DE2206779A1 - METHOD OF BULK FOOD SECURITY - Google Patents

Description

FARBENFABRIKEN BAYER AG

LEVERKUSEN-Bayerwerk * «. - _r "

¹ '■■ i't'3. ■ Central area ·. ■>

Patents, trademarks and licenses

Er / Gk method for mass breeding of FMD viruses

The present invention relates to a new method of mass breeding FMD viruses.

The following methods of multiplying FMD viruses have already become known:

1) Virus breeding on the tongue mucous membrane of living cattle (0. Waldmänn, G. PyI, K.O. Hobohm and H. Möhlmann ZbI. Bakt. 1. Orig. 148, 1, (1941).

2) Virus cultivation on surviving epithelial cells of isolated bovine tongue mucosa in vitro (H.S. Frenkel, Bull. Off. Int »Epiz. 28, 155, (1947) and 39, 91, (1953).

3) Virus replication in the surviving, enzymatically digested Tissue from isolated ruminant stomach in vitro (K.O. Hobohm, H.U. Rahneberg, H.S. Fiege,

German Offenlegungsschrift 1,937,760, DBP

4) Virus multiplication in single-layer primary cell cultures of Calf kidneys (B. Ubertini, B.L. Nardelli, A. Dal Prato,

Le A 14 244

309833/1005

G. Panin and G. Santero, ZbI. Vet. Med. 1965 , 93-101).

5) Virus replication in single-layer and suspension BHK (Baby hamster kidney) cell cultures (Macpherson and Stoker, Virology 16, 147, (1962).

Virus obtained in this way is suitable for the production of FMD vaccines. These vaccines immunize hoofed animals (cattle, pigs, sheep, goats) against foot and mouth disease (FMD),

However, the above methods have a number of disadvantages:

Virus breeding on the tongue mucous membrane of living cattle (Waldmann method is used in most of the FMD vaccines) Countries of the world banned for epidemic police reasons.

The cultivation of foot-and-mouth disease virus according to the Frenkel process comes across because of the need for continuous supply sufficient amounts of isolated tongue mucosa from freshly slaughtered cattle in many places to considerable difficulties. The FMD virus cultivation in tissue cultures, in particular on so-called rionolayer cultures, is costly and time-consuming for large-scale production.

Foot-and-mouth disease vaccine from FMD viruses on heterologous cell lines cultured, e.g. BHK cell lines, harbor the danger of repeated vaccinations and even initial vaccinations cause allergic diseases in the vaccinees.

Virus breeding only takes place in surviving enzymatically disrupted tissues of ruminant stomachs in vitro

Le A 14 244 - 2 -

309833/1005

the virus synthesis slowly at »(30 - 42 hours) and the virus -1

small amount.

Virus yield is r> ro ml with 10 "to 10 infectious doses.

It has been found that by a combination virus culture method, which is characterized in that nan.sowohl insulated bovine tongue epithelium as well as surviving one Tissues from ruminants' stomachs used in a combined culture process for virus replication, the above Disadvantages of the FMD virus breeding methods known from the literature are avoided.

The method according to the invention is suitable for the large-scale production of foot-and-mouth disease virus for the manufacture of foot-and-mouth disease vaccines. It is simple. It is cheaper than pure breeding using the Frenkel method. It is both the Frenkel method as well also pure breeding in surviving, enzymatically digested Ruminant stomach tissues superior in terms of virus yield. The combination breeding process significantly reduces the difficulty in obtaining sufficient quantities of bovine tongue epithelium, which exists in many places. In the method according to the invention, the FMD virus is grown on homologous tissue. The ones made from it Vaccines do not cause any allergic symptoms. They are effective, durable and well tolerated. That The method according to the invention thus represents a great enrichment for veterinary medicine.

To carry out the process according to the invention, cattle tongues and ruminant stomachs are used. One uses in the method according to the invention preferably stomachs of cattle, namely pillars and stomach floor of the rumen as well as the epithelial cell-containing mucous membrane of the bovine tongue.

