Development and efficacy test of a live, attenuated Mycoplasma hyorhinis vaccine candidate strain.

Comparison of infection sequence of porcine reproductive and respiratory syndrome virus (PRRSV) and Mycoplasma hyorhinis to potentiate PRRSV-induced pneumonia.

Mycoplasma hyorhinis is a ubiquitous pathogen of swine that causes polyserositis and polyarthritis and is also associated with conjunctivitis, meningitis, pneumonia, and abortions. This microorganism is a high prevalence pathogen in Chinese swine herds. However, few studies on M. hyorhinis have been reported in Chongqing, China. The overuse of antimicrobials has led to an increased risk of antimicrobial resistance, but a series of Chinese herbal monomers exhibited antibacterial activity to drug-resistant bacteria, including mycoplasmas. The aim of the study was to determine the prevalence, sequence types, growth kinetics, susceptibility to antimicrobials and Chinese herbal monomers, and relationships between the phenotypes and genotypes in terms of the resistance of M. hyorhinis to fluoroquinolones, macrolides and lincomycin. A total of 28 M. hyorhinis strains were recovered from the lungs of 404 slaughtered pigs. The isolates belonging to 11 novel STs, ST226, ST227, ST228, ST229, ST230, ST260, ST261, ST262, ST263, ST264 and ST265, were clustered separately from other reference isolates in the database. The growth kinetic of each isolate was generated, and the maximum color changing unit (CCU) values of isolates varied from 1012 to 1020 CCU/ml. In vitro susceptibility testing showed that the isolates were inhibited by low concentrations of tiamulin (MIC: ≤ 0.25 μg/ml), doxycycline (MIC: ≤ 0.25–1 μg/ml), ciprofloxacin (MIC: ≤ 0.25–2 μg/ml), florfenicol (MIC: 0.5–2 μg/ml), kanamycin (MIC: ≤ 0.25–4 μg/ml), enrofloxacin (MIC: 0.5–4 μg/ml) and berberine hydrochloride (MIC range: 1–8 μg/ml). However, the MICs of erythromycin were high (MIC: 32–≥128 μg/ml) for all isolates. The MICs of tylosin, tilmicosin and lincomycin for 50%, 60.7% and 46.4% of isolates were equal to or >16, 32 and 8 μg/ml, respectively. No correlation was detected between resistance to fluoroquinolones and QRDRs. However, the high MICs of tylosin, tilmicosin and lincomycin were most likely attributed to an A1553G mutation in domain V of the 23S rRNA. Our findings demonstrated the diversity of STs among M. hyorhinis isolates in Chongqing. The high MICs of M. hyorhinis isolates to macrolides and lincomycin suggested that the use of lincomycin for the treatment of M. hyorhinis infections should be carefully evaluated.

Mycoplasmas are pathogens causing infectious diseases in various animals, including humans. They are also common contaminants of cell culture. Although it is suggested that mycoplasmas alter nucleic acid metabolism of host cells through their nucleases, the actual impact of the nucleases on host cell RNAs is unknown. Here we report that Mycoplasma hyorhinis, a common laboratory contaminant species, promotes cleavage of host cell transfer RNAs (tRNAs) through a membrane-associated nuclease. When infected by M. hyorhinis, scraping of cells as well as cell lysis induced a marked cleavage of host RNAs. Further analysis suggested that the protein encoded by the membrane nuclease A (mnuA) gene is responsible for the host RNA cleavage. MnuA protein demonstrates DNase and RNase activities dependent on Ca2+/Mg2+ ions. Purified MnuA protein acts as an atypical sugar non-specific nuclease: while it possesses broad DNase activities, its RNase activity is highly specific to tRNAs in live cells. Mutational analysis shows that the nuclease activity is mediated by a domain which is highly similar to DNase I. Furthermore, M. hyorhinis promoted cleavage of host tRNAs under amino acid deprivation, suggesting that M. hyorhinis infection may alter RNA metabolism in host cells under certain physiological stress. As mnuA genes are conserved in various mycoplasma species, our findings provide novel insights into the effects of MnuA nucleases on host cell RNAs under mycoplasma infection.

