Glucuronidase Method to Determine Mastitis Levels in Goat Milk

Rubén Oliszewski, Martha Núñez de Kairúz, Silvia González, and Guillermo Oliver

1. Introduction

Mastitis is a general term that refers to the inflammation of the mammary gland. It is the most common illness in dairy farms and it has different causes, mainly a great number of germs that infect the gland.

These infectious diseases induce gross variations in milk composition, reflected by physical, chemical, and bacteriological changes. They produce milk jellification, a decrease in important components such as lactose, casein, and fats and minerals such as calcium, phosphorus, and potassium and increases in other unimportant technological components, such as serum proteins and chlorides; all these affect the cheese efficiency and the starter culture action (1).

Assuming that cheese making is the principal use of goat milk in industry, an evaluation of the quality of the milk used as the raw material is of fundamental importance. It is impossible to obtain quality products by using milk with an anomalous chemical composition (2).

Somatic cell count (SCC) is the indicator most used for mastitis detection. These cells, which are contained in milk, can be grouped into three types: epithelial cells, blood cells, and cytoplasmatic particles. During an attack of mastitis, the immune defenses of the udder are activated, polynucleated leukocytes pass from the blood toward the mammary gland in large numbers, and the number of somatic cells in the milk increases (2).

The level of somatic cells in goat milk is characterized by great variability between different countries and between regions of the same country. Different authors show averages between 750,000 and 5,400,000 cells/mL (1-4). These values differ greatly between cow and goat milk, mainly because normally nonleukocytic cell-like particles can be found as a result of the particular apocrine secretion process in the goat mammary gland. These particles are large fragments of cytoplasm originating from the distal portion of alveolar secretory cells and are of similar size (5-30 ^m in diameter) to milk leukocytes. They contain abundant RNA-positive granular material (associated with dilated cisternae of the rough endoplasmic reticulum), large amounts of protein, and some lipids, but no DNA (5,6). Thus it is important to use techniques that disregard these other substances and allow only a count of somatic cells (7,8).

The afflux of polymorphonuclear leukocytes and macrophages is one of the essential body defenses against clinical and subclinical mastitis. These cells are used as a base for the SCC method. However, their secretion products can also be used. These cells begin the phagocytic process with the secretion of hydrolytic enzymes, which, together with other factors, participate in the degradation of tissue constituents in the inflammatory process. One of these enzymes is p-glucuronidase (7). It has proved to be the most significant selectively released enzyme in this process (9,10).

A p-glucuronidase test was recently standardized for goat milk and was shown to be reliable, measuring only the presence of the enzyme in milk, without the other substances that are picked up by the SCC method (1). In this chapter, we describe the procedure used in our laboratory to determine p-glucuronidase levels in milk.

2. Materials

2.1. p-Glucuronidase Method

2.1.1. Standard Curve

1. 1 mM ^-Nitrophenol solution: 13.9 mg _p-nitrophenol in 100 mL acetate buffer, to prepare a standard curve.

3. Spectrophotometer at 410 nm, for optical density (OD) assay to determine the extinction coefficient.

2.1.2. Enzyme Assay

1. Milk samples collected and immediately cooled to 4°C.

2. 40 mM pnPG: 1.5 g _p-nitrophenol |3-D-glucuronide in 100 mL of distilled water.

2.2. Somatic Cell Count

2. Clean microscope slides.

3. Micropipet, with appropriate tips, suitable for rapid and convenient transfer of 0.01 mL of milk.

4. Slide drier: heat source regulated at 40-45°C.

5. Forceps or slide holder: required for dipping and holding slides.

6. Slide storage: clean, dust-free insect-proof boxes, cases, or files.

7. Microscope: for computing single strip factor (SSF).

8. Immersion oil.

9. Carnoy's fixative: prepared by combining 60 mL chloroform, 20 mL glacial acetic acid, and 120 mL 100% ethyl alcohol.

10. Pyronin Y-methyl green stain (PYMG ) for goat milk: prepared by combining 1.0 g pyronin Y, 0.56 g methyl green, and 196 mL water. Filter through Whatman no. 1 paper before use. Stain is light-sensitive; store in brown bottle.

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