Proteomic and immunoproteomic insights into the exoproteome of Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia.

Porcine pleuropneumonia caused by Actinobacillus pleuropneumoniae affects pig health status and the swine industry worldwide. Despite the extensive number of studies focused on A. pleuropneumoniae infection and vaccine development, a thorough analysis of the A. pleuropneumoniae exoproteome is still missing. Using a complementary approach of quantitative proteomics and immunoproteomics we gained an in-depth insight into the A. pleuropneumoniae serotype 2 exoproteome, which provides the basis for future functional studies. Label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed 593 exoproteins, of which 104 were predicted to be virulence factors. The RTX toxins ApxIIA and ApxIIIA -were found to be the most abundant proteins in the A. pleuropneumoniae serotype 2 exoproteome. Furthermore, the ApxIVA toxin was one of the proteins showing the highest abundance, although ApxIVA is commonly assumed to be expressed exclusively in vivo. Our study revealed several antigens, including proteins with moonlight functions, such as the elongation factor (EF)-Tu, and proteins linked to specific metabolic traits, such as the maltodextrin-binding protein MalE, that warrant future functional characterization and might present potential targets for novel therapeutics and vaccines. Our Ig-classes specific serological proteome analysis (SERPA) approach allowed us to explore the development of the host humoral immune response over the course of the infection. These SERPAs pinpointed proteins that might play a key role in virulence and persistence and showed that the immune response to the different Apx toxins is distinct. For instance, our results indicate that the ApxIIIA toxin has properties of a thymus-independent antigen, which should be studied in more detail.

Comparison of metabolic adaptation and biofilm formation of Actinobacillus pleuropneumoniae field isolates from the upper and lower respiratory tract of swine with respiratory disease.

Most outbreaks of disease due to infection with Actinobacillus (A.) pleuropneumoniae are caused by pigs already pre-colonised in tonsillar tissue, where the pathogen is protected from exposure to antibiotic substances administered for treatment. As it has been shown recently under experimental conditions, A. pleuropneumoniae displays host tissue-specific metabolic adaptation. In this study, pairs of A. pleuropneumoniae field isolates were recovered from lung as well as from tonsillar and nasal tissue from 20 pigs suffering from acute clinical signs of pleuropneumonia and showing characteristic pathological lung alterations. Metabolic adaptation to the porcine lower and upper respiratory tract of 32 A. pleuropneumoniae serotype 2 field isolates was examined using Fourier transform infrared (FTIR) spectroscopy as a high resolution metabolic fingerprinting method. All strains showed metabolic adaptations to organ tissue reflected by hierarchical cluster analysis of FTIR spectra similar to those previously observed under experimental conditions. Notably, differences in antimicrobial resistance patterns and minimal inhibitory concentrations of isolates from different tissues in the same animal, but not in biofilm production capability in a microtiter plate assay were found. Overall, biofilm formation was observed for 71 % of the isolates, confirming that A. pleuropneumoniae field isolates are generally able to form biofilms, although rather in a serotype-specific than in an organ-specific manner. A. pleuropneumoniae serotype 6 isolates formed significantly more biofilm than the other serotypes. Furthermore, biofilm production was negatively correlated to the lung lesion scores and tonsillar isolates tended to be more susceptible to antimicrobial substances with high bioavailability than lung isolates.

Actinobacillus pleuropneumoniae triggers IL-10 expression in tonsils to mediate colonisation and persistence of infection in pigs.

Actinobacillus pleuropneumoniae (APP) persisting in clinically healthy pigs may be the causative agent of sudden outbreaks of severe respiratory disease in swine herds. During the course of acute disease, the pathogen is eliminated from inflamed lung tissue, which is characterized by the expression of pro-inflammatory cytokines and an influx of neutrophils. However, if clearance by the porcine immune system fails, APP may switch to a persistent form. At later stages of infection, the pathogen may reside in tonsillar tissue without being eliminated by the host immune defence. To better understand the host immune response at different stages of infection, expression pattern of cytokines in tonsils and lung were recorded. In contrast to lung tissue, in which APP presence was associated with a pronounced pro-inflammatory character, APP presence in the tonsils elicited an increased IL-10 expression. In both organs of infected animals, a marked reciprocal correlation of the pro-inflammatory IL-17A and the anti-inflammatory IL-10 was found, supporting the idea that both cytokines are produced in highly associated, but reciprocal differentiated cell types, possibly APP-specific Th17 subsets. It appears that a persistent phenotype of APP triggers the anti-inflammatory immune response in tonsillar tissue in an attempt to evade the porcine immune defence.

