Pleuropneumonia In Pigs Research Articles

Rhein kills Actinobacillus pleuropneumoniae, reduces biofilm formation, and effectively treats bacterial lung infections in mice.

Background. Actinobacillus pleuropneumoniae, a member of the Pasteurellaceae family, is known for its highly infectious nature and is the primary causative agent of infectious pleuropneumonia in pigs. This disease poses a considerable threat to the global pig industry and leads to substantial economic losses due to reduced productivity, increased mortality rates, and the need for extensive veterinary care and treatment. Due to the emergence of multi-drug-resistant strains, Chinese herbal medicine is considered one of the best alternatives to antibiotics due to its unique mechanism of action and other properties. As a type of Chinese herbal medicine, Rhein has the advantages of a wide antibacterial spectrum and is less likely to develop drug resistance, which can perfectly solve the limitations of current antibacterial treatments.Methods. The killing effect of Rhein on A. pleuropneumoniae was detected by fluorescence quantification of differential expression changes of key genes, and scanning electron microscopy was used to observe the changes in A. pleuropneumoniae status after Rhein treatment. Establishing a mouse model to observe the treatment of Rhein after A. pleuropneumoniae infection.Results. Here, in this study, we found that Rhein had a good killing effect on A. pleuropneumoniae and that the MIC was 25 µg ml-1. After 3 h of action, Rhein (4×MIC) completely kills A. pleuropneumoniae and Rhein has good stability. In addition, the treatment with Rhein (1×MIC) significantly reduced the formation of bacterial biofilms. Therapeutic evaluation in a murine model showed that Rhein protects mice from A. pleuropneumoniae and relieves lung inflammation. Quantitative RT-PCR (Quantitative reverse transcription polymerase chain reaction is a molecular biology technique that combines both reverse transcription and polymerase chain reaction methods to quantitatively detect the amount of a specific RNA molecule) results showed that Rhein treatment significantly downregulated the expression of the IL-18 (Interleukin refers to a class of cytokines produced by white blood cells), TNF-α, p65 and p38 genes. Along with the downregulation of genes such as IL-18, it means that Rhein has an inhibitory effect on the expression of these genes, thereby reducing the activation of inflammatory cells and the production of inflammatory mediators. This helps reduce inflammation and protects tissue from further damage.Conclusions. This study reports the activity of Rhein against A. pleuropneumoniae and its mechanism, and reveals the ability of Rhein to treat A. pleuropneumoniae infection in mice, laying the foundation for the development of new drugs for bacterial infections.

The dose regimen formulation of doxycycline hydrochloride and florfenicol injection based on ex vivo pharmacokinetic-pharmacodynamic modeling against the Actinobacillus pleuropneumoniae in pigs

Doxycycline hydrochloride and florfenicol combination (DoxHcl&FF) is an effective treatment for respiratory diseases. In the study, our objective was to evaluate the activity of DoxHcl&FF against Actinobacillus pleuropneumoniae (APP) in porcine pulmonary epithelial lining fluid (PELF) and the optimal dosage scheme to avoid the development of resistance. The DoxHcl&FF was administered intramuscularly (IM) at 20 mg/kg, and the PELF was collected at different time points. The minimum inhibitory concentration (MIC) and time-mortality curves were also included in the study. Based on the sigmoid Emax equation and dose equations, the study integrated the in vivo pharmacokinetic data of infected pigs and ex vivo pharmacodynamic data to obtain the area under concentration time curve (AUC0-24h)/MIC values in PELF and achieve bacteriostatic activity, bactericidal activity and the virtual eradication of bacteria. The study showed that the combination of DoxHcl and FF caused no significant changes in PK parameters. The peak concentration (Cmax) of FF in healthy and diseased pigs was 8.87 ± 0.08 μg/mL and 8.67 ± 0.07 μg/mL, the AUC0-24h were 172.75 ± 2.52 h·μg/mL and 180.22 ± 3.13 h·μg/mL, the Cmax of DoxHcl was 7.91 ± 0.09 μg/mL and 7.99 ± 0.05 μg/mL, and the AUC0-24h was 129.96 ± 3.70 h·μg/mL and 169.82 ± 4.38 h·μg/mL. DoxHcl&FF showed strong concentration-dependent tendencies. The bacteriostatic, bactericidal, and elimination activity were calculated as 5.61, 18.83 and 32.68 h, and the doses were 1.37 (bacteriostatic), 4.59 (bactericidal) and 7.99 (elimination) mg/kg. These findings indicated that the calculated recommended dose could assist in achieving more precise administration, increasing the effectiveness of DoxHcl&FF treatment for APP infections.

