Bluetongue
OIE Reference Laboratory Reports
Activities in 2011
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 332440
Fax: / +39 0861 332251
e-mail address: /
website: / www.izs.it
Name (including Title and Position) of Head of Laboratory (Responsible Official): / Rossella Lelli
DVM
Director
Istituto G. Caporale, Teramo, Italy
Name(including Title and Position) of OIE Reference Expert: / Giovanni Savini
DVM, PhD
Head of the Virology Department
Name (including Title and Position) 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 / 19887
VNT / Antibody / Type / 53,418
KC + VERO cell culture
/ Virus isolation / Group / 18ECE+VERO cell culture
/ Virus isolation / Group / 18VERO cell culture
/ Virus isolation / Group / 17VERO cell culture
/ Virus titration / Group / 550Serotype specific PCR real time
/ Virus identification / Type / 33PCR real time
/ Agent identification / Group / 4.161Virus typing
/ Virus identification / Type / 2Microscopic examination
/ Culicoides imicola identification / Species / 4.896PCR
/ Culicoides obsoletus and Culicoides scoticus identification / Obsoletus complexspecies / 364
PCR
/ Culicoides dewulfi: identificazione /Species
/ 1Microscopic examination
/ Other species of Culicoides /Species
/ 406Microscopic examination
/ Culicoides spp. /Species
/ 5.0442. 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 2011, BTV C-ELISA reagents sufficient for 382,050 tests have been produced and supplied to Italian laboratories.
RNA of BTV-16 collected during the 2011 Italian surveillance program together with the BTV-1 typed from Tunisia samples were aliquoted and stocked. They were also cloned and sequenced in order to better understand the isolate origin.
From the 2011 National Surveillance Program 1464 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 / 2ml / 1ml (IRVT, Tunisia)
BTV-2 / 2ml / 1ml (IRVT, Tunisia)
BTV-3 / 0.3ml
BTV-4 / 2ml / 1ml (IRVT, Tunisia)
BTV-5 / 0.3ml
BTV-6 / 2ml / 1ml (IRVT, Tunisia)
BTV-7 / 0.3ml
BTV-8 / 2ml / 1ml (IRVT, Tunisia)
BTV-9 / 2ml / 1ml (IRVT, Tunisia)
BTV-10 / 0.3ml
BTV-11 / 0.3ml
BTV-12 / 0.3ml
BTV-13 / 0.3ml
BTV-14 / 0.3ml
BTV-15 / 2ml / 1ml (IRVT, Tunisia)
BTV-16 / 2ml / 1ml (IRVT, Tunisia)
BTV-17 / 0.3ml
BTV-18 / 0.3ml
BTV-19 / 0.3ml
BTV-20 / 0.3ml
BTV-21 / 0.3ml
BTV-22 / 0.3ml
BTV-23 / 0.3ml
BTV-24 / 0.3ml
BTV reference strains / BTV-1 / 180ml / 1ml (IRVT, Tunisia)
BTV-2 / 180ml / 1ml (IRVT, Tunisia)
25ml (OVI, South Africa)
BTV-3 / 40ml
BTV-4 / 180ml / 1ml (IRVT, Tunisia)
BTV-5 / 40mll
BTV-6 / 180ml / 1ml (IRVT, Tunisia)
BTV-7 / 40ml
BTV-8 / 200ml / 5 ml (University of Scotland, Glasgow, UK)
1.5 (Huzong University, Whuan City, China)
BTV-9 / 190ml / 1ml (IRVT, Tunisia)
0.5ml (University of Scotland, Glasgow, UK)
25ml (OVI, South Africa)
BTV-10 / 40ml
BTV-11 / 40ml
BTV-12 / 40ml
BTV-13 / 40ml
BTV-14 / 40ml
BTV-15 / 180ml / 1ml (IRVT, Tunisia)
BTV-16 / 180ml / 1ml (IRVT, Tunisia)
BTV-17 / 40ml
BTV-18 / 40ml
Type of reagent / Amount supplied nationally
(including for own use) / Amount supplied to other countries
BTV reference strains / BTV-19 / 40ml
BTV-20 / 40ml
BTV-21 / 40ml
BTV-22 / 40ml
BTV-23 / 40ml
BTV-24 / 40ml
Control positive serum for c-ELISA / 1400ml
Control negative serum for c-ELISA / 8000ml
c-ELISA antigen for 382,050 tests / 400ml
VP7 monoclonal antibody / 12ml / 0.5ml (IRVT, Tunisia)
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
a) Establishment and maintenance of a network with other OIE Reference Laboratories designated for the same pathogen or disease and organisation of regular inter-laboratory proficiency testing to ensure comparability of results
