WO2017147130A2

WO2017147130A2 - Direct fed microbial for prevention of shrimp disease

Info

Publication number: WO2017147130A2
Application number: PCT/US2017/018843
Authority: WO
Inventors: David Drahos; Seth D'IMPERIO
Original assignee: Novozymes A/S
Priority date: 2016-02-23
Filing date: 2017-02-22
Publication date: 2017-08-31
Also published as: CN108603166A; WO2017147130A3; ECSP18071568A

Abstract

The present invention relates to Bacillus subtilis strains and their use for treatment of shrimp disease such as Early Mortality Syndrome.

Description

DIRECT FED MICROBIAL FOR PREVENTION OF SHRIMP DISEASE

Reference to a deposit of biological material

This application contains a reference to a deposit of biological material, which deposit i incorporated herein by reference. For complete information see Example 1.

Reference to sequence listing

This application contains a Sequence Listing in computer readable form. The computer readable form is incorporated herein by reference.

Index to sequence listing:

SEQ ID NO: 1 is 16S rDNA of Bacillus subtilis strain 014VRQ having deposit accession number NRRL B-67221.

SEQ ID NO: 2 is 16S rDNA of Bacillus subtilis strain MF1048 having deposit accession number NRRL B-67220.

FIELD OF THE INVENTION

The present invention relates to Bacillus subtilis strains and their use for treatment of shrimp disease such as Early Mortality Syndrome. BACKGROUND OF THE INVENTION

Early Mortality Syndrome (EMS) also known as acute hepatopancreatic necrosis disease (AHPND) is a relative new shrimp disease that is presently disrupting production in shrimp producing countries such as China, Thailand, Vietnam and Mexico. Outbreaks of EMS naturally occur in the first 30 days after stocking a freshly arranged shrimp pond, and the rate of mortality can pass beyond 70%.

EMS is caused by a bacterial agent(s), typically transmitted orally, that colonize in the shrimp gastrointestinal tract and produce toxin(s) that causes tissue destruction and dysfunction of the shrimp digestive organ known as the hepatopancreas. EMS is caused by specific strains of Vibrio, in particular Vibrio paralnaemolyticus. The Vibrio EMS strain is a highly virulent pathogen, causing up to 80% shrimp mortality in China, Thailand, and Vietnam.

EMS will require a different approach than the current strategies against white spot virus. Use of antibiotics to control microbial developments is not desirable due to potential development of antibiotic resistance.

SUMMARY OF THE INVENTION

The present invention relates to Bacillus subtilis strain 014VRQ and Bacillus subtilis strain MF1048 having ability to inhibit, control, prevent and/or ameliorate EMS. In five trials involving replicated and controlled treatments, strains 014VRQ and MF1048 typically provided 50-60% shrimp survival compared to 0-10% survival with non-inoculated Control shrimp within 3 days of disease exposure.

Thus the invention provides an isolated Bacillus subtilis strain selected from the group consisting of

(a) Bacillus subtilis strain 014VRQ having deposit accession number NRRL B-67221 or a strain having all of the identifying characteristics of the Bacillus subtilis strain NRRL B-67221 or a mutant thereof; and

(b) Bacillus subtilis strain MF1048 having deposit accession number NRRL B-67220 or a strain having all of the identifying characteristics of the Bacillus subtilis strain NRRL B-67220 or a mutant thereof.

The invention further relates to compositions comprising one or more of the isolated Bacillus subtilis strains according to the invention and a carrier.

In another aspect the invention relates to a method for treating a shrimp disease such as EMS (also known as AHPND for Acute Hepatopancreatic Necrosis Disease) comprising the step of administering one or more of the isolated Bacillus subtilis strains according to the invention or the composition according to the invention to the shrimp.

In a further aspect, the invention relates to the use of Bacillus subtilis strain selected from the group consisting of:

(b) Bacillus subtilis strain MF1048 having deposit accession number NRRL B-67220 or a strain having all of the identifying characteristics of the Bacillus subtilis strain NRRL B-67220 or a mutant thereof;

for treatment of a virulent Vibrio parahaemolyticus associated disease such as EMS in shrimps.

DEFINITIONS:

In general, the terms and phrases used herein have their art-recognized meaning, which can be found by reference to standard texts, journal references, and context known to those skilled in the art. The following definitions are provided to clarify their specific use in context of the disclosure.

As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

The term "about" means a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 % to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

As used herein, the terms "aquaculture", "aquaculturing", "aquafarm", and "aquafarming" can be used interchangeably and refer to the cultivation, breeding, raising, production, propagation and/or harvesting of an aquatic or marine animal, generally in an artificial environment such as a tank (e.g., an aquarium), a pond, a pool, a paddy, a lake, etc., or in an enclosed or fenced off portion of the animals natural habitat, such as a pond, a pool, a paddy, a lake, an estuary, an ocean, a marsh (e.g., a tidal marsh), a lagoon (e.g., a tidal lagoon), etc.

As used herein, the terms "aquatic animal", "marine animal" or "aquatic and/or marine animals" refer to organisms that live in an aquatic or marine environment. Non-limiting examples include fish, e.g., osteichthyes (including, but not limited to catfish, tilapia, trout, salmon, perch, bass, tuna, wahoo, tuna, swordfish, marlin, grouper, sturgeon, snapper, eel and walleye) and chondrichthyes (including, but not limited to sharks, rays, and skates), crustaceans (including, but not limited to crabs, lobsters, crayfish, shrimp, krill, and prawn) and mollusks (including, but not limited to snails, slugs, conch, squid, octopus, cuttlefish, clams, oysters, scallops, and mussels). In a particular embodiment, the aquatic animal is shrimp or prawn.

As used herein, the terms "administer", "administered", or "administering", is intended to mean bringing an aquatic animal such as shrimp into contact with a Bacillus strain or a composition as described herein. A preferred form of administration is oral administration such as administration via water or via animal feed.

As used herein, the term "control" or "controlling" as in. e.g., the phrase: the "control" of pathogenic microorganisms, or "controlling" pathogenic microorganisms, or as in the phrase: "controlling" species of pathogenic microorganisms, refers to any means for preventing infection by pathogenic microorganisms, reducing the number of pathogenic microorganisms, killing the pathogenic microorganisms, or elimination the pathogenic microorganisms as defined herein. Indeed, "control" or "controlling" as used herein refers to any indicia of success in prevention, killing, elimination, reduction or amelioration of one or more pathogenic bacteria.

Throughout this disclosure, unless the context requires otherwise, the words "comprise," "comprises," and "comprising" will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.

The term "consisting of" means including, and limited to, whatever follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and that no other elements may be present. The term "consisting essentially of" means including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they materially affect the activity or action of the listed elements.

As used herein, the terms "spore" and "endospore" are interchangeable and have their normal meaning which is well known and understood by those of skill in the art. As used herein, the term spore refers to a microorganism in its dormant, protected state.

As used herein, the term "isolated" means that the one or more bacterial strains described herein are in a form or environment which does not occur in nature, that is, the one or more bacterial strains are at least partially removed from one or more or all of the naturally occurring constituents with which it is associated in nature.

As used herein, the term "blend" means more than one of the bacterial strains described herein.

As used herein, the term "pathogenic microorganism" means any microorganism that can adversely affect the health, yield, or environment of one or more aquatic animals.

As used herein, the term "health" refers to the state or condition of an organism or one of its parts.

As used herein, the terms "effective amount", "effective concentration", or "effective dosage" are defined as the amount, concentration, or dosage of the bacterial strain(s) sufficient to improve the health or yield of an aquatic animal such as shrimp. The actual effective dosage in absolute numbers depends on factors including: the state of health of the aquatic animal in question; whether the aim is prevention or reduction of a pathogenic organism, to improve overall health, gut health, etc.; other ingredients present, and also the surface or aqueous environment in question. In an embodiment an effective dosage of bacteria, e.g., of the one or more of the Bacillus subtilis strains disclosed, would be in the range from 1x10² to 1x10¹² CFU/g of the composition, preferably 1x10⁴ to 1x10⁹ CFU/g of the composition, more preferably 1x10⁵ to 1x10⁸ CFU/g of the composition, and even more preferably 1x10⁶ to 5x10⁸ CFU/g of the composition. Further, in an embodiment the ratio between the bacteria strain or blends concerned herein and the undesired microorganism(s) in question may be between 1 : 100,000 and 100,000: 1 (strain/blend:undesired microorganism), preferably 1 : 10,000 to 10,000: 1 , more preferably 1 : 1 ,000 to 1 ,000: 1 , more preferably 1 : 100 to 100: 1 , even more preferably 1 : 10 to 10: 1. The "effective amount", "effective concentration", or "effective dosage" of the bacterial strains may be determined by routine assays known to those readily skilled in the art.

