The epidemiology of Spotty Liver Disease (SLD) was investigated by assaying 1,840 samples collected from layer chickens and the environment in poultry farms across Australia for the presence of Campylobacter hepaticus, the agent responsible SLD in chickens. A C. hepaticus specific PCR and bacterial culture were used. Results showed that birds could be infected with C. hepaticus up to 8 weeks before clinical SLD was manifested. In addition, birds could be infected long before laying starts, as young as 12 weeks old, but the peak period for SLD outbreaks was when the birds were 26-27 weeks old. Campylobacter hepaticus DNA was detected in motile organisms such as wild birds and rats and so these organisms may be vectors for C. hepaticus dissemination. Moreover, water, soil, mites, flies, and dust samples from SLD infected farms were also found to be PCR-positive for C. hepaticus DNA. However, it still remains to be determined whether these environmental sources carry any viable C. hepaticus. The indications from this study are that environmental sources are a likely transmission source of C. hepaticus. Therefore, biosecurity practices need to be strictly followed to prevent the spread of SLD amongst and between flocks. Also, a rapid, molecular detection method such as PCR should be used as to monitor for C. hepaticus presence in flocks before clinical disease is apparent, and therefore inform the use of biosecurity and therapeutic measures to help prevent SLD outbreaks.
Chickens infected with Campylobacter jejuni or Campylobacter coli are largely asymptomatic, however, infection with the closely related species, Campylobacter hepaticus, can result in Spotty Liver Disease (SLD). C. hepaticus has been detected in the liver, bile, small intestine and caecum of SLD affected chickens. The survival and colonization mechanisms that C. hepaticus uses to colonize chickens remain unknown. In this study, we compared the genome sequences of 14 newly sequenced Australian isolates of C. hepaticus, isolates from outbreaks in the United Kingdom, and reference strains of C. jejuni and C. coli, with the aim of identifying virulence genes associated with SLD. We also carried out global comparative transcriptomic analysis between C. hepaticus recovered from the bile of SLD infected chickens and C. hepaticus grown in vitro. This revealed how the bacteria adapt to proliferate in the challenging host environment in which they are found. Additionally, biochemical experiments confirmed some in silico metabolic predictions. We found that, unlike other Campylobacter sp., C. hepaticus encodes glucose and polyhydroxybutyrate metabolism pathways. This study demonstrated the metabolic plasticity of C. hepaticus, which may contribute to survival in the competitive, nutrient and energy-limited environment of the chicken. Transcriptomic analysis indicated that gene clusters associated with glucose utilization, stress response, hydrogen metabolism, and sialic acid modification may play an important role in the pathogenicity of C. hepaticus. An understanding of the survival and virulence mechanisms that C. hepaticus uses will help to direct the development of effective intervention methods to protect birds from the debilitating effects of SLD.
A novel species of Campylobacter was isolated from bile samples of chickens with spotty liver disease in Australia, making it the second novel species isolated from chickens with the disease, after Campylobacter hepaticus was isolated and described in 2016. Six independently derived isolates were obtained. They were Gram-stain-negative, microaerobic, catalase-positive, oxidase-positive and urease-negative. Unlike most other species of the genus Campylobacter , more than half of the tested strains of this novel species hydrolysed hippurate and most of them could not reduce nitrate. Distinct from C. hepaticus , many of the isolates were sensitive to 2,3,5-triphenyltetrazolium chloride (0.04%) and metronidazole (4 mg ml−1), and all strains were sensitive to nalidixic acid. Phylogenetic analysis using 16S rRNA and hsp60 gene sequences demonstrated that the strains formed a robust clade that was clearly distinct from recognized Campylobacter species. Whole genome sequence analysis of the strains showed that the average nucleotide identity and the genome blast distance phylogeny values compared to other Campylobacter species were less than 86 and 66%, respectively, which are below the cut-off values generally recognized for isolates of the same species. The genome of the novel species has a DNA G+C content of 30.6 mol%, while that of C. hepaticus is 27.9 mol%. Electron microscopy showed that the cells were spiral-shaped, with bipolar unsheathed flagella. The protein spectra generated from matrix-assisted laser desorption/ionization time of flight analysis demonstrated that they are different from the most closely related Campylobacter species. These data indicate that the isolates belong to a novel Campylobacter species, for which the name Campylobacter bilis sp. nov. is proposed. The type strain is VicNov18T (=ATCC TSD-231T=NCTC 14611T).
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