Internship Report on Vaccine Production at FMD and VRI

INTERNSHIP REPORT 2019
Organizations:
Foot and Mouth Disease Research Centre (FMD),
Lahore
Veterinary Research Institute (VRI), Lahore
Submitted By:
SAFIA BIBI
(FA-2017/M.Sc.Zoology/016)
Session:
(2017-2019)
An internship report submitted to Lahore Garrison University in
partial fulfillment of the requirement for the degree of M.Sc
Zoology
Sumission date:
30 june, 2019
DEPARTMENT OF BIOLOGY
LAHORE GARRISON UNIVERSITY

i
LETTER OF UNDERTAKING
I Safia Bibi (Roll no# Fa-17/MscZoo/016). Session Fall 2017-19. Department of Biology
in Lahore Garrison University (LGU). I have completed my internship at Foot and Mouth
Disease Research Centre (FMD), and Veterinary Research Institute (VRI) Lahore. I hareby
declare that this internship has been genuinely conducted for theprescribed duration of time
and in case of any misstatement or false description the undersigned may be held
responsible.
---------------------------
Signature of student
---------------------------------
Internship Supervisor
Dr. Azam Ali Nasir
Veterinary Research Institute (VRI), Lahore
---------------------------------
Internship Supervisor
Dr. Reehan.
Foot and Mouth Disease Research Centre (FMD), Lahore
---------------------------------
Class Coordinator
Dr. Fouzia
Senior lecturer
Department Of Biology
Lahore Garrison University (LGU)
---------------------------------
Head Of Department
Col (Retd) Dr. Muhammad Amjad
Department Of Biology
Lahore Garrison University (LGU)

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DEDICATIONS
First of all I am thankful to Almighty Allah for all
that he blessed me with.
I dedicate this work to my beloved parents all my
teachers, my friends and all those who helped and
encouraged me to complete my work.

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ACKNOWLEDGEMENT
All praise to Almighty Allah the Merciful.
The internship opportunity I had with [FMD&RC] and [VRI] was a great chance for
learning and professional development. I am also grateful for having a chance to meet so
many wonderful people and professionals who led me though this internship period.
I would first of all acknowledge honourable H.O.D Col (Retd.) Dr. Muhammad Amjad.
I am very happy to say thankful to Dr. Fouzia Qamar coordinator for her great support. She
helped me a lot during my whole work and she guided me a lot to complete my work
accurately and on time.
I express my deepest thanks to Ex-Director Dr. Zafarullah and Present-Director Dr. Sajjad
for taking part in useful decision & giving necessary advices and guidance and arranged
all facilities to make life easier. I choose this moment to acknowledge their contribution
gratefully.
It is my radiant sentiment to place on record my best regards, deepest sense of gratitude to
Dr. Azam Ali Nasir (Internship supervisor at VRI), Dr. Reehan (Internship supervisor at
FMD&RC), and all the staff for their careful and precious guidance which were extremely
valuable for my study both theoretically and practically.
I perceive this opportunity as a big milestone in my career development. I’ll strive to use
gained skills and knowledge in the best possible way, Hope to continue cooperation with
all of you in the future,
Sincerely,
Safia Khan
Fa-17/M.Sc.-Zoo/016

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EXECUTIVE SUMMARY
The venues of my internship is “FOOT AND MOUTH DISEASE RESEARCH CENTRE
(FMD & RC)” and “VETERNARY RESEARCH INSTITUTE (VRI)”.
The different type of animal vaccines and medicines are produced at both these institutes.
At FMD & RC the FMD vaccine is produced to treat the FMD disease caused by
apthovirus. There are four sections in FMD vaccine production first of all at media section
GMEM media is prepared it is then send to cell culture section for culturing and then to
virus section in this DMEM media is prepared then the inoculation of virus is done after
that the media is harvested and sent for inactivation of virus the last section is formulation
and bottling section at which the vaccine is formulated and packed and ready to use.
There is another section R & D section which performs different types of diagnostic tests
e.g., ELISA, 146S, PCR etc.
My internship at “VETERINARY RESEARCH INSTITUTE (VRI)” was a good
experience. Veterinary Research Institute Lahore is a premier research organization in the
country which comes under Government of the Punjab.
Many vaccines are prepared in this organization like HS Vaccine, PPR Vaccine in cell
culture section, LaSota and Mukteswar Vaccines in poultry vaccine section and many
others.
Media and Anaerobe Section deals with the preparation of media cultures and their bulk
storage. Water purity test are performed in this section. Different media are prepared in
this section like, Nutrient Agar, MacConkey Agar, Sabouraud Agar In anaerobe section
vaccine against ENTEROTOXEMIA, BLACK QUARTER and LAMB DYSENTRY was
prepared.
In Poultry Vaccine Section ND LaSota Vaccine and Mukteswar Vaccine was prepared. All
the process of vaccine production was observed.
Preparation of HS vaccine was observed under HS Section. In this section vaccine was
prepared against the disease named as HEMORRHAGIC SEPTICEMA. Different tests
were performed for vaccine which show that this vaccine is ready for commercialization
i.e. sterility and efficacy test.
In Cell Culture Section PPR Vaccines was prepared by the use of Vero cell lines. These
cell lines were prepared from green monkey which was imported from France. Propagation
of cells was observed, development of monolayer and production of CPEs under inverted
microscope was observed

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TABLE OF CONTENTS
Sr.
No.
Topic Page
No.
1. Training program 1
2. FMD Introduction 1
3. FMD vaccine production 4
4. Media Section 5
5. Cell Culture Section 7
6. Virus Section 9
7. Formulation & Bottling 15
8. R & D Section 17
9. Media & Anaerobe Section 19
10. Poultry Vaccine Section 24
11. HS section 29
12. Cell Culture Section 34
13. Reflective Journal Entries 38
14. Sample handling 42
15. Critical & SWOT analysis 44-45
16. Conclusion 46
17. Recommendations 47
18. References 48
19. Annexures 51

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LIST OF FIGURES
Figure No Figures Page no.
Figure 1.1 Diseased animal 1
Figure 1.2 Lab test 2
Figure 1.3 Vaccine 3
Figure 2.1 GMEM powdered media 5
Figure 2.2 Filtration 6
Figure 2.3 BHK 21 cells 7
Figure 2.4 Cell line passage 8
Figure 2.5 DMEM powdered media 9
Figure 2.6 Media formation 10
Figure 2.7 Filtration 11
Figure 2.8 CPE 12
Figure 2.9 Passage 13
Figure 2.10 Sodium hiosulfate 15
Figure 2.11 Bottling 15
Figure 2.12 FMD vaccine 16
Figure 3.1 ELISA 17
Figure 3.2 Results 18
Figure 4.1 Media section 20
Figure 4.2 Blood agar 21
Figure 4.3 Nutrient agar 21
Figure 4.4 MacConkey agar 22
Figure 4.5 Sabouraud agar 22
Figure 4.6 Thioglycollate agar 23
Figure 5.1 Diseased chick 24
Figure 5.2 Virus innoculation. 24
Figure 5.3 Candling 25
Figure 5.4 Drilling 25
Figure 5.5 Incubation 26
Figure 5.6 Chilling 27
Figure 5.7 Harvesting 27
Figure 5.8 Newcastle Disease vaccine 28
Figure 5.9 Mukteswar vaccine 28
Figure 6.1 PPR vaccine 29
Figure 6.2 Master seed activation 30
Figure 6.3 Gram staining 31
Figure 6.4 ALUM precipitated vaccine 32
Figure 6.5 Packaging 33
Figure 7.1 Vero cell line 34
Figure 7.2 PPR vaccine 34
Figure 7.3 Sterilization 35

