DK144083B - PROCEDURE FOR THE PREPARATION OF AN AGENT FOR THE PREVENTION OF GASTRO-INTESTINAL DISORDERS OF PIGS - Google Patents

Description

(19) DENMARK

| J | (12) SUBMISSION WRITING out m083B

DIRECTORATE OF THE PATENT AND TRADEMARKET SYSTEM

(21) Application No. 2657/71 (51) IntCI.3 A 61 K 39/02 (22) Filing Day 1. June 1971 // A 23 K 1/18 (24) Race day 1 Jun. 1971 (41) Aim. available Dec. 4; 1971 (44) Posted 7 Dec. 1981 (86) International application no.

(86) International filing day (85) Continuation day - (62) Master application no. -

(30) Priority 3 Jun. 1970, 26746/70, GB

(71) Applicant UNILEVER N.V., Rotterdam, NL.

(72) Inventor Jan Roland Hill, GB: Raymond Kenworthy, GB: Philip

Porter, GB.

(74) The engineering firm Budde, Schou & Co.

(54) Process for the preparation of an agent for the prevention of gastrointestinal disorders in pigs.

The present invention relates to a method of preparing a means for preventing gastrointestinal disorders in pigs, and this method is of the kind in which pathogenic porcine gut-infecting bacteria are cultivated, separating the bacterial OQs from the culture and killing the separated bacteria on a such a way that endotoxins are released from the cells of the bacteria.

3 Gastro - intestinal disorders in pigs often arise from the presence in the intestine of one or more pathogenic strains

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[- of the bacterium Escherichia coli, in particular strains of the following serotypes: * 3 2 144083

Weybridqe classification International serotype classification_ G7 08: K87 (B) K88a, b (L)

E65 045: K

E57 0138: K81 (B) E4 0139: K82 (B) E68II 0141: K85a, b-> K85a, c (B) G1253 0147: K89 (B) K88a, c (L) 'Abbotstown' 0149; K91 K88a, e (L) When the pig's environment or diet changes, e.g. when moved or weaned, the change in conditions leaves the pig's system in a state favorable to the propagation of the pathogenic E. coli strains, and the gastrointestinal disorders that may result from it impair the general health and weight gain of the animal, often with fatal outcome.

The present invention is based on the recognition that the porcine gut can be stimulated to produce antibodies against pathogenic E. coli strains already present by introducing into the gut (and thus not in the blood stream as by injection) the endotoxins of one or more of the above E. coli serotypes, practically free of living E. coli organisms.

The process according to the invention, which is of the kind already described, is in accordance with the above-mentioned property in that it produces endotoxins from one or more of the pathogenic E. coli serotypes 08, 045, 0138, 0139, 0141, 0147 and 0149, after which the released endotoxins are mixed with usual feed ingredients, preferably at pH 5-6.

When the material to be introduced into the pig intestine is specified as "endotoxins", it is not intended to exclude the presence of exotoxins or of the cellular material in which the endotoxins are contained in the living bacterium; it is merely intended that the endotoxins are of primary importance in achieving the desired immunological effect, while this is not the case for the exotoxins and cell debris. However, in some cases it will be appropriate to allow either exotoxins or cell debris or both 3 U4083 to remain associated with the endotoxins, firstly, thereby saving you the trouble of separating them, and secondly, allowing you to utilize their antigenic ability.

