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African swine fever
José Manuel Sánchez-Vizcaíno2
1Ministerio
de Educación y Ciencia, INIA, Centro de Investigacion en Sanidad Animal (CISA)
Valdeolmos, 28130-Madrid, SPAIN
Tel.: (+34-91) 620.2300/2036/2061; Fax: (+ 34-91) 620.2247
Contact: Laboratory expert: Marisa Arias, Technical Director, Carmina Gallardo (ASF Coordinator). ASF EU
Reference laboratory coordinator: [email protected]; [email protected];
Web page: www.inia.es; http://www.asf-referencelab.info/
2Ministerio de Educación y Ciencia, Facultad de Veterinaria, Universidad Complutense de Madrid, Avda. Puerta de
Hierro s/n 28040, Madrid, SPAIN
Tel. (+34-91) 394.4082; Fax: (+34-91) 394.3908
jmvizcaí[email protected]; web page: http://www.sanidadanimal.info
Summary of general activities related to the disease
1.
Test(s) in use/or available for the specified disease at your laboratory
Antibody detection
Test
For
Specificity
Surveillance
Research
Total
ELISA
Antibody
Group
271
66
337
Immunoblotting
Antibody
Group
40
45
85
IIF
Antibody
Group
Virus detection
Analysis of 153 samples from Kenya, Uganda and Burkina Faso; were performed by PCR and virus isolation in
primary culture cells. The virus isolation is based on the incubation of sample material on susceptible primary cell
cultures of porcine origin, monocytes and macrophages, and confirmation by Hemoadsorption effect, and PCR.
2.
Production and distribution of diagnostic reagents
Spain (surveillance programmer): Antigen for ELISA, 2,472,000 doses; Immunoblotting (IB) strips, 4,775; ASFpositive reference serum, 240 ml; ASF-negative reference serum, 208 ml; ASF-limit reference serum 91.5 ml; plates
for ELISA 15,060; protein-A peroxidase 170 mg
Germany: Antigen for ELISA, 8,000 doses; positive and negative reference serum 1 ml ; protein-A peroxidase 1 mgr;
80 plates
Italy: Antigen for ELISA, 160,000 doses; positive reference serum 15 ml; negative reference serum 25 ml; FITCConjugate 2,5 ml .
Romania: Antigen for ELISA, 32,000 doses; positive and negative reference serum 3 ml/each; Immunoblotting strips
(IB) 50; FITC-Conjugate 1 ml .
Poland: positive, limit and negative reference serum 1 ml/each; Immunoblotting strips (IB) 57
Armenia: Antigen for ELISA 8,000 doses; positive, limit and negative reference serum 1 ml/each; ; protein-A
peroxidase 1 mgr; 80 plates; Immunoblotting strips (IB) 145.
China. Elisa test for antibodies detection.
Annual reports of OIE Reference Laboratories and Collaborating Centres, 2007
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African swine fever
Activities specifically related to the mandate
of OIE Reference Laboratories
3.
International harmonisation and standardisation of methods for diagnostic testing or the
production and testing of vaccines
In November 2006 a panel of sera and tissues for the IV EU ASF-inter-laboratory comparison test were sent to
National Reference Laboratories (NRLs) of EU Member States. Samples panel was sent also to RLs of other
European, American and African countries upon request. Test panels consisted of seven lyophilised porcine serum
samples and three lyophilised homogenised tissues. The samples had to be analysed using all methods that are
routinely employed in the examining laboratory (e.g. virus isolation, antigen ELISA, PCR analyses, antibody
ELISA). In addition, ASF diagnostic reagents as antigenic strips using for immunoblotting were included in each
parcel in order NRLs could perform the serological confirmatory technique. The main idea was to harmonize the
laboratories in the standardization of the techniques for ASF antibody and virus detection. Samples were sent to 27
National Reference Laboratories (NRLs) including 23 laboratories from 21 NRL from EU Member States
(Austria, Belgium, Czech Republic, Denmark, Estonia, Finland, France (2), Germany, Greece, Hungary, Ireland,
Italy (2), Latvia, Netherlands, Poland, Portugal, Romania, Slovakia, Slovenia, Sweden, and UK, 2 NRLs of
European countries not EU members (Norway and Switzerland), 1 RL from USA and 1 from South Africa. The
results were received from 27 participants during 2007.
