IL33614A - Mastitis immuno-globulin,its preparation and use for treating mastitis in cows - Google Patents

Description

Kastitis immuno-glo¾ulin, Its preparation and use for treating mastitis in cows AORICURA LABOBATORIA IIMIT.RD C:-31898 THIS INVENTION relates to the treatment of cows, sheep ewes and she-goa.ts, either therapeutically or prophylicaticall , against mastitis and to the products used in the treatment and to production of these products.

Mastitis is one of the most important erosion diseases of cows. Apart from the heavy losses which the milk farmer, and consequently the dairy industry, suffers as a result of damaged udders and lower milk production, the use of infected milk is a serious public health problem . It is therefore not surprising that mastitis today, as in the past 10 to 15 years, enjoys the intensive attention of researchers throughout the world. Nevertheless, there has been a progressive increase in the incidence and degree of . the infectious disease-complex, even though antibiotic remedies are used in large quantities and preventative precautions, such as hygiene and the use of vaccines, are taken 33614-2 Active immunity by way of systematic vaccination of the cow with a vaccine is generally of little value.

These vaccines can produce circulating antibodies in the blood, but, the sera agglutinin and precipitin in the blood cannot enter or be taken up by the udder and are thus of no value in preventing mastitis.

Antibiotics are generally only valuable as additives to the vaccines and alone are incapable of controlling mastitis due to various shortcomings. Narrow-spectrum antibiotics, for example . penicillin , are of minor value today because their action is confined to a certain group of organisms, for example sensitive, gram-positive organisms, while mastitis is a complex infection. This criticism applies to Penicillin G, the semi-synthetic Penicillins and other narrow-spectrum antibiotics.

The available wide-spectrum antibiotic remedies or combinations of narrow-spectrum antibiotics are not always effective and are also generally too expensive to be used as standard control measures. Not all wide-spectrum antibiotics can be mixed because1 their actions are. antagonistic, while other antibiotics are not suitable for intra-udder administration. , The injudicious and continuous use of an ibio ic remedies has resulted in supra-infections (i.e. infections in addition to the mastitis treated) as well as the development of resistant forms of organisms which were previously sensitive. This applies to both the narrow-spectrum and wide-spectrum antibiotic remedies.

In addition, the excretion of antibiotics in milk is a serious public health problem.

Mastitis also occurs in other animals, particularly sheep ewes and she-goats and, where applicable, the term "cows" as used in this specification and claims is to be interpreted as including sheep ewes and she-goats.

The present invention relates to a method of. treating mastitis in cows (as just defined) and to produces for the treatment which can be prepared economically.

Accordingly the present invention provides a method of treating, either therapeutically or prophylactically , mastitis in cows (as hereinbefore defined) which comprises the intramammary introduction of mastitis iminuno-globulin (as hereinafter defined), into the cows. _ - A modification of this basic method of treatment uses the mastitis immuno-globulin in conjunction with one or more other anti-microbial agents, instead of using the mastitis immuno-globulin by itself.

In the method of treatment, and in its modification , it has been found preferable to use a polyvalent mastitis immuno-globulin as hereinafter defined, and especially to use a polyvalent heterologous mastitis immuno-globulin.

The invention also provides an antibody product, namely mastitis immuno-globulin, and compositions c ontaining it and one or more other antimicrobial agents.

In order that the invention may be understood more readily, the following definitions are given.

By "gamma-globulin" as used in this specification is meant that globulin fraction of the plasma of the blood of cows (as hereinbefore defined) which in neutral or alkeline solution has the lowest electrophoretic mobility, "Mastitis immuno-globulin" (often hereinafter shortened to "immunoglobulin") is gamma-globulin (as just defined) having antibody activity against mastitis and obtained from the blood o 33614-2 a cow which is suffering from chronic mastitis arid/or is in a hyper-immune state with respect to mastitis.

"Polyvalent mastitis immuno-globulin" is mastitis immunoglobulin (as just defined) having antibody activity against many of the bacteria and/or toxins responsible for mastitis. "Non-specific mastitis immuno-globulin" is mastitis immuno-globulin (as defined above) or polyvalent mastitis immuno-globulin (as just defined) having antibody activity against bacteria and/or toxins responsible for mastitis but which bacteria and/or toxins may or may not be involved in the particular case of mastitis being treated.

