To the Editor:Sporolactobacillus laevolacticus, formerly known as Bacillus laevolacticus, is a gram-positive, acid-tolerant, catalase-positive, facultatively anaerobic and mesophilic bacteria initially isolated from the rhizosphere of wild plants  but his procalcitonin level (0.19 µg/L) was increased, which suggested that the infection had not been cured. A cellulitis abscess was suspected, and surgical cleaning and drainage was performed on March 10  and fibrinogen (8.35 g/L), which indicated inflammation. His leukocyte count was normal . However, a surgical sample inoculated into a blood culture bottle grew gram-positive bacilli after 4 days.S. laevolaticus (score 1.88). Identification was confirmed by PCR amplification of the 16S RNA gene (S. laevolaticus strain (GenBank accession no. AB362648) by BLAST analysis (http://www.ncbi.nlm.nih.gov).Subculture colonies were identified by using matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry  as S. laevolaticus strain was susceptible to amoxicillin, amoxicillin/clavulanate, imipenem, metronidazole, clindamycin, and vancomycin. The antimicrobial drug regimen was then changed to clindamycin and trimethoprim/sulfamethoxazole, and the patient showed an excellent clinical outcome. The patient was considered clinically cured 7 weeks later (The ks later , panel CS. laevolacticus has been studied for its capacity to survive extreme conditions and for its fermentation system (S. laevolacticus. Since September 2009, we have used MALDI-TOF mass spectrometry in North Hospital for routine identification of bacterial species isolated from clinical samples (S. laevolaticus can be responsible for human infections and suggests that this bacterial species could be an emerging opportunistic pathogen responsible for human infections.The bacterial species was accurately identified by using MALDI-TOF and then confirmed by using a 16S RNA PCR. Because the bacterium was originally isolated from a plant rhizosphere and the patient was hospitalized with an open wound in the foot and bacteremia, we speculate that the infection was the direct result of close extended contact between the wound and soil infected with the bacteria. This case confirms that


Culex torrentium, Cx. pipiens, and Culiseta morsitans collected in an area of Sweden where SINV disease is endemic. The infection rate in Cx. torrentium mosquitoes was exceptionally high , defining Cx. torrentium as the main enzootic vector of SINV in Scandinavia.We isolated Sindbis virus (SINV) from the enzootic mosquito vectors  Togaviridae, genus Alphavirus) is an etiologic agent for outbreaks of rash and long-lasting polyarthritis  in Cx. pipiens/Cx. torrentium and used for DNA extraction, enabling identification of the individual specimens to species by using a previously described molecular method  baited with carbon dioxide; the traps were set within the regular mosquito surveillance area of the River Dalälven floodplains in central Sweden  and from 74 pools of mosquitoes pooled by collection trap and week (290 Cs. morsitans); pool sizes ranged from 1 to 19 mosquitoes. The mosquitoes were processed for RNA extraction, real-time reverse transcription PCR (rRT-PCR), and virus isolation on Vero cells as previously described  or smaller pools of (Culiseta) mosquitoes were ultimately tested for SINV RNA. The second rRT-PCR showed 16 samples positive for SINV RNA. One of the positive Cx. torrentium pools, which contained samples from 10 mosquitoes, included samples from 3 SINV RNA–positive mosquitoes. Thus, 11 of 301 individual Cx. torrentium and 3 of 367 individual Cx. pipiens mosquitoes were positive for SINV RNA. For Cs. morsitans mosquitoes, 2 of 74 pools were positive for SINV RNA  × 1,000; differences between the species were tested for significance by using a χ2 test. The actual species-specific IR differed significantly between species (p = 0.01): 36.5, 8.2, and 21 infections/1,000 mosquitoes for Cx. torrentium, Cx. pipiens, and mixed species (Cx. pipiens and Cx. torrentium), respectively. Cs. morsitans mosquitoes were tested in pools, so we calculated the minimum IR (MIR) as  × 1,000; the calculated MIR was 6.9 infections/1,000 Cs. morsitans mosquitoes. A comparison of MIR and maximum-likelihood estimates of infection gave similar estimates.Because individual SINV was successfully isolated from all 16 rRT-PCR–positive mosquito samples . Part ofCx. torrentium and Cx. pipiens, reliably identified to species level. The significantly higher SINV-I IR observed for field-caught Cx. torrentium than Cx. pipiens mosquitoes is a key addition to the previous findings of the extreme susceptibility of Cx. torrentium mosquitoes to SINV-I (Cx. torrentium mosquitoes could transmit the virus upon refeeding on a susceptible animal (Cx. torrentium mosquitoes translates to 36.0 mosquitoes/1,000 being able to transmit SINV-I. In contrast, because only one third of infected Cx. pipiens mosquitoes can transmit SINV-I upon refeeding, the observed natural IR of 8.2 infections/1,000 Cx. pipiens mosquitoes translates to only 2.0 mosquitoes/1,000 being able to transmit the virus (We describe information on the actual occurrence and IR of SINV-I in the enzootic mosquito vectors Cx. torrentium is among the highest ever reported for mosquitoes. This could partly be attributed to the fact that single mosquitoes were analyzed, as compared with the more common technique of pooling. This higher IR would have remained undetected if only pooled mosquitoes were analyzed, even though our original pools consisted of only 10 individual mosquitoes. Cx. pipiens mosquitoes are generally considered a secondary enzootic vector of SINV because, as in Sweden, they are less frequently found infected in nature in South Africa, Israel, and Saudi Arabia, where Cx. univittatus mosquitoes are the main SINV vector (The observed SINV-I IRs are very high for both species, and the IR for Cx. torrentium mosquitoes in Sweden, the outstanding vector competence results for the species in the laboratory (Culex species in SINV-endemic areas of Europe (Cx. torrentium can now be identified as the main enzootic vector of SINV-I in Scandinavia. In areas of Sweden and Finland where clinical SINV-I infections are most prevalent, Cx. torrentium mosquitoes account for >90% of the Cx. pipiens/Cx. torrentium population; in central Europe, where the virus is more uncommon in mosquitoes and no human cases have been observed, both species are equally common (Cx. torrentium mosquitoes may be a prerequisite for the intense enzootic transmission of SINV-I that is needed to increase the risk for spillover infections in humans. Whether the Cx. torrentium mosquitoes are also to be considered a vector of other mosquitoborne bird viruses remains to be investigated. In continental Europe, West Nile virus and Usutu virus are emerging, and it is unknown if Cx. torrentium has a vector role for these viruses is unknown because of the lack of transmission experiments and isolation attempts from reliably identified female Cx. pipiens/Cx. torrentium mosquitoes. Knowledge from such experiments and isolation attempts would be especially valuable for northern and central Europe where Cx. torrentium is the dominating candidate enzootic vector species for bird-associated viruses (Because of the exceptionally high SINV IR for field-caught


Saccharina japonica, one of the most economically important seaweeds. The 537-Mb assembled genomic sequence covered 98.5% of the estimated genome, and 18,733 protein-coding genes are predicted and annotated. Gene families related to cell wall synthesis, halogen concentration, development and defence systems were expanded. Functional diversification of the mannuronan C-5-epimerase and haloperoxidase gene families provides insight into the evolutionary adaptation of polysaccharide biosynthesis and iodine antioxidation. Additional sequencing of seven cultivars and nine wild individuals reveal that the genetic diversity within wild populations is greater than among cultivars. All of the cultivars are descendants of a wild S. japonica accession showing limited admixture with S. longissima. This study represents an important advance toward improving yields and economic traits in Saccharina and provides an invaluable resource for plant genome studies.Seaweeds are essential for marine ecosystems and have immense economic value. Here we present a comprehensive analysis of the draft genome of  Kelps are ecologically and economically important seaweeds. Here the authors sequence the genome of Saccharina japonica to gain insights into the evolutionary adaptation of polysaccharide biosynthesis, iodine concentration and antioxidation mechanisms and the population genetics of kelp domestication. S. japonica, one of the most common Laminariales alga along the northwest coasts of the Pacific OceanS. japonica sea farming has evolved rapidly to make it the most common seaweed in the worldS. japonica reached 7.9 million tons (dry weight) and had a market value of more than US $1.3 billion in 2012  belong to the phylum Stramenopiles, a phylogenetic lineage that is distantly related to terrestrial plants and animals012 ref. .Ectocarpus siliculosusS. japonica in this study. Here we report a draft genome sequence of the female gametophytes of the artificially cultivated S. japonica strain Ja and the resequencing of seven wild populations and nine representative cultivars of Saccharina species. Comparative genomic analyses of these data provide novel insight into the evolutionary adaptation and the functional diversification of the polysaccharide biosynthesis and iodine concentration mechanisms of S. japonica. The Saccharina genomic sequence that was obtained represents an important advance toward securing bioproducts and biofuels from macroalgae and provides an invaluable resource for plant gene and genome evolution studies.Despite the ecological, economic and evolutionary importance of kelps, we currently have a very limited knowledge of their genetic architecture and metabolism, including their iodine concentration system and alginate-producing pathway, a limitation that hinders both genetic research and mariculture practices. Furthermore, years of interspecific hybridization and biomass yield-targeted artificial selection have not only degeneratted the economic characteristics of these kelps but have also narrowed their genetic variation. The recently sequenced small filamentous model brown alga S. japonica strain Ja cultured in Qingdao, China. The DNA sequencing reads were obtained using both Roche and Illumina technologies and were assembled after filtering out the low-quality and duplicated reads. A total of 84 Gb of high-quality Illumina reads and 10 Gb of PacBio long reads  with a scaffold N50 length of 