Campylobacter Hepaticus Research Articles

Development of tools for the genetic manipulation of Campylobacter and their application to the N-glycosylation system of Campylobacter hepaticus, an emerging pathogen of poultry.

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.

Descriptive epidemiology of spotty liver disease in Australian cage-free brown egg layer chicken flocks with a scratch area

Spotty Liver Disease (SLD), caused by Campylobacter hepaticus or C. bilis infection in adult female chickens continues to emerge as a major disease problem in cage-free production systems. Free range production has become the predominant system in Australian egg production and SLD is widespread in these farms. Previous studies have identified having a scratch area as a key determinant for SLD occurrence. An Australia-wide survey of egg production flocks with scratch areas was conducted regarding SLD including 48 individual flocks. Descriptive information on the facilities and flock management practices was reported. The incidence of SLD, age of first outbreak, initial mortality rate, duration of elevated mortality, and magnitude and duration of any associated egg production decline are described. Recurrence of SLD in the same flock was also reported and discussed. Therapies applied were recorded and assessed across SLD severity and duration. SLD occurred in 66.7% of layer flocks whose facility included a scratch area. Recurrent SLD outbreaks occurred in 31% of flocks experiencing SLD. Antibiotic medication reduced duration of mortality and egg production decline. Antibiotic therapy was associated with reduced duration of mortality and a less severe and shorter duration of egg production drops compared to untreated flocks. PCR detection of C. hepaticus in cloacal swabs and house dust samples and a serological ELISA test were compared and evaluated as diagnostic aids or as possible predictors of SLD outbreaks. The ELISA showed substantial agreement with detection of C. hepaticus in cloacal swabs by PCR. Examining composite house dust samples by PCR for C. hepaticus DNA appeared to be the most convenient and cost-effective aid to diagnosis and as a putative predictor for SLD outbreaks.

Investigation into the efficiency of diverse N-linking oligosaccharyltransferases for glycoengineering using a standardised cell-free assay.

The application of bacterial oligosaccharyltransferases (OSTs) such as the Campylobacter jejuni PglB for glycoengineering has attracted considerable interest in glycoengineering and glycoconjugate vaccine development. However, PglB has limited specificity for glycans that can be transferred to candidate proteins, which along with other factors is dependent on the reducing end sugar of glycans. In this study, we developed a cell-free glycosylation assay that offers the speed and simplicity of a 'yes' or 'no' determination. Using the assay, we tested the activity of eleven PglBs from Campylobacter species and more distantly related bacteria. The following assorted glycans with diverse reducing end sugars were tested for transfer, including Streptococcus pneumoniae capsule serotype 4, Salmonella enterica serovar Typhimurium O antigen (B1), Francisella tularensis O antigen, Escherichia coli O9 antigen and Campylobacter jejuni heptasaccharide. Interestingly, while PglBs from the same genus showed high activity, whereas divergent PglBs differed in their transfer of glycans to an acceptor protein. Notably for glycoengineering purposes, Campylobacter hepaticus and Campylobacter subantarcticus PglBs showed high glycosylation efficiency, with C. hepaticus PglB potentially being useful for glycoconjugate vaccine production. This study demonstrates the versatility of the cell-free assay in rapidly assessing an OST to couple glycan/carrier protein combinations and lays the foundation for future screening of PglBs by linking amino acid similarity to glycosyltransferase activity.

Developing a selective culturing approach for Campylobacter hepaticus.

