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ABSTRACT Various species of campylobacters cause significant disease problems in both humans and animals. The continuing development of tools and methods for genetic and molecular manipulation of campylobacters enables the detailed study of bacterial virulence and disease pathogenesis. Campylobacter hepaticus is an emerging pathogen that causes spotty liver disease (SLD) in poultry. SLD has a significant economic and animal welfare impact as the disease results in elevated mortalities and significant decreases in egg production. Although potential virulence genes of C. hepaticus have been identified, they have not been further studied and characterized, as appropriate genetic tools and methods to transform and perform mutagenesis studies in C. hepaticus have not been available. In this study, the genetic manipulation of C. hepaticus is reported, with the development of novel plasmid vectors, methods for transformation, site-specific mutagenesis, and mutant complementation. These tools were used to delete the pglB gene, an oligosaccharyltransferase, a central enzyme of the N-glycosylation pathway, by allelic exchange. In the mutant strain, N-glycosylation was completely abolished. The tools and methods developed in this study represent innovative approaches that can be applied to further explore important virulence factors of C. hepaticus and other closely related Campylobacter species.IMPORTANCESpotty liver disease (SLD) of layer chickens, caused by infection with Campylobacter hepaticus, is a significant economic and animal welfare burden on an important food production industry. Currently, SLD is controlled using antibiotics; however, alternative intervention methods are needed due to increased concerns associated with environmental contamination with antibiotics, and the development of antimicrobial resistance in many bacterial pathogens of humans and animals. This study has developed methods that have enabled the genetic manipulation of C. hepaticus. To validate the methods, the pglB gene was inactivated by allelic exchange to produce a C. hepaticus strain that could no longer N-glycosylate proteins. Subsequently, the mutation was complemented by reintroduction of the gene in trans, on a plasmid vector, to demonstrate that the phenotypic changes noted were caused by the mutation of the targeted gene. The tools developed enable ongoing studies to understand other virulence mechanisms of this important emerging pathogen.

ABSTRACT: Spotty liver disease (SLD) is recognized to be caused by infection with Campylobacter hepaticus in adult layer hens farmed in cage-free environments. SLD is an emerging disease as cage-free egg production increases in popularity in response to desires for improved welfare of poultry. Outbreaks of SLD are frequently experienced around peak egg production in flocks, commonly between 25 and 40 wk of age. The disease becomes manifest with increased exposure and access of the birds to the feces of the flock. This study follows from a previous epidemiological survey of free-range and barn flocks in Australia which identified the presence of a scratch area within the laying house as a major risk factor for the occurrence of SLD. However, that survey also observed SLD occurrence in 45% of houses with a fully slatted floor (no scratch area). The present study describes a further analytical survey aimed at identification of risk factors for SLD in houses with fully slatted flooring. A comprehensive questionnaire was completed for 49 cage-free flocks from point of lay until 40 wk of age across Australia, retrieving information on house design, bird breed, flock size, stocking densities, bird growth, and performance and the occurrence of SLD.Multiple logistic regression model building was used to separate factors and identify important management factors that may be amenable to modify the occurrence of SLD in egg layers.Key determinants of SLD identified from the analyses were that houses with mechanical ventilation (such as tunnel ventilation) have some protection from SLD and an increase of an extra 1 bird/m2 of nest space increased odds of occurrence of SLD by 1.172 times. A recommendation to not exceed 112 brown egg layer hens/m2 of nest space in naturally ventilated houses with a full slat floor was suggested. A delay in birds reaching 60% hen day production (HD) by 1 wk is suggested as a possible predictor for a subsequent outbreak of SLD.

