Patent Description:
It has been known that the aquatic environment in which grows up and cultivates fishes, favors the emergence of diseases. Many measures have been taken for controlling or preventing these diseases, and until now the vaccination, is one of the tools more used for the control of bacterial diseases in fishes. This due to vaccinations show some advantages that begin from their good effectiveness preventing and correcting infections to their low impact in the environment and in the public health, allowing the obtaining of a clean product. In general, this doesn't happen with antibiotics, and among its adverse effects can be mentioned: development of the bacterial resistance; accumulation of antibiotics in the muscle of the fish; contamination of aquatic environments among others.

Nevertheless, the vaccination has disadvantages like it requires of a big manipulation of the fishes, so its practice requires of being careful for not stressing the fishes and doing a little bit effective de vaccination or cause mortality, also adverse effects can be shown like the existence of adherences that can lead to mortalities and decrease in rates of fishes growing. In general, the diseases increase under stress conditions, and certainly, in the intensive production systems used for fish farming, those conditions, are always present. We don't have to forget that aquaculture is the fastest growing sector in the production of food, being one of the main economic activities of this century and with important projections of being the main source of animal protein for human consumption in accordance with the last studies of the FAO.

The losses of production are large and the damages devastating, in the intensive system of production, when the diseases appear.

On the other hand, it is known that efficient vaccination doesn't exist in the prevention of some relevant intracellular pathogens, this is the case of Pisciricketssia salmonis, SAV virus, Francisella noatunensis, Renibacterium salmoninarum, IPNv, in early stages, previous to the vaccination shot. This is due to the intracellular nature of the pathogens, which requires of the activation of an immune answer of the type Th1, activating mainly lymphocytes T cytotoxic, the ones that are in charge of destroying infective cells. The combination of bioactive molecules formed by Fuicoidans + Andrographolide, that causes the production by part of the macrophages of cytokines like the IL-<NUM> and IFN, that promote the differentiation of lymphocytes Th0 to Th1, this particularity in their action mechanism, transform the combination in a new tool for prevention and control of intracellular pathogens in salmonids fishes.

The diseases cause in the fishes, symtoms like erosions, warts, eruptions or spots in the skin, flap fraying, inflammation of the abdomen, erratic swimming, bleedings, injuries and ulcers in the pancreas, esophagus, muscle spasms and ascites, among others.

Between the intracellular microorganisms for fishes, we can find: pisciricketssia; viruses such as viral hemorrhage septicemia virus (VHS), infectious pancreatic necrosis virus (IPNv), infectious hematopoietic necrosis virus (IHNV), salmon alphavirus (SAV), infectious salmon anemia (ISAv), and bacteria like Francicella sp and Renibacterium salmoninarum.

Between diseases cause by these pathogens, that are important in aquaculture we can mention piscirickettsiosis, viral hemorrhage septicemia, infectious pancreatic necrosis, infectious hematopoietic necrosis, sickness of pancreas and sleeping disease, infectious salmon anemia, francisellosis y renibacteriosis.

In particular, since <NUM>, in the south of Chile, a disease with high mortalities in salmon was detected, called "Sindrome del salmon coho" (salmon coho syndrome), "Sindrome de Huito" (syndrome huito) or piscirickettsiosis (<NPL>on) and which affected various species of salmonids. At the beginning it was found only in the coho salmon, but after affected all the salmonids species cultivated in Chile, causing until a <NUM>% of mortality in some places (<NPL>. The disease was found mainly in sea water and estuarine (<NPL>. <NPL>; <NPL>) and rarely in sweet water (<NPL>).

The etiologic agent corresponded to the first isolated rickettsia and characterized from aquatic animals and was called Piscirickettsia salmonis (P. This pathogen is an obligate intracellular parasite, cytopathic for different salmon cell lines and some warm-water fishes. It is Gram negative, pleomorphic, usually coconut, in pairs or in ring shapes and of a size between <NUM>-<NUM> in diameter (<NPL>).

The clinical signs of the piscirickettsiosis are characterized by swimming on the surface, slowly, erratically and sometimes in a corkscrew way. In addition lethargy, anorexia, shock against the walls of the cage raft, marring and darkening have been described. The most relevant external macroscopic lesions described, include: peeling, branchial pallor, equimotic and petechial bleeding at the base of the fins, nodules an ulcers in the skin up to <NUM> diameter (<NPL>; <NPL>; <NPL>. Hematocrit levels reveal severe anemia (<NPL>.

In the analysis of necropsy of the abdominal cavity, the presence of ascites, renomegaly and splenomegaly was found frequently, subcapsular nodules of creamy to yellowish color in the liver, the presence of a pseudomembrane over the heart and petechial bleeding in the stomach, pyloric blind, intestine, swimming bladder, muscle and visceral fat (<NPL>. In most cases, the intestine was filled with a yellowish mucous content and the stomach with a seromucous transparent liquid (<NPL>); The latter gives the impression that the fish has been swallowed some water (<NPL>).

From the histopathological point of view, the main injuries were necrosis in different organs and tissues, being the most affected the kidney, liver, spleen and intestine. It was also common to see vascular injuries similar to the mentioned for rickettsias in mammals, like the endothelial necrosis and thrombus formation.

In addition, macrophages containing organisms within the cytoplasm, intravascular coagulation, perivascular inflammation, pericarditis, endocarditis, chronic inflammatory injury in the layer of the intestine, increased granule cell numbers of the intestinal granular stratum and lamellar hyperplasia and fusion were found. (<NPL>, <NPL>.

For the diagnosis, smear and tissue staining methods are recommended with Gram, Giemsa, acridine barabha (<NPL>; <NPL>; <NPL>) and / or toludine blue (<NPL>). These techniques are suitable for initial routine identification. The indirect immunofluorescence method (IFAT), developed by Lannan et al. (<NPL>), is nowadays one of the most sensitive and specific methods for the detection of piscirickettsiosis. This technique has been modified by the use of microwaves (<NPL>), markedly decreasing the incubation times of the first and second antibody, without varying the specificity and sensitivity.

Another important disease is infectious pancreatic necrosis (IPN), a disease caused by a birnavirus which affects several wild and cultivated aquatic organisms (<NPL>). Salmonidae are mainly affected, so this disease has a significant impact on salmon and trout farming, due to a high mortality of offspring and fry. IPN is on the list of fish diseases of the World Organization for Animal Health (OIE) in its International Aquatic Animal Health Code and should be notified (World Organization for Animal Health (OIE). International health code for Aquatic Animals, France: OIE, <NUM>).

