Pleuropneumoniae Infection Research Articles

Evaluation the kill rate and mutant selection window of danofloxacin against Actinobacillus pleuropneumoniae in a peristaltic pump model

BackgroundActinobacillus 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.MethodsFor 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.ResultsIn 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.ConclusionThese kill rate and MSW results will provide valuable guidance for the use of DAN to treat A. pleuropneumoniae infections.

Halicin: A New Horizon in Antibacterial Therapy against Veterinary Pathogens.

It is crucial to discover novel antimicrobial drugs to combat resistance. This study investigated the antibacterial properties of halicin (SU3327), an AI-identified anti-diabetic drug, against 13 kinds of common clinical pathogens of animal origin, including multidrug-resistant strains. Employing minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assessments, halicin demonstrated a broad-spectrum antibacterial effect. Time-killing assays revealed its concentration-dependent bactericidal activity against Escherichia coli ATCC 25922 (E. coli ATCC 25922), Staphylococcus aureus ATCC 29213 (S. aureus ATCC 29213), and Actinobacillus pleuropneumoniae S6 (APP S6) after 4 h of treatment at concentrations above the MIC. Halicin exhibited longer post-antibiotic effects (PAEs) and sub-MIC effects (PA-SMEs) for E. coli 25922, S. aureus 29213, and APP S6 compared to ceftiofur and ciprofloxacin, the commonly used veterinary antimicrobial agents, indicating sustained antibacterial action. Additionally, the results of consecutive passaging experiments over 40 d at sub-inhibitory concentrations showed that bacteria exhibited difficulty in developing resistance to halicin. Toxicology studies confirmed that halicin exhibited low acute toxicity, being non-mutagenic, non-reproductive-toxic, and non-genotoxic. Blood biochemical results suggested that halicin has no significant impact on hematological parameters, liver function, and kidney function. Furthermore, halicin effectively treated respiratory A. pleuropneumoniae infections in murine models. These results underscore the potential of halicin as a new antibacterial agent with applications against clinically relevant pathogens in veterinary medicine.

Antimicrobial susceptibility and resistome of Actinobacillus pleuropneumoniae in Taiwan: a next-generation sequencing analysis

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.

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

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

Hyper-production of porcine contagious pleuropneumonia subunit vaccine proteins in Escherichia coli by developing a bicistronic T7 expression system.

The ApxII toxin and the outer membrane lipoprotein (Oml) of Actinobacillus pleuropneumoniae are important vaccine antigens against porcine contagious pleuropneumonia (PCP), a prevalent infectious disease affecting the swine industry worldwide. Previous studies have reported the recombinant expression of ApxII and Oml in Escherichia coli; however, their yields were not satisfactory. Here, we aimed to enhance the production of ApxII and Oml by constructing a bicistronic expression system based on the widely used T7 promoter. To create efficient T7 bicistronic expression cassettes, 16 different fore-cistron sequences were introduced downstream of the T7 promoter. The expression of three vaccine antigens Oml1, Oml7, and ApxII in the four strongest bicistronic vectors were enhanced compared to the monocistronic control. Further optimization of the fermentation conditions in micro-well plates (MWP) led to improved production. Finally, the production yields reached unprecedented levels of 2.43gL-1 of Oml1, 2.59gL-1 of Oml7, and 1.21gL-1 of ApxII, in a 5 L bioreactor. These three antigens also demonstrated well-protective immunity against A. pleuropneumoniae infection. In conclusion, this study establishes an efficient bicistronic T7 expression system that can be used to express recombinant proteins in E. coli and achieves the hyper-production of PCP vaccine proteins.

Tea Polyphenols Protects Tracheal Epithelial Tight Junctions in Lung during Actinobacillus pleuropneumoniae Infection via Suppressing TLR-4/MAPK/PKC-MLCK Signaling.

