Patent Application
20240407371
The present invention relates to methods for the killing, inactivating, or inhibiting of pathogens or pests using a lipopeptide biosurfactant.
Diseases in agricultural or aquacultural production species, both plants and animals, can be triggered by a multitude of different infectious agents from multicellular parasites to virus particles.
In WO2019101739 it was described how a lipopeptide biosurfactant, obtained from the bacterium Pseudomonas fluorescens strain H6, was used to effectively treat white spot disease in fish caused by ciliate parasite Ichthyophthirius multifiliis (ICH). Previous treatment of ICH included several chemical agents less friendly to the environment such as malachite green, sodium percarbonate, copper sulphate, formalin, peracetic acid and totrazuril. Also, certain plant extracts had attracted attention due to their potential effect on certain life stages of ICH including plant derived compounds comprising cynatratoside-C, sanguinarine, dihydrosanguinarine, dihydrochelerythrine, and pentagalloylglucose. However, the long-term residual impact on the environment, fish, and humans remains unsolved and there is a persistent need for developing new compounds and regimes for the safe treatment of agricultures or aquacultures against infestations.
Controlling of undesired species have been shown previously using various methods.
Liu et al. 2015 discloses that that some Pseudomonas species isolated from healthy salmon eggs produces more biosurfactant than Pseudomonas species isolated from Saprolegnia-infected salmon eggs, and that some Pseudomonas species such as H6 produces a viscosin-like lipopeptide surfactant which inhibits the growth of Saprolegnia diclina on salmon eggs in vitro. However this viscosin-like lipopeptide surfactant offers no significant protection of salmon eggs against Saprolegniosis.
Lamar et al. 2018 discloses control of the scuticociliatosis-causing parasite, Philasterides dicentrarchi using the saposin-like antibacterial peptide Nk-lysin or shortened analogues thereof.
Park Seong Bin et al. 2014 discloses the control of the ciliates that cause scuticociliatosis in olive flounder, including P. dicentrarchi and Miamiensis avidus, using a combination technique involving the disinfectant and surfactant, benzalkonium chloride, and bronopol.
Al-Jubury A et al. 2018 discloses that the Pseudomonas H6 lipopeptide surfactant is able to control the ciliate I. multifilis at various life-cycle stages and suggests its development for application as an antiparasitic control agent in aquaculture.
Jensen Hannah Malene et al. 2020 discloses that the Pseudomonas H6 lipopeptide surfactant is able to control gill amoebae in freshwater rainbow trout.
Parama et al. 2007 discloses the effects of the cysteine proteinases isolated from P. dicentrarc on the phagocytic functions of turbot pronephric leucocytes. Further, Parama et al. 2007 teaches that the pro-inflammatory cytokine IL-1 beta is expressed in fish infected with P. dicentrarchi like those infected with other ciliate parasites, I. multifiliis or the monogenean Gyrodactylus derjavin.
However, it remains unknown in the art which mechanism-of-action the lipopeptide biosurfactant of the art excerpts on Ichthyophthirius multifiliis or Saprolegnia diclina, which treatment regimes can be used to treat these pests in live fish in aquaculture and if lipopeptide biosurfactant of the art is effective against other types of pathogen pests sharing little or no commonalities. For example Saprolegnia is an oomycete mold which differs significantly in genotype, phenotype, habitat, life-cycle and reproduction from Ichthyophthirius which is a Ciliate.
Many microbial and simple pathogens or pests having short life spans and generation times are evolutionary much further apart compared with longer living organisms, such as humans. While humans have a generation time of approximately 25 years, many microbial or simple organisms can have generation times of days, weeks, or months, and therefore evolutionary divergence of microbes and simple organisms happens much faster than for higher life forms. In addition the anthropocentric structure of taxonomic classification creates finer divisions between taxonomic groups the more closely related they are to humans, whilst conversely grouping those that are unrelated. Accordingly, while e.g. Ichthyophthrius and Tetrahymena are both single cell organisms, they are in fact extremely biologically and genetically divergent, and the methodology for controlling such divergent species is unpredictable a priori of any known established mechanism of action.
Against this background art, the inventors of the present invention have now found that a lipopeptide surfactant, such as the lipopeptide surfactant from Pseudomonas fluorescens strain DSMZ-34058, can effectively be used to kill, inactivate, or inhibit a range of pathogens or pests or combination of pathogen or pests, which causes disease or poisoning upon infecting production species in agriculture and aquaculture and accordingly, the invention disclosed herein provides for a method for the killing, inactivating, or inhibiting of one or more pathogens or pests selected from
Moreover, it has now been been found that the killing effect on Ichthyophthrius seems to be associated with effects on cilia as well on the membrane which mechanism of action would differ completely the MoA on Saprolegnia because the latter is an oomycete which do not possess cilia.
In some aspects, the present disclosure provides for a method for the killing, inactivating, or inhibiting of one or more pathogens or pests selected from
Surprisingly, it has been shown that the lipopeptide surfactant is effective in natural habitat of these pathogens and in production species of agriculture and aquaculture, using an improved dosing regime performing better that the known pesticides such as formalin and/or Cu based pesticides currently used in agriculture and aquaculture and moreover the lipopeptide biosurfactant is biologically degradable in nature and much less toxic damaging to the ecology of the habitat.
In a further aspect as described herein is a lipopeptide biosurfactant for use in the treatment of an infection in a subject by one or more pathogens or pests selected from
In a further aspect as described herein is a lipopeptide biosurfactant for use in the treatment of an infection in a subject by one or more pathogens or pests selected from
In a further aspect disclosed herein is a bacterial isolate for use in the treatment of an infection in a subject by one or more pathogens or pests selected from
In a further aspect disclosed herein is a bacterial isolate for use in the treatment of an infection in a subject by one or more pathogens or pests selected from
The figures included herein are illustrative and simplified for clarity, and they merely show details which are essential to the understanding of the invention, while other details may have been left out. Throughout the specification, claims and drawings the same reference numerals are used for identical or corresponding parts. In the figures and drawing include herein:
All publications, patents, and patent applications referred to herein are incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In the event of a conflict between a term herein and a term in an incorporated reference, the term herein prevails and controls.
The features and advantages of the present invention is readily apparent to a person skilled in the art by the below detailed description of embodiments and examples of the invention with reference to the figures and drawings included herein.
The terms “substantially” or “approximately” or “about”, if used herein refers to a reasonable deviation around a value or parameter such that the value or parameter is not significantly changed. These terms of deviation from a value should be construed as including a deviation of the value where the deviation would not negate the meaning of the value deviated from. For example, in relation to a reference numerical value the terms of degree can include a range of values plus or minus 10% from that value. For example, deviation from a value can include a specified value plus or minus a certain percentage from that value, such as plus or minus 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from the specified value.
The term “and/or” as used herein is intended to represent an inclusive “or”. The wording X and/or Y is meant to mean both X or Y and X and Y. Further the wording X, Y and/or Z is intended to mean X, Y and Z alone or any combination of X, Y, and Z.
The term “isolated” as used herein about a compound, refers to any compound, which by means of human intervention, has been put in a form or environment that differs from the form or environment in which it is found in nature. Isolated compounds include but is no limited to compounds of the invention for which the ratio of the compounds relative to other constituents with which they are associated in nature is increased or decreased. In an important embodiment the amount of compound is increased relative to other constituents with which the compound is associated in nature. In an embodiment the compound of the invention may be isolated into a pure or substantially pure form. In this context a substantially pure compound means that the compound is separated from other extraneous or unwanted material present from the onset of producing the compound or generated in the manufacturing process. Such a substantially pure compound preparation contains less than 10%, such as less than 8%, such as less than 6%, such as less than 5%, such as less than 4%, such as less than 3%, such as less than 2%, such as less than 1%, such as less than 0.5% by weight of other extraneous or unwanted material usually associated with the compound when expressed natively or recombinantly. In an embodiment the isolated compound is at least 90% pure, such as at least 91% pure, such as at least 92% pure, such as at least 93% pure, such as at least 94% pure, such as at least 95% pure, such as at least 96% pure, such as at least 97% pure, such as at least 98% pure, such as at least 99% pure, such as at least 99.5% pure, such as 100% pure by weight.
The term “host cell” refers to any cell type that is susceptible to transformation, transfection, transduction, or the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. Host cell encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.
The term “comprise” and “include” as used throughout the specification and the accompanying items as well as variations such as “comprises”, “comprising”, “includes” and “including” are to be interpreted inclusively. These words are intended to convey the possible inclusion of other elements or integers not specifically recited, where the context allows.
The articles “a” and “an” are used herein refers to one or to more than one (i.e. to one or at least one) of the grammatical object of the article. By way of example, “an element” may mean one element or more than one element.
Terms like “preferably”, “commonly”, “particularly”, and “typically” are not utilized herein to limit the scope of the itemed invention or to imply that certain features are critical, essential, or even important to the structure or function of the itemed invention. Rather, these terms are merely intended to highlight alternative or additional features that can or cannot be utilized in a particular embodiment of the present invention.
The term “cell culture” as used herein refers to a culture medium comprising a plurality of host cells of the invention. A cell culture may comprise a single strain of host cells or may comprise two or more distinct host cell strains. The culture medium may be any medium that may comprise a recombinant host, e.g., a liquid medium (i.e., a culture broth) or a semi-solid medium, and may comprise additional components, e.g., a carbon source such as dextrose, sucrose, glycerol, or acetate; a nitrogen source such as ammonium sulfate, urea, or amino acids; a phosphate source; vitamins; trace elements; salts; amino acids; nucleobases; yeast extract; aminoglycoside antibiotics such as G418 and hygromycin B.
The term “freshwater fish” as used herein refers to fish living at least during a certain stage of its life cycle in freshwater. Example of freshwater fish includes salmonids (e.g. rainbow trout (Oncorhynchus mykiss), aquaculture (such as salmonids (exemplified by rainbow trout (Oncorhynchus mykiss), cyprinids (e.g. grass carp (Ctenopharyngodon idella), black carp (e.g. Mylopharyngodon piceus), silver carp (Hypophthalmichthys molitrix), common carp (Cyprinus carpio), bighead carp (Hypophthalmichthys nobilis), catla (Indian carp, Catla catla), crucian carp (Carassius carassius), roho labeo (Labeo rohita)), tilapia (e.g. Nile tilapia (Oreochromis niloticus)), milkfish (Chanos chanos), catfish (e.g. Amur catfish (Silurus asotus)), Wuchang bream (Megalobrama amblycephala), northern snakehead (Channa argus).
The term “marine fish” as used herein refers to fish species living at least a part of their life in marine waters. Examples are fish raised for aquaculture in mariculture systems such as gilthead seabream (Sparus auratus) and seabass (Dicentrarchus labrax). In addition, a long range of ornamental fish species used in marine aquaria is covered by the term.
The term “lipopeptide biosurfactant” as used herein refers to a compound or composition of compounds comprising a lipid connected to a peptide, preferably a cyclic peptide having surfactant properties. Surfactant properties are to be understood as lowering the surface tension (or interfacial tension) between two liquids, between a gas and a liquid, or between a liquid and a solid. The lipopeptide biosurfactant of the invention is preferably an organic compound or composition of organic compounds that are amphiphilic.
