Publications
230 results found
Daniels-Treffandier H, de Nie K, Marsay L, et al., 2016, Impact of Reducing Complement Inhibitor Binding on the Immunogenicity of Native Neisseria meningitidis Outer Membrane Vesicles., PLOS One, Vol: 11, ISSN: 1932-6203
Neisseria meningitidis recruits host human complement inhibitors to its surface to down-regulate complement activation and enhance survival in blood. We have investigated whether such complement inhibitor binding occurs after vaccination with native outer membrane vesicles (nOMVs), and limits immunogenicity of such vaccines. To this end, nOMVs reactogenic lipopolysaccharide was detoxified by deletion of the lpxl1 gene (nOMVlpxl1). nOMVs unable to bind human complement factor H (hfH) were generated by additional deletions of the genes encoding factor H binding protein (fHbp) and neisserial surface protein A (NspA) (nOMVdis). Antibody responses elicited in mice with nOMVdis were compared to those elicited with nOMVlpxl1 in the presence of hfH. Results demonstrate that the administration of human fH to mice immunized with fHbp containing OMVlpxl1 decreased immunogenicity against fHbp (but not against the OMV as a whole). The majority of the OMV-induced bactericidal immune response (OMVlpxl1 or OMVdis) was versus PorA. Despite a considerable reduction of hfH binding to nOMVdis, and the absence of the vaccine antigen fHbp, immunogenicity in mice was not different from nOMVlpxl1, in the absence or presence of hfH (serum bactericidal titers of 1:64 vs 1:128 after one dose in the nOMVdis and nOMVlpxl1-immunized groups respectively). Therefore, partial inhibition of fH binding did not enhance immunity in this model.
Bosse JT, Chaudhiri RR, Li Y, et al., 2016, The complete genome sequence of MIDG2331, a genetically tractable serovar 8 clinical isolate of Actinobacillus pleuropneumoniae, Genome Announcements, Vol: 4, ISSN: 2169-8287
We report here the complete annotated genome sequence of a clinical serovar 8 isolate Actinobacillus pleuropneumoniae MIDG2331. Unlike the serovar 8 reference strain 405, MIDG2331 is amenable to genetic manipulation via natural transformation as well as conjugation, making it ideal for studies of gene function.
Qin W, Wang L, Zhai R, et al., 2016, Trimeric autotransporter adhesins contribute to Actinobacillus pleuropneumoniae pathogenicity in mice and regulate bacterial gene expression during interactions between bacteria and porcine primary alveolar macrophages, Antonie Van Leeuwenhoek International Journal of General and Molecular Microbiology, Vol: 109, Pages: 51-70, ISSN: 1572-9699
Actinobacillus pleuropneumoniae is an important pathogen that causes respiratory disease in pigs. Trimeric autotransporter adhesin (TAA) is a recently discovered bacterial virulence factor that mediates bacterial adhesion and colonization. Two TAA coding genes have been found in the genome of A. pleuropneumoniae strain 5b L20, but whether they contribute to bacterial pathogenicity is unclear. In this study, we used homologous recombination to construct a double-gene deletion mutant, ΔTAA, in which both TAA coding genes were deleted and used it in in vivo and in vitro studies to confirm that TAAs participate in bacterial auto-aggregation, biofilm formation, cell adhesion and virulence in mice. A microarray analysis was used to determine whether TAAs can regulate other A. pleuropneumoniae genes during interactions with porcine primary alveolar macrophages. The results showed that deletion of both TAA coding genes up-regulated 36 genes, including ene1514, hofB and tbpB2, and simultaneously down-regulated 36 genes, including lgt, murF and ftsY. These data illustrate that TAAs help to maintain full bacterial virulence both directly, through their bioactivity, and indirectly by regulating the bacterial type II and IV secretion systems and regulating the synthesis or secretion of virulence factors. This study not only enhances our understanding of the role of TAAs but also has significance for those studying A. pleuropneumoniae pathogenesis.
