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Growing evidence suggests an association between Mycoplasma infections and the development and progression of prostate cancer (PCa). In this study, we report that chronic and persistent M. hyorhinis infection induced robust TNF-α secretion from PCa cells. TNF-α secreted from M. hyorhinis-infected PCa cells subsequently led to activation of the NF-κB pathway. Chronic M. hyorhinis infection induced gene expression of pro-inflammatory cytokines and chemokines in a NF-κB-dependent manner and promoted cell proliferation, migration, and invasion in PCa cells. The elimination of M. hyorhinis in PCa cells significantly blocked TNF-α secretion, gene expression of cytokines and chemokines, migration, and invasion in PCa cells, suggesting M. hyorhinis-induced TNF-α plays an important role to promote malignant transformation of PCa. Furthermore, second mitochondria-derived activator of caspases (SMAC) mimetics potentiated caspase activation and cell death in M. hyorhinis-infected PCa by antagonizing inhibitor of apoptosis proteins (IAPs) activity. Tissue microarray analysis indicated that TNF-α is co-expressed in M. hyorhinis-infected human patient tissues. Findings from this study advance our understanding of the mycoplasma-oncogenesis process and suggest the potential for new approaches for preventions, diagnosis, and therapeutic approaches against prostate cancers.

Abstract Background Accurate measurement of disease associated with endemic bacterial agents in pig populations is challenging due to their commensal ecology, the lack of disease-specific antemortem diagnostic tests, and the polymicrobial nature of swine diagnostic cases. The main objective of this retrospective study was to estimate temporal patterns of agent detection and disease diagnosis for five endemic bacteria that can cause systemic disease in porcine tissue specimens submitted to the Iowa State University Veterinary Diagnostic Laboratory (ISU VDL) from 2017 to 2022. The study also explored the diagnostic value of specific tissue specimens for disease diagnosis, estimated the frequency of polymicrobial diagnosis, and evaluated the association between phase of pig production and disease diagnosis. Results S. suis and G. parasuis bronchopneumonia increased on average 6 and 4.3%, while S. suis endocarditis increased by 23% per year, respectively. M. hyorhinis and A. suis associated serositis increased yearly by 4.2 and 12.8%, respectively. A significant upward trend in M. hyorhinis arthritis cases was also observed. In contrast, M. hyosynoviae arthritis cases decreased by 33% average/year. Investigation into the diagnostic value of tissues showed that lungs were the most frequently submitted sample, However, the use of lung for systemic disease diagnosis requires caution due to the commensal nature of these agents in the respiratory system, compared to systemic sites that diagnosticians typically target. This study also explored associations between phase of production and specific diseases caused by each agent, showcasing the role of S. suis arthritis in suckling pigs, meningitis in early nursery and endocarditis in growing pigs, and the role of G. parasuis, A. suis, M. hyorhinis and M. hyosynoviae disease mainly in post-weaning phases. Finally, this study highlighted the high frequency of co-detection and -disease diagnosis with other infectious etiologies, such as PRRSV and IAV, demonstrating that to minimize the health impact of these endemic bacterial agents it is imperative to establish effective viral control programs. Conclusions Results from this retrospective study demonstrated significant increases in disease diagnosis for S. suis, G. parasuis, M. hyorhinis, and A. suis, and a significant decrease in detection and disease diagnosis of M. hyosynoviae. High frequencies of interactions between these endemic agents and with viral pathogens was also demonstrated. Consequently, improved control programs are needed to mitigate the adverse effect of these endemic bacterial agents on swine health and wellbeing. This includes improving diagnostic procedures, developing more effective vaccine products, fine-tuning antimicrobial approaches, and managing viral co-infections.

Background Accurate measurement of disease associated with endemic bacterial agents in pig populations is challenging due to their commensal ecology, the lack of disease-specific antemortem diagnostic tests, and the polymicrobial nature of swine diagnostic cases. The main objective of this retrospective study was to estimate temporal patterns of agent detection and disease diagnosis for five endemic bacteria that can cause systemic disease in porcine tissue specimens submitted to the Iowa State University Veterinary Diagnostic Laboratory (ISU VDL) from 2017 to 2022. The study also explored the diagnostic value of specific tissue specimens for disease diagnosis, estimated the frequency of polymicrobial diagnosis, and evaluated the association between phase of pig production and disease diagnosis. Results S. suis and G. parasuis bronchopneumonia increased on average 6 and 4.3%, while S. suis endocarditis increased by 23% per year, respectively. M. hyorhinis and A. suis associated serositis increased yearly by 4.2 and 12.8%, respectively. A significant upward trend in M. hyorhinis arthritis cases was also observed. In contrast, M. hyosynoviae arthritis cases decreased by 33% average/year. Investigation into the diagnostic value of tissues showed that lungs were the most frequently submitted sample, However, the use of lung for systemic disease diagnosis requires caution due to the commensal nature of these agents in the respiratory system, compared to systemic sites that diagnosticians typically target. This study also explored associations between phase of production and specific diseases caused by each agent, showcasing the role of S. suis arthritis in suckling pigs, meningitis in early nursery and endocarditis in growing pigs, and the role of G. parasuis, A. suis, M. hyorhinis and M. hyosynoviae disease mainly in post-weaning phases. Finally, this study highlighted the high frequency of co-detection and -disease diagnosis with other infectious etiologies, such as PRRSV and IAV, demonstrating that to minimize the health impact of these endemic bacterial agents it is imperative to establish effective viral control programs. Conclusions Results from this retrospective study demonstrated significant increases in disease diagnosis for S. suis, G. parasuis, M. hyorhinis, and A. suis, and a signifcant decrease in detection and disease diagnosis of M. hyosynoviae. High frequencies of interactions between these endemic agents and with viral pathogens was also demonstrated. Consequently, improved control programs are needed to mitigate the adverse effect of these endemic bacterial agents on swine health and wellbeing. This includes improving diagnostic procedures, developing more effective vaccine products, fne-tuning antimicrobial approaches, and managing viral co-infections. [ABSTRACT FROM AUTHOR]

Organizations like the Clinical and Laboratory Standards Institute (CLSI) or the European Committee of Antimicrobial Susceptibility Testing (EUCAST) provide standardized methodologies for antimicrobial susceptibility testing of a wide range of nonfastidious and fastidious bacteria, but so far not for Mycoplasma spp. of animal origin. Recently, a proposed method for the standardized broth microdilution testing of Mycoplasma hyorhinis using commercial Sensititre microtiter plates was presented. In this study, we evaluated this broth microdilution method with 37 field isolates and tested their susceptibility toward the following antimicrobial agents: doxycycline, enrofloxacin, erythromycin, florfenicol, gentamicin, marbofloxacin, tetracycline, tiamulin, tilmicosin, tulathromycin, and tylosin. The isolates originated from different countries, isolation sites, and years. The broth microdilution method was carried out using a modified Friis broth as the culture and test medium. For macrolides and lincosamides, a bimodal distribution with elevated MIC values could be observed for almost half of the tested field isolates, deducing reduced susceptibility toward these substances. With a recently published protocol, we were able to test a variety of field isolates, and consistent data could be obtained. Using this method, monitoring studies of Mycoplasma hyorhinis isolates can be carried out in a comparable manner, and the observed susceptibility profiles can be screened for possible changes in MIC values in the future. [ABSTRACT FROM AUTHOR]

