Imperial College London

ProfessorPaulLangford

Faculty of MedicineDepartment of Infectious Disease

Professor of Paediatric Infectious Diseases
 
 
 
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Contact

 

+44 (0)20 7594 3359p.langford Website

 
 
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Location

 

236Wright Fleming WingSt Mary's Campus

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Summary

 

Publications

Publication Type
Year
to

200 results found

Cohen LM, Bossé JT, Stegger M, Li Y, Langford PR, Kielland C, Klem TB, Gulliksen SM, Ranheim B, Grøntvedt CA, Angen Øet al., 2021, Comparative genome sequence analysis of actinobacillus pleuropneumoniae serovar 8 isolates from Norway, Denmark, and the United Kingdom indicates distinct phylogenetic lineages and differences in distribution of antimicrobial resistance genes, Frontiers in Microbiology, Vol: 12, Pages: 1-13, ISSN: 1664-302X

Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia, a disease of major impact on pig health, welfare, and productivity globally. Serovar 8 (APP) is the predominant clinical serovar in Norway and the United Kingdom (UK), and has been isolated from clinical cases in Denmark. The primary objective of this study was to characterize the genetic variability of isolates of A. pleuropneumoniae APP8 in the Norwegian population. The secondary objectives were to determine the within-host variability of APP8; to compare the APP8 bacterial populations in Norway, Denmark, and the UK, including antimicrobial resistance (AMR) gene profiles and to assess the effect of national differences in antimicrobial drug use and restricted animal movement on the occurrence of resistance. Isolates of APP8 from the UK (n=67), Denmark (n=22), and Norway (n=123) collected between 1983 and 2020 were compared using whole genome sequencing. To investigate genetic variability within individual hosts, an additional 104 APP8 isolates from the lungs of six Norwegian pigs were compared. Very low within-host variation was observed (≤ 2 single nucleotide polymorphisms). The phylogeny of 123 Norwegian APP8 isolates from 76 herds revealed some within-herd genetic variation, but substantial geographical clustering. When inferring the relatedness of the three international APP8 collections, the topology highlighted the existence of two distinct monophyletic branches characterized by the Norwegian and UK isolates, respectively. Three Danish isolates were scattered across the UK branch, whereas the remaining 19 Danish isolates clustered in two monophyletic groups nested in the Norwegian branch. Coalescence analysis, performed to estimate the divergences from a common ancestor, indicated a last common ancestor several centuries ago. The phylogenetic analyses also revealed striking differences in occurrence of AMR genes, as these were 23-times more prevalent among the UK isolates

Journal article

Stringer O, Bosse J, Lacoutre S, Gottschalk M, Fodor L, Angen Ø, Velazquez E, Penny P, Lei L, Langford P, Li Yet al., 2021, Rapid detection and typing of Actinobacillus pleuropneumoniae serovars directly from clinical samples: combining FTA® card technology with multiplex-PCR, Frontiers in Veterinary Science, Vol: 8, Pages: 1-9, ISSN: 2297-1769

Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, is highly contagious and responsible for high morbidity, mortality and economic losses in the swine industry worldwide, but quick serotyping and diagnosis are still not widely available. In this study, we sought to validate the use of Whatman FTA® cards for collection and processing of A. pleuropneumoniae isolates, or porcine lung tissue samples, for direct use in diagnostic multiplex PCRs.We have optimized the processing of 3 mm discs punched from FTA® cards loaded with cultured A. pleuropneumoniae, or imprinted on lesioned regions of lung tissue, with only three distilled water washes before addition into our APP-mPCR assay for rapid, low-cost identification and serotyping. DNA captured on FTA® cards generated the same diagnostic PCR results as DNA extracted using commercial kits for 85 A. pleuropneumoniae clinical isolate cultures and 22 lung samples. Additionally, bacterial DNA bound to FTA® cards was detectable by PCR after six months of storage at 37°C.This study provides simple, efficient, rapid and practical sample processing for detection and molecular serotyping of A. pleuropneumoniae.

Journal article

Langford P, Stringer O, Li Y, Bosse Jet al., 2021, Application of the MISTEACHING(S) disease susceptibility framework to Actinobacillus pleuropneumoniae to identify research gaps: an exemplar of a veterinary pathogen, Animal Health Research Reviews, ISSN: 1466-2523

Historically, the MISTEACHING (microbiome, immunity, sex, temperature, environment, age, chance, history, inoculum, nutrition, genetics) framework to describe the outcome of host-pathogen interaction, has been applied to human pathogens. Here, weshow, using Actinobacillus pleuropneumoniaeas an exemplar, that the MISTEACHING framework can be applied to a strict veterinary pathogen enabling the identification of major research gaps, and the formulation of hypotheses whose study will lead to a greater understanding of pathogenic mechanisms, and/or improved prevention/therapeutic measures. We also suggest that the MISTEACHING framework should be extended with the inclusion of a “strain” category, to become MISTEACHINGS. We conclude that the MISTEACHINGS framework can be applied to veterinary pathogens, whether they be bacteria, viruses, or parasites, and hope to stimulate others to use it to identify research gaps and to formulate hypotheses worthy of study with their own pathogens.

