Characterization and application of monoclonal antibodies against Mycoplasma hyorhinis pyruvate dehydrogenase E1 complex subunit alpha.

Mycoplasma hyorhinis is commonly found in the respiratory tract of pigs and is the etiological agent of polyserositis. The metabolic enzymes of M. hyorhinis may play important roles in host-pathogen interactions. We immunized BALB/c mice with sodium deoxycholate-extracted antigens (DOC-Ags) and screened 10 hybridomas that secreted antibodies against various M. hyorhinis proteins. Pyruvate dehydrogenase E1 complex subunit alpha (PDHA) was identified as the protein that reacted with five of the 10 monoclonal antibodies (mAbs). Sequence analysis indicated that PDHA was highly conserved among M. hyorhinis strains, but not among other mycoplasmas. We predicted the three-dimensional structure of PDHA and identified three epitopes ((277)RTEEEEK(283), (299)KDKKYITDE(307), and (350)LKEQKQHAKDY(360)). The mAb 1H12 we generated was used to detect M. hyorhinis PDHA in vitro and in piglets infected with M. hyorhinis. We observed that PDHA was mainly located in the epithelial cells of the lungs. Our results indicate that the mAbs we generated could be used to further investigate the structure and function of M. hyorhinis PDHA. In addition, they could be used in the differential diagnosis of M. hyorhinis and other mycoplasmas.

Associations among Haemophilus parasuis, Mycoplasma hyorhinis, and porcine reproductive and respiratory syndrome virus infections in pigs with polyserositis.

The objective of this study was to determine the associations among Haemophilus parasuis, Mycoplasma hyorhinis, and porcine reproductive and respiratory syndrome (PRRS) virus (EU-field strain) infections in 95 pigs with polyserositis. A significant association between H. parasuis and M. hyorhinis was identified. H. parasuis and M. hyorhinis were significantly more often detected in PRRS virus positive pigs.

Association entre infections avecHaemophilus parasuis, Mycoplasma hyorhinis,et le virus du syndrome dysgénésique et respiratoire du porc chez les porcs atteints de polysérosite. L'objectif de l'étude était d'étudier l'association de Haemophilus parasuis, Mycoplasma hyorhinis, et du SDRP (souche européenne-sauvage) chez 95 porcs atteints de polysérosite. Une association significative a été mise en évidence entre H. parasuis et M. hyorhinis. H. parasuis et M. hyorhinis ont été significativement plus fréquemment détectés chez SDRP positif porcs.(Traduit par les auteurs).

Development of a loop-mediated isothermal amplification assay for rapid detection of Mycoplasma hyorhinis.

BACKGROUND: To establish a method for sensitive and rapid diagnosis of Mycoplasma hyorhinis in clinical specimens, a simple, sensitive loop-mediated isothermal amplification (LAMP) assay was designed and evaluated. METHODS: Three sets of four special primers, recognizing distinct sequences of the target, were designed for sensitive, specific amplification of nucleic acid under isothermal conditions. The LAMP assay was carried out using 35 clinical specimens of bronchoalveolar lavage fluid (BALF) from pigs. For comparison, these specimens were also tested using conventional PCR, real-time PCR, and nested PCR assays. RESULTS: After optimization of the reaction condition and reaction system, the LAMP reaction successfully detected Mycoplasma hyorhinis within 40 minutes at 61 degrees C. The LAMP assay achieved a sensitivity of 10(1) copies per microL at 61 degrees C in 40 minutes, compared to real-time PCR and nested PCR, and was over 10(3) times more sensitive than conventional PCR. In the test for the specificity of the LAMP assay, only Mycoplasma hyorhinis genomic DNA was positive and no other microorganisms were positive with the primers, indicating that the LAMP assay is specific to Mycoplasma hyorhinis. Mycoplasma hyorhinis was detected in 32 samples using the LAMP and real-time PCR assays and in 27 and 11 samples using the nested PCR assay and conventional PCR assay, respectively. All the positive samples detected by real-time PCR, nested PCR and conventional PCR assays were positive in the LAMP assay. CONCLUSIONS: The LAMP assay is inexpensive, easy to perform, shows a rapid reaction and does not require complex instruments like PCR. Therefore, LAMP is a simple, accurate, fast, and economical assay suitable as an alternative in veterinary practices.

