FIELD OF THE INVENTION
The purpose of this invention is to develop a synergistic formulation comprising commercially available natural antimicrobial compounds against Early Mortality Syndrome (EMS) causing bacterial pathogen Vibrio parahaemolyticus. Although quite a few phytochemical compounds have already been reported for their antimicrobial activity against several bacterial pathogens, their efficacy at the commercial level is yet to be standardized. Hence, the present invention is indented to evaluate the synergistic activity of certain phytochemicals and their effective formulations with the viable cost to control the virulence of EMS-specific V. parahaemolyticus infections in shrimpaquaculture.
BACKGROUND FOR THE INVENTION
Cultivation of Litopenaeus monodon and L. vannamei is becoming the preferable choice of aquaculture worldwide. Despite the apparent success, in terms of production and economy, currently, this sector is experiencing a steep decrease in the rate of production and heavy economic loss worldwide due to the outbreaks of microbial diseases. In the year 2012, Ruwandeepika et al. reported that the bacterial infectious diseases commonly ensue at all stages of the shrimp life cycle and confer severe economic losses. For instance, in the year 2015, Zorriehzahra and Banaederakhshan stated that the shrimp farming is most commonly affected by bacterial diseases especially EMS which was also known as "Acute Hepatopancreatic Necrosis Disease" (AHPND). It is an emerging and most devastating disease caused by a bacterial pathogen, which most commonly causes infection on farmed penaeid shrimp. EMS typically targets the shrimp between the age group of 20 to 40 days old post-larvae and results in 100% mortality within a week. The syndrome is more often fatal to shrimp rather than other aquatic animals. Though various technologies were well standardized for control of bacterial diseases, the control of EMS in early stages of shrimp larvae still remains unexplored.

In this fact, many researchers described that a wide range of antibiotics is being used to treat established bacterial infections. Although control of bacterial diseases in aquaculture has been partially achieved through the administration of antibiotics, the indiscriminate use of antibiotics has resulted in the widespread occurrence of antibiotic-resistant bacteria in the aquatic environment and posing severe threats to both aquatic animals and human health (Cabello, 2006). Therefore, the recent research interest in disease management has been focused towards finding alternatives to antibiotics, which are more effective and environment-friendly. Thus, a novel alternative control strategy is highly essential to combat EMS.
The use of natural compounds in combination is a robust emerging strategy to reduce microbial infections and to potentiate the biological activity of each other. Al-Yaqout et al. (2014) have patented an anesthetic and therapeutic composition of thyme oil against aquatic bacterial pathogens (Patent No: US 2014/0044812 Al). Based on these facts, the current study is aimed to formulate an effective synergistic formulation with an enhanced antimicrobial activity against EMS specific V. parahaemolyticus using non-antibiotic phytochemical compounds.
SUMMARY OF THE INVENTION
The present invention relates to finding suitable formulations to control the devastating disease viz., EMS, caused by V. parahaemolyticus in shrimp especially in the cultures of L. vannamei, which ruins the shrimp aquaculture industry worldwide.
In the present invention, the antimicrobial potential of various commercially available phytotherapeutic compounds was evaluated against EMS disease. Further, in order to enhance their antimicrobial activity, a combinatorial approach was followed to formulate effective formulations with environmental friendly and economically viable qualities. Further a formulated feed comprising the present synnergistic formulation was developed.
Thus, 20 different commercially available compounds such as catechin, eugenol, furfural, hexadecanoic acid, curcumin, geraniol, quercitin, menthol, pyrogallol, vanillin, tannic acid, citral, mangiferin, morin, naringin, rosmarinic acid, linalool, phytol,

