Exosomal miR-181a-5p reduce Mycoplasma gallisepticum (HS strain) infection in chicken by targeting PPM1B and activating the TLR2-mediated MyD88/NF-κB signaling pathway.

Mycoplasma gallisepticum (MG) is one of the most important pathogens that causes chronic respiratory disease (CRD) in chickens. Exosomes secreted from cells have been well demonstrated to deliver miRNAs to recipient cells to modulate cellular functions. The purpose of this study is to explore the underlying functions and mechanisms of exosomal miR-181a-5p in MG-HS infection. In this study, we found that miR-181a-5p expression in vivo and in vitro was significantly up-regulated after MG-HS infection. It was also upregulated in exosomes, which were derived from MG-HS-infected type-II pneumocytes cells (CP-II). In addition, exosomes secreted by MG-HS-infected CP-II were able to transfer miR-181a-5p to recipient chicken embryo fibroblast cells (DF-1), resulting in a significant upregulation of miR-181a-5p expression in recipient DF-1 cells. We further identified that Mg2+/Mn2+-dependent protein phosphatase 1B (PPM1B) was the target gene of miR-181a-5p. Overexpression of miR-181a-5p or knockdown of PPM1B activated the nuclear factor-κB (NF-κB) signaling pathway, whereas inhibition of miR-181a-5p and overexpression of PPM1B led to the opposite results. Besides, up-regulation of miR-181a-5p significantly increased the expression of toll-like receptor 2 (TLR2), myeloid differentiation factor 88 (MyD88), tumor necrosis factors alpha (TNF-α) and interleukin-1ß (IL-1ß), whereas inhibition of miR-181a-5p showed a contrary result. Up-regulation of miR-181a-5p promoted cell proliferation, cell cycle progression and inhibited apoptosis to resist MG-HS infection. Moreover, overexpression of miR-181a-5p significantly negative regulated the expression of Mycoplasma gallisepticum adhesin protein (pMGA1.2) by directly inhibiting PPM1B. Thus, we concluded that exosomal miR-181a-5p from CP-II cells activated the TLR2-mediated MyD88/NF-κB signaling pathways by directly targeting PPM1B to promote the expression of pro-inflammatory cytokines for defending against MG-HS infection in recipient DF-1 cells.

Effects of immunosuppression on the efficacy of vaccination against Mycoplasma gallisepticum infection in chickens.

Immunosuppression can increase the susceptibility of chickens to other disease-causing pathogens and interfere with the efficacy of vaccination against those pathogens. Chicken anaemia virus (CAV) and infectious bursal disease virus (IBDV) are common causes of immunosuppression in chickens. Immunosuppression was induced by experimental infection with either CAV or IBDV to assess the effect of immunosuppression on the efficacy of vaccination with Mycoplasma gallisepticum strain ts-304 against infection with virulent M. gallisepticum, a common bacterial pathogen of chickens worldwide. Birds were experimentally infected with either CAV or IBDV at 1 week of age, before vaccination and challenge with M. gallisepticum to examine the effect of immunosuppression at the time of vaccination, or at 6 weeks of age, after vaccination against M. gallisepticum but before challenge with virulent M. gallisepticum, to investigate the effect of immunosuppression at the time of challenge. All birds were vaccinated with a single dose of the ts-304 vaccine at 3 weeks of age and experimentally challenged with the virulent M. gallisepticum strain Ap3AS at 8 weeks of age. In immunosuppressed chickens there was a reduction in protection offered by the ts-304 vaccine at two weeks after challenge, as measured by tracheal mucosal thicknesses, serum antibody levels against M. gallisepticum, air sac lesion scores and virulent M. gallisepticum load in the trachea. Immunosuppressed birds with detectable serum antibodies against M. gallisepticum were less likely to have tracheal lesions. This study has shown that immunosuppression caused by infection with CAV or IBDV can interfere with vaccination against mycoplasmosis in chickens.

Levels of pathogen virulence and host resistance both shape the antibody response to an emerging bacterial disease.

