Characterisation of the tracheal transcriptional response of chickens to chronic infection with Mycoplasma synoviae.

Mycoplasma synoviae causes infectious synovitis and respiratory tract infections in chickens and is responsible for significant economic losses in the poultry industry. Effective attachment and colonisation of the trachea is critical for the persistence of the organism and progression of the disease it causes. The respiratory tract infection is usually sub-clinical, but concurrent infection with infectious bronchitis virus (IBV) is known to enhance the pathogenicity of M. synoviae. This study aimed to explore differentially expressed genes in the tracheal mucosa, and their functional categories, during chronic infection with M. synoviae, using a M. synoviae-IBV infection model. The transcriptional profiles of the trachea were assessed 2 weeks after infection using RNA sequencing. In chickens infected with M. synoviae or IBV, only 1 or 8 genes were differentially expressed compared to uninfected chickens, respectively. In contrast, the M. synoviae-IBV infected chickens had 621 upregulated and 206 downregulated genes compared to uninfected chickens. Upregulated genes and their functional categories were suggestive of uncontrolled lymphoid cell proliferation and an ongoing pro-inflammatory response. Genes associated with anti-inflammatory effects, pathogen removal, apoptosis, regulation of the immune response, airway homoeostasis, cell adhesion and tissue regeneration were downregulated. Overall, transcriptional changes in the trachea, 2 weeks after infection with M. synoviae and IBV, indicate immune dysregulation, robust inflammation and a lack of cytotoxic damage during chronic infection. This model provides insights into the pathogenesis of chronic infection with M. synoviae.

Transcriptomic analysis of the effects of tylosin on the protective immunity provided by the Mycoplasma gallisepticum vaccine Vaxsafe MG ts-304.

The antimicrobial tylosin is commonly used to control mycoplasma infections, sometimes in combination with vaccination. However, the efficacy of a live mycoplasma vaccine, when combined with subsequent antimicrobial treatment, against the effects of subsequent infection with a virulent strain is unknown. This study employed differential gene expression analysis to evaluate the effects of tylosin on the protection provided by the live attenuated Vaxsafe MG ts-304 vaccine, which has been shown to be safe and to provide long-term protective immunity against infection with Mycoplasma gallisepticum. The transcriptional profiles of the tracheal mucosa revealed significantly enhanced inflammation, immune cell proliferation and adaptive immune responses in unvaccinated, untreated birds and in unvaccinated birds treated with tylosin 2 weeks after infection with virulent M. gallisepticum. These responses, indicative of the typical immune dysregulation caused by infection with M. gallisepticum, were less severe in the unvaccinated, tylosin-treated birds than in the unvaccinated, untreated birds. This was attributable to the effect of residual levels of tylosin in the tracheal mucosa on replication of virulent M. gallisepticum. These responses were not detected in vaccinated, tylosin-treated birds or in vaccinated, untreated birds after infection. The tracheal mucosal transcriptional profiles of these birds resembled those of unvaccinated, untreated, uninfected birds, suggesting a rapid and protective secondary immune response and effective vaccination. Overall, these results show that, although tylosin treatment reduced the duration of immunity, the initial protective immunity induced by Vaxsafe MG ts-304 lasted for at least 22 weeks after vaccination, even after the administration of tylosin for 16 weeks following vaccination.

Th-1 cytotoxic cell-mediated response predominates in the tracheal mucosa following Mycoplasma synoviae infection of MS-H-vaccinated chickens.

