Bluetongue
OIE Reference Laboratory Reports
Activities in 2010
Name of disease (or topic) for which you are a designated OIE Reference Laboratory: / BluetongueAddress of laboratory: / Via Campo Boario
64100, Teramo
Italy
Tel.: / (+39-0861) 33.24.40
Fax: / (+39-0861) 33.22.51
e-mail address: /
website: /
Name of Head of Laboratory (Responsible Official): / Rossella Lelli
Name of OIE Reference Expert: / Giovanni Savini
Name of writer of this report
(if different from above):
Part I:Summary of general activities related to the disease
1.Test(s) in use/or available for the specified disease/topic at your laboratory
Test / For / Specificity / TotalC-ELISA / Antibody / Group / 21,006
VNT / Antibody / Type / 46,840
KC + VERO cell culture
/ Virus isolation / Group / 34ECE+VERO cell culture
/ Virus isolation / Group / 34VERO cell culture
/ Virus isolation / Group / 34VERO cell culture
/ Virus titration / Group / 70Serotype specific PCR real time
/ Virus identification / Type / 40PCR real time
/ Agent identification / Group / 2,926Virus typing
/ Virus identification / Type / 33Microscopic examination
/ Culicoides imicola identification / Species / 5,796PCR
/ Culicoides obsoletus and Culicoides scoticus identification / Obsoletus complex species / 507PCR
/ Culicoides dewulfi: identificazione /Species
/ 20Microscopic examination
/ Other species of Culicoides /Species
/ 406Microscopic examination
/ Culicoides spp. /Species
/ 5,7962.Production and distribution of diagnostic reagents
Production and testing of bluetongue (BT) virus (BTV) antigen and sheep antisera, to be used in serological tests and diagnostic kits, have continued throughout the year.
In the 2010, BTV C-ELISA reagents sufficient for 418.140 tests have been produced and supplied to Italian laboratories.
Isolates of BTV-1 collected during the 2010 Sardinian outbreaks together with the BTV-16 typed from Turkish samples were aliquoted and stocked. They were also cloned and sequenced in order to better understand the isolate origin.
From the 2010 National Surveillance Program 2,877 new sera were collected, aliquoted, lyophilised and stored.
Type of reagent / Amount supplied nationally(including for own use) / Amount supplied to other countries
Control positive serum for BTV / BTV-1 / 1.2 ml / 30 ml (IAH, Pirbright, UK)
BTV-2 / 1.2 ml / 30 ml (IAH, Pirbright, UK)
BTV-3 / 0.05 ml
BTV-4 / 1.2 ml
BTV-5 / 0.05 ml
BTV-6 / 1.2 ml
BTV-7 / 0.05 ml
BTV-8 / 1.2 ml
BTV-9 / 1.2 ml
BTV-10 / 0.05 ml
BTV-11 / 0.05 ml / 30 ml (IAH, Pirbright, UK)
BTV-12 / 0.05 ml
BTV-13 / 0.05 ml
BTV-14 / 0.05 ml
BTV-15 / 1.2 ml
BTV-16 / 1.2 ml
BTV-17 / 0.05 ml
BTV-18 / 0.05 ml
BTV-19 / 0.05 ml
BTV-20 / 0.05 ml
BTV-21 / 0.05 ml
BTV-22 / 0.05 ml
BTV-23 / 0.05 ml
BTV-24 / 0.05 ml
Type of reagent / Amount supplied nationally
(including for own use) / Amount supplied to other countries
BTV reference strains
/ BTV-1 / 164 ml / 1 ml (IDAH, Bucharest, Romania)BTV-2 / 154 ml
BTV-3 / 34 ml
BTV-4 / 184 ml
BTV-5 / 34 ml
BTV-6 / 354 ml
BTV-7 / 34 ml
BTV-8 / 180 ml / 5 ml (University of Scotland, Glasgow, UK)
BTV-9 / 160 ml
BTV-10 / 34 ml
BTV-11 / 34 ml
BTV-12 / 34 ml
BTV-13 / 34 ml
BTV-14 / 50 ml
BTV-15 / 200 ml
BTV-16 / 230 ml
BTV-17 / 34 ml
BTV-18 / 34 ml
BTV-19 / 34 ml
BTV-20 / 34 ml
BTV-21 / 20 ml
BTV-22 / 34 ml
BTV-23 / 34 ml
BTV-24 / 34 ml
Part II: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
The ninth inter-laboratory proficiency testing for BT ELISA including 37 Italian laboratories and the Croatian Veterinary Institute of Split, Croatia, was organised. All ring trial records (subscriptions, data submission, final report) were handled through an on-line internet interface.
The fourth inter-laboratory proficiency testing for RT-PCR including 13 Italian laboratories was also organised. As for the serology, all ring trial records were handled through an on-line internet interface.
