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Veterinary Services Act (Cap. 437) Animal Health Conditions Governing The Movement And Import From Third Countries Of Euqidae Rules, 2005 (L.N. 88 Of 2005 )

VETERINARY SERVICES ACT (CAP.437)
Animal Health Conditions governing the Movement and Import from Third Countries of Equidae Rules, 2005
IN exercise of the powers conferred under articles 8 and 9 of the Veterinary Services Act, the Minister for Rural Affairs and the Environment has made the following rules>-

Chapter 1> GENERAL PROVISIONS

1. (1) The title of these rules is the Animal Health ConditionsTitle, scope and governing the Movement and Import from Third Countries of Equidaeapplicability. Rules, 2005
(2) The scope of these rules is to implement the rules found under European Union Council Directive 90#426#EEC of the 2t6h. June
1990 on animal health conditions governing the movement and import from third countries of equidae.
(3) These rules lay down animal health conditions for the movement between Malta and Member States and import from third countries of live equidae.
2. For the purposes of these rules –
(a) “the Commission” means the European Commission<

Definitions.

(b) “the Community” means the European Community as established under the Treaty establishing the European Community<
(c) “the Competent authority” means the Veterinary Services within the territory of Malta as provided under article 2 of the Veterinary Services Act<
(d) “compulsorily notifiable diseases” means the diseases listed in Schedule A, to these rules<
(e) “equidae” means wild or domesticated animals of the equine (including zebras) or asinine species or the offspring of crossings of those species<

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(f) “equidae for breeding and production” means equidae other than equidae for slaughter and registered equidae<
(g) “equidae for slaughter” means equidae intended to be transported either directly or after transit through a market or an approved marshalling centre to the slaughterhouse for slaughter<
(h) “holding” means an agricultural or training establishment, a stable or, generally speaking, any premises or facilities in which equidae are habitually kept or bred, for whatever use<
(i) “Member State” means a State which is a Member within the European Community<
(j) “Member State or third country free from African horse sickness” means any Member State or third country in which there has been no clinical, serological (in unvaccinated equidae) or epidemiological evidence of African horse sickness on the territory concerned in the previous two years and in which there have been no vaccinations against the disease during the previous 12 months<
(k) “official veterinarian” means the veterinarian designated by the competent central authority of a Member State or of a third country<
(l) “registered equidae” means any equidae registered as defined in Directive 90#427#EEC, identified by means of an identification document issued by the breeding authority or any other competent authority of the country where the animal originated which manages the studbook or register for that breed of animal or any international association or organisation which manages horses for competition or racing<
(m) “temporary admission” means the status of a registered animal originating in a third country and admitted into Community territory for a period of less than 90 days to be fixed by the Commission in accordance with the procedure of the Community, depending on the health situation in the country of origin<
(n) “Third country” means a State which is not a Member within the European Community<
(o) “Veterinary Services” means the competent authority within Malta as established under article 2 of the Veterinary Services Act.

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Chapter 2> RULES FOR THE MOVEMENT OF EQUIDAE

3. Veterinary Services shall authorise the movement of equidaeAuthorisations of registered in Malta or send equidae to another Member State only wheremovement of they satisfy the conditions laid down in rules 4 and 5. However, the exemptions.
Veterinary Services at destination may grant general or limited exemption in respect of movement of equidae which -
(a) are being ridden or taken, for sporting or recreational purposes, along roads situated near internal borders of the Community<
(b) are taking part in cultural or similar events or in activities organised by authorised local bodies situated near internal borders of the Community<
(c) are intended solely for temporary pasturing or work near internal borders of the Community. When making use of such authorisation, Veterinary Services shall inform the Commission of the content of the exemptions granted.
4. (1) Equidae must show no clinical sign of disease at Rules for inspection
inspection. Inspection must be carried out in the 48 hours prior to theirof equidae.
embarkation or loading. In the case of registered equidae, however, this inspection shall, without prejudice to regulation 6, be required for intra- Community trade only.
(2) Without prejudice to the requirements of sub-rule (5) regarding compulsorily notifiable diseases, the official veterinarian must, at the time of inspection, be satisfied that there are no grounds in particular on the basis of declarations by the owner or breeder for concluding that the equidae have been in contact with equidae suffering from an infectious or contagious disease during the 15 days immediately preceding inspection.
(3) The equidae must not be intended for slaughter under a national programme of contagious or infectious disease eradication.
(4) The equidae must be identified in the following manner> (i) in the case of registered horses, by means of an
identification document, as provided for in Directive 90#427#EEC,
which must certify in particular that Article 5 (5) and (6) have been complied with. The official veterinarian will have to suspend the validity of this document for the period of the prohibitions provided for in Article 5(5). The document should, following the

