221,089 research outputs found
Streptococcus equi subsp. equi and Streptococcus equi subsp. zooepidemicus
Get PDFThe bacterium Streptococcus equi subsp. equi (S. equi) is the causative agent of the highly contagious upper respiratory disease "strangles" in horses. The ancestor of S. equi, Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) is considered an opportunistic commensal of the equine upper respiratory tract but it is also known to cause disease in several animal species and occasionally in humans. Periodically, S. zooepidemicus alone is isolated from suspected strangles cases. This leads to a clinical dilemma of whether the horse has strangles despite failure to recover S. equi or whether S. zooepidemicus is actually the organism responsible for the clinical disease. The current "gold standard" of bacteriological culture for detection of S. equi may fail in as many as 40% of suspected strangles cases. Results presented in this thesis show that it is possible to increase detection of S. equi up to 90% in acute strangles outbreaks by using a nasopharyngeal lavage in combination with a nasal swab sample and analyzing the samples by real-time PCR directly from the sampling material. Using the same techniques, this thesis also demonstrates that in some strangles-like outbreaks S. zooepidemicus alone is responsible for clinical disease.
Determining genetic relationships between different strains of S. equi and S. zooepidemicus is important in epidemiological investigations of outbreaks in both horses and humans. Sequencing of the SeM protein gene in S. equi was useful in establishing relationships between strains isolated from Swedish strangles outbreaks.
Characterization of human and equine isolates of S. zooepidemicus revealed zoonotic transmission of certain strains of S. zooepidemicus from healthy horses that caused severe disease in humans. A human isolate of S. zooepidemicus was closely related to a S. zooepidemicus strain isolated from a large disease outbreak in horses, suggesting that certain strains of S. zooepidemicus may be disease-causing in both humans and horses.
Characterization of a disease-causing strain of S. zooepidemicus (ST-24) in an outbreak of upper respiratory disease in Icelandic horses suggested that certain strains of S. zooepidemicus may not act solely as opportunistic pathogens, but may be more adapted to infect the upper respiratory tract in horses
Genomic evidence for the evolution of Streptococcus equi : host restriction, increased virulence, and genetic exchange with human pathogens
Get PDFThe continued evolution of bacterial pathogens has major implications for both human and animal disease, but the exchange of genetic material between host-restricted pathogens is rarely considered. Streptococcus equi subspecies equi (S. equi) is a host-restricted pathogen of horses that has evolved from the zoonotic pathogen Streptococcus equi subspecies zooepidemicus (S. zooepidemicus). These pathogens share approximately 80% genome sequence identity with the important human pathogen Streptococcus pyogenes. We sequenced and compared the genomes of S. equi 4047 and S. zooepidemicus H70 and screened S. equi and S. zooepidemicus strains from around the world to uncover evidence of the genetic events that have shaped the evolution of the S. equi genome and led to its emergence as a host-restricted pathogen. Our analysis provides evidence of functional loss due to mutation and deletion, coupled with pathogenic specialization through the acquisition of bacteriophage encoding a phospholipase A(2) toxin, and four superantigens, and an integrative conjugative element carrying a novel iron acquisition system with similarity to the high pathogenicity island of Yersinia pestis. We also highlight that S. equi, S. zooepidemicus, and S. pyogenes share a common phage pool that enhances cross-species pathogen evolution. We conclude that the complex interplay of functional loss, pathogenic specialization, and genetic exchange between S. equi, S. zooepidemicus, and S. pyogenes continues to influence the evolution of these important streptococci.Peer reviewe
The Epidemiology of Streptococcus equi subspecies equi in New Zealand in Relation to Vaccine Efficacy
No full textStrangles is caused by host restricted pathogen Streptococcus equi subspecies equi (S. equi) and is the most frequently diagnosed equine disease. This highly contiguous disease accounts for approximate 30% of recorded incidents of equine disease annually (Harrington, Sutcliffe, & Chanter, 2002). Strangles is characterized by abscessation of the lymph nodes of the head and neck. The severity of this disease varies largely depending on the immune status of the affected animal (Sweeney et al., 2005). Diagnosis of strangles can be complicated by the presence of other beta haemolytic streptococci, especially the closely related S. zooepidemicus. Moreover, the current method used to detect S. equi in the carriage state – via samples from the guttural pouches of apparently healthy horses after recovery from strangles - is not always practical.
