Antiviral and antibacteria agents based on quaternary ammonium compound complexed with boric acid and its derivatives
10899776 · 2021-01-26
Assignee
Inventors
Cpc classification
C07F5/05
CHEMISTRY; METALLURGY
Y02A40/81
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
A23L33/30
HUMAN NECESSITIES
A23V2002/00
HUMAN NECESSITIES
International classification
A23L33/00
HUMAN NECESSITIES
C07F5/05
CHEMISTRY; METALLURGY
Abstract
The present document describes compounds resulting from the complexation of quaternary ammonium compounds with boric acid and/or its derivatives, and methods of making the same, and methods of using the same for the treatment of pathogenic infections.
Claims
1. An ion of formula I, or a pharmaceutically acceptable salts thereof, and stereoisomers thereof: ##STR00006## wherein R.sup.1 is C.sub.1-6 alkylene, R.sup.2, R.sup.3, and R.sup.4 are independently C.sub.1-6 alkyl optionally substituted with one or more OH, and R.sup.5 is selected from OB(OH).sub.2, ##STR00007## with the proviso that when R.sup.1 is CH.sub.2CH.sub.2 and R.sup.2, R.sup.3, and R.sup.4 are CH.sub.3, R.sup.5 is different than OB(OH).sub.2.
2. The ion of claim 1, or a pharmaceutically acceptable salts thereof, and stereoisomers thereof, wherein R.sup.1 is CH.sub.2CH.sub.2.
3. The ion of claim 1, or a pharmaceutically acceptable salts thereof, and stereoisomers thereof, wherein any one of R.sup.2, R.sup.3, and R.sup.4 is independently CH.sub.3, CH.sub.2CH.sub.3, or CH.sub.2CH.sub.2CH.sub.3.
4. The ion of claim 1, or a pharmaceutically acceptable salt thereof, wherein R.sup.1 is CH.sub.2CH.sub.2, and R.sup.2, R.sup.3, and R.sup.4 is independently CH.sub.3.
5. The ion of claim 1, or a pharmaceutically acceptable salt thereof, wherein said ion is selected from the following ions: ##STR00008## or a combination thereof.
6. A mixture of the ion of claim 1, or a pharmaceutically acceptable salt thereof, comprising the following-ions of formula I,: ##STR00009## in combination with the following ion ##STR00010##
7. A pharmaceutical composition comprising an ion of any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
8. The pharmaceutical composition of claim 7, for use in the treatment of a pathogenic infection in a subject.
9. The pharmaceutical composition of claim 7, for use in the treatment of a pathogenic infection in a crustacean in need thereof.
10. The pharmaceutical composition of claim 7, wherein said subject is selected from the group consisting of a mammal, a fish, a bird, and a crustacean.
11. The pharmaceutical composition of claim 10, wherein: said mammal is selected from the group consisting of a human, a bovine, an equine, and an ungulate; said fish is selected from the group consisting of a hagfish, a lamprey, a cartilaginous fish and a bony fish; said bird is selected from the group consisting of chicken, a turkey, and a fowl; and said crustacean is selected from the group consisting of a shrimp, a crab, a lobster, a langouste.
12. The pharmaceutical composition of claim 8, wherein said pathogenic infection is caused by a virus, a microorganism, or combinations thereof.
13. The pharmaceutical composition of claim 12, wherein: said virus is one of more of White Spot Syndrome Virus (WSSV), Taura Syndrome Virus (TSV), Yellow Head Virus (YHV), Infectious Hypodermal and Haematopoietic Necrosis (IHHNV), Spherical Baculovirus, Spawner-isolated Mortality Virus Disease, Spring Viremia of Carp (SVC caused by Rhabdoviruses), Koi Herpes Virus (KHV), Large Mouth Bass Virus (LMBV), and Baculovirus penaei (BP); and said microorganism is one or more of Vibrio parahaemolyticus, Vibrio harveyi, V. splendidus, V. parahaemolyticus, V. alginolyticus, V. anguillarum, V. vulnificus, V. campbelli, V. fischeri, V. damsella, V. pelagicus, V. orientalis, V. ordalii, V. mediterrani, V. logei, an Enterobacteriacae, such as an Escheria coli, a Salmonella, a Shigella; Clostridium botulinum, Listeria monocytogenes.
14. The pharmaceutical composition of claim 7, wherein said pharmaceutical composition is a dietary composition.
15. A method of treating or preventing a pathogenic infection in a fish or a crustacean in need thereof comprising: administering a therapeutically effective amount an ion of claim 1, or a pharmaceutically acceptable salt thereof.
16. The method of claim 15, wherein said crustacean is selected from the group consisting of a shrimp, a crab, a lobster, and a langouste, and wherein said fish is selected from the group consisting of fish a hagfish, a lamprey, a cartilaginous fish and a bony fish.
17. The method of claim 15, wherein said pathogenic infection is caused by a virus, a microorganism, or combinations thereof.
18. The method of 17, wherein said virus is one of more of White Spot Syndrome Virus (WSSV), Taura Syndrome Virus (TSV), Yellow Head Virus (YHV), Infectious Hypodermal and Haematopoietic Necrosis (IHHNV), Spherical Baculovirus, Spawner-isolated Mortality Virus Disease, Spring Viremia of Carp (SVC caused by Rhabdoviruses), Koi Herpes Virus (KHV), Large Mouth Bass Virus (LMBV), and Baculovirus penaei (BP) and said microorganism is one or more of Vibrio parahaemolyticus, Vibrio harveyi, V. splendidus, V. parahaemolyticus, V. alginolyticus, V. anguillarum, V. vulnificus, V. campbelli, V. fischeri, V. damsella, V. pelagicus, V. orientalis, V. ordalii, V. mediterrani, V. logei, an Enterobacteriacae.
19. The method of claim 18, wherein said Enterobacteriacae is one or more of an Escheria coli, a Salmonella, a Shigella.
20. The method of claim 15, wherein administering is by feeding said ion, or said pharmaceutically acceptable salt thereof, to said crustacean.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1) Further features and advantages of the present disclosure will become apparent from the following detailed description, taken in combination with the appended drawings, in which:
(2)
(3)
(4)
(5)
(6)
(7)
(8)
(9)
(10)
(11)
(12)
(13)
(14)
(15)
(16)
(17)
DEFINITIONS
(18) Alkyl, as well as other groups having the prefix alk, such as alkoxy and alkanoyl, means carbon chains which may be linear or branched, and combinations thereof, unless the carbon chain is defined otherwise. Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, sec- and tert-butyl, pentyl, hexyl, heptyl, octyl, nonyl, and the like. Where the specified number of carbon atoms permits, e.g., from C3-10, the term alkyl also includes cycloalkyl groups, and combinations of linear or branched alkyl chains combined with cycloalkyl structures. When no number of carbon atoms is specified, C1-6 is intended.
(19) Aryl means a mono- or polycyclic aromatic ring system containing carbon ring atoms. The preferred aryls are monocyclic or bicyclic 6-10 membered aromatic ring systems. Phenyl and naphthyl are preferred aryls. The most preferred aryl is phenyl.
(20) The term cycloalkyl is a subset of alkyl and means a saturated carbocyclic ring having a specified number of carbon atoms. Examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and the like. A cycloalkyl group generally is monocyclic unless stated otherwise. Cycloalkyl groups are saturated unless otherwise defined Cycloalkanes consist of only carbon (C) and hydrogen (H) atoms and are saturated because there are no multiple CC bonds to hydrogenate (add more hydrogen to). A general chemical formula for cycloalkanes would be C.sub.nH.sub.2(n+1g) where n=number of C atoms and g=number of rings in the molecule. For those cycloalkanes that have one ring in their molecules, cycloalkanes can be treated as isomers of their alkene counterparts, for example, cyclopropane and propene both have the chemical formula C.sub.3H.sub.6. Cycloalkanes with a single ring are named analogously to their normal alkane counterpart of the same carbon count: cyclopropane, cyclobutane, cyclopentane, cyclohexane, etc. The larger cycloalkanes, with greater than 20 carbon atoms are typically called cycloparaffins.
(21) The term composition as used herein is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts. Such term in relation to pharmaceutical composition is intended to encompass a product comprising the active ingredient(s) and the inert ingredient(s) that make up the carrier, as well as any product which results, directly or indirectly, from combination, complexation or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients. Accordingly, the pharmaceutical compositions of the present invention encompass any composition made by admixing a compound of the present invention and a pharmaceutically acceptable carrier. By pharmaceutically acceptable or acceptable it is meant the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
(22) The terms administration of, administer and/or administering a compound should be understood to mean providing, to dispense, to mete out, give to, a compound of the invention of the invention to the individual or subject in need of treatment by any suitable means, such as oral administration, parenteral, rectal, transdermal, etc. According to an embodiment for administration to crustacean, the compounds and composition of the present invention may be provided through the food administered to the subject.
(23) Compounds of Formula I may contain one or more asymmetric centers and can thus occur as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures and individual diastereomers. The present invention is meant to comprehend all such isomeric forms of the compounds of Formula I.