ABSTRACT: Spotty Liver Disease (SLD) is a serious problem in laying hens farmed in cage-free systems. The causative organism, Campylobacter hepaticus, is regarded as having a fecal-oral method of transmission and hence may build up and spread readily in housing systems which allow ease of direct contact of hens with the flock's fecal material. The epidemiology of SLD has not been thoroughly investigated. An initial cross-sectional analytical epidemiological survey of SLD in free range and barn layer systems was conducted in Australia over 2019 to 2021.The survey involved rearing flocks (n = 32) which were then followed through into laying flocks (n = 24) up to 40 wk of age. Cloacal swabs were collected during rearing and lay for C. hepaticus detection by PCR. Flocks were classified as “Cases” (n = 18) where clinical SLD according to the case definition was observed or “Controls” (n = 6) which were clinically unaffected. No C. hepaticus was detected in cloacal swabs from rearing houses whereas the organism was detected in 18 Case flocks in lay and from 2 Control flocks in lay. All layer houses that incorporated a scratch area (n = 13) were categorized as Cases. Thus, having a scratch area is a key determinant for SLD and no analyses of further contributory factors from these flocks were able to be made. Of the remaining 11 flocks which had floors fully covered by slats, 5 were Cases (45%). Further risk factor analysis was compromised by this small sample size and identification of other significant associations was not possible. A larger survey investigating flocks laying in houses with fully slatted floors was undertaken to further the understanding of SLD epidemiology and is reported in a companion paper.

ABSTRACT: A Campylobacter species was first described as the etiological agent of Spotty Liver Disease (SLD) in 2015 and subsequently named as Campylobacter hepaticus in 2016. The bacterium predominantly affects barn and/or free-range hens at peak lay, is fastidious and difficult to isolate, which has impeded elucidation of its sources, means of persistence and transmission. Ten farms from South-Eastern Australia, of which 7 were free range entities participated in the study. A total of 1,404 specimens from layers and 201 from environmental sources, were examined for the presence of C. hepaticus. In this study, our principal findings included the continuing detection of C. hepaticus infection in a flock following an outbreak, indicating a possible transition of infected hens to asymptomatic carriers, that was also characterized by no further occurrence of SLD in the flock. We also report that the first outbreaks of SLD on newly commissioned free-range farms affected layers ranging from 23 to 74 wk of age, while subsequent outbreaks in replacement flocks on these farms occurred during the more conventional peak lay period (23–32 wk of age). Finally, we report that in the on-farm environment, C. hepaticus DNA was detected in layer feces, inert elements such as stormwater, mud, soil, as well as in fauna such as flies, red mites, Darkling beetles, and rats. While in off-farm locations, the bacterium was detected in feces from a variety of wild birds and a canine.

Campylobacter hepaticus is the aetiological agent of Spotty Liver Disease (SLD). SLD can cause significant production loss and mortalities among layer hens at and around peak of lay. We previously developed an enzyme linked immunosorbent assay (ELISA), SLD-ELISA1, to detect C. hepaticus specific antibodies from bird sera using C. hepaticus total proteins and sera pre-absorbed with Campylobacter jejuni proteins. The high specificity achieved with SLD-ELISA1 indicated the presence of C. hepaticus specific antibodies in sera of infected birds. However, some of the reagents used in SLD-ELISA1 are time consuming to prepare and difficult to quality control. This understanding led to the search for C. hepaticus specific immunogenic proteins that could be used in recombinant forms as antibody capture antigens in immunoassay design. In this study, an immunoproteomic approach that combined bioinformatics analysis, western blotting, and LC MS/MS protein profiling was used, and a fragment of filamentous hemagglutinin adhesin (FHA), FHA1,628−1,899 with C. hepaticus specific antigenicity was identified. Recombinant FHA1,628−1,899 was used as antigen coating on ELISA plates to capture FHA1,628−1,899 specific antibodies in sera of infected birds. SLD-ELISA2, based on the purified recombinant FHA fragment, is more user-friendly and standardizable than SLD-ELISA1 for screening antibody responses to C. hepaticus exposure in hens. This study is the first report of the use of FHA from a Campylobacter species in immunoassays, and it also opens future research directions to investigate the role of FHA in C. hepaticus pathogenesis and its effectiveness as a vaccine candidate.