Quantifying variation in the ability to fight infection among free-living hosts is challenging and often constrained to one or a few measures of immune activity. While such measures are typically taken to reflect host resistance, they can also be shaped by pathogen effects, for example, if more virulent strains trigger more robust immune responses. Here, we test the extent to which pathogen-specific antibody levels, a commonly used measure of immunocompetence, reflect variation in host resistance versus pathogen virulence, and whether these antibodies effectively clear infection. House finches (Haemorhous mexicanus) from resistant and susceptible populations were inoculated with > 50 isolates of their novel Mycoplasma gallisepticum pathogen collected over a 20-year period during which virulence increased. Serum antibody levels were higher in finches from resistant populations and increased with year of pathogen sampling. Higher antibody levels, however, did not subsequently give rise to greater reductions in pathogen load. Our results show that antibody responses can be shaped by levels of host resistance and pathogen virulence, and do not necessarily signal immune clearance ability. While the generality of this novel finding remains unclear, particularly outside of mycoplasmas, it cautions against using antibody levels as implicit proxies for immunocompetence and/or host resistance.

A Mycoplasma gallisepticum Glycerol ABC Transporter Involved in Pathogenicity.

MalF has been shown to be required for virulence in the important avian pathogen Mycoplasma gallisepticum To characterize the function of MalF, predicted to be part of a putative ABC transporter, we compared metabolite profiles of a mutant with a transposon inserted in malF (MalF-deficient ST mutant 04-1; ΔmalF) with those of wild-type bacteria using gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry. Of the substrates likely to be transported by an ABC transport system, glycerol was detected at significantly lower abundance in the ΔmalF mutant, compared to the wild type. Stable isotope labeling using [U-13C]glycerol and reverse transcription-quantitative PCR analysis indicated that MalF was responsible for the import of glycerol into M. gallisepticum and that, in the absence of MalF, the transcription of gtsA, which encodes a second transporter, GtsA, was upregulated, potentially to increase the import of glycerol-3-phosphate into the cell to compensate for the loss of MalF. The loss of MalF appeared to have a global effect on glycerol metabolism, suggesting that it may also play a regulatory role, and cellular morphology was also affected, indicating that the change to glycerol metabolism may have a broader effect on cellular organization. Overall, this study suggests that the reduced virulence of the ΔmalF mutant is due to perturbed glycerol uptake and metabolism and that the operon including malF should be reannotated as golABC to reflect its function in glycerol transport.IMPORTANCE Many mycoplasmas are pathogenic and cause disease in humans and animals. M. gallisepticum causes chronic respiratory disease in chickens and infectious sinusitis in turkeys, resulting in economic losses in poultry industries throughout the world. Expanding our knowledge about the pathogenesis of mycoplasma infections requires better understanding of the specific gene functions of these bacteria. In this study, we have characterized the metabolic function of a protein involved in the pathogenicity of M. gallisepticum, as well as its effect on expression of selected genes, cell phenotype, and H2O2 production. This study is a key step forward in elucidating why this protein plays a key role in virulence in chickens. This study also emphasizes the importance of functional characterization of mycoplasma proteins, using tools such as metabolomics, since prediction of function based on homology to other bacterial proteins is not always accurate.

Duration of protective immunity induced by Mycoplasma gallisepticum strain ts-304 vaccine in chickens.

Mycoplasma gallisepticum (MG) is an important pathogen of poultry worldwide, causing chronic respiratory disease in chickens and turkeys. MG ts-304 is a GapA positive clone recovered from Vaxsafe MG (strain ts-11) that has been shown to be safe in chickens when delivered by the eye drop route to 3-week-old specific-pathogen-free chickens and to confer protection against challenge at 4 weeks after vaccination, as measured by tracheal mucosal thickness and air sac lesion scores. In this study, specific pathogen-free chickens (SPF) were vaccinated with a single dose of the MG ts-304 vaccine (106.0 colour changing units) at 3 weeks of age and experimentally challenged by aerosol with the virulent M. gallisepticum strain Ap3AS at 40, 48 and 57 weeks after vaccination. There were no significant differences in tracheal mucosal thickness 2 weeks after challenge between chickens challenged at the three time points, or between the vaccinated birds after challenge and unvaccinated/unchallenged control birds. Thus there was clear evidence that the immunity conferred by vaccination with the MG ts-304 vaccine resulted in significant protection against tracheitis in chickens that extended to, but was highly likely to exceed, 57 weeks after vaccination and that similar long term protective immunity could be expected to be conferred by a vaccine dose lower than that used in this study.

Differential house finch leukocyte profiles during experimental infection with Mycoplasma gallisepticum isolates of varying virulence.

