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Pathobiology and Impact of
Chicken Anemia Virus
Haroldo Toro, DVM, PhD
Professor
Department of Pathobiology
Chicken infectious anemia virus
1979 1979 Japan Japan (Yuasa)(Yuasa)
1981 1981 Germany Germany (von (von BülowBülow))
1981 1981 Sweden Sweden ((EngstromEngstrom))
1989 1989 USA USA (McNulty; (McNulty;
Rosenberger)Rosenberger)
Worldwide distributionWorldwide distribution
H. Toro 2009
2
Serological evidence of CAV in the U.S.
at least since 1959
0,000
0,200
0,400
0,600
0,800
1,000
1,200
59 59 60 60 61 62 63 63 63 63 63 63 63 63 63 63 63 63 64 64 64 64 64 64 64 64 64 64 64 64 64 64 64 64 64 64
S/N
ra
tio
(6
20
nm
)
Individual Serum / Year
Toro, H., S. Ewald, and F.J. Hoerr (2006). Avian Diseases, 50:124-126.
H. Toro 2009
CAV is heat stable and unaffected by ether,
chloroform or low pH. Most disinfectants are
relatively ineffective. Eradication NOT possible.
H. Toro 2009
3
Chicken Anemia VirusFamily: Circoviridae
Genus: Girovirus
Genome: Single stranded circular DNA
Envelope: Non
Morphology: icosahedral particles of 22-26 nm
Viral proteins
• VP1 is the only capsid protein
• VP2 has phosphatase activity and participates in the capsid formation as a chaperon by allowing VP1 to fold
• VP3, or apoptin induces cell death by apoptosis
Replication: Lymphoid cells (MSB1, & other)
Serotypes: 1
Transmission: Vertical & horizontal
CAVCAV2298 nt2298 nt
VP2VP2
VP1VP1
VP3VP3
Van Santen et al, 2004
H. Toro 2009
CAVCAV2298 nt2298 nt
VP2VP2
VP1VP1
VP1 HV region
VP3VP3
CAVCAV genomegenome
VP3VP3 VP1VP1
VP2VP2 HV Region
Portion Sequenced
823 nt
2298 nt
H. T. 2009
Van Santen, V., F.J. Hoerr, K. Joiner, C. Murray, N. Petrenko, & H. Toro (2004). Avian Diseases
48:494-504.
4
Sequence Origin (Year) 22 75 97 139 14405A Alabama (98) Q I L Q Q26 Alabama (98) Q I L Q Q55 Alabama (98) Q I L Q Q57B Alabama (98) Q I L Q Q58 Alabama (98) Q I L Q Q69B Alabama (98) Q I L Q Q73 Alabama (98) Q I L Q Q05B Alabama (98) N I L Q Q28B Alabama (98) N I L Q Q57A Alabama (98) N I L Q Q70 Alabama (98) N V M K E03-4876 Alabama (03) N V M K E28A Alabama (98) H I L K E52 Alabama (98) H V M K E69A Alabama (98) H V M K E01-4201 Alabama (01) H V M K ECIA-1 U.S. N I L Q Q704 Australia H I L Q QTR20 Japan H I L Q QL028 U.S. H T L Q Q26P4 U.S. H V M K EA2B Japan H V M K ECux-1 Germany H V M K DCAU269/7 Australia H V M K EAustralian Australia H V M K E
Amino acid positionHV regionPolymorphisms in
VP1 amino acid sequence among Alabama CAV sequences
V - Valine (Val)
L - Leucine (Leu)
I - Isoleucine (Ile)
M - Methionine (Met)
H - Histidine (His)
K - Lysine (Lys)
Q - Glutamine (Gln)
N - Asparagine (Asn)
E - Glutamic Acid (Glu)
D - Aspartic Acid (Asp)
T - Threonine (Thr)
Van Santen, V., F.J. Hoerr, K. Joiner, C. Murray, N. Petrenko, & H. Toro (2004). Avian Diseases 48:494-504.
H. Toro 2009
CAV signs: Uneven pullets; mortality
usually < 20%.
H. Toro 2009
5
0
50
100
150
200
250
300
350
400
0 7 14 21 28
Weight (g)
Days p.i.
Weights
Uninfected
CAV oral
CAV i.m.
CAV signs: Reduced Weight Gain (SPF chicks
inoculated at 1 day of age by the IM or oral routes with CAV isolate 03-4876).
