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Milk ring test


Milk ring test


In lactating animals, the MRT can be used for screening herds for brucellosis. In large herds (>100 lactating cows), the sensitivity of the test becomes less reliable. False-positive reactions may occur in recently vaccinated cattle (less than 4 months) or in samples containing abnormal milk, such as colostrum or that due to mastitis.

MRT antigen is prepared from concentrated, killed B. abortus strain 99 or 1119-3 cell suspension, grown as described previously. It is centrifuged at, for example, 23,000 g for 10 minutes at 4C, followed by resuspension in haematoxylin-staining solution. Various satisfactory methods are in use; one example is as follows: 100 ml of 4% (w/v) haematoxylin (Cl No. 75290) dissolved in 95% ethanol is added to a solution of ammonium aluminium sulphate (5 g) in 100 ml of distilled water + 48 ml of glycerol. 2 ml of freshly prepared 10% (w/v) sodium iodate is added to the solution. After standing for 30 minutes at room temperature, the deep purple solution is added to 940 ml of 10% (w/v) ammonium aluminium sulphate in distilled water. The pH of this mixture is adjusted to 3.1, and the solution must be aged by storage at room temperature in the dark for 45- 90 days.

Before use, the staining solution is shaken and filtered through cotton wool. The packed cells are suspended in the staining solution at the rate of 1 g per 30 ml stain, and held at room temperature for 48 hours (some laboratories prefer to heat at 80C for 10 minutes instead). The stained cells are then deposited by centrifugation, and washed three times in a solution of sodium chloride (6.4 g), 85% lactic acid (1.5 ml) and 10% sodium hydroxide (4.4 ml) in 1.6 litres of distilled water, final pH 3.0. The washed cells are resuspended at the rate of 1 g in 27 ml of a diluent consisting of 0.5% phenol saline, adjusted to pH 4.0 by the addition of 0.1 M citric acid (approximately 2.5 ml) and 0.5 M disodium hydrogen phosphate (approximately 1 ml) and maintained at 4C for 24 hours. The mixture is filtered through cotton wool, the pH is checked, and the PCV is determined and adjusted to approximately 4%. The sensitivity of the antigen is then repeatedly checked against that of a previously standardised batch using a panel of samples of varying degrees of reaction prepared by diluting a positive working standard serum (calibrated against the ISABS) in negative milk (7). The antigen must be stored at 4C and not frozen.

The pH of the antigen should be between 3.3 and 3.7 and its colour should be dark blue. There might be a little free stain in the supernatant of a centrifuged sample. When diluted in milk from a brucellosis-free animal, the antigen must produce a uniform coloration of the milk layer with no deposit and no coloration of the cream layer.

The test is performed by adding 30 l of antigen to a 1 ml volume of whole milk that has been stored for at least 24 hours at 4C. The height of the milk column in the tube must be at least 25 mm. If bulk tank samples from large herds are to be examined, the volume of milk should be increased to 3 ml. The milk samples must not have been frozen, heated or subjected to violent shaking. The milk/antigen mixtures are incubated at 37C for 1 hour, together with positive and negative control samples. A strongly positive reaction is indicated by formation of a dark blue ring above a white milk column. Any blue layer at the interface of milk and cream should be considered positive as it might be significant, especially in large herds. The test is considered to be negative if the colour of the underlying milk remains homogeneously dispersed in the milk column. If the milk at the bottom of the tube becomes gradually whitened, the result is regarded as inconclusive and the test should be repeated.