S.L. 437.60 Art. 19National reference library.

The competent authority may designate a national reference laboratory for one or more of the diseases of equidae listed in Schedule A and shall stipulate the functions, tasks and procedures regarding collaboration with laboratories responsible for diagnosing infectious diseases of equidae in other Member States. SCHEDULE A Compulsorily notifiable diseases following diseases are compulsorily notifiable: Dourine Glanders encephalomyelitis (of all types, including VEE) Infectious anaemia

Anthrax African horse sickness Vesicular stomatitis.

SCHEDULE B a HEALTH INFORMATION ( ) ....................... undersigned, certify (b) that the equine animal described above meets the requirements: has been examined today and shows no clinical sign of disease; is not intended for slaughter under a national programme of contagious or infectious disease eradication; does not come from the territory or part of the territory of a Member State/third country which is the subject of restrictions for reasons of c African horse sickness ( ), or - it comes from the territory or part of the territory of a Member State which was subject to prohibition for animal health reasons and has undergone, with satisfactory results, the tests provided for in Article 5 (3) of Directive 90/426/=+EEC in the quarantine station of .............................................................. between c ................................. and .......................................... ( ) - it is not vaccinated against African horse sickness, or it was vaccinated against African horse sickness on .................................... (c) (d) ; has not come from a holding which was subject to prohibition for animal health reasons nor had contact with equidae from a holding which was subject to prohibition for animal health reasons: - during six months in the case of equidae suspected of having contracted dourine, beginning on the date of the last actual or possible contact with a sick animal. However, in the case of a stallion, the prohibition shall apply until the animal is castrated, - during six months in the case of glanders or equine encephalomyelitis, beginning on the day on which the equidae suffering from the disease in question are slaughtered, - in the case of infectious anaemia, until the date infected animals having been slaughtered, the remaining have shown a negative reaction to two Coggins tests three months apart, - during six months from the last case, in the case stomatitis, - during one month from the last case, in the case of - during fifteen days from the last case, in the case of - if all the animals of species susceptible to the disease the holding have been slaughtered and the premises during thirty days, beginning on the day on which were destroyed and the premises disinfected, except anthrax, where the period of prohibition is fifteen days;

(e) to the best of my knowledge, it has not been in contact suffering from an infectious or contagious disease in the prior to this declaration. Date Place Stamp and signature official veterinarian

(1) Name in block capitals and capacity.

(f) at the time of inspection it was fit to be transported on journey in accordance with the provisions of Directive 91/628/EEC.

--------------------------------------------a ( ) This information is not required where there is a bilateral agreement with Article 6 of Directive 90/426/EEC.

(b) Valid for 10 days. c ( ) Delete whichever does not apply. d ( ) The vaccination date must be entered in the passport. SCHEDULE C MODEL

Health certificate for trade between Member States of the EEC

......................................................

State of dispatch: .................................................................................... responsible: ............................................................................................ Department responsible: .......................................................................

Number of equidae: Identification of equidae:

equidae Species: Breed, Age, Sex Method of identification 2 horse, ass, mule, and identification ( ) hinny and destination of animal/s: is/are to be sent from: ................................................................ (Place of export) .................................................................................................................... (Member State and place of destination) address of consignor: ............................................................................ ............................................................................................................................. address of consignee: ............................................................................ ............................................................................................................................. a information ( )

undersigned, certify that the animal/s described above meet/s the following has/have been examined today and show/s no clinical sign of disease; is/are not intended for slaughter under a national programme of infectious disease eradication; it/they does/do not come from the territory or part of the territory of a Member State/third country which is the subject of restrictions for reasons of African horse sickness, or it/they come/s from the territory or part of the territory of a Member State which was the subject of restrictions for reasons of African horse sickness and has/have undergone, with satisfactory results, the tests provided for in Article 5 (3) of Directive 90/426/EEC in the quarantine station of ............................................. between ................................. and ...............................; it/they is/are not vaccinated against African horse sickness, or it/they was/were vaccinated against African horse sickness on b ............................... ( ); has/have not come from a holding which was subject to prohibition for animal health reasons nor had contact with equidae from a holding subject to prohibition for animal health reasons: - during six months in the case of equidae suspected of having dourine, beginning on the date of the last actual or possible a sick animal. However, in the case of a stallion, the prohibition apply until the animal is castrated, - during six months in the case of glanders or equine encephalomyelitis, beginning on the day on which the equidae suffering from question are slaughtered, - in the case of infectious anaemia, until the date on which, animals having been slaughtered, the remaining animals negative reaction to two Coggins tests carried out three months - during six months from the last case, in the case of vesicular - during one month from the last case, in the case of rabies, - during fifteen days from the last case, in the case of anthrax, - if all the animals of species susceptible to the disease holding have been slaughtered and the premises desinfected days, beginning on the day on which the animals were destroyed premises desinfected, except in the case of anthrax, where prohibition is fifteen days;

5. to the best of my knowledge, it/they has/have not been in equidae suffering from an infectious or contagious disease in the fifteen this declaration. 6. at the time of inspection it/they was/were fit to be transported intended journey in accordance with the provisions of Directive 91/628/EEC V. This certificate is valid for ten days.

