CN104138597B - A kind of Vibrio anguillarum bivalent vaccine and preparation and application thereof - Google Patents
A kind of Vibrio anguillarum bivalent vaccine and preparation and application thereof Download PDFInfo
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Abstract
The present invention relates to aquatic animal disease prevention techniques, a kind of Vibrio anguillarum (Vibrio anguillarum) bivalent vaccine and preparation and application thereof.Vaccine is Vibrio anguillarum (Vibrio anguillarum) 01 serotype and the bacteria suspension of 02 serotype two strain bacterium mixing, described Vibrio anguillarum (Vibrio anguillarum) 01 serotype and 02 serotype two strain bacterium are preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation date December in 2012 07 day, preserving number is respectively CGMCC No.6942 and CGMCC No.6944.Bivalent inactivated vaccine of the present invention can protect susceptible Fish from the infection of pathogenic Vibrio anguillarum effectively.The bivalent vaccine of the present invention has widened vaccine protection domain, and effective stimulus cultured fishes produce specific immune response reaction, and effectively provide the immunoprotective effec to disease.The Vibrio anguillarum bivalent vaccine of the preparation present invention, is used that the cost of material is low, to animal and Environmental security.
Description
Technical field
The present invention relates to aquatic animal disease prevention techniques, a kind of Vibrio anguillarum (Vibrio
Anguillarum) bivalent vaccine and preparation and application thereof.
Background technology
Vibrio anguillarum is one of important pathogen causing aquatic animal septicemia, and the vibriosis being induced by is China's water
Producing the principal disease of cultured fishes, the cultured fishes of infection relate to the weights such as turbot, Paralichthys olivaceus, Cynoglossus semilaevis, Lateolabrax japonicus (Cuvier et Va-lenciennes) (Lateolabracis), Sciaenops ocellatus
Want breed variety, cause the heavy economic losses of cultured fishes industry.The sick means of prevention of Vibrio anguillarum be mainly Drug therapy and
Vaccine immunity.Drug therapy relates to the use of antibiotic and chemicals, easily causes the resistance to of aquatic products drug residue and antibacterial
The property of medicine, destroys ecological environment balance, human health is formed potential hazard.Application band due to antibiotic and other chemicalses
The harm come, is worldwide very restricted, and vaccine immunity is excellent in terms of specificity, safety and protected effect
Treat in antibiotic etc, have proved to be the diseases prevention and treatment means of a kind of effective and safe.Inactivation fish vaccine safety good,
Production cost is low, and country has had multiple business-like inactivated vaccine to be applied to the salmon of scale in Norway, Spain, North America etc.
Fish, Squaliobarbus ourriculus, cat fish etc. cultivate, to reducing antibiotic usage, ensureing that the Fish economy of countries and regions plays huge impetus.
Along with the factors such as cultivation temperature rising, environmental pollution, intensive culture, the pathogenic microorganism speed of mutation of aquatic animal and frequency
Accelerating, there is increase trend, for the disease being continually changing and cause of disease kind, need to carry out in time disease in cause of disease kind and serotype
Sick epidemiological study, specifies cause of disease kind and Changing Pattern thereof, develops and apply effective novel vaccine.According to reported literature,
Vibrio anguillarum has 23 kinds of serotypes, has 01,02,03 serotype to what Fish were caused a disease, occurs in that 01a, 01b, 02a, 02b etc. cause at present
Sick blood serum subtype, the pathogenic of cultured fishes is also had been reported that by other serotypes such as 04,05 serotype.Due to blood serum subtype bacterium
Strain and the appearance of new pathogenic serotypes bacterial strain, existing Vibrio anguillarum vaccine can not provide effectively protection or protective rate to cultivated animals
Low, need to carry out epidemiological study according to the vibriosis of different regions different breeding kind, specify pathogenic strain and serotype thereof,
Development can provide the Vibrio anguillarum vaccine of effectively protection.
Summary of the invention
Present invention aim at as overcoming existing Vibrio anguillarum vaccine that aquatic animal is not provided that to protect completely, protecting
The shortcoming that power is low provides a kind of Vibrio anguillarum bivalent vaccine and preparation and application thereof.
