ABSTRACT
Actinobacillus pleuropneumoniae is responsible for porcine pleuropneumonia, a highly contagious lung infection. The control of this respiratory disease remains heavily reliant on antibiotics, with phenicols being one of the primary classes of antibiotics used in pig farming. In the present study, we describe three isolates (B2278, B2176 and B2177) of A. pleuropneumoniae resistant to florfenicol attributed to the presence of the floR gene, which were obtained from two pig farms in Italy. Florfenicol susceptibility tests indicated that B2176 exhibited an intermediate susceptibility profile, while B2177 and B2278 were resistant. All three isolates belonged to serovar 6 and tested positive for the presence of the floR gene. Whole genome sequencing analysis revealed that isolates B2176, B2177 and B2278 harbored genes encoding the toxins ApxII and ApxIII, characteristic of strains with moderate virulence. Moreover, phylogenetic analysis demonstrated that these isolates were closely related, with single nucleotide polymorphisms (SNPs) ranging from 8 to 19. The floR gene was located on a novel 5588â¯bp plasmid, designated as pAp-floR. BLASTN analysis showed that the pAp-floR plasmid had high nucleotide identity (99â¯%) and coverage (60â¯%) with the pMVSCS1 plasmid (5621â¯bp) from Mannheimia varigena MVSCS1 of porcine origin. Additionally, at least under laboratory conditions, pAp-floR was stably maintained even in the absence of direct selective pressure, suggesting that it does not impose a fitness cost. Our study underscores the necessity of monitoring the spread of florfenicol-resistant A. pleuropneumoniae isolates in the coming years.
Subject(s)
Actinobacillus Infections , Actinobacillus pleuropneumoniae , Anti-Bacterial Agents , Drug Resistance, Bacterial , Swine Diseases , Thiamphenicol , Animals , Actinobacillus pleuropneumoniae/drug effects , Actinobacillus pleuropneumoniae/genetics , Actinobacillus pleuropneumoniae/isolation & purification , Actinobacillus pleuropneumoniae/classification , Thiamphenicol/analogs & derivatives , Thiamphenicol/pharmacology , Swine , Italy/epidemiology , Swine Diseases/microbiology , Anti-Bacterial Agents/pharmacology , Actinobacillus Infections/microbiology , Actinobacillus Infections/veterinary , Drug Resistance, Bacterial/genetics , Phylogeny , Microbial Sensitivity Tests , Whole Genome Sequencing , Farms , Pleuropneumonia/microbiology , Pleuropneumonia/veterinary , Plasmids/genetics , Bacterial Proteins/genetics , Polymorphism, Single Nucleotide , Virulence/geneticsABSTRACT
A swine production system had 3 sections located a few kilometers apart. Sections A and C contained several thousand sows and nursery and finishing pigs. Section B, located between the other 2 sections, was the smallest and had 6 finishing sites and 2 sow sites. The entire system was infected with porcine reproductive and respiratory syndrome virus, Mycoplasma hyopneumoniae, and Actinobacillus pleuropneumoniae. Section B was depopulated, cleaned, disinfected, and repopulated with negative gilts. Despite extreme measures, recontamination occurred for each pathogen, with aerosol considered the most plausible contamination source.
Transmission suspectée d'agents pathogènes porcins par aérosol : un cas de terrainUn système de production porcine comportait 3 sections situées à quelques kilomètres l'une de l'autre. Les sections A et C contenaient plusieurs milliers de truies et de porcs en maternité et en finition. La section B, située entre les 2 autres sections, était la plus petite et comptait 6 sites de finition et 2 sites de truies. L'ensemble du système était infecté par le virus du syndrome reproducteur et respiratoire porcin, Mycoplasma hyopneumoniae et Actinobacillus pleuropneumoniae. La section B a été dépeuplée, nettoyée, désinfectée et repeuplée de cochettes négatives. Malgré des mesures extrêmes, une recontamination s'est produite pour chaque agent pathogène, les aérosols étant considérés comme la source de contamination la plus plausible.(Traduit par Dr Serge Messier).