Leukocyte differentials are a useful tool for assessing systemic immunological changes during pathogen infections, particularly for non-model species. To date, no study has explored how experimental infection with a common bacterial pathogen, Mycoplasma gallisepticum (MG), influences the course and strength of haematological changes in the natural songbird host, house finches. Here we experimentally inoculated house finches with MG isolates known to vary in virulence, and quantified the proportions of circulating leukocytes over the entirety of infection. First, we found significant temporal effects of MG infection on the proportions of most cell types, with strong increases in heterophil and monocyte proportions during infection. Marked decreases in lymphocyte proportions also occurred during infection, though these proportional changes may simply be driven by correlated increases in other leukocytes. Second, we found significant effects of isolate virulence, with the strongest changes in cell proportions occurring in birds inoculated with the higher virulence isolates, and almost no detectable changes relative to sham treatment groups in birds inoculated with the lowest virulence isolate. Finally, we found that variation in infection severity positively predicted the proportion of circulating heterophils and lymphocytes, but the strength of these correlations was dependent on isolate. Taken together, these results indicate strong haematological changes in house finches during MG infection, with markedly different responses to MG isolates of varying virulence. These results are consistent with the possibility that evolved virulence in house finch MG results in higher degrees of immune stimulation and associated immunopathology, with potential direct benefits for MG transmission. RESEARCH HIGHLIGHTS House finches show a marked pro-inflammatory response to M. gallisepticum infection. Virulent pathogen isolates produce stronger finch white blood cell responses. Among birds, stronger white blood cell responses are associated with higher infection severity.

The effect of Mycoplasma gallisepticum infection on energy metabolism in chicken lungs: Through oxidative stress and inflammation.

Mycoplasma gallisepticum (Mg) causes chronic respiratory disease (CRD) in chickens. However, the effect of Mg infection on energy metabolism in chicken lungs is still unknown. The present study was aimed to investigate the effect of Mg infection on energy metabolism in chicken lungs. Four-weeks-old white leghorn chickens were randomly divided into control group (L1) and Mg infection group (L2). Histopathology, transmission electron microscopy, qRT-PCR and Western blot were used to determine the hallmarks of ultrastructural analysis, inflammation and energy metabolism. Results revealed that Mg infection induced oxidative stress in the chicken lungs and serum cytokine activities were enhanced at the three time points. Chickens infected with Mg revealed abnormal morphology and cellular damage including increased inflammatory cells infiltrate, cellular debris and exudate, mitochondrial and DNA damage in the lungs. The mRNA and protein expression level of inflammation-related genes were significantly increased in L2 group, showing that Mg induced inflammation in chicken lungs. In addition, ATPase activities were reduced in L2 group compared to L1 group. Meanwhile, the expression of energy metabolism related genes were decreased at both mRNA and protein level at all assessed time points, which showed that Mg infection weakened energy metabolism in chicken lungs. In summary, the data suggested that Mg infection induced oxidative stress, inflammation and energy metabolism dysfunction in the chicken lungs, exploring new therapeutic targets and providing a reference for comparative veterinary medicine.

GroEL Protein (Heat Shock Protein 60) of Mycoplasma gallisepticum Induces Apoptosis in Host Cells by Interacting with Annexin A2.

Mycoplasma gallisepticum is an avian respiratory and reproductive tract pathogen that has a significant economic impact on the poultry industry worldwide. Although membrane proteins of Mycoplasma spp. are thought to play crucial roles in host interactions, very few have had their biochemical function defined. In this study, we found that the GroEL protein (heat shock protein 60) of Mycoplasma gallisepticum could induce apoptosis in peripheral blood mononuclear cells, and the underlying molecular mechanism was further determined. The GroEL gene from Mycoplasma gallisepticum was cloned and expressed in Escherichia coli to facilitate the functional analysis of recombinant protein. The purified GroEL protein was shown to adhere to peripheral blood mononuclear cells (PBMCs) and DF-1 cells and cause apoptosis in PBMCs. A protein pulldown assay coupled with mass spectrometry identified that annexin A2 possibly interacted with GroEL protein. Coimmunoprecipitation assays confirmed that GroEL proteins could bind to annexin A2, and confocal analysis further demonstrated that GroEL colocolized with annexin A2 in HEK293T cells and PBMCs. Moreover, annexin A2 expression was significantly induced by a recombinant GroEL protein in PBMCs, and knocking down annexin A2 expression resulted in significantly reduced apoptosis. Taken together, these data suggest that GroEL induces apoptosis in host cells by interacting with annexin A2, a novel virulence mechanism in Mycoplasma gallisepticum Our findings lead to a better understanding of molecular pathogenesis in Mycoplasma gallisepticum.

gga-miR-146c Activates TLR6/MyD88/NF-κB Pathway through Targeting MMP16 to Prevent Mycoplasma Gallisepticum (HS Strain) Infection in Chickens.