Done at ..........................................., date ..........................................

............................................................... (signature of the official veterinarian)

.......................................................

(Name in capital capacity of signing veterinarian) SCHEDULE D African horse sickness diagnosis the enzyme-linked immunosorbent assays (ELISA) described below obtained from the European Community Reference Laboratory or the OIE Laboratories for African horse sickness. Competitive elisa for the detection of antibodies to african horse sickness (prescribed test) ELISA is used to detect specific AHSV antibodies in sera from any equidae. The broad spectrum, polyclonal, immune anti-AHSV guinea-pig (hereinafter "guinea-pig antiserum") is serogroup specific and is able to detect serotypes of AHS virus.

principle of the test is the interruption of the reaction between AHSV antigen guinea-pig antiserum by a test serum sample. AHSV antibodies in the test will compete with those in the guinea-pig antiserum resulting in a the expected colour (following the addition of enzyme labelled anti-antibody and substrate). Sera can be tested at a single dilution of 1 in 5 method) or may be titrated (serum titration method) to give dilution end-Inhibition values higher than 50% may be regarded as positive. protocol described hereinafter is used in the Regional Reference African horse sickness in Pirbright, United Kingdom. procedure Preparation of plates 1.1.1.1. Coat ELISA plates with AHSV antigen extracted from infected cell cultures and diluted in carbonate/bicarbonate buffer, pH 9,6. Incubate the ELISA plates overnight at 4°C. 1.1.1.2. Wash plates three times by flooding and emptying the wells with phosphate buffered saline (PBS), pH 7,2 to 7,4 pH, and blot dry on adsorbent paper. Control wells 1.1.2.1. Titrate the positive control sera in a twofold dilution series, from 1 in 5 to 1 in 640, across column 1 in blocking buffer (PBS containing 0,05% (v/v) Tween-20, 5,0% (w/v) skimmed-milk powder (Cadbury's MarvelTM) and 1% (v/v) adult bovine serum) to give a final volume of 50 µl/well 1.1.2.2. Add 50 µl of the negative control serum at a dilution of 1 in 5 (10 µl serum + 40 µl blocking buffer) to wells A and B of column 2. 1.1.2.3. Add 100 µl/well of blocking buffer to wells C and D of column 2 (blank). 1.1.2.4. Add 50 µl of blocking buffer to wells E, F, G and H of column 2 (guinea pig control). Spot test method 1.1.3.1. Add a 1 in 5 dilution of each test serum in blocking buffer to duplicate wells of columns 3 to 12 (10 µl sera + 40 µl blocking buffer). or Serum titration method 1.1.4.1. Prepare a twofold dilution series of each test sample (1 in 5 to 1 in 640) in blocking buffer across eight columns (3 to 12). then 1.1.5. Add 50 µl of guinea pig antisera, pre-diluted in blocking all wells except the blank wells of the ELISA plate contain a final volume of 100 µl). 1.1.5.1. Incubate for 1 hour at 37°C on an orbital shaker. 1.1.5.2. Wash plates three times and blot dry as before. 1.1.5.3. Add 50 µl of rabbit anti-guinea-pig horseradish

(HRP) conjugate prediluted in blocking buffer 1.1.5.4. Incubate for 1 hour at 37°C on an orbital shaker. 1.1.5.5. Wash plates three times and blot dry as before. 1.1.6. Chromogen Prepare the chromogen OPD (OPD = ortho-phenyldiamine) according to the manufacturers instructions (0,4 mg/ml distilled water) just before use. Add substrate (hydrogen H2O2) to give a final concentration of 0,05% (v/v) (1 30% solution of H2O2). Add 50 µl of the OPD solution and leave plates on the bench for 10 minutes at ambient Stop the reaction by the addition of 50 µl/well of 1M (H2SO4). 1.1.7. Reading Read spectrophotometrically at 492 nm.

