European Union Reference Laboratory For African Swine Fever

Transcription

1 European Union Reference Laboratory For African Swine Fever THE EU RL ROLE AND ACTIVITIES: IMPROVING KNOWLEDGE FOR LABORATORY DIAGNOSTICS. Dr. Marisa Arias Animal Health Research Centre, CISA- INIA 28130, Valdeolmos, Madrid, Spain Pulawy, 26 March, 2014

2 CISA-INIA (1993), Valdeolmos, Madrid, SPAIN BIOSAFETY LEVEL 3 and 4 (Agriculture) FACILITY Research, Development and Innovation of novel tools for preparation, prevention, surveillance control and eradication of emerging and transboundary infectious diseases of livestock.

3 40 BSL-3 laboratories. 2 BSL-4 (Agri) - CISA Valdeolmos -

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

5 EUROPEAN UNION REFERENCE LABORATORY FOR AFRICAN SWINE FEVER ( May 2002) FAO REFERENCE CENTRE (November 2013)

6 CONTENT - ASF some characteristics. - ASF Diagnosis - The EURL Duties Interactions within EURL and NRLs Research activities: Improving knowledge of the epidemiology situation worldwide ASF Diagnostic tools are adapted to the different scenarios? Technology Transfer Some GAPS and NEEDs in affected Regions with special atention to GAPS in Diagnosis.

7 MAIN CHARACTERISTICS - Haemorrhagic disease of swine, caused by a large complex virus, High economic Impact - High mortality rates associated with acute and peracute forms belonging to the former OIE List A

8 ASF PIGS (D & W) and SOFT TICKS Very resistant to inactivation in E DNA virus, Genetic variability 22 genotypes. In Eastern Europe genotype II

9 ASF EURL: MAIN DUTY. AFRICAN SWINE FEVER DIAGNOSIS Virus detection techniques. Virus Isolation. Identification. Serological Tests EURL Mission: keeping abreast of developments in African swine fever surveillance, epizootiology and prevention throughout the world.

10 Virological Tests VIRUS ISOLATION AND IDENTIFICATION Haemoadsorption autorosette (HA) test with peripheral blood leukocytes from infected pigs DETECTION OF VIRUS ANTIGENS 1. Direct immuno fluorescent test (DIFT/FAT) OIE PRESCRIBED 2. ELISA for antigen detection VALIDATED METHODS Detection of the 22 genotypes of ASFV isolates

11 Virological Tests DETECTION OF THE VIRUS GENOME by PCR OIE PRESCRIBED VALIDATED METHODS Detection of the 22 genotypes of ASFV isolates

12 Virological tests NEW VALIDATED PCR TESTS FOR ASFV TaqMan real-time PCR Tignon et al, 2011 UPL real-time PCR Fernández-Pinero et al, 2012 Tetracore real-time PCR COMMERCIAL kit for ASFV

13 ASF DIAGNOSTICS TOOLS Antibody Detection Techniques - Immunoblotting test (IB) OIE PRESCRIBED - Indirect Immunofluorescence test (IFA) VALIDATED METHOD

14 Antibody Detection Techniques NEW COMMERCIAL ELISAs Specificity 100% Sensitivity 89% PRELIMINARY EVALUATION AT EURL Specificity 87,88% Sensitivity 95,8% STANDARDIZATION OF NEW TEST Indirect Immunoperoxidase test (IPT) DIAGNOSTIC SENSITIVITY 98% DIAGNOSTIC SPECIFICITY 98% EURL VALIDATED METHOD

15 FIRST ASF PENSIDE TEST FOR IN SITU DETECTION OF SPECIFIC ANTIBODIES IN BLOOD and SERUM. on-site use 99% Diagnostic Sensitivity 99.9% Diagnostic Specificity Antibodies could be detected in blood and serum samples from 10 days after infection. 10 minutes VALIDATED PENSIDE TEST, based on Immuno chromatographic test INGENASA. REGISTERED