Spotty liver disease (SLD) caused by Campylobacter hepaticus affects the health and productivity of layer hens and is a disease of concern in poultry. In this study, blood and cloacal swab samples were collected from 709 birds across 11 free-range layer farms from different regions of Australia. The prevalence of C. hepaticus specific antibodies and DNA was assessed using a C. hepaticus specific ELISA and PCR and its correlation with mortalities and changes in egg production was analyzed to better understand the seroprevalence of C. hepaticus in Australian free-range layer farms. C. hepaticus specific antibodies were detected from birds in four of the five farms that had no history of SLD with seroprevalence as high as 41% in one of the farms. Seroprevalence of anti-C. hepaticus antibodies among flocks that had an active or previous SLD outbreak varied between 2 and 64%. C. hepaticus DNA was detected from birds in three farms with no known SLD history and five farms with confirmed SLD outbreaks. A good correlation was observed between the ELISA and PCR results with a Pearson correlation coefficient value of 0.85 (p-value = 0.001). No correlation was observed between the flock size or flock age and ELISA or PCR outcomes, and no significant difference between the seroprevalence of anti-C. hepaticus antibodies among flocks with or without a known history of SLD was established (p = 0.143). This study demonstrates the usefulness of C. hepaticus specific ELISA and PCR in identifying the occurrence of mild or sub-clinical SLD and provides a broader and more complete understanding of SLD epidemiology that will inform future research aimed at the development of methods to control SLD, such as appropriate biosecurity measures, vaccines, and feed additives.

Spotty Liver Disease (SLD) is a serious infectious disease which occurs mainly in laying chickens in free range production systems. SLD outbreaks can increase mortality and decrease egg production of chickens, adversely impact welfare and cause economic hardship for poultry producers. The bacterium Campylobacter hepaticus is the primary cause of the disease. This study aimed to identify the effects of C. hepaticus on chicken gut microbiota and gut structure. Three C. hepaticus strains (HV10T, NSW44L and QLD19L), isolated from different states of Australia, were used in the study. Chickens at 26-weeks post-hatch were orally dosed with one of the C. hepaticus strains (challenged groups) or Brucella broth (unchallenged or control group). Six days after the challenge, birds were necropsied to assess liver damage, and caecal content and tissue samples were collected for histology, microbiology, and 16S rRNA gene amplicon sequencing to characterize the composition of the bacterial microbiota. Strain C. hepaticus NSW44L produced significantly more disease compared to the other C. hepaticus strains and this coincided with more adverse changes observed in the caecal microbiota of the birds challenged with this strain compared to the control group. Microbial diversity determined by Shannon and Simpson alpha diversity indices was lower in the NSW44L challenged groups compared to the control group (p = 0.009 and 0.0233 respectively, at genus level). Short-chain fatty acids (SCFAs) producing bacteria Faecalibacterium, Bifidobacterium and Megamonas were significantly reduced in the challenged groups compared to the unchallenged control group. Although SLD-induction affected the gut microbiota of chickens, their small intestine morphology was not noticeably affected as there were no significant differences in the villus height or ratio of villus height and crypt depth. As gut health plays a pivotal role in the overall health and productivity of chickens, approaches to improve the gut health of the birds during SLD outbreaks such as through diet and keeping the causes of stress to a minimum, may represent significant ways to alleviate the impact of SLD.

