Lung microdialysis and in vivo PK/PD integration of cefquinome against Actinobacillus pleuropneumoniae in a porcine experimental lung infection model.

This study aim to explore the application of microdialysis in pharmacokinetic (PK) and pharmacodynamic (PD) integration of cefquinome against Actinobacillus pleuropneumoniae in a porcine experimental lung infection model. The model was established via intratracheal inoculation where average bacterial counts (CFU) in the lungs of infected pigs reached 6.57 log10 CFU/g after 3 h. The PK profiles of unbound cefquinome in lung dialysates were determined following intramuscular injection of single doses of 0.125, 0.25, 0.5, 1, 2, and 4 mg/kg. Lung dialysate samples were collected using microdialysis at a flow rate of 1.5 µL/min until 24 h. The PD studies were conducted over 24 h based on 10 intermittent dosing regimens and total daily doses ranged from 0.25 to 4 mg/kg and dosage intervals included 12 and 24 h. The lung tissue was collected after 24 h of treatment and homogenized for bacterial counts. The relationships between PK/PD parameters derived from lung dialysates and drug efficacy were analyzed using an inhibitory sigmoid Emax model. The percentage of time the free drug concentration exceeded the minimum inhibitory concentration (%fT > MIC) was the PK/PD index best describing the antimicrobial activity (R2 = 0.96) in the porcine experimental infection model. The %fT > MIC values required to achieve net bacterial stasis, 1, 2 and 3 log10 CFU/g reductions in the lung were 22.45, 28.86, 37.62, and 56.46%, respectively. Cefquinome exhibited time-dependent characteristics against A. pleuropneumoniae in vivo. These results provide valuable insights into the application of microdialysis in PK/PD integration model studies and optima regimen of cefquinome for the treatment of porcine respiratory diseases caused by A. pleuropneumoniae.

Characterization of Actinobacillus pleuropneumoniae biovar 2 isolates reportedly reacted with the serovar 4 antiserum, and development of a multiplex PCR for O-antigen typing.

We have analyzed the capsule (CPS) and the lipopolysaccharide O-Antigen (O-Ag) biosynthesis loci of twelve Spanish field isolates of Actinobacillus pleuropneumoniae biovar 2, eleven of them previously typed serologically as serovar 4 and one non-typable (NT) (Maldonado et al., 2009, 2011). These isolates have the common core genes of the type I CPS locus, sharing >98% identity with those of serovar 2. However, the former possesses the O-Ag locus as serovar 4, and the latter possesses the O-Ag locus as serovar 7. The main difference found between the CPS loci of the 11 isolates and that of serovar 2 reference strain S1536 are two deletions, one of an 8 bp sequence upstream of the coding sequence and one of 111 bp sequence at the 5' end of the cps2G gene. The deletion mutations mentioned lead to a defect in the production of CPS in these isolates, which contributed to their previous mis-identification. In order to complement the serotyping of A. pleuropneumoniae in diagnostics and epidemiology, we have developed a multiplex PCR for the comprehensive O-Ag typing of all A. pleuropneumoniae isolates.

Biological characteristics of mechanosensitive channels MscS and MscL in Actinobacillus pleuropneumoniae.

