NO791690L - PROCEDURE FOR IMMUNIZATION OF FISH - Google Patents

Description

The present invention relates to a bivalent vaccine

for protection against vibrosis. The vaccine is obtained from two serotypes of Vibrio anguillarum. The vaccination of fish with the vaccine is carried out by easy immersion.

Vibriosis, a bacterial disease of all marine fish is a serious worldwide problem in connection with all intensively farmed marine fish and is one of the limiting factors in salmonid farming. The disease can occur at any time, and has been reported at water temperatures as low as 4°C. However, it is most prevalent in hot summer months. Outbreaks can be expected when water temperatures reach 14-20°C.

Vibriosis is caused by the organism Vibrio anguillarum.

This bacterium is a gram-negative motile rod, usually

slightly curved when originating from fresh isolates. The size of the cells is within the range of 0.5 x 1.4 ym to 10 x 2.7 ym.

The motile organisms can often be seen in wet sections of fresh spleen preparations from infected fish at 900 times magnification. It is usually a salt-demanding organism, but can be found in brackish water areas with salinity levels down to 5 parts per thousand.

Vaccination against vibriosis is steadily gaining popularity among fish farmers as a prophylactic control method. However, the lack of an appropriate and effective delivery method for mass immunization of fish has limited the use of vaccines to control vibriosis. Although various techniques are available for mass inoculation, e.g. Oral administration, injection, pressurized syringe vaccination (usually with a pressure of approx. 6.3-7 kg/cm ) and methods with forced fluid inlet such as vacuum infiltration and hyperosmotic infiltration have, apart from injection, provided non-quantitative vaccine doses .

Incorporation of the vaccine bacteria into the diet is e.g. a desirable delivery system due to its ease. However, the degree and duration of immunity achieved is usually less than by injection. This is possibly due to the difficulty in ensuring uniform dosage uptake in the diet. With regard to the injection technique, the inherent deficiency is obvious. Although the fish can be injected individually on a limited scale, individual injection of millions of small fish is not practically feasible. The shortcomings of pressure spray vaccine are the inapplicability to small fish weighing less than 4 g and possible hypersensitivity to the vaccine by the user due to the accompanying aerosol mist that occurs with this technique. A serious shortcoming of forced fluid flow is the traumatic physiological response of the fish to stresses induced by these pressure methods.

It has now been found that mass inoculation of marine fish against vibriosis can be easily achieved without the above-mentioned shortcomings by simple immersion without pressure in a bivalent vaccine obtained from Vibrio anguillarum.

The object of the invention is thus a vaccine solution usable for immunizing marine fish against vibriosis.

A further object of the invention is to produce such a vaccine comprising bacteria obtained from two serotypes of Vibrio anguillarum as immunogenic agents.

A further object of the invention is to produce a method for immunizing marine fish by simple immersion without pressure in the above-mentioned vaccine solution.

The main active agents found to be associated with vibriosis are two serotypes of vibrio anguillarum designated as Vibrio anguillarum 775 or Type I and Vibrio sp. 1669

or Type II (see, e.g., T.P.T. Evenul,"J. Fish. Res. Board Can." 28, 517 (1971) and L.W. Harrel et al., in "Fish Bull.", USA, 74, 447 (1976 )). When the live virulence of these two agents is administered to bony fish, the clinical symptoms of vibriosis including death appear. Administration of these two live virulent agents can thus

is used as a method to challenge a potential vaccine to test the immunogenic properties against it. The vaccine according to the invention has accordingly been found to be effective when immunizing sensitive fish exposed to infectious doses of the two agents. It is thus believed that the vaccine according to the invention can be used to significantly control attacks by vibriosis on fish.

The two virulent bacterial agents, serotype I (775)

and serotype II (1669) used for the preparation of the vaccine described below was obtained from the National Marine Fisheries Service, Seattle, Washington.

Vibrio Anguillarum 77 5, Type I, was isolated from coho salmon (Oncorhynchus kisutch) in April 1973 at the National Marine Service, Manchester site, Washington. The isolate used herein was obtained from the National Marine Fisheries Service on July 16, 1976. This is a typical marine vibrio; gram negative, motile curved rods, oxidase positive, anaerogenic enzymes, and sensitive to the vibriostatic compound 0/129 (2,4-diamino-6-7-di-isopropyl pteridine phosphate).

