Novel DNA Markers for Identification of Actinobacillus pleuropneumoniae

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.

Kill Rate and Evaluation of Ex Vivo PK/PD Integration of Cefquinome Against Actinobacillus pleuropneumoniae

We wished to study the detailed and precise antibacterial activity of cefquinome against Actinobacillus pleuropneumoniae (APP) in vitro and ex vivo. We analyzed the relationships between kill rate and cefquinome concentration in broth and between pharmacokinetic/pharmacodynamic (PK/PD) parameters and antibacterial effect in serum and tissue cage fluid (TCF) of piglets. Cefquinome exhibited time-dependent antibacterial activity against APP according to the kill rate. The maximum kill rate was 0.48 log10 CFU/mL/h at the 0-9-h period in broth. In the ex vivo PK/PD study, the maximum concentration (Cmax), time to reach the maximum concentration (Tmax), terminal half-life (T1/2β), and area under the concentration time curve (AUCinfinity) were 5.65 μg/ml, 0.58 h, 2.24 h, and 18.48 μg·h/ml in serum and 1.13 μg/ml, 2.60 h, 12.22 h, and 20.83 μg·h/ml in TCF, respectively. The values of area under the curve during 24 h/minimum inhibitory concentration (AUC24h/MIC) for bacteriostatic, bactericidal, and bacterial eradication effects were 18.94, 246.8, and 1013.23 h in serum and 4.20, 65.81, and 391.35 h in TCF, respectively. Our findings will provide a valuable basis for optimization of dosage regimens when applying cefquinome to treat APP infection.

Streptococcus pluranimalium 2N12 Exerts an Antagonistic Effect Against the Swine Pathogen Actinobacillus pleuropneumoniae by Producing Hydrogen Peroxide

Actinobacillus pleuropneumoniae is the causal agent of porcine pleuropneumonia, a highly contagious and often deadly respiratory disease that causes major economic losses in the swine industry worldwide. The aim of the present study was to investigate the hydrogen peroxide (H2O2)-dependent antagonistic activity of Streptococcus pluranimalium 2N12 (pig nasal isolate) against A. pleuropneumoniae. A fluorimetric assay showed that S. pluranimalium produces H2O2 dose- and time-dependently. The production of H2O2 increased in the presence of exogenous lactate, suggesting the involvement of lactate oxidase. All 20 strains of A. pleuropneumoniae tested, belonging to 18 different serovars, were susceptible to H2O2, with minimal inhibitory concentrations and minimal bactericidal concentrations ranging from 0.57 to 2.3 mM. H2O2, as well as a culture supernatant of S. pluranimalium, killed planktonic cells of A. pleuropneumoniae. Treating the culture supernatant with catalase abolished its bactericidal property. H2O2 was also active against a pre-formed biofilm-like structure of A. pleuropneumoniae albeit to a lesser extent. A checkerboard assay was used to show that there were antibacterial synergistic interactions between H2O2 and conventional antibiotics, more particularly ceftiofur. Based on our results and within the limitations of this in vitro study, the production of H2O2 by S. pluranimalium could be regarded as a potential protective mechanism of the upper respiratory tract against H2O2-sensitive pathogens such as A. pleuropneumoniae.

Dynamic immune response characteristics of piglets infected with Actinobacillus pleuropneumoniae through omic

Porcine infectious pleuropneumonia is characterized by a high-rate of carriage and mixed infection with other pathogens. The host immune response induced by Actinobacillus pleuropneumoniae (APP) is the basis for elucidating pathogenesis and controlling disease. However, there is currently no comprehensive and dynamic data characterising the host immune response. In this study, piglets were infected with APP and differentially expressed proteins of bronchoalveolar lavage fluid (BALF) and peripheral serum were identified by iTRAQ-LC-MS/MS, and differentially expressed genes of peripheral blood mononuclear cells (PBMC) by RNA-seq. The results of the integrated analysis of serum, BALF and PBMC showed significant metabolism and local immune responses in BALF, the general immune response in PBMC mainly involves cytokines, while that in serum mainly involves biosynthesis, phagosome, and complement and coagulation cascades. Furthermore, immune responses in PBMCs and serum were rapid and maintained compared to the lung where metabolism and cell adhesion activities were enriched. Some innate immunity pathways of the cellular response to ROS, neutrophil mediated immunity, granulocyte activation and leukocyte cell-cell adhesion were identified as central points, connecting multiple signaling pathways to form an integrated large network. At 24 h post-infection, 14 molecules were up regulated in BALF, 10 of which were shared with PBMC, but at 120 h, 20 down-regulated molecules were identified in BALF, 11 of them still up- regulated in PBMC. We conclude that, the immune response in the lung is different from that in blood, but there is a similarity in response in PBMC and serum.

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.

Identification of FtpA, a Dps-like protein involved in anti-oxidative stress and virulence in Actinobacillus pleuropneumoniae

