Fowl Adenovirus:Using Serology to Control

Your Flocks

Rafael Monleon DVM, MSpVM, ACPV, PASBusiness Unit Manager (Poultry)

9th September 2014

Adenovirus• Double stranded DNA; Non-enveloped

• Reproduce in nucleus

• Produce intranuclear inclusion bodies

Adenovirus

• Very resistant

• pH changes

• Disinfectants

• 60-70C for 30 minutes

• Formaldehyde and some other disinfectants have been somehow effective

Adenovirus

• Worldwide distribution

• Ubiquitous in poultry farms

• Chicken

• Turkeys,

• Ducks, geese, quail

• Found also in wild birds

Familiy Adenoviridae• Genus Aviadenovirus: Group I Avian Adenoviruses

• Fowl Adenovirus - 12 Serotypes / 5 Species

• Inclusion Body Hepatitis (IBH)

• Hydropericardium Syndrome (Angara Disease)

• Gizzard Erosion and Proventiculitis

• Genus Siadenovirus: Group II Avian Adenoviruses• Hemorraghic Enterititis (turkeys), Marble Spleen Disease

(pheasants), Splenomegaly (chickens)

• Genus Atadenovirus: Group III Avian Adenoviruses• Egg Drop Syndrome (EDS)

Classification FAV

FamilyAdenoviridae

GenusAviadenovirus

AD Group I

A

B

C

D

E

FAV-1

FAV-5

FAV-11

FAV-8b

FAV-2FAV-3FAV-9

FAV-4FAV-10

FAV-6FAV-7FAV-8a

CELO, 112, QBV, Ote, H1

340, TR22, M2, Tipton, IBH-2A

KR5, 506, H2, K31, 61, J2-A C2B, M11, CFA20, SA2, C-2B

SR48, 685, H3, P7-A, GA1-1, Z7 SR49, 75, H5, 75-1A-1 A02, 90, CFA19, A2-A

CR119, 168

UF71, 380

YR36, X11, X11-A, 122 TR59, 58, CFA40, T8-A 764, VRI-33, B-3A ICTV Classification

Fowl Adenovirus (FAV)Epidemiology

• Antibodies to FAV can be found in many poultry flocks

• FAV can be isolated from healthy as well as clinical birds

• Vertical transmission important in spread or “seeding” farms with several serotypes

• Birds can be infected with more than one serotype

• Protection is primarily serotype specific• Some Cross-protection Between Serotypes

• 3&4 / 2&11

• Birds can shed a serotype while having antibodies to another serotype

Fowl Adenovirus (FAV)Epidemiology

Fowl Adenovirus (FAV)Transmission

• Horizontal Transmission• Usually >15-20d• Litter, Equipment, Personnel• Recurrent Problem in Poorly C+D Houses• Commonly related with immunosuppression

Fowl Adenovirus (FAV)Transmission

• Vertical Transmission• Approx. >3d – 18-20d

• Breeders not seroconverted before lay• Infection During Lay > Virus shedding for 4-8 weeks• When ALL breeders seroconvert shedding stops –

Vertical transmission stops• Reactivation of latent virus might occur with stress• “Clean House Syndrome”

• Generally Seen High Levels Biosecurity• “Dirty” Houses – Re-used litter – Less Problems

Dynamics of FAV Infection Scenario I

16 W 40 W25 W8 W

FAV Infection

Point of Lay

NO TRANSMISSION OF ACTIVE VIRUS TO PROGENY

%Positives

(seroconversion)

VERY IMPORTANT TO ACHIEVE SEROCONVERSION BEFORE LAY

Dynamics of FAV Infection Scenario II

12

16 W 40 W25 W

%Positives

(seroconversion)

8 W

FAVInfection

Point of Lay

TRANSMISSION OF ACTIVE VIRUS TO PROGENY FOR A PERIOD OF ~4 – 8 WEEKS

LOSSES DUE TO LATE SEROCONVERSION

FAV - VERTICAL TRANSMISSIONOffspring Mortality

AGE

FAVOffspring Mortality – Vertical Transmission

FARM Dead% Cum.

