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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