For achieving the above object, the technical solution used in the present invention is:
A kind of Vibrio anguillarum bivalent vaccine, vaccine is Vibrio anguillarum (Vibrio anguillarum) 01 serotype and 02 serotype
The bacteria suspension of two strain bacterium mixing, described Vibrio anguillarum (Vibrio anguillarum) 01 serotype and 02 serotype two strain bacterium preservation
In China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation date December in 2012 07 day, preserving number divides
Not Wei CGMCC No.6942 and CGMCC No.6944, depositary institution address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.
Described vaccine is the bacteria suspension being dissolved in phosphate buffered saline(PBS)；Vibrio anguillarum (Vibrio in described bacteria suspension
Anguillarum) 01 serotype and 02 serotype two strain bacterium are in the ratio mixing that cell quantity ratio is 1: 1.
By Vibrio anguillarum (Vibrio anguillarum) 01 serotype and activation culture, the fermentation respectively of 02 serotype two strain bacterium
Cultivate and after formalin-inactivated, by Vibrio anguillarum (Vibrio anguillarum) 01 serotype after inactivation and 02 serotype two strain bacterium
Killed cells be mixed in sterile phosphate buffer saline solution in the ratio that quantity ratio is 1: 1, be bivalent inactivated vaccine.
The preparation method of Vibrio anguillarum bivalent vaccine, by Vibrio anguillarum (Vibrio anguillarum) 01 serotype and 02 serum
After type two strain bacterium activation culture, fermentation culture and formalin-inactivated respectively, by the Vibrio anguillarum (Vibrio after inactivation
Anguillarum) killed cells of 01 serotype and 02 serotype two strain bacterium is mixed in aseptic phosphorus in the ratio that quantity ratio is 1: 1
In acid buffering saline solution, it is bivalent inactivated vaccine.
The using method of Vibrio anguillarum bivalent vaccine, it is characterised in that: described bivalent inactivated vaccine is used for preparing preventing and treating cultivation
The vaccine that Fish Vibrio anguillarum is sick.It is 10 that above-mentioned bivalent inactivated vaccine is administered to dose6～108Cells/ tail；Or it is dense to soak administration
Degree is 107～109Cells/ml, soak time 10～30min.
Vibrio anguillarum bacterial strain 01 serotype and 02 serotype two strain bacterium that the present invention uses all are preserved in China General Microbiological
Culture presevation administrative center, preservation date December in 2012 07 day, preserving number is respectively CGMCC No.6942 and CGMCC
No.6944, the Cultured Flounder, Paralichthys Olivaceus occurring Vibrio anguillarum sick from China separates with turbot, and its Physiology and biochemistry, 16SrRNA genic system are sent out
The feature educating the features such as analysis, serology, PCR qualification and Vibrio anguillarum meets (seeing Tables 1 and 2 and Figure 1A, B, C).
Table 1 vibrio CGMCC No.6942 and CGMCC No.6944 physiological and biochemical property
+: positive ,-: it is negative,
Table 2 Vibrio anguillarum serotype feature
+, positive；-, negative
Advantage for present invention: the present invention uses Vibrio anguillarum bacterial strain 01 serotype and 02 serotype two strain bacterium to prepare
Bivalent vaccine has widened the protection domain of Vibrio anguillarum vaccine, uses this vaccine and can resist Vibrio anguillarum 01 serotype and 02 serum simultaneously
The infection of type bacterial strain.The Vibrio anguillarum bivalent vaccine of the present invention is prepared with ablation method, can be effectively retained the antigen of bacterium surface certainly
Fixed bunch, stimulating cultured fishes to produce specific immune response, the antibacterial simultaneously inactivated does not exists infects in cultivated animals and environment
, the most there is not threat to environment and the mankind, there is good safety in the danger of other animals.The Vibrio anguillarum bivalence of the present invention
Production of vaccine low cost, can carry out immunity to animal by the way of injection and immersion, simply, conveniently, safely, effectively, suitable
Conjunction aquaculture production needs, and has business development and is worth.
Accompanying drawing explanation
Figure 1A, B, C are the 16Sr rna gene sequence of the Vibrio anguillarum CGMCC No.6942 and CGMCC No.6944 of the present invention
Row and the phylogenetic systematics cladogram set up based on 16Sr rna gene sequence.Wherein A, Vibrio anguillarum CGMCC No.6942；B, eel
Vibrio CGMCC No.6944；C, 16Sr rna gene sequential system auxology cladogram.