V-ASF inter-laboratory comparison test 2007: A panel of biological samples for ring testing of serological and
virological diagnostic methods were analysed and prepared as Community Reference Laboratory and sent during
November-December 2007 to 26 countries participating: Austria, Belgium, Canada, Cyprus, Czech Republic,
Denmark, Estonia, Finland, France (2), Germany, Hungary, Ireland, Italy (2), Latvia, Netherlands, Norway,
Poland, Portugal, Romania, Slovakia, Slovenia, South Africa, Switzerland, UK, and USA.
Continuation of the collaboration between the ASF OIE reference laboratory and ASF affected countries in the EU
(Italy) and Africa for the molecular characterization of ASFV isolates to survey incidence of ASF.
Updating and maintenance of a sequence data bank of selected constant and variable regions of different ASFV
isolates constructed by the OIE Reference Laboratory (http://webainia.inia.es/cisa/asfv/index.asp).
Updating and maintenance of the web site of the Community Reference Laboratory (CRL) for African Swine
Fever (http://www.asf-referencelab.info/).
Distribution of laboratory protocols (ELISA, IB, PCR and isolation test) to several countries as requested.
In December 2007 we received the visit of Dr Hongfei Zhu to revised the PCR and the ELISA test for ASF
detection.
4.
Preparation and supply of international reference standards for diagnostic tests or vaccines
International standards, positive, limit and negative reference sera, soluble antigen for ELISA, and IB strips have
been produced and supplied to the countries mentioned in Summary of general activities (2).
5.
Research and development of new procedures for diagnosis and control
“Development of new diagnostic assays and epidemiological surveillance of viral pathogens of livestock in SubSaharan and North Africa.” A collaborative Project between the International Livestock Research Institute (ILRI)
in Nairobi (Kenya) and Centro de Investigation En Sanidad Animal (CISA-INIA).
Preparation of the new Project: “Evaluating and controlling the risk of African swine fever in the EU”. Project
funded by 7th Frame Programme EU. Other participants in the project: Faculdade de Medicina Veterinária,
Universidade Técnica de Lisboa, Portugal; Gulbenkian Institute for Science, Lisbon, Portugal; Facultad de
Veterinária, Universidad Complutense de Madrid, Spain; Centro de Biología Molecular “Severo Ochoa”, Consejo
Superior de Investigaciones Científicas, Madrid, Spain; CIRAD-EMVT, Campus International de Baillarguet
Montpellier, France; Instituto Zooprofilattico Sperimentale dell'Umbria e delle Marche, Perugia, Italy; FriedrichLoeffler-Institut, Federal Research Institute for Animal Health, Insel-Riems, Germany; The Royal Veterinary
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Annual reports of OIE Reference Laboratories and Collaborating Centres, 2007
African swine fever
College UK; The National Veterinary Institute, Uppsala, Sweden;Exotic Diseases Division, Onderstepoort
Veterinary, Republic of South Africa; Laboratoire Central de Pathologie Animale, Côte d’Ivoire; Veterinary and
Agrochemical Research Centre, Belgium.
“Caracterización de nuevos inmunógenos del virus de la peste porcina africana: estudio de la respuesta inmune
inducida en cerdo” funded by Spanish Government AGL 2004-07857-C03-02. Other participants: Fundacio
Centre de Recerca en Sanitat Animal (CRESA, Spain) and Biotechnology Department –INIA, Spain.
