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)

THE EUROPEAN COMMISSION,
Having regard to the Treaty on the Functioning of the European Union,
Having regard to Council Directive 2009/156/EC of 30 November 2009 on animal health conditions governing the movement and importation from third countries of equidae, and in particular Article 20 thereof,
OJ L 192, 23.7.2010, p. 1.
Whereas:
(1) Annex IV to Directive 2009/156/EC sets out diagnostic methods for African horse sickness to be used, when necessary, for testing equidae prior to their movement within the Union or imports from non-EU countries.
(2) Since the adoption of Directive 2009/156/EC, laboratory capacities to carry out advanced, highly sensitive and efficient tests for the diagnosis of African horse sickness have developed. In parallel, the Chapter related to the diagnosis of African horse sickness in the Manual of Diagnostic Tests and Vaccines for Terrestrial Animals of the World Organisation for Animal Health (OIE) has been amended to reflect that development.
http://www.oie.int/fileadmin/Home/eng/Health_standards/tahm/2.05.01_AHS.pdf
(3) As part of their 2014 work programme, the European Union Reference Laboratory for African horse sickness produced a report on the technical assessment of the diagnostic methods described in Annex IV to Directive 2009/156/EC. The assessment, which was presented to the Commission in May 2015, concluded that the competitive enzyme-linked immunosorbent assay (ELISA) is no longer available, indirect ELISA is not in common use but could be provided in 4-6 months after the request and that the blocking ELISA is commercially available and commonly used for analysis of samples during the Proficiency Test exercises organised by the European Union Reference Laboratory for African horse sickness.
Council Directive 92/35/EEC of 29 April 1992 laying down control rules and measures to combat African horse sickness (OJ L 157, 10.6.1992, p. 19).
(4) In addition, the report points out that the nucleic acid recognition by reverse-transcription polymerase chain reaction (RT-PCR) methods have advantages over serological diagnostic methods, because they allow for the detection of the disease at an early stage of infection. In addition, most of the national reference laboratories of the European Union Member States use real-time RT-PCR methods, including for the diagnosis of African horse sickness, which have proven to be fit for purpose in the annual Proficiency Test exercises performed from 2009 to 2014. The report also indicates that outside the Union there are a number of OIE reference laboratories and other laboratories with specific African horse sickness expertise that have implemented at least one of the real-time RT-PCR methods for the detection of African horse sickness genome.
(5) On the 24-25 November 2015, the Joined Workshop of African Horse Sickness/Bluetongue European Union Reference Laboratories together with national reference laboratories held in Ascot, United Kingdom, recommended the inclusion in Annex IV to Directive 2009/156/EC of real time reverse transcription (RRT)-polymerase chain reaction (PCR) methods for the detection of the African horse sickness virus.
(6) Although all available real-time RT-PCR methods for the African horse sickness genome detection are sufficiently sensitive, the procedure described by Agüero et al. (2008) is the most widely used by laboratories. The method described by Guthrie et al. (2013) was specifically designed to ensure that horses from areas at risk of African horse sickness can be transported safely after the minimum quarantine period required in accordance with the Terrestrial Animal Health Code of the OIE.
Agüero M., Gomez-Tejedor C., Angeles Cubillo M., Rubio C., Romero E. and Jimenez-Clavero A. (2008). Real-time fluorogenic reverse transcription polymerase chain reaction assay for detection of African horse sickness virus. J. Vet. Diagn. Invest., 20, 325-328.
Guthrie AJ, MacLachlan NJ, Joone C, Lourens CW, Weyer CT, Quan M, Monyai MS, Gardner IA. Diagnostic accuracy of a duplex real-time reverse transcription quantitative PCR assay for detection of African horse sickness virus. Journal of Virological Methods. 2013;189(1):30-35.
http://www.oie.int/fileadmin/Home/eng/Health_standards/tahc/current/chapitre_ahs.pdf
(7) It is therefore appropriate to incorporate in Annex IV to Directive 2009/156/EC methods for agent identification and for the detection of antibody as complementary methods for a rapid diagnosis of African horse sickness.
(8) Annex IV to Directive 2009/156/EC should be therefore amended by deletion of the competitive ELISA test and by an updating the procedures for the indirect and blocking ELISA tests in accordance with Chapter 2.5.1. of the Manual of Diagnostic Tests and Vaccines for Terrestrial Animals of the OIE, Edition 2016 based on the Version adopted by the World Assembly of Delegates of the OIE in May 2012. At the same time, real-time RT-PCR procedures as described by Agüero et al. (2008) as well as by Guthrie et al. (2013) should be included in that Annex to make those agent identification tests available for the purpose of pre-movement testing.
See footnote 2.
(9) Directive 2009/156/EC should therefore be amended accordingly.
(10) The measures provided for in this Decision are in accordance with the opinion of the Standing Committee on Plants, Animals, Food and Feed,
HAS ADOPTED THIS DECISION:

