Serotype diversity and antimicrobial susceptibility profiles of Actinobacillus pleuropneumoniae isolated in Italian pig farms from 2015 to 2022.

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.

A candidate competitive ELISA based on monoclonal antibody 3A8 for diagnosis of contagious bovine pleuropneumonia.

For the development of a competitive ELISA (cELISA) to detect serum antibodies against the Mycoplasma mycoides subsp. Mycoides (Mmm) (strain PG1), the causative agent of contagious bovine pleuropneumonia (CBPP), all the proteins of this pathogen were analyzed. Then, a specific extracellular region of a transmembrane protein with the potential for diagnosis was identified. After that, a monoclonal antibody (Mab) named 3A8 was obtained using this extracellular region as an immunogen. Finally, a cELISA was established with the extracellular domain of this transmembrane protein as the coating antigen, Mab 3A8 as the competitive antibody, and HRP-labeled goat anti-mouse IgG as the enzyme-labeled antibody. This established method was used to detect the antibody dynamic regularity of goats which are artificially immunized Mmm and was also compared with a commercial ELISA kit. Further, the sera of 1011 different cattle from border provinces of China were monitored using a candidate Mab 3A8 cELISA. The detection results of known background sera used in this study indicate that a candidate diagnostic marker was successfully identified by analyzing all the coding proteins of Mmm in this research, and the cELISA established based on the Mab 3A8 against this protein can detect CBPP-positive serum with specificity and has no cross-reaction with other related epidemic disease-positive sera. In addition, we tested the sera collected from the border areas of China using the established ELISA, and no positive sample was detected. The research protocol of the CBPP cELISA established in this study is different from the traditional method, which can greatly reduce the investment of manpower and capital and save development time. We believe that this study's protocol could serve as a reference for the development of detection methods for mycoplasma and other complex pathogens. KEY POINTS: • A Mmm-specific diagnostic marker was obtained based on protein characteristics. • A cELISA was established for CBPP serum antibody detection. • The serological investigation was conducted for CBPP in the border areas of China.

Seroprevalence of contagious bovine pleuropneumonia (CBPP) in cattle from Karamoja region, North-eastern Uganda.

BACKGROUND: Contagious bovine pleuropneumonia [CBPP] is a transboundary animal disease of cattle caused by Mycoplasma mycoides subsp. mycoides [Mmm]. CBPP causes severe economic losses to livestock producers in sub-Saharan Africa mainly due to high mortality, morbidity, reduction in productivity as well as livestock trade restrictions. This study aimed at determining seroprevalence of Mmm in cattle from Karamoja region, north-eastern Uganda; data that are required to design and implement risk based CBPP control program. METHODS: We randomly collected blood samples from 2,300 cattle spread across Karamoja region. Serum was extracted and screened for antibodies against Mycoplasma mycoides subsp. mycoides [Mmm] using the competitive enzyme linked immunosorbent assay [cELISA]. RESULTS: A quarter [25.4%; 95% CI: 23.7-27.3] of the screened cattle [n = 2,300] were sero-positive for Mmm. Amudat and Kaabong districts recorded the lowest [12.3%] and highest [30.7%] Mmm seroprevalence respectively. Increasing age, overnight stay in cattle kraals and location [certain districts, villages, herds and sub counties] of the cattle herds, the factors that promote animal commingling, were the most significant risk factors of seroconversion with Mmm. CONCLUSION: Results from this study indicated a higher seroprevalence of Mmm in Karamoja region cattle herds. This could be due to the increased frequency of CBPP outbreaks in recent years. To be effective, CBPP vaccination programs should target high risk herds along the international borders and other hotspot areas [e.g., parishes or sub counties] where cattle commingling is high.

Efficacy, Safety, and Complications of Pigtail Thoracostomy for the Treatment of Pediatric Pleuropneumonia.

