Download Role of viral load in the pathogenesis of chicken anemia virus

Journal of General Virology (2005), 86, 1327–1333
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Communication
DOI 10.1099/vir.0.80599-0
Role of viral load in the pathogenesis of chicken
anemia virus
Jianming Tan and Gregory A. Tannock
Department of Biotechnology and Environmental Biology, RMIT University, PO Box 71,
Bundoora, Victoria 3083, Australia
Correspondence
Gregory A. Tannock
[email protected]
Received 10 September 2004
Accepted 25 January 2005
The pathogenesis of strain 3711 of the chicken anemia virus (CAV), propagated in chickens,
and two preparations of strain 3711 that had been adapted to grow to high titre in cells of the
MDCC-MSB1 line were studied in chicken embryos and/or chickens. Highest viral loads in
infected chickens, as measured by a microplate DNA-hybridization assay, were detected in the
thymus, clotted blood and pancreas, and the lowest in the duodenum. The CAV DNA copy
number in the organs of chicken embryos was significantly lower than in chickens. Route
of infection was an important determinant of the course of disease in chickens, with clinical signs
appearing earlier in birds infected by the intramuscular than those infected by the oral route;
there was a direct relationship between viral load in particular organs and the extent of clinical
signs. No reduction in the pathogenicity for chickens was noted for strain 3711 after 65 or
129 passages in the MDCC-MSB1 cell line.
Chicken anemia virus (CAV) is a small, non-enveloped,
single-stranded DNA virus, first isolated by Yuasa et al.
(1979), and was assigned as the only member of a new
genus, Gyrovirus, within the family Circoviridae (Pringle,
1999). The virus causes aplastic anaemia, generalized
lymphoid atrophy and increased mortality after infection
of day-old susceptible chickens (Bisgaard, 1983; Engström
& Luthman, 1984; Yuasa et al., 1987; Vielitz & Landgraf,
1988; Chettle et al., 1989). Infection of older birds with
CAV results in subclinical disease and reduced resistance
to other pathogens. Dual infection of chickens with other
immunosuppressive viruses increases the severity of CAV
signs and decreases the resistance of older birds to infection
(Yuasa et al., 1980b; Otaki et al., 1988a, b; Rosenberger &
Cloud, 1989; De Boer et al., 1994; Cui et al., 1999; Toro et al.,
2000). CAV can be transmitted between chickens by both
the horizontal and vertical routes. Horizontal spread usually
occurs via the oral–faecal route, but infection via the respiratory route has also been demonstrated in experimentally
infected birds (Rosenberger & Cloud, 1989). Vertical spread
through hatched chickens is considered to be the most
important means of dissemination of the virus (Yuasa &
Yoshida, 1983; Yuasa et al., 1983; Hoop, 1992, 1993).
The growth of CAV in chicken embryos was first described
by von Bülow & Witt (1986). Most studies of CAV pathogenesis have involved inoculation of chickens by the intramuscular (i.m.) route, which has been shown to be the
most sensitive for inducing clinical signs in susceptible
day-old chickens (Rosenberger & Cloud, 1989; Yuasa, 1989).
The size of the CAV inoculum influences the severity
of anaemia and other clinical signs and the proportion
of chickens infected (Yuasa et al., 1980a; Rosenberger &
0008-0599 G 2005 SGM
Cloud, 1989; McNulty et al., 1990). In the present study,
the growth of strain 3711, an Australian vaccine virus
prepared from infected chickens (Connor et al., 1991), was
examined in various tissues of specific-pathogen-free (SPF)
chicken embryos by using a microplate DNA-hybridization
assay for estimation of the CAV DNA copy number. These
studies were extended to SPF chickens, where its growth
was compared with that of other preparations of strain
3711 after passage in cells of the MDCC-MSB1 line. The
relationship between the occurrence of clinical signs and
viral load was also measured.
CAV vaccine strain 3711 was supplied by Dr Gordon Firth,
Intervet (Australia) Pty Ltd, as a liver homogenate in PBS
after three passages in SPF chickens and was shown to be
free from other avian pathogens. Before use, the virus preparation was clarified, sterilized by filtration and diluted in
PBS to contain 40 50 % chicken infectious doses (CID50).
Strain 3711 was also adapted to grow to high titre (>106?0
TCID50 in 0?1 ml) in cells of the MDCC-MSB1 line
(Tannock et al., 2003). Viruses used in the present study
were passaged 65 and 129 times.