Mycoplasma gallisepticum (MG), a pathogen that infects chickens and some other birds, triggers chronic respiratory disease (CRD) in chickens, which is characterized by inflammation. The investigation of microbial pathogenesis would contribute to the deep understanding of infection control. Since microribonucleic acids (miRNAs) play a key role in this process, gga-mir-146c, an upregulated miRNA upon MG infection, was selected according to our previous RNA-sequencing data. In this paper, we predicted and validated that MMP16 is one of gga-miR-146c target genes. Results show that MMP16 is the target of gga-miR-146c and gga-miR-146c can downregulate MMP16 expression within limits. gga-miR-146c upregulation significantly increased the expression of TLR6, NF-κB p65, MyD88, and TNF-α, whereas the gga-miR-146c inhibitor led to an opposite result. gga-miR-146c upregulation effectively decreased apoptosis and stimulated DF-1 cells proliferation upon MG infection. On the contrary, gga-miR-146c inhibitor promoted apoptosis and repressed the proliferation. Collectively, our results suggest that gga-miR-146c upregulation upon MG infection represses MMP16 expression, activating TLR6/MyD88/NF-κB pathway, promoting cell proliferation by inhibiting cell apoptosis, and, finally, enhancing cell cycle progression to defend against host MG infection.

Immunologic Pathways in Protective versus Maladaptive Host Responses to Attenuated and Pathogenic Strains of Mycoplasma gallisepticum.

Mycoplasmas are small bacterial commensals or pathogens that commonly colonize host mucosal tissues and avoid rapid clearance, in part by stimulating inflammatory, immunopathogenic responses. We previously characterized a wide array of transcriptomic perturbations in avian host tracheal mucosae infected with virulent, immunopathologic Mycoplasma gallisepticum; however, mechanisms delineating these from protective responses, such as those induced upon vaccination, have not been thoroughly explored. In this study, host transcriptomic responses to two experimental M. gallisepticum vaccines were assessed during the first 2 days of infection. Relative to virulent infection, host metabolic and immune gene responses to both vaccines were greatly decreased, including early innate immune responses critical to disease development and subsequent adaptive immunity. These data specify host genes and potential mechanisms contributing to maladaptive versus beneficial host responses-information critical for design of vaccines efficacious in both limiting inflammation and enabling pathogen clearance.

Characterization of Mycoplasma gallisepticum pyruvate dehydrogenase alpha and beta subunits and their roles in cytoadherence.

Mycoplasma gallisepticum is a causative agent of chronic respiratory disease in chickens, typically causing great economic losses. Cytoadherence is the critical stage for mycoplasma infection, and the associated proteins are important for mycoplasma pathogenesis. Many glycolytic enzymes are localized on the cell surface and can bind the extracellular matrix of host cells. In this study, the M. gallisepticum pyruvate dehydrogenase E1 alpha subunit (PDHA) and beta subunit (PDHB) were expressed in Escherichia coli, and their enzymatic activities were identified based on 2,6-dichlorophenol indophenol reduction. When recombinant PDHA (rPDHA) and recombinant PDHB (rPDHB) were mixed at a 1:1 molar ratio, they exhibited strong enzymatic activity. Alone, rPDHA and rPDHB exhibited no or weak enzymatic activity. Further experiments indicated that both PDHA and PDHB were surface-exposed immunogenic proteins of M. gallisepticum. Bactericidal assays showed that the mouse anti-rPDHA and anti-rPDHB sera killed 48.0% and 75.1% of mycoplasmas respectively. A combination of rPDHA and rPDHB antisera had a mean bactericidal rate of 65.2%, indicating that rPDHA and rPDHB were protective antigens, and combining the two sera did not interfere with bactericidal activity. Indirect immunofluorescence and surface display assays showed that both PDHA and PDHB adhered to DF-1 chicken embryo fibroblast cells and adherence was significantly inhibited by antisera against PDHA and PDHB. Adherence inhibition of M. gallisepticum to DF-1 chicken embryo fibroblast cells was 30.2% for mouse anti-rPDHA serum, 45.1% for mouse anti-rPDHB serum and 72.5% for a combination of rPDHA and rPDHB antisera, suggesting that rPDHA and rPDHB antisera may have synergistically interfered with M. gallisepticum cytoadherence. Plasminogen (Plg)-binding assays further demonstrated that both PDHA and PDHB were Plg-binding proteins, which may have contributed to bacterial colonization. Our results clarified the enzymatic activity of M. gallisepticum PDHA and PDHB and demonstrated these compounds as Plg-binding proteins involved in cytoadherence.