Background: Mycoplasma hyorhinis is a prevalent respiratory pathogen in swine, causing significant economic loss to pig producers. There is growing evidence that respiratory pathogen infections have a large impact on intestinal microecology. To study the effect of M. hyorhinis infection on gut microbial composition and metabolome profile, pigs were infected with M. hyorhinis. Metagenomic sequencing analysis was performed of fecal samples and a liquid chromatography/tandem mass spectrometry (LC–MS/MS) analysis of gut digesta was made. Results: Pigs infected with M. hyorhinis had enriched Sutterella and Mailhella, and depleted Dechloromonas, Succinatimonas, Campylobacter, Blastocystis, Treponema, and Megasphaera. The pigs infected with M. hyorhinis also had greater abundances of bacterium_0_1xD8_71, Ruminococcussp_CAG_353, Firmicutes_bacterium_CAG_194, Firmicutes_bacterium_CAG_534, bacterium_1xD42_87, and lower abundances of Chlamydia_suis, Megasphaera_elsdenii, Treponema_porcinum, Bacteroidessp_CAG_1060, Faecalibacterium_prausnitzii. Metabolomic analysis revealed that some lipids and lipid‐like molecules increased in the small intestine, whereas most lipids and lipid‐like molecule metabolites decreased in the large intestine. These altered metabolites induce changes in intestinal sphingolipid metabolism, amino acid metabolism, and thiamine metabolism. Conclusion: These findings demonstrate that infection with M. hyorhinis can alter the gut microbial composition and metabolite structure in pigs, which may further affect amino acid metabolism and lipid metabolism in the intestine. © 2023 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Growing evidence suggests an association between Mycoplasma infections and the development and progression of prostate cancer (PCa). In this study, we report that chronic and persistent M. hyorhinis infection induced robust TNF-α secretion from PCa cells. TNF-α secreted from M. hyorhinis-infected PCa cells subsequently led to activation of the NF-κB pathway. Chronic M. hyorhinis infection induced gene expression of pro-inflammatory cytokines and chemokines in a NF-κB-dependent manner and promoted cell proliferation, migration, and invasion in PCa cells. The elimination of M. hyorhinis in PCa cells significantly blocked TNF-α secretion, gene expression of cytokines and chemokines, migration, and invasion in PCa cells, suggesting M. hyorhinis-induced TNF-α plays an important role to promote malignant transformation of PCa. Furthermore, second mitochondria-derived activator of caspases (SMAC) mimetics potentiated caspase activation and cell death in M. hyorhinis-infected PCa by antagonizing inhibitor of apoptosis proteins (IAPs) activity. Tissue microarray analysis indicated that TNF-α is co-expressed in M. hyorhinis-infected human patient tissues. Findings from this study advance our understanding of the mycoplasma-oncogenesis process and suggest the potential for new approaches for preventions, diagnosis, and therapeutic approaches against prostate cancers.

Introduction: Mycoplasma hyorhinis is an emerging swine pathogen with high prevalence worldwide. The main lesions caused are arthritis and polyserositis, and the clinical manifestation of the disease may result in significant economic losses due to decreased weight gain and enhanced medical costs. We aimed to compare two challenge routes to induce M. hyorhinis infection using the same clinical isolate. Methods: Five-week-old, Choice hybrid pigs were inoculated on 2 consecutive days by intravenous route (Group IV-IV) or by intravenous and intraperitoneal routes (Group IV-IP). Mock-infected animals were used as control (control group). After the challenge, the clinical signs were recorded for 28 days, after which the animals were euthanized. Gross pathological and histopathological examinations, PCR detection, isolation, and genotyping of the re-isolated Mycoplasma sp. and culture of bacteria other than Mycoplasma sp. were carried out. The ELISA test was used to detect anti-M. hyorhinis immunoglobulins in the sera of all animals. Results: Pericarditis and polyarthritis were observed in both challenge groups; however, the serositis was more severe in Group IV-IV. Statistically significant differences were detected between the challenged groups and the control group regarding the average daily weight gain, pathological scores, and ELISA titers. Additionally, histopathological scores in Group IV-IV differed significantly from the scores in the control group. All re-isolated strains were the same or a close genetic variant of the original challenge strain. Discussion: Our results indicate that both challenge routes are suitable for modeling the disease. However, due to the evoked more severe pathological lesions and the application being similar to the hypothesized natural route of infection in Group IV-IV, the two-dose intravenous challenge is recommended by the authors to induce serositis and arthritis associated with M. hyorhinis infection. [ABSTRACT FROM AUTHOR]

Bacterial and/or viral co-infections are very common in swine production and cause severe economic losses. Mycoplasma hyopneumoniae, Mycoplasma hyorhinis and Streptococcus suis are pathogenic bacteria that may be found simultaneously in the respiratory tracts of pigs. In the present study, the interactions of S. suis with epithelial and phagocytic cells in the presence or absence of a pre-infection with M. hyopneumoniae and/or M. hyorhinis were studied. Results showed relatively limited interactions between these pathogens. A previous infection with one or both mycoplasmas did not influence the adhesion or invasion properties of S. suis in epithelial cells or its resistance to phagocytosis (including intracellular survival) by macrophages and dendritic cells. The most important effect observed during the co-infection was a clear increment in toxicity for the cells. An increase in the relative expression of the pro-inflammatory cytokines IL-6 and CXCL8 was also observed; however, this was the consequence of an additive effect due to the presence of different pathogens rather than a synergic effect. It may be hypothesized that if one or both mycoplasmas are present along with S. suis in the lower respiratory tract at the same time, then increased damage to epithelial cells and phagocytes, as well as an increased release of pro-inflammatory cytokines, may eventually enhance the invasive properties of S. suis. However, more studies should be carried out to confirm this hypothesis. [ABSTRACT FROM AUTHOR]

Achieving food security involves the maintenance of intensive production systems and large animal populations where infectious diseases are the most important challenge that need to be addressed. Respiratory infections in pigs are a formidable threat to swine farmers worldwide. It is multifactorial and is caused by the interplay between bacterial pathogens, viral agents and environmental factors. The present study aimed to identify mycoplasmal agents causing respiratory infections in pigs. A total of 43 samples, comprising of nasal swabs, and necropsy samples with pneumonic lesions were collected. The DNA extracted from the samples were subjected to Mycoplasma genus-specific, followed by species-specific PCR. Twenty five samples were positive for Mycoplasma spp., but further molecular detection performed through species-specific primers, revealed that 12 samples were positive for M. hyorhinis. None of the samples were positive for M. hyopneumoniae. The identity of the amplicons were confirmed by nucleic acid sequencing and BLAST analysis. The evolutionary relationship between the detected organisms was also studied by phylogenetic analysis. This study hints at the significance of M. hyorhinis in causing respiratory infections in swine and also suggests that M. hyopneumoniae may not be a significant health hazard for swine populations of north Kerala.

Conducting antimicrobial susceptibility testing (AST) in a comparable manner requires the availability of a standardized method. Organizations, such as the Clinical and Laboratory Standards Institute (CLSI) or the European Committee on Antimicrobial Susceptibility Testing (EUCAST), provide standardized protocols for a range of fastidious bacteria but not for Mycoplasma hyorhinis. We developed a broth microdilution method for testing M. hyorhinis in a standardized and harmonized way using a modified Friis broth devoid of antimicrobial or otherwise bacterial growth-inhibiting agents. The type strain M. hyorhinis DSM 25591 was chosen to establish the methodology. The antimicrobial agents of interest were doxycycline, enrofloxacin, erythromycin, florfenicol, gentamicin, marbofloxacin, tetracycline, tiamulin, tilmicosin, tulathromycin, and tylosin, tested by using commercial SensititreTM microtiter plates. In addition, the suitability of the methodology was evaluated via variation of the individual ingredients of the modified Friis broth by either using different batches or choosing other distributors. Despite these alterations, the method provided reliable results. We obtained repeatable minimal inhibitory concentrations for all six tested field isolates and the M. hyorhinis type strain. With this newly proposed method, we aim to provide an improved AST method for diagnostic laboratories and monitoring purposes with better comparability between times and countries. In addition, this new method will allow for an improvement of targeted treatments using antimicrobial agents and thereby reduce the options for resistance development. [ABSTRACT FROM AUTHOR]