“African Swine Fever Virus: development of vaccines and epidemiological investigations”. Funded by Welcome
Trust. Other participants in the project: The Royal Veterinary College UK; Institute for Animal Health Pirbright
Lab UK; Exotic Diseases Division, Onderstepoort Veterinary South Africa; Biotechnology Department INIA;
Centro de Biología Molecular “Severo Ochoa”, Consejo Superior de Investigaciones Científicas, Madrid, Spain;
CIRAD-EMVT, Campus International de Baillarguet Montpellier, France; Gulbenkian Institute for Science,
Lisbon, Portugal; Central Veterinary Laboratory , Democratic Republic of Congo (DRC); Dep. Medicina
Preventiva (Microbiologia), Facultad de Medicina, Universidad Autónoma de Madrid, Spain.
The development and standardization of a real-time PCR assay using a specific TaqMan probe for its application
in the molecular diagnosis of ASF is in progress under Spanish project: “Potenciación de los métodos de lucha
contra la Peste Porcina Africana, el diagnóstico de enfermedades exóticas y las actuaciones ante emergencias
sanitarias”, INIA project OT01-002, Spanish governmental funds.
6.
Collection, analysis and dissemination of epizootiological data relevant to international disease
control
A total of 153 samples from were received from Kenya, Burkina Faso and Uganda. Samples from Kenya were
collected in several districts of the country from the outbreak occurred in Kenya in February 2007. The samples
collected were 10 sera and 11 tissues. 20 sera, 26 tissues and 4 bloods samples from different regions of Burkina
Faso, were also received from the outbreaks occurred. From different districts of Uganda, a total of 40 bloods, 36
sera and 6 tissues were analysed.
For the detection of antibodies, 66 sera were tested by ASF serodiagnosis tests described in the OIE Manual
diagnosis. 44 blood, 43 tissues (spleen, lung, kidney, etc), and 63 sera were analyzed for the detection of ASFV
genomic DNA by PCR and sequencing and virus isolation in primary culture cells. The molecular data will be
included in the Molecular Data Bank and compared with sequences available from ASFV isolates from different
origins (Europe, other African countries, America) and collected in different years.
7.
Provision of consultant expertise to OIE or to OIE Member Countries
None
8.
Provision of scientific and technical training to personnel from other OIE Member Countries
Scientific and technical training was performed through the “XVI International course on Animal Exotic Diseases”
that were held from 5th to 30th November 2007 at CISA-Valdeolmos and organized by the International
Cooperation Spanish Agency AECI and INIA (Spain). Different conferences on ASF epidemiology, molecular
biology, control and eradication strategies and clinical and laboratorial diagnosis as well as laboratory practices on
serological and virological diagnosis on ASF were performed. The Course also included an in vivo
experimentation with African Swine Fever and Classical Swine Fever, clinical and pathological observation and
necropsy. More than 20 professionals coming from the Agriculture Ministry, University and/or other Public
Institutions from Argentina; Brazil, Chile, Colombia, Peru, Mexico, Uruguay and Spain participated in the
International Course.
9.
Provision of diagnostic testing facilities to other OIE Member Countries
Nil.
Annual reports of OIE Reference Laboratories and Collaborating Centres, 2007
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African swine fever
10. Organisation of international scientific meetings on behalf of OIE or other international bodies
Nil.
11. Participation in international scientific collaborative studies
Nil.
12. Publication and dissemination of information relevant to the work of OIE (including list of
scientific publications, internet publishing activities, presentations at international conferences)

Presentations at meetings
Carmina Gallardo, Dufton M. Mwaengo, Virginia Pelayo, Edward Okoth, Joseph M. Macharia and Richard
Bishop. "Molecular Epidemiology of ASFV isolates recovered from samples collected in Kenya during 2006-2007
suspected outbreaks”. Annual Meeting of National African Swine fever Laboratories. Hannover (Germany) 6 th
June 2007.
G.M. De Mia, C. Gallardo, E. Martìn, C. Pellegrini, M. Arias. "Molecular Characterization of ASFV strains
causing outbreaks in Sardinia”. Annual Meeting of National African Swine fever Laboratories. Hannover
(Germany) 6th June 2007.
