An Experimental Dermal Oedema Model for Apx Toxins of Actinobacillus pleuropneumoniae.

In-vivo models of Actinobacillus pleuropneumoniae (App) infection in pigs are required for the development of vaccines and investigations of pathogenicity. Existing models cause severe respiratory disease with pulmonary oedema, dyspnoea and severe thoracic pain, and careful monitoring and early intervention with euthanasia is, therefore, needed to avoid unnecessary suffering in experimental animals. As a potential replacement for the existing respiratory infection model, an in-vivo protocol was evaluated using intradermal or subcutaneous injection of different App strains and Apx toxins into the abdominal skin of pigs. High concentrations of serovar 1 and serovar 10 App induced diffuse visible dermal oedema and inflammation. Injection of Apx toxins alone did not adequately produce macroscopic lesions, although an influx of inflammatory cells was seen on histopathology. ApxI-producing strains of App induced more inflammation than ApxII- and ApxIII-producing strains. Induction of skin lesions by injection of App or Apx toxins was not sufficiently repeatable or discrete for a robust experimental model that could be used for assessment of novel interventions.

Rationally designed mariner vectors for functional genomic analysis of Actinobacillus pleuropneumoniae and other Pasteurellaceae species by transposon-directed insertion-site sequencing (TraDIS).

Comprehensive identification of conditionally essential genes requires efficient tools for generating high-density transposon libraries that, ideally, can be analysed using next-generation sequencing methods such as Transposon Directed Insertion-site Sequencing (TraDIS). The Himar1 (mariner) transposon is ideal for generating near-saturating mutant libraries, especially in AT-rich chromosomes, as the requirement for integration is a TA dinucleotide, and this transposon has been used for mutagenesis of a wide variety of bacteria. However, plasmids for mariner delivery do not necessarily work well in all bacteria. In particular, there are limited tools for functional genomic analysis of Pasteurellaceae species of major veterinary importance, such as swine and cattle pathogens, Actinobacillus pleuropneumoniae and Pasteurella multocida, respectively. Here, we developed plasmids, pTsodCPC9 and pTlacPC9 (differing only in the promoter driving expression of the transposase gene), that allow delivery of mariner into both these pathogens, but which should also be applicable to a wider range of bacteria. Using the pTlacPC9 vector, we have generated, for the first time, saturating mariner mutant libraries in both A. pleuropneumoniae and P. multocida that showed a near random distribution of insertions around the respective chromosomes as detected by TraDIS. A preliminary screen of 5000 mutants each identified 8 and 14 genes, respectively, that are required for growth under anaerobic conditions. Future high-throughput screening of the generated libraries will facilitate identification of mutants required for growth under different conditions, including in vivo, highlighting key virulence factors and pathways that can be exploited for development of novel therapeutics and vaccines.

Complete genome for Actinobacillus pleuropneumoniae serovar 8 reference strain 405: comparative analysis with draft genomes for different laboratory stock cultures indicates little genetic variation.

We report here the complete genome sequence of the widely studied Actinobacillus pleuropneumoniae serovar 8 reference strain 405, generated using the Pacific Biosciences (PacBio) RS II platform. Furthermore, we compared draft sequences generated by Illumina sequencing of six stocks of this strain, including the same original stock used to generate the PacBio sequence, held in different countries and found little genetic variation, with only three SNPs identified, all within the degS gene. However, sequences of two small plasmids, pARD3079 and p405tetH, detected by Illumina sequencing of the draft genomes were not identified in the PacBio sequence of the reference strain.

Evaluation of the recombinant proteins RlpB and VacJ as a vaccine for protection against Glaesserella parasuis in pigs.

BACKGROUND: Glaesserella parasuis, the causative agent of Glӓsser's disease, is widespread in swine globally resulting in significant economic losses to the swine industry. Prevention of Glӓsser's disease in pigs has been plagued with an inability to design broadly protective vaccines, as many bacterin based platforms generate serovar or strain specific immunity. Subunit vaccines are of interest to provide protective immunity to multiple strains of G. parasuis. Selected proteins for subunit vaccination should be widespread, highly conserved, and surface exposed. RESULTS: Two candidate proteins for subunit vaccination (RlpB and VacJ) against G. parasuis were identified using random mutagenesis and an in vitro organ culture system. Pigs were vaccinated with recombinant RlpB and VacJ, outer membrane proteins with important contributions to cellular function and viability. Though high antibody titers to the recombinant proteins and increased interferon-γ producing cells were found in subunit vaccinated animals, the pigs were not protected from developing systemic disease. CONCLUSIONS: It appears there may be insufficient RlpB and VacJ exposed on the bacterial surface for antibody to bind, preventing high RlpB and VacJ specific antibody titers from protecting animals from G. parasuis. Additionally, this work confirms the importance of utilizing the natural host species when assessing the efficacy of vaccine candidates.

