Treatment of Parasitic Infections of Fish Surfaces

Abstract

The invention relates to the use of bacterial lipopeptide biosurfactants in the treatment of white spot disease in fresh water and marine fish. Particularly useful for treatment of white spot disease are viscosin-like lipopeptide biosurfactants obtainable from the Pseudomonas fluorescens strain H6, massetolide or a derivative thereof and putisolvin or a derivative thereof.

Claims

1-15. (canceled)

16. A method for treating white spot disease in fish using a lipopeptide biosurfactant.

17. A method according to claim 16, wherein the lipopeptide biosurfactant is selected from a viscosin lipopeptide or a derivative thereof.

18. A method according to claim 17, wherein the viscosin lipopeptide is a viscosin-like lipopeptide obtainable from the Pseudomonas fluorescens strain H6.

19. A method according to claim 16, wherein the lipopeptide biosurfactant is selected from a massetolide lipopeptide or a derivative thereof.

20. A method according to claim 19, wherein the massetolide lipopeptide a massetolide surfactant obtainable from the Pseudomonas fluorescens strain SS101.

21. A method according to claim 16, wherein the lipopeptide biosurfactant is selected from a putisolvin lipopeptide or a derivative thereof.

22. A method according to claim 21, wherein the putisolvin lipopeptide is a putisolvin biosurfactant obtainable from Pseudomonas putida 267.

23. A method according to claim 16, wherein the method is for treating an Ichthyophthirius multifiliis infection.

24. A method according to claim 16, wherein the method comprises administering the lipopeptide biosurfactant to the fish.

25. A method according to claim 24, wherein the method comprises administering the lipopeptide biosurfactant as a bacterial culture, wherein the bacterial culture produces the lipopeptide biosurfactant.

26. A method according to claim 25, wherein the bacterial culture comprises Pseudomonas fluorescens strain H6.

27. A method according to claim 16, wherein the method comprises administering the lipopeptide biosurfactant to fish tank water in a concentration of 10-1000 .Math.g/ml.

28. A method according to claim 16, wherein the method comprises performing the treatment several times.

29. A method according to claim 16, wherein the method comprises treating white spot disease in fish using a composition, wherein the composition comprises the lipopeptide biosurfactant.

30. A method according to claim 29, wherein the lipopeptide biosurfactant is a lipopeptide obtainable from a Pseudomonas species.

31. A method according to claim 30, wherein the lipopeptide is obtainable from Pseudomonas fluorescens strain H6.

32. A method according to claim 29, wherein the composition comprises the lipopeptide biosurfactant in a concentration of 10 - 1000 .Math.g/ml.

33. A method according to claim 29, wherein the composition comprises one or more carriers, and wherein the composition comprises a slow-release form which sheds the lipopeptide biosurfactant over a prolonged period.

34. A method according to claim 16, further comprising preparing the composition comprising the lipopeptide biosurfactant, wherein the preparation comprises acidic precipitation of the lipopeptide biosurfactant.

35. A method according to claim 34, further comprising redissolution and drying of the precipitate.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0053] FIG. 1A is a graph showing effects of Pseudomonas fluorescens H6 lipopeptide biosurfactant (PS) on Ichthyophthirius multifiliis tomonts.

[0054] FIG. 1B is a graph showing the effects of Pseudomonas fluorescens H6 lipopeptide biosurfactant (PS) on Ichthyophthirius multifiliis tomocysts.

[0055] FIG. 1C is a graph showing the effects of Pseudomonas fluorescens H6 lipopeptide biosurfactant (PS) on Ichthyophthirius multifiliis theronts.

[0056] FIG. 2 are photos of tomonts released from the fish skin and tomocysts with enclosed tomites of Ichthyophthirius multifiliis affected by PS. A) Tomonts without PS. B) Tomocysts with enclosed tomites after 30 sec exposure to PS. C) Tomont showing a membrane disruption and release of cytoplasm following 15 min of PS exposure. D) Tomocyst with enclosed dead tomites in the cyst center after 15 min PS exposure.