IntroductionMycoplasma hyorhinis is an emerging swine pathogen with high prevalence worldwide. The main lesions caused are arthritis and polyserositis, and the clinical manifestation of the disease may result in significant economic losses due to decreased weight gain and enhanced medical costs. We aimed to compare two challenge routes to induce M. hyorhinis infection using the same clinical isolate.MethodsFive-week-old, Choice hybrid pigs were inoculated on 2 consecutive days by intravenous route (Group IV-IV) or by intravenous and intraperitoneal routes (Group IV-IP). Mock-infected animals were used as control (control group). After the challenge, the clinical signs were recorded for 28 days, after which the animals were euthanized. Gross pathological and histopathological examinations, PCR detection, isolation, and genotyping of the re-isolated Mycoplasma sp. and culture of bacteria other than Mycoplasma sp. were carried out. The ELISA test was used to detect anti-M. hyorhinis immunoglobulins in the sera of all animals.ResultsPericarditis and polyarthritis were observed in both challenge groups; however, the serositis was more severe in Group IV-IV. Statistically significant differences were detected between the challenged groups and the control group regarding the average daily weight gain, pathological scores, and ELISA titers. Additionally, histopathological scores in Group IV-IV differed significantly from the scores in the control group. All re-isolated strains were the same or a close genetic variant of the original challenge strain.DiscussionOur results indicate that both challenge routes are suitable for modeling the disease. However, due to the evoked more severe pathological lesions and the application being similar to the hypothesized natural route of infection in Group IV-IV, the two-dose intravenous challenge is recommended by the authors to induce serositis and arthritis associated with M. hyorhinis infection.

Abstract Glaesserella parasuis, Mycoplasma hyorhinis, and Mycoplasma hyosynoviae are important porcine pathogens responsible for polyserositis, polyarthritis, meningitis, pneumonia, and septicemia causing significant economic losses in the swine industry. A new multiplex quantitative polymerase chain reaction (qPCR) was designed on one hand for the detection of G. parasuis and the virulence marker vtaA to distinguish between highly virulent and non‐virulent strains. On the other hand, fluorescent probes were established for the detection and identification of both M. hyorhinis and M. hyosynoviae targeting 16S ribosomal RNA genes. The development of the qPCR was based on reference strains of 15 known serovars of G. parasuis, as well as on the type strains M. hyorhinis ATCC 17981T and M. hyosynoviae NCTC 10167T. The new qPCR was further evaluated using 21 G. parasuis, 26 M. hyorhinis, and 3 M. hyosynoviae field isolates. Moreover, a pilot study including different clinical specimens of 42 diseased pigs was performed. The specificity of the assay was 100% without cross‐reactivity or detection of other bacterial swine pathogens. The sensitivity of the new qPCR was demonstrated to be between 11–180 genome equivalents (GE) of DNA for M. hyosynoviae and M. hyorhinis, and 140–1200 GE for G. parasuis and vtaA. The cut‐off threshold cycle was found to be at 35. The developed sensitive and specific qPCR assay has the potential to become a useful molecular tool, which could be implemented in veterinary diagnostic laboratories for the detection and identification of G. parasuis, its virulence marker vtaA, M. hyorhinis, and M. hyosynoviae.

Simple Summary: This study mainly aims to investigate the impact and the etiology of swine polyserositis, focusing on three causative agents: Glaesserella parasuis, Streptococcus suis and Mycoplasma hyorhinis. Results indicate that the etiology of polyserositis is an intricate puzzle of pathogens, and that each pig herd likely represents a unique scenario. Therefore, the correct diagnosis of polyserositis can be really challenging and often makes the implementation of therapeutic and control strategies frustrating. Polyserositis mostly affects 4–8 weeks old piglets and is usually caused by Glaesserella parasuis, and/or Streptococcus suis, and/or Mycoplasma hyorhinis. The present study aimed to investigate the prevalence and etiology of polyserositis in a tricky pig herd. The concurrent effect of vaccination for Glässer's disease was also assessed. A total of 46 sows and 387 piglets were herein investigated, subdivided into three groups based on their immune status (i.e., vaccination of sows and piglets). All the piglets found spontaneously dead between the 2nd and 16th week of age were recorded and necropsied. Whenever polyserositis was diagnosed, biomolecular investigations were carried out to detect the above-mentioned pathogens. Mycoplasma hyorhinis was detected most frequently (n = 23), often as the only causative agent (n = 15), whereas S. suis was observed in 8 cases (6 as the only pathogen). Moreover, Glaesserella parasuis was demonstrated in 6 piglets, always in combination with Mycoplasma hyorhinis and/or Streptococcus suis. Vaccination did not significantly affect mortality rates. Overall, our data indicate that polyserositis is likely caused by an intricate puzzle of pathogens, even when dealing with a small herd and during a short time span. That makes it challenging to achieve the correct diagnosis and to properly manage this health issue. [ABSTRACT FROM AUTHOR]

Abstract Mycoplasma hyorhinis may cause systemic inflammation of pigs, typically polyserositis and arthritis, and is also associated with several types of human cancer. However, the pathogenesis of M. hyorhinis colonizing and breaching the respiratory barrier to establish systemic infection is poorly understood. Glycolytic enzymes are important moonlighting proteins and virulence-related factors in various bacteria. In this study, we investigated the functions of a glycolytic critical enzyme, enolase in the infection and systemic spread of M. hyorhinis. Bacterial surface localization of enolase was confirmed by flow cytometry and colony hybridization assay. Recombinant M. hyorhinis enolase (rEno) was found to adhere to pig kidney (PK-15) cells, and anti-rEno serum significantly decreased adherence. The enzyme was also found to bind host plasminogen and fibronectin, and interactions were specific and strong, with dissociation constant (K D) values of 1.4 nM and 14.3 nM, respectively, from surface plasmon resonance analysis. Activation of rEno-bound plasminogen was confirmed by its ability to hydrolyze plasmin-specific substrates and to degrade a reconstituted extracellular matrix. To explore key sites during these interactions, C-terminal lysine residues of enolase were replaced with leucine, and the resulting single-site and double-site mutants show significantly reduced interaction with plasminogen in far-Western blotting and surface plasmon resonance tests. The binding affinities of all mutants to fibronectin were reduced as well. Collectively, these results imply that enolase moonlights as an important adhesin of M. hyorhinis, and interacts with plasminogen and fibronectin. The two lysine residues in the C-terminus are important binding sites for its multiple binding activities.

Organizations like the Clinical and Laboratory Standards Institute (CLSI) or the European Committee of Antimicrobial Susceptibility Testing (EUCAST) provide standardized methodologies for antimicrobial susceptibility testing of a wide range of nonfastidious and fastidious bacteria, but so far not for Mycoplasma spp. of animal origin. Recently, a proposed method for the standardized broth microdilution testing of Mycoplasma hyorhinis using commercial Sensititre microtiter plates was presented. In this study, we evaluated this broth microdilution method with 37 field isolates and tested their susceptibility toward the following antimicrobial agents: doxycycline, enrofloxacin, erythromycin, florfenicol, gentamicin, marbofloxacin, tetracycline, tiamulin, tilmicosin, tulathromycin, and tylosin. The isolates originated from different countries, isolation sites, and years. The broth microdilution method was carried out using a modified Friis broth as the culture and test medium. For macrolides and lincosamides, a bimodal distribution with elevated MIC values could be observed for almost half of the tested field isolates, deducing reduced susceptibility toward these substances. With a recently published protocol, we were able to test a variety of field isolates, and consistent data could be obtained. Using this method, monitoring studies of Mycoplasma hyorhinis isolates can be carried out in a comparable manner, and the observed susceptibility profiles can be screened for possible changes in MIC values in the future.