Development of a new multiplex quantitative PCR for the detection of Glaesserella parasuis, Mycoplasma hyorhinis, and Mycoplasma hyosynoviae.

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.

Genome analysis of a Mycoplasma hyorhinis strain derived from a primary human melanoma cell line.

The complete genome of Mycoplasma hyorhinis strain MCLD has been sequenced and annotated. This genome differs by the inversion of a 14.4-kb and a 3.7-kb fragment and the deletion of a 9.9-kb fragment from M. hyorhinis strain HUB-1, isolated from swine respiratory tract. The genome revealed 778 coding sequences (CDSs), with a limited number of vlp genes encoding variable surface lipoproteins.

Mycoplasma hyorhinis infection in gastric carcinoma and its effects on the malignant phenotypes of gastric cancer cells.

BACKGROUND: Mycoplasma hyorhinis infection has been postulated to play a role in the development of several types of cancer, but the direct evidence and mechanism remained to be determined. METHODS: Immunohistochemistry assay and nested polymerase-chain reaction (PCR) were performed to examine the mycoplasma hyorhinis infection in gastric cancer tissues. Statistical analysis was used to check the association between mycoplasma infection and clinicopathologic parameters. Transwell chamber assay and metastasis assay were used to evaluate mycoplasma hyorhinis' effects on metastasis in vitro and in vivo. Mycoplasma hyorhinis-induced extracellular signal-regulated kinase (ERK) and epidermal growth factor receptor (EGFR) activation were investigated by Western blot. RESULTS: Mycoplasma hyorhinis infection in gastric cancer tissues was revealed and statistical analysis indicated a significant association between mycoplasma infections and lymph node metastasis, Lauren's Classification, TNM stage, and age of the patients. Mycoplasma hyorhinis promoted tumor cell migration, invasion and metastasis in vitro and in vivo, which was possibly associated with the enhanced phosphorylation of EGFR and ERK1/2. The antibody against p37 protein of Mycoplasma hyorhinis could inhibit the migration of the infected cells. CONCLUSIONS: The infection of mycoplasma hyorhinis may contribute to the development of gastric cancer and Mycoplasma hyorhinis-induced malignant phenotypes were possibly mediated by p37.

Development of in situ hybridization for the detection of Mycoplasma hyorhinis in formalin-fixed paraffin-embedded tissues from naturally infected pigs with polyserositis.

The aim of this study was to develop in situ hybridization for detection of Mycoplasma hyorhinis in formalin-fixed, paraffin-wax-embedded tissues from pigs with polyserositis. M. hyorhinis was isolated from the spleen (2 pigs) and pericardium (1 pig). M. hyorhinis DNA was detected 16 out of 20 pigs with polyserositis. In situ hybridization produced a distinct positive signal for the M. hyorhinis p37 gene in inflammatory cells in the polyserositis. In situ hybridization developed in the present study present diagnostic tools capable of detection of M. hyorhinis in formalin-fixed, paraffin-wax-embedded tissues from the naturally infected pigs.

Mycoplasma hyorhinis and Mycoplasma hyosynoviae dual detection patterns in dams and piglets.