Vibrio sp. 1669, Type II, was isolated from coho salmon in November 1975 at Domesa Farms, Nanchester, Washington. The isolate used herein was obtained from the National Marine Fisheries Service on August 2, 1976. This is also a typical vibrio, but differs from Type I as follows: (1) Serologically different; (2) Arginine - Type II alkaline reaction on Møller<1>'s medium is negative in contrast to Type I's positive reaction; and (3) Type II does not produce acid from arabinose as Type I does. Furthermore, the average generation time is somewhat slower.

Biochemical and serological profiles of types I and II

is given by L. W. Harrell et al., ibid., in Table I on page 44.8.

In general, the present invention requires that after separate growth of Vibrio anguillarum Type I and Type II cells in a suitable liquid nutrient medium, a vaccine is prepared by killing the cells, preferably by chemical inactivation methods, although others such as e.g. heat can be used, and then combination of the two inactivated bacterial preparations with or without previous adjustment of the organism count to roughly equal levels.

Cells of Vibrio anguillarum can be cultured in any sterile liquid medium that can fulfill all the metabolic requirements such as Bacto tryptic soy broth or vibrio broth (see Example VII) or Bacto Furunculosis media. Preferred temperatures for growth are 20 to 28°C and for maximum yield the cultures should be aerated during growth. The yield can also be increased by adding glucose after a 10 hour incubation period to replace the carbon source.

In the present application, a preferred embodiment of the invention is described with reference to the development of a bivalent vaccine from the two above-mentioned isolates of Vibrio anguillarum Type I and Type II. However, the invention is not intended to be limited by these particular isolates.

Injections of each serotype from the two above-mentioned isolates into susceptible sockeye salmon (a Pacific salmon) at 15°C confirmed virulence. Each virulent serotype was then re-isolated by culturing the infected kidney sample from the dying fish, obtained by inserting a sterile needle into the fish kidney, on vibrio agar (see Example V) at 20°C for 24 hours. The colonies were checked for purity and typical serological and biochemical reactions confirmed for each serotype to thereby provide a pure subculture

on vibrio agar for each serotype. A master seed was obtained by then growing each serotype in atomizing desiccators (see Example VI) at 20°C for 24 hours followed by lyophilization in 2 ml vials which were then stored at -70°C.

For each serotype, a working seed was prepared by aseptically adding 0.5 ml of vibriokraft (see Example VII) to a vial of lyophilized master seed. The contents of the dehydrated culture were then added to a 3 liter flask containing 1500 ml of vibriokraft» "Working seed" was incubated with agitation at 22°C for 24 hours to give sufficient growth of the virulent organism. Growth was measured by recording the turbidity. After 24 hours of incubation, an optical density of at least 0.8 was reached using a 13 mm tube in a "Spectronic 20" spectrophotometer at 525 nm.

The 1,500 ml "working seed" specified above provided the inoculum for the respective production culture for each serotype: The 1,500 ml was added to 1,200 liters of vibrio power in a 1,800 liter stainless steel fermenter. The production culture was incubated at 22°C for 48 hours under constant agitation and aeration using sterile air through a purge unit at a rate of 0.25 cubic feet, per minute. The pH value of the production culture was maintained between 7.2 and 7.4 by the addition of ION sodium hydroxide. Prior to inactivation of the live virulent organisms, each culture was examined for purity by the conventional procedure of gram staining, culture and serological reaction to specific antisera.

The production cultures were then chemically inactivated by addition of 0.3% vol/vol formalin followed by incubation at 4°C for 48 hours. The inactivated bacterial preparation constituted bacterial material for each serotype. Each bacteria was tested for sterility before use, e.g. by standard procedures of incubation of vibrio kraft and thioglycollate kraft for 14 days at 22°C and 37°C to observe organism growth. 1.2 g/litre oxytetracycline hydrochloride was then added to each bacterial medium as a preservative. The stock solution of oxytetracycline hydrochloride was prepared by using 24 g per liter of distilled water and sterilizing the solution at 121°C for 30 minutes.

