Abstract
Malignant catarrhal fever (MCF) is a sporadic but fatal lymphoproliferative viral
disease of cattle, deer and other ruminants. The causative agents are highly-cellassociated herpesviruses of the subfamily gammaherpesvirinae including
Alcelaphine herpesvirus-1 (A1HV-1) and Ovine herpesvirus-2 (OvHV-2). The
identification of MCF-affected animals is based on the detection of antibodies to
A1HV-1 in a variety of assays including indirect-immunofluorescence tests (IFAT)
and a competitive-inhibition enzyme-linked immunosorbent assay (CI-ELISA),
although histopathological examination remains the most recognised diagnostic
procedure. PCR assays are also available for the detection of MCFV DNA.
The aim of this project was to develop a serological assay based on
recombinant antigens derived from the OvHV-2 genome and validation of the EL1SA
test using clinical serum samples. Eight OvHV-2 genes were selected for expression
in three bacterial systems. Four genes could be expressed in at least one system, but
only a fragment of the Ov8 protein and ORF65 were found to be recognised by antiMCF sera. Additionally Ov2.5, expressed in mammalian cells, was not recognised by
anti-MCF sera. Three genes were expressed in an in vitro transcription-translation
system (Ov8, Ov8.5 and ORF65). Protein purification, using epitope-tag affinity
purification and conventional chromatography, was unsuccessful so that these
polypeptides could not be evaluated as recombinant EL1SA antigens, requiring
further work on large scale expression and purification.
Due to the lack of recombinant antigen expression, crude antigen preparations
from cultured A1HV-1 and OvFlV-2 infected cells were analysed. Antigens suitable
for the detection of MCF virus specific serum antibodies could not be extracted from
an OvHV-2 infected cell line but A1F1V-1 WC11 antigen could be used to
differentiate MCF-positive and -negative sera. An ELISA based on this crude antigen
was established, designated WC11 -ELISA. Initial validation revealed over 93%
concordance between WC11-ELISA and commercial ELISA, while a comparison
with serological assays, a PCR test and final veterinary evaluations, showed good
agreement between tests. The WC11-ELISA showed 57% agreement to PCR while
commercial CI-ELISA showed 66% agreement for the same samples. This indicates
the value of the ELISA as an economical, rapid and specific serological assay for the
detection of antibodies against MCFV.
ction of antibodies against MCFV.
Potentially antigenic components of the ELISA lysate were analysed by
western blotting in comparison with other bovine herpesviruses, and fractionation by
gel filtration showed clear antigenic bands in the eluent. Proteomic analysis of
antigenic bands identified proteins encoding viral thymidine kinase (ORF21), a virus
capsid protein (ORF5), ORF52 (homologue to tegument protein HSV UL49) and a
viral antigen (ORF54) which encodes a dUTPase.
The simplicity of the WC11-ELISA and its lack of cross-reactivity make this
assay a reliable, accessible and low cost tool for routine MCFV diagnosis and
epidemiological studies.
