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Genome-wide analysis of a avirulent and reveal the strain induces pro-tective immunity against challenge with virulent Streptococcus suis Serotype 2
Jing Wang1,
Youjun Feng2,
Changjun Wang3,
Feng Zheng3,
Bachar Hassan4,
Liming Zhi1,
Wenjuan Li1,
Yi Yao1,
Elaine He5,
Shibo Jiang7 &
Jiaqi Tang6
BMC Microbiology volume 17, Article number: 67 (2017) Cite this article
The Erratum to this article has been published in BMC Microbiology 2017 17:152
It was previously reported in China that two recent large-scale outbreaks of Streptococcus suis serotype 2 (S. suis 2) infections in human were caused by two highly virulent S. suis 2 strains, from which a novel genomic island (GEI), associated with disease onset and progression and designated 89 K, was identified. Here, an avirulent strain, 05HAS68, was isolated from a clinically healthy pig.
By comparing the genomes of this avirulent strain with virulent strains, it was found that massive genomic rearrangements occurred, resulting in alterations in gene expression that caused enormous single gene gain and loss. Important virulent genes were lost, such as extracellular protein factor (ef) and suilysin (sly) and larger mutants, such as muramidase-released protein (mrp). Piglets vaccinated with the avirulent strain, 05HAS68, had increased TNF-α and IFN-γ levels in the peripheral blood and were fully protected from challenge infection with the most virulent S. suis 2 strain, 05ZYH33. Transfusion of T cells and plasma from vaccinated pigs resulted in protection of recipient animals against the 05ZYH33 challenge.
These results suggest that analysis genome of the avirulent strains are instrumental in the development of vaccines and for the functional characterization of important of genetic determinants.
The genus Streptococcus is one of the most diverse gram-positive pathogen for both humans and livestock. A few multivalent streptococcal conjugate vaccines have been introduced and have significantly reduced the invasiveness of these pathogens. Recently however, there are some strains that have become resistant to these new types of vaccine [1]. S. suis is one of the most serious pathogens, causing meningitis, septicemia, arthritis and other diseases in pigs [2] and is responsible for substantial economic losses in the swine industry. S. suis has also been associated with meningitis, endocarditis and other diseases in humans [3]. At present, 32 serotypes, based on capsular antigens, have been identified. Serotype 2 is considered the most virulent and is frequently isolated from both swine and humans [4]. The mechanisms involved in the pathogensis and virulence of serotype 2 (S.suis 2) are poorly understood. S. suis 2, followed closely by S. pneumonia, has been found to have the greatest number of gene gain and loss under positive selection pressure [5]. Prevention and control of diseases caused by this pathogen is hampered by the lack of an effective vaccine that generates a protective response.
Several candidate vaccines have been investigated. First, vaccines against bacteria that cause disease mainly by production of virulence protein factors were considered such as suilysin (SLY), muramidase-released protein (MRP) and extracellular protein factor (EF). These proteins have been shown to protect pigs from homologues and heterologous serotype 2 strains; however, a substantial number of virulent strains in some geographic regions do not express these proteins [6, 7]. Second, capsule polysaccharide is another type of virulence factor found in S. suis. However, a vaccine based on capsular material was unsatisfactory because polysaccharide and lipoligosaccharide antigens are similarly variable and are unlikely to provide long-lived immunity, since they are T-cell-independent antigens that are incapable of stimulating immunological memory [8]. Third, some immunity protection has been reported using killed whole cells; however the heat-killed bacteria are rapidly eliminated by chemokine-activated phagocytes and are unable to activate protective long-lasting or immunological memory responses [9]. S.suis vaccines, either polysaccharide, conjugate or killed bacteria, are currently being tested in trials in order to overcome the rise in worldwide antibiotic resistance, However, given the reasons stated above, none of these vaccines are expected to provide full immunological coverage in a near future. It is generally accepted that live-attenuated or avirulent vaccines are suitable for inducing cell-mediated immunity without affecting humoral immunity. Attenuated and/or avirulent live S.suis strains have been tested, though the results have been equivocal [10, 11]. This concern has prompted exploration into alternative possibilities with better therapeutic potentials against diseases caused by S.suis.
At present, the few known factors related to pathogenesis have not been found to induce global protection and are of limited use. Comparing and analyzing genome sequences between virulent and avirulent strains of S.suis 2 may predict ORFs, specify unique genes and reveal the presence of some previously uncharacterized genes predicted to involve in virulence and gene regulation. This discovery will provide strategies for vaccine development and new approaches in the prevention and treatment of diseases caused by S.suis 2.
In this study, an avirulent S.suis 2 strain, designated 05HAS68, was isolated from a healthy pig in the Jiangsu area of China. Genome analysis of strain should significantly increase our understanding of different virulence related genes and genomic islands of highly virulent strains, 05ZYH33 and 98HAH33, which come from the same affected areas. The genomic analysis will increase our understanding the pathogenesis and evolution of these strains. Using this information, we evaluated the ability of the avirulent strain, 05HAS68, to provide protective immunity against a challenge with S.suis 2 virulent strain, 05ZYH33, taken from a patient with streptococcal toxic shock syndrome (STSS).
Bacterial strains and growth
A single-colony isolate of the S. suis 2 strain was assigned the unique strain designation, 05HAS68. This isolate originated from clinically-healthy pigs in the affected areas of Jiangsu in China. This strain and the reference strain, 05ZYH33 were grown in Todd-Hewitt broth (THB) (Difco Laboratories, Detroit,MI) medium or and plated on THB agar plates containing 5% (vol/vol) sheep blood at 37 °C.
Identification of Streptococcus suis serotype
Serotyping was performed by slide agglutination using specific antisera (Statens Serum Institute, Copenhagen, Denmark) and was positive for serotype 2. Briefly, 2 μl of the broth was mixed with 8 μl of 0.1 M PBS (pH 7.4) buffer containing 1: 500 diluted serotype-specific antibody or 3% BSA, incubated at room temperature for 30 min, and visualized at 400 × optical microscope.
Experimental infections of piglets
Three-week-old male specific-pathogen-free (SPF) - piglets from the experimental animal center of Medicinal Research Institute of Nanjing Military Command, were divided into groups, six piglets per group, and were used to evaluate protective effect tests and or specific T cells transfer protective effect tests (6 piglets/group in random). The avirulent strain 05HAS68 was used as the experimental group and virulent strain 05ZYH33 and 98HAH33 was as positive control. All of the SPF piglets were inoculated intravenously at a dose of 1.8 × 109 CFU per piglet through the ear with different bacterial suspensions. For the virulence studies, three groups of piglets were directly injected with 05HAS68, 05ZYH33, or 98HAH12, respectively. To evaluate the protective effect of 05HAS68, two groups of piglets were simultaneously inoculated with 05HAS68, and were given the same dose of bacterial suspensions for booster on day 7. Subsequently, these two groups were challenged with 05ZYH33 or 98HAH12 on 14. All animal experiments were performed in a biosafety level 3 facilities and approved by the ethical committee of PLA Hospital No. 454.
Genome sequencing and comparative genomics
The 05HAS68 S. suis genome was sequenced and assembled using the routine random shotgun method [12]. Prediction of putative coding sequences (CDSs) and gene annotation were done using the NCBI's Microbial Genome Annotation Tools and Genome Annotation Pipeline (http://www.ncbi.nlm.nih.gov/genome/browse/reference/). The predictions and the slices were then searched against the NCBI Entrez Protein Cluster database (https://www.ncbi.nlm.nih.gov/proteinclusters/browse/stats/) as well as proteins from all complete microbial genomes. The genome sequence feature information was visualized using the CGView genome visualization program ([13] http://stothard.afns.ualberta.ca/). Annotation was supplied from both sets and supplemented with information from the Conserved Domain Database (https://www.ncbi.nlm.nih.gov/cdd) and from Clusters of Orthologous Groups [14]. The protein sets of strains 98HAH33, P1/5 and 05HAS68 were compared using FASTA3. The sequence alignment was produced by Mauve Genome software [14] and the phylogenetic tree was built using CLC Genomics Workbench 3.2 software (https://www.qiagenbioinformatics.com/genomics/). Comparison of S. suis genome structures was done as follows: pair wise comparisons of the P1/7, 05ZYH33 and 05HAS68 displayed chromosomes was done using the Artemis Comparison Tool (ACT) [15, 16] and a dot matrix was used to show the relatedness of genome sequences generated with MUMmer [17]. The 05HAS68, 98HAH33, 05ZYH33 and P1/7 CRISPR defense systems were analyzed using CRT1.1 software [18]. Identification of the bacterial CRISPR-associated proteins (Cas) adjacent to the CRISPR loci and comparative analysis of proteins from putative prophages, viruses (bacteriophage and viruses of archaea), and plasmid-like sequences was done using the Non-Redundant GenBank databases (https://www.ncbi.nlm.nih.gov/genbank/).
Light and transmission electron microscopy (TEM)
To examine the strains morphological differences between 05HAS68 and 05ZYH33, the methods were performed according to our described previously [4]. The samples from agar-grown bacteria (05HAS68 and 05ZYH33 strain) were prepared by conventional methods. Briefly, bacterial cell were fixed in 5% glutaraldehyde for 2 h, post fixed with 2% osmium tetroxide for 2 h, dehydrated in a graded series id acetone washes, and embedded in Epon-812 epoxy resin. Thin sections were post stained with uranyl acetate and lead citrate and then examined with a JEM-1010 TEM (Jeol Ltd., Tokyo, Japan) at an accelerating voltage of 100KV.
