Abstract:
A method to stimulate the immune system of fish that comprises administering to the fish a β-1,3-glucan having a β-1,3-linked main chain with β-1,6-linked single glucose side chains.

Description:
This application is a continuation-in-part application of the application Ser. No. 07/313,033 having a filing date of Feb. 21, 1989, now U.S. Pat. No. 5,147,862. 
    
    
     BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     This invention relates to a method to enhance the efficacy of a fish vaccine and to a method to stimulate the immune system of a fish and to a composition and an agent useful in these methods. 
     2. Discussion of Background 
     Fish rearing in fish farms is increasingly evident. The advantages of aquaculture in comparison to fishing wild stocks is as apparent as the keeping of cattle compared with the hunting of wild animals. However, there are problems. The fish must be kept at a very high density and this means that the fish are susceptible to disease and, in particular, to the rapid spread of disease through the fish farm. Aquaculture techniques are improving all the time and the use of antibiotics, usually administered in the food, has been a major advantage in reducing disease. Nevertheless the use of antibiotics in this way can lead to the development of resistant strains of fish pathogens. There is also a popular sentiment against the use of antibiotics, growth hormones and the like in livestock and fish to be consumed by humans. 
     There has been some success in the use of vaccines, notably against diseases such as vibriosis and enteric redmouth. But there are still diseases for which vaccines are not effective or are of inadequate efficacy. 
     It is known in medicine that the efficacy of certain vaccines can be improved by the administration of compounds in addition to the vaccines. 
     It is known that certain glucans can be useful in curing human cancer by an apparent immunomodulating effect. Glucans are the anhydrides of glucose derivatives, for example cellulose, starch, dextrin and glycogen. 
     Relevant patents relating to glucans known to applicant include U.S. Pat. Nos. 4,098,661 issued Jul. 4, 1978, British Patent 1,061,043 dated Oct. 23, 1963 and Canadian Patent 968,286 issued May 27, 1975. 
     It has now been observed that certain glucans exhibit appreciable, advantageous effect in the use of vaccines for treating fish diseases. Of particular interest are β-1,3-glucans and particularly the compounds schizophyllan (SPG) and scleroglucan (SG). These water soluble β-1,3-glucans are reported to have triple helical structures. Schizophyllan is prepared by the precipitation in a culture filtrate of the fungi Schizophyllum commune, using acetone, ethanol or other water miscible solvent as a precipitant. Scleroglucan is obtained in a similar manner from a culture filtrate of a selected species of Sclerotium. The above compounds are represented by the following structural formulae: ##STR1## 
     SUMMARY OF THE INVENTION 
     The present invention is thus directed to the immunostimulating and vaccine enhancing effects of β-1,3-glucans and is based on the surprising observation that these compounds are effective in the treatment of fish and in vaccine prophylaxis in fish. 
     Accordingly, in a first aspect, the present invention is a method to stimulate the efficacy of a fish vaccine that comprises administering to a fish treated with the vaccine a β-1,3-glucan having a β-1,3-linked main chain with β-1,6-linked single glucose side chains. 
     In a second aspect the invention is a method to stimulate the immune system of fish that comprises administering to the fish a β-1,3-glucan as defined above. 
     In yet a further aspect the invention is a composition able to enhance the efficacy of a fish vaccine and comprising a β-1,3-glucan having β-1,3-linked main chain with β-1,6-linked single glucose side chains with an antigen/β-1,3-glucan weight ratio of from 0.015 to 0.15. Preferably the antigen source is a vaccine. 
     In yet a further aspect the invention is an agent to administer to a fish to stimulate the immune system of the fish comprising an effective amount of β-1,3-glucan having a β-1,3-linked main chain with β-1,6-linked single glucose side chains and carrier acceptable to the fish. 
     The carrier will typically be saline for injectable agents and food for agents to be administered by mouth. With advantage alpha-cellulose may be mixed with the glucan prior to mixing with food. 
    
