Hyopneumoniae Strain Research Articles

Efficacy of three innovative bacterin vaccines against experimental infection with Mycoplasma hyopneumoniae

New vaccine formulations that include novel strains of Mycoplasma hyopneumoniae and innovative adjuvants designed to induce cellular immunity could improve vaccine efficacy against this pathogen. The aim of this experimental study was to assess the efficacy of three experimental bacterin formulations based on M. hyopneumoniae field strain F7.2C which were able to induce cellular immunity. The formulations included a cationic liposome formulation with the Mincle receptor ligand trehalose 6,6-dibehenate (Lipo_DDA:TDB), a squalene-in-water emulsion with Toll-like receptor (TLR) ligands targeting TLR1/2, TLR7/8 and TLR9 (SWE_TLR), and a poly(lactic-co-glycolic acid) micro-particle formulation with the same TLR ligands (PLGA_TLR). Four groups of 12 M. hyopneumoniae-free piglets were primo- (day (D) 0; 39 days of age) and booster vaccinated (D14) intramuscularly with either one of the three experimental bacterin formulations or PBS. The pigs were endotracheally inoculated with a highly and low virulent M. hyopneumoniae strain on D28 and D29, respectively, and euthanized on D56. The main efficacy parameters were: respiratory disease score (RDS; daily), macroscopic lung lesion score (D56) and log copies M. hyopneumoniae DNA determined with qPCR on bronchoalveolar lavage (BAL) fluid (D42, D56). All formulations were able to reduce clinical symptoms, lung lesions and the M. hyopneumoniae DNA load in the lung, with formulation SWE_TLR being the most effective (RDSD28–D56 −61.90%, macroscopic lung lesions −88.38%, M. hyopneumoniae DNA load in BAL fluid (D42) −67.28%). Further experiments raised under field conditions are needed to confirm these results and to assess the effect of the vaccines on performance parameters.

Comparison of the sensitivity of laryngeal swabs and deep tracheal catheters for detection of Mycoplasma hyopneumoniae in experimentally and naturally infected pigs early and late after infection

Detection of Mycoplasma hyopneumoniae infection in live pigs is a critical component to measure the success of disease control or elimination strategies. However, in vivo diagnosis of M. hyopneumoniae is difficult and the imperfect sensitivity of diagnostic tools has been deemed as one of the main challenges. Here, the sensitivity of laryngeal swabs and deep tracheal catheters for detection of M. hyopneumoniae early and late after infection was determined using inoculation status as a gold standard in experimentally infected pigs and a Bayesian approach in naturally infected pigs. Three-hundred and twenty 8-week old seeder pigs were intra-tracheally inoculated with M. hyopneumoniae strain 232 and immediately placed with 1920 contact pigs to achieve a 1:6 seeder-to-contact ratio. A subset of seeders and contacts were longitudinally sampled at 7, 28, 97, and 113 days post-inoculation (dpi) and at 28, 56, 84, and 113 days post-exposure (dpe), respectively, using laryngeal swabs and deep tracheal catheters. Samples were tested for M. hyopneumoniae by a species-specific real-time PCR. The sensitivity of deep tracheal catheters was higher than the one obtained in laryngeal swabs at all samplings (seeders: 36% higher than laryngeal swabs at 7 dpi, 29% higher at 97 dpi, and 44% higher at 113 dpi; contacts: 51% higher at 56 dpe, 42% higher at 84 dpe, and 32% higher at 113 dpe). Our study indicates that deep tracheal catheters were a more sensitive sample than laryngeal swabs. The sensitivity of both sample types varied over time and by exposure method, and these factors should be considered when designing diagnostic strategies.

Pharmacokinetic/Pharmacodynamic Integration of Doxycycline Against Mycoplasma hyopneumoniae in an In Vitro Model.