As already mentioned, introduction into the intestine ("administration") of the endotoxins of only one of the above E. coli serotypes has some beneficial effect insofar as the ability of the aforementioned serotype will thereby be impaired. but it is preferable to administer the endotoxins of all the serotypes, and accordingly, by the method of the invention, it is particularly convenient that endotoxins from all the seven E. coli serotypes mentioned are incorporated into the feed. The endotoxins to be administered can be obtained by culturing each serotype, samples of which can be obtained from the Central Veterinary Laboratory, Ministry of Agriculture and Fischeries, New Haw, Weybridge, Surrey, UK, and reaching the growth of the microorganism and thus its formation. of endotoxins has progressed appropriately, the reproductive microorganisms are killed and the endotoxins released, which can be done by boiling or autoclaving. The entire sterilized cultures, each containing exotoxins and cell debris as well as endotoxins, can then be combined and administered, but rather than sterilizing the entire cultures, it is preferable that the bacteria be separated, as is the case by the process of the invention, suitably by centrifugation. and treated in a small volume of an aqueous medium, e.g. water or saline solution, to kill the bacteria and release the endotoxins.

The simplest and most economical way to kill the separated bacteria consists of heating. If the heating is sufficiently long-lasting, e.g. 1 hour to 100 ° C, 20 minutes to 125 ° C, most of the endotoxin is released to be associated with the cell walls of the killed bacteria and released in the medium in which the heating is performed. The residual bound endotoxins can, as it is found, be dissolved by treatment with an enzyme such as trypsin, lysozyme, pepsin, lipase or mixtures thereof. The typical treatment results are summarized in Table I, showing the relative amounts (titer) of endotoxins released in the water by an initial treatment to kill the bacteria followed by an enzyme treatment to release endotoxin left behind. bound to the bacterial cell walls.

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In practice, of course, the administration of the endotoxins to the pig occurs by mouth, either simply by forced administration of sterile culture material or with the drinking water or as a constituent of a pig feed, suitably a feed containing the protein, the carbohydrate source, f. eg. cereals, and the minerals and minerals that are essential constituents of pig food.

Such pig feed constituents such as herring flour, yeast, wheat flour, crushed oats, ground barley and skimmed milk powder have been found to bind to E. coli endotoxins at pH values of 5.9 and above, as shown in Table II.

Table II

Titer of bound endotoxin per unit weight component at pH =

Dietary component 5.3 5.9 6.3 6.8 7.2

Wheat flour 0 480 480 480 560

Oatmeal 000 320 560

Painted barley 0 0 320 320 480

Herring flour 0 480 480 480 560 yeast 0 320 480 480 560 t

Skimmed milk powder j 0 320 480 480 560

It has also been found that proteolytic enzymes such as trypsin is capable of releasing the endotoxin bound in this way. Thus, at a pH of 7.2, trypsin will release approx. half of the above bound endotoxin content in yeast and skimmed milk powder.

In addition, E. coli endotoxins have been found to precipitate from aqueous systems at pH values below 5 as shown in Table III.

144083 7

Table III

pH Titer for standard value endotoxin after adjusting to the specified pH value 7.0 320 6.5 320 6.0 320 5.5 320 5.0 320 4.5 160 4.0 80 3.2 40 2.5 40

It is convenient to incorporate the endotoxins into a feed by first preparing a premix of the endotoxins with one of the feed ingredients (suitably skim milk) and water, after which this premix is mixed with the other ingredients of the feed. Using this method, it is desirable, for the results set forth in Tables II and III, to adjust the pH of the pre-mix to 5-6 to ensure that at least part of the content of endotoxins in the feed immediately after the sinking is made available for stimulation of the immune system in the intestine of the pig. Thus, in the process of the invention, it is convenient that a premix of the endotoxins be prepared with one or more nutrients at a pH of 5-6 and that this premix is then mixed with the other nutrients.

The invention is particularly important for improving the infection resistance of pigs at the weaning stage, where they are particularly vulnerable to stress. By administering the endotoxin material before the pig is weaned, starting, for example, when it is 4-10 days old, and continuing until weaning, the pig intestine can be stimulated to form appropriate antibodies so that, at the time when the pig taken from the sow is a sufficiently large antibody circulation in the gut to cover any E. coli proliferation or at least to reduce the severity of any disease state caused by it. The endotoxin material can for this purpose be incorporated into a so-called "rigid" diet, ie. a feed specially formulated to meet the nutritional requirements for pigs at the weaning stage. In this case, the feed is given to the pigs, taking care to prevent the sow from eating it, usually by making it available only through a narrow opening.

