ANIMAL HEALTH CONDITIONS GOVERNING THE MOVEMENT AND IMPORT FROM THIRD COUNTRIES OF EQUIDAE RULES

SUBSIDIARY LEGISLATION 437.60

ANIMAL HEALTH CONDITIONS GOVERNING

THE MOVEMENT AND IMPORT FROM THIRD

COUNTRIES OF EQUIDAE RULES

1st April, 2005

LEGAL NOTICE 88 of 2005, as amended by Legal Notice 260 of 2010.

Chapter 1: GENERAL PROVISIONS

(1) The title of these rules is the Animal Health Conditions

governing the Movement and Import from Third Countries of

Equidae Rules.

(2) The scope of these rules is to implement the rules found

under European Union Council Directive 90/426/EEC of the 26th

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.

Compulsorily notifiable diseases

The following diseases are compulsorily notifiable

Glanders

Equine encephalomyelitis (of all types, including VEE)

Infectious anaemia

African horse sickness

Vesicular stomatitis.

(1) Unless stated otherwise in these rules the definitions in

the Act shall continue to apply.

(2) For the purpose of these rules and unless the context

otherwise requires

"the Act” means the Veterinary Services Act;

"assembly centre" means collection centres and markets, at

which under the supervision of the official veterinarian, equidae

originating from different holdings are grouped together to form

consignments of animals for national movement;

"the Commission" means the European Commission;

"the Community" means the European Community as established

under the Treaty establishing the European Community;

"the competent authority" m eans the Veterinary Services

Authority in Malta as established by the Act;

"compulsorily notifiable diseases" means the diseases listed in

Schedule A;

"equidae" means wild or domesticated animals of the equine

(including zebras) or asinine species or the offspring of crossings

of those species;

"equidae for breeding and production" means equidae other than

equidae for slaughter and registered equidae;

"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;

"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;

" M em b e r St at e " m e an s a st a t e w h i ch i s a m em b e r o f t he

European Union;

"Member State of dispatch" means such Member State from

where equidae are transported or transmitted or are intended to be

transported or transmitted through an approved market or assembly

centre to the place of destination;

" M e m b e r St a t e o r t h i r d c o u n t r y f r e e f r o m A f r i c a n h o r se

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 twelve

months;

"official veterinarian" means the veterinarian designated by the

competent authority of Malta or by the competent authority of a

Member State or of a third country;

"place of arrival or destination" means such Member State to

which equidae are transported from their holding or establishment

of origin either directly or through an approved market or assembly

centre in the Member State of dispatch or from a third country;

"registered equidae" means any equidae registered as defined

under the Trade with Member States in Equidae (Zootechnical and

Genealogical Conditions) Regulations, identified by means of an

identification document issued by the breeding authority or any

ot her competent author ity of the country where the animal

originated, which manages the studbook or register for that breed

of animals or any international association or organisation which

manages horses for competition or racing;

"temporary admission" means the status of a registered animal

originating in a third country and admitted into the Community for

a period of less than ninety 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;

"third country" means a state which is not a member of European

Union;

"trade" means trade between Member States or between a third

country and a Member State in animals and their products, a

business or occupation to make profit or gain, particularly in

wholesale or retail sales and includes dealings in a particular

business activity.

Chapter 2: RULES FOR THE MOVEMENT OF EQUIDAE

HEALTH INFORMATION ( a )

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,

ANIMAL HEALTH CONDITIONS GOVERNING THE MOVEMENT

AND IMPORT FROM THIRD COUNTRIES OF EQUIDAE

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 fifteen 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 thirty 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 fifteen 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 fiftten days

prior to this declaration.

Date

Place

Stamp and signature of the

official veterinarian ( 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.

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

with Article 6 of Directive 90/426/EEC.

Valid for 10 days.

Delete whichever does not apply.

The vaccination date must be entered in the passport.

ANIMAL HEALTH CONDITIONS GOVERNING THE MOVEMENT

AND IMPORT FROM THIRD COUNTRIES OF EQUIDAE

The competent authority shall authorise the movement of

equidae registered in Malta or send equidae to another Member

State only where they satisfy the conditions laid down in rules 4

and 5. However, the competent authority 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, the competent authority

shall inform the Commission of the content of the

exemptions granted.

