Classifications

• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N59/00—Biocides, pest repellants or attractants, or plant growth regulators containing elements or inorganic compounds
  • A01N59/14—Boron; Compounds thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K33/00—Medicinal preparations containing inorganic active ingredients
  • A61K33/22—Boron compounds

Definitions

• Embodiments of the present disclosure relate to methods for administering an antimicroorganism composition to an aquatic animal to treat chitin-containing microorganism infection or colonization.
• the methods may include dissolving an anti- microorganism composition in a carrier to give an anti-microorganism solution, and contacting an aquatic animal with the antimicroorganism solution.
• the anti-microorganism composition may include boric acid (BA).
• kits for administering an antimicroorganism composition to an aquatic animal to treat chitin-containing microorganism infection or colonization may include a container having the antimicroorganism composition including boric acid (BA), and package material that may include instructions directing the administering the anti-microorganism composition to the aquatic animal to treat the chitincontaining microorganism infection or colonization.
• the antimicroorganism composition is dissolved in a carrier to give an anti-microorganism solution.
• Embodiments of the present disclosure also relate to methods for treating chitincontaining microorganism infection or colonization on an aquatic animal.
• the methods may include contacting the aquatic animal with marine or freshwater containing BA in a sufficient quality to inhibit proliferation of the chitin-containing microorganism.
• Embodiments of the present disclosure also relate to systems for farming an aquatic animal.
• the systems may include marine or freshwater containing BA in a sufficient quality to inhibit proliferation of chitin-containing microorganisms.
• the aquatic animal may be selected from the group consisting of a fish, an aquatic mammal, an aquatic bird, an aquatic reptile, an amphibian, and an aquatic invertebrate. In certain embodiments, the aquatic animal may be a fish.
• the fish may be a farmed fish including at least one of a brown trout, an Atlantic salmon, a rainbow trout, a coho salmon, a channel catfish, a pike, an arctic char, an eel, a roach, a carp, a sturgeon, a kissing gourami, a guppy, a swordfish, a tilapia, or a platyfish, or any fish kept as pet-fish in aquaria, or zebrafish kept in experimental units or as pet fish.
• the fish may include at least one of a fish egg, a Juvenile fish, a fry, a fingerling, an adult fish or an off-spring of the fish.
• the aquatic animal may have the chitin-containing microorganism infection or colonization.
• the chitincontaining microorganism infection may be associated with an oomycete including at least one of a Saprolegnia, an Aphanomyces, or Branchiomyces.
• the chitin-containing microorganism infection may be associated with a Saprolegnia.
• the chitin-containing microorganism infection may be associated with an ectoparasites.
• the ectoparasites comprises a sea louse.
• the sea louse may be a sea louse of at least one of a genera Lepeophtheirus, Caligus, Caligus rogercresseyi, Caligus clemensi, Caligus chiastos, Caligus epidemicus, Caligus elongates, or Lepeophtheirus salmonis.
• the BA in the anti-microorganism solution may have a concentration in the antimicroorganism solution of between about 0.2 g/L and 1 g/L. In particular embodiments, the BA in the anti-microorganism solution may have a concentration in the anti- microorganism solution of between about 0.1 g/L and 0.2 g/L. In particular embodiments, the BA in the antimicroorganism solution may have a concentration in the anti- microorganism solution that is not less than about 0.2 g/L.
• the BA in the antimicroorganism solution may have a concentration in the antimicroorganism solution of between about 0.2 g/L and 0.5 g/L. In particular embodiments, the BA in the antimicroorganism solution may have a concentration in the anti-microorganism solution of between about 0.5 g/L and 1 g/L.
• the anti-microorganism solution may also contain (propionic acid) PA, and a concentration of PA in the anti-microorganism solution of not less than 0.2 g/L.
• the anti-microorganism solution may contain PA, and a concentration of PA in the antimicroorganism solution of between about 0.2 g/L and 1 g/L.
• the contacting the aquatic animal with the antimicroorganism solution may include contacting the aquatic animal with the anti-microorganism solution for a period of time of between about 24 hours and 96 hours.
• the contacting the aquatic animal with the anti-microorganism solution may include contacting the aquatic animal with the anti-microorganism solution for a predetermined number of times within 24 hours. In these instances, the predetermined number may be at least one
• the methods may include removing the aquatic animal from the anti-microorganism solution.
• the methods may also include contacting the aquatic animal with water not containing the anti-microorganism composition.
• FIG. 1 shows effect of boric acid on the germination and colonization of
• FIG. 2 shows Saprolegnia zoosporangia in the non-treated control before (al) and after (a2) spore release.
• FIG. 3 is fluorescence microscopy showing the viability of boric acid treated Saprolegnia spores and zoosporangia with SYTO 9 stain.
• FIG. 4 shows microscopical examination of infected dead eggs used as a source of infection after the termination of the continuous exposure experiment.
• FIG. 5 illustrates mortality, infection, hatching rates and fry viability in boric acid treated groups and controls at the end of the first experiment (Continuous exposure).
