4. DESCRIPTION (Description shall start from next page.)

Description

FIELD OF INVENTION

1. The present invention is related to new novel nanopreparation with silver metal nanoparticles for any fish diseases. 2. The present invention relates to the field of Nanoscience for disease treatment of all types of fishes by one time treatment at low concentration. 3. The present invention relates to the field of Nanoscience for disease prevention of all types of fishes by one time treatment with nanoformulation at low concentration.

CLAIM OF COMPLETE APPLICATION RIGHTS
A. Claims

1. The method of claim 1 that the silver metal compounds are used for treatment either for curing /prevention of fish diseases.

2. The method of claim 1 that various nanoparticles are synthesized for treatment either for curing /prevention of fish diseases.

3. The method of claim 1 various reducing and stabilizing agents used for synthesis for treatment either for curing /prevention of fish diseases.

4. The method of claim 2 that individual nanopreparations are used as nanosolution for treatment either for curing /prevention of fish diseases.

5. The method of claim 3 that various combinations are mixed for nanosolution preparation for treatment either for curing /prevention of fish diseases .

6. The method of claim 4 that nanosolutions are used in liquid form to suit the method and mode of treatment for treatment either for curing /prevention of fish diseases

B.Claims
1. Method of synthesis of metal 1 nanopreparations using silver nanoparticles in various combinations for treatment either for curing /prevention of fish diseases.

C.Claims
What is claimed is:

1. A method of mixing various silver metal compounds in water and adding various reducing and stabilizing agents like citrate,starch and plant extracts Mixing at room temperature or heating for one minute to ten minutes depending on the nanoparticle formation..

2. A method of treatment either for curing /prevention of fish diseases by nanosolution comprising any silver nanoparticles comprising the steps of:

• Mixing the solution in water in a container tank to form an uniform suspension
• Treating the fishes by bathing method with nanosolution for treatment either for curing /prevention of fish diseases.
• The method according to claim 1 further comprises killing all microbial infections including protozoan and fungal diseases that are present on fishes surface and immediate surrounding areas.

TECHNCIAL FIELD OF INVENTION

1. The present invention is related to new novel nanopreparation with silver metal nanoparticles for any fish diseases.

2. The present invention relates to the field of nanoscience for disease treatment of all types of fishes by one time treatment at low concentration.

3. The present invention relates to the field of nanoscience for disease prevention of all types of fishes by one time treatment with nanoformulation at low concentration.

BACKGROUND OF THE INVENTION

Prevalence of fish diseases has negative economic impact on aquaculture Goldfishes are one of the most popular pets in the world. White spot and red spot are the very common goldfish diseases. White Spot Disease {Ichthyophthiriasis - Ich) is actually caused by protozoa Ichthyophthirius multifiliis with Salt-like specks on the bodyfins and excessive slime, having problems of breathing, clamped fins and with a loss of appetite. Epizootic ulcerative syndrome (EUS) or 'red-spot' disease caused by pathogenic fungus, Aphanomyces invadans, is ulcerative syndromes which sometimes extends into the abdominal cavity and also develop unilateral or bilateral cloudiness in the cornea offish

In aquaculture, chemotherapeutic agents such as commercial antibiotics and disinfectants (chemicals) (Derwa 2004 ) are commonly employed for disease management, although this is not applicable due to high cost, environmental hazards, and the antibiotic resistance developed by many pathogens (Khalifa et al 1983 ) and also there are three phases to the life cycle of these protozoa. Ich is susceptible to treatment at only one stage of the life cycle, Vaccination of goldfish Carassius auratus against Ichthyophthirius multifiliis (Ich) was performed with live theronts, trophonts, and sonicated trophonts and with Ich antigens (Osman et al 2009 a,b, He et al 1997, Dickerson & Clark 1998, Ling et al 1993, Dalgaard et al 2002, Wang & Dickerson 2002, Xu et al 2004, Lobb 1987, Cain et al 2000, Houghton & Matthews 1990, Ando 1983, Marvao et al 1994). Protection of various fishes against Ichthyophthirius
multifiliis by immunization was attempted by several authors (He et al 1997, Dickerson & Clark 1998, Osman et al 2009a,b, Ling et al 1993, Dalgaard et al 2002, Wang & Dickerson 2002, Xu et al 2004, Lobb 1987, Cain et al 2000, Houghton & Matthews 1990). Fish develop protective immunity against the parasite following immunization with Ich antigens (Ando 1983, Marvao et al 1994).

