Genes required for survival and proliferation of Mycoplasma bovis in association with host cells.

Mycoplasma bovis is an important emerging pathogen of cattle and bison, but our understanding of the genetic basis of its interactions with its host is limited. The aim of this study was to identify genes of M. bovis required for interaction and survival in association with host cells. One hundred transposon-induced mutants of the type strain PG45 were assessed for their capacity to survive and proliferate in Madin-Darby bovine kidney cell cultures. The growth of 19 mutants was completely abrogated, and 47 mutants had a prolonged doubling time compared to the parent strain. All these mutants had a similar growth pattern to the parent strain PG45 in the axenic media. Thirteen genes previously classified as dispensable for the axenic growth of M. bovis were found to be essential for the growth of M. bovis in association with host cells. In most of the mutants with a growth-deficient phenotype, the transposon was inserted into a gene involved in transportation or metabolism. This included genes coding for ABC transporters, proteins related to carbohydrate, nucleotide and protein metabolism, and membrane proteins essential for attachment. It is likely that these genes are essential not only in vitro but also for the survival of M. bovis in infected animals. IMPORTANCE: Mycoplasma bovis causes chronic bronchopneumonia, mastitis, arthritis, keratoconjunctivitis, and reproductive tract disease in cattle around the globe and is an emerging pathogen in bison. Control of mycoplasma infections is difficult in the absence of appropriate antimicrobial treatment or effective vaccines. A comprehensive understanding of host-pathogen interactions and virulence factors is important to implement more effective control methods against M. bovis. Recent studies of other mycoplasmas with in vitro cell culture models have identified essential virulence genes of mycoplasmas. Our study has identified genes of M. bovis required for survival in association with host cells, which will pave the way to a better understanding of host-pathogen interactions and the role of specific genes in the pathogenesis of disease caused by M. bovis.

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.

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.

SdrA, an NADP(H)-regenerating enzyme, is crucial for Coxiella burnetii to resist oxidative stress and replicate intracellularly.

Coxiella burnetii, the causative agent of the zoonotic disease Q fever, is a Gram-negative bacterium that replicates inside macrophages within a highly oxidative vacuole. Screening of a transposon mutant library suggested that sdrA, which encodes a putative short-chain dehydrogenase, is required for intracellular replication. Short-chain dehydrogenases are NADP(H)-dependent oxidoreductases, and SdrA contains a predicted NADP+ binding site, suggesting it may facilitate NADP(H) regeneration by C. burnetii, a key process for surviving oxidative stress. Purified recombinant 6×His-SdrA was able to convert NADP+ to NADP(H) in vitro. Mutation to alanine of a conserved glycine residue at position 12 within the predicted NADP binding site abolished significant enzymatic activity. Complementation of the sdrA mutant (sdrA::Tn) with plasmid-expressed SdrA restored intracellular replication to wild-type levels, but expressing enzymatically inactive G12A_SdrA did not. The sdrA::Tn mutant was more susceptible in vitro to oxidative stress, and treating infected host cells with L-ascorbate, an anti-oxidant, partially rescued the intracellular growth defect of sdrA::Tn. Finally, stable isotope labelling studies demonstrated a shift in flux through metabolic pathways in sdrA::Tn consistent with the presence of increased oxidative stress, and host cells infected with sdrA::Tn had elevated levels of reactive oxygen species compared with C. burnetii NMII.

Mycoplasma bovis Membrane Protein MilA Is a Multifunctional Lipase with Novel Lipid and Glycosaminoglycan Binding Activity.

The survival, replication, and virulence of mycoplasmas depend on their ability to capture and import host-derived nutrients using poorly characterized membrane proteins. Previous studies on the important bovine pathogen Mycoplasma bovis demonstrated that the amino-terminal end of an immunogenic 226-kDa (P226) protein, encoded by milA (the full-length product of which has a predicted molecular weight of 303 kDa), had lipase activity. The predicted sequence of MilA contains glycosaminoglycan binding motifs, as well as multiple copies of a domain of unknown function (DUF445) that is also found in apolipoproteins. We mutagenized the gene to facilitate expression of a series of regions spanning the gene in Escherichia coli Using monospecific antibodies against these recombinant proteins, we showed that MilA was proteolytically processed into 226-kDa and 50-kDa fragments that were both partitioned into the detergent phase by Triton X-114 phase fractionation. Trypsin treatment of intact cells showed that P226 was surface exposed. In vitro, the recombinant regions of MilA bound to 1-anilinonaphthalene-8-sulfonic acid and to a variety of lipids. The MilA fragments were also shown to bind heparin. Antibody against the carboxyl-terminal fragment inhibited the growth of M. bovisin vitro This carboxyl end also bound and hydrolyzed ATP, suggestive of a potential role as an autotransporter. Our studies have demonstrated that DUF445 has lipid binding activity and that MilA is a multifunctional protein that may play multiple roles in the pathogenesis of infection with M. bovis.

