DE2001671B2 - Polyvalent mastitis immunoglobulin and its use in combating mastitis - Google Patents

Description

The invention relates to a novel polyvalent mastitis immunoglobulin obtained from the blood of a variety from cows suffering from various types of chronic mastitis, as well as the same Use in the control of mastitis in cows, ewes and goats.

Mastitis is one of the most common diseases in cows. In addition to the heavy ones Losses suffered by dairy farmers and the dairy industry as a result of sick udders and reduced milk production also include the use of a Mastitis-infected milk poses a serious health problem.

It is therefore not surprising that in the last IO Up to 15 years of age, research in the field of combating mastitis will be increased considerably Although antibiotics have been used in large quantities for the prophylaxis and therapy of mastitis and Even if hygienic measures are applied and vaccinations are carried out, these are becoming more widespread Illness continues to grow.

Active immunity through systematic vaccination of the animals affected by this disease, in particular cows, ewes and goats, can be achieved not achieve according to the current state of the art. Vaccines can be circulating in the blood Produce antibodies, however, the serum agglutinin or precipitin in the blood from the udder cannot be absorbed, so that mastilis diseases of the udder cannot be prevented.

Antibiotics are only of value as additives as they are not able to fight mastitis on their own, for the following reasons:

Narrow-band antibiotics, such as / .. B. penicillin, are of little value today because their effects are limited to certain microorganisms, for example gram-positive microorganisms, while mastitis is a complicated infectious disease cannot be fought. This applies both to penicillin G, the semi-synthetic penicillin, and to other narrow-band antibiotics.

The broad spectrum antibiotics currently available or combinations of narrow spectrum antibiotics and broad spectrum antibiotics are also not always effective and also far too expensive to be used on a large scale. On top of that, not all broad spectrum antibiotics are compatible with one another

ίο because of their antagonistic effect. Again, other broad spectrum antibiotics are not suitable for one Administration to the udders of cows, ewes and goats. Also, the widespread use of Antibiotics against infections result in the development of resistant microorganisms that were previously sensitive to these antibiotics. This applies to both narrow-spectrum antibiotics as well as for Brek ^L .andantibiotika. Furthermore, the content of antibiotics in milk represents that intended for human consumption, a first Health problem.

The above problem of mastitis occurs not only in cows but also in ewes and goats. Therefore, if subsequently from

2 ^r "cows" are mentioned, then "ewes" and "mother goats" are always to be understood.

The object of the invention was therefore to develop a method with the help of which it is possible to economical way those with cows, ewes and

κι To combat mastitis occurring in mother goats both prophylactically and therapeutically.

It has now been found that this object can be achieved according to the invention by a new one polyvalent mastitis immunoglobulin targeting the

i-> the manner described below is obtained and at the prophylactic and therapeutic control of mastitis can be ended.

The invention relates to a new polyvalent mastitis immunoglobulin obtained from the

<ii) Blood from a variety of cows raised on different Suffering types of chronic mastitis, the production of which is characterized by the following, per se known process steps:

a) centrifuging and coagulating the Blutpias * ^r> mas,

b) Precipitation of the blood serum from the plasma by gradually adding ammonium sulfate up to a final concentration of 25% (NH ₄ ) JSO ₄ ,

c) centrifuging off the main precipitate of the "-Globu- '" lins,

d) purifying the precipitate obtained according to (c) by dialysis and centrifugation,

e) concentrating the immunoglobulin obtained to a solids content of 15 to 16% and

"f) Standardizing the concentrated immunoglobulin by agglutination.

The invention also relates to the use of the mastitis vaccine obtained as indicated above

w) munglobulins together with those for injection solutions usual packaging means in the fight of mastitis.

The term "mastitis immunoglobulin" used here is to be understood as meaning a γ-globulin on which

hl immune bodies adhere to mastitis. The use according to the invention is that the polyvalent Mastitis immunoglobulin of the invention, optionally together with one or more antimicrobial

effective means and together with conventional formulations for injection solutions in the form an injection solution is introduced, preferably injected, into the udders of cows, ewes and goats. In this way, the Combat mastitis in cows, ewes and goats effectively and economically both prophylactically and therapeutically by administering the polyvalent according to the invention Mastitis immunoglobulin.

The polyvalent mastitis immunoglobulin according to the invention contains complete and / or incomplete antibodies which are suitable for heterologous microorganisms, causing mastitis are specific. It can preferably be in a freeze-dried form. When it is administered in the form of injection solutions, it can also contain antimicrobial agents, preferably antibiotics, in addition to customary formulations such as diluents and preservatives. Rs can be infused or injected into the udders of the infected dams (cows, sheep, goats) are introduced. The introduction takes place preferably through the teat canal. The treatment using the polyvalent mastitis immunoglobulin according to the invention can be prophylactic or be done therapeutically.

The invention is explained in more detail below with reference to a preferred embodiment

For example, if a cow's udder has been infected with a specific gram-positive microorganism that causes mastitis, the Microorganism with an immunoglobulin according to the invention, which the antibody "jr this specific gram-positive microorganisms can be effectively combated. Is the concentrate'm the specific Antibody in the immunoglobulin high enough to produce a pharmacological effect can Immunoglobulin can be used alone without further additives. But it is also possible to use an immunoglobulin that does not contain antibodies against the microorganisms that cause mastitis. In this However, it is necessary to take the immunoglobulin together with an antibiotic or a chemo ■ therapeutic agent that is effective against the mastitis-causing microorganism to use, with yourself the microorganism must be in a non-resistant state.

