CN108210920A - A kind of preparation method and applications of Salmonella anatis inactivated vaccine - Google Patents
A kind of preparation method and applications of Salmonella anatis inactivated vaccine Download PDFInfo
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- CN108210920A CN108210920A CN201611147699.XA CN201611147699A CN108210920A CN 108210920 A CN108210920 A CN 108210920A CN 201611147699 A CN201611147699 A CN 201611147699A CN 108210920 A CN108210920 A CN 108210920A
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- A61K2039/521—Bacterial cells; Fungal cells; Protozoal cells inactivated (killed)
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Abstract
The invention discloses a kind of preparation methods of Salmonella anatis inactivated vaccine, are as follows：The meat duck of infection Salmonella anatis is chosen out of local meat duck farm, isolates Salmonella anatis；Salmonella anatis is enlarged culture, is inactivated, 4 DEG C save backup；Prepare adjuvant：Adjuvant is made of the raw material of following parts by weight：0.8 1.2 parts of sorbester p17,1.5 2.5 parts of Tween 80,1.5 2.5 parts of propolis weigh sorbester p17, Tween 80 and propolis by weight ratio, are uniformly mixed；The Salmonella anatis of inactivation is diluted to its concentration to be added in bacterium solution more than 5 × 109 CFU/mL, then by adjuvant, is uniformly mixed, you can obtains inactivation Salmonella anatis vaccine.The Salmonella anatis inactivated vaccine of the offer of the present invention is with strong points, and to local popular Salmonella strains good immune effect, preparation process is easy to operate, and cost is relatively low, easily stored, suitable for there is the scale farm in laboratory.
Description
Technical field
The invention belongs to biological product technical fields, and in particular to a kind of preparation method of Salmonella anatis inactivated vaccine and
It is applied.
Background technology
Salmonella is enterobacteriaceae member, more than 2500 serotypes, nearly all animal extensive in distributed in nature
It can infect.
Duckling is easy to infection salmonella, especially goes out several days ducklings to three week old after shell, the death rate up to 10~
60%.Salmonella is distributed very wide, the poor duck of most of duck generally existing, particularly environmental sanitations in nature, because
This, there are many chance in duck group's contagion source.In addition, studies have found that salmonella can post in the caecum of many healthy ducks only
Occupy, when feeding management is not good at, environmental sanitation is severe, there was an abrupt change in weather, long-distance transport when stress when, cause duck resistance decline or
Enteric flora disturbance promotes salmonella to enter internal organs and causes morbidity, thus has the title of " opportunistic illnesses ".
If scale livestock farming infection salmonella, except removing causes morbidity dead, often for a long time with poison and interval
Toxin expelling is just easy to feel when people contacts these Fit First animals or eats by salmonella-polluted animal products
Dye, gently then causes self limiting diarrhea, heavy then can cause systemic disease（Such as typhoid）, it can be seen that, salmonella is suitable
Important zoonosis.
Salmonella serogroup is so various, so in terms of vaccine immunity, although multivalence fire extinguishing vaccine application range is very
Extensively, effect is also good, still, still has the serotypes in many cultivation areas with immune vaccine on not, leading to immune effect
Undesirable, this is also existing market common problem.Therefore, developing effective local vaccine has great reality meaning
Justice.
Invention content
The technical problem to be solved by the present invention is to local vaccine serotypes not to be inconsistent, to solve the above problems, the present invention provides
A kind of preparation method and applications of Salmonella anatis inactivated vaccine.
The purpose of the present invention is what is realized in the following manner：
A kind of preparation method of Salmonella anatis inactivated vaccine, is as follows：
（1）The meat duck of infection Salmonella anatis is chosen out of local meat duck farm, isolates Salmonella anatis；
（2）By step（1）Isolated Salmonella anatis is enlarged culture, and inactivation, 4 DEG C save backup；
（3）Prepare adjuvant：The adjuvant is made of the raw material of following parts by weight：0.8-1.2 parts of Arlacel-80, Tween-80
1.5-2.5 parts, 1.5-2.5 parts of propolis weighs Arlacel-80, Tween-80 and propolis by weight ratio, is uniformly mixed；
（4）The Salmonella anatis of inactivation is diluted to its concentration more than 5 × 109 CFU/mL, then by step（3）The adjuvant of preparation
It is added in bacterium solution, is uniformly mixed, you can obtain inactivation Salmonella anatis vaccine；
Wherein, the mass ratio of Salmonella anatis bacterium solution and adjuvant is（2-4）：（6-8）.