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slaughter of the registered horse, be returned to the authority which issued it<
(ii) for equidae for breeding and production, identification by a method to be established by the Commission in accordance with the relevant procedure of the Community. Until such time as this method is in use, the officially approved national identification methods shall remain applicable, provided that they are notified by the Veterinary Services to the Commission and the other Member States within three months of the date on which these regulations are adopted.
(5) In addition to the requirements laid down in rule 5, the equidae must not come from a holding which has been the subject of one of the following prohibition orders -
(i) if all the animals of species susceptible to the disease located on the holding have not been slaughtered, the period of prohibition concerning the holding of origin must be at least -
(a) 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,
(b) 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,
(c) in the case of infectious anaemia, until the date on which, the infected animals having been slaughtered, the remaining animals have shown a negative reaction to two Coggins tests carried out three months apart,
(d) six months in the case of vesicular stomatitis,
(e) one month from the last recorded case, in the case of rabies,
(f) 15 days from the last recorded case, in the case of anthrax<
(ii) if all the animals of species susceptible to the disease located on the holding have been slaughtered and the premises disinfected, the period of prohibition shall be 30 days, beginning

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on the day on which the animals were destroyed and the premises disinfected, except in the case of anthrax, where the period of prohibition is 15 days.
The Veterinary Services may derogate from these prohibition measures for hippodromes and racecourses, and shall notify the Commission of the nature of any derogation granted.
(6) Where Veterinary Services draws up or has drawn up a voluntary or compulsory control programme for a disease to which equidae are susceptible, they may present the programme to the Commission outlining in particular –
(a) the distribution of the disease on its territory<
(b) the reasons for the programme, taking into consideration the significance of the disease and its cost#benefit advantages<
(c) the geographical area in which the programme will be implemented<
(d) the status categories to be applied to establishments, the standards which must be attained for each species and the test procedures to be used, the programme monitoring procedures<
(e) the action to be taken if, for any reason, a holding loses its status<
(f) the measures to be taken if the results of the tests carried out in accordance with the provisions of the programme are positive<
(g) the non-discriminatory nature of trade in Malta with respect to intra-Community trade. The programmes presented by the Veterinary Services shall be examined by the Commission. Where appropriate it shall approve them in accordance with the relevant procedure of the Community. Any additional guarantees, general or specific, which may be required in intra-Community trade may be defined in accordance with the same procedure. Such guarantees must not exceed those required by Malta in its own territory. Programmes submitted by Veterinary Services may be amended or supplemented in accordance with the same procedure.

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Dispatch of equidae when not free from African horse sickness.

5. (1) When Malta is not free of African horse sickness within the meaning of a Member State or Third Country free from African horse sickness in rule 2 hereof, Veterinary Services may authorise to dispatch equidae from that part of the territory of Malta which is considered to be infected within the meaning of sub-rule (2) of this rule only under the conditions set out in sub-rule (3) hereof.
(2) (i) A part of the territory of Malta shall be considered to be infected with African horse sickness if>
(a) clinical, serological (in unvaccinated animals) and, or epidemiological evidence has revealed the presence of African horse sickness in the past two years, or
(b) vaccination against African horse sickness has been carried out in the past 12 months.
(ii) The part of the territory considered to be infected with African horse sickness must comprise as a minimum>
(a) a protection zone with a radius of at least 100 km around any centre of infection<
(b) a surveillance zone at least 50 km extending beyond the protection zone, in which no vaccination has been carried out in the last 12 months.
(iii) The rules controlling the combat measures relating to the territories and zones referred to in paragraphs (a) and (b) and the relevant derogation are specified in Directive 92#35#EEC.
(iv) All vaccinated equidae found in the protection zone must be registered and identified in accordance with Article 6 (1) of Directive 92#35#EEC. The identification document and, or health certificate shall carry a clear reference to such vaccination.
(3) Malta may dispatch from its territory referred to in sub- rule 2 (b) only equidae which meet the following requirements>
(i) they must be dispatched only during certain periods of the year, having regard to the activity of vector insects as has been determined by the Community<
(ii) they must show no clinical symptom of African horse sickness on the day of the inspection referred to in rule 4 (1)<

