CN102740878A

CN102740878A - Streptococcal combi-vaccine

Info

Publication number: CN102740878A
Application number: CN2010800471565A
Authority: CN
Inventors: L.拉布里; B.舍汉; Y.S.李; W.F.仙
Original assignee: Intervet International BV
Current assignee: Intervet International BV
Priority date: 2009-10-19
Filing date: 2010-10-18
Publication date: 2012-10-17
Also published as: CN107648600A; TWI495478B; BR112012009309B1; WO2011048041A1; BR112012009309A2; TW201119671A

Abstract

The present invention relates to a combination vaccine for the protection of fish against streptococcal infection, the use of streptococci for the manufacture of such a vaccine, to methods for the preparation of such a combination vaccine and to a kit-of-parts comprising such a vaccine.

Description

The streptococcus combination-vaccine

The present invention relates to be used to protect fish not receive the combination-vaccine of streptococcal infection, streptococcus is used to make the purposes of this type of vaccine, is used to prepare the method for this type of combination-vaccine and the test kit (kit-of-parts) of the each several part composition that comprises this type of vaccine.

Through the past many decades, what the visible Fish of worldwide consumed rolls up.This is about cold water fish for example salmon, flatfish, halibut and morrhua, and the tropical fish situation of Asia jewfish, tilapia, milk fish, amberjack, amberjack, cabrilla and Rachycentron canadum for example.Therefore, the number of fish farm and the increase of size have been seen, so that satisfy cumulative market demand.

As known by for example animal husbandry, a large amount of animals that closely live in together are subject to various sickness influences, or even the disease of knowing hardly or not seeing, or or even the disease of the unknown before large-scale commercial applications is cultured.This is real for fish culture equally.

The example of notorious commercially important fish diseases substance be Vibrio anguillarum ( Vibrio anguillarum), Mermaid luminous bacillus kill the fish subspecies ( Photobacterium damselaeSubspecies Piscicida), the ocean subdue bacillus ( Tenacibaculum maritimum), the Flavobacterium species ( Flavobacterium sp.), the Flexibacter species ( Flexibacter sp.), the Streptococcus species ( Streptococcus sp.), the Ge Shi Lactococcus ( Lactococcus garviae), blunt tarda ( Edwardsiella tarda), the Silurus asotus fish tarda ( E. ictaluri), viral necrosis virus, irido virus and Koi herpesvirus.

Several species of antibacterial Streptococcus cause infection in the known fish of in fish, more specifically in aquatic products industry, raising at present.The example of these type of Streptococcus species be Streptococcus iniae ( Streptococcus iniae), difficulty distinguish streptococcus ( S. difficile), streptococcus agalactiae ( S. agalactiae), streptococcus dysgalactiae ( S. dysgalactiae) and the sea dog streptococcus ( S. phocae).

Recently in the suggestion of the proper naming method of distinguishing streptococcus and streptococcus agalactiae about difficulty, there has been some variation.People such as Vandamme (Int. J. Syst. Bacteriology 47:81-85 (1995)) have advised that difficulty distinguishes that streptococcus in fact is the non-hemolytic streptococcus agalactiae.

Streptococcus iniae is frequently found in tilapia, rainbow trout, European jewfish and bream, Asia jewfish, Sciaenops ocellatus, Fugu ocellatus, Paralichthys olivaceus, amberjack and hybridization willow perch.Streptococcus iniae infects the influence of aquatic products industry annual above 100,000,000 dollar.

Difficulty distinguish streptococcus tilapia, madai and even ray in frequently find.

Although in many fish species, find, tilapia is infected in the main at present discovery of streptococcus agalactiae.

The vaccine that is used for resisting the streptococcal infection of fish is known in the art.

Many streptococcus vaccines are based on killed full cell.Streptococcus agalactiae vaccine is for example described in U.S. Patent application US 2005/0208077.Streptococcus iniae vaccine is for example in U.S. Pat 6,379, describes in 677.Being used to resist the vaccine that Streptococcus iniae infects also is purchased and can gets.Example is Norvax Strep Si, the vaccine that infects and sold by Intervet Int. B.V. to Streptococcus iniae.People such as Eldar (Vaccine 13:867-870 (1995)) have described based on killed full cell and have distinguished streptococcic vaccine to difficulty.

In order to improve the more specifically understanding of the streptococcus disease in tilapia in tropical fish, inventor's J disease investigation of having carried out being widely current in the main tilapia production country in Asia and Latin America.Through 8 years in the past, these researchs obtained almost 500 streptococcus separators that reclaim in about 50 places from 13 countries.Use standard biological chemistry and bacteriology's identification method to identify separator, and analyze (based on the not weighting of difference percentage to cell mean) through cluster (cluster) subsequently.What is interesting is that in almost 500 the streptococcus separators that from tilapia, reclaim, 82% is accredited as streptococcus agalactiae, and 18% is accredited as Streptococcus iniae.

Streptococcus iniae is the important fish diseases substance that causes disease and mortality rate in the fish species of many oceans and the fresh water cultivation in the torrid zone and subtropical zone.Comprise that at multiple fish species the vaccine that the antagonism Streptococcus iniae infects in the tilapia is obtainable, and have lot of documents about the pathogenesis of this biology in multiple fish species.

