WO2010109470A1

WO2010109470A1 - Formulations and methods for enhancing non specific immunity and disease resistance in tilapia

Info

Publication number: WO2010109470A1
Application number: PCT/IL2010/000262
Authority: WO
Inventors: Ilan Ben-Gal; Ariav Raanan
Original assignee: Aqua Pro Ltd.
Priority date: 2009-03-26
Filing date: 2010-03-25
Publication date: 2010-09-30

Abstract

The invention discloses a bioactive pharmaceutical composition (BPC) useful in controlling or preventing diseases in Tilapia. The aforementioned pharmaceutical composition comprises a mixture of live bacteria and biologically active compounds (BAC).

Description

FORMULATIONS AND METHODS FOR ENHANCING NON SPECIFIC IMMUNITY AND DISEASE RESISTANCE IN TILAPIA

FIELD OF THE INVENTION

Disclosed herein are compositions and methods useful in aquaculture. More particularly compositions comprising a concentrated mixture of live bacteria specifically selected for enhancement of non-specific immunity, growth and disease resistance in Tilapia is disclosed.

BACKGROUND

Because of their large size, rapid growth, and palatability, a number of tilapiine cichlids are at the focus of major aquaculture efforts, specifically various species of Oreochromis, Sarotherodon, and Tilapia, collectively known colloquially as tilapias. Like other large fish, they are a good source of protein and a popular target for artisanal and commercial fisheries. Originally, the majority of such fisheries were in Africa, but accidental and deliberate introductions of tilapia into freshwater lakes in Asia have led to outdoor aquaculturing projects countries with a tropical climate such as Papua New Guinea, the Philippines, and Indonesia. In temperate zone localities, tilapiine farming operations require energy to warm the water to the tropical temperatures these fish require. One method involves warming the water using waste heat from factories and power stations.

Tilapias have an omnivorous diet, mode of reproduction (the fry do not pass through a planktonic phase), tolerance of high stocking density, and rapid growth. In some regions the fish can be put out in the rice fields when rice is planted, and will have grown to edible size (12-15 cm, 5-6 inches) when the rice is ready for harvest. One recent estimate for the FAO (Food and Agriculture Organization) puts annual production of tilapia at about 1.5 million tonnes, a quantity comparable to the annual production of farmed salmon and trout. Unlike salmon, which rely on high-protein feeds based on fish or meat, commercially important tilapiine species eat a vegetable or cereal based diet. Tilapias raised in inland tanks or channels are considered safe for the environment, since their waste and disease should be contained and not spread to the wild.

Fish farmers in the U.S. reported that disease related mortality represents the major economic loss to the aquaculture industry. Historically, antibiotics and chemicals have been used to treat diseases in aquatic animals. However, given the fact that diseased fish eat poorly, only a limited number of FDA approved and efficacious drugs/chemicals are available for treatment, and the increasing problem of emerging drug-resistant pathogens and the resultant food and environmental contamination, disease prevention is a better means of controlling infectious diseases. Streptococcal (Strep) diseases of fish can cause significant mortality in aquaculture. Some aquatic Strep species may cause disease in humans in unusual circumstances. In addition to bacteria in the genus Streptococcus, there are several other closely related groups of bacteria that can cause similar disease, including Lactococcus, Enterococcus and Vagococcus. For purposes of this fact sheet, all of these bacteria and the disease itself will be referred to as Strep.

Strep infections in fish can cause high mortality rates (> 50%) over a period of 3 to 7 days. Some outbreaks, however, are more chronic in nature and mortalities may extend over a period of several weeks, with only a few fish dying each day. Keys to disease prevention in fish include: a) maintaining good water quality; b) providing proper nutrition; c) keeping the environment clean; and d) quarantining new fish before adding them to an existing collection.

Nutrition has proved to be a key factor in maintaining fish health. All essential nutrients are required in diets in adequate quantity to promote growth, sustain health, and maintain the ability of fish to withstand stress and resist disease-causing agents. Dietary modulation of stress and immune responses has been accomplished in numerous terrestrial animals as well as in some fish species using various approaches, including use of additives and supplementation of certain essential nutrients at levels above the minimum requirements for growth.

A long felt and unmet need would be met if compositions were provided for adding to Tilapia feed to improve the general well being of Tilapia populations in aquaculture. A further long felt and unmet need would be met if compositions were provided for adding to Tilapia feed to improve the growth rate of Tilapia populations in aquaculture. A further long felt and unmet need would be met if compositions were provided for adding to Tilapia feed to improve the resistance to disease of Tilapia populations in aquaculture. A further long felt and unmet need would be met if compositions were provided for adding to Tilapia feed to strengthen or maintain the immune systems of Tilapia populations in aquaculture. SUMMARY OF THE INVENTION

It is one object of the present invention to provide a bioactive pharmaceutical composition (BPC) useful in controlling or preventing diseases in Tilapia, wherein said pharmaceutical composition comprises a mixture of live bacteria and biologically active compounds (BAC).

It is another object of the present invention to provide the BPC as defined above, wherein said live bacteria are selected from a group consisting of Lactobacillus acidophilus, Lactobacillus casei, Bifido bacterium bifidium, Enterococcus faecium,

Bacillus subtilis.

It is another object of the present invention to provide the BPC as defined above, wherein said BAC is selected from a group consisting of rice mill by -product and

Calcium carbonate.

It is another object of the present invention to provide the BPC as defined above, wherein said BPC and/or BAC comprises at least one immunomodulator especially useful for improving the non-specific immune response to a range of aquatic pathogens in Tilapia.

It is another object of the present invention to provide the BPC as defined above, wherein said immunomodulator is selected from a group consisting of non-specific

Immunostimulators .

It is another object of the present invention to provide the BPC as defined above, wherein said immunomodulator is adapted to enhance the immune response in aquatic animals with a weak or developing immune system.

It is another object of the present invention to provide the BPC as defined above, wherein said BPC is adapted to provide biological activities, said activities selected from a group consisting of anti-bacterial activity, anti-protozoal activity, increased growth rates, decreased Feed Conversion Rate (FCR), decreased mortality rate, improving water quality parameters activity or any combination thereof.

It is another object of the present invention to provide the BPC as defined above, wherein said BPC is especially adapted for controlling aquatic pathogenic diseases caused by bacteria or parasites.

It is another object of the present invention to provide the BPC as defined above, wherein said BPC is adapted for controlling aquatic bacterial infections selected from a group consisting of Streptoccocus spp. infections and Aeromonas spp. infections. It is another object of the present invention to provide the BPC as defined above, wherein said BPC is adapted to control intestinal Coccidial infections in Tilapia.