Currently there are two vaccines for strangles available in NZ. One is a non-encapsulated and attenuated strain of S. equi, Pinnacle® IN (Ford Dodge, USA), which is administered intranasally. Another is an inactivated bacterin, Equivac® S (Pfizer, NZ), used intramuscularly. However, the efficacy of these two vaccines to the three NZ S. equi strains is not clear. Also the level of induced antibodies with the sera of inoculated animals is unkown.
In this study, an enzyme-linked immuno-sorbent assay (ELISA) and an indirect fluorescent-antibody assay (IFA) were developed to detect specific antibodies in sera from horses either infected with S. equi, or vaccinated with Pinnacle or Equivac S, or unvaccinated. Four peptides synthesized by GenScript (GenScript, USA) were used for the ELISA study. Of these, three peptides targeted the N-terminal variable regions of S. equi strains 99, 100 and the vaccine strain. The other was a PEPK repeats peptide, corresponding to a region present in the N-terminal region of the S. equi protein SzPSe. Both ELISA and IFA results showed that relatively high levels of antibodies were induced following vaccination or infection. Also, the induced antibodies demonstrated cross-reactivity to all three S. equi strains tested. ELISA for the PEPK peptide produced higher ODs than seen with the three SeM peptides, suggesting that there were more antibodies against PEPK peptide within the sera. The IFA results showed a difference in titre between pre-vaccinated horses and post-vaccinated horses; whilst the ELISA results did not. This may be due to serological reactivity between those four peptides and ‘natural antibodies’ induced by exposure to the closely related S. zooepidemicus. Further evidence in support of this result was seen in western blotting, which showed that ‘natural antibodies’ in serum from pre-vaccinated horses reacted serologically with proteins from S. equi. IFA was shown to be the better method for the diagnosis of strangles and for the study of the antibody responses after vaccination or infection.
ELISA and IFA results also showed that the observed antibody levels induced by Pinnacle and Equivac S were similar, indicating the comparable efficacy of these two vaccines. All of the vaccinated horses had high levels of pre-existing antibodies within their sera, even one year after vaccination. This may suggest that an increase in the interval of booster time could be acceptable. However, it is acknowledged that antibody titres do not necessarily equal protection from infection. Western blotting results indicated that a stronger immunity was formed after natural infection than seen in horses after. This is because after infection the entire immune system (both humoral and cell-mediated) will be stimulated
Potentially virulence-related extracellular proteins of Streptococcus equi
Get PDFEquine strangles, a disease of the upper respiratory tract caused by the bacterium Streptococcus equi subspecies equi, is one of the most commonly diagnosed and serious diseases in horses. However, the molecular basis of S. equi subsp. equi infection is poorly understood and there are no safe and effective vaccines on the market. The main objective of this study was to identify and characterise extracellular proteins used by S. equi subsp. equi to initiate infection and cause disease. Extracellular proteins, which can be secreted or cell surface-located, play an important role in the initiation of infection and in continued bacterial survival inside the host. Adhesins are a specific class of virulence-related proteins that are used by the bacteria to attach to host tissues. This study focused on a number of cell-surface anchored proteins that specifically adhere to collagen and fibronectin, two major extracellular matrix proteins of vertebrates. The binding characteristics of the fibronectin-binding protein FNEB were compared to two previously studied fibronectin-binding proteins, FNE and SFS. A follow-up investigation showed that FNE and FNEB are part of a family of six similar proteins encoded by S. equi subsp. equi. One of the four novel proteins identified (FNEE) was shown to bind to fibronectin and all four to collagen. In another study, a secreted immunoglobulin-specific protease was characterised. This enzyme could be used by the bacteria to interfere with the immune response of the infected horse. A useful tool in this research was the public S. equi subsp. equi genome database, which can be used to identify homologues to virulence-related proteins of other pathogens. An applied objective of the present study was to identify potential components for a future vaccine against strangles. One of the collagen-binding proteins characterised (CNE) is currently being used in vaccination trials as a component of a protein subunit vaccine for horses
Investigation of the molecular basis of virulence of Streptococcus equi
No full textEquine strangles is a purulent lymphadenitis of the head and neck and is caused by Streptococcus equi ssp equi remaining a worldwide, endemic infection that represents around 30% of all annually recorded incidents of equine disease. Despite much effort, current vaccination strategies have proved largely ineffective towards S. equi, ssp equi, with the current focus based on ‘reverse vaccinology’, using genome sequence data of S. equi ssp equi to identify surface exposed and secreted proteins.
