DESC:TITLE OF INVENTION
Vaccine for Infectious Bursal Disease

FIELD OF INVENTION
The present invention generally relates to the Vaccine for Infectious bursal disease (IBD) in poultry.

BACKGROUND OF INVENTION
Infectious bursal disease (IBD) is an acute, highly contagious viral infection in chicken manifested by inflammation and subsequent atrophy of the bursa of Fabricius, various degrees of necrosis and immunosuppression. IBD infection depends on age and the breed of chicken and virulence of the virus. Infections may be subclinical or clinical. Infections before 3 weeks of age are usually subclinical. Chickens are most susceptible to clinical disease at 3– 6 weeks of age when immature B cells populate the bursa and maternal immunity has waned, but severe infections have occurred in Leghorn chickens up to 18 weeks of age.

Early subclinical infections are the most important form of the disease because of economic losses. They cause severe, long-lasting immunosuppression due to destruction of immature lymphocytes in the bursa of Fabricius, thymus, and spleen. The humoral (B cell) immune response is the most severely affected; the cell-mediated (T cell) immune response is affected to a lesser extent. Chickens immunosuppressed by early IBDV infections do not respond well to vaccines and are predisposed to infections with normally less pathogenic viruses and bacteria. Common diseases are usually exacerbated by IBDV infections. Some strains of IBDV can cause subclinical infections in older birds (3–6 weeks old), which leads to losses from poor feed efficiency and longer times to market. In these cases, the immunosuppression is usually transient, and convalescent birds may recover most or all of their humoral immune function. However, secondary infections that occur during the transient immunosuppression can cause significant economic losses.

Different types of vaccines are already available for the prevention of this disease; however, it has been observed recently, that currently available vaccines are unable to provide protection against IBD in the field condition always. Therefore, there is a need for a vaccine which will provide higher protection against such strains. This the present disclosure discloses the isolation of the virulent strain of virus and attenuated the field virus by serial passage in chicken embryos and the vaccine developed.

OBJECT OF INVENTION
The object of this invention is to generate a live and inactivated vaccine with higher strength against the Infectious Bursal Disease in poultry.

SUMMARY OF THE INVENTION
This summary is provided to introduce a selection of concepts, in a simple manner, which are further described in detailed description of the invention. This summary is neither intended to identify the key or essential inventive concept of the subject matter, nor to determine the scope of the invention.

According to the present invention, an attenuated vaccine effective against Infectious Bursal Disease in poultry, comprising of an infectious bursal disease (IBD) virus strain of west Bengal India; a stabilizer 1; and a stabilizer 2. The vaccine of 2.2 ml comprises of 1.76 ml of the IBD virus and 0.22ml of each of the stabilizers. Preferably the stabilizer 1 is either 20% lactose or 20 % milk. Preferably, the stabilizer 2 is either 20 % NZ amine or 20 % peptone. More preferably the IBD virus concentration per dose for the live vaccine is 1X10 3 EID50.

A further aspect of the present invention provides for an inactivated vaccine against the Infectious Bursal Disease in poultry, comprising of an IBD virus strain of West Bengal India and an adjuvant. Preferably the adjuvant is made from mineral oil, Tween 80 and span 80. More Preferably the concentration of the virus per dose for the inactivated vaccine is 1 X 10 6.5 EID 50.
To further clarify the advantages and features of the present invention, a more particular description of the invention will follow by reference to specific embodiments thereof, which are illustrated in the appended figures. It is to be appreciated that these figures depict only typical embodiments of the invention and are therefore not to be considered limiting in scope. The invention will be described and explained with additional specificity and detail with the appended figures.

BRIEF DESCRIPTION OF DRAWINGS
The invention will be described and explained with additional specificity and detail with the accompanying figures in which:

Figure 1 illustrates the flow diagram for vaccine with living egg based production procedure.
Figure 2 illustrates the antibody titre of individual bird at 35 DPV, tested with IDEXX ELISA kit.
Figure 3 illustrates the antibody titre Geometric Mean of each group at 35 DPV, tested in IDEXX ELISA kit.