Mort. Age of Disease

B9 705 11.73% 0-6 -2-1 wk.

A7 1,155 16.02% 0-6 -2-0 wk.

A8 1,769 24.83% 0-4-1-6 wk.

A2 83 1.43% 0-4-1-1 wk.

A3 291 4.98% 0-4-2-1 wk.

A4 535 9.22% 1-0-2-0 wk.

A5 510 8.56% 0-6-2-0 wk.

Total

5,048 Average10.97%  

Fowl Adenovirus (FAV)Inclusion Body Hepatitis

• First Described in 1960’s in USA• “the 3 days disease”• 3d ↑ mortality, 3d plateau, and 3d drop in mortality

• Depression, Weakness, Jaundice, Convulsions, Anorexia, Death

• Enlarged, dystrophic friable liver with redish-yellowish color

• Pale necrotic pancreas, muscle hemorrhages, bursal atrophy

• Breeder Mortality, Drops in Egg Production & Hatchability Reported

Fowl Adenovirus (FAV)Inclusion Body Hepatitis

• Multiple serotypes involved

• Serotype 2, 8b, 9, 11 common

• Traditionally Associated with IBD/CAV (ImS) • Now Considered Primary As Well

Mortality Associated

with FAV(21D)

Dias Mortalidad

1 3

2 5

3 6

4 2

5 5

6 335

7 63

8 26

9 18

10 18

11 12

12 10

13 14

14 16

15 12

16 14

17 7

18 10

19 14

20 10

21 19

Cumulative Mortality5.79%

Caribe @ 21D

India – Mortality >10%

INDIACUMULATIVE MORTALITY

>10%

Fowl Adenovirus (FAV)Hepatitis-Hydropericardium Syndrome

••• Mainly Serotype 4 (FAV-4 / Species C)

• Broilers / Breeders

• Increase of pericardial fluid (hydropericardium); hepatitis

• Generally 3 weeks or older

• Variable mortality but can be very high 40%+

• Sometimes associated with Immunosuppression

Fowl Adenovirus (FAV)Gizzard Erosion and Ulceration (GEU)

• Excessive erosion and ulceration of gizzard lining

• Mostly Species A (FAV-1) – Also reported FAV-8

• GEU experimentally reproduced

• Low mortality, uneven growth, increased FCR

• Also seen pancreatitis, proventiculitis, Hepatitis

• Rule-outs / Comorbidity: Mycotoxins, Biogenic Amines (Histamine, Gizzerosine, etc.), Vitamine B6 deficiency

Fowl Adenovirus (FAV)Gizzard Erosion and Ulceration (GEU)

Schade, 2013

Fowl Adenovirus (FAV)Diagnostic Tools

• Histopathology – Gold Standard

• Intranuclear Inclusion Bodies

• liver, pancreas, proventiculus or gizzard

Fowl Adenovirus (FAV)Diagnostic Tools

• Virus Isolation / PCR / In-Situ Hybridization

• VI+PCR > Detecting Presence of Virus

• Sequencing > Determine Serotype /Species

• HRM (Alternative)

• PCR+ / VI+ Does not Equal Disease

Fowl Adenovirus (FAV)Diagnostic Tools

• Serology• VN - serotype specific antibody detection

• Serotype specific

• Tedious, expensive, complicated, few places to perform

• ELISA – group specific antibody detection• Fowl adenovirus specific (Avian Adenovirus Group I)

• CANNOT DIFFERENCIATE BETWEEN SEROTYPES

• Easy to reproduce, inexpensive, automation, mass testing

• Interpretation of ELISA meaningful when comparing healthy vs. clinical birds and/or having meaningful baseline / guidelines

