Spotty Liver Disease

I. INTRODUCTION

Spotty Liver Disease (SLD) is characterized by the occurrence of multiple grey/white spots in the liver. It causes mortalities and reduction in egg output and is prevalent within the layer industry in Australia, especially within the free range sector of the industry (Grimes & Reece, 2011). The disease is less commonly found in barn and cage birds and parent stock (Scott, 2016). The recent identification of Campylobacter hepaticus, as the causative agent (Van et al., 2016), and the development of an experimental disease induction method (Van et al., 2017a), provide the tools to facilitate the study of disease pathogenesis and the evaluation of experimental vaccines.

The development of specific and sensitive PCR detection methods allows the detection of C. hepaticus in the gut of diseased birds. C. hepaticus occurs throughout the gut, increasing in abundance down the gut. To date we have only detected C. hepaticus in the gut of birds from sheds that have clinical signs of disease. C. hepaticus could not be detected in the gut of birds from other sheds, on the same farms, that have not had a history of clinical disease. Currently, we do not have a highly selective culture method for C. hepaticus so the organism can only be isolated from samples such as liver and bile, that are not infected with other bacteria, as C. hepaticus is slow growing and hence is rapidly overgrown by any other contaminating bacteria.

II. IDENTIFICATION OF THE CAUSATIVE AGENT

Disease cases with similar clinical presentations as modern day SLD were reported in the USA in the 1950’s (Tudor, 1954; Delaplane et al., 1955). Bacteria described as “vibrios” were cultured from diseased birds, initially by passage in chicken embryos and subsequently cultured on rich agar media of various compositions. In one case, cultured bacteria were fairly comprehensively characterised for fermentation and enzymatic activities; however the bacterial genus was not identified and no subsequent study of the isolates has been reported (Peckham, 1958). Other researchers have suggested the possible involvement of a number of bacterial species, including Campylobacter jejuni, Campylobacter coli, Clostridium sordellii, and Helicobacter pullorum (Burnens et al., 1996; Forsyth et al., 2005; Jennings et al., 2011). The Campylobacter and Helicobacter species would be consistent with the previous findings of “vibrio” like bacteria, but in no cases could the disease be experimentally reproduced with the candidate cultured bacteria.

Crawshaw et al. recovered a number of bacterial isolates from SLD affected hens from UK flocks and identified them as campylobacters (Crawshaw et al., 2015). Van et al. isolated similar bacteria from Australian cases of SLD and went on to fully characterise the organism and identified it as a new species that they named Campylobacter hepaticus (Van et al., 2016). The role of C. hepaticus as the etiological agent of SLD was confirmed by its ability to induce lesions, typical of clinical cases of disease, in experimentally infected birds (Van et al., 2017a).

III. ISOLATION OF C. HEPATICUS FROM CLINICAL SAMPLES

C. hepaticus was first isolated from the livers of layer birds with typical indications of SLD. The two groups who have reported successful isolation of the organism have used slightly different culturing methods (Crawshaw et al., 2015; Van et al., 2016). In both cases aseptically collected internal fragments of liver were macerated in Preston broth and incubated under microaerophilic conditions at 37o C; the UK group for 7 days and the Australian group for 2 days. Samples from the pre-enrichment step were plated onto 5% sheep blood agar (SBA) (UK group) or Brucella agar with 5% horse blood (BAB) (Australian group) and again incubated microaerophilically for several days. The Australian isolates produced clearly visible colonies within 3-5 days whereas the UK group reported that some isolates required up to 7 days before growth was obvious. An easier route to isolation of C. hepaticus from diseased birds, taken by both groups, is the direct plating of bile onto either SBA or BAB and incubation under microaerophilic conditions at 37°C for several days.

There is currently no highly selective media available for C. hepaticus isolation and so strains have only been recovered from tissue samples that only carry the target organism. When other bacteria are present any potential C. hepaticus colonies are rapidly overgrown by more rapidly multiplying bacteria.

