Council Directive 2009/156/EC of 30 November 2009 on animal health conditions governing the movement and importation from third countries of equidae (codified version) (Text with EEA relevance) (c. 156)

[F1ANNEX IV U.K. AFRICAN HORSE SICKNESS DIAGNOSIS

Textual Amendments

F1 Substituted by Commission Implementing Decision (EU) 2016/1840 of 14 October 2016 amending Annex IV to Council Directive 2009/156/EC as regards methods for African horse sickness diagnosis (notified under document C(2016) 6509) (Text with EEA relevance).

PART A U.K. Serological tests

1. Indirect ELISA for the detection of antibodies to African horse sickness virus (AHSV) U.K.

The conjugate used in this method is a horseradish peroxidase anti-horse gamma-globulin reacting with the serum of horses, mules and donkeys. The method described by Maree & Paweska (2005) (1) uses protein G as conjugate that also reacts with zebra serum.

The antigen may be provided by the Centro de Investigación en Sanidad Animal (CISA), Spain, within 4 to 6 months of request.

1.1. Test procedure U.K.

1.1.1. Solid phase U.K.

1.1.1.1. Coat ELISA plates with recombinant AHSV-4 VP7 diluted in carbonate/bicarbonate buffer, pH 9,6. Incubate plates overnight at 4 °C. U.K.

1.1.1.2. Wash the plates five times with distilled water containing 0,01 % (v/v) Tween 20 (washing solution). Gently tap the plates onto absorbent material to remove any residual wash. U.K.

1.1.1.3. Block the plates with phosphate buffered saline (PBS) pH 7,2 + 5 % (w/v) skimmed milk (Nestlé Dry Skim Milk ^TM ), 200 μl/well, for 1 hour at 37 °C. U.K.

1.1.1.4. Remove the blocking solution and gently tap the plates onto absorbent material. U.K.

1.1.2. Test samples U.K.

1.1.2.1. Serum samples to be tested, and positive and negative control sera, are diluted 1 in 25 in PBS + 5 % (w/v) skimmed milk + 0,05 % (v/v) Tween 20, 100 μl per well. Incubate for 1 hour at 37 °C. U.K.

For titration, make a twofold dilution series from 1 in 25 (100 μl/well), one serum per plate column, and do the same with positive and negative controls. Incubate for 1 hour at 37 °C.

1.1.2.2. Wash the plates five times with distilled water containing 0,01 % (v/v) Tween 20 (washing solution). Gently tap the plates onto absorbent material to remove any residual wash. U.K.

1.1.3. Conjugate U.K.

1.1.3.1. Dispense 100 μl/well of horseradish-peroxidase (HRP) -conjugated anti-horse gamma-globulin diluted in PBS + 5 % milk + 0,05 % Tween 20, pH 7,2. Incubate for 1 hour at 37 °C. U.K.

1.1.3.2. Wash the plates five times with distilled water containing 0,01 % (v/v) Tween 20 (washing solution). Gently tap the plates onto absorbent material to remove any residual wash. U.K.

1.1.4. Chromogen/Substrate U.K.

1.1.4.1. Add 200 μl/well of chromogen/substrate solution (10 ml of 80,6 mM DMAB (dimethyl aminobenzaldehyde) + 10 ml of 1,56 mM MBTH (3-methyl-2-benzo-thiazoline hydrazone hydrochlorid) + 5 μl H ₂ O ₂ ). U.K.

Colour development is stopped by adding 50 μl of 3N H ₂ SO ₄ after approximately 5 to 10 minutes (before the negative control begins to be coloured).

Other chromogens such as ABTS (2,2′-Azino-bis-[3-ethylbenzothiazoline-6-sulphonic acid]), TMB (tetramethyl benzidine), or OPD (ortho-phenyldiamine) can also be used.

1.1.4.2. Read the plates at 600 nm (or 620 nm). U.K.

1.2. Interpretation of the results U.K.

1.2.1. Calculate the cut-off value by adding 0,06 to the value of the negative control (0,06 is the standard deviation derived with a group of 30 negative sera). U.K.

1.2.2. Test samples giving absorbance values lower than the cut-off are regarded as negative. U.K.

1.2.3. Test samples giving absorbance values greater than the cut-off + 0,15 are regarded as positive. U.K.

1.2.4. Test samples giving intermediate absorbance values are considered to be inconclusive and a second technique must be employed to confirm the result.] U.K.

(1)

[F1Maree S. and Paweska J.T. (2005). Preparation of recombinant African horse sickness virus VP7 antigen via a simple method and validation of a VP7-based indirect ELISA for the detection of group-specific IgG antibodies in horse sera. J. Virol. Methods, 125 (1), 55-65.]