• Baselines / Guidelines

• Launched in 2011• Worldwide distributed FAV ELISA kit• Fowl Adenovirus Serotype 1 • Indirect ELISA, dilution 1:100• Specificity > 98 %• Filter 405 nm• Positive Cuttof S/P ≥ 0.5

• Positive Titer >1070

Biochek FAV ELISA

Detects Different SpeciesS/P RATIO Positive Cut-off >0.5

Biochek, Unpublished

FAV A FAV B FAV D FAV E FAV E0

1

2

3

4

5

6

7

S/P

AGP vs. Histopathology vs. ELISAELISA Positive Titer 1070

BioChek, Unpublished

FAV ELISA SPECIFICITYPOSITIVE CUT-OFF >0.5 (n=102) SPF

0 20 40 60 80 100 120

-0.1

-8.32667268468867E-17

0.0999999999999999

0.2

0.3

0.4

0.5

0.6

100% SPECIFICITY

BioChek, Unpublished

BioChek FAVBreeders

Birds in production generally positive Although poor seroconversion often occur

Monitoring is useful to confirm seroconversion, before entering production in troublesome areas BB should be 100% positive > 12-18W

High antibody titers do not necessarily correlate with protection Protection is SEROTYPE specific

BB 26 wks Poor SeroconversionAMT = 15200, CV % 53 AMT = 23467, CV % 20

BB 18 wks Good Seroconversion

FAV ELISA

Flock A Flock B0

5000

10000

15000

20000

25000

30000

0

5

10

15

20

25

30

35

40

AMTCV

FAV ELISAMONITORING SEROCONVERSION BREEDER FLOCKS

POOR SEROCONVERSION

GOOD SEROCONVERSION

FLOCK AT RISK OF INFECTION

BB 17W

BB 10W

BB 29WBB 29W

POOR SEROCONVERSION GOOD SEROCONVERSION

FAV ELISA Challenged BB Flocks (Vertical Transmission)

1 2 30

5000

10000

15000

20000

25000

30000

35000

512 5124096

8192

32768

16384 25 wks29 wks

Adapted Grafl et. al, 2012Titers converted from log2

WEAK SEROCONVERTIONPRE-LAY

WITH SUBSEQUENTINFECTION AND VERTICAL

TRANSMISSION

0w 4w 9w 15w 26w0

5000

10000

15000

20000

25000

30000

35000

0

20

40

60

80

100

120

140

AMTCV

FAV ELISA Vaccinated @ 8 weeks

VACCINATION

FAV ELISA BB 12w old / Vaccinated @ 8 weeks

A B C D E0

5000

10000

15000

20000

25000

30000

0

10

20

30

40

50

60

70

80

90

100

23143 2209823993

22398 22422

AMTCV

Flock

FAV ELISABB Flocks / Vaccinated @ 12-13wks

25w 60w0

2000400060008000

10000120001400016000

1800020000

010203040

50607080

90100

AMTCV

Flock A Flock B

LOW AMT / HIGH CVSEROTYPE?/PRIMING?

BioChek FAVDay Old Chicks

• Biochek FAV ELISA can demonstrate antibodies in DOC

• Samples in group 0 (negative) can be easily infected

• Positive titers do not necessarily correlate with protection

• Protection is SEROTYPE specific – Not GROUP specific

• Some examples of serology of DOC field data

• DOC offspring from vaccinated breeders

• FAV Serotype 4 – Inactivated Vaccine x1/x2

FAV ELISAMonitoring MAB DOC

10/20 NEGATIVEDOC AT RISK

BioChek FAVBroilers

• Limited data available

• Many healthy broiler flocks will test low positive (MT < 5000) at > 35D

• Clinical flocks have MT > 6500

• Useful to compare healthy non-clinical birds with clinically affected birds

• Baselines

FAV ELISANaturally Infected Broilers 45D-46D

A B C D0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

0

10

20

30

40

50

60

70

80

90

100

AMTCV

FLOCK

Tite

r

(%)

NON-CLINICAL

IBH Broilers Case History

First symptoms at 13-16D

Depressed, crouching position, lethargic with acute mortality

Peak mortality 3-5D after first symptoms, disappearing after 6-7D.

