RESUMO
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.
Assuntos
Infecções por Actinobacillus , Actinobacillus pleuropneumoniae , Antibacterianos , Farmacorresistência Bacteriana , Doenças dos Suínos , Tianfenicol , Animais , Actinobacillus pleuropneumoniae/efeitos dos fármacos , Actinobacillus pleuropneumoniae/genética , Actinobacillus pleuropneumoniae/isolamento & purificação , Actinobacillus pleuropneumoniae/classificação , Tianfenicol/análogos & derivados , Tianfenicol/farmacologia , Suínos , Itália/epidemiologia , Doenças dos Suínos/microbiologia , Antibacterianos/farmacologia , Infecções por Actinobacillus/microbiologia , Infecções por Actinobacillus/veterinária , Farmacorresistência Bacteriana/genética , Filogenia , Testes de Sensibilidade Microbiana , Sequenciamento Completo do Genoma , Fazendas , Pleuropneumonia/microbiologia , Pleuropneumonia/veterinária , Plasmídeos/genética , Proteínas de Bactérias/genética , Polimorfismo de Nucleotídeo Único , Virulência/genéticaRESUMO
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.
Assuntos
Infecções por Actinobacillus , Actinobacillus pleuropneumoniae , Antibacterianos , Testes de Sensibilidade Microbiana , Filogenia , Doenças dos Suínos , Sequenciamento Completo do Genoma , Actinobacillus pleuropneumoniae/genética , Actinobacillus pleuropneumoniae/efeitos dos fármacos , Actinobacillus pleuropneumoniae/classificação , Actinobacillus pleuropneumoniae/isolamento & purificação , Suínos , Animais , Infecções por Actinobacillus/microbiologia , Infecções por Actinobacillus/veterinária , Infecções por Actinobacillus/transmissão , Doenças dos Suínos/microbiologia , Doenças dos Suínos/transmissão , Antibacterianos/farmacologia , Pleuropneumonia/microbiologia , Pleuropneumonia/veterinária , Genótipo , Genoma Bacteriano , Farmacorresistência Bacteriana/genética , Espanha/epidemiologiaRESUMO
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.
Assuntos
Anticorpos Monoclonais , Pleuropneumonia , Animais , Bovinos , Proteínas de Membrana , China , Ensaio de Imunoadsorção Enzimática , CabrasRESUMO
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.
Assuntos
Infecções por Actinobacillus , Actinobacillus pleuropneumoniae , Pleuropneumonia , Doenças dos Suínos , Tianfenicol/análogos & derivados , Suínos , Animais , Sorogrupo , Testes de Sensibilidade Microbiana/veterinária , Enrofloxacina , Fazendas , Estudos Retrospectivos , Pleuropneumonia/epidemiologia , Pleuropneumonia/veterinária , Pleuropneumonia/microbiologia , Antibacterianos/farmacologia , Sulfametoxazol/farmacologia , Trimetoprima/farmacologia , Itália/epidemiologia , Doenças dos Suínos/epidemiologia , Doenças dos Suínos/microbiologia , Infecções por Actinobacillus/epidemiologia , Infecções por Actinobacillus/veterinária , Infecções por Actinobacillus/microbiologia , Sorotipagem/veterináriaRESUMO
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.
Assuntos
Doenças dos Bovinos , Mycoplasma mycoides , Mycoplasma , Pleuropneumonia Contagiosa , Pleuropneumonia , Pneumonia por Mycoplasma , Bovinos , Animais , Uganda/epidemiologia , Estudos Soroepidemiológicos , Pleuropneumonia/veterinária , Pleuropneumonia Contagiosa/epidemiologia , Pneumonia por Mycoplasma/veterináriaRESUMO
Vaccination is the most cost-effective tool to control contagious bovine pleuropneumonia. The vaccines currently used in Africa are derived from a live strain called T1, which was attenuated by passage in embryonated eggs and broth culture. The number of passages is directly correlated to the degree of attenuation of the vaccinal strains and inversely correlated to their immunogenicity in cattle. Current quality control protocols applied to vaccine batches allow the assessment of identity, purity, and titers, but cannot assess the level of genetic drift form the parental vaccine strains. Deep sequencing was used to assess the genetic drift generated over controlled in vitro passages of the parental strain, as well as on commercial vaccine batches. Signatures of cloning procedures were detected in some batches, which imply a deviation from the standard production protocol. Deep sequencing is proposed as a new tool for the identity and stability control of T1 vaccines.