Le A 14 244 - 3 -

309833/1005

/ χ

The beef tongues were prepared according to the method of f'ronkol (UM Fronkfil, Bull. Off. Int. 'EpI z. 20, 155, (1947) and ^, <) λ _t (1 ^f J53); Jr.oUm! r.chnr ontlbiotikahultigor citjftokühltor frtlzlOmiM / r KiiM.Mi! ii! i * ilt. UrimltLclbur after removal of the midday meal with laughed children, the pillars and the zottonf'roio part of the stomach floor of the rumen are removed. Dioao To J. are washed with lukewarm water, free of layers of fat, for 2 minutes on both sides at a distance of approx. 30 cm. irradiated with ultraviolet light and bundled in an antibiotic-containing isotonic ice-cold saline solution.

Both the bovine tongue epithelia obtained by the Frenkel method as well as the rumen parts isolated as described are minced in a meat grinder (hole size 12-20 mm) and washed with isotonic cold saline three times. After that, the shredded rumen tissue and the crushed tongue epithelia shaken for one hour in an isotonic saline solution containing antibiotics.

The isotonic saline solution containing antibiotics is used advantageously a physiological salt solution of the following composition:

NaCl - 8 gr., KCl - 0.4 gr., NaHCO ₃ - 0.35 gr., Penbillin 0.5 gr., Chloramphenicol - 0.25 gr., Nystatin - 0.075 gr., Aqua dest. ad 1000 ml.

After treatment with the isotonic anti-biotic Saline solution, the supernatant is decanted and the tissue pulp is 1/10 of the volume intended for the total batch infected with KKS virus suspension.

Le A 14 244 - 4 -

309833/1005

ALo ΜΚΓ'-Virua, with dom das orfindungagomUaao demonstrators
! leads to ^ y; wordon konn, k'ommon allo bokannton Viruntypori In
Question: e.g. the European virus types O, A and C, dlo oUdirr.crJkanlochen virus types O, A and C, the African types- • <n IJAT 1, 2 and 3 as well as the aalatlüchon types, wio zH Ariln 1 ui; d all subtype the mentioned basic virus type.

After an adsorption time of 20 minutes, the infected Rumen tissue and tongue epithelium under sterile conditions in the breeding fermentor prepared for virus breeding

brought.

The fermenter already contains the required proportion of culture medium for cultivation. Used as a culture medium a physiological nutrient solution adjusted to a pH of 7.4 to 7.6 with the following composition is preferred:

Na Cl
KCl

Mg Cl ₂ * 6K ₂ O Ma H ₂ PO - " ^Λ
Na HCO ₃
Ma ₂ HPO ₄ '
Glucose
Peptone

6H ₂ O -

~ H ₂ 0

- 720 gr.

- 18 gr.

18 gr. 9 gr. - 11.1 gr.

200 gr. - 48.06 gr. - 100 gr. - 240 gr.

- 300 gr.

Casein hydrolyzate - 300 gr.Hemin solution (0.02 %) - 3.6 ml

DL - methionine 12.8 gr. DL - tryptophan 8.0 gr. DL - threonine 2.5 gr.

L - cystine HCl 5.2 gr.

L - thyroxine 0.0009 gr. D-Ca pantothenate 0.032 gr. Niacin 0.16 gr. Insulin 80 IU
Chloramphenicol 10 gr. Pericillin 32 gr. Neomycin sulfate 10 gr. Polymixin B 0.5 gr. Nystatin 0.75. Aqua dest. ad 100 liters

Le A 14 244

! NSPECTED

309833/1005

The breeding approach is adjusted and after the addition of the seed virus-tissue suspension with glycine buffer at a pH from 7.4 to 7.6 was stirred at 37 ⁰ C and 25 revolutions per minute.

Glycocolla buffers of the following composition are preferably used :

Glycocolla - 158 gr., Na Cl - 1-4 gr., Na OH - 51 gr., Aqua dest. ad 1000 ml.

At the same time, sterile filtered pure oxygen (2 liters per minute) is introduced into the cultivation mixture. Of the The pH of the cultivation mixture is constantly monitored and, if necessary, by adding glycocolla buffer to the pH value adjusted from 7.4 - 7.6.