Total mortality at slaughter between 8 – 30%

Enlarged, dystrophic liver with yellowish color and crumbly texture

Nephritis

Intracellular Inclusion bodies in liver and kidney tissues

CESAC, SPAIN

CESAC, SPAIN

FAV ELISANon-clinical vs. Clinically Affected Flocks

Non-Clinical Clinically Affected0

2000

4000

6000

8000

10000

12000

14000

16000

0

10

20

30

40

50

60

70

80

90

100

AMTCV

CESAC, SPAIN

FAV ELISA Clinically Affected Broiler Flock Over Time

16d 38d0

2000

4000

6000

8000

10000

12000

14000

16000

0

20

40

60

80

100

120

140

AMTCV

START CLINICAL SYMPTOMS

SLAUGHTER

Control of Disease

• Prevent Immunosuppression

• IBD, CAV, REV, MD, Others

• Biosecurity• Cleaning and Disinfection + Downtime

• Beware “Clean / New House Syndrome”

• Some benefit with formaldehyde

Control of Disease

• Seroconversion of Breeders• Once neutralizing antibodies are present shedding generally stops

(exceptions in some isolates)

• Natural Exposure – Litter, Faeces• Beware what you bring in (i.e. Mycoplasma / Salmo)

• Live Un-attenuated Vaccine• Serotype 8b (Australia)

• Inactivated Vaccines - Serotypes 4, 5 & 8 available

• Autogenous Vaccines

48

Promoting Sero-conversion

49

FAV Inactivated Vaccines

Poultrymed (accessed 5/09/14)

Control of Disease

• Supportive Treatment• BLOOD BIOCHEMICAL CHANGES IN BIRDS WITH

INCLUSION BODY HEPATITIS AND THE EFFECT OF SUPPORTIVE TREATMENTS DURING OUTBREAKS

• D. VenneA and Y. ChorfiB – Western Poultry Diseases Conference 2011

• Treatment: • Treatment consisted in the addition of 125 g of sodium bicarbonate

plus 400 g of sugar in 576 L of drinking water.

Application BioChek FAV ELISA

• SPF Monitoring Confirming Negative Status

• Monitoring BREEDERS Natural Seroconversion

• 12-18W of age > 90% POSITIVE

• Vaccination Monitoring of BREEDERS• After Vaccination with FAV vaccines

• >90% POSITIVE

• Disease Monitoring BREEDERS

Application BioChek FAV ELISA

• DOC Titer – Monitoring of Mab Transfer

• Disease Monitoring BROILERS > 35D

• Suggested Horizontal Transmission Protection

• Compare clinical vs non-clinical serology

• AMT > 6500 associatied clinical disease

• Non-clinical flocks have AMT < 5000

• Vaccination Monitoring of BROILERS

Baselines BioChek FAV ELISA Test Kit

Provisional Baselines

Vaccine AMT CV %POS SUSPECT TITER CV

Broiler Breeders Inactivated / Natural SC 12000-30000 <35% >90% >15000-18000 <35%

Broilers None <5000 >40% >6500 <35%

These guidelines are based on our experience and information from our clients. BioChek does not accept any responsibility for the results using these guidelines.

These guidelines are subject to change without notice

Summary

• FAV are world wide distributed viruses that affect poultry in a wide array of manifestions that vary from mild to very severe forms

• FAV diagnosis is attained by combination of: clinical history, serology, and molecular techniques.• ELISA has been proved to be an useful tool

• Prevention of FAV starts by achieving seroconversion of parent flocks prior to lay and avoiding immunosupression

THANK YOU

rafaelmonleon@biochek.com