Preclinical and clinical trials of a new Ukrainian inactivated vaccine against actinobacillary pleuropneumonia in pigs

Preclinical and clinical trials of a new Ukrainian inactivated vaccine against actinobacillary pleuropneumonia in pigs

Comparative Efficacy in Challenge Dose Models of a Toxin Expressing Whole-Cell Vaccine against Eight Serovars of Actinobacillus pleuropneumoniae in Pigs.

Actinobacillus pleuropneumoniae is a major economically significant bacterial respiratory pig pathogen, and whole cell vaccines are used to prevent disease. However, there is little data available on multi-serovar whole cell vaccine protection. Therefore, we determined the protective efficacies of a whole-cell A. pleuropneumoniae serovar 1 and 2 vaccine comprising ApxI-III toxins (C-vaccine, Coglapix®, Ceva, France) against serovars 1, 2, 4, 5, 6, 7, 9/11, and 13. The infection doses used induced disease representative of endemic field conditions, and standard protocols were used for all studies. Protection against homologous serovars 1 and 2 significantly reduced lung lesion scores (LLS) compared to positive controls: p = 0.00007 and p = 0.00124, respectively. The protection against heterologous serovars 4, 5, 6, 7, 9/11, and 13 also significantly reduced LLS: range p = 2.9 × 10-10 to p = 0.00953. As adjudged by the estimated random effect, reproducibility between studies was high. A highly significant serovar-independent reduction of pathological lung lesions by the C-vaccine was found for all the serovars tested (1, 2, 4, 5, 6, 7, 9/11, and 13). We conclude that the C-vaccine gives high serovar-independent protection against disease and is suitable for this use in the field.

The two-component system CpxA/CpxR is critical for full virulence in Actinobacillus pleuropneumoniae.

Actinobacillus pleuropneumoniae, a major bacterial porcine respiratory tract pathogen causing pig pleuropneumonia, has resulted in high economic losses worldwide. The mutation of the two-component system CpxAR strongly impacted the virulence of A. pleuropneumoniae, but the underlying regulatory mechanism remained unclear. Here, we found that CpxAR positively regulated the cpxDCBA gene cluster involved in polysaccharide capsule export. A capsular layer was confirmed in wild-type cells by transmission electron microscopy, whereas cpxAR and cpxD mutants were non-capsulated. The mutants for polysaccharide capsule export gene cpxD exhibited non-capsulated and were strongly impaired in virulence for mice, indicating a major role of CPS export system in virulence. We then demonstrated that CpxR directly regulated the transcription of the CPS export gene cluster cpxDCBA. Taken together, our data suggested that CpxAR is a key modulator of capsule export that facilitates A. pleuropneumoniae survival in the host.

Outer Membrane Vesicles of Actinobacillus pleuropneumoniae Exert Immunomodulatory Effects on Porcine Alveolar Macrophages.

