Plasmodium vivax is a complex protozoan parasite with over 6,500 genes and stage-specific differential expression. Much of the unique biology of this pathogen remains unknown, including how it modifies and restructures the host reticulocyte. Using a recently published P. vivax reference genome, we report the proteome from two biological replicates of infected Saimiri boliviensis host reticulocytes undergoing transition from the late trophozoite to early schizont stages. Using five database search engines, we identified a total of 2000 P. vivax and 3487 S. boliviensis proteins, making this the most comprehensive P. vivax proteome to date. PlasmoDB GO-term enrichment analysis of proteins identified at least twice by a search engine highlighted core metabolic processes and molecular functions such as glycolysis, translation and protein folding, cell components such as ribosomes, proteasomes and the Golgi apparatus, and a number of vesicle and trafficking related clusters. Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.8 enriched functional annotation clusters of S. boliviensis proteins highlighted vesicle and trafficking-related clusters, elements of the cytoskeleton, oxidative processes and response to oxidative stress, macromolecular complexes such as the proteasome and ribosome, metabolism, translation, and cell death. Host and parasite proteins potentially involved in cell adhesion were also identified. Over 25% of the P. vivax proteins have no functional annotation; this group includes 45 VIR members of the large PIR family. A number of host and pathogen proteins contained highly oxidized or nitrated residues, extending prior trophozoite-enriched stage observations from S. boliviensis infections, and supporting the possibility of oxidative stress in relation to the disease. This proteome significantly expands the size and complexity of the known P. vivax and Saimiri host iRBC proteomes, and provides in-depth data that will be valuable for ongoing research on this parasite’s biology and pathogenesis. Plasmodium vivax is a serious neglected disease that can result in extreme morbidity and possible death [P. vivax and the closely related simian malaria model species P. cynomolgi [Malaria caused by le death –3. It isle death . Additioynomolgi –7, can iynomolgi –9.Plasmodium vivax is phylogenetically distant from the pathogen causing the more lethal form of malaria, Plasmodium falciparum [P. vivax, but outside the African continent the proportion of P. vivax infections has been reported as 41% [lciparum , thus itlciparum . About 4d as 41% . The prod as 41% .Plasmodium vivax merozoites invade reticulocytes [P. falciparum, which has some preference for reticulocytes but invades RBCs (red blood cells) of all ages. These differences are largely unexplored at the molecular level, and are intriguing since under normal circumstances most CD71 high reticulocytes are known to reside in the bone marrow [P. falciparum trophozoite and schizont infected RBC (iRBC) that display knob-like surface structures linked to variant adhesive proteins associated with cytoadhesion and virulence [P. vivax and P. cynomolgi synthesize elaborate caveolae-vesicle complexes (CVCs), which have been observed all along the host cell membrane, with external caveolae cup-like structures and associated cytoplasmic vesicular and tubular structures [95, associated with the caveolae and the cytoplasmic face of the tubules [Plasmodium falciparum iRBC strongly adhere to endothelial cells deep within the venular vasculature, a process linked to severe disease [P. vivax iRBCs, in particular relating to the schizont stage, has only recently been proposed [ulocytes –13, requulocytes  with a sulocytes , which rulocytes –17. Thise marrow . After iirulence , P. vivaructures –20 contaructures . The CVC tubules –22. Plas disease ; endotheproposed –26 and rP. vivax and P. cynomolgi express members of the multigene Plasmodium interspersed repeat (pir) family, encoding PIR proteins with multiple predicted localizations  [P. falciparum and Plasmodium knowlesi, which are encoded by the var and SICAvar multigene families, respectively [P. falciparum erythrocyte membrane protein 1 (PfEMP1) [P. vivax has such mechanisms, involving VIR or other proteins expressed at the surface, remains to be more deeply explored.Another intriguing difference across the species is that  as vir) –31, but ectively –35. The (PfEMP1)  and schi(PfEMP1) ). WhetheP. vivax and P. falciparum, the expressed proteome during the pathogen's life cycle in both mosquito vectors and primate hosts would be expected to differ between stages [For both n stages –39. Eachn stages . ExperimS. boliviensis [P. vivax infections with blood draws timed to enrich individual life cycle stages, allowing increased association of identified proteins with individual developmental stages and disease processes. Using this animal model and 2D lc/ms/ms, and searches based on the P. vivax  genome with 5459 genes [P. vivax and 3209 S. boliviensis monkey host proteins in 71–91% enriched trophozoite-stage preparations [P. vivax trophozoite-stage proteins and 485 Aotus monkey host proteins in a pooled sample enriched for 70% trophozoites, and 727 P. vivax schizont-stage and 310 Aotus monkey host proteins in a pooled sample enriched for 91% schizonts [P. vivax proteins from a single patient [P. vivax proteins from cultured schizont-stage enriched iRBCs from a multi-patient pool also containing gametocytes [Many of these challenges can be overcome by use of nonhuman primate (NHP) models, such as the Bolivian squirrel monkey, iviensis , 41–43, 59 genes , we havearations . Another patient , 154 fro patient , and 314P. vivax blood-stage cycle, including modeling potential, we have developed two biological replicate trophozoite-schizont transition-stage proteomes from P. vivax  iRBCs purified from infections of two S. boliviensis monkeys. These are the first proteomes focusing on the actively growing transition period between the P. vivax trophozoite and schizont stages, including large late-stage trophozoites and early multi-nucleated (2–4) schizonts. Using methods based on four 2D lc/ms/ms runs per proteome and analysis with five database search engines [P. vivax and 3487 S. boliviensis host RBC proteins at a ~2% false discovery rate (FDR) for each search engine. We identified a number of post-translational modifications including oxidized host and parasite proteins suggestive of stress responses, as also reported in the trophozoite-enriched proteome [P. vivax proteins and DAVID functional annotation clustering identified host and/or pathogen enriched protein clusters that are predicted to be important, and perhaps essential, for a variety of iRBC growth and development biological and pathogenic processes.With the goal of continuing to expand knowledge of the  engines , 49 we iproteome , and a nSaimiri boliviensis monkeys were acquired by the Yerkes National Primate Research Center (YNPRC), an Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) internationally-certified institution, from the Keeling Center for Comparative Medicine and Research, UT MD Anderson Cancer Center, supported by the National Institutes of Allergy and Infectious Diseases.The animals were socially housed in pairs at the YNPRC, and all housing was in compliance with Animal Welfare Act regulations as well as the Guide for the Care and Use of Laboratory Animals. Standard procedures for splenectomy, monitoring the clinical conditions of the animals, collecting biological samples, treatment, and euthanasia were approved by Emory University’s Institutional Animal Care and Use Committee (IACUC) with the approval # YER-2003225. All nonhuman primates used in this study were provided regular environmental enrichment opportunities consisting of daily feeding enrichment, provision of manipulanda, and physical enrichment. Subjects were regularly monitored for any behavioral signs of distress by the YNPRC behavior management personnel. Animals were trained using positive reinforcement to allow blood collections from the ear without sedation.Anesthesia was performed with Ketamine (5–10 mg/kg IM) or Telazol . Euthanasia was performed per the recommendations of the "AVMA Guidelines for the Euthanasia of Animals: 2013 edition". Intravenous (iv) injection of barbiturates is an acceptable method of euthanasia for nonhuman primates, per these recommendations, and was approved by Emory’s IACUC. Animals are first anesthetized with either ketamine or telazol and when possible a catheter is placed in a peripheral vessel to ensure delivery. Pentobarbital 100mg/kg is injected intravenously and the animal is monitored and auscultated for cessation of heart beat and breathing.P. vivax Sal-I blood-stage infections were initiated in S. boliviensis monkeys using procedures discussed previously [S. boliviensis monkeys were inoculated with cryopreserved and reconstituted P. vivax  iRBC monkey-adapted stocks acquired from the Centers for Disease Control and Prevention (CDC) and monitored daily; 0.5–1.0 ml of blood with a parasitemia of 0.5–1% was transferred from donor to recipient monkeys. These iRBCs had been passaged previously at the CDC in splenectomized S. boliviensis to ensure adequate peak parasitemias  and high iRBC yields; thus, splenectomies were performed in this study prior to infection to ensure comparable yields. The spleen modulates iRBC parasite variant antigen surface expression in P. knowlesi and P. falciparum infections [P. vivax protein.Two independent eviously . Brieflyfections –53; but,The parasite density was estimated from microscopy analyses of thin blood smears, counting 2000 RBCs. Blood with respectively 0.5% and 0.9% parasitemia was collected from SB3256 and SB5115 monkeys into sodium heparin tubes and processed through ADP-glass beads and a Plasmodipur filter using standard procedures to remove platelets and white blood cells, respectively. The infected blood was then layered onto a 52% Percoll gradient to concentrate and further purify the iRBCs; platelets were less than 0.1% of the original platelet count after purification. The late-stage trophozoite/early-stage, 2–4 nucleated schizont/gametocyte differential microscopy readings for Proteomes 1 and 2 respectively were 44%/56%/0% and 40%/60%/<1%. These iRBC samples were frozen at -80 C and thawed at a later date for generation of tryptic peptides and subsequent proteomic analysis.Plasmodium vivax  iRBC proteins and peptides were prepared for analysis using the FASP-II protocol [Two biological replicate proteomes were analyzed. protocol  and analprotocol . Four 2Dprotocol .P. vivax reference genome [S. boliviensis fasta protein database , combined with common contaminants such as the trypsin used for protein digestion, human cytokeratins, etc. [P. vivax Indonesian clinical isolate [P. vivax reference genome [Database search engines