Differential antigenicity of individual Mycoplasma hyorhinis variable lipoproteins.

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.

Evaluation of colonization, variable lipoprotein-based serological response, and cellular immune response of Mycoplasma hyorhinis in experimentally infected swine.

Mycoplasma hyorhinis (Mhr) is a commensal of the upper respiratory tract that can be shed by nasal secretions and transmitted by direct contact in neonatal and nursery pigs. Lesions associated with Mhr infection include polyserositis and arthritis; however, systemic Mhr disease pathogenesis is not well characterized. This study aimed to investigate the immunopathogenesis and bacterial dissemination pattern of Mhr using single and multiple inoculation approaches in a caesarian-derived colostrum-deprived (CDCD) pig model. Animals in three treatment groups were inoculated once (Mhr 1; n = 12) or four (Mhr 2; n = 8) times with Mhr or sham-inoculated (NC group; n = 3) nasally and by tonsillar painting. Inoculum consisted of a triple cloned Mhr field isolate (4.5 × 107 CFU/mL) in Friis medium. Clinical signs were evaluated daily during the study. Serum and oral fluid antibody (IgA and IgG) response and cellular immune response were assessed using a recombinant chimeric VlpA-G-based indirect ELISA and by ELISpot, respectively. The presence of Mhr in oral fluids, nasal and oropharyngeal swabs were evaluated by qPCR. At 6 wpi, pigs were euthanized and evaluated for gross lesions consistent with Mhr and bacterial colonization in tonsils by qPCR. No clinical signs or gross lesions consistent with Mhr-associated disease were observed throughout the study. For Mhr 2 group, the presence of IgA and IgG in serum and oral fluids were detected at 2 and 4 weeks post-inoculation (wpi), respectively, while in Mhr 1, only IgA was detected in oral fluids at 6 wpi. The proportion of animals shedding Mhr in nasal secretions varied from 20 to 40 % in the Mhr 1 and 62.5-100% in the Mhr 2 group. However, the proportion of animals shedding Mhr in oropharyngeal swabs was consistent through the study (60 %) in Mhr 1 and fluctuated from 20 % to 87.5 % in Mhr 2 group. The lack of clinical signs and the presence of Mhr specific humoral response and bacterial colonization indicates that the multiple inoculation experimental model may mimic subclinical natural infection in the field. In addition, the humoral and transient cellular response did not result in bacterial clearance. Based on these results, animals would have to be exposed multiple times to mount a detectable immune response.

Efficacy in pigs of a new inactivated vaccine combining porcine circovirus type 2 and Mycoplasma hyorhinis.

Coinfection with porcine circovirus type 2 (PCV2) and Mycoplasma hyorhinis (Mhr) can induce more-severe disease than a single infection with either. We evaluated the efficacy of a new vaccine combining inactivated PCV2 and Mhr, in a model of PCV2 and Mhr infection. Twenty-five 35-day-old PCV2- and Mhr-free pigs were randomly divided into five groups, with five pigs in each group. The pigs in groups 1 and 2 were vaccinated with the combined vaccine and then challenged with Mhr or PCV2, respectively. The pigs in groups 3 and 4 were not vaccinated and then challenged with PCV2 or Mhr, respectively, and group 5 was used as the unvaccinated unchallenged control. Two weeks after booster immunization via the intramuscular route, all the pigs except those in control group 5 were challenged with PCV2 or Mhr. All the pigs were euthanized 28 days after challenge. The pigs in vaccinated groups 1 and 2 showed a significant increase in weight after challenge with PCV2 or Mhr (P < 0.001), with an average daily gain (ADG) of 0.315 kg compared with unvaccinated groups 3 and 4 (0.279 kg). Mhr was isolated from the unvaccinated pig lungs after Mhr challenge, whereas it was not isolated from the vaccinated pigs. No PCV2 or Mhr was detected with PCR or histochemical staining in vaccinated groups 1 and 2. A statistical analysis showed that the PCV2 and Mhr combined vaccine providing protected against PCV2 infection causing viremia and inguinal lymphadenopathy (5 pigs protected out 5) or against Mhr infection causing fiber inflammation (4 pigs out 5). Thus, we have developed an effective combined vaccine for the prevention and control of PCV2 or Mhr infections in swine herds, this will help reduce prevalence of PCV2 and Mhr coinfections.