Various species of campylobacters cause significant disease problems in both humans and animals. The continuing development of tools and methods for genetic and molecular manipulation of campylobacters enables the detailed study of bacterial virulence and disease pathogenesis. Campylobacter hepaticus is an emerging pathogen that causes spotty liver disease (SLD) in poultry. SLD has a significant economic and animal welfare impact as the disease results in elevated mortalities and significant decreases in egg production. Although potential virulence genes of C. hepaticus have been identified, they have not been further studied and characterized, as appropriate genetic tools and methods to transform and perform mutagenesis studies in C. hepaticus have not been available. In this study, the genetic manipulation of C. hepaticus is reported, with the development of novel plasmid vectors, methods for transformation, site-specific mutagenesis, and mutant complementation. These tools were used to delete the pglB gene , an oligosaccharyltransferase, a central enzyme of the N- glycosylation pathway, by allelic exchange. In the mutant strain, N -glycosylation was completely abolished. The tools and methods developed in this study represent innovative approaches that can be applied to further explore important virulence factors of C. hepaticus and other closely related Campylobacter species., Importance: Spotty liver disease (SLD) of layer chickens, caused by infection with Campylobacter hepaticus , is a significant economic and animal welfare burden on an important food production industry. Currently, SLD is controlled using antibiotics; however, alternative intervention methods are needed due to increased concerns associated with environmental contamination with antibiotics, and the development of antimicrobial resistance in many bacterial pathogens of humans and animals. This study has developed methods that have enabled the genetic manipulation of C. hepaticus . To validate the methods, the pglB gene was inactivated by allelic exchange to produce a C. hepaticus strain that could no longer N -glycosylate proteins. Subsequently, the mutation was complemented by reintroduction of the gene in trans , on a plasmid vector, to demonstrate that the phenotypic changes noted were caused by the mutation of the targeted gene. The tools developed enable ongoing studies to understand other virulence mechanisms of this important emerging pathogen., Competing Interests: D.M.A. is an employee of Bioproperties Pty Ltd, a veterinary vaccine business that manufactures and sells live vaccines for production animals. Therefore, he has a commercial interest in understanding the biology of veterinary pathogens such as Campylobacter hepaticus. All other authors declare no conflict of interest.

ABSTRACT: Spotty liver disease (SLD) is a serious condition affecting extensively housed laying hens. The causative bacterium was described in 2015 and characterized in 2016 and named Campylobacter hepaticus. Antibiotics are the only tool currently available to combat SLD. However, antimicrobial resistance has already been detected, so finding therapeutic alternatives is imperative. Isoquinoline alkaloids (IQA), such as sanguinarine and chelerythrine, have been shown to have immunomodulatory effects. It has been hypothesized that IQA could ameliorate some of the deleterious effects of SLD. This study aimed to address that hypothesis in an experimental disease induction model. Birds were fed with diets containing 2 different doses of an IQA containing product, 100 mg of product/kg of feed (0.5 ppm of sanguinarine) and 200 mg of product/kg of feed (1.0 ppm of sanguinarine). Two additional groups remained untreated (a challenged positive control and an unchallenged negative control). After 4 wk of treatment, birds from all groups except the negative control group were exposed to C. hepaticus strain HV10. The IQA treated groups showed a reduction in the number of miliary lesions on the liver surface and reduced lesion scores compared with untreated hens. A significant reduction of egg mass was detected 6 d after exposure to C. hepaticus in the untreated group (P = 0.02). However, there was not a significant drop in egg-mass in the IQA groups, especially those fed with a high dose of IQA (P = 0.93). IQA supplementation did not produce significant changes in intestinal villus height and crypt depth but did result in a significant reduction in the proinflammatory cytokine, interleukin-8, in the blood (P < 0.01). Microbiota analysis showed that IQA treatment did not alter the alpha diversity of the cecal microbiota but did produce changes in the phylogenetic structure, with the higher dose of IQA increasing the Firmicutes/Bacteroidetes ratio.Other minor changes in production indicators included an increase in feed consumption (P < 0.01) and an increase in body weight of the treated hens (P < 0.0001). The present study has demonstrated that IQA confers some protection of chickens from the impact of SLD.

Spotty liver disease (SLD) causes substantial egg production losses and chicken mortality; therefore, it is a disease that concerns Australian egg farmers. Over the last few decades, much research has been conducted to determine the etiologic agents of SLD and to develop potential therapeutics; however, SLD still remains a major issue for the chicken industries globally and remained without the elucidation of potentially multiple pathogens involved. To help fill this gap, this study was aimed at understanding the viral diversity of bile samples from which the SLD-causing bacterium, Campylobacter hepaticus, has been isolated and characterised. The collected samples were processed and sequenced using high-throughput next-generation sequencing. Remarkably, this study found 15 galliform chaphamaparvoviruses (GaChPVs), of which 14 are novel under the genus Chaphamaparvovirus. Among them, nine were complete genomes that showed between 41.7% and 78.3% genome-wide pairwise similarities to one another. Subsequent phylogenetic analysis using the NS1 gene exhibited a multiple incursion of chaphamaparvovirus lineages, including a novel lineage of unknown ancestral history in free-range laying chickens in Australia. This is the first evidence of circulating many parvoviruses in chickens in Australia, which has increased our knowledge of the pathogen diversity that may have an association with SLD in chickens.