cinnamaldehyde, and crystal violet were purchased from Sigma-Aldrich, St. Louis, MO, USA and were screened against the growth of V. parahaemolyticus. Among these, eight different compounds such as citral, curcumin, geraniol, pyrogallol, quercetin, tannic acid, vanillin, and crystal violet showed killing effect at their minimal concentrations against V. parahaemolyticus. Further, to evaluate the combinatorial effect of all these known antimicrobial compounds, a total of 28 different combinations were designed to investigate their synergistic activity. Among the 28 different combinations, eight combinations showed synergistic effect such as curcumin + tannic acid, tannic acid + geraniol, curcumin + geraniol, curcumin + quercetin, geraniol + quercetin, pyrogallol + vanillin, curcumin + menthol and menthol + citral against the growth of V. parahaemolyticus. Out of eight combinations tested, pyrogallol + vanillin effectively inhibited the growth of V. parahaemolyticus. Then the third compound crystal violet with higher synergistic score was decided to added with pyrogallol + vanillin combination in order to further enhance the activity.
BRIEF DESCRIPTION OF THE DRAWINGS
The foregoing summary, as well as the following detailed description of the invention will be better understood when read in conjunction with the appended drawings. For the purpose of assisting in the explanation of the invention, there are shown in the drawings embodiments which are presently preferred and considered illustrative. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities shown therein.
Figure 1: Shows synergistic antimicrobial analysis of pyrogallol and vanillin against V. parahaemolyticus. Green highlighted box represent the higher synergistic concentration.
Figure 2: Shows effect of test formulation on the growth of V. parahaemolyticus (MIC -Pyrogallol+ Vanillin + CV - (25+50+2) jig/mL).

Figure 3: Shows effect of test formulation on preformed biofilm of V. parahaemolyticus (MIC - Pyrogallol+ Vanillin + CV - (25+50+2) jig/mL).
Figure 4: Shows microscopic analyses. A) Light microscopic analysis B) CLSM (MIC -Pyrogallol + Vanillin + CV - (25+50+2) jig/mL).
Figure 5: Shows effect of test formulation on swarming motility of V. parahaemolyticus (MIC-Pyrogallol+ Vanillin + CV - (25+50+2) jig/mL).
Figure 6: Shows toxicity study of Pyrogallol, Vanillin and CV alone and in combination using the L. vannamei
Figure 7: Shows experimental stage of L. vannamei (In this study, the in vivo experiments were performed using the animals stages of PL-40 and size 4.5 cm ±0.26).
Figure 8: Shows In vivo challenging study for test formulation (Pyrogallol + Vanillin + CV) against V. parahaemolyticus infection on L. vannamei
Figure 9: Shows impact of test formulation on the bacterial colonization in shrimp Hepatopancreas (HP)
Figure 10: In field trial analysis. Impact of test formulation on the bacterial count in pond water.
DETAILED DESCRIPTION OF THE INVENTION
For the purposes of the following detailed description, it is to be understood that the invention may assume various alternative variations and step sequences, except where expressly specified to the contrary. Moreover, other than in any operating examples, or

where otherwise indicated, all numbers expressing, for example, quantities of ingredients used in the specification are to be understood as being modified in all instances by the term "about". It is noted that, unless otherwise stated, all percentages given in this specification and appended claims refer to percentages by weight of the total composition.
Thus, before describing the present invention in detail, it is to be understood that this invention is not limited to particularly exemplified systems or process parameters that may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to limit the scope of the invention in any manner.
The use of examples anywhere in this specification including examples of any terms discussed herein is illustrative only, and in no way limits the scope and meaning of the invention or of any exemplified term. Likewise, the invention is not limited to various embodiments given in this specification.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the case of conflict, the present document, including definitions will control.
It must be noted that, as used in this specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, a reference to a "solvent" may include two or more such solvents. The terms "preferred" and "preferably" refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the invention As used herein, the terms "comprising" "including," "having,"

"containing," "involving," and the like are to be understood to be open-ended, i.e., to mean including but not limited to.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains.
The term "EMS" as used herein refers to a new emerging disease in shrimp, first reported in 2009, was initially named early mortality syndrome (EMS).
The expression "Combinatorial therapy" as used herein refers to the use of combinations of commercially available phytochemical compounds and antibiotics as therapeutic agents to microbial infections.
The term "Vibrios" as used herein refers to the major disease-causing bacterial pathogen and are ubiquitous in the marine environment.
The term "Antibiotics" as used herein refers to the chemical substances which kill or inhibit the growth of bacteria by interfering with the cell wall, nucleic acid, protein synthesis and other enzymatic pathways.
The term "Phytochemicals" as used herein refers to the biologically active chemical substances extracted from natural resources.
The term "Virulence" as used herein refers to the ability of an agent of infection to produce disease. The virulence of a microorganism is a measure of the severity of the disease caused by them.
The term "Pathogens" as used herein refers to either bacteria, fungi or virus which cause the infectious disease to the host cell.

