DESC:TECHNICAL FIELD
The present disclosure relates to the field of aquaculture. Particularly, the invention is in the field of bio-feed formulations for white spot syndrome virus (WSSV) infection in crustaceans comprising crustacean feed and polyclonal IgY antibody against the pathogen white spot syndrome virus, Whispovirus.

BACKGROUND OF THE DISCLOSURE
WSSV is an extremely virulent shrimp pathogen that was first detected in Taiwan in 1992 and has spread vastly to cause significant outbreaks in various parts of the world. WSSV has invaded almost all Asian countries and was first noted in India in 1994 on the east coast, and the following year on the west coast, affecting the industry in India, and resulting in immense economic loss. WSSV emerges to be significantly lethal due to its high mortality rates and is majorly contagious due to its viral transmissible mechanism causing widespread infection along with broad-spectrum potential hosts and vectors. WSSV, therefore, happens to be one of the most prevalent pathogens that causes infected shrimps to develop white spots ranging from 0.5–3.0 mm in diameter on the exoskeleton, appendages, and inside the epidermis.

WSSV is an ovoid-to-bacilliform virus with a rod-shaped nucleocapsid and a tail-like appendix at one end of the virion and its size ranges between 210 and 420 nm in length and 70–167 nm in diameter. WSSV belongs to the Family of Nimaviridae and is the lone member of the genus, Whispovirus due to its unusual genetic and morphological features. It possesses double-stranded circular DNA with an estimated size of ~290Kbp and is composed of 5 major virion proteins: VP28, VP19 reside in the envelope, VP26, VP2, and VP15 are present in nucleocapsid.

VP28 is the major envelope protein encoded by wsv421 gene which may play a crucial role in the initial steps of WSSV infection as an attachment protein, binding the virus to shrimp cells, and enabling it to enter into the cytoplasm of the host cell. VP26, encoded by the wsv311 gene is likely to function as a linker protein whose N-terminus anchors in the envelope, while the C-terminus is bound to the nucleocapsid. In addition, VP26 is capable of binding to actin or actin-associated proteins of the host cell thereby enabling the transport of WSSV to the site of transcription and replication after cellular internalization. VP28 and VP26 naturally form projected trimmers in the viral envelope and exhibit an architecture similar to the structure of other viral envelope fusion proteins which gain entry into the host cells through the cytoplasm by forming a fusion of their lipid envelope with the host cell membrane. The other major envelope proteins and nucleocapsid proteins of WSSV involved in the systemic infection of shrimps are the 6 WSSV envelope proteins (VP31, VP36A, VP36B, VP110, VP187, and VP281), which are reported to be involved in the process of virus binding to cell surface integrin’s which have emerged as an attachment or post-attachment receptors in host cells. VP35 is a major capsid protein that contains two potential clusters of four amino acids (24KRKR27, 53KRPR56) resembling a nuclear localization signal (NLS) which enables the virus to import its DNA into the host cell nucleus for replication.

The function of VP28 protein in the infection of shrimp cells is that it acts as an attachment protein and a mediator for viral transport into the host cell. VP28 possesses five potential sites for N-linked glycosylation, two sites for O-glycosylation, and nine possible phosphorylation sites. However, the protein consists of a strong hydrophobic region at the N-terminus, including a putative transmembrane region. Further, VP28 along with VP26 adopt a b-barrel architecture with a protruding N-terminal area, which allows them to interact with the host receptor or fuse with the host cell membrane for efficient transfer of viral particles. These biological properties of VP28 enable it to play the role of an important medium for host-pathogen interaction. Moreover, a previous study highlights the sequence of events occurring during WSSV infection where the position of VP28 protein was monitored using laser scanning confocal microscopy post-binding. It was recorded that VP28-EGFP (envelope protein VP28 fused with enhanced green fluorescence protein) was observed in the whole of the host cell cytoplasm 3 hours post-adsorption. This suggests that VP28 not only facilitates virus attachment but also assists the virus to penetrate the cytoplasm which then becomes uncoated to release virions.

WSSV exhibits horizontal transmission where the ingestion of infected tissue, direct exposure of body surfaces to virus particles in the water, or injection of a cell-free extract of infected tissue has been reported to spread infection. Besides, the WSSV virus is passed from an infected female parent to her F1 progeny which is a typical example of vertical transmission. However, the eggs die from the infection before maturation aligning with the lethality of the virus. The rotifer, Brachionus urceus, exists almost in every water body and is an example of a passive host of WSSV which in turn becomes a potential WSSV carrier for shrimps. In addition, polychaetes act as a passive vector of WSSV in aquatic systems. Further, studies have shown that seabirds serve as a feasible source of viral transmission. These properties confer the characteristic contagious nature of the virus which needs to be addressed to cope with the leading economic loss in the aquaculture industries.

Analysing the underlying socioeconomic importance of shrimp farming, the development of solid measures to combat WSSV has captured significant attention globally. One of the potential strategies to efficiently fight viral infections is through virus neutralization techniques. The envelope spikes on the virion binds to the neutralizing antibodies, which in turn prevents the attachment of the virus to the cell surface, cell entry, or virus uncoating. The neutralization technique also might serve as a passive immunization strategy. Because the major envelope protein, VP28 occurs abundantly in WSSV and conclusively evident that it is the first molecule to interact with the host cell during infection, followed by its function as an attachment protein and virus transport mediator, VP28 has been chosen as the target protein.

Production of IgY (chicken IgG) in a hen's egg yolk is an efficient and economical method to raise polyclonal antibodies. Bleeding of hens is not necessary for collecting IgY and purification of IgY is relatively simple. Chicken IgG (or IgY) in the egg follicle is passed by receptors in large amounts into the yolk. The IgY concentration in the yolk is comparable to the concentration of IgY in the serum; 6-13 mg/ml. Laying hens are highly cost-effective as producers of antibodies compared with other mammals traditionally used for such production. Also, chicken antibodies have biochemical advantages over mammalian antibodies due to the phylogenetic differences between avian and mammalian species, resulting in increased sensitivity and expanded antibody repertoire.

During WSSV infection, clinical signs will be obvious when the virus pathogenesis begins in the direct exposure of body surfaces to virus particles in the water bodies majorly. Therefore, the neutralization of the virus in the initial stage which is the direct exposure of body surfaces to virus particles in the water bodies is important to reduce the disease pathogenesis and to save the animal. The present invention provides a feed formulation containing anti-WSSV VP28 chicken IgY for imparting anti-WSSV activity in the body surfaces and the water bodies. The formulation was prepared to overcome the larger area of the water bodies and other difficulties so that the antibodies are available for the shrimp’s body surfaces to impart its neutralizing effect.