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(iii) (a) if they have not been vaccinated against African horse sickness, they must have undergone and reacted negatively to a complement fixation test for African horse sickness as described in Schedule D, on two occasions, with an interval of between 21 and 30 days between the two tests, the second of which must have been carried out during the 10 days prior to dispatch,
(b) if they have been vaccinated, they must not have undergone vaccination during the previous two months and must have undergone the fixation test described in Schedule D at the aforementioned intervals without having recorded an increase in the antibody count. Other monitoring methods recognised by the Commission may be used
(iv) they must have been kept in a quarantine station for a minimum period of 40 days prior to dispatch<
(v) they must have been protected from vector insects during the period of quarantine and during transportation from the quarantine station to the place of dispatch.
6. Veterinary Services may grant, to equidae coming fromDerogation in case
Member States which implement an alternative control system providingof inspection.
guarantees equivalent to those laid down in rule 4(5) as regards movements within their territory of equidae and registered equidae, in particular by means of the identification document, one other derogation from the provisions of the second sentence of rule 4(1) and the second indent of rule 8(1) (ii) on a reciprocal basis. The Veterinary Services shall notify the Commission thereof.
7. (1) The equidae must be transported, as soon as possible, Transportation of
from the holding of origin either directly or via an approved market orequidae.
marshalling centre as defined in Article 2 (2) (o) of Directive
64#432#EEC to the place of destination by means of transport or containers which have been regularly cleansed and disinfected with a disinfectant at intervals to be fixed by the Veterinary Services. The means of transport must be designed in such a way that equidae droppings, litter or fodder cannot escape from such means during transportation. Transportation must be effected in such a way that the health and wellbeing of the equidae can be protected effectively.
(2) The Veterinary Services may, at destination, on a general or restricted basis, grant a derogation from some of the requirements of rule 4 (5) for any animal bearing a special mark indicating that it is scheduled for slaughter, provided that the health certificate mentions such derogation. In the case of granting such a derogation, equidae for

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slaughter must be transported directly to the designated slaughterhouse and be slaughtered within five days of arrival at the slaughterhouse.
(3) The official veterinarian must record the identification number or identification document number of the slaughtered animal and forward to the competent authority of the place of dispatch, at the latter’s request, an attestation to the effect that the animal has been slaughtered.

Identification document and certificates.

Application of Veterinary Framework Act and Council Directive

90#425#EEC.

On-the-spot inspections.

8. (1) The Veterinary Services shall verify that>
(i) registered equidae which leave their holdings are accompanied by the identification document laid down in rule 4 (4) together if they are intended for intra-Community trade with the attestation provided for in Schedule B<
(ii) equidae for breeding, production and slaughter are, during their transportation to a Member State, accompanied by a health certificate complying with Schedule C to these rules. Equidae for breeding, production and slaughter at destination Malta are accompanied by a health certificate complying with Schedule C thereof. The certificate, or in the case of an identification document, the form containing the health particulars, must, without prejudice
to rule 6, be drawn up during the 48 hours preceding their embarkation or else no later than the last working day prior to it, in at least one of the official languages of Malta and of the Member State of destination. The duration of validity of the certificate is
10 days. The certificate must consist of a single sheet.
(2) Imports of equidae, other than registered equidae, may be covered by a single health certificate per consignment rather than by the individual certificate referred to in sub-rule paragraph (1)(ii) hereof.
9. The rules laid down in Article 15 of the Veterinary Framework Act and in Council Directive 90#425#EE concerning veterinary and zootechnical checks in intra-Community trade in certain live animals and products with a view to the completion of the internal market, shall apply in particular to checks at origin, to the organisation of, and follow- up to, the checks to be carried out at destination, and to the safeguard measures to be implemented.
10. Veterinary experts from the Commission may, to the extent necessary to ensure uniform application of these rules and in co-operation with the Veterinary Services, carry out on-the-spot inspections. Veterinary Services shall give the experts all the assistance necessary to carry out their task.

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Chapter 3> RULES FOR IMPORTS FROM THIRD COUNTRIES

11. Equidae imported from a third country into the CommunityConditions for
through the territory of Malta must satisfy the conditions laid down inimported equidae
rules 12 to 16.

country.