Differential innate immune responses induced by Mycoplasma hyopneumoniae and Mycoplasma hyorhinis in various types of antigen presenting cells.

Mycoplasma (M.) hyopneumoniae is the etiological agent of enzootic pneumonia in pigs and is closely related to M. hyorhinis, which can be isolated from the healthy mucosal surfaces of the upper respiratory tract. In rare cases it can also cause arthritis and polyserositis. Since the innate immune system is an important first line of defense and promotes adaptive immune responses, we characterized the innate immune response of various antigen presenting cells (APCs) to M. hyopneumoniae and M. hyorhinis, which differ in their pathogenicity in vivo. Porcine peripheral blood mononuclear cells were infected with different multiplicities of infection (MOI) of live and inactivated porcine mycoplasmas. Both Mycoplasma species induced strong tumour necrosis factor (TNF) responses in monocytes, with a stronger activation by M. hyorhinis. This higher stimulatory activity was also confirmed for CD40 upregulation. Conventional and plasmacytoid dendritic cells (cDC and pDC, respectively) did not or poorly respond to mycoplasmas in terms of TNF expression but more efficiently in terms of CD40 upregulation. Again, these responses were generally stronger with M. hyorhinis than with M. hyopneumoniae. Both Mycoplasma species also activated B cells in terms of CD25 upregulation, proliferation, and IgM secretion. Interestingly, while the induction of CD25 and in particular proliferation was higher with M. hyorhinis, the IgM secretion did not differ between the two species with the exception of the highest dose of M. hyopneumoniae,which appeared to suppress IgM responses. Taken together, our results provide a comparative analysis of innate immune response with different porcine APCs and demonstrate Mycoplasma species-dependent differences, which could relate to their different pathogenicity in vivo.

Early detection and differential serodiagnosis of Mycoplasma hyorhinis and Mycoplasma hyosynoviae infections under experimental conditions.

Mycoplasma hyorhinis (MHR) and Mycoplasma hyosynoviae (MHS) are common opportunistic pathogens in the upper respiratory tract and tonsils of swine. The identification of the specific species involved in clinical cases using conventional diagnostic methods is challenging. Therefore, a recombinant chimeric polypeptide based on the seven known variable lipoproteins (A-G) specific of MHR and a cocktail of surface proteins detergent-extracted from MHS cultures were generated and their suitability as antemortem biomarkers for serodiagnosis of MHR- and MHS-infection were evaluated by ELISA. M. hyorhinis and MHS ELISA performance, evaluated using serum samples collected over a 56-day observation period from pigs inoculated with MHR, MHS, M. hyopneumoniae, M. flocculare, or Friis medium, varied by assay, targeted antibody isotype, and cutoffs. The progressions of MHR and MHS clinical diseases were evaluated in relation to the kinetics of the isotype-specific antibody response in serum and bacterial shedding in oral fluids during the observation period. In pigs inoculated with MHR, bacterial DNA was detected in one or more of the 5 pens at all sampling points throughout the study, IgA was first detected at DPI 7, one week before the first clinical signs, with both IgA and IgG detected in all samples collected after DPI 14. The peak of MHS shedding (DPI 8) coincided with the onset of the clinical signs, with both IgA and IgG detected in all serum samples collected ≥ DPI 14. This study demonstrated, under experimental conditions, that both ELISAs were suitable for early detection of specific antibodies against MHR or MHS. The diagnostic performance of the MHR and MHS ELISAs varied depending on the selected cutoff and the antibody isotype evaluated. The high diagnostic and analytical specificity of the ELISAs was particularly remarkable. This study also provides insights into the infection dynamics of MHR-associated disease and MHS-associated arthritis not previously described.

Duration of immunity for an inactivated Mycoplasma hyorhinis vaccine in pigs.

