diagnostic tools and main activities of the eu rl. : an ...jan 26, 2011 · eu rl main activities...

S

DDIAGNOSTIC IAGNOSTIC TTOOLS and main OOLS and main activities of the eu rl. An STATE activities of the eu rl. An STATE

OF ARTOF ART

Berlin, 26 January 2011Berlin, 26 January 2011

EU REU Reference eference LLaboratoryaboratory

IncubationIncubation period range: 4period range: 4--19 days.19 days.

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

-- NOT VACCINE AVAILABLE. NOT VACCINE AVAILABLE.

Control of the disease is mainly Control of the disease is mainly

based onbased on Early Detection Early Detection and and

Strict Sanitary Measures Strict Sanitary Measures Recognition of the Recognition of the disease in the fielddisease in the field

Laboratory Diagnosis Laboratory Diagnosis

LABORATORY DIAGNOSIS IS ESSENTIAL FOR THE CONTROL LABORATORY DIAGNOSIS IS ESSENTIAL FOR THE CONTROL

OF ASF OF ASF

ASF LABORATORY DIAGNOSISASFASF LLABORATORY ABORATORY DDIAGNOSISIAGNOSIS

Identification of the Agent and isolation Identification of the Agent and isolation

•• Isolation in cells culturesIsolation in cells cultures: : HaemoadsorptionHaemoadsorption‘‘autorosetteautorosette’’ (HA) test(HA) test with with peripheral blood leukocytes from infected pigsperipheral blood leukocytes from infected pigs

•• Direct immunofluorescent testDirect immunofluorescent test (DIF)(DIF)

•• Antigen ELISA Antigen ELISA

••Low sensitivity in subacuteLow sensitivity in subacute and chronic formsand chronic forms

Significant lack of sensitivity after first week pi. (because tSignificant lack of sensitivity after first week pi. (because the antibody he antibody

appearance) Give a significant number of false negative resultsappearance) Give a significant number of false negative results. .

Antigen DetectionAntigen Detection

•• VVIRUS IRUS DDETECTIONETECTION

PCR DETECTION USING DIAGNOSTIC PRIMERSPCR DETECTION USING DIAGNOSTIC PRIMERSPCR DETECTION USING DIAGNOSTIC PRIMERS

89000 bp

0 50 100 150 200 kb

P72

88733 bp86793 bp

AMPLIFLIES 257 bpAMPLIFLIES 257 bp AMPLIFLIES 278 bpAMPLIFLIES 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 1ASF 1--22 A12IA12I--VV

Real time King et al, 2003, Real time King et al, 2003,

Others recently validatedOthers recently validated

VVIRUSIRUS DDETECTIONETECTION BYBY PCRPCRASF LABORATORY DIAGNOSISASFASF LLABORATORY ABORATORY DDIAGNOSISIAGNOSIS

••ELISAELISA teststests

••Inmunoblotting ( IB) testInmunoblotting ( IB) test

••Indirect immunofluorescent test Indirect immunofluorescent test (IIF)(IIF)

Indirect Indirect ““in Housein House”” ELISA (OIE)ELISA (OIE)

AANTIBODY NTIBODY DDETECTIONETECTION

““In HouseIn House”” ELISAsELISAs

Commercial ELISA, Ingezim K3Commercial ELISA, Ingezim K3

PositivePositive

•• Indirect Immunoperoxidase Test Indirect Immunoperoxidase Test

CO

NFIR

MA

TO

RY T

ESTS

SC

RE

EN

ING

ASF LABORATORY DIAGNOSISASFASF LLABORATORY ABORATORY DDIAGNOSISIAGNOSIS

Validated

Validated

ELISA in eastern european countriesELISA in eastern european countries

http://www.asf-referencelab.info/http://www.asf-referencelab.info/

web page of the EU RL for ASFweb page of the EU RL for ASF

General informationGeneral information

Current situation and Current situation and

EpidemiologyEpidemiology

Video about clinical signs and Video about clinical signs and

lesionslesions

Publications and advances Publications and advances

Legislation,Legislation,

Diagnostic Procedures, SOPsDiagnostic Procedures, SOPs

Events, meetingsEvents, meetings

Sequece Data Bank, Etc,Sequece Data Bank, Etc,……

Which are the diagnostic tools

currently used within European

Union and colaborating countries in

surveillance and control-eradication

programmes?