Host-pathogen interplay at primary infection sites in pigs challenged with Actinobacillus pleuropneumoniae.

BACKGROUND: Actinobacillus (A.) pleuropneumoniae is the causative agent of porcine pleuropneumonia and causes significant losses in the pig industry worldwide. Early host immune response is crucial for further progression of the disease. A. pleuropneumoniae is either rapidly eliminated by the immune system or switches to a long-term persistent form. To gain insight into the host-pathogen interaction during the early stages of infection, pigs were inoculated intratracheally with A. pleuropneumoniae serotype 2 and humanely euthanized eight hours after infection. Gene expression studies of inflammatory cytokines and the acute phase proteins haptoglobin, serum amyloid A and C-reactive protein were carried out by RT-qPCR from the lung, liver, tonsils and salivary gland. In addition, the concentration of cytokines and acute phase proteins were measured by quantitative immunoassays in bronchoalveolar lavage fluid, serum and saliva. In parallel to the analyses of host response, the impact of the host on the bacterial pathogen was assessed on a metabolic level. For the latter, Fourier-Transform Infrared (FTIR-) spectroscopy was employed. RESULTS: Significant cytokine and acute phase protein gene expression was detected in the lung and the salivary gland however this was not observed in the tonsils. In parallel to the analyses of host response, the impact of the host on the bacterial pathogen was assessed on a metabolic level. For the latter investigations, Fourier-Transform Infrared (FTIR-) spectroscopy was employed. The bacteria isolated from the upper and lower respiratory tract showed distinct IR spectral patterns reflecting the organ-specific acute phase response of the host. CONCLUSIONS: In summary, this study implies a metabolic adaptation of A. pleuropneumoniae to the porcine upper respiratory tract already during early infection, which might indicate a first step towards the persistence of A. pleuropneumoniae. Not only in lung, but also in the salivary gland an increased inflammatory gene expression was detectable during the acute stage of infection.

Frequency of Th17 cells correlates with the presence of lung lesions in pigs chronically infected with Actinobacillus pleuropneumoniae.

Porcine contagious pleuropneumonia caused by Actinobacillus pleuropneumoniae (APP) remains one of the major causes of poor growth performance and respiratory disease in pig herds. While the role of antibodies against APP has been intensely studied, the porcine T cell response remains poorly characterized. To address this, pigs were intranasally infected with APP serotype 2 and euthanized during the acute phase [6-10 days post-infection (dpi)] or the chronic phase of APP infection (27-31 dpi). Lymphocytes isolated from blood, tonsils, lung tissue and tracheobronchial lymph nodes were analyzed by intracellular cytokine staining (ICS) for IL-17A, IL-10 and TNF-α production after in vitro stimulation with crude capsular extract (CCE) of the APP inoculation strain. This was combined with cell surface staining for the expression of CD4, CD8α and TCR-γδ. Clinical records, microbiological investigations and pathological findings confirmed the induction of a subclinical APP infection. ICS-assays revealed the presence of APP-CCE specific CD4+CD8αdim IL-17A-producing T cells in blood and lung tissue in most infected animals during the acute and chronic phase of infection and a minor fraction of these cells co-produced TNF-α. APP-CCE specific IL-17A-producing γδ T cells could not be found and APP-CCE specific IL-10-producing CD4+ T cells were present in various organs but only in a few infected animals. The frequency of identified putative Th17 cells (CD4+CD8αdimIL-17A+) in lung and blood correlated positively with lung lesion scores and APP-specific antibody titers during the chronic phase. These results suggest a potential role of Th17 cells in the immune pathogenesis of APP infection.