Plasma Ceruloplasmin and Fibrinogen during Enzyme Therapy of Mastitis in Dairy Cows

Bake‰ J , J . I l lek: Plasma Ceruloplasmin and Fibrinogen during Enzyme Therapy of Mastitis in Dairy Cows. Acta Vet. Brno, 2006, 75: 241-246. The aim of this study was to demonstrate the benefits of topical administration of proteolytic enzymes used in the symptomatic mastitis therapy. Eleven lactating cows with clinical signs of mastitis in one udder quarter at least were divided into two groups. Group A was given an antibiotic alone cefotaxime (250 mg, intramammary administration, 6 doses in total). Group B was given a lower dose of the same antibiotic (cefotaxime, 100 mg, intramammary administration, 5 doses in total) and a blend of enzymes in one drug form, i.e. both the dose and total number of administrations were lower in this group. Before and after the therapy, milk samples were collected and subjected to microbiological examination, and blood samples were withdrawn to determine fibrinogen and ceruloplasmin as acute phase proteins. Before the beginning of the therapy, common mastitis pathogens were detected in milk of both groups; after the end of the therapy, the test results were negative. Mean plasma concentrations of fibrinogen in Group A increased significantly from 6.0 ± 1.4 to 7.4 ± 1.1 g·l-1, in Group B a non-significant decrease was observed, from 6.5 ± 0.86 g·l-1 to 6.4 ± 0.9 g·l-1. However, differences between the groups were significant (p < 0.05). In Group A, mean plasma ceruloplasmin concentration increased significantly from 0.9 ± 0.2 to 1.3 ± 0.18 μmol·l-1, and in Group B it decreased non-significantly from 0.9 ± 0.36 to 1.1 ± 0.26 μmol·l-1, and the difference between the groups was non-significant. In the present study, a favourable effect of repeated topical application of a mixture of proteolytic enzymes was demonstrated. The enzyme therapy resulted in a decreased therapeutic dose of the antibiotic, and suppressed an increase in the levels of inflammation markers under investigation. Bromelain, cefotaxime, chymotrypsin, papain Particularly during acute mastitis, the mammary gland shows typical signs of inflammatory process. In order to provide the shortest and most effective therapy of acute mastitis, it is necessary to support causal therapy, focused on the causative agent (antiinfectives) using symptomatic therapy to suppress the inflammation (antiflogistics). In practice, glucocorticoids have been widely used, non-steroid antiflogistics (NAF) and enzymes have been used less frequently. Many studies demonstrated that the effects of enzymes are comparable with those of NAF (Salamberidze et al. 2002). The systemic effect is the most pronounced in glucocorticoids, even when it comes to undesirable effects. Unlike enzymes, glucocorticoides show an anti-inflammatory effect accompanied by immunosuppression. Positively adjuvant effects of enzyme therapy include the potentiation of antibiotic effect at a site of inflammation, an increase in the acute cell response and an anti-oedema effect of trypsin, chymotrypsin and bromelain, and an oedema-protective effect of papain (Kasserol ler and Wenning 2003). Doses of antibiotics, when combined with the enzyme therapy, can be decreased to a half of the monotherapeutic ones (Suchich et al. 1997), and at the same time the regression of clinical disease signs is accelerated (Tinozzi and Venegoni 1978; Luert i and Vignal i 1978; Neubauer 1961). Proteases ACTA VET. BRNO 2006, 75: 241–246 Address for correspondence: PharmDr. Josef Bake‰ Chief pharmacist, Hospital Pharmacy Podfiipská nemocnice s poliklinikou Roudnice n.L., s.r.o. Alej 17. listopadu 1101, Czech Republic Phone: +420 606 603 578 E-mail: lekarnik@seznam.cz http://www.vfu.cz/acta-vet/actavet.htm change the spectrum of adhesive molecules in the cell membrane (Wald et al. 2001), which favourably influences the production of different types of cytokines. Proteases increase the clearance of anti-inflammatory cytokines (Desser et al. 1999; Desser et al. 2001; Xiang et al. 2001), as well as plasminogen concentration in blood. They also decrease fibrin formation and dissolve the existing thrombi. Decreased thrombocyte aggregation helps to maintain correct microcirculation (Nouza et al. 1999). The aim of the present study was to demonstrate the beneficial effects of topical administration of proteolytic enzymes used as symptomatic therapy of mastitis with decreased total antibiotic doses. For the evaluation, we have chosen the results of development of ceruloplasmin and fibrinogen dynamics and microbiological examination of collected milk and blood plasma samples. Materials and Methods A field part of the study was performed on the farm Tû‰any u Brna, owned by the company AGRO MONET a.s. Moutnice. The study included 11 lactating cows that met the following criteria: clinical signs of inflammation present in at least one quarter, 4-12 years of age, an apparent and unplanned decrease in the volume of milk produced, positive results of barn mastitis test, changes in milk quality. Right after the allocation to the study, each lactating cow was placed in quarantine. Collected milk was discharged until the end of withdrawal period of a drug used. Lactating cows were randomly allocated to two groups. Group A received only a broad-spectrum ß-lactam antibiotic, cephalosporin of 3rd generation – cefotaxime at a single dose of 250 mg, made by galenic modification of Taxcef inj. 1 × 1 g (Ranxbay, UK) in the sterile suspension-ointment basis. The ointment was applied twice a day for 3 days intramammarily, only to inflamed quarters. Group B received the same antibiotic (cefotaxime 100 mg) and a mixture of enzymes in one drug form. The enzyme mixture was made from 3 coated tablets of the registered enzyme preparation Wobenzym drg. (Mucos, Germany), modified galenically. Proportions of proteolytic activities were as follows: 46.15% papain, 18.46% trypsin, 0.77% chymotrypsin a 34.61% bromelain. The preparation was applied only into inflamed quarters. The following protocol was used: first and second day one dose BID, third day: one dose SID. Both the dose and number of administrations were lower, as compared with Group A. In both groups, the drugs were administered in the form of intramammary sterile suspension ointment at a dose of 10 cm3, by a disposable sterile injector. A conic tip of the injector was carefully inserted into the teat of an affected quarter. By pressing the injector, all its contents were squeezed into the quarter. This was always done after thorough milking and teat tip disinfection. The preparation was kept refrigerated at 2 8 °C. It was taken out of the refrigerator about 15 min before the administration. Before and 12 24 hours after the therapy, milk and blood samples were collected. Milk samples were obtained by squirting the middle flow of milk from all the productive quarters to sterile glass test tubes. The samples were examined at the microbiological laboratory of the State Veterinary Institute Brno for the presence of Streptococcus agalactiae, Str. dysgalactiae, Str. uberis, Staphylococcus aureus, E. coli and Klebsiella. Blood was withdrawn from the tail vein into plastic vacuum sterile tubes and was used to determine fibrinogen and ceruloplasmin as acute phase proteins. Fibrinogen was measured by the turbidimetric analysis, using the set FIBRINOGEN Boehringer Mannheim. Ceruloplasmin levels in the serum were measured (Henry et al. 1974) using the biochemical analyser COBAS MIRA S, at the laboratory of clinical biochemistry at the Clinic of Ruminant Diseases of the Faculty of Veterinary Medicine, University of Veterinary and Pharmaceutical Sciences Brno. For the statistical analysis of levels of both acute phase proteins, F-test and paired and non-paired t-tests were used. The difference was regarded as statistically significant at p < 0.05.