OBJECTIVE: Pigtail thoracostomy (PT) has become the mainstay technique for the drainage of pediatric pleuropneumonic effusions (PLPe). However, its efficacy and complication profile has been questioned when compared with video-assisted thoracoscopic surgery and larger bore traditional tube thoracostomy. The aim of this study was to assess the efficacy, safety, and complications associated with PT. METHODS: A cross-sectional study at a freestanding tertiary children's hospital. We extracted the medical records of all children aged younger than 18 years treated with PT for PLPe from June 2016 to June 2020. The primary efficacy outcome was treatment failure defined as the need for a repeat drainage procedure, thoracostomy, or video-assisted thoracoscopic surgery. Secondary efficacy outcomes were length of hospital stay (LOS) and duration of in situ PT. The primary safety outcomes were adverse events during or after insertion. We also recorded any associated complications. RESULTS: During the study period, 55 children required PT. The median age was 25 months (interquartile range, 14-52) and 58.2% were boys. Eight (14.4%) were bacteremic or in septic shock. There were no adverse events related to insertion. Forty-two (76.3%) children were treated with fibrinolysis. There were 2 (3.6%) treatment failures. The median LOS and PT durations were 13 and 4 days (interquartile ranges, 10-14.8, 3-6.7), respectively. Eight (14.4%) children experienced complications that were nonoperatively managed. CONCLUSIONS: Our findings suggest that PT drainage offers a safe and highly effective option for managing PLPe and carries a very low failure rate.

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.

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 ß-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.

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.

Rapid detection of Actinobacillus pleuropneumoniae targeting the apxIVA gene for diagnosis of contagious porcine pleuropneumonia in pigs by polymerase spiral reaction.

Actinobacillus pleuropneumoniae is the primary aetiological agent of contagious porcine pleuropneumonia associated with serious economic impact on pig husbandry worldwide. Diagnosis of the disease by existing techniques including isolation and identification of bacteria followed by serotyping, serological techniques, conventional PCR, real-time PCR and LAMP assays are cumbersome, time-consuming, costly and not suitable for rapid field application. A novel isothermal polymerase chain reaction (PSR) technique is standardized for all the reagents, incubation time and incubation temperature against A. pleuropneumoniae. The sensitivity of the assay was determined against various dilutions of purified DNA and total bacterial count. The specificity of the assay was determined against 11 closely related bacterial isolates. The relative sensitivity and specificity were compared with bacterial isolation, conventional PCR and real-time PCR assays. The PSR assay for specific detection was standardized at 64°C for 30 min of incubation in a water bath. The result was visible by the naked eye after centrifugation of the reaction mixture or after incorporation of SYBR Green dye as yellowish-green fluorescence. The technique was found to be 100% specific and equally sensitive with real-time PCR and 10 times more sensitive than conventional PCR. The PSR assay could be applicable in the detection of the organisms in porcine nasal swabs spiked with A. pleuropneumoniae. This is the first-ever report on the development of PSR for specific detection of A. pleuropneumoniae and can be applied for early diagnosis at the field level.

Sero- and apx-typing of German Actinobacillus pleuropneumoniae field isolates from 2010 to 2019 reveals a predominance of serovar 2 with regular apx-profile.

Serotyping is the most common method to characterize field isolates of Actinobacillus (A.) pleuropneumoniae, the etiological agent of porcine pleuropneumonia. Based on serology, many farms seem to be infected and antibodies against a wide variety of serovars are detectable, but, so far it is unknown to what degree respective serovars contribute to outbreaks of clinical manifest disease. In this study, 213 German A. pleuropneumoniae field isolates retrieved for diagnostic purposes from outbreaks of porcine pleuropneumonia between 2010 and 2019 were genetically serotyped and analyzed regarding their apx-toxin gene profile using molecular methods. Serotyping revealed a prominent role of serovar 2 in clinical cases (64% of all isolates) and an increase in the detection of this serovar since 2010 in German isolates. Serovar 9/11 followed as the second most frequent serovar with about 15% of the isolates. Furthermore, very recently described serovars 16 (n = 2) and 18 (n = 8) were detected. Most isolates (93.4%) showed apx-profiles typical for the respective serovar. However, this does not hold true for isolates of serovar 18, as 75% (n = 6) of all isolates of this serovar deviated uniformly from the "typical" apx-gene profile of the reference strain 7311555. Notably, isolates from systemic lesions such as joints or meninges did not harbor the complete apxICABD operon which is considered typical for highly virulent strains. Furthermore, the extremely low occurrence (n = 1) of NAD independent (biovar II) isolates in German A. pleuropneumoniae was evident in our collection of clinical isolates.