The term “in vitro” as used herein refers to medical procedures, tests, and experiments that are performed outside of a living organism. An in vitro study occurs in a controlled environment, such as a test tube or petri dish.
The term “in vivo” as used herein refers to tests, experiments, and procedures that are performed in or on a whole living organism, such as a person, laboratory animal, or plant.
All methods described herein can be performed in any suitable order of steps unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.
The term “Cyanobacteria” as used herein are also referred to as blue-green algae. Blue-green algae are a type of prokaryotes that obtain energy via photosynthesis. The main difference between bacteria and cyanobacteria/blue-green algae is that the bacteria are mainly heterotrophs while the cyanobacteria are autotrophs. Furthermore, bacteria do not contain chlorophyll while blue-green algae contain chlorophyll-a.
All percentages, ratios and proportions herein are by weight, unless otherwise specified. A weight percent (weight %, also as wt. %) of a component, unless specifically stated to the contrary, is based on the total weight of the composition in which the component is included (e.g., on the total amount of the reaction mixture).
The present invention provides for methods for the killing, inactivating, or inhibiting of one or more pathogens or pests selected from
The present disclosure also provides for methods for the killing, inactivating, or inhibiting of one or more pathogens or pests selected from
The lipopeptide biosurfactant of the invention can be any lipopeptide surfactant compound or composition of compounds that procides treatment of the pathogens or pests selected herein. Suitable lipopeptide surfactants include surfactin and derivatives thereof, daptomycin and derivatives thereof, massetolide and derivatives thereof, viscosin and derivatives thereof, thanamycin and derivatives thereof and putisolvin and derivatives thereof. In one embodiment the lipopeptide biosurfactant of the invention is a) a viscosin or viscosin-like lipopeptide or a derivative thereof; b) a massetolide or a derivative thereof and/or c) a putisolvin or a derivative or any combination of the foregoing.
Viscosin-like lipopeptides are lipopeptides that are structurally and/or functionally similar to viscosin (IUPAC: (4R)-5-[[(3S,6R,9S,12R,15S,18R,21R,22R)-3-[(2S)-butan-2-yl]-6,12-bis(hydroxymethyl)-22-methyl-9,15-bis(2-methylpropyl)-2,5,8,11,14,17,20-heptaoxo-18-propan-2-yl-1-oxa-4,7,10,13,16,19-hexazacyclodocos-21-yl]amino]-4-[[(2S)-2-[[(3R)-3-hydroxydecanoyl]amino]-4-methylpentanoyl]amino]-5-oxopentanoic acid).
In some embodiments, the viscosin-like lipopeptide comprises, similarly to viscosin, a circular peptide and a fatty acid covalently connected to the circular peptide. In some embodiments, the viscosin-like lipopeptide comprises 9 amino acids according to the general formula: L-Leucine-X1-D-Allothreonine-X2-X3-D-Serine-L-Leucine-D-Serine-X4; wherein any one of X1, X2, X3, and X4 can be any amino acid independently of each other, such as any proteinogenic amino acid, but in some embodiments;
In some embodiments, X1 is D-Glutamate and X2-X4 are as defined according to the general formula above.
In some embodiments, X1 is D-Glutamine and X2-X4 are as defined according to the general formula above.
In some embodiments, X2 is D-Valine and X1, X3-X4 are as defined according to the general formula above.
In some embodiments, X2 is D-Isoleucine and X1, X3-X4 are as defined according to the general formula above.
In some embodiments, X2 is D-Alloisoleucine and X1, X3-X4 are as defined according to the general formula above.
In some embodiments, X3 is L-Leucine and X1-X2, X4 are as defined according to the general formula above.
In some embodiments, X3 is D-Leucine and X1-X2, X4 are as defined according to the general formula above.
In some embodiments, X4 is L-Isoleucine and X1-X3 are as defined according to the general formula above.
In some embodiments, X4 is L-Leucine and X1-X3 are as defined according to the general formula above.
In some embodiments, X4 is L-Valine and X1-X3 are as defined according to the general formula above.
In some embodiments, the viscosin-like lipopeptide comprises 9 amino acids being 3×leucine, 2×serine, valine, threonine, isoleucine, and glutamic acid. In some embodiments, the viscosin-like lipopeptide comprises 9 amino acids being 3×leucine, 2×serine, valine, threonine, isoleucine, and glutamic acid.
In some embodiments, the hydroxyl group of the D-Allothreonine is bound to the carboxyl group of the amino acid residue defined by “X4”, such as to form an ester. In some embodiments, the D-Allothreonine is bound to X4 via its hydroxyl group of its side chain.
In some embodiments, the L-Leucine bound to X1 in the general formula described above is covalently connected to a fatty acid. In some embodiments, the fatty acid is C3-hydroxylated. In some embodiments, the fatty acid has a length of from 10 to 16 carbon atoms, such as from 10 to 11, such as from 11 to 12, such as from 12 to 13, such as from 13 to 14, such as from 14 to 15, such as from 15 to 16 carbon atoms. In some embodiments, the fatty acid is C3-hydroxylated and has a length of from 10 to 16 carbon atoms. In some embodiments, the fatty acid is 3-OH-decanoic acid. In preferred embodiments, the fatty acid is covalently attached via its carboxyl functionality.
In some embodiments, the viscosin-like lipopeptide of the present disclosure has a molecular weight of from 1111 to 1168 g/mol. In some embodiments, the viscosin-like lipopeptide has a molecular weight of from 1111 to 1168 g/mol and comprises the general formula as described above.
In some embodiments, the viscosin-like lipopeptide has a molecular weight of from 1111 to 1168 g/mol and comprises the general formula as described above, wherein L-leucine which is connected to X1 is also connected to a fatty acid, for example a fatty acid as disclosed herein, which is optionally C3-hydroxylated.
In some embodiments, the viscosin-like lipopeptide of the present disclosure is selected from the group consistinf of: Viscosin, Massetolide A, B, C, D, E, F, G, and H, WLIP (“White Line Inducing Principle”, Pseudophomin A and B, Viscosinamide A, B, C, and D, and Pseudodesmin A and B.
In some embodiments, the present disclosure provides a combination of one or more viscosin-like lipopeptides as disclosed herein. In such embodiments, the lipopeptide biosurfactant comprises one or more of said viscosin-like lipopeptides, optionally in a predefined ratio.
In some embodiments, the lipopeptide surfactant is provided in a composition comprising a combination of one or more viscosin-like lipopeptides as defined herein, wherein a single viscosin-like lipopeptide as defined herein accounts for at least 50% of the total combination of viscosin-like lipopeptides, such as from 50% to 99% of the total combination of viscosin-like lipopeptides, such as from 50% to 60%, such as from 60% to 70%, such as from 70% to 80%, such as from 80% to 90%, such as from 90% to 95%, such as from 95% to 99%.
In some embodiments, the single viscosin-like lipopeptide accounting for at least 50% of the total combination of viscosin-like lipopeptides comprises the general formula as described above, wherein L-leucine which is connected to X1 is also connected to a fatty acid, for example a fatty acid as disclosed herein, which is optionally C3-hydroxylated.
In some embodiment the lipopeptide surfactant is derived from a microbial source, such as a bacterium, a fungus or an achaea. Generally, the biosynthetic pathway encoding the lipopeptide surfactant within a given microbial strain leads to a single main lipopeptide surfactant and minor amounts of structurally related derivatives of the main lipopeptide surfactant. Useful and known bacterial lipopeptide biosurfactants includes for example massetolide A, massetolide B, massetolide C, massetolide D, massetolide E, massetolide F, massetolide G and massetolide H. Other useful and known bacterial lipopeptide biosurfactants includes putisolvin I and putisolvin II.
A further particularly useful viscosin-like lipopeptide biosurfactant is produced by and obtainable from the bacterium Pseudomonas fluorescens strain H6. Pseudomonas fluorescens strain H6 is also described in Liu et al. 2015 and a sample of the Pseudomonas fluorescens strain H6 was deposited on Nov. 1, 2017 under the Regulations of the Budapest Treaty in the CBS collection of the Westerdijk Fungal Biodiversity Institute with deposit number CBS 143505, which deposit is available by reference in the WO2019101739.
A further particularly useful viscosin-like lipopeptide biosurfactant is produced by and obtainable from the bacterium Pseudomonas fluorescens strain SDW1. A sample of the Pseudomonas fluorescens strain SDW1 was deposited on 6 Oct. 2021 under the Regulations of the Budapest Treaty in the DSMZ—German Collection of Microorganisms and Cell Cultures GmbH under deposit number DSMZ-34058.
In a particular embodiment the lipopeptide surfactant indeed comprises a viscosin or viscosin-like lipopeptide isolated from Pseudomonas fluorescens strain H6 or SDW1. In other embodiments lipopeptide biosurfactant of the invention comprises a massetolide, such as a massetolide lipopeptide surfactant obtainable from Pseudomonas fluorescens strain SS101 or a derivative thereof. In addition or in the alternative, the lipopeptide biosurfactant of the invention can also comprise a putisolvin, such as the putisolvin biosurfactant obtainable from Pseudomonas putida 267 or a derivative thereof. In addition or in the alternative, the lipopeptide biosurfactant of the invention can also comprise a viscosin lipopeptide, such as the viscosin lipopeptide obtainable from Pseudomonas fluorescens SBW25 or a derivative thereof. The isolation and characterization of the lipopeptide biosurfactant of Pseudomonas fluorescens strain H6 or SDW1 can be done as described by Liu et al. (2015) and is found to be clearly distinguished from the well-known lipopeptide biosurfactants of related strains such as the massetolide lipopeptide obtained from Pseudomonas fluorescens SS101 (described in De Bruijn et al (2008)), the viscosin lipopeptide obtained from Pseudomonas fluorescens SBW25 and the putisolvin lipopeptide obtained from Pseudomonas putida 267 (described in Kruijt et al (2008)).
The lipopeptide biosurfactant may be incorporated into a composition comprising a single lipopeptide biosurfactant compound or two or more lipopeptide compounds. Such compostion can further include one or more carriers, agents, adjuvants, additives and/or excipients. The composition of the invention can further be formulated into a desirable final dry or liquid formula such as a slow-release form capable of releasing the lipopeptide biosurfactant(s) over a prolonged period of time to a surrounding medium. Suitable formulations include spray dried, lyophilized, granulated extruded, liquid stabilized formulas,
In particular embodiments the lipopeptide biosurfactant is isolated from a microbial source such as strains of Pseudomonas including Pseudomonas fluorescens strain H6 or SDW1, Pseudomonas fluorescens strain SS101, Pseudomonas fluorescens strain SBW25 and/or Pseudomons putida strain 267, as a composition which contain only a limited amount of ammonia, such as below 10 mg ammonia/g LS. Ammonia, can advantageously be used to promote microbial production of the lipopeptide biosurfactant, but is undesirable in composition to be used for treatment of infections in subject because ammonia generally is toxic to fish.
