DE2001671C3 - Plyvalent mastits immunoglobulin and its use in the fight against mastits - Google Patents

Description

a) centrifuging and coagulating the blood plasma,

IO

15th

b) Precipitation of the blood serum from the plasma by the gradual addition of ammonium sulfate to to a final concentration of 25% (NH ^ ijSO. »,

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 customary formulation agents in the fight against mastitis.

so

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 J5 cows, ewes and goats.

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

It is therefore not surprising that in the last 10 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.

An active immunity by 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 so that mastitis diseases of the udder cannot be prevented. so

Antibiotics are only available as additives from WeFt as they are are unable to fight mastitis on their own for the following reasons:

Narrow band antibiotics such as B. penicillin, are of little value today because their effect on certain t> 5 Microorganisms is restricted, for example to gram positive microorganisms, while mastitis is a complicated infectious disease that represents so cannot be fought. This applies to both Penicillin G, the semi-synthetic penicillin, as well as 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 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 has led to the development of resistant microorganisms previously used for these antibiotics were sensitive. This applies to both narrow-band antibiotics and Brcitb-ondantibiotika. Furthermore, the content of antibiotics in milk represents that intended for human consumption poses an early health problem.

The above problem of mastitis occurs not only in cows but also in ewes and goats. Therefore, if subsequently from "Cows" are always to be understood as meaning "ewes" and "mother goats".

The object of the invention was therefore to develop a method with the help of which it is possible to economical way to combat mastitis occurring in cows, ewes and 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 obtained in the manner set out below and used in the prophylactic and therapeutic control of mastitis can be used.

The invention relates to a new polyvalent mastitis immunoglobulin obtained from the Blood from a large number of cows suffering from various types of chronic mastitis, the production of which is characterized by the following, per se known process steps:

a) centrifuging and coagulating the blood plasma,

b) Precipitation of the blood serum from the plasma by the gradual addition of ammonium sulfate up to a final concentration of 25% (NHtJjSO ^,

c) centrifuging off the main precipitate of the α-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.

The invention also relates to the use of the mastitis immunoglobulin obtained as indicated above together with that 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 Adhere immune bodies 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 customary formulations for injection solutions in the form of an injection solution into the udders of cows, ewes and goats, preferably injected. In this way, mastitis in cows, ewes and mother goats can be combated effectively and economically, both prophylactically and therapeutically by administering the polyvalent mastitis immunoglobulin according to the invention.

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 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. It can be infused or injected into the udders of the diseased dams (cows, sheep, goats) are introduced. The introduction takes place preferably through the teat canal. Treatment using the polyvalent mastitis immunoglobulin of the present invention may be prophylactic or be done therapeutically.

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

For example, if the udder of a cow with a The specific gram-positive microorganism causing mastitis may have been infected Microorganism with an immunoglobulin according to the invention, which the antibody for these specific gram-positive microorganism can be effectively combated. Is the concentration 'of 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 use that does not contain antibodies against the microorganisms that cause mastitis In this one However, it is necessary to take the immunoglobulin together with an antibiotic or a chemotherapeutic agent that is used against the mastitis-causing agent Microorganism is effective to use, the microorganism must be in a non-resistant state.

For example, if it is a cow that has mastitis and has an antimicrobial Means have been treated in which no or only one If there is little effect, then the cow can use immunoglobulin in conjunction with the one or the other other chemotherapeutic agents.

A number of experiments carried out on cows infected with mastitis has showed 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 the fact that, 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 described in greater detail below explained

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

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 The microorganism has been infected with antibodies against this microorganism can develop even if the animal is vaccinated originating from the microorganism causing the specific disease state, 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 obtained from a hyperimmune animal is from has 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.

jo A hyperimmune serum is made in a universal way by artificial immunization of animals by repeated injection of a specific microorganism or its toxoid at short time intervals Using increasingly higher doses made The time intervals between injections vary from 4 to 14 days, usually at least 4 to 8 injections are required. The injections can be made either subcutaneously, intramuscularly, or intravenously.