Journal article

Bao C, Liu B, Zhu R, Xiao J, Li Z, Jiang H, Wang B, Langford PR, Fei R, Li N, Lei Let al., 2021, IFN-gamma(-/-) Mice Resist Actinobacillus pleuropneumoniae Infection by Promoting Early Lung IL-18 Release and PMN-I Accumulation, INFECTION AND IMMUNITY, Vol: 89, ISSN: 0019-9567

Journal article

Stringer O, Bosse J, Lacouture S, Gottschalk M, Fodor L, Angen Ø, Velazquez E, Penny P, Lei L, Langford P, Li Yet al., 2021, Proposal of Actinobacillus pleuropneumoniae serovar 19, and reformulation of previous multiplex PCRs for capsule-specific typing of all known serovars, Veterinary Microbiology, Vol: 255, ISSN: 0378-1135

Two serologically and molecularly non-typeable isolates of the porcine lung pathogen Actinobacillus pleuropneumoniae have been identified from diseased swine in two different continents. Genome sequencing was carried out to identify their diagnostically relevant genotypes. Both isolates are biovar 1 and encode genes for production of ApxIV and ApxII (apxIICA structural genes, and apxIBD export genes). They both possess the same novel type II capsule locus (most similar to serovar 1, but with two capsule genes not previously found in A. pleuropneumoniae) but differ in their O-Ag loci. Strain 7213384-1 from Denmark, which we propose as the reference strain for serovar 19, has a serogroup 3/6/8/15 O-Ag locus; the Canadian isolate A08-13 has a serogroup 4/7 O-Ag locus. We have expanded the second of our two previously described A. pleuropneumoniae mPCRs to include capsule gene-specific primers for definitive detection of serovars 13-14 and 16-19.

Journal article

Gliddon H, Kaforou M, Alikian M, Coote D, Zhou C, Oni T, Anderson ST, Brent AJ, Crampin AC, Eley B, Heyderman R, Langford PR, Kern F, Ottenhoff THM, Hibberd ML, French N, Wright V, Dockrell HM, Coin L, Wilkinson R, Levin Met al., 2021, Identification of reduced host transcriptomic signatures for tuberculosis disease and digital PCR-based validation and quantification, Frontiers in Immunology, Vol: 12, ISSN: 1664-3224

Recently, host whole blood gene expression signatures have been identified for diagnosis of tuberculosis (TB). Absolute quantification of the concentrations of signature transcripts in blood have not been reported, but would facilitate diagnostic test development. To identify minimal transcript signatures, we applied a transcript selection procedure to microarray data from African adults comprising 536 patients with TB, other diseases (OD) and latent TB (LTBI), divided into training and test sets. Signatures were further investigated using reverse transcriptase (RT)—digital PCR (dPCR). A four-transcript signature (GBP6, TMCC1, PRDM1, and ARG1) measured using RT-dPCR distinguished TB patients from those with OD (area under the curve (AUC) 93.8% (CI95% 82.2–100%). A three-transcript signature (FCGR1A, ZNF296, and C1QB) differentiated TB from LTBI (AUC 97.3%, CI95%: 93.3–100%), regardless of HIV. These signatures have been validated across platforms and across samples offering strong, quantitative support for their use as diagnostic biomarkers for TB.

Journal article

Liu H, Lei S, Jia L, Xia X, Sun Y, Jiang H, Zhu R, Li S, Qu G, Gu J, Sun C, Feng X, Han W, Langford PR, Lei Let al., 2021, Streptococcus suis serotype 2 enolase interaction with host brain microvascular endothelial cells and RPSA-induced apoptosis lead to loss of BBB integrity, Veterinary Research, Vol: 52, Pages: 30-30, ISSN: 1994-4659

Host proteins interacting with pathogens are receiving more attention as potential therapeutic targets in molecular medicine. Streptococcus suis serotype 2 (SS2) is an important cause of meningitis in both humans and pigs worldwide. SS2 Enolase (Eno) has previously been identified as a virulence factor with a role in altering blood brain barrier (BBB) integrity, but the host cell membrane receptor of Eno and The mechanism(s) involved are unclear. This study identified that SS2 Eno binds to 40S ribosomal protein SA (RPSA) on the surface of porcine brain microvascular endothelial cells leading to activation of intracellular p38/ERK-eIF4E signalling, which promotes intracellular expression of HSPD1 (heat-shock protein family D member 1), and initiation of host-cell apoptosis, and increased BBB permeability facilitating bacterial invasion. This study reveals novel functions for the host-interactional molecules RPSA and HSPD1 in BBB integrity, and provides insight for new therapeutic strategies in meningitis.

