FOOT AND MOUTH DISEASE REASEARCH CENTRE LAHORE

Requirement for the degree of

DVM (Doctor of veterinary medicine )
Riphah college of veterinary science Lahore
( 2019)
Muhammad Ahsan Ahmad
Roll NO 105 Group H (Section2)
FMDRC Lahore Cantt
FOOT AND MOUTH DISEASE

Research Centre Lahore

Zarrar Shaheed Road Lahore Cantt
FOOT & MOUTH DISEASE RESEARCH CENTRE,
LAHORE
 INTRODUCTION

• Foot and Mouth disease Research centre

Lahore is a premier Institute working under the
Livestock and Dairy Development Punjab
Lahore.
• Established in 1964 as a section of Veterinary
Research institute Lahore on 23 acres of land at
Barki road Lahore.
• Declared as an independent Centre financially
and administratively in july2001.
 Main Functions of FMDRC

• Production of cell culture trivalent(O, A, Asia-

1) Foot And Mouth disease vaccine.
• Diagnosis of foot and mouth disease outbreak
and typing of virus isolates.
• Research and development.
• Production of hyper immune serum.
• In-service Training internship.
 Sections In FMDRC
• Media Section.
• Cell culture section.
• Virus culture section.
• Research and development section.
• Formulation Filling and Storage section
• Hyperimmune against FMD Serum.
 Media Section
• Preparation of Growth media GMEM
Media(Glass glow minimum essential
medium).
• Washing of glass ware,
• Sterilization of equipment or Lab ware etc.
 Preparation of GMEM Media for
propagation of BHK21 cell line
 Cell Culture Section
• Observation of cell culture / roller flasks
having BHK21 cells.( Baby Hamster Kidney
cells).
• Propagation of BHK21 cells line into cell
culture flasks
• BHK21 cells under Inverted Microscope.
• Preservation of BHK21 cell line.
• Revival Of BHK21 cell line.
Observing The BHK21 cells on Inverted
microscope
Propagation Of BHK21 Cells
Detachment of cells with Trypsin
versene solution
Pouring the GMEM Media
 Virus Culture section

• Preparation OF DMEM Media(Dulbecco’s

Modified Eagle media).
• Infection of BHK 21 cell line by O, A, Asia-1
virus)
• Harvesting of FMD virus.
• Inactivation of FMD virus.(BEI)
• In process Quality Control( Sterility Test, Safety
test, TCID ,).
50
Preparation of DMEM Media For
Maintenance And Infection and
Harvesting of FMD virus
Discard The Media
Infection the BHK21 cells with DMEM
Media
Incubate At 37 degree for 24 hrs
 Quality Control R&D
• Adaptation of Field virus isolates on BHK21
cell line.
• Quality Control test including Safety ,
Sterility,Potency . Serotyping Sandwich ELISA
,VNT and PCR.
• Maintenance of Experimental colony (Guinea
pigs. Mice colony).
• Propagation of BHK21 cell line.
 Adaptation OF FMD Virus (ON BHK21
Cells).
• Materials:-
• FMD Virus epithelium or vesicular fluid.
• BHK21 Cell line.
• DMEM. Media
• Incubator
• Safety cabinet
• Procedure:-
• Grind the tissue of FMD virus with sterile sand
in pestle mortar .and centrifuge at 2000rpm
for 10 mints.
• Filter the inoculation with 0.2 u.m syringe
filter and inoculate on BHK-21 cell line.
• Place infected cell line in CO2 incubator.
Observe the CPE for next 24-48hours .Make
serial passages for the isolation of FMD virus
on BHK21 cells.
Safety And Sterility Test of Vaccine
 Potency Test of FMD Vaccine
• Purpose:- To demonstrate the potency of a batch produced
in manufacturing.
• Procedure:-
• Use 7 guinea pigs of equal age and size .
• Vaccinate 7 guinea pigs with 0.5ml sub/cut .Keep 2 negative
control without vaccination in the same housing.
• Collect blood for serum collection at 28 days and prepare
serum.
• Store the serum in -20 degree
• Use the serum in the Virus neutralization test to determine
the antibody level .
 Typing of FMD virus serotype by ELISA:-

• Scope:-The test can be used to determine the

different types of FMD virus in the field.
• Equipment:-
• Micro plates positive and negative control.
• ELISA diluents buffer
• PBS
• Conjugate .
• Substrate.Chromogen solution.
• Stop solution (H2SO4).
Procedure:-
1. Dilute samples in 1.5ml tube by adding diluents
buffer.
2. Add 50ul each samples in 12 columns of the row
3. Add 50ul well of diluents buffer in all wells.
4. Incubate plates for 1 hour at room temperature.
5. Washing with 1X washing solution .Fill the wells
with 200ul washing solution and incubate for 3
mints.
6. Add 50ul conjugate each well and incubate at
room temperature for one hour.
7. Then 4 times washing and incubate for 5 mints.
8. Add 50ul stop solution each well.Read the OD
wavelength.
Performing Serotyping ELISA
 VNT (Virus neutralization test)
Scope:-
To determine the level of
:-

neutralizing antibodies against FMD

virus in serum.
PCR (Polymerase chain
reaction)
Scope:- The PCR used to amplify
genome fragments of FMDV in
diagnostic materials including
epithelium, milk, and serum
samples.
Guinea Pigs Area
Vaccine Filling and Bottling Section
• Formulation of vaccine.
• Filling of vaccine.
• Labeling printing and packing of vaccine.
• Storage of vaccine.
 Formulation of vaccine
• Purpose:- To inoculate standard dose of FMD
virus in vaccine.
• Montanide oil.
• Virus.
• Glycol buffer.
• Thiomersal(preservative).
• Then homogenize these materials at 12000rpm
for half an hour.
Filling and labeling of Vaccine
FMD Vaccine
Packing and storage room
 Serum Section
• Production of Hyperimmune serum against
FMD.
FMD Hyper immune Serum Vial 50ml
FMD Hyper immune serum production
per year
 Procedure
1. The bulls are inoculated with FMD disease
virus serotype 0.at zero day 1.5cc Virus S/C.
2. After 21 days 2ndinoculation with FMD
disease virus serotype A 1.5 cc virus S/C.
3. After 21 days 3rd inoculation with FMD
disease virus serotype ASIA 1. S/C.
4. After 21 days 4 inoculation with mix serotype
O.A .ASIA 1. 1.5CC S/C .
5. Bleeding is done at 21 -30 days and a shot
with mix serotype is given after 7 days.
6. 1 % working solution of potassium oxalate
is added to blood as an anticoagulant. Plasma
is collected after 24 hours in plasma vessels.
7 . 1 % calcium chloride is added to remove
clotting factor.
8. Then specially placed plasma weights are
placed after 1 hour of calcium chloride.
9 . Serum is collected after 24 hours.
10 . Carbolic acid is added as preservative.
11. Then add antibiotics in
serum(streptomycin and pencillin.)
12. Bottling is done after washing of bottles in
0.5% carbolic acid solution.
Bleeding of Animal
Plasma Vessels Tank