Abstract:
A method of treating protozoan infections in fish comprising introducing a sufficient quantity of one or more photoactive dyes to an aqueous environment containing one or more fish infected with protozoa such that the resulting concentration of the one or more photoactive dyes in the aqueous environment is toxic to at least some of the protozoa.

Description:
RELATED APPLICATION DATA 
     The present application claims priority from U.S. Provisional Patent Application No, 60/223,915, titled “METHODS OF TREATMENT OF PROTOZOAN INFECTIONS IN FISH”, filed on Aug. 9, 2000, the entire contents of which are incorporated by reference herein. 
    
    
     FIELD OF THE INVENTION 
     The present invention relates to the field of aquaculture. More particularly, the present invention relates to the field of the therapeutic treatment of parasitic infections in fish. More particularly to the use of photoactive dyes such as Phloxine B as a method of controlling protozoan infections in fish populations. More particularly, the invention relates to the use of photoactive dyes such as Phloxine B for the treatment and/or prevention and of parasitic diseases in fish populations such as the disease Ick which is caused by the infection of fish with the external protozoan,  Ichthyophthirius multifiliis.    
     BACKGROUND OF THE INVENTION 
     Aquaculture in the United States represents a relatively small segment of agricultural production, however the industry is relatively young and growing rapidly. Per capita consumption of fish food products in the United States has increased more than 50 percent since 1970. Furthermore, ornamental fish distribution has more than doubled since 1986. Food fish and ornamental fish markets combined contribute nearly $2 billion per year to the retail United-States economy. It has been estimated that these trends will continue in the new millenium. 
     Although the demand for food fish and ornamental fish products has increased, natural fish harvest populations have not increased and, in many cases, have declined. As such, aquaculture of fish species has increased to meet consumer demand. Among food fish, catfish, salmonid, and cichlid species dominate commercial aquaculture in the US. Catfish aquaculture, estimated at nearly $770 million in farm sales for 1999, accounts for about half of total annual US production. Among ornamental species, guppies, swordtails, and mollies dominate the ornamental fish market in the US. Ornamental aquaculture in Florida alone is valued at over $52 million. 
     Aquaculture of fish has been plagued by disease since its inception. Disease outbreaks are particularly prevalent when culturing large numbers of fish in crowded conditions. Many parasites and diseases can spread and quickly kill entire fish populations. Although the economic impact of infectious diseases is difficult to determine, it is estimated that annual losses in the US catfish industry may exceed $20 million. 
     A majority of fish losses in the aquaculture industry can be attributed to protozoan infections. Protozoans can infect both external and internal portions of the fish including the gills, fins, skin, and digestive organs. External protozoa of major concern to aquaculturists include members of the genus Costia, Chilodon, Scyphidia, Trichodina, Epistylis, Carchesium, and Trichophrya. The external ciliate,  Ichthyophthirius multifiliis , causes white spot disease known as Ick or Ich. Ick is difficult to control and is often observed in crowded cultures of catfish and warm-water aquarium fish. Characteristic signs of Ick infection include the presence of grayish-white warts on the external surfaces of the fish and behavior changes such as flashing, jumping, or thrashing erratically in the water. 
     The life cycle of  I. multifiliis  is indirect. An adult stage called a trophozoite invades the skin or gills of fish and feeds directly on tissue fluids. Trophozoites, the form of Ick found on fish, are large protozoans and can easily be distinguished by light microscopy. Trophozoites are identified by a clearly visible C-shaped nucleus. After several days, depending on water temperature, the trophozoite releases from the fish, encysts, and undergoes fission to produce the next stage of its life cycle. After the cyst ruptures, an infective form called a tomite is released. As many as 2,000 tomites can be produced from a single cyst. Tomites are highly ciliated, pear-shaped (30-45 nm diameter), and are actively mobile in seeking out a new host. Prior therapeutic efforts to control Ick infections have focused on treating free-swimming tomites and unattached trophozoites. These therapeutic treatments are ineffective in controlling Ick trophozoites once they have attached to the fish. 
     As of 1999, only five drugs have been approved by the FDA for use in the aquaculture industry. Use of these drugs for particular disease conditions is highly regulated particularly regarding the use of these drugs with food fish. Formalin, oxytetracycline, and sulfadimethoxine have been approved to treat catfish diseases. Although not approved for food fish, potassium permanganate and copper sulfate are also used to treat Ick infections of ornamental fish. Other non-approved drugs, including malachite green and quinine, have been demonstrated effective against Ick. Toxicity and safety concerns have hindered FDA approval of many of these treatments. Malachite green, for example, is both mutagenic and teratogenic, and its use is restricted in many countries. Formalin, although approved, has also been demonstrated as a potential carcinogen to fish. Other drugs, such as potassium permanganate, have been demonstrated to cause gill injury at effective concentrations. 