Assuntos
Doenças dos Bovinos , Mycoplasma mycoides , Pleuropneumonia Contagiosa , Pleuropneumonia , Animais , Bovinos , Vacinas Bacterianas/genética , África , Vacinas Atenuadas/genética , Controle de Qualidade , Sequenciamento de Nucleotídeos em Larga Escala , Pleuropneumonia Contagiosa/prevenção & controle , Mycoplasma mycoides/genéticaRESUMO
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.
Assuntos
Infecções por Actinobacillus , Actinobacillus pleuropneumoniae , Pleuropneumonia , Doenças dos Roedores , Doenças dos Suínos , Animais , Suínos , Camundongos , Pleuropneumonia/microbiologia , Pleuropneumonia/veterinária , Biofilmes , Actinobacillus pleuropneumoniae/metabolismo , Proteína Receptora de AMP Cíclico/genética , Pulmão/microbiologia , Infecções por Actinobacillus/veterinária , Infecções por Actinobacillus/microbiologia , Doenças dos Suínos/microbiologiaRESUMO
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.
Assuntos
Drenagem , Tempo de Internação , Pleuropneumonia , Toracostomia , Humanos , Masculino , Feminino , Toracostomia/métodos , Toracostomia/efeitos adversos , Estudos Transversais , Pré-Escolar , Lactente , Pleuropneumonia/cirurgia , Tempo de Internação/estatística & dados numéricos , Drenagem/métodos , Estudos Retrospectivos , Resultado do Tratamento , Complicações Pós-Operatórias/epidemiologia , Criança , Cirurgia Torácica Vídeoassistida/métodos , AdolescenteRESUMO
Ethiopia is one of the largest African countries where livestock farming represent a relevant resource for the economy and the livelihood of the population. Contagious bovine pleuropneumonia (CBPP) is among the transboundaries animal disease that is hindering cattle farming in Ethiopia. Due to the limited resources of veterinary services, disease control and surveillance is discontinuous and occasional field investigations of target areas contribute to depict disease spreading in the country. The study was conducted to determine the seroprevalence, at herd and animal level, and identify the risk factors involved in CBPP diffusion and persistence in the Borana pastoral zone. A total of 498 serum samples were collected from 120 cattle herds and tested using competitive Enzyme-Linked Immunosorbent Assay (c-ELISA). Of 120 herds sampled, 37 (30.83%; (95% CI = 22.73-39.91%) were tested positive to CBPP antibody. Out of 498 sera samples tested 46 (9.24%; 95% CI = 6.84-12.13%) were positive. The highest prevalence was observed in Teltele (12/95; 12.90%; 95% CI = 6.7-21%) followed by Yabello (12/104; 11.54%; 95% CI = 6.1-19.3%) and Arero (10/91; 10.99%; 95% CI = 95% CI = 5.4-19.3%), whereas the lowest prevalence was observed in Gomole (5/101; 6.42%; 95% CI = 1.6-11.2%) and Dubluk (7/109; 4.95%; 95% CI = 2.6-12.8%) districts and statistically not significant (p > 0.05). Results of multivariate logistic regression analysis revealed that, age, herd movement and herd size of the animals had statistically significant effect on sero-positivity to CBPP (p < 0.05). Sex, season and body condition were not significantly (p > 0.05) associated with the occurrence of CBPP. The study confirms that CBPP is persistent in the territory and remain as a major problem that affects health and productivity of cattle. Therefore, awareness creation to the pastoralists in the study area about the effect of CBPP and designing appropriate control methods has a paramount importance to improve the health and productivity of cattle production in the area.
Assuntos
Doenças dos Bovinos , Pleuropneumonia Contagiosa , Pleuropneumonia , Pneumonia por Mycoplasma , Animais , Bovinos , Etiópia/epidemiologia , Prevalência , Estudos Soroepidemiológicos , Pleuropneumonia/veterinária , Doenças dos Bovinos/epidemiologia , Pleuropneumonia Contagiosa/epidemiologia , Estudos Transversais , Pneumonia por Mycoplasma/veterináriaRESUMO
Background: Emerging infectious diseases pose a significant threat to both human and animal populations. Rapid de novo identification of protective antigens from a clinical isolate and development of an antigen-matched vaccine is a golden strategy to prevent the spread of emerging novel pathogens. Methods: Here, we focused on Actinobacillus pleuropneumoniae, which poses a serious threat to the pig industry, and developed a general workflow by integrating proteosurfaceomics, secretomics, and BacScan technologies for the rapid de novo identification of bacterial protective proteins from a clinical isolate. Results: As a proof of concept, we identified 3 novel protective proteins of A. pleuropneumoniae. Using the protective protein HBS1_14 and toxin proteins, we have developed a promising multivalent subunit vaccine against A. pleuropneumoniae. Discussion: We believe that our strategy can be applied to any bacterial pathogen and has the potential to significantly accelerate the development of antigen-matched vaccines to prevent the spread of an emerging novel bacterial pathogen.