Can obtain the information of much less for the pathogenic streptococcus agalactiae of fish.

Streptococcus agalactiae is a so-called B group B streptococcus (GBS).It is the important pathogen of humans and animals.Although more relevant usually with the disease among people and the Niu host, the pathogenic streptococcus agalactiae of fish early obtained proof to 1966, at that time non-hemolytic B group B streptococcus be accredited as golden shiner (the America bream ( Notemigonus crysoleucas)) in 2 kinds of epizootic reasons.

Today, along with the reinforcement of aquatic products industry, streptococcus agalactiae is known to be that marine products and fresh water are cultivated mortality rate and the major reason of sickness rate in species and the particularly tilapia.Like what find through the inventor, the labor of tilapia streptococcus agalactiae separator is illustrated in the existence of 2 different in multiple biochemistry and phenotypic characteristic uniquenesses bunch.These unique bunch is called biotype; And on this basis, can between ' classics ' streptococcus agalactiae (hereinafter referred to as the streptococcus agalactiae biotype 1) and usually non-β hemolytic streptococcus agalactiae (hereinafter referred to as the streptococcus agalactiae biotype 2), make differentiation.This a kind of bacterial strain in back is previous identify awkward distinguish streptococcus ( S. difficile/ S. difficilis), but be re-classified as the non--beta hemolysis property variant of streptococcus agalactiae subsequently.

Streptococcus agalactiae in the tilapia of culturing infects known remarkable sickness rate, mortality rate and the economic loss of causing at present.The infection of streptococcus agalactiae cause multiple internal organs particularly brain septicemia with build crowd (colonization), cause clinical symptoms.The clinical sign that streptococcus agalactiae infects comprises unusual swimming, ' C ' body position and inappetence.Streptococcus agalactiae is popular everywhere in temperate zone and torrid areas, and the inventor is from being recovered to it Europe, Central America and Latin America and Asia ill tilapia everywhere.

2 streptococcus agalactiae biotypes cause trickle different disease syndrome, wherein biotype 1 infect from childhood to the production cycle fish everywhere that grows up, and in bigger fish, cause disease with preponderating biology 2.

In the inventor's up to now Epidemiological study, for almost 500 the streptococcus separators from 13 countries, the great majority in all streptococcus separators of tilapia are streptococcus agalactiae biotypes 2.The inventor has identified streptococcus agalactiae biotype 2 in the most ill fish that produces country from main tilapia, said country comprises Indonesia, China, Vietnam, Philippine, Ecuador, Honduras, Mexico and Brazil.

The analysis of isolating biotype 1 bacterial strain discloses and has 2 serotypes from fish: serotype Ia bacterial strain and serotype III bacterial strain (Suanyuk, people such as N., Aquaculture 284:35-40 (2008)).

Yet 2 bacterial strains have and make it become the identical biochemical characteristics of biotype 1 bacterial strain, and they all are hemolytics.

The most important thing is, 2 bacterial strains contain the proteinic gene of the bonded α-C of coded surface ( Bca).For this protein, show extensively that in 1991 it induces the protective immunity (Michel, people such as J.L., Inf. & Immun. 59:2023-2028 (1991)) to streptococcus agalactiae.

Even having produced monoclonal antibody to the specific protective α-C protein epitope in the B group B streptococcus, it can kill B group B streptococcus (Madoff, people such as L.C., Inf. & Immun. 59:204-210 (1991)).

α-C protein finds in streptococcus agalactiae serotype Ia bacterial strain usually, but uncommon on the surface of people's streptococcus agalactiae serotype III bacterial strain.Yet the result is that it is present on the surface of isolating up to now all fish diseases originality streptococcus agalactiae serotype III bacterial strains usually.

Therefore; Can positively reach a conclusion based on the for example existing whole-cell vaccines of the serotype of Mullet separator Ia serotype, can induce to known streptococcus agalactiae serotype Ia bacterial strain and the protective immune response of the streptococcus agalactiae serotype III bacterial strain of having found in the recent period.

Find surprisingly at present; Although it has relevant (joined) biotype, has relevant beta hemolysis property characteristic and the α with relevant surface combination-C protein; But the whole-cell vaccines based on known Mullet (Ia) bacterial strain only provide the partial protection to the streptococcus agalactiae serotype III bacterial strain of having found since recent really, and vice versa.

This unexpected discovery is still unheeded up to now.

Same unexpected consequence is that present vaccine is not enough to effectively avoid or treat the streptococcus agalactiae infection in the fish.

One of the object of the invention is a combination-vaccine of protecting fish not infected by streptococcus agalactiae through being provided for; Solution to this problem is provided, and said combination-vaccine has the characteristic that it comprises 2 streptococcus agalactiae biotype 1 bacterial strains of immunogenicity amount: one belongs to serotype Ia and one and belongs to serotype III.

Therefore first embodiment of the present invention relates to and is used to protect fish not receive the combination-vaccine of streptococcal infection, and wherein said vaccine comprises the streptococcus agalactiae biotype 1 serotype Ia cell of immunogenicity amount and the streptococcus agalactiae biotype 1 serotype III cell and the pharmaceutically acceptable carrier of immunogenicity amount.

