DE2250315A1 - ORAL AGENT FOR KAELBER - Google Patents

Description

PATENT LAWYERS

DR. ING. A. VAN DER WERTH D «. FRANZ LEATHER

21 HAMBURG 90 8 MUNICH 80

WILSTORFER STR. 33 · TEL. (CMIII 770861 LUCILE-GRAHN.-STR. SS ■ TEL. COB I Il 47 29 47

Munich, October 3, 1972 Case 0.105

Unilever N.V, Museumpark 1, Rotterdam, Netherlands

Oral preparation for calves

The invention "relates to the rearing of calves and more particularly the creation of conditions to reduce their susceptibility to diseases of the intestinal tract and in particular to such Illnesses, which result from one that is contracted after the first few days of life Infection are to be lessened.

One such disease is "Salmonellosis", which results from the entry of one or more strains of Salmonella dublin and Salmonella typhimurium into the intestines of calves. Another, but usually less serious, intestinal disease that occurs in calves is the result of infection by pathogenic strains of Bacterium Escherichia coli, and in particular those strains which have the serotype designations 08, 09, 015 ₅ 078, 0114, OI37 and 0139 (International Serotype Classification)

own. 3098 16/1176

DKUTICHE SANK AC. HARBURG 93/20 »13 POST CHECK. HAMBURG 117330 TELEORAtMMEi LEATHER PATENT MÖNCHEN

When the environment or the feed of calves is changed. for example during their transport, inclusion in the Stall or when taking away from the mother, the associated stressful situations cause the calves to face Multiplication of pathogenic organisms are less resilient, and there is a high probability that any Salmonella or E. coli pathogens that have been ingested with food, severe diarrhea which can be very serious in S. dublin and S. typhinurium. In calves bought for rearing and which usually become infected about one to three weeks after purchase, the death rates from infection can be reduced with Salmonella strains are $ 0% or more.

The object of the invention is to provide a means against this.

It has now surprisingly been found that the intestines of calves are used to generate antibodies for pathogenic organisms like the above mentioned strains of Salmonella and E. coli can be stimulated by the endotoxins of the organisms introduced into the intestine in a substantially free form from living organisms, d. H. different from that Introduced into the bloodstream, such as by injection.

The object according to the invention is achieved by an oral gown for rearing calves which is characterized in that it contains the endotoxins of a pathogenic bacterium which has an infectious effect on the intestinal tract of calves.

Kit labeled "Endotoxins" for materials to be introduced into the intestines of calves is not intended to imply the presence or absence of exotoxins in these materials

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Presence of the cellular material in which the endotoxins are entrapped in the living bacterium is excluded This should only be understood to mean that endotoxins see in the achievement of the desired immunological Effect have the main meaning, while exotoxins and Residues of the cells do not have this desired immunological effect. ”However, it can be more convenient if necessary be either exotoxins or cell debris or both with to have the endotoxins present together, initially to save the work of separating them and then also, to take advantage of the antigenic effects they may have. .

The introduction to the intestine, i.e. H. the application, the endotoxins from only one of the aforementioned strains of Salmonella or only from one of the aforementioned strains E. coli has a certain beneficial effect in that the maturity of this tribe to cause harm is diminished will. However, it is preferred to use the endotoxins of both S. dublin and S. typhimurium and the endotoxins of all To apply strains of E. coli. The endotoxins to be applied can be made by growing any of these strains, wherein a sample of each strain from the Central Veterinary Laboratory, Ministry of Agriculture and Fisheries, New Haw, Weybridge, Surrey, United Kingdom or from the National Collection of Type Cultures, and as the growth of the Microorganism and thus the production of endotoxins up to a suitable extent by killing the multiplying Microorganisms and release of the endotoxins are obtained. The latter can be done by boiling or treating in an autoclave can be achieved. The entire, sterilized cultures, of which "every exotoxins and the cell debris as well as the endotoxins

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can then be combined and applied.