• FIG. 6 shows microscopical examination of the infected dead eggs, used as a source of infection, by the end of the intermittent exposure experiment.
• FIG. 7 illustrates mortality, infection, hatching rates and number of live fry obtained by the end of the experiment in boric acid treated groups and nontreated control in response to intermittent exposure to BA.
• FIG. 8 illustrates mortality, infection, hatching rates and fry viability in egg groups intermittently exposed to boric acid (third experiment) compared to non-treated control in response to intermittent exposure to BA.
• FIG. 9 illustrates mortality patterns recorded every other day among treated salmonid yolk sac fry and control ones during the outbreak of saprolegniosis.
• FIG. 10 illustrates cumulative mortalities in salmonid yolk sac fry in both boric acid treated and non-treated control groups by the end of the natural outbreak of saprolegniosis.
• Embodiments of the present contemplate a use of boric acid (BA) or combination of boric acid and propionic acid (PA) for treating chitincontaining microorganism (e.g., Saprolegnia, sea lice, and etc.) infection or colonization.
• BA boric acid
• PA propionic acid
• the present disclosure relates, in part, to the demonstration that boric acid or combination of BA and PA can inhibit germination and growth of Saprolegnia and decrease survival rates of sea lice.
• BA may decrease Saprolegnia spore activity and mycelial growth in certain low concentrations, and completely inhibit germination and growth of Saprolegnia in certain high concentrations.
• the articles “a” and “an” are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article.
• an element means one element or more than one element.
• “about” is meant a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
• aquatic animal refers to any animal that spends all or some of the life in marine or fresh water.
• An "aquatic animal” can be, but is not limited to, a mammal such as a seal, sea lion, walrus, manatee, dugong, porpoise, dolphin, cetaceous or non-cetaceous whale, otter, or beaver; a bird such as, but not limited to, a web-footed bird such as a duck, goose, swan, gull, cormorant, penguin, a wading bird such as a coot, moor hen, flamingo, stork, heron; an aquatic reptile such as, but not limited to, an alligator, cayman, crocodile, turtle, snake or lizard; an amphibian such as, but not limited to, frogs, toads, newts and salamanders, neotenous larva or larvae thereof; fish
• carrier refers to any pharmaceutically acceptable solvent of antibiotics, chelating agents and pH buffering agents that will allow the antimicrobial composition of the present disclosure to be administered directly to an aquatic animal.
• a “carrier” as used herein therefore, refers to such solvent as, but is not limited to, water, saline, physiological saline, ointments, creams, oil-water emulsions or any other solvent or combination of solvents and compounds known to one of skill in the art that is pharmaceutically and physiologically acceptable to the recipient animal.
• carrier further includes vitamin E or the like that may comprise an oily film over the site of application on the surface of an animal.
• derivative is meant a chemical compounds (e.g., borates) that has been derived from the basic structure (e.g., boric acid) by any reaction, for example, by conjugation or complexing with other chemical moieties and/or structures.
• basic structure e.g., boric acid
• fish refers to any marine or freshwater fish species maintained in a tank, aquarium, pool, pond, aquaculture facility, fish farm, or any means other than the natural environment of the fish species.
• fish also refers to species and individuals thereof captured, rescued or taken from their native habitat and which may require treatment for microbial infestations.
• Fish species to which the methods of the present disclosure may be applied include, but are not limited to, ornamental fish, zebrafish, goldfish, koi, oscar, cichlids, tropical fish and fish for human or animal food such as, but not limited to, catfish, and salmonids such as trout, or salmon.
• examples of fish may include a brown trout, an Atlantic salmon, a rainbow trout, a coho salmon, a channel catfish, a pike, an arctic char, an eel, a roach, a carp, a sturgeon, a kissing gourami, a guppy, a swordfish, or a platyfish.
• Fish also refers to a fish of different stages between birth and adulthood, for example, eggs, juvenile fish, growing fish or a mature fish.
• disease refers to a pathological condition recognizable as an abnormal condition of an animal.
• a "fish disease” is a pathological condition of fish that may be fatal or benign such as, but not limited to, ulcers, fin rot, dropsy, Malawi bloat disease, gill disease and columnaris, Saprolegnia infections, see louse infections, or saddlepatch disease.
• anti-viral response level in response to viral infection is decreased relative to an unmodified or differently modified cell by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500%, or at least 1000%.
• An "increased” or “enhanced” amount is typically a "statistically significant” amount, and may include an increase that is 1.1, 1.2, 1.3, 1.4, 1.5, 1.6 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, or 50 or more times (e.g., 100, 500, 1000, 10 000 times or more) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) an amount or level described herein.
• a “decreased,” “inhibiting,” or “reduced” or “lesser” amount is typically a “statistically significant” amount, and may include a decrease that is about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, or 50 or more times (e.g., 100, 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) an amount or level described herein.
