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AVIAN INFLUENZA VACCINATIONAND CHALLENGE TEST USING

SUBTIPE H5N1 LOCAL ISOLATE

Fedik A. Rantam

Virology and Immunology Laboratory,Microbiology Departmen and POSKO-AI Airlangga University Surabaya

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INTRODUCTION

Avian Influenza virus very difficult to eliminate

Spreading in all region in Indonesia

The big problem for public health not in

Indonesia only but in round the world

Causing economic loss

Serious disease in the poultry

Vaccination is the best alternative in Indonesiato decrease mortality, productivity and spreading

virus to animal and to human

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PROBLEMS

What kind the immunogen can induce

antibody with high titer

When the vaccination program to do

How to increase the animal protectivity

again AI-virus using vaccination

How is the strategy to decrease virusspreading

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MECHANISM OF IMMUN

RESPONS

Antigen,

Immunogen(Vaccine

materials)

Target:

Machrophage,

Langerhans cellDendritic cell

T cell, B cell

Mediated immunity

Output:

1. High antibody titer2. Low antibody titer

3. Zero antibody titer

Doses/Concen

tration,

Genetic,

Envinronment

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Kill Vaccine

Vaccination

Non

specific

responsimmune

Specific

Responsimmune

Antibody

MemoryCell

No

Memory

CellMachrophage

, dendritic

cell,langerhans

cell, etc

T cell and B

cell

Resistance to Agent

Cytokin

(Chemokine,

monokin)

Cytokin

(Limphokin),

Th1, Th2, T reg.

Defence I Defence II

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Antibody After Immunisation

IgM

IgG

IgM

IgG

Fisrt

imunisation

booster

AntibodyTit

er

Days after immunisation

Negative

Phase

IgG

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When, How The Doses, Route

Vaccination

Chicken 5 - 7 days old

Aplication route s.c. or i.m. have no

significant

The Doses minimal 107 or 5-10 g/ ml

0 hr 34 d

10 d 20 d 30 d

IgG

IgM

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SIGNALING TRANSDUCTION INCELLULAR IMMUNE RESPONS

Goose MuskafPegeon

Duck Chicken

Peripheral blood mononuclear cells (PBMCs) from

animal experiment were reacted using toll likereceptors as signaling reseptor.

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SIGNALING IMMUNERESPONS (TLRs) IN GOOSE

PBMCs from Gooseafter immunizationusing AI Sub TypeH5N1

Showed that the innateimmunity as nonspecificimmune response afterreceive signal throughTLR and then immunesystem became moreactive to induce specificimmune response as well

as B cell and T cell0

5000

10000

15000

20000

25000

30000

35000

40000

V1 V2 V3 V4

Homolog

Heterol 1

Heterol 2

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SIGNALING IMMUNERESPONS (TLRs) IN MUSKAF

PBMCs from Muskaf

after immunization using

AI Sub Type H5N1

Showed that the innate

immunity as nonspecificimmune response after

receive signal through

TLR and then immune

system became moreactive to induce specific

immune respons as wellas B cell and T cell

0

10000

20000

30000

40000

50000

60000

V1 V2 V3 V4

Homolog

Heterol 1

Heterol 2

Negative Phase

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SIGNALING IMMUNERESPONS (TLRs) IN PEGEON

PBMCs from Pegeon after

immunization using AI

Sub type H5N1

Showed that the innateimmunity as nonspecific

immune response after


TLR and then immune


immune respons as well


5000

10000

15000

20000

25000

30000

V1 V2 V3

Homolog

Heterol 1

Heterol 2

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SIGNALING IMMUNERESPONS (TLRs) IN DUCK

PBMCs from Duck after

immunization using AI

Sub typeH5N1

Showed that the innateimmunity as nonspecific



TLR and then immune




20000

30000

40000

50000

60000

70000

V1 V2 V3 V4

Homolog

Heterol 1

Heterol 2

Negative Phase

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SIGNALING IMMUNERESPONS (TLRs) IN CHICKEN

PBMCs from Chicken

after immunization using

AI Sub type H5N1Showed that the innate

immunity as nonspecific



TLR and then immune



as B cell and T cell

0

1000

2000

3000

4000

5000

6000

V1 V2 V3

Homolog

Heterol 1

Heterol 2

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REACTIVITY ADAPTIVE IMMUNITYUSING MARKER CD4,CD8,CD14 IN

CHICKEN

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GOOSE

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REACTIVITY ADAPTIVE IMMUNITYUSING MARKER CD4,CD8,CD14 IN DUCK

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MUSKAF

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Antibody titer and sheedding virus fromtrachea and duck cloaca After Challenge

Ab-Titer

3 weeks

post

vaccinati

on

2 days

postchallenge

4 days

postchallenge

6 days

postchallenge

sc im vr vr v

r

v

r

v

r

vr

A 28 27 + + + + - -

A 26 24 + + + + - -

A 24 24 + + + + - -

Challenge Test

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Antibody titer and sheedding virus fromMuskaf trachea and cloaca after

challengeAb-

Titer 3

weeks

post

vaccination

2 days

postchallenge

4 days

postchallenge

6 days

postchallenge

sc im v

r

v

r

v

r

v

r

v

r

v

r

A 27 26 + + + + + +

B 24 24 + + + + + +

C 23 24 + + + + + +

Challenge Test

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Antibody titer and sheedding virus fromGoose trachea and cloaca After