The causal agent in a virus of the bimavirus family that is icosahedral in shape, approximately <NUM> in diameter (<NPL>, <NPL>). It contains a genome composed of two double-stranded ribonucleic acid (RNA) segments. Segment A codifies for two structural proteins (VP2 and VP3) and a non-structural protease, while segment B codifies for an RNA polymerase (<NPL>). The VP2 protein stimulates the production of neutralizing monoclonal antibodies of a specific type (<NPL>) and it is thought that it contains all epitopes recognized by these antibodies (<NPL>). IPNV penetrates through gills and mouth, or through the sensory pores of the lateral line system (<NPL>, <NPL>). The vertical transmission has been verified in rainbow trout (Oncorhychus mykiss) and brown trout (Salmo Trutta); in other species, cases have been observed associated with infected sexual eggs or fluids, which probably correspond to a vertical infection by external contamination (<NPL>). It has been proposed that vertical infection is also associated with the concentration of viral particles (<NPL>). After a period of undetectable viremia, at four days post-infection aproximately necrotic areas are observed in the exocrine pancreas and other organs (<NPL>); however, the viral distribution may be variable in the different organs Eléout et al. , (<NPL>) observed that the virus could be found in several organs with the exception of the pancreas, which it may be associated to a different level of tissue tropism that present different isolates viral.

During clinical disease, mortality is inversely proportional to the age of the affected animals (<NPL>). The most characteristic disease pattern is shown in rainbow trout, brook trout, brown trout, Atlantic salmon and several species of Pacific salmon (<NPL>). In offsprings that have normally completed the first feeding, the disease outbreak is usually less explosive, reaching losses of <NUM>% or more over a period of two months. The losses in larger animals can be between <NUM> and <NUM>% ><NPL>).

Generally, affected fishes show anorexia and irregular swimming (swimming on corkscrew way with ataxia lapses). These fishes change to a dark color (hyperpigmentation) and have moderate exophthalmos and abdominal distention. Gills and haemorrhages in the ventral area, the fins included, are also pale. The fishes are thin and have witish "hanging feces" (<NPL>).

According to the main findings of necropsy in offspring, spleen, heart, liver and kidneys are shown pale, and most of the time no food is found in the digestive tract. Petechial haemorrhages are seen in visceras. In some cases, food is found in small amounts, confined to the distal and rectum part of the intestine. Ascitic fluid is frequently seen in the abdominal cavity. In the stomach and intestine can be observed a cohesive milky mucus, among other findings (<NPL>).

The main injuries found in the histopathological study include foci of coagulative necrosis in the pancreas, kidney and intestines. Pancreatic tissue is shown degenerated, even in the acinar areas, with release of the zymogen granules. The nuclei of the acinar cells are observed pyknotics and of variable sizes. In many cases no infiltration of inflammatory cells is shown.

In the stomach and intestine, there are variable processes of degeneration and necrosis (<NPL>) mucosal detachment (catarrhal enteritis) into the intestinal lumen where epithelial cells with eosinophilic and hyaline cytoplasm and swollen can be observed, many times with their nucleus fragmented, making accumulations of basophilic material, distributed in cellular periphery, showing of a process of apoptosis (<NPL>). In the liver, it is possible to find areas of focal or generalized necrosis, which are usually severe in salmon, whereas in rainbow trout are more moderate or insignificant (<NPL>).

The virulence, which is the relative ability of the pathogen to cause disease, is a manifestation of the interaction between the adverse effects produced by components of the virus and the defense mechanisms developed by the cells to try to eliminate the infection; however, the result of such interactivity is always determined by the virus through its virulence factors, a function that can be applied by any component of the viral particle (<NPL>). The differences in the level of virulence shown between different strains of IPNV have been attributed to their genetic variation (<NPL>), and with the property of the virus to modify cell signaling pathways through viral proteins encoded by its A segment, able to manipulate cellular machinery to facilitate viral synthesis and avoid the answer of the defense (<NPL>; <NPL>). In this sense, the viral proteins considered as the main virulence factors of IPNV are VP2, a component of the outer cover of the viral capsid that participates in the recognition of the virus to the cells; the VP5 protein of inconclusive function, since it has been shown not to be necessary for establishing infection and the viral multiplication, but with anti-apoptotic activity that apparently has no relation in the establishment of the carrier state. Recently, it has been reported that over expression of the VP3 induce apoptosis in cultured cells, but it is difficult to detect in an infection with fully viral particles. However, Vp4 and Vp1 have not been associated with adverse effects at the cellular level, but proteins of similar activity in other viruses have been observed with implication in the pathogenicity of the strains (<NPL>; <NPL>; <NPL>).

During the infection process, the host expresses a varied response aimed at attempting to prevent infection or dissemination of the agent. For this, the non-specific defense system is the most important as a protection measure for fishes, and within this, the interferon system (IFN) is one of the first lines from defense of viral infection through inducing the synthesis of proteins having antiviral activity; in the case of IPNV, the Mxl protein and the kinase protein dependent of double stranded RNA (PKR) have been shown to have antiviral activity (<NPL>). However, it has been reported that some viruses may inhibit or modulate the antiviral response exerted by IFN (<NPL>), which it is supposed also for IPNV (<NPL>), but it has not been evaluated.

IPNV shows high antigenic and genotypic variability, features that influence the virus-cell interaction, the virulence and the development of the carrier state; however, the mechanisms involved in these processes are not fully determined (<NPL>).

The procedure for the identification of NPI, as recommended by the OIE, is based on the isolation of VNPI in cell culture, followed by the immunological identification of the isolation by immunofluorescence tests (<NPL>), serum neutralisation (<NPL>) y ELISA (<NPL>).

The diagnosis of clinical cases is generally based on the histology and immunological evidence of VNPI in the infected tissues. These cases are confirmed by the isolation and immunological identification of the virus by means of such tests (<NPL>). Serological tests in order to identify antibodies against VNPI in infected fishes have not yet been recognized by the OIE (<NUM>), due to insufficient knowledge of the humoral immune response of the fishes to this virus (<NPL>).

The detection of VNPI in cell lines is consistent and simple, mainly in lines belonging from homologous species. This is because <NUM>) the virus is present in elevated titles in tissues; <NUM>) The isolation can be performed from non-diseased animals; <NUM>) there is no phase in which the virus cannot be isolated; <NUM>) the time required for isolation and identification of the agent is from two to three weeks, which is not critical for the presentation of an epizootic, and <NUM>) high sensitivity and cytopathic effect can be seen easily. The cell lines used for the isolation of VNPI include the RTG-<NUM> (rainbow trout gonad), CHSE-<NUM> (chinook salmon embryo) and BF-<NUM> (bluegill fry) (<NPL>).