(24) Compounds of Formula I may be separated into their individual diastereoisomers by, for example, fractional crystallization from a suitable solvent, for example methanol or ethyl acetate or a mixture thereof, or via chiral chromatography using an optically active stationary phase. Absolute stereochemistry may be determined by X-ray crystallography of crystalline products or crystalline intermediates which are derivatized, if necessary, with a reagent containing an asymmetric center of known absolute configuration.
(25) Alternatively, any stereoisomer of a compound of the general structural Formula I may be obtained by stereospecific synthesis using optically pure starting materials or reagents of known absolute configuration.
(26) If desired, racemic mixtures of the compounds may be separated so that the individual enantiomers are isolated. The separation can be carried out by methods well known in the art, such as the coupling of a racemic mixture of compounds to an enantiomerically pure compound to form a diastereomeric mixture, followed by separation of the individual diastereomers by standard methods, such as fractional crystallization or chromatography. The coupling reaction is often the formation of salts using an enantiomerically pure acid or base. The diasteromeric derivatives may then be converted to the pure enantiomers by cleavage of the added chiral residue. The racemic mixture of the compounds can also be separated directly by chromatographic methods utilizing chiral stationary phases, which methods are well known in the art.
DETAILED DESCRIPTION
(27) In embodiments, there are disclosed compounds formed by the complexation of boric acid and its derivatives and quaternary ammonium compounds.
(28) Boric Acid and its Derivatives
(29) Boric acid end its salts were registered in 1983 for control of cockroaches, ants, grain weevils and several beetles. They were also used as herbicide, fungicide and wood preservative, even as an insect repellent in insulation. As an insecticide, boric acid acts as a stomach poison for ants, cockroaches, silverfish and termites, and as abrasive to the insects exoskeleton. As an herbicide, boric acid causes desiccation or interrupts photosynthesis in plants.
(30) Boron is a naturally-occurring element in the earth's crust and background levels even circulate in the human bloodstream. According to United States Environmental Protection Agency (US EPA. 1993. Prevention, Pesticides, and Toxic Substances. EPA-738-F-93-006), boric acid and its salts will not pose unreasonable risks or adverse effects to humans or the environment. Available studies indicate that (ethnical boric acid is practically nontoxic to birds, fish and aquatic invertebrates, and relatively nontoxic to beneficial insects. Moreover, the amount of boric acid and its salts used as pesticides are relatively small and significant lower than amounts of boron presented naturally in soil and water.
(31) Borates that may be used in the present invention include but are not limited to orthoborate, metaborate, triborate, tetraborate, pentaborate, etc. or combination thereof. Also included are boric acid derivatives such as boronic acids (alkyl- (e.g. myristyl, palmityl, stearyl), alkenyl- or aryl-substituted boric acid such as phenyl boronic acid, 4 pyridine boronic acid, etc,) or organoborates (alkyl, alkenyl or aryl ester borate such as phenyl ester boric acid) or combination thereof.
(32) Quaternary Ammonium Compound and Choline
(33) Quaternary ammonium compounds (cations), also known as quats, are positively charged polyatomic ions of the structure NR.sub.4.sup.+, R being an alkyl group or an aryl group. Unlike the ammonium ion (NH.sub.4.sup.+) and the primary, secondary, or tertiary ammonium cations, the quaternary ammonium cations are permanently charged, independent of the pH of their solution. Quaternary ammonium salts or quaternary ammonium compounds (called quaternary amines in oilfield parlance) are salts of quaternary ammonium cations with an anion.
(34) According to an embodiment, the quaternary ammonium compound is choline and derivatives thereof, which is a water-soluble nutrient, usually grouped within the B-complex vitamins, that plays key roles in many biological processes. Choline generally refers to the various quaternary ammonium salts containing the N,N,N-trimethylethanol ammonium cation. The cation appears in the head groups of phosphatidylcholine and sphingomyelin, two classes of phospholipid that are abundant in cell membranes. Choline is the precursor molecule for the neurotransmitter acetylcholine, which is involved in many functions including memory and muscle control. Choline must be consumed through the diet for the body to remain healthy. It is used in the synthesis of the constructional components in the body cell membranes. Despite the perceived benefits of choline, dietary recommendations have discouraged people from eating certain high-choline foods, such as egg and fatty meats. The 2005 National Health and Nutrition Examination Survey stated that only 2% of postmenopausal women consume the recommended intake for choline.
(35) According to another embodiment, quaternary ammonium compound is preferably, but not limited to, choline chloride (2-hydroxyethyl trimethyl ammonium chloride) or its derivatives possessing on the alkyl chain at least a free hydroxyl group, i.e. (2-Hydroxyethyl) triethylammonium chloride, (2-hydroxypropyl)trimethyl ammonium chloride; (2,3-dihydroxypropyl)trimethyl ammonium chloride, etc.
(36) Therefore, in embodiments, there is disclosed an antibacterial and antiviral compound based on borate or borate derivatives which are complexed with a quaternary ammonium.
(37) In embodiments, there is disclosed a compound of formula I, or a pharmaceutically acceptable salts thereof, and stereoisomers thereof:
(38) ##STR00004##
wherein
(39) R.sup.1, R.sup.2, R.sup.3, and R.sup.4 are independently selected from alkyl, cycloalkyl, or aryl, optionally substituted with one or more OH, and
(40) each of the R.sup.1, R.sup.2, R.sup.3, and R.sup.4 may be optionally connected to
(41) another of the R.sup.1, R.sup.2, R.sup.3, and R.sup.4;
(42) R.sup.5 is selected from BO.sub.2, BO.sub.3, BO.sub.4, B.sub.2O.sub.3, B.sub.2O.sub.4, B.sub.3O.sub.5, B.sub.3O.sub.7, B.sub.4O.sub.7, B.sub.4O.sub.9B.sub.5O.sub.8, OBR.sup.6R.sup.7, R.sup.9BR.sup.6R.sup.7;
(43) R.sup.6 is selected from H, OH, alkyl, alkenyl, aryl, OR.sup.8;
(44) R.sup.7 is absent or selected from H, OH, alkyl, alkenyl, aryl, and OR.sup.8;
(45) R.sup.8 is selected from alkyl, alkenyl, aryl;
(46) R.sup.9 is selected from alkyl, alkenyl, and aryl.
(47) According to an embodiment, the R.sup.1 may be C.sub.1-6, alkyl, linear or branched. According to an embodiment, the R.sup.1 may be CH.sub.2CH.sub.2.
(48) According to another embodiment, the R.sup.2, R.sup.3, and R.sup.4 may be C.sub.1-3 alkyl. According to another embodiment, the R.sup.2, R.sup.3, and R.sup.4 may be independently CH.sub.3, CH.sub.2CH.sub.3, or CH.sub.2CH.sub.2CH.sub.3.
(49) According to another embodiment, the R.sup.5 is B.sub.5O.sub.8.
(50) Indeed, according to an embodiment, boric acid or its derivatives complexed with choline are preferably used in the present invention, due to several advantages such as naturally occurring; low toxicity; inexpensive and easy to manufacture.
(51) According to another embodiment, the compound of formula I, or a pharmaceutically acceptable salts thereof, and stereoisomers thereof. may be selected from the following compounds:
(52) ##STR00005##
and combinations thereof.
(53) The invention includes the compounds as shown, and also includes (where possible) individual diastereomers, enantiomers, and epimers of the compounds, and mixtures of diastereomers and/or enantiomers thereof including racemic mixtures. Although the specific stereochemistries disclosed herein are preferred, other stereoisomers, including diastereomers, enantiomers, epimers, and mixtures of these may also be useful in treating pathogenic infections. Inactive or less active diastereoisomers and enantiomers are useful for scientific studies relating to the target pathogens and the mechanisms of action of the compounds of the present invention.
(54) The compounds disclosed herein may be used in pharmaceutical compositions comprising (a) the compound(s) or pharmaceutically acceptable salts thereof, and (b) a pharmaceutically acceptable carrier. The compounds may be used in pharmaceutical compositions that include one or more other active pharmaceutical ingredients. The compounds may also be used in pharmaceutical compositions in which the compound of Formula I or a pharmaceutically acceptable salt thereof is the only active ingredient.
(55) The compounds disclosed herein may be used in compositions comprising (a) the compound(s) or acceptable salts thereof, and (b) an acceptable carrier. The compounds may be used in compositions that include one or more other active ingredients. The compounds may also be used in compositions in which the compound of Formula I or an acceptable salt thereof is the only active ingredient.
(56) According to another embodiment, there is disclosed a use of a compound of the present invention, or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for treatment of a pathogenic infection in a subject.
(57) Also disclosed is the use of a compound of the present invention, or a pharmaceutically acceptable salt thereof, or the composition of the present invention, for treatment of a pathogenic infection in a subject.
(58) According to another embodiment, there is disclosed a method of treating or preventing a pathogenic infection in a subject in need thereof comprising administering a therapeutically effective amount a compound of the present invention, or a pharmaceutically acceptable salt thereof, or the composition of the present invention, to the subject.