Generation and Evaluation of a Glaesserella (Haemophilus) parasuis Capsular Mutant.

Glaesserella (Haemophilus) parasuis is a commensal bacterium of the upper respiratory tract in pigs and also the causative agent of Glässer's disease, which causes significant morbidity and mortality in pigs worldwide. Isolates are characterized into 15 serovars by their capsular polysaccharide, which has shown a correlation with isolate pathogenicity. To investigate the role the capsule plays in G. parasuis virulence and host interaction, a capsule mutant of the serovar 5 strain HS069 was generated (HS069Δcap) through allelic exchange following natural transformation. HS069Δcap was unable to cause signs of systemic disease during a pig challenge study and had increased sensitivity to complement killing and phagocytosis by alveolar macrophages. Compared with the parent strain, HS069Δcap produced more robust biofilm and adhered equivalently to 3D4/31 cells; however, it was unable to persistently colonize the nasal cavity of inoculated pigs, with all pigs clearing HS069Δcap by 5 days postchallenge. Our results indicate the importance of the capsular polysaccharide to G. parasuis virulence as well as nasal colonization in pigs.

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.

We report here the draft genome sequences of the type strains of Actinobacillus indolicus (46K2C) and Actinobacillus porcinus (NM319). These NAD-dependent bacterial species are frequently found in the upper respiratory tract of pigs and are occasionally associated with lung pathology.

Antimicrobial susceptibility and genetic profile of Mycoplasma hyopneumoniae isolates from Brazil.

Mycoplasma hyopneumoniae is the etiologic agent of porcine enzootic pneumonia, responsible for major production losses worldwide. The bacteria have a limited metabolism and need to obtain molecules from the growth environment, which causes multiple difficulties for in vitro culture. These limitations have a negative influence on the ability to carry out research for the development of the rational use of antimicrobials and vaccines. The objective of this investigation was to evaluate the genetic profile and in vitro susceptibility of field isolates of M. hyopneumoniae to different antimicrobials. All 16 isolates obtained from the samples presented 100% of identity in the partial sequence of 16S rRNA gene when compared to M. hyopneumoniae. A dendrogram was created using the PCR results of the genes related to pathogenicity, and the isolates were distributed into four clusters, suggesting genetic variability among four different isolates circulating on the same farm. The minimum inhibitory concentration of the isolates was higher for the antimicrobials tylosin (< 0.001-16 mg/L) and spiramycin (< 0.001-16 mg/L) than for enrofloxacin (< 0.001-0.125 mg/L) and tiamulin (< 0.001-0.125 mg/L). Our results demonstrate the genetic variability among M. hyopneumoniae isolates from pigs of the same farm, with differences in their susceptibility to antimicrobial agents.

Publisher Correction: Genomic signatures of human and animal disease in the zoonotic pathogen Streptococcus suis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Mycoplasma hyopneumoniae evades phagocytic uptake by porcine alveolar macrophages in vitro.

Mycoplasma hyopneumoniae, the agent of porcine enzootic pneumonia (EP), is able to persist in the lung tissue and evade destruction by the host for several weeks. To understand the mechanism of pathogen survival, phagocytic uptake of M. hyopneumoniae by primary porcine alveolar macrophages was investigated. Intracellular location and survival of the pathogen were explored using gentamicin survival assays, flow cytometry and confocal microscopy of M. hyopneumoniae 232 labelled with green fluorescent protein (GFP). Following 1 h and 16 h of co-incubation, few viable M. hyopneumoniae were recovered from inside macrophages. Flow cytometric analysis of macrophages incubated with M. hyopneumoniae expressing GFP indicated that the mycoplasmas became associated with macrophages, but were shown to be extracellular when actin-dependent phagocytosis was blocked with cytochalasin D. Confocal microscopy detected GFP-labelled M. hyopneumoniae inside macrophages and the numbers increased modestly with time of incubation. Neither the addition of porcine serum complement or convalescent serum from EP-recovered pigs was able to enhance engulfment of M. hyopneumoniae. This investigation suggests that M. hyopneumoniae evades significant uptake by porcine alveolar macrophages and this may be a mechanism of immune escape by M. hyopneumoniae in the porcine respiratory tract.