Streptococcus equi shares much of its genome with the genetically closely related, S. equi ssp zooepidemicus. Despite this close genetic association, S. equi ssp zooepidemicus exhibits very distinct pathogenicity variations. S. equi ssp zooepidemicus can infect a wide
variety of vertebrate hosts showing a high degree of antigenic variability and a large amount of strain variability whereas S. equi is largely equine specific and processes a limited number of strain types. Understanding the molecular basis of virulence of these contrasting organisms therefore remains a key requirement if a suitable vaccination.
Identification of potential virulence factors has been greatly aided by the availability
of the full genome sequence of S. equi ssp equi (4047) and S. equi ssp zooepidemicus (H70).
In this study, we identified and investigated a range of putative virulence factors including the covalently attached surface protein SEQ2190, the secreted phospholipase
enzymes SlaA (SEQ0849) and SlaB (SEQ2155) and finally the lipoprotein, acid phosphatase SeLppC (SEQ0346) and its orthologue SzLppC (SZO16870). In all cases, using molecular
biology techniques the coding region representative for each of these proteins was cloning into expression constructs, expressed and purified and further investigations carried out.
Although efforts to obtain a 3-dimensional structure of SEQ2190 were unsuccessful, bioinformatic investigations have identified SEQ2190 as a unique protein in S. equi ssp equi
with a putative structure suggesting a role in bacteria-host interaction.
SlaA was demonstrated to be an active sPLA2 enzyme, active against a 1, 2-dithio analog of diheptanoyl phosphatidylcholine but not 2-deoxy-2-thio- Arachidonoyl
phosphatidylcholine. It was also demonstrated to have a specific requirement for the divalent ion, Ca2+ for activity, an optimum temperature higher than expected (40°C), a Km of 14.40 ± 7.866 mM and a specific activity of 5.06x10-2 ± 3.01x10-3 μmol/min/mg. SlaA was also shown to react to equine post-infection convalescent serum.
Although some characterisation had been previously undertaken regarding whole cell SeLppC extracts, in this study we produced expression constructs of SeLppC and its
orthologous pseudogene (SzLppC) found in the genome strain of S. equi ssp zooepidemicus. We used purified SeLppC and SzLppC to demonstrate activity against pNPP and two
biologically significant substrates (5’AMP and 5’UMP) and recorded the Km values for each. We also identified SeLppC to not possess a specific activity for the divalent ion Cu2+, a lower than expected pH optimum and a higher than expected temperature range. Furthermore we also demonstrated that SzLppC is not a pseudogene, indicating an error within the S. equi spp zooepidemicus genome strain
Genome specialization and decay of the strangles pathogen, Streptococcus equi, is driven by persistent infection
No full textThe authors would like to thank the Horserace Betting Levy Board for funding the analysis of the eqbE mutant (ref: Vet/prj/758). The Horse Trust funded the collection of isolates from UK outbreaks of strangles (G1606). We thank the core sequencing and informatics teams at the Sanger Institute for their assistance and The Wellcome Trust for its support of the Sanger Institute Pathogen Genomics and Biology groups. SRH, JP and MTGH were supported by Wellcome Trust grant 098051.Strangles, the most frequently diagnosed infectious disease of horses worldwide, is caused by Streptococcus equi. Despite its prevalence, the global diversity and mechanisms underlying the evolution of S. equi as a host-restricted pathogen remain poorly understood. Here we define the global population structure of this important pathogen and reveal a population replacement in the late 19th or early 20th century. Our data reveal a dynamic genome that continues to mutate and decay, but also to amplify and acquire