As used herein, the term "aquatic animal feed" or "aquatic animal feed ingredient" refers to any compound, preparation, or mixture suitable for, or intended for intake by an aquatic animal such as shrimp.

As used herein, the term "vegetable protein(s)" refers to any compound, preparation or mixture that includes at least one protein derived from or originating from a vegetable, including modified proteins and protein-derivatives. As used herein, the terms "pellets" and/or "pelleting" refer to solid rounded, spherical and/or cylindrical tablets or pellets and the processes for forming such solid shapes, particularly feed pellets and solid extruded animal feed. As used herein, the terms "extrusion" or "extruding" are terms well known in the art and refer to a process of forcing a composition, as described herein, through an orifice under pressure.

As used herein, the term "composition" refers to a composition comprising a carrier and at least one bacterial strain as described herein. The compositions described herein may be mixed with an animal feed(s) and referred to as a "mash feed." Alternatively, the composition is, e.g., sinking feed pellets.

DETAILED DESCRIPTION OF THE INVENTION

Bacterial strains

Described and disclosed herein are bacterial strains for improving the well-being, general condition or health and/or yields of aquatic animals, improving, e.g., the weight of aquatic animals, improving the overall gut health of aquatic animals, and/or controlling pathogenic microorganisms (e.g., Vibrio, Vibrio harveyi, Vibrio parahaemolyticus, Vibrio cam bellii, Vibrio fischeri, Photobacterium and/or Photobacterium phosphoreum). It is an object of the embodiments disclosed herein, that the bacterial strains provided throughout will improve the health of aquatic animals and/or survival rate of the aquatic animals. In a preferred embodiment the aquatic animal is shrimp.

In a preferred embodiment the invention relates to an isolated Bacillus subtilis strain selected from the group consisting of:

In one embodiment the Bacillus subtilis strain has a 16S rDNA sequence selected from the group consisting of:

(a) 16s rDNA having at least 98.00%, e.g., at least 98.05%, at least 98.10%, at least 98.15%, at least 98.20%, at least 98.25%, at least 98.30%, at least 98.35%, at least 98.40%, at least 98.45%, at least 98.50%, at least 98.55%, at least 98.60%, at least 98.65%, at least 98.70%, at least 98.75%, at least 98.80%, at least 98.85%, at least 98.90%, at least 98.95%, at least 99.00%, at least 99.05%, at least 99.10%, at least 99.15%, at least 99.20%, at least 99.25%, at least 99.30%, at least 99.35%, at least 99.40%, at least 99.45%, at least 99.50%, at least 99.55%, at least 99.60%, at least 99.65%, at least 99.70%, at least 99.75%, at least 99.80%, at least 99.85%, at least 99.90%, at least 99.95% or 100% sequence identity to SEQ ID NO: 1 ; and

(b) 16s rDNA having at least 98.00%, e.g., at least 98.05%, at least 98.10%, at least 98.15%, at least 98.20%, at least 98.25%, at least 98.30%, at least 98.35%, at least 98.40%, at least 98.45%, at least 98.50%, at least 98.55%, at least 98.60%, at least 98.65%, at least 98.70%, at least 98.75%, at least 98.80%, at least 98.85%, at least 98.90%, at least 98.95%, at least 99.00%, at least 99.05%, at least 99.10%, at least 99.15%, at least 99.20%, at least 99.25%, at least 99.30%, at least 99.35%, at least 99.40%, at least 99.45%, at least 99.50%, at least 99.55%, at least 99.60%, at least 99.65%, at least 99.70%, at least 99.75%, at least 99.80%, at least 99.85%, at least 99.90%, at least 99.95% or 100% sequence identity to SEQ ID NO: 2.

Preferably the Bacillus subtilis strain has activity against shrimp pathogens such one or more Vibrio strains and/or Photobacterium strains. In a preferred embodiment the Vibrio strain is selected from the group consisting of Vibrio fischeri, Vibrio vulnificus, Vibrio fluvialis, Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio mimicus, Vibrio cholera, Vibrio harveyi, or any combination thereof. In a preferred embodiment the Photobacterium strain is selected from the group consisting of Photobacterium phosphoreum, Photobacteria damselae subspecies damselae, Photobacterium damselae subspecies piscicida or any combination thereof.

In a preferred embodiment the Bacillus subtilis strain is isolated. In another preferred embodiment the Bacillus subtilis strain is on spore form.

Compositions

In an aspect of the invention, a composition is disclosed comprising an animal feed ingredient and one or more bacterial strains according to the invention. In another embodiment, the one or more bacterial strains are stable strains when the strains are subjected to an extrusion process having a pressure of 1 bar to 35 bar; the strains are subjected to an extrusion process wherein the extrusion process temperatures are temperatures from 80°C to 120°C; and the strains that control one or more pathogenic microorganisms.

In a more particular embodiment, the one or more bacterial strains are selected from the group consisting of:

In a particular embodiment the composition comprising one or more of the isolated Bacillus subtilis strains selected from the group consisting of (a) Bacillus subtilis strain 014VRQ having deposit accession number NRRL B-67221 or a strain having all of the identifying characteristics of the Bacillus subtilis strain NRRL B-67221 or a mutant thereof; and

and a carrier. The carrier can, e.g., be calcium carbonate, other carriers mentioned elsewhere herein or other carriers described in the prior art.

In a preferred embodiment the composition comprises a Bacillus subtilis strain having a 16S rDNA sequence selected from the group consisting of:

(b) 16s rDNA having at least 98.00%, e.g., at least 98.05%, at least 98.10%, at least 98.15%, at least 98.20%, at least 98.25%, at least 98.30%, at least 98.35%, at least 98.40%, at least 98.45%, at least 98.50%, at least 98.55%, at least 98.60%, at least 98.65%, at least 98.70%, at least 98.75%, at least 98.80%, at least 98.85%, at least 98.90%, at least 98.95%, at least 99.00%, at least 99.05%, at least 99.10%, at least 99.15%, at least 99.20%, at least 99.25%, at least 99.30%, at least 99.35%, at least 99.40%, at least 99.45%, at least 99.50%, at least 99.55%, at least 99.60%, at least 99.65%, at least 99.70%, at least 99.75%, at least 99.80%, at least 99.85%, at least 99.90%, at least 99.95% or 100% sequence identity to SEQ ID NO: 2;

or the composition comprises one Bacillus subtilis strain having a 16S rDNA sequence consisting of (a) and a second Bacillus subtilis strain having a 16S rDNA sequence consisting of (b);

and a carrier.

In a preferred embodiment the one or more of the isolated Bacillus subtilis strains in the composition are on spore form.

The composition is preferably an animal feed or an animal feed additive such as an aquatic animal feed or an aquatic animal feed additive such as a shrimp feed or a shrimp feed additive. In one embodiment the animal feed is feed pellets such as sinking feed pellets. In a specific embodiment the feed pellets are surface coated with one or more of the Bacillus subtilis strains according to the invention. In a preferred embodiment the composition comprises from 10⁴ to 10⁸ CFU/g feed such as from 10⁵ to 10⁷ CFU/g feed or such as from 5 x 10⁶ to 9 x 10⁶ CFU/g feed of the Bacillus subtilis strains according to the invention.