After 18 to 24 hours, depending on the type of virus, virus cultivation is terminated and the suspension is cooled ^{to 4 ° C.} Afterward. the virus-containing tissues (rumen tissue and tongue epithelium) are crushed using a colloid mill in order to recover the viruses in the cells. The tissue pulp thus disrupted is extracted together with the virus-containing culture medium for one hour at 4 ° C. with slow stirring (25 rpm). Mertogan (Thimerosal) is added to the tissue suspension at a concentration of 1: 40,000 or 0.2 to 1 % chloroform.

After extraction, the virus suspension is placed in a continuous flow centrifuge centrifuged of the Sharpies or Westfalia type. The clear virus extract obtained in this way can be used quantitatively Determination of its FMD virus antigen content can be used as an antigen for the production of FMD vaccines.

Le A 14 244-6

309833/1005

The bootlmmung dos Vlrusgohaltes, namely dos Gohaltoo on infoktiijijom Antigon and an Complomontbindondon Antimony follows quantitatively.

Olmlt an inrokbiUuom antigen is on MUuoon wlo JToI κ b

boo tiniint:

Jo 10 four to seven day old Mäuso received in Äthor-

-4 - ¹ S -fi -7 anesthesia 0.10 ml. The virus dilution 10, 10 ^J , 10, 10 ',

Q _Q

10 and 10 injected intraperitoneally. The duration of the experiment is 7 days. Fatalities before 8 hours of poat infection are not counted, since the incufoationuzoit is at least 48 hours. For the individual dilutions, according to B. Behrens, Naunyn - Schmiedeberg ¹ S Arch. Exp-. Peth. Pharmac. 140 , 237, (1920) and G. Kärber idem. 162 , 489, (1931) determined the 50 # endpoints. The content of infectious FMD antigen (_ = virus titer) is given in MsLDcq / hIL.

The content of complement-binding antigen is determined serologically in the complement binding reaction (KBR) according to the "50% method" (Traub, E. and Möhlmann H. ZbI. Bakt. I. Orig. I50 , 289 (143 a) and ZbI. Bakt I. OrIg. 300 (1943b), Camargo, NF, Eichhorn BA, Devine JM and Telloz Giron, A., Proc. 87th Ann. Meet Amer. Vet. Med. Assoc. 195O i 207-211.) Under spectrophotometric evaluation determined quantitatively.

Le A 12 244 - 7 -

309833/1005

Example 1 :

15 kilograms of purified, low-germ using UV radiation Made rumen tissue and 15 kilograms of purified tongue epithelium, which has been rendered germ-free by means of UV radiation, are stored in isotonic, antibiotic-containing salt solution collected and minced in the meat grinder.

The tissue pulp thus obtained is washed with physiological saline solution and then shaken for 1 hour with isotonic saline solution containing antibiotics. The isotonic, antibiotic-containing salt solution is then removed by decanting and the tissue pulp with 30 kilograms of a MKS-VIrus suspension of the South American virus type C, the virus titer of which is MsLD ^ ₀ 10 ^ mI. is _> infected.

After an adsorption time of 20 minutes, the infected tissue pulp is placed under sterile conditions in the fermenter filled with 240 kg of culture medium. The culture medium was warmed to 37 ° C. beforehand. The culture mixture is adjusted to a pH of 7.5 with 250 ml of glycocolla buffer and stirred at 37 ° C. and 25 revolutions per minute. During the entire cultivation process, sterile filtered pure oxygen (2 liters per minute) is introduced into the tissue suspension. The pH value is constantly monitored and readjusted to a pH value of 7.4 for 5 hours pi and 8 hours pi by adding 300 ml each and 14 hours pi by adding 400 ml of glycocolla buffer. Samples are taken from 12 hours pi at intervals of 2 hours to 22 hours pi for the quantitative determination of the content of infectious and complement-binding antigen. Cultivation is terminated after 22 hours and the batch is cooled ^{to 4 ° C.} The tissue parts of the breeding approach

Le A 14 244 - 8 -

309833/1005

¹ J 'ί - / Ι ^ι 2 ⁹ ΐ ^Ί 6779

received on July ,

were by means of a colloid mill for the EndviruiJoxtrftkU.Qn aufßoachloaoon. The ao bohandolto Gowobobrol becomes /.urwumrimi with the vlrus-containing cultivation mode for one hour at 4 ° C extracted with slow stirring (25 U / ml.).