Particularly during acute mastitis, the mammary gland shows typical signs of inflammatory process.In order to provide the shortest and most effective therapy of acute mastitis, it is necessary to support causal therapy, focused on the causative agent (antiinfectives) using symptomatic therapy to suppress the inflammation (antiflogistics).In practice, glucocorticoids have been widely used, non-steroid antiflogistics (NAF) and enzymes have been used less frequently.Many studies demonstrated that the effects of enzymes are comparable with those of NAF (Salamberidze et al. 2002).The systemic effect is the most pronounced in glucocorticoids, even when it comes to undesirable effects.Unlike enzymes, glucocorticoides show an anti-inflammatory effect accompanied by immunosuppression.Positively adjuvant effects of enzyme therapy include the potentiation of antibiotic effect at a site of inflammation, an increase in the acute cell response and an anti-oedema effect of trypsin, chymotrypsin and bromelain, and an oedema-protective effect of papain (Kasseroller and Wenning 2003).Doses of antibiotics, when combined with the enzyme therapy, can be decreased to a half of the monotherapeutic ones (Suchich et al. 1997), and at the same time the regression of clinical disease signs is accelerated (Tinozzi and Venegoni 1978;Luerti and Vignali 1978;Neubauer 1961).Proteases change the spectrum of adhesive molecules in the cell membrane (Wald et al. 2001), which favourably influences the production of different types of cytokines.Proteases increase the clearance of anti-inflammatory cytokines (Desser et al. 1999;Desser et al. 2001;Xiang et al. 2001), as well as plasminogen concentration in blood.They also decrease fibrin formation and dissolve the existing thrombi.Decreased thrombocyte aggregation helps to maintain correct microcirculation (Nouza et al. 1999).
The aim of the present study was to demonstrate the beneficial effects of topical administration of proteolytic enzymes used as symptomatic therapy of mastitis with decreased total antibiotic doses.For the evaluation, we have chosen the results of development of ceruloplasmin and fibrinogen dynamics and microbiological examination of collected milk and blood plasma samples.

