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.

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.

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.

Enhancement of Campylobacter hepaticus culturing to facilitate downstream applications.

Campylobacter hepaticus causes Spotty Liver Disease (SLD) in chickens. C. hepaticus is fastidious and slow-growing, presenting difficulties when growing this bacterium for the preparation of bacterin vaccines and experimental disease challenge trials. This study applied genomic analysis and in vitro experiments to develop an enhanced C. hepaticus liquid culture method. In silico analysis of the anabolic pathways encoded by C. hepaticus revealed that the bacterium is unable to biosynthesise L-cysteine, L-lysine and L-arginine. It was found that L-cysteine added to Brucella broth, significantly enhanced the growth of C. hepaticus, but L-lysine or L-arginine addition did not enhance growth. Brucella broth supplemented with L-cysteine (0.4 mM), L-glutamine (4 mM), and sodium pyruvate (10 mM) gave high-density growth of C. hepaticus and resulted in an almost tenfold increase in culture density compared to the growth in Brucella broth alone (log10 = 9.3 vs 8.4 CFU/mL). The type of culture flask used also significantly affected C. hepaticus culture density. An SLD challenge trial demonstrated that C. hepaticus grown in the enhanced culture conditions retained full virulence. The enhanced liquid culture method developed in this study enables the efficient production of bacterial biomass and therefore facilitates further studies of SLD biology and vaccine development.

Isoquinoline alkaloids induce partial protection of laying hens from the impact of Campylobacter hepaticus (spotty liver disease) challenge.

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.

Evaluation of Residues in Hen Eggs After Exposure of Laying Hens to Water Containing Per- and Polyfluoroalkyl Substances.

Per- and polyfluoroalkyl substances (PFAS) have been used in aqueous film-forming foams used in firefighting, resulting in soil and groundwater contamination and leading to human exposure via animal products grown in contaminated areas. The present study reports the relationship between PFAS intake by hens and the PFAS concentrations in the edible parts of eggs. Laying hens were exposed via drinking water to different concentrations of 4 PFAS compounds (perfluorooctane sulfonate [PFOS], perfluorohexane sulfonate [PFHxS], perfluorooctanoic acid [PFOA], and perfluorohexanoic acid) over 61 d. Egg PFAS residues were assessed for a further 30 d after exposure ceased. The target concentrations of PFAS were 0, 0.3, 3, 30, and 300 µg/L for the treatment groups T1-T5, respectively; and PFAS residues were determined from the eggs collected every second day. There was a linear correlation between the PFAS concentrations in the drinking water of hens and those detected in the egg, which could be useful in estimating PFAS concentrations in the egg by measuring water concentrations. Exposure of hens to drinking water with PFAS concentrations below the Australian Government Department of Health limits (PFOS and PFHxS, 0.07 µg/L; PFOA, 0.56 µg/L), and with no other sources of PFAS exposure, is unlikely to result in egg PFAS concentrations that would exceed the 10% limit set by Food Standards Australia New Zealand for human consumption. Environ Toxicol Chem 2021;40:735-743. © 2020 SETAC.

Development of an enzyme-linked immunosorbent assay for detecting Campylobacter hepaticus specific antibodies in chicken sera - a key tool in Spotty Liver Disease screening and vaccine development.

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.

Ionic liquids based on azolate anions.

Compartmentalized molecular level design of new energetic materials based on energetic azolate anions allows for the examination of the effects of both cation and anion on the physiochemical properties of ionic liquids. Thirty one novel salts were synthesized by pairing diverse cations (tetraphenylphosphonium, ethyltriphenylphosphonium, N-phenyl pyridinium, 1-butyl-3-methylimidazolium, tetramethyl-, tetraethyl-, and tetrabutylammonium) with azolate anions (5-nitrobenzimidazolate, 5-nitrobenzotriazolate, 3,5-dinitro-1,2,4-triazolate, 2,4-dinitroimidazolate, 4-nitro-1,2,3-triazolate, 4,5-dinitroimidazolate, 4,5-dicyanoimidazolate, 4-nitroimidazolate, and tetrazolate). These salts have been characterized by DSC, TGA, and single crystal X-ray crystallography. The azolates in general are surprisingly stable in the systems explored. Ionic liquids were obtained with all combinations of the 1-butyl-3-methylimidazolium cation and the heterocyclic azolate anions studied, and with several combinations of tetraethyl- or tetrabutylammonium cations and the azolate anions. Favorable structure-property relationships were most often achieved when changing from 4- and 4,5-disubstituted anions to 3,5- and 2,4-disubstituted anions. The most promising anion for use in energetic ionic liquids of those studied here, was 3,5-dinitro-1,2,4-triazolate, based on its contributions to the entire set of target properties.