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Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia, a challenging respiratory disease for the global swine industry. Variations in the serotypes associated with clinical disease have been observed in different regions worldwide. This study aimed to provide an updated epidemiological assessment of A. pleuropneumoniae serotypes in Spain, incorporating bacterial characterization through serotyping and toxinotyping. Serotypes 9/11, 2, 4, 5, 17, and 13 were frequently identified in diseased animals. Furthermore, qPCR of lung samples from an outbreak, even when samples were pooled, emerged as a robust diagnostic tool, enabling the rapid detection of A. pleuropneumoniae and their serotypes without the need for microbiological isolation. This technology also facilitates serotype monitoring of apparently healthy herds through the testing of oral fluids. The study revealed the frequent simultaneous presence of diverse serotypes within a farm. Serotypes 1, 7, 10, 12, 18, and 19 were frequently found in subclinically infected animals but were rarely detected in acute pleuropneumonia outbreaks in the current study. These results provide valuable information for interpreting the potential virulence of the different serotypes in Spain. However, other predisposing factors and the immune status of the herds such as type of vaccines used when appropriate, should be carefully considered before drawing definitive conclusions. Nevertheless, the study offers valuable insights that underscore the necessity for detailed regional data to contribute toward a comprehensive understanding of the disease dynamics and toward formulating effective control measures for porcine pleuropneumonia.

Actinobacillus pleuropneumoniae (APP) is a bacterium frequently associated with porcine pleuropneumonia. The acute form of the disease is highly contagious and often fatal, resulting in significant economic losses for pig farmers. Serotype diversity and antimicrobial resistance (AMR) of APP strains circulating in north Italian farms from 2015 to 2022 were evaluated retrospectively to investigate APP epidemiology in the area. A total of 572 strains isolated from outbreaks occurring in 337 different swine farms were analysed. The majority of isolates belonged to serotypes 9/11 (39.2%) and 2 (28.1%) and serotype diversity increased during the study period, up to nine different serotypes isolated in 2022. The most common resistances were against tetracycline (53% of isolates) and ampicillin (33%), followed by enrofloxacin, florfenicol and trimethoprim/sulfamethoxazole (23% each). Multidrug resistance (MDR) was common, with a third of isolates showing resistance to more than three antimicrobial classes. Resistance to the different classes and MDR varied significantly depending on the serotype. In particular, the widespread serotype 9/11 was strongly associated with florfenicol and enrofloxacin resistance and showed the highest proportion of MDR isolates. Serotype 5, although less common, showed instead a concerning proportion of trimethoprim/sulfamethoxazole resistance. Our results highlight how the typing of circulating serotypes and the analysis of their antimicrobial susceptibility profile are crucial to effectively manage APP infection and improve antimicrobial stewardship.

Contagious caprine pleuropneumonia (CCPP) is a serious disease of goats, occasionally sheep and wild ruminants, caused by Mycoplasma capricolum subspecies capripneumoniae (Mccp). The disease is characterized by severe serofibrinous pleuropneumonia, very high morbidity (∼100%), and mortality (80-100%). CCPP affects goats in more than 40 countries of the world thereby posing a serious threat to goat farming around the globe. The characteristic clinical signs of CCPP are severe respiratory distress associated with sero-mucoid nasal discharge, coughing, dyspnea, pyrexia, pleurodynia, and general malaise. In later stages, severe lobar fibrinous pleuropneumonia, profuse fluid accumulation in pleural cavity, severe congestion of lungs and adhesion formation is observed. Mycoplasmal antigen interactions with host immune system and its role in CCPP pathogenesis are not clearly understood. CCPP is not a zoonotic disease. Diagnosis has overcome cumbersome and lengthy conventional tests involving culture, isolation, and identification by advanced serological (LAT, cELISA) or gene-based amplification of DNA (PCR, RFLP, and hybridization) and sequencing. The latex agglutination test (LAT) is rapid, simple, and better test for field and real-time diagnosis applicable to whole blood or serum and is more sensitive than the CFT and easier than the cELISA. Moreover, the studies on antibiotic sensitivity and exploration of novel antibiotics (fluoroquinolones, macrolides) can help in better therapeutic management besides preventing menace of antibiotic resistance. Re-visiting conventional prophylactic measures focussing on developing novel strain-based or recombinant vaccines using specific antigens (capsular or cellular) should be the most important strategy for controlling the disease worldwide.

Background Actinobacillus pleuropneumoniae is a prevalent respiratory pathogen causing substantial economic losses in swine production worldwide. The bacterium's ability to rapidly develop antimicrobial resistance (AMR) poses a significant challenge to effective treatment and control. In Poland, limited data on A. pleuropneumoniae serotype distribution and AMR patterns hinder evidence-based treatment strategies. This study examined the serotype diversity and AMR patterns of A. pleuropneumoniae isolates from porcine pleuropneumonia outbreaks in northeastern Poland between 2019 and 2024, providing crucial information for regional veterinary practices and antimicrobial stewardship efforts. Results Analysis of 119 isolates from 67 farms demonstrated the predominance of serotype 2 (65.5%), followed by serogroups 3, 6, 8 (18.5%) and 1, 9, 11 (15.1%). This distribution differs from recent trends in other European countries, suggesting regional epidemiological variations. High resistance rates were observed for tylosin (55.5%), gentamicin (36.1%), doxycycline (32.8%), and sulfamethoxazole/trimethoprim (26.1%). Multidrug resistance fluctuated between 14.3% and 21.9% over the study period, with no clear linear trend. From 2022 onwards, strains exhibiting resistance to seven or more antimicrobials, including cephalosporins, emerged, marking a significant shift in resistance profiles. Temporal analysis revealed diverse resistance patterns, with significant increases in some antimicrobials (e.g., sulfamethoxazole/trimethoprim, p  = 0.001) and stability in others (e.g., tetracycline, p  = 0.890). Notably, several antimicrobials, including florfenicol and colistin, maintained 100% efficacy against all isolates throughout the study period. Conclusions The findings highlight the dynamic nature of AMR development in A. pleuropneumoniae and underscore the need for ongoing surveillance in the region. The emergence of highly resistant strains, particularly those resistant to cephalosporins, raises concerns about future treatment options. These results can guide evidence-based treatment strategies and enhance antimicrobial stewardship efforts in regional swine production. Furthermore, the study emphasizes the importance of local AMR data in guiding antimicrobial use policies and the need for a coordinated approach to combat AMR in veterinary medicine.