12. In order to be imported, the equidae must come from a thirdWhen equidae are
country or part of a third country appearing on a list drawn up by theimported.
Commission.
13. (1) The equidae must come from third countries> (i) free from African horse sickness<
(ii) which have been free for two years from Venezuelan equine encephalomyelitis (VEE)<
(iii) which have been free for six months from dourine and glanders.
(2) Veterinary Services shall apply for a decision of the
Commission that may>

Conditions for the importation of equidae.

(i) decide that the provisions of sub-rule (1) shall apply to only a part of the territory of a third country. In the event that the African horse sickness requirements apply on a regional basis, at the very least the measures laid down in rules 5 (2) and (3) must be complied with<
(ii) require additional guarantees for diseases alien to the
Community.
14. Before the day of loading for transportation to Malta, theOther conditions for
equidae must have remained without interruption in the territory or partransportation.
of the territory of a third country or, in the event of regionalisation, in the part of the territory defined pursuant to rule 13 (2) (i) for a period determined in rule 15. They must come from a holding placed under veterinary supervision.
15. (1) Importation of equidae from the territory of a thirdOther qualifications country or part thereof as defined in accordance with rule 13 (2) (i) onfor importation of the list drawn up in accordance with rule 12 shall be authorised only ifCountries.
the equidae, over and above the requirements of rule 13 -
(i) comply with the animal health requirements adopted, with reference to the species in question and the categories of

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equidae, by the Community for imports of equidae from that country. The reference basis for fixing animal health conditions in accordance with sub-rule (1) shall be the standards laid down in rules 4 and 5< and
(ii) in the case of a third country not free of vesicular stomatitis or viral arteritis for at least six months, the equidae must meet the following requirements>
(a) they must come from a holding which has been free of vesicular stomatitis for at least six months and they must have reacted negatively to a serological test prior to dispatch<
(b) in the case of viral arteritis, male equidae must notwithstanding regulation 18 have reacted negatively to a serological test or to a virus isolation test or to any other test recognised by the Community.
The requirement relating to equine viral arteritis shall apply to male equidae with the exception of>
(a) equidae vaccinated against equine viral arteritis under official supervision with a vaccine approved by the competent authority and which have reacted with a negative result at 1 in 4 to a serum neutralisation test for that disease. The equidae shall be vaccinated on the date on which the blood specimen is taken or within the following 15 days on condition that they have been
isolated during that period. The test shall be carried out and certified, and the result and vaccination certified, under official
veterinary supervision. Vaccination shall be repeated at regular intervals. In the case of registered equidae, vaccinations and the results of serological tests shall be entered in the identification document (passport),
(b) equidae vaccinated against equine viral arteritis under official supervision with a vaccine approved by the competent authority and which have undergone two serum neutralisation tests for that disease at an interval of at least 10 days between the age of
180 and 270 days without an increase in antibodies being established. The equidae shall be isolated under official supervision from the time the first blood specimen is taken up to vaccination. The tests shall be carried out and certified, and the results and vaccinations certified, under official veterinary supervision. Vaccination shall be repeated at regular intervals. In the case of

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registered equidae, vaccinations and the results of serological tests shall be entered in the identification document (passport),
(c) equidae less than 180 days old,
(d) equidae for slaughter sent directly to a slaughterhouse accompanied by a certificate as provided for in Annex I to Commission Decision 93#196#EEC animal health conditions and veterinary certification for imports of equidae for slaughter.
(2) Without prejudice to Directive 92#160#EEC, Malta shall authorise imports of equidae for slaughter from a third country appearing in Part I of the Annex to Decision 79#542#EEC and which are clearly and indelibly marked by a hot-branded ‘S’ of not less than 3 cm size on the hoof of the left front leg>
(i) and which, if sent directly to a slaughterhouse to be slaughtered within five days after arrival at the slaughterhouse and not more than five days after arrival in the Community, meet the requirements of Annex I to the Decision 93#196#EEC on animal health conditions and veterinary certification for imports of equidae for slaughter. However, where equidae are subjected to a sea- voyage of more than eight days, the Veterinary Services of Malta may decide that such equidae may be slaughtered within 21 days of arrival at the slaughterhouse, provided they remain at the slaughterhouse under the daily supervision of the official veterinarian. Malta Veterinary Services shall notify the Commission of such cases<
(ii) or which, if they are passing through a market or a marshalling centre before being slaughtered, meet the requirements of Annex II to the European Commission Decision 93#196#EC.
(3) Without prejudice to Decision 92#160#EEC, Malta shall authorise imports of registered equidae and equidae for breeding –
(i) coming from a third country appearing in Annex I to Commission Decision 93#197#EC on animal health conditions and veterinary certification for imports of registered equidae and equidae for breeding and production, and
(ii) conforming to the requirements laid down in the appropriate specimen animal health certificate set out in Annex II to Commission Decision 93#197#EC.

