Genome Reduction for Niche Association in Campylobacter Hepaticus, A Cause of Spotty Liver Disease in Poultry

Abstract

The term "spotty liver disease" (SLD) has been used since the late 1990s for a condition seen in the UK and Australia that primarily affects free range laying hens around peak lay, causing acute mortality and a fall in egg production. A novel thermophilic SLD-associated Campylobacter was reported in the United Kingdom (UK) in 2015. Subsequently, similar isolates occurring in Australia were formally described as a new species, Campylobacter hepaticus. We describe the comparative genomics of 10 C. hepaticus isolates recovered from 5 geographically distinct poultry holdings in the UK between 2010 and 2012. Hierarchical gene-by-gene analyses of the study isolates and representatives of 24 known Campylobacter species indicated that C. hepaticus is most closely related to the major pathogens Campylobacter jejuni and Campylobacter coli. We observed low levels of within-farm variation, even between isolates collected over almost 3 years. With respect to C. hepaticus genome features, we noted that the study isolates had a ~140 Kb reduction in genome size, ~144 fewer genes, and a lower GC content compared to C. jejuni. The most notable reduction was in the subsystem containing genes for iron acquisition and metabolism, supported by reduced growth of C. hepaticus in an iron depletion assay. Genome reduction is common among many pathogens and in C. hepaticus has likely been driven at least in part by specialization following the occupation of a new niche, the chicken liver.

Keywords: Campylobacter hepaticus; genome reduction; niche adaptation; poultry; spotty liver disease.

Figure 1

Relationships between C. hepaticus and other Campylobacter species based on gene-by-gene analyses. Maximum likelihood trees based on concatenated nucleotide sequences of (A) 52 rMLST genes from 86 isolates comprising 25 Campylobacter species; and (B) 646 core genes identified by cgMLST in ≥90% of 59 isolates comprising 11 closely related thermophilic Campylobacter species. Bootstrap values <80 (A) or <100 (B) are shown for major nodes. Gray shading indicates taxa with multiple representative isolates. Subspecies are marked with colored circles, as indicated in the legend. The insets provide magnified views of the relationships among C. hepaticus isolates. ^*, farm 1; ^**, farm 2; ▴, farm 4; ▾, farm 5.

Figure 2

Phylogeny of the UK Campylobacter hepaticus isolates. Maximum likelihood tree constructed by reference to the whole genome sequence of isolate HV10. UK C. hepaticus isolates: S10-0209, S11-010, S11-5013, S12-1018, S11-0036, S11-0038, S12-002, S11-0071, and S12-0322.

Figure 3

BRIG (BLAST Ring Image Generator) comparison of C. hepaticus isolates with C. jejuni NCTC 11168. Similarities between the reference genome C. jejuni NCTC 11168 and C. hepaticus isolates are presented as concentric rings. Inner circle: C. jejuni NCTC 11168 (purple ring). The 10 UK C. hepaticus isolates and HV10 are presented in the outer concentric rings.

Figure 4

Comparison of the functional subsystems in the UK C. hepaticus (red) and 5 C. jejuni reference genomes (blue) as generated by RAST. References: M1, NCTC 11168, PT14, R14, and 4031. The open circles represent the outliers. Only subsystems with significant differences (student's t-test) are shown. The names of some subsystems are shortened: Cell Division, Cell Division and Cell Cycle; Cell signaling, Regulation and Cell signaling; Nucleosides, Nucleosides and Nucleotides; Iron acquisition, Iron acquisition and metabolism; Virulence, Virulence, Disease and Defense; Fatty Acids, Fatty Acids, Lipids, and Isoprenoids; Motility, Motility and Chemotaxis; Cell Wall, Cell Wall and Capsule; Cofactors, Cofactors, Vitamins, Prosthetic Groups, Pigments; Amino Acids, Amino Acids and Derivatives.

Figure 5

Reduced growth of C. hepaticus isolates in iron depleted conditions. Growth curves for 3 UK C. hepaticus isolates and C. jejuni NCTC 11168 grown in Brain Heart Infusion broth either regular or untreated (NT) or iron-depleted (−Fe).