DE2538994A1 - BACTERIAL VACCINE AND METHOD FOR MANUFACTURING IT - Google Patents

Description

Bacterial vaccine and process for its manufacture

The invention relates to "bacterial vaccines from Escherichia coli, which are effective against enteric coliform bacteria infection in animals, for example in piglets, protect, and a process for their production; it relates in particular to live vaccines, that are made from selected strains that are genetically modified (altered) to produce the desired, contain antigens transferred by a plasmid.

E. coli-derived vaccines (vaccines) have so far been produced by obtaining living cells, for example by washing them off a solid medium, for preparation a live bacterial vaccine, as well as by killing or permanent inactivation of bacterial cultures, for example with formaldehyde or with the application of heat. Even

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a live broth culture has already been used as a vaccine. Some of these vaccines couldn't be a good one Protection can be demonstrated and in others the potential pathogenic effects were unacceptably high.

In US patent application 256,977 there is partial formalinization (Treatment with formalin) of living E. coli cells to greatly reduce the titer (number) of viable cells and to "numb" the remaining viable cells Cells so that they can no longer grow and multiply for a few days. The vaccine you received exhibited a large part of the antigenicity (antigen content) of a live vaccine, without the one with one having such a vaccine associated infectivity and pathogenicity. It also became a polyvalent antigenicity proven. However, efforts were made to further reduce the infectivity and pathogenicity while maintaining a still high antigenicity or protective effect.

It has now been found that even better results in this direction can be achieved by using artificial Strains (artificially created strains) of E. coli that have been modified to give them the desired antigenic components (by plasmid transfer), select and give these live strains a partial formalinization treatment (Treatment with formalin). This treatment is so controlled that a considerable decrease in the number (des Titers) of viable cells (e.g., a decrease of 20 to 75% * usually about 25 to about 40%, based on) on the percentage of change in the exponential number), with the remaining living cells delaying one Have the ability to reproduce. It has been found that this vaccine has a consistent high protective effect and is polyvalent Has antigenicity and that the occurrence of an infection or pathogenic effects in the selected Strains is remarkably rare.

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The selection of the starting strains is important. To the goal of To achieve a further reduction in infectivity and pathogenicity, strains are selected that are practically non-pathogenic are or have low infectivity and pathogenicity and are capable of being recipients of transmissible Plasmids to act. However, these strains exhibit deficiency in certain antigens or antigenic components apparently exhibited a low level of the desired antigenicity. It has now recently become possible to use a plasmid (a small complete ring of DNA that can multiply within a bacterial cell) on the genetic makeup an E. coli cell. The plasmid should have the ability to add foreign antigens or antigen components encode (encipher) and incorporate them into the E. coli cell. As a rule, they are naturally occurring, unchanged Transfer plasmids. The details of this transfer of the plasmids have already been described in the literature (See e.g. Orskov and Orskov, "J. Bacteriol." £ 1, p. 69 to 75 (1966)).

In the following examples, the invention is described using pig and cattle strains, but it is by no means limited to that. Strains that have been modified with a plasmid include K12 and P235 »but can other strains can also be used in the same way. In which Strain K12 is a laboratory strain with a very low level of infectivity to domestic animals, i.e. it is practically non-pathogenic. It can also be mixtures of two or more strains are used, in particular to achieve an improved polyvalent antigenic effect, The vaccines can also be mixed for better protection against heterologous strains.

The plasmids to be transferred are selected so that they meet the following criteria: (1) They must be based on the

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desired host bacteria to be transmissible and (2) they should lead to increased immune sensitivity, the results in a better protective effect on the treated animal. The protective effect of a selected strain-transferred Plasmid combination cannot normally be predicted will. However, some antigens are known to impart properties to E. coli cells that enhance their potential to cause a disease, such as its ability to to stick to the wall of the small intestine, seems to increase. Antibodies raised against these antigens in vivo should be the adhesion of E. coli to the intestinal epithelial cells impede.