Van Santen, V., F.J. Hoerr, K. Joiner, C. Murray, N. Petrenko, & H. Toro (2004). Avian Diseases 48:494-504.
H. Toro 2009
CAV signs: Anemia resulting from infection
of hemocytoblasts in the bone marrow
H. Toro 2009
6
0
5
10
15
20
25
30
35
40
45
0 7 14 21 28
Hematrocrit (%)
Days p.i.
Hematocrits
Uninfected
CAV oral
CAV i.m.
CAV signs: Anemia (SPF chicks infected at day 1 of age with CAV isolate
03-4876 via the oral or IM routes).
Van Santen, V., F.J. Hoerr, K. Joiner, C. Murray, N. Petrenko, & H. Toro (2004). Avian Diseases
48:494-504.
H. Toro 2009
CAV signs: Hemorrhages
H. Toro 2009
7
Trombocytes in peripheral blood of chickens inoculated with strain Gifu-1
0
5
10
15
20
25
30
35
40
0 4 8 12 16 20 24 28 32 36 40
Th
rom
bo
cyte
s x
103m
m3
Days after Inoculation
Control
CAV
Tanigushi et al., (1983)H. Toro 2009
CAV: Thymic lesions
Control
H. Toro 2009
8
Control
Experimental infection with CAV 10343 in 10Experimental infection with CAV 10343 in 10--ww--old broiler old broiler
breedersbreeders
Toro et al. (1997) Avian Pathology 26:485-499. H. Toro 2009
Lymphocyte depletion: Infection of T lymphocytoblasts in the thymic
cortex
12
3 4
H. Toro 2009
9
CAV: Inclusion bodies in the thymus (HE & EM).
H. Toro 2009
Lymphocyte depletion: Cortical
lymphocyte/parenchyma ratio
(CL/P)
Morphometry using ImageJ software version
1.29X (Public Domain, http://rsb.info.nih.gov/ij/)
H. Toro 2009
10
Thymic lymphocyte depletion: cortical lymphocyte/parenchyma
ratio (CL/P). SPF chicks infected at 1 day of age with CAV 03-
4876 by the oral or IM routes.
CAV: 7 days PI
Control CAV-I.M. CAV-Oral0.00
0.25
0.50
0.75
a
aa
Inoculation route
CL
/P r
ati
o
CAV: 10 days PI
Control CAV-I.M. CAV-Oral0.00
0.25
0.50
0.75
aa
b
Inoculation route
CL
/P r
ati
o
Van Santen, V., F.J. Hoerr, K. Joiner, C. Murray, N. Petrenko, & H. Toro (2004). Avian Diseases 48:494-504.
H. Toro 2009
CAV: 14 days PI
Control CAV-I.M. CAV-Oral0.00
0.25
0.50
0.75
a
b
c
Inoculation route
CL
/P r
ati
o
CAV: 28 days PI
Control CAV-I.M. CAV-Oral0.00
0.25
0.50
0.75
aa a
Inoculation route
CL
/P r
ati
o
Thymic lymphocyte depletion: cortical lymphocyte/parenchyma ratio
(CL/P). SPF chicks infected at 1 day of age with CAV 03-4876 by the
oral or IM routes.
Van Santen, V., F.J. Hoerr, K. Joiner, C. Murray, N. Petrenko, & H. Toro (2004). Avian Diseases 48:494-504.
H. Toro 2009
11
CAV DNA by qPCR in thymuses of SPF chickens inoculated with CAV
at day 1 of age via the IM or oral routes (Error bars +/- 1 SD).
Viral isolation from
all birds at day 28
post infection.
Van Santen, V., F.J. Hoerr, K. Joiner, C. Murray, N. Petrenko, H. Toro (2004). Avian Diseases 48:494-504.
H. Toro 2009
CAV genomes by qPCR in cecal tonsils or Harderian glands
of SPF chickens inoculated with CAV at day 1 of age via the
IM or oral routes (Error bars +/- 1 SD).
Van Santen, V., F.J. Hoerr, K. Joiner, C. Murray, N. Petrenko, H. Toro (2004). Avian Diseases 48:494-504.
