FIELD OF THE INVENTION

The present invention relates to the treatment of parasitic infections causing scuticociliatosis in marine organisms.

BACKGROUND OF THE INVENTION

Diseases in fish are an economic challenge in fish production industry targeting fish for consumption as well as ornamental fish. One economically important disease is scuticociliatosis in cultured turbot, caused by the histiophagous scuticociliate Philasterides dicentrarchi (Ciliophora, Scuticociliatia) which is a serious threat to the aquaculture industry, causing high mortalities and substantial economic losses.

P. dicentrarchi may enter fish farms continuously through the feeder channels and they increase in number in fish tanks as the temperature and organic matter levels rise, conditions that increase the risk of fish suffering from scuticociliatosis.

P. dicentrarchi was found to induce severe systemic infections in marine organisms susceptible to scuticociliatosis, in particular in fish like turbot, sea bass, olive flounder, fine flounder, sharks, sea dragons and sea horses because of its ability to penetrate and spread throughout fish organs. The endoparasitic location of this parasite makes treatment of scuticociliatosis difficult. At present, vaccination may be the most effective method of preventing disease outbreaks. However, experiments with different serotypes of this scuticociliate did not reveal any cross protection. Until a universal vaccine is obtained, other methods must be developed to reduce the fish mortality associated with scuticociliatosis.

Many compounds have been reported to be toxic to P. dicentrarchi or to Miamiensis avidus, a scuticociliate parasite with many morphological similarities to the former. A large group of these compounds have also been found to show antiprotozoal activity. However, many of the compounds evaluated are toxic to aquatic life and should not be used to reduce the number of ciliates in water or on the surface of infected fish. They also cannot be used to treat infected fish. In this respect, compounds with antiprotozoal activity but that also do not harm aquatic organisms are required.

Lama R et al., "Turbot (Scophthalmus maximus) Nk-lysin induces protection against the pathogenic parasite Philasterides dicentrarchi via membrane disruption", Fish & Shellfish Immunology, 2018, describes that Turbot Nk-lysin has the ability to inhibit proliferation of the Philasterides dicentrarchi parasite.

Park Seong Bin et al., "Combination treatment against scuticociliatosis by reducing the inhibitor effect of mucus in olive flounder, Paralichthys olivaceus", Fish & Shellfish Immunology, 2014, describes a combination treatment, using benzalkonium chloride and bronopol to overcome a protective effect of mucus to scuticociliates.

W02019101739A1 describes the use of lipopeptide biosurfactants for use in the treatment of white spot disease in fresh water and marine fish.

Al-Jubury A et al., "Impact of Pseudomonas H6 surfactant on all external life cycle stages of the fish parasitic ciliate Ichthyophthirius multifiliis", Journal of Fish Diseases, 2018, describes that P. fluorescens H6 lipopeptide biosurfactant has a strong in vitro antiparasitic effect on the fish pathogenic ciliate Ichthyophtirius multifilis.

Jensen Hannah Malene et al., "Gilt amoebae from freshwater rainbow trout (Oncorhynchus mykissy. In vitro evaluation of antiparasitic compounds against Vannella sp. "Journal of Fish Diseases, 2020, describes an in vitro determination of antiparasitic effects (dose-response studies) on a monoculture of Vannella sp. using various compounds, including sodium chloride, hydrogen peroxide, peracetic acid, formalin, aqueous garlic and oregano extracts and a Pseudomonas H6 surfactant.

Yiying Liu et al., "Diversity of Aquatic Pseudomonas Species and Their Activity against the Fish Pathogenic Oomycete Saprolegnia", PLOS ONE, 2015 describes that among Pseudomonas species isolated from salmon eggs, significantly more biosurfactant producers were retrieved from healthy salmon eggs than from Saprolegnia-infected eggs, and that Pseudomonas isolate H6 significantly reduced salmon egg mortality caused by S. diclina.

PARAMA et al., "Scuticociliate cysteine proteinases modulate turbot leucocyte functions", Fish & Shellfish Immunology, 2007, describes an investigation of the effects exerted by cysteine proteinases isolated from the histiophagous ciliate Philasterides dicentrarchi on the phagocytic functions of turbot pronephric leucocytes.

SUMMARY OF THE INVENTION

According to the present invention it was surprisingly found that lipopeptide surfactants can be used in the treatment of scuticociliatosis in marine organisms susceptible to scuticociliatosis. Marine organisms known to be susceptible to scuticociliatosis include teleosts, seahorses, sharks and certain crustaceans.

