ASF DIAGNOSIS TOOLS - FLI

ASF DIAGNOSIS TOOLS

Prof. José M. Sánchez- Vizcaí[email protected]

ASF RREFERENCE EFERENCE LLABORATORYABORATORY

Berlin, 2011

ASFV: A old friend 1978-

MAIN WORK:DIAGNOSIS and ERADICATION MODELS

ASF RREFERENCE EFERENCE LLABORATORYABORATORY

ASF 2007-2011

Agenda: A rapid View

1) LAB. DIAGNOSIS:

a) Key points of Asf diagnosisb) Tests Available

2) ASF PROTECTION: Vaccine

3) EARLY DETECTION AND CONTINGENCY PLAN

4) THINGS TO DO

ASF Laboratory DIAGNOSIS: KEY POINTS

• No Vaccine Available

•• NNo o NNeutralizing eutralizing AAntibodiesntibodies

Antibodies = INFECTIONAntibodies = INFECTIONINFECTION

•• Viremia for Long period of TimeViremia for Long period of Time

•Antibodies PersistDuring Month , Even Years

• SEVERAL TEST AVAILABLE. In Good Condition

Abs good infectious marker!!!!

From 7From 7--12 dpi.12 dpi.

AbAb

Infection Clinical Carriers

VIRUSVIRUS

ASF DIAGNOSIS. Key points

INFECTION DISEASES/DEAD CARRIERS

12 h to 1 week 1 – 3 weeks 4 Weeks to 2 years

ASF LABORATORY TESTsASF LABORATORY TESTs

•• Identification of the AgentIdentification of the Agent

•• Virus Isolation in cell culturesVirus Isolation in cell cultures: : TIME: 3 to 10 DaysTIME: 3 to 10 Days

HaemoadsorptionHaemoadsorption‘‘autorosetteautorosette’’ (HA) test(HA) test with peripheral blood leukocytes with peripheral blood leukocytes

from infected pigs. from infected pigs.

Mandatory in first notification in free countryMandatory in first notification in free country

ONLY Reference Labs!!ONLY Reference Labs!!

PCR: CONVENTIONAL and REAL TIMEPCR: CONVENTIONAL and REAL TIME

108 bp-

257 bp-

1 2 3 4 5 6 7 8 9 10 11 12 13 14 C M V

PPCPPC--3/4 + PPA3/4 + PPA--

1/21/2

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-

257 bp-

MOST COMMONLY

USED

TIME: 5 to 6 H

ASF LABORATORY DIAGNOSISASF LABORATORY DIAGNOSIS

DNA DetectionDNA Detection

King et al.,

2003Agüero et al., 2003

Agüeroet al.,

2004

Antigen DetectionAntigen Detection


• Direct immunofluorescence (DIF)

Easy to use

Personal trained needed to interpretate the results

••Low sensitivity in Low sensitivity in subacutesubacute

••and chronic and chronic formsformsSignificant lack of sensitivity due to AgSignificant lack of sensitivity due to Ag--Ab complex formation. Not recomended for analysis of serum Ab complex formation. Not recomended for analysis of serum

and tissue and tissue ––homogenated samples after first week pi. due to false negative rhomogenated samples after first week pi. due to false negative results. esults.

NEGATIVE

POSITIVE

TIME 75 MINUTES


• Immunochromatography – TIME 30 MINUTES

• Pen side tests

Easy to use

No special equipment needed


POSITIVE

SAMPLE

NEGATIVE

SAMPLE

PPA-CROM ANTIGEN DETECTION

• Immunochromatography

Good working with high levels of virus

Lower sensibility than PCR



10-1 10-2 10-3 10-4 C+C-10-5

ORIGINAL

10-2

10-1

EASY TO USE

RAPID RESULTS

No training need

• ELISA DAS

TIME 3 HOURS

Antigen detection in spleen samples



INGEZIM PPA DAS

ASF LABORATORY Ab detectionASF LABORATORY Ab detection

••ELISAELISA teststests

••Immunoblotting Immunoblotting

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

Indirect ELISA (OIE)Indirect ELISA (OIE)

Antibody Detection

In House In House

ELISAsELISAsCommercial ELISA, Ingezim PPA Commercial ELISA, Ingezim PPA

COMPACTCOMPACTMOST COMMONLY USED. TIME 3 H

CONFIRMATORY TESTCONFIRMATORY TEST

TIME 3 H

Infection Clinical Carriers

ASF DIAGNOSIS. Key points

INFECTION DISEASES/DEAD CARRIERS

1 week 2 – 3 weeks 4 Weeks to 2 years

108 bp-

257 bp-

1 2 3 4 5 6 7 8 9 10 11 12 13 14 C M V108 bp-

257 bp-

1 2 3 4 5 6 7 8 9 10 11 12 13 14 C M V

• NO INACTIVATED VACCINE

• NO ATENUATED VACCINE. ONLY PARCIAL PROTECTION

• NO RECOMBINAT VACCINE: NO TARGET GENES

•NO DNA

•Gene Delection

•Subunit: partial

ANTIBODIES ARE RELATED WITH SOME

TYPE OF PROTECTION

AS WELL AS

IN CHRONIC AND ENDEMIC ASF INFECTION

ASF PROTECTION: NO VACCINE

Eradication without vaccine is

possible but not easy. Spain

ASF. Spain: 1985-1995

IN DOOR

EARLY DETECTION

ASF CONTROL

CONTINGENCY PLANS

ASF EARLY DETECTION:

FIELD TRAINING: Farmers and Vets

FARMERS INFORMATION: Risk points

MANUAL for a FAST RESPONSE

1. A good contingency plan adapted to the risk and scenario2. NOTIFICATION SYSTEM

3. ZONING OF AFECTED AREAS4. BAN ON ANIMALS MOVEMENTS

5. LABORATORY CONFIRMATION

6. PROCEDURE FOR DESTRUCTIONS OF CARCASSES7. DEPOPULATION

8. CLEANING AND DISINFECTION9. SEROLOGICAL CONTROLS

10.STUDY WILD BOAR AND/OR VECTORS

11.SENTINEL ANIMALS12.REPOPULATION

Eradication’s Key action: Our experience

a)Aproved programe with farmers. Good information

b)Early detection and good contingen plansc)Detection of positives and carriers animals by serology

d)Elimination of all positives and carriers animals

e)Improvements of biosecurity in farms (in side and outside)f)Include good Restrictions areas

g)Control of movements. Identificationsh)Control of ticks (Elisa for ticks)

i)Economical compensation

THINGS TO DO

• A good early detection program

• A good contingency plan, adapted to the Risk.

• A good control program adapted to the

different scenarios.

• Better knowledge of ticks and WB: Control &

distribution.

• Good collaboration. OIE Lab and our expertise

is yours.

AGRADECIMIENTOS Carmen BulboaDeborah KukielkaFacundo LinaresLuis Martín OteroBeatriz MartínezMarta MartínezLina Mur Ana C. PérezBelén Rivera Rocío SánchezAlmudena SánchezVíctor RodríguezRaquel Vargas Marina Vicente

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ASF DIAGNOSIS TOOLS - FLI