Assuntos
Actinobacillus pleuropneumoniae , Pleuropneumonia , Animais , Humanos , Suínos , Antígenos de Bactérias , Vacinas Bacterianas , Proteínas de Bactérias , Pleuropneumonia/microbiologia , Pleuropneumonia/prevenção & controleRESUMO
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.
Assuntos
Infecções por Actinobacillus , Actinobacillus pleuropneumoniae , Ascomicetos , Infecções por Mycoplasma , Pleuropneumonia , Pneumonia , Doenças dos Suínos , Animais , Camundongos , Suínos , Neutrófilos , Pneumonia/veterinária , Pleuropneumonia/veterinária , Infecções por Mycoplasma/veterinária , Infecções por Actinobacillus/veterinária , PulmãoRESUMO
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.
Assuntos
Infecções por Actinobacillus , Actinobacillus pleuropneumoniae , Mycoplasma , Pleuropneumonia , Doenças dos Suínos , Suínos , Animais , Pleuropneumonia/microbiologia , Pleuropneumonia/veterinária , Sorogrupo , Abscesso/patologia , Abscesso/veterinária , Infecções por Actinobacillus/microbiologia , Infecções por Actinobacillus/veterinária , Doenças dos Suínos/microbiologia , Pulmão/patologiaRESUMO
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.
Assuntos
Infecções por Actinobacillus , Actinobacillus pleuropneumoniae , Pleuropneumonia , Doenças dos Suínos , Animais , Suínos , Sorogrupo , Actinobacillus pleuropneumoniae/genética , Infecções por Actinobacillus/epidemiologia , Infecções por Actinobacillus/veterinária , Infecções por Actinobacillus/diagnóstico , Doenças dos Suínos/epidemiologia , Doenças dos Suínos/diagnóstico , Pleuropneumonia/epidemiologia , Pleuropneumonia/veterinária , Pleuropneumonia/diagnóstico , PolissacarídeosRESUMO
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.
Assuntos
Infecções por Actinobacillus , Actinobacillus pleuropneumoniae , Actinobacillus , Infecções por Mycoplasma , Pleuropneumonia , Doenças dos Suínos , Animais , Suínos , Camundongos , Pleuropneumonia/microbiologia , Receptor 4 Toll-Like/metabolismo , Junções Íntimas , Pulmão/microbiologia , Infecções por Actinobacillus/tratamento farmacológico , Infecções por Actinobacillus/microbiologia , Chá/metabolismo , Doenças dos Suínos/microbiologiaRESUMO
Contagious caprine pleuropneumonia (CCPP) is a serious contagious disease of goats, sheep and wild ruminants caused by Mycoplasma capricolum subspecies capripneumoniae. The disease is known for its high mortality, morbidity and economic losses. A cross-sectional study using multistage cluster sampling technique was conducted in Amhara region from January 2019 to July 2019 to estimate seroprevalence and identify risk factors of CCPP occurrence in the region. A total of 2080 goats from 61 villages and 12 districts of the region were tested for CCPP serostatus using Competitive Enzyme-Linked Immunosorbent Assay (C-ELISA). A multilevel mixed-effect logistic regression model was used to identify risk factors of CCPP seropositivity at animal and flock-level. The serum sample results revealed an overall animal level seroprevalence of 5.1% (95% CI: 3.8-6.6) and flock-level prevalence of 26.0% (95% CI: 19.7-33.4). At individual animal level, presence of other health problems (OR = 45.9 (95% CI: 25.3-83.4)), age (adult age (OR = 6.2 (95% CI:3.4-11.4)) and old age (OR = 13.1 (95% CI: 6.2-27.8))), and breed type (Afar (OR= 32.3 (95% CI: 2.9-366.1)), Central highland (OR=13.7 (95% CI: 1.3-140.6)), and western highland (OR=16.2 (95% CI: 1.4-185.7))) were identified as risk factors for CCPP seropositivity. In contrast, contact with other flocks (OR = 59.9 (95% CI: 6.1-585.6)), presence of trade route (OR = 3.1 (95% CI: 1.0-9.1)) and presence of sheep (OR = 2.6 (95% CI: 1.2-5.7)) were flock-level risk factors for CCPP seropositivity. Generally, CCPP appears to be common among goats of Amhara region. Goat flocks dominated with older age animals; breeds of Afar, central highland, and western highland; raise with sheep; have contact with other flocks; and kept along trade routes are more at risk for CCPP. Hence, awareness creation to the producers, movement control, and regular prophylactic vaccination should be considered to control CCPP in Amhara region.