Article 1 Annex IV to Directive 2009/156/EC is replaced by the text set out in the Annex to this Decision.
Article 2 This Decision is addressed to the Member States.
Done at Brussels, 14 October 2016.
For the Commission
Vytenis Andriukaitis
Member of the Commission
ANNEX

‘ANNEX IV

AFRICAN HORSE SICKNESS DIAGNOSIS

PART A Serological tests
The serological method described hereinafter are enzyme-linked immunosorbent assays (ELISA) based on point 2 of Section B in Chapter 2.5.1 of the Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, Edition 2016 as adopted by the World Assembly of Delegates of the OIE in May 2012.
The VP7 viral protein is an immuno-dominant major antigen of the African horse sickness virus (AHSV) and is conserved across the nine AHSV serotypes. Recombinant AHSV-VP7 proteins have been shown to be stable and innocuous and suitable to be used as antigens in ELISA procedures for determination of AHSV antibodies with a high degree of sensitivity and specificity (Laviada et al., 1992b; Maree and Paweska, 2005). The indirect ELISA and the blocking ELISA are the two AHS-VP7 ELISA tests suitable for serological diagnosis of African horse sickness (AHS).
Laviada M.D., Roy P. and Sanchez-Vizcaino J.M (1992b). Adaptation and evaluation of an indirect ELISA and immunoblotting test for African horse sickness antibody detection. In: Bluetongue, African Horse Sickness and Related Orbiviruses: Proceedings of the Second International Symposium. Walton T.E. & Osburn B.l., Eds. CRC Press, Boca Raton, Florida, USA, 646-650.

1. Indirect ELISA for the detection of antibodies to African horse sickness virus (AHSV)
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) uses protein G as conjugate that also reacts with zebra serum.
Maree 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.
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

1.1.1. Solid phase
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.
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.
1.1.1.3. Block the plates with phosphate buffered saline (PBS) pH 7,2 + 5 % (w/v) skimmed milk (Nestlé Dry Skim MilkTM), 200 μl/well, for 1 hour at 37 °C.
1.1.1.4. Remove the blocking solution and gently tap the plates onto absorbent material.

1.1.2. Test samples

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.
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.

1.1.3. Conjugate
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.
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.

1.1.4. Chromogen/Substrate

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 H2O2).
Colour development is stopped by adding 50 μl of 3N H2SO4 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).

1.2. Interpretation of the results
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).
1.2.2. Test samples giving absorbance values lower than the cut-off are regarded as negative.
1.2.3. Test samples giving absorbance values greater than the cut-off + 0,15 are regarded as positive.
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.

2. Blocking ELISA for the detection of antibodies to African horse sickness virus (AHSV)
The competitive blocking ELISA is designed to detect specific AHSV antibodies in sera from animals of any equine species, i.e. horses, donkeys, zebra and their crosses, preventing the problem of specificity experienced occasionally using the indirect ELISAs.
The principle of the test is the blocking of the reaction between the recombinant VP7 protein absorbed to the ELISA plate and a conjugated AHS-VP7 specific monoclonal antibody (Mab). Antibody in the test sera will block the reaction between the antigen and the Mab resulting in a reduction in colour. Because the Mab is directed against the VP7, the assay will give a high level of sensitivity and specificity.
The competitive blocking ELISA is commercially available.