252,007 bp  and a contig N50 length of 58,867 bp  , the L1 and the CRE elements and the RTE-2 and the RTEX-1 retrotransposons identified in S. japonica shared a high sequence similarity (>95%), indicating that they were recently inserted into the S. japonica genome and are likely to be active in the amplification (11E. siliculosus possesses a significant fraction of RTE elements (1.6%) that share high sequence similarity with those found in S. japonica, whereas the RTEs identified in two green algal genomes (Volvox carteri and Chlamydomonas reinhardtii) were relatively divergent. A phylogenetic analysis of the RTE elements showed that they were not consistent with the species tree, indicating the lateral gene transfer of the RTE elements in these organisms. (By combining homology-based and  (22.7%) . Among tfication . Previouganisms. .S. japonica genome, we performed deep transcriptome sequencing of both female gametophytes and sporophytes, which generated 11.3 Gb of RNA sequencing data. By combining homologue-based, ab initio and transcriptome-based approaches, we predicted 18,733 protein-coding genes (gene models) in the S. japonica genome, which is greater than the number of genes predicted in E. siliculosusE. siliculosus . More than 90.7% of the predicted coding sequences were supported by transcriptome sequencing data (≥5 reads), indicating the high accuracy of the gene predictions in the sequenced S. japonica genome  could be aligned to the assembled genome. Among the annotated genes, 86.1% of the encoded proteins had homologues in the NCBI non-redundant protein database, with 70.1% of the putative proteins showing best-hit matches to E. siliculosus. Previous studies have revealed that E. siliculosus possesses an integrated virus (EsV-1) in its genome9Saccharina genome. However, using PFAM domain searches, we identified 121 putative proteins in S. japonica containing the FNIP repeat domain (PF05725), which is ∼22 residues long and has previously only been found in Dictyostelium and a few double-stranded DNA viruses. Similar FNIP repeat domains have also found in the E. siliculosus genome. A phylogenetic analysis based on the FNIP repeat domain revealed that the closest relatives of these FNIP sequences came from a giant virus . Because of insufficient evidence of protein similarities to other algae, most of these newly gained families in Phaeophyceae were classified as proteins with unknown function. It should be noted that 102 annotated gained families were found to be significantly enriched (P<10−5) in the protein kinase or HeH or peptidase CA super family domains  families were found to have more gene copies in S. japonica  than in E. siliculosus , but only 652 families were identified to be the opposite . These results indicate that the majority of the amplified genes found in S. japonica resulted from recent duplication events , including those families involved in iodine concentration and the biogenesis and remodelling of cell wall polysaccharides, for example, vanadium-dependent haloperoxidases (vHPOs) that catalyse the oxidation of halidesD-mannuronate residues into L-guluronate for alginate biosynthesis and alpha--fucosyltransferase (GT23 family) that polymerize GDP-fucose into the elongated fucan chainS. japonica, into oligosaccharides that respond to tissue damage and provide protection against pathogens by triggering defence responses such as an initial oxidative burstEctocarpusE. siliculosus, and are related to the development of the sporophyte and gametophyte generationsFucus vesiculosusPhyscomitrellaS. japonica.families  with 10 Laminaria, the identified vHPOs comprise two large multigenic families encoding vanadium-dependent bromoperoxidases (vBPOs) and iodoperoxidases (vIPOs)Brown macroalgal species are the most well-known effective iodine accumulators among all living organisms and are major contributors to the global biogeochemical iodine cycle, displaying an average iodine content of 1% (up to 5%) of their dry weight, representing ∼30,000 times the concentration of this element in seawaterS. japonica genome, 17 vBPO and 59 vIPO genes were identified and annotated, and a phylogenetic analysis revealed that all of the vHPO genes form a monophyletic group sharing a common ancestor with the vCPO (vanadium-dependent chloroperoxidase) genes of fungi, after which they evolved independently in red and brown algae  and the amount of vHPOs were expressed in the juvenile sporophytes, followed by the distal blades (53). These results agree well with the largest quantity of iodine elements being found in the juvenile sporophytes and distal blades in L. digitataS. japonica  and were clustered on seven scaffolds Notably, in caffolds , indicatE. siliculosus hypothesized that this evolutionary history involved HGT in  and a wild isolate  and 96-K-small InDels in the cultivars and an average of 2.27 M SNVs and 274-K-small InDels in the wild populations. In addition, considering the large genetic distance among S. latissima and S. sp. E and the reference Ja, we first built draft genome assemblies for S. latissima and S. sp. E and then employed NUMMERSaccharina cultivars. A log likelihood STRUCTURE analysisS. japonica and S. longissima (C5). The number of groups identified within the wild Saccharina individuals increased with an increasing number of K, with up to four groups at K=5, which was consistent with the phylogenetic analysis , wild kelps show greater morphological plasticity in response to harsh intertidal environments32E. siliculosus, S. japonica shows several unique characteristics, such as more complex differentiation, large blades, a higher polysaccharide content and striking iodine concentrating abilities. In this study, some of the mechanisms underlying these characteristics were identified using comparative genomics. S. japonica can grow up to dozens of metres with large blades, and its cell wall polysaccharide content is much higher than Ectocarpus. We speculated that the expansion of the cellulose synthase, mannuronan C-5-epimerase and alpha--fucosyltransferase gene families may be responsible for the formation of the extensible cell walls of S. japonica that support its large blade structureS. japonica, but it also play important roles in the defence system, in which it can be hydrolysed into oligosaccharides by endo-1,3-beta-glucanase. In addition, laminarin and its derived oligosaccharides can even activate the defence response and protect against pathogens in terrestrial plants5S. japonica. The expansion of the endo-1,3-beta-glucanase and vHPO gene families in S. japonica may provide it with more complex defence systems than Ectocarpus. Moreover, tissue differentiation was more complex in S. japonica than in Ectocarpus with respect to such events as the emergence of the holdfast, stipe and blades. In addition to several other expanded gene families, including Cupin-like proteinBrown algae are valuable economic and ecological resources and are an important evolutionary lineage because of the development of specialized tissues and organs outside of plants and metazoans, which makes their genomes exciting resources for comparative investigations in this field. The S. japonica genome is the first sequenced reference genome from kelps and the second genome from brown algae. When compared with filamentous Saccharina individuals, providing the basic materials for population genetic studies. The Saccharina cultivars that are currently farmed in China were mainly bred from the descendants of wild S. japonica that were introduced from northern Japan. According to our results, only limited crossbreeds were derived from the hybridization of S. japonica and L. longissima. Therefore, the restricted germplasm base and continuous selfing of these kelps have resulted in very low genetic diversity within Saccharina cultivars. The obtained genomic information on wild Saccharina species can not only be used to increase the genetic diversity through hybridization, but it can also provide a large source of candidate genes for further functional studies aimed at improving quality and yield. There are other important issues that remain for the further exploitation of these kelps, such as the many biological features that are unique to Saccharina, that need to be studied in detail at the molecular level. In addition, the identification of SNVs in representative wild and cultivated individuals will provide opportunities for marker-assisted breeding.This work also presents the first comprehensive resequencing data for wild and cultivated S. japonica Ja and DNA libraries with 300-bp inserts were constructed for the other 16 Saccharina individuals. These DNA libraries were paired-end sequenced on an Illumina HiSeq 2000 sequencer. Two-large-insert 454 libraries (8 and 16 kb) were also constructed for S. japonica Ja and were sequenced using 454 pyrosequencing. For PacBio library construction, genomic DNA was sheared to 8 kb using an ultrasonicator and was converted into the proprietary SMRTbell library format using an RS DNA Template Preparation Kit. SMRTbell templates were subjected to standard SMRT sequencing on the PacBio RS system according to the manufacturer's protocol.DNA libraries harbouring 180-bp, 300-bp, 500-bp, 800-bp, 3-kb and 5-kb inserts were subsequently constructed for We used the paired-end reads from the short-insert-size libraries  to assemble the genome into contigs using SOAPdenovo2 ref.  with theE. siliculosus were referred to as authentic Saccharina contigs. For each contig, the GC content and the open reading frame (ORF) density  were calculated. The candidate bacterial contigs showing an ORF density of ≥60% or a biased GC content (>60 or <40%) were filtered. The assembly statistics are shown in http://124.16.129.28:8080/saccharina/.To remove potential bacterial contamination, the assembled contigs were first subjected to BLASTX searches against the NCBI non-redundant protein database. The contigs with the best-hit matches to bacteria were referred to as candidate bacterial contigs, and the contigs with the best-hit matches to S. japonica genome using TOPHAT v1.3.2 . These RNA libraries were paired-end sequenced on an Illumina HiSeq 2000 sequencer. RNA sequencing reads were mapped against the 3.2 ref.  with ann3.2 ref.  was empl3.2 ref.  was usedhttp://www.repeatmasker.org/RepeatModeler.html) and RepARKS. japonica genome using default parameters. RepeatModeler is a de novo repeat family identification and modelling package containing two de novo repeat-finding programs (RECONhttp://www.repeatmasker.org). To address this problem, we only retained the sequences that were longer than 800 bp that were produced by RepARK in the new library and used the RepeatModeler-detected short repeats (≤800 bp) as