genes despite the organism having lost its natural competence and become host-restricted. The lifestyle of S. equi within the horse is defined by short-term acute disease, strangles, followed by long-term carriage. Population analysis reveals evidence of convergent evolution in isolates from post-acute disease samples, as a result of niche adaptation to persistent carriage within a host. Mutations that lead to metabolic streamlining and the loss of virulence determinants are more frequently found in carriage isolates, suggesting that the pathogenic potential of S. equi reduces as a consequence of long term residency within the horse post-acute disease. An example of this is the deletion of the equibactin siderophore locus that is associated with iron acquisition, which occurs exclusively in carrier isolates, and renders S. equi significantly less able to cause acute disease in the natural host. We identify several loci that may similarly be required for the full virulence of S. equi, directing future research towards the development of new vaccines against this host-restricted pathogen.Peer reviewe
Characterisation of the PrtM maturase of Streptococcus equi; a proven virulence factor in strangles
No full textStreptococcus equi subspecies equi (S. equi) is the pathogen responsible for the prevalent and highly contagious equine disease called strangles. Strangles has been reported worldwide as a cause of a high level of animal suffering and economic loss. S. equi is susceptible to many antibiotics in vitro, but relapse due to insufficient vascularity often renders such treatment ineffective. Getting effective and universally accepted vaccines against S. equi have been slow mainly because of safety concerns. It was previously reported that colonization of air interface organ cultures, after inoculation with a mutant strain (ΔprtM138-213) deficient in the putative maturase lipoprotein (PrtM, a homologue of the pneumococcal PpmA) was less than that seen in cultures which were infected with wild-type S. equi strain 4047 (Hamilton et al, 2006), indicating that PrtM is a major virulence factor in strangles. It has also been
demonstrated by in vitro and in vivo studies that many streptococcal adhesins, for example, serve as colonization or virulence factors and this makes them attractive targets for therapeutic and preventive strategies against streptococcal infections (Nobbs et al., 2009). S. equi adhesins or other colonization factors may be substrates for PrtM.
Understanding PrtM is key to designing drugs or vaccines against the equine S. equi infection and strangles. In this research, advanced biomolecular techniques were systematically applied to investigate and characterise PrtM, and to evaluate its potential as a therapeutic or vaccine target. Bioinformatics, microbiological, biochemical and molecular biology techniques were used in screening the S. equi WT 4047 and Mutant (prtM138-213) strains to evaluate the immunogenicity and conservation of PrtM. Proteomics techniques: two-dimensional gel electrophoresis and mass spectrometry were employed in evaluating the cell-associated and secreted protein extracts of both the S. equi WT 4047 and mutant (prtM138-213) strains. In this study, genetic engineering technology involving targeted domain knock-out and/or knock-in, was employed in producing the central domain recombinant protein and mutant (prtM138-213) revertants. Following cloning, over-expression and purification of the full length and central domain, biochemical data on the PrtM protein (kcat/KM for S. equi 4047 PrtM full length recombinant protein = 5.84 x 106 /M/s) were derived via enzyme(peptydylprolyl isomerase - PPIase) assay; and crystallography was applied in an attempt to derive structural data on the PrtM protein. Advanced biomolecular techniques (including Western blots and Proteomics) were employed in screening the complemented mutants.