In an embodiment, the compositions described herein can be of any form so long as the carrier is able to support the one or more bacterial strains, regardless of form (e.g., a vegetative state or a dormant state), and the composition is suitable for intake by an aquatic animal such as shrimp. In certain embodiments, the composition may be in the form of a liquid, a slurry, a solid, or a powder (wettable powder or dry powder). In a particular embodiment, the composition disclosed herein, regardless of form, e.g., a liquid, slurry, or powder (e.g., wettable powder or dry powder), is suitable for use as an ingredient in an animal feed. In a more particular embodiment, the compositions described herein are suitable for use as an ingredient in a pelleted animal feed. In still a more particular embodiment, the compositions described herein are suitable for use as an ingredient in an animal feed produced via an extrusion process.

Carrier(s) in the composition:

The carriers described herein will allow the one or more bacterial strains described herein to remain efficacious (e.g., capable of improving aquatic animal well-being, general condition, health or survival rate) and viable once formulated. Non-limiting examples of carriers described herein include liquids, slurries, or solids (including wettable powders or dry powders).

In one embodiment, the carrier is a liquid carrier. Non-limiting examples of liquids useful as carriers for the compositions disclosed herein include water, aqueous solutions, or nonaqueous solutions. In one embodiment, the carrier is water. In another embodiment the carrier is an aqueous solution, such as sugar water. In another embodiment, the carrier is a non-aqueous solution. If a liquid carrier is used, the liquid (e.g., water) carrier may further include growth media to culture the one or more bacterial strains. Non-limiting examples of suitable growth media for the deposited bacterial strains include arabinose-gluconate (AG), yeast extract mannitol (YEM), G16 media, or any media known to those skilled in the art to be compatible with, and/or provide growth nutrients to the one or more bacterial strains.

In another embodiment, the carrier is a slurry.

In another embodiment, the carrier is a solid. In a particular embodiment the solid is a powder. In one embodiment the powder is a wettable powder. In another embodiment, the powder is a dry powder. In another embodiment, the solid is a granule. Non-limiting examples of solids useful as carriers for the compositions disclosed herein include calcium carbonate, sodium bicarbonate, sodium chloride, peat, wheat, wheat chaff, ground wheat straw, bran, vermiculite, cellulose, starch, soil (pasteurized or unpasteurized), gypsum, talc, clays (e.g., kaolin, bentonite, montmorillonite), and silica gels. In a particular embodiment, the carrier is calcium carbonate. In another embodiment, the carrier is sodium bicarbonate. Bacterial Strain(s) in the composition:

The composition as described herein comprises one or more bacterial strains according to the invention. In an embodiment, the one or more bacterial strains are stable when the strains are subjected to a feed manufacturing process. In a particular embodiment, the one or more strains are stable when the strains are subjected to an extrusion process for pelleting.

In one embodiment, the one or more bacterial strains are stable when subjected to pressures achieved during an extrusion process for pelleting. In a particular embodiment, the one or more bacterial strains are stable at pressures ranging from 1 bar to 40 bar, particularly 10 bar to 40 bar, more particularly 15 bar to 40 bar, even more particularly 20 bar to 40 bar, still even more particularly 35 bar to 37 bar, even still more particularly 36 bar.

In a particular embodiment, the one or more bacterial strains are stable at high temperatures. In particular, the bacterial strains are stable when they are subjected to temperatures achieved during an extrusion process for pelleting. In an even more particular embodiment, the one or more bacterial strains are stable at temperatures ranging from 80°C to 120°C, particularly temperatures ranging from, 90°C to 120°C, even more particularly temperatures ranging from 95°C to 120°C.

In a further particular embodiment the extrusion stability of the bacterial strain(s) is determined by extrusion at 100°C or 1 10°C, exhibiting 50% or more survival at 100°C or 25% or more survival at 1 10°C.

In an even more particular embodiment, the one or more bacterial strains are stable when the strains are subjected to an extrusion process wherein the extruder has a die diameter of 0.5 mm to 5.0 mm.

In another embodiment, the one or more bacterial strains control one or more pathogenic microorganisms.

In still another embodiment, the one or more bacterial strains are stable strains when the strains are subjected to an extrusion process having a pressure of 1 bar to 40 bar; the strains are subjected to an extrusion process wherein the extrusion process temperatures are temperatures from 80°C to 120°C; and the strains control one or more pathogenic microorganisms.

In a particular embodiment, the one or more bacterial strains will be present in a quantity between 1x10² and 1x10¹² CFU/g of the composition, particularly 1x10⁴ and 1x10⁹ CFU/g of the composition, and more particularly 1x10⁵ and 5x10⁸ CFU/g of the composition. In a more particular embodiment the one or more bacterial strains will be present in a quantity between 1x10⁶ and 1x10⁸ CFU/g of the composition.

The fermentation of the one or more bacterial strains may be conducted using conventional fermentation processes, such as, aerobic liquid-culture techniques, shake flask cultivation, and small-scale or large-scale fermentation (e.g., continuous, batch, fed-batch, solid state fermentation, etc.) in laboratory or industrial fermentors, and such processes are well known in the art. Notwithstanding the production process used to produce the one or more bacterial strains, the one or more bacterial strains may be used directly from the culture medium or subject to purification and/or further processing steps (e.g., a drying process).

Following fermentation, the one or more bacterial strains may be recovered using conventional techniques (e.g., by filtration, centrifugation, etc.). The one or more bacterial strains may alternatively be dried (e.g., air-drying, freeze drying, or spray drying to a low moisture level, and storing at a suitable temperature, e.g., room temperature).

In an embodiment, the one or more bacteria disclosed herein are stable and retain a sufficient effective amount of activity when used. Methods for producing stabilized microorganisms are known in the art. In one embodiment, the one or more bacteria disclosed herein are present in the composition in the form of a stable spore.

Optional Ingredients in the composition:

The compositions described herein may further comprise one or more optional ingredients that are suitable for consumption by an aquatic animal such as shrimp. Non-limiting optional ingredients include enzymes. Such ingredients are known to those skilled in the art.

Enzymes

It is further envisioned that the compositions described herein optionally include one or more enzymes as described herein. In a particular embodiment, the one or more enzymes may be any enzyme or combination of different enzymes that are suitable to be given to an animal, meaning that it in one way or the other will be good for the animal nutritionally to eat the enzyme. Accordingly, when reference is made to "an enzyme" this will in general be understood to include one or more feed enzymes. In a particular embodiment it is not construed as including enzymes which has a therapeutic function in medical sense.

The feed enzymes should be feed/food grade, thus meaning that they may not be harmful to the aquatic animal and be a feed/food grade meaning that it should comply with recommended purity specifications for food grade enzymes. In a particular embodiment this means that the enzyme complies with recommended purity specifications for food grade enzymes given by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) and the Food Chemical Codex (FCC).

The enzyme shall in a particular embodiment comprise less than 30 coliform bacteria per gram and comprise a viable count of less than 50,000/g.

In another embodiment, the compositions described herein optionally include one or more enzymes. Enzymes can be classified on the basis of the handbook Enzyme Nomenclature from NC-I UBMB, 1992), see also the ENZYME site at the internet: expasy.cn/enzyme/. ENZYME is a repository of information relative to the nomenclature of enzymes. It is primarily based on the recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUB-MB), Academic Press, Inc., 1992, and it describes each type of characterized enzyme for which an EC (Enzyme Commission) number has been provided (Bairoch. 2000, The ENZYME database, Nucleic Acids Res. 28: 304-305). This IUB-MB Enzyme nomenclature is based on their substrate specificity and occasionally on their molecular mechanism; such a classification does not reflect the structural features of these enzymes.

Another classification of certain glycoside hydrolase enzymes, such as endoglucanase, xylanase, galactanase, mannanase, dextranase, lysozyme and galactosidase is described in Henrissat et al, 2014, "The carbohydrate-active enzymes database (CAZy) in 2013", Nucl. Acids Res. (D1): D490-D495; see also cazy.org.

