DE2133654A1 - Process for the production of a vaccine from Bacteroides nodosus and its use - Google Patents

Description

Γ'Λ i I: i TANWA Ln:

Pl '.' Or. D.i. DiV. J. RE1TSTÖTTER

Thing. wolfram dünte

D.W KARL GEORG LÖSCH

It IU) OO Mill.'I-HiM 1. ">. Tj AU Li ₁ Ί ι" ι. / \ l · '· I. ■ ·;' .. Γ-UiiNHU F lOlllll :! ■ '<···

Munich, the '<' 6. M / 11609

New Zealand Inventions Development Authority-Display Center Building, Sturdee Street, Wellington, New Zealand

Process for the production of a vaccine from Bacteroides nodosus

and its use

The present invention relates to the production of a culture of Bacteroides nodosus for use in a vaccine is suitable that against B. nodosus (foot rot f ektiori.) 'at Sheep and other ruminants is effective. '

Foot rot, an infectious disease that causes lameness in sheep, is ubiquitous in most sheep farming areas observed in the world, especially in areas with temperate climates and moderate to high annual rainfall. In areas where the infection is often endemic to herds, the disease becomes an acute economic one

1098 83/1840

BATH ORIGINAL

M / 11609 j

Problem and can lead to losses in the wool or meat ■ production and entail considerable costs for the treatment of the infected animals.

According to the traditional method of fighting the disease in herds, the infected animals are either used for Single treatment or isolated for slaughter. In practice, the known therapeutic methods are laborious, 'cost-' playful and insecure, so that a complete eradication of the disease on goods is seldom achieved.

As the bacteria that cause sheep foot rot, B. nodosus, 19A-1 were identified for the first time (W.I.B. Beveridge, Australia, Council of Scientific * & Industrial Research Bulletin No. 140) the question was also considered whether sheep can be protected from infection by using vaccines could. However, it was believed that because of the chronic and superficial nature of the disease, the prospects for Control through immunization is low. Beveridge noted, however, that sheep that were killed with crops of B. nodosus when exposed to infection were milder disease symptoms developed than un-. vaccinated animals. He then suggested that alternative measures should be taken In order to eradicate the disease, prophylactic vaccination should prove ineffective to give. · ■,

The effect of the 'vaccination by attenuating the disease Later foot rot infections, as originally observed by Beveridge, was supported by recent research in Australia (J.R. Egerton and D.H. Burrell, Aust. Vet. J. 46, pp. 517-22, 197p) and later in New Zealand (T.M. Skerman, N, Z. Vet. J.) in press, confirmed. It has also been found that vaccinated sheep are more resistant to the disease than untreated animals

- 2 T09883 / 184 0

ι Μ / 11609 _ο ^:

«Λ!

and that the inoculation of sheep already infected with foot rot has an important healing effect. If at the

Description of the present invention so to the protective! Antigens are referred to, it means that they are both! have healing and prophylactic properties. j

So far, however, there is still no practical method to; for the production of foot rot vaccines on a commercial scale. -i

Large scale vaccine production depends primarily on satisfactory in vitro methods of growing the specific local organisms under discussion. In general ^{: it} is necessary that the bacteria be grown under conditions which produce cells and / or cell by-products which produce a sufficient degree of protective response in the vaccinated animal. It is also desirable that the selected components of the culture media be the cheapest and most readily available and that the basic culture methods can be adapted to large scale vaccine production. The only published method for breeding B. nodosus is to use a liquid medium consisting of an extract of finely ground sheep's hooves, and an enzyme. Compound digest of pancreas or trypsin (JH Thomas, Aust. Vet. J. 39 (11), 434, 1963. DS Roberts, Brit. J. exp. Path. 48 (6), 665, 1967). The medium is placed in test tubes or small bottles, which are then inoculated and incubated in standard anaerobic vessels under an atmosphere of 90% hydrogen and 10 % carbon dioxide. In ^; In a modification of this process, a two-phase medium is prepared by allowing a quantity of agar medium to solidify in the bottles before the sterile one

- 3 -

109883/1840

2 13365A

M / 11609 ' j.

liquid medium is added (J.R. Egerton and D.H. Burreil, Aust. Vet. J. 46, 'S. 577-22, 1970). ·

It is obvious, however, that the nature of the main ingredients of this medium, the laborious production that they require and the limited volume of culture that is cultivated can become, together, significant obstacles to the production of significant quantities of * B. nodosus cultures in an economical and reproducible way. '