Abstract Background Gastric cancer is the third leading cause of cancer-related deaths worldwide and has been associated with infections that may promote tumour progression. Accordingly, we analysed the presence of Mollicutes, Mycoplasma hyorhinis, Fusobacterium nucleatum and Helicobacter pylori in gastric cancer tissues and evaluated their correlation with clinicopathological factors. Methods Using a commercial kit, DNA were extracted from 120 gastric samples embedded in paraffin: 80 from patients with gastric cancer and 40 from cancer free patients, dating from 2006 to 2016. Mollicutes and H. pylori were detected by PCR; F. nucleatum and M. hyorhinis were detected by qPCR, together with immunohistochemistry for the latter bacteria. Results Mollicutes were detected in the case and control groups (12% and 2.5%) and correlated with the papillary histologic pattern (P = 0.003), likely due to cell transformation promoted by Mollicutes. M. hyorhinis was detected in the case and control group but was not considered a cancer risk factor. H. pylori was detected at higher loads in the case compared to the control group (8% and 22%, P = 0.008) and correlated with metastasis (P = 0.024), lymphatic invasion (P = 0.033), tumour of diffused type (P = 0.028), and histopathological grading G1/G2 (P = 0.008). F. nucleatum was the most abundant bacteria in the case group, but was also detected in the control group (26% and 2.5%). It increased the cancer risk factor (P = 0.045, OR = 10.562, CI95% = 1.057–105.521), and correlated with old age (P = 0.030) and tumour size (P = 0.053). Bacterial abundance was significantly different between groups (P = 0.001). Conclusion Our findings could improve the control and promote our understanding of opportunistic bacteria and their relevance to malignant phenotypes.

Mycoplasma hyorhinis may cause systemic inflammation of pigs, typically polyserositis and arthritis, and is also associated with several types of human cancer. However, the pathogenesis of M. hyorhinis colonizing and breaching the respiratory barrier to establish systemic infection is poorly understood. Glycolytic enzymes are important moonlighting proteins and virulence-related factors in various bacteria. In this study, we investigated the functions of a glycolytic critical enzyme, enolase in the infection and systemic spread of M. hyorhinis. Bacterial surface localization of enolase was confirmed by flow cytometry and colony hybridization assay. Recombinant M. hyorhinis enolase (rEno) was found to adhere to pig kidney (PK-15) cells, and anti-rEno serum significantly decreased adherence. The enzyme was also found to bind host plasminogen and fibronectin, and interactions were specific and strong, with dissociation constant (KD) values of 1.4 nM and 14.3 nM, respectively, from surface plasmon resonance analysis. Activation of rEno-bound plasminogen was confirmed by its ability to hydrolyze plasmin-specific substrates and to degrade a reconstituted extracellular matrix. To explore key sites during these interactions, C-terminal lysine residues of enolase were replaced with leucine, and the resulting single-site and double-site mutants show significantly reduced interaction with plasminogen in far-Western blotting and surface plasmon resonance tests. The binding affinities of all mutants to fibronectin were reduced as well. Collectively, these results imply that enolase moonlights as an important adhesin of M. hyorhinis, and interacts with plasminogen and fibronectin. The two lysine residues in the C-terminus are important binding sites for its multiple binding activities. [ABSTRACT FROM AUTHOR]

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Mycoplasma hyorhinis is a highly prevalent pathogen in pig farms worldwide, causing polyserositis and polyarthritis, resulting in great economic losses. Previous genotyping and pathogenic studies have revealed significant genetic and antigenic diversity among M. hyorhinis strains. While there are reports on M. hyorhinis vaccine development, the cross-protection between different M. hyorhinis strains has not been clarified. In this study, two M. hyorhinis strains (HEF-16 and JS-54), belonging to different sequence types, were inactivated to produce vaccines. Pigs were vaccinated respectively and subsequently infected with strain HEF-16. The protection against challenge with homologous or heterologous strains was determined and compared. Both vaccinated groups of pigs exhibited a high antibody titer two weeks after the first vaccination, and significant decreases in pathogen load in joints, along with an increase in average daily weight gain compared to the challenged group after M. hyorhinis challenge. Pigs immunized with the HEF-16-derived vaccine showed a significant reduction in joint swelling and lameness, similar to pigs immunized with the JS-54-derived vaccine. At necropsy, animals in the challenged group exhibited moderate-to-severe polyserositis and arthritis, whereas pathological changes were greatly reduced in animals from the vaccinated groups. No significant differences were observed in clinical symptoms nor pathological damages between the two vaccinated groups. Overall, our study demonstrates the effective protection of the inactivated M. hyorhinis vaccines against challenges with homologous or heterologous strains in commercial pigs. This indicates a promising clinical application prospect for inactivated bacterin vaccines in preventing M. hyorhinis-related diseases in pig farms., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024. Published by Elsevier Ltd.)

Mycoplasma hyorhinis (M. hyorhinis) lacks a cell wall and resists multiple antibiotics. We describe here the striking > 90% inhibitory effect of hemin, a natural inducer of the cytoprotective enzyme heme oxygenase‐1 (HO‐1), on M. hyorhinis replication in chronically infected LNCaP prostate cancer cells. The role of HO‐1 in interrupting M. hyorhinis replication was confirmed by HO‐1‐specific siRNA suppression of hemin‐induced HO‐1 protein expression, which increased intracellular M. hyorhinis DNA levels in LNCaP cells. Proteomic analysis and transmission electron microscopy of hemin‐treated cells confirmed the complete absence of M. hyorhinis proteins and intact microorganisms, respectively, strongly supporting these findings. Our study is the first to our knowledge suggesting therapeutic potential for activated HO‐1 in cellular innate responses against mycoplasma infection.

Bacterial and/or viral co-infections are very common in swine production and cause severe economic losses. Mycoplasma hyopneumoniae, Mycoplasma hyorhinis and Streptococcus suis are pathogenic bacteria that may be found simultaneously in the respiratory tracts of pigs. In the present study, the interactions of S. suis with epithelial and phagocytic cells in the presence or absence of a pre-infection with M. hyopneumoniae and/or M. hyorhinis were studied. Results showed relatively limited interactions between these pathogens. A previous infection with one or both mycoplasmas did not influence the adhesion or invasion properties of S. suis in epithelial cells or its resistance to phagocytosis (including intracellular survival) by macrophages and dendritic cells. The most important effect observed during the co-infection was a clear increment in toxicity for the cells. An increase in the relative expression of the pro-inflammatory cytokines IL-6 and CXCL8 was also observed; however, this was the consequence of an additive effect due to the presence of different pathogens rather than a synergic effect. It may be hypothesized that if one or both mycoplasmas are present along with S. suis in the lower respiratory tract at the same time, then increased damage to epithelial cells and phagocytes, as well as an increased release of pro-inflammatory cytokines, may eventually enhance the invasive properties of S. suis. However, more studies should be carried out to confirm this hypothesis.

While male infertility has been associated with Mycoplasma infections, few studies have investigated the association between Mycoplasma infection and male infertility. Therefore, this study aimed at addressing this issue. Semen samples were collected from 136 patients (68 infertile men and 68 fertile men) in the Central Laboratory of Yazd, Iran. Of semen samples collected from 68 infertile and 68 fertile men, 13 (19.12%) and 2 (2.94%) cases were positive for Mycoplasma spp. using PCR, respectively. Among Mycoplasma-infected infertile men, 10 and 6 men showed abnormal sperm morphology and motility, respectively. None of the positive samples for Mycoplasma spp. was positive for M. hominis and one of the positive samples for Mycoplasma spp. belonged to Mycoplasma hyorhinis (strain NBRC 14858). The presence of Mycoplasma spp. was significantly higher in infertile men (p =.003). Mycoplasma infection was relatively high in infertile men. The surprising issue was the absence of M. hominis and the presence of M. hyorhinis strain NBRC 14858 in the semen of infertile men. Therefore, investigating reproductive tract infections caused by other Mycoplasma spp. should be taken into consideration in male infertility. What is already known on this subject?Mycoplasma hyorhinis has been mostly reported as a cause of animal respiratory tract infections and the development of various cancers. Information on the association of M. hyorhinis with male infertility is not yet available. What do the results of this study add? This study shows that the presence of M. hyorhinis in the semen of infertile men may be associated with infertility. This study shows that the investigation of unpredictable species of genus Mycoplasma such as M. hyorhinis in the semen of infertile men is essential. What are the implications of these findings for clinical practice and/or further research? The results of the present study indicate that in addition to M. genitalium and M. hominis, studies on the role of M. hyorhinis in reproductive tract infections and infertility should be expanded. [ABSTRACT FROM AUTHOR]