Nowadays, many methods of detection have been developed by means of the technique of reverse-reaction transcription in the polymerase chain reaction (RT-PCR, by their acronysms in English: reverse transcriptase-polymerase chain reaction) (<NPL>). However, the sensitivity of this technique has not been greater than that of the cell culture, so that isolation of the virus and serological confirmation are the processes of choice for identifying VNPI.

On the other hand, the use of Andrographis paniculata is known in the treatment of a wide range of pathologies (<NPL>); including diseases such as the common cold, dysentery, fever, tonsillitis, diarrhea, liver diseases, herpes, among others (<NPL>). It has anti-inflammatory and antimicrobial properties, (<NPL>), antithrombotic (<NPL>), hepatoprotective (<NPL>), anti AIDS activity (<NPL>), antidiabetic, (<NPL>) and antitumorals (<NPL>).

The plant is extremely bitter in each of its parts (<NPL>), however, the aerial portions of Andrographis paniculata are used to extract the active principles phytochemicals. The extract contains diterpene, flavonoide, and stigmasters (<NPL>). Being the andrographolide, the first and best isolated active principle, which has been chemically defined as a bicyclic diterpene lactone (<NPL>.

Different of preclinical and clinical studies have been performed to identify the pharmacological properties of the components of Andrographis paniculata, for this, both the raw extract of the plant and the purified andrographolide have been used, like the purified Andrographolide. In preclinical studies, the plant extract has shown several activities, such as: hepatoprotective effect administered intraperitoneally in rats (<NPL>). antidiarrheal activity in animal models treated with enterotoxin of Escherichia coli (<NPL>), and immunostimulatory capacity in mice BALB/c (<NPL>). Another active principle related, but less studied, is the <NUM>-deoxyandrgrapholide which has demonstrated a hypotensive effect (<NPL>) and anti-inflammatory and antipyretic properties (<NPL>).

Other species that may be used instead of Andrographis paniculata are: Andrographis affinis Nees, Andrographis beddomei, Andrographis echioides Nees, Andrographis elongata, Andrographis humifusa, Andrographis lineata Nees, Andrographis macrobotrys Nees, Andrographis paniculata Nees, Andrographis neesiana, Andrographis ovata, Andrographis paniculata Nees, Andrographis Rothii, Andrographis serpyllifolia, Andrographis viscosula Nees, Andrographis viscosula var, explicata y Andrographis wightiana.

Also, in a study conducted at the Research Cluster for Health, Southern Cross University of Lismore, Australia, it is reported that extracts of brown algae containing fucoidan, have been shown to have modulating effect of immunity. This study aimed to determine whether marine algae containing a mixture of extracts from three different species of brown algae is safe to administer, and if it has, a biological potential as an immunomodulator. The results allowed to conclude that the complex of nutrients was safe to administer and furthermore that the preparation proved to have potential as an immune modulator efectively (<NPL>).

Numerous studies indicate that sulfated polyanions, mainly heparin, dextran sulfate, lambda and kappa carrageenan xylogalactans, xylomanans and fuicoidans, possess potent therapeutic activity against viral diseases like as other anticoagulant and antitumor properties (<NPL>).

The mechanism of antiviral action of sulfated polysaccharides is primarily inhibiting the entry of enveloped viruses, such as Herpesvirus (HSV), inside of the host cell, by competence for the cell surface receivers (<NPL>. <NPL>; <NPL>). There is a number of receivers including the heparin sulfate receiver, expressed in various cell types, which provide the incoming points for the Herpes virus. Antiviral activity, in part, is due to the similarity of sulfated polyanions to heparin sulfate molecules in mammals (Campadelli-Fiume et al.

Fucoidan was tested against several viruses involved in DNA and RNA, such as HSV, HCMV, VSV and HIV, and proved to be a potent and selective inhibitor of these viruses, regardless of their type of nucleic acid (<NPL>; <NPL>).

As an example, various immuno-stimulating compositions containing andrographolide or derivatives or fuicoidans are disclosed for use in humans, see like an example the following patent literature: <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>. Also, immuno-stimulating pharmaceutical compositions of fucoidans and chitosan, and fuicoidans and lactobacillus, are known see like an example <CIT> and <CIT>.

Also, vaccines for fish comprising fuicoidans are known, see <CIT>.

The prior art also discloses various immunostimulatories, nutritional compositions or others to treat and prevent diseases in humans, which comprise terpenes or their derivatives and flavonoids and their derivatives, see like a way of example, the following patent literature: <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT> and <CIT>.

The prior art also discloses compositions comprising terpenes or their derivatives and flavonoids and their derivatives in order to restore the color of cultured fishes or to pigment them, promote their growth or the foods that enhance their immunity, see like a way of example the following patent literature: <CIT>, <CIT>, <CIT> and <CIT>.

Likewise, antiviral or sporozoal compositions are known, comprising terpenes or their derivatives and flavonoids and their derivatives, are known to treat fishes diseases, see like a way of example the following patent literature: <CIT> and <CIT>.

However, the present invention relates to a synergistic immunostimulating composition for fishes comprising fuicoidans and andrographolide.

Regarding the fuicoidans it is important to point out that they belong from different species of brown algae, and they differ in accordance to the orders corresponding to brown algae.

Thus, the fucal order comprises the binding of Fucose units which varies according to the species to be analyzed but mainly shows glycosidic bonds of the type (<NUM>→<NUM>) or (<NUM>→<NUM>) and the sulfated groups can be located in the positions C-<NUM>, C-<NUM> or C-<NUM>. Examples of algae from which fucoids can be obtained from the fucal order are: Fucus vesiculosus, Fucus evanescens, Fucus distichus, Fucus serratus, Pelvetia wrightii, Ascophyllum nodosum, Himanthalia Lorea, Bifurcaria bifurcata, Sargassum stenophylfum, Hizikia fusiforme y Durvillaea antárctica.

In the order Laminare and another brown algae, the binding of Fucose units varies by species but mainly shows glycosidic bonds of the type (<NUM>→<NUM>) or (<NUM>→<NUM>) and the sulfated groups can be located in the positions C-<NUM> or C-<NUM>. It is also mentioned that the Galactan fraction present is given by bonds (<NUM>→<NUM>) and (<NUM>→<NUM>) with sulphated groups especially in the position C-<NUM>. Examples of algae from which fucoids of laminar order and other brown algae can be obtained are: Lessonia nigrescens, Lessonia trabeculata, Lessonia vadosa, Macrocystis pyrifera, Undaria pinnatifida, Padina pavonia, Laminaria angustata, Laminaria japónica, Ecklonia kurome, Adenocytis utricularis, Dictyota menstrua/is, Spatog/ossum schroederi y Chordaria flagellifonnis.

