Current status of serological and nucleic acid based diagnostic techniques for ASF
Nairobi July 19th-22nd 2011Current Status of Serological and Nucleic acid-based Diagnostic techniques for ASF: EU REFERENCE LABORATORY FOR AFRICAN SWINE FEVER Current Status of Serological and Nucleic Dr. Marisa Arias acid-based Diagnostic ASF WORKSHOP techniques for ASF Nairobi July, 2011
SPAIN …In the 80´s Classical swine fever: Erradicated with vaccination in 1986. Foot and Mouth Disease: Erradicated with vaccination in 1987. African Swine Fever - CISA Valdeolmos -
Research and development in Animal infectiuos diseases, and the development of new diagnostic tools for prevention, diagnosis, control and eradication of animal infectious diseases of obligatory report.1993: CISA-INIA,Valdeolmos BIOSAFETY LEVEL 3 and 3+ FACILITY
SPAIN - Second Pig Producing Country in the European Union (EU) - More than 4.000 million Euros /year.census 2011: 2,7 Million Sows 2011: 29,5 millions pigs SPAIN 38% Export market. SPAIN European Leader in the pig sector. - CISA Valdeolmos -
CONTENT1.- ASF : The Diagnostic Tools. Current Status2.- ASF Diagnostic tools usually employed in the EU and third countries3.- ASF Diagnostic tools are adapted to the different scenarios?4.- Advances in ASF Diagnosis . The ASFRISK EU project .
- Very Complex Disease, cause by a big complex virus . - NOT VACCINE AVAILABLE. Control of the disease is mainly based on Early Detection and Strict Sanitary Measures Recognition of the disease in the field Incubation period range: 4-19 days. Laboratory DiagnosisLABORATORY DIAGNOSIS IS ESSENTIAL FOR THECONTROL OF ASF
ASF LABORATORY DIAGNOSIS• VIRUS DETECTION Identification of the Agent and isolation • Isolation in primary cells cultures: Haemoadsorption‘autorosette’ (HA) test with peripheral blood leukocytes from infected pigsOIE Validated Antigen Detection • Direct immunofluorescent test (DIF) • Antigen ELISA Low sensitivity in subacute and chronic forms Significant lack of sensitivity after first week pi. (because the antibody appearance) Give a significant number of false negative results.
ASF LABORATORY DIAGNOSISVIRUS DETECTION BY PCR PCR DETECTION USING DIAGNOSTIC PRIMERS 0 50 100 150 200 kb P72 86793 bp 88733 bp 86500 87000 87500 88000 88500 ASF 1-2 A12I-V 89000 bp AMPLIFLIES 257 bp AMPLIFLIES 278 bp . Aguero M, Fernandez J, Romero L, Sanchez Mascaraque C, Arias M, Sanchez-Vizcaino JM. J Clin Microbiol. 2003 Sep;41(9):4431-4. and OIE Manual, 2008. OIE Validated OIE Validated Real time King et al, 2003 Others recently validated.
ASF LABORATORY DIAGNOSISANTIBODY DETECTION •ELISA tests SCREENING OIE Validated Indirect “in House” ELISA (OIE) Commercial ELISA, Ingezim K3 OIE Validated “In House” ELISAs ELISA in eastern european countries OIE Validated CONFIRMATORY TESTS •Indirect immunofluorescent test (IIF) •Inmunoblotting ( IB) test OIE Validated • Indirect Immunoperoxidase Test Validated by EU RL Positive
DIAGNOSIS Which are the diagnostic toolscurrently used within EuropeanUnion and colaborating countries in surveillance and control- eradication programmes?
ANNUAL INTERLABORATORY COMPARISON TEST FOR NATIONAL REFERENCE LABORATORIES FOR EU MEMBER STATES • NRL EU MS: • Austria, Bulgaria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, Participants France,Germany, Hungary, Ireland, Italy, Latvia, Lithuania, Netherlands, Poland, Portugal, Romania, Spain, Slovakia, Slovenia, 39 Laboratories from Sweden and UK. • 4 Other Laboratories from EU Member States participating: 4 34 countries • 7 NRLs of European countries not EU members; Norway, Switzerland, Croatia, Russia,Serbia., Belarus • 4 NRLs non-European countries; USA, Canada and South Africa, ChinaTest Panel : 9 serum samples and 6 Tissue samples
LABS RESULTS; ASF virus detection PCR PROCEDURESReal Time Conventional
ILCTS INFORMATION. CONCLUSIONSAntibody (Ab) Detection Techniques NRLs employed at least one Ab detection technique. INGENASA ELISA K3 is the choice Technique for ASF antibody detection. 74% of NRLs employ antibody confirmatory tests → Immunoblotting (IB) is a choice confirmatory procedure for ASF antibody detection The need for the use of a confirmatory technique (IB,IIF, IPT) by NRLs is strongly emphasized .