Which are the diagnostic tools Which are the diagnostic tools

currently used within European currently used within European

Union and colaborating countries in Union and colaborating countries in

surveillance and controlsurveillance and control--eradication eradication

programmes? programmes?

DIAGNOSISDIAGNOSISDIAGNOSIS

ANNUAL INTERLABORATORY COMPARISON TEST FOR NATIONAL REFERENCE LABORATORIES FOR

EU MEMBER STATES

AANNUAL NNUAL IINTERLABORATORY NTERLABORATORY CCOMPARISON OMPARISON TTEST FOR EST FOR

NNATIONAL ATIONAL RREFERENCE EFERENCE LLABORATORIES FORABORATORIES FOR

EU MEU MEMBER EMBER SSTATES TATES

20102010--2011: Belarus and Russia incorporated2011: Belarus and Russia incorporated.

LABS RESULTS; LABS RESULTS; ASF antibody detectionASF antibody detection

1 technique 1 technique 2 techniques2 techniques 3 techniques3 techniques

ELISAELISA

27.3%27.3%

69.7%69.7%

3%3%

ELISA+IBELISA+IB20/23 20/23 →→87%87%

ELISA+IFAELISA+IFA3/23 3/23 →→9%9%

ELISA+IPTELISA+IPT1/23 1/23 →→4%4%

ELISA+IB+IPTELISA+IB+IPT

87,88

9,09 12,12

29/33 3/33 4/33

INGENASA IN HOUSE CRL-ELISA

LABS RESULTS; LABS RESULTS; ASF antibody detectionASF antibody detection

LABS RESULTS; LABS RESULTS; ASF virus detectionASF virus detection

50%50% 47%47%

3%3%

1 technique 17/341 technique 17/34 2 techniques 16/342 techniques 16/34 3 techniques 1/343 techniques 1/34

ELISA+VI+PCRELISA+VI+PCR

34/34 34/34 →→ 100%100%

PCR PROCEDURESPCR PROCEDURESPCR PROCEDURES

LABS RESULTS; LABS RESULTS; ASF virus detectionASF virus detection

ConventionalConventionalReal TimeReal Time

AgAgüüero et al ero et al

20032003

AgAgüüero et al 2004 ero et al 2004

(MULTIPLEX)(MULTIPLEX)OIE 2004OIE 2004 In houseIn house Not Not

informatin informatin

providedprovided

King et al King et al

20032003

King et al 2003 King et al 2003

modifiedmodifiedZsack et al Zsack et al

20052005In houseIn house Not information Not information

providedprovided

EU RL Main ActivitiesEU RL Main Activities

Caucasus Region, Italy and Africa

1.1.Reagents for Diagnosis, standardized protocols, Molecular Reagents for Diagnosis, standardized protocols, Molecular

characterization, genotyping, of circulating strains. characterization, genotyping, of circulating strains. In collaboration with In collaboration with Russian Federation, FAO, Italy, International Livestock ResearchRussian Federation, FAO, Italy, International Livestock Research InstituteInstitute--ILRIILRI-- and Vet. and Vet.

Services of Kenya, Uganda, Tanzania, CongoServices of Kenya, Uganda, Tanzania, Congo--Brazaville, Ruanda, Nigeria, Togo, Burkina Brazaville, Ruanda, Nigeria, Togo, Burkina

Fasso, Ghana, Fasso, Ghana, Côte d'Ivoire, etc. Côte d'Ivoire, etc.

2. Clinical and 2. Clinical and pathological characterization by pathological characterization by In vivo experiments: In vivo experiments:

ASFV circulating isolates from outbreaks in Caucasus Regions andASFV circulating isolates from outbreaks in Caucasus Regions and

those exhibits major variation is eastern African countries. those exhibits major variation is eastern African countries.