MODEL

Health certificate for trade between Member States of the EEC

EQUIDAE

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

Member State of dispatch: ....................................................................................

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

Territorial Department responsible: .......................................................................

I.

Number of equidae

II.

Identification of equidae

Number of equidae

Species

Breed, Age, Sex

Method of identification

and identification ( 2 )

horse, ass, mule,

hinny

III.

Origin and destination of animal/s

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

(Place of export)

to: ....................................................................................................................

(Member State and place of destination)

Name and address of consignor: ............................................................................

Name and address of consignee: ............................................................................

Health information ( a )

IV.

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

ANIMAL HEALTH CONDITIONS GOVERNING THE MOVEMENT

AND IMPORT FROM THIRD COUNTRIES OF EQUIDAE

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 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 fifteen 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 fifteen 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 fifteen 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 ).

This certificate is valid for ten days.

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

(signature of the official veterinarian) ( 3 )

(Signature)

(Name in capital letters and

capacity of signing veterinarian) ( c )

ANIMAL HEALTH CONDITIONS GOVERNING THE MOVEMENT

AND IMPORT FROM THIRD COUNTRIES OF EQUIDAE

(1) Equidae must show no clinical sign of disease at

inspection. Inspection must be carried out in the forty-eight hours

prior to their 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 fifteen 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 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 competent authority to the Commission and the

other Member States within three months of the date

on which these rules 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

ANIMAL HEALTH CONDITIONS GOVERNING THE MOVEMENT

AND IMPORT FROM THIRD COUNTRIES OF EQUIDAE

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) fifteen 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

thirty 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

fifteen days.

The competent authority may derogate from these

prohibition measures for hippodromes and racecourses, and shall

notify the Commission of the nature of any derogation granted.

(6) Where the competent authority draws up or has drawn up a

voluntary or compulsory control programme for a disease to which

equidae are susceptible, it 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 competent authority 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 competent

authority may be amended or supplemented in

accordance with the same procedure.

African horse sickness diagnosis

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.

1. Competitive elisa for the detection of antibodies to african horse sickness

virus (AHSV) (prescribed test)

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.

Preparation of plates

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.

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

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 Marvel TM ) and 1% (v/v)

adult bovine serum) to give a final volume of 50 µ l/well

Add 50 µ l of the negative control serum at a dilution of 1 in

5 (10 µ l serum + 40 µ l blocking buffer) to wells A and B of

column 2.

Add 100 µ l/well of blocking buffer to wells C and D of

column 2 (blank).

Add 50 µ l of blocking buffer to wells E, F, G and H of

column 2 (guinea pig control).

Spot test method

Add a 1 in 5 dilution of each test serum in blocking buffer

to duplicate wells of columns 3 to 12 (10 µ l sera + 40 µ l

blocking buffer). or

Serum titration method

Prepare a twofold dilution series of each test sample (1 in 5

ANIMAL HEALTH CONDITIONS GOVERNING THE MOVEMENT

AND IMPORT FROM THIRD COUNTRIES OF EQUIDAE

to 1 in 640) in blocking buffer across eight wells of single

columns (3 to 12). then

Add 50 µ l 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 µ l).

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 µ l of rabbit anti-guinea-pig horseradish peroxidase

(HRP) conjugate prediluted in blocking buffer to each well.

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.

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 µ l 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 µ l/well of 1M sulphuric acid

(H2SO4).

Reading

Read spectrophotometrically at 492 nm.

1.2. Expression of results

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.

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.

ANIMAL HEALTH CONDITIONS GOVERNING THE MOVEMENT

AND IMPORT FROM THIRD COUNTRIES OF EQUIDAE

-ve cont = negative control.

+ve con = positive control.

GP cont = guinea pig control

1:5

1:5

1:10

1:10

1:20

1:20

1:40

1:40

1:80

1:80

1:160

1:160

1:320

1:320

1:640

1:640

-ve cont = negative control.

+ve con = positive control.

GP cont = guinea pig control

2. Indirect elisa for the detection of antibodies to african horse sickness virus

(AHSV) (prescribed test)

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

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.

Solid phase

ELISA plates are coated with recombinant AHSV-4 VP7

diluted in carbonate/bicarbonate buffer, pH 9,6. Incubate

plates overnight at 4°C.

