CS272959B1 - Method of medium antigen preparation for poultry's mark's disease diagnostics by means of precipitation test - Google Patents
Method of medium antigen preparation for poultry's mark's disease diagnostics by means of precipitation test Download PDFInfo
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- 239000000427 antigen Substances 0.000 title claims abstract description 35
- 102000036639 antigens Human genes 0.000 title claims abstract description 35
- 108091007433 antigens Proteins 0.000 title claims abstract description 35
- 201000010099 disease Diseases 0.000 title claims abstract description 20
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 title claims abstract description 20
- 238000000034 method Methods 0.000 title claims abstract description 16
- 244000144977 poultry Species 0.000 title claims abstract description 7
- 238000001556 precipitation Methods 0.000 title claims abstract description 7
- 238000002360 preparation method Methods 0.000 title description 6
- 241000700605 Viruses Species 0.000 claims abstract description 25
- 238000004113 cell culture Methods 0.000 claims abstract description 19
- 239000000725 suspension Substances 0.000 claims abstract description 10
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims abstract description 5
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims abstract description 5
- 235000011130 ammonium sulphate Nutrition 0.000 claims abstract description 5
- 210000002257 embryonic structure Anatomy 0.000 claims abstract description 4
- 239000011521 glass Substances 0.000 claims abstract description 4
- 238000005119 centrifugation Methods 0.000 claims abstract 2
- 239000001963 growth medium Substances 0.000 claims description 15
- 239000002609 medium Substances 0.000 claims description 15
- 238000004519 manufacturing process Methods 0.000 claims description 10
- 208000015181 infectious disease Diseases 0.000 claims description 6
- 238000000464 low-speed centrifugation Methods 0.000 claims description 6
- 239000002244 precipitate Substances 0.000 claims description 4
- 230000000644 propagated effect Effects 0.000 claims description 3
- 238000003756 stirring Methods 0.000 claims description 3
- 150000001413 amino acids Chemical class 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims description 2
- 150000003839 salts Chemical class 0.000 claims description 2
- 235000013343 vitamin Nutrition 0.000 claims description 2
- 229940088594 vitamin Drugs 0.000 claims description 2
- 229930003231 vitamin Natural products 0.000 claims description 2
- 239000011782 vitamin Substances 0.000 claims description 2
- 239000012888 bovine serum Substances 0.000 claims 2
- 238000003745 diagnosis Methods 0.000 claims 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 claims 1
- 239000007788 liquid Substances 0.000 claims 1
- 239000002953 phosphate buffered saline Substances 0.000 claims 1
- 239000002002 slurry Substances 0.000 claims 1
- 230000000391 smoking effect Effects 0.000 claims 1
- 241000287828 Gallus gallus Species 0.000 abstract description 5
- 241000272525 Anas platyrhynchos Species 0.000 abstract description 4
- 238000000502 dialysis Methods 0.000 abstract description 4
- 150000003467 sulfuric acid derivatives Chemical class 0.000 abstract description 2
- 239000001166 ammonium sulphate Substances 0.000 abstract 1
- 230000035755 proliferation Effects 0.000 abstract 1
- 230000000384 rearing effect Effects 0.000 abstract 1
- 208000024891 symptom Diseases 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 16
- 241001465754 Metazoa Species 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 239000006285 cell suspension Substances 0.000 description 4
- 239000002054 inoculum Substances 0.000 description 4
- 210000002966 serum Anatomy 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 229910019142 PO4 Inorganic materials 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 210000002308 embryonic cell Anatomy 0.000 description 2
- 210000003746 feather Anatomy 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
- 235000021317 phosphate Nutrition 0.000 description 2
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 2
- 239000002356 single layer Substances 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 208000030289 Lymphoproliferative disease Diseases 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 230000003467 diminishing effect Effects 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 210000000578 peripheral nerve Anatomy 0.000 description 1
- 230000002085 persistent effect Effects 0.000 description 1
- 230000002797 proteolythic effect Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 210000003491 skin Anatomy 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 210000001835 viscera Anatomy 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
IAND
CS 272 959 BICS 272,959 BI
Vynález sa týká sposobu přípravy médiového antigénu na diagnostiku Markovej choro-by hydiny precipitačným testom.The present invention relates to a method of preparing a media antigen for diagnosing Mark's disease in poultry by a precipitation assay.
