Treatment of parasitic infections in fish

Abstract

The present invention relates to the use of bacterial lipopeptide biosurfactants in the treatment of white spot disease in freshwater and marine fish. Particularly useful for the treatment of white spot disease are mucin-like lipopeptide biosurfactants obtainable from Pseudomonas fluorescens strain H6, massetolide or its derivatives and putisolvin or its derivatives.

Description

Technical Field

[0001] The present invention relates to the treatment of parasitic infections causing white spot disease in fish. Background Art

[0002] Diseases in fish, such as freshwater fish, caused by parasites on and within their bodies (gills, skin, fins), are an economic challenge in the fish production industry targeting both consumer and ornamental fish. An important disease is white spot disease of freshwater fish, particularly caused by the parasitic ciliate Ichthyophthirius multifiliis, which causes high morbidity and mortality in wild and farmed fish worldwide.

[0003] Currently, several chemicals are being used to control white spot disease in fish, such as freshwater fish.

[0004] Following the ban on the use of malachite green, an organic dye previously used to treat the disease, fish farmers have used a number of compounds to control the disease, including sodium percarbonate, copper sulfate, formalin, peracetic acid, and trotazole. However, these compounds may have adverse environmental effects. Plant extracts have also attracted considerable attention due to their effects on certain life cycles of parasites, and recently purified plant-derived compounds including cynatratoside-C, sanguinarine, dihydrosanguinarine, and dihydrochelerythrine, pentagalloylglucose have shown some antiparasitic effects in laboratory experiments. However, despite encouraging reports on the antiparasitic effects of natural, semisynthetic, and synthetic plant-derived products, questions remain regarding their residual and toxic effects on the environment, fish, and humans.

[0005] White spot disease causes serious economic losses in freshwater and marine aquaculture. Therefore, there is a need for an effective and safe treatment for white spot disease in fish. Summary of the invention

[0006] According to the present invention, it has surprisingly been found that lipopeptide surfactants can be used to treat white spot disease in fish.

[0007] Therefore, the present invention relates to the use of lipopeptide surfactants in treating fish white spot disease. In particular, the present invention relates to the use of bacterial lipopeptide surfactants in treating fish white spot disease.

[0008] Furthermore, the treatment was considered safe as fish showed no immediate or late adverse symptoms after several hours of incubation at effective lipopeptide biosurfactant concentrations.

[0009] Therefore, surfactant lipopeptides can be used as antiparasitic control agents in aquaculture fish.

[0010] To this end, fish or fish tank water may be treated with the isolated surfactant lipopeptide, or with a composition or formulation containing the lipopeptide.

[0011] Alternatively, the fish may be treated with a bacterial isolate, wherein the bacteria is capable of producing the lipopeptide biosurfactant. DETAILED DESCRIPTION

[0012] The present invention relates to lipopeptide biosurfactants for use in treating white spot disease in fish, such as freshwater fish and marine fish.

[0013] Examples of parasites that cause white spot disease in fish are the parasites of the family Ichthyophthiriidae, in particular Ichthyophthirius multifiliis, which causes freshwater white spot disease. A further example is the parasite Cryptocaryon irritans (originally classified as Ichthyophthirius marinus), which causes marine white spot disease.

[0014] Thus, in one specific embodiment, the present invention relates to lipopeptide biosurfactants for use in treating Cryptocaryon irritans, such as those caused by Ichthyophthirius multifida infection and Cryptocaryon irritans infection.

[0015] More specifically, the present invention relates to lipopeptide biosurfactants for use in the prevention of Cryptocaryon irritans, such as caused by Ichthyophthirius multifida infection in freshwater fish and Cryptocaryon irritans in marine fish.

[0016] As used herein, the term "freshwater fish" refers to fish that live in freshwater during at least some stage of their life cycle. Suitable examples are fish kept for food (e.g. salmon (e.g. rainbow trout (Oncorhynchus mykiss)), carp (e.g. grass carp (Ctenopharyngodon idella)), black carp (Mylopharyngodon piceus), silver carp (Hypophthalmichthys molitrix), common carp (Cyprinus carpio), bighead carp (Hypophthalmichthys nobilis), catla (Catla catla), crucian carp (Carassius carassius), rohu (Labeo rohita), other fish including tilapia (e.g. Nile tilapia (Oreochromis niloticus)), milkfish (Chanos chanos), catfish (e.g. Silurus asotus), Wuchang fish (Megalobrama amblycephala), snakehead fish (Channa argus), and species of remote ornamental fish kept for long periods in aquariums.