     There are arguably no effective long-term treatments for fish infected with the protozoan parasite,  Ichthyophthirus multifiliis . Prior treatments are ineffective in controlling the complete life cycle of Ick and have the potential to be harmful to fish or other animals, including humans. Current chemical treatments are costly and may also be labor intensive due to clean-up procedures required both before and after treatment. Last, available treatments are not practical for treating large numbers of infected fish such as those cultured in indoor hatcheries or in ponds. Phloxine B, by contrast, is non-toxic to most animal species, does not accumulate or pose an environmental threat, and is relatively inexpensive compared to other drug treatments. 
     The limited number of approved treatments for protozoan infections results in large production losses each year. Ick alone is responsible for nearly 50% all catfish losses reported during the Spring and Summer months of pond farm production and as mush as 80% of ornamental industry losses annually. The use of Phloxine B for treating Ick infections in fish may dramatically reduce these annual losses. Furthermore, the innovative use of a chemical currently approved by the FDA may reduce the need for extensive environmental and human safety testing before approval. 
     The limited number of approved and/or effective treatments for Ick infections presents a problem for the aquaculture industry. Costs, however, for approving a single therapeutic by the FDA can typically exceed $50 million. As such, pharmacological suppliers are reluctant to sponsor expensive research and testing campaigns for new drug therapy. Currently, researchers are examining alternative uses for chemicals that have already been FDA approved. For example, oxytetracycline was marketed as a human medicinal and then later approved for use in the aquaculture industry to control certain bacterial infections. Alternative uses for FDA approved chemicals can provide not only an alternative treatment for fish diseases but also reduce the costs associated with extensive FDA research and testing. 
     Research examining the effectiveness of photodynamic (or photoactive) dyes as pesticides has been conducted since the early seventies. The USDA&#39;s Agricultural Research Service (USDA-ARS) have identified at least twenty photodynamic dyes that are toxic to insects, many of which are used in the human cosmetic industry. The pesticide SureDye™ was discovered through a joint effort conducted by the USDA&#39;s Animal and Plant Health Inspection Service (USDA-APHIS) and Photodye International, Inc. Specifically, studies demonstrated that Phloxine B, as the active ingredient in SureDye™, was effective in the control of various Diptera species. Other photoactivated dyes such as rose Bengal and acridine orange have been shown to be toxic to fire ants and  E. coli , respectively. It has been suggested that dye-light therapy may also be effective against the herpes simplex virus. 
     Phloxine B, is FDA approved and has been used in human cosmetics for nearly 30 years. Phloxine B exists as a powder at room temperature and melts or decomposes at higher temperatures. The compound is a halogenated xanthene dye (see FIG. 1) with a molecular weight of 691.91 g and a water solubility of greater than 120 mg/l. 
     Due to its hydrophilic nature, bioaccumulation of Phloxine B residues is hypothesized as unlikely. Exposure to sunlight results almost immediate degradation and detoxification (&lt;1 hr), hence the dye is not considered a potential long-term environmental hazard. Metabolites and other degradation products also appear non-toxic. According to the FDA (1982), Phloxine B is relatively non-toxic to humans when ingested (≦1.25 mg/kg body weight). Although potentially a mild skin and eye irritant, the risk of lethal exposure to humans is unlikely. Studies performed on rats, dogs, and mice have also demonstrated the limited adverse effects of Phloxine B (LD 50  (mg/kg) rat 8400, dog &gt;4600, mouse 310). 
     The mechanism of action of Phloxine B is not well understood. It has been proposed that the dye collects visible light energy, converting ground state oxygen to a reactive, toxic, single oxygen molecule. Transformation of energy to the short-lived radical results in a series of reactions that eventually result in the formation of a longer-lived, metastable, triplet oxygen molecule. The excess energy of the triplet radical contributes to the oxidation of other chemicals, eventually returning the oxygen to the stable ground state. Photodynamic reactivity of Phloxine B is mediated by several physical and chemical factors including light wavelength and intensity, temperature, pH, and concentration of the dye itself. Because the transfer of light energy to oxygen is facilitated by halogens, the toxicity of the dye directly increases with increasing halogenation. 