Proteome analysis
The S. suis strains, 05HAS68 and 05ZYH33, were grown at 37 °C in 400 mL THB, until they reached the mid-exponential phase (OD600 0.8). The bacterial cells were harvested by centrifugation at 5000 × g for 10 min and washed with phosphate-buffered saline. Samples were then homogenized on ice in 1 ml of lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS, 30 mM Tris-Cl, pH 8.5, protease inhibitor mixture) using a glass homogenizer. After sonication on ice for 10 s using an ultrasonic processor, the samples were centrifuged for 30 min at 20,000 × g to remove particulate material. Aliquots (280 μl) of the samples were loaded onto 13-cm Immobiline DryStrip gels (pH 3 to 10; GE Healthcare Biosciences Co.). The first-dimensional electrophoresis conditions and second-dimensional sodium dodecyl sulfate- polyacrylamide gel electrophoresis separation were done as described previously. The separated proteins were transferred to PVDF membranes by electrotransfer. After transfer, membranes were blocked with 1% w/v BSA in PBS (pH 7.4) for 1.5 h at room temperature. The membranes were incubated with sera taken from swine during the convalescent-phase of 05ZYH33 infection or from swine that had been vaccinated with 05HAS68. The sera (1:1000 dilution) was incubated for 1 h at room temperature and subsequently washed three times with 0.05% w/v Tween-20 in PBS for 10 min. Immunodetection was performed with Staphylococcal Protein A-HRP (SPA-HRP, Boster; 1:1000 dilution) at room temperature. Before and after the addition of the secondary antibody, the membranes were washed three times with 0.05% w/v Tween-20 in PBS for 15 min. The membranes were then washed with 50 mM Tris-HCl buffer (pH 7.4) and developed with tetra hydrochloride (DAB, AMRESCO) substrate until optimum color development was observed.
To compare the abundance of protein spots on the 2-dimensional electrophoresis gels, the stained gels were scanned and analyzed with an Image-Master instrument. Each protein spot was analyzed using Malanie viewer 7 software (http://world-2dpage.expasy.org/melanie/).
Lymphocyte preparation, culture and T cell adoptive transfer
The lymphocytes were isolated from the heparinized blood from SPF pigs vaccinated with the avirulent strain, 05HAS68. Briefly, lymphocytes (>85% purity) were prepared by sequential centrifugation on Ficoll and Percoll gradients. After isolation, cells (1 × 106 cells/ml) were incubated with heat-inactive 05HAS68 cells (1 × 106 cells/ml) for 3 days at 37 °C in a 5% CO2 atmosphere. The specific T lymphoblasts were prepared by sequential centrifugation on Ficoll gradients. After washing, the cells were resuspended in RPMI1640 and 1.8 × 109 cells or mixed with 5 ml plasma from the piglets vaccinated with avirulent 05HAS68 and were then transferred to naïve piglets (n = 6) via injection into the ear vein.
TNF-α and IFN-γ quantification by ELISA
Plasma or sera from the pigs was collected following centrifugation and stored at −80 °C until ELISA analysis. TNF-α and IFN-γ was quantified using a swine immunoassay kit (JINMEI BITECH, Beijing, China), as specified by the manufacturer. Sample dilutions giving optical density readings in the linear portion of the appropriate standard curve were used to quantify the level of each cytokine. Standard and sample dilutions were added to duplicate wells on each ELISA plate, and all analyses were performed at least three times for each individual stimulation assay. The A450 of the plates was determined using a microplate reader (Thermo Scientific Multiskan GO, USA). For all comparisons, was considered P < 0.05 to be significant. Statistical analyses were performed using SPSS 11.0 for Windows
Quantitative results were expressed as the means ± SEM. The data were analyzed using IBM SPSS Statistics 19. The unpaired two-tailed Student's t-test or Mann-Whitney test was used to compare means between different groups. One-way ANOVA with Bonferroni's post-test was considered appropriate for multiple comparisons. P-values less than 0.05 were considered significant.
Basic biological features
S. suis strain 05HAS68, isolated from a healthy pig was identified by a quelling reaction and agglutination test. The bacterial body of the 05HAS68 strain showed agglutination and swelled after incubation with S.suis type 2 capsular antiserum (Fig. 1a). This result indicated that the 05HAS68 strain belongs to S.suis serotype 2. A slight difference in growth was observed between the virulent 05ZYH33 and avirulent 05HAS68 strains. The 05HAS68 strain grew at a slower rate than the 05ZYH33 strain in THB and the mean chain length of 05HAS68 was longer than that of 05ZYH33 under the same growth conditions (Fig. 1b). Hemolytic analysis on sheep blood agar plates indicated that 05HAS68 tends to induce diminutive hemolytic zones, compared with 05ZYH33 (Fig. 1c). Similar capsular thickness was observed between the 05HAS68 and 05ZYH33 strains (Fig. 1d).
Identified as S. suis serotype 2 using an agglutination test and the biological features of 05HAS68. a Strain 05HAS68 serotype identification a. Strain 05HAS68 incubated with 3% BSA 0.1 M PBS as control; b. Strain 05HAS68 incubated with coagglutination reagent of specificanti-capsular type 2 antibody. Comparison of basic biological characteristics between strain 05HAS68 and 05ZYH33 (b-d): b), Light microscope morphology of S. suis strains using Gram-staining. c Hemolytic phenotype of the strains on blood agar plates. d Transmission electron micrographs of bacteria cultured in THS
General genomic characterization
The strain 05HAS68 genome is a single, circular chromosome of 2,188,363 bp (Fig. 2a) with a GC content of 41.18%. Here, 1041 genes (82.2%) have homologs in all 3 genomes. Strain 05HAS68 shares 32 genes (1.7%) with 98HAH12 and 26 genes (1.4%) with P1/7. Finally, 275 genes (14.7%) are unique to 05HAS68 (Fig. 2b).
Circular representation of the 05HAS68 genome and in silico comparisons between S. suis genomes. a The outer circle shows the scale (bp). From in to out indicate that circle 1: GC-skew (blue: > 0; light coral: < 0), circle 2: G + C content (avg = 41%), circle 3: duplicated genes, circle 4: colinear blocks (blue: unique to strain68; purple: colinear with strain05), circle 5: unique genes (inner: on plus strand; outer: on minus strand), circle 6: gene density (avg = 85%, win_size = 5 kb; blue: below avg level; red: above avg level), circle 7: cds (color: see COG legend; inner: on plus strand; outer: on minus strand), circle 8: coordinates. b The 05HAS68, 98HAH33, and Sanger protein sets were compared using FASTA3. Numbers under the strain name indicate genes that are not shared with the other strains; values in parentheses are the number of proteins in each strain (excluding frame shifted and degenerated genes). Numbers in the intersections indicate genes shared by two or three strains. These are displayed in the color corresponding to the strains used as the query. Numbers in any given intersection are slightly different due to gene duplications in some strains. c Phylogenetic tree of S.suis serotype 2 strains based on genomic sequences. The CRISPR antiviral system of 05HAS68 (d) and 89/1591 (e). f Two-dimensional gel electrophoresis profile of silver stained whole-cell proteins from S.suis 2 strain 05HAS68 (a) and 05ZYH33 (b) grown to the mid-exponential phase. In the 3D image displayed, the important virulence proteins EF, MRP and SYL are indicated by color arrow grey, black and white
Except the 89 K large insertions in 05ZYH33 and 98HAH33, few differences were found in virulent strain P1/7 that has been reported by us. The 05HAS68 genome is similar in size, number of open reading frames (ORF), GC content, gene length and density to strains 05ZYH33 and 98HAH33, which were sequenced by our lab 2 years ago. However, the order of genes and genome fragments, gene components and a number of the CDS are quite different compared to the virulent strain (Table 1). Furthermore, the capsule plays an important role in virulence and dividing serotypes and 14 genes that control capsular biosynthesis were found to be identical between 05HAS68, 05ZYH33, 98HAH33 and P1/7.
Table 1 Genome features of 05HAS, 05ZYH33, 98HAH12 and P1/7
Two-component gene regulatory systems (TCSs) have been well studied in many pathogenic bacteria and are known to regulate expression of many key virulence factors, such as extracellular toxins [19,20,21,22,23]. The two genomes of the S.suis 2 Chinese virulent strains, 05ZYH33 and 98HAH33, have 15 of the same conserved TCSs. Except that two of them are located in the specific 89 K GEI of the Chinese virulent strains [12], are the same as those in strain P1/7. However, only nine of 15 TCSs were found in the avirulent strain, 05HAS68 (Table 2). The results of sequence and proteomic analysis indicated that some major putative virulence factors, such as EF and SLY, are absent in strain 05HAS68 (Table 3, Fig. 2f). Analysis of the virulence factor MRP, in both strains 05HAS68 and 89/1591 using clustalw and antigenic peptide prediction, showed a large number of mutants on amino acid sequences and antigenic epitopes (Additional file 1: Figure S1).
Table 2 Analysis of virulence-associated genes from 05HAS68 and 05ZYH33 strains
Table 3 Two component signal transduction systems identified in the genome of different S. suis 2 strains
Since it is hard to find the real ancestor of S.suis 2, the last common ancestor (LCA) of the S.suis genome was reconstructed by identifying the homologous genes among the 05ZYH33, 98HAH33, 05HAS68, P1/7 and 89/1591 strains, and assuming that the genes of the LCA were still present in at least two strains and that the special genes in each strain were genes, which were gained. The genes were included in order to construct the LCA genome and to study evolutionary phenomenon, such as rearrangement, duplication, and gain or deletion in each ongoing genome based on the available information, though this arbitrary classification cannot produce the exact genome. In total, 1920 orthologous clusters (ORs) were obtained in these five strains, and cumulative study showed that about 37 orthologous genes may have been reduced with the addition of more strains (Fig. 2b, Additional file 1: Table S1, Table 2). The actual gene number may vary in different strains due to gene duplication. Compared to the LCA, the five strains were missing 192, 181, 223, 235, and 434 ORs, but had added 80, 336, 121, 9, and 155 genes. To evaluate the relationships among strains, a dendrogram was constructed based on noncore genic differences. As expected, strain 05HAS68 and strain 89/1591 from North America, was the most closely related pair (Fig. 2c).
The clustered regularly interspaced short palindromic repeat (CRISPR) presents an interesting genetic marker for comparative and evolutionary analysis of closely related bacterial strains [24, 25]. Analysis of the S.suis 2 05HAS68 complete genome sequence revealed a locus of 1767655–1768150 bp, having a typical CRISPR organization: eight repeats of 36 bp, separated by unique sequence spacers of 30 bp. A clustering of the cas1, cas6 and cas7 genes (stc: str0658, str0659 and str0660) was localized upstream of the locus. A leader sequence, which contained an AT-rich sequence, was located 5'to the CRISPR loci, directly adjoining the first repeat (Fig. 2d). A similar structure was found in contig 269 of 89/1519 (gi|80977822|gb|AAFA02000001.1|), though one repeat of 36 bp was absent (Fig. 2e). CRISPRs were not found with CRISPR recognition tool (CRT) when examining strains 05ZYH33, 98HAH33 and P1/7 [18].