Fig. 2 is that the PCR of the Vibrio anguillarum CGMCC No.6942 and CGMCC No.6944 of the present invention identifies collection of illustrative plates.(qualification side
Method is with reference to Xiao Peng, Mo Zhaolan, Mao Yunxiang, Wang Chunling, Zou Yuxia, Li
Jie.2009.Detection of Vibrio anguillarum by PCR amplification of the emp A
Gene.J Fish Dis32,293-296.), 1, Vibrio anguillarum CGMCC No.6942；2, Vibrio anguillarum CGMCC No.6944；3, eel
Vibrio ATCC43305 (01 serotype)；4, Vibrio anguillarum ATCC43306 (02 serotype)；5, Vibrio anguillarum ATCC43307 (03 serum
Type)；6, Vibrio anguillarum ATCC43308 (04 serotype)；7, Vibrio anguillarum ATCC43309 (05 serotype)；8, Vibrio anguillarum M3；9, eel arc
Bacterium SMP1；10, Vibrio anguillarum M5；11, Vibrio anguillarum MHK3；12, negative control；13, Vibrio anguillarum MHK7；14, Vibrio anguillarum SMW1；15,
Algin vibrio；16, vibrio parahaemolytious；17, Vibro harveyi；18, vibrio fluvialis；19, Vibrio splindidus；20, slow Ai Dehuashi
Bacterium；21, staphylococcus aureus 22；Bacillus subtilis；23, escherichia coli；24, negative control；M, DNA marker.
Detailed description of the invention
Embodiment 1: the Vibrio anguillarum pathogenicity to Paralichthys olivaceus (Paralichthys olivaceus)
1) median lethal dose(LD 50) (LD of Vibrio anguillarum infectable infection Paralichthys olivaceus50)
Body weight is that the healthy test fish of 10-12g is supported temporarily in 1000L aquarium, is equipped with flow circuit water and processes and purify system
System, temperature of cultivation maintains 18-20 DEG C.After supporting 4 weeks temporarily, it is randomized to 40L and tests aquarium, often organize 20 tail fishes, continue to support temporarily 2
Week.Test aquarium respectively changes water once morning and afternoon every day, and quantity of exchanged water is 1/2, maintains water temperature 18-20 DEG C.
When Experimental infection, take Vibrio anguillarum CGMCC No.6942 alive or CGMCC No.6944 cell uses nothing respectively
It is 10 that bacterium PBS buffer solution is diluted to series concentration5-109The bacteria suspension of cfu/ml, every dilution factor infectable infection one group experiment fish,
Every tail fish belly portion's intramuscular injection 0.1mL, matched group injection 0.1mlPBS.Observing 14 days after infection, the death of every day often organized in record
Fish number, calculates accumulative death toll and mortality rate, calculates median lethal dose(LD 50) LD by the method for Reed and Muench (1938)50Value (ginseng
Examine document: Reed LJ, Muench H.1938.A simple method of estimating fifty percent
Endpoints.Am J Epidemiol27:493-497.).
Find out from table 1.1, Vibrio anguillarum CGMCC No.6942 and the LD of CGMCC No.6944 infectable infection50It is respectively 6.4
×105Cfu/ tail and 3.2 × 106Cfu/ tail.
The pathogenicity of table 1.1 Vibrio anguillarum infectable infection Paralichthys olivaceus
2) Vibrio anguillarum soaks the median lethal dose(LD 50) (LD infecting Paralichthys olivaceus50)
Body weight is that the healthy test fish of 5-7g is supported temporarily in 1000L aquarium, is equipped with flow circuit water and processes and cleaning system,
Temperature of cultivation maintains 18-20 DEG C.After supporting 4 weeks temporarily, it is randomized to 30L and tests aquarium, often organize 20 tail fishes, continue to support 2 weeks temporarily.
Test aquarium respectively changes water once morning and afternoon every day, and quantity of exchanged water is 1/2, maintains water temperature 18-20 DEG C.