STATEMENT OF THE DISCLOSURE
Accordingly, the object of the invention is to provide a feed formulation for white spot syndrome virus (WSSV) infection in crustaceans, comprising of polyclonal IgY antibody formulation and crustacean feed; wherein the polyclonal IgY antibody formulation and crustacean feed are present in the ratio of 1:1000; wherein the polyclonal IgY antibody formulation comprises polyclonal anti-WSSV VP28 IgY antibody as the active pharmaceutical ingredient (API).

Another object of the invention is to provide a method of application of the feed formulation in aquatic systems wherein crustaceans are cultivated wherein the feed formulation comprises polyclonal IgY antibody formulation and crustacean feed; wherein the polyclonal IgY antibody formulation and crustacean feed are present in the ratio of 1:1000; wherein the polyclonal IgY antibody formulation comprises polyclonal anti-WSSV VP28 IgY antibody as the active pharmaceutical ingredient.

Another object is to provide a formulation for white spot syndrome virus (WSSV) infection in crustaceans, comprising of polyclonal anti-WSSV VP28 IgY antibody, wherein the polyclonal IgY antibody formulation comprises polyclonal anti-WSSV VP28 IgY antibody as the active pharmaceutical ingredient.

Another object of the invention is to provide a method of preparation of feed formulation comprising preparation of polyclonal anti-WSSV VP28 IgY antibody from egg yolk of hen/chicken immunized with WSSV-VP28 recombinant protein.

Yet another object is to provide a method of application of the formulation in aquatic systems wherein crustaceans are cultivated wherein the formulation comprises of polyclonal anti-WSSV VP28 IgY antibody, wherein the polyclonal IgY antibody formulation comprises polyclonal anti-WSSV VP28 IgY antibody as the active pharmaceutical ingredient (API).

BRIEF DESCRIPTION OF THE ACCOMPANYING FIGURES
In order that the present disclosure may be readily understood and put into practical effect, reference will now be made to exemplary embodiments as illustrated with reference to the accompanying figures. The figures together with the detailed description below, are incorporated in and form part of the specification and serve to further illustrate the embodiments and explain various principles and advantages, where:

FIGURE 1 depicts the vector construct for the expression of recombinant protein WSSV-VP28, in accordance with an embodiment to the present disclosure.

FIGURE 2a depicts an immunoblot, wherein the band corresponds to recombinant protein WSSV-VP28 expressed by the vector construct of Figure 1 in recombinant microbial cells (E.coli BL21 DE3 star) and purified in accordance with an embodiment of the present disclosure.

FIGURE 2b depicts an immunoblot of purified recombinant protein WSSV-VP28 with anti-His antibody, in accordance with an embodiment of the present disclosure.

FIGURE 3a depicts an immunoblot of polyclonal anti-WSSV-VP28 IgY antibody prepared and purified from egg yolk of chicken, immunized with recombinant protein WSSV-VP28, in accordance with an embodiment of the present disclosure.

FIGURE 3b depicts an immunoblot of polyclonal anti-WSSV-VP28 IgY antibody with anti-chicken IgY HRPO conjugate antibody prepared and purified from egg yolk of chicken, immunized with recombinant protein WSSV-VP28, in accordance with an embodiment of the present disclosure.

FIGURE 4a depicts an immunoblot of a positive strain sample and VP28 protein sample with polyclonal anti-WSSV-VP28 IgY antibody from egg yolk of chicken, immunized with recombinant protein WSSV-VP28.

FIGURE 4b depicts an immunoblot of WSSV-positive strain samples isolated from WSSV-infected shrimp lysates having various concentrations of WSSV proteins with polyclonal anti-WSSV-VP28 IgY antibody from egg yolk of chicken, immunized with recombinant protein WSSV-VP28. The blot shows that the polyclonal anti-WSSV-VP28 IgY antibody can bind even with lowest concentration of WSSV protein.

FIGURE 5 illustrates a plot depicting the in-vitro neutralization study of WSSV by polyclonal anti-WSSV-VP28 IgY antibody raised against recombinant protein WSSV-VP28 using competitive ELISA. The WSSV-specific polyclonal anti-WSSV-VP28 IgY antibody completely neutralized the WSSV virus when incubated at a concentration starting from 5 µL.

FIGURE 6a illustrates a plot depicting the survival rates of shrimps injected with WSSV mixed with anti-WSSV-VP28 IgY antibody raised against recombinant protein WSSV-VP28 at different time intervals in the in-vivo neutralization study. More than 90% survival was observed in 4mg IgY group and more than 70% survival was observed in 2mg IgY group.

FIGURE 6b illustrates a plot depicting the average survival rates of 4 consecutive studies of shrimps injected with WSSV mixed with anti-WSSV-VP28 IgY antibodies raised against recombinant protein WSSV-VP28 at different time intervals for the in-vivo neutralization study. The results showed 90%-100% survival rate in the 4mg polyclonal anti-VP28 IgY antibody group.

FIGURE 7 illustrates a plot representing the amount of anti-WSSV-VP28 IgY antibody against recombinant protein WSSV-VP28 in gut content and gut of shrimp fed with feed formulation comprising anti-WSSV-VP28 IgY antibodies against recombinant protein WSSV-VP28 (30 mg/g of feed) at different time intervals (hours) by competitive ELISA. Values are based on four trials. B – Blank, NC - Negative Control, PC – Positive Control, Feed – IgY formulated feed. FG – Foregut, FG cont – Content of foregut, MG – Midgut, MG cont – Content of midgut, HG – Hindgut, HG cont – Content of hindgut, in accordance with an embodiment of the present disclosure.

FIGURE 8 illustrates a plot representing the amount of anti-WSSV-VP28 IgY antibody against recombinant protein WSSV-VP28 in the faecal matter of shrimps fed with feed formulation comprising anti-WSSV-VP28 IgY antibodies against recombinant protein WSSV-VP28 (30 mg/g of feed) at different time intervals (hours) by competitive ELISA where faecal samples are collected at different time intervals by competitive ELISA, in accordance with an embodiment of the present disclosure.

FIGURE 9 depicts the agarose gel analysis to detect the presence or absence of WSSV in faecal matter of shrimps injected with anti-WSSV-VP28 IgY antibodies (IgY) treated WSSV and untreated WSSV, wherein lane 1 indicates Gill tissue from shrimp injected with WSSV treated with IgY, Lane 2 – Head soft tissue from shrimp injected with WSSV treated with IgY, Lane 3 – Gill tissue from shrimp injected with untreated WSSV, Lane 4 – Head soft tissue from shrimp injected with untreated WSSV, in accordance with an embodiment of the present disclosure.

FIGURE 10 illustrates a plot representing the stability of anti-WSSV-VP28 IgY antibody formulation at 2 – 8 ? by competitive ELISA, in accordance with an embodiment of the present disclosure.

FIGURE 11 represents a plot representing the stability of anti-WSSV-VP28 IgY antibody formulation at different pH levels (6, 7, and 8) by competitive ELISA against WSSV and WSSV specific recombinant protein VP28. Results showed that anti-WSSV-VP28 IgY antibody is stable in varying pH conditions.

