Genetic Manipulation of Campylobacter hepaticus and the Role of N-glycosylation in Spotty Liver Disease
Creators: name: McDonald, Jamieson
Publisher: name: RMIT University
Year: 2025
Subjects: classificationCode: 300304
subject: Animal protection (incl. pests and pathogens)
classificationCode: 300903
subject: Veterinary bacteriology

description: Spotty Liver disease (SLD) is a poultry disease with global economic and animal welfare implications. Free-range hens infected with the causative organism Campylobacter hepaticus can experience decreased egg production and, in severe cases, mortality. The global demand for egg production has rapidly increased in the past three decades and continues to rise. Thus, the egg production industry needs new intervention strategies in the form of vaccines to reduce the incidence and spread of SLD.

At the commencement of this thesis's work, knowledge of C. hepaticus pathogenicity, key virulence factors, and vaccination prospects were limited. While several genes had been identified to be suspected key virulence factors, their functional roles in the development of SLD had not been characterised due to the lack of available genetic tools and methods for mutagenesis studies in this emerging pathogen. Consequently, targeted intervention strategies in the form of vaccines against C. hepaticus remained largely unexplored. This thesis aims to develop genetic tools and methods to identify and characterise C. hepaticus virulence factors that contribute to the pathogenesis of SLD to aid in vaccine development against SLD.

The functionality of the C. hepaticus N-glycosylation system was confirmed by glycoproteomic analysis (Chapter 2). Proteins were found to be modified with an N-linked heptasaccharide. A large proportion of these glycoproteins were highly conserved with Campylobacter jejuni, playing critical roles in host colonisation. Some glycoproteins were unique to C. hepaticus, possibly required for niche adaptation and survival. Importantly, this chapter provided the first evidence that C. hepaticus N-glycosylation may function as an essential virulence factor but lacked the genetic tools to functionally characterise the individual genes involved in the system.

Chapter 3 describes the development of genetic tools and methods for the genetic manipulation of C. hepaticus. These tools were used to investigate the pglB gene and its product's role in the N-glycosylation of proteins. This chapter reports the first successful generation of targeted genetic mutants in C. hepaticus, specifically pglB, which abolished C. hepaticus’s ability to N-glycosylate proteins. Successful complementation of the pglB mutant demonstrated the value of the developed tools and provided direct evidence of the role of PglB in protein N-glycosylation. More importantly, this work provides the methods and tools to characterise other suspected virulence factors involved in SLD pathogenesis.

Chapter 4 characterises, validates, and assesses the immunological response of a novel S. Typhimurium vaccine engineered to express the Campylobacter heptasaccharide as a part of S. Typhimurium endogenous LPS. Administration of the vaccine strain demonstrated sufficient attenuation of the live vector and significantly increased IgY antibody titres specific to the Campylobacter heptasaccharide antigen. Introduction of the heterologous heptasaccharide into S. Typhimurium endogenous LPS had not significantly impacted IgY antibody responses raised against S. Typhimurium, highlighting the vaccine's bi-valent immunological potential to vaccinate against two common pathogens of free-range poultry.

In conclusion, this thesis presents a systematic approach from identifying a suspected key virulence factor of C. hepaticus to its functional investigation and translational application in vaccine development. The work presented significantly advances our understanding of C. hepaticus N-glycosylation. The novel tools and methods developed during this study have fundamental implications for investigating other suspected key virulence determinants in C. hepaticus, their role in SLD pathogenesis, and in developing targeted vaccine control strategies against this economically significant poultry disease.