The present invention relates to a synergistic immunostimulating composition for fishes comprising fuicoidans and andrographolide. More preferably, the present invention relates to a synergistic immunostimulating composition for fishes comprising fuicoidans and andrographolide, and allowing effective control and prevention of infections produced by intracellular microorganisms. Still more particularly, the present invention relates more closely to a synergistic composition comprising fuicoidans and andrographolide, and allowing effective control and prevention of piscirickettsiosis and infectious pancreatic necrosis (IPN) in fishes.

A veterinary composition for use in fishes comprising an aqueous extract of seaweed with a percentage of fucoidans of <NUM>% and an extract of Andrographis sp plant with a percentage of total andrographolide of <NUM>%, where the fucoidan: andrographolide ratio is of <NUM>:<NUM>, wherein said aqueous extract of seaweed is selected from an aqueous extract of selected brown algae Fucus vesiculosus, Fucus evanescens, Fucus distichus, Fucus serratus, Pelvetia wrightii, Ascophyllum nodosum, Himanthalia Lorea, Bifurcaria bifurcata, Sargassum stenophyllum, Hizikia fusiforme, Durvillaea antarctica, Lessonia nigrescens, Lessonia trabeculata, Lessonia vadosa, Macrocystis pyrifera, Undaria pinnatifida, Padina pavonia, Laminaria angustata, Laminaria japonica, Ecklonia kurome, Adenocytis utricularis, Dictyota menstrua/is, Spatoglossum schroederi and Chordaria flagelliformis.

The present invention is related with a synergistic immunostimulant veterinary composition for use in fishes comprising an aqueous extract of seaweed with a percentage of fucoidans of <NUM>% and an extract of Andrographis sp plant with a percentage of total andrographolide of <NUM>%, where the fucoidan: andrographolide ratio is of <NUM>:<NUM>, wherein said aqueous extract of seaweed is selected from an aqueous extract of selected brown algae Fucus vesiculosus, Fucus evanescens, Fucus distichus, Fucus serratus, Pelvetia wrightii, Ascophyllum nodosum, Himanthalia Lorea, Bifurcaria bifurcata, Sargassum stenophyllum, Hizikia fusiforme, Durvillaea antarctica, Lessonia nigrescens, Lessonia trabeculata, Lessonia vadosa, Macrocystis pyrifera, Undaria pinnatifida, Padina pavonia, Laminaria angustata, Laminaria japonica, Ecklonia kurome, Adenocytis utricularis, Dictyota menstrua/is, Spatoglossum schroederi and Chordaria flagelliformis.

In another embodiment the invention relates to the subject matter of any of the claims <NUM> to <NUM>.

Another aspect of the invention relates to the subject matter of claim <NUM> and dependent claims thereof <NUM> and <NUM>.

The present invention is related mainly to a synergistic immunostimulating composition for fishes comprising fucoidians and andrographolide, which allows an effective control and prevention of infections produced by intracellular microorganisms.

The present invention is related more particularly to a synergistic composition comprising fuicoidans and andrographolide, which allows an effective control and prevention of piscirickettsiosis, viral haemorrhagic septicemia, infectious pancreatic necrosis, infectious haematopoietic necrosis, pancreas disease and sleeping disease, infectious anemia of salmon, francicellosis and renibacteriosis.

The dried brown algae were first pulverized by freezing them in liquid N<NUM>, and using a porcelain mortar, to obtain a <NUM> micron algae powder.

Ten grams of the ground dried algae mixture were extracted with <NUM> of distilled water with continuous stirring for <NUM> hours at <NUM>. The algal tissue was removed by simple filtration. The aqueous extract was centrifuged until a clarified solution. The solution is precipitated by the addition of <NUM> volumes of ethanol. The precipitate was recovered by centrifugation and dried in an electric oven at <NUM>.

For the preparation of the extract of andrographolide, dried leaves of Andrographis sp were used, the extraction was carried out using an <NUM>% v/v water-ethanol mixture, the final extract is a composition containing <NUM>% of the native extract and <NUM>% of maltodextrin.

A mechanical mixture of both dry extracts was performed in a ratio of <NUM>/<NUM> of algae extract and extract of Andrographis sp. , respectively. For this, a KitchenAid Heavy Duty mixer (Model KS5SS, USA) was used with stainless steel container, adjustable speed and capacity > <NUM>,<NUM>. The selected mixing speed was, according to the equipment, level <NUM> equivalent to ± <NUM> rpm of the upper shaft. The mixing time was <NUM> minutes, the mixture was prepared according to the proportions of ingredients and the densities of these were determined through a gravimetric method with results of <NUM> grs/cm<NUM>.

The SHK-<NUM> cell line, derived from Salmo salar's kidney, was used. The assay was started when the cells shown <NUM>% of confluence, and then they were stimulated with algae extract (<NUM>% of Fuicoidan).

The treatment was applied independently to SHK-<NUM> cells, in L15 medium supplemented with <NUM>% fetal bovine serum at a dose of <NUM>µg/ml, the cells were incubated at <NUM> during <NUM> hours of stimulation time.

After the treatment, the cell supernatant was discarded and the cells were lysed with <NUM>µl of TRK lysis buffer and stored in tubes of <NUM> at -<NUM>. RNA extraction from the cells was performed using the RNA extraction kit (Omega-bio-tek), according to the manufacturer's protocol.

Once the total RNA was obtained, the mRNAs were transformed into cDNA by means of the reverse transcription reaction, which was performed in a total volume of <NUM>µl of solution, divided in two parts. The first reaction was performed in a mixture containing <NUM>µl of oligo-dT (<NUM>µg/ml) for analysis of gene expression of the markers <NUM>µl of dNTPs (<NUM>); <NUM>µl of total RNA (<NUM>µg) and <NUM>µl of nuclease-free water and it was incubated for <NUM> at <NUM> to elute secondary structures of the mRNAs. Subsequently, a second mixture comprised of <NUM>µl of M-MLV reverse transcriptase (<NUM> U), <NUM>µI of 5X enzyme buffer and <NUM>µl of recombinant RNAseOUT ribonuclease inhibitor (<NUM> U) was added to the solution, in a total volume of <NUM>µl and it will be incubated for <NUM> at <NUM>. Finally for the inactivation of reverse transcriptase, the reaction mixture was incubated at <NUM> during <NUM>. The synthesized cDNA was stored at -<NUM> for its subsequent amplification for PCR or its quantification by real-time (qPCR) for IFN-I and IL-<NUM> genes relative to EF-1α expression using the primers listed in Table <NUM> below:.