Genome feature with large scale arrangement
Whole-genome alignments of 05HAS68 with 05ZYH33 or a Sanger P1/7 display included all commonly used rearrangement operations that affect a permutation, including reversals (also called inversions), transpositions, and block interchanges (i.e., generalized transpositions). Large chromosomal rearrangements, including both inversions and translocations and at least seven large inversions rearrangement locations were observed, when comparing 05HAS68 to the 05ZYH33 and P1/7 strains. These inversions seemed to create an "X-shaped" like alignment, which likely resulted from chromosomal inversions that pivoted around the origin and terminus many times (Fig. 3a). However, in the S.suis serotype 2 strains, the devil of account genes from phages, but no useful information was acquired to hint that contain prophage in these strain chromosome with prophage prediction software phage finder [26], except a few transposases local in these fragment upstream or downstream such as on sequence of 05HAS68 chromosome site at bases 630703 to 788878 (IS111A), bases 868905 to 920358 (IS204) or bases 1772301 to 1887337. Despite numerous rearrangements and IS elements, the 05HAS68 genome has remained remarkably stable during over a period of 2 years of cultivation in vitro (data not shown).
Whole-genome alignments between the P1/7 and 05ZYH33 strains with the 05HAS68 strain are shown by using MUMmer or ACT. a Dotmatch analysis in MUMmer. 05HAS68 or 05ZYH33 genome sequnce was plased X or Y axes. The red dot indicates that the match DNA sequence is in the same direction and the blue dot indicates the opposite direction. b The G + C content of genomes are shown by 20000-bp sliding window plot and the thin grey line tracing the average G + C content
Most of the genes from prophages were found in these genomes differed in their DNA sequences, suggesting that they were acquired (and then often degraded) after separation of the bacterial lineages. However, at the DNA sequence level the genomes could still be aligned over essentially the entire chromosome (data not shown).
Structural characterization of a GEI
We previously reported that a candidate GEI of ~89 kb in length, which is related to virulence, was investigated at the genomic level [12]. This GEI was found to be site-specifically integrated in the 3' end of the ORF L7/12 of S.suis 05ZYH33 and 98HAH33. This was compared with a strain very closely related to P1/7, which does not carry any element integrated in the 3' end of L7/12. As one novel type GEIs, they contain 15 bp (TTATTTAAGAGTAAC) insertion sequence (IS), which was present in 5' terminal of highly conserved gene L7/L12. This interesting mosaic structure suggests that this region of the genome is a hot spot for lateral GEI transfer. The franking 89 kb genomic island from this IS element in the chromosome of S.suis 2 strains 05ZYH33 and 98HAH33 was noticeable. The IS element also emerged in the 89/1591 strain genome. Since the genomic sequence of strain 89/1591 is unfinished, a novel genomic island, named 55 k, was predicted and assembled from four contigs (gb: AAFA02000004.1, gb: AAFA02000007.1, gb: AAFA02000008.1, gb: AAFA02000134.1). Comparing and mapping this IS element between strains 05ZYH33 and 05HAS68 showed that a large 112 k DNA fragment was inserted in the hot spot. In these regions, G + C content differ significantly from that of the overall G + C content of the host chromosomes (Fig. 3b). Besides franking the fragment, one of the IS elements was located in the 112 k fragment and divided it into 48 k and 64 k. This result suggests a potential tandem integration of GEIs. GEIs 89 k, 112 k and 64 k have identical features, which were found to be integrated at the 3' end of the chromosome L7/L12 gene, presented integrase at 3' terminal and replication initiator A at 5' terminus of the GEI and mosaic structures of several individual acquisitions (Fig. 4). As previously reported, the 89 k GEI carries some genes or gene clusters expressing virulence related proteins and regulators [12] and the 48 k GEI carries a novel system responsible for L-fucose metabolism and the 64 k genetic loci encodes galactose metabolism. Both the 48 k and 64 k GEIs carry two components (VirB4 and VirD4, respectively) from the type IV secretory pathway. Analysis of the 48 k GEI construct showed that it was truncated to lose the replication initiator A gene at the end of the 5' terminus.
Analysis of site-specific genomic island. Colored column represents gene homologues. Green = RPL7/12, black bar = 15 bp repeat sequence, red = integrase, blue = replication initiator, blue = 89 k, violet = 2-component signal transduction systems, histidine kinase (deep) and DNA binding regulatory protein (light)
Evaluation of protective immunity of a piglet against S. suis strain 05ZYH33
Piglets were vaccinated with either the 05HAS68 or 05ZYH33 strain or were used as a control. After two vaccinations given intravenously, via the ear vein, the piglets were challenged with 1 × 108 CFU of the 05ZYH33 virulent strain administered intraperitoneally (i.p.). All of the piglets (100%) immunized with the 05HAS68 avirulent live vaccine strain survived, whereas 100% of unvaccinated control piglets died after challenge with 05ZYH33. The time to death/survival ratio of piglets immunized with the strain 05HAS68 was significantly (P < 0.001) greater than that of nonimmunized piglets (Fig. 5a).
Comparison of whole-cell proteins extracted from S.suis 2 strain 05ZYH33 by 2-dimensional Western blotting using anti-sera pooled from pigs in the convalescent-phase, which had been clinically infected with the S.suis 2 strain 05ZYH33 (a) or vaccinated with 05HAS68 (b). The MRP, EF and SLY proteins are indicated by red, yellow and green arrows
A 2-D Western blot approach was used to compare and visualize the reaction of the whole 05ZYH33 bacteria proteome to swine sera from convalescent-phase piglets with 05ZYH33 infection or piglets vaccinated with 05HAS68. After vaccination in the field trial, the same specific antibodies against the 05ZYH33 strain were induced and compared with the convalescent phase 0f 05ZYH33 infection except with anti-EF, MRP and SLY (Fig. 5). The Wilcoxon signed ranks test was used to analyze the level of serum specific IgG to S.suis in anterior-posterior vaccination 05HAS68. The result showed that the level of anti-S.suis specific IgG in serum from piglets vaccinated with 05HAS68 was significantly increased P (Z) < 0.05. The reactive IgG titers were more than 1: 5000.
The plasma levels of TNF-α and IFN-γ, TNF-α and IFN-γ were not significantly raised (p < 0.05) 2 days after vaccination with the 05HAS68 strain. Compared with naïve piglets, the levels of TNF-α or IFN-γ in the peripheral blood of 05HAS68 vaccinated piglets challenged with 05ZYH33 were significantly increased (p < 0.01) (Fig. 6).
Level of cytokine IFN-γ and TNF-α from different phases of plasma: Group A indicates the cytokine value from naïve piglets. Group B and C indicates cytokine values from plasma after 48 h after vaccination with strain 05HAS68 or from piglets (n = 6) that had been vaccinated twice with strain 05HAS68 and then challenged by an intravenous injection of virulent strain 05ZYH33 2 days later. Values are arithmetic means ± standard errors of the means. *, P < 0.05; **, P < 0.01
Specific T cells transfer protection
Active specific T cells were obtained from the peripheral blood of piglets, from fraternal multiple births that was vaccinated with the live 05HAS68 avirulent strain, and activated via heat inactivation in vitro. These T cells were then transferred to 05ZYH33-infected piglets. The results showed that these T cells could significantly delay the time of death (48–72 h) in the 05ZYH33-infected piglets. Interestingly, the results in piglets injected with the mixture of these T cells and plasma from an immunized animal were similar to those observed in vaccinated piglets, which survived without any obvious clinical symptoms until the end of the experiment (14 days after infection with the 05ZYH33 virulent strain, Fig. 7b).
Observed survival time of piglets. a Protection induced by vaccination of piglets with the live avirulent S.suis serotype 2 strain 05HAS68. (a) and (b) The piglets were challenged intravenously with stain 05ZYH33 and 98HAH12 as positive control.; groups (c) and (d) on day 7 after vaccination with 109 colony forming unit (CFU) of avirilent 05HAS68, the piglets were injected with the same bacterial concentrations of 05ZYH33 or 98HAH12, separately; (e) The piglets were injected only with stain 05HAS68 as negative control. b Observed protection by transfer of active specific T cell against S.suis to thepiglets (4 piglets/group). (a) The piglets were challenged intravenously with stain 05ZYH33 as positive control; (b) the piglets were transfused intravenously active specific T cell (1.8 × 109 cells) from the piglets were vaccined with stain 05HAS68 or (c) active specific T cell (1.8 × 109 cells) and plasma (5 ml) mixture from the piglets were vaccined with stain 05HAS68; (d) the piglets were transfused intravenously T cells (1.8 × 109 cells) and plasma (5 ml) mixture from healthy naïve piglets as negative controls. Each data point represents one piglet
Chinese isolates of highly pathogenic S.suis 2 caused two recent large-scale human epidemics and caused a large number of pig deaths in China. The mechanisms involved in the pathogenesis and virulence of S.suis 2 are not completely understood. Attempts to control the infection have been hampered by lack of an effective vaccine. The identification and characterization of a potential live avirulent vaccine is one of the strategies and prerequisites for development of an effective vaccine.
At least 10% of swine in Germany carry an S. suis strain [27], and it is likely that the percent is higher in China. As the most prevalent among the invasive porcine and human isolates, the complete genome sequences from a few virulent S.suis 2 strains have been reported, including P1/7, 89/1591, 05ZYH33 and 98HAH33; however, the characterized genomes of avirulent strains, which comprise the overwhelming majority of S.suis 2 strains, are almost completely unknown.