IV. CULTURING AND CHARACTERISATION OF C. HEPATICUS

Following primary isolation, C. hepaticus can be reliably grown on BAB but grows poorly in liquid culture without blood supplementation. It grows at 37°C and 42°C but not at 25°C and does not grow under aerobic conditions. Electron microscopy (EM) showed that C. hepaticus has typical Campylobacter morphology. Cultures consist mainly of S-shaped cells and longer helical cells, but some cocci forms are also present (Figure 1). Some cells have bipolar unsheathed flagella while many appear to have single polar flagella or no flagella; the variation observed under EM may be due to the sensitivity of the flagella to mechanical breakage as the scanning EM appears to show a lot of broken flagella fragments. Whole genome sequencing and comparison to the genomes of other Campylobacter species indicated that C. hepaticus is most closely relate to Campylobacter jejuni and Campylobacter coli.

Figure 1 - Panels A and B: transmission electron micrographs of isolated C. hepaticus cells. Note the long bipolar flagella shown in Panel B. Panels C and D: scanning electron micrographs of surface of a colony of C. hepaticus cells. Note in panel D the variation in cell length, ranging from the S-shaped cell in the top centre of the panel to the long helical cell in the centre of the panel.

V. EXPERIMENTAL REPRODUCTION OF DISEASE

Early attempts to induce pathology used some of the embryo passaged or cultured bacteria isolated from US cases in the 1950’s. The “vibrio” bacteria caused death and lesions in challenged chicken embryos and, in some cases, signs of clinical disease were reproduced in inoculated adult bird (Hofstad et al., 1958; Peckham, 1958; Sevoian et al., 1958). More contemporary attempts to reproduce clinical disease, using the recent UK isolates in specific pathogen free chicks, resulted in microscopically visible lesions in the liver of challenged birds (Crawshaw et al., 2015).

It is only with the use of the Australian C. hepaticus isolates in birds coming into lay that full-blown disease typical of field cases of SLD has been successfully reproduced following experimental infections (Van et al., 2017a). Those studies fulfilled Koch’s postulates (Grimes, 2006) to unequivocally demonstrate that C. hepaticus causes SLD (Van et al., 2017a). Disease induction was achieved by inoculating birds from flocks with no history of SLD with 10⁹ to 10¹⁰ CFU of C. hepaticus HV10^T via direct oral gavage. The severity of disease in the 24 challenged birds varied from no macroscopically obvious disease in one bird to severe disease covering the entire surface of all lobes of the liver in a few birds. Most of the birds had moderate numbers of macroscopically obvious lesions on the liver surface. No long term trials to investigate the effect of experimental disease challenge on egg output have yet been reported. From both the success of the oral gavage in the disease induction experiments and the findings of SLD in cage facilities (relatively rare compared with free-range operations) where it is usually birds on the lower layers that suffer disease, it is concluded that natural SLD infection probably occurs via the faecal-oral route.

VI. C. HEPATICUS GENOME

Whole genome sequencing has shown that the genomes of 14 Australian isolates range in size from 1.48 to 1.53 Mb (unpublished data). Sequencing of 10 British isolates showed a wider range of genome sizes from 1.50 to 1.80 Mb (Petrovska et al., 2017). The type strain, HV10^T (=NCTC 13823^T; =CIP 111092^T), has a genome of 1,520,669 nucleotides and is predicted to encode 1494 protein coding sequences and 52 RNA coding genes (unpublished results). Overall whole genome comparison, on a single nucleotide polymorphism gene-by-gene basis of the core genome, showed that the Australian type-strain isolate differed from the three sub-clades of the British isolates. The Australian isolate had a lower GC content; 27.9% compared with an average of 28.4% for the British isolates (Petrovska et al., 2017). The C. hepaticus isolates have smaller genomes than typically found for the closely related species, C. jejuni and C. coli, with approximately 140 fewer genes encoded, including a notable reduction in the number of genes encoding products predicted to be involved in iron acquisition and general metabolism. There were also fewer putative virulence, disease, and defence subsystem genes predicted in the genomes of C. hepaticus. C. hepaticus genomes encoded more genes involved in carbohydrate, fatty acid, lipid, and isoprenoid metabolism than typically found in C. jejuni genomes (Petrovska et al., 2017). Petrovska et al. (2107) have suggested that the reduced genome of C. hepaticus may result from the more specialised lifestyle that C. hepaticus has compared to C. jejuni, in particular, the reduction in iron acquisition may result from specialised niche adaptation to the iron rich environment within the liver.