After extraction, the virus suspension is placed in an olnor centrifuge vim type Woatfalia centrifuged. Dor no orhaltoncn clear VlruuuuiJi) onsion is Mortogan in one concentration of 1: 40,000 added. f

The virus titers and the titers of the complement-binding antigono developed in the course of the 22 hour breeding process as follows:

time / hour p. i. MsLD ₅₀ 96Hamoiysenema
dilution mung in, Anxiety 1: 5 1 : 10, 1:20 12th
14th
16 ₁₀ -6.0
10 ~ ⁶ ' ⁸
₁₀ -7.25 15th
22nd
41 0
0
0 0
0
0 18th 10 " ⁷ » ⁵ 100 38 0 20th ₁₀ -8.0 100 85 15th 22nd ΙΟ " ⁸ ' ⁵ 100 1 00 68 3eisr> iel 2:

According to Example 1, bovine tongue epithelium and rumen tissue are used a suspension prepared and infected with the South American FMD virus type 0, subtype 0.-Campos (Vlrustitor ') infected.

Le A 14 244 - 9 -

309833/10 0 5

I) Io V Lrna :: UchLung is abßobrochon after 20 Gtundon. ΙλΙ.η Knt.wi f: Klun / -r dor Vlruotltor and doo complomontb.Inflondon Ti ~ lor ι. iiit as fol ^ t:

Zolt /. "Td. P. 1.

MgLD

50

% Hiimolyuohoinrnung in antimony · dilution

1: 5

1: 10

20th

12 14 16

20th

₁₀ -5.5 0 0 0 ΙΟ " ^{6 '25} 21 0 0 ΊΟ " ⁷ ' ⁵ 38 0 O ΙΟ " ⁸ ' ⁰ 100 72 10 ₁₀ -9.5 100 100 82

example 3 '

According to Example 1, a tissue suspension is produced from rumen tissue and bovine tongue epithelium and inoculated with the South American virus type A, subtype A _oc - (virus titer MaLD ₁ 10 "^{/> o} / ml.

'50

After 24 hours, the cultivation is stopped and the Vlru3 is harvested. The development of the virus titre and the titera of the complement-binding antigen is as follows:

Time / hour p. i.

MsLD

Hemolysis inhibition in antigen dilution

20th

12 14 16

ΙΟ ' ⁴ ' ⁵ 0 0 0 10-5.0 10 0 0 10-6.25 10 0 0

Le A 14 244

-10-

309833/1005

Zoit / ntd. pi MaLD ^ _n ? UHUmolyaohemruunß in antigen

VordUnnun / -;

1: 5 1: 10 1:: · ()

18 10 "'» ^D 63 5 O

20 10 " ⁸ ' ⁰ 100 10 0

22 1O " ^{9 '25} 100 75 10

24 10 ~ ⁸ '75 ιoo 100 87

The virus suspension obtained from Example 1, 2 and 3 are used as an FMD virus antigen in a trivaleal FMD Vukzino used.

The vaccine protects cattle against artificial infection with the 3 virus types O ₁ -Canpos, Apc and C.

Le A 14 244 - 11 - '

309933/1005

Claims (2)

Patent claims: 1. A method for mass breeding of foot-and-mouth disease virus, characterized in that FMD virus is reproduced in a suspension of surviving cells of bovine tongue epithelia in combination with surviving cells of ruminant stomachs. 2. The method according to claim 1, characterized in that * bovine tongue epithelia are used in combination with rumen pillars and rumen floors of beef stomachs. Le A 14 244 - 12 - 309833/1005