Such immuno-globulins contain antibodies against mastitis which may be either complete antibodies, incomplete antibodie or both. "Complete antibodies" are effective against both a given strain of bacteria causing mastitis and the toxin produced : thereby. "Incomplete antibodies" are effective against either the given strain of bacteria or the toxin produced thereby. As many types of bacteria may be involved it is, of course, possible for both complete and incomplete antibodies to be present in a given sample of these mastitis immuno-globulins. . ' . · ' ■ The invention also provides a process for preparing mastitis immuno-globulin (as hereinbefore defined ) which compr ses - . (a) mixing blood obtained from a cow which is suffering from chronic mastitis and/or is in a hyperimmune state with respect to mastitis, with an anticoagulant and an antihaemolytic ; (b) removing the blood cells by centrifuging to form a plasma; (c) converting the plasma to a gel by adding calcium chloride solution; (d) straining the gel to produce a serum; (e) precipitating the mastitis irnmuno-globulin from the serum by adding ammonium sulphate solution; (f) filtering off the mastitis irnmuno-globulin and removing ammonium sulphate by dialysis; (g) removing large-molecule protein impurities by further . centrifuging. 33614-2 The invention further provides for the blood or serum, from which. the mastitis immunoglobulin is obtained to be pooled from a number of the cows and to produce therefrom a polyvalent mastitis immuno-globulin (as hereinbefore defined). It will. of course be appreciated that the pooling may, in fact, be carried out, either partly or completely, at any stage of the process, and, if partly carried out, completed at some later stage.

The basic method of treatment and its modification are best illustrated by examples. If the udder of a cow is infected by, for example, a specific gram-positive organism which causes mastitis, ^the organism may be treated effectively in any one of several possible ways. An immuno-globulin may be used which contains antibody specific to that gram-positive . organism and if the concentration of that specific antibody in the immuno-globulin is high enough to be pharmacologically effective, the immuno-globulin may be used alone, i.e. without the use of any additives. An alternative treatment uses an immuno-globulin that does not contain antibodies specific to that organism but which does contain antibodies generally effective against masti is-causing organisms i.e. a non-specific mastitis immuno-globulin. ' Such immunoglobulin may be obtained for example as follows j 33614-2 A specific bacteria , e.g. staph, aureus phage type 1 is grown in or on a medium that would promote maximum production of a and/or β toxin or bacterial cells. A cow (or one group .of cows) is hyper-immunised with the purified toxin and another cow (or a different group of cows) with the bacterial cell components (anti-bacterial immunity). The. cows are bled and from the pooled blood of the two cows (or groups of cows) monovalent mastitis immuno-globulin is obtained by the procedure described below in Example 1..

Generally in this latter case it will be necessary to use the non-specific mastitis immuno-globulin in conjunction with an anti-bio.tic or other chemotherapeutic agent that is effective against the specific mastitis-causing organism when it is in a non-resistant state with respect to the anti biotic or chemotherapeutic agent.

A series of experiments conducted on cows infected with mastitis has suggested that the immuno-globulin increases the sensitivity of the organisms to the action of the antimicrobial agent by immobilisation and growth inhibition of the micro-organisms in conjunction with increased phagocytosis. 33614-2 Accordingly it is thought that, if an organism has built up a resistance to the drug, on administration of the drug in conjunction with a non-specific mastitis immuno-globulin , the immuno-globulin breaks down the resistance of the organism to that antibiotic which will in turn be effective against the organism and so enhance the therapeutic effect of the immuno-globulin, i.e. the combination is synergistic; Generally, in practice, the immuno-globulin is used with a preservative, a diluent (as hereinafter defined), and is mixed with an anti-niicr,obial agent as referred to above.

• As a further example of the method of treatment, if a cow infected with mastitis has been treated with, for example an antimicrobial agent, with little or no effect, the cow may be treated successfully with immuno-globulin in conjunction with that antimicrobial agent or another cheirio-therapeutic agent.

In general, when treating mastitis, it is preferable to analyse a sample of the infected milk to ascertain which microorganisms (for example gram-positive or gram-negative or a yeast or fungus) are causing the mastitis. An appropria antibiotic or anti-fungal compound can then be added to the immuno-globulin before introduction into the udder. 33614-2 A detailed description of the manner in which the. immuno-globulin may be obtained is set out below.

Blood used in the preparation.

The blood serum of normal animals contains a large quantity of protein material, inter alia, albumin and globulin. The globulin consists of different components including the gamma-globulin which contains any anti-bodies, which are present. These antibodies may develop'' in the body of an animal if that animal becomes infected. They may also develop if the animal is vaccinated with a vaccine derived from the organism causing a specific disease conditio or when the animal is suitably injected with large doses of a specific infectious organism at short intervals. These latter injections generally produce a hyper-immune state.

The serum obtainable from an animal in such ει hyper-imuiune state has some prophylactic value and can be used for establishing passive immunity. This serum may also be used for the production of the immuno-globulin.

Hyper-immune serum is usually produced from the blood of animals which have been artificially immunised by repeated injections of a specific bacterium or its toxoid at short intervals using increasingly higher doses. : The interval between injections varies from - Ik days and a minimum of 4 - 8 injections is usually required; the subcutaneous, intra-muscular or intraveneous routes or combinations thereof are used for the injections. 33614-2 Λ blood sample is taken 8 to Ik days af er the. last injection and the serum thereof tested for the presence of specific agglutinating antibodies and for their levels. If the serum is unsatisfactory, the whole process of hyper-imiiiunisation is repeated. In order to stimulate the production of higher levels of agglutinating antibody, adjuvants are used ( for example Freund's complete or incomplete ad uvants). With an adjuvant, the agglutinating, but not . necessarily the immunising, antibodies can be increased up to 10 times.

