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Textual Amendments
F1 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.
the OIEISS,
the weak positive OIE ELISA standard serum (OIEELISA WP SS),
the strong positive OIE ELISA standard serum (OIEELISA SP SS),
the negative OIE ELISA standard serum (OIEELISA N SS).
the weak positive OIE ELISA standard serum (OIEELISA WP SS),
the strong positive OIE ELISA standard serum (OIEELISA SP SS),
the negative OIE ELISA standard serum (OIEELISA N SS).
The technique used and the interpretation of results must have been validated in accordance with the principles laid down in Chapter 1.1.4 of the OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, Sixth Edition, 2008, and must include at least laboratory and diagnostic studies.
a 1/150 pre-dilution (1) of the OIEISS or a 1/2 pre-dilution of the OIEELISA WP SS or a 1/16 pre-dilution of the OIEELISA SP SS made up in a negative serum (or in a negative pool of sera) must give a positive reaction;
a 1/600 pre-dilution of the OIEISS or a 1/8 pre-dilution of the OIEELISA WP SS or a 1/64 pre-dilution of the OIEELISA SP SS made up in a negative serum (or in a negative pool of sera) must give a negative reaction;
the OIEELISA N SS must always give a negative reaction.
a 1/150 pre-dilution of the OIEISS or a 1/2 pre-dilution of the OIEELISA WP SS or a 1/16 pre-dilution of the OIEELISA SP SS made up in a negative serum (or in a negative pool of sera) and again diluted in negative sera by the number of samples making up the pool must give a positive reaction;
the OIEELISA N SS must always give a negative reaction;
the test must be adequate to detect evidence of infection in a single animal of the group of animals, of which samples of serum have been pooled.
a 1/ 1 000 pre-dilution of the OIEISS or a 1/16 pre-dilution of the OIEELISA WP SS or a 1/125 pre-dilution of the OIEELISA SP SS made up in a negative serum (or in a negative pool of sera) and again diluted 1/10 in negative milk must give a positive reaction;
the OIEELISA N SS diluted 1/10 in negative milk must always give a negative reaction;
the test must be adequate to detect evidence of infection in a single animal of the group of animals, of which samples of milk or whey have been pooled.
bovine serum: 56 to 60 °C for 30 to 50 minutes,
porcine serum: 60 °C for 30 to 50 minutes.
control of the anti-complementary effect of the serum;
control of the antigen;
control of sensitised red blood cells;
control of the complement;
control using a positive serum of sensitivity at the start of the reaction;
control of the specificity of the reaction using a negative serum.
The OIEISS contains 1 000 international CFT units (ICFTU) per ml. If the OIEISS is tested in a given method the result is given as a titre (i.e. highest direct dilution of the OIEISS giving 50 % haemolysis, T OIEISS ). The test result for the test serum given as titre (T TESTSERUM ) must be expressed in ICFTU per ml. In order to convert the expression of a titre into ICFTU, the factor (F) necessary to convert a titre of an unknown test serum (T TESTSERUM ) tested by that method into the ICFTU expression can be found from the formula:
F = 1 000 × 1/T OIEISS
and the content of international CFT units per ml of test serum (ICFTU TESTSERUM ) from the formula:
ICFTU TESTSERUM = F × T TESTSERUM
A serum containing 20 or more ICFTU per ml shall be considered to be positive.
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.
negative reaction: coloured milk, colourless cream;
positive reaction:
identically coloured milk and cream, or
colourless milk and coloured cream.
serum (20-30 μl) is mixed with an equal volume of antigen on a white tile or enamel plate to produce a zone approximately 2 cm in diameter. The mixture is rocked gently for four minutes at ambient temperature, and then observed in a good light for agglutination;
an automated method may be used but must be at least as sensitive and accurate as the manual method.
Any visible reaction shall be considered to be positive, unless there has been excessive drying round the edges.
Positive and negative working standards shall be included in each series of tests.
Formaldehyde must not be used.
Antigens may be delivered in the concentrated state provided the dilution factor to be used is indicated on the bottle label.
EDTA may be added to the antigen suspension to 5 mM final test dilution to reduce the level of false positives to the serum agglutination test. Subsequently the pH of 7.2 must be readjusted in the antigen suspension.
It may also be advisable to compare the reactivity of new and previously standardised batches of antigen using a panel of defined sera.
At least three dilutions must be prepared for each serum. Dilutions of suspect serum must be made in such a way that reading of the reaction at the positivity limit is made in the median tube (or well for the microplate method).
The degree of Brucella agglutination in a serum must be expressed in IU per ml.
A serum containing 30 or more IU per ml is considered to be positive.
The FPA shall be standardised so that:
the OIEELISA SP SS and OIEELISA WP SS consistently give positive results.
a 1/8 pre-dilution of the OIEELISA WP SS or a 1/64 pre-dilution of the OIEELISA SP SS made up in a negative serum (or in a negative pool of sera) always gives a negative reaction;
the OIEELISA N SS always gives a negative reaction.
The following shall be included in each batch of tests: a strong positive, a weak positive, a negative working standard serum (calibrated against the OIE ELISA Standard Sera).] ] ]
Textual Amendments
F1 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.