Journal article

Asai M, Sheehan G, Li Y, Robertson B, Kavanagh K, Langford P, Newton Set al., 2021, Innate immune responses of Galleria mellonella to Mycobacterium bovis BCG challenge identified using proteomic and molecular approaches, Frontiers in Cellular and Infection Microbiology, Vol: 11, ISSN: 2235-2988

The larvae of the insect Galleria mellonella, have recently been established as a non-mammalian infection model for the Mycobacterium tuberculosis complex (MTBC). To gain further insight into the potential of this model, we applied proteomic (label-free quantification) and transcriptomic (gene expression) approaches to characterise the innate immune response of G. mellonella to infection with Mycobacterium bovis BCG lux over a 168 h time course. Proteomic analysis of the haemolymph from infected larvae revealed distinct changes in the proteome at all time points (4, 48, 168 h). Reverse transcriptase quantitative PCR confirmed induction of five genes (gloverin, cecropin, IMPI, hemolin, and Hdd11), which encoded proteins found to be differentially abundant from the proteomic analysis. However, the trend between gene expression and protein abundance were largely inconsistent (20%). Overall, the data are in agreement with previous phenotypic observations such as haemocyte internalization of mycobacterial bacilli (hemolin/β-actin), formation of granuloma-like structures (Hdd11), and melanization (phenoloxidase activating enzyme 3 and serpins). Furthermore, similarities in immune expression in G. mellonella, mouse, zebrafish and in vitro cell-line models of tuberculosis infection were also identified for the mechanism of phagocytosis (β-actin). Cecropins (antimicrobial peptides), which share the same α-helical motif as a highly potent peptide expressed in humans (h-CAP-18), were induced in G. mellonella in response to infection, giving insight into a potential starting point for novel antimycobacterial agents. We believe that these novel insights into the innate immune response further contribute to the validation of this cost-effective and ethically acceptable insect model to study members of the MTBC.

Journal article

Siris S, Gladstone CA, Guo Y, Pinder CL, Shattock RJ, McKay PF, Langford PR, Bidmos FAet al., 2021, Isolating Pathogen-Specific Human Monoclonal Antibodies (hmAbs) Using Bacterial Whole Cells as Molecular Probes., Methods Mol Biol, Vol: 2183, Pages: 9-18

The immunoglobulin capture assay (ICA) enables the enrichment for pathogen-specific plasmablasts from individuals with a confirmed adaptive immune response to vaccination or disseminated infection. Only single recombinant antigens have been used previously as probes in this ICA and it was unclear whether the method was applicable to complex probes such as whole bacterial cells. Here, we describe the enrichment of plasmablasts specific for polysaccharide and protein antigens of both Streptococcus pneumoniae and Neisseria meningitidis using whole formalin-fixed bacterial cells as probes. The modified ICA protocol described here allowed for a pathogen-specific hmAb cloning efficiency of >80%.

Journal article

Menikou S, McArdle AJ, Li M-S, Kaforou M, Langford PR, Levin Met al., 2020, A proteomics-based method for identifying antigens within immune complexes, PLoS One, Vol: 15, ISSN: 1932-6203

A novel approach to recover and identify immune complexes (ICs) was developed using size exclusion chromatography (SEC) and affinity chromatography on immunoglobulin binding columns (HiTrap Protein G). The purification process was monitored by 1D SDS-PAGE, protein staining, Western blotting and, finally, liquid chromatography tandem mass spectrometry (LC MS/MS) was used to identify the recovered antigens. This approach was applied to serum with artificially created immune complexes (ICs) comprising vaccine antigen (influenza) and antibody, which led to recovery and identification of influenza peptides within the recovered ICs. This approach was compared with the established method for IC detection and recovery, polyethylene glycol (PEG) precipitation, followed by LC MS/MS. Both approaches successfully enabled capture, recovery and characterization of immunoglobulins and influenza antigen(s) in complex with the immunoglobulins. However, PEG precipitation has the advantage of simplicity and is more suited for large scale studies.

