Genomic and Pathogenicity Studies on Campylobacter Hepaticus, The Agent Responsible for Spotty Liver Disease in Chickens

Campylobacter hepaticus currently causes significant economic losses to the poultry industry, as it is the cause of spotty liver disease (SLD) in chickens (Van et al., 2016, 2017a). The clinical manifestations of SLD include the formation of gray/white lesions in the liver, an increase in mortality rate in a flock, and reduction in egg production. It has been sporadically reported over the last 60 years, first from the United States, then in other countries, including Australia. The disease has become increasingly common in Australia over the last decade and is now considered one of the most significant health challenges in the egg industry (Grimes and Reece, 2011). Campylobacter hepaticus was identified and characterised from Australian cases of SLD in 2016 (Van et al., 2016). This work built on the report of isolation of a novel Campylobacter isolated from UK cases of SLD (Crawshaw et al., 2015). In 2017, C. hepaticus was definitively shown to be the cause of SLD (Van et al., 2017a, Van et al., 2016, Van et al., 2017b).

C. hepaticus is most closely related to the foodborne pathogens C. jejuni and C. coli. It is anticipated that C. hepaticus must harbour genes which are responsible for its ability to cause SLD in chickens, but these are yet to be discovered.

To investigate the bacterium’s biology and pathogenicity, we compared the whole genome sequences of 16 Australian isolates of C. hepaticus, together with nine C. hepaticus isolates from outbreaks in the United Kingdom. We also investigated the plasmid content of these Australian isolates and carried out animal challenge studies with selected Australian isolates to determine their comparative levels of virulence.

C. hepaticus was isolated from bile and/or liver samples from outbreaks throughout Australia using the methods described in Van et al. (2017a). DNA of 16 Australian isolates was extracted and sequenced on an Illumina MiSeq Sequencer at RMIT University (Van et al., 2016), whereas the sequencing data of nine isolates from UK was obtained online (Petrovska et al 2017). Genomes were assembled using the A5Miseq pipeline (Coil et al., 2015), producing draft genomes for all isolates. For the type strain HV10, a complete finished genome was obtained using sequence data from both Illumina and PacBio platforms (accession CP031611.1). All assemblies and read sets were deposited in NCBI (Bioproject PRJNA485661). The Compare Sequence feature of the SEED viewer was used to compare sequence identity of the annotated genes of all strains.

Sequence assembly contigs with genes annotated as suspected plasmid elements were Blasted against the NCBI database (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Significant matches were identified with >98% coverage and identity to previously characterised plasmids from other Campylobacter sp. Selected isolates identified as carrying plasmids bearing antibiotic resistant genes were grown on horse blood agar plates supplemented with antibiotic to confirm their antibiotic resistant characteristics.

Two C. hepaticus challenge trials of 26-week old Hy-Line layer hens were carried out to investigate the comparative levels of virulence of selected isolates. The animal experimentation 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 Van et al., 2017a. The birds were challenged by direct oral gavage with 1 x 109 CFU or 108 CFU of the relevant C. hepaticus strain in 1 ml of Brucella broth. Isolates from Victoria (HV10), Queensland (19L) and New South Wales(44L) were used in the first trial. A second animal trial was performed using HV10 and 44L isolates to confirm the findings of the first trial. Unchallenged control chickens were given 1 ml of uninoculated Brucella broth. Birds were scored as SLD positive if the liver had typical SLD lesions. Fisher’s exact test was used to find statistically significant differences in the number of SLD positive birds.

Genome sizes of the C. hepaticus isolates ranged from 1.481-1.535 Mb and the GC content ranged from 27.9 to 28.2%. The genomes contained 1,472 to 1,555 predicted protein coding sequences. By comparison to the HV10 reference genome, the UK isolates showed some variation in genome sequences and the Northern QLD isolates 19L and 54L showed higher variation than the other Southern Australian isolates. The protein analysis coincides with the genome analysis with regards to the comparative relationship between isolates (Fig. 1).