In recent research at Wallaeevill Animal Research _; Center for the metabolism of B. nodosus, the main nutritional needs of these organisms were determined. ■ Some combination of these requirements seems; to be peculiar to this species. Deiflgemäß a specific '· ■ designed culture medium that meets the special needs of growth of these bacteria. All components of this culture medium are products available on the market, so that the need to purchase large quantities of complex natural materials, such as sheep's hooves, is completely eliminated. In addition, large reproducible batches of cultures can be grown, since the volume of dormant cultures that can be obtained depends only on the size of the

. available container. The same medium can also be used in an apparatus designed for the continuous culture of anaerobic microorganisms. As well as . using both stationary and continuous culture techniques, it has been found that the culture medium provides very high yields of cells, soluble cell components, and proteolytic enzymes. ,

The subject of the present invention is the creation of a method for the cultivation of B. nodosus organisms, which can be used in the manufacture of vaccines against B. nodosus (foot rot) infections in sheep and

- 4 109883/184 0

M / 11609 ^,

Other ruminants are effective, the disadvantages!

of existing procedures or at least a useful

lent alternative forms. ' \

Accordingly, the present invention provides a method for producing a vaccine against Bacteroides nodosus Beveridge (Mraz) infection in sheep or other re-; cattle is effective and the anaerobic cultivation of a strain ^: of said microorganisms in a nutrient medium which contains L-arginine or salts thereof, wherein antigens are obtained from the culture medium which cause protection against B. nodosus infection and the protective antigens obtained are incorporated into a vaccine by conventional methods. ■ ''

In particular, according to this method, a culture medium is used which consists of a liquid broth medium, the water, a pancreatic digest of casein, preferably trypticase, a bovine extract, preferably LAB-LEMCO (registered trademark No. 49 289 in New Zealand), proteose peptone, yeast extract , Sodium carbonate, a reducing agent, preferably sodium thioglycollate, L-arginine, preferably free base of L-arginine or L-arginine monohydrocbloride, · ' ^!

Serine, lysine, carbon dioxide and soluble starch in one pH of approximately 7.8 anaerobically, in the presence of carbon

contains dioxide. · *

(a) Trypticase creates a source of peptide nitrogen,

which is essential for the growth of B. nodosus. Other

Enzyme digests of casein -can be used instead but of those tested, trypticase consistently gives the most growth.

109883/1840

M / 11609

(b) For the purposes of the present invention, cysteine hydrochloride (0.04% w / v), sodium formaldehyde sulfoxylate (0.03% w / v), or ascorbic acid (0.25 % w / v) are equally effective reducing agents such as sodium thioglycollate or thioglycolic acid. The latter two have the advantage that they can be treated with the medium in an autoclave. , * * <'.

(c) L-arginine, either as free base L-arginine or as, L-arginine monohydrochloride, is an essential specific nutrient for B. nodosus. It occurs in almost every nutrient broth, but only in an amount that is far below the optimum. Experiments have shown that the addition of precisely defined amounts of L-arginine to a nutrient broth, which is otherwise the optimum, increases the growth yield of bacteria by 10 to 100 times. COp, which is the gas phase in the culture vessels, is also an essential nutrient. Na ₀ CO _x is added to the medium as a buffer for the acidity caused by dissolved COp. Serine strongly stimulates the growth of B. nodosus. Certain strains of B. nodosus are also stimulated by lysine, it is included in the medium for general use.

(d) The growth of B. nodosus in the medium is through further addition of carbohydrates is obviously not encouraged. However, the final yield will be bacterial growth increased when the medium contains a small amount of soluble starch. The added strength also makes it easier recovering the bacterial cells from the culture Centrifuge..

(e) Although the above anaerobic culture method was developed to obtain batch cultures of B. nodosus, the same medium and conditions for growing continuous cultures by methods as essentially for anaerobic CO ^ -using bacteria from P.N. Hobson, J. Gen. Microbiol. 38, 161, 1965, P.N. Hobson and R. Summers,

- 6 - ... 109883/1 840

Μ / 11609 «Τ,

J. Gen. Microbiol. 47, 53, 1967 was used.

All B. nodosus strains used were from clinical Sheep foot rot cases in New Zealand. Isolated.