Enteric coli bacterial infection in piglets is characterized by the presence of large numbers of Escherichia coli cells in the anterior portion of the small intestine. With a few exceptions, only those E. coli cells which have the plasmid-controlled ability to synthesize enterotoxins are able to cause enteric disease. The majority, but not all, of the enteropathogenic compounds. coli contain the antigen K88. The synthesis of this antigen is controlled by the plasmid so that the ability to produce enterotoxin or the K88 antigen can be genetically transferred to non-pathogenic E. coli. The antigens K88 convey the ability to adhere to the mucosal membrane of the small intestine, thereby amplify the E. such Enteropathogenizitat coli bacteria (potentiate) which can form _t enterotoxin. It is already known that some enteropathogenic lamb and calf coli contain an antigen (KcO or K99) which apparently has similar biological properties.

The administration of such live bacteria which contain a plasmid-borne antigenic material, in particular by the oral route, is potentially dangerous as the E88-A.ntigen-

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plasmid could be transmitted in vivo to the non-pathogenic Making bacteria pathogenic. The administration of the organisms modified by the plasmid by intramuscular Paths is probably not that dangerous and the danger is increased by using the live formalization process considerably reduced.

Suitable for plasmid transfer to the selected strains among other things antigens of the type K88 (to which the two types K88 ac and K83 ab belong) and KcO (K99) and those produced in this way Artificial or modified strains are suitable for conversion into a vaccine using the partial formalinization treatment. Other antigens that meet these criteria can also be used. These three transferred antigens were identified in those described below Examples used.

The bacteria are grown in a broth culture or on a solid medium such as nutrient agar. In the formalinization treatment (Treatment with formalin) can live cells the bacteria can be recovered from the medium, for example by centrifugation or washing off the solid agar, or it the broth culture itself can be used. The cell concentration at the beginning of the treatment is expediently about

8 10

10 to 10 living (viable) organisms per ml, but this is not critical. Nutrients or extracellular by-products need not be present during treatment or in the vaccine. The concentration of formalin can be within the range of, about 0.02 to about 0.08 v / v. -% (0.008 to 0.032 wt .- / vol .-% formaldehyde), preferably about 0.03 to 0.06% formalin. The incubation time with formalin can be up to about 24 hours, about 10 to about 20 hours with 0.03 to 0.06% formalin being very suitable. The vaccine can be lyophilized, combined with adjuvants, and otherwise adapted for administration or handling.

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The following examples are intended to explain the invention in more detail. but without limiting it to ". The underlined part of the strain names is that transmitted by a plasmid Antigen component in the host organism. The transfer of the plasmid and the isolation of the modified host strains is carried out essentially as described by Orskov et al., supra, "It basically involves the incubation of the host strains (smaller proportion) with donor strains, which the desired Plasmid contain (larger proportion), applying the incubated mixture to a growth of the donor strain suppressive medium, isolating and multiplying the cells of the host strain which assimilates the desired plasmid Has. Except for the strains described below, ΚΊ2 was also the strain P255 modified by plasmid transfer, see above that it contained the plasmid K99 and / or an enterotoxin plasmid, these modified strains are also suitable for vaccine production.

example 1

Some of the strains were formalinized from 5 strains of E. coli Live vaccines made with three of the strains modified to be one transmitted by a plasmid Contained antigen. After 15 hours of incubation at 37 ° C in Tryptic Soy Broth was added to Pormalin down to 0.04 vol .- / vol .-% and after 15 hours of incubation the living bacteria were counted with the normalin. The results obtained are shown in Table I below specified.

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'Table I.

CD O CO OD

organism Bacteria "per ml - before the formalin addition
■■; .- '■ ^be - after 1.5 hours in
incubation at 37 ° ^ with
0.04 % formalin -_ formalinized alive
+ Adjuvant 1: 1
(Friend's incomplete) K12
K12 K88ab
K12 K88ac
EWl
K12 K99. ent 1.4 χ 10p
1.0 χ 10 ^a
1.4 χ 10?
8.0 χ 10
1.8 χ 10?
1.2 χ 10 *
1.4 χ 10? -
1.6 χ 10 *
1.2 χ 10?
8.0 χ 10 * 2.8 χ loZ
1.4 χ ΙΟ ⁷
1.0 χ loZ
8.0 χ 10 °
2.6 χ loZ
3.4 χ 10
2.0 χ Ιοί
2.4 x 10
2.6 χ loZ
4.4 χ 10 6.0 χ loS
3.0 χ 10
1.0 χ loZ
7.0 χ 10
4.0 χ io5
1.1 χ 10 '
1.2 χ loi
2.1 x 10
1.0 χ loZ
8.0 χ 10