ABSTRACTOuter membrane vesicles (OMVs) are spontaneously released by Gram-negative bacteria, including Actinobacillus pleuropneumoniae, which causes contagious pleuropneumonia in pigs and leads to considerable economic losses in the swine industry worldwide. A. pleuropneumoniae OMVs have previously been demonstrated to contain Apx toxins and proteases, as well as antigenic proteins. Nevertheless, comprehensive characterizations of their contents and interactions with host immune cells have not been made. Understanding the protein compositions and immunomodulating ability of A. pleuropneumoniae OMVs could help illuminate their biological functions and facilitate the development of OMV-based applications. In the current investigation, we comprehensively characterized the proteome of native A. pleuropneumoniae OMVs. Moreover, we qualitatively and quantitatively compared the OMV proteomes of a wild-type strain and three mutant strains, in which relevant genes were disrupted to increase OMV production and/or produce OMVs devoid of superantigen PalA. Furthermore, the interaction between A. pleuropneumoniae OMVs and porcine alveolar macrophages was also characterized. Our results indicate that native OMVs spontaneously released by A. pleuropneumoniae MIDG2331 appeared to dampen the innate immune responses by porcine alveolar macrophages stimulated by either inactivated or live parent cells. The findings suggest that OMVs may play a role in manipulating the porcine defense during the initial phases of the A. pleuropneumoniae infection.IMPORTANCE Owing to their built-in adjuvanticity and antigenicity, bacterial outer membrane vesicles (OMVs) are gaining increasing attention as potential vaccines for both human and animal use. OMVs released by Actinobacillus pleuropneumoniae, an important respiratory pathogen in pigs, have also been investigated for vaccine development. Our previous studies have shown that A. pleuropneumoniae secretes OMVs containing multiple immunogenic proteins. However, immunization of pigs with these vesicles was not able to relieve the pig lung lesions induced by the challenge with A. pleuropneumoniae, implying the elusive roles that A. pleuropneumoniae OMVs play in host-pathogen interaction. Here, we showed that A. pleuropneumoniae secretes OMVs whose yield and protein content can be altered by the deletion of the nlpI and palA genes. Furthermore, we demonstrate that A. pleuropneumoniae OMVs dampen the immune responses in porcine alveolar macrophages stimulated by A. pleuropneumoniae cells, suggesting a novel mechanism that A. pleuropneumoniae might use to evade host defense.

Rapid detection of Actinobacillus pleuropneumoniae targeting the apxIVA gene for diagnosis of contagious porcine pleuropneumonia in pigs by polymerase spiral reaction.

Actinobacillus pleuropneumoniae is the primary aetiological agent of contagious porcine pleuropneumonia associated with serious economic impact on pig husbandry worldwide. Diagnosis of the disease by existing techniques including isolation and identification of bacteria followed by serotyping, serological techniques, conventional PCR, real-time PCR and LAMP assays are cumbersome, time-consuming, costly and not suitable for rapid field application. A novel isothermal polymerase chain reaction (PSR) technique is standardized for all the reagents, incubation time and incubation temperature against A. pleuropneumoniae. The sensitivity of the assay was determined against various dilutions of purified DNA and total bacterial count. The specificity of the assay was determined against 11 closely related bacterial isolates. The relative sensitivity and specificity were compared with bacterial isolation, conventional PCR and real-time PCR assays. The PSR assay for specific detection was standardized at 64°C for 30 min of incubation in a water bath. The result was visible by the naked eye after centrifugation of the reaction mixture or after incorporation of SYBR Green dye as yellowish-green fluorescence. The technique was found to be 100% specific and equally sensitive with real-time PCR and 10 times more sensitive than conventional PCR. The PSR assay could be applicable in the detection of the organisms in porcine nasal swabs spiked with A. pleuropneumoniae. This is the first-ever report on the development of PSR for specific detection of A. pleuropneumoniae and can be applied for early diagnosis at the field level.

Revealing Genomic Insights of the Unexplored Porcine Pathogen Actinobacillus pleuropneumoniae Using Whole Genome Sequencing.