The term B.nodosus used for "description of the present invention is further defined in the index Bergeyana, an annotated, alpha betis go _t list 'of names of taxa of bacteria. Editors RE Buchanan, JG Holt, EF Lessell Jr., publishers E. & F. Livingstone Ltd. London, 1966. ι ^

ι cultures of microorganisms used according to the invention are deposited with the New Zealand National Reference Culture Collection, Animal Research Center, Wallaceville, and have the Received allocation numbers 30 000 and 3-0 001.

The key point of the present invention is that in breeding B. nodosus in a common Broth medium the addition of a suitable amount of L-arginine greatly increases the growth yield.

Commercially it is of great importance that a culture medium for B. nodosus be formulated from materials that are commercially available.

The vaccines are made either from bacterial cells or from soluble products of the cultures of one or more strains of B. nodosus after treatment with formalin or andecren bactericidal agents and adding a suitable adjuvant manufactured.

Hereinafter, a preferred embodiment of the present invention ₄ will be described.

Liquid medium is produced as follows: (Although special manufacturers for components of the medium indicated, this does not preclude the use of ingredients from other commercial sources.)

109883 / 18AO

M / 11609

1 liter of medium contains:

213365A

1. Trypticase (BBL, Division of Bioquest, Maryland, U.S.A.) 1.0 to 50.0 g, preferably 15.0 g.

2. LAB-LEMCO (Oxo Ltd., London, England) 1.0 g to 50 "0 g, preferably 5.0 g. ·

3. Proteose Peptone (Difco Laboratories, Detroit, U.S.A.) '

1.0 g to 50.0 g, preferably 5.0 g.

4. Yeast extract (Difco Laboratories, Detroit, U.S.A.), 0.1 g to 10.0 g, preferably 2.0 g.

5. sodium thioglycollate (B.D.H. Limited, Poole, England); 0.1 g to 5.0 g, preferably 1.0 g, alternatively, thioglycolic acid (97%) (B.D.H. Limited, Poole, England)

6. L-arginine monohydrochloride (B.D.H. Limited, Poole, England) (alternatively, L-arginine free base (B.D.H. Limited,

Poole, England)), 1 to 50 g, preferably 5.0 g. ·

7. DL-Serine (B.D.H. Limited, Poole, England) 0.1 g to 5 ° g, preferably 1.5 g.

8. L-lysine hydrochloride (B.D.H. Limited, Poole, England); 0.1 g to 5.0 g, preferably 1.0 g. »

9. Soluble starch (B.D.H. Limited, Poole, England),., 0.1 g to 20.0 g, preferably 0.5 g. <

The starch (point 9) is separated into about 200 ml of boiling dissolved in distilled water, filtered through Whatman # 3 paper and the filtrate added to the remainder of the medium.

10988 3/1840

t -

M / 11609 -J

The pH of the medium is adjusted to about 7.6 to 7.8 with 4 η NaOH, and it is made up with distilled water! 1 liter. i

Glass bottles with screw caps, which have perforated metal caps with rubber or Nebpren inserts, are suitable vessels for growing batch cultures. They are about half filled with medium and the closures are ·; loosely screwed on before treatment in the autoclave at 121 ° C, which lasts at least 15 minutes. When the autoclave has reached atmospheric pressure again, the bottles are removed while they are still hot and the closures are immediately tightened. The resulting partial vacuum is equalized to atmospheric pressure with 100% sterile, oxygen-free CO ₂ , C point 10), which is introduced through the bottle cap. 20% w / v filtered through a Seitz filter. Na-pCO ^ solution, (item 11), is also added to a final concentration of 0.2% w / v. added in the medium.

The batch medium is then ready to be inoculated.

To grow cultures in the continuous culture apparatus, large amounts of liquid medium are prepared and similarly sterilized in flasks. The medium-filled flasks are allowed to cool under a stream of sterile COp before the leads are connected to the device, such as. by PN Ilobson and R. Summers (J. Gen. Microbiol. 47, 53, 1967). Oxygen-free CO ₂ is also added to the fermentation vessel, either as 100% Cp ₂ or as a mixture of CO ₂ with nitrogen gas, depending on the particular growth rate of the microorganisms in the vessel.

109883/1840

BATH ORIGINAL

M / 11609 &

The inoculum is made as follows:

In an ampoule, the freeze-dried living cells of a or contains several authentic strains of Bacteroides nodosus, 0.05 ml sterile nutrient broth (Oxo Ltd., London, England) given. The resulting cell suspension is placed on plates of an agar medium of similar composition wj.e the 'of the above

th liquid medium, without ingredients 5, 7, 8, 9, 10 and 11, but with 15.0 g agar (Dav; Ls) per liter.