• germination or colonization associated with Saprolegnia spores in marine or freshwater containing BA is decreased relative to marine or freshwater without containing BA by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500%, or at least 1000%.
• marine or “freshwater” refer to the natural environment of an aquatic animal.
• marine refers to any environment relating to the oceans or seas wherein the water is saline.
• freshwater refers to, but is not limited to, lakes, ponds, rivers, streams, brooks or any other low salinity water.
• statically significant it is meant that the result was unlikely to have occurred by chance. Statistical significance can be determined by any method known in the art.
• p-value which is the frequency or probability with which the observed event would occur, if the null hypothesis were true. If the obtained p-value is smaller than the significance level, then the null hypothesis is rejected. In simple cases, the significance level is defined at a p-value of 0.05 or less.
• substantially or “essentially” means nearly totally or completely, for instance, 95%, 96%, 97%, 98%, 99% or greater of some given quantity.
• treatment is defined as the application or administration (e.g., a bath) of a therapeutic agent to a subject (e.g. a fish), or application or administration of the therapeutic agent to an isolated tissue (e.g., fish eggs) or cell line from a patient, who has a disease (e.g., Saprolegnia infections), a symptom of disease or a predisposition toward a disease, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease, the symptoms of disease, or the predisposition toward disease.
• a disease e.g., Saprolegnia infections
• Treatment of a subject in whom no symptoms or clinically relevant manifestations of a disease or disorder have been identified is preventive or prophylactic therapy, whereas clinical, curative, or palliative "treatment” of a subject in whom symptoms or clinically relevant manifestations of a disease or disorder have been identified generally does not constitute preventive or prophylactic therapy.
• Treatment of Saprolegnia infections in a fish includes inhibiting or preventing colonization of infectious stages of Saprolegnia or killing of infectious and/or multiplying stages of Saprolegnia like hyphae.
• Treating Saprolegnia infections in a fish and preventing Saprolegnia infection progression can include alleviating or preventing symptoms, disorders or clinical disease associated with Saprolegnia infections, thereby curing an infection and restituting the health of the fish or through prophylactic treatment, prevent clinical manifestation of disease to occur.
• Each form of treatment may be considered a distinct aspect of the disclosure.
• chitin-containing microorganism infection refers to any pathological or non-pathological presence of at least a chitin-containing microorganism on or in an aquatic animal, and which may be treated by an antimicroorganism composition containing bath or dip of the animal.
• chitin-containing microorganism colonization refers to microorganisms carrying chitin (deacetylated chitosan) as part of their cell membrane or their exoskeleton.
• chitin-containing microorganism refers to organisms carrying a coat, membrane, cell wall or exoskeleton containing chitin or chitosan-derived material, for example, Saprolegnia or Sea louse.
• microorganism refers to any bacteria, fungus, oomycete or Arthropode, for example, Saprolegnia or Sea louse.
• boric acid refers to an organic compound with the formula H3BO3 or any chemical compound containing parts or traces of H3BO3
• propionic acid refers to an organic compound with the formula CH3CH2COOH or any chemical compound containing parts or traces of propionic acid
• Embodiments of the present disclosure relate to methods for administering an antimicroorganism composition to an aquatic animal to treat chitin-containing microorganism infection or colonization.
• the methods may include dissolving an anti- microorganism composition in a carrier to give an anti-microorganism solution, the anti- microorganism composition including boric acid (BA), and contacting an aquatic animal with the antimicroorganism solution.
• BA boric acid
• Embodiments of the present disclosure also relate to kits for administering an antimicroorganism composition to an aquatic animal to treat chitin-containing microorganism infection or colonization.
• the kits may include a container having the antimicroorganism composition including boric acid (BA), the anti-microorganism dissolved in a carrier to give an anti-microorganism solution, and package material that may include instructions directing the administering the anti-microorganism composition to the aquatic animal to treat the chitin-containing microorganism infection or colonization.
• Embodiments of the present disclosure also relate to methods for treating chitincontaining microorganism infection or colonization on an aquatic animal. The methods may include contacting the aquatic animal with marine or freshwater containing BA in a sufficient quality to inhibit proliferation of the chitin-containing microorganism.
• Embodiments of the present disclosure also relate to systems for farming an aquatic animal.
• the systems may include marine or freshwater containing BA in a sufficient quality to inhibit proliferation of chitin-containing microorganisms.
• the aquatic animal may be selected from the group consisting of a fish, an aquatic mammal, an aquatic bird, an aquatic reptile, an amphibian, and an aquatic invertebrate. In certain embodiments, the aquatic animal may be a fish.
• the fish may be a farmed fish including at least one of a brown trout, an Atlantic salmon, a rainbow trout, a coho salmon, a channel catfish, a pike, an arctic char, an eel, a roach, a carp, a sturgeon, a kissing gourami, a guppy, a swordfish, or a platyfish or fish kept as pet-fish, or zebrafish kept as petfish or for medical research.