Mycoplasma hyorhinis (M. hyorhinis) is an important pathogen in the pig industry, especially during the nursery period. Multi-locus sequence typing (MLST) is a specific method used to identify many bacterial species. At present, 108 MLST schemes of M. hyorhinis have been reported around the world. This study aimed to investigate the variable multi-locus sequence typing (MLST) schemes of M. hyorhinis in pig herds from bacterial stock at the large animal hospital, Faculty of Veterinary Science, Chulalongkorn University, since 2010. Bacteria were collected from 98 deceased pigs sent for autopsy at this veterinary hospital. Samples were collected from at least one lesion per pig located in the joint capsule, lung, thoracic cavity, abdominal cavity, and pericardium. Bacteria were cultured and confirmed the species by PCR. MLST was detected by PCR and DNA sequencing. Sequence data were reported to GenBank and PubMLST databases. In this study, the positive results of M. hyorhinis were found on 75 samples, while 23 samples gave negative results. The highest population of this pathogen was shown on the joint organ but no significant difference with the other organs. Only nine positive samples could be cultured, purified, and sent for sequencing. Sequencing results revealed 6 MLST schemes, while 5 of them were defined as new ST types (ST71-75) defined for the first time in Thailand. A diverse array of MLST in this location, some of which are novel, implied that bacteria might adapt to their environment. MLST information might play a role in vaccine development and preventative strategies. [ABSTRACT FROM AUTHOR]

ABSTRACT Lung inflammation induced by Mycoplasma hyorhinis infection accounts for significant economic losses in the swine industry. Increasing evidence suggests that there is cross talk between the lungs and the gut, but little is known about the effect of the lung inflammation caused by M. hyorhinis infection on gut microbiota and intestinal barrier function. Here, we investigated changes in the fecal microbiotas of pigs with M. hyorhinis infection and the microbial regulatory role of such infection in intestinal barrier function. We infected pigs with M. hyorhinis and performed 16S rRNA gene sequencing analyses of fecal samples, data-independent acquisition (DIA) quantitative proteomic analyses of intestinal mucosa, and analyses of barrier dysfunction indicators in serum. We found that pigs with M. hyorhinis infection exhibit lung and systemic inflammation, as reflected by the histopathological changes and activation of the TLR4/MyD88/NF-κB p65 signaling pathway in lung tissue, as well as the increased concentrations of serum inflammatory cytokines. Gut microbiotas tended to become disturbed, as evidenced by the enrichment of opportunistic pathogens. The increased diamine oxidase activities and d-lactate concentrations in serum and the decreased relative mRNA expression of Occludin, ZO-1, and Mucin2 indicated the impairment of intestinal barrier function. Quantitative proteomic analyses showed a variety of altered proteins involved in immunomodulatory and inflammatory functions. There was a positive correlation between the abundance of opportunistic pathogens and inflammatory-cytokine concentrations, as well as intestinal immunomodulatory proteins. Our results suggest that lung inflammation induced by M. hyorhinis infection can contribute to the dysbiosis of gut microbiota and intestinal barrier dysfunction, and dysbiosis of gut microbiota was associated with systemic inflammation and intestinal immune status. IMPORTANCE Cumulative evidence suggests that bacterial pneumonia may contribute to the dysbiosis of the gut microbiota and other gastrointestinal symptoms. Our experiment has demonstrated that lung inflammation induced by M. hyorhinis infection was associated with gut microbiota dysbiosis and intestinal barrier dysfunction, which may provide a theoretical basis for exploring the gut-lung axis based on M. hyorhinis infection.

ABSTRACT Mycoplasma hyorhinis is a widespread pathogen in pig farms worldwide. Although the majority of M. hyorhinis-colonized pigs have no apparent clinical disease, the pathogen can induce diseases such as polyserositis, arthritis, and eustachitis in some cases. To explore the mechanisms for the occurrence of these diseases, we challenged 4 groups of Bama miniature pigs with M. hyorhinis isolated from pigs without clinical symptoms (non-clinical origin [NCO] strain) or with typical clinical symptoms (clinical origin [CO] strain) and investigated the impacts of different strains and inoculation routes (intranasal [IN], intravenous [IV] + intraperitoneal [IP], and IV+IP+IN) on disease induction. Another group of pigs was set as a negative control. Pigs inoculated with the CO strain through a combined intravenous and intraperitoneal (IV+IP) route showed a significant decrease in average daily weight gain (ADWG), serious joint swelling, and lameness compared with the pigs in the negative-control group. Furthermore, this group developed moderate-to-severe pericarditis, pleuritis, peritonitis, and arthritis, as well as high levels of IgG and IgM antibodies. Pigs inoculated IV+IP with the NCO strain developed less marked clinical, pathological changes and a weaker specific antibody response compared with the pigs inoculated with the CO strain. The challenging results of the NCO strain via different routes (IV+IP, IV+IP+IN, and IN) indicated that the combined route (IV+IP) induced the most serious disease compared to the other inoculation routes. Intranasal inoculation induced a smaller decrease in ADWG without obvious polyserositis or arthritis. These data suggest that differences in both strain virulence and inoculation route affect the consequences of M. hyorhinis infection. IMPORTANCE Mycoplasma hyorhinis is a widespread pathogen in pig farms worldwide. The mechanisms or conditions that lead to the occurrence of disease in M. hyorhinis-infected pigs are still unknown. The objective of this study was to evaluate the impact of differences in the virulence of strain and the inoculation route on the consequences of M. hyorhinis infection.

Abstract Background Mycoplasma hyorhinis (M. hyorhinis) is a bacterium commonly found in the upper respiratory tract of healthy pigs and an agent of polyserositis and polyarthritis. Moreover, it can carry antibiotic resistance genes (Wu et al, Vet. Microbiol. 76: 25–30, 2000). Economic losses caused by M. hyorhinis can be reduced by antibiotic therapy, however, isolation and antimicrobic susceptibility profile are rarely performed. Case presentation The present report describes a case of pericarditis caused by M. hyorhinis in a weaned piglet with respiratory symptoms and reduced growth performance. At post mortem examination, the main macroscopic finding was a severe fibrinous pericarditis and M. hyorhins was the only agent isolated from the pericardial fluid. In this strain, Minimum Inhibitory Concentration (MIC) determination revealed resistance to various antimicrobial molecules such as erythromycin, tylosin and tilmicosin. Conclusion This paper highlights the importance of including M. hyorhins in the differential diagnosis of polyserositis in swine. Moreover, due the possible presence of multidrug resistance, the determination of antimicrobial susceptibility pattern should be performed on a regular basis.

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. [ABSTRACT FROM AUTHOR]

Conducting antimicrobial susceptibility testing (AST) in a comparable manner requires the availability of a standardized method. Organizations, such as the Clinical and Laboratory Standards Institute (CLSI) or the European Committee on Antimicrobial Susceptibility Testing (EUCAST), provide standardized protocols for a range of fastidious bacteria but not for Mycoplasma hyorhinis. We developed a broth microdilution method for testing M. hyorhinis in a standardized and harmonized way using a modified Friis broth devoid of antimicrobial or otherwise bacterial growth-inhibiting agents. The type strain M. hyorhinis DSM 25591 was chosen to establish the methodology. The antimicrobial agents of interest were doxycycline, enrofloxacin, erythromycin, florfenicol, gentamicin, marbofloxacin, tetracycline, tiamulin, tilmicosin, tulathromycin, and tylosin, tested by using commercial SensititreTM microtiter plates. In addition, the suitability of the methodology was evaluated via variation of the individual ingredients of the modified Friis broth by either using different batches or choosing other distributors. Despite these alterations, the method provided reliable results. We obtained repeatable minimal inhibitory concentrations for all six tested field isolates and the M. hyorhinis type strain. With this newly proposed method, we aim to provide an improved AST method for diagnostic laboratories and monitoring purposes with better comparability between times and countries. In addition, this new method will allow for an improvement of targeted treatments using antimicrobial agents and thereby reduce the options for resistance development.