4. Evaluation of diagnostic tools in epidemiological 4. Evaluation of diagnostic tools in epidemiological

situations in Caucasus, west and eastern Africa. situations in Caucasus, west and eastern Africa.

5. Training on Diagnostic Techniques and virus 5. Training on Diagnostic Techniques and virus

characterization . characterization .

3. Epidemiology and Prevalence studies, in certain East , and We3. Epidemiology and Prevalence studies, in certain East , and West African st African

countries countries

Molecular techniquesMolecular techniquesCurrent European circulating ASFV isolates in Caucasus and RussiCurrent European circulating ASFV isolates in Caucasus and Russiaa

In collaboration with In collaboration with

�� The Russian FederationThe Russian FederationDenis Kolvasov, andhis team at Denis Kolvasov, andhis team at

National Institute Veterinary Virology and National Institute Veterinary Virology and

Microbiology, Pokrov,Microbiology, Pokrov,

�� The FAO.The FAO.

Molecular EMolecular EpidemiologypidemiologyP72P72--genotyping, p54 and p30genotyping, p54 and p30

Genotype IIGenotype II

All of them classified into domesticAll of them classified into domestic--pig cycle genotype II and pig cycle genotype II and

related to Mozambique, Madagascar and Zambia isolates related to Mozambique, Madagascar and Zambia isolates

GeorgiaGeorgia

June 2007June 2007

100% sequence homogeneity between Caucasus ASFV isolates., suggesting a single

introduction in Europe in 2007.

Main ActivitiesMain Activities


1.1.Molecular characterization and genotyping, of circulating strainMolecular characterization and genotyping, of circulating strains. In s. In

collaboration with Russian Federation, FAO, Italy, Internationalcollaboration with Russian Federation, FAO, Italy, International

Livestock Research InstituteLivestock Research Institute--ILRIILRI-- and Vet. Services of Kenya, and Vet. Services of Kenya,

Uganda, Tanzania, CongoUganda, Tanzania, Congo--Brazaville, Ruanda, Nigeria, Togo, CoBrazaville, Ruanda, Nigeria, Togo, Co´́te de te de

Voire, Voire,








3. Epidemiology studies, in East , Central and West Africa 3. Epidemiology studies, in East , Central and West Africa


In vivo experiments at the Animal Facility BSL3 at CISA

-- ASFV isolates from Caucasus Regions ASFV isolates from Caucasus Regions

(genotype II) (genotype II)

Hyperemia

Mucosal nasal discharges

Nasal hemorrhages

Azerbaijan, Az08D Az08D

ArmeniaArmenia: : Arm 07Arm 07

--ASFV isolates from Eastern Africa: ASFV isolates from Eastern Africa:

••Domestic cycle (genotype IX) Domestic cycle (genotype IX)

••Sylvatic cycle (genotype X)Sylvatic cycle (genotype X)

��The infected animals developedThe infected animals developed Acute form of the disease Acute form of the disease showing showing

typical clinical signs and lesions associated to typical clinical signs and lesions associated to ASFV virulent strainsASFV virulent strains..

��Viremia (Viremia (PCR + on Blood samples PCR + on Blood samples ) was detectable ) was detectable by OIEby OIE-- prescribed prescribed

virological diagnostic techniquesvirological diagnostic techniques at early times post infection at early times post infection from 3 dpi from 3 dpi

and was maintained during the whole infection. and was maintained during the whole infection.

��Most experimental animals Most experimental animals (more than 80% (more than 80% of pigs 2of pigs 2--3 month of age ) 3 month of age )

died between 6died between 6--9 dpi. 9 dpi. without an specific antibody response.without an specific antibody response.

��Initial antibody Initial antibody response detectable by OIEresponse detectable by OIE-- prescribed serological prescribed serological

diagnostic techniquesdiagnostic techniques was developed in the second week of infection. was developed in the second week of infection.