ChallengeAb

titer 3

weeks

post

vaccine

2 days

postchallenge

4 days

postchallenge

6 days

postchallenge

sc im v

r

v

r

v

r

v

r

v

r

v

r

A 28 211 + + + + + +

B 24 27 + + + + + +

C 23 24 + + + + + +

Challenge Test

A tib d tit d h ddi i f

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Antibody titer and sheedding virus fromPegeon trachea and cloaca After

ChallengeAb titer 3

weeks post

vaccination

2 days

postchallenge

4 days

postchallenge

6 days

postchallenge

sc im vr vr vr vr vr vr

A 27 26 - - - - - -

B 25 23 - - - - - -

C 25 25 - - - - - -

NB : Control group without vaccination virus were sheedded

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Antibody titer and sheedding virusfrom Kampung Chicken trachea and

cloacaVa

cci

ne

Ab

titer in

3rd

week

2 days

postchallenge

4 days

postchallenge

6 days

postchallenge

sc im vr

vr

vr

vr

vr

vr

A 27 27 + + + + + +

B 26 25 + + + + + +

C 25 25 + + + + + +

Challenge Test

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Antibody titer and sheedding virusfrom Chicken (broiler) trachea and

cloacaVa

cci

ne

Ab

titer in

3rd

week

2 days

postchallenge

4 days

postchallenge

6 days

postchallenge

sc im vr

vr

vr

vr

vr

vr

A 27 27 + + + + + +

B 26 26 + + + + + +

C 26 25 + + + + + +

Challenge Test

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ANATOMI PATHOLOGY POSTCHALLENGE TEST

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When sheedding Virus fromtrachea and cloaca ?

Titer

Ab

mgg

Ke-3

Hari ke-2

postchallenge

Hari ke-4

postchallenge

Hari ke-6

postchallenge

sc i

m

cl

o

v

r

c

l

o

tr

k

cl

o

tr

k

Ho

mo

log

27 27 + + - + - +

He

ter

ol1

26 25 + + - + - +

He

ter

ol

2

25 25 + + - + - +

Challenge Test

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Reisolation AI virus from different animal and organ withinfetious doses (106 EID50/anl) using intranasal and oral

Spp

Vaksin Trakea/par

u

Hati Limpa Ginjal Pankrea

s

Usus

Muskaf A - - - - -

B - - - + -

C - - - - -

C@ + - - + + +

Control - - - - - -

Chicken A + + + + + +

B + + + + + +

C + + + + + +

Positive

Control

+ + + + + +

Sentinel

Control

+ + + + + +

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Spreading Virus in Visceral Organ

Spleen C Spleen C Kidney B

Proven A Kidney A Lung B

Immunochemistry

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GAMBARAN HISTOPATOLOGI

Proventrikulitis JantungParu

Pangkreas Ginjal

Hati

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CONCLUSION

The homolog epitop with the viruschallenge given more better to induceimmune response and resistant

Using vaccination can decrease mortalityrate

All of animal experiment post challenge

released virus through cloaca andtrachea, but released virus from trchealonger than cloaca

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MATURNUWUN

Our future

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GAMBARAN HISTOPATOLOGIOTAK

Proliferasi vasculer

pada otak, degenerasi

neuron

Reisolasi virus AI dari berbagai hewan dan organ dengan

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Reisolasi virus AI dari berbagai hewan dan organdengandosis infeksi (106 EID50/ekor) dengan cara intranasal dan

oral

Spp

Vaksin Trakea/par

u

Hati Limpa Ginjal Pankrea

s

Usus

Mentok H5N1 - - - - - td

H5N2 - - - + - td

H5N9 - - - - - td

H5N9@ + - - + + +

Kontrol - - - - - -

Bebek Kontrol - - - - - -

Ayam H5N1 + + + + + +

H5N2 + + + + + +

H5N9 + + + + + +

Kontrol + + + + + +

Kontrol

*

+ + + + + +

Ket: @ hewan mati setelah 7 hari infeksi, * penularan alami hewan dicampur hewan sakit,

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TERJADI SHEEDDING VIRUS ATAUTIDAK ?

HEWAN COBA SEBELUM

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HEWAN COBA SEBELUMDIINFEKSI VIRUS

HEWAN COBA DIINFEKSI

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HEWAN COBA DIINFEKSIVIRUS

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AYAM MATI KURANG DARI 48

JAMSETELAH DIINFEKSI

KONDISI AYAM SETELAH

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KONDISI AYAM SETELAHINFEKSI AI KURANG DARI 48

JAM

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TIGA HARI SETELAH INFEKSI AI

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PROFIL ANTIBODY HUMORALTERHADAP AI DENGAN UJI ELISA

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