Mycoplasma hyorhinis (homotypic synonym of Mesomycoplasma hyorhinis) is a pathobiont from the upper respiratory tract of pigs. Under unclear circumstances, it can disseminate systemically and cause disease. Although some studies have reported different infectious capabilities among strains, no factors have been directly linked to virulence. This study aimed to analyze the core and accessory genes of all available M. hyorhinis strains (pangenome) to identify potential virulence markers. We characterized the pangenome of 110 strains, including isolates from healthy (nasal cavity) and diseased (systemic organs, nasal cavity or lung) animals. Comparative analyses were performed according to the clinical background. Although most putative virulence genes were shared, we identified several genes absent in most health-associated strains related to DNA-processing mechanisms, including hsdM-hsdR restriction-modification system and various helicases. Furthermore, the particular analysis of variable lipoprotein (vlp) genes revealed a similar presence in all strains but higher number of repeats in region III of vlpF in strains isolated from systemic lesions. Genome-scale metabolic models were used to infer the metabolic capabilities of the strains, showing highly conserved predicted reactomes, including growth capabilities and auxotrophies. In conclusion, although all strains may carry genes enabling disease, nasal strains from healthy animals lacked some DNA-processing genes and showed distinct vlp patterns. Additional genomes, especially from strains isolated from healthy animals, would be needed to confirm these findings.

Evaluation of chicken serum and chicken egg yolk as novel supplements for the in vitro cultivation of Mycoplasma hyorhinis

Polyserositis, meningoencephalitis, arthritis and omphalitis due to Salmonella Agona infection in a pig.

Mycoplasma hyorhinis is an important respiratory pathogen in swine, yet optimal culture conditions for high-yield propagation remain undefined. This study compared horse serum (HS) and chicken serum (CS) at graded concentrations (10%, 20%, 30%) for their ability to support in vitro growth of four clinical M. hyorhinis isolates (strains A, B, C, and D). Cultures were prepared in modified Friis medium, and growth performance was assessed by final titer (color changing unit, CCU/mL) and time-to-detection at 102 and 104 CCU/mL. All media supported growth, but HS consistently outperformed CS in both yield and growth kinetics. The highest titers (109 CCU/mL) and shortest detection times (3.6–6 days) were observed in 20% HS for most strains. Increasing HS concentration to 30% reduced yield for several strains, suggesting a concentration-dependent inhibitory effect. CS demonstrated limited but strain-dependent growth support, with comparable performance to HS for strain B at lower thresholds. These findings identify 20% HS as an optimal supplement for efficient M. hyorhinis cultivation, while highlighting the potential of CS as a cost-effective alternative under certain conditions, with implications for diagnostic reagent production and vaccine development.

Ensuring the safety and efficacy of veterinary vaccines requires reliable methods for detecting microbial contamination, particularly from Mycoplasma species, which pose a significant risk in cell-culture-derived vaccines. In the Republic of Korea, polymerase chain reaction (PCR) is predominantly used for Mycoplasma testing due to its faster turnaround compared to culture-based methods. However, in combination with vaccines containing Erysipelothrix rhusiopathiae and classical swine fever virus, PCR is rendered ineffective because of cross-reactivity between Mycoplasma universal primers and E. rhusiopathiae, resulting in non-specific amplification. This limitation necessitates reliance on the labor-intensive culture method, underscoring the need for more accurate and efficient alternatives. This study aimed to develop and validate next-generation sequencing (NGS)-based methods for detecting Mycoplasma contamination in veterinary vaccines and to compare their performance with that of PCR. Five species, including Acholeplasma laidlawii (genus Acholeplasma) and four Mycoplasma species—Mycoplasma fermentans, Mycoplasma orale, Mycoplasma hyorhinis, and Mycoplasma synoviae–were spiked into samples containing E. rhusiopathiae, a common vaccine component. Two NGS-based approaches were evaluated: (1) a reference-mapping method incorporating two-step alignment and de novo assembly, and (2) a 16S rRNA-based metabarcoding analysis using DADA2 and Qiime2. The reference-mapping method effectively filtered non-specific reads and accurately reconstructed Mycoplasma-derived contigs, whereas the metabarcoding approach enabled taxonomic profiling with quantitative resolution. The detection limits of NGS-based methods were substantially lower than those of PCR, demonstrating improvements of up to 100-fold depending on the species. Notably, omission of the initial mapping step resulted in excessive non-specific contig formation, highlighting the importance of the dual-step reference-mapping strategy. Although metabarcoding provided valuable abundance data, it was more prone to non-specific hits due to limited read overlap. In conclusion, the reference-mapping method demonstrated superior sensitivity, specificity, and quantification compared to both conventional PCR and metabarcoding, supporting its use as a robust tool for vaccine quality control. Implementing NGS-based detection methods could significantly enhance the safety and effectiveness of veterinary vaccines, ultimately enhancing vaccine quality control.

The current report on drug development focuses on "The new dimension" of cancer hallmarks.

Detection of phosphatidylserine exposure on Mycoplasma hyorhinis by flow cytometry.

