DE2250315C2 - Use of a calf feed - Google Patents

Description

The invention relates to the use of a feed for calves, whereby in particular the susceptibility of calves against diseases of the intestinal tract and especially against such diseases, which are the result of an infection after the first few days of life is reduced.

One such disease is "Salmonellosis", which is caused by the entry of one or more bacterial strains of Salmonella dublin and Salmonella typhimurium originate in the intestines of calves. One more, however, usually less serious bowel disease that occurs in calves is the result of a Infection by pathogenic strains of Bacterium Escherichia coli, and in particular those strains which the serotype designations 08, 09, 015, 078, 0114, 0137 and 0139 (International Serotype Classification) own.

If the environment or the feed of calves is changed, for example during their transport, the Taking them into the barn or taking them away from the mother cause the associated stressful situations, that the calves are less resistant to the reproduction of pathogenic organisms and there is a high possibility that some Salmonella or E. Coli, which were ingested with food, severe diarrhea, which in S. dublin and S. typhinurium can be very serious cause. For calves that have been bought for rearing and which ones Usually infected about one to three weeks after purchase, death rates from infection can be with Salmonella strains are 30% or more.

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

The invention therefore relates to the use of a feed for calves which, in addition to the usual feed ingredients a from inactivated cells of one or more cultured pathogenic bacterial types, which are involved in the triggering of diseases of the intestinal tract of calves contains.

Are calf feeds containing antibody material derived from inactivated enteropathogenic bacteria known US-PS 36 51 214 relates to the production of oral vaccines from bacteria that are responsible for gastroenteritis are responsible in calves. This well-known vacein is made from killed bacteria, and it does can be mixed with feed. Although this kills the bacteria and removes endotoxins Bacteria are released, such released endotoxins are discarded according to the known procedure.

The present invention is based on the finding that endotoxins, which from inactivated (killed) Cells of certain strains of enteropa'.hogen bacteria calves can be given orally via their feed to keep them effective to immunize against gastrointestinal diseases. The invention thus offers a possibility of Reinforcement of the natural resistance of calves to these endemic diseases, thereby the meat and miichproduktionsleistur.g under modern Intensive housing conditions are increased. Although older calves also benefit significantly the invention, it is of particular importance for the very young calf. In the first days of life after setting, the young calf acquires passive immunity to infection by E. coli and others gastro-enteric bacteria, as it absorbs the respective antibodies contained in the cow's colostrum

The use according to the invention of a calf feed in addition to the usual feed ingredients one of inactivated cells of one or more cultured pathogenic bacteria types that are involved in triggering are involved in diseases of the intestinal tract of calves, contains antigenic material, to reduce the susceptibility of the calves to diseases of the intestinal tract, is characterized by that endotoxins obtained by culturing are used as the antigen material of pathogenic bacteria that have an infectious effect on the intestinal tract of calves and kill the bred ones Bacterial cultures releasing the endotoxins and that the calf feed in such an amount for It is provided that at least 1 to 10 hemagglutination inhibitory units of the endotoxins of each bacterial type are ingested per calf per day.

In the older patent P 21 '25 641 7 a pig feed similar composition claimed.

With the designation »endotoxins« for the materials to be introduced into the intestines of calves it is not said that in these materials the presence of endotoxins or the presence of the Cell material in which the endotoxins are entrapped in the living bacterium must be excluded target. This is only to be understood as meaning that endotoxins are used to achieve the desired immulological effect the main importance, while endotoxins and residues of the cells have this desired immunological Do not have any effect. However, it may be more convenient to use either endotoxins or Having cell debris or both with the endotoxins present first of all to get the job done to save their separation 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 previously mentioned strains of E. coli has to this extent a some beneficial effect as the ability of this tribe to cause harm is diminished. However it is preferred to remove the endotoxins from both S. dublin as well as from S. typhimurium and the endotoxins of all strains of E. coli. The ones to be applied Endotoxins can be obtained by growing each of these strains, taking a sample of each strain from Central Veterinary Laboratory, Ministry of Agriculture and Fisheries, New Haw, Weybridge, Surrey UK or from the National Collection of Type Cultures, and as you progress the growth of the microorganism and thus the production of endotoxins up to a suitable one Extent obtained by killing the multiplying microorganisms and releasing the endotoxins will. The latter can be achieved by boiling or treatment in an autoclave. The whole, sterilized cultures, each of which contains exotoxins and cell residues as well as endotoxins, can then be combined and applied.