     Studies have demonstrated that photoactive dyes may damage DNA, nucleotides, prokaryotic and eukaryotic cell membranes, viral membranes, and cytosolic proteins. The FDA approved photoactive compound, SureDye™, is administered to insects via food baits. Although the target of toxicity is unknown, researchers suggest that external tissues, not internal organs, are the regions of immediate toxicity. It has been suggested that humans and other animals are protected from the effects of ingesting phototoxic dyes since the internal organs function in relative darkness and, if absorbed, would be rapidly eliminated by the intestines and liver. 
     SUMMARY OF THE INVENTION 
     The present Invention comprises the use of photoactive dyes such as Phloxine B, which is chemically known as 2′, 4′, 5′ 7′-tetrabromo-4, 5, 6, 7-tetrachlorofluorescein, disodium salt and which is registered as D&amp;C (Drug and Cosmetic) Red Dye #28, as a treatment for fish infected with external protozoans such as  Ichthyophthirius multifiliis . It has been discovered that photoactive dyes such as Phloxine B are readily absorbed by external protozoans such as  Ichthyophthirius multifiliis . It has also been discovered that, after absorption by translucent protozoa such as  Ichthyophthirius multifiliis , photoactive dyes may be photoactivated by exposing the translucent external protozoa to light. Moreover, it has been discovered that exposure of translucent protozoans to light after absorption of photoactive dyes such as Phloxine B is toxic to the protozoan, presumably due to photoactivation of the photoactive dye. 
     There are many advantages over the prior art of using photoactive dyes such as Phloxine B as a treatment for fish infected with external protozoans. For example, photactive dyes such as Phloxine B are non-toxic to most animal species, do not accumulate or pose an environmental threat, and are relatively inexpensive. Moreover, because exposure to sunlight results in almost immediate degradation and detoxification, any long-term environmental hazards are unlikely. 
     According to one embodiment of the invention, there is disclosed a method of treating protozoan infections in fish including introducing a quantity of photoactive dye, such as Phloxine B, to an aqueous environment containing one or more fish infected with protozoa. According to one aspect of the invention, the method includes introducing under low-light conditions a quantity of photoactive dye to an aqueous environment containing one or more fish infected with protozoa. 
     According to yet another embodiment of the invention, there is disclosed a method of treating protozoan infections in fish including introducing a quantity of photoactive dye to an aqueous environment containing one or more fish infected with protozoan such that the resulting concentration of the photoactive dye in the aqueous environment is toxic to the protozoan. According to one aspect of the invention, the method includes introducing under low-light conditions a quantity of photoactive dye to an aqueous environment containing one or more fish infected with protozoan such that the resulting concentration of the photoactive dye in the aqueous environment is toxic to the protozoan. 
     According to yet another embodiment of the invention, there is disclosed a method of treating protozoan infections in fish including the steps of: (a) introducing a quantity of photoactive dye to an aqueous environment containing one or more fish infected with protozoa; and (b) repeating step “(a)” one or more times. According to one aspect of the invention, the method includes the steps of: (a) introducing a quantity of photoactive dye under low-light conditions to an aqueous environment containing one or more fish infected with protozoa; and (b) repeating step “(a)” one or more times. 
     According to yet another embodiment of the invention, there is disclosed a method of treating protozoan infections in fish including the steps of: (a) introducing a quantity of photoactive dye to an aqueous environment containing one or more fish infected with protozoan such that the resulting concentration of the photoactive dye in the aqueous environment is toxic to the protozoan; and (b) repeating step (a) one or more times. According to one aspect of the invention, the method includes the steps of: (a) introducing a quantity of photoactive dye under low-light conditions to an aqueous environment containing one or more fish infected with protozoan such that the resulting concentration of the photoactive dye in the aqueous environment is toxic to the protozoan; and (b) repeating step (a) one or more times. 
     According to yet another embodiment of the invention, there is disclosed a method of treating protozoan infections in fish including the steps of: (a) introducing a quantity of photoactive dye under low-light conditions to an aqueous environment containing one or more fish infected with protozoan; (b) allowing sufficient time for protozoan absorption of the photoactive dye under low-light conditions; and (c) photoactivating the absorbed photoactive dye. According to one aspect of the invention, the method includes the steps of: (a) introducing a quantity of photoactive dye under low-light conditions to an aqueous environment containing one or more fish infected with protozoan; (b) allowing sufficient time for protozoan absorption of the photoactive dye under low-light conditions; (c) photoactivating the absorbed photoactive dye; and (d) repeating steps (a)-(c) one or more times. 