(59) According to an embodiment, the pathogenic infection may be a bacterial infection, a viral infection, a fungal infection, a parasite infection, or a combination thereof. Examples of bacterial infections include but are not limited to Vibrionaceae infection, a Enterobacteriacae infection, a Clostridium botulinum infection, a Listeria monocytogenes infection. Examples of viral infection include but are not limited to a Taura Syndrome Virus (TSV) infection, a Yellow Head Virus (YHV) infection, a Infectious Hypodermal and Haematopoietic Necrosis (IHHNV) virus infection, a Spherical Baculovirus infection, a Spawner-isolated Mortality Virus Disease infection, a Rhabdovirus infection, a Koi Herpes Virus (KHV) infection, a Large Mouth Bass Virus (LMBV) infection, a Baculovirus penaei (BP) infection, Herpesviridae infection, a Retroviridae infection, a Filoviridae infection, and an HIV infection.
(60) According to an embodiment, the subject may be a mammal, a fish, a bird, and a crustacean. The mammal may be a human, a bovine, an equine, an ungulate, etc. The fish may be a hagfish, a lamprey, a cartilaginous fish and a bony fish. Said bird may be a chicken, a turkey, a fowl. Said crustacean may be a shrimp, a crab, a lobster, a langouste, etc.
(61) According to another embodiment, there is disclosed a method of treating or preventing a pathogenic infection in a crustacean in need thereof comprising administering a therapeutically effective amount a compound of any one of the present invention, or a pharmaceutically acceptable salt thereof, or the composition of the present invention, to the crustacean.
(62) The present invention will be more readily understood by referring to the following examples which are given to illustrate the invention rather than to limit its scope.
(63) These examples further illustrate the method of production of borate derivatives and method to complex with quaternary ammonium compound, preferably choline (2-hydroxyethyl)trimethyl ammonium chloride (
EXAMPLE 1
Preparation of Choline/Borate Complex
(64) The choline/borate complex (
(65) 1-1Complexation of Choline Chloride with Boric Acid
(66) An amount of 123.66 g of boric acid is dispersed in 400 mL of distilled water at 90 C. The solution is under strong stirring. After dispersion of Boric acid, an amount of 279.24 g of choline chloride is slowly added in the boric acid solution and the temperature is maintained at 60 C. When choline chloride is added, the solution is cloudy, but become clear after 1 h heating, always under strong stirring (600 rpm). The reaction is continued during at least 2 h.
(67) 1-2Crystallization of the Choline/Borate Complex
(68) At the end of the reaction, the temperature is gradually reduced in order to cool down slowly the solution, while gentle stirring (200 rpm). When the temperature reached between 35-40 C., the solution is transferred in a plastic container and the stirring is reduced at 150 rpm to promote crystal formation. At this moment, some ice can be added into the water bath to accelerate the cooling-down processing. Once the temperature is about of 15 C., the solution is refrigerated between 4-6 C. for 24 hours. The crystals are then collected by decantation or by filtration using a 24-cm Whatman filter paper No 1 under a negative pressure (vacuum) about of 40 kPa. The collected crystals are dried in an oven at a temperature ranging from about 35-40 C. for at least 24 h.
EXAMPLE 2
Preparation of Choline/Tetraborate Complex
(69) The complexation of choline and tetraboric acid is prepared under identical conditions as described previously in EXAMPLE 1, except that disodium tetraborate decahydrate is used instead of boric acid.
EXAMPLE 3
Preparation of Choline/Pentaborate Complex
(70) The synthesis of choline/pentaborate complex could be prepared in two ways. Firstly, one approach consists in preparing first choline/borate complex which reacts with disodium tetraborate decahydrate to form choline/pentaborate complex. Alternatively, another approach consists in primarily synthesizing sodium pentaborate followed the complexing with choline.
(71) 3.1Method I:
(72) 3-1-1Preparation of Choline/Borate Complex
(73) The complexation of choline and boric acid is prepared under identical conditions as described previously in EXAMPLE 1 (
(74) 3-1-2Complexation of Choline/Borate with Disodium Tetraborate Decahydrate
(75) When the reaction of complexation choline/boric acid is achieved, the temperature of the solution is reduced to about 60 C. Then, an amount of 127.39 g of disodium tetraborate decahydrate is added in the solution, under strong stirring in order to react with choline/boric acid previously obtained (
(76) 3-2Method II
(77) 3-2-1Preparation of Sodium Pentaborate
(78) A volume of 800 mL of distilled water is introduced in a glass tempering beaker and heated at the temperature of about 60 C. When the set temperature is reached, an amount of 350 g of disodium tetraborate decahydrate and of 340 g of boric acid are introduced in the beaker alternately, portion by portion (approximately 20 g for each), under gentle stirring. Dissolution of all chemicals is ensured before adding the next portion, without stopping stirring.
(79) Once all chemicals are completely added, the temperature of the solution is maintained at 60 C., always under stirring for at least 30 min. To obtain sodium pentaborate powders, the solution is slowly cooled down with moderate stirring (about of 200 rpm). When the temperature reaches 25 C., the solution is refrigerated at 4 C. during 24 h. The precipitate is collected by filtration in a plastic container and stored at 40 C. at least 24 h before use.
(80) 3-2-2Complexation of Pentaborate with Choline
(81) An amount of 300 g of sodium pentaborate crystals (previously obtained) is introduced in a glass tempering beaker containing 350 mL distilled water, under) gentle stirring at a temperature 552 C. After completely dissolving, an amount of 140 g of choline chloride is slowly added in the pentaborate solution, always under stirring and the reaction is started for at least 1 h (
(82) The choline/pentaborate complex crystals are obtained by refrigeration under identical conditions as described previously for preparation of sodium pentaborate. Indeed, the choline/pentaborate solution is slowly cooled down with moderate stirring (about of 200 rpm). When the temperature reaches 25 C., the solution is refrigerated at 4 C. during 24 h. The precipitate is collected by filtration in a plastic container and stored at 40 C. at least 24 h before use. The predicted structure of choline/pentaborate complex is presented in
EXAMPLE 4
Characterization of Complex of Choline/Borate and its Derivatives
(83) 4-1Determination of Quaternary Ammonium Compound in the Complex
(84) 4-1-1Spectrophotometric Assay Based on Dragendorff Reagent
(85) In order to estimate the quaternary ammonium compounds (QAC) of choline derivatives, a spectrophotometric assay based on Dragendorff reagent is used as described by Stumpf (Stumpf, D. K. 1984. Plant. Physiol., 75, 273-274) with slight modifications. The principle consists in using bismuth nitrate and sodium iodide which form a complex and cause the precipitation of QAC with a development of a brick red color in the reaction medium. Practically, Dragendorff reagent is prepared by mixing equal volumes of bismuth nitrate 0.35 M in acetic acid 20% (v/v) and sodium iodide 2.45 M (in distilled water). Then, an amount of 100 L of mixture is added in 10 L of sample. The standard curve is made by placing 1.0-8.0 L of a stock solution of choline (0.500 g/ml) into plastic 1.5 mL microcentrifuge tubes. Sample and standard solutions are centrifuged for 3 min at 10,000 g and the supernatants are completely removed. The obtained precipitates are then dissolved in 1 mL of 2.45 M Nal solution under strong stirring for at least 15 min and diluted by adding 99 mL of 0.49 M Nal. Finally, the samples are recorded by spectrophotometry at 420 nm (maximum absorption) using solution of 0.49 M Nal as a blank.
(86) Generally, the quaternary ammonium compounds are detected in all samples which the ratio of choline/borate (tetraborate or pentaborate) complex is 1:1 (equimolar complex). No significant difference is observed for choline/pentaborate complexes obtained from method 1 and 2, as described in EXAMPLE 3.
(87) 4-1-2Elemental Analysis
(88) Analysis of the element compositions of choline/borate or its derivatives complexes allows confirming the structure of the complex formed between choline and different borate or its derivatives. No significant difference is observed between the ratio of choline/pentaborate obtained by the method 1 and 2, as described in EXAMPLE 3.
(89) 4-1-3FTIR Analysis
(90) FTIR spectra are recorded on a Spectrum One (Perkin Elmer, Canada) instrument equipped with a Universal Attenuated Total Reflectance (UATR) device. All samples including borate or its derivatives and borate or its derivative complexes are recorded under powder (20 mg) forms in the spectral region (4000-650 cm.sup.1) with 24 scans/min at a 4 cm.sup.1 resolution.
(91)
(92) For planar boric acid FTIR spectrum, the stretching vibration of the BOH bands are observed at 3220 cm.sup.1. For absorption bands located at 1430 and 1195 cm.sup.1, they are respectively assigned for the asymmetric BO stretching vibration and the in-plane BOH bending. With regard for pentaborate, similar observation for the stretching vibration of the BOH bands is noticed at 3220 cm.sup.1. However, a new absorption band appeared at about 3380 cm.sup.1 and is possibly due to HOH (free OH from water). Additionally, BO absorption bands contributed for the asymmetric BO stretching vibration and the in-plane BOH bending are shifted to 1375 and 1140 cm.sup.1. A new band also observed at 1300 cm.sup.1 and could be due to the BOB stretching vibration.