In vivoIn vivo Experimentation Experimentation

ASFV circulating isolates in Armenia and ASFV circulating isolates in Armenia and

Azerbaijan (2007Azerbaijan (2007--2009) 2009)











characterization genotyping, of circulating strains. characterization genotyping, of circulating strains. In collaboration with In collaboration with Russian Federation, FAO, Italy, International Livestock ResearchRussian Federation, FAO, Italy, International Livestock Research InstituteInstitute--ILRIILRI-- and Vet. and Vet.


Fasso, Ghana, Fasso, Ghana, Côte d'Ivoire, etc. Côte d'Ivoire, etc.

3. Epidemiology and Prevalence studies, in certain East , and We3. Epidemiology and Prevalence studies, in certain East , and West Africa st Africa

20042004--2010 Epidemiology and prevalence study of 2010 Epidemiology and prevalence study of African Swine Fever in Kenya and UgandaAfrican Swine Fever in Kenya and Uganda


Surveillance program in Kenya and Uganda Surveillance program in Kenya and Uganda

Prevalence study of ASFV inPrevalence study of ASFV in wild pigs (warhogs, wild pigs (warhogs,

bushpigs) and ticks (Kenya) bushpigs) and ticks (Kenya) and their role in theand their role in the

transmission of the disease.transmission of the disease.

By sampling collection in domestic pigs , wild suids, ticks. By sampling collection in domestic pigs , wild suids, ticks.

--A significant percentage of ASF Virus in samples (40% of sampliA significant percentage of ASF Virus in samples (40% of sampling)., and in contrast ng)., and in contrast

Low seroprevalence in domestic pigs without clinical signs. Low seroprevalence in domestic pigs without clinical signs.

NIGERIA






44. Evaluation of diagnostic tools in epidemiological . Evaluation of diagnostic tools in epidemiological







Fasso, Togo, Ghana, Fasso, Togo, Ghana, Côte d'Ivoire, etc. Côte d'Ivoire, etc.

2. Epidemiology and Prevalence studies, in certain East , and We2. Epidemiology and Prevalence studies, in certain East , and West Africa st Africa

Are the current ASF diagnostic tools Are the current ASF diagnostic tools adapted adapted

to all epidemiological situations?to all epidemiological situations?

3 different Transmission cycles3 different Transmission cycles

Genetic variability of ASFV: Genetic variability of ASFV: 22 genotypes22 genotypes

European, American, and West African

isolates

↑ HOMOLOGY OF SEQUENCE

European, American, and West African European, American, and West African

isolatesisolates

↑↑ HOMOLOGY OF SEQUENCEHOMOLOGY OF SEQUENCE

East African isolates

↑ VARIABILITY OF SEQUENCE.East African isolatesEast African isolates

↑↑ VARIABILITY OF SEQUENCE.VARIABILITY OF SEQUENCE.

First conclusion: VALIDATED PCR TECHNIQUES for VALIDATED PCR TECHNIQUES for

Virus Detection ARE SENSITIVE For Circulating Virus Detection ARE SENSITIVE For Circulating

Isolates and Genotypes Isolates and Genotypes

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

different virus isolates.

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

different virus isolates.different virus isolates.

�Evaluation of the capability and competence of capability and competence of

formal OIE serological diagnostic tests. formal OIE serological diagnostic tests.

East African isolatesEast African isolatesP72 genotype VIII, IX and XP72 genotype VIII, IX and X

Genome variabilityGenome variability

�Evaluation of the capability and competence of capability and competence of

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

1.1. Analysis of Analysis of 816816 FIELD SERUMFIELD SERUM

samples collected from different samples collected from different

epidemiological situations since epidemiological situations since

20032003--20092009

2.2. Analyses of Analyses of 166 experimental166 experimental serum serum

samples from pigs inoculated with samples from pigs inoculated with

diferent genotypes (diferent genotypes (I, II, IX, XI, II, IX, X))

