Actinobacillus pleuropneumoniae encodes multiple phase-variable DNA methyltransferases that control distinct phasevarions.

Actinobacillus pleuropneumoniae is the cause of porcine pleuropneumonia, a severe respiratory tract infection that is responsible for major economic losses to the swine industry. Many host-adapted bacterial pathogens encode systems known as phasevarions (phase-variable regulons). Phasevarions result from variable expression of cytoplasmic DNA methyltransferases. Variable expression results in genome-wide methylation differences within a bacterial population, leading to altered expression of multiple genes via epigenetic mechanisms. Our examination of a diverse population of A. pleuropneumoniae strains determined that Type I and Type III DNA methyltransferases with the hallmarks of phase variation were present in this species. We demonstrate that phase variation is occurring in these methyltransferases, and show associations between particular Type III methyltransferase alleles and serovar. Using Pacific BioSciences Single-Molecule, Real-Time (SMRT) sequencing and Oxford Nanopore sequencing, we demonstrate the presence of the first ever characterised phase-variable, cytosine-specific Type III DNA methyltransferase. Phase variation of distinct Type III DNA methyltransferase in A. pleuropneumoniae results in the regulation of distinct phasevarions, and in multiple phenotypic differences relevant to pathobiology. Our characterisation of these newly described phasevarions in A. pleuropneumoniae will aid in the selection of stably expressed antigens, and direct and inform development of a rationally designed subunit vaccine against this major veterinary pathogen.

Development of a novel glycoengineering platform for the rapid production of conjugate vaccines.

Conjugate vaccines produced either by chemical or biologically conjugation have been demonstrated to be safe and efficacious in protection against several deadly bacterial diseases. However, conjugate vaccine assembly and production have several shortcomings which hinders their wider availability. Here, we developed a tool, Mobile-element Assisted Glycoconjugation by Insertion on Chromosome, MAGIC, a novel biotechnological platform that overcomes the limitations of the current conjugate vaccine design method(s). As a model, we focused our design on a leading bioconjugation method using N-oligosaccharyltransferase (OTase), PglB. The installation of MAGIC led to at least twofold increase in glycoconjugate yield via MAGIC when compared to conventional N-OTase based bioconjugation method(s). Then, we improved MAGIC to (a) allow rapid installation of glycoengineering component(s), (b) omit the usage of antibiotics, (c) reduce the dependence on protein induction agents. Furthermore, we show the modularity of the MAGIC platform in performing glycoengineering in bacterial species that are less genetically tractable than the commonly used Escherichia coli. The MAGIC system promises a rapid, robust and versatile method to develop vaccines against serious bacterial pathogens. We anticipate the utility of the MAGIC platform could enhance vaccines production due to its compatibility with virtually any bioconjugation method, thus expanding vaccine biopreparedness toolbox.

Long-chain LPS-based enzyme-linked immunosorbent assay to detect swine herds infected by Actinobacillus pleuropneumoniae serotype 17.

Actinobacillus pleuropneumoniae serotype 17, one of the two most recent serotypes described, has been isolated from diseased pigs in North America. Yet, no serological test for surveillance has been developed so far. An enzyme-linked immunosorbent assay (ELISA) using the long-chain lipopolysaccharide antigen (LC-LPS) of this serotype is described. As predicted by previous genetic data on the O-antigen locus, cross reactions were observed between this serotype and serotypes 3, 6, 8, and 15. Although animals infected by serotype 17 would be detected using the current serotype 3 LC-LPS ELISA, better results may be obtained when plates are coated with the antigen purified from the homologous serotype.