Mycoplasma hyorhinis and M. hyosynoviae are agents associated with arthritis in pigs. This study investigated the tonsillar detection patterns of M. hyorhinis and M. hyosynoviae in a swine population with a history of lameness. The plausibility of dual PCR detection of these agents in dams at one and three weeks post-farrowing and their offspring at the same time was determined. The association between M. hyorhinis and M. hyosynoviae detection in piglets and potential development of lameness in wean-to-finish stages was evaluated by correlating individual piglet lameness scores and PCR detection in tonsils. Approximately 40% of dams were detected positive for M. hyorhinis and M. hyosynoviae at both one and three weeks post-farrowing. In first parity dams, M. hyorhinis was detected in higher proportions (57.1% and 73.7%) at both weeks of sampling compared to multi-parity dams. A lower proportion of first parity dams (37.5%) were detected positive at week one with M. hyosynoviae and an increase in this proportion to 50% was identified in week three. Only 8.3% of piglets were detected positive for M. hyorhinis in week one compared to week three (50%; p<0.05). The detection of M. hyosynoviae was minimal in piglets at both weeks of sampling (0% and 0.9%). Lameness was scored in pigs 5-22 weeks of age, with the highest score observed at week 5. The correlation between PCR detection and lameness scores revealed that the relative risk of developing lameness post-weaning was significantly associated with detection of M. hyorhinis in piglets at three weeks of age (r = 0.44; p<0.05).The detection pattern of M. hyorhinis and M. hyosynoviae in dams did not reflect the detection pattern in piglets. Results of this study suggest that positive detection of M. hyorhinis in piglets pre-weaning could act as a predictor for lameness development at later production stages.

Molecular epidemiology of Mycoplasma hyorhinis porcine field isolates in the United States.

Mycoplasma hyorhinis is one of the causative agents of polyserositis and arthritis in post-weaning pigs. Here we describe the development of a multi-locus sequence typing (MLST) protocol for the characterization of M. hyorhinis field isolates. A total of 104 field isolates from different geographical locations, swine production systems, and clinical backgrounds, were analyzed. Twenty-seven genes, including housekeeping and those encoding surface proteins, were evaluated to index diversity. Genes encoding surface proteins were included to increase the discriminatory power of the MLST. Four target gene fragments were selected to be included in the final MLST-s (surface) protocol: pdhB, p95, mtlD and ung. Within each locus the nucleotide variation ranged from 1.4% to 20%. The 104 field isolates were classified into 39 distinct sequence types (STs). Multiple STs were found within the same production system and within the same pig. The majority of STs grouped strains from the same production system; however, cases existed where multiple systems shared a ST, indicating potential relationships between pig flows. The majority of the nucleotide changes observed in these genes generated synonymous changes, while non-synonymous changes were exclusively in the mtlD gene fragment, suggesting that this protein is undergoing selection. Molecular typing of M. hyorhinis will primarily aid swine practitioners with pig flow management and identifying sources of infection during outbreaks.

Swine conjunctivitis outbreaks associated with Mycoplasma hyorhinis.

Conjunctivitis is an uncommon finding in commercial swine herds, and the etiology of the disease is rarely studied. We investigated cases of conjunctivitis in 3 wean-to-finish swine farms. Eye swabs and tissues were obtained from clinically affected pigs (8-22 wk of age), from unaffected pigs in contact with affected pen-mates, and from age-matched pigs from an unaffected herd. Real-time PCR (rtPCR) testing for Mycoplasma hyorhinis demonstrated consistent detection and high bacterial load in samples from affected herds (clinically affected animals and non-clinical pen-mates). Ct values in affected pigs were 18.9-25.3; values were 36.4-38.6 in unaffected pigs from unaffected herds. Additionally, M. hyorhinis was identified within inflamed palpebral conjunctivae by in situ hybridization. The association of rtPCR and in situ detection of M. hyorhinis, along with the lack of detection of other potential pathogens and noninfectious causes, suggests the involvement of M. hyorhinis in the etiology and pathogenesis of the reported swine conjunctivitis.

Effect of storage conditions on detection of mycoplasma in biopharmaceutical products.

