Year

2014

Degree Name

Doctor of Philosophy

Department

School of Biological Sciences

Abstract

Mycoplasma hyopneumoniae is the causal agent of mycoplasmal pneumonia; a mild chronic respiratory disease of pigs that is characterised by a dry, non-productive cough and the sequential development of microscopic and macroscopic lesions of the respiratory tract and lung. Strains of M. hyopneumoniae vary in pathogenicity; however, a major driver of lung pathology is the host immune response to infection. Although the underlying inflammatory mechanisms are ill-defined, secretion of pro-inflammatory cytokines is purported to play a role in host-mediated lung tissue damage. Enhanced activation of M. hyopneumoniae surface-bound plasminogen to plasmin, a potent stimulus and chemoattractant of pro-inflammatory cells, also raises the question of whether pathogen-exploitation of the host plasminogen activation system contributes to pathogenicity. Commercial inactivated whole-cell preparations (bacterins) can reduce clinical signs of disease and regulate the secretion of pro-inflammatory cytokines, but they do not prevent the colonisation of swine by M. hyopneumoniae, thus necessitating alternative vaccination strategies. A prerequisite for M. hyopneumoniae pathogenesis is adherence to ciliated respiratory epithelia; a multifactorial process involving an array of adhesins including those belonging to the P97/P102 paralog family. Of the P97/P102 paralog family of proteins, only P97 has been investigated as a potential vaccine candidate. Whilst partial protection against lung pathology was observed in vaccinated pigs, it was hypothesised that administration of multiple antigens from the P97/P102 paralog family may result in a superior level of protection against M. hyopneumoniae infection.

In this study, two Australian M. hyopneumoniae field strains (Hillcrest and Beaufort) were compared for their ability to induce mycoplasmal pneumonia. The optimum strain for use in a subsequent vaccine trial was determined by combining clinical observations with pathologic and histopathologic examination of tissue samples from challenged pigs. Pig serological and mucosal responses to recombinant subunit vaccines containing functional domains of eight of the P97/P102 paralogs formulated with Alhydrogel® or Montanide™ Gel01 were then compared with a commercial bacterin (Suvaxyn® M. hyo) following experimental challenge with the more virulent of the two field isolates. Cytokine levels (interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α) in tracheobronchial lavage fluid (TBLF) were assessed and a M. hyopneumoniae-specific quantitative PCR assay confirmed the presence of mycoplasmal DNA in the TBLF of challenged pigs. Damage to the ciliated respiratory epithelium of challenged pigs was evaluated by scanning electron microscopy of fixed tracheal samples collected six weeks post-infection (wpi) with the Hillcrest strain. Finally, TBLF collected from Suvaxyn®-vaccinated and unvaccinated pigs were examined for plasmin activity and these results were correlated with individual cytokine responses and bacterial load data.

The results of the challenge study indicated that a single dose of the Hillcrest strain induced significantly higher coughing scores and gross and histological lesions symptomatic of mycoplasmal pneumonia during the first 3-4 weeks after challenge, relative to the less virulent Beaufort strain. As such, M. hyopneumoniae Hillcrest was employed as a challenge strain in the subsequent vaccine trial. While mycoplasmal pneumonia was successfully induced during the vaccine trial using the Hillcrest strain, the vaccine formulations varied in their efficacy in protecting against disease. Despite inducing significant systemic immune responses, the Alhydrogel® and Montanide™ Gel01 formulations failed to stimulate significant post-vaccination mucosal immunity, and were unable to regulate the secretion of IL-1β, IL-6 and TNF-α in the porcine airways in response to M. hyopneumoniae challenge. In contrast, Suvaxyn® M. hyo administration induced significant mucosal immunity and regulated the secretion of proinflammatory cytokines, resulting in superior reduction of clinical signs of disease and bacterial load in the porcine airways relative to immunisation with the recombinant formulations. The Alhydrogel® and Montanide™ Gel01 formulations did, however, offer comparable protection against damage to epithelial cilia. Plasmin activity in the porcine airways was significantly enhanced in unvaccinated pigs, but not Suvaxyn® M. hyo vaccinates, during experimental infection, and was positively correlated with IL-1β, IL-6 and TNF-α responses and bacterial load in the TBLF of challenged animals. In conclusion this study highlights the impact of M. hyopneumoniae strain virulence on the course of clinical disease, and the importance of selecting an appropriate strain for use in experimental challenge systems to evaluate vaccine efficacy. The results also emphasize the importance of pro-inflammatory cytokine responses and plasmin activity as mediators of lung tissue damage, and illustrate that plasminogen activation in the porcine respiratory tract is ameliorated by administration of the partially protective bacterin Suvaxyn® M. hyo. Finally, this study highlights the need for further research into the P97/P102 paralogs as protective antigens and optimisation of vaccine strategies that will induce protective immunity against M. hyopneumoniae infection.

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