16 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, organising periodic comparative tests of diagnostic procedures at Community level, collecting and collating data and information on the methods of diagnosis used and the results of tests carried out, characterising isolates of the virus by the most up-to-date methods available to allow greater understanding of the epizootiology of African swine fever, keeping abreast of developments in African swine fever surveillance, epizootiology and prevention throughout the world, retaining expertise on the virus causing African swine fever and other pertinent viruses to enable rapid differential diagnosis;

17 2. Preparation and distribution of ASF reference standards, biological material and reagents for ASF diagnosis. harmonization of ASF diagnostic techniques at NRLs. EU MEMBER STATES THIRD COUNTRIES COUNTRY INT. ORGANIZATIONS TOTAL ASF REFERENCE STANDARDS ASF-Ag ASF-PC ASF-NC ASF-LC ASF-IB ASF-DNA ASF-FITC (doses) (ml) (ml) (ml) (strips) (nº) (ml) AUSTRIA BELGIUM FRANCE GREECE ITALY SPAIN (surveillancep , POLAND BELARUS TANZANIA MOZAMBIQUE ,5 RUSSIA UKRAINE UGANDA WRL (Spain) WRL (South Afri , EU MEMBER STATES THIRD COUNTRIES COUNTRY INT. ORGANIZATIONS TOTAL ASF BIOLOGICAL MATERIAL ASFV-isolates ASF-IPT VERO cells (ml) (fixedplates) (ml) DENMARK 1-1 GREECE ITALY (Sardinia) NETHERLANDS 1-1 SWEDEN 1-1 SPAIN ARGENTINA BELARUS KENYA RUSSIA WRL (Spain)

18 URL duties -Council Directive 2002/60/EC, Annex V- 3. Preparation and distribution of REFERENCE PANELS (non-infectious) for the implementation, validation and internal verification of diagnostic techniques in NRLs. 12 reference sample sera antibody detection techniques. 16 reference sample panel (serum and tissues) for DNA extraction methods purposes. 14 reference DNAs PCR techniques.

19 SUMMARY OF ACTIVITIES OF THE ASF-EURL IN EASTERN EUROPEAN COUNTRIES 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)

20 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, organising periodic comparative tests of diagnostic procedures at Community level, collecting and collating data and information on the methods of diagnosis used and the results of tests carried out, characterising isolates of the virus by the most up-to-date methods available to allow greater understanding of the epizootiology of African swine fever, keeping abreast of developments in African swine fever surveillance, epizootiology and prevention throughout the world,

21 EUROPEAN UNION REFERENCE LABORATORY for African Swine Fever THE CISA-INIA ROLE Harmonization of the ASF diagnosis in the NRLs INTERLABORATORY COMPARISON TESTS (ILCT) Blind samples, 12/14serum samples and 6/10 tissue samples resembling different escenarios, (outbreaks, chronic animals, endemic situation )

22 41/41 Labs DIAGNOSTIC TOOLS IN USE : ANTIBODY DETECTION 19.51% (8 Labs) 41.46% (17 Labs) ELISA

23 41/41 Labs DIAGNOSTIC TOOLS IN USE : VIRUS DETECTION PCR ELISA-AG VI ,7 36,6

24 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, organising periodic comparative tests of diagnostic procedures at Community level, collecting and collating data and information on the methods of diagnosis used and the results of tests carried out, characterising isolates of the virus by the most up-to-date methods available to allow greater understanding of the epizootiology of African swine fever, To perform research activities and whenever possible coordinate research activities directed towards an improved control of ASF ( Through funds from different sources,: EURL, ILRI-INIA, ASFRISK, ASFORCE EU project, etc,..)

