Enhancement of Campylobacter hepaticus culturing to facilitate downstream applications

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.

Spotty liver disease adversely affect the gut microbiota of layers hen

“…C. hepaticus HV10 T , C. hepaticus NSW44L and C. hepaticus QLD19L were grown on Brucella agar (Becton Dickinson) with 5% horse blood (HBA) and incubated at 37 °C in microaerobic conditions using CampyGen gas packs (Oxoid) for 3 days ( 6 ). To prepare the inoculum for the chicken challenge, the culture was grown in Brucella broth supplemented with l-cysteine (0.4 mM), and l-glutamine (4 mM) and sodium pyruvate (10 mM) (modified Brucella broth) in 75 cm 2 tissue culture flasks at 37°C for 48 h in microaerophilic conditions and used directly for the challenge, as previously described ( 14 ).…”

“…The animal experiment was approved by the Wildlife and Small Institutions Animal Ethics Committee of the Victorian Department of Economic Development, Jobs, Transport and Resources (approval number 14.16). The challenges were carried out as described in ( 14 ). In brief, the control chickens were given 1 mL of modified Brucella broth, the challenged groups were dosed with 1 x 10 9 CFU of the relevant C. hepaticus strain in 1 mL of modified Brucella broth.…”