Subject(s)
Actinobacillus pleuropneumoniae , Aerosols , Mycoplasma hyopneumoniae , Porcine respiratory and reproductive syndrome virus , Swine Diseases , Animals , Swine , Swine Diseases/transmission , Swine Diseases/microbiology , Swine Diseases/virology , Mycoplasma hyopneumoniae/isolation & purification , Actinobacillus pleuropneumoniae/isolation & purification , Porcine respiratory and reproductive syndrome virus/isolation & purification , Actinobacillus Infections/veterinary , Actinobacillus Infections/transmission , Actinobacillus Infections/microbiology , Pneumonia of Swine, Mycoplasmal/transmission , Female , Porcine Reproductive and Respiratory Syndrome/transmission , Animal HusbandrySubject(s)
Actinobacillus pleuropneumoniae , Serogroup , Streptococcal Infections , Streptococcus suis , Swine Diseases , Animals , Swine Diseases/microbiology , Swine , Quebec/epidemiology , Streptococcus suis/isolation & purification , Streptococcal Infections/veterinary , Streptococcal Infections/microbiology , Actinobacillus pleuropneumoniae/isolation & purification , Actinobacillus pleuropneumoniae/classification , Actinobacillus pleuropneumoniae/genetics , Actinobacillus Infections/veterinary , Actinobacillus Infections/microbiologyABSTRACT
BACKGROUND: Actinobacillus pleuropneumoniae is a serious pathogen in pigs. The abundant application of antibiotics has resulted in the gradual emergence of drugresistant bacteria, which has seriously affected treatment of disease. To aid measures to prevent the emergence and spread of drug-resistant bacteria, herein, the kill rate and mutant selection window (MSW) of danofloxacin (DAN) against A. pleuropneumoniae were evaluated. METHODS: For the kill rate study, the minimum inhibitory concentration (MIC) was tested using the micro dilution broth method and time-killing curves of DAN against A. pleuropneumoniae grown in tryptic soy broth (TSB) at a series drug concentrations (from 0 to 64 MIC) were constructed. The relationships between the kill rate and drug concentrations were analyzed using a Sigmoid Emax model during different time periods. For the MSW study, the MIC99 (the lowest concentration that inhibited the growth of the bacteria by ≥ 99%) and mutant prevention concentration (MPC) of DAN against A. pleuropneumoniae were measured using the agar plate method. Then, a peristaltic pump infection model was established to simulate the dynamic changes of DAN concentrations in pig lungs. The changes in number and sensitivity of A. pleuropneumoniae were measured. The relationships between pharmacokinetic/pharmacodynamic parameters and the antibacterial effect were analyzed using the Sigmoid Emax model. RESULTS: In kill rate study, the MIC of DAN against A. pleuropneumoniae was 0.016 µg/mL. According to the kill rate, DAN exhibited concentration-dependent antibacterial activity against A. pleuropneumoniae. A bactericidal effect was observed when the DAN concentration reached 4-8 MIC. The kill rate increased constantly with the increase in DAN concentration, with a maximum value of 3.23 Log10 colony forming units (CFU)/mL/h during the 0-1 h period. When the drug concentration was in the middle part of the MSW, drugresistant bacteria might be induced. Therefore, the dosage should be avoided to produce a mean value of AUC24h/MIC99 (between 31.29 and 62.59 h. The values of AUC24h/MIC99 to achieve bacteriostatic, bactericidal, and eradication effects were 9.46, 25.14, and > 62.59 h, respectively. CONCLUSION: These kill rate and MSW results will provide valuable guidance for the use of DAN to treat A. pleuropneumoniae infections.