In 2011 the activities related to the OIE Bluetongue reference laboratories network (OIE-BTNet), continued. The Istituto G. Caporale (ICT) has managed the Secretariat and has continuously updated the contents of the website (http://oiebtnet.izs.it/btlabnet/). The network membership, which included the OIE Bluetongue Reference Laboratories, OIE collaborating centres (OIE-CC) on Diagnosis, Epidemiology and Control of Animal Diseases in Tropical Regions - CIRAD/EMVT; OIE-CC on Animal Disease Surveillance Systems and Risk Analysis – USDA-APHIS-VS-CEAH; OIE-CC on Veterinary Training, Epidemiology, Food Safety and Animal Welfare – ICT, and members of OIE ad hoc group on bluetongue, .has been expanded and a group of vector experts has been created. This new group included 9 experts from Australia, France, England, Italy, South Africa Spain and US.
In 2011 the Secretariat published two new numbers of Blue Tongue Newsletter which have been distributed to all network’s members. The Newsletter aims to disseminate the most valuable news on BT and stimulate discussion on recent and new aspects related to disease’s epidemiology.
A collaborative study involving all OIE BT reference labs for validating the qRT-PCR, one of the methods most commonly used for BT diagnosis, also started. The method selected for validation was that based on NS3 (segment 10) gene as described by Hoffmann et al. This method was chosen as it is the only qRT-PCR capable of detecting all known BTV serotypes and strains currently circulating. A validation protocol designed based on that used by the Australian labs in their last validation trial has been prepared. Each Lab is using their own isolates to validate the test and, if they have the possibility of getting the 24 original reference strains from the OVI, they will use the South African reference strains for the validation.
b) Organisation of inter-laboratory proficiency testing with laboratories other than OIE Reference Laboratories for the same pathogens and diseases to ensure equivalence of results
The laboratory successfully participated to the Annual 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).
The tenth inter-laboratory proficiency testing for BT ELISA including 37 Italian laboratories was organised. A panel of 30 serum samples were prepared and distributed to the participants. All ring trial records (subscriptions, data submission, final report) were handled through an on-line internet interface and data collection and analysis are currently in progress.
The fifth inter-laboratory proficiency testing for RT-PCR including 13 Italian laboratories and the Institut de la Recherche Vétérinaire de Tunisie (IRVT) was also organised. A panel of 20 blood samples were prepared and distributed to all participants. As for the serology, all ring trial records were handled through an on-line internet interface and data collection and analysis are currently in progress.
4. Preparation and supply of international reference standards for diagnostic tests or vaccines
All 24 reference BTV serotypes and relative antisera are available in the laboratory. Cell lines including VERO, BHK21, BSR, Kc (cells from Culicoides), C6/36, CP TRT are also accessible . The laboratory has the facilities to produce anytime large amount of standard materials depending on request. The laboratory also produces c-ELISA antigen, positive and negative control sera as well as VP7 monoclonal antibody. For details please see table on paragraph 2.
5. Research and development of new procedures for diagnosis and control
Several research projects are currently in progress including:
- Development and standardization of an immunometric assay for the serological diagnose of BT. The goal of the study is to assess a c-ELISA based on recombinant VP7 protein. The novel assay intends to replace the “old” whole virus based-assay. VP7 is a serogroup-specific protein thus being shared by all existing serotypes. Experimental work has been completely done and a final report is currently in preparation.
- Development and validation of a qRT-PCR assay for the rapid detection of BT genome targeting the NS1 gene of all known serotypes from different specimens (blood, internal organs, insects). Experimental work has been successfully completed and a final report is currently in progress.
