DD216954A1 - METHOD FOR VIRULENT WEAKNESSING IN BACTERIA OF THE GENUS PASTEURELLA, SPECIES P. MULTOCIDA - Google Patents

Abstract

The method for Virulenzabschwaechung in bacteria of the genus Pasteurella, P. multocida, pursues the goal of achieving a lasting Virulenzabschwaechung in these bacteria. This is achieved by repeatedly passaging the bacteria in a chemically defined iron deficient medium. The invention is of particular importance in vaccine production.

Description

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Method for virulence attenuation in bacteria of the genus Pasteurella, species P. muttocida Field of the invention

The invention relates to a method for virulence attenuation of bacteria of the genus Pasteurella, species Pasteurella multocida (P.multocida).

P. multocida plays an essential role as a causative agent of acute and chronic diseases of pets, especially in the complex of enzootic pneumonia of the calf and the pig. The production of an effective, well-tolerated vaccine contributes greatly to the health of the young stock and to the achievement of good breeding results.

Characteristic of the known technical solutions

The prerequisite for the production of live vaccines is the availability of avirulent bacteria to prevent or reduce harmful side effects in the vaccine application. Virulence attenuation of bacteria is possible through the use of chemical detergents or high-energy radiation types (eg UV light), which cause lasting damage to the bacteria.

As is known, it is possible to cultivate P. multocida in good yield both in complex and in chemically defined nutrient media {Tables 1 and 2).

As an essential micronutrient for microorganisms, iron is of particular importance.

It can be assumed that in the hitherto commonly used nutrient media there was always sufficient iron for the nutrition of the bacteria.

Object of the invention

The invention pursues the virulence attenuation in bacteria of the genus Pasteurella, species P. multocida.

Explanation of the essence of the invention

The invention has for its object to describe a method by which a Virulenzabschwächung in bacteria

of the genus Pasteurella, species P.multocida. ^:

Surprisingly, it was found that P. multocida loses its virulence on continuous passage in an iron-deficient medium and that P.multocida can also be cultured in a chemically defined iron deficiency medium of the following composition without restriction of multiplication and passability (Table 3). The passaging is carried out by inoculating 10VoI.% Of the bacterial culture in the next culture medium (tubes standing culture) and incubation at 37 ⁰ C. The investigated stem material is listed in Table 4.

Experimental Evidence of Virulence Attenuation Results from Mouse Exposure by Dose Determination, which is 50%

kills the mice (LD ₅₀ , reported in live germs per mouse) (Table 5). ^; , ^:

Comparative studies in ferrous nutrient media demonstrate the undiminished virulence (Table 6).

The stability of the virulence attenuation which occurred during the cultivation of the bacteria in the iron-deficient medium was tested in the originally highly virulent strain 383 and in the originally moderately virulent strain 35 by subsequent keeping with normal iron supply in chemically defined medium, by culturing on blood agar plates and by passage over the test animal mouse ,

The passaging takes place as already described for the liquid culture media in cultures on blood agricultural plates by direct inoculation of the seeds with an inoculation loop and incubation at 37 ⁰ C and in mice passages by injection of each 0.5 ml of bacteria culture ip and subsequent liver failure line on a blood agricultural plate.

After every 150 previous passages in the iron deficiency medium and subsequently 25 passages in chemically defined medium or on blood agar plates, P.multocida continued to be avirulent. After 150 previous passages in the iron-deficient medium and subsequently 25 passages through the mouse, the virulence slowly increased again, but without reaching the originally high virulence. Comparative studies in iron-containing, chemically defined nutrient media as well as in complex nutrient media demonstrate the undiminished virulence even after frequent passage

(Table 6). '

embodiment

Bacteria of the genus Pasteurella, species P. multocida. Strain 383, from the strain collection of the Institute for Bacterial Epidemiology Jena, was continuously cultivated in an iron-deficient medium (Table 3). The passaging was carried out by inoculating 0.5 ml of bacterial culture in 5 ml of culture medium as a tube culture and incubation at 37 ⁰ C for 18 to 24 hours. '

To determine the virulence of the bacterial suspension after 18 to 24 hours was diluted with culture medium so that dilutions in the range of 10 "^ ¹ to 10 ~ templates ^10. These dilutions were applied to groups of 10 white mice with a body weight of 20g intraperitoneally. The Deaths were counted within 10 days of injection, and the virulence (LD ₅₀ ) is calculated as the dose that kills 50% of the test animals or from the number of germs required to kill a mouse. f

Compared to bacteria of the genus Pasteurella, Species P. multocida. Strain 383, which was not passaged in the iron-deficient medium (LD _S0 value: 1 germ per mouse), showed a virulence attenuation after 150 passages in iron-deficient medium (LD 50 value:> 5 × 10 ⁵ germs per mouse).