For example, if it is a cow that has mastitis and has an antimicrobial If the agent has been treated with little or no effect, the cow can use Immunoglobulin be treated in conjunction with one or the other chemotherapeutic agent.

A number of experiments carried out on cows infected with mastitis has shown that the immunoglobulin or γ-globulin according to the invention increases the sensitivity of the organism causing the mastitis to the action of antimicrobial agents increased. This effect is based on dali when opposed to a microorganism the active ingredient has become resistant when the active ingredient is administered in conjunction with γ-globulin or Immunoglobulin this breaks the resistance of the microorganism to this active ingredient, so that the active ingredient can develop its effect against this microorganism again. In this way, the The therapeutic effect of γ-globulin or immunoglobulin is improved. Usually this is y-globulin or immunoglobulin together with a preservative, a diluent and / or a other antimicrobial agents as mentioned above are used

The manner in which the immunoglobulin according to the invention is produced is detailed below explained

The blood serum of normal animals contains a large amount of proteins, including albumin and

ίο globulin. The globulin consists of different

Components, for example the y-globulin, with the Antibodies, if any, are coupled. Antibodies can be found in an animal's body

if this animal develop with the desired Microorganism has been infected Antibodies against this microorganism can then also develop develop when the animal has been vaccinated with a vaccine derived from the microorganism that causes the specific disease condition, or Then, when the animal is continuously at short intervals with large doses of the specific, the infection causing microorganism is injected. In the latter case, a hyperimmune state achieved The serum produced by

r-, a hyperimmune animal, is of considerable therapeutic value and can also be used to achieve passive immunity. This serum can be used to produce the immunoglobulin according to the invention.

to A hyperimmune serum is made in a universal way by artificial immunization of animals repeated injection of a specific microorganism or its toxoid at short time intervals Using increasingly higher doses made.

i> The time intervals between injections vary from 4 to 14 days, with a minimum of 4 to 8 injections usually required. The injections can be made either subcutaneously, intramuscularly, or intravenously.

W 8 to 14 days after the last injection, a A blood sample is taken, after which the serum is checked for the presence of specific agglutinating antibodies as well as their contents examined. If the blood sample is unsatisfactory then the whole procedure will be the

4ϊ hyperimmunization repeated. To stimulate the To generate higher levels of agglutinating antibodies, adjuvants are used, e.g. B. Freund's complete or incomplete adjuvants. With With the help of adjuvants, the agglutinating,

vi but not necessarily the immunizing ones Antibodies can be increased up to 10 times. Is the The content of agglutinating antibodies in the blood sample of the examined animal is then sufficient the animal is allowed to bleed to death in a sterile manner.

Vi The main advantages of artificial hyperimmunization are as follows:

a) There is only one type of microorganism per animal (Serotype or variant) or just a toxoid used. In the animal is therefore generally

^M) a monovalent homologous hyperimmune serum

generated.

In the case of, for example, Staphylococcus aureus as one of the many causes of mastitis some h ^r> hundred serotypes and more than 80 are known Phagotypen.

For the production of a polyvalent hyperimmune cream against a group of homologous microorganisms

men (like ζ, Β. Staphylococcus aureus) should at least 20 to 30 animals are used while producing a polyvalent hyperimmune serum at least 100 animals should be used against the multitude of heterologous bacteria.

b) To prevent the death of animals that are hyperimmunized, cultures are killed or toxoids are used. By killing the pathogenic bacteria, large parts of the natural virulent factors, about which little is known to date, what destroys the formation of incomplete antibodies. The same is true when using toxoids.

c) The response to artificial immunization varies from animal to animal.

d) Died in an artificial hyperimmunization Usually large numbers of animals in shock, especially during the later stages of immunization, whereby the size of the doses used plays a role

These disadvantages are avoided by the fact that the blood of a large number of animals from different Areas suffering from chronic mastitis is collected. Such a collected hyperimniotic serum contains complete natural antibodies against heterologous groups of bacteria as well as against the heterologous types within a group, responsible for the different manifestations of mastitis are responsible. It was found that the blood was that of animals slaughtered in a switch house collected can be a satisfactory source of blood from naturally infected animals. It was also found that the polyvalent heterologous hyperimmune serum obtained from the Blood obtained from animals suffering from chronic mastitis is better than that obtained from the blood of a single animal was obtained.

The heterologous hyperimmune serum from which the immunoglobulin according to the invention is produced should, can in an expedient manner in the manner described below from the collected blood be won.

Unlike the blood of horses, the blood of cattle is produced when it is naturally produced can coagulate, very little serum (10 to 20%), with hemolysis occurring very easily. For elimination this problem turns the collected blood into sodium citrate (anticoagulant) and dextrose (antihemolytic) in in an amount necessary to achieve a final concentration of 0.5% each. The plasma wii'd separated from the blood cells by centrifugation. In this way, at least 60% Plasma won. The serum is obtained from the plasma by adding CaCb in one concentration of 3.75 g / l to the plasma, resulting in the formation of a gel. The gel comes in small portions divided and sifted through a cheesecloth to remove the recovered fibrin and fibrinogen from the serum separate. The serum yield is ± 50% of the original volume.