Rapid Antagonistic Coevolution in an Emerging Pathogen and Its Vertebrate Host.

Host-pathogen coevolution is assumed to play a key role in eco-evolutionary processes, including epidemiological dynamics and the evolution of sexual reproduction [1-4]. Despite this, direct evidence for host-pathogen coevolution is exceptional [5-7], particularly in vertebrate hosts. Indeed, although vertebrate hosts have been shown to evolve in response to pathogens or vice versa [8-12], there is little evidence for the necessary reciprocal changes in the success of both antagonists over time [13]. Here, we generate a time-shift experiment to demonstrate adaptive, reciprocal changes in North American house finches (Haemorhous mexicanus) and their emerging bacterial pathogen, Mycoplasma gallisepticum [14-16]. Our experimental design is made possible by the existence of disease-exposed and unexposed finch populations, which were known to exhibit equivalent responses to experimental inoculation until the recent spread of genetic resistance in the former [14, 17]. Whereas inoculations with pathogen isolates from epidemic outbreak caused comparable sub-lethal eye swelling in hosts from exposed (hereafter adapted) and unexposed (hereafter ancestral) populations, inoculations with isolates sampled after the spread of resistance were threefold more likely to cause lethal symptoms in hosts from ancestral populations. Similarly, the probability that pathogens successfully established an infection in the primary host and, before inducing death, transmitted to an uninfected sentinel was highest when recent isolates were inoculated in hosts from ancestral populations and lowest when early isolates were inoculated in hosts from adapted populations. Our results demonstrate antagonistic host-pathogen coevolution, with hosts and pathogens displaying increased resistance and virulence in response to each other over time.

Metabolite profiling of Mycoplasma gallisepticum mutants, combined with bioinformatic analysis, can reveal the likely functions of virulence-associated genes.

Mycoplasma gallisepticum is an economically important pathogen of commercial poultry. An improved understanding of M. gallisepticum pathogenesis is required to develop better control methods. We recently identified a number of M. gallisepticum mutants with defects in colonization and persistence in chickens using signature-tagged transposon mutagenesis. Loss of virulence was associated with mutations in a putative oligopeptide/dipeptide (opp/dpp) ATP-binding cassette (ABC) transporter (where the transposon was inserted into the MGA_0220 (oppD1) gene and two hypothetical proteins (encoded by MGA_1102 and MGA_0588), one of which (MGA_1102) contains a putative peptidase motif. To further characterise the function of these proteins, we compared the metabolome of each transposon mutant with that of wild type bacteria. Two independent LC/MS analyses revealed consistent significant decreases in the abundances of several amino acids and the dipeptide alanyl-glycine (Ala-Gly) in the MGA_0220 mutant, consistent with this protein being a peptide transporter. Similarly, lysine and Ala-Gly were significantly decreased in the MGA_1102 mutant, consistent with our bioinformatic analysis suggesting that MGA_1102 encodes a membrane-located peptidase. Few differences were observed in metabolite levels in the MGA_0588 mutant, suggesting that the disrupted protein has a non-metabolic role. Overall, this study indicates that metabolomics is a useful tool in the functional analysis of mutants.

Quantitative host resistance drives the evolution of increased virulence in an emerging pathogen.

Emergent infectious diseases can have a devastating impact on host populations. The high selective pressures on both the hosts and the pathogens frequently lead to rapid adaptations not only in pathogen virulence but also host resistance following an initial outbreak. However, it is often unclear whether hosts will evolve to avoid infection-associated fitness costs by preventing the establishment of infection (here referred to as qualitative resistance) or by limiting its deleterious effects through immune functioning (here referred to as quantitative resistance). Equally, the evolutionary repercussions these different resistance mechanisms have for the pathogen are often unknown. Here, we investigate the co-evolutionary dynamics of pathogen virulence and host resistance following the epizootic outbreak of the highly pathogenic bacterium Mycoplasma gallisepticum in North American house finches (Haemorhous mexicanus). Using an evolutionary modelling approach and with a specific emphasis on the evolved resistance trait, we demonstrate that the rapid increase in the frequency of resistant birds following the outbreak is indicative of strong selection pressure to reduce infection-associated mortality. This, in turn, created the ecological conditions that selected for increased bacterial virulence. Our results thus suggest that quantitative host resistance was the key factor underlying the evolutionary interactions in this natural host-pathogen system.

