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ABSTRACT Various species of campylobacters cause significant disease problems in both humans and animals. The continuing development of tools and methods for genetic and molecular manipulation of campylobacters enables the detailed study of bacterial virulence and disease pathogenesis. Campylobacter hepaticus is an emerging pathogen that causes spotty liver disease (SLD) in poultry. SLD has a significant economic and animal welfare impact as the disease results in elevated mortalities and significant decreases in egg production. Although potential virulence genes of C. hepaticus have been identified, they have not been further studied and characterized, as appropriate genetic tools and methods to transform and perform mutagenesis studies in C. hepaticus have not been available. In this study, the genetic manipulation of C. hepaticus is reported, with the development of novel plasmid vectors, methods for transformation, site-specific mutagenesis, and mutant complementation. These tools were used to delete the pglB gene, an oligosaccharyltransferase, a central enzyme of the N-glycosylation pathway, by allelic exchange. In the mutant strain, N-glycosylation was completely abolished. The tools and methods developed in this study represent innovative approaches that can be applied to further explore important virulence factors of C. hepaticus and other closely related Campylobacter species.IMPORTANCESpotty liver disease (SLD) of layer chickens, caused by infection with Campylobacter hepaticus, is a significant economic and animal welfare burden on an important food production industry. Currently, SLD is controlled using antibiotics; however, alternative intervention methods are needed due to increased concerns associated with environmental contamination with antibiotics, and the development of antimicrobial resistance in many bacterial pathogens of humans and animals. This study has developed methods that have enabled the genetic manipulation of C. hepaticus. To validate the methods, the pglB gene was inactivated by allelic exchange to produce a C. hepaticus strain that could no longer N-glycosylate proteins. Subsequently, the mutation was complemented by reintroduction of the gene in trans, on a plasmid vector, to demonstrate that the phenotypic changes noted were caused by the mutation of the targeted gene. The tools developed enable ongoing studies to understand other virulence mechanisms of this important emerging pathogen.

Various species of campylobacters cause significant disease problems in both humans and animals. The continuing development of tools and methods for genetic and molecular manipulation of campylobacters enables the detailed study of bacterial virulence and disease pathogenesis. Campylobacter hepaticus is an emerging pathogen that causes spotty liver disease (SLD) in poultry. SLD has a significant economic and animal welfare impact as the disease results in elevated mortalities and significant decreases in egg production. Although potential virulence genes of C. hepaticus have been identified, they have not been further studied and characterized, as appropriate genetic tools and methods to transform and perform mutagenesis studies in C. hepaticus have not been available. In this study, the genetic manipulation of C. hepaticus is reported, with the development of novel plasmid vectors, methods for transformation, site-specific mutagenesis, and mutant complementation. These tools were used to delete the pglB gene , an oligosaccharyltransferase, a central enzyme of the N- glycosylation pathway, by allelic exchange. In the mutant strain, N -glycosylation was completely abolished. The tools and methods developed in this study represent innovative approaches that can be applied to further explore important virulence factors of C. hepaticus and other closely related Campylobacter species., Importance: Spotty liver disease (SLD) of layer chickens, caused by infection with Campylobacter hepaticus , is a significant economic and animal welfare burden on an important food production industry. Currently, SLD is controlled using antibiotics; however, alternative intervention methods are needed due to increased concerns associated with environmental contamination with antibiotics, and the development of antimicrobial resistance in many bacterial pathogens of humans and animals. This study has developed methods that have enabled the genetic manipulation of C. hepaticus . To validate the methods, the pglB gene was inactivated by allelic exchange to produce a C. hepaticus strain that could no longer N -glycosylate proteins. Subsequently, the mutation was complemented by reintroduction of the gene in trans , on a plasmid vector, to demonstrate that the phenotypic changes noted were caused by the mutation of the targeted gene. The tools developed enable ongoing studies to understand other virulence mechanisms of this important emerging pathogen., Competing Interests: D.M.A. is an employee of Bioproperties Pty Ltd, a veterinary vaccine business that manufactures and sells live vaccines for production animals. Therefore, he has a commercial interest in understanding the biology of veterinary pathogens such as Campylobacter hepaticus. All other authors declare no conflict of interest.