New ASF serological diagnostic tools

Negative field serum samples East, west, central, Africa Negative field serum samples East, west, central, Africa

and Europeanand European

C.OC.O

ID sera

Abso

rban

ce value

OD.492

IPT IPT

C.OC.O

Positive field samplesPositive field samples

OIEOIE

++

EXPERIMENTAL INFECTION WITH GENOTYPE II

OIE +OIE +

OIE +OIE +

EXPERIMENTAL INFECTION WITH GENOTYPE X

Are the current ASF diagnostic tools Are the current ASF diagnostic tools adapted adapted

to all epidemiological situations?to all epidemiological situations?The current ASF serological diagnostic tools The current ASF serological diagnostic tools

ARE ADAPTED TO ALL EPIDEMIOLOGICAL ARE ADAPTED TO ALL EPIDEMIOLOGICAL

SITUATIONS SITUATIONS

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








5. Trainingand/or Collaboration on Diagnostic 5. Trainingand/or Collaboration on Diagnostic

Techniques and virus characterization . Techniques and virus characterization .




Fasso, Togo, Ghana, Fasso, Togo, Ghana, Côte d'Ivoire, etc. Côte d'Ivoire, etc.

2. Epidemiology studies, in East , Central and West Africa 2. Epidemiology studies, in East , Central and West Africa

WWorkshoporkshop forfor NRLs NRLs on viral on viral DDetection etection TTechniques,echniques,NovNov--Dec, 2009. New on going in 2011Dec, 2009. New on going in 2011

Trainings at EU RL by MS (including all eastern Trainings at EU RL by MS (including all eastern

countries of EUcountries of EU

Assisting neighboring countries

through TAIEX (EC) to improve

their technical standards

Research Vet. Institute UKRAINE (April 2010)

National Reference Laboratory BELARUS (January 2011)

Assisting neighboring countriesAssisting neighboring countries

through TAIEX (EC) to improvethrough TAIEX (EC) to improve

their technical standardstheir technical standards

Research Vet. Institute Research Vet. Institute UKRAINEUKRAINE (April 2010)(April 2010)

National Reference Laboratory National Reference Laboratory BELARUS BELARUS (January (January 2011)2011)

Trainings and Collaborations at EU RLTrainings and Collaborations at EU RL

-National Institute of Veterinary Virology and

Microbiology, Pokrov, Russia,

- Contact and collaborative studies, 2008

- Collaboration study on “ASFV

epidemiological characterization of current

circulating ASFV isolates ” . 2009.

--National Institute of Veterinary Virology and National Institute of Veterinary Virology and

Microbiology, Pokrov, Russia, Microbiology, Pokrov, Russia,

-- Contact and collaborative studies, 2008Contact and collaborative studies, 2008

-- Collaboration study on Collaboration study on ““ASFV ASFV

epidemiological characterization of current epidemiological characterization of current

circulating ASFV isolates circulating ASFV isolates ”” . . 2009.2009.

EEuropeanuropean UUnion nion RReference eference LLaboratory aboratory

TRAINING ON ASF diagnostic techniquesTRAINING ON ASF diagnostic techniquesTRAINING ON ASF diagnostic techniques

-Venue: Nigeria, Ruanda, Kenya Nigeria, Ruanda, Kenya

--(funds: INIA(funds: INIA--ILRI)ILRI)

10 days Trainings Courses, on Diagnostic techniques 10 days Trainings Courses, on Diagnostic techniques

-Venue: UgandaUganda : 25 attendances from Vet Services of Kenya, Tanzania and Uganda.

(funds:funds: ASFRISK RTD,EC, IASFRISK RTD,EC, INIANIA--ILRIILRI))

20072007--20102010

-Venue: Tanzania, tanzania, Kenya, Ruanda and Uganda .30 attendances. (funds: (funds:

ASFRISK RTD,ECASFRISK RTD,EC INIAINIA--ILRI) ILRI)

-To personnel from Asia: :

-China-(Lanzhou Veterinary Institute,

CAAS),

-National Reference Laboratory

-( October 2010)

-Vietnam (NVRI, Hanoi).