Assuntos
Doenças das Cabras , Pleuropneumonia Contagiosa , Pleuropneumonia , Pneumonia por Mycoplasma , Animais , Ovinos , Cabras , Etiópia/epidemiologia , Pleuropneumonia/veterinária , Estudos Soroepidemiológicos , Estudos Transversais , Doenças das Cabras/epidemiologia , Pleuropneumonia Contagiosa/epidemiologia , Pleuropneumonia Contagiosa/prevenção & controle , Pneumonia por Mycoplasma/veterinária , Fatores de RiscoRESUMO
Acute pleuropneumonia in swine, caused by Actinobacillus pleuropneumoniae, is characterized by a high and sustained fever. Fever creates an adverse environment for many bacteria, leading to reduced bacterial proliferation; however, most pathogenic bacteria can tolerate higher temperatures. CpxAR is a two-component regulation system, ubiquitous among Gram-negative bacteria, which senses and responds to envelope alterations that are mostly associated with protein misfolding in the periplasm. Our previous study showed that CpxAR is necessary for the optimal growth of Actinobacillus pleuropneumoniae under heat stress. Here, we showed that mutation of the type IV pilin gene apfA rescued the growth defect of the cpxAR deletion strain under heat stress. RNA sequencing (RNA-seq) analyses revealed that 265 genes were differentially expressed in the ΔcpxAR strains grown at 42°C, including genes involved in type IV pilus biosynthesis. We also demonstrated direct binding of the CpxR protein to the promoter of the apf operon by an electrophoretic mobility shift assay and identified the binding site by a DNase I footprinting assay. In conclusion, our results revealed the important role of CpxAR in A. pleuropneumoniae resistance to heat stress by directly suppressing the expression of ApfA. IMPORTANCE Heat acts as a danger signal for pathogens, especially those infecting mammalian hosts in whom fever indicates infection. However, some bacteria have evolved exquisite mechanisms to survive under heat stress. Studying the mechanism of resistance to heat stress is crucial to understanding the pathogenesis of A. pleuropneumoniae during the acute stage of infection. Our study revealed that CpxAR plays an important role in A. pleuropneumoniae resistance to heat stress by directly suppressing expression of the type IV pilin protein ApfA.
Assuntos
Actinobacillus pleuropneumoniae , Pleuropneumonia , Doenças dos Suínos , Animais , Actinobacillus pleuropneumoniae/genética , Proteínas de Fímbrias/genética , Proteínas de Fímbrias/metabolismo , Fímbrias Bacterianas/genética , Fímbrias Bacterianas/metabolismo , Resposta ao Choque Térmico , Óperon , Pleuropneumonia/microbiologia , Suínos , Doenças dos Suínos/metabolismoRESUMO
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.