(93) When boric acid or pentaborate are complexed with choline, similar FTIR spectral profiles are observed. In the spectral regions 3500-3000 cm.sup.1, the stretching vibrations of the OH bands are observed at 3380 and 3220 cm.sup.1 which are respectively assigned for the absorption band of HOH and the absorption band of BOH. In the spectral region 1500-1000 cm.sup.1, the stretching vibrations for BO are located at 1375 and 1300 cm.sup.1 and the in-plan BOH bending is noticed at 1140 cm.sup.1. A slight shoulder observed at 1410 cm.sup.1 seems to contribute to quaternary ammonium CH.sub.3N.sup.+ (from choline). Also, the absorption bands at 1080 and 1010 cm.sup.1 are respectively attributed to CN and CO stretching vibrations.
EXAMPLE 5
Toxicity Evaluation of Choline/Borate and Choline/Borate Derivative Complexes for Shrimp Litopenaeus vannamei
(94) 5-1Collection and Maintenance of Experimental Animals
(95) Shrimp, Litopenaeus vannamei (post-larvae about 12-16 day-old), are obtained from a commercial hatchery in La Paz (BCS, Mexico) and maintained in 1000 L fiber glass tanks with air-lift biological filters at room temperature in water with a salinity of 35 parts per thousand (ppt).
(96) Natural seawater is used in all the experiments. It is obtained from the Ensenada de La Paz after removing the sand and other suspended particles in sea water. Finally, the seawater is sterilized with UV-lamp before use for the experiments. The tanks are individually aerated through air stones connected to a high-volume air blower. Partial water changes are made once a week to maintain the water quality. The shrimps are initially fed Artemia sp and are weaned onto a commercial diet (containing 35% crude protein, Purina Brand) when they reached 20 days of age (post-larvae 20 day-old). Daily, temperature and pH value are recorded; salinity is measured with a Salinometer (Aquafauna, Japan) and dissolved oxygen is estimated by the Winkler method (Strickland and Parsons, 1972).
(97) 5-2Toxicity Evaluation of Choline/Borate and Choline/Borate Derivative Complexes
(98) White shrimp (L. vannamei) from intensive culture ponds are selected and acclimated to the experimental conditions during one week before starting the trial. Indeed, shrimp are placed in 1000 L circular tanks with filtered sea water at 38 parts per thousand (ppt) and constant aeration, fed ad libitum with a commercial pellet containing 35% crude proteins.
(99) After the acclimatation period, different concentrations (1, 5 and 10 mg/g) of choline/borate or its derivatives are physically mixed with commercial pellet feeds and fed in the identical conditions (ad libitum) during 15 days. Globally, there are 6 groups are investigated in this study: 1. Boric acid; 2. Tetraborate; 3. Pentaborate; 4. Choline/borate complex; 5. Choline/tetraborate complex; 6. Choline/pentaborate complex.
(100) Results show that there is no significant difference between group of control and groups treated with boric acid or its derivatives complexed with choline. These results suggest that no toxicity are apparent for white shrimp L. vannamei for doses of boric acid or its derivative complexes lower than 10 mg/g of shrimp feed.
EXAMPLE-6
Preparation for Challenge Studies
6-1Preparation for Bacterial Assays
6-1-1Bacterial Strains and Culture Conditions
(101) Virulent bacterial strains Vibrio parahaemolyticus CAIM 170 (Collection of Aquatic Important Microorganisms, CIAD, Mexico) are used in this study (Roque et al., 1998). These strains are maintained in Tryptone Soy Broth (TSB) containing 2.5% (w/v) NaCl and 15% (v/v) of glycerol at 80 C. Prior to use, a cryovial is thawed and inoculated into 5 mL of TSB with 2.5% (w/v) NaCl and incubated overnight at. 37 C. in a rotary shaker (200 rpm) for activation. Thereafter, a volume of 2 mL of the overnight culture is transferred to 100 mL of TSB and reincubated in the similar activation conditions (37 C., 200 rpm).
(102) Density of bacteria is measured by spectrophotometry at 600 nm for every 30 min. At the same time, an aliquot is withdrawn for viable count determination by plating serial dilutions on Tryptone Soy Agar with 1% NaCl. The plates are incubated for 24 h at 37 C. A growth curve was prepared by plotting the viable count (x-axis) against optical density at 600 nm values (y-axis). This graph was used to determine the viable cell count during further spiking studies. For the challenge, control groups are included in all trials: A positive control group of shrimp is treated with pathogen V. parahaemolyticus; A negative control group of shrimp, instead of receiving the pathogen, is treated with a sterile saline solution (without pathogens).
(103) Preliminary trials showed that a V. parahaemolyticus (VP) suspension of 110.sup.6 colony-forming units (cru)/mL could kill 60% of the shrimp population in 24 h and about of 70% in 96 h, whereas a group treated with a non-virulent V. parahaemolyticus strain showed the cumulative shrimp mortality at 96 h is less than 10%.
(104) 6-1-2Optimization of Real-Time Polymerase Chain Reaction Assay for Quantitative Determination of V. parahaemolyticus
(105) The primers used In this assay are Vp-ToxR q-PCR (Vibrio parahaemolyticus ToxR gene quantitative Polymerase Chain Reaction) 176 F (forward primer, SEQ ID NO:1: GGA AGT TTT AAC CCG TAA CGA GC) and 176 R (reverse primer, SEQ ID NO: 2: GGT ACA AAT GAG TTG ATA GCC TCG) and designed as described by Untergasser et al. (2007) using the software Primer3Plus, with the following parameters: Primer length: 18-24 bp; GC content: 35-65% Melting Temperature. (Tm): 58C-60 C.; Product size: 80-250 bp (Wang, X. and Seed, B. 2007. In: Real-time PCR. Derek M. Tevfik. Ed. Taylor & Francis. New York. USA. p. 93-105).
(106) Thermodynamic parameters of each primer are evaluated to check primer-dimer and secondary structure using the software Oligoevaluator (Sigma-Aldrich) and Primer digital (Kalendar. R., Lee, D., Schulman, A. H. 2011. Genomics, 98, 137-144). These primers yielded a 176 by amplicon and are suspended in nuclease free water to make a working solution of 10 pmol/L.
(107) To establish the standard curve, serial dilutions are done with the DNA extracted and purified from culture of V. parahaemolyticus CALM 170 using the following mixture: 5 L of SSO-Fast supermix evagreen (BIORAD, USA); 1 L of Vp-ToxR q-PCR 176F (10 pmol/L); 1 L of Vp-ToxR q-PCR176R (10 pmol/L); 2 L of DNAse-free water and 1 L of template as DNA.
(108) Quantitative PCR is performed on a Rotor gene 6000 Real-Time PCR system (Qiagen). Amplification conditions are carried out as follows: initial activation at 50 C. for 2 min; initial denaturation at 95 C. for 10 min and followed by 45 cycles of denaturation at 95 C. for 15 s primer annealing at 60 C. for 20 s and elongation at 72 C. for 30 s and a final elongation at 72 C. for 5 min. A melting curve analysis is performed at the end of the amplification using the following conditions: 65 C.-80 C. (1 C./s).
(109) The data are analyzed using the software Rotor Gene-Q Pure Detection (1.7 Build 94). 6-1-3Enrichment Media and Samples for Detection of V. parahaemolyticus
(110) In this study, an enrichment medium is used to evaluate their effect on detection of V. parahaemolyticus. The enrichment medium used is alkaline peptone water (APW; 1% peptone, 1% NaCl, pH 8.5; Tyagi et al., 2009). Practically, an amount of 5 g of shrimp is collected and placed on ice. Immediately, these shrimp are homogenized in phosphate buffer at pH 7.5 with a Polytron homogenizer, under sterile conditions. A volume of 1 mL of homogenized material is inoculated in APW medium and incubated overnight at 37 C. for 24 h in a rotary shaker at 200 rpm.
(111) 6-1-4V. parahaemolyticus DNA Extraction from APW Medium
(112) A volume of 1 mL of APW medium is collected in a microcentrifuge tube. The tube containing medium is centrifuged at 10,000 g for 10 min and the pellet is collected and washed with miliQ sterile water and suspended in 400 L in miliQ sterile water. Further centrifugation at 10,000 g for 5 min and the tubes are incubated at 98 C. for 20 min before centrifuged at 5,000 g for 5 min. The supernatant is collected in a new microcentrifuge tube and stored at 20 C.