Markova choroba hydiny je virusové lymfoproliferatívne ochorenie kury domácej, cha-rakterizované výskytom nádorov vo viscerálnych orgánoch, svalovine, koži, 3alej lymfoid-nými infiltráciami v periférnych nervoch a vysokou mortalitou, ktorá niekedy dosahuje až50 najčastejšie 10 až 20 %.Mark's disease of poultry is a viral lymphoproliferative disease of the domestic chicken characterized by the appearance of tumors in visceral organs, muscle, skin, 3 further lymphoid infiltrations in the peripheral nerves, and high mortality, sometimes reaching up to 50 most commonly 10-20%.
Kultivačně médium a buňky z kultúr infikovaných vírusom Markovéj choroby obsahujúspecifické antigény označené A, B, C, dokázatel'né precipitačným testom v agarovom géli. V kultivačnom médiu je přítomný najma A antigén, ktorý sa používá na dokaž precipitač-ných protilátok proti virusu Markovej choroby v krvi, ako následok přítomnosti infekciev chove. Okrem toho sa A antigén tvoří aj v buňkách epítelu peřových folikulov kurčiatinfikovaných vírusom Markovej choroby. Na přípravu A antigénu pre diagnostické účely savyužívajú najčastejšie dva sposoby s použitím virulentného virusu Markovej choroby: 1. Antigén sa získává z kultivačného média buňkových kultúr infikovaných virulent-ným vírusom Markovej choroby. 2. Na přípravu antigénu sa používajú koncové časti peria alebo koža zvierat infiko-vaných virulentným vírusom Markovej choroby.Culture medium and cells from cultures infected with Mark's disease virus contain specific antigens designated A, B, C, proven by agar gel precipitation assay. In particular, an A antigen is used in the culture medium, which is used to prove blood antibodies of the Mark's disease virus as a result of the presence of infections in the breed. In addition, A antigen is also produced in the epithelial cells of the follicle follicles of chicken infected with the Mark disease virus. For the preparation of A antigen for diagnostic purposes, the two most commonly used methods are the use of virulent virus of Mark's disease: 1. The antigen is obtained from the culture medium of cell cultures infected with the virulent virus of Mark's disease. 2. For the preparation of the antigen, the feathers or skin of the animals infected with the virulent virus of Mark's disease are used.
Uvedené postupy majú následovně nevýhody. Pri prvom sposobe přípravy virulentný vi-rus pasážovaním v buňkových kultúrach mění svoje vlastnosti tak, že postupné strácaschopností tvorby A antigénu. Při získávání antigénu z koze infikovaných zvierat - tietosú trvalým zdrojom nákazy šířením infekcie do prostredia - čo je nežiadúce z hfadiskatlmenia ophorenia v populácii kury domácej. Okrem toho, antigén připravený z kože obsa-huje mnoho nedefinovaných zložiek a vyžaduje Salši čistenie.These processes have the following disadvantages. In the first process of preparing virulent viruses by passaging in cell cultures, it changes its properties by gradually degrading the A antigen formation. In obtaining antigen from goat-infected animals - a persistent source of infection by spreading infection into the environment - which is undesirable from the view of diminishing the burn in the domestic chicken population. In addition, the skin-prepared antigen contains many undefined components and requires saline purification.