The streptococcus agalactiae cell of immunogenicity amount is the amount that causes that immunne response is required, compares with not vaccinated fish, and it can reduce the severity of disease at least.

Pharmaceutically acceptable carrier can be the same simple with water or buffer agent or emulsion or oil-in-water or water-in-oil emulsion.

Although streptococcus agalactiae biotype 1 serotype Ia cell and streptococcus agalactiae biotype 1 serotype III cell can obtain for the technical staff easily, the example of streptococcus agalactiae serotype III bacterial strain has been preserved in state-run microbial preservation center (France), Pasteur's Institute on October 15th, 2009 under registration number CNCM I-4232; No. 25, Docteur Roux street, F-75724 Paris postcode 15, France (Collection Nationale de Cultures de Microorganisms (CNCM); Institut Pasteur, 25 Rue du Docteur Roux, F-75724 Paris Cedex 15; France), has the special Internaional, Inc of InterVideo, No. 35, dimension nurse Kyle Korver street; 5831 AN, Bock Si Meier, Holland (Intervet International B.V.; Wim de K rverstraat 35; 5831 AN, Boxmeer, The Netherlands) title and address.

In combination-vaccine according to the present invention, antibacterial can be to live attenuation form or for example exist as vaccine (bacterin) with inactivated form.The fact that still exists of the immunogenic properties of antibacterial importantly.This can easily guarantee through using full bacteria preparation.As stated, as long as the immunogenic properties of antibacterial still exists, the antibacterial in the preparation be that live, killed or even fragmented (fragmented) (for example through pushing it) through French press be not very important.

The antibacterial of attenuation alive is very suitable, because they confirm to carry the immunogenicity characteristic.And the antibacterial of the attenuation of living has the advantage above vaccine, and they can need not adjuvant and easily be given.In addition, their self replications are stopped by immune system until them to a certain degree, therefore can give the cell than low number.

On the other hand, when these antibacterials during with the form of vaccine, the immunogenicity characteristic also is present on the antibacterial.And vaccine has the advantage of the antibacterial that surpasses the attenuation of living, because they are very safe.

Therefore, in the preferred form of this embodiment, the present invention relates to wherein, the streptococcus agalactiae cell is the combination-vaccine of deactivation.Preferably, cell is with the form of vaccine.

Vaccine is defined as the antibacterial with inactivated form herein.The method that is used for deactivation seems that the activity for vaccine is incoherent.The conventional method that is used for deactivation for example all is a heat treatment well-known in the art, is applicable with processing such as formalin, divinyl imines, thimerosal.Use for example French press (French Press), provide by means of the bacteria inactivation of physiological stress to be used to make same suitable raw material (starting material) according to vaccine of the present invention.

Can begin preparation by bacterial cultures according to the well-known technology of skilled practitioner according to vaccine of the present invention.

The survey article that relates to fish vaccine and manufacturing thereof is Sommerset for example, I., Kross y, B., Biering, E. and Frost, P. in Expert Review of Vaccines4:89-101 (2005); Buchmann, K., Lindenstr m, T. and Bresciani, in J. Acta Parasitologica46:71-81 (2001), Vinitnantharat, S., Gravningen, K. and Greger, E. in Advances in veterinary medicine41:539-550 (1999) and Anderson, D.P. In Developments in Biological Standardization90:257-265 (1997).

In addition, skilled practitioner will find guidance among the embodiment hereinafter.

Basically the antibacterial and the pharmaceutically acceptable carrier that comprise effective dose according to vaccine of the present invention.

The cell concentration of using will depend on the existence of route of administration, adjuvant and use constantly.

In addition, those skilled in the art particularly find enough guidances among the embodiment in the information that list of references that preceding text are mentioned and hereinafter provide.

In general, generally speaking can use through injection based on the vaccine constructed in accordance of vaccine, its dosage is 10 ³-10 ¹⁰, preferred 10 ⁶-10 ¹⁰, more preferably 10 ⁸-10 ¹⁰Individual antibacterial.Although be that immunology is suitable, because business reason surpasses 10 ¹⁰The dosage of individual antibacterial will be less captivation.

For the amount of bacteria constructed in accordance and vaccine that is used for the per os application, the example of hereinafter will provide abundant guidance.

Being suitable for being used for the pharmaceutically acceptable carrier example that vaccine according to the present invention uses is for example PBS etc. of sterilized water, saline, aqueous buffer solution.In addition, can comprise other additives, the adjuvant that for example is described below, stabilizing agent, antioxidant and other according to vaccine of the present invention.

In preferably appearing, the vaccine that particularly comprises vaccine according to vaccine of the present invention can also contain immunostimulation material, so-called adjuvant.Generally speaking, adjuvant comprises the material of strengthening host's immunne response with non-specific mode.Many different adjuvants are known in the art.Be generally used for fish and the Crustacean adjuvant example in culturing and be muramyldipeptide, lipopolysaccharide, several kinds of glucosans and polysaccharide and Carbopol (R).The extensive overview ot of adjuvant that is suitable for fish and Crustacean vaccine is through Jan Raa (Reviews in Fisheries Science 4 (3): provide in summary file 229-288 (1996)).