Thus the composition of the invention may also comprise at least one other enzyme selected from the group comprising of phytase (EC 3.1.3.8 or 3.1.3.26); xylanase (EC 3.2.1.8); galactanase (EC 3.2.1.89); alpha-galactosidase (EC 3.2.1.22); protease (EC 3.4); phospholipase A1 (EC 3.1.1.32); phospholipase A2 (EC 3.1.1.4); lysophospholipase (EC 3.1.1.5); phospholipase C (3.1.4.3); phospholipase D (EC 3.1.4.4); amylase such as, for example, alpha- amylase (EC 3.2.1.1); lysozyme (EC 3.2.1.17); arabinofuranosidase (EC 3.2.1.55); beta- xylosidase (EC 3.2.1.37); acetyl xylan esterase (EC 3.1.1.72); feruloyl esterase (EC 3.1.1.73); cellulase (EC 3.2.1.4); cellobiohydrolases (EC 3.2.1.91); beta-glucosidase (EC 3.2.1.21); pullulanase (EC 3.2.1.41) and beta-glucanase (EC 3.2.1.4 or EC 3.2.1.6), or any mixture thereof.

In a particular embodiment, the composition of the invention comprises a phytase (EC

3.1.3.8 or 3.1.3.26). Examples of commercially available phytases include Bio-Feed™ Phytase (Novozymes), Ronozyme® P, Ronozyme® NP and Ronozyme® HiPhos (DSM Nutritional Products), Natuphos™ (BASF), Finase® and Quantum® Blue (AB Enzymes), OptiPhos® (Huvepharma) Phyzyme® XP (Verenium/DuPont) and Axtra® PHY (DuPont). Other preferred phytases include those described in, e.g. , WO 98/28408, WO 00/43503, and WO 03/066847.

In a particular embodiment, the composition of the invention comprises a xylanase (EC 3.2.1.8). Examples of commercially available xylanases include Ronozyme® WX and Ronozyme® G2 (DSM Nutritional Products), Econase® XT and Barley (AB Vista), Xylathin® (Verenium), Hostazym® X (Huvepharma) and Axtra® XB (Xylanase/beta-glucanase, DuPont) In a particular embodiment, the composition of the invention comprises a protease (EC

3.4). Examples of commercially available proteases include Ronozyme® ProAct (DSM Nutritional Products).

Animal Feed

In certain embodiments the compositions described herein are suitable for use in animal feed(s) such as shrimp feed(s). The characteristics of the compositions described herein allow its use as a component which is well suited for inclusion with an animal feed. In particular embodiments, the compositions described herein are mixed with an animal feed ingredient and/or animal feed(s) and referred to as a mash feed. In certain embodiments, the mash feed is subsequently pelletized.

The animal feed may comprise any ingredient suitable for intake by aquatic animals (such as shrimp), e.g., comprising sources of protein, lipids, carbohydrates, salts, minerals and vitamins. The animal feed ingredients may be selected, and mixed in any proportions, suitable to meet the nutritional needs of the aquatic animals to be fed with the feed and/or to keep the raw material cost of the feed within desired limits and/or to achieve other desired properties of the feed. Non-limiting examples of animal feed ingredients may include one or more of the following materials: plant derived products, such as seeds, grains, leaves, roots, tubers, flowers, pods, husks, oil, soybean meal, soy protein isolate, potato protein powder, wheat, barley, corn, soybean oil, and corn gluten meal; animal derived products, such as fish meal, fish oil, milk powder, skim milk powder, bone extract, meat extract, blood extract, and the like; additives, such as minerals, vitamins, aroma compounds, and feed enhancing enzymes.

In particular embodiments, the animal feed may comprise 0-80% maize; and/or 0-80% sorghum; and/or 0-70% wheat; and/or 0-70% barley; and/or 0-30% oats; and/or 0-40% soybean meal; and/or 0-10% fish meal; and/or 0-20% whey.

The animal feed may comprise vegetable proteins. In particular embodiments, the protein content of the vegetable proteins is at least 10, 20, 30, 40, 50, 60, 70, 80, or 90% (w/w). Vegetable proteins may be derived from vegetable protein sources, such as legumes and cereals, for example, materials from plants of the families Fabaceae (Leguminosae), Cruciferaceae, Chenopodiaceae, and Poaceae, such as soy bean meal, lupin meal, rapeseed meal, and combinations thereof.

In a particular embodiment, the vegetable protein source is material from one or more plants of the family Fabaceae, e.g., soybean, lupine, pea, or bean. In another particular embodiment, the vegetable protein source is material from one or more plants of the family Chenopodiaceae, e.g., beet, sugar beet, spinach or quinoa. Other examples of vegetable protein sources are rapeseed, and cabbage. In another particular embodiment, soybean is a preferred vegetable protein source. Other examples of vegetable protein sources are cereals such as barley, wheat, rye, oat, maize (corn), rice, and sorghum.

In another embodiment, the animal feed may optionally comprise one or more suitable animal feed additives. Non-limiting examples of suitable animal feed additives include enzyme inhibitors, fat-soluble vitamins, water soluble vitamins, trace minerals, macro minerals, and combinations thereof.

In another embodiment, the animal feed may further optionally comprise one or more feed-additive ingredients. Non-limiting examples of feed-additive ingredients include coloring agents, aroma compounds, stabilizers, anti-microbial peptides (non-limiting examples of anti- microbial peptides (AMP's) are CAP18, Leucocin A, Tritrpticin, Protegrin-1 , Thanatin, Defensin, Ovispirin such as Novispirin (Robert Lehrer, 2000), and variants, or fragments thereof which retain antimicrobial activity), anti-fungal polypeptides (AFP's) (non-limiting examples include the Aspergillus giganteus, and Aspergillus niger peptides, as well as variants and fragments thereof which retain antifungal activity, as disclosed in WO 94/01459 and PCT/DK02/00289), and/or at least one other enzyme selected from amongst phytases EC 3.1.3.8 or 3.1.3.26; xylanases EC 3.2.1.8; galactanases EC 3.2.1.89; and/or beta-glucanases EC 3.2.1.4.

In still another embodiment, the animal feed may still further optionally include one or more fat- and water soluble vitamins, trace minerals and macro minerals. Usually fat- and water-soluble vitamins, as well as trace minerals form part of a so-called premix intended for addition to the feed, whereas macro minerals are usually separately added to the feed.

Non-limiting examples of fat-soluble vitamins include vitamin A, vitamin D3, vitamin E, and vitamin K, e.g., vitamin K3.

Non-limiting examples of water-soluble vitamins include vitamin B12, biotin and choline, vitamin B1 , vitamin B2, vitamin B6, niacin, folic acid and panthothenate, e.g. , Ca-D- panthothenate.

Non-limiting examples of trace minerals include boron, cobalt, chloride, chromium, copper, fluoride, iodine, iron, manganese, molybdenum, selenium, zinc, etc.

Non-limiting examples of macro minerals include calcium, magnesium, potassium, sodium, etc.

Method of treatment

In one embodiment, a method for improving the well-being, general condition or health of an aquatic animal is disclosed comprising administering to an aquatic animal such as shrimp one or more bacterial strains in an effective amount. In a particular embodiment, the method comprises contacting the gut of an aquatic animal with one or more bacterial strains. In an even more particular embodiment, the bacterial strains can control and/or inhibit species of Vibrio, Photobacterium and/or combinations thereof.

The invention relates to a method for treating a shrimp disease such as EMS comprising the step of administering one or more of the isolated Bacillus subtilis strains selected from the group consisting of:

(b) Bacillus subtilis strain MF1048 having deposit accession number NRRL B-67220 or a strain having all of the identifying characteristics of the Bacillus subtilis strain NRRL B-67220 or a mutant thereof to shrimp. The invention further relates to a method for treating a shrimp disease such as EMS comprising the step of administering one or more of isolated Bacillus subtilis strains having a 16S rDNA sequence selected from the group consisting of:

The method preferably comprises a daily administration for at least 2 days such as 3, 4,

5, 6, 7, 8, 9, 10 or more than 10 days (coherent or non-coherent days).