The laboratory also participated to the “sixth European Union (EU) ring trial for BT ELISAs and RT-PCR” organised by the Institute for Animal Health, Pirbright Laboratory, Pirbright, UK, (European Community Reference Laboratory for BT). In the 2010 proficiency testthe serumneutralisation assay was also included. The Istituto G. Caporale was actively involved in supplying the serum panel and analysing the results of the trial.
4.Preparation and supply of international reference standards for diagnostic tests or vaccines
The attenuation process of the BTV-16 on either VERO cells and BHK21has been continued,
Antiserum and virus stocks were continuously refilled and supplied to other Member Countries when requested.
A new antigen derived from the surnatant of BTV infected tissue culture has been prepared and is now under testing.
Four batches of VP7 recombinant protein have been produced from insect cell lines infected with Baculovirus.
Monoclonal antibodies as well as purified-inactivated antigen have been produced as reagents of the c- ELISA Kit.
Antisera against BTV-14 and BTV-20 have been produced from guinea pigs.
BTV-1 reference strain has been sent to the Institute of Diagnosis and Animal Health, Bucharest, Romania.
Five different strains of BTV-8 have been sent to the Faculty of Veterinary Medicine, Institute of Comparative Medicine, University of Glasgow, Scotland, UK.
Cell lines (2x25 cm2 flasks) from Culicoides sonorensis (Kc) and Aedes albopictus (C6/36) have been supplied to the Central Veterinary Control and Research Institute of Ankara, Turkey.
During the 2010, 14 light traps for catching Culicoides spp. were built and sold to France, Netherlands and Croatia.
5.Research and development of new procedures for diagnosis and control
Works on the Italian Administration-funded projects coordinated by personnel of the OIE reference laboratory continued throughout 2010 as follows:
- The program for validating the manually executed real time RT-PCR based on NS1 gene for detecting all BTV serotypes from insects, organs and blood samples was completed. The system is now under automatization and the validation program for this new step is in progress.
- Two commercial(IDVET, Montpellier, France; Adiagene-VMRD, Sain Breuk, France) competitive ELISA kits for detecting BTV antibodies from serum samples have been validated.
- A trial funded by the Italian Ministry of Health which aims to study the transplacental transmission of different strains of BTV-8 in cows started. Animals have been selected according to their gestation period and the facilities for the animals have been set up.
- Validation program of the commercial kit “VetMAX bluetongue virus reagents” of the Applied Biosystem for detecting BTV RNA from blood and tissue samples has been completed.
- The laboratory part of the Project on “New expressing vector for the bluetongue virus” has been completed. In this project an ORF virus has been used to express the VP2 and VP5 gene segments of BTV in order to immunize susceptible animals. During the 2010, sheep have also been selected and the facilities to allocate the animals have been prepared. The project has been realised with the partnership of the Federal Research Institute for Animal Health, Institute of Immunology, Friedrich-Loeffler-Institute, Tuebingen, Germany and Pfizer Research and Development Department.
- The activities ofproject on “the use of “landscape analysis and Remote Sensing” techniques to investigate the microclimatic and environmental factors associated to the C. obsoletus s.s., C. scoticus, C. dewulfi and C. montanus distribution, continued. The project aims to identify the most important factors which determine the presence of C. obsoletus s.s., C. scoticus, C. dewulfi and C. montanus in the Italian environment.
6.Collection, analysis and dissemination of epizootiological data relevant to international disease control
Relevant data on BTV control were collected, analysed and disseminated through the use of a web-GIS system and the establishment an OIE Bluetongue reference laboratories network (OIE-BTNet). In 2010 the activities related to to the establishment of an OIE Bluetongue reference laboratories network (OIE-BTNet), aiming to improve the knowledge of the bluetongue epidemiological situation and develop a wider bluetongue surveillance network, as a tool for international animal disease control and to facilitate international trade of animals, continued. The website ( contents have been kept updated. Information on 609 BTV isolates, including genetic sequences data, were provided in the “BTV strain” section. The web-GIS application collected around 1,500 references related to BTV worldwide occurrence from 1943 to date. In July 2010, the first number of the Blue Tongue Newsletter has been published and distributed to all network’s members. The Newsletter which is prepared by the personnel of the Institute “G. Caporale” in charge of the Secretariat is planned to be published every 4-6 months and aims to disseminate the most valuable news on BT and stimulate discussion on recent and new aspects related to disease’s epidemiology. In December, the second number of the Newsletter was publishe and distributed to the network.
According to the Italian BT surveillance program, insects collected from nearly 5,000 catches originating from permanent (249) and mobile light traps (24) in a total of 320 municipalities located in all Italian regions, were coordinated and examined. These, together with all relevant virological, serological and epidemiological data on bluetongue in Italy, were published through a web-based information system on a weekly basis (website:
7.Provision of consultant expertise to OIE or to OIE Members
Personnel of the OIE reference lab:
participated at the ad hoc Group on Scientific Partnerships of OIE Laboratories, Paris, France, 6-7 May, 2010.
at the EU Task Force Meeting on Animal Health Surveillance, Bruxelles, Belgium, 16-18 May 2010.