The minimum doses are in the range 1-5 units (regarding the measurement of a unit, see the following examples) of the endotoxins of each E. coli serotype per pigs per day, but it has been observed that pigs given 1000 times this amount showed no ill effects. A very suitable amount for incorporation into a pig feed is 10 to 10 units of the endotoxins of each serotype per day. Thus, in the process of the invention, it is particularly convenient for the endotoxins 45 of each E. coli serotype to be incorporated in an amount of 10 to 10 haemagglutination units per ml. kg. feed.

The following examples are intended to further illustrate the process of the invention.

Example 1.

Preparation and Isolation of Endotoxins

Endotoxins are prepared from each of the seven E. coli serotypes mentioned above, generally following the method set forth by Westphal, Luderitz and Bister (1952) in Z. Nature. Forsch. 7, 148.

A freeze-dried culture of E. coli is reconstituted in pep-ton water and incubated at 37 ° C for 6 hours. After appropriate growth, the culture of purity is checked on a washed sheep blood / agar plate and then used for grafting nutrient agar (Oxoid) slabs in Roux flasks. The culture is grown at 37 ° C overnight, after which the bacteria are harvested in sterile distilled water.

The bacterial suspension obtained is aseptically distributed in Universal bottles of 30 ml and centrifuged at 4000 rpm for 10 minutes.

The overlying liquid is removed and the remaining cake of bacteria is resuspended in 4 ml of sterile distilled water and lyophilized.

To isolate the endotoxins, 0.5 g of freeze-dried E. coli is suspended in 5 ml of 0.15 M NaCl, and 10 ml of 90% aqueous phenol is added as a lysing agent. The mixture is heated to 68 ° C for 30 minutes with continuous stirring, then centrifuged at 4000 rpm to compact the cell debris.

The aqueous phase is removed and cooled to 4 ° C, then slowly added 10 volumes of ice-cold ethanol. The resulting precipitate is redissolved in water and nucleic acids are precipitated from the solution by the addition of 2 volumes of ethanol and removed. From the residual solution, the endotoxins are precipitated by the addition of an additional 8 volumes of ethanol, which are separated by centrifugation, washed with ice-cold ethanol and redissolved in 0.15 M NaCl.

This solution of endotoxins will hereinafter be referred to as Reagent 1.

EE £ ¥ CiQ2_å £ _§SdQtQ2iner a. Preparation of antiserum

Specific hyperimmune sera ("antisera") are produced in white New Zealand rabbits against washed, heat killed (100 ° C, 21/2 hours) organisms of each of the E. coli serotypes.

Subsequently, suspensions of the heat-killed organisms are prepared in 0.15 M NaCl (about 3 x 10 organisms per ml) and these suspensions are injected intravenously. The immunization schedule begins at an injection volume of 0.1 ml and is continued every 4 days, doubling the volumes to 1.6 ml. The rabbits are drawn for blood 10 days after completion of the immunization schedule, and the blood obtained is centrifuged, collecting the top layer of hype immune serum. This antiserum will hereinafter be referred to as Reagent 2.

b. Measurement of antibody (anti-endotoxin) activity in antiserum Sheep erythrocytes are sensitized by treatment of a 5% suspension of washed packed cells with an equal volume of endotoxin solution Reagent 1 at 37 ° C for 30 minutes. , after which the sensitized cells are separated by centrifugation, washed free of excess endotoxins and resuspended in 0.15 M NaCl to form a 2.5% suspension of endotoxin-sensitized sheep erythrocytes. This suspension will hereinafter be referred to as Reagent 3.