Actinobacillus pleuropneumoniae is an important respiratory pathogen that can cause porcine contagious pleuropneumonia (PCP), resulting in significant economic losses in swine industry. Microorganisms are subjected to drastic changes in environmental osmolarity. In order to alleviate the drastic rise or fall of osmolarity, cells activate mechanosensitive channels MscL and MscS through tension changes. MscL not only regulates osmotic pressure but also has been reported to secrete protein and uptake aminoglycoside antibiotic. However, MscL and MscS, as the most common mechanosensitive channels, have not been characterized in A. pleuropneumoniae. In this study, the osmotic shock assay showed that MscL increased sodium adaptation by regulating cell length. The results of MIC showed that deletion of mscL decreased the sensitivity of A. pleuropneumoniae to multiple antibiotics, while deletion of mscS rendered A. pleuropneumoniae hypersensitive to penicillin. Biofilm assay demonstrated that MscL contributed the biofilm formation but MscS did not. The results of animal assay showed that MscL and MscS did not affect virulence in vivo. In conclusion, MscL is essential for sodium hyperosmotic tolerance, biofilm formation, and resistance to chloramphenicol, erythromycin, penicillin, and oxacillin. On the other hand, MscS is only involved in oxacillin resistance.IMPORTANCEBacterial resistance to the external environment is a critical function that ensures the normal growth of bacteria. MscL and MscS play crucial roles in responding to changes in both external and internal environments. However, the function of MscL and MscS in Actinobacillus pleuropneumoniae has not yet been reported. Our study shows that MscL plays a significant role in osmotic adaptation, antibiotic resistance, and biofilm formation of A. pleuropneumoniae, while MscS only plays a role in antibiotic resistance. Our findings provide new insights into the functional characteristics of MscL and MscS in A. pleuropneumoniae. MscL and MscS play a role in antibiotic resistance and contribute to the development of antibiotics for A. pleuropneumoniae.

Corrigendum: Animal-based factors prior to infection predict histological disease outcome in porcine reproductive and respiratory syndrome virus- and Actinobacillus pleuropneumoniae-infected pigs.

[This corrects the article DOI: 10.3389/fvets.2021.742877.].

Examination of the Virulence of Actinobacillus pleuropneumoniae Serovar 16 in Pigs.

Different virulence variants of A. pleuropneumoniae are involved in the etiology of porcine pleuropneumonia. The purpose of the present trial was examination of the virulence of the Actinobacillus pleuropneumoniae A-85/14 strain, the type strain of serovar 16, in an animal challenge experiment. Thirty 12-week-old piglets seronegative for A. pleuropneumoniae were allocated into three trial groups each of 10 animals, and they were infected intranasally with 106, 107, or 108 colony forming units (cfu) of the strain, respectively. Clinical signs were recorded twice a day, and the animals were euthanized 6 days after the infection. Typical clinical signs and postmortem lesions of porcine pleuropneumonia were seen in the animals of each trial group; however, they were generally mild, and no significant differences could be seen between the three groups. Even 106 colony forming units of A. pleuropneumoniae A-85/14 strain could induce clinical signs and lesions. Based on these results, the type strain of serovar 16 of A. pleuropneumoniae must be regarded as a typical pathogenic strain of the species.

The cAMP receptor protein gene contributes to growth, stress resistance, and colonization of Actinobacillus pleuropneumoniae.

Porcine infectious pleuropneumonia (PCP) is a severe disease of porcine caused by Actinobacillus pleuropneumoniae (APP). The spread of PCP remains a threat to the porcine farms and has been known to cause severe economic losses. The cAMP receptor protein (CRP) serves as a pivotal player in helping bacteria adapt to shifts in their environment, particularly when facing the challenges posed by bacterial infections. In this study, we investigated the role of CRP in APP. Our results revealed that crp mutant (Δcrp) strains were more sensitive to acidic and osmotic stress resistance and had lower biofilm formation ability than wild-type (WT) strains. Furthermore, the Δcrp strains showed deficiencies in anti-phagocytosis, adhesion, and invasion upon interaction with host cells. Mice infected with the Δcrp strains demonstrated reduced bacterial loads in their lungs compared to those infected with the WT strains. This study reveals the pivotal role of crp gene expression in regulating pleuropneumonia growth, stress resistance, iron utilization, biofilm formation, phagocytosis, adhesion, invasion and colonization. Our discoveries offer novel perspectives on understanding the development and progression of APP infections.

Intranasal B5 promotes mucosal defence against Actinobacillus pleuropneumoniae via ameliorating early immunosuppression.