The convergence of nanotechnology with nanomedicine has added new hope in the therapeutic and pharmaceutical field. The fisheries and aquaculture industry can be revolutionized by using nanotechnology with new tools like rapid disease detection, enhancing the ability of fish to absorb drugs like hormones, vaccines and nutrients etc. rapidly. Nanoparticles possess exceptional physical and chemical properties Silver nanoparticles are among the most commercialized nanoparticles due to their antimicrobial potential. Silver nanoparticles based cosmetics, therapeutic agents and household products are in wide use.

SUMMARY OF THE INVENTION

1. This inventive subject matter relates to novel nano metal solution and methods for using such compounds for treating all fish diseases including white spot and red spot diseases The present invention relates to new metal nanoparticles like silver substituted with various stabilizing agents like starch, polyol etc for curing white spot and red spot diseases and other infections of fishes.

2. It is an object of the present invention to provide a nanosolution wherein nanoparticles of silver in combination with various stabilizing agents like starch,citrate, various plant extracts like polyol etc.

3. It is an object of the present invention to provide therapeutics and protection of fishes against any infection including red spot and white spot diseases using the nanopreparartions

4. The invention disclosed herein relates to the formulation of nanosolution for protecting and/ or curing any infection in fishes.

5. In one embodiment the silver metal compounds are selected and another embodiment the reducing agents are selected and in another embodiment the stabilizing agents either plant or nonplant sources are selected. Another embodiment mixes the nanopreparations in various combinations for treating all infections including protozoan and fungal infection in fishes. Another embodiment mixes the
nanopreparations for preventing any infections including protozoan,fungal etc in fishes.

Methods Synthesis of silver nanoparticles
To starch solution, aqueous solution of silver nitrate was added and autoclaved for 5 min. This preparation containing silver nanoparticles were used for further analysis. Characterization of silver nanoparticle.

Silver nanoparticles synthesized were characterized by UV-visible absorption spectroscopy, Fluorescence spectroscopy, Transmission electron microscopy and FITR.

In vivo Fish study
In order to study the uptake, toxicity and effect on diseased condition, Gold fishes were kept in oxygenated, dechlorinated tap water at 37°C. The standard method recommended by the Animal welfare committee was followed. For each group, 3 fishes were maintained in 60 liters of water. Experimental (Diseased) groups were treated by bathing method in 20ml of water with silver nanoparticles at a concentration of l0ng for 20 seconds every day for seven days. Control fish (normal and diseased) were exposed to dechlorinated tap water without the addition of particles. After the treatment period, whole body X-Ray was taken. Blood samples were collected from live fish by cutting caudal peduncle for biochemical and UV visible spectroscopy, FTIR analysis. Toxicity study Biochemical assay:

Blood samples (approximately 2 ml) were drawn from the caudal vein. Before collecting blood samples no anesthetic was applied to fish as it may affect blood parameters. Total protein, albumin, phosphorus, calcium and other levels in blood plasma were assayed using commercially available kits (Roche). Enzyme assays:

Blood biochemistry analysis such as alkaline phosphatase activity, Na+ K+ ATPase activity, GSH level, Catalase assay and LPO activity were done following the methods of Ronner etal (1977); Beutler et al (1963 ); Beers & Sizer (1952) and Ohkawa et al (1979) respectively.