[0017] As referred to herein, "marine fish" refers to fish that live in marine waters for at least a portion of their lives. Examples include aquaculture fish that are farmed in marine aquaculture systems, such as gilthead sea bream (Sparus auratus) and sea bass (Dicentrarchus labrax). In addition, the term covers remote aquarium fish used in marine aquariums.

[0018] As referred to herein, the expression "lipopeptide biosurfactant" relates to a molecule consisting of a lipid linked to a peptide (usually a cyclic peptide) having surfactant properties (i.e., reducing the surface tension of a fluid). Lipopeptide biosurfactants can be produced by bacteria. Typically, the biosynthetic pathway encoding a lipopeptide surfactant in a given bacterial strain results in a single major lipopeptide surfactant and a small number of structurally related derivatives of the major lipopeptide surfactant.

[0019] Known bacterial lipopeptide biosurfactants include, for example, surfactin and its derivatives, daptomycin and its derivatives, massetolide and its derivatives, mucin and its derivatives, thanamycin and its derivatives, and putisolvin and its derivatives.

[0020] Suitable massetolide lipopeptide surfactants for use according to the invention are massetolide A, massetolide B, massetolide C, massetolide D, massetolide E, massetolide F, massetolide G and massetolide H.

[0021] Treatment of white spot disease in fish includes, inter alia, prevention of white spot disease by preventing the development of tomont and tomocyst, and more specifically, prevention of white spot disease by preventing the development of free-living theronts of organisms that cause white spot disease in fish, such as Ichthyophthirius multifida and Cryptocaryon irritans.

[0022] Another mucin-like lipopeptide biosurfactant obtained from Pseudomonas fluorescens strain H6 has recently been reported to kill zoospores of the oomycete fish pathogen Saprolegnia diclina (de Bruijn et al. 2007; Liu et al. 2015) and may therefore be useful for controlling Saprolegnia infections.

[0023] Surprisingly, we have found that this mucin-like lipopeptide biosurfactant of Pseudomonas fluorescens strain H6 can also be suitably used to treat white spot disease in fish, such as infections caused by Ichthyophthirius multifida.

[0024] In a specific embodiment, the present invention relates to a bacterial lipopeptide biosurfactant obtainable from Pseudomonas fluorescens strain H6 or a derivative thereof for use in treating Ichthyophthirius multifida infection in fish.

[0025] In another embodiment, the present invention is directed to a composition comprising at least one lipopeptide biosurfactant for treating white spots in fish, particularly freshwater and marine fish.

[0026] In another embodiment, the present invention is directed to a composition comprising at least one lipopeptide biosurfactant for treating Ichthyophthirius multifida infection in fish, particularly freshwater fish.

[0027] In another embodiment, the present invention is directed to a composition comprising a bacterial lipopeptide biosurfactant obtainable from Pseudomonas fluorescens strain H6 for use in treating white spot in fish, particularly freshwater and marine fish.

[0028] In another embodiment, the present invention relates to a composition comprising a bacterial lipopeptide biosurfactant obtainable from Pseudomonas fluorescens strain H6 or a derivative thereof for use in treating Ichthyophthirius multifida infection in fish, particularly freshwater fish.

[0029] Suitable compositions according to the invention may comprise essentially one or more lipopeptides, for example one or more lipopeptides and one or more carriers, or may, for example, comprise a sustained release form (ie a particle) which releases the lipopeptide biosurfactant over a prolonged period of time.

[0030] In a specific embodiment, a suitable composition according to the invention can be prepared from a lyophilized solution of one or more lipopeptides by dissolving the lyophilized solution in water, such as sterile distilled water.

[0031] Alternatively, the lipopeptide biosurfactant can be administered to fish as a bacterial culture that is capable of producing the lipopeptides in aquaculture.