A blood sample is taken 8 to 14 days after the last injection, after which the serum is applied to the The presence of specific agglutinating antibodies and their contents are examined. The blood sample is examined unsatisfactory, then the whole procedure becomes the 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,

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

The main advantages of artificial hyperimmunization are as follows:

a) There is only one type of microorganism (serotype or variant) or only one toxoid per animal A monovalent homologous hyperimmune serum is therefore generally used in the animal generated.

In the case of, for example, Staphylococcus aureus One of the many causes of mastitis is several hundred serotypes and more than 80 phagotypes known.

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

men (like ζ. Β, Staphylococcus aureus) should at least 20 Ws 30 animals are used while producing a polyvalent hyperimmune serum at least 100 animals should be used against the large number 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 applies to the use of 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 pooled hyperimmune 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, came. in an expedient manner in the following can be obtained from the collected blood in the manner described.

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 (antihemolyfic) in in an amount necessary to achieve a final concentration of 0.5% each. The plasma is 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 blood volume.

The serum is then checked for the presence of 40 representative bacterial cultures of specific agglutinins !! tested. Examples of such cultures are Staphylococcus aureus, Streptococcus agalactiae, Klebsieila pneumoniae, Pseudomonas aeruginosa, E. coli 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 ^ SOr solution into an equal volume of serum. In this way, a final concentration of 25% (NH ₁ ) 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 with a 2% BaClr solution for the presence of (NH _{4 I 2} SO ₄ tested The (NH ₄ I ₂ SO ₄ free concentrated immunoglobulin is centrifuged at 1500 rpm to remove protein contaminants 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-globe concentration, performed immunoelectrophoresis,

b) then the total protein concentration is determined

c) a growth inhibition test is carried out,

d) it becomes the agglutination antibody content certainly

e) a sterility test is carried out and
f) a security test is carried out

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

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:

■ 40 20 ml of the end product per quarter of a cow that delivers less than 11 1 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 The form tested is that double the amount of the recommended dose to a young calf or quarter of a normal udder.

The polyvalent mastitis immunoglobulin of the present invention is preferably placed on the market in freeze-dried form, and optionally along with a diluent (water) that contains 0.25% phenol as a preservative for a prophylactic treatment of mastitis used while for therapeutic treatment of a clinical Mastitis per dose 0.25% phenol + 200 000 IU penicillin G and 250 mg dihydrostreptoTiycin used will.

When carrying out a test, three mastitis infected cows were given three different antibiotics.

bo tika treated There was no improvement in the Disease detected. After that, the cows were given a combination of the antibiotics and the Treated mastitis immunoglobulin according to the invention. Within 3 days there was an improvement in the

b5 Disease detected, all cows were dead after 10 days completely healed '.

The results obtained according to the invention show that the treatment is at least for a limited period

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 20 liters of 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 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 ι », ammonium sulfate is used to make the serum to fail. Sample precipitations are carried out using varying amounts of ammonium sulfate. The protein precipitate is analyzed by electrophoresis. The following results are obtained:

calculated on that
Serum volumes

(iesamlprotcin albumin

8.6

9.5

10.6

0.64
0.24
0.23

<7-ll-cilohulin % Total α-globulin % Total % Total 18.1 V 74.1 7.8 1.56 15.4 6.40 82.1 2.5 1.46 6.8 7.80 91.0 2.2 0.72 9.62

25 g of ammonium sulfate per 100 ml of serum are used to carry out the main precipitation of the globulin. The globulin is centrifuged 8 hours later, placed in bags made of regenerated cellulose film and ^{dialyzed at 4 C} with running tap water for a period of 5-7 days and then with a 0.8% aqueous NaCl solution for a period of 2 days . In this way the ammonium sulphate is removed. The product obtained is concentrated to a solids content of 15-16%, filtered and then standardized by agglutination. Growth inhibition tests are then 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 which contains i ^ mi water, i mi this solution is in a transferred to a second tube containing 199 ml of water.

Table I.

while ! ml of this solution can be poured into a third tube containing 199 ml of water, etc. A total of 13 pipes are used.

