Dr.T.V.Rao MD

Dr.T.V.Rao MD 1

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�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 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

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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

Changed situation

�Rapid techniques have emerged

�Screening for Blood transfusion

�Treatment availableDr.T.V.Rao MD 4

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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

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Microscopy�Electron Microscope / Immune Electron Microscopy

�Light microscope –Inclusion bodies eg Negri Body in Rabies

�Fluorescent Microscope -Fluorescent antibody technique

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Demonstration of Viral Antigens

�Precipitation on gel eg HBsAg

�Immunofluorescence

�Counter Immuno Electro Phoresis (CIEP)

�Enzyme Linkes Immuno Sorbant Assay (ELISA)

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Isolation of Virus

� Laboratory animals

� Fertilized Hen’s Egg

�Chorioallantoic membrane

�Allantoic cavity

�Amniotic cavity

�Yolk sac

�Organ/Tissue/Cell Culture

�Growth identified by serological method like neutralization.

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Embryonated Egg

Cell Lines/ Tissue

cultures

Animal inoculation

Chorioallantioc membrane (CAM)

Allantoic cavity

Amniotic cavity

Yolk Sac

Primary

Diploid/ Secondary

Continuous

Suckling mice

Virus CultureVirus CultureVirus CultureVirus Culture

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��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

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�� 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:

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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.

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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.

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Only Embryonated Eggs Are Suitable for Growing Virus

�Inoculated eggs are candled daily to see the chicken embryos inside.

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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.

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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.

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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� Maintenance-easier� Less labor� Readily available

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Advantages of Fertilized Eggs are

� Free from bacteriaand many latentviruses.

� Free from specificand non specificfactors of defense.

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Structure and Utility of Fertilized Egg

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Routes of Injecting the Fertilized Eggs

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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.

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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.

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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.

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�� 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.� Cotton wool.� 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

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�� 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

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�� 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

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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.

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Routes of Egg Inoculation

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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.

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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.

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Piercing the Chorioallantoic Membrane

�Little holes are drilled through the egg shell for infection of the chorio-allantoic membrane.

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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.

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Piercing the Shell with Needle

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Injecting Infective Material with Needle

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Overview of Inoculating Sites

�� 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:

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ALLANTOIC ROUTE –INOCULATION SITE DETERMINATION

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Amniotic cavity:

�The amniotic sac is mainly inoculated for primary isolation of influenza a virus and the mumps virus.

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Amniotic Route of Inoculation

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Yolk sac:

�It is inoculated for the cultivation of some viruses as well as for some bacteria like Chlamydia and Rickettsia.

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YOLK SAC ROUTE

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Influenza Vaccine Development in Fertilized Eggs

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Influenza Vaccine Traditional Methods- Influenza Examining the infected eggs Vaccine

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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.

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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

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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.

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Eggs are Used in Mass Scale Development of Vaccines

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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.

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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.

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��Created by Dr.T.V.Rao MD for ‘e’

learning for Medical and Paramedical students in the

Developing world

�Email

�doctortvrao@gmail.com

Dr.T.V.Rao MD 52