The current study reports on an avirulent S.suis 2 strain, 05HAS68, which has similar basic biological features as Chinese isolates of highly pathogenic S.suis 2, with the exception of hemolytic activity in vitro. Proteome detection indicated that the 05HAS68 strain lacks some major putative virulence factors, such as EF and SLY. MRP as an important virulent factor, compare the amino acid sequences of MRP from strain 05HAS68, P1/7, 89/1591, and 05ZYH33, the protein from strain 05HAS68 and 89/1591 emerge large mutant. Analysis and prediction suggest that this mutant causes a change of potential biological function and antigenic epitopes. Analysis of the 05HAS68 genome indicates that it lacks six of 15 TCSs compared with the 05ZYH33 strain. At least three of the missing six TCSs relate to the virulence of S.suis 2. Comparing the genic contents of the five strains suggests that the total number of pan-genes in the S.suis 2 species is around 3880 and the total number of core genes is around 1290. The pan-genome size of S.suis 2 has not been estimated very well since only five sequences are currently available. When the genomes were analyzed together, fewer than 70% of all core-genes were conserved among all strains. When the genomes of individual strains were evaluated, 15 to 35% of the core-genes were noncore. Strain 05HAS68 and the typical North American virulent strain, 89/1591, belong to one branch of the noncore genes tree and strains 05ZYH33 and P1/7 are on another branch. This suggests that strains 05HAS68 and 05ZYH33 are different than the most recent common ancestor.
Previous research presented a comparative genomics approach to assess levels of recombination and positive selection pressure in the core-genome and pan-genome size for each of the Streptococcus taxonomic groups. The results suggested that Streptococcus is the greatest positive selection pressure, has the largest number of loci uniquely selected, and the lineage that had the greatest amount of gene gain and loss. This positive selection pressure means that S.suis 2 is likely to shuttle back and forth between pig and human hosts. We certainly concur with these authors that much of gene gain and loss of S.suis 2 likely reflects species-specific adaptation [5].
Comparing present genomes to the ancestor genome reveals clues to their virulence and environmental adaptation via four major evolutionary paths including genome rearrangement, deletion, duplication and horizontal gene transfer. The flexibility of genomes and examples of DNA rearrangement and their roles in gene regulation have been documented in different prokaryotes [28,29,30]. The whole-genome alignments between P1/7, 05ZYH33 and 05HAS68 displayed numerous DNA fragment inversions and transpositions in their chromosomes. The DNA rearrangements altering gene expression has been reported [29]. These inversions and transpositions chequered with S.suis 2 strains whether change the expression of virulent or relate genes, which deserve our further study… Previous studies from our laboratory, using two genomic sequences (05ZYH33 and 98HAH33) from clinical strains, identified novel genes that are not present in the Sanger P1/7 strain and suggested a GEI insert in the chromosome. In the current study, the genomes of four S.suis 2 strains were analyzed, and a similar GEI structure was found in strains 05HAS68 and 89/1591. Numerous integrative elements (e.g., prophages and/or integrative conjugative elements) integrate into the 3' end of genes encoding tRNAs, though their sequences remain unmodified by the integration. Other integrative elements (e.g., most of the conjugative transposons) integrate into several or numerous sites [31]. Only a few elements site-specifically integrate into the 3' end of protein-encoding genes. The substitution sequence is then generally only similar to the original one [28]. Similar to one novel type GEI, these sequences insert into a chromosome by site-specific integration at the 3' terminal dependent 15 bp direct repeat sequence of an L7/L12 gene and require a functional integrase gene. It is very interesting that these similar GEIs carrying gene clusters, which are very different in function. A few of the strain-specific genes are in the 89 k GEI, although they do not show the classical features of pathogenicity islands, are flanked by insertion elements and display an atypical nucleotide composition, suggesting possible acquisition through horizontal transfer for adoptive evolutionary response to new hosts. Our current knowledge of the impact of mobile genetic elements, as GEIs, in their hosts comes primarily from pathogenicity islands in which bacteriophages, plasmids and transposons act as carriers of genes encoding toxins, effector proteins, cell wall modification enzymes, fitness factors, and antibiotic and heavy metal resistance determinants in pathogenic bacteria. Much less is known about the diversity and role of GEIs in nonpathogens, in which these elements may enable their hosts to adapt to changing environmental conditions or colonize new ecological niches [32]. As avirulent bacterium colonize in the nasopharynges of healthy pigs only, the 05HAS68 genomic 48 k GEI carries a novel L-fuculose metabolic system in S.suis 2 to adapt to this environmental parameter and the 64 k GEI carries a homeo-gene cluster, which is a classical lactose metabolism system. The 48 and 64 k GEI of strain 05HAS68 couple and assemble a larger 112 GEI. As the 48 k construct emerged it was truncated and lost the replication initiator A gene at the end of the 5' terminus. It has been suggested that the 112 k construct evolved by site-specific accretion from a composite structure of GEIs. Although the 112 k GEI from avirulent strain 05HAS68 exhibits a partial similarity to 89 and 55 k GEIs from virulent strains, the virulent strains also carry large niche-specific genes that could putatively affect the survival of strain 05HAS68 in natural habitats. In addition, when strain 05ZYH33 were repeatedly inoculated and cultured for a long time in THB medium, the 89 k GEI was lost from the chromosome and the circular 89 k DNA was detected in the cells [33]. This supports a general hypothesis that GEI contributes to host fitness. Under the same culture conditions, a loss of 112, 48, or 64 k GEI has not been observed in the 05HAS68 strain. Possible reasons are either that the cells cultured in THB had some similarities with their primary habitat or that fitness was not lost due to interference by the CRISPR defense system; however, the reason(s) are not fully understood.
Indeed, to date, no successful vaccine against S.suis has been produced and heat-killed bacterial preparations have failed to induce protection. Because S.suis 2 commands such a large genic diversity, it is almost impossible to depend on 1 or even several virulence factors from one S.suis 2 strain to induce complete immunological protection for each of the S.suis 2 pathogenic strains. The heat-killed bacteria can be quickly cleaned by phagocytes and are not enough to induce specific immunological protection. In the current study, the live cells of the avirulent strain, 05HAS68, could be used for the immunization of swine, the natural host. It was completely cleared in the peripheral blood of the host until about 30 h, and thus had enough time to induce specific immunological protection. Recent studies suggest that the leading vaccine candidate, which primarily stimulates antibody-mediated humoral immunity, may not suffice. T cell-dependent cellular immunity comprises a second means by which vaccines prime long-lived protection against virulent bacterial pathogens [34].
Our results showed that the high titer IgG serum from piglets immunized against the avirulent strain could recognize most proteins of the tested virulent strain. Analysis of cytokines in the serum after 05HAS68 vaccination and 05ZYH33 challenge indicated that the levels of TNF-α and IFN-γ were significant increased. It has been reported that CD4 responses are associated with strong cell-mediated inflammatory responses, which may be favorable for pathogen elimination from the host [35]. The CD4 T cell is a major cellular source of TNF-α and IFN-γ. Mice lacking IFN-γ have been shown to be more susceptible to pneumococcal infection and administration of IFN-γ can enhance survival of mice after pneumococcal challenge [36, 37]. However, TNF-α is a double-edged sword; it is a proinflammatory cytokine that can promote protective immune responses against a variety of pathogens, but may also cause inflammatory host injury under certain conditions [38].
This study found that co-transference of active T cells and plasma from pigs of vaccinated with strain 05HAS68 provided global protection to pigs challenged with the 05ZYH33 virulent strain; however transferring one of the two were insufficient for providing immunological protection. This result suggests that plasma contains specific antibodies and that the bactericidal activity of T cells against S. suis is very important, because T cell immunity may also provide antibody-independent protection against S.suis 2 infection. Results of this study suggest that a naïve avirulent live cellular vaccine that promotes both humoral and cellular immunity will most effectively combat the highly virulent 05ZYH33 strain and has important implications for the development of an effective vaccine to protect pigs against S. suis infection.
In conclusion, comparison of S.suis 2 genomes showed massive scale arrangement, which can potentially alter genes expression, an enormous amount of single gene gain and loss, and GEIs that carry different functional gene cluster inserts for S.suis 2 species. As S.suis 2 has the largest genic diversity, it is almost impossible to depend on using one or even several virulence related substances as vaccines to induce global immune protection for all S.suis 2 pathogenic strains. In view of these results, that specific protection was induced from humoral and cellular immunity via vaccination with the avirulent strain, 05HAS68, it is suggested that research strategies for vaccine design and development be reconsidered using model strains for testing.
Artemis Comparison Tool
BSA:
Bovine serum albumin
CDS:
Coding sequences
CRISPR:
Clustered regularly interspaced short palindromic repeats
EF:
Extracellular protein factor
ELISA:
Enzyme linked immunosorbent assay
FASTA:
It is a DNA and protein sequence alignment software package first described
GEI:
Genomic island
IFN:
LCA:
Last common ancestor
MRP:
Muramidase-released protein
OD values:
Optical density values
Ors:
Orthologous clusters
PBS:
Phosphate-buffered saline
PVDF:
Polyvinylidene fluoride, or polyvinylidene difluoride
Sly:
Suilysin
SPA-HRP:
Staphylococcal protein A labeled with horseradish peroxidase
STSS:
Streptococcal toxic shock syndrome
TCSs:
Two-component gene regulatory systems
TNF:
Tumor necrosis factor
Hsu HE, Shutt KA, Moore MR, Beall BW, Bennett NM, Craig AS, et al. Effect of pneumococcal conjugate vaccine on pneumococcal meningitis. N Engl J Med. 2009;360(3):244–56.
Fittipaldi N, Sekizaki T, Takamatsu D, Domínguez-Punaro Mde L, Harel J, Bui NK, et al. Significant contribution of the pgdA gene to the virulence of Streptococcus suis. Mol Microbiol. 2008;5:1120–35.
Gottschalk M, Segura M, Xu J. Streptococcus suis infections in humans: the Chinese experience and the situation in North America. Anim Health Res Rev. 2007;8(1):29–45.
Tang J, Wang C, Feng Y, Yang W, Song H, Chen Z, et al. Streptococcal toxic shock syndrome caused by Streptococcus suis serotype 2. PLoS Med. 2006;3(5): e151.
Lefébure T, Stanhope MJ. Evolution of the core and pan-genome of Streptococcus: positive selection, recombination, and genome composition. Genome Biol. 2007;8(5):R71.