Severe bilateral pleuropneumonia caused by Legionella sainthelensi: a case report.

BACKGROUND: Legionella spp. are ubiquitous freshwater bacteria responsible for rare but potentially severe cases of Legionnaires' disease (LD). Legionella sainthelensi is a non-pneumophila Legionella species that was first isolated in 1980 from water near Mt. St-Helens (USA). Although rare cases of LD caused by L. sainthelensi have been reported, very little data is available on this pathogen. CASE PRESENTATION: We describe the first documented case of severe bilateral pleuropneumonia caused by L. sainthelensi. The patient was a 35-year-old woman with Sharp's syndrome treated with long-term hydroxychloroquine and corticosteroids who was hospitalized for an infectious illness in a university hospital in Reunion Island (France). The patient's clinical presentation was complicated at first (bilateral pneumonia, multiloculated pleural effusion, then bronchopleural fistula) but her clinical condition eventually improved with the reintroduction of macrolides (spiramycin) in intensive care unit. Etiological diagnosis was confirmed by PCR syndromic assay and culture on bronchoalveolar lavage. CONCLUSIONS: To date, only 14 documented cases of L. sainthelensi infection have been described worldwide. This pathogen is difficult to identify because it is not or poorly detected by urinary antigen and molecular methods (like PCR syndromic assays that primarily target L. pneumophila and that have only recently been deployed in microbiology laboratories). Pneumonia caused by L. sainthelensi is likely underdiagnosed as a result. Clinicians should consider the possibility of non-pneumophila Legionella infection in patients with a compatible clinical presentation when microbiological diagnostic tools targeted L. pneumophila tested negative.

Emergency Physician-Administered Sedation for Thoracostomy in Children With Pleuropneumonia.

BACKGROUND: Thoracostomy drainage is sometimes required in children with pleuropneumonia who have large parapneumonic effusion. This procedure is usually performed under sedation. The aim was to report sedation adverse events (SAEs) in pneumonia patients sedated for thoracostomy by pediatric emergency physicians. METHODS: A retrospective cohort study was conducted. The medical records of all emergency department patients who underwent thoracostomy between January 1, 2012, and December 31, 2018, were extracted. Study outcomes were SAEs that required intervention. RESULTS: Pigtail catheters were placed by chest surgeons in 28 children with a median age of 2 years (interquartile range [IQR], 1-5 years). All the thoracostomies were successfully performed under sedation performed by 11 pediatric emergency physicians. The median amount of fluid drained after catheter insertion was 200 mL (IQR, 100-500 mL). The median pleural fluid PH was 7.0 (IQR, 6.9-7.3), and the median white blood cell count was 34,600 per mm3 (IQR, 11,800-109,000 per mm3). Thirteen patients (46.4%) were sedated with a total median dose of 3 mg/kg of ketamine (IQR, 2-4 mg/kg) and 0.2 mg/kg of midazolam (IQR, 0.2-0.3 mg/kg); 11 patients (39.3%) were treated with 1 mg/kg of ketamine (IQR, 0.5-2 mg/kg) and 3 mg/kg of propofol (IQR, 2-4 mg/kg). Four patients (14.3%) were treated exclusively with 4 mg/kg of ketamine (IQR, 3-5 mg/kg). Nine oxygen desaturations required intervention; 1 was associated with laryngospasm and 1 with apnea. All the SAEs were successfully managed. No cases of hypotension, bradycardia, airway obstruction, or pulmonary aspiration were recorded. CONCLUSIONS: The first series of pneumonia patients sedated for thoracostomy by pediatric emergency physicians is reported. Sedation was safely performed in this cohort.

