Second Workshop of NRLs for Parasites - Rome 14-15 June 2007

The Community Reference The Community Reference Laboratory for Parasites and Laboratory for Parasites and

the the NRLsNRLs

Patrizia RossiPatrizia Rossi

Second Workshop of NRLs for Parasites - Rome 14-15 June 2007

The Community The Community ReferenceReferenceLaboratoryLaboratory forfor Parasites (CRLP)Parasites (CRLP)

The CRLP wasThe CRLP was established by the European Commission for established by the European Commission for a period of five years (2006a period of five years (2006--2011)2011)The CRLP is located in the Unit of Gastrointestinal and The CRLP is located in the Unit of Gastrointestinal and Tissue Parasitic Diseases of the Department of Infectious, Tissue Parasitic Diseases of the Department of Infectious, Parasitic and Parasitic and ImmunomediatedImmunomediated Diseases of the Diseases of the Istituto Istituto Superiore di SanitSuperiore di Sanitàà (Rome, Italy)(Rome, Italy)The Unit has been accredited since July 2006, according to The Unit has been accredited since July 2006, according to the ISO/IEC 17025 international standardthe ISO/IEC 17025 international standardThe Unit performs research, diagnosis, surveillance, and The Unit performs research, diagnosis, surveillance, and control activities on:control activities on:

helminthichelminthic zoonoseszoonoses (e.g., (e.g., trichinellosistrichinellosis, , echinococcosisechinococcosis, , cysticercosiscysticercosis, , anisakiasisanisakiasis, and , and taeniasistaeniasis) ) protozoan protozoan zoonoseszoonoses (e.g., cryptosporidiosis, (e.g., cryptosporidiosis, giardiasisgiardiasisand toxoplasmosis)and toxoplasmosis)

Second Workshop of NRLs for Parasites - Rome 14-15 June 2007

The Staff of the CRL The Staff of the CRL forfor Parasites Parasites ––Unit of Gastrointestinal and Tissue Parasitic Unit of Gastrointestinal and Tissue Parasitic

DiseasesDiseases

Director of CRL Dr. E. Pozio

Immunology and serology

Dr. M.A. Gomez Morales

MolecularDiagnosis

and Epidemiologyof Helminths Dr. G. La Rosa

Technical staffDr. A. LudovisiDr. S. CherchiMr. M. Amati

Quality Assuranceand

Web site ManagerDr. P. Rossi

Technical staffDr. A. CasulliDr. G. MarucciMr. D. Tonanzi

MolecularEpidemiology

Cryptosporidiumand GiardiaDr. S. Cacciò

Cellular biologyof Toxoplasma

Dr. F. Spano

Cellular biologyof

CryptosporidiumDr. F. Tosini

Animal house Mr. C. Bagnoli

Technical staffDr. A. Possenti

Technical staffDr. M. Lalle

Information Technology

Support ProviderMr. F. Galati

Second Workshop of NRLs for Parasites - Rome 14-15 June 2007

The Staff of theThe Staff of theCRL CRL forfor ParasitesParasites

Second Workshop of NRLs for Parasites - Rome 14-15 June 2007

FiveFive yearyear work work programmeprogramme of of CRLP CRLP -- 11

(2006(2006--20112011))

General tasksGeneral tasksTechnical and scientific support to the CommissionTechnical and scientific support to the CommissionCoCo--operation with international organisations and operation with international organisations and NRLsNRLsDocumentation services, interchange of informationDocumentation services, interchange of information

Development and validation of analytical Development and validation of analytical methodsmethods

Development of diagnostic methodsDevelopment of diagnostic methodsProduction and collection of Reference MaterialsProduction and collection of Reference MaterialsConfirmatory methodsConfirmatory methods

Second Workshop of NRLs for Parasites - Rome 14-15 June 2007

FiveFive yearyear work work programmeprogramme of the of the CRLP CRLP -- 22

(2006(2006--2011)2011)