Li Y, Martinez G, Gottschalk M, Lacouture S, Willson P, Dubreuil JD, et al. Identification of a surface protein of Streptococcus suis and evaluation of its immunogenic and protective capacity in pigs. Infect Immun. 2006;74(1):305–12.
Wisselink HJ, Vecht U, Stockhofe-Zurwieden N, Smith HE. Protection of pigs against challenge with virulent Streptococcus suis serotype 2 strains by a muramidase-released protein and extracellular factor vaccine. Vet Rec. 2001;148(15):473–7.
Newcombe J, Eales-Reynolds LJ, Wootton L, Gorringe AR, Funnell SG, Taylor SC, et al. Infection with an avirulent phoP mutant of Neisseria meningitidis confers broad cross-reactive immunity. Infect Immun. 2004;72(1):338–44.
Segura M, Stankova J, Gottschalk M. Heat-killed Streptococcus suis capsular type 2 strains stimulate tumor necrosis factor alpha and interleukin-6 production by murine macrophages. Infect Immun. 1999;67(9):4646–54.
Busque P, Higgins R, Caya F, Quessy S. Immunization of pigs against Streptococcus suis serotype 2 infection using a live avirulent strain. Can J Vet Res. 1997;61(4):275–9.
Wisselink HJ, Stockhofe-Zurwieden N, Hilgers LA, Smith HE. Assessment of protective efficacy of live and killed vaccines based on a non-encapsulated mutant of Streptococcus suis serotype 2. Vet Microbiol. 2002;84(1-2):155–68.
Chen C, Tang J, Dong W, Wang C, Feng Y, Wang J, et al. A glimpse of streptococcal toxic shock syndrome from comparative genomics of S. suis 2 Chinese isolates. PLoS One. 2007;2(3): e315.
Grant JR, Stothard P. The CGView Server: a comparative genomics tool for circular genomes. Nucleic Acids Res. 2008;36:181–4.
Tatusov RL, Galperin MY, Natale DA, Koonin EV. The COG database: a tool for genome-scale analysis of protein functions and evolution. Nucleic Acids Res. 2000;28(1):33–6.
Darling AC, Mau B, Blattner FR, Perna NT. Mauve: multiple alignment of conserved genomic sequence with rearrangements. Genome Res. 2004;14(7):1394–403.
Carver TJ, Rutherford KM, Berriman M, Rajandream MA, Barrell BG, Parkhill J. ACT: the Artemis comparison tool. Bioinformatics. 2005;21:3422–3.
Kurtz S, Phillippy A, Delcher AL, Smoot M, Shumway M, Antonescu C, Salzberg SL. Versatile and open software for comparing large genomes. Genome Biol. 2004;5(2):R12.
Bland C, Ramsey TL, Sabree F, Lowe M, Brown K, Kyrpides NC, et al. CRISPR recognition tool (CRT): a tool for automatic detection of clustered regularly interspaced palindromic repeats. BMC Bioinformatics. 2007;8:209.
Li M, Wang C, Feng Y, Pan X, Cheng G, Wang J, et al. SalK/SalR, a two-component signal transduction system, is essential for full virulence of highly invasive Streptococcus suis serotype 2. PLoS One. 2008;3(5): e2080.
Pan X, Ge J, Li M, Wu B, Wang C, Wang J, et al. The orphan response regulator CovR: a globally negative modulator of virulence in Streptococcus suis serotype 2. J Bacteriol. 2009;191(8):2601–12.
McKessar SJ, Hakenbeck R. The two-component regulatory system TCS08 is involved in cellobiose metabolism of Streptococcus pneumoniae R6. J Bacteriol. 2007;189(4):1342–50.
Mitrophanov AY, Churchward G, Borodovsky M. Control of Streptococcus pyogenes virulence: modeling of the CovR/S signal transduction system. J Theor Biol. 2007;246(1):113–28.
Lévesque CM, Mair RW, Perry JA, Lau PC, Li YH, Cvitkovitch DG. Systemic inactivation and phenotypic characterization of two-component systems in expression of Streptococcus mutans. Lett Appl Microbiol. 2007;45(4):398–404.
Sorek R, Kunin V, Hugenholtz P. CRISPR--a widespread system that provides acquired resistance against phages in bacteria and archaea. Nat Rev Microbiol. 2008;6(3):181–6.
Marraffini LA, Sontheimer EJ. CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA. Science. 2008;322(5909):1843–5.
Derrick EF. Phage_Finder: automated identification and classification of prophage regions in complete bacterial genome sequences. Nucleic Acids Res. 2006;34(20):5839–51.
Baums CG, Verkühlen GJ, Rehm T, Silva LM, Beyerbach M, Pohlmeyer K, et al. Prevalence of Streptococcus suis genotypes in wild boars of Northwestern Germany. Appl Environ Microbiol. 2007;73:711–7.
Savic DJ, Ferretti JJ. Novel genomic rearrangement that affects expression of the Streptococcus pyogenes streptolysin O (slo) gene. J Bacteriol. 2003;185(6):1857–69.
Borst P, Greaves DR. Programmed gene rearrangements altering gene expression. Science. 1987;235:658–67.
Burrus V, Roussel Y, Decaris B, Guédon G. Characterization of a novel integrative element, ICESt1, in the lactic acid bacterium Streptococcus thermophilus. Appl Eernviron Microbiol. 2000;66(4):1749–53.
Vernikos GS, Parkhill J. Resolving the structural features of genomic islands: a machine learning approach. Genome Res. 2008;18(2):331–42.
Mavrodi DV, Loper JE, Paulsen IT, Thomashow LS. Mobile genetic elements in the genome of the beneficial rhizobacterium Pseudomonas fluorescens Pf-5. BMC Microbiol. 2009;9(8):1471–2180.
Li M, Shen X, Yan J, Han H, Zheng B, Liu D, Cheng H, Zhao Y, Rao X, Wang C. GI-type T4SS-mediated horizontal transfer of the 89K pathogenicity island in epidemic Streptococcus suis serotype 2. Mol Microbiol. 2011;79:1670–83.
Malley R, Trzcinski K, Srivastava A, Thompson CM, Anderson PW, Lipsitch M. CD4+ T cells mediate antibody-independent acquired immunity to pneumococcal colonization. Proc Natl Acad Sci U S A. 2005;102(13):4848–53.
van Rossum AM, Lysenko ES, Weiser JN. Host and bacterial factors contributing to the clearance of colonization by Streptococcus pneumoniae in a murine model. Infect Immun. 2005;73(11):7718–26.
Nakamatsu M, Yamamoto N, Hatta M, Nakasone C, Kinjo T, Miyagi K, et al. Role of interferon-gamma in Valpha14+ natural killer T cell-mediated host defense against Streptococcus pneumoniae infection in murine lungs. Microbes Infect. 2007;9(3):364–74.
Zhang Q, Bagrade L, Bernatoniene J, Clarke E, Paton JC, Mitchell TJ, et al. Low CD4 T cell immunity to pneumolysin is associated with nasopharyngeal carriage of pneumococci in children. J Infect Dis. 2008;197(9):1354.
Aggarwal BB. Signalling pathways of the TNF superfamily: a double-edged sword. Nat Rev Immunol. 2003;3(9):745–56.
The authors wish to think Dr. Ming Li and Chen Chen for helpful discussions. The authors also acknowledge the laboratory post-graduate of Dan Hu and Xiaodan Fen in the Department of Epidemiology, Research Institute for Medicine of Nanjing Com-mand. We thank LetPub (www.letpub.com) for its linguistic assistance during the preparation of this manuscript.
This work was supported by grants to J.T. from the National Natural Science Foundation of China (#81171527 and 81371768), to C.W. from the National Natural Science Foundation of China (#81471920, 31170124 and 81172794), to Feng Zheng from the National Natural Science Foundation of China (#31300119) and to J.W. from the Key Issue of Medical and Health Foundation of Nanjing Military Command (#12Z17 and 09Z015).
The genomic sequences of 05HAS68 in this study were submitted to GenBank (http://www.ncbi.nlm.nih.gov/nuccore/821177616) under accession numbers GenBank: CP002007.2.
JW, YF, SJ, FZ and CW conducted the experiments and prepared the manuscript. LZ, YY, WL critically revised the manuscript and contributed to data analysis and interpretation. SJ, BH, EH and JT conceived of the study, isolated the bacterium and edited the manuscript. All authors read and approved the final manuscript.
The experimental piglets used in this study were reviewed and approved by the Committee on the Ethics of Animal Experiments of PLA Hospital No. 454 in accordance with the relevant guidelines and regulations. The welfare of the animals was adequately protected, and efforts were made to minimize the suffering and distress of the animals.
Translational Medicine Center, PLA Hospital No. 454, Nanjing, 210002, China
Jing Wang, Liming Zhi, Wenjuan Li & Yi Yao
Department of Medical Microbiology and Parasitology, Zhejiang University School of Medicine, Hangzhou, Zhejiang, 310058, China
Youjun Feng
Department of Epidemiology, Medicinal Research Institute, Nanjing Military Command, Nanjing, 210002, China
Changjun Wang & Feng Zheng
University of North Carolina, Chapel Hill, NC, USA
Bachar Hassan
The Warren Alpert Medical School of Brown University, Providence, RI02912, USA
Elaine He
PLA Research Institute of Clinical Laboratory Medicine, Nanjing General Hospital, Nanjing Military Command, Nanjing, 210002, China
Jiaqi Tang
Lindsley F. Kimball Research Institute, New York Blood Center, New York, NY, 10065, USA
Shibo Jiang
Changjun Wang
Feng Zheng
Liming Zhi
Wenjuan Li
Yi Yao
Correspondence to Jing Wang or Jiaqi Tang.
An erratum to this article is available at http://dx.doi.org/10.1186/s12866-017-1061-z.
