Nairobi July 19th-22nd 2011
Current 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

40 BSL-3 laboratories.
- CISA Valdeolmos -

BSL3+ Laboratories

BSL-3 ANIMAL FACILITY
21 BSL-3 Animal Rooms
Two Corridors
Pneumatic doors
Diferential pressure - 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.
census
2011: 2,7 Million Sows
2011: 29,5 millions pigs SPAIN
38% Export market.
SPAIN European Leader in the pig sector. - CISA Valdeolmos -

CONTENT
1.- ASF : The Diagnostic Tools. Current Status
2.- ASF Diagnostic tools usually employed in the
EU and third countries
3.- 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 Diagnosis
LABORATORY DIAGNOSIS IS ESSENTIAL FOR THE
CONTROL 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 pigs
OIE 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 DIAGNOSIS
VIRUS 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 DIAGNOSIS
ANTIBODY 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

http://asf-referencelab.info

DIAGNOSIS
Which are the diagnostic tools
currently used within European
Union 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, China
Test Panel : 9 serum samples and 6 Tissue samples

LABS RESULTS; ASF antibody detection
1 technique 2 techniques 3 techniques
74%
21%
5%
ELISA + IB
ELISA 89%

LABS RESULTS; ASF virus detection
1 technique 14/38 2 -3 techniques 24/38
63%
37%
PCR

LABS RESULTS; ASF virus detection
1 technique 14/38 2 -3 techniques 24/38
63%
37%

LABS RESULTS; ASF virus detection
PCR PROCEDURES
Real Time Conventional

ILCTS INFORMATION. CONCLUSIONS
Antibody (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 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 detection
without PCR or VI→ URL strongly encourages to incorporate
PCR techniques as the first choice.
 From the results of ILCT: The use of virus detection techniques
in serum samples in addition to tissue samples is recommended.

RECOMMENDATIONS
Serum samples are good target
samples for ASF diagnosis.
Antibody and virus detection techniques
should be performed simultaneously in
serum samples for a reliable diagnosis.

RECOMMENDATIONS cont.
Bear in mind the limits of each diagnostic technique (especially
antigen detection techniques -DIF and ELISA-) and their
feasibility 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 M
257 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 LABORATORY
DIAGNOSIS
In certain affected areas of Africa and some Eastern Europe
countries:
-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.
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:
Nigeria
Chad
Central African Rep.
Kenya
Tanzania
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 to
Lisbon, 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 EPIDEMIOLOGY
Complex epidemiological situation in eastern
regions of Africa
INIA-ILRI Studies
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

History ASF
ASF validated serological diagnostic tests are based
on 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 the
ELISA) using new Antigens obtained from different virus
isolates.

Evaluation of the capability and competence
of formal OIE serological diagnostic tests.
East African isolates
P72 genotype VIII, IX and X
Genome variability
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 SERUM
samples collected from different
epidemiological situations since
2003-2009
2. Analyses of 166 experimental
serum samples from pigs
inoculated with diferent
genotypes (I, II, IX, X)

Positive field samples
C.O

New ASF serological diagnostic tools
Negative field serum samples from east, west, central
Africa and from Europe
Absorbance value OD.492
IPT
C.O
ID sera

Are the current ASF diagnostic tools
The current ASF serological diagnostic
adapted to all epidemiological
tools ARE ADAPTED TO ALL
situations?
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",
ASFR I SK .
Grant Agreement no. 211691
April 2008-September 2011

Task 2: DIAGNOSIS Updating and improvement of
current molecular and
OF ASF 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 affected
countries/labs with limited resources

MOLECULAR
DIAGNOSIS OF ASF
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.

PCR techniques
Improved ASFV real-time PCR
using 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 kit
BioSeeq-Vet (Smiths Detection):
•Fully portable equipment for field testing
•Cartridge including DNA extraction and
amplification
•Expensive system
•Currently in stand-by

LAMP ASSAY for ASFV
(loop-mediated isothermal amplification)
•DNA amplification at a constant
temperature (60-65ºC)
•High specificity, all ASFV p72 genotypes
detected (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 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. 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 ELISA
based on ASFV recombinant protein. Currently it is being
transferred to INGENASA to develop an ELISA commercial
prototype.
-Development, standardization and validation of an IgM ELISA
for the detection of ASFV-anti-IgM antibodies. Currently it is
being transferred to INGENASA to develop an ELISA
commercial prototype.
VALI DATED

SEROLOGICAL
DIAGNOSIS OF ASF
Pen-side test for Ab detection
INGEZIM PPA CROM is based on the •Results within minutes VALIDATED
technique of Direct Immunochromatography •Easy use and interpretation
which 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 ON
years
• Expensive system VALI DATED

Task 2: DIAGNOSIS OF
ASF
Conclusion:
A range of valuable molecular and serological tools
has been produced within the ASFRISK project, being
now offered to improve and complement the
available ASF diagnostic tools, suitable for use in
well-equipped international and national reference
laboratories, in basic regional and local laboratories,
or even for rapid on site application.

ASFRISK project
www.asfrisk.eu
THANK
YOU!

Our Gratitude/Ahsante
TO OUR HOST
COUNTRY:
AND BecA- ILRI
AND
SCIRO sponsor of this Event

IN HONOR OF
ISABEL MINGUEZ-TUDELA
Senior Scientific Officer
European Commision,
DG RESEARCH