The result of the agglutination tests is shown in Table I. In this table, 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 point out that such a product, as is to be erenen from the ι aDeiien. has a polyvalent nature.

Microorganism 50 100 200 400 800 1600 3200 6400 12 800 25600 51200 102400 Ver same F .. coli 2 + 2+ 2+ 2+ 2+ N N N N N N N N Staph. aureus 3+ 3 + 2+ 2+ 2+ + N N N N N N N E. coli 3+ 2 + 2+ 2+ 2+ 2+ 2+ 2+ 2+ 2+ 2+ 2+ N + Bacillus sp. 3+ 3 + 2+ 2+ 2+ N N N N N N N N E. coli 4+ 3+ 3+ 3+ 3+ 2+ + N N N N N N Staph. aureus 3+ 3+ 2+ N N N N N N N N N N -C-Bacillus 3+ 3+ 3+ 2+ 2+ + + N N N N N N Staph. aureus 3+ 3+ 2+ 2+ + + N N N N N N N Staph. aureus 4+ 4+ 4+ 4+ 2+ 2+ 2+ + + + N N N Staph. Epidermidis 3+ 3+ 3+ 2+ 2+ N N N N N N N N Staph. aureus 4+ 4+ 4+ 3+ 3+ 2+ + + + N N N N Strept Agalactiae 3+ 3+ 3+ 3+ 3+ 2+ 2+ + + N N N N Staph. Epidermidis 3+ 3+ 3+ 3+ 2+ 2+ 2+ 2+ 2+ N N N N Pseudomonas 4+ 4+ 4+ 4+ 3+ 3+ 2+ + N N N N N

continuation

ίο

Microorganism

100 200 400 3200 6400 12800 25600 51200 IO24OO comparison

Staph. aureus
Staph. aureus
Staph. aureus
Staph. aureus
Staph. Epidermidis
Staph. Epidermidis
Streptococcus
Staph. aureus
Staph. aureus
Staph. Epidermidis

3+ 3+ 3+ 3+

4+ 4+ 4+ 3+

3+ 3+ 3+ 3+

3+ 3+ 3+ 3+

3+ 3+ 3t-

3+

3+ 3 + 2+ 2 +

3+ 3 + 2 + 2 +

3+ 3+ 3+ 3+ 3+ 3+ 3+ 3+

3+ 2+

Staph. Epidermidis 3+ 3+

2+ 2 +

2+

3+ 3+

2+ 2+ 2+

N N + N

2+

2+

N 2 t-

2+

N N N N 2+ 2+ + N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N

Table Il

Microorganism Agglutination Product, Conc Serum before 50% con trated the off centered product precipitation 800 1600 - Bacillus 200 6 400 51200 Streptococcus 400 102 400 102 400 Staph. aureus 3,200 25 600 12 800 Staph. aureus 3,200 6 400 25 600 Staph. aureus 3,200 6 400 12 800 E. coli 6 400 800 1600 E. coli 200 12 800 3,200 + Bacillus 6 400 3,200 12 800 + Bacillus 6 400 100 800 Staph. Epidermidis 400 200 6 400 Staph. Epidermidis 1 600 200 400 Staph. aureus 3,200 400 800 Staph. aureus 1600 1? M 800 1600 Staph. aureus 1600 400 6400 Staph. aureus 3,200 400 102 400 Staph. Epidermidis 25 600 6 400 102 400 Staph. Epidermidis 12 800 800 25 600 Staph. aureus 3,200 800 102 400 Staph. aureus 25 600 6 400 51200 Pseudomonas 25 600 12 800 102 400 Streptococcus 25 600 800 51200 Staph. aureus 25 600 1600 25 600 Staph. Epidermidis 12 300

The antibacterial activity of the product of Example 1 obtained above is shown in Table HI emerged. The microorganisms used are the standard microorganisms that make up approximately 90% of all Cases of mastitis are responsible In this table mean:

The heading (1) means that in the pipe

visible growth.

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 with one 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 is obtained compared to the case that this two antibiotics without immunoglobulin are used.