Actinobacillus pleuropneumoniae (APP) is an important pathogen of the porcine respiratory disease complex, which leads to huge economic losses worldwide. We previously demonstrated that Pichia pastoris-producing bovine neutrophil ß-defensin-5 (B5) could resist the infection by the bovine intracellular pathogen Mycobacterium bovis. In this study, the roles of synthetic B5 in regulating mucosal innate immune response and protecting against extracellular APP infection were further investigated using a mouse model. Results showed that B5 promoted the production of tumour necrosis factor (TNF)-α, interleukin (IL)-1ß, and interferon (IFN)-ß in macrophages as well as dendritic cells (DC) and enhanced DC maturation in vitro. Importantly, intranasal B5 was safe and conferred effective protection against APP via reducing the bacterial load in lungs and alleviating pulmonary inflammatory damage. Furthermore, in the early stage of APP infection, we found that intranasal B5 up-regulated the secretion of TNF-α, IL-1ß, IL-17, and IL-22; enhanced the rapid recruitment of macrophages, neutrophils, and DC; and facilitated the generation of group 3 innate lymphoid cells in lungs. In addition, B5 activated signalling pathways associated with cellular response to IFN-ß and activation of innate immune response in APP-challenged lungs. Collectively, B5 via the intranasal route can effectively ameliorate the immune suppression caused by early APP infection and provide protection against APP. The immunization strategy may be applied to animals or human respiratory bacterial infectious diseases. Our findings highlight the potential importance of B5, enhancing mucosal defence against intracellular bacteria like APP which causes early-phase immune suppression.

Naringin's Alleviation of the Inflammatory Response Caused by Actinobacillus pleuropneumoniae by Downregulating the NF-κB/NLRP3 Signalling Pathway.

Actinobacillus pleuropneumoniae (APP) is responsible for causing Porcine pleuropneumonia (PCP) in pigs. However, using vaccines and antibiotics to prevent and control this disease has become more difficult due to increased bacterial resistance and weak cross-immunity between different APP types. Naringin (NAR), a dihydroflavonoid found in citrus fruit peels, has been recognized as having significant therapeutic effects on inflammatory diseases of the respiratory system. In this study, we investigated the effects of NAR on the inflammatory response caused by APP through both in vivo and in vitro models. The results showed that NAR reduced the number of neutrophils (NEs) in the bronchoalveolar lavage fluid (BALF), and decreased lung injury and the expression of proteins related to the NLRP3 inflammasome after exposure to APP. In addition, NAR inhibited the nuclear translocation of nuclear factor kappa-B (NF-κB) P65 in porcine alveolar macrophage (PAMs), reduced protein expression of NLRP3 and Caspase-1, and reduced the secretion of pro-inflammatory cytokines induced by APP. Furthermore, NAR prevented the assembly of the NLRP3 inflammasome complex by reducing protein interaction between NLRP3, Caspase-1, and ASC. NAR also inhibited the potassium (K+) efflux induced by APP. Overall, these findings suggest that NAR can effectively reduce the lung inflammation caused by APP by inhibiting the over-activated NF-κB/NLRP3 signalling pathway, providing a basis for further exploration of NAR as a potential natural product for preventing and treating APP.

Discovery of a Novel Integrative Conjugative Element ICEAplChn2 Related to SXT/R391 in Actinobacillus pleuropneumoniae.

Objective: The objective of this study was to characterize ICEAplChn2, a novel SXT/R391-related integration and conjugation element (ICE) carrying 19 drug resistance genes, in a clinical isolate of Actinobacillus pleuropneumoniae from swine. Methods: Whole genome sequencing (WGS) of A. pleuropneumoniae CP063424 strain was completed using a combination of third-generation PacBio and second-generation Illumina. The putative ICE was predicted by the online tool ICEfinder. ICEAplChn2 was analyzed by PCR, conjugation experiments, and bioinformatics tools. Results: A. pleuropneumoniae CP063424 strain exhibited high minimum inhibitory concentrations of clindamycin (1,024 mg/L). The WGS data revealed that ICEAplChn2, with a length of 167,870 bp and encoding 151 genes, including multiple antibiotic resistance genes such as erm(42), VanE, LpxC, dfrA1, golS, aadA3, EreA, dfrA32, tetR(C), tet(C), sul2, aph(3)″-lb, aph(6)-l, floR, dfrA, ANT(3″)-IIa, catB11, and VanRE, was found to be related to the SXT/R391 family on the chromosome of A. pleuronipneumoniae CP063424. The circular intermediate of ICEAplChn2 was detected by PCR, but conjugation experiments showed that it was not self-transmissible. Conclusions: To our knowledge, ICEAplChn2 is the longest member with the most resistance genes in the SXT/R391 family. Meanwhile, ATP-binding cassette superfamily was found to be inserted in the ICEAplChn2 and possessed a new insertion region, which is the first description in the SXT/R391 family.

