EGG INOCULATIONPrinciples Practice and Vaccine Development : EGG INOCULATIONPrinciples Practice and Vaccine Development Dr.T.V.Rao MD Dr.T.V.Rao MD 1 Towards Developing Better Skills in Microbiology : Egg inoculation continues to be a Important Student Exercise in Several Post Graduate Examinations in Medical Microbiology for evaluation. The Students should develop the Necessary skills to be familiar with the exercise in Virology Towards Developing Better Skills in Microbiology Dr.T.V.Rao MD 2 Viruses are Different From Other Microbes : Viruses are obligate intracellular parasites. They depend totally on their host cells for their existence. Their total host dependence makes it extremely difficult to get good insight of them natural conditions, because the internal characteristics of the host cells are likely to interfere with the observations. Due to these reasons, it has been found desirable that viruses are cultivated or grown in the laboratory itself. Viruses are Different From Other Microbes Dr.T.V.Rao MD 3 Difficulties in Diagnosis of Viral Infections : Difficulties in Diagnosis of Viral Infections Can not be seen under light microscope
Can not be cultivated easily
Do not grow on culture media
Treatment was not available
Rapid techniques have emerged
Screening for Blood transfusion
Treatment available Dr.T.V.Rao MD 4 Techniques used : Techniques used Microscopy
Detection of Viral Antigen
Growing and detecting viruses in
Tissue / Organ / Cell culture
Fertilized hen’s egg
Laboratory animal inoculation eg mice
Detection of antibody in serum
IgG – Rising titer in paired sample
IgM – Indicates current / recent infection Dr.T.V.Rao MD 5 Microscopy : Microscopy Electron Microscope / Immune Electron Microscopy
Light microscope – Inclusion bodies eg Negri Body in Rabies
Fluorescent Microscope -Fluorescent antibody technique Dr.T.V.Rao MD 6 Demonstration of Viral Antigens : Demonstration of Viral Antigens Precipitation on gel eg HBsAg
Counter Immuno Electro Phoresis (CIEP)
Enzyme Linkes Immuno Sorbant Assay (ELISA) Dr.T.V.Rao MD 7 Isolation of Virus : Isolation of Virus Laboratory animals
Fertilized Hen’s Egg
Growth identified by serological method like neutralization. Dr.T.V.Rao MD 8 Slide 9: Embryonated Egg Cell Lines/ Tissue cultures Animal inoculation Chorioallantioc membrane (CAM) Allantoic cavity
Amniotic cavity Yolk Sac Primary Diploid/ Secondary Continuous Suckling mice Virus Culture Dr.T.V.Rao MD 9 Embryonated Hen’s Egg Cultivation of Viruses and Bacteria : Chorioallantoic membrane (CAM) – visible lesions called pocks. Each infectious virus particle forms one pock. e.g. Variola, Vaccinia virus
Allantoic cavity – Influenza virus (vaccine production) & paramyxoviruses
Amniotic cavity – primary isolation of Influenza virus
Yolk sac – Chlamydia, Rickettsia & some viruses Embryonated Hen’s Egg Cultivation of Viruses and Bacteria Dr.T.V.Rao MD 10 Embryonated eggs: : The Embryonated hen’s egg was first used for cultivation of viruses by Good Pasteur and Burnet (1931). Cultivation of viruses in organized tissues like chick embryo necessitates a different type of approach.. For all practical purposes they all themselves behave as tissue cultures. The process of cultivation of viruses in embryonated eggs depends on the type of egg which is used. The egg used for cultivation must be sterile and the shell should be intact and healthy. Embryonated eggs: Dr.T.V.Rao MD 11 Burnet as Director of the Hall Institute, 1944-1965 : Burnet as Director of the Hall Institute, 1944-1965 F.M. Burnet in the laboratory in the early 1950's, was experimenting on influenza virus genetics, using the developing hen's egg. Dr.T.V.Rao MD 12 Burnet Wins Nobel Prize : Burnet Wins Nobel Prize Burnet was confirmed by the award of the 1960 Nobel Prize to him and Peter Medawar for the discovery of immunological tolerance, a discovery in immunology of minor importance compared with the clonal selection theory. Dr.T.V.Rao MD 13 Only Embryonated Eggs Are Suitable for Growing Virus : Only Embryonated Eggs Are Suitable for Growing Virus Inoculated eggs are candled daily to see the chicken embryos inside. Dr.T.V.Rao MD 14 Eggs are Used for Mass Vaccine Production in Influenza : Eggs are Used for Mass Vaccine Production in Influenza Animals and chick embryo were the first method that was used to cultivate virus. This method is rarely used as it is not convenient. However, when preparing for bulk virus, (e.g. antigen or vaccine production) the usage of chick embryo is useful. Dr.T.V.Rao MD 15 Advantages of Fertile Eggs : Advantages of Fertile Eggs Fertile chicken eggs provide a convenient, space-saving incubator for many kinds of animal viruses. Different viruses can be injected into an egg at different sites and the egg can be easily observed for viral replication throughout the development of the chicken embryo. Dr.T.V.Rao MD 16 Advantages of Using Embryonated Eggs : Advantages of Using Embryonated Eggs Isolation and cultivation of many avian and few mammalian viruses
Ideal receptacle for virus to grow
Sterile & wide range of tissues and fluids
Cost- much less
Readily available Dr.T.V.Rao MD 17 Advantages of Fertilized Eggs are : Advantages of Fertilized Eggs are Free from bacteria and many latent viruses.