Acute fibrinous pleuropneumonia and septicaemia caused by Bibersteinia trehalosi in neonatal calves in New Zealand.

Case history: In July and August 2019, 15/40, ≤48-hour-old calves became acutely ill. The calves were all born on-farm, transferred to pens soon after birth, and fed with "gold" colostrum. The hygiene, biosecurity and ventilation in the pens were poor. Of the 15 calves, 11 died or were euthanised and four calves, ≤48-hour-old, that became acutely ill later in the outbreak were treated with cefquinome, a fourth-generation cephalosporin, and recovered. Clinical findings: The affected calves presented with acute recumbency, lethargy, tachypnoea, tachycardia, increased lung sounds, inability to stand or feed, and dehydration without pyrexia. Pathological findings: Gross findings in a calf that died naturally included fibrinous pleuropneumonia, marked oedematous expansion of the interlobular septa, especially in the ventral lung lobes, fibrinous polyserositis and fibrinous polyarthritis. A second calf that was euthanised had strikingly similar lung lesions. Histologically, the pulmonary interlobular septa of both calves were prominently expanded by oedema, dilated lymphatics and the infiltration of numerous neutrophils and macrophages interspersed with small Gram-negative rod bacteria. Likewise, the visceral pleura showed fibrinopurulent inflammation with numerous small Gram-negative rods. Microbiological findings: Microbial culture and matrix-assisted laser desorption ionisation time-of-flight (MALDI-TOF) mass spectrometry identified Bibersteinia trehalosi in the lung, stifle joint and peritoneal cavity of the first calf and lung of the second. Diagnosis: B. trehalosi acute fibrinous pleuropneumonia and septicaemia. Clinical relevance: This is the first report of the clinical findings and histological lesions of B. trehalosi pleuropneumonia and septicaemia in calves in New Zealand. The pathogen is isolated with increasing frequency from cases of bovine respiratory disease in dairy cows, feedlot cattle and calves in the United Kingdom and North America. The importance of microbial culture in cases such as this with unusual lung lesions in calves <48 hours of age, cannot be over emphasised. Cefquinome was administered to all remaining heifer calves and four calves that became ill later in the outbreak recovered after cefquinome treatment.

Candidate genes and gene markers for the resistance to porcine pleuropneumonia.

Actinobacillus (A.) pleuropneumoniae is one of the most important respiratory pathogens in global pig production. Antimicrobial treatment and vaccination provide only limited protection, but genetic disease resistance is a very promising alternative for sustainable prophylaxis. Previous studies have discovered multiple QTL that may explain up to 30% of phenotypic variance. Based on these findings, the aim of the present study was to use genomic sequencing to identify genetic markers for resistance to pleuropneumonia in a segregating commercial German Landrace line. 163 pigs were infected with A. pleuropneumoniae Serotype 7 through a standardized aerosol infection method. Phenotypes were accurately defined on a clinical, pathological and microbiological basis. The 58 pigs with the most extreme phenotypes were genotyped by sequencing (next-generation sequencing). SNPs were used in a genome-wide association study. The study identified genome-wide associated SNPs on three chromosomes, two of which were chromosomes of QTL which had been mapped in a recent experiment. Each variant explained up to 20% of the total phenotypic variance. Combined, the three variants explained 52.8% of the variance. The SNPs are located in genes involved in the pathomechanism of pleuropneumonia. This study confirms the genetic background for the host's resistance to pleuropneumonia and indicates a potential role of three candidates on SSC2, SSC12 and SSC15. Favorable gene variants are segregating in commercial populations. Further work is needed to verify the results in a controlled study and to identify the functional QTN.

Contagious Bovine Pleuropneumonia: A Comprehensive Overview.