Développement d'un test sérologique ELISA en utilisant l'antigène de la chaine O du LPS afin de détecter les troupeaux infectés par Actinobacillus pleuropneumoniae sérotype 17. Le récemment décrit Actinobacillus pleuropneumoniae sérotype 17 a été isolé chez des porcs malades en Amérique du Nord. Pourtant, aucun test sérologique n'a été mis au point à ce jour. Nous avons développé un test ELISA utilisant l'antigène lipopolysaccharidique à longue chaîne (LC-LPS) de ce sérotype. Tel que prévu par l'analyse du locus de l'antigène O du LPS, des réactions croisées ont été observées entre ce sérotype et les sérotypes 3, 6, 8 et 15. Bien que les animaux infectés par le sérotype 17 pourraient être détectés en utilisant le sérotype 3 LC-LPS ELISA, plus de sensibilité pourrait être obtenue en utilisant l'antigène purifié à partir du sérotype homologue.(Traduit par les auteurs).

A computational strategy for the search of regulatory small RNAs in Actinobacillus pleuropneumoniae.

Bacterial regulatory small RNAs (sRNAs) play important roles in gene regulation and are frequently connected to the expression of virulence factors in diverse bacteria. Only a few sRNAs have been described for Pasteurellaceae pathogens and no in-depth analysis of sRNAs has been described for Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, responsible for considerable losses in the swine industry. To search for sRNAs in A. pleuropneumoniae, we developed a strategy for the computational analysis of the bacterial genome by using four algorithms with different approaches, followed by experimental validation. The coding strand and expression of 17 out of 23 RNA candidates were confirmed by Northern blotting, RT-PCR, and RNA sequencing. Among them, two are likely riboswitches, three are housekeeping regulatory RNAs, two are the widely studied GcvB and 6S sRNAs, and 10 are putative novel trans-acting sRNAs, never before described for any bacteria. The latter group has several potential mRNA targets, many of which are involved with virulence, stress resistance, or metabolism, and connect the sRNAs in a complex gene regulatory network. The sRNAs identified are well conserved among the Pasteurellaceae that are evolutionarily closer to A. pleuropneumoniae and/or share the same host. Our results show that the combination of newly developed computational programs can be successfully utilized for the discovery of novel sRNAs and indicate an intricate system of gene regulation through sRNAs in A. pleuropneumoniae and in other Pasteurellaceae, thus providing clues for novel aspects of virulence that will be explored in further studies.

Characterization of the Actinobacillus pleuropneumoniae SXT-related integrative and conjugative element ICEApl2 and analysis of the encoded FloR protein: hydrophobic residues in transmembrane domains contribute dynamically to florfenicol and chloramphenicol efflux.

OBJECTIVES: To characterize ICEApl2, an SXT-related integrative and conjugative element (ICE) found in a clinical isolate of the porcine pathogen Actinobacillus pleuropneumoniae, and analyse the functional nature of the encoded FloR. METHODS: ICEApl2 was identified in the genome of A. pleuropneumoniae MIDG3553. Functional analysis was done using conjugal transfer experiments. MIDG3553 was tested for susceptibility to the antimicrobials for which resistance genes are present in ICEApl2. Lack of florfenicol/chloramphenicol resistance conferred by the encoded FloR protein was investigated by cloning and site-directed mutagenesis experiments in Escherichia coli. RESULTS: ICEApl2 is 92660 bp and contains 89 genes. Comparative sequence analysis indicated that ICEApl2 is a member of the SXT/R391 ICE family. Conjugation experiments showed that, although ICEApl2 is capable of excision from the chromosome, it is not self-transmissible. ICEApl2 encodes the antimicrobial resistance genes floR, strAB, sul2 and dfrA1, and MIDG3553 is resistant to streptomycin, sulfisoxazole and trimethoprim, but not florfenicol or chloramphenicol. Cloning and site-directed mutagenesis of the floR gene revealed the importance of the nature of the hydrophobic amino acid residues at positions 160 and 228 in FloR for determining resistance to florfenicol and chloramphenicol. CONCLUSIONS: Our results indicate that the nature of hydrophobic residues at positions 160 and 228 of FloR contribute dynamically to specific efflux of florfenicol and chloramphenicol, although some differences in resistance levels may depend on the bacterial host species. This is also, to our knowledge, the first description of an SXT/R391 ICE in A. pleuropneumoniae or any member of the Pasteurellaceae.