[0032] Thus, in another embodiment, the present invention relates to bacterial isolates, wherein the bacteria are capable of producing lipopeptide surfactants for use in treating white spot in fish, particularly freshwater and marine fish.

[0033] In another embodiment, the present invention relates to bacterial isolates, wherein the bacteria are capable of producing a lipopeptide surfactant for use in treating Ichthyophthirius multifida infection in fish, particularly freshwater fish.

[0034] In another embodiment, the present invention relates to a bacterial isolate of Pseudomonas fluorescens strain H6 for use in the treatment of white spot in fish, particularly freshwater and marine fish.

[0035] In another embodiment, the present invention relates to bacterial isolates, wherein the bacteria are capable of producing massetolide and / or derivatives for use in the treatment of white spot disease in fish, particularly freshwater fish.

[0036] In another embodiment, the present invention relates to bacterial isolates, wherein the bacteria is capable of producing putisolvin and / or derivatives for use in the treatment of white spot disease in fish, particularly freshwater fish.

[0037] In another embodiment, the present invention relates to a bacterial isolate of Pseudomonas fluorescens strain H6 or a derivative thereof, or a derivative thereof, for use in treating Ichthyophthirius multifida infection in fish.

[0038] In accordance with the provisions of the Budapest Treaty, the Pseudomonas fluorescens strain H6 sample was deposited in the CBS library of the Westerdijk Fungal Biodiversity Institute on November 1, 2017, with the deposit number CBS 143505.

[0039] (2015) have described the isolation and characterization of a lipopeptide biosurfactant from Pseudomonas fluorescens strain H6. This mucin-like lipopeptide biosurfactant was found to be distinct from well-known lipopeptide biosurfactants from related strains, such as the massetolide lipopeptide obtainable from Pseudomonas fluorescens SS101, the mucin lipopeptide obtainable from Pseudomonas fluorescens SBW25, and the putisolvin lipopeptide obtainable from Pseudomonas putida 267.

[0040] The results showed that these lipopeptide biosurfactants have a strong in vitro antiparasitic effect on the fish pathogenic ciliate Ichthyophthirius multifida. Ichthyophthirius multifida is a pathogenic ciliate with different life cycle stages, the infectious theront stage, the trophont stage in the fish epidermis, the tomont (the stage reached by the trophozoite after leaving the fish body surface in the water), and the tomocyst containing the larvae released into the water as the tomocyst. The life cycle stages including the theront, the tomont, and the tomocyst were found to be sensitive to the H6 lipopeptide biosurfactant. The tomont was the most sensitive, showing 100% mortality within 30 minutes at low concentrations of 10 μg / ml and 13 μg / ml. The tomont was the most resistant, but was still killed quickly at higher concentrations of 100 μg / mL and 1000 μg / mL. The tomont is generally resistant to chemical and drug treatments due to the surrounding protective cyst wall. Surprisingly, they were sensitive to a lipopeptide biosurfactant from Pseudomonas fluorescens strain H6, which, even at low concentrations of 10 and 13 μg / mL, killed the enclosed tomites within minutes.

[0041] Therefore, this lipopeptide biosurfactant of Pseudomonas fluorescens strain H6 can be used as an antiparasitic control agent in aquaculture fish, especially aquaculture trout (e.g. rainbow trout).

[0042] As mentioned above, the present invention also relates to the use of other lipopeptide biosurfactants in the control of white spot disease. For example, it has been shown that massetolide and putisolvin also show significant in vitro activity against various developmental stages of the parasite that causes white spot disease in fish.

[0043] Therefore, in a specific embodiment, the present invention relates to the use of a lipopeptide biosurfactant selected from the following: (a) mucin-like lipopeptides (e.g., lipopeptide biosurfactants obtainable from Pseudomonas fluorescens strain H6) or derivatives thereof, (b) massetolide (e.g., massetolide surfactant obtainable from Pseudomonas fluorescens strain SS101) or derivatives thereof, and (c) putisolvin (e.g., putisolvin biosurfactant obtainable from Pseudomonas putida 267) or derivatives thereof for treating white spot disease in fish, in particular in freshwater fish and marine fish, more particularly in treating white spot disease caused by the pathogenic ciliate Ichthyophthirius multifida.