Podlá navrhovaného postupu médiový antigén sa připravuje v buňkových kultúrachz kuřácích alebo kačacích embryonálnych buniek infikovaných apatogénnym vírusom Marko-vej choroby označeným MDV-KOL-A, uloženým v zbierke výrobných kmeňov národného podnikuBioveta v Nitře pod číslom BN-MDV-06-1985. Uvedený virus bol izolovaný z chovu hydinybez zjevných klinických príznakov ochorenia, V buňkových kultúrach sa vel'mi dobré množíbez predchádzajúcej adaptácie. Tvorba antigénu do kultivačného média je stabilně aj popřekonaní 25 sériových pasáží. Sposob přípravy médiového antigénu na diagnostiku Marko-vej choroby hydiny je založený na tom, že kmen MDV-KOL-A virusu Markovej choroby, ktorýje neškodný pre kuru domácu, sa pomnožuje na primárných alebo sekundárných kultúrachkuřácích alebo kačacích embryonálnych buniek narastených na skle, plastickéj hmotě, kul-tivovaných štacionárnym sposobom alebo v otáčaných ffašiach, připadne na mikronosičoch.According to the proposed method, the media antigen is prepared in cell cultures of smokers or duck embryonic cells infected with the apatogenic Marko disease virus designated MDV-KOL-A, deposited in a collection of production strains of a national enterprise of Bioveta in Nitra under the number BN-MDV-06-1985. Said virus has been isolated from poultry breeding with no apparent clinical signs of disease. In cell cultures, it is very good to reproduce without prior adaptation. The production of antigen into the culture medium is steadily overcome by 25 serial passages. The method of preparing the media antigen for diagnosing Marko's disease of poultry is based on the fact that the strain of MDV-KOL-A virus of the Mark's disease, which is harmless to domestic chicken, is propagated on primary or secondary cultures of smokers or duck embryonic cells grown on glass, plastic mass , cultured in a rotary bottle or in a rotary bottle, may be on microcarriers.
Na kultiváciu buniek pre produkciu médiového antigénu sa použijú vhodné živné médiá obo-hatené o kultivačně sérum. Buňkové kultúry sa infikujú vírusom Markovej choroby, ktorýje viazaný na živé buňky. To znamená, že inokulum představuje suspenzia živých buniek,ktoré obsahujú pomnožený produkčný virus. Infikujú sa 24 až 48hodínové kultúry bunieknarastené na kultivačnom povrchu tak, že suspenzia infikovaných buniek sa přidá do ras-tového média. Rovnako je možné suspenziu infikovaných buniek pridať do suspenzie buniekv rastovom médiu a súčasne naliatí do kultivačnej nádoby. Virus sa pomnožuje v bunkovejkultúre a v jednovrstvé buniek vyvolává viditelné změny - rozpad buniek. Tieto změny akpostihnu viac ako polovicu buniek, kultivačně médium sa zleje a vyčíri nízkoobrátkovoucentrifugáciou. Antigén sa připraví vyzrážaním po přidaní krystalického síranu amonného do média pri chladničkovej teplote. Precipitát sa zahustí nízkoobrátkovou centrifugácioua následné dialyzuje proti fosforečnanmi tlmenému fyziologickému roztoku chloridu sodné-ho. Získaný antigén po dialýze sa upraví tak, aby výsledná koncentrácia dosiahla 25 ažlOOnásobok povodného objemu. Antigén sa skladuje pri teplotách nižších ako O °C krátko-dobo, ale je možné ho aj lyofilízovatí po rozplnení do ampuliek. Výhodou uvedeného postupu přípravy médiového antigénu je, že sa používá namiestovirulentného prirodzene apatogénny virus Markovej choroby, ktorý aj po sériovom pasážo- c CS 272 959 Bl 2 vání v buňkových kultúrach si zachovává schopností produkcie médiového antigénu. Ďalej,že na přípravu antigénu nie je potřebné používatí živé zvieratá infikované virulentnýmvírusom Markovej choroby, ktoré sú potom trvalým zdrojom infekcie pre spoluumiestnenévnímavé zvieratá, čo má význam najma z hřadiska tlmenia nákazy. Příprava antigénu z ko-ze a z koncových častí peria je sice nenáročná, ale jednoduchým postupom sa nedosiahnetaká čistota antigénu ako pri přípravě z buňkových kultúr.To cultivate the cells for the production of the media antigen, appropriate nutrient media, encompassed by culture serum, are used. Cell cultures are infected with the Mark's disease virus, which is bound to living cells. That is, the inoculum represents a suspension of living cells that contain an enriched production virus. 