The method preferably comprises a daily dose of 1.5x10E6 CFU of the Bacillus subtilis strain according to the invention per aquatic animal such as per shrimp at 5 g each. The method comprises in another embodiment a daily dose of from 0.9x10E6 to 6.0x10E6 CFU of the Bacillus subtilis strain according to the invention per aquatic animal such as per shrimp at 5 g each. The method comprise in yet another embodiment a daily dose of from 0.1x10E6 to 1.0x10E7 CFU of the Bacillus subtilis strain according to the invention per aquatic animal such as per shrimp at 5 g each such as a dose selected from the group consisting of from 0.1x10E6 to 0.5x10E6, from 0.5x10E6 to 1.0x10E6, from 1.0x10E6 to 2.0x10E6, from 2.0x10E6 to 3.0x10E6, from 3.0x10E6 to 4.0x10E6, from 4.0x10E6 to 5.0x10E6, from 5.0x10E6 to 6.0x10E6, from 6.0x10E6 to 7.0x10E6, from 7.0x10E6 to 8.0x10E6, from 8.0x10E6 to 9.0x10E6, from 9.0x10E6 to 1.0x10E7 CFU of the Bacillus subtilis strain according to the invention per aquatic animal such as per shrimp at 5 g each, or any combination of these intervals.

In another preferred embodiment the treatment results in survival of at least 20% more of the shrimps compared to the control (no administration of the isolated Bacillus subtilis strains according to the invention) such as at least 30% more, such as at least 40% more, such as at least 50% more or such as at least 60% more survival. The invention also relates to an isolated Bacillus subtilis strains selected from the group consisting of:

(b) Bacillus subtilis strain MF1048 having deposit accession number NRRL B-67220 or a strain having all of the identifying characteristics of the Bacillus subtilis strain NRRL B-67220 or a mutant thereof

for use as a medicament.

The invention also relates to an isolated Bacillus subtilis strain having a 16S rDNA sequence selected from the group consisting of:

(a) 16s rDNA having at least 98.00%, e.g., at least 98.05%, at least 98.10%, at least 98.15%, at least 98.20%, at least 98.25%, at least 98.30%, at least 98.35%, at least 98.40%, at least 98.45%, at least 98.50%, at least 98.55%, at least 98.60%, at least 98.65%, at least 98.70%, at least 98.75%, at least 98.80%, at least 98.85%, at least 98.90%, at least 98.95%, at least 99.00%, at least 99.05%, at least 99.10%, at least 99.15%, at least 99.20%, at least 99.25%, at least 99.30%, at least 99.35%, at least 99.40%, at least 99.45%, at least 99.50%, at least 99.55%, at least 99.60%, at least 99.65%, at least 99.70%, at least 99.75%, at least 99.80%, at least 99.85%, at least 99.90%, at least 99.95% or 100% sequence identity to SEQ ID NO: 1 ; and (b) 16s rDNA having at least 98.00%, e.g., at least 98.05%, at least 98.10%, at least

98.15%, at least 98.20%, at least 98.25%, at least 98.30%, at least 98.35%, at least 98.40%, at least 98.45%, at least 98.50%, at least 98.55%, at least 98.60%, at least 98.65%, at least 98.70%, at least 98.75%, at least 98.80%, at least 98.85%, at least 98.90%, at least 98.95%, at least 99.00%, at least 99.05%, at least 99.10%, at least 99.15%, at least 99.20%, at least 99.25%, at least 99.30%, at least 99.35%, at least 99.40%, at least 99.45%, at least 99.50%, at least 99.55%, at least 99.60%, at least 99.65%, at least 99.70%, at least 99.75%, at least 99.80%, at least 99.85%, at least 99.90%, at least 99.95% or 100% sequence identity to SEQ ID NO: 2.

The invention further relates to isolated Bacillus subtilis strains selected from the group consisting of:

for treatment of a shrimp disease such as EMS. The invention also relates to an isolated Bacillus subtilis strain having a 16S rDNA sequence selected from the group consisting of:

for treatment of a shrimp disease such as EMS.

In another embodiment the invention relates to use of a Bacillus subtilis strain selected from the group consisting of:

In still yet another embodiment of the method, the one or more bacterial strains are present in the form of a stable spore. In still a further embodiment of the method, the stable spore will germinate in the gut of the aquatic animal.

In a particular embodiment, the method comprises administering to an aquatic animal such as shrimp one or more bacterial strains described herein, wherein the bacterial count of the one or more bacterial strains is between 1x10² and 1x10¹² CFU/g of the composition, particularly 1x10⁴ and 1x10⁹ CFU/g of the composition, and more particularly 1x10⁵ and 5x10⁸ CFU/g of the composition. In a more particular embodiment the bacterial count of the one or more bacterial strains described herein is between 1x10⁶ and 1x10⁸ CFU/g of the composition. In an even more particular embodiment, the method comprises the step of contacting the gut of an aquatic animal such as shrimp with one or more bacterial strains wherein the one or more bacterial strains are selected from the group consisting of:

In a particular embodiment, the method comprises the step of contacting the gut of an aquatic animal such as shrimp with one or more of the bacterial strains described herein wherein the one or more bacterial strains improve the health of the aquatic animal by increasing the weight or the aquatic animal. In still another embodiment, the one or more bacterial strains improve the health of the aquatic animal by enhancing the immune system and/or immune response of the aquatic animal. In yet another embodiment, the one or more bacterial strains improve the health of the aquatic animal by improving the overall health of the gut of the aquatic animal. Non-limiting examples of improved gut health include reducing gut inflammation, increasing the length and/or surface area of intestinal villi, increasing intestinal crypt depth, improving nutrient absorption by the gut, maintaining and/or improving healthy gut microflora, or combinations thereof. In still yet another embodiment the one or more bacterial strains improve the health of the aquatic animal by controlling pathogenic microorganisms. In a particular embodiment, the one or more bacterial strains improve the health of the aquatic animal by controlling pathogenic microorganisms in the gut of the aquatic animal.

In a further embodiment, the one or more bacterial strains that control pathogenic microorganisms belonging to the genus Vibrio and/or Photobacterium strains. In a more particular embodiment, the one or more bacterial strains that control pathogenic microorganisms are selected from the group consisting of Vibrio fischeri, Vibrio vulnificus, Vibrio fluvialis, Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio mimicus, Vibrio cholera, Vibrio harveyi, Photobacterium phosphoreum, Photobacteria damselae subspecies damselae, Photobacterium damselae subspecies piscicida or any combination thereof.

In a particular embodiment, the aquatic animal is a fish (e.g., catfish, tilapia, trout, salmon, perch, bass, tuna, wahoo, tuna, swordfish, marlin, grouper, sturgeon, snapper, eel and walleye, sharks, rays, and skates), a crustacean (e.g., crabs, lobsters, crayfish, shrimp, krill, and prawn) a mollusk (e.g., snails, slugs, conch, squid, octopus, cuttlefish, clams, oysters, scallops, and mussels), and combinations thereof. In an even more particular embodiment, the aquatic animal is a fish, a shrimp, a lobster, an eel, a crayfish, a prawn, an oyster, a mussel, a cockle, or a combination thereof. In an even more particular embodiment, the aquatic animal is a catfish, a tilapia, a shrimp or combinations thereof.

The invention also relates to use of the bacterial strains according to the invention in aquaculturing.

Manufacturing Processes

In still another embodiment, methods described herein comprise administering to an aquatic animal and/or contacting the gut of an aquatic animal such as shrimp with a composition described herein. In a particular embodiment, the composition is an ingredient in an animal feed (i.e., a mash feed) as described herein.

In a preferred embodiment bacterial spores according to the invention are blended into a Calcium carbonate carrier (such as #1491 Vicron 45-3FG), to form the product (target is preferably 1x10e8 CFU/g to 1x10e10 CFU/g such as, e.g., 1x10e9 CFU/g) from an initial spray- dried spore concentrate (usually at 5-9x10e9 CFU/g to 5-9x10e11 such as, e.g., CFU/g 5- 9x10e10 CFU/g). This formulation is typically stable for at least 2 years at RT (23°C). For use, the product is typically suspended 1 :5 g/g to 1 :20 g/g such as 1 : 10 g/g in, e.g., water or vegetable oil to provide a suspension of, e.g., 1x10e8 CFU/ml. This material is preferably sprayed evenly on the pre-formed feed pellets, e.g., at a 5% to 20% v/g rate such as a 10% v/g rate, and dried, e.g., overnight at 21-30°C. The resultant recoverable level of spores according to the invention on the pellets is preferably 6-8 x 10e5 CFU/g feed to 6-8 x 10e8 CFU/g feed such as, e.g., about 6-8 x 10e6 CFU/g feed. In a specific embodiment the bacterial concentration on the feed pellets is from 1 x 10e5 CFU/g feed to 8 x 10e8 CFU/g feed such as from 1 x 10e5 CFU/g feed to 8 x 10e5 CFU/g feed, such as from 8 x 10e5 CFU/g feed to 1 x 10e6 CFU/g feed, such as from 1 x 10e6 CFU/g feed to 1 x 10e7 CFU/g feed, such as from 1 x 10e7 CFU/g feed to 1 x 10e8 CFU/g feed, or any combination of these intervals.