If the agglutinating titre is satisfactory the animal is bled sterilely, to obtain blood from which serum can be prepared.

To produce a polyvalent hyper-immune serum against one group of homologous organisms (e.g. Staph, aureus) at least 20, (e.g. at least J'O) , cows should be used, whereas at least 100 cows should be used to produce a polyvalent hyper-immune serum against the majority of the heterologous bacteria involved in mastitis.

The blood used may be pooled from a large number of animal suffering from chronic mastitis and/or in a hyper-immune state with respect to the disease, which animals have come from different areas. Pooled hyper-immune serum or pooled blood from such animals, contains complete natural antibodies against heterologous groups of bacteria and the heterologous types within a given group responsible for the various forms of the disease. It has been found that abattoir-slaughtered animals can be a satisfactory source of the pooled blood of naturally infected animals.

Preparation of serum The heterologous hyper-immune serum from which the desired immuno-globulin is to be obtained may be conveniently produced from. pooled blood as described below; the immunoglobulin produced is thus a polyvalent mastitis immuno-globulin.

Unlike horse blood, the blood from bovines, when allowed" to clot naturally, . produces very little serum ( 10 to 20 by volume) and haemolyses very easily. To avoid these problems the pooled blood is collected in sodium citrate (i.e. an anticoagulant) and dextrose (i.e. an antihaemolytic ) to a final concentration of 0.5 by weight of each. The plasma is separated from the blood cells by centrifuging. This produces at least 60 by volume plasma. The serum is obtained from the plasma by adding CaCl^ solution with a concentration of 3· 85 g/litre; this results in the formation of a gel.

The gel is cut into small portions and strained through a cheese-cloth to separate the clotted fibrin and fibrinogen , from the serum. The. serum yield is about 50 of the original volume of the blood used.

- The serum is then tested for specific agglutinins using hO representative bacterial isolates, for example : Staph, aureus, Strept. agalactiac, Klebsiella pneumoniae, Pseudomonas aeruginosa, E. coli and Salmonella. Satisfactory serum is then used to produce the immuno-globulin of. the invention. .

Extraction of Immuno-globulin .

The gamma-globulin to which the antibodies are attached (i.e. mastitis immuno-globulin) is precipitated from the serum with (NH )2S0 , by slowl dripping a 50 by weight (NH^^SO^ gamma-globulin precipitate is recovered by filtration, transferred to a special cellulose dialysis casing and dialysed against cold, running water to remove the (NH^^SO^. Dialysing lasts 6 -8 days, after which time the dialysate is tested with .2$ by · weight BaCl^ solution for the presence of (NH^^SO^. The (NH^ ) ^SO^-free concentrated immuno-globulin is centrifuged at 1500 r.p.m. to remove large-molecule protein impurities and is preferably sterilised by filtration.

Standardisation of the immuno-globulin may be achieved by the following methods : (a) immuno-electrophoresis for determining the gamma-globulin , i.e. mastitis immunoglobulin, concentration; (b) determination of the total protein concentration ; (c) a growth-inhibition test; (d) agglutinating antibody titre; and in addition the following were carried out- : (e) a sterility test and, (f) a' safety test.

Depending on the results of (a), (b) , (c) and (d) the concentrated product is diluted in a phosphate-bu er saline solu ion to the final conce tration required. This final concentration is generally 10-12 (w/v) protein, more than 0 by weight of which is gamma-globu lin with a minimum., of 6'l00 U agglutinins per ml (i.e. agglutination is produced at a dilution of 1 : 6400 ) and an in vitro growth-inhibi ion concentration at a dilution of 1 : 10 (i.e. 1ml immunoglobulin and 'Ti-T- sterile water or saline).

Preferably, the immuno-globulin is present as the pure concentrated polyvalent product containing, 15·5ο (w/v) protein solids preferably with more than of J*-glotaulin, and minimum agglutinin values of 6400U and and minimum growth concentration against various Staphylococci, Streptococci, E. coli, Klebsiella, Acrobacter and Pseudomonas micro-organisms at a dilution of 1 : 100. The recommended dose is : 20 ml. of the final product per quarter of a cow producing less than 3 gallons per day and 0ml. for higher producing animals .

Sterility tests were carried out on blood agar plates and the safety of the product tested by injecting double the recommended dose into a young calf or into one quarter of a normal udder.