Mycoplasma hyorhinis (Mhr) is a pathogen of pigs causing polyserositis and polyarthritis. The most susceptible population are nursery pigs of approximately 7 weeks of age, although we have shown that clinical signs can persist into finishing aged animals after a late-nursery infection. We have previously demonstrated the efficacy of a novel inactivated Mhr vaccine for the reduction of lameness and polyserositis in caesarian-derived colostrum-deprived (CDCD) pigs vaccinated at 3 weeks and challenged with Mhr at 6 weeks of age. Here we evaluated the duration of immunity (DOI) of the same vaccine. Vaccine or placebo was administered to CDCD pigs at 3 weeks of age. Pigs were challenged with Mhr at either 10 weeks of age (=7 week DOI) or 13 weeks of age (=10 week DOI). In the 7 week DOI, vaccination provided significant reductions in lameness (p = 0.0018), arthritis (p = 0.0002), and pericarditis (p = 0.0312) versus the placebo control. In the 10 week DOI, a significant reduction in arthritis (p = 0.0320) was observed in the vaccine group as compared to the placebo group. Both vaccine groups showed a significant increase (p < 0.0001) in the post-challenge average daily gain (ADG), gaining 0.2 kg/day more than their respective placebo groups.

A p37-based ELISA used to monitor anti- Mycoplasma hyorhinis IgG in serum from pigs immunized with inactivated M. hyorhinis vaccines.

Mycoplasma hyorhinis is an important pathogen of swine that can often occur as a respiratory coinfection with viral pathogens, but can also cause arthritis and polyserositis in infected animals. To date, no assay is available to assess the serologic response to M. hyorhinis vaccines, to our knowledge. We used recombinantly expressed M. hyorhinis p37 protein to monitor the magnitude of the IgG response in vaccinated animals. The assay was able to distinguish animals vaccinated with M. hyorhinis from those vaccinated with the other important Mycoplasma species: M. hyopneumoniae and M. hyosynoviae. When formulated with an ideal adjuvant, inactivated vaccines designed to protect animals against M. hyorhinis induced a measurable and dose-dependent antibody response against the p37 protein. Additionally, the protein appears to be highly conserved between strains of M. hyorhinis isolated in the United States. The specificity of the assay as well as the conservation and immunogenicity of the p37 protein make it an ideal candidate antigen for use in measuring the immune response against M. hyorhinis after vaccination in weaned pigs.

Reduction of mycoplasmal lesions and clinical signs by vaccination against Mycoplasma hyorhinis.

Porcine mycoplasmal pneumonia is a significant disease problem in the swine industry. The causative agents include Mycoplasma hyopneumoniae and Mycoplasma hyorhinis. M. hyopneumoniae is the major pathogen contributing to the porcine respiratory disease complex, but is difficult to isolate from the respiratory tract and tonsils, whereas M. hyorhinis is not. Although M. hyorhinis is commonly detected in the lungs, the role of M. hyorhinis as a cause of pneumonia remains unclear. Current vaccines for porcine mycoplasmal pneumonia only include M. hyopneumoniae, not M. hyorhinis. M. hyopneumoniae vaccines are widely used, but disease still occurs because of poor vaccine efficacy and possibly the presence of M. hyorhinis. In this study, an inactivated vaccine containing a mixture of M. hyorhinis and M. hyopneumoniae was generated and evaluated for safety, immunogenicity, and protective efficacy against challenge with M. hyorhinis in pigs. The inactivated vaccine induced an antibody response and reduced pneumonic lesions in the lungs and tracheas compared with the non-vaccinated group.

Efficacy of an inactivated Mycoplasma hyorhinis vaccine in pigs.