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.

Analysis of antibody response to an epitope in the haemagglutinin subunit 2 of avian influenza virus H5N1 for differentiation of infected and vaccinated chickens.

The H5N1 subtype of highly pathogenic avian influenza virus has been circulating in poultry in Indonesia since 2003 and vaccination has been used as a strategy to eradicate the disease. However, monitoring of vaccinated poultry flocks for H5N1 infection by serological means has been difficult, as vaccine antibodies are not readily distinguishable from those induced by field viruses. Therefore, a test that differentiates infected and vaccinated animals (DIVA) would be essential. Currently, no simple and specific DIVA test is available for screening of a large number of vaccinated chickens. Several epitopes on E29 domain of the haemagglutinin H5N1 subunit 2 (HA2) have recently been examined for their antigenicity and potential as possible markers for DIVA in chicken. In this study, the potential of E29 as an antigen for DIVA was evaluated in detail. Three different forms of full-length E29 peptide, a truncated E29 peptide (E15), and a recombinant E29 were compared for their ability to detect anti-E29 antibodies. Preliminary ELISA experiments using mono-specific chicken and rabbit E29 sera, and a mouse monoclonal antibody revealed that the linear E29 peptide was the most antigenic. Further examination of the E29 antigenicity in ELISA, using several sera from experimentally infected or vaccinated chickens, revealed that the full-length E29 peptide had the greatest discrimination power between infected and vaccinated chicken sera while providing the least non-specific reaction. This study demonstrates the usefulness of the HPAI H5N1 HA2 E29 epitope as a DIVA antigen in HPAI H5N1-vaccinated and -infected chickens.RESEARCH HIGHLIGHTS E29 (HA2 positions 488-516) epitope is antigenic in chickens.Antibodies to E29 are elicited following live H5N1 virus infection in chickens.E29 epitope is a potential DIVA antigen for use in ELISA.

De novo NAD synthesis is required for intracellular replication of Coxiella burnetii, the causative agent of the neglected zoonotic disease Q fever.

Coxiella burnetii is an intracellular Gram-negative bacterium responsible for the important zoonotic disease Q fever. Improved genetic tools and the ability to grow this bacterium in host cell-free media has advanced the study of C. burnetii pathogenesis, but the mechanisms that allow it to survive inside the hostile phagolysosome remain incompletely understood. Previous screening of a transposon mutant library for replication within HeLa cells has suggested that nadB, encoding a putative l-aspartate oxidase required for de novo NAD synthesis, is needed for intracellular replication. Here, using genetic complementation of two independent nadB mutants and intracellular replication assays, we confirmed this finding. Untargeted metabolite analyses demonstrated key changes in metabolites in the NAD biosynthetic pathway in the nadB mutant compared with the WT, confirming the involvement of NadB in de novo NAD synthesis. Bioinformatic analysis revealed the presence of a functionally conserved arginine residue at position 275. Using site-directed mutagenesis to substitute this residue with leucine, which abolishes the activity of Escherichia coli NadB, and expression of WT and R275L GST-NadB fusion proteins in E. coli JM109, we found that purified recombinant WT GST-NadB has l-aspartate oxidase activity and that the R275L NadB variant is inactive. Complementation of the C. burnetii nadB mutant with a plasmid expressing this inactive R275L NadB failed to restore replication to WT levels, confirming the link between de novo NAD synthesis and intracellular replication of C. burnetii This suggests that targeting this prokaryotic-specific pathway could advance the development of therapeutics to combat C. burnetii infections.