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Figure 7.4 CPE 36
Figure 7.5 Lyophilization 37

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LIST OF ABBREVIATIONS
1. HS Hemorrhagic Septicemia
2. BHI Brain Heart Infusion
3. QCL Quality Control Lab
4. PPR Pestedes Petites Ruminants
5. GMEM Glasgow’s Minimum Essential Medium
6. DMEM Dulbecco’s Modified Eagle Medium
7. CPEs Cytopathic Effects
8. ND New Castle’s Disease
9. BQM Black Quarter Vaccine
10. RCM Reinforced Clostridial Medium
11. APV Alum Precipitated Vaccine
12. OAV Oil Adjuvant Vaccine

x
LIST OF INSTRUMENTS
Serial No Instrument (Model) Made
1 Binocular Microscope
(CX21FS1)
Made in China
2 Centrifuge BMS
(H9R5P4)
Made in Canada
3 Micropipette
(N137471)
Made in Germany
4 Multichannel Micropipette
(KR600P401)
Made in Japan
5 Steam Boiler
(MSS-STR-EQP-003)
Made in Pakistan
6 Gas Sterilizer
(MSS-PHL-EQP-001)
Made in Pakistan
7 Incubator
(PVS-MUK-EQP-07)
Made in Denmark
8 Candler
(PVS-MUK-EQP-015)
Made in Pakistan
9 Drilling Machine
(PVS-MUK-EQP-012)
Made in Pakistan
10 Homogenizer
(HSII-VPDR-EQP-007)
Made in Pakistan

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OVERVIEW OF THE ORGANIZATION
Veterinary Research Institute (VRI) overview:
The Veterinary Research Institute was established in 1962 on up-gradation from
Bureau of Disease Investigation & Biological Production, Lahore. It is premier research
organization in the country which is controlled by Government of the Punjab. Playing a
“Role of Savior” towards maintenance of animal’s health & creating conditions suitable
for the poultry & livestock sector. Undertaking research on important animal diseases
prevalent throughout the country and produces sera, vaccines and diagnostic agents for
effective control of major epizootic and enzootic diseases in the country.
The nature of work in Veterinary Research Institute, Lahore is basically a combination of
biologics production and applied research. The institute has controlled significantly
towards prevention and control of prevailing, newly emerging and re-emerging diseased of
livestock and poultry.
MOTTO: “Prevention is better than cure”.
Objectives:
The main objectives of the institute are:
1) Large scale production of standard biologics for control of infectious disease of
livestock and poultry.
2) Research studies in the related disciplines of animal health and biologics produced in
VRI.
3) Studies related to obscure diseases of livestock and newly emerging disease of poultry.
4) Development of modern techniques for vaccine production and disease production.
5) In-service training for the members of Veterinary profession in advance techniques.
6) Training of the farmers to familiarize them in livestock health problem and vaccination
schedule etc.
Foot and Mouth Disease Research Institute (FMD&RC)
overview:
The Foot and Mouth Disease Research Centre (FMD & RC) Lahore is a premier
Research Organization in the country administratively controlled by Livestock and Dairy
Development Department, Government of Punjab.
The institute was separated in 2001 from Veterinary Research Institute Lahore and is
situated at Zarrar Shaheed Road Lahore Cantt. It has a complex of partly air conditioned
laboratories and animal houses extended over a total area of 23 acres.

xii
Main functions:
1. The production of cell culture trivalent foot and mouth disease vaccine.
2. Diagnosis of Foot and Mouth Disease outbreaks and typing of virus isolates.
3. Research and development
4. Production of hyper immune serum.
5. In service training/internship.
Different sections:
1. Cell culture section
2. Media section
3. Virus culture section
4. Research and development section
5. Labeling packing and bottling section.
6. Hyper immune FMD serum section.

0
PLAN OF INTERNSHIP PROGRAM
With reference to DVRI diary no 1242 to 1245 dated 20-03-2019. The
tentative schedule for internship program was as under.
Serial no. Section Duration
1. FMD & RC 1/4/2019 to 15/5/2019
2. Media and Anaerobe Section 16/5/2019 to 31/5/2019
3. Poultry Vaccine Section 1/6/2019 to 10/6/2019
4. HS Section 11/6/2019 to 20/6/2019
5. Cell Culture & Flury Section 21/6/2019 to 28/6/2019
6. Evaluation/Exam 1st
July

1
TRAINING PROGRAM
CHAPTER#1
INTRODUCTION TO FOOT AND MOUTH DISEASE (FMD).
1.1: What is FMD?
The organism which causes FMD is an aphthovirus of the family Picornaviridae.
There are seven strains (A, O, C, SAT1, SAT2, SAT3 (South African Territories), and
Asia1) which are endemic in different countries worldwide (AA., 2004.). Each strain
requires a specific vaccine to provide immunity to a vaccinated animal.
Figure 1.1: Diseased animal
1.2: How FMD is isolated?
FMD virus is isolated from the samples on cell culture.
1.3: FMD diagnosis:
 Diagnosis cannot be made reliably on the basis of clinical signs.
 Diagnostic techniques used include serologic tests.
 Virus isolation (VI) and neutralization (VN) test.
 Electron microscope (EM) inoculation studies.
 Polymerase chain reaction (PCR) tests have been developed and are frequently
utilized (Alexandersen S Z. Z., 2003).
1.4: Transmission and spread:
 Contaminated pens/buildings or contaminated animal transport vehicles.
 Contaminated materials such as hay, feed, water, milk.
 Contaminated clothing, footwear, or equipment.
 Virus-infected meat or other contaminated animal products (if fed to animals when
raw or improperly cooked) (Alexandersen S M. N., 2005).

2
 Infected aerosols (spread of virus air currents).
1.5: Public health risk:
FMD is not readily transmissible to humans and is not a public health risk.
1.6: Clinical signs:
 Fever
 Blisters in the mouth and on feet
 Drop in milk production
 Weight loss
 Loss of appetite
 Cows may develop blisters on teats
1.7: Sample Collection:
 Epithelium samples from un-ruptured vesicles/lesions.
 If a good lesion is present –carefully remove the EPITHELIUM with FORCEPS.
 A GOOD Sample should be 5 g.
 Sample is packed, labeled and marked
 Try to sample TWO OR THREE animals each into a SEPARATE BOTTLE. (Arzt
J, 2010. ).
1.8: Types of samples:
 Epithelium Tissue
 Vesicular fluid /Saliva
 Probang(Throat washings)
 Myocardial tissue (fatal cases) (Arzt J, 2010. ).
 Whole Blood.
Figure 2.2: Lab test

3
1.9: Treatment:
Treatment is not given. Affected animals will recover. However because of the loss
of production and the infectious state of the disease, infected animals are usually culled.
1.10: Prevention and control:
 Control over people’s access to livestock and equipment.
 Controlled introduction of new animals into existing herds.
 Regular cleaning and disinfection of livestock pens, buildings etc.
 Monitoring and reporting of illness (Arzt J, 2010. ).
 Appropriate disposal of manure and dead carcasses.
1.11: Vaccination:
Vaccination can be used to reduce the spread of FMD or protect specific
animals. Vaccination with one serotype does not protect the animal against other serotypes,
and may not protect the animal completely or at all from other strains of the same serotype
(Alexandersen S Z. Z., 2003)
Figure 3.3: Vaccine.