H. Toro 2009
12
CAV antibodies (ELISA) in SPF chickens inoculated at 1-d with
CAV 03-4876 via the oral or IM routes
0
0,2
0,4
0,6
0,8
1
1,2
1,4
1,6
7 10 14 28
S/N
rati
o
Days post-inoculation
Controls
Oral
IM
H. Toro 2009
Van Santen, V., F.J. Hoerr, K. Joiner, C. Murray, N. Petrenko, H. Toro (2004). Avian Diseases 48:494-504.
Conclusions
� Peak CAV genome concentrations in the thymus occur 10 and
14 days after IM and oral exposure respectively.
� High concentrations of CAV genomes until 28 days despite of
seroconversion occurring at day 14 after infection.
� Lymphocyte repopulation of the thymus occurs at day 28 in
spite of the presence of the virus.
� Orally infected chicks do not develop signs of disease. Body
weight, hematocrits and histological lesions are less severe.
This is consistent with a delayed increase of CAV genomes in
the thymus.
H. Toro 2009
13
Broiler Whole Bird Condemnation (Region). USDA
National Agricultural Statistics Service
SW Mid-West S.
East
Mid-Atlantic
S. Central
% % SeptoxSeptox 0.1900.190 0.2320.232 0.2270.227 0.2080.208 0.1950.195
% Airsac 0.041 0.046 0.087 0.082 0.084
% I.P. 0.012 0.006 0.042 0.043 0.017
%Leukosis 0.000 0.000 0.001 0.023 0.001
% Bruise 0.003 0.001 0.009 0.005 0.002
% Other 0.008 0.006 0.007 0.005 0.021
Total 0.173 0.291 0.373 0.365 0.320
H. Toro 2009
Problem
• HACCP: Zero tolerance
for birds passed with
SepTox
– Company inspectors,
USDA monitored
• Small birds, condemned
for Septicemia-Toxemia
0.5% average/week
(Hoerr, 2003)
H. Toro 2009
14
Plant Visit
• Representative condemned broilers
• Examination and necropsy
• Specimen collection
– Histopathology
– Virology
(Hoerr, 2003)
H. Toro 2009
Category No. Birds
Total number of small birds
examined
35
Obvious external lesions
(not further examined)
19/35
No external lesions:
Necropsy Findings
16/35
Ascites 2
Hepatitis 3
Yellow discoloration 1
Tibial dyschondroplasia (TD) 1
Air sacculitis 4
Small bursa of Fabricius 1616
Small thymus 1313
Thymus hemorrhage 4
35 Small “Sep-
Tox” broilers,
41-d-old
(Hoerr, 2003)H. Toro 2009
15
Histopatology:
– Lymphocyte depletion in the thymus
Virology:
– PCR(+) CAV.
– RT-PCR (+) IBDV
– CAV 03-4876 isolation in MSB1.
H. Toro 2009
Conclusion
CAV and IBDV contribute to a significant
portion of condemnations at chicken
slaughter plants
H. Toro 2009
16
Association between Viral
Immunodeficiency and Infectious
Bronchitis Virus
H. Toro 2009
Problem
Numerous IBV isolates were obtained in
Alabama between 1997 and 2003 from
broiler flocks with respiratory disease in spite
of IBV vaccination in the region.
Toro, H., V. L. van Santen, L. Li, S.B. Lockaby, E. van Santen, F. J. Hoerr (2006). Avian
Pathology 35:455-464.
H. Toro 2009
17
Alabama Broilers 1997-2002: 322 Respiratory cases. Inclusion
criteria: IBV isolation; histologic scoring of bursa and thymus;
known age of the broilers
0
5
10
15
20
25
0
1
2
3
4
10 20 30 40 50
No. IB
V Case
s
Bursa &
Thymus Depletion Sco
re
Age of Chickens
Bursa Bpred Thymus Tpred IBV IBVpred
Toro, H., V. L. van Santen, L. Li, S.B. Lockaby, E. van Santen, F. J. Hoerr (2006). Avian Pathology 35:455-464.
H. Toro 2009
Chickens (%) with audible tracheal rales at different time points
after IBV isolate 98/4614 nasal and ocular inoculation at 7 days of
age. Chickens were infected with CAV and IBDV at day 1 of age.
Number of chicks/group: 26 (days 4 and 8 DPI), and 21 (day 13 DPI).
CAV (03-4876) intramuscularly. IBDV (APHIS strain) orally.