FIGURE 12 illustrates a plot representing the stability of anti-WSSV-VP28 IgY antibody formulation exposed to extracts prepared from different parts of the digestive tract (Fore gut, Mid gut and Hind gut) by competitive ELISA against WSSV and WSSV specific recombinant protein VP28. Results showed that anti-WSSV-VP28 IgY antibody is stable upon exposure to gut contents.

FIGURE 13 illustrates a plot representing the stability of anti-WSSV-VP28 IgY antibody formulation at different salinities (0, 5, 10, 20, and 25 ppt) at different time intervals (days) by competitive ELISA against WSSV and WSSV-specific recombinant protein VP28. Results showed that anti-WSSV-VP28 IgY antibody is stable under varying salinity conditions.

FIGURE 14 demonstrates a plot representing the detection of anti-WSSV-VP28 IgY antibody in shrimp gill, gut, sediment, and water in the open pond challenge studies.

Sequences:
SEQ ID NO: 1 depicts the DNA sequence of a fragment encoding the VP28_WSSV protein.
atggatctttctttcactctttcggtcgtgtcggccatcctcgccatcactgctgtgattgctgtatttattgtgatttttaggtatcacaacactgtgaccaagaccatcgaaacccacacaggcaatatcgagacaaacatggatgaaaacctccgcattcctgtgactgctgaggttggatcaggctacttcaagatgactgatgtgtcctttgacagcgacaccttgggcaaaatcaagatccgcaatggaaagtctgatgcacagatgaaggaagaagatgcggatcttgtcatcactcccgtggagggccgagcactcgaagtgactgtggggcagaatctcacctttgagggaacattcaaggtgtggaacaacacatcaagaaagatcaacatcactggtatgcagatggtgccaaagattaacccatcaaaggcctttgtcggtagctccaacacctcctccttcacccccgtctctattgatgaggatgaagttggcacctttgtgtgtggtaccacctttggcgcaccaattgcagctaccgccggtggaaatcttttcgacatgtacgtgcacgtcacctactctggcactgagaccgagtaa

SEQ ID NO: 2 depicts the protein sequence of a fragment encoding the VP28_WSSV protein.
MDLSFTLSVVSAILAITAVIAVFIVIFRYHNTVTKTIETHTGNIETNMDENLRIPVTAEVGSGYFKMTDVSFDSDTLGKIKIRNGKSDAQMKEEDADLVITPVEGRALEVTVGQNLTFEGTFKVWNNTSRKINITGMQMVPKINPSKAFVGSSNTS

DETAILED DESCRIPTION OF THE DISCLOSURE
Unless otherwise defined, all terms used in the disclosure, including technical and scientific terms, have meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. By means of further guidance, term definitions are included for better understanding of the present disclosure.

As used herein, the singular forms ‘a’, ‘an’ and ‘the’ include both singular and plural referents unless the context clearly dictates otherwise.

The term ‘comprising’, ‘comprises’ or ‘comprised of’ as used herein are synonymous with ‘including’, ‘includes’, ‘containing’ or ‘contains’ and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps.

The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints.

The term ‘about’ as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of ±10% or less, preferably ±5% or less, more preferably ±1% or less and still more preferably ±0.1% or less of and from the specified value, insofar such variations are appropriate to perform the present disclosure. It is to be understood that the value to which the modifier ‘about’ refers is itself also specifically, and preferably disclosed.
Reference throughout this specification to ‘some embodiments’, ‘one embodiment’, or ‘an embodiment’ means that a particular feature, structure, or characteristic described in connection with the embodiment may be included in at least one embodiment of the present disclosure. thus, the appearances of the phrases ‘in some embodiments’, ‘in one embodiment’ or ‘in an embodiment’ in various places throughout this specification may not necessarily all refer to the same embodiment. It is appreciated that certain features of the disclosure, which are for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination.

The terms “bio-feed formulation” and “feed formulation” are interchangeably employed throughout the specification.