Doxycycline is a broad-spectrum antibacterial drug. It is used widely to treat diseases caused by Mycoplasma species. We investigated the antibacterial activity of doxycycline against the Mycoplasma hyopneumoniae strain ATCC25934. The minimum inhibitory concentration (MIC) of doxycycline against M. hyopneumoniae determined by a microdilution method was 0.125 μg/ml. Static time–kill curves with constant drug concentrations (0–64 MIC) showed that a bacteriostatic effect occurred if the doxycycline concentration reached 4 MIC. Doxycycline produced a maximum antimycoplasmal effect (reduction of 2.76 log10CFU/ml) at 64 MIC within 48 h. The effect of doxycycline against M. hyopneumoniae was analyzed by a sigmoid E max model, and there was high correlation between the kill rate and doxycycline concentration (R 2 = 0.986). A one-compartment open model with first-order absorption was adopted and was used to simulate doxycycline pharmacokinetics in porcine plasma. The dynamic time–concentration curve showed that the area under the curve at 24 h (AUC24 h) and C max (peak concentration) after each drug administration was 1.78–48.4 μg h/ml and 0.16–3.41 μg/ml, respectively. The reduction of M. hyopneumoniae (log10CFU/ml) for 1, 2.5, 5, 7.5, 10, 15, 20, and 30 mg/kg body weight was 0.16, 1.29, 1.75, 2.94, 3.35, 3.91, 4.35, and 5.77, respectively, during the entire experiment, respectively. When the dose was >10 mg/kg body weight, continuous administration for 3 days could achieve a bactericidal effect. The correlation coefficient of AUC24 h/MIC, C max/MIC, and %T > MIC (the cumulative percentage of time over a 24-h period that the drug concentration exceeds the MIC) with antibacterial effect was 0.917, 0.923, and 0.823, respectively. Doxycycline showed concentration-dependent activity, and the value of AUC24 h/MIC and C max/MIC required to produce a drop of 1 log10CFU/ml was 164 h and 9.89, respectively.

Intracellular changes of a swine tracheal cell line infected with a Mycoplasma hyopneumoniae pathogenic strain

Mycoplasma hyopneumoniae is the etiological agent of enzootic pneumonia (EP), a widespread disease that causes major economic losses to the pig industry. The swine host response plays an important role in the outcome of M. hyopneumoniae infections. The whole proteome of newborn pig trachea (NPTr) epithelial cells infected with the M. hyopneumoniae pathogenic strain 7448 was analyzed using an LC-MS/MS approach to shed light on intracellular processes triggered in response to the pathogen. Overall, 853 swine protein species were identified, 156 of which were differentially represented in response to M. hyopneumoniae 7448 infection in comparison with non-infected control cells. These differentially represented proteins were categorized by function. Fifty-seven of them were assigned to the immune system and/or response to stimulus functional subcategories. Comparative expression analysis of these immune-related proteins in NPTr cells infected with attenuated or non-pathogenic mycoplasmas (M. hyopneumoniae J strain and M. flocculare, respectively) revealed proteins whose abundance was altered only in response to the pathogenic M. hyopneumoniae 7448 strain. Among these proteins, calcium homeostasis and endoplasmic reticulum stress-related biomarkers were detected, providing evidence of molecular mechanisms that might lead to swine cell apoptosis.

Tn-sequencing of Mycoplasma hyopneumoniae and Mycoplasma hyorhinis mutant libraries reveals non-essential genes of porcine mycoplasmas differing in pathogenicity

Mycoplasma hyopneumoniae and Mycoplasma hyorhinis are two phylogenetically related species colonizing the respiratory tract of pigs but differing in pathogenicity, the basis of which is not well resolved. We hypothesize that genes belonging to the species-specific portion of the genome and being non-essential during ideal laboratory growth conditions encode possible virulent determinants and are the driver of interspecies differences. To investigate this, transposon mutant libraries were generated for both species and a transposon sequencing (Tn-seq) method for mycoplasmas was established to identify non-essential genes. Tn-seq datasets combined with bidirectional Blastp analysis revealed that 101 out of a total 678 coding sequences (CDS) are species-specific and non-essential CDS of M. hyopneumoniae strain F7.2C, while 96 out of a total 751 CDS are species-specific and non-essential CDS in the M. hyorhinis strain JF5820. Among these species-specific and non-essential CDS were genes involved in metabolic pathways. In particular, the myo-inositol and the sialic acid pathways were found to be non-essential and therefore could be considered important to the specific pathogenicity of M. hyopneumoniae and M. hyorhinis, respectively. Such pathways could enable the use of an alternative energy source providing an advantage in their specific niche and might be interesting targets to knock out in order to generate attenuated live vaccines.

Intra-Species and Inter-Species Differences in Cytokine Production by Porcine Antigen-Presenting Cells Stimulated by Mycoplasma hyopneumoniae, M. hyorhinis, and M. flocculare.