Comparing of the MRPs from four strains of S.suis 2. (A) Alignment of the amino acids sequences of MRP. (B) Phylogenetic comparison of MRP proteins. Figure S2 A novel system responsible for L-fucose metabolism from the GEI of 48 k. (A) L-fucose metabolic gene cluster. (B) Illustration of L-fucose metabolic pathway. Table S1 Prediction of antigenic peptides of MRPs from four strains of S.suis 2. (DOCX 552 kb)
Wang, J., Feng, Y., Wang, C. et al. Genome-wide analysis of a avirulent and reveal the strain induces pro-tective immunity against challenge with virulent Streptococcus suis Serotype 2. BMC Microbiol 17, 67 (2017). https://doi.org/10.1186/s12866-017-0971-0
Insertion Sequence
Cluster Regularly Interspaced Short Palindromic Repeat
Avirulent Strain
Live Vaccine Strain
Microbial genetics, genomics and proteomics


I am a descendant of a Hanley/Hanly family from. Cashel and Emly Parish. My ggggrandfather was Richard Hanley and he came to Ontario Canada with a group of brothers and sisters or cousins in the years 1840-1845. They include Johanna Hanley, Matthew Hanley and wife Ann Lahey and their children Cornelius, Ellen and Mary; Patrick Hanley, Michael Hanly, Bridget Hanley and John Hanley.They all settled together as farmers. Matthew and Ann's three children were baptized at Emly and Matthew and Ann were married at Emly. Michael was married at Emly and baptized his eldest son Dennis there. I believe that we are related to the Cornelius Hanley family in Emly graveyard.
I am visiting Ireland for the first time next week. My maternal great grandfather was Michael Joy, born in 1874 in Emly. He emigrated with his wife to Liverpool at some stage - my grandmother appears in the 1901 Census in Liverpool, aged 6. We don't have a birth certificate for her and aren't sure if she was born in Ireland or Liverpool. Hope to find out more when we visit Emly next week.
I am third great granddaughter of Patrick Kean, son of Patrick Kean and Eleanor Beardon, baptised in Parish of Emly on 17th March 1826. He left Ireland in 1856 arriving in Melbourne, Australia in February 1857. He died in Melbourne 8th May 1880. He was married to Maria Glennon of Athlone, Co. Roscommon. He had five children. One of his daughter, Deborah Maria Kean was my great grandmother. She married John Dacy (Deasy). Pleased to correspond with any other researchers and to learn more of the Kean family. They have been the most elusive of all my ancestors. Proudly Irish fifth generation Australian.
My maternal Great-Great grandparents came from your parish and I recently found records to verify my Great-Great Grandfathers Thomas Quinlan's baptism. I am overjoyed! Having visited Ireland twice, I must have been within a stones throw ofyour parish and didnt know.
This is for Patrick Lonergan who posted a query on 14th April 2016. I believe your father was my uncle. My mother was Mary Lonergan and she had a brother named Patrick. Jo Hogan was my aunt, my grandfather was Ailbe Lonergan and grandmother Bridget Lonergan. I can put you in touch with my cousin who lives in Ireland (daughter of Teresa Lonergan (sister of Jo) if you haven't had any other response. I came across your post quite by chance.
My mother was Mai Moloney who lived at Lackelly in the early 1900's. Her family house was burned down by the Black and Tans. She left for England in the 1920's. Her brother Danny stayed in Ireland and raised a family. I would love to make contact with my cousins or family.


Best chiropractor Edinburgh for older individuals will be from a chiropractic centre that understands the differing needs of older people. While every patient requiring chiropractic treatment is different, and will have distinctive health needs, there are some specific aspects of care that should be present in a chiropractor who treats older patients.
Firstly, the Edinburgh chiropractor that you approach should have an in-depth understanding of the specific age related health and wellness problems related to aging. They should be understanding of the physical, emotional and mental difficulties associated with age related pain and illness and work holistically to treat problems. Your chiropractor Edinburgh based should also understand the different individual physical needs of older patients and not make assumptions based purely on age. This means ensuring that they still take a full history and complete all necessary tests before beginning any treatments. If you are looking for chiropractic care to relieve age related pain or conditions, then the best way to ensure that you use a chiropractor that fits this description is to ask questions, read reviews of city wide chiropractors, and talk to people who have used local chiropractic services. If you are nervous about your initial visit to the clinic, or concerned that you may not ask the right questions, then take a family member or friend with you who can help fill in the gaps and give you a second opinion if needed. Always ensure that the chiropractor you are considering has experience of spinal manipulation in older individuals and that they are a member of the General Chiropractic Council.
If you are looking for the best chiropractor Edinburgh wide, you can find them at the best clinic in Edinburgh, Capital Chiropractic. We take pride in our level of commitment to all our patients, particularly our older ones who need that little extra support. To see how we can help you or an older relative, visit http://www.capitalchiropractic.co.uk.


text:
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redpajama_set_name: RedPajamaGithub


text:
Ergebnis gesprochen von "Tina Brown" in Alle Kategorien
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The Palace Papers
Inside the House of Windsor, the Truth and the Turmoil
Von: Tina Brown
Gesprochen von: Tina Brown
'Never again', became Queen Elizabeth II's mantra shortly after Diana's death. More specifically, there could never be 'another Diana'—a member of the family whose global popularity upstaged, outshone and posed an existential threat to the British monarchy. Picking up where The Diana Chronicles left off, The Palace Papers reveals how the royal family reinvented itself after the traumatic years when Diana's blazing celebrity ripped through the House of Windsor like a comet.
Unterhaltend (natürlich) und auch informativ
Von Anonymer Hörer Am hilfreichsten 17.10.2022
4.5 out of 5 stars 19 Bewertungen
Inside the House of Windsor - the Truth and the Turmoil
Tina Brown has been observing and chronicling the British monarchy for three decades, and her sweeping account is full of powerful revelations, newly reported details, and searing insight gleaned from remarkable access to royal insiders. Stylish, witty, and erudite, The Palace Papers will irrevocably change how the world perceives and understands the royal family.
125 Years of Literary History
Von: The New York Times, Tina Jordan - editor, Noor Qasim - editor
Gesprochen von: Robert Petkoff, Tina Jordan, Gary Shteyngart, und andere
Since its first issue on October 10, 1896, The New York Times Book Review has brought the world of ideas to the reading public. It is the publication where authors have been made, and where readers first encountered the classics that have enriched their lives. Now the editors have curated the Book Review's dynamic 125-year history, which is essentially the story of modern American letters. Brimming with remarkable reportage, this book collects interesting reviews, never-before-heard anecdotes about famous writers, and spicy letter exchanges.
Gesprochen von: Robert Petkoff, Tina Jordan, Gary Shteyngart, Jodi Picoult, Junot Diaz, Sloane Crosley, Jenna Wortham, Tayari Jones, Roxane Gay, Sarah Weinman, Jericho Brown, Dominic Hoffman
4 out of 5 stars 1 Bewertung
The Vanity Fair Diaries
The Vanity Fair Diaries is the story of an Englishwoman barely out of her 20s who arrives in New York City with a dream. Summoned from London in hopes that she can save Condé Nast's troubled new flagship Vanity Fair, Tina Brown is immediately plunged into the maelstrom of the competitive New York media world and the backstabbing rivalries at the court of the planet's slickest, most glamour-focused magazine company. She survives the politics, the intrigue, and the attempts to derail her by a simple stratagem: succeeding.
The Vanity Fair Diaries: 1983-1992
The Vanity Fair Diaries is the story of an Englishwoman barely out of her 20s who arrives in New York City with a dream. Summoned from London in hopes that she can save Condé Nast's troubled new flagship Vanity Fair, Tina Brown is immediately plunged into the maelstrom of the competitive New York media world and the backstabbing rivalries at the court of the planet's slickest, most glamour-focused magazine company.
Memory Hacks - How to Hack Your Memory and Remember Almost Anything
Spieldauer: 29 Min.
Have you always wanted to have perfect memory? Perfect or picture perfect memory as it's sometimes called is something that most people dream about. But, actually it's a skill that with practice any one can learn how to have perfect recall no matter the subject. Imagine studying and being able to recall every note? Or, going to work and being able to remember everything that happened in the last meeting without having to take any notes? How would having a memory like that change your life?
Regulärer Preis: 4,95 € oder -1 Guthaben
Verkaufspreis: 4,95 € oder -1 Guthaben
We have all at some point in time, had the nasty green monster inside of us, emerge. Jealousy can ruin our relationships and cause us intense grief and despair. That is why the author of, How to Deal With Jealousy, has created this exceptional audiobook. As you listen to the meaningful advice and tips you will discover: Asking yourself if women like men who are jealous? The answer, of course, will be, NO! That jealousy can ruin intimate relationships. Analyze whether you suffer from neighbor envy. Asking yourself if you have ever been guilty of going through your spouse's belongings?
Efficiency Hacks - Productivity Hacks That Speed up Your Efficiency
Picture this: you send off your loved ones and step into a quiet house. There is no chaotic rush hour traffic, pesky co-workers, or mindless meetings. Instead, you sit in the comfort of your home and immerse yourself in a peaceful and productive workday. This sounds like a dream, right? No, this can be your reality. In Efficiency Hacks: Productivity Hacks That Speed Up Your Efficiency, the author takes you down a motivational journey to finding yourself living out the dream of working from home.
How to Persevere
Even When You Really Want to Quit
Von: Mark Bogdanovic
In Accepting Rejection: The Cornerstone of Perseverance, a self-help motivational book, the author offers tips and tricks on how to find perseverance to build and grow your life. Dealing with rejection is a part of life. For some of us, we have to learn how to move forward after the rejection. Broken into easy to follow sections, this audio book offers real-world situations and experiences that we all face in our everyday lives. Knowing how to push through your own limitations and building a successful ladder to reach your dreams is something we all desire.
Leadership Hacks
Simple Concepts to Become a Better Leader
When you communicate with others at your place of work, do you find people coming to you for advice? Is your voice heard when important decisions need to be made? Some people are privileged to have the character trait of being a leader. But, do you use your leadership skills for other positive purposes? If you have solutions to most problems, try using that ability in your community.