Alternatively, however, instead of sterilizing the The bacteria are separated from whole cultures, usually by centrifugation, and in a small volume of one aqueous medium, e.g. B. in water or a saline solution, to kill the bacteria and to release the endotoxins be treated.

The simplest and most economical way to kill the separated bacteria is by heating. If the heating lasts long enough, e.g. B. one hour at 100 ° C., 20 minutes at 125 ^° C., a large part of the endotoxin is released from its connection with the cell walls of the killed bacteria and released into the medium in which the heating is carried out. The remaining, bound endotoxins can optionally be brought into solution by treatment with an enzyme such as trypsin, as described e.g. B. in the Dutch patent 7 107 558 is described.

In practice, the endotoxins are administered to the calves orally; H. over the mouth, either just by compulsory input with the sterilized culture material or via the drinking water or in solid or liquid form mixed with one or more of the vital components of the calves' feed, d. H. Protein, fat, carbohydrate, Vitamins and mineral elements, e.g. B. Calcium and Phosphorus. In this way, the endotoxins for the application with skimmed milk powder, cereals or mineral elements be mixed, or they can be added to a complete feed, i.e. H. a feed which

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contains all of the vital components of food such as protein, fat, etc.

The invention is in increasing resilience of particular importance in relation to infection of calves if they are removed from the mother and as a result are particularly exposed to stressful situations. By starting entering the endotoxin material at an early stage Age, suitably from four to ten days, and preferably at the last time the dam was fed shortly thereafter and by continuation for a reasonable period of time, advantageously up to the age of four up to six weeks, the intestines of the calves can be stimulated to generate suitable antibodies so that at the time at which the calf is exposed to a high risk of infection, a sufficiently high circulation of antibodies is present in the intestine, either to become more pathogenic with the multiplication of any ingested food To cope with bacteria, or at least the severity of any disease condition that is still developing. to discontinue. For this purpose, the endotoxin material can be added to a so-called "milk substitute", i. H.. in the diet, which is specially formulated to meet the nutritional needs of calves, if they are taken away from the mother. It can also be added to the first dry food that is placed in Combination with milk or a milk substitute is given when weaning. It is beneficial to have the endotoxins in the form of a dry, solid premix with one or more of the food ingredients protein, fat, or carbohydrate to make mineral elements available, whereby the consumer this premix with the remaining ingredients of the feed mixed.

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If necessary, endotoxins of the Salmonella strains can be combined with feed containing those of the strains of E. coli getting produced.

The minimum dose rates are in the range of 1 to 10 Einhei th of the endotoxins of each bacterial type per calf per Day, referring to the following examples for the measurement of a unit. However, it was found that calves given 1000 times this amount showed no signs of disease. A very suitable one The value for an entry in a feed for calves is 10 to 1000 units of each endotoxins Serotype per kg of feed.

The invention is explained in more detail below with the aid of examples:

Example 1 .

Endotoxins can be produced from the Salmonella strains and the seven serotypes of E. coli by the following method, which is generally carried out using the method of Westphal, Luderitz and Bister, Z. Naturforsch. 2. 0952), p. 148.

A freeze-dried culture of the microorganism is swollen in peptone water and incubated for 6 hours bei'37 ^'0 C. After a suitable growth, the culture is examined for its integrity on a washed sheep blood agar plate and then used for inoculating oblique nutrient agar (Oxoid) in Roux bottles. The culture is allowed to ^{grow overnight at 37 °} C. and the bacteria are collected in sterile, distilled water. The bacterial suspension obtained is aseptically placed in 30 ml universal bottles and at 4000 rpm for 10 minutes

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centrifuged. The supernatant liquid is removed and the remaining bacterial pellet is placed in 4 ml of sterile, distilled water resuspended and then freeze-dried. ·.