Quality assurance and quality control including the organisationQuality assurance and quality control including the organisation and and implementation of proficiency testsimplementation of proficiency tests

Maintenance of the quality assurance system (QAS) to ISO/IEC 170Maintenance of the quality assurance system (QAS) to ISO/IEC 1702525Organisation of proficiency testsOrganisation of proficiency testsOrganisation of ring trialsOrganisation of ring trials

Technical and scientific support to Technical and scientific support to NRLsNRLs and third countriesand third countriesAnalytical support and trainingAnalytical support and trainingMissions to Missions to NRLsNRLs and diffusion of scientific informationand diffusion of scientific informationPreparation and delivery of reference materialsPreparation and delivery of reference materialsAnalysis of official samplesAnalysis of official samplesOrganisation of workshops and training coursesOrganisation of workshops and training coursesCollaboration with laboratories responsible for foodborne parasiCollaboration with laboratories responsible for foodborne parasites in third tes in third countriescountries

Research activities on foodborne parasites and related infectionResearch activities on foodborne parasites and related infections in s in animalsanimals

Second Workshop of NRLs for Parasites - Rome 14-15 June 2007

Activities carried out by the CRLP Activities carried out by the CRLP ((JulyJuly 2006 2006 –– MayMay 2007) 2007) -- 11

Validation of an indirect ELISA test for the detection Validation of an indirect ELISA test for the detection of antiof anti--TrichinellaTrichinella antibodies in swine serumantibodies in swine serum

InterlaboratoryInterlaboratory comparison of the indirect ELISA testcomparison of the indirect ELISA test

Validation of a method for the identification of Validation of a method for the identification of TrichinellaTrichinella muscle larvae at species level by Multiplex muscle larvae at species level by Multiplex PCRPCR

Development of a Development of a ““Guideline for the detection of Guideline for the detection of Trichinella Trichinella larvae at the slaughterhouse in a Quality larvae at the slaughterhouse in a Quality Assurance SystemAssurance System””

Second Workshop of NRLs for Parasites - Rome 14-15 June 2007

Activities carried out by the CRLP Activities carried out by the CRLP ((JulyJuly 2006 2006 –– MayMay 2007) 2007) -- 22

Development of a Guideline for the Development of a Guideline for the ““Identification and Identification and development of sampling methods and design of suitable development of sampling methods and design of suitable protocols for monitoring of protocols for monitoring of TrichinellaTrichinella infection indicator infection indicator speciesspecies””

Provision of Provision of TrichinellaTrichinella sppspp. reference strains to laboratories . reference strains to laboratories from European and third countries from European and third countries ((http://http://www.iss.it/site/Trichinellawww.iss.it/site/Trichinella//))

Development of the CRLP web site (Development of the CRLP web site (http://www.iss.it/crlp/http://www.iss.it/crlp/))

First workshop of First workshop of NRLsNRLs for parasites (Rome, 24 Novemberfor parasites (Rome, 24 November20062006))

InterlaboratoryInterlaboratory comparison of the magnetic stirred digestion comparison of the magnetic stirred digestion method for the detection of method for the detection of TrichinellaTrichinella larvae in meatlarvae in meat

Second Workshop of NRLs for Parasites - Rome 14-15 June 2007

The The NationalNational ReferenceReference

LaboratoriesLaboratories forfor ParasitesParasites -- 11

C. EconomidesC. EconomidesA. A. PhylactouPhylactou

State State VeterinaryVeterinary LaboratoryLaboratoryNicosiaNicosia

AllAll food food borneborneparasitesparasitesCyprusCyprus

M. HalachevaM. HalachevaNationalNational DiagnosticDiagnostic and and

ResearchResearch VeterinaryVeterinaryInstituteInstitute, Sofia, Sofia

TrichinellaTrichinellaBulgariaBulgaria

C. MaddoxC. Maddox--HyttelHyttelThe The DanishDanish InstituteInstitute forfor

Food and Food and VeterinaryVeterinaryResearchResearch, Copenaghen, Copenaghen

AllAll food food borneborneparasitesparasitesDenmarkDenmark

B. KoudelaB. KoudelaV. V. SvobodovaSvobodova

University of Veterinary and University of Veterinary and Pharmaceutical Sciences, Pharmaceutical Sciences, Dept of Parasitology, BrnoDept of Parasitology, Brno