ABSTRACTActinobacillus pleuropneumoniae (APP) is the causative agent of pleuropneumonia in pigs, one of the most relevant bacterial respiratory diseases in the swine industry. To date, 19 serotypes have been described based on capsular polysaccharide typing with significant virulence dissimilarities. In this study, 16 APP isolates from Spanish origin were selected to perform antimicrobial susceptibility tests and comparative genomic analysis using whole genome sequencing (WGS). To obtain a more comprehensive worldwide molecular epidemiologic analyses, all APP whole genome assemblies available at the National Center for Biotechnology Information (NCBI) at the time of the study were also included. An in-house in silico PCR approach enabled the correct serotyping of unserotyped or incorrectly serotyped isolates and allowed for the discrimination between serotypes 9 and 11. A pangenome analysis identified the presence or absence of gene clusters to be serotype specific, as well as virulence profile analyses targeting the apx operons. Antimicrobial resistance genes were correlated to the presence of specific plasmids. Altogether, this study provides new insights into the genetic variability within APP serotypes, correlates phenotypic tests with bioinformatic analyses and manifests the benefits of populated databases for a better assessment of diversity and variability of relatively unknown pathogens. Overall, genomic comparative analysis enhances the understanding of transmission and epidemiological patterns of this species and suggests vertical transmission of the pathogen, including the resistance genes, within the Spanish integrated systems.IMPORTANCE Pleuropneumonia is one of the most relevant respiratory infections in the swine industry. Despite Actinobacillus pleuropneumoniae (APP) being one of the most important pathogens in the pig production, this is the first comparative study including all available whole genome sequencing data from NCBI. Moreover, this study also includes 16 APP isolates of Spanish origin with known epidemiological relationships through vertical integrated systems. Genomic comparisons provided a deeper understanding of molecular and epidemiological knowledge between different APP serotypes. Furthermore, determination of resistance and toxin profiles allowed correlation with the presence of mobile genetic elements and specific serotype, respectively.

Isolation of Biotype 1 Serotype 12 and Detection of Actinobacillus pleuropneumoniae from Wild Boars.

Actinobacillus pleuropneumoniae is a major pathogen of swine, which can cause severe pleuropneumonia in pigs, but sometimes the disease can be generalized. Diseases caused by A. pleuropneumoniae are frequent all over the world, resulting in high losses among domestic pigs. However, our knowledge on the occurrence of A. pleuropneumoniae in wild boars and feral pigs is limited. We aimed to examine the carriage of A. pleuropneumoniae by hunted wild boars. The presence of A. pleuropneumoniae was examined in tonsils of 68 hunted wild boars collected at a game processing unit. An in-house designed species-specific PCR test was used to detect the gene of Apx IV toxin, and the samples were inoculated on a modified selective agar. A. pleuropneumoniae was detected in 10 animals (14.7%) by PCR and one A. pleuropneumoniae serotype 12 strain was isolated. The antibiotic resistance pattern of the strain resembled field strains that were isolated from farmed pigs in Hungary. This is the first case for the detection of A. pleuropneumoniae not only using PCR or ELISA, but also its isolation, identification, and serotyping.

In silico capsule locus typing for serovar prediction of Actinobacillus pleuropneumoniae.

Actinobacillus pleuropneumoniae is a causative agent of pleuropneumonia in pigs of all ages. A . pleuropneumoniae is divided into 19 serovars based on capsular polysaccharides (CPSs) and lipopolysaccharides. The serovars of isolates are commonly determined by serological tests and multiplex PCR. This study aimed to develop a genomic approach for in silico A. pleuropneumoniae typing by screening for the presence of the species-specific apxIV gene in whole-genome sequencing (WGS) reads and identifying capsule locus (KL) types in genome assemblies. A database of the A . pleuropneumoniae KL, including CPS synthesis and CPS export genes, was established and optimized for Kaptive. To test the developed genomic approach, WGS reads of 189 A . pleuropneumoniae isolates and those of 66 samples from 14 other bacterial species were analysed. ariba analysis showed that apxIV was detected in all 189 A . pleuropneumoniae samples. These apxIV-positive WGS reads were de novo assembled into genome assemblies and assessed. A total of 105 A . pleuropneumoniae genome assemblies that passed the quality assessment were analysed by Kaptive analysis against the A . pleuropneumoniae KL database. The results showed that 97 assemblies were classified and predicted as 13 serovars, which matched the serovar information obtained from the literature. The six genome assemblies from previously nontypable isolates were typed and predicted as serovars 17 and 18. Notably, one of the two “Actinobacillus porcitonsillarum” samples was apxIV positive, and its genome assembly was typed as KL03 with high identity and predicted as A . pleuropneumoniae serovar 3. Collectively, a genomic approach was established and could accurately determine the KL type of A . pleuropneumoniae isolates using WGS reads. This approach can be used with high-quality genome assemblies for predicting A . pleuropneumoniae serovars and for retrospective analysis.