Participated, as invited speaker, to the Workshop on Eradication and Control of Bluetongue organised by the European Commission's DG Enlargement within the framework of the TAIEX Instrument on the 29th-30th of March, Belgrade, Serbia.
Participated, as invited speaker, to the Workshop on Bluetongue organised by the European Commission's DG Enlargement within the framework of the TAIEX Instrument on the 4th of November, Ankara, Turkey.
has been invited by the European Food Safety Authority (EFSA) to be a member of the Working Group on “Bluetongue Monitoring and Surveillance”, opinion of the Scientific Panel on Animal Health and Welfare on request from the Commission “to provide scientific advice on the expected prevalence (design prevalence) under different circumstances and to provide an updated scientific assessment of the size of the geographical relevant area for the purpose of monitoring and surveillance programmes for bluetongue.” The first meeting (teleconference) washeld on the 17th ofDecember 2010.
was invited to be a member of the ad-hoc Scientific Advisory Group (SAG) - Bluetongue vaccine, organised by the European Medicines Agency on the 10th (teleconference) and the 17th of May 2010, London, UK.
was invited to be a member ad-hoc Scientific Advisory Group (SAG) - Bluetongue vaccine-Follow up which will meet in London next year.
participated with other experts from 9 different Institutions, to review the “Disease and Product analysis document” on bluetongue within the Discontool project organised by the International Federation for Animal Health Europe
participated, as invited speaker, to the Bluetongue Information Day, organised by the Istituto Zooprofilattico Sperimentale del Piemonte and by Piemonte Veterinary Service, lecturing on “Bluetongue in Italy, epidemiological situation and future perspectives”, Turin, Italy, 26 March, 2010.
reviewed the Bluetongue chapter of the “Manual of Diagnostic Tests and Vaccines for Terrestrial Animals (Terrestrial Manual)”.
The laboratory also contributed to the EU Workshop on “Annual meeting of National Reference Laboratories for bluetongue”, Madrid,Spain, 01-03 December 2010, presenting the analyisis of the data of the SN proficiency test and the results on the “Cross placental transmission of different BTV-8 strains in ewes”.
8.Provision of scientific and technical training to personnel from other OIE Members
Within the framework of the collaboration between the Institute “G. Caporale” and the Veterinary Authorities of the Mediterranean Countries:
an OIE twinning project on bluetongue between the Institute “G. Caporale” and the InstitutdelaRechercheVétérinairedeTunisie(IRVT) was prepared, signedand approved by the OIE, The project aimed to train the personnel of the IRVT on the bluetongue diagnosis. The first stage on serology has been completed on December 2010.
anOIE regional cooperation project on bluetongue between the Institute “G. Caporale”, the InstitutdelaRechercheVétérinairedeTunisie(IRVT) and the Institut de Recherche Agronomique du Liban – FANAR (IRAL) was also prepared, signed by the competent authorities and approved by the OIE. In this program personnel of the IRAL will be trained on the laboratory diagnosis of bluetongue. The first stage on virology will be next January.
Within the framework of the MIUR (Italian Ministry of Universities and Research) project “Innovative eLearning system for the agricultural sector in the Mediterranean countries: recognition, diagnosis, control and prevention of Arthropod-borne diseases”, the course on BT was conducted between October and December involving Veterinarians and personnel involved on animal health.
Within the agreement with the Ministry of Agriculture, Waterand Forestry and the Department of Veterinary Services of Namibia, the reference laboratory supported the Central Veterinary Laboratory of Windhoek in the application of bluetongue diagnostic protocols.
Continuous advices on BT entomological surveillance and laboratory diagnosis have been given to the Croatian Veterinary Institute, Zagreb and Split, Croatia; Ministry of Rural Affairs and the Environment (MRAE), Valletta, Malta; Kosovo Central Veterinary Laboratory, Prishtina, Kosovo; Veterinary Faculty, University of Slovenia, Ljubljana, Slovenia; Institute for Diagnosis and Animal Health, Bucharest, Romania, State Veterinary Institute, Zvolen, Slovakia, State Veterinary Institute, Jihlava, Czech Republic. Kimron Veterinary Institute, Beit Dagan. Israel
9.Provision of diagnostic testing facilities to other OIE Members
Within the framework of the agreement for the co-operation between the Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale” and the Department of Veterinary Services of the Republic of San Marino for assistance in Bluetongue surveillance signed in 2007, the surveillance activities continued.
The following table summarises the official diagnostic services provided and the Member Countries where the samples originated.