A Unique Capsule Locus in the Newly Designated Actinobacillus pleuropneumoniae Serovar 16 and Development of a Diagnostic PCR Assay.

Actinobacillus pleuropneumoniae causes pleuropneumonia, an economically significant lung disease of pigs. Recently, isolates of A. pleuropneumoniae that were serologically distinct from the previously characterized 15 serovars were described, and a proposal was put forward that they comprised a new serovar, serovar 16. Here we used whole-genome sequencing of the proposed serovar 16 reference strain A-85/14 to confirm the presence of a unique capsular polysaccharide biosynthetic locus. For molecular diagnostics, primers were designed from the capsule locus of strain A-85/14, and a PCR was formulated that differentiated serovar 16 isolates from all 15 known serovars and other common respiratory pathogenic/commensal bacteria of pigs. Analysis of the capsule locus of strain A-85/14 combined with the previous serological data show the existence of a sixteenth serovar-designated serovar 16-of A. pleuropneumoniae.

Identification and characterization of serovar-independent immunogens in Actinobacillus pleuropneumoniae.

Despite numerous actions to prevent disease, Actinobacillus pleuropneumoniae (A. pleuropneumoniae) remains a major cause of porcine pleuropneumonia, resulting in economic losses to the swine industry worldwide. In this paper, we describe the utilization of a reverse vaccinology approach for the selection and in vitro testing of serovar-independent A. pleuropneumoniae immunogens. Potential immunogens were identified in the complete genomes of three A. pleuropneumoniae strains belonging to different serovars using the following parameters: predicted outer-membrane subcellular localization; ≤ 1 trans-membrane helices; presence of a signal peptide in the protein sequence; presence in all known A. pleuropneumoniae genomes; homology with other well characterized factors with relevant data regarding immunogenicity/protective potential. Using this approach, we selected the proteins ApfA and VacJ to be expressed and further characterized, both in silico and in vitro. Additionally, we analysed outer membrane vesicles (OMVs) of A. pleuropneumoniae MIDG2331 as potential immunogens, and compared deletions in degS and nlpI for increasing yields of OMVs compared to the parental strain. Our results indicated that ApfA and VacJ are highly conserved proteins, naturally expressed during infection by all A. pleuropneumoniae serovars tested. Furthermore, OMVs, ApfA and VacJ were shown to possess a high immunogenic potential in vitro. These findings favour the immunogen selection protocol used, and suggest that OMVs, along with ApfA and VacJ, could represent effective immunogens for the prevention of A. pleuropneumoniae infections in a serovar-independent manner. This hypothesis is nonetheless predictive in nature, and in vivo testing in a relevant animal model will be necessary to verify its validity.

Galleria mellonella is an effective model to study Actinobacillus pleuropneumoniae infection.

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 10(4) to 10(7) 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.

Identification of dfrA14 in two distinct plasmids conferring trimethoprim resistance in Actinobacillus pleuropneumoniae.

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.

Multiplex PCR assay for unequivocal differentiation of Actinobacillus pleuropneumoniae serovars 1 to 3, 5 to 8, 10, and 12.

An improved multiplex PCR, using redesigned primers targeting the serovar 3 capsule locus, which differentiates serovars 3, 6, and 8 Actinobacillus pleuropneumoniae isolates, is described. The new primers eliminate an aberrant serovar 3-indicative amplicon found in some serovar 6 clinical isolates. Furthermore, we have developed a new multiplex PCR for the detection of serovars 1 to 3, 5 to 8, 10, and 12 along with apxIV, thus extending the utility of this diagnostic PCR to cover a broader range of isolates.

Development of a self-replicating plasmid system for Mycoplasma hyopneumoniae.