Actinobacillus pleuropneumoniae (APP) is the causative pathogen of porcine pleuropneumonia, a highly contagious respiratory disease in the pig industry. The increasingly severe antimicrobial resistance in APP urgently requires novel antibacterial alternatives for the treatment of APP infection. In this study, we investigated the effect of tea polyphenols (TP) against APP. MIC and MBC of TP showed significant inhibitory effects on bacteria growth and caused cellular damage to APP. Furthermore, TP decreased adherent activity of APP to the newborn pig tracheal epithelial cells (NPTr) and the destruction of the tight adherence junction proteins β-catenin and occludin. Moreover, TP improved the survival rate of APP infected mice but also attenuated the release of the inflammation-related cytokines IL-6, IL-8, and TNF-α. TP inhibited activation of the TLR/MAPK/PKC-MLCK signaling for down-regulated TLR-2, TLR4, p-JNK, p-p38, p-PKC-α, and MLCK in cells triggered by APP. Collectively, our data suggest that TP represents a promising therapeutic agent in the treatment of APP infection.

Adh Promotes Actinobacillus pleuropneumoniae Survival in Porcine Alveolar Macrophages by Inhibiting CHAC2-Mediated Respiratory Burst and Inflammatory Cytokine Expression.

Actinobacillus pleuropneumoniae (A. pleuropneumoniae) causes porcine pleuropneumonia that seriously endangers pig's health. Adh, located in the head region of trimeric autotransporter adhesion of A. pleuropneumoniae, affects bacterial adhesion and pathogenicity. However, how Adh mediates A. pleuropneumoniae immune invasion is still unclear. Here, we established the A. pleuropneumoniae strain L20 or L20 ΔAdh-infected porcine alveolar macrophages (PAM) model, and applied protein overexpression, RNA interference, qRT-PCR, Western blot and immunoflourescence techniques to dissect the effects of Adh on PAM during A. pleuropneumoniae infection. We found that Adh could increase the A. pleuropneumoniae adhesion and intracellular survival in PAM. Gene chip analysis of piglet lungs further showed that Adh significantly induced cation transport regulatory-like protein 2 (CHAC2) expression, whose overexpression suppressed the phagocytic capacity of PAM. Furthermore, CHAC2 overexpression dramatically increased glutathione (GSH) expression, decreased reactive oxygen species (ROS), and promoted A. pleuropneumoniae survival in PAM, while the knockdown of CHAC2 reversed these phenomena. Meanwhile, CHAC2 silence activated the NOD1/NF-κB pathway, resulting in an increase in IL-1β, IL-6, and TNF-α expression, whereas this effect was weakened by CHAC2 overexpression and addition of NOD1/NF-κB inhibitor ML130. Moreover, Adh enhanced the secretion of LPS of A. pleuropneumoniae, which regulated the expression of CHAC2 via TLR4. In conclusion, through a LPS-TLR4-CHAC2 pathway, Adh inhibits respiratory burst and inflammatory cytokines expression to promote A. pleuropneumoniae survival in PAM. This finding may provide a novel target for the prevention and treatment of A. pleuropneumoniae.

Explorative Field Study on the Use of Oral Fluids for the Surveillance of Actinobacillus pleuropneumoniae Infections in Fattening Farms by an Apx-Real-Time PCR.

Simple SummaryOral fluid sampling (OFS) is an animal friendly and easy way for surveillance purposes in domestic swine populations, especially concerning respiratory diseases. In case of Actinobacillus (A.) pleuropneumoniae surveillance, measures are usually combined with burdensome sampling for animals and humans. In the present study, we evaluated the suitability of oral fluids (OFs) for surveillance purposes of A. pleuropneumoniae infections in fattening pigs using an Apx-toxin real-time PCR. We were able to demonstrate that the examination of OFs by an Apx-toxin real-time PCR is suitable for A. pleuropneumoniae surveillance in the field in an animal friendly and easy way. These results might contribute to an increased compliance of laboratory diagnostic measures on pig farms and thereby to increased animal welfare due to less burdensome sampling and improved animal health.Oral fluids (OFs) represent a cost effective and reliable tool for surveillance purposes, mostly regarding viruses. In the present study, we evaluated the suitability of OFs for surveillance purposes concerning Actinobacillus (A.) pleuropneumoniae infections in fattening pigs under field conditions. OFs were examined with an Apx-toxin real-time PCR that detects the genes encoding for Apx I-, Apx III-, and Apx IV-toxin. For this purpose, we conducted a pen-wise collection of OFs over one fattening period from fattening pigs of two farms (farm A and B) with a known history of A. pleuropneumoniae infection. Lung lesions were determined at slaughter to estimate the extend of pulmonary lesions and pleural affection. Apx III- and Apx IV-toxin DNA were present in the OFs of both farms whereas Apx I-toxin DNA was present on farm A only. We were able to detect Apx I-, Apx III-, and Apx IV-toxin DNA in different patterns directly after introduction of the new pigs in the farms and over the entire study period. In summary, or results indicate the suitability of OFS for the early detection and surveillance of A. pleuropneumoniae in fattening farms.