Autoimmune-Disease-Prone NOD Mice Help To Reveal a New Genetic Locus for Reducing Pulmonary Disease Caused by Mycoplasma pulmonis.

Mycoplasmas are bacterial pathogens of a range of animals, including humans, and are a common cause of respiratory disease. However, the host genetic factors that affect resistance to infection or regulate the resulting pulmonary inflammation are not well defined. We and others have previously demonstrated that nonobese diabetic (NOD) mice can be used to investigate disease loci that affect bacterial infection and autoimmune diabetes. Here we show that NOD mice are more susceptible than C57BL/6 (B6) mice to infection with Mycoplasma pulmonis, a natural model of pulmonary mycoplasmosis. The lungs of infected NOD mice had higher loads of M. pulmonis and more severe inflammatory lesions. Moreover, congenic NOD mice that harbored different B6-derived chromosomal intervals enabled identification and localization of a new mycoplasmosis locus, termed Mpr2, on chromosome 13. These congenic NOD mice demonstrated that the B6 allele for Mpr2 reduced the severity of pulmonary inflammation caused by infection with M. pulmonis and that this was associated with altered cytokine and chemokine concentrations in the infected lungs. Mpr2 also colocalizes to the same genomic interval as Listr2 and Idd14, genetic loci linked to listeriosis resistance and autoimmune diabetes susceptibility, respectively, suggesting that allelic variation within these loci may affect the development of both infectious and autoimmune disease.

Characterisation of the antigenic epitopes in the subunit 2 haemagglutinin of avian influenza virus H5N1.

Monitoring avian influenza (AI) infection and detecting silent infection in vaccinated chickens has been challenging due to the lack of effective serological diagnostic assays to differentiate between vaccinated and infected animals. Very few studies have identified suitable proteins in AI virus that can be used in successfully differentiating infected from vaccinated animals (DIVA). An HA2 peptide: HA2 position 197-201 (HA position 488-516) described by Khurana et al. (J Virol 85(23):12455-12463, 2011), was shown to have DIVA ability by differentiating H5N1-infected human sera in ELISA. In order to explore the capacity of the HA2 protein, as a DIVA reagent in chickens, four overlapping recombinant HA2 proteins, were expressed in E. coli and tested for reaction with H5N1 sera obtained from infected and vaccinated chickens. Recombinant protein HA2_B2 (380-461) was able to generate a detectable reaction with both H5N1 infected and vaccinated chicken sera but recombinant protein HA2_B4 (483-565) reacted strongly only with sera obtained from chickens infected with live virus, confirming its suitability as a DIVA antigen. Further analysis of the HA2 using several overlapping peptides suggested that positions 380-461 and 483-565 were antigenic in mouse and chicken. This study, for the first time, identified novel antigenic epitopes on the H5N1 HA2 subunit. Two epitopes, found in the HA2 ectodomain, have never been described for AIV infection in any animal species. Also one HA2 epitope was found to have high potential as a DIVA antigen.

Mycoplasma bovis antibody dynamics in naturally exposed dairy calves according to two diagnostic tests.

BACKGROUND: Inexpensive and convenient diagnostic tests for use in clinical work and for the surveillance of infection with Mycoplasma bovis are in demand. The objective of this longitudinal field study was to gain knowledge about the dynamics of antibodies against M. bovis in sera from naturally exposed calves with and without different clinical signs, measured by two different ELISA tests. RESULTS: A total of 83 calves were subject to between one and five blood samples and clinical examinations using a standard protocol during five herd visits to each of four outbreak dairy herds. The blood samples were analysed for the presence of antibodies against M. bovis using the commercial IgG ELISA test BioX K302 (BioX) and an in-house indirect IgG ELISA test (MilA ELISA). Linear mixed models were used to describe and compare the antibody dynamics as measured by the two tests in relation to the disease status and age of the animals. The BioX ELISA response was below the recommended cut-off (37 ODC%) for the entire study period in many of the calves. The estimated mean ODC% increased slowly but did not reach the recommended individual animal cut-off in three of the four herds. The highest estimated ODC% was not reached until the calf was 110-130 days old. The MilA ELISA response rose above the recommended cut-off (135 antibody units (AU)) in almost all calves, and in two herds, the estimated mean was above the individual animal cut-off shortly after the birth of the calf. The highest estimated antibody concentration was reached when the calf was approximately 60 days old. Disease status of the calf was not significantly associated with the results of either test. CONCLUSIONS: We conclude that the BioX ELISA cannot be recommended for use in calves below 3 months of age. The MilA ELISA was able to detect antibodies shortly after birth (i.e. from approximately 3 weeks of age and onwards) and is therefore a more sensitive test for M. bovis exposure in young calves. Neither ELISA seemed able to differentiate between calves with arthritis and/or otitis media, and respiratory disease.

