EU REFERENCE LABORATORY FOR AFRICAN SWINE FEVER

Dr. Marisa Arias

Current Status of Serological and Nucleic acid-based Diagnostic techniques for ASF:

Current Status of Serological and Nucleic acid-based Diagnostic techniques for ASF

Nairobi July 19th-22nd 2011 Nairobi July 19th-22nd 2011

ASF WORKSHOP Nairobi July, 2011

…In the 80´sClassical swine fever: Erradicated withvaccination in 1986.

- CISA Valdeolmos -

Foot and Mouth Disease: Erradicated withvaccination in 1987.

SPAIN

African Swine Fever

BIOSAFETY LEVEL 3 and 3+ FACILITY

1993: CISA-INIA,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.

40 BSL-3 laboratories.

- CISA Valdeolmos -

BSL3+ Laboratories

21 BSL-3 Animal RoomsTwo Corridors

Pneumatic doorsDiferential pressure

BSL-3 ANIMAL FACILITY

- CISA Valdeolmos -

BSL-3 ANIMAL FACILITY

EU REFERENCE LABORATORY FOR AFRICAN SWINE FEVER

SPAIN- Second Pig Producing Country in

the European Union (EU)- More than 4.000 million Euros /year.

European Leader in the pig sector.

2011: 2,7 Million Sows2011: 29,5 millions pigs38% Export market.

SPAIN

SPAIN- CISA Valdeolmos -

census

CONTENT

2.- ASF Diagnostic tools usually employed in the EU and third countries

1.- ASF : The Diagnostic Tools. Current Status

4.- Advances in ASF Diagnosis . The ASFRISK EU project .

3.- ASF Diagnostic tools are adapted to the different scenarios?

Incubation period range: 4-19 days.

- Very Complex Disease, cause by a big complex virus .

- NOT VACCINE AVAILABLE.

Control of the disease is mainlybased on Early Detection and Strict Sanitary Measures

Recognition of the disease in thefield

Laboratory Diagnosis

LABORATORY DIAGNOSIS IS ESSENTIAL FOR THE CONTROL OF ASF

ASF LABORATORY DIAGNOSIS

Identification of the Agent and isolation• Isolation in primary cells cultures: Haemoadsorption‘autorosette’ (HA)

test with peripheral blood leukocytes from infected pigs

• Direct immunofluorescent test (DIF)• Antigen ELISA

Significant lack of sensitivity after first week pi. (because the antibodyappearance) Give a significant number of false negative results.

Antigen Detection

• VIRUS DETECTION

Low sensitivity in subacute and chronic forms

OIE Validated

PCR DETECTION USING DIAGNOSTIC PRIMERS

89000 bp

0 50 100 150 200 kb

P7288733 bp86793 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.

86500 87000 87500 88000 88500

ASF 1-2 A12I-V

Real time King et al, 2003

VIRUS DETECTION BY PCRASF LABORATORY DIAGNOSIS

OIE Validated

OIE Validated

Others recently validated.

•ELISA tests

•Inmunoblotting ( IB) test

•Indirect immunofluorescent test (IIF)

Indirect “in House” ELISA (OIE)

ANTIBODY DETECTION

“In House” ELISAs

Commercial ELISA, Ingezim K3

Positive

• Indirect Immunoperoxidase Test

CO

NFI

RM

ATO

RY

TES

TSS

CR

EE

NIN

G

ASF LABORATORY DIAGNOSIS

OIE Validated

OIE Validated

ELISA in eastern european countries

OIE Validated

OIE Validated

Validated by EU RL

http://asf-referencelab.info

Which are the diagnostic toolscurrently used within EuropeanUnion and colaborating countries

in surveillance and control-eradication programmes?

DIAGNOSIS

Participants39 Laboratories from

34 countries

• NRL EU MS:• Austria, Bulgaria, Belgium, Cyprus, Czech

Republic, Denmark, Estonia, Finland,France,Germany, Hungary, Ireland, Italy,Latvia, Lithuania, Netherlands, Poland,Portugal, Romania, Spain, Slovakia, Slovenia,Sweden and UK.

• 4 Other Laboratories from EU Member States participating: 4

• 7 NRLs of European countries not EU members; Norway, Switzerland, Croatia, Russia,Serbia., Belarus

• 4 NRLs non-European countries; USA, Canada and South Africa, China

ANNUAL INTERLABORATORY COMPARISON TEST FOR NATIONAL REFERENCE LABORATORIES FOR

EU MEMBER STATES

Test Panel : 9 serum samples and 6 Tissue samples

21%

74%

5%

1 technique 2 techniques 3 techniques

LABS RESULTS; ASF antibody detection

ELISAELISA + IB

89%

LABS RESULTS; ASF virus detection

37%63%

1 technique 14/38 2 -3 techniques 24/38

PCR

LABS RESULTS; ASF virus detection

37%63%

1 technique 14/38 2 -3 techniques 24/38

PCR PROCEDURES

LABS RESULTS; ASF virus detection

ConventionalReal Time

ILCTS INFORMATION. CONCLUSIONSAntibody (Ab) Detection Techniques

The need for the use of a confirmatorytechnique (IB,IIF, IPT) by NRLs is

strongly emphasized .