In an even more particular embodiment, the feed mash is pelleted. Pelleting processes are known in the art. In a particular embodiment, the pellets are manufactured through a pelleting process. In another embodiment the pellets are manufactured through an extrusion process.

In an embodiment, the feed mixture (mash feed) may be prepared by mixing the composition comprising the one or more bacterial strains with desired feed components. In a particular embodiment the mash feed may be conditioned or unconditioned.

In one embodiment the mash feed is conditioned prior to the pelleting process. In a particular embodiment, the mash feed is led to a conditioner e.g., a cascade mixer with steam injection. The feed is in the conditioner heated up to a specified temperature such as, e.g., 60- 120°C, e.g., 60°C, 70°C, 80°C, 90°C, 100°C, 105°C, 1 10°C, 115°C, or 120°C by injecting steam, measured at the outlet of the conditioner. The residence time can be variable from seconds to minutes and even hours. Such as 5 seconds, 10 seconds, 15 seconds, 30 seconds, 1 minute, 2 minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4, hours, 5 hours, 6 hours, 7 hours, 8, hours, 9, hours, 10 hours, 1 1 hours, 12 hours, and up to 24 hours and beyond. In a particular embodiment, the process temperature during steam treatment is at least 60°C. In a more particular embodiment, the process temperature during steam treatment is at least 70°C. In an even more particular embodiment, the process temperature during steam treatment is at least 80°C. In a most particular embodiment, the process temperature during steam treatment is at least 90°C.

In another embodiment, the pelleting process is an extrusion process. Typical, extrusion processes for manufacturing feed pellets are known to those skilled in the art. Extrusion or pelletized products, wherein the feed mixture (mash feed) is pressed to pellets or under pressure is extruded through a small opening and cut into particles which are subsequently dried. Such particles usually have a considerable size because of the material in which the extrusion opening is made (usually a plate with bore holes) sets a limit on the allowable pressure drop over the extrusion opening. Also, very high extrusion pressures when using a small opening increase heat generation in the mash feed. (Michael S. Showell (editor); Powdered detergents; Surfactant Science Series; 1998; vol. 71 ; page 140-142; Marcel Dekker).

In a particular embodiment, the mash feed is led to an extruder to form pellets of variable length from the extrudate. The extrusion apparatus may be any screw-type extruder known in the art. In a particular embodiment, the extruder is a double screwed extruder, e.g., a Werner & Pfleiderer Type continua 37" extruder. Extrusion parameters (e.g., capacity, screw speed, die diameter, drying temperatures, drying time, etc.) are dependent upon the particular extrusion process and/or extrusion apparatuses employed.

In an embodiment, the screw speed of the extruder is 1-1 ,000 RPM. In a more particular embodiment, the screw speed of the extruder is 100 RPM. In an even more particular embodiment, the screw speed of the extruder is 150 RPM. In yet an even more particular embodiment, the screw speed of the extruder is 200 RPM. In still an even more particular embodiment, the screw speed of the extruder is 250 RPM. In still yet an even more particular embodiment, the screw speed of the extruder is 300 RPM.

In an embodiment, the die diameter is 0.5-5.0 mm. In a more particular embodiment, the die diameter is 0.5 mm. In an even more particular embodiment, the die diameter is 1.0 mm. In yet an even more particular embodiment, the die diameter is 1.5 mm. In a most particular embodiment, the die diameter is 2.0 mm.

The pellets are placed then dried for a specified time, e.g., at least 15 minutes, preferably

20 minutes, at temperatures of 60-100°C, preferably 90-100°C, more preferably 90°C, even more preferably 95°C, even still more preferably 100°C. PREFERRED EMBODIMENTS

Preferred embodiments of the invention are given in the set of items herein below. 1. An isolated Bacillus subtilis strain selected from the group consisting of

2. An isolated Bacillus subtilis strain which has a 16S rDNA sequence selected from the group consisting of:

(a) 16s rDNA having at least 98.00%, e.g., at least 98.05%, at least 98.10%, at least

98.15%, at least 98.20%, at least 98.25%, at least 98.30%, at least 98.35%, at least 98.40%, at least 98.45%, at least 98.50%, at least 98.55%, at least 98.60%, at least 98.65%, at least 98.70%, at least 98.75%, at least 98.80%, at least 98.85%, at least 98.90%, at least 98.95%, at least 99.00%, at least 99.05%, at least 99.10%, at least 99.15%, at least 99.20%, at least 99.25%, at least 99.30%, at least 99.35%, at least 99.40%, at least 99.45%, at least 99.50%, at least 99.55%, at least 99.60%, at least 99.65%, at least 99.70%, at least 99.75%, at least 99.80%, at least 99.85%, at least 99.90%, at least 99.95% or 100% sequence identity to SEQ ID NO: 1 ; and

3. The Bacillus subtilis strain according to item 1 or 2, wherein the strain has activity against shrimp pathogens, and/or inhibits/limits growth of shrimp pathogens. 4. The Bacillus subtilis strain according to item 3, wherein the shrimp pathogen is a Vibrio strain. 5. The Bacillus subtilis strain according to item 4, wherein the Vibrio strain is selected from the group consisting of Vibrio fischeri, Vibrio vulnificus, Vibrio fluvialis, Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio mimicus, Vibrio cholera, Vibrio harveyi or any combination thereof. 6. The Bacillus subtilis strain according to item 3, wherein the shrimp pathogen is a Photobacterium strain.

7. The Bacillus subtilis strain according to item 6, wherein the Photobacterium strain is selected from the group consisting of Photobacterium phosphoreum, Photobacteria damselae subspecies damselae, Photobacterium damselae subspecies piscicida or any combination thereof.

8. The Bacillus subtilis strain according to any of items 1 to 7, wherein the Bacillus subtilis strain results in a shrimp survival of at least 15% after 104 hours (after feeding shrimps a standard diet containing the Vibrio parahaemolyticus pathogen and the Bacillus subtilis strain as described in Example 2) such as a shrimp survival of at least 20%, at least 25% or at least 30% after 104 hours.

9. The Bacillus subtilis strain according to any of items 1 to 8, wherein the strain is stable when subjected to pressures achieved during an extrusion process for pelleting and the temperature during said extrusion process is 105°C or lower, such as ranging from 80°C to 105°C.

10. The Bacillus subtilis strain according to any of items 1 to 9, wherein the strain is stable at pressures ranging from 1 bar to 40 bar such as from 10 bar to 40 bar, from 15 bar to 40 bar, from 20 bar to 40 bar, from 35 bar to 37 bar, or at 36 bar, and the temperature is 105°C or lower, such as ranging from 80°C to 105°C.

11. The Bacillus subtilis strain according to any of items 1 to 10, wherein the strain is stable when subjected to temperatures achieved during an extrusion process for pelleting such as at temperatures ranging from 80°C to 105°C, from 90°C to 105°C, or from 95°C to 105°C.

12. The Bacillus subtilis strain according to any of items 1 to 11 , wherein the strain is stable when subjected to an extrusion process having a pressure of 1 bar to 40 bar, a temperature from 80°C to 105°C, and the strain controls one or more pathogenic microorganisms.

