INTRODUCTION

Mastitis is one of the most important diseases affecting cattle production. This problem is reduces milk quality and quantity and ha a negative impact in dairy business ¹. This pathology is defined as inflammation of the mammary gland, due to no infectious (traumatic) and infectious agents, including bacteria such as Staphylococcus aureus, Escherichia coli, Streptococcus dysgalactiae, S. uberis, Arcanobacterium pyogenes, Klebsiella spp., mycoplasmas, yeasts and algae ². All these microorganisms are capable to colonize the udder, increasing the number of somatic cells, mainly neutrophils and epithelial cells.

Sub-clinical mastitis is one of the main problems of milk production. In a herd with an average production of 8500 kg/305 days and cell counts greater than 200,000 cells/ml, losses can reach US $22 (USD) per animal/year. During subclinical presentation, the udder shows no signs of inflammation and milk may appear normal. In contrast, in clinical mastitis the infected quarter is inflamed, some cows manifest tenderness and milk is visibly altered ³. In severe clinical cases (acute mastitis), the cow shows generalized symptoms including fever, elevated heart rate, loss of appetite and significant reduction in milk production with a dramatic increase in somatic cell production ⁴, classic signs of inflammation can be found as well as alterations in milk, identified as lumpy (dairy clots due to the inflammatory process). When the disease becomes chronic, inflammation can affect the immune system ⁵ and causes resistance to almost any treatment. Additionally, can result in anatomopatolhogical and physiological problems, such as the formation of fibrous tissue, cell destruction and necrosis ⁶.

The bases for the Control of Contagious Mastitis in the UK consist of a five-point plan for prevention and management, also mentions the importance of using antimicrobials during the stages of lactation and drying when necessary ⁷. However, field veterinarians know that antibiotics sometimes are not enough and that it is necessary to treat the inflammation using NSAIDs and glucocorticoids, and the treatment of particular cases with proteolytics (chymotrypsin) that have proven highly effectiveness.

The major intramammary antimicrobial treatments that have been used with different success rates to control bovine mastitis include beta-lactams such as natural G penicillins (sodium, potassium, procaine and benzathine), synthetic ones such as ampicillin, amoxicillin and anti-staphylococcus drugs (cloxacillin, oxacillin, nafcillin, meticillin); cephalosporins (Cefapirin, cefalonium, cephalothin, cefoperazone, ceftiofur, cefquinome); aminoglycosides such as dihydrostreptomycin, gentamicin and neomycin; aminocoumarins (novobiocin); fluoroquinolones (norfloxacin); tetracyclines (oxytetracycline); macrolides (erythromycin); lincosamides such as lincomycin and pirlimycin; chloramphenicol, among others.

Additionally, anti-inflammatory treatments in cases of mastitis is usually common and necessary. Anti-inflammatory drugs such as carprofen can be used to accelerate recovery of mammary glands in concomitant therapies with antibacterial drugs. Meloxicam is also recommended. These medicines help to decrease somatic cell counts better than with a single antimicrobial treatment ⁸. During the inflammatory process, the release of bacterial endotoxin causes systemic symptoms such as elevated body temperature, decreased ruminal motility, and even increased heart and respiratory rates. Therefore, using drugs that not only have anti-inflammatory but also antiendotoxemic properties can speed up the recovery process. Among the commonly used drugs in the United States that have these characteristics, flunixin meglumine also has analgesic, antiinflammatory and antipyretic properties ⁹ that accelerates gland recovery and bacterial elimination. Others, such as ketoprofen, also have a recognized anti-endotoxemic action ¹⁰, and its analgesic effect is important to improve udder health and milk production ¹¹.

Additionally, glucocorticoids have been employed in parenteral ¹² and intramammary ¹³ formulas due to greater anti-inflammatory action compared to non-steroidal drugs.

Meanwhile, proteolytic enzymes have been considered as an effective treatment for bovine mastitis ¹⁴. Among the mean enzymes there are trypsin, chymotrypsin and papain, which have fibrinolytic and proteolytic activity on inflamed tissues ¹⁵. Bovine pancreatic chymotrypsin is the active ingredient in Quimotripsyn(r) - Virbac Laboratories, whose action increases plasmin, a proteolytic enzyme of endothelial origin that is responsible for lysing excess of fibrin from the inflammatory process and is considered as the key of fibrinolysis ¹⁶.

