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The present study aimed to investigate the optimal inactivants and inactivation conditions for preparing inactivated vaccines of Mycoplasma hyopneumoniae and Mycoplasma hyorhinis. Mycoplasma inactivation was performed using formaldehyde, thimerosal, β-propiolactone (BPL), and binary ethylenimine (BEI) and compared. The results showed that M. hyopneumoniae was completely inactivated when incubated with 0.01 % formaldehyde for 24 h or 0.02 % formaldehyde for 12 h at 37 °C, with 0.0008 % thimerosal for 12 h at 37 °C, with 0.02 % BPL for 24 h or 0.1 % BPL for 12 h at 4 °C, or with 0.004 % BEI for 24 h or 0.5 % BEI for 12 h at 37 °C. M. hyorhinis was completely inactivated when incubated with 0.01 % formaldehyde for 24 h or 0.02 % formaldehyde for 12 h at 37 °C, with 0.004 % thimerosal for 24 h or 0.02 % thimerosal for 12 h at 37 °C, with 0.1 % BPL for 12 h at 4 °C, or with 0.004 % BEI for 24 h or 0.5 % BEI for 12 h at 37 °C. Next, the immunogenicity of the mycoplasmas after inactivation was evaluated by immunizing BALB/c mice. Immunization of mice with a high dose (106 color-changing units [CCU] per dose) of M. hyopneumoniae and M. hyorhinis vaccines inactivated with all inactivants led to high levels of serum IgG antibodies. M. hyopneumoniae vaccines inactivated with formaldehyde induced significantly higher titers of antibodies than vaccines inactivated with other inactivants, whereas M. hyorhinis vaccines inactivated with BEI induced significantly higher titers of antibodies than vaccines inactivated with thimerosal. However, in mice immunized with a low dose of mycoplasmas (104 CCU per dose), only M. hyopneumoniae vaccines inactivated with formaldehyde and BEI and M. hyorhinis vaccines inactivated with formaldehyde, BPL, and BEI led to significant levels of serum IgG antibodies. Among these groups, the antibody levels in the formaldehyde-inactivated vaccine group were higher than those in the other inactivant groups. This study provides a reliable basis for inactivation during large-scale production of Mycoplasma hyopneumoniae and Mycoplasma hyorhinis inactivated vaccines.

The destruction of the respiratory barrier caused by Mesomycoplasma ( Mycoplasma ) hyopneumoniae plays a pivotal role in facilitating secondary infections by other respiratory pathogens. However, the pathogenesis of M. hyopneumoniae breaching the respiratory barrier to establish infection remains largely elusive. In this study, the role of Mhp390 encoded by M. hyopneumoniae in invasion of the respiratory tract barrier, including extracellular matrix and tracheal epithelial cells, were investigated through the Transwell assay. Our finding indicated that M. hyopneumoniae may exploit the host fibrinolytic system via Mhp390 to accumulate activated plasmin outside its membrane, thereby breaching the respiratory tract barrier and facilitating the progression of infection. Furthermore, the key functional domains within Mhp390 involved in its interaction with host plasminogen were determined by using truncated mutation techniques. Collectively, these findings will enhance our understanding of the mechanism underlying respiratory barrier invasion by M. hyopneumoniae thereby providing new theoretical basis for the development of novel vaccines and effective control strategies against secondary infection.

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.

Vaccination against Mycoplasma hyopneumoniae is still carried out worldwide, but unfortunately current commercial vaccines only provide partial protection. Therefore, two M. hyopneumoniae strains were genetically modified by transposon-mediated gene disruption of mmsA and mnuA , encoding methylmalonate semialdehyde dehydrogenase and membrane nuclease A, respectively. We investigated how immune responses elicited by these genetically modified M. hyopneumoniae strains protected pigs against challenge infection. An endotracheal single dose vaccination with genetically modified M. hyopneumoniae strain 1 (Δ mmsA ) or 2 (Δ mnuA ), or physiological saline solution (Control) was followed by challenge infection. Piglets from Δ mnuA had a higher respiratory disease score post-vaccination, but this group coughed significantly less after challenge. Significantly fewer DNA copies of the challenge strains were observed in broncho-alveolar lavage fluid (BAL) from Δ mnuA after challenge. Two weeks post-challenge, significantly more BAL IgG and BAL IgA was observed in Δ mnuA , but at euthanasia significantly more IgA and less pro-inflammatory cytokines were detected in BAL from both vaccinated groups. Furthermore, a significantly lower percentage of IFN-γ + and TNF-α + IFN-γ + CD8 + T cells was observed after administration of Δ mnuA. The percentage of IFN-γ + CD8 + T cells was significantly lower in Δ mmsA at euthanasia. To conclude, the results of this exploratory study show that a single endotracheal administration of Δ mnuA resulted in coughing post-vaccination, but reduced clinical signs post-challenge and challenge strain DNA load in BAL. Therefore, a strain mutated in the mnuA gene might be an interesting mutant strain that could be promising as a potential live vaccine candidate strain as it can reduce M. hyopneumoniae infection burden under field conditions.

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 K D 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.