The product is preferably in a free-dried form, which is in use, mixed with a diluent (generally water or a phosphate buffer saline solution) containing 0.25 by weight phenol as a preservative, for the preventative (prophylactic) treatment of mastitis; whereas, for the treatment of clinical mastitis, it is used mixed with 0.25% by weight phenol plus 200,000 I.U. Penicillin G and 250 mg. Dihydrostreptomycin per dose. As used in this speci ication the term "diluent" does not of course include that combination of substances additionally present when the immuno-globulin is in a hyperimmune serum. 33614-2 Examples of antibiotics or combinations thereof which may be used in place of the combination of Penicillin G and Dihydrostreptomycin are : (a) Penicillin G, by itself, (b) a semi-synthetic penicillin,, (c) F Furazolidone (d) Chloramphenicol, (e) a combination of Penicillin G and streptomycin, (f) a combination of Penicillin G .and Dihydrostreptomycin sulphate, (g) a combination of Erythromycin and Novobiocin, or (h) a combination of Neomycin and Bacitracin.

Similar compositions to those just described, but without the antibiotics, can be used to produce passive immunity against bovine mastitis and have the advantage that antibiotic is not being introduced into the animal. In this connection, it is recommended that the milk, obtained within 24 hours of the treatment . from a quarter or quarters which have been treated with an immuno-globulin plus an antibiotic, should preferably be discarded. Where no antibiotic has been used the milk need not be discarded.

Healthy cows treated with the compositions of the invention show only mild invitation which disappears after 3 to 5 days.

The compositions of the invention are preferabl stored, for example in a refrigerator, at h°C .

The treatment of mastitis in a cow using the composition of theinvention may be, as has .already been stated, either therapeutic, i.e. a curative treatment, or prophylactic, i.e. creation of active and/or passive immunity. The composition may be introduced by injection into the udder, preferably by injection through the teat canal or, alternatively, by infusion through the teat canal.

A comparison between the efficacy of antibiotic treatment and a treatment using mastitis immu o- lobulin and an antibiotic was carried out. Three infected cows were treated with three different antibiotics, no improvement was noticed The cows were then treated with a combination of mastitis irnmuno-globulin and Penicillin G. A response was noticed within three days and all the cows were completely cured a ter ten days .

The results obtained in this comparison showed that the treatment of the invention also produces at least a limited period of passive immunity, to mastitis, for example, 6 to 8 weeks. Further, it was found that artificial or natural infection of the cow during such a period of passive immuriit acted as a booster which indicated that a resultant active immunity was present.' It will, of course, be appreciated that the irnmuno-globulin for treatment of sheep and goats 33614-2 may be obtained from these animals themselves and that it is not essential that the blood of cows, here bovine femals, be used for this purpose. This specification and claims should therefore be read in the light of this' and thus, where applicable, the term "cow" should be understood to include or to be replaced by 'fewe!! or "she-goat" as appropriate.

The following Examples illustrate the invention.

E X A M P L E I Isolation of a sample of . mastitis immuno- lobulin .

A total of 20 litres of blood was collected from a number of cows suffering from different types of chronic mastitis. 170 ml. of a 50$ w/v solution of sodium .citrate in 5 (w/v) aqueous dextrose solution were added to the pooled blood.

The mixture was transported to the laboratory and there centrifuged in a continuous-type centrifuge at 3000 r.p.m. for 30 minutes to separate the red blood cells from the plasma.

The plasma was treated with a solution of calcium chloride containing 3·¾5 grams of calcium chloride per litre to coagulate: it. The coagulate gel formed was broken up and suspended on mutton cloth for the serum to drip out and be collected.

Ammonium sulphate was then used to precipitate the serum.

Practical precipitation was effected using different amounts of ammonium sulphate and the protein precipitate was analysed by electrophoresis. The results were as follows : Grams of ammonium sulphate per 100 mis. of serum were used for the bulk precipitation of the fc* - globulin, which was centrifuged off 8 hours later, placed into sacs of regenerated cellulose film and dialysed at 4°C, with running tap water for 5 - 7 days, and then with 0.8 by weight aqueous NaCl solution for 2 days to remove the ammonium sulphate. The resultant product was concentrated, to ■ - l6 (w/v) solids, filtered and then standardised by known agglutination and growth inhibition tests using different degrees of dilution of the product. This filtered product is herein after referred to as "the product of Example I". 1 ml. of the above product was added to a first tube containing 49. ml. water to give a solution with dilution 50; this solution was then diluted so that the dilution doubled at each stage of the ' dilutio . A total of 13 tubes were used, one ©f which was a: control. , 33614-2 The results of the agglutination, tests are given in Table I; the dilution is the number of parts, by volume, of water containing 1 part of the product.

Also + means little agglutination 2+ means strong agglutination 3+ means very strong agglutination + means very strong agglutionation indeed and N means no agglutination.