Hathroubi S, Hancock MA, Bossé JT, et al., 2015, Surface polysaccharide mutants reveal that absence of O antigen reduces biofilm formation of Actinobacillus pleuropneumoniae., Infection and Immunity, Vol: 84, Pages: 127-137, ISSN: 1098-5522
Actinobacillus pleuropneumoniae is a Gram-negative bacterium belonging to the Pasteurellaceae family and the causative agent of porcine pleuropneumonia, a highly contagious lung disease causing important economic losses. Surface polysaccharides including lipopolysaccharides (LPS) and capsular polysaccharides (CPS) are implicated in the adhesion and virulence of A. pleuropneumoniae, but their role in biofilm formation is still unclear. In this study, we investigated the requirement of these surface polysaccharides in biofilm formation by A. pleuropneumoniae serotype 1. Well characterized mutants were used: an O-antigen LPS mutant, a truncated core-LPS mutant with an intact O-antigen, a capsule mutant and a poly-N-acetylglucosamine (PGA) mutant. We compared the amount of biofilm produced by the parental strain and the isogenic mutants using static and dynamic systems. Compared to the parental or other strains, the biofilm of the O-antigen and the PGA mutants was dramatically reduced and it had less cell-associated PGA. Real-time PCR analyses revealed a significant reduction in the level of pgaA, cpxR and cpxA mRNA in the biofilm cells of the O-antigen mutant compared to the parental strain. Specific binding between PGA and LPS was consistently detected by surface plasmon resonance but the lack of O-antigen did not abolish these interactions. In conclusion, the absence of the O-antigen reduces the ability of A. pleuropneumoniae to form a biofilm and this is associated with a reduction in the expression and the production of PGA.
Baig A, Weinert LA, Peters SE, et al., 2015, Whole genome investigation of a divergent clade of the pathogen Streptococcus suis, Frontiers in Microbiology, Vol: 6, ISSN: 1664-302X
Li G, Xie F, Zhang Y, et al., 2015, Role of (p)ppGpp in Viability and Biofilm Formation of Actinobacillus pleuropneumoniae S8., PLOS One, Vol: 10, ISSN: 1932-6203
Actinobacillus pleuropneumoniae is a Gram-negative bacterium and the cause of porcine pleuropneumonia. When the bacterium encounters nutritional starvation, the relA-dependent (p)ppGpp-mediated stringent response is activated. The modified nucleotides guanosine 5'-diphosphate 3'-diphosphate (ppGpp) and guanosine 5'-triphosphate 3'-diphosphate (pppGpp) are known to be signaling molecules in other prokaryotes. Here, to investigate the role of (p)ppGpp in A. pleuropneumoniae, we created a mutant A. pleuropneumoniae strain, S8ΔrelA, which lacks the (p)ppGpp-synthesizing enzyme RelA, and investigated its phenotype in vitro. S8ΔrelA did not survive after stationary phase (starvation condition) and grew exclusively as non-extended cells. Compared to the wild-type (WT) strain, the S8ΔrelA mutant had an increased ability to form a biofilm. Transcriptional profiles of early stationary phase cultures revealed that a total of 405 bacterial genes were differentially expressed (including 380 up-regulated and 25 down-regulated genes) in S8ΔrelA as compared with the WT strain. Most of the up-regulated genes are involved in ribosomal structure and biogenesis, amino acid transport and metabolism, translation cell wall/membrane/envelope biogenesis. The data indicate that (p)ppGpp coordinates the growth, viability, morphology, biofilm formation and metabolic ability of A. pleuropneumoniae in starvation conditions. Furthermore, S8ΔrelA could not use certain sugars nor produce urease which has been associated with the virulence of A. pleuropneumoniae, suggesting that (p)ppGpp may directly or indirectly affect the pathogenesis of A. pleuropneumoniae during the infection process. In summary, (p)ppGpp signaling represents an essential component of the regulatory network governing stress adaptation and virulence in A. pleuropneumoniae.