0 25 50 75 100
4.0
8.0
13.0
16.0IBV
IBV+CAV+IBDV
Chickens (%)
Days a
fter
IBV
Ino
cu
lati
on
H. Toro 2009
Toro, H., V. L. van Santen, L. Li, S.B. Lockaby, E. van Santen, F. J. Hoerr (2006). Avian Pathology 35:455-464.
18
IBV RNA in lachrymal fluids
CAV + IBDV + IBVIBV
11 dpi
14 dpi
16 dpi
20 dpi
24 dpi Not done
28 dpi Not done
Individual
samples
Toro, H., V. L. van Santen, L. Li, S.B. Lockaby, E. van Santen, F. J. Hoerr (2006). Avian Pathology
35:455-464.H. Toro 2009
IBV RNA in the trachea
9 DPI
1 30
90
0
27
,00
0
Dilu
tio
n:
1 30
90
0
27
,00
0
CAV + IBDV + IBVIBV
CAV + IBDV + IBVIBV
14 DPI
19 DPI
Undiluted
individual
samples
Dilutions of
pooled
samples
Toro, H., V. L. van Santen, L. Li, S.B. Lockaby, E. van Santen, F. J. Hoerr (2006). Avian Pathology 35:455-464.H. Toro 2009
19
•ELISA absorbance is > mean absorbance for uninfected control + 3 standard deviations. Total number
•of samples with detectable IBV-antibodies higher (P<0.05) in IBV alone (Fisher’s exact test)
Incidence of detectable*
IBV-specific IgA
0,00
0,02
0,04
0,06
0,08
2 5 8 11 14 17 20 23 26 29
Mean A
bsorb
ance
DPI
IBV
CAV + IBDV +IBV
Uninfected
Mean absorbance vs. day
post IBV inoculation
IBV-specific IgA in
tears measured by
ELISA
5 8 11 14 16 20 24 28
IBV 50% 100% 100% 90% 100% 100% 100% 83%
CAV + IBDV +IBV 0% 22% 60% 90% 100% 80% 100% 83%
0%
20%
40%
60%
80%
100%
% C
hic
ken
s w
ith
de
tect
ab
le
an
ti-I
BV
Ig
A in
te
ars
DPI
H. Toro 2009
Conclusions
� Increased IBV isolations coincide with
lymphocytic depletion of bursa and/or thymus
� Immunodeficient chickens (CAV & IBDV) show:
� Increased and more severe IBV clinical signs
� Longer IBV persistence in trachea and lachrymal fluid
� Reduced local IBV-specific IgA response
� More severe and persistent pathological changes due
to IBV in the trachea
H. Toro 2009
20
Influence of CAV and/or IBDV on the IBV-specific
IgA response in the HG.
Ginkel van, F.W., V.L. van Santen, S. Gulley, H. Toro. Avian Diseases, 52: 608-617, 2008
H. Toro 2009
Conclusion
Mucosal immunity has been an important goal for the
prevention of respiratory viral infection in chickens.
Viral immunodeficiency by CAV and/or IBDV reduces B
and T helper cells at the HG, a relevant mucosal
effector site.
H. Toro 2008
Ginkel van, F.W., V.L. van Santen, S. Gulley, H. Toro. Avian Diseases, 52: 608-617, 2008
21
Association CAV and Fowl
Adenovirus type 1
H. Toro 2009
Inclusion body hepatitis/hydropericardium syndrome
field outbreak in 55 day-old brown layer pullets
Toro, H., C. Gonzalez, L. Cerda, M. Hess, E. Reyes, and C. Geisse (2000). Avian Diseases 44: 51-58.
H. Toro 2009
22
Outbreak of IBH/HP syndrome in 55-d-old brown layer pullets
Toro, H., C. Gonzalez, L. Cerda, M. Hess, E. Reyes, and C. Geisse (2000). Avian Diseases 44: 51-58.
H. Toro 2009
Inclusion Body Hepatitis/Hydropericardium
Syndrome (IBH/HP)
Some FAV serotype 4 isolates have shown to act as
primary pathogens in outbreaks of IBH/HP (Mazaheri et
al., 1998).
Many FAV isolates obtained from outbreaks of
IBH/HP have shown low pathogenicity when
experimentally inoculated in healthy chickens (e.g.