It is another object of the present invention to provide the BPC as defined ^"above, wherein said BPC is characterized by having a nitrifying activity useful for improving defined water quality parameters in Tilapia aquaculture.

It is another object of the present invention to provide the BPC as defined above, wherein said BPC is adapted for increasing the growth rates of Tilapia by about 12% to about 15%.

It is another object of the present invention to provide the BPC as defined above, wherein said BPC is adapted for decreasing the Feed Conversion Rate (FCR) in

Tilapia by about 15%.

It is another object of the present invention to provide the BPC as defined above, wherein said BPC is adapted for decreasing the mortality rate of Tilapia by about

30%.

It is another object of the present invention to provide the BPC as defined above, wherein said BPC is provided as a formulation for premix of Tilapia feed.

It is another object of the present invention to provide the BPC as defined above, wherein said BPC is adapted for incorporating into aquatic animal feed.

It is another object of the present invention to provide the BPC as defined above, wherein said BPC is incorporated into said aquatic animal feed, mixed at the feed mill or on the farm at an inclusion rate of about 0.3%.

It is another object of the present invention to provide the BPC as defined above, wherein said probiotic bacteria is used as a mixture of live probiotic bacteria.

It is another object of the present invention to provide the BPC as defined above, wherein said BPC comprises a fermentation product of dehydrated live bacteria.

It is another object of the present invention to provide the BPC as defined above, wherein said BPC comprises probiotic bacteria selected from bacteria strains including but not limited to strains of Lactobacillus acidophilus, Lactobacillus casei,

Bifido bacterium bifidium, Enterococcus faecium, Bacillus subtilis.

It is another object of the present invention to provide the BPC as defined above, wherein said BPC comprises regulative compounds and additives selected from a group consisting of rice mill by- products and calcium carbonate. It is another object of the present invention to provide the BPC as defined above, wherein said BPC is adapted to reduce the need for antimicrobial compounds by 50% in Tilapia.

It is another object of the present invention to provide a method of controlling or preventing diseases in Tilapia, said method comprising steps of feeding said Tilapia with a bioactive pharmaceutical composition (BPC), wherein said BPC comprises a mixture of live bacteria and biologically active compounds (BAC), said mixture adapted to control said diseases.

It is another object of the present invention to provide the method as defined above, additionally comprising steps of selecting said live bacteria from a group consisting of

Lactobacillus acidophilus, Lactobacillus casei, Bifido bacterium bifidium,

Enterococcus faecium, Bacillus subtilis.

It is another object of the present invention to provide the method as defined above, additionally comprising steps of selecting said BAC from a group consisting of rice mill by-product and Calcium carbonate.

It is another object of the present invention to provide the method as defined above, additionally comprising steps of formulating said BPC and/or BAC with at least one immunomodulator especially useful for improving the non-specific immune response in Tilapia.

It is another object of the present invention to provide the method as defined above, additionally comprising steps of selecting said immunomodulator from a group consisting of non-specific Immunostimulators.

It is another object of the present invention to provide the method as defined above, wherein said method comprises steps of obtaining said BPC adapted to provide biological activities, said activities selected from a group consisting of anti-bacterial activity, anti-protozoal activity, increased growth rates, decreased Feed-Conversion

Rate (FCR), decreased mortality rate activity, improving water quality parameters activity or any combination thereof.

It is another object of the present invention to provide the method as defined above, additionally comprising steps of obtaining said BPC adapted to controlling aquatic pathogenic diseases caused by bacteria or parasites in Tilapia.

It is another object of the present invention to provide the method as defined above, wherein said method comprises steps of selecting said bacteria from a group consisting of Streptoccoucus spp. and Aeromonas spp. infections. It is another object of the present invention to provide the method as defined above, wherein said method comprises steps of administering BPC to said Tilapia, said BPC especially adapted for controlling intestinal Coccidial infections.

It is another object of the present invention to provide the method as defined above, wherein said method comprises steps of administering said BPC characterized by having a nitrifying activity useful for improving defined water quality parameters, to said Tilapia.

It is another object of the present invention to provide the method as defined above, wherein said method comprises steps of administering said BPC adapted for increasing the growth rates of said Tilapia by about 12% to about 15%.

It is another object of the present invention to provide the method as defined above, wherein said method comprises steps of administering said BPC adapted for decreasing the mortality rates of said Tilapia by about 30%.

It is another object of the present invention to provide the method as defined above, wherein said method comprises steps of administering said BPC adapted to decrease the Feed Conversion Rate (FCR) of said Tilapia by about 15%.

It is another object of the present invention to provide the method as defined above, wherein said method comprises steps of providing said BPC as a formulation for premix of Tilapia feed and administering said feed to said Tilapia.

It is another object of the present invention to provide the method as defined above, wherein said method comprises steps of formulating said BPC with a mixture of live probiotic bacteria.

It is another object of the present invention to provide the method as defined above, wherein said method comprises steps of formulating said BPC with a fermentation product of dehydrated live bacteria.

It is another object of the present invention to provide the method as defined above, wherein said method comprises steps of selecting said probiotic bacteria from strains of bacteria including but not limited to strains of Lactobacillus acidophilus,

Lactobacillus casei, Bifido bacterium bifidium, Enterococcus faecium, Bacillus subtilis.

It is another object of the present invention to provide the method as defined above, wherein said method comprises steps of obtaining said BPC comprising regulative compounds and additives selected from a group consisting of rice mill by- products and Calcium carbonate, formulating them into said BPC and administering said BPC to said Tilapia.

It is another object of the present invention to provide the method as defined above, wherein said method comprises steps of incorporating said BPC into aquatic animal feed and administering said feed to said Tilapia.

It is another object of the present invention to provide the method as defined above, wherein said method comprises steps of incorporating said BPC into said aquatic animal feed, mixing at the feed mill or on the farm at an inclusion rate of about 0.3%.

It is another object of the present invention to provide a protocol useful for controlling or preventing diseases hi Tilapia, said protocol defined by administering in a predetermined manner, a bioactive pharmaceutical composition (BPC) to a predetermined starter population of Tilapia in an aquacultural facility so as to improve parameters concerned with biological activities of said starter population.

It is another object of the present invention to provide the protocol as defined above, wherein said BPC comprises a mixture of live bacteria and biologically active compounds (BAC).

It is another object of the present invention to provide the method as defined above, wherein said aquacultural facility selected from a group consisting of vat, tank, cage, pond, stream, or lake.