AllAll food food borneborneparasitesparasites

CzechCzechRepublicRepublic

P. DornyP. DornyC. C. MarleenMarleen

PrinsPrins LeopoldLeopold InstituteInstitute forforTropicalTropical SciencesSciences, ,

AntwerpenAntwerpen

Trichinella, Taenia, Trichinella, Taenia, EchinococcusEchinococcus

BelgiumBelgium and and LuxembourgLuxembourg

K.K. SchSchööpfpfInstituteInstitute of of VeterinaryVeterinaryMedicine, Medicine, InnsbruckInnsbruck

AllAll food food borneborneparasitesparasitesAustriaAustria

Contact Contact personperson/s/sInstitutionInstitutionParasite/sParasite/sCountryCountry

Second Workshop of NRLs for Parasites - Rome 14-15 June 2007

The The NationalNational ReferenceReference

LaboratoriesLaboratories forfor ParasitesParasites -- 22

A. KA. KäärssinrssinI.I. NurmojaNurmoja

EstonianEstonian VeterinaryVeterinaryand Food and Food LaboratoryLaboratory, ,

TartuTartuAllAll food food borneborne parasitesparasitesEstoniaEstonia

A. OksanenA. OksanenFinnishFinnish Food Food SafetySafety

AuthorityAuthority, , OuluOuluAllAll food food borneborne parasitesparasitesFinlandFinland

F.F. J.J. ConrathsConrathsFriedrichFriedrich--LoefflerLoeffler

InstitutInstitut, , GreifswaldGreifswald--InselInsel RiemsRiems

EchinococcusEchinococcus

K.K. HorstHorstBundesanstaltBundesanstalt fur fur ErnahrungErnahrung und und

LebennsmittelKarlsruheLebennsmittelKarlsruheAnisakidaeAnisakidae

K.K. NoecklerNoecklerL.L. EllerbroekEllerbroek

BundesinstitutBundesinstitut fur fur RisikobewertungRisikobewertung, , BerlinBerlinTrichinellaTrichinella

GermanyGermany

F.F. CliquetCliquetAFFSA AFFSA -- LERRPAS, LERRPAS,

MalzevilleMalzevilleEchinococcusEchinococcus

P. BoireauP. BoireauI.I. VallVallééee

AFFSA AFFSA -- LERPAZ, LERPAZ, Maison Maison AlfortAlfortTrichinellaTrichinella, , AnisakidaeAnisakidae

FranceFrance

Contact Contact personperson/s/sInstitutionInstitutionParasite/sParasite/sCountryCountry

Second Workshop of NRLs for Parasites - Rome 14-15 June 2007

The The NationalNational ReferenceReference

LaboratoriesLaboratories forfor ParasitesParasites -- 33

S. BoutsiniS. BoutsiniCentreCentre of of AthensAthens

VeterinaryVeterinary InstituteInstitute, , Dep.Dep. ParasitolParasitol., ., AthensAthens

ToxoplasmaToxoplasma, Trichinella, TrichinellaGreeceGreece

T.T. SrSrééterterCentralCentral VeterinaryVeterinaryInstituteInstitute, Budapest, BudapestAllAll food food borneborne parasitesparasitesHungaryHungary

C. MasalaC. MasalaIstituto Istituto ZooprofilatticoZooprofilattico

SperimentaleSperimentale of of SassariSassari

EchinococcusEchinococcus

S. CaracappaS. CaracappaIstituto Istituto ZooprofilatticoZooprofilattico

SperimentaleSperimentale of of PalermoPalermo

AnisakidaeAnisakidae

E. PozioE. PozioIstituto Superiore di Istituto Superiore di SanitSanitàà, Rome, RomeTrichinellaTrichinella

ItalyItaly

J.J. FerrisFerrisDep.Dep. of of AgricultureAgriculture and and

Food Food LaboratoriesLaboratories, , BackwestonBackweston,,

AllAll food food borneborne parasitesparasitesIrelandIreland

Contact Contact personperson/s/sInstitutionInstitutionParasite/sParasite/sCountryCountry

