ASF DIAGNOSIS UPDATE. oie - ASF REFERENCE LABORATORY, MADRID. Prof. José M. Sánchez- Vizcaíno

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1 ASF DIAGNOSIS UPDATE oie - ASF REFERENCE LABORATORY, MADRID Prof. José M. Sánchez- Vizcaíno jmvizcaino@visavet.ucm.es

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3 BSL-2 LABORATORIES

4 BSL-3 LABORATORIES

5 BSL-3 BOXES MONITORING Video Environmental conditions

6 ASFV: A old friend OIE ASF REFERENCE LABORATORY MAIN WORK: DIAGNOSIS TEST & REAGENTS EPIDEMIOLOGY-CONTROL and ERADICATION

7 AGENDA: ASF. Characteristic related with diagnosis Diagnostic test available. How are the available test working?? Sample for a good ASF diagnosis New development ARE WE AWARE?. ARE WE READY?

8 AFRICAN SWINE FEVER ONE OF THE MOST COMPLEX DISEASES AFFECTING LIVESTOCK

9 ASF A REEMERGING THREAT An early detection programs prepared

10 ASF EPIDEMIOLOGY: Historical distribution 1970 s Portugal, 1957, 60-94, 99 Spain, France, 1964, Italy, 1967,69,93 Malta, 1978, Belgium, 1985, Nedherlands, 1986 Lisbon 1957, 1960 Kenya, 1921 Cuba, 1971, 1980 Brasil, 1978 R. Dom., 1978, Haití, 1979 Eradicated in 1994 in Portugal and 1995 in Spain

11 SFV is in good form Out of Control Single introduction Asfv, genotype II Mozambique, Madagascar, Zambia 2007 More ASFv More affected areas Tolerance pigs? Countries affected by ASFV since 1997

12 EXPERIMENTAL INFECTION: ASFv Ken05/K2 10 Exotic domestic swine (Landrace) from Kitengela (6- month old) 29 Indigenous domestic swine (local breed) from Homa Bay district (6- month old)

13 European pigs Presence of ASFV Positive to Ab against ASFV The viremia was detectable at 14dpi with a high viral load (as it was the case in European breeds). LABORATORY RESULTS Delay in the seroconversion in local breeds. At 28 dpi 66% did not present antibody response. Local breed Presence of ASFV Negative to Ab against ASFV Only 4 of 19 animals presented Ab

14 AFRICAN SWINE FEVER VIRUS (ASFV) Asfarviridae family Very complex DNA virus, big syze, large genome: kbp Very complex molecular structure Genetic variability Replication in macrophages NO production of neutralizing antibodies 200 nm More than 100 structural proteins 22 genotypes (VP72) ONLY TYPE I & II are Active out Africa VERY DIFFICULT AND EXPENSIVE CONTROL

15 Differents Forms: Only acute is circulanting SYMPTOMS and LESIONS: Acute form Acute forms, % mortality

16 Clinical Signs: Easily Confused WITH: Classical Swine Fever Erysipelas Salmonellosis Actinobacillus (App) Other Septicaemic conditions PDNS LABORATORY DIAGNOSIS IS NEEDED

17 EARLY DETECTION (FIELD & LABs) FAST RESPONCE. CONTIGENCY PLAN THE ASF CONTROL CHALLENGE

18 ASF EARLY DETECTION NEEDED: FIELD: Risk information ASF Information LABs: Good conection with field Good test and procedure TRAINING: FIELD AND LABORATORY

19 LACK OF EQUILIBRIUM LAB. DETECTION 4-12 Hours 1 to 4 Months FIELD DETECTION

20 IS in GOOD FORM for THE 22 Genotypes Virus or viral proteins ASF Abs P72 pp62 P32 (30) P54 p P12 Virus DNA

21 ASF Laboratory DIAGNOSIS: KEY POINTS No Vaccine Available Antibodies = INFECTION No Neutralizing Antibodies ASFV specific antibodies do not neutralise virus in the classical concept of neutralizationonly a partial neutralization in vitro has been demonstrated. Viremia for Long period of Time Antibodies Persist During Month, Even Years From 7-12 dpi. Abs good infectious marker Antigen Antibody Immunocomplex Formation

22 ASF DIAGNOSIS. Key points INFECTION DISEASES/DEAD CARRIERS 12 h to 1 week 1 3 weeks 4 Weeks to 2 years Both Ab and Ag detection should be carry out VIRUS Ab Infection Clinical Carriers

23 ASF LABORATORY DIAGNOSIS Identification of the Agent Some ASFv No HA Isolation in cells cultures: Haemoadsorption autorosette (HA) test with peripheral blood leukocytes from infected pigs. TIME: 3 to 10 Days. ONLY R.Labs Antigen Detection. TIME 75 MIN. Direct immunofluorescent test (DIF) Low sensitivity in subacute and chronic forms

24 ASF DNA DETECTION PCR: CONVENTIONAL and REAL TIME TIME: 5 to 6 H 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 257 bp- 257 bp C M V 257 bp- 108 bp- CSF + ASF (MULTIPLEX) MOST COMMONLY USED

25 ASF LABORATORY DIAGNOSIS for Ab detection ELISA tests Indirect ELISA (OIE) Commercial ELISA, Ingezim K3 MOST COMMONLY USED. TIME 3 H Indirect immunofluorescent test (IIF) Indirect Immunoperoxidase Test Inmunoblotting (confirmation test)