The term "Biofilms" as used herein refers to a self-developed hydrated matrix made up of microbial cells and biomolecules such as polysaccharides, proteins, nucleic acids, and lipids.
The term "MIC" as used herein refers to the Minimum Inhibitory Concentration (MIC) and defined as the lowest concentration of phytochemicals, alone or in combination with other compounds, which visibly inhibits the growth of bacteria.
The term "FIC" as used herein refers to the Fractional Inhibitory Concentration. The degree of synergy between natural phytochemical drugs is often expressed in terms of the FIC.
The FIC index (FIG) was calculated using the following formula: FIC index = FICA + FICB FICA = [A] / MICA; FICB = [B] / MICB
Here [A] and [B] represents the MIC of compound A and B in combination, respectively. The FIC index obtained was interpreted as follows: <0.5 denoting synergy; 0.5-0.75 denoting partial synergy; 0.76-1 denoting an additive effect; 1-4 denoting indifference; and >4 denoting antagonism.
Problem to be solved by the Invention
As discussed under the background of the invention, the problem identified in the present invention is the lack of an eco-friendly , economical and natural formulation in effectively treating the devastating disease EMS caused by V. parahaemolyticus in shrimp.
The objective of the present invention

The present invention is intended to provide an alternate non-antibiotic Combinatorial/Synergistic antimicrobial phytochemical formulations against V. parahaemolyticus causing EMS in shrimp.
Solution provided
It was surprisingly observed by the present inventors that the synergetic formulation according to the present invention satisfies the need in the field. Particularly, the present synergetic formulation was found to be highly effective in reducing or eliminating EMS caused by V. parahaemolyticus in shrimp. Thus a synergetic formulation comprising pyrogallol+ vanillin + crystal violet in a concentration of about 0.625 |ig/mL to about 5ug/mL : about 6.25 to about 50ug/mL: about 0.05 to about 0.4ug/mL was achieved.
The present formulation is found to be economical, user friendly, and highly effective with no side effects. In aquaculture, this considered to be a good tool for targeting bacterial challenges and act as a good alternative to antibiotic usage. Especially in critical phases such as disease occurrence, it exhibits its potential in treating the infections and thereby decreases the mortality rates. Under standard conditions, it protects the shrimp health, shortens the time to harvest and therefore increases the productivity. Most interestingly, the application this formulation will prevent the selectve pressure for the development of antibiotic resistance among aquatic microbial pathogens and bioaccumulation of antibiotic residues in the edible tissues of cultured animals. Hence, it is worth to mention that this formulation not only enhances the survival of the shrimp, also it promotes the productivity and quality of the shrimp.
It was studied by the present inventors that when phytochemicals compounds are combined in the manner disclosed herein the description provided enhanced efficacy compared to the same compounds used in single. It is a novel approach, which totally ruled out the utilization of antibiotics, it reduces the production costs and enhances the growth and quality of the shrimp. It is interesting to note that it is highly stable over the years, has