     In accordance with these discoveries, it is an object of the present invention to provide a method for the control of external protozoan in fish populations. Other objects of the present invention will become readily apparent from the following description. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1 illustrates the chemical structure of Phloxine B. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The present invention now will be described more fully. This invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. 
     Photoactive dyes such as Phloxine B may be readily absorbed by protozoans. Because many protozoans such as  Ichthyophthirius multifiliis  are translucent it is possible to photoactivate the dyes after absorption by the protozoans. Photoactiviation of dyes after absorption by protozoans may be lethal to the protozoans. Therefore, photoactive dyes may be used as an effective treatment for fish infected with external translucent protozoans. 
     Examples of photoactive dyes which may be used to practice the present invention include, but are not limited to Azure A, Azure B, Methylene Blue, NewMethyleneB1 N, Toluidine Blue, Methylene Green, Thionin, Brilliant Cresylblue, Rhodamine B, Thioflavine T, Eosine Y, Erythrosine B, Phloxine B, Pyronine Y, Rhodamine 6 G, Rose Bengal, D&amp;C Orange 5, Pyronine B, Neutral Red, Safranin O, Auramine O, and Alizarin Red S. Included in this list are several dyes which are used as food, cosmetic, or as dyes for cloth, leather, wool, cotton, and the like, which have been approved by the FDA for use as food, drug, or cosmetic. Those skilled in the art may be aware of other photoactive dyes which could be used to practice the present invention. 
     By way of example only, the present invention may be practiced by adding a quantity of photoactive dye to an aqueous environment containing a population of fish infected with external translucent protozoans. The photoactive dye may be added to the aqueous environment in a dry powdered form or as a liquid, such as from a stock solution of known concentration. A single species of photoactive dye may be added to the aqueous environment or combinations of different photoactive dyes may be used. For example, SureDye™, which is composed of a 1:1 molar mixture of Phloxine B (D&amp;C Red Dye No. 28-69% by weight) and Uranine (D&amp;C Yellow Dye No. 8-31% by weight), may be used. 
     The photoactive dyes are preferably introduced to the aqueous environment under low light conditions. Low light conditions may be necessary to prevent substantial deactivation or degradation of the photoactive dyes prior to absorption by the protozoans. In a preferred embodiment, the photoactive dye is added to the aqueous environment at night-time. 
     In a preferred embodiment, after addition of the photoactive dye to the aqueous environment, sufficient time should be provided to allow the protozoans to absorb the photoactive dye before exposure to light. In a preferred embodiment, the photoactive dye is added at night and the protozoans are allowed to uptake the dye over a period of several hours before exposure to daylight. 
     After the photoactive dye is absorbed by the external, translucent protozoans, the protozoans are exposed to light. Presumably, as a result of the photoactivation of the absorbed dye, those protozoan which have absorbed the photoactive dye and are exposed to light will be killed. 
     The effectiveness of treatments of photoactive dyes on parasite control in a particular fish population can be determined by those skilled in the art by monitoring the general behavior of the fish (swimming, feeding, etc.) and the appearance of the parasite by microscopic examination. For example, live Ick trophozoites may be monitored using image analysis microscopy using a Image-Pro Plus V. 4.0.0.13, available from Media Cybernetics, L.P. 
     In one embodiment of the invention, the photoactive dye, Phloxine B, is added as a dry powder to an aquarium containing fish infected with Ick. The Phloxine B is added to achieve a final concentration of around 5 mg/L. The Phloxine B is added to the aquarium in darkness and at least eight hours of absorption is allowed before photoactivation. Photoactivation is achieved by exposing the Ick infected fish to light from artificial lamps. Microscopic examination suggests that Phloxine B is effective against the formerly invulnerable trophozoite stage when attached to the fish host. Although this particular embodiment is performed in a laboratory setting, those skilled in the art will understand that the teachings can be adapted to a commercial operation without undue experimentation. 
     The invention is described further in the following non limiting examples: 
     EXAMPLE 1 
     An in vivo study was conducted involving the direct dosing of Ick parasites with Phloxine B. 1 μl of Ick theronts, both free-swimming tomites and non-ruptured cysts, and 9 μl of a Phloxine solution containing either 1000, 100, 10, 1 and 0 ppm of Phloxine B were placed into 100×15 mm tri-plate petri dishes providing 3 replicates for each treatment. 1 set of the plates was left exposed to ambient light (6 mE m −2 s −1 ) for 5 hr (LIGHT EXPERIMENT), while the other set was kept in the dark for 4 hr then exposed to ambient light for 1 hr (DARK EXPERIMENT). Percent mortality was assessed, then both light and dark plates received a second dose of Phloxine B at the same concentration as before and treated in the same manner. The results of the experiment are shown in the tables below: 
     