Mycoplasma hyorhinis and M. flocculare are commonly co-isolated with M. hyopneumoniae (primary agent of swine enzootic pneumonia) in gross pneumonia-like lesions, but their involvement in the disease process remains unknown. T cells play an immuno-pathological role during mycoplasmal infections. Dendritic cells (DCs) are major antigen-presenting cells involved in T cell activation and differentiation. In this study, we investigated cytokine (IL-6, IL-8, IL-10, IL-12, and TNF-α) production by porcine bone-marrow-derived DCs (BM-DCs) stimulated by M. hyopneumoniae, M. hyorhinis, and/or M. flocculare. Results showed that cytokine production levels were relatively homogenous for all evaluated M. hyopneumoniae strains in contrast to M. hyorhinis and M. flocculare strains. The most noteworthy inter-species differences were the overall (i) lower IL-12 production capacity of M. hyopneumoniae, and (ii) higher TNF-α production capacity of M. flocculare. Co-stimulation of BM-DCs showed that M. hyorhinis dominated the IL-12 production independently of its association with M. hyopneumoniae or M. flocculare. In addition, a decreased BM-DC production of TNF-α was generally observed in the presence of mycoplasma associations. Lastly, M. flocculare association with M. hyopneumoniae increased BM-DC ability to secrete IL-10. A higher cytotoxicity level in BM-DCs stimulated by M. hyorhinis was also observed. Overall, this study demonstrated that the combination of M. hyorhinis or M. flocculare with M. hyopneumoniae may participate to the modulation of the immune response that might affect the final disease outcome.

Differential responses to stress of two Mycoplasma hyopneumoniae strains

Mycoplasma hyopneumoniae is a respiratory pathogen, causing porcine enzootic pneumonia. To survive in the porcine respiratory tract, M. hyopneumoniae must cope with both oxidative and heat stress imposed by the host. To get insights into M. hyopneumoniae stress responses and pathogenicity mechanisms, the protein profiles of two M. hyopneumoniae strains, pathogenic 7448 strain and non-pathogenic strain J, were surveyed under oxidative (OS) or heat (HS) stress. M. hyopneumoniae strains were submitted to OS (0.5% hydrogen peroxide) or HS (temperature shifts to 42 °C) conditions and protein profiling was carried out by LC-MS/MS and label-free quantitative analyses. Data are available via ProteomeXchange with identifier PXD012742. Qualitative and quantitative differences involving 40–60 M. hyopneumoniae proteins were observed for both strains when comparing bacteria exposed to OS or HS to non-treated controls. However, no differences in abundance were found in proteins classically related to stress responses, as peroxidases and chaperones, suggesting that these proteins would be constitutively present in both strains in the tested conditions. Interestingly, under stress conditions, more virulence-related proteins were detected in M. hyopneumoniae 7448 differentially represented proteins than in M. hyopneumoniae J, suggesting that stress may trigger a differential response of the corresponding genes, shared by both strains.

Characterization of Mycoplasma hyopneumoniae strains in vaccinated and non-vaccinated pigs from Spanish slaughterhouses

This study aimed to describe Mycoplasma hyopneumoniae (M. hyopneumoniae) genetic variability in vaccinated (V) and non-vaccinated (NV) slaughtered pigs showing cranio-ventral pulmonary consolidation (CVPC). Ten V and 10 NV fattening farms with respiratory problems associated to M. hyopneumoniae were selected. Lung lesions of one batch per farm were scored at slaughterhouse and the enzootic pneumonia (EP)-index was calculated. Moreover, three lungs showing the most extensive CVPC per farm were sampled and tested for M. hyopneumoniae detection by real-time (rt)-PCR. Positive samples with cycle threshold ≤30 were selected to be genotyped by sequencing of four loci (P97, P146, H1 and H5). Typing profiles (TP) were assigned considering four or two (P97, P146) loci. Five commercial vaccines for M. hyopneumoniae (VS) and two reference strains (RF) were also genotyped. The EP-index (mean ± SD) in NV farms (3.8 ± 1.9) was not significantly different from V ones (2.2 ± 1.3). From the 60 selected lungs, 46 (76.7%) were M. hyopneumoniae positive by rt-PCR (25/30 and 21/30 from NV and V farms, respectively), and 43 (93.5%) of those were successfully genotyped. A total of 24 different TP(12 in V and 12 in NV farms) or 17 TP(9 in V and 9 in NV farms, being one TP in both farm types) were identified by analyzing four or two loci, respectively. One to three TP per farm were detected, being different from VS and RF. Interestingly, farms with same breeding origin had the same TP using two loci, but such link was not found using four loci. Therefore, high inter-farm and limited intra-farm M. hyopneumoniae genetic variability were detected, but variability depended on the number of studied loci.