Lameness and polyserositis in pigs caused by Mycoplasma hyorhinis are generally treated with antibiotics and may require multiple doses. The costs of these antibiotics combined with economic losses from culling and reduced feed conversion due to lameness are hardships to the swine producer. In this study we have demonstrated efficacy of an inactivated M. hyorhinis vaccine administered to three-week old caesarian-derived colostrum-deprived piglets. Three doses of vaccine (high, medium, and low) were evaluated and compared to a placebo control. Mycoplasma hyorhinis challenge occurred three weeks after vaccination. Pigs were observed for lameness and respiratory distress for three weeks following challenge. Pigs were then euthanized and a gross pathological evaluation for polyserositis and arthritis was performed. A minimum immunizing dose of vaccine was defined as containing at least 7.41 × 107 CCU of M. hyorhinis per 2.0 mL dose as represented by the medium dose vaccine. This vaccine provided significant reductions in lameness and pericarditis with preventive fractions of 0.76 (95% CI [0.26, 0.92]) and 0.58 (95% CI [0.31, 0.74]), respectively, compared to the placebo control group. A significant increase in post-challenge weight gain (P < .0001) was also achieved with this vaccine, with an average daily gain (ADG) of 0.92 lbs/day compared to 0.57 lbs/day in the placebo group.

Mycoplasma hyorhinis is a potential pathogen of porcine respiratory disease complex that aggravates pneumonia caused by porcine reproductive and respiratory syndrome virus.

The porcine respiratory disease complex (PRDC) caused by numerous bacterial and viral agents has a great impact on pig industry worldwide. Although Mycoplasma hyorhinis (Mhr) has been frequently isolated from lung lesions from pigs with PRDC, the pathological importance of Mhr may have been underestimated. In this study, 383 serum samples obtained from seven herds with a history of PRDC were tested for specific antibodies to Mhr, Mycoplasma hyopneumoniae (Mhp), and porcine reproductive and respiratory syndrome virus (PRRSV). Seropositive rates of PRRSV were significantly correlated with those of Mhr (correlation coefficient, 0.862; P-value, 0.013), but not with those of Mhp (correlation coefficient, -0.555; P-value, 0.196). In vivo experiments demonstrated that pigs co-infected with Mhr and PRRSV induced more severe lung lesions than pigs infected with Mhr or PRRSV alone. These findings suggest that Mhr is closely associated with pneumonia caused by PRRSV and provide important information on Mhr pathogenesis within PRDC. Therefore, effective PRDC control strategies should also consider the potential impact of Mhr in the pathogenesis of PRDC.

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.

Mycoplasma hyorhinis-Contaminated Cell Lines Activate Primary Innate Immune Cells via a Protease-Sensitive Factor.

Mycoplasma are a frequent and occult contaminant of cell cultures, whereby these prokaryotic organisms can modify many aspects of cell physiology, rendering experiments that are conducted with such contaminated cells problematic. Chronic Mycoplasma contamination in human monocytic cells lines has been associated with suppressed Toll-like receptor (TLR) function. In contrast, we show here that components derived from a Mycoplasma hyorhinis-infected cell line can activate innate immunity in non-infected primary immune cells. Release of pro-inflammatory cytokines such as IL-6 by dendritic cells in response to Mycoplasma hyorhinis-infected cell components was critically dependent on the adapter protein MyD88 but only partially on TLR2. Unlike canonical TLR2 signaling that is triggered in response to the detection of Mycoplasma infection, innate immune activation by components of Mycoplasma-infected cells was inhibited by chloroquine treatment and sensitive to protease treatment. We further show that in plasmacytoid dendritic cells, soluble factors from Mycoplasma hyorhinis-infected cells induce the production of large amounts of IFN-α. We conclude that Mycoplasma hyorhinis-infected cell lines release protein factors that can potently activate co-cultured innate immune cells via a previously unrecognized mechanism, thus limiting the validity of such co-culture experiments.

[Serological course investigations of Haemophilus parasuis and Mycoplasma hyorhinis in three pig farms]. / Serologische Verlaufsuntersuchungen auf Haemophilus parasuis und Mycoplasma hyorhinis in drei schweine-haltenden Betrieben.

