F1F2F3ANNEX CBRUCELLOSIS
2.IMMUNOLOGICAL TESTS
2.4.Milk ring test (MRT)
2.4.1.
The antigen represents a bacterial suspension in phenol-saline (NaCl 0,85 % (m/v) and phenol at 0,5 % (v/v)) stained with haematoxylin. The antigen must be stored at 4 °C and not frozen.
2.4.2.
The antigen sensitivity must be standardised in relation to the OIEISS in such a way that the antigen produces a positive reaction with a 1/500 dilution of the OIEISS in negative milk, while a 1/1 000 dilution must be negative.
2.4.3.
The ring test must be made on samples representing the contents of each milk churn or the content of each bulk tank from the farm.
2.4.4.
The milk samples must not have been frozen, heated or subjected to violent shaking.
2.4.5.
The reaction must be carried out using one of the following methods:
on a column of milk of at least 25 mm height and on a volume of milk of 1 ml to which either 0,03 ml or 0,05 ml of one of the standardised stained antigens has been added,
on a column of milk of at least 25 mm height and on a volume of milk of 2 ml to which 0,05 ml of one of the standardised stained antigens has been added,
on a volume of milk of 8 ml to which 0,08 ml of one of the standardised stained antigens has been added.
2.4.6.
The mixture of milk and antigens must be incubated at 37 °C for 60 minutes, together with positive and negative working standards. A subsequent 16- to 24-hours incubation at 4 °C increases the sensitivity of the test.
2.4.7.
Interpretation of results:
- (a)
negative reaction: coloured milk, colourless cream;
- (b)
positive reaction:
identically coloured milk and cream, or
colourless milk and coloured cream.
Substituted by Council Directive 97/12/EC of 17 March 1997 amending and updating Directive 64/432/EEC on health problems affecting intra-Community trade in bovine animals and swine.