Development of growth medium and epidemiological studies of the bacteria causing spotty liver disease in chickens

Spotty Liver Disease (SLD) causes losses in egg production and mortality in laying hens. Recently, an increasing number of reports of the presence of SLD outbreaks have been published worldwide and the disease is now known as one of the common diseases in the poultry industry. The disease was first described many decades ago, but the definitive etiological agent was only recently confirmed and named Campylobacter hepaticus. As a newly identified bacterium, studies to refine the in vitro growth conditions of C. hepaticus, its survival in stress conditions, and investigate disease epidemiology, have not yet been conducted. Such issues need to be addressed to advance our understanding of how to develop a high-density liquid culture method for C. hepaticus, to enable downstream experiments; the pathways of C. hepaticus ingression to chicken sheds; and the inter-related issue of the response of C. hepaticus to environmental stress. All these issues are important to address if methods to control SLD are to be devised. The standard bacteriological media used for primary isolation and characterisation of the bacterium produced low C. hepaticus cell densities, resulting in difficulties to conduct reproducible assays designed to explore the biology of the organism. The low growth limited the production of bacterial biomass available for use in animal infection models and the production of experimental bacterins. Thus, the development of a higher density liquid culture method for C. hepaticus would be of great benefit and the work undertaken to address this need is presented in Chapter 2. This study used genomic analysis and in vitro experiments to develop a high density culturing method for C. hepaticus. Genomic analysis indicated that C. hepaticus lacks genes for the biosynthesise of L-cysteine, L-lysine and L-arginine, but experiments showed that only the addition of exogenous L-cysteine enhanced the growth of C. hepaticus in Brucella broth. In addition, it was found that the addition of L-glutamine, L-serine and pyruvate could significantly improve the growth of C. hepaticus. The best growth achieved for C. hepaticus was in Brucella broth supplemented with L-cysteine (0.4 mM), L-glutamine (4 mM) and sodium pyruvate (10 mM) in large surface-area-to-volume vessels under microaerobic static conditions at 37oC. The use of the enhanced liquid culture method resulted in a 10-fold increase in culture density compared to the growth in Brucella broth alone The enhanced liquid culture method developed in this study was used to produce C. hepaticus cultures for an SLD challenge trial. C. hepaticus grown in the enhanced culture conditions had comparable virulence compared to bacteria grown and harvested from standard horse blood agar plates - the previous method used to produce bacteria for challenge trials. The method developed in this study enables the relatively efficient production of bacterial biomass compared to previously used methods and, therefore, facilitates further studies of SLD biology and vaccine development.  The study then focused on the epidemiology of C. hepaticus (Chapter 3). In 2 years, more than 1800 chicken and environmental samples were collected from different chicken farms across Australia to study the seasonality of SLD outbreaks, the age distribution of affected flocks, the genetic diversity of C. hepaticus isolates, and possible routes of transmission. SLD outbreaks occurred in many farms across Australia and mostly in laying hens when they were reaching peak lay (26-27 weeks old). It was found 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, while still on rearing farms. Environmental samples including water, soil, dust, insects and wild bird faeces were positive by PCR for C. hepaticus indicating environmental sources may be important in the transmission of C. hepaticus. Therefore, biosecurity practices need to be strictly followed to prevent the spread of SLD amongst and between flocks. The whole genomes of C. hepaticus isolates recovered in this study shared high similarity to the reference genome, C. hepaticus HV10T. Two isolates were identified to harbour a plasmid with 99% identity to Campylobacter jejuni subsp. jejuni 81- 176 plasmid, pTet, containing a tetracycline-resistant gene, tet (O). This study provides information relevant to the assessment of SLD risk. Although C. hepaticus DNA was detected by PCR of environmental samples such as soil, wild bird faeces, and water, acquired the isolation of C. hepaticus was unsuccessful using standard horse blood agar plates. Other Campylobacter species have been shown to survive under stressful conditions, such as those found in the environment, by entering a viable but non-culturable (VBNC) state. It was hypothesised that C. hepaticus may also be capable of entering a VBNC state. Chapter 4 reports on studies aimed to enhance the understanding of how C. hepaticus responds to environmental stress and changes to a viable but non-culturable form and the potential role of water in the transmission of C. hepaticus to chickens. It was shown that C. hepaticus could be cultured using standard HBA from water for up to 4-5 days and from an isotonic solution, Ringer¿s solution, for 9-12 days at room temperature. At 4oC, C. hepaticus was culturability for 19-22 days in distilled water. This survival may be a key to the spread of C. hepaticus within flocks via drinking water. In Ringer¿s solution at 4oC, C. hepaticus exhibited culturability for 53 to 65 days, depending on the strain. C. hepaticus couldn¿t be recovered from Ringer¿s solution after 65 days of incubation, using standard HBA media. However, viable cells of C. hepaticus could still be detected using MycolightTM Green dye and fluorescence microscopy, indicating that C. hepaticus could enter a VBNC state. The morphology changed from spiral to cocci in the VBNC state. A rich nutrient broth supplemented with Vitox, FBP (ferrous sulfate hydrate 0.05%, sodium metabisulfite 0.05%, and sodium pyruvate 0.05%), and 0.4 mM L-cysteine was developed in this study and successfully used to resuscitate the VBCN C. hepaticus cells. This is a significant finding as it indicates that C. hepaticus may be capable of long-term survival in the environment and hence the detection of C. hepaticus DNA in environmental samples may indicate possible sources and routes of transmission of C. hepaticus. It is hypothesised that standing water (puddles) on the range of free-range farms may be an issue that requires special attention. Biosecurity controls should be considered to minimise the presence of C. hepaticus in the water supply. Among the bacterial isolates that were recovered from bile samples of SLD affected chickens, several isolates were found which were positive for SLD-specific PCR but had a different genome compared to C. hepaticus and other Campylobacter species. Chapter 4 describes the study to confirm this is a new species, including the whole genome sequence, biochemical properties, and morphologies of these isolates. Phylogenetic analysis based on 16S rRNA gene sequences showed that the new bacterium belongs to the genus Campylobacter and forms a robust clade similar but separate from C. hepaticus, C. jejuni, C. coli and other Campylobacter species. The whole-genome sequence of the novel species showed less than 84% of similarity to other Campylobacter spp., which is below the generally accepted cut-off values for isolates of the same species. The strains were unidentifiable from the current database of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF), further demonstrating that they are different from the known Campylobacter species. The bacterium is Gram-negative, urase-negative, catalase and oxidase-positive, grows under microaerobic conditions at 37oC and 42oC. Electron microscopy indicated that it is spiral-shaped with bipolar unsheathed flagella. The novel species¿s name was proposed to be Campylobacter bilis [bi.lis. Gr. fem. n., referring to bile, from which the bacterium was first isolated]. Strain VicNov18 was proposed as the type strain for the species and was deposited at ATCC and NCTC (= ATCC TSD-231T= NCTC 14611T).