long losting activity, doesnot require any storage specifications and more ecofriendly in nature
The embodiment of the present invention comprises a synergistic formulation comprising
pyrogallol, vanillin and crystal violet.
The formulation is an anti microbial formulation with an anti vibriosis activity.
The anti microbial activity is particularly against V. parahaemolyticus.
The formulation is for the control of shrimp disease.
The formulation comprises pyrogallol, vanillin and crystal violet in a weight ratio of about
3.13:31.25:0.25 to about 25:250:2
The formulation comprises of about 0.625 |ig/mL to about 5ug/mL of pyrogallol.
The formulation comprises of about 6.25 |ig/mL to about 50ug/mL of vanillin
The formulation comprises of about 0.05 |ig/mL to about 0.4 |ig/mL of crystal violet.
The formulation is for the control of shrimp disease Early Mortality Syndrome [EMS].
The minimum inhibitory concentration of the formualtion has a microbial inhibitory
activity of about 55 % to about 65%.
The minimum inhibitory concentration of the formulation showed a shrimp survival rate of
about 70% to about 80%.
Accordingly, the first aspect of the present invention relates to the screening of about 20 different commercially available phytochemical compounds against the growth of V.parahaemolyticus. These compounds include catechin, eugenol, furfural, hexadecanoic acid, curcumin, geraniol, quercetin, pyrogallol, vanillin, tannic acid, citral, mangiferin, morin, naringin, rosmarinic acid, 2,6-ditert-butyl-4-methylphenol, linalool, phytol, cinnamaldehyde, and crystal violet. From the above list of compounds, 8 compounds were found to have high killing effect of V.parahaemolyticus at its minimal concentration. These 8 compounds identified were citral, curcumin, geraniol, pyrogallol, quercetin, tannic acid, vanillin, and crystal violet (Table-1). Hence, the compounds as it alone and in combinations were taken for further experimental analysis.

In the second aspect of the invention, to evaluate the combinatorial effect of the 8 selected compounds, 28 different combinations were formulated and tested as mentioned in Table -2. Among the 28 combinations tested, 8 combinations showed synergistic anti microbial activity such as curcumin + tannic acid, tannic acid + geraniol, curcumin + geraniol, curcumin + quercetin, geraniol + quercetin, pyrogallol + vanillin, curcumin + menthol and menthol + citral against the growth of V. parahaemolyticus (Table-2).
In the third aspect of the invention, the highly effective synergistic anti V. parahaemolyticus combination at 50, and 100 |ig/mL concentration was selected comprising Pyrogallol and vanillin as shown in Figure 1.
Pyrogallol (A) + Vanillin (B) = (50 + 100) [ig/mh
MIC A = 200 ^g/mL
MIC B = 800 ^g/mL
FIC index = FICA + FICB
FIC A =50/200 = 0.25
FICB = 100/800 = 0.125
FIC index = 0.25 + 0.125 = 0.375
0.375 < 0.5 - Synergism
In the fourth aspect of the present inevntion pyrogallol and vanillin is combined with a third compound - crystal violet with higher synergistic score in order to further enhance the activity which effectively inhibited the growth of V.parahaemolyticus. Hence, 2 |ig/mL concentration of Crystal violet was added, which surprisingly reduced the concentration of pyrogallol + vanillin upto 50% (Table 3). Hence, the MIC of the formulation (pyrogallol + vanillin+CV) is 25 + 50 + 2 |ig/mL (Figure 2).
The active formulation - pyrogallol + vanillin + crystal violet at MIC and 2xMIC was effectively eradicated the preformed biofilm of V. parahaemolyticus to the level of 60% and 87%, respectively (Figure 3).

The following examples, without limiting the scope of the claimed invention, illustrate the composition, its physical characteristics and its synergy compared to the conventional dust suppressant composition.
EXPERIMENTAL DETAILS
General Information and Materials: All the ingredients of the present composition were procured from the commercial suppliers as shown below:
Pyrogallol - Sisco Research Laboratories Pvt. Ltd.(SRL) (India)
Vanillin - Rankem & Kemphasol (Mumbai)
Crystal violet - Sisco Research Laboratories Pvt. Ltd.(SRL) (India)
EXAMPLE 1
Screening of phytochemicals for its antimicrobial efficacy against V.
parahaemolyticus (Determination of MIC)
Initially, the antimicrobial activity at their MIC value of phytochemicals against V. parahaemolyticus was determined by broth dilution method as described by the Clinical and Laboratory Standards Institute (CLSI, 2006). In the experiment, the assays were performed in a 24-well microtitre plate (MTP) with 1 ml of LB broth in each well supplemented with phytochemicals in two-fold increased concentration ranging between 25 - 1280 ug/mL. Then, 1% cell suspension (1 x 106 cells/mL) of V. parahaemolyticus was inoculated and incubated for 24 h at 30°C. Behind incubation time, the optical density (OD) of the grown culture was measured at 600 nm. Here, the lowest concentration of compounds which showing the complete growth inhibition of the bacteria was considered as MIC value(s). The results are shown in Table -1.
Table-1 List of commercially available phytochemicals screened for antimicrobial potential against V. parahaemolyticus