4
CHAPTER#2:
VACCINE PRODUCTION
Foot and Mouth Disease Research Institute Units:
1. Media section
2. Cell culture section
3. Virus culture section
4. Vaccine formulation and bottling section
2.1: Media section:
2.1.1: GMEM media preparation:
It is abbreviated as Glasgow’s Minimum Essential Medium (GMEM) and
is also called as growth media. It was previously called as HS solution. It’s a combination
of different salts (Pay TW, 1987. ).
It is used to provide nutrients to the cells such as BHK21 cells for its growth.BHK21 cells
are only observed under inverted microscope and not any other microscope (Alexandersen
S M. N., 2005).
2.1.2: Material Required:
 GMEM (stored in refrigerator) powdered form.
 Distilled water.
 Tryptose phosphate broth (TPB).
 Sodium bicarbonate.
 Adult bovine serum 10%.
 Antibiotics (Benzyl penicillin Sodium + Streptomycin sulfate).
2.1.3: Equipment:
 Magnetic stirrer with hot plate.
 Flat bottom flask.
 Aspirated bottle.
 Filtration assembly.
 Biosafety cabinet 2
2.1.4: Procedure:
 Took 10liter distilled/autoclaved water.

5
 Took 125gm GMEM powdered form it can make 10liter of media on standard
125g is poured in 10liter of water.
Figure 2.1: GMEM powdered form
 Took two flat bottom flask each filled with 5liter water.
 The flat bottom flask was sterilized and autoclaved before use. Its was a six liter
flask and 5liter water was filled into it.
 Put the flask on hot plate magnetic stirrer and stirred well.
 Poured powdered GMEM in the flask each with 5liter of water.
 Poured 22-30gm tryptose phosphate broth (TPB) in it. It was a cell culture
supplement for BHK-21 cells used for vaccine production (AA., 2004.).
 Added 10% Adult bovine serum. The serum is always kept in the frozen state.
When used it is thawed. It is more vulnerable to contamination so a lot of care is
taken in its use to prevent contamination (Francis MJ, 1983. ).
 Added two vials of each two antibiotics in the 10liter water:
 Benzyl penicillin Sodium
 Streptomycin sulfate
 (Both the antibiotics were in the powdered form).
 The antibiotics were used to stop contamination of the media and to protect it from
the bacteria and viruses. But the media can then also be contaminated if care is not
taken (Zhang ZD, 2001. ).
 With the help of Magnetic stirrer the media in the flat bottom flask was stirred well
and after half an hour the stirring was stopped. Kept the media on the stirrer until
whole the ingredients were dissolved well.
 When all the ingredients were dissolved well then the media was ready for the next
step which was filtration (TR., 2003. ).
2.1.5: Filtration:
Filtration is the last step in the GMEM growth media preparation. Different types
of filtration assemblies with 0.2 micron filter were used in the filtering for example
membrane filters, Seitz filters, cartridge filters, capsule filters etc. (Sumption K, 2012. ). A

6
6 litter flask of GMEM media is filled with 5 litter media and was filtered in the biosafety
cabinet into autoclaved sterilized flasks (Reed LJ, 1938. ).
The filtration process involves the the following steps:
 Took aspirator flask/autoclaved bottles of 10liter
 Placed the GMEM media in the biosafety cabinet.
 The filtration assembly was also fitted in the biosafety cabinet.
 One pipe from the filtration assembly was placed in the GMEM media flask
(Poulin MC, 2006. ).
 The other pipe was fitted in the aspirator bottle.
 The filtration process begins when the pipes were fitted and it was continued until
whole the media was filtered.
 The filtered media was collected in the flask. Then the GMEM media was ready
for use as growth media.
Figure 2.2: Filteration
2.1.6: Storage:
Then the filtered media was incubated (stored) in the incubator at 4ºC-37ºC for 2-
3 days for later use (Perry BD, 2007. ).
2.1.7: Contamination:
Checked the media properly for contamination under inverted microscope.

7
2.2: Cell Culture Section:
The section is used in culturing of BHK21 cells. The GMEM media prepared in the
media section is used for the growth and culturing of cell. Cell line passage is done in this
section.
2.2.1: Introduction to cell line passage:
A passage number is the number of times a cell culture has been sub-cultured.
BHK (Baby hamster kidney) cells line is one of the most commonly used cell lines.
The most widely used line is a sub clone (clone 13) of BHK21, usually called as BHK
21(C13) (Pay TW, 1987. ), which has been involved in the commercial manufacture of foot
and mouth disease vaccines. Take cell culture and start propagation/cell line passage with
3-4 small flasks using BHK21 as culture (Orsel K, 2005.).
Figure 2.3: BHK 21 cells
2.1.2: Steps:
 Took BHK21 cell culture from the incubator.
 Checked growth under inverted microscope, BHK21 cells were seen elongated and
monolayer.
 Took GMEM growth media from incubator.
 Turned on UV light in biosafety cabinet for at least 30 minutes before starting
passage in the cabinet (Mackay D, 2004. ).
 Turned off UV light and opened the biosafety cabinet
 Placed both cell culture and broth media into the biosafety cabinet
 Took 3 small flasks and label date on them.
 Removed the cell culture from the flask.
 Added small amount of trypsin to the cell culture flask to remove the adherent cells
from the surface.
 Kept for 2-3 minutes in the incubator (Francis MJ, 1983. ).

8
 Then remove the trypsin or you can also proceed without removing it.
 Added 200-300ml of growth media into it.
 Shaked it well.
 Poured it equally in 3-4 small sized flasks. It depends on the growth in the cell
culture if the cell growth is maximum then pour it in 3-4 or more flasks or in 2-3
flask when growth is minimum.
 If one flask is used then the growth will be observed after 24hours when 2 flasks
used then growth is seen after 48 hours and so on.
 Closed the lid and remove the 3-4 flasks from the biosafety cabinet and placed them
in the incubator.
 Placed each flask horizontally
 Then cleaned the cabinet with disinfectant e.g. Ethanol
 Closed the cabinet and turn on UV light.
Figure 2.4: cell line passage

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2.3: Virus Section:
In this section we perform the following tasks
1. DMEM media preparation.
2. Virus inoculation/infection.
3. Harvesting.
4. Inactivation.
2.3.1: DMEM media preparation:
It is a maintenance media. It is abbreviated as Dulbecco’s Modified Eagles Medium
(DMEM). It is prepared to give infection to the cells and to inoculate virus in the media.
The inoculated viruses are O, A, Asia1. These strains of viruses are found in Pakistan
(Grant CF, 2012.).
This media is previously called Earlse media.
Materials required:
 Dulbecco’s Modified Eagles Medium (DMEM)
 Distilled water 10liter
 Sodium bicarbonate
 Virus (O, A, Asia1) 2ml.
 Adult bovine serum 2%
 Antibiotics (Benzyl penicillin Sodium + Streptomycin sulfate).
Figure 2.5: DMEM powdered form.
Equipment:
 Magnetic stirrer with hot plate.
 Flat bottom flask.
 Aspirated bottle.
 Filtration assembly.