Mycoplasma synoviae is a pathogen of poultry that causes upper respiratory tract disease. MS-H is a live attenuated temperature-sensitive vaccine that effectively control M. synoviae infection in chickens. However, the mechanisms underpinning protection have not been described previously. In this study, specific-pathogen-free chickens were vaccinated at 3 weeks of age with MS-H vaccine and challenged with field strain M. synoviae 94011/v-18d at 6 weeks of age. Tracheal mucosal inflammation was characterised by the assessment of thickness, histopathological lesions, cellular infiltrates and cytokine transcription. Tracheal lesion scores of unvaccinated-challenged (-V+C) birds were higher than that of vaccinated-challenged (+V+C) birds. +V+C birds displayed early upregulation of IL-4, consistent with a Th-2-skewed response, followed by a later increase in IFN-γ transcription, indicating transition to a Th-1-skewed response. -V+C birds displayed a concurrent early Th-2 and Th-17 response characterised by increase expression of IL-4 and IL-17A respectively, and late T regulatory response characterised by increased IL-10 transcription. +V+C chickens had more cytotoxic T cells (CD8+ T cells) at 7- and 21 days post-challenge (dpc), while -V+C chickens had higher numbers of infiltrating CD4+CD25+ at 7 and 21 dpc. Overall, these observations suggest that the immune response in +V+C chickens had an inflammation characterised by an early Th-2 skewed response followed closely by a Th-1 response and infiltration of cytotoxic T cells, while the response in -V+C chickens was an early Th-2/Th-17-skewed response closely followed by a T regulatory response.

Effects of administration of tylosin on the duration of protective immunity induced by the novel Mycoplasma gallisepticum vaccine strain, Vaxsafe MG ts-304, in chickens.

Prophylactic use of antimicrobials after administration of live vaccines is a common practice in the poultry industry, but the impact of this on the efficacy and duration of protection induced by the vaccines is unknown. The effect of treatment with tylosin on the efficacy of vaccination with the live attenuated M. gallisepticum strain, Vaxsafe MG ts-304, was examined. This vaccine has previously been shown to provide protection for at least 57 weeks. Ten-week-old specific-pathogen-free chickens were vaccinated with Vaxsafe MG ts-304 and then treated with tylosin at a therapeutic dose in drinking water from 6 weeks after vaccination. Tylosin was withdrawn 5 days before challenge with M. gallisepticum strain Ap3AS at 6, 10, 14, 18 or 22 weeks after vaccination. Air sac lesions, tracheal mucosal thickening and the concentrations of serum antibodies against M. gallisepticum were assessed at 2 weeks after challenge. The protection induced by the vaccine in the 6 weeks before initiation of tylosin treatment persisted for 18 weeks after vaccination, with lesions only observed in the air sacs of vaccinated birds that had been treated with tylosin after challenge at 22 weeks after vaccination. Concentrations of serum antibodies against M. gallisepticum began to decrease in vaccinated birds that had been treated with tylosin from 16 weeks after vaccination. This study has suggested that treatment of chickens with tylosin after vaccination with a live attenuated mycoplasma vaccine reduces the duration of protective immunity afforded by the vaccine.

Tracheal cellular immune response in chickens inoculated with Mycoplasma synoviae vaccine, MS-H or its parent strain 86079/7NS.

Mycoplasma synoviae causes respiratory tract disease in chickens characterised by mild to moderate lymphoplasmacytic infiltration of the tracheal mucosa. MS-H (Vaxsafe1 MS, Bioproperties Pty Ltd.) is an effective live attenuated vaccine for M. synoviae, but the immunological basis for its mechanism of protection has not been investigated, and the phenotypes of lymphocytes and associated cytokines involved in the local adaptive immune response have not been described previously. In this study, specific-pathogen-free chickens were inoculated intra-ocularly at 3 weeks of age with either M. synoviae vaccine strain MS-H or vaccine parent strain 86079/7NS (7NS), or remained uninoculated. At 2-, 7- and 21 days post-inoculation (dpi), tracheal mucosal pathology, infiltrating lymphocytes subsets and transcription levels of mRNA encoding 8 cytokines were assessed using light microscopy, indirect immunofluorescent staining and RT-qPCR, respectively. After inoculation, tracheal mucosal thickness, tracheal mucosal lesions, and numbers of infiltrating CD4+CD25- cells, B-cells, and macrophages were greater in MS-H- and 7NS-inoculated chickens compared with non-inoculated. Inoculation with 7NS induced up-regulation of IFN-γ, while vaccination with MS-H induced up-regulation of IL-17A, when compared with non-inoculated birds. Both inoculated groups had a moderate infiltrate of CD4+CD25+ T cells in the tracheal mucosa. These findings reveal that the tracheal local cellular response after MS-H inoculation is dominated by a Th-17 response, while that of 7NS-inoculated chickens is dominated by a Th-1 type response.