Plates made from sterile molten medium are not dried for more than 5 minutes at 37 ° C. After they are inoculated by slipping on, they are immediately placed in anaerobic containers (such as Gallenkamp anaeroiae culture containers, which are equipped with room temperature catalysts), which are successively evacuated and filled three times with a mixture of 90% H ₂ and 10 % CO _2. The cultures are incubated ^{at 37 °} C. for three to four days.

Test tubes which contain 10 ml of sterile liquid medium, which has been prepared as described above, but without Na ₂ CO ,, (point 11), and which have either been freshly prepared or previously stored under hydrogen, with a "loopful" growth of a 2 to 3 day old agar culture inoculated. The test tubes are then incubated for 24 to 72 hours at 37 ^° C. in anaerobic vessels under 90 % H ₂ and 10 % CO _2. The resulting `` log phase growth '' is used to inoculate bottles of the larger batch cultures by injecting 0.01 to 0.1 volumes with an aseptic hypodermic syringe through the closures.

In continuous culture containers, growth is initiated in the same way. All cultures are incubated at 37 ⁰ C.

- 10 -

10988 3/1840

Κ / 11609 ΛΊ

3.0 ml of a 24 to 48 hour old inoculum that is approximately 10 'contains bacterial cells and has been inoculated into a 300 ml batch of liquid medium as described above, yield a yield of 3 to 6 χ 10 or more cells per ml, in 18 to 48 hours, depending on the particular strain used.

Manufacture of the vaccine

• ι \

Cultures obtained according to the invention as described above are suitable for further processing into vaccines as follows:

A. Cell vaccine

The bacterial cells in the culture are killed by adding a suitable bactericidal agent such as 0.01 vol. Formalin. The culture fluid is centrifuged to isolate the cells, which are then resuspended in a phosphate-buffered saline solution, which has a pH of 6.8 and contains 0.5 % formalin or another suitable preservative, where a final concentration is obtained

VIl R

from 3 x 10 to 3 x 10 per ml, preferably from 3 x 10 yields up to 3 x 10 cells per ml. The cell suspension is then made with any suitable tool, such as Freud's incomplete adjuvant, by mixing 35 parts ^ the above selected cell suspension with 1 part of a lipophilic Emulsifying agent such as polyoxyethylene sorbitan monooleate and emulsifying the resulting aqueous phase with a oil phase, such as 9 parts of ARLACEL A (registered trademark No. 53371 in New Zealand) and 55 parts of Drageol F. combined. This is then the cell vaccine.

B. Supernatant vaccine

Supernatant from the centrifuged culture is used treated to carry the extracellular antigenic material through

- 11 109883/1840

BATH ORIGINAL

M / 11609

Methods such as dialysis, ultrafiltration or joint or Focus on coprecipitation. The resulting concentrate is incomplete in a suitable adjuvant such as Freud's Adjuvant, recoru & ituated, increasing a 2 to 100 fold Concentration of the original volume of the protruding. . Culture liquid results. This is then the supernatant vaccine.

C. Combined cell and supernatant vaccine

Aqueous, formalin-treated cell suspensions, &quot; which are like are prepared as described under A, are added to konfe centered supernatant liquid, which as under B and combined with a suitable adjuvant, such as Freud's Incomplete Adjuvant, so that they

" 7'11 result in a final cell concentration of 10 to 10 cells, preferably 10 to 10 cells per ml plus a 2 to 100 fold concentrate of supernatant fluid. This is the combined cell and supernatant vaccine.

• ι - ·

An effective dose, preferably in a volume of 1.0 ml of one of the vaccines obtained as above is injected intramuscularly or subcutaneously into sheep. The dose can, preferably can be repeated after 14 days, but also at any time after the first or sensitizing dose. The after-" The results show that sheep which have been treated in this way show a high level of immunity compared to those who have not been vaccinated Control animals when exposed to foot rot infection. In addition, the vaccine shows an important one therapeutic effect when injected into sheep already showing clinical signs of foot rot.

The following examples using the preferred method for making bacterial cells and products thereof are intended to further illustrate the invention, but not to limit it.

- 12 109883 / 18AO

M / 11609 &

Example 1 (test l)

Eleven sheep were vaccinated intramuscularly in a hind leg with the adjuvant cell vaccine prepared as described above. Each tier ¹
(Group 1).