Assuntos
Infecções por Actinobacillus , Actinobacillus pleuropneumoniae , Mycoplasma , Pleuropneumonia , Doenças dos Suínos , Suínos , Animais , Pleuropneumonia/veterinária , Infecções por Actinobacillus/veterinária , Proteômica , Proteoma/metabolismo , Antígenos T-Independentes/metabolismo , Cromatografia Líquida , Proteínas de Bactérias/metabolismo , Espectrometria de Massas em Tandem , Fatores de Virulência/metabolismo , Fatores de Alongamento de PeptídeosRESUMO
Outer membrane vesicles (OMVs) are spontaneously released by Gram-negative bacteria, including Actinobacillus pleuropneumoniae, which causes contagious pleuropneumonia in pigs and leads to considerable economic losses in the swine industry worldwide. A. pleuropneumoniae OMVs have previously been demonstrated to contain Apx toxins and proteases, as well as antigenic proteins. Nevertheless, comprehensive characterizations of their contents and interactions with host immune cells have not been made. Understanding the protein compositions and immunomodulating ability of A. pleuropneumoniae OMVs could help illuminate their biological functions and facilitate the development of OMV-based applications. In the current investigation, we comprehensively characterized the proteome of native A. pleuropneumoniae OMVs. Moreover, we qualitatively and quantitatively compared the OMV proteomes of a wild-type strain and three mutant strains, in which relevant genes were disrupted to increase OMV production and/or produce OMVs devoid of superantigen PalA. Furthermore, the interaction between A. pleuropneumoniae OMVs and porcine alveolar macrophages was also characterized. Our results indicate that native OMVs spontaneously released by A. pleuropneumoniae MIDG2331 appeared to dampen the innate immune responses by porcine alveolar macrophages stimulated by either inactivated or live parent cells. The findings suggest that OMVs may play a role in manipulating the porcine defense during the initial phases of the A. pleuropneumoniae infection. IMPORTANCE Owing to their built-in adjuvanticity and antigenicity, bacterial outer membrane vesicles (OMVs) are gaining increasing attention as potential vaccines for both human and animal use. OMVs released by Actinobacillus pleuropneumoniae, an important respiratory pathogen in pigs, have also been investigated for vaccine development. Our previous studies have shown that A. pleuropneumoniae secretes OMVs containing multiple immunogenic proteins. However, immunization of pigs with these vesicles was not able to relieve the pig lung lesions induced by the challenge with A. pleuropneumoniae, implying the elusive roles that A. pleuropneumoniae OMVs play in host-pathogen interaction. Here, we showed that A. pleuropneumoniae secretes OMVs whose yield and protein content can be altered by the deletion of the nlpI and palA genes. Furthermore, we demonstrate that A. pleuropneumoniae OMVs dampen the immune responses in porcine alveolar macrophages stimulated by A. pleuropneumoniae cells, suggesting a novel mechanism that A. pleuropneumoniae might use to evade host defense.
Assuntos
Infecções por Actinobacillus , Actinobacillus pleuropneumoniae , Pleuropneumonia , Animais , Infecções por Actinobacillus/veterinária , Infecções por Actinobacillus/microbiologia , Actinobacillus pleuropneumoniae/genética , Proteínas da Membrana Bacteriana Externa/genética , Vacinas Bacterianas , Imunidade , Macrófagos Alveolares , Peptídeo Hidrolases , Pleuropneumonia/veterinária , Pleuropneumonia/microbiologia , Pleuropneumonia/prevenção & controle , Proteoma , Superantígenos , SuínosRESUMO
Actinobacillus pleuropneumoniae (APP) is the causative agent of pleuropneumonia in pigs, one of the most relevant bacterial respiratory diseases in the swine industry. To date, 19 serotypes have been described based on capsular polysaccharide typing with significant virulence dissimilarities. In this study, 16 APP isolates from Spanish origin were selected to perform antimicrobial susceptibility tests and comparative genomic analysis using whole genome sequencing (WGS). To obtain a more comprehensive worldwide molecular epidemiologic analyses, all APP whole genome assemblies available at the National Center for Biotechnology Information (NCBI) at the time of the study were also included. An in-house in silico PCR approach enabled the correct serotyping of unserotyped or incorrectly serotyped isolates and allowed for the discrimination between serotypes 9 and 11. A pangenome analysis identified the presence or absence of gene clusters to be serotype specific, as well as virulence profile analyses targeting the apx operons. Antimicrobial resistance genes were correlated to the presence of specific plasmids. Altogether, this study provides new insights into the genetic variability within APP serotypes, correlates phenotypic tests with bioinformatic analyses and manifests the benefits of populated databases for a better assessment of diversity and variability of relatively unknown pathogens. Overall, genomic comparative analysis enhances the understanding of transmission and epidemiological patterns of this species and suggests vertical transmission of the pathogen, including the resistance genes, within the Spanish integrated systems. IMPORTANCE Pleuropneumonia is one of the most relevant respiratory infections in the swine industry. Despite Actinobacillus pleuropneumoniae (APP) being one of the most important pathogens in the pig production, this is the first comparative study including all available whole genome sequencing data from NCBI. Moreover, this study also includes 16 APP isolates of Spanish origin with known epidemiological relationships through vertical integrated systems. Genomic comparisons provided a deeper understanding of molecular and epidemiological knowledge between different APP serotypes. Furthermore, determination of resistance and toxin profiles allowed correlation with the presence of mobile genetic elements and specific serotype, respectively.