AVIAN INFLUENZA VACCINATION AND CHALLENGE TEST USING SUBTIPE H5N1 LOCAL ISOLATE

Fedik A. Rantam Virology and Immunology Laboratory, Microbiology Departmen and POSKOAI Airlangga University Surabaya

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 Indonesia to decrease mortality, productivity and spreading virus to animal and to human

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 virus spreading

MECHANISM OF IMMUN RESPONS

Target:

Antigen, Immunogen (Vaccine materials)

Machrophage,
Langerhans cell Dendritic cell T cell, B cell Mediated immunity

Output: 1. High antibody titer

2. Low antibody titer

3. Zero antibody titer

Doses/Concen tration, Genetic, Envinronment

Resistance to Agent
Vaccination Defence I Kill Vaccine Non specific respons immune Specific Respons immune Antibody Defence II Memory Cell

Machrophage , dendritic cell, langerhans cell, etc Cytokin (Chemokine, monokin)

T cell and B cell

No Memory Cell

Cytokin (Limphokin), Th1, Th2, T reg.

Antibody After Immunisation

IgG

Antibody Titer

Fisrt imunisation IgM

IgG

booster

IgG IgM

Days after immunisation Negative Phase

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
IgG IgM 0 hr 10 d 20 d 30 d 34 d

SIGNALING TRANSDUCTION IN CELLULAR IMMUNE RESPONS

Goose Muskaf Pegeon

Duck

Chicken

Peripheral blood mononuclear cells (PBMCs) from animal experiment were reacted using toll like receptors as signaling reseptor.

SIGNALING IMMUNE RESPONS (TLRs) IN GOOSE

PBMCs from Goose after immunization using AI Sub Type H5N1 Showed that the innate immunity as nonspecific immune response after receive signal through TLR and then immune system became more active to induce specific immune response as well as B cell and T cell

40000 35000 30000 25000 20000 15000 10000 5000 0 V1 V2 V3 V4 Homolog Heterol 1 Heterol 2

SIGNALING IMMUNE RESPONS (TLRs) IN MUSKAF

PBMCs from Muskaf after immunization using AI Sub Type H5N1 Showed that the innate immunity as nonspecific immune response after receive signal through TLR and then immune system became more active to induce specific immune respons as well as B cell and T cell

60000 50000 40000 30000 20000 10000 0 V1 V2 V3 V4 Homolog Heterol 1 Heterol 2

Negative Phase

SIGNALING IMMUNE RESPONS (TLRs) IN PEGEON

PBMCs from Pegeon after immunization using AI Sub type H5N1 Showed that the innate immunity as nonspecific immune response after receive signal through TLR and then immune system became more active to induce specific immune respons as well as B cell and T cell

30000 25000 20000 15000 10000 5000 0 V1 V2 V3 Homolog Heterol 1 Heterol 2

SIGNALING IMMUNE RESPONS (TLRs) IN DUCK

PBMCs from Duck after immunization using AI Sub type H5N1 Showed that the innate immunity as nonspecific immune response after receive signal through TLR and then immune system became more active to induce specific immune respons as well as B cell and T cell

70000 60000 50000 40000 30000 20000 10000 0 V1 V2 V3 V4 Homolog Heterol 1 Heterol 2

Negative Phase

SIGNALING IMMUNE RESPONS (TLRs) IN CHICKEN

PBMCs from Chicken after immunization using AI Sub type H5N1 Showed that the innate immunity as nonspecific immune response after receive signal through TLR and then immune system became more active to induce specific immune respons as well as B cell and T cell

6000 5000 4000 3000 2000 1000 0 V1 V2 V3 Homolog Heterol 1 Heterol 2

REACTIVITY ADAPTIVE IMMUNITY USING MARKER CD4,CD8,CD14 IN CHICKEN

REACTIVITY ADAPTIVE IMMUNITY USING MARKER CD4,CD8,CD14 IN GOOSE

REACTIVITY ADAPTIVE IMMUNITY USING MARKER CD4,CD8,CD14 IN DUCK

REACTIVITY ADAPTIVE IMMUNITY USING MARKER CD4,CD8,CD14 IN MUSKAF

Antibody titer and sheedding virus from trachea and duck cloaca After Challenge
Ab-Titer 3 weeks post vaccinati on sc im 2 days postchallenge 4 days postchallenge 6 days postchallenge