ILCTS INFORMATION. CONCLUSIONS contVIRUS DETECTION→ NRLs participating (34)countries employ at least one virus detection technique. PCR as the choice procedure for ASFV detection, by 97%.Virus Isolation employed by 50% Not recommended the use of Ag-ELISA for ASFV detectionwithout PCR or VI→ URL strongly encourages to incorporatePCR techniques as the first choice. From the results of ILCT: The use of virus detection techniquesin serum samples in addition to tissue samples is recommended.
RECOMMENDATIONS Serum samples are good target samples for ASF diagnosis.Antibody and virus detection techniquesshould be performed simultaneously inserum samples for a reliable diagnosis.
RECOMMENDATIONS cont.Bear in mind the limits of each diagnostic technique (especiallyantigen detection techniques -DIF and ELISA-) and theirfeasibility for each epidemiological situation. 97% Ag 18% 50% VI PCR Direct immunofluorescent test (DIF) 8 8 8 Antigen ELISA LOW SENSITIVITY IN SUBACUTE AND CHRONIC FORMS DUE TO ASF SPECIFIC ANTIBODY PRESENCE - giving false negative rsults-
RECOMMENDATIONS cont. 0 dpi 1 dpi 2 dpi 3 dpi 4 dpi 7 dpi B S B S B S B S B S B S M257 bp- In case of clinical suspicion, if PCR is not available, any other Ag detection technique, such as DIF or Ag ELISA, should be performed , ALWAYS using serological tests simultaneously.
SOME GAPS IDENTIFIED IN LABORATORYDIAGNOSISIn certain affected areas of Africa and some Eastern Europecountries:-Regional labs lacks of infrastructure and/or expertise for areliable ASF diagnostic service.-Some of the existing regional laboratories poses limitedcapacity and in most of them, the Direct fluorescent test isthe preferred assay for virus detection. Antibody Detection techniques Points for should be incorporated together the Collaboration : virus detection techniques . • Training to improve expertise • Improvement of technical standards. • Support in Validation of ASF Virus and Antibody techniques. • Support in any matter concerning ASF diagnosis that could be required.
ASF Current situation 2010-2011 EUROPE Continuing Armenia Russia(2010)Zambia, Uganda, Togo, Namibia,Mozambique, Malawi, Madagascar,Guinea-Bissau, Ghana, Congo Rep,Cammeroon, Burkina-Fasso, Benin,Angola.AFRICA Continuing 2011:NigeriaChadCentral African Rep.KenyaTanzania Endemic since 1978 in Sardinia (Italy) Endemic in more than 20 Subsaharan African countries
ASF EPIDEMIOLOGY 22 genotypes described in Africa. ASF Genotyping Standarized procedures - P72 genotyping (C-terminal end) - P54 genotyping (full gene) - CVR subtyping Bastos et al 2003, Lubisi et. al 2005; Boshoff et. al 2007, Gallardo et al. 2009.
1957 Angola: genotype I toLisbon, spreading Europe and c/s Am. ASF EPIDEMIOLOGY 2007 Eastern Africa: genotype II Li Caucasus Region and RF s Cuba 1971, 1980 b Dom. Rep 1978 o Haiti 1978 n 195 7, 60 Brasil 1978 Related ASF-West Africa viruses Georgia June 2007
ASF EPIDEMIOLOGYComplex epidemiological situation in easternregions of Africa INIA-ILRI StudiesSignificant number of pigs with nonevident ASF clinical signs have beenobserved showing a high amount ofvirus presence and lack of ASF-antibody response
History ASFASF validated serological diagnostic tests are basedon the use of genotype I isolates Lisbon 1957, 60 Cuba 1971, 1980 Dom. Rep 1978 Haiti 1978 Related ASF-West Africa viruses Brasil 1978 (genotype I)
DIAGNOSIS Are the ASF diagnostic tools adapted to the different scenarios?
Are the current ASF serological diagnostic tools adapted to all epidemiological situations? Different Transmission cycles East African isolates ↑ VARIABILITY OF SEQUENCE.
Evaluation of the capability and competence of formal OIE serological diagnostic tests.STRATEGY: To Develop new serological diagnostic tools(ELISA and IPT as specific confirmatory test for each of theELISA) using new Antigens obtained from different virusisolates.
Evaluation of the capability and competence of formal OIE serological diagnostic tests. East African isolates P72 genotype VIII, IX and X Genome variabilitySTRATEGY: To Develop New serological diagnostic tools (ELISA and IPT asconfirmatory test) using new Antigens obtained from different virusisolates.
1. Analysis of 816 FIELD SERUM samples collected from different epidemiological situations since 2003-20092. Analyses of 166 experimental serum samples from pigs inoculated with diferent genotypes (I, II, IX, X)
New ASF serological diagnostic tools Negative field serum samples from east, west, central Africa and from EuropeAbsorbance value OD.492 IPT C.O ID sera
Are the current ASF diagnostic toolsThe current ASF serological diagnostic adapted to all epidemiological tools ARE ADAPTED TO ALL situations? EPIDEMIOLOGICAL SITUATIONSThe results obtained using new Ags based on current and variable circulatingASFV strains were 100%according to those obtained using OIE prescribedantibody detection techniques.
