosicoli, the bacteriophage size was proportional to genome size. Hypothetical proteins encoded in this region were shared between 95/1000 and B2904, however WesB contained four unique hypothetical genes. The B2904 pP2 bacteriophage possessed a unique ankyrin repeat protein (B2904_orf1943). An adenine-specific DNA methyltransferase gene was present only in the WesB pP3 bacteriophage (wesB_0711), adjacent to the DNA methylase gene found in B. pilosicoli bacteriophages , but absent from those of B. murdochii 56-150T. Two separate novel bacteriophages regions were found in B2904 (pP4) and WesB (pP5). The ~29 Kb pP4 bacteriophage contained seven phage proteins (B2904_orf133 – B2904_orf180), six predicted proteins with homology to sequences of other Brachyspira species, and 35 unique hypothetical genes. The ~28 Kb pP5 bacteriophage (wesB_0301 – wesB_0341) shared all the components of the pI1 bacteriophage of B. intermedia PWS/AT, suggesting transfer of the bacteriophage in an inter-species HGT event. Interestingly, pP5 was flanked by VSH-1 components , and hence the VSH-1 GTA may be responsible for mediating the HGT event. Two nuclease genes (wesB_0306 and wesB_0308) and a number of unique hypothetical genes in pP5 were not identified in pI1. Clustered regularly interspaced short palindromic repeats (CRISPR), which provide bacteria with acquired resistance to bacteriophages [B. murdochii 56-150T, which suggests a role for bacteriophages in Brachyspira pathogenicity. B. pilosicoli B2904 and B. intermedia PWS/AT did however possess a bacteriophage resistance protein  which has been implicated in protecting against bacteriophages [Outside of bacteriophage regions, four, 61 and 31 MGEs were identified in 95/1000, B2904 and WesB, respectively, correlating with the extent of genomic rearrangements. The types and copy number of all MGEs in the ostridium, and supostridium. In WesBand pM3) ,28, was B. hyodysenteriae and B. pilosicoli has revealed that these species share many metabolic capabilities [Brachyspira genomes was extended by application of Biolog PM™ technology for phenotypic determination of carbon source utilisation of the B. pilosicoli strains. The utilisation of 178 unique carbon compounds .High proportions of the sm Table , and froriae WA1 , were foactivity , howeveractivity  was alsobacteria . Of the B. pilosicoli as a sole carbon source  cycle identified in this species [B. pilosicoli as a sole carbon source; these amino acids are primary products of ammonia assimilation used in peptidoglycan, LOS and outer membrane protein biosynthesis [B. pilosicoli strains possessed genes for glutamate dehydrogenase , which catalyses the reversible synthesis of glutamate from α-ketoglutarate and ammonium. Since α-ketoglutarate was able to sustain B. pilosicoli, the presence of a transporter for α-ketoglutarate and not glutamate may explain this phenotype. The ability to utilise certain amino acids as an energy source may have become redundant in Brachyspira, which typically occupy the nutrient-rich lower GI tract, and thus associated features may have been lost through reductive evolution.Of the COG categories related to metabolism, the greatest proportion of the genome was related to amino acid transport and metabolism Table . Phenoty species , rather ynthesis , hence tB. pilosicoli strains were able to utilise three purine and two pyrimidine nucleosides tested as a sole carbon source , no long chain fatty acids tested were utilised by B. pilosicoli strain B2904 and the near complete genome of strain WesB. Together with the previously reported 95/1000 genome, this allowed the first intra-species genome comparison within the genus Brachyspira. Our feature-based analysis revealed a high level of similarity between the three strains and identified genes that we suggest different strains of the spirochaete may have lost in a process of reductive genome evolution. Sequence-based comparisons showed the majority of sequence was shared between the strains, with few unique regions; however, genome rearrangements were observed around the oriC. MGEs were found associated to areas of rearrangements, and these features may be a factor that has driven or is driving reductive evolution. Novel bacteriophages were identified in the newly-sequenced genomes, which displayed evidence of intra- and inter-species HGT, and these may have key practical applications for use in genetic