[0044] In the above uses, the lipopeptide according to the present invention can be administered to fish (eg, fish tank water) at a concentration of 10-1000 μg / mL, such as 10-100 μg / mL of lipopeptide biosurfactant.

[0045] Therefore, in a specific embodiment, the present invention relates to the use of a lipopeptide biosurfactant selected from the following: (a) mucin-like lipopeptides (e.g., lipopeptide biosurfactants obtainable from Pseudomonas fluorescens strain H6), (b) massetolide (e.g., massetolide biosurfactant obtainable from Pseudomonas fluorescens strain SS101) or a derivative thereof, and (c) putisolvin (e.g., Putisolvin biosurfactant obtainable from Pseudomonas putida 267) or a derivative thereof, at a concentration of 10-1000 μg / mL (e.g., 10-100 μg / mL).

[0046] The treatment may be a one-time treatment after infection is confirmed. Alternatively, the treatment may be performed several times, such as once a day or once a week.

[0047] The use of the verb "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 specification and claims, the words "comprises," "comprising," etc. are to be understood in an inclusive sense, rather than an exclusive or exhaustive sense; that is, meaning "including but not limited to."

[0048] The indefinite article preceding an element does not exclude the presence of a plurality of such elements.

[0049] The terms "treatment" and "prevention" are used interchangeably and all refer to the prevention and cure of disease.

[0050] The various aspects discussed in this patent can be combined to provide additional benefits. In addition, those skilled in the art will appreciate that the embodiments can be combined, and more than two embodiments can also be combined. In addition, some features can form the basis of one or more divisional applications.

[0051] Notice

[0052] The project leading to this patent application has received funding from the European Union's Horizon 2020 research and innovation programme under grant number 634429. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1. Effect of Pseudomonas fluorescens H6 lipopeptide biosurfactant (PS).

[0055] Three life stages of free-living Ichthyophthirius multifleuroides were incubated in water with a range of PS concentrations and compared with parasites in control water without PS. Parasite mortality was recorded at 0, 15, 30, 45, 75, 60, 120 and 180 min after treatment. A. Precyst, B. Cyst, C. Predator.

[0056] Figure 2 Cyst precursors released from fish skin and cysts with enclosed larvae of Ichthyophthirius spp. affected by PS. A) Cyst precursors without PS. B) Cyst with enclosed larvae after 30 seconds of exposure to PS. C) Cyst precursors showing membrane rupture and cytoplasm release after 15 minutes of PS exposure. D) Cyst with enclosed dead larvae in the cyst wall after 15 minutes of PS exposure.

[0057] Figure 3Effect of biosurfactants on predator populations. The biosurfactants tested were massetolide obtained from Pseudomonas fluorescens SS101, a putisolvin-like biosurfactant obtained from Pseudomonas putida 267, and a mucin-like biosurfactant from Pseudomonas H6. In this figure, the following data are included: (1) water control; (2) 0.15 mg / ml mucin (H6); (3) 0.015 mg / ml mucin (H6); (4) 0.15 mg / ml of massetolide (SS101); (5) 0.015 mg / ml of massetolide (SS101); (6) 0.15 mg / ml of putisolvin (267); (7) 0.015 mg / ml of putisolvin (267).

[0058] Example

[0059] Materials and methods

[0060] Parasites:

[0061] The Danish strain of Ichthyophthirius multifida was established in a laboratory population of rainbow trout originally raised in a disease-free recirculating system (Xueqin et al. (2012)).

[0062] Parasites were collected from infected rainbow trout raised in a Danish commercial trout farm (Jylland, western Denmark) as described previously (Aihua & Buchmann, (2001)). Live infected fish were transported to the University of Copenhagen.

[0063] Ichthyophthirius multifle parasites were isolated from euthanized (300 mg / L tricaine methanesulfonate, MS222, Sigma-Aldrich, Denmark) fish in fresh water (22°C) petri dishes at room temperature by recovering the fins and gills. This induced the release of superficial trophozoites from the fish tissues as cyst precursors. Some of these were isolated and used directly in the lipopeptide biosurfactant exposure studies. Others were further incubated and converted into cysts (24 hours) each containing hundreds of larvae. These subpopulations were used for exposure and other subpopulations were further incubated until they released predators within 24-30 hours. These were isolated and similarly used for in vitro evaluation of the effects of lipopeptide biosurfactants.

