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Dr. Marisa Arias

Animal Health Research Centre , CISA- INIA 28130, Valdeolmos, Madrid, Spain

Budapest, 4-5 December, 2012

European Union Reference Laboratory For

African Swine Fever

EU RL ASSISTANCE TO EASTERN EUROPEAN

COUNTRIES: EXPERIENCES, GAPS AND NEEDS TO

IMPROVE LABORATORY DIAGNOSTICS.

40 BSL-3 Labs.

- CISA Valdeolmos -

2 BSL-4 (Agri)

BIOSAFETY LEVEL 3 and 4 (Agriculture) FACILITY

R& D on Animal infectious diseases of obligatory report: : New diagnostic tools and

strategies for surveillance, prevention, control and eradication.

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BSL-3 ANIMAL FACILITY

21 BSL-3 Animal Rooms Two Corridors Pneumatic doors Diferential pressure

EUROPEAN UNION REFERENCE LABORATORY FOR

AFRICAN SWINE FEVER

FAO REFERENCE LABORATORY

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Incubation period range: 4-19 days.

- NOT VACCINE AVAILABLE.

Control of the disease is mainly

based on Early Detection and

Strict Sanitary Measures

Recognition of the disease in the field

Laboratory Diagnosis

LABORATORY DIAGNOSIS IS ESSENTIAL FOR THE

CONTROL OF ASF

ASFV genotype II

2007-2012 African Swine Fever is

moving without control

Real Risk of

introduction in

neighboring regions

22 Genotypes in Africa

ASF EPIDEMIOLOGY

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Collect and Collate epidemiology data and information of

the disease,

Roadmap for a reliable and efficient diagnosis according

the situation and support NRLs to fit for purpose.

Harmonization of the ASF diagnosis in the NRLs

Coordinate Actions to achieve that goals.

EUROPEAN UNION REFERENCE

LABORATORY for African Swine Fever

THE CISA-INIA ROLE

Characterization of ASFV

Isolates

INIA-ILRI

INIA-ILRI

FAO

ASFRISK

FAO

MOLECULAR EPIDEMIOLOGY WORLDWIDE

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SUMMARY OF ACTIVITIES OF THE ASF-EURL IN EASTERN EUROPEAN COUNTRIES 2007-2012

Epidemiological investigation by laboratory analysis of

field samples collected in Caucasus countries.

ASF diagnosis and molecular characterization of samples

received through FAO from the outbreaks occurred

in Azerbaijan on January 22, 2008.

SUMMARY OF ACTIVITIES OF THE ASF-EURL IN EASTERN EUROPEAN COUNTRIES 2007-2012

Epidemiological investigation by laboratory analysis of

field samples collected in Caucasus countries.

Molecular

characterization of

Caucasus ASFV

isolates (2007-2010) in collaboration with

the NIVVM Pokrov.

100% HOMOLOGY

P72, p54 and p30 genes

A single introduction

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SUMMARY OF ACTIVITIES OF THE ASF-EURL IN EASTERN EUROPEAN COUNTRIES 2007-2012

Epidemiological investigation by laboratory analysis of

field samples collected in Caucasus countries.

Confirmatory ASF diagnosis and molecular

characterization of samples received from the

outbreak occurred in Ukraine on July

2012.

- ASFV isolates employed:

Azerbaijan, Az08D

Armenia: Arm 07

CLINICAL AND PATHOLOGICAL CHARACTERIZATION

ASFV circulating isolates from outbreaks in Caucasus Regions. In Vivo Experiments:

INITIAL ANTIBODY RESPONSE : SECOND WEEK of infection in surviving pigs.

VIRULENT STRAIN

CLINICAL PICTURE : Acute Form

HIGH MORTALITY RATE from 6-9 dpi.