Vaccine can also comprise so-called " vehicle ".Vehicle is that antibacterial adheres to it and need not covalently bound with it chemical compound.This type of vehicle is biological example microcapsule, little alginate, liposome and macrosols, all is known in the art.

Wherein to be embedded into this type of vaccine of the special form in the vehicle be so-called ISCOM (European patent EP 109.942, EP 180.564, EP 242.380) to antigen part.

In addition, vaccine can comprise one or more suitable surface active cpds or emulsifying agent for example Span or Tween.

But the oily adjuvant that is suitable in water in oil emulsion, using is a for example mineral oil or metabolism oil.Mineral oil is Bayol for example , Marcol And Drakeol

The example of non-mineral oil adjuvant is Montanide-ISA-763-A for example.

But metabolism oil is for example Oleum Arachidis hypogaeae semen and soybean oil of vegetable oil for example, and animal oil is fish oil squalane and Squalene and tocopherol and derivant thereof for example.

Suitable adjuvant is for example w/o emulsion, o/w emulsion and w/o/w dual emulsion.

Based on the example of the nano-particle adjuvant of water is Montanide-IMS-2212 for example.

Usually, vaccine is mixed with stabilizing agent, for example have the protein of propensity for degradation not to be degraded, strengthen the pot-life of vaccine, or improve lyophilization efficient with protection.Useful stabilizing agent is SPGA (people such as Bovarnik for example; J. Bacteriology 59:509 (1950)); Carbohydrate is sorbitol, mannitol, trehalose, starch, sucrose, dextran or glucose for example; Protein is albumin or casein or its catabolite and buffer agent alkali metal phosphate for example for example.

In addition, vaccine can be suspended in physiology's acceptable diluent.

Much less, adjuvantization, adding vehicle chemical compound or proteinic other modes of diluent, emulsifying or stabilisation also embody in the present invention.

Known when giving as water-in-oil emulsion, inactivated vaccine immunogenicity that show to improve of vaccine for example particularly.

Therefore, in the more preferably form of this embodiment, the present invention relates to wherein, vaccine is the combination-vaccine of water-in-oil emulsion.

From the acceptable viewpoint of pharmacy, the mineral oil of more and more being unwilling to use.

Therefore, being more preferably in the form of this embodiment, the present invention relates to the combination-vaccine that its medium oil is non-mineral oil.

In the most preferred form of this embodiment, the present invention relates to the combination-vaccine that its medium oil is Montanide ISA 763 A.

Can use many methods of application, it all is known in the art.According to vaccine of the present invention preferably via injecting, immerse, flood or be administered orally in fish.

The per os application is attractive method of application with for example intraperitoneal application especially.

In general: if vaccine can improve through adding adjuvant, method of application is incited somebody to action preferably intraperitoneal approach so.From the immunology viewpoint, be very effective vaccination approach with the intraperitoneal vaccination of vaccine, particularly because its allows mixing of adjuvant.

Application program can establishing criteria vaccination practice carry out optimization.The technical staff will understand how to accomplish this point, or he will find guidance in above-mentioned file.

The age of treating vaccinated fish is not crucial, although clear and definite be the stage of hoping as far as possible early, promptly before possibly being exposed to pathogen, be directed against the vaccination of fish pathogenic bacteria.

Yet the vaccination of minimum fish is difficulty and consuming time.In general, if desired or hope that the fish more than 5 grams can carry out vaccination by means of injection.

For oral administration, vaccine preferably be used for the suitable carrier that per os uses and mix, said carrier is for example alpha-cellulose or plant or animal origin different oily of cellulose, food or metabolizable material.Equally, attractive mode is that vaccine administration to high concentration living feed is biological, feeds to target animals fish for example living feed is biological subsequently.The living feed that is used for preferred especially food carrier according to the oral delivery of vaccine of the present invention and is packing vaccine is biological.

Suitable living feed biology comprises the non-selective filter feeder of plankton appearance (filter feeder), the member of preferred wheel animalcule, artemia ( Artemia) etc.It is highly preferred that Penaeus seu panulirus genus artemia species.

According to embodiment, the cross protection level between streptococcus agalactiae biotype 1 and the streptococcus agalactiae biotype 2 is very low, or non-existent as follows.

Therefore, the combination-vaccine of another kind of preferred form comprises antigen or this type of antigenic genetic stocks of encoding of streptococcus agalactiae biotype 2 cells of streptococcus agalactiae biotype 2 cells, the immunogenicity amount of immunogenicity amount in addition according to the present invention.

In addition, will benefit from antigen or the other existence of this type of antigenic genetic stocks of encoding of Streptococcus iniae of Streptococcus iniae cell, the immunogenicity amount of immunogenicity amount according to vaccine of the present invention.

Therefore, antigen or this type of the antigenic genetic stocks of encoding of Streptococcus iniae that comprises Streptococcus iniae cell, the immunogenicity amount of immunogenicity amount according to the combination-vaccine of another kind of preferred form once more of the present invention in addition.