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.

Spotty Liver Disease (SLD) is an emerging disease of serious concern in the egg production industry, as it causes significant egg loss and mortality in layer hens. The causative agent is a newly identified Gram-negative bacterium, Campylobacter hepaticus, and knowledge about C. hepaticus pathogenesis and the potential for vaccine development is still in its infancy. Current detection methods for SLD, such as PCR and culturing, only detect an active infection and will not give any indication of a past infection from which the bacteria have been cleared. An immunological assay, on the other hand, can provide information on previous infections and therefore is crucial in vaccine development against SLD. In the present study, we have developed the first immunoassay capable of detecting C. hepaticus-specific antibodies present in the sera of infected birds. The assay uses C. hepaticus total protein extract (TPE) as the antigen coating on enzyme-linked immunosorbent assay (ELISA) plates. The cross reactivity of C. hepaticus antibodies with closely related C. jejuni and C. coli antigens was successfully overcome by pre-absorbing the sera using C. jejuni cell extracts. The assay was validated using sera samples from both naturally- and experimentally-infected birds, birds vaccinated with formalin-killed bacteria, and serum samples from SLD-negative birds (control group). The optimized ELISA assay had 95.5% specificity and 97.6% sensitivity. The immunoassay provides a useful tool for monitoring the exposure of poultry flocks to C. hepaticus infection and can be used to direct and support vaccine development. HIGHLIGHTS The first immunoassay developed for Spotty Liver Disease (SLD). A useful method for detecting C. hepaticus-specific antibodies in birds. Highly specific (95.5%) and sensitive (97.6%) assay. A key tool for use in epidemiological studies and vaccine development. [ABSTRACT FROM AUTHOR]

Spotty liver disease (SLD) is recognized to be caused by infection with Campylobacter hepaticus in adult layer hens farmed in cage-free environments. SLD is an emerging disease as cage-free egg production increases in popularity in response to desires for improved welfare of poultry. Outbreaks of SLD are frequently experienced around peak egg production in flocks, commonly between 25 and 40 wk of age. The disease becomes manifest with increased exposure and access of the birds to the feces of the flock. This study follows from a previous epidemiological survey of free-range and barn flocks in Australia which identified the presence of a scratch area within the laying house as a major risk factor for the occurrence of SLD. However, that survey also observed SLD occurrence in 45% of houses with a fully slatted floor (no scratch area). The present study describes a further analytical survey aimed at identification of risk factors for SLD in houses with fully slatted flooring. A comprehensive questionnaire was completed for 49 cage-free flocks from point of lay until 40 wk of age across Australia, retrieving information on house design, bird breed, flock size, stocking densities, bird growth, and performance and the occurrence of SLD. Multiple logistic regression model building was used to separate factors and identify important management factors that may be amenable to modify the occurrence of SLD in egg layers. Key determinants of SLD identified from the analyses were that houses with mechanical ventilation (such as tunnel ventilation) have some protection from SLD and an increase of an extra 1 bird/m2 of nest space increased odds of occurrence of SLD by 1.172 times. A recommendation to not exceed 112 brown egg layer hens/m2 of nest space in naturally ventilated houses with a full slat floor was suggested. A delay in birds reaching 60% hen day production (HD) by 1 wk is suggested as a possible predictor for a subsequent outbreak of SLD. [ABSTRACT FROM AUTHOR]