Results: Among these, eight different compounds such as citral, curcumin, geraniol, menthol, pyrogallol, quercetin, tannic acid, and vanillin showed efficient killing activity at their minimal concentrations against V. parahaemolyticus (Table 1). Hence, the compounds as it alone and in combinations were taken for further experimental analysis.

EXAMPLE: 2
Evaluation of synergistic antimicrobial potential of phytochemicals against V.
parahaemolyticus by checkerboard method.
The degree of synergy between phytochemicals is often expressed in terms of the fractional inhibitory concentration (FIC). Notably, FIC value is calculated from the value of MIC in combination was divided by MIC at the individual. A checkerboard technique was used to examine the synergism between two active compounds. In this method, MIC was determined, for the test compounds in their paired combinations (Isenberg 2007) in two¬fold increased concentrations up to the MIC of alone. Then 1% cell suspension (106 cells/mL) of V. parahaemolyticus was inoculated and incubated for 24 h at 30°C. Afterwards, the OD of the grown culture was measured at 600 nm. The lowest concentration at which no visible growth occurred was recorded to be the MIC value of the individual and combined test compounds. FIC was calculated from the MIC of test compound A and the MIC of test compound A in combination with test compound B. Therefore, FIC of A = MIC of test compound A in combination/MIC of test compound A alone.
The FIC of test compound B was calculated in the same manner and the sum of the two FIC compounds combined to give the SFIC index. SFIC index = FIC of test compound A + FIC of test compound B
The calculated FIC index was used to detect the nature of the interaction between the two test compounds and the interaction either synergism or indifference or antagonism type.
The values published by the American Society of Microbiology were used to decide the nature of the interaction FICI < 0.5 synergy, 0.5 < FICI < 1 partial synergy, FICI = 1 additive, 2 < FICI < 4 indifferent, and 4 < FICI antagonism (Botelho 2000).Table -2; and Figure 1 and 2.
Table-2 List of phytochemicals selected for synergistic effect against V. parahaemolyticus

Results: In 28 different combinations tested, eight combinations showed a synergistic effect against the growth of V. parahaemolyticus (Table 2). This includes curcumin + tannic acid, tannic acid + geraniol, curcumin + geraniol, curcumin + quercetin, geraniol + quercetin, pyrogallol + vanillin, curcumin + menthol and menthol + citral. Among these eight combinations, the pyrogallol + vanillin combination exhibited effective inhibitions in the growth of V. parahaemolyticus at a ratio of 1:2 [50 and lOOug/mL concentration] respectively (Figure 1).
Pyrogallol (A) + Vanillin (B) = (50 + 100) [ig/mh
MICA = 200 ^g/mL
MICB= 800 ^g/mL
FIC index = FICA + FICB
FIC A = 50/200 = 0.25
FICB = 100/800 = 0.125
FIC index = 0.25 + 0.125 = 0.375
0.375 < 0.5 - Synergism
Further, a third compound crystal violet with higher synergistic score was decided to added with pyrogallol + vanillin combination in order to further enhance the activity.