Subject(s)
Actinobacillus Infections , Actinobacillus pleuropneumoniae , Anti-Bacterial Agents , Fluoroquinolones , Microbial Sensitivity Tests , Actinobacillus pleuropneumoniae/drug effects , Actinobacillus pleuropneumoniae/genetics , Anti-Bacterial Agents/pharmacology , Fluoroquinolones/pharmacology , Animals , Actinobacillus Infections/veterinary , Actinobacillus Infections/drug therapy , Swine , Drug Resistance, Bacterial , Swine Diseases/drug therapy , Swine Diseases/microbiology , MutationABSTRACT
Actinobacillus pleuropneumoniae (APP) causes significant economic losses to the swine industry. Antibiotic treatment can be challenging due to its clinical urgency and the turnover of antimicrobial susceptibility results from the diagnostic laboratory. The aim of this study was to evaluate the vertical transmission of APP within integrated systems as a criterion for optimising antimicrobial treatment in the field, using whole genome sequencing (WGS). Additionally, the genetic variability of Spanish APP isolates has been assessed to decipher antimicrobial resistance (AMR) determinants, toxin presence, serotype, and phenotype/genotype concordance of AMR. A total of 169 isolates from clinical cases of porcine pleuropneumonia with known antimicrobial susceptibility profiles were sequenced. Additionally, 48 NCBI assemblies were included to perform a phylogenetic analysis. Phylogenetic analysis revealed high association between phylogenetic clusters, serotypes, and presence of toxins that are associated within vertically integrated systems by its epidemiological link. Concordance between presence of AMR determinants (genotype) vs in-vitro antimicrobial susceptibility pattern (phenotype) was acceptable for amoxicillin, florfenicol, oxytetracycline, and enrofloxacin using epidemiological cut-off values (ECOFFs), but low concordance was observed for doxycycline and trimethoprim-sulfamethoxazole (T/S). On the other hand, using CLSI clinical breakpoints (CBPs), concordance was acceptable for florfenicol and enrofloxacin and not evaluated for doxycycline, oxytetracycline, trimethoprim-sulfamethoxazole (T/S), and amoxicillin because no CBP are available for them. Finally, WGS has demonstrated the clonality between isolates that shared a common origin (grandmother's farm) and resistance phenotype, suggesting vertical transmission of this pathogen and supporting the use of the epidemiological approach as a good criterion to optimise the antimicrobial use.
Subject(s)
Actinobacillus Infections , Actinobacillus pleuropneumoniae , Anti-Bacterial Agents , Microbial Sensitivity Tests , Phylogeny , Swine Diseases , Whole Genome Sequencing , Actinobacillus pleuropneumoniae/genetics , Actinobacillus pleuropneumoniae/drug effects , Actinobacillus pleuropneumoniae/classification , Actinobacillus pleuropneumoniae/isolation & purification , Swine , Animals , Actinobacillus Infections/microbiology , Actinobacillus Infections/veterinary , Actinobacillus Infections/transmission , Swine Diseases/microbiology , Swine Diseases/transmission , Anti-Bacterial Agents/pharmacology , Pleuropneumonia/microbiology , Pleuropneumonia/veterinary , Genotype , Genome, Bacterial , Drug Resistance, Bacterial/genetics , Spain/epidemiologyABSTRACT
Actinobacillus pleuropneumoniae (APP) causes porcine pleuropneumonia (PCP), which is clinically characterized by acute hemorrhagic, necrotizing pneumonia, and chronic fibrinous pneumonia. Although many measures have been taken to prevent the disease, prevention and control of the disease are becoming increasingly difficult due to the abundance of APP sera, weak vaccine cross-protection, and increasing antibiotic resistance in APP. Therefore, there is an urgent need to develop novel drugs against APP infection to prevent the spread of APP. Naringin (NAR) has been reported to have an excellent therapeutic effect on pulmonary diseases, but its therapeutic effect on lung injury caused by APP is not apparent. Our research has shown that NAR was able to alleviate APP-induced weight loss and quantity of food taken and reduce the number of WBCs and NEs in peripheral blood in mice; pathological tissue sections showed that NAR was able to prevent and control APP-induced pathological lung injury effectively; based on the establishment of an in vivo/in vitro model of APP inflammation, it was found that NAR was able to play an anti-inflammatory role through inhibiting the MAPK/NF-κB signaling pathway and exerting anti-inflammatory effects; additionally, NAR activating the Nrf2 signalling pathway, increasing the secretion of antioxidant enzymes Nqo1, CAT, and SOD1, inhibiting the secretion of oxidative damage factors NOS2 and COX2, and enhancing the antioxidant stress ability, thus playing an antioxidant role. In summary, NAR can relieve severe lung injury caused by APP by reducing excessive inflammatory response and improving antioxidant capacity.