Materials and Methods
A field part of the study was performed on the farm Tû‰any u Brna, owned by the company AGRO MONET a.s.Moutnice.The study included 11 lactating cows that met the following criteria: clinical signs of inflammation present in at least one quarter, 4-12 years of age, an apparent and unplanned decrease in the volume of milk produced, positive results of barn mastitis test, changes in milk quality.Right after the allocation to the study, each lactating cow was placed in quarantine.Collected milk was discharged until the end of withdrawal period of a drug used.Lactating cows were randomly allocated to two groups.Group A received only a broad-spectrum ß-lactam antibiotic, cephalosporin of 3 rd generation -cefotaxime at a single dose of 250 mg, made by galenic modification of Taxcef inj. 1 × 1 g (Ranxbay, UK) in the sterile suspension-ointment basis.The ointment was applied twice a day for 3 days intramammarily, only to inflamed quarters.
Group B received the same antibiotic (cefotaxime 100 mg) and a mixture of enzymes in one drug form.The enzyme mixture was made from 3 coated tablets of the registered enzyme preparation Wobenzym drg.(Mucos, Germany), modified galenically.Proportions of proteolytic activities were as follows: 46.15% papain, 18.46% trypsin, 0.77% chymotrypsin a 34.61% bromelain.The preparation was applied only into inflamed quarters.The following protocol was used: first and second day -one dose BID, third day: one dose SID.Both the dose and number of administrations were lower, as compared with Group A. In both groups, the drugs were administered in the form of intramammary sterile suspension ointment at a dose of 10 cm 3 , by a disposable sterile injector.A conic tip of the injector was carefully inserted into the teat of an affected quarter.By pressing the injector, all its contents were squeezed into the quarter.This was always done after thorough milking and teat tip disinfection.The preparation was kept refrigerated at 2 -8 °C.It was taken out of the refrigerator about 15 min before the administration.Before and 12 -24 hours after the therapy, milk and blood samples were collected.Milk samples were obtained by squirting the middle flow of milk from all the productive quarters to sterile glass test tubes.The samples were examined at the microbiological laboratory of the State Veterinary Institute Brno for the presence of Streptococcus agalactiae, Str.dysgalactiae, Str.uberis, Staphylococcus aureus, E. coli and Klebsiella.Blood was withdrawn from the tail vein into plastic vacuum sterile tubes and was used to determine fibrinogen and ceruloplasmin as acute phase proteins.Fibrinogen was measured by the turbidimetric analysis, using the set FIBRINOGEN Boehringer Mannheim.Ceruloplasmin levels in the serum were measured (H e n r y et al. 1974) using the biochemical analyser COBAS MIRA S, at the laboratory of clinical biochemistry at the Clinic of Ruminant Diseases of the Faculty of Veterinary Medicine, University of Veterinary and Pharmaceutical Sciences Brno.For the statistical analysis of levels of both acute phase proteins, F-test and paired and non-paired t-tests were used.The difference was regarded as statistically significant at p < 0.05.