Contagious bovine pleuropneumonia (CBPP) is a respiratory disease of cattle that is listed as notifiable by the World Organization for Animal Health. It is endemic in sub-Saharan Africa and causes important productivity losses due to the high mortality and morbidity rates. CBPP is caused by Mycoplasma mycoides subsp. mycoides (Mmm) and is characterized by severe fibrinous bronchopneumonia and pleural effusion during the acute to subacute stages and by pulmonary sequestra in chronic cases. Additional lesions can be detected in the kidneys and in the carpal and tarsal joints of calves. Mmm infection occurs through the inhalation of infected aerosol droplets. After the colonization of bronchioles and alveoli, Mmm invades blood and lymphatic vessels and causes vasculitis. Moreover, Mmm can be occasionally demonstrated in blood and in a variety of other tissues. In the lung, Mmm antigen is commonly detected on bronchiolar and alveolar epithelial cells, in lung phagocytic cells, within the wall of blood and lymphatic vessels, inside necrotic areas, and within tertiary lymphoid follicles. Mmm antigen can also be present in the cytoplasm of macrophages within lymph node sinuses, in the germinal center of lymphoid follicles, in glomerular endothelial cells, and in renal tubules. A complete pathological examination is of great value for a rapid presumptive diagnosis, but laboratory investigations are mandatory for definitive diagnosis. The purpose of this review is to describe the main features of CBPP including the causative agent, history, geographic distribution, epidemiology, clinical course, diagnosis, and control. A special focus is placed on gross and microscopic lesions in order to familiarize veterinarians with the pathology and pathogenesis of CBPP.

Link between Heterotrophic Carbon Fixation and Virulence in the Porcine Lung Pathogen Actinobacillus pleuropneumoniae.

Actinobacillus pleuropneumoniae is a capnophilic pathogen of the porcine respiratory tract lacking enzymes of the oxidative branch of the tricarboxylic acid (TCA) cycle. We previously claimed that A. pleuropneumoniae instead uses the reductive branch in order to generate energy and metabolites. Here, we show that bicarbonate and oxaloacetate supported anaerobic growth of A. pleuropneumoniae Isotope mass spectrometry revealed heterotrophic fixation of carbon from stable isotope-labeled bicarbonate by A. pleuropneumoniae, which was confirmed by nano-scale secondary ion mass spectrometry at a single-cell level. By gas chromatography-combustion-isotope ratio mass spectrometry we could further show that the labeled carbon atom is mainly incorporated into the amino acids aspartate and lysine, which are derived from the TCA metabolite oxaloacetate. We therefore suggest that carbon fixation occurs at the interface of glycolysis and the reductive branch of the TCA cycle. The heme precursor δ-aminolevulinic acid supported growth of A. pleuropneumoniae, similar to bicarbonate, implying that anaplerotic carbon fixation is needed for heme synthesis. However, deletion of potential carbon-fixing enzymes, including PEP-carboxylase (PEPC), PEP-carboxykinase (PEPCK), malic enzyme, and oxaloacetate decarboxylase, as well as various combinations thereof, did not affect carbon fixation. Interestingly, generation of a deletion mutant lacking all four enzymes was not possible, suggesting that carbon fixation in A. pleuropneumoniae is an essential metabolic pathway controlled by a redundant set of enzymes. A double deletion mutant lacking PEPC and PEPCK was not impaired in carbon fixation in vitro but showed reduction of virulence in a pig infection model.

Establishment and comparison of Actinobacillus pleuropneumoniae experimental infection model in mice and piglets.

Actinobacillus pleuropneumoniae (APP) causes porcine pleuropneumonia, a disease responsible for substantial losses in the worldwide pig industry. In this study, outbred Kunming (KM) and Institute of Cancer Research (ICR) mice were evaluated as alternative mice models for APP research. After intranasal infection of serotype 5 reference strain L20, there was less lung damage and a lower clinical sign score in ICR compared to KM mice. However, ICR mice showed more obvious changes in body weight loss, the amount of immune cells (such as neutrophils and lymphocytes) and cytokines (such as IL-6, IL-1ß and TNF-α) in blood and bronchoalveolar lavage fluid (BALF). The immunological changes observed in ICR mice closely mimicked those found in piglets infected with L20. While both ICR and KM mice are susceptible to APP and induce pathological lesions, we suggest that ICR and KM mice are more suitable for immunological and pathogenesis studies, respectively. The research lays the theoretical basis for determine that mice could replace pigs as the APP infection model and it is of significance for the study of APP infection in the laboratory.