Jovita Fernández, Carmina Gallardo, Covadonga Pérez, Alicia Simón, Ana Robles, Marisa Arias. "Evaluation of
PCR fast Format for the rapid diagnosis of ASF”. Annual Meeting of National African Swine fever Laboratories.
Hannover (Germany) 6th June 2007.
C. Gallardo, E. Blanco, A.L.Reis, R.M.E Parkhouse, A Leitão. "Validation of four recombinant ASFV proteins
providing reproducible targets for serodiagnosis”. Annual Meeting of National African Swine fever Laboratories.
Hannover (Germany) 6th June 2007.
Miguel Ángel Jiménez-Clavero, Carmina Gallardo, Jovita Fernández and Marisa Arias. : “Future trends on ASF
research”. Annual Meeting of National African Swine fever Laboratories. Hannover (Germany) 6 th June 2007.

Presentations at international conferences
Marisa Arias. “African swine fever”. 2nd International Symposium on Animal Disease Control in the 21st Century
Greifswald-Insel Riems (Alemania). April 23-25, 2007.
Carmina Gallardo, Dufton M. Mwaengo, Virginia Pelayo, Edward Okoth, Joseph M. Macharia and Richard
Bishop. “Molecular epidemiology of African swine fever virus (ASFV) isolates recovered from samples collected
in Kenya during 2006-2007 suspected outbreaks”. First annual meeting of EPIZONE. Lublin, Poland, May 30 thJune 1st , 2007.
Jovita Fernández, Carmina Gallardo, Raquel Nieto, Ana Robles, Covadonga Pérez, Marisa Arias. “Evaluation of
PCR fast format for the rapid diagnosis of African Swine Fever (ASF)”. First Annual Meeting of the Network of
Excellence EPIZONE. Lublin, Poland, May 30 th- June 1st , 2007.
Carmina Gallardo, Richard Bishop, Dufton M. Mwaengo, Elena Martín, Joseph M. Macharia and Marisa Arias.
“New approaches in molecular and serological diagnosis of African swine fever (ASF) on new emerging
variants”. 13th International Symposium for the World Association of Veterinary Laboratory Diagnosticians.
Melbourne, Australia, November 11th-14th, 2007.
Carmina Gallardo, Ana.L.Reis, R.M.E Parkhouse, A Leitão, Esther Blanco. “Valoración del potencial diagnóstico
de cuatro proteínas recombinantes del virus de la peste porcina africana, como antigenos en ensayos
serológicos.” IX National Congress in Virology, Zaragoza. April 11-14, 2007.
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Annual reports of OIE Reference Laboratories and Collaborating Centres, 2007
African swine fever
Carmina Gallardo, Raquel Martin, Rose Ademun, Noelina Nantima, Richard Bishop and Esther Blanco.
“Caracterización molecular de aislados del virus de la peste porcina africana obtenidos a partir de muestras
recogidas en Uganda en el 2003”. IX National Congress in Virology, Zaragoza. April 11-14, 2007.
Jordi Arguilaget, Eva Perez, Carmina Gallardo, Javier Salguero, Mariano Perez-Filgueira, Joan Pujols, Miquel
Nofrarias, Esther Blanco, JM Escribano, Natalia Fernandez-Borges & Fernando Rodríguez. “Protección parcial
frente a VPPA en ausencia de anticuerpos”. IX National Congress in Virology, Zaragoza. April 11-14, 2007

Scientific publications in peer-reviewed journals
Carmina Gallardo, Esther Blanco, Ana Luísa Reis, R.M.E. Parkhouse and Alexandre Leitão. Standardization and
validation of new African swine fever serodiagnosis test using the ASFV recombinant proteins pB602L, p54,
pK205R and histone-like protein expressed in E. coli, submitted.
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Annual reports of OIE Reference Laboratories and Collaborating Centres, 2007
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