ES2321791B2 - PROCEDURE FOR OBTAINING ANTI-PSEUDOMONAS ANGUILLISEPTICA VACCINE (PA-VAC) FOR THE PREVENTION OF PSEUDOMONIASIS DISEASE IN CULTIVATED FISH. - Google Patents

Abstract

Procedure for obtaining the anti-vaccine
Pseudomonas anguilliseptica (PAV-Vac) for the prevention of pseudomoniasis disease in cultured fish.

Procedure for obtaining the PA-Vac anti- Pseudomonas anguilliseptica vaccine, for the prevention of pseudomoniasis disease in cultured fish produced by the microorganism Pseudomonas anguilliseptica . The procedure is characterized by including in the vaccine the formalin-inactivated bacterial cells of two strains of anguilliseptic Pseudomonas with different geographical and host origin (CECT 899T and CECT 7245), representatives of the two serotypes described in this bacterial species. The vaccine has very effective levels of protection of fish against pseudomoniasis. The vaccine is applicable in the aquaculture sector.

Description

Procedure for obtaining an anti- Pseudomonas anguilliseptica (PA-Vac) vaccine for the prevention of pseudomoniasis disease in cultured fish.

Procedure for obtaining an anti- Pseudomonas anguilliseptica (PA-Vac) vaccine for the prevention of pseudomoniasis disease in different species of farmed fish. The vaccine is based on the combination of strains, causing mortalities in fish, isolated in different geographical areas and from different hosts, which differ in their antigenic and genetic characteristics.

Pseudomoniasis, produced by Pseudomonas anguilliseptica, is one of the main emerging diseases in marine aquaculture due to the economic losses that it causes, especially in eel, gold and sea bass crops. Other affected species are turbot, sea bream and cod.

Since 1997 epizootics of pseudomoniasis have been detected with some frequency in various Spanish fish farms, mainly affecting gilthead seabream and to a lesser extent. All strains isolated from these epizootics have high biochemical and antigenic homogeneity (Lopez-Romalde et al., 2003a: Journal of Aquatic Animal Health, 5: 39-47; López-Romalde et al., 2003b: Veterinary Microbiology 94: 325- 333). However, antigenic differences have been detected with the strains of this pathogen isolated from eel in other European countries and in Japan (López-Romalde et al. 2003b op. Cited ). Studies of genetic characterization have also demonstrated the existence of two major genetic groups, which correspond to the serological groups (López-Romalde et al ., 2003ª op. Cited ).

Preparation

From two logs in the logarithmic phase of growth of two strains of Pseudomonas anguilliseptica , CECT 899 and CECT 7245, two-liter flasks containing a liter of triptyse soybean culture medium (TSC: tryptone 17 g / L; peptone; of soybean 3 g / L; glucose 2.5 g / L; bipotassium phosphate 2.5 g / L; sodium chloride 10 g / L; pH 7.3 ± 0.2) supplemented with 0.5% chloride sodium

Incubation is performed at 25 ° C for 48 hours. When the culture reaches an optical density of 1 (Absorban-
cia 550) (approximately 10 10 cells / mL), formalin is added to a final concentration of 0.35% to kill the bacteria, and it is kept under stirring for three more hours, after which flasks are transferred at 4 ° C. The two strains used are mixed in a proportion of 50%.

Sterility control is carried out sowing the vaccine mixture in TSA-1 plates (Tryptone soy agar supplemented with 0.5% sodium chloride) and in thioglycolate tubes, incubating for 72 hours at 25 and 37 ° C respectively.

The specificity control is carried out by slide agglutination, using cells as antigen Whole used in the manufacture of the vaccine.

The vaccine should be stored at 4 ° C until utilization.

Administration mode

This vaccine should be given by injection. intraperitoneal To do this, fish are inoculated with 0.1 mL / fish (in fish smaller than 100 g) or 0.2 mL / fish (in fish larger than 100 g) of undiluted vaccine mixture.

The strains of Pseudomonas anguilliseptica used in this vaccine are CECT 899 T (type strain of the species) isolated from eel in Japan and TW P1 (Spanish isolated from gilthead) (deposited in the Spanish Type CECT Collection as CECT 7245) .

The efficacy of this vaccine was evaluated in turbot and gilt at both laboratory scale and scale countryside. The power, expressed as Survival Rate Relative (RSP) has been in both cases greater than 90%.

Claims (4)

1. Composition of the PA-Vac anguilliseptic anti- Pseudomonas vaccine that includes the formalin-inactivated cells of two strains of this pathogen: the type strain of the bacterial species (CECT 899 T), isolated from eel in Japan and the Spanish isolated from golden TW P1 (CECT 7245), which differ in their serological and genetic characteristics. 2. Method for obtaining the composition of claim 1, characterized by obtaining the bacterial cells in liquid cultures of Tryticasa soy broth medium (TSC) supplemented with 0.5% sodium chloride, by its inactivation with formalin at a final concentration 0.35% for 4 hours at room temperature and because the final concentration of bacterial cells is 10 10 cells / mL. 3. Method according to claim 2, characterized in that the mixing ratio of both strains in the vaccine is 50%. 4. PA-Vac anguilliseptic anti- Pseudomonas vaccine obtained by the method of claims 2 and 3 for the prevention of pseudomoniasis disease in cultured fish.