( Where the name of a micro-organism is repeated it is to be understood that an tigenically different strains of it are involved . ) Agglutination tests for the serum before precipitation, the immuno-globulin (50$ (w/v) concentrated and fully concentrated) are shown in Table II.

It can be seen from the Tables that the product is polyvalent, i.e. is a polyvalent mastitis immuno-globulin. - 1 -2 T A B L E II AGGLUTINATION MICRO-ORGANISM Serum before Product 50 w/M Concentrated Precipitation Concentrated Product -Bacillus 200 800 1/600 Streptococcus 400 6400 51,200 Staph* aureus 3200 102400 102, 00 Staph, aureus 3200 256ΟΟ 12,800 Staph, aureus 3200 6400 25,600 E. coli 6400 6400 12,800 E.coli 200 800 1,600 + Bacillus 6400 12800 3, 200 + Bacillus 6400 32ΟΟ 12,800 Staph. Epider- midis 400 100 800 Staph. Epider- midis 1600 200 6,400 Staph, aureus 3200 . 200 400 Staph. aureus 1600 400 800 Klebsiella 3200 32ΟΟ 25,600 Staph.aureus . 1600 800 1,600 Staph. aureus 3200 400 6,400 Staph. epider- midis 256ΟΟ 400 102, 400 Staph. epider-r&n rnidis 12800 6400 102, 00 Staph.aureus 32ΟΟ 800 ! 25 i 00 Staph. aureus 256ΟΟ 800 102,400 Pseudomonas 256ΟΟ 6400 51, 200 Streptococcus 256ΟΟ 12800 102, 400 Staph. aureus 256ΟΟ 800 ' 51, 200 Staph. epider- midis 12800 160ό 25,600 - E !X A M P L E 2 The anti-bacterial activity of the product of Example 1 is shown in Table 11. The micro-organisms used in the determination of the anti-bacterial activity are the standard micro-organisms responsible for about 90% of cases of mastitis .

In this Table : Heading (l) means growth visible in tube; Heading (2) means growth only visible on blood agar plate + etc and N have the same meanings as in Table I; Ig stands for immuno-globulin ; P stands for Penicillin G at a concentration of 5 gamma per ml of sample; S stands for dihydrostrepomycin at a concentration of 125 gamma per ml of sample.

It can be seen that the immuno-globulin itself is bacteriostatic and that when mixed with a combination of penicillin and dxhydrostreptomycin, an increased bactericidal effect is obtained compared with when these two antibiotics are present together but in the absence of the immuno-globulin.

T A B L E III In Table .III bacterial growth was- evaluated in terms of the turbidity of the growth medium.

E A M P L E ■ 3 The product of Example I, when freeze-dried , can be stored in bottles. To prepare a composition for application this product may be mixed with 20 ml of sterile water and 0.25$ by weight of phenol (as a preservative), 200,000 International Units of Penicillin G and 250 mg. of dihydro-streptomycin sulphate.

Generally 10 - 50 ml of this composition, but containin no antibiotics, can be infused per quarter of an udder, and generally gives at least 6 - 8 weeks protection by way of passive immunity against mastitis.

Any other suitable antibiotic substance can be used instead of, or i additio to, the Penicillin G and di-hydrostreptomycin . 1 33614-2 E Λ M P L E Results obtained for some cows treated for mastitis are shown in Table Groups IV, V, VI, VII, VIII, IX and X.

In these Tables : "Quarter" means quarter of an udder (RF. RH. LF . LH. standing for right front, right hind, left front and left hind respectively); "Prod." stands for milk production per day; "CMT" stands for California Mastitis Test; "CI" is the chloride content of the milk; y ■ "Pol." stands for the number of Polymorphonuclear Leucocytes/ml of milk; "Mon." stands for the number of Mononuclear Leucocytes/ ml of milk; "Tot." stands for total number of Leucocytes; "Tot/ml" stands for total number of bacterial isolates/ ml . of milk ; "a" means "unaccountable" when used when a number is otherwise expected. 33614-2 T A B L E IV Chronic Mastitis - Mixed Infection Before Treatment T A B L E IV(a) Before treatment with Immunoglobulin. days after treatment of RF and LP with Immuno Globulin (Z doses at 12 nr. intervals), plus 0.25$ by weight Phenol, 200,000 I.U. Penicillin G and 250 mg Dihydrostreptomycin per dose.