Six out of 16 Australian isolates contained plasmids. Five isolates from Vic (ACE1, ACE8659, ACEM3A, 84B, 27L) contain a C. jejuni pCJDM210-like plasmid (>98% identity) and one isolate from South Australia (SAJune18) contained a C. coli pCC31-like plasmid (100% identity). Both plasmid types contain a tetracycline resistant gene, tet(O). These plasmid-containing isolates grew on horse blood agar plates containing 30 µg/mL of tetracycline, whereas plasmid negative isolates such as HV10 and NSW44L isolates did not grow.

Two animal trials were carried out to examine the relative degree of virulence of a number of isolates. In the first trial, at the same challenge dose of 109 CFU, the isolate HV10 (Victoria) showed a similar level of virulence as the isolate 54L (QLD), as judged by the percentage of challenged birds that had visible liver lesions. At a challenge dose of 108 CFU, strain NSW44L (NSW) showed a higher level of virulence than the strain HV10, in terms of disease severity and percentage of SLD-positive birds, as all 8 birds of the former group contained 200-1000 SLD typical white spots, while only half of the latter group of birds have that similar disease severity. A second trial was carried out to confirm the greater pathogenicity of the NSW44L strain compared to the HV10 strain. As expected, when birds were challenged with the same dose, NSW44L caused disease in more birds than the HV10 strain (p=0.03) (Table 1).

Genome comparison between NSW44L and HV10 showed 12 genes were present in NSW44L but absent in HV10 (eight of these genes were hypothetical proteins, and the remaining were genes encoding trimethylamine-N-oxide reductase, beta lactamase, possible lipoprotein, and YidD), plus there were various genes with less than 100% identity between the two isolates.

Genome and proteome comparison showed that the isolates from the Southern region (Vic, NSW and SA) were more closely related to each other than to the isolates from the Northern region (QLD). This indicates that C. hepaticus clonal populations are geographically confined, although more isolates need to be collected to confirm this finding.

The genome sequences of all isolates were examined for plasmid content as plasmids may play an important role in dissemination of antibiotic resistance genes. Plasmids were found in isolates from Victoria and SA. They contained two different types of plasmid; isolates from Victoria contained C. jejuni pCJDM210L-like plasmids, and the SAJune18 isolate from SA contained a C. coli pCC31-like plasmid. Both plasmids contained a tetracycline resistant gene, tet(O). Selected isolates were also shown to phenotypically express tetracycline resistance as they grew well on tetracycline containing plates in the laboratory. It seems that both C. jejuni and C. coli can pass antibiotic resistance plasmids to C. hepaticus, as the UK isolates also have the C. coli pCC31-like plasmid (Petrovska et al., 2017), which has 89% query coverage and 100% identity to Australian pCC31-like plasmids. The presence of Tet resistant plasmid in C. hepaticus isolated from SA might be the reason why the flock from which this strain was isolated did not respond to chlortetracycline treatment (Kapil Chousalkar, personal communication). The C. coli pCC31 plasmid has been shown to be conjugative (Batchelor et al., 2004), therefore C. hepaticus plasmids could be disseminated to important human and animal pathogenic bacteria.

Three isolates from different states were selected for the first challenge trial (HV10 from Victoria, NSW 44L from NSW, and 19L from QLD) and it was shown that the NSW44L strain was more virulent than the type strain HV10, while HV10 showed a similar level of virulence as the isolate 54L. Genome comparison between HV10 and NSW44L isolates showed some unique genes within the NSW44L isolate and these may play a role in the greater virulence of NSW44L. However, to confirm this finding, substantial further work is needed, for example the construction and testing of targeted mutants of potential virulence genes to determine their involvement in disease pathogenesis.

ACKNOWLEDGEMENTS: SLD related research at RMIT University and Scolexia Pty Ltd is supported by grants from Australian Eggs, Poultry Hub Australia and the Innovation Connections scheme of the Commonwealth Government.