It is another object of the present invention to provide the method as defined above, wherein said biological activities selected from a group consisting of anti-bacterial activity, anti-protozoal activity, increased growth rates, decreased Feed-Conversion

It is another object of the present invention to provide the method as defined above, wherein said growth rate is about 12% to about 15% higher in said predetermined starter population of Tilapia in said aquaculture facility administered by said protocol compared to similar predetermined starter population of Tilapia in said aquaculture facility which has not been administered by said protocol.

It is another object of the present invention to provide the method as defined above, wherein said mortality rate is about 30% lower in said predetermined starter population of Tilapia in said aquacultural facility administered by said protocol compared to similar predetermined starter population of Tilapia in said aquacultural facility which has not been administered by said protocol. It is another object of the present invention to provide the method as defined above, wherein said Feed Conversion Rate (FCR) values are about 15% lower in said predetermined starter population of Tilapia in said aquacultural facility administered by said protocol compared to similar predetermined starter population of Tilapia in said aquacultural facility which has not been administered by said protocol. It is another object of the present invention to provide the method as defined above, wherein said BPC is characterized by having a nitrifying activity useful for improving defined water quality parameters by about 25% in said predetermined starter population of Tilapia in said aquacultural facility administered by said protocol compared to water quality parameters obtained from a similar aquaculture which has not been administered by said protocol.

It is another object of the present invention to provide the method as defined above, wherein said protocol is administered to said Tilapia such that less than about 50% of anti microbial compounds are required to be administered to said Tilapia to achieve the same yield of Tilapia as is achieved in an aquaculture of Tilapia which has not been administered by said protocol.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to understand the invention and to see how it may be implemented in practice, a plurality of embodiments is adapted to now be described, by way of non- limiting example only, with reference to the accompanying drawings; wherein: FIG. 1 is photographic representation of a fish pond at Ein-Hamifratz earthen fish . ponds as a preferred embodiment of the invention;

FIG. 2 is a graphic representation of the Feed Conversion Rate (FCR) of Tilapia in treatment and control ponds;

FIG. 3 is a graphic representation of the survival rate of Tilapia in treatment and control ponds;

FIG. 4 is a graphic representation of the average net weight gain of Tilapia in treatment and control ponds;

FIG. 5 is a graphic representation of the average net weight gain per Dunam of Tilapia in treatment and control ponds;

FIG. 6 is a graphic representation of the growth rate of Tilapia in treatment and control ponds; FIG. 7 is a photographic representation of the vats accommodating Red Tilapia aurea fingerlings as preferred embodiments of the invention;

FIG. 8 is a graphic illustration of the mortality rates in treatment and control Red

Tilapia aurea groups;

FIG. 9 is a graphic representation of the effect of probiotic feed additives on ulcerative disease of Koi (Cyprinus carpio);

FIG. 1OA is a photographic illustration of typical ulceration in Koi {Cyprinus carpio) in accordance with embodiments of the current invention; and,

FIG. 1OB is a photographic illustration of severe ulceration in Koi (Cyprinus carpio) in accordance with embodiments of the current invention.

DETAILED DESCRIPTION OF THE INVENTION

It is herein acknowledged that a core principle of the invention is the provision and disclosure of a composition comprising a concentrated mixture of live bacteria specifically selected for enhancement of non-specific immunity, growth and disease resistance in Tilapia. Factors Contributing to Disease in Fish

Most infectious diseases offish are opportunistic. This means that the simple presence of the pathogen in the environment of the fish is inadequate to cause a disease outbreak. Other factors usually come into play such that either the pathogen has an advantage over the host, or the immune system of the host is compromised in some way, increasing its susceptibility to the pathogen. This phenomenon is often precipitated by "stress". Stress often plays a significant role in outbreaks of infectious disease in fish populations. Some stressors that have been associated with Streptococcus outbreaks include high water temperatures (e.g., during the summer), high stocking densities, harvesting or handling, and poor water quality, such as high ammonia or nitrite concentrations.

Bacteria that cause disease in fish are often categorized based upon their perceived or documented virulence (ability to cause disease). However, individual bacterial strains often differ in their virulence, and many bacteria have a wide spectrum of virulence, with true opportunists being the least virulent and primary pathogens being considered the most virulent. For example, the most common bacterial pathogen in freshwater fish, Aeromonas hydrophila, is considered an opportunistic pathogen, because it is relatively common in the aquaculture environment (hence termed "environmental bacteria") and typically does not cause disease in healthy, well- maintained fish populations.

Streptococcus (Strep), on the other hand, does not seem to be a truly opportunistic pathogen, as it can be more aggressive than many other environmental bacteria. Ideally, after identification of the bacterium in a sick fish, a sensitivity test should be conducted to select the most effective antibiotic to use. Typically, Gram-positive bacteria, including Strep, are susceptible to erythromycin. One oral dosage rate that has proven effective in the field is 1.5 grams of erythromycin per pound of food, fed for 10 to 14 days. Amoxicillin has also been shown to be effective against Strep in experimentally infected tilapia and sunshine bass at an oral dosage rate of 80 mg/kg body weight (3.6 grams per pound of food) fed for 8 to 12 days (Darwish and Ismaiel 2003; Darwish and Hobbs 2005). However, sensitivity testing will confirm whether or not one of these is the antibiotic of choice when an outbreak is in progress. Consequently, the assistance of a fish health specialist or diagnostic laboratory is highly recommended. There are restrictions concerning the antibiotics which can be legally used to treat food or game fish. A fish health specialist should be consulted before attempting to treat fish with a potentially illegal drug that could result in tissue residues. A fish health specialist can also recommend the proper dosage regime for a particular antibiotic and situation. Prevention

Prevention of disease is always preferable and more profitable than treatment of disease outbreaks. Preventive medicine programs should be designed to minimize stress (see IFAS Extension Circular 919, Stress - Its Role in Fish Diseases); maintain the best water quality possible (see IFAS Extension Circular 715, Management of Water Quality for Fish); and minimize exposure to infectious agents by following appropriate disinfection and sanitation protocols (see IFAS Extension fact sheet VM- 87, Sanitation Practices for Aquaculture Facilities). Although Strep does seem to occur more frequently at warmer temperatures, it can occur at any time of the year. Studies in marine systems in Japan (Kitao et al. 1979) indicate that Strep may be present in salt water and mud, with higher incidences in the water during summer months. This implies that, in aquaculture systems some Streptococcus that occur naturally in the environment may become endemic (established within the farm) and cause disease on a periodic basis. As a result, Strep infections may be a cyclic, recurring problem especially during periods of high stress. Immunostimulators added to the feed, such as beta-glucans and nucleotides, have been shown to increase survival for infected redtail black shark (ornamental fish) populations (Russo et al., 2006).