Journal article

Crispim JS, da Silva TF, Sanches NM, da Silva GC, Pereira MF, Rossi CC, Li Y, Terra VS, Vohra P, Wren BW, Langford PR, Bossé JT, Bazzolli DMSet al., 2020, Serovar-dependent differences in Hfq-regulated phenotypes in Actinobacillus pleuropneumoniae., Pathogens and Disease, Vol: 78, Pages: 1-12, ISSN: 2049-632X

The RNA chaperone Hfq regulates diverse processes in numerous bacteria. In this study, we compared phenotypes (growth rate, adherence, response to different stress conditions, and virulence in Galleria mellonella) of wild-type (WT) and isogenic hfq mutants of three serovars (1, 8 and 15) of the porcine pathogen A. pleuropneumoniae. Similar growth in rich broth was seen for all strains except Ap1∆hfq, which showed slightly reduced growth throughout the 24 hour time course, and the complemented Ap8∆hfqC mutant had a prolonged lag phase. Differences were seen between the three serovar WT strains regarding adherence, stress response and virulence in G. mellonella, and deletion of hfq affected some, but not all of these phenotypes, depending on serovar. Complementation by expression of cloned hfq from an endogenous promoter only restored some WT phenotypes, indicating that complex regulatory networks may be involved, and that levels of Hfq may be as important as presence/absence of the protein regarding its contribution to gene regulation. Our results support that Hfq is a pleiotropic global regulator in A. pleuropneumoniae, but serovar-related differences exist. These results highlight the importance of testing multiple strains/serovars within a given species when determining contributions of global regulators, such as Hfq, to expression of complex phenotypes.

Journal article

Bao C, Jiang H, Zhu R, Liu B, Xiao J, Li Z, Chen P, Langford PR, Zhang F, Lei Let al., 2020, Differences in pig respiratory tract and peripheral blood immune responses to Actinobacillus pleuropneumoniae, VETERINARY MICROBIOLOGY, Vol: 247, ISSN: 0378-1135

Journal article

Zhu R, Bao C, Liu B, Xiao J, Sun C, Feng X, Langford PR, Li Y, Lei Let al., 2020, iTRAQ-based quantitative proteomic analysis of peripheral blood serum in piglets infected withActinobacillus pleuropneumoniae, AMB EXPRESS, Vol: 10, ISSN: 2191-0855

Journal article

Asai M, Li Y, Spiropoulos J, Cooley W, Everest D, Robertson BD, Langford PR, Newton SMet al., 2020, A novel biosafety level 2 compliant tuberculosis infection model using a ΔleuDΔpanCD double auxotroph of Mycobacterium tuberculosis H37Rv and Galleria mellonella, Virulence, Vol: 11, Pages: 811-824, ISSN: 2150-5594

Mammalian infection models have contributed significantly to our understanding of the host-mycobacterial interaction, revealing potential mechanisms and targets for novel antimycobacterial therapeutics. However, the use of conventional mammalian models such as mice, are typically expensive, high maintenance, require specialised animal housing, and are ethically regulated. Furthermore, research using Mycobacterium tuberculosis (MTB), is inherently difficult as work needs to be carried out at biosafety level 3 (BSL3). The insect larvae of Galleria mellonella (greater wax moth), have become increasingly popular as an infection model, and we previously demonstrated its potential as a mycobacterial infection model using Mycobacterium bovis BCG. Here we present a novel BSL2 complaint MTB infection model using G. mellonella in combination with a bioluminescent ΔleuDΔpanCD double auxotrophic mutant of MTB H37Rv (SAMTB lux) which offers safety and practical advantages over working with wild type MTB. Our results show a SAMTB lux dose dependent survival of G. mellonella larvae and demonstrate proliferation and persistence of SAMTB lux bioluminescence over a 1 week infection time course. Histopathological analysis of G. mellonella, highlight the formation of early granuloma-like structures which matured over time. We additionally demonstrate the drug efficacy of first (isoniazid, rifampicin, and ethambutol) and second line (moxifloxacin) antimycobacterial drugs. Our findings demonstrate the broad potential of this insect model to study MTB infection under BSL2 conditions. We anticipate that the successful adaptation and implementation of this model will remove the inherent limitations of MTB research at BSL3 and increase tuberculosis research output.