Antibiotic susceptibility testing of Mycoplasma hyopneumoniae field isolates from Central Europe for fifteen antibiotics by microbroth dilution method.

Mycoplasma hyopneumoniae infections are responsible for significant economic losses in the swine industry. Commercially available vaccines are not able to inhibit the colonisation of the respiratory tract by M. hyopneumoniae absolutely, therefore vaccination can be completed with antibiotic treatment to moderate clinical signs and improve performances of the animals. Antibiotic susceptibility testing of M. hyopneumoniae is time-consuming and complicated; therefore, it is not accomplished routinely. The aim of this study was to determine the in vitro susceptibility to 15 different antibiotics of M. hyopneumoniae isolates originating from Hungarian slaughterhouses and to examine single-nucleotide polymorphisms (SNPs) in genes affecting susceptibility to antimicrobials. Minimum inhibitory concentration (MIC) values of the examined antibiotics against 44 M. hyopneumoniae strains were determined by microbroth dilution method. While all of the tested antibiotics were effective against the majority of the studied strains, high MIC values of fluoroquinolones (enrofloxacin 2.5 μg/ml; marbofloxacin 5 μg/ml) were observed against one strain (MycSu17) and extremely high MIC values of macrolides and lincomycin (tilmicosin, tulathromycin and lincomycin >64 μg/ml; gamithromycin 64 μg/ml; tylosin 32 μg/ml and tylvalosin 2 μg/ml) were determined against another, outlier strain (MycSu18). Amino acid changes in the genes gyrA (Gly81Ala; Ala83Val; Glu87Gly, according to Escherichia coli numbering) and parC (Ser80Phe/Tyr; Asp84Asn) correlated with decreased antibiotic susceptibility to fluoroquinolones and a SNP in the nucleotide sequence of the 23S rRNA (A2059G) was found to be associated with increased MIC values of macrolides. The correlation was more remarkable when final MIC values were evaluated. This study presented the antibiotic susceptibility profiles of M. hyopneumoniae strains circulating in the Central European region, demonstrating the high in vitro efficacy of the tested agents. The observed high MIC values correlated with the SNPs in the examined regions and support the relevance of susceptibility testing and directed antibiotic therapy.

Fructose-1,6-bisphosphate aldolase encoded by a core gene of Mycoplasma hyopneumoniae contributes to host cell adhesion

Mycoplasma hyopneumoniae is an important respiratory pathogen that causes great economic losses to the pig industry worldwide. Although some putative virulence factors have been reported, pathogenesis remains poorly understood. Herein, we evaluated the relative abundance of proteins in virulent 168 (F107) and attenuated 168L (F380) M. hyopneumoniae strains to identify virulence-associated factors by two-dimensional electrophoresis (2-DE). Seven proteins were found to be ≥ 1.5-fold more abundant in 168, and protein–protein interaction network analysis revealed that all seven interact with putative virulence factors. Unexpectedly, six of these virulence-associated proteins are encoded by core rather than accessory genomic elements. The most differentially abundant of the seven, fructose-1,6-bisphosphate aldolase (FBA), was successfully cloned, expressed and purified. Flow cytometry demonstrated the surface localisation of FBA, recombinant FBA (rFBA) mediated adhesion to swine tracheal epithelial cells (STEC), and anti-rFBA sera decreased adherence to STEC. Surface plasmon resonance showed that rFBA bound to fibronectin with a moderately strong KD of 469 nM. The results demonstrate that core gene expression contributes to adhesion and virulence in M. hyopneumoniae, and FBA moonlights as an important adhesin, mediating binding to host cells via fibronectin.