Campylobacter hepaticus, the causative agent of Spotty Liver Disease (SLD) is an important disease in cage-free egg producing chickens causing mortality and production drops. C. hepaticus is a slow growing Campylobacter easily overgrown by fecal bacteria. It is currently only reliably isolatable from bile samples. A selective media for isolation from feces or environment would assist diagnosis and impact assessment. Growth of five Australian C. hepaticus isolates was studied using Horse blood agar (HBA), sheep blood agar (SBA), Bolton, Preston and Brain Heart Infusion (BHI) base media. Blood and/or bile were added to Bolton, Preston and BHI medias. C. jejuni was used as a positive control. Plates were incubated in duplicate under microaerophilic conditions at 42°C for 10 days and examined at days 3-5 and 7-10 of incubation. Each isolate was examined for sensitivity to 14 antimicrobials using HBA sensitivity plates. Growth was inhibited by BHI and by added bile, while blood improved growth. Further replicates using SBA, HBA, Bolton and Preston media showed best growth on Bolton agar with blood. All five C. hepaticus isolates were resistant to trimethoprim and vancomycin, while four were also resistant to rifampicin and bacitracin. Media based upon Bolton plus blood supplemented with vancomycin and trimethoprim might be used as the most appropriate media for selective growth of C. hepaticus. The addition of bile to media for C. hepaticus isolation and growth will inhibit growth and is not advised.

Identification of epidemiological risk factors for spotty liver disease in cage-free layer flocks in houses with fully slatted flooring in Australia

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.

Scratch area as an epidemiological risk factor for Spotty Liver Disease in cage-free layers in Australia

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.

Comparative genomic analysis of Campylobacter hepaticus genomes associated with spotty liver disease, Georgia, United States.

Campylobacter hepaticus has re-emerged as an important cause of disease in egg laying birds worldwide, resulting in morbidity, mortality, and significant losses in eggs for the breeding and table egg laying industries. Although birds may appear asymptomatic, the disease is characterized by spots on the liver of birds and histopathological analysis reveals multifocal fibrogranulocytic necrotizing hepatitis microscopically. The re-emergence of C. hepaticus may be linked with housing practices as the disease appears more prevalent in pasture raised birds with outside exposure. Here we describe, the whole genome sequences and comparative analysis of four C. hepaticus genomes associated with an outbreak on pasture raised breeders from a farm in Georgia, United States. All four genomes were relatively similar in size and virulence genes harbored. Using these genomes, comparison with current C. hepaticus genomes available in NCBI and other databases and other members of the Campylobacter species was carried out. Using current tools available, virulence gene factor content was compared, and it was found that different tools lead to different numbers of factors identified. The four genomes from this study were relatively similar to C. hepaticus HV10 the type strain from Australia but differed from the other sequenced US strains from Iowa and Florida. C. hepaticus was found to have an overall lower gene content for genes associated with virulence and iron acquisition compared to other Campylobacter genomes and appears to cluster differently than UK genomes on phylogenetic analysis, suggesting the emergence of two lineages of C. hepaticus. This analysis provides valuable insight into the emerging pathogen C. hepaticus, its virulence factors and traits associated with disease in poultry production in the US, potentially providing insight into targets for its control and treatment for laying birds. Our analysis also confirms genes associated with iron acquisition are limited and the presence of the multidrug efflux pump CmeABC in C. hepaticus which may promote survival and persistence in the host niche - the chicken liver/bile. One unique aspect of this study was the finding of a close genetic relationship between C. hepaticus and Campylobacter fetus species and evidence of genome reduction in relation to host niche specificity.