Subject(s)
Actinobacillus Infections , Actinobacillus pleuropneumoniae , Acute Lung Injury , Flavanones , Kelch-Like ECH-Associated Protein 1 , NF-E2-Related Factor 2 , NF-kappa B , Animals , Mice , Actinobacillus Infections/veterinary , Actinobacillus Infections/drug therapy , Actinobacillus pleuropneumoniae/drug effects , Acute Lung Injury/drug therapy , Acute Lung Injury/prevention & control , Flavanones/therapeutic use , Flavanones/pharmacology , Heme Oxygenase-1 , Kelch-Like ECH-Associated Protein 1/metabolism , Membrane Proteins , NF-E2-Related Factor 2/metabolism , NF-kappa B/metabolism , Signal Transduction/drug effectsABSTRACT
Actinobacillus pleuropneumoniae infection causes a high mortality rate in porcine animals. Antimicrobial resistance poses global threats to public health. The current study aimed to determine the antimicrobial susceptibilities and probe the resistome of A. pleuropneumoniae in Taiwan. Herein, 133 isolates were retrospectively collected; upon initial screening, 38 samples were subjected to next-generation sequencing (NGS). Over the period 2017-2022, the lowest frequencies of resistant isolates were found for ceftiofur, cephalexin, cephalothin, and enrofloxacin, while the highest frequencies of resistant isolates were found for oxytetracycline, streptomycin, doxycycline, ampicillin, amoxicillin, kanamycin, and florfenicol. Furthermore, most isolates (71.4%) showed multiple drug resistance. NGS-based resistome analysis revealed aminoglycoside- and tetracycline-related genes at the highest prevalence, followed by genes related to beta-lactam, sulfamethoxazole, florphenicol, and macrolide. A plasmid replicon (repUS47) and insertion sequences (IS10R and ISVAp11) were identified in resistant isolates. Notably, the multiple resistance roles of the insertion sequence IS10R were widely proposed in human medicine; however, this is the first time IS10R has been reported in veterinary medicine. Concordance analysis revealed a high consistency of phenotypic and genotypic susceptibility to florphenicol, tilmicosin, doxycycline, and oxytetracycline. The current study reports the antimicrobial characterization of A. pleuropneumoniae for the first time in Taiwan using NGS.
Subject(s)
Actinobacillus Infections , Actinobacillus pleuropneumoniae , Anti-Bacterial Agents , High-Throughput Nucleotide Sequencing , Microbial Sensitivity Tests , Swine Diseases , Actinobacillus pleuropneumoniae/drug effects , Actinobacillus pleuropneumoniae/genetics , Taiwan/epidemiology , Anti-Bacterial Agents/pharmacology , Animals , Swine Diseases/microbiology , Swine Diseases/epidemiology , Swine , Actinobacillus Infections/veterinary , Actinobacillus Infections/microbiology , Retrospective Studies , Drug Resistance, Multiple, Bacterial/genetics , Drug Resistance, Bacterial/geneticsABSTRACT
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.
Subject(s)
Actinobacillus Infections , Actinobacillus pleuropneumoniae , Anthraquinones , Anti-Bacterial Agents , Animals , Anthraquinones/pharmacology , Anthraquinones/therapeutic use , Actinobacillus pleuropneumoniae/drug effects , Actinobacillus pleuropneumoniae/genetics , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , Mice , Actinobacillus Infections/drug therapy , Actinobacillus Infections/microbiology , Actinobacillus Infections/veterinary , Swine , Disease Models, Animal , Female , Drugs, Chinese Herbal/pharmacology , Drugs, Chinese Herbal/therapeutic use , Lung/microbiology , Lung/pathology , Swine Diseases/drug therapy , Swine Diseases/microbiologyABSTRACT
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.