24-48 hour cultures of cells grown on the culture surface are infected so that the suspension of infected cells is added to the growth medium. It is also possible to add the suspension of infected cells to the cell suspension in the growth medium and simultaneously pour into the culture vessel. The virus propagates in the cell culture and causes visible changes in the monolayer cells - cell disintegration. These changes affect more than half of the cells, the culture medium is decanted and clarified by low-speed centrifugation. The antigen is prepared by precipitation after addition of crystalline ammonium sulfate to the medium at refrigerator temperature. The precipitate is concentrated by low-speed centrifugation and subsequently dialysed against phosphates buffered with saline. The dialysis antigen obtained is adjusted to give a final concentration of 25-100 times the flood volume. The antigen is stored at temperatures below 0 ° C for a short time, but can also be lyophilized after filling into ampoules. An advantage of said process of preparing a media antigen is that it uses a non-virulent, naturally apatogenic Mark's virus, which retains the ability to produce media antigen even after serial passage of CS 272 959 B1 in cell cultures. Furthermore, it is not necessary to use live animals infected with the virulent virus of the Mark disease, which are then a permanent source of infection for co-incipient animals, which is of particular importance for disease control, for the preparation of antigen. The preparation of the antigen from the cage and the end feather parts is undemanding, but the purity of the antigen as in the preparation from cell cultures is not easy.
Uvedené příklady predmet vynálezu nijako neobmedzujú, ani nevyčerpávajú: 1. Sposob kultivácie:The examples are not intended to limit or limit the invention in any way: 1. Culture method:
Suspenzia buniek, připravená pomocou proteolytických fermentov z lldňových kuřá-cích resp. 13dňových kačacích zárodkov v rastovom médiu s definovaným množstvom solí,aminokyselin a vitamínov s prídavkom 5 % hmot séra určeného pre tkanivové kultúry sakultivuje na skle, plastickej hmotě, štacionárnym sposobom alebo v otáčaných ftíašiach,připadne v suspenzii na mikronosičoch. 2. Sposob infekcie buňkových kultúr produkčným vírusom:Cell suspension prepared by proteolytic ferment from 1 day smokers and 1 day smokers respectively. 13-day duck embryos in a growth medium with a defined amount of salts, amino acids and vitamins with the addition of 5% by weight of the tissue culture serum are cultured on glass, plastic, stationary or rotary pads, optionally in suspension on microcarriers. 2. Cell virus infection method by production virus:
Inokulum představuje suspenzia živých buniek infikovaných produkčným vírusom, kto-ré sa přidávájú do kultivačněj nádoby s buňkami v pomere 1:3 až 1:6 (suspenzia buniekz jednej kultivačněj nádoby sa přidává do 3 až 6 nádob s buňkami). Buňkové kultúry při-pravené a pěstované podtía bodu 1 sa infikujú produkčným vírusom nasledovnými spSsobmi: a. Infekcia narastenej jednovrstvý buniek:The inoculum represents a suspension of live cells infected with the production virus which is added to the cell culture vessel in a ratio of 1: 3 to 1: 6 (cell suspension in one culture vessel is added to 3 to 6 cell containers). The cell cultures prepared and cultured under point 1 are infected with the production virus by the following methods: a.
