US20200353035A1 - Treatment of parasitic infections of fish surfaces - Google Patents

Treatment of parasitic infections of fish surfaces Download PDF

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Publication number
US20200353035A1
US20200353035A1 US16/764,437 US201816764437A US2020353035A1 US 20200353035 A1 US20200353035 A1 US 20200353035A1 US 201816764437 A US201816764437 A US 201816764437A US 2020353035 A1 US2020353035 A1 US 2020353035A1
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fish
lipopeptide
treatment
biosurfactant
derivative
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Inventor
Irene De Bruijn
Josephus Maria Raaijmakers
Kurt Buchman
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Kobenhavns Universitet
Nederlands Instituut Voor Ecologie (nioo-Knaw)
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Kobenhavns Universitet
Nederlands Instituut Voor Ecologie (nioo-Knaw)
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Publication of US20200353035A1 publication Critical patent/US20200353035A1/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • C12R1/39—
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/38—Pseudomonas
    • C12R2001/39—Pseudomonas fluorescens

Definitions

  • the present invention relates to the treatment of parasitic infections causing white spot disease in fish.
  • White spot disease causes severe economic losses in fresh water and sea water aquaculture. Accordingly, there is a need for an effective and safe remedy against white spot disease in fish.
  • lipopeptide surfactants can be used in the treatment of white spot disease in fish.
  • the present invention relates to the use of lipopeptide surfactants in the treatment of white spot disease in fish.
  • the present invention relates in particular to the use of bacterial lipopeptide surfactants in the treatment of white spot disease.
  • surfactant lipopeptides may find application as an antiparasitic control agent in aquacultured fish.
  • the fish or fish tank water may be treated with the isolated surfactant lipopeptide, or with compositions or formulations containing this lipopeptide.
  • the fish may be treated with the bacterial isolate wherein the bacteria are capable to produce said lipopeptide biosurfactant.
  • the present invention relates to a lipopeptide biosurfactant for use in the treatment of white spot disease in fish, such as in fresh water fish and in marine fish.
  • Examples of parasites causing white spot disease in fish are parasites of the family termed Ichthyophthudidae, and in particular Ichthyophthirius multifiliis causing freshwater white spot disease.
  • a further example is the parasite Cryptocaryon irritans (originally classified as Ichthyophthirius marinus ) causing marine white spot disease.
  • the present invention relates to lipopeptide biosurfactant for use in the treatment of white spot disease, such as caused by Ichthyophthirius multifiliis infection and Cryptocaryon irritans infection.
  • the present invention relates to lipopeptide biosurfactant for use in the prevention of white spot disease, such as caused by Ichthyophthirius multifiliis infection in fresh water fish and Cryptocaryon irritans in marine fish.
  • fresh water fish relates to fish living at least during a certain stage of its life cycle in fresh water.
  • suitable examples are fish raised for consumption in aquaculture (such as salmonids (exemplified by rainbow trout ( Oncorhynchus mykiss )), cyprinids (exemplified by grass carp ( Ctenopharyngodon idella ), black carp ( Mylopharyngodon piceus ), silver carp ( Hypophthalmichthys molitrix ), common carp ( Cyprinus carpio ), bighead carp ( Hypophthalmichthys nobilis ), catla (Indian carp, Catla calla), crucian carp ( Carassius carassius ), roho labeo ( Labeo rohita )), other fish families including tilapia (exemplified by nile tilapia ( Oreochromis niloticus )), milkfish ( Chanos
  • marine fish relates 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 ornamental fish species used in marine aquaria is covered by the term.
  • 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 are massetolide A, massetolide B, massetolide C, massetolide D, massetolide E, massetolide F, massetolide G and massetolide H.
  • Treatment of white spot disease in fish in particular comprises the prevention of white spot disease by preventing the development of the tomonts and tomocysts and more in particular by preventing the development of the free-living theronts of the organisms causing white spot disease in fish, like Ichthyophthirius multifiliis and Cryptocaryon irritans.
  • a further viscosin-like lipopeptide biosurfactant obtainable from the bacterium Pseudomonas fluorescens 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.
  • this vicosin-like lipopeptide biosurfactant of Pseudomonas fluorescens strain H6 can also suitably be used for the treatment of white spot disease in fish such as infections caused by Ichthyophthirius multifiliis.
  • the present invention relates to a bacterial lipopeptide biosurfactant obtainable from the Pseudomonas fluorescens strain H6 or a derivative thereof for use in the treatment of Ichthyophthirius multifiliis infection in fish.
  • the present invention relates to a composition comprising at least one lipopeptide biosurfactant for use in the treatment of white spot in fish, in particular in fresh water fish and in marine fish.
  • the present invention relates to a composition comprising at least one lipopeptide biosurfactant for use in the treatment of Ichthyophthirius multifiliis infection in fish, in particular in fresh water fish.
  • the present invention relates to a composition
  • a composition comprising a bacterial lipopeptide biosurfactant obtainable from the Pseudomonas fluorescens strain H6 for use in the treatment white spot in fish, in particular in fresh water fish and in marine fish.
  • the present invention relates to a composition
  • a composition comprising a bacterial lipopeptide biosurfactant obtainable from the Pseudomonas fluorescens strain H6 or a derivative thereof for use in the treatment of Ichthyophthirius multifiliis infection in fish, in particular in fresh water fish.
  • a composition suitable according to the present invention may predominantly comprise the lipopeptide or lipopeptides, for example the one or more lipopeptides 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.
  • a slow-release form i.e. granules
  • composition suitable according to the present invention may be prepared from a freeze-dried solution of the lipopeptide or lipopeptides by dissolving the freeze-dried solution in water, such as sterile distilled water.
  • the lipopeptide biosurfactant may be administered to the fish as a bacterial culture, which is capable to produce the lipopeptide in aquaculture.
  • the invention relates to a bacterial isolate of the Pseudomonas fluorescens strain H6 for use in the treatment of white spot in fish, in particular in fresh water fish and in marine fish.
  • 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 white spot disease in fish, in particular in fresh water fish.
  • this lipopeptide biosurfactant of Pseudomonas fluorescens strain H6 may find further application as an antiparasitic control agent in aquacultured fish, in particular in aquacultured trout, such as in rainbow trout.
  • the present invention relates to the use of a lipopeptide biosurfactant selected from (a) a viscosin-like lipopeptide (such as lipopeptide biosurfactant obtainable from the Pseudomonas fluorescens 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 10-1000 ⁇ g/ml, such as in a concentration of 10-100 ⁇ g/ml, in the treatment of white spot disease in fish, in particular in fresh water fish, and more particular in the treatment of white spot disease caused by the pathogenic ciliate Ichthyophthirius multifiliis.
  • a viscosin-like lipopeptide such as
  • treatment and “control” or “controlling” may be used interchangeably and may all relate to prevention and curing of diseases.
  • FIG. 3 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.
  • 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, 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 until they released theronts within 24-30 h. These were isolated and similarly used for in vitro evaluation of lipopeptide biosurfactant effects.
  • a lipopeptide biosurfactant of Pseudomonas fluorescens strain H6 was extracted according to the method described by Liu et al. (2015).
  • Pseudomonas fluorescens strain H6 was grown on Pseudomonas agar plates (20 ml plates) for 48 h at 25 C. 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 (4 C) 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.
  • 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.
  • the final concentrations of the lipopeptide biosurfactant in the wells were 1000, 100, 20, 13, 10, 7, 5, 2.5, 2 and 1 ⁇ /mL and all concentrations were tested in triplicate for each parasite stage.
  • the volume added into each well was 100 ⁇ L composed by mixing 50 ⁇ L of lipopeptide biosurfactant solution with 50 ⁇ L of fresh water containing parasites.
  • a Leica MZ 95 dissection microscope (magnification 6-40 ⁇ ) was used for monitoring motility of tomonts, tomocysts and theronts.
  • Motility was recorded as presence of ciliary activity and cell movements of free theronts, free tomonts and tomites enclosed in tomocysts.
  • Non-motile and lysed tomites, theronts and tomonts were considered dead.
  • Ichthyophthirius multifiliis tomonts were only sensitive to the two highest concentrations (1000 and 100 ⁇ g/mL PS), which killed all parasites within 15 min ( FIG. 1A ).
  • Cytoplasmic movements inside the tomonts 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 ).
  • 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 ).
  • Tomocysts were phenotypically not affected at PS concentrations of 0 and 7 ⁇ g/mL.
  • Ichthyophthirius multifiliis theronts showed a high sensitivity towards PS and when exposed to 1000 and 100 ⁇ g/mL PS theronts showed 100% mortality within 5 min ( FIG. 10 ). In 20 ⁇ g/mL PS, less than 20% survival was seen at this time point and the remaining theronts were killed after 30 min.
  • 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.
  • 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.
  • 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 ).
  • 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).