The above-mentioned bacteria-containing preparations can be used as they are in the production of the bivalent vaccines mentioned herein. However, it may be desirable to have the bacterial content in each preparation adjusted to essentially the same amount of serotype, i.e. the number of organisms per ml. Accordingly, each bacterial preparation was diluted with 0.85% saline to achieve an optical density of 1.0 as described above, which was found to be equivalent to approx. 1 x 10 9 organisms per ml. For serotype I, 4 ml of saline per 1 ml of bacterial medium and for serotype II with 2 ml of salt solution per 1 ml of bacterial medium to achieve an optical density of 1.0.

The bivalent vaccine was prepared by mixing equal volumes of each bacterial preparation (1:1) to obtain a preparation containing approx. 5 x 10 8 organisms per ml of each serotype with an optical density of 1.0.

For use as comparative controls, each was removed

the monovalent bacterial media diluted with the same volume of normal saline (1:1) to similarly obtain 5 x 10<8>

organisms per ml. The following three bacterial media were thus available for testing. 1. Bivalent vaccine Types I & II...' 5 x 10 8 organisms/ml of each type. 2. Monovalent bacteria Type I ... 5 x 10 8 organisms/ml Type I. 3. Monovalent bacteria Type II ... 5 x 10 8 organisms/ml Type II..

Each of the three bacterial media was then further diluted with water or water containing sodium chloride to a maximum concentration of 0.85% (normal saline) in tenfold increments as follows: IO-1 (1:10), 10~<2>( 1:100), 10~<3>(1:1000) and 10~4 (1:10,000). These dilutions were prepared to compare the potency of each preparation and to determine whether interferences in immunization could be detected by combining the two vibrios serotypes.

Antigenic substances prepared by the method indicated in the preceding description were evaluated in the following samples.

Example I

Separate groups of approximately 80 sockeye salmon weighing 8 g each were inoculated by immersing each group of fish in one of the tenfold diluted saline solutions of bacterial medium described above for 2 minutes. The final immersion ratio was 4 54 g fish per liter of bacterial medium. Controls consisted of immersing a separate group of fish in normal saline alone for 2 minutes. All fish were then held for 24 days at 10°C to induce immunity.

About. half of the fish from each group were then exposed to Type I and the other to Type II Vibrio anguillarum. These solutions were in each case a 24 hour culture

of each serotype grown in vibrio broth at 2 2°C. Each culture was then diluted 1000-fold with normal saline as a bath. The fish was placed in this bath for approx. 17 minutes. The exact level for each serotype was:

Type I ... 4.9 x 10 organisms per ml.

Type II... 2.1 x 10 organisms per ml.

After the exposure to these bacteria, the fish were kept at 16°C for 14 days. The cause of death was confirmed by isolation of organisms from the kidney of dead fish on vibrio agar followed by standard techniques such as column morphology, grain staining, section agglutination and sensitization to vibriostat 0/129.

The results obtained from these samples are shown in Tables 1 and 2.

Fish immunized with Type II or bivalent bacterial medium were protected from Type II exposure, but immunization with Type I did not result in significant protection against Type II exposure (Table 1). The bivalent Type J/Type II bacterial medium was as effective as the monovalent •Type II bacterial medium for protecting fish from vibriosis. There was no antigenic interference. Dilutions of 10 ^ to 10 -3 showed very effective protection and 10 -4 dilutions.

was less protective.

Exposure to Type I resulted in high losses of Type II vaccines while Type I and bivalent groups were protected (Table 2). The 19% mortality for the exposed control group was significantly lower than 41%, 45% respectively. 57% mortality in the three lowest dilutions of Type II vaccines (P=0.05). No antigenic interference was observed in the dilutions in the bivalent group. The protection from Type I preparations

-4

was ig^en smaller at the 10 ~ dilutions.

A comparison of all dilutions of monovalent versus bivalent immersion medium did not show antigenic competition. Effectiveness was observed with a single dip without significant loss of potency. Immersion of fish in bivalent bacterial medium appears to be an effective means of immunization.