Up-Regulation of miR-130b-3p Activates the PTEN/PI3K/AKT/NF-κB Pathway to Defense against Mycoplasma gallisepticum (HS Strain) Infection of Chicken.

Mycoplasma gallisepticum (MG) is the pathogen of chronic respiratory disease (CRD), hallmarked by vigorous inflammation in chickens, causing the poultry industry enormous losses. miRNAs have emerged as important regulators of animal diseases. Previous miRNA sequencing data has demonstrated that miR-130b-3p is up-regulated in MG-infected chicken embryo lungs. Therefore, we aimed to investigate the function of miR-130b-3p in MG infection of chickens. RT-qPCR results confirmed that miR-130b-3p was up-regulated both in MG-infected chicken embryo lungs and chicken embryonic fibroblast cells (DF-1 cells). Furthermore, functional studies showed that overexpression of miR-130b-3p promoted MG-infected DF-1 cell proliferation and cell cycle, whereas inhibition of miR-130b-3p weakened these cellular processes. Luciferase reporter assay combined with gene expression data supported that phosphatase and tensin homolog deleted on chromosome ten (PTEN) was a direct target of miR-130b-3p. Additionally, overexpression of miR-130b-3p resulted in up-regulations of phosphatidylinositol-3 kinase (PI3K), serine/threonine kinase (AKT), and nuclear factor-κB (NF-κB), whereas inhibition of miR-130b-3p led to the opposite results. Altogether, upon MG infection, up-regulation of miR-130b-3p activates the PI3K/AKT/NF-κB pathway, facilitates cell proliferation and cell cycle via down-regulating PTEN. This study helps to understand the mechanism of host response to MG infection.

gga-miR-451 Negatively Regulates Mycoplasma gallisepticum (HS Strain)-Induced Inflammatory Cytokine Production via Targeting YWHAZ.

Mycoplasma gallisepticum (MG) is the most economically significant mycoplasma pathogen of poultry that causes chronic respiratory disease (CRD) in chickens. Although miRNAs have been identified as a major regulator effect on inflammatory response, it is largely unclear how they regulate MG-induced inflammation. The aim of this study was to investigate the functional roles of gga-miR-451 and identify downstream targets regulated by gga-miR-451 in MG infection of chicken. We found that the expression of gga-miR-451 was significantly up-regulated during MG infection of chicken embryo fibroblast cells (DF-1) and chicken embryonic lungs. Overexpression of gga-miR-451 decreased the MG-induced inflammatory cytokine production, including tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and interleukin-6 (IL-6), whereas inhibition of gga-miR-451 had the opposite effect. Gene expression data combined with luciferase reporter assays demonstrated that tyrosine3-monooxygenase/tryptophan5-monooxygenase activation protein zeta (YWHAZ) was identified as a direct target of gga-miR-451 in the context of MG infection. Furthermore, upregulation of gga-miR-451 significantly inhibited the MG-infected DF-1 cells proliferation, induced cell-cycle arrest, and promoted apoptosis. Collectively, our results demonstrate that gga-miR-451 negatively regulates the MG-induced production of inflammatory cytokines via targeting YWHAZ, inhibits the cell cycle progression and cell proliferation, and promotes cell apoptosis. This study provides a better understanding of the molecular mechanisms of MG infection.

Comparative Evaluation of the Pathogenicity of Mycoplasma gallinaceum in Chickens.