used a PvP01 e genome  derived ns, etc. . Althoug isolate , it was e genome , due to e genome .Data analysis utilized five search engines. Andromeda   used preP. vivax or S. boliviensis biology. Different search engines used different algorithms for protein grouping; proteins are thus presented as individual proteins independent of groups, with information on individual search engine results presented in Identification at least twice by a search engine (similar to ) was reqP. vivax genome to date. Identifications by SEQUEST from separate searches for oxidative modifications and basic modifications  were combined.To streamline the modification identification protocol used previously for trophozoite-enriched proteomes , post-trS. boliviensis proteins is generally listed when available. The source of the functional annotation, when not obvious from the protein description, is listed in a separate annotation column. The "transcription" functional category includes transcription factors, RNA polymerase complex proteins, and proteins involved in RNA polyadenylation, capping and splicing, and RNA transport to the cytoplasm. The "translation" category includes ribosome assembly proteins, ribosomal proteins, and proteins involved in elongation on tRNA and termination.Functional annotation for piecharts, of proteins identified at least twice by a search engine, was from PlasmoDB , UniprotP. vivax proteins used PlasmoDB [GO term enrichment (relative to the whole genome) for PlasmoDB  release PlasmoDB  is listeS. boliviensis identified proteins, we used Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.8 Bioinformatics Resources [Saimiri boliviensis proteins were first blasted against the human genome using BlastP [For analysis of esources –75. Saimg BlastP  with theS. boliviensis and P. vivax proteins combined from both proteomes; in total, 2000 P. vivax and 3487 S. boliviensis proteins were identified by at least one search engine.A summary of proteins identified in Proteomes 1 and 2 is listed in P. vivax and S. boliviensis proteins are listed in P. vivax protein, glyceraldehyde-3-phosphate dehydrogenase (GPDH), is ca. 15-fold more abundant than the same (but distinct) S. boliviensis protein. Other abundant P. vivax proteins include a number of additional glycolytic enzymes , the four core histones comprising a nucleosome, two heat shock proteins (HSP), ribosomal protein P2, elongation factor 1 alpha, the CVC protein PHIST/CVC-8195 (PVP01_0119200) [P. falciparum [S. boliviensis proteins include five hemoglobin chains, ribosomal and ubiquitin-related proteins, the Ig superfamily hypothetical protein C17orf99 homolog, cytoplasmic actin, flavin reductase, two carbonic anhydrase enzymes, and the proinflammatory cytokine IL-36 beta. Nine different complement proteins are identified at least twice by a search engine; of these three  have a relative abundance in the range of 0.15–0.21, and six  have a relative abundance in the range of 0.02–0.07).The relative abundances of 0119200) , and an lciparum . The mosP. vivax and 1376 S. boliviensis proteins. Individual protein functional categories are listed in P. vivax and 64 S. boliviensis proteins annotated as mitochondrial proteins. The largest P. vivax category, including ca. 22% of this proteome, consists of hypothetical proteins without functional annotation. Other major categories include translation, metabolism, proteolysis, transcription and trafficking. Two additional significant categories include Plasmodium exported proteins and PIR/VIR proteins. Saimiri boliviensis iRBC proteins were more evenly divided among a number of categories, the largest being metabolism, signaling, proteolysis, transcription and translation. About half of the identified cytoskeletal proteins were linked to actin. For both organisms, the functional pie charts are similar to those from our previously reported trophozoite-enriched stage [P. vivax hypothetical proteins. Identification of PIR/VIR proteins was also substantially increased (to 45) compared to analysis with the Sal-I database, which could reflect stage or isolate specific differences though also likely reflects the improved assembly and annotation of the telomeres in this reference sequenceed stage ; use of P. vivax proteins  and the Benjamini-Hochberg FDR listed for the top term of each cluster. The clusters are grouped into sets of similar clusters, representing vesicles and trafficking, the cytoskeleton, oxidation, pathogen processes, and homeostasis. The most highly enriched single cluster includes extracellular exosomes for both Proteomes 1 and 2, with an 80-154-fold enrichment. The cytoskeleton set of clusters consists of 8–12 individual clusters for Proteomes 1 and 2, including enrichment of e.g. the actin cytoskeleton, microtubules, spectrin-associated cytoskeleton, and adherens junctions. Analysis of both proteomes highlights a set of oxidation-related clusters, which includes response to oxidative stress and glutathione metabolic processes, and in the more oxidized Proteome 1, antioxidant activity, cell redox homeostasis, hydrogen peroxide metabolic processes and oxidation clusters were prominently identified. Both proteomes contain the ~10–30 fold enriched cluster "symbiosis encompassing mutualism through parasitism"; Proteome 1 also contains clusters for pathogenic E. coli infection and three viral infection-related clusters. Details of the clusters are presented in S. boliviensis enriched protein annotation clusters related to protein folding and the response to unfolded proteins, macromolecular complexes (e.g. the proteasome (both proteomes), and ribosome, spliceosome, and nucleosome in Proteome 1), metabolism, RNA-related clusters (particularly translation and splicing), and a set of cell death clusters including apoptotic mitochondrial changes, regulation of neuron apoptotic processes, and regulation or negative regulation of (programmed) cell death. Additional clusters in Proteome 1 include regulation of apoptotic signaling pathway and the mitochondrion. Details of each individual cluster are presented in P. vivax proteins from the trophozoite-schizont transition proteomes that are annotated as, or predicted to be potentially involved in cytoadhesion, and identified at least twice by a search engine. These include four merozoite rhoptry proteins [proteins , 79–80 aproteins . An addiproteins  were ideS. boliviensis proteins are also annotated as involved in cytoadhesion, including the transferrin receptor , shown to be an important marker (when at high levels) of reticulocytes preferentially invaded by P. vivax [P. yoelli [Sixteen P. vivax  and P. y. yoelli . Other pP. vivax and S. boliviensis trophozoite-stage enriched proteins in two biological replicates [S. boliviensis hemoglobin, actin, and the P. vivax CVC protein PHIST/CVC-81-95 [P. vivax reference genome sequence [We previously reported significant oxidation and nitration of both plicates , includiVC-81-95 . Table 4sequence  for resiMycobacterium smegmatis proteome prepared using the same FASP-II protocol, electrosprayed under identical conditions [S. boliviensis proteins or subunits with at least one high confidence oxidized residue and 144 oxidized P. vivax proteins or subunits.nditions , and ananditions , and trpP. vivax and 3487 S. boliviensis trophozoite-schizont transition proteins by at least one of five search engines, using two biological replicates, expanding identifications from previous P. vivax trophozoite and schizont proteomes, and enhancing the modeling potential of this parasite’s biology by the broad research community. All data has been deposited in PlasmoDB. In addition to analysis of each biological replicate with four separate 2D lc/ms/ms runs, and use of higher amounts of tryptic peptides than our P. vivax trophozoite proteome published in 2015 with 1375 P. vivax and 3209 S. boliviensis proteins [We have identified 2000 proteins , analysiproteins , 66, 44 proteins , which mS. boliviensis host transferrin receptor protein 1  was identified in the trophozoite-enriched as well as the current proteomes, a point worth noting given P. vivax’s known preference of invading CD71 high reticulocytes and open questions surrounding the parasite-host receptor-ligand and invasion requirements [P. vivax schizont stage–with up to 16 merozoites–would be valuable for the characterization of late-stage schizont proteins including a predominance of merozoite proteins that are important for egress from their host RBCs and invasion of reticulocytes to propagate the blood-stage infection as well as development of new sexual stage forms for infection of the Anopheles mosquito vector and continued transmission. While genome and transcriptome studies  can predict this parasite species and stage-specific proteomes and help generate hypotheses, proteomic studies provide the evidence of protein expression. Future time course analyses with integrated omics  will help develop a deeper appreciation of the parasite’s unique biology, over ~48 hours between the invasion, takeover, and destruction of the reticulocyte with the release of new merozoite progeny.Interestingly, the irements , 16, 85.P. vivax trophozoite-schizont stage protein is GPDH, ca. 15-fold more abundant than the same (but distinct) S. boliviensis protein, and ca. 6–10 fold more abundant than additional P. vivax glycolytic enzymes. Elevated levels of the P. vivax glycolytic enzymes are consistent with 50–100 fold increased glucose consumption in P. falciparum-infected erythrocytes [P. vivax proteins include heat shock proteins (HSPs), histones H4 and H2B, and elongation factor-1-alpha, as well as the P. vivax caveolae vesicle complex PHIST protein CVC-81-95 reported originally to be a main protein of the CVCs [P. falciparum schizont-stage proteome also includes as highly abundant proteins GPDH and other glycolytic enzymes, histones H4 and H2B, three HSPs and elongation factor-1-alpha [The most abundant hrocytes , as glychrocytes . Enzymeshrocytes . Other athe CVCs –22. The -1-alpha .S. boliviensis protein identified is hemoglobin, whose alpha subunit is ca. 160-fold above levels of the next most abundant non-hemoglobin protein, GPDH. Two host carbonic anhydrases are identified here at relatively high levels. Carbon dioxide is essential for iRBC growth, pyrimidine biosynthesis and control of intracellular pH [2 transport across the RBC plasma membrane. This parasite enzyme has been noted as a potential malaria drug target [P. vivax malaria [P. falciparum iRBC [P. knowlesi [As expected, the most abundant lular pH ; carbonig target –90; the  malaria  and comprum iRBC –93 and Pknowlesi .P. vivax GO-term enrichment. DAVID functional annotation clustering and PlasmoDB-based GO-term enrichment focus on gene sets enriched for particular molecular functions, biological processes or cellular locations.Protein function is presented using several