Campylobacteriosis in Poultry: A Review

Campylobacter is common in poultry, including layer and broiler chickens, geese, ducks, and turkeys. This review aimed to emphasize the prevalence of campylobacteriosis, recent poultry diagnoses, and strict prevention measures. Campylobacter species colonize the intestines of poultry and waterfowl but are generally nonpathogenic in poultry. However, they are the most common bacterial cause of sporadic human enteritis in both developed and developing countries. The main species responsible for campylobacteriosis is Campylobacter jejuni, followed by Campylobacter coli. A number of other Campylobacter species, such as Campylobacter lari, fetus, upsaliensis, and hyointestinalis are rarely associated with campylobacteriosis. Campylobacter hepaticus is the species linked to spotty liver disease in layers and breeder chickens, and it may be the etiological agent of the disease previously known as avian vibrionic hepatitis. The most prevalent infection source for Campylobacter is environmental contamination from poultry droppings. However, some Campylobacter species can be transmitted vertically, either on the surface of eggs or via trans-ovarian transmission in addition to consumption of contaminated feed or water. Due to the non-specific clinical signs such as diarrhea and weight loss, diagnosing campylobacteriosis in poultry requires culture or polymerase chain reaction tests. Little is known about the available vaccine or effective antibiotic treatment due to the rapid development of antibiotic resistance. Therefore, strict biosecurity measures play a crucial role in preventing Campylobacter infection in commercial poultry. These measures include decontaminating housing between flocks, preventing the entry of rodents, wild birds, and animals, and eradicating insects. To control campylobacteriosis and reduce infection risks, it is important to implement efficient on-farm biosecurity measures, conduct regular inspections of workers at meat processing plants and poultry farms, and ensure thorough preparation of chicken meat and eggs before consumption. These measures are vital in minimizing the Campylobacter transmission from both broiler and laying chickens, thereby reducing the risk of foodborne diseases caused by contaminated food.

Investigation of Spotty Liver Disease and Campylobacter hepaticus in Layer Flocks-A Field Study.

Campylobacter hepaticus (C. hepaticus) was recently discovered as the causative agent of Spotty Liver Disease (SLD). SLD affects laying hens and causes significant economic losses in egg production in several countries throughout the world. Field observations reveal that cases of SLD appear with a high risk of reoccurrence, specifically in free-range and organic brown-feathered layer lines. Possible factors contributing to the development of SLD still have to be elucidated. In this field study, one free range (Flock 1) and one organic flock (Flock 2) of brown laying hens kept on farms with a history of clinical SLD were monitored for C. hepaticus colonization, clinical signs, and egg production from 16 to 79 wk of age on the first farm and from 17 to 83 wk of age on the other. The flocks showed a significant drop in egg production at 32 to 39 or 56 wk of age, respectively, which was associated with macroscopically visible liver lesions typical for SLD. Interestingly, in both cases observed clinical disease was linked to a stressful event: heat stress for Flock 1 and respiratory symptoms for Flock 2. C. hepaticus was detected by PCR during the acute phase of the disease in Flock 1. At 50 wk after the initial clinical outbreak had waned, C. hepaticus was still able to be isolated by culture in this flock. This clearly demonstrates that C. hepaticus persists either in the birds or their environment. We speculate that this long persistence may favor chronic SLD in affected flocks and the reoccurrence of SLD in subsequent flocks. Clinically less severe SLD outbreaks may be observed after re-exposure of clinically recovered flocks.

First Report of Campylobacter hepaticus Isolation in Laying Hens and Broiler Breeders with Spotty Liver Disease in Costa Rica.

Poultry producers in Costa Rica have informally reported a spotty liver disease-like syndrome for more than 20 yr. However, despite many attempts, the infectious agent responsible for this syndrome had not been identified. Therefore, following current knowledge of spotty liver disease diagnosis, we invited veterinarians and poultry producers to submit samples to the diagnostic laboratories of the Veterinary Medicine School, Universidad Nacional, to identify the infectious agent of this syndrome. Veterinarians and poultry producers were instructed to collect gallbladders and livers aseptically and send them for pathology examinations and bacterial cultures in less than 24 hr after collection. Samples were processed for standard histopathologic studies and cultured under aerophilic, anaerobic, and microaerophilic conditions. Campylobacter-like colonies were isolated and identified by biochemical and PCR tests. Here we report for the first time the isolation, biochemical characterization, and molecular confirmation of Campylobacter hepaticus in laying hens and broiler breeders with spotty liver disease in Costa Rica.

Campylobacter hepaticus in the Production Environment and Stagnant Water as a Potential Source of C. hepaticus Causing Spotty Liver Disease in Free-Range Laying Hens in Georgia, United States.