Mycoplasma hyopneumoniae is a prevalent swine respiratory pathogen that is a major cause of economic loss to pig producers. Control is achieved by a combination of antimicrobials, vaccination and management practices, but current vaccines offer only partial control and there is a need for improved preventative strategies. A major barrier to advances in understanding the pathogenesis of M. hyopneumoniae and in developing new vaccines is the lack of tools to genetically manipulate the organism. We describe the development and optimisation of the first successful plasmid-based system for the genetic manipulation of M. hyopneumoniae. Our artificial plasmids contain the origin of replication (oriC) of M. hyopneumoniae along with tetM, conferring resistance to tetracycline. With these plasmids, we have successfully transformed M. hyopneumoniae strain 232 by electroporation, generating tetracycline resistant organisms. The persistence of extrachromosomal plasmid and maintenance of plasmid DNA over serial passages shows that these artificial plasmids are capable of self-replication in M. hyopneumoniae. In addition to demonstrating the amenability of M. hyopneumoniae to genetic manipulation and in optimising the conditions necessary for successful transformation, we have used this system to determine the minimum functional oriC of M. hyopneumoniae. In doing so, we have developed a plasmid with a small oriC that is stably maintained over multiple passages that may be useful in generating targeted gene disruptions. In conclusion, we have generated a set of plasmids that will be valuable in studies of M. hyopneumoniae pathogenesis and provide a major step forward in the study of this important swine pathogen.

Transposon mutagenesis in Mycoplasma hyopneumoniae using a novel mariner-based system for generating random mutations.

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.

Identification of novel small RNAs in extracellular vesicles produced by Actinobacillus pleuropneumoniae.

Extracellular vesicle (EV) production by bacteria is an important mechanism for microbial communication and host-pathogen interaction. EVs of some bacterial species have been reported to contain nucleic acids. However, the role of small RNAs (sRNAs) packaged in EVs is poorly understood. Here, we report on the RNA cargo of EVs produced by the pig pathogen Actinobacillus pleuropneumoniae, the causal agent of porcine pleuropneumonia, a disease which causes substantial economic losses to the swine industry worldwide. The EVs produced by aerobically and anaerobically grown bacteria were only slightly different in size and distribution. Total cell and outer membrane protein profiles and lipid composition of A. pleuropneumoniae whole cell extracts and EVs were similar, although EVs contained rough lipopolysaccharide compared to the smooth form in whole cells. Approximately 50% of Galleria mellonella larvae died after the injection of EVs. RNAseq, RT-PCR, protection from nuclease degradation, and database searching identified previously described and 13 novel A. pleuropneumoniae sRNAs in EVs, some of which were enriched compared to whole cell content. We conclude that A. pleuropneumoniae EVs contain sRNAs, including those known to be involved in virulence, and some with homologs in other Pasteurellaceae and/or non-Pasteurellaceae. Further work will establish whether the novel sRNAs in A. pleuropneumoniae EVs play any role in pathogenesis.

Bolaamphiphile Analogues of 12-bis-THA Cl2 Are Potent Antimicrobial Therapeutics with Distinct Mechanisms of Action against Bacterial, Mycobacterial, and Fungal Pathogens.