methods. Piecharts give a broad overview of the proteome and highlight some significant areas such as hypothetical proteins, exported proteins, and PIR proteins less readily observed by P. vivax infected host cell membrane [P. vivax PHIST protein CVC-8195 [P. yoelii exosomes have in fact been shown to contain parasite proteins, and these may be involved in cell-cell communication and immune modulation [P. vivax cluster from Proteome 2, is important for secreted proteins; 69 exported proteins, related proteins, or Plasmodium exported proteins were also identified. This retrograde transport can involve COPI-coated vesicles, the Golgi apparatus, and Golgi-associated vesicles [P. vivax GO-term clusters, as well as protein folding in the ER. Gautier et al. [Little is known about the restructuring of the host reticulocyte and the development of numerous caveolae vesicle complexes (CVCs), which include parasite-encoded proteins positioned all along the membrane –22. In aCVC-8195  may wellCVC-8195 , 95–96; dulation . Proteindulation . Retrogrvesicles , all of r et al.  identifir et al. –103, apir et al. , and apir et al. –106 iRBCr et al. . ROS andr et al.  and may r et al. .P. vivax cytoskeletal proteins  were also identified.A second area of protein annotation cluster enrichment in Proteomes 1 and 2 includes DAVID-derived host cytoskeleton-related clusters, e.g. the actin cytoskeleton, microtubules, the spectrin-associated cytoskeleton, contractile fibers, and adherens junctions. When orthochromatic erythroblasts enucleate to form reticulocytes, cytoskeletal proteins, including the spectrin cytoskeleton, microtubules, myosin and actin  are reta2+ entry, ceramide formation, caspase stimulation, energy depletion, and oxidative stress [Plasmodium falciparum infection of erythrocytes can induce oxidative stress and trigger eryptosis [P. vivax anti-apoptotic protein bax inhibitor 1, which could inhibit bax-mediated apoptosis signaling and premature iRBC death, as well as 64 host and 15 parasite mitochondrial proteins. It is possible that some of these proteins are contaminants from platelets not completely removed during iRBC purification; however in a proteome of human reticulocytes isolated from cell culture, 6 apoptosis proteins partition after orthochromatic erythroblast enucleation into the reticulocyte at a level of 5% or higher, as do 105 mitochondrial proteins and 198 nuclear or nucleolar proteins [P. vivax iRBC biology and the normal host response to parasitism.A third set of enriched clusters includes host protein DAVID-identified cell death-related clusters in both proteomes, e.g. regulation of (programmed) cell death, apoptotic mitochondrial changes, and regulation of neuron apoptotic processes. Reticulocytes are enucleated; however anucleate cells can undergo forms of apoptosis. Anucleate platelet apoptosis has some features in common with nucleated cell apoptosis, e.g. expression of pro- and anti-apoptotic proteins, depolarization of the mitochondrial inner membrane and cytochrome c release, surface exposure of phosphatidyl serine, activation of caspase 3, platelet shrinkage and fragmentation . Red bloe stress . Plasmodryptosis ; prematuryptosis . Reticulryptosis , 116. Weryptosis . We alsoproteins . It is tP. vivax and S. boliviensis proteins in the two transition proteomes, as before for a trophozoite proteome [We evaluated oxidized proteome , as theyproteome , which dproteome , which wS. boliviensis functional annotation clusters for response to oxidative stress, antioxidant activity, cell redox homeostasis, oxidation, hydrogen peroxide metabolic processes, glutathione metabolic processes, and P. vivax cell redox homeostasis and antioxidant enriched GO-term clusters in the more heavily oxidized Proteome 1  or those close to the site of ROS (reactive oxygen species) or RNS (reactive nitrogen species) generation. Highly reactive hydroxyl radicals with a short lifetime (ca. 2 ns) will derivatize proteins only within ca. 20 Å of their source . Our obs2O2 and O2 [2O2 and O2 by superoxide dismutase. The Fenton reaction, utilizing Fe2+, produces OH. radicals from H2O2 [S. boliviensis actin at Y240, may in fact result in altered function, as noted earlier [ROS can be generated by degradation of hemoglobin in food vacuoles , the hig2 and O2 ; in the rom H2O2 , which c earlier .P. falciparum trophozoite food vacuoles [P. falciparum, to a food vacuole-associated cytochrome b5 [Plasmodium vivax trophozoite and schizont iRBC also contain food vacuoles; thus, the S. boliviensis cytochrome b5 identified in the transition proteome could potentially contribute to NO generation. Nitric oxide can also be generated by reaction of deoxyhemoglobin with the erythrocyte cytosolic metabolite nitrite [The significant protein nitration we observe could result from induction of the pathophysiological NO/ONOO cycle, some elements of which are observed here . Howevervacuoles –124; thehrome b5 –124. Pla nitrite –131, whi nitrite .P. vivax iRBC by other events. The hemoglobin catabolite hemozoin, produced in iRBC food vacuoles, increases iNOS (inducible nitric oxide synthase) in macrophages [It is also possible that NO production is initiated in rophages . Inductirophages  in iRBC rophages , 132 prorophages .P. falciparum is due in part to strong iRBC adhesion to the endothelium in the deep venular vasculature [Plasmodium vivax iRBC may adhere to endothelial and other cell types under static and flow conditions [P. vivax schizont iRBC observed in the blood may be due to enhanced schizont adhesion compared to other stages of asexual development [P. vivax trophozoite-schizont transition proteome for proteins that might be involved in adhesion. Enhanced sequestration of P. falciparum iRBC may involve altered expression of endothelial cell adhesion receptors linked to oxidative stress [P. vivax as well.Virulence of culature . Plasmodnditions , adhere nditions ; or undenditions . Lower lelopment . As iRBCe stress . In viewP. vivax analogs of P. falciparum erythrocyte binding or adhesion proteins, as well as 29 additional MAAP-predicted adhesins [S. boliviensis proteins are implicated in cell adhesion processes, including thrombospondin-3, fermitin family homolog 3, protocadherin 10-like, vitronectin, talin-2, neuroplastin, retinoschisin and PDZ domain-containing protein 2. Thus both host and/or parasite proteins may potentially be involved in adhesion.The proteome identified here contains 45 PIR/VIR proteins identified at least twice by a search engine; we recognize that details of the expressed VIR proteins (e.g. sequences outside of identified tryptic peptides) could differ between the PvP01 isolate and Sal-I strain, and that a Sal-I genome with improved subtelomere sequencing may enable the identification of additional VIR proteins. Individual VIR proteins could have multiple functions, including immune evasion by adherence to spleen barrier cells to escape clearance by spleen macrophages , 136, adadhesins . SurfaceP. vivax trophozoite-schizont transition iRBC proteome using a new P. vivax reference genome, identifying 2000 P. vivax and 3487 host S. boliviensis proteins. Over 22% of P. vivax proteins still have no functional annotation, highlighting a large gap in the basic molecular understanding of P. vivax biology. Oxidation and nitration observed in the trophozoite stage-enriched proteome appear also to be present at the transition to the schizont stage and could reflect iRBC oxidative stress. GO-term enrichment analysis highlighted a number of parasite metabolic processes and molecular functions including glycolysis, translation and protein folding, cell components such as ribosomes, proteasomes and the Golgi apparatus, and a number of vesicle- and macromolecule/organelle trafficking-related clusters. DAVID analysis of S. boliviensis proteins included cytoskeletal actin-, tubulin- and spectrin-related clusters, oxidative processes and response to oxidative stress, vesicle and trafficking-related clusters including exosomes and exocytosis, macromolecular complexes such as the proteasome and ribosome, metabolism, translation, and cell death. Host and parasite proteins potentially involved in cell adhesion were also identified. These identifications may provide details for a deeper understanding of P. vivax biology as well as hypotheses for the functional involvement of individual proteins in iRBC biology, parasitism, immunity and pathogenesis.In this paper, we have examined the S1 TableP. vivax proteins are indicated by accession numbers beginning with PvP01_ and S. boliviensis proteins are identified by accession numbers consisting of nine numbers. Crux did not include protein annotations with identifications. XID is the identification number of the protein; Xrep is the number of search engines identifying the protein. Proteins identified by all 5 engines are listed in the worksheet "5x of 5x"; proteins identified by only one of 5 engines are in the worksheet "1x of 5x" etc. Avg. Xrep is the average number of times a search engine identified a protein from within the four individual 2D lc/ms/ms runs for each proteome. For Maxquant, all four individual 2D lc/ms/ms runs were analyzed at once , thus Avg. Xrep is 1 for all proteins. For Sequest, all four 2D lc/ms/ms runs were analyzed twice Click here for additional data file.S2 TableP. vivax proteins are indicated by accession numbers beginning PvP01_, and S. boliviensis proteins are identified by accession numbers consisting of nine numbers. XID is the identification number of the protein; Xrep is the number of search engines identifying the protein. Proteins identified by all 5 engines are listed in the worksheet "5x of 5x"; proteins identified by only one of 5 engines are in the worksheet "1x of 5x" etc. Xrep is the number of search engines identifying the protein; Avg. Xrep (as for This table shows Proteome 2 identifications using 5 different search engines. p as for  is the a(XLS)Click here for additional data file.S3 TableP. vivax and S. boliviensis proteins identified at least once by a search engine, obtained by combining This table contains all of the (XLS)Click here for additional data file.S4 TableP. vivax and S. boliviensis proteins, for all proteins identified by Mascot using the exponentially multiplied protein abundance index (emPAI) [This table contains the relative abundance of  (emPAI) . Values (XLS)Click here for additional data file.S5 TableP. vivax and S. boliviensis. Proteins included have been identified at least twice by a search engine in the combined proteomes. The functional categories are listed to the right of the proteins, which are identified by accession number. Xrep represents identification of the protein in one or both proteomes; Avg. Xrep is the average number of times a single search engine identified a protein from within the four individual 2D lc/ms/ms runs for each proteome, and