- The performance of several commercial kits has been evaluated on a panel of sera which included samples from naturally infected animals as well as from animals vaccinated with either inactivated or live modified vaccines. The kits under validation are c-ELISA assay manufactured by VMRD (Pullman, WA USA), a BT one step, and a DIVA assay, that permits the discrimination between vaccinated and infected animals both manufactured by the IDVET Innovative Diagnostics (Montpelllier, France). The performance of a BTV-8 specific ELISA also produced by IDVET was also assessed by using sera from naturally and experimentally infected animals as well as from BTV8 vaccinated animals.
- The laboratory part of the Project on “New expressing vector for the bluetongue virus” has been completed. 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. 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. In 2011, several groups of sheep have been vaccinated with different doses of the new vaccine and challenged with the European BTV-8 strain. Clinical signs were recorded and viraemia monitored. The laboratory testing is still in progress.
- Together with the Friedrich-Loeffler-Institut, Greifswald-Insel Riems, Germany, and the Institute of Animal Health, Pirebright England, blood samples collected from sheep and cattle infected with BTV-8 or BTV-6 at different time from infection, have been tested with various techniques in order to compare the virus isolation methods between the labs.
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).
According to the Italian BT surveillance program, insects collected from nearly 5,044 catches originating from permanent (249) and mobile light traps (24) in a total of 246 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: www.izs.it).
7. Maintenance of a system of quality assurance, biosafety and biosecurity relevant to the pathogen and the disease concerned
The laboratory is accredited according to UNI EN 45001 norms, now to ISO/IEC 17025 by SINAL (National System for the Accreditation of Test Laboratories) since 1995. All procedures from the reagent preparation to the diagnostic test execution are performed in accordance to the ISO 17025 and the compliance is yearly controlled by the National Authority for the accreditation of testing laboratories.
In 2011, the Institute “G. Caporale” has completely restructured the BSL3 laboratories. Procedures on infected material manipulation, appropriate use of instruments and worker behaviour have been prepared by ICT personnel and approved by the quality assurance and bio-safety Units of the Institute. An insect proof facility with HEPA filtered air stream has also been built in order to perform trials on vector borne diseases.
8. Provision of consultant expertise to OIE or to OIE Member Countries
Personnel of the OIE reference lab:
ü participated, as invited speaker, to the Workshop on Bluetongue, organised by the European Commission's DG Enlargement within the framework of the TAIEX Instrument (44065) on the 7th-8 th of June, Kiev, Ukraine. Lecturers on “BTV: clinical signs, anatomo-pathological lesions and differential diagnosis” and on “BTV: vaccination strategy and overview of vaccines” were presented.
ü participated, as invited speaker, to the Workshop on Modern field diagnostic on Bluetongue organised by the European Commission's DG Enlargement within the framework of the TAIEX Instrument (45097) on the 4th – 5th of October, Pokrov, Russia. Lecturers on “Experience obtained from Bluetongue outbreaks and surveillance: the example of Italy”, on “Control of Bluetongue in the EU: vaccination strategy and overview of vaccines” and on “Laboratory diagnosis of Bluetongue: current status” were presented.
ü Was 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 opinion of the panel has been published in Arjan Stegeman (chair), Annette Bøtner, Giovanni Savini, Jordi Casal, Rudolf Meiswinkel, Yves van der Stede, Jean-Philippe Amat and Andrea Gervelmeyer, Sofie Dhollander, Diane Lefebvre, Francesca Riolo and Jane Richardson. 2011. Scientific Opinion on bluetongue monitoring and surveillance, EFSA Journal 2011;9(6):2192 [61 pp.]. doi:10.2903/j.efsa.2011.2192
ü was invited to be a member of the ad-hoc Scientific Advisory Group (SAG) - Bluetongue vaccine, organised by the European Medicines Agency to analyse and discuss the results of a commercial BTV-8 inactivated vaccine (Bovilis), London, UK, 7th of February 2011.
ü participated to the to the EU Workshop on “Annual meeting of National Reference Laboratories for bluetongue”, Bruxelles, Belgium, 28-29 November 2011, presenting data on the “Italian situation in 2011 and "BTV-8 vaccination: the Italian experience”.,
ü reviewed the Bluetongue chapter of the “Manual of Diagnostic Tests and Vaccines for Terrestrial Animals (Terrestrial Manual)”.
9. Provision of scientific and technical training to personnel from other OIE Member Countries