Immune responses to vaccination and infection with Mycoplasma gallisepticum in turkeys.

Infection with Mycoplasma gallisepticum induces severe lymphoproliferative lesions in multiple sites along the respiratory tract in chickens and turkeys. These immunopathological responses have been well-characterized in chickens, but have not been studied closely in turkeys. The aim of the study described here was to examine the immune responses of turkeys after live vaccination and infection with M. gallisepticum. In a strain comparison study, the mean log10 antibody titre of birds exposed to an aerosol culture of M. gallisepticum strain Ap3AS was found to be significantly higher at day 14 than that of birds exposed to strain 100809/31. In a dose-response study, there was a significant difference in the mean log10 antibody titre between birds exposed to mycoplasma broth and birds exposed to the highest dose of strain Ap3AS at day 7 after exposure. Immunohistochemical analysis of the tracheal mucosa and the air sacs revealed similar patterns of distribution of CD4+ and CD8+ lymphocytes to those seen in the tracheal mucosa of chickens, implicating these cell types in the pathogenesis of respiratory mycoplasmosis in turkeys. Turkeys that had been vaccinated with M. gallisepticum GapA+ ts-11 had significantly higher antibody titres than unvaccinated birds at both 7 and 14 days after challenge with strain Ap3AS. Vaccination with GapA+ ts-11 protected against the lymphoproliferative response to infection with virulent M. gallisepticum in both the tracheal mucosa and the air sacs, suggesting that this strain may be a useful vaccine candidate for use in turkeys.

Evaluation of an IgG Enzyme-Linked Immunosorbent Assay as a Serological Assay for Detection of Mycoplasma bovis Infection in Feedlot Cattle.

Mycoplasma bovis is a pathogen of emerging significance in cattle throughout the world that is causing a range of diseases, including mastitis, arthritis, and pneumonia. The limited availability and efficacy of current diagnostic and prophylactic tools for its control and its increasing antimicrobial resistance are contributing to its increasing importance in beef and dairy cattle. We have developed an indirect IgG enzyme-linked immunosorbent assay (ELISA) based on a recombinant fragment of the MilA protein and have shown its potential as an effective diagnostic tool. To more comprehensively estimate the diagnostic sensitivity and specificity of this IgG ELISA for detection of infection with M. bovis in cattle and to define a suitable cutoff for use in the field, we further assessed its performance in experimentally infected calves in a closed beef herd and by applying Bayesian latent class modeling to laboratory testing results from 7,448 cattle entering Australian feedlots. The most effective cutoff points were estimated to be 68.6 antibody units (AU) for experimentally infected calves and to be 58.7 AU for a closed adult herd. Under field conditions, in feedlot cattle the globally optimal cutoff was estimated to be 105 AU. At this cutoff, the diagnostic sensitivity was 94.3% (95% probability interval [PI], 89.9% to 99.6%) with a diagnostic specificity of 94.4% (95% PI, 90.3% to 99.6%). Applying this 105 AU cutoff, 13.1% of cattle were seropositive for infection with M. bovis on entry into feedlots, and 73.5% were seropositive when followed up approximately 6 weeks later suggesting a high risk of infection shortly after entry into feedlots.

Evaluation of a conserved HA274-288 epitope to detect antibodies to highly pathogenic avian influenza virus H5N1 in Indonesian commercial poultry.

