Enhancement of Campylobacter hepaticus culturing to facilitate downstream applications

Canh Phung,Thi Thu Hao Van,Peter C Scott,Timothy B Wilson,José A Quinteros,Robert J Moore

doi:10.1038/s41598-021-00277-8

Abstract

Campylobacter hepaticus causes Spotty Liver Disease (SLD) in chickens. C. hepaticus is fastidious and slow-growing, presenting difficulties when growing this bacterium for the preparation of bacterin vaccines and experimental disease challenge trials. This study applied genomic analysis and in vitro experiments to develop an enhanced C. hepaticus liquid culture method. In silico analysis of the anabolic pathways encoded by C. hepaticus revealed that the bacterium is unable to biosynthesise l-cysteine, l-lysine and l-arginine. It was found that l-cysteine added to Brucella broth, significantly enhanced the growth of C. hepaticus, but l-lysine or l-arginine addition did not enhance growth. Brucella broth supplemented with l-cysteine (0.4 mM), l-glutamine (4 mM), and sodium pyruvate (10 mM) gave high-density growth of C. hepaticus and resulted in an almost tenfold increase in culture density compared to the growth in Brucella broth alone (log10 = 9.3 vs 8.4 CFU/mL). The type of culture flask used also significantly affected C. hepaticus culture density. An SLD challenge trial demonstrated that C. hepaticus grown in the enhanced culture conditions retained full virulence. The enhanced liquid culture method developed in this study enables the efficient production of bacterial biomass and therefore facilitates further studies of SLD biology and vaccine development.

Highlights

Campylobacter hepaticus causes Spotty Liver Disease (SLD) in chickens
In silico pathway analysis of the C. hepaticus genome predicted that the bacterium lacks some of the genes encoding enzymes required for the biosynthesis of l-cysteine, l-lysine and l-arginine
C. hepaticus was predicted to be unable to biosynthesise l-lysine and l-arginine, the addition of these amino acids to Brucella broth did not significantly increase the culture densities that could be achieved for C. hepaticus compared to densities supported by unmodified Brucella broth

Summary

Introduction

Campylobacter hepaticus causes Spotty Liver Disease (SLD) in chickens. C. hepaticus is fastidious and slow-growing, presenting difficulties when growing this bacterium for the preparation of bacterin vaccines and experimental disease challenge trials. In previous studies, when a large C. hepaticus biomass was required, such as for SLD induction experiments, C. hepaticus was cultured on Brucella agar supplemented with 5% defibrinated horse blood (HBA), incubated for 3 days and harvested. C. hepaticus had to be harvested from dozens of Petri dishes to produce sufficient biomass for a modestly sized animal trial[10] This methodology is time–consuming, uses a lot of resources, and is prone to contamination. Necessary substrates for the growth of Bukholderia glumae were defined using the Pathcomp tool in KEGG16 The objective of this project was to identify and evaluate compounds that are required or that could enhance the growth of C. hepaticus in liquid culture, by analysing the metabolic pathways of this species. This study applied genomic analysis and in vitro experiments to develop an enhanced C. hepaticus liquid culture method

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Conclusion