The step（2）The specific steps are：By step（1）Isolated Salmonella anatis is inoculated into ordinary nutrient agar
On tablet, select neat in edge, surface is glossy, moistening, smooth single plant bacterium colony be inoculated into LB meat soups, shaken at 37 DEG C
10h adds in the Formalin inactivation that mass fraction is 0.4% after count of bacteria, carries out steriling test, sterile inspection afterwards for 24 hours
After testing qualification, 4 DEG C save backup.
The step（3）In adjuvant be made of the raw material of following parts by weight：1 part of Arlacel-80,2 parts of Tween-80,
2 parts of propolis.
The step（4）The mass ratio of middle Salmonella anatis bacterium solution and adjuvant is 3：7.
A kind of duck Salmonella inactivated vaccine, the duck Salmonella inactivated vaccine are husky with any ducks of claim 1-4
What the preparation method of Men Shi inactivated vaccines was prepared.
A kind of application of duck Salmonella inactivated vaccine, the duck Salmonella inactivated vaccine described in claim 5 are preventing or are controlling
Treat the application in Salmonella anatis infectious disease.
Relative to the prior art, beneficial effects of the present invention are as follows：
（1）Salmonella anatis inactivated vaccine provided by the invention is more preferable using propolis adjuvant absorbability, generates immune effect faster
More preferably, Arlacel-80, Tween-80 emulsibility are good, and vaccine stability is good, are suitble to storage, transport.
（2）The Salmonella anatis inactivated vaccine of the offer of the present invention is with strong points, to local popular Salmonella strains
Good immune effect, preparation process is easy to operate, and cost is relatively low, easily stored, suitable for there is the scale farm in laboratory.
Specific embodiment
Embodiment 1：
A kind of preparation method of Salmonella anatis inactivated vaccine, is as follows：
（1）The meat duck of infection Salmonella anatis is chosen out of local meat duck farm, isolates Salmonella anatis；
（2）By step（1）Isolated Salmonella anatis is enlarged culture, and inactivation, 4 DEG C save backup；
（3）Prepare adjuvant：The adjuvant is made of the raw material of following parts by weight：0.8-1.2 parts of Arlacel-80, Tween-80
1.5-2.5 parts, 1.5-2.5 parts of propolis weighs Arlacel-80, Tween-80 and propolis by weight ratio, is uniformly mixed；
（4）The Salmonella anatis of inactivation is diluted to its concentration more than 5 × 109 CFU/mL, then by step（3）The adjuvant of preparation
It is added in bacterium solution, is uniformly mixed, you can obtain inactivation Salmonella anatis vaccine；
Wherein, the mass ratio of Salmonella anatis bacterium solution and adjuvant is（2-4）：（6-8）.
Step（2）The specific steps are：By step（1）Isolated Salmonella anatis is inoculated into General nutrition agar plate
On, select neat in edge, surface is glossy, moistening, smooth single plant bacterium colony be inoculated into LB meat soups, 10h is shaken at 37 DEG C, pass through
The Formalin inactivation that mass fraction is 0.4% is added in after crossing count of bacteria, carries out steriling test afterwards for 24 hours, steriling test is qualified
Afterwards, it saves backup for 4 DEG C.
Step（3）In adjuvant be made of the raw material of following parts by weight：1 part of Arlacel-80,2 parts of Tween-80, propolis 2
Part.
Step（4）The mass ratio of middle Salmonella anatis bacterium solution and adjuvant is 3：7.
A kind of duck Salmonella inactivated vaccine, the duck Salmonella inactivated vaccine are husky with any ducks of claim 1-4
What the preparation method of Men Shi inactivated vaccines was prepared.
A kind of application of duck Salmonella inactivated vaccine, the duck Salmonella inactivated vaccine described in claim 5 are preventing or are controlling
Treat the application in Salmonella anatis infectious disease.
