DE1810438A1 - Prophylactic or therapeutic preparation - Google Patents

Description

Patent attorneys Dipl.-Ing. E Veickmann, 1810438

Dipping. H. Weickmann, Dipl.-Phys. Dr. K. Fincke Dipl.-Ing. F. A / Weickmann, Dipl.-Chem. B. Huber

.j, _} 8 MUNICH 27, DEN

MÖHLSTRASSE 22, CALL NUMBER 48 3921/22

Oasis TL-14

TWYiOED LABORATORIES LIMITED, London / England

Prophylactic or therapeutic preparation

The invention relates to prophylactic and therapeutic preparations, in particular the manufacture of a substance suitable for administration by injection or through ingestion is suitable for newborn and young calves.

Passive immunity against a wide variety of diseases v / ird on The newborns of numerous species are transmitted via the colostrum, i.e. the mammary gland secretions of the mother in the immediate vicinity Postpartum period. Young cows, sheep, goats, horses and pigs all receive their first immunity in this manner and food ingestion and absorption of adequate amounts a suitable colostrum is extremely desirable to protect the young animal in the first few weeks of life before its own developed immunological system. This is a period during which the animals are subject to modern breeding methods are particularly prone to infection.

Nature has developed a method through which, for example the pregnant cow in the late stages of pregnancy the antibodies present in her own blood in hers Concentrated colostrum, so that it is present to the calf at birth be available. Those present in the cow at that time Antibodies are known for their immunological history

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true, i.e. are produced by the cow in response to the stimulus resulting from the presence of various microorganisms in their environment.

The protective substances in colostrum are relatively large protein molecules, which are sometimes referred to as immune bodies or immunoglobulins. The passage of these large molecules through the intestines of the young animals is limited to a short period after birth, after which no more absorption occurs due to changes occurring in the intestine. In addition, Ψ the concentration of immunoglobulins in colostrum drops very quickly after birth and in the case of the cow, for example, the composition of the secretion changes within a few days to what is called normal milk, which contains only small amounts of immune bodies of the colostrum and the receptivity of the newborn animal only for a short time.

Modern livestock methods often result in the production of calves that have received insufficient protection. The calves are often taken away from their mothers, without that they have taken adequate amounts of colostrum at a time to be overall length, where the inclusion of the immune body was possible. More often, however, calves that have ingested sufficient amounts of colostrum are placed shortly after birth in environments where the natural stimulus differs significantly from that under which the calves were born. Since the immunity conferred on the calves is a reflection of the mother's immunity since she was pregnant, as a result of her immunological history, such calves may have little resistance to infection in their new environment.

Calves can be protected ¹ by a suitable colostrum is supplied within a few hours after birth, with the food to them, or by suitable Immunoglobulihpräpärate be injected. Colostrum itself has certain disadvantages as an injectable material, but immunoglobulin-containing fractions.

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You can isolate colostrum from ordinary colostrum and you can do this Satisfactory to be injected * Some calves are unable to absorb immune bodies without sufficient Obtaining suitable colostrum with food have recorded - and not within a few hours after the "birth" and in such cases allows an injectable Preparation a protection;

An object of the invention is to provide a method in the cows are actively immunized during the gestation period, to stimulate the production of specific antibodies against the microorganisms used, so that in the colostrum obtained one or more substances are present with improved and specific immunological activity, which young animals be made available or administered as required can.

In this process, normal cows become a specific one Time during gestation immunized to the immunological system of the animal to the formation of a high level of special Stimulate antibodies in colostrum just before birth. All suitable Ahtigeria. Microorganisms can be used and the active immunization is established so that maximum Blood antibody levels are synchronized with the expected calving date;

This is where the natural ability of the cow to produce these antibodies to concentrate in their colostrum, which is collected after the calving is done; The length of time after the birth while;, which is colostrum of a corresponding immunoglobulin content can be collected depending on the rating of the Interesting value of the various collections, but in general the. two first milkings taken after calving,

Measurements from your period of 15 to 18 hours of birth are combined * The combined colostrum is defatted and that., casein, removed using conventional methods, - where a colostrum whey is obtained that

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development of an injectable preparation, hyperimmunoglobulin contains, is sterilized, so that one is an injectable preparation of immunoglobulins, which receives more immunoglobulin contains, according to the injected microorganism, than is found in normal colostrum. The product can be used in calves Orally administered a few hours after birth, as long as the bowel is permeable, or can be administered by injection will. In any case, the immune bodies of the Colostrum preparation quickly in the bloodstream of the calves and a. Protecting the immune body level is achieved if a prepa ™ rat of sufficient potency is used. Except for those through immunization The immunoglobulins produced by the cow are of course also the antibodies from the cow's own natural system.