--To personnel from Asia: To personnel from Asia: ::

--ChinaChina--(Lanzhou Veterinary Institute, (Lanzhou Veterinary Institute,

CAAS), CAAS),

--National Reference Laboratory National Reference Laboratory

--( October 2010) ( October 2010)

--Vietnam Vietnam (NVRI, Hanoi).(NVRI, Hanoi).

Trainings at EU RL Trainings at EU RL Within ASFRISK project Within ASFRISK project

(DG Research. EC)(DG Research. EC)

Demonstration and hands Demonstration and hands ––on on

exercisesexercises

TRAINING ON ASF EPIDEMIOLOGY AND DIAGNOSIS TRAINING ON ASF EPIDEMIOLOGY AND DIAGNOSIS

September 2010: Venue: Lanzhou, China .

(funds: (funds: ASFRISK RTDASFRISK RTD) )

NRLs should perform at least one virus detection technique in

addition to the antibody detection techniques in order to

ensure an adequate ASF diagnosis. Virus detection techniques

should be available in all NRLs.

NRLs should perform NRLs should perform at leastat least one virus detectionone virus detection technique in technique in

addition to the antibody detection techniquesaddition to the antibody detection techniques in order to in order to

ensure an adequate ASF diagnosis. Virus detection techniques ensure an adequate ASF diagnosis. Virus detection techniques

should be available in all NRLs. should be available in all NRLs.

EU RL encourages to incorporate PCR techniques as EU RL encourages to incorporate PCR techniques as

first choice, whenever be possible. first choice, whenever be possible.

RRECOMMENDATIONS at the ECOMMENDATIONS at the Diagnostic Laboratories Diagnostic Laboratories

to be prepared in case of an ASF to be prepared in case of an ASF

outbreak : outbreak :

Bear in mind Bear in mind the limits of each diagnostic techniquethe limits of each diagnostic technique (especially (especially

antigen detection techniques antigen detection techniques --DIF and ELISADIF and ELISA--) and their feasibility for ) and their feasibility for

each epidemiological situation. each epidemiological situation.

-- Antigen ELISAAntigen ELISA

Recommended for screening of farms with clinical symtomps of Recommended for screening of farms with clinical symtomps of

haemohrragic disease. Not recomended for haemohrragic disease. Not recomended for individual individual

diagnosis.diagnosis.

LOW SENSITIVITY IN SUBACUTE AND CHRONIC FORMS DUE TO ASF LOW SENSITIVITY IN SUBACUTE AND CHRONIC FORMS DUE TO ASF

SPECIFIC ANTIBODY PRESENCE SPECIFIC ANTIBODY PRESENCE -- giving false negative rsultsgiving false negative rsults--

–– Direct immunofluorescent test (DIF)Direct immunofluorescent test (DIF)

TO BE USED ALWAYS TOGETHER WITH AN AB DETECTION TECHNIQUETO BE USED ALWAYS TOGETHER WITH AN AB DETECTION TECHNIQUE

RRECOMMENDATIONS to Diagnostic ECOMMENDATIONS to Diagnostic Laboratories to be prepared in case of an Laboratories to be prepared in case of an

ASF outbreak : ASF outbreak :

GAP ANALYSIS IN LABORATORY DIAGNOSISGAPGAP ANALYSIS IN LABORATORY DIAGNOSISANALYSIS IN LABORATORY DIAGNOSIS

-Regional labs lacks of infrastructure and/or expertise for a

reliable 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 rcountries:

Points for

Collaboration : Points for

Collaboration :

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

, ,

European Union REFERENCE LABORATORY FOR AFRICAN SWINE

FEVEREEuropeanuropean UUnion nion RREFERENCE EFERENCE LLABORATORY FOR ABORATORY FOR AAFRICAN FRICAN SSWINE WINE

FFEVEREVER

Thank you for your attention!Thank you for your attention!Thank you for your attention!

diagnostic tools and main activities of the eu rl. : an ...jan 26, 2011 · eu rl main activities...

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