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 Biosafety cabinet 2
Procedure:
 Took 10 liter of autoclaved distilled water in the flask.
 Took two flat bottom flask each with 6 liter capacity and fill 5liter in each making
a total of 10liter.
 Took 125gm Dulbecco’s Modified Eagles Medium (DMEM) and it is in the
powdered form and it is a combination of different salts (Blink EJ, 2005. ).
 On standard for 10 litter of water the 125gm DMEM is used to prepare media.
 Kept water on the magnetic stirrer in the round bottom flask.
 Stirred the solution to dissolve the powdered DMEM completely in the water.
 Added 2% adult bovine serum to the solution. The serum is always kept in the
frozen state. When used it is thawed. It is more vulnerable to contamination so a lot
of care is taken in its use to prevent contamination.
 Added Sodium bicarbonate also known as baking soda up to 22 gm to the solution
 Added two vials of each two antibiotics in the 10liter water:
 Benzyl penicillin Sodium &Streptomycin sulfate (Both the antibiotics are in the
powdered form).
 Both injections were added in the powdered for to the media. As the media was in
the liquid form so the injections were added in the powdered form.
 The antibiotics are used to stop contamination of the media and to protect it from
the bacteria and viruses. But the media can then also be contaminated if care is not
taken.
 At the end virus was also added to the media which needed to be infected each
injection was inoculated individually. There were three strains of virus in Pakistan
O, A, Asia1. The three viruses were not used at the same time in the media
preparation instead used seperately.
Figure 2.6: Media formation
 “At one time only one type of virus is used in the DMEM media preparation”. For
example if we want to prepare media for O virus then we will add 2ml of O virus
in the DMEM media preparation.
 “For 100ml of DMEM media only 2ml of virus is added” (Inamine A, 2005.).

11
 Mixed all the ingredients well and stirred on the hot plate magnetic stirrer for up
to 30 minutes.
 When all the ingredients were dissolved then the media was ready for filtration.
Filtration:
Filtered it by passing through the 0.2 micron filters in the biosafety cabinet.
Different types of filtration assemblies are used for filtration. Media is filtered through the
suction pump e.g., through Millipore filter assembly. Aspiration bottle of 10 liter which is
packed and autoclaved is used to keep the filtered media (Shenoy GN, 2012.).
The filtration process involves the the following steps:
 Took aspirator flask/autoclaved bottles of 10liter
 Placed the DMEM media in the biosafety cabinet.
 The filtration assembly was also fitted in the biosafety cabinet.
 One pipe from the filtration assembly was placed in the DMEM media flask.
 The other pipe was fitted in the aspirator bottle.
 The filtration process begin when the pipes were fitted and it was continued until
whole the media was filtered.
 The filtered media was collected in the flask. Then the DMEM media (maintenance
media) was ready for the next step which was inoculation/infection of BHK 21
cells.
Figure 2.7: Filtration
2.3.2: Inoculation/Infection of BHK21 cell line with FMD virus:
Purpose:
Harvesting of virus on BHK21 cell line to produce FMD vaccine. This process can
be applied for the harvesting of different types of FMD virus O, A, Asia 1 (Mulcahy G,
1990. ). These viruses are used in FMD vaccine production.
Materials/Chemicals:
1. Distilled water

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2. DMEM
3. BHK21 cell line
4. Adult bovine serum
5. Virus O A Asia 1
6. Antibiotics
Equipment’s:
1. Filter apparatus
2. Safety cabinet
3. Incubator
4. Aspirator bottle
5. Media storage bottles.
Procedure:
 Liquefied each type of FMD virus (O, A, Asia1)
 Aspirated each serotype into 10ml sterile disposable syringe transferred to a sterile
caped glass test tube (Fazilleau N, 2007. ).
 Filtered through 0.2 micron filter.
 Prepared maintenance media
 Filtered it through the filter apparatus
 Added 5% bovine capsule to the maintenance media (DMEM).
 Took a cell culture flask with monolayer BHK21 cells.
 Replaced growth media (GMEM) in each flask with the maintenance media
(DMEM)
 Then added 2ml of virus per 100ml of DMEM for infection
 Each cultured flask was incubated at 37ºC for 24 hours
 All serotypes showed cell destruction (CPE) within 18-24 hours (Clarke ET, 2013.).
Figure 2.8: Cytopathic effect

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2.3.3: Harvesting of virus after CPE:
Purpose:
It means keeping all the cells together. After CP effect is observed the flask with
virus is poured into the vessels/buckets e.g., into a 10 liter vessel. To use propagated virus
as seed for vaccine production.
Equipment’s:
 Inverted microscope
 Deep freezer -20ºC
 Vessels/Polyfluralkoxy bottles
Figure 2.9: Passage
Procedure:
 Examined the CPE under inverted microscope
 There was a giant cell formation of infected monolayer
 On complete destruction of cell, contents of cell culture flask containing the same
serotype were pooled separately.
 Processed each type of virus serotype for biological titration to determine tissue
culture infective dose TCID 50.
 Poured the antigen in vessels/Polyfluralkoxy bottles.
 Store at -20ºC.
2.3.4: Inactivation of FMD virus:
Purpose:
To kill live FMD virus for vaccine production
Chemicals:
 2-Bromoethyl amine (BEA)

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 Sodium thiosulfate
 Sodium hydroxide
Equipment:
 Filter paper
 Fume hood
 Autoclave
 Dispenser
Inactivation procedure:
 BEI chemical was used for inactivation of virus.
 BEA was present in the powdered form it was converted to the liquid form. Taken
20.49g/liter in water.
 Added 7g sodium hydroxide (NaOH in distilled form).
 Performed inactivation process in a second vessel inactivation method
 Added BEI to the solution immediately after harvesting.
 Added at a ratio of 15ml BEI per liter antigen.
 Stirred it well.
 Kept it at -4C for the virus to act.
 Kept for 24 hours.
 BEI was toxic so after 24 hours added sodium thiosulfate to neutralize its effect.
On standard 100ml/liter sodium thiosulfate is added.
 Stored the antigen immediately at -20ºC until further use. At -20ºC it can be stored
for long period of time.
 If the antigen will be used within one week it can also be stored at 4ºC.
2.3.5: Composition of stock solution for BEI:
Material:
 1 liter of distilled water
 Sodium hydroxide NaOH 7g
 BEA powdered form 20.49g
 Sodium thiosulfate is added 100ml
Procedure:
Mix all the ingredients in a closed vessel or fume hood.
On standard sodium thiosulfate is added 100ml/liter. It is used to neutralize the effect of
BEI.
Stir at 37ºC for 30 minutes.
BEI should be prepared fresh before inactivation.