25 IMPROVING KNOWLEDGE ON EPIDEMIOLOGY AND DIAGNOSIS IN AFRICA

26 Africa, More than 20 countries of Subsaharan Africa endemic

27 Improve knowledge about the epidemiological situation of ASF in Africa Description of the epidemiological situation in African countries based on epidemiological findings and sample collection. FAO INIA-ILRI Sampling and characterisation of currently circulating field strains, epidemiology of wild suids, reservoirs (ticks).. ASFRISK EU Improve understanding of virus spread and maintenance in West and east African countries FAO INIA-ILRI

28 WEST AFRICA Evidence of a high incidence of the disease Cross-virus circulation between neighbouring countries Togo, Ghana, Benin and Nigeria in Maritime areas, as well as in bordering areas among Ghana and Burkina Faso. Domestic pig-cycle associated genotype I.

29 ASF endemic country Republic of the Congo WEST AFRICA Movement of a virus genotype previously associated with virulent ASF in Eastern Africa, towards western Africa, where the viruses have been always classified in p72 genotype I, to date, There would be the possibility of future outbreaks of disease caused by novel viruses in western Africa. Confirming the continuing spread of ASFV.,

30 Main Activities Epidemiology and prevalence study of African Swine Fever in Kenya and Uganda NIGERIA WHAT S GOING ON? Surveillance program in Kenya and Uganda By sampling collection in domestic pigs, wild suids, ticks. Prevalence study of ASFV in wild pigs (warhogs, bushpigs) and ticks (Kenya) and their role in the transmission of the disease. -A significant percentage of ASF Virus in samples (40% of sampling)., and in contrast Low seroprevalence in domestic pigs without clinical signs.

31 CLINICAL AND PATHOLOGICAL CHARACTERIZATION OF AFRICAN STRAINS BY IN VIVO EXPERIMENTS ASFV circulating isolates from circulating strains, particularly those exhibiting major variation is eastern African countries. -ASFV isolates from Eastern Africa (Kenya): Domestic cycle (genotype IX). Sylvatic cycle (genotype X) Viremia: from 4-7dpi; High virulent. No Ab response before died in the second week pi Viremia: from 4-7dpi Pigs died between (CC)dpi Two pigs (C7,C8)survive to infection

32 C7-C8 SURVIVAL PIGS D0 ASF-Ken05 Tk1(10 UHA ) D46 (D02ªinoculation) ASF-Ken05 Tk1(10 4UHA ) D24 2ª inoculation SACRIFIED Ab + 2nd challenge The in vivo experiments with circulating eastern Africa strains confirmed the sensitivity of Serological (OIE ELISA, and Ingenasa ELISA) and virological diagnostic techniques (OIE Conventional and Real Time PCR currently used at the MS).

33 Are the diagnostic tools adapted to the different scenarios? Three different Transmission cycles East African isolates VARIABILITY OF SEQUENCE.

34 Evaluation of serological diagnostic tools in epidemiological situations of Europe, west and eastern Africa. STRATEGY: to develop New serological diagnostic tools (ELISA and IPT as confirmatory test using new Antigens obtained from a number of virus isolates of several specific genotypes.

35 The current Evaluation ASF of serological diagnostic diagnostic tools in tools epidemiological ARE ADAPTED situations TO ALL EPIDEMIOLOGICAL in Caucasus, west and eastern SITUATIONS Africa.

36 Reseach Activities Comparative study of African swine fever virus in African and European domestic pigs. Clinical and pathological characterization of Kenyan isolates.

37 EXPERIMENTAL DESIGN Selected DOMESTIC PIG BREEDS 29 Indigenous domestic swine (local breed) from Homa Bay district (6-month old) 10 Exotic domestic swine (Landrace) from Kitengela (6-month old)

38 COMPARATIVE RESULTS

39 CONCLUSIONS Different behavior in ASFV infection, in ASF clinical signs. A clear delay of onset of ASF in indigenous pigs Indigenous pigs: No external clinical signs related to ASF the disease could be easily unrecognized in field conditions. Several animals even died without fever. The laboratory was essential to confirm the presence of ASF by PCR technique the viremia was detectable by conventional methods as expected as it was the case in European breeds. Delay in the seroconversion in indigenous pigs. Several animals at 30 dpi not seroconverted. INDIGENOUS DOMESTIC PIGS: TOLERANT PIGS

