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Studies of big liver and spleen disease virus of broiler breeder hens

Payne, Christine Jayne (2001) Studies of big liver and spleen disease virus of broiler breeder hens. PhD thesis, Murdoch University.

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Big liver and spleen disease (BLSD) is a disease of broiler breeders first recognised in Australia in 1980. The disease occurs in sexually mature hens and manifests as decreased egg production in association with a slight increase in mortality rates in affected flocks. The predominant lesions in sick hens or dead hens are splenomegaly and hepatomegaly. Previous to the studies reported in this thesis, the cause of the disease had not been identified, although a viral agent was suspected, and agar gel immunodiffusion (AGIO) tests had detected a disease-specific antigen in liver tissue of chickens during the early stages of the disease. The objective of the research reported in this thesis was the identification and characterisation of the putative virus associated with BLSD, tentatively designated big liver and spleen disease virus (BLSV).

Utilising the disease-specific antigen present in the liver of affected hens, which was purified by affinity chromatography, an antibody-detection enzyme-linked immunosorbent assay (ELISA) was developed that enabled the detection of disease-specific antibodies in affected and recovered chickens, presumed to be antibodies against BLSV. The antibody-detection ELISA was more sensitive than the AGIO used previously, and better suited to testing large numbers of samples. This ELISA antigen also stimulated a strong antibody response when used to immunise chickens and inoculated chickens demonstrated a rapid antigen clearance following challenge with infectious BLSV. A monoclonal antibody (mAb 1H4) against the BLSD-specific antigen was produced, and this monoclonal antibody was used to develop a BLSV-antigen-detection ELISA (ACELISA), and an immunoperoxidase technique for the detection of a BLSV-specific protein in cell cultures and tissue sections of affected chickens. BLSV was successfully cultivated in an in ovo system, which provided a means to quantitate infectious virus.

These techniques were then used to study physicochemical characteristics of the virus. Chloroform or ether treatment failed to inactivate infectivity, showing that BLSV was nonenveloped. The buoyant density was estimated to be 1.2 g/ml in continuous sucrose density gradients and the BLSV particle size was estimated to be between 50 and 100 nm by membrane filtration studies.

To further characterise the virus, molecular biological tools were applied to amplify and clone cDNA representing a fragment of the viral nucleic acid, and determine the genomic sequence of this fragment. The sequence obtained was submitted to a database for comparison to other known viral genomes to identify possible familial relationships. There was 62% similarity between BLSV sequence and a region of open reading frame 1 (ORF1) of hepatitis E virus (HEV; Accession no. X98292). A possible relationship between BLSV and human enterically-transmitted HEV, a non-enveloped, single-stranded, positive-sense RNA virus, was introduced as a hypothesis based on the sequence data.

The improved techniques developed were used to study the transmission and epidemiology of the virus. BLSV transmission studies in commercial flocks and in experimental conditions supported the hypothesis that BLSV is an enterically-transmitted virus. BLSV spread readily between chickens within individual sheds, from shed to shed on affected farms, and between experimentally infected and in-contact chickens. Hens experimentally inoculated by intravenous, oral, or conjunctival routes showed serological evidence of BLSV infection, and aerosol transmission occurred over limited distances. BLSV-antigen, detected by AC-ELISA, and infectivity were demonstrated in faeces from experimentally inoculated chickens.

Item Type: Thesis (PhD)
Murdoch Affiliation(s): Division of Veterinary and Biomedical Sciences
Notes: Note to the author: If you would like to make your thesis openly available on Murdoch University Library's Research Repository, please contact: Thank you.
Supervisor(s): Wilcox, Graham and Ellis, Trevor
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