Each amplification reaction was performed using <NUM>µl of cDNA as the annealing, <NUM> (Table <NUM>) primers, <NUM>µl MgCl<NUM> (<NUM>), <NUM>µl Lightcycler® Fast Start DNA Master SYBR Green amplification mixture in a volume of <NUM>µl. The reaction was carried out in a LightCycler® <NUM> thermal cycler. The program consists of the following steps: initial denaturation at <NUM> for <NUM>, followed by a PCR reaction of <NUM> cycles each composed of denaturation at <NUM> for <NUM> seconds, mating at <NUM> for <NUM> seconds and extension at <NUM> for <NUM> seconds. Subsequently a cycle to obtain the melting curve for <NUM> at <NUM>, and finally a cooling cycle at <NUM> for <NUM>. For the relative quantification, it was performed with a standard curve, consisting of reactions containing dilutions of the purified PCR product of known concentration for the gene of interest. After obtaining and quantifying the PCR product corresponding to each gene, dilutions were made in a range of <NUM><NUM> to <NUM><NUM> numbers of copies/µl for each gene under study, for the subsequent calculation of the efficiency of the reaction, where the following relation, E = <NUM>(-<NUM>/slope)-<NUM> will be used. For the calculation of relative expression by the qPCR technique amplification reactions of the ELF-<NUM> gene cDNA were performed on each RNA sample from cells treated with the different stimuli in vitro. Then, the expression changes were calculated using the comparative CT method (Pfaffl, <NUM>).

The SHK-<NUM> cell line, derived from Salmo's kidney, was used. The assay was started when the cells showed <NUM>% confluency, where they were stimulated with an extract of Andrographis sp (<NUM>% total Andrographolide).

The treatment was applied independently to SHK-<NUM> cells in L15 medium supplemented with <NUM>% fetal bovine serum at a dose of <NUM> of the Andrographis sp extract, the cells were incubated at <NUM> for <NUM> hours of stimulation time.

After the treatment was finished, the cell supernatant was discarded and the cells were lysed with <NUM>µl of TRK lysis buffer and stored in <NUM> tubes at -<NUM>. RNA extraction from the cells was performed using the RNA extraction kit (Omega-bio-tek), in accordance to the manufacturer's protocol.

Once the total RNA was obtained, the mRNAs were transformed into cDNA by means of the reverse transcription reaction, which was performed in a total volume of <NUM>µl of solution, divided into two parts. The first reaction was performed in a mixture containing <NUM>µl of oligo-dT (<NUM>µg/ml) for analysis of gene expression of the <NUM>µl markers of dNTPs (<NUM>); <NUM>µl of total RNA (<NUM>µg) and <NUM>,<NUM>µl of water free of nucleases and incubated for <NUM> at <NUM> to remove secondary structures from the mRNAs. After this a second mixture comprised of <NUM>µl of M-MLV reverse transcriptase (200U), 4µl of 5X enzyme buffer and <NUM>. 5µl of RNAsaOUT <NUM> U recombinant ribonuclease inhibitor was added to this solution, in a total volume of <NUM>µl andit will be incubated for <NUM> at <NUM>. Finally for inactivation of reverse transcriptase, the reaction mixture was incubated at <NUM> for <NUM>. The synthesized cDNA was stored at -<NUM> for subsequent PCR amplification or quantification by real-time PCR (qPCR) for the IFN-<NUM> and IL-<NUM> genes relative to the expression of EF-1α using the primers indicated in Table <NUM> below:.

Each amplification reaction was performed using <NUM>µl CDNA as a template, primers of <NUM> (Table <NUM>), <NUM>µl MgCl <NUM> (<NUM>), <NUM>µl Lightcycler® Fast Start DNA Master SYBR Green amplification mixture in a volume of <NUM>µl. The reaction was carried out in a LightCycler® <NUM> thermal cycler. The program consisted on the following steps: initial denaturation at <NUM> during <NUM>, followed by a PCR reaction of <NUM> cycles each one composed of denaturation at <NUM> during <NUM>, mating at <NUM> during <NUM> and an extension at <NUM> during <NUM>. Subsequently a cycle to obtain the melting curve for <NUM> at <NUM>. And finally a cooling cycle at <NUM> during <NUM>. The relative quantification was performed with a standard curve, consisting of reactions containing dilutions of the purified PCR product of a concentration known for the gene of interest. After obtaining and quantifying the PCR product corresponding to each gene, successive dilutions were performed in a range of <NUM><NUM> to <NUM><NUM> numbers of copies/µl for each gene under study, for the subsequent calculation of the efficiency of the reaction, where the following relation, E = <NUM>(-<NUM>/slope)-<NUM> will be used. For the calculation of relative expression by the qPCR technique amplification reactions of the ELF-<NUM> gene cDNA were performed on each RNA sample from cells treated with the different stimuli in vitro. Then, the expression changes were calculated using the comparative CT method (Pfaffl, <NUM>).

The results are illustrated in <FIG> and <FIG>.

The SHK-<NUM> cell line, derived from Salmo salar's kidney, was used. The assay was started when the cells had <NUM>% confluence, where they were stimulated with an extract of Andrographis sp and an extract of brown algae.

The treatment was applied at the same time to SHK-<NUM> cells, in a half of L15 supplemented with <NUM>% fetal bovine serum at doses of <NUM>µg/ml of brown algae extract (<NUM>% total fuicoidans) and <NUM> extract of Andrographis sp (<NUM>% total andrographolide) cells were incubated at <NUM> during <NUM> hours of stimulation time.

After treatment, the cell supernatant was discarded and cells were lysed with <NUM>µl of TRK lysis buffer and stored in <NUM> tubes at -<NUM>. RNA extraction from the cells was performed using the RNA extraction kit (Omega-bio-tek), according to the manufacturer's protocol.

Once the total RNA was obtained, the mRNAs were transformed into cDNA by means of the reverse transcription reaction, which was performed in a total volume of <NUM>µl of solution, divided in two parts. The first reaction was performed in a mixture containing <NUM>µl of oligo-dT (<NUM>µg/ml) for analysis of gene expression of the <NUM>µl markers of dNTPs (<NUM>); <NUM>µl of total RNA (<NUM>µg) and <NUM>µl of water free of nucleases and it was incubated during <NUM> at <NUM> to remove secondary structures from the mRNAs. Subsequently, to this solution was added a second mixture comprised of <NUM>µl of M-MLV reverse transcriptase (<NUM> U), <NUM>µl of 5X enzyme buffer and <NUM>µl of recombinant RNAseOUT ribonuclease inhibitor (<NUM> U), in a total volume of <NUM>µl and it will be incubated during <NUM> at <NUM>. Finally for inactivation of reverse transcriptase, the reaction mixture was incubated at <NUM> during <NUM>. The synthesized cDNA was stored at -<NUM> for further amplification by PCR or quantification by real-time PCR (qPCR) for the IFN-I and IL-<NUM> genes relative to EF-1α expression using the primers indicated in Table <NUM> below:.