40 ASF EPIDEMIOLOGY IMPROVING KNOWLEDGE ON EPIDEMIOLOGY AND DIAGNOSIS WAHID OIE IN EUROPE Georgia June 2007 New ASFV p72 genotype II associated to those circulating in East African countries

41 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 and Armenia

42 SUMMARY OF ACTIVITIES OF THE ASF-EURL IN EASTERN EUROPEAN COUNTRIES Epidemiological investigation by laboratory analysis of field samples collected in Caucasus countries. Molecular characterization of Caucasus ASFV isolates ( ) in collaboration with the NIVVM Pokrov. 100% HOMOLOGY P72, p54 and p30 genes A single introduction

43 In Vivo studies. Azerbaijan: Az08D Armenia: Arm 07 Ukraine: Ukr12/ZAPO Lithuania on going VIRULENT ISOLATES ACUTE CLINICAL FORMS HIGH MORTALITY, from 6-9 dpi. ANTIBODY RESPONSE FROM SECOND WEEK OF INFECTION.

44 Confirmatory ASF diagnosis and molecular characterization of samples received from the outbreak occurred in UKRAINE, 2012, BELARUS 2013, Isolation and characterization of Lithuanian (2014) and Polish (2014) isolates

45 URL duties(council Directive 2002/60/EC, Annex V): To perform research activities and whenever possible coordinate research activities directed towards an improved control of ASF (ILRI-INIA Agreement, ASFRISK, National funds) To make the necessary arrangements for training or re-training experts in laboratory diagnosis with a view to harmonising diagnostic techniques. To have trained personnel available for emergency situations occurring within the Community

46 TRAINING COURSES, EU MS and Eastern countries. 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.

47 TECHNOLOGY TRANSFER Short-Term Trainings EU NRLs : Netherlands, Poland, Italy, Greece, from Croatia Belarus, Russia and Belarus January 2011, 2013 EPI-LAB training course, CISA/UCM, June, 2011 Ukraine, June 2012,2013 NRLs Eastern Countries: Russia, Belarus, Ukraine,. NRLs ASIA: China, Vietnam. Russia June 2008, 2009,2012

48 MISSIONS from 2011 IMPROVING KNOWLEDGE OF THE DISEASE, and TECHNOLOGY TRANSFER ON SITE. Training courses on African swine fever (ASF) diagnostic IN EUROPE - NRLs from EU Bulgary (2011), Greece (2012), Lithuania (2014), Poland (2014) - EASTERN EUROPE: RusSia (2011), Ukraine (2011), Armenia (2012), Kazahjistán (2013) Belarus (2012, 2013) Next:.. Eastern countries (LoAs FAO)

49 MISSIONS :. In Africa Kenya (2006, 2007, 2008), Tanzania (2008), Uganda (2009), Nigeria (2009), Ruanda (2010), Burundi (2010/11), SouthÁfrica (2011) In China 2011(Lanzhou, NRL Kingdao)

50 THREAT OF ASF SPREAD IN EASTERN EUROPE: URGENT NEED FOR INTERNATIONAL COLLABORATION. Budapest, December EXPERT WORKSHOP ON ASF, GLOBAL RESEARCH ALLIANCE, PLUM ISLAND (PIADC), N.YORK, USA, April 2013 (RESOLAB).Sixth Annual Coordination Meeting, Dakar, Senegal, December EKE, EXPERT KNOWLEDGE ELICITATION PARMA, January, 2014 ASF GLOBAL ALLIANCE, ROME, ITALY FAO Headquarters November, 2013

51 SOME NEEDS IN EASTERN EUROPE Continue improving knowledge on ASF Epidemiology. 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

52 FROM OUR EXPERIENCE: More information about : PREVALENCE STUDIES in certain regions, APPROPRIATE SAMPLING STRATEGY CHARACTERISTICS OF THE VIRUS ISOLATES CIRCULATING in the regions.