    
     BRIEF DESCRIPTION OF THE DRAWING 
     FIG. 1 relates cumulative mortality to time in certain comparative experiments described in the Experimental Work below. 
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     Experimental Work 
     Experimental procedures were developed to determined the ability of certain β-1,3-glucans to enhance the performance of formalin killed bacterin and stimulate the non-specific immune system of salmonid fishes such that they can show increased survival during a virulent challenge. The model system chosen for the challenge was Aeromonas salmonicida the etiological agent of furunculosis, because of the reported involvement of cell mediated immunity in this disease. Cell mediated immunity plays a major role in immune protection against bacterial kidney disease (BKD), another disease that plagues aquaculture fish, but the course of infection is long, necessitating the use of a furunculosis model system. 
     EXAMPLE 1 
     Two systems of challenge were used. First immersion, secondly cohabitation. The mortality figures were then adjusted by calculating relative potency so that the comparisons could be made between the two challenge groups. Relative potency showed good reproducibility between tanks and lentinan and schizophyllan enhanced vaccines were both found to have similar level of potency within and between tanks. The relative potency was determined according to Table 1. Relative potency is defined as percentage mortality in the control divided by the percentage mortality in the vaccinates. 
     The results are set out in Table 1. 
     
                       TABLE 1______________________________________Enhancement of efficacy ofinjection-delivered vaccine by the β-1,3-Glucans,Lentinan and Schizophyllan (SPG).Challenge                         *RelativeMethod      Treatment    % Mort.  Potency______________________________________Immersion   Lentinan + Ag                    60%      1.6       SPG + Ag     64%      1.5       Antigen only 96%      1.0Cohabitation       Lentinan + Ag                    52%      1.6       SPG + Ag     52%      1.6       Antigen only 84%      1.0______________________________________ Ag: Antigen *Relative Potency is defined as follows: ##STR2## 
    
     Because of the similarity in structure function the following experimental work was conducted with one compound, schizophyllan. This compound is less costly to produce than lentinan. It can be produced by batch fermentation but lentinan must be extracted from the fruiting body of the mushroom Lentinus edodes, which is a popular food in Japan and elsewhere, making the compound lentinan expensive. 
     EXAMPLE 2 
     In the next phase of experiments SPG was obtained in three different forms. First a purified and extensively depolymerized form (SPG-P). Secondly a crude extract with only slight depolymerization (SPG-C) and, thirdly, a crude preparation of native SPG in culture broth containing pulverized mycelia (SPG-M). 
     These forms of SPG were administered to fish by intraperitoneal (i.p.) injection in an admixture containing A. salmonicida bacterin and one each of the three different forms of SPG. Fish were then challenged by immersion in virulent A. salmonicida and the resulting mortalities were monitored. The results are in Table 2. 
     
                       TABLE 2______________________________________Differences in Efficacy of various molecularweight forms of SPG.Treatment % mortality   % morality                             pooledreceived  replicate 1   replicate 2                             % mort.______________________________________SPG-M1 + Ag      6.7%         26.7%     16.7%SPG-C2 + Ag     60.0%          3.3%     31.7%SPG-P3 + Ag     56.7%         66.7%     61.7%Ag only   33.3%         20.0%     26.7%saline    76.7%         66.6%     71.7%______________________________________ 1 SPGM designates the crudest form of SPG. This is a preparation of colloidaly pulverized mycelia containing the highest molecular weight of SPG. 2 SPGC is a crude form of SPG that has been somewhat purified and somewha depolymerized. 3 SPGP is the purified and depolymerized form of SPG. 
    