[0064] Pseudomonas fluorescens H6 lipopeptide biosurfactant (PS):

[0065] Lipopeptide biosurfactants from Pseudomonas fluorescens strain H6 were extracted according to the method described by Liu et al. (2015).

[0066] Pseudomonas fluorescens strain H6 was grown on Pseudomonas agar plates (20 ml plates) at 25°C for 48 hours. Cells of strain H6 were collected from the agar plates and suspended in sterile demineralized water (5-10 ml per plate) and vortexed to homogenize the cell suspension. The cell suspension was then centrifuged twice at 9,000 rpm (4°C) for 10 minutes and the supernatant was filtered with a 0.2 μm filter. The lipopeptide biosurfactant present in the cell-free culture supernatant was precipitated by acidifying the supernatant to pH 2.0 with 9% (v / v) HCl. Precipitate on ice for 1 hour. The precipitate was collected by centrifugation at maximum speed and washed continuously 3 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 dissolve the precipitate. The resulting solution was freeze-dried.

[0067] A 10 mg / mL stock solution was prepared by dissolving the product in sterile distilled water, and then a dilution series was prepared for parasite exposure.

[0068] In vitro incubation and exposure:

[0069] Glass plates (6 mm thickness) each had 30 wells (25 mm diameter, 3 mm depth, maximum water capacity 2000 μL) for incubation of parasite life stages (predators, precysts and cysts).

[0070] The final concentrations of lipopeptide biosurfactant in the wells were 1000, 100, 20, 13, 10, 7, 5, 2.5, 2, and 1 μg / mL, and all concentrations were tested in triplicate for each parasite stage.

[0071] The number of parasites in each well was: 20-25 predators, 2 precysts and 2 cysts.

[0072] Make up a volume of 100 µL per well by mixing 50 µL of the lipopeptide biosurfactant solution with 50 µL of fresh water containing parasites.

[0073] The experiments were performed at room temperature (22°C) and the motility of the parasites was recorded at 0, 15, 30, 45, 75, 60 and 90 minutes. The experiments were performed in triplicate.

[0074] Monitoring the Effects of Lipopeptide Biosurfactants on Parasite Motility:

[0075] A Leica MZ 95 dissecting microscope (magnification 6-40X) was used to monitor the movements of cyst precursors, cysts, and predators.

[0076] Movement was recorded as ciliary activity and cellular movements of free predators, free cyst precursors, and larvae enclosed in cysts.

[0077] Inactive and lysed larvae, predators, and cyst precursors were considered dead.

[0078] Sensitivity of fish to lipopeptide biosurfactants:

[0079] Rainbow trout (2 × 3) were exposed to plastic fish tanks (total volume 3 L) containing 1 L of lipopeptide biosurfactant solution and 3 rainbow trout per tank to lipopeptide biosurfactant concentrations (10 μg / mL and 13 μg / mL) found to be effective against all tested parasite life stages within 90 min. Three control fish were maintained under the same conditions but without lipopeptide biosurfactant.

[0080] Fish were monitored in the lipopeptide biosurfactant solution for 3 h after exposure and then transferred to an 80 L container containing pure water only and observed for 7 days for any adverse behavioral signs (balance disturbance, lethargy, anorexia).

[0081] Data analysis

[0082] Since no significant differences between triplicate wells were observed in parasite survival (three different parasite life stages at different lipopeptide biosurfactant concentrations), these data were pooled.

[0083] The survival rates of different parasite life stages were visualized in Kaplan–Meier plots and statistically tested by Dunn's multiple comparison test at a probability level of 5%.

[0084] All statistical analyses and graphs in this study were performed using Graph Pad Prism version 5.

[0085] Example 1

[0086] Effects of lipopeptide biosurfactant from Pseudomonas fluorescens strain H6 on Ichthyophthirius multifida in vitro

[0087] Cyst precursor

[0088] Ichthyophthirius multifida cyst precursors were only sensitive to the two highest concentrations (1000 μg / mL and 100 μg / mL PS), which killed all parasites within 15 min ( Figure 1A ).