53 ASF DIAGNOSIS INFECTION DISEASE/DEAD CARRIERS Dinamic of ASF Infection VIRUS VIRUS AND ANTIBODIES ANTIBODIES DNA/Ag Detection Techniques DNA and antibody Detection Techniques 0 dpi CLINICAL SIGNS 7 dpi 35 dpi CARRIERS

54 DIAGNOSTIC TESTS GAP: Expand the catalogue of sample choices VALIDATION OF NEW SAMPLE TYPES Oral fluids and herd diagnostics meat juice: diaphragmatic muscle juice. (virus: PCR, Antibodies: OIE ELISA,IPT) solid supports (filter papers) for transporting and testing validate type of samples from wild species in different tests biological samples: more studies for best sample

55 Potential use of ORAL FLUID SAMPLES as diagnostic tool for ASF antibody detection

56 Validation of dried blood storage filter papers for application in ASF wild boar sero-surveillance programmes. ASF Antibody stability study onto the filter papers storing of positive, limit and negative DB samples at room temperature for 7, 15, 30, 90 and 180 days for further analyses AFTER 30 DAYS Comparable sensitivity and specificity values by the analyses of the samples using the IPT. OIE ELISAs decreasing specificity to 68%. INGENASA ELISAs decreasing sensitivity to 75%) Usefulness and feasibility of the filter paper blood collection method for testing of ASF antibodies using the IPT test.

57 DIAGNOSTICS TESTS GAP: On site Diagnosis DEVELOPMENT AND VALIDATION OF NEW TESTS ASF DIAGNOSIS in endemic areas MAY BE NOT AN EASY TASK - New diagnostic tests adapted to the different scenarios, - Antigen detection acompanying the Antibody detection Penside tests To get a complete picture of the infection status on site, in the farm.

58 GAP: DIAGNOSTICS TESTS Due to the ever-increasing number of ASF diagnostic tests, trusting results is becoming more difficult. Control policies based on surveillance data require confidence in the veracity of the data. FIELD VALIDATION DATA NEEDED TO EXPAND FOR ALL TESTS

59 DIAGNOSTICS TESTS GAP: There is not enough genetic information to develop new good molecular based technologies and nanotechnologies INCREASE GENOME SEQUENCING To Improve molecular epidemiology,opening the possibility of other genomic regions with enhanced discriminatory value for: Classification of virus strain diversity/epidemiology /Geographical distribution/evolution/new introductions To Design more efficient diagnostic tools based on detection of genetic material and alerting diagnostics for new variants

60 NEW EU PROJECT. : ASFORCE THEME [KBBE ] Targeted research effort on ASF 18 partners from Europe, FAO, and Private Companies. (Portugal, Bulgary, France, Germany, Italy, Russia, Spain, UK,) - Pig-wild boar- argasidae interactions relevant for ASF epidemiology. - Prevention, control and eradication models for ASF - Development of protection tools against ASF, carrier studies and Penside tests. - Training and knowledge transfer

61 SOME NEEDS IN EUROPE (EU MS and Non-MS) BE READY WITH AN ASF LABORATORY CONTINGENCY PLANS (LCPs) Designed to put into place in case of an ASF emergency Full equipped laboratories with a range of technical standards; experienced staff; SOPs (standard operation procedures) etc. LCP should address the issue of rapid scaling up of virus diagnostic and serological diagnostic capacity. Guideline on biosecurity requirements. - CISA Valdeolmos

62 SUMARIZING: FIELD: Improve knowledge LABORATORY: Good conection with field Good tests and procedures TRAINING: FIELD AND LABORATORY TRANSFER OF TECHNOLOGY: TRAINING COURSES AT RL

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65 Thank You Very Much for your Attention ASF EU REFERENCE LABORATORY To support you in your needs

66 THANK YOU NRL NVRI,PULAWY ASF REFERENCE LABORATORY, UCM, Madrid-Spain CISA-INIA TEAM