When Experimental infection, take Vibrio anguillarum CGMCC No.6942 alive or CGMCC No.6944 respectively with filtering sea
It is 10 that water is diluted to series concentration6-109The bacteria suspension of cfu/ml, puts into often organizing experiment fish, and 30min is soaked in inflation；Matched group
Fish inflation is soaked in filtering sea 30min.Observing 14 days after infection, the dead fish number of every day often organized in record, calculates accumulative death
Number and mortality rate, calculate median lethal dose(LD 50) LD50Value.
Finding out from table 1.2, Vibrio anguillarum CGMCC No.6942 and CGMCC No.6944 soaks the LD infecting Paralichthys olivaceus50It is respectively
3.3×107Cfu/ml and 7.5 × 107cfu/ml。
Table 1.2 Vibrio anguillarum soaks the pathogenicity infecting Paralichthys olivaceus
Embodiment 2: the Vibrio anguillarum pathogenicity to turbot (Scophthalmus maximus)
1) median lethal dose(LD 50) (LD of Vibrio anguillarum infectable infection turbot50)
Body weight is that the healthy test fish of 9-12g is supported temporarily in 1000L aquarium, is equipped with flow circuit water and processes and purify system
System, temperature of cultivation maintains 16-18 DEG C.After supporting 4 weeks temporarily, it is randomized to 30L and tests aquarium, each aquarium 10 tail fish, continue
Continuous the most foster 2 weeks.Test aquarium respectively changes water once morning and afternoon every day, and quantity of exchanged water is 1/2, maintains water temperature 16-18 DEG C.
When Experimental infection, take Vibrio anguillarum CGMCC No.6942 alive or CGMCC No.6944 cell uses nothing respectively
It is 10 that bacterium PBS buffer solution is diluted to series concentration5-109The bacteria suspension of cfu/ml, every dilution factor infectable infection one group experiment fish,
Every tail fish belly portion's intramuscular injection 0.1mL, matched group injection 0.1ml PBS.Observing 14 days after infection, the death of every day often organized in record
Fish number, calculates accumulative death toll and mortality rate, calculates median lethal dose(LD 50) LD50Value.
Find out from table 2.1, Vibrio anguillarum CGMCC No.6942 and the LD of CGMCC No.6942 infectable infection turbot50Respectively
It is 2.4 × 105Cfu/ tail and 1.2 × 106Cfu/ tail.
The LD of table 2.1 Vibrio anguillarum infectable infection turbot (Scophthalmus maximus)50
2) Vibrio anguillarum soaks the median lethal dose(LD 50) (LD infecting turbot50)
Body weight is that the healthy test fish of 6-8g is supported temporarily in 1000L aquarium, is equipped with flow circuit water and processes and cleaning system,
Temperature of cultivation maintains 16-18 DEG C.After supporting 4 weeks temporarily, it is randomized to 30L and tests aquarium, often organize 20 tail fishes, continue to support 2 weeks temporarily.
Test aquarium respectively changes water once morning and afternoon every day, and quantity of exchanged water is 1/2, maintains water temperature 16-18 DEG C.
When Experimental infection, take Vibrio anguillarum CGMCC No.6942 alive or CGMCC No.6944 cell was used respectively
It is 10 that filter sea water is diluted to series concentration6-109The bacteria suspension of cfu/ml, puts into often organizing experiment fish, and 30min is soaked in inflation；Right
It is soaked in filter sea water 30min according to group fish inflation.Observing 14 days after infection, the dead fish number of every day often organized in record, calculates accumulative dead
Die and count and mortality rate, calculate median lethal dose(LD 50) LD50Value.
Finding out from table 2.2, Vibrio anguillarum CGMCC No.6942 and CGMCC No.6944 soaks the LD infecting turbot50Respectively
It is 3.2 × 107Cfu/ml and 8.3 × 107cfu/ml。
Table 2.2 Vibrio anguillarum soaks the LD infecting turbot50
Embodiment 3: prepared by Vibrio anguillarum inactivated vaccine
A. take that Vibrio anguillarum CGMCC No.6942 and CGMCC No.6944 is seeded to containing 1.5%NaCl (w/v) respectively is general
In logical broth bouillon, it is placed in the shaking flask of 25-30 DEG C cultivation 24-36 hour；Gained culture is separately added into final concentration of
The formaldehyde of 0.3-0.5%, is placed in the shaking flask of 25-30 DEG C cultivation 24-36 hour；
B. above-mentioned gained culture is centrifuged respectively under the speed of 4000-5000rpm/min 10-20min, collects respectively
Precipitum, and suspending precipitation with PBS phosphate buffer solution more respectively, regulation bacterial cell concentration to～109Cells/ml, point
Wei Vibrio anguillarum bacterial strain CGMCC No.6942 inactivated vaccine and Vibrio anguillarum CGMCC No.6944 inactivated vaccine.