manipulation. This is the first analysis of the spirochaete in a high-throughput phenotype screening tool, allowing correlation between genotype and phenotype. Future work will focus on the application of this technology to a wider range of Brachyspira species to validate genome differences, potentially providing a means by which these phenotypes can be used for rapid screening to infer genotypes and improve current diagnostic methods. With the increasing availability of Brachyspira genome sequences, such technology should facilitate the validation of metabolic models based on genome sequence.In this study, we report the genome of TM; PTS: Phosphotransferase system; RNA: Ribonucleic acid; rRNA: Ribosomal RNA; SNP: Single nucleotide polymorphisms; SOAP: Short oligonucleotide alignment program; T: Thymine; TCA: Tricarboxylic acid; tmRNA: Transfer-messenger RNA; tRNA: Transfer RNA.A: Adenine; AIS: Avian intestinal spirochaetosis; ABC: ATP-binding cassette; ACT: Artemis comparison tool; ATP: Adenosine triphosphate; bp: Base pair; C: Cytosine; CDS: Coding DNA sequence; COG: Cluster of Orthologous Genes; CRISPR: Clustered regularly interspaced short palindromic repeats; CTAB: Cetyltrimethylammonium bromide; DNA: Deoxyribonucleic acid; FABA: Fastidious anaerobe blood agar; G: Guanine; GI: Gastrointestinal; GTA: Gene transfer agent; GTR: General time reversible; HGT: Horizontal gene transfer; iCORN: Iterative correction of reference nucleotides; IMAGE: Iterative mapping and assembly for gap elimination; Indels: Insertions/deletions; ISE: Insertion sequence element; KAAS: KEGG automatic annotation server; Kb: Kilobase; KEGG: Kyoto encyclopedia of genes and genomes; KO: KEGG orthology; LOS: Lipooligosaccharide; LPS: Lipopolysaccharide; Mb: Megabase; MCL: Markov clustering algorithm; MGE: Mobile genetic element; MLST: Multi locus sequence typing; NCBI: National center for biotechnology information; ORF: Open reading frame; PCR: Polymerase chain reaction; PFGE: Pulse-field gel electrophoresis; PID: Percentage identity; PIS: Porcine intestinal spirochaetosis; PM: Phenotype MicroArrayThe authors declare that they have no competing interests.B. pilosicoli B2904. JP and AKT were involved in genome sequencing of the WesB. DJH, TL and NDP were involved in the genome sequencing of B. aalborgi 513T, B. alvinipulli C1T and B. intermedia HB60. LJM, MLB, DJH, MIB and ACD conceived and designed comparative genomic studies. LJM and MLB performed comparative genomic analysis and analysed the data. LJM, MA, MJW and RML conceived and designed phenotyping experiments. LJM and MA conducted phenotyping experiments. LJM wrote the manuscript. All authors read and approved the final manuscript.LJM, DJH and RML conceived the study. LJM and ACD performed the genome sequencing and gap closure of Type and copy number of mobile genetic elements (MGE) in the genomes ofB. pilosicoli 95/1000, B2904 and WesB. A combination of protein markov cluster analysis and reciprocal blast searches against the conserved domain database (CDD) was used to determine the copy number of each type of MGE across the three B. pilosicoli genomes, using a cut-off e-value of 1e-20. The ORF number, position, and size of all MGEs identified in each of the B. pilosicoli genomes is displayed.Click here for fileConserved and shared protein clusters between the six genome-sequencedBrachyspira strains. B. hyodysenteriae WA1 (H), B. intermedia PWS/AT (I), B. murdochii 56-150T (M) and B. pilosicoli 95/1000 (Pa), B2904 (Pb) and WesB (Pc)a strains we included in the protein cluster analysis. A cut-off e-value of 1e-20 was used.Click here for fileComparison of the utilisation of unique carbon sources byB. pilosicoli95/1000, B2904 and WesB. Biolog Phenotype MicroArray™ (PM) technology was employed for these studies and OmniLog apparatus was used to detect formazan formation and hence, respiration due to utilisation of the carbon source; +, able to utilise the compound; -, unable to utilise the compound.Click here for file