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.

Comparative genomics of 26 complete circular genomes of 18 different serotypes of Actinobacillus pleuropneumoniae.

Actinobacillus pleuropneumoniae is a Gram-negative, rod-shaped bacterium of the family Pasteurellaceae causing pig pleuropneumonia associated with great economic losses worldwide. Nineteen serotypes with distinctive lipopolysaccharide (LPS) and capsular (CPS) compositions have been described so far, yet complete circular genomes are publicly available only for the reference strains of serotypes 1, 4 and 5b, and for field strains of serotypes 1, 3, 7 and 8. We aimed to complete this picture by sequencing the reference strains of 17 different serotypes with the MinION sequencer (Oxford Nanopore Technologies, ONT) and on an Illumina HiSeq (Illumina) platform. We also included two field isolates of serotypes 2 and 3 that were PacBio- and MinION-sequenced, respectively. Genome assemblies were performed following two different strategies, i.e. PacBio- or ONT-only de novo assemblies polished with Illumina reads or a hybrid assembly by directly combining ONT and Illumina reads. Both methods proved successful in obtaining accurate circular genomes with comparable qualities. blast-based genome comparisons and core-genome phylogeny based on core genes, SNP typing and multi-locus sequence typing (cgMLST) of the 26 circular genomes indicated well-conserved genomes across the 18 different serotypes, differing mainly in phage insertions, and CPS, LPS and RTX-toxin clusters, which, consistently, encode serotype-specific antigens. We also identified small antibiotic resistance plasmids, and complete subtype I-F and subtype II-C CRISPR-Cas systems. Of note, highly similar clusters encoding all those serotype-specific traits were also found in other pathogenic and commensal Actinobacillus species. Taken together with the presence of transposable elements surrounding these loci, we speculate a dynamic intra- and interspecies exchange of such virulence-related factors by horizontal gene transfer. In conclusion, our comprehensive genomics analysis provides useful information for diagnostic test and vaccine development, but also for whole-genome-based epidemiological studies, as well as for the surveillance of the evolution of antibiotic resistance and virulence genes in A. pleuropneumoniae .

Optimal regimens based on PK/PD cutoff evaluation of ceftiofur against Actinobacillus pleuropneumoniae in swine

BackgroundActinobacillus pleuropneumoniae formerly known as Haemophilus pleuropneumoniae, can cause pleuropneumoniae in pigs, which lead to significant mortality. Ceftiofur was the first cephalosporin antibiotic used in animals, which was effective against gram-negative and gram-positive bacterium. This study aimed to formulate a rational dosage strategy and review the preceding recommended dosage based on PK/PD modeling and Establish Clinical breakpoint of ceftiofur against Actinobacillus pleuropneumoniae based on the pharmacodynamic-pharmacokinetic cutoff.ResultsThe epidemiologic cutoff value was 0.125 μg/mL. The results of the pharmacodynamic study showed that the MICs of BW39 were 0.5 μg/mL and 1 μg/mL in vitro and ex-vivo, respectively. The minimal bactericidal concentrations (MBCs) under in vitro and ex vivo conditions were both 1 μg/mL. The time-killing profiles of ceftiofur against BW39 were time-dependent with a partly concentration-dependent pattern. Based on the inhibitory sigmoid Emax model, the AUC24 h/MIC values for the bacteriostatic, bactericidal, and elimination effects in serum were 45.73, 63.83, and 69.04 h for healthy pigs separately. According to the Monte Carlo simulation, the COPD was calculated as 2 μg/mL, and the optimized dosage regimen of ceftiofur against Actinobacillus pleuropneumoniae to achieve bacteriostatic, bactericidal, and elimination effects over 24 h was 2.13, 2.97, and 3.42 mg/kg for the 50% target attainment rate (TAR) and 2.47, 3.21, and 3.70 mg/kg for the 90% TAR respectively.ConclusionsIn conclusion, we reveal the EOFF and PK/PD cutoff values of ceftiofur against A. pleuropneumoniae in piglets. However, with the paucity of clinical data for ceftiofur to establish a clinical cutoff against A. pleuropneumoniae, the PK/PD cutoff value of 2 μg/mL will be recommended as surrogate. According to the PK/PD data and the MIC distribution in China, the single bactericidal dose was 3.21 mg/kg for the 90% target, which would be more able to cure Actinobacillus pleuropneumoniae and avoid the emergence of resistance for clinical ceftiofur use in piglet.