Differential secretome profiling of a swine tracheal cell line infected with mycoplasmas of the swine respiratory tract

Mycoplasma hyopneumoniae and Mycoplasma flocculare are genetically similar. However, M. hyopneumoniae causes porcine enzootic pneumonia, while M. flocculare is a commensal bacterium. M. hyopneumoniae and M. flocculare do not penetrate their host cells, and secreted proteins are important for bacterium-host interplay. Thus, the secretomes of a swine trachea cell line (NPTr) infected with M. hyopneumoniae 7448 (a pathogenic strain), M. hyopneumoniae J (a non-pathogenic strain) and M. flocculare were compared to shed light in bacterium-host interactions. Medium from the cultures was collected, and secreted proteins were identified by a LC-MS/MS. Overall numbers of identified host and bacterial proteins were, respectively, 488 and 58, for NPTr/M. hyopneumoniae 7448; 371 and 67, for NPTr/M. hyopneumoniae J; and 203 and 81, for NPTr/M. flocculare. The swine cells revealed different secretion profiles in response to the infection with each M. hyopneumoniae strain or with M. flocculare. DAMPs and extracellular proteasome proteins, secreted in response to cell injury and death, were secreted by NPTr cells infected with M. hyopneumoniae 7448. All three mycoplasmas secreted virulence factors during NPTr infection, but M. hyopneumoniae 7448 secreted higher number of adhesins and hypothetical proteins, that may be related with pathogenicity. SignificanceThe enzootic pneumonia caused by mycoplasmas of swine respiratory tract has economic loss consequences in pig industry due to antibiotic costs and pig weight loss. However, some genetically similar mycoplasmas are pathogenic while others, such as Mycoplasma hyopneumoniae and Mycoplasma flocculare, are non-pathogenic. Here, we conducted an infection assay between swine cells and pathogenic and non-pathogenic mycoplasmas to decipher secreted proteins during host-pathogen interaction. Mycoplasma response to cell infection was also observed. Our study provided new insights on secretion profile of swine cells in response to the infection with pathogenic and non-pathogenic mycoplasmas. It was possible to observe that pathogenic M. hyopneumoniae 7448 secreted known virulence factors and swine cells responded by inducing cell death. Otherwise, M. hyopneumoniae J and M. flocculare, non-pathogenic mycoplasmas, secreted a different profile of virulence factors in response to swine cells. Consequently, swine cells altered their secretome profile, but the changes were not sufficient to cause disease.

Comparative proteomics of two Mycoplasma hyopneumoniae strains and Mycoplasma flocculare identified potential porcine enzootic pneumonia determinants

ABSTRACT Mycoplasma hyopneumoniae and Mycoplasma flocculare are genetically similar bacteria, which coinhabit the porcine respiratory tract. These mycoplasmas share most of the known virulence factors, but, while M. hyopneumoniae causes porcine enzootic pneumonia (PEP), M. flocculare is a commensal species. To identify potential PEP determinants and provide novel insights on mycoplasma-host interactions, the whole cell proteomes of two M. hyopneumoniae strains, one pathogenic (7448) and other non-pathogenic (J), and M. flocculare were compared. A cell fractioning approach combined with mass spectrometry (LC-MS/MS) proteomics was used to analyze cytoplasmic and surface-enriched protein fractions. Average detection of ~ 50% of the predicted proteomes of M. hyopneumoniae 7448 and J, and M. flocculare was achieved. Many of the identified proteins were differentially represented in M. hyopneumoniae 7448 in comparison to M. hyopneumoniae J and M. flocculare, including potential PEP determinants, such as adhesins, proteases, and redox-balancing proteins, among others. The LC-MS/MS data also provided experimental validation for several genes previously regarded as hypothetical for all analyzed mycoplasmas, including some coding for proteins bearing virulence-related functional domains. The comprehensive proteome profiling of two M. hyopneumoniae strains and M. flocculare provided tens of novel candidates to PEP determinants or virulence factors, beyond those classically described.