Vaccines may be useful for facilities that have continual or cyclic outbreaks of Streptococcus. Autogenous vaccines (vaccines developed for a specific facility, targeting a specific bacteria isolated from a disease outbreak at that facility) have been shown to be effective under certain conditions. Commercial vaccines may also be available for use within the next few years.

Examples of Strep species that have been associated with disease in fish include Streptococcus difficilis, isolated from fish in Israel; S. milleri, isolated from the kidney samples of koi observed with external ulcerations; and S. parauberis, isolated from farmed turbot in northern Spain (Austin and Austin 1999). Streptococcus iniae is another species that has been isolated from diseased fish and mammals (Austin and Austin 1999). Other Gram-positive bacteria that are closely related to Strep and which have been associated with disease in fish include, Lactococcus garvieae, isolated from diseased eels and yellowtail in Japan (Austin and Austin 1999); L. piscium; and Vagococcus salmoninarum, isolated from diseased rainbow trout. As used herein the term "immunomodulator" refers to a drug which affects the immune system. There are two types of such drugs based on their effects: immunosuppressants and immunostimulators.

The term "immunostimulator" used herein refers to substances (drugs and nutrients) that stimulate the immune system by inducing activation or increasing the activity of any of its components. Specific immunostimulators are those which provide antigenic specificity in immune response, such as vaccines or any antigen. Nonspecific immunostimulators are those which act irrespective of antigenic specificity to augment immune response of other antigen or stimulate components of the immune system without antigenic specificity, such as adjuvants and non-specific immunostimulators such as hormones and nutrients.

Immunomodulators play a valuable role in Tilapia where their use includes improving the non-specific immune response to a range of pathogens and enhancing the immune response in aquatic animals with a weak or developing immune system. It is within the scope of the present invention to provide the aforementioned composition comprising such immunimodulators. The term "defined water quality parameters" used herein refers in a non-limiting manner to any physical, chemical and biological characteristics of water (in relationship to a set of standards) such as temperature, dissolved oxygen, ammonia and nitrite levels, pH, carbon dioxide, chlorine, phosphates and turbidity. The term "aquaculture facility" used herein refers in a non-limiting manner to vat, tank, cage, stream, pond or lake.

The term "biological activities" used herein refers to activities selected from a group consisting of anti-bacterial activity, anti-protozoal activity, increased growth rates, decreased Feed-Conversion Rate (FCR), decreased mortality rate activity, improving water quality parameters activity or any combination thereof.

In order to understand the invention and to see how it may be implemented in practice, a plurality of preferred embodiments will now be described, by way of non- limiting example only, with reference to the following examples, wherein all percentages are denoted for weight percents.

EXAMPLE 1

The Effect of Probiotic Feed Additives on Growth and Performance of Tilapia in earthen fish ponds in Ein Hamifrats Fisheries

Materials and Methods

The herein described clinical trial was conducted in Ein-Hamifratz earthen fish ponds.

The experiment included the following fish ponds:

Pond "B", treatment pond, total area -45 Dunam, Dunam=l,000 m².

Pond "E", control pond, total area - 40 Dunam.

The average depth of the ponds was 2 meters. Furthermore, the ponds contained automatic feeding tank and paddlewheels for water aeration as shown in Fig. 1.

Tilapia fry (Oreochromis nilotica X aureus) produced at the Ein-Hamifratz hatchery was used in the trial.

Water quality parameters, such as temperature, dissolved oxygen, ammonia and nitrite levels were monitored during the period of the clinical trial in treatment and control ponds.

The fish in treatment pond ("B") were supplemented with the present invention's

Probiotic feed fish additive at the dose of 2 kg per ton of feed, while, pond "E" was supplemented with normal fish feed as a control. The control fish were fed with fish pellets manufactured by T.L.C. feed mill, code number 8218 (30% protein, 4% fat, 4.5 mm extruded floating pellets) at a ratio of 1.5-3.0% of the daily biomass, for a period of 161 days. The Initial stocking data in the ponds are showed in Table 1.

Table 1: Initial stocking data in ponds

Results

1. Tilapia performance - The Feed Conversion Rate (FCR), survival rate, final weight, weight gain per Dunam and the growth rate of Tilapia fed by the Probiotic supplemented diets, in comparison to control Tilapia are presented in Table 2 and in Figs. 2-6.

Table 2: Tilapia treated by Probiotic Feed Additives versus control

*FCR: Food Conversion Rate. Feed conversion ratio (FCR) is calculated from the number of kilos of feed that are used to produce one kilo of whole fish.

The above results show that, by feeding Tilapia with diet supplemented with the novel probiotic additives of the invention, an improvement in Tilapia performance is achieved, especially, in FCR, in yield per Dunam and in survival rates.

2. Disease diagnosis - lower morbidity levels of disease causing organisms such as Centrocestus (Digenea parasite), Trichodina, Gyrodactylus and Sessiline are expected in treatment ponds fed by the Probiotic supplemented diets in comparison to control ponds.

3. Analysis of economic profitability Tilapia value:

Value of 1.0 Kg Tilapia: $ 3.00 USD

Net increment in body weight (37 kg/Dunam X 45 Dunam Pond "B" =1,665 kg

Value = 1,665 Kg. X $ 3.00 USD/Kg. = $ 4995 USD

Treatment cost:

Estimated cost of Probiotic feed additive: $ 15 USD/Kg.

Dosage of Probiotic Feed additive: 2 Kg. per Ton of Feed.

Estimated cost of Probiotic feed additive per ton of Tilapia feed: $ 30 USD

Food used during 161 days trial in treated pond "B": 41.3 ton

Treatment cost: 41.3 ton food X $ 30 USD/Ton - $ 1239 USD.

Net profit: Pond "B" = $ 4995 - $ 1239 = $ 3756 USD

Thus, it can be concluded that the use of the novel Probiotic Feed Additives in Tilapia aquaculture is effective and advantageous.

EXAMPLE 2

The Effect of Probiotic Feed Additives on health and disease resistance of Red Tilapia aurea challenged with Gram (+) Streptococcus spp. and sub-optimal environmental conditions

The goals of this study were to evaluate the effect of the Probiotic Feed Additives on health and disease resistance of Red Tilapia aurea challenged with Gram (+) Streptococcus spp. and sub-optimal environmental conditions. The evaluation included the following parameters:

• Reaction to disease causing organisms: o Bacteria o Parasites o Fungus

• Morbidity rates

• Mortality rates Experimental protocol:

12 groups of Red Tilapia aurea fmgerlings were stocked for a period of 90 Days in aquaculture facility (vats) illustrated in Fig. 7. Of the above groups:

6 groups of control fish (vat # 1-6) were fed with Red Tilapia feed, R.M.C. 6 groups of treatment fish (vat # 6-12) were fed with R.M.C. supplemented with recommended dose of Probiotic Feed Additive of the invention.