Do rastového média 24 až 48hodinovej kultúry buniek sa přidá inokulum a buňkové kultúrysa Sálej inkubujú bez výměny média pri teplote 38 °C počas 72 až 120 hodin. b. Buňková suspenzia v rastovom médiu s hustotou asi 10^.ml-·'- buniek sa zmieša s inokulom buniek infikovaných produkčným vírusom a súčasne vleje do kultivačnej nádo-by. Kultivácia sa robí pri 38 °C počas 72 až 120 hodin. 3. Zber média, ktoré obsahuje antigén: 24 připadne 48 hodin po infekcii je možné povodně rastové médium vyměnit za médiumrovnakého zloženia, ale so zníženým obsahom (0,5 až 1,0 % hmot.) kultivačného séra. Vi-rus sa pomnožuje v bunkovej kultúre tak, že dochádza počas 72 až 120 hodin k rozsiahle-mu rozpadu buniek a tým k uvďňovaniu A antigénu do média. Ak změny postihujú viac ako50 % jednovrstvý buniek, médium sa zleje a egalizuje zo všetkých kultivačných nádob. 4. Izolácia médiového antigénu:Inoculum is added to the growth medium of 24-48 hour cell culture and cell cultures are incubated without exchange of medium at 38 ° C for 72-120 hours. b. A cell suspension in growth medium with a density of about 10 µm cells is mixed with the inoculum of cells infected with the production virus and simultaneously fed into the culture vessel. Cultivation is performed at 38 ° C for 72 to 120 hours. 3. Collecting antigen containing antigen: 24 days after infection, it is possible to exchange the growth medium with medium of the same composition but with a reduced content (0.5 to 1.0% by weight) of the culture serum. The virus is propagated in cell culture so that there is extensive cell disintegration within 72 to 120 hours, thereby introducing A antigen into the medium. If the changes affect more than 50% of the monolayer, the medium is decanted and leveled from all culture vessels. 4. Isolation of media antigen:
Egalizované médium sa vyčíri nízkoobrátkovou centrifugáciou. Vyčírené médium sazleje a za stálého miešania sa přidává krystalický síran amonný v množstve 30 g na100 ml média. Miešanie pri laboratórnej teplote sa robí až do rozpustenia celého množ-stva síranu. Precipitácia antigénu z média potom Sálej pokračuje pri teplote 2 až 8 °Cpočas 24 až 72 hodin. Precipitát od média sa oddělí nízkoobrátkovou centrifugáciou adialýzou sa zbaví síranov. Dialýza proti fosforečnanmi tlmenému fyziologickému roztokuchloridu sodného sa robí tak, že precipitát sa nerozpúšťa, ale priamo vkládá do dia-lyzačnej hadice, čím sa dosiahne po dialýze dostatočná koncentrácia - 25 až lOOnásobokpóvodného objemu. Ďalšiu koncentráciu je možné dosiahnuť lyofilizáciou s tým, že lyofi-lizát sa rozpúšťa v menšom množstve destilovanej vody, ako bolo povodně množstvo anti-génu před lyofilizáciou.The medium is clarified by low speed centrifugation. The clarified medium is seeded and crystalline ammonium sulfate (30 g per 100 ml of medium) is added with stirring. Stirring at room temperature is continued until all of the sulfate is dissolved. Antigen precipitation from the medium is then continued at 2-8 ° C for 24-72 hours. The precipitate from the medium is separated by low speed centrifugation by adialysis to remove the sulphates. Dialysis against phosphates buffered with physiological sodium chloride solution is made so that the precipitate does not dissolve but is directly introduced into the diaphragm tube to provide a sufficient concentration of 25-25 times the original volume after dialysis. Further concentration can be achieved by lyophilization, with the lyophilizate being dissolved in a smaller amount of distilled water than the flood of anti-gene prior to lyophilization.
Claims (5)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CS971286A CS272959B1 (en) | 1986-12-22 | 1986-12-22 | Method of medium antigen preparation for poultry's mark's disease diagnostics by means of precipitation test |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CS971286A CS272959B1 (en) | 1986-12-22 | 1986-12-22 | Method of medium antigen preparation for poultry's mark's disease diagnostics by means of precipitation test |
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| Publication Number | Publication Date |
|---|---|
| CS971286A1 CS971286A1 (en) | 1990-07-12 |
| CS272959B1 true CS272959B1 (en) | 1991-02-12 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CS971286A CS272959B1 (en) | 1986-12-22 | 1986-12-22 | Method of medium antigen preparation for poultry's mark's disease diagnostics by means of precipitation test |
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| Country | Link |
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| CS (1) | CS272959B1 (en) |
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1986
- 1986-12-22 CS CS971286A patent/CS272959B1/en unknown
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| Publication number | Publication date |
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| CS971286A1 (en) | 1990-07-12 |
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