The serum is then checked for the presence of 40 representative bacterial cultures tested by specific aggregates. examples for such cultures are Staphylococcus aureus, Streptococcus agalactiae, Klebsieila pneumoniae, Pseudomonas aeruginosa, E. ei ii and Salmonella. A satisfactory one Serum is then used to obtain the desired immunoglobulin

The ^ -globulin to which the antibodies are linked is _{precipitated from the serum using ammonium sulfate (NH 4 I 2} SO ₄ by slowly dropping a 50% (NH ^ SCV solution into an equal volume of serum. In this way, a final concentration of 25% (NFU) ZSO ₄ achieved The (NH ^ SCVy-globulin precipitate is separated by filtration and placed in a special cellulose cell

ίο transferred by dialyzing it against cold running water to remove the (NH ₄ I ₂ SO _4. The dialysis lasts 6 to 8 days. Then the dialysate is checked for the presence of (NH ₄ J ₂ SO ₄ tested The _{concentrated immunoglobulin free of (NH 1} I ₂ SO ₄ is centrifuged at 1500 rpm to remove protein impurities consisting of large molecules and then sterilized by filtration

The standardization is carried out according to the invention as follows:

a) To determine the y-globulin concentration is performed immunoelectrophoresis,

b) then the total protein concentration certainly

²⁾ c) a growth inhibition test is carried out,

d) the agglutination antibody content is determined

e) a sterility test is carried out and
^{J {)} f) a security test is carried out

Depending on the results of process steps (a), (b), (c) and (d) the concentrated product in a phosphate buffer salt solution to the required final concentration

J5 concentration diluted. The final preparation contains 10 to 12% protein, more than 90% y-globulin and at least 1: 6400 agglutinins and it has an in vitro growth inhibiting concentration of 1:10 (dilution per ml). The recommended dose is as follows:

20 ml of the end product per quarter of a cow that produces less than 11 l of milk per day, and 40 ml for more cows producing milk.

The sterility tests are carried out on blood agar plates while the safety of the products is in

• n the form is tested that double the amount of recommended dose is injected into a young calf or the quarter of a normal udder.

The polyvalent mastitis immunoglobulin of the present invention is preferably in freeze-dried

> o Form placed on the market and, if applicable along with a diluent (water) that contains 0.25% phenol as a preservative for a prophylactic treatment of mastitis used while for the therapeutic treatment of a clinical

Vy see mastitis per dose 0.25% plienol + 200 000 IU penicillin G and 250 mg dihydrostreptomycin are used.

When a test was carried out, three cows infected with mastitis were given three different antibiotics treated. No improvement in the disease was found. After that the cows were with a combination of the antibiotics and the mastitis immunoglobulin according to the invention treated. Within 3 days there was an improvement in the

tvi disease ('established, after 10 days all cows were completely healed.

The results obtained according to the invention show that the treatment at least for one limited, ·

Period leads to passive immunity to mastitis. In addition, the artificial or natural infection of a cow during such a period of time results in passive immunity as reinforcement, from which the active immunity obtained can be seen.
The following examples illustrate the invention.

Example I.
Isolation of the antibody

A total of 201 blood are collected from a number of cows of different types suffer from chronic mastitis. 170 ml of a 50% solution of sodium citrate in a 50% aqueous solution Dextrose are added. The mixture is transferred to the laboratory and in a continuous operating centrifuge at 3000 rpm for a period of 30 minutes to separate the red Blood cells centrifuged from the plasma.

The plasma is treated with 3.75 g calcium chloride per liter for coagulation. The formed coagulum gel is broken open and placed on a cloth to allow the serum to drain and be collected. then ammonium sulfate is used to precipitate the serum. Using varying amounts test precipitations are carried out on ammonium sulphate. The protein precipitate is determined by electrophoresis analyzed. The following results are obtained:

Serum volume

(icsamtprotein X.ft albumin 7ο licsamt (7-ll-Cilohulin 7 »υ esa mi α-glohulin ■ "■ (icsam ι μ '».5 μ 7.8 μ 18.1 G 74.1 10.6 0.64 2.5 1.56 ί. \ 4 6.40 82.1 0.24 2.2 1.46 ft.8 7.80 91.0 0.23 0.72 9.62

25 g of ammonium sulfate per 100 ml of serum are added used to carry out the main precipitation of de * α-globulin. The globulin is centrifuged 8 hours later, in bags made of regenerated cellulose film given and at 4 ° C with running tap water for a period of 5 - 7 days and then with a 0.8% aqueous N ^ CI solution dialyzed for a period of 2 days. In this way the ammonium sulfate is removed. The product obtained is concentrated to a solids content of 15-16%, filtered and then standardized by agglutination. Then growth inhibition tests are carried out at various degrees of dilution of the product.

This test method is known. In the present case 1 ml of the product is placed in a first tube containing 199 ml of water. 1 ml of this solution is in a transferred to a second tube containing 199 ml of water. while I ml of this solution into a third tube · containing 199 ml of water, etc. A total of 13 tubes are used.

The result of the agglutination tests is shown in Table I. Iu of this table refers to the Dilution to the number of parts of water that contains 1 part of the product.

+ means agglutination while
N shows that no agglutination has taken place.

Agglutination tests of serum before precipitation, immunoglobulin (50% concentration) and the fully concentrated immunoglobulin are summarized in Table II. It's on it to indicate that such a product, as can be seen from the tables. has a polyvalent nature.

