Pathobiology and Impact of Chicken Anemia Virus
Haroldo Toro, DVM, PhD Professor Department of Pathobiology
Chicken infectious anemia virus
1979 Japan (Yuasa) 1981 Germany (von BülowBülow)) 1981 Sweden ((EngstromEngstrom)) 1989 USA (McNulty; Rosenberger)
Worldwide distribution
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1 Serological evidence of CAV in the U.S. at least since 1959
1,200
1,000
0,800
0,600
0,400 S/N S/N ratio (620nm)
0,200
0,000 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 Individual Serum / Year
Toro, H., S. Ewald, and F.J. Hoerr (2006). Avian Diseases, 50:124-126.
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CAV is heat stable and unaffected by ether, chloroform or low pH. Most disinfectants are relatively ineffective. Eradication NOT possible.
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2 Chicken Anemia Virus Family: 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 CAV VP2 Replication: Lymphoid cells (MSB1, & other) 2298 nt VP3 Serotypes: 1 VP1 Transmission: Vertical & horizontal
Van Santen et al, 2004
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2298 nt
CAV genome VP3 VP1 CAV VP2 2298 nt VP3 HV Region VP1 VP2
Portion Sequenced 823 nt VP1 HV region
Van Santen, V., F.J. Hoerr, K. Joiner, C. Murray, N. Petrenko, & H. Toro (2004). Avian Diseases 48:494-504.
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3 Amino acid position HV region Polymorphisms in Sequence Origin (Year) 22 75 97 139 144 VP1 amino acid 05A Alabama (98) Q I L Q Q 26 Alabama (98) Q I L Q Q sequence among 55 Alabama (98) Q I L Q Q 57B Alabama (98) Q I L Q Q Alabama CAV 58 Alabama (98) Q I L Q Q 69B Alabama (98) Q I L Q Q sequences 73 Alabama (98) Q I L Q Q 05B Alabama (98) N I L Q Q 28B Alabama (98) N I L Q Q 57A Alabama (98) N I L Q Q 70 Alabama (98) N V M K E 03-4876 Alabama (03) N V M K E 28A Alabama (98) H I L K E 52 Alabama (98) H V M K E 69A Alabama (98) H V M K E V - Valine (Val) L - Leucine (Leu) 01-4201 Alabama (01) H V M K E I - Isoleucine (Ile) CIA-1 U.S. N I L Q Q M - Methionine (Met) 704 Australia H I L Q Q H - Histidine (His) K - Lysine (Lys) TR20 Japan H I L Q Q Q - Glutamine (Gln) L028 U.S. H T L Q Q N - Asparagine (Asn) E - Glutamic Acid (Glu) 26P4 U.S. H V M K E D - Aspartic Acid (Asp) A2B Japan H V M K E T - Threonine (Thr) Cux-1 Germany H V M K D CAU269/7 Australia H V M K E Australian Australia H V M K E
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%.
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4 CAV signs: Reduced Weight Gain (SPF chicks inoculated at 1 day of age by the IM or oral routes with CAV isolate 03-4876).
Weights
400
350
300
250 Uninfected
200 CAV oral Weight (g) Weight 150 CAV i.m.
100
50
0 0 7 14 21 28 Days p.i.
Van Santen, V., F.J. Hoerr, K. Joiner, C. Murray, N. Petrenko, & H. Toro (2004). Avian Diseases 48:494-504.
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CAV signs: Anemia resulting from infection of hemocytoblasts in the bone marrow
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5 CAV signs: Anemia (SPF chicks infected at day 1 of age with CAV isolate 03-4876 via the oral or IM routes).
Hematocrits
45
40
35
30 Uninfected 25 CAV oral 20
Hematrocrit (%) Hematrocrit CAV i.m. 15
10
5
0 0 7 14 21 28 Days p.i.