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.

HtrA of Actinobacillus pleuropneumoniae is a virulence factor that confers resistance to heat shock and oxidative stress

Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia, which is a severe and often fatal disease that results in significant economic loss. The means by which A. pleuropneumoniae survives within the host are not clear. High temperature requirement A (HtrA) proteases have been shown to affect cell viability during stressful conditions and are virulence factors in many bacterial species. In this study, we examined the biological role of HtrA during A. pleuropneumoniae infection by analyzing the impact of htrA mutation on virulence-associated phenotypes. We found that htrA mutation had a dramatic impact on stress tolerance. The htrA mutant (ΔhtrA) displayed a lethal phenotype at elevated temperature (42 °C). Further, ΔhtrA exhibited increased susceptibility to H2O2-induced oxidative stress when compared to the parental strain (SLW01) and a complementation strain (ΔhtrA-Compl). Animal infection assays demonstrated that absence of HtrA led to decreased in vivo colonization ability, and ΔhtrA is less virulent in pigs relative to SLW01. Furthermore, pig competitive infection assays demonstrated fewer blood associated CFUs with ΔhtrA infection than with SLW01. These results demonstrate HtrA plays a significant role in the survival and growth of A. pleuropneumoniae during stressful conditions, and that immune escape and invasiveness are important to the process of A. pleuropneumoniae infection.

A Combinatorial Vaccine Containing Inactivated Bacterin and Subunits Provides Protection Against Actinobacillus pleuropneumoniae Infection in Mice and Pigs.

Actinobacillus pleuropneumoniae (APP) is the etiological agent of porcine contagious pleuropneumonia (PCP) that causes great economic losses in the swine industry. Currently, vaccination is still a commonly used strategy for the prevention of the disease. Commercially available vaccines of this disease, including inactivated bacterins and subunit vaccines, have clinical limitations such as side effects and low cross-protection. In this study, a combinatorial vaccine (Bac-sub) was developed, which contained inactivated bacterial cells of a serovar 1 strain and three recombinant protoxins (rApxIA, rApxIIA, and rApxIIIA). Its side effects, immune protection, and cross-protection were evaluated and compared with a commercial subunit vaccine and a commercial trivalent bacterin in a mouse infection model. The results revealed that the Bac-sub vaccine showed no obvious side effects, and induced higher levels of Apx toxin-specific IgG, IgG1, and IgG2a than the commercial vaccines after booster. After a challenge with virulent strains of serovars 1, 5, and 7, the Bac-sub vaccine provided greater protection (91.76%, 100%, and 100%, respectively) than commercial vaccines. Much lower lung bacterial loads (LBLs) and milder lung lesions were observed in the Bac-sub-vaccinated mice than in those vaccinated with the other two vaccines. The protective efficacy of the Bac-sub vaccine was further evaluated in pigs, which showed that vaccinated pigs displayed significantly milder clinical symptoms and lung lesions than the unvaccinated pigs after the challenge. Taken together, Bac-sub is a safe and effective vaccine that could provide high protection against A. pleuropneumoniae infection in both mice and pigs.

Application of M-cell targeting ligand Co4B enhances Ag-specific mucosal and systemic immunity and protection against MERS-CoV infection after intranasal immunization