     It is seen from the above that the crudest form of SPG (SPG-M) was the most effective. With a decrease in molecular weight there was a decline in the level of enhancement to the vaccine. 
     EXAMPLE 3 
     A further series of experiments was conducted to assess the efficacy of SPG as a non-specific chemoprophylactant. It was found that when injected i.p. at the rate of 20 mg/kg 20 days prior to challenge, fish were protected for at least 40 days after injection to a similar extent to fish that received an i.p. delivered vaccine consisting of antigen alone. These results are summarized in FIG. 1, which graphically relates cumulative percentage mortality to time. The curve is a mortality curve showing saline-injected fish compared with fish injected with SPG alone and with antigen alone. Fish injected with SPG alone show a similar level of protection to those vaccinated with antigen only. This protection seems to last about 20 days after challenge. The fish were injected with the SPG and the antigen 20 days before day zero of the curve of FIG. 1. 
     Experimental procedures were developed to determine the ability of certain β-1,3-glucans to enhance the performance of formalin killed bacterin and stimulate the non-specific immune system of salmonid fishes. 
     EXAMPLE 4 
     A virulent challenge was conducted by using Aeromonas salmonicida the etiological agent of furunculosis by means of immersion. Diet containing crude schizophyllane (hereinafter referred to simply as SPG-M) and Aeromonas salmonicida vaccine (hereinafter referred to simply as vaccine) was orally administered to fish which was attacked by 5.8×10 5  cfu/mL of Aeromonas salmonicida etiological agent, in an amount of 2% of the weight for 7 days, and the mortality was examined. The composition for the diet is shown in Table 3 and mortalities relative to the ratio of SPG-M to vaccine are shown in Table 4. The diet for control contains no SPG-P or vaccine. Further, data for single use of vaccine are also shown in Table 4. 
     It is evident from Table 4 that when SPG-M and vaccine are used in combination, the mortality is reduced as compared with the single use of vaccine even though the dose of the vaccine is reduced to one third. The ratio of antigen to SPG-M is preferably from 0.015 to 0.15. 
     
                       TABLE 3______________________________________Composition for dietComposition         Amount (g/kg diet)______________________________________Steam Dried Herring Meal               558.66Dried Whey           84.6Blood Flour          48.17Euphasids (whole frozen)                21.02Wheat Middling      126.19Vitamin Supplement   2.41Mineral Supplement   7.27Choline Chloride (60%)                4.60Ascorbic Acid        1.84Permapell            13.81Herring Oil          91.38SPG-M + Vaccine      0-20.15α-Cellulose    40.05-19.90______________________________________ 
    
     
                                           TABLE 4__________________________________________________________________________Enhancement of efficacy of orally administered vaccine by SPGContents of SPG and Ag       Mortality (%)in diet (g/kg)       Repricate 1             Repricate 2                   Repricate 3                         Pooled Mortality (%)__________________________________________________________________________Control     50.0  45.0  62.5  52.5Ag 0.45     37.5  25.0  20.0  27.5Ag 0.15     45.0  40.0  60.0  48.3SPG 0.1 + Ag 0.15       25.0  12.5  12.5  16.7SPG 0.1 + Ag 0.15       5.0   0.0   0.0   1.7SPG 10.0 + Ag 0.15       5.0   10.0  2.5   5.8SPG 20.0 + Ag 0.15       20.0  25.0  12.5  19.2__________________________________________________________________________ Note 1. SPG: SPGM Note 2. Ag: Antigen 
    
     EXAMPLE 5 
     The same operation was conducted as in Example 4 except that scleroglucan (hereinafter referred to simply as SG) instead of SPG-M. 
     The results thus obtained are shown in Table 5. It is evident from Table 5 that when SG and vaccine are used in combination, the mortality is reduced as compared with the single use of vaccine even though the dose of the vaccine is reduced to one third. The ratio of antigen to SG is preferably from 0.015 to 0.15. 
     