It can be seen from this that the number of living (viable) bacteria decreased by 10 to 1 Cr organisms per ml after the 15 hour treatment. The addition of an equal volume of the adjuvant after formalinization resulted in a further smaller decrease in the number of bacteria (the bacterial titer). Details regarding the Freunds adjuvants are from EG Anderson, RC Masters and DU Mowat, "Res. Vet. Sei." Be taken in 1971, Volume 12, pages 342-350, and pages 351-357. The use of a Freunds adjuvant is not critical; other adjuvants can also be used.

It has been found to be appropriate to administer at least one dose of the vaccine containing an adjuvant, because the protective effect achieved in this way has usually been improved. As not all adjuvants were found to be very beneficial the adjuvant should be selected so that it gives an improved protective effect in the particular system.

Example 2

Live formalinized vaccines were produced from 4 bacterial samples based on the K12 strain. 3 samples of K12 had been modified by plasmid transfer to contain the three antigens K99, K88ac and K88ab .

The samples were grown in a broth culture and the

The culture concentration was adjusted to about 10 viable organisms per ml. Formalin was added to a concentration of about 0.04 vol / vol. -% and the mixture was incubated at 37 ° C for 15 hours. After incubation, the solution was ^{stored at -7O 0} G and used as a vaccine immediately after thawing.

23 sows were intramuscularly with the before piglets partially formalinized vaccines from the 4 strains of

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E. coli y / ie vaccinated below:

28th day before piglet: 2 ml + 2 ml Freund's incomplete adjuvant 14th day before piglet: 2 ml
7th day before piglet: 2 ml

Before ingesting the colostrum (the first milk), the piglets were

at an age of less than 5 hours

10
infected with 2 × 10 organisms of the virulent heterologous strain E. coli 014-9: K91 (B); K88ac (L): H10. The results obtained are given in Table II below.

Table II

Mortality rate in piglets from young sows which inoculated with plasmid-borne antigens in the live formalinized form containing E. coli.

for d ^ le vaccination
used trunk number
d.Mother
pigs Number of live pigs
- on days
0 2 7 21 17th
58
31 *
18th
33 *
21
15th
55 15th
63
31 *
18th
29 *
31
15th
55 15th
63
30 *
21
28 *
33
15th
55 K12
Mortality%
K12 K88ac 5
5
4th
4th
I. 40
38
42
33 Mortality%
K12 K88ab Mortality%
K12 K99 {KcO) ent
Mortality%

* Significantly different (p ~ 7 0.05) compared to the comparison values (K12) and compared to the K12 K99 group.

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It can be seen that there was a significant decrease in mortality with vaccines from the plasmid-borne K88 organisms. Since K99 is a bovine antigen, the decrease in the percentage of mortality of the piglets was less. In the fourth cross column of Table II, the word " ent " refers to the plasmid-borne component responsible for enterotoxin production. In some cases it may be useful to transfer the enterotoxin antigen by plasmid to host strains that do not have this antigen.

Example 3

Two cows were vaccinated with the partially formalinized strain K12 and two cows were vaccinated with the partially formalinized strain K12 K99 4 · »2 and 1 week before the expected calving.

Ί2 ^ ¹
vaccinates. The calves were with 10 - ^. coli organisms of the strain B44- subjected to infection. The two suckling and cow-raised calves vaccinated with K12 died of severe enteric ooli-bacterial infection, while the two suckling calves raised by cows vaccinated with K12 K99 did not become ill. The vaccine from the plasmid-borne antigen strain was effective against the heterologous calf organism (B44-). The vaccine was given intramuscularly and no adjuvant was present.

Example 4-

Vaccines were prepared from 6 strains by partial formation as indicated above. A mixture of the two strains K12 K88ac + EW1 was used in a test. The K12 K88ac strain was grown both in a treated broth culture and on agar by washing the cells with a phosphate-buffered saline solution and treating the resulting suspension.