Identification of novel small RNAs in extracellular vesicles produced by Actinobacillus pleuropneumoniae.

Extracellular vesicle (EV) production by bacteria is an important mechanism for microbial communication and host-pathogen interaction. EVs of some bacterial species have been reported to contain nucleic acids. However, the role of small RNAs (sRNAs) packaged in EVs is poorly understood. Here, we report on the RNA cargo of EVs produced by the pig pathogen Actinobacillus pleuropneumoniae, the causal agent of porcine pleuropneumonia, a disease which causes substantial economic losses to the swine industry worldwide. The EVs produced by aerobically and anaerobically grown bacteria were only slightly different in size and distribution. Total cell and outer membrane protein profiles and lipid composition of A. pleuropneumoniae whole cell extracts and EVs were similar, although EVs contained rough lipopolysaccharide compared to the smooth form in whole cells. Approximately 50% of Galleria mellonella larvae died after the injection of EVs. RNAseq, RT-PCR, protection from nuclease degradation, and database searching identified previously described and 13 novel A. pleuropneumoniae sRNAs in EVs, some of which were enriched compared to whole cell content. We conclude that A. pleuropneumoniae EVs contain sRNAs, including those known to be involved in virulence, and some with homologs in other Pasteurellaceae and/or non-Pasteurellaceae. Further work will establish whether the novel sRNAs in A. pleuropneumoniae EVs play any role in pathogenesis.

Brazilian Clinical Strains of Actinobacillus pleuropneumoniae and Pasteurella multocida: Capsular Diversity, Antimicrobial Susceptibility (In Vitro) and Proof of Concept for Prevention of Natural Colonization by Multi-Doses Protocol of Tildipirosin.

One hundred Actinobacillus pleuropneumoniae (App) and sixty Pasteurella multocida subsp. multocida serogroup A (PmA) isolates were recovered from porcine pneumonic lungs collected from eight central or southern states of Brazil between 2014 and 2018 (App) or between 2017 and 2021 (PmA). A. pleuropneumoniae clinical isolates were typed by multiplex PCR and the most prevalent serovars were 8, 7 and 5 (43, 25% and 18%, respectively). In addition, three virulence genes were assessed in P. multocida isolates, all being positive to capA (PmA) and kmt1 genes, all negative to capD and toxA, and most of them (85%) negative to pfhA gene. The susceptibility of both pathogens to tildipirosin was investigated using a broth microdilution assay. The percentage of isolates susceptible to tildipirosin was 95% for App and 73.3% for PmA. The MIC50 values were 0.25 and 1 µg/mL and the MIC90 values were 4 and >64 µg/mL for App and PmA, respectively. Finally, a multiple-dose protocol of tildipirosin was tested in suckling piglets on a farm endemic for both pathogens. Tildipirosin was able to prevent the natural colonization of the tonsils by App and PmA and significantly (p < 0.0001) reduced the burden of Glaesserella parasuis in this tissue. In summary, our results demonstrate that: (i) tildipirosin can be included in the list of antibiotics to control outbreaks of lung disease caused by App regardless of the capsular type, and (ii) in the case of clinical strains of App and PmA that are sensitive to tildipirosin based on susceptibility testing, the use of this antibiotic in eradication programs for A. pleuropneumoniae and P. multocida can be strongly recommended.

