Novel molecular markers of disease-association among strains of Streptococcus suis: a genomic approach

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Wileman, Thomas Mathew  ORCID logo

This thesis focuses on the use of a genomic approach to identify novel molecular markers to differentiate Streptococcus suis (S. suis) isolates into two populations, i) disease-associated and ii) non-disease associated. S. suis is a Gram-positive coccus that is considered one of the most important zoonotic bacterial pathogens of swine responsible for significant economic losses to swine production worldwide. Importantly, S. suis is not only an invasive pathogen but also a very successful coloniser of mucosal surfaces; often endemic in swine populations sampled.

The widescale use of antibiotics to control and prevent the various clinical manifestations caused by S.suis has become unsustainable, due to increases in antibiotic resistance and government pressures. Other popular control strategies, such as the development of efficacious vaccines, are hindered by differences in virulence not only between but also within S. suis serotypes, as well as, the lack of a detailed understanding of the role in pathogenesis of many proposed virulence-factors. As a result, the detection of S. suis in asymptomatic swine herds is of little practical value in predicting the likelihood of future clinical relevance.

This thesis aims to further understanding of the role the S. suis genome has in pathogenesis. The value of future surveillance and preventative health management lies in the detection of strains that genetically have increased potential to cause disease in presently healthy animals. The first results chapter of this thesis (chapter 3) describes the use of genome-wide associations studies, a so-far unexploited method for S. suis, to identify genetic markers associated with the observed clinical phenotype i) invasive disease or ii) asymptomatic carriage on the palatine tonsils of swine. Chapter 4 then describes the analyses used to select three genetic markers to pathotype S. suis - differentiate isolates of the same species based on their ability to cause disease; going on to describe the design and evaluation of a multiplex-PCR tool targeting the three newly defined "pathotyping markers" in comparison to existing methods used to characterise S. suis.

These findings were taken further by using the pathotyping markers to screen material scrapped from the palatine tonsils of swine with no obvious signs of streptococcal disease. This produced an interesting result - the production of both invasive disease-associated and non- disease associated multiplex-PCR amplicons from the same experimental sample. Unsurprising in itself, what was surprising is the frequency with which this observation was found. Picking single colonies from solid agar plates is a crippling bottleneck of existing S. suis diagnostics, and its removal has the potential to improve the sensitivity of surveillance and preventive health management programs. Chapter 5 describes investigation of this surprising observation and indicates that classic culture-based methods of detection are not sensitive enough to confidently report the presence (or absence) of invasive disease-associated S. suis strains.

This thesis concludes with the description of efforts to address the lack of a comprehensive understanding of S. suis virulence/'virulence-associated' factors. Chapter 6 describes the design of an isogenic mutant knocking out the invasive disease-associated pathotyping marker, SSU1589 (also known as virA). That is then evaluated in simple in vitro and in vivo experimental models in order to understand the role Type I restriction modification proteins have in S. suis pathogenesis.

In conclusion, this thesis furthers our understanding that differences in the S. suis genome are an important factor in S. suis pathogenesis, and describes the identification and evaluation of novel genetic markers for the detection and control of invasive disease-associated S. suis strains in intensive pig production systems.

Tucker, Alexander William
Maskell, Duncan John
Streptococcus suis, Pathotype, Virulence
Doctor of Philosophy (PhD)
Awarding Institution
University of Cambridge
This PhD research project was a BBSRC Knowledge Transfer Network (KTN) CASE studentship co-funded by Zoetis, a company committed to expanding its portfolio of diagnostic support to enable ongoing responsible use of antimicrobials in the UK and global pig industry. Significant contribution was also received from BQP Ltd, a major pig integrator focused on reducing streptococcal disease in their pig breeding system. This research also contributed to the BBSRC Longer and Larger (LoLa) project, grant number: BB/G019274/1, that had the aim to create single platform diagnostics and multivalent vaccines against Actinobacillus pleuropneumoniae, Haemophilus parasuis, Mycoplasma hyopneumoniae and Streptococcus suis - all important bacterial pathogens commonly recovered from the upper respiratory tract of swine.