CAV DNA extracted from tissue samples by the method
of Todd et al. (1992) was amplified with the forward
primer 59-CAGTGAATCGGCGCTTAGC-39 and the reverse
primer 59-GCTCGTCTTGCCATCTTACAG-39. Standard
PCR mixtures (50 ml) contained 2?0 mM each dNTP,
2?0 mM MgCl2, 0?2 mM forward and reverse primers,
1?0 U AmpliTaq DNA polymerase (Roche Molecular
Systems) and 5 ml template DNA. Each reaction was
allowed to take place for 35 cycles, each consisting of 30 s
at 95 uC, 30 s at 55 uC and 1 min at 72 uC.
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J. Tan and G. A. Tannock
A 266 bp probe within the 452 bp segment was amplified by
PCR using primers 59-GACCATCAACGGTGTTCAG-39
and 59-CTCGCTTACCCTGTACTCG-39. The PCR product
was purified and then labelled with Photoprobe biotin
(Vector Laboratories), according to procedures described by
Forster et al. (1985) and Hibma et al. (1994).
The microplate DNA-hybridization assay was based on the
method described by Inouye & Hondo (1990). Briefly, the
amplified DNA segment (452 bp) was purified (Todd et al.,
1992) and coated onto a microplate well at 37 uC for 2 h.
After washing three times, 5 ng heated–denatured biotin
probe (266 bp) and 4?5 mg salmon-sperm DNA were
added per well and incubated at 42 uC overnight. After a
further three washes, 100 ml horseradish peroxidaseconjugated streptavidin (1 : 500) was added to each well
and the plate was held at room temperature for 1 h. The
wells were again washed and 100 ml TMB substrate
(3,39,5,59-tetramethylbenzidine; KPL) was added per well
and incubated for 30 min. The reaction was then stopped
by addition of 50 ml 2 M H2SO4 per well and A450 was
read. The DNA-hybridization assay could detect 0?005–
0?025 ng of the 452 bp CAV segment; bands containing
5–10 ng of the 452 bp segment could be detected by a
standard PCR (Tan, 2002). Novak & Ragland (2001) also
demonstrated that a competitive DNA-hybridization assay
in microtitre plates for the detection of CAV was more
sensitive than in situ hybridization, dot-blot and ELISA
assays.
The Mr of the 452 bp segment was estimated to be
2?98326105 and its concentration was determined by
A260 measurement. The copy number of the 452 bp segment was calculated according to the following formula
(Mackay, 1999):
DNA copy number=Avogadro’s number (6?0261023)6
mass (g)/Mr.
For internal standardization, serial 10-fold dilutions of the
purified 452 bp segment were prepared in parallel. A 50 ml
aliquot of each dilution, containing 101–1010 copies of the
452 bp segment, was amplified by the standard PCR and
the products were quantified by microplate DNA hybridization using the biotin probe. A good linear correlation
between A450 and copy number for the 452 bp segment was
observed (y=0?1751x20?0561). As each 452 bp segment
represented a single CAV genome, the number of CAV
DNA templates in samples could be estimated by using the
standard PCR to amplify the 452 bp segment, followed by
DNA hybridization with the biotin probe and extrapolation
from the standard curve. For the assay of tissue extracts,
standard preparations containing 104–107 copies of the
452 bp segment were run in parallel. Assays were considered
valid if the mean A450 of each standard preparation fell
within ±1SD of the expected values.
Strain 3711, prepared from infected chickens, was inoculated to 25 5-day-old SPF chicken embryos via the yolk
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sac. Fifteen eggs were inoculated with PBS as controls and
all were examined at days 16–20. Deaths occurred in 12
infected embryos (48 %) and in one from the control group
(6 %). Of the 12, four appeared normal, four were stunted
and four were stunted with unilateral abnormalities of the
eye. The organs from six individual surviving embryos at
day 20 were harvested and extracts were prepared for testing
for CAV viral load by the microplate DNA-hybridization
assay. The CAV copy number for the various organs was
3?6–8?6 log10 (g tissue)21. CAV DNA could be detected in
all thymus and pancreas samples [mean viral loads, 8?60±
1?08 and 5?60±0?81 log10 copies (g tissue)21, respectively].
For the brain, bursa, kidney, liver and lung samples, CAV
DNA was detected in one or two of three samples tested
[4?85±0?75 or 7?43±0?62 log10 copies (g tissue)21]. Only
one of three duodenum samples tested was positive and
the viral copy number [4?60 log10 copies (g tissue)21] was
lower than that in other organs.