Free from specific and non specific factors of defense. Dr.T.V.Rao MD 18 Structure and Utility of Fertilized Egg : Structure and Utility of Fertilized Egg Dr.T.V.Rao MD 19 Routes of Injecting the Fertilized Eggs : Routes of Injecting the Fertilized Eggs Dr.T.V.Rao MD 20 Slide 21: Dr.T.V.Rao MD 21 Cultivation of Virus in Eggs : Cultivation of Virus in Eggs To cultivate viruses in eggs, the procedure adopted should be very simple. The eggs are kept in incubator and embryos of 7-12 days old are used. The egg containing embryo usually has an air apace at the larger end. The position of this sac is first determined. Dr.T.V.Rao MD 22 Begin you Exercise with Candling Eggs : Begin you Exercise with Candling Eggs Candling is the process of holding a strong light above or below the egg to observe the embryo. A candling lamp consists of a strong electric bulb covered by a plastic or aluminum container that has a handle and an aperture. The egg is placed against this aperture and illuminated by the light. If you do not have a candling lamp, improvise. Try using a torch. Dr.T.V.Rao MD 23 Marking the inoculation site: : Marking the inoculation site: 1. Hold the blunt end of the egg against the aperture of the candling lamp and note the position of the head of the embryo.
2. Turn the egg a quarter turn away from the head.
3. Draw a line on the shell marking the edge of the air sac.
4. Draw an X approximately 2 mm above this line.
5. The X marks the inoculation site. Dr.T.V.Rao MD 24 Materials Needed for Egg Inoculation : Eggs: 9-day old or 10-day old embryonated eggs. Candle the eggs and mark the inoculation sites as described in Section 5. Eggs should be placed in an egg rack with the inoculation site uppermost.
Egg shell punch.
A 70 percent alcohol solution in water.
Syringe 1 mL.
Needles preferably 25 gauge, 16 mm.
Stationery tape (also called cello or sticky tape) or melted wax to seal the inoculation site.
Inoculum. This must be free of microbial contamination.
Discard tray. Materials Needed for Egg Inoculation Dr.T.V.Rao MD 25 Inoculation of the Allantoic cavity : 1. Use cotton wool and 70 percent alcohol to swab the end of the eggs to be inoculated. Allow the alcohol to evaporate.
2. Swab the eggshell punch with 70 percent alcohol solution. Place used cotton wool in discard tray.
3. Pierce a hole in the end of the egg at the marked inoculation site.
4. Attach needle to 1 mL syringe.
5. Draw inoculum into 1 mL syringe. Inoculation of the Allantoic cavity Dr.T.V.Rao MD 26 Inoculation of the Allantoic cavity : 6 Keeping the needle and syringe vertical, place the needle through the hole in the eggshell. The needle will need to penetrate approximately 16 mm into the egg to reach the allantoic cavity.