Pathotyping the Zoonotic Pathogen Streptococcus suis: Novel Genetic Markers To Differentiate Invasive Disease-Associated Isolates from Non-Disease-Associated Isolates from England and Wales.

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.

Differential susceptibility to tetracycline, oxytetracycline and doxycycline of the calf pathogens Mannheimia haemolytica and Pasteurella multocida in three growth media.

For clinical isolates of bovine Mannheimia haemolytica and Pasteurella multocida, this study reports minimum inhibitory concentration (MIC) differences for tetracycline, oxytetracycline and doxycycline between cation-adjusted Mueller-Hinton broth (CAMHB), foetal bovine serum (FBS) and Roswell Park Memorial Institute (RPMI) medium. MICs were determined according to CLSI standards and additionally using five overlapping sets of twofold dilutions. Matrix effect: (a) free drug MICs and minimum bactericidal concentrations (MBC) for all drugs were significantly higher in FBS than in CAMHB for both pathogens (p < 0.001); (b) MICs and MBCs were higher for CAMHB and FBS compared to RPMI for P. multocida only. Net growth rate for P. multocida in CAMHB was significantly slower than in FBS and higher than in RPMI, correlating to MIC and MBC ranking. Drug effect: doxycycline MICs and MBCs were significantly lower (p < 0.001) in both CAMHB and FBS than tetracycline and oxytetracycline for both pathogens. Only for M. haemolytica were oxytetracycline MIC and MBC significantly lower than tetracycline, precluding the use of tetracycline to predict oxytetracycline susceptibility in this species. Determining potencies of tetracyclines in a physiological medium, such as FBS, is proposed, when the objective is correlation with pharmacokinetic data for dosage determination.

Comparative sequence analysis of the capsular polysaccharide loci of Actinobacillus pleuropneumoniae serovars 1-18, and development of two multiplex PCRs for comprehensive capsule typing.

Problems with serological cross-reactivity have led to development of a number of PCRs (individual and multiplex) for molecular typing of Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia. Most of these assays were developed for detection of specific amplicons within capsule biosynthetic genes before the availability of complete sequences for the different serovars. Here we describe comparative analysis of the complete capsular loci for all 18 serovars of A. pleuropneumoniae, and development of two multiplex PCRs for comprehensive capsule typing of this important pig pathogen.

Proposal of serovars 17 and 18 of Actinobacillus pleuropneumoniae based on serological and genotypic analysis.

The aim of this study was to investigate isolates of Actinobacillus pleuropneumoniae previously designated serologically either as non-typable (NT) or as 'K2:07', which did not produce serovar-specific amplicons in PCR assays. We used whole genome sequencing to identify the capsule (CPS) loci of six previously designated biovar 1 NT and two biovar 1 'K2:O7' isolates of A. pleuropneumoniae from Denmark, as well as a recent biovar 2 NT isolate from Canada. All of the NT isolates have the same six-gene type I CPS locus, sharing common cpsABC genes with serovars 2, 3, 6, 7, 8, 9, 11 and 13. The two 'K2:O7' isolates contain a unique three-gene type II CPS locus, having a cpsA gene similar to that of serovars 1, 4, 12, 14 and 15. The previously NT isolates share the same O-antigen genes, found between erpA and rpsU, as serovars 3, 6, 8, and 15. Whereas the 'K2:O7' isolates, have the same O-antigen genes as serovar 7, which likely contributed to their previous mis-identification. All of the NT and 'K2:O7' isolates have only the genes required for production of ApxII (apxIICA structural genes, and apxIBD export genes). Rabbit polyclonal antisera raised against representative isolates with these new CPS loci demonstrated distinct reactivity compared to the 16 known serovars. The serological and genomic results indicate that the isolates constitute new serovars 17 (previously NT) and 18 (previously 'K2:O7'). Primers designed for amplification of specific serovar 17 and 18 sequences for molecular diagnostics will facilitate epidemiological tracking of these two new serovars of A. pleuropneumoniae.