Subject(s)
Actinobacillus Infections , Actinobacillus pleuropneumoniae , Pleuropneumonia , Swine Diseases , Thiamphenicol/analogs & derivatives , Swine , Animals , Serogroup , Microbial Sensitivity Tests/veterinary , Enrofloxacin , Farms , Retrospective Studies , Pleuropneumonia/epidemiology , Pleuropneumonia/veterinary , Pleuropneumonia/microbiology , Anti-Bacterial Agents/pharmacology , Sulfamethoxazole/pharmacology , Trimethoprim/pharmacology , Italy/epidemiology , Swine Diseases/epidemiology , Swine Diseases/microbiology , Actinobacillus Infections/epidemiology , Actinobacillus Infections/veterinary , Actinobacillus Infections/microbiology , Serotyping/veterinaryABSTRACT
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 8â¯bp sequence upstream of the coding sequence and one of 111â¯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.
Subject(s)
Actinobacillus Infections , Actinobacillus pleuropneumoniae , Swine Diseases , Animals , Swine , Serogroup , Multiplex Polymerase Chain Reaction/veterinary , O Antigens/genetics , Actinobacillus Infections/veterinary , Serotyping/veterinaryABSTRACT
Porcine contagious pleuropneumonia, caused by Actinobacillus pleuropneumoniae, has resulted in significant economic losses to the swine industry. Although antibiotics are commonly employed to control this disease, their widespread use or misuse can lead to the development of antibiotic resistance in A. pleuropneumoniae. Consequently, it is crucial to conduct antimicrobial susceptibility testing on clinical isolates. In our study, we identified one strain of A. pleuropneumoniae with resistance to florfenicol and extracted a 5919 bp plasmid named pAPPJY, which confers florfenicol resistance. Sequence analysis revealed that the plasmid contains four open reading frames, namely rep, antioxin vbha family protein, floR, and a partial copy of lysr. Although a few variations in gene position were observed, the plasmid sequence exhibits a high degree of similarity to other florfenicol-resistant plasmids found in Glaesserella parasuis and A. pleuropneumoniae. Therefore, it is possible that the pAPPJY plasmid functions as a shuttle, facilitating the spread of florfenicol resistance between G. parasuis and A. pleuropneumoniae. In addition, partial recombination may occur during bacterial propagation. In conclusion, this study highlights the horizontal transmission of antibiotic resistance among different bacterial species through plasmids, underscoring the need for increased attention to antibiotic usage.
Subject(s)
Actinobacillus Infections , Actinobacillus pleuropneumoniae , Swine Diseases , Thiamphenicol/analogs & derivatives , Animals , Swine , Anti-Bacterial Agents/pharmacology , Actinobacillus pleuropneumoniae/genetics , Microbial Sensitivity Tests , Plasmids , Actinobacillus Infections/drug therapy , Actinobacillus Infections/veterinary , Swine Diseases/drug therapy , Swine Diseases/microbiologyABSTRACT
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.
Subject(s)
Actinobacillus Infections , Actinobacillus pleuropneumoniae , Pleuropneumonia , Rodent Diseases , Swine Diseases , Animals , Swine , Mice , Pleuropneumonia/microbiology , Pleuropneumonia/veterinary , Biofilms , Actinobacillus pleuropneumoniae/metabolism , Cyclic AMP Receptor Protein/genetics , Lung/microbiology , Actinobacillus Infections/veterinary , Actinobacillus Infections/microbiology , Swine Diseases/microbiologyABSTRACT
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.
Subject(s)
Actinobacillus Infections , Actinobacillus pleuropneumoniae , Swine Diseases , Swine , Animals , Macrophages, Alveolar , Bacterial Proteins , Actinobacillus pleuropneumoniae/genetics , Caspase 3 , Apoptosis , Acylation , Necrosis/veterinary , Actinobacillus Infections/veterinary , Hemolysin ProteinsABSTRACT
We conducted whole-genome sequencing to investigate the serotypes, the presence of virulence and antimicrobial resistance genes, and the genetic relationships among isolates of Actinobacillus. pleuropneumoniae derived from diseased pigs. Serotype 2 (71.2%) was the most common, but the prevalence of serotypes 6 (13.6%) and 15 (6.8%) increased. Existing vaccines are considered ineffective on the isolates belonging to serotypes 6 and 15. The phylogenetic tree based on core genome single nucleotide polymorphisms showed that the isolates were clustered by serotype. Of the isolates, 62.5% did not have an antimicrobial resistance gene, including a florfenicol resistance gene, but 32.2% had a tetracycline resistance gene. The antimicrobial resistant phenotype and genotype were almost identical. The plasmid-derived contigs harbored resistance genes of aminoglycosides, tetracyclines, ß-lactams, phenicols, or sulfonamides. It has been suggested that isolates with different genetic properties from vaccine strains are circulating; however, antimicrobial resistance may not be widespread.