The fish from both groups were stocked at the 30 Gram range, every vat was stocked with 30 fish.

All vats were supplied with warm (23°), chemically balanced well water for a period of 45 Days. During this period, all vats were operating on "open flow" system which provided optimal environmental conditions for the fish.

Following 45 days of feeding and optimal environmental conditions, all vats were exposed to sub-optimal environmental conditions including increased water temperatures, increased levels of ammonia, increased levels of Nitrite, decreased levels of oxygen and increased levels of suspended solids.

In addition, during this period each vat was stocked with 5 large Red Tilapia aurea

(+500 Gram) with characteristic clinical symptoms of Streptoccoucus infection.

The Fish in all vats were monitored for additional period of 45 Days:

• The presence of disease causing organisms (parasites, bacteria or fungal infections) and clinical symptoms of Streptoccoucus were continuously assessed during the experiment. Daily assessment included the following water quality parameters:

1. Ammonia

2. Nitrite

3. PH

4. Temperature

5. Oxygen

• A daily assessment of mortality and morbidity rates was conducted.

• Veterinary assessments were conducted on a weekly basis. This assessment included a detailed clinical evaluation of 3-5 fish per vat.

Results:

The mortality rates in treatment groups supplemented with the Probiotic Feed Additives of the present invention in comparison to control groups is presented in Table 3 and in Fig. 8. Table 3: Mortality rates in treatment and control groups (Day 1-45 Post Challenge)

The above results show that treating Tilapia with Probiotic Feed Additives of the present invention reduces fish mortality by two fold in comparison to control fish, indicating the effectiveness of the Probiotic Feed Additive in Tilapia survival.

EXAMPLE 3

The effect of Probiotic Feed Additives on treatment of Carp Ulcerative Disease

Syndrome (Atypical Aeromonas salmonicidά)

Introduction- Carp Ulcerative Disease:

Ulcerative Disease, induce heavy economic loss in Cyprinus carpio populations. Atypical

Aeromonas salmonicida has been determined as the main causative agent, however some strains of A. hydrophϊla can induce an identical condition.

Ulcerative Disease of Carp is frequently related to the development of primary skin lesions, caused by ectoparasites (especially leeches and lice) which injure the skin, or to small wounds caused by bad management, usually during sorting the fish.

Bacteria can infect only debrided areas of skin, such as lessions and wounds, thus the main advantage of an intact skin is the formation of an impermeable barrier to said bacteria.

Reduced body condition after a long winter can favour the outbreak of Ulcerative

Disease. The clinical signs of Ulcerative Disease include centrally necrotic areas on the body surface surrounded by a haemorrhagic halo, subsequently, there are deep ulcers with a diameter of up to about 4 cm including skin subcutis and underlying muscles.

The carp's general behaviour is often normal, but these open surface areas may induce an ionic imbalance due to loss through the unprotected surface areas. There is often spontaneous healing but deep, constricted scars are developed and there maybe curvature of the vertebrae and body axis as a consequence. Experimental Protocol and initial results:

1. Heavily Ulcerated Koi (Cyprinus carpiό) were stocked in ponds # 4 and # 5 of the Gan - Shmuel Ornamental Fish farm:

Pond # 4, treatment . pond: 10,000 heavily Ulcerated Koi were stocked on

01/04/2005, their feeding was based on R.M.C. Koi Carp feed, supplemented with the recommended dose of "Common Carp Probiotic Formula".

Pond # 5, control pond: 6000 heavily Ulcerated Koi were stocked on 23/03/2005, their feeding was based on R.M.C. Koi Carp feed.

In both ponds the final ratio of Ulcerated fish was 90%.

Reference is now made to Fig. 10 presenting typical ulceration in Koi (Fig.10A) and severe ulceration in Koi (Fig. 10B) as embodiments of the invention.

2. Pond # 5: one month after stocking the fish (on 24/3/2005), a 10 day protocol of medicated feed was administered. This protocol was repeated monthly.

Feed was medicated with T.M.S. (potentiated Sulfa) at a ratio of lOOmg Active Ingredient per Day, based on 1% of Body Wt. per Day.

3. Fish in both ponds were evaluated for presence of Ulcerative disease during a 90 day period. The Ulceration disease rates in treatment pond # 4, supplemented with Probiotic feed additives and in control pond # 5, administered with medicated protocol are presented in Table 4 and Fig. 9.

Table 4: Ulceration rate (%

The ulcerated fish treated with the novel Probiotic feed additives showed a significant reduction in ulceration disease rates relative to ulcerated fish treated with the known medicated feed (containing T.M. Sulfa), which did not cause any improvement in Carp disease.

Thus the above results indicate that the Probiotic feed additives are effective in treatment and curing of ulceration disease syndrome in Carp. It is herein disclosed that the present invention provides a bioactive pharmaceutical composition (BPC) useful in controlling or preventing diseases in Tilapia. It is a core aspect of the invention that the aforementioned pharmaceutical composition comprises a mixture of live bacteria and biologically active compounds (BAC). It is herein, further acknowledged that a preferred embodiment of the invention provides the aforementioned mixture comprising in a non limiting manner at least some of the following;

• Dehydrated Lactobacillus acidophilus fermentation product

• Dehydrated Lactobacillus casei fermentation product

• Dehydrated Bifido bacterium bifidium fermentation product

• Dehydrated Enterococcus faecium fermentation product

• Bacilus subtilis

• Rice mill by product

• Calcium carbonate

Thus according to preferred embodiments of the invention, live bacteria of the BPC are selected from a group consisting of Lactobacillus acidophilus, Lactobacillus casei, Bifido bacterium bifidium, Enterococcus faecium, Bacillus subtilis. It is herein a further aspect of the invention to disclose the BAC selected from a group consisting of rice mill by -product and Calcium carbonate.

It is a further aspect of the invention to disclose the BPC and/or BAC comprises at least one immunomodulator especially useful for improving the non-specific immune response to a range of aquatic pathogens in Tilapia.

It is a further aspect of the invention to provide the immunomodulator selected from a group consisting of non-specific Immunostimulators.