Example II

In this test, the bivalent vaccine described above was diluted 10-fold with water instead of saline. Such a 10-fold dilution is the preferred field dose for commercial fisheries. In addition, the 1:10 aqueous bacterial medium was further diluted 1:1 with water, whereby a 1:20 aqueous bacterial medium corresponding to half of the recommended field dose was obtained. "Sockeye" salmon with an average weight of 2.6 g were dipped in these solutions for 2 min. and then exposed to Type I or Type II Vibrio anguillarum for 20 minutes as indicated in the following Tables 3 and 4. The cumulative mortality rate was reported.

Example III

This example shows another aqueous immersion vaccination test using chinook salmon with an average weight of 2.5 g. The 1:10 and 1:20 dilutions of bivalent bacterial medium were prepared as in Example II. The fish were immersed in these dilutions for 2 minutes and then exposed to the doses indicated in Table 5 of Vibrio anguillarum Type I for 20 minutes. The percentage of cumulative mortality was listed.

Example IV

This example shows that immersion times of less than 2 min. can be used to effectively immunize fish with the bivalent bacterial medium according to the invention. Sockeye salmon with an average weight of 2.5 g were immersed in the aqueous 1:10 and 1:20 dilutions described in Example II for periods of 5, 15, 30, 60 and 120 seconds. About. 40 fish were tested in each group. A separate group was placed in water for 120 seconds as a test. After immersion in the bacterial medium dilution, the fish were kept at 10°C for approx. 30 days before it was exposed to impact. This exposure was carried out with Vibrio anguillarum Type I at two different levels (8.4 x 10 5 and 1.7 x 10 5 organisms per ml). These solutions were prepared from 1 24 hour force culture of Vibrio anguillarum Type I containing 8.4 x 10 g of organisms per ml, diluted with water to 1:1000 or 1:5000. The fish were immersed in the exposure solution for 20 minutes and then kept at 18°C for 10 days. Dead fish were removed from each group daily. Standard procedures were used to confirm vibriosis as the cause of death. Percentage mortality and the relative percentage survival RPS were calculated. These results are shown in table 6.

The effectiveness of the immersion time was determined by comparing the loss of immunized fish with the loss of unvaccinated controls. The mortality for non-immunized controls was 9 6.4% at 8.4 x 10 5 organisms per ml impact level and 74.4% at 1.7 x 10 organisms per ml levels. Mortality was therefore satisfactory for potency testing at each exposure level. Survival in all groups included all immersion times tested and both immunization levels and was 82.2% or greater (ie, statistically significant) such that all test groups satisfactorily passed the potency test. The protection was approx. the same in all groups and in general fish immersed for 5 seconds were as well protected as those immersed for 120 seconds.

TABLE 6. Comparison of mortality and survival for "sockeye" salmon immunized by immersion in bivalent bacterial medium at different time intervals and at two dosages of bacterial medium.

a = 8.4 x 10 organisms per ml

b = 1.7 x IO<5>organisms per ml

c = RPS = relative percent survival

Example V

The following recipe produced 1000 ml of Vibrio Agar.

The ingredients were mixed in suitable containers, e.g. 1500 ml amounts in a 3 liter flask and then sterilized, e.g. by autoclaving at 121°C.

Example VI

The following recipe produced 4000 ml of mist desiccant.

Distilled water, q.s. ad 1000 ml

Sterile horse serum, q.s. ad 4000 ml

The first four ingredients are combined and sterilized, e.g. by filtering through a 0.22 ym membrane filter. After sterilization, sterile horse serum was added and the sterile medium dispensed into suitable sterile containers. The medium, which is examined for possible contamination before use, is stored at 2-8°C until use or for a period of no longer than 120 days.

Example VII

The following recipe gives approx. 1 liter of vibrio power.

Distilled water, q.s. ad 1000 ml

B. Antifoam agent ..5 ml.

The components in part A are combined and poured into suitable containers. The antifoam agent, "Medical Antifoam C Emulsion" in part B was then added with stirring. Containers and the finished mixture were then sterilized, and e.g. by autoclaving at 121°C.