15
Sodium thiosulfate (Na2S2O3) preparation:
 On standard dissolve 22-48g of sodium thiosulfate in 1 liter of distilled water (Pay
TW, 1987. ).
 Then it is filtered with filter paper.
 Autoclaved it.
 Then it was ready.
 After the use of BEI it was poured to the solution after 24 hrs.
 After sodium thiosulfate sterility and safety was also given to the vaccine for our
own satisfaction.
 Sterility is given with nutrient agar/ broth etc. while safety is given in mice, pigs
etc.
Figure 2.10: Sodium hiosulfate
2.3.6: Vaccine formulation and bottling section:
 Added 50% of inactivated virus and 50% of mordant oil in homogenizer machine.
 Mixed it well in the machine. Freeze it at -20⁰C for 1 day.
 Then sent it to filling and bottling section for packing.
Figure 2.11: Bottling

16
2.3.7: Seeding of virus:
After the appearance of cytopathic effect the slides were observed carefully under
inverted microscope. Then 3-4 or more slides which show good CPE were stored in the
freezer (Deelder AM, 1974).
Marked date of harvesting on the flask. Also marked the type of virus inoculated in it e.g.,
O virus. These can be stored for long period of time.
2.3.8: Failure of FMD vaccine:
There are only three strains of viruses used in this vaccine which are O, A, Asia 1.
These strains have further many subtypes for e.g., O virus has further subtypes PAN Asia
2 and Mannisa etc. so if the type of strain used in the vaccine differs from those present in
the animal then the vaccine failure occurs.
2.3.9: FMD vaccine:
After the above procedure the vaccine is prepared and is ready to use. It is a trivalent
inactivated oil based FMD vaccine. It has three strains of virus O, A, Asia1. The strains are
grown on BHK 21 cells.
Figure 2.12: FMD Vaccine

17
CHAPTER#3:
R & D SECTION AT FOOT AND MOUTH DISEASE RESEARCH
CENTRE (FMD & RC).
3.1: ROLE:
 The adaptation and isolate of FMD virus on BHK 21 cells.
 Sterility test of FMD vaccine.
3.2: Elisa Test:
Scope:
Elisa test can be used to determine the different type of fmd virus in the field.
Equipment:
Micro plate with positive and negative control, Elisa dilution buffer, PBS, Substrate
chromogen solution, stop solution (H2SO4).
Figure 3.1: ELISA
Procedure:
 Diluted samples in 1.5ml tube by adding diluent buffer.
 Added 50μl each sample in 12 columns of the row: two replicates for each type
(Bout D, 1975).
 Added 50μl well of diluent buffer in all wells.
 Incubated plate for 1 hr. at room temperature,

18
 Washing with 1x washing solution. Filled the well with 200micl washing solution
and incubate for 3 minutes.
 Added 50micl conjugate each well and incubated at room temperature for 1 hr.
(Bartlett A, Sep 1975 ).
 Then four times washed and incubated for 5 minutes.
 Added 50μl stop solution in each well.
 Read the OD wavelength.
Figure 4
Figure 3.2: Obtaining results

19
CHAPTER#4.
Media & Anaerobe section:

20
4.1: Introduction:
 Different types of media required for the growth/maintenance such as nutrient agar,
nutrient broth is prepared in this section.
 The media prepared in this section is supplied to all the sections of VRI on demand.
 The sterilization of the vaccines, glassware and all other equipment’s is also done
in this section.
Figure 4.1: Media section
4.2: Classification of Bacterial Culture Media:
4.2.1: On the basis of consistency:
 Solid media
 Semi solid media
 Liquid broth media
4.2.2: On the basis of composition:
 The synthetic or chemically defined media (exact composition is known)
 The non-synthetic or chemically undefined media
4.2.3: Common media in routine use:
The following are the types of media which are prepared in the media and anaerobe
section.
 Blood agar
 Nutrient broth
 Nutrient agar

21
 Tryptic soy broth
 MacConkey agar
 Sabouraud agar
 Thioglycollate media
 Blood Agar:
 Blood agar is an enriched media.
 It is used for determining the hemolytic capabilities of an organism. Some
bacteria produce exo-enzymes that lyse red blood cells and degrade
hemoglobin; these are called hemolysins.
 Because of this reason blood agar is used for the growth of fastidious
bacteria such as Streptococci (Bhattacharya S, 2002). (annexe#1)
Figure 4.2: Blood agar
 Nutrient agar:
This medium was used for the cultivation and maintenance of non-
fastidious species for microbe’s culture, supporting the growth of wide range of non-
fastidious organisms. (Annexe#2)
Figure 4.3: Nutrient agar

22
It is widely used media because; it can grow wide variety of bacteria, fungi because it
contains many types of nutrients for bacterial growth.
 MacConkey agar:
It is used in the differentiation of lactose fermenting from lactose non-
fermenting gram-negative bacteria. It is used for the isolation of coliforms and intestinal
pathogens in water, dairy products and biological specimens.
It is a selective and differential media. This media is used for the differentiation and
isolation of non- fastidious gram-negative bacteria. (Annexe#3).
Figure 4.4: MacConkey agar
 Sabouraud Dextrose agar:
The medium is often used with antibiotics for the isolation of pathogenic
fungi from material containing large numbers of other fungi or bacteria.
This medium is primarily used for the cultivation, isolation, and identification of yeast
and molds. (Annexe#4).
Figure 4.5: Sabouraud agar

23
 Thioglycollate media:
It is a multipurpose, enriched, differential medium used primarily to
determine the oxygen requirements of microorganisms (Fernández Andreu CM, 1998).
Sodium thioglycollate in the medium consumes oxygen and permits the growth of
obligate anaerobes (Krulwich TA, 2011). It is the enriched medium mostly used in
diagnostic bacteriology.
Figure 4.6: Thioglycollate agar

24
Chapter#5.
NEWCASTLE DISEASE/POULTRY VACCINE SECTION
5.1: Introduction:
In this section vaccine for disease in poultry is produced the production of LaSota
and Mukteswar vaccine was observed. The disease for which vaccine is produced is
Newcastle Disease.
NewCastle disease is an infection of domestic poultry and other bird species with virulent
NewCastle disease virus (NDV) (Clarke ET, 2013.). Severity depends on the virulence of
the infecting virus and host susceptibility.
Figure 5.1: Diseased chick
5.2: NewCastle disease vaccine production:
5.2.1: Virus inoculation:
Almost 10 folds dilution of working seed of NewCastle disease virus
(laSota strain) was prepared in sterilized Phosphate-buffered saline (PBS) containing
antibiotics (D. J. Alexander, 2001.). Mix it well.
Figure 5.2: Virus innoculation.

25
Candling:
 Eggs were examined at 9th
day of incubation.
 Embryonated eggs were collected to proceed further.
 Holes were made in embryonated eggs with the help of sterilized nail. (Candling)
 Alive embryo was used for further process otherwise it can’t be used further.
Figure 5.3: Candling
Surface Sterilization:
 After candling pyodine was used to disinfect the surface of embryonated eggs in
sterilize environment.
Drilling:
 A hole was made on allatonic cavity with the help of sterile thumb nails.
Figure 5.4: Drilling

26
Inoculation of virus:
 Virus dilutions were inoculated in embryonated eggs through the hole 0.1ml/egg
by using 1cc disposable syringe (K. Murulitharan, 2013.).
 At the time of inoculation anti-biotic (penicillin) and anti-fungal were added
(Amphoteric) to avoid any type of contamination.
Sealing of eggs:
 Melted Paraffin wax was used to seal the eggs which prevent leakage from eggs.
Incubation:
 Eggs were placed in incubator at 37⁰C (99-102F) for 96-120hrs almost 4 days (L.
M. Kim D. J., 2008.).
 After 24 hours eggs were observed to check that eggs were alive or dead if dead
discarded them if alive then placed them back in incubator.
 Eggs were then kept again in the incubator and taken out of the incubator after 96
hours.
Figure 5.5: Incubation
5.2.2: Harvesting of ND (LaSota strain virus):
Chilling:
 After 96hrs the eggs were removed from the incubator in order to avoid bleeding
from blood vessels embryonated eggs were placed in freezer at 4⁰C for 2-3 hrs. (L.
M. Kim D. L., 2008).
 The purpose of chilling is to shrink the blood vessels.

