pathobiology and impact of chicken anemia virus - · pdf file1 pathobiology and impact of...

1

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).


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


0.25

0.50

0.75

aa

b

Inoculation route

CL

/P r

ati

o


H. Toro 2009

CAV: 14 days PI


0.25

0.50

0.75

a

b

c

Inoculation route

CL

/P r

ati

o

CAV: 28 days PI


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.


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).


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


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

pathobiology and impact of chicken anemia virus - · pdf file1 pathobiology and impact of...

Documents