Pratt AJ, DiDonato M, Shin DS, et al., 2015, Structural, functional and immunogenic insights on Cu,Zn Superoxide Dismutase pathogenic virulence factors from Neisseria meningitidis and Brucella abortus, Journal of Bacteriology, Vol: 197, Pages: 3834-3847, ISSN: 1098-5530
Bacterial pathogens Neisseria meningitidis and Brucella abortus pose threats to human and animal health worldwide, causing meningococcal disease and brucellosis, respectively. Mortality from acute N. meningitidis infections remains high despite antibiotics, and brucellosis presents alimentary and health consequences. Superoxide dismutases are master regulators of reactive oxygen, general pathogenicity factors and therefore therapeutic targets. Cu,Zn superoxide dismutases (SODs) localized to the periplasm promote survival by detoxifying superoxide radicals generated by major host antimicrobial immune responses. We discovered that passive immunization with an antibody directed at N. meningitidis SOD (NmSOD) was protective in a mouse infection model. To define the relevant atomic details and solution assembly states of this important virulence factor, we report high-resolution and X-ray scattering analyses of NmSOD and SOD from B. abortus (BaSOD). The NmSOD structures revealed an auxiliary tetrahedral Cu-binding site bridging the dimer interface; mutational analyses suggested that this metal site contributes to protein stability, with implications for bacterial defense mechanisms. Biochemical and structural analyses informed us about electrostatic substrate guidance, dimer assembly and an exposed C-terminal epitope in the NmSOD dimer. In contrast, the monomeric BaSOD structure provided insights for extending immunogenic peptide epitopes derived from the protein. These collective results reveal unique contributions of SOD to pathogenic virulence, refine predictive motifs for distinguishing SOD classes and suggest general targets for anti-bacterial immune responses. The identified functional contributions, motifs, and targets distinguishing bacterial and eukaryotic SOD assemblies presented here provide a foundation for efforts to develop SOD-specific inhibitors or vaccines against these harmful pathogens. IMPORTANCE: By protecting microbes against reactive oxygen insults
Howell KJ, Peters SE, Wang J, et al., 2015, Development of a Multiplex PCR Assay for Rapid Molecular Serotyping of Haemophilus parasuis., Journal of Clinical Microbiology, Vol: 53, Pages: 3812-3821, ISSN: 1098-660X
Haemophilus parasuis causes Glässer's disease and pneumonia in pigs. Indirect hemagglutination (IHA) is typically used to serotype this bacterium, distinguishing 15 serovars with some nontypeable isolates. The capsule loci of the 15 reference strains have been annotated, and significant genetic variation was identified between serovars, with the exception of serovars 5 and 12. A capsule locus and in silico serovar were identified for all but two nontypeable isolates in our collection of >200 isolates. Here, we describe the development of a multiplex PCR, based on variation within the capsule loci of the 15 serovars of H. parasuis, for rapid molecular serotyping. The multiplex PCR (mPCR) distinguished between all previously described serovars except 5 and 12, which were detected by the same pair of primers. The detection limit of the mPCR was 4.29 × 10(5) ng/μl bacterial genomic DNA, and high specificity was indicated by the absence of reactivity against closely related commensal Pasteurellaceae and other bacterial pathogens of pigs. A subset of 150 isolates from a previously sequenced H. parasuis collection was used to validate the mPCR with 100% accuracy compared to the in silico results. In addition, the two in silico-nontypeable isolates were typeable using the mPCR. A further 84 isolates were analyzed by mPCR and compared to the IHA serotyping results with 90% concordance (excluding those that were nontypeable by IHA). The mPCR was faster, more sensitive, and more specific than IHA, enabling the differentiation of 14 of the 15 serovars of H. parasuis.