Abstract Middle East respiratory syndrome is associated with severe pneumonia and poses a global public health threat. Authorized vaccines against the causative agent of MERS-CoV are urgently needed. In order to optimize the efficacy of recombinant vaccines, adjuvants should be required. Therefore, we previously identified a ligand Co4B as an adjuvant to enhance Ag delivery to the nasal mucosa immune compartment and promote Ag-specific mucosal and systemic immune responses via intranasal administration against A. pleuropneumoniae infection in a murine model. Based on the pathogenesis of MERS-CoV which infects host following respiratory route; thus, we generated a recombinant protein conjugated with Co4B to the MERS-CoV spike protein receptor-binding domain (S-RBD) to develop mucosal subunit vaccine candidate. Ag-specific mucosal IgA and systemic IgG antibody responses, together with virus-neutralizing capacities, were highly elicited in C57BL/6 and human dipeptidyl peptidase 4-transgenic (hDPP4-Tg) mice intranasally immunized with Co4B-conjugated S-RBD compared to those immunized with originally S-RBD. Ag-specific T cell-mediated immunity was also induced in the spleen and lungs of mice intranasally immunized with Co4B-conjugated S-RBD. Intranasal immunization of hDPP4-Tg mice with Co4B-conjugated S-RBD prominently reduced immune cell infiltration into the tissues of virus-challenged mice. Finally, Co4B-conjugated S-RBD-immunized mice demonstrated were improved protection against infection, more likely to survive, and exhibited less body weight loss. Collectively, our results suggest that Co4B-conjugated S-RBD could be used as an improved subunit vaccine candidate against MERS-CoV infection. This work was supported by the Ministry of Science and ICT No. 2020K1A4A7A02095058 and the NRF funded by the Ministry of Education No. 2019R1I1A3A01062224. Ms. Y. L. Yang and Ms. B.-H. Cho were supported by the BK21 FOUR program of the Department of Bioactive Material Sciences.

DEVELOPMENT OF MULTILOCUS SEQUENCE TYPING (MLST) OF ACTINOBACILLUS PLEUROPNEUMNIAE

Porcine pleuropneumonia caused by Actinobacillus pleuropneumoniae (AP) infection leads to considerable financial loss in the swine industry worldwide. Multilocus sequence typing (MLST), a definitive molecular typing technique that is highly reproducible and is capable of comparing data across laboratories, has not been studied on AP. Therefore, the aim of this work is to develop a MLST assay to characterize AP isolates collected from Taiwan. A total of 85 AP isolates collected from pleuropneumonia of diseased pigs with respiratory symptoms and seven reference isolates from other countries were included for comparison. Seven housekeeping genes (recF, gly, rho, tpi, pyk, recN, and rpo) were selected for sequencing. Capsule types that determine serotypes and Apx toxins were analyzed by the polymerase chain reaction (PCR). The sequencing results showed that the 7 housekeeping genes differentiated the 92 isolates into 14 sequence types that belonged to three major clonal complexes and five singletons by eBURST. The 85 isolates assigned to detection of capsule types and Apx toxins showed that serotype 1 with Apx I, II, IV (55.3%) followed by serotype 5 with Apx I, II, IV (29.4%) were the most prevalent in Taiwan. Notably, serotype 15 was identified for the first time. Clonal complexes based on ST profiles from MLST analysis were highly associated with the distribution of capsule types, suggesting that MLST scheme was sufficient to identify DNA samples directly from AP. Therefore, MLST might be a useful tool for identification and further epidemiological assay of AP.

Characterization of monoclonal antibodies against antigenic epitopes of Actinobacillus pleuropneumoniae ApxIVA

Porcine pleuropneumonia caused by Actinobacillus pleuropneumoniae is a highly contagious disease that leads to enormous economic losses in pig industry, worldwide. Of the many virulence factors produced by the causative bacterium, ApxA exotoxins have been considered as the most important contributor to the disease. The toxins are classified into four different types; ApxIA, ApxIIA, ApxIIIA and ApxIVA. Uniquely, ApxIVA is expressed across all serotypes of A. pleuropneumoniae only during in vivo infection in pigs. Active research focusing on resolving the precious roles and mechanisms of the toxins is still at its primitive stage. In this study, we report the development of monoclonal antibodies against the two major antigenic epitopes that were characterized in our previous study incorporating the in silico predictions and protein modeling analyses. Recombinant proteins of the selected epitopes were expressed and purified after molecular cloning of the corresponding partial genes in E. coli expression system. Subsequently, we generated hybridomas with lymphoid cells from the rats immunized with the recombinantly expressed proteins of Apx. Consequently, hybridomas exhibiting strong productivity of the monoclonal antibodies were selected for downstream verifications that tested for reactivity and specificity using Western blot and ELISA. Our results strongly suggest the potential application of the monoclonal antibodies developed in this study as useful reagents to further elaborate the mechanism of the A. pleuropneumoniae infection in pig.