Actinobacillus pleuropneumoniae, surface proteins and virulence: a review.

Actinobacillus pleuropneumoniae (App) is a globally distributed Gram-negative bacterium that produces porcine pleuropneumonia. This highly contagious disease produces high morbidity and mortality in the swine industry. However, no effective vaccine exists to prevent it. The infection caused by App provokes characteristic lesions, such as edema, inflammation, hemorrhage, and necrosis, that involve different virulence factors. The colonization and invasion of host surfaces involved structures and proteins such as outer membrane vesicles (OMVs), pili, flagella, adhesins, outer membrane proteins (OMPs), also participates proteases, autotransporters, and lipoproteins. The recent findings on surface structures and proteins described in this review highlight them as potential immunogens for vaccine development.

A multiplex real-time RT-PCR system to simultaneously diagnose 16 pathogens associated with swine respiratory disease.

AIMS: Swine respiratory disease (SRD) is a major disease complex in pigs that causes severe economic losses. SRD is associated with several intrinsic and extrinsic factors such as host health status, viruses, bacteria, and environmental factors. Particularly, it is known that many pathogens are associated with SRD to date, but most of the test to detect those pathogens can be normally investigated only one pathogen while taking time and labor. Therefore, it is desirable to develop rapidly and efficiently detectable methods those pathogens to minimize the damage caused by SRD. METHODS AND RESULTS: We designed a multiplex real-time RT-PCR (RT-qPCR) system to diagnose simultaneously 16 pathogens, including nine viruses and seven bacteria associated with SRD, on the basis of single qPCR and RT-qPCR assays reported in previous studies. Multiplex RT-qPCR system we designed had the same ability to single RT-qPCR without significant differences in detection sensitivity for all target pathogens at minimum to maximum genomic levels. Moreover, the primers and probes used in this system had highly specificity because the sets had not been detected pathogens other than the target and its taxonomically related pathogens. Furthermore, our data demonstrated that this system would be useful to detect a causative pathogen in the diagnosis using oral fluid from healthy pigs and lung tissue from pigs with respiratory disorders collected in the field. CONCLUSIONS: The rapid detection of infected animals from the herd using our system will contribute to infection control and prompt treatment in the field.

Native ApxIIA secreted by Actinobacillus pleuropneumoniae induces apoptosis in porcine alveolar macrophages dependent on concentration and acylation.

Actinobacillus pleuropneumoniae is an important swine respiratory pathogen causing substantial economic losses to the global pig industry. The Apx toxins of A. pleuropneumoniae belong to the RTX toxin family and are major virulence factors. In addition to hemolysis and/or cytotoxicity via pore-forming activity, RTX toxins, such as ApxIA of A. pleuropneumoniae, have been reported to cause other effects on target cells, e.g., apoptosis. A. pleuropneumoniae ApxIIA is expressed by most serotypes and has moderate hemolytic and cytotoxic activities. In this study, porcine alveolar macrophages (3D4/21) were stimulated with different concentrations of purified native ApxIIA from the serotype 7 strain AP76 which only secretes ApxIIA. By observation of nuclear condensation via fluorescent staining and detection of apoptosis and necrosis by flow cytometry, it was found that high and low concentrations of native ApxIIA mainly caused necrosis or apoptosis of 3D4/21 cells, respectively. ApxIIA purified from an AP76 mutant with a deleted acetyltransferase gene (apxIIC) did not induce necrosis nor apoptosis. Western blot analysis using specific antibodies showed that a cleaved caspase 3 and activated capase 9 was detected after treatment of cells with a low concentration of native ApxIIA, while general or specific inhibitors of caspase 3, 8, 9 blocked these effects. ApxIIA-induced apoptosis of macrophages may be a mechanism of A. pleuropneumoniae to escape host immune clearance.

Antimicrobial Resistance of Actinobacillus pleuropneumoniae, Streptococcus suis, and Pasteurella multocida Isolated from Romanian Swine Farms.