NRLs employed at least one Ab detection technique.

INGENASA ELISA K3 is the choice Technique for ASF antibodydetection.

74% of NRLs employ antibody confirmatory tests →Immunoblotting (IB) is a choice confirmatory procedure for ASF antibodydetection

ILCTS INFORMATION. CONCLUSIONS cont

VIRUS 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.

Antibody and virus detection techniquesshould be performed simultaneously in serum samples for a reliable diagnosis.

Serum samples are good target samples for ASF diagnosis.

RECOMMENDATIONS

Bear in mind the limits of each diagnostic technique (especially antigen detection techniques -DIF and ELISA-) and their feasibility for each epidemiological situation.

Antigen ELISA

LOW SENSITIVITY IN SUBACUTE AND CHRONIC FORMS DUE TO ASF SPECIFIC ANTIBODY PRESENCE - giving false negative rsults-

RECOMMENDATIONS cont.

8 8 8

97%18% 50%

Direct immunofluorescent test (DIF)

PCRVI

Ag

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.

RECOMMENDATIONS cont.

257 bp-

0 dpi 1 dpi 2 dpi 3 dpi 4 dpi 7 dpi

MB B B B B BS S S S S S

SOME GAPS IDENTIFIED IN LABORATORY DIAGNOSIS

-Regional labs lacks of infrastructure and/or expertise for a reliable ASF diagnostic service.-Some of the existing regional laboratories poses limited capacity and in most of them, the Direct fluorescent test is the preferred assay for virus detection.

• 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.

In certain affected areas of Africa and some Eastern Europe countries:

Points for Collaboration :

Antibody Detection techniques should be incorporated together the virus detection techniques .

ASF Current situation 2010-2011 EUROPE ContinuingArmeniaRussia

Endemic since 1978 in Sardinia (Italy)

Endemic in more than 20 SubsaharanAfrican countries

(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

ASF EPIDEMIOLOGY

22 genotypes describedin Africa.

ASF Genotyping

Bastos et al 2003, Lubisi et. al 2005;Boshoff et. al 2007, Gallardo et al. 2009.

Standarized procedures- P72 genotyping (C-terminal end)- P54 genotyping (full gene)- CVR subtyping

Related ASF-West Africa viruses

Lisbon

1957, 60

Cuba 1971, 1980

Dom. Rep 1978

Haiti 1978

Brasil 1978

GeorgiaJune 2007

ASF EPIDEMIOLOGY 1957 Angola: genotype I to Lisbon, spreading Europe and c/s Am.

2007 Eastern Africa: genotype IICaucasus Region and RF

Complex epidemiological situation in eastern regions of Africa

Significant number of pigs with non evident ASF clinical signs have been observed showing a high amount of virus presence and lack of ASF-antibody response

ASF EPIDEMIOLOGY

INIA-ILRI Studies

History ASF

Related ASF-West Africa viruses (genotype I)

Lisbon 1957, 60

Cuba 1971, 1980

Dom. Rep 1978

Haiti 1978

Brasil 1978

ASF validated serological diagnostic tests are basedon the use of genotype I isolates

Are the ASF diagnostictools adapted to the different scenarios?

DIAGNOSIS

Different Transmission cycles

East African isolates↑ VARIABILITY OF SEQUENCE.

Are the current ASF serological diagnostic tools adapted to all epidemiological situations?

STRATEGY: To Develop new serological diagnostic tools (ELISA and IPT as specific confirmatory test for each of the ELISA) using new Antigens obtained from different virus isolates.

Evaluation of the capability and competenceof formal OIE serological diagnostic tests.

East African isolatesP72 genotype VIII, IX and X

Genome variability

Evaluation of the capability and competenceof formal OIE serological diagnostic tests.

STRATEGY: To Develop New serological diagnostic tools (ELISA and IPT as confirmatory test) using new Antigens obtained from different virus isolates.

1. Analysis of 816 FIELD SERUMsamples collected from different epidemiological situations since 2003-2009

2. Analyses of 166 experimentalserum samples from pigs inoculated with diferent

genotypes (I, II, IX, X)

C.O

Positive field samples

New ASF serological diagnostic tools

Negative field serum samples from east, west, central Africa and from Europe

C.O

ID sera

Abs

orba

nce

valu

e O

D.4

92

IPT

Are the current ASF diagnostic tools adapted to all epidemiological

situations?

