Exp. No. 11 Cultivation of Virus – Egg Inoculation method (Demonstration)

 

 Cultivation of Virus – Egg Inoculation method (Demonstration)

Introduction

            Embryonated eggs are utilized as a laboratory host system for primary isolation and propagation of a variety of different viruses, including the avian coronaviruses, infectious bronchitis virus (IBV), turkey coronavirus (TCoV), and pheasant coronavirus. They have been extensively utilized for propagation of these viruses for research purposes and, in the case of IBV, for commercial production of vaccines. In addition, embryonated eggs provide a potential host system for studies aimed at identifying other, novel coronavirus species.

            The embryonated egg is comprised of the developing embryo and several supporting membranes which enclose cavities or “sacs” within the egg. The shell membrane lies immediately beneath the shell; this is a tough fibrinous membrane that forms the air sac in the region of the blunt end of the egg. In contrast to the shell membrane, chorioallantoic, amniotic, and yolk membranes are comprised largely of epithelium, and represent potential sites of coronaviral replication. The chorioallantoic membrane (CAM) lies directly beneath the shell membrane; this is a highly vascular membrane that serves as the respiratory organ of the embryo. The CAM is the largest of the embryo membranes, and it encloses the largest cavity within the egg, the allantoic cavity; in the embryonated chicken egg, this cavity contains approximately 5–10 ml of fluid, depending upon the stage of embryonation. The amniotic membrane encloses the embryo and forms the amniotic cavity; in the embryonated chicken egg, this cavity contains approximately 1 ml fluid. The yolk sac is attached to the embryo and contains the nutrients the embryo utilizes during embryonic development and the immediate post-hatch period. The developing embryo and its membranes (CAM, amniotic, yolk) provide the diversity of cell types that are needed for successful replication of a wide variety of different viruses. Embryonated eggs may be inoculated by depositing virus directly onto the CAM, or by depositing virus within allantoic, amniotic, and yolk sacs. For avian coronaviruses, inoculation of eggs by allantoic or amniotic routes has been shown to provide these viruses with access to specific cell types that support their replication.

Anatomy of Embryonated Egg

            The term embryonating egg identifies any eggs in which an embryo is developing. It is common to refer to fertile egg which have been incubated as being 3,5,10 days etc. This does not refer to the time that has been exposed since the egg was laid but to the time that it has been incubated. Although certain early primitive developmental stages occur of fertilization and while the shell wall forms around egg contents. This development does not continues after bird lay eggs. Subsequently incubated artificially further development. This potential does not lost so long as eggs are not subject to extreme of temperature, it is this characteristic of suspended development which make it possible for avian to production their natural habitat. Clutch of eggs before setting down to incubate them, same principle of control storage which make it possible for common hatcheries to hatch thousand of chicks on special day.

Shell and Shell Membrane

If using sharp pointed forceps gently pick away shell of egg; a thin white membrane seen, so closely attached to shell itself that if broken piece of shell examined most would find it difficult to accept the fact that there is a membrane underneath the shell. Scrapping of inner shell of eggshell with forceps show this to be true. This is shell membrane. Outermost hard calcareous whitish covering of egg is egg shell. The shell itself is only are of the several system of avian eggs within function as an exchange system across its surface. Gaseous and liquid molecule passes in both directions. This is why the egg are incubated in presence of humidity and adequate circulation. If an egg is incubated in an atmosphere too low in humidity it will use its moisture. The content will be dehydrated and the death of the embryo will result. Same is true for circulation. If embryonating eggs are tightly packed for too long a period of time while under incubator the impairment of circulation will result in embryo death. The shell and shell membrane are not just inert covering of a living content but themselves function to maintain life formed within it.

Air Space

All normal eggs have a rounded end and opposite to this is the end formed by more acute angle of shell that is pointed end under the shell and shell membrane of the blunt rounded end its an empty space. The egg contents are prevented from entering this area by layering of several membranes, which act as a barrier. The space is called air shell or air space. Its function both in respiration and pressure adjustment.

Chorioallantoic sac/ membrane (CAM)

Attached to developing embryo and originating hindgut, is a sac whose function is to remove wastes. As the embryo increases in size the sac also enlarges until it surrounds the embryo much like a double layered umbrella. Membrane which forms the sac is usually called chorioallantoic membrane together with the content of sac is designated as CAS or allantoic cavity. The fluid content in sac is known as allantoic fluid. It is necessary to differentiate between CAM inoculation when intend is to place the virus on/in membrane CAS or AF inoculation where intend is to place the virus in the allantoic fluid, allantoic cavity formed by CAM.