Journal article

Hau SJ, Luan S-L, Loving CL, Nicholson TL, Wang J, Peters SE, Seilly D, Weinert LA, Langford PR, Rycroft AN, Wren BW, Maskell DJ, Tucker AW, Brockmeier SLet al., 2020, Evaluation of the recombinant proteins RlpB and VacJ as a vaccine for protection againstGlaesserella parasuisin pigs, BMC VETERINARY RESEARCH, Vol: 16

Journal article

Mashbat B, Bellos E, Hodeib S, Bidmos F, Thwaites RS, Lu Y, Wright VJ, Herberg JA, Klobassa DS, Zenz W, Hansel TT, Nadel S, Langford PR, Schlapbach LJ, Li M-S, Redinbo MR, Di YP, Levin M, Sancho-Shimizu Vet al., 2020, A rare mutation in SPLUNC1 underlies meningococcal disease affecting bacterial adherence and invasion, Clinical Infectious Diseases, Vol: 70, Pages: 2045-2053, ISSN: 1058-4838

BackgroundNeisseriameningitidis (Nm) is a nasopharyngeal commensal carried by healthy individuals. However, invasive infections occurs in a minority of individuals, with devastating consequences. There is evidence that common polymorphisms are associated with invasive meningococcal disease (IMD) but the contribution of rare variants other than complement has not been determined.MethodsWe identified familial cases of IMD in the UK meningococcal disease study and the European Union Life-threatening Infectious Disease Study. Candidate genetic variants were identified by whole exome sequencing of two patients with familial IMD. Candidate variants were further validated by in vitro assays.ResultsExomes of two siblings with IMD identified a novel heterozygous missense mutation in BPIFA1/SPLUNC1. Sequencing of 186 other non-familial cases identified another unrelated IMD patient with the same mutation. SPLUNC1 is an innate immune defence protein expressed in the nasopharyngeal epithelia, however, its role in invasive infections is unknown. In vitro assays demonstrated that recombinant SPLUNC1 inhibits biofilm formation by Nm, and impedes Nm adhesion and invasion of human airway cells. The dominant negative mutant rSPLUNC1 (p.G22E) showed reduced anti-biofilm activity, increased meningococcal adhesion and invasion of cells compared with wild type SPLUNC1.ConclusionsA mutation in SPLUNC1 affecting mucosal attachment, biofilm formation and invasion of mucosal epithelial cells is a new genetic cause of meningococcal disease.

Journal article

Eberle KC, Hau SJ, Luan S-L, Weinert LA, Stasko JA, Wang J, Peters SE, Langford PR, Rycroft AN, Wren BW, Maskell DJ, Tucker AW, Brockmeier SLet al., 2020, Generation and Evaluation of a Glaesserella (Haemophilus) parasuis Capsular Mutant, INFECTION AND IMMUNITY, Vol: 88, ISSN: 0019-9567

Journal article

Singh Khara J, Mojsoska B, Mukherjee D, Langford P, Robertson B, Ee PLR, Newton Set al., 2020, Ultra-short antimicrobial peptoids show propensity for membrane activity against multi-drug resistant Mycobacterium tuberculosis, Frontiers in Microbiology, Vol: 11, Pages: 1-11, ISSN: 1664-302X

Tuberculosis (TB) results in both morbidity and mortality on a global scale. With drug resistance on the increase, there is an urgent need to develop novel anti-mycobacterials. Thus, we assessed the anti-mycobacterial potency of three novel synthetic peptoids against drug-susceptible and multi-drug resistant (MDR) Mycobacterium tuberculosis in vitro using Minimum Inhibitory Concentration, killing efficacy and intracellular growth inhibition assays, and in vivo against mycobacteria infected BALB/c mice. In addition, we verified cell selectivity using mammalian cells to assess peptoid toxicity. The mechanism of action was determined using flow cytometric analysis, and microfluidic live-cell imaging with time-lapse microscopy and uptake of propidium iodide. Peptoid BM 2 demonstrated anti-mycobacterial activity against both drug sensitive and MDR M. tuberculosis together with an acceptable toxicity profile that showed selectivity between bacterial and mammalian membranes. The peptoid was able to efficiently kill mycobacteria both in vitro and intracellularly in murine RAW 264.7 macrophages, and significantly reduced bacterial load in the lungs of infected mice. Flow cytometric and time lapse fluorescence microscopy indicate mycobacterial membrane damage as the likely mechanism of action. These data demonstrate that peptoids are a novel class of antimicrobial which warrant further investigation and development as therapeutics against TB.

Journal article

Li G, Zhao Q, Luan T, Hu Y, Zhang Y, Li T, Wang C, Xie F, Zhang W, Langford PR, Liu Set al., 2020, Basal Level Effects of (p)ppGpp in the Absence of Branched Chain Amino Acids in Actinobacillus pleuropneumoniae., J Bacteriol