must be at least 2. The protein descriptions from the database used for identification are also listed. Some details from additional annotation (e.g. from Uniprot) are listed in the Annotation column. Proteins are placed into broad functional categories of interest, e.g. cytoskeleton(-related) as indicated. More than one functional category may be listed when this is supported by available information.This table shows the functional categorization of proteins identified in combined trophozoite-schizont transition Proteomes 1 and 2 for both (XLS)Click here for additional data file.S6 TableP. vivax and S. boliviensis are listed in layer 1, and details of peptide Sequest scores, and modifications of individual peptides, are listed in layer 2 for each proteome. Proteins containing only methionine oxidized to met sulfoxide, which can occur in solutions exposed to air, are excluded. Proteome 1 contains 52 oxidized S. boliviensis and 83 oxidized P. vivax proteins or subunits, Proteome 2 has 38 oxidized S. boliviensis and 86 oxidized P. vivax proteins or subunits; the combined proteome contains 74 different oxidized S. boliviensis and 143 different oxidized P. vivax proteins or subunits.This table lists all Sequest-identified proteins containing at least one high confidence (Percolator posterior error probability of 0.01 or less) oxidized peptide, in trophozoite-schizont transition Proteomes 1 and 2. Proteins from both (XLS)Click here for additional data file.S7 TableP. vivax proteins from proteome 1.Enrichments were obtained using PlasmoDB. The table lists all GO terms, including GO_biological process, GO_molecular function, and GO_cellular component, enriched for (XLS)Click here for additional data file.S8 TableP. vivax proteins from proteome 2.Enrichments were obtained using PlasmoDB. The table lists all GO terms, including GO_biological process, GO_molecular function, and GO_cellular component, enriched for (XLS)Click here for additional data file.S9 TableS. boliviensis proteins identified at least twice by a search engine. Each cluster is composed of a set of similar GO terms . The overall cluster is generally named for the top term or a consensus of individual terms; each GO term has a p-value and Benjamini-Hochberg false discovery rate calculated by DAVID 6.8. Annotation clusters for Proteomes 1 and 2 are in separate tabs.This table presents results for all DAVID-derived annotation clusters of (XLS)Click here for additional data file.S10 TableP. vivax and S. boliviensis proteins identified in trophozoite-schizont transition Proteomes 1 and/or 2 at least twice by a search engine, that are either annotated as involved in cell adhesion, or predicted to be adhesins [This table lists adhesins . We also(XLS)Click here for additional data file.


Stenotrophomonas maltophilia ubiquitously occurs in the hospital environment. This opportunistic pathogen can cause severe infections in immunocompromised hosts such as hematopoietic stem cell transplantation (HSCT) recipients. Between February and July 2016, a cluster of four patients on the HSCT unit suffered from S. maltophilia bloodstream infections (BSI).S. maltophilia isolates. Environmental and blood culture samples were subjected to whole genome sequence (WGS)-based typing.For epidemiological investigation we retrospectively identified the colonization status of patients admitted to the ward during this time period and performed environmental monitoring of shower heads, shower outlets, washbasins and toilets in patient rooms. We tested antibiotic susceptibility of detected S. maltophlilia BSI, three were found to be colonized previously. In addition, retrospective investigations revealed two patients being colonized in anal swab samples but not infected. Environmental monitoring revealed one shower outlet contaminated with S. maltophilia. Antibiotic susceptibility testing of seven S. maltophlia strains resulted in two trimethoprim/sulfamethoxazole resistant and five susceptible isolates, however, not excluding an outbreak scenario. WGS-based typing did not result in any close genotypic relationship among the patients’ isolates. In contrast, one environmental isolate from a shower outlet was closely related to a single patient’s isolate.Of four patients with S. maltophilia on a HSCT ward but uncoverd that sanitary installations can be an actual source of S. maltophilia transmissions.WGS-based typing successfully refuted an outbreak of  Stenotrophomonas maltophilia, an intrinsically multidrug resistant gram negative pathogen, is widely distributed in aqueous habitats such as, sink drains, endoscopes and hemodialysis water within clinical settings [settings , 2. Usuasettings . Recipiesettings .S. maltophilia is reported on oncologic wards [S. maltophilia isolates [In recent years, an increasing incidence of ic wards . Moreoveic wards , 7. In aisolates –11. Thesisolates  and the isolates –17. Moreisolates .S. maltophilia bacteremia in recipients of allogeneic stem cell grafts using WGS-based typing.Here we evaluated a cluster of The 1500-bed University Hospital Münster includes two HSCT-units each comprising 10 patient rooms, which are all HEPA-filtered and equipped with separated bathrooms for each patient  were defined as one or more blood cultures positive for S. maltophilia obtained from patients with clinical signs of infection  according to the critieria given by the European Centre for Disease Prevention and Control [S. maltophilia BSI were detected in patients  in the HSCT ward between February and July 2016. As this number exceeds the average baseline of 0.5 S. maltophilia BSI per year detected during 2011–2015, an epidemiological investigation was initiated. Routine environmental screening of aqueous habitats  within patients’ rooms in the HSCT unit, normally focusing on multidrug resistant Pseudomonas aeruginosa, was expanded to S. maltophilia. Patients colonized with S. maltophilia, coincidentally detected in anal/rectal swabs or stool samples (as screening of HSCT-patients concentrates on multidrug resistant Pseudomonas aeruginosa), were retrospectively identified. Moreover, hand hygiene and surface disinfection measures were intensified. Control . Four S.Positive blood culture samples were detected using an automated blood culture system . Environmental samples were plated on Columbia sheep blood agar  and MacConkey agar  after enrichment in Tryptic Soy Broth (Becton Dickinson) for 24 h at 36 °C. Subsequent species identification was performed by Matrix-Assisted Laser Desorption/Ionization-Time of Flight-Mass Spectrometry . Susceptibility testing for trimethoprim-sulfamethoxazole (TMP-SMX) was performed using disk diffusion method in accordance with the European Committee on Antimicrobial Susceptibility Testing (EUCAST) standards and interpreted using EUCAST clinical breakpoints (version 6).S. maltophilia strains isolated from blood cultures, the isolates were subjected to WGS using the Illumina MiSeq platform  as described previously [+ software version 2.0 beta , all coding regions were extracted and compared in a gene-by-gene approach  using S. maltophilia K279a strain (GenBank accession number AM743169.1) as a reference sequence. Instead of a published cgMLST scheme, which is not yet available, this ad hoc scheme was used to differentiate the cluster. SeqSphere+ software was used to display the clonal relationship in a minimum-spanning tree (MST). For backwards compatibility with classical molecular typing, i. e. MLST, the MLST sequence types (ST) were extracted from the WGS data in silico.To determine the clonal relationship of eviously . RetrospS. maltophilia is displayed in Fig. S. maltophilia bacteremia, three  were previously tested positive for S. maltophilia in stool samples or anal swabs. Additionally, two colonized patients  admitted to ward 2 were uncovered by the retrospective analysis (Table S. maltophilia infection. Susceptibility testing revealed all patient isolates to be susceptible to TMP-SMX, except isolate P3. The environmental isolate was resistant to TMP-SMX (Table Chronological order and spacial distribution of isolated S. maltophilia BSI suffered from acute myeloid leukemia as the underlying disease. P2 and P5, who were only colonized by S. maltophilia, suffered from chronic lymphatic leukemia and myeloproliferative neoplasm. Except P1, all patients had already received allogenic SCT at the time of S. maltophilia detection. P1 and P6 developed an acute respiratory distress syndrome. P1, P3, and P6 died due to S. maltophilia bacteremia.All patients with S. maltophilia strains isolated from P3 and E1 showed a similar genotype with only four alleles difference, suggesting a relationship between these two isolates. P3 and E1 harbored the MLST ST 94. All other isolates harbored new MLST STs.We analyzed four bloodstream isolates and the environmental isolate by WGS. Comparison via cgMLST, based on 1876 genes present in all isolates, revealed no genetical relationship among isolates originating from patients Fig. . In contS. maltophilia has emerged as an important nosocomial pathogen associated with increased mortality rates in patients suffering from hematological malignancies and receiving hematopoietic stem cell transplantations [S. maltophilia BSI in patients partially suffering from pulmonary dysfunction associated with the infection due to this pathogen [ntations , 21. In pathogen . Susceptpathogen , excludeS. maltophilia BSI. Due to its multidrug resistant nature, selection of this pathogen can easily occur in patients after receiving broad-spectrum antibiotics [S. maltophilia infections occur frequently in combination with the distinct immunocompromised status of HSCT recipients [S. maltophilia within hospital settings [A number of different sources are possibly responsible for the increasing number of ibiotics . Moreovecipients . On the settings .S. maltophilia could not yet be shown in detail. In this study we documented a genetical relation between an environmental and a patient blood stream infection isolate by analyzing WGS data. Although there was no local proximity, S. maltophilia could be transmitted from the environment to a patient or vice versa, at least giving the possibility of further spread via aqueous habitats in the HSCT unit.To what extent these habitats are origins for hospital-acquired colonizations or infections of S. maltophilia. Additionally, WGS- based typing documented sanitary installations in HSCT units to be an actual source or result of transmission between environmental and human habitats. Hence, in addition to classical infection control strategies, monitoring of aqueous habitats has to be established within the rooms of HSCT recipients in order to prevent transmission of these multidrug resistant organisms, as recently also shown for P. aeruginosa [WGS- analysis can be used to precisely refute an outbreak of ruginosa .