Further, those skilled in the art will appreciate that elements in the figures are illustrated for simplicity and may not have necessarily been drawn to scale. Furthermore, the figures may show only those specific details that are pertinent to understanding the embodiments of the present invention so as not to obscure the figures with details that will be readily apparent to those skilled in the art having the benefit of the description herein.

DETAILED DESCRIPTION
For the purpose of promoting an understanding of the principles of this disclosure, reference will now be made to the embodiment illustrated in the figures and specific language will be used to describe them. It will nevertheless be understood that no limitation of the scope of the disclosure is thereby intended. Such alterations and further modifications in the illustrated system, and such further applications of the principles of the invention as would normally occur to those skilled in the art are to be construed as being within the scope of the present invention.

It will be understood by those skilled in the art that the foregoing general description and the following detailed description are exemplary and explanatory of the invention and are not intended to be restrictive thereof.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. The system, methods, and examples provided herein are only illustrative and not intended to be limiting.

The present invention relates to a vaccine that has been formulated from the attenuated strain of IBD which maybe in the form of live vaccine or inactivated vaccine.
Following examples further describes the invention in stepwise manner.

EXAMPLE 1
The different stages in development of the vaccine, i.e, vaccine isolation, purification, preparation and inactivation are described below.

Step 1: Vaccine Isolation.
The vaccine virus is isolated from the field and purified. The isolated virus strain is serially passaged in the chicken embryo to adapt the virus in the different system of the growth substrate [unnatural] in order to attenuate it. Initially the bursa tissue from the dead chicken are triturated, centrifuged supernatant is collected and passed through the filter and inoculated in the developing chicken embryos for virus isolation. The same procedure is then repeated. Eventually the virus loses its ability to produce the disease in the chicken and induces a strong immune response against the IBD and the birds are protected from the newly evolved field virus. The virus is isolated from the infected chicken which are found in the West Bengal region in India and the filed isolates of the virus have the Genbank accession numbers KT630855.1 & KT630847.1.

Step 2: Vaccine purification.
The original virus is isolated from the bursa of the Fabricious of a chicken that died of Infectious Bursal Disease [IBD]. The bursal tissue is triturated to make a 10% solution in PBS and filtered through 0.2-micron filter and inoculated into a set of 10 days old embryonated Specific Pathogen Free [SPF] chicken eggs by chorioallantoic membrane [CAM] route and incubated for 5 to 7 days, the embryos die on 5th day having characteristic oedematous & haemorrhagic lesions. The embryos and the allantoic fluid are collected aseptically, triturated and centrifuged, a 10% suspension is made and inoculated into another set of 10 days old embryonated Specific Pathogen Free [SPF] chicken eggs; likewise, 3 passages are performed. After 3 consecutive passages the virus suspension are serially diluted up to 10-6 dilution in PBS and each dilution of the virus is inoculated into a set of 5 SPF 10 days’ old embryos and incubated for 7 days. The embryos that died at 10-5 dilution are considered to be the high tittered purified virus, which are serially passaged up to 40 passage level for evaluation of the virus as vaccine.

Step 3: Live Vaccine preparation.
The live vaccine is prepared by titrating the 40th passage virus [supernatant of triturated embryos in allantoic fluid] using EID50. The antigen is adjusted to contain 1 X 103.5 EID50/ dose of virus and then mixed with NZ Amine and Lactose stabilizers. It is then freeze dried to prepare the live vaccine.

Step 4 : Inactivated Vaccine preparation.
For the inactivated vaccine, the virus production is done according to the standard procedure wherein the virus is inoculated in a set of 10 days old embryonated Specific Pathogen Free [SPF] chicken eggs by allantoic cavity [ AC] route and incubated for 3 days. The embryos that died between 24-48 hours post inoculation are discarded and all the live embryos are transferred in the cold room and are left overnight, to kill the embryos. The embryos are harvested and the allantoic fluids are also collected, then the embryo & allantoic fluids are triturated, centrifuged at 4000 rpm and supernatant is collected, the vaccine virus is inactivated by the addition of formaldehyde to have a final volume of 0.5%. A formulation for the antigen 1X 106.5 EID50/ dose with 30 aqueous and 70 mineral oil ratio of Arlacel 83 and tween 80 as emulsifier for the water in oil vaccine preparation.