Spotty liver disease (SLD) has emerged as an important cause of disease in egg-producing flocks in countries such as the United Kingdom and Australia and has emerged in the United States. The organisms implicated in SLD include Campylobacter hepaticus and, more recently, Campylobacter bilis. These organisms have been found to cause focal lesions on the livers of infected birds. Campylobacter hepaticus infection results in reduced egg production, decreased feed consumption resulting in reduced egg size, and increased mortality of highly valuable hens. In the fall of 2021, birds from two flocks (A and B) of organic pasture-raised laying hens were submitted to the Poultry Diagnostic Research Center at the University of Georgia with a history suspicious of SLD. Postmortem examination of Flock A found 5/6 hens had small multifocal lesions on the liver and were PCR positive for C. hepaticus from pooled swab analysis of samples of the liver and gall bladder. Necropsy of Flock B found 6/7 submitted birds had spotty liver lesions. In pooled bile swabs, 2/7 hens from Flock B were also PCR positive for C. hepaticus. A follow-up visit to Flock A was scheduled 5 days later, as well as a visit to a flock where SLD has not been reported (Flock C), which was used as a comparative control. Samples of the liver, spleen, cecal tonsil, ceca, blood, and gall bladder were collected from six hens per house. Additionally, feed, water nipples, and environmental water (stagnant water outside the house) were collected from the affected farm and the control farm. To detect the organism, all samples collected were subjected to direct plating on blood agar and enrichment in Preston broth with incubation under microaerophilic conditions. After multiple phases of bacterial culture purification from all samples, single bacterial cultures displaying characteristics of C. hepaticus were tested by PCR to confirm identity. From Flock A, liver, ceca, cecal tonsils, gall bladder, and environmental water were PCR positive for C. hepaticus. No positive samples were detected in Flock C. After another follow-up visit, 10 wk later, Flock A was PCR positive for C. hepaticus from gall bladder bile and feces and one environmental water sample displayed a weak positive reaction for C. hepaticus. Flock C was PCR negative for C. hepaticus. To gain more knowledge about C. hepaticus prevalence, a survey of 6 layer hens from 12 different layer hen flocks between the ages of 7 to 80 wk, raised in different housing systems, were tested for C. hepaticus. The 12 layer hen flocks were culture and PCR negative for C. hepaticus. Currently, there are no approved treatments for C. hepaticus and no vaccine is available. The results of this study suggest that C. hepaticus may be endemic in some areas of the United States, and free-range laying hens may be exposed from the environment/stagnant water in areas where they range.

Complete Genome Sequence of Campylobacter hepaticus RBCL71delta, Associated with Spotty Liver Disease in Organic Pasture-Raised Laying Hens in Georgia, USA.

Spotty liver disease (SLD) is as an important cause of disease in egg-producing flocks. The organisms implicated include Campylobacter hepaticus and Campylobacter bilis. Here, we report the complete genome sequence of C. hepaticus strain RBCL71delta and three additional strains, isolated from a layer flock in Georgia, USA.

Detection, characterization, and persistence of Campylobacter hepaticus, the cause of spotty liver disease in layer hens

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.

Characterisation of N-linked protein glycosylation in the bacterial pathogen Campylobacter hepaticus

Campylobacter hepaticus is an important pathogen which causes Spotty Liver Disease (SLD) in layer chickens. SLD results in an increase in mortality and a significant decrease in egg production and therefore is an important economic concern of the global poultry industry. The human pathogen Campylobacter jejuni encodes an N-linked glycosylation system that plays fundamental roles in host colonization and pathogenicity. While N-linked glycosylation has been extensively studied in C. jejuni and is now known to occur in a range of Campylobacter species, little is known about C. hepaticus glycosylation. In this study glycoproteomic analysis was used to confirm the functionality of the C. hepaticus N-glycosylation system. It was shown that C. hepaticus HV10T modifies > 35 proteins with an N-linked heptasaccharide glycan. C. hepaticus shares highly conserved glycoproteins with C. jejuni that are involved in host colonisation and also possesses unique glycoproteins which may contribute to its ability to survive in challenging host environments. C. hepaticus N-glycosylation may function as an important virulence factor, providing an opportunity to investigate and develop a better understanding the system’s role in poultry infection.