7. Inject 0.1 mL of inoculum into the egg.
8. Withdraw the needle from the egg.
9. Seal the hole in the shell with stationery tape or melted wax.
10. Discard the used needles and syringes.
11. Place the inoculated eggs into a second incubator. Check the temperature and humidity of incubate Inoculation of the Allantoic cavity Dr.T.V.Rao MD 27 Piercing a hole in the egg shell : Piercing a hole in the egg shell A dental drill can be used if it is available. In most laboratories a tool called an eggshell punch can be improvised using materials that are cheap and easy to procure. Dr.T.V.Rao MD 28 Routes of Egg Inoculation : Routes of Egg Inoculation Dr.T.V.Rao MD 29 Inoculating the Specimens : Inoculating the Specimens The rest of the embryo then gets exposed and ready for use. Virus suspension to be cultivated is taken in dropper and gently spread over the exposed embryo. After inoculation is thus completed, the open area of the shell is sealed eggs are incubated for one week as in hatching. The virus particles infect the membrane at random and create pock marked appearance against the transparent background. This indicate viral basis. Dr.T.V.Rao MD 30 Chorioallantoic membrane (CAM): : Chorioallantoic membrane (CAM): CAM is inoculated mainly for growing poxvirus. Herpes simplex virus is also grown. Virus replication produces visible lesions, grey white area in transparent Cam. Each pock is derived from a single virion. Pocks produced by different virus have different morphology. Under optimal conditions, each infectious virus particle can form one pock. Pock counting, therefore can be used for the assay of pock forming virus such as vaccinia. Dr.T.V.Rao MD 31 Piercing the Chorioallantoic Membrane : Piercing the Chorioallantoic Membrane Little holes are drilled through the egg shell for infection of the chorio-allantoic membrane. Dr.T.V.Rao MD 32 Can be used in few Fungal Infection : Can be used in few Fungal Infection They provide a complex environment, including phagocytic cells, to study fungal host-pathogen interaction, but are of a lower developmental stage than adult mice. Dr.T.V.Rao MD 33 Slide 34: Dr.T.V.Rao MD 34 Piercing the Shell with Needle : Dr.T.V.Rao MD 35 Piercing the Shell with Needle Injecting Infective Material with Needle : Dr.T.V.Rao MD 36 Injecting Infective Material with Needle Overview of Inoculating Sites : Dr.T.V.Rao MD 37 Overview of Inoculating Sites Allantoic cavity: : Inoculation into the allantoic cavity provides a rich yield of influenza and some paramyxoviruses. Allantoic inoculation is employed for growing the influenza virus for vaccine production. Other allantoic vaccines include Yellow fever (17D strain), and rabies vaccines. Duck eggs are bigger and have a longer incubation period then hen’s egg. They therefore provide a better yield of rabies virus and were used for the preparation of the inactivated non-neural rabies vaccines. Allantoic cavity: Dr.T.V.Rao MD 38 ALLANTOIC ROUTE – INOCULATION SITE DETERMINATION : ALLANTOIC ROUTE – INOCULATION SITE DETERMINATION Dr.T.V.Rao MD 39 Amniotic cavity: : Amniotic cavity: The amniotic sac is mainly inoculated for primary isolation of influenza a virus and the mumps virus. Dr.T.V.Rao MD 40 Amniotic Route of Inoculation : Amniotic Route of Inoculation Dr.T.V.Rao MD 41 Yolk sac: : Yolk sac: It is inoculated for the cultivation of some viruses as well as for some bacteria like Chlamydia and Rickettsia. Dr.T.V.Rao MD 42 YOLK SAC ROUTE : YOLK SAC ROUTE Dr.T.V.Rao MD 43 Influenza Vaccine Development in Fertilized Eggs : Influenza Vaccine Development in Fertilized Eggs Dr.T.V.Rao MD 44 Influenza Vaccine Traditional Methods- Influenza Examining the infected eggs Vaccine : Influenza Vaccine Traditional Methods- Influenza Examining the infected eggs Vaccine Dr.T.V.Rao MD 45 How Vaccines are Produced in Eggs : How Vaccines are Produced in Eggs In egg culture, flu viruses are injected into chicken egg embryos, where they multiply. After several days of incubation a machine opens the egg and harvests the virus, which is then purified and chemically killed.On average it takes one or two eggs to produce a single dose of annual flu vaccine.In cell culture, the virus is grown in animal or human cells, which are available in unlimited supply. Dr.T.V.Rao MD 46 How the Reassortant Vaccines for Influenza Produced in Eggs : How the Reassortant Vaccines for Influenza Produced in Eggs The egg is inoculated with a mixture of the epidemic influenza virus strain (red) and a standard strain (green) that can replicate in chicken eggs. Both strains replicate themselves, but as they do so their genetic material becomes mixed, producing hybrid viruses known as reassortants Dr.T.V.Rao MD 47 Eggs as Tools for Developing Influenza Vaccines : Eggs as Tools for Developing Influenza Vaccines Influenza vaccine manufacture in eggs, computer artwork. Fertilized chicken eggs can be used to produce vaccines against influenza viruses. The reassortants are analyzed, and those which have the epidemic strain surface proteins but other genes of the standard strain will be selected. These are injected into different eggs to replicate before harvesting. Dr.T.V.Rao MD 48 Eggs are Used in Mass Scale Development of Vaccines : Eggs are Used in Mass Scale Development of Vaccines Dr.T.V.Rao MD 49 Egg Allergies and Vaccines : Egg Allergies and Vaccines No suitable cell culture system exists and egg inoculation is the method of choice. Influenza virus vaccines are still cultivated in eggs, and hence people with egg allergies cannot tolerate the influenza vaccines. Dr.T.V.Rao MD 50 Follow all the Biosafety Considerations : Follow all the Biosafety Considerations All procedures involving the manipulation of infectious materials are conducted within biological safety cabinets, specially designed hoods, or other physical containment devices, or by personnel wearing appropriate personal protective clothing and equipment. Dr.T.V.Rao MD 51 Slide 52: Created by Dr.T.V.Rao MD for ‘e’ learning for Medical and Paramedical students in the Developing world
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