substitute sequences. After applying RepeatMasker to mask the S. japonica genome with this new repeat library, we further simplified the library by removing the sequences with fewer than 50 hits in the S. japonica genome. The RepeatClassifier module in the RepeatModeler package was used to classify the identified repetitive elements based on Repbase.RepeatModeler (ams RECON. RepARK p). The average number of substitutions per site for each fragmented repeat was estimated using the one-parameter Jukes–Cantor model (−3/4ln(1−4/3p)).We first built a consensus sequence for each abundant TE using a combination of RepeatModeler and CAP3 ref. . In totaab initio and transcriptome-based approaches were integrated to predict protein-coding genes in the S. japonica genome. For the ab initio gene predictions, Augustus (version 2.5.5)Arabidopsis thaliana, Ectocarpus siliculosus, Chlamydomonas reinhardtii and Phaeodactylum tricornutum were downloaded from Phytozome and used to align to the S. japonica genome with Exonerate v. 2.2.0  against the NCBI non-redundant protein sequence database. The annotation information on the best BLAST hit derived from the database was transferred to the gene set. Motifs and protein domains were determined with InterProScan v5 ; 1 was Rhizaria (Bigelowiella natans); 1 was Glaucophyta (Cyanophora paradoxa); 5 were Rhodophyta ; 3 were Chlorophyta ; and 2 were plants .We identified gene families in The global protein identities of each BLAST match were calculated using InParanoidS. japonica genome before or after its divergence from E. siliculosus, synonymous (Ks) and non-synonymous (Ka) substitution rates were calculated using KaKs_CalculatorS. japonica genome.A Dollo analysis was conducted on the homologue clusters of the 25 algal and plant proteomes using the Dollop tool in the PHYLIP packageS. japonica genome were conducted with BLASTp  using 8 vBPOs and 3 vIPOs present in L. digitata and E. siliculosus as reference sequences. All of the hits were determined by NCBI BLASTp using the default parameters. The phylogenetic tree was constructed by the neighbour-joining algorithm of the MEGA 5.0 programhttp://cran.r-project.org/web/packages/gplots/index.html). The analogous set of genes involved in the polysaccharide biosynthesis metabolism pathways for mannitol, trehalose, cellulose, laminarin, alginate, sulfate fucan and sucrose in the 14 algal genomes were identified and annotated based on KEGG and previous functional classifications92966S. japonica and distinguish the differences in cell wall polysaccharides from other algae, we performed CAZymes screening in S. japonica and the other 13 algal genomes  were aligned to the draft assembly using BWA v.0.7.5. We employed the default parameters to align the reads, with the exception of the ‘-q 15' parameter, which was used to trim the low-quality regions at the 3′ ends of the reads before mapping. Bcftools in the SAMtools50de novo assembled separately using SOAPdenovoS. japonica Ja assembly. After filtering the multiple mapped regions, NUCmer was employed to call single-nucleotide variants and short InDels.The paired-end reads from the two distantly related species, E and C14 were S. japonica accessions. The neighbour-joining method in MEGA5 was applied to construct a phylogenetic tree based on the p-distance method with 1,000 bootstrap replicates.Homozygous single-nucleotide polymorphism (SNPs) were used to calculate the genetic distances between different K, representing the number of putative ancestral clusters of allelic similarity. We employed an admixture model with correlated allele frequencies to assign individuals to the K clusters. A 10,000 step burn-in period for Monte Carlo Chain searches, followed by 20,000 replicate runs, was applied at each K from 2 to 6.We used STRUCTURE software (version 2.3.2) to investigate population structure across different values of N.Y. and F.Z. conceived the project, designed content and managed the project; X.Z. and D.L. coordinated the project; X.Z. and X.F. prepared DNA and RNA samples; D.X., D.W., Y.W., Z.G. and Z.G. provided algal materials and conducted experiments; J.W. constructed DNA libraries and performed sequencing; F.Z. and P.J. performed raw data analysis; F.Z. and W.S. performed the genome assembly; Y.Z. identified and annotated repeats; J.Q. and F.Z. performed transcriptome analysis, gene prediction and annotation; X.F. performed mitochondrial and chloroplast genome assembly and annotation; X.Z., X.F., G.Y., L. Z., J.Q. and F.Z. performed comparative genomic analyses; F.Z. performed read mapping and SNP identification; and M.M. and F.Z. performed phylogenetic and population genetic analyses. X.Z., F.Z., J.Q., X.F., M.M., Y.Z. and Y.N. wrote the manuscript. Z.Z. and S.C. revised the manuscript. All authors commented on the manuscript.Accession codes. This whole genome shotgun project has been deposited in GenBank under the accession code JXRI00000000.How to cite this article: Ye, N. et al. Saccharina genomes provide novel insight into kelp biology. Nat. Commun. 6:6986 doi: 10.1038/ncomms7986 (2015).Supplementary Figures 1-15, Supplementary Tables 1-8, Supplementary Notes 1-2, Supplementary Methods and Supplementary ReferencesThe 41 ortholog genes used for constructing phylogenetic treeGene families predicted from the S. japonica and the other 24 genomesGain and loss gene families obtained using Dollo analysisSelected genes with decreased Tajimas D values in cultivated S. japonica individualsSelected genes with elevated Tajimas D values in cultivated S. japonica individualsSelected genes with decreased Tajimas D values in wild S. japonica individualsSelected genes with elevated Tajimas D values in wild S. japonica individuals


Ostrinia nubilalis and is suggested to indirectly decrease Fusarium spp. infestation in maize ears. To evaluate this effect, co-occurrence of insect and fungal pests on MON 810 maize was studied. During 2009, exceptionally high maize ear infestation occurred in Julianna-major (Hungary). From investigation of some thousands of maize ears, the majority of the larval damage originated from Helicoverpa armigera larvae, while O. nubilalis larvae contributed significant damage only at a single plot. Fusarium verticillioides infection appeared only in a small portion (∼20–30%) of the insect damaged cobs. H. armigera and O. nubilalis larvae feeding on F. verticillioides mycelia can distribute its conidia with their fecal pellets. MON 810 maize showed 100% efficacy against O. nubilalis in the stem, but lower efficacy against O. nubilalis and H. armigera in maize ears. The ∼Cry1Ab toxin content of maize silk, the entry site of H. armigera, was lower than that in the leaves/stem/husk leaves of MON 810. Fusarium-infected MON 810 cobs are rarely found and only after larval damage by O. nubilalis. H. armigera larvae could not tolerate well F. verticillioides infected food and attempted to move out from the infected cobs. For further feeding they re-entered the maize ears through the 8–12 husk leaves, but in the case of the MON 810 variety, they usually could not reach the kernels. Apical damage on cobs resulted in only a minor (about one-tenth of the cob) decrease in yield.MON 810 maize was developed against  Helicoverpa zea (Boddie)  in North America, Spodoptera frugiperda (J. E. Smith)  in South America, Helicoverpa armigera (Hübner)  in Eastern Europe and Sesamia nonagrioides (Lefebvre)  in Western Europe [O. nubilalis is a stem and maize ear pest, while larvae of European noctuid species damage only maize ears. In the Pannonian Region, where maize ear damage mostly originates from O. nubilalis and H. armigera, strong infestation by O. nubilalis occurs only about once every ten years [H. armigera and O. nubilalis larvae occurred only in 2009. Presently, after soil-disinfection pesticides (formerly used against Melolonthidae and Elateridae larvae) have been withdrawn, Hungarian farmers do not use chemical insecticides in maize production. The low frequency of maize ear damage caused by lepidopterous larvae may question the economic usefulness of MON 810 maize in this region [Fusarium spp. infestation, therefore, fusariotoxin contamination of such kernels is lower. A certain professional debate exists in this field: some groups claim that maize ear rot is associated with feeding damage caused by O. nubilalis and H. zea larvae in the U.S. [O. nubilalis only [MON 810 maize, expressing a transgene encoding a truncated version of the lepidopteran-specific Cry1Ab toxin of estation ,3 resultestation . Differen Europe . O. nubien years . In the s region . Presentlis only . The aim2.2.1.F. verticillioides originating from a single microconidium (storage: 10 °C) [O. nubilalis and H. armigera were established. For a stock colony of O. nubilalis, adults were collected during 2004 in Kéty (Hungary). Larvae were fed on a semi-synthetic diet [H. armigera, larvae were collected during 2008 in Zsámbék (Hungary). Larvae were fed on a different semi-synthetic diet [Potato dextrose agar was used for stock culture of : 10 °C) . Laborattic diet . For bre2.2.H. armigera were collected in a maize field . The fecal samples contained F. verticillioides (∼80%) and F. proliferatum (∼20%) microconidia, thus further experiments were focused on F. verticillioides. RbGUM selective media was used for Fusarium species isolation . Insect and plant samples under field conditions were collected and examined using RbGUM media [In a preliminary examination, fecal pellets of F. verticillioides mycelia. To prepare a F. verticillioides infected disc, sweet maize cobs were cut to 3 cm thick discs. Discs were dipped into an aqueous solution (200 mL) including a stock culture of F. verticillioides and 0.1% Tween 20. The final concentration was ∼80,000 microconidia/mm3. After four days (incubation: 22–25 °C), when the distribution of mycelia was at least ∼50% on the surface of a single disc, second or third instar larvae were transferred onto them. Larvae were fed on sweet maize discs with or without F. verticillioides mycelia until the next molt.Lepidopteran larvae reared for the field experiments in maize ear isolators were fed on sweet maize (Jubilee) discs with or without 2.3.H. armigera larvae having consumed F. verticillioides; (ii) H. armigera larvae not having consumed F. verticillioides; (iii) O. nubilalis larvae having consumed F. verticillioides; (iv) O. nubilalis larvae not having consumed F. verticillioides mycelia; and (v) untreated control  consisting of F. verticillioides mycelia on RbGUM media disc (5 mm diameter) fixed on maize silk was also used. After two weeks, maize ear isolators were collected and their content was carefully checked.Field work was done at the Ecological Research Station, Plant Protection Institute