Serological test for poultry (HI Test)

Mohamed Nabeh
Scientific office Director EM
DVM,MSCs, PhD Student of Poultry Diseases
AHRI
Mohamed Nabeh D. PhD Poultry
Disease

Sampling
Antigen titration
HI Procedures
Reporting
Interpretation
Disease

Sampling material
Disease

Sampling
serum
Blood
Disease

Disease

Sampling
size
• 10-20 Random
sample
• 1-5/1000 bird
Blood
2-3 ml in sterile
syringe not
contain anti-
coagulant
0.5-0.75 ml
serum
Preservation
4 c for short
time
-20 c for long
period
Disease

 Broiler
 Layer
 (floor)
 Cage system
 Breeders
During a disease outbreak when clinical signs
of the disease are first observed, followed by
collection from the same birds 3 to 5 weeks
later.
Disease

Sampling time
1day for MDA
7-10 days after live
vaccine
15-21 days after killed
vaccine
Disease

Sampling
interpretations
Turbidity
Putrefied (protelytic enzymes)
haemolysis
Bad
•Straw clear yellow
serumGood
Disease

if sample in -20 c
Shack sample well after thawing
Sample preservation
Disease

HI test
Disease

HI test material
Common Micropipette Sizes Volume Range
P50 multichannel 5-50 uL
P50 uni 5-50 uL
P100 10-100 uL
P1000 100-1000 u
Disease

Preparation of 4HA units conc.
From the virus antigen to be
used in HI test.
Disease

HA UNIT
It is the least amount of the virus
which is capable to haemagglutinate
1% RBCs solution in suspension
Disease

Preparation of washed 1% RBCs
RBCs taken from a minimum
of 3 SPF or SNA chickens on
4% sodium citrate or heparin
Cells should be washed three times in PBS
Centrifuge 3000 rpm/10 min
Disease

Antigen HA Titeration
Disease

Dispense washed 1%RBCs into each well of a plastic V-
bottomed microtitre plate
Make two fold serial dilutions of 25μl volume of the
antigen across the plate.
Dispense 25μl antigen in 1st well
Dispense 50μl PBS into each well of a plastic V-bottomed
microtitre plate
Disease

+
RBCsAG
Disease

HI Test procedure
4- Add 25μl of virus (4HA unit) to each well till the well no. 11
3- Make two fold serial dilutions of 25μl volume of the serum
across the plate till the well no. 11 .
2- Place 25μl of serum in the first well of the plate.
1- Dispense 25μl PBS into each well of a plastic V-bottomed microtitre plate
Disease

9- Wells that show inhibition should have RBCs stream at the same
rate as positive control wells (containing 25μl PBS and 25μl RBCs only).
8- Tilt the plate, determine HI titre (highest dilution of serum causing
complete inhibition of 4HAU of antigen).
7- Mix gently, then allow the RBCs to settle for about 20-30 min. at room temperature
6- Add 25μl of 1% chicken RBCs to each well.
5- Leave for 30 minutes at room temperature.
Disease

+
+
RBCs
ABAG
Disease

It is the least amount of the virus
which is capable to
haemagglutinate 1% RBCs
solution in suspension
Disease

Elution
Separation of the RBCs from the
agglutinating virus after attachment
and sedimentation of RBCs.
Disease

Types of elution
Reversible
Irreversible
Disease

Reversible
Due to change of PH alteration the bonding
strength between viral peplomers and RBCs receptors .
PH readjusted regains its ability to binds with RBCs
Disease

Irreversible:
Virus contain two surface peplomers
•A-haemagglutinin :responsible for binding with
RBCs.
•B- neuramidinase: for destruction of RBCS
receptors
•(NDV and Influenza)
Disease

1- Virus type
HA virus :a- Attach to RBCS
to form a network
B-sufficient virus titer
should be available to
induce HA as if the virus
titer increases the HA and
elution activities rise directly
Disease

2- RBCS
A-RBCS types :
each HA virus has
ability to agglutinate
one or more types
of RBCS
Ex: NDV RBCS
chicken
Parainfluenza-3
guinea pig RBCS
Disease

2- RBCS
•B-Duration of cell storage :
•prolonged storage cause
deterioration of their receptors , so
must be fresh or stored at 4c for
short period.
Disease

3- Environment
•A- Temperature :The
lower the temperature
reduce HA speed.
• B- PH :
• Any change in PH results
affect inhibition of HA
processes as well as
elution Ex : NDV 7.3.
Disease

C- Salt concentration :
suitable quantities of the salts(NaCl)
,very important in effective binding
between the virus and RBCs by
formation of chlorine bridge.
Disease

D- presence of non specific inhibitors:
HA may be inhibit due to presence of
non specific inhibitors in the reaction
buffer derived from serum remnants
resulting from insufficient washing of
RBCS during preparation.
Disease

Non specific inhibitors present
in the serum (e.x. sialic acid
residues) which react with
antigen resulting in nonspecific
inhibition and leading to false
interpretation.
Sera from chicken have low or
undetectable level of non-
specific inhibitors.
Disease

Heat treatment
(56C/30min).
game birds
(pheasant, partridge,
ect), quail, ostriches
and guinea fowl
RDE (receptor
destroying enzyme).
RBCs 10%.
Disease

Interpret the results of
different
haemagglutination tests.
Disease

HI test report
Disease

Thank you
Disease

Serological test for poultry (HI Test)

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