Spotty Liver Disease (SLD) is a serious problem in laying hens farmed in cage-free systems. The causative organism, Campylobacter hepaticus , is regarded as having a fecal-oral method of transmission and hence may build up and spread readily in housing systems which allow ease of direct contact of hens with the flock's fecal material. The epidemiology of SLD has not been thoroughly investigated. An initial cross-sectional analytical epidemiological survey of SLD in free range and barn layer systems was conducted in Australia over 2019 to 2021.The survey involved rearing flocks (n = 32) which were then followed through into laying flocks (n = 24) up to 40 wk of age. Cloacal swabs were collected during rearing and lay for C. hepaticus detection by PCR. Flocks were classified as "Cases" (n = 18) where clinical SLD according to the case definition was observed or "Controls" (n = 6) which were clinically unaffected. No C. hepaticus was detected in cloacal swabs from rearing houses whereas the organism was detected in 18 Case flocks in lay and from 2 Control flocks in lay. All layer houses that incorporated a scratch area (n = 13) were categorized as Cases. Thus, having a scratch area is a key determinant for SLD and no analyses of further contributory factors from these flocks were able to be made. Of the remaining 11 flocks which had floors fully covered by slats, 5 were Cases (45%). Further risk factor analysis was compromised by this small sample size and identification of other significant associations was not possible. A larger survey investigating flocks laying in houses with fully slatted floors was undertaken to further the understanding of SLD epidemiology and is reported in a companion paper. [ABSTRACT FROM AUTHOR]

A Campylobacter species was first described as the etiological agent of Spotty Liver Disease (SLD) in 2015 and subsequently named as Campylobacter hepaticus in 2016. The bacterium predominantly affects barn and/or free-range hens at peak lay, is fastidious and difficult to isolate, which has impeded elucidation of its sources, means of persistence and transmission. Ten farms from South-Eastern Australia, of which 7 were free range entities participated in the study. A total of 1,404 specimens from layers and 201 from environmental sources, were examined for the presence of C. hepaticus. In this study, our principal findings included the continuing detection of C. hepaticus infection in a flock following an outbreak, indicating a possible transition of infected hens to asymptomatic carriers, that was also characterized by no further occurrence of SLD in the flock. We also report that the first outbreaks of SLD on newly commissioned free-range farms affected layers ranging from 23 to 74 wk of age, while subsequent outbreaks in replacement flocks on these farms occurred during the more conventional peak lay period (23–32 wk of age). Finally, we report that in the on-farm environment, C. hepaticus DNA was detected in layer feces, inert elements such as stormwater, mud, soil, as well as in fauna such as flies, red mites, Darkling beetles, and rats. While in off-farm locations, the bacterium was detected in feces from a variety of wild birds and a canine. [ABSTRACT FROM AUTHOR]

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.

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 acutemortality 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. [ABSTRACT FROM AUTHOR]

Campylobacter hepaticus was recently identified as the aetiological agent of Spotty Liver Disease (SLD). SLD causes significant health and productivity losses in the Australian egg industry and the disease is present in other countries. Following the isolation and characterization of C. hepaticus, molecular tools and refined culturing methods have been developed to identify the pathogen. It is suspected that the application of these tools will lead to identification of the pathogen in many poultry production systems throughout the world. As C. hepaticus has only recently been identified, little is known about the mechanisms of pathogenesis and, hence, new research needs to be directed towards understanding SLD epidemiology and C. hepaticus virulence mechanisms to inform efforts to develop intervention strategies. [ABSTRACT FROM AUTHOR]