C. by Vibrio anguillarum (Vibrio anguillarum) the CGMCC No.6942's and CGMCC No.6944 after inactivation
Killed cells is mixed in sterile phosphate buffer saline solution in the ratio that quantity ratio is 1: 1, is bivalent inactivated vaccine.
Broth medium composition containing 1.5%NaCl (w/v) is: 10g tryptone, 5g yeast extract, 15g
NaCl, distilled water 1000ml, pH7.6.
Embodiment 4: the Cross immunogenicity of Paralichthys olivaceus is tested by Vibrio anguillarum univalent vaccine
Taking the healthy test fish that body weight is 10-13g and be randomly divided into 3 groups, often group 2 is parallel, each parallel group of 30 tail fishes.Exempt from
Epidemic disease step is: 2 parallel group carries out immunity with Vibrio anguillarum CGMCC No.6942 vaccine, use Vibrio anguillarum CGMCC for 2 parallel group
No.6944 vaccine carries out immunity, and immunization ways is lumbar injection 0.1ml/ tail, and immunizing dose is～108Cells/ tail；Residue two
Individual parallel group of lumbar injection 0.1ml PBS is as comparison.Temperature of cultivation 18-20 DEG C.After immunity 4 weeks, from each immune group and comparison
Group Paralichthys olivaceus takes 3 tail fishes at random and prepares serum from tail venous blood sampling, take described each group of determination of serum its with inactivation Vibrio anguillarum CGMCC
No.6942 cell and the agglutination titer of inactivation Vibrio anguillarum CGMCC No.6944 cell；With the wild type Vibrio anguillarum CGMCC lived
No.6942 and CGMCC the No.6944 remaining immune group of counteracting toxic substances and matched group Paralichthys olivaceus respectively, counteracting toxic substances mode is abdominal muscles injection
0.1ml bacteria suspension, counteracting toxic substances dosage～108Cfu/ tail.Adding up immune group and matched group death toll in 14 days, Computation immunity is protected
Rate.Immune protective rate RPS computing formula is: RPS%=(matched group mortality rate-immune group mortality rate)/matched group mortality rate ×
100%.
The table 3.1 Vibrio anguillarum univalent vaccine immunity Paralichthys olivaceus serum antibody titer of 4 weeks
Find out from table 3.1, inactivate epidemic disease with Vibrio anguillarum CGMCC No.6942 inactivated vaccine and Vibrio anguillarum CGMCC No.6944
Seedling inoculation Paralichthys olivaceus respectively is after 4 weeks, and immunity fish serum produces significant antibody titer, titer to the cell antigen of homotype serum
Scope is 1: 853～1: 1024；The antibody titer producing the antigen of special-shaped serum is relatively low, and titer scope is 1: 27～1: 43.
The table 3.2 Vibrio anguillarum CGMCC No.6942 vaccine immunological cross protected effect to Paralichthys olivaceus
The table 3.3 Vibrio anguillarum CGMCC No.6944 vaccine immunological cross protected effect to Paralichthys olivaceus
Finding out from table 3.2, table 3.3,01 serotype Vibrio anguillarum CGMCC resisted by Vibrio anguillarum CGMCC No.6942 vaccine
It is 100% that No.6942 infects the immune protective rate of Paralichthys olivaceus, resists 02 serotype Vibrio anguillarum CGMCC No.6944 and infects Paralichthys olivaceus
Immune protective rate is 40%；Vibrio anguillarum CGMCC No.6944 vaccine is resisted 01 serotype Vibrio anguillarum CGMCC No.6942 and is infected tooth
The immune protective rate of Flounder is 35%, and the immune protective rate resisting 02 serotype Vibrio anguillarum CGMCC No.6944 infection Paralichthys olivaceus is
100%.This result shows to use unit price Vibrio anguillarum vaccine immunity Paralichthys olivaceus homologous serotype Vibrio anguillarum can be infected offer 100%
Immunoprotection, and special-shaped serotype Vibrio anguillarum is infected offer 35～the immunoprotection of 45%.