Antimicrobial resistance is an important health issue in human and veterinary medicine. The aim of this study was to monitor the antimicrobial resistance of three of the most important bacteria involved in porcine respiratory disease. A total of 465 isolates were tested during the 2017-2022 period for antimicrobial susceptibility for Actinobacillus pleuropneumoniae (n = 137), Streptococcus suis (n = 207), and Pasteurella multocida (n = 121) by disk diffusion method. The results were interpreted by CLSI breakpoints, where available. High rates of susceptibility (from 90 to >99%) were observed for cefquinome, ceftiofur, amoxicillin + clavulanic acid, amoxicillin, penicillin, ampicillin, florfenicol, enrofloxacin, marbofloxacin, and trimethoprim-sulfamethoxazole. A. pleuropneumoniae isolates showed high resistance to streptomycin (77%), gentamycin (45%), tilmicosin (39%) erythromycin (33%), oxytetracycline (19%), and tetracycline (18%). For S. suis, the highest rates of resistance were observed for streptomycin (98%), tetracycline (75%), oxytetracycline (72%), doxycycline (52%), and erythromycin (51%). P. multocida presented a high rate of resistance for streptomycin (63%), tilmicosin (29%), oxytetracycline (13%), and tetracycline (14%). Bacteria isolates maintained high susceptibility against antimicrobial agents usually used against the mainly respiratory tract pathogens of swine. Resistance for streptomycin, tetracycline, oxytetracycline, and tilmicosin was high for all the tested pathogens.

The two-component system CpxAR is required for the high potassium stress survival of Actinobacillus pleuropneumoniae.

Introduction: Actinobacillus pleuropneumoniae is an important respiratory pathogen, which can cause porcine contagious pleuropneumonia and lead to great economic losses to worldwide swine industry. High potassium is an adverse environment for bacteria, which is not conducive to providing turgor pressure for cell growth and division. Two-component system CpxAR is an important regulatory system of bacteria in response to environmental changes, which is involved in a variety of biological activities, such as antibiotic resistance, periplasmic protein folding, peptidoglycan metabolism and so on. Methods: However, little is known about the role of CpxAR in high potassium stress in A. pleuropneumoniae. Here, we showed that CpxAR is critical for cell division of A. pleuropneumoniae under high potassium (K+) stress. Results: qRT-PCR analysis found that CpxAR positively regulated the cell division genes ftsEX. In addition, we also demonstrated that CpxR-P could directly bind the promoter region of the cell division gene ftsE by EMSA. Discussion: In conclusion, our results described a mechanism where CpxAR adjusts A. pleuropneumoniae survival under high-K+ stress by upregulating the expression of the cell division proteins FtsE and FtsX. These findings are the first to directly demonstrate CpxAR-mediated high-K+ tolerance, and to investigate the detailed molecular mechanism.

Optimizing tylosin dosage for co-infection of Actinobacillus pleuropneumoniae and Pasteurella multocida in pigs using pharmacokinetic/pharmacodynamic modeling.

