GB2176705A - Live vaccine effective against sheep and goat listeriosis - Google Patents
Live vaccine effective against sheep and goat listeriosis Download PDFInfo
- Publication number
- GB2176705A GB2176705A GB08615114A GB8615114A GB2176705A GB 2176705 A GB2176705 A GB 2176705A GB 08615114 A GB08615114 A GB 08615114A GB 8615114 A GB8615114 A GB 8615114A GB 2176705 A GB2176705 A GB 2176705A
- Authority
- GB
- United Kingdom
- Prior art keywords
- cultivation
- vaccine
- listeriosis
- effective against
- fermenter
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 229960005486 vaccine Drugs 0.000 title claims abstract description 33
- 206010024641 Listeriosis Diseases 0.000 title claims abstract description 13
- 241001494479 Pecora Species 0.000 title claims description 10
- 241000283707 Capra Species 0.000 title claims description 9
- 239000002028 Biomass Substances 0.000 claims abstract description 11
- 230000001681 protective effect Effects 0.000 claims abstract description 7
- 239000001888 Peptone Substances 0.000 claims abstract description 5
- 229930006000 Sucrose Natural products 0.000 claims abstract description 5
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims abstract description 5
- 229960004793 sucrose Drugs 0.000 claims abstract description 5
- 230000002238 attenuated effect Effects 0.000 claims abstract description 4
- 239000001828 Gelatine Substances 0.000 claims abstract description 3
- 235000013681 dietary sucrose Nutrition 0.000 claims abstract description 3
- PXEDJBXQKAGXNJ-QTNFYWBSSA-L disodium L-glutamate Chemical compound [Na+].[Na+].[O-]C(=O)[C@@H](N)CCC([O-])=O PXEDJBXQKAGXNJ-QTNFYWBSSA-L 0.000 claims abstract description 3
- 229920000159 gelatin Polymers 0.000 claims abstract description 3
- 235000019322 gelatine Nutrition 0.000 claims abstract description 3
- 235000013923 monosodium glutamate Nutrition 0.000 claims abstract description 3
- 229940073490 sodium glutamate Drugs 0.000 claims abstract description 3
- 238000000034 method Methods 0.000 claims description 21
- 238000002360 preparation method Methods 0.000 claims description 7
- 241000186781 Listeria Species 0.000 claims description 4
- 238000004519 manufacturing process Methods 0.000 claims description 3
- 238000003756 stirring Methods 0.000 claims description 3
- 238000007865 diluting Methods 0.000 claims description 2
- 239000005720 sucrose Substances 0.000 claims description 2
- 230000001580 bacterial effect Effects 0.000 abstract description 7
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 abstract description 3
- 239000008103 glucose Substances 0.000 abstract description 3
- 238000004108 freeze drying Methods 0.000 description 5
- 206010010741 Conjunctivitis Diseases 0.000 description 3
- 238000000855 fermentation Methods 0.000 description 3
- 230000004151 fermentation Effects 0.000 description 3
- 230000003068 static effect Effects 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000700198 Cavia Species 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 238000011109 contamination Methods 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 239000007789 gas Substances 0.000 description 2
- 230000002163 immunogen Effects 0.000 description 2
- 230000007935 neutral effect Effects 0.000 description 2
- 238000000859 sublimation Methods 0.000 description 2
- 230000008022 sublimation Effects 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 235000019687 Lamb Nutrition 0.000 description 1
- 239000003708 ampul Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000002310 elbow joint Anatomy 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 244000005706 microflora Species 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 239000001397 quillaja saponaria molina bark Substances 0.000 description 1
- 229930182490 saponin Natural products 0.000 description 1
- 150000007949 saponins Chemical class 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000002255 vaccination Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/0208—Specific bacteria not otherwise provided for
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
- A61K2039/522—Bacterial cells; Fungal cells; Protozoal cells avirulent or attenuated
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Attenuated strains of Listeriosis monocitogenes deposited in "NBPMKK" under numbers 251 and 252 are cultivated on meat-peptone bouillon in a fermenter, the bouillon containing 8-20 g/l glucose. Cultivation is at 35-37 DEG C and pH from 7 to 7.6, with air passed through preferably at 3-5 l/min, and a stirrer operated at 300 to 1000 r.p.m. The amount of innoculum is from 1:20 to 1:100 of the working volume of the fermenter. After termination of the cultivation, depending on the density of the biomass obtained, the latter is diluted with protective medium and then it may be homogenised and lyophilised. A biomass having a density of 12 milliard/ cm<3> living bacterial cells may be obtained. For lyophilising of the vaccine, a protective medium containing 2 to 17% by weight sodium glutamate, 5 to 20% saccharose and 0.5 to 5% by weight denatured gelatine is presently used.
Description
SPECIFICATION
Method for the preparation of live vaccine effective against sheep and goat listeriosis
This invention relates to a method for the preparation of a live vaccine effective against sheep and goat listeriosis.