Results
In Group A, 18 quarters were affected in total.Mean total dose per one quarter was at least 1400 mg cefotaxime.In Group B, 18 quarters were affected in total.Mean total dose per one quarter was 500 mg cefotaxime and 1870 mg total enzyme.Results of microbiological examination are listed in Table 1.Before the therapy the respective pathogenes were found in milk samples from all the cows monitored, but with Str.agalactiae prevailing (7 samples); bacteriological examination of the samples obtained 24 hours after the therapy showed negative results (Table 1).In Group A, mean plasma concentrations of fibrinogen were 6 ± 1.4 g•l -1 before the therapy and 7.4 ± 1.1•g -1 after the therapy.In Group B, mean initial and final plasma fibrinogen concentrations were 6.5 ± 0.86•g -1 and 6.4 ± 0.9•g -1 , respectively.Fibrinogen levels on the initial and final days within one group, compared by the paired t-test (Table 2a), were significant only in Group A where an increase in fibrinogen levels by 27% on average on the 4th day, as compared with the initial values, was found.Changes in Group B were non-significant, although most cows showed an apparent decrease as compared with the initial values.When comparing percentage changes of the indicators under study between the groups (Table 2b), Group B showed a marked decrease compared to Group A. In Group A, mean ceruloplasmin concentrations were 0.9 ± 0.2 µmol•l -1 before the therapy, and 1.3 ± 0.18 µmol•l -1 thereafter.In Group B, mean ceruloplasmin concentrations were 0.9 ± 0.36 µmol•l -1 before the therapy and 1.1 ± 0.26 µmol•l -1 thereafter.Ceruloplasmin concentrations (µmol•l -1 ) were compared between the first and last day within the groups using the paired t-test (Table 3a).Statistical significance was achieved only in Group A where ceruloplasmin concentrations increased by 49% on average compared to initial values.Changes observed in Group B were not significant, although in all cows an increase from the initial values was obvious.No statistical significance was found in percentage changes of the variable under study between the groups (Table 3b).formation of fibrin matrix that enables the movement of fibroblasts and other cells and stimulates their production during the healing of damaged tissue ad secundam.Its biological half-time is 2.4 -4.2 days (Masopust 1998).Unlike Group A in which a significant increase of fibrinogen concentration was observed, Group B (subjected to the enzyme therapy) showed a decrease, although a non-significant one.This might have been a response to direct or indirect anti-inflammatory effect of enzyme therapy, and would have corresponded with previously published results that attributed the enzyme therapy antioedema, fibrinolytic, anti-aggregation and therefore analgesic effects on the tissue affected with an inflammatory process (Kasseroler andWenning 2003, Guggenbichler 1988).It is also possible that the suppression of increase of fibrinogen concentration is due to a direct fibrinolytic ability of some proteolytic enzymes (Nouza et al. 1999).
Ceruloplasmin is an inflammation marker, too, and it is degraded more rapidly than it is produced during the inflammation.The half-time of ceruloplasmin is 2-3 days (Masopust 1998).Therefore its decrease, even in case a therapeutic used exerts an anti-inflammatory effect, is delayed.In Group A we found a significant increase as compared to the initial values.No significant differences were found in Group B. In spite of that, in both groups the final values were higher (by 42% in Group A, by 23% in Group B) than the initial values.We assume that in Group A, the drug used affected directly the infectious agent, without an ability to directly influence the signs of inflammation which under normal circumstances diminish gradually, only during the healing process.By the moment of breakpoint in the process of damage and repair there is a limited persisting growth of marker values.Apparently, in Group B, the process of persisting growth was indirectly hindered by antiinflammatory effects of enzyme therapy.A significant effect on levels of acute phase proteins under study suggests a systemic effect of the proteolytic enzyme blend administered intramammarily.
In conclusion, it can be summarized that macromolecular substances of proteolytic enzyme nature, applied topically, exert a systemic effect, and a therapeutical approach combining enzymes and antibiotics not only enables to decrease doses of antibiotics, providing the same efficacy as the antibiotic monotherapy, but also may contribute to a reduction in the inflammation intensity.
We assume that the administration of antimicrobials along with selected proteases (bromelain, chymotrypsin) requires further investigations.This may help us find the most suitable therapy for infectious inflammations and related risks.

Table 1 .
Initial and final results of bacteriological examination in individual cows

Table 2a .
Initial and final values of fibrinogen and evaluation by paired t-test

Table 2b .
Comparison of percentage changes in fibrinogen levels between the groups by non-paired t-test

Table 3a .
Initial and final values of ceruloplasmin and evaluation by paired t-test

Table 3b .
Comparison of percentage changes in ceruloplasmin concentrations between the groups by non-paired t-test