[0057] FIG. 3 is a graph showing the effect of biosurfactants on number of theronts. The biosurfactants tested were massetolide obtained from Pseudomonas fluorescens SS101, the putisolvin-like biosurfactant from Pseudomonas putida 267 and the viscosin-like biosurfactant of Pseudomonas H6. In this figure the following data are included (1) water control; (2) 0.15 mg/ml viscosin (H6); (3) 0.015 mg/ml viscosin (H6); (4) 0.15 mg/ml massetolide (SS101); (5) 0.015 mg/ml massetolide (SS101); (6) 0.15 mg/ml putisolvin (267); (7) 0.015 mg/ml putisolvin (267).

EXAMPLES

Materials and Methods

Parasites

[0058] A Danish strain of Ichthyophthirius multifiliis was established in a laboratory population of rainbow trout originally raised in a disease free recirculation system (Xueqin et al. (2012)).

[0059] Parasites were collected from infected rainbow trout reared in a Danish commercial trout farm (Jutland, western part of Denmark) as previously described (Aihua & Buchmann, (2001)). Infected live fish were transported to the University of Copenhagen.

[0060] Ichthyophthirius multifiliis parasites were isolated at room temperature by placing fins and gills, recovered from a fish euthanized (300 mg/L of tricaine methanesulfonate, MS222,

[0061] Sigma-Aldrich, Denmark), in Petridishes with freshwater (22° C.). This induced release of epidermal trophonts to leave the fish tissues as tomonts. Some were isolated and used directly for lipopeptide biosurfactant exposure studies. Others were incubated further and transformed into tomocysts each containing several hundreds of tomites (24 h). A subpopulation of these were used for exposure and others were incubated further untill they released theronts within 24-30 h. These were isolated and similarly used for in vitro evaluation of lipopeptide biosurfactant effects.

Pseudomonas Fluorescens H6 Lipopeptide Biosurfactant (PS):

[0062] A lipopeptide biosurfactant of Pseudomonas fluorescens strain H6 was extracted according to the method described by Liu et al. (2015).

[0063] Pseudomonas fluorescens 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-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 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.

[0064] 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.

In Vitro Incubation and Exposure:

[0065] Glass plates (thickness 6 mm) each with 30 concave wells (diameter 25 mm, depth 3 mm, maximum water capacity 2000 .Math.L) were used for incubation of parasite life stages (theronts, tomont and tomocysts).

[0066] The final concentrations of the lipopeptide biosurfactant in the wells were 1000, 100, 20, 13, 10, 7, 5, 2.5, 2 and 1 .Math.g/mL and all concentrations were tested in triplicate for each parasite stage.

[0067] The number of parasites in each well was for theronts 20-25, for tomonts 2 and for tomocysts 2.

[0068] The volume added into each well was 100 .Math.L composed by mixing 50 .Math.L of lipopeptide biosurfactant solution with 50 .Math.L of fresh water containing parasites.

[0069] The experiment was performed at room temperature (22° C.) and parasite motility was recorded at 0, 15, 30, 45, 75, 60 and 90 min. The experiments were conducted in triplicate.

Monitoring Effect of Lipopeptide Biosurfactant on Motility of Parasites

[0070] A Leica MZ 95 dissection microscope (magnification 6-40X) was used for monitoring motility of tomonts, tomocysts and theronts.

[0071] Motility was recorded as presence of ciliary activity and cell movements of free theronts, free tomonts and tomites enclosed in tomocysts.

[0072] Non-motile and lysed tomites, theronts and tomonts were considered dead.

Sensitivity of Fish to Lipopeptide Biosurfactant

[0073] Rainbow trout (2x3) were exposed in plastic fish tanks (total volume 3 L), each containing 1 L of lipopeptide biosurfactant solution and three rainbow trout, to concentrations of lipopeptide biosurfactant (10 and 13 .Math.g/mL) which were found effective for all tested parasite life stages within 90 min. Three control fish were kept under the same conditions but without lipopeptide biosurfactant.

