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    1. EGG INOCULATION Principles Practice and Vaccine Development Dr.T.V.Rao MD Dr.T.V.Rao MD 1

    2. 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

    3. 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

    4. 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 available Dr.T.V.Rao MD 4

    5. Techniques used Microscopy Detection of Viral Antigen Growing and detecting viruses in Tissue / Organ / Cell culture Fertilized hens 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

    6. 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

    7. Demonstration of Viral Antigens Precipitation on gel eg HBsAg Immunofluorescence Counter Immuno Electro Phoresis (CIEP) Enzyme Linkes Immuno Sorbant Assay (ELISA) Dr.T.V.Rao MD 7

    8. Isolation of Virus Laboratory animals Fertilized Hens Egg Chorioallantoic membrane Allantoic cavity Amniotic cavity Yolk sac Organ/Tissue/Cell Culture Growth identified by serological method like neutralization. Dr.T.V.Rao MD 8

    9. Dr.T.V.Rao MD 9

    10. 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 Hens Egg Cultivation of Viruses and Bacteria Dr.T.V.Rao MD 10

    11. The Embryonated hens 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

    12. 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

    13. 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

    14. 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

    15. 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

    16. 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

    17. 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 Dr.T.V.Rao MD 17

    18. 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

    19. Structure and Utility of Fertilized Egg Dr.T.V.Rao MD 19

    20. Routes of Injecting the Fertilized Eggs Dr.T.V.Rao MD 20

    21. Dr.T.V.Rao MD 21

    22. 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

    23. 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

    24. 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

    25. 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 Dr.T.V.Rao MD 25

    26. 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

    27. 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

    28. 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

    29. Routes of Egg Inoculation Dr.T.V.Rao MD 29

    30. 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

    31. 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

    32. 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

    33. 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

    34. Dr.T.V.Rao MD 34

    35. Dr.T.V.Rao MD 35 Piercing the Shell with Needle

    36. Dr.T.V.Rao MD 36 Injecting Infective Material with Needle

    37. Dr.T.V.Rao MD 37 Overview of Inoculating Sites

    38. 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 hens 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

    39. ALLANTOIC ROUTE INOCULATION SITE DETERMINATION Dr.T.V.Rao MD 39

    40. 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

    41. Amniotic Route of Inoculation Dr.T.V.Rao MD 41

    42. 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

    43. YOLK SAC ROUTE Dr.T.V.Rao MD 43

    44. Influenza Vaccine Development in Fertilized Eggs Dr.T.V.Rao MD 44

    45. Influenza Vaccine Traditional Methods- Influenza Examining the infected eggs Vaccine Dr.T.V.Rao MD 45

    46. 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

    47. 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

    48. 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

    49. Eggs are Used in Mass Scale Development of Vaccines Dr.T.V.Rao MD 49

    50. 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

    51. 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

    52. 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