The (p)ppGpp-mediated stringent response (SR) is a highly conserved regulatory mechanism in bacterial pathogens, enabling adaptation to adverse environments and linked to pathogenesis. Actinobacillus pleuropneumoniae can cause damage to the lungs of pigs, it's only known natural host. Pig lungs are known to have a low concentration of free branched chain amino acids (BCAAs) compared to plasma. We had investigated the role for (p)ppGpp in viability and biofilm formation of A. pleuropneumoniae Now, we sought to determine whether (p)ppGpp was a trigger signal for the SR in A. pleuropneumoniae in the absence of BCAAs. Combining transcriptome and phenotypic analyses of wild type (WT) and relAspoT double mutant (which does not produce (p)ppGpp), we found that (p)ppGpp could repress de novo purine biosynthesis and activate antioxidant pathways. There was a positive correlation between GTP and endogenous hydrogen peroxide content. Furthermore, the growth, viability, morphology and virulence were altered by the inability to produce (p)ppGpp. Genes involved in the biosynthesis of BCAAs were constitutively up-regulated regardless of the existence of BCAAs without accumulation of (p)ppGpp beyond basal level. Collectively, our study shows that the absence of BCAAs was not a sufficient signal to trigger the SR in A. pleuropneumoniae (p)ppGpp-mediated regulation in A. pleuropneumoniae is different to that described for the model organism Escherichia coli. Further work will establish whether the (p)ppGpp-dependent SR mechanism in Apleuropneumoniae is conserved among other veterinary pathogens, especially those in the Pasteurellaceae family.IMPORTANCE(p)ppGpp is a key player in reprogramming transcriptomes to respond to nutritional challenges. Here, we present a transcriptional and phenotypic differences of A. pleuropneumoniae grown in different chemically defined media in the absence of (p)ppGpp. We show that the deprivation of branch-chain amino acids (BCAAs) does not elicit a cha

Journal article

Bosse JT, Li Y, Fernandez Crespo R, Angen O, Holden MTG, Weinert LA, Maskell DJ, Tucker AW, Wren BW, Rycroft AN, Langford PR, Consortium Bet al., 2020, Draft genome sequences of the type strains of Actinobacillus indolicus (46K2C) and Actinobacillus porcinus (NM319), two NAD-dependent bacterial species found in the respiratory tract of pigs, Microbiology Resource Announcements, Vol: 9, Pages: 1-3, ISSN: 2576-098X

We report here the draft genome sequences of the type strains of Actinobacillus indolicus (46K2CT) and Actinobacillus porcinus (NM319T). These NAD-dependent bacterial species are frequently found in the upper respiratory tract of pigs and are occasionally associated with lung pathology.

Journal article

Lewin A, Hamilton S, Witkover A, Langford P, Nicholas R, Chataway J, Bangham CRMet al., 2019, Free serum haemoglobin is associated with brain atrophy in secondary progressive multiple sclerosis [version 2; peer review: 3 approved], Wellcome Open Research, Vol: 1, ISSN: 2398-502X

Background: A major cause of disability in secondary progressive multiple sclerosis (SPMS) is progressive brain atrophy, whose pathogenesis is not fully understood. The objective of this study was to identify protein biomarkers of brain atrophy in SPMS. Methods: We used surface-enhanced laser desorption-ionization time-of-flight mass spectrometry to carry out an unbiased search for serum proteins whose concentration correlated with the rate of brain atrophy, measured by serial MRI scans over a 2-year period in a well-characterized cohort of 140 patients with SPMS. Protein species were identified by liquid chromatography-electrospray ionization tandem mass spectrometry. Results: There was a significant (p<0.004) correlation between the rate of brain atrophy and a rise in the concentration of proteins at 15.1 kDa and 15.9 kDa in the serum. Tandem mass spectrometry identified these proteins as alpha-haemoglobin and beta-haemoglobin, respectively. The abnormal concentration of free serum haemoglobin was confirmed by ELISA (p<0.001). The serum lactate dehydrogenase activity was also highly significantly raised (p<10-12) in patients with secondary progressive multiple sclerosis. Conclusions: The results are consistent with the following hypothesis. In progressive multiple sclerosis, low-grade chronic intravascular haemolysis releases haemoglobin into the serum; the haemoglobin is subsequently translocated into the central nervous system (CNS) across the damaged blood-brain barrier. In the CNS, the haemoglobin and its breakdown products, including haem and iron, contribute to the neurodegeneration and consequent brain atrophy seen in progressive disease. We postulate that haemoglobin is a source of the iron whose deposition along blood vessels in multiple sclerosis plaques is associated with neurodegeneration. If so, then chelators of haemoglobin, rather than chelators of free serum iron, may be effective in preventing this neurodegeneration.