12-Bis-THA Cl2 [12,12'-(dodecane-1,12-diyl)-bis-(9-amino-1,2,3,4-tetrahydroacridinium) chloride] is a cationic bolalipid adapted from dequalinium chloride (DQC), a bactericidal anti-infective indicated for bacterial vaginosis (BV). Here, we used a structure-activity-relationship study to show that the factors that determine effective killing of bacterial, fungal, and mycobacterial pathogens differ, to generate new analogues with a broader spectrum of activity, and to identify synergistic relationships, most notably with aminoglycosides against Acinetobacter baumannii and Pseudomonas aeruginosa, where the bactericidal killing rate was substantially increased. Like DQC, 12-bis-THA Cl2 and its analogues accumulate within bacteria and fungi. More hydrophobic analogues with larger headgroups show reduced potential for DNA binding but increased and broader spectrum antibacterial activity. In contrast, analogues with less bulky headgroups and stronger DNA binding affinity were more active against Candida spp. Shortening the interconnecting chain, from the most lipophilic twelve-carbon chain to six, improved the selectivity index against Mycobacterium tuberculosis in vitro, but only the longer chain analogue was therapeutic in a Galleria mellonella infection model, with the shorter chain analogue exacerbating the infection. In vivo therapy of Escherichia coli ATCC 25922 and epidemic methicillin-resistant Staphylococcus aureus 15 (EMRSA-15) infections in Galleria mellonella was also achieved with longer-chain analogues, as was therapy for an A. baumannii 17978 burn wound infection with a synergistic combination of bolaamphiphile and gentamicin. The present study shows how this class of bolalipids may be adapted further to enable a wider range of potential applications. IMPORTANCE While we face an acute threat from antibiotic resistant bacteria and a lack of new classes of antibiotic, there are many effective antimicrobials which have limited application due to concerns regarding their toxicity and which could be more useful if such risks are reduced or eliminated. We modified a bolalipid antiseptic used in throat lozenges to see if it could be made more effective against some of the highest-priority bacteria and less toxic. We found that structural modifications that rendered the lipid more toxic against human cells made it less toxic in infection models and we could effectively treat caterpillars infected with either Mycobacterium tuberculosis, methicillin resistant Staphylococcus aureus, or Acinetobacter baumannii. The study provides a rationale for further adaptation toward diversifying the range of indications in which this class of antimicrobial may be used.

JMM Profile: Actinobacillus pleuropneumoniae: a major cause of lung disease in pigs but difficult to control and eradicate.

The Gram-negative bacterium Actinobacillus pleuropneumoniae is the causative agent of pleuropneumonia in pigs, its only known natural host. Typical symptoms of peracute disease include fever, apathy and anorexia, and time from infection to death may only be 6 h. Severe lung lesions result from presence of one or two of the ApxI-III toxins. Control is through good husbandry practice, vaccines and antibiotic use. Culture and presence of the species-specific apxIV gene by PCR confirms diagnosis, and identification of serovar, of which 19 are known, informs on appropriate vaccine use and epidemiology.

Novel DNA Markers for Identification of Actinobacillus pleuropneumoniae.

Actinobacillus pleuropneumoniae causes porcine pleuropneumonia, an important disease in the pig industry. Accurate and sensitive diagnostics such as DNA-based diagnostics are essential for preventing or responding to an outbreak. The specificity of DNA-based diagnostics depends on species-specific markers. Previously, an insertion element was found within an A. pleuropneumoniae-specific gene commonly used for A. pleuropneumoniae detection, prompting the need for additional species-specific markers. Herein, 12 marker candidates highly conserved (99 - 100% identity) among 34 A. pleuropneumoniae genomes (covering 13 serovars) were identified to be A. pleuropneumoniae-specific in silico, as these sequences are distinct from 30 genomes of 13 other Actinobacillus and problematic [Actinobacillus] species and more than 1700 genomes of other bacteria in the Pasteurellaceae family. Five marker candidates are within the apxIVA gene, a known A. pleuropneumoniae-specific gene, validating our in silico marker discovery method. Seven other A. pleuropneumoniae-specific marker candidates within the eamA, nusG, sppA, xerD, ybbN, ycfL, and ychJ genes were validated by polymerase chain reaction (PCR) to be specific to 129 isolates of A. pleuropneumoniae (covering all 19 serovars), but not to four closely related Actinobacillus species, four [Actinobacillus] species, or seven other bacterial species. This is the first study to identify A. pleuropneumoniae-specific markers through genome mining. Seven novel A. pleuropneumoniae-specific DNA markers were identified by a combination of in silico and molecular methods and can serve as additional or alternative targets for A. pleuropneumoniae diagnostics, potentially leading to better control of the disease. IMPORTANCE Species-specific markers are crucial for infectious disease diagnostics. Mutations within a marker sequence can lead to false-negative results, inappropriate treatment, and economic loss. The availability of several species-specific markers is therefore desirable. In this study, 12 DNA markers specific to A. pleuropneumoniae, a pig pathogen, were simultaneously identified. Five marker candidates are within a known A. pleuropneumoniae-specific gene. Seven novel markers can be used as additional targets in DNA-based diagnostics, which in turn can expedite disease diagnosis, assist farm management, and lead to better animal health and food security. The marker discovery strategy outlined herein requires less time, effort, and cost, and results in more markers compared with conventional methods. Identification of species-specific markers of other pathogens and corresponding infectious disease diagnostics are possible, conceivably improving health care and the economy.