Bombyx mori) and wild (Antheraea pernyi) silk fibers were characterised over a wide spectral range from THz 8 cmAntheraea pernyi moths. Low energy T-ray bands at 243 and 229 cmAntheraea pernyi silk fibers. Systematic analysis and assignment of the observed spectral bands is presented. Water solubility and biodegradability of silk, required for bio-medical and sensor applications, are directly inferred from specific spectral bands.Domestic ( Such coSilk II)  as well Silk II) ,10.Mechanisms controlling crystallisation of protein coils via ordered hydrogen bonding and their unzip-decomposition at different annealing temperatures, laser and electron beam exposure conditions are of interest for applications in material science and bio-medical fields ,12,13. SPhoto-thermal control of ecovered . However0−7 m2/s , likewisHere, transmittance measurements of silk over a broad spectral range from THz wavelengths (T-ray) until deep UV are reported and spectral signatures of the constituent components of silk: T, down to ∼77 K temperature.The THz/Far-IR Beamline at the Australian synchrotron was used to characterise silk in the 40–600 cmFor the largest T-ray wavelengths in the 8–80 cmr pulses ,24. The ick ZnTe .A, can be unambiguously deduced, calculated as T is the field transmittance corresponding to the ratio of the Fourier magnitudes. The fields transmitted by a reference and by the sample are alternatively acquired 50 times to give the standard deviation shown as error bars in The amplitude and phase-resolved fields transmitted through the silk samples, as well as through a reference (air), are Fourier transformed to give frequency-dependent amplitudes as well as their phases. If the sample thickness is well defined and only the first Fresnel coefficient can be considered, the phase difference of these Fourier transforms is directly related to the real part of the index of refraction, whereas the ratio of the field magnitudes yields the absorption coefficient. In the case of silk fibers, however, the sample thickness is not well defined, so only the absorbance, Shorter wavelength characterisation was carried out with a UV-Vis spectrometer  by measuring total transmittance and reflectance of fibers in a 150 mm integrating sphere geometry. Photoluminescence excitation spectra were collected using a fluorescence spectrometer LS55 (PerkinElmer), whereas a FT-IR spectrometer  was used for far-IR-to-near-IR transmittance measurements. Spectral ranges of the selected tools allowed for continuously covering an unprecedentedly wide spectral range from T-rays to deep-UV.d for which analytical solutions are known. Mie scattering has very strong polarization and angular dependence [The measured spectral properties over a large range of wavelengths have different contributions of Rayleigh scattering, which is proportional to pendence , which mBombyx mori and wild Antheraea pernyi species of silk worms. A. pernyi silkworm cocoons were obtained from Liaoning province, China. B. mori was collected from the silk rearing house in Jiangsu province, China. The silk fiber from B. mori has superior elasticity and toughness due to the way the disordered fibroin matrix is reinforced by glycine and alanine based A. pernyi has a different primary structure of fibroin, composed of alternating appearances of large repetitive poly-alanine blocks and glycine rich regions [B. mori but has distinct similarities to spider dragline silk [A. pernyi survives at the lowest temperatures among all silk moths. Raw cocoons were degummed three times using 0.5% NaA. pernyi cocoon, but 30 min for B. mori cocoon [Silk samples were prepared from domestic β-sheets . However regions . This isine silk . Furtheri cocoon . FinallyB. mori silk fibers was done by first dissolving them in a 1:8:2 molar ratio ternary mixture of CaClFibroin extraction from degummed m resist .Silk fibroin samples spectroscopically investigated in this work allow for comparisons between Different spectral ranges of silk absorbance/transmittance have been probed using different methods; however, there was always an overlap between two adjacent spectral ranges. Absorbance at the longest T-ray wavelengths at 8–80 cmA. pernyi silk fibers.The terahertz frequency range is associated with low frequency macromolecular motions strongly related to the dynamics and conformational changes of proteins and peptides . Hence, A, spectra of different silk samples. Sericin-free crystalline fibers of B. mori and A. pernyi silk have slightly different spectral positions of the absorption bands. Amorphous water soluble fibroin had a spectrally broader absorption at ∼115 cmB. mori and A. pernyi silk fibers as well as amorphous fibroin from B. mori subjected to hot plate annealing at 250 B. mori) and ∼229 ± 15 cmA. pernyi) silk  silk a,b. Thiscrystals . Hence, m−1 band c.A. pernyi silk  technique. The N–H stretching, alkyl, and Amide bands represent the most prominent spectral features for all three samples. Some absorbance peaks of significant interest are the 961 cmB. mori derived white silk fibers exhibit different, slightly subdued features at 975 and 998 cmld silks . Conversβ-sheets ,43. The brations . Its widB. mori exhibiting a peak at 1232 cmB. mori silk has a strong line at 1340 cmThe Amide III band ranging from 1200 cmrmations . The com268 cm−1 , with B. β-turns ; howeverk I form . Signaturmations . The speAla-Glyn , with inThe three distinct varieties of fibroin-based structures show distinct variations in the Amide I and Amide II regions that dominate the spectra. Each of these lines has a complex spectral composition due to conformational variations . The majd chains . In addi fibroin , which i species .In order to shed further light on the internal structure of silk transmittance, IR imaging was performed for natural (not degummed) sericin rich brown silk fibers embedded in a thin KBr pallet. Spectroscopic maps were acquired at the 1660 cmhe fiber . The fibsurement .B. mori and wild A. pernyi silk fibers from the shortest UV wavelengths to near-IR spectral range, deduced from total hemispherical reflectance and transmittance spectra.Fibroin based compounds have a low molar absorptivity in the visible to near-infrared spectral regions. Furthermore, the effects of water vapour are negligible at such wavelengths. r(C–H) and r(C–H). The next group of four vibration combinations has similarly been tied to In the near-IR, absorbance lines in silk fibers can be attributed to the combination or overtone modes of various vibrations of the chemical bonds of the constituent peptides . An examB. mori over the wild silk variety observed in mid-IR spectra is corroborated by results in the near-IR.The spectral range between 4500–4900 cmAmide II 55 cm−1. The pronounced peak at 5167 cmbrations . SpectraB. mori and A. pernyi fiber specimens. In both cases, a strong PL excitation band centered at the excitation wavelength of 15,800 cmSignificant absorption starts to dominate the spectra at UV wavelengths. ) region ,51. A reelengths . The bonallenges .A. pernyi fibers, photoluminescence was relatively stronger than for the B. mori fibers and an additional PL band exists for the longer ∼26,315 cmA. pernyi silks, with significant variations in UV spectral response of sericin from different silks [In crystalline nt silks . The broal stage .Absorption band assignments for the wild, domestic and spider silks in IR spectral range have been analysed using a multivariate approach  and werel region . Typicalide bond . HoweverTemperature of 77 K corresponds to the thermal energy of 53.5 cmarrowing , it was B. mori silk at 1085, 1232, and 1667 cmB. mori fibroin films, provide a closer correspondence [A. pernyi fibers, where alanine and glycine rich regions are segregated, further reinforces this assignment. The low energy bands are also found at the silk-I polypeptide recognisably by signatures at about 1415, 1105, 950, 930, 865, 260, and 230 cmA. pernyi fibers at 20 cmA. pernyi silk has a smaller amount of B. mori silk which has 60%–65% crystallinity [It has been demonstrated that Raman active bands observed in scattering of - moiety , the quapondence . The pea230 cm−1 . The speresponse . Since Aβ-sheets  as compaallinity , the polallinity . In addiSpectral absorbance of silk over ∼3.8 decades in frequency (brations  in domesHigh transmittance of silk up to 35,714 cmlatforms , implant


Anaplasmataceae is a member of the order Rickettsiales; it includes the genera Anaplasma, Ehrlichia, Neorickettsia and Wolbachia. Anaplasmosis and ehrlichiosis traditionally refer to diseases caused by obligate intracellular bacteria of the genera Anaplasma and Ehrlichia. The aim of this study was to identify and estimate the prevalence of Anaplasmataceae species infecting domestic animals and ticks in Corsica.Corsica is a French island situated in the Mediterranean Sea. The island provides suitable natural conditions to study disease ecology, especially tick-borne diseases and emerging diseases in animals and ticks. The family Anaplasmataceae bacteria were based on the 23S rRNA, rpoB and groEl genes.In this study, 458 blood samples from sheep, cattle, horses, goats, dogs, and 123 ticks removed from cattle, were collected in Corsica. Quantitative real-time PCR screening and genetic characterisation of Rhipicephalus bursa (118) and Hyalomma marginatum marginatum (5). Molecular investigation showed that 32.1% (147/458) of blood samples were positive for Anaplasmataceae infection. Anaplasma ovis was identified in 42.3% (93/220) of sheep. Anaplasma marginale was amplified from 100% (12/12) of cattle and two R. bursa (2/123). Several potentially new species were also identified: Anaplasma cf. ovis, “Candidatus Anaplasma corsicanum”, “Candidatus Anaplasma mediterraneum” were amplified from 17.3% (38/220) of sheep, and Anaplasma sp. marginale-like was amplified from 80% (4/5) of goats. Finally, one R. bursa tick was found to harbour the DNA of E. canis. All samples