A further batch of 25 embryos was infected similarly with
another batch of CAV vaccine and 15 chickens (60 %)
hatched. Three surviving chickens from infected and control groups were euthanized at days 5, 10, 15, 20 and 25
after hatch and individual organ samples were collected
and tested for virus load. Hatched chickens inoculated in
ovo showed clinical signs, characterized by moderate to
severe depression, anorexia, anaemia and dehydration at
days 5 and 10 after hatch. One bird from the infected group
died at day 15. Fig. 1 shows that the mean body weight of
the infected group was lower than that of the controls at
day 10 (Student’s t-test; P<0?05), but the greatest differences were seen at days 15–25 (P<0?01). The thymus :
and bursa : body-weight ratios of inoculated chickens were
significantly lower than those of the control group at days
5–15 (P<0?01). However, for the spleen : body-weight
ratio, significant differences were noted only between
inoculated and control birds at day 25 (P<0?05).
A significant decline in the haematocrit value (HV) of
infected birds compared with controls could be detected at
days 5, 10, 15 and 20 after hatch (P<0?01). HV levels had
returned to near-normal values (27 %; von Bülow & Schat,
1997; Todd, 2000) by day 25, although, even then, the
difference between the two groups was still significant
(P<0?05). The decrease in HV preceded the appearance
of antibody to CAV by 10–15 days and the highest titre
was reached by day 25 (Fig. 1). Macroscopic lesions,
including the appearance of pale bone marrow, atrophy of
the thymus and paleness of comb and muscle, were present
in infected birds. Atrophy was most severe in the bone
marrow by days 5 and 10 and in the thymus by days 10
and 15 after hatch. These lesions were evident until day
20, but no subcutaneous or intramuscular haemorrhages
were present. Neither clinical signs nor gross lesions were
detected in the controls.
Extracted DNA was tested by the microplate DNAhybridization assay. The results (Table 1) indicate that all
tested tissues were positive, but highest viral loads were
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Journal of General Virology 86
Role of viral load in the pathogenesis of CAV
Fig. 1. Mean body and organ weights, HV and antibody titre of hatched chickens inoculated as 5-day embryos with strain
3711 prepared in chickens ($) and control (#).
Table 1. CAV load in different organs of hatched chickens infected as 5-day-old embryos with 40 CID50 strain 3711
prepared by growth in chickens
Values are means±SD log10 DNA copy number (g tissue)21. The minimum level for detection was 3?1 log10 copies (g tissue)21.
Organ
Brain
Bursa
Clotted blood
Duodenum
Kidney
Liver
Lung
Pancreas
Spleen
Thymus
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Time after hatching (days)
5
10
15
20
25
3?60±0
5?60±0
8?10±1?0
<3?1
6?85±0?8
7?85±0?3
8?10±0
10?85±0?8
9?25±0?3
9?85±2?3
3?35±0?8
7?93±1?3
10?26±1?0
5?60±0
7?85±0?3
9?43±1?0
5?35±2?3
10?60±1?0
8?43±0?8
10?93±1?3
4?76±0?9
8?10±1?5
9?60±1?7
4?10±0
6?60±0
9?53±0?2
4?85±1?8
10?85±0?8
7?60±1?0
10?10±1?5
5?60±0
6?60±1?0
9?35±0?3
4?60±0
7?85±1?8
9?35±1?3
6?35±0?8
7?60±1?9
8?43±2?0
11?60±1?0
3?60±0
7?60±1?0
8?10±1?5
<3?1
7?60±0?5
9?25±0?8
4?60±0
7?85±0?8
8?10±0?5
8?60±1?0
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J. Tan and G. A. Tannock
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Journal of General Virology 86
Fig. 2. (a) Mean body and thymus weight, HV and antibody titre of chickens; (b) viral load in different organs of chickens inoculated with strain 3711
prepared in chickens by the i.m. (X) and oral (%) routes and by contact transmission (m). Results for strain 3711 after 65 ($) and 129 passages (&) in
cells of the MDCC-MSB1 line. The limit for detection was a copy number of 3?1 log10 (g tissue)”1.
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Role of viral load in the pathogenesis of CAV
present in the thymus [8?60–11?60 log10 copies (g tissue)21].