Mortierella alpina isolate CDC-B6842. M. alpina is a nonpathogenic member of the Mucoromycotina subphylum of fungi that is an important model for understanding the molecular mechanisms of lipid production and metabolism.We report the draft genome sequence of  Mucoromycotina consist of a diverse group of organisms with industrial, biotechnological, and clinical relevance. They have been studied for their use in commercial applications, such as with biodiesel and other lipid-based products , according to the manufacturer’s instructions. The genomic DNA was sequenced at the University of Maryland School of Medicine, Institute for Genome Sciences, Genomics Resource Center (http://www.igs.umaryland.edu) using a combination of paired-end libraries (average insert of 483 bp) and mate-pair (3-kb) libraries on the Illumina HiSeq 2000. A total of 44.5 million 100-bp reads were generated. The draft genome data were assembled using the MaSuRCA genome assembler . Briefly, repeat annotation was performed using RepeatModeler (http://www.repeatmasker.org) and RepeatMasker  eukaryotic annotation pipeline protocol 1.0 at the Institute for Genome Sciences, Informatics Resource Center (atMasker . Genes ws: CEGMA , GeneMars: CEGMA , Augustus: CEGMA , and SNAs: CEGMA . Augustuwith AAT , using cwith AAT . A totalwith AAT .AZCI00000000. The version described in this paper is the first version, AZCI01000000.This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession no.


Clostridium septicum infection, especially bacteremia. We reviewed retrospectively all positive blood cultures for this organism at a 300-bed general hospital over 4 years. Four of 15 cases were associated with concurrent colon carcinoma. C. septicum infection was the presenting feature of previously undiagnosed large bowel malignancy in three patients. We report this small case series to alert clinicians to the diverse spectrum and diagnostic difficulties of this rare, potentially catastrophic association. Although commonly associated with necrotizing skin or soft tissue infections, this bacterium can present with nonspecific or atypical symptoms. All patients with positive blood cultures for C. septicum, even without clinical suspicion of large bowel malignancy, should undergo colonoscopy to evaluate for colon carcinoma.An association exists between colon carcinoma and  Streptococcus bovis infection and colon carcinoma, but there is another link between Clostridium septicum and large bowel malignancies [C. septicum has been associated with hematological malignancies, immunosuppression, neutropenia, and diabetes mellitus [C. septicum septicemia associated with large bowel malignancy to alert clinicians to the diverse, potentially misleading features of this life-threatening association.There is a connection between certain bacterial infections and malignancy. The best-known relationship is between gnancies –6. Additmellitus , 3. We rC. septicum was performed over a 4-year period in a 300-bed general medical surgical hospital in Providence, RI. All patients with bacteremia caused by this organism were identified. Hospital charts were then assessed for any association with large bowel malignancy.A retrospective review of blood culture results for C. septicum, 4 had biopsy-documented colon carcinoma. The age range was from 73 to 81, including 3 men and 1 woman. Each patient had fever of 1–7 days duration. One patient had a history of colon cancer resected surgically in the prior year. Evaluation of this patient documented unsuspected local recurrence of cancer in the colon. In the remaining 3 cases, symptoms due to clostridial infection were the presenting features of previously undiagnosed cancer. Only 2 of 4 had symptoms or signs suggesting abdominal pathology. The tumor was located in the cecum in each case. One patient had bacterial aortitis. No patient had gas gangrene, a necrotizing skin, or soft tissue infection (C septicum infection.Of 15 patients with positive blood cultures for nfection . No patiS. bovis, C. septicum is not part of the normal bowel flora and is a rare cause of infection, accounting for as few as 1.3% of all blood cultures positive for clostridium [C. septicum infections are associated with gastrointestinal malignancy. In a review of 320 cases dating back to 1969, 39% had a gastrointestinal malignancy [C. septicum sepsis is prohibitive, reported to range from 45% to 70% [Unlike stridium . Our reslignancy . Anotherlignancy . Another% to 70% .C. septicum is a gram positive, anaerobic, spore-forming rod which grows normally in soil. The postulated mechanism of infection in colon cancer involves disruption of the normal mucosal barrier due to tumor-induced ulceration, followed by bloodstream invasion. Anaerobic glycolysis in the tumor may provide an acidic and hypoxic environment facilitating spore germination [C. septicum can spread hematogenously. Mucosal disruption can be caused by tumor necrosis, bowel perforation, surgery, radiation, or a medical procedure such as colonoscopy or barium enema [mination . Cecal tmination . Subsequum enema , 2, 5, 6C. septicum is diverse ( C. perfringens, is thus more likely to infect healthy tissue. Nonspecific symptoms of fever or abdominal pain are common [The clinical spectrum of  diverse , most coe common . C. septicum aortitis, 21 of 23 cases were associated with colonic adenocarcinoma or polyps [Aortitis, a rare manifestation occurring in one of our patients, can present with nonspecific symptoms such as fever or abdominal pain. Diagnosis is most commonly made by CT scan showing a soft tissue prominence surrounding a normal aorta initially and later development of peri-aortic gas. It may also present as life-threatening aortic aneurysmal rupture. In a review ofr polyps . This inr polyps , 11. A rC. septicum-associated large bowel malignancy may be delayed or missed. Clinical manifestations are commonly nonspecific, mimicking more common disorders. At times, no clinical clue to a colon malignancy is present. Some clinicians may be unaware of the association. Bacterial sepsis may be the initial feature of previously undiagnosed and unsuspected large bowel carcinoma.The diagnosis of C. septicum sepsis had associated large bowel malignancy. All patients with blood cultures positive for this organism should undergo colonoscopy, as in our 4 patients. Unexplained fever in patients with known large bowel cancer should prompt consideration of this organism. Because malignant tumors can occur as long as several years after infection, follow-up evaluation is imperative [C. septicum can present with nonspecific symptoms or a variety of other infections and should prompt consideration of malignancy. In our small series, 4 of 15 cases of


Azadirachta indica (neem) tree is a source of a wide number of natural products, including the potent biopesticide azadirachtin. In spite of its widespread applications in agriculture and medicine, the molecular aspects of the biosynthesis of neem terpenoids remain largely unexplored. The current report describes the draft genome and four transcriptomes of A. indica and attempts to contextualise the sequence information in terms of its molecular phylogeny, transcript expression and terpenoid biosynthesis pathways. A. indica is the first member of the family Meliaceae to be sequenced using next generation sequencing approach.The A. indica were sequenced using multiple sequencing platforms and libraries. The A. indica genome is AT-rich, bears few repetitive DNA elements and comprises about 20,000 genes. The molecular phylogenetic analyses grouped A. indica together with Citrus sinensis from the Rutaceae family validating its conventional taxonomic classification. Comparative transcript expression analysis showed either exclusive or enhanced expression of known genes involved in neem terpenoid biosynthesis pathways compared to other sequenced angiosperms. Genome and transcriptome analyses in A. indica led to the identification of repeat elements, nucleotide composition and expression profiles of genes in various organs.The genome and transcriptomes of A. indica genome and transcriptomes will provide a model for characterization of metabolic pathways involved in synthesis of bioactive compounds, comparative evolutionary studies among various Meliaceae family members and help annotate their genomes. A better understanding of molecular pathways involved in the azadirachtin synthesis in A. indica will pave ways for bulk production of environment friendly biopesticides.This study on  Azadirachta indica A. Juss (neem) is an evergreen tree native to the Indian subcontinent.Authors declare no competing interests.BP conceived, planned, designed and directed the project; NMK and SP designed the analytical workflow and guided the process; NMK, SP, PJ, PG, RC, and SV analyzed the data, DSA, AKH and BK produced the Solexa Illumina sequencing data. LV, JN produced the capillary sequencing data; NV, KD and KR produced and helped in the generation of pyro-sequencing data, BK and JN performed the neem genomic DNA cloning experiment; NMK, SP and BP wrote the manuscript. All authors read and approved the final manuscript.Table S1. Description of sequencing read libraries used for genome and transcriptome assembly. Table