Biological distribution pattern
In order to study the pattern of uptake and tissue distribution in fish, X-Ray detection was done after one month of silver nanoparticle treatment The quantity of silver nanoparticles present in all tissues were obtained from the UV-Visible absorption spectra at 420 nm specific for silver nanoparticles. Statistical

Analysis
Statistical analysis was performed using the statistical package. The data were analyzed using STDEV (Microsoft Excel, Microscoft Corporation, USA.) .The data are expressed as Mean ± SD.

Results Silver nanoparticles synthesized were found to be stable in solution at room temperature and showed no signs of aggregation. The UV visible spectrum of synthesized silver nanoparticles showed absorption maximum at 419( Fig.l). The band at 419 nm can be attributed to Mie scattering which responds only to silver metal (Aoki, Chen, Yang & Naga Sawa 2003). Transmission Electron Micrograph of starch stabilized silver nanoparticles shows the presence of particles at an average size range of 5 to 10 nm (Fig.2). The silver nanoparticles were also found to be mono dispersed uniformly. The fluorescence spectrum showed maximum fluorescence at 413-417nm (Fig. 3).

Bathing of diseased fish at a concentration of l0ng for 20 seconds for 7 days completely cured the red and white spot diseases (Fig. 4).Approximately 3 fold increases in body weight was observed (Fig. 5) and the fish is still alive and healthy. Interestingly during the period of study, the fishes did not show any significant changes in behavior that might have indicated the neurotoxic effects.

Fish serum may reflect status of many biochemical processes in the metabolism. Changes in serum biochemistry in response to metalnanoparticle exposure were studied in freshwater ornamental gold fish Carassius auratus. Fish were exposed to lOng concentration for 7 days to determine changes in the levels of biochemical parameters: silver nano particles in blood serum and whole body.

Various biochemical parameters analyzed in blood of control and experimental fish showed significant difference (Fig. 6). There was significant increase in sugar, albumin, protein cholesterol, bilirubin and
phosphorus. All other components like creatinine, urea, and calcium decreased. The enzyme activities such as LPO, Na+ K+ 158 ATPase, alkaline phosphatase, and catalase increased in contrast to GSH content which showed reduction in their activities in experimental fish than control fish (Fig. 7).

In the present, study silver nanoparticles were detected in all organs including brain as observed in whole body X-Ray (Fig. 8). Silver nanoparticles were seen in blood even after two months of treatment. The fluorescence spectrum of whole body tissue (F of other protein and a shift in the absorption spectrum and florescence spectrum. To determine the change in chemical bonding between the synthesized silver nanoparticles and the particles present in vivo in association with proteins, FTIR measurements of both samples were performed (Fig. 10). The whole body homogenate exhibited two very intense absorption bands in the 1560-1500cm-1 and 1350-1300cm-1 region of the spectrum arising from asymmetric and symmetric stretching vibrations of the highly polar nitrogen - oxygen bonds. For N substituted compounds, such as proline, only the- NH2 stretching vibrations are involved and these appear at lower frequency. A relatively prominent band between 2200 and 2000cm-1 is found in the spectrum of most aminoacids and their hydrochloride salts and can be clearly seen in the spectrum of valine at 2130cm-1.

There is an additional band for O-H stretching vibration near 3200cm-1 Discussion Nanoparticles have diverse applications in life sciences such as drug development, protein detection and gene delivery. Drug targeting through nanoparticles may improve therapies yet a thorough understanding of the feature that regulates the effect of carrier nanoparticle is needed to translate this approach into the clinical application. Hence the present study was carried out to understand the toxicity and curing property of Silver nanoparticles against White spot and Red spot Diseases of gold fish.