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Young mothers were vaccinated with the vaccines obtained in this way: K12 K88ac , grown in a broth and on agar, K12 K88ab , K12 K88ac + 0 156 EV17 (ΕΪ71). For comparison, tests were also carried out with a killed K12 K88ac vaccine (bacterin) and with partially formalinized vaccines from K88-negative strains as follows: K12, K12 K99 ,. 0 157 KV17 (SV / 1) and 09 K35 · The vaccine was administered intramuscularly in three doses, the first dose containing an adjuvant as in Example 2. The piglets that were born were infected with at 5 hours or less and before ingestion of the colostrum

ΊΟ
2 χ 10 Organism K88ac: H10 E.coli.

10
2 χ 10 organisms of the heterologous strain P155 "from Ο14 · 9 * .Κ91:

Piglets from a second group of young mothers, who had been vaccinated with some of these vaccines

10 subjected to infection with 1 χ 10 E. coli, serotype 09 K35 · The results thereby obtained are in the following Tables III and IIIA given.

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- ic: -

Table III • Protection Test Results - P155 Infection

vaccine use
dete ouj
ge courage ~
terschwf bgg
rPerke
Ii- ^: living ". .. _N
Animals ( ^% mortality)
2 days 7 days 21 days 15 (63)
15 (55)
2 (96)
5 (74) 15 (63)
15 (55)
2 (96)
5 (74) K12
K12 K99
EWl
09 K35 no
4th
4th
2 40
33
47
19th 17 (57)
15 (55)
12 (74)
6 (68) 37 (73) 37 (73) Total amount of
K88 plasmid, -
free tribes 15th 139 50 (64) 31 (18)
29 (31)
32 (20)
27 (18) 30 (21)
28 (33)
31 (22)
25 (24) K12 K88ac 5
4th
5
4th 38
42
40
33 31 (18)
33 (21)
35 (12)
28 (15) 119 (22) 113 (26) Kl 2 K88ab 18th 153 127 (17) 20 (61) 18 (65) K12 K88ac + EWl 5 51 26 (49) K12 K88ac (grown on agar
tet) Total amount of
K88 plasmid ~
Stäiiuae. K12 K88ac killed with
0.4 % iOimalin

σ co αο

■ Table IHA Protection Test Results - 09 K55 Infection

vaccine reuse
; e boy
lutter -born
no
piglet 3- living
Animals (% mortality) after
² days 7 days 21 days 0 (100)
0 (100)
18 (10) 9 (100)
0 (100)
18 (10) K12
K12 K88ac * -EWl NJ N) N) 14th
22nd
20th 2 (86)
0 (100)
18 (10) 09 K35

Milk, colostrum and serum samples from all young dams vaccinated with K88-carrying antigens prevented in vitro the adhesion of E. coli to isolated epithelial cells. None of the young sows exhibited this inhibition factor in the serum samples before vaccination, but all had it in the serum after vaccination and / or in the colostrum after vaccination. Both the live formalinized and the killed formalinized antigens induced the Formation of these non-stick antibodies, but only those that are alive formalinized antigens gave significant protection (see Table III). The protection achieved by the live formalinized vaccine is therefore surprising and not explainable because if it were completely due to the non-stick antibodies, it formalinized to the point of being killed Vaccine should also provide protection, but it did not.

There was no antibacterial activity (bactericidal activity or reproduction inhibition) against the heterologous Infectious organism found in the colostrum of one of the vaccinated young mother pigs, but it became an antibacterial one Activity against the homologous vaccine organisms after vaccination established.

Summary £ BeispjLel_ fO__

1.) Live formalinized, K88 plasmid-transferred antigens resulted in good protection against strong infection (challenge) with heterologous E. coli FI55 carrying K88, but not with respect to the heterologous K88-ve E. coli 09 K35;

2.) Live formalinized, enteropathogenic K88-free vaccines showed good protection against the homologous vaccine strain of the organisms (09 K35), but they did not give any. · Protection against K88 infectious organisms;

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3.) K88 plasmid-transferred antigens killed with formalin gave no protection against heterologous K88-carrying E. coli;

4.) Organisms grown on agar were just as suitable for vaccine production as those grown in a Broth were grown, i.e. the components of the broth culture were not required for vaccine production.

