BG65739B1 - STRAIN No.322 LACTOCOCCUS GARVIEAE, VACCINE MADE OF IT AGAINST STREPTOCOCCOSIS IN VERTEBRATE FISH AND PROCESS FOR ITS PREPARATION - Google Patents

Abstract

The strain is filed in NBIMCC under No. 8353. It finds application in the biotechnologies for the preparation of degraded vaccine against streptococcosis in vertrebral fish. It is cultivated on solidand liquid nutritive medium. The vaccine is used for the immunization only of healthy populations. It is administered interperitoneally, by immersion or perorally.

Description

(54) LACTOCCOCUS GARVIEAE, A VACCINE FOR STRAPTOPHOCOSE IN ITS SPIRITUAL FISH AND A METHOD FOR PREPARING IT

Technical field

The invention relates to a strain of Lactoccocus garvieae, a killed vaccine from it against vertebrate streptococcosis and a method for its preparation.

BACKGROUND OF THE INVENTION

Lactoccocus garvieae is also known as Enteroccocus seriolicida and Streptoccocus garvieae, with group N. streptococcal antigen. This bacterium is the leading cause of the so-called. fish streptococcosis. The epizootic (animal epidemic) caused by it was first described in Japan in 1974 in yellow-tailed fish - Seriola quinqueradiata (R. Kushida, E. Satali, Enteroccocus seriolicida and Streptoccocus iniae, In: Fish Diseases and Disorders , Vol 3; Viral, Bacterial and Fungal Infection, pp. 303-317, eds PTK Woo and DW Bruno, Cab International 1999). The disease subsequently spread to different countries, incl. Far East, USA, South Africa, Australia, Israel and Europe, with the same bacterium affecting more than twenty species of fish: cultivated, freshwater and marine species. The bacterium is also isolated from seawater, silt in ponds, from the gut of wild fish - the Spanish mackerel (Skomber Japonicus) and the black scraper (Novodonmodestis). The infection is systemic. It occurs with hemorrhagic septicemia and exophthalmos. Mortality rates are as high as 50-80%, leading to huge economic losses. The economic losses of aquaculture fish require the creation of a vaccine for fish streptococcosis. In addition to the direct enormous economic losses from fish mortality and reduced weight, strepcococcosis from diseased fish also causes disease in humans.

It is known from the literature that Lactoccocus garvieae was used for which no data were published. An experimental vaccine was prepared from it. In experimental conditions, rainbow trout were vaccinated with it, resulting in an 80-90% survival rate and up to 70-80% in aquaculture (Bercovier HAD, Ghittino CAU, Eldar A. Immunization with bacterial antigens: infections with streptococci and related organisms, Dev Biol Stand 1997; 90: 153-159).

Also known is a vaccine derived from Lactoccocus garvieae strain for which no data have been published. However, the results of its use show a mortality rate of 13.5-15.6% compared to the control group of unvaccinated fish, in which the mortality rate is 52-68% (John S. Clark and Patrick D. Smith, Aquaculture Vaccines Ltd. Prevention of Streptococcus in Tilapia by Vaccination). However, when administered to fish in cages in Bulgaria, this vaccine did not produce the expected results, due to the difference in the strain causing the disease in our country.

It is an object of the invention to provide a strain, a vaccine thereof against Lactoccocus garvieae and a method of producing it, which vaccine will provide a survival rate of more than 95% of the immunized fish, suitable for self-administration or in combination with immunostimulants, and to reduce mortality fish to technological.

SUMMARY OF THE INVENTION

The task is solved by strain Lactoccocus garvieae, deposited in NBPMCC - Sofia, Republic of Bulgaria under No. 8353 of 19.02.2004.

The strain is obtained by isolating from the brain of a streptococcosis patient rainbow trout Lactoccocus garvieae cultured routinely on standard enriched culture media under aerobic conditions for 24 h at 2042 ° C. As standard enriched medium, either blood agar or cerebral agar medium or trypticase-soy broth is used.

The strain is characterized by the basic characteristics of the species, the strain Lactoccocus garvieae, as follows:

- Cultivated on blood agar under aerobic conditions at a temperature range of 10 to 42 ° C. On blood agar, the colonies were smooth, convex, 1-1.5 mm in diameter, white to off-white and opaque, non-hemolytic after cultivation, and with alpha-hemolysis while storing the crops for 2-3 days at room temperature.

