ABSTRACT
The live attenuated temperature sensitive vaccine strain MS-H (Vaxsafe® MS, Bioproperties Pty. Ltd., Australia) is widely used to control disease associated with M. synoviae infection in commercial poultry. MS-H was derived from a field strain (86079/7NS) through N-methyl-N'-nitro-N-nitrosoguanidine (NTG)-induced mutagenesis. Whole genomic sequence analysis of the MS-H and comparison with that of the 86079/7NS have found that MS-H contains 32 single nucleotide polymorphisms (SNPs). Three of these SNPs, found in the obgE, oppF and gapdh genes, have been shown to be prone to reversion under field condition, albeit at a low frequency. Three MS-H reisolates containing the 86079/7NS genotype in obgE (AS2), obgE and oppF (AB1), and obgE, oppF and gapdh (TS4), appeared to be more immunogenic and transmissible compared to MS-H in chickens. To investigate the influence of these reversions in the in vitro fitness of M. synoviae, the growth kinetics and steady state metabolite profiles of the MS-H reisolates, AS2, AB1 and TS4, were compared to those of the vaccine strain. Steady state metabolite profiling of the reisolates showed that changes in ObgE did not significantly influence the metabolism, while changes in OppF was associated with significant alterations in uptake of peptides and/or amino acids into the M. synoviae cell. It was also found that GAPDH plays a role in metabolism of the glycerophospholipids as well as an arginine deiminase (ADI) pathway. This study underscores the role of ObgE, OppF and GAPDH in M. synoviae metabolism, and suggests that the impaired fitness arising from variations in ObgE, OppF and GAPDH contributes to attenuation of MS-H.
Subject(s)
Mycoplasma Infections , Mycoplasma synoviae , Poultry Diseases , Animals , Mycoplasma Infections/veterinary , Mycoplasma synoviae/genetics , Chickens , Mutation , Mutagenesis , Vaccines, Attenuated/genetics , Poultry Diseases/prevention & controlABSTRACT
Infections caused by Mycoplasma synoviae are major welfare and economic concerns in poultry industries worldwide. These infections cause chronic respiratory disease and/or synovitis in chickens and turkeys leading to reduced production and increased mortality rates. The live attenuated vaccine strain MS-H (Vaxsafe® MS), commonly used for protection against M. synoviae infection in many countries, contains 32 single nucleotide variations compared to its wildtype parent strain, 86079/7NS. Genomic analysis of vaccine strains reisolated from flocks following the administration of MS-H has identified reversions to the original 86079/7NS sequence in the obgE, oppF and gapdh genes. Here, three MS-H field reisolates containing the 86079/7NS genotype in obgE (AS2), obgE and oppF (AB1), and obgE, oppF and gapdh (TS4), as well as the vaccine MS-H and the parental strain 86079/7NS were experimentally inoculated to chickens. The strains were assessed for their ability to infect and elicit immune responses in the recipient chickens, as well as in naïve in-contact chickens. Despite the loss of temperature sensitivity phenotype and colonization of the reisolates in the lower respiratory tract, there was no significant differences detected in the microscopic mucosal thickness of the middle or lower trachea of the inoculated chickens. Concurrent reversions in ObgE, OppF and GAPDH proteins were associated with higher gross air sac lesion scores and increased microscopic upper-tracheal mucosal thickness in chickens directly inoculated with the reisolates following intratracheal administration of a virulent strain of infectious bronchitis virus. The gross air sac lesions of the chickens in-contact with those inoculated with reisolates were not significantly different to those of chickens in-contact with MS-H inoculated chickens, suggesting that horizontal transmission of the reisolates in the poultry flock will not lead to higher pathogenicity or clinical signs. These results suggest a significant role of GAPDH and/or cumulative effect of ObgE, OppF and GAPDH on M. synoviae pathogenicity. Future experiments will be required to investigate the effect of single mutations in gapdh or oppF gene on pathogenicity of M. synoviae.