1.2. Expression of results 1.2.1. Using a software package print out the optical density and the percentage inhibition (PI) for test and control the mean value recorded in the four guinea pig control data expressed as OD and PI values are used to determine the test has performed within acceptable limits. The limits (UCL) and lower control limits (LCL) for control are between OD values 1,4 and 0,4 respectively. point titre for the positive control based on 50% PI 240 (within a range from 1 in 120 to 1 in 480). Any plate conform to the above criteria must be rejected. However, positive control serum titre is greater than 1 in 480 samples are still negative then the negative test samples accepted. The duplicate negative control serum wells and the duplicate wells should record PI values between + 25% and between + 95% and + 105%, respectively. Failure to limits does not invalidate the plate but does suggest that colour is developing. 1.2.2. The diagnostic threshold (cut-off value) for test sera 50%). Samples recording PI values greater than 50% positive. Samples recording PI values lower than 50% as negative. Samples that record PI values above and below the threshold duplicate wells are considered doubtful. Such samples tested in the spot test and by titration. Positive samples titrated to provide an indication of the degree of positivity. layout

2 3 4 5 6 7 8 9 10 11 12 Test sera +ve cont 31 32 33 34 35 36 37 38 39 40 -ve cont 31 32 33 34 35 36 37 38 39 40 Blank Blank GP cont. GP cont. GP cont. 1 2 3 4 5 6 7 8 9 10 GP cont. 1 2 3 4 5 6 7 8 9 10 negative control. positive control. guinea pig control --------------------------------------------------------------------------------------------------

2 3 4 5 6 7 8 9 10 11 12 Test sera +ve cont 1:5 1:5 -ve cont 1:10 1:10 Blank 1:20 1:20 Blank 1:40 1:40 GP cont. 1:80 1:80 GP cont. 1:160 1:160 GP cont. 1:320 1:320 GP cont. 1:640 1:640 negative control. positive control. guinea pig control --------------------------------------------------------------------------------------------------Indirect elisa for the detection of antibodies to african horse sickness virus (prescribed test) described hereinafter is in accordance with the test description in Chapter OIE Manual of Standards for Diagnostic Tests and Vaccines, fourth recombinant VP7 protein has been used as antigen for AHS virus antibody with a high index of sensitivity and specificity. Other advantages are and not infective. procedure

Solid phase 2.1.1.1. ELISA plates are coated with recombinant AHSV-4 VP7 diluted in carbonate/bicarbonate buffer, pH 9,6. Incubate plates overnight at 4°C. 2.1.1.2. Wash the plates five times with distilled water containing 0,01 % (v/v) Tween 20 (washing solution). plates onto absorbent material to remove any 2.1.1.3. Block the plates with phosphate buffered saline

(w/v) skimmed milk (Nestlé Dry Skim Milk well, for 1 hour at 37°C. 2.1.1.4. Remove the blocking solution and gently tap absorbent material. 2.1.2. Test samples 2.1.2.1. Serum samples to be tested, and positive control sera, are diluted 1 in 25 in PBS skimmed milk + 0,05% (v/v) Tween 20, 100 Incubate for 1 hour at 37°C. For titration, make a twofold dilution series (100 µl/well), one serum per plate column, and with positive and negative controls. Incubate 37°C. 2.1.2.2. Wash the plates as described in step 2.1.1.2. 2.1.3. Conjugate 2.1.3.1. Dispense 100 µl/well of horseradish-peroxidase conjugated anti-horse gamma-globulin diluted milk + 0,05% Tween 20, pH 7,2. Incubate 37°C. 2.1.3.2. Wash the plates as described in step 2.1.1.2. 2.1.4. Cromogen/Substrate 2.1.4.1. Add 200 µl/well of chromogen/substrate solution 80,6 mM DMAB (dimethyl aminobenzaldehyde) 1,56 mM MBTH (3-methyl-2-benzo-thiazoline hydrochlorid) + 5 µl H2O2) Colour development is stopped by adding H2SO4 after approximately 5 to 10 minutes negative control begins to be coloured). Other chromogens such as ABTS (2,2'-Azino-bis-[3-ethylbenzothiazoline-6-sulphonic acid]), TMB benzidine), or OPD (ortho-phenyldiamine) used. 2.1.4.2. Read the plates at 600 nm (or 620 nm). 2.2. Interpretation of the results

2.2.1. Calculate the cut-off value by adding 0,6 to the value control (0,6 is the standard deviation derived with negative sera). 2.2.2. Test samples giving absorbance values lower than regarded as negative. 2.2.3. Test samples giving absorbance values greater than the are regarded as positive. 2.2.4. Test samples giving intermediate absorbance values are a second technique must be employed to confirm the result.