at Julianna-major (the north-western outskirts of Budapest). Maize ear isolators were placed on MON 810 or near isogenic plants at the beginning of silking time . Isolators were placed on artificially infected cobs using third or fourth larval stadia. The stem was cut above the maize ear at the height of maize silk, an appropriately treated larva was placed on it, and the fine mesh maize ear isolator  was placed on the entire maize ear and was tightly closed at the bottom. Treatments were: (i)  control . Repetit2.4.Cry1Ab toxin content in the main parts of the maize ears was determined by a commercial sandwich immunoassay, Abraxis Bt-Cry1Ab/Ac ELISA kit  carried out in 96-well microplates according to manufacturer-provided protocol. ELISA signals were detected on an iEMS microtiter plate reader . Cry1Ab protoxin calibrators (Abraxis) were put on every microplate at concentrations between 0.25 and 4 ng/mL, assays were used for determination of analyte concentration by linear regression. Activated Cry1Ab toxin concentrations were calculated from detected protoxin concentration values with Cry1Ab activated toxin/protoxin cross-reactivity, 56.4%, previously determined for the Abraxis Bt-Cry1Ab/Ac ELISA kit ,12.2.5.ca. 100–400) of maize ears were collected from every assortment. Upon removal of husk leaves, cobs were carefully investigated for symptoms and severity of larval and fungal damage . Four replicates were used resulting in 457 to 1,339 cobs investigated per a single plot. Data without transformation were analyzed using Statistica ver. 5.5 program (ANOVA and Tukey test). Insect and plant samples were also collected for Fusarium spp. identification in the laboratory.A MON 810 (DK-440 BTY) and its near isogenic line (DK-440) were investigated during October 5–8, 2009, in Julianna-major. Depending on the plot size, some hundreds  in different sizes  infected by 3.3.1.Fusarium verticillioides was identified in laboratory fungal rearing tests (using Fusarium-selective RbGUM media) from plant and fecal pellet samples collected by the maize cob isolators in the field experiments. Thus, F. verticillioides infection was verified in fecal pellets of H. armigera and O. nubilalis, husk leaves, ripening kernels and cobs from maize plants damaged by H. armigera and O. nubilalis, as well as stem tunnels caused by O. nubilalis. Insect-transmitted infestation of F. verticillioides (manifested in the occurrence of pink ear rot) appeared to be dependent on the choice of the plant part by the insect to feed on, and on the varying subsequent survival rates due to different Cry1Ab toxin exposures. In contrast, no infestation by F. verticillioides was observed in the untreated control, and none was produced by fixing F. verticillioides mycelia on corn silk, either (H. armigera larvae used (third and fourth stadia) chose maize ears for feeding. The majority of O. nubilalis larvae (third and fourth stadia) preferred to feed on the fresh wound on the stem, where it had been cut prior to the placement of the maize ear isolators on isogenic maize. A small proportion (10–15%) of O. nubilalis larvae tended to feed on husk leaves on MON 810 maize . F. verticillioides infection was transmitted only by larvae having been fed previously on its mycelia  having been fed on F. verticillioides mycelia could transmit Fusarium infestation; the rate was only 20–42%. All of the O. nubilalis larvae died on MON 810 maize, although only this species could transmit F. verticillioides infection feeding on the base of maize ears. Some H. armigera larvae (third and fourth stadia) could survive on MON 810 maize, feeding on husk leaves  were observed in all three, closely located maize fields investigated. Only 20–30% of cobs with larval damage were also infected by ird case . Indepen damages .O. nubilalis, the tunnels were found mostly on stems, and only a quarter of the damage occurred on maize ears  of apical damage was related to O. nubilalis , older H. armigera larvae might move (∼10%) toward the middle of the cob, seeking a drier environment, or come out from the maize ear at the top and choose another maize ear. In this latter case, older H. armigera larvae choose husk leaves to reach the middle of the cob. The most frequent damage type by H. armigera larvae occurs on the top of the cob by feeding on ripening seeds. An ample amount of fecal pellets may be found at places of earlier seeds. Fecal pellets of H. armigera are sometimes covered by mycelia of different fungi. In our case, plant pathogenic F. verticillioides was the most abundant  was H. armigera larvae—the most frequent damage type . O. nubilalis larvae could tolerate F. verticillioides mycelia much better. Nonetheless, H. armigera and O. nubilalis larvae feeding on F. verticillioides mycelia for a short period can spread fungal microconidia, which can survive the digestive channel of these insects. F. verticillioides was readily identified from the fecal pellets of both species. H. armigera larvae usually attempt to escape from moldy tunnels. During this period, the larva can transmit F. verticillioides. Evaluating co-occurrence of insect and fungal infection on the isogenic maize line, only 20–40% of the larva feeding on F. verticillioides infected seeds was found to transmit maize pink ear rot  [It was apparent from the laboratory experiments that young larvae of eir food . In a la ear rot . Similareriments . In cont as well . Incidens effect –18. In as effect ,20, infe events) .4.2.O. nubilalis larvae, which usually feed on the base of the leaves after hatching and subsequently make a tunnel into the stem , while only a moderate concentration  is produced in the stem [O. nubilalis larvae was seen in the stem of MON 810  ∼Cry1Ab toxin distribution in MON 810 plants modifies survivorship of a single larva depending on where it attempts to enter the plant; (ii) O. nubilalis larvae prefer feeding in stems, while H. armigera larvae feed on corn ears only.Laboratory experiments using third and fourth larval stages of H. armigera larvae tend to change feeding place in case of F. verticillioides infection, when they attempt to reach kernels via husk leaves; (iv) early infestation caused mostly apical maize ear damage; (v) only in 20–30% of cases was larval damage followed by a F. verticillioides infection; (vi) eventual yield loss is only about one-tenth of the corn ear apical infection; (vii) MON 810 maize effectively controls maize ear infection by H. armigera and O. nubilalis, but some larvae may survive leading to faster development of Cry1Ab-resistance in the future.Field experiments with natural infestation indicated: (iii)


Rhodnius prolixus and Triatoma dimidiata are the main domestic triatomine species known to transmit T. cruzi. However, there are multiple reports of T. cruzi transmission involving secondary vectors. In this work, we carried out an eco-epidemiological study on Margarita Island, located in the Caribbean region of Colombia, where Chagas disease is associated with non-domiciliated vectors.In Colombia, Trypanosoma cruzi in this area, we designed a comprehensive, multi-faceted study including the following: (i) entomological evaluation through a community-based insect-surveillance campaign, blood meal source determination and T. cruzi infection rate estimation in triatomine insects; (ii) serological determination of T. cruzi prevalence in children under 15 years old, as well as in domestic dogs and synanthropic mammals; (iii) evaluation of T. cruzi transmission capacity in dogs and Didelphis marsupialis, and (iv) genetic characterization of T. cruzi isolates targeting spliced-leader intergene region (SL-IR) genotypes.To understand the transmission dynamics of Triatoma maculata, and 71.6 % of them were infected with T. cruzi. Blood-meal source analysis showed that T. maculata feeds on multiple hosts, including humans and domestic dogs. Serological analysis indicated 2 of 803 children were infected, representing a prevalence of 0.25 %. The prevalence in domestic dogs was 71.6 % (171/224). Domestic dogs might not be competent reservoir hosts, as inferred from negative T. cruzi xenodiagnosis and haemoculture tests. However, 61.5 % (8/13) of D. marsupialis, the most abundant synanthropic mammal captured, were T. cruzi-positive on xenodiagnosis and haemocultures.Out of the 124 triatomines collected, 94 % were T. maculata and dogs in T. cruzi persistence in this region and presents evidence that D. marsupialis are a reservoir mediating peridomestic-zoonotic cycles. This picture reflects the complexity of the transmission dynamics of T. cruzi in an endemic area with non-domiciliated vectors where active human infection exists. There is an ongoing need to control peridomestic T. maculata populations and to implement continuous reservoir surveillance strategies with community participation.This study reveals the role of peridomestic The online version of this article (doi:10.1186/s13071-015-1100-2) contains supplementary material, which is available to authorized users. Trypanosoma cruzi, its aetiological agent, is a multi-host parasite adaptable to hundreds of mammalian species in diverse ecological niches that uses numerous routes of transmission [T. cruzi is defined as a heterogeneous taxon and subdivided into six Discrete Typing Units (DTUs), named TcI to TcVI [T. cruzi to domestic transmission cycles is carried out by the presence of triatomine vector species adapted to human dwellings and by the random house invasion of sylvatic adult triatomines, mechanisms by which persistent Chagas transmission occurs along the American continent [Chagas disease is a zoonosis endemic to the Americas. smission . T. cruz to TcVI , and mor to TcVI . Moreove to TcVI . The incontinent .Rhodnius prolixus and Triatoma dimidiata are the main domestic species known to transmit T. cruzi [T. cruzi transmission involving secondary or non-domestic adventitious household-invading triatomines, such as R. pallescens, Eratyrus cuspidatus, T. venosa and T. maculata; which suggest the importance of the sylvatic cycle and a close relationship between the zoonotic cycles of T. cruzi and human populations [In Colombia, T. cruzi . Howeverulations –10.T. cruzi transmission in conventional vectorial or oral transmission [T. maculata, was often considered to be of little epidemiological relevance due to its low infection rates and apparently ornithophilic preferences [T. maculata in peridomestic human dwellings [T. maculata in transmission cycles from distinct geographical areas, suggests the ability of this vector to adapt to a variety of environments and its distinct ecological behaviours have different epidemiological implications [T. maculata might vary according to its ability to feed on different mammals and by