Veterinary Biosciences - Research Publications
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ItemVirulence factors of Mycoplasma synoviae: Three genes influencing colonization, immunogenicity, and transmissibility(FRONTIERS MEDIA SA, 2022-11-25)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.
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ItemDifferential Response of the Chicken Trachea to Chronic Infection with Virulent Mycoplasma gallisepticum Strain Ap3AS and Vaxsafe MG (Strain ts-304): a Transcriptional Profile(AMER SOC MICROBIOLOGY, 2020-05)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.
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ItemEffects of immunosuppression on the efficacy of vaccination against Mycoplasma gallisepticum infection in chickens(ELSEVIER, 2021-09)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.
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ItemTranscriptomic Analysis of Long-Term Protective Immunity Induced by Vaccination With Mycoplasma gallisepticum Strain ts-304(FRONTIERS MEDIA SA, 2021-02-02)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.
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ItemA novel transposon construct expressing PhoA with potential for studying protein expression and translocation in Mycoplasma gallisepticum(BMC, 2012-07-08)BACKGROUND: Mycoplasma gallisepticum is a major poultry pathogen and causes severe economic loss to the poultry industry. In mycoplasmas lipoproteins are abundant on the membrane surface and play a critical role in interactions with the host, but tools for exploring their molecular biology are limited. RESULTS: In this study we examined whether the alkaline phosphatase gene (phoA ) from Escherichia coli could be used as a reporter in mycoplasmas. The promoter region from the gene for elongation factor Tu (ltuf) and the signal and acylation sequences from the vlhA 1.1 gene, both from Mycoplasma gallisepticum , together with the coding region of phoA , were assembled in the transposon-containing plasmid pISM2062.2 (pTAP) to enable expression of alkaline phosphatase (AP) as a recombinant lipoprotein. The transposon was used to transform M. gallisepticum strain S6. As a control, a plasmid containing a similar construct, but lacking the signal and acylation sequences, was also produced (pTP) and also introduced into M. gallisepticum . Using a colorimetric substrate for detection of alkaline phosphatase activity, it was possible to detect transformed M. gallisepticum . The level of transcription of phoA in organisms transformed with pTP was lower than in those transformed with pTAP, and alkaline phosphatase was not detected by immunoblotting or enzymatic assays in pTP transformants, eventhough alkaline phosphatase expression could be readily detected by both assays in pTAP transformants. Alkaline phosphatase was shown to be located in the hydrophobic fraction of transformed mycoplasmas following Triton X-114 partitioning and in the membrane fraction after differential fractionation. Trypsin proteolysis confirmed its surface exposure. The inclusion of the VlhA lipoprotein signal sequence in pTAP enabled translocation of PhoA and acylation of the amino terminal cysteine moiety, as confirmed by the effect of treatment with globomycin and radiolabelling studies with [14C]palmitate. PhoA could be identified by mass-spectrometry after separation by two-dimensional electrophoresis. CONCLUSION: This is the first study to express PhoA as a lipoprotein in mycoplasmas. The pTAP plasmid will facilitate investigations of lipoproteins and protein translocation across the cell membrane in mycoplasmas, and the ease of detection of these transformants makes this vector system suitable for the simultaneous screening and detection of cloned genes expressed as membrane proteins in mycoplasmas.
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ItemEvaluation of the Capacity of PCR and High-Resolution Melt Curve Analysis for Identification of Mixed Infection with Mycoplasma gallisepticum Strains(PUBLIC LIBRARY SCIENCE, 2015-05-13)Pathogenicity and presentation of Mycoplasma gallisepticum (MG) infection may differ from one strain to another and this may have implications on control measures. Infection of individual birds with more than one MG strain has been reported. A PCR followed by high resolution melt (HRM) curve analysis has been developed in our laboratory and routinely used for detection and differentiation of MG strains. However the potential of this test for identification of MG strains in a mixed specimen has not been evaluated. In the present study, the capability of PCR-HRM curve analysis technique, targeting vlhA and pvpA genes was assessed for identification of individual MG strains in a mixed population. Different DNA ratios of two MG strains from 1 to 10(-4) ng were tested with some generated conventional and normalized curves distinct from those of individual strains alone. Using genotype confidence percentages (GCP) generated from HRM curve analysis, it was found that vlhA PCR-HRM was more consistent than pvpA PCR-HRM for the detection of MG ts-11 vaccine strain mixed with any of the MG strains 6/85, F, S6 or a field isolate. The potential of vlhA PCR-HRM to detect mixed MG strains in a specimen was found to be primarily dependent on quantity and proportion of the target DNAs in the mixture. This is the first study examining the capacity of PCR-HRM technique for identification of individual MG strains in a mixed strain population.