Subject(s)
Actinobacillus Infections , Actinobacillus pleuropneumoniae , Swine Diseases , Swine , Animals , Actinobacillus pleuropneumoniae/genetics , Japan/epidemiology , Phylogeny , Anti-Bacterial Agents/pharmacology , Whole Genome Sequencing/veterinary , Swine Diseases/epidemiology , Actinobacillus Infections/veterinaryABSTRACT
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.
Subject(s)
Actinobacillus Infections , Actinobacillus pleuropneumoniae , Ascomycota , Mycoplasma Infections , Pleuropneumonia , Pneumonia , Swine Diseases , Animals , Mice , Swine , Neutrophils , Pneumonia/veterinary , Pleuropneumonia/veterinary , Mycoplasma Infections/veterinary , Actinobacillus Infections/veterinary , LungABSTRACT
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.
Subject(s)
Actinobacillus Infections , Actinobacillus pleuropneumoniae , Mycoplasma , Pleuropneumonia , Swine Diseases , Swine , Animals , Pleuropneumonia/microbiology , Pleuropneumonia/veterinary , Serogroup , Abscess/pathology , Abscess/veterinary , Actinobacillus Infections/microbiology , Actinobacillus Infections/veterinary , Swine Diseases/microbiology , Lung/pathologyABSTRACT
Three Actinobacillus pleuropneumoniae isolates from clinical cases of porcine pleuropneumonia were positive by capsular serovar 12-specific PCR assay, but not reactive to antiserum prepared against serovar 12 using the rapid slide agglutination (RSA) test. The isolates were positive for apxIICA, apxIIICA, apxIBD, apxIIIBD, and apxIVA in the PCR toxin gene assay, which is the profile seen in serovars 2, 4, 6, 8, and 15, and reacted with antisera against serovars 3, 6, 8, 15, and 17. Nucleotide sequence analysis revealed that genes involved in the biosynthesis of capsular polysaccharide of the 3 isolates were identical or nearly identical to those of serovar 12. However, genes involved in the biosynthesis of O-polysaccharide of the 3 isolates were highly similar to those of reference strains of serovars 3, 6, 8, 15, 17, and 19. In agreement with results from the RSA test, transmission electron microscopic analysis confirmed the absence of detectable capsular material in the 3 isolates. The existence of nonencapsulated A. pleuropneumoniae serovar K12:O3 would hamper precise serodetection.
Subject(s)
Actinobacillus Infections , Actinobacillus pleuropneumoniae , Pleuropneumonia , Swine Diseases , Animals , Swine , Serogroup , Actinobacillus pleuropneumoniae/genetics , Actinobacillus Infections/epidemiology , Actinobacillus Infections/veterinary , Actinobacillus Infections/diagnosis , Swine Diseases/epidemiology , Swine Diseases/diagnosis , Pleuropneumonia/epidemiology , Pleuropneumonia/veterinary , Pleuropneumonia/diagnosis , PolysaccharidesABSTRACT
Actinobacillus pleuropneumoniae (APP) is a gram-negative pathogenic bacterium responsible for porcine contagious pleuropneumonia (PCP), which can cause porcine necrotizing and hemorrhagic pleuropneumonia. Actinobacillus pleuropneumoniae-RTX-toxin (Apx) is an APP virulence factor. APP secretes a total of four Apx toxins, among which, ApxI demonstrates strong hemolytic activity and cytotoxicity, causing lysis of porcine erythrocytes and apoptosis of porcine alveolar macrophages. However, the protein interaction network between this toxin and host cells is still poorly understood. TurboID mediates the biotinylation of endogenous proteins, thereby targeting specific proteins and local proteomes through gene fusion. We applied the TurboID enzyme-catalyzed proximity tagging method to identify and study host proteins in immortalized porcine alveolar macrophage (iPAM) cells that interact with the exotoxin ApxI of APP. His-tagged TurboID-ApxIA and TurboID recombinant proteins were expressed and purified. By mass spectrometry, 318 unique interacting proteins were identified in the TurboID ApxIA-treated group. Among them, only one membrane protein, caveolin-1 (CAV1), was identified. A co-immunoprecipitation assay confirmed that CAV1 can interact with ApxIA. In addition, overexpression and RNA interference experiments revealed that CAV1 was involved in ApxI toxin-induced apoptosis of iPAM cells. This study provided first-hand information about the proteome of iPAM cells interacting with the ApxI toxin of APP through the TurboID proximity labeling system, and identified a new host membrane protein involved in this interaction. These results lay a theoretical foundation for the clinical treatment of PCP.