Wash the plates five times with distilled water containing

ANIMAL HEALTH CONDITIONS GOVERNING THE MOVEMENT

AND IMPORT FROM THIRD COUNTRIES OF EQUIDAE

0,01 % (v/v) Tween 20 (washing solution). Gently tap the

plates onto absorbent material to remove any residual wash.

Block the plates with phosphate buffered saline (PBS) + 5%

(w/v) skimmed milk (Nestlé Dry Skim Milk TM ), 200 µ l/

well, for 1 hour at 37°C.

Remove the blocking solution and gently tap the plates onto

absorbent material.

Test samples

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 µ l per well.

Incubate for 1 hour at 37°C.

For titration, make a twofold dilution series from 1 in 25

(100 µ l/well), one serum per plate column, and do the same

with positive and negative controls. Incubate for 1 hour at

37°C.

Wash the plates as described in step 2.1.1.2.

Conjugate

Dispense 100 µ l/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.

Wash the plates as described in step 2.1.1.2.

Cromogen/Substrate

Add 200 µ l/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 µ l H2O2)

Colour development is stopped by adding 50 µ l 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.

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

2.2. Interpretation of the results

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).

Test samples giving absorbance values lower than the cut-off are

regarded as negative.

Test samples giving absorbance values greater than the cut-off + 0,15

are regarded as positive.

Test samples giving intermediate absorbance values are doubtful and

a second technique must be employed to confirm the result.

3. Blocking elisa for the detection of antibodies to african horse sickness virus

(AHSV) (prescribed test)

ANIMAL HEALTH CONDITIONS GOVERNING THE MOVEMENT

AND IMPORT FROM THIRD COUNTRIES OF EQUIDAE

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.

T he p ri ncip al o f the test i s th e in ter rup ti on of the react ion bet ween 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.

ELISA plates

Coat ELISA plates with recombinant AHSV-4 VP7 diluted

in carbonate/bicarbonate buffer, pH 9,6. Incubate overnight

at 4°C.

Wash the plates five times with phosphate buffered saline

(PBS) containing 0,05% (v/v) Tween 20 (PBST).

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.

Test samples and controls

For screening

dilute test sera and controls 1 in 10 directly on the plate in

PBST to give a final volume 100 µ l/well. Incubate for 1

hour at 37°C.

For titration

prepare a twofold dilution series of test sera and positive

controls (100 µ l/well) from 1 in 10 to 1 in 1 280 across

eight wells. Negative control is tested at 1 in 10 dilution.

Conjugate

Add 50 µ l/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.

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

Chromogen/Substrate

Add 100 µ l/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 µ l/well

of 2% (w/v) SDS (sodium dodecyl sulphate).

Reading

Read at 405 nm in an ELISA reader.

3.2. Interpretation of the results

ANIMAL HEALTH CONDITIONS GOVERNING THE MOVEMENT

AND IMPORT FROM THIRD COUNTRIES OF EQUIDAE

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.

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.

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.

(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, the competent authority 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) only under the conditions set out in sub-rule (3).

(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 twelve 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 subrule

(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);

(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 twenty-one and thirty days between

the two tests, the second of which must have

been carried out during the ten 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 forty 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.

The competent authority may grant, to equidae coming

from Member States which implement an alternative control system

providing 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

competent authority shall notify the Commission thereof.

(1) The equidae shall be transported, as soon as possible,

from the holding of origin either directly or via an approved market

or marshalling centre defined as "assembly centre" under these

rules to the place of destination in vehicles or containers which

have been regularly cleansed and disinfected with a disinfectant at

intervals to be fixed by the Member State of dispatch. The vehicles

shall be designed in such a way that equidae droppings, litter or

fodder cannot escape from the vehicle during transportation.

Transportation shall be effected in such a way that the health and

well-being of the equidae can be protected effectively.

(2) The competent authority 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 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.

(1)

The competent authority 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 forty-eight 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 ten 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 (1)(ii).

The rules laid down in article 15 of the 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.

Veterinary experts from the Commission may, to the extent

necessary to ensure uniform application of these rules and in co-

operation with the competent authority, carry out on-the-spot

inspections. The competent authority shall give the experts all the

assistance necessary to carry out their task.