Each animal was injected 1 ml with 1 × 10 bacteria

For comparison, another 10 sheep were treated with an adjuvant ' supernatant vaccine. Each animal was injected with 1 ml and the concentrated supernatants with 100 ml Culture contained (group 2).

For comparison, 9 sheep were also combined with an adjuvant Cell and protruding vaccine. Each Animal was injected with 1 ml of 1 × 10 bacteria plus concentrated supernatant from 100 ml of culture contained (group 3). '

For comparison, 10 sheep were vaccinated with no adjuvant or other vaccine. (Group 4)

In order to determine the effectiveness of the vaccine. These Sheep infected with viable B. nodosus cultures produced according to the preferred embodiment, exposed.

Λ t

5 weeks after exposure to infection, 9 out of 10 unvaccinated sheep (group 4) developed foot rot, compared to 2 out of 11 from group 1, one out of 10 from group 2 and 2 out of 9 from group 3. In the vaccinated groups was significantly less foot rot than in the unvaccinated group.

- 13 -

109883/1 840

M / 11609 / l {

Example 2 (test 2)

70 sheep were subcutaneously in the neck with adjuvant cell vaccine, prepared as described above. Each animal was injected with 1 ml, which was about 1 χ 10 Contained bacteria. 14 days later, each animal was again injected 1 ml with about 1 × 10 bacteria.

There were others for comparison. 70 sheep with the'vors going inoculated adjuvant cell vaccine described. Every animal 1 ml was injected only once, containing about 1 × 10 bacteria contained. This was done on the same day as the first vaccination of the twice vaccinated group.

In comparison, a further 70 sheep were not vaccinated with either an adjuvant or any other vaccine.

Thereafter, the group of 210 sheep was flocked into one flock and one 4 weeks after the first injection exposed to natural infection by adding 12 infected sheep.

The foot rot infection did not spread for several weeks after the animals were exposed to the infection. However, 10 weeks after the animals were exposed to infection, 43 out of 70 unvaccinated sheep had _% foot rot, compared to 12 out of 70 animals vaccinated once and 5 out of 70 animals vaccinated twice. There was significant prevention of infection in both vaccinated groups.

- 14 -

109883/1840

M / 11609

B ο is ρ ie 1 3 (Test 3)

Thirty-seven ewes with foot rot were subcutaneously in the neck with the adjuvant cell vaccine prepared as described above was vaccinated. Each animal was injected with 2 ml,

7 :

ao

which contained about 1 χ 10 bacteria. 7 days later each animal in turn injected 2 ml with about 1 × 10 bacteria. In comparison, other 20 sheep with foot rot were Adjuvant preparation without added bacteria injected on the same day, on which the first vaccination was carried out in the twice-vaccinated group.

Six weeks after the first injection, 33 of the 37 Ewes injected twice with the bacteria-containing vaccine no longer had foot rot compared with 3 out of 20 Ewes injected with the preparation without bacteria had been. The vaccine containing "bacteria" had one far greater numbers of animals cured than the preparation to which no bacteria were added.

These results show that 3rd nodosus vaccines prepared according to the preferred method described above provide important prophylactic protection against sheep foot rot. The vaccines have also been shown to have therapeutic effects when administered to animals manifestly afflicted with the disease. ^IV

- 15 109883/1840

Claims (17)