T A B L E IV(b) 33614-2 Table Group V I T A B L E V I Before Treatment T A B L E V(a) 60 Hours after treatment of all 4 quarters with Immuno-Globulin plus Phenol 0.25$ by weight ; dose: 50 ml per quarter T A B L E V(b) 8 Days after one treatment with Immuno- Globuli Isolate Cells /ml (10:) Quarter Identifica Clinical Prod CMT PH CI Pol MonITot tion/ml Minute (lb) 4+ 6.85 124.15 1000 0 1000 1. B-haenio- RF Fibrosis lytic staph, aureus/ 800. 2. Staph, epider- midis/2 RH It >" 28 . 2+ 6.75 117.30 000 300 900 B-haemo- lytic staph. aureus LF ti + 6.70 102.80 500 500 1000 B-haemo- lytic staph, aureus LH + 6.70 9 . ^O OO 600 900 ■ A B L E V(c) 22 Days after first treatment with immuno-globulin (alone) and lb days after second treatment with immuno-globulin plus 0.25 Phenol by weight, 200,000 I.U. Penicillin G and 250 mg Dihydrostreptomycin per dose. 361 - T A B L E Vl(a) Ik days after 2nd treatment T A B L E VI (b) 21 days after 3rd Treatment Table Group VII T A B L E VII Chronic Mastitis - Before Treatment T A B L E Vll(a) 3 Days after treatment of RF, RH , LF with immuno-globulin ; dose: 50 ml per quarter T A B L E Vll(b) 6 Days after Treatment T A B L E VIl(c) l6 Days after Treatment Cell-5/ml( 103) Isolate Quarter Clinical Prod CMT PH CI IdentifiPol Mon Tot cation/ml RF Localised (lb) Fibrosis 6.65 88.50 0 200 200 No growth RH II 27 6.6Ο 88.50 200 50 25Ο No growth LF Normal - 6.6Ο 78.10 0 0 0 No growth LH Normal - 6.60 74.55 50 150 I50 No growth able roup T A B L E VIII Before Treatment.

T A B L E VIII (a) 60 hours after 2nd treatment of RF and RH with an immunoglobulin which is a mixture of two different batches of imniuno-globulin ; dose 5 ml and 72 hours after treatment of LF and LH with Immuno-Globulin (25 ml of the immuno-globulin mixture, 200,000 I.TJ. Penicillin G and 0 mg. Dihyclros trep to- myc: LnJ Cells/mi(i0->) Isolate Quarter Clinical Prod CMT pH 01 Identifi¬ Pol Mon Tot cation/ml RF Fibrosis "(lb) 4+ 6.80 128.40 1000 400 1400 No growth.

RH Minute 4+ 6.80 II3.6O 800 200 1000 Staph. aureus Fibrosis (B.haemolytic 60 LF Fibrosis 4+ 6.70 102.80 1200. 800 2000 No growth + Atrophy . LH Hardening 4+ 6.70 I34. O 800 400 1200 No growth . -2 T A B L E Vlll(b) Days after last, treatment T A B L E VIII (c) days after last treatment Isolate Quarter Cells/ml Clinical Prod (l03) CMT pH CI Identifi¬ Pol Mon Tot cation/ml RF Normal (lb) - 6.6Ο 78.10 0 100 100 No growth (fibrosis, dry) RH Normal > 32 - 6.65 78.10 50 200 25Ο No growth LF . Normal - 6.65 88.70 0 I O I O No growth (Atrophy) . LH Normal - 6.68 92.30 100 100 200 No growth (Fibrosis dry) Table -Group IX 3361 -2 T A B L E Chronic Mastitis Treatment TABLE IX.(a) 3 days after one treatment of RF with an immuno-globulin which. as one of the two batches used in Table VIIl(a); dose 25ml, 2nd Treatment of RF with T A B L E IX(b) 14 Days after treatment of RF with the immuno-globulin; dose 25 ml ! Isolate Cells/ml(l03) Quarter Clinical Prod CMT pH Cl Identifi¬ Pol Mon Tot cation/ml RF Fibrosis (lb) - 6.7O 92.80 100 200 300 No growth (dry) RH Normal 3 -■ 6.65 88.10 0 100 100 No growth LF II 6.65 81.20 50 50 100 No growth lh II - 6.6O 78.10 0 200 200 No growth T A B L E X Table. Group X 61 -2 Treatment of an artificially caused · mastitis .

Before treatment and 14 days after exposure of RF T A B L E X(a) l6 days after exposure of RF (Pseudorrionas) and LF ( Staph . aureu s ) and 12 hours after first treatment of RF and RH with an immuno-globulin which is a mi ure' f two different batches of immuno-globulin ; one of the batches was one of those used in the Table VIIX(a) (not the one used in Table IX(a) ) ; dose : . mi' per quarter Isolate Quarter Clinical Cells/ml(l03) CMT pH Cl Identifi- Pol iMonjTot cation/rnl RF Improvement ; - 6.80 Too litt:e milk No growth j acute mastitis interstitial oedema; lumps RH Larger than LF 6.70 88.75 Uncoimtatlie No growth and LH,milk normal LF Normal 6.70 74.55 Uncovantatile Staph. aureus 330 Strept . aga- lactiae LH Normal 6.55 67.45 100 25O 350 ¾ureaih(l v ■ SomoYysis 2. Staph. , aureus (B ) 3. Staph »epi- 1—d_e—rn.iitlxs . 14-2 T A B L E X(b) 4 days after 1st treatment of RF and RH; 2 days after 2nd treatment of RF and RH and 1st treatment of LF and LH with the immuno-globulin used in the above Table. Dose per treatment 25 'ml/quarter T A B L E X(c) 17 days after last treatment ' of all four quarters.