Accordingly, the present invention relates to the use of a lipopeptide surfactants in the treatment of scuticociliatosis in marine organisms susceptible to scuticociliatosis. The present invention relates in particular to the use of bacterial lipopeptide surfactants in the treatment of scuticociliatosis.

Furthermore, such treatment is considered safe as marine organisms susceptible to scuticociliatosis seem to suffer no adverse immediate or late signs following several hours of incubation in the effective concentrations of lipopeptide biosurfactant.

Accordingly a lipopeptide biosurfactant may find application as a scuticociliatosis controlling agent in aquacultured fish such as in aquacultured turbot, sea bass and flounder as well as in ornamental fish.

To this end, the marine organisms susceptible to scuticociliatosis may be treated with the (isolated) lipopeptide biosurfactant, or with compositions or formulations containing the lipopeptide biosurfactant in order to prevent scuticociliatosis or to treat marine organisms inflicted with scuticociliatosis. Alternatively, the marine organisms susceptible to scuticociliatosis may be treated with a bacterial isolate wherein the bacteria are capable to produce the lipopeptide biosurfactant in order to prevent scuticociliatosis or to treat marine organisms inflicted with scuticociliatosis.

DETAILED EMBODIMENTS OF THE INVENTION

The present invention relates to a lipopeptide biosurfactant for use in the treatment of scuticociliatosis in a marine organism, especially in a marine organism susceptible to scuticociliatosis and in particular in fish and more in particular in turbot. Accordingly, in a particular embodiment, the present invention relates to a lipopeptide biosurfactant for use in the treatment of scuticociliatosis, such as caused by Philasterides dicentrarchi infection.

In a further embodiment, the present invention relates to a lipopeptide biosurfactant for use in the prevention of scuticociliatosis in marine organisms, such as turbot, sea bass and flounder, and such as scuticociliatosis as caused by Philasterides dicentrarchi.

In further embodiments, the present invention relates to a use of the lipopeptide biosurfactant, or of a composition comprising the lipopeptide biosurfactant, for the treatment (or “prevention”) of scuticociliatosis in a marine organism, especially in a marine organism susceptible to scuticociliatosis and in particular in fish and more in particular in turbot.

In embodiments, the marine organism may be a marine organism susceptible to scuticociliatosis. Further, in embodiments, the marine organism may comprise a marine animal. In further embodiments, the marine organism may comprise a marine fish, especially a flatfish, or especially a marine fish selected from the group comprising a turbot, a sea bass, an olive flounder, a fine flounder, a shark, a sea dragon and a sea horse, such as a farmed fish selected from the group comprising the turbot, the sea bass, the olive flounder and the fine flounder, or such as an aquarium fish selected from the group comprising the shark, the sea dragon, and the sea horse. In further embodiments, the marine organism may comprise a crustacean. The term “marine organism” may herein also refer to a plurality of (different) marine organisms, especially to a plurality of marine organisms of the same species.

As referred herein the expression “lipopeptide biosurfactant” relates to a molecule consisting of a lipid connected to a peptide, generally a cyclic peptide, with surfactant properties (i.e. lowering surface tension of fluids). Lipopeptide biosurfactants can be produced by bacteria. Generally, the biosynthetic pathway encoding the lipopeptide surfactant within a given bacterial strain leads to a single main lipopeptide surfactant and minor amounts of structurally related derivatives of the main lipopeptide surfactant.

Known bacterial lipopeptide biosurfactants are for example 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.

Suitable massetolide lipopeptide surfactants for use according to the present invention may be lipopeptides belonging to the viscosin group of lipopeptides, including viscosin, massetolide A, massetolide B, massetolide C, massetolide D, massetolide E, massetolide F, massetolide G and massetolide H.

Treatment of scuticociliatosis according to the present invention in particular comprises also the prevention of scuticociliatosis in marine organisms susceptible to scuticociliatosis.

A further viscosin-like lipopeptide biosurfactant obtainable from the bacterium Pseudomonas sp. strain H6 was recently reported to kill zoospores of the oomycete fish pathogen Saprolegnia diclina (de Bruijn et al. (2007); Liu et al. (2015)) and thus might be useful to control Saprolegnia infections.