ANTIBIOTIC RESISTANCE OF ACTINOBACILLUS PLEUROPNEUMONIAE IN SWINE: PROBLEMS AND SOLUTIONS

Porcine pleuropneumonia is an infectious contagious disease caused by bacteria Actinobacillus pleuropneumoniae. Currently, the disease is widespread in many countries with well-developed pig production. The disease causes significant economic damage to farms due to the large mortality and expenses for treatment of diseased pigs and implementation of veterinary and sanitary measures. Due to increased number of Actinobacillus pleuropneumoniae cases in pigs, and the emergence of actinobacillus-resistant forms, it is necessary to perform a more thorough study and discussion of this problem. The disease epidemic surveillance is based on continuous monitoring aimed at porcine Actinobacillus pleuropneumoniae identification, confirmation and registration, determination of its characteristics and trends in development of sensitivity to antimicrobial preparations. The article addresses the topic of antibiotic use and the antibiotic resistance of microorganisms, which is actual not only for veterinary medicine but also for medicine. The model of swine Actinobacillus pleuropneumoniae was used to study the reasons of antibiotic resistance. Possible approaches to overcoming the resistance of actinobacilli to antibiotics have been discussed. The prospects for the use of antibiotics were discussed in detail to cope with this problem. Targeted surveillance, aimed at monitoring and collecting information on the prescription of antibiotics is of great importance for the solution of the problem of antibiotic resistance. The information obtained from the monitoring can be used for development of the plan and strategy for the use of antibacterial preparations (preparation selection, dose, route of administration, frequency, number of courses), development and implementation of more effective approaches to the treatment of Actinobacillus pleuropneumoniae in pigs, control of the antibiotic-resistant bacteria occurrence and spread.

Comparative Genomics of the First and Complete Genome of "Actinobacillus porcitonsillarum" Supports the Novel Species Hypothesis.

“Actinobacillus porcitonsillarum” is considered a nonpathogenic member of the Pasteurellaceae family, which phenotypically resembles the pathogen Actinobacillus pleuropneumoniae. Previous studies suggested that “A. porcitonsillarum” may represent a new species closely related to Actinobacillus minor, yet no full genome has been sequenced so far. We implemented the Oxford Nanopore and Illumina sequencing technologies to obtain the highly accurate and complete genome sequence of the “A. porcitonsillarum” strain 9953L55. After validating our de novo assembly strategy by comparing the A. pleuropneumoniae S4074T genome sequence obtained by Oxford Nanopore Technology combined with Illumina reads with a PacBio-sequenced S4074T genome from the NCBI database, we performed comparative analyses of the 9953L55 genome with the A. minor type strain NM305T, A. minor strain 202, and A. pleuropneumoniae S4074T. The 2,263,191 bp circular genome of 9953L55 consisted of 2168 and 2033 predicted genes and proteins, respectively. The lipopolysaccharide cluster resembled the genetic organization of A. pleuropneumoniae serotypes 1, 9, and 11, possibly explaining the positive reactions observed previously in serotyping tests. In contrast to NM305T, we confirmed the presence of a complete apxIICABD operon in 9953L55 and 202 accounting for their hemolytic phenotype and Christie-Atkins-Munch-Petersen (CAMP) reaction positivity. Orthologous gene cluster analysis provided insight into the differential ability of strains of the A. minor/“porcitonsillarum” complex and A. pleuropneumoniae to ferment lactose, raffinose, trehalose, and mannitol. The four strains showed distinct and shared transposable elements, CRISPR/Cas systems, and integrated prophages. Genome comparisons based on average nucleotide identity and in silico DNA-DNA hybridization confirmed the close relationship among strains belonging to the A. minor/“porcitonsillarum” complex compared to other Actinobacillus spp., but also suggested that 9953L55 and 202 belong to the same novel species closely related to A. minor, namely, “A. porcitonsillarum.” Recognition of the taxon as a separate species would improve diagnostics and control strategies of pig pleuropneumonia.