[0074] Fish were monitored in the lipopeptide biosurfactant solution for 3 h after exposure whereafter they were transferred to 80 L tanks containing only pure water and observed for any adverse behavioural signs (balance disturbances, lethargia, anorexia) for 7 days.

Data Analysis

[0075] As no significant differences between the triplicate wells were observed with regard to parasite survival (three different parasite life stages in different lipopeptide biosurfactant concentrations), data from these were pooled.

[0076] Survival of the different parasite life stages was visualized in a Kaplan-Meier plot and statistically tested by Dunn’s multiple comparison test with a probability level of 5%.

[0077] All the statistical analyses and graphs in this study were performed by using Graph Pad Prism Version 5.

Example 1

In Vitro Effects Pseudomonas Fluorescens Strain H6 Lipopeptide Biosurfactant on Ichthyophthirius Multifiliis

Tomonts

[0078] Ichthyophthirius multifiliis tomonts were only sensitive to the two highest concentrations (1000 and 100 .Math.g/mL PS), which killed all parasites within 15 min (FIG. 1A).

[0079] Cytoplasmic movements inside the tomonts (FIG. 2A) initially increased when exposed to the lipopeptide biosorfactant, whereafter a disruption of the membrane followed and finally cytoplasm was released into the surroundings of the tomonts (FIG. 2C).

[0080] A 10 .Math.g/mL PS concentration had no effect on this parasite life stage within the observation period tested.

Tomocysts

[0081] Ichthyophthirius multifiliis tomocysts, with their enclosed tomites (FIG. 2B), showed a different sensitivity to the lipopeptide biosurfactant when compared to tomonts (FIG. 1B).

[0082] At the highest tested concentration of 1000 .Math.g/mL PS, the majority of tomites in the tomocysts (83%) were immotile after 15 min of exposure (FIG. 1B). When exposed to 100 .Math.g/mL PS, immobilization was observed for 83% tomites after 30 min. After 60 min, all tomocysts with their content of tomites were dead (FIG. 2D).

[0083] Dead tomites were concentrated at the center of the tomocyte because tomites in the tomocyst moved away from the periphery immediately after PS addition (FIG. 2D).

[0084] The effect of 13 and 10 .Math.g/mL PS was slightly lower; nevertheless, all parasites were killed within 75 min and 90 min, respectively.

[0085] Tomocysts were phenotypically not affected at PS concentrations of 0 and 7 .Math.g/mL.

Theronts

[0086] Ichthyophthirius multifiliis theronts showed a high sensitivity towards PS and when exposed to 1000 and 100 .Math.g/mL PS theronts showed 100% mortality within 5 min (FIG. 1C). In 20 .Math.g/mL PS, less than 20% survival was seen at this time point and the remaining theronts were killed after 30 min.

[0087] Concentrations of 13 and 10 .Math.g/mL PS were lethal for theronts within 60 min.

[0088] Concentrations of 7 .Math.g/mL PS and lower had no visual effect even after 90 min.

Example 2

Sensitivity of Fish to Pseudomonas Lipopeptide Biosurfactant

[0089] Rainbow trout showed no immediate or late adverse reactions when exposed for 3 h to PS concentrations of 10 and 13 .Math.g/mL. No toxic effects on fish could be detected.

Example 3

Comparison of In Vitro Activity of Various Biosurfactants Against Tomonts and Theronts

[0090] Collection of I. multifillis was performed as described above. Extracts of the biosurfactant massetolide A was obtained from Pseudomonas fluorescens SS101 as described in De Bruijn et al (2008), the putisolvin-like biosurfactant from Pseudomonas putida 267 as described in Kruijt et al (2008) and the viscosin-like biosurfactant of Pseudomonas H6 as described above. A stock solution of 15 mg/mL was prepared for each surfactant by dissolving the product in sterile distilled water whereafter a dilution series was prepared for parasite exposures that was performed as described in above. Concentrations of 0.15 and 0.015 mg/ml were tested for all three biosurfactants against tomonts (one replicate) and theronts (in duplicate) and mortality recorded every 15 min up to 1 h of exposure. Significant differences were calculated for the theront mortality data by analysis of variance followed by Dunnet’s posthoc analyses (p< 0.05).