Surprisingly, it was discovered that this vicosin-like lipopeptide biosurfactant of Pseudomonas sp. strain H6 can also suitably be used for the treatment of scuticociliatosis in marine organisms susceptible to scuticociliatosis such as infections caused by Philasterides dicentrarchi .

In a particular embodiment, the present invention relates to a bacterial lipopeptide biosurfactant obtainable from the Pseudomonas sp. strain H6 or a derivative thereof for use in the treatment of Philasterides dicentrarchi infection in a marine organism susceptible to scuticociliatosis.

In a further embodiment, the present invention relates to a composition for use in the treatment of scuticociliatosis in a marine organism, wherein the composition comprises a lipopeptide biosurfactant.

In a further embodiment, the present invention relates to a composition for use in the treatment of Philasterides dicentrarchi infection in a marine organism susceptible to scuticociliatosis, wherein the composition comprises a lipopeptide biosurfactant.

In a further embodiment, the present invention relates to a composition comprising a bacterial lipopeptide biosurfactant obtainable from the Pseudomonas sp. strain H6 for use in the treatment of scuticociliatosis in the marine organism. In a further embodiment, the present invention relates to a composition for use in the treatment of Philasterides dicentrarchi infection in marine organisms susceptible to scuticociliatosis, wherein the composition comprises a bacterial lipopeptide biosurfactant obtainable from Pseudomonas sp. strain H6 or a derivative thereof.

A composition suitable according to the present invention may predominantly comprise the lipopeptide biosurfactant or lipopeptide biosurfactants, for example the one or more lipopeptide biosurfactants together with one or more carriers, or may for example comprise a slow-release form (i.e. granules) which sheds the lipopeptide biosurfactant(s) over a prolonged period.

In a particular embodiment, the composition suitable according to the present invention may be prepared from a freeze-dried solution of the lipopeptide biosurfactant (or lipopeptide biosurfactants) by dissolving the freeze-dried solution in water, such as sterile distilled water.

Alternatively, the lipopeptide biosurfactant may be administered to the marine organism susceptible to scuticociliatosis as a bacterial culture, which is capable to produce the lipopeptide biosurfactant in aquaculture.

Accordingly, in a further embodiment, the invention relates to a bacterial isolate wherein the bacteria are capable to produce the lipopeptide surfactant for use in the treatment scuticociliatosis in the marine organism.

In a further embodiment, the invention relates to a bacterial isolate wherein the bacteria are capable to produce lipopeptide surfactant for use in the treatment of Philasterides dicentrarchi infection in the marine organism susceptible to scuticociliatosis and particular in fish, such as turbot, sea bass and flounder.

In a further embodiment, the invention relates to a bacterial isolate of the Pseudomonas sp. strain H6 for use in the treatment of scuticociliatosis in the marine organism and particular in fish, such as turbot, sea bass and flounder.

In a further embodiment, the invention relates to a bacterial isolate wherein the bacteria are capable to produce a massetolide and/or derivatives for use in the treatment of scuticociliatosis in the marine organism and particular in fish, such as turbot, sea bass and flounder. In a further embodiment, the invention relates to a bacterial isolate wherein the bacteria are capable to produce a putisolvin and/or derivatives for use in the treatment of scuticociliatosis in the marine organism and particular in fish, such as turbot, sea bass and flounder.

In a further embodiment, the invention relates to a bacterial isolate of the Pseudomonas sp. strain H6 or a derivative thereof for use in the treatment of Philasterides dicentrarchi infection in the marine organism susceptible to scuticociliatosis.

A sample of the Pseudomonas sp. strain H6 has been deposited on November 1, 2017 under the Regulations of the Budapest Treaty in the CBS collection of the Westerdijk Fungal Biodiversity Institute with deposit number CBS 143505. The sample has been deposited with proposed taxonomic designation “Pseudomonas fluorescens H6”.

The isolation and characterization of the lipopeptide biosurfactant of Pseudomonas sp. strain H6 has been described by Liu et al. (2015). This viscosin-like lipopeptide biosurfactant was found to be clearly distinguished from the well-known lipopeptide biosurfactants of related strains such as the massetolide lipopeptide which can be obtained from Pseudomonas fluorescens SS101, the viscosin lipopeptide which can be obtained from Pseudomonas fluorescens SBW25 and the putisolvin lipopeptide which can be obtained from Pseudomonas putida 267.