Genotyping Mycoplasma hyopneumoniae isolates based on multi-locus sequence typing, multiple-locus variable-number tandem repeat analysis and analysing gene p146

Mycoplasma hyopneumoniae is a swine pathogen bacterium, causing significant economic losses worldwide. Epidemiological investigations based on molecular typing methods support the prevention and eradication strategies for the control of M. hyopneumoniae, through tracing the spreading of the pathogen. The present study describes the genotyping of 44 M. hyopneumoniae strains isolated from Hungarian, Czech and Slovakian porcine lung samples by multi-locus sequence typing (MLST), multiple-locus variable-number tandem repeat analysis (MLVA) and analysing gene p146, and the evaluation of the used methods. The resolution of the three-gene (adk, rpoB, tpiA) and the seven-gene (efp, metG, pgiB, recA, adk, rpoB, tpiA) based MLST systems was identical with 27 sequence types. MLVA utilising loci P97-RR1 and Locus1 extended with the serine repeat numbers of gene p146 showed the highest resolution power among the studied methods differentiating 40 genotypes. The independent analysis of gene p146 revealed 31 different types among the isolates.High variability of M. hyopneumoniae strains was detected by the used typing methods. The results confirmed that utilization of the minimal MLST is suitable for phylogenetic analyses of M. hyopneumoniae strains. The MLVA method extended with the evaluation of serine repeat numbers of gene p146 is adequate for the resolution of genetic relationships within MLST groups. Examination of the p146 gene is suitable to complement both MLST and MLVA methods in order to refine closer genetic relationships.

Assessment of the in vitro growing dynamics and kinetics of the non-pathogenic J and pathogenic 11 and 232 Mycoplasma hyopneumoniae strains

Information on the in vitro growth of pathogenic and non-pathogenic Mycoplasma hyopneumoniae (M. hyopneumoniae) strains is scarce and controversial. Despite its limitations, the colour changing units (CCU) assay is still considered the golden standard titration technique for M. hyopneumoniae culture. Thus, the aims of the present study were: (1) to describe the growth dynamics and kinetics of pathogenic and non-pathogenic M. hyopneumoniae strains, and (2) to monitor the strains’ daily growth by ATP luminometry, CCU, colony forming units (CFU), and DNA quantification by real time quantitative PCR (qPCR) and by fluorescent double-stranded DNA (dsDNA) staining, to evaluate them as putative titration methodologies. The growth of the non-pathogenic J (ATCC®25934™) type strain and the pathogenic 11 (ATCC®25095™) reference strain and 232 strain was modelled by the Gompertz model. Globally, all three-strain cultures showed the same growing phases as well as similar maximal titres within a particular technique, but for CFU. However, the J strain displayed the fastest growing. During the logarithmic phase of growing, CCU, ATP and M. hyopneumoniae copy titres were strongly and linearly associated, and correlation between techniques could be reliably established. In conclusion, real-time culture titration by means of ATP or molecular assays was useful to describe the in vitro growth of the tested strains. Knowledge about the in vitro growth behaviour of a specific strain in a specific medium may provide several advantages, including information about the time required to reach maximal titres by the culture. Noteworthy, the obtained results refers to the three strains used, so extrapolation to other M. hyopneumoniae strains or culture conditions should be made cautiously.

Elongation Factor Thermo Unstable (EF-Tu) Moonlights as an Adhesin on the Surface of Mycoplasma hyopneumoniae by Binding to Fibronectin.

Mycoplasma hyopneumoniae is a colonizing respiratory pathogen that can cause great economic losses to the pig industry worldwide. Although putative virulence factors have been reported, the pathogenesis of this species remains unclear. Here, we used the virulent M. hyopneumoniae strain 168 to infect swine tracheal epithelial cells (STEC) to identify the infection-associated factors by two-dimensional electrophoresis (2-DE). Whole proteins of M. hyopneumoniae were obtained and compared with samples cultured in broth. Six differentially expressed proteins with an increase in abundance of ≥1.5 in the cell infection group were successfully identified. A String network of virulence-associated proteins showed that all the six differential abundance proteins were involved in virulence of M. hyopneumoniae. One of the most important upregulated hubs in this network, elongation factor thermo unstable (EF-Tu), which showed a relatively higher expression in M. hyopneumoniae-infected STEC and obtained a higher score on mass spectrometry was successfully recombined. In addition to its canonical enzymatic activities in protein synthesis, EF-Tu was also reported to be located on the cell surface as an important adhesin in many other pathogens. The cell surface location of EF-Tu was then observed in M. hyopneumoniae with flow cytometry. Recombinant EF-Tu (rEF-Tu) was found to be able to adhere to STEC and anti-rEF-Tu antibody enclosed M. hyopneumoniae decreased adherence to STEC. In addition, surface plasmon resonance (SPR) analysis showed that rEF-Tu could bind to fibronectin with a specific and moderately strong interaction, a dissociation constant (KD) of 605 nM. Furthermore, the block of fibronectin in STEC also decreased the binding of M. hyopneumoniae to the cell surface. Collectively, these data imply EF-Tu as an important adhesin of M. hyopneumoniae and fibronectin as an indispensable receptor on STEC. The binding between EF-Tu with fibronectin contributes to the adhesion of M. hyopneumoniae to STEC.HIGHLIGHTS Elongation factor thermo unstable (EF-Tu) exists on the cell surface of M. hyopneumoniae.EF-Tu moonlights as an adhesin of M. hyopneumoniae.The adhesive effect of EF-Tu is partly meditated by fibronectin.