Embodiment 2：
（1）The meat duck of local meat duck farm suspected infection salmonellosis is chosen, takes a serious histoorgan of fritter lesion
（About 0.5g）, it is placed in LB meat soups, 37 DEG C of 160 r/ min shaking table shakes 6h, and LB meat soups is taken to become muddy sample, are inoculated into Kerma (unit of kinetic energy)
On good salmonella color culture medium, 37 DEG C of constant incubator cultures for 24 hours, take red or aubergine bacterium colony, and purifying culture obtains
One plant of bacterium, dyed, differential medium, biochemical test are accredited as salmonella.
（2）By step（1）Obtained salmonella is inoculated on General nutrition agar plate, selects neat in edge, surface
Glossy, moistening, smooth single plant bacterium colony are inoculated into LB meat soups, and 200rpm shakes 10h at 37 DEG C, are added after count of bacteria
Enter the Formalin inactivation that mass fraction is 0.4%, carry out steriling test afterwards for 24 hours, after steriling test qualification, 4 DEG C save backup.
Steriling test method：Inactivated bacterial liquid is taken to be inoculated on a small quantity on LB tablets respectively, 37 DEG C of cultures for 24 hours, should give birth to without bacterium
It is long.
（3）Prepare adjuvant：The adjuvant is made of the raw material of following parts by weight：Arlacel-80 0.8g, Tween-80
1.5g, propolis 1.5g weigh Arlacel-80, Tween-80 and propolis by weight ratio, are uniformly mixed；
（4）The Salmonella anatis of inactivation is diluted to its concentration more than 5 × 109 CFU/mL, then by step（3）The adjuvant of preparation
It is added in bacterium solution, is uniformly mixed, you can obtain inactivation Salmonella anatis vaccine；
Wherein, the mass ratio of Salmonella anatis bacterium solution and adjuvant is 2：6.
Embodiment 3：
（1）The meat duck of local meat duck farm suspected infection salmonellosis is chosen, takes a serious histoorgan of fritter lesion
（About 0.5g）, it is placed in LB meat soups, 37 DEG C of 160 r/ min shaking table shakes 6h, and LB meat soups is taken to become muddy sample, are inoculated into Kerma (unit of kinetic energy)
On good salmonella color culture medium, 37 DEG C of constant incubator cultures for 24 hours, take red or aubergine bacterium colony, and purifying culture obtains
One plant of bacterium, dyed, differential medium, biochemical test are accredited as salmonella.
（2）By step（1）Obtained salmonella is inoculated on General nutrition agar plate, selects neat in edge, surface
Glossy, moistening, smooth single plant bacterium colony are inoculated into LB meat soups, and 200rpm shakes 10h at 37 DEG C, are added after count of bacteria
Enter the Formalin inactivation that mass fraction is 0.4%, carry out steriling test afterwards for 24 hours, after steriling test qualification, 4 DEG C save backup.
Steriling test method：Inactivated bacterial liquid is taken to be inoculated on a small quantity on LB tablets respectively, 37 DEG C of cultures for 24 hours, should give birth to without bacterium
It is long.
（3）Prepare adjuvant：The adjuvant is made of the raw material of following parts by weight：Arlacel-80 0.85g, Tween-80
1.6g, propolis 1.6g weigh Arlacel-80, Tween-80 and propolis by weight ratio, are uniformly mixed；
（4）The Salmonella anatis of inactivation is diluted to its concentration more than 5 × 109 CFU/mL, then by step（3）The adjuvant of preparation
It is added in bacterium solution, is uniformly mixed, you can obtain inactivation Salmonella anatis vaccine；
Wherein, the mass ratio of Salmonella anatis bacterium solution and adjuvant is 2：7.
Embodiment 4：
（1）The meat duck of local meat duck farm suspected infection salmonellosis is chosen, takes a serious histoorgan of fritter lesion
（About 0.5g）, it is placed in LB meat soups, 37 DEG C of 160 r/ min shaking table shakes 6h, and LB meat soups is taken to become muddy sample, are inoculated into Kerma (unit of kinetic energy)
On good salmonella color culture medium, 37 DEG C of constant incubator cultures for 24 hours, take red or aubergine bacterium colony, and purifying culture obtains
One plant of bacterium, dyed, differential medium, biochemical test are accredited as salmonella.