Actinobacillus pleuropneumoniae: The molecular determinants of virulence and pathogenesis.

Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, is responsible for high economic losses in swine herds across the globe. Pleuropneumonia is characterized by severe respiratory distress and high mortality. The knowledge about the interaction between bacterium and host within the porcine respiratory tract has improved significantly in recent years. A. pleuropneumoniae expresses multiple virulence factors, which are required for colonization, immune clearance, and tissue damage. Although vaccines are used to protect swine herds against A. pleuropneumoniae infection, they do not offer complete coverage, and often only protect against the serovar, or serovars, used to prepare the vaccine. This review will summarize the role of individual A. pleuropneumoniae virulence factors that are required during key stages of pathogenesis and disease progression, and highlight progress made toward developing effective and broadly protective vaccines against an organism of great importance to global agriculture and food production.

Serologic monitoring of herds with and without bacterin vaccination for Actinobacillus pleuropneumoniae

Objective: Investigate diagnostic serology for Actinobacillus pleuropneumoniae (APP) infections in naturally infected and vaccinated pigs. Materials and methods: The APP status of 12 farms (A-L) was established by lung cultures and isolate serotyping. Screening enzyme-linked immunosorbent assay (ELISA) detected antibodies to ApxIV antigen or multiple APP serotypes. Serotype-specific ELISAs were conducted for serotypes 5 and 7. Seven groups of farm F pigs (serotype 7) were moved to farm K (serotype 5). Autogenous vaccines (V1/V2) prepared from APP serotype 5 cultures from farm K and a commercial, killed APP vaccine (V3) containing serotypes 1, 7, and 15 were used to vaccinate pigs in each group twice or thrice at 3-week intervals, commencing at 10 weeks of age. Blood samples were analyzed with ELISAs specific for serotype 5 and ApxI and ApxII toxins. Serum titers were compared using an analysis of variance. Results: Serotypes 5, 7, 12, or 15 were present in lung cultures. The ApxIV screening ELISA and mix-serotype ELISA regularly detected serotypes 5, 7, and 15. Serotype 12 infections were detected in the mix-serotype ELISA, but not in the ApxIV assays. The serotype 5 or 7 specific ELISA regularly detected herd infections with the relevant serotype. Serotype 5 titers of pigs vaccinated with V1/V2 thrice were higher than those dosed twice with the equivalent volume (P < .05). Pigs receiving V3 showed no serotype 5 antibody response. The ApxI and II titers in V1/V2-vaccinated pigs were higher than controls. Implications: Screening and serotype-specific ELISAs verified APP status. Repeated serotype-specific autogenous APP vaccine doses provided a strong antibody response.

Методичні підходи щодо бактеріологічної діагностики актинобацилярної плевропневмонії свиней

Методичні підходи щодо бактеріологічної діагностики актинобацилярної плевропневмонії свиней

Benchmarking of anatomopathological lesions assessed at slaughter and their association with tail lesions and carcass traits in heavy pigs

Recording lesions at the abattoir provides information for benchmarking pig health and welfare conditions. The study involved 79 batches of Italian heavy pigs, mainly tail-docked. The aim of the study was to identify the prevalence of anatomopathological lesions and to identify relationships with carcass traits. The scoring of enzootic pneumonia-like lesions (EP), chronic pleuritis (CP), dorso-caudal pleuritis, white spots on the liver and tail lesions (TLs) was carried out. Relationships among the lesions, the season of slaughter, slaughtering age and carcass traits were tested using logistic regression. Enzootic pneumonia-like lesions were observed on 30.20% (±14.94) of the lungs with an average score of 0.91 (±0.77). The prevalence of CP was 38.07% (±15.86) with an average score of 0.83 (±0.34) and an A. pleuropneumoniae index (APPI) of 0.71 (±0.39). The prevalence of pericarditis and peritonitis was 5.5% (±3.73) and 1.09% (±0.64), respectively. White spots on the liver showed a prevalence of 12.94% (±23.60) and an average score of 0.02 (±0.04). The prevalence of TLs was 34.08% (±11.21); 29.64% (±11.21) showed moderate damage, and 4.44% (±5.02) severe damage, not associated with pulmonary lesions or carcass traits. Dorso-caudal pleuritis, suggestive of previous Actinobacillus pleuropneumoniae infection, remained similar and was strongly associated with a higher lean meat percentage. Additional studies are necessary to better understand this association. The results showed that preventive measures against Actinobacillus pleuropneumoniae are deemed necessary to reduce dorso-caudal pleuritis and to improve pig health and carcass value. Highlights Cardiopulmonary lesions and tail lesions are indicative of poor welfare and loss of profit in the pork chain and could share common risk factors; Dorso-caudal pleuritis, suggestive of previous Actinobacillus pneumoniae infection, was associated with carcass traits; Tail lesions were not associated with lung lesions or carcass traits. Both lesions can share similar risk factors but have different aetiologies.