Hence, 2 |ig/mL concentration of CV was added, which surprisingly reduced the concentration of pyrogallol + vanillin upto 50% (Table 3). Hence, the MIC of the formulation (pyrogallol + vanillin+CV) is 25 + 50 + 2 |ig/mL (Figure 2).
Table-3
Furthermore, the active formulation- pyrogallol + vanillin + crystal violet at MIC and 2xMIC was effectively eradicated the preformed biofilm of V. parahaemolyticus to the level of 60% and 87%, respectively (Figure 3).
EXAMPLE: 3
Preformed biofilm eradication assay
Eradication efficiency of the test formulation pyrogallol+vanillin+crystal violet against preformed biofilm formation of V. parahaemolyticus was studied. Initially, V. parahaemolyticus was allowed to form biofilm for 24h, in a sterile 24 well MTP in which one mL of fresh media supplemented with 1% inoculum of V. parahaemolyticus. Then

planktonic cells were removed and refilled with fresh media supplemented with MIC and 2xMICs of pyrogallol + vanillin+ crystal violet and incubation was extended up to 24h. After the incubation, the planktonic cells were discarded and the wells were washed with sterile distilled water and allowed to air dry. The adhered biofilm cells on the walls of MTP wells were stained with 0.4% crystal violet for 10 min. After staining, the unbound stain was washed with distilled water and the wells were destained using 20% glacial acetic acid (GAA) for 30 min and OD570 nm was measured using a UV spectrometer (Hitachi U- 2800, Tokyo, Japan). The intensity of dye after washing is directly proportional to the number of cells present in the biofilm. The control was maintained without the addition of test formulation. The percentage of biofilm inhibition was calculated using the formula: % of inhibition = [(Control ODs70nm -Treated ODs70nm) / Control OD570nm]x 100.
Results: The active formulation pyrogallol + vanillin + crystal violet at MIC and 2xMICs have effectively eradicated the preformed biofilm of V. parahaemolyticus to the level of 60% and 87%, respectively. The results are shown in Figure 3.
EXAMPLES
Microscopic analyses of preformed biofilm of V. parahaemolyticus
In microscopic analysis, V. parahaemolyticus was allowed to form biofilms on glass slides (1 x 1 cm) for 24 h at 30QC. Then the slides were treated with MIC and 2xMICs of pyrogallol + vanillin+ crystal violet and incubated as previously mentioned conditions. Then the slides were washed with sterile PBS and processed as below.
In light microscopic analysis, the control and treated glass slides were stained with 0.4% CV for 5 mins and washed with sterile distilled water. Then the stained slides were imaged under light microscope (Nikon Eclipse Ti 100, Japan) at 400x magnification (Santhakumari etal. 2016).
In Confocal Laser Scanning Microscopic (CLSM) analysis, the control and treated slides were stained with 0.1% acridine orange in dark for 5 mins and subsequently washed with

sterile distilled water. Then the stained slides were imaged under 488nm argon laser in CLSM (Zeiss LSM 710, Carl Zeiss, Germany) and processed with Zeiss LSM Image Examiner (Version 4.2.0.121).
Results: The light microscope, as well as CLSM images, clearly revealed a visual reduction of surface coverage and thickness of biofilm on the slides treated with the formulation consisting pyrogallol + vanillin+ crystal violet, than that of untreated controls. The results are shown in Figure 4(a) &(b).
EXAMPLE: 5 Motility assessment
The effect of test formulation on swarming motility of V. parahaemolyticus was determined by placing 5 uL of V. parahaemolyticus cell suspension in the centre of the swarm agar plates (1% peptone, 0.5% NaCl, 0.5% agar and 0.5% of filter-sterilized d-glucose) with and without active test formulation. Then the plates were then incubated at 30°C in an upright position for 24 h and observed for reduction in swimming and swarming migration zones (Srinivasan etal. 2016).
Results: The results clearly showed that the pyrogallol + vanillin+ crystal violet in a ratio of 12.5:25:l(25+50+2ug/mL) effectively reduced the swarming motility of V. parahaemolyticus. Results are shown in Figure 5.
EXAMPLE: 6
Effects of the test formulation (Pyrogallol+ Vanillin + Crystal violet) on growth and
survival of shrimp (white shrimp, Litopenaeus vannamei)
The toxicity of the compound was preliminarily assessed by measuring the survival rate of shrimp grown in 5 liters of UV sterilized seawater in the presence and absence of pyrogallol+ vanillin + Crystal violet (25+50+2 ug/mL).