It is of particular interest to interrogate the genome of C. hepaticus for potential toxins that may be important in disease pathogenesis, in particular the pathology observed in the liver. To date no obvious toxin encoding genes have been identified although it should be noted that, like all genomes, the C. hepaticus genome contains many genes for which a function could not be predicted. There are only a few genes in other Campylobacter species that have been identified as encoding toxins that may play some role in disease pathogenesis. In C. jejuni cytolethal distending toxin may play a role in disease pathogenesis but all the genes involved in its synthesis are absent from C. hepaticus (Petrovska et al., 2017).

VII. MOLECULAR ASSAYS

Whole genome sequencing of C. hepaticus supplied the DNA sequence information required to develop polymerase chain reaction (PCR) methods to specifically and sensitively detect C. hepaticus (Van et al., 2017b). Such methods are particularly useful for C. hepaticus given its slow growing and fastidious nature and the current inability to selectively culture the organism. It is only with the use of PCR detection methods that the bacterium can be identified and quantified in microbially complex samples such as samples from the gut. Application of the PCR assay (Figure 2A) to gut samples showed that C. hepaticus was present within the gut and increased in abundance along the length of the small intestine (Van et al., 2017b). The detection of sheading of C. hepaticus in the faeces supports the contention that SLD is likely to be transmitted via the faecal-oral route. In the future the PCR assay could be used to investigate the epidemiology of bacterial acquisition and spread within a flock and the presence of the bacterium within the environment. It will important to identify the source of infection so that preventative measures can be taken to reduce the incidence of disease in the Australian flock. A multiplex PCR assay that can detect and differentiate three Campylobacter species commonly found in chickens has also been developed (Figure 2B), further facilitating the epidemiological investigation of Campylobacter carriage in chickens.

Figure 2 - Panel A: PCR identification of C. hepaticus in microbially complex samples. Lane 1: Molecular size standards, EasyLadder (Bioline); Lanes 2 and 3: DNA extracts from chicken caecum samples from birds with clear clinical indications of SLD; Lane 4: Negative control – chicken caecal DNA from a bird from a flock with no history of SLD. Panel B: Multiplex identification of C. hepaticus, C. jejuni and C. coli in caecum samples from chickens (lanes 2-8); lane 1, no DNA negative control; lane 9, spiked DNA positive control; lane 10, Molecular size standards, EasyLadder (Bioline).

VIII. CONCLUSIONS

Recent research has identified C. hepaticus as the etiological agent of SLD. Further, characterisation of C. hepaticus and the development of an experimental disease induction model have provided the basic tools that can now be used to investigate and develop treatment options for SLD. The disease model can be used to test the efficacy of experimental vaccine formulations under controlled conditions and the model can also be used to test the effectiveness of other potential interventions such as prebiotics, fatty acids, phytobioics, and probiotics. Development of treatment options would be also be advanced by elucidating basic mechanisms of pathogenesis and understanding how C. hepaticus traffics from the gut to the liver. In the future, epidemiological studies, facilitated by the application of the developed molecular assays, may inform management practices that could be modified to reduce disease incidence.

Presented at the 29th Annual Australian Poultry Science Symposium 2018. For information on the latest and future editions, click here.