Mycoplasma contamination affects many different aspects of cell culturing, resulting in unreliable experimental results and potentially harmful biological products. Therefore, the specificity, sensitivity, and reliability of detecting mycoplasma contamination are important aspects of quality control in biotechnological products. In this study, Mycoplasma hyorhinis was adopted as a model strain to evaluate the effects of storage on the viability of Mycoplasma species in cell culture samples. Medium X was compared with conventional media 243 and 988 for the ability to detect M. hyorhinis. The 10(1) CFU/ml of M. hyorhinis was inoculated into medium X prepared using the same lots of components and preserved for 7 d, 1 mo, and 2 mo. M. hyorhinis grew readily and typically on agar plates prepared within 1 mo. The viable mycoplasmas in samples containing different initial titers (10(1) and 10(6) CFU/ml) after storage at 4 degrees C and -30 degrees C were analyzed. During storage, viable organisms were found with little or no reduction in titers after storage for 8 wk at -30 degrees C under aerobic and anaerobic conditions. A reduction in titers of 3 log10 occurred after 4 wk storage for high-dose cultures (10(6) CFU/ml) at 4 degrees C. The titers of viable organisms were diminished over 8 wk at 4 degrees C under aerobic and anaerobic conditions.

Infection dynamics of Mycoplasma hyorhinis in three commercial pig populations.

Mycoplasma hyorhinis is one of the causative agents of polyserositis and arthritis in postweaning pigs. Knowledge regarding colonisation frequency and age distribution in modern pig production is lacking. The objective of this study was to estimate the prevalence of M hyorhinis colonisation in different age groups across three commercial pig populations. Nasal swabs were collected from sows, piglets and nursery pigs of different ages. Oral fluids were collected from nursery pigs. Necropsies were performed to assess the presence of M hyorhinis-associated disease. M hyorhinis was detected in 5/60 sows in herd A, 3/60 in herd B and none in herd C. In herd A and B, the prevalence was low in preweaning piglets (∼8 per cent) and high in postweaning pigs (∼98 per cent). A total of 7/8 oral fluids tested PCR positive in herds A and B, while 1/8 tested positive in herd C. In herd C, the preweaning and postweaning prevalence was low. In herds A and B, necropsied pigs had polyserositis lesions where M hyorhinis was detected by PCR. This study showed that prevalence of M hyorhinis colonisation varies with pig age and across farms. Information generated will aid in the design and implementation of control and prevention strategies.

Bacteria arise at the border of mycoplasma-infected HeLa cells, containing cytoplasm with either malformed cytosol, mitochondria and endoplasmic reticulum or tightly adjoined smooth vacuoles.

A study with transmission electron microscopy of mycoplasma-contaminated HeLa cells using five cell donors referred to as donors A, B, C, D and E, observations are herein presented. Experiments performed with cells from donors B, C and D, revealed the presence of Mycoplasma hyorhinis after PCR and sequencing experiments. Bacteria probably originated from a cytoplasm with compacted tiny granular particles replacing the normal cytosol territories, or from the contact with the cytoplasm through a clear semi-solid material. The compact granularity (CG) of the cytoplasm was crossed by stripes of smooth and rough endoplasmic reticulum cisternae. Among apparently normal mitochondria, it was noted, in variable proportions, mitochondria with crista-delimited lucent central regions that expand to and occupied the interior of a crista-less organelle, which can undergo fission. Other components of the scenarios of mycoplasma-induced cell demolition are villus-like structures with associated 80-200 nm vesicles and a clear, flexible semi-solid, process-sensitive substance that we named jam-like material. This material coated the cytoplasmic surface, its recesses, irregular protrusions and detached cytoplasmic fragments. It also cushioned forming bacteria. Cyst-like structures were often present in the cytoplasm. Cells, mainly apoptotic, exhibiting ample cytoplasmic sectors with characteristic net-like profile due to adjoined vacuoles, as well as ovoid or elongated profiles, consistently appeared in all cells from the last four cell donors. These cells were named "modified host cells" because bacteria arose in the vacuoles. The possibility that, in some samples, there was infection and/or coinfection of the host cell by another organism(s) cannot be ruled out.