Regarding to this, it is important to mention the relationship between the inflammatory process and coagulation, since fibrinogen and fibrin directly affect the production of cytokines and other proinflammatory substances (TNF-α, IL-1β, monocyte chemoattractant protein) in endothelial and mononuclear cells. If there is a fibrinogen deficiency, there would be an alteration in macrophage aggregation and a deficit in immune response, but in the other hand, an excess of fibrinogen would result in anatomo pathological and physiological changes in the affected organ ¹⁷. Acute inflammatory processes involve fibrin formation, which is histologically recognized due to an abundant leukocytes and fibrin accumulation. This process should not be confused with fibrosis, which can be chronic ¹⁸.

During the inflammatory process in the mammary gland, many tissue factors are activated and stimulate the coagulation cascade and production of thrombin ¹⁹. The final phase results in an increase of fibrin and the reduced activity of the fibrinolytic system ²⁰. There have been discovered different processes and mechanisms related to the fibrinolysis suppression on regulatory molecules that activate and inhibit plasminogen (precursor of plasmin) during inflammation, TNF-α and IL-1β ²¹. Plasmin is derived from plasminogen, which lyses the excess of fibrin produced during the inflammatory process.

When these cytokines (TNF-α and IL-1β) are released into circulation, plasminogen activators are also released from endothelial cells. These activators are known as "tissue-type plasminogen activators" and "urokinase type plasminogen activators" ²¹. However, it is necessary the release of plasminogen for the fibrinolysis and the release of plasmin is offset by an inhibitor that activates the inflammatory process ²². The resulting effect is a complete inhibition on fibrinolysis and results in an inadequate removal of fibrin which contributes to microvascular thrombosis. In humans, increasing plasmin concentration in blood has been suggested as an effective treatment to dissolve thrombi and has decreased mortality in patients ²³.

The use of chymotrypsin activates the production of plasminogen-plasmin and reduces the severity of infections caused by Staphylococcus aureus^24,25, which is considered as one of the main pathogens in bovine mastitis. Now days this mechanism is not yet understood, but it is recognized that elevated plasmin levels can reduce the virulence of S. aureus strains ²⁶. It is suggested that plasmin is an important regulator in tissue remodeling and inflammation processes. The present study included cattle with acute mastitis, the use of chymotrypsin was challenged together with beta-lactam antibiotics in order to assess effectiveness on somatic cell count (SCC), and the semiological findings were compared to a group of animals treated only with antibiotics.

MATERIALS AND METHODS

Area and study population. The study was conducted on a dairy farm in the savannah of Bogota in the town of Sopó, Cundinamarca, at 2600 m above sea level and an average temperature of 14°C. The animals used in this study were Holstein Friesian cattle with an average weight of 550 kg. The cows were milked twice daily in a mechanical milking parlor. A total of 14 cows were evaluated and a total of 46 mammary glands quarter were diagnosed with lumps. In order to establish the severity of mastitis, there was executed the Californian Mastitis Test to determine the changes of the gland before and after treating with chymotrypsin.

Treatment. Once the animals were identified according to inclusion criteria, they were treated with chymotrypsin at a total dosage of 10 ml per animal intramuscularly every 24 hours for 3 applications. Additionally, the animals received 15 mg/kg amoxicillin IM every 24 hours for 3 days. There was a total of 3 control animals and a group of 10 with the same characteristics that only received amoxicillin and clavulanic acid, without chymotrypsin.

Laboratory methods. The Californian Mastitis Test (CMT) was executed using a sample of milk of each gland quarter in an special container before washing and blunting the gland. Three ml of milk were taken from each one on a black CMT tray and an equal amount of CMT reagent (Masti-Test(r) - Laboratorios SFC LTDA) was added, stirring for 5 seconds until the reaction was obtained. To classify the degree of mastitis according to the CMT test, the criteria described by Kivaria et al ²⁷ was developed and explained in Table 1. The CMT was performed minutes before treatment and seven days later.