Actinobacillus pleuropneumoniae is the primary etiological agent of porcine contagious pleuropneumonia, a devastating respiratory disease that causes substantial economic losses to the global swine industry. The emergence of multidrug-resistant strains with enhanced virulence poses increasing challenges to disease control and necessitates comprehensive genomic characterization to inform targeted intervention strategies. This study aimed to characterize the genomic features, antimicrobial resistance profile, and pathogenic potential of a novel A. pleuropneumoniae strain isolated from a severe outbreak in China, with particular focus on identifying unique resistance mechanisms and virulence determinants. We isolated strain APPFJLYC01 from lung and bronchial tissues of pigs during a severe pleuropneumonia outbreak in Fujian Province, China (incidence rate 30%, mortality rate 56%). Complete genome sequencing was performed using combined PacBio Sequel II and DNBSEQ platforms, followed by comprehensive bioinformatics analysis including virulence factor identification, antibiotic resistance gene profiling, phylogenetic analysis, and comparative genomics. Pathogenicity was evaluated through experimental infection of 3-week-old piglets with subsequent clinical, pathological, and histopathological examinations. The genome of APPFJLYC01 strain is 2,308,741 bp in size, encoding 2,149 genes. Notably, it contains 190 virulence factor homologs and 10 resistance genes. Phylogenetic analysis based on CorePan revealed that APPFJLYC01 shares a close evolutionary relationship with strain JL03, potentially due to their shared geographical origin in China. Pathogenicity evaluation in piglets confirms its high virulence. These findings highlight APPFJLYC01 as a multidrug-resistant and highly virulent strain, providing insights for controlling porcine pleuropneumonia.

Due to the increase in bacterial resistance, improving the anti-infectious immunity of the host is rapidly becoming a new strategy for the prevention and treatment of bacterial pneumonia. However, the specific lung immune responses and key immune cell subsets involved in bacterial infection are obscure. Actinobacillus pleuropneumoniae (APP) can cause porcine pleuropneumonia, a highly contagious respiratory disease that has caused severe economic losses in the swine industry. Here, using high-dimensional mass cytometry, the major immune cell repertoire in the lungs of mice with APP infection was profiled. Various phenotypically distinct neutrophil subsets and Ly-6C + inflammatory monocytes/macrophages accumulated post-infection. Moreover, a linear differentiation trajectory from inactivated to activated to apoptotic neutrophils corresponded with the stages of uninfected, onset, and recovery of APP infection. CD14 + neutrophils, which mainly increased in number during the recovery stage of infection, were revealed to have a stronger ability to produce cytokines, especially IL-10 and IL-21, than their CD14 − counterparts. Importantly, MHC-II + neutrophils with antigen-presenting cell features were identified, and their numbers increased in the lung after APP infection. Similar results were further confirmed in the lungs of piglets infected with APP and Klebsiella pneumoniae infection by using a single-cell RNA-seq technique. Additionally, a correlation analysis between cluster composition and the infection process yielded a dynamic and temporally associated immune landscape where key immune clusters, including previously unrecognized ones, marked various stages of infection. Thus, these results reveal the characteristics of key neutrophil clusters and provide a detailed understanding of the immune response to bacterial pneumonia.

Actinobacillus pleuropneumonia (APP) is the etiological agent of porcine pleuropneumonia, characterized by acute hemorrhagic fibrinous pleuropneumonia and chronic fibrinous necrotizing pleuropneumonia. The acute manifestation of APP is marked by a high fatality rate, leading to substantial economic repercussions to the global swine industry. Current vaccination strategies against APP primarily include bacterin vaccines, subunit vaccines, and live attenuated vaccines. However, their immediate protective efficacy after immunization, particularly for emergency scenarios, remains poorly characterized. In this study, pigs were immunized with APP live attenuated (gene-deleted) vaccine HB04M (serovar 7) via intramuscular or intranasal route at 3 and 7 days prior to challenge with a virulent heterologous serotype strain (serovar 5) at a lethal dose. The findings revealed that pigs receiving intramuscular vaccination 7 days pre-challenge demonstrated a significantly effective immunological defense, achieving a 100% survival rate with minimal lung injury. Intramuscular vaccination 3 days and intranasal vaccination 7 days pre-challenge both exhibited 80% protection, while intranasal vaccination 3 days pre-challenge offered only 60% protection against the challenge. The immediate protection observed 3 days post-immunization was correlated with the rapid vaccine-induced IFN-γ response, while protection at 7 days post-immunization was enhanced by the synergistic effects of HB04M-induced antibodies and IFN-γ. Overall, HB04M demonstrated significant protection against a lethal dose of the heterologous strain as early as 3 days post-immunization, with intramuscular vaccination delivering nearly complete protection by 7 days post-immunization. These findings suggest that HB04M could serve as an effective emergency vaccination strategy during APP outbreaks in pig farms, providing timely protection to mitigate morbidity and mortality.

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 + CD8α dim 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 + CD8α dim IL-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.

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.

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.