from horses and dogs were negative for Anaplasmataceae infection.Two tick species were collected in the present study: Anaplasmataceae species infecting animals and ticks in Corsica and contributes toward the identification of current Anaplasmataceae species circulating in Corsica.To our knowledge, this study is the first epidemiological survey on  Anaplasma and Ehrlichia are obligate intracellular bacteria transmitted by arthropods, mainly ticks, from one vertebrate host to another. Transmission usually occurs transstadially [Anaplasma and Ehrlichia can cause a persistent infection in vertebrate hosts, which allows these hosts to be reservoirs [Anaplasmataceae infection in domestic animals were known as early as the beginning of the twentieth century. However, wide interest in studying these bacteria arose when discovering species pathogenic for humans [Anaplasma phagocytophilum was known to cause disease in domestic ruminants in Europe and the USA decades before its identification in humans [A. marginale results in the development of mild to severe anaemia and occurs in tropical and subtropical regions, including South and Central America, the United States, southern Europe, Africa, Asia and Australia [Babesia and Anaplasma infections in India was estimated to be $57 million [A. marginale has spread up to the northern latitudes of Switzerland, Austria and Hungary [Anaplasma centrale, a less pathogenic organism but closely related to A. marginale, was reported in cattle in Sicily, Italy [Anaplasma ovis is an intraerythrocytic pathogen of sheep, goats and wild ruminants [A. ovis human infection was reported in a 27-year-old woman in Cyprus [Bacteria from the genera tadially , 2, althtadially . In the tadially , 5. Anapservoirs , 5. Probr humans . Anaplasn humans . The firn humans –11. Boviustralia . In Indiustralia . The eco million . In Euro Hungary . Anaplasy, Italy , and froy, Italy . Anaplasuminants . It is tuminants . Ovine auminants . The hisuminants , and in uminants . In 2007Anaplasmataceae largely interconnected in an epidemiological network involving animals, vectors and humans [Anaplasmataceae bacteria in Corsica in domestic animals has never been reported. The aim of this study was to screen for the presence and the prevalence of Anaplasmataceae species infecting and currently circulating in domestic animals and their ticks in this region.The management of vector-borne diseases requires increased communication between physicians and veterinarians, particularly when physicians are dealing with patients with unexplained febrile illnesses in an endemic area were pathogen like d humans . Corsicad humans . Recentld humans  and Westd humans . Our main = 5) were all sampled on one farm; they had anaemia and a drop in milk production. In addition, blood samples were collected from horses at a different ranch. Dogs sampled in the present study included hunting dogs, sheep dogs, military working dogs and some pet dogs. Animals were examined with the assistance of their owners. Blood samples were collected by a veterinarian. After transport to the laboratory in Marseille, all samples were stored at -80 °C.From 2014 to 2015, EDTA blood samples were obtained from domestic animals on different farms from 14 different areas situated on the east coast of Corsica, France  using a commercial EZ1 DNA Tissue Kit (Qiagen) according to the manufacturer’s instructions. DNA was extracted from 200 μl of blood from all animal samples. Ticks were recovered from ethanol, rinsed with distilled water and dried on sterile filter paper in a laminar-flow hood. Each tick was cut in half lengthways (the blades were discarded after each tick was cut). DNA was individually extracted from one half, and the remaining halves of the ticks were frozen at -80 °C for subsequent studies, as previously described .Anaplasmataceae in Corsican ticks and domestic animals, DNA from ticks and blood were initially screened by a qPCR targeting the 23S rRNA gene. This qPCR has been reported to amplify most bacteria belonging to the family Anaplasmataceae [Anaplasmataceae species in domestic animals or ticks, positive samples were tested by PCR using Anaplasma genus-specific primers targeting the 525 bp fragment of the RNA polymerase subunit beta (rpoB) gene, and Ehrlichia genus-specific primers targeting the 590 bp fragment of the heat shock protein (groEl) gene [For the molecular identification of the species of selected ticks, the DNA samples were subjected to standard PCR to amplify a 360-base-pair (bp) fragment of the mitochondrial 12S rRNA gene  gene   using the DNA sequencing BigDye Terminator Kit (Perkin-Elmer) according to the manufacturer’s instructions. The obtained sequences were assembled using ChromasPro 1.7 software  and the sequences of primers were removed. Sequences obtained in this study were aligned with other ticks or on MEGA7 , with thRhipicephalus bursa, and 3 as Hyalomma marginatum. Thirty-five damaged ticks, including 32 engorged ticks, were only morphologically identified to the genus level as follows: 29 ticks Rhipicephalus sp., 2 Hyalomma sp., and 4 ticks were not identified. Two or three specimens from each tick identified at a species level were selected randomly, and all 35 damaged/engorged ticks were subjected to molecular identification. After 12S rDNA amplification and blast analysis, the six morphologically identified specimens were confirmed to be R. bursa and Hy. marginatum marginatum. From the 35 damaged ticks, 32 ticks were identified as R. bursa, and 3 were identified as Hy. m. marginatum. All 12S rDNA sequences of the R. bursa were identical to each other and showed 100% identity with R. bursa from Italy , and 99% identity with R. bursa ticks reported from Spain (KC243834) , Israel (KT391046), Morocco (AF150034) and Yemen (HE819515)  were identified as Hy. m. marginatum; two were engorged females, and three were male.In total, 123 ticks were collected. Eighty-five removed ticks were identified as Anaplasmataceae DNA was detected in three R. bursa of the 123 ticks examined (2.4%). After the 23S rRNA gene sequencing of the Anaplasmataceae DNA present in the three ticks, A. marginale was identified in two ticks. The two sequences of A. marginale were identical to each other and showed 100% homology with the A. marginale strain Dawn (CP006847) and Gypsy Plains (CP006846) reported from Australia, and 99% with the A. marginale strain Florida (CP001079) and St. Maries (CP000030) reported from the USA  reported from the USA , cattle 100% (12/12) and goats 80% (4/5), whereas all blood samples from horses and dogs were negative. Identification of bacterial species was achieved by amplification followed by sequencing of the portion of the 23S rRNA gene. Seventy-one percent (93/131) of Anaplasmataceae-positive sheep samples were infected by A. ovis. The 23S sequences obtained were identical to each other and showed 100% identity with A. ovis strain KMND Niayes-14 reported in sheep from Senegal [Anaplasma. In 13/131 (9.9%) of infected sheep, the obtained sequences were identical to each other and showed only 96% identity with the A. ovis strain KMND Niayes-14. Due to the absence of additional data on this Anaplasma and genetic relatedness to A. ovis, we refer to this genotype here as Anaplasma cf. ovis. There were 3/131 (2.3%) sheep infected by another genotype of Anaplasma. These three sequences had 96–98% identity to each other and showed 91–94% identity with the A. phagocytophilum strain Norway Variant 2, reported from sheep in Norway (CP015376). We are provisionally calling this incompletely characterised bacterium “Candidatus Anaplasma corsicanum”. Finally, a third genotype was found infecting 22/131 (16.8%) sheep, sampled only in 2015. All sequences of this genotype were identical to each other and showed 95% identity with the A. centrale strain Israel (CP001759) reported from Israel  for Anaplasma similar to A. marginale. All sequences were identical to each other and showed 99% homology with the A. marginale strain Dawn (CP006847), A. centrale strain Israel (CP001759) and 99% with A. ovis strain KMND Niayas-14 (KM021411)  Fig. .Anaplasmataceae bacteria was performed by amplification/sequencing of a portion of the rpoB gene (for Anaplasma-positive samples) or groEL gene (for Ehrlichia-positive samples). RpoB sequences from A. ovis-positive samples were also identical to each other and showed 100% identity with A. ovis strain KMND Nayes-14. rpoB sequences from two A. marginale-positive R. bursa ticks and the four other sequences obtained from cattle blood samples were identical to each other and showed 100% identity with A. marginale strain Dawn (CP006847) and Gypsy Plains (CP006846) and 99% with A. marginale strain Florida (CP001079) and St. Maries (CP000030). For the E. canis identified in one R. bursa tick, the DNA sample was amplified using groEl Ehrlichia genus-specific primers and sequenced. The sequence showed 99% homology with the E. canis strain Jack (CP000107) , Dog2 (CP006618), JM (CP006617) and HZ (CP000235). RpoB sequences of “Ca. Anaplasma mediterraneum” presented 84% identity with A. centrale strain Israel (CP001759). Finally, Anaplasma cf. marginale from four goats had rpoB sequences that shared 98% identity with A. ovis strain KMND Niayes-14 (KX155494) and strain RhburBas11 (KX155495), 93% with A. marginale strain Florida (CP001079), St. Maries (CP000030), 89% strain Dawn (CP006847) and Gypsy Plains (CP006846), and 87% with A. centrale strain Israel (CP001759)  , Anaplasma cf. ovis OVCF115 (KY498326), “Ca. Anaplasma corsicanum” OVCF72 (KY498327), “Ca. Anaplasma corsicanum” OVCF81 (KY498328), “Ca. Anaplasma corsicanum” OVCF65 (KY498329), “Ca. Anaplasma mediterraneum” OVCFO215 (KY498330), Anaplasma cf. marginale CpCF01 (KY498331), Anaplasma marginale BvCF13 (KY498332), A. marginale Rh.burCF08 (KY498334), A. marginale Rh.burCF10 (KY498335), Ehrlichia canis Rh.burCF07 (KY498333); (ii) for the rpoB gene: Anaplasma