IntroductionRespiratory diseases have a substantial impact on swine production worldwide. Understanding the relationship between gross lung lesions and the presence of infectious agents is crucial for developing effective disease control strategies that target both primary and secondary pathogens.Material and MethodsA cross-sectional study was conducted on 22 pig farms in western Poland. Cranioventral pulmonary consolidation (CVPC) in slaughtered pigs was assessed, and 20 lung tissue samples were collected from each herd. The presence of common bacterial and viral respiratory pathogens was identified using PCR-based methods.ResultsThe disorder was observed in 79.3% (95% confidence interval 75.3–82.8) of slaughtered pigs across all examined herds. The most frequently detected pathogens at both the herd and individual animal levels were Glaesserella parasuis, Mycoplasma hyopneumoniae and porcine circovirus 2. Co-infections involving two or more respiratory pathogens were prevalent, occurring in 100% of herds and 87.7% of individual pigs. Mean CVPC scores were significantly higher in pigs infected with Mycoplasma hyopneumoniae, Mycoplasma hyorhinis and porcine reproductive and respiratory syndrome virus 1.ConclusionThese findings highlight the multifactorial nature of respiratory infections in pigs. Effective control measures should consider the high prevalence of co-infections and their impact on lung lesion severity to improve overall herd health and productivity.

Mastitis is the inflammation of the mammary gland parenchyma and many microorganisms, mostly bacteria are associated with the condition. A study was conducted to assess whether mycoplasma is associated with cases of mastitis that are refractory to antibiotic treatment as the condition caused by the microbe is reported to be difficult to treat. A total of 71 milk samples were collected from antibiotic-resistant mastitic cows in the northern districts of Kerala. Total DNA was extracted from all the samples by phenol-chloroform method with prior sample lysis and subjected to Mycoplasma spp. and Mycoplasma bovis specific Polymerase Chain Reaction (PCR) and the amplicons obtained were sequenced. Mycoplasma spp. was detected in two (2.81 per cent) samples and no samples were positive for M. bovis. Sequencing of PCR amplicons revealed the presence of Mycoplasma hyorhinis in one of the samples. The identity of the Mycoplasma spp. in the other sample could not be conclusively proved as the nucleotide sequence obtained matched with that of M. bovis, M. arginini, and uncultured Mycoplasma spp. detected in nasal swabs of animals. Both the milk samples from which mycoplasma was detected were positive for multidrug-resistant gram positive and gram-negative bacteria and from one sample fungi could be isolated. From the results obtained, though mycoplasma was detected, it could not be proven that it is the sole cause of the condition. Also, the presence of mycoplasma is not wide spread in cases of refractory mastitis in northern Kerala.

As a prevalent swine pathogen worldwide, Mycoplasma hyorhinis (M. hyorhinis, Mhr) is associated with various diseases, including multiple serositis, pneumonia, arthritis, and otitis media. It is also linked to the porcine respiratory disease complex (PRDC). M. hyorhinis prevalence in 2022 Chinese lung samples was assessed by species-specific PCR, followed by isolation and purification of field strains, followed by genetic characterization via multilocus sequence typing (MLST). Pathogenicity evaluation of three isolates (ZZ-1, GD-1 and AH-1) was evaluated using controlled piglet infection trials. Mhr detection in clinical lung samples showed 31.77% prevalence. Three isolates (ZZ-1/ST166, GD-1/ST167, AH-1/ST144) were characterized by MLST. Piglet infection trials confirmed Mhr-induced polyserositis, pneumonia, and arthritis, with strain-dependent virulence variation observed. This study confirms M. hyorhinis as a high-prevalence pathogen (31.77%) in Chinese swine herds. Animal infection models demonstrated virulence variation among different Mhr strains. These findings contribute to identifying and assessing the threats posed by different strains to pig health, guiding the development of clinical prevention and control strategies.