Count of somatic cell was done using an electronic DeLaval(r) counter with a measuring range from 10000 to 4000000 cells/ml. These samples were collected individually in a tube after eliminating the first spurts of milk.

RESULTS

Table 2 shows the results according to the degree of Mastitis found in the CMT and somatic cell count before and after treatment with Quimotripsyn(r) and amoxicillin + clavulanic acid. For statistical analysis of somatic cell counts, there was developed an analysis of variance.

Of the 46 cows treated with antibiotics and chymotrypsin, clinical improvement and decreased somatic cell count was observed in 70% of cases with statistically significant differences.

In 39 (84.7%) cases, significant clinical differences were observed, mainly between what was observed before therapy and the absence of lumps in milk and a significant decrease in inflammation 7 days after starting treatment. Statistically significant differences were observed in the same number of cases in somatic cell count (p<0.01) during the same period of time. The remaining number also decreased somatic cell counts and mastitis, although the response was moderate compared to that observed in the treatment group in 84.7% of the cases (Table 2).

Additionally, 84.7% of the animals that were treated with chymotrypsin showed an initial recovery within the first 24 hours of treatment, which could mainly be observed in body temperature. The physical examination results at 24 hours after the start of the chymotrypsin treatment showed a decrease in inflammation and even a rise in milk quality, as seen in a decrease of existing clots. It is noteworthy that the physical examination was conducted every 24 hours post-treatment for a total of 10 days.

The following are the results of the control group (Table 3).

The control group showed a clinical response in the CMT test in 70% of cases. In the control group no significant statistical differences regarding somatic cell count were observed before and after treatment.

DISCUSSION

This study was carried out based on scientific support of the action of chymotrypsin ¹⁵, a drug that is quite often used in bovine mastitis ¹⁴, but there were not found studies or case reports that prove the effect of this drug and clinical effectiveness.

To explain these results using pharmacodynamics, it must be understood that antibiotic treatments alone or used together with anti-inflammatory drugs can sometimes be ineffective or may take time to obtain the expected results, especially due to increased antimicrobial resistance ²⁸. In particular, it is difficult for antibacterial substances to access some tissues, even more when inflammation is present. Mastitis treatment requires the use of drugs to accelerate recovery by breaking the fibrin capsule, with the consequent increase in the spread of antimicrobials to the inflamed tissue. Considering this, a proteolytic drug with fibrinolytic properties should be used in cases of acute bovine mastitis to facilitate penetration of antibiotics into the inflamed and infected tissue, thereby accelerating recovery of the gland.

It is known that somatic cell count is an important indicator of the degree of mastitis and a post-treatment decrease is reliable evidence of recovery. Based on this and the clinical signs observed before and after treatment as well as measurements provided by the CMT, it can be concluded that there is a positive udder health effect when chymotrypsin is applied. Significant differences in somatic cell counts between treated and control groups as well as the physical evidence of recovery (visible results on physical examination 24 hours after treatment) are important indicators that should be considered for veterinarians when treating cases of bovine mastitis.

The relationship between fast recovery of the affected glands and production improvement is directly proportional, and therefore applying a specific and opportune treatment for each type of mastitis (subclinical, acute clinical, reoccurring and/or chronic) is recommended. Treating subclinical and even clinical mastitis with chymotrypsin has advantages such as: reduction of risking evolution of subclinical mastitis to clinical phase, and milk from treated animals can be sold and milk quality can be improved by reducing the presence of somatic cells.

Additionally, considering the properties of chymotrypsin, it can also be used in vascular inflammation processes, edemas, hematomas, abscesses, adhesions, post-vaccination fibromas, mastitis, acrobustitis, metritis, respiratory infections, which would be cause for further investigation. When it is impossible to assess the degree of fibrosis, even during acute inflammation there is an accumulation of leukocytes and fibrin ²⁹ that leads to fibrosis of the gland. This is the key point for chymotrypsin to act by decreasing the production of fibrin, thereby allowing more effective distribution of antibiotical drugs. Therefore it is important to conclude that the use of chymotrypsin could be an adjunct in the treatment of acute mastitis.

Finally, it is also vital to clarify that it was not possible to have a control group that was completely free of treatment, due to field conditions and animal welfare. Not treating animals with acute mastitis is related to professional ethics issues.