To isolate the endotoxins, 0.5 S of the freeze-dried material is suspended in 5 ml of 0.15M NaCl, and 10 ml of 90% aqueous phenol are added as a lysing gown. The mixture is heated to 68 ⁰ JO minutes C.unter lasting Hühren, then it is centrifuged at 4000 rpm for compressing the cell residues. The aqueous phase is removed and cooled ^{to 4 ° C. 1} , 10 volumes of ice-cold ethanol are slowly added to this. The precipitate formed in this way is redissolved in water and the nucleic acids are precipitated and removed from the solution by adding 2 volumes of ethanol. The endotoxins are precipitated from the remaining solution by adding a further 8 volumes of ethanol. They are separated by centrifugation, washed with ice-cold ethanol and redissolved in 0.15M NaCl. This solution of the endotoxins is referred to as reagent 1 in the following.

Example 2

a) Production of antiserum

Specific, hyperimmune sera (antisera) are generated in white New Zealand rabbits against washed, heat-killed (100 ^° C., 2.5 hours) organisms of each of the serotypes of E. coli and Salmonella.

Suspensions of the heat-killed organisms are prepared in 0.15M NaCl (about 3 χ 10 "organisms / ml), and

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injected intravenously. The immunization program begins with an injection volume of 0.1 ml and is applied to each continued on the fourth day, doubling the volume to 1.6 ml. From the rabbits will be ten days after completion of the program, the blood is removed and this blood is centrifuged. The upper layer of the hyperimmune serum is collected. This antiserum is called reagent 2.

b) Kessung the antibody (anti-endotoxin) activity in the antiserum

Erythrocytes from sheep are sensitized by treating a 5% suspension of washed, packed cells with an equal volume of the endotoxin solution reagent 1 at 37 ^° C. for 30 minutes. The sensitized cells are separated by centrifugation, washed free of excess endotoxins and resuspended in 0.15M NaCl to form a 2.5% suspension of endotoxin-sensitized sheep erythrocytes. This suspension is referred to as reagent 3.

The reagent 2, i.e. the antiserum, is successively diluted with 0.15 ^ NaCl to produce a series of solutions of equal volume "(1 vol.) with an antibody concentration of 1/51, 1/10, 1/20, 1/40 ... 1 / (5 x 2 ⁿ " ¹ ) that of reagent, and 1/5 volume of reagent 3 is added to each of these solutions. Hemagglutination occurs in the stronger antiserum solutions, but not in the weaker solutions, and the endpoint of the ^{titration carried out at A- 0} C is considered to be the solution in which the hemagglutination just occurs. In a typical procedure, the endpoint is at the twelfth solution, that is, the solution which has an antibody concentration of 1 / (5 χ 2 ^ ¹ ) that of reagent 2.

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11 Therefore, reagent 2 can have an antiserum titer of 5 x 2 (· ■ 10.240) can be attributed.

c) Measurement of the endotoxin concentration in solution of unknown concentration

The solution to be examined (Y) is successively diluted with 0.15M NaGl to produce a series of solutions of equal volume (3 vol.) With endotoxin concentrations 1/3, 1/6, 1/12, 1/24 etc. that of solution Y to obtain. 1 vol. Of reagent 2 (antiserum), which has been diluted so that it has a titer of 20 (see above under b)), and then after a few minutes 1 vol. Of reagent 3 (sensitized sheep erythrocytes) are added to each of these solutions. are added, ultimately endotoxin concentrations of 1/5 »i / 10, 1/20 1 / (5 x 2"") of those of solution Y are obtained. Hemagglutination is inhibited in the stronger endotoxin solutions, but occurs in the weaker ones, and the end point of the titration is assumed for the solution in which hemagglutination is just prevented. In a typical procedure, this was done in the sixth solution with an endotoxin concentration »1 / (5 x 2 ^) that of solution Y. This means that solution Y has a titer of 5 x 2 C ^a 160), which is 160 units Endotoxin / ml is equivalent, see example 3)

Example 3

A. Manufacture of raw endotoxin material

The following procedure was separated for each of the Salmonella strains and "each of the serotypes of E. coli mentioned above.