The use of filamentous hemagglutinin adhesin to detect immune responses to Campylobacter hepaticus infections in layer hens.

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.

Campylobacter bilis, the second novel Campylobacter species isolated from chickens with Spotty Liver Disease, can cause the disease

Spotty Liver Disease (SLD) is a significant disease of commercial layer hens. It can cause up to 10 % flock mortalities and reduce egg production by 25 %. Campylobacter hepaticus has been identified as the main cause of the disease, although it also appears that predisposing factors, such as some form of stress, may increase the likelihood of clinical disease occurring. Recently, a newly identified species, Campylobacter bilis, was isolated from bile samples of clinical SLD affected chickens. To investigate the pathogenic potential of C. bilis two independent isolates were used in infection trials of layer hens. Within 6 days of oral challenge birds developed typical SLD liver lesions, demonstrating that both strains induced SLD. C. bilis could be recovered from all the challenged birds that developed SLD. Thus, each of the steps in Koch’s postulates have been fulfilled, confirming that C. bilis is an additional cause of SLD. A PCR method was developed which can specifically detect C. bilis from samples with complex microbiota. The identification of this newly discovered Campylobacter species as a second cause of SLD and the provision of a rapid method to detect the SLD causing bacterium will help with SLD vaccine development and epidemiology, thus assisting in the control of this important disease of poultry.

Unravelling Bile Viromes of Free-Range Laying Chickens Clinically Diagnosed with Spotty Liver Disease: Emergence of Many Novel Chaphamaparvoviruses into Multiple Lineages.

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.

Prevalence of Campylobacter hepaticus specific antibodies among commercial free-range layers in Australia.

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 adversely affect the gut microbiota of layers hen.

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.

Species diversity of dominant etiologically significant bacteria circulating in industrial poultry

The problem of the spread of bacterial infections of birds associated with opportunistic microorganisms occupies a significant place in the modern infectious pathology of birds.The aim of this work is to determine the species diversity of the dominant etiologically significant bacteria circulating in industrial poultry farming. Based on the results for the period 2019 to 2020, we carried out bacteriological studies of pathological material from birds in poultry farms of various technological directions.The dominant species are Escherichia coli, which accounts for 50.7%, Enterococcus faecalis - 25.4%, and Proteus mirabilis - 8.4%. Also, thermophilic Campylobacter spp. - 4.3% were isolated from the organs, including such epidemiologically dangerous species as Campylobacter jejuni. The microorganisms were virulent and differed in their cultural and biochemical properties.Cultures such as Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa, Enterococcus faecalis are isolated from many organs, which indicates the generalization of the infectious process, the culture of thermophilic Campylobacter spp. were isolated from the liver, which indicates the organotropy of these types of microorganisms.The species composition of the allocated microflora is determined by the epizootic situation in each individual farm, which results in a different percentage of the dominant species.Pure cultures of thermophilic campylobacter were obtained on selective Preston agar with cultivation for 7 days at a temperature of 42 ° C under microaerophilic conditions. However, culturing Campylobacter hepaticus is difficult because it is sensitive to a number of antimicrobial agents used in Campylobacter enrichment media and selective agar.The emergence of new Campylobacter species requires a revision of phenotypic identification algorithms. MALDI TOF mass spectrometry databases need to be updated and specific primers developed to identify new Campylobacter species.Significant variability of indicators of indication of one or another microflora from the liver and gallbladder indicates significant differences in the etiology of bacterial infections of birds.