Rapid Detection of Actinobacillus pleuropneumoniae From Clinical Samples Using Recombinase Polymerase Amplification.

Actinobacillus pleuropneumoniae (APP) is the causative agent of porcine pleuropneumonia, resulting in high economic impact worldwide. There are currently 19 known serovars of APP, with different ones being predominant in specific geographic regions. Outbreaks of pleuropneumonia, characterized by sudden respiratory difficulties and high mortality, can occur when infected pigs are brought into naïve herds, or by those carrying different serovars. Good biosecurity measures include regular diagnostic testing for surveillance purposes. Current gold standard diagnostic techniques lack sensitivity (bacterial culture), require expensive thermocycling machinery (PCR) and are time consuming (culture and PCR). Here we describe the development of an isothermal point-of-care diagnostic test - utilizing recombinase polymerase amplification (RPA) for the detection of APP, targeting the species-specific apxIVA gene. Our APP-RPA diagnostic test achieved a sensitivity of 10 copies/µL using a strain of APP serovar 8, which is the most prevalent serovar in the UK. Additionally, our APP-RPA assay achieved a clinical sensitivity and specificity of 84.3 and 100%, respectively, across 61 extracted clinical samples obtained from farms located in England and Portugal. Using a small subset (n = 14) of the lung tissue samples, we achieved a clinical sensitivity and specificity of 76.9 and 100%, respectively) using lung imprints made on FTA cards tested directly in the APP-RPA reaction. Our results demonstrate that our APP-RPA assay enables a suitable rapid and sensitive screening tool for this important veterinary pathogen.

Identification of small RNAs associated with RNA chaperone Hfq reveals a new stress response regulator in Actinobacillus pleuropneumoniae.

The RNA chaperone Hfq promotes the association of small RNAs (sRNAs) with cognate mRNAs, controlling the expression of bacterial phenotype. Actinobacillus pleuropneumoniae hfq mutants strains are attenuated for virulence in pigs, impaired in the ability to form biofilms, and more susceptible to stress, but knowledge of the extent of sRNA involvement is limited. Here, using A. pleuropneumoniae strain MIDG2331 (serovar 8), 14 sRNAs were identified by co-immunoprecipitation with Hfq and the expression of eight, identified as trans-acting sRNAs, were confirmed by Northern blotting. We focused on one of these sRNAs, named Rna01, containing a putative promoter for RpoE (stress regulon) recognition. Knockout mutants of rna01 and a double knockout mutant of rna01 and hfq, both had decreased biofilm formation and hemolytic activity, attenuation for virulence in Galleria mellonella, altered stress susceptibility, and an altered outer membrane protein profile. Rna01 affected extracellular vesicle production, size and toxicity in G. mellonella. qRT-PCR analysis of rna01 and putative cognate mRNA targets indicated that Rna01 is associated with the extracytoplasmic stress response. This work increases our understanding of the multilayered and complex nature of the influence of Hfq-dependent sRNAs on the physiology and virulence of A. pleuropneumoniae.

Application of the MISTEACHING(S) disease susceptibility framework to Actinobacillus pleuropneumoniae to identify research gaps: an exemplar of a veterinary pathogen.

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, we show, using Actinobacillus pleuropneumoniae as an exemplar, that the MISTEACHING framework can be applied to a strict veterinary pathogen, enabling the identification of major research gaps, 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, fungi, 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.

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.

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 than among the Norwegian isolates. An increased understanding of the effects of population strategies is helpful in surveillance and control of infectious diseases.