the local fauna composition, the rate of host-vector contact, and the transmissibility competence of domestic, synanthropic and wild mammals that serve as blood meal sources for this species [The epidemiological importance of some secondary vectors in Colombia has been supported by showing their involvement in smission –13. Amonferences , 7, 9. Hwellings , 14, 15.ications . The eco species –19.T. cruzi transmission near homes and the local risk for emergence of human cases [T. cruzi transmission cycles to humans. Dogs-as-sentinels programmes have been proposed for evaluating transmission risk and exposure to T. cruzi as it may indicate T. cruzi transmission hotspots [Several studies have highlighted the importance of peridomestic habitats in the re-infestation process by domestic and secondary vectors, oral outbreaks and emergence or re-emergence of Chagas disease in a region , 19, 20.hotspots , 21.R. prolixus, such as the regions of Boyacá and Tolima [T. cruzi transmission dynamics in areas where only non-domiciliated triatomines are present. Aiming to understand this, we performed a multi-faceted eco-epidemiological study involving the following: vector identification; blood-meal source determination; natural infection rate estimation; seroprevalence estimation in children, dogs and synanthropic mammals; and T. cruzi genotyping. Our results demonstrate the role of T. maculata as vector, dogs as bioindicators of T. cruzi persistence in peridomestic ecotopes, and D. marsupialis as a reservoir connecting the zoonotic/peridomestics ecotopes. These factors constitute the principal eco-epidemiological contributors to the risk of human infection in this region.In Colombia, it has been estimated that 0,96 % of the population living in endemic areas is infected, representing approximately 437,960 people , althougd Tolima –26. HoweT. maculata and R. pallescens [The study was carried out between 2010 and 2012 in seven locations from two municipalities in lowland Margarita Island in the Bolivar department of the Caribbean plains bioregion of Colombia. Sampled localities were Tierrafirme (TF), La Rinconada (LR), La Loma de Simon (LS) and Guataca (GU) located in rural zones of the Mompós municipality; and Talaigua Nuevo (TN), Talaigua Viejo (TV) and El Vesubio (EV) located in the Talaigua Nuevo municipality  from Mompós municipality, the Association of Community Mothers of Talaigua Nuevo (ACMTN), leaders of Community Action Committees (CAC) and the Families in Action Program (FAP), summoned by Talaigua Nuevo town hall. Workshops and individual visits to houses were organized to provide information about Chagas disease and vectors. Local residents were then instructed to collect triatomines found inside their houses and neighbourhoods in plastic vials/bags labelled with their name, address and date of capture. All participants received biosafety training and gloves for self-protection when manipulating the insects.T. cruzi infection determination and blood-meal source analysis.Receiving stations for deposit and storage of triatomine insects were established in schools at all localities and in the office of the health secretary in the TN city hall. Insects were gathered from the school or health secretary each month. Additionally, the houses that delivered nymphs were visited by technical staff to verify the presence of insect colonies. Collected insects were sent to a laboratory in Medellin for species identification, T. cruzi infection by direct microscopy and molecular methods. Faeces were obtained by abdominal compression and diluted in 300 μL of sterile PBS pH 7.2. A 10-μL aliquot was examined under an optical microscope at 400X for flagellated forms. The remaining sample was used for DNA extraction and T. cruzi genetic typing (see below).All triatomines received alive were inspected for T. maculata, 40 adults were selected according to their apparent nutritional status . We carried out a cytochrome b gene high-resolution melting (HRM) analysis from insect faeces following standard procedures reported elsewhere [D. marsupialis), mouse (Mus musculus L), cow (Bos taurus), goat (Capra aegagrus), dog (Canis lupus familiaris) sheep (Ovis aries), horse , pig (Sus scofa), rabbit (Oryctolagus cuniculus), cat (Felis catus), rat (Rattus norvegicus), donkey (Equus asinus), human (Homo sapiens L) and chicken . These species were selected by DNA availability, proximity to human environments, and ecoepidemiological importance.To identify blood-meal sources of lsewhere , 29. Brilsewhere . Reactiolsewhere . The staA. butyracea palm trees. Data management and statistical analyses were conducted using SPSS V.15.0 and Epi-info V 2000. A screening  was performed using 2 x 2 contingency tables, using dogs infected and exposure variables as dependent variables.A questionnaire was provided to dog owners to assess epidemiological data and risk factors associated with Chagas disease . For eacT. cruzi prevalence of ~15 % and a margin of error of ~5 %  were analysed, assuming an estimated Small wild and synanthropic mammals were captured using Tomahawk® and Sherman® live traps arranged linearly. The capture points were established with intercalated traps. The traps were baited with a mixture of peanut, banana, oat and fish, and sets of 15 live traps were placed at 20 m intervals. The traps were placed in peridomestic areas that we defined as the total area around the house, including permanent or temporary structures built and used by humans or their domestic animals. The total capture effort was 360 trap-nights for each town. Eight collection periods were conducted over two consecutive years (2010 and 2011). Wild mammals were anesthetized intramuscularly (9:1 ketamine clorohidrate 10 % and xylazine 2 %) and had their blood collected by cardiac puncture. All of the tested mammals were returned to the wild at the capture site after the experimental procedure.T. cruzi antibodies was conducted using an Indirect Immunofluorescence Antibody Test (IFAT) and an Enzyme-Linked Immunosorbent Assay . The test cut-off was determined as of 1/40 for IFAT and optical absorbance ≥0.200 (mean +/− 3SD) for the ELISA test. Animals were defined as seropositive when samples were reactive to both tests.Detection of anti-T. cruzi and Leishmania spp. in serum were also assayed with antigens derived from a mixture of L. infantum and L. panamensis using IFAT and the Rapid Test for Diagnosis of Canine Visceral Leishmaniasis  [Cross-reactions and/or mixed infection by zil-DPP) , 32.T. cruzi infection was 1/40 when the IFAT results for L. infantum was lower than 1/40 and the DPP result was negative. On the other hand, in dogs positive for L. infantum, positive T. cruzi infection was considered only when the IFAT titer was 1/80 or higher. For L. infantum infection, the adopted IFAT cut-off value was 1/40 when the infection was also confirmed by DPP and 1/80 when the DPP assay was negative [The IFAT cut-off value adopted for negative , 32.T. cruzi sero-positive dogs showing high antibody titers were selected because higher IgG titers occur in the initial phases of infection [T. cruzi epimastigote forms for three months. Xenodiagnostic tests were performed using 20 fourth or fifth instar R. prolixus nymphs per dog and 5–10 nymphs for small mammals. R. prolixus faeces were subsequently examined four times in the next 60 days by direct observation at 400X.Small mammals were anesthetized (9:1 ketamine cloridrate 10 % and xylazine 2 %) to obtain blood samples, which were examined using xenodiagnosis and haemoculture. Similarly, a group of 20 nfection . These dDidelphis marsupialis was collected by direct manipulation and observed under the MO in search of moving flagellates. The positive sample was then stored at 4 °C, for T. cruzi detection by molecular markers and posterior genetic typing (see below).Additionally, secretions from the scent gland of a captured The animal protocol was reviewed and approved by the Institutional Animal Care and Use Committee of the University of Antioquia, Colombia .T. maculata and R. pallescens captured in domiciliary and peridomiciliary habitats and from the R. prolixus used in xenodiagnosis using a phenol-chloroform method [D. marsupialis that was positive in MO.Total DNA was extracted from faeces of m method . DNA wasT. cruzi molecular detection was performed by direct PCR of a 180 bp fragment of the satellite DNA using the primers TcZ1 and TcZ2 [and TcZ2  and a 33and TcZ2 . PCR proand TcZ2 .T. cruzi strains obtained in this work belong to, a 350-bp fragment of the Splice leader intergenic region (SL-IR) was amplified using the primers TC2, TC1 and TCC and PCR conditions as reported in Souto et al. [D. marsupialis (including the sample from the scent gland secretion) and seven samples isolated from T. maculata captured in peridomiciliary environments.To determine which TcI genotypes the o et al. . All ampFor TcI genotype identification, representative sequences of each TcI SL-IR group –40 were With previous written informed consent of one or both parents and following the requirements of the University of Antioquia Ethics committee (License 08-012-185), blood samples were obtained from 803 children under 15 years of age. The procedure was carried out in local hospitals and rural schools by trained personnel. Approximately 5 mL of whole blood was collected by venipuncture. Blood was centrifuged and serum was stored at −30 °C until processing.T. cruzi IgG was detected by two serological tests: (i) all samples were subject to one initial screening by ELISA (enzyme-linked immunoabsorbent assay) based on crude parasites extracted from two T. cruzi isolates . The optical density (OD) values of previously confirmed positive and negative controls were used to define the limits for seropositivity in this assay. OD values higher than 2 SD above the OD average for negative controls were considered ELISA-positive. (ii) All ELISA-positive samples were later evaluated by indirect inmunofluorescence (IFAT) with a titer of 1/40 used as the positive cut off. Samples were considered positive if both tests were reactive.Anti-T. maculata  while the rest were R. pallescens (n = 6) and E. cuspidatus (n = 1). Active search in peridomiciliary environments from localities in TF and TN provided only 10 specimens , whereas most insects (mostly adults) were collected by the community, who brought them to receiving stations at all localities. All specimens were captured in peridomicile structures (mainly in pigeon and chicken coops) and no domestic infestation was detected. Of the 106 T. maculata specimens evaluated for T. cruzi, 76 were positive by PCR, which represents an average prevalence for all localities of 71.6 %  of R. pallescens were positive, and the only E. cuspidatus