26 ASF LABORATORY DIAGNOSIS Antibodies Detection Immunochromatography Pen side tests Easy to use No special equipment needed TIME 15 MINUTES PPA-CROM ANTIGEN DETECTION POSITIVE SAMPLE NEGATIVE SAMPLE

27 PROTOCOLS & VIDEOS

28 SAMPLE FOR ASF DIAGNOSIS Limpho nodes Spleen Kidney Always: Simultaneous detection of antigen and antibodies Lung Blood and serum Oral Fluid

29 Material and methods: ORAL FLUID COLLECTION PROCEDURE 1. TRAINING PIGS 2. PIGS CHEWING THE ROPE

30 Material and methods: ORAL FLUID COLLECTION PROCEDURE 3. OBTAINING ORAL FLUID

31 Results Oral fluid VS serum Optimization of serological techniques for the use of oral fluid samples OIE- indirect ELISA ORAL FLUIDVS SERUM IMMUNUNOPEROXIDASE TEST ORAL FLUID VS SERUM C+ CL C- Optimization of the protocol for OF samples: Double antigen concentration (1/800) Use of undiluted OF samples Overnight incubation at 4ºC of OF samples 5x concentration of the conjugate ASFV BL Optimization of the protocol for OF samples: OF samples were used 40x more concentrated than sera

32 PRELIMINARY RESULTS SERUM VS ORAL FLUID In both groups similar results were found: Seroconversion in serum samples: 8-11 days Seroconversion in oral fluid samples: days * Seroconversion in oral fluid samples related with Ab titres in serum 1/2500 Serum titer >1/2500 Positive OF

33 How are the available test working with the 22 serotypes? Evaluate the capability and competence of formal OIE diagnostic tests with the current and most variable circulating ASFv strains.

34 Genetic variability of ASFV: 22 genotypes European, American, and West African isolates HOMOLOGY OF SEQUENCE East African isolates VARIABILITY OF SEQUENCE. First conclusion: VALIDATED PCR TECHNIQUES for Virus Detection ARE SENSITIVE For Circulating Isolates and Genotypes

35 How about antibodies detection?? ASF validated serological diagnostic tests are based on the use of antigens belonging to genotype I ASFV isolates. Lisbon 1957, 60 Related ASF-West Africa viruses (genotype I) Cuba 1971, 1980 Dom. Rep 1978 Haiti 1978 Brasil 1978

36 ASFV antigenic proteins variability; comparative study based on recent ASFV circulating strains p72 p54 p72 p54 p30

37 Comparative study (OIE prescribed serological techniques versus new Cp-Ags based ELISAs and IPT) Analyses of 1062 field sera collected from endemic areas in Africa and Sardinia since 2004 until experimental sera from experimental infections using current circulating ASFV isolates, I, II, X, IX.

38 Are the The current ASF ASF diagnostic serological tools diagnostic adapted to tools all epidemiological ARE ADAPTED TO ALL EPIDEMIOLOGICAL situations? SITUATIONS

39 NEW DEVELOPMENTS PEN-SIDE AND FRONT LINE ANTIBODY DETECTION TESTS FOR ASF DIAGNOSIS Development of a simple and robust rapid test for detection of specific ASFV antibodies

40 PENSIDE PROCEDURE Penside immunochromatography INGEZIM PPA CROM TECHNICAL BASIS & PRODUCT APPLICATION conjugate pad sample pad absorbent pad nitrocellulose test line membrane Test Line: VP72 protein of ASFV adsorbed on the nitrocellulose membrane Control Line: -control protein MAb adsorbed on the nitrocellulose membrane. control line Red latex microparticles covalently coated with VP72 protein. Blue latex microparticles covalently coated with a control protein.

41 NEW PCR and PORTABLE PCR Development and validation of new nucleic acid-based assays for sensitive, specific and rapid detection of currently circulating ASFV strains. Further adaptation to commercial portable PCR machines.

42 RESULTS Tetracore real-time PCR kit for ASFV EVALUATED and VALIDATED T-COR 4 TM Only commercial kit available at present Valid in any real-time PCR machine Can be used with portable PCR machine for on-site application Good sensitivity and specificity Dried reagents ready for use

43 LAMP ASSAY DNA amplification at a constant temperature (60-65ºC) High specificity, all ASFV p72 genotypes detected. Slightly lower sensitivity LAMP ASSAY for ASFV (loop-mediated isothermal amplification) No need of sophisticated equipment, lowcost technique Rapid DNA amplification First-line diagnostic tool: penside test Adaptation of the LAMP assay to a commercial kit VALIDATED Easy acquisition and implementation in the lab Dried reagents ready for use: storage at RT On-site application

44 TO TAKE HOME: 1) ASFv is in good form and spreading. 2) Only virus that induced acute form are circulating. 3) Many good tests for laboratory diagnosis for all 22 genotypes are available. (field condition) 4) Always the simultaneously detection of Ag and Ab should be performed 5) Ab against ASFV are detected on oral fluid samples 6) The best tool for ASF control is the early detection. 7) More efforts should be made for increase field detection (more and better information, field test, etc) 8) A Fast Responce need a good Contigency plan.

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