Chapter 3: RULES FOR IMPORTS FROM THIRD COUNTRIES

Equidae imported from a third country into the Community

through the territory of Malta must satisfy the conditions laid down

in rules 12 to 16.

(1) The importation of equidae in Malta as a Member

State of destination shall only be authorised from such third

countries that appear on a list or lists to be drawn up or amended in

accordance with the procedure referred to in rule 15. Taking into

account the health situation and the guarantees provided by the

third country for equidae, it may be decided in accordance with the

procedure referred to in rule 15 that the authorisation provided for

in this sub-rule shall apply to the whole territory of the third

country or to only part of its territory. For such purpose and on the

basis of the relevant international standards, account shall be taken

of how the third country applies and implements those standards, in

particular the principle of regionalisation, within its own territory

and in relation to its sanitary requirements for importation from

other third countries and other Member States.

(2) When the lists provided for in sub-rule (1) are drawn up or

amended, particular account shall be taken of

(a) the health status of the equidae, other domestic

animals and wildlife in the third country, with

particular regard to exotic animal diseases and any

aspects of the general health and the environmental

situation in the third country which may pose a risk to

the health and environmental status of the Community;

(b) the legislation of the third country in relation to animal

health and welfare;

(c) the organisation of the competent authority of the third

country and its inspection services, the powers of

those services, the supervision to which they are

subject, and the means at their disposal, including staff

and laboratory capacity, to apply national legislation

effectively;

(d) the assurances which the competent authority of the

third country can give regarding compliance or

equivalence with the relevant animal health conditions

applicable in the Community;

(e) whether the third country is a member of the Office

International des Epizooties (OIE) and the regularity

and rapidity of the information supplied by the third

country relating to the existence of infectious or

contagious diseases of equidae in its territory, in

particular those diseases listed by the OIE and in

Schedule A;

(f)

the guarantees given by the third country to directly

inform the Commission and the Member States

(i)

within twenty-four hours, of the confirmation of

the occurrence of infectious diseases of equidae

listed in Schedule A and of any change in the

vaccination policy concerning such diseases;

(ii) within an appropriate period, of any proposed

changes in the national sanitary rules concerning

equidae, in particular regarding the importation

of equidae;

(iii) at regular intervals, of the animal health status

of its territory concerning equidae;

(g) any experience of previous imports of live equidae

from the third country and the results of any import

controls carried out;

(h) the results of Community inspections and, or audits

carried out in the third country, in particular the results

of the assessment of the competent authorities thereof

or, where the Commission so requests, the report

submitted by the competent authorities thereof on the

inspections which they have carried out; and

(i)

the rules on the prevention and control of infectious or

contagious animal diseases in force in the third

country and their implementation, including rules on

importation of equidae from other third countries.

(3) Special import conditions for each third country or group of

third countries, having regard to the animal health situation

concerning equidae in the third country or countries concerned

shall be established in accordance with the procedure referred to in

rule 15.

(4) Detailed rules for the application of this rule and criteria for

including third countries or parts of third countries in the lists

provided for in sub-rule (1) may be adopted in accordance with the

procedure referred to in rule 15.

(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) The competent authority shall apply for a decision of the

Commission that may

(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.

Before the day of loading for transportation to Malta, the

equidae must have remained without interruption in the territory or

p a r t o f t h e t e r r i t o r y o f a t h i r d c o u n t r y o r, i n t h e e v e n t o f

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.

(1) Importation of equidae from the territory of a third

country or part thereof as defined in accordance with rule 13(2)(i)

on the list drawn up in accordance with rule 12 shall be authorised

only if 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 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 rule 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 fifteen 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 ten 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

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

competent authority of Malta may decide that such

equidae may be slaughtered within twenty-one days of

arrival at the slaughterhouse, provided they remain at

the slaughterhouse under the daily supervision of the

official veterinarian. The competent authority in Malta

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 93/96/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.

(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. The

competent authority 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.

(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, the competent authority may, on animal

health grounds, designate the slaughterhouse to which such equidae

must be taken.

Without prejudice to Decision 92/160/EEC, the competent

authority shall authorise the re-entry of registered horses for racing,

competition and cultural events after temporary export where such

horses

(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.

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.