13. A composition comprising one or more of the isolated Bacillus subtilis strains according to any of items 1 to 12 and a carrier. 14. A composition comprising one or more isolated Bacillus subtilis strains having a 16S rDNA sequence selected from the group consisting of:

(b) 16s rDNA having at least 98.00%, e.g., at least 98.05%, at least 98.10%, at least 98.15%, at least 98.20%, at least 98.25%, at least 98.30%, at least 98.35%, at least 98.40%, at least 98.45%, at least 98.50%, at least 98.55%, at least 98.60%, at least 98.65%, at least 98.70%, at least 98.75%, at least 98.80%, at least 98.85%, at least 98.90%, at least 98.95%, at least 99.00%, at least 99.05%, at least 99.10%, at least 99.15%, at least 99.20%, at least 99.25%, at least 99.30%, at least 99.35%, at least 99.40%, at least 99.45%, at least 99.50%, at least 99.55%, at least 99.60%, at least 99.65%, at least 99.70%, at least 99.75%, at least 99.80%, at least 99.85%, at least 99.90%, at least 99.95% or 100% sequence identity to SEQ ID NO: 2 and a carrier.

15. The composition according to any of items 13 to 14, wherein the one or more of the isolated Bacillus subtilis strains is in spore form. 16. The composition according to any of items 13 to 15, wherein the carrier is selected from the group consisting of liquids, slurries and/or solids.

17. The composition according to any of items 13 to 16, wherein the composition is an animal feed such as an aquatic animal feed such as shrimp feed or an animal feed additive such as an aquatic animal feed additive such as shrimp feed additive.

18. The composition according to item 17, wherein the animal feed is feed pellets such as sinking feed pellets or extruded pellets such as floating pellets. 19. The composition according to item 18, wherein the feed pellets are surface coated with the Bacillus subtilis strains according to any of items 1 to 12. 20. The composition according to any of items 13 to 19, wherein the composition comprises from 10⁴ to 10⁸ CFU/g feed such as from 10⁵to 10⁷ CFU/g feed or such as from 5 x 10⁶to 9 x 10⁶ CFU/g feed of the Bacillus subtilis strain. 21. A method for treating a shrimp disease such as Early Mortality Syndrome (EMS) comprising the step of administering the one or more of the isolated Bacillus subtilis strains according to any of items 1 to 12 or the composition according to any of items 13 to 20 to the shrimp. 22. A method for treating a virulent Vibrio parahaemolyticus associated disease such as EMS in shrimps comprising the step of administering the one or more of the isolated Bacillus subtilis strains according to any of items 1 to 12 or the composition according to any of items 13 to 20 to the shrimp. 23. The method according to item 21 or 22, wherein the administering comprises a daily administration for at least 2 days such as 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 days (coherent or non-coherent days).

24. The method according to any of items 21 to 23, wherein the treatment results in survival of at least 10% more of the shrimps compared to the control (no administration of the isolated Bacillus subtilis strains according to any of items 1 to 12 or the composition according to any of items 13 to 20), such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, or such as at least 70% higher survival. 25. The isolated Bacillus subtilis strain according to any of items 1 to 12 or the composition according to any of items 13 to 20 for use as a medicament.

26. The isolated Bacillus subtilis strain according to any of items 1 to 12 or the composition according to any of items 13 to 20 for use in the treatment of a shrimp disease such as EMS.

27. The isolated Bacillus subtilis strain according to any of items 1 to 12 or the composition according to any of items 13 to 20 for use in the treatment of a virulent Vibrio parahaemolyticus associated disease such as EMS in shrimp. 28. Use of one or more Bacillus subtilis strain(s) selected from the group consisting of:

29. Use of one or more isolated Bacillus subtilis strains having a 16S rDNA sequence selected from the group consisting of:

(b) 16s rDNA having at least 98.00%, e.g., at least 98.05%, at least 98.10%, at least 98.15%, at least 98.20%, at least 98.25%, at least 98.30%, at least 98.35%, at least 98.40%, at least 98.45%, at least 98.50%, at least 98.55%, at least 98.60%, at least 98.65%, at least 98.70%, at least 98.75%, at least 98.80%, at least 98.85%, at least 98.90%, at least 98.95%, at least 99.00%, at least 99.05%, at least 99.10%, at least 99.15%, at least 99.20%, at least 99.25%, at least 99.30%, at least 99.35%, at least 99.40%, at least 99.45%, at least 99.50%, at least 99.55%, at least 99.60%, at least 99.65%, at least 99.70%, at least 99.75%, at least 99.80%, at least 99.85%, at least 99.90%, at least 99.95% or 100% sequence identity to SEQ ID NO: 2 for treatment of a virulent Vibrio parahaemolyticus associated disease such as EMS in shrimps. 30. The use according to item 28, wherein the Bacillus subtilis strain has a 16S rDNA sequence selected from the group consisting of:

(b) 16s rDNA having at least 98.00%, e.g., at least 98.05%, at least 98.10%, at least 98.15%, at least 98.20%, at least 98.25%, at least 98.30%, at least 98.35%, at least 98.40%, at least 98.45%, at least 98.50%, at least 98.55%, at least 98.60%, at least 98.65%, at least 98.70%, at least 98.75%, at least 98.80%, at least 98.85%, at least 98.90%, at least 98.95%, at least 99.00%, at least 99.05%, at least 99.10%, at least 99.15%, at least 99.20%, at least 99.25%, at least 99.30%, at least 99.35%, at least 99.40%, at least 99.45%, at least 99.50%, at least 99.55%, at least 99.60%, at least 99.65%, at least 99.70%, at least 99.75%, at least 99.80%, at least 99.85%, at least 99.90%, at least 99.95% or 100% sequence identity to SEQ ID NO: 2; or

wherein one Bacillus subtilis strain has a 16S rDNA sequence consisting of (a) and a second Bacillus subtilis strain has a 16S rDNA sequence consisting of (b).

31. Use of the isolated Bacillus subtilis strain according to any of items 1 to 12 or the composition according to any of items 13 to 20 in the manufacture of a medicament.

32. Use of the isolated Bacillus subtilis strain according to any of items 1 to 12 or the composition according to any of items 13 to 20 in the manufacture of a medicament for the treatment of a shrimp disease such as EMS.

33. Use of the isolated Bacillus subtilis strain according to any of items 1 to 12 or the composition according to any of items 13 to 20 in the manufacture of a medicament for the treatment of a virulent Vibrio parahaemolyticus associated disease such as EMS in shrimp. EXAMPLES

Example 1

Deposit of Biological Material

The following biological material has been deposited under the terms of the Budapest Treaty with the Agricultural Research Service Patent Culture Collection (NRRL), Northern Regional Research Center, 1815 University Street, Peoria, Illinois, 61604, USA, and given the following accession numbers:

The Bacillus subtilis strain 014VRQ was isolated from a soil sample from Virginia, USA on May 5, 2006 by Michael Frodyma from Novozymes North America Inc. The Bacillus subtilis strain MF1048 was isolated from a soil sample from Virginia, USA on October 4, 2005 by Michael Frodyma from Novozymes North America Inc.

The strain has been deposited under conditions that assure that access to the culture will be available during the pendency of this patent application to one determined by foreign patent laws to be entitled thereto. The deposit represents a substantially pure culture of the deposited strain. The deposit is available as required by foreign patent laws in countries wherein counterparts of the subject application, or its progeny are filed. However, it should be understood that the availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action.

Example 2

Table 1 shows the relative survival of eight shrimp feed diets each containing one of 8 Bacillus candidate strains. Shrimp fed either Bacillus subtilis strain NRRL B-67221 (014VRQ) or Bacillus subtilis strain NRRL B-67220 (MF1048) had significantly greater survival than six other Bacillus strains. Three tanks per treatment, with ten shrimp in each 10 gallon tank (30 shrimp per treatment) were used to compare shrimp survival rates over a 104-hour period. All shrimp were fed standard amounts of commercial shrimp feed pellets which had been treated with the indicated Bacillus organisms by surface spray directly on the feed pellet 24 hours prior to feeding. Specifically, a spore-suspension in water of each treatment bacterium was prepared at a preferable concentration of 0.6 x 10E8 CFU/ml to 1.2 x 10E8 CFU/ml. The suspension was sprayed evenly onto dry feed pellets (sinking) at a preferable rate of 1.0 ml spore suspension to 10.0 g of feed pellets, and allowed to dry overnight at 4-23°C. The resulting recoverable bacterial concentration on the pellets was 0.6-1.2 x 10E7 CFU/g-feed. The treated pellets (after subsequent pathogen inoculation; see below) were added to each specified Tank at a rate of 3% (w/w) of shrimp body weight per day. For all shrimp in this study, with an average weight of 5 g/shrimp, the target inoculation was 0.15 g-feed/shrimp/day, or 1.5 x 10E6 CFU/shrimp per day. At test initiation (Time 0 hrs.), all shrimp (100%) were healthy and similar in size.