Each amplification reaction was performed using <NUM>µl of cDNA as a template, primers of <NUM> (Table <NUM>), <NUM>µl MgCl (<NUM>), <NUM>µl Lightcycler® Fast Start DNA Master SYBR Green amplification mixture in a volume of <NUM>µl. The reaction was carried out in a LightCycler® <NUM> thermal cycler. The program consisted of the following steps: initial denaturation at <NUM> during <NUM>, followed by a PCR reaction of <NUM> cycles each composed of denaturation at <NUM> during <NUM>, mating at <NUM> during <NUM> sec and extension at <NUM> during <NUM> sec. Subsequently a cycle to obtain the melting curve during <NUM> at <NUM>, and finally a cooling cycle at <NUM> during <NUM>. The relative quantification was performed with a standard curve, consisting of reactions containing dilutions of the purified PCR product of known concentration for the gene of interest. After obtaining and quantifying the PCR product corresponding to each gene, successive dilutions were performed in a range of <NUM><NUM> to <NUM><NUM> number of copies/µl for each gene under study, for the subsequent calculation of the efficiency of the reaction, where the following relation, E = <NUM>(-<NUM>/slope)-<NUM> will be used. For the calculation of the relative expression by the qPCR technique amplification reactions of the ELF-<NUM> gene cDNA were performed in each RNA sample from cells treated with the different stimuli in vitro. Then, the expression changes were calculated using the comparative CT method (Pfaffl, <NUM>).

The SHK-<NUM> cell line, derived from Salmo salar's kidney, was used. The assay was started when the cells showed a <NUM>% of confluence, where they were stimulated with a combination that will ensure a concentration of the brown algae extract of <NUM>µg/ml and <NUM> of the extract of Andrographis sp, as well as the individual stimulation with a concentration of <NUM>µg/ml extract of brown algae, and individual stimulation with a concentration of <NUM> of the extract of Andrographis sp. The treatments were applied independently to SHK-<NUM> cells in L15 supplemented with <NUM>% fetal bovine serum at the above-mentioned doses, cells were incubated at <NUM> during <NUM> hrs incubation.

Infection with P. salmonis was carried out after the incubation period. For the challenge, a strain of Piscirickettsia salmonis PPT005 grown on a SHK-<NUM> cell line, which was originally isolated from a dying population of Atlantic salmon (Salmo salar) from a farm salmond center near Puerto Montt, Chile, using CHSE-<NUM> cells. The cells were infected and incubated at <NUM> in L-<NUM> medium supplemented with <NUM>% SFB. The bacteria were harvested from the infected cells when they had a <NUM>% cytopathic effect (CPE). Finally, functional assays were performed by sowing <NUM><NUM> bacteria per mL in SHK-<NUM> cells at <NUM>% of confluence.

The lenght of the challenge test was <NUM> days, time required to obtain a <NUM>% cytopathic effect in the control monolayers.

After the treatment was complete, the supernatants were harvested and the surviving cells were lysed with <NUM>µl of TRK lysis buffer and stored in tubes of <NUM> at -<NUM>.

<NUM> of each culture supernatant from SHK-<NUM> cells was taken with <NUM> days of infection. These supernatants were centrifuged at <NUM> xg during <NUM> to remove the cell residue, and then at <NUM>,<NUM> xg for <NUM> to recover the bacteria in an Eppendorf <NUM> centrifuge. All the sediments were processed by the Chelex method for obtaining the DNA to amplify. Briefly, they were suspended in <NUM>µl of <NUM>% Instagene p/v (Chelex <NUM>, Bio-Rad), they were shaken at maximum speed in a vortex and centrifuged at <NUM>,<NUM> xg during <NUM> in an Eppendorf centrifuge to collect the pearls of Chelex in the sediment and DNA in the supernatant. For the estimation of bacterial numbers, standard numbers were used like reference with known numbers of P. salmonis copies, obtained by cloning the ITS of this bacterium in the vector pCR® <NUM> TOPO TA (Invitrogen). The standards of <NUM><NUM> to <NUM><NUM> number of copies and the DNAs obtained by Chelex were amplified in parallel. Each reaction was performed in a volume of <NUM>µl with <NUM>µg of each DNA sample, and <NUM> of the ITS primers (Marshall et al <NUM>) labeled with FAM (<NUM>-<NUM>). For amplification an initial denaturation of <NUM> minutes at <NUM> was performed, followed by <NUM> cycles with the following segments: denaturation at <NUM> during <NUM> seconds, alignment at <NUM> during <NUM> seconds and an extension at <NUM> during <NUM> seconds, and then a final extension of <NUM> at <NUM>. From the amplification curves obtained for each sample, the Ct (crossing threshold) values of the copy number standards were estimated, in accordance to the method described by Phaffi, <NUM>, the results are plotted in <FIG>. In the case of the suriing cells a RNA extraction from the cells was performed using the RNA extraction kit (Omega-bio-tek), in accordance to the manufacturer's protocol.

Once the total RNA was obtained, the mRNAs were transformed to cDNA by means of the reverse transcription reaction, which was performed in a total volume of <NUM>µl of solution, divided into two parts. The first reaction was carried out in a mixture containing <NUM>µl of oligo- <1T (<NUM>µg/ml) for analysis of gene expression of the markers <NUM>µl of dNTPs (<NUM>); <NUM>µl of total RNA (<NUM>µg) and <NUM>,<NUM>µl of water free of nucleases and it was incubated during <NUM> at <NUM> to remove secondary structures from the mRNAs. After, a second mixture comprised of <NUM>µl of M-MLV reverse transcriptase (<NUM> U), <NUM>µl of 5X enzyme buffer and <NUM>µl of recombinant RNAseOUT ribonuclease inhibitor (<NUM> U) was then added to this solution, in a total volume of <NUM>µl and it will be incubated during <NUM> at <NUM>. Finally for inactivation of the reverse transcriptase, the reaction mixture was incubated at <NUM> during <NUM>. The synthesized cDNA was stored at -<NUM> for subsequent PCR amplification or quantification by real-time PCR (qPCR) for the IFN-<NUM> and IL-<NUM> genes related to EF-1a expression using the primers indicated in table <NUM> below:.