i. Acute toxicity study
The acute toxicity effect of the test formulation, Pyrogallol+ Vanillin + Crystal violet was studied using white shrimp L. vannamei as an aquatic animal model system.
In in vivo toxicity analysis, each aquaria contains 5 L of UV sterilized seawater with 10 numbers of L. vannamei with equal size (4.5 cm ±0.26) and weight as shown in Figure 7. After 7 days of acclimatization, animals were exposed to different concentrations of test formulation such as 100% (PG+ VA+ CV), 50% (PG+ VA+ CV) and 10% PG+ 50%VA+10% CV, in order to study the toxic effect. In addition to these, the individual compounds of test formulation were also tested for their toxicity effect against L. vannamei. The rate of mortality and survival of animals were calculated after 96 h.
Results: The results showed that 100% of survival even after treated with individual compounds such as pyrogallol, vanillin and CV at 25 ug/mL, 50 ug/mL, and 2 |ig/mL respectively. Wherein, MIC and 50% MIC of test formulation treated groups showed 75% and 80% survival of white shrimp after 96 hrs, respectively. There was a slight decrease in the survival rate of shrimp was observed at MIC or sub-MICs of test formulation supplemented in pond water. Hence, the formulation has been reformulated by reducing the concentrations of incredients, which resulted in reduced toxicity to the shrimps. In the revised formulation the incredients were used at ratio of 10% pyrogallol + 50% vanillin + 10% CV from the original MIC value. End up with this trail has made it suitable dosage for the cultivation of shrimp culture in ponds. Interestingly, the final formulation consisting of pyrogallol (2.5 ug/mL) + vanillin (25 ug/mL) + CV (0.2 ug/mL) has not showed any toxic and lethal effects to the shrimps at this particular concentration. Hence, the final formulation was designed with pyrogallol + vanillin + crystal violet at the concentraions of 2.5, 25 and 0.2 ug/mL, respectively. Formulation: Pyrogallol (2.5 ppm) + Vanillin (25 ppm) + CV (0.2 ppm).
ii. In vivo challenging study

For the assessment of in vivo disease control efficacy of test formulation against V. parahaemolyticus infection, the survival assay was performed using L. vannamei. In this experiment, a group of 10 healthy shrimp (equal size and weight) was taken in plastic tubs with 5 L of UV sterilized seawater. The animals were acclimatized for 7 days and challenged with V. parahaemolyticus (108 CFU/mL) and incubated for 12 h along with uninfected control. After 12 h, the infected shrimp were transferred into the aquaria contains 5 L of fresh UV sterilized sea water with and without test formulation (PG+VA+CV). The rate of mortality was monitored for every 1 h up to 96 h along with an experimental control group.
Results: The obtanied results established that V. parahaemolyticus infected shrimp upon supplemented with pyrogallol + vanillin + crystal violet showed increased survival rate compared to the untreated control. Hence, it is concluded that, Pyrogallol, Vanillin and Crystal violet at the concentration of 2.5 ppm, 25 ppm and 0.2 ppm respectively is sufficient to control the infection and to rescue the shrimps from the lethal effect of V. parahaemolyticus (Figure 8).
EXAMPLE: 7
Impact of test formulation on the bacterial colonization in shrimp Hepatopancreas (HP)
The efficacy of the test fomuation (Pyrogallol+ Vanillin + Crystal violet: 2.5+25+0.2 ppm) on bacterial colonization in shrimp's HP region was evaluated by spread plate method. The HP regions of test formulation treated and untreated infected shrimp were collected and grained with sterile distilled water. Then the samples were spreaded on TCBS agar plates. After incubation, the control and treated TCBS plates were observed for V. paraharmolyticus colony formations.
Results: As shown in figure 9, a significant reduction in number of colonies was observed in the formulation treated sample than that of untreated control. The obtanied results