An unexpected observation was the very high load in the
pancreas [7?60–10?85 log10 copies (g tissue)21]. Organs
with moderate loads were the liver [7?85–9?53 log10 copies
(g tissue)21], spleen [7?60–9?25 log10 copies (g tissue)21],
bursa [5?60–8?10 log10 copies (g tissue)21], kidney
[6?60–7?85 log10 copies (g tissue)21] and lung [4?60–8?10
log10 copies (g tissue)21]. Lower loads were present in the
brain and duodenum [3?35–5?60 and 3?10–5?60 log10 copies
(g tissue)21]. Viral loads in the organs of chickens were
significantly higher than in those of embryos. The very high
load in clotted blood is partly attributable to viraemia
and partly due to the growth of CAV in the bone marrow.
The presence of CAV DNA in most organs is probably a
consequence of viraemia and not an indication that virus
replication had taken place. The high CAV load in the
pancreas suggests that it, like the thymus, is a primary
target organ. Smyth et al. (1993) described the presence
of CAV antigen in the pancreas of infected birds and
similar findings have been reported for coxsackievirus
B4 virus, which produces infections of the pancreas and a
focal myositis of mice (Pallansch & Roos, 2001).
In total, 90 day-old SPF chickens were divided randomly
into six groups of 15 chickens. Five groups were inoculated
with different CAV preparations by different routes. Chickens
in the contact transmission group were wing-tagged and
housed together in the same cage with the group that was
inoculated with the chicken-propagated strain 3711 by the
i.m. route; the sixth control group was inoculated by the
i.m. route with PBS.
All birds infected with CAV by i.m. inoculation developed
characteristic anaemia by days 10–12 post-infection (p.i.),
with peak signs being present at days 15–18. Affected birds
appeared anorexic and depressed, with pallor of the comb
and wattles and ruffled feathers. Only one bird in the group
died, at day 20. Birds in groups inoculated by the oral route
developed the same clinical signs by day 15 and no deaths
occurred during the experimental period, confirming earlier
findings (Rosenberger & Cloud, 1989; Yuasa, 1989) that the
i.m. route is most efficient for inducing clinical disease.
Neither clinical signs nor deaths occurred in the contact
group.
Birds inoculated by the i.m. route showed a significant
decline in weight gain after i.m. inoculation at days 10 and
25, compared with the controls (Fig. 2a; P<0?05), and
greater differences were seen at days 15 and 20 (P<0?01).
Similar results were obtained in the groups inoculated
orally at days 15, 20 and 25 (P<0?01), but there was no
decline in body weight for the contact group. Only thymic
atrophy was observed in i.m.- and orally infected chickens
and the thymus : body-weight ratios were greater than
for the control group at days 15–20 (P<0?01) and 25
(P<0?05). Lower HVs were observed in groups infected
by i.m. inoculation. HVs of <27 % were observed at days
10–20 but, in 53 % of cases, increases to near-normal values
had occurred by day 25 (Fig. 2a). For groups inoculated
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orally, HVs of <27 % were seen up to day 20. Most birds
were still anaemic by day 25 and recovery to normal values
occurred in one-third of birds. There were no changes in
the mean HV of the contact group. Overall, no differences
in pathogenesis could be detected between the groups
inoculated with chicken- or cell culture-grown viruses.
A blocking ELISA (Tannock et al., 2003) was used to test
the antibody status of chickens from each group. Antibody
to CAV could not be detected in any group before 10 days.
In the group inoculated by the i.m. route, antibody could
be detected in 75 and 89 % of birds at days 15 and 20,
respectively, and 100 % by day 25. By contrast, only 44 and
67 % of birds in the groups infected by the oral route
developed antibody by days 20 and 25 and only one bird
from the contact group by day 25 (Fig. 2a).
In the groups inoculated by the i.m. route, CAV DNA
could be detected in all organs tested at days 5–25 p.i., with
highest copy numbers being present in samples obtained
from clotted blood, pancreas and thymus (Fig. 2b). Peak
levels [10?10–11?60 log10 copies (g tissue)21] were obtained
at days 10–20. Intermediate levels [3?35–9?76 log10 copies
(g tissue)21] were present in samples from the brain,
bursa, kidney, liver, lung and spleen and lowest levels
[3?10–5?60 log10 copies (g tissue)21] from the duodenum.