Alternatively, however, instead of sterilizing the entire cultures, the bacteria can also be separated off, usually by centrifugation, and in a small volume of aqueous medium, e.g. B. in water or a saline solution to kill the bacteria and release the endotoxins.

The easiest 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 71 07 538 is described.

In practice, the endotoxins are applied with the usual feed ingredients, e.g. B. Protein, Fat, carbohydrate, vitamins and mineral elements, e.g. B. Calcium and Phosphorus. In this way can use the endotoxins for 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 contains all the vital components of food such as protein, fat, etc.

The invention is particularly useful in increasing the resistance to infection in calves Meaning if this has been taken away from the mother and as a result especially stressful situations are exposed. By starting the input of the endotoxin material at an early age, suitably with four to ten days, and preferably at the last feeding time with the mother or shortly thereafter and by continuation for a reasonable period of time, advantageously until an age from four to six weeks, the intestines of the calves can be stimulated to generate suitable antibodies, so that at the time when the calf is exposed to a severe risk of infection, a sufficiently high risk Circulation of antibodies in the intestine is present either with the multiplication of any coping with ingested pathogenic bacteria, or at least the severity of any Disease state that develops nonetheless. The endotoxin material can too for this purpose they can be added to a so-called "milk substitute", d. H. in the food, which special is formulated to meet the nutritional needs of calves when they are of the Mother to be taken away. It can also be put into the first, dry food, which

ίο in conjunction with milk or a milk substitute 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, carbohydrate or mineral Elements to be made available, whereby the consumer this premix with the remaining ingredients of the feed mixed.

If necessary, endotoxins of the Salmonella strains can be used along with those of the strains of E.

coli containing feed.

The minimum dose rates are in the range of 1 to 10 units of the endotoxins of each bacterial type per calf per day, reference being made to the following examples for the measurement of a unit. It However, it was found that calves to which 100 times Amount of this who '- ^ s was given, no symptoms of disease showed. A very suitable value for input into a feed for calves is 10 to 1000 units of the endotoxins of each serotype per kg of feed.

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

example 1
Production and isolation of endotoxin

Endotoxins can be derived from the Salmonella strains and the seven serotypes of E. coli according to the following Method are produced, which in general with the method of Westphal, Luderitz and Bister, Z. Naturforsch. 7 (1952), p. 148.

A freeze-dried culture of the microorganism is swollen in peptone water and kept at 37 ° C. for 6 hours incubated. After appropriate growth, the culture is checked for purity on a washed sheep blood agar plate and then used to inoculate slanting agar (Oxoid) in Roux bottles. The culture is grown 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 centrifuged at 4000 rpm for 10 minutes. The protruding Fluid is removed and the remaining bacterial pellet is placed in 4 ml of sterile, distilled Resuspended water and then freeze dried.

To isolate the endotoxins, 0.5 g of the freeze-dried Suspended material in 5 ml of 0.15 M NaCl, and there are 10 ml of 90% aqueous phenol added as a lysing agent. The mixture is heated to 68 ° C for 30 minutes while stirring continuously, it is then centrifuged at 4000 rpm to compact the cell debris. The aqueous phase is removed and cooled to 4 ° C., 10 volumes of ice-cold ethanol are slowly added. The precipitate thus formed is redissolved in water and the nucleic acids are removed from the solution

precipitated and removed by adding 2 volumes of ethanol. The endotoxins are made from the residual solution precipitated by adding a further 8 volumes of ethanol. They are separated by centrifugation, with Washed with ice-cold ethanol and redissolved in 0.15 M NaCl. This solution of the endotoxins is in the hereinafter referred to as reagent 1.