clearly evidenced the disease rescue efficacy of this formulation (Pyrogallol+ Vanillin + Crystal violet: 2.5+25+0.2 ppm) against V. parahaemolyticus infection in shrimp.
EXAMPLE: 8
Infield trial of the test formulation on bacterial load in natural pond water
In order to determine the effect of test formulation in reducing the density and growth of V. parahaemolyticus and other vibrios natuarlly present in the pond water, the in field trial was made with commercial aquaculture pond water. Initially, 100 L of natural pond water sample was collected from a commercialshrimp farm in Sirkazhi, Tamilnadu. In this experiment, the test formulation at the selected concentrations of 10% PG + 50% VA + 10% CV (2.5+25+0.2 ug/mL) was added to the pond water. Then the water samples were collected at different time intervals as 0, 12 , 24 and 48 h. And 100 ul of samples from each treated and untreated pond water at mentioned intervals were spread on TCBS agar plate and incubated for 16 h at 30°C and CFU was calculated.
Results: The obtained results showed that the test formulation (Pyrogallol+ Vanillin + Crystal violet: 2.5+25+0.2 ppm) was effectively reduced the density of Vibrio sp, present in the pond water sample, in a time-dependent manner (Figure 10).
CONCLUSION
In the present study, a screening of commercially available phytochemicals belonging to different compound categories were randomly selected and tested for its antibacterial efficacy against V. parahaemolyticus. Further the combinatorial usage of phytochemicals with potent antimicrobial effect comprising, pyrogallol, vanillin and crystal violet exhibited effective inhibitions in the growth of V. parahaemolyticus at 25 +50+2 |ig/mL concentration respectively (Fig.3).
The invivo efficacy of this active formulation (Pyrogallol, vanillin and crystal violet), when applied in shrimp pond water in a concentration of 10% pyrogallol, 50% vanillin

and 10% crystal violet controlled the infection and rescued the shrimp from the lethal effect of V. parahaemolyticus (Figure 8). The obtained results clearly evidenced the in vivo bactericidal effect of this formulation against V. parahaemolyticus infection.
Thus the present invention delineates novel synergistic formulations comprising commercially available phytochemicals against EMS specific V. parahaemolyticus. Further, it accentuates the high pharmaceutical and economical values of individual phytochemicals and an effective formulation comprising of pyrogallol, vanillin and crystal violet to treat EMS associated V.parahaemolyticus infection in shrimp aquaculture. The formulation of the present invention is eco-friendly without any side effects to the cultured organisms. It is user-friendly and very economical and highly effective in controlling EMS.

WE CLAIM
1. A synergistic formulation comprising:
i. Pyrogallol,
ii. Vanillin and iii. Crystal violet.
2. The formulation as claimed in claim 1 wherein the said formulation is an anti microbial formulation.
3. The formulation as claimed in claims 1 and 2 wherein the antimicrobial activity is anti vibriosis activity.

4. The formulation as claimed in claims 1 to 3 wherein the microorganism is V. parahaemolyticus.
5. The formulation as claimd in claim 1 to 4 wherein the said formulation is for the control of shrimp disease.
6. The formulation as claimd in claims 1 wherein the formulation comprises
pyrogallol+vanillin+crystal violet in a weight ratio of about 3.13:31.25:0.25 to about
25:250:2
7. The formulation as claimed in cliams 1 wherein the formulation comprises of about 0.625 |ig/mL to about 5ug/mL of pyrogallol.
8. The formulation as claimd in claim 1 wherein the formulation comprises ofabout 6.25 |ig/mL to about 50ug/mL of Vanillin

9. The formulation as claimd in claim 1 wherein the formulation of about 0.05 |ig/mL to
about 0.4 |ig/mL of crystal violet.
10. The formulation as claimed in claim 1 to 9 wherein the shrimp disease is Early Mortality Syndrome [EMS].
11. The formulation as claimed in claim 1 to 10 wherein the minimum inhibitory concentration of the formualtion has a microbial inhibitory activity of about 55 % to about 65%.
12. The formulation as claimed in claim 1 to 11 wherein the minimum inhibitory concentration of the formulation showed a shrimp survival rate of about 70% to about 80%.