Embodiment 5: the Vibrio anguillarum bivalent vaccine immuning effect test to Paralichthys olivaceus
1) immune efficacy of Vibrio anguillarum bivalent vaccine injecting immune Paralichthys olivaceus
Taking the healthy test fish that body weight is 9-12g and be randomly divided into 4 groups, often group 2 is parallel, every parallel group of 40 tail fishes.By upper
The bivalent vaccine of the Vibrio anguillarum CGMCC No.6942 and CGMCC No.6944 that state preparation carries out immunity in the way of lumbar injection,
Every endnote penetrates 0.1ml, and immunizing dose is respectively～107Cells/ tail～108Cells/ tail ,～109Cells/ tail, each concentration
Two parallel group.Two parallel control groups inject the PBS of 0.1ml/ tail respectively.Temperature of cultivation 18-20 DEG C.After immunity 4 weeks, from respectively
Immune group and matched group Paralichthys olivaceus take 3 tail fishes at random and prepare serum from tail venous blood sampling, take described each group of determination of serum antibody titer.
Residue test Paralichthys olivaceus uses wild type Vibrio anguillarum CGMCC No.6942 and CGMCC No.6944 with abdominal muscles injection system respectively
Carrying out counteracting toxic substances, counteracting toxic substances dosage is～108Cfu/ tail.Adding up immune group and matched group death toll in 14 days, Computation immunity is protected
Rate.
Table 4.1 Vibrio anguillarum bivalent vaccine injecting immune Paralichthys olivaceus serum titer
Finding out from table 4.1, after Vibrio anguillarum bivalent inactivated vaccine inoculation immunity Paralichthys olivaceus 4 weeks, immunity fish serum is to two
The Vibrio anguillarum cell antigen planting serotype produces the antibody titer of 1: 128～1: 683, and valence value increases with the increase of immunizing dose
Add.
The immune protective rate of table 4.2 Vibrio anguillarum bivalent vaccine injecting immune Paralichthys olivaceus
From table 4.2 it can be seen that bivalent vaccine is with 106～108The concentration injecting immune Paralichthys olivaceus of cells/ tail, to 01 serum
Type and 02 serotype Vibrio anguillarum infect and have significant immune protective effect, and immune protective rate is 77%～100%, exempting from of vaccine
Epidemic disease protective rate increases along with the increase of immunizing dose.
2) immune efficacy of Vibrio anguillarum bivalent vaccine immersion immunity Paralichthys olivaceus
Taking the healthy test fish that body weight is 5-7g and be randomly divided into 4 groups, often group 2 is parallel, every parallel group of 60 tail fishes.By above-mentioned
The bivalent vaccine of the Vibrio anguillarum CGMCC No.6942 and CGMCC No.6944 that prepare gained is configured to concentration with filtering sea
107cells/ml、108cells/ml、109The vaccine solution of cells/ml.Often group fish is soaked in the vaccine solution of variable concentrations
10min, matched group fish soaks 10min at filtering sea, immersion group be all provided with matched group two parallel, water temperature keeps 18-20 DEG C.
After immunity 4 weeks, take 5 tail fishes at random from each immune group and matched group Paralichthys olivaceus and prepare serum from tail venous blood sampling, take each group of serum and survey
Determine itself and inactivation Vibrio anguillarum CGMCC No.6942 cell and the agglutination titer of inactivation Vibrio anguillarum CGMCC No.6944 cell.Residue
Test Paralichthys olivaceus carries out soaking counteracting toxic substances, concentration with the viable bacteria of wild type Vibrio anguillarum CGMCC No.6942 and CGMCC No.6944 respectively
For～109Cfu/ml, the time is 30min.Immune group and matched group death toll, Computation immunity protective rate is added up in 14 days.