Example 2

Investigation of endotoxins
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 dissolved in 0.15 M NaC! (about 3 χ 10 ^Λ organisms / ml) and injected intravenously. The immunization program begins with an injection volume of 0.1 ml and continues to work every fourth day, doubling the volume to 1.6 ml. The blood is taken from the rabbits ten days after the end of the program and this blood is centrifuged. The top layer of the hyperimmune serum is collected. This antiserum is called reagent 2.

b) Measurement of antibodies (anti-endotoxins)
Activity in the antiserum

solutions of the same volume (3 vol.) with the endoioxin concentrations 1/3, 1/6, 1/12, 1/24 etc. of those of solution Y to be obtained. 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 volume of reagent 3 (sensitized sheep erythrocytes) are added to each of these solutions , with endotoxin concentrations of 1 / 5.1 / 10, 1/20 ... 1 / (5 χ 2 "- ^! ) of those obtained in solution Y. 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 barely prevented. In a typical procedure, this was done with the sixth solution with an endotoxin concentration = 1 / (5 χ 2 ^s ) that of solution Y. From this it turns out that the solution Y has a titer of 5 χ 2 ⁵ (= 160), which is equivalent to 160 units of endotoxin / ml, see Example 3).

Sheep erythrocytes 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 off by centrifugation, washed free of excess endotoxins and resuspended in 0.15MNaCl to form a 2.5% suspension of endotoxin-sensitized sheep erythrocytes. This suspension is referred to as reagent 3.

The reagent 2, ie the antlerum, is successively diluted with 0.15 M NaCl to make a series of solutions of the same volume (1 vol.) With an antibody concentration of 1/5. 1/10, 1/20, 1/40 ... 1 / (5 χ 2 "- ') that of reagent 2, and to each of these solutions 1/5 volume of reagent 3 is added. In the stronger In antiserum solutions, hemagglutination occurs, but not in the weaker solutions, and the endpoint of the ^{titration carried out at 4 13} C is considered to be the solution in which hemagglutination just occurs the solution, which has an antibody concentration of 1/5 χ 2 ") that of reagent 2.

Therefore, reagent 2 can be assigned an antiserum titer of 5 χ 2 "(-10,240).

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

The zuuntersuchu.de solution (Y) is diluted one after the other with 0.15 M NaCl to produce a series of Lö-Example 3
A. Production of raw endotoxin material

The following procedure was carried out separately for each of the Salmonella strains and each of the Seroiypes of E. coli mentioned above.

(i) The bacterium was grown from a deposited stock culture on washed blood agar plates im Case of E. coli and streaked on nutrient agar plates in the case of Salmonella and 24 hours at Incubated at 37 ° C. The plates were then appropriately examined for strain purity.

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

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

The entire culture obtained under (ii) was used to inoculate 1.5 ml of Oxoid nutrient broth No. 2, and the nutrient liquid was incubated with shaking at 37 ° C. for 24 hours. Each final culture obtained in this manner contained 10 ⁹ - 10 ¹⁰ viable 8akterien / ml. Samples of the cultures of E. coli, when they had been treated with steam for 2 hours at 125 ° C. and subjected to the test method of Example 2), gave a titer of 2560, which corresponds to 2560 units of endotoxin per ml of the steamed 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; π a titer of 1280. Each of the new cultures, ie 7 species of E. coli and one of each Salmonella serotypes were centrifuged to separate the bacteria, which were then partially resuspended in the separated supernatant fluid to obtain a suspension having a concentration 30 times that of the culture before centrifugation. Each suspension was separately steamed for 2 hours in an autoclave at 125 ° C to kill the bacteria, then the new steamed suspensions were combined.

7th
B. Manufacture of the oral composition for calves

The obtained under A., combined, steamed material (1 part by weight) was 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 77 added fat 17.1 Whey powder 5 Glucose 0.3 precipitated lime 0.4 Dicalcium phosphate .Track minerals and 0.2 Vitamins on carriers

In the product obtained, the concentration was of endotoxins:

Endotoxins from everyone

E. coli serotype 100 units / kg

of the feed

Endotoxins from everyone

Salmonella serotype 50 units / kg

of the feed

30th

40

50

Claims (1)

Claim: Use of a calf feed that is inactivated in addition to the usual feed ingredients Cells of one or more cultured pathogenic bacterial types that are involved in the triggering of Diseases of the intestinal tract of calves are involved, contains antigenic material derived from Reduction of the calves' susceptibility to diseases of the intestinal tract, characterized in that that endotoxins obtained by culturing pathogens on the intestinal tract are used as antigenic material of calves infectious bacteria and killing of the cultivated bacterial cultures with release the endotoxins and that the calf feed is made available in such an amount will that at least 1 to 10 hemagglutination units of everyone's endotoxins. Bacterial type per calf per day.