A peptide enzyme linked immunosorbent assay (ELISA) based on an epitope in the haemagglutinin (HA) of avian influenza virus H5N1, amino acid positions 274-288 (HA274-288) was evaluated for detection of H5N1-specific antibodies. An optimized ELISA based on the tetrameric form of the HA274-288 epitope designated MP15 gave low background with non-immune chicken sera and detected vaccinated and infected birds. The HA274-288 epitope was highly conserved in Indonesian H5N1 strains and antibody responses were detected in the majority of the vaccinated chickens regardless of the H5N1 strain used for vaccination. The HA274-288 epitope was also conserved in the majority of H5N1 strains from the neighbouring Asian region, and other H5 subtypes potentially allowing for a wider use of the MP15 ELISA in H5N1 vaccinated and infected flocks. The MP15 ELISA results correlated significantly with haemagglutination inhibition (HI) test results and test sensitivity and specificity were 87% and 92%, respectively. The MP15 ELISA titres were significantly higher than the HI titres in all immune sera allowing for sera to be tested at a single dilution of 1:400 which is of advantage in routine surveillance. The study indicated that the MP15 ELISA is potentially useful for serological detection of H5N1 vaccinated or infected poultry and to have some advantages over the standard HI test for routine monitoring of flocks' immunity after vaccination.

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.

Evaluation of the safety and efficacy of the novel Mycoplasma gallisepticum vaccine, Vaxsafe MG304, after spray-vaccination of 1-day-old specific pathogen-free chicks.

Mycoplasma gallisepticum causes chronic respiratory disease in poultry. A novel vaccine, Vaxsafe MG304 (the ts-304 strain), has greater protective efficacy in chickens than the Vaxsafe MG (strain ts-11) vaccine when delivered by eye drop at 3 weeks of age. Applying this vaccine in the hatchery to 1-day-old birds, using mass administration methods, would improve animal welfare and reduce labour costs associated with handling individual birds. This study assessed the protection provided by vaccination with Vaxsafe MG304 after administration to 1-day-old chicks. Chicks were administered a single dose of the vaccine to assess the efficacy of either a high dose (107.0 colour changing units, CCU) or a low dose (105.7 CCU) after eye drop or spray (in water or gel) administration against experimental challenge with virulent M. gallisepticum strain Ap3AS at 7 weeks of age. The vaccine was able to colonise the palatine cleft of chicks after vaccination by eye drop (at both doses) or by spray (in water or gel) (at the high dose). The high dose of vaccine, when delivered by eye drop or spray, was shown to be safe and induced a serological response and protective immunity (as measured by tracheal mucosal thickness and air sac lesion scores) against challenge. Vaccination of 1-day-old chicks with Vaxsafe MG304 by eye drop induced protective immunity equivalent to vaccination at 3 weeks of age. Vaxsafe MG304 was also protective when applied by both coarse- and gel spray methods at the higher dose and is therefore a suitable live attenuated vaccine for use in 1-day-old chicks.

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.

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.

Efficient disruption of the function of the mnuA nuclease gene using the endogenous CRISPR/Cas system in Mycoplasma gallisepticum.

Mycoplasmas are important animal pathogens, but the functions and roles of many of their genes in pathogenesis remain unclear, in large part because of the limited tools available for targeted mutagenesis in these bacteria. In this study we used the Mycoplasma gallisepticum CRISPR/Cas system to target a nuclease gene, MGA_0637 (mnuA), which is predicted to play a role in survival and virulence. Our strategy used simultaneous targeting of the ksgA kasugamycin resistance gene, as a mutation in this gene would not interfere with replication but would confer a readily detectable and selectable phenotype in transformants. A guide RNA plasmid, pKM-CRISPR, was constructed, with spacers targeting the ksgA and mnuA genes transcribed under the control of the vlhA1.1 promoter in a backbone plasmid carrying the oriC of M. imitans, and this plasmid was introduced into electrocompetent M. gallisepticum strain S6 cells. PCR assays targeting the ksgA gene, followed by Sanger sequence analyses of the phenotypically resistant transformants, detected polymorphisms within the targeted region of ksgA, confirming the activity of the endogenous CRISPR/Cas system. The nuclease activity of the kasugamycin resistant colonies was then assessed using zymogram assays. The complete or partial loss of nuclease activity in the majority of kasugamycin resistant isolates transformed with the CRISPR plasmid confirmed that the endogenous CRISPR/Cas system had effectively interfered with the function of both ksgA and mnuA genes. Sanger sequencing and RT-qPCR analyses of the mnuA gene suggested that the M. gallisepticum CRISPR/Cas system can be programmed to cleave both DNA and RNA.