Egg Inoculation in Virology


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Egg Inoculation in Virology

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Egg Inoculation in Virology

  1. 1. Dr.T.V.Rao MD Dr.T.V.Rao MD 1
  2. 2. Towards Developing Better Skills in Microbiology Egg inoculation continues to be a Important Student Exercise in SeveralPost Graduate Examinations in Medical Microbiology for evaluation. TheStudents should develop the Necessaryskills to be familiar with the exercise in Virology Dr.T.V.Rao MD 2
  3. 3. Viruses are Different From Other MicrobesViruses are obligate intracellular parasites. Theydepend totally on their host cells for theirexistence. Their total host dependence makes itextremely difficult to get good insight of themnatural conditions, because the internalcharacteristics of the host cells are likely tointerfere with the observations. Due to thesereasons, it has been found desirable that virusesare cultivated or grown in the laboratory itself. Dr.T.V.Rao MD 3
  4. 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 availableChanged situation Rapid techniques have emerged Screening for Blood transfusion Treatment available Dr.T.V.Rao MD 4
  5. 5. Techniques usedMicroscopyDetection of Viral AntigenGrowing and detecting viruses in Tissue / Organ / Cell culture Fertilized hen’s egg Laboratory animal inoculation eg miceDetection of antibody in serum IgG – Rising titer in paired sample IgM – Indicates current / recent infection Dr.T.V.Rao MD 5
  6. 6. MicroscopyElectron Microscope /Immune ElectronMicroscopyLight microscope –Inclusion bodies egNegri Body inRabiesFluorescentMicroscope -Fluorescent antibodytechnique Dr.T.V.Rao MD 6
  7. 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. 8. Isolation of VirusLaboratory animalsFertilized Hen’s Egg Chorioallantoic membrane Allantoic cavity Amniotic cavity Yolk sacOrgan/Tissue/CellCultureGrowth identified byserological method like Dr.T.V.Rao MD 8neutralization.
  9. 9. Virus CultureEmbryonated Egg Chorioallantioc membrane (CAM) Allantoic cavity Amniotic cavity Yolk SacCell Lines/ Tissue Primarycultures Diploid/ Secondary ContinuousAnimal inoculation Suckling mice Dr.T.V.Rao MD 9
  10. 10. Embryonated Hen’s Egg Cultivation of Viruses and BacteriaChorioallantoic membrane (CAM) – visible lesionscalled pocks. Each infectious virus particle formsone pock. e.g. Variola, Vaccinia virusAllantoic cavity – Influenza virus (vaccineproduction) & paramyxovirusesAmniotic cavity – primary isolation of Influenza virusYolk sac – Chlamydia, Rickettsia & some viruses Dr.T.V.Rao MD 10
  11. 11. Embryonated eggs:The Embryonated hen’s egg was first used for cultivationof viruses by Good Pasteur and Burnet (1931).Cultivation of viruses in organized tissues like chickembryo necessitates a different type of approach.. For allpractical purposes they all themselves behave as tissuecultures. The process of cultivation of viruses inembryonated eggs depends on the type of egg which isused. The egg used for cultivation must be sterile and theshell should be intact and healthy. Dr.T.V.Rao MD 11
  12. 12. Burnet as Director of the Hall Institute, 1944-1965F.M. Burnet in thelaboratory in theearly 1950s, wasexperimenting oninfluenza virusgenetics, using thedeveloping hensegg. Dr.T.V.Rao MD 12
  13. 13. BurnetWins 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. 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. 15. Eggs are Used for Mass Vaccine Production in InfluenzaAnimals and chick embryowere the first method thatwas used to cultivate virus.This method is rarely usedas it is not convenient.However, when preparingfor bulk virus, (e.g. antigenor vaccine production) theusage of chick embryo isuseful. Dr.T.V.Rao MD 15
  16. 16. Advantages of Fertile EggsFertile chicken eggsprovide a convenient,space-saving incubator formany kinds of animalviruses. Different virusescan be injected into an eggat different sites and theegg can be easily observedfor viral replicationthroughout thedevelopment of the chickenembryo. Dr.T.V.Rao MD 16
  17. 17. Advantages of Using Embryonated EggsIsolation and cultivation ofmany avian and few mammalianvirusesIdeal receptacle for virus togrowSterile & wide range of tissuesand fluidsCost- much lessMaintenance-easierLess labor Readily available Dr.T.V.Rao MD 17
  18. 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. 19. Structure and Utility of Fertilized Egg Dr.T.V.Rao MD 19
  20. 20. Routes of Injecting the Fertilized Eggs Dr.T.V.Rao MD 20
  21. 21. Dr.T.V.Rao MD 21
  22. 22. Cultivation of Virus in Eggs To cultivate viruses in eggs,the procedure adoptedshould be very simple. Theeggs are kept in incubatorand embryos of 7-12 daysold are used. The eggcontaining embryo usuallyhas an air apace at the largerend. The position of this sacis first determined. Dr.T.V.Rao MD 22
  23. 23. Begin you Exercise with Candling EggsCandling is the process ofholding a strong light above orbelow the egg to observe theembryo. A candling lampconsists of a strong electricbulb covered by a plastic oraluminum container that has ahandle and an aperture. Theegg is placed against thisaperture and illuminated bythe light. If you do not have acandling lamp, improvise. Tryusing a torch. Dr.T.V.Rao MD 23
  24. 24. Marking the inoculation site:1. Hold the blunt end of the eggagainst the aperture of thecandling lamp and note theposition of the head of theembryo.2. Turn the egg a quarter turnaway from the head.3. Draw a line on the shellmarking the edge of the air sac.4. Draw an X approximately 2mm above this line.5. The X marks the inoculationsite. Dr.T.V.Rao MD 24
  25. 25. Materials Needed for Egg InoculationEggs: 9-day old or 10-day old embryonated eggs. Candle the eggsand mark the inoculation sites as described in Section 5. Eggs shouldbe 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 sealthe inoculation site.Inoculum. This must be free of microbial contamination.Discard tray. Dr.T.V.Rao MD 25
  26. 26. Inoculation of the Allantoic cavity1. Use cotton wool and 70 percent alcohol to swabthe end of the eggs to be inoculated. Allow thealcohol to evaporate.2. Swab the eggshell punch with 70 percent alcoholsolution. Place used cotton wool in discard tray.3. Pierce a hole in the end of the egg at the markedinoculation site.4. Attach needle to 1 mL syringe.5. Draw inoculum into 1 mL syringe. Dr.T.V.Rao MD 26
  27. 27. Inoculation of the Allantoic cavity6 Keeping the needle and syringe vertical, place the needlethrough the hole in the eggshell. The needle will need topenetrate approximately 16 mm into the egg to reach theallantoic 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. Checkthe temperature and humidity of incubate Dr.T.V.Rao MD 27
  28. 28. Piercing a hole in the egg shellA dental drill can beused if it is available. Inmost laboratories a toolcalled an eggshellpunch can beimprovised usingmaterials that are cheapand easy to procure. Dr.T.V.Rao MD 28
  29. 29. Routes of Egg Inoculation Dr.T.V.Rao MD 29
  30. 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. 31. Chorioallantoic membrane (CAM):CAM is inoculated mainly forgrowing poxvirus. Herpes simplexvirus is also grown. Virusreplication produces visiblelesions, grey white area intransparent Cam. Each pock isderived from a single virion.Pocks produced by different virushave different morphology. Underoptimal conditions, each infectiousvirus particle can form one pock.Pock counting, therefore can beused for the assay of pock formingvirus such as vaccinia. Dr.T.V.Rao MD 31
  32. 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. 33. Can be used in few Fungal InfectionThey provide a complexenvironment, includingphagocytic cells, to studyfungal host-pathogeninteraction, but are of a lowerdevelopmental stage thanadult mice. Dr.T.V.Rao MD 33
  34. 34. Dr.T.V.Rao MD 34
  35. 35. Piercing the Shell with Needle Dr.T.V.Rao MD 35
  36. 36. Injecting Infective Material with Needle Dr.T.V.Rao MD 36
  37. 37. Overview of Inoculating Sites Dr.T.V.Rao MD 37
  38. 38. Allantoic cavity:Inoculation into the allantoic cavity provides a rich yieldof influenza and some paramyxoviruses. Allantoicinoculation is employed for growing the influenza virusfor vaccine production. Other allantoic vaccines includeYellow fever (17D strain), and rabies vaccines. Duck eggsare bigger and have a longer incubation period then hen’segg. They therefore provide a better yield of rabies virusand were used for the preparation of the inactivated non-neural rabies vaccines. Dr.T.V.Rao MD 38
  40. 40. Amniotic cavity: The amniotic sacis mainlyinoculated forprimary isolationof influenza avirus and themumps virus. Dr.T.V.Rao MD 40
  41. 41. Amniotic Route of Inoculation Dr.T.V.Rao MD 41
  42. 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. 43. YOLK SAC ROUTE Dr.T.V.Rao MD 43
  44. 44. Influenza Vaccine Development in Fertilized Eggs Dr.T.V.Rao MD 44
  45. 45. Influenza Vaccine Traditional Methods- Influenza Examining the infected eggs Vaccine Dr.T.V.Rao MD 45
  46. 46. How Vaccines are Produced in EggsIn egg culture, flu viruses areinjected into chicken eggembryos, where they multiply.After several days of incubationa machine opens the egg andharvests the virus, which is thenpurified and chemically killed.On average it takes one or twoeggs to produce a single dose ofannual flu vaccine.In cell culture, the virus is grownin animal or human cells, whichare available in unlimitedsupply. Dr.T.V.Rao MD 46
  47. 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. 48. Eggs as Tools for Developing Influenza VaccinesInfluenza vaccinemanufacture in eggs, computerartwork. Fertilized chickeneggs can be used to producevaccines against influenzaviruses. The reassortants areanalyzed, and those whichhave the epidemic strainsurface proteins but othergenes of the standard strainwill be selected. These areinjected into different eggs toreplicate before harvesting. Dr.T.V.Rao MD 48
  49. 49. Eggs are Used in Mass Scale Development of Vaccines Dr.T.V.Rao MD 49
  50. 50. Egg Allergies and VaccinesNo suitable cell culturesystem exists and egginoculation is the methodof choice. Influenza virusvaccines are stillcultivated in eggs, andhence people with eggallergies cannot toleratethe influenza vaccines. Dr.T.V.Rao MD 50
  51. 51. Follow all the Biosafety ConsiderationsAll procedures involving themanipulation of infectiousmaterials are conductedwithin biological safetycabinets, specially designedhoods, or other physicalcontainment devices, or bypersonnel wearingappropriate personalprotective clothing andequipment. Dr.T.V.Rao MD 51
  52. 52. Created by Dr.T.V.Rao MD for ‘e’ learning for Medical and Paramedical students in the Developing world Email Dr.T.V.Rao MD 52
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