ABSTRACT
The Mycoplasma synoviae live attenuated vaccine strain MS-H (Vaxsafe MS; Bioproperties Pty., Ltd., Australia) is commonly used around the world to prevent chronic infections caused by M. synoviae in birds and to minimize economic losses in the poultry industry. MS-H is a temperature-sensitive strain that is generated via the chemical mutagenesis of a virulent M. synoviae isolate, 86079/7NS. 32 single nucleotide polymorphisms have been found in the genome of MS-H compared to that of 86079/7NS, including 25 in predicted coding sequences (CDSs). There is limited information on the stability of these mutations in MS-H in vitro during the propagation of the vaccine manufacturing process or in vivo after the vaccination of chickens. Here, we performed a comparative analysis of MS-H genomes after in vitro and in vivo passages under different circumstances. Studying the dynamics of the MS-H population can provide insights into the factors that potentially affect the health of vaccinated birds. The genomes of 11 in vitro laboratory passages and 138 MS-H bird reisolates contained a total of 254 sequence variations. Of these, 39 variations associated with CDSs were detected in more than one genome (range = 2 to 62, median = 2.5), suggesting that these sequences are particularly prone to mutations. From the 25 CDSs containing previously characterized variations between MS-H and 86079/7NS, 7 were identified in the MS-H reisolates and progenies examined here. In conclusion, the MS-H genome contains individual regions that are prone to mutations that enable the restoration of the genotype or the phenotype of wild-type 86079/7NS in those regions. However, accumulated mutations in these regions are rare. IMPORTANCE Preventative measures, such as vaccination, are commonly used for the control of mycoplasmal infections in poultry. A live attenuated vaccine strain (Vaxsafe MS; MS-H; Bioproperties Pty. Ltd., Australia) is used for the prevention of disease caused by M. synoviae in many countries. However, information on the stability of previously characterized mutations in the MS-H genome is limited. In this study, we performed a comparative analysis of the whole-genome sequences of MS-H seeds used for vaccine manufacturing, commercial batches of the vaccine, cultures minimally passaged under small-scale laboratory and large-scale manufacturing conditions, MS-H reisolated from specific-pathogen-free (SPF) chickens that were vaccinated under controlled conditions, and MS-H reisolated from vaccinated commercial poultry flocks around the world. This study provides a comprehensive assessment of genome stability in MS-H after in vitro and in vivo passages under different circumstances and suggests that most of the mutations in the attenuated MS-H vaccine strain are stable.
Subject(s)
Mycoplasma synoviae , Poultry Diseases , Animals , Vaccines, Attenuated/genetics , Chickens , Bacterial Vaccines , Mycoplasma synoviae/genetics , Genomics , Poultry Diseases/prevention & controlABSTRACT
An amendment to this paper has been published and can be accessed via the original article.
ABSTRACT
BACKGROUND: Genomic comparison of Mycoplasma synoviae vaccine strain MS-H and the MS-H parental strain 86,079/7NS established a preliminary profile of genes related to attenuation of MS-H. In this study we aimed to identify the stability of mutations found in MS-H after passage in experimental or field chickens, and to evaluate if any reverse mutation may be associated with changes in characteristics of MS-H in vitro or in vivo. RESULTS: Whole genome sequence analysis of 5 selected MS-H field reisolates revealed that out of 32 mutations reported previously in MS-H, 28 remained stable, while four found to be reversible to the wild-type. Each isolate possessed mutations in one to three of the genes obg, oppF1 and gap and/or a non-coding region. Examination of the 4 reversible mutations by protein modeling predicted that only two of them (in obg and oppF1 genes) could potentially restore the function of the respective protein to that of the wild-type. CONCLUSIONS: These results suggest that the majority of the MS-H mutations are stable after passage in vaccinated chickens. Characterisation of stable mutations found in MS-H could be utilised to develop rapid diagnostic techniques for differentiation of vaccine from field strains or ts- MS-H reisolates.