Table I. 50 100 200 400 800 1600 3,200 6400 12 800 25 600 51200 102400 Ver Microorganism same 2 + 2 + 2 + 2+ 2+ N N N N N N N N E. coli 3+ 3 + 2+ 2 + 2+ + N N N N N N N Staph. aureus 3+ 2+ 2+ 2+ 2+ 2+ 2+ 2+ 2+ 2+ 2+ 2+ N E. coli 3+ 3 + 2+ 2+ 2+ N N N N N N N N + Bacillus sp. 4+ 3+ 3+ 3+ 3+ 2+ + N N N N N N E. coli 3 + 3+ 2+ N N N N N N N N N N Staph. aureus 3+ 3+ 3+ 2+ 2+ + + N N N N N N -C-Bacillus 3+ 3+ 2+ 2+ + 4- N N N N N N N Staph. aureus 4+ 4+ 4+ 4+ 2+ 2+ 2+ + + + N N N Staph. aureus 3+ 3+ 3+ 2+ 2+ N N N N N N N N Staph. Epidermidis 4+ 4+ 4+ 3+ 3+ 2+ + + + N N N N Staph. aureus 3+ 3+ 3+ 3+ 3+ 2+ 2+ + + N N N N StrepL Agaiactiae 3+ 3+ 3+ 3+ 2+ 2+ 2+ 2+ 2+ N N N N Staph. Epidermidis 4+ 4 + 4+ 4+ 3+ 3+ 2+ + N N N N N Pseudomonas

50 9 KX) 200 4 (X) 20th 01 671 M (X) 12 8 (X) 10 51 2 (X) 1024 (X) Ver same continuation 3+ 34- 34 · 34- 4- N 25 MX) N N N Microorganism 44 44- 44- 34- X (X) INH) .12 (K) 24- 24- 24- 4- N 34 · 34- 34- 34- 4- N N N N N Staph. aureus 34- 34- 34- 34- + 4- + 24- 24 24- N N N S'iph. aureus 34- + 4- 4- 34- 34- 2+ N N N N N N Sttph. aureus 3 + 3 + 34- 34- 2+ 24- 24- 4- 4- N N N N Staph. aureus 34 34- 34- 24- 3 + 3+ 2 ^ N N N N N N Staph. Fpidermidis 34- 2+ 24- 24- 4- 4th N N N N N N N Staph. Fpidermidis 34- 34- 34- 34- 24- 4th 4- N N N N N N Strcpto-coccus 34- 3+ 34- 34- 2 + 4- 4- 4- 4- N N N N Staph. aureus 1 + 1 + + + 4- 4- N N N N N N N Staph. aureus N N N N Staph. Epidcrmidis 24- 24- 4- N Qt'inh Prviil Armi / lic M. N N

Tahclle Il Agglutination Product, Conc Microorganism Serum before 50% con trated the exit centered product babble 800 1600 200 6 400 51 200 - Bacillus 400 102 400 102 400 Streptococcus 3,200 25 600 12 800 Staph. aureus 3,200 6 400 25 600 Staph. aureus 3,200 6 400 12 800 Staph. aureus 6 400 800 1600 E. coli 200 12 800 3,200 E. coli 6 400 3,200 I2 8OO 4- Bacillus 6 400 100 800 4- Bacillus 400 200 6 400 Staph. Epidcrmidis I 6 (X) 200 400 Staph. Epidermidis 3,200 400 800 Staph. aureus 1600 3,200 25 600 Staph. aureus 3,200 800 1600 Klebsiella I 600 400 6 400 Staph. aureus 3,200 400 102 400 Staph. aureus 25 600 6 400 102 400 Staph. Epidcrmidis 12 800 800 25 600 Staph. Epidermidis 3,200 800 102 400 St; iph. aureus 25 600 6400 51 200 Staph. aureus 25 600 12 800 102 400 Pseudomonas 25 600 800 51200 Streptococcus 25 600 1600 25 600 Staph. aureus 12 800 Staph. Epidermidis

The antibacterial activity of the above obtained Product of Example 1 is shown in Table III. The microorganisms used are the Standard microorganisms responsible for approximately 90% of all mastitis cases.

In this table:

The heading (1) means the growth visible in the pipe.

The headline (2) means growth, that only is visible on blood agar plates

+ and N have the same meanings as in

Table I.

Ig stands for immunoglobulin.

P stands for penicillin G at a concentration of 5 gamma per 0.025 mm.

S stands for dihydrostreptomycin at a concentration of 125 gamma per 0.025 mm.

It can be seen that the immunoglobulin is bacteriostatic, it will with penicillin and dihydrostreptomycin mixed, then an increased bactericidal effect compared with the case that these two antibiotics are used without immunoglobulin.