Recombinant tandem epitope vaccination provides cross protection against Actinobacillus pleuropneumoniae challenge in mice

Actinobacillus pleuropneumoniae (A. pleuropneumoniae/APP) is the pathogen that causes porcine contagious pleuropneumonia. Actinobacillus pleuropneumoniae is divided into 18 serovars, and the cross protection efficacy of epitopes is debatable, which has resulted in the slow development of a vaccine. Consequently, epitope-based vaccines conferring Actinobacillus pleuropneumoniae cross protection have rarely been reported. In this study, B cell epitopes in the head domain of trimeric autotransporter adhesin were predicted, and 6 epitopes were selected. Then, the predicted epitopes (Ba1, Bb5, C1, PH1 and PH2) were connected by linkers to construct a recombinant tandem antigen (rta) gene. The RTA protein encoded by the recombinant rta gene was expressed, and finally the ICR mice were immunized with the RTA protein with or without inactivated Actinobacillus pleuropneumoniae (serovars 1 and 5b) and challenged with Actinobacillus pleuropneumoniae to evaluate the protective effect of the epitope-based vaccine and combined vaccine. The mice in the RTA-immunized group and RTA plus inactivated Actinobacillus pleuropneumoniae vaccine group had a significant improvement in clinical symptoms and a higher level of antibody in the serum than those in the control group. The RTA immune group had a 40% survival rate after Actinobacillus pleuropneumoniae infection, whereas the combination of RTA and inactivated Actinobacillus pleuropneumoniae produced very strong cross immune protection in mice, at least 50% (RTA IB1 + C5) and at most 100% (RTA IB5 + C1), whereas no cross immunoprotection was found in the solo Actinobacillus pleuropneumoniae immune group. Overall, the combination of the RTA protein and inactivated bacteria significantly enhanced the cross protection effects. This implies that RTA protein in combination with a suitable inactivated Actinobacillus pleuropneumoniae strain could be a candidate vaccine for porcine contagious pleuropneumonia.

The antimicrobial peptide MPX kills Actinobacillus pleuropneumoniae and reduces its pathogenicity in mice

Actinobacillus pleuropneumoniae is the causative agent of highly contagious and fatal respiratory infections, causing substantial economic losses to the global pig industry. Due to increased antibiotic resistance, there is an urgent need to find new antibiotic alternatives for treating A. pleuropneumoniae infections. MPX is obtained from wasp venom and has a killing effect on various bacteria. This study found that MPX had a good killing effect on A. pleuropneumoniae and that the minimum inhibitory concentration (MIC) was 16 μg/mL. The bacterial density of A. pleuropneumoniae decreased 1000 times after MPX (1 × MIC) treatment for 1 h, and the antibacterial activity was not affected by pH or temperature. Fluorescence microscopy showed that MPX (1 × MIC) destroyed the bacterial cell membrane after treatment for 0.5 h, increasing membrane permeability and releasing bacterial proteins and Ca2+, Na+ and other cations. In addition, MPX (1 × MIC) treatment significantly reduced the formation of bacterial biofilms. Quantitative RT-PCR results showed that MPX treatment significantly upregulated the expression of the PurC virulence gene and downregulated that of ApxI, ApxII, and Apa1. In addition, the Sap A gene was found to play an important role in the tolerance of A. pleuropneumoniae to antimicrobial peptides. Therapeutic evaluation in a murine model showed that MPX protects mice from a lethal dose of A. pleuropneumoniae and relieves lung inflammation. This study reports the use of MPX to treat A. pleuropneumonia infections, laying the foundation for the development of new drugs for bacterial infections.