The aim of the study was to investigate the infection dynamic of Haemophilus (H.)parasuis and Mycoplasma (M.) hyorhinis in 3 farms. A total of 61 piglets were clinically investigated at 1., 3., 5., 7., 9., 11., 14., 18. and 22. weeks of life and a blood sample was taken from each piglet as well as from the sows. The serum samples were tested using ELISA for antibodies against H. parasuis and M. hyorhinis. Clinical signs indicating polyserositis were seen in farm 1 and 3. For both pathogens, a decline of the maternal antibodies could be detected up to the 5th or 7th week of life. The duration of persistence depended on the level of the maternal antibodies. In farm 1, all animals were tested positive for antibodies against H. parasuis during the fattening period. In farm 3, several sows were tested positive in the M. hyorhinis ELISA, therefore, positive results in sows can indicate a higher infection dynamic during the fattening period. For H. parasuis as well as for M. hyorhinis a significant correlation between the level of the antibodies in the sows and their piglets could be seen.

Mycoplasma hyorhinis induces proinflammatory responses in mice lymphocytes.

Mycoplasmas are frequent contaminants of cultured cells, leading to alterations in cellular gene expression, protein synthesis, signal transduction, and metabolic pathways. Mycoplasma hyorhinis, the major contaminant of tissue cultures, has been implicated in a variety of diseases in swine. Most human and animal mycoplasmas remain attached to the surface of epithelial cells. Nonetheless, we have recently shown that M. hyorhinis is able to invade nonphagocytic melanoma cells. In the present study, we show by confocal laser scanning microscopy, that by exposing mice splenocytes to intact M. hyorhinis, intracellular mycoplasmas were detected. Mycoplasmal components were not detected within splenocytes after exposure to heat inactivated M. hyorhinis or to a purified M. hyorhinis lipoprotein (LPP) fraction. However, incubation of the splenocytes with intact M. hyorhinis cells, heat inactivated cells or M. hyorhinis LPP fraction induced accelerated cell proliferation and the secretion of interferon gamma and interleukin 17. Thus, M. hyorhinis and its LPPs can be added to the list of infectious agents causing direct stimulation of proinflammatory responses by mammalian lymphocytes.

Mycoplasma hyorhinis activates the NLRP3 inflammasome and promotes migration and invasion of gastric cancer cells.

BACKGROUND: Mycoplasma hyorhinis (M.hyorhinis, M.hy) is associated with development of gastric and prostate cancers. The NLRP3 inflammasome, a protein complex controlling maturation of important pro-inflammatory cytokines interleukin (IL)-1ß and IL-18, is also involved in tumorigenesis and metastasis of various cancers. METHODOLOGY/PRINCIPAL FINDINGS: To clarify whether M.hy promoted tumor development via inflammasome activation, we analyzed monocytes for IL-1ß and IL-18 production upon M.hy challenge. When exposed to M.hy, human monocytes exhibited rapid and robust IL-1ß and IL-18 secretion. We further identified that lipid-associated membrane protein (LAMP) from M.hy was responsible for IL-1ß induction. Applying competitive inhibitors, gene specific shRNA and gene targeted mice, we verified that M.hy induced IL-1ß secretion was NLRP3-dependent in vitro and in vivo. Cathepsin B activity, K(+) efflux, Ca(2+) influx and ROS production were all required for the NLRP3 inflammasome activation by M.hy. Importantly, it is IL-1ß but not IL-18 produced from macrophages challenged with M.hy promoted gastric cancer cell migration and invasion. CONCLUSIONS: Our data suggest that activation of the NLRP3 inflammasome by M.hy may be associated with its promotion of gastric cancer metastasis, and anti-M.hy therapy or limiting NLRP3 signaling could be effective approach for control of gastric cancer progress.

Detection of antibodies directed at M. hyorhinis p37 in the serum of men with newly diagnosed prostate cancer.

