Development of immunological assays to assist the control of spotty liver disease in layer hens
Creators: name: Muralidharan, Chithralekha
Publisher: name: RMIT University
Year: 2024
Subjects: classificationCode: 300903
subject: Veterinary bacteriology
classificationCode: 310701
subject: Bacteriology

description: Spotty Liver Disease (SLD) is an emerging disease of serious concern in poultry and causes significant egg loss and mortality in layer hens, predominantly in free-range production systems. The causative agent was identified and named as a Gram-negative bacteria, Campylobacter hepaticus, six years ago. Knowledge about host immune responses, pathogenicity, reinfection potential, and vaccination prospects of this novel Campylobacter species is still in its infancy. The egg production industry worldwide has an urgent need for new control measures, such as vaccines, to prevent the onset and spread of SLD. Current detection methods for SLD, such as PCR and culturing can only detect an active infection and will not give any indication of a past infection from which the bacteria have been cleared. Immunological assays, on the other hand, can provide information on previous infections and therefore are crucial in assessing previous exposure of flocks and the effectiveness of infection trials and vaccines to be developed against SLD. The overarching aim of the work reported in this thesis was to develop immunological assays to detect C. hepaticus specific antibodies/antigens that may be present in sera, tissue, or body fluids of infected birds, and provide useful tools that can be used for monitoring the exposure of poultry flocks to C. hepaticus infection and to direct and support vaccine development.
 
Firstly, an enzyme-linked immunosorbent assay (ELISA) for detecting C. hepaticus specific antibodies in the sera of infected birds, SLD-ELISA1, was developed. The assay uses C. hepaticus total protein extract (TPE) as the antigen coating on ELISA plates. The cross-reactivity of C. hepaticus antibodies with closely related Campylobacter antigens was successfully overcome by pre-absorbing the sera with C. jejuni proteins. The assay was validated using sera samples from both naturally and experimentally infected birds. The optimized ELISA assay had 95.5% specificity and 97.6% sensitivity. 
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 to develop a second ELISA, named SLD-ELISA2. 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), FHA1628-1899 with C. hepaticus specific antigenicity was identified. Recombinant FHA1628-1899 was used as antigen coating on ELISA plates to capture FHA1628-1899 specific antibodies in sera of infected birds. SLD-ELISA2 is more user-friendly and standardizable than SLD-ELISA1 for screening antibody responses to C. hepaticus exposure in hens. This study also opened future research directions to investigate the role of FHA in C. hepaticus pathogenesis and its effectiveness as a vaccine candidate.

SLD-ELISA2 was then used to assay the prevalence of anti-C. hepaticus antibodies in Australian free-range layer farms. 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 analysed to better understand the seroprevalence of C. hepaticus in Australian free-range layer farms. C. hepaticus specific antibodies were detected in four out of five farms that had no history of SLD, with seroprevalence as high as 41% in one of the farms. The seroprevalence of anti-C. hepaticus antibodies varied between 2 and 64% in farms with active or previous SLD outbreaks. 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 seroprevalence of anti-C. hepaticus antibodies and C. hepaticus DNA with a Pearson correlation coefficient value of 0.85. There was no significant difference between the seroprevalence of anti-C. hepaticus antibodies among flocks with or without a known history of SLD (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.

Finally, a sandwich ELISA was developed using unconjugated (capture antibody) and HRP-conjugated (detection antibody) polyclonal antibodies generated against two recombinant FHA protein fragments FHA623-953 (F2) and FHA1628-1899 (F4). The optimal concentration of unconjugated anti-FHA-F2F4 polyclonal antibody coating was determined to be 16 µg/ml. C. coli and C. jejuni antigens did not cross-react with the polyclonal antibody pair and demonstrated the specificity of the assay. The limit of detection (LOD) of C. hepaticus whole cells was 0.6 x 107 cfu/ml and the LOD of total protein extracts from C. hepaticus was 1 µg/ml. The LOD of purified recombinant proteins was 0.005 µg/ml. When bile samples were used, only one out of five C. hepaticus culture positive bile samples tested positive in SLD-ELISA3 (20% assay sensitivity). SLD-ELISA3 did not distinguish liver extracts from C. hepaticus-positive and negative birds and indicated its limitation to test liver or other tissue samples. This is likely because the level of C. hepaticus in the tissue is lower than the detection limit. However, the polyclonal antibody produced for SLD-ELISA3 could be used to develop immunohistochemical assays for use on tissue sections to localise the surface exposed or secreted C. hepaticus FHA proteins.

The novel ELISAs developed in this study are valuable diagnostic tools for detecting the exposure of layer chickens to C. hepaticus to help in flock and environmental monitoring and management to reduce the impact of SLD. As C. bilis, a new bacterial species, has also been recently confirmed to cause SLD in several farms in Australia, the effectiveness of the assays developed in this study in detecting C. bilis antibodies needs to be evaluated.