Each amplification reaction was performed using as a template <NUM>µl of cDNA, <NUM> primers (Table <NUM>), <NUM>µl MgCl2 (<NUM>), <NUM>µl of Lightcycler® Fast Start DNA Master SYBR Green amplification mixture in a volume of 10µl. The reaction was carried out in a LightCycler® <NUM> thermal cycler. The program consisted of the following steps: initial denaturation at <NUM> during <NUM>, followed by a PCR reaction of <NUM> cycles each one composed of denaturation at <NUM> during <NUM>, mating at <NUM> during <NUM> and extension at <NUM> during <NUM>. Subsequently a cycle to obtain the melting curve during <NUM> at <NUM>, and finally a cooling cycle at <NUM> during <NUM>. Relative quantification was performed with a curve standard, consisting of reactions containing dilutions of the purified PCR product of known concentration for the gene of interest. After obtaining and quantifying the PCR product corresponding to each gene, successive dilutions were performed in a range of <NUM><NUM> to <NUM><NUM> number of copies/µ! for each gene under study, for the subsequent calculation of the efficiency of the reaction, where the following relationship, E = <NUM> (-<NUM>/slope)-<NUM>. For the calculation of the relative expression by the qPCR technique amplification reactions of the ELF-<NUM> gene cDNA were performed on each RNA sample of cells treated with the different stimuli in vitro. Then, the expression changes were calculated using the comparative CT method (Pfaffl, <NUM>).

A study was performed on salmonid fishes cell lines, where the composition of the present invention was evaluated. Cell lines exposed to the combination of fuicoidans + Andrographolide, cell lines exposed only to fuicoidans, and subsequently, molecular markers relevant in Th0 to Th1 differentiation such as IL-<NUM> and IFN-<NUM> were evaluated. The separate fuicoidans and the combination of these ones + Andrographolide are significantly different in surviving cells following a challenge with IPNV. At the same time, there is a decrease in the copy numbers of the pathogen agents significantly higher than the cells that received the composition of the present invention in relation to the use of only the fuicoidans or the aqueous extract of brown algae.

The studies in fishes were carried out in ponds, where the composition of the present invention was an aqueous extract of seaweed with a reference percentage of <NUM>% fuicoidans obtained from Macrocystes Pyrifera in combination with an extract from the Andrographis sp plant with a <NUM> % of total andrographolide in a proportion of <NUM>% and <NUM>%, respectively, the fish feed or diet was incorporated, in a dose in the range of <NUM> to <NUM> Kh per ton of food. Preferably, at a dose of <NUM> per ton of food.

We used <NUM> specimens of rainbow trout (Oncorhynchus mykiss) with average weight of <NUM>-<NUM>. , however, additional fishes were available to achieve a coefficient of variation of less than or equal to <NUM>%.

Samples of <NUM> fishes were taken to be analyzed in the laboratory by real-time RT-PCR technique, to discard the presence of IPNv, BKD and SRS.

An exploratory sampling was done to know the average weight of the population and to carry out the selection of the fishes chosen for the test. Only animals presenting the required weight, good condition of adaptation to the saline environment and sanitary condition approved by the veterinarian (free of IPNv, SRS and BKD) were included.

With the selection data, all animals with weight outside the selection range, as well as those with peeling or those that their condition was not appropriate for the present study, were excluded when marking.

The fishes were marked with pittags <NUM> days before the beginning of the test, proceeding as follows:.

Six ponds of <NUM> rn<NUM>were formed from fishes previously marked with pittags. In each pond <NUM> fishes were deposited and of which the code of the chip was read, creating a database that associated, initial weight and length and pond number. The database allowed to follow the traceability of the tagged fishes, related to productive indexes and subsequent challenge with the pathogen. Simultaneously <NUM> ponds with <NUM> fishes each were formed, according to the same procedure, which were kept until the challenge stage, see <FIG>.

The fishes were kept in the ponds during <NUM> days as acclimatization period, under controlled conditions; average temperature of <NUM> (± <NUM>), salinity in a range of <NUM>-<NUM> ppt, oxygen <NUM>-<NUM>% saturation and pH of <NUM>-<NUM>. The environmental parameters were monitored daily.

During this stage, the ponds were fed with diet without the present composition, manually at <NUM>-<NUM>% PC, with a <NUM>% ration in the morning.

Daily, the unconsumed food of each pond was recovered, to later estimate the actual feeding rate of each group.

F existing mortality, it was extracted and recorded in the corresponding pond, and necropsy was carried out by trained personnel from the fish farming.

The fishes were fed <NUM>-<NUM>% pc/day, during acclimatization, treatment administration and challenge stage with pathogen. The food was administered manually, delivering <NUM>% of the ration during the morning. It should be noted that during the acclimatization and challenge a commercial diet without additives was administered.

The amount of food supplied was adjusted regularly according to the expected growth rate for the species and mortality. On a daily basis, unconsumed food was collected from the ponds, thus obtaining the actual feed intake for the estimation of subsequent productive indexes.

During the development of the test, tissue samples were taken, considering the kidney, proximal intestine and blood samples for plasma collection. The number of samples and sample time are shown in Table <NUM> below.

Kidney samples were taken in two <NUM><NUM> pieces, which were immersed in <NUM> eppendorf tubes (individually for each fish) containing <NUM>µL of later RNA (Ambion). These samples were labeled and refrigerated (<NUM>-<NUM>'C) during <NUM>, then cooled to - <NUM>.

The proximal intestine was destined to histological analysis, for which they were deposited in falcon tubes with buffered formalin. In this case, the number of samples per pond (<NUM>) was placed in the same tube, labeled with date, pond number and treatment.

Blood samples were deposited immediately after collection in eppendorf tubes prepared with heparin (<NUM> IU). They were then centrifuged to remove the plasma, which was placed in a new tube (previously labeled) and stored in the ultra-freezer (-<NUM>) until taking off them.

The food with the composition of the present invention was distributed to three ponds (triplicate), as indicated in Table <NUM>, during <NUM> consecutive days. The remaining ponds (controls) continued their feeding with a standard diet throughout the evaluation period. Administration of treatment was as described above.

During this time, temperature, pH, salinity and oxygen were monitored daily. If there was mortality at this stage, it was taken out and recorded in the corresponding pond, performing an anatomopathological test.

Control corresponds to the commercial diet.

The immuno-modulating agent corresponds to the composition of the present invention.

Then, a challenge was performed with P. Table <NUM> details the specifications of the P. salmonis isolate that was used in the inoculation of Trojan fishes.