Potential role of intratumor bacteria in mediating tumor resistance to the chemotherapeutic drug gemcitabine.

Growing evidence suggests that microbes can influence the efficacy of cancer therapies. By studying colon cancer models, we found that bacteria can metabolize the chemotherapeutic drug gemcitabine (2',2'-difluorodeoxycytidine) into its inactive form, 2',2'-difluorodeoxyuridine. Metabolism was dependent on the expression of a long isoform of the bacterial enzyme cytidine deaminase (CDDL), seen primarily in Gammaproteobacteria. In a colon cancer mouse model, gemcitabine resistance was induced by intratumor Gammaproteobacteria, dependent on bacterial CDDL expression, and abrogated by cotreatment with the antibiotic ciprofloxacin. Gemcitabine is commonly used to treat pancreatic ductal adenocarcinoma (PDAC), and we hypothesized that intratumor bacteria might contribute to drug resistance of these tumors. Consistent with this possibility, we found that of the 113 human PDACs that were tested, 86 (76%) were positive for bacteria, mainly Gammaproteobacteria.

Mycoplasma-associated multidrug resistance of hepatocarcinoma cells requires the interaction of P37 and Annexin A2.

Mycoplasma infection has been reported to be associated with cancer migration, invasion, epithelial-mesenchymal transition as well as the resistance to nucleoside analogues chemotherapeutic drugs. In this study, we found that the sensitivity of hepatocarcinoma cells to Cisplatin, Gemcitabine and Mitoxantrone was increased by mycoplasma elimination. Similar to the effect of anti-mycoplasma agent, interrupting the interaction between Mycoplasma hyorhinis membrane protein P37 and Annexin A2 of host cells using the N-terminal of ANXA2 polypeptide enhanced the sensitivity of HCC97L cells to Gemcitabine and Mitoxantrone. Meanwhile, we did not observe any changes in expression or distribution of multidrug resistance associated transporters, ATP-Binding Cassette protein B1, C1 and G2, on the removal of mycoplasma. These results suggest that mycoplasma induces a resistance to multiple drugs in hepatocarcinoma cells which required the interaction of P37 and Annexin A2. The pathway downstream this interaction needs to be explored.

[Detection of Haemophilus parasuis and Mycoplasma hyorhinis in swine and association of those pathogens with clinical and pathological-anatomic findings]. / Nachweis von Haemophilus parasuis und Mycoplasma hyorhinis bei Schweinen sowie Assoziation dieser Erreger mit klinischen und pathologisch-anatomischen Befunden.

In routine diagnostic it is difficult to detect a Haemophilus parasuis infection causing a polyserositis. In the presented study, a detection method by means of PCR, from collective swabs of the serosal surfaces from animals, with and without fibrinous serositis, was investigated. A significant association was detected between post mortal findings of fibrinous serositis and molecular biological detection of Haemophilus parasuis and Mycoplasma hyorhinis. However, no significant association existed between cultural detection of Haemophilus parasuis in bronchoalveolar lavage fluid and pathological-anatomical ascertainable fibrinous serositis. A significant association between Haemophilus parasuis and Mycoplasma hyorhinis could be determined in the animals that were included in this study.

[Influenza outbreak in weaners with involvement of Mycoplasma hyorhinis and Haemophilus parasuis. A case report]. / Influenza-Ausbruch bei Aufzuchtferkeln unter Beteiligung von Mycoplasma hyorhinis und Haemophilus parasuis. Ein Fallbericht.