S2. Classification of repeats in the neem genome. Table S3. Transcript annotations from similarity-based analyses. Table S4. Mapping neem RNA-Seq reads to other plant genomes. Table S5. Transcript mapping statistics using PASA. Table S6. Gene ontology based functional categorization of annotated transcripts. Table S7. Expression levels of genes associated with metabolism in neem compared to other plant species. Click here for fileFigure S1. kMer frequency curve. Figure S2. Frequency histograms for symmetric 4-mers. Figure S3. Genome scaffold mapping between neem and other species. Figure S4.Gene ontology based functional categorization of annotated transcripts. Figure S5. Expression density profiles of neem organ transcriptomes. Figure S6. Gene prediction statistics. Figure S7. GC-correlated expression density profiles of neem organ transcriptomes. Figure S8.AT and GC density profiles in genomes and transcriptomes of neem and other plant species.Click here for fileText S1. Supplementary methods. Click here for fileGene sequences from different species used to perform phylogenetic analysis.Click here for filePhylogenetic file generated using rbcL and rbcS genes of 24-plant species.Click here for filePhylogenetic file generated using rbcL gene of Meliaceae family members.Click here for file


Objective. To report the first case of bilateral ovarian endometriomas, leading to nonprogress of labour, successfully excised during cesarean section. Design. Case report. Setting. Department of Obstetrics & Gynecology of Dr. RPGMC Tanda, Kangra, India. Patients. A primigravida in labour at term gestation. Interventions. Surgical management. Main Outcome Measures. Description and treatment of a pregnant woman with bilateral ovarian endometriomas during cesarean section. Results. Successful excision of ovarian endometriomas and reconstruction of the ovaries during cesarean section. Conclusion. Management of incidentally detected endometriomas during cesarean section should be individualized, taking into account the symptoms, size, bilaterality, and adhesion with adjacent organs. Endometriosis is an estrogen-dependent condition in which ectopic endometrial glands and stroma are found outside the uterus. Most endometrial deposits are found in the pelvis  .The inciA 32-year-old primigravida at 38 weeks of gestation reported to delivery suite in active labour. She had inadequately supervised antenatal period. No ultrasound scan was done during pregnancy. This was a spontaneous conception after six years of primary infertility. History was also significant for dyspareunia and severe dysmenorrhoea for six years. Her vaginal examination was notable for tender ill-defined mass palpable through posterior fornix. Despite adequate uterine contractions, labour was prolonged and patient was taken up for emergency cesarean section. Lower segment cesarean section was done and a live born healthy male baby weighing 2.7 kg was delivered. Intra-operatively, bilateral ovarian endometriomas measuring 5 × 5 cm each were found stuck in the cul-de-sac. Both the masses were adherent to posterior uterine wall and sigmoid colon, thus partially obliterating the cul-de-sac . AccordiPregnancy has long been considered as having beneficial effect on the course of endometriosis. As Beecham stated, “Nature (since the beginning of time) has employed an efficient prophylactic and curative measure for endometriosis, that is, pregnancy” . The obs


Anaplasma (A.) platys, the causative agent of infectious canine cyclic thrombocytopenia, is endemic in countries of the Mediterranean basin. However, few reports are available from the Balkans. This case report describes a dog, which was imported from Croatia to Germany in May 2010. One month later the dog was presented to a local veterinarian in Germany due to intermittent/recurrent diarrhoea. Diagnostic tests were performed to identify infections caused by Anaplasma spp., Ehrlichia spp., Hepatozoon canis, Babesia spp., Leishmania spp., Borrelia burgdorferi and/or Dirofilaria immitis.It is known that A. platys with a species-specific real-time PCR. Additionally, an infection with Babesia (B.) vogeli was also detected (PCR and serology). No specific antibodies against Anaplasma antigen were detectable. Although the dog showed no specific clinical signs, thrombocytopenia, anaemia and elevated C-reactive protein (CRP) were observed. Sequencing of a 1,348-bp partial ribosomal