（2）By step（1）Obtained salmonella is inoculated on General nutrition agar plate, selects neat in edge, surface
Glossy, moistening, smooth single plant bacterium colony are inoculated into LB meat soups, and 200rpm shakes 10h at 37 DEG C, are added after count of bacteria
Enter the Formalin inactivation that mass fraction is 0.4%, carry out steriling test afterwards for 24 hours, after steriling test qualification, 4 DEG C save backup.
Steriling test method：Inactivated bacterial liquid is taken to be inoculated on a small quantity on LB tablets respectively, 37 DEG C of cultures for 24 hours, should give birth to without bacterium
It is long.
（3）Prepare adjuvant：The adjuvant is made of the raw material of following parts by weight：Arlacel-80 0.9g, Tween-80
1.8g, propolis 1.8g weigh Arlacel-80, Tween-80 and propolis by weight ratio, are uniformly mixed；
（4）The Salmonella anatis of inactivation is diluted to its concentration more than 5 × 109 CFU/mL, then by step（3）The adjuvant of preparation
It is added in bacterium solution, is uniformly mixed, you can obtain inactivation Salmonella anatis vaccine；
Wherein, the mass ratio of Salmonella anatis bacterium solution and adjuvant is 2：8.
Embodiment 5：
（1）The meat duck of local meat duck farm suspected infection salmonellosis is chosen, takes a serious histoorgan of fritter lesion
（About 0.5g）, it is placed in LB meat soups, 37 DEG C of 160 r/ min shaking table shakes 6h, and LB meat soups is taken to become muddy sample, are inoculated into Kerma (unit of kinetic energy)
On good salmonella color culture medium, 37 DEG C of constant incubator cultures for 24 hours, take red or aubergine bacterium colony, and purifying culture obtains
One plant of bacterium, dyed, differential medium, biochemical test are accredited as salmonella.
（2）By step（1）Obtained salmonella is inoculated on General nutrition agar plate, selects neat in edge, surface
Glossy, moistening, smooth single plant bacterium colony are inoculated into LB meat soups, and 200rpm shakes 10h at 37 DEG C, are added after count of bacteria
Enter the Formalin inactivation that mass fraction is 0.4%, carry out steriling test afterwards for 24 hours, after steriling test qualification, 4 DEG C save backup.
Steriling test method：Inactivated bacterial liquid is taken to be inoculated on a small quantity on LB tablets respectively, 37 DEG C of cultures for 24 hours, should give birth to without bacterium
It is long.
（3）Prepare adjuvant：The adjuvant is made of the raw material of following parts by weight：Arlacel-80 0.95g, Tween-80
2g, propolis 2g weigh Arlacel-80, Tween-80 and propolis by weight ratio, are uniformly mixed；
（4）The Salmonella anatis of inactivation is diluted to its concentration more than 5 × 109 CFU/mL, then by step（3）The adjuvant of preparation
It is added in bacterium solution, is uniformly mixed, you can obtain inactivation Salmonella anatis vaccine；
Wherein, the mass ratio of Salmonella anatis bacterium solution and adjuvant is 3：6.
Embodiment 6：
（1）The meat duck of local meat duck farm suspected infection salmonellosis is chosen, takes a serious histoorgan of fritter lesion
（About 0.5g）, it is placed in LB meat soups, 37 DEG C of 160 r/ min shaking table shakes 6h, and LB meat soups is taken to become muddy sample, are inoculated into Kerma (unit of kinetic energy)
On good salmonella color culture medium, 37 DEG C of constant incubator cultures for 24 hours, take red or aubergine bacterium colony, and purifying culture obtains
One plant of bacterium, dyed, differential medium, biochemical test are accredited as salmonella.
（2）By step（1）Obtained salmonella is inoculated on General nutrition agar plate, selects neat in edge, surface
Glossy, moistening, smooth single plant bacterium colony are inoculated into LB meat soups, and 200rpm shakes 10h at 37 DEG C, are added after count of bacteria
Enter the Formalin inactivation that mass fraction is 0.4%, carry out steriling test afterwards for 24 hours, after steriling test qualification, 4 DEG C save backup.