Kroll JS, Pratt AJ, DiDonato M, et al., 2015, Structural, Functional, and Immunogenic Insights on Cu,ZnSuperoxide Dismutase Pathogenic Virulence Factors from Neisseriameningitidis and Brucella abortus., Journal of Bacteriology, ISSN: 1098-5530
Siggins MK, Gill SK, Langford PR, et al., 2015, PHiD-CV induces anti-Protein D antibodies but does not augment pulmonary clearance of nontypeable Haemophilus influenzae in mice, Vaccine, Vol: 33, Pages: 4954-4961, ISSN: 1873-2518
BackgroundA recently-licensed 10-valent pneumococcal conjugate vaccine (PHiD-CV; Synflorix, GSK) uses Protein D from Haemophilus influenzae as a carrier protein. PHiD-CV therefore has the potential to provide additional protection against nontypeable H. influenzae (NTHi). NTHi frequently causes respiratory tract infections and is associated with significant morbidity and mortality worldwide and there is currently no vaccine.MethodsWe developed mouse models of NTHi infection and influenza/NTHi superinfection. Mice were immunized with PHiD-CV, heat-killed NTHi, or a 13-valent pneumococcal conjugate vaccine that did not contain Protein D (PCV13; Prevenar, Pfizer) and then infected intranasally with NTHi.ResultsInfection with NTHi resulted in weight loss, inflammation and airway neutrophilia. In a superinfection model, prior infection with pandemic H1N1 influenza virus (strain A/England/195/2009) augmented NTHi infection severity, even with a lower bacterial challenge dose. Immunization with PHiD-CV produced high levels of antibodies that were specific against Protein D, but not heat-killed NTHi. Immunization with PHiD-CV led to a slight reduction in bacterial load, but no change in disease outcome.ConclusionsPHiD-CV induced high levels of Protein D-specific antibodies, but did not augment pulmonary clearance of NTHi. We found no evidence to suggest that PHiD-CV will offer added benefit by preventing NTHi lung infection.
Shaw AG, Sim K, Randell P, et al., 2015, Late-onset bloodstream infection and perturbed maturation of the gastrointestinal microbiota in premature infants, PLOS One, Vol: 10, ISSN: 1932-6203
Chen Y, Liu T, Langford P, et al., 2015, <i>Haemophilus parasuis</i> induces activation of NF-κB and MAP kinase signaling pathways mediated by toll-like receptors, MOLECULAR IMMUNOLOGY, Vol: 65, Pages: 360-366, ISSN: 0161-5890
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- Citations: 31
Bosse JT, Li Y, Atherton TG, et al., 2015, Characterisation of a mobilisable plasmid conferring florfenicol and chloramphenicol resistance in Actinobacillus pleuropneumoniae, Veterinary Microbiology, ISSN: 1873-2542
Wang L, Qin W, Yang S, et al., 2015, The Adh adhesin domain is required for trimeric autotransporter Apa1-mediated <i>Actinobacillus pleuropneumoniae</i> adhesion, autoaggregation, biofilm formation and pathogenicity, VETERINARY MICROBIOLOGY, Vol: 177, Pages: 175-183, ISSN: 0378-1135
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- Citations: 20
Wang L, Qin W, Yang S, et al., 2015, The Adh adhesin domain is required for trimeric autotransporter Apa1-mediated Actinobacillus pleuropneumoniae adhesion, autoaggregation, biofilm formation and pathogenicity, Veterinary Microbiology, Vol: 177, Pages: 175-183, ISSN: 0378-1135
Actinobacillus pleuropneumoniae is a causative agent of porcine pleuropneumonia, which is a highly contagious endemic disease of pigs. Adhesion is a critical first step in the infection process. Trimeric autotransporter adhesions (TAAs) have been identified as novel virulence factors; however, little is known on their roles in A. pleuropneumoniae pathogenicity. Here, our data show that YadA-like head region (Adh) of Apa1 was the optimal adhesion functional domain via segment expression and adhesion assays in vitro. Additionally, Adh induced partial protection against A. pleuropneumoniae 5b L20 and serotypes 1, 3, and 5a in mice. The deletion of Adh gene significantly decreased autoaggregation, biofilm formation and adherence to host cells in vitro. Furthermore, with delaying of clinical symptoms, reducing production of pro-inflammatory cytokines and lessening the lung injury after infection, Adh deletion strain (5b. ϕAdh) significantly reduced the pathogenicity to piglets. To elucidate the mechanism of lung injury, the differentially expressed genes in the lung tissues of piglets infected with the 5b L20 or 5b. ϕAdh strains were investigated using microarray analysis and validated by qRT-PCR. Compared with the 5b L20 infected piglets, 495 genes were differentially expressed in 5b. ϕAdh infected lung tissue (221 upregulated and 274 downregulated). Especially, the antigen processing and presentation gene IFI30 was increased following infection with the 5b. ϕAdh strain. Thus, Adh may enhance pathogenicity by depressing host immune recognition. We conclude that the head domain of the A. pleuropneumoniae trimeric autotransporter Apa1 regulates autoagglutination, biofilm formation, adhesion to host cells and pathogenicity.