RNA gene revealed highest homology to A. platys sequences from Thailand, Japan and France.Haematological examination of a blood smear revealed basophilic inclusions in thrombocytes, which were confirmed as A. platys was detected for first time in a dog imported from Croatia. As the dog was also co-infected by B. vogeli, unique serological and haematological findings were recorded. Thrombocytopenia, anaemia and elevated values of C-reactive protein were the laboratory test abnormalities observed in this case. A. platys infections should be considered in dogs coming from Croatia and adjacent regions. Anaplasma platys (formerly Ehrlichia platys) was first identified and described in 1978 in Florida (USA) as a Rickettsia-like, platelet-specific organism in dogs with infectious canine cyclic thrombocytopenia (ICCT) [Ehrichia canis, the microorganism was first proposed as E. platys [A. phagocytophilum and A. marginale, which led to reclassification and designation as A. platys [. platys . Sequenc. platys ,4.A. platys organisms infect peripheral blood platelets and form basophilic inclusions in the cells, so-called morulae, which contain one or more subunits [A. platys infection with ecchymotic haemorrhagia was reported to be caused by a Greek strain [Rhipicephalus sanguines, as in several studies A. platys-DNA was detected in this tick species and co-infections in dogs with E. canis and B. vogeli, two pathogens that share the same vector, reinforce this speculation [R. sanguineus, however, could not be proven so far [A. platys has been described in both American continents , Asia , Austrants . No abnormalities were found during the clinical assessment.9/L; reference range 150-500 × 109/L). The differential and absolute white blood cell (WCB) counts were within the usual range  value 19.1) was accompanied by a B. vogeli-positive PCR . PCR testing for other Babesia , Hepatozoon canis, Ehrlichia spp.  and Leishmania spp. were negative. Serological assays as the SNAP 4Dx test , microplate ELISA for antibodies against Leishmania infantum and IFA on A. phagocytophilum, E. canis, and Leishmania-antigen produced negative results. In contrast, a B. canis-specific ELISA was positive (a low level of antibodies at 20.2 test units was detected).Blood analysis indicated anaemia with erythrocytes at 4.20 T/L (reference range 6-9 T/L), haemoglobin of 9.4 g/dL (reference range 15-19 g/dL) and haematocrit of 32% (reference range 38-55%). The anaemia was classified as normocytic at the upper limit to macrocytic  as well as hypochromic due to a mean corpuscular hemoglobin concentration (MCHC) lower than the reference range . Thrombocytopenia was registered as well  was initiated for four weeks. After three weeks of Doxycycline therapy, a single injection of imidocarb dipropionate (6 mg/kg) was administered subcutaneously. In the following hours after the application the general health condition of the dog worsened, described as an anaphylactic reaction by the in-clinic veterinarian. Despite the immediate use of infusions and the administration of parenteral atropine, the dog died the next day.Blood was collected on the day of the presentation in the clinic. CBC with blood smear review was performed on the sample paying particular attention to blood parasites and hemotropic bacteria, as was complete serum chemistry (IDEXX Vet Med Lab). CRP concentrations were measured by means of a validated CRP immunoturbidimetric assay.A. platys (groEL), A. phagocytophilum (msp2), B. vogeli, B. canis, B. rossi, B. gibsoni (hsp 70), B. conradae (ITS2), H. canis (18S rRNA), Leishmania spp. (GP63), E. canis, E. chaffeensis, E. ewingii (dsb).Total DNA was extracted from whole blood by using QIAamp DNA Blood Mini kit  according to the manufacturer's instructions. Real-time PCR at IDEXX Vet Med Lab was performed using the LightCycler 480 (Roche) with proprietary forward and reverse primers and hydrolysis probes. Target genes for pathogen detection using real-time PCR were as follows: A. phagocytophilum IFA , a microplate L. infantum ELISA , an E. canis IFA , and a microplate B. canis ELISA . In regard to B. vogeli, positive reactions were documented earlier utilizing a B. canis IFA test [® 4Dx®) was used as described elsewhere [® 4Dx® detects antibodies against A. phagocytophilum (p44), Borrelia burgdorferi sensu lato (C6), E. canis  as well as antigen of Dirofilaria immitis.Serological examinations for