Clear and definite is, also will benefit to be used to make another kind of fish invasive organism or fish pathogenic virus, this quasi-microorganism or the antigen of virus or the existence of this type of antigenic genetic stocks of encoding of the another kind of immunogenicity amount of vaccine according to vaccine of the present invention.

Therefore; The another kind of preferred form of first embodiment relates to according to combination-vaccine of the present invention, and wherein said vaccine comprises the another kind of fish invasive organism of immunogenicity amount or antigen or this type of antigenic genetic stocks of encoding of fish pathogenic virus, this quasi-microorganism or virus in addition.

Preferably, said other microorganisms or virus are selected from following fish diseases substance: Vibrio anguillarum, Mermaid luminous bacillus kill the fish subspecies, bacillus, Flavobacterium species, Flexibacter species, Ge Shi Lactococcus, blunt tarda, Silurus asotus fish tarda, streptococcus dysgalactiae, viral hemorrhagic septicemia, VHS virus, viral necrosis virus, irido virus, Cyprinus carpio ramaninjana toxemia and Koi herpesvirus are subdued in the ocean.

Therefore; With more preferred form, other microorganisms or virus are selected from following fish diseases substance: Vibrio anguillarum, Mermaid luminous bacillus kill the fish subspecies, bacillus, Flavobacterium species, Flexibacter species, Ge Shi Lactococcus, blunt tarda, Silurus asotus fish tarda, streptococcus dysgalactiae, viral hemorrhagic septicemia, VHS virus, viral necrosis virus, irido virus, Cyprinus carpio ramaninjana toxemia and Koi herpesvirus are subdued in the ocean.

The streptococcus agalactiae biotype 1 serotype III cell that another embodiment of the invention relates to streptococcus agalactiae biotype 1 serotype Ia cell and the immunogenicity amount of immunogenicity amount is used to make and is used to protect fish not receive the purposes of the vaccine of streptococcal infection.

Another one embodiment of the present invention relates to and is used to prepare the method according to combination-vaccine of the present invention, and wherein the sort of method comprises streptococcus agalactiae biotype 1 serotype Ia cell and the streptococcus agalactiae biotype 1 serotype III cell of immunogenicity amount and the step of pharmaceutically acceptable carrier of mixed immunity originality amount.

Another embodiment once more of the present invention relates to the test kit that each several part is formed; It has the characteristic that it comprises at least 2 bottles, and wherein these bottles comprise the streptococcus agalactiae biotype 1 serotype Ia cell of immunogenicity amount and the streptococcus agalactiae biotype 1 serotype III cell and the pharmaceutically acceptable carrier of immunogenicity amount together.The test kit that this type of each several part is formed for example also comprises the test kit of the each several part composition that comprises at least 2 bottles; One of them bottle comprises the streptococcus agalactiae biotype 1 serotype Ia cell of immunogenicity amount and the streptococcus agalactiae biotype 1 serotype III cell of immunogenicity amount, and another bottle comprises biotype 2 cells and pharmaceutically acceptable carrier.

Embodiment:

Embodiment 1

Vaccination

Vaccine:

Vaccine-1:TI1428 streptococcus agalactiae biotype 1, serotype III is also referred to as Sa1 (X)

Medicament forms: Water-In-Oil: 30% water and 70%Montanide ISA 763 oil

Antigen concentration: with the streptococcus agalactiae 1 (X) of 1.36E+8 cell/ml

Vaccine-2:TI1422 streptococcus agalactiae biotype 1, serotype Ia is also referred to as Sa1 (Y)

Medicament forms: Water-In-Oil: 30% water and 70%Montanide ISA 763 oil

Antigen concentration: with the streptococcus agalactiae 1 (Y) of 1.36E+8 cell/ml

Standard vaccine dilution buffer liquid (SVDB)

Table 1 provides the description of the attack bacterial strain of use.

Table 1: the attack bacterial strain that is used to attack

The bacterial strain coding

The streptococcus agalactiae bacterial strain

Agglutination * with the anti-TI1425 of multi-clone rabbit

Agglutination * with the anti-TI513 of multi-clone rabbit

The serotype name

Biochemical analysis

PCR

TI1428

Streptococcus agalactiae biotype 1

+

-

III

Streptococcus agalactiae

TI1422

Streptococcus agalactiae biotype 1

-

+

1a

Streptococcus agalactiae

TI016

Streptococcus agalactiae biotype 2

-

+

1b

Difficulty is distinguished streptococcus

Streptococcus agalactiae

* standardized microscope slide agglutination, TI1425 produces to biotype 1 serotype III, and TI513 produces to biotype 2.

Animal

Species: tilapia (mouth incubate the fish species ( OreochromisSp))

Average weight when arriving: < 0.5g

Average weight 23g when the experiment beginning.

Management

Water

Salinity: about 6 ppt after vaccination, fresh water after attack

Temperature: after vaccination about 27 ℃ ± 2 ℃, back 32 ℃ ± 2 ℃ in attack

Groove: 500L after vaccination, 250L after attack.

Feedstuff

After vaccination, fish is raised with 2-4 % body weight/day.Adjust the feed rate of each processed group weekly.After attack, if accept feedstuff, fish is raised with its body weight/day of 1%-3% so.