Complete Genome Sequence of Mycoplasma hyopneumoniae Strain KM014, a Clinical Isolate from South Korea.

ABSTRACTMycoplasma hyopneumoniae is the etiological agent of swine enzootic pneumonia, resulting in considerable economic losses in the swine industry. A few genome sequences of M. hyopneumoniae have been reported to date, implying that additional genome data are needed for further genetic studies. Here, we present the annotated genome sequence of M. hyopneumoniae strain KM014.

Efficacy of one dose vaccination against experimental infection with two Mycoplasma hyopneumoniae strains

BackgroundMycoplasma hyopneumoniae (M. hyopneumoniae) is the primary agent of enzootic pneumonia in pigs. Pigs are often infected with different M. hyopneumoniae strains. This study assessed the efficacy of vaccination against experimental infection with two genetically different M. hyopneumoniae strains in weaned piglets. At 33 days of age (D0), 45 M. hyopneumoniae-free piglets were randomly assigned to three different groups: 1) negative control group (NCG; n = 5): not vaccinated, not infected, 2) positive control group (PCG; n = 20): not vaccinated, infected, and 3) vaccination group (VG; n = 20): single vaccination with an inactivated whole-cell M. hyopneumoniae vaccine (Hyogen®, Ceva) (D1), infected. The PCG and VG were endotracheally inoculated with 7 × 107 CCU in 7 ml of the highly virulent M. hyopneumoniae strain F7.2C (D24) and 7 × 107 CCU in 7 ml low virulent strain F1.12A (D25). A respiratory disease score (RDS) was assessed from D24 until D53. At D53 (euthanasia), macroscopic lung lesions (MLL) were scored, log copies of M. hyopneumoniae DNA (qPCR) and IL-1 and IL-6-concentrations (ELISA) on bronchoalveolar lavage fluid were determined.ResultsThe RDS and MLL at euthanasia were respectively 0, 1.20 and 0.55 (P < 0.001) and 0, 7.56 and 0.68 (P < 0.001) for NCG, PCG and VG, respectively. The qPCR results for PCG and VG were 3.99 and 1.78 log copies (P < 0.001), respectively, with a significant difference between PCG and VG. The IL-1 and IL-6 results at euthanasia for NCG, PCG and VG were 17.61, 1283.39 and 53.04 pg/ml (P < 0.001) and 148.10, 493.35 and 259.80 pg/ml (P = 0.004), respectively with a significant difference between PCG and VG.ConclusionsVaccination with Hyogen® in pigs was efficacious against an experimental challenge with both a low and highly virulent M. hyopneumoniae strain as the vaccinated pigs coughed significantly less, and showed significantly less lung lesions compared to the non-vaccinated challenged pigs: the vaccinated animals showed a 52.9% lower RDS and 91.0% lower MLL compared to the PCG. In the bronchoalveolar lavage fluid collected at the necropsy of the vaccinated pigs, a significantly lower amount of M. hyopneumoniae-DNA and a significantly lower IL-1 and IL-6 concentration was found compared to the pigs of the PCG.