Bacteria have evolved a variety of enzymes to eliminate endogenous or host-derived oxidative stress factors. The Dps protein, first identified in Escherichia coli , contains a ferroxidase center and protects bacteria from reactive oxygen species damage. There is a lack of knowledge of the role of Dps-like proteins in bacterial pathogenesis. Actinobacillus pleuropneumoniae causes pleuropneumonia, a respiratory disease of swine. The A. pleuropneumoniae ftpA gene is up-regulated during a shift to anaerobiosis, in biofilms and, as found in this study, also by H 2 O 2 . An A. pleuropneumoniae ftpA deletion mutant (△ ftpA ) had increased H 2 O 2 sensitivity, less intracellular viability in macrophages, and decreased virulence in a mouse infection model. Expression of ftpA in an E. coli dps mutant restored wild-type resistance to H 2 O 2 . FtpA possesses a conserved ferritin domain containing a ferroxidase site. Recombinant rFtpA bound and oxidized Fe 2+ reversibly. Under aerobic conditions, compared with the wild-type strain, the viability of an △ ftpA mutant was reduced after extended culture, transition from anaerobic to aerobic conditions, and upon supplementation with Fenton reaction substrates. Under anaerobic conditions, additional H 2 O 2 resulted in a more severe growth defect of △ ftpA than under aerobic conditions. Therefore, by oxidizing and mineralizing Fe 2+ , FtpA alleviates oxidative damage mediated by intracellular Fenton reactions. Furthermore, by mutational analysis, two residues were confirmed to be critical for Fe 2+ binding and oxidization, as well as for A. pleuropneumoniae H 2 O 2 resistance. Taken together, this study demonstrates that A. pleuropneumoniae FtpA is a Dps-like protein, playing critical roles in oxidative stress resistance and virulence. IMPORTANCE As a ferroxidase, Dps of Escherichia coli can protect bacteria from reactive oxygen species damage, but its role in bacterial pathogenesis has received little attention. In this study, FtpA of the swine respiratory pathogen A. pleuropneumoniae was identified as a new Dps-like protein. It facilitated A. pleuropneumoniae resistance to H 2 O 2 , survival in macrophages, and infection in vivo . FtpA could bind and oxidize Fe 2+ through two important residues in its ferroxidase site and protected the bacteria from oxidative damage mediated by the intracellular Fenton reaction. These findings provide new insights into the role of the FtpA-based antioxidant system in the pathogenesis of A. pleuropneumoniae , and the conserved Fe 2+ binding ligands in Dps/FtpA provide novel drug target candidates for disease prevention.

Animal-Based Factors Prior to Infection Predict Histological Disease Outcome in Porcine Reproductive and Respiratory Syndrome Virus- and Actinobacillus pleuropneumoniae-Infected Pigs

A large variety of clinical manifestation in individual pigs occurs after infection with pathogens involved in porcine respiratory disease complex (PRDC). Some pigs are less prone to develop respiratory disease symptoms. The variation in clinical impact after infection and the recovery capacity of an individual animal are measures of its resilience. In this paper, we examined which ones of a range of animal-based factors (rectal temperature, body weight, skin lesion scores, behavior, natural antibody serum levels, serum levels of white blood cells, and type of T and granulocyte subsets) when measured prior to infection are related to disease severity. These animal-based factors and the interaction with housing regimen of the piglets (conventional or enriched) were modeled using linear regression to predict disease severity using a dataset acquired from a previous study using a well-established experimental coinfection model of porcine reproductive and respiratory syndrome virus (PRRSV) and Actinobacillus pleuropneumoniae. Both PRRSV and A. pleuropneumoniae are often involved in PRDC. Histological lung lesion score of each animal was used as a measure for PRDC severity after infection. Prior to infection, higher serum levels of lymphocytes (CD3+), naïve T helper (CD3+CD4+CD8−), CD8+ (as well as higher relative levels of CD8+), and memory T helper (CD3+CD4+CD8+) cells and higher relative levels of granulocytes (CD172a) were related to reduced disease severity in both housing systems. Raised serum concentrations of natural IgM antibodies binding to keyhole limpet hemocyanin (KLH) were also related to reduced disease severity after infection. Increased levels of skin lesions at the central body part (after weaning and before infection) were related to increased disease severity in conventional housing systems only. High resisters showed a lower histological lung lesion score, which appeared unrelated to sex. Body temperature, behavior, and growth prior to infections were influenced by housing regimen but could not explain the variation in lung lesion scores after infection. Raised basal lymphocyte counts and lower skin lesion scores are related to reduced disease severity independent of or dependent on housing system, respectively. In conclusion, our study identifies intrinsic animal-based measures using linear regression analysis that predicts resilience to infections in pigs.

Comparison of Protectivity and Safety of Two Vaccines against Actinobacillus pleuropneumoniae in a Field Study

Actinobacillus pleuropneumoniae causing porcine pleuropneumoniae is responsible for lowered productivity and reduction of performance indicators such as daily weight gain and increase of losses in the swine industry worldwide. To control the disease, vaccination is used to reduce clinical signs and production losses. A randomized, blinded field trail was conducted to compare two licensed A. pleuropneumoniae vaccines in 600 finishing pigs in terms of lung lesions, mortality, medication, weight gain and safety, in a farm in northeast Germany. After weaning, pigs were allocated randomly in two groups resulting in group sizes of 300 individuals. Nursery pigs were vaccinated at the age of 7 to 10 weeks either with a A. pleuropneumoniae bacterin, containing ApxI-III toxoids (group 1), or with a subunit purified A. pleuropneumoniae toxoid vaccine (group 2). Blinded lung lesion scoring at slaughter following the Ceva Lung Program methodology revealed a significantly lower proportion of lungs affected with pleurisy in group 1 compared to group 2. Weighing of the animals did not show a significant difference (p = 0.092); however, at the end of finishing animals of group 1 showed a 1.59 kg higher weight (100.40 ± 10.15 kg) compared to animals in group 2 (98.81 kg ± 11.56 kg). Mortality and antimicrobial medication were significantly lower in group 1 compared to group 2 (13 losses and 17 antimicrobial medications in group 2, 4 losses and 1 antimicrobial medications in group 1). Injection site and systemic adverse reactions were recorded on both days of vaccination and did not differ significantly between the groups (p > 0.05). In this study, the efficacy of vaccination with a commercially available A. pleuropneumoniae bacterin containing ApxI-III toxoids was superior to that of a commercially available A. pleuropneumoniae subunit toxoid vaccine in preventing pulmonary lesions associated with A. pleuropneumoniae infection.Grzegorz Woźniakowski