Weinert LA, Chaudhuri RR, Wang J, et al., 2015, Erratum: Genomic signatures of human and animal disease in the zoonotic pathogen Streptococcus suis, Nature Communications, Vol: 6, ISSN: 2041-1723
Bosse J, Li Y, Walker S, et al., 2015, Identification of dfrA14 in two distinct plasmids conferring trimethoprimresistance in Actinobacillus pleuropneumoniae, Journal of Antimicrobial Chemotherapy, Vol: 70, Pages: 2217-2222, ISSN: 1460-2091
Objectives The objective of this study was to determine the distribution and genetic basis of trimethoprim resistance in Actinobacillus pleuropneumoniae isolates from pigs in England.Methods Clinical isolates collected between 1998 and 2011 were tested for resistance to trimethoprim and sulphonamide. The genetic basis of trimethoprim resistance was determined by shotgun WGS analysis and the subsequent isolation and sequencing of plasmids.Results A total of 16 (out of 106) A. pleuropneumoniae isolates were resistant to both trimethoprim (MIC >32 mg/L) and sulfisoxazole (MIC ≥256 mg/L), and a further 32 were resistant only to sulfisoxazole (MIC ≥256 mg/L). Genome sequence data for the trimethoprim-resistant isolates revealed the presence of the dfrA14 dihydrofolate reductase gene. The distribution of plasmid sequences in multiple contigs suggested the presence of two distinct dfrA14-containing plasmids in different isolates, which was confirmed by plasmid isolation and sequencing. Both plasmids encoded mobilization genes, the sulphonamide resistance gene sul2, as well as dfrA14 inserted into strA, a streptomycin-resistance-associated gene, although the gene order differed between the two plasmids. One of the plasmids further encoded the strB streptomycin-resistance-associated gene.Conclusions This is the first description of mobilizable plasmids conferring trimethoprim resistance in A. pleuropneumoniae and, to our knowledge, the first report of dfrA14 in any member of the Pasteurellaceae. The identification of dfrA14 conferring trimethoprim resistance in A. pleuropneumoniae isolates will facilitate PCR screens for resistance to this important antimicrobial.
Menikou S, Sancho-Shimizu V, Levin Z, et al., 2015, Identification of the agent causing Kawasaki Disease via throat swab cultures, 11th International Kawasaki Disease Symposium (IKDS), Publisher: LIPPINCOTT WILLIAMS & WILKINS, ISSN: 0009-7322
Menikou S, Sancho-Shimizu V, Levin Z, et al., 2015, Identification of the agent causing Kawasaki Disease via throat swab cultures, 11th International Kawasaki Disease Symposium (IKDS), Publisher: LIPPINCOTT WILLIAMS & WILKINS, ISSN: 0009-7322
Weinert LA, Chaudhuri RR, Wang J, et al., 2015, Genomic signatures of human and animal disease in the zoonotic pathogen Streptococcus suis, Nature Communications, Vol: 6, ISSN: 2041-1723
Sim K, Shaw AG, Randell P, et al., 2015, Dysbiosis anticipating necrotizing enterocolitis in very premature infants, Clinical Infectious Diseases, Vol: 60, Pages: 389-397, ISSN: 1537-6591
Background. Necrotizing enterocolitis (NEC) is a devastating inflammatory bowel disease of premature infants speculatively associated with infection. Suspected NEC can be indistinguishable from sepsis, and in established cases an infant may die within hours of diagnosis. Present treatment is supportive. A means of presymptomatic diagnosis is urgently needed. We aimed to identify microbial signatures in the gastrointestinal microbiota preceding NEC diagnosis in premature infants.Methods. Fecal samples and clinical data were collected from a 2-year cohort of 369 premature neonates. Next-generation sequencing of 16S ribosomal RNA gene regions was used to characterize the microbiota of prediagnosis fecal samples from 12 neonates with NEC, 8 with suspected NEC, and 44 controls. Logistic regression was used to determine clinical characteristics and operational taxonomic units (OTUs) discriminating cases from controls. Samples were cultured and isolates identified using matrix-assisted laser desorption/ionization–time of flight. Clostridial isolates were typed and toxin genes detected.Results. A clostridial OTU was overabundant in prediagnosis samples from infants with established NEC (P = .006). Culture confirmed the presence of Clostridium perfringens type A. Fluorescent amplified fragment-length polymorphism typing established that no isolates were identical. Prediagnosis samples from NEC infants not carrying profuse C. perfringens revealed an overabundance of a Klebsiella OTU (P = .049). Prolonged continuous positive airway pressure (CPAP) therapy with supplemental oxygen was also associated with increased NEC risk.Conclusions. Two fecal microbiota signatures (Clostridium and Klebsiella OTUs) and need for prolonged CPAP oxygen signal increased risk of NEC in presymptomatic infants. These biomarkers will assist development of a screening tool to allow very early diagnosis of NEC.Clinical Trials Registration. NCT01102738.