2.1. Test procedure

2.1.1. Solid Phase
2.1.1.1. Coat ELISA plates with 50-100 ng of recombinant AHSV-4 VP7 diluted in carbonate/bicarbonate buffer, pH 9,6. Incubate overnight at 4 °C.
2.1.1.2. Wash the plates three times with phosphate buffered saline (PBS) 0,1× containing 0,135 M NaCl and 0,05 % (v/v) Tween 20 (PBST). Gently tap the plates on to absorbent material to remove any residual wash.

2.1.2. Test samples and controls

2.1.2.1. Serum samples to be tested, and positive and negative control sera, are diluted 1 in 5 in diluent containing 0,35 M NaCl, 0,05 % (v/v) Tween 20 and 0,1 % Kathon, 100 μl per well. Incubate for 1 hour at 37 °C.
For titration, make a twofold dilution series of the test sera from 1 in 10 to 1 in 280 across 8 wells (100 μl/well), one serum per plate column, and do the same with positive and negative controls. Incubate for 1 hour at 37 °C.
2.1.2.2. Wash the plates five times with phosphate buffered saline (PBS) 0,1× containing 0,135 M NaCl and 0,05 % (v/v) Tween 20 (PBST). Gently tap the plates on to absorbent material to remove any residual wash.

2.1.3. Conjugate
2.1.3.1. Dispense 100 μl/well of horseradish peroxidase-conjugated Mab anti-VP7. In advance, this Mab has been diluted 1/5 000-1/15 000 in a 1/1 solution of StabiliZyme Select® Stabilizer (SurModics. Reference: SZ03) in distilled water. Incubate for 30 minutes at 37 °C.
2.1.3.2. Wash the plates five times with phosphate buffered saline (PBS) 0,1× containing 0,135 M NaCl and 0,05 % (v/v) Tween 20 (PBST). Gently tap the plates on to absorbent material to remove any residual wash.

2.1.4. Chromogen/Substrate
Add 100 μl/well chromogen/substrate solution, i.e. 1 ml of ABTS (2,2′-Azino-bis-[3-ethylbenzothiazoline-6-sulphonic acid]) 5 mg/ml + 9 ml of substrate buffer (0,1 M Phosphate-Citrate buffer of pH 4 containing 0,03 % H2O2), and incubate for 10 minutes at room temperature. Colour development is stopped by adding 100 μl/well of 2 % (w/v) SDS (sodium dodecyl sulphate).

2.1.5. Reading
Read at 405 nm in an ELISA reader.

2.2. Interpretation of the results

2.2.1. Determine the blocking percentage (BP) of each sample by applying the following formula, where “Abs” stands for antibodies:
BP=Abscontrol−−AbssampleAbscontrol−−Abscontrol+×100
2.2.2. Samples showing a BP value higher than 50 % should be considered as positive for AHSV antibodies.
2.2.3. Samples showing a BP value lower than 45 % should be considered as negative for AHSV antibodies.
2.2.4. Samples showing a BP value between 45 % and 50 % should be considered as inconclusive and must be retested. If the result is again inconclusive, the animals should be retested on samples taken not earlier than two weeks after the sample which was considered to be inconclusive was taken.

PART B Identification of the agent

Real-time Reverse-Transcription Polymerase Chain Reaction (rRT-PCR)
Agent identification tests based on nucleic acid methods must detect reference strains from the nine virus serotypes of the AHSV.
The method described in point 2.1 is based on point 1.2 of Section B in Chapter 2.5.1 of the Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, Edition 2016 as adopted by the World Assembly of Delegates of the OIE in May 2012.
Any RT-PCR detection method used for the testing of samples, either blood or spleen, in the context of Directive 2009/156/EC must perform equal to or exceed the sensitivity of the methodologies described in point 2.
Inactivated virus of serotypes 1 to 9 reference strains may be obtained from the European Union Reference Laboratory or the OIE Reference Laboratory for African horse sickness, Algete, Spain.