Subject(s)
Actinobacillus Infections , Actinobacillus pleuropneumoniae , Swine Diseases , Swine , Animals , Actinobacillus pleuropneumoniae/genetics , Macrophages, Alveolar/metabolism , Exotoxins/pharmacology , Apoptosis , Membrane Proteins/metabolism , Bacterial Proteins/genetics , Actinobacillus Infections/veterinary , Actinobacillus Infections/microbiology , Hemolysin Proteins/toxicity , Swine Diseases/microbiologyABSTRACT
Actinobacillus pleuropneumoniae is an important swine respiratory pathogen. Previous studies have suggested that growth as a biofilm is a natural state of A. pleuropneumoniae infection. To understand the survival features involved in the biofilm state, the growth features, morphology and gene expression profiles of planktonic and biofilm A. pleuropneumoniae were compared. A. pleuropneumoniae in biofilms showed reduced viability but maintained the presence of extracellular polymeric substances (EPS) after late log-phase. Under the microscope, bacteria in biofilms formed dense aggregated structures that were connected by abundant EPS, with reduced condensed chromatin. By construction of Δpga and ΔdspB mutants, polymeric ß-1,6-linked N-acetylglucosamine and dispersin B were confirmed to be critical for normal biofilm formation. RNA-seq analysis indicated that, compared to their planktonic counterparts, A. pleuropneumoniae in biofilms had an extensively altered transcriptome. Carbohydrate metabolism, energy metabolism and translation were significantly repressed, while fermentation and genes contributing to EPS synthesis and translocation were up-regulated. The regulators Fnr (HlyX) and Fis were found to be up-regulated and their binding motifs were identified in the majority of the differentially expressed genes, suggesting their coordinated global role in regulating biofilm metabolism. By comparing the transcriptome of wild-type biofilm and Δpga, the utilization of oligosaccharides, iron and sulfur and fermentation were found to be important in adhesion and aggregation during biofilm formation. Additionally, when used as inocula, biofilm bacteria showed reduced virulence in mouse, compared with planktonic grown cells. Thus, these results have identified new facets of A. pleuropneumoniae biofilm maintenance and regulation.
Subject(s)
Actinobacillus Infections , Actinobacillus pleuropneumoniae , Swine Diseases , Animals , Swine , Mice , Actinobacillus pleuropneumoniae/genetics , Biofilms , Transcriptome , Virulence , Actinobacillus Infections/veterinary , Actinobacillus Infections/microbiology , Swine Diseases/microbiologyABSTRACT
We describe phenotypic and genetic characterization of an atypical Japanese Actinobacillus pleuropneumoniae isolate OT761. Nucleotide sequence analysis revealed that gene clusters involved in capsular polysaccharide and O-polysaccharide (O-PS) biosynthesis of the isolate were nearly identical to those of serovar 2 reference strain. The main difference found between the O-PS loci is the shortening of 31 amino acids from the C terminus of WcaJ in the atypical isolate due to a 93 bp deletion at the 3' end of wcaJ gene. Immunoblot analysis revealed that this isolate could not produce O-PS. Taken together, our results showed that the C-terminal domain of the A. pleuropneumoniae WcaJ plays a critical role in enzyme function of WcaJ involved in the biosynthesis of O-PS.