Tracheal pooling for Mycoplasma hyopneumoniae ( M. hyopneumoniae ) DNA detection allows for decreased diagnostic cost, one of the main constraints in surveillance programs. The objectives of this study were to estimate the sensitivity of pooled-sample testing for the detection of M. hyopneumoniae in tracheal samples and to develop probability of M. hyopneumoniae detection estimates for tracheal samples pooled by 3, 5, and 10. A total of 48 M. hyopneumoniae PCR-positive field samples were pooled 3-, 5-, and 10-times using field M. hyopneumoniae DNA-negative samples and tested in triplicate. The sensitivity was estimated at 0.96 (95% credible interval [Cred. Int.]: 0.93, 0.98) for pools of 3, 0.95 (95% Cred. Int: 0.92, 0.98) for pools of 5, and 0.93 (95% Cred. Int.: 0.89, 0.96) for pools of 10. All pool sizes resulted in PCR-positive if the individual tracheal sample Ct value was < 33. Additionally, there was no significant decrease in the probability of detecting at least one M. hyopneumoniae -infected pig given any pool size (3, 5, or 10) of tracheal swabs. Furthermore, this manuscript applies the probability of detection estimates to various real-life diagnostic testing scenarios. Combining increased total animals sampled with pooling can be a cost-effective tool to maximize the performance of M. hyopneumoniae surveillance programs.

Mycoplasma hyopneumoniae is the most costly pathogen for swine production. Although several studies have focused on the host-bacterium association, little is known about the changes in gene expression of swine cells upon infection. To improve our understanding of this interaction, we infected swine epithelial nptr cells with M. hyopneumoniae strain J to identify differentially expressed mRNAs and miRNAs. The levels of 1,268 genes and 170 miRNAs were significantly modified post-infection. Up-regulated mRNAs were enriched in genes related to redox homeostasis and antioxidant defense, known to be regulated by the transcription factor NRF2 in related species. Down-regulated mRNAs were enriched in genes associated with cytoskeleton and ciliary functions. Bioinformatic analyses suggested a correlation between changes in miRNA and mRNA levels, since we detected down-regulation of miRNAs predicted to target antioxidant genes and up-regulation of miRNAs targeting ciliary and cytoskeleton genes. Interestingly, most down-regulated miRNAs were detected in exosome-like vesicles suggesting that M. hyopneumoniae infection induced a modification of the composition of NPTr-released vesicles. Taken together, our data indicate that M. hyopneumoniae elicits an antioxidant response induced by NRF2 in infected cells. In addition, we propose that ciliostasis caused by this pathogen is partially explained by the down-regulation of ciliary genes.

Many bacterial moonlighting proteins were originally described in medically, agriculturally, and commercially important members of the low G + C Firmicutes. We show Elongation factor Tu (Ef-Tu) moonlights on the surface of the human pathogens Staphylococcus aureus (Sa Ef-Tu ) and Mycoplasma pneumoniae (Mpn Ef-Tu ), and the porcine pathogen Mycoplasma hyopneumoniae (Mhp Ef-Tu ). Ef-Tu is also a target of multiple processing events on the cell surface and these were characterised using an N-terminomics pipeline. Recombinant Mpn Ef-Tu bound strongly to a diverse range of host molecules, and when bound to plasminogen, was able to convert plasminogen to plasmin in the presence of plasminogen activators. Fragments of Ef-Tu retain binding capabilities to host proteins. Bioinformatics and structural modelling studies indicate that the accumulation of positively charged amino acids in short linear motifs (SLiMs), and protein processing promote multifunctional behaviour. Codon bias engendered by an A + T rich genome may influence how positively-charged residues accumulate in SLiMs.

Mycoplasma hyopneumoniae is the etiologic agent of porcine enzootic pneumonia, responsible for major production losses worldwide. The bacteria have a limited metabolism and need to obtain molecules from the growth environment, which causes multiple difficulties for in vitro culture. These limitations have a negative influence on the ability to carry out research for the development of the rational use of antimicrobials and vaccines. The objective of this investigation was to evaluate the genetic profile and in vitro susceptibility of field isolates of M. hyopneumoniae to different antimicrobials. All 16 isolates obtained from the samples presented 100% of identity in the partial sequence of 16S rRNA gene when compared to M. hyopneumoniae. A dendrogram was created using the PCR results of the genes related to pathogenicity, and the isolates were distributed into four clusters, suggesting genetic variability among four different isolates circulating on the same farm. The minimum inhibitory concentration of the isolates was higher for the antimicrobials tylosin (< 0.001–16 mg/L) and spiramycin (< 0.001–16 mg/L) than for enrofloxacin (< 0.001–0.125 mg/L) and tiamulin (< 0.001–0.125 mg/L). Our results demonstrate the genetic variability among M. hyopneumoniae isolates from pigs of the same farm, with differences in their susceptibility to antimicrobial agents.

Mycoplasma ( M .) hyopneumoniae is the main pathogen of porcine enzootic pneumonia (PEP). Its controlling is challenging, and requires alternative strategies. This study aimed to develop an oral vaccine against M. hyopneumoniae using a nanostructured mesoporous silica (SBA-15) as an adjuvant, and compare its effect with an intramuscular (IM) commercial vaccine (CV). Fifty 24 day-old M . hyopneumoniae -free piglets composed five equal groups for different immunization protocols, consisting of a CV and/or oral immunization (OI). Control piglets did not receive any form of immunization. All piglets were challenged with M. hyopneumoniae strain 232 on D49 by tracheal route. IgA antibody response in the respiratory tract, bacterial shedding and serum IgG were evaluated. The piglets were euthanized on 28 (D77) and 56 (D105) days post-infection. Lung lesions were macroscopically evaluated; lung fragments and bronchoalveolar fluid (BALF) were collected for estimation of bacterial loads by qPCR and/or histopathology examination. All immunization protocols induced reduction on Mycoplasma -like macroscopic lung lesions. IgA Ab responses anti- M. hyopneumoniae , the expression of IL-4 cytokine and a lower expression of IL-8 were induced by CV and OI vaccines, while IgG was induced only by CV. Oral immunization using silica as a carrier-adjuvant can be viable in controlling M. hyopneumoniae infection.