ESTERASE E.C 3.1 1

Milk from mastitic quarters shows a 10 fold higher A-esterase activity than does that from healthy quarters ( Forster et 1961 ). Kitchen et al ( 1980 ) reported that milk

from quarters with a cell count > 1,000,000 cells per ml, had an A-esterase activity three times that of milk from quarters with < 500,000 cells per ml.

The infusion of streptococci ( Booth et 1%5 ) or of staphylococci ( Marquardt et

1966 ) into the udder causes a modest elevation of A-esterase activity.

The A-esterase activity is related to the severity of the mastitis. Estimates of the correlation coefficient between the somatic cell count and the A-esterase activity range

from 0.66 to 0.81 ( Marquardt and Forster, 1964; Marquardt et 1966; Kitchen et

1980 ).

The average blood plasma to milk ratio for A-esterase is 1800:1, suggesting that the

plasma is the major source of this enzyme in the milk, and that this assay could be

used as an indicator of leakage between blood and milk compartments ( Marquardt and

Forster, 1966; Marquardt et 1%6 ). This suggestion is supported by the finding that

A-esterase is highly correlated with bovine serum albumin ( Kitchen et 1980 ).

Mammary gland tissue is a poor source of A-esterase in comparison with the blood

serum ( Kitchen et 1980 ).

The enzyme has been purified and characterized by Kitchen et al ( 1973 ) who found it to occur in milk as a lipoprotein with a molecular weight of approximately 440,000 daltons. This would suggest that the diffusion of A-esterase from the blood to milk might be slower than that of smaller proteins, thus limiting its value as an indicator of altered permeability.

The activity of A-esterase in milk is affected by the stage of lactation and by -the breed of cow, although the activity does not vary significantly between milk fractions ( Forster

et 1961; Marquardt and Forster, 1966 ). Although this assay is time consuming,

because of the need for an ultracentrifugation step, an improved assay procedure could make the determination of A-esterase a useful diagnostic test for subclinical mastitis ( Kitchen, 1981 ).

CARBOXYLESfERASE E.C. 3.1.1.1

Although little attention has been paid to the activity of the enzyme carboxylesterase in response to mastitis, a higher activity has been associated with the disease. Fitzgerald et

al ( 1981 ) reported marked increases in the enzyme carboxylesterase following the

infusion of bacterial endotoxin. The activity is also elevated during naturally occurring mastitis. The correlation between carboxylesterase and the somatic cell count has been

LIPASE E.C. 3.1.1.3

Conflicting reports on the effect of mastitis on the milk lipase activity have been published. In an early study, little difference was observed between the lipase activity

of normal and of mastitic milk ( Forster et 1961 ).

Tallamy and Randolph ( 1969 ) reported that milk with a Wisconsin Mastitis Test score of > 20 mm showed a small but significantly higher lipase activity than did milk with a Wisconsin Mastitis Test score of < 10 mm, with similar findings being reported by Randolph and Erwin ( 1974 ). However, Deeth and Fitzgerald ( 1975 ) reported a lower lipase activity in mastitic than in normal milk. The conflicting results have been attributed to the presence of inhibitors and of activators of this enzyme in the milk ( Kitchen, 1981 ).

PROTEINASE ENZYMES

Indirect evidence for the existence of proteinase enzymes in mastitic milk has been provided by several studies. High cell count milk contains a lower concentration of both alpha and beta caseins than does normal milk, and this is associated with an

increase in the concentration of kappa casein ( Haenlein et 1973 ). Anderson and

Andrews ( 1977 ) confirmed the presence of kappa casein in milk from mastitic cows and suggested that this might be due to the high levels of proteolytic enzym� which are present in the milk.

The proteolysis of casein in mastitic milk has been attributed to a plasmin-like enzyme which probably originates from the blood plasma ( Barry and Donnelly, 1981 ). De Rham and Andrews ( 1982 ), who had found the proteolytic activity of mastitic milk to be 5 to 10 fold higher that that of normal milk, reported that plasmin accounted for approximately one third of the total proteinase activity of the milk following infusion of udders with endotoxin.

Different proteinases are associated with various milk fractions ( De Rham and Andrews, 1982 ).

Cows with clinical mastitis or those recetvmg endotoxin infusions have a very high proteinase activity ( Andrews, 1 983 ). The plasmin is thought to originate from the blood serum, while the remaining proteinases may come from the leukocytes.

The electrophoretic techniques used to demonstrate these enzymes are not conducive to large scale monitoring for subclinical mastitis.

B-GLUCURONIDASE E.C. 3.2.1.31

The activity of the enzyme iS-glucuronidase is higher in mastitic than in normal milk. Kitchen ( 1976 ) reported that the B-glucuronidase activity of milk with a cell count of > 1,500,000 cells per ml was approximately four fold higher than that of milk containing < 500,000 cells per ml.

The activity of iS-glucuronidase correlates well with the milk somatic cell count ( r = 0.71 ) suggesting that this enzyme could be useful for estimating the somatic cell count ( Kitchen, 1976 ). Obara ( 1985 ) reported a correlation coefficient of only 0.59 between iS-glucuronidase activity and somatic cell count, but when the variables were transformed to log derivatives this figure increased to 0.72. Nagahata et al ( 1987 ) reported a very close association between an indirect indicator of the somatic cell count ( the California Mastitis Test ) and the B-glucuronidase activity ( r = 0.92 ).

Milk macrophages are a rich source of iS-glucuronidase ( Nagahata et 1987 ).

The B-glucuronidase activity is related to the presence of both major and of minor pathogens, as well as to the somatic cell count ( Peridigon et 1986 ). These authors suggested that iS-glucuronidase might be a reliable indicator of the inflammatory process, since it is specific to leukocyte lysosomes.

The activity of iS-glucuronidase may be determined by spectrophotometry, although this method includes a four hour incubation period ( Kitchen, 1976 ). A more rapid method involving an incubation period of only 10 minutes, using the fluorimetric determination of B-glucuronidase, has been described ( Nagahata et 1987 ).

a-MANNOSIDASE E.C. 3.2.1 .24

The primary source of milk a-mannosidase activity is unclear. Mellors and Harwalkar ( 1968 ) showed that the a-mannosidase activity is associated with casein micelles in milk, and suggested that it is released from the lysosomes of damaged leukocytes. However, the cell debris fraction was not found to contain significant activity of this enzyme. This finding is reflected in a low correlation between the somatic cell count and a-mannosidase activity ( Kitchen, 1976; Obara et 1983 ). Kitchen ( 1976 ) reported a similar activity for a-mannosidase in milk from normal and from mastitic quarters.

The activity of a-mannosidase would appear to be of little value in the detection of