From the description given above and the examples given, it is obvious that the present invention offers a method for producing a bivalent vaccine for immunizing fish against vibrio diseases and which comprises: a. To inoculate a liquid bacteriological culture that satisfies all metabolic requirements for the bacteria Vibrio anguillarum with the vibrioserotype Vibrio anguillarum 77 5; b. inoculation of a second such culture medium with . the vibrios serotype Vibrio sp. 1669; c. incubating each inoculated culture medium at a temperature and for a period of time sufficient to yield a substantial number of bacterial cells;

d. inactivating each culture medium; and

e. thoroughly mixing the two inactivated culture media.

Previous trial results also show that the best protection against vibriosis is achieved when the present vaccine comprises an immunologically effective amount of the two described bacterial media components compared to each of the monovalent bacterial media components alone because cross-protection was not demonstrated. The concentration of each bacterium in the present biva-4 5

lean vaccine should be at least 10 and preferably from 10 to 10<9>. inactivated organisms per ml of vaccine. Higher concentrations are also suitable/but are considered unnecessary with regard to the effectiveness of the final product. The most preferred concentration for each bacterial medium is from 10 6 to 10 8 organisms per ml. It should be pointed out that the concentration of each bacterial medium in the present bivalent vaccine need not be the same.

As previously shown, the method of immunization comprises simple immersion of the fish in the bivalent vaccine for a period of time sufficient to. to confer immunity, usually at least 5 seconds and preferably from 5 to 120 seconds, although longer periods may be tolerated.

For field use, the present bivalent vaccine is preferably used in the form of a concentrate which can be easily diluted, e.g. with water or salt solution, to the desired effective concentration of bacterial medium. According to this, the present invention includes the production as described above of such concentrated vaccines as e.g. by suitable dilution, e.g. 1:1 to 1:20 (vaccine:diluent) in an immersion bath with a concentration of at least 10 4 of each of the inactivated organisms per ml bath.

The temperature in the immersion bath is not essential and the limits are determined by the fish. However, the onset of immunity may vary directly with temperature. E.g. it is noted that the immunity is achieved after approx. 40 days at 4°C while it is noted at 5-10 days at 18°C. The preferred temperature for the immersion bath is from 8 to 15°C.

It should be obvious that the present invention is not limited to immunization only of those fish that are. stated above, but includes other bony fish such as rainbow trout, goldfish, flounder, various types of salmon, eel, etc.

In addition, it should be obvious that the invention is not limited to the particular embodiment or the methods. where described, but includes all such modified forms as may. lie within the framework of the present requirements.

Claims (8)

1. Method for immunizing fish against vibrio disease, characterized in that it comprises immersing the fish in a bivalent vaccine comprising an immunologically effective amount of killed organisms Vibrio anguillarum 775 and Vibrio sp. 1669 for a period of time sufficient to give the fish immunity. 2. Method according to claim 1, characterized by immersing the fish in a bivalent vaccine comprising an immunologically effective amount of killed organisms Vibrio anguillarum 775 and Vibrio sp. 1669 in a concentration of at least 10 4 of each organism per ml of vaccine for at least 5 seconds. 3. Method according to claim 2, characterized in that the concentration is from 10 6 to 10 8 of each organism per ml of vaccine. 4. Method according to claim 2, characterized in that the immersion time is from 5 to 120 seconds. 5. Bivalent vaccine for immunization of fish against vibrio diseases, characterized by an immunologically effective amount of killed organisms Vibrio anguillarum 775 and Vibrio sp. 1669. 6. Bivalent vaccine according to claim 1, characterized by killed organisms Vibrio anguillarum 775 and Vibrio sp. 1669 in a concentration of at least 10^ of each organism per ml of vaccine. 7. Vaccine according to claim 6, characterized by 6 8 that the concentration is from 10 to 10 of each organism per ml of vaccine. 8. Method for producing a bivalent vaccine for immunizing fish against vibrio diseases, characterized in that it includes: a. To inoculate a liquid bacteriological culture that satisfies all metabolic requirements of the bacterium Vibrio anguillarum with the vibrios serotype Vibrio anguillarum 775; b. inoculation of a second such culture medium with Vibrio serotype Vibrio sp. 1669; c. incubating each inoculated culture medium at a temperature and for a period of time sufficient to yield a substantial number of bacterial cells; d. inactivating each culture medium; and e. intimately mixing the two inactivated culture media.