The inoculum was administered with TCID50/ml determined by the Karber Spearman method by the laboratory ADL Diagnostic Chile Ltda. In addition, the purity of the inoculum was evaluated, considering ISAv, IPNv, BKD, F. psycrophilum RT-PCR analysis and bacteriological cultures in medium TSA and TSA / s at <NUM> and <NUM>° of incubation.

At the end of the administration of the diet with the composition of the present invention, the fishes were redistributed to perform the challenge. Three ponds of <NUM><NUM> were formed, considering the mixture of treated and untreated fishes at random in the new ponds, as indicated in table <NUM>. At the time of the new distribution the pittag was read, assigning to each chip the group and pond, see <FIG>.

The challenge was achieved by cohabitation, which involved introducing fishes infected with P. salmonis, trojans, into healthy fishes ponds (treated and controlled), as indicated in Table <NUM>, considering an infection pressure of <NUM>%. The inoculum was administered intraperitoneally to the trojan group at a rate of <NUM>/fish. The inoculation was performed according to the following procedure:.

Subsequently, the fishes were left in the ponds waiting for the appearance of mortality. During this stage, the feeding was carried out in accordance to the point <NUM> and daily environmental parameters such as temperature, salinity, oxygen and pH were registered. Mortality was identified according to the number of pittag from the database, registering daily.

The challenge lasted for <NUM> days, period of time that, the accumulated mortality of the control group was expected to reach <NUM>-<NUM>%, thus ending the test.

The mortality recorded during the days of challenge was sent to the diagnostic laboratory to be analyzed by anatomopathological observations. In parallel, molecular analyzes were performed by real-time RT-PCR, for IPN and SRS viruses, to <NUM>% of the total, considering <NUM> trojans, <NUM> of the treated group and <NUM> of the control group, to confirm the presence of the pathogen.

Weight and length were measured at day <NUM> (Beginning Acclimatization), at <NUM> days of treatment administration and at the end of the challenge with P. salmonis, on the <NUM>% of the fish in each group. From the data, condition factor (K), feed rate (SFR), specific growth rate (SGR), % growth, thermal growth rate (GF3) and food conversion rate (FCRb).

Below are summarized in tables some of the productive variables such as average body weight, condition factor K, coefficient of variation and weight gain at the end of treatment administration. As can be seen, the final weight (see Fig. <NUM>) increased in all ponds.

Table <NUM> summarizes the production parameters obtained during the treatment administration period (Diet with the present composition of the present invention). From the table can be seen that the growth indicators (% growth, SGR and SFR) were similar between the treated group and the control group. The data from each group did not present significant differences in weight, obtained at the end of the administration (p> <NUM>), in the SGR specific growth rate (p> <NUM>) and in the thermal growth rate GF3 (p> <NUM>).

Table <NUM> shows the biomass increase and cumulative growth (%) post-administration of the commercial diet with the composition of the present invention. The increase in biomass fluctuated from <NUM> to <NUM> and the accumulated growth of <NUM> to <NUM>% between the different test ponds.

The specific growth rate (SGR) ranged varied from <NUM> to <NUM> among different replicates, however, no differences were observed between the group to which a commercial diet was supplied with the composition of the present invention and the group to which only the commercial diet has been supplied. The same happened for the growth rate term (GF3), with a range of <NUM> to <NUM> behaving similarly in both groups.

From the results obtained, T test was performed for independent samples, not observing significant differences (p> <NUM>) for the growth variable, between the control group and the group treated with experimental additive.

For the specific rate of thermal growth and specific rate, the same analysis was applied, not registering significant differences (p <<NUM>) between the treated group and the control group.

In the challenge stage with P. salmonis, post challenge mortality was analyzed. To do this, during the period of cohabitation the rainbow trout groups presented a similar percentage of cumulative mortality among replicates in the group of trojans.

In the control group and the one administered with food of the composition of the present invention, it was higher in one of the replicates (TK C9), whereas replicate <NUM> and <NUM> (TK C10 and C11) had similar mortality, however, the trend was similar between the replicates, where the control group had higher mortality than the treated groups. <FIG> and <FIG> show the evolution of daily and accumulated mortality per pond.

In order to confirm the reason of mortality of the challenged groups, dead fishes were analyzed by molecular techniques (RT-PCR real time) with a total of <NUM> samples, obtaining <NUM>% of positive cases with presence of P. salmonis, and <NUM> positive samples for IPNV, in the different groups evaluated (see Table <NUM>). The average Ct for the control group was <NUM> and for the experimental group <NUM>.

In addition to molecular analysis, necropsy of the mortality was performed, external and internal lesions associated with SRS were seen. In general, the most recurrent injuries were ulcerative injuries on the skin, fin-hemorrhages, congestive intestinal serous, congestive brain, renomegaly, congestive adipose tissue, splenomegaly, and congestive pyloric blinds.

Table <NUM> shows the average weight, condition factor (K) and percentage of growth obtained at the end of the challenge for the control group and the treated group. As noted, the group treated with the composition of the present invention obtained higher average weight, condition factor and cumulative % growth at the end of the challenge.

For the interpretation of the efficacy results of the treatment, the relative percentage of survival (RPS) was calculated, based on the mortality recorded during the challenge. The RPS is the ratio between the cumulative mortality of treated fishes at the time the cumulative mortality of control (untreated) fishes reach <NUM>-<NUM>%. The RPS is expressed according to the following formula: <MAT>.

Also, the cumulative mortality of the control group was calculated at day <NUM> post challenge and at the end of the study (day <NUM>). The RPS for the group treated by pond and as a group is presented below in Tables <NUM> and <NUM>.

As seen in the tables, the RPS at day <NUM> was <NUM>% and then decreasing to day <NUM> post challenge with <NUM>%.

Claim 1:
A veterinary composition for use in fishes comprising an aqueous extract of seaweed with a percentage of fucoidans of <NUM>% and an extract of Andrographis sp plant with a percentage of total andrographolide of <NUM>%, wherein:
Said aqueous extract of seaweed is selected from an aqueous extract of selected brown algae Fucus vesiculosus, Fucus evanescens, Fucus distichus, Fucus serratus, Pelvetia wrightii, Ascophyllum nodosum, Himanthalia Lorea, Bifurcaria bifurcata, Sargassum stenophyllum, Hizikia fusiforme, Durvillaea antarctica, Lessonia nigrescens, Lessonia trabeculata, Lessonia vadosa, Macrocystis pyrifera, Undaria pinnatifida, Padina pavonia, Laminaria angustata, Laminaria japonica, Ecklonia kurome, Adenocytis utricularis, Dictyota menstrualis, Spatoglossum schroederi and Chordaria flagelliformis; and the algae extract and extract of Andrographis sp. ratio is of <NUM>:<NUM>.