20 01 H 1% 5% will. To make a International Units Peni- polyvalent product before, before- Protein solids are present. E-coli, Klebsieila, Aerobacter and lie is similar to the one mentioned, however 2 671 Immunity to 12th Immune + Penicillin comparison streptomyein I. 2 I 2 Animals <an antibiotic can for an infusion in can be 10-50 ml a veal fattening Milk that comes out of one with then used then Administration can use the product Dihydrostreptomycin sulfate preferably more than 80% in the form of «-globulin, > The minimum aegglutinin values are 1: 6400 and - £ Pseudomonas, at 1: 100. Does not contain antibiotics, can be used for passive N penicillin Dihydro- only in the serum + 2+ the teat canal are used, l immunity. Antibiotic treated will. Solos one Table IH I 2 I with 25 ml of sterile water and 0.25% phenol (as i_ during 153% (g / V) - * ■■ the minimum growth concentrations against ver .: A preparation, c 4- Dihydro streptomycin serum 10% OK N + 2 + The preparations are preferably in one Treatment against one per quarter up to one to recommend it. Microorganism 150 + N Protective agent), 200 0OC , The immunoglobulin is conveniently located as different microorganisms, such as staphy- N 3-t- 2+ Stored at 4 "C and cooler tis as well as to generate a passive Udders are infused, generally creating a after treatment too Ident ifi 7ieru η 2 Number of immunoglobulin in serum 580 + N cillin G and 250 mg V- pure concentrated i, lococci, streptococci, + N N 2 + the udder through In general Protection is achieved for at least 6-8 weeks. no antibiotic Ιν1 & 1ΙΙ.ΙΙΙ £ Ί \ * Ι UIIg Organisms 123 + N be mixed. + + 5 u P / I25S N N 2+ for one It is recommended that not discarded too per train 700 ++ N I 2 2+ + Immunoglobulin plus one Mastitis can be treated. put ml 240 2+ N. N N N + 2+ Udder quarter is obtained, X)? + N N 10% N N 3 + 3+ Time span of 24 hours 60 2+ N N N + N + 3+ vp-throw. Will 520 2+ N. 1 2 N N N + 2+ needs the milk Staph. aureus 270 2+ N. + N + N N 2 + 4+ Staph. epidermis 320 2+ + N + N N 3 + 3+ Staph. epidermis 250 3+ N + N N 2+ 3+ Streptococcus 1000 2+ 2+ + + N N N N 2+ Staph. epidermis 1500 f 2+ N + N + + 2+ 800 2+ N + N N N N + Staph. epidermis 280 N 2+ + N + 2 + 3+ Staph. aureus 200 2+ + + 2 + 2 + 3 + 4+ Staph. aureus 300 ++ N + 2 + 2+ 3+ Staph. aureus 114 3+ N. N 3 + N N 3+ 3-t- Staph. aureus 1000 2+ 3+ 2+ + + 2+ + Bacillus Sp. 2190 2+ N. 2+ N N N N 3+ Pseudomonas 1000 3+ 2+ 2+ N + 2 + 3+ Staph. aureus 2500 3+ 3+ N + N N + j + F .. coli 390 3+ 3+ 2+ 2 + N + 3+ Staph. aureus 2500 3+ 3+ + N + N N N N 3+ Staph. aureus 1850 N 3+ + + N N 2+ Staph. aureus 1000 2+ N N + N N E. coli 400 2+ 2+ 3+ 2+ Calf mastitis can be used and Staph. aureus At; 2+ + has the advantage that the E. coli The freeze-dried product of Example 1 can 2+ N N is administered. Staph. aureus stored in Raschen 3 + N N - C-Bacillus Preparation for a 3+ N N Staph. aureus 3+ Staph. aureus 3+ Staph. epidermidis N + Streptococcus 3+ > ρ i e I 2 55 60 65

Sample of infected milk to be analyzed 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 treating cows 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, KH, LF and LH mean right front, right rear, 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 Polyhorphonucleur Leucocytes / 0.025 mm

Mon. means Mononucleur Leucocytes / 0.025 mm Tot means the total number of leukocytes