Country / Test / No. of samplesTunisia / BTV Typing RT- PCR / 26
BTV sequencing / 26
Virus neutralisation / 83
EHDV RT-PCR / 26
KC + VERO cell culture / 26
ECE+VERO cell culture / 26
VERO cell culture / 26
Morocco / Serumneutralisation / 149
San Marino / C-ELISA / 240
Serumneutralisation / 240
Turkey / BTV typing / 1
Virus neutralisation / 1
Croatia / Serumneutralisation / 29
Real time RT-PCR / 15
ECE+VERO cell culture / 3
Brazil / Serumneutralisation / 3
Virus isolation / 6
Real time RT-PCR / 6
KC + VERO cell culture / 6
ECE+VERO cell culture / 6
VERO cell culture / 6
10.Organisation of international scientific meetings on behalf of OIE or other international bodies
The 3rd“OIE Bluetongue Reference Laboratory Network Meeting Workshop”,has been organised to discuss on the validation methods. It will be held in Teramo on the 21st of January 2011.
11.Participation in international scientific collaborative studies
Activities on the project “Oral susceptibility of European and South African Culicoides species for European and South African bluetongue virus isolates” continued in collaboration with the Agricultural Research Council – Onderstepoort Veterinary Institute in South Africa, within the framework of the “Executive programme of scientific and technological cooperation between Italy and South Africa”, signed in 2007.
Within the Med_Reo_Net Project (a project which aimed to create a Surveillance Networkof Reoviruses, Bluetongue and African Horse Sickness, in the Mediterranean basin and Europe), the 2010 activities were focused on developing molecular diagnostic tests to identify species of Culicoides within the subgenus Avaritia. The results were presented at the WP2 Meeting “Regional surveillance of vectors” held in Tunis, Tunisia, 4th-6th of May 2010.
STEREO2 - EPISTIS – Within the project on remote sensing tools to study the epidemiology and space/time dynamics of diseases sponsored by Ministry for Science Policy of Belgium(STERO II Project), spread models able to characterize and define the vector distribution based on geneticcharacteristics were developed. In collaboration with “l’ Universitè Libre de Bruxelles”, models which investigate the space/time dynamics of C. imicola through the analysis of 7 years of entomological surveillance were set up. Beside l’ Universitè Libre, the project also involves “AVIAGIS”, the Agriculture and Veterinary Information and Analysis. With this partner, information on the soil through the satellite images as well as on the optimal habitat of the C. imicola through the landscape analysis continued.
In collaboration with the Faculty of Veterinary Medicine, Institute of Comparative Medicine, University of Glasgow, Scotland, UK within the project on the “identification of the Molecular determinants of BTV virulence”, studies on the pathogenicity of some field and vaccine BTV strains using mouse model continued.
Within the framework of the ERA-Net on Emerging and Major Infectious Diseases of Livestock EMIDA, the Istituto G. Caporale, together with other 9 dedicated laboratories of 9 different countries, prepared and submitted a proposal on “Combating orbivirus infections of livestock: understanding of the molecular basis for protein function / virus phenotype, molecular epidemiologyand improving diagnostic assays.”. The project has been approved by the EMIDA expert evaluation panel and will start next year.
Together with the Friedrich-Loeffler-Institut, Greifswald-Insel Riems, Germany, and the Institute of Animal Health, PirebrightEngland , blood samples collectedfrom sheep and cattle infected with BTV-8 or BTV-6 at different time from infection, will be tested with various techniques in order to compare the virus isolation methods between the labs.
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 international conferences and meetings
Savini, G., Di Gennaro, A.P., Paoletti, M., Migliaccio,P.,Monaco, F. Cross placental transmission of some bluetongue isolates other than BTV-8. 4th European Congress of Virology, 7-11 April 2010, Villa Erba, Cernobbio, Como, Italy.
Radaelli M.C., Vitale N., Chiavacci L., Masoero L., Goria M., Savini G. 2010 Bluetongue Virus Serotype 8 in Wild Ruminants in North-western Italy. The first Biennial Congress of the EAVLD, Lelystad, Olanda, 15- 17 September 2010
Navillod F.V., D’Ottavio M., Domenis L., Botti V., Robetto S., Mancini M., Mignone W., Bertolla A., Ragionieri M, Savini G., Orusa R., Guidetti C., 2010. Caso di sieropositività da BTV 8 in un lama in Valle d’Aosta. XII Congresso Nazionale SIDiLV. Genova, 27-29 October 2010.
Radaelli M.C., Vitale N., Chiavacci L., Masoero L., Goria M., Ariello D., Pignata L., Savini G. "Bluetongue sierotipo 8 nei cervi del parco regionale della Mandria (Piemonte)". 2010. Atti del XII Congresso Nazionale S.I.Di.L.V. Genova, 27-29 October 2010