The current ASF serological diagnostic tools ARE ADAPTED TO ALL

EPIDEMIOLOGICAL SITUATIONS

The results obtained using new Ags based on current and variable circulating ASFV strains were 100%according to those obtained using OIE prescribed antibody detection techniques.

ADVANCES IN AFRICAN SWINE

FEVER DIAGNOSIS.Research Activities under ASFRISK EU project.

"Evaluating and controlling the risk of African sw ine fever in the EU", ASFRISK.

Grant Agreement no. 211691April 2008-September 2011

Task 2: DIAGNOSIS OF ASF

Updating and improvement of current molecular and serological techniques

To 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 affectedcountries/labs with limited resources

MOLECULAR APPROACHES:

-New improved and affordable Real-time PCR assays using commercial universal probes (UPL) (CISA-INIA).

-LATE-PCR technique adapted to commercial portable PCR machines for rapid on-site diagnosis (SVA).

- Isothermal Amplification assays for rapid detection of ASFV (QUB, SVA). Adaptation to on site diagnosis by a simple, inexpensive and portable platform.

MOLECULAR DIAGNOSIS OF ASF

PCR techniques

Improved ASFV real-time PCR using commercial UPL probe

Aprox. 4.5€/sample(DNA extraction 3€+PCR)

•Highly sensitive method

•Reproducible results

•All p72 genotypes detected(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

VALIDATED

ASFV LATE-PCR(linear after the exponential PCR)

•Sensitive and specific method

•Compatible with any real-time PCRequipment

BioSeeq-Vet (Smiths Detection):

•Fully portable equipment for field testing

•Cartridge including DNA extraction andamplification

•Expensive system

•Currently in stand-by

Adaptation of LATE-PCR assay to a commercial kit

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

•First-line diagnostic tool: pen-side test

•Easy acquisition and implementation in the lab

•Dried reagents ready for use: storage at RT

•Portatil. On-site application

Aprox. 4.5€/sample(DNA extraction 3€+LAMP)

Adaptation of the LAMP assay to a commercial kit

UNDER VALIDATION

PCR techniques

Tetracore real-time PCR kit for ASFV

•Unique commercial kit available at present

•Dried reagents ready for use

•Valid in any real-time PCR machine

•Combined to portable PCR machine can be employed for an on-site application

•Good sensitivity and specificity

Aprox. 12000 €

Aprox. 10€/sample (PCR)

T-COR 4TM

PCR techniques

ASFV conventional PCR

Aprox. 4€/sample(DNA extraction 3€+PCR)

•Reference method in OIE Manual

•All p72 genotypes detected

•Fully validated and widely usedin NRLs

Adaptation of the conventionalPCR test to a commercial kit

•Easy and fast implementation

•Reagents ready for use

•Intended format as jellified reagents: storage at 4ºC

•Currently under validation

OIE VALIDATED

257 bp-

0 dpi 1 dpi 2 dpi 3 dpi 4 dpi 7 dpi

MB B B B B BS S S S S S

257 bp-

0 dpi 1 dpi 2 dpi 3 dpi 4 dpi 7 dpi

MB B B B B BS S S S S S

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.

VALIDATED

SEROLOGICAL DIAGNOSIS OF ASF

•Results within minutes

•Easy use and interpretation

•High sensitivity and specificity

•Real on-site application

•Currently under validation for blood samples

INGEZIM PPA CROM is based on the technique of Direct Immunochromatography which uses a Monoclonal Antibody (MAb) specific of VP73 of ASFV.

VALIDATED

3.5€/sample

Pen-side test for Ab detection

SAMPLE COLLECTION ON FILTER PAPER

3MM filter paper

FTA cards

•Easy for sampling collection

•Transport and storage at roomtemperature

•Infrastructure only filter paper

•Personnel: vets are not required on site

• Chemically-treated filter paper

• Pathogens inactivation

• Viral genome detection overyears

• Expensive system

• Standard chromatography filterpaper

• Antibody and genome detection

• Low-cost system

UNDER VALIDATION

VALIDATED

Conclusion: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.

Task 2: DIAGNOSIS OF ASF

ASFRISK projectwww.asfrisk.eu

THANK YOU!

Our Gratitude/AhsanteTO OUR HOST COUNTRY:

AND BecA- ILRI

SCIRO sponsor of this Event

AND

IN HONOR OF

ISABEL MINGUEZ-TUDELA Senior Scientific OfficerEuropean Commision, DG RESEARCH