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
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6 Trombocytes in peripheral blood of chickens inoculated with strain Gifu-1
40 35 30
25 Control 20 CAV 15 Thrombocytes x 103mm3 x Thrombocytes 10 5 0 0 4 8 12 16 20 24 28 32 36 40 Days after Inoculation Tanigushi et al., (1983) H. Toro 2009
CAV: Thymic lesions
Control
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7 Experimental infection with CAV 10343 in 1010--ww--ololdd broiler breeders
Control
Toro et al. (1997) Avian Pathology 26:485-499. H. Toro 2009
Lymphocyte depletion: Infection of T lymphocytoblasts in the thymic cortex
1 2
3 4
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8 CAV: Inclusion bodies in the thymus (HE & EM).
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Lymphocyte depletion: Cortical lymphocyte/parenchyma ratio (CL/P)
Morphometry using ImageJ software version 1.29X (Public Domain, http://rsb.info.nih.gov/ij/ )
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9 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 CAV: 10 days PI
0.75 0.75 a a a a a 0.50 0.50 CL/P ratio CL/P CL/P CL/P ratio 0.25 0.25 b
0.00 0.00 Control CAV-I.M. CAV-Oral Control CAV-I.M. CAV-Oral Inoculation route Inoculation route
Van Santen, V., F.J. Hoerr, K. Joiner, C. Murray, N. Petrenko, & H. Toro (2004). Avian Diseases 48:494-504.
H. Toro 2009
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: 14 days PI CAV: 28 days PI
0.75 0.75
a a a a 0.50 0.50 c CL/P ratio CL/P CL/P ratio CL/P 0.25 0.25 b
0.00 0.00 Control CAV-I.M. CAV-Oral Control CAV-I.M. CAV-Oral Inoculation route Inoculation route
Van Santen, V., F.J. Hoerr, K. Joiner, C. Murray, N. Petrenko, & H. Toro (2004). Avian Diseases 48:494-504.
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10 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.
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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.
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11 CAV antibodies (ELISA) in SPF chickens inoculated at 1-d with CAV 03-4876 via the oral or IM routes
1,6 1,4 1,2 1 Controls 0,8 Oral
S/N ratio S/N 0,6 IM 0,4 0,2 0 7 10 14 28 Days post-inoculation
Van Santen, V., F.J. Hoerr, K. Joiner, C. Murray, N. Petrenko, H. Toro (2004). Avian Diseases 48:494-504. H. Toro 2009
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.
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12 Broiler Whole Bird Condemnation (Region). USDA National Agricultural Statistics Service
SW Mid-West S. Mid- S. East Atlantic Central % Septox 0.190 0.232 0.227 0.208 0.195 % 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
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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)
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13 Plant Visit
• Representative condemned broilers • Examination and necropsy • Specimen collection – Histopathology – Virology
(Hoerr, 2003) H. Toro 2009
Category No. Birds 35 Small “Sep- Total number of small birds 35 Tox” broilers, examined Obvious external lesions 19/35 41-d-old (not further examined) No external lesions: 16/35 Necropsy Findings 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 (Hoerr, 2003) H. Toro 2009
14 Histopatology: – Lymphocyte depletion in the thymus
Virology: – PCR(+) CAV. – RT-PCR (+) IBDV – CAV 03-4876 isolation in MSB1.
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Conclusion
CAV and IBDV contribute to a significant portion of condemnations at chicken slaughter plants
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15 Association between Viral Immunodeficiency and Infectious Bronchitis Virus
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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.
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16 Alabama Broilers 1997-2002: 322 Respiratory cases. Inclusion criteria: IBV isolation; histologic scoring of bursa and thymus; known age of the broilers
Bursa Bpred Thymus Tpred IBV IBVpred 4 25
20 3
15 2 10 No. IBVCases No. 1 5 Bursa & Thymus Depletion Depletion Score Thymus & Bursa
0 0 10 20 30 40 50 Age of Chickens
Toro, H., V. L. van Santen, L. Li, S.B. Lockaby, E. van Santen, F. J. Hoerr (2006). Avian Pathology 35:455-464.
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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.
IBV 16.0 IBV+CAV+IBDV
13.0
Inoculation 8.0 Days after IBV Days
4.0
0 25 50 75 100 Chickens (%) Number of chicks/group: 26 (days 4 and 8 DPI), and 21 (day 13 DPI). CAV (03-4876) intramuscularly. IBDV (APHIS strain) orally.