Journal article

Weinert LA, Chaudhuri RR, Wang J, Peters SE, Corander J, Jombart T, Baig A, Howell KJ, Vehkala M, Valimaki N, Harris D, Tran TBC, Nguyen VVC, Campbell J, Schultsz C, Parkhill J, Bentley SD, Langford PR, Rycroft AN, Wren BW, Farrar J, Baker S, Hoa NT, Holden MTG, Tucker AW, Maskell DJ, Bosse JT, Li Y, Maglennon GA, Matthews D, Cuccui J, Terra Vet al., 2019, Publisher Correction: Genomic signatures of human and animal disease in the zoonotic pathogen Streptococcus suis (vol 6, 6740, 2015), Nature Communications, Vol: 10, ISSN: 2041-1723

Journal article

Asai M, Li Y, Singh Khara J, Robertson B, Langford P, Newton Set al., 2019, Galleria mellonella: a novel infection model for screening potential anti-mycobacterial compounds against members of the Mycobacterium tuberculosis complex, Frontiers in Microbiology, Vol: 10, ISSN: 1664-302X

Drug screening models have a vital role in the development of novel antimycobacterial agents which are urgently needed to tackle drug-resistant tuberculosis (TB). We recently established the larvae of the insect Galleria mellonella (greater wax moth) as a novel infection model for the Mycobacterium tuberculosis complex. Here we demonstrate its use as a rapid and reproducible screen to evaluate antimycobacterial drug efficacy using larvae infected with bioluminescent Mycobacterium bovis BCG lux. Treatment improved larval survival outcome and, with the exception of pyrazinamide, was associated with a significant reduction in in vivo mycobacterial bioluminescence over a 96 hour period compared to the untreated controls. Isoniazid and rifampicin displayed the greatest in vivo efficacy and survival outcome. Thus G. mellonella, infected with bioluminescent mycobacteria, can rapidly determine in vivo drug efficacy, and has the potential to significantly reduce and/or replace the number of animals used in TB research.

Journal article

Wileman TM, Weinert LA, Howell KJ, Wang J, Peters SE, Williamson SM, Wells JM, Langford PR, Rycroft AN, Wren BW, Maskell DJ, Tucker AW, Bosse JT, Li Y, Cuccui J, Terra VS, Beddow J, Maglennon GA, Chaudhuri RRet al., 2019, Pathotyping the zoonotic pathogen streptococcus suis: Novel genetic markers to differentiate invasive disease-associated isolates from non-disease-associated isolates from England and Wales, Journal of Clinical Microbiology, Vol: 57, ISSN: 0095-1137

Streptococcus suis is one of the most important zoonotic bacterial pathogens of pigs, causing significant economic losses to the global swine industry. S. suis is also a very successful colonizer of mucosal surfaces, and commensal strains can be found in almost all pig populations worldwide, making detection of the S. suis species in asymptomatic carrier herds of little practical value in predicting the likelihood of future clinical relevance. The value of future molecular tools for surveillance and preventative health management lies in the detection of strains that genetically have increased potential to cause disease in presently healthy animals. Here we describe the use of genome-wide association studies to identify genetic markers associated with the observed clinical phenotypes (i) invasive disease and (ii) asymptomatic carriage on the palatine tonsils of pigs on UK farms. Subsequently, we designed a multiplex PCR to target three genetic markers that differentiated 115 S. suis isolates into disease-associated and non-disease-associated groups, that performed with a sensitivity of 0.91, a specificity of 0.79, a negative predictive value of 0.91, and a positive predictive value of 0.79 in comparison to observed clinical phenotypes. We describe evaluation of our pathotyping tool, using an out-of-sample collection of 50 previously uncharacterized S. suis isolates, in comparison to existing methods used to characterize and subtype S. suis isolates. In doing so, we show our pathotyping approach to be a competitive method to characterize S. suis isolates recovered from pigs on UK farms and one that can easily be updated to incorporate global strain collections.

Journal article

Asai M, Li Y, Khara J, Gladstone C, Robertson B, Langford P, Newton Set al., 2019, Use of the invertebrate Galleria Mellonella as an infection model to study the Mycobacterium tuberculosis complex, Jove-Journal of Visualized Experiments, Vol: 148, ISSN: 1940-087X

Tuberculosis is the leading global cause of infectious disease mortality and roughly a quarter of the world’s population is believed to be infected with Mycobacterium tuberculosis. Despite decades of research, many of the mechanisms behind the success of M. tuberculosis as a pathogenic organism remain to be investigated, and the development of safer, more effective antimycobacterial drugs are urgently needed to tackle the rise and spread of drug resistant tuberculosis. However, the progression of tuberculosis research is bottlenecked by traditional mammalian infection models that are expensive, time consuming, and ethically challenging.Previously we established the larvae of the insect Galleria mellonella (greater wax moth) as a novel, reproducible, low cost, high-throughput and ethically acceptable infection model for members of the M. tuberculosis complex. Here we describe the maintenance, preparation, and infection of G. mellonella with bioluminescent Mycobacterium bovis BCG lux. Using this infection model, mycobacterial dose dependent virulence can be observed, and a rapid readout of in vivo mycobacterial burden using bioluminescence measurements is easily achievable and reproducible. Although limitations exist, such as the lack of a fully annotated genome for transcriptomic analysis, ontological analysis against genetically similar insects can be carried out. As a low cost, rapid, and ethically acceptable model for tuberculosis, G. mellonella can be used as a pre-screen to determine drug efficacy and toxicity, and to determine comparative mycobacterial virulence prior to the use of conventional mammalian models. The use of the G. mellonella-mycobacteria model will lead to a reduction in the substantial number of animals currently used in tuberculosis research.