If 20th 2 5% 01 67 1 2 12th IG penicillin 2 comparison + ι P / I25S Dihydro
streptomycin only
serum Table III number of I. + Immune + 2 + Microorganism Organisms
per train Immunoglobulin im N serum penicillin
Dihydro N identification put ml N streptomycin N N N + in the serum + N + 10% N I. 1 2 1% N 10% + 5 t N f 2 + Kl I. N 2 + 150 I. 2 1 N 3+ 2 + Staph. aureus 580 + N N N + N N N + 2 + Staph. epidermis 123 + N + N N N N + 2+ Staph. epidermis 700 + N N + N + Strepto-c occus 240 + + + + N N ^ ' Staph. epidermis 2+ N + N Kl + f 2+ ι *. 1 "J. JU -> ι 2 + Xl ₍ + 3 + N 3t .3ld | fll. 6 | fiu6riiii.S 60 Z. ' 2 + ¹ N N 3+ Staph. epidermis 520 2 + N N + + N N N N 2+ Staph. aureus 270 2+ 2 + N 2+ N 2+ 4+ Staph. aureus 320 2 + + N N + N N 3 + 3+ Staph. aureus 250 2+ + 3 + N + 2 + 3+ Staph. aureus 1000 3+ N + 2 + 2+ N N 2+ + Bacillus Sp. 1500 2 + 3+ Ί f. 2+ + 2+ Pseudomonas 800 + N 2 + N N N N + Staph. aureus 280 2+ 2+ + N + 2+ 3+ E. coli 200 N 3+ 2 + N N 3+ 4+ Staph. aureus 300 2 + 3 + N N 2 + + 3+ Staph. aureus 114 + 3 + + + N 3+ N 3+ Staph. aureus 1000 3+ 3 + + N 2+ N + N 2+ E. coli 2190 2+ N 3+ N N N 3 + Staph. aureus 1000 2+ 2+ N 2+ Η N 2+ 3+ E. coli 2500 3+ 2+ Ν + 3+ Staph. aureus 390 3+ 3 + 2+ N 3+ -C-Bacillus 2 yes 3 + 3 + + N 3+ Staph. aureus 1850 3+ 3+ N N 2+ Staph. aureus 1000 N 3+ N Staph. epidermidis 400 2+ + N N Streptococcus 2 + 3+ Example ; 2

The freeze-dried product of Example I can be stored in bottles. To make a To prepare for administration, the product can be mixed with 25 ml of sterile water and 0.25% phenol (as Protective agent), 200,000 international units of penicillin G and 250 mg of dihydrostreptomycin sulfate mixed The immunoglobulin is expediently in the form of a pure, concentrated polyvalent product, preferably greater than 80% in the form of α-globulin while 15.5% (w / v) protein solids are present. The minimum agglutinin values are at! : 6400 and the minimum growth concentrations against various Microorganisms such as, for example, Staphylococci, Streptococci Exoli, Klebsiella, Aerobacter -jnd Pseudomonas, at ί: i00.

A preparation similar to the one mentioned, however does not contain antibiotics, can be used for passive immunity to calf mastitis and has the advantage that no antibiotic is administered to the animals.

The preparations are preferably in one

ü Coolers stored at 4 ° C and can be used for infusion into the udder through the teat canal for treatment against calf mastitis as well as for generating passive immunity.
Generally 10-50 ml per quarter can do one

W) udder to be infused, creating generally a Protection is achieved for at least 6-8 weeks.

It is recommended that milk made from one treated with immunoglobulin plus an antibiotic Quarter of the udder is obtained up to a period of 24 hours after treatment discard. If no antibiotic is used, the milk does not need to be discarded. Should one Mastitis is treated, then it is to be recommended.

to analyze a sample of the infected milk in a laboratory to determine which microorganisms (e.g. gram positive or gram negative or yeast or fungi) that cause mastitis. then a suitable antibiotic or a suitable antifungal compound can be added to the immunoglobulin before the Infusion can be added.

The results obtained in pre-treatment for mastitis are as follows Tables IV, V, VI, VII, VIII, IX and X are summarized. In these tables, the designations are as follows Meanings:

Quarter means the quarter of an udder (RF, RH,

LF and LH mean right front, right back.

left front or left rear)

Prod means milk production per day

CMT means California Mastitis Test

Cl is the chloride content of the milk

Pole. means Polyhorphonucieur Leucocytes /

0.025 mm

Mon. means Mononucleur Leucocytes / 0.025 mm

Dead means the total number of leukocytes

Dead / ml means the total number of bacterial isolates

Table IV

Chronic mastitis - mixed infection
Before the treatment

Four - I Clinical

Rl- Hard interstiticlle
(Fibrosis)

RH normal

LF hard interstitial *

Edema (fibrosis)

LH normal

Piod CMT pll Cl / ellcn / niKK) ') isolate identification / ml

Pol Mon Tot

25th

(Ib) 4+ 6.8 142.0 950 450 1400 1st staph. aureus / 200

2. gram + Bacillus /> 2000

6.7

6.60

42.8 250 2 (X) 450 Staph. epidermidis / 200

4+ 6.75 127.8 1350 500 1850 1st staph. aureus / 190

2Strept. agalactia / 2000

78.1 150 150 300 Steph. epidermidis / 80

Table IV (a)

Before treatment with immunoglobulin

10 days after treatment of RF and LF with immunoglobulin (2 doses) at 12 hour intervals plus 0.25%

Phenol, 200,000 I.U. penicillin G and 250 mg dihydrostrcptomycin per dose

quarter Clinically Prod CMT pH Clinically Prod CMT pH Cl /ell.·-
Pn 'ml (K)' ')
'(in Cells / ml (10 ^; )
PoI Mon 100
150
200
50 Dead Isolate ltlen ', w' ./ ierjng / ml Isolate identification / ml RF
RH
LF
LH Fibrosis (localized)
normal
Fibrosis (dry)
normal (Ib)
28 3+ 6.8
6.6
6.7
6.6 Fibrosis (normal)
normal
Fibrosis (normal)
normal (Ib)
36 I I I I 6.7
6.65
6.7
66 117.15
74.8
102.95
71.0 2000
50
50
100 400
150
200
200 100
0
0
0 2400
200
250
300 Staph. aureus / 300
no growth
no growth
no growth no growth
no growth
no growth
no growth Tabel IV (b) 14 days after the second treatment of all four quarters
G and 250 mg dihydrostreptomycin per dose with immunoglobulin plus 0, 25% phenol. 200,0001. U. Penicillin quarter Cl Toi RF
RH
LF
LH 92.8
78.1
88.4
71.0 200
150
500
50