Table 4.3 Vibrio anguillarum bivalent vaccine immersion immunity Paralichthys olivaceus serum titer
Finding out from table 4.3, after Vibrio anguillarum bivalent inactivated vaccine immersion immunity Paralichthys olivaceus 4 weeks, the serum of immunity fish is to two kinds
The antibody that the Vibrio anguillarum cell antigen of serotype produces is 1: 6～1: 32, and antibody titer concentration increases with the increase of immunizing dose
Add.
Table 4.4 Vibrio anguillarum bivalent vaccine soaks the immune protective rate of Paralichthys olivaceus
Find out from table 4.4,107～109Inoculation 10min, Vibrio anguillarum bivalent vaccine opposing 01 is soaked under cells/ml concentration
It is 23%-73% that serotype and 02 serotype wild type Vibrio anguillarum soak the immune protective rate infected；Along with soaking inoculum density
Increasing, the immune protective rate of vaccine also increases.
Comparison sheet 4.1 and the data of table 4.3, the serum antibody titer of injecting immune Paralichthys olivaceus is significantly higher than immersion immunity Paralichthys olivaceus
Antibody titer.Comparison sheet 4.2 and the data of table 4.4, bivalent vaccine carries than immersion way immunity with injection system immunity Paralichthys olivaceus
For higher immunoprotection.
Embodiment 6: the Vibrio anguillarum bivalent vaccine immuning effect test to turbot
1) immune efficacy of Vibrio anguillarum bivalent vaccine injecting immune turbot
Take the healthy test fish that body weight is 12-15g and be randomly divided into 4 groups, often group set two parallel, every parallel group of 40 tail fishes.
The bivalent vaccine of the Vibrio anguillarum CGMCC No.6942 and CGMCC No.6944 of above-mentioned gained is carried out in the way of lumbar injection
Immunity, every endnote penetrates 0.1ml, and injection dosage is respectively～107Cells/ tail～108Cells/ tail ,～109Cells/ tail.Two
The PBS of individual parallel control group fish injection 0.1ml/ tail.Temperature of cultivation 16-18 DEG C.After immunity 4 weeks, from each immune group and matched group
Turbot takes 3 tail fishes at random, takes each group of determination of serum antibody titer.Residue test turbot uses wild type Vibrio anguillarum respectively
CGMCC No.6942 and CGMCC No.6944 carries out counteracting toxic substances with abdominal muscles injection system, and injection dosage is 0.1ml, and counteracting toxic substances is dense
Degree～108Cfu/ tail.Immune group and matched group death toll, Computation immunity protective rate is added up in 14 days.
Table 5.1 Vibrio anguillarum bivalent vaccine injecting immune turbot serum titer
Finding out from table 5.1, Vibrio anguillarum bacterium bivalent inactivated vaccine inoculation immunity turbot is after 4 weeks, the serum of immunity fish
The Vibrio anguillarum cell antigen of two kinds of serotypes is produced significant antibody titer, titer scope 1: 171～1: 853, antibody titer
Concentration increases with the increase of immunizing dose.
The immune protective rate of table 5.2 Vibrio anguillarum bivalent vaccine injecting immune turbot
From table 5.2 it can be seen that bivalent vaccine is with 106～108The concentration injecting immune turbot of cells/ tail, to 01 blood
Clear type and 02 serotype Vibrio anguillarum infect and have significant immune protective effect, and immune protective rate is 72%～100%, vaccine
Immune protective rate increases along with the increase of immunizing dose.
2) immune efficacy of Vibrio anguillarum bivalent vaccine immersion immunity turbot
Taking the healthy test fish that body weight is 3-5g and be randomly divided into 4 groups, often group sets two parallel group, often organizes 60 tail fishes.By upper
The bivalent vaccine of the Vibrio anguillarum CGMCC No.6942 and CGMCC No.6944 that state preparation is configured to concentration with filtering sea
107cells/ml、108cells/ml、109The vaccine solution of cells/ml.Often group fish is soaked in the vaccine solution of variable concentrations
30min, every concentration set two parallel；Two parallel control group fishes soak 30min at filtering sea.Water temperature keeps 16-18 DEG C.Exempt from
After epidemic disease 4 weeks, take 5 tail fishes at random from each immune group and matched group turbot and prepare serum from tail venous blood sampling, take each group of serum and survey
Determine antibody titer.Residue test turbot is soaked with wild type Vibrio anguillarum CGMCC No.6942 and CGMCC No.6944 respectively
Bubble counteracting toxic substances, concentration is～108Cfu/ml, soak time is 30min.Immune group and matched group death toll, meter is added up in 14 days
Calculate immune protective rate.