In some cases, the pathogen or pest is contacted with the lipopeptide biosurfactant in an aqueous solution comprising an effective amount of the lipopeptide biosurfactant to kill, inactivate, or inhibit the pathogen or pest. Such aqueous solutions can be is a hypersaline (hyper saline lakes or trapped tidal coastal water bodies), a marine (sea water), a brackish (mixture of sea water and fresh water eg from rivers) or a freshwater solution (waters of lakes, rivers, bogs, puddles etc.).
In some cases, the pathogen or pest is a flagellated protist including dinoflagellate. Flagellated protists, particularly in the form of microalgae species can given the right conditions grow excessively in hypersaline, marine, brackish and/or freshwater environments known as a Harmful Algal Bloom (HAB), also for marine environments known as “red tide”. HAB is an algal bloom that causes negative impacts to other organisms including humans via production of natural algae-produced toxins, mechanical damage to other organisms, or by other means. HABs are sometimes defined as only those algal blooms that produce toxins, and sometimes as any algal bloom that can result in severely lower oxygen levels in natural waters, killing organisms in marine or fresh waters. More specifically, species causing HAB usually have one or more of the following properties and harmful impacts: i) non-toxic species but high biomass blooming species that can directly or indirectly kill marine organisms by deoxygenation of water bodies, or by their physical effects, ii) species producing toxins involved in food poisoning in humans with either neurological or gastrointestinal symptoms, iii) species causing no damage to humans but which are harmful to fish and marine invertebrates by mechanical effects. Such blooms can last from a few days to many months. After the bloom dies, the microbes that decompose the dead algae use up more of the oxygen, generating a “dead zone” which can cause fish die-offs. When these zones can cover a large area for an extended period of time, where neither fish nor plants are able to survive.
Described herein is an environment friendly solution to the problem of Harmful Algal Bloom, using natural fermented compounds killing, inactivating, or inhibiting the microalgaes causing the HAB. In a separate aspect the solution is a method for the killing, inactivating, or inhibiting of one or more flagellated protists comprising contacting the flagellated protists in an aqueous solution with an effective amount of a lipopeptide biosurfactant. The lipopeptide biosurfactants described herein is less toxic than the toxins naturally produced by the HAB microalgae.
In some embodiments the aqueous solution is a hypersaline solution, a marine solution, a brackish solution or a freshwater solution, and the flagellated protist is a micro algae, optionally a harmful microalgae capable of causing Harmful Algal Bloom (HAB) in hypersaline, marine solution, brackish or freshwater environments. The flagellated protist is suitably selected from one or more of the classes Dinophyceae, Pelagophyceae, Raphidophyceae, or Prymnesiophyceae. The Dinophyceae is suitably of the order Actiniscales, Akashiwales, Amphilothales, Apodiniales, Blastodiniales, Brachidiniales, Dinophysales, Dinotrichales, Gonyaulacales, Gymnodiniales, Haplozoonales, Nannoceratopsiales, Peridiniales, Phytodiniales, Prorocentrales, Ptychodiscales, orThoracosphaerales or a combination thereof. Within this embodiment the Gonyaulacales can be of the family Ostreopsidaceae, optionally of the genus Alexandrium, optionally of the species A. tamarense; and/or optionally of the genus Gambierdiscus, optionally the species G. toxicus. Additionally or alternatively, the Gymnodiniales can be of the family Kareniaceae; optionally of the genus Karenia; optiojally of the species K. brevis. The Pelagophyceae can be of the order Pelagomonadales or Sarcinochrysidales or a combination thereof, where the Pelagomonadales suitably is of the genus Aureococcus, optionally the species A. anophagefferens. The Raphidophyceae can be of the order Actinophryida, Chattonellales, Commatiida, or Raphidomonadales or a combination thereof, where the Chattonellales suitably is of the genus Heterosigma, optionally the species H. akashiwo. The Prymnesiophyceae can be of the order Coccolithales, Coccosphaerales, Isochrysidales, Phaeocystales, Prymnesiales, Syracosphaerales, or Zygodiscales, where the Prymnesiales suitably is of the genus Prymnesium, optionally the species P. parvum.
In some embodiments the aqueous solution is freshwater, and the pathogen or pest is:
In some embodiments the aqueous solution is freshwater and the pathogen or pest is:
In other embodiments the aqueous solution is hypersaline, marine, or brackish marine and the pathogen or pest is
In other embodiments the aqueous solution is hypersaline, marine, or brackish marine and the pathogen or pest is
In further embodiments the pathogen or pest is causing disease in mollusks and the pathogen or pest is:
In still other cases the pathogen or pest is a virus selected from the genus Whispovirus causing white spot syndrome in whiteleg shrimp
Additionally or alternatively the pathogen or pest can also be a plant pathogen or pest, such as
Additionally or alternatively the pathogen or pest can also bea human pathogen or pest such as a flatworm selected from the genus Schistosoma causing the disease schistosomiasis in humans and wherein the pathogen or pest is contacted with the lipopeptide biosurfactant in an amount effective of preventing and/or inhibiting the proliferation of the flatworm.
Additionally or alternatively the pathogen or pest can also be a plant pathogen or pest, such as
Additionally or alternatively the pathogen or pest can also be a human pathogen or pest such as a sporozoan selected from the genus Cryptosporidium contaminating water causing the disease cryptosporidiosis in humans and wherein the pathogen or pest is contacted with the lipopeptide biosurfactant as a disinfectant in an amount effective of preventing and/or inhibiting the excystation of the oocysts.
Additionally or alternatively the pathogen or pest can also be an avian pathogen or pest such as a sporozoan selected from the genus Eimeria causing the disease coccidiosis in avians and wherein the pathogen or pest is contacted with the lipopeptide biosurfactant as a disinfectant in an amount effective of preventing and/or inhibiting the sporulation of the oocysts.
The effective amount of the lipopeptide biosurfactant is suitably a concentration in the aqueous solution of from 5 μg/mL to 1000 mg/L. In some embodiments, in particular in the treatment of micro-algae causing red/green tide, the concentration of the lipopeptide biosurfactant in the aqueous solution is preferably between 0.1 to 1000 mg/L, optionally 0.5 to 500 mg/L, optionally 1 to 100 mg/L, optionally 2 to 50 mg/l, optionally 5 to 25 mg/L.
Also described herein are lipopeptide biosurfactants effective in treating infections in a subject resulting by one or more pathogens or pests selected from Ciliates, which is not Ichthyophthirius multifiliis; Flagellated protists, including dinoflagellates; Flatworms; Amoebae; Bacteria, including cyanobacteria; Viruses; Oomycetes; and/or Fungi.
In some embodiments the subject to be treated is a fish. The fish may be a hypersaline, a marine, a brackish or a freshwater fish, preferably useful for farming for consumption or as an ornamental fish.
For freshwater fish, the lipopeptide surfactant of the invention has been found to be effective in the treatment of
For freshwater fish, the lipopeptide surfactant is in some embodiments effective in the treatment of trichodiniasis, where the pathogen or pest is a ciliate parasite of the genus Trichodina;
For marine fish, the lipopeptide surfactant of the invention has been found to be effective in the treatment of
For marine fish, the lipopeptide surfactant is in some embodiments effective in the treatment of
In some embodiments the subject to be treated is a mollusk. The mollusk may be a marine or a freshwater mollusk useful for farming and consumption. For mollusks the lipopeptide surfactant of the invention has been found to be effective in the treatment of
In some embodiments the subject to be treated is a crustacean. The crustacean may be a marine or a freshwater crustacean preferably useful for farming and consumption. For crustaceans such as shrimps, in particular whiteleg shrimps, the lipopeptide surfactant of the invention has been found to be effective in the treatment of white spot syndrome, where the pathogen or pest is a virus of the genus Whispovirus.
In some embodiments the subject to be treated is a cultivated plant. For plants the lipopeptide surfactant of the invention has been found to be effective in the treatment of
In some embodiments the subject to be treated is an animal. For animals, including humans, the lipopeptide surfactant of the invention has been found to be effective in the treatment of schistosomiasis, where the pathogen or pest is a flatworm of the genus Schistosoma, and wherein the pathogen or pest is contacted with a lipopeptide biosurfactant in an amount effective of killing and/or preventing and/or inhibiting the proliferation of the flatworm.
The lipopeptide biosurfactant useful for the treatment of diseases in animals or plants are described supra.
The lipopeptide surfactant of the invention is applied to the subject in a suitable manner enabling effective treatment of a subject. Where the subject to be treated is aquacultured species, fish, mollusks etc, the lipopeptide biosurfactant of the invention can be added to the water where the subject is kept and cultured. Alternatively, the lipopeptide biosurfactant can also be produced in situ by adding and cultivating a microbial source producing the lipopeptide biosurfactant to the water where the subject is kept and cultured. The lipopeptide biosurfactant can also be administered by spiking the lipopeptide biosurfactant of the invention into a feed for the subject which is then ingested by the subject. Concentrations of the lipopeptide biosurfactant in the water should be kept between 5 to 1000 μg/ml, such as 10 to 100 μg/ml of the lipopeptide biosurfactant. In some embodiments suitable concentrations can range from 30 to 70 μg/ml, especially about 50 μg/ml. In other embodiments further embodiments suitable concentrations can be at least 10 μg/ml, especially at least 30 μg/ml, such as at least 50 μg/ml. In still further embodiments suitable concentrations can be up to 500 μg/ml, especially up to 200 μg/ml, such as up to 100 μg/ml.
Where the subject is a plant the lipopeptide biosurfactant can be applied administering a powder or a solution comprising the lipopeptide biosurfactant of the invention to the plant, or alternatively administering a microbial source producing the lipopeptide biosurfactant in situ on the plant to be treated.
Where the subject is an animal the lipopeptide biosurfactant of the invention can be administered enterally, parenterally or topically.
Suitable administration of the lipopeptide surfactant of the invention includes one-time administration or repeated administrations and can also include bolus administrations to achieve peak concentrations, optionally supplemented with lower concentration maintenance administrations.
In some aspects, the a lipopeptide biosurfactant as defined herein is used in a closed or semi-closed water flow system that comprises a bacterial water filter, whereby the lipopeptide biosurfactant works on a target pathogen but the bacterial water filter is unharmed.
1. A method for the killing, inactivating, or inhibiting of one or more pathogens or pests selected from
Chemicals used in the examples herein e.g. for buffers and substrates are commercial products of at least reagent grade. (NH4)2SO4, Na2HPO4, KH2PO4, NaCl, HCl, NaOH, NH3·H2O and corn steep liquor are used in the fermentation medium and purification reagent.