Reagent 2 (antiserum) is serially diluted with 0.15 M NaCl to give a series of solutions of equal volume (1 vol) and with an antibody concentration of 1/5, 1/10, 1/20, 1/1. ... 1 / (5 x 2) of Reagent 2's concentration, and to each of these solutions is added 1/5 volume of Reagent 3. In the stronger antiserum solutions, but not in the weaker ones, hemagglutination occurs and the end point of the titration (carried out at 4 ° C) is taken as the solution in which just a hemagglutination takes place. In a typical method, this end point may be at the twelfth solution, i.e. the solution having an antibody concentration of 1 / (5 x 2 3 ") of Reagent 2's concentration. Reagent 2 is then said to have an antiserum titer of 5 x 2 ^ (= 10,240).

c. Measuring the concentration of endotoxins in a solution of unknown concentration_

The solution (Y) to be measured is serially diluted with 0.15 M NaCl to obtain a series of solutions of equal volume (3 vols) and with endotoxin concentrations of 1/3, 1/6, 1/12, 1/24 etc. of the solution Y's concentration. To each of these solutions, add 1 volume of Reagent 2 (antiserum) diluted to a titer of 20 (see b. Above) and then (after a few minutes) 1 volume of Reagent 3 (sensitized sheep erythrocytes) so that final concentrations of endotoxins are obtained. of solution Y's. Hemagglutination is inhibited in the stronger endotoxins solutions but occurs in the weaker ones and the end point of the titration is taken. as the solution in which the hemagglutination is just inhibited, in a typical method this may be at the sixth solution, i.e., an endotoxin concentration of 1 / (5 x 2) of solution Y's. a titer of 5 x 2 ^ (= 160), corresponding to 160 units of endotoxin per ml (see Example 3).

144083 11

Example 2.

A. Preparation of Endotoxin Materials

The method described below was followed separately for each of the E. coli serotypes previously mentioned.

(I) The bacterium is ironed from a storage culture on washed blood agar plates and incubated at 37 ° C for 24 hours, after which the plates are checked in the usual manner for purity of the strain.

(II) Colonies of the bacterium are transferred from the plates to 5Q ml of Oxoid Nutrient Broth No. 2 (catalog no. CM 67) and the culture liquid is kept at 37 ° C for 24 hours.

(III) The entire culture obtained in (II) was used to inoculate 1.5 liters of Oxoid Nutrient Broth No. 2, and the culture fluid was incubated with shaking at 37 ° C for 24 hours.

Each end culture thus prepared contained ca. Viable bacteria per 100 and a sample of the culture, after steam treatment at 125 ° C for 2 hours and subjected to the test method described in Example 2, gave a titer of 2560, corresponding to 2560 endotoxin units per ml. ml of steam treated culture.

The cultures of all the serotypes were pooled and the total product steamed for 2 hours in an autoclave (125 ° C) to kill the bacteria.

B. Production of pig feed

The entire sterilized culture fluid obtained as described under A is adjusted to a pH of 5 and incorporated in a weight ratio of 15:85 into a conventional pig wean feed of the following composition: 12%

Roll-dried skimmed milk 20

White fish meal 25

Rolled oatmeal (oat flakes) 36 Sugar (sucrose) 10 Dried yeast (75% un extracted) 5.5

Cod liver oil 2

Sodium chloride 0.5

Mineral supplement 1.0

The resulting mixed feed is dried at 65 ° C to a moisture content of approx. 15%.

Example 3

The final culture of each serotype obtained in the manner described under A in Example 2 is centrifuged to separate the bacteria which is then resuspended in water and heated to 125 ° C for 30 minutes. The killed bacteria are then treated for 3 hours at 37 ° C with a mixture of the enzymes lysozyme and pepsin, and the total endotoxin material from the killing and enzyme treatment is tested. The endotoxin material tested from all the serotypes is pooled, adjusted to a pH of 5 and added to skim milk. The mixture is then incorporated into a conventional pig feed of the composition set forth in Example 2, so as to obtain a concentration of 10 endotoxin units of each serotype per ml. kg of feed.