captured specimen was not infected.A total of 124 insects were collected during this study, from 48 houses throughout all seven localities , followed by dogs (20 %), and humans (10 %). Additionally, 15 % of T. maculata had mixed chicken and dog blood sources in several localities sampled   dogs were seropositive for only Leishmania spp. Three dogs presented specific positive reactions against L. infantum as determined by the DPP test.The mean and standard deviation age of dogs was 3.65 ± 2.2 years (range from 6 months to 13 years). A total of 171 dogs (70.1 %) were seropositive for d) Table . Co-infeT. cruzi infection rate according to the dogs’ sex, age (dogs ≤ 3 years old and older dogs) or the others variables evaluated in bivariate analysis used to identify risk factors. Overall, despite the high rate of T. cruzi infection in dogs, all haemocultures were negative and the presence of flagellates was not detected by direct observation of R. prolixus faeces from xenodiagnosis. However, T. cruzi DNA was successfully amplified by PCR in 45 % (9/20) of insects faeces from the xenodiagnosis bug pools.We found no significant difference in the D. marsupialis and ten Rattus rattus were captured in peridomestic areas.Thirteen D. marsupialis were positive for T. cruzi .Of the 23 synanthropic mammals tested using xenodiagnosis and haemoculture, eight zi Table . InteresT. cruzi isolates from D. marsupialis and T. maculata were DTUI. A comparison of each sequence with reference sequences for each DTUI genotype showed that most isolates were TcIb, whereas the sample obtained from the opossum gland secretion was TcIa   the household-invading behaviour of infected adults, b) transmission in children less than fourteen years old, and c) an epidemiological association between the presence of this species in houses and human seropositivity [T. maculata in Colombia, Venezuela and Brazil [T. maculata is a generalist species with the ability to feed on different hosts, including humans. (iii) We found infected T. maculata that had fed on dogs and humans. This ecological behaviour has not been reported in other endemic regions such as Venezuela and Brazil, where peridomestic T. maculata is found with low or null T. cruzi infection, which is attributed to its ornithophilic preference [T. cruzi vector [T. cruzi transmission cycle in the peridomiciles, including T. maculata¸ dogs and synanthropic D. marsupialis. This is especially relevant for (re) interpreting the routes of canine infection because dogs may become infected by ingesting infected T. maculata [T. maculata populations. Peridomestic insects were found carrying sylvatic, peridomestic and domestic genotypes, such as TcIa, TcIb, and TcId, demonstrating the active participation of T. maculata in different T. cruzi transmission cycles. A limitation of this dataset is the impossibility of genotyping from the blood of positive dogs and humans due to limited amount of DNA shields in such samples. Subsequent studies in the area should direct the efforts in characterizing the genotypes of these hosts to be able to establish the ultimate relationship and intensity of transmission among triatomine vectors, dogs and humans in the area.Traditionally, the apparent ornithophily preference of n plains , 10, 42.n plains , our grositivity . The pred Brazil , 16, 43.eference , 14. Thieference , could ei vector . (iv) Wemaculata  or ingesmaculata . (v) TheT. cruzi within households has been assessed in several studies that established that dogs may play different roles in different epidemiological scenarios, and should therefore be considered to be unique within a certain spatiotemporal scale. In Argentina, Gran Chaco dogs are highly infected by reported triatomine vector transmission, and therefore are considered the domestic reservoirs in this area [T. cruzi [T. maculata contact corroborated with our blood meal source determination, suggesting that this relationship could explain the maintenance of T. cruzi in peridomestic habitats in this area. Moreover, the importance of a secondary reservoir could change according to other factors, such as health conditions, including immune suppression and concomitant parasitic infection [The importance of dogs in the transmission and maintenance of his area . A diffehis area , 19, 31.T. cruzi . SimultaT. cruzi , 48. TheT. cruzi and may eventually transmit it. In addition, molecular diagnosis allowed us to confirm T. cruzi infection in areas with co-circulation of related protozoan species that might cross-react in serologic tests. In the study area, the presence of T. rangeli and Leishmania infantum have been detected [The finding of PCR-positive xenodiagnosis insects gives further support to the fact that seropositive dogs were actually infected with detected .T. cruzi transmission risk to humans is an important tool of surveillance programmes in endemics areas. Serological tests show the exposure of dogs to T. cruzi infection, revealing the presence of parasites in areas close to humans and therefore the epidemiological risk of Chagas disease [R. prolixus, such as the Boyaca and Tolima departments, suggested a moderate T. cruzi transmission cycle [T. cruzi transmission cycle close to the human home. This finding is supported by the fact that 19 of 30 dogs under one year of age were seropositive, which indicates the occurrence of active transmission. Almost all (~90 %) of the infected dogs are used for house protection, and therefore restricted to peridomestic areas. In this sense, the low prevalence observed in children could be explained by the absence of triatomines and dogs inside the houses. However, a proportion of T. maculata fed with human blood, confirmed that a high risk of T. cruzi transmission could be related to the human infections reported here [The role of domestic dogs as bioindicators of  disease , 50. In on cycle –25. We rted here .Trypanosoma cruzi blood-forms in peridomiciliary-collected D. marsupialis support their role as an amplifier reservoir of T. cruzi transmission in the peridomestic area. D. marsupialis is a recognized synanthropic animal, and as it approaches human dwellings, it may serve as a bridge between wild and domestic transmission cycles [T. cruzi isolates from both opossums and vectors were mostly TcIb, a genotype previously found only in wild R. pallescens captured in an A. butyracea palm tree and in periodomestic T. maculata [. Another remarkable result is the positive infection detected in the scent gland secretion of one D. marsupialis individual captured in the TN locality. Infective T. cruzi-forms present in scent glands might confer a vectorial role for D. marsupialis as parasite transmission to humans and dogs can occur through food or skin contaminated with scent gland secretions [. This sample was genotyped as TcIa, a genotype with a strong association with domestic cycles and reported to be the second most frequent genotype in oral outbreaks in Colombia [D. marsupialis was not reported to be infected with T. cruzi in areas of Colombia where oral outbreaks occurred [D. marsupialis is able to maintain T. cruzi infection in the glands without parasitemia [The presence of n cycles –53. T. cmaculata . Anothercretions , 55. ThiColombia . Howeveroccurred . Our resasitemia .D. marsupialis as reservoirs in environments with a high degree of human activity [T. cruzi enzootic cycle or as a link between the enzootic and domestic cycles [T. maculata could be a consequence of the high efficiency of D. marsupialis in introducing many T. cruzi genotypes in peridomestic areas [D. marsupialis as a blood source for T. maculata, which could be a result of our small sample size if D. marsupialis is not a frequent host, because the technique detects only the most recent blood meal [Some authors have highlighted the competence of activity , 56, 57.c cycles , 19, 31.ic areas , 59. HowR. prolixus and T. dimidiata as main vectors in the area, T. cruzi transmission is well established around homes, both in triatomine vectors and the canine population. Moreover, this complete T. cruzi eco-epidemiological profile shows that in an endemic area in Colombia, active transmission is associated with secondary vectors such as T. maculata and wild synantropic reservoirs that may also link sylvatic and domestic habitats. These results emphasize the need for continuous surveillance in areas without the main Chagas vectors, including the use of dogs-as-sentinels, active community participation in entomological surveillance programmes and ad hoc community-based interventions that organize and clear peridomestic areas to remove D. marsupialis.We show that despite the absence of


Salicornia europaea, a salt mash euhalophyte, is a suitable model plant to study salt adaptation mechanisms. S. europaea is also a vegetable, forage, and oilseed that can be used for saline land reclamation and biofuel precursor production on marginal lands. Despite its importance, no miRNA has been identified from S. europaea thus far.microRNAs (miRNAs) are implicated in plant development processes and play pivotal roles in plant adaptation to environmental stresses. S. europaea. Two hundred and ten conserved miRNAs comprising 51 families and 31 novel miRNAs (including seven miRNA star sequences) belonging to 30 families were identified. About half (13 out of 31) of the novel miRNAs were only detected in salt-treated samples. The expression of 43 conserved and 13 novel miRNAs significantly changed in response to salinity. In addition, 53 conserved and 13 novel miRNAs were differentially expressed between the shoots and roots. Furthermore, 306 and 195 S. europaea unigenes were predicted to be targets of 41 conserved and 29 novel miRNA families, respectively. These targets encoded a wide range of proteins, and genes involved in transcription regulation constituted the largest category. Four of these genes encoding laccase, F-box family protein, SAC3/GANP family protein, and NADPH cytochrome P-450 reductase were validated using 5′-RACE.Deep sequencing was performed to investigate small RNA transcriptome of S. europaea, which may play important roles in salt tolerance of this euhalophyte. The S. europaea salt-responsive miRNAs and miRNAs that target transcription factors, nucleotide binding site-leucine-rich repeat proteins and enzymes involved in lignin biosynthesis as well as carbon and nitrogen metabolism may be applied in genetic engineering of crops with high stress tolerance, and genetic modification of biofuel crops with high biomass and regulatable lignin biosynthesis.Our results indicate that specific miRNAs are tightly regulated by salinity in the shoots and/or roots of The online version of this article (doi:10.1186/s12870-015-0451-3) contains supplementary material, which