Scientific
We wish to thank Lisa Smith, Wendy Forbes and Kim Brett
for their technical assistance.
Full-length infectious clone of a
pathogenic Australian isolate of
chicken anaemia virus
References
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like toxin (stx1) and (stx2), intimin (eaeA), and enterohemorrhagic Escherichia
coli (EHEC) hemolysin (EHEC hlyA) genes in animal feces by multiplex PCR.
Appl Environ Microbiol 1999;65:868-872.
K BROWN
Department of Microbiology and
Immunology,
2. Bettelheim KA, Bensink JC, Tambunan HS. Serotypes of verotoxin-producing
(Shiga toxin-producing) Escherichia coli isolated from healthy sheep. Comp
Immunol Microbiol Infect 2000; 23:1-7.
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3. Beutin L, Geier D, Steinrück H, Zimmermann S, Scheutz F. Prevalence and
some properties of Verotoxin (Shiga-like toxin)-producing Escherichia coli in
GF BROWNING
PC SCOTT
Faculty of Veterinary Science,
The University of Melbourne,
Victoria 3010
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a
BS CRABB
Department of Microbiology and
Immunology,
The University of Melbourne,
Victoria 3010
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hicken anaemia virus (CAV) causes both clinical and
subclinical infection in chickens throughout the world.
C
CAV-induced anaemia results from destruction of haemopoietic
precursor cells in the bone marrow and infection is also associ-
ated with marked immunosuppression due to the depletion of
thymocytes, particularly young T cells.1,2 CAV is a distinct
member of the Circoviridae family of viruses that are charac-
terised by their small, single-stranded, circular DNA genome.
In studies in the Netherlands, Japan and Northern Ireland, the
full-length CAV genome has been cloned into plasmid vectors
in a form that is infectious when transfected into in vitro
cultured cells.3-6 Such molecular clones are important tools for
analysing the function of the virus’s genes and for dissection of
the pathogenesis of disease caused by CAV. The purpose of this
study was to derive an infectious molecular clone of a virulent
Australian isolate of CAV to further studies of the virus in
Australia.
The CAV isolate used in this study (CAU269/7) was
obtained from D O’Rouke and T Bagust (Faculty of Veterinary
Science, The University of Melbourne) and was originally
isolated from a commercial breeder flock in Australia.7 The
virus was cultured in the Marek’s disease virus transformed
lymphoblastoid cell line, MDCC-MSB1,2 based on the method
described by McNulty et al.8 Inoculation of CAU269/7 into
MDCC-MSB1 cells produced cytopathic effects consistent with
that of other reported CAV isolates9 and was characterised by
the appearance of enlarged, misshapen cells within 40 h. Total
cell degeneration was apparent within 96 h after infection.
Infected cells were stained for the CAV-specific protein, VP3, in
an indirect immunofluorescence assay (IFA) following the
method of Renshaw et al10 using the mouse derived monoclonal
antibody JCU/CAV/1C1 (JCU TropBio, Townsville,
Queensland). Infected cells showed strong fluorescence relative
to mock-infected control cells.
18. Piérard D, Stevens D, Moriau L, Lior H, Lauwers S. Isolation and virulence
factors of verocytotoxin-producing Escherichia coli in human stools. Clin
Microbiol Infect 1997;3:531-540.
19. Mackenzie, AMR, Lebel P, Orrbine et al. Sensitivities and specificities of
premier E. coli O157 and premier EHEC enzyme immunoassays for diagnosis
of infection with verotoxin (shiga-like toxin)-producing Escherichia coli. J Clin
Microbiol 1998;36:1608-1611.
20. Cameron S, Walker C, Beers M, Rose N, Anear E. Enterohaemorrhagic
Escherichia coli outbreak in South Australia associated with consumption of
mettwurst. Comm Dis Intel 1995;19:70-71.
To assess the pathogenicity of the Australian isolate
CAU269/7 for chickens, culture medium (~1x105.5
TCID50/mL) from CAV-infected cells was used to inoculate
yolk sacs of fifteen CAV-free, 7-day-old, specific-pathogen-free
(SPF) chicken embryos (SPAFAS, Parkville, Victoria). In
parallel, fifteen 7-day-old chicken embryos were mock-infected
with CAV-free culture medium. Thirteen days postinoculation
(day 20; 1 day before hatch), five embryos from each group
(Accepted for publication 3 April 2000)
a
Author for correspondence
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