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US16/764,437 2017-11-21 2018-11-20 Treatment of parasitic infections of fish surfaces Abandoned US20200353035A1 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
EP17202669.2 2017-11-21
EP17202669 2017-11-21
PCT/EP2018/081923 WO2019101739A1 (fr) 2017-11-21 2018-11-20 Traitement d'infections parasitaires touchant les surfaces des poissons

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PCT/EP2018/081923 A-371-Of-International WO2019101739A1 (fr) 2017-11-21 2018-11-20 Traitement d'infections parasitaires touchant les surfaces des poissons

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EP (1) EP3713419A1 (fr)
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WO (1) WO2019101739A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112889717A (zh) * 2021-02-24 2021-06-04 中山大学 一种利用罗非鱼生物防控刺激隐核虫感染的方法

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
NL2029375B1 (en) * 2021-10-11 2023-05-04 Univ Santiago Compostela Treatment of parasitic infections of fish
CA3232499A1 (fr) 2021-10-11 2023-04-20 Malte Jarlgaard HANSEN Procede d'elimination, d'inactivation ou d'inhibition d'algues ou d'algues bleu vert nuisibles susceptibles de provoquer une proliferation d'algues nuisibles (hab)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE10328003B4 (de) * 2003-01-27 2010-02-04 Alpha-Biocare Gmbh Mittel gegen einzellige Parasiten von Fischen

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112889717A (zh) * 2021-02-24 2021-06-04 中山大学 一种利用罗非鱼生物防控刺激隐核虫感染的方法

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CN111787937A (zh) 2020-10-16
JP2021510379A (ja) 2021-04-22
JP7422669B2 (ja) 2024-01-26
EP3713419A1 (fr) 2020-09-30
WO2019101739A1 (fr) 2019-05-31
US20230107426A1 (en) 2023-04-06

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