The vaccines according to the invention can be administered to dairy cows / ground and impart the milk to these cows, as has been found immunity to newborn animals when administered orally. Fractions containing immunogenic or suitable antibodies this milk was also suitable for this purpose, such as cheese whey.

Example 5

Protection of weaned pigs against enteric coli bacterial infection with cheese whey which was produced by γοη vaccinated cows.

Six weeks before the presumed calving date, young cows were inoculated intramuscularly with 8 ml of a vaccine prepared by concentrating overnight broth cultures of K12 K88ab and K12 K88ac to 50 % and adding them together in equal amounts. The vaccine was added to an aluminum hydroxide gel adjuvant in the amount of 3 parts of vaccine to 1 part of adjuvant.

4 weeks after vaccination, the cows were given intramamma vaccination subjected at 2 ml per gland, the vaccine containing no adjuvant. This was repeated 1 week later, Blood samples were taken before the first vaccination and at the time of calving. Colostrum samples were taken when calving.

The milk was obtained and the milk collected between the 18th and the day after calving was used to make

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Cheeseiao Ike used. The whey was used in anti-hai "t and Protective tests used in biotic pigs.

5-day-old gnotobiotic pigs were infected through a gastric tube with 10 organisms P155 cLes strain serotype 014-9 of E. coli. They were then fed 4 daily with 30 ml twice concentrated (by reverse osmosis) cheese whey made from milk collected between the 18th and 35th day after calving from 88 vaccinated young cows. A control group was fed 2 concentrated (by reverse osmosis) whey from non-vaccinated animals. The rest of the feed was The survival time was measured. The following Table IV shows the results of the protection test: Based on previous tests, a 50% increase in survival time showed protection in conventional

1-day-old pigs sufficient to withstand

10 '

Infection with 10 enteropathogenic E. coli to be resistant.

Table IV

Survival time of gnotobiotic piglets (4 per isolator group) (Hours.)

, fed lined insulator K.88. -Whey normal whey 1 42 22nd 22nd 22nd 2 50 26th 93 26th 3 44 32 44 32; 4th 44 24! 93 44 i Average 54 28.5

Significant protection occurred in the group fed with K88 whey (p> 0.05).

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Table V below shows the results of non-stick tests when using serum colostrum and cheese whey from vaccinated and non-vaccinated cows.

Anti-adhesion antibodies were present in the whey and in the colostrum and serum after vaccination of the cattle vaccinated with K88. The fourth cross column in the "Vaccine" column in Table V below relates to a mixed vaccine, ie vaccine K12 K88ac + vaccine K12 K88ab.

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Table V

Non-stick

CD CD CO 00 ro cj-

C OC-

before the serum - after this Colostrum 1/10 4 χ concentrated 1/1 vaccine Vaccinate Vaccinate ■ ») emoll-re_ i) —— 1/5 1 / 2.5 not applicable t * '-
K12 *
K12 K88ac * * + K12 K88ab ** ' *

Non-stick, tested against E. coli carrying K88ac Non-stick, tested against E. coli carrying K88ab +) = no liability

I I

ro cn LO OO CD CD

Example 6

Ewes were given live formalinized vaccines

of the following types:

K12 comparator vaccine

K12 K99 produced from E. coli strain E12 to which the K99 plasmid was transferred,

B42 ß 'in E. coli, serotype 09. K35. K99, isolated from Calves and pathogenic to calves; it contained the antigen K99, which is known for the adhesion of E. coli on the intestinal mucous wall of calves is responsible

B44 is an E. coli of the serotype 09 · K30., Which is the Has lost E99 antigen.

(/ O 50 °) intramuscularly vaccinated ewes were 1 to 2 years old and had been thrown no lambs "You were once with '4 ml of a mixture of live formalxnisiertem vaccine (50%) and Ereundsincomplete adjuvant. 3 of the ewes in each group were given a second inoculation with 2 ml of the vaccine 25 days after the first vaccination. All ewes were inoculated with 2 ml of the vaccine 40 days after the first inoculation.

Lambs were born 1 to 3 months after the last vaccination and were given orally between 12 and 20 hours of age

11
5 x 10 E. coli of the strain B42 infected. The received

Results are given in Table VI below.