E X A M P L E 5 An ewe of German Merino breed, suffering from mastitis or "blue udder" whose udder was hard, swollen t · and sensitive was used. The ewe was treated With immunoglobulin from the pooled blood of slaughtered cows i.e. bovine females; a single dose, administered once only, of 2 ml of the product of Example 1 was used per quarter. After two days the udder of the ewe had returned to normal.: The experiment was repeated (with another ewe) with the same satisfactory results. I * Similar experiments were conducted on the goats and once again the mastitis was cured.

No detailed tables have been provided for these experi-ments as only single treatments, and at reduced dosage compared with those for cows, were required.

E X A M P L E 6 33614/2 i . ' A specific bacteria, e.g. staphV aureus \ phagetyp® 1 is grown in or on a medium that would promote maximum production of a and/or β toxi or bacterial cells. A cow (or one group of cows) is hyper-immunised with the purified toxin and another cow (or a different group of cows) with the bacterial cell components (anti-bacterial immunity). The cows are bled and from the pooled blood of the two cows (or groups of cows) monovalent mastitis immunoglobulin is obtained b the procedure described i Example 1.

Claims (9)

1. CLAIMS 1. Mastitis immuno-globulin as hereinbefore defined .

2. Mastitis immuno-globulin according to Claim 1 which is a polyvalent mastitis immunoglobulin (as hereinbefore defined). 3· Mastitis iiiiiiiuno-globulin according to claim 1 or 2 containing complete antibodies (as hereinbefore defined) . h . Mastitis immuno-globulin according to claim 1 or 2 containing incomplete antibodies (as hereinbefore defined). 5. Mastitis immuno-globulin according to claim 1 or 2 containing both complete and incomplete antibodies. 6. Mastitis immuno-globulin according to claim 3 or 5 n which the complete antibodies are specific to heterologous organisms causing mastitis. 7. Mastitis immuno-globulin according to any of claims 1 to 6 which comprises 10 - 12$ (weight for volume) .protein, more than 90 by weight of which is gamma" globulin (as hereinbefore defined) , and has a minimum of ^100 U agglutinins per ml and an in vitro growth inhibition concentration at a dilution of 1 : 10. 8* Mastitis immuno-globulin according to any of claims 1 to 7 which also. , comprises a preservative. Ί . ■ 9. Mastitis immuno-globulin according to claim 8 in which the preservative is phenol. 33614-2 10. Mastitis immuno-globulin according to any of claims 1 to 9 which also comprises a diluent as hereinbefore defined. 11. Mastitis itnmuno-globulin according to claim 10 a wherein the diluent is water or/phosphate -buf er saline solution. 12. Mastitis immuno-globulin according to any of claims 1 to 11 in a freeze dried form. 1

3. Mastitis imrnurio-gl.obulin according to any of the pi"oceding claims which also comprises one or more other antimicrobial agents. 1 . Mastitis immuno-globulin according to claim 13 in which the other antimicrobial agent is an antibiotic. Ί 15. Mastitis imnvuno-globulin according to claim Ik in which the antibiotic 'is : (a) Penicillin G, (b) a s.erni- sy thetic penicillin, - A3 - 33614-2 (c) F Furazolidone, (d) Chloramphenicol, (e) a combination of Penicillin G and stre tomycin, (f) a combination of Penicillin G and dihydrostreptoinycin , (g) a combination of Penicillin G and dihydrostreptomycin sulphate, (h) a combination of erythromycin and novobiocin, or (i) a combination of neomycin and bacitracin, l6. Mastitis immuno-globulin according to claim 1 which also contains an antibiotic. 17. Mastitis immuno-globulin according to claim l6 wherein the antibiotic is Penicillin G. 18. Mastitis immuno-globulin according to any of claims 1 to 17 substantially as hereinbefore described. 19· Mastitis immuno-globulin according to any of claims 1 to l7 substantially as described in any of Examples 1 to 5 20. A process for preparing mastitis immuno-globulin (as hereinbefore defined) which comprises - - k - 33614-2 (a) mixing blood obtained from a cow which is suffering from chronic mastitis and/or is in a hyper-'immune state with respect to mastitis with an anticoagulant and an anti- haemolytic; (b) removing the blood cells by centrifuging to form a plasma; (c) convertin the plasma to a gel by adding Ί calcium chloride solution;. (d) straining the gel to produce a serum; (e) precipitating the mastitis immuno-globulin from the serum by adding ammonium sulphate solution; (f) filtering off the mastitis immuno-globulin and removing ammonium sulphate by dialysis; (g) removing largeyiiiolecule protein impurities by further centrifuging. 21. A process according to claim 20 wherein the cow is suffering from chronic mastitis. 22. A process according to claim 20 wherein the hype immune state has been reached by natural infection. 23. A process according to claim 20 wherein the hype immune state has been reached by artificial infection. - h5 - 2