1. Extraction of viral RNA
To assure a good reaction it is necessary to extract from the sample an AHSV RNA of high quality. The extraction of nucleic acids from clinical samples can be performed by a variety of in-house and commercially available methods.
Commercial kits use different approaches for RNA isolation. Most are based on one of the following procedures:
Phenol-chloroform extraction of nucleic acids;
Adsorption of nucleic acids to filter system;
Adsorption of nucleic acids to magnetic beads system.
An example of an in-house RNA extraction is given below:
1.1. 1 g of tissue sample is homogenised in 1 ml of denaturing solution (4 M guanidium thiocyanate, 25 mM sodium citrate, 0,1 M 2-mercaptoethanol, 0,5 % sarcosyl).
1.2. After centrifugation, 1 μg of yeast RNA, 0,1 ml of 2 M sodium acetate pH 4, 1 ml of phenol and 0,2 ml of chloroform/isoamyl alcohol mixture (49/1) are added to the supernatant.
1.3. The suspension is vigorously shaken and cooled on ice for 15 minutes.
1.4. After centrifugation, the RNA present in the aqueous phase is phenol extracted, ethanol precipitated and resuspended in sterile water.

2. Real-time RT-PCR Procedure

2.1. Group-specific real-time RT-PCR by Agüero et al., 2008
Agüero M., Gomez-Tejedor C., Angeles Cubillo M., Rubio C., Romero E. and Jimenez-Clavero A. (2008). Real-time fluorogenic reverse transcription polymerase chain reaction assay for detection of African horse sickness virus. J. Vet. Diagn. Invest., 20, 325-328.
This group-specific real-time RT-PCR targets VP7 of the AHSV and is able to detect all known AHSV serotypes and strains currently circulating. It has been employed with very good results by the participating national reference laboratories of the European Union Member States in the proficiency tests annually organised by the European Union Reference Laboratory for the period 2009-2015. Moreover, in an international ring trial organised in 2015 in the framework of the OIE reference laboratories network this protocol was ranked very high amongst others.
Primer and probe sequences for the detection of AHSV species viruses:

— forward Primer
5′-CCA-GTA-GGC-CAG-ATC-AAC-AG-3′

— reverse Primer
5′-CTA-ATG-AAA-GCG-GTG-ACC-GT-3′

— MGB-TaqMan probe
5′-FAM-GCT-AGC-AGC-CTA-CCA-CTA-MGB-3′
2.1.1. Primer stock concentration is diluted to a working concentration of 8 μM (“primer working stock 8 μM”) whereas probe is diluted to a working concentration of 50 μM (“probe working stock 50 μM”). A test plate layout should be designed and loaded into the real time PCR machine software. Using the layout as a guide, 2,5 μl of each primer working stock 8 μM is added to each well that will contain RNA samples, positive and/or negative controls (final concentration of the primer will be 1 μM in the 20 μl RT-PCR mix). The plate is held on ice.
2.1.2. 2 μl of isolated RNA (test samples and positive control), or 2 μl of RNAse-free water in negative reaction controls, is mixed with forward and reverse primers. This mixture is denatured by heating at 95 °C for 5 minutes, followed by rapid cooling on ice for at least 5 minutes.
2.1.3. An appropriate volume of real time one-step RT-PCR master mix for the number of samples to be tested is prepared following manufacturer's instructions. 0,1 μl of probe working stock 50 μM is added to each well containing RNA samples (final concentration of the probe will be 0,25 μM in each well containing RNA samples). 13 μl of real time one-step RT-PCR master mix is distributed in each well on the PCR plate containing the denatured primers and RNA.
2.1.4. The plate is placed in a real time thermal cycler programmed for reverse transcription and cDNA amplification/fluorescence detection. Amplification conditions consist of a first reverse-transcription step at 48 °C for 25 minutes, followed by 10 minutes at 95 °C (“hot start”) and 40 cycles of 15 seconds at 95 °C, 35 seconds at 55 °C and 30 seconds at 72 °C (or 40 cycles at 97 °C for 2 seconds and 55 °C for 30 seconds if reagents and thermocycler allowing fast reactions are used). Fluorescence data are acquired at the end of the 55 °C step.
2.1.5. The assay is considered not valid if atypical amplification curves are obtained, and must be repeated.
Samples are considered positives, if the Ct value (cycle number at which the fluorescence generated within a reaction crosses the fluorescence threshold) is lower than or equal to the defined Ct threshold (35) within 40 PCR cycles (Ct ≤ 35).
Samples are considered inconclusive, if the Ct value is higher than the defined Ct threshold (35) within 40 PCR cycles (Ct > 35).
Samples are considered negative, if a horizontal amplification curve is obtained which does not cross the threshold line within 40 PCR cycles.

