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Chapter 3 Materials and Methods

CHAPTER 3
MATERIALS AND METHODS
Avian influenza (AI) is a serious disease of poultry, resulting in severe mortality in
chickens and major disruption to production and trade (FAO, 2004). AI is further
classified into 15 hemagglutinin (H1-15) and 9 different neuraminidase (N1-9) subtypes.
AI viruses can of low (LP) and high pathogenisity (HP) for chickens and related poultry
species. LPAI viruses cause respiratory and gastrointestinal infections without infecting
the meat. By contrast, HPAI viruses produce infection of respiratory and gastrointestinal
tracts, produce a viremia and virus is present in the meat and internal contents of eggs
during the acute stages of the infection (Swayne, 2004). Avian influenza (AI) viruses
have been circulating periodically among domestic poultry over the past 100 years
(Stubbs et.al., 1948; Swayne and Suarez, 2000; Webster and Kawaoka, 1994). Vaccines
have been used as a tool in LPAI and HPAI eradication strategy. AI vaccines can prevent
clinical signs and death in poultry, increase resistance of birds to infection, and decrease
the amount of virus shed in the environment (Swayne, 2004).

The present study was designed to evaluate the comparative immunological response of
commercial oil based and liposomal vaccines of avian influenza. Commercial oil based
and Liposomal AI vaccine (MediExcel Pharmaceuticals, Islamabad, Pakistan) were
evaluated in commercial Layers. The present studies were designed to evaluate the
possible role of vaccine in restoring the commercial losses of the flocks by reducing
mortality and production losses with liposomal vaccine against avian influenza.

Preparation of liposomal vaccine

Immunogen-containing liposomes was prepared by mixing, 20 mg of sphingomyelin with
8mg of cholesterol and dissolved in 3 ml of chloroform and 3 ml of ether was added. The
mixture was bathed and sonicated to get a uniform water in oil emulsion with 10 mg of

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immunogen already dissolved in a balance salt solution (BSS) in 5.5 mM glucose, 0.4
mM KH2PO4, 1.2 mM Na2HPO4.7HPO4.7H2O, 1.3 mM CaCl2. 2H2O, 5.4 mM KCl, 136
mM NaCl, 1 mM MgCl2. 6H2O, 0.8 mM MgSO4.7H2O.

• Liposomes were made up to 2 ml with BSS and treated with 0.1 % osmium
tetrodxide (final concentration) in saline.

• The suspension was held for 30 minutes at room temperature and then dialyses it
extensively against a continuous flow of deionized, double distilled autoclaved
water.

• Liposomes were separated from free immunogen by centrifugation at 1000 g.

• Liposome suspension was made up in BSS to appropriate incorporated
immounogen concentration for injection.

IMMUNIZATION

Thirty commercial layers were partitioned in isolated bio-secure room and were divided
randomly and were given the same keeping and feeding facilities. Layers were divided
into three groups, A, B and C with 10 birds in each group. Each group was marked with a
specific color, as each group had to receive a different antigen preparation. Group A
served as control, Group B was immunized with conventional AI oil-based vaccines 0.5
ml/ bird and Group C was immunized with AI Liposomal vaccines 0.5 ml/ bird through
sub/cut injection. Blood samples were taken and serum was separated at day 0,
7,14,21,28 and day 35. Each time at least 4 samples were taken for antibody titration
through HA, HI and AGPT.

TITERATION OF THE ANTIBODY

PREPARATION OF ERYTHROCYTIC SUSPENSION FOR HI

To prepare red blood cell (RBC) suspension, blood samples were collected from
apparently healthy 4-6 week old broiler chickens. The blood samples were obtained in 10
ml sterile disposable syringes to which 2 ml of anticoagulant (2.5 % Sodium Citrate) was
added prior drawing the blood. Approximately 8 to 10 ml of blood was obtained from the
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subclavian vein of the birds. After collection the syringes containing blood and
anticoagulant were gently shaken to allow mixing of blood and sodium citrate to prevent
clotting. The blood samples were then poured into centrifugal tubes, which were spun at
1500 rpm for 5 minutes. The supernatant was discarded and 5 ml of PBS was added to
each tube containing packed RBCs. The samples were again centrifuged at the above
mentioned speed for the same period of time, the supernatant was discarded and the same
quantity of PBS was added. The process of washing blood samples was repeated thrice.
After which 0.5 % RBCs suspension was prepared in PBS (pH 7.2) and stored at 40C for
use in HA and HI tests.