• the fish may include at least one of a fish egg, a Juvenile fish, a fry, a fingerling, an adult fish or an off-spring of the fish.
• the aquatic animal may have the chitincontaining microorganism infection or colonization.
• the chitin-containing microorganism infection may be associated with an oomycete including at least one of a Saprolegnia, an Aphanomyces, or Branchiomyces.
• the chitincontaining microorganism infection may be associated with a Saprolegnia.
• the chitin-containing microorganism infection may be associated with an ectoparasites.
• the ectoparasites comprises a sea louse.
• the sea louse may be a sea louse of at least one of a genera Lepeophtheirus, Caligus, Caligus rogercresseyi, Caligus clemensi, Caligus chiastos, Caligus epidemicus, Caligus elongates, or Lepeophtheirus salmonis.
• the BA in the anti-microorganism solution may have a concentration in the anti-microorganism solution of between about 0.2 g/L and 1 g/L. In particular embodiments, the BA in the anti-microorganism solution may have a concentration in the anti-microorganism solution of between about 0.1 g/L and 0.2 g/L. In particular embodiments, the BA in the anti-microorganism solution may have a concentration in the antimicroorganism solution that is not less than about 0.2 g/L. In particular embodiments, the BA in the anti-microorganism solution may have a concentration in the anti- microorganism solution of between about 0.2 g/L and 0.5 g/L. In particular embodiments, the BA in the anti-microorganism solution may have a concentration in the anti- microorganism solution of between about 0.5 g/L and 1 g/L.
• BA may decrease Saprolegnia spore activity and mycelial growth at concentrations more than 0.2 g/L, and BA may completely inhibit germination and growth of Saprolegnia at a concentration of lg/L.
• BA may also control saprolegniosis on Atlantic salmon eyed eggs at concentrations ranging from 0.2-1.4 g/L during continuous exposure, and at 1.0-4.0 g/L during intermittent exposure.
• BA may also control saprolegniosis on salmon yolk sac fry at a concentration of 0.5 g/L boric acid during intermittent exposure in response to a natural outbreak of saprolegniosis.
• Treatment of sea lice copepodits with 0.4 g/L of boric acid may reduce swimming activity and lower survival rate of the sea lice.
• the anti-microorganism solution may also contain (propionic acid) PA, and a concentration of PA in the anti-microorganism solution of not less than 0.2 g/L.
• the antimicroorganism solution may contain PA, and a concentration of PA in the antimicroorganism solution of between about 0.2 g/L and 1 g/L.
• the contacting the aquatic animal with the antimicroorganism solution may include contacting the aquatic animal with the anti-microorganism solution for a period of time of between about 24 hours and 96 hours.
• the contacting the aquatic animal with the anti-microorganism solution may include contacting the aquatic animal with the anti-microorganism solution for a predetermined number of times within 24 hours. In these instances, the predetermined number may be at least one of 1, 2, 3, 4, 5, 6, or 8.
• the aquatic animal may be removed from the antimicroorganism solution.
• the aquatic animal may then contacted with water not containing the anti-microorganism composition.
• Chitin is a long-chain polymer of a N-acetylglucosamine, derived from glucose. Chitin is the main component of the cell walls of fungi and oomycetes (e.g., Saprolegnia sp.) that infect freshwater fish, the exoskeletons of arthropods (e.g., crustaceans and copepods). For example, Caligidae parasites of fish (i.e., external parasites) can be found on mucus and skin of wild and farmed fish. The structure of chitin is comparable to the polysaccharide cellulose and forms crystalline nanofibrils. By function it may be compared to the protein keratin.
• Chitin is the building block of exoskeletons that is the external skeleton that supports and protects the body of an animal like grasshoppers and cockroaches, crustaceans (e.g., crabs and lobsters), and arthropods (e.g., copepods).
• arthropods may include Caligidae parasites of fish (e.g., sea louse (Lepeophtheirus salmonis) of salmonids and Caligus sp. of fish).
• Exoskeletons contain rigid and resistant components that fulfil a set of functional roles including protection, excretion, sensing, support, feeding and acting as a barrier against desiccation in terrestrial organisms. Exoskeletons have a role in defense from pests and predators, support, and in providing an attachment framework for musculature. Exoskeletons contain in addition to chitin also calcium carbonate which makes them harder and stronger.
• Saprolegnia spp. are generally termed "watermolds" and share common features with fungi and algae.
• the term saprolegniasis refers to any disease of fishes or fish eggs caused by species of the family Saprolegniaceae (Oomycotina). Symbiotic associations between fish and Saprolegnia spp. have been known for decades and the first description dates back to 1748 where saprolegniasis was reported in roach (Rutilus rutilus L.) in England. Since then saprolegniasis has been detected in a growing number of species in or on various fishes from all over the world. Particular interest has been paid from countries and regions with growing aquaculture industry since saprolegniasis causes high economic losses in fish and ova.
• Saprolegnia infection is traditionally known as "fungal infection” in fish, and is typical seen in the freshwater stage of salmonids. Saprolegnia infections are visible to the naked eye as white patches on the skin of the fish or as "cotton wool” on fish eggs. The "fungal" patches may consist of one or more species of Saprolegnia and may become grayish due to the presence of bacteria and debris.