Detection and quantification of lipopeptide surfactant (LS) was performed on Agilent Technologies 1200 series High Performance Liquid Chromatogram (HPLC) equipped with Luna® 5 μm C18(2) 100 Å, LC Column 150×2 mm (Part No. #00F-4252-BO, Phenomenex). Mobile phases of water with 0.1% (v/v) trifluoroacetic acid (TFA) (Solution A) and acetonitrile (ACN) with 0.085% (v/v) TFA (Solution B) were used. The absorbance at 210 nm was used to detect LS. One to five microliters of sample were injected onto the column. The flow rate was set at 0.9 ml/min. The HPLC running program was started with 90% of the solution A and 10% of the solution B, after running 2 minutes, the solution B was increased to 80% with gradient and the solution A reduced to 20%. After 8 minutes, the solution B was increased to 100% and kept running for 1 minute, between the 9 minutes to 10 minutes, the solution B was reduced back from 100% to 10% and the solution A was increased from 0% to 90%. LS purified by AnalytiCon Discovery GmbH (Germany) using preparative HPLC was used as standard LS with >99.5% purity for quantification.
In all in vivo efficacy tests, fish were acclimatized, regarding the light/dark cycle and temperature, to the experimental conditions for 14 days prior to performing the experiments.
A strain of Pseudomonas sp., strain LMG 5329 available from Belgiun Coordinated Collections of Microorganisms (BCCM)—see: https://bccm.belspo.be/catalogues/lmg-strain-details?NUM=5329&COLTYPE=&LIST1=STRNUM&TEXT1=5329&LIST2=SPECIES&TEXT2=&LIST3=ORIG SUBST&TEXT3=&LIST4=ORIGIN&TEXT4=&LIST5=ALL%20FIELDS&TEXT5=&CONJ=OR&RANGE=20 was fermentated and lipopeptide biosurfactant was isolated and purified using the methodology of Liu et al 2015 and/or in WO2019101739. This strain is also known from Rokni-Zadeh et al 2013 and Oni et al 2020 to produce the lipopeptide White Line Inducing Factor (WLIP). HPLC analysis of the resulting isolate revealed a major product peak of lipopeptide (
A stock solution of the lipopeptide surfactant (LS) was prepared prior to the tests by dissolving the freeze-dried LS in distilled water and preparing a dilution series ranging from 5 to 500 μg/ml for pathogen or pest exposures. Negative control (pathogen or pest and distilled water), blank control (distilled water and media), and turbidity control (LS and media) were included in all tests.
Lipopeptide biosurfactant of Pseudomonas fluorescens strain H6 was extracted according to the method described by Liu et al. (2015) and/or WO2019/101739. The Pseudomonas fluorescens strain H6 deposited under CBS 143505 was grown on Pseudomonas agar plates for 48 hours at 25° C. Cells of strain H6 were collected from the agar plates. Cells were collected from the agar plates and suspended in sterile de-mineralized water and mixed to homogenize the cell suspension. Cell suspensions were then centrifuged twice for 10 min at 9,000 rpm at 4° C. and the supernatant was filter-sterilised with 0.2 um filters. The lipopeptide biosurfactant present in the cell-free culture supernatant was precipitated by acidification of the supernatant with 9% (v/v) HCl to pH 2.0. Precipitation was allowed for 1 hour on ice. The precipitate was collected by centrifugation at 12000 g, 4° C. for 15 min speed and washed with acidified (pH 2.0) demineralized water. Demineralized water was added to the washed precipitate and the pH was adjusted to 8.0 with 0.2 M NaOH to allow the precipitate to dissolve. The resulting solution of lipopeptide biosurfactant was freeze-dried in a vacuum freeze dryer. A HPLC analysis chromatogram is shown in
A stock solution of the lipopeptide surfactant (LS) was prepared prior to the tests by dissolving the freeze-dried LS in distilled water and preparing a dilution series ranging from 5 to 500 μg/ml for pathogen or pest exposures. Negative control (pathogen or pest and distilled water), blank control (distilled water and media), and turbidity control (LS and media) were included in all tests.
Lipopeptide biosurfactant of Pseudomonas fluorescens strain SDW1 was extracted according to the method described by Liu et al. (2015) and/or WO2019/101739. The Pseudomonas fluorescens strain SDW1 was grown on Pseudomonas agar plates for 48 hours at 25° C. Cells of strain SDW1 were collected from the agar plates. Cells were collected from the agar plates and suspended in sterile de-mineralized water and mixed to homogenize the cell suspension. Cell suspensions were then centrifuged twice for 10 min at 9,000 rpm at 4° C. and the supernatant was filter-sterilised with 0.2 um filters. The lipopeptide biosurfactant present in the cell-free culture supernatant was precipitated by acidification of the supernatant with 9% (v/v) HCl to pH 2.0. Precipitation was allowed for 1 hour on ice. The precipitate was collected by centrifugation at 12000 g, 4° C. for 15 min speed and washed with acidified (pH 2.0) demineralized water. Demineralized water was added to the washed precipitate and the pH was adjusted to 8.0 with 0.2 M NaOH to allow the precipitate to dissolve. The resulting solution of lipopeptide biosurfactant was freeze-dried in a vacuum freeze dryer.
A stock solution of the lipopeptide surfactant (LS) was prepared prior to the tests by dissolving the freeze-dried LS in distilled water and preparing a dilution series ranging from 5 to 500 μg/ml for pathogen or pest exposures. Negative control (pathogen or pest and distilled water), blank control (distilled water and media), and turbidity control (LS and media) were included in all tests.
Ciliates are propagated in a T-25 flask containing a suitable culture medium and harvested 1-2 days after reaching peak density (quantified by hemacytometer). Volumes of 100 μl of diluted ciliate culture are aliquoted into tubes containing 100 μl of LS from example 1, 2 and 3 at concentrations ranging from 5 to 160 μg/ml (total volume 200 μl), vortexed, and transferred into 12-well cell culture plates together with controls (each sample in quadruplicate). Plates are covered, incubated at room temperature (22-25° C.), and observed at 0, 15, 30, 45, 60, 90, and 120 minutes for motility and MIC confirmation under a stereomicroscope based on observations of ciliate motility. Non-motile and lysed ciliates are considered dead.
LS concentrations between 10 and 30 μg/ml and above are lethal for tested ciliates within 30-60 min of exposure, whereas concentrations lower than 10 μg/ml shows little or no effect on the pathogens or pests.
Flagellated protists/dinoflagellates are propagated in a T-25 flask containing a suitable culture medium and harvested 1-2 days after reaching peak density (quantified by hemacytometer). Volumes of 100 μl of diluted flagellated protist culture are aliquoted into tubes containing 100 μl of LS from example 1, 2 and 3 at concentrations ranging from 5 to 160 μg/ml (total volume 200 μl), vortexed, and transferred into 12-well cell culture plates together with controls (each sample in quadruplicate). Plates are covered, incubated at room temperature (22-25° C.), and observed at 0, 15, 30, 45, 60, 90, and 120 minutes for motility and MIC confirmation under a dissecting microscope based on observations of flagellated protist motility. Non-motile flagella and lysed cells are considered dead.
LS concentrations between 10 and 30 μg/ml and above are lethal for tested flagellated protists/dinoflagellates within 30-60 min of exposure, whereas concentrations lower than 10 μg/ml shows no effect on the pathogens or pests.
To provide the source of flatworms (monogeneans) for tests, a laboratory infection is established under controlled conditions. To obtain parasites, fish from laboratory infection are anaesthetized and live parasites are removed from the fish surface and transferred into 12-well cell culture plates. Volumes of 200 μl of LS from example 1, 2 and 3 at concentrations ranging from 5 to 160 μg/ml are added into the plate wells together with controls (each sample in quadruplicate). Plates are incubated at room temperature and observed for survival and egg production under a stereomicroscope after 1 h, 2 h, 3 h, 4 h, 8 h, 12 h, and 24 h of exposure. Parasite showing no signs of motion and failing to respond to tactile stimulus are considered dead (Trasviña-Moreno et al. 2017).
LS concentrations between 10 and 30 μg/ml and above are lethal for tested flatworm within 30-60 min of exposure, whereas concentrations lower than 10 μg/ml shows no effect on the pathogens or pests.
Isolated amoebae from infected fish are cultured on malt yeast agar (0.01% malt, 0.01% yeast, 2% Bacto agar, 0.2 am filtered sea water with 35% salinity) overlaid with 0.2 am filtered sea water. Plates are incubated at 18° C. (Cano et al. 2019). Amoebae are subcultured by transferring amoebae by pipetting from the agar plate to wells in a 24-well cell culture plate containing malt yeast agar (1 ml in each well) and moistened with Neff's amoeba saline (Jensen et al. 2020). Subcultures are allowed to establish over weeks and cell counting is performed in a haemocytometer. When dense amoeba layers (more than 20 live amoebae in a 20 μl) is achieved, volumes of 500 μl of LS from example 1, 2 and 3 at concentrations ranging from 5 to 500 μg/ml are added into the plate wells together with controls (each sample in quadruplicate). Plates are incubated for 24 h, and viability of amoeba are recorded at 5 min, 30 min, 1 h, 2 h, 24 h. Amoebae with shrunken appearance, lack of movements, and no cytoplasmic activity are considered dead (Jensen et al. 2020).
After 24 h, all amoebae exposed to 250 μg/ml and above are dead. Both live and dead amoebae are observed in wells exposed to 125 μg/ml.
Lipopeptide biosurfactant of DSMZ 34058 (hereinafter referred to as LP34058) obtained in example 3 was tested for impacts on nitrifying bacteria in biofilters.
Biofilter bacteria in fish tanks have the key function of nitrifying the ammonia derived from fish feces and leftover food and converting it into the less toxic nitrite and subsequently nitrate.
To evaluate the effect of LP34058 on nitrifying bacteria, biofilters from an earlier fish challenge study treated with low dose (6.3 mg active compound/L) and high dose (12.6 mg active compound/L) of LP34058 were used for a new study targeting the water quality. In this study the total ammonia nitrogen (TAN), nitrite and nitrate were measured for 15 consecutive days using test strips.
The study consisted of 6 arms: A) No fish, no biofilter; B) No fish+biofilter not exposed to LP34058; C) Six fish+biofilter previously exposed to “low dose” LP34058; D) No fish+biofilter, previously exposed to “low dose” LP34058”); E) Six fish+biofilter, previously exposed to “high dose” LP34058; and F) No fish+biofilter, previously exposed to “high dose” LP34058. Feed was added every second day, and no water exchange was performed during the study.
No fish from arms C and E showed any signs of intoxication, indicating good water quality and full functionality of biofilter. These observations were in accordance with the concentrations of TAN, nitrite and nitrate measured (
The study confirms that LP34058 does not affect biofilter performance.
Oomycete/fungus isolates are grown on agar dish containing glucose and yeast extract until covering the dish. A small section of agar is cut out and inverted onto a polycarbonate filter membrane and incubated until mycelium reaches the edge of the membrane. Mycelia and filters are transferred in quadruplicate to sterile petri dishes containing different concentrations of LS from example 1 and 2 ranging from 10 to 320 μg/ml. For each concentration, a control dish containing distilled water only (no LS) and a control dish containing no oomycete is included. Membranes are kept in LS solution for 1 day, and then washed with distilled water and transferred to an agar plate. Agar plates are then incubated at respective conditions regarding temperature and incubation time. Following incubation, hyphal growth is measured, and morphological abnormalities are evaluated under stereomicroscope.