How to Be More Daring, Bold and Audacious
Are you tired of feeling like everyone in your life walks over you? Do you catch yourself backing down from conflict? If you answered yes to these questions, then you need to check out this audiobook. In "How to Be More Daring, Bold, and Audacious", the author walks you through easy steps to help you take back your life and your voice. In this easy to follow audiobook, you will: Learn to pick your battles when dealing with conflict. Understand that be by being more daring, you are opening yourself to take more risks. Have newfound confidence.
Dealing with Problems and Obstacles
How to Master Your Life
Could you imagine your life without obstacles? If the answer to that question is yes, then this audiobook is just for you. In Dealing with problems and obstacles, the author takes you down a path to self-discovery to welcome our everyday challenges and see the benefits in each obstacle presented to us. The feeling of elation one receives when overcoming a difficult challenge drives people to want to continue, even when life gets difficult. In this audiobook you will learn the necessary tools that will: Teach you that failure is just an obstacle, not an end.
The Subtle Art of Not Caring
Do you ever find yourself worrying about what others think about you? Do you ever make important life decisions based on how others view you? Do you take everything people say and do personally? We all do... it's something our society has created inside each of us. If you find yourself trapped inside the opinions of others, you will find, "I Don't Care - The Subtle Art of Not Caring", a transformative guide to helping you care less about judgments made by those around you.
Forget the Mistake, Remember the Lesson
How to Bounce Back from Your Mistakes
In Forget the Mistake, Remember the Lesson, learn how to bounce back from mistakes you make in your personal and financial life. Be prepared to accept your imperfections and grow as a person and leader as you enhance your listening skills and to learn from those around us. The author offers insightful how-to tips and advice on correcting those pesky mistakes and owning up to your faults.With detailed subheadings, readers can journey through the audiobook to learn how to: Give yourself permission to make mistakes, Help others bounce back from the same mistakes you have made.
Simple Ways to Stop Procrastinating
Feel like you just can't get anything done? Struggling with deadlines? Or, do you want to become more motivated in life and unlock your true potential? If the answer to these questions is yes, then you are struggling with procrastination. With Stop Procrastinating, an easy to follow guide on how to stop procrastinating, you'll discover tips and tricks to end that pesky habit once and for all. The solution is easy and outlined in several paragraphs with detailed subheadings outlining each helpful section.
Smart and Simple Ways to Motivate Yourself
In I Can: Smart and Simple Ways to Motivate Yourself, a self-help motivational audiobook, author Tina Brown offers smart and simple ways to motivate yourself. Broken into easy to follow sections, this book offers modern ideas for the business savvy reader. With a business theme evident, readers will find connections to their everyday lives and favorite products and past times - which makes this self-help book stand out from the rest.
Setting Smart Goals: How to Think Big and Achieve Your Goals
Today, the entire human race is trying to set smart goals, as only through setting up a smart system will you be able to achieve your dreams. Goals are a huge part of our everyday life, as they help us achieve the plans we have set in place. These goals can be about your everyday life and work, or about your professional work. Either way, it all comes down to accomplishing something. If you do not set goals your life will be chaotic, and all over the place.
Being People Person: How to Be a People Person When You Aren't
A people's person is someone who is good at being around other individuals. Some have this tendency naturally, while others have to put in more effort to become a people's person. You might have found that it is not easy for you to talk to random strangers as your friend, but that is something that you can work on. The right place to start is by making a commitment to yourself, as this is a long track and you will need to be persistent.
Tips to Boost Confidence Right Now
Easy Ways to Increase Your Confidence
Similar to finding the perfect pair of pants, or getting into an IVY League school, gaining confidence is an elusive thing. However, without confidence, it's too easy to fall behind in life. As it leads to self-doubt, and negative thoughts. This does not only damage you on a personal level, but increases the chances of depression, and anxiety. As when you keep your thoughts and ideas to yourself, you are unable to have meaningful conversations, or make an impact. Fortunately, just because you lack in confidence right now, does not mean you have to keep living like this.
Beauport Anthology: A Collection of Dramatic Monologues of Gloucester's Historical Characters (1600-1900)
Von: Dr Jay DiPrima
Gesprochen von: Bradley Royds, Gordon Baird, James Buhrendorf, und andere
Spieldauer: 2 Std. und 1 Min.
Hear the stories from the colorful characters of Gloucester in their own words! From the Sachem Masconnomet to the renowned Fitz Henry Lane, Howard Blackburn, General Benjamin Butler, John and Judith Sargent Murray, and many others. Relive the early history of America's oldest seaport through the mouths of its salty characters.
Gesprochen von: Bradley Royds, Gordon Baird, James Buhrendorf, Duncan Nelson, David Adams, Nora Messier, Tina Greel, Talia Brown, Jay DiPrima
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redpajama_set_name: RedPajamaCommonCrawl


To meet online is increasingly becoming the norm. In fact, online meetings have been increasingly gaining popularity over the last several years, thanks to services like Google Hangouts. At the same time, there are commercial solutions that are available as well.
We are happy to announce the availability of Techniq Meeting, a simple, easy to use, online web conferencing tool. Participants can use webcams to see each other and their headsets to speak via VOIP.
The presenter needs to install Java in order to screen share. Other than this no installs are required. The presenter can however, share docs, ppts etc. during the presentation rather easily.
The presenter can also record the session. For now, we allow up to 10 attendees per session. Registrants can manage their contacts and easily invite other attendees from their contacts.
And guess what,this service is free!
Give it a test drive, try it today and do send us your feedback!


text:
This Week's TV: Ease on Down the Road—The Wiz Is Coming to Television!
Gordon Jackson
Filed to: What to watchFiled to: What to watch
Doctor Who turns everything upside down. The Flash and Arrow cross over to battle Vandal Savage. Minority Report ends. Plus the live-action debut of Red Tornado! And lo, The Wiz! live on television! All this, and much, much more, on This Week's TV!
Be Cool, Scooby-Doo! (6:30PM, CARTOON)
A pterodactyl haunts the Scooby gang— on Christmas! The science vs. religion debate has never been more intense.
Regular Show (7:30PM, CARTOON)
A full week of new episodes! Every day at 7:30.
Gotham (8PM, FOX)
The two-fisted fall finale! Can the full force of the GCPD's firearms take on the spooky monk-cult, what with their hand daggers?
Supergirl (8PM, CBS)
Red Tornado makes his debut! And yes, he does look a bit like Kryten from Red Dwarf, perhaps.
Minority Report (9PM, FOX)
Series finale! Back into the milk bath it goes...
Uncle Grandpa (6:30PM, CARTOON)
It's Secret Santa-time aboard the UG RV. Who gets Pizza Steve?
The Muppets (8PM, ABC)
"After a show-stopping duet with Miss Piggy on "Up Late with Miss Piggy," Joseph Gordon-Levitt joins Scooter, Pepe and the gang for poker night; The Great Gonzo gears up to perform his dream stunt; and Dave Grohl challenges Animal to a drum-off."
The Flash (8PM, CW)
The highly anticipated Flash/Arrow crossover introducing the immortal Neanderthal, Vandal Savage!
Scream Queens (9PM, FOX)
A Black Monday-set slasher episode! That's such an intuitive idea, I'm not sure how it hasn't been done already…
Agents of SHIELD (9PM, ABC)
"Ward's campaign for revenge brings the S.H.I.E.L.D. team to their knees, and Coulson proves he will do anything to settle the score."
iZombie (9PM, CW)
The iZombie cast eat po' boy sandwiches in this oddly selected clip from "The Hurt Stalker"— a Babineaux-centric episode in which the murder victim is his own heretofore unmentioned stalker!
Adventure Time (6:30PM, CARTOON)
The day of secret tapes is upon us.
Arrow (8PM, CW)
The back-half of the Flash crossover concludes!
Supernatural (9PM, CW)
A whole episode apparently inspired by Drop Dead Fred!
American Horror Story (10PM, FX)
John closes in on the Ten Commandments Killer!
Teen Titans Go! (6:30PM, CARTOON)
"Fed up with never getting presents from Santa, the Titans head to the North Pole to take themselves off the Naughty List, by any means necessary."
The Vampire Diaries (8PM, CW)
The Heretics are throwing a Murder Party in honor of Mary Louise & Nora's anniversary. That's very sweet.
The Wiz Live! (8PM, NBC)
With a cast that includes Common, Queen Latifah, Mary J. Blige, Stephanie Mills, Ne-Yo, and David Allan Grier as the Cowardly Lion, this is everything we've ever wanted. Talking to the Hollywood Reporter, director Kenny Leon explains why Queen Latifah is the Wiz this time around:
"There's no reason she can't be a woman — it's a dream!" he laughed, as he told The Hollywood Reporter during a New York City press day for the NBC musical. Seizing the opportunity to say something "about the treatment of women," he and book writer Harvey Fierstein included a new backstory for Latifah's character. "She didn't have the greatest relationship with her husband and that's how she ended up here. All of the women in this story, they make their own decisions in collaboration with men, but the men are not speaking for them, and that's the way it should be anyway."
The Originals (9PM, CW)
"After learning that Cami's (Leah Pipes) life is in danger and Aurora (guest star Rebecca Breeds) may be to blame, Klaus (Joseph Morgan) is forced to engage in another one of her devious games and follows a series of clues she's left behind for him. Meanwhile, Elijah (Daniel Gillies), Freya (Riley Voelkel) and Hayley (Phoebe Tonkin) take drastic measures against Tristan (guest star Oliver Auckland) when they discover he holds a valuable piece of information they need. Elsewhere, Aya (guest star Tracy Ifeachor) gives Marcel (Charles Michael Davis) an ultimatum that leaves him with a difficult decision to make, while tensions between Hayley and Jackson cause her to reevaluate her involvement in Mikaelson family matters."
Haven (10PM, SYFY)
"Plans for building a new barn are complicated when an old trouble brings the Haven Police Station to life. It's up to Audrey and Nathan to find a way to regain control, but their efforts are hampered when a mysterious killer leaves a wake of bodies right under their noses."
Just three episodes left! Naturally, it's time to build a barn.
Grimm (9PM, NBC)
Nick vs The Rat King!
Z Nation (10PM, SYFY)
"It's discovered that the Z-vaccine doesn't work; Vasquez gets caught trying to kill Escorpion; Murphy uses mental manipulation to help Roberta."