A method for the preparation of sheep and goat listeriosis vaccine is described by I. Ivanov in "Study of the active immunopropylaxis of sheep listeriosis", Proceedings of the Vllth International
Symposium on problems of Listeriosis, 1979. In this method the strains Listeria monocitogenes 1/2a
No. 326 and 4B No. 125 deposited in "PBPMKK" under Nos. 251 and 252 respectively are statically cultivated in meat-peptone bouillon or meatpeptone agar. The biomass obtained from both strains is used to produce native or lyophilised vaccine.
This method for the preparation of the vaccine has the following disadvantages. The bacterial suspension obtained in the static cultivating of the bouillon has a low density which makes it difficult to prepare a lyophilised vaccine. Production of cultures on agar is labour intensive with a high percentage of rejects due to contamination with other bacteria.
There is thus no practical possibility of producing the vaccine industrially. Furthermore, with static cultivating, many of the bacterial cells perish during the cultivation. After lyophilising, the survival rate for listeriosis cells amounts to only 30 to 50%.
According to the present invention, there is provided a method for the preparation of a live bivaccine effective against listeriosis in sheep and goats, which comprises cultivating together attenuated Listeria monocitogenes 1/2a and 4B strains deposited in "NBPMKK" under the Nos. 251 and 252 respectively, on meat-peptone bouillon containing 8 to 20 gll sucrose, cultivation being carried out in a fermenter through which air is passed and in which stirring takes place, the cultivation temperature and pH being from 35 to 37"C and from 7 to 7.6 respectively, with the innoculum occupying 120th to 100th of the working volume of the fermenter, terminating cultivation and optionally diluting the biomass obtained with a protective medium.After ending the cultivation, depending on the density of the biomass obtained, it is used as such or it may be diluted with a protective medium and again used as such or homogenised and lyophilised to be reconstituted subsequently for use.
The fermentation cultivating is carried out as follows: the necessary innoculum is obtained from vaccine strains Listeria monocitogenes 1/2a and 4B and is introduced into the fermenter. A meatpeptone bouillon with 8 to 20 g/l glucose is lyophilisation is subsequently to take place contair 2 to 70% by weight sodium glutamate, 5 to 20% by weight saccharose and 0.5 to 5% by weight denatured gelatine.
During the lyophilisation process, the temperature of the product during the period of sublimation of water is maintained below -30"C.
After ending this process, the temperature is increased to 40 C. The vacuum in the chamber is from 0.1 to 0.05 mm/Hg. The temperature of the condenser is below 45"C. After termination of drying, the vacuum is released by admitting a neutral gas which fills the vaccine containers.
When preparing live bi-vaccine effective against sheep and goat listeriosis by the method of the invention, a biomass is produced which is uniform possesses high density, contains a high percentagi of live cells free from the risk of contamination by secondary microflora and which preserves the initi properties of the vaccine's attenuated strains after lyophilising. Moreover, the method according to tP invention can be automatised. The vaccine obtaine shows a survival rate for the cells of both serotypez which is not lower than 80% and has been found tc have a period of validity of as much as one year.
Hence a product is produced which is stable and which results in high live cell content, immunogen characteristics, harmlessness and purity, as well ac containing the two strains used in equal quantities
The method thus developed permits the manufacture of vaccine on industrial scale.
The following Examples illustrate the invention:
EXAMPLE 1
Fermentation Cultivation of Strains 1/2a and 4B of
L.monocitogenes Lyophiiised samples containing vaccine strains were suspended and cultivated in a meat-peptone bouillon with 1% by weight glucose. The ampoule! were incubated for 24 hours and then sub-cultures were prepared and introduced into the fermenter ii an amount of 1/20 of the working volume of the fermenter which was 7 litres. The cultivating was effected under the following conditions: air fed through at the rate of 3 to 5 I/min; stirrer operated 300 rpm; temperature 37"C and pH 7.2.
At every stage of the operation, the purity was checked by means of a preparation coloured after "Gram" into which samples were innoculated, cultivation occurring in Petri dishes with meatpeptone bouillon.
At the end of the fermentation cultivating, all reproduction was interrupted by having deceleration of the rate of multipiication take place
A biomass with a density of up to 12 milliard/cm3 living bacterial cells was obtained. The density of the bacterial suspension of sub-culture and determined by the density of the biomass obtained in which the ratio of the two vaccine strengths was 1:1. After homogenising the biomass with the protective medium, the vaccine thereby produced was poured into bottles (vials) or metal containers suited to the needs of the consumer.
EXAMPLE 2
Lyophilisation of a Vaccine
The vaccine produced in the preceding Example was frozen in advance in a static or rotary freezer depending on the form of packaging containing the vaccine. The vaccine was then hydrophilised in a iyophilising chamber. During the process, the temperature of the product during the period of sublimation was maintained below -30 C. After termination of this process, the temperature was increased to 40"C. The vaccine in the chamber was 0.1 to 0.05 mm/Hg. The temperature in the condenser was below -45 C. After termination of the drying, the vacuum was broken by admission of a neutral gas (nitrogen) with which the containers were filled.The product obtained was stored in a dry, dark place at a temperature of 4 to 8"C. Make-up solvent used for reconstituting the vaccine was a physiological solution containing 0.1 g/l saponin and of pH 7.2. After reconstituting the vaccine by placing the lyophilisation product in suspension, the vaccine contained not less than 1 milliard/cm3 living bacterial cells in total belonging to both serotypes in approximately equal quantities.