In vivo testing of novel vaccine prototypes against Actinobacillus pleuropneumoniae.

Actinobacillus pleuropneumoniae (A. pleuropneumoniae) is a Gram-negative bacterium that represents the main cause of porcine pleuropneumonia in pigs, causing significant economic losses to the livestock industry worldwide. A. pleuropneumoniae, as the majority of Gram-negative bacteria, excrete vesicles from its outer membrane (OM), accordingly defined as outer membrane vesicles (OMVs). Thanks to their antigenic similarity to the OM, OMVs have emerged as a promising tool in vaccinology. In this study we describe the in vivo testing of several vaccine prototypes for the prevention of infection by all known A. pleuropneumoniae serotypes. Previously identified vaccine candidates, the recombinant proteins ApfA and VacJ, administered individually or in various combinations with the OMVs, were employed as vaccination strategies. Our data show that the addition of the OMVs in the vaccine formulations significantly increased the specific IgG titer against both ApfA and VacJ in the immunized animals, confirming the previously postulated potential of the OMVs as adjuvant. Unfortunately, the antibody response raised did not translate into an effective protection against A. pleuropneumoniae infection, as none of the immunized groups following challenge showed a significantly lower degree of lesions than the controls. Interestingly, quite the opposite was true, as the animals with the highest IgG titers were also the ones bearing the most extensive lesions in their lungs. These results shed new light on A. pleuropneumoniae pathogenicity, suggesting that antibody-mediated cytotoxicity from the host immune response may play a central role in the development of the lesions typically associated with A. pleuropneumoniae infections.

Use of Proteins Identified through a Functional Genomic Screen To Develop a Protein Subunit Vaccine That Provides Significant Protection against Virulent Streptococcus suis in Pigs.

Streptococcus suis is a bacterium that is commonly carried in the respiratory tract and that is also one of the most important invasive pathogens of swine, commonly causing meningitis, arthritis, and septicemia. Due to the existence of many serotypes and a wide range of immune evasion capabilities, efficacious vaccines are not readily available. The selection of S. suis protein candidates for inclusion in a vaccine was accomplished by identifying fitness genes through a functional genomics screen and selecting conserved predicted surface-associated proteins. Five candidate proteins were selected for evaluation in a vaccine trial and administered both intranasally and intramuscularly with one of two different adjuvant formulations. Clinical protection was evaluated by subsequent intranasal challenge with virulent S. suis While subunit vaccination with the S. suis proteins induced IgG antibodies to each individual protein and a cellular immune response to the pool of proteins and provided substantial protection from challenge with virulent S. suis, the immune response elicited and the degree of protection were dependent on the parenteral adjuvant given. Subunit vaccination induced IgG reactive against different S. suis serotypes, indicating a potential for cross protection.

Zoonotic potential of Salmonella enterica carried by pet tortoises.

The prevalence of Salmonella in chelonians is not known in the UK and it is not clear whether such Salmonella strains would be pathogenic for human beings. Some strains, such as members of the Arizonae subgroup, may be unable to cause anything more than very mild disease. To determine the carriage of Salmonella in pet tortoises, cloacal swabs were taken for culture. Salmonella enterica Group D was isolated from 5 of the 89 samples. All five were from the same household of seven tortoises. Salmonella isolates were shown by PCR to carry the invA and spiC genes associated with pathogenicity islands 1 and 2. Each isolate carried both genes indicating they had the genetic basis for disease and enterocyte invasion in human beings. The study indicates a low rate of asymptomatic carriage among the general population of pet tortoises. However, it does suggest that those Salmonella strains colonising the tortoise can carry Salmonellapathogenicity island (SPI)-1 and SPI-2 conferring the potential to cause disease in human beings and other animals.

Patterns of antimicrobial resistance in Streptococcus suis isolates from pigs with or without streptococcal disease in England between 2009 and 2014.