15th V Prod CMT 20th 01 67 1 Cells / ml (! 0 ^J ) Mon Dead 16 Clinically Prod CMT pH CMT pH Cl Cells / ml (10 ³ ) Mon Clinically Prod CMT Mon Dead Isolate identification / ml Cl Cells / ml (I0 ¹ ) Mon ToI Isolate [dcnfi'fizicrung / 'mi 030 139 / M treatment pole countable pole _ 0 1800 pole 100 300 Tabel Clinically (Ib) 4+ not normal size, hard (Ib) 4+ 6.85 4+ 6.85 142.00 _ - Small fibrosis (Ib) 2100 Staph. aureus / 430 95.85 200 100 100 no growth Before the Isolate identification / ml (interstitial edema) 1 1 4+ 6.80 124.95 - - Small fibrosis - 1200 no growth 71.00 0 200 400 no growth quarter Acute edematosis pH Cl countable 1 / 4+ 6.70 117.30 - - Small fibrosis .14 - 300 900 no growth 84.50 200 50 100 no growth Mastitis in all
4 quarters 4+ not l.Staph. aurejs / 2400 4+ 6.70 2+ 6.75 110.75 - Small fibrosis no growth 78.10 50 no growth RF painful, 6th 6.85 142.6 countable 2. StrepL agalactiae / 2 V (b) 500 Difficult to walk. 4+ not countable laughing at a treatment with immunoglobulin + 6.70 Cells / ml (ΙΟ ³ ) no feeding 4+ not 1. Strep, agalactiae / 800 Clinically Prod Cl pole 600 Dead Isolate identification / ml RH 6.80 127.30 2nd staph. epidermidis / 2O0 + 6.70 1000 1000 Staph. epidermidis / 800 Small fibrosis (Ib) 124.15 1. B hemolytic LF 6.80 124.80 l.Staph. epidermidis / 14 days staph. aureus / 800 LH 6.70 113.60 1000 600 900 2nd staph. epidermidis / 2 2nd staph. aureus / 1000 Small fibrosis 117.30 B-hemolytic 28 500 1000 staph. aureus Tabel 60 hours after treatment of all 4 quarters with immunoglobulin plus phenol (0.25%), dose 50mI per fourth Small fibrosis 102.80 B-hemolytic Fourth! 300 900 staph. aureus Small fibrosis 99.40 B-hemolytic RF staph. aureus RH after the first treatment with immunoglobulin (alone) and after LF Immunogloboline plus 0.25% phenol, 200,000 I. the second treatment mi V (i) LH quarter J. Penicillin G and 250mg dihydrostreptomycin per dose Tabel pH 8 days ι RF quarter RII 6.65 LF 6.60 RF LH 6.65 V (c) (..60 RH LF LH Tabel 22 days

\ 7 VI 16 VI (a) (Ib) Vl (b) (Ib) VII CMT Clinical Prod (Ib) (MT 20th 01 671 Cells / ml (10 ³ ) Mon Mon Mon Mon Dead 18th treatment after the second treatment after the third treatment 11 pole countable 100 100 1500 Tabel Clinical Prod Clinical Prod 20th Clinical Prod 3+ Fibrosis with internal 3+ not 1400 150 Before the siiticllcn edema countable 200 Isolate identification / ml quarter Chronic fibrosis (Ib) Fibrosis (smaller) Fibrosis (small) 3+ (hard) pH Cl not 100 250 8 (M) Fibrosis (smaller) Fibrosis (small) Fihrosis mil inter- .Vf 1000 2500 l.Staph. aureus / a RF Chronic fibrosis Fibrosis (small) 3+ sliticllcn edema 6.75 106.50 1500 countable 2nd staph. epidermidis / ff Fibrosis (smaller) Fibrosis (small) 3+ (hard* not / cllcn / ml (K) ') 0 l.Staph. epidermidis / ur RH Chronic fibrosis Fibrosis (smaller) normal - 6.70 102.95 Cells / ml (ΙΟ ¹ ) pole SO 2.StrepL agalactiae / ff Distinct fibrosis normal Cells / ml (I0 ³ ) pole 2000 StrepL agalactiae / a LF CMT 6.80 131.35 pole 0 Dead StrepL agalactiac / ff LH 6.85 134.90 150 150 250 Tabel - 1200 0 950 2600 14 days 2+ 250 Isolate li'entirizierung / ml quarter pH Cl 250 350 + 100 4200 Staph. aureus / 40 RF 4+ 6.70 88.75 0 Staph. aureus / 80 RH 6.80 110.05 (a + B haemoIytic) no growth g LF CMT 6.80 113.60 Dead Staph. aureus / 4000 ί LH 7.00 138.45 100 Tabel - 300 21 days - 200 Isolate Idcntill / icrung / ml ■: quarter - pH Cl 500 no growth ί RF 6.70 92.30 no growth ii RH Chioric Mastitis - Before Treatment 6.70 192.95 no growth 3 LF quarter 6.70 99.40 lot Staph. epidermidis / 220 j < LII 6.75 110.05 3500 Tabel RF Isoliil Idenlifi / icrung / ml 'i pH Cl 1750 RII Staph. aurcus / 2050 6.75 117.15 KH) LF M) Staph. au rc u s 5 (MK) III 0.70 127.80 ditto 271 Staph. cpidcrmidis / 50 6.60 95.85 no growth (. M) 78.10