• the disease was previously controlled by the use of malachite green, an organic dye which has proved very efficient in controlling all infectious stages of Saprolegnia spp. The use of malachite green has been banned in Norway and other parts of the world due to its potential toxicological effects. This has increased the incidence of Saprolegnia infections in the aquaculture all over the world, resulting in huge economic losses.
• Saprolegnia spp. belong to the class Oomycetes, which is a group of fungi-like pathogens in the kingdom Straminiphila. Oomycetes have their phylogenetic roots with the chromophyte algae (which includes the diatoms, chrysophytes and brown seaweeds) rather than with the main evolutionary line of chitin containing fungi.
• the Oomycetes are subdivided in orders and comprises several pests, like Phytophthora infestans causing the potato late blight, Aphanomyces astasi causing crayfish plaque, several fish pathogens from the genera Aphanomyces, Achlya and Saprolegnia and at least one species (Pythium insidiosum) with the potential of infecting humans and other mammals.
• Phytophthora infestans causing the potato late blight
• Aphanomyces astasi causing crayfish plaque
• several fish pathogens from the genera Aphanomyces Achlya and Saprolegnia
• at least one species Pythium insidiosum
• Taxonomic classification of Saprolegnia is shown in Table 1. Table 1: Taxonomic classification of Saprolegnia
• Saprolegnia spp. are characterized by the growth of delicate, nonseptate hyphae and asexual reproduction by secondary zoospore discharge one by one in rapid succession through one exit pore in the sporangium.
• 8-10 individual species have been implicated as causing saprolegniasis of salmonid fishes (Hughes 1994), including S.australis, S.delica, S.diclina, S.ferax, S.monoica, S.parasitca, S.salmonis, S.shikotsuensis and S.tortulosa.
• Saprolegnia species are the fish pathogenic Saprolegnia parasitica and the less pathogenic species Saprolegnia diclina.
• Saprolegnia species are separated by the presence of sexual reproduction and characteristics of the gonads, i.e. the oogonia and the antheridia.
• the taxonomy is complex, and in particular the so-called "Saprolegnia parasitica-diclina complex" has led to a lot of confusion.
• Saprolegnia species that grows on fish as parasites do not normally produce sexual structures during laboratory conditions. Absence of sexual reproductive structures one of the primary distinguishing characteristics of Saprolegnia parasitica.
• Saprolegnia parasitica any Saprolegnia growing on a living fish, and not producing sexual reproductive structures, is, by definition, Saprolegnia parasitica.
• Saprolegnia species isolated from fish and with clusters of longspines in the secondary zoospore should be termed Saprolegnia parasitica as a practical approach.
• Saprolegnia is homothallic, meaning that one single individual contains both male and female sex organs.
• the male and the female sex organs, the antheridium and the oogonium, respectively, are developed from the hyphae.
• Meiosis occurs to produce male nuclei and female eggs.
• the antheridia grow toward the oogonia and produce fertilization tubes that penetrate the oogonia. Fertilization occurs when the male nuclei travel down these tubes to the female eggs and fuse with the female nuclei. This produces several thick-walled zygotes, called oospores.
• the number of oospores per oogonium is not constant, ranging from one to four in some species, to over fourty in others. Each oospore germinates into a new hypha which will produce a zoosporangium. From the zoosporangium the asexual reproduction, which is the main type of reproduction, occurs.
• the pyriform primary zoospores, which are released from the zoosporangia are weak swimmers and function simply to disperse the spores from the immediate vicinity of the sporangium and parent colony. These primary zoospores settle down to produce 5-10 ⁇ thin-walled cysts.
• This primary cyst acts as a miniature sporangium and releases a reniform secondary zoospore, which is the main motile stage.
• the secondary zoospore can maintain motility for many hours, even days, until it also encysts to produce a secondary cyst (syn. "encysted zoospore", “zoospore cyst” or “cystospore”). Then, they usually germinate into a new mycelium, on which sexual reproduction occurs, thus starting the reproduction cycle anew.
• the secondary cysts can also release new secondary-like zoospores which are able to encyst again.
• Polyplanetism contributes to the fungus ' pathogenicity by helping it to make several attempts locating a suitable culture medium to live on before settling down for good. After they have encysted the secondary zoospores develop hairs for attachment. It has been suggested that these hairs are also used for buoyancy to decrease sedimentation rate and for fungal-host recognition response and are important for pathogenesis.
• Saprolegnia spp. are distributed worldwide in rivers and freshwater reservoirs. Over the last decades, saprolegniasis have been reported frequently from literally all continents. I nternational transfer of fish and eggs as part of the aquaculture industry is a possible risk factor when it comes to spread of different Saprolegnia species and strains throughout the world.
• Saprognia infection is the single largest cause of economic losses in aquaculture and worldwide this disease is second only to bacterial diseases in economic importance. Fifty percent per year losses have been reported in elver (Angullia anguilla) and in coho salmon (Oncorhynchus kisutch) culture in Japan. I n the south-eastern United States, major financial losses occur in channel catfish farming due to a condition called "winter kill" caused by Saprolegnia infections.