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ItemThe performance of three immune assays to assess the serological status of cattle experimentally exposed to Mycoplasma bovis(MDPI, 2018-03-08)© 2018 by the authors. Mycoplasma bovis is associated with several clinical syndromes of cattle. Currently, limited information is available on the sensitivity (Se) and specificity (Sp) of serological assays used for the detection of M. bovis-specific antibodies. Consequently, it is difficult to critically evaluate the outcomes of studies that use these assays. Therefore, the current study used bovine sera sourced from M. bovis exposure studies from three countries to estimate the Se and Sp of two commercial M. bovis enzyme-linked immunosorbent assays (ELISA), BIO K302 and BIO K260, and Western blotting. Western blotting had the highest Se estimate of 74% (95% confidence interval (CI): 16-98%), compared to the BIO K302: 47% (95% CI: 10-87%) and BIO K260: 28% (95% CI: 1-92%). However, for Sp, the BIO K302: 96% (95% CI: 87-99%) and the BIO K260: 100% (95% CI: 93-100%) out-performed Western blotting: 88% (95% CI: 56-98%). Western blotting was the best assay for detecting seroconversion, correctly identifying 61% (95% CI: 29-86%) of exposed animals compared to 35% for BIO K302 (95% CI: 21-54%) and 8% for BIO K260 (95% CI: 0-87%). While none of the methods assessed had high Se and Sp, the availability of these estimates will aid in the interpretation of studies that use these assays. The results of this study highlight the difficulties encountered when using serology to detect exposure to M. bovis in cattle.
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ItemReproduction of respiratory mycoplasmosis in calves by exposure to an aerosolised culture of Mycoplasma bovis(Elsevier, 2017-10-01)© 2017 Mycoplasma bovis is an important pathogen of cattle, causing pneumonia, arthritis and otitis media in young calves, and mastitis in lactating cows, resulting in increased morbidity and, in some instances, mortality. The objective of this study was to evaluate the survival of a M. bovis isolate following nebulisation and to establish whether respiratory disease similar to that seen in the field could be induced in calves by exposing them to an aerosolised culture of M. bovis. A group of eight M. bovis-free calves 14–28 days old were exposed to an aerosolised culture of a field isolate of M. bovis that had originally been recovered from a joint lesion in a calf. Three weeks after aerosol exposure necropsies were conducted on all calves. Lung lesions were seen in 7 of 8 calves exposed to the aerosol of M. bovis, whilst calves exposed to the culture medium alone did not develop lesions. Two calves in the infected group had detectable concentrations of serum antibody against M. bovis on day 7 post infection and 4 calves had detectable concentrations of serum antibody against M. bovis on day 21 post infection when tested by MilA IgG ELISA. M. bovis was reisolated from the upper trachea of 6 of the 8 infected calves. The infection method described here appeared to induce lung lesions typical of naturally occurring disease associated with infection with M. bovis and should be applicable to testing the safety and efficacy of attenuated vaccine candidates to control disease caused by this pathogen.
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ItemNo Preview AvailableHistochemical and morphometric characterization of broncho-pneumonia in calves caused by infection with Mycoplasma bovis(Elsevier, 2012-07-06)The aim of this study was to identify morphometric histological features of pneumonia caused by Mycoplasma bovis in calves. Eight three-month-old calves were infected with M. bovis and samples of their lung tissue, three weeks after exposure, compared to samples from four uninfected calves. In the uninfected animals the goblet cells were clustered in the crypt area of the epithelial folds, while in the infected calves they had migrated towards the tips of the folds and were distributed evenly throughout the folds. In infected lung tissue there was goblet cell hyperplasia and metaplasia in the bronchioles and an increased epithelial height. Goblet cell mucin in uninfected calves was acidic, but in infected calves most goblet cells contained neutral mucins. These morphometric and histochemical bronco-epithelial changes may be able to be used as markers of the severity of bovine respiratory mycoplasmosis. © 2012 Elsevier B.V.
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ItemEvaluation of an IgG enzyme-linked immunosorbent assay as a serological assay for detection of mycoplasma bovis infection in feedlot cattle(American Society for Microbiology, 2016-05-01)Copyright © 2016, American Society for Microbiology. All Rights Reserved. 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.