Formulating a therapeutic strategy that can effectively combat concurrent infections of Actinobacillus pleuropneumoniae (A. pleuropneumoniae) and Pasteurella multocida (P. multocida) can be challenging. This study aimed to 1) establish minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), time kill curve, and post-antibiotic effect (PAE) of tylosin against A. pleuropneumoniae and P. multocida pig isolates and employ the MIC data for the development of epidemiological cutoff (ECOFF) values; 2) estimate the pharmacokinetics (PKs) of tylosin following its intramuscular (IM) administration (20 mg/kg) in healthy and infected pigs; and 3) establish a PK-pharmacodynamic (PD) integrated model and predict optimal dosing regimens and PK/PD cutoff values for tylosin in healthy and infected pigs. The MIC of tylosin against both 89 and 363 isolates of A. pleuropneumoniae and P. multocida strains spread widely, ranging from 1 to 256 µg/mL and from 0.5 to 128 µg/mL, respectively. According to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) ECOFFinder analysis ECOFF value (≤64 µg/mL), 97.75% (87 strains) of the A. pleuropnumoniae isolates were wild-type, whereas with the same ECOFF value (≤64 µg/mL), 99.72% (363 strains) of the P. multicoda isolates were considered wild-type to tylosin. Area under the concentration time curve (AUC), T1/2, and Cmax values were significantly greater in healthy pigs than those in infected pigs (13.33 h × µg/mL, 1.99 h, and 5.79 µg/mL vs. 10.46 h × µg/mL, 1.83 h, and 3.59 µg/mL, respectively) (p < 0.05). In healthy pigs, AUC24 h/MIC values for the bacteriostatic activity were 0.98 and 1.10 h; for the bactericidal activity, AUC24 h/MIC values were 1.97 and 1.99 h for A. pleuropneumoniae and P. multocida, respectively. In infected pigs, AUC24 h/MIC values for the bacteriostatic activity were 1.03 and 1.12 h; for bactericidal activity, AUC24 h/MIC values were 2.54 and 2.36 h for A. pleuropneumoniae and P. multocida, respectively. Monte Carlo simulation lead to a 2 µg/mL calculated PK/PD cutoff. Managing co-infections can present challenges, as it often demands the administration of multiple antibiotics to address diverse pathogens. However, using tylosin, which effectively targets both A. pleuropneumoniae and P. multocida in pigs, may enhance the control of bacterial burden. By employing an optimized dosage of 11.94-15.37 mg/kg and 25.17-27.79 mg/kg of tylosin can result in achieving bacteriostatic and bactericidal effects in 90% of co-infected pigs.

High-dimensional analysis reveals an immune atlas and novel neutrophil clusters in the lungs of model animals with Actinobacillus pleuropneumoniae-induced pneumonia.

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.

Histopathological lesions of Actinobacillus pleuropneumoniae serotype 8 in infected pigs.

This study aimed to report, for the first time, histopathological lesions caused by an outbreak of acute Actinobacillus pleuropneumoniae serotype 8 infections in two farms in Cyprus. Lung tissue samples were collected from two different affected farms (a total of eight samples) for bacterial culture, multiplex polymerase chain reaction (PCR)-based serotyping and histopathological evaluation. Severe respiratory clinical signs, vomiting, anorexia, sudden deaths, a morbidity rate of around 25.00% and a mortality rate of over 60.00% in the fattening stage were reported. Macroscopic lesions included acute to subacute fibrotic, hemorrhagic and necrotizing pneumonia with occasionally encapsulated nodule-like abscesses and fibrous pleuritis. Histopathological evaluation revealed fibrous exudate between alveolar spaces and connective tissue, areas of necrosis mixed with alveolar macrophages, lymphocytes, plasma cells and necrotic leukocytes surrounding colonies of cocci. The bronchial and bronchiolar epithelia were degenerated and replaced by eosinophilic cell debris mixed with inflammatory cells. Several arteries and capillaries were clotted and/or infiltrated by inflammatory cells. In conclusion, these A. pleuropneumoniae serotype 8 cases were accompanied by acute illness, death and more pronounced bronchitis and bronchiolitis.

Pulmonary lesions with asteroid bodies in a pig experimentally infected with Actinobacillus pleuropneumoniae serovar 15.

Five pigs experimentally infected with Actinobacillus pleuropneumoniae serovar 15 isolated in our previous study were pathologically examined. One pig died at 2 days post inoculation (dpi) and four pigs were euthanized at 7 dpi. Autopsy revealed fibrinohemorrhagic pleuropneumonia in all pigs. Histopathologically, the lesions were characterized by extensive hemorrhage and necrosis, fibrin deposition, and multifocal abscesses composed of numerous neutrophils including oat cells and numerous Gram-negative bacilli. In one survived pig, asteroid body formation was confirmed in the lung. The bacteria within the abscesses and asteroid bodies were immunohistochemically positive for antiserum raised against A. pleuropneumoniae serovar 15. This is the first report describing porcine pleuropneumonia with asteroid bodies in a pig experimentally infected with A. pleuropneumoniae serovar 15.