                                           TABLE 5__________________________________________________________________________Enhancement of efficacy of orally administered vaccine by SGContents of SG and Ag       Mortality (%)in diet (g/kg)       Repricate 1             Repricate 2                   Repricate 3                         Pooled Mortality (%)__________________________________________________________________________Control     57.5  45.0  62.0  54.8Ag 0.45     40.0  25.0  20.0  28.3Ag 0.15     50.0  40.0  60.0  50.0SG 0.1 + Ag 0.15       25.0  17.5  12.0  18.2SG 0.1 + Ag 0.15       2.5   5.0   0.0   2.5SG 10.0 + Ag 0.15       5.0   10.0  5.0   6.7SG 20.0 + Ag 0.15       12.5  20.0  20.0  17.5__________________________________________________________________________ Note 1. SG: Scleroglucan Note 2. Ag: Antigen 
    
     EXAMPLE 6 
     SPG-P or SG was injected i.p. twice to a salmon at the rate of 20 mg/kg every two days. In two days after the last injection, the pronephoros of the salmon was excised and the living cell suspension of the phagocyte (about 1×10 7  cells/ml) was prepared. A killing baker&#39;s yeast suspension having about 2-2.5 times the cell number of the phagocyte was mixed to the suspension of the phagocyte, and incubated at 17° C. for one hour. Then, a part of the suspension mixture was smeared on a slide glass, and dried and dyed with a Wright solution, followed by observation by means of a microscope. 
     The phagocytic index (PI) was calculated in accordance with the following formula: ##EQU1## 
     As control, saline was administered instead of the glucans to salmon and PI was calculated in the same manner as above. 
     The results are shown in Table 6. It is evident from Table 6 that the PI values for SPG-P and SG are high as compared with control, such showing stimulation of the non-specific immune system. 
     
                       TABLE 6______________________________________Change in phagocytic activity of salmon&#39;spronephoros cells by SPG-P and SG  Number of fish tested               Phagocytic Index (PI)______________________________________Control  10              2.35 ± 0.75 (a)SPG-P    10             10.53 ± 3.24 (b)SG       10             12.51 ± 4.72 (b)______________________________________ 
    
     EXAMPLE 7 
     SPG-M or SG was mixed to a diet to have the composition as shown in Table 7 (i.e. 1 g/kg diet). Then, the diet was orally administered to salmon in an amount of 2% of the weight for 7 days. In two days after the administration, the pronephoros of the salmon was excised and PI was calculated in the same manner as in Example 6. As control, a diet containing no SPG-M or SG was used. 
     The results thus obtained are shown in Table 8. It is evident from Table 8 that the PI values for SPG-M and SG are high as compared with control, such showing stimulation of the non-specific immune system. 
     
                       TABLE 7______________________________________Composition for dietComposition         Amount (g/kg diet)______________________________________Steam Dried Herring Meal               558.66Dried Whey           84.6Blood Flour          48.17Euphasids (whole frozen)                21.02Wheat Middling      126.19Vitamin Supplement   2.41Mineral Supplement   7.27Choline Chloride (60%)                4.60Ascorbic Acid        1.84Permapell            13.81Herring Oil          91.38SPG-M/SG             1.00 (0) *α-Cellulose    39.05 (40.05) *______________________________________ * The value in the parenthesis is for control. 
    
     
                       TABLE 8______________________________________Change in phagocytic activity of salmon&#39;spronephoros cells by SPG-M and SG  Number of fish tested               Phagocytic Index (PI)______________________________________Control  10             2.81 ± 0.95 (a)SPG-M    10             9.53 ± 3.36 (b)SG       10             10.36 ± 3.47 (b)______________________________________ 
    
     Thus the results show that β-1,3-glucans are effective both as means of stimulating the immune system of fish and as means of improving the efficacy of vaccines. 
     The results show effect against A. salmonicida, the causative agent in furunculosis for which published vaccination attempts have yielded poor results. But the same effect could, it is believed, be achieved against bacterial kidney disease (BKD) caused by the organism Renibacterium salmoninarum which is also a pathogen of salmonid fish. 
     The above experimental results are directed to fish belonging to the family salmonidae. However, the experimental results are equally applicable to other species, for example ornamental fishes, pet, hobby fishes, carp, sea bream and the like. In particular there is nothing that would lead the skilled worker to believe that the compositions are not effective for all fish but the invention is of particular interest in the treatment of aquaculture fish. There is a substantial homogeneity in the immune systems of all fish species.