After the lyophilisation, the vaccine was checked forthe number of living bacterial cells by cultivation thereof on innoculation into Petri dishes. Safety with guinea pigs and immunogenic properties were determined. No conjunctivitis appeared on conjunctival treatment of guinea pigs according to the method of Anton, in which there is initial dropping of vaccine into the eyes with administration after at least 12 days in like manner of cultures from the pathogenic strains
L.monocitogenes 1/2a and 4B. In contrast to the controls not treated initially with the vaccine, there was a clearly expressed conjunctivitis with pus formation. No conjunctivitis was observed with animals previously given the vaccine in the aforesaid manner.
EXAMPLE 3
The vaccine of each of Examples 1 and 2 was applied once to a sheep, a goat and to a lamb and a kid aged over 6 weeks in a dose of 2 ml. The animals were injected hypodermically behind the elbow joint. Immunity was obtained 12 days after the vaccination and lasted for 10 months.
Claims (7)
1. A method for the preparation of a live bivaccine effective against listeriosis in sheep and goats, which comprises cultivating together attenuated Listeria monocitogenes 1/2a and 4B strains, deposited in "NBPMKK" under the numbers 251 and 252 respectively, on meat-peptone bouillon containing S20 g/l sucrose, cultivating being carried out in a fermenter through which air is passed and in which stirring takes place, the cultivation temperature and pH being from 35--37"C and from 7 to 7.6 respectively with the innoculum occupying 1/20 to 1/100 of the working volume of the fermenter, terminating cultivation and optionally diluting the biomass obtained with a protective medium.
2. A method as claimed in claim 1, wherein, during cultivation, air is passed at a rate of 3 to 5 I/minute.
3. A method as claimed in claim 1 or 2, wherein stirring of the bouillon takes place at 300 to 1000 r.p.m. during cultivation.
4. A method as claimed in any preceding claim, wherein the biomass obtained at termination of cultivation, is homogenised and then lyophilised.
5. A method as claimed in any preceding claim, wherein the protective medium contains from 2 to 17% by weight sodium glutamate, 5 to 20% by weight saccharose and from 0.5 to 5% by weight denatured gelatine.
6. A method for the production of a live bi-vaccine effective against listeriosis in sheep and goats, substantially as described in either of Examples 1 and 2.
7. A live bi-vaccine effective against listeriosis, whenever produced by the process claimed in any preceding claim.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| BG7076185A BG41848A1 (en) | 1985-06-20 | 1985-06-20 | Method for preparing living bivaccin against listheriosis illness of sheeps and goats |
Publications (3)
| Publication Number | Publication Date |
|---|---|
| GB8615114D0 GB8615114D0 (en) | 1986-07-23 |
| GB2176705A true GB2176705A (en) | 1987-01-07 |
| GB2176705B GB2176705B (en) | 1989-07-26 |
Family
ID=3915796
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| GB8615114A Expired GB2176705B (en) | 1985-06-20 | 1986-06-20 | Method for the preparation of live vaccine effective against sheep and goat listeriosis |
Country Status (5)
| Country | Link |
|---|---|
| BG (1) | BG41848A1 (en) |
| DD (1) | DD267864A7 (en) |
| FR (1) | FR2583641B1 (en) |
| GB (1) | GB2176705B (en) |
| NO (1) | NO167123C (en) |
-
1985
- 1985-06-20 BG BG7076185A patent/BG41848A1/en unknown
-
1986
- 1986-06-19 NO NO862448A patent/NO167123C/en unknown
- 1986-06-20 DD DD29151386A patent/DD267864A7/en not_active IP Right Cessation
- 1986-06-20 FR FR8608926A patent/FR2583641B1/en not_active Expired - Fee Related
- 1986-06-20 GB GB8615114A patent/GB2176705B/en not_active Expired
Also Published As
| Publication number | Publication date |
|---|---|
| FR2583641A1 (en) | 1986-12-26 |
| GB2176705B (en) | 1989-07-26 |
| BG41848A1 (en) | 1987-09-15 |
| GB8615114D0 (en) | 1986-07-23 |
| DD267864A7 (en) | 1989-05-17 |
| FR2583641B1 (en) | 1990-03-09 |
| NO862448L (en) | 1986-12-22 |
| NO167123B (en) | 1991-07-01 |
| NO167123C (en) | 1991-10-09 |
| NO862448D0 (en) | 1986-06-19 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PCNP | Patent ceased through non-payment of renewal fee |
Effective date: 19940620 |