[0089] When exposed to the lipopeptide biosurfactant, cytoplasmic motility within the cyst precursors increased ( Figure 2 A), followed by membrane rupture and cytoplasm release around the cyst precursor ( Figure 2 C).

[0090] A PS concentration of 10 μg / mL had no effect on this parasite life stage during the observation period tested.

[0091] cyst

[0092] Ichthyophthirius multifleuroides cysts and their enclosed larvae ( Figure 2 B), showing different sensitivity to lipopeptide biosurfactants compared to encapsulated precursors ( Figure 1B ).

[0093] At the highest tested concentration of 1000 μg / mL PS, most (83%) larvae in the cysts were no longer active after 15 min of exposure ( Figure 1B When exposed to 100 μg / mL PS, 83% of the larvae were immobilized after 30 min. After 60 min, all cysts containing larvae were dead ( Figure 2 D).

[0094] The dead larvae were mainly concentrated in the center of the cyst because after the addition of PS, the larvae in the cyst immediately moved away from the periphery ( Figure 2 D).

[0095] The effects of 13 μg / mL and 10 μg / mL PS were slightly lower; however, all parasites were killed within 75 and 90 minutes, respectively.

[0096] The cyst phenotype was not affected at PS concentrations of 0 and 7 μg / mL.

[0097] Predator

[0098] The predators of Ichthyophthirius multifida were highly sensitive to PS. When exposed to 1000 μg / mL and 100 μg / mL PS, the mortality rate of the predators was 100% within 5 minutes ( Figure 1C ). In the case of 20 μg / mL PS, less than 20% survival was observed at this time point, and the remaining predators were killed after 30 minutes.

[0099] PS concentrations of 13 μg / mL and 10 μg / mL were lethal to predators within 60 minutes, while PS concentrations of 7 μg / mL and lower had no detectable effect even after 90 minutes.

[0100] Example 2

[0101] Susceptibility of fish to Pseudomonas lipopeptide biosurfactants

[0102] When rainbow trout were exposed to 10 μg / mL and 13 μg / mL PS concentrations for 3 h, no immediate or late adverse effects were shown. No toxic effects on fish were found.

[0103] Example 3

[0104] Comparison of the in vitro activity of various biosurfactants against encapsulated precursors and predators

[0105] Ichthyophthirius multifle was collected as described above. Extracts of the biosurfactant massetolide A were obtained from Pseudomonas fluorescens SS101 as described in De Bruijn et al. (2008); putisolvin-like biosurfactants were obtained from Pseudomonas putida 267 as described in Kruijt et al. (2008), and mucin-like biosurfactants from Pseudomonas H6 were obtained as described above. A 15 mg / mL stock solution was prepared for each surfactant by dissolving the product in sterile distilled water, and then a dilution series for parasite exposure was prepared as described above. Concentrations of 0.15 mg / ml and 0.015 mg / ml were tested for all three biosurfactants on cyst precursors (one replicate) and predators (in duplicate), and mortality was recorded every 15 minutes until 1 h of exposure. Significant differences (p<0.05) in predator mortality data were calculated by analysis of variance followed by Dunnet post hoc analysis.

[0106] result

[0107] At a concentration of 0.15 mg / mL, mucin, massetolide, and putisolvin biosurfactants extracted from Pseudomonas H6, Pseudomonas fluorescens SS101, and Pseudomonas putida 267, respectively, caused 100% mortality in the first 5 minutes of exposure to predators. At a concentration of 0.015 mg / ml, massetolide and mucin-like biosurfactants caused 90% and 50% mortality in predators, respectively, within 15 minutes, while putisolvin had no effect at this concentration ( Figure 3 ).

[0108] After exposure to the colistin-like biosurfactant of Pseudomonas H6, precysts were killed within 15 minutes at a concentration of 0.1 mg / ml. Precysts exposed to massetolide at 1.5 mg / mL and 0.15 mg / mL were lethal within 15 minutes after exposure, while putisolvin killed precysts within the first 15 minutes at a concentration of 1.5 mg / mL, but no effect was observed at a concentration of 0.15 mg / mL (Table 1).