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(i) The bacterium was streaked from a deposited stock culture on washed blood agar plates in the case of E. coli and on nutrient agar plates in the case of Salmonella and ^{incubated for 24 hours at 37 °} C. The plates were then examined in a suitable manner for the purity of the strain.

(ii) Colonies of the bacterium were removed from the plates in 50 ml Oxoid nutrient broth No. 2 (Catalog No. CM 67) transferred.

The broth or nutrient liquid was ^{kept at 37 °} C. for 24 hours.

(iii) The whole culture obtained in (ii) was used to inoculate 1.5 mL of Oxoid Nutrient Broth No. 2, and the nutrient liquid was incubated with shaking for 24 hours at ⁰ C $ 7. Each final culture obtained in this way contained 10 ^ - 1 0 ^Λ0 viable bacteria / ml. Samples of the cultures of E. coli were, if they had been treated for 2 hours at 125 ⁰ Q with steam and the examination method of Example 2) were subjected to a titer of 2560, which corresponds to 2560 units of endotoxin per mL of the steam-treated culture. Samples of the cultures of S. dublin and S. typhimurium were steamed and examined in the same way as the cultures of E. coli and gave a titer of 1280. Each of the new cultures, ie 7 types of E. coli and each one of the Salmonella bacteria Serotypes were centrifuged to separate the bacteria, which were then partially resuspended in the separated supernatant mother liquor to obtain a suspension having a concentration 30 times that of the culture before centrifugation. Each suspension was separated for 2 hours

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Treated in an autoclave at 125 ⁰ C to kill the bacteria with steam, then the new suspensions treated with steam were combined.

B. Manufacture of the oral composition for calves

The combined, steamed material obtained under A. (1 part by weight) was mixed with 19 parts of whey powder mixed, and the resulting mixture was placed in a common milk substitute of the following composition:

Wt%

Skimmed milk powder with added 77 fat 17.1 Whey powder 5 Glucose 0.5 precipitated lime 0.4 Dicalcium phosphate. "

Trace minerals and vitamins

on carrier 0.2

In the product obtained, the concentration of endotoxins was:

Qedem serotype endotoxins

of E. coli ■ 100 units / kg des

Feed

Endotoxins of feathers serotype ■ 50 units / kg des of Salmonella feed

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Claims (9)

Patent claims. fly Oral agent for calves, characterized in that that it contains the endotoxins of a pathogenic bacterium which has an infectious effect on the intestinal tract of calves. 2. Oral agent according to claim 1, characterized ge k ennzeich net that it contains the endotoxins of Salmonella dublin. 3 · Oral agent according to claim 1, characterized net that it is the endotoxins of Salmonella typhimurium contains. 4. Oral agent according to claim 1, characterized in that it is marked net that it is the endotoxins of Salmonella dublin and of Contains Salmonella typhimurium. 5. Oral agent according to claim 1, characterized in that it is e k e η η ζ e i c h net that it is the endotoxins of one or more of the Serotypes 08, 09, 015, 078, 0114, 0137 and / or 0139 of Contains E. coli. 6. Oral agent according to claim 5 »marked thereby net that there are the endotoxins of all seven serotypes of Contains E. coli. 7 * Oral agent according to claim 1, characterized ■ n e t that it is the endotoxins of Salmonella dublin, of Salmonella typhimurium and of one or more of the above contains serotypes of E. coli mentioned. 309816/1176 8. Oral agent nach.- one of claims i-bls 7 in porin of a feed for calves, thereby drawing g.e k e η η, that it is the endotoxins of each serotype in an amount of 10 to 1ÖÖ0 units per kg of Feed contains 9. Oral agent according to one of claims 1 to 8, characterized characterized in that it-additionally protein, Contains fat, carbohydrate, vitamins and mineral elements. ' ■ - ■ '\' ■ '"■ 309B1S / 1176- _orae , _{NAL 1NSPECTED}