Respiratory disease (RD) is a worldwide leading threat to the pig industry, but there is still limited understanding of the pathogens associated with swine RD. In this study, we conducted a nationwide genomic surveillance on identifying viruses, bacteria, and antimicrobial resistance genes (ARGs) from the lungs of pigs with RD in China. By performing metatranscriptomic sequencing combined with metagenomic sequencing, we identified 21 viral species belonging to 12 viral families. Among them, porcine reproductive and respiratory syndrome virus, influenza A virus, herpes virus, adenovirus, and parvovirus were commonly identified. However, emerging viruses, such as Getah virus and porcine respiratory coronaviruses, were also characterized. Apart from viruses, a total of 164 bacterial species were identified, with Streptococcus suis, Mycoplasma hyorhinis, Mycoplasma hyopneumoniae, Glaesserella parasuis, and Pasteurella multocida being frequently detected in high abundances. Notably, Escherichia coli, Enterococcus faecalis, Staphylococcus aureus, and Klebsiella pneumoniae were also highly detected. Our further analysis revealed a complex interaction between the identified pathogens in swine RD. We also conducted retrospectively analyses to demonstrate the prevalent viral genotypes or bacterial serotypes associated with swine RD in China. Finally, we identified 48 ARGs, which conferred resistance to 13 predicted antimicrobial classes, and many of these ARGs were significantly associated with a substantial number of mobile genetic elements, including transposons (e.g., tnpAIS1, tnpA1353, int3, and ISCau1) and plasmids (e.g., Col(BS512), Col(YC)]. These findings will contribute to further understanding the etiology, epidemiology, and microbial interactions in swine RD, and may also shed a light on the development of effective vaccines.IMPORTANCEIn this study, we identified viruses and bacteria from the lungs of pigs with RD in China at a nationwide farm scale by performing metatranscriptomic sequencing combined with metagenomic sequencing. We also demonstrated the complex interactions between different viral and/or bacterial species in swine RD. Our work provides a comprehensive knowledge about the etiology, epidemiology, and microbial interactions in swine RD and data reference for the research and development of effective vaccines against the disease.

Mycoplasma hyorhinis is a frequently observed contaminant in cell cultures, and its detection and purification pose considerable challenges. Fragments or other cell components are similar in size to those of Mycoplasma; therefore, distinguishing them is difficult. In this study, we used Hoechst staining in combination with carboxyfluorescein succinimidyl ester (CFSE) to label Mycoplasma. The trigger threshold was set in the Hoechst Blue channel rather than in the default forward scatter channel, utilizing the differences in DNA content between Mycoplasma and fragments. Subsequently, we identified and isolated it at single-cell resolution via flow cytometry and successfully sorted infectious Mycoplasma in cell culture. Simultaneously, we validated the accuracy and feasibility of this approach using polymerase chain reaction, fluorescence confocal microscopy, and cryo-electron microscopy. This methodology enabled the diagnosis of Mycoplasma at extremely low concentrations, significantly enhancing the detection efficiency and facilitating the isolation and purification of parasitic Mycoplasma in cell culture instead of pure Mycoplasma culture in artificial media for subsequent studies.

Successful retrieval of Aerococcus viridans from porcine clinical specimens has been rarely described, and data has only been obtained from a few swine-producing countries. Therefore, the aim of this study was the isolation of A. viridans recovered from a specimen originating from a commercial pig farm located in Poland. Seven dead 12-week-old pigs weighing 24-26 kg with joint swelling of the hind legs were selected on a modern farrow-to-nursery farm in Poland in October 2023. The research material was joint swabs from one affected limb amputated through the proximal part of the femur. Bacteria were isolated using matrix-assisted laser desorption/ionisation-time-of-flight mass spectrometry. Antimicrobial susceptibility testing of isolates of Staphylococcus aureus, A. viridans and Trueperella pyogenes was performed by disc diffusion and also by minimal inhibitory concentration evaluation in the case of A. viridans. Two pooled samples were screened by PCR for Streptococcus suis, Actinobacillus pleuropneumoniae, Actinobacillus suis, Mycoplasma hyorhinis, M. hyosynoviae and Glaesserella parasuis. Five samples were positive for bacteria by culture and five isolates were recovered. Staphylococcus aureus was identified in three samples and T. pyogenes and A. viridans in one each. Pooled samples were G. parasuis-, M. hyorhinis-, M. hyosynoviae-, Actinobacillus pleuropneumoniae-, Actinobacillus suis- and S. suis-negative in the PCR. The A. viridans isolated was susceptible to beta-lactams and gentamicin. Ten representative nucleotide contigs from the 500 obtained showed similarity as high as 97.5% to GenBank reference strains. To the best of our knowledge, this is the first identification of A. viridans in clinically affected Polish pigs. It elevates the importance of uncommon pathogens, including A. viridans, in the development of lameness in pigs. This research emphasises the role of modern diagnostic tools for accurate identification of swine pathogens. Further research would prove beneficial to elucidate the mechanisms of A. viridans infection, its prevalence, pathogenicity and virulence factors.