According to the invention, a method for the formation of increased specific immunoglobulin levels is therefore created by injection a pregnant cow with a microorganism, collecting the colostrum and isolating the globulin fraction therefrom.

Isolation can be done by degreasing the colostrum, removal of the casein to form a colostrum and a sterilizing sieve tion take place with the formation of a preparation, which hyperimmunoglobulin contains.

The invention also provides a prophylactic or therapeutic one Preparation containing hyperimmune ostrum globulin. In other words, the prophylactic or therapeutic preparation contains colostral immunoglobulins, one of which or several are present in an increased amount or are as a result of administration of the corresponding microorganism to the Cows before birth.

The invention further provides a method for the prevention or treatment of diseases in calves which consists in the administration of a prophylactic or therapeutic preparation containing hyperimmunoglobulin by injection or orally. 909828/1537

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Colostrum whey produced according to the known process tends to form a granular sediment on standing. This material appears to be composed essentially of calcium phosphate to exist and is troublesome for an injectable material. the Use of citric acid in reducing pH leads to the presence of citrate ions in solution and prevents it such precipitates due to the complexing effect on calcium ions. By adding sodium citrate to the finished product the precipitate is completely eliminated. This can be done while maintaining the isotonic state, when the "skimmed milk" is diluted with water before the casein is removed. In vivo experiments on abortion have shown that these preparations can be injected quite safely in large quantities. 10 ml of the final preparation were applied to mice Hyperimmunoglobulin per kg body weight injected intravenously without the occurrence of symptoms. This matches with Volumes in excess of 300 ml in a newborn calf.

Optionally, the colostrum whey preparation, which can be different Contains components other than immune bodies, further fractionated to produce purer products with increased immunoglobulin concentration, this can be done by conventional methods, for example by precipitation the immunoglobulin-containing fractions with ammonium sulfate, Discard the supernatant (which contains lactose and various low molecular weight components) and redissolve the Precipitation. The ammonium sulfate can be removed from the resulting solution by dialysis.

The final preparation of the human immunoglobulin can optionally freeze-dried and offered in this Porm. The reconstitution before the injection can be done to the original volume or to a lower one in order to be more concentrated Manufacture product. Water or an aqueous solution, which are compatible components, can be used as the reconstitution fluid which must be injected at the same time. Oral administration can do that

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Preparation in water or another suitable feed material be dissolved or dispersed.

The immunization procedure can be used to Increase levels of specific antibodies known to be of value in protecting young calves and that Product can be used therapeutically if necessary.

One of the most important organisms in causing disease in newborn calves the p-influenza 3 virus (P.I.3) in association with the calf virus pneumonia complex. The following describes the production of a hyperimmunoglobulin product, which is obtained from cow colostrum with elevated levels of antibodies to this virus by using of P.I.3 as a microorganism in the method according to the invention. The manufacture of products with increased levels of others Antibodies can be produced in the same way from cows which have been immunized with the corresponding microorganisms. Such important disease-causing microorganisms are viruses such as adenoviruses, reoviruses and foot-and-mouth disease virus or Bacteria, such as the strains of B. coli, Salmonella, Brucella, Pasteurella, Staphylococcus and Streptococcus.

It is possible to immunize cows with more than one microorganism, causing increased levels of multiple antibodies can be obtained in the final preparation. In many cases, preparations of Cows that fight against individual certain but different microorganisms immunized, are mixed with one another to produce an end product with a predetermined protective value.

As a result of the general immaturity of the immunological system Cattle in the perinatal period is a vaccination of calves usually unsatisfactory at birth. Parental passive immunity enables them to respond to natural stimuli or stimuli. To withstand attacks during the first lifetime, this one

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However, immunity does not last. Developed after a few weeks their own antibody-producing system. Routine vaccination of very young calves is usually carried out by the Residual levels of parental immunity are complicated and a high level of such immunity at the time of vaccination prevents the success of this treatment. In addition, the administration induces des Immunoglobulinpräparateε for young calves none Stress, such as that often caused by a vaccine, which makes the body susceptible to other infections. Peat calves are usually slaughtered around 12 weeks of age and provide prophylactic treatment for the newborn calf With a colostrum preparation with a satisfactory protective effect, the vaccination should be superfluous.