• Saprolegnia parasitica form cotton-wool-like tufts on the integument.
• the early lesions are grey-white in colour and normally appear as circular colonies.
• the lesions are not randomly located, as head, fins and gills are more susceptible to infection.
• moribund fish may in severe cases have as much as 80% of the body surface area covered by Saprolegnia.
• Virulent strains of Saprolegnia spp. can cause very high mortalities (up to 100%) in many different salmonid species, and hyphae from Saprolegnia spp. are usually restricted to the integument and superficial musculature.
• Histopatological changes beneath the superficial mycelia mat include dermal necrosis and oedema during the early stage, and deeper myofibrillar necrosis and extensive haemorrhage in the more progressive lesions.
• the tissue damage is probably caused by extracellular enzymes secreted by the advancing hyphae including hyphae penetration of the basement membrane.
• any Saprolegnia species produces toxins which could cause cellular damage at sites remote from the sites of hyphal invasion. It is generally accepted that the ultimate cause of death is the severe haemodilution caused by haemorrhage and by the destruction of the waterproof properties of the fish integument.
• Saprolegnia spp. may infect both fish and ova of all types of salmonids.
• the economic importance of brown trout (Salmo trutta), Atlantic salmon (Salmo salar L), rainbow trout (Oncorhynchus mykiss (Walbaum)) and coho salmon (Oncorhynchus kisutch) is the main reason that saprolegniasis has been subject to such strong focus in these species.
• Saprolegnia spp. can also infect a number of other teleost species.
• the sea louse (plural sea lice) is a copepod within the order Siphonostomatoida, family Caligidae. There are more than 550 species in 37 genera, including approximately 162 Lepeophtheirus and 268 Caligus species. Sea lice are marine ectoparasites (external parasites) that feed on the mucus, epidermal tissue, and blood of host marine fish. The genera Lepeophtheirus and Caligus parasitize marine fish, in particular those species that have been recorded on farmed salmon. Lepeophtheirus salmonis and various Caligus species are adapted to saltwater and are major ectoparasites of farmed and wild Atlantic salmon. Several antiparasitic drugs have been developed for control purposes. Since L.
• salmonis is the major sea louse of concern and has the most known about its biology and interactions with its salmon host. Caligus rogercresseyi has become a major parasite of concern on salmon farms in Chile. Recent evidence is also emerging that L. salmonis in the Atlantic Ocean has sufficient genetic differences from L. salmonis from the Pacific, showing that Atlantic and Pacific L. salmonis may have independently co-evolved with Atlantic and Pacific salmonids.
• L. salmonis has high specificity for salmonids, like farmed Atlantic salmon (Salmo salar). Lepeophtheirus salmonis can to some degree parasitize other salmonids, like brown trout (sea trout, Salmo trutta), arctic char (Salvelinus alpinus), and Pacific salmon species. Pacific L. salmonis can also develop on three-spined stickleback (Gasterosteus aculeatus) the life-cycle will not be completed. Temperature, light and currents depend the survival of sea lice (at different stages). Sea lice cannot live in freshwater and die and fall off anadromous fish such as salmonids as they return to freshwater. Atlantic salmon migrate to and swim upstream in the fall to reproduce, while the smolts return to saltwater the second spring.
• Lepeophtheirus salmonis is approximately twice the size of most Caligus spp. (e.g. C. elongatus, C. clemensi, etc.).
• the body of sea lice consists of 4 regions: cephalothorax, fourth leg-bearing segment, genital complex, and abdomen. All species of lice have mouth parts shaped as a siphon or oral cone.
• the second antennae and oral appendages are modified to hold the parasite on the fish (attached stages).
• the adult females are significantly larger than males and develop a large genital complex which makes up the majority of the body mass.
• Two egg strings of 500 to 1000 eggs that get darker as it matures are approximately the same length as the female's body.
• Sea lice cause physical and enzymatic damage at attachment sites resulting in abrasion-like lesions that vary in severity depending and size. It is not clear whether stressed fish are particularly prone to infestation. Sea lice infection causes a generalized chronic stress response in fish. This can decrease the immune responses and render fish more susceptible to other diseases. Infection also impacts growth. The degree of damage is also dependent on the species of sea lice, the developmental stages that are present, and the number of sea lice on a fish.
• BA H3BO3, M 61.83 g/mol
• BA was used as a source for borate to test its effect on the germination of Saprolegnia spores and the growth of Saprolegnia hyphae (in vitro).
• BA was also tested for control of Saprolegnia infection of eyed salmon eggs and yolk sac fry (in vivo) and its safety in vivo was also assessed.
• Propionic acid PA; CH3CH2COOH
• BA was diluted to desired concentration in Sterilized aquarium Water (SAW). It was tested initially at 0.01, 0.1 and 1.0 g /L to find out the preliminary minimum inhibitory concentration (MIC) intervals in vitro. Once the concentration range able to inhibit Saprolegnia spore germination and arrest hyphae growth was determined, the test intervals were narrowed down and concentrations between 0.1 and 1.0 g/L were tested at incremental increase of O.lg/L.
• SAW Sterilized Aquarium Water
• Table 2 Grades for the germination and colonization of Saprolegnia spores on sesame seeds Grade 0: no germination no growing mycelia on the sesame seeds