The Vibrio parahaemolyticus pathogen was introduced to each test group via feed inoculation. A starter V. parahaemolyticus culture (OD₆oo at 0.5) can be grown in various commercial media, such as Tripticase Soy Broth (TSB), Luria Broth (LB), or other suitable media. For these studies, V. parahaemolyticus was inoculated into Trypticase Soy Broth supplemented with an additional 2% NaCI (TSB2+) and cultured for 18 hours in an incubator that was set to agitate at 250 rpm at 30°C (Inoculate). V. parahaemolyticus strain concentrations grown throughout the treatment were measured using standard plate counting methods to insure equivalency for all inoculum time points.

Inoculate (18-hr culture with V. parahaemolyticus pathogen) was applied to feed previously treated with the test Bacilllus spores (as described above) at a 1 : 1 ratio (milliliters inoculate to gram feed). The Inoculate was permitted to absorb to the feed for three minutes before it was introduced to the shrimp tank. Shrimp survival was assessed every eight and 24 hours during the test duration. Prior to each feeding, solid waste was suctioned out of the water to reduce nitrogenous waste that may contribute to poor water conditions and dead shrimp were removed if found. Feed inoculated with V. parahaemolyticus was introduced to the shrimp tanks every 24 hours until 50% mortality of the shrimp was observed in the Control group (no added Bacillus bacteria). After that time, only standard (non-disease) feed was added to all tanks.

Shrimp Survival (%)

Table 1 : Shrimp survival after feeding standard diets containing the Vibrio parahaemolyticus pathogen (High Level) plus various Bacillus strains.

Table 2 shows the relative survival of four shrimp feed diets each containing Bacillus subtilis strain NRRL B-67221 (014VRQ) from four separate spore lot preparations (014VRQ- NA; 014VRQ-HA; 014VRQ-NASD; and 014VRQ-ASD). In is study, all four Bacillus spore preparations demonstrated disease survival of 40% to 50% compared with the non-treated, disease-inoculated Control (NTC+). Shrimp survival was 100% with feed not inoculated with the Vibrio disease and not treated with the Bacillus strains (NTC-). Shrimp Survival (%)

Table 2: Shrimp survival after feeding standard diets containing the Vibrio parahaemolyticus pathogen (moderate level) plus Bacillus strain 014VRQ (NRRL B-67221) from four different spore lots preparations.

Claims

1. An isolated Bacillus subtilis strain selected from the group consisting of

(a) Bacillus subtilis strain 014VRQ having deposit accession number NRRL B-67221 or a strain having all of the identifying characteristics of Bacillus subtilis strain NRRL B-67221 or a mutant thereof; and

(b) Bacillus subtilis strain MF1048 having deposit accession number NRRL B-67220 or a strain having all of the identifying characteristics of Bacillus subtilis strain NRRL B-67220 or a mutant thereof.

(b) 16s rDNA having at least 98.00%, e.g., at least 98.05%, at least 98.10%, at least 98.15%, at least 98.20%, at least 98.25%, at least 98.30%, at least 98.35%, at least 98.40%, at least 98.45%, at least 98.50%, at least 98.55%, at least 98.60%, at least 98.65%, at least 98.70%, at least 98.75%, at least 98.80%, at least 98.85%, at least 98.90%, at least 98.95%, at least 99.00%, at least 99.05%, at least 99.10%, at least 99.15%, at least 99.20%, at least 99.25%, at least 99.30%, at least 99.35%, at least 99.40%, at least 99.45%, at least 99.50%, at least 99.55%, at least 99.60%, at least 99.65%, at least 99.70%, at least 99.75%, at least 99.80%, at least 99.85%, at least 99.90%, at least 99.95% or 100% sequence identity to SEQ ID NO: 2. 3. The Bacillus subtilis strain of claim 1 or 2, wherein the strain has activity against shrimp pathogens, and/or inhibits/limits growth of shrimp pathogens.

4. The Bacillus subtilis strain of any of claims 1 to 3, wherein the Bacillus subtilis strain results in a shrimp survival of at least 15% after 104 hours (after feeding shrimps a standard diet containing the Vibrio parahaemolyticus pathogen and the Bacillus subtilis strain as described in Example 2) such as a shrimp survival of at least 20%, at least 25% or at least 30% after 104 hours.

5. A composition comprising one or more of the isolated Bacillus subtilis strains of any of claims 1 to 4 and a carrier. 6. A composition comprising one or more isolated Bacillus subtilis strains having a 16S rDNA sequence selected from the group consisting of:

98.15%, at least 98.20%, at least 98.25%, at least 98.30%, at least 98.35%, at least 98.40%, at least 98.45%, at least 98.50%, at least 98.55%, at least 98.60%, at least 98.65%, at least 98.70%, at least 98.75%, at least 98.80%, at least 98.85%, at least 98.90%, at least 98.95%, at least 99.00%, at least 99.05%, at least 99.10%, at least 99.15%, at least 99.20%, at least 99.25%, at least 99.30%, at least 99.35%, at least 99.40%, at least 99.45%, at least 99.50%, at least 99.55%, at least 99.60%, at least 99.65%, at least 99.70%, at least 99.75%, at least 99.80%, at least 99.85%, at least 99.90%, at least 99.95% or 100% sequence identity to SEQ ID NO: 2 and a carrier. 7. The composition of claim 5 or 6, wherein the one or more of the isolated Bacillus subtilis strains is in spore form.

8. The composition of any of claims 5 to 7, wherein the carrier is selected from the group consisting of liquids, slurries and/or solids.

9. The composition of any of claims 5 to 8, wherein the composition is an animal feed such as an aquatic animal feed such as shrimp feed or an animal feed additive such as an aquatic animal feed additive such as shrimp feed additive. 10. A method for treating a shrimp disease such as Early Mortality Syndrome (EMS) comprising the step of administering the one or more of the isolated Bacillus subtilis strains of any of claims 1 to 4 or the composition of any of claims 5 to 9 to the shrimp.

11. The method of claim 10, wherein the treatment results in survival of at least 10% more of the shrimps compared to the control (no administration of the isolated Bacillus subtilis strains of any of claims 1 to 4 or the composition of any of claims 5 to 9), such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, or such as at least 70% higher survival.

12. The isolated Bacillus subtilis strains of any of claims 1 to 4 or the composition of any of claims 5 to 9 for use as a medicament.

13. The isolated Bacillus subtilis strains of any of claims 1 to 4 or the composition of any of claims 5 to 9 for use in the treatment of a shrimp disease such as EMS.

14. Use of one or more Bacillus subtilis strain(s) selected from the group consisting of:

15. Use of one or more isolated Bacillus subtilis strains having a 16S rDNA sequence selected from the group consisting of:

(b) 16s rDNA having at least 98.00%, e.g., at least 98.05%, at least 98.10%, at least 98.15%, at least 98.20%, at least 98.25%, at least 98.30%, at least 98.35%, at least 98.40%, at least 98.45%, at least 98.50%, at least 98.55%, at least 98.60%, at least 98.65%, at least 98.70%, at least 98.75%, at least 98.80%, at least 98.85%, at least 98.90%, at least 98.95%, at least 99.00%, at least 99.05%, at least 99.10%, at least 99.15%, at least 99.20%, at least 99.25%, at least 99.30%, at least 99.35%, at least 99.40%, at least 99.45%, at least 99.50%, at least 99.55%, at least 99.60%, at least 99.65%, at least 99.70%, at least 99.75%, at least 99.80%, at least 99.85%, at least 99.90%, at least 99.95% or 100% sequence identity to SEQ ID NO: 2 for treatment of a virulent Vibrio parahaemolyticus associated disease such as EMS in shrimps.