Dead / ml means the total number of bacterial isolates

Tabel IV K'hiisch I < infection pll Cl Cells / ml (K) ')
Pol Mon 450 ToI Isolation identification runp / ml Hard intcrstiticllc
(Fibrosis) • rod CMT 6.8 142.0 950 2 (X) 1400 I. Staph. aurcus / 200
2. gram +
Bacillus /> 2000 "! ironic mastitis mixtures
Before treatment normal lh) 4+ 6.7 92.8 250 500 450 Slaph. cpidcrmidis / 200 quarter Hard interstiticlle
Edema (fibrosis) - 6.75 127.8 1350 150 1850 I. Staph. aurcus / 190
2. Strepl. agalactia / 2000 RF normal 4+ 6.60 78.1 150 300 Steph. epidermidis / 80 RII IV (a) - LF LII Tabel

Before treatment with immunoglobulin

10 days after the treatment of RF and LF "with immunoglobulin (2 doses) in 12 hour intervals plus 0.25", Phenol. 200,000 I.U. penicillin G and 250 mg dihydrostreptomycin per dose

,,. , ϊ-i!, Γμ.ϊ *;! *, (Ib) 3+ 28 6.8 C: , -Il. " Mon ToI Staph. aureus / 300 Fibrosis (localized) - 6.6 117.15 pole 400 2400 no growth RF normal 6.7 74.8 2000 150 200 no growth RII Fibrosis (dry) 6.6 102.95 50 200 250 no growth LF normal 71.0 50 200 300 LIl IV (b) 100 Tabel

14 days after the second treatment of all four quarters with immunoglobulin plus 0.25% phenol. 200,0001. U. Penicillin G and 250 mg of dihydrostreptomycin per dose

quarter Clinically Prod CMl pH Cl Cells / ml (I0 ^: Mon Dead I soles Identification / ml pole 100 200 RF Fibrosis (normal) (Ib) 6.7 92.8 100 150 150 no growth RH normal 6.65 78.1 0 200 200 no growth LF Fibrosis (normal) 6.7 88.4 0 50 50 no growth LH normal 6.6 71.0 0 no growth

15th

16

Tabel V Prod CMT pH Clinically Prod CMT pH Cl Lines / ml (ΙΟ ³ ) Isolate identification / ml Cells / ml (10- ') Isolate identification / ml Before the Pol Mon Tot Pol Mon ToI quarter treatment (Ib) 4+ 6.85 normal size, hard (Ib) 4+ 6.85 142.6 not countable l.Staph. aureus / 2400 1800 Staph. aureus / 430 Clinically (interstitial edema) 17th 4+ 6.80 ZStrept agalactiae / 2 2100 no growth RF ι / 4+ 6.70 - - 1200 no growth Acute edematosis 4+ 6.80 4+ 6.70 127.30 not countable 1. Strep, agalactiae / 800 900 no growth Mastitis in all
4 fourths 6th 2nd staph. epidermidis / 200 RH painful, 4+ 6.80 124.80 not countable Staph. epidermidis / 800 Walking difficult 4+ 6.70 113.60 not countable l.Staph. epidermidis / LF no feeding 1000 LH 2nd staph. aureus / 1000 60 hours after treatment of all 4 quarters rrit Immunogiobuhn plus phenol (0.25%), dose 50ml per quarter Tabel quarter Cl RF 142.00 RH 124.95 LF 117.30 LH 110.75 V (a)

Table V (b)

8 days after immunoglobulin treatment

Clinical quarter

Prod CMT pH

Cl cells / ml (Iu ³ )

Pol Mon Tot

Isolate identification / ml RF Small fibrosis RH Small fibrosis LF Small fibrosis LH Small fibrosis