27
Figure 5.6: Chilling
Harvesting:
 After chilling harvesting of eggs for vaccine production was performed.
 Harvest the egg by cutting egg shell of air sac in sterile environment.
 To cut the egg shell sterile scissor was used.
 Air sac removed with the help of sterile forceps.
 Harvested virus was pooled in sterile glass flask.
 Stored in freezer at -20⁰C.
Figure 5.7: Harvesting
Pooling:
 Harvested fluid was transferred in round bottom flask.
 As a stabilizer skimmed milk was added with anti-biotic.
 To make homogenized solution vaccine mixed properly. Vaccine was dispensed in
sterilized vials with the help of filling syringes.
 After that capping was done and sent to freeze drying section for lyophilization.

28
Vaccine formed:
 Safety & sterility tests performed
 Bottled with batch no and date
 Stored in freezer at -20C.
Figure 5.8: Newcastle Disease vaccine
5.3: Mukteswar vaccine production:
 Same procedure is for Mukteswar Vaccine but still there was a main difference
and the difference was that infection was given at 10th
day of incubation while in
LaSota vaccine infection was given at 9th
day (OIE, 2012.).
Figure 5.9: Mukteswar vaccine

29
Chapter#6.
HEMORRHAGIC SEPTICEMIA SECTION
6.1: Introduction:
Hemorrhagic septicemia is one of the most important diseases of cattle and
buffalo in Pakistan and causes heavy losses in Livestock. It is considered number one
killer of buffalo. HS is caused by two serotypes of G –ve, non-motile, coccobacillus
bacteria named as Pasteurella multocida (Dowling, 2002).
6.2: Signs &Symptoms:
Majority of cases are acute in nature with death occurring from 6-24 hours in
cattle and buffalo after the appearance of signs. Signs include:
 Dullness
 Reluctant to move
 High body temperature
 Serous nasal discharge
 Salivation
 Difficult respiration and animal dies within few hours
6.3: Vaccine preparation:
Vaccination is done to prevent the occurrence of disease. Vaccines produced at
veterinary research institute the process is given as:
Figure 6.1: HS vaccine
6.3.1: Collection of samples:
Samples include blood, lung, liver, heart collected from cattle’s of different age and
sex suffering from HS disease.

30
6.3.2: Examination of sample:
For master seed preparation the collected samples were cultures according to the
standard methods. The sample was inoculated into blood agar, MacConkey agar slant, etc.
for better growth. The media was then incubated at 37⁰C in the incubator to check the
growth (Hodgson, 1993).
6.3.3: Preparation of master seed:
 Two different isolates of P. multocida were obtained from the heart blood of calf
with in 2 to 3 hours of it’s death.
 The P. multocida were inoculated separately in blood agar, nutrient broth/agar.
 Then heart blood stored at -20◦C in 1 ml aliquote in an eppendroof .
 This master seed used for each batch of vaccine.
6.3.4: Maintenance of master seed:
 Blood agar slants were used to maintain the stock culture.
 The P. multocida were inoculated in slant by streaking and incubated at 37⁰C for
24hrs.
 A sterile mineral oil was obtained.
 Equal volume of it with 80% glycerin is taken in Eppendorf tube mixed and stored
at -80⁰C (Horadagoda, 1999).
 Safety was check in albino mice.
6.3.5: Activation of master seed:
 The P. multocida isolates were inoculated into mice through subcutaneous rout.
 The mice were observed at 2hr interval for up to 24 hours.
 The died or sick mice were necropsied.
 The heart blood was smeared stained by gram staining and observed under
microscope.
Figure 6.2: Master seed activation

31
Gram Staining:
1) Made the slides Grease or oil free.
2) Labelled the smear it may be helpful to clearly designate the smear.
3) Heat fixed the smear
4) Placed slide on staining tray. (annexe#5)
5) Gently flooded smear with crystal violet for 1 minute.
6) Tilted the slide slightly and gently rinsed with tap water. (Dowling, 2002).
7) Gently flooded the smear with Gram iodine for 1 minute.
8) Tilted the slide slightly and gently rinsed with tap water or distilled water.
9) The smear will appear as a purple circle on the slide.
10) Decolorized using 95% ethyl alcohol or acetone for 5 to 10 seconds until the
alcohol runs almost clear. Be careful not to over-decolorize.
11) Immediately rinsed with water.
12) Gently flooded with safranin to counter-stain for 45 seconds.
13) Tilted the slide slightly and gently rinse with tap water or distilled water.
14) Blot dried the slide with paper.
15) Viewed the smear using a light-microscope under oil-immersion.
16) Gram-negative bacteria stained pink/red and
17) Gram-positive bacteria stained blue/purple.
Figure 6.3: Gram staining
6.3.6: Working Seed Preparation:
 The heart blood of dead mice was collected and cultured in round bottom flask.
 BHI was added to the flask.
 Incubated for 15-18hrs.
 Heart blood was then cultured on nutrient agar.

32
6.3.7: Biochemical test:
 Culture media was then checked microscopically after incubation.
 Different types of Biochemical test were performed for all the isolates of P.
multocida for purity.
 This working seed was then stored for 5 days at 4⁰C.
6.3.8: Inactivation and Pooling:
 Add formalin with the conc. of 0.5% to terminate the growth.
 Sterilized tanks were used for pooling of formalized media.
6.3.7: Preparation of vaccine:
 Two types of vaccines were prepared from this mass culture.
a) Alum precipitated hemorrhagic septicemia vaccine (APHSV):
 The inactivated mass culture was diluted in saline solution.
 Aluminum potassium sulphate was admixed with the diluted culture to achieve its
0.5 percent concentration.
 The pH was adjusted to 7 .2.
 The aluminized culture was incubated at 25°C for 24 hours.
Figure 6.4: ALUM precipitated vaccine
a) Oil-based hemorrhagic septicemia vaccine (OBHSV):
 One part of the formalin inactivated dense culture was admixed with 4 part of the
oil-base at 8000-15000rpm.
 The oil montanide ISA 50base was composed thiomersal Na and Bacterin.
 The mixture was blended for 3 minutes.
 Safety of the vaccines was evaluated in rabbits.