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Table 1: Chemically defined nutrient medium for Pasteurelia "906", pH 7.8 to 8

substance g / IH ₂ O (dist.) Na ₂ HPO ₄ -12H ₂ O 32.3 KH ₂ PO ₄ 1.36 NaCl *. 1.19 MgSO ₄ -7H ₂ O 0.25 glucose 6 L-Arginine HCL 0.2 L-aspartic acid 1.6 L-Cysin 0.12 L-glutamic acid 0.15 DL-serine 0.2 (Or L-serine 0.1 L-isoleucine 0,065 L-leucine, 0,065 L-phenylalanine » 0.085 L-tyrosine 0.09 Ca Pantotheriat 0,002 nicotinamide 0.005 Vitamin B ₁ (thiamine · HCl) 0.001 Iron (III) citrate 0.001 orotic 0,015 Table 2: Partially defined medium "803" for P.multocida (g / 1) Na ₂ HPO ₄ -12H ₂ O. ; 32.3 KH ₂ PO ₄ . · 1.36 NaCl - 1,19 MgSO ₄ -7H ₂ O 0.25 Glucose 6 L-arginine-HCl 0.1 L-aspartic acid 1.6 L-cystine 0.06 L-glutamic acid 0,075 L-serine 0.05 (or DL-serine 0.1) orotic 0,015 nicotinamide 0.005 Ca pantothenate 0,002 Vitamin B, (thiamine · HCl) 0.001 Yeast extract * .5 H ₂ O ad 11, pH 7.8-8 *

mmol

* Supplement V _s (Dessau) or Difco yeast extract.

Table 3: Chemically defined nutrient medium for pasteurelia "902", pH 7.8 to 8 (ironmilk medium).

Substance <g / IH ₂ O (dist.)

90 10 20

1 30 1 12

0.5

1)

0.5

0.5

0.5

0.1

mmol

Na ₂ HPO ₄ -12H ₂ O

KH ₂ PO ₄

MgSO ₄ -7H ₂ O

glucose

L-arginine-HCl

L-aspartic acid

L-cystine:

L-glutamic acid

DL-serine

(Or L-serine

L-isoleucine

L-leucine

L-phenylalanine

L-Tyrosine

Ca pantothenate

nicotinamide

Vitamin B ₁ (thiamine HCI)

orotic

32.3 1.36 1.19 0.25 6

0.2 1.6

• 0.12 0.15 0.2 0.1

0.065 0.065 0.085 0.09 0.002 0.005 0.001 0.015 90 10 20

1 30 1 12 '

0.5

1)

0.5

0.5

0.5

0.5

0.1

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Table 4: Investigated stock material

Strain serotype.after

Namioka / CARTER

Host animal origin

Mäusevirulenz

35Ve: A

Buniall

Table 5: strain

Beef 5: A

6: B 2: B 2: D 6: E District Institute for 6-10 ⁶

Veterinary Dresden, GDR Chick National Institute of

Animal Health, Tokyo Pig Institute of bacterial

Animal disease research Jena, GDR cattle Institute for bacterial

Animal Disease Research Jena, GDR cattle Vet.-med. research Institute

»Pasteur" Bucharest, SRR Pig Institute of bacterial

Animal Disease Research Jena, GDR Bovine National Institute of

Animal Health, Tokyo

Dependence of virulence on the number of passages in iron deficient medium 902 (LD ₈₀ ) Passenger number

100

Buniall

Table 6:

tribe

95

1 32

6-10 ⁵ 13 52

LD ₅₀ after passage of P. multocida in some media with normal iron content

Passengen number 0

17 i '' > 1 10 " 2 10 ⁸ > 5- 10 ⁹ 10 > 1 10 ^e 3 > 1 10 ⁶ 9 10 ⁷ > 5 x 10 ⁹ 9 10 ⁵ > 1 · 10 ⁸ 1340 > 1 10 ⁸

901

803

803 + S *

VA 3 95 1 1 9 383 1 8th 2 40 D33 32 45 19 8th Buniall 2 13 2 2

100 803 803 906 40 41 16 28 15 25 25 70 28- 20 10 ⁵ 7

Culture medium 803 + S: medium 803 with addition of 10% serum. Table 7: Verification of virulence return

150 passages in

and 25 mouse passages 3-10 ⁶

3-10 ⁶

95 65

13 32

Culture conditions and 25 passages in 906 LD ₆₀ 383 35 Remarks 0 passages in 902 and 25 passages on blood plate 1 6-10 ^s highly virulent / moderately virulent 150 passages in 902 > 2 · 10 ⁸ avirulent / •) 'avirulent 150 passages in 902 > 3 · 10 ^β > 3 · 10 ⁸ avirulent / avirulent j 150 passages in 902 > 2 · 10 ⁸ ,> 1 · 10 ⁸ avirulent / avirulent

weakly virulent / weakly virulent

Claims (2)

-} - 248 576 claims for invention: 1. Method for reducing virulence in chemically defined media in bacteria of the genus Pasteurella, Species P. multocida, characterized in that the cultivation is carried out repeatedly in iron deficient media - is. · _: . 2. The method according to item 1, characterized in that 150 passages are made in iron deficient medium. For this 6 pages tables