(Ib) 4+ 6.85 124.15 1000 0 1000 IB hemolytic

staph. aureus / 800

2nd staph. epidermidis / 2

2+ 6.75 117.30 600 300 900 B-hemolytic staph. aureus

+ 6.70 102.80 500 500 1000 B-hemolytic

staph. aureus

+ 6.70 99.40 300 600 900 B-hemolytic

staph. aureus

Tabel V (c) plus 0.25% Clinically Fibrosis Phenol, 200,000 I. U. Penicillin G ι CMT pH Cl 14 days after the second treatment with Mon Dead Isolate Identification / ml 130 241/28 22 days Fibrosis Prod jnd 250mg dihydrostreptomycin per dose 100 300 Small Fibrosis 6.65 95.85 Cells / ml (10- ') 100 100 no growth Small Fibrosis (Ib) 6.60 71.00 pole 200 400 no growth Small ΐΛ 6.65 84.50 200 50 100 no growth Small 6.60 78.10 0 no growth after the first treatment with immunoglobulin (alone) and 200 Immunogloboline 50 quarter RF RH LF LH

Table VI Clinical Prod (Ib) Via) (Ib) VI (b) (Ib) VII CMT Clinical Prod (Ib) CMT 20th 01 671 Cells / rol (10 ³ ) Mon 1000 Mon Mon Mon Dead 18th I. Before treatment after the second treatment after the third treatment TJ PoI not countable not countable 100 100 1500 Staph. aureus / 2050 I. quarter Chronic fibrosis Clinical Prod 20th Clinical Prod Li. 3+ Fibrosis with inter- 3+ 1400 150 V 17th 16 stitial edema 200 Isolate identification / ml RF Chronic fibrosis Fibrosis (smaller) Fibrosis (small) 3+ (hard) Lj PH CI not countable 100 250 800 Staph. aureus 5000? Fibrosis (smaller) Fibrosis (small) Fibrosis with inter- 3+ 2500 1st staph. anreus / a ditto 273 RH Chronic fibrosis Fibrosis (small) 3+ stitial edema 6.75 106.50 1500 Cells / ml (10 ³ ) 2nd staph. epidermidis / a Distinct fibrosis Fibrosis (smaller) Fibrosis (small) 3+ (hard) pole Cells / ml (1O ³ ) 0 1st staph. epidermidis / ff Staph. epidermidis / 50 LF Fibrosis (smaller) normal - 6.70 102.95 150 Cells / ml (10 ³ ) pole 50 ZStrept agalactiae / a no growth LH normal - 1200 pole 2000 StrepL agalactiae / ff Tabel CMT 6.80 131.35 0 Dead StrepL agalacträe / or 14 days 6.85 134.90 250 150 250 quarter - 0 950 2600 2+ 250 Isolate identification / ml RF pH Cl 350 RH + 100 4200 Staph. aureus / 40 4+ 6.70 88.75 0 Staph. aureus / 80 LF 6.80 110.05 (a + B hemolytic) LH no growth Tabel CMT 6.80 113.60 Dead Staph. aureus / 4000 21 days 7.00 138.45 100 quarter - 300 - 200 Isolate identification / ml RF - pH Cl 500 j RH - no growth LF 6.70 92.30 no growth LH Chronic Mastitis - Before Treatment 6.70 192.95 no growth Tabel quarter 6.70 99.40 Dead Staph. epidermidis / 220 6.75 110.05 3500 RF Isolate identification / ml pH Cl 1750 RH 6.75 117.15 100 LF 50 LH 6.70 127.80 6.60 95.85 6.60 78.10