Campylobacter hepaticus is the aetiological agent of Spotty Liver Disease (SLD). SLD can cause significant production loss and mortalities among layer hens at and around peak of lay. We previously developed an enzyme linked immunosorbent assay (ELISA), SLD-ELISA1, to detect C. hepaticus specific antibodies from bird sera using C. hepaticus total proteins and sera pre-absorbed with Campylobacter jejuni proteins. The high specificity achieved with SLD-ELISA1 indicated the presence of C. hepaticus specific antibodies in sera of infected birds. However, some of the reagents used in SLD-ELISA1 are time consuming to prepare and difficult to quality control. This understanding led to the search for C. hepaticus specific immunogenic proteins that could be used in recombinant forms as antibody capture antigens in immunoassay design. In this study, an immunoproteomic approach that combined bioinformatics analysis, western blotting, and LC MS/MS protein profiling was used, and a fragment of filamentous hemagglutinin adhesin (FHA), FHA 1,628-1,899 with C. hepaticus specific antigenicity was identified. Recombinant FHA 1,628-1,899 was used as antigen coating on ELISA plates to capture FHA 1,628-1,899 specific antibodies in sera of infected birds. SLD-ELISA2, based on the purified recombinant FHA fragment, is more user-friendly and standardizable than SLD-ELISA1 for screening antibody responses to C. hepaticus exposure in hens. This study is the first report of the use of FHA from a Campylobacter species in immunoassays, and it also opens future research directions to investigate the role of FHA in C. hepaticus pathogenesis and its effectiveness as a vaccine candidate., Competing Interests: JQ, AA, TW, and PS were employed by Scolexia Pty Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Muralidharan, Quinteros, Anwar, Wilson, Scott, Moore and Van.)

Spotty liver disease (SLD) causes substantial egg production losses and chicken mortality; therefore, it is a disease that concerns Australian egg farmers. Over the last few decades, much research has been conducted to determine the etiologic agents of SLD and to develop potential therapeutics; however, SLD still remains a major issue for the chicken industries globally and remained without the elucidation of potentially multiple pathogens involved. To help fill this gap, this study was aimed at understanding the viral diversity of bile samples from which the SLD-causing bacterium, Campylobacter hepaticus , has been isolated and characterised. The collected samples were processed and sequenced using high-throughput next-generation sequencing. Remarkably, this study found 15 galliform chaphamaparvoviruses (GaChPVs), of which 14 are novel under the genus Chaphamaparvovirus . Among them, nine were complete genomes that showed between 41.7% and 78.3% genome-wide pairwise similarities to one another. Subsequent phylogenetic analysis using the NS1 gene exhibited a multiple incursion of chaphamaparvovirus lineages, including a novel lineage of unknown ancestral history in free-range laying chickens in Australia. This is the first evidence of circulating many parvoviruses in chickens in Australia, which has increased our knowledge of the pathogen diversity that may have an association with SLD in chickens.

Spotty liver disease (SLD) caused by Campylobacter hepaticus affects the health and productivity of layer hens and is a disease of concern in poultry. In this study, blood and cloacal swab samples were collected from 709 birds across 11 free-range layer farms from different regions of Australia. The prevalence of C. hepaticus specific antibodies and DNA was assessed using a C. hepaticus specific ELISA and PCR and its correlation with mortalities and changes in egg production was analyzed to better understand the seroprevalence of C. hepaticus in Australian free-range layer farms. C. hepaticus specific antibodies were detected from birds in four of the five farms that had no history of SLD with seroprevalence as high as 41% in one of the farms. Seroprevalence of anti- C. hepaticus antibodies among flocks that had an active or previous SLD outbreak varied between 2 and 64%. C. hepaticus DNA was detected from birds in three farms with no known SLD history and five farms with confirmed SLD outbreaks. A good correlation was observed between the ELISA and PCR results with a Pearson correlation coefficient value of 0.85 ( p -value = 0.001). No correlation was observed between the flock size or flock age and ELISA or PCR outcomes, and no significant difference between the seroprevalence of anti- C. hepaticus antibodies among flocks with or without a known history of SLD was established ( p = 0.143). This study demonstrates the usefulness of C. hepaticus specific ELISA and PCR in identifying the occurrence of mild or sub-clinical SLD and provides a broader and more complete understanding of SLD epidemiology that will inform future research aimed at the development of methods to control SLD, such as appropriate biosecurity measures, vaccines, and feed additives., Competing Interests: Authors JH, AA, and PS were employed by Scolexia Pty Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Muralidharan, Huang, Anwar, Scott, Moore and Van.)