(113) 6-2Preparation for White Spot Syndrome Virus (WSSV) Assays
(114) 6-2-1WSSV Stock and In Vivo Titration
(115) The virus is isolated from WSSV-infected adult L. vannamei shrimp obtained from commercial farms located in Sinaloa, Mexico in 2013. Virus stocks were purified from infected shrimp homogenates by improved differential centrifugation as described previously by Du et al, (Du, H. H., Fu, L. L., Xu, Y. X., Kil, Z. S., Xu. Z. R. 2007. Aquaculture, 262:532-534) and then stored at 80 C. For assays, the WSSV stock is serially diluted to prepare solutions containing various target copy numbers determined by competitive PCR.
(116) A volume of 20 L of different dilutions of the tissue homogenate (containing WSSV in 330 mM of NaCl) is injected intramuscularly in fourth or fifth abdominal segment of L. vannamei using an insulin needle. The mortality is recorded twice a day and dead shrimp are tested by PCR to highlight the presence of WSSV.
(117) A dilution of WSSV containing 110.sup.6 copies is appropriated to use in subsequent experiments (Du et at. 2006). In the present study, this dilution containing 110.sup.6 copies is approximately corresponding to 3% of WSSV suspension which can kill 50% of shrimps in 48 h, and 100% of shrimps in 96 h; on the other hand, a virus dilution at 1% of WSSV suspension can kill 80% of the shrimps in 120 h.
(118) 6-2-2Detection of WSSV Using q-PCR Technique
(119) After 48, 72 and 96 h after infection, haemolymph is collected from the ventral sinus using a sterile syringe containing 500 L of anticoagulant solution (pH 7.3, at 4 C.) as described by Vargas-Albores et al. (Vargas-Albores, F., Guzmn, M. A., Ochoa, J. L. 1993. Comma. Biochem. Physiol., Part A, 106, 299-303): 450 mM NaCl; 10 mM KCl; 10 mM HEPES; 10 mM EDTA
(120) The collected haemolymph is centrifuged at 12,000 g for 20 min at 4 C. and the precipitate is resuspended in TRIzol reagent. According to the manufacturer's instructions, total RNA is extracted and the pellets of total RNA are resuspended in 15 L of RNAse free water. Total RNA is quantified using Nanodrop 1000 (Thermoscientific) using an amount of 1 g of total RNA treated with DNAse I (Invitrogen). The cDNA synthesis is performed using Go Script Reverse Transcription System (Promega). The sequence of used primer WSV230F is SEQ ID NO:3: GCT GGT GGG GGA TGA TAC TA, and that of primer WSV230R is SEQ ID NO:4: GTC TCC CGT CAC CGT CTT TA, as described by Gomez-Anduro et al. (Gomez-Anduro, G. A., Barillas-Mury, C. V., Peregrino-Uriarte, A. B., Gupta, L, Gollas-Galvam, T., Hernandez-Lopez, J., Yepiz-Plascencia, G. 2006, Develop. Comp. Immunol., 30: 893-900). The q-PCR is performed as follows: 5 L of SSO-fast qPCR supermixes evaGreen (BIORAD, USA); 1 L of WSV230F (10 pmol/L) and 1 L of WSV230R (1.0 pmol/L); 2 L of DNAse-free water; 1 L of template as cDNA from hemocytes.
(121) Initial denaturation at 95 C. for 10 min followed by 40 cycles of denaturation at 95 C. for 15 s, primer annealing at 58 C. for 20 s, elongation at 72 C. for 30 s and a final elongation at 72 C. for 5 min. A melting curve analysis is performed at the end of the amplification using the following conditions: 70 C.-95 C. (1 C./s). The data is analyzed using the software Rotor-Gene-Q Pure Detection (1.7 Build 94).
(122) 6-3Challenge Studies
(123) 6-3-1Experimental System
(124) A static experimental system is used in consisting of 24, 20-1 fiber glass tanks. Each tank containing filtered and UV-sterilized seawater (at 35 ppt., pH 8.0) is individually aerated by an air stone at 27 C.
(125) 6-3-2Experimental Procedure
(126) 6-3-2-1Acclamation
(127) An amount of twenty (20) shrimp are introduced to each tank filled with 30-L of UV-sterilized seawater. The shrimp are left to adapt to the system (acclamation) during 2 days. Shrimp is'fed with pelletized commercial feed mixed with different quantities of choline/pentaborate complex during 28-days for further challenges with WSSV and a pathogenic Vibrio parahaemolyticus strain,
(128) 6-3-2-2Challenge Test
(129) The shrimp are either challenged in triplicate by injection with a known concentration of White Spot Syndrome Virus (1-3% viral suspension) or a pathogenic Vibrio parahaemolyticus (110.sup.6 cfu/mL) strain. There are two control groups: 1. Positive control groupsShrimp that are not previously exposed with any borate or its derivatives and are either injected with a known concentration of WSSV or pathogenic VP; 2. Negative control groupsShrimp are not fed with borate or its derivatives and are not treated with any pathogens; however, shrimp are injected with a saline solution, instead pathogens.
(130) After the pathogen challenge, treated and control shrimp groups continued receiving the corresponding feed. The shrimp mortality percentage is recorded daily. Shrimps are observed for 96 to 120 h after each challenge and recorded water temperature and mortality. Shrimp samples are taken on the first day, during challenge and 72 h post-challenge. All shrimp samples are stored at 80 C. in RNA latter for further q-PCR analysis of pathogen loadings and expression of immune related genes.
(131) 6-3-2-3Determination of Mortality Shrimp
(132) Dead shrimp are recorded daily during 120 hours. All dead shrimp samples are stored at 80 C. in RNA latter for further q-PCR analysis of pathogen loading and expression of immune related genes.
(133) 6-4Results of Challenge Studies with Boric Acid or its Derivatives Used Alone or Complexed with Choline
(134) Shrimp challenged with virulent V. parahaemolyticus (
(135) In contrast, high survival rates (>60%, p<0.05) are observed for choline/borate, choline/tetraborate and choline/pentaborate complexes,
(136) Similar observation for shrimp challenged with WSSV (infected with 1% of WSSV suspension homogenate), the shrimp survival for the negative (not infected with pathogen and not treated with any borate complexes) control group are 100% after 96 h. For positive (pathogen infected, but not treated with any borate complexes) control group, the survival rate is about 30%. Similar survival rate for borate, tetraborate or pentaborate (not complexed with choline) are observed at different doses (1, 5 and 10 mg/g of commercial pellets). In contrast, high survival rates (>60%) are observed for choline/borate, choline/tetraborate and choline/pentaborate complexes.
(137) It is worth mentioning that for all complexes tested with V. parahaemolyticus, the best survival (>80%) results are obtained with choline/pentaborate complex supplemented at dose of 5 mg/g of commercial pellet feed. Consequently, the choline/pentaborate complex is selected for subsequent studies.
(138) 6-4-1Results of Choline/Pentaborate Complex Challenged with Vibrio parahaemolyticus
(139) For shrimp infected with V. parahaemolyticus (110.sup.6 cfu/mL) and treated with choline/pentaborate complex at dose 5 mg/g of feed, the survival (
(140) 6-4-1Results of Choline/Pentaborate Complex Challenged with WSSV
(141) There are no statistical differences between replicates for the same treatments (p>0.05), as the standard deviation between samples is lower than 3%.
(142) Results show that there are statistical differences in shrimp challenged with a 3% WSSV suspension. Survival after 48 hours for shrimp from the positive control (infected) is about of 80% and for shrimp fed with choline/pentaborate complex at dose 5 mg/g is 95% survival (statistically different p<0.05). After 96 h post-challenge, only 10% of the positive control shrimp are alive. There is a marginal improvement for shrimp fed with choline/pentaborate complex at doses the 1 and 5 mg/g, where the survivals rates are about of 40%.
(143) In contrast, when shrimp are challenged with 1% WSSV suspension (
(144) The use of functional feeds that contain antipathogenic compounds is considered fundamental in the strategy to prevent early infectious diseases in shrimp, such as Early Mortality Syndrome (EMS) and White Spot Syndrome Virus (WSSV). Further, some antimicrobial compounds, such as the ones tested in this trial, appear to be able to disrupt bacterial communication that activate certain genes associated to the release of toxins. This quorum quenching represents a significant alternative to the use of antibiotics.
(145) Data analysis shows that the choline/pentaborate complex formulation supplemented at a 5 mg/g in feed diets is effective in reducing impact of WSSV infection when following standardized injection protocols. This represents a major breakthrough for the control of a disease that affects a significant number of commercial shrimp production operations, from hatcheries to grow out facilities. Though several products have been developed to prevent and treat WSSV, such as genetic vaccines, these have proven ineffective in commercial operations, impractical to apply for lack of effective delivery mechanisms, or expensive. Choline/pentaborate complex is not toxic, simple to manufacturing, stable, easy to administer and relatively inexpensive.
(146) The use of choline/pentaborate complex as additive in commercial diets provides an efficient mechanism for microbial, activity control in the shrimp gut that may contribute to the solution of the mass mortalities associated to V. parehemolyticus and WSSV in commercial shrimp farms.