The present invention provides a feed formulation for white spot syndrome virus (WSSV) infection in crustaceans, comprising of polyclonal IgY antibody formulation and crustacean feed; wherein the polyclonal IgY antibody formulation and crustacean feed are present in the ratio of 1:1000; wherein the polyclonal IgY antibody formulation comprises polyclonal anti-WSSV VP28 IgY antibody as the active pharmaceutical ingredient (API).
In various embodiments of the feed formulation, the polyclonal IgY antibody formulation comprises polyclonal anti-WSSV VP28 IgY antibody and at least two of the excipients comprising sucrose, starch, tween 20 or tween 80; wherein the polyclonal anti-WSSV VP28 IgY antibody and the excipients are in the ratio ranging from 14:1:0.02 to 15:2:3.
In an embodiment of the feed formulation, the polyclonal IgY antibody formulation comprises polyclonal anti-WSSV VP28 IgY antibody, sucrose, and tween 20 or tween 80.
In various embodiments of the feed formulation, the polyclonal anti-WSSV VP28 IgY antibody ranges from 70 to 75%; sucrose ranges from 5 to 10%; tween 20 or tween 80 ranges from 0.1 to 1% in the polyclonal IgY antibody formulation.
In an embodiment of the feed formulation, the polyclonal IgY antibody formulation is in liquid form.
In various embodiments of the feed formulation, the polyclonal IgY antibody formulation is blended along with water in the range of 0.01% to 0.1% (w/v).
In an embodiment of the feed formulation, the polyclonal IgY antibody formulation comprises polyclonal anti-WSSV VP28 IgY antibody, sucrose, and starch.
In various embodiments of the feed formulation, the polyclonal anti-WSSV VP28 IgY antibody ranges from 70 to 75%; sucrose ranges from 5 to 10%; starch ranges from 1 to 15% in the polyclonal IgY antibody formulation.
In an embodiment of the feed formulation, the polyclonal IgY antibody formulation is in solid powder form.
In various embodiments of the feed formulation, the polyclonal IgY antibody formulation is blended along with crustacean feed in the range of 0.001% to 0.01% (w/w).
In various embodiments of the feed formulation, the crustaceans comprise prawns, crabs, lobsters, crayfish, shrimps such as seed shrimp, opossum shrimps, mantis shrimp, preferably shrimps.
In a preferable embodiment of the feed formulation, wherein VP28 is a recombinant protein comprising a sequence of SEQ.ID. NO.2 encoded by a nucleotide sequence comprising a sequence of SEQ.ID.NO. 1.
In a particular embodiment of the feed formulation, the recombinant VP28 protein comprising a sequence of SEQ.ID. NO.2 is expressed in prokaryotic microbial cells using a bacterial expression vector comprising the full-length VP28 nucleotide sequence of SEQ.ID NO. 1.
In various embodiments of the feed formulation, the polyclonal IgY antibody formulation is stable for more than 18 months at 2 - 8ºC.
In various embodiments of the feed formulation, the formulation is stable at salinity up to 25ppt, and at pH ranging from 6 to 8.
In various embodiments of the feed formulation, wherein the crustacean feed comprises animal protein, starch, minerals and vitamins in specific proportions.
In various embodiments of the feed formulation, wherein the crustacean feed is preferably shrimp feed and comprises animal protein, starch, minerals and vitamins in specific proportions.
In various embodiments of the feed formulation, the animal protein of the feed formulation comprises but not limited to fish meat etc. Further it comprises starch, vitamins and minerals; wherein the vitamins comprise fat-soluble vitamins (A, D, E and K) and water-soluble vitamins (B1, B2, B6, B12, C, Biotin, Folacin and Inositol); wherein the minerals comprise macro minerals comprising but not limited to calcium, magnesium, sodium and potassium and micro minerals comprising but not limited to iron, copper, zinc, selenium, manganese.
In various embodiments of the feed formulation, the feed formulation further comprises additives comprising supplements, phyto-compounds, immune-stimulants, probiotics, binders.
In an embodiment of the present disclosure, the presence of polyclonal anti-WSSV VP28 IgY antibody in the digestive tract of shrimps fed with the feed formulation is detected at least after 12 hours of feeding by competitive ELISA against the recombinant VP28 protein.
In an embodiment of the present disclosure, the presence of polyclonal anti-WSSV VP28 IgY antibody in the digestive tract of shrimps fed with the feed formulation was detected at least after 12, 24, 36, and 48 hours of feeding by competitive ELISA against the recombinant VP28 protein.
In an embodiment of the present disclosure, the presence of polyclonal anti-WSSV VP28 IgY antibody in the faecal matter of shrimps was detected after 12 hours of feeding by competitive ELISA against the recombinant VP28 protein.
In an embodiment of the present disclosure, the presence of polyclonal anti-WSSV VP28 IgY antibody in the faecal matter of shrimps was detected after 12, 24, 36, 48, 60, 72, 84, and 96 hours of feeding by competitive ELISA against the recombinant VP28 protein.
In an embodiment of the present disclosure, no WSSV virus was found to be present in the faecal matter of shrimps, injected intramuscularly with WSSV virus incubated with polyclonal anti-WSSV VP28 IgY antibody, by PCR.
In an embodiment of the present disclosure, the polyclonal anti-WSSV VP28 IgY antibody provided vide the feed formulation was stable at different pH levels 6, 7 and 8 even after 24 hours of incubation, confirmed by competitive ELISA against both the recombinant VP28 protein and the WSSV virus itself.
In an embodiment of the present disclosure, the WSSV-specific polyclonal anti-WSSV VP28 IgY antibody (IgY) provided vide the feed formulation was stable even after exposing it to the extracts prepared from the different parts of the digestive tract (fore gut, mid gut and hind gut) for at least 1 hour of incubation, confirmed using competitive ELISA against both the recombinant VP28 protein and the WSSV virus itself.
In an embodiment of the present disclosure, the WSSV-specific polyclonal anti-WSSV VP28 IgY antibody provided vide the feed formulation was stable at different salinities 0, 5, 10, 20, and 25 parts-per-thousand (ppt) even after 1, 3, 5, and 10 days of incubation, confirmed using competitive ELISA against both the recombinant VP28 protein and the WSSV virus itself.
In an embodiment of the present disclosure, the feed formulation was applied to aquatic systems such as open pond environments, which showed no changes in WSSV-specific IgY’s properties when analyzed with samples like shrimps, pond water, and sludge at regular intervals from day 0 till harvest (week 16-17), using competitive ELISA against the recombinant VP28 protein.
Another aspect of the invention discloses a method of preparation of feed formulation comprising the steps of:
? preparation of recombinant VP28 protein by culturing a recombinant prokaryotic cell comprising a bacterial expression vector comprising polynucleotide of SEQ ID. No.1 encoding the recombinant VP28 protein comprising amino acid sequence of SEQ ID. No.2 under a condition that the polynucleotide is expressed, recovering and purifying the recombinant VP28 protein from the recombinant prokaryotic cell or a medium in which the recombinant prokaryotic cell has been cultured;
? immunizing the chicken with a formulation comprising the recombinant VP28 protein and adjuvant;
? isolation and purification of polyclonal anti-VP28 IgY antibodies from yolk of egg of the immunized chicken by de-lipidation and other suitable methods comprising gradient precipitation;
? freeze drying or spray drying of the purified polyclonal anti-VP28 IgY antibodies to form polyclonal IgY antibody formulation;
? optionally blending and homogenization of at least two excipients with purified polyclonal anti-VP28 IgY antibodies to form polyclonal IgY antibody formulation, wherein the excipients comprise sucrose, starch, tween 20, or tween 80, and storing the formulation at 4?.
? mixing of polyclonal IgY antibody formulation with water and/or crustacean feed.
In an embodiment of the method of preparation of feed formulation, the vector for the expression of the recombinant VP28 protein is selected from bacterial expression vector pET28a comprising the polynucleotide encoding the recombinant VP28 protein which is transformed into BL21 (DE3) for synthesizing the coding sequence of WSSV VP28.
In an embodiment of the method of preparation of feed formulation, the chickens were immunized with the purified WSSV VP28 recombinant protein on days 0, 21, 35, 49, 63, 77, and 100 which is emulsified with adjuvants. The adjuvant selected is oil. 50 to 100 µg of purified WSSV VP28 recombinant protein was used per immunization to elicit an immune response. Anti-WSSV VP28 IgY from chicken egg yolk was purified on day 70 (from the initial immunization day) onwards.
In an embodiment, the stability of the freeze-dried polyclonal anti-VP28 IgY antibody was more than 18 months when stored at 2 to 8?.
In an embodiment of the present disclosure, the WSSV-specific polyclonal anti-VP28 IgY antibody raised against the WSSV-specific recombinant protein VP28 exhibits binding capacity even at low concentrations of WSSV from 50 µg - 3.125µg.
In an embodiment of the present disclosure, the WSSV-specific polyclonal anti-VP28 IgY antibody raised against the WSSV-specific recombinant protein VP28 completely neutralizes the WSSV virus when incubated at a concentration starting from 5 µL in the in-vitro condition.
In an embodiment of the present disclosure, the WSSV viral stock incubated with polyclonal anti-VP28 IgY antibody raised against the WSSV-specific recombinant protein VP28 and injected via intramuscular routes into shrimps showed at least 90% survival rate in the 4mg polyclonal anti-VP28 IgY antibody group in the in-vivo neutralization study.
In an embodiment of the present disclosure, the WSSV viral stock incubated with polyclonal anti-VP28 IgY antibody raised against the WSSV-specific recombinant protein VP28 and injected via intramuscular routes into shrimps showed at least 90%, 91%, 92% 93%, or 94% to 99% survival rate in the 4mg polyclonal anti-VP28 IgY group in the in-vivo neutralization study.
In an embodiment of the present disclosure, the WSSV viral stock incubated with polyclonal anti-VP28 IgY antibody raised against the WSSV-specific recombinant protein VP28 and injected via intramuscular routes into shrimps showed at least a 70% survival rate in the 2mg polyclonal anti-VP28 IgY antibody group in the in-vivo neutralization study.
In an embodiment of the present disclosure, the WSSV viral stock incubated with polyclonal anti-VP28 IgY antibody raised against the WSSV-specific recombinant protein VP28 and injected via intramuscular routes into shrimps showed at least 70%, 71%, 72% 73%, or 74% to 99% survival rate in the 4mg polyclonal anti-VP28 IgY antibody group in the in-vivo neutralisation study.
In an embodiment of the present disclosure, a repeated study of the WSSV viral stock with polyclonal anti-VP28 IgY antibody raised against the WSSV-specific recombinant protein VP28 incubated and injected via intramuscular routes showed a 100% survival rate from the 4mg polyclonal anti-VP28 IgY antibody group in the in-vivo neutralization study.
Yet another aspect of the invention discloses a formulation for white spot syndrome virus (WSSV) infection in crustaceans, comprising of polyclonal anti-WSSV VP28 IgY antibody wherein the polyclonal IgY antibody formulation comprises polyclonal anti-WSSV VP28 IgY antibody as the Active Pharmaceutical Ingredient (API).
In various embodiments of the formulation, the polyclonal IgY antibody formulation comprises polyclonal anti-WSSV VP28 IgY antibody and at least two of the excipients comprising sucrose, starch, tween 20 or tween 80;
In various embodiments of the formulation, the polyclonal anti-WSSV VP28 IgY antibody and the two excipients are in the ratio of 14:1:.02 to 15:2:3.
In various embodiments of the formulation, the polyclonal anti-WSSV VP28 IgY antibody ranges from 70 to 75%; sucrose ranges from 5 to 10%; tween 20 or tween 80 ranges from 0.1 to 1%.
In various embodiments of the formulation, the formulation is blended along with water in the range of 0.01% to 0.1% (w/v).
In various embodiments of the formulation, the polyclonal anti-WSSV VP28 IgY antibody ranges from 70 to 75%; sucrose ranges from 5 to 10%; starch ranges from 1 to 15%.
In various embodiments of the formulation, the formulation is blended along with crustacean feed in the range of 0.001% to 0.01% (w/w).
In various embodiments, the formulation is either a powder or a liquid. In various embodiments, the formulation can be either added as a food supplement or as a water supplement that can be employed as a dip solution for shrimps against WSSV.
In various embodiments of the formulation, the formulation contains one or more pharmaceutically acceptable excipients including any suitable stabilizers, preservatives, buffering agents, surfactant, carriers, or fillers, and includes any pharmaceutical agent that does not itself induce the virus neutralization, not harmful to the organism receiving the composition, and that which may be administered without undue toxicity.

It is to be understood that the foregoing description is illustrative not a limitation. While considerable emphasis has been placed herein on features of this disclosure, it will be appreciated that various modifications can be made, and that many changes can be made in the preferred embodiments without departing from the principles of the disclosure. Those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the embodiments as described herein. Similarly, additional embodiments and features of the present disclosure will be apparent to one of ordinary skill in art based upon the description provided herein.

Descriptions of well-known/conventional methods/steps and techniques are omitted so as to not unnecessarily obscure the embodiments herein. Further, the disclosure herein provides examples illustrating the above-described embodiments, and in order to illustrate the embodiments of the present disclosure, certain aspects have been employed. The examples used herein for such illustration are intended merely to facilitate an understanding of ways in which the embodiments may be practiced and to further enable those of skill in the art to practice the embodiments. Accordingly, the following examples should not be construed as limiting the scope of the embodiments herein.

EXAMPLES

Materials
The bacterial vector pET28a and prokaryotic host E.coli BL21 DE3 star were obtained from commercial sources Genscript and Invitrogen respectively. Rest of the chemicals were obtained from Sigma Aldrich. The chicken was sourced from Venkateshwara hatcheries, Chennai, Tamil Nadu. Shrimp was sourced and its related studies were conducted in Aquatic Animal Health Laboratory, C. Abdul Hakeem College, Tamil Nadu. Shrimp feed (CPF Shrimp feed) was obtained from commercial sources, and it preferably comprises animal protein such as fish meat, starch, minerals, vitamins etc.
Example 1
Production of recombinant WSSV-VP28 protein
The full-length WSSV-VP28 protein gene (SEQ.ID.NO.1) was cloned in the correct reading frame in a bacterial expression vector - pET28a (Genscript) (Figure1), for high-level expression of recombinant WSSV-VP28 protein in E. coli. BL21 DE3 star. The right-size recombinant clones were selected for protein expression. Small-scale expression results showed that all the recombinant VP28 clones selected were expressing the target WSSV-VP28 protein of approximately 24 kDa at different levels when analysed by SDS– PAGE whereas in the control sample (without inducer) there was no expression of the target protein (data not shown). The expression of recombinant protein was confirmed by Western blot probed with anti-His6 MAb. The best clone was chosen for medium-scale expression and purification. The WSSV-VP28 recombinant protein was expressed and purified. SDS-PAGE using 12% gel and Western blot analysis using anti-His6 MAb gave a single band of ~24 kDa (Figure 2a & 2b). LPS and HCP contamination in the WSSV proteins were checked and kept minimal to prevent nonspecific reactivity.
Example 2
Development of WSSV immune chicken IgY formulation
Immunization of Chicken:
The WSSV-VP28 recombinant protein was used for immunizing the chicken. The formulation used for immunization contained oil as an adjuvant (Freund’s adjuvants, Sigma Aldrich). Polyclonal anti-WSSV-VP28 IgY antibody (IgY) was obtained from yolk of immunized hen’s eggs. Obtained IgY was purified. The egg yolk was de-lipidated using chloroform and IgY was purified by gradient precipitation using ammonium sulphate. 5 to 10 mg/ml of purified IgY was obtained from each egg.
Freeze drying:
The purified polyclonal anti-WSSV-VP28 IgY antibody (IgY) was subjected to freeze drying/lyophilization, after purification under optimized conditions. Later it was re-suspended in PBS (phosphate buffer saline) buffer according to the required concentration, as per the need.
Example 3
Analysis of WSSV-specific VP28 IgY
The WSSV-specific polyclonal anti-WSSV-VP28 IgY antibody after lyophilization was subjected to analytical analysis. The lyophilized powder was taken and about 10 µg was analyzed in SDS-PAGE with reducing dye. The WSSV-specific polyclonal anti-WSSV-VP28 IgY antibody had the required bands at both the heavy-chain & light chain regions (Figure 3a). These bands were further immunoblotted for confirmation with anti-Chicken IgY HRPO conjugate antibody (Figure 3b).
To demonstrate that the WSSV-specific polyclonal anti-WSSV-VP28 IgY antibody is specific to the WSSV, an antigen sample from various strains of the virus is immunoblotted against WSSV-specific polyclonal anti-WSSV-VP28 IgY antibody. Three different strains of the WSSV virus along with the VP28 recombinant purified protein formed bands when immunoblotted with polyclonal anti-WSSV-VP28 IgY antibody (Figure 4a). To further analyze the effectiveness of the binding, the WSSV-infected shrimp lysate was taken and the total protein concentration loaded to the SDS gels was varied and immunoblotted. It is found that the polyclonal anti-WSSV-VP28 IgY antibody formed bands with various high to low concentrations (50 – 3.125µg) of the total protein proving its effectiveness in binding to the WSSV virus even at lower concentrations (Figure 4b). The results proved that polyclonal anti-WSSV-VP28 IgY antibody’s specificity in binding to the WSSV antigen.
Example 4
In-vitro neutralization of WSSV by IgY raised against recombinant protein WSSV-VP28
Viral Neutralisation
The WSSV viral stock was prepared to a working concentration in phosphate buffer followed by dilutions of the polyclonal anti-WSSV-VP28 IgY antibody raised against WSSV-specific recombinant protein WSSV-VP28 (2.5, 5 and 10µg) in the same buffer. The WSSV viral solution was mixed with equal volumes of each of the diluted polyclonal anti-WSSV-VP28 IgY antibody solution after which the mixture was incubated for 1-3 hours at 37? to form virus-polyclonal anti-WSSV-VP28 IgY antibody mixtures. For control purposes, the virus without neutralization was also incubated alongside.
Competitive ELISA:
ELISA plates were coated with VP28 recombinant protein beforehand. Blocking of non-specific binding sites was done with skimmed milk. The pre-incubated virus- polyclonal anti-WSSV-VP28 IgY antibody mixture was added to the designated wells and incubated. The detection of the virus- polyclonal anti-WSSV-VP28 IgY antibody complex was done by anti-chicken IgY antibody to measure the absorbance (Optical density at 550nm). The percentage of inhibition was calculated and plotted on a graph which clearly shows that the neutralisation of polyclonal anti-WSSV-VP28 IgY antibody with WSSV prevented the polyclonal anti-WSSV-VP28 IgY antibody from binding to the WSSV specific recombinant protein VP28 (Figure 5). The WSSV was completely neutralized by polyclonal anti-WSSV-VP28 IgY antibody at the concentration of 5 µg onwards.
Example 5
In-vivo neutralization of WSSV by IgY raised against recombinant protein WSSV-VP28 in shrimps
A high titer WSSV viral stock without dilution was mixed with 2mg and 4mg of polyclonal anti-WSSV-VP28 IgY antibody (IgY), which was incubated for 1-3 hours. The mix was inoculated through intramuscular routes into the disease free/healthy shrimps. The control groups received the viral dose without polyclonal anti-WSSV-VP28 IgY antibody for comparison. The shrimps were released into separate tanks to avoid cross-contamination. The survival and symptoms like signs of infection, and abnormal behaviours were also monitored. More than 90% survival was observed in 4mg polyclonal anti-WSSV-VP28 IgY antibody group and more than 70% survival was observed in 2mg polyclonal anti-WSSV-VP28 IgY antibody group (Table 1). This proves that the WSSV-specific polyclonal anti-WSSV-VP28 IgY antibody neutralises the virus efficiently even in the direct challenge by intramuscular route. The comparison graph of the study to better understand the perspective was done (Figure 6a). The results indicated significant survivability of shrimp which is equivalent to that of negative control group.

Table 1: Survival percentage of shrimps post infection with neutralised IgY
Experiment Percentage of survival of shrimp at different days of post infection of WSSV (%)
1 2 3 4 5 6 7
Negative control 100 100 100 100 100 100 100
Positive control 100 67 34 0 0 0 0
WSSV +IgY (2 mg) 100 100 100 100 87.9 73.3 73.3
WSSV +IgY (4 mg) 100 100 100 100 100 100 92
IgY alone (4 mg) 100 100 100 100 100 100 100

The above experiment was repeated four more times to calculate the average survival of shrimp injected with WSSV (>108 copy numbers of the virus per ml) mixed with polyclonal anti-WSSV-VP28 IgY antibody raised against WSSV specific recombinant protein VP28 at different time intervals (Table 2). Totally 15 shrimps were taken in each of the groups and the average survival values were based on four trials (Figure 6b). This repeated study further proved that the WSSV specific polyclonal anti-WSSV-VP28 IgY antibody (IgY) formulation neutralized 100% virus when 4 mg of the IgY was mixed with 1 ml of WSSV infected shrimp lysate containing >108 copy numbers of the virus per ml in the lysate.
Table 2: Survival percentage of shrimps post infection with neutralised IgY in 4 consecutive studies
Experiment Average percentage of survival of shrimp at different days of post-infection with WSSV in 4 consecutive studies (%)
1 2 3 4
Negative control 100 100 100 100
Positive control 100 22.5 0 0
WSSV +IgY (1 mg) 100 98 73.3 46
WSSV +IgY (2 mg) 100 100 86.5 62
WSSV +IgY (4 mg) 100 100 100 100
WSSV +IgY (8 mg) 100 100 100 100
IgY alone (4000 µg) 100 100 100 100

Example 6
Preparation of polyclonal anti-WSSV-VP28 IgY antibody formulation and feed formulation comprising the same.
A formulation of polyclonal anti-WSSV-VP28 IgY antibody specific to WSSV in shrimp ponds were made considering factors such as stability, efficacy, and its ease of practical application. Based on these factors, the following two formulations were made.
Liquid Formulation
The ingredients of the formulation along with its composition is given in Table 3.
Table 3: Liquid Formulation of polyclonal anti-WSSV-VP28 IgY antibody.
S.No Raw Materials / Ingredients Quantity
1 Polyclonal anti-WSSV-VP28 IgY antibody (freeze dried/spray dried/liquid) 5 mg/ml
2 Sucrose 5% w/v
3 Tween 20 or 80 0.1% v/v
4 Phosphate-Buffered Saline (PBS) (Buffer) -

The required polyclonal anti-WSSV-VP28 IgY antibody powder was reconstituted in PBS, to which sucrose was added and mixed gently. Tween 20 or 80 was also added to the mixture for improved stability. The final volume was made up with PBS, mixed well, and filter sterilized to obtain a sterile and homogenous liquid formulation. Said liquid polyclonal anti-WSSV-VP28 IgY antibody formulation was stored at 4?. The liquid formulation was sprayed onto or mixed with the shrimp feed in the ratio of 1:1000, to ensure proper and even distribution of the formulation. Additionally, the polyclonal anti-WSSV-VP28 IgY antibody liquid formulation was generally prepared at 0.01% to 0.1% (w/v) in water and was employed as dip solution for the shrimps. The dosage of polyclonal anti-WSSV-VP28 IgY antibody to be administered depends on multiple factors such as shrimp size, health status, and intended objective.
Powder Formulation
The ingredients for the formulation along with its composition is given in Table 4.
Table 4: Powder Formulation of polyclonal anti-WSSV-VP28 IgY antibody
S.No Raw Materials / Ingredients Quantity
1 Polyclonal anti-WSSV-VP28 IgY antibody (freeze dried/spray dried) 5 mg/100 mg
2 Sucrose 5% w/w
3 Starch 1 - 15% w/w

The freeze dried/spray dried polyclonal anti-WSSV-VP28 IgY antibody was mixed with sucrose gently to obtain an even mixture. The polyclonal anti-WSSV-VP28 IgY antibody powder mixture was then mixed with the carrier substance, starch. A thorough and proper mixing was done to achieve a consistent and homogenous powder. The formulation was immediately packed in containers, sealed and stored at 4?. The powder formulation was mixed with the shrimp feed at the required ratios and even distribution was ensured to facilitate consistent intake by the shrimps. The polyclonal anti-WSSV-VP28 IgY antibody powder formulation was generally coated with the shrimp feed at a ratio of 1:1000 using appropriate binder, wherein the amount of polyclonal anti-WSSV-VP28 IgY antibody ranges from 0.001% to 0.01% (w/w) to form the feed formulation. The dosage of polyclonal anti-WSSV-VP28 IgY antibody employed depends on factors such as shrimp size, health status, and intended objective.
Example 7
Presence of WSSV specific IgY in shrimp after feeding
Digestive Tract of Shrimp
The shrimps (healthy & disease free) were fed with feed formulation comprising polyclonal anti-WSSV-VP28 IgY antibody (30mg/g of shrimp feed) and the presence of IgY in the digestive tract (the gut and its contents) were examined at different periods post-feeding for a total of four trials. The different time intervals chosen for this study were 12, 24, 36, and 48 hours. At these particular intervals, shrimps were taken and sacrificed to dissect their digestive tract. Fore gut, mid gut, and hind gut and their contents were then homogenized to extract any IgY present in the tissues and a competitive ELISA was performed against the recombinant VP28 protein. A positive control (polyclonal anti-WSSV-VP28 IgY antibody), negative control (buffer) and the feed mixed with polyclonal anti-WSSV-VP28 IgY antibody sample were all taken into consideration for comparison of the efficiency of the WSSV specific IgY test sample. The presence of WSSV specific IgY in shrimps were analysed by the competitive ELISA. The results confirmed the presence of WSSV-specific IgY in the digestive tract and its contents of shrimps fed with feed formulation comprising polyclonal anti-WSSV-VP28 IgY antibody (Figure 7).
Faecal Tract of Shrimp
The shrimps (healthy & disease free) were fed with feed formulation comprising polyclonal anti-WSSV-VP28 IgY antibody (30mg/g of shrimp feed) and the presence of IgY in the faecal matter was examined at different periods post-feeding. The different time intervals chosen for this study was 12, 24, 36, 48, 60, 72, 84, and 96 hours. At these particular intervals, fresh faecal samples were collected and homogenised in a buffer solution (PBS) for analysis. A competitive ELISA was performed against the recombinant VP28 protein to find the presence of WSSV specific IgY. A positive control (WSSV specific IgY), negative control (buffer) and the feed mixed with WSSV specific IgY sample were all taken into consideration for comparison of the efficiency of the WSSV specific IgY test sample. The presence of WSSV-specific IgY in shrimps were analyzed by the competitive ELISA. The results confirmed the presence of WSSV-specific IgY in the faecal matter after 12 hours of post-feeding of feed formulation comprising polyclonal anti-WSSV-VP28 IgY antibody which gradually increased and reached a highest concentration at 84 hours post-feeding and found till 96 hours(Figure 8).
Example 8
Absence of WSSV in faecal matter of shrimps injected with IgY treated WSSV
The shrimps (healthy & disease free) were injected with polyclonal anti-WSSV-VP28 IgY antibody treated WSSV and with untreated WSSV. After 24 hours the fresh faecal matter from the shrimps were collected and homogenised in a buffer solution (PBS) for analysis. Extraction of DNA from the homogenised faecal sample was done. Using WSSV specific primers, PCR analysis was done to amplify the WSSV DNA if present. The PCR products were then analysed on agarose gel (Figure 9). The study additionally proved that polyclonal anti-WSSV-VP28 IgY antibody neutralized WSSV efficiently and no trace amounts of WSSV were found.
Example 9
Stability studies of IgY
Stability of IgY formulation in 2 – 8 ?
The anti-WSSV specific polyclonal anti-WSSV-VP28 IgY antibody formulation was stored at 2 – 8 ?, and at continuous intervals, the formulation was tested for its stability using competitive ELISA with VP28 recombinant protein. The ELISA graph shows that the anti-WSSV specific polyclonal anti-WSSV-VP28 IgY antibody formulation was stable for about 720 days (2 years) (Figure 10). Thus, it provides us with a conclusion that the WSSV specific polyclonal anti-WSSV-VP28 IgY antibody formulation can be stored at 2 – 8 ? for more than a year before mixing it with the feed.
Stability of IgY formulation at different pH levels
The anti-WSSV specific polyclonal anti-WSSV-VP28 IgY antibody formulation was incubated for 24 hours in different pH (6, 7, and 8) buffers. After the incubation, the exposed IgY antibody was analysed using competitive ELISA with both WSSV virus and VP28 recombinant protein. The ELISA graph shows no difference in the binding efficiency with WSSV and VP28 recombinant protein between the exposed and unexposed polyclonal anti-WSSV-VP28 IgY antibody formulation (Figure 11).
Stability of IgY formulation exposed to extracts prepared from the different parts of digestive tract
The anti-WSSV specific polyclonal anti-WSSV-VP28 IgY antibody was incubated with extracts prepared form the different parts of the digestive tract of shrimp (Fore gut, Mid gut and Hind gut) for 1 hr. After the incubation, the exposed IgY antibody was analysed using competitive ELISA with both WSSV virus and VP28 recombinant protein. The ELISA graph showed no difference in the binding efficiency with WSSV and VP28 recombinant protein between the exposed and unexposed IgY antibodies (Figure 12).
Stability of IgY at different salinities (0, 5, 10, 20, and 25 ppt)
The anti-WSSV specific polyclonal anti-WSSV-VP28 IgY antibody formulation was exposed to different salinities (0, 5, 10, 20 and 25ppt) at different time intervals (1, 3, 5 and 10 days of post incubation). After the incubation, the exposed IgY antibody was analyzed using competitive ELISA with both WSSV virus and VP28 recombinant protein. The ELISA graph shows no difference in the binding efficiency with WSSV and VP28 recombinant protein between the exposed and unexposed IgY antibodies (Figure 13).
Example 10
Evaluating feed formulation comprising IgY formulation in open pond environments
An open pond was selected for the study to evaluate the WSSV-specific polyclonal anti-WSSV-VP28 IgY antibody formulation on shrimps for protection against WSSV infection. The WSSV-specific polyclonal anti-WSSV-VP28 IgY antibody formulation was mixed with the shrimp feed (CPF Shrimp feed) like a supplement and was fed to shrimps in an open pond environment from day 0 till harvest (usually 16-17 weeks). The dosage of polyclonal anti-WSSV-VP28 IgY antibody formulation was at 0.001% of the shrimp feed with a dose interval of 3 days per week. Samples were regularly collected at weekly intervals such as shrimps, pond water and sludge. All the samples were analyzed by competitive ELISA method to detect WSSV-specific IgY presence in them. The data from the harvest week’s samples against competitive ELISA with both WSSV virus and VP28 recombinant protein are provided (Figure 14). The study’s results proved the presence of WSSV-specific WSSV-specific polyclonal anti-WSSV-VP28 IgY in all the samples revealing the stable nature of the polyclonal anti-WSSV-VP28 IgY antibody formulation in an open pond environment when fed as a feed formulation. Further the polyclonal anti-WSSV-VP28 IgY antibody formulation when fed as a feed formulation offers protection against WSSV infection to shrimps and increases their survival rate.
Additional embodiments and features of the present disclosure will be apparent to one of ordinary skill in art based on the description provided herein. The embodiments herein provide various features and advantageous details thereof in the description. Descriptions of well-known/conventional methods and techniques are omitted so as to not unnecessarily obscure the embodiments herein.

The foregoing description of the specific embodiments reveal the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore, while the embodiments in this disclosure have been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the embodiments as described herein.

Throughout this specification, the term ‘combinations thereof’ or ‘any combination thereof’ or ‘any combinations thereof’ are used interchangeably and are intended to have the same meaning, as regularly known in the field of patents disclosures.

While considerable emphasis has been placed herein on the particular features of this disclosure, it will be appreciated that various modifications can be made, and that many changes can be made in the preferred embodiments without departing from the principles of the disclosure. These and other modifications in the nature of the disclosure or the preferred embodiments will be apparent to those skilled in the art from the disclosure herein, whereby it is to be distinctly understood that the foregoing descriptive matter is to be interpreted merely as illustrative of the disclosure and not as a limitation.

Advantages:

The claimed feed formulation confers protection to crustaceans, shrimps against WSSV infection. The feed formulation is stable, easy to apply, cost-effective and confers protection by neutralizing the WSSV in the initial stage of viral infection, which is the direct exposure of crustacean body surfaces or oral exposure of infection in guts to virus particles in the water bodies. The feed formulation and the antibody present in it are stable, can withstand changes in pH and salinity and are not affected by the gut contents of crustaceans. The feed formulation was prepared to overcome the larger area of the water bodies and other limitations such as salinity and pH variations so that the antibody in the feed formulation remains stable and is available for the shrimp’s body surfaces and gut organelles to impart its neutralizing effect against WSSV infection. Thus, the claimed feed formulation confers protection against the WSSV virus infection and increases the survival rate of crustaceans thereby helping their farming, cultivation and economy. ,CLAIMS:1. A feed formulation for white spot syndrome virus (WSSV) infection in crustaceans, comprising of polyclonal IgY antibody formulation and crustacean feed; wherein the polyclonal IgY antibody formulation and crustacean feed are present in the ratio of 1:1000; wherein the polyclonal IgY antibody formulation comprises polyclonal anti-WSSV VP28 IgY antibody as the active pharmaceutical ingredient (API).
2. The feed formulation as claimed in claim 1, wherein the polyclonal IgY antibody formulation is in liquid form.
3. The feed formulation as claimed in claims 1 and 2, wherein the polyclonal IgY antibody formulation is blended with water in the range of 0.01% to 0.1% (w/v).
4. The feed formulation as claimed in claim 1, wherein the polyclonal IgY antibody formulation is in powder form.
5. The feed formulation as claimed in claim 4, wherein the polyclonal IgY antibody formulation is blended with crustacean feed in the range of 0.001% to 0.01% (w/w).
6. The feed formulation as claimed in claim 1, wherein the crustaceans comprise prawns, crabs, lobsters, crayfish, shrimps such as seed shrimp, opossum shrimps, mantis shrimp, preferably shrimp.
7. The feed formulation as claimed in claim 1, wherein VP28 is a recombinant protein comprising a sequence of SEQ.ID. NO.2 encoded by a nucleotide sequence comprising a sequence of SEQ.ID.NO. 1.
8. The feed formulation as claimed in claim 1, wherein recombinant VP28 protein comprising a sequence of SEQ.ID. NO.2 is expressed in prokaryotic microbial cells using a bacterial expression vector comprising the full-length VP28 nucleotide sequence of SEQ.ID NO. 1.
9. The feed formulation as claimed in claim 1, wherein the polyclonal IgY antibody formulation is stable for more than 18 months at 2 - 8ºC.
10. The feed formulation as claimed in claim 1, wherein the formulation is stable at salinity upto 25ppt, and at pH ranging from 6 to 8.
11. The feed formulation as claimed in claim 1, wherein the crustacean feed comprises of animal protein, starch, minerals and vitamins.
12. The feed formulation as claimed in claims 1-11, further comprises additives comprising supplements, phyto-compounds, immune-stimulants, probiotics, binders.
13. A method of preparation of feed formulation as claimed in claim 1 comprising the steps of:
? preparation of recombinant VP28 protein by culturing a recombinant prokaryotic cell comprising a bacterial expression vector comprising polynucleotide of SEQ ID. No.1 encoding the recombinant VP28 protein comprising amino acid sequence of SEQ ID. No.2 under a condition that the polynucleotide is expressed, recovering and purifying the recombinant VP28 protein from the recombinant prokaryotic cell or a medium in which the recombinant prokaryotic cell has been cultured;
? immunizing the chicken with a formulation comprising the recombinant VP28 protein and adjuvant;
? isolation and purification of polyclonal anti-VP28 IgY antibodies from yolk of egg of the immunized chicken by de-lipidation and other suitable methods;
? freeze drying or spray drying of the purified polyclonal anti-VP28 IgY antibodies to form polyclonal IgY antibody formulation;
? mixing of polyclonal IgY antibody formulation with water and/or crustacean feed.
14. A formulation for white spot syndrome virus (WSSV) infection in crustaceans, comprising of polyclonal anti-WSSV VP28 IgY antibody, wherein the polyclonal IgY antibody formulation comprises polyclonal anti-WSSV VP28 IgY antibody as the active pharmaceutical ingredient.
15. The formulation as claimed in claim 14, wherein the formulation is blended with water in the range of 0.01% to 0.1% (w/v).
16. The formulation as claimed in claim 14, wherein the formulation is blended with crustacean feed in the range of 0.001% to 0.01% (w/w).