Results

[0091] The viscosin, massetolide and putisolvin biosurfactants extract from Pseudomonas sp H6, Pseudomonas fluorescens SS101 and Pseudomonas putida 267, respectively, elicited 100% mortality of theronts during the first 5 min exposure at a concentration 0.15 mg/mL. At a concentration of 0.015 mg/ml, massetolide and viscosin-like biosurfactant elicited 90% and 50% mortality of theronts within 15 min, whereas putisolvin had no effect at this concentration (FIG. 3).

[0092] Tomonts were killed at 0.1 mg/ml within 15 minutes upon exposure of the viscosin-like biosurfactant of Pseudomonas H6. Tomonts exposed to 1.5 and 0.15 mg/mL of massetolide were lethal within 15 min of exposure, whereas putisolvin killed tomonts at 1.5 mg/mL within the first 15 min, but no effects were observed at a concentration 0.15 mg/mL (Table 1).

TABLE-US-00001 Effect of biosurfactants on number of tomonts. The biosurfactants included were massetolide obtained from Pseudomonas fluorescens SS 101, the putisolvin-like biosurfactant from Pseudomonas putida 267. number of tomonts concentration time water control massetolide putisolvin 1.5 mg/ml 0 min 2 2 2 15 min 2 0 0 30 min 2 0 0 45 min 2 0 0 60 min 2 0 0 0.15 mg/ml 0 min 2 2 2 15 min 2 0 2 30 min 2 0 2 45 min 2 0 2 60 min 2 0 2

Conclusions

[0093] The viscosin-like biosurfactant of Pseudomonas H6 showed clear inhibitory effect on the free-living tomont and theront life stages of Ich at concentrations 100 and 10-20 ug/ml, respectively.

[0094] Structurally related biosurfactant massetolide (produced by Pseudomonas fluorescens SS101) showed similar activity as the viscosin-like biosurfactant from Pseudomonas H6 towards theronts and tomonts. Putisolvin, which is structurally more distant from the visocosin-like biosurfactant of H6, is less active at lower concentrations against theronts and not active against tomonts.

References

[0095] 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.

[0096] De Bruijn et al (2008): De Bruijn I, De Kock MJD, De Waard P, Van Beek TA, Raaijmakers JM. “Massetolide A Biosynthesis in Pseudomonas fluorescens.” J Bacteriol 190, 2777-2789 (2008).

[0097] Liu et al. (2015): Yiying Liu, Elzbieta Rzeszutek, Menno van der Voort, Cheng-Hsuan Wu, Even Thoen, Ida Skaar, Vincent Bulone, Pieter C. Dorrestein, Jos M. Raaijmakers, Irene de Bruijn “Diversity of Aquatic Pseudomonas Species and Their Activity against the Fish Pathogenic Oomycete Saprolegnia” PLOSOne; DOI:10.1371/journal.pone.0136241; published 28, August 2016.

[0098] Xueqin et al. (2012): Xueqin J, Kania PW and Buchmann K. “Comparative effects of four feed types on white spot disease susceptibility and skin immune parameters in rainbow trout, Oncorhynchus mykiss (Walbaum)” J Fish Dis. 2012 Feb;35(2):127-35.

[0099] Aihua, L., Buchmann, K. (2001). “Temperature- and salinity-dependent development of a Nordic strain of Ichthyophthirius multifiliis from rainbow trout.” J Appl Ichthyol 17, 273-276.

[0100] Kruijt et al (2008): Kruijt M, Tran H, Raaijmakers JM. “Functional, genetic and chemical characterization of biosurfactants produced by plant growth-promoting Pseudomonas putida 267.” Journal of applied microbiology 107, 546-556 (2009).