• FIG. 1 (a) Grade 3: Profuse Saprolegnia hyphal growth on sesame seeds in the non- treated control group (water).
• Grade 3 germinating spores with mycelial growth on the sesame seeds (boric acid ⁇ 0.2 g/L).
• Grade 2 reduced germination rate and minimal mycelial growth on the sesame seeds (boric acid 0.2- 0.4 g/L).
• Grade 1 germinating spores without growing mycelia on the sesame seeds (boric acid 0.50.9 g/L).
• Grade 0 no germinating spores, no growing mycelia on the sesame seeds (boric acid >1 g/L).
• Grade 0 positive control group (bronopol), no spore germination with absence of the growing mycelia on sesame seeds.
• FIG. 3 is a fluorescence microscopy showing the viability of boric acid treated Saprolegnia spores and zoosporangia with SYTO 9 stain.
• FIG. 3 c-1 and c-2) Growing hyphae were treated for 24h with BA and left in SAW for another 24h after treatment was terminated. Thin hyphae can be seen projecting out from seeds in c-1.
• FIG. 3c-2 shows a close-up of zoosporangium treated as in FIG. 3 c-1.
• FIG. 3 d-1 and d2) Growing hyphae were treated for 96h in BA and then left in SAW for 24h after treatment was terminated. There is no colonization on seeds after 96h BA treatment and the zoosporangium FIG. 3 (d-2) appears with condensed staining and a thin wall.
• Results obtained during the second week were similar to what seen during the first week where no infection was recorded in any of the concentrations tested except 0.2 g/L. At this concentration, slight attachment was observed between the egg-shell of the live eggs and the infected dead egg (source of infection). This did not cause the foetus to die as eggs hatched and yolk sac fry swam away from the eggshell.
• VIO 2741 parasitica (VIO 2741) were packed in a piece of gauze and fixed inside the main tubes that supplied the water to all containers.
• Treatment with BA was carried out as follows. The water flow was stopped and each group, except the non-treated control group, was treated with BA at concentrations of 1, 2, 3, and 4 g/L. The treatment period was 4 hours/day after which water flow was resumed. Dead eggs were inspected daily over 14 days for their colonization with Saprolegnia spores and the presence of growing hypha. Number of dead eggs per group and hatching rates were recorded.
• FIG. 6 demonstrates microscopical examination of the infected dead eggs, used as a source of infection, by the end of the intermittent exposure experiment.
• Non-treated control group FIG. 6 (a) compared to treated ones intermittently exposed to boric acid treatment at different concentrations
• the abundance of the growing mycelia in the non-treated control and colonization of neighboring ones result in death of the neighboring ones.
• a method for administering an anti-microorganism composition to an aquatic animal to treat chitin-containing microorganism infection or colonization comprising: dissolving an anti-microorganism composition in a carrier to provide an antimicroorganism solution, the anti-microorganism composition including boric acid (BA); and contacting an aquatic animal with the anti-microorganism solution.
• a kit for administering an anti-microorganism composition to an aquatic animal to treat chitin-containing microorganism infection or colonization comprising: a container having the anti-microorganism composition including boric acid (BA), the antimicroorganism composition dissolvable in a carrier to provide an anti-microorganism solution; and package material that includes an instruction directing administering of the anti-microorganism composition to the aquatic animal to treat the chitincontaining microorganism infection or colonization .
• BA boric acid
• the fish is a farmed fish including at least one of a brown trout, an Atlantic salmon, a rainbow trout, a coho salmon, a channel catfish, a pike, an arctic char, an eel, a roach, a carp, a sturgeon, a kissing gourami, a guppy, a swordfish, or a platyfish.
• sea louse is a sea louse of at least one of a genera Lepeophtheirus, Caligus, Caligus rogercresseyi, Caligus clemensi, Caligus chiastos, Caligus epidemicus, Caligus elongates, or
• the antimicroorganism solution contains propionic acid (PA), and a concentration of the PA in the anti-microorganism solution of not less than about 0.2 g/L.
• PA propionic acid
• antimicroorganism solution contains propionic acid (PA), and a concentration of the PA
• PA in the anti-microorganism solution of between about 0.2 g/L and about 1 g/L. 20. The method or kit of any one of embodiments 1 and 2, wherein the contacting the aquatic animal with the anti-microorganism solution comprises contacting the aquatic animal with the anti-microorganism solution for a period of time of between about 24 hours and about 96 hours.
• a method for treating chitin-containing microorganism infection or colonization on an aquatic animal comprising: contacting the aquatic animal with marine or fresh water containing boric acid (BA)in a sufficient quality to inhibit proliferation of the chitincontaining microorganism.
• a system for farming an aquatic animal comprising: a farming system for farming the aquatic animal, the farming system including marine or fresh water containing boric acid (BA) in a sufficient quality to inhibit proliferation of chitin-containing microorganisms.
• the fish is a farmed fish including at least one of a brown trout, an Atlantic salmon, a rainbow trout, a coho salmon, a channel catfish, a pike, an arctic char, an eel, a roach, a carp, a sturgeon, a kissing gourami, a guppy, a swordfish, or a platyfish.
• sea louse is a sea louse of at least one of a genera Lepeophtheirus, Caligus, Caligus rogercresseyi, Caligus clemensi, Caligus chiastos, Caligus epidemicus, Caligus elongates, or Lepeophtheirus salmonis.