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Identification of equidae.

Equidae on arrival in Malta.

Re-entry of registered horse for racing.

16. (1) The equidae must be identified in accordance with rule
4 (4) and accompanied by a certificate drawn up by an official veterinarian of the exporting third country. This certificate must>
(i) be issued on the day of loading of the animals for dispatch to the Member State of destination or, in the case of registered horses, on the last working day before embarkation<
(ii) be drawn up in at least one of the official languages of Malta or the Member State of destination and one of those of the Member State in which the import inspection is carried out<
(iii) accompany the animals in the original<
(iv) attest to the fact that the animals satisfy the requirements of Directive 90#426#EEC and those laid down pursuant to that Directive with regard to importation of equidae from third countries<
(v) consist of a single sheet<
(vi) be made out for a single consignee or, in the case of animals for slaughter, for a consignment, provided the animals are properly marked and identified. Veterinary Services shall inform the Commission if they make use of this option.
(2) The certificate must be drawn up on a form complying with a model established by the Community.
17. (1) Immediately upon arrival in Malta as Member State of destination, equidae for slaughter shall be taken to a slaughterhouse, either directly or after transition through a market or a marshalling centre, and, in accordance with animal health requirements, be slaughtered within a period of time specified in rule 15.
(2) Without prejudice to any special conditions which may be adopted by the Community, Veterinary Services may, on animal health grounds, designate the slaughterhouse to which such equidae must be taken.
18. Without prejudice to Decision 92#160#EEC, Veterinary Service shall authorise the re-entry of registered horses for racing, competition and cultural events after temporary export where such horses>

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(a) return from third countries appearing in Parts I and II of
the special column for equidae in the Annex to Decision
79#542#EEC to which they have been temporarily exported either directly or after transit through other countries of the same group in Annex I to the Decision 93#195#EEC on animal health conditions and veterinary certification of the re-entry of registered horses for racing, competition and cultural events after temporary export,
(b) comply with the requirements laid down in the specimen animal health certificate set out in Annex II to the Decision
93#195#EEC.

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The following diseases are compulsorily notifiable:

- Dourine

- Glanders

- Equine encephalomyelitis (of all types, including VEE)

- Infectious anaemia

- Rabies

- Anthrax

- African horse sickness

- Vesicular stomatitis.

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Passport No. ………………..

I, the undersigned, certify (b) that the equine animal described above meets the following requirements: (a) it has been examined today and shows no clinical sign of disease;

(b) it is not intended for slaughter under a national programme of contagious or infectious disease eradication;

(c) it 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 African horse sickness (c), 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 …………………………………. and ………………………………………..(c) ;

- it is not vaccinated against African horse sickness, or it was vaccinated against African horse sickness on ……………………………… (c) (d) ;

(d) it 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 on which, the infected animals having been

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slaughtered, the remaining animals have shown a negative reaction to two Coggins tests carried out three months apart,

- during six months from the last case, in the case of vesicular stomatitis,

- during one month from the last case, in the case of rabies,

- during 15 days from the last case, in the case of anthrax,

- if all the animals of species susceptible to the disease located on the holding have been slaughtered and the premises disinfected during 30 days, beginning on the day on which the animals were destroyed and the premises disinfected, except in the case of anthrax, where the period of prohibition is 15 days;

(e) to the best of my knowledge, it has not been in contact with equidae suffering from an infectious or contagious disease in the 15 days prior to this declaration.

(1) Name in block capitals and capacity.

(f) at the time of inspection it was fit to be transported on the intended journey in accordance with the

provisions of Directive 91/628/EEC(4)

(a) This information is not required where there is a bilateral agreement in accordance 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.

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EQUIDAE

No:......................................................................................................................................................................

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

Member State of dispatch :

..................................................................................................................................................................... Ministry responsible:

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

Territorial Department responsible:

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

The animal/s is/are to be sent from:

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

(Place of export)

to:

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

(Member State and place of destination)

Name and address of consignor:

............................................................................................................................................……………………

…………............................................................................................................................................................