The following examples further illustrate the invention.

example 1

P.I.3 virus

A pregnant cow was injected intramuscularly with a killed bovine strain of the PI3 virus in an emulsion as a carrier 7 weeks and 3 weeks before the expected calving date. From two milkings, 9 liters of colostrum were collected from the cow during 15 hours after birth. The colostrum was combined and the cream or the cream in a continuous separator at 4O ⁰ G centrifugation. The obtained non-fat skimmed milk was mixed with an equal volume of distilled water

and the pH value is reduced to 4.5 by slowly adding a 2 molar citric acid solution while stirring. That precipitated casein was stirred for about 1 hour and, after stirring in Gelite "Hyflo Supercel" (Johns-Manville, New York) filtered off as filter aid (25 g oil / l diluted skimmed milk). The yellow piltrate (13.0 1) was obtained by adding 2N sodium hydroxide solution adjusted to pH 6.0. The osmotic pressure of the solution was now 180 m. The isotonic pressure was restored by stirring in 168 g of solid sodium tricitrate, so that a

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osmotic pressure of 303 m osmoles and a pH of 6.3 were obtained. The solution was then placed on Oarlson-IOrd cellulose asbestos pillows pre-filtered (Carlson-Ford Ltd., Ashton-under-Lyne, England), first through variety No. 6, then through variety S9, and finally sterilized by filtering over EKS variety. The sterile fluid containing the hyperimmunoglobulin (i.e. immunoglobulins with increased P.I.3 antibody levels) was aseptically filled into suitable containers. The P.I.3 antibody level in the immunoglobulin product is at least 4 times the size of control samples collected and examined at the same time from non-immunized animals became.

Example 2

Adenovirus

Three pregnant cows were injected intramuscularly with a killed vaccine containing aluminum hydroxide as an additive and produced from a strain of bovine type 3 adenovirus. The injections were given for 8 weeks and 4 weeks before the expected calving date and the colostrum and the colostrum of some non-vaccinated control cows that were kept on the same farm and where calving was expected at the same time, was collected and how ' treated as described in Example 1 for P.I.3 virus. A hyperimmune gluboline was obtained, i.e. immunoglobulins with an increased Adenovirus antibody levels. Examination of the antibody level found that the colostrum of all three vaccinated cows was at least 4 times higher than that of the control samples.

Example 3
Reοvirus

Three pregnant cows were actively immunized by means of an inactivated one Vaccine containing aluminum hydroxide as an additive and made from a strain of reovirus type 1

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had been established. "The immunization took place about 7% and 3 'Labor before the expected calving date and colostrum probes were collected and treated as described in the case of B.I.3 virus «- - Similar colostrum samples were obtained from non-vaccinated control cows' collected, which were kept on the same farm in the same period and were processed in the same way like-those of the vaccinated cows. One received a hyper * iMQtJBglbbüliri, i.e. immunoglobulins with an increased reovirus antibody mirror. The tests of these samples for the specific reovirus antibody level showed that this was in the Sampled the vaccinated egg was at least four times as large as in the control samples. *)how

Example 4

E _fc GOli (a tribe)

A.' monovalent vaccine was obtained from a strain of S. coli Ö? 8IC8; O! Produced and after inactivation an adjuvant from ifeölsicMsityp incorporated. Three pregnant cows then die with This vaccine was actively immunized for about 7 weeks Mid -3 "weeks before the expected calving date. Bach dem The colostrum samples from these three vaccinated animals were calved those of some other non-vaccinated control die were collected on the same farm during the same period and described as in Example 1

A hyperimmunoglobulin, i.e. Immunomit, was obtained an increased E. coli 078K80 antibody level, "Analysis of the samples from the vaccinated and control animals d © K specific; Antibody levels to the specific E. eQli strain from which the vaccine was made, showed that all samples from the vaccinated animals had at least 4 antibody levels compared to the control samples.