Tabel 19th
VIHa) Prod 20th 01 671 20th pH CI Cells / ml (! Ο *)
Pol Mon 150 100
300
100
50 200
50
0
150 - Dead Isolal identification / ml 3 days ι (Ib)
30th PtH, LF with Ig No. 13, dose 50 ml per quarter 6.80
6.70
6.70
6.65 117.15
102 ^ 5
113.60
81.65 100 800 2800
3300
1200
250 no growth
no growth
no growth
no growth quarter after treating RF, CMT 600 RF
RH
LF
LH Clinically 4+
4+
4+ Cells / ml (Iß ³ )
Pol Mon Cells / ml (10 ³ )
Pol Men Zcllen / mldO ³ )
Pol Mon 600 Tabel Distributed fibrosis
(no edema)
Distributed fibrosis
(no edema)
normal
normal Prod Pi! Ci 1950
350
200
50 0
200
0
50 2000 Dead ! salad identification / rn! 6 days (Ib)
29 6.70
6.65
6.60
6.60 106.5
92 ^
92 ^
78.1 1200 2950
650
3 € 0
100 no growth
no growth
no growth
no growth p fourth! VU (b) CMT 900 1 ^rf
1 RH
I LF
S ^LH after treatment 2+
2+ 1800 I table Clinically Prod pH Cl Dead Isolal identification / ml 1 16 days Fibrosis (dry)
Fibrosis (dry)
normal
normal (Ib)
27 6.65
6.60
6.60
6.60 88.50
88.50
78.10
74.55 200
250
0
150 no growth
no growth
no growth
no growth I quarter CMT Egg ^RF
I RH
I LF
Nj LH
\ VII (C) - ii table after treatment Prod pH Cl Dead Isolal identification / ml r 'front Clinically (Ib) 6.95 134.9 2000 Strept. agalactiae / 3200 h quarter Localized fibrosis
Localized fibrosis
normal
normal 14th CMT 6.90 142.05 2000 l. StrepL agalactiae / 1100
2nd staph. aureus
(Size a-hemol) / 200 I ^RF VIII 4+ 6.75 85.10 1500 Staph. aureus
(CL ff-hemol) / 130 i \ RH
/ I
S. treatment 4+ 6.85 134.90 2400 I. Strept. agalactiae / 1800
2nd staph. aureus
(gr. a-hemol) / 70
3rd staph. aureus
(Cl. Ff-haemolvtisch / 50) LF Clinically 2+ LH Hard ones
Fibrosis 4+ normal
(small fibrosis) Fibrosis atrophy Hard ones
Fibrosis

21 VIII (a) 2001 671 Clinically Prod Prod Prod CMT Clinically Prod CMT pH RH with Ig 17/18 22nd Cells / ml (10 ³ ) M on Mon Mon Mon Dead Isolal identification / ml pole 400 200 100 250 1400 Tabel Fibrosis (Ib) (Ib) (Ib) 4+ Fibrosis (edema) (Ib) 4+ 6.80 Dose 25 mL and 72 hours after loading 1000 200 150 1000 no growth RF and small fibrosis 4+ (Lump in the 6.80 17/18), 200 0001. U, penicillin G and 250 mg dihydrastrepto- 800 200 Staph. aureus treatment of LF and LH with chromoglobulin (25 ml Ig 16 Milk) 800 150 150 250 2000 (B. nImoIytisch / 60) mycin Fibrosis + atrophy 25th 32 4+ normal 30th - 6.70 Cl 1200 400 100 50 1200 no growth quarter Hardening 4+ normal - 6.70 800 300 0 no growth 60 hours after the second treatment of VIII (b) normal 128.40 RF after the last treatment 113.60 Cells / ml (Hr ') RH Clinically after the last treatment CMT pH pole Cells / ml (ία ¹ ) / rush / ml (ίο ') Dead Isolal Identifying / ml Clinically 102.80 200 pole pole 400 LF Fibrosis (dry) ± 6.70 134.90 50 0 850 200 Staph. epidermidis / 20 LH normal normal - 6.70 B. haolytic Tabel (Fibrosis dry) 150 50 300 Staph. aureus 15 days Atrophy + fibrosis normal - 6.70 CI 0 100 no growth quarter (dry) Normal (atrophy) 50 100 0 350 Fibrosis (dry) normal - 6.70 78.10 100 no growth RF VIII (C) (Tweak fibrosis) 85.10 RH IX CMT pH 81.65 Dead Isolal identification / ml LF 100 _ 6.60 88.75 no growth LH 250 Tabel - 6.65 150 no growth 30 days - 6.65 Cl 200 no growth quarter - 6.68 no growth 78.10 RF Chronic Mastitis - Before Treatment 78.10 RH quarter Pll 88.70 Dead Isolate Idcnlifi / icrung / ml LF 92.30 1100 LH RF 6.75 Staph. aureus / 990 Tabel 350 RH 6.65 Cl 50 no growth LF 6.625 100 no growth LH 6,625 124.50 no growth 81.65 74.55 71.00