33
 Two ml of each of the vaccines was injected to each of the five rabbits and the
reaction was recorded for 7 days (Reed LJ, 1938. ).
 No mortality indicated the safety of the vaccines.
6.3.8: Filling of vaccine:
 50ml of vaccine was filled in each bottle.
6.3.9: Labeling and packaging of vaccine:
 Labeling with batch no, production and expiry date is done.
 All the vaccine batches are packed in shrink machine with shrink shopper.
Figure 6.5: Packaging
6.3.10: Storage and supply system:
 Vaccine batches are stored in special cold rooms with 4C temperature.
 It is supplied according to demand.
 Cold chain supply system is used for its supply

34
Chapter#7.
CELL CULTURE SECTION.
7.1: PPR vaccine introduction:
Peste des petits ruminants (PPR) is a highly contagious and economically important
viral disease affecting goats, sheep and wild ruminants. The disease is endemic in Pakistan.
Mass vaccination is the only useful mean to control the disease.
Vero cell lines derived from, the kidney of an African green monkey in the 1960s (Bout D,
1975). Vero cells are one of the most common mammalian continuous cell lines used in
research it produced a monolayer of cell. In cell culture section PPR vaccine was prepared.
PPR vaccine is a vaccine that is used against viral infection.
Figure 7.1: Vero cell line
7.2: Preparation of PPR Vaccine:
Cell lines are produced in cell culture flasks. Cells from these flasks were
propagated, given infection and vaccine was prepared.
Figure 7.2: PPR vaccine

35
7.2.1: Washing of glassware:
 Washing was done with warm tape water.
 Soaked the glassware overnight in 1-2% detergent solution.
 Cleaned and brush the outer and inner surface.
 Washed thoroughly with tape water.
7.2.2: Sterilization of glassware and labware:
 Pippete were packed in copper container and sterilize in hot air oven at 180C for 2
hrs.
 Petri dishes wrapped in brown paper, tied and sterilize in hot air oven at 180C for
2 hrs. Cover measuring bottles and cylinders with aluminum foil (Clarke ET,
2013.).
 Plastic bottles, eppendrophs, screw cap bottles, rubber cork and filter apparatus
were sterilized.
Figure 7.3: Sterilization
7.2.3: Filtration:
 All the cell culture media was filtered with the filtration assembly.
 Point zero micron filters are used.
 Different types of filtration assemblies are used.
7.2.4: Propagation of cells:
 Disinfected the biological safety cabinet 2 with 70% ethanol
 GMEM and DMEM media enriched by 10% TPB was prepared.
 Amphotericin-B Gentamycin was added in the media.
 Cells washing was done with PBS (Phosphate Buffer Saline)
 Trypsin solution was added so that the cells detach from the surface and incubate
at 37º C For 5-10 min.

36
 Media was removed from cell culture flask (parent flask) to another empty flask
(roux flask).
 Strain of PPR virus infection was given to roux flask.
 Culture flask was labelled with date of passage, cell line and passage number and
incubated at 37C
 Cells required 48-72 hours for complete growth.
 After ¾ days monolayer was observed showed the growth of virus. (annexe#6)
7.2.5: Observation of Cell Culture and CPEs:
 Observed the cells daily.
 At 3-5 day Virus started replicating inside cells and start producing CPEs.
 Cells started detaching and become shiny, irregular and clump together.
Figure 7.4: CPE
7.2.6: Harvesting:
 First harvesting was done when CPE reaches 40-50% of cell layer (Deelder AM,
1974)
 Final harvesting was done when CPE reaches 70-80% of cell layer
 It took about 10 days, during this time cells were observed under inverted
microscope.
7.2.7: Pooling:
 Media from all flasks were collected in bottle that had a capacity of 1-2 and
labeled and then stored at -70 C in blast freezer.
 Stabilizer was added in viral suspension in equal amount and stored at -70
 The stabilizer stops the activity of virus and cell culture.

37
7.2.8: Filling:
 In cold conditions homogenize the wet vaccine.
 All the bottles were then poured in to round bottom flask with the addition of
antibiotic like streptomycin and benzyl penicillium sodium.
 Skimmed milk 10% was added as stabilizer. Flask was put on magnetic stirrer and
filled in the vials by using a syringe. Each vial contains 2ml.
7.2.9: Lyophilization:
 After filling vials were then sent to freeze drying section for lyophilization (Bout
D, 1975).
 The freeze dried vials were then sent to quality control department for sterility
and safety testing.
Figure 7.5: Lyophilization
7.2.10: Supplying:
 Prepared vaccine was then supplied to the supply chain according to demand.

38
REFLECTIVE JOURNAL ENTRIES
FMD & RC SECTION
Serial no. Date Day Work Impressi
on
1.
1/4/2019 Monday University day -
2.
2/4/2019 Tuesday University day -
3.
3/4/2019 Wednesday Visit of all sections of
FMD & RC
Excellent
4.
4/4/2019 Thursday General introductory
lecture about FMD
vaccine
Excellent
5.
5/4/2019 Friday Assignment on FMD
disease
Good
6.
6/4/2019 Saturday Holiday -
7.
7/4/2019 Sunday Holiday -
8.
9.
10.
10/4/2019 Wednesday Cell line passage observed Excellent
11.
11/4/2019 Thursday Assignment on cell culture
and its types
Good
12.
12/4/2019 Friday Cell line passage
performed
Excellent
13.
14.
15.
16.
17.
17/4/2019 Wednesday Microscopy practical
performance.
Excellent
18.
18/4/2019 Thursday Visit to SLISA & 146S
LAB and its introduction
Excellent
19.
19/4/2019 Friday Media section visit and
observed media
preparation, filtration
Excellent
20.
21.

39
22.
23.
24.
24/4/2019 Wednesday ELISA performance
observed
Excellent
25.
25/4/2019 Thursday 146S test observed Excellent
26.
26/4/2019 Friday Cell culture section visit,
assignments
Good
27.
28.
29.
30.
31.
1/5/2019 Wednesday Virus section, DMEM
media preparation.
Good
32.
2/5/2019 Thursday Virus inactivation,
harvesting
Good
33.
3/5/2019 Friday Master seed preparation,
sterility and safety.
Good
34.
35.
36.
37.
38.
8/5/2019 Wednesday safety test performed on
mice and guini pigs
Good
39.
9/5/2019 Thursday Assignment -
40.
10/5/2019 Friday harvesting of virus Excellent
41.
42.
43.
44.
45.
15/5/2019 Wednesday Formulation and bottling
section visit, vaccines
labelled bottled and stored
Excellent

40
MEDIA AND ANAEROBE SECTION
Serial no. Date Day Work Impression
1.
16/5/2019 Thursday Visit of the section Good
2.
17/5/2019 Friday Types of media Good
3.
4.
5.
20/5/2019 Monday Plugs making for roux
flask
Excellent
6.
21/5/2019 Tuesday Observation of media
formation
Good
7.
22/5/2019 Wednesday University Exams -
8.
23/5/2019 Thursday University Exams -
9.
24/5/2019 Friday University Exams -
10.
11.
12.
27/5/2019 Monday University Exams -
13.
28/5/2019 Tuesday University Exams -
14.
29/5/2019 Wednesday Media filtration Good
15.
30/5/2019 Thursday Blood agar media
preparation
Excellent
16.
31/5/2019 Friday University Exams -
POULTRY VACCINE SECTION:
1.
2.
3.
3/6/2019 Monday Visit of the section Good
4.
4/6/2019 Tuesday Eid-ul-Fitar -
5.
5/6/2019 Wednesday Eid-ul-Fitar -
6.
6/6/2019 Thursday Eid-ul-Fitar -
7.
7/6/2019 Friday Eid-ul-Fitar -

41
8.
9.
10.
10/6/2019 Monday Detailed Lecture Excellent
HEMORRHAGIC SEPTICEMIA (HS) SECTION
1.
11/6/2019 Tuesday Visit to the section Good
2.
12/6/2019 Wednesday Gram staining Good
3.
13/6/2019 Thursday Observed Mice
dissection
Excellent
4.
14/6/2019 Friday Gram staining practical
performance
Good
5.
6.
7.
17/6/2019 Monday Haemagglutination test
observed
Excellent
8.
18/6/2019 Tuesday Culture, labelling,
injection of vaccine
Excellent
9.
19/6/2019 Wednesday Presentation on
Haemagglutination test
Good
10.
20/6/2019 Thursday Packaging of vaccine Good
CELL CULTURE AND FLURY SECTION
1.
21/6/2019 Friday Visit of section & lecture
about the section
Excellent
2.
3.
4.
24/6/2019 Monday Cell line passage
practically done
Good
5.
25/6/2019 Tuesday PPR vaccine formation,
labelling
Good
6.
26/6/2019 Wednesday Assignment -
7.
27/6/2019 Thursday Report writing -