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Name and address of consignee:

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

.......................................................................................................................................................................…

…………………….

I, the undersigned, certify that the animal/s described above meet/s the following requirements:

1. it/they has/have been examined today and show/s no clinical sign of disease;

2. it/they is/are not intended for slaughter under a national programme of contagious or infectious disease eradication;

3. - 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);

4. it/they has/have 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,

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- in the case of infectious anaemia, until the date on which, the infected animals having been slaughtered, the remaining animals have shown a negative reaction to two Coggins tests carried out three months apart,

- during six months from the last case, in the case of vesicular stomatitis,

- during one month from the last case, in the case of rabies,

- during 15 days from the last case, in the case of anthrax,

- if all the animals of species susceptible to the disease located on the holding have been slaughtered and the premises desinfected during 30 days, beginning on the day on which the animals were destroyed and the premises desinfected, except in the case of anthrax, where the period of prohibition is 15 days;

5. to the best of my knowledge, it/they has/have not been in contact with equidae suffering from an infectious or contagious disease in the 15 days prior to this declaration.

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

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

Stamp

.........…………………………………………………

(signature of the official veterinarian) (3)

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

…………

(Signature)

(Name in capital letters and capacity of signing veterinarian) (c)

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Reagents for the enzyme-linked immunosorbent assays (ELISA) described below may be obtained from the

European Community Reference Laboratory or the OIE Reference Laboratories for African horse sickness.

Competitive ELISA is used to detect specific AHSV antibodies in sera from any species of equidae. The broad spectrum, polyclonal, immune anti-AHSV guinea-pig serum (hereinafter "guinea-pig antiserum") is serogroup specific and is able to detect all known serotypes of AHS virus.

The principle of the test is the interruption of the reaction between AHSV antigen and a guinea-pig antiserum by a test serum sample. AHSV antibodies in the test serum sample will compete with those in the guinea-pig antiserum resulting in a reduction in the expected colour (following the addition of enzyme labelled anti-guinea-pig antibody and substrate). Sera can be tested at a single dilution of 1 in 5 (spot test method) or may be titrated (serum titration method) to give dilution end-points. Inhibition values higher than 50 % may be regarded as positive.

The test protocol described hereinafter is used in the Regional Reference Laboratory for African horse sickness in Pirbright, United Kingdom.

1.1.1. 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.

1.1.2. Control wells

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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).

1.1.3. 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

1.1.4. Serum titration method

then

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 wells of single columns (3 to 12).

1.1.5. Add 50 µl of guinea pig antisera, pre-diluted in blocking buffer, to all wells except the blank wells of the ELISA plate (all wells now 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 peroxidase (HRP) conjugate pre- diluted in blocking buffer to each well.

1.1.5.4. Incubate for 1 hour at 37 °C on an orbital shaker.

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1.1.5.5. Wash plates three times and blot dry as before.

1.1.6. Chromogen

Prepare the chromogen OPD (OPD = ortho-phenyldiamine) solution according to the manufacturers instructions (0,4 mg/ml in sterile distilled water) just before use. Add substrate (hydrogen peroxide

= H2O2) to give a final concentration of 0,05 % (v/v) (1 in 2000 of a 30 % solution of H2O2). Add

50 µl of the OPD solution to each well and leave plates on the bench for 10 minutes at ambient temperature. Stop the reaction by the addition of 50 µl/well of 1M sulphuric acid (H2SO4).

1.1.7. Reading

Read spectrophotometrically at 492 nm.

1.2.1. Using a software package print out the optical density (OD) values, and the percentage inhibition (PI) for test and control sera based on the mean value recorded in the four guinea pig control wells. The data expressed as OD and PI values are used to determine whether the test has performed within acceptable limits. The upper control limits (UCL) and lower control limits (LCL) for the guinea pig control are between OD values 1,4 and 0,4 respectively. The end-point titre for the positive control based on 50 % PI should be 1 in 240 (within a range from 1 in 120 to 1 in 480). Any plate that fails to conform to the above criteria must be rejected. However, if the positive control serum titre is greater than 1 in 480 and the test samples are still negative then the negative test samples can be accepted.

The duplicate negative control serum wells and the duplicate blank wells should record PI values between + 25 % and - 25 %, and between + 95 % and + 105 %, respectively. Failure to be within these limits does not invalidate the plate but does suggest that background colour is developing.