In office where manage for example be transferred to groove, weigh before, make fish hunger at least 12 hours, and before injection, make fish hunger at least 48 hours.

Groove

All grooves (tank) in moist chamber (wet lab) facility have unique number coding, and thisly are coded in experiment and use from start to finish with on all chartings.After vaccination, fish is placed the 500L groove.By means of vertically placing the intermediary net of groove, in two with groove.The two halves of groove are identified through groove numbering and letter (A and B).This numbering is fixed for groove.Fish is under fire raised in vertical 2 nets 250L groove separately of placing through 2, and said net is divided into 3 compartments with groove.Each compartment is identified through groove numbering and alphabetical A, B or C.This numbering is fixed for groove.

Animal is dispensed to processed group

285 fishes altogether of similar size are used for this experiment.Is that a component is 3 groups with fish with 95 fishes, 2 vaccination groups and 1 matched group.

Vaccination

Carry out vaccination through the IP injection.With fish with AQUI-S anesthesia until calmness, and between the tip of the end of pectoral fin and pectoral fin, locate to carry out IP greatly and inject with 0.05 ml.After injection, immediately fish is transferred to the groove that their distribute and recovers.Contrast fish use same program is injected with the SVDB of similar volume.Table 2 is described the group and the groove after vaccination that use and is distributed.

Table 2: the groove of processing time harmony in the exterior after vaccination distributes

Attack the preparation of inoculum

TI 1422 and TI 1428 bacterial strains are from < the 50 ℃ of refrigerated glycerol original seeds recoveries, and hatching about 24 hours at 26-32 ℃ subsequently on TSA.

For TI 1422, growth-gen is collected among the TSB, until reaching OD ₆₆₀0.134-0.147.Use 0.9% aseptic NaCl, carry out 10 times of dilutions until 10 ^-60.4% this preparation is seeded to the TSB of more volume.Culture medium (broth) was hatched 16-17 hour at 32 ℃.Work as OD ₆₆₀Reach at 0.879-0.904 o'clock,, culture is used for preparation attacks suspension through culture is diluted 200 times in 0.9% aseptic NaCl.For attacking in the 3rd week and the 6th week, be determined as 1.0E+7 CFU/ml and 1.3E+7 respectively at the CFU of the resulting suspension that is used for attacking.

For TI 1428, growth-gen is collected among the SGM, until reaching OD _660nm0.162-0.170.Preparation is inoculated in the 100ml SGM with 1%v/v, and hatches at 32 ℃ subsequently.After hatching in about 16 hours, the OD of culture _660nmBe 1.339-1.362.Through it is diluted 100 times in 0.9% aseptic NaCl, culture is used for preparation attacks suspension.For attacking in the 3rd week and the 6th week, be determined as 9.0E+7 CFU/ml and 7.4E+7 respectively at the CFU of the resulting suspension that is used for attacking.

For TI016 (streptococcus agalactiae biotype 2 (being also referred to as Sa2)), obtain 1ml and attack the seed bottles from≤-50 ℃ of refrigerators, thaw and its content is inoculated in the 100ml SGM.Culture is placed on 32 ℃ the orbital shaker, wherein hunting speed is made as 150 RPM.After hatching in 21 hours-22 hours, culture has OD ₆₆₀0.181-0.177.Through it is diluted 1000 times in 0.9% aseptic NaCl, culture is used for preparation attacks suspension.For attacking in the 3rd week and the 6th week, be determined as 9.9E+5 CFU/ml and 4.2E+5 respectively at the CFU of the resulting suspension that is used for attacking.

Plate is paved in standard coating through the bacterial suspension of 10 times of dilutions of 100 μ l aliquots on TSA, and hatches 24-48 hour at 32 ℃ subsequently, is determined at all and attacks the colony forming unit number in the culture.

Attack

When the 3rd week and the 6th week, before attack, make fish hunger at least 48 hours, be emptied completely to guarantee gastrointestinal, thereby and stop owing to inject damage for internal organs.Use AQUI-S that they are anaesthetized, and carry out attack through the IP injection.For each attack time point, 15 fishes carry out the IP injection with the above-mentioned attack suspension of 0.1ml.After injection, immediately fish is shifted the groove (half groove) that is back to their distribution and recovers (about distributing referring to table 3).

Table 3: for the 3rd week and the 6th week attacking experimental group, fish number/group and the groove that after attack, distributes

X: only with the sero-fast agglutination of Sa1, Y: only with the sero-fast agglutination of Sa2.

Outcome evaluation

Through calculating relative protection percentage ratio (RPP) value, assessment vaccine protection fish does not receive the ability of multiple attack bacterial strain.The RPP value is calculated according to following formula:

* infected fish comprises from wherein isolating attacks the biological dead fish of the observation period process, collecting, and attacks the biological fish of when research finishes, collecting from wherein isolating.

Water parameter and the mortality rate after vaccination

After vaccination, the 8th day the time, find 1 the fish death of (TI 1422) group from vaccinated Sa1 (Y).Fish is dissected (cannibalised).From (plated) internal organs of bed board, do not observe growth.The cause of the death can't be confirmed.Do not observe Deviant Behavior in the experimental period process after vaccination.