There were no significant differences in load between
chicken- or cell culture-passaged viruses (Fig. 2b). Peak
loads in the thymus, pancreas and blood of chickens
inoculated by the i.m. route occurred at day 15 and by
the oral route by day 20, although the extent of each was
similar. CAV was present in thymus and clotted blood at
day 5 p.i. and other organs of chickens inoculated by the
oral route by day 10. Highest loads were present in the
thymus, clotted blood and pancreas. In the contact group,
CAV DNA could not be detected in any organ before day
10. Fewer birds were positive and the viral loads [3?10–
7?76 log10 copies (g tissue)21] in individual organs were
significantly lower than those in birds inoculated by the
i.m. [3?10–11?60 log10 copies (g tissue)21] and oral [3?10–
10?60 log10 copies (g tissue)21] routes.
Apart from route of inoculation, the ability to produce
anaemia after experimental infection is related directly to
the virus dose (Yuasa et al., 1979; Yuasa, 1989; McNulty
et al., 1990). The present study demonstrates, for the first
time, a relationship between viral load and the extent of
clinical disease. All chickens in groups inoculated by the
i.m. and oral routes or by inoculation of embryos showed
typical clinical signs. Chickens infected by contact transmission did not show signs and exhibited similar increases
in total weight and in the weights of immune organs to
those of the controls. Viral loads in all organs of the
chickens infected by contact were significantly lower than
those infected by the i.m., oral or in ovo routes (Fig. 2b).
There were no differences in pathogenicity between preparations of strain 3711 that were propagated in chickens
or passaged to different levels in the MDCC-MSB1 cell
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J. Tan and G. A. Tannock
line (Fig. 2a). By contrast, Todd et al. (1995, 1998) found
that the Cux-1 strain became substantially less pathogenic
after 173 and 320 passages in MDCC-MSB1 cells and that
the pathogenicity of attenuated viruses could be restored
after 10 passages in young chickens. The reasons for these
differences are unclear, but could be related to undefined
genetic differences between strain 3711 and the Cux-1
strain. There were also no significant differences with
respect to viral load, the appearance of clinical signs,
reductions in weight gain, declines in HV or levels of
antibody induced between chickens inoculated in ovo at
day 5 of embryogenesis and others inoculated at day 1
after hatch (Table 1; Figs 1–2). However, mortality rates
following egg inoculation were high (48 and 40 %). In
support of this, decreases in the bursa : and spleen : bodyweight ratios occurred in hatched chickens inoculated
in ovo, but not in chickens inoculated at day 1 (Tan, 2002).
There have been several recent reports on the use of realtime PCR for measuring viral loads of CAV in infected
chickens (Markowski-Grimsrud et al., 2002) and for the
measurement of viral DNA copy number in isolated
lymphocytes (Markowski-Grimsrud & Schat, 2003). This
test has also been adapted to measurement of CAVneutralizing antibody (van Santen et al., 2004a). An
advantage of the test used in the present study is its low
cost and suitability for the simultaneous processing of
large numbers of samples without the need for an expensive, dedicated instrument.
Overall, these findings confirm that the bone marrow and
thymus are the most important target organs in the pathogenesis of CAV infection. The destruction of haemocytoblasts in bone marrow results in a severe depletion of
erythroid and myeloid cells and produces anaemia; the
destruction of T-lymphocyte progenitor cells in the thymus
results in depletion of mature cytotoxic and helper T cells
and induces severe atrophy (Adair, 2000). Virus is spread
to other organs by viraemia and lymphoid foci develop in
the liver, kidney and lung. However, the reason for higher
viral loads in the pancreas is unclear. N.B. A paper on
the measurement of peak CAV loads by real-time PCR in
chickens infected by the i.m. and oral routes appeared at
the time that our paper was submitted, and showed similar
results (van Santen et al., 2004b).
References
Adair, B. M. (2000). Immunopathogenesis of chicken anemia virus
infection. Dev Comp Immunol 24, 247–255.
Cui, Z. Z., Jin, W. J., Liu, Y. L., Xu, Y. M., Li, Y. W. & Zhu, C. G. (1999).
Multiple infections with immunosuppressive viruses in chickens in
China. In Proceedings of the 1999 International Conference and
Exhibition on Veterinary Poultry, Beijing, China, pp. 153–157.
De Boer, G. F., van Roozelaar, D. J., Moormann, R. J., Jeurissen,
S. H. M., van den Wijngaard, J. C., Hilbink, F. & Koch, G. (1994).
Interaction between chicken anemia virus and live Newcastle disease
virus vaccine. Avian Pathol 23, 263–275.