Steriling test method：Inactivated bacterial liquid is taken to be inoculated on a small quantity on LB tablets respectively, 37 DEG C of cultures for 24 hours, should give birth to without bacterium
It is long.
（3）Prepare adjuvant：The adjuvant is made of the raw material of following parts by weight：Arlacel-80 1.0g, Tween-80
2.1g, propolis 2.1g weigh Arlacel-80, Tween-80 and propolis by weight ratio, are uniformly mixed；
（4）The Salmonella anatis of inactivation is diluted to its concentration more than 5 × 109 CFU/mL, then by step（3）The adjuvant of preparation
It is added in bacterium solution, is uniformly mixed, you can obtain inactivation Salmonella anatis vaccine；
Wherein, the mass ratio of Salmonella anatis bacterium solution and adjuvant is 3：7.
Embodiment 7：
（1）The meat duck of local meat duck farm suspected infection salmonellosis is chosen, takes a serious histoorgan of fritter lesion
（About 0.5g）, it is placed in LB meat soups, 37 DEG C of 160 r/ min shaking table shakes 6h, and LB meat soups is taken to become muddy sample, are inoculated into Kerma (unit of kinetic energy)
On good salmonella color culture medium, 37 DEG C of constant incubator cultures for 24 hours, take red or aubergine bacterium colony, and purifying culture obtains
One plant of bacterium, dyed, differential medium, biochemical test are accredited as salmonella.
（2）By step（1）Obtained salmonella is inoculated on General nutrition agar plate, selects neat in edge, surface
Glossy, moistening, smooth single plant bacterium colony are inoculated into LB meat soups, and 200rpm shakes 10h at 37 DEG C, are added after count of bacteria
Enter the Formalin inactivation that mass fraction is 0.4%, carry out steriling test afterwards for 24 hours, after steriling test qualification, 4 DEG C save backup.
Steriling test method：Inactivated bacterial liquid is taken to be inoculated on a small quantity on LB tablets respectively, 37 DEG C of cultures for 24 hours, should give birth to without bacterium
It is long.
（3）Prepare adjuvant：The adjuvant is made of the raw material of following parts by weight：Arlacel-80 1.05g, Tween-80
2.2g, propolis 2.2g weigh Arlacel-80, Tween-80 and propolis by weight ratio, are uniformly mixed；
（4）The Salmonella anatis of inactivation is diluted to its concentration more than 5 × 109 CFU/mL, then by step（3）The adjuvant of preparation
It is added in bacterium solution, is uniformly mixed, you can obtain inactivation Salmonella anatis vaccine；
Wherein, the mass ratio of Salmonella anatis bacterium solution and adjuvant is 3：8.
Embodiment 8：
（1）The meat duck of local meat duck farm suspected infection salmonellosis is chosen, takes a serious histoorgan of fritter lesion
（About 0.5g）, it is placed in LB meat soups, 37 DEG C of 160 r/ min shaking table shakes 6h, and LB meat soups is taken to become muddy sample, are inoculated into Kerma (unit of kinetic energy)
On good salmonella color culture medium, 37 DEG C of constant incubator cultures for 24 hours, take red or aubergine bacterium colony, and purifying culture obtains
One plant of bacterium, dyed, differential medium, biochemical test are accredited as salmonella.
（2）By step（1）Obtained salmonella is inoculated on General nutrition agar plate, selects neat in edge, surface
Glossy, moistening, smooth single plant bacterium colony are inoculated into LB meat soups, and 200rpm shakes 10h at 37 DEG C, are added after count of bacteria
Enter the Formalin inactivation that mass fraction is 0.4%, carry out steriling test afterwards for 24 hours, after steriling test qualification, 4 DEG C save backup.
Steriling test method：Inactivated bacterial liquid is taken to be inoculated on a small quantity on LB tablets respectively, 37 DEG C of cultures for 24 hours, should give birth to without bacterium
It is long.
（3）Prepare adjuvant：The adjuvant is made of the raw material of following parts by weight：Arlacel-80 1.1g, Tween-80
2.3g, propolis 2.3g weigh Arlacel-80, Tween-80 and propolis by weight ratio, are uniformly mixed；
（4）The Salmonella anatis of inactivation is diluted to its concentration more than 5 × 109 CFU/mL, then by step（3）The adjuvant of preparation
It is added in bacterium solution, is uniformly mixed, you can obtain inactivation Salmonella anatis vaccine；
Wherein, the mass ratio of Salmonella anatis bacterium solution and adjuvant is 4：6.