ovis OVCF02 (KY498325), Anaplasma cf. ovis OVCF115 (KY498336), “Ca. Anaplasma corsicanum” OVCF72 (KY498338), “Ca. Anaplasma corsicanum” OVCF81 (KY498339), “Ca. Anaplasma corsicanum” OVCF65 (KY498340), “Ca. Anaplasma mediterraneum” OVCFO215 (KY498341), Anaplasma cf. marginale CpCF01 (KY498342), Anaplasma marginale BvCF13 (KY498343), A. marginale Rh.burCF08 (KY498344), A. marginale Rh.burCF10 (KY498345); (iii) For the groEl gene: Ehrlichia canis Rh.burCF07 (KY498324). For tick species, the 12S rRNA sequences were submitted under the following accession number: Hy. m. marginatum (KY595783) and R. bursa (KY595784).All sequences obtained in the present study were submitted to GenBank under the following accession numbers: (i) for the 23S rRNA gene: Anaplasma infection may likely be neglected because of its unknown economic importance in small ruminants. To our knowledge, the present study is the first report of the incidence of Anaplasmataceae species in ticks and animals in Corsica. Furthermore, the presence and molecular traits of six species belonging to the genus Anaplasma from ruminants and ticks infecting cattle, and one Ehrlichia, are shown. The typical Mediterranean environment of Corsica with hot summers, along with the geographical location, favours the spread of seasonal tick infestations. Two tick species were collected and confirmed by the morphological and molecular investigation as R. bursa and Hy. m. marginatum ; only two engorged ticks were found to harbour A. marginale.Livestock farming in Corsica is an important economic activity involving approximately 150,000 sheep, 48,000 goats, 40,000 pigs and 70,000 cattle . The sig rufipes . While Hng birds . Recentli et al. . Rhipiceh Africa , 34. Rhironments . Howeverronments , 35. Cornal host . This sp A. ovis . DNA of se ticks , 35. HerPortugal . It is lEhrlichia canis was amplified from one non-engorged R. bursa female. In Europe, E. canis is associated with the presence of the brown dog tick R. sanguineus [E. canis has also been reported from R. bursa collected from goats in Sardinia, Italy [Cediopsylla inaequalis collected from red foxes in Sicily, Italy [E. canis from D. marginatus collected from dogs, Ixodes canisuga collected from red foxes, and I. ricinus collected from vegetation in Hungary [R. bursa [R. bursa and the other arthropod species in the transmission of E. canis remains unknown.nguineus . Howevera, Italy  and Cediy, Italy . In othe Hungary , 42. DomR. bursa ; howeverHy. m. marginatum ticks were positive for Anaplasmataceae infection. However, in Spain, Hy. m. marginatum has been identified as a potential biological vector for A. marginale [Babesia caballi, causing babesiosis in horses and Theileria annulata infection under laboratory conditions [None of the five arginale . These tnditions . Other sAnaplasma spp. in our study was surprisingly high in ruminants. Based on the 23S rRNA gene molecular investigations, the individual prevalence observed was 59.5% in sheep, 100% in cattle, and 80% in goats. However, none of the canine or equine blood samples was positive. Genetic characterisation using 23S rRNA and the rpoB genes identified A. ovis, A. marginale, and several potentially new species, all belonging to the genus Anaplasma. These data confirm the relevance of ruminants as important hosts and reservoirs of different Anaplasma species in the Mediterranean ecosystem. The prevalence of Anaplasma spp. in ruminants examined by us was lower than the prevalence data reported from Sardinia [A. marginale infection in cattle was higher than that observed in cattle in Sicily [The prevalence of Sardinia . The pren Sicily ; howevern Sicily .A. ovis, and three potentially new species, “Ca. Anaplasma corsicanum”, “Ca. Anaplasma mediterraneum”, and Anaplasma cf. ovis. In the Mediterranean area, A. ovis is reported to be endemic to Sicily [A. ovis has also been reported from Portugal, Hungary [Anaplasma ovis infection in the mouflon and the European roe deer has been reported from Cyprus and southern Spain, respectively [A. ovis in Europe is R. bursa [A. ovis DNA was amplified from I. ricinus removed from cattle in Hungary [Haemaphysalis sulcata removed from mouflons in Cyprus [Melophagus ovinus) and deer ked (Lipoptena cervi) in Hungary [A. ovis was amplified from foxes, and a flea, Xenopsylla cheopis, removed from these foxes [A. ovis remains unclear. Anaplasmosis in sheep is usually subclinical. This bacterium can lead to severe infection with severe illness in sheep; severe illness can occur in some extreme conditions, such as the association with more than one parasitic disease or other stress factors [Sheep, goats, and cattle sampled in this study manifested poor health. In sheep, most clinical manifestations observed were relapsing fever, drop in milk production and mortality. Molecular and phylogenetic analysis of sequences amplified from sheep blood samples were identified o Sicily , 48. Thio Sicily , 49. In  Hungary  and Slov Hungary . Anaplasectively , 51. TheR. bursa . However Hungary , Haemaphn Cyprus , and these foxes . The rol factors .A. marginale. In this farm, the farmer reported mortality in his livestock. Anaemia and icterus were most observed in other cattle  compared to the prevalence of A. ovis; however, 80% (4/5) of the goats sampled in this study were infected by Anaplasma cf. marginale. The importance of this amplified Anaplasma species remains to be understood.The potentially new species “sma Fig. . Phylogevis Fig. . Interesriations . The obsA. marginale in cattle and A. ovis and the others amplified Anaplasma spp. in sheep and goats suggest that the mortalities can be linked to these Anaplasma species. However, other tick- or vector-borne diseases can also lead to mortalities like Piroplasmosis [Anaplasmataceae species amplified in the present study should be considered with caution do to the possible implication of other pathogens.Mortalities in animals were reported by the farmers in the sheep and cattle herds. Unfortunately, we did not have access to body tissue of fluid from the dead animals to perform a post-mortem diagnosis. The different reported symptoms and the results found in the present study with the high prevalence of lasmosis , 55. In lasmosis . The invA. phagocytophilum in animal or tick samples. Anaplasma platys and E. canis were also not found in dogs, although E. canis was found in Rh. bursa collected from a cow.In our study, we did not find Anaplasma species, whereas R. bursa seems to be a vector of A. marginale in cattle. The prevalence of Anaplasma spp. infection was high. The use of quantitative real-time PCR complemented with sequencing and genetic characterisation using two genes, rpoB and groEl, revealed an interesting diversity of Anaplasma spp. infection in small ruminants and R. bursa, including potentially new species and E. canis in one R. bursa tick. Nevertheless, characterisation studies are needed to ascertain the pathogenesis and/or the zoonotic potential of the strains and their significance for animals and public health.The present study demonstrates that ruminants in Corsica are a reservoir for multiple


Heteroglaux blewitti has been the subject of a century-old debate. The current classifications based on non-phylogenetic comparisons of morphology place the small owls of Asia into three genera, namely, Athene, Glaucidium, and Heteroglaux. Based on morphological and anatomical data, H. blewitti has been alternatively hypothesized to belong within Athene, Glaucidium, or its own monotypic genus Heteroglaux. To test these competing hypotheses, we sequenced six loci (~4300 bp data) and performed phylogenetic analyses of owlets. Mitochondrial and nuclear trees were not congruent in their placement of H. blewitti. However, both mitochondrial and nuclear combined datasets showed strong statistical support with high maximum likelihood bootstrap (>/ = 90) and Bayesian posterior probability values (>/ = 0.98) for H. blewitti being nested in the currently recognized Athene group, but not sister to Indian A. brama. The divergence of H. blewitti from its sister taxa was between 4.3 and 5.7 Ma coinciding with a period of drastic climatic changes in the Indian subcontinent. This study presented the first genetic analysis of H. blewitti, a Critically Endangered species, and addressed the long debate on the relationships of the Athene-Heteroglaux-Glaucidium complex. We recommend further studies with more data and complete taxon sampling to understand the biogeography of Indian Athene species.Range-restricted species generally have specific niche requirements and may often have unique evolutionary histories. Unfortunately, many of these species severely lack basic research, resulting in poor conservation strategies. The phylogenetic relationship of the Critically Endangered Forest Owlet  Endemic and endangered species are often ecologically specialized and there is an urgent need to better understand the ecology and phylogenetic history of endangered species to uncover features that might be crucial for conservation. Such species could be viewed as model species to understand evolutionary processes in the landscape of their occurrence –3. HowevHeteroglaux blewitti , Spotted Owlet Athene brama  and Jungle Owlet Glaucidium radiatum  were not included in the most recently published owl phylogenies . g injury . The capH. blewitti. We compared genetic data collected from eggshells  and used the same criteria mentioned above to ensure species identity. In case of A. brama and G. radiatum, we collected fallen feathers below known nest-sites outside Protected Areas or shed feathers from injured bird rescued by NGOs. We trapped G. radiatum individuals in mist nets (in Chhattisgarh). We sampled spatially non-overlapping and distant nest-sites to avoid resampling the same individuals. For both the species, for ensuring species identity, we followed the same approach as for H. blewitti. We sequenced target genes from a