IntroductionPathogen introduction and transmission at the farm, regional, or national level are associated with reduced animal welfare and negative impacts on herd economics. Ongoing infectious disease surveillance, active or passive, is therefore of high importance. For optimal resolution, each pig is sampled individually, for example by collecting blood or nasal swabs. In recent years, oral fluids have become very useful for population surveillance at the pen level. Another alternative is sampling the air to capture pathogens circulating across the entire barn via bioaerosols.ObjectiveThis study aimed to examine the potential utility of bioaerosol metagenomics for pathogen detection on pig farms.MethodsBioaerosols via automated air sampler, and oral fluid via pen-based ropes, were collected from each of two Scottish indoor pig farms. All samples were subjected to conventional routine bacterial isolation. Total genomic nucleic acids were extracted for PCR screening for three pig DNA viruses, three bacterial Mycoplasma species and an RNA virus. Illumina shotgun metagenomic sequencing was also conducted.ResultsOral fluids contained more DNA compared to bioaerosol samples. DNA integrity exhibited limited impact on PCR or sequence yield. While Streptococcus suis could be cultured from a single oral fluid sample, reads mapped to S. suis were detectable in all metagenomic samples. Other bacterial pig pathogens, including Mycoplasma hyorhinis, M. hyopneumoniae and M. hyosynoviae, were detected in oral fluid and aerosols by PCR and metagenomics. One of the two farms was PRRSV positive, and the virus was detectable via PCR in oral fluids but not in bioaerosols. Antimicrobial resistance (AMR) gene profiles had less variation between bioaerosols and oral fluids. Some identified AMR genes had strikingly similar abundance overall.ConclusionOverall, these findings indicate that there is potential utility of bioaerosol metagenomics for pathogen surveillance on pig farms; however, more research is needed for technical and cost optimization to allow for routine pathogen detection on livestock farms.

Domestic swine (Sus scrofa domesticus) are important translational models for cardiovascular transplant studies. This can be attributed to the anatomic and physiologic similarities of their cardiovascular system to humans. Transplant studies frequently employ clinically relevant immunosuppression regimens to prevent organ rejection postoperatively. Immunosuppression can lead to opportunistic infection, including presentations that are novel or poorly described in immunocompetent hosts. In this study, we describe the first case of Mycoplasma hyorhinis -induced endocarditis affecting the pulmonary valve in a juvenile, immunosuppressed pig following a partial heart transplantation procedure. Clinical signs of infection began at 15 d postoperation, were consistent with a variety of infectious agents, including Mycoplasma hyorhinis, and included lethargy, respiratory signs, and elevated white blood cell counts. By 28 d post procedure, lameness and soft tissue swelling around the left tarsus developed. Joint fluid obtained by arthrocentesis was PCR positive for Mycoplasma hyorhinis and negative for other tested pathogens. Despite antimicrobial treatment, the transplanted pulmonary valve developed leaflet thickening, stenosis, and insufficiency starting at 30 d after the procedure. At 86 d posttransplantation, the pig reached experimental endpoints and was humanely euthanized for necropsy and histopathology. The pulmonary valve had numerous dark red vegetative expansions of all 3 leaflets. Postmortem testing of a vegetative lesion was positive for Mycoplasma hyorhinis, confirming the etiologic agent responsible for endocarditis. Mycoplasma hyorhinis -induced endocarditis of an orthotopic transplanted pulmonary valve has yet to be described in swine. This case report demonstrates that infections following immunosuppression may present with novel or undercharacterized clinical signs.

The Mycoplasma hyorhinis (Mhr) variable lipoprotein (Vlp) family, comprising Vlps A, B, C, D, E, F, and G, are highly variable in expression, size, and cytoadhesion capabilities across Mhr strains. The ‘Vlp system’ plays a crucial role in cytoadhesion, immune evasion, and in eliciting a host immunologic response. This pilot study described the development of Vlp peptide-based ELISAs to evaluate the antigenic reactivity of individual Vlps against Mhr antisera collected throughout a longitudinal study focused on Mhr strain 38983, reproducing Mhr-associated disease under experimental conditions. Specifically, serum samples were collected at day post-inoculation 0, 7, 10, 14, 17, 21, 24, 28, 35, 42, 49, and 56 from Mhr- and mock (Friis medium)-inoculated cesarean-derived, colostrum-deprived pigs. Significant Mhr-specific IgG responses were detected at specific time points throughout the infection, with some variations for each Vlp. Overall, individual Vlp ELISAs showed consistently high accuracy rates, except for VlpD, which would likely be associated with its expression levels or the anti-Vlp humoral immune response specific to the Mhr strain used in this study. This study provides the basis and tools for a more refined understanding of these Vlp- and Mhr strain-specific variations, which is foundational in understanding the host immune response to Mhr.