Growth after the vaccination

Provide through 6 all observation period being grown among Fig. 1 in vaccination and matched group.

Mortality rate is similar in vaccination and matched group.

Effect

Mortality rate after attack

Attack for Sa1-X, Sa1-Y and Sa2, the mortality rate that after attacking with the 3rd week of multiple attack bacterial strain, obtains is respectively in Fig. 2,3 and 4 illustrated.

After the attack of the 3rd week, the mortality rate in matched group is like (after Sa1-X, Sa1-Y and Sa2 attack, being respectively 87%, 93% and 80% cumulative mortality %) of expection.

After attacking with the Sa2 bacterial strain, mortality rate very high in 2 vaccine group (67% and 87%).After attacking with Sa1-X, when comparing with the allogenic Sa1-Y vaccination of serology group (60 mortality rate %), mortality level is in the homologous Sa1-X vaccination of serology group lower (27%).

After attacking with Sa1-Y, make identical observation: when comparing with the allogenic Sa1-X vaccination of serology group (40 mortality rate %), mortality level is lower in the homologous Sa1-Y vaccination of serology group (6.7%).About the be confirmed death general introduction of rate % of accumulation, referring to table 4.

Table 4: after the attack of the 3rd week, accumulate the general introduction (n=15) of the rate % that is confirmed death.

Attack for Sa1-X, Sa1-Y and Sa2, the mortality rate that obtains after attacking in the 6th week with multiple attack bacterial strain is respectively in Fig. 5,6 and 7 illustrated.About the be confirmed death general introduction of rate % of accumulation, referring to table 4.

After the attack of the 6th week, the mortality rate in matched group is like (after Sa1-X, Sa1-Y and Sa2 attack, being respectively 86.7%, 60% and 66.7% cumulative mortality %) of expection.

The observation of when the 3rd week, carrying out is confirmed after attacking in the 6th week: after attacking with the Sa2 bacterial strain, and mortality rate very high in 2 vaccine group (66.7% and 73.3%).After attacking with Sa1-X, when comparing with the allogenic Sa1-Y vaccination of serology group (26.7 mortality rate %), mortality level is in the homologous Sa1-X vaccination of serology group lower (6.7%).

After attacking with Sa1-Y, obtain identical observation: when comparing with the allogenic Sa1-X vaccination of serology group (53.3 mortality rate %), mortality level is in the homologous Sa1-Y vaccination of serology group lower (6.7%).About the be confirmed death general introduction of rate % of accumulation, referring to table 5.

About every day mortality rate and isolating again details in appendix 3, provide.

Table 5: after the attack of the 6th week, accumulate the general introduction (n=15) of the rate % that is confirmed death.

The RPP value

Calculate the RPP value and expression in table 6.

Table 6: the 3rd and 6 whens week after vaccination, the RPP value after streptococcus agalactiae is attacked.

When the 3rd week attacking bacterial strain execution attack with Sa2, irrelevant with vaccine, very weak extremely do not exist (for Sa1 (X) vaccine, RPP 16.7%, for Sa1 (Y) vaccine, RPP-8.3%) of protection.This is confirmed after observing and attacking in the 6th week, wherein for the invisible protection of arbitrary vaccine (RPP 0% (Sa1-X vaccine) and-10%Sa1 (Y) vaccine).

When carrying out homology Sa1-X vaccination/when Sa1-X attacks, obtain good protection (the 3rd and 6 when all, RPP is respectively 69.2% and 92.3%).Yet,, protect very low (the 3rd and 6 when all, RPP is respectively 57.1% and 11.1%) when carrying out allos Sa1-X vaccination/when Sa1-Y attacks.

When carrying out homology Sa1-Y vaccination/when Sa1-Y attacks, obtain identical observation.When the fish of Sa1-Y vaccination is attacked the bacterial strain attack with Sa1-Y, observe good protection (when the 3rd and 6 weeks, RPP is respectively 92.9% and 88.9%).Yet, when the fish of Sa1-Y vaccination is attacked with the Sa1-X bacterial strain, when attacking relatively, protect lower (when the 3rd and 6 weeks, RPP is respectively 30.8% and 69.2%) with homology.

Conclusion

In a word, we can say acquisition protection between the different streptococcus agalactiae bacterial strain of biochemistry: streptococcus agalactiae biotype 1 vaccine is to the infection cross protection by streptococcus agalactiae biotype 2.Yet streptococcus agalactiae biotype 1 vaccine provides the protection of attacking to streptococcus agalactiae biotype 1.However, when streptococcus agalactiae biotype 1 vaccine was attacked (X-Y and Y-X) with the allogenic attack bacterial strain of serology, visible protection reduced.For the homologous attack of serology (X-X and Y-Y), splendid protection can be provided.Therefore, the combination-vaccine that comprises streptococcus agalactiae biotype 1 serotype Ia cell and streptococcus agalactiae biotype 1 serotype III cell provides the splendid protection to 2 kinds of streptococcus agalactiae biotype 1 serotypes.

The abbreviation of using

The IP intraperitoneal

The Ppt one thousandth

The RPP percentage ratio of surviving relatively

The TSA tryptose soya agar

The TSB pancreas peptone soybean broth

SGM streptococcus growth culture medium.