Second Workshop of NRLs for Parasites - Rome 14-15 June 2007

The The NationalNational ReferenceReference

LaboratoriesLaboratories forfor ParasitesParasites -- 44

NOT YET DESIGNEDNOT YET DESIGNEDMaltaMalta

M. M. KirjusinaKirjusinaG. G. JaunbauereJaunbauere

Laboratory of Food and Laboratory of Food and Environmental Environmental

Investigations, RigaInvestigations, Riga

TrichinellaTrichinella, , EchinococcusEchinococcus, ,

AnisakidaeAnisakidaeLatviaLatvia

J. MiliusJ. MiliusR. R. BubulieneBubuliene

National Veterinary National Veterinary Lab., VilniusLab., VilniusAll food borne parasitesAll food borne parasitesLithuaniaLithuania

J.J. Van der GiessenVan der GiessenNationalNational InstituteInstitute of of Public Public HealthHealth and and

EnvironmentEnvironment, , BilthovenBilthovenAll food borne parasitesAll food borne parasitesNetherlandsNetherlands

R. NovaisR. NovaisInstitutoInstituto NacionalNacional de de SaSaúúdede, , LisboaLisboaAll food borne parasitesAll food borne parasitesPortugalPortugal

T. T. CencekCencekJ. J. OsekOsek

National Vet. Research National Vet. Research Inst., Dep. Inst., Dep. ParasitolParasitol..

PulawyPulawyAll food borne parasitesAll food borne parasitesPolandPoland

Contact Contact personperson/s/sInstitutionInstitutionParasite/sParasite/sCountryCountry

Second Workshop of NRLs for Parasites - Rome 14-15 June 2007

The The NationalNational ReferenceReference

LaboratoriesLaboratories forfor ParasitesParasites -- 55

K. HuntK. HuntVeterinary Laboratories Veterinary Laboratories

Agency, Agency, WeybridgeWeybridgeAll food borne parasitesAll food borne parasites

United United KingdomKingdom

Nicolae StefanNicolae StefanInstitute for Diagnosis and Institute for Diagnosis and Animal HealthAnimal Health, , BucarestBucarestAll food borne parasitesAll food borne parasitesRomaniaRomania

F. TovarF. TovarCentro Centro NacionalNacional de de Alimentation, Alimentation, MajadahondaMajadahonda

TrichinellaTrichinella, , AnisakidaeAnisakidae, , EchinococcusEchinococcus

J. PosediJ. PosediA. A. VerglesVergles RatajRataj

University of LjubljanaUniversity of LjubljanaVeterinary Faculty, Veterinary Faculty,

LjubljanaLjubljanaAll food borne parasitesAll food borne parasitesSloveniaSlovenia

V. V. BeladiBeladiččovovááD. D. ValentovaValentova

State Veterinary and Food State Veterinary and Food Institute, BratislavaInstitute, BratislavaAll food borne parasitesAll food borne parasites

Slovak Slovak RepublicRepublic

F.F. Garrido AbellanGarrido AbellanR. R. GonzalesGonzales CanoCano

Laboratorio Laboratorio CentralCentral de de SanidadSanidad AnimalAnimal de Santa de Santa

FeFe, , GranadaGranadaAll food borne parasitesAll food borne parasites

SpainSpain

D. ChristenssonD. ChristenssonE. E. OstermanOsterman LindLind

National Veterinary National Veterinary Institute, UppsalaInstitute, UppsalaAll food borne parasitesAll food borne parasitesSwedenSweden

Contact Contact personperson/s/sInstitutionInstitutionParasite/sParasite/sCountryCountry