Table 5.3 Vibrio anguillarum bivalent vaccine immersion immunity turbot serum titer
Table 5.3 is found out, Vibrio anguillarum bivalent inactivated vaccine immersion immunity turbot is after 4 weeks, and turbot serum is to two kinds of serum
The Vibrio anguillarum cell antigen of type produces antibody titer, and titer scope 1: 8～1: 32, antibody titer concentration is with the increase of immunizing dose
And increase.
The immune protective rate of table 5.4 Vibrio anguillarum bivalent vaccine immersion immunity turbot
Showing from table 5.4 result, Vibrio anguillarum bivalent vaccine is 107～109Inoculation turbot is soaked under cfu/ml concentration
30min, the immune protective rate resisting 01 serotype and the infection of 02 serotype Vibrio anguillarum is 20%-71%；Immune protective rate along with
The increase of immersion immunity concentration and improve.
Comparison sheet 5.1 and the data of table 5.3, the serum antibody titer of injecting pathway immunity turbot is significantly higher than immersion way
The antibody titer of footpath immunity turbot.Comparison sheet 5.2 and the data of table 5.4, Vibrio anguillarum bivalent vaccine is big with injection system immunity
Brill provides higher immunoprotection than immersion way immunity.
Claims (4)
1. a Vibrio anguillarum bivalent inactivated vaccine, it is characterised in that: vaccine is Vibrio anguillarum (Vibrio anguillarum) 01 blood
Clear type and the bacteria suspension of 02 serotype two strain bacterium mixing, described Vibrio anguillarum (Vibrio anguillarum) 01 serotype and 02 blood
Clear type two strain bacterium is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation date in December, 2012
07, preserving number was respectively CGMCC No.6942 and CGMCC No.6944.
2. the Vibrio anguillarum bivalent inactivated vaccine as described in claim 1, it is characterised in that: described vaccine is for being dissolved in phosphate-buffered salt
Bacteria suspension in solution；Vibrio anguillarum (Vibrio anguillarum) 01 serotype and 02 serotype two strain bacterium in described bacteria suspension
Mix than the ratio for 1:1 in cell quantity.
3. the Vibrio anguillarum bivalent inactivated vaccine as described in claim 1 or 2, it is characterised in that: by Vibrio anguillarum (Vibrio
Anguillarum) after 01 serotype and 02 serotype two strain bacterium activation culture, fermentation culture and formalin-inactivated respectively, will inactivation
After Vibrio anguillarum (Vibrio anguillarum) 01 serotype and the killed cells of 02 serotype two strain bacterium by quantity ratio for 1:1
Ratio be mixed in sterile phosphate buffer saline solution, be bivalent inactivated vaccine.
4. the preparation method of the Vibrio anguillarum bivalent inactivated vaccine described in a claim 1, it is characterised in that: by Vibrio anguillarum
(Vibrio anguillarum) 01 serotype and 02 serotype two strain bacterium activation culture, fermentation culture and formalin-inactivated respectively
After, by the killed cells of Vibrio anguillarum (Vibrio anguillarum) 01 serotype after inactivation and 02 serotype two strain bacterium by number
Amount is mixed in sterile phosphate buffer saline solution than the ratio for 1:1, is bivalent inactivated vaccine.
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| CN106047763A (en) * | 2016-07-18 | 2016-10-26 | 中国水产科学研究院黄海水产研究所 | Vibrio anguillarum O3 serotype bacterial strain and application thereof |
| CN107596363A (en) * | 2017-09-29 | 2018-01-19 | 中国水产科学研究院黄海水产研究所 | A kind of Vibrio anguillarum tervalence inactivated vaccine and its application |
| CN110840841B (en) * | 2019-11-01 | 2021-07-02 | 中国水产科学研究院黄海水产研究所 | Turbot oil emulsion vaccine and application thereof |
| CN113583894B (en) * | 2021-06-24 | 2024-02-13 | 中国水产科学研究院南海水产研究所 | Lateolabrax joint pathogenicity vibrio harveyi strain and inactivated vaccine thereof |
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