antibody detection were performed using an IFA test . Furtherlsewhere  followin4. After an initial denaturation step at 94°C the reactions cycled 30 times at 94°C for 30 sec, 58°C for 45 sec, 68°C for 90 sec and finally incubated at 68°C for 5 min in a Mastercycler pro . The PCR products were purified with a peqGOLD Gel extraction Kit  and submitted for sequencing to Eurofins MWG Operon . The PCR product was sequenced three times in both directions. A 1,348-bp partial ribosomal RNA sequence was deposited to GenBank™ under accession number JQ396431.A 1,400-bp fragment of the 16S ribosomal RNA gene was amplified from total blood DNA by using forward (AGAGTTTGATCCTGGCTCAG) and reverse (CGGCTACCTTGTTACGACTT) Anaplasmataceae-specific primers, which were modified according to a study published elsewhere . The reaA. platys and B. vogeli-co-infected dogs [A. platys and B. vogeli found in the dog. Infections with A. platys are difficult to detect, as in most cases the clinical manifestations are not clearly evident. B. vogeli infections usually cause subclinical to mild or moderate changes in adult dogs, however, concomitant conditions or additional pathogens present at the same time can exacerbate the course of infection [With the exception of intermittent/recurrent diarrhoea the dog had no clinical signs, but anaemia and thrombocytopenia were evident in the haemogram Table . Both arted dogs  and are nfection .A. platys has a cyclic character and is considered the result of the destruction of blood platelets by the proliferating pathogen during initial phase of infection, which probably triggers immunologic mechanisms in the subsequent course of the infection [B. vogeli-infected dogs, regenerative haemolytic anaemia is a well-known feature, but not all naturally infected dogs become anaemic. Furthermore, B. vogeli infections do not show a homogenous clinicopathological pattern [B. vogeli infections, even though the decrease of platelets in blood is a consistent haematological abnormality in B. canis infections [A. platys and B. vogeli infections leading to significantly lower numbers of platelets compared to single pathogen infection [A. phagocytophilum, H. canis, Ehrlichia spp. and Leishmania spp., which may occur also in Croatia and could take further influence on haematological parameters [E. canis in Croatia.Thrombocytopenia due to a monoinfection with nfection . In B. v pattern ,30. Indefections . The thrnfection . Both, trameters -34. On tA. platys and B. vogeli. Acute phase response as a part of the innate host defence system is linked to early innate responses for any pathological processes or diseases as reviewed elsewhere [B. canis [E. canis [L. infantum [A. phagocytophilum [A. platys and B. vogeli exist.As representatives for acute phase proteins C-reactive protein  and albumin (negative acute phase protein) were evaluated, which in fact showed abnormal values. While CRP was elevated to up to 38.2 mg/L (reference range 0-9.7 mg/L), the serum albumin was decreased to 2.7 g/dL (reference range 3.2-4.7 g/dL) indicating systemic involvement likely due to a co-infection of lsewhere ,36. In dlsewhere . An incrB. canis , E. caniE. canis , L. infatophilum . Thus faA. phagocytophilum antigen failed to detect antibodies in the present case, but an ELISA based on B. canis antigen produced a positive result. However, the antibody level was low. It is known, that IFA tests with B. canis as a substrate cross-react with antibodies against B. vogeli and B. rossi, but homologous species antigens will cause stronger reactions [B. vogeli infection and follow-up serological examinations after initial PCR-positive results, as shown previously [A. platys was based on cross-reaction with A. phagocytophilum antigen. It has previously been shown, that the serum samples from naturally infected A. platys dogs from USA and China react positively with A. phagocytophilum antigen on the SNAP 4Dx [A. platys infected animals in Portugal react well by means of IFA with A. phagocytophilum antigen [A. platys mono-infection occurs between 13 to 19 days post infection as shown with strains collected in US and Greece [A. phagocytophilum-infections previously [A. platys and E. canis it was documented, that A. platys-specific antibodies were detectable for the first time on average 27 days (range 14-35 days) post infection [A. platys and B. vogeli might have induced a delay in the humoral