Embodiment 9：
（1）The meat duck of local meat duck farm suspected infection salmonellosis is chosen, takes a serious histoorgan of fritter lesion
（About 0.5g）, it is placed in LB meat soups, 37 DEG C of 160 r/ min shaking table shakes 6h, and LB meat soups is taken to become muddy sample, are inoculated into Kerma (unit of kinetic energy)
On good salmonella color culture medium, 37 DEG C of constant incubator cultures for 24 hours, take red or aubergine bacterium colony, and purifying culture obtains
One plant of bacterium, dyed, differential medium, biochemical test are accredited as salmonella.
（2）By step（1）Obtained salmonella is inoculated on General nutrition agar plate, selects neat in edge, surface
Glossy, moistening, smooth single plant bacterium colony are inoculated into LB meat soups, and 200rpm shakes 10h at 37 DEG C, are added after count of bacteria
Enter the Formalin inactivation that mass fraction is 0.4%, carry out steriling test afterwards for 24 hours, after steriling test qualification, 4 DEG C save backup.
Steriling test method：Inactivated bacterial liquid is taken to be inoculated on a small quantity on LB tablets respectively, 37 DEG C of cultures for 24 hours, should give birth to without bacterium
It is long.
（3）Prepare adjuvant：The adjuvant is made of the raw material of following parts by weight：Arlacel-80 1.15g, Tween-80
2.4g, propolis 2.4g weigh Arlacel-80, Tween-80 and propolis by weight ratio, are uniformly mixed；
（4）The Salmonella anatis of inactivation is diluted to its concentration more than 5 × 109 CFU/mL, then by step（3）The adjuvant of preparation
It is added in bacterium solution, is uniformly mixed, you can obtain inactivation Salmonella anatis vaccine；
Wherein, the mass ratio of Salmonella anatis bacterium solution and adjuvant is 4：7.
Embodiment 10：
（1）The meat duck of local meat duck farm suspected infection salmonellosis is chosen, takes a serious histoorgan of fritter lesion
（About 0.5g）, it is placed in LB meat soups, 37 DEG C of 160 r/ min shaking table shakes 6h, and LB meat soups is taken to become muddy sample, are inoculated into Kerma (unit of kinetic energy)
On good salmonella color culture medium, 37 DEG C of constant incubator cultures for 24 hours, take red or aubergine bacterium colony, and purifying culture obtains
One plant of bacterium, dyed, differential medium, biochemical test are accredited as salmonella.
（2）By step（1）Obtained salmonella is inoculated on General nutrition agar plate, selects neat in edge, surface
Glossy, moistening, smooth single plant bacterium colony are inoculated into LB meat soups, and 200rpm shakes 10h at 37 DEG C, are added after count of bacteria
Enter the Formalin inactivation that mass fraction is 0.4%, carry out steriling test afterwards for 24 hours, after steriling test qualification, 4 DEG C save backup.
Steriling test method：Inactivated bacterial liquid is taken to be inoculated on a small quantity on LB tablets respectively, 37 DEG C of cultures for 24 hours, should give birth to without bacterium
It is long.
（3）Prepare adjuvant：The adjuvant is made of the raw material of following parts by weight：Arlacel-80 1.2g, Tween-80
2.5g, propolis 2.5g weigh Arlacel-80, Tween-80 and propolis by weight ratio, are uniformly mixed；
（4）The Salmonella anatis of inactivation is diluted to its concentration more than 5 × 109 CFU/mL, then by step（3）The adjuvant of preparation
It is added in bacterium solution, is uniformly mixed, you can obtain inactivation Salmonella anatis vaccine；
Wherein, the mass ratio of Salmonella anatis bacterium solution and adjuvant is 4：8.