Mycoplasma hyorhinis is a common pathogen of swine and is also associated with various human tumors. It causes systemic inflammation, typically polyserositis and polyarthritis, in some infected pigs. However, the pathogenic mechanism of M. hyorhinis remains unclear. DnaK is a highly conserved protein belonging to the heat-shock protein 70 family of molecular chaperones, which plays important roles as a moonlighting protein in various bacteria. In the present study, we identified the surface exposure of M. hyorhinis DnaK. Two virulent strains expressed more DnaK on their surface than the avirulent strain. Thereafter, the potential moonlighting functions of DnaK were investigated. Recombinant M. hyorhinis DnaK (rMhr-DnaK) was found to be able to adhere to swine PK-15 cells and human NCI-H292 cells. It also bound to four extracellular matrix components—fibronectin, laminin, type IV collagen, and vitronectin—in a dose-dependent manner. ELISA demonstrated an interaction between rMhr-DnaK and plasminogen, which was significantly inhibited by a lysine analog, ε-aminocaproic acid. rMhr-DnaK-bound plasminogen was activated by tissue-type plasminogen activator (tPA), and the addition of rMhr-DnaK significantly enhanced the activation. Finally, a DnaK-specific antibody was detected in the serum of pigs immunized with inactivated vaccines, which indicated good immunogenicity of it. In summary, our findings imply that DnaK is an important multifunctional moonlighting protein in M. hyorhinis and likely participates extensively in the infection and pathogenesis processes of M. hyorhinis. [ABSTRACT FROM AUTHOR]

Abstract Background Mycoplasma hyorhinis is an invader of the upper respiratory tract in swine that is considered to have ubiquitous distribution. It is mainly known for causing polyserositis and polyarthritis in weaned piglets, even though the mechanisms of systemic spread are not fully understood. Mycoplasma hyorhinis has also been associated with other diseases in pigs such as pneumonia or otitis media, but so far has not been known to cause central nervous disorders. This case series reports the isolation of Mycoplasma hyorhinis from cerebrospinal fluid and/ or meningeal swabs from piglets originating from four different piglet producing farms in Austria. Case presentation On farm 1, coughing, stiff movement and central nervous signs occurred in nursery piglets. Mycoplasma hyorhinis was the only pathogen isolated from meningeal swabs from two piglets showing central nervous signs. Fibrinopurulent leptomeningitis was only observed in one piglet. Only one of two nursery piglets from farm 2 showed mild central nervous signs but no histologic lesions; Mycoplasma hyorhinis was isolated from cerebrospinal fluid of the piglet with neurologic signs. Mycoplasma hyorhinis was isolated from cerebrospinal fluid of all three investigated piglets from farm 3, all of which showed central nervous signs and purulent leptomeningitis. Further, Streptococcus suis was isolated from the cerebrospinal fluid of one piglet. Fibrinopurulent leptomeningitis was detected in two piglets from farm 4 that had died overnight without showing any clinical signs and Mycoplasma hyorhinis was isolated from meningeal swabs from both piglets. Conclusion While causality has yet to be proven by experimental infection and in situ detection of the pathogen in histologic sections, the findings of this study and the absence of other pathogens suggest Mycoplasma hyorhinis as a potential causative agent of meningitis in swine.

Mycoplasma hyorhinis (M. hyorhinis) is an important pathogen in the pig industry, especially during the nursery period. Multi-locus sequence typing (MLST) is a specific method used to identify many bacterial species. At present, 108 MLST schemes of M. hyorhinis have been reported around the world. This study aimed to investigate the variable multi-locus sequence typing (MLST) schemes of M. hyorhinis in pig herds from bacterial stock at the large animal hospital, Faculty of Veterinary Science, Chulalongkorn University, since 2010. Bacteria were collected from 98 deceased pigs sent for autopsy at this veterinary hospital. Samples were collected from at least one lesion per pig located in the joint capsule, lung, thoracic cavity, abdominal cavity, and pericardium. Bacteria were cultured and confirmed the species by PCR. MLST was detected by PCR and DNA sequencing. Sequence data were reported to GenBank and PubMLST databases. In this study, the positive results of M. hyorhinis were found on 75 samples, while 23 samples gave negative results. The highest population of this pathogen was shown on the joint organ but no significant difference with the other organs. Only nine positive samples could be cultured, purified, and sent for sequencing. Sequencing results revealed 6 MLST schemes, while 5 of them were defined as new ST types (ST71-75) defined for the first time in Thailand. A diverse array of MLST in this location, some of which are novel, implied that bacteria might adapt to their environment. MLST information might play a role in vaccine development and preventative strategies.

Mycoplasma hyorhinis is a common pathogen of swine and is also associated with various human tumors. It causes systemic inflammation, typically polyserositis and polyarthritis, in some infected pigs. However, the pathogenic mechanism of M. hyorhinis remains unclear. DnaK is a highly conserved protein belonging to the heat-shock protein 70 family of molecular chaperones, which plays important roles as a moonlighting protein in various bacteria. In the present study, we identified the surface exposure of M. hyorhinis DnaK. Two virulent strains expressed more DnaK on their surface than the avirulent strain. Thereafter, the potential moonlighting functions of DnaK were investigated. Recombinant M. hyorhinis DnaK (rMhr-DnaK) was found to be able to adhere to swine PK-15 cells and human NCI-H292 cells. It also bound to four extracellular matrix components—fibronectin, laminin, type IV collagen, and vitronectin—in a dose-dependent manner. ELISA demonstrated an interaction between rMhr-DnaK and plasminogen, which was significantly inhibited by a lysine analog, ε-aminocaproic acid. rMhr-DnaK-bound plasminogen was activated by tissue-type plasminogen activator (tPA), and the addition of rMhr-DnaK significantly enhanced the activation. Finally, a DnaK-specific antibody was detected in the serum of pigs immunized with inactivated vaccines, which indicated good immunogenicity of it. In summary, our findings imply that DnaK is an important multifunctional moonlighting protein in M. hyorhinis and likely participates extensively in the infection and pathogenesis processes of M. hyorhinis.

Background: Gastric cancer is the third leading cause of cancer-related deaths worldwide and has been associated with infections that may promote tumour progression. Accordingly, we analysed the presence of Mollicutes, Mycoplasma hyorhinis, Fusobacterium nucleatum and Helicobacter pylori in gastric cancer tissues and evaluated their correlation with clinicopathological factors. Methods: Using a commercial kit, DNA were extracted from 120 gastric samples embedded in paraffin: 80 from patients with gastric cancer and 40 from cancer free patients, dating from 2006 to 2016. Mollicutes and H. pylori were detected by PCR; F. nucleatum and M. hyorhinis were detected by qPCR, together with immunohistochemistry for the latter bacteria. Results: Mollicutes were detected in the case and control groups (12% and 2.5%) and correlated with the papillary histologic pattern (P = 0.003), likely due to cell transformation promoted by Mollicutes. M. hyorhinis was detected in the case and control group but was not considered a cancer risk factor. H. pylori was detected at higher loads in the case compared to the control group (8% and 22%, P = 0.008) and correlated with metastasis (P = 0.024), lymphatic invasion (P = 0.033), tumour of diffused type (P = 0.028), and histopathological grading G1/G2 (P = 0.008). F. nucleatum was the most abundant bacteria in the case group, but was also detected in the control group (26% and 2.5%). It increased the cancer risk factor (P = 0.045, OR = 10.562, CI95% = 1.057–105.521), and correlated with old age (P = 0.030) and tumour size (P = 0.053). Bacterial abundance was significantly different between groups (P = 0.001). Conclusion: Our findings could improve the control and promote our understanding of opportunistic bacteria and their relevance to malignant phenotypes. [ABSTRACT FROM AUTHOR]

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.