It was shown that this lipopeptide biosurfactant has a strong in vitro antiparasitic effect on the fish pathogenic ciliate Philasterides dicentrarchi.

Accordingly, this lipopeptide biosurfactant of Pseudomonas sp. strain H6 may find further application as an antiparasitic control agent in aquacultured fish, such as in aquacultured turbot, sea bass and flounder.

In a particular embodiment, the present invention relates to the use of a lipopeptide biosurfactant selected from (a) a viscosin-like lipopeptide biosurfactant (such as lipopeptide biosurfactant obtainable from the Pseudomonas sp. strain H6) or a derivative thereof, (b) a massetolide (such as a massetolide surfactant obtainable from Pseudomonas fluorescens strain SSI 01) or a derivative thereof, and (c) a putisolvin (such as the putisolvin biosurfactant obtainable from Pseudomonas putida 267) or a derivative thereof in the treatment of scuticociliatosis in the marine organism, like in the marine fish, such as turbot, sea bass and flounder, and more particular in the treatment of scuticociliatosis caused by the pathogenic ciliate Philasterides dicentrarchi .

In embodiments, the composition may comprise the lipopeptide biosurfactant at a concentration selected from the range of 10-100 pg/ml, such as a concentration selected from the range of 30 - 70 pg/ml, especially about 50 pg/ml. In further embodiments, the composition may comprise the lipopeptide biosurfactant at a concentration of at least 10 pg/ml, especially at least 30 pg/ml, such as at least 50 pg/ml. In further embodiments, the composition may comprise the lipopeptide biosurfactant at a concentration of at most 500 pg/ml, especially at most 200 pg/ml, such as at most 100 pg/ml.

In the applications mentioned above, the lipopeptide biosurfactant according to the present invention may be administered to the fish (such as to the fish tank water) in a concentration of 10-100 pg/ml, such as of 30-70 pg/ml, of the lipopeptide biosurfactant.

In embodiments, the lipopeptide biosurfactant may be provided to (sea) water, especially to a body of water comprising the (sea) water, wherein the marine organism is exposed to the water. In particular, in embodiments, the marine organism may reside in a body of water, especially in an aquacultural body of water, and the lipopeptide biosurfactant may be provided to water in the body of water. Accordingly, in a particular embodiment, the present invention relates to the use of a lipopeptide biosurfactant selected from (a) a viscosin-like lipopeptide biosurfactant (such as lipopeptide biosurfactant obtainable from the Pseudomonas sp. strain H6) (b) a massetolide (such as a massetolide biosurfactant obtainable from Pseudomonas fluorescens strain SS101) or a derivative thereof, and (c) a putisolvin (such as the putisolvin biosurfactant obtainable from Pseudomonas putida 267) or a derivative thereof in a concentration of 10-100 pg/ml, such as in a concentration of about 50 pg/ml, in the treatment of scuticociliatosis in the marine organism, and in particular in (marine) fish, such as in turbot, sea bass and flounder, and more in particular in the treatment of scuticociliatosis caused by the pathogenic ciliate Philasterides dicentrarchi.

The treatment may be a one-time treatment after infection has been identified. Alternatively the treatment may be performed several times, such as once a day or once a week.

The present invention also relates to a process for the treatment or prevention of scuticociliatosis in marine organisms (such as teleosts, like turbot, sea bass, olive flounder, and fine flounder) susceptible to scuticociliatosis by administering a lipopeptide biosurfactants as defined above.

In embodiments, the marine organism(s) may comprise a farmed fish selected from the group comprising turbot, sea bass, and flounder, such as olive flounder and/or fine flounder.

In further embodiments, the marine organism(s) may comprise an aquarium fish selected from the group comprising a sea horse, a sea dragon, and a shark.

In a further aspect, the invention may provide a feed composition comprising the lipopeptide biosurfactant. In particular, low toxicity of the lipopeptide biosurfactant to the marine organism (see below) may facilitate providing the lipopeptide biosurfactant to the marine organism as a feed component.

Use of the verb "to comprise" and its conjugations does not exclude the presence of elements or steps other than those stated in a claim. Unless the context clearly requires otherwise, throughout the description and the claims, the words “comprise”, “comprising”, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”.

The article "a" or "an" preceding an element does not exclude the presence of a plurality of such elements.