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• Example 5 ■ '^;'■-> ϊ. ~ .Ν "± ΐίΌ £?: - ~; ;; ■ <".: ■ ^{;: ■. ■ - ·:} - ■ - ■■ "' · ' ■ _; - ■ - '-: μ y-; ·' νΆ π;: '.! .. · .. ν .- · - · - . ^r . ■ · ·. ■ E. coli (three strains) ^:;: "" ■ = ■ '■. ■ - '·>;*:> ü-. ·. ^; '·.:. ·

A trivalent emulsion-type adjuvant killed vaccine was prepared from three different E. coli strains (078K80, O137K79 and O35K) and three pregnant p. Fours were actively immunized with it approximately 8 and 4 per cent before calving. Shortly after calving, the colostrum samples were collected from each of the vaccinated cows, as well as from some non-vaccinated control animals that had been kept for the same period and on the same farm, and subjected to the treatment described in Example 1, with the production of Hyperimmunoglobulin, ie "an immunoglobulin with an increased level of E. coli 078K80, E. cold O157K79 and R. coil.: O35K antibodies. The examination of the fertile sterile coli of the control animals revealed that the 'specific antibody level to each' of the ¹ in. dem :. Vaccine used three E. coli-atäminen- · bei'-deh: ^ elmprt - ^ - n- · ^ "- · animals, at least four times as -gro ^ 'were -'A / ie in the Zontrali- _: '- animals. - ■ · ■ - · '■

Example 6

Pasteurella

A killed vaccine with a smulsion-type adjuvant was made from Pasteurella multoeida. Two pregnant cows were expected about 6 weeks and 2 weeks before the expected Vaccinated calving with it. Other pregnant cows expected to calve at the same time as vaccinated cows and which were kept on the same yard served as controls. Each of the calving, the colostrum of all was vaccinated Animals and the control animals collected and processed as described in Example 1 with the formation of hyperimmunoglobulin, i.e. an immunoglobulin with an elevated Pasteurella multoclda antibody mirror. 3The examination these treated Kölostrum samples on the special antikaipef mirror

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against the Pasteurella strain of the vaccine showed that each of the vaccinated cows at least four times as high a level compared to the control animals.

Example 7

Salmonella

Two pregnant cows were about 4 to 5 weeks and then again 2 to 5 "weeks before birth with a heat-inactivated strain vaccinated by Salmonella dublin, which contains sodium alginate as Ad- * contained juvant.

The colostrum from these immunized cows was collected shortly after calving. The serological examination showed that im Comparison with the colostrum of a non-vaccinated control animal which was kept on the same farm at the same time was, the colostrum of the immunized cows had high levels of antibodies against Salmonella dublin showed such specific antibodies, while the colostrum of the control animal not contained at all.

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Claims (9)

- 12 claims 1. Prophylactic or therapeutic preparation, marked by a content of hyperimmune colostrum globulin. 2. Preparation according to claim 1, characterized in that it contains one or more colostruin immunoglobulins in an increased amount as a result of the administration of the appropriate microorganism to cows prior to calving. 3. A preparation according to claim 2, characterized in that the Microorganism consists of one or more viruses and / or bacteria. 4. Preparation according to claim 1, characterized in that it contains immunoglobulins with an increased P.I.3 antibody level. 5. A preparation according to claim 1, characterized in that it contains immunoglobulins with increased adenovirus antibody levels » 6. A preparation according to claim 1, characterized in that it Contains immunoglobulins with raised levels of reovirus antibodies. 7. A preparation according to claim 1, characterized in that it contains immunoglobulins with an increased E ₀ coli antibody level. 8. A preparation according to claim 1, characterized in that it contains immunoglobulins with an increased Pasteurella antibody level contains. 9. A preparation according to claim 1, characterized in that it contains immunoglobulins with an increased Salmonella antibody level contains. 909828/1537 10. A method for producing the preparation according to claim 1 to 9 »characterized in that a pregnant cow Microorganism is injected, the colostrum is collected and the G-lobulin fraction is isolated from it. 13. The method according to claim 10, characterized in that the isolation by degreasing the colostrum, removing the Caseins to form a colostrum whey and sterilize he follows. 12. The method according to claim 10, characterized in that the microorganism from one or more viruses and / or Bak- f series exists. 13. The method according to claim 12, characterized in that the virus from p-Influenza 3 virus, adenovirus and / or reovirus consists. 14. The method according to claim 12, characterized in that the bacteria from one or more strains of E. coli, Pasteurella and / or Salmonella exist. 15 · Use of the preparation according to claims 1 to 9 for contraception or treatment of disease in calves by oral administration or injection of a prophylactic or therapeutic amount. 9 0 9828/15 3 7