       
         
               
             
               
               
               
             
               
               
               
               
             
           
               
                   
               
               
                 LIGHT EXPERIMENT (Ambient Light) 
               
             
          
           
               
                 Phloxine B TREATMENT 
                 1 DOSE 
                 2 DOSES 
               
               
                   
               
             
          
           
               
                 1 
                 ppm 
                 No effect 
                 No effect 
               
               
                 10 
                 ppm 
                 No effect 
                 No effect 
               
               
                 100 
                 ppm 
                 20% Mortality 
                  80% Mortality 
               
               
                 1000 
                 ppm 
                 80% Mortality 
                 100% Mortality 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
             
               
               
               
               
             
           
               
                   
               
               
                 DARK EXPERIMENT (Lab Conditions) 
               
             
          
           
               
                 Phloxine B TREATMENT 
                 1 DOSE 
                 2 DOSES 
               
               
                   
               
             
          
           
               
                 1 
                 ppm 
                 No effect 
                 No effect 
               
               
                 10 
                 ppm 
                 No effect 
                 No effect 
               
               
                 100 
                 ppm 
                 No effect 
                 10% Mortality 
               
               
                 1000 
                 ppm 
                 40% Mortality 
                 95% Mortality 
               
               
                   
               
             
          
         
       
     
     EXAMPLE 2 
     A light and dark experiment were set up following the protocol in Example 1, but exposure times were doubled—8 hr dark then 2 hr light or 10 hr light. The results indicated that longer exposure time increased the effectiveness of the dye. 
     EXAMPLE 3 
     An experiment was designed to test the effect of light intensity on the effectiveness of Phloxine B treatment of Ick. The previous experiments described in Examples 1 and 2 were conducted in the lab. The ambient light intensity in the lab was very low, 6 μmol m −2 s −1 . An experiment was conducted as following the protocol of Example 1, except the experiment was conducted in a greenhouse to better mimic natural light conditions. The results from the Light Experiment under Greenhouse conditions are shown in the table below: 
     
       
         
               
             
               
               
             
               
               
               
             
           
               
                   
               
               
                 LIGHT EXPERIMENT (Greenhouse Conditions) 
               
             
          
           
               
                 TREATMENT 
                 1 DOSE 
               
               
                   
               
             
          
           
               
                 1 
                 ppm 
                 No effect 
               
               
                 10 
                 ppm 
                 No effect 
               
               
                 100 
                 ppm 
                  50% Mortality 
               
               
                 1000 
                 ppm 
                 100% Mortality 
               
               
                   
               
             
          
         
       
     
     Conclusions drawn from the experiments described in the Examples 1-3 above were that increased exposure time and increased light intensity both increased the effectiveness of the Phloxine B dye as a treatment for Ick. At low light intensities, multiple doses also increased the dye&#39;s effectiveness. It was also observed that the dye was taken up both free-swimming and encysted organisms. 
     Many modifications and other embodiments of the invention will come to mind to one skilled in the art to which this invention pertains having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the invention is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.