Engström, B. E. & Luthman, M. (1984). Blue wing disease of
chickens: experimental infection with a Swedish isolate of chicken
anaemia agent and an avian reovirus. Avian Pathol 13, 23–32.
Forster, A. C., McInnes, J. L., Skingle, D. C. & Symons, R. H. (1985).
Non-radioactive hybridization probes prepared by the chemical
labelling of DNA and RNA with a novel reagent, photobiotin.
Nucleic Acids Res 13, 745–761.
Hibma, M. H., Ely, S. J. & Crawford, L. (1994). A non-radioactive
assay for the detection and quantitation of a DNA binding protein.
Nucleic Acids Res 22, 3806–3807.
Hoop, R. K. (1992). Persistence and vertical transmission of chicken
anaemia agent in experimentally infected laying hens. Avian Pathol
21, 493–501.
Hoop, R. K. (1993). Transmission of chicken anaemia virus with
semen. Vet Rec 133, 551–552.
Inouye, S. & Hondo, R. (1990). Microplate hybridization of amplified
viral DNA segment. J Clin Microbiol 28, 1469–1472.
Mackay, I. (1999). Development and evaluation of a quantitative
PCR for the determination of cytomegalovirus load. In Abstracts of
the XIth International Congress of Virology, Sydney, Australia,
VP65.23, p. 63.
Markowski-Grimsrud, C. J. & Schat, K. A. (2003). Infection with
chicken anaemia virus impairs the generation of pathogen-specific
cytotoxic T lymphocytes. Immunology 109, 283–294.
Markowski-Grimsrud, C. J., Miller, M. M. & Schat, K. A. (2002).
Development of strain-specific real-time PCR and RT-PCR assays for
quantitation of chicken anemia virus. J Virol Methods 101, 135–147.
McNulty, M. S., Connor, T. J. & McNeilly, F. (1990). Influence of virus
dose on experimental anaemia due to chicken anaemia agent. Avian
Pathol 19, 167–171.
Novak, R. & Ragland, W. L. (2001). Competitive DNA hybridization
in microtitre plates for chicken anaemia virus. Mol Cell Probes 15,
1–11.
Otaki, Y., Nunoya, T., Tajima, M., Kato, A. & Nomura, Y. (1988a).
Depression of vaccinal immunity to Marek’s disease by infection
with chicken anaemia agent. Avian Pathol 17, 333–347.
Otaki, Y., Tajima, M., Saito, K. & Nomura, K. (1988b). Immune
response of chicks inoculated with chicken anemia agent alone or in
combination with Marek’s disease virus or turkey herpesvirus.
Nippon Juigaku Zasshi 50, 1040–1047.
Pallansch, M. A. & Roos, R. P. (2001). Enteroviruses: polioviruses,
coxsackieviruses, echoviruses, and newer enteroviruses. In Fields
Virology, 4th edn, pp. 723–775. Edited by D. M. Knipe & P. M.
Howley. Philadelphia, PA: Lippincott Williams & Wilkins.
hemorrhagic disorder in Danish broilers. Nord Vet Med 35, 397–407.
Pringle, C. R. (1999). Virus taxonomy at the XIth International
Congress of Virology, Sydney, Australia, 1999. Arch Virol 144,
2065–2070.
Chettle, N. J., Eddy, R. K., Wyeth, P. J. & Lister, S. A. (1989). An
Rosenberger, J. K. & Cloud, S. S. (1989). The effects of age, route of
outbreak of disease due to chicken anaemia agent in broiler chickens
in England. Vet Rec 124, 211–215.
Connor, T. J., McNeilly, F., Firth, G. A. & McNulty, M. S. (1991).
exposure, and coinfection with infectious bursal disease virus on the
pathogenicity and transmissibility of chicken anemia agent (CAA).
Avian Dis 33, 753–759.
Biological characterisation of Australian isolates of chicken anaemia
agent. Aust Vet J 68, 199–201.
Smyth, J. A., Moffett, D. A., McNulty, M. S., Todd, D. & Mackie, D. P.
(1993). A sequential histopathologic and immunocytochemical study
Bisgaard, M. (1983). An age related and breeder flock associated
1332
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Fri, 12 May 2017 10:11:50
Journal of General Virology 86
Role of viral load in the pathogenesis of CAV
of chicken anemia virus infection at one day of age. Avian Dis 37,
324–338.
comparison of the oral and the intramuscular routes of infection.