Second Workshop of NRLs for Parasites - Rome 14-15 June 2007

Legal Status of Legal Status of NRLsNRLs

3; 10%

14; 47%13; 43%

Governmental

Academic

Not stated

Second Workshop of NRLs for Parasites - Rome 14-15 June 2007

Main field of activity Main field of activity of the of the NRLsNRLs

5

10

10

246

8101214

16

Food Microbiology Veterinary microbiology General Microbiology

n. of

NRLs

Second Workshop of NRLs for Parasites - Rome 14-15 June 2007

Type and number of analysis Type and number of analysis carried out by carried out by

the the NRLsNRLs

0

5

10

15

20

foodstuff animal human environmental

400 (10-1700)

200(2-2000)

28000(50-78000)

150(3-500)

10 16

710

n.

of

NRLs

Second Workshop of NRLs for Parasites - Rome 14-15 June 2007

ConfirmationConfirmation and and typingtyping of of parasite parasite isolatesisolates sentsent bybyperipheralperipheral laboratorieslaboratories

0

5

10

15

20

food human animal environmental

10

4

10

3

30 (3-200) 11 (2-100) 60 (10-300) 3 (3-10)n.

of

NRLs

Second Workshop of NRLs for Parasites - Rome 14-15 June 2007

LaboratoriesLaboratories and and methodsmethods accreditedaccreditedaccordingaccording toto the ISO/IEC 17025the ISO/IEC 17025

12 accredited NRLsAccredited Methods

1. Trichinoscopy (5 laboratories)2. ER 2075/2005 Reference detection method (3 Laboratories)

1. Magnetic stirrer method for pooled meat sample digestion for Trichinella (4 Laboratories)

3. D. repens and D. immitis from blood samples4. Diagnostics of dourine (Trypanosoma equiperdum)- complement fixation

test5. McMaster6. Tritrichomonas diagnostics7. Methods of parasitological examination of a marine fish and fish production

(freshen, cooled and frozen marine fish)8. Method of definition of common for man and animal freshwater fish

helmint-zoonoses9. honey bees (diagnosis of nosematosis, acarapidosis, varroatosis)10. Copro-antigen ELISA (if test kits available from manufacturer)11. Multiplex PCR 12. ELISA13. Western blot

Second Workshop of NRLs for Parasites - Rome 14-15 June 2007

MethodsMethods usedused forfor the detection ofthe detection ofparasites in parasites in differentdifferent matricesmatrices -- 11

Intestinal Scraping (Echinococcus sp.) (1)

Smear (1)

Baermann (1)

Serology (1)

Propagation (1)

PCR (1)

stereomicroscopy, sieving (1)

IFT (1)

Parasitological (1)Larvoscopy (1)ELISA (1)Concentration (1)

Staining (1)Faecal examinations (1)Histology (1)IMS (1)

CFT, DAT (1)ELISA (1)Morphological examination (1)Filtration (1)

Morphological examination (2)

Macroscopic examination (2)

FITC for Cryptosporidium and Giardia detection (1)

Stereo microscopy (1)

PCR (3)Necropsy (3) Intestine scraping (1)Necropsy, visual examination (1)

Immunological method (6)

Flotation & sedimentation (6)

Molecular (PCR) (3) Trichinoscopy (1)

Microscopy (6)Digestion (7) Microscopy (6)Artificial digestion (7)

Isolation (n. lab)Detection (n. lab)Isolation (n. lab)Detection (n. lab)

Animal samplesFoodstuffs

Second Workshop of NRLs for Parasites - Rome 14-15 June 2007

MethodsMethods usedused forfor the detection ofthe detection ofparasites in parasites in differentdifferent matricesmatrices -- 22

Microscopy (1)

PCR (1)PCR (1)

FITC labelled mAbagainst Cryptosporidium and Giardia (1)

Filtration (1)Morphology (1)Digestion (1)

Floating on Zinc Chloride (1)