Update onMycoplasma hyopneumoniaeinfections in pigs: Knowledge gaps for improved disease control

Mycoplasma hyopneumoniae (M.hyopneumoniae) is the primary pathogen of enzootic pneumonia, a chronic respiratory disease in pigs. Infections occur worldwide and cause major economic losses to the pig industry. The present paper reviews the current knowledge on M.hyopneumoniae infections, with emphasis on identification and analysis of knowledge gaps for optimizing control of the disease. Close contact between infected and susceptible pigs is the main route of M.hyopneumoniae transmission. Management and housing conditions predisposing for infection or disease are known, but further research is needed to better understand M.hyopneumoniae transmission patterns in modern pig production systems, and to assess the importance of the breeding population for downstream disease control. The organism is primarily found on the mucosal surface of the trachea, bronchi and bronchioles. Different adhesins and lipoproteins are involved in the adherence process. However, a clear picture of the virulence and pathogenicity of M.hyopneumoniae is still missing. The role of glycerol metabolism, myoinositol metabolism and the Mycoplasma Ig binding protein-Mycoplasma Ig protease system should be further investigated for their contribution to virulence. The destruction of the mucociliary apparatus, together with modulating the immune response, enhances the susceptibility of infected pigs to secondary pathogens. Clinical signs and severity of lesions depend on different factors, such as management, environmental conditions and likely also M.hyopneumoniae strain. The potential impact of strain variability on disease severity is not well defined. Diagnostics could be improved by developing tests that may detect virulent strains, by improving sampling in live animals and by designing ELISAs allowing discrimination between infected and vaccinated pigs. The currently available vaccines are often cost-efficient, but the ongoing research on developing new vaccines that confer protective immunity and reduce transmission should be continued, as well as optimization of protocols to eliminate M.hyopneumoniae from pig herds.

Metabolic modeling of energy balances in Mycoplasma hyopneumoniae shows that pyruvate addition increases growth rate.

ABSTRACT Mycoplasma hyopneumoniae is cultured on large‐scale to produce antigen for inactivated whole‐cell vaccines against respiratory disease in pigs. However, the fastidious nutrient requirements of this minimal bacterium and the low growth rate make it challenging to reach sufficient biomass yield for antigen production. In this study, we sequenced the genome of M. hyopneumoniae strain 11 and constructed a high quality constraint‐based genome‐scale metabolic model of 284 chemical reactions and 298 metabolites. We validated the model with time‐series data of duplicate fermentation cultures to aim for an integrated model describing the dynamic profiles measured in fermentations. The model predicted that 84% of cellular energy in a standard M. hyopneumoniae cultivation was used for non‐growth associated maintenance and only 16% of cellular energy was used for growth and growth associated maintenance. Following a cycle of model‐driven experimentation in dedicated fermentation experiments, we were able to increase the fraction of cellular energy used for growth through pyruvate addition to the medium. This increase in turn led to an increase in growth rate and a 2.3 times increase in the total biomass concentration reached after 3–4 days of fermentation, enhancing the productivity of the overall process. The model presented provides a solid basis to understand and further improve M. hyopneumoniae fermentation processes. Biotechnol. Bioeng. 2017;114: 2339–2347. © 2017 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals, Inc.

Determinants for swine mycoplasmal pneumonia reproduction under experimental conditions: A systematic review and recursive partitioning analysis

One of the main Mycoplasma hyopneumoniae (M. hyopneumoniae) swine experimental model objectives is to reproduce mycoplasmal pneumonia (MP). Unfortunately, experimental validated protocols to maximize the chance to successfully achieve lung lesions induced by M. hyopneumoniae are not available at the moment. Thus, the objective of this work was to identify those factors that might have a major influence on the effective development of MP, measured as macroscopic lung lesions, under experimental conditions. Data from 85 studies describing M. hyopneumoniae inoculation experiments were compiled by means of a systematic review and analyzed thereafter. Several variables were considered in the analyses such as the number of pigs in the experiment, serological status against M. hyopneumoniae, source of the animals, age at inoculation, type of inoculum, strain of M. hyopneumoniae, route, dose and times of inoculation, study duration and co-infection with other swine pathogens. Descriptive statistics were used to depict M. hyopneumoniae experimental model main characteristics whereas a recursive partitioning approach, using regression trees, assessed the importance of the abovementioned experimental variables as MP triggering factors. A strong link between the time period between challenge and necropsies and lung lesion severity was observed. Results indicated that the most important factors to explain the observed lung lesion score variability were: (1) study duration, (2) M. hyopneumoniae strain, (3) age at inoculation, (4) co-infection with other swine pathogens and (5) animal source. All other studied variables were not relevant to explain the variability on M. hyopneumoniae lung lesions. The results provided in the present work may serve as a basis for debate in the search for a universally accepted M. hyopneumoniae challenge model.