Yolk Sac and Yolk

Not only there is a CAS extending from developing embryo but structure called the yolk sac completely surround the yolk sac contents of the egg through its membrane and blood vessels, metabolites of yolk are transferred to embryos. As the embryo matures contents of yolk sac noticeably diminishes until the sac is approximately 1 cm. in diameter about three days before embryo hatch. At this state the embryo has attained such size that it nearly fills the egg and except for small opening in its abdomen from which yolk is produced the embryo is perfectly formed. In the final days remaining to hatching time the opening of abdomen fills slowly and the contents will be absorbed in the digestive system of embryo.

Amniotic Sac

This vary with membrane system is most easily seen in embryo of 4 to 9 days. It appears as translucent clear liquid field stretched over developing embryo. Infact it is completely surrounding embryo except for areas where chorioallantoic sac and yolk sac are attached. It’s involvement with embryo and its function can just be seen by gently removing the entire contents of an egg into a petridish and remaining the intact structure. This membrane and its contents serve to protect the embryo against the physical damage and also function as an area of exchange. As the embryo enlarge the membrane stretches to accommodate the increase in size and serve less and less in the role of buffer against physical motion. By the time the embryo has hatched the membrane is thinly stretched and barely visible as its surrounds to fully developed form.

 

Image showing different locations of an embryonated eggs and the type of virus that could be inoculated

Aim

To demonstrate the inoculation of virus in different parts of an embryonated egg and its cultivation.

Materials and Methods

1.  Fertile eggs are obtained, preferably, from specific-pathogen-free (SPF) flocks (e.g., Charles River/SPAFAS).

2.      Disinfectant: 70 % ethanol, 3.5 % iodine, 1.5 % sodium iodide.

3.      Use a scissor and a forceps to cut open the top of the embryonated egg. Prior to use, disinfect the tip of the engraving tool to prevent contamination of the egg.

4.      Plastic cement, glue, tape, or nail varnish are used to seal holes in egg shells after inoculation.

5.      Egg flats.

6.      Egg candlers are available from a variety of commercial sources.

7.   A suitable egg incubator is needed; these are available from a variety of commercial sources. Commercially available egg incubators generally are equipped with heat source, humidifier, and a timer-based mechanical turning system.

8.      Sterile scissors and forceps.

9.      Sterile pipettes or 5 ml syringes with 1 in., 18 gauge needles.

10.  Sterile plastic tubes, e.g., 12 × 75 mm snap-cap tubes or microcentrifuge tubes.

 Procedure

Embryonated eggs from avian species other than chickens and turkeys may be utilized; these are inoculated essentially as described for chicken and turkey eggs, primarily by making adjustments in the length of time embryos are incubated before inoculation. Embryonated chicken eggs are inoculated by the allantoic route at approximately the middle of the 21-day embryonation period, at 8–10 days of embryonation; they are inoculated by the amniotic route late in the incubation period, at 14–16 days of embryonation. Turkey and duck eggs have a 28-day embryonation period and generally are inoculated by the allantoic route at 11–14 days of embryonation, and by the amniotic route at 18–22 days of embryonation.

Embryonated chicken and turkey eggs are incubated at a temperature of 38–39 °C with a relative humidity of 83–87 %. They should be turned several times per day to ensure proper embryo development and to prevent development of adhesions between the embryo and its membranes. Fertile eggs may be stored for brief periods with minimal loss of viability. Ideally, fertile eggs are stored at a temperature of 19 °C with a relative humidity of approximately 70 %. Alternatively, eggs may be stored at room temperature; these should be tilted at 45°, and daily alternated from side to side to minimize loss of embryo viability.

Determination of embryo position

Embryo position was determined so that inoculation into specific membrane or cavity can be done. Choose position, which will place inoculation route. A little to one side or the other of larger vessel still keeping as close vesicle to desired route.

Efficiency in candling

It is generally more efficient to make after an hole in shell at time after candling the exception to may be an unusually large number of egg being found dead then remove first thedied egg free candle and then make a desired hole.

Piercing the shell

The size of opening necessary for inoculation varies considerably depending on the route employed and for this purpose the dental drill is suitable. With this type of device simple holes may be cut with an ordinary drill attachment, while whole segment may be cut from shell with a rotating carborandum disc. The segment are then removed gently piercing of with a sterile scalpel whatever the size of opening; the actual drilling process should cut through the shell only leaving the underline shell membrane intact; the latter must never be pierced by drill.

Techniques of embryo inoculation

The methods of inoculation are as follows

1)      Allantoic sac inoculation

2)      Amniotic sac inoculation

3)      Yolk sac inoculation

4)      Chorioallantoic membrane (CAM) inoculation

5)      Intraembryonic

6)      Intracerebral

7)      Intravenous

The first four methods are in common use and will be described briefly. The other three methods require considerable experience and skill to perform and are limited to special work.