Cubillos et al., 1986; Hidalgo et al., 1994; Cowen et al., 1996; Toro et al.,1999)
H. Toro 2009
23
Cumulative mortality in a 10 day period after intramuscular
inoculation with CAV 10343 and fowl adenovirus 341
0
10
20
30
40
50
60
CAV+FAV CAV FAV Control
Toro, H., C. González, L. Cerda, M. Hess, E. Reyes, and C. Geisse (2000). Avian Diseases 44: 51-58.
H. T. 2009
Conclusion
Some FAV isolates, showing low pathogenicity when
inoculated in immunocompetent chickens, cause
IBH/HP in chickens with CAV -induced
immunodeficiency.
H. Toro 2009
24
Association CAV and Infectious
Bursal Disease Virus
H. T. 2009
�Chickens inoculated with CAV and IBDV show a
prolonged acute phase (Cloud et al. 1992a); lower in vitro
responses (Cloud et al. 1992b); CAV at high titers from dually
infected chickens (Imai et al, 1999).
�CAV persists in the chicken in spite of the presence of
neutralizing antibody (van Santen et al., 2005)
�T cells are needed in IBDV protection (Rautenschlein & Sharma,
2002).
H. Toro 2009
25
Broiler breedersBroiler breeders
• 89/03 In ovo
• Bursal SVS 510 Water
• Primevac IBD-3 Water
• Breedervac Inject
• Bursa Guard Inject
• CAV Wing Web
Broiler chickensBroiler chickens
• 89/03 In ovo
Effects of CAV and IBDV inoculation in broilers
Mix of CAV (10 5.6 TCID50/ml) and IBDV AL2 (10 5.0 CID50/ml) via
the drinking water on days 3 and 14 of age.
Toro, H., V. L. van Santen, F. J. Hoerr, C. Breedlove (2009). Avian Diseases 53:94-102.
H. Toro 2009
Broilers: Bursa lymphocyte
depletion
Bursa Index
15 20 25 30 35 40 450.0
0.5
1.0
1.5
2.0
2.5
3.0
Days of age
Bursa histomorphometry
15 20 25 30 35 40 450.3
0.4
0.5
0.6
0.7
Days of age
Control vs CAV+AL2 (P<0.05)
Control vs AL2 (P>0.05)
Control
CAV
AL2
CAV+AL2
Control vs CAV+AL2 (P< 0.05) Control
vs AL2 (P< 0.05)
Toro, H., V. L. van Santen, F. J. Hoerr, C. Breedlove (2009). Avian Diseases 53:94-102. H. Toro 2009
26
Broiler chickens: Thymus lymphocyte
depletion Means, SD, & linear regression
15 20 25 30 35 40 452.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
Days of ageCAV+AL2 vs Control (P=0.01)
AL2 vs Control (P>0.05)
Control
CAV
AL2
CAV+AL2
Th
ym
us h
isto
mo
rph
om
etr
y
Toro, H., V. L. van Santen, F. J. Hoerr, C. Breedlove (2009). Avian Diseases 53:94-102. H. Toro 2009
Day
20-
AL2
Day
20-
CAV+A
L2
Day
30-
AL2
Day
30-
CAV+A
L2
0
2
4
6
8
10
12
14
IB
DV
RN
A (
Rela
tive a
mo
un
t)
Day 20
Day 30
AL2-only CAV + AL2
1 2 3 4 5 6 1 2 3 4 5 6
IBDV RNA in broilers exposed to IBDV or CAV+IBDV on days
3 and 14 of age.
Relative intensities of bands of IBDV RT-PCR product obtained by image analysis were normalized to the
relative number of copies of ß-actin mRNA in each RNA sample obtained by qRT-PCR. Mean ratio of IBDV
RNA to ß-actin mRNA for AL2 chickens at 20 days was set to 1.
RT-PCR detection of IBDV RNA. On 20 d of age
4/6 AL2-only exposed chickens and all (6/6)
chickens exposed to CAV+AL2 were IBDV
positive. Day 30 all birds were positive.
Relative levels of IBDV RNA in bursae
of AL2+CAV infected chickens at 20 d
was different than other groups
(P<0.01).
H. Toro 2009
27
Conclusions
� CAV inoculated orally in broilers from CAV
vaccinated breeders did not persist through day 40
of age but was responsible for damage to the
immune system.
� CAV influenced IBDV replication.
� Maternal antibodies and IBDV in ovo vaccination
confers incomplete protection against CAV and IBDV
AL2 challenge.
H. Toro 2009