[0109] Table 1. Effect of biosurfactants on the number of encapsulated precursors. The biosurfactants included were massetolide obtained from Pseudomonas fluorescens SS101, putisolvin-like biosurfactant obtained from Pseudomonas putida 267.

[0110]

[0111] in conclusion

[0112] -The mucin-like biosurfactant of Pseudomonas sp. H6 showed significant inhibitory effects on the free-living procyst and predator life stages of Ichthyophthirius multifleurus at concentrations of 100 μg / ml and 10-20 μg / ml, respectively.

[0113] - The structurally related biosurfactant massetolide (produced by Pseudomonas fluorescens SS101) showed similar activity against predators and pre-encysts as the mucin-like biosurfactant of Pseudomonas H6. Putisolvin, which is structurally distant from the mucin-like biosurfactant of H6, showed less activity against predators at lower concentrations and no activity against pre-encysts.

[0114] References

[0115] de Bruijn et al. (2007): de Bruijn L, de Kock MJD, Yang M, de Waard P, vanBeek 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.

[0116] De Bruijn et al (2008): De Bruijn L, De Kock MJD, De Waard P, Van Beek TA, Raaijmakers JM. "Massetolide A Biosynthesis in Pseudomonas fluorescens." JBacteriol 190, 2777-2789 (2008).

[0117] Liu et al.(2015):Yiying Liu,Elzbieta Rzeszutek,Menno of the V00rt,Cheng-Hsuan Wu,Even Thoen,lda Skaar,Vincent Bulone,Pieter C.Dorrestein,JosM.Raaijmakers,the teachers Bruijn“Diversity of Aquatic Pseudomonas Species andTheir Activity against the Fish Pathogenic Oomycete Saprolegnia′′PLOSOne:DOl:10.1371 / journal.pone.0136241,published 28.August2016.

[0118] Xueqin et al.(2012):Xueqin J,Kania PW and Buchmann K.′′Comparativeeffects of four feed types on white spot disease susceptibility and skin immune parameters in rainbow trout,Oncorhynchus mykiss(Walbaum)”J FishDis.2012Feb:35(2):127-35.

[0119] Aihua,L.,Buchmann,K.(2001).“Temperature-and salinity-dependentdevelopment of a Nordic strain of lchthyophthirius multifiliis from rainbowtrout.” J Appl lchthyol 17,273-276.

[0120] Kruijt et al(2008):Kruijt M,Tran H,Raaijmakers JM.“Functional,geneticand chemical characterization of biosurfactants produced by plant growth-promoting Pseudomonas putida 267.″Joumal of applied microbiology 107,546-556(2009).

[0121]

Claims

1. Use of a lipopeptide biosurfactant selected from (a) a mucin-like lipopeptide obtained from Pseudomonas fluorescens H6, (b) massetolide obtained from Pseudomonas fluorescens SS101 and (c) putisolvin obtained from Pseudomonas putida 267 in the preparation of a medicament for treating infection by Ichthyophthirius multifiliis in fish.

2. The use according to claim 1, wherein the fish is a freshwater fish.

3. Use of a composition for preparing a medicament for treating Ichthyophthirius multifida infection in fish, wherein the composition comprises at least one lipopeptide biosurfactant selected from (a) a mucin-like lipopeptide obtained from Pseudomonas fluorescens H6, (b) massetolide obtained from Pseudomonas fluorescens SS101, and (c) putisolvin obtained from Pseudomonas putida 267.

4. Use of a bacterial isolate selected from (a) Pseudomonas fluorescens H6, (b) Pseudomonas fluorescens SS101 and (c) Pseudomonas putida 267 in the preparation of a medicament for treating infection by Ichthyophthirius multifida in fish, wherein the bacteria produces a lipopeptide biosurfactant selected from (a) mucin-like lipopeptides, (b) massetolide and (c) putisolvin.

5. The use according to claim 4, wherein the bacterial isolate Pseudomonas fluorescens H6 is deposited in the CBS repository of the Westerdijk Fungal Biodiversity Institute under the provisions of the Budapest Treaty with the deposit number CBS143505, and produces a mucin-like lipopeptide biosurfactant.

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