OIE Reference Laboratory Reports s14

OIE Reference Laboratory Reports Activities in 2011

Name of disease (or topic) for Swine vesicular disease which you are a designated OIE Reference Laboratory:

Address of laboratory: Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia Romagna (IZSLER) Via A. Bianchi 7/9, Brescia, ITALY

Tel.: (+39 030) 2290310

Fax: (+39 030) 2290369

e-mail address: [email protected]

website: www.izsler; www.cerves.it

Name (including Title and Dr. Stefano Cinotti Position) of Head of Laboratory IZSLER General Director (Responsible Official):

Name(including Title and Dr. Emiliana Brocchi Position) of OIE Reference Head of the National Reference Centre for Expert: Vesicular Diseases Head of the Dept of Biotechnology

Name (including Title and Position) of writer of this report (if different from above):

Annual reports of OIE Reference Laboratories and Collaborating Centres, 2011 1 Swine vesicular disease

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

Surveillance& Test For Specificity Diagnosis

ELISA 1) Antibody (screening) all antibodies 100,738 a)

IgG-specific ELISA2) Antibody IgG class 1,135

IgM-specific ELISA2) Antibody IgM class 1,135

VNT Antibody neutralizing antibody 1,174

IB-RS2 cell culture Virus isolation SVD live virus 99

RT-PCR Genome SVDV 3D-fragment (all genotypes) 2,614

Sandwich ELISA3) Antigen SVDV (all antigenic variants) 58

Sequencing Molecular analysis 3D 6 b) IRES 16 c)

1) 5B7 Monoclonal antibody-based competitive ELISA (prescribed test for screening) 2) Isotype specific indirect ELISA, using conjugated anti-swine IgG and anti-swine IgM monoclonal antibodies 3) Sandwich ELISA based on monoclonal antibodies. a) In addition, approximately 270,000 further samples were analysed by other regional laboratories in compliance of the national surveillance programme using ELISA kits supplied by the National/OIE reference laboratory. b) N. 6 sequences of representative isolates from outbreaks occurred in 2011. c) Including isolates from 2011 and from previous years.

2. Production and distribution of diagnostic reagents

Amount supplied nationally Amount supplied to other Type of reagent (including for own use) countries

Kit for antibody detection ELISA for testing of 506,000 samples Poland: for testing of 8,600 samples

5B7 catching monoclonal Ab 23 ml Russia, Canada, Taiwan, Belgium

5B7 peroxidase-conjugated MAb 20 ml Russia, Canada, Belgium

SVDV inactivated antigen 400 ml Private Company for kit preparation

All diagnostic reagents used to perform ELISA assays for antibody and antigen detection as described above were developed and are regularly produced within the laboratory. During 2011 assembled reagents to perform 5B7- competitive ELISA (prescribed test for antibody screening) have been supplied on national basis to 11 Regional laboratories in sufficient amounts for testing of 403,000 sera, while reagents for further 105.000 tests were used by the laboratory itself to accomplish the national serosurveillance program for SVD. Diagnostic reagents for the 5B7-competitive ELISA were also supplied to Russia, Canada, Taiwan, Belgium as specified above. In addition, inactivated SVDV antigen, has been supplied to a private Company for introduction into a commercial kit for antibody detection ELISA.

2 Annual reports of OIE Reference Laboratories and Collaborating Centres, 2011 Swine vesicular disease

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

There are only two OIE Reference Laboratories for SVD in the world. The two laboratories have been collaborating for almost 20 years on different aspects of the disease and the virus.

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 participated at the 2011 combined proficiency testing exercise for foot-and-mouth disease and swine vesicular disease that is annually organised by Pirbright Laboratory of the Institute for Animal Health to fulfil its function as the European Union Reference Laboratory for FMD and SVD and the FAO World Reference Laboratory for FMD. The international ring trial was addressed to all NRL of the European Community as well as to countries of strategic importance for EU, in addition other interested laboratories are invited to participate. The scope of the exercise was to monitor the proficiency of all serological and virological assays currently used for SVD and FMD diagnosis, including differential diagnosis and case interpretation. Samples included a panel of sera that were tested by VNT, 5B7- competitive ELISA and IgG/IgM specific ELISAs, as well as epithelium and faecal suspensions, that enabled monitoring of ELISA for antigen detection, virus isolation and different genome amplification procedures. The OIE reference expert of the laboratory participated as a member at the Advisory Board for the SVD/FMD 2011 proficiency testing scheme.

 As National Reference Centre for SVD, the laboratory maintains supervision on the performance of 11 Italian regional Laboratories that regularly carry out the 5B7-competitive ELISA for the national surveillance programme. Monitoring is maintained through organisation of national inter-laboratory trials and confirmation of positive results. Testing of a single serum dilution (1/7.5) instead of two dilutions (1/7.5 and 1/22.5) was adopted for the screening test in all regional laboratories, after demonstration of the accuracy and harmonisation of results elaborated from a panel of 84 representative sera, examined in a national ring test. A new panel of 25 sera has been prepared for the ring trial planned in 2012.

 Since vaccination against SVD is not practised, no need for vaccine production and quality control exists.

4. Preparation and supply of international reference standards for diagnostic tests or vaccines

A weak positive reference standard serum, called EU-RS4, has been established as a weak positive serum that must be detected by any serological test; this primary standard is available at the EURL. A Secondary Standard equivalent to the EU-Primary Standard is available at this OIE Reference laboratory; it was regularly provided to all laboratories that received kits or reagents for 5B7-competitive ELISA.

Reference materials are also the immunological components (catching and conjugated 5B7 Monoclonal Antibody) and the two additional internal controls (strong positive and negative serum controls) that are systematically produced and supplied to perform the OIE prescribed screening ELISA for antibody detection. These materials could constitute an international bank of diagnostic reagents.

In addition, the OIE reference laboratory has gathered during several years a collection of known field positive sera and field positive faeces samples from SVD outbreaks, as well as SVDV isolates, that represent a biobank of unique reference materials