immunological response in this patient.The serological tests (IFA and SNAP 4Dx) based on eactions . Furthereviously , was notSNAP 4Dx ,42. Furt antigen . Therefod Greece . As the eviously . The othIntermittent diarrhoea was a reason for the presentation of the dog. The possible cause for the diarrhoea remains unknown, as no faecal sample was examined. But, it seems unlikely that intermittent diarrhoea alone could have led to hypoalbuminemia in the dog due to substantial gastrointestinal loss of fluids.B. canis infection, which had been preselected due to renal involvement and treated with imidocarb dipropionate, four dogs died spontaneously and the kidneys of all animals of the group showed degenerative changes of mainly the proximal convoluted tubules as well as necrosis of the whole tubule in some cases [B. vogeli and A. platys infections. Nevertheless, it is recommended to treat simultaneously even mildly azotaemic dogs with proper intravenous fluid therapy when imidocarb dipropionate is applied and lower the dose of the drug (e.g. 3 mg/kg) in patients suspected to have renal involvement, in order to decrease the risk of renal insufficiency [The dog died shortly after imidocarb dipropionate administration. However, the cause of death stays speculative, because no post-mortem examination was performed. An anaphylactoid reaction was observed in this case. Such reactions were described after imidocarb dipropionate administration in rare cases , while sme cases . The autme cases . In the ficiency .A. platys 16S rRNA sequences generated in Thailand (EF139459), Japan (AY077619) and France (AF303467). The analysis of 16S rRNA sequences confirms the previous results, in which little genetic diversity was observed in 16S rRNA sequences of A. platys strains from different geographic locations [Sequencing of the 16S rRNA gene produced a 1,348-bp sequence, which was identical to other ocations ,48.A. platys and B. vogeli infection is well known from previous reports and represents a large fraction of co-infections among tick borne pathogens in dogs [A. platys and B. vogeli [B. vogeli in Croatia and other southern European countries is well documented [A. platys in moderate climate zones and simultaneous coinfections with Babesia spp. allow the assumption that Rhipicephalus sp. e.g. Rhipicephalus sanguineus ticks may serve as the natural vector. On the other hand, this tick species has a wide genetic diversity [A. platys. The detection of A. platys in non-engorged questing adult Rhipicephalus turanicus tick in Israel [Rhipicephalus species actually serve as vectors for this agent.The mixed  in dogs ,26,49. T. vogeli . The occcumented . The nativersity  making in Israel  raises tA. platys infection in a dog imported from Croatia. Based on the owner's declaration, the dog has never travelled abroad before importation to Germany and the dog was presumably infected in Croatia. The infection in Germany is unlikely due to the following reasons: the vectors Rhipicephalus spp. ticks are commonly not present in Germany, even though ticks imported from abroad with travelled dogs can survive in homes with moderate temperatures [B. vogeli and A. platys from the region where the dog spent its final days.To our knowledge this is the first report of eratures . The dogA. platys infection was detected for the first time in a dog imported from Croatia. A co-infection with B. vogeli probably induced the untypical serological results. The major clinical manifestations were thrombocytopenia, anaemia and elevated values of C-reactive protein. A. platys infection should be considered in dogs living in or returning after travel from this area and showing abnormal clinicopathologic findings described in this report.ICCT: Infectious canine cyclic thrombocytopenia; CRP: C-reactive protein; IFA: Immunofluorescence antibody assay; WCB: White blood cell; ELISA: Enzyme linked immunosorbent assay; Ct: Cycle threshold; CBC: Complete Blood Count; SID: Once daily.The authors have no competing interests.VD wrote the manuscript and performed sequencing and analysis of 16S rRNA. NP performed serological and clinicopathologic examinations, case consultation and correction of manuscript. H-JB performed diagnostic PCRs and correction of manuscript. AM performed anamnesis, clinical examination and treatment. RKS supervised and revised the manuscript. All authors read and approved the final version of the manuscript.