Exposure to LS show almost complete growth-inhibitory activity at 80 μg/ml. Hyphal growth is partially inhibited at the concentration of 40 μg/ml and below. Larger hyphal diameter is observed in oomycetes grown under exposure with LS with higher number of branches compared to the control.
Three experimental groups are established having 2 tanks per group each containing 5 fish subjects, divided as follows:
Prior to testing the treatment of fish with the LS, parasites are introduced into the tank water of group B and C. The time from introduction of the parasite to testing treatment effect depends on the parasite species and its life cycle and varies from hours to days. When the infection of the fish by the parasite is established, group C is treated with LS from example 1 and 2.
For infections with ciliates, flagellated protists/dinoflagellates, flatworms, and amoeba, the fish receive a bath exposure of LS at the effective concentration (based on the results from MIC tests) for 6 h.
Afterwards, fish are transferred to a tank with plain water. After the treatment, the fish subjects are euthanized and checked under a stereomicroscope for the infection status in the gills, fins, and skin.
After the course of treatment, fish subjects in group C has no detectable parasite infection, while fish in group B had parasite infection.
Three experimental groups are established having 2 tanks per group each containing 5 fish subjects, divided as follows:
Prior to testing the treatment of bacterium-infected fish with the LS, the fish in group B and C are challenged by bath exposure to the bacteria in 5 L water volume for 6 h, and then, the tank water level is raised up to 20 L diluting the bacterial concentration. The fish swim in the bacterial solution for 18 h after which water is totally replaced with bacterium free water. Subsequently, fish from group C are subjected to daily baths of 2-3 hours with LS from example 1 and 2 at the effective concentration for 4 days, consecutively.
Upon termination of the experiment, headkidney swabs from fish in group B and C are collected and cultured on blood agar to re-isolate the bacteria from group B to confirm that the bacterium causing disease was identical to the challenge strain, and also to confirm the absence of bacterial infection in treated fish with LS.
After the course of treatment, no bacterial infection is detected in the fish treated with LS.
For infection model with oomycete (Saprolegnia/Achlya) salmon eggs are used. Live eggs are placed in 97-98° C. distilled water and incubated for 80-150 s. The dead eggs are drained and placed on potato dextrose agar plates previously grown with oomycete isolates and incubated overnight at 25° C. Afterwards, the eggs are transferred into a 24-well cell culture plate and incubated for 1 day at 15-25° C. until colonization of oomycete hyphae is visible.
Five experimental groups are divided as follows:
Each experimental group is divided in two incubation units each containing three perforated cups with ≈50 live salmon eggs per cup. In the infection groups, two of the dead infected eggs are added to each cup. The infected eggs receive treatment with LS and malachite green every 2-3 days for 90-120 min with aeration. Then, the treated water is removed, eggs are rinsed, and fresh water is replaced. Hyphal expansion on eggs is measured every 48 h and hyphal attachment is evaluated by lifting infection inocula and counting the number of eggs attached to the hyphal patch at 18 days post infection (Liu et al. 2014, Liu et al. 2015).
Exposure to LS showed partial growth-inhibitory activity at 40 μg/ml and almost complete inhibitory effect at 80 μg/ml.
Lipopeptide biosurfactant of DSMZ 34058 (hereinafter referred to as LP34058) obtained in example 3 was tested for effectiveness in inhibiting the growth rate and cell yield of marine harmful microalgae species. The tests were conducted on the microalgae strains following the basic outline of OECD Method No. 201, incorporated herein by reference. Briefly, a single parent culture for each strain, except for Gambierdiscus toxicus (Strain no.: CCMP3466), was grown to mid/late exponential phase in a 500 mL glass flask, this single culture was divided equally across 12 sterile glass test tubes; triplicate test tubes with maintenance media (as a control) and triplicate test tubes with LP34058 in maintenance media at each of ˜16.7, ˜66.7 and ˜333.3 mg/L (active Lipopeptide biosurfactant concentrations of ˜5, ˜20 and ˜100 mg/L concentrations) with a final volume of 18 mL for each replicate. CCMP3466 does not grow well in glass test tubes, and thus grown in a 500 mL glass flask and transferred to sterile 50 mL slant neck tissue culture flasks laying on their side for the test experiment. Culture test tubes/t-flasks were then incubated at the standard growth temperature and irradiance (50-100 μmol photons m-2 s-1) for each strain. Cell growth was assessed by daily readings of in vivo chlorophyll fluorescence (except CCMP3466) and direct counts of cell abundance for 96 h. Cell abundance samples were counted using an Improved Neubauer Hemocytometer, with ˜200 cells counted for each time point.
Growth rates from the in vivo chlorophyll fluorescence and daily cell count estimates were calculated over the entire experimental period using a standard equation (eq. 1) for each replicate in the control and treatments.
where N2 and N1 are fluorescence readings/cell counts at times T2 (96 h) and T1 (0 h). Time course plots of fluorescence and cell counts were plotted and examined to confirm that the control treatment for all phytoplankton strains were growing exponentially throughout the entire test period.
Cell yield, final minus initial cell abundance, was calculated for each strain, as the other evaluation metric noted in OECD Method No. 201. For each strain, differences between the cell count based growth rates and cell yield in each treatment versus the respective control were statistically assessed using a 1-way ANOVA followed by Tukey's test if the normality assumption was met, or the Holm-Sidak Test if it was not. All pairwise comparisons were evaluated against a probability value of 0.05.
LP34058 was fully dissolved in Milli-Q water to make a 10.04 mg/ml stock solution. The LP34058 solution was sterile filtered (0.2 μm syringe filter) and added to the test tube containing each strain as outlined in Table 13-1.
The following marine phytoplankton strains from the NCMA Culture Collection were used in these growth assays (Table 13-2).
Alexandrium
tamarense
Aureococcus
anophagefferens
Gambierdiscus
toxicus
Heterosigma
akashiwo
Karenia
brevis
Prymnesium
parvum
All strains grew in the control treatments. Based upon the examination of the time course plots, all strains were in exponential growth throughout the experiment (ie., linear increase in fluorescence/cell count with time when plotted as the natural log). Growth rates were all significantly greater than zero (Table 13-2). As predicted, growth rates were much lower than those of the ‘green weeds’ recommended in the OECD 201 method, and thus fold-increases in biomass ranged from 1.8-7.4, averaging 3.9. Gambierdiscus toxicus (CCMP3466) grew the slowest and showed the least fold-increase in biomass (1.84), regardless, it was very clear that the LP34058 treatments had the desired effect on this strain. Responses to the LP34058 treatment varied between strains, but all strains were susceptible to LP34058 with at least one treatment concentration, showing a lethal effect and loss of cells.
For all strains, a consistent growth pattern was observed between in vivo chlorophyll fluorescence and cell abundance time courses in the controls and treatments (
For most strains, even the lowest LP34058 treatment concentration, 16.67 mg LP34058/L & 5 mg active component/L, resulted in a significant reduction in growth rate and cell yield (Table 13-3), the one exception being Aerococcus anophagefferens (CCMP1984). A. anophagefferens did not show any negative impact at the lowest LP34058 concentration, while the mid-level concentration appeared to be sublethal as the culture recovered in the middle of the test and continued to grow but at a slower rate. The highest LP34058 concentration was clearly toxic resulting in significant cell loss. It is worth noting that the majority of the treatments were found to have large negative growth rates, meaning cells were not only not growing, but their cell membrane integrity was being compromised leading to the lysis of cells and thus the dramatic loss of cells; near quantitative loss in many cases. This was very evident for G. toxicus and A. tamarense as few live cells were observed in the LP34058 treatments, but empty thecae were very abundant confirming that there had been live cells in the culture at the beginning of the experiment. For H. akashiwo, K. brevis, and P. parvum the cells become increasingly ‘ratty’ looking, with cell membranes clearly being compromised and the substantial increase in detritus associated with the lysis of cells. Furthermore, for most strains, the LP34058 treatments led to increased settling of cells in the test tube compared to the respective controls, and in some cases, it was very difficult to get the cells resuspended. Overall, it appears that the LP34058 compound showed the desired response—significant reduction in harmful algae growth and cell abundance. The reduced data are shown in Table 13-3, and includes calculated growth rates for controls and treatments, percent inhibition calculations, and statistical analyses.
LD50 values (i.e. the concentration at which 50% of algal cells will be killed after 96 hrs) were estimated using the AAT Bioquest online calculator2 (incorporated herein by reference), with the results given in Table 13-4. Given that for most strains, all LP34058 treatments results in near total cell loss even at the lowest treatment concentration estimated LD50's should be considered with a great degree of caution.
However, for A. tamarense (CCMP1771) and A. anophagefferens (CCMP1984), these estimates appear reasonably robust.
A. tamarense (CCMP1771)
A. anophagefferens (CCMP1984)
G. toxicus (CCMP3466)
H. akashiwo (CCMP3149)
K. brevis (CCMP2281)
P. parvum (CCMP3037)
Lipopeptide biosurfactant of DSMZ 34058 (hereinafter referred to as LP34058) obtained in example 3 was tested for effectiveness in inhibiting the growth rate and cell yield of marine harmful microalgae species and freshwater/brackish water cyanobacteria species by the National Center for Marine Algae and Microbiota (NCMA) at Bigelow Laboratory. The tests were conducted on the strains following the basic outline of OECD Method No. 201, incorporated herein by reference. Briefly, a single parent culture for each strain was grown to mid/late exponential phase in a 500 mL glass flask, this single culture was divided equally across sterile glass test tubes; triplicate test tubes with maintenance media (as a control) and triplicate test tubes with LP34058 in maintenance media at each of concentrations listed in Table 14-1. Culture test tubes/t-flasks were then incubated at the standard growth temperature and irradiance (50-100 μmol photons m-2 s-1) for each strain. Cell growth was assessed by readings of in vivo chlorophyll fluorescence and direct counts of cell abundance for 72 h.
Cell abundance samples were counted using an Improved Neubauer Hemocytometer or a Palmer Maloney counting chamber.
Growth rates from the in vivo chlorophyll fluorescence and cell count estimates were calculated over the entire experimental period using a standard equation (eq. 1) for each replicate in the control and treatments.
Where N2 and N1 are fluorescence readings/cell counts at times T2 (72 h) and T1 (0 h). Time course plots of fluorescence and cell counts were plotted and examined to confirm that the control treatment for all phytoplankton strains were growing exponentially throughout the entire test period. Cell yield, final minus initial cell abundance, was calculated for each strain, as the other evaluation metric noted in OECD Method No. 201. For each strain, differences between the cell count based growth rates and cell yield in each treatment versus the respective control were statistically assessed using a 1-way ANOVA followed by Tukey's test if the normality assumption was not met, or the Holm-Sidak Test if it was. All pairwise comparisons were evaluated against a probability value of 0.05.