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 IBV RNA in lachrymal fluids
Individual samples IBV CAV + IBDV + IBV
11 dpi 14 dpi 16 dpi 20 dpi
24 dpi Not done
28 dpi Not done
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 Dilutions of pooled IBV CAV + IBDV + IBV samples 900 27,000 900 27,000 1 30 30 1 Dilution: 9 DPI
Undiluted individual samples IBV CAV + IBDV + IBV 14 DPI
19 DPI
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
18 IBV 0,08 CAV + IBDV +IBV IBV-specific IgA in Uninfected tears measured by 0,06
ELISA 0,04
0,02 Mean absorbance vs. day Absorbance Mean 0,00 post IBV inoculation 2 5 8 11 14 17 20 23 26 29
DPI
100%
80%
60%
40% Incidence of detectable* 20% 0% IBV-specific IgA 5 8 11 14 16 20 24 28 anti-IBV anti-IBV IgAin tears IBV 50% 100% 100% 90% 100% 100% 100% 83% % Chickens with Chickens detectable % CAV + IBDV +IBV 0% 22% 60% 90% 100% 80% 100% 83%
DPI
•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) 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
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19 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
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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.
Ginkel van, F.W., V.L. van Santen, S. Gulley, H. Toro. Avian Diseases, 52: 608-617, 2008
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20 Association CAV and Fowl Adenovirus type 1
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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.
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21 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.
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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)
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22 Cumulative mortality in a 10 day period after intramuscular inoculation with CAV 10343 and fowl adenovirus 341
60
50
40
30
20
10
0 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.
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23 Association CAV and Infectious Bursal Disease Virus
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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).
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24 Effects of CAV and IBDV inoculation in broilers
5.6 5.0 Mix of CAV (10 TCID 50 /ml) and IBDV AL2 (10 CID 50 /ml) via the drinking water on days 3 and 14 of age.
Broiler breeders • 89/03 In ovo • Bursal SVS 510 Water • PrimevacIBD-3 Water • Breedervac Inject • Bursa Guard Inject • CAV Wing Web
Broiler chickens • 89/03 In ovo
Toro, H., V. L. van Santen, F. J. Hoerr, C. Breedlove (2009). Avian Diseases 53:94-102. H. Toro 2009
Broilers: Bursa lymphocyte Control CAV depletion AL2 CAV+AL2
Bursa Index Bursa histomorphometry 3.0 0.7
2.5 0.6 2.0
1.5 0.5
1.0 0.4 0.5
0.0 0.3 15 20 25 30 35 40 45 15 20 25 30 35 40 45 Days of age Days of age
Control vs CAV+AL2 (P<0.05) Control vs CAV+AL2 (P< 0.05) Control Control vs AL2 (P>0.05) 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
25 Broiler chickens: Thymus lymphocyte
depletion Means, SD, & linear regression
6.5 6.0 Control 5.5 CAV 5.0 AL2 4.5 CAV+AL2 4.0 3.5 3.0
Thymus histomorphometry Thymus 2.5 15 20 25 30 35 40 45 Days of age CAV+AL2 vs Control ( P=0.01) AL2 vs Control ( P>0.05)
Toro, H., V. L. van Santen, F. J. Hoerr, C. Breedlove (2009). Avian Diseases 53:94-102. H. Toro 2009
IBDV RNA in broilers exposed to IBDV or CAV+IBDV on days 3 and 14 of age. RT-PCR detection of IBDV RNA. On 20 d of age Relative levels of IBDV RNA in bursae 4/6 AL2-only exposed chickens and all (6/6) of AL2+CAV infected chickens at 20 d chickens exposed to CAV+AL2 were IBDV was different than other groups positive. Day 30 all birds were positive. (P<0.01).
14 AL2-only CAV + AL2 12 1 2 3 4 5 6 1 2 3 4 5 6 10 8 Day 20 6 4 2 Day 30 0 IBDV RNA (Relative amount) 2
Day 20-AL2 Day 30-AL2
Day 20-CAV+AL Day 30-CAV+AL2 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. H. Toro 2009
26 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.
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