A) Allantoic sac inoculation

Materials - Embryonated 9-10 days incubated eggs, inoculum or virus suspension, tuberculin syringe with 27 gauge^ZA to 1 inch needle, rubber cork with a pin, egg candler, rubber bulb, tincture iodine, molten wax etc

Procedure

1.      Candle the egg and mark the boundary of air sec with pencil.

2.      Select an area of the chorioallantoic membrane free of large blood vessels about 3mm below the base of the air sac.

3.      In this area make a pencil mark at the point for inoculation and other mark on air sac.

4.      Apply tincture of iodine at the sites selected for making holes.

5.      Rubber cork with a pin dipped in tincture iodine is used for making holes on the air sac as well as on other selected site.

6.      Using a 1-ml tuberculin syringe fitted with a needle through a hole in the side of egg to a depth of about % inch and deposits the inoculum.

7.      Withdraw the needle and inoculate the next egg or keep the syringe in sterile tube from where it was removed.

For use of 0.1ml or more of inoculum the hole over the air sac is a necessary air vent to accommodate the inoculum within the egg and to prevent the inoculum from escaping through the hole on the side of the egg. Seal the holes in the eggs with molten wax.

Amniotic sac inoculation Procedure

1.      Candle 12-13 days old egg and make a pencil mark on the shell below the base of the air sac to locate the embryo.

2.      Draw a circle parallel to and above 5 mm, above the base of air sac.

3.      Using a small corborundum disc cut through the shell at the circle but do not pierce the shell membrane.

4.      Apply tincture iodine to the groove cut by the disc and allow to dry.

5.      Using forceps, remove the cap of shell over the air sac to expose the shell membrane.

6.      Apply a few drops of sterile saline solution to the shell membrane over the embryo to make the membrane transparent. To cover more than about % of the shell membrane will interfere with the respiration and may result in the death of the embryo. 12

7.      Using a 1 ml tuberculin syringe fitted with a 27 gauge, 1 inch needle, insert the needle into the amniotic cavity and inject the inoculum.

8.      Close the opening in the shell over the air sac by sealing a disc of sterile heavy paper to the shell with molten wax.

C. Yolk sac inoculation Procedure 

1.      Candle 5-7 days old egg and mark the position of embryo and yolk sac.

2.      Apply tincture iodine and disinfect the site of inoculation.

3.      Drill a small hole through the shell at the upper extremity of the shell over the air sac.

4.      Use a 20-22 gauge 1% to YA inch needle and insert it perpendicular through the hole to the depth of at least 1 inch and deposit the inoculum.

5.      Seal the hole in the egg with molten wax.

D) Chorioallantoic membrane (CAM) inoculation

Procedure

1.      Candle 10-12 days old egg and make a pencil mark on the shell below the base of the air sac. Then select a site on the side of embryo about 3mm above & parallel to the base of the air sac for making the whole without puncturing the CAM.

2.      Mark the second site for making hole over the air sac. Apply tincture iodine on both the sites.

3.      Make the hole on both the sites taking care not to puncture the CAM with the help of rubber cork containing small pin.

4.      For producing an artificial air sac over the CAM, air is sucked with the help of rubber bulb from the hole in air sac. Thus air will pass through the opening made over embryo side permitting the chorioallantoic membrane to drop from the shell membrane at this point. The embryo membranes and fluid will fill normal air sac, thus creating an artificial air sac on the side of egg

5.      Using a 1 ml tuberculin syringe with a 27 gauge, Y2 inch needle, insert the needle through the shell membrane over the artificial air sac and deposit the inoculum on the CAM.

6.      Seal the holes with molten wax

Post inoculation procedure – Sealing

The egg is sealed immediately after inoculation to prevent desiccation and maintain sterility. Simple drill holes are readily sealed with a piece of adhesive tape which is water proof and needs no sterilization before use. Large “windows” in the shell are best sealed with paraffin wax and a glass coverslip. A shoulder of wax is first built up round the neck, which is then covered with paraffin wax and a glass coverslip; a shoulder of wax is first built up round the hole which is then covered with a warm, sterile cover subsequent cooling of the glass serves to seal the hole.

Incubation

Temperature

            The optimum temperature for the development of the embryo (38- 39°C) is too high for proper replication of most vi ruses and incubation temperature is accordingly reduced during the post incubation period to about 37°C, the exact temperature depending upon the viru s being studied. The lower temperature has no significant effect upon the embryo during the few days. It is usually under observation.

Humidity

The same strict precautions concerning humidity of 65% during the pre-inoculation period also observed after the egg has been inoculated. Air circulation Forced air circulation by fan should be available to insure balanced temperature distribution throughout incubation.