Mycoplasma gallinaceum is not among the most pathogenic mycoplasmas affecting poultry, but its continuous re-isolation from flocks in South Africa displaying typical signs of mycoplasmosis prompted us to revisit its role in respiratory disease. Specific-pathogen-free white leghorn chickens were co-challenged with either M. gallinaceum (MGC) and QX-like infectious bronchitis virus (IBV), or the more virulent Mycoplasm gallisepticum (MG) and IBV. No clinical signs were observed apart from sneezing in chickens challenged with IBV, MGC + IBV, and MG + IBV. On postmortem examination, one bird each in the MGC + IBV and IBV groups developed peritonitis or airsacculitis, respectively. In the tracheas, the MG + IBV group showed the most severe ciliary damage with a mean ciliostatic score of 32.40 compared to scores of 26.83 and 20.4 for the MGC + IBV and IBV groups, respectively. Corresponding tracheal lesions were recorded. Quantitation of the challenge pathogens by quantitative real-time PCR and real-time reverse transcriptase-PCR determined that MGC was shed in much higher titers from the trachea than MG, when co-infected with IBV. Interestingly, the presence of both MG and MGC appeared to enhance IBV replication in the tracheas of infected chickens, whereas the presence of IBV suppressed MG and MGC proliferation in the trachea. In general, the nonpathogenicity of M. gallinaceum in chickens was confirmed, but it was able to aggravate respiratory disease and pathogen proliferation with virulent QX-like IBV.

Incomplete host immunity favors the evolution of virulence in an emergent pathogen.

Immune memory evolved to protect hosts from reinfection, but incomplete responses that allow future reinfection may inadvertently select for more-harmful pathogens. We present empirical and modeling evidence that incomplete immunity promotes the evolution of higher virulence in a natural host-pathogen system. We performed sequential infections of house finches with Mycoplasma gallisepticum strains of various levels of virulence. Virulent bacterial strains generated stronger host protection against reinfection than less virulent strains and thus excluded less virulent strains from infecting previously exposed hosts. In a two-strain model, the resulting fitness advantage selected for an almost twofold increase in pathogen virulence. Thus, the same immune systems that protect hosts from infection can concomitantly drive the evolution of more-harmful pathogens in nature.

TLR2/MyD88/NF-κB signaling pathway regulates IL-1ß production in DF-1 cells exposed to Mycoplasma gallisepticum LAMPs.

Mycoplasma gallisepticum (M. gallisepticum) is one of the most important pathogens that cause chronic respiratory disease in chickens. M. gallisepticum-derived lipid-associated membrane proteins (LAMPs) are thought to be one of the major factors in mycoplasma pathogenesis and are potent inducers of the host innate immune response. However, the interaction of pathogenic M. gallisepticum-derived LAMPs with Toll-like receptors (TLRs) and the signaling pathways responsible for activating inflammation and NF-κB have not been fully elucidated. In this study, we found that IL-1ß expression was induced in DF-1 cells stimulated with M. gallisepticum LAMPs. Subcellular localization experiments using immunofluorescence assays (IFAs) showed p65 translocation from the cytoplasm to the nucleus in DF-1 cells following stimulation with M. gallisepticum LAMPs. Phosphorylation of p65 was detected in LAMP-stimulated DF-1 cells. Treatment with an NF-κB-specific inhibitor showed that NF-κB is required for M. gallisepticum LAMP-induced IL-1ß expression. In addition, the results indicated that TLR2 and myeloid differentiation primary-response protein 88 (MyD88)-dependent signaling pathways were involved in the activation of NF-κB by M. gallisepticum LAMPs. Together, these results provide evidence that M. gallisepticum LAMPs activate IL-1ß production through the NF-κB pathway via TLR2 and MyD88.

Resident Microbiome Disruption with Antibiotics Enhances Virulence of a Colonizing Pathogen.

There is growing evidence that symbiotic microbes play key roles in host defense, but less is known about how symbiotic microbes mediate pathogen-induced damage to hosts. Here, we use a natural wildlife disease system, house finches and the conjunctival bacterial pathogen Mycoplasma gallisepticum (MG), to experimentally examine the impact of the ocular microbiome on host damage and pathogen virulence factors during infection. We disrupted the ocular bacterial community of healthy finches using an antibiotic that MG is intrinsically resistant to, then inoculated antibiotic- and sham-treated birds with MG. House finches with antibiotic-disrupted ocular microbiomes had more severe MG-induced conjunctival inflammation than birds with unaltered microbiomes, even after accounting for differences in conjunctival MG load. Furthermore, MG cultures from finches with disrupted microbiomes had increased sialidase enzyme and cytadherence activity, traits associated with enhanced virulence in Mycoplasmas, relative to isolates from sham-treated birds. Variation in sialidase activity and cytadherence among isolates was tightly linked with degree of tissue inflammation in hosts, supporting the consideration of these traits as virulence factors in this system. Overall, our results suggest that microbial dysbiosis can result in enhanced virulence of colonizing pathogens, with critical implications for the health of wildlife, domestic animals, and humans.