is available to authorized users. Caenorhabditis elegans in 1993 [microRNAs (miRNAs) are a class of endogenous small non-coding RNAs (sRNAs) that are 21–24 nt in length; they regulate gene expression at transcriptional and post-transcriptional levels [miRNAs participate in diverse plant growth and development processes, including leaf morphogenesis and polarity, floral differentiation and development, root initiation and development, vascular development, and phase transition . In addiSalinity is one of the most severe and wide-ranging abiotic stresses that adversely affect plant growth and limit the yields of major crops worldwide. Thus far, soil salinity has been an increasing agricultural problem. More than 800 million ha of the world’s land area, which account for over 6% of the land worldwide, are estimated to be affected by salinity FAO, 2008). Elucidating the mechanisms of plant responses to salinity is an important topic for genetic engineering of crops to improve salt tolerance and ultimately improve crop yield and quality. As sessile organisms, plants have developed various adaptive mechanisms to improve their resistance against salt stress. Over the past decades, numerous studies have focused on revealing the complex mechanisms underlying plant tolerance to salt stress. Salt tolerance is a complex trait controlled by multiple genes, which are strictly regulated at several levels under salinity conditions . In addi08. EluciThellungiela salsuginea [Salicornia brachiata [In contrast to glycophytes, halophytes can thrive in highly saline conditions and are good candidate materials to study salt adaptation mechanisms in plants. The biomass production of halophytes with seawater irrigation varies from 10 ton/ha to 20 ton/ha, which is equivalent to that of conventional crops . Thus, hlsuginea  and SaliSalicornia europaea, a salt marsh euhalophyte belonging to Chenopodiaceae, is one of the most salt-tolerant plant species worldwide [S. europaea has been recognized as a model plant to study the molecular mechanisms of halophytes in surviving under salinity conditions. Salicornia seeds contain high levels of unsaturated oils and proteins [S. europaea [S. europaea miRNAs has been reported to date. Taking advantage of S. europaea transcriptome data in our previous study [S. europaea through high-throughput sequencing technology and bioinformatics analysis in the present study. Conserved and novel miRNAs of S. europaea were identified, and their targets were predicted. The expression profiles of miRNAs during salt treatment and between the shoots and roots were also investigated. This study contributed in elucidating the molecular mechanisms of salt tolerance in S. europaea. Specific miRNAs in S. europaea may be applied in breeding stress-tolerant plants and genetically engineering plants with improved properties, which are suitable for growing on marginal lands.orldwide . S. europroteins ,23; henceuropaea -28. Howeus study , we globS. europaea seedlings treated with 200 mM NaCl for 0 h, 12 h, and 7 d . High-throughput sequencing was then performed to identify S. europaea miRNAs responsive to salt stress. Each library generated more than 13 million raw sRNA readouts. After removing low-quality sequences, adaptor contaminants, poly (A) sequences, RNAs smaller than 18 nt, and other artifacts, we obtained about five million unique high-quality sRNAs from each library  [S. europaea mRNA transcriptome sequences. Thereafter, the known non-coding RNAs, including rRNAs, tRNAs, snRNAs, and snoRNAs, were annotated and removed. The remaining sRNA sequences were used for BLASTn search against the known plant miRNAs in the public miRNA database miRBase [In our previous study, we acquired n 500 bp . In the .org.cn) . About 3S. europaea contained a large and complex sRNA population. The sRNA singleton rate of S. europaea (77% in average) is similar to that of Arabidopsis thaliana (65%), Oryza sativa (82%), and Cunninghamia lanceolata (74%) [Although some sRNAs were abundant and appeared hundreds of thousands times in our database, the majority of sRNAs were only sequenced few times. For example, 4,503,775 (79%) sRNAs were sequenced only once in S-0 h Table , indicatta (74%) .The majority of total sRNA reads ranged from 20 nt to 24 nt in length, which are the typical size range for Dicer-derived products . For allS. europaea miRNAs have been reported. In this study, we conducted a local BLASTn search using S. europaea unique sRNA candidates against all plant miRNAs in the miRBase ; this database contains 7,385 miRNAs across 72 plant species [S. europaea [S. europaea  varied from 0.45 to 1.02 with an average value of 0.85  were uniquely expressed in salt-treated shoots or roots. Furthermore, the expression levels of these novel miRNAs were relatively low  were identical to the sequences obtained from Illumina sequencing. Ten of these novel miRNAs contained less than six mismatched nucleotides, and three had more than six mismatched nucleotides  and 13 novel miRNAs (belonging to 12 families) significantly changed in response to salt treatment in S. europaea , indicating the reliability of the high-throughput data  and one candidate novel miRNA (miR5-3p); we analyzed these miRNAs by using stem-loop qRT-PCR, which is a specific, sensitive, accurate, and reliable method to measure individual miRNAs . Almost S. europaea, we predicted putative targets by using web-based psRNATarget program with default settings (http://plantgrn.noble.org/psRNATarget/?function=3) [S. europaea mRNA transcriptome database were used as a custom target database, whereas 210 conserved and 31 novel mature miRNAs were used as a custom miRNA database. A total of 306 S. europaea unigene sequences were predicted as putative targets of 41 conserved miRNA families  . A totalva Table A. Genes va Table B. Most oS. europaea mRNA sequences.We were unable to predict the targets for 10 conserved and one novel miRNA families because of insufficient S. europaea, we amplified the predicted target genes through rapid amplification of 5′ cDNA ends (5′-RACE). Four unigene sequences were verified to be targets of four S. europaea miRNAs  were down-regulated under salinity conditions  and two growth response factor (GRF) proteins were predicted to be their targets. Gene regulation under salt stress is mediated by multiple transcriptional cascades, in which a transcription factor gene is induced to activate or repress downstream targets important for salt resistance. These S. europaea miRNAs may define several of these cascades, in which they act as crucial molecules at an upstream mode in the transcriptional regulatory networks of salt stress signal transduction; as a result, the morphological adaptation of S. europaea can be achieved under salt conditions.A potential regulatory network of salt-responsive miRNAs in s Figure . First, ze roots ,9,17. Inze roots ,47, and ze roots ,49; both Z. mays ,37. In tlsuginea . The tarlsuginea -53. In SS. europaea. Previous studies have shown that UBP16 is required for salt tolerance in Arabidopsis by modulating Na+/H+ antiporter activity and serine hydroxymethyltransferase1 (SHM1) stability and activity [S. europaea. In Arabidopsis, miR393, which targets F-box proteins and a basic-helix-loop-helix family protein, is strongly up-regulated by 300 mM NaCl [T. salsuginea [P. tomentosa [OsmiR393 found that they are more sensitive to salinity and alkaline stresses [Second, some salt-responsive miRNAs target genes involved in protein turnover processes. Seu-miR396h/I, which targets ubiquitin-specific protease (UBPs) that functions in protein degradation, was suppressed during long-term salt treatment of activity . The sup mM NaCl . Howeveromentosa . Analysistresses . ConsideMesembryanthemum crystallinum (ice plant), iPGAM is induced under saline and drought conditions [S. europaea.Third, some other salt-regulated miRNAs target genes encoding metabolic enzymes. One target of seu-miR396h/i encodes sucrose phosphate synthase (SPS), which plays a major role in sucrose biosynthesis. SPS is activated in leaf tissues under osmotic stress in darkness, and thus may function to facilitate sucrose formation for osmoregulation . Moreovenditions . In addinditions . The regS. europaea miRNAs are related to direct responses to stresses and the downstream signaling processes. These miRNAs include seu-miR1, which could target glyoxal oxidase, Rca6, and nucleotide binding site–leucine-rich repeat (NBS-LRR)-type resistance protein; these proteins are involved in stress and/or disease response. Seu-miR2 could also target leucine-rich repeat family protein and phosphoinositide 3-kinase, which are implicated in the signaling pathway.Fourth, specific targets of salt-responsive S. europaea-specific miRNAs; these miRNAs may play key roles in ion homeostasis or accumulation of compatible molecules under salt conditions. A potassium efflux antiporter was the candidate target of seu-miR15, which was only detected in control samples and R-7 d. The genes encoding putative K+ efflux transporter (KEA) from higher plants were first classified in A. thaliana. Thus far, plant KEA function has not been reported despite the importance of its homologs in E. coli for bacterial survival during exposure to toxic metabolites [AtKEA2) is highly expressed in the leaves, stems, and flowers, but not in the roots. AtKEA2 functions as K+/H+ antiporter and can modulate monovalent cations and pH homeostasis in plant chloroplast or plastids [+ antiporter may play important roles in maintenance of Na+ and K+ homeostasis in S. europaea, which is essential for plant survival under saline conditions. A nitrate transporter was predicted to be the target of seu-miR24, which was only detected in S-7 d. Nitrate is an essential nutrient required for plant growth and also acts as a signal that regulates plant development. Nitrate uptake and its distribution at the whole plant level depend on the combined activity of nitrate transporters; a sodium/nitrate cotransport system has been reported in some halotolerant cyanobacteria [Zostera marina L. [S. europaea miRNAs, such as seu-miR24, are involved in regulation of nitrate transport and distribution under saline conditions, which is important for plant development under salt conditions.Finally, many transporters or ion channels are predicted to be targets of specifically conserved and abolites . Arabidoplastids . Therefobacteria  and in tarina L. , in whicmiR156b [miR156 (Corngrass 1) [miR156 in rice, maize, and Brachypodium results in morphological alterations, including increased number of branches, thus providing a favorable phenotype for improved biomass production [S. europaea miRNAs targeting transcription factors   in switcoduction ,66. In tmiR319 leads