EXAMPLE 2
Evaluation of the prepared inactivated IBD oil emulsion vaccine:
Testing the quality control of the prepared Inactivated IBD vaccine including sterility and safety test are carried out according to the procedure described in Indian Pharmacopeia.

Sterility test: The sterility test is performed to confirm as to whether the prepared vaccine is free from bacterial, mycoplasma and fungal contamination by inoculation into the appropriate media and incubated according to the standard operational procedure described in Indian Pharmacopeia. The vaccine is found to be sterile.

Safety test in chicks: Safety of the prepared inactivated IBDV oil emulsion vaccine is examined in a group of twenty, 3 weeks old SPF chickens, inoculated with 1 ml (double dose) of the vaccine subcutaneous at the neck. These chicks are observed for 2 weeks for any signs of local reaction or appearance of any clinical signs. No local or systemic reaction is observed therefore the vaccine is found to be safe.

Potency Test: Three groups of SPF chicken at 3 weeks of age are taken. Out of that, two groups of 20 birds are inoculated subcutaneously with 0.5 ml of the inactivated vaccine and another group is inoculated subcutaneously with PBS [Placebo]. Group 1 is inoculated with experimental vaccine and group 2 is vaccinated with regular vaccine of IBD. Group 3 is kept as non-vaccinated control.
After 35 days of vaccination; serum samples are collected from all the three groups of chicken and serum samples are analysed to observe the humoral immune response induced by the vaccines by using IDEXX ELISA kit.

As Illustrated in Figure 2 and 3, the Antibody titre of individual bird at 35 DPV and the Geometric mean tested using the IDEXX ELISA kit clearly shows that the all the birds vaccinated with experimental vaccine generated good immune response which is comparable to the regular commercially available inactivated IBD vaccine.

While specific language has been used to describe the invention, any limitations arising on account of the same are not intended. As would be apparent to a person skilled in the art, various working modifications may be made to the method in order to implement the inventive concept as taught herein.
The figures and the foregoing description give examples of embodiments. For example, order of processes described herein may be changed and are not limited to the manner described herein. Moreover, the actions of any flow diagram need not be implemented in the order shown; nor do all of the acts need to be necessarily performed. Also, those acts that are not dependent on other acts may be performed in parallel with the other acts. The scope of embodiments is by no means limited by these specific examples.
,CLAIMS:CLAIMS
We claim:
1. An attenuated vaccine effective against Infectious Bursal Disease in poultry, comprising of:
-An infectious bursal disease (IBD) virus strain of west Bengal India;
-A stabilizer 1; and
-A stabilizer 2.

2. The vaccine as claimed in claim 1 wherein 2.2 ml of the vaccine comprises of 1.76 ml of the IBD virus and 0.22ml of each of the stabilizers.

3. The vaccine as claimed in claim 1 or 2, wherein the Stabilizer 1 is selected from the group of 20% lactose and 20% milk.

4. The vaccine as claimed in any of the claims 1 to 3, wherein the stabilizer 2 is selected from the group of 20 % NZ amine and 20% peptone.

5. The vaccine as claimed in any of the claims 1 to 5, wherein the IBD virus concentration per dose for the live vaccine is 1X10 3 EID50.

6. The vaccine according to any of the claims 1 to 6, wherein the vaccine is in lyophilized form.

7. An inactivated vaccine effective against Infectious Bursal Disease in poultry, comprising of:
-An infectious bursal disease (IBD) virus strain of west Bengal India; and
-An adjuvant.

8. The vaccine as claimed in claim7, wherein the adjuvant is made from mineral oil, Tween 80 and span 80.

9. The vaccine as claimed in any of the claims 7 or 8, wherein the virus concentration per dose for the inactivated vaccine is 1 X 10 6.5 EID 50.