Measures of tracheal lesions are more discriminatory and reproducible indications of chronic respiratory disease caused by Mycoplasma gallisepticum in poultry.

Mycoplasma gallisepticum is the primary causative agent of chronic respiratory disease in poultry, and vaccination is the measure most commonly used for its control. Pathological changes caused by M. gallisepticum are mainly observed in the trachea and air sacs, but assessment of air sac lesions is subjective. Standardized parameters for evaluation of pathological changes, and their reproducibility and discrimination in uninfected and infected groups, are critical when assessing the efficacy of M. gallisepticum vaccination. This study reviewed and critically appraised the published literature on evaluation of vaccine efficacy against pathological changes caused by M. gallisepticum in poultry in the trachea and air sacs. A search of four electronic databases, with subsequent manual filtering, identified 23 eligible papers published since 1962 describing the assessment of histopathological changes in the trachea using tracheal lesion scores and/or measurement of tracheal mucosal thicknesses and assessment of gross air sac lesions using lesion scores. Measurement of tracheal lesions proved a more reliable and robust method of assessing disease induced by M. gallisepticum when compared to assessment of air sac lesions, highlighting the importance of including assessment of tracheal lesions as the primary outcome variable in vaccine efficacy studies. In addition, this study also identified the necessity for use of a standardized model for evaluation and reporting on M. gallisepticum vaccines to minimize variations between vaccine efficacy studies and to allow direct comparisons between them.RESEARCH HIGHLIGHTS Tracheal and air sac lesions have been used to assess M. gallisepticum vaccine efficacy.The specific parameters and statistical tests used to compare tracheal and air sac lesions vary greatly.Measures of tracheal lesions are more discriminatory than measures of air sac lesions.A standardized model is needed to evaluate vaccines against infection with M. gallisepticum.

Mucosal immune responses in the trachea after chronic infection with Mycoplasma gallisepticum in unvaccinated and vaccinated mature chickens.

Tracheitis associated with the chronic respiratory disease in chickens caused by Mycoplasma gallisepticum is marked by infiltration of leukocytes into the mucosa. Although cytokines/chemokines are known to play a key role in the recruitment, differentiation, and proliferation of leukocytes, those that are produced and secreted into the trachea during the chronic stages of infection with M. gallisepticum have not been described previously. In this study, the levels of transcription in the trachea of genes encoding a panel of 13 cytokines/chemokines were quantified after experimental infection with the M. gallisepticum wild-type strain Ap3AS in unvaccinated chickens and chickens vaccinated 40-, 48- or 57-weeks previously with the novel attenuated strain ts-304. These transcriptional levels in unvaccinated/infected and vaccinated/infected chickens were compared with those of unvaccinated/uninfected and vaccinated/uninfected chickens. Pathological changes and subsets of leukocytes infiltrating the tracheal mucosa were concurrently assessed by histopathological examination and indirect immunofluorescent staining. After infection, unvaccinated birds had a significant increase in tracheal mucosal thickness and in transcription of genes for cytokines/chemokines, including those for IFN-γ, IL-17, RANTES (CCLi4), and CXCL-14, and significant downregulation of IL-2 gene transcription. B cells, CD3+ or CD4+ cells and macrophages (KUL01+ ) accumulated in the mucosa but CD8+ cells were not detected. In vaccinated birds, the levels of transcription of the genes for IL-6, IL-2, RANTES and CXCL-14 were significantly lower after infection than in the unvaccinated/infected and/or unvaccinated/uninfected birds, while the transcription of the IFN-γ gene was significantly upregulated, and there were aggregations of B cells in the tracheal mucosa. These observations indicated that M. gallisepticum may have suppressed Th2 responses by upregulating secretion of IFN-γ and IL-17 by CD4+ cells and induced immune dysregulation characterized by depletion of CD8+ cells and downregulation of IL-2 in the tracheas of unvaccinated birds. The ts-304 vaccine appeared to induce long-term protection against this immune dysregulation. TAKE AWAY: The ts-304 vaccine-induced long-term protection against immune dysregulation caused by M. gallisepticum Detection of B cells and plasma cells in the tracheal mucosa suggested that long-term protection is mediated by mucosal B cell memory Infection of unvaccinated birds with M. gallisepticum resulted in CD8+ cell depletion and downregulation of IL-2 in the tracheal mucosa, suggestive of immune dysregulation Infection of unvaccinated birds with M. gallisepticum resulted in upregulation of IFN-γ and infiltration of CD4+ cells and antigen presenting cells (B and KUL01+ cells) into the tracheal mucosa, suggesting enhanced antigen processing and presentation during chronic infection Th2 responses to infection with M. gallisepticum may be dampened by CD4+ cells through upregulation of IFN-γ and IL-17 during chronic infection.