4. A process according to any of claims 20 to 3 wherein the blood is obtained from a pre-selected slaughtered cow . 25^ A process according to any of claims 20 to 23 wherein the blood is obtained by bleeding a living cow. 26. A process according to any of claims 20 ΐο 25 wherein the blood used in (a) is obtained by pooling the blood of a number of cows. 27· A process according to any of claims 20 to 25 wherein the serum used in (e) is obtained by pooling the sera derived from a number of cows. 28. A process according to claim 26 or 27 wherein at least 20 cows are used. 29. A process according to claim 28 wherein at least 100 cows are used. . 30. A process according to any of claims 20 to 29 wherein the anticoagulant in (a) is sodium citrate and the antihaemolytic is. dextrose. - 4.6 - 3-Ί-. Λ process according to any of claims 20 to 30 which comprises a further step, (h), in which the mastitis iniiiiuno-globulin is sterilised by filtration.. 32 . A process according to any of claims 20 to 30 wherein the mastitis immurio-globulin is freeze-dried. 33 · process according to any of claims 20 to 32 wherein the mastitis immuno-globulin is subsequently mixed with a diluent as hereinbefore defined. 3^ . A process according to claim 33 wherein the diluent is water or 'a phosphate buffer saline solution. .35 · A process according to claim 33 or' 3¾ wherein the diluent contains a. preservative. 36 . A process according to claim 35 wherein the preservative is phenol. 37 . A process according to any of claims 20 to 3 wherein the mastitis immuno-globulin is mixed with one or more other antimicrobial agent. 33614- Ί 38. A process according to claim 37 wherein the other antimicrobial agent is an antibiotic. 39· A process according to claim 38 wherein the antibiotic is : (a) Penicillin G; (b) a semi-synthetic penicillin; (c) F Furazolidone; (d) Chloramphenicol; (e) A combination of Penicillin G and streptomycin; (f) a combination of Penicillin G and dihydrostreptomycin; (g) a combination of Penicillin G and dihydrostreptomycin sulphate; (li) a · combination of erythromycin and novobiocin; or (i) a combination of neomycin and bacitracin.. ■40. A process according to claim 20 in which the cow is in a hyper-immune state w.ith respect to mastitis. 4l. A process according to claim 20 substantially as hereinbefore described. . A process according to claim 20 substantially as described in Example 1 or 3 · 33614-2 3. Mastitis itiimuno-globulin as defined in any of claims 1 to 12, 18, or 19» whenever prepared by a process as claimed in any of claims 20 to 36, l or 42. 44. Mastitis iiiirnu o-globulin as defined in claim 1 whenever prepared by a process as claimed in claim 40. '

5. Mastitis immuno-globulin as defined in any of claims 13 to 17 whenever prepared by a process as claimed in any of claims 37 to 39. 4

6. Mastitis immuno-globulin according to claim 44 which also contains an antibiotic. 4

7. Mastitis immuno-globulin according to claim 46 wherein the antibiotic is Penicillin G.. 4

8. Λ method of treating, either prophylactically or therapeutically, mastitis in cows (as hereinbefore defined) which comprises the intra-mammary introduction ■ of mastitis immuno-globulin (as hereinbefore defined) into the cows. 4

9. A method according toclaim 48, used therapeutically. 50. A method according to claim 48 or 49 in which the introduction is effected by injection into the body of the udder. 33614-2 51. A method according to claim 48 or 49 in whifch the introduction is effected by injection through the teat canal. 52. A method according to claim 48 or 9 wherein the introduction is by infusion through the teat canal. 53· A method according to any of claims 48 to 1 wherein the mastitis irnmuno-globulin is a mastitis immunoglobulin as defined in any of claims 1 to 12, 18, 19 or Ό.· . 54. A method according to claim 48 or 5 wherein the mastitis irnmuno-globulin is a mastitis irnmuno-globulin as defined in .claim 1 or 44. , 55. A method according to any of claims 48 to 1» wherein the mastitis irnmuno-globulin is a mastitis immunoglobuli as defined in any of claims 13 to 17 or 45. 56. A method according to claim 48 or 52 wherein the mastitis irnmuno-globulin is a mastitis irnmuno-globulin as defined in claim l6 or 46. 57 . A method according to claim 8 or 52 wherein the mastitis iiiimuno- lobulin is a mastitis immunoglobulin as defined in claim 17. or 7 . 58 . A method according: to claim 8 substantially as hereinbefore described. 59· A method according to claim 48 substantially as described in Example 3 to 5«