Position of egg

The position of the egg after inoculation depends upon the location in the shell of the largest aperture. The later must remain uppermost, so that there is the least risk of the egg content being lost. Thus after inoculation by the amniotic route the egg is incubated in a horizontal position. Contrary to the pre-inoculation procedure; the egg must not be turned at any time after inoculation.

Examination schedule

Inoculated egg should be candled with same frequency until determination made concerning length of time necessary to observe the length of the inoculation. The proformed schedule is make examination 14 approximately 18-20 hours but never late then 24 hours. The examination is to find which are dead or dying from trauma of inoculation. Some eggs may die within the time period due to virus inoculation. Its even balance of egg also inoculated with some virus suspension will provide sufficient material to harvest. Its surface generally not necessary to concerned about harvesting material from their early death. The so eggs are always best, discarded. If for some reason an excessive concentration of virus suspension are inoculated this killed the eggs within this incidental death period. The best approach is to return another sample of original material and re-inoculate the egg but with the higher duration of suspension make as well as subsequent examination there after 24 hour interval at least where embryo dead or dying remove them and refrigerate immediately. Total duration of candling period can be determined by

1.      Nature of virus

2.      Response pattern of embryo

3.      Tissues/ cell harvested

For example yolk is to be harvested the embryo should be removed before 12th day of incubation. The surviving embryo should still be examined for death or gross pathology. Changes initially candling for examination for eggs can be continued until hatching time but practically 15th day of inoculation until can be seen. Because of the increased size of embryo and hence opacity.

Common embryo response to virus inoculation –

Embryo can respond to virus infection in many ways. A few responses which may be noticed following inoculation of the embryonated eggs. Some of the changes and lesions, which may be induced by the virus growth, are listed below.

1.      Embryo death

2.      Hemorrhages of subcutaneous tissues, feather follicles; occipital region.

3.      Congestion of vessels of the wings and feet of the entire embryo.

4.      Growth of the embryo is stunted.

5.      Tucking of the embryo.

6.      Decreased amount of amniotic fluid.

7.      Increased amount of allantoic fluid.

8.      Thickening andoedema of chrioallantoic membrane.

9.      Pocks or areas of leucocytic infiltration, often with central necrosis.

10.  Microscopic lesions.

11.  Formation of inclusion bodies.

            It is desirable to refrigerate the embryos for 4-5 hours before examination. This will reduce hemorrhage into the fluid if the embryo is still alive. If material is to be saved for further passage or for vaccine production it is important to disinfect the shell with alcohol or tincture of iodine before opening the egg so as to avoid bacterial contamination. Gross embryo abnormalities as stunning destroying of muscles and limb body disfunction Histopathological changes can be seen only under microscope. Lesion of extra cellular membrane may be pock formation hemorrhagic oedema etc

Harvesting material from inoculated eggs

Collection of specimen from embryonatinq chicken eggs –

Extra embryonic fluids and yolk are collected with 5ml or 10ml syringe fitted with a 20 gauge, 1 inch needle. The membrane and embryo are collected with forceps.

Allantoicfuid 

1.      Apply tincture iodine or another suitable disinfectant to the shell over the air sac. Break the shell over the air sac with forceps and remove the shell to within 5-10 mm of the base of the air sac.

2.      Insert the needle into the allantoic cavity and aspirate the fluid. The amount collected per egg will vary, but on an average 5-10ml can be 18 collected from an egg. Collect the allantoic fluid in individual container or pooled them.

Amniotic fluid 

1.      Remove the shell and shell membrane from air sac end of the egg.

2.      Collect the amniotic fluid as described above. Apply few drop of saline solution to the shell membrane to render it partially transparent. Instead of applying saline solution the shell membrane and the chorioallantoic membrane may be removed from the base of air sac to permit a better view of the amnion.

3.      Using another syringe and needle insert the needle into the amniotic cavity and aspirate the fluid. Collect the fluid individually or pooled into a proper container.

Yolk

For harvesting yolk from embryos insert a 20 gauge YA inch needle, perpendicularly through the air sac as was done for inoculation by this method and aspirate the yolk in the syringe. Collect the yolk into a proper container.

Chorioallantoic membrane 

1.      Apply tincture of iodine or 70% of alcohol to the shell surface over the air sac. Break the shell with forceps and remove the shell.

2.      Using forceps, remove the shell membrane from the upper pole of the chorioallantoic membrane. Rupture the chorioallantoic membrane. Invert the egg and deposit the embryo, yolk sac and extra embryonic fluid and membrane in a petri dish.

3.      If the chorioallantoic membrane adheres to the shell membrane in the shell, then strip it with another forceps. Put the membrane in a petri dish containing normal saline or phosphate buffer solution. Wash it thoroughly, drain. Collect in a container.

Result

Sample inoculation and extraction methods from different layers of embryonated eggs were demonstrated and then be asked to perform in groups.