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- The strain grows at 42 ° C and very sparingly at 45 ° C; develops into standard broth with pH 9.6 but not into broth with 6.5% NaCl.

- Identification based on the enzymatic performance of the strain with the BBL Crystal Identification Panel - Rapid Gram Positive ID System (Becton & Dickinson) indicates that it is more than 95% likely to belong to the species Lactoccocus garvieae.

- It is resistant to the antibiotic clindamycin, which distinguishes it from other closely related bacteria.

- The strain differs from other known Lactoccocus garvieae strains (NBPMK No. 8066 and killed L. garvieae from a commercial lactococcal vaccine of Aquaculture Vaccines Ltd, Essex, UK) in the presence of additional surface antigens, which is detected by hyperimmune mouse murine strains and indirect immunofluorescence response. The presence of these additional antigens in the strain explains the high efficacy of the vaccine it produces.

To obtain primary and working seed batches, the bacterial strain is propagated on solid culture medium for 18-24 h, at a temperature of 20-40 ° C, after which it is washed with serum-glutamate protective medium and lyophilized. The resulting lyophilisate was stored in ampoules at 2-8 ° C.

The vaccine contains bacteria killed by heat and formalin bacteria strain Lactoccocus garvieae, deposited in NBPMCK - Sofia, Republic of Bulgaria under No. 8353 of February 19, 2004, suspended in saline, with a density of 0.5-10 x 10 ⁹ cells / ml and preservative merthiolate 0.01-0.1 mg / ml, preferably 0.04-0.06 mg / ml.

To obtain the subculture, the lyophilisate from the ampoule was resuspended in a sterile casein fermentation broth. It was sown in a flask containing the same culture medium and cultured under continuous shaking at 100300 rpm / min for 18-24 h at 20-40 ° C.

The vaccine is obtained in liquid or solid media.

For preparation in liquid culture medium, the resulting subculture is introduced into a fermenter containing sterile casein fermentation broth at a ratio of 1: 20-1: 100.

It is cultured in a routine manner, maintaining the dissolved oxygen level 5-50% automatically, pH 6.8-7.6 and temperature 28-38 ° C for 8-16 hours. At the beginning of the stationary phase, cultivation is stopped and bacterial density is determined spectrophotometrically.

For preparation in solid culture medium, the subculture was seeded in the medium and cultured for 18-24 h at 2838 ° C. Bacterial growth was washed with saline and bacterial density was determined spectrophotometrically.

After being prepared in solid or liquid culture medium, the bacteria are killed by heating for 20-60 minutes at 54-58 ° C and formalin is introduced in an amount of 0.1 -0.5%, preferably 0.2-0.4% . The bacterial culture is examined for the absence of live bacterial cells. The sterile bacterial suspension is diluted with sterile saline to the required density and, by the routine method, merthiolate is added in an amount of 0.01 -0.1, preferably 0.04-0.06 mg / ml. After repeated sterility checks, the vaccine is packaged in primary packs.

Standard blood agar or standard heart-brain agar or casein fermentation broth containing 2% agar-agar is used as the solid medium.

The vaccine is given by injection, immersion and orally at a water temperature in the reservoir between 8 and 15 ° C.

To induce protective immunity at a water temperature of 8- 10 ° C, it is necessary to administer the vaccine at least 4 weeks before the expected onset of the disease in the population. Immunization after 2-3 weeks is recommended for intense and prolonged immunity.

Injectable administration: The vaccine is injected in a volume of 0.1 ml per unit of fish intraperitoneally (i. P.), With the fish pre-anesthetized.

Immersion administration: The vaccine is diluted with water in a suitable container in a ratio of 1 l vaccine to 9 l water. The fish is removed from the reservoir in which it is reared and immersed in the vaccine vessel for 1 minute, then returned to the reservoir. 11 vaccines are used for the immune system

65739 100 kg fish fish.

Oral administration: The vaccine is suspended, mixed with food immediately before each fish feeding. The fish are fed the vaccine for 5-7 days and after a rest of 5-7 days they are fed again for 5-7 days. The dose for 10-14 days is 0.3 ml / fish distributed in the food for those days.

Advantages

An original strain of Lactoccocus garvieae was isolated and characterized. A killed vaccine was created by him against Lactoccocus garvieae infection in vertebrate fish, a method for its preparation and administration. The advantage of the vaccine is its high efficiency - over 95%. The vaccine created is suitable for injection, immersion and oral administration, alone or in combination with immunostimulators. The vaccine builds up intense and prolonged immunity sufficient during the epidemic season to protect the population from disease and mortality. This immunity coincides with the technological period for the cultivation of fish and provides a weight sufficient for the realization of production. This vaccine is very effective in preventing vertebrate farmed fish in our country, incl. trout.

Examples of carrying out the invention

The following examples illustrate the invention without limitation.

Example 1. A strain of Lactoccocus garvieae

Lactoccocus garvieae strain deposited in NBPMCK-Sofia, Republic of Bulgaria under No. 8353 of 19.02.2004.

The strain is characterized by the basic characteristics of the species Lactoccocus garvieae, as follows:

- Cultivated on blood agar under aerobic conditions at a temperature range of 10 to 42 ° C. On blood agar, the colonies were smooth, convex, 1-1.5 mm in diameter, white to off-white and opaque., Non-hemolytic after cultivation, and with alpha-haemolysis, keeping the crops for 2-3 days at room temperature.

- The strain grows at 42 ° C and very sparingly at 45 ° C; develops into standard broth with pH 9.6 but not into broth with 6.5% NaCl.

- Identification based on the enzymatic performance of the strain with the BBL Crystal panel - Rapid Gram Positive ID

System (Becton & Dickinson) shows over 95% probable affiliation with Lactoccocus garvieae.

- It is resistant to the antibiotic clindamycin, which distinguishes it from other closely related bacteria.

- The strain differs from other known Lactoccocus garvieae strains (NBPMK No. 8066 and killed L. garvieae from a commercial lactococcal vaccine of Aquaculture Vaccines Ltd., Essex, UK) in the presence of additional surface antigens, which is detected by hyperimmune mouse murine serum strain and indirect immunofluorescence reaction. The presence of these additional antigens in the strain explains the high efficacy of the vaccine it produces.

The strain was obtained by isolating from the brain of a patient with streptococcosis rainbow trout Lactoccocus garvieae and cultured routinely on standard enriched culture medium blood agar under aerobic conditions for 24 h at 30 ° C.

In a variant of the method, the cerebral agar medium is used as a standard enriched nutrient medium.

In a variant of the method, trypticase-soy broth is used as a standard enriched nutrient medium.

Work is done on the system of primary and working seeding series. A new ampoule of lyophilized starting strain was used for each cultivation.

To obtain primary and working seed batches, the bacterial strain is propagated on solid culture medium for 22 h at 30 ° C, after which it is washed with serum-glutamate protective medium and lyophilized. The resulting lyophilisate was stored in ampoules at 6 ° C,

Example 2. Composition of the vaccine

The vaccine vessel contains the heat and formalin bacteria killed by strain Lactoccocus garvieae, deposited in NBPMCC under No. 8353 of 19.02.2004, suspended in saline, at a density of 3 x 10 'cells / ml and preservative merthiolate 0.05 mg / ml. .

Example 3. Method for the preparation of the vaccine.

Example 3.1. Obtaining a subculture.

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To obtain a subculture, the lyophilisate from the strain ampoule was sterile opened and resuspended in a sterile casein fermentation broth. It was sown in an Erlenmeyer flask containing the same culture medium and cultured under continuous shaking of a rotary shaker at 200 rpm / min, 4 cm deviation for 20 h at 30 ° C.

Example 3.2. Preparation in a liquid culture medium.

The resulting subculture was introduced into a fermentor containing sterile casein fermentation broth at a ratio of 1:50. It is cultured in a routine manner, maintaining automatically a dissolved oxygen level of 10%, a pH of 7.2 and a temperature of 30 ° C for 12 hours. At the beginning of the stationary phase, cultivation was stopped and bacterial density was determined spectrophotometrically. The bacterial suspension obtained in the fermenter is killed by heating for 30 minutes at 56 ° C and formalin is added in an amount of 0.3%.

The pH is adjusted with sodium hydroxide.

Bacterial culture is assayed for the absence of live bacterial cells by routine methods.

The sterile bacterial suspension is diluted with sterile saline to the required density and, according to the routine method, merthiolate 0.05 mg / ml is added.

After repeated sterility checks, the vaccine is packed in sterile containers.

Example 3.3. Obtaining in solid nutrient medium.

Example 3.3.1. The subculture was seeded in solid culture medium and cultured for 20 h at 30 ° C. Standard blood agar is used as the solid medium. Bacterial growth was washed with saline and bacterial density was determined spectrophotometrically. Bacteria were killed by heating for 30 min at 56 ° C, after which formalin was introduced at 0.3%.

The necessary routine control tests are performed to demonstrate the absence of microbial contamination and live cells from the original strain.

The sterile bacterial suspension is diluted with sterile saline to the required density. 0.05 mg / ml merthiolate was added. After repeated sterility checks, the vaccine was routinely packaged under sterile conditions in sterile containers.

Example 3.3.2. In a variant of the method, the subculture is cultured in solid culture medium with standard cardiovascular agar, the cultivation being carried out in the same manner as in Example 3.3.1.

Example 3.3.3. In a variant of the method, the subculture is cultured in solid culture medium with a standard casein fermentation broth containing 2% agar-agar, the cultivation being carried out in the same manner as in Example 3.3.1.

Example 3.4. Storage of the vaccine ..

The bacterial strain was stored in lyophilized form, in ampoules, at 6 ° C. The vaccine is stored in containers under the same conditions for up to 30 months.

Example 4. Use and administration of the vaccine.

The vaccine is used for injection, immersion and oral immunization of vertebrate fish. It is applied at a water temperature of 10 ° C in the reservoir. To induce protective immunity, at 10 ° C water temperature, the vaccine is administered 4 weeks before the expected onset of the disease in the population. Only healthy populations are immunized, avoiding stress on fish as much as possible. The vaccine is also used for immunization. For intense and prolonged immunity, fish are immunized after 2 weeks.

Example 4.1. Injectable application.

The vaccine is injected in a volume of 0.1 ml / piece of fish intraperitoneally (i. P.), With the fish pre-anesthetized.

Example 4.2. Immersion application.

The vaccine is diluted with water in a suitable container of a ratio of 1 l of vaccine to 9 l of water. The fish is removed from the reservoir in which it is reared and immersed in the vaccine vessel for 1 minute, then returned to the reservoir. 11 vaccines are used to immunize 100 kg of fish.

Example 4.3. Oral use.

The vaccine is mixed with food mis

65739 Bl immediately before each fish feeding. The fish are fed the vaccine for 6 days and after a rest of 6 days they are fed again for 6 days. The dose for 12 days is 0.3 ml / ί of fish distributed in the food for those days.

Example 5. Field experience.

Example 5.1. Injectable application.

The field experience was performed on rainbow trout grown in a pond, in cages, each with 50,000 specimens. We identified five of the fish cages as a control group, and in another ten cages we immunized the fish with the vaccine once intraperitoneally at a dose of 0.1 ml / fish. The investigations were carried out in April, when the water temperature in the reservoir reached 12 ° C. We reported the results in December. In non-immunized fish cages, a massive manifestation of streptococcosis was reported, with typical disease manifestations, as well as a delay in body weight gain, with mortality reaching 80%. No fish with symptoms of streptococcosis were observed in the immunized fish cages and the mortality rate remained within the technological marriage limit of 2%.

Example 5.2. Immersion application.

For the purpose of the experiment, we identified three cages with 300 kg of fish each, the two cages designated for the cultivation of immunized fish and the third for control. The vaccine was diluted with water in a suitable container of a ratio of 3 l vaccine to 27 l water. We removed the fish from the first cage in which it was farmed and immersed it in the vaccine vessel for 1 min, then returned the fish back to the same cage. We used 1 1 vaccine to immunize 300 kg of fish. We also vaccinated the other 300 kg of fish in the second cage. The fish from the third tank designated for control were submerged in water only with no vaccine. The investigations were carried out in April, when the water temperature in the reservoir reached 12 ° C. We reported the results in December. In the non-immunized fish cage, a massive manifestation of streptococcosis was reported, with typical disease manifestations, as well as a delay in body weight gain. The death rate reached 85%. No fish with symptoms of streptococcosis were observed in the immunized fish cages and the mortality rate remained within the technological marriage limit of 2%.

Example 5.3. Oral use.

The field experience was performed on rainbow trout grown in a reservoir, in three cages, each with 10,000 specimens. We identified one of the fish cages as a control, and in the other two cages we immunized the fish with the vaccine. The investigations were carried out in April, when the water temperature in the reservoir reached 12 ° C. In all three cages we fed the fish with the same food, and we also added a vaccine to the fish of the first and second cages. We mixed the vaccine with food just before each fish meal. The fish were fed the vaccine for 6 days and after a rest of days we were fed again for 6 days. The dose for 12 days is 0.3 ml per 1 fish distributed in the food for those days. We reported the results in December. The non-immunized fish cage reported a massive manifestation of streptococcosis, with typical disease manifestations, as well as a delay in body weight gain. The mortality rate reached 83%. No fish with symptoms of streptococcosis were observed in the immunized fish cages and the mortality rate remained within the technological marriage limit of 2%.

Use of the invention

The invention is used in biotechnology for the production of killed vertebrate streptococcosis vaccine, and the resulting vaccine is immunized with intraperitoneally, immersion or orally healthy populations.

Claims

Claims (8)

1. Lactoccocus garvieae strain deposited in NBPMCC - Sofia, Republic of Bulgaria under No 8353 of 19.02.2004 as a vaccine ingredient. 2. A vertebrate streptococcosis vaccine containing bacteria from the Lactoccocus garvieae strain, characterized in that it contains heat-killed and formalin bacteria from the Lactoccocus garvieae strain, per. No. 8353 of 02/19/2004, suspended in saline, with a density of 0,5-10 x 10 ⁹ cells / m < ^{1 &gt}; and preservative. 3. Vertebrate streptococcosis vaccine according to claim 2, characterized in that 0.01-0.1 mg / ml, preferably 0.04-0.06 mg / ml, is used as a preservative. 65739 Bl 4, A method of producing a vaccine, characterized in that the Lactoccocus garvieae strain is isolated from the brain of a streptococcosis rainbow trout patient, cultured on standard enriched culture media under aerobic conditions for 24 h at 20-42 ° C, after which the bacterial strain is propagated on a solid culture medium for 18-24 h, at a temperature of 20-40 ° C and washed with serum-glutamate protective medium, lyophilized and, for the preparation of subculture, lyophilisate is resuspended in a sterile casein fermentation bu flax is seeded in a flask containing the same culture medium and cultured under continuous shaking at 100-300 o6 / min for 18-24 h at a temperature of 20-40 ° C, after which the vaccine is obtained in liquid or in solid media, after which the bacterial density is determined spectrophotometrically, the bacterial suspension is killed by heating for 20-60 minutes at a temperature of 54-58 ° C, formalin is introduced in an amount of 0.1-0.5%, preferably 0, 20.4%, after which the bacterial culture is examined for the absence of live bacterial cells, sterile bacterial sus the pension is diluted with saline to the required density and a preservative added. 5. A method for producing a vaccine according to claim 4, characterized in that in a variant of the method the vaccine is prepared in a liquid culture medium by introducing the resulting subculture into a fermenter containing sterile casein fermentation broth at a ratio of 1: 20-1 : 100 and cultured in a routine manner, maintaining the dissolved oxygen level 5-50% automatically, pH 6.8-7.6 and temperature 28-3 8 ° C for 8-16 h and at the beginning of the stationary phase of cultivation is terminated and bacterial density is determined by spectrophotometry. o. A method of producing a vaccine according to claim 4, characterized in that in a variant of the method the vaccine is obtained in solid culture medium by subculturing into the medium, cultured for 18-24 hours at a temperature of 28-38 ° C, the resulting bacterial growth was washed with saline and bacterial density was determined spectrophotometrically. A method for preparing a vaccine according to claims 4 and 6, characterized in that a standard blood agar or standard heart-brain agar or casein fermentation broth containing 2% agar-agar is used as a solid nutrient medium for the preparation of the vaccine. . A method for producing a vaccine according to claim 4, characterized in that the strain is cultured on standard enriched culture medium blood agar or cerebral cardiac agar medium or trypticase-soy broth. Publication of the Patent Office of the Republic of Bulgaria 1797 Sofia, 52-B Dr. GM Dimitrov Blvd.