Antimicrobial resistance in Streptococcus suis, a global zoonotic pathogen of pigs, has been mostly studied only in diseased animals using surveys that have not evaluated changes over time. We compared patterns of resistance between S. suis isolates from clinical cases of disease (CC) and non-clinical case (NCC) pigs in England, collected over two discrete periods, 2009-2011 and 2013-2014. Minimum inhibitory concentrations (MIC) of 17 antimicrobials (nine classes) were determined on 405 S. suis isolates categorised by sampling period and disease association to assess changes in resistance over time and association with disease. First, isolates were characterized as resistant or susceptible using published clinical breakpoints. Second, epidemiological cut-offs (ECOFF) were derived from MIC values, and isolates classified as wild type (WT) below the ECOFF and non-wild type (NWT) above the ECOFF. Finally, isolate subsets were analysed for shifts in MIC distribution. NCC isolates were more resistant than CC isolates to cephalosporins, penams, pleuromutilins, potentiated sulphonamides and tetracyclines in both study periods. Resistance levels among CC isolates increased in 2013-2014 relative to 2009-2011 for antimicrobials including aminoglycosides, cephalosporins, fluoroquinolones, pleuromutilins, potentiated sulphonamides and tetracyclines. The prevalence of isolates categorised as NWT for five or more classes of antimicrobials was greater among NCC than CC isolates for both time periods, and increased with time. This study used standardised methods to identify significant shifts in antimicrobial resistance phenotypes of S. suis isolated from pigs in England, not only over time but also between isolates from known clinical cases or disease-free pigs.

"Pathotyping" Multiplex PCR Assay for Haemophilus parasuis: a Tool for Prediction of Virulence.

Haemophilus parasuis is a diverse bacterial species that is found in the upper respiratory tracts of pigs and can also cause Glässer's disease and pneumonia. A previous pangenome study of H. parasuis identified 48 genes that were associated with clinical disease. Here, we describe the development of a generalized linear model (termed a pathotyping model) to predict the potential virulence of isolates of H. parasuis based on a subset of 10 genes from the pangenome. A multiplex PCR (mPCR) was constructed based on these genes, the results of which were entered into the pathotyping model to yield a prediction of virulence. This new diagnostic mPCR was tested on 143 field isolates of H. parasuis that had previously been whole-genome sequenced and a further 84 isolates from the United Kingdom from cases of H. parasuis-related disease in pigs collected between 2013 and 2014. The combination of the mPCR and the pathotyping model predicted the virulence of an isolate with 78% accuracy for the original isolate collection and 90% for the additional isolate collection, providing an overall accuracy of 83% (81% sensitivity and 93% specificity) compared with that of the "current standard" of detailed clinical metadata. This new pathotyping assay has the potential to aid surveillance and disease control in addition to serotyping data.

Whole Genome Sequencing for Surveillance of Antimicrobial Resistance in Actinobacillus pleuropneumoniae.

The aim of this study was to evaluate the correlation between antimicrobial resistance (AMR) profiles of 96 clinical isolates of Actinobacillus pleuropneumoniae, an important porcine respiratory pathogen, and the identification of AMR genes in whole genome sequence (wgs) data. Susceptibility of the isolates to nine antimicrobial agents (ampicillin, enrofloxacin, erythromycin, florfenicol, sulfisoxazole, tetracycline, tilmicosin, trimethoprim, and tylosin) was determined by agar dilution susceptibility test. Except for the macrolides tested, elevated MICs were highly correlated to the presence of AMR genes identified in wgs data using ResFinder or BLASTn. Of the isolates tested, 57% were resistant to tetracycline [MIC ≥ 4 mg/L; 94.8% with either tet(B) or tet(H)]; 48% to sulfisoxazole (MIC ≥ 256 mg/L or DD = 6; 100% with sul2), 20% to ampicillin (MIC ≥ 4 mg/L; 100% with blaROB-1), 17% to trimethoprim (MIC ≥ 32 mg/L; 100% with dfrA14), and 6% to enrofloxacin (MIC ≥ 0.25 mg/L; 100% with GyrAS83F). Only 33% of the isolates did not have detectable AMR genes, and were sensitive by MICs for the antimicrobial agents tested. Although 23 isolates had MIC ≥ 32 mg/L for tylosin, all isolates had MIC ≤ 16 mg/L for both erythromycin and tilmicosin, and no macrolide resistance genes or known point mutations were detected. Other than the GyrAS83F mutation, the AMR genes detected were mapped to potential plasmids. In addition to presence on plasmid(s), the tet(B) gene was also found chromosomally either as part of a 56 kb integrative conjugative element (ICEApl1) in 21, or as part of a Tn7 insertion in 15 isolates. Our results indicate that, with the exception of macrolides, wgs data can be used to accurately predict resistance of A. pleuropneumoniae to the tested antimicrobial agents and provides added value for routine surveillance.