Pereira MF, Rossi CC, de Queiroz MV, et al., 2015, Galleria mellonella is an effective model to study Actinobacillus pleuropneumoniae infection, Microbiology, Vol: 161, Pages: 387-400, ISSN: 1350-0872
Actinobacillus pleuropneumoniae is responsible for swine pleuropneumonia, a respiratory disease that causes significant global economic loss. Its virulence depends on many factors, such as capsular polysaccharides, RTX toxins and iron-acquisition systems. Analysis of virulence may require easy-to-use models that approximate mammalian infection and avoid ethical issues. Here, we investigate the potential use of the wax moth Galleria mellonella as an informative model for A. pleuropneumoniae infection. Genotypically distinct A. pleuropneumoniae clinical isolates were able to kill larvae at 37 °C but had different LD50 values, ranging from 104 to 107 c.f.u. per larva. The most virulent isolate (1022) was able to persist and replicate within the insect, while the least virulent (780) was rapidly cleared. We observed a decrease in haemocyte concentration, aggregation and DNA damage post-infection with isolate 1022. Melanization points around bacterial cells were observed in the fat body and pericardial tissues of infected G. mellonella, indicating vigorous cell and humoral immune responses close to the larval dorsal vessel. As found in pigs, an A. pleuropneumoniae hfq mutant was significantly attenuated for infection in the G. mellonella model. Additionally, the model could be used to assess the effectiveness of several antimicrobial agents against A. pleuropneumoniae in vivo. G. mellonella is a suitable inexpensive alternative infection model that can be used to study the virulence of A. pleuropneumoniae, as well as assess the effectiveness of antimicrobial agents against this pathogen.
Wang L, Qin W, Zhai R, et al., 2015, Differential gene expression profiling of <i>Actinobacillus pleuropneumoniae</i> during induction of primary alveolar macrophage apoptosis in piglets, MICROBIAL PATHOGENESIS, Vol: 78, Pages: 74-86, ISSN: 0882-4010
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- Citations: 16
Howell KJ, Weinert LA, Chaudhuri RR, et al., 2014, The use of genome wide association methods to investigate pathogenicity, population structure and serovar in Haemophilus parasuis., BMC Genomics, Vol: 15, Pages: 1179-1179, ISSN: 1471-2164
BACKGROUND: Haemophilus parasuis is the etiologic agent of Glasser's disease in pigs and causes devastating losses to the farming industry. Whilst some hyper-virulent isolates have been described, the relationship between genetics and disease outcome has been only partially established. In particular, there is weak correlation between serovar and disease phenotype. We sequenced the genomes of 212 isolates of H. parasuis and have used this to describe the pan-genome and to correlate this with clinical and carrier status, as well as with serotype. RESULTS: Recombination and population structure analyses identified five groups with very high rates of recombination, separated into two clades of H. parasuis with no signs of recombination between them. We used genome-wide association methods including discriminant analysis of principal components (DAPC) and generalised linear modelling (glm) to look for genetic determinants of this population partition, serovar and pathogenicity. We were able to identify genes from the accessory genome that were significantly associated with phenotypes such as potential serovar specific genes including capsule genes, and 48 putative virulence factors that were significantly different between the clinical and non-clinical isolates. We also show that the presence of many previously suggested virulence factors is not an appropriate marker of virulence. CONCLUSIONS: These genes will inform the generation of new molecular diagnostics and vaccines, and refinement of existing typing schemes and show the importance of the accessory genome of a diverse species when investigating the relationship between genotypes and phenotypes.