Fish serum may reflect status of many biochemical processes in the metabolism. Heavy metals, as environmental stressors, may alter serum biochemical parameters in fishes. In order to evaluate the toxic effect of silver nanoparticles, Starch capped silver nanoparticles were introduced in to fishes and various biochemical parameters were analyzed in blood of healthy control and white and red spot diseased fishes. Increased concentration of GSH content and an increased capacity to maintain glutathione in the reduced state play an important role in LPS stimulated macrophages and endothelial cells in the detoxification of H2O2 and thus in the protection against oxidative stress as observed by Inoue (1994);

Jaeschke (1995); Kaplowitz & Tsukamoto(1996); Bautista et al ( 1990). Maintenance of cellular GSH could be the result of elevated activity of catalase and glutathione reductase or of augmented supply of
NADPH. Elevated GSH may protect cellular proteins or could also directly interact with ROS generated by activated Kupffer and endothelial cells (Jaeschke 1993, Mc Cord 1993). To neutralize the impact of ROS, both enzymatic and nonenzymatic antioxidants are activated (Lopez-Torres et al 1993, Filho 1996). All metal exposures increased cholesterol concentration in the serum Calcium level decreased only in Cu-exposed fish, and Cl~ level decreased in Ag-exposed fish.

(Oner et al 2008)

The Na+ K+ -ATPase helps maintain resting potential, active transport, and regulate cellular volume. It also functions as signal transducer/integrator to regulate MAPK pathway, ROS, as well as intracellular calcium. For most animal cells, the Na+ K+ -ATPase is responsible for 1/3 of the cell's energy expenditure. For neurons, the Na+K+-ATPase is responsible for 2/3 of the cell's energy expenditure. Na+ K+ - ATPase activities increased in response to increased external salinity. Progressive transfer to seawater enhances intestinal and branchial Na+ K+ -ATPase activity in non-anadromous rainbow trout (Fuentes et al 1997), the electrogenic cationic pump Na+ K+ - ATPase is essential for trans-cellular movement of water and ions. It was previously shown that angiotensin II (Ang II) modulates the activity of the Na+ K+ -ATPase in the eel gill and kidney, suggesting a role in NaCl homeostasis for this hormone. Ang II also modulates the activity of Na+ K+ -ATPase in the eel kidney via mobilization of intracellular calcium (Ca ) and protein kinase C (PKC) activation (Marsigliante et al 2000).

Our study showed very significant increase in alkaline phosphatase activity compared to control revealing the advantages of silver nanoparticles in maintaining the body physiology and health (Schiele et al 1998). Under conditions of oxidative stress, cells increase the levels of some of these antioxidant enzymes, most commonly MnSOD and catalase (Bannister et al 1987, Hasset & Cohen 1989, Heffner & Repine 1989). The increased catalase activity and decreased GSH content in the present study indicate the basal level oxidative stress maintenance condition of the animal because of the presence of silver nanoparticles in the body. Endogenous antioxidant defense systems, though scavenge and minimize the formation of oxygen free radicals, are not fully effective especially in pathological conditions demanding use of exogenous antioxidants.

The reduction of lipid peroxidation in the gills of fish exposed to the high concentration of N10 silver, despite a large accumulation of silver in the gills was reported by Tessa M. Scown et al (2010) in contrast with several studies, which demonstrated the potential for silver NPs to generate ROS and cause oxidative stress. Rahman et al (2009) found that 25-nm silver particles induced the expression of oxidative stress-related genes in the mouse brain after iv injection of 100, 500, and 1000 mg/kg, and in vitro studies have shown that silver NPs have the capacity to generate ROS (Carlson et al 2008, Hsin, et al 2008) and cause increased lipid peroxidation (Arora et al (2008). Both liver and gill tissues have the ability to up regulate survival genes and DNA repair mechanisms (Diehl 2000, Hansen et al 1996) when an organism is exposed to environmental stressors, which may also explain the reduced lipid per oxidation seen in response to silver nitrate in the liver and to the high concentration of 10-nm silver particles in the gills. Silver nanoparticles were distributed in all organs as observed in whole body X-Ray (Fig. 7) similar to that observed by Raynal, et al ( 2004); Briley-Saebo et al (2006 ) and Quan-Yu Cai et al, (2007 ) in the case of iron oxide nanoparticles and AUNP-PEG nanoparticles, Oberdorster et al (2004 );