Journal article

Menikou S, Langford P, Levin M, 2019, Kawasaki disease: the role of immune complexes revisited, Frontiers in Immunology, Vol: 10, ISSN: 1664-3224

Kawasaki disease (KD) is an inflammatory disease in children associated with vasculitis affecting predominantly the coronary arteries and is now the most common cause of acquired heart disease in children in developed countries. The etiology of KD is unknown but epidemiological studies implicate an infectious agent or toxin, which causes disease in genetically predisposed individuals. The presence of immune complexes (ICs) in the serum of children with KD was established in numerous studies during the 1970s and 80s. More recent genetic studies have identified variation in Fcγ receptors and genes controlling immunoglobulin production associated with KD. In this review we link the genetic findings and IC studies and suggest a key role for their interaction in pathophysiology of the disease.

Journal article

Bosse J, Li Y, Stringer O, Langford Pet al., 2019, Serotyping of Actinobacillus pleuropneumoniae field isolates by PCR, Publisher: Pig33 (www.pig333.com)

Working paper

Gliddon HD, Kaforou M, Alikian M, Habgood-Coote D, Zhou C, Oni T, Anderson ST, Brent AJ, Crampin AC, Eley B, Kern F, Langford PR, Ottenhoff THM, Hibberd ML, French N, Wright VJ, Dockrell HM, Coin LJ, Wilkinson RJ, Levin Met al., 2019, Identification of reduced host transcriptomic signatures for tuberculosis and digital PCR-based validation and quantification, Publisher: Cold Spring Harbor Laboratory

<jats:title>Abstract</jats:title><jats:p>Recently, host whole blood gene expression signatures have been identified for diagnosis of tuberculosis (TB). Absolute quantification of the concentrations of signature transcripts in blood have not been reported, but would facilitate the development of diagnostic tests.</jats:p><jats:p>To identify minimal transcript signatures, we applied a novel transcript selection procedure to microarray data from African adults comprising 536 patients with TB, other diseases (OD) and latent TB (LTBI), divided into training and test sets. Signatures were validated using reverse transcriptase (RT) - digital PCR (dPCR).</jats:p><jats:p>A four-transcript signature (<jats:italic>GBP6</jats:italic>, <jats:italic>TMCC1</jats:italic>, <jats:italic>PRDM1</jats:italic>, <jats:italic>ARG1</jats:italic>) measured using RT-dPCR distinguished TB patients from those with OD (area under the curve (AUC) 93.8% (CI<jats:sub>95%</jats:sub> 82.2 – 100%). A three-transcript signature (<jats:italic>FCGR1A, ZNF296, C1QB</jats:italic>) differentiated TB from LTBI (AUC 97.3%, CI<jats:sub>95%</jats:sub>: 93.3 – 100%), regardless of HIV.</jats:p><jats:p>These signatures have been validated across platforms and across samples offering strong, quantitative support for their use as diagnostic biomarkers for TB.</jats:p>

Working paper

Bao C-T, Xiao J-M, Liu B-J, Liu J-F, Zhu R-N, Jiang P, Li L, Langford PR, Lei L-Cet al., 2019, Establishment and comparison of Actinobacillus pleuropneumoniae experimental infection model in mice and piglets, MICROBIAL PATHOGENESIS, Vol: 128, Pages: 381-389, ISSN: 0882-4010

Journal article

Jiang H, Zhu R, Liu H, Bao C, Liu J, Eltahir A, Langford PR, Sun D, Liu Z, Sun C, Gu J, Han W, Feng X, Lei Let al., 2018, Transcriptomic analysis of porcine PBMCs in response to Actinobacillus pleuropneumoniae reveals the dynamic changes of differentially expressed genes related to immuno-inflammatory responses, ANTONIE VAN LEEUWENHOEK INTERNATIONAL JOURNAL OF GENERAL AND MOLECULAR MICROBIOLOGY, Vol: 111, Pages: 2371-2384, ISSN: 0003-6072

Journal article

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