23 24

Table IX (a)

3 days after treatment with IG 18, dose 25 ml for RF. Second treatment of RF with IG 18.25 ml

quarter Clinically Prod CMT pH Cl Cells / ml (K) ') M on Dead Isohlt identification / ml pole 500 1150 RF Fibrosis
(clean milk) (Ib) 4+ 6.70 113, CO 650 100 150 Staph. aureus / 340 RH normal 28 6.60 74.55 50 150 150 no growth LF normal - 6.65 67.45 0 100 150 no growth LH normal 6.60 78.10 50 no growth

Table IX (b)

14 days after treating RF with IG 18, 25 ml

quarter Clinically Pnid CMT pH Cl / express · · n / ml (cl Ί To « UoIaI identification / ml pole Mon iOO 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

\ iertel Clinically Prod CMT pH Cl 7ellen / ml ι Ι0Ί Dead Isolate identification / ml Pol Mon RF Acute painful
edema mastitis,
very hard less
Milk, moist
jelly (Ib) 6.95 142.0 not countable Pseudomonas RH Enlarged and
edemaic 8th 6.80 13S not countable 1100 Pseudomonas LF Clinically normal 6.75 117 - 650 Staph. aureus Strejrt.
non-agalactiae LH normal + 6.7Ö 113 250 400 Staph. aureus
Staplx epidermidis

25th

26th

Tabel X (a) Clinical CMT pH Cl aureus) and 12 ! milk Hours after the first Improvement, acute mastitis,
interstitial edema,
Lump 6.80 to Cells / ml (10 ')
Pol Mon Tot countable Isolate identification / ml Greater than LF and LII,
Milk normal 6.70 88.75 less countable no growth normal 6.70 74.55 not 250 350 no growth 16 days after treatment for RF (Pseudomonas) and LF (Staph .;
treatment of RF and RH with Ig 17/20, dose 25 ml per quarter normal 6.55 67.45 not Staph. aureus / 330
Strept. agalactiae quarter 100 I. Staph. aureus
(major hemolysis / 390)
2nd staph. aureus (B)
3rd staph. epidermidis RF RH LF LH

Table X (b)

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

Four things clinical

Prnd CMT pH

Cl

/ ellen / ml (IO '( Pol Mon To!

Isolation identification / ml

RF Mild edema (fibrosis) (± 80% normal)

RH slight edema LF normal LH normal

(Ib) + 17

7.0 138

6.80 124

6.75 124

6.70 113

not countable

no growth

4000 - 4000 no growth

1200 200 1400 no growth

800 400 1200 no growth

Table X (c)

17 days after the last treatment of all 4 quarters

Clinical quarter Prod CMT pH Cl

Cells / ml MO " ¹ ) Pol Mon Toi

Isolate identification / ml

RF RH LF LH

Normal Normal Normal Normal

(Ib) -28

6.70 113.60 50 150 200 no growth

6.70 113.60 100 100 200 no growth

6.70 106.50 0 100 100 Staph. epidermidis / 40

6.70 102.40 0 50 50 no growth

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

Numerous experiments have shown that healthy

understand is so one nevertheless assumes that the cows that are treated with the antibody, only

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

education of macroorganisms in connection with one have completely disappeared.

Example 3

Another suitable antibiotic substance can be can be used instead of oiler in addition to the Sabsumz given in Example 1.

In general, an in vivo efficiency index of approximately 40% is achieved with penicillin G, with semi-synthetic penicillin or a combination of

Penicillin G plus streptomyein 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 udder being hard, swollen and tender.

A single D'yiis of 2 ml was added per quarter administered once.

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 ίο mother goats. Even in these cases the same mastitis-healing successes were achieved.

Claims (1)

Patent claims: 1. PolyvaJentes Mastitis Immunoglobulin, obtained from the blood of a large number of cows raised on s suffer from different types of chronic mastitis, characterized by the in itself known process steps: