WO2008084125A2

WO2008084125A2 - Method for the preparation of a scuticociliatosis vaccine for farmed marine fish

Info

Publication number: WO2008084125A2
Application number: PCT/ES2007/070217
Authority: WO
Inventors: José Manuel LEIRO VIDAL; Manuel Luis Sanmartin Duran; Jesús LAMAS FERNANDEZ; Juan Luis Barja Perez; María VAZQUEZ RUIZ DE OCENDA; Santiago Cabaleiro Martinez
Original assignee: Universidad De Santiago De Compostela; Cluster De La Acuicultura De Galicia
Priority date: 2007-01-12
Filing date: 2007-12-28
Publication date: 2008-07-17
Also published as: WO2008084125A3; ES2306592A1

Abstract

Method for preparing a vaccine against the principal pathogenous ciliates causing scuticociliatosis (genera Philasterides/Miamiensis and Uronema) in farmed marine fish. The vaccine consists of trophozoites of the ciliates inactivated with formaldehyde and emulsified in an oil-based adjuvant. The vaccine administered to fish experimentally infected with the ciliates at concentrations from 10 4 to 108 cells per millilitre, inactivated with a concentration of formaldehyde less than 1% and emulsified in adjuvant at concentrations between 10 and 90%, led to a high degree of protection.

Description

PROCEDURE FOR THE PREPARATION OF A VACCINE AGAINST ESCUTICOCILIATOSIS IN CROP MARINE FISH.

The object of the invention relates to the preparation of a vaccine that eliminates the risk of infection by ciliated parasites responsible for infections in marine fish. Specifically, a vaccine for use in the control of a systemic disease caused by ciliated protozoa of the Order Philasterida (according to the classification of the Phylum Ciliophora proposed by Denis Lyan in 2002) responsible for large mortalities in fish culture plants.

Ciliates are free-living organisms that live in water, soil, and also on the surface or inside of animal hosts using different symbiotic relationships. Scuticociliatosis, produced by histiophage ciliates, is an emerging infectious disease in the culture of marine fish such as, for example, flounder, Paralichthys olivaceus, in Korea (Dis. Aquat. Org. 62: 233-238, 2004; Dis, Aquat. Org. 62: 239-249, 2004; Dis. Aquat. Org. 47: 49-55, 2001): and in the turbot, Scophthalmus maxims, in Europe (Dis. Aquat Org. 18: 5-9, 1994; J Fish Dis. 23: 33-37, 2004; Dis. Aquat Org, 46: 47-55, 2001). The species of scuticociliado responsible for the infections in cultivated turbot trees is farmed in the northwest of Spain has been identified as Philasterides dicentrarchi by Iglesias and collaborators in 2001 (Dis. Aquat Org. 46: 47-55).

The scuticociliado P. dicentrarchi is an facultative parasite that produces a fatal scuticociliatosis in marine fish such as sea bass Dicentrarchus labrax (Eur. J. Protistol. 31: 327-340, 1995), or turbot. Infected fish show skin ulcers, darkening of the skin, alterations in swimming, exophthalmia and abdominal distention as a result of the accumulation of ascitic fluid in the body cavity. Ciliates appear in all the organs and tissues of infected turbots, including blood and ascites fluid. Hispatological studies demonstrated the presence of severe encephalitis and meningitis (associated with different degrees of liquefaction of the brain), hepatic parenchymal necrosis, severe edema of the intestinal wall, degeneration of muscle fibers, hyperplasia of the branchial epithelium, and vascular and perivascular inflammation .

Several investigations carried out in other areas of Spain, Portugal, France and Korea have also detected the presence of scuticociliate infection outbreaks (Fol. Parásitol. 51: 177-187, 2004) in the turbot produced by species, initially framed in the genera Philasterides / Miamiensis; although recent studies have demonstrated that both species are synonymous (Parasitology 132: 555-564.2006) ^,

Although Philasterides / Miamiensis ciliates can be destroyed by formalin treatments when it is in e! external environment (Aquaculture 217: 73-80, 2003), however, there is no effective therapeutic treatment to date when the parasite is located inside the fish causing systemic disease.

An attractive alternative to solve the problem of the absence of pharmacological treatments against infection would be the development of prophylactic tools capable of preventing infection by these pathogens. Traditionally, vaccine development has been the main preventive means to control many infectious diseases. To date, there is no commercial vaccine available! for the control of these ciliates, which together with the lack of effective therapeutic measures against this infection, makes the economic losses caused by these in flatfish aquaculture very considerable. There is, therefore, the need to develop effective vaccines capable of preventing infections by these pathogens and overcoming the previously mentioned drawbacks.

The invention describes an effective method to control infections by scuticociliated protozoa in the absence of other pharmacological resources to alleviate the systemic infection once established in the fish. The solution provided is based on the development of a vaccine capable of inducing effective protection by increasing resistance to infection, especially in predisposed fish, such as fish in intensive culture.

Embodiment

For the preparation of the vaccine, isolates of Philasterides / Miamiensis / Uronema have been used from naturally infected fish that have been identified based on their morphological and biometric characteristics. This identification was subsequently confirmed based on the genetic characterization based on the analysis of the nucleotide sequence of the genomic DNA corresponding to the gene that builds the small subunit of the ribosomal RNA (Parasitology 132; 555-564, 2006).

The ciliates were isolated under aseptic conditions of ascitic fluid, obtained by means of abdominalinocentesis, from marine fish with scuticociliatosis. The ciliates were concentrated by centrifugation of the ascetic fluid at 1000 g for 5 minutes, washed twice by resuspension / centrifugation in incomplete L-15 medium prepared as indicated below. The L-15 medium (Leibovitz) containing L-glutamine was obtained from Sigma-Aldrich Chimie (Germany) and was prepared according to the manufacturer's instructions, was sterilized by 0.22 μm filtration, and the pH was adjusted to 7.2. For To prepare 1 liter of complete L-15 medium, each vial of L-15 medium concentrated in 640 ml of double distilled water was dissolved, 90 mg of adenosma, histidine and uridine, 150 mg of guanosine 5 g of glucose, 250.mL of a lipid solution (the lipid solution was prepared by dissolving 1.6 mg / mL of L-α-lechina and 0.8 mg / mL of Tween 80 is double distilled water, heating the solution at 80 ° C for 1 hour), the final pH of the medium was adjusted to 72 and sterile filtered by 0.22 UIXL Under sterile conditions, this medium was supplemented with 100 mL of synthetic serum (final concentration of 10%), previously inactivated by incubation for 30 minutes at 56 ° C and A sterile antibiotic solution consisting of 100 units of penicillin G, 0.1 mg / mL of streptomycin sulfate and 0.25 mg / mL of amphotericin B was added, an aliquot of the resulting suspension was fixed with glurataldehyde (final concentration 0.25%), and the secilia suspension count two was performed in a hemocitometer. After counting, the density of

adjusted to 10 ° trophozoites / mL with incomplete L-15 medium and this suspension was used for experimental infections.

Fish of 50-100 g were inoculated intraperitoneally with 10 ³ ciliates / g of fish and kept at a constant temperature of 17-18 ° C. After 10-15 days or, alternatively, when the presence of symptoms of the infection was observed, the ciliates were isolated from the ascites fluid and the trophozoites were cultured in the laboratory for the production of the vaccine or for its long term maintenance.

Then, prior to the preparation of the vaccine, a contamination control was carried out by other microorganisms. To this end, the ascitic fluid containing the ciliates was grown in 25 cm ² culture bottles in complete L-15 medium for 24 hours at 23 ° C. Once verified that e! culture was axenic, inoculation was carried out in 230 cm ³ culture bottles containing 150 mL of complete L-15 medium, for maintenance in the laboratory, and for vaccine preparation, in 500 mL flasks containing 150 mL of complete L-15 medium and a 1/100 suspension of a stock of cells obtained from animal tissues that, unvaguely disintegrated and nitrated by 100 μn meshes, were subjected to an autoclave cycle of 121 ° C for 20 minutes. For the maintenance in the laboratory of the ciliates, the cultures were incubated at 18 ° C, while for the production of the vaccine, the incubation temperature of the cultures was 23 ° C. The advantage of the addition of animal cells and the increase in temperature at 23 ° C is related to a very significant increase in the growth kinetics of ciliates. In addition, the addition of heat-inactivated animal tissues to the culture presents undoubted advantages both from a point of view. Economic as food security. After a week in culture and, after checking the absence of the cells used as a supplement in the culture medium, the ciliates were isolated for the production of the vaccine. Under these culture conditions, the density of ciliates usually reaches values of about 10 ⁶ trophozoites / mL. At this time, the flask contents were centrifuged in 250 mL bottles at 1000 g for 15 minutes and the precipitate was washed a once with incomplete L-15 medium, counting the precipitate ciliates that were resuspended in the same medium at a concentration of 2 x 10 ⁷ ciliates / mL. The ciliates were washed once more at 1000 g for 5 minutes and finally resuspended in PBS containing formalin at a concentration of less than 1%, incubating for 1 hour at 4 ° C. After the inactivation, the emulsion was carried out with the oil-based adjuvant. To 1 volume of adjuvant, 1 volume of a solution of inactivated ciliates at a concentration between 10 ⁸ and 10 ⁴ cells / mL in PBS was added. The emulsion was carried out in test tubes of 15 mL of polypropylene or glass by gentle agitation. It is sufficient to form a stable emulsion that was stored at 4 ° C until use.

The vaccine should be given by injection to the following fish: turbot, flounder, flounder, sea bream, sea bass, parracho and hirami. The fish, weighing between 5-100 g, are inoculated with an antigenic load of 10 ⁴ - 10 ⁸ ciliates / fish. The vaccination is administered in two doses, the second dose being administered between 30-90 days after the first dose.

EXAMPLE 1

For the optimization of the vaccine we have carried out a complete 2x3 factorial design analyzing the influence of three experimental factors included in the production of the vaccine such as: a) the number of ciliates / mL; b) the percentage of formaldehyde used for inactivation; c) the percentage of adjuvant used for the emulsion of the vaccine on the experimental survival variable (percentage of accumulated mortality after 18 days after the experimental infection), Table 1. The vaccination protocol and determination of efficacy (mortality) used is schematized in Figure 1 and the observed and modeled effects of the factors analyzed on the experimental variable studied (cumulative mortality rate) are shown in Table 2.

The results presented in Table 2 indicate that the R ² statistical test explains 94.7946% of the variability in mortality. The adjusted R ² test that would be more suitable for comparing models with different number of independent variables, would explain 84.383% of the mortality variability. The average absolute error of 3.16 indicates the average value of the residuals. Table 1.- Summary of the coding system used in the factorial design to investigate the effects of the concentration of ciliates (A), formaldehyde (B) and adjuvant (C) used in the preparation of the vaccine.

Table 2.- Protection prediction values for the anti-Philasterides / Miamiensis / Uromma vaccine generated using the experimental model and the ANOVA analysis of variance. The table includes: (1) the experimental values observed; (2) the prediction will stop the expected values; (3) studentized residual values; (4) regression coefficients; (5) significance values with a probability of less than 90% (P <0.1)

R ² - 94.7946%

R ² (adjusted for degrees of freedom) ^: = 84.3338% Standard error of the estimates = 8.75309

Average absolute error = 3.16

Durbin-Watson statistic = 1, 37318

TO; ciliates concentration / mL; B: formalin (%}; C: adjuvant (%)

The Durbín-Watson statistical test compares the residual values to analyze the existence of a significant correlation based on the order in which the values are indicated: values below 1, 4 indicate that there is no influence on the order. After the analysis of variance (ANOVA) it is observed that 3 factors have values of P <0.1, indicating that SOB sigíwficatiΛ'smeϊite different from 0 COQ \ m 90% άe nivd of confidence.

Table 3 indicates the influence of the data based on the existence of experimental values greater than 3 times the average value of the observation, or that have an unusually high value in the DFITS statistic. In this case, no average data has an influence value equal to or less than 0.4 which indicates that there is no value with 3 times the average value of observer Is; however, there is a data with a very high value of DFITS Io that indicates that it can be a point of influence in the model. -

Figure 2 shows the frequency histogram indicating the influence of each factor on the response (mortality; M). The dashed line indicates the level of significance of 90%. Factor A: concentration of ciliates / mL; Factor B; formaldehyde concentration (%}; Factor C: Adjuvant concentration (%} The histogram shows that the factor that has the greatest influence at the time of vaccine protection is the concentration of parasites (Factor A). adjuvant and the combination of the concentration of ciliates and the concentration of the inactivating agent (CA) also have a significant influence, but of much less relevance.

The influence of the analyzed factors on the protection of the vaccine can be analyzed graphically in the following response curves constructed with the significant regression coefficients. In Figure 3 (a, b and c) the "response surfaces derived from the Regression analysis of the data obtained in the factory design! to investigate the combined effect of the factors concentration of ciliates / ml, percentage of formaldehyde and percentage of adjuvant on mortality (M in percentage) of vaccinated and experimentally infected fish ", using the following equation: M (%) = 18.8 + 19,625 A + 7,875C - 7,875 A. It can be seen that, as the concentration of ciliates in the vaccine increases (Figure 3a), greater protection against infection is obtained, this protection also increases when it decreases The concentration of adjuvant (Figure 3b), and is independent of the concentration of formaldehyde that is used to inactivate the ciliates (at least in the range of inactivating agent used) (Figure 3a, b).

EXAMPLE 2

Fish were vaccinated following e! experimental scheme indicated in figure 4; determining the levels of protection and the production of antibodies.

Initially, no mortality was observed in the fish infected with the ciliates in the first 6 days, but from this day the fish died. On day 18 post-infection in vaccinated fish with a single antigen dose in adjuvant, maximum protection of 57% was obtained against unvaccinated fish (with an accumulated mortality of 80%). However, there were no significant differences in the levels of protection with respect to other groups of fish vaccinated solely with the antigen or in fish immunized exclusively with the adjuvant (Figure 5).

When a second dose of vaccine was administered, it was observed that mortality levels decreased very significantly in the vaccinated fish with the antigen preparation emulsified in adjuvant with respect to the vaccinated fish with a single dose obtaining a significantly higher 77% survival rate. than that presented in fish vaccinated exclusively without adjuvant or with antigen (figure 6).

When the efficacy time of the vaccine was evaluated in fish vaccinated with two antigen doses, it was observed that fish vaccinated with ciliates emulsified in adjuvants, presented a greater degree of protection than fish vaccinated exclusively with adjuvant or that unvaccinated fish (figure 7).

The levels of antibodies obtained in the fish increased very significantly in the vaccinated fish with two antigen doses in combination with the adjuvant with respect to those vaccinated with a single dose (Figure 8).

Finally, the influence of the vaccination on fish growth was analyzed. As seen in Figure 9, there are no differences in the growth of vaccinated fish compared to unvaccinated fish.

Claims

1.- Procedure for the preparation of a vaccine against Philasterides / Miamiensis / Uronema, composed of trophozoites of these ciliates inactivated with formalin and emulsified in an oil-based adjuvant.

2. Method according to claim 1, characterized by the culture of Philasterides / Miamiensis / Uronema in Leibovitz L-15 medium supplemented with animal tissue cells.

3. Method according to claim 1, characterized in that the final concentration of ciliates in the vaccine preparation is between 10 ⁴ -10 ⁸ cells per milliliter of vaccine.

4. Method according to claim 1, characterized in that the final concentration of the formalin inactivating agent in the vaccine is less than 1%.

5. Method according to claim 1, characterized in that the final concentration of the adjuvant in the vaccine is 10-90%.

6. Method according to claims 1 and 5, characterized in that a stable adjuvant emulsion / aqueous preparation is obtained by stirring.

7. Vaccine, prepared according to the procedure of the preceding claims, for use in the prevention of scuticociliatosis of marine farmed fish such as, for example, turbot, flounder, flounder, sea bream, sea bass, sea bass, hirami.