LP34058 was made up at 10.04 mg/ml in Milli-Q water. The compound fully dissolved and no ‘pellet’ was observed at the bottom and thus no sodium hydroxide was added (per dilution protocol provided by Sundew). The LP34058 solution was sterile filtered (0.2 μm syringe filter) and added to the test tube containing each strain as outlined in Table 14-1.
10.04
0.6
18
334.52
10.04
0.12
18
66.90
10.04
0.03
18
16.73
The following marine phytoplankton strains from the NCMA Culture Collection were used in these growth assays (Table 14-2).
Alexandrium
tamarense
Heterosigma
akashiwo
Karenia brevis
Prymnesium parvum
Prorocentrum lima
Chattonella marina
Nostoc sp.
Aphanizomenon sp.
Microcystis cf.
aeruginosa
All strains grew in the control treatments, although growth rates were low to moderate. Based upon the examination of the time course plots, all strains were in exponential growth throughout the experiment (ie., linear increase in fluorescence/cell count with time when plotted as the natural log). As predicted, growth rates were much lower than those of the ‘green weeds’ recommended in the OECD 201 method, and thus fold-increases in biomass in the control treatments was <3-fold. Responses to the LP34058 treatment varied between strains, but all strains were susceptible to LP34058 with at least one treatment concentration, showing a lethal effect and loss of cells (Tables 14-3 & 14-4).
In this experiment, with the finer temporal resolution of 0.5-1 h measurements highlighted that there appeared to be an autofluorescence in the LP34058 compound. Without further study we cannot determine which compound may be autofluorescing, understand its decay characteristics, and ultimately its impact on fluorescence in the Chlorophyll emission wavelength range. Thus, for this experiment, while we include the chlorophyll a fluorescence data, it should be interpreted with care.
For most strains in the ‘low test concentration’ range, even the lowest LP34058 treatment concentration resulted in a substantial reduction in growth rate and cell yield (Table 14-4). The same was not the case with the cyanobacteria (Table 14-3), where two of the three strains tested did not show a significant impact of the LP34058 treatment. Why these two strains were not sensitive to the LP34058 compound is unknown. For CCMP2764 (Nostoc sp.) it appears that the LP34058 compound had the effect of disrupting the compounds holding filaments together, but not the cell walls themselves, thus there was an impact on growth morphology, see next subsection, but not on growth rate.
Higher LP34058 concentrations were clearly toxic resulting in significant cell loss for many non-cyanobacteria strains. It is worth noting that the majority of the treatments were found to have large negative growth rates, meaning cells were not only not growing, but their cell membrane integrity was being compromised leading to the lysis of cells and thus the dramatic loss of cells; near quantitative loss in many cases. This was very evident for A. tamarense as few live cells were observed, and counted, in the LP34058 treatments but empty thecae were very abundant confirming that there had been live cells in the culture at the beginning of the experiment. For H. akashiwo, K. brevis, and P. parvum the cells become increasingly ‘ratty’ looking, with cell membranes clearly being compromised and the substantial increase in detritus associated with the lysis of cells. Furthermore, for most non-cyanobacteria strains, the LP34058 treatments led to increased settling of cells in the test tube compared to the respective controls, and in some cases, it was very difficult to get the cells resuspended. Overall, it appears that the LP34058 compound showed the desired response—significant reduction in harmful algae growth and cell abundance.
The reduced data are shown in Table 14-3 and 14-4, and includes calculated growth rates for controls and treatments, percent inhibition calculations, and statistical analyses.
In this section, we briefly summarize strain specific growth observations.
CCMP2962—this strain became very sticky in the LP34058 treatments, perhaps due to release of polysaccharides upon the cell membrane being compromised. As a result, the culture would form very large aggregates, even if the cells were still clearly cells, making it very difficult to count cells accurately. In the higher concentrations, those aggregates became increasingly difficult to resuspend.
CCMP3413 & CCMP2764—these strains are filamentous cyanobacteria making them difficult to count even in the control (filament lengths vary a little bit), but in the LP34058 treatments, the filaments all were disrupted. So, while in the control there were 15-20 cells/filament, in the LP34058 treatments, it was pretty consistent that filaments were disrupted into pairs of cells. Thus, to assess ‘growth’ required a lot more counting of partial filaments and scaling those back to the whole filaments in the control samples. The counts are presented as filaments/mi not cells/mi.
CCMP2281—this strain is of note as the LP34058 treatments led to very rapid cell lysis at >1 mg/L active ingredient concentration.
CCMP1771—this strain settled to the bottom of the culture tube in the LP34058 treatments but not the control, an observation made in the prior analysis, although it appeared more pronounced in this experiment. Perhaps this is why it was less sensitive in this experiment than in the prior experiment, as cells looked generally healthy when resuspended.
LD50 values for growth inhibition were estimated using the AAT Bioquest online calculator2, with the results given in Table 14-5. Given that for most strains, all treatments results in significant cell loss estimated LD50's should be considered with caution. LD50s are presented as concentration of the active ingredient in the LP34058. Values for all but one strain tested at the low-test concentration range resulted in LD50 values that could be calculated. The dinoflagellate Prorocentrum lima showed substantial reduction in growth, but the resulting LD50 exceeded the test concentration range and should be considered qualitative. For the cyanobacteria, an LD50 could be calculated for Nostoc sp., but not for Aphanizomenon sp. While an LD50 could be calculated for M. aeruginosa, the value exceeded the test concentration range and thus should be considered qualitative.
For some of the strains, this experiment was a retest at a lower concentration range. For those strains, we combined data from both experiments to estimate an LD50 value. For CCMP1771 & CCMP2281, the calculated LD50s have a significant degree of uncertainty. For CCMP1771, it was less sensitive than in the first experiment, and for CCMP2281 it was more sensitive than in the first experiment. CCMP3149 & CCMP3037 showed very good agreement between the experiments.
A. tamarense (CCMP1771)
H. akashiwo (CCMP3149)
K. brevis (CCMP2281)
P. parvum (CCMP3037)
P. lima (CCMP684)
C. marina (CCMP2962)
0.6
Nostoc
sp. (CCMP3413)
7.9
Aphanizomenon
sp.
No reasonable
(CCMP2764)
LD50
139.6
Lipopeptide biosurfactant of DSMZ 34058 (hereinafter referred to as LP34058) obtained in example 3 was used to test the effects on Chilodonella uncinata reproduction and survival using in vitro bioassays.
Survival and growth of protozoa Chilodonella uncinata (C. uncinata) under assay conditions was observed for, at minimum, 24 hours prior to assay start. Peak growth time points for C. uncinata cultures were validated prior to assay start. Cultures of C. uncinata were incubated at 15° C. and split three days prior to assay start. Cultures were observed microscopically for peak growth phase confirmation immediately prior to sampling. Samples were taken from culture surface and 100 μL was pipetted in quadruplicate into six rows of a transparent 96-well microtitre plate. The number of C. uncinata was observed microscopically and recorded for each well. Individual wells had to contain, at minimum, 15 C. uncinata to be utilized for the assay and wells containing fewer C. uncinata were replaced with an additional replicate.
A stock solution of 10 mg/ml LP34058 was prepared utilizing Sonneborn's Paramecium Media as a diluent, immediately prior to assay start. Stock solution was diluted to twice the effective concentrations listed in Table 15-1. Each LP34058 dilution, and Sonneborn's Paramecium Media alone as a negative control, was pipetted in quadruplicate into a well containing a pre-recorded number of C. uncinata. Number of motile C. uncinata were observed and recorded immediately post-addition of either diluted LP34058 or Sonneborn's Paramecium Media, and at 15, 30, 45, 60, 90, and 120 minutes post-addition. A final observation was performed at 24 hours.
The microtitre plate was incubated at 20° C. between observations. Each count was independently verified between two operators and had to be within ±5 count of one another to be accepted. Only motile C. uncinata were counted.
Concentrations of LP34058 50 μg/ml (20 μg AC/ml), 125 μg/ml (50 μg AC/ml), and 250 μg/ml (100 μg AC/ml) were immediately lethal to C. uncinata and these groups were reduced to zero survivors, which was maintained until assay termination. Exposure to 25 μg/ml (10 μg AC/ml) LP34058 was 79% lethal immediately, and 100% lethal by 15 minutes. Exposure to 12.5 μg/ml (5 μg AC/ml) was 30% lethal immediately, 76% lethal by 15 minutes, and 93% lethal by 30 minutes, after which the remaining survivors ceased to decline and remained stable until 24 hours. Negative control (no LP34058) C. uncinata population increased over the duration of the assay (Average Δcount 68) (Table 15-2).
Survival of C. uncinata was not affected by addition of diluent or by the conditions of the assay, as represented by the increase of the negative control population over 24 hours. Though 7% of C. uncinata exposed to 12.5 μg/ml (5 μg AC/ml) survived for the duration of the assay, efficacy was observed through the 93% decline in viability and the failure of remaining C. uncinata to replicate over a 24-hour period. Therefore, of the tested conditions, the minimum lethal dose of LP34058 against C. uncinata is 25 μg/ml (10 μg AC/ml) and the minimum effective dose is 12.5 μg/ml (5 μg AC/ml).
aToo many to accurately count.
This experiment demonstrated that LP34058, at concentrations between 12.5 (5 μg AC/mi) and 250 μg/ml (100 g AC/mi), was highly efficacious against C. uncinate.
Lipopeptide biosurfactant of DSMZ 34058 (hereinafter referred to as LP34058) obtained in example 3 was tested for biocidal effect on two sporozoans Eimeria and Cryptosporidium species.
The negative control group did not contain LP34058 and enabled observation of the chosen organisms without the test item being present. Two reference items were used which are known to kill the intended species at the manufacturers recommended dosage.
Eimeria
Cryptosporidium parvum
The Eimeria culture was isolated from partridge intestines to provide a source of fresh unsporulated oocysts prior to the start of the in vitro phase of the study.
Cryptosporidium parvum
Live, viable C. parvum oocysts were purchased from Waterborne Inc, USA and shipped prior to the start of the in vitro study. The strain was a calf passaged strain.
Distilled water was used in all solutions, unless otherwise stated all solutions was stored at 4° C.
Cryptosporidium parvum
The oocysts for each organism were exposed to the 5 treatment options, negative control, positive control, LP34058 (1) (17 μg/mL (5 μg AC/mL)), LP34058 (2) (67 μg/mL (20 μg AC/mL)) and LP34058 (3) (333 μg/mL (100 μg AC/mL)). Three replicates of each were conducted with a 24-hour contact time. Test assays were compared to the positive and negative controls.
Counts were recorded as the percentage of sporulated vs unsporulated in the first 100 oocysts. Sporulated were counted as containing 4 sporozoites, any abnormalities in appearance noted. These counts inform the claim for biocidal effects of LP34058.
Cryptosporidium parvum
Counts were recorded as the percentage of visible free sporozoites vs oocysts in the first 100 oocysts, (4 free sporozoites equal to one oocyst). Any abnormalities were recorded. These counts inform the claim for biocidal effects of LP34058.