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.

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 Response of the Chicken Trachea to Chronic Infection with Virulent Mycoplasma gallisepticum Strain Ap3AS and Vaxsafe MG (Strain ts-304): a Transcriptional Profile.

Mycoplasma gallisepticum is the primary etiological agent of chronic respiratory disease in chickens. Live attenuated vaccines are most commonly used in the field to control the disease, but current vaccines have some limitations. Vaxsafe MG (strain ts-304) is a new vaccine candidate that is efficacious at a lower dose than the current commercial vaccine strain ts-11, from which it is derived. In this study, the transcriptional profiles of the trachea of unvaccinated chickens and chickens vaccinated with strain ts-304 were compared 2 weeks after challenge with M. gallisepticum strain Ap3AS during the chronic stage of infection. After challenge, genes, gene ontologies, pathways, and protein classes involved in inflammation, cytokine production and signaling, and cell proliferation were upregulated, while those involved in formation and motor movement of cilia, formation of intercellular junctional complexes, and formation of the cytoskeleton were downregulated in the unvaccinated birds compared to the vaccinated birds, reflecting immune dysregulation and the pathological changes induced in the trachea by infection with M. gallisepticum Vaccination appears to protect the structural and functional integrity of the tracheal mucosa 2 weeks after infection with M. gallisepticum.

Transcriptomic Analysis of Long-Term Protective Immunity Induced by Vaccination With Mycoplasma gallisepticum Strain ts-304.

Live attenuated vaccines are commonly used to control Mycoplasma gallisepticum infections in chickens. M. gallisepticum ts-304 is a novel live attenuated vaccine strain that has been shown to be safe and effective. In this study, the transcriptional profiles of genes in the tracheal mucosa in chickens challenged with the M. gallisepticum wild-type strain Ap3AS at 57 weeks after vaccination with ts-304 were explored and compared with the profiles of unvaccinated chickens that had been challenged with strain Ap3AS, unvaccinated and unchallenged chickens, and vaccinated but unchallenged chickens. At two weeks after challenge, pair-wise comparisons of transcription in vaccinated-only, vaccinated-and-challenged and unvaccinated and unchallenged birds detected no differences. However, the challenged-only birds had significant up-regulation in the transcription of genes and enrichment of gene ontologies, pathways and protein classes involved in infiltration and proliferation of inflammatory cells and immune responses mediated through enhanced cytokine and chemokine production and signaling, while those predicted to be involved in formation and motor movement of cilia and formation of the cellular cytoskeleton were significantly down-regulated. The transcriptional changes associated with the inflammatory response were less severe in these mature birds than in the relatively young birds examined in a previous study. The findings of this study demonstrated that vaccination with the attenuated M. gallisepticum strain ts-304 protects against the transcriptional changes associated with the inflammatory response and pathological changes in the tracheal mucosa caused by infection with M. gallisepticum in chickens for at least 57 weeks after vaccination.