Bosse JT, Soares-Bazzolli DM, Li Y, et al., 2014, The Generation of Successive Unmarked Mutations and Chromosomal Insertion of Heterologous Genes in <i>Actinobacillus pleuropneumoniae</i> Using Natural Transformation, PLOS ONE, Vol: 9, ISSN: 1932-6203
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- Citations: 18
Bosse JT, Li Y, Angen O, et al., 2014, Multiplex PCR Assay for Unequivocal Differentiation of <i>Actinobacillus pleuropneumoniae</i> Serovars 1 to 3, 5 to 8, 10, and 12, JOURNAL OF CLINICAL MICROBIOLOGY, Vol: 52, Pages: 2380-2385, ISSN: 0095-1137
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- Citations: 35
Anderson ST, Kaforou M, Brent AJ, et al., 2014, Diagnosis of Childhood Tuberculosis and Host RNA Expression in Africa, New England Journal of Medicine, Vol: 370, Pages: 1712-1723, ISSN: 1533-4406
Rossi CC, Pereira MF, Langford PR, et al., 2014, A BOX-SCAR fragment for the identification of Actinobacillus pleuropneumoniae, FEMS MICROBIOLOGY LETTERS, Vol: 352, Pages: 32-37, ISSN: 0378-1097
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- Citations: 3
Khara JS, Wang Y, Ke X-Y, et al., 2014, Anti-mycobacterial activities of synthetic cationic α-helical peptides and their synergism with rifampicin, BIOMATERIALS, Vol: 35, Pages: 2032-2038, ISSN: 0142-9612
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- Citations: 85
Maglennon GA, Cook BS, Deeney AS, et al., 2013, Transposon mutagenesis in Mycoplasma hyopneumoniae using a novel mariner-based system for generating random mutations., Veterinary Research, Vol: 44, ISSN: 1297-9716
Mycoplasma hyopneumoniae is the cause of enzootic pneumonia in pigs, a chronic respiratory disease associated with significant economic losses to swine producers worldwide. The molecular pathogenesis of infection is poorly understood due to the lack of genetic tools to allow manipulation of the organism and more generally for the Mycoplasma genus. The objective of this study was to develop a system for generating random transposon insertion mutants in M. hyopneumoniae that could prove a powerful tool in enabling the pathogenesis of infection to be unraveled. A novel delivery vector was constructed containing a hyperactive C9 mutant of the Himar1 transposase along with a mini transposon containing the tetracycline resistance cassette, tetM. M. hyopneumoniae strain 232 was electroporated with the construct and tetM-expressing transformants selected on agar containing tetracycline. Individual transformants contained single transposon insertions that were stable upon serial passages in broth medium. The insertion sites of 44 individual transformants were determined and confirmed disruption of several M. hyopneumoniae genes. A large pool of over 10 000 mutants was generated that should allow saturation of the M. hyopneumoniae strain 232 genome. This is the first time that transposon mutagenesis has been demonstrated in this important pathogen and could be generally applied for other Mycoplasma species that are intractable to genetic manipulation. The ability to generate random mutant libraries is a powerful tool in the further study of the pathogenesis of this important swine pathogen.
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