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.

Involvement of NF-κB in regulation of Actinobacillus pleuropneumoniae exotoxin ApxI-induced proinflammatory cytokine production in porcine alveolar macrophages

Actinobacillus pleuropneumoniae is a crucial respiratory pathogen that causes fibrinous, hemorrhagic, necrotizing pleuropneumonia in pigs. A. pleuropneumoniae exotoxins (ApxI to IV) are the major virulence factors contributing to A. pleuropneumoniae pathogenesis. Previously, we demonstrated that ApxI induces the expression of proinflammatory cytokines in porcine alveolar macrophages (PAMs) via the mitogen-activated protein kinases (MAPKs) p38 and cJun NH2-terminal kinase (JNK). Nonetheless, the role of nuclear factor (NF)-κB—a transcription factor widely implicated in immune and inflammatory responses—in ApxI-elicited cytokine production has yet to be defined. In the present study, we examined the involvement of NF-κB in ApxI-elicited production of interleukin (IL)-1β, IL-8, and tumor necrosis factor (TNF)-α in PAMs and investigated the correlation between NF-κB and MAPK (p38 and JNK) pathways in this event. The results of Western blot analysis, confocal microscopy, and a DNA binding activity assay revealed that the classical NF-κB pathway was activated by ApxI, as evidenced by the decreased levels of IκB and subsequent NF-κB translocation and activation in ApxI-stimulated PAMs. Moreover, the blocking of ApxI-induced NF-κB activation significantly attenuated the levels of mRNA and protein secretion of IL-1β, IL-8, and TNF-α in PAMs. Notably, the attenuation of JNK activation by a specific inhibitor (SP600125) reduced ApxI-induced NF-κB activation, whereas a p38 blocker (SB203580) had no effect on the NF-κB pathway. Further examination revealed that the level of phosphorylation at serine 536 on the NF-κB p65 subunit was dependent on JNK activity. Collectively, this study, for the first time, demonstrates a pivotal role of NF-κB in ApxI-induced IL-1β, IL-8, and TNF-α production; JNK, but not p38, may positively affect the activation of the classical NF-κB pathway.

Enriched Housing Reduces Disease Susceptibility to Co-Infection with Porcine Reproductive and Respiratory Virus (PRRSV) and Actinobacillus pleuropneumoniae (A. pleuropneumoniae) in Young Pigs.

Until today, anti-microbial drugs have been the therapy of choice to combat bacterial diseases. Resistance against antibiotics is of growing concern in man and animals. Stress, caused by demanding environmental conditions, can reduce immune protection in the host, influencing the onset and outcome of infectious diseases. Therefore psychoneuro-immunological intervention may prove to be a successful approach to diminish the impact of diseases and antibiotics use. This study was designed to investigate the effect of social and environmental enrichment on the impact of disease, referred to as “disease susceptibility”, in pigs using a co-infection model of PRRSV and A. pleuropneumoniae. Twenty-eight pigs were raised in four pens under barren conditions and twenty-eight other pigs were raised in four pens under enriched conditions. In the enriched pens a combination of established social and environmental enrichment factors were introduced. Two pens of the barren (BH) and two pens of the enriched housed (EH) pigs were infected with PRRSV followed by A. pleuropneumoniae, the other two pens in each housing treatment served as control groups. We tested if differences in disease susceptibility in terms of pathological and clinical outcome were related to the different housing regimes and if this was reflected in differences in behavioural and immunological states of the animals. Enriched housed pigs showed a faster clearance of viral PRRSV RNA in blood serum (p = 0.014) and histologically 2.8 fold less interstitial pneumonia signs in the lungs (p = 0.014). More barren housed than enriched housed pigs developed lesions in the lungs (OR = 19.2, p = 0.048) and the lesions in the barren housed pigs showed a higher total pathologic tissue damage score (p<0.001) than those in enriched housed pigs. EH pigs showed less stress-related behaviour and differed immunologically and clinically from BH pigs. We conclude that enriched housing management reduces disease susceptibility to co-infection of PRRSV and A. pleuropneumoniae in pigs. Enrichment positively influences behavioural state, immunological response and clinical outcome in pigs.