to enhanced salt and drought tolerance in creeping bentgrass [P. tomentosa [S. europaea, miR319a-3p was suppressed in R-12 h and R-7 d . rRNA, tRNA, snoRNA and snRNA sequences were downloaded from NCBI and Rfam 9.0 (http://rfam.sanger.ac.uk/), while coordinates of genomic repeats were obtained from RepeatMasker (http://www.repeatmasker.org/PreMaskedGenomes.html). The perfectly aligned sRNA was annotated as rRNA/tRNA/snRNA/snoRNA, miRNA, repeat element, exon-sense, exon-antisense, intron-sense or intron-antisense and stored in separate files, based on the annotation of the sequence which it overlapped in S. europaea transcriptome. Matching sRNAs without annotation were stored in the un-annotated file.The raw data were processed with BGI sRNA analysis pipeline to filter out artifact sequences. Non-redundant sRNAs ranging from 18 to 30 nt were collected and stored in the Clean file, which were mapped to S. europaea mRNA transcriptome database were utilized in local BLASTn analysis  against the mature and precursor sequences of miRNAs in miRBase version 20.0 (http://www.mirbase.org/) [S. europaea mRNA transcriptome database using MIREAP with default parameters (http://sourceforge.net/projects/mireap/). BLASTn searches against all nucleotide sequences in NCBI databases were performed to investigate whether these potential miRNA precursors were conserved in other plant species. Putative precursors homologous to known plant rRNAs, tRNAs or mRNAs were excluded. MFOLD was used to predict the secondary structures of the candidate miRNA precursor sequences, utilizing default parameters  [To identify conserved miRNAs, unique sRNAs from sRNA library and contigs from se.org/) . The uniny.edu/) . Only thny.edu/) .P-value. Fold change = Log2  or Log2 (root/shoot). A 2 × 2 contingency table was used to perform Pearson’s chi-squared test for significance of miRNA expression from two samples. miRNAs with P-value less than 0.05 and fold change greater than 1 or less than −1 were considered to be significantly altered [All miRNAs were normalized to transcript expression levels per million reads (RPM). If the raw read of one miRNA in a library was zero, the normalized RPM was adjusted to 0.01. Normalized miRNA reads with values less than 1 were excluded from the differential analysis. The remaining normalized reads were used to calculate the change in miRNA expression and  altered .S. europaea seedlings as described above. cDNAs were synthesized from 2 μg of purified total RNA in 25-μL reactions, containing 200 U M-MLV reverse transcriptase  and 1 μg Oligo d(T), according to the manufacturer’s protocol. Thirty-five pairs of primers for S. europaea precursor sequences were designed  for all of the mature miRNAs and U6 snRNA, which was chosen as a reference controle . The custom plant transcript databases include 57151 unigene sequences from S. europaea mRNA transcriptome database [Web-based psRNATarget program was used to identify putative targets for conserved and novel miRNAs  from equally mixed 6 RNA poles was used to synthesize 5′- RACE-ready cDNAs with N-15 random primer mix and BD Smart RACE cDNA Amplification Kit  according to the manufacturer’s instruction. The first round of PCR involved 10×UPM, outer gene-specific primers and PCR Polymerase (TransGen). The product was diluted 50 times and then used as template for the second round of PCR, which involved NUP and outer/inner gene-specific primers. Amplicons were separated from the gel, then cloned into T1-simple vector (TransGen) and sequenced. The outer and inner gene-specific primers were listed in Additional file http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE62521) [The data sets supporting the results of this article are available in the Gene Expression Omnibus repository under accession no GSE62521 (SE62521) .


Hancornia speciosa, commonly known as mangaba, is used by pregnant women for diabetes treatment, but their effects and possible maternal biochemical repercussions are unknown.To evaluate the effect of Hancornia speciosa aqueous extract treatment on biochemical parameters in serum and oxidative stress in liver of diabetic and non-diabetic pregnant rats.Diabetes was induced by streptozotocin (40 mg/Kg) in virgin female Wistar rats. After diabetes induction, rats were mated. The pregnant diabetic rats were put in four experimental groups, with n minimum=12 animals/group: Non-diabetic; Non-diabetic Treated; Diabetic and Diabetic Treated. Hancornia speciosa leaf aqueous extract (600 mg/kg) was daily administered in all gestational period. On days 0, 7, 14 and 21 the glycemia were measured. On day 21 of pregnancy, all rats were anesthetized and killed. The blood and liver were collected. The biochemical parameters were analyzed for blood  and also in liver  for oxidative stress. Analysis of variance followed by Tukey's test were used. Differences were considered statistically significant when p<0.05.After treatment with H. speciosa extract, non-diabetic and diabetic rats presented no glycemic changes. Therefore, all the experimental groups showed increasing in glutathione levels compared to control group. Both diabetic groups presented higher levels of triglycerides and cholesterol and ALT actives, and also decreasing in serum protein levels compared to non-diabetic animals. Moreover, the treatment with H. speciosa in diabetic group increased HDL-cholesterol and decreased malondialdehyde (MDA) levels compared to diabetic group.The aqueous extract from H. speciosa leaves failed to modify the maternal hyperglycemia, biochemical parameters and stress oxidative.


Arcanobacterium pluranimalium strain  following necropsy from a juvenile giraffe. The species identity could be confirmed by phenotypical investigations and by MALDI-TOF MS analysis, by sequencing the 16S rDNA, pluranimaliumlysin encoding gene pla, and glyceraldehyde-3-phosphate dehydrogenase encoding gene gap with sequence similarities to A. pluranimalium reference strain DSM 13483T of 99.2%, 89.9%, and 99.1%, respectively. To our knowledge, the present study is the first phenotypic and genotypic characterization of an A. pluranimalium strain isolated from a giraffe.The present study was designed to characterize phenotypically and genotypically an  Arcanobacterium was described by Collins et al. 1982 [ Arcanobacterium haemolyticum, Arcanobacterium hippocoleae, Arcanobacterium phocae, and Arcanobacterium pluranimalium. More recently, Arcanobacterium canis and Arcanobacterium phocisimile, two species which were most closely related to A. haemolyticum, were described as novel species of this genus [Genusis genus , 4. A. pluranimalium was performed with two strains isolated from a dead harbour porpoise and a dead fallow deer [ A. pluranimalium could also be isolated from a dog with pyoderma [ A. pluranimalium strains recovered from various specimens were identified phenotypically and by using various molecular targets [The original species characterization oflow deer . In the pyoderma , from ovpyoderma . More re targets . A. pluranimalium strain following necropsy from a juvenile giraffe by various phenotypic properties, by MALDI-TOF MS analysis, and genotypically by sequencing 16S rDNA and the A. pluranimalium-specific target genes pla and gap.The present study was focused on the characterization of anGiraffa camelopardalis reticulata) of the present study was born in 2013. The giraffe was not accepted by its mother or wet nurse and did not accept hand rearing attempts and, because of general weakness, was euthanized three days after birth. The subsequent postmortem analysis revealed an acute hyperemia of lung and liver and a focal emphysema of the lung. The acute pneumonia was caused by a bacterial infection associated with aspirated foreign bodies.The 80.5 kg female giraffe ; A. pluranimalium grew only in low numbers (+). The A. pluranimalium strain 4868, originally obtained from the spleen, was used for further studies. The bacterial strain was investigated phenotypically and by MALDI-TOF analysis [ pla with the oligonucleotide primer pla-F: 5′-GTT GAT CTA CCA GGA TTG ACG C-3′ and pla-R: 5′-TTG TCG GGG TGT CCA TTG CC-3′ and gene gap with the oligonucleotide primer gap-F 5′-TTG ACC GAC AAC AAG ACC CT-3′ and gap-R 5′-CCA TTC GTT GTC GTA CCA AG-3′as described [ A. pluranimalium DSM 13483T.Bacteriological investigations yielded the isolation ofanalysis  and genoescribed . AlignmeA. pluranimalium 4868 investigated in the present study was identified by determination of hemolysis and CAMP-like hemolytic reactions, by using a commercial identification system as well as various other phenotypical tests. The CAMP-like hemolytic reactions with Staphylococcus aureusβ-hemolysin, Rhodococcus equi, and Arcanobacterium haemolyticum as indicator strains are known as typical characteristics of this species [ A. pluranimalium [ A. pluranimalium 4868 of the present study was catalase negative. This was observed previously for A. pluranimalium of bovine origin [ species . Compara Arcanobacterium and Trueperella , MA pla and the glyceraldehyde-3-phosphate dehydrogenase encoding target gene gap revealed a sequence similarity of 99.2%, 89.9%, and 99.1% to the respective sequences of A. pluranimalium DSM 13483T. All three sequences of A. pluranimalium 4868 were deposited in GenBank . A typical dendrogram of the sequencing results of the genes pla and gap is shown in Figures  plo of T. pyogenes, which appeared to be a constant characteristic of all investigated T. pyogenes [ pla of A. pluranimalium seems to be also constantly present in all strains of this species and could be used, as described previously [ A. pluranimalium. More recently, Moser et al. 2013 [ pla as novel target for molecular identification of this species.Sequencing 16S rDNA, the potentially cytolytic toxin pluranimaliumlysin encoding target genepyogenes , pla of  gap had already been described for molecular identification of staphylococcal species [ A. haemolyticum strain isolated from a donkey [ gap could also be used as novel target for identification of A. pluranimalium. Further studies will give information about the constant presence and sequence similarities of both target genes pla and gap, respectively.Sequencing of gene species  and morea donkey . In the  A. pluranimalium of the present study, which was isolated from various organs of the giraffe together with in high number appearing E. coli, remains unclear. Since, beside aspiration pneumonia, no other pathological findings could be detected, this might represent the route of infection. However, the isolation of this bacterial species from giraffe and the hitherto described origin harbor porpoise, fallow deer, dog, sheep, and cow emphasizes the species name A. pluranimalium.The clinical importance of