Avian Dis 48, 494–504.
Tan, J. (2002). The use of newer methodologies to study the pathogenesis of the chicken anemia virus. PhD thesis, RMIT University,
Melbourne, VIC, Australia.
Vielitz, E. & Landgraf, H. (1988). Anaemia-dermatitis of broilers:
field observations on its occurrence, transmission and prevention.
Avian Pathol 17, 113–120.
Tannock, G. A., Tan, J., Mawhinney, K. A., Todd, D., O’Rourke, D. &
Bagust, T. J. (2003). A modified blocking ELISA for the detection of
von Bülow, V. & Witt, W. (1986). Vermehrung des Erregers der
antibody to chicken anaemia virus using an Australian strain. Aust
Vet J 81, 428–430.
Todd, D. (2000). Circoviruses: immunosuppressive threats to avian
species: a review. Avian Pathol 29, 373–394.
Todd, D., Mawhinney, K. A. & McNulty, M. S. (1992). Detection and
aviären infektiosen Anämie (CAA) in embryonierten Huhnereiern.
Zentbl Vetmed Reihe B 33, 664–669 (in German).
von Bülow, V. & Schat, K. A. (1997). Infectious anaemia. In Diseases
of Poultry, 10th edn, pp 739–756. Edited by B. W. Calnek, H. J.
Barnes, C. W. Beard, L. R. McDougald & Y. M. Saif. Ames, IA: Iowa
State University Press.
differentiation of chicken anemia virus isolates by using the
polymerase chain reaction. J Clin Microbiol 30, 1661–1666.
Yuasa, N. (1989). CAA: review and recent problems. In Proceedings
Todd, D., Connor, T. J., Calvert, V. M., Creelan, J. L., Meehan, B. M. &
McNulty, M. S. (1995). Molecular cloning of an attenuated chicken
Yuasa, N. & Yoshida, I. (1983). Experimental egg transmission of
chicken anemia agent. Natl Inst Anim Health Q (Tokyo) 23, 99–100.
anaemia virus isolate following repeated cell culture passage. Avian
Pathol 24, 171–187.
Yuasa, N., Taniguchi, T. & Yoshida, I. (1979). Isolation and
of 38th Western Poultry Disease Conference, Tempe, Arizona, 14–20.
characteristics of an agent inducing anemia in chicks. Avian Dis
23, 366–385.
Todd, D., Connor, T. J., Creelan, J. L., Borghmans, B. J., Calvert,
V. M. & McNulty, M. S. (1998). Effect of multiple cell culture passages
Yuasa, N., Noguchi, T., Furuta, K. & Yoshida, I. (1980a). Maternal
on the biological behaviour of chicken anaemia virus. Avian Pathol
27, 74–79.
antibody and its effect on the susceptibility of chicks to chicken
anemia agent. Avian Dis 24, 197–201.
Toro, H., Gonzalez, C., Cerda, L., Hess, M., Reyes, E. & Geissea, C.
(2000). Chicken anemia virus and fowl adenoviruses: association to
Yuasa, N., Taniguchi, T., Noguchi, T. & Yoshida, I. (1980b). Effect of
induce the inclusion body hepatitis/hydropericardium syndrome.
Avian Dis 44, 51–58.
van Santen, V. L., Kaltenboeck, B., Joiner, K. S., Macklin, K. S. &
Norton, R. A. (2004a). Real-time quantitative PCR-based serum
neutralization test for detection and titration of neutralizing antibodies to chicken anemia virus. J Virol Methods 115, 123–135.
van Santen, V. L., Joiner, K. S., Murray, C., Petrenko, N., Hoerr,
F. J. & Toro, H. (2004b). Pathogenesis of chicken anemia virus:
http://vir.sgmjournals.org
infectious bursal disease virus infection on incidence of anemia by
chicken anemia agent. Avian Dis 24, 202–209.
Yuasa, N., Taniguchi, T., Imada, T. & Hihara, H. (1983). Distribution
of chicken anemia agent (CAA) and detection of neutralizing
antibody in chicks experimentally inoculated with CAA. Natl Inst
Anim Health Q (Tokyo) 23, 78–81.
Yuasa, N., Imai, K., Watanabe, K., Saito, F., Abe, M. & Komi, K.
(1987). Aetiological examination of an outbreak of haemorrhagic
syndrome in a broiler flock in Japan. Avian Pathol 16, 521–526.
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