BACKGROUND: Recent epidemiologic, genetic, and molecular studies suggest infection and inflammation initiate certain cancers, including cancers of the prostate. Over the past several years, our group has been studying how mycoplasmas could possibly initiate and propagate cancers of the prostate. Specifically, Mycoplasma hyorhinis encoded protein p37 was found to promote invasion of prostate cancer cells and cause changes in growth, morphology and gene expression of these cells to a more aggressive phenotype. Moreover, we found that chronic exposure of benign human prostate cells to M. hyorhinis resulted in significant phenotypic and karyotypic changes that ultimately resulted in the malignant transformation of the benign cells. In this study, we set out to investigate another potential link between mycoplasma and human prostate cancer. METHODS: We report the incidence of men with prostate cancer and benign prostatic hyperplasia (BPH) being seropositive for M. hyorhinis. Antibodies to M. hyorhinis were surveyed by a novel indirect enzyme-linked immunosorbent assay (ELISA) in serum samples collected from men presenting to an outpatient Urology clinic for BPH (N = 105) or prostate cancer (N = 114) from 2006-2009. RESULTS: A seropositive rate of 36% in men with BPH and 52% in men with prostate cancer was reported, thus leading us to speculate a possible connection between M. hyorhinis exposure with prostate cancer. CONCLUSIONS: These results further support a potential exacerbating role for mycoplasma in the development of prostate cancer.

Mycoplasma contamination revisited: mesenchymal stromal cells harboring Mycoplasma hyorhinis potently inhibit lymphocyte proliferation in vitro.

BACKGROUND: Mesenchymal stromal cells (MSC) have important immunomodulatory effects that can be exploited in the clinical setting, e.g. in patients suffering from graft-versus-host disease after allogeneic stem cell transplantation. In an experimental animal model, cultures of rat T lymphocytes were stimulated in vitro either with the mitogen Concanavalin A or with irradiated allogeneic cells in mixed lymphocyte reactions, the latter to simulate allo-immunogenic activation of transplanted T cells in vivo. This study investigated the inhibitory effects of rat bone marrow-derived MSC subsequently found to be infected with a common mycoplasma species (Mycoplasma hyorhinis) on T cell activation in vitro and experimental graft-versus-host disease in vivo. PRINCIPAL FINDINGS: We found that M. hyorhinis infection increased the anti-proliferative effect of MSC dramatically, as measured by both radiometric and fluorimetric methods. Inhibition could not be explained solely by the well-known ability of mycoplasmas to degrade tritiated thymidine, but likely was the result of rapid dissemination of M. hyorhinis in the lymphocyte culture. CONCLUSIONS: This study demonstrates the potent inhibitory effect exerted by M. hyorhinis in standard lymphocyte proliferation assays in vitro. MSC are efficient vectors of mycoplasma infection, emphasizing the importance of monitoring cell cultures for contamination.

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.

The percentage of CD133+ cells in human colorectal cancer cell lines is influenced by Mycoplasma hyorhinis infection.

BACKGROUND: Mollicutes contamination is recognized to be a critical issue for the cultivation of continuous cell lines. In this work we characterized the effect of Mycoplasma hyorhinis contamination on CD133 expression in human colon cancer cell lines. METHODS: MycoAlert and mycoplasma agar culture were used to detect mycoplasma contamination on GEO, SW480 and HT-29 cell lines. Restriction fragment length polymorphism assay was used to determine mycoplasma species. All cellular models were decontaminated by the use of a specific antibiotic panel (Enrofloxacin, Ciprofloxacin, BM Cyclin 1 and 2, Mycoplasma Removal Agent and MycoZap). The percentage of CD133 positive cells was analyzed by flow cytometry on GEO, SW480 and HT-29 cell lines, before and after Mycoplasma hyorhinis eradication. RESULTS: Mycoplasma hyorhinis infected colon cancer cell lines showed an increased percentage of CD133+ cells as compared to the same cell lines rendered mycoplasma-free by effective exposure to antibiotic treatment. The percentage of CD133 positive cells increased again when mycoplasma negative cells were re-infected by Mycoplasma hyorhinis. CONCLUSIONS: Mycoplasma hyorhinis infection has an important role on the quality of cultured human colon cancer cell lines giving a false positive increase of cancer stem cells fraction characterized by CD133 expression. Possible explanations are (i) the direct involvement of Mycoplasma on CD133 expression or (ii) the selective pressure on a subpopulation of cells characterized by constitutive CD133 expression.In keeping with United Kingdom Coordinating Committee on Cancer Research (UKCCCR) guidelines, the present data indicate the mandatory prerequisite, for investigators involved in human colon cancer research area, of employing mycoplasma-free cell lines in order to avoid the production of non-reproducible or even false data.