Polyserositis mostly affects 4–8 weeks old piglets and is usually caused by Glaesserella parasuis, and/or Streptococcus suis, and/or Mycoplasma hyorhinis. The present study aimed to investigate the prevalence and etiology of polyserositis in a tricky pig herd. The concurrent effect of vaccination for Glässer’s disease was also assessed. A total of 46 sows and 387 piglets were herein investigated, subdivided into three groups based on their immune status (i.e., vaccination of sows and piglets). All the piglets found spontaneously dead between the 2nd and 16th week of age were recorded and necropsied. Whenever polyserositis was diagnosed, biomolecular investigations were carried out to detect the above-mentioned pathogens. Mycoplasma hyorhinis was detected most frequently (n = 23), often as the only causative agent (n = 15), whereas S. suis was observed in 8 cases (6 as the only pathogen). Moreover, Glaesserella parasuis was demonstrated in 6 piglets, always in combination with Mycoplasma hyorhinis and/or Streptococcus suis. Vaccination did not significantly affect mortality rates. Overall, our data indicate that polyserositis is likely caused by an intricate puzzle of pathogens, even when dealing with a small herd and during a short time span. That makes it challenging to achieve the correct diagnosis and to properly manage this health issue.

Mycoplasma hyorhinis (M. hyorhinis) lacks a cell wall and resists multiple antibiotics. We describe here the striking > 90% inhibitory effect of hemin, a natural inducer of the cytoprotective enzyme heme oxygenase‐1 (HO‐1), on M. hyorhinis replication in chronically infected LNCaP prostate cancer cells. The role of HO‐1 in interrupting M. hyorhinis replication was confirmed by HO‐1‐specific siRNA suppression of hemin‐induced HO‐1 protein expression, which increased intracellular M. hyorhinis DNA levels in LNCaP cells. Proteomic analysis and transmission electron microscopy of hemin‐treated cells confirmed the complete absence of M. hyorhinis proteins and intact microorganisms, respectively, strongly supporting these findings. Our study is the first to our knowledge suggesting therapeutic potential for activated HO‐1 in cellular innate responses against mycoplasma infection. [ABSTRACT FROM AUTHOR]

Antimicrobial susceptibility testing (AST) should be conducted in a standardized manner prior to the start of an antimicrobial treatment. For fastidious bacteria, such as porcine Mycoplasma (‘Mesomycoplasma’) spp., specifically M. hyorhinis, neither guidelines or standards for the performance of AST, nor quality control strains for the validation of AST results are approved by organizations like the Clinical and Laboratory Standards Institute (CLSI) or the European Committee on Antimicrobial Susceptibility Testing (EUCAST). The CLSI- and EUCAST-approved quality control strains Enterococcus faecalis ATCC 29212 and Staphylococcus aureus ATCC 29213 were chosen to validate AST by broth microdilution using modified Friis broth, developed as growth medium for porcine Mycoplasma (‘Mesomycoplasma’) spp. The antimicrobial agents doxycycline, enrofloxacin, erythromycin, florfenicol, gentamicin, marbofloxacin, tetracycline, tiamulin, tilmicosin, tulathromycin, and tylosin were examined using customized SensititreTM microtiter plates. Minimal inhibitory concentrations, determined after 24, 48, and 72 h, were mostly within the CLSI-approved quality control ranges for defined antimicrobial agents. We propose the use of the combination of E. faecalis ATCC 29212 and S. aureus ATCC 29213 as surrogate quality control strains for the validation of future AST results obtained for M. hyorhinis by broth microdilution using modified Friis broth. [ABSTRACT FROM AUTHOR]

The control of Mycoplasma hyorhinis infection relies mainly on antimicrobial therapy. However, the antibiotic susceptibility testing of the bacteria is usually not performed before applying the treatment, and thus therapeutic failures are not uncommon. In the case of M. hyorhinis, several antibiotic-resistance-related single nucleotide polymorphisms (SNPs) are known but assays for their detection have not been described yet. The aims of the present study were to investigate macrolide- and lincomycin-resistance-related SNPs in Hungarian M. hyorhinis isolates and to develop mismatch amplification mutation assays (MAMA) to detect the identified resistance markers. Minimal inhibitory concentrations (MIC) of different drugs and whole genome sequences of 37 M. hyorhinis isolates were used to find the resistance-related mutations. One MAMA assay was designed to detect the mutation of the 23S rRNA gene at nucleotide position 2058 (Escherichia coli numbering). For further evaluation, the assay was challenged with 17 additional isolates with available MIC data and 15 DNA samples from clinical specimens. The genotypes of the samples were in line with the MIC test results. The developed assay supports the practice of targeted antibiotic usage; hence it may indirectly reduce some bacterial resistance-related public health concerns. [ABSTRACT FROM AUTHOR]

Mycoplasma hyorhinis (Mhr) and M. hyosynoviae (Mhs) are commensal organisms of the upper respiratory tract and tonsils but may also cause arthritis in pigs. In this study, 8-week-old cesarean-derived colostrum-deprived (CDCD) pigs (n = 30; 3 groups, 10 pigs per group, 2 pigs per pen) were inoculated with Mhr , Mhs , or mock-inoculated with culture medium and then pen-based oral fluids were collected at different time points over the 56 days of the experimental study. Oral fluids tested by Mhr and Mhs quantitative real-time PCRs revealed Mhr DNA between day post inoculation (DPI) 5–52 and Mhs DNA between DPI 5–15. Oral fluids were likewise tested for antibody using isotype-specific (IgG, IgA, IgM) indirect ELISAs based on a recombinant chimeric polypeptide of variable lipoproteins (A-G) for Mhr and Tween 20-extracted surface proteins for Mhs. Mhr IgA was detected at DPI 7 and, relative to the control group, significant (p < 0.05) antibody responses were detected in the Mhr group between DPI 12–15 for IgM and DPI 36–56 for both IgA and IgG. In the Mhs group, IgM was detected at DPI 10 and significant (p < 0.05) IgG and IgA responses were detected at DPI 32–56 and DPI 44–56, respectively. This study demonstrated that oral fluid could serve as an effective and convenient antemortem sample for monitoring Mhr and Mhs in swine populations. • Mycoplasma hyorhinis and Mycoplasma hyosynoviae have emerged as important contributors of arthritis and lameness in growing pigs. • The infection dynamics of M. hyorhinis and M. hyosynoviae was evaluated in relation to the antibody response and bacterial shedding in oral fluids as antemortem specimen. • Oral fluid could serve as an effective and convenient antemortem sample for monitoring M. hyorhinis and M. hyosynoviae in swine populations. [ABSTRACT FROM AUTHOR]

We assessed the bacterial agents found in 8–12-wk-old post-weaning pigs with arthritis. The bodies of 178 post-weaning pigs from 90 farms (average of 2 pigs/farm) with recurrent problems of lameness and swollen joints in a high-density breeding area were submitted for autopsy and sampled for further bacterial investigation. The most common articular gross lesions and histopathologic findings were serofibrinous (95 of 178; 53%) or serous (65 of 178; 37%) arthritis; suppurative lesions were less frequent (18 of 178; 10%). In 133 of 178 (74.7%) cases, a bacterial agent was detected in joints. Mycoplasma hyorhinis was the most common bacterium detected (82 of 133; 61.6%). Haemophilus parasuis and Streptococcus spp. were observed in 27 of 133 (20.3%) and 24 of 133 (18.0%) cases, respectively. Other bacteria in the 113 cases, considered less important, in order of their low frequency, were Mycoplasma spp. (13; 9.8%), Trueperella pyogenes (11; 8.2%), Mycoplasma hyosynoviae (4; 3.0%), Staphylococcus spp. (3; 2.2%), Escherichia coli (2; 1.5%), and Actinobacillus spp. (2; 1.5%). Our results highlight the primary role of M. hyorhinis compared to other microorganisms involved in young pigs with arthritis. [ABSTRACT FROM AUTHOR]

Abstract Background Mycoplasma hyorhinis (Mhr) is the etiologic agent of lameness and polyserositis in swine. P37 is a membrane protein of Mhr that may be an important immunogen and is a potential target for diagnostic development. However, there is little information concerning Mhr P37 protein epitopes. A precise analysis of the P37 protein epitopes should extend our understanding of the antigenic composition of the P37 protein and the humoral immune responses to Mhr infection. Investigating the epitopes of Mhr P37 will help to establish a detection method for Mhr in tissue and provide an effective tool for detecting Mhr infection. Results Western blot and indirect immunofluorescence assays (IFA) confirmed that the expressed P37 protein was recognized by Mhr-positive porcine and mouse sera. Furthermore, the P37 protein was purified using affinity chromatography and used to immunize mice for hybridoma cell fusion. Four monoclonal antibodies (mAbs) found to be positive for Mhr were detected in infected lung tissue. A panel of truncated P37 proteins was used to identify the minimal B cell linear epitopes of the protein based on these mAbs. The core epitope was determined to be 206KIKKAWNDKDWNTFRNF222 . Conclusions In this study, we identified 17 critical amino acids that determine the epitope of the P37 protein of Mhr. This study identified mAbs that could provide useful tools for investigating the Mhr P37 antigenic core epitope (amino acids 206–222) and detecting Mhr-specific antigens in infected tissue.