The term “treatment” may herein specifically relate to the prevention and curing of diseases. Hence, in embodiments, the treatment may comprise prevention. In further embodiments, the treatment may comprise curing.

The various aspects discussed in this patent can be combined in order to provide additional advantages. Further, the person skilled in the art will understand thatiembodiments can be combined, and that also more than two embodiments can be combined. Furthermore, some of the features can form the basis for one or more divisional applications.

The term “plurality” refers to two or more.

The terms “substantially” or “essentially” herein, and similar terms, will be understood by the person skilled in the art. The terms “substantially” or “essentially” may also include embodiments with “entirely”, “completely”, “all”, etc. Hence, in embodiments the adjective substantially or essentially may also be removed. Where applicable, the term “substantially” or the term “essentially” may also relate to 90% or higher, such as 95% or higher, especially 99% or higher, even more especially 99.5% or higher, including 100%.

The term “comprise” also includes embodiments wherein the term “comprises” means “consists of’.

The term “and/or” especially relates to one or more of the items mentioned before and after “and/or”. For instance, a phrase “item 1 and/or item 2” and similar phrases may relate to one or more of item 1 and item 2. The term "comprising" may in an embodiment refer to "consisting of but may in another embodiment also refer to "containing at least the defined species and optionally one or more other species".

Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

The devices, apparatus, or systems may herein amongst others be described during operation. As will be clear to the person skilled in the art, the invention is not limited to methods of operation, or devices, apparatus, or systems in operation.

It should be noted that the above-mentioned embodiments illustrate rather than limit the invention, and that those skilled in the art will be able to design many alternative embodiments without departing from the scope of the appended claims.

In the claims, any reference signs placed between parentheses shall not be construed as limiting the claim.

Use of the verb "to comprise" and its conjugations does not exclude the presence of elements or steps other than those stated in a claim. Unless the context clearly requires otherwise, throughout the description and the claims, the words “comprise”, “comprising”, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”.

The article "a" or "an" preceding an element does not exclude the presence of a plurality of such elements. The invention may be implemented by means of hardware comprising several distinct elements, and by means of a suitably programmed computer. In a device claim, or an apparatus claim, or a system claim, enumerating several means, several of these means may be embodied by one and the same item of hardware. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. In yet a further aspect, the invention (thus) provides a software product, which, when running on a computer is capable of bringing about (one or more embodiments of) the method as described herein.

The invention also provides a control system that may control the device, apparatus, or system, or that may execute the herein described method or process. Yet further, the invention also provides a computer program product, when running on a computer which is functionally coupled to or comprised by the device, apparatus, or system, controls one or more controllable elements of such device, apparatus, or system.

The invention further applies to a device, apparatus, or system comprising one or more of the characterizing features described in the description and/or shown in the attached drawings. The invention further pertains to a method or process comprising one or more of the characterizing features described in the description and/or shown in the attached drawings.

The various aspects discussed in this patent can be combined in order to provide additional advantages. Further, the person skilled in the art will understand that embodiments can be combined, and that also more than two embodiments can be combined. Furthermore, some of the features can form the basis for one or more divisional applications.

NOTIFICATION

The project leading to this patent application has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No 634429.

EXAMPLES

Materials and methods

Unless specified otherwise, all experimental procedures are carried out using the materials and methods specified in this section. Ciliate culture

P. dicentrarchi (isolate Ii), obtained from an outbreak of scuticociliatosis in cultured turbot in Galicia (Iglesias et al. (2001)), was routinely cultured at 18 °C in complete sterile LI 5 medium (Leibovitz, PAA Laboratories GmbH, 1% salinity, pH 7.2) containing 90 mg/L each of adenosine, cytidine and uridine, 150 mg/L of guanosine, 5 g/L of glucose, 400 mg/L of L-a-phosphatidylcholine, 200 mg/L of Tween 80, 10% of heat-inactivated foetal bovine serum (FBS) and 10 mL/L of 100X antibiotic-antimycotic solution (100 units/mL of penicillin G, 0.1 mg/mL of streptomycin sulphate and 0.25 mg/mL of amphotericin B), as described by Iglesias et al. (2003). Alternatively, ciliates were cultured in sea water with autoclaved marine bacteria (10 ⁶ cells/mL) supplied as food at 21 °C. The ciliates were cultured under normoxic conditions in tissue culture flasks fitted with vented caps that enabled aeration of the culture medium.