It is a further aspect of the invention to provide the immunomodulator adapted to enhance the immune response in aquatic animals with a weak or developing immune system.

It is a further aspect of the invention to disclose the BPC adapted to provide biological activities. The aforementioned activities selected from a group consisting of antibacterial activity, anti-protozoal activity, increased growth rates, decreased Feed Conversion Rate (FCR), decreased mortality rate, improving water quality parameters activity or any combination thereof.

It is a further aspect of the invention to disclose the BPC especially adapted for controlling aquatic pathogenic diseases caused by bacteria or parasites. The aforementioned aquatic bacterial infections selected from a group consisting of

Streptoccocus spp. infections and Aeromonas spp. infections.

According to another embodiment of the invention, the BPC is adapted to control intestinal Coccidial infections in Tilapia.

According to one embodiment of the invention the BPC is characterized by having a nitrifying activity useful for improving defined water quality parameters in aquaculture.

According to another embodiment of the invention, the BPC is adapted for increasing the growth rates of Tilapia by about 12% to about 15%.

According to another embodiment of the invention the BPC is adapted for decreasing the Feed Conversion Rate (FCR) in Tilapia by about 15%.

According to another embodiment of the invention, the BPC is adapted for decreasing the mortality rate of Tilapia by about 30%.

According to another embodiment of the invention, the BPC is provided as a formulation for premix of Tilapia feed.

According to another embodiment of the invention the BPC is adapted for incorporating into aquatic animal feed.

According to another embodiment of the invention the BPC is incorporated into said aquatic animal feed, mixed at the feed mill or on the farm at an inclusion rate of about

0.3%.

According to another embodiment of the invention, the probiotic bacteria are used as a mixture of live probiotic bacteria.

According to another embodiment of the invention the BPC comprises a fermentation product of dehydrated live bacteria.

According to another embodiment of the invention the BPC comprises probiotic bacteria selected from bacteria strains including but not limited to strains of

Lactobacillus acidophilus, Lactobacillus casei, Bifido bacterium bifidium,

Enterococcus faecium, Bacillus subtilis.

According to another embodiment of the invention the BPC comprises regulative compounds and additives selected from a group consisting of rice mill by- products and calcium carbonate.

According to another embodiment of the invention the BPC is adapted to reduce the need for antimicrobial compounds by 50% in Tilapia aquaculture. It is herein disclosed a method of controlling or preventing diseases in Tilapia. The aforementioned method comprises steps of feeding the Tilapia with a bioactive pharmaceutical composition (BPC). The BPC comprises a mixture of live bacteria and biologically active compounds (BAC). The disclosed mixture is adapted to control the aforesaid diseases.

It is herein further disclosed that the aforementioned method additionally comprises steps of selecting the live bacteria from a group consisting of Lactobacillus acidophilus., Lactobacillus casei, Bifido bacterium bifidium, Enterococcus faecium,

Bacillus subtilis.

It is herein further disclosed that the aforementioned method additionally comprises steps of selecting the BAC from a group consisting of rice mill by-product and

Calcium carbonate.

It is herein further disclosed that the aforementioned method additionally comprises steps of formulating the BPC and/or BAC with at least one immunomodulator especially useful for improving the non-specific immune response in Tilapia.

It is herein further disclosed that the aforementioned method additionally comprises steps of selecting said immunomodulator from a group consisting of non-specific

Immunostimulators.

It is herein further disclosed that the aforementioned method comprises steps of obtaining the BPC adapted to provide biological activities. The aforesaid activities selected from a group consisting of anti-bacterial activity, anti-protozoal activity, increased growth rates, decreased Feed-Conversion Rate (FCR), decreased mortality rate activity, improving water quality parameters activity or any combination thereof.

It is herein further disclosed that the aforementioned method additionally comprises steps of obtaining the BPC adapted to controlling aquatic pathogenic diseases caused by bacteria or parasites in Tilapia.

It is herein further disclosed that the aforementioned method comprises steps of selecting the bacteria from a group consisting of Streptoccoucus spp. and Aeromonas spp. infections.

It is herein further disclosed that the aforementioned method comprises steps of administering BPC to the Tilapia. The disclosed BPC especially adapted for controlling intestinal Coccidial infections. It is herein further disclosed that the aforementioned method comprises steps of administering the BPC characterized by having a nitrifying activity useful for improving defined water quality parameters, to the Tilapia.

It is herein further disclosed that the aforementioned method comprises steps of administering the BPC₅ adapted for increasing the growth rates of the Tilapia by about

12% to about 15%.

It is herein further disclosed that the aforementioned method comprises steps of administering the BPC adapted for decreasing the mortality rates of the Tilapia by about 30%.

It is herein further disclosed that the aforementioned method comprises steps of administering the BPC adapted to decrease the Feed Conversion Rate (FCR) of the

Tilapia by about 15%.

It is herein further disclosed that the aforementioned method comprises steps of providing the BPC as a formulation for premix of Tilapia feed and administering the feed to the Tilapia.

It is herein further disclosed that the aforementioned method comprises steps of formulating the BPC with a mixture of live probiotic bacteria.

It is herein further disclosed that the aforementioned method comprises steps of formulating the BPC with a fermentation product of dehydrated live bacteria.

It is herein further disclosed that the aforementioned method comprises steps of selecting the probiotic bacteria from strains of bacteria including but not limited to strains of Lactobacillus acidophilus, Lactobacillus casei, Bifido bacterium bifidium,

Enterococcus faecium, Bacillus subtilis.

It is herein further disclosed that the aforementioned method comprises steps of obtaining the BPC comprising regulative compounds and additives selected from a group consisting of rice mill by- products and Calcium carbonate, formulating them into the BPC and administering the BPC to the Tilapia.

It is herein further disclosed that the aforementioned method comprises steps of incorporating the BPC into aquatic animal feed and administering the feed to the

Tilapia.

It is herein further disclosed that the aforementioned method comprises steps of incorporating the BPC into the aquatic animal feed, mixing at the feed mill or on the farm at an inclusion rate of about 0.3%. It is a further aspect of the invention to disclose a protocol useful for controlling or preventing diseases in Tilapia. The aforementioned protocol defined by administering in a predetermined manner, a bioactive pharmaceutical composition (BPC) to a predetermined starter population of Tilapia in an aquacultural facility so as to improve parameters concerned with biological activities of the starter population. The BPC further comprises a mixture of live bacteria and biologically active compounds

(BAC).