EXAMPLE-7
Highlighting of Expression of Immune Related Genes in Shrimp Litopenaeus vannamei by Choline/Pentaborate Complex
(147) 7-1RNA Extraction
(148) RNA is extracted using TRIzol and the Qiagen RNeasy Mini Kit (Qiagen, Germantown, Md., USA). Shrimp samples immersed in TRIzol are placed in the FastPrep-24 (MP Biomedicals LLC, Solon, Ohio, USA) and homogenized during 4 C. for 40 s pulses at 6 m/s. A volume of 200 L of chloroform is added to a 1.5 mL RNase free tube containing the homogenate material and inverted 15 times. Samples are incubated at room temperature for 3 min before centrifuged at 8000 g for 15 min at 4 C. using the Eppendorf centrifuge. The supernatant is carefully transferred into a new 1.5 mL RNase free tube and the extraction continued following the manufacturer's protocol.
(149) 7-2Assessing RNA Quality and Quantity
(150) The quality and quantity of RNA are determined by the NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, Mass., USA) and Bio-Rad (Bio-Rad, Hercules, Calif., USA) using denatured gel electrophoresis with formaldehyde and buffer MOPS 1 (Sambrook, J. 1989. in: Molecular cloninga laboratory manual, 2nd ed. J. Sambrook, E. F. Fritsch, T. Maniatis Eds. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press).
(151) 7-3Microarray and Scanning
(152) Microarrays are designed based on publically available gene sequences and ESTs determined from cDNA libraries representing multiple tissues of male and female L. vannamei shrimp, and/or of various physiological conditions.
(153) In total, a length of oligonucleotides about of 61440-mer is included in this study: 39592 shrimp ESTs; 1216 Quality control spots; 112 positive control Alexa-555 dye; 80 positive control Alexa 647 dye; 20320 Unigenes; 16 astringent positive control alexa-555: 16 astringent positive control Alexa-647 dye and 88 negative control empty are submitted to the MYcroarray (University of Michigan, Chemical Engineering Department) for printing on aminosaline glass slides. The exposure and reference sample RNAs are adjusted to final volume 19 L with oligo d(T) (2 g/L) and random primers. (1 g/L) in water molecular biology grade. The samples are incubated at 70 C. for 10 min and are then chilled in ice. The samples are mixed finally with 5 Reaction Buffer Superscript II kit (Invitrogen, Life technologies, Carlsbad Calif., USA): 25 mM MgCl.sub.2; aminoallyl-dNTP with dUTP (3:1); 0.1 M DTT and 200 U/L Superscript II Reverse Transcriptase. The mix is incubated at 25 C. for 10 min and then at 42 C. for 2 hours. RNA hydrolysis is made with 5 L of NaOH 1 N and 1 L of EDTA 0.5 M. The mixture is incubated at 65 C. for 10 min, and then added 25 L of HEPES 1 M at pH 7.5. The choline/pentaborate complex exposed samples (n=6) are labeled with Alexa-647 dye Molecular Probes (Life Technologies, Eugene Oreg., USA) fluorescent dye and the reference samples (n=6) are labeled with Alexa-555 dye Molecular Probes (Life Techynolooles, Eugene Oreg., USA). The aminoallyl-DNA (aa-DNA) is purified using QIAquick (Qiagen, Duesseldorf, Germany). A volume of 7 L of sodium acetate 3 M and 400 L binding buffer supplied by the Kit are added. The mixture is allowed to stand 5 min according to the manufacturer's instructions for purification. The eluted aa-DNA is concentrated in a Vacufuge until dry and is resuspended in 4.5 L of sodium bicarbonate 100 mM (pH 9.0). The reaction is conducted in darkness at room temperature overnight. The aa-DNA labeled is purified using QIAquick (Qiagen, Duesseldorf, Germany) and eluted with 50 uL water. The aa-DNA labeled is quantified in a Nanodrop 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, Mass., USA).
(154) After completely drying in a Vacufuge concentrator, the aa-DNA labeled is resuspended in hybridation mixture containing: 0.1 optical density units of aa-DNA labeled from each sample; SSC (5) buffer; 0.1% SDS; 1 buffer TE. The hybridation mixture is desaturated at 94 C. for 5 min and finally at 65 C. for 30 s. The mixture is placed in the microarray slide previously placed in a chamber for microarray hybridization and covered using a RNase-free plastic coverslip (Hybrislip, Schleicher & Schuell, HS404022 mm; HS222222 mm via Thomas Scientific). The hybridation chamber is incubated at 42 C. overnight.
(155) The slide is placed in a 50 mL conical tube for the first wash and a volume of 50 mL of SSC (1) buffer and of SDS 0.05% are added for 1 min. The washing is repeated twice using a volume of 50 mL of SSC (0.06) for 2 min. The slide is centrifuged at 1500 rpm during 5 min.
(156) The microarray slide is scanned in GenePix 4100A Microarray Scanner (Molecular Devices, Silicon Valley, Calif., USA) at 5 m resolution on the channels PMT 647 and PMT 555. The data of the scanning is analyzed in GenArise v2.7 software to obtain the data of select genes that are significantly differentially expressed onto control and treated samples. The genes are classified into categories and assigned a numerical value zScore base in the data normalization. The EST, Nucleotide and Unigenes sequences from the microarray are downloaded using Batch-Entrez at http://www.ncbi.nlm.nih.gov/sites/batchentrez and loaded in Blast2GO as FASTA files (Conesa, A., Gtz, S., Garcia-Gmez, J. M., Terol, J., Taln, M., & Robles, M. 2005. Bioinformatics, 21, 3674-3676), performing a BlastX using an e-Value of 110.sup.3 against Non-Redundant Database and functional annotation from gene ontology Database.
(157) 7-4Analysis of Genes by RT-qPCR
(158) RT q-PCR is performed on the 8 samples of shrimp treated with choline/pentaborate complex and a similar number of samples of shrimp non-treated for the microarray analysis (n=16) in order to validate 7 genes of interest (GOIs) and 3 reference genes. Primer of manganese superoxide dismutase (MnSOD) [Lv MnSOD q-PCR 149F (forward primer, SEQ ID NO: 5: GGG CTT CAT TAA CAA CCT AAT TGC), and Lv MnSOD q-PCR 149R (reverse primer, SEQ ID NO: 6: GGG CTT CAT TAA CAA CCT AAT TGC)]; and reference gene ribosomal protein L8 [Lv L8pro q-PCR 166F (forward primer, SEQ ID NO: 7: TAG GCA ATG TCA TCC CCA TT) and Lv L8pro q-PCR 166R (reverse primer, SEQ ID NO: 8: TCC TGA AGG AAG CTT TAC ACG)] are taken from Gomez-Anduro et al. (2006). Primer design is performed in the online application primer3plus (Untergasser, A., Nijveen, H., Rao, W., Bisseling, T., Geurts, R., Leunissen, J. A. M. 2007, Nucleic Acids Research, 35: 71-74) based in the following features: 18-24 nt length; GC content 35-65% product size 80-250 bp; and 2 GC clamp (Wang, X. and Seed, B., 2007). The primers are evaluated in the online application Oligo Evaluator Sigma Aldrich (St' Louis, Mo.) to check thermodynamic characteristics based in self-aligned primer and primer-dimer structures. The primers selected are 2 Kcal/mol and evaluated using an in silico PCR software Primer Digital (Kalender et al., 2011). RNA extracts used for the microarray hybridizations are converted to cDNA using promega GoScript Reverse Transcription System (Promega Corporation, Madison, Wis., USA) and SsoFast EvaGreen Supermix (Bio-Rad, Hercules, Calif., USA) for RT-qPCR. Fluorescence is detected by using the Rotor gene 6000 Real-Time PCR detection system (Corbette). A three-step cycling protocol is used with primer specific annealing temperatures. The RT q-PCR cycle is 95 C. for 10 min; followed by 40 cycles of 95 C. for 15 s and 20 s at the primer specific annealing temperature (see Table 1); 72 C. for 25 s after which the plate is read. The annealing temperature range for the primers is 56-62 C. Melt curve analysis is performed to determine product specificity over the temperature range of 65-95 C. in 1 C. increments, and read every 5 s. The same six samples treated and non-treated shrimps (n=12) used for microarray are assayed in triplicate for RT q-PCR, in addition to triplicates of negative RT controls and negative template controls (NTC). The geNorm software (Vandensompele et al., 2002) identified 3 genes: -actin, elongation factor 1- and ribosomal protein L8 to be the most stable and meeting the selection criteria of M value of <1.25, representing the average expression stability and V value1.5, indicating pair wise variation.