42
SAMPLES HANDLING
SAMPLE#1: GMEM Media Preparation:
Fig:1 Pour all the ingredients in the round
bottom flask and put on the stirrer.
Fig: 2 filter the media through micron
filters
SAMPLE#2: Cell Line Passage:
Fig:1 Take BHK 21
cells culture from
incubator
Fig:2 examine BHK 21 cell
culture under inverted
microscope
Fig: 3 take GMEM media from the
incubator for passage. Take other
3 small flask and mention date on
them

43
Fig: 4 remove cell culture from the flask
and add trypsin to remove adherent cells.
Fig: 5 remove trypsin. Add growth media
into it. Shake well then pour equally into ¾
small flask and store in incubator
SAMPLE#3: Infection of BHK21 cell line with FMD virus:
Fig: 1 BHK 21 cells before infection
with virus are elongated shape
Fig: 2 BHK 21 cells after infection with
O, A, Asia 1 virus it become rounded
shape

44
CRITICAL ANALYSIS
Veterinary research institute seemed a very good institute near me. It enhanced my
understandings academic knowledge and skills. My internship experience at this institute
is great. It polished my knowledge and experience. This institute not only provided the
opportunity and experience but also help to learn the applications of theories in actual
application.
It improved my general knowledge about the equipment used during my training program.
Not only staff members doctors but also all the other workers helped to understand
important procedures. They behaved well with us in a good Manner.
I have learned a lot of techniques at FMD & RC. The staff members contributed a lot and
helped me in learning thing. The internship & research supervisor at FMD & RC was a
good person. He provide us with hands on practice.
The media formation learned were a good thing. It enabled us to deal with media.
At VRI the staff members also contributed a lot. Many skills were learned at VRI. At the
PV section the learning techniques were so interesting. It was a work of joy at this section
because it deals with embryonated eggs.
At the hemorrhagic septicemia (HS) section. Necroscopy of dead mice was observed and
gram staining was also performed. In addition to techniques the box for vaccine were also
prepared. It was indeed a good experience for making boxes.
The thing I feel bad is that we were not able to perform mice necroscopy at HS section due
to shortage of time.
At all the section techniques were learned.
A big problem that VRI is facing is related to the funding. A very small amount of funds
are given to this research institute due to which it still lacks behind from some of the latest
technology.
Government should show special interest in this institute as many important procedures
and vaccines are produced there. Latest machinery should be provided to reduce the work
load and increase the qualities of products produces there.

45
SWOT ANALYSIS
Strengths:
 The strength of Veterinary Research Institute (VRI) is that it has trained and
qualified staff.
 All the staff members perform their work with great loyalty.
 There are experienced doctors and Veterinary researchers who work with passion
in their field.
 All the staff members and workers have cooperation between them.
 This institute provides transport facility to its workers.
 A hostel is made for the internees and houses built for the doctors.
 All the necessary resources are provided to the workers.
Weaknesses:
 The main weakness of the institute is the lack of advanced facilities.
 There is lack of apparatus.
 There is no cafeteria within the institute there should be a cafeteria for the visitors
and internees.
 On the gate of VRI there is a large number of waste dumped and also there an open
sewage line which may create the environment dirty so the chances of
contamination of vaccine can occur it should be checked by the government.
Opportunities:
 VRI offered opportunities to learn for the internees and for the researchers.
 Students from far off places come here for education purpose.
 It produce a good variety of vaccines for the animals and serves the farmers by
providing a variety of vaccine in small cost.
Threats:
 Threats are usually those shortcomings or identifiable measures or activities that
can cause long term harms to the statusxistence or well being of any Institute or the
organization.
 VRI though is an institute that harbors very important research activities but yet it
needs to have lot more to attain the status of a state of an art institute. Lack of
coordination and improper distribution of resources can impose a serious threat to
its existence and long term functioning. If not properly managed and resources or
facilities are not administered efficiently, the centre can face serious challenges of
remaining functional and getting evolved into a centre of excellence in veterinary
sciences.

46
CONCLUSION
My Internship at VRI has been an excellent and great learning experience. It is concluded
that vaccine production and different techniques has been learnt at both VRI and FMD &
RC.
During this internship program, I studied a lot of animals diseases and there consequences
in economy of Pakistan. Observed and practiced full procedure of vaccine production i.e.
LaSota vaccine, HS Vaccine, and PPR Vaccine.
Whatsoever it is believed that the time spent there was well worth. All the labs are kept
clean and safety rules are followed strictly.
Veterinary officers and trained staff helped to understand important procedures of vaccine
production. VRI enhanced academic skills. It is the only research institute producing
vaccine at government level. Millions of doses produce monthly. It is a non-profit institute
saving our country from great economic loss.
A lot of the tasks and activities performed in the lab during internship were well familiar
with the subject of zoology.
Needless to say that all the staff of this organization is well trained and highly qualified
putting all their effort towards the success of organization.
My internship was a great experience at both VRI and FMD & RC.

47
RECOMMENDATION
 Appoint more staff workers in each section to cope with the busy routine of staff
members.
 Doctors should come on time.
 Lab should follow Biosafety levels according to their work.
 Collaboration with internees and training staff or coordinators.
 Should use technologies to save time.
 Safety measures should be following by everyone.
 Gloves, lab coats, face mask, shoe covers, head caps, must be wear before doing work.
 Only personnel should allow in the work place.
 The doctors should have command on their work. Labs should be cleaned and sterilized
and must fumigated once a week.
 Vaccine tank should be cover before filling to avoid contamination.
 Veterinary schools or institutions must offer postgraduate training to the student and
after few years they become veterinarians and work for their nation.
 Labs are well furnished but some latest technologies should be introduced in the labs.

48
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51
ANNEXURES
Annexe#1:
Blood Agar Composition:
Peptone 0.5%
Beef extract/yeast
extract
0.3%
Agar 1.5%
NaCl 0.5%
Distilled water 100 ml
Sheep Blood 5%
pH 7.2 to 7.6 (7.4)
Annexe#2:
Nutrient Agar Composition:
Distilled Water 1000 ml
Beef Extract 3.0 g
Peptone 5.0 g
Agar 15.0 g
Final pH 6.8 +/- 0.2.
 Composition of Nutrient Broth: It contains same ingredients except agar.
Annexe#3:
MacConkey Agar composition:
Peptone 17 g
Lactose 10g
Bile salts 1.5g
NaCl 5g
Neutral red 5g
Crystal violet 0.001g
Agar 13.5 g
pH 7.1
Proteose 3 g
Distilled Water 1000ml

52
Annexe#4:
Sabouraud dextrose agar media composition:
Mycological peptone 10 g
Distilled Water 1000ml
Dextrose 40 g
Agar 15 g
pH 5.6 at 250
C
Annexe#5:
Gram staining:
Annexe#6:
Propagation of cells:

Internship Report on Vaccine Production at FMD and VRI

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