Embodiment 11：
（1）The meat duck of local meat duck farm suspected infection salmonellosis is chosen, takes a serious histoorgan of fritter lesion
（About 0.5g）, it is placed in LB meat soups, 37 DEG C of 160 r/ min shaking table shakes 6h, and LB meat soups is taken to become muddy sample, are inoculated into Kerma (unit of kinetic energy)
On good salmonella color culture medium, 37 DEG C of constant incubator cultures for 24 hours, take red or aubergine bacterium colony, and purifying culture obtains
One plant of bacterium, dyed, differential medium, biochemical test are accredited as salmonella.
（2）By step（1）Obtained salmonella is inoculated on General nutrition agar plate, selects neat in edge, surface
Glossy, moistening, smooth single plant bacterium colony are inoculated into LB meat soups, and 200rpm shakes 10h at 37 DEG C, are added after count of bacteria
Enter the Formalin inactivation that mass fraction is 0.4%, carry out steriling test afterwards for 24 hours, after steriling test qualification, 4 DEG C save backup.
Steriling test method：Inactivated bacterial liquid is taken to be inoculated on a small quantity on LB tablets respectively, 37 DEG C of cultures for 24 hours, should give birth to without bacterium
It is long.
（3）Prepare adjuvant：The adjuvant is made of the raw material of following parts by weight：Arlacel-80 1g, Tween-80 2g, bee
Glue 2g weighs Arlacel-80, Tween-80 and propolis by weight ratio, is uniformly mixed；
（4）The Salmonella anatis of inactivation is diluted to its concentration more than 5 × 109 CFU/mL, then by step（3）The adjuvant of preparation
It is added in bacterium solution, is uniformly mixed, you can obtain inactivation Salmonella anatis vaccine；
Wherein, the mass ratio of Salmonella anatis bacterium solution and adjuvant is 3:7.
Stability experiment：
The Salmonella anatis inactivated vaccine of Example 9-11 carries out stability experiment respectively：
1. ordinary temperature stability is tested
The Salmonella anatis inactivated vaccine of Example 9-11 is placed in room temperature 0d, 30d, 60d, 120d and 180d observation.Observation knot
Fruit shows appearance as before, no situations such as changing colour, crystallizing, illustrates that the invention sample has good stability at normal temperatures.
2. 4 DEG C of observation experiments
The Salmonella anatis inactivated vaccine of Example 9-11 is placed at a temperature of 4 DEG C, respectively the 0d after preparation, 7d, 15d, 21d
It is observed with 30d, observation indicate that, appearance as before, no situations such as changing colour, crystallizing, illustrates the invention sample in 4 DEG C of temperature
Stability inferior is good.
3. anti-freezing stability
The Salmonella anatis inactivated vaccine of embodiment 9-11 is placed in -10 DEG C of refrigerators and preserves 10d, restores to room temperature to observe.Knot
Fruit shows the Salmonella anatis inactivated vaccine of embodiment 9-11, and appearance as before, no situations such as changing colour, crystallizing, illustrates the hair
Bright sample has good stability at a temperature of -10 DEG C.
Clinical effect trial：
Illustrate beneficial effects of the present invention below by way of experimental data：
1. testing program
The healthy 7 age in days duckling 100 of selection, is randomly divided into 5 groups：
A groups, 20, according to Salmonella anatis inactivated vaccine prepared by embodiment 1, only, leg muscle is immunized 0.5ml/；
B groups, 20, according to Salmonella anatis inactivated vaccine prepared by embodiment 2, only, leg muscle is immunized 0.5ml/；
C groups, 20, according to Salmonella anatis inactivated vaccine prepared by embodiment 3, only, leg muscle is immunized 0.5ml/；
D groups, with physiological saline 0.5ml/ only, 7 days after A-D groups are immune, use salmonella infection by 20；
E groups, 20, blank assay control is not immune not infect.
2. evaluation index
2.1 clinical symptoms periodically observe and record the clinical symptoms of chicken, such as appetite, breathing, the state of mind daily；
2.2 pathologic findings dissect dead duck, observe pathological change.
2.3 efficacy determinations are cured, are improved, is effective, is dead.
3. result
3.1 clinical symptoms
After healthy duckling is immunized, mental symptom, feeding situation have no significant change.Morbidity duck mainly shows after infection salmonella
Color loose stool is stained with for spiritual depressed, loose random, loss of appetite, drinking-water increase, anus, the phenomena of mortality occurs in severe infections person.