It is herein further disclosed that the aquacultural facility selected from a group consisting of vat, tank, cage, pond, stream,

It is a further aspect of the invention to disclose the aforementioned protocol sufficient to achieve a growth rate of a predetermined starter population of Tilapia in an aquacultural facility selected from a group consisting of vat, tank, cage, pond, stream, or lake, about 12 to about 15% higher compared with the growth rate of a similar predetermined starter population of Tilapia in the aquacultural facility, which has not been administered by the aforementioned protocol.

It is a further aspect of the invention to disclose the aforementioned protocol sufficient to achieve Feed Conversion Rate (FCR) values in a population of Tilapia in an aquacultural facility about 15% higher than the FCR values obtained in a population of Tilapia in the aquacultural facility, which has not been administered by the aforementioned protocol.

It is a further aspect of the invention to disclose the aforementioned protocol such that the mortality rate of the population of Tilapia is about 30% lower than the mortality rate of a similar population of Tilapia in an aquacultural facility, which has not been administered by the aforementioned protocol.

It is a further aspect of the invention to disclose a protocol for controlling the presence of pathogenic microorganisms selected from a group consisting of Bacteria and

Parasites in the aquatic environment prior to and during the production cycle of

Tilapia in a commercial aquaculture.

It is a further aspect of the invention to disclose a protocol for reducing the clinical effect of Streptococcus spp. and Aeromonas spp. in Tilapia. The aforementioned protocol comprising steps of administering the BPC mixture to a population of

Tilapia, thereby reducing the incidence of intestinal Coccidial infections in the aforementioned Tilapia population compared with the incidence of Intestinal Coccidial infections in a similar Tilapia population which has not been administered with the aforementioned protocol.

It is a further aspect of the invention to disclose a protocol for nitrifying the water in an aquaculture adapted for Tilapia production. The aforementioned protocol comprises steps of administering the BPC mixture to the Tilapia.

It is a further aspect of the invention to disclose the aforementioned protocol wherein the nitrifying effect improves water quality parameters in Tilapia by about 25% compared to water quality parameters obtained from a similar aquaculture which has not been administered with the aforementioned protocol.

It is a further aspect of the invention to disclose a protocol comprising administering the BPC mixture to Tilapia, such that less than about 50% of anti microbial compounds are required to be administered to the aforementioned Tilapia to achieve the same yield of Tilapia as is achieved in Tilapia which has not been administered with the aforementioned protocol.

REFERENCES

FAO Fishery Information, Data and Statistics Service (1993). "Aquaculture production (1985-1991)". FAO Fisheries Circular (FAO) 815: 20-21.

Petr, T. (2000): Interactions between fish and aquatic macrophytes in inland waters. A review. FAO Fisheries Technical Papers 396.

Trewavas, Ethelwynn (1983): Tilapiine fishes of the genera Sarotherodon, Oreochromis and Danakilia. Published by the British Museum (Natural History), London. 583 pages. ISBN 0-565-00878-1

Darwish, A.M. and Ismaiel, A. A. 2003. Laboratory efficacy of amoxicillin for the control of Streptococcus Mae infection in sunshine bass. Journal of Aquatic Animal Health 15:209-214.

Darwish, A.M. and Hobbs, M.S. 2005. Laboratory efficacy of amoxicillin for the control of Streptococcus Mae infection in blue tilapia. Journal of Aquatic Animal Health 17(2): 197-202.

Kitao, T., Aoki, T. and Iwata, K. 1979. Epidemiological study on streptococcosis of cultured yellowtail {Seriola quinqueradiata) — I. Distribution of Streptococcus sp. in seawater and muds around yellowtail farms. Bulletin of the Japanese Society of Scientific Fisheries, 45: 567-72.

Austin, B. and Austin, D.A. 1999. Third ed. Chapter 2: Characteristics of the diseases. In Bacterial Pathogens: Diseases of Farmed and Wild Fish. Springer-Praxis, Praxis Publishing, Ltd. Chichester, cUK. pp 13-15.

Russo, R., H. Mitchell, and R. P. E. Yanong. 2006. Characterization of Streptococcus iniae isolated from ornamental cyprinid fishes and development of challenge models. Aquaculture 256: 105-110.

Claims

1. A bioactive pharmaceutical composition (BPC) useful in controlling or preventing diseases in Tilapia, wherein said pharmaceutical composition comprises a mixture of live bacteria and biologically active compounds (BAC).

2. The BPC according to claim 1, wherein said live bacteria are selected from a group consisting of Lactobacillus acidophilus, Lactobacillus casei, Bifϊdo bacterium bifidium, Enterococcus faecium, Bacillus subtilis.

3. The BPC according to claim 1, wherein said BAC is selected from a group consisting of rice mill by -product and Calcium carbonate.

4. The BPC according to claim 1, wherein said BPC and/or BAC comprises at least one immunomodulator especially useful for improving the non-specific immune response to a range of aquatic pathogens in Tilapia.

5. The BPC according to claim 4, wherein said immunomodulator is selected from a group consisting of non-specific ϊmmunostimulators.

6. The BPC according to claim 4, wherein said immunomodulator is adapted to enhance the immune response in aquatic animals with a weak or developing immune system.

7. The BPC according to claim 1, wherein said BPC is adapted to provide biological activities, said activities selected from a group consisting of anti-bacterial activity, anti-protozoal activity, increased growth rates, decreased Feed Conversion Rate (FCR), decreased mortality rate, improving water quality parameters activity or any combination thereof.

8. The BPC according to claim 1, wherein said BPC is especially adapted for controlling aquatic pathogenic diseases caused by b.acteria or parasites.

9. The BPC according to claim 8, wherein said BPC is adapted for controlling aquatic bacterial infections selected from a group consisting of Streptoccocus spp. infections and Aeromonas spp. infections.

10. The BPC according to claim 8, wherein said BPC is adapted to control intestinal

Coccidial infections in Tilapia.

11. The BPC according to claim 1, wherein said BPC is characterized by having a nitrifying activity useful for improving defined water quality parameters in Tilapia aquaculture.

12. The BPC according to claim 1, wherein said BPC is adapted for increasing the growth rates of Tilapia by about 12% to about 15%.

13. The BPC according to claim I₅ wherein said BPC is adapted for decreasing the

Feed Conversion Rate (FCR) in Tilapia by about 15%.

14. The BPC according to claim 1, wherein said BPC is adapted for decreasing the mortality rate of Tilapia by about 30%.

15. The BPC according to claim 1, wherein said BPC is provided as a formulation for premix of Tilapia feed.

16. The BPC according to claim 1, wherein said BPC is adapted for incorporating into aquatic animal feed.

17. The BPC according to claim 1, wherein said BPC is incorporated into said aquatic animal feed, mixed at the feed mill or on the farm at an inclusion rate of about 0.3%.