19 20

Table VII (a)

3 days after treatment of RF, RH, LF with Ig No. 13, dose 50 ml per quarter

quarter Clinically RF Distributed fibrosis (no edema) RH Distributed fibrosis (no edema) LF normal LH normal

Prod CMT pH

Cl cells / ml (ΙΟ ¹ )

Pol Mon Tot

Isoluc identification / ml

(Ib) 4+

4+ 4+

30th

6.80 117.15
6.70 102.95

2800 no growth 3300 no growth

6.70 113.60 - - 1200 no growth 6.65 81.65 100 150 250 no growth

Table VII (b)

6 days after treatment

quarter Clinically (dry) Prod CMT P " Cl Cells / ml (I0 ¹ ) Mon Dead IsoUit Ident (dry) pole 100 2950 RF Fibrosis (Ib) 2+ 6.70 106.5 1950 300 650 no growth RH Fibrosis 2+ 6.65 92.3 350 100 300 no growth LF normal 29 - 6.60 92.3 200 5Π 100 no growth LH normal - 6.60 78.1 50 no growth

Table VII (c)

16 days after treatment

quarter Clinically Prod CMT PH Cl Cells / ml (ΙΟ ¹ )
Pol Mon 200
50
0
150 - ToI Isolal Idcntifi / .icrung / nil RF
RH
LF
LH Localized fibrosis
Localized fibrosis
normal
normal (Ib)
27 - 6.65
6.60
6.60
6.60 88.50
88.50
78.10
74.55 0
200
0
50 800 200
250
0
150 no growth
no growth
no growth
no growth Tabel VIII 600 Before the treatment / cllcn / ml (K) ')
Pol Mon 600 quarter Clinically Prod CMT pH (Ί 2000 Dead Isolation identification / ml RF Hard ones
Fibrosis (Ib) 4+ 6.95 134.9 1200 2000 Strept. agalactiae / 3200 RH normal
(little F'hrosis) 14th 4+ 6.90 142.05 900 2000 I. Strept. agalactiae / llOO
2nd staph. aureus
(Gr. Ff-Haemol) / 200 LF Fibrosis atrophy 2+ 6.75 85.10 1800 1500 Staph. aurcus LH Hard ones
Fibrosis 4+ 6.85 1. (4.90 2400 I. Strept. agalactiac / 1800
2nd staph. aureus
(gr.ff-ll; imol) / 70
3rd staph. aureus
(Cl. Ff-hemolytic / 50)

21

22nd

Table VIII (a)

60 hours after the second treatment of RF and RH with Ig 17/18, dose 25 ml, and 72 hours after treatment of LF and LH with immunoglobulin (25 ml Ig 17/18), 200,0001. U. penicillin G and 250 mg dihydrostreptomycin

quarter Clinically Prod CMT pH Ci Cells / ml (Iu ³ ) Mon Dead Isolate identification / ml pole 400 1400 RF Fibrosis (Ib) 4+ 6.80 128.40 1000 200 1000 no growth RH small fibrosis 4+ 6.80 113.60 800 Staph. aureus 16 800 2000 (B. hemolytic / 60) LF Fibrosis + atrophy 4+ 6.70 102.80 1200 400 1200 no growth LH Hardening 4+ 6.70 134.90 800 no growth

Table Viii (b)

15 days after the last treatment

quarter Clinically Prod CMT 25th - after the last treatment Prod CMT pH Cl Cells / ml (Iu ³ ) Mon Mon Dead Isclat identification / ml Clinically pole 200 400 RF Fibrosis (dry) (Ib) ± 6.70 78.10 200 150 200 Stap! I. epidermidis / 20 RH normal - 6.70 85.10 50 B. hemolytic 150 300 Staph. aureus LF Atrophy + fibrosis 6.70 81.65 150 no growth (dry) 300 350 LH Fibrosis (dry) 6.70 88.75 50 no growth Tabel VIII (C) 30 day " Cells / ml (10 ') quarter pH Cl pole Dead Isolate identification / ml

RF normal

(Fibrosis dry)

RH normal LF normal (atrophy) LH normal

(Fibrosis dry)

(Ib) 32

6.60 78.10

6.65 78.10

6.65 88.70

6.68 92.30

0 100 100 no growth

50 200

0 150

100 100

250
150
200

no growth no growth no growth

Table IX

Chronic Mastitis - Before Treatment

quarter Clinically Prod CMT pH Cl Cells / .iMIO ³ ) Dead Hol Mon 1100 RF Fibrosis (edema) (Ib) 4+ 6.75 124.50 850 250 (Lump in the Milk) 350 RH normal 30th - 6.65 81.65 100 250 50 LF normal - 6.625 74.55 0 50 100 LH normal 6,625 71.00 100 0

Isolate Idcnlifl / icrung / ml

no growth no growth

23 24

Table IX (a)

3 days after treatment with IG 18. Dose 25 ml for RF. / wide treatment of RF with IG 18.25ml

Clinical quarter

RF 'fibrosis (clean milk)