HAEMAGLUTINATION TEST
Inactivated AI Antigen H7 strain was used for HA and HI tests according to the
following procedure.
I. a volume of 50 microlitre (µl) of PBS (pH 7.2) was dispensed in all wells of
rows A 1-12 and B 1-12 of 96 round bottom microtitre plate (Titertek,
Germany).
II. In first well (1A and 1B) of each row, 50 µl of AI antigen was added and
mixed with PBS.
III. Using micro-diluter tow-fold dilutions of the AI antigens were prepared.
IV. Using a micropipette 50 µl of 0.5 % RBCs suspension was added to each of
the wells of micropipette plate.
V. Well 12 was kept as control as only the diluent (PBS) and RBCs suspension
was added to this well.
VI. This microtitre plate was shaken gently to allow mixing of the contents of the
wells. The plate was then incubated at room temperature. The results were
recorded after 10-15 minutes when the RBCs in well 12 settled in the form of
a button at the bottom.
The results of HA test were interpreted as follows:
• Positive: the bottom of the well covered by finely clumped RBCs.

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• Negative: a small sharply outlined button of RBCs (bead formation) on the
bottom of the well.
• Doubtful: a ring formed by the unagglutinated RBCs disrupting the thin layer of
clumped cells coating the bottom of the well.
The HA titre was the reciprocal of the highest dilution exhibiting haemaglutination.

HAEMAGLUTINATION INHIBITION (HI) TEST
The HI test was conducted accortding to the procedure described by Javed et. al., 1986.
I. A volume of 50 microliter (µl) of PBS (pH 7.2) was dispensed in al wells of
rows A1 through H1 of 96 round bottom microtitre plate (Titertek, Germany).

II. A volume of 50 miocrolitre (µl) of PBS (pH7.2) was dispensed in all
remaining wells of all rows A2-12 through H2-12 of 96 round bottom
microtitre plate (Titertek, Germany).

III. 20 µl serum samples were pipetted in first well (A1 through H1) of each row.

IV. Sera were serially diluted by picking 50 µl from one well mixing in the next
well and transferring to the next and discarding 50 µl from the last well.

V. 50 µl of 10 HA stock antigen of AI was added in each well and mixed with
PBS, the plate was incubated for 30 minutes.

VI. Using a micropipette 50 µl of 0.5 % RBCs suspension was added to each of
the wells of microtitre plate, the plate was incubated for 30 minutes at 370C.

VII. Wells of 2 rows were kept as control negative as only the diluent (PBS) and
RBCs suspension was added to this well.

VIII. This microtitre plate was shaken gently to allow mixing of the contents of the
wells. The plate was then incubated at room temperature. The results were
recorded after 10-15 minutes when the RBCs in well 12 settled in the form of
a button at the bottom.

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The results were recorded as follows:

• Positive: clear button formation on the bottom of the wells.
• Negative; formation of uniform thin layer of finely clumped RRBCs

AGAR GEL PRECIPITATION TEST

PREPARTION OF AGAR GEL PLATE

Noble Agar gel was prepared by adding 0.9 % of Noble agar, 8 % of sodium chloride,
0.01 % of Sodium azide in distilled water. All ingredients were mixed in distilled water
and then heated to boil until a uniform suspension was obtained. Then uniform
suspension of the agar gel was cooled to 45 oC and poured into Petri-plate. The plate after
pouring was kept at room temperature until gel was solidified. The solidified gel was
transferred to refrigerator until use (Abbas et.al., 2004).

PUNCHING OF WELLSs

Well guiding plate was used for punching wells in the agar gel plate. The diameters of
wells were 5.3 mm and distances between wells were 2.4 mm (Abbas et.al., 2004). The
lid of plate was removed and template was placed on the agar gel plate. Care was taken
that the template shoµl d not touch the surface of agar gel. A gel borer was used to punch
wells agar gel. The punched gel in the wells was removed with great care with the help of
suction pump using a sterilized micropipette tip. Bottom of all wells was sealed with 20
µl of melted agar. This was done to minimize the leakage of antigen and antiserum
between gel and glass plate.

CHARGING OF WELLS

For qualitative assay the five wells were punched in a circle around a center well.
Unknown samples of antigens were dispensed in the peripherals wells while known
sample of antibodies was dispensed in the center well. The plate was incubated for 96
hours in humid chamber. Between wells of specific antigen and antibodies a precipitation
line was developed. The dilutions of these samples can be used laterally in experiment.

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For quantitative assay wells were punched in row and column fashion. Numbers of wells
were equal in all rows and columns. Two fold dilutions of antibodies were made in
micro-titer plate. The diluted antibodies were transferred into the wells of first and third
row and undiluted antigen was dispensed into the second row. The charged agar plate
was transferred to a humid chamber at room temperature and kept there for 96 hours.
Appearance of precipitation line was recorded.

STATISTICAL ANALYSIS

All the data for the experiment was analyzed by using the “M Stat C” developed by
Russel D. Fred and Scott P. Eisensmith, Crop and Soil Science department, Michigan
State University, USA (1995). Significant differences among different treatments were
analyzed with Duncan’s Mµl tiple Range Test (DMR-T). The statistical model was
completely randomized design with six replicates. Level of significance was P<0.01 in all
analysis.

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