The number of intact/viable oocysts/cysts were counted, and the percentage estimated by counting a total of 100 oocysts/cysts. In addition to these observations any abnormalities in terms of size, appearance and damage were observed and recorded where necessary. The oocysts/cysts were slightly flattened under the pressure of a cover slip. Results were obtained via light and/or phase contrast microscopy.
Data was presented as the mean±S.E.M. A t-test or one-way ANOVA followed by Tukey's test to detect significance among treatment groups. P<0.05 considered to be statistically significant. Results
The results showed that the mean number of oocysts sporulated after 120 hours incubation at 28° C. in the negative control samples was 29.3%. No oocysts were observed to have sporulated in the positive control samples. The mean percentage of sporulated oocysts in all the LP34058 dilutions were not significantly different to the number in the negative control.
Eimeria oocysts counted, also see FIG. 18.
Cryptosporidium parvum
The number of cysts that successfully excysted in the negative control was 87.9%, the results below show that the positive control, and all 3 dilutions of LP34058 inhibited excystation. In the positive control a mean of 31.7% of cysts excysted, and in the 67 μg/ml LP34058 dilution a mean of only 9.8% of cysts excysted.
Cryptosporidium parvum oocysts, also see FIG. 19.
The results showed that LP34058 had no significant biocidal effects on Eimeria oocysts. The results for the 3 dilutions were similar (plateaued) indicating that increasing the concentration of the LP34058 would not give a different result.
All 3 dilutions of LP34058 inhibited excystation of the Cryptosporidium parvum oocysts. The 67 μg/ml LP34058 dilution showed the greatest inhibition of excystation, 9.8% of oocysts excysted compared to 87.9% in the negative control.
Lipopeptide biosurfactant of DSMZ 34058 (hereinafter referred to as LP34058) obtained in example 3 was tested for effectiveness in inhibiting the growth of some plant pathogenic fungi and plant and animal pathogenic oomycetes. A simple plate assay was performed, whereby LP34058 was added in liquid culture medium resulting in different concentrations of 0, 17, 33, 67, 167, 333 and 665 μg/ml medium. The growth of the fungal or oomycete culture were assessed over time. The following oomycetes/fungi cultures were used in these growth assays (Table 17-1).
Aphanomyces astaci
Aphanomyces sp.
Saprolegnia parasitica
Pythium catenulatum
Pythium dissotocum
Phytophthora palmivora
Phytophthora ramorum
Phytophthora cryptogea
Fusarium oxysporum sp. gladioli
Fusarium oxysporum f.sp. lycopersici
Verticillium dahliae
Same medium (24 g/L Potato Dextrose medium (PD) in small Petri dishes (5.5 cm diameter)) was used for all strains to be able to compare results between different strains.
LP34058 stock solution was prepared freshly for each test by dissolving LP34058 in water with addition of 0.2M NaOH (sodium hydroxide). The LP34058 solution was added to PD to provide the final concentrations of 0, 17, 33, 67, 167, 333, and 665 μg/ml.
The isolates were grown on PD-agar plates at 24° C. This provided sufficient material for the growth and LP34058 inhibition experiments.
An agar plug (1 cm diameter cut with a cork borer) of freshly grown mycelia on PDA plates was transferred to 5 ml liquid PD (containing various amounts of LP34058) in 5.5 cm Petri dishes. The plates were incubated at 12° C. for 2 weeks.
Images of the cultures were taken on day 4, 7, 9 and 11 after transfer to the liquid medium with and without LP34058.
The plant pathogenic oomycetes Phytophthora ramorum (
The isolates showing some reduction in growth, especially at higher concentrations of LP34058, were the animal pathogenic oomycetes including Aphanomyces astaci (
In addition, the plant pathogenic Pythium species showed a very clear reduction in growth especially with high concentrations of LP34058. Pythium catenulatum (
The inhibition experiments demonstrated that LP34058, at high concentrations, has a modest to good effect against the growth of the animal pathogenic oomycetes Aphanomyces astaci and Saprolegnia parasitica, as well as an unknown Aphanomyces species isolated from an aquarium fish shop. The plant pathogenic fungi and Phytophthora species seemed relatively unaffected by LP34058, whereas the tested plant pathogenic Pythium species (i.e. P. dissotocum and P. catenulatum) were considerably inhibited at high concentrations of LP34058. P. dissotocum was the most sensitive isolate, where inhibition of growth was seen at concentrations of 167 ag/ml and higher.
In this study, a lipopeptide biosurfactant of DSMZ 34058 (hereinafter referred to as LP34058) obtained in example 3 was used to test the effects on survival of two strains of Tetrahymena (T. thermophila and T. pyriformis) using in vitro bioassays. The T. thermophila B2086 II (Resource Identification Citation: TSC_SD00709) and T. pyriformis GL-C(Resource Identification Citation: TSC_SD00707) strain used in the bioassays was purchased from the Tetrahymena Stock Center at Cornell University.
A single parent culture of each strain was grown to mid/late exponential phase in a 10 mL Modified Neff media (Cassidy-Hanley 2012) with penicillin and streptomycin (250 ag/ml) in a T25 cell flask incubated at 30° C. From the surface of the parent culture, 1 ml was collected and diluted to an appropriate cell density. After the dilution, 5 μL was pipetted to each well of a 96-well microtiter plate and the number of Tetrahymena cells for each well was determined just prior to the test using a microscope. Only individual wells containing 5-15 cells was utilized for the assay.
A stock solution of 10 mg/ml LP34058 was prepared utilizing Modified Neff media as a diluent, immediately prior to assay start. Stock solution was diluted to twice the effective concentrations listed in Table 18-1. For each LP34058 treatment and Modified Neff media alone (i.e., negative control), 5 μL was pipetted into each of four wells containing a pre-recorded number of T. thermophila. Cell survival was assessed and recorded by direct counts of cell abundance under microscope at 0, 15, 30, 45 and 60 min after addition of LP34058 solution or at 0 and 60 min after addition of Modified Neff media in the negative control treatment. T. thermophila survival was determined by assessing mobility and ciliary movement, with non-motile and lysed cells considered as dead. The 96-well microtiter plate was incubated at room temperature between observations.
Tetrahymena thermophila
LP34058 concentrations of >31.5 mg active compound/L immediately killed all T. thermophila and no surviving cells were observed after 0 min. Cells in these high concentrations were almost all lysed with no intact T. thermophila visible after 60 min (see
The LD50 value for mortality after 60 min was estimated to be 22.3 mg active compound/L using the AAT Bioquest online calculator.
Tetrahymena pyriformis
T. pyriformis exposed to >25.2 mg active compound/L was all lysed and killed after 15 min with cells exposed to 18.9 mg active compound/L instantly exhibiting high mortality, see
The LD50 value for mortality after 60 min was estimated to be 15.3 mg active compound/L using the AAT Bioquest online calculator.
LP34058 was instantly lethal to both Tetrahymena strain at concentrations above 31.5 mg active compound/L with no or very low survival observed at 25.2 mg active compound/L after 60 min. The T. pyriformis strain was found to be less tolerant to LP34058 than T. thermophila as 18.9 mg active compound/L resulted in almost 100% mortality in the former but showed no killing effect on the latter. This was also apparent in the lower LD50 estimated for T. pyriformis (15.3 mg active compound/L) compared to T. thermophila (22.3 mg active compound/L).
Lipopeptide biosurfactant of DSMZ 34058 (hereinafter referred to as LP34058) obtained in example 3 was tested for biocidal effect on Giardia lamblia cysts as a disinfectant.
The negative control group did not contain LP34058 and enabled observation of the chosen organism without the test item or any parasiticide being present. A reference item was used which is known to kill the intended pathogen at the manufacturers recommended dosage.
Giardia lamblia overview
Live, viable G. lamblia cysts were purchased from Waterborne Inc, USA and shipped prior to the start of the in vitro study.
Giardia lamblia cysts were exposed to the 5 treatment options, negative control, positive control, LP34058 (1) (17 μg/mL (5 μg AC/mL)), LP34058 (2) (67 μg/mL (20 μg AC/mL)) and LP34058 (3) (333 μg/mL (100 μg AC/mL)). Three replicates of each were conducted with a 24-hour contact time. Test assays were compared to the positive and negative controls.
Counts were recorded as the number of cysts present in 5 μL of sample. No excystation of the cysts was observed, therefore the percentage of excyzoites vs encysted cysts in the first 100 cysts could not be recorded, however lysing of cysts was observed. These counts inform the claim for biocidal effects of LP34058.
Fresh parasites were sourced, and the assay repeated, counts were recorded as the number of cysts present in 20 μL of sample (a larger aliquot of sample was examined than in the previous testing to better assess the number of cysts remaining at the end of the assay).
Distilled water was used in all solutions, unless otherwise stated all solutions was stored at 4° C. Bovine serum was heat-inactivated by placing at 56° C. for 30 minutes. Peptone digest, yeast extract, and bovine serum were lot tested.
Alternatively, Sodium Bicarbonate can be substituted for 0.3 g of 3% KH2PO4 and 0.65 g K2HPO4·3H20. As media components for Giardia media solution 1 solubilise and combine, media should turn from opaque into a clear solution.
This media must be made fresh and used immediately on day of preparation.
The number of intact/viable cysts were counted, and the percentage estimated in a count of 100 cysts. In addition to these observations any abnormalities in terms of size, appearance and damage were observed and recorded where necessary. It should be noted that the cysts were slightly flattened because of being under the pressure of a cover slip and not because of treatment. Results were obtained via light and/or phase contrast microscopy.
Data was presented as the mean±S.E.M. A t-test or one-way ANOVA followed by Tukey's test to detect significance among treatment groups. P<0.05 is considered to be statistically significant.
Excystation of the G. lamblia cysts did not occur in any of the samples. Results shown in table 2 indicate that the number of cysts present in a 5 μL aliquot were significantly decreased in the positive control compared to the negative control. There was a positive dose-response lysis with increasing concentration of LP34058, though none of the dilutions appeared as effective as the positive control.
The mean number of G. lamblia cysts counted in 5 μl of sample is shown in
Repeat Testing of G. lamblia
The number of G. lamblia cysts in the positive control were significantly reduced by 99.7% in the 20 μL of sample examined, compared to the negative control. The number of cysts lysed in the 333 μg/mL LP34058 solution was decreased compared to the negative control (although not significantly). Results for all dilutions of LP34058 were significantly different to the positive control. No excystment occurred in any to the test samples.
The mean number of G. lamblia cysts counted in 20 μL of sample is shown in
The G. lamblia assay showed a positive lysis dose-response with increasing concentration of LP34058, though none of the dilutions appeared as effective as the positive control. No excystment occurred in the negative control samples.
The repeat G. lamblia assay results were consistent with the first assay, no excystment occurred in the negative control, but lysis was seen in both positive control and LP34058 assays.
This pilot study suggests that LP34058 could have a role to play in control of environmental or water contamination with G. lamblia.
Lipopeptide biosurfactant of DSMZ 34058 (hereinafter referred to as LP34058) obtained in example 3 was tested for effectiveness against the enveloped virus causing Viral hemorrhagic septicemia (VHS).