Kiruba Daniel et al (2010), Kiruba Daniel,et al (2011a) , Nimrodh Anantha et al Umapathi (2011) in the case of silver nanoparticles in mice and rat and also experiments, Zebra fish (Kiruba Daniel et al 2011), on medaka fish using fluorescent solid latex nanoparticles confirmed a homogeneous distribution of the particles (Kashiwada 2006). Silver nanoparticles were seen in blood even after two weeks of injection in rat. Increased half life of ultra small particles was shown by Quan-Yu Cai et al(2007 ) with reference to gold nanoparticles. Similar results were observed in the present study also (Fig. 8) The nanoparticles were detected in the brain indicating that silver nanoparticles have the ability to penetrate blood brain barrier as observed in Zebra fish (Asharani et al 2008, Kiruba Daniel et al 201 lb), mice and rat model (Kiruba Daniel et al. 2010, 201 la ). It was suggested that the nanoparticles could enter the cells through many routes, some of which include diffusion or endocytosis through the skin of embryos.( Kashiwada 2006, Bai Wei et al 2010; Zhu et al 2008). Both nanocopper and nanosilver exposures increased metal content associated with gill tissue, though silver concentrations were much higher following nanosilver exposures suggesting that intact silver nanoparticles are associated with the gill. The concentrations were analysed using an Fourier transform infra-red (FTIR) technique (Fig. 9)

White Spot Disease and red-spot disease are the most common fish diseases. In aquaculture, chemotherapeutic agents are not preferred due to high cost, environmental hazards, and the antibiotic resistance developed by many pathogens (Khalifa, Al-Khayat & Al-Rijab 1983). Protection of goldfish against Ichthyophthirius multifiliis by immunization with a recombinant vaccine was attempted by He et al (1997). Goldfish immunized with live theronts by immersion or injection acquired high levels of immunity and protection against infection with Ich. (Osman et al 2009 a,b).

Metal nanoparticles are applied in biology as biosensors therapeutics and other diverse applications in life sciences Among noble metal nanoparticles, silver nanoparticles have received considerable attention due to their attractive physicochemical properties and in addition the strong toxicity that silver exhibits in various chemical forms to a wide range of microorganism. The therapeutic potential of silver nanoparticles was studies against red spot and white spot diseases. Our study has shown that 20 second bathing with silver nanoparticles at l0nM concentration for seven days was sufficient to completely cure the red and white spot diseases and to give total life long protection.

Comparative toxicity of several metal oxide nanoparticle aqueous suspensions to Danio rerio early developmental stage was reported earlier. The embryo toxicity test revealed that nano-ZnO killed Danio rerio embryos (50 and l00mg/L), retarded the embryo hatching (1-25 mg/L), and reduced the body length of larvae. The embryo toxicity of nano-Cu at 0.01 and 0.05 mg/L showed no significant difference from Cu2+ at the corresponding concentrations (0.006 and 0.03 mg/L), but 0.1 mg/L nano-Cu had a greater toxicity than 0.06 mg/L Cu2+( Bai Wei,et al 2010)As nanoparticle concentration increased, the number of normally developed Danio rerio decreased, while the number of dead Danio rerio increased (Zhu et al 2008, Wei Bai et al 2010, Cristina Ispas et al (2010) and Kerry J.Lee et al (2007).
Results showed that silica, zinc, and nickel nanoparticles in aqueous suspensions delayed zebra fish embryo and larva development, decreased their survival and hatching rates, and caused tissue damage.( Karl Fent et al (2010),Wei Bai et al 2010; Cristina Ispas et al 2010, Robert J. Griffitt et al 2009).
But the real time study of transport and biocompatablity in early embryonic development in Zebra fish embryo single silver nanoparticles (5-46nm) showed at 0.19nM concentration showed no toxicity.(Kerry J.Lee et al 2007) and no toxicity was observed at 160 ng concentration in the adult Zebra fish ( Kiruba Daniel et al 201 lb).