It has been proven from this research that the PrtM of S. equi 4047 is involved in adaptation to NaCl stress and in regulating sensitivity to antibiotics; PrtM may have roles in speeding up the synthesis of hyaluronic acid and in the folding or remodeling of HPr Kinase. The parvulin-type structure of PrtM elucidated by bioinformatics analysis, the cross reactions of the WT and mutant with a number of antisera, the observation that PrtM may be a multisubstrate foldase due to the detectable and significant differences in the proteomes of the WT, mutant and complemented mutants, the dimeric protein formed by the full length recombinant protein of S. equi 4047 WT, and the PPIase-Chaperonine activities of PrtM, all observed from this study, validate PrtM of S. equi 4047 as a viable and novel therapeutic target which pharmaceutical industries should extensively evaluate for the prevention and treatment of S. equi infection and strangles
Equitable access to quality injury care; Equi-Injury project protocol for prioritizing interventions in four low- or middle-income countries : a mixed method study
Get PDFFunding This research was funded by the NIHR (award number 133135) using UK aid from the UK Government to support global health research. The views expressed in this publication are those of the authors and not necessarily those of the NIHR or the UK governmentPeer reviewe
Structural characterisation of the virulence-associated protein VapG from the horse pathogen Rhodococcus equi
No full textVirulence and host range in Rhodococcus equi depends on the variable pathogenicity island of their virulence plasmids. Notable gene products are a family of small secreted virulence-associated proteins (Vaps) that are critical to intramacrophagic proliferation. Equine-adapted strains, which cause severe pyogranulomatous pneumonia in foals, produce a cell-associated VapA that is necessary for virulence, alongside five other secreted homologues. In the absence of biochemical insight, attention has turned to the structures of these proteins to develop a functional hypothesis. Recent studies have described crystal structures for VapD and a truncate of the VapA orthologue of porcine-adapted strains, VapB. Here, we crystallised the full-length VapG and determined its structure by molecular replacement. Electron density corresponding to the N-terminal domain was not visible suggesting that it is disordered. The protein core adopted a compact elliptical, anti-parallel β-barrel fold with β1-β2-β3-β8-β5-β6-β7-β4 topology decorated by a single peripheral α-helix unique to this family. The high glycine content of the protein allows close packing of secondary structural elements. Topologically, the surface has no indentations that indicate a nexus for molecular interactions. The distribution of polar and apolar groups on the surface of VapG is markedly uneven. One-third of the surface is dominated by exposed apolar side-chains, with no ionisable and only four polar side-chains exposed, giving rise to an expansive flat hydrophobic surface. Other surface regions are more polar, especially on or near the α-helix and a belt around the centre of the β-barrel. Possible functional significance of these recent structures is discussed
The Identification, Isolation and Partial Characterisation of VapA-encoding virulence plasmids in Rhodococcus equi found in New Zealand.
No full textThis thesis describes the identification, isolation and partial characterisation of VapA-encoded virulence plasmids present in Rhodococcus equi (R. equi) found in foals in New Zealand. R. equi infection associated with the presence of virulence plasmids causes severe pyogranulomatous pneumonia in foals and, if left untreated, it can be lethal. Foals are thought to be infected when they ingest or breathe in bacteria present in soil, dust and faecal particles after which the bacteria multiply inside macrophages and cause pneumonia. Studies have shown that virulence is associated with the presence of the vapA gene in a plasmid that encodes a VapA protein. Twelve different types of VapA-encoding virulence plasmids have been described so far. Epidemiological studies of America, Australia, Korea, Japan and some parts of Europe have revealed that more than one subtype can exist in a farm or even a country. The aim of the study was to investigate if more than one subtype of VapA-encoded plasmids exists in a farm in New Zealand.
Nasal swabs for the analysis were obtained from the Auckland Veterinary Centre in Papakura. Based on colony morphology and 16S primers, successful identification of R. equi was possible, VapA primers assisted with distinguishing bacteria that carried VapA-encoded plasmids. Since success with plasmid isolations was modest, characterization of plasmids was conducted by sequence analysis of an approximately 1-kb variable region present in VapA-encoded plasmids. Sequence analysis and restriction digestion patterns revealed two distinct patterns of sequences from the variable region which were divided into two groups: PI and PII.
Results indicated that PI sequences were significantly similar to published 85-kb type I plasmids and must also be from 85-kb type I plasmids but more detailed analysis of other regions is required to confirm the results. PII sequences on the other hand were distinctly different from PI sequences and displayed poor matches with published plasmid sequences. This is the first attempt at characterising VapA-encoded virulence plasmids from R. equi isolates in New Zealand. This work will contribute towards increasing our knowledge regarding the unique characteristics of the VapA-encoding plasmids, which could define transmission characteristics, possible sources of infection in disease outbreaks and vaccine development
- …