1.2.2. The diagnostic threshold (cut-off value) for test sera is 50 % (PI 50 %). Samples recording PI values greater than 50 % are recorded as positive. Samples recording PI values lower than 50 % are recorded as negative.

Samples that record PI values above and below the threshold for the duplicate wells are considered doubtful. Such samples may be re-tested in the spot test and by titration. Positive samples may also be titrated to provide an indication of the degree of positivity.

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The test described hereinafter is in accordance with the test description in Chapter 2.1.11 of the OIE Manual of Standards for Diagnostic Tests and Vaccines, fourth edition, 2000.

The recombinant VP7 protein has been used as antigen for AHS virus antibody determination with a high index of sensitivity and specificity. Other advantages are that it is stable and not infective.

2.1.1. 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). Gently tap the plates onto absorbent material to remove any residual wash.

2.1.1.3. Block the plates with phosphate buffered saline (PBS) + 5 % (w/v) skimmed milk (Nestlé Dry Skim MilkTM), 200 µl /well, for 1 hour at 37 °C.

2.1.1.4. Remove the blocking solution and gently tap the plates onto absorbent material.

2.1.2. Test samples

2.1.2.1. 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.

2.1.2.2. Wash the plates as described in step 2.1.1.2.

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2.1.3. Conjugate

2.1.3.1. 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.

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 (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.

2.1.4.2. Read the plates at 600 nm (or 620 nm).

2.2.1. Calculate the cut-off value by adding 0,6 to the value of the negative control (0,6 is the standard deviation derived with a group of 30 negative sera).

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

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

2.2.4. Test samples giving intermediate absorbance values are doubtful and a second technique must be employed to confirm the result.

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The blocking ELISA is designed to detect specific AHSV antibodies in sera from any susceptible species. VP7 is the major, antigenic, viral protein of AHSV, and is conserved within the nine serotypes. Because the monoclonal antibody (Mab) is also directed against the VP7, the assay will give a high level of sensitivity and specificity. Further, the recombinant VP7 antigen is completely innocuous and therefore guarantees a high degree of safety.

The principal of the test is the interruption of the reaction between the recombinant VP7, as the antigen bound to the ELISA plate and the conjugated Mab specific for the VP7. Antibody in the test sera will block the reaction between the antigen and the Mab resulting in a reduction in colour.

The test described hereinafter is carried out in the European Community Reference Laboratory for African horse sickness in Algete, Spain.

3.1.1. ELISA plates

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

3.1.1.2. Wash the plates five times with phosphate buffered saline (PBS) containing 0,05

% (v/v) Tween 20 (PBST).

3.1.1.3. Stabilise the plate by treatment with a stabilising solution (in order to allow long term storage at 4 °C without loss of activity) and blot dry onto adsorbent material.

3.1.2. Test samples and controls

3.1.2.1. For screening:

dilute test sera and controls 1 in 10 directly on the plate in PBST to give a final volume 100 µl/well. Incubate for 1 hour at 37 °C.

3.1.2.2. For titration:

prepare a twofold dilution series of test sera and positive controls (100 µl/well) from 1 in 10 to 1 in 1 280 across eight wells. Negative control is tested at 1 in 10 dilution.

3.1.3. Conjugate

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Add 50 µl /well of pre-diluted horseradish-peroxidase (HRP) -conjugated Mab (monoclonal antibodies specific for VP7) to each well and mix gently to ensure homogeneity. Incubate for

30 minutes at 37 °C.

3.1.4. Wash the plates five times with PBST and blot dry as above.

3.1.5. Chromogen/Substrate

Add 100 µl /well of chromogen/substrate solution (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).

3.1.6. Reading

Read at 405 nm in an ELISA reader.

3.2.1. Assay validation

The test is valid when the optical density (OD) of negative control (NC) is higher than 1,0 and the

OD of positive control (PC) is lower than 0,2.

3.2.2. Cut-off calculation

Positive cut-off = NC – ((NC – PC)x 0,3) Negative cut-off = NC – ((NC – PC)x 0,2)

Where, NC is the OD of the negative control and PC the OD of positive control.

3.2.3. Interpretation of results

Samples with OD lower than positive cut-off should be considered as positives to AHSV antibodies. Samples with OD higher than negative cut-off should be considered negatives for AHSV antibodies. Samples with OD between these two values should be considered doubtful and the animals re- sampled after two to three weeks.