Concurrent host-pathogen gene expression in the lungs of pigs challenged with Actinobacillus pleuropneumoniae.

BackgroundActinobacillus pleuropneumoniae causes pleuropneumonia in pigs, a disease which is associated with high morbidity and mortality, as well as impaired animal welfare. To obtain in-depth understanding of this infection, the interplay between virulence factors of the pathogen and defense mechanisms of the porcine host needs to be elucidated. However, research has traditionally focused on either bacteriology or immunology; an unbiased picture of the transcriptional responses can be obtained by investigating both organisms in the same biological sample.ResultsHost and pathogen responses in pigs experimentally infected with A. pleuropneumoniae were analyzed by high-throughput RT-qPCR. This approach allowed concurrent analysis of selected genes encoding proteins known or hypothesized to be important in the acute phase of this infection. The expression of 17 bacterial and 31 porcine genes was quantified in lung samples obtained within the first 48 hours of infection. This provided novel insight into the early time course of bacterial genes involved in synthesis of pathogen-associated molecular patterns (lipopolysaccharide, peptidoglycan, lipoprotein) and genes involved in pattern recognition (TLR4, CD14, MD2, LBP, MYD88) in response to A. pleuropneumoniae. Significant up-regulation of proinflammatory cytokines such as IL1B, IL6, and IL8 was observed, correlating with protein levels, infection status and histopathological findings. Host genes encoding proteins involved in iron metabolism, as well as bacterial genes encoding exotoxins, proteins involved in adhesion, and iron acquisition were found to be differentially expressed according to disease progression. By applying laser capture microdissection, porcine expression of selected genes could be confirmed in the immediate surroundings of the invading pathogen.ConclusionsMicrobial pathogenesis is the product of interactions between host and pathogen. Our results demonstrate the applicability of high-throughput RT-qPCR for the elucidation of dual-organism gene expression analysis during infection. We showed differential expression of 12 bacterial and 24 porcine genes during infection and significant correlation of porcine and bacterial gene expression. This is the first study investigating the concurrent transcriptional response of both bacteria and host at the site of infection during porcine respiratory infection.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1557-6) contains supplementary material, which is available to authorized users.

The RNA Chaperone Hfq Promotes Fitness of Actinobacillus pleuropneumoniae during Porcine Pleuropneumonia

Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia, an economically important disease of pigs. The hfq gene in A. pleuropneumoniae, encoding the RNA chaperone and posttranscriptional regulator Hfq, is upregulated during infection of porcine lungs. To investigate the role of this in vivo-induced gene in A. pleuropneumoniae, an hfq mutant strain was constructed. The hfq mutant was defective in biofilm formation on abiotic surfaces. The level of pgaC transcript, encoding the biosynthesis of poly-β-1,6-N-acetylglucosamine (PNAG), a major biofilm matrix component, was lower and PNAG content was 10-fold lower in the hfq mutant than in the wild-type strain. When outer membrane proteins were examined, cysteine synthase, implicated in resistance to oxidative stress and tellurite, was not found at detectable levels in the absence of Hfq. The hfq mutant displayed enhanced sensitivity to superoxide generated by methyl viologen and tellurite. These phenotypes were readily reversed by complementation with the hfq gene expressed from its native promoter. The role of Hfq in the fitness of A. pleuropneumoniae was assessed in a natural host infection model. The hfq mutant failed to colonize porcine lungs and was outcompeted by the wild-type strain (median competitive index of 2 × 10(-5)). Our data demonstrate that the in vivo-induced gene hfq is involved in the regulation of PNAG-dependent biofilm formation, resistance to superoxide stress, and the fitness and virulence of A. pleuropneumoniae in pigs and begin to elucidate the role of an in vivo-induced gene in the pathogenesis of pleuropneumonia.