(159) The other primers are:
(160) TABLE-US-00001 Ferritin:FerritinForward,SEQIDNO:9: CAAGCGAACCTCTGGAAATC, and FerritinReverse,SEQIDNO:10: TGGCAAATCCAGGTAGAGC. Toll-likereceptor:LvTollForward,SEQIDNO:11: GCCCTAAATGATGGATGAC, and LvTollReverse,SEQIDNO:12: GCCAAGGGAAAAAGAAAT. ElongationFactor1-:LvEF1AForward, SEQIDNO:13: CCACACTGCTCACATTGC, and LvEF1AReverse,SEQIDNO:14: GAAGGTCTCCACGCACAT. B-Actin:LvACTBForward,SEQIDNO:15: TGGGACGACATGGAGAAG, and LvACTBReverse,SEQIDNO:16: GGGGGTGTTGAAGGTCTC. Pre-amylase1:LvPAMYForward,SEQIDNO:17: CCGTCTCCTATAAACTCGTCACTC, and LvPAMYReverse,SEQIDNO:18: TCGCCGTAGTTTTCAATGTTC. Trypsinogen1:LvTryForward,SEQIDNO:19: TCGTCGGAGGAACTGACG, and LvTryReverse,SEQIDNO:20: TGCCCTCATCCACATCCT. LipaseDigestive1:LvLIPForward,SEQIDNO:21: TCCTGGCTCACACACCTG, and LvLIPReverse,SEQIDNO:22: GTCCTTCAGCGAGCCTTG. CathepsinL:LvCPLForward,SEQIDNO:23: CGTCCTTCCAGTTCTACCAT, and LvCPLReverse,SEQIDNO:24: ATCTGGATGTAGCCCTTGTT.
7-5Statistical Analysis
(161) At termination of the exposure experiment, survival and mortality data are analyzed using TOXSTAT probit (adapted from Stephan, 1977) analysis software to calculate a LC.sub.50 with a 95% confidence interval. Microarray gene expression values are analyzed using a one-way ANOVA and 100 permutations to detect significantly at p-value of 0.05. A Tukey post-hoc test is also used to identify significantly differentiated genes affected by the choline/pentaborate complex treatment. A user defined k-means cluster analysis (n=4) is run with a Pearson centered distance matrix and 100 iterations using GeneSpring (Agilent, Mississauga, ON, Canada). The mean CNRQ values from the RT q-PCR analysis of GOI for treated and non-treated shrimps are compared to identify significant differences in relative abundance using a one-way ANOVA with multiple test corrections and significant p-value<0.05. The CNRQ values are log2 transformed and compared to microarray loge transformed expression ratios.
(162) 7-6Results for Analysis of Genes by RT q-PCR
(163) 7-6-1Treatment of L. vannamei Shrimp with Choline/Pentaborate Complex
(164) The two shrimp groups fed with a commercial feed (control group), and the other group, fed with the same commercial feed, but supplemented with choline/pentaborate complex at dose 5 mg/g of feed are maintained in the bioassay laboratory for up to 28 days.
(165) It is of interest to mention that the shrimp fed with the commercial diet supplemented 5 mg of choline/pentaborate complex had a 20% increment in mean weight, when compared with the shrimps control group (
(166) 7-6-2DNA Microarray Analysis of Shrimp Treated with Choline/Pentaborate Complex
(167) The shrimp DNA microarray contains 60000 spots. Table 2 summarizes the classification of differential gene expression based in the patterns of absorbance source in two channels: Alexa-555 and Alexa-647 dye, and the number of Up- and Down-regulated genes in the microarray analysis.
(168) TABLE-US-00002 TABLE1 PrimersequencesusedintheRT-qPCR Align Melt NCBI Temp Peak Size Accession PrimerID GeneName ( C.) Sequence(5-3) ( C.) (bp) AY955373.1 LvFerritinF Ferritin 58 CAAGCGAACCTCTGGAAATC 83.5 230 LvFerritinR TGGCAAATCCAGGTAGAGC FE147224.1 LvTollF Toll-like 62 GCCCTAAATGATGGATGAC 88.2 151 Receptor GCCAAGGGAAAAAGAAAT GU136229.1 LvEF1AF ElongationFactor 56 CCACACTGCTCACATTGC 85.5 151 1- LvEF1AR GAAGGTCTCCACGCACAT JF288784.1 LvACTBF -actin 60 TGGGACGACATGGAGAAG 86.7 150 LvACTBR GGGGGTGTTGAAGGTCTC X77318.1 LvPAMYF Pre-amylase1 58 CCGTCTCCTATAAACTCGTCACTC 88.5 259 LvPAMYR TCGCCGTAGTTTTCAATGTTC JQ277721.1 LvTryF Trypsinogen1 58 TCGTCGGAGGAACTGACG 89.2 210 LvTryR TGCCCTCATCCACATCCT FJ619564.1 LvLIPF LipaseDigestive1 PENDING TCCTGGCTCACACACCTG PENDING 231 LvLIPF GTCCTTCAGCGAGCCTTG X99730.1 LvCPLF CathepsinL PENDING CGTCCTTCCAGTTCTACCAT PENDING 166 LvCPLR ATCTGGATGTAGCCCTTGTT
(169) TABLE-US-00003 TABLE 2 Statistical analysis of the number of Up- and Down-regulated genes in the DNA shrimp microarray analysis Expression Levels (Score) Number of spots All Spots 60,000 Spots available to analysis 57,193 Without signal 37 >2 1,650 1.5 to 2 1,984 1 to 1.5 3,662 No regulated genes (1 to 1) 43,950 1.5 to 1 3,221 1.5 to 2 1,259 <2 1,434
7-6-3Gene Ontology Analysis
(170) Gene ontology (GO) is commonly used to categorize gene products and standardize their representation across species. In order to eliminate redundancy only the 2-fold Up- and Down-regulated differentially expressed genes are submitted to Blast2GO suite for the assignment of several functional groups based on GO terminology. There were 583 Up-regulated genes in samples from shrimp fed for 14 days with the feed supplemented with choline/pentaborate complex at dose 5 mg/g, expressed genes fall into the followed biological processes: 1. Cellular Processes (20%); 2. Metabolic Process (18%); 3. Single-organism Process (17%); 4. Biological Regulation (9%); 5. Cellular Component Organization or Biogenesis (7%); 6. Localization (7%); 7. Response to Stimulus (6%); 8. Multicellular Organismal Process (6%); 9. Developmental Process (6%) and Signaling (4%) (
(171) The Molecular Functions of the 583 for 2-fold Up-regulated expressed genes fall into four categories: 10. Binding Activity (44%); 11. Catalytic Activity (43%); 12. Transporter Activity (7%); 13. Structural Molecule Activity (6%) (
(172) The Fisher's exact test from GO terms of the 2-fold Up-regulated genes (shrimps treated with choline/pentaborate complex) versus all genes (shrimp control group) from DNA microarray shows that choline/pentaborate complex treated shrimps have gene with increased expression in the categories described as follows: 14. Ion Transport (8.15%); 15. Monovalent Inorganic Cation Transport (5.92%); 16. Cytoskeletal Protein Binding (5.55%); 17. Hydrogen Transport (4.81%); 18. Proton Transport (4.81%); 19. Actin Cytoskeleton (4.62%); 20. Calcium Ion Binding (4.25%); 21. Monosaccharide Metabolic Process (4.10%); 22. Positive Regulation of Biosynthetic Process (3.70%); 23. Positive Regulation of Cellular Biosynthetic Process (3.70%) (
(173) Regarding the Functional Annotation of the 2-fold Down-regulated expressed genes from the DNA microarray of the Biological Processes, it is found that there were 262 genes with a 2-fold Down-regulated expression in shrimp fed for 14 days with the feed supplemented with choline/pentaborate complex 5 mg/g, expressed genes fall into the followed Biological Processes: 24. Cellular Process (20%); 25. Metabolic Process (20%); 26. Response to Stimulus (5%); 27. Single-organism Process (17%); 28. Multicellular Process (6%); 29. Signaling (3%); 30. Localization (8%); 31. Biological Regulation (9%); 32. Developmental Process (5%); 33. Cellular Component Organization or Biogenesis (7%) (
(174) The Molecular Function of the 262 genes in the 2-fold Down-regulated expressed genes fall into four categories: 34. Binding (44%); 35. Catalytic Activity (40%); 36. Transporter Activity (9%); 37. Structural Molecule Activity (4%); 38. Enzyme Regulator Activity (3%) (
(175) The Fishers exact test from GO terms of the 2-fold Down-regulated genes (shrimps treated with choline/pentaborate complex) versus all genes (shrimp control group) from DNA microarray showed that shrimp treated with choline/pentaborate complex over-represented gene categories described as follows: 1. Regulation of protein metabolic process (10.56%); 2. Transporter activity (10.56%), extracellular region (9.75%); 3. Extracellular region part (5.69%), extracellular space (4.87%); 4. Translation factor activity, nucleic acid binding (4.47%); 5. Photoreceptor cell differentiation (2.43%); 6. Gas transport (2.03%); 7. Oxygen transporter activity (2.03%); 8. Starch metabolic process (2.03%); 9. Sucrose metabolic process (2.03%); 10. Toll-like Receptor for signaling pathway (1.62%) (
7-6-4Comparative Analysis of Metabolic Pathways
(176) The L. vannamei transcriptomic sequences from the shrimp DNA microarray are compared to ESTs and nucleotide sequences from Drosophila present in the NCBI database in order to detect the presence of proteins that are over-expressed in different Metabolic Pathways. The results of the 2-fold Up-regulated genes, related to genes and enzyme expressed into each group are summarized in Table 3. Similarly, the results of the 2-fold Down-regulated genes, related to genes and enzyme expressed into each group are summarized in Table 4.