The first born lamb was in group K12 and became him 10 'administered to organisms without adverse effects,

Finally, the lambs were given 5 x ^ O organisms and the first lamb was deducted from all calculations. The three lambs in the K12 control group all received one severe diarrhea lasting 2 to 7 days. In appropriate

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Sometimes 4 of the 7 lambs in group B4-4 received severe diarrhea dated 2 "to 6 days. 2 of the 6 lambs in group B4-2 received mild diarrhea lasting 1 to 4 days and none of the 5 lambs in group K12 K99 showed any adverse clinical sign after infection with the BA-2 organisms.

There were no differences between the ewe lambs that received 2 or 5 doses of vaccine. The lambs were clinically examined for infection every 10 days. The observations were made without knowledge of the previous vaccination history of the respective ewes whose lamb was examined.

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Table VI

Responsiveness to infection of lambs from vaccinated maternal farms it 11

pp sheep with

_χ

11

_{tribal m2} (09.K35.K99)

CD O CD OO 1 "O CT

Vaccine vaccinated lambs days / diarrhea / · lambs / mortality in

Ewes born lambs diarrhea %

K12. K99

B. 42 (09. K35.K99)

B. 44 (09. K30) K12

4 *

5 3 **

0
1.0

2.1
4.7

0
2

4th
3

0 43 .0 I. 0 33 .0 ro
O 1 (3/7) (1/3)

* a ewe was not pregnant ** a lamb was stillborn ^

** one lamb was only infected with 10 'organisms

Ul CO OO CD CD

Claims (12)

1y Method for the production of a live bacteria vaccine from Escherichia coli, characterized in that one a) Bacterial strains with a low level of infectivity and Degree of pathogenicity generated that is so genetically modified v / ground that they have additional antigen by plasmid transfer containing forming plasmids, the antigens being selected so that they confer increased protection, b) the bacteria modified by a plasmid in suspension brings into contact with formalin and incubates the modified bacteria in the formalin until the number of living cells is decreased and the remaining living cells have a retarded ability to multiply, and c) the live formalinized bacterial vaccine with a low pathogenicity and infectivity and with a increased protective effect compared to the vaccines from the Host strains not modified with a plasmid wins. 2. The method according to claim 1, characterized in that the antigen transferred by a plasmid is of the type K88ab, K88ac, K99 or Enterctoxin used. 3. The method according to claim. 1, characterized in that one a K12 or P235 type was used as the bacterial strain . 4-, The method according to claim 1, characterized in that the Formalin concentration about 0.03 to about 0.06%, the number of Starting organisms per ml about 10 to about 10 and the incu- 609825/0844 r of) ίΚ7 REQUIRED '"" P 25 38 994.0 Munich, the Canadian Pat. Devel ... D / Hn - C2928 - 22 bation time be about 10 to about 20 hours. 5. The method according to claim 1, characterized in that one of a mixture of strains, of which at least one by a plasmid has been modified, a composite Manufactures vaccine. 6. The method according to claim 1, characterized in that one by combining separately formalinized stems from at least one of which has been modified by a plasmid, produces a composite vaccine. 7. Live formalinized bacterial vaccine, characterized in that that it contains at least one Escherichia coli strain or it consists of containing a plasmid-transferred antigenic material. 8. Vaccine according to claim 7, characterized in that the antigen transmitted by a plasmid is one of the type K88ab, K88ac, K99 or Enterotoxin. 9. Inoculation drink according to claim 7, characterized in that it the bacterial strain is of the type K12 or P235. 10. Vaccine according to claim 7, characterized in that it contains a selected adjuvant. 11. Milk composition or an antibody-containing fraction thereof, in particular for the treatment of newborns Animals exposed to E. coli infections, characterized in that that they made off with the E. coli vaccine of the claims 7 to 10 vaccinated cows have been obtained. 609825 / UÖ44 nachträp'l- ^ h changed T>) n / A protective antibody contains - i ¥ ^r action thereof during the wu ^ siiii ^ feeTi ^ protection or treatment period stäj 12. Kilch composition or containing an antibody Fraction thereof characterized in that it is obtained from cows vaccinated with the E. coli vaccine of claims 7 "to 10 has been won. 60982b / üd44