Cytoplasmic glycoengineering of Apx toxin fragments in the development of Actinobacillus pleuropneumoniae glycoconjugate vaccines.

BACKGROUND: Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia and represents a major burden to the livestock industry. Virulence can largely be attributed to the secretion of a series of haemolytic toxins, which are highly immunogenic. A. pleuropneumoniae also encodes a cytoplasmic N-glycosylation system, which involves the modification of high molecular weight adhesins with glucose residues. Central to this process is the soluble N-glycosyl transferase, ngt, which is encoded in an operon with a subsequent glycosyl transferase, agt. Plasmid-borne recombinant expression of these genes in E. coli results in the production of a glucose polymer on peptides containing the appropriate acceptor sequon, NX(S/T). However to date, there is little evidence to suggest that such a glucose polymer is formed on its target peptides in A. pleuropneumoniae. Both the toxins and glycosylation system represent potential targets for the basis of a vaccine against A. pleuropneumoniae infection. RESULTS: In this study, we developed cytoplasmic glycoengineering to construct glycoconjugate vaccine candidates composed of soluble toxin fragments modified by glucose. We transferred ngt and agt to the chromosome of Escherichia coli in order to generate a native-like operon for glycoengineering. A single chromosomal copy of ngt and agt resulted in the glucosylation of toxin fragments by a short glycan, rather than a polymer. CONCLUSIONS: A vaccine candidate that combines toxin fragment with a conserved glycan offers a novel approach to generating epitopes important for both colonisation and disease progression.

Carbon source utilisation and evaluation of the Biolog system in the identification of Actinobacillus pleuropneumoniae.

Sixty-eight Actinobacillus pleuropneumoniae strains were isolated from porcine acute pleuropneumonia cases from different parts of Hungary between 2000 and 2014. A total of 41 isolates were identified as A. pleuropneumoniae bio-type I and 27 strains as biotype II based on cultural, morphological and biochemical characteristics. The aim of this study was to evaluate metabolic fingerprinting in the species-level identification of A. pleuropneumoniae isolates. Utilisation of carbon sources by these field isolates and six reference strains was characterised by the Biolog system (GN2 Microplate, MicroLog3 Version 4.20.05 software). Twenty-nine field strains were correctly identified by the Biolog system as A. pleuropneumoniae, 36 strains as A. lignieresii, two strains as H. paraphrohaemolyticus and one strain as A. equuli after 24 h of incubation. Among the six A. pleuropneumoniae reference strains the Biolog system identified one strain as A. pleuropneumoniae, four as A. lignieresii and one as H. paraphrohaemolyticus. There was no correlation between biotypes and serotypes of A. pleuropneumoniae and the carbon source utilisation pattern and species identification by the Biolog system. our data indicate that the efficacy of the Biolog system used here could be improved by including phenotypes of more A. pleuropneumoniae strains representing a wider geographical occurrence into the database.

Comparative evaluation of different therapeutic protocols for contagious caprine pleuropneumonia in Himalayan Pashmina goats.