In a closed farrow-to-finish piglet producing farm 80% of 7-week-old piglets displayed respiratory disease with a 5% mortality rate. In addition to purulent bronchopneumonia in combination with interstitial pneumonia predominantly in the apical and middle lobes, fibrinous serositis was present in the thoracic and abdominal cavities. Further investigations succeeded in confirming the non-pandemic strain of porcine influenza A virus (FLUAVsw) subtype H1avN1. The molecular genetic studies on Mycoplasma (M.) hyopneumoniae and porcine reproductive and respiratory syndrome virus were negative, whereas M. hyorhinis and Haemophilus parasuis were isolated from serous membranes. The possible importance of the underrated M. hyorhinis as a cofactor for viral infections should be emphasized and we demonstrated that the cause of apical lobe pneumonia is not restricted to M. hyopneumoniae. Mother pigs had been vaccinated with an influenza vaccine covering the subtype H1avN1. Only 33% of the examined piglets had maternal antibodies in the 7th week of life. The difficulty of prophylaxis of infections by FLUAVsw in weaners due to lack of vaccine authorization for piglets before their 56th day is reflected by this observation.

Mycoplasma Infection Alters Cancer Stem Cell Properties in Vitro.

Cancer cell lines can be useful to model cancer stem cells. Infection with Mycoplasma species is an insidious problem in mammalian cell culture. While investigating stem-like properties in early passage melanoma cell lines, we noted poorly reproducible results from an aliquot of a cell line that was later found to be infected with Mycoplasma hyorhinis. Deliberate infection of other early passage melanoma cell lines aliquots induced variable and unpredictable effects on expression of putative cancer stem cell markers, clonogenicity, proliferation and global gene expression. Cell lines established in stem cell media (SCM) were equally susceptible. Mycoplasma status is rarely reported in publications using cultured cells to study the cancer stem cell hypothesis. Our work highlights the importance of surveillance for Mycoplasma infection while using any cultured cells to interrogate tumor heterogeneity.

[Detection and classification of Mycoplasma in cell lines].

OBJECTIVE: To detect the rate of Mycoplasma infection in cell lines and further determine its types. METHODS: We performed nest PCR amplification of Mycoplasma's conserved regions (16S-23S) and sequenced the spacer with different length between conserved regions. RESULTS: Within the tested 22 cell lines, 17 (77.3%) showed Mycoplasma infections, of which 5 had two or more types of Mycoplasma. M. fermentans and hyorhinis were more frequently detected within the types of infected Mycoplasma. CONCLUSION: The high rate of Mycoplasma infection in cell lines makes it necessary for researchers to pay more attention to its influence on research data when using cell lines as models. Establishment of detection and classifying techniques make it possible to further study the pathogenesis of different types of Mycoplasma.

Quantitative real-time polymerase chain reaction for detecting Mycoplasma hyosynoviae and Mycoplasma hyorhinis in pen-based oral, tonsillar, and nasal fluids.

Mycoplasma (M.) hyorhinis and M. hyosynoviae are pathogens known to cause disease in pigs post-weaning. Due to their fastidious nature, there is increased need for culture-independent diagnostic platforms to detect these microorganisms. Therefore, this study was performed to develop and optimize quantitative real-time PCR (qPCR) assays to rapidly detect M. hyorhinis and M. hyosynoviae in pen-based oral fluids as well as nasal and tonsillar fluids as proxies for samples used in swine herd surveillance. Two methods of genomic DNA extraction, automated versus manual, were used to compare diagnostic test performance. A wean-to-finish longitudinal study was also carried out to demonstrate the reproducibility of using pen-based oral fluids. Overall, pen-based oral and tonsillar fluids were more likely to be positive for both types of bacteria whereas only M. hyorhinis was detected in nasal fluids. DNA extraction protocols were shown to significantly influence test result. Although the initial detection time somewhat differed, both organisms were repeatedly detected in the longitudinal study. Overall, this study evaluated two qPCR methods for rapid and specific detection of either mycoplasma. Results from the present investigation can serve as a foundation for future studies to determine the prevalence of the two microorganisms, environmental load, and effectiveness of veterinary interventions for infection control.