IMS (1)Staining (1)Concentration (1)

Morphological and visual examination (2)

Visual examination(1)

Immunological (2) (antibody detection; ELISA IFT, serology)

Larvoscopy (1)

Microscopy (3) Flotation and sedimentation (2)

Microscopy (4)Flotation and sedimentation (2)

IsolationDetectionIsolationDetection

Environmental samplesHuman samples

Second Workshop of NRLs for Parasites - Rome 14-15 June 2007

ActivitiesActivities of of NRLsNRLs –– 11

0

2

4

6

8

10

12

n of N R Ls

food and feed

human samples

animal samples

environmental samples

Trichinella Echinococcus spAnisakidae

T. gondiiSarcocystis sp

Cryptosporidium spGiardia

Taenia sp

Second Workshop of NRLs for Parasites - Rome 14-15 June 2007

ActivitiesActivities of of NRLsNRLs –– 22

Urban foxesToxocara spp.Ascaris sppTrichuris sppAll parasites species in veterinary parasitologyMicrosporidia

Environmental (3 labs)

Generic (1 lab)

Zoo (2 labs)

Wild (8 labs)

1.Vermifugation of foxes, domestic animal, urban foxes2.Surveillance of foxes for Trichinella: 300 per year (start 2007)3.Control of echinococcosis2004 – 2008

Trypanosoma equiperdumToxocaraTicksLeishmaniaHorse intestinal parasitesMicrosporidiaNon-zoonotic parasitesAll parasites species in veterinary parasitology

Domestic (7 labs)

Animal

No ToxocaraLeishmaniaMalariaAscaris

Human (1 lab)

Urban foxesFish parasitesMicrosporidiaSeashore and marine fish

Food and feed (3 labs)

Special programsOther parasitesType of samples

Second Workshop of NRLs for Parasites - Rome 14-15 June 2007

Expertise of Expertise of NRLsNRLs

0

2

4

6

8

10

12

Animal modelsExp. Infections

Immune resp.Mol. Biology

Ab PreparationAg preparation

SurveillanceOutbreak inv. GIS analysis

n.

of

NRLs

Second Workshop of NRLs for Parasites - Rome 14-15 June 2007

FacilitiesFacilities of of NRLsNRLs

1.from 1992 (2 labs)2.last 10 years (2 labs)3.starting from 2006 (1 lab) 5 labs

Database availability

1100 (400-2000)3 labsWild animal sera4 labs

Animalisolates

1400 (100-2500)3 labs

Domestic animalsera

1 labHumanisolates

0noHuman sera 100 (10-250)8 labsParasites

Approxn.

Serum collections from parasite disease

Approx. n.

Parasite collections

Type of samples

Second Workshop of NRLs for Parasites - Rome 14-15 June 2007

NeedsNeeds expressedexpressed byby NRLsNRLs -- 11

Identification of Diphyllobothriumspp.

Identification of zoonoticparasites (teory and practice)

Transfer of technology

Training of personel

Special trainings and education (GIS, cell culture, …)

Sensitive 1-larva multiplex PCR forTrichinella spp

Practical workshop for detection of Echinococcus and Cysticercus - detection of parasites from the meat inspection samples

Training

Serological methods for detection of Trichinella spp.

Methodics for detection of fish parasites

Reliable extractionprotocols

Rapid test for diagnosis of leishmaniosis and dirofilariosis

Standardisedmethods

Sensitive copro-PCR forEchinococcus

Developmentof diagnostic

methods

Third(5 labs)

Second(6 labs)

First(9 labs)

Second Workshop of NRLs for Parasites - Rome 14-15 June 2007

NeedsNeeds expressedexpressed byby NRLsNRLs -- 22

International data bases

TechnicianWorkable budget

Parasites info-netStaffLaboratory facilities

Others

Cooperation possibility in the field of Echinococcus spp., Toxoplasma gondiidiagnosis

International ring trials

Cooperation/ring trials

Third(5 labs)

Second(6 labs)

First(9 labs)