18. The BPC according to claim 1, wherein said probiotic bacteria is used as a mixture of live probiotic bacteria.

19. The BPC according to claim 1, wherein said BPC comprises a fermentation product of dehydrated live bacteria.

20. The BPC according to claim 1, wherein said BPC comprises probiotic bacteria selected from bacteria strains including but not limited to strains of Lactobacillus acidophilus, Lactobacillus casei, Bifido bacterium bifidium, Enterococcus faecium, Bacillus subtilis.

21. The BPC according to claim 1, wherein said BPC comprises regulative compounds and additives selected from a group consisting of rice mill byproducts and calcium carbonate.

22. The BPC according to claim 1, wherein said BPC is adapted to reduce the need for antimicrobial compounds by 50% in Tilapia.

23. A method of controlling or preventing diseases in Tilapia, said method comprising steps of feeding said Tilapia with a bioactive pharmaceutical composition (BPC), wherein said BPC comprises a mixture of live bacteria and biologically active compounds (BAC), said mixture adapted to control said diseases.

24. The method according to claim 23, additionally comprising steps of selecting said live bacteria from a group consisting of Lactobacillus acidophilus, Lactobacillus casei, Bifido bacterium bifidium, Enterococcus faecium, Bacillus subtilis.

25. The method according to claim 23, additionally comprising steps of selecting said

BAC from a group consisting of rice mill by-product and Calcium carbonate.

26. The method according to claim 23, additionally comprising steps of formulating said BPC and/or BAC with at least one immunomodulator especially useful for improving the non-specific immune response in Tilapia.

27. The method according to claim 26, additionally comprising steps of selecting said immunomodulator from a group consisting of non-specific Immunostimulators.

28. The BPC according to claim 23, wherein said method comprises steps of obtaining said BPC adapted to provide biological activities, said activities selected from a group consisting of anti-bacterial activity, anti-protozoal activity, increased growth rates, decreased Feed-Conversion Rate (FCR), decreased mortality rate activity, improving water quality parameters activity or any combination thereof.

29. The method according to claim 23, additionally comprising steps of obtaining said

BPC adapted to controlling aquatic pathogenic diseases caused by bacteria or parasites in Tilapia.

30. The method according to claim 29, wherein said method comprises steps of selecting said bacteria from a group consisting of Streptoccoucus spp. and Aeromonas spp. infections.

31. The method according to claim 29, wherein said method comprises steps of administering BPC to said Tilapia, said BPC especially adapted for controlling intestinal Coccidial infections.

32. The method according to claim 23, wherein said method comprises steps of administering said BPC characterized by having a nitrifying activity useful for improving defined water quality parameters, to said Tilapia.

33. The method according to claim 23, wherein said method comprises steps of administering said BPC adapted for increasing the growth rates of said Tilapia by about 12% to about 15%.

34. The method according to claim 23, wherein said method comprises steps of administering said BPC adapted for decreasing the mortality rates of said Tilapia by about 30%.

35. The method according to claim 23, wherein said method comprises steps of administering said BPC adapted to decrease the Feed Conversion Rate (FCR) of said Tilapia by about 15%.

36. The method according to claim 23, wherein said method comprises steps of providing said BPC as a formulation for premix of Tilapia feed and administering said feed to said Tilapia.

37. The method according to claim 23, wherein said method comprises steps of formulating said BPC with a mixture of live probiotic bacteria.

38. The method according to claim 23, wherein said method comprises steps of formulating said BPC with a fermentation product of dehydrated live bacteria.

39. The method according to claim 23, wherein said method comprises steps of selecting said probiotic bacteria from strains of bacteria including but not limited to strains of Lactobacillus acidophilus, Lactobacillus casei, Bifϊdo bacterium bifidium, Enterococcus faecium, Bacillus subtilis.

40. The method according to claim 23, wherein said method comprises steps of obtaining said BPC comprising regulative compounds and additives selected from a group consisting of rice mill by- products and Calcium carbonate, formulating them into said BPC and administering said BPC to said Tilapia.

41. The method according to claim 23, wherein said method comprises steps of incorporating said BPC into aquatic animal feed and administering said feed to said Tilapia.

42. The method according to claim 23, wherein said method comprises steps of incorporating said BPC into said aquatic animal feed, mixing at the feed mill or on the farm at an inclusion rate of about 0.3%.

43. A protocol useful for controlling or preventing diseases in Tilapia, said protocol defined by administering in a predetermined manner, a bioactive pharmaceutical composition (BPC) to a predetermined starter population of Tilapia in an aquacultural facility so as to improve parameters concerned with biological activities of said starter population.

44. The protocol according to claim 43, wherein said BPC comprises a mixture of live bacteria and biologically active compounds (BAC).

45. The protocol according to claim 43, wherein said aquacultural facility selected from a group consisting of vat, tank, cage, pond, stream, or lake.

46. The protocol according to claim 43, wherein said biological activities selected from a group consisting of anti-bacterial activity, anti-protozoal activity, increased growth rates, decreased Feed-Conversion Rate (FCR), decreased mortality rate activity, improving water quality parameters activity or any combination thereof.

47. The protocol according to claim 46, wherein said growth rate is about 12% to about 15% higher in said predetermined starter population of Tilapia in said aquaculture facility administered by said protocol compared to similar predetermined starter population of Tilapia in said aquaculture^' facility which has not been administered by said protocol.

48. The protocol according to claim 46, wherein said mortality rate is about 30% lower in said predetermined starter population of Tilapia in said aquacultural facility administered by said protocol compared to similar predetermined starter population of Tilapia in said aquacultural facility which has not been administered by said protocol.

49. The protocol according to claim 46, wherein said Feed Conversion Rate (FCR) values are about 15% lower in said predetermined starter population of Tilapia in said aquacultural facility administered by said protocol compared to similar predetermined starter population of Tilapia in said aquacultural facility which has not been administered by said protocol.

50. The protocol according to claim 46, wherein said BPC is characterized by having a nitrifying activity useful for improving defined water quality parameters by about 25% in said predetermined starter population of Tilapia in said aquacultural facility administered by said protocol compared to water quality parameters obtained from a similar aquaculture which has not been administered by said protocol.

51. The protocol according to claim 43, wherein said protocol is administered to said

Tilapia such that less than about 50% of anti microbial compounds are required to be administered to said Tilapia to achieve the same yield of Tilapia as is achieved in an aquaculture of Tilapia which has not been administered by said protocol.