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ANTIBODIES

CLINICAL SIGNS CARRIERS

VIRUS

ASF DIAGNOSIS INFECTION DISEASE/DEAD CARRIERS

DNA and antibody Detection

Techniques

VIRUS AND ANTIBODIES

7 dpi 35 dpi 0 dpi

Dinamic of ASF Infection

DNA/Ag Detection

Techniques

Antibody

detection techniques

Virus detection

techniques

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VALIDATED

PENSIDE TEST, based on Immuno chromatographic test

INGENASA. REGISTERED

FIRST PENSIDE TEST FOR “IN SITU” DETECTION OF

SPECIFIC ANTIBODIES IN BLOOD and SERUM AGAINST ASFV.

99% Diagnostic Sensitivity

99.9% Diagnostic Specificity

Antibodies could be detected in

blood and serum samples from 10

days after infection. 10 minutes

Useful for veterinary services

Evaluation of diagnostic tools in

epidemiological situations in Caucasus,

west and eastern Africa.

The current ASF serological and virological

diagnostic tools ARE ADAPTED TO ALL

EPIDEMIOLOGICAL SITUATIONS

Three different

Transmission cycles

East African isolates

↑ VARIABILITY OF SEQUENCE.

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URL duties(Council Directive 2002/60/EC, Annex V):

• To coordinate, in consultation with the Commission, the methods employed in the Member States for diagnosing African swine fever, specifically by:

– typing, storing and supplying strains of African swine fever virus and supplying standardised sera, and other reference reagents to the national laboratories in order to standardise the tests and reagents employed in the Member States,

– building up and holding an African swine fever virus collection,

– collecting and collating data and information on the methods of diagnosis used and the results of tests carried out,

organising periodic comparative tests of diagnostic procedures at Community level,

EUROPEAN UNION REFERENCE LABORATORY

Participants→ 42 Labs from 35 countries

ILCTs: Blind samples ( 16 serum, and tissue samples) representing the different scenarios

worldwide, acute/ subacute/ chronic/ endemic, at early, middle or late stages of infection.

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1

22.0

63.4

14.6

ANALYSIS OF NRL´S RESULTS; ASF antibody detection

All participating countries and all NRLs from Member States

performed the ASF serological diagnosis.

Techniques:

78% used confirmatory tests.

INGENASA ELISA K3 as a choice procedure (87,8%).

ANALYSIS OF NRL´S RESULTS; VIRUS DETECTION

40% 52,5%

7,5%

1 technique 2 techniques 3 techniques

THE PCR TEST WAS THE CHOICE TECHNIQUE BY ALL NRLs

PARTICIPATING IN THE VIROLOGICAL ASSAY for ASFV detection in

tissues and serum samples.

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-What are the ASF diagnostic techniques employed by NRLs:

- TYPE of techniques in use and procedures employed,

- The PERFORMANCE of ASF diagnostic techniques employed,

if it ‘fit for purpose’ to give a correct diagnosis

ANALYSIS OF DEVIATIONS,

ACTIONS TAKEN BY THE EURL

ILCTs allows the EU RL to know:

- EVALUATION OF THE ASF DIAGNOSIS TECHNIQUES (sensitivity, specificity,

reagents.....)

- Additional PROVISION OF ASF REFERENCE PANELS, SOPS, REAGENTS,.

- TRANSFER OF TECHNOLOGY: • Specific short term trainings at the EURL • EURL Missions. and Training at the NRLs, implementation of new

diagnostic tecniques

USE OF BLIND

SAMPLES

Workshop for NRLs on Diagnostic Techniques, December ,2009. for

diagnostic preparedness for ASF in the NRLs with special emphasis

on those from Eastern European countries

Trainings at EU RL by MS (Eastern Countries of EU)

15 participants representatives of NRLs from 15 EU MS: Estonia, Latvia, Lithuania, Poland, Bulgaria, Romania, Hungary, Sweden, Austria, Slovakia, Slovenia, Czech Republic, Ireland, Italy and Cyprus.

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THE EU REFERENCE

LABORATORY AND EASTERN

EUROPEAN COUNTRIES NON-MS

INTERACTIONS

SUMMARY OF ACTIVITIES OF THE ASF-EURL IN EASTERN EUROPEAN COUNTRIES 2007-2012

1. Participation in the Interlaboratory comparison tests (ILCTs) annually prepared by the URL → In order to harmonize

the current ASF diagnostic techniques employed in Eastern European countries, the

URL opened the possibility, since 2008, to participate in the ILCTs to non EU-NRLs

belonging to the Eastern European countries Russia, Armenia, Georgia, Belarus and Ukraine.