3.2 pathological change
Morbidity duckling dissect is as it can be seen that liver enlargement, unsharp border have tiny canescence necrosis point, and intestinal mucosa is congested, bleeding,
There is white downright bad point on surface, and meropia phleboedesis is big.
3.3 clinical efficacy
Clinical efficacy the results are shown in Table 1, and as shown in Table 1, A, B, C group attack poison after being immunized using Salmonella anatis inactivated vaccine, obtain
The protective rate arrived is 95%, 95%, 100%；D group positive controls, the death rate reach 40%, E group blank controls.Thus explanation uses this
The vaccine immunity prepared is invented, local Salmonella anatis is infected with good effect to control.
What has been described above is only a preferred embodiment of the present invention, it is noted that for those skilled in the art,
Under the premise of general idea of the present invention is not departed from, several changes and improvements can also be made, these should also be considered as the present invention's
Protection domain.
Claims (6)
1. a kind of preparation method of Salmonella anatis inactivated vaccine, it is characterised in that：It is as follows：
（1）The meat duck of infection Salmonella anatis is chosen out of local meat duck farm, isolates Salmonella anatis；
（2）By step（1）Isolated Salmonella anatis is enlarged culture, and inactivation, 4 DEG C save backup；
（3）Prepare adjuvant：The adjuvant is made of the raw material of following parts by weight：0.8-1.2 parts of Arlacel-80, Tween-80
1.5-2.5 parts, 1.5-2.5 parts of propolis weighs Arlacel-80, Tween-80 and propolis by weight ratio, is uniformly mixed；
（4）The Salmonella anatis of inactivation is diluted to its concentration more than 5 × 109CFU/mL, then by step（3）The adjuvant of preparation adds
Enter into bacterium solution, be uniformly mixed, you can obtain inactivation Salmonella anatis vaccine；
Wherein, the mass ratio of Salmonella anatis bacterium solution and adjuvant is（2-4）：（6-8）.
2. the preparation method of Salmonella anatis inactivated vaccine according to claim 1, it is characterised in that：The step（2）
The specific steps are：By step（1）Isolated Salmonella anatis is inoculated on General nutrition agar plate, and it is whole to select edge
Together, surface is glossy, moisten, smooth single plant bacterium colony is inoculated into LB meat soups, 10h is shaken at 37 DEG C, after count of bacteria
The Formalin inactivation that mass fraction is 0.4% is added in, carries out steriling test afterwards for 24 hours, after steriling test qualification, 4 DEG C of preservations are standby
With.
3. the preparation method of Salmonella anatis inactivated vaccine according to claim 1, it is characterised in that：The step（3）
In adjuvant be made of the raw material of following parts by weight：1 part of Arlacel-80,2 parts of Tween-80,2 parts of propolis.
4. the preparation method of Salmonella anatis inactivated vaccine according to claim 1, it is characterised in that：The step（4）
The mass ratio of middle Salmonella anatis bacterium solution and adjuvant is 3：7.
5. a kind of duck Salmonella inactivated vaccine, it is characterised in that：The duck Salmonella inactivated vaccine is with any institutes of claim 1-4
What the preparation method of duck Salmonella inactivated vaccine stated was prepared.
6. a kind of application of duck Salmonella inactivated vaccine, it is characterised in that：Duck Salmonella inactivated vaccine described in claim 5 exists
Application in prevention or treatment Salmonella anatis infectious disease.
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| CN109663125A (en) * | 2019-01-30 | 2019-04-23 | 山东省农业科学院畜牧兽医研究所 | A kind of chicken intestinal diorder salmonella inactivated vaccine and its application |
| CN114042152A (en) * | 2021-11-30 | 2022-02-15 | 山东滨州博莱威生物技术有限公司 | Duck enteritis salmonellosis inactivated vaccine and preparation method thereof |
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| CN109663125A (en) * | 2019-01-30 | 2019-04-23 | 山东省农业科学院畜牧兽医研究所 | A kind of chicken intestinal diorder salmonella inactivated vaccine and its application |
| CN114042152A (en) * | 2021-11-30 | 2022-02-15 | 山东滨州博莱威生物技术有限公司 | Duck enteritis salmonellosis inactivated vaccine and preparation method thereof |
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