2.2. Group-specific real-time RT-PCR by Guthrie et al., 2013
Guthrie AJ, MacLachlan NJ, Joone C, Lourens CW, Weyer CT, Quan M, Monyai MS, Gardner IA. Diagnostic accuracy of a duplex real-time reverse transcription quantitative PCR assay for detection of African horse sickness virus. Journal of Virological Methods. 2013;189(1):30-5.
Real-time RT-PCR using fluorescence resonance energy transfer (FRET) probes to detect nucleic acid of AHSV.
The AHSV RT-PCR assay described was designed using sequences from a wide variety of currently circulating field strains of AHSV (Quan et al., 2010). It also incorporates a proprietary synthetic external control assay to verify proper functioning of the assay components.
Quan, M., Lourens, C.W., MacLachlan, N.J., Gardner, I.A., Guthrie, A.J., 2010. Development and optimisation of a duplex real-time reverse transcription quantitative PCR assay targeting the VP7 and NS2 genes of African horse sickness virus. J. Virol. Methods 167, 45-52.
Kits for the one-step real-time PCR are available commercially. Below are some basic steps as described by Guthrie et al. (2013), which can be modified depending upon local/case-specific requirements, kits used and equipment available.
Primer and probe sequences for the detection of AHSV species viruses:

— forward Primer
5′-AGA-GCT-CTT-GTG-CTA-GCA-GCC-T-3′

— reverse Primer
5′-GAA-CCG-ACG-CGA-CAC-TAA-TGA-3′

— MGB-TaqMan probe
5′-FAM-TGC-ACG-GTC-ACC-GCT-MGB-3′
2.2.1. Primer and probe mix stock solutions are made up in a 25× concentration at 5 μΜ for the forward and reverse primers and 3 μΜ for the probe. A test plate layout should be designed and loaded into the real-time PCR machine software. Using the layout as a guide, 5 μl of RNA samples, including test samples and positive and negative controls, are added to appropriate wells of the plate following the layout.
2.2.2. The RNA is denatured by heating at 95 °C for 5 minutes, followed by rapid cooling on ice for at least 3 minutes.
2.2.3. An appropriate volume of real-time one-step RT-PCR master mix for the number of samples to be tested is prepared, following the manufacturer's instructions. 1 μl of 25× primer probe mix stock solution (from point 2.2.1 above) is included in the master mix to give a final concentration in each well of 200 nM for each primer and 120 nM of the probe. 20 μl of the master mix is distributed in each well on the PCR plate containing the denatured RNA.
2.2.4. The plate is placed in a real-time thermal cycler programmed for reverse transcription and cDNA amplification/fluorescence detection as suggested by the manufacturers. Amplification conditions consist of, for example, a first reverse-transcription step at 48 °C for 10 minutes, followed by 10 minutes at 95 °C and 40 cycles of 15 seconds at 95 °C and 45 seconds at 60 °C.
2.2.5. Samples are considered positives, if the normalised fluorescence for the AHSV RT-PCR assay exceeds a 0,1 threshold within 36 PCR cycles in all replicates of a sample.
Samples are considered inconclusive, if the normalised fluorescence for the AHSV RT-PCR assay exceeds a 0,1 threshold between 36 and 40 PCR cycles in any replicate of a sample.
Samples are considered negative, if the normalised fluorescence for the AHSV RT-PCR assay did not exceed a 0,1 threshold within 40 PCR cycles in all replicates of a sample and if the normalised fluorescence for the proprietary synthetic external control assay exceeded a 0,1 threshold within 33 PCR cycles.’