Different concentrations of LP34058 were incubated in a serial dilution of VHS virus. A stock solution of the LP34058 was prepared by dissolving 1.9 mg of LP34058 in 600 μl RNase/DNase free water plus 18.75 μl of 0.2M NaOH followed by extensive vortexing and centrifuge (14000 rpm) for 5 min. To prepare different testing concentrations of LP34058 (320 μg/ml, 160 μg/ml, 80 μg/ml, 40 μg/ml, 20 μg/ml, and 10 μg/ml), 168.4 μl of stock solution was diluted in 1 ml transport media (320 μg/ml) and other concentrations were made by adding 250 μl of the previous concentration to 500 μl transport media to reach to the lowest concentration. Earlier studies on the virus titration showed that the available viral stock loose strength against the host cell on 10−6 dilution, therefore, viral dilutions until 10−5 were selected. Serial dilutions of the virus (10−1,10−2,10−3,10−4,10−5) were prepared in a 96 well plate and incubated with LP34058 at 15° C. for 3 h. To investigate the potential effect of NaOH on the virus, the virus was incubated with NaOH at the same plate for the same period. After 3 h, the mixture of virus and LP34058 was inoculated to two 96 wells plates coated with EPC (epithelioma papulosum cyprini) cells for 24 h. Inoculated plates were placed at 15° C. for 72 h.
Plates were assessed for both CPE (cytopathic effect) and GFP (green fluorescent protein) for each well, it means any damage in cells compared to control cells and also the presence of virus characterized by GFP was evaluated. For LP34058 concentrations between 80 and 10 μg/ml, clear CPE and GFP was observed meaning that LP34058 could not deactivate the virus at this range of concentrations. For concentrations 320 and 160 μg/ml (in particular 160 μg/ml) GFP was only detected in a few wells, and CPE was positive but weak. This might indicate a toxic effect of high concentrations of LP34058 on VHS virus. Damage on cells with LP34058 only (320 and 160 μg/ml) and NaOH only was observed in negative controls.
High concentrations of LP34058 (320 and 160 μg/ml) might damage or deactivate the VHS virus. However, this effect might be due to the toxic effects of LP34058 on cells.
Lipopeptide biosurfactant of DSMZ 34058 (hereinafter referred to as LP34058) obtained in example 3 was tested for effectiveness against Ichthyobodo (Costia) infecting Asian sea bass (Lates calcarifer).
Twenty Asian sea bass were treated (in duplicate, 10 fish per tank) via immersion administration with a dose of LP34058 (20 mg/L), for 4 hours in tanks with 30 L of fresh water. Fish were held at 24±1° C. and the average weight at the time of treatment was approximately 1 g. To standardize the scoring for the analysis 5 fish were euthanized before the treatment and the Ichthyobodo infestation was evaluated following the instructions in table 21-1.
The results during standardization showed that 5 out of 5 fish presented severe infection. After confirmation of the presence of the parasite, the bay preparation for treatment was started. 10 morbid fish were placed in the control bucket and 10 morbid fish were placed in the treatment bucket.
The immersion treatment lasted for 4 hours; the treatment tanks were supported with oxygen stones and monitored for any kind of clinical signs.
After 4 hours fish were euthanized, and gill health conditions was assessed by wet mount analysis. The analyst received samples not knowing which bags (5 fish/bag) contained fish from control group or the treatment group (blind analysis). The infection level in each fish was quantified according to table 21-1 and presented in Table 21-2.
In the control group, 3 out of 10 fish presented Ichthyobodo infection (30% prevalence). The p value (t-test) was 0.093918. In the group exposed to LP34058 no Ichthyobodo was found. The control group had an average score of 0.7 vs 0 for the treated group.
Besides the absence of Ichthyobodo in the treated group, some changes in the tissue were observed, cells appeared swollen, however this test is not sensitive enough to evaluate tissue changes.
The control group showed 50% of parasite prevalence with different severities, while the group treated with LP34058 did not show parasites after treatment.
Wet mount gives an idea of the presence of the parasites, however the changes observed in tissue cannot be evaluated only with wet mounts. Histopathology is recommended to prove the safety of the product regarding gill health.
Even though the fish reacted well to the treatment (no mortalities), It is recommended first to evaluate the impact of the treatment in gill tissue through histopathology in fish without any pathology.
Lipopeptide biosurfactant of DSMZ 34058 (hereinafter referred to as LP34058) obtained in example 3 was tested for effectiveness to control saprolegniosis by assessing survival of Atlantic salmon (Salmo solar L.) eggs challenged with Saprolegnia diclina.
LP34058 was used at four different concentrations to prepare immersion baths using freshwater. 1300 developing eyed Atlantic salmon eggs were used for the study to test the efficacy of LP34058 administration against saprolegniosis.
The study utilized 13 treatment groups each having 100 eyed eggs divided into four replications housed in four wells of 6-well plates. Each 6-well plate was submerged in a plastic container containing 2 L freshwater; treatment group details in Table 23-1. Eggs were challenged with S. diclina by introducing a pre-colonized trojan egg (
Ca. 50 eyed Atlantic salmon eggs were heat killed at 60° C. for 1 minute in a small beaker with 50 ml freshwater followed by cooling down at 4° C. for 60 mins. Then, the eggs were transferred to S. diclina grown petri dishes using a pair of forceps and incubated at 15° C. for three days to ensure colonization (
Ca. 1,300 Atlantic salmon eyed eggs were treated with 1665 ppm 37% formalin for 15 minutes before distributing into 6-well plates. Only healthy-looking live eggs were recruited in the study. Well plates were carefully submerged into the experimental containers having 2 L freshwater according to Table 1. The containers were stored in an incubator with aeration where temperature was maintained at 4±2° C. throughout the study period.
According to Table 23-2, 85.25 and 682.03 mg of LP34058 were weighed and mixed in chilled distilled water to prepare stock solutions for 1 hr and continuous immersion baths. To ensure LP34058 is fully dissolved, stock solutions were freshly prepared and stored at 4-6° C. for at least 1 hr prior to immersion baths preparation for the target dose (Table 23-2). Both stock solution and immersion baths were freshly prepared on 1st, 5th and 9th day post challenge during the study. In addition, formalin bath was prepared by mixing 416.25 μL 37% formaldehyde in 250 mL freshwater.
Any dead eggs resulting after disinfection, handling and treatment distribution were replaced with healthy eggs the following day post egg distribution. Trojan eggs from S. diclina petri dishes (
Nine days post challenge 100% eggs in treatments H, I, J, and K were dead. Thus, the continuous immersion treatments including controls e.g., L, and M, were terminated by carefully counting number of apparently live eggs, hatched eggs, dead eggs without entanglement, dead eggs entangled to trojan, uninfected eggs and total number of eggs entangled to each trojan. One hour immersion treatments were terminated in a similar way ten days post challenge.
Parametric data yielded from the study were analyzed by one-way ANOVA followed by Tukey's multiple comparisons test where significant differences were observed. For each analysis, normality of residuals was tested to ensure model fitness.
The study compared the efficacy and toxicity of four concentrations of LP34058 immersion treatments on Atlantic salmon eggs challenged with S. diclina. When LP34058 was administered utilizing one hour baths every calendar day for 10 days, a significant reduction in entanglement was observed in A (21.3 mg/L), C (85.3 mg/L), and D (170.5 mg/L) compared to the Sap-control (Trt A, P=0.002; Trt C, P=0.001; Trt D, P<0.0001) while formalin treated eggs did not suffer any sign of saprolegniosis (
Continuous immersion treatments were proven to be highly toxic on eggs resulting in 100% mortality of eggs in all LP34058 treatments. Saprolegnia growth was not affected in these continuous immersion treatments despite the toxic effect on eggs. Mycelial entanglements were evident which spread from the trojans into surrounding dead eggs (
The study demonstrated encouraging results of LP34058 in providing protection against saprolegniosis when administered in 1 hr immersion method. Although the highest dose in 1 hr immersion method was proven to be toxic on eggs, the lowest dose i.e., Trt A (21.3 mg/L) demonstrated promising efficacy in reducing mycelial entanglement and higher survival in eggs compared to the Saprolegnia control treatment. Eggs in Trt B (42.6 mg/L) suffered highest entanglement than the rest of the LP34058 doses which may have resulted due to instability of the active ingredient in this treatment. However, similar survival rates observed among the LP34058 treatments A-C demonstrate the safety of the product on eggs at these lower doses. Despite the protection, all LP34058 treatments suffered some mortalities and henceforth, mycelia grew from trojan to colonize the dead eggs, especially in the continuous immersion treatments. With access to abundant nutrient from the dead eggs, mycelia could have actively deterred any negative effect enabling them to grow and colonize the eggs in presence of LP34058 (Liu et al. 2015). Furthermore, eggs often respond to acute toxicity by hatching early to escape the condition which was evident in the highest dose in 1 hr exposure and continuous treatments further demonstrating the toxic effect of LP34058 at the given doses and exposure method. Nonetheless, it remains unclear whether the mode of action of LP34058 is to actively prevent Saprolegnia from growing, or passively to provide a layer of protection to the eggs since a dose-depended protection was not observed during the study. Therefore, investigations on further lower doses of LP34058 in in vivo and in vitro studies are deemed necessary before the product may be suitable for use in commercial hatchery condition in the treatment of saprolegniosis.
LP34058 when administered in continuous exposure method was found to be highly toxic on the eggs which might be related to the negative effect of the active ingredient in the product. In addition, continuous exposure treatments also yielded higher incident of hatching and larval mortality which could be the outcome of LP34058 toxicity. However, the mechanism of toxicity remains to be fully understood. Large amounts of lipid molecules present in Atlantic salmon eggs allow for essential membrane fluidity and ion exchange during development which might have been affected by the active ingredient in continuous exposure method resulting in the toxicity (Cornet et al. 2021). LP34058 may have contributed to degradation of the chorion layer exposing the premature larvae to mortality.
The study demonstrated that 1 hr daily exposure of eggs to the lowest LP34058 dose (21.3 mg/L) was more efficacious than the higher doses. However, formalin treatment showed the best results in terms of survival and entanglement. Atlantic salmon eggs were highly susceptible to the toxic effect of LP34058 when administered by continuous immersion method which caused 100% mortality. Despite the toxicity, Saprolegnia mycelia actively colonized dead eggs in these treatments. Therefore, the doses of LP34058 on or below 21.3 mg/L yet high enough to control the Saprolegnia mycelia, in, preferably 1 hr, immersion bath holds the most promising potential against saprolegniosis.
To demonstrate the distinctiveness of two different ciliates, the characteristics of Ich and Tetrahymena were compared:
Tetrahymena thermophila cells were generally more tolerant to copper sulphate,
T. thermophila (=1.38 mM), Ich theronts (=0.17 mM) and tomonts (0.34 mM)
T. thermophila
T. thermophila
T. thermophila
| Number | Date | Country | Kind |
|---|---|---|---|
| 21202024.2 | Oct 2021 | EP | regional |
| 22172778.7 | May 2022 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2022/078154 | 10/10/2022 | WO |