Asharani et al (2008) reported that the Ag-nanoparticles treated embryos showed a normal cardiac morphology, with atria and ventricle differentiated normally with proper orientation with time. Also higher concentrations of Ag-nanoparticles resulted in significant growth retardation, which could be due to delay or inhibition of cell division. Ag+ ions have been reported to have toxicity in aquatic species. Ag+ ions
treatment induced ion regulatory impairment and increased mortality in rainbow trout eggs.

In our study young Guppies were let to swim in silver nanoparticle solution for one hour and they are still alive and healthy. The above results clearly indicated that there was no toxicity developed against starch stabilized silver nanoparticles and it could penetrate all tissues including the brain through BBB Life time protection can be given to diseased fishes or healthy young ones at a very low concentration by simple bathing method. This is the first report on silver nanoparticle therapy against protozoan infection in fishes.

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Figure Legend Figurel UV-Visible Spectrum of silver nanoparticles Figure2 Transmission Electron Microscopic image of starch stabilized silver nanoparticles Figure3 The fluorescence spectrum of the synthesized silver nanoparticles Figure 4 Curing property of silver nanoparticles. Red Spot diseased fish before (1) and after (3) treatment, White Spot diseased fish before (2) and after (4) treatment
Figure5 Body weight of silver nanoparticle treated (pink) experimental fish and Control (blue) fish
Figure6 Fold Difference in l.Urea 2.Creatinine 3.Sugar 4.Albumin 5.protein 6.Cholesterol 7. Bilirubin 8.

Calcium 9. Phosphorous content between control fish and Silver nanoparticle treated fish . Figure7 Comparison of various enzyme activities l.LPO 2.GSH 3. Na+K+ATPase 4. Alkaline phosphatase 5.Catalase in control and silver nanoparticle treated fish.

Control fish Treated fish Figure8 Whole body X-Ray image of Control (1), Red spot disease treated (2) and White Spot disease treated fish (3). Figure9 The fluorescence spectrum of whole body tissue of Red spot disease treated & White Spot disease treated fish compared with control fish.

FigurelO The FTIR pattern of control and experimental fish with silver nanoparticle pattern as an insert. Orange arrows indicate changes in FTIR pattern compared to control.

5. CLAIMS (not applicable for provisional specification. Claims should start with the preamble — "l/we Claim" on separate page)
CLAIM OF COMPLETE APPLICATION RIGHTS

We claim A. Claims
1. The method of claim 1 that the silver metal compounds are used for treatment either for curing /prevention of fish diseases.

2. The method of claim 1 that various nanoparticles are synthesized for treatment either for curing /prevention of fish diseases.

3. The method of claim 1 various reducing and stabilizing agents used for synthesis for treatment either for curing /prevention of fish diseases.

4. The method of claim 2 that individual nanopreparations are used as nanosolution for treatment either for curing /prevention of fish diseases.

5. The method of claim 3 that various combinations are mixed for nanosolution preparation for treatment either for curing /prevention of fish diseases .

6. The method of claim 4 that nanosolutions are used in liquid form to suit the method and mode of treatment for treatment either for curing /prevention of fish diseases

B.Claims

1. Method of synthesis of metal 1 nanopreparations using silver nanoparticles in various combinations for treatment either for curing /prevention of fish diseases. C.Claims 1. Method of treating all types of fishes for treatment either for curing /prevention of fish diseases

What is claimed is:

1. A method of mixing various silver metal compounds in water and adding various reducing and stabilizing agents like citrate,starch and plant extracts Mixing at room temperature or heating for one
minute to ten minutes depending on the nanoparticle formation..

2. A method of treatment either for curing /prevention of fish diseases by nanosolution comprising any silver nanoparticles comprising the steps of:

• Mixing the solution in water in a container tank to form an uniform suspension

• Treating the fishes by bathing method with nanosolution for treatment either for curing /prevention of fish diseases.

The method according to claim 1 further comprises killing all microbial infections including protozoan and fungal diseases that are present on fishes surface and immediate surrounding areas.