In summary, the NRLs from Belarus, Ukraine and Russia have been actively participating since 2010 receiving the panel of samples and sending the results on time to the URL.

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SUMMARY OF ACTIVITIES OF THE ASF-EURL IN EASTERN EUROPEAN COUNTRIES 2007-2012

Preparation and distribution of SOPs, ASF reference standards, biological material and reagents for ASF diagnosis. ASF- OIE reference standards: • ASF semipurified antigen (ASF-Ag) for the OIE indirect ELISA

• ASF reference positive (ASF-PC), limit (ASF-LC) and negative (ASF-NC) sera.

• ASF Immunoblotting strips (ASF-IB) for the OIE-IB confirmatory technique.

• Fixed ASFV Ba71V VERO cells 96- wells immuno-plates (ASF-IPT) for the

standarization of the URL-Immunoperoxidase technique (IPT)

SUMMARY OF ACTIVITIES OF THE ASF-EURL IN EASTERN EUROPEAN COUNTRIES 2007-2012

Preparation and distribution of SOPs, ASF reference

standards, biological material and reagents for ASF

diagnosis.

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SUMMARY OF ACTIVITIES OF THE ASF-EURL IN EASTERN EUROPEAN COUNTRIES 2007-2012

Preparation and distribution of the URL-ASF reference material for internal validation and verification of ASF diagnostic tests.

• 12 reference sample sera → antibody

detection techniques.

• 16 reference sample panel (serum and tissues)

→ for DNA extraction methods purposes.

• 14 reference DNAs → PCR techniques.

SUMMARY OF ACTIVITIES OF THE ASF-EURL IN EASTERN EUROPEAN COUNTRIES 2007-2012

TRANSFER OF TECHNOLOGY THROUGH…

1. Organization of specific short term trainings at the EURL

on ASF diagnostic techniques and production/quality control of

ASF reference standards.

Trainings on “ASFV epidemiological characterization” for the National

Research Institute of Veterinary, Virology and Microbiology (Pokrov,

Russia).

26 August -September 2008. 26-30 November 2012. 11-18 December 2009.

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SUMMARY OF ACTIVITIES OF THE ASF-EURL IN EASTERN EUROPEAN COUNTRIES 2007-2012

TRANSFER OF TECHNOLOGY. Short Term Trainings

Belarus, January 2011 Russia and Belarus

EPI-LAB training course, CISA/UCM, June, 2011

Ukraine, June 2012

SUMMARY OF ACTIVITIES OF THE ASF-EURL IN EASTERN EUROPEAN COUNTRIES 2007-2012

TRANSFER OF TECHNOLOGY THROUGH…

2. Assisting EU member states and Eastern European countries in ASF

diagnosis through EURL Missions.

Pokrov (Russia) 20 to 25 April 2009.

Pokrov (Russia) 4 – 5 October 2011.

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SUMMARY OF ACTIVITIES OF THE ASF-EURL IN EASTERN EUROPEAN COUNTRIES 2007-2012

TRANSFER OF TECHNOLOGY MISSIONS

DG SANCO “Workshops on ASF

epidemiology and diagnosis”

14-18 May 2012. Yerevan, Armenia

21-25 Nov 2011. Poltava, Ukraine

SUMMARY OF ACTIVITIES OF THE ASF-EURL IN EASTERN EUROPEAN COUNTRIES 2007-2012

TRANSFER OF TECHNOLOGY. MISSIONS

19-23 Nov 2012. Minsk, Belarus

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EPIDEMIOLOGY LABORATORY DIAGNOSIS

FROM OUR EXPERIENCE , WE HAVE LEARNED:

UCM CISA –INIA

EU RL, and EPI

Group WRL for ASF , UCM,

Madrid, Spain

SOME GAPS

Information on ASF Epidemiology and Prevalence.