RH normal LF normal Ll! normal

l'rod CMT pll Cl / ellen / ml (ΙΟΊ Isoliil lclcnlifi / ierunp / nil

l'ol Mon Tot

28

(Ib) 4+ 6.70 113.60 650 500 1150 Staph. aureus / 340

6.60 74.55 50 KX) 150 no growth 6.65 67.45 0 150 150 no growth

6.60 78.10 50 100 150 no growth

Table IX (b)

14 days after treating RF with IG 18, 25 ml

quarter Clinically l'rod (MT pll (I. / ell en / ml (ΙΟΊ Toi Isolate identification / ml pole Mon 300 RF Fibrosis (dry) (Ib) 6.70 92.8 100 200 100
100 no growth RH
LF normal
normal 33 6.65
6.65 88.1
81.2 0
50 100
50 200 no growth
no growth LH normal 6.60 78.1 0 200 no growth

Table X

Treatment of artificially created mastitis

See immunity to mastitis. Before treatment and 14 days after treatment for RF

Clinical quarter Prod CMT pH Cl

Cells / ml (10) Pol Mon Tot

Isolal identification / ml

RF Acute painful edema mastitis, very hard, less Milk, moist jelly

RH Enlarged and edemaic

LF Clinically normal

LH normal

(Ib) -

6.95 142.0 not countable

Pseudomonas

6.80 138 not countable Pseudomonas

6.75 117 - - 1100 staph. aureus strept

p.or.-agaiactiae

6.70 113 250 400 650 Staph. aureus

Staph. epidermidis

25th

2fi

Table X (a)

16 days after treatment for RF (Pseudomonas) and LF (Staph. Aurcus) and 12 hours after the first treatment of RF and RH with Ig 17/20, dose 25 ml per quarter

Clinical quarter

(MT pll Cl

Cells / ml (K) ') isolate identification / ml

Pol Mon Tot

RF improvement, acute mastitis, - 6.80 to

interstitial edema, lumps

little milk

RlI Greater than IJ and LH. Milk normal

normal

6.70 88.75 not countable
6.70 74.55 not countable

no growth

no growth

Staph. aureus / 330 Strept. agalactiac

lh [normal

Table X (b)

(O:> b /, 45 ίου, ου 3) u i.Mapn. aurcus

(large haemolysc / 390)

2nd staph. aureus (B) 3rd staph. epidermidis

4 days after the first treatment of RF 'and RH. 2 days after the second treatment of RF and RH and first Treatment of LF and LII with Ig 17/20. Dose per treatment 25 ml / quarter.

Clinical quarter

I'rod CMI pll Cl

/ cells / ml (10) Isohii identification / ation / ml

l'ol Mon Tot

RF Mild edema (fi'urosis) (± 80% normal)

RH slight edema LF normal LH normal

me (Ib) + 7.0 138 not countable 4000 no growth iall 1400 me 17 ₊ 6, S0 124 4000 - 1200 no growth + 6.75 124 1200 200 no growth + 6.70 113 800 400 no growth

Table X (c)

17 days after the last treatment of all 4 quarters

Clinical quarter

RF
RH
LF
LH

Normal Normal Normal Normal

I'rod CMT pH Cl / cllcn / ml (K) ¹ ) Mon Dead Isolation identification / ml l'ol 150 200 (Ib) 6.70 113.60 50 100 200 no growth 6.70 113.60 100 100 100 no growth 28 6.70 106.50 0 50 50 Staph. epidermidis / 40 6.70 102.40 0 no growth

Even if the effect of the antibody is due to the increased phagocytosis

Numerous experiments have shown that healthy

is understood, it is nevertheless assumed that the cows that are treated with the antibody are only

Effect on immobilization and growth inhibition show slight irritation after 3 to 5 days

education of macroorganisms in connection with one have completely disappeared.

example

Another suitable antibiotic substance can generally be penicillin G an in

instead of or in addition to the vivo efficiency index of approximately 40% given in Example 1, halved Substance to be used. synthetic penicillin or a combination of

Penicillin G plus streptomycin an index of approximately 50%, with F furazolidone, a combination of erythromycin and noviobiocin, or a combination from neomycin and bocitracin an index of about 70%; and with chloramphenicol an index of about 90% achieved.

Using an immunoglobulin obtained from blood collected from slaughtered cows was won, an experiment was made using a German merino ewe carried out.

The ewe suffered from mastitis or "blue udder",

the filter had, swollen and tender.

A single dose of 2 ml was given at one time per quarter.

After 2 days the udder returned to its original condition.

This experiment was repeated and the same satisfactory results were obtained.

Similar experiments were carried out using mother goats. Even in these cases the same mastitis-healing successes were achieved.

Claims (2)

Patent claims: 1. Polyvalent Mastitis Immunoglobulin, obtained from the blood of a variety of cows infected with different types of chronic mastitis suffer, characterized by the process steps known per se: a) centrifuging and coagulating the blood plasma, b) Precipitation of the blood serum from the plasma by gradually adding ammonium sulfate up to a final concentration of 25% (NH ₄ I ₂ SO ₄ , c) centrifuging off the main precipitate of the «-globulin, d) purifying the precipitate obtained according to (c) by dialysis and centrifugation, e) concentrating the immunoglobulin obtained to a solids content of 15 to 16% and f) Standardizing the concentrated immunoglobulin by agglutination. 2. Use of the mastitis immunoglobulin obtained according to claim 1 together with the for Injection solutions common formulations in the fight against mastitis.