vouchered tissue of A. superciliaris obtained from the Field Museum of Natural History , following the manufacturer’s protocol with a few modifications. We added 20 μl of 1% Dithiothreitol (DTT) in the lysis reaction. To ensure a higher concentration of DNA and minimize loss, we eluted DNA twice in separate vials, each time in 100 μl of AE buffer, instead of the recommended one elute of 200 ul and used the first elute for further analysis. We amplified two mitochondrial genes , a nuclear exon (RAG-1), and three nuclear introns . These genes were chosen based on previously available data on other species of aucidium , 9. We c studies –42. We sWe viewed and manually edited the sequences in Chromas Lite 2.1.1  and aligned them using the software Geneious v7.0.6  . We downA. noctua) in the LDH dataset; therefore, we did not include the dataset in the final combined analysis. The concatenated dataset contained <9% missing data. We used codon-specific DNA substitution models , nuclear (RAG1 + TGFB2 + MYO), and concatenated (CYTB + COI + RAG1 + TGFB2 + MYO). There was a missing in-group taxon  as outgroup taxa since this is the family closest to the Strigidae family with an estimated known divergence time for the Strigidae / Tytonidae split (54–83 Ma)  blewitti. The species’ diurnal, ambushing predatory nature might be an adaptation to maximally utilize the available niche, given the presence of other co-distributed crepuscular and nocturnal owlets such as A. brama and G. radiatum in the same area. Further information on the dietary preference of H. blewitti and its ecological interactions with other species would help understand its adaptations. Nevertheless, our study provides another line of evidence to the role of climatic fluctuations in the diversification of Indian birds.Perhaps the Plio-Pleistocene climatic fluctuations and the subsequent retraction of evergreen forests , 76, durn spaces , 78. Thiet al. (2008) for A. otus / O. leucotis split (16.7–19.3 Ma)  blewitti does not directly impact the IUCN status of the species, its ranking in international conservation listings that use phylogenetic information may change. The Evolutionary Distinct and Globally Endangered (EDGE) listing will perhaps no longer carry the same evolutionary distinctness score for the species  blewitti is a species of Central Indian old growth dry deciduous forests, occurring in protected as well as non-protected areas  blewitti is under severe threat of habitat loss due to large-scale logging, timber harvesting, and land-use change  blewitti and A. brama  in this study, hybridization cannot be wholly ruled out. In the rapidly changing human-dominated landscape of the Central India, circumstances are similar to other owls such as Barred and Spotted Owls [ed areas . Across e change , 77, 78.bitation . Althougted Owls  and Nortted Owls , where hWith this first molecular phylogenetic study of this Critically Endangered species, we demonstrate that crucial information can only be obtained through capture-based sampling that strengthens and supports ecological data collected through conventional methods. Capture-based genetic studies still do not find support from conservation managers in India , 83, butAthene / Heteroglaux clade and highlight the need for studies predicting species responses to climate change.The new information provided here will facilitate both the taxonomic revision of the S1 FigH. blewitti is the only range-restricted, rare owlet among Indian owlets.(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 FigBlue line: the consensus tree (primary hypothesis), magenta line: the next most popular tree after consensus (secondary hypothesis), green lines: tertiary hypotheses.(TIF)Click here for additional data file.S4 FigHB: H. blewitti; ASUP: A. superciliaris; ATHNB: A. brama; ANOCT: A. noctua; ACUN: A. cunicularia; GLRAD: G. radiatum and GCUCU: G. cuculoides.The species code used can be referred from (TIF)Click here for additional data file.S5 FigHB: H. blewitti; ASUP: A. superciliaris; ATHNB: A. brama; ANOCT: A. noctua; ACUN: A. cunicularia; GLRAD: G. radiatum and GCUCU: G. cuculoides.The species code used can be referred from (TIF)Click here for additional data file.S6 FigHB: H. blewitti; ASUP: A. superciliaris; ATHNB: A. brama; ANOCT: A. noctua; ACUN: A. cunicularia; GLRAD: G. radiatum and GCUCU: G. cuculoides.The species code used can be referred from (TIF)Click here for additional data file.S7 FigHB: H. blewitti; ASUP: A. superciliaris; ATHNB: A. brama; ANOCT: A. noctua; ACUN: A. cunicularia; GLRAD: G. radiatum and GCUCU: G. cuculoides.The species code used can be referred from (TIF)Click here for additional data file.S8 FigHB: H. blewitti; ASUP: A. superciliaris; ATHNB: A. brama; ANOCT: A. noctua; ACUN: A. cunicularia; GLRAD: G. radiatum and GCUCU: G. cuculoides.The species code used can be referred from (TIF)Click here for additional data file.S9 FigHB: H. blewitti; ASUP: A. superciliaris; ATHNB: A. brama; ACUN: A. cunicularia; GLRAD: G. radiatum and GCUCU: G. cuculoides.The species code used can be referred from (TIF)Click here for additional data file.S10 FigHB: H. blewitti; ASUP: A. superciliaris; ATHNB: A. brama; ANOCT: A. noctua; ACUN: A. cunicularia; GLRAD: G. radiatum and GCUCU: G. cuculoides.The species code used can be referred from the (TIF)Click here for additional data file.S11 FigHB: H. blewitti; ASUP: A. superciliaris; ATHNB: A. brama; ANOCT: A. noctua; ACUN: A. cunicularia; GLRAD: G. radiatum and GCUCU: G. cuculoides.The species code used can be referred from the (TIF)Click here for additional data file.S1 TableBNHS: Bombay Natural History Society museum; JCT: Jivdaya Charitable Trust (rescued bird); CKV: C.K. Vishnudas.(DOCX)Click here for additional data file.S2 TableTm: Optimal annealing temperature.(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 TableNA: Not available/Not used.(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.


Mycobacterium riyadhense in Saudi Arabia. In 18 months, 12 new cases of M. riyadhense infection were observed, predominantly among Saudi nationals, as a cause of pulmonary disease. M. riyadhense may be emerging as a more common pathogen in Saudi Arabia.We explored in detail the nationwide existence of  Mycobacterium, including several human pathogens. M. riyadhense is a slow-growing NTM identified as a cause of pulmonary and extrapulmonary illnesses in humans from Riyadh, Saudi Arabia  appear to be emerging globally, but the definitive reasons for this are unclear. Advances in diagnostic technologies have led to the identification of >160 species of i Arabia . M. riyaM. riyadhense in clinical settings in Saudi Arabia, we conducted a prospective study on a nationwide collection of isolates. Suspected NTM isolates reported as M. tuberculosis complex or Mycobacterium species with nonspecific banding pattern by line probe assays were subjected to different conservative gene sequencing to identify M. riyadhense.To explore the presence of M. riyadhense on the basis of previous studies  (https://blast.ncbi.nlm.nih.gov) and the EzTaxon database. We followed stringent identification criteria, requiring similarity >99% between isolate and reference strain for species confirmation.During April 2014–September 2015, we collected 458 NTM isolates, with clinical and epidemiological data, from all 9 national referral laboratories in different provinces of Saudi Arabia. We formulated the isolate enrollment strategy to suspect M. riyadhense had a 99%–100% match with 3 database strains . On the other hand, rpoB and hsp65 sequences also showed 99%–100% similarity with other sequences . The other closest species observed during the analysis were M. alsense, M. szulgai, and M. angelicum (98% similarity with 16S rRNA gene sequences); M. genavens and M. simulans (96% similarity with hsp65 gene sequences); and M. lacus, M. intracellulare, and M. malmoense (94% similarity with rpoB gene sequences). Two isolates that matched inclusion criteria could not be identified as M. riyadhense; BLAST analysis showed the closest matching species as M. lacus DSM 44577(T), with 89% similarity. Two 16S rRNA gene sequences from this study were deposited in GenBank (accession nos. KX898970 and KX898971).We identified 14 isolates that fit the inclusion criteria; most were reported from the Central province, Riyadh, in Saudi Arabia, but the reason is unclear. Microbiological analysis showed slow-growing mycobacteria producing rough white colonies on LJ medium within 3–4 weeks of incubation at 37°C. Primary sequencing of the 16S rRNA gene showed 12 cases of M. riyadhense infection, including pulmonary and extrapulmonary invasive infections, over a period of 18 months. Demographically, Saudi citizens dominated; 11 of 12 case-patients were male, and mean age was 50 years. Geographic distribution of cases showed 10 cases from Riyadh  and 2 from Dammam (Eastern province). Clinical data revealed 9 cases with pulmonary involvement and 3, including a pediatric case, with lymphadenitis. Of note, 75% of the respiratory cases were clinically relevant according to American Thoracic Society criteria for NTM pulmonary disease. Most patients recovered with isoniazid, rifampin, and ethambutol therapy (We identified 12 clinical cases of  therapy .M. riyadhense. Without an accurate diagnosis, treatment is delayed. In this study, most of the patients were treated with standard TB regimens; some of them received clarithromycin, which did not appear to be highly effective (M. riyadhense disease has been developed, likely due to its status as a rare species. In the cases reported here, patients generally responded well to the initial therapies, but drug resistance may challenge the empirical treatment used. A strain resistant to isoniazid is already reported from South Korea (M. riyadhense as a more common pathogen.The lack of advanced molecular diagnostic tools in clinical laboratories in Saudi Arabia impedes the accurate identification of