Information on ASF diagnostic techniques in use, and

/or validation in certain regions/countries.

Coordination of Actions to put in place get a reliable

diagnosis

From the EU/FAO Reference

Laboratory CISA-INIA point of view:

Information on the Characteristics of the virus isolates

circulating in some regions .

Lack of sensitivity of some “in house”diagnostic techniques.

Confirmatory techniques in some countries.

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SOME NEEDS IN EASTERN EUROPE

• Continue improving knowledge on ASF Epidemiology.

• Identification of Risk Factors for ASF introduction and

spreading

(maps of pig density, wild boar distribution and ticks,,,).

• Maps for Risk Assessment

• Models for Control and Eradication programmes adapted

to the scenarios.

A good Diagnosis of ASF is important but not enough,

Information for implementing good ASF Control estrategies

is needed to get eradication

SOME NEEDS IN EASTERN EUROPE

AFFECTED REGIONS

• Prevalence studies through appropriate sampling strategy, and the application of prescribed ASF diagnostic tools to improve sensitivity and specificity of the ASF diagnosis.

Achieve Harmonization in diagnosis

through VALIDATED diagnostic techniques.

•Continue molecular characterization studies of isolates from new outbreaks and from endemic areas.

Active cooperation with affected countries that could require support

in the characterization of circulating isolates by International

Reference Laboratories

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Designed to put into place in case of an ASF emergency

• IMPROVEMENT OF LABORATORY CONTINGENCY PLANS

(LCPS)

LCP should address the issue of rapid scaling up of virus

diagnostic and serological diagnostic capacity.

Guideline on biosecurity requirements.

Full equipped laboratories with a range of technical standards;

experienced staff; SOPs (standard operation procedures) etc.

SOME NEEDS IN EASTERN EUROPE

•EDUCATION: Be ready to detect the clinical

picture

SOME NEEDS IN EASTERN EUROPE

IMPROVE EXPERTISE

FOLLOW UP on SITE

•TRAINING and TECHNOLOGY TRANSFER at NRLs and

at Province/District laboratories,

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SUMARIZING: ASF EARLY DETECTION NEED…

FIELD:

• Risk information

• ASF Information

LABORATORY:

• Good conection with field

• Good tests and procedures

TRAINING: FIELD AND LABORATORY

Coordinating Actions to be taken through a National

Reference Laboratory Network

Coordination of Actions within International

Reference Laboratories (EURL/OIE/FAO/ other

Laboratories supporting an area).

Region/Province

Laboratories

SUMARIZING: An IMPORTANT NEED…

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Coordinating Actions to be taken through a

National Reference

Laboratory Network

Coordination of Actions : International

Reference Laboratories

VETERINARY

SERVICES

Region/Province

Laboratories

Regional

Vet. Services

NATIONAL

VETERINARY

SERVICES

SUMARIZING: An IMPORTANT NEED…

MORE RESEARCH ON

AFRICAN SWINE FEVER

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WORKSHOP, Nairobi, 2011

EU RL, Improving knowledge in Africa,,…

NEW EU PROJECT. : ASFORCE

THEME [KBBE.2012.1.3-02]

Targeted research effort on ASF

18 partners from Europe, FAO , and Private Companies. (Portugal, Bulgary, France, Germany, Italy, Russia, Spain , UK)

- Development of protection tools against ASF (vaccines) ,

carrier studies and Penside tests.

- Prevention, control and eradication models for ASF

- Pig-wild boar- argasidae interactions relevant for ASF

epidemiology.

- Training and knowledge transfer

ISSUES FOCUSED TO EUROPE:

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Our Gratitude TO OUR HOST

COUNTRY…

THE ORGANIZERS…

Cote d`Ivoire

Congo Rep Tanzania

SouthAfrica

Cameroon

WRL for ASF ,

UCM, Madrid,

Spain

ACKNOWLEDGEMENTS

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ACKNOWLEDGEMENTS

EU and FAO Reference Laboratory and

Epidemiology CISA-INIA TEAMS

http://asf-referencelab.info

Thank You Very

Much for your

Attention