Culture of the RAW 264. 7 cell line

The RAW 264.7 murine macrophage cell line, acquired from the American Type Culture Collection (ATCC®, USA), was used as a control for toxicity. The cell line was routinely cultured in Iscove’s Modified Dulbecco’s Medium (IMDM; Sigma, St. Louis, USA) supplemented with 3.024 mg/L NaHCCh, 10% foetal bovine serum and 10 mL/L of 100X antibiotic-antimycotic solution. Cells were maintained sub-confluent at 37 °C in humidified air containing 5% CO2. Cultured cells were used when the confluence reached 75%.

Pseudomonas sp. H6 lipopeptide biosurfactant (PS):

A lipopeptide biosurfactant of Pseudomonas sp. strain H6 was extracted according to the method described by Liu et al. (2015), which is hereby herein incorporated by reference.

Pseudomonas sp. strain H6 was grown on Pseudomonas agar plates (20 ml plates) for 48 h at 25C. Cells of strain H6 were collected from the agar plates and suspended in sterile de-mineralized water (5-10 ml per plate), and vortexed to homogenize the cell suspension. Cell suspensions were then centrifuged twice for 10 min at 9,000 rpm (4C) and supernatant filter-sterilized 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) HC1 to pH 2.0. Precipitation was allowed for 1 h on ice. The precipitate was collected by centrifugation at maximum speed and washed three consecutive times 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 was freeze-dried.

A stock solution of 10 mg/mL was prepared by dissolving the product in sterile distilled water whereafter a dilution series was prepared for parasite exposures.

Determination of anti-ciliate activity

The toxicity of PS H6 surfactant to P. dicentrarchi was tested as follows. The Pseudomonas H6 lipopeptide (bio-)surfactant (PS) was prepared, lyophilized and stored at -20°C as described by Liu et al. (2015). A stock solution of PS was prepared in distilled water (1000 ppm) and diluted in culture medium or in sea water at the final concentrations used in each assay and sterile filtered (through 0.2 pm filters) before being added to the parasites or to RAW 264.7 cells.

The anti-ciliate activity was assayed as previously described (Iglesias et al. (2002)), with minor modifications as described in Leiro et al. (2004). Ciliates were concentrated by centrifugation at 650 * g for 5 min and resuspended in medium at the concentration required for the assays. AH experiments were carried out in triplicate. Wells containing ciliates in culture medium or sea water without the lipopeptide biosurfactant were assayed as negative controls. In these assays, the lipopeptide biosurfactants were diluted in 1 ml of culture medium or sea water and added to the wells of 24-well sterile culture plates containing 2.5* 10 ⁴ ciliates per well, at final concentrations of 3.23, 6.25, 12.5, 25, 50 and 100 pM in culture medium. The effect of the lipopeptide biosurfactants on the motility and morphology of the ciliates was monitored periodically (at 30 minutes, 1, 2, 3, 24 and 48 h) under an inverted optical microscope. Ciliates were considered dead when they lysed or were round and immotile. To estimate the total number of ciliates per well, the ciliates were inactivated by addition of 0.25% glutaraldehyde and counted in a haemocytometer under an optical microscope (Iglesias et al (2002)). The lowest dose of the selected lipopeptide biosurfactants resulting in 100% ciliate mortality (LCioo) was assessed at 1, 2 and 3 h after incubation of parasites with sample dilutions (from 0 to 100 pg/mL, in 10 pg intervals). The concentration that inhibits 50% of ciliate growth (GI50), in comparison with the control group, was calculated with Excel software (Microsoft Office; Microsoft), by plotting the dose-response data (log dose-percent of growth inhibition) and applying linear regression (y = mx + b) for data fitting: the values were then calculated from the equation log GI50 = (50 - b)/m, where m is the slope yi- yi/xi- X2 and b is the intercept of the line.