vr

vr

v r +

v r +

v r -

vr

28

27

26

24

24

24

Challenge Test

Antibody titer and sheedding virus from Muskaf trachea and cloaca after challenge
AbTiter 3 weeks post vaccina tion sc A 27 im 26 2 days postchallenge 4 days postchallenge 6 days postchallenge

v r +

v r +

v r +

v r +

v r +

v r +

B
C

24
23

24
24

+
+

+
+

+
+

+
+

+
+

+
+

Challenge Test

Antibody titer and sheedding virus from Goose trachea and cloaca After Challenge
Ab titer 3 weeks post vaccine sc A B C 28 24 23 im 211 27 24 2 days postchallenge 4 days postchallenge 6 days postchallenge

v r + + +

v r + + +

v r + + +

v r + + +

v r + + +

v r + + +

Challenge Test

Antibody titer and sheedding virus from Pegeon trachea and cloaca After Challenge
Ab titer 3 weeks post vaccination sc im 2 days postchallenge vr vr 4 days postchallenge vr vr 6 days postchallenge vr vr

27

26

25

23

25

25

NB : Control group without vaccination virus were sheedded

Antibody titer and sheedding virus from Kampung Chicken trachea and cloaca
Va cci ne Ab titer in 3rd week 2 days postchallenge 4 days postchallenge 6 days postchallenge

sc

im

v r
+

v r
+

v r
+

v r
+

v r
+

v r
+

27

27

26

25

Challenge Test
C 25 25 + + + + + +

Antibody titer and sheedding virus from Chicken (broiler) trachea and cloaca
Va cci ne Ab titer in 3rd week 2 days postchallenge 4 days postchallenge 6 days postchallenge

sc

im

v r
+

v r
+

v r
+

v r
+

v r
+

v r
+

27

27

26

26

26

25

Challenge Test

ANATOMI PATHOLOGY POST CHALLENGE TEST

When sheedding Virus from trachea and cloaca ?

Titer Ab mgg Ke-3 sc i m Hari ke-2 postchallenge Hari ke-4 postchallenge Hari ke-6 postchallenge

cl o

v r

c l o

tr k

cl o

tr k

Ho mo log
He ter ol 1

27

27

26

25

Challenge Test

He ter ol 2

25

25

Reisolation AI virus from different animal and organ with infetious doses (106 EID50/anl) using intranasal and oral
Vaksin Spp Trakea/par u Hati Limpa Ginjal Pankrea s Usus

Muskaf

A
B C C@ Control

+ + + + + +

+ + + + +

+ + + + +

+ + + + + + +

+ + + + + + + + + + + +

Chicken

A B C Positive Control Sentinel Control

Spreading Virus in Visceral Organ

Spleen C Spleen C Kidney B

Proven A

Kidney A

Lung B

Immunochemistry

GAMBARAN HISTOPATOLOGI
Proventrikulitis

Paru

Jantung

Pangkreas

Ginjal

Hati

CONCLUSION
The homolog epitop with the virus challenge given more better to induce immune response and resistant Using vaccination can decrease mortality rate All of animal experiment post challenge released virus through cloaca and trachea, but released virus from trchea longer than cloaca

MATURNUWUN

Our future

GAMBARAN HISTOPATOLOGI OTAK

Proliferasi vasculer pada otak, degenerasi neuron

Reisolasi virus AI dari berbagai hewan dan organ dengan dosis infeksi (106 EID50/ekor) dengan cara intranasal dan oral
Vaksin Spp Trakea/par u Hati Limpa Ginjal Pankrea s Usus

Mentok

H5N1
H5N2 H5N9 H5N9@ Kontrol

+ + + + +

+ + + +

+ + + +

+ + + + + +

+ + + + +

td
td td + + + + +

Bebek Ayam

Kontrol H5N1 H5N2 H5N9 Kontrol

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

TERJADI SHEEDDING VIRUS ATAU TIDAK ?

HEWAN COBA SEBELUM DIINFEKSI VIRUS

HEWAN COBA DIINFEKSI VIRUS

AYAM MATI KURANG DARI 48 JAM SETELAH DIINFEKSI

KONDISI AYAM SETELAH INFEKSI AI KURANG DARI 48 JAM

TIGA HARI SETELAH INFEKSI AI

PROFIL ANTIBODY HUMORAL TERHADAP AI DENGAN UJI ELISA