Abstract Background We previously identified that Mycoplasma hyorhinis infection promotes gastric cancer cell motility. The β‐catenin signaling pathway is critical to determining malignant cancer cell phenotypes; however, the association between M hyorhinis and the β‐catenin signaling pathway is unclear. Methods We performed subcellular fractionation and immunofluorescence staining to observe β‐catenin accumulation in the nucleus. The expression of downstream β‐catenin genes was detected by quantitative RT‐PCR. Gastric cancer cell motility was examined by transwell chamber migration and wound healing assays, and a co‐immunoprecipitation assay was used to detect the proteins associated with the membrane protein p37 of M hyorhinis. Results We found that M hyorhinis infection promoted nuclear β‐catenin accumulation and enhanced the expression of downstream β‐catenin genes. M hyorhinis‐promoted gastric cancer cell motility was counteracted by treatment with the β‐catenin inhibitor XAV939 or β‐catenin knockdown. We further detected a protein complex containing LRP6, GSK3β, and p37 in M hyorhinis‐infected cells. M hyorhinis also induced LRP6 phosphorylation in a GSK3β‐dependent fashion. Knockdown of LRP6 or GSK3β abolished M hyorhinis‐induced cell motility. Conclusion Our results reveal that the β‐catenin signaling pathway could be activated by M hyorhinis infection, thereby contributing to M hyorhinis‐induced gastric cancer cell motility.

The objective of this study was to develop a TaqMan probe-based, sensitive, specific duplex real-time PCR assay for simultaneous detection of Mycoplasma hyopneumoniae and Mycoplasma hyorhinis. The specific primers and probes, labeled with FAM and Texas Red, respectively, were designed to amplify the p97 gene of M. hyopneumoniae and p37gene of M. hyorhinis. The duplex real-time PCR reaction mixtures were established and optimized and the sensitivity, specificity and reproducibility of the assay were assessed. The sensitivity of the duplex real-time PCR was found to be 10 copies/μL for both M. hyopneumoniae and M. hyorhinis, respectively. There was no cross reaction with other common viral and bacterial pathogens. The concentration of standard coefficient of variation of Ct values was less than 5%, indicating a good reproducibility. Clinical samples (n = 937) were tested by the duplex real-time PCR assay, including broncho-alveolar lavage fluids, nasal swabs, tissues and cell culture supernatant. Duplex real-time PCR for simultaneous detection of M. hyopneumoniae and M. hyorhinis was highly sensitive and can be utilized for diagnosing clinical samples. It is timeefficient and economic, thereby providing a new approach to control both M. hyopneumoniae and M. hyorhinis.

Purpose: To explore the relationship between Mycoplasma hyorhinis infection and tyrosine kinase inhibitor (TKI) resistance in lung adenocarcinoma patients. Methods: Mycoplasma hyorhinis infection can be verified with the monoclonal antibody PD4, which specifically recognizes a distinct protein of M. hyorhinis. Immunohistochemistry (IHC), using PD4 to detect M. hyorhinis, was performed on paraffin-embedded lung adenocarcinoma tissues of patients who had epidermal growth factor (EGFR) mutations and had received oral TKI. The number of patients enrolled in our study was 101. Assessments following TKI treatment were performed until objective disease progression or stable disease at the cutoff date was reached. In all of the patients, the primary endpoint was investigator-assessed progression-free survival (PFS). Results: Immunohistochemistry revealed that 61 of 101 cases (60.4%) of lung adenocarcinoma were positive for M. hyorhinis, which comprised of 31 low-positive cases and 30 high-positive cases; the remaining 40 cases (39.6%) were negative. The median PFS was significantly longer in the negative group [18 months (95% CI 14.15–21.85)] than in the low-positive group [10 months (95% CI 7.70–12.30); hazard ratio (HR) 4.095, 95% CI 2.254–7.438; p < 0.001] and in the high-positive group [4 months (95% CI 2.85–5.15); HR 31.703, 95% CI 14.425–69.678; p < 0.001]. The results of the subgroup analysis were satisfactory. The PFS benefit with negative M. hyorhinis infection was consistent across subgroups. Conclusions: In this retrospective, exploratory analysis, M. hyorhinis infection significantly reduced PFS. With increased levels of M. hyorhinis infection, the progression of the disease was more advanced, likely due to the hydrolysis of TKI by M. hyorhinis. A strong correlation was found between M. hyorhinis infection and TKI resistance in lung adenocarcinoma. This study provides potent evidence that M. hyorhinis hydrolyses TKI and will assist in the research of related mechanisms in the future. Implications for cancer survivors: It provides an option to improve the efficacy of TKI, including strategies to decrease M. hyorhinis infection, thereby reducing long-term distress in TKI resistance patients with EGFR mutations. [ABSTRACT FROM AUTHOR]

Background: Mycoplasma hyorhinis (M. hyorhinis) is a bacterium commonly found in the upper respiratory tract of healthy pigs and an agent of polyserositis and polyarthritis. Moreover, it can carry antibiotic resistance genes (Wu et al, Vet. Microbiol. 76: 25–30, 2000). Economic losses caused by M. hyorhinis can be reduced by antibiotic therapy, however, isolation and antimicrobic susceptibility profile are rarely performed. Case presentation: The present report describes a case of pericarditis caused by M. hyorhinis in a weaned piglet with respiratory symptoms and reduced growth performance. At post mortem examination, the main macroscopic finding was a severe fibrinous pericarditis and M. hyorhins was the only agent isolated from the pericardial fluid. In this strain, Minimum Inhibitory Concentration (MIC) determination revealed resistance to various antimicrobial molecules such as erythromycin, tylosin and tilmicosin. Conclusion: This paper highlights the importance of including M. hyorhins in the differential diagnosis of polyserositis in swine. Moreover, due the possible presence of multidrug resistance, the determination of antimicrobial susceptibility pattern should be performed on a regular basis. [ABSTRACT FROM AUTHOR]

Five annotated genomes of Mycoplasma hyorhinis were analyzed for clarifying evolutionary dynamics driving the overall codon usage pattern. Information entropy used for estimating nucleotide usage pattern at the gene level indicates that multiple evolutionary dynamics participate in forcing nucleotide usage bias at every codon position. Moreover, nucleotide usage bias directly contributes to synonymous codon usage biases with two different extremes. The overrepresented codons tended to have A/T in the third codon position, and the underrepresented codons strongly used G/C in the third position. Furthermore, correspondence analysis and neutrality plot reflect an obvious interplay between mutation pressure and natural selection mediating codon usage in M. hyorhinis genome. Due to significant bias in usages between A/T and G/C at the gene level, different selective forces have been proposed to contribute to codon usage preference in M. hyorhinis genome, including nucleotide composition constraint derived from mutation pressure, translational selection involved in natural selection, and strand‐specific mutational bias represented by different nucleotide skew index. The systemic analyses of codon usage for M. hyorhinis can enable us to better understand the mechanisms of evolution in this species. [ABSTRACT FROM AUTHOR]