Spotty Liver Disease (SLD) is a serious infectious disease which occurs mainly in laying chickens in free range production systems. SLD outbreaks can increase mortality and decrease egg production of chickens, adversely impact welfare and cause economic hardship for poultry producers. The bacterium Campylobacter hepaticus is the primary cause of the disease. This study aimed to identify the effects of C. hepaticus on chicken gut microbiota and gut structure. Three C. hepaticus strains (HV10 T , NSW44L and QLD19L), isolated from different states of Australia, were used in the study. Chickens at 26-weeks post-hatch were orally dosed with one of the C. hepaticus strains (challenged groups) or Brucella broth (unchallenged or control group). Six days after the challenge, birds were necropsied to assess liver damage, and caecal content and tissue samples were collected for histology, microbiology, and 16S rRNA gene amplicon sequencing to characterize the composition of the bacterial microbiota. Strain C. hepaticus NSW44L produced significantly more disease compared to the other C. hepaticus strains and this coincided with more adverse changes observed in the caecal microbiota of the birds challenged with this strain compared to the control group. Microbial diversity determined by Shannon and Simpson alpha diversity indices was lower in the NSW44L challenged groups compared to the control group ( p = 0.009 and 0.0233 respectively, at genus level). Short-chain fatty acids (SCFAs) producing bacteria Faecalibacterium, Bifidobacterium and Megamonas were significantly reduced in the challenged groups compared to the unchallenged control group. Although SLD-induction affected the gut microbiota of chickens, their small intestine morphology was not noticeably affected as there were no significant differences in the villus height or ratio of villus height and crypt depth. As gut health plays a pivotal role in the overall health and productivity of chickens, approaches to improve the gut health of the birds during SLD outbreaks such as through diet and keeping the causes of stress to a minimum, may represent significant ways to alleviate the impact of SLD., Competing Interests: Authors AA, TW, and PS were employed by Scolexia Pty Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Van, Lee Nen That, Perera, Anwar, Wilson, Scott, Stanley and Moore.)

Spotty liver disease (SLD) is a serious condition affecting extensively housed laying hens. The causative bacterium was described in 2015 and characterized in 2016 and named Campylobacter hepaticus. Antibiotics are the only tool currently available to combat SLD. However, antimicrobial resistance has already been detected, so finding therapeutic alternatives is imperative. Isoquinoline alkaloids (IQA), such as sanguinarine and chelerythrine, have been shown to have immunomodulatory effects. It has been hypothesized that IQA could ameliorate some of the deleterious effects of SLD. This study aimed to address that hypothesis in an experimental disease induction model. Birds were fed with diets containing 2 different doses of an IQA containing product, 100 mg of product/kg of feed (0.5 ppm of sanguinarine) and 200 mg of product/kg of feed (1.0 ppm of sanguinarine). Two additional groups remained untreated (a challenged positive control and an unchallenged negative control). After 4 wk of treatment, birds from all groups except the negative control group were exposed to C. hepaticus strain HV10. The IQA treated groups showed a reduction in the number of miliary lesions on the liver surface and reduced lesion scores compared with untreated hens. A significant reduction of egg mass was detected 6 d after exposure to C. hepaticus in the untreated group (P = 0.02). However, there was not a significant drop in egg-mass in the IQA groups, especially those fed with a high dose of IQA (P = 0.93). IQA supplementation did not produce significant changes in intestinal villus height and crypt depth but did result in a significant reduction in the proinflammatory cytokine, interleukin-8, in the blood (P < 0.01). Microbiota analysis showed that IQA treatment did not alter the alpha diversity of the cecal microbiota but did produce changes in the phylogenetic structure, with the higher dose of IQA increasing the Firmicutes/Bacteroidetes ratio. Other minor changes in production indicators included an increase in feed consumption (P < 0.01) and an increase in body weight of the treated hens (P < 0.0001). The present study has demonstrated that IQA confers some protection of chickens from the impact of SLD. [ABSTRACT FROM AUTHOR]

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., (Copyright © 2020 Phung, Vezina, Anwar, Wilson, Scott, Moore and Van.)

[This corrects the article on p. 354 in vol. 7, PMID: 28848714.].