ADVANCES IN AFRICAN SWINE FEVER DIAGNOSIS.Research Activities under ASFRISK EUproject. "Evaluating and controlling the risk of African sw ine fever in the EU", ASFR I SK . Grant Agreement no. 211691 April 2008-September 2011
Task 2: DIAGNOSIS Updating and improvement of current molecular and OF ASF serological techniquesTo increase the available diagnostic techniques in well-equipped reference laboratories To offer simple and rapid first-line tools at the pen-side To assist with affordable techniques to affected countries/labs with limited resources
MOLECULAR DIAGNOSIS OF ASFMOLECULAR APPROACHES:-New improved and affordable Real-time PCR assays using commercialuniversal probes (UPL) (CISA-INIA).-LATE-PCR technique adapted to commercial portable PCR machines forrapid on-site diagnosis (SVA).- Isothermal Amplification assays for rapid detection of ASFV (QUB,SVA). Adaptation to on site diagnosis by a simple, inexpensive andportable platform.
PCR techniquesImproved ASFV real-time PCRusing commercial UPL probe VALI DATED•Highly sensitive method•Reproducible results Aprox. 4.5€/sample•All p72 genotypes detected (DNA extraction 3€+PCR)(19 genotypes tested) Adaptation of the real-time PCR test to a commercial kit •Easy and fast implementation •Reagents ready for use •Intended format as lyophilised reagents: storage at RT, pen-side application •Currently under validation
ASFV LATE-PCR (linear after the exponential PCR) •Sensitive and specific method •Compatible with any real-time PCR equipment Adaptation of LATE-PCR assay to a commercial kitBioSeeq-Vet (Smiths Detection):•Fully portable equipment for field testing•Cartridge including DNA extraction andamplification•Expensive system•Currently in stand-by
LAMP ASSAY for ASFV (loop-mediated isothermal amplification)•DNA amplification at a constanttemperature (60-65ºC)•High specificity, all ASFV p72 genotypesdetected (19 genotypes tested)•No need of sophisticated equipment,low-cost technique•Rapid DNA amplification Aprox. 4.5€/sample•First-line diagnostic tool: pen-side test (DNA extraction 3€+LAMP) UNDER VALI DATI ON Adaptation of the LAMP assay to a commercial kit •Easy acquisition and implementation in the lab •Dried reagents ready for use: storage at RT •Portatil. On-site application
PCR techniques Tetracore real-time PCR kit for ASFV Aprox. 12000 € T-COR 4TM•Unique commercial kit available atpresent•Dried reagents ready for use•Valid in any real-time PCR machine•Combined to portable PCR machine can beemployed for an on-site application•Good sensitivity and specificity Aprox. 10€/sample (PCR)
PCR techniques 0 dpi 1 dpi 2 dpi 3 dpi 4 dpi 7 dpi B S B S B S B S B S B S M ASFV conventional PCR 257 bp- 257 bp-•Reference method in OIE Manual•All p72 genotypes detected Aprox. 4€/sample•Fully validated and widely used OI E VALI DATED (DNA extraction 3€+PCR)in NRLs Adaptation of the conventional PCR test to a commercial kit •Easy and fast implementation •Reagents ready for use •Intended format as jellified reagents: storage at 4ºC •Currently under validation
SEROLOGICAL DIAGNOSIS OF ASF-Development, standardization and validation of new ELISAbased on ASFV recombinant protein. Currently it is beingtransferred to INGENASA to develop an ELISA commercialprototype.-Development, standardization and validation of an IgM ELISAfor the detection of ASFV-anti-IgM antibodies. Currently it isbeing transferred to INGENASA to develop an ELISAcommercial prototype. VALI DATED
SEROLOGICAL DIAGNOSIS OF ASFPen-side test for Ab detectionINGEZIM PPA CROM is based on the •Results within minutes VALIDATEDtechnique of Direct Immunochromatography •Easy use and interpretationwhich uses a Monoclonal Antibody (MAb)specific of VP73 of ASFV. •High sensitivity and specificity •Real on-site application 3.5€/sample •Currently under validation for blood samples
SAMPLE COLLECTION ON FILTER PAPER •Easy for sampling collection •Transport and storage at room temperature •Infrastructure only filter paper •Personnel: vets are not required on site 3MM filter paper • Standard chromatography filter paper FTA cards • Antibody and genome detection • Low-cost system• Chemically-treated filter paper• Pathogens inactivation• Viral genome detection over UNDER VALI DATI ONyears• Expensive system VALI DATED
Task 2: DIAGNOSIS OF ASFConclusion:A range of valuable molecular and serological toolshas been produced within the ASFRISK project, beingnow offered to improve and complement theavailable ASF diagnostic tools, suitable for use inwell-equipped international and national referencelaboratories, in basic regional and local laboratories,or even for rapid on site application.