Therapeutic management of contagious caprine pleuroneumonia (CCPP) involves mostly the use of oxytetracycline followed by enrofloxacin and rarely tylosin. In many parts of the world including India, the former antibiotics are commonly available than the latter. Therefore, prolonged use of the same leads to the development of antibiotic resistance and decreased efficacy of drug. Besides, inflammatory and allergic pathogenesis of CCPP envisages combination therapy. In this study, we evaluated the effectiveness of the combination therapy using different antibiotics (oxytetracycyline @ 10: group I, enrofloxacin @ 5 group II, and tylosin: group III, @ 10 mg/kg body weight), along with anti-inflammatory (meloxicam @ 0.5 mg/kg) and anti-allergic (pheneramine maleate @ 1.0 mg/kg) drugs. These drugs were given intramuscularly at the interval of 48 h for four times in three test groups (n = 10) of Pashmina goats, viz. groups I, II, and III, respectively, affected with CCPP. Group IV (n = 10) was kept as healthy control when group V (n = 10) treated with oxytetracycline @ 10 mg/kg alone was used as positive control. Clinical signs, clinical parameters, pro-inflammatory cytokine (tumor necrosis factor alpha (TNF-α)), and oxidative stress indices (total oxidant status (TOS), total antioxidant status (TAS)) were evaluated at hours 0, 48, 96, and 144 of experimental trial. Tylosin-based combination therapy resulted in a rapid and favorable recovery resulting in restoration of normal body temperature (102.46 ± 0.31 °F), respiration rate (16.30 ± 0.79 per minute), and heart rate (89.50 ± 2.63 per minute) compared to the oxytetracycline (102.95 ± 0.13, 21.30 ± 1.12, 86.00 ± 2.33, respectively) and enrofloxacin (102.97 ± 0.19, 21.00 ± 1.25, 90.00 ± 2.58, respectively) treated groups. By hour 144, all the groups showed restoration of clinical parameters of normal health and diminishing signs of CCPP, viz. fever, dyspnea, coughing, nasal discharge, weakness, and pleurodynia. Significant (P ≤ 0.05) decrease in levels of TNF-α and non-significant (P > 0.05) decrease in levels of TOS and an increase in levels of TAS were noted from hour 0 to 144 in all the test groups. Within the groups, no significant (P > 0.05) change was noted in TNF-α, TOS, and TAS levels; however, TNF-α levels were comparatively lower in group III. Hematological parameters did not differ significantly (P > 0.05). From these findings, it can be inferred that tylosin-based combination therapy is relatively better for early, rapid, and safe recovery besides minimizing inflammatory and oxidative cascade in CCPP affected Pashmina goats compared to oxytetracycline- and enrofloxacin-based therapies.

Activation of Porcine Alveolar Macrophages by Actinobacillus pleuropneumoniae Lipopolysaccharide via the Toll-Like Receptor 4/NF-κB-Mediated Pathway.

Actinobacillus pleuropneumoniae is the causative agent of porcine contagious pleuropneumonia. Overproduction of proinflammatory cytokines, like interleukin-1ß (IL-1ß), IL-6, tumor necrosis factor alpha, and resistin, in the lung is an important feature of A. pleuropneumoniae infection. These proinflammatory cytokines enhance inflammatory and immunological responses. However, the mechanism that leads to cytokine production remains unclear. As a major virulence factor of A. pleuropneumoniae, lipopolysaccharide (LPS) may act as a potent stimulator of Toll-like receptor 4 (TLR4), triggering a number of intracellular signaling pathways that lead to the synthesis of proinflammatory cytokines. Porcine alveolar macrophages (PAMs) are the first line of defense against pathogenic microbes during pathogen invasion. The results of the present study demonstrate that A. pleuropneumoniae LPS induces PAMs to produce inflammatory cytokines in time- and dose-dependent manners. Moreover, PAMs were activated by A. pleuropneumoniae LPS, resulting in upregulation of signaling molecules, including TLR4, MyD88, TRIF-related adaptor molecule, and NF-κB. In contrast, the activation effects of A. pleuropneumoniae LPS on PAMs could be suppressed by specific inhibitors, like small interfering RNA and Bay11-7082. Taken together, our data indicate that A. pleuropneumoniae LPS can induce PAMs to produce proinflammatory cytokines via the TLR4/NF-κB-mediated pathway. These findings partially reveal the mechanism of the overproduction of proinflammatory cytokines in the lungs of swine with A. pleuropneumoniae infection and may provide targets for the prevention of A. pleuropneumoniae-induced pneumonia. All the data could be used as a reference for the pathogenesis of respiratory infection.