Assessment of the toxicity of PS H6 surfactant on RA W 264. 7 macrophage cell line

RAW 264.7 cells (1 x 10 ⁵ cells/well) were incubated in 100 pL of Iscove's Modified Dulbecco's Medium (IMDM, Thermo (Spain)) medium with a specific concentration (corresponding to the LC50 value in the anti-ciliate assay on P. dicentrarchi, see below) of PS H6 surfactant in a 96-well sterile culture plate (Corning) for 3 and 24 h at 37 °C and 5% CO2. Cell viability was then estimated by the MTT (3-(4,5-dimethylthiazol-2-yl)- 2,5- diphenyl tetrazolium bromide) assay, as previously described by Denizot et al. (1986), with minor modifications. Briefly, 50 pL of the MTT solution (1 mg/mL in phosphate buffered saline; pH 7.2) was added to each well, and plates were incubated at 37°C and with 5% CO2 for 4 h. The yellow tetrazolium salt of MTT is reduced by mitochondrial dehydrogenases in metabolically active cells to form insoluble purple formazan crystals, which are then dissolved by the addition of DMSO (100 pL/well). Prior to the addition of MTT or DMSO, the plates were centrifuged at 800 g and the supernatant was removed. The absorbance was measured at 540 nm in a microplate reader (Multiskan™, Thermo, USA), and the relative cell viability was determined as the mean percentage of viable cells relative to untreated cells.

Control cells with only IMDM were also included in the assay.

EXAMPLE 1

Effect of Pseudomonas H6 surfactant on P. dicentrarchi

The toxicity of the Pseudomonas H6 biosurfactant (PS) to P. dicentrarchi was investigated. The ciliates were cultured in sea water and exposed to several concentrations of PS H6 lipopeptide biosurfactant for 1, 2 and 3 h, yielding a lethal concentration 50 (LC50 values of respectively 16.90, 9.0 and 7.8 pg/mL). Incubation of the ciliate with several concentrations of PS H6 lipopeptide biosurfactant revealed a dose-dependent response, with death of all the parasites exposed to 50 pg/mL of PS H6 lipopeptide biosurfactant after 3h. It was also shown that the PS H6 lipopeptide biosurfactant was much less toxic to the RAW 264.7 macrophage cell line than to the parasite. At a concentration of 7.8 pg/mL (LC50 for the ciliate), about 88% of RAW 264.7 cells were viable after 3 h of incubation with the PS H6 lipopeptide biosurfactant, i.e., (only) about 12% of RAW 264.7 cells were killed within 3 h of incubation.

References de Bruijn et al. (2007): de Bruijn I, de Kock MJD, Yang M, de Waard P, van Beek TA, Raaijmakers JM. “Genome-based discovery, structure prediction and functional analysis of cyclic lipopeptide antibiotics in Pseudomonas species.” Molecular Microbiology. 2007; 63(2):417-28.

Denizot et al. (1986): Denizot, F.; Lang, R. Rapid colorimetric assay for cell growth and survival. J. Immunol. Methods 1986, 89, 271-277.

Iglesias et al. (2001): Iglesias, R.; Parama, A.; Alvarez, M. F.; Leiro, J.; Fernandez, J.; Sanmartin, M.L. Philasterides dicentrarchi (Ciliophora, Scuticociliatida) as the causative agent of scuticociliatosis in farmed turbot, Scophthalmus maximus in Galicia (NW Spain). Dis. Aquat. Organ. 2001, 46, 47-55.

Iglesias et al. (2002): Iglesias, R.; Parama, A.; Alvarez, M.F.; Leiro, J.; Sanmartin, M.L. Antiprotozoal s effective in vitro against the scuticociliate fish pathogen Philasterides dicentrarchi. Dis. Aquat. Organ. 2002, 49, 191-19.

Iglesias et al. (2003): Iglesias, R.; Parama, A.; Alvarez, M. F.; Leiro, J.; Aja, C.; Sanmartin, M.L. In vitro growth requeriments for the fish pathogen Philasterides dicentrarchi (Ciliophora, Scuticociliatida). Vet. Parasitol. 2003, 111, 19-30.

Liu et al. (2015): Liu, Y.; Rzeszutek, E.; van der Voort, M.; Wu, C.H.; Thoen, E.; Skaar, I.; Bulone, V.; Dorrestein, P.C.; Raaijmakers JM.; de Bruijn I. Diversity of Aquatic Pseudomonas Species and Their Activity against the Fish Pathogenic Oomycete Saprolegnia. PLoS One 2015, 10(8), e0136241.

Leiro et al. (2004): Leiro, J.; Arranz, J. A.; Parama, A.; Alvarez, M.F.; Sanmartin, M.L. In vitro effects of the polyphenols resveratrol, mangiferin and epigallocatechin-3 -gallate on the scuticociliate fish pathogen Philasterides dicentrarchi. Dis. Aquat. Organ. 2004, 59 (2), 171-174. PCT

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