M / 11609 4Q claims 1. Process for the preparation of a vaccine against Bacteroides nodosus Beveridge (Mraz) infection in sheep and other ruminants is characterized by that one strain of said microorganism in a nutrient medium containing L-arginine or salts thereof, ^ anaerobic ' cultured, antigens which induce protection against Bacteroides nodosus infection are obtained from the culture medium and incorporating the protective antigens obtained into a vaccine by conventional methods. 2. Culture medium for Bacteroides nodosus, characterized in that it is a nutrient medium for anaerobic bacteria including a suitable source of growth-promoting Contains peptides for B. nodosus, an organic reducing agent, carbon dioxide and L-arginine or salts thereof. 3. Culture medium according to claim 2, characterized in that that the source of growth promoting peptides is an enzymatic digest of casein. 4. Culture medium according to claim 2 or 3, characterized in that it 'contains an aqueous nutrient broth which,. (a) an enzymatic digest of casein, (b) a meat extract and / or digest, (c) an organic reducing agent, (d) a yeast extract, (e) amino acids, - 16 109883/1840 M / 11609 JT, (f) L-arginine, (g) sodium carbonate, (h) soluble starch, (i) carbon dioxide, -j. I-Iulöurr .-. Eiiu; :: according to claim 4, characterized in that (a) the enzymatic digest of casein trypticase ("BBL, Division of Bioquest, Maryland, U.S.A.), (b) the meat extract LAB-LEMCO '(Oxo Ltd., London, England), (c) the organic reducing agent sodium thioglycollate, (B.D.H. Ltd., Poole, England), (d) yeast extract yeast extract (Difco Laboratories, Detroit, U.S.A.), (e) the amino acids DL-serine (B.D.H., Poole, England) and L-lysine hydrochloride (B.D.H. Ltd, Poole, England), (f) Sodium Carbonate Seitz-filtered 20% w / v. Na ₂ CO-Z solution, (g) soluble starch soluble starch (B.D.H. Limited, Poole, England), (h) the meat digest proteose peptone (Difco Laboratories, Detroit, U. SA), ^Λ (i) the CO ₂ sterile oxygen-free CO ₂ 6. Culture medium according to claim 2 to 5, characterized in that the L-arginine is derived from the free base of L-arginine (BDH Limited, Poole, England). - 17 - 109883 / 18AO BATH ORIGINAL M / 11609 IQ 7. Culture medium according to claim 2 to 5, characterized in that that the L-arginine from L-arginine monohydrochloride (B.D.H. Limited, Poole, England). 8. Culture medium according to claim 2 to 5, characterized in that that the L-arginine is derived from an L-arginine salt is. · 9 · Formulation of a culture medium for Bacteroides nodosus according to 'claims 2 to 8, characterized in that to 1 liter of water (a) 1.0 g to 50.0 g trypticase, (b) 1.0 g to 50.0 g Lab Lemco, (c) 1.0 g to 50.0 g proteose peptone, (d) 0.1 g to 10.0 g yeast extract, (e) 0.1 g to 5.0 g sodium thioglycollate, (f) 1 to 50 g L-arginine, (g) 0.1 g to 5 g DL-serine, (h) 0.1 g to 5 g L-lysine hydrochloride, (i) 0.1 g to 20 g soluble starch are given and (j) up to a final concentration in the medium of 0.2 % w / v. Na ₂ CO ^ -LOsU) Ig is added and (k) the surface of the solution is exposed to sterile, oxygen-free COp at atmospheric pressure. 10. Formulation of a culture medium for Bacteroides nodosus according to claim 9, characterized in that instead of sodium thioglycollate 0.1 to 5.0 ml of 97% thioglycolic acid be used. - 18 -.
109883 / 18A0 11. Formulation of a culture medium for Bacteroides nodocuG according to claim 9, characterized in that to 1 liter water (a) 15.0 g trypticase, (b) 5.0 g LAB-LEMCO, (c) £ 5.0 proteose peptone, (d) 2.0 g yeast extract, (e) 1.0 g sodium thioglycollate, (f) 1.5 ζ DL-serine, (g) 1.0 g L-Lysine Hydrο Chloride,
(h) 0.5 g soluble starch are given. 12. Formulation of a culture medium for Bacteroides nodosus according to claim 9, characterized in that to 1 liter of water is given 5.0 g of L-arginine free base. 13. Formulation of a culture medium for Bacteroides nodosus according to claim 9, characterized in that 5.0 g of L-arginine monohydrochloride are added to 1 liter of water. 14. Formulation of a culture medium for Bacteroides nodosus according to claim 9, characterized in that to 5.0 g of an L-arginine salt are added to 1 liter of water. 15. Formulation of a culture medium for Bacteroides nouosus according to claim 11, characterized in that the 1.0 g of r-sodium thioglycollate with 0.65 ml of 97% thioglycolic acid be replaced. - 19 - 109883/1840. BATH ORIGINAL M / 11609 16. Process for the preparation of a vaccine against Bacteroides nodosus infection in sheep or others Ruminant is effective, gekennzeiclinet that 'one strain of said microorganisms in a nutrient culture medium according to one of claims 2 to 13> the L-arginine or contains salts thereof, cultivated anaerobically, antigens obtained from the culture medium / which protect against Bacteroides induce nodosus infection and recover the protective "Incorporates antigens into a vaccine by conventional methods;" 17 - Vaccine, characterized in that it is produced by a * method according to claim 1 or sixteenth BATH ORIGINAL