(177) TABLE-US-00004 TABLE 3 Metabolic pathways of DNA shrimp microarray analysis of the 2-Up regulated genes in L. vannamei treated with the choline/pentaborate complex supplemented (5 mg/g) commercial feed. Metabolic Pathway # Genes # Enzymes Oxidative phosphorylation 18 6 Purine metabolism 14 7 Glycolysis 8 8 Amino sugar and nucleotide Sugar 8 7 metabolism Glutathione metabolism 7 5 Pyruvate metabolism 6 6 Valine, leucine and isoleucine 6 6 degradation Cytochrome P450 6 3 Pentose phosphate pathway 5 4
(178) TABLE-US-00005 TABLE 4 Metabolic pathways of DNA shrimp microarray analysis of the 2-Down regulated genes in L. vannamei treated with the choline/pentaborate supplemented (5 mg/g) commercial feed Metabolic Pathway # Genes # Enzymes Oxidative phosphorylation 6 4 Pentose phosphate pathway 5 5 Starch and sucrose metabolism 5 2 Purine metabolism 4 3 Phenylpropanoid biosynthesis 3 1 Aminoacyl-tRNA biosynthesis 3 3 Glycolysis/Gluconeogenesis 3 3 Pentose and glucuronate 3 2 interconversions Phenylalanine metabolism 3 1 Ascorbate and aldarate metabolism 3 2
(179) 7-6-5Data Mining of the Immune Related Genes
(180) A search of the DNA microarray analysis, with GO term: 0006955 (immune response), revealed the presence of an elevated number of relevant molecules for the immune response that are over-expressed after shrimp are fed for 14 days with the feed supplemented with choline/pentaborate complex 5 mg/g. The main components related to immune response in L. vannamei that are 2-fold Up-regulated are described in Table 5.
(181) 7-6-6Validation of Specific Genes by RT-VCR
(182) Selected genes related to immune and digestive proteins presented in the shrimp DNA microarray (Table 1) are further validated by RT q-PCR, and the results are illustrated in the
(183) TABLE-US-00006 TABLE 5 Data mining of immune related genes from the 2 Up-regulated in the shrimp DNA Microarray # NCBIID Protein Description 1 40958211 interleukin Enhancer Binding Factor 2 52863030 Probable Protein Brick1-like 3 171595417 Actin-related Protein 3 Isoform X2 4 171649044 Protein Toll 5 171484743 Srsf Protein Kinase 2 6 171604948 Profilin 7 171640969 Histone Acetyltransferase P300-Partial 8 171533428 Protein Red 9 171638050 Serine Threonine-protein Phosphatase 2a 65 Kda Regulatory Subunit A Alpha Isoform-like 10 171601498 Ubiquitin-40s Ribosomal Protein S27a 11 171578425 Ubiquitin Family Protein 12 171527123 Protein Lsm14 Homolog A isoform X2 13 171576197 Beta--Glucan-binding Protein 14 171497789 Sam Domain And Hd Domain-containing Protein 1 15 171616000 Cathepsin C 16 171660130 Gluaosidase 2 Subunit Beta 17 171644991 Calmodulin 18 171655889 Cytoplasmic Partial 19 171508897 Dipeptidyl Peptidase 1 20 171534712 Profilin 21 171606038 Dna-directed Rna Polymerases And III Subunit Rpabc6 22 171616286 Dna-directed Rna Polymerase III Subunit Rpc6 23 171649684 ExocystComplex Component2-like 24 171524287 Ubiquitin-40s Ribosomal Protein S27a 25 171488738 Ubiquitin-conjugating Enzyme E2 26 171580467 Ubiquitin C 27 171533219 Lrr And Pyd Domains-containing Protein 10 28 171626089 A Chain Orally Active 2-amino Thionopyrimidine Inhibitors Of Tho Hsp90 Chaperone 29 156637483 Lipopolysaccharide And Beta-glucan Binding Protein 30 89258160 Ecdysteroid-regulated Protein 31 68271149 Dead Box Helicase Partial 32 1907112 Bone Morphogenetic Protein 6 33 29838465 Beta-glucan-binding Protein
(184) Globally, commercial shrimp diets supplemented with 5 mg choline/pentaborate complex by gram of feed are effective to stimulate the non-specific immune system of shrimp and to improve natural resistance of L. vannamei to WSSV and Vibrio parahaemolyticus infections.
(185) Choline/pentaborate complex supplementation can increase the immunologic reactivity of shrimps as revealed by the number of immune related genes expressed in the DNA shrimp microarray analysis. There are at least 33 immune related genes that are over-expressed (Table 5). Some of these genes are further validated by RT q-PCR, and our results revealed that the expression of some of these genes, such as the Toll-Like Receptor and SOD increased more than 200 fold (
(186) As arthropod species, shrimp mainly rely on the innate immune system, which consists of humoral and cellular responses against viral infections. The direct or indirect recognition of pathogens or pathogen-associated molecular patterns by germ line-encoded proteins called pattern recognition receptors (PRRs) that tightly related to Toll-like Receptors leads to rapid humoral and cellular immune responses (Li, F., Xiang, J. 2013, Dev. Comp. Immunol. 39, 11-26).
(187) It is of interest to mention that invertebrates do not possess an adaptive immune system based on highly specific antibodies and antigen receptors. They must rely on efficient immune defense to protect them against invaders. It has been proven that hemocytes are key cells for innate invertebrate defense reactions. One important immune defense reaction of crustacean hemocytes is phagocytosis when the organism is attacked by microorganisms or viruses. During the course of phagocytosis, the host oxidases (e.g. NADPH oxidase, and particularly prophenol oxidase that is involved in the shrimp clotting system and in the innate immunity) get activated which in turn enhances the glycolytic reactions (explaining amylase gene over-expression) that will increase the consumption of oxygen, and induce the production of a mass of reactive oxygen species (ROS) such as superoxide anion (O.sub.2.sup.
(188) Besides over-expression of immune related genes in shrimp treated with diets supplemented with choline/pentaborate complex, other biological processes are modulated too in L. vannamei, including the expression of genes associated to: Response to stimuli (43), Metabolic process (166), Cellular process (159), Biogenesis (59), Developmental process (40), Biological regulation (73) Localization (66), and Signaling (26) (
(189) Shrimp diets supplemented with choline/pentaborate complex at concentration of 5 mg/g of feed are effective in reducing the impact of WSSV infection when following standardized administration protocols. This represents a major breakthrough for the control of a disease that affects a significant number of commercial shrimp production operations. The inclusion of choline/pentaborate complex used as additive in commercial diets provides an efficient mechanism for stimulation of the shrimp immune system that contribute to improve natural resistance of L. vannamei to WSSV and Vibrio parahaemolyticus infections.
EXAMPLE-8
Other Quaternary Ammonium Compounds Used to Complex with Boric Acid or its Derivatives
(190) 8-1Complexation of (2-Hydroxyethyl)Triethylammonium with Boric Acid (or its Derivatives)
(191) The complexation of (2-hydroxyethyl)triethylammonium and boric acid (or its derivatives) is prepared under identical conditions as described previously in EXAMPLE 1, except that (2-hydroxyethyl)triethylammonium is used, instead of (2-hydroxyethyl)trimethylammonium (
(192) 8-2Complexation of (2,3-Dihydroxyethyl)Triethylammonium with Boric Acid (or its Derivatives)
(193) Since choline ([2-hydroxyethyl]trimethyl ammonium) possesses a hydroxyl group, the complexation could be less stable, because there is a hydroxyl groups involved in formation of the complex with boric acid (or its derivatives). Consequently, the use of choline having two hydroxyl groups such as (2,3-dihydroxypropyl)trimethylammonium chloride seems more stable probably due to its contribution of two hydroxyl groups in complexing with boric acid (or its derivatives).
(194) The complexation of choline and boric acid is prepared under identical conditions as described previously in EXAMPLE 1, except that (2,3-dihydroxypropyl) trimethylammonium is used, instead of (2-hydroxyethyl)trimethylammonium (
(195) 8-3Complexation of Choline with Phenylboronic Acid
(196) The complexation of choline and phenylboronic acid is prepared under identical conditions as described previously in EXAMPLE 1, except that phenylboronic acid is used, instead of boric acid (
(197) 8-4Complexation of Choline with Myristylboronic Acid
(198) The complexation of choline and phenylboronic acid is prepared under identical conditions as described previously for Complexation of choline with phenylboronic acid, except that Myristylboronic acid is used, instead of phenylboronic acid (
(199) While preferred embodiments have been described above and illustrated in the accompanying drawings, it will be evident to those skilled in the art that modifications may be made without departing from this disclosure. Such modifications are considered as possible variants comprised in the scope of the disclosure.