Tim & Eric's Bedtime Stories: Tornado: (11PM, CARTOON)
Ash vs. The Evil Dead (9PM, STARZ)
We're halfway through the first season! Now that Ash knows what to do with the Necronomicon, he decides to visit a rustic, Twin Peaks-esque diner.
Doctor Who (9PM, BBC AMERICA)
Season finale! Doctor Who does a Western on Gallifrey, in "Hell Bent"!
Is Me/Ishildur the Hybrid? Will we finally get some closure on The Valeyard? Is the Doctor truly half-human on his mother's side? And why did he really leave Gallifrey in the first place? You may-or-may-not get the answers!
Personally, I'm crossing my fingers for The Could've Been King and his army of Meanwhiles and Neverweres.
The Librarians (8PM, TNT)
"When people begin suffering the effects of accidents and sins in which they have not actually been involved, the team tracks the connection to the perpetually young Dorian Gray, whose magic now works on selfies."
Dorian Grey's magic now works on selfies. You want high concept? The Librarians has it in spades.
Once Upon a Time (8PM, ABC)
Season finale!
The Last Man on Earth (9:30PM, FOX)
"Along with most human life, the virus wiped out normal traditions and holidays. As Christmas approaches, Carol spearheads a "Secret Santa" gift exchange to spread some festive cheer."
Recent from Gordon Jackson
The Most Interesting Stars Rumored for Marvel's Eternals Deny They're in Marvel's Eternals
Seems Like the Power Rangers Movies Are Already Being Rebooted
Another Wild Rumor About the Cast of Marvel's Eternals


This report has summarized and showed what I have done and learn during the whole process of this Final Year Project. The objective of this project is to detect pedestrians or any object in a captured video in a real time case by using the image analysis techniques. Besides that, user can also choose an image to upload to the website thereafter the detection box, detection value and processing time will be shown on the image. In the server, we limit to a few detection objects such as car, human, dog, bicycle and etc. it does not have everything in the server due to the time taken to retrieve an image data from the server. In other words, if there are too many data in the server itself, it will slow down the process time taken to retrieve an image data. In the pedestrian detection, the images are in a range of different poses, backgrounds, genders and heights. There are several methods that can be used for detection. The detection methods are from tradition methods to modern methods. They are HoG Feature + SVM, DPM, R-CNN, Fast R-CNN, Faster R-CNN, YOLO, SSD and YOLOv2.


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David LaChapelle in Rome
One of the world's most controversial and startling photographers is back in Rome for the first time in over 15 years: David LaChapelle. His greatest works can be seen in the Palazzo delle Esposizioni until September 13th.
David LaChapelle is one the most appreciated and influential photographers in the world. He is famous for his provocative and shocking images, which may be controversial, but always inspire some emotion. You cannot walk away from one of his photos without developing an opinion or attitude.
Rome played a huge role in LaChapelle's creative development. His private visit of the Sistine Chapel in 2006 inspired the photographer to create his series called " The Deluge," which led to a meaningful turning point in his artistic path. After "The Deluge", the American photographer began to produce artwork with new aesthetical and conceptual features.
His early work is filled with religious themes (Jesus in male model form), abundant nudity, artistic icons, and pop culture references that range from Louis Vuitton handbags to Jeff Koons' sculptures. LaChapelle also uses major celebrities in his pieces: the Kardashian family members, Courtney Love, Michael Jackson, Naomi Campbell, etc.
To allow the public to understand the "origins" of LaChapelle's work before "The Deluge," the exhibition also provides a selection of the photos that first brought him publicity. One of the sections of the exhibition which I found most interesting is the projection space, where you can watch short "behind the scene" videos and understand how photo production works.
LaChapelle's work is exhibited in the most important public and private international collections and in many museums, among those: Musée D'Orsay, Paris; the Brooklyn Museum, New York; the Museum of Contemporary Art, Taipei; the Tel Aviv Museum of Art, Tel Aviv; Los Angeles County Museum of Art (LACMA), Los Angeles; The National Portrait Gallery, London; Fotografiska Museet, Stockholm and The National Portrait Gallery in Washington DC. Right now LaChapelle's photos can be seen in Museum of Contemporary Art in Santiago, the National Portrait Gallery in London, the Bayerische Staatsoper Portrait Gallery in Munich, and the Palazzo delle Esposizioni in Rome.
The exhibition is open until September 13th; don't miss the chance to see the greatest works of David LaChapelle in real life!
Visit many other exhibitions while studying in John Cabot University!
Прочитайте этот блог на русском языке!
Anastasiia Komarova
Hometown: Moscow, Russia
« Student Leadership Challenge at John Cabot UniversityFrom Republic to Empire: Key Moments in Rome's Transformation »
© 2019 John Cabot University Blog|ProPhoto theme


By Steve Owens. Allows the user to create 4 different types of address files: Holiday, Birthday, Home and Commercial Address.
Machine language. 8K. By Bob Bishop. From 1978. One the first assembly language assembler for the Apple II line of computers.
By Tom Thorpe and Bob Philp.
Business Utility-1 and -2 are two programs on real estate, banking, securities, leasing, investment analysis, and statistics. Rapid access to 10 frequently occurring business decisions in areas requiring speed and accurate analysis of data. Applesoft, cassette $24.95 each.
By Paul Justin. In Integer BASIC. 24K. (c) 1978.
By Bob Bishop in 1978. For 8K machines. This software allows you to edit files of various types. It also includes different options to renumber lines, add, modify and delete entries of your lists.
A 256-byte software to dump your Apple II memory.
By Bob Bishop. Applesoft II 32K.
By Bob Bishop. SAUCER INVASION is a high resolution graphics game for the Apple II microcomputer. The object of the game is to destroy as many alien flying saucers in the time alloted using the ground to air missles at your command.
By Bob Bishop. SPACE MAZE is a high resolution graphics game for the Apple II microcomputer. The object of the game is to maneuver your space craft through a maze as fast as you can without touching the walls of the maze.
By Bob Bishop. Star war is a high resolution graphics game for the Apple II microcomputer. The object of the game is to destroy the enemy space craft in the time allotted using the amunition at your disposal.
The standard against which other versions have to be compared.


Any amount of experience provides you with at least something to share. Writing is a journey that never ends and along that way comes learning and growing. That's why I cherish every bit of advice I've received, and I hope to, in return, help you find something you can hold onto. That's why I've created something special for all my fans.
When you subscribe below, not only do you get exclusive access to special announcements and other great writing features, you also get my Guide to Editing and Revising for free. With 30 pages full of all my experiences, tricks, and thoughts, you'll find valuable information enclosed that many writers have had to go through.
I've learned from other writers (and my own mistakes) and I hope to help you avoid some of the same issues. Maybe you are even facing some of the same problems today but haven't found the solution. It's my desire to share all that I can with you on your path as a writer, and I hope you'll share what you can with me!


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Roshnee Scheme 2022 for Net Zero Carbon Emissions by 2070 – India's COP-26 Commitment
January 11, 2022 by sohansajit
In the upcoming Union Budget 2022, central government is likely to bring Roshnee Scheme to fulfill India's COP-26 commitments. At COP-26, Prime Minister Narendra Modi had pledged that India will achieve net zero carbon emissions by the year 2070. The progress towards net 0 carbon emissions will be marked by India meeting half of its energy needs from renewable energy by 2030. In this article, we will tell you about complete details of Roshnee Scheme.
Roshnee Scheme 2022 for Net Zero Carbon Emissions by 2070
The arrival of Omicron variant of COVID-19 and subsequent surge in COVID-19 cases starting early January 2022 slashed India's hopes of recovering from the pandemic, as the country – like the rest of the world – continues to be consumed by the efforts to contain the spread of virus. The Union Budget 2022, therefore, is likely to be strong on measures to implement past budget announcements along with carrying on to the commitments India made in Glasgow COP-26 Climate summit.
Union Budget 2022 may announce a new Roshnee Scheme to cut India's carbon emissions by 1 billion tonnes. The Roshnee scheme may also aim to reduce Indian economy's carbon intensity to less than 45 per cent by 2030. India will achieve net zero carbon emissions by the year 2070.
Roshnee Yojana to Fulfill India's COP-26 Commitment
Roshnee Scheme or Roadmap for Sustainable and Holistic Approach through National Energy Efficiency Scheme is likely to run for nine years till 2030. It may initially have a corpus of around Rs. 5,000 crore with a total outlay of around Rs. 10,000 crore till 2030 and is aimed at helping India meet the climate commitments made at the Conference of Parties (COP-26) summit in Glasgow.
Climate Finance Announced at COP-26 Climate Meeting in Glasgow
India expects developed countries to provide climate finance of $1 trillion at the earliest. PM Modi at COP-26 Climate meet at Glasgow, Scotland mentioned "It is necessary that as we track the progress made in climate mitigation, we should also track climate finance."
At COP-26, Prime Minister Narendra Modi had pledged that India will achieve net-zero carbon emissions by 2070. The progress towards net-zero carbon emissions will be marked by India meeting half of its energy needs from renewable energy by 2030. India has also aimed to raise non-fossil fuel power generation capacity to 500GW by the end of 2020s.
According to documents presented by government in the parliament last year in 2021 Union Budget, projects of about 47,000 MW of capacity with an investment of Rs. 2.14 lakh Crore (approximately) have been awarded by Solar Energy Corporation of India Limited. Out of this almost 8,300 MW of capacity has been commissioned and the numerous projects are under various stages of implementation.
Central Government Schemes 2022केंद्र सरकारी योजना हिन्दीPopular Schemes in Central:PM Awas Yojana Gramin (PMAY-G)Narendra Modi Schemes ListSECC-2011 BPL List | नयी Final BPL लिस्ट
Source / Reference Link: https://english.jagran.com/business/budget/budget-2022-govt-likely-to-bring-roshnee-scheme-to-fulfill-indias-cop26-commitments-10037667
Categories Sarkari Yojana Tags Carbon, Commitment, COP26, Emissions, Indias, Net, Roshnee, Scheme Post navigation
[Apply] Kaushalya Karnataka Online Application Form 2022 at www.kaushalkar.com
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