Legend

Fig. 1: (n=15 removes when the 3rd week: n=45 and the 6th week: n=50) through growth after attacking the back vaccination of 6 observation periods in week.

Fig. 2: the 3rd when week after vaccination; After the attack of carrying out with Sa1-X (TI1428); The accumulative perception (%) that in vaccination group (Vacc Sa1 (X), Vacc Sa1 (Y)) and matched group (Ctr), confirms (n=15) is included in the positive of attacking when afterwards the observation period finishes and separates fish (pos) again.

Fig. 3: the 3rd when week after vaccination; After the attack of carrying out with Sa1-Y (TI1422); The accumulative perception (%) that in vaccination group (Vacc Sa1 (X), Vacc Sa1 (Y)) and matched group (Ctr), confirms (n=15) is included in the positive of attacking when afterwards the observation period finishes and separates fish (pos) again.

Fig. 4: the 3rd when week after vaccination; After the attack of carrying out with Sa2 (Y) (TI 016); The accumulative perception (%) that in vaccination group (Vacc) and matched group (Ctr), confirms (n=15) is included in the positive of attacking when afterwards the observation period finishes and separates fish (pos) again.

Fig. 5: the 6th when week after vaccination; After the attack of carrying out with Sa1-X (TI1428); The accumulative perception (%) that in vaccination group (Vacc) and matched group (Ctr), confirms (n=15) is included in the positive of attacking when afterwards the observation period finishes and separates fish (pos) again.

Fig. 6: the 6th when week after vaccination; After the attack of carrying out with Sa1-Y (TI1422); The accumulative perception (%) that in vaccination group (Vacc) and matched group (Ctr), confirms (n=15) is included in the positive of attacking when afterwards the observation period finishes and separates fish (pos) again.

Fig. 7: in the 6th when week after vaccination, after attacking with Sa2 (Y) (TI 016), the accumulative perception (%) of confirmation (n=15) comprises that the positive separates fish (pos) again in vaccination group (Vacc) and matched group (Ctr).

Claims

1. be used to protect fish not receive the combination-vaccine of streptococcal infection, it is characterized in that said vaccine comprises the streptococcus agalactiae biotype 1 serotype Ia cell of immunogenicity amount and the streptococcus agalactiae biotype 1 serotype III cell and the pharmaceutically acceptable carrier of immunogenicity amount.

2. according to the combination-vaccine of claim 1, it is characterized in that said streptococcus agalactiae cell is deactivation.

3. according to the combination-vaccine of claim 1 or 2, it is characterized in that said vaccine is a water-in-oil emulsion.

4. according to the combination-vaccine of claim 3, it is characterized in that said oil is non-mineral oil.

5. according to the combination-vaccine of claim 4, it is characterized in that said non-mineral oil is Montanide ISA 763.

6. according to the combination-vaccine of claim 1-5, it is characterized in that said vaccine comprises antigen or this type of antigenic genetic stocks of encoding of the streptococcus agalactiae biotype 2 of streptococcus agalactiae biotype 2 cells of immunogenicity amount, immunogenicity amount in addition.

7. according to the combination-vaccine of claim 1-5, it is characterized in that said vaccine comprises antigen or this type of antigenic genetic stocks of encoding of the Streptococcus iniae of the Streptococcus iniae cell of immunogenicity amount, immunogenicity amount in addition.

8. according to the combination-vaccine of claim 1-7, it is characterized in that said vaccine comprises the another kind of fish invasive organism of immunogenicity amount or antigen or this type of antigenic genetic stocks of encoding of fish pathogenic virus, this quasi-microorganism or virus in addition.

9. according to Claim 8 combination-vaccine, it is characterized in that said another kind of microorganism or virus are selected from following fish diseases substance: Vibrio anguillarum, Mermaid luminous bacillus kill the fish subspecies, bacillus, Flavobacterium species, Flexibacter species, Ge Shi Lactococcus, blunt tarda, Silurus asotus fish tarda, streptococcus dysgalactiae, viral hemorrhagic septicemia, VHS virus, viral necrosis virus, irido virus, Cyprinus carpio ramaninjana toxemia and Koi herpesvirus are subdued in the ocean.

10. the streptococcus agalactiae biotype 1 serotype III cell of the streptococcus agalactiae biotype 1 serotype Ia cell of immunogenicity amount and immunogenicity amount is used to make and is used to protect fish not receive the purposes of the vaccine of streptococcal infection.

11. be used to prepare method, it is characterized in that difficulty that said method comprises streptococcus agalactiae biotype 1 serotype Ia cell and the immunogenicity amount of mixed immunity originality amount distinguishes the step of streptococcus biotype 1 serotype III cell and pharmaceutically acceptable carrier according to the combination-vaccine of claim 1-9.

12. the test kit that each several part is formed; Said test kit is characterised in that it comprises at least 2 bottles, and said bottle comprises the streptococcus agalactiae biotype 1 serotype Ia cell of immunogenicity amount and the streptococcus agalactiae biotype 1 serotype III cell and the pharmaceutically acceptable carrier of immunogenicity amount together.