Landscapes

• Life Sciences & Earth Sciences (AREA)
• Health & Medical Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Inorganic Chemistry (AREA)
• General Health & Medical Sciences (AREA)
• Veterinary Medicine (AREA)
• Pest Control & Pesticides (AREA)
• Animal Behavior & Ethology (AREA)
• Pharmacology & Pharmacy (AREA)
• Public Health (AREA)
• Medicinal Chemistry (AREA)
• Agronomy & Crop Science (AREA)
• Epidemiology (AREA)
• Plant Pathology (AREA)
• Engineering & Computer Science (AREA)
• Dentistry (AREA)
• Wood Science & Technology (AREA)
• Zoology (AREA)
• Environmental Sciences (AREA)
• Farming Of Fish And Shellfish (AREA)
• Fodder In General (AREA)

Applications Claiming Priority (1)

Publications (1)

Family

ID=54014767

Family Applications (1)

Country Status (3)

• 2015
  • 2015-05-19 EP EP15756844.5A patent/EP3297441A1/de not_active Withdrawn
  • 2015-05-19 WO PCT/EP2015/061029 patent/WO2016184507A1/en active Application Filing
  • 2015-05-19 CA CA2986275A patent/CA2986275A1/en not_active Abandoned

Also Published As

Similar Documents

Legal Events