Viral Inoculation: Fertilized Chicken Egg

Viral Inoculation: Fertilized Chicken Egg

viral inoculation

Hey Friends, Today i will discuss about the inoculation procedure of embryonated egg. This is very interesting topic  So lets see about Viral Inoculation; Inoculation Of Embryonated Egg.

Inoculation Of Embryonated Egg.

EMBRYONATED EGG

State of a fertilized egg

Containing an embryo

Foetus in its early stages of developments

Especially before it has reached a distinctively recognizable form.

Eggs used in Virology

HEN’S EGG

DUCK’S EGG

TURKEY’S EGG
Selection of egg

Must be sterile

Shell should be intact and healthy.

Should be obtained from non-vaccinated, disease-free flocks

Process of artificial incubation

process viral inoculation

Incubation – 38 – 39°C and 60 – 70% humidity.

Need to be turned at least twice a day or

Rolled continually in a specially designed egg incubator.

Viral Inoculation; Inoculation Of Embryonated Egg.

EGG INCUBATOR

Artificially hatched – controlled and favourable conditions

Maintain favourable incubation/ environment – constant temperature over a specified period.

Electrically heated – thermostat

Intelligent control system – correct measurement of heat quantity ,

Adjusting hatching control temperature constantly

Variation of temperature – ambient to 70° C

Controlled by “JUMO”/ EGO” German Capillary thermostat having accuracy of + 0.5° c.

Capacity: 50 to 2000 eggs

ADVANTAGES OF EMBRYONATED EGG

Ideal for viruses to grow, offers several sites for virus cultivation

Isolation and cultivation of many avian viruses and few mammalian viruses

Sterile and wide range of tissues and fluids

Economical and Readily available

Maintenance easier

Less labour (not need feeding and caging)

They do not have immune mechanism like animals to counteract virus infection.

DISADVANTAGES

Some viruses do not show growth on primary inoculation into the egg.

Slight amount of bacterial contamination in the inoculum may kill the embryo.

Eggs may be contaminated with mycoplasma and latent fowl viruses which may interfere with the growth of other virus.

CANDLING OF EGG:

Process of holding a strong beam of light above or below the egg to observe the embryo.

Done in a darkened room or area shielded by curtains

Use candling box

CANDLING BOX
Consists of A candling lamp a strong electric bulb covered by a plastic or aluminium container with handle and aperture.

PROCEDURE OF CANDLING

The egg is placed against the aperture and illuminated by light.

Under the candling lamp, the embryo appears as a dark shadow with the head as a dark spot (eye).

Incubated eggs are candled daily to see the chicken embryos inside.

Live Embryonated Egg – Healthy embryos will respond to the light by moving.

Dead Embryo – Candling will reveal a small dark area and disrupted blood vessels.

Viral Inoculation; Inoculation Of Embryonated Egg.

MARKING OF AIRSAC

Hold the blunt end of the egg against the aperture of the candling lamp and note the position of the head of the embryo.

Draw a line on the shell marking the edge of the air sac.

Draw an x approximately 2 mm above this line.

ROUTES OF INOCULATION

Allantoic cavity

Yolk sac

Chorio- allantoic membrane (CAM)

Amniotic cavity

Viral Inoculation; Inoculation Of Embryonated Egg.

Materials required for egg inoculation

Egg

Egg holders

Egg shell punch

Cotton wool

70% alcohol

Syringe 1 ml

Needles preferably 23-25 gauge

Stationery tape or melted wax to seal the inoculation site

Inoculum

Discard tray
1. ALLANTOIC INOCULATION
STEPS:-
Candle the eggs mark the airsac.

Puncture the shell over the centre of the air sac.

Insert a 23- gauge needle, 1-1/2 inches in length on a 1 ml syringe, into the egg through the puncture in the shell at a

45 angle to the long axis of the egg and away from the embryo.
Inject O.2ml of fluid into the egg

Seal the puncture with – Nail polish/cellophone tape

Position the eggs and incubate at 37o C.

Harvesting of Allantoic Fluid

Eggs must be chilled to obtain allantoic fluid free of RBCs.

Clean the upper half of the shell with 70% alcohol.

Cut away the shell above the air space.

Peel away the white opaque shell membrane lining the air space, exposing the transparent allantoic membrane directly beneath.

Tear the allantoic membrane with sterile forceps.

Attach a ballpoint needle to a syringe and insert the needle into the cavity.

Remove the fluid by suction.

Culture the harvested fluid in a suitable medium for a sterility check.

2. YOLK SAC INOCULATION
STEPS:-

Candle the egg and mark the position of the airsac

Puncture the shell over the centre of the air cell

Insert a 22 gauge needle,2 inches in length on a syringe, into the egg via the puncture

Point the needle straight down for depth of about 1-1/2 inches.

Express 0.5 ml of inoculum into the yolk sac.

Seal the puncture with nail polish or cellophane tape.

Incubate the eggs.

Harvesting Of Yolk Sac

Disinfect the upper half of the shell.

Remove the shell, shell membrane and underlying chorio-allantoic membrane.

Lift the embryo up with sterile forceps to expose the attached yolk sac.

Pull the yolk sac free with another pair of forceps and place it in a sterile petridish.

3. Chorio- Allantoic Membrane (CAM) Inoculation
STEPS:-

11- 14 days old eggs required :- Viruses inoculated – HERPES SIMPLEX VIRUS, POX VIRUS AND ROUS SARCOMA VIRUS.

Candle the egg and locate an area on the side of the egg that is free of large blood vessels. Mark this area with a pencil.

Grind a hole through the shell, but not the shell membrane , at the site marked.

Puncture a second hole at the air sac end, this time penetrating the outer shell membrane.

Place a drop of sterile physiological saline on the side hole and gently tease apart the fibers of the shell membrane with a 27- gauge needle.

When the shell membrane has been penetrated, the drop of saline will be drawn into the egg as a result of separation of the CAM and shell membrane.

Apply negative pressure to the air space opening by means of mouth suction with a rubber tube. As the air is removed the CAM will drop from the shell around the side hole, creating an artificial airspace , outline the limits of artificial airspace. Express 0.2 ml of inoculum through the side opening onto the CAM.

Seal openings with cellophane tape.

Gently rotate the egg to spread the inoculum over the entire CAM under the false air space.

Incubate the eggs on side with false air space upward.

Harvesting the CAM Membrane

Disinfect the shell.

With sterile scissors, cut through the shell along the longitudinal axis, about 1/3 down from the upper surface.

Gently remove the shell to a discard pan.

With sterile forceps,lift the CAM, cut free.

Place the CAM in sterile saline and float free for examination.
4. AMNIOTIC CAVITY INOCULATION
STEPS:-

9-10 days old egg required.

Candle the egg and mark the position of the embryo and the outline of the airspace on the shell.

Punch a hole through the shell at the edge of the airspace directly above the embryo.

Using a 23- gauge, 1 inch needle on a syringe make a short jab through the punched hole, towards the embryo.

When the amniotic membrane is penetrated, the embryo will be seen to follow the movements of the needle.

Express upto 0.2 ml of inoculum.

Seal the puncture with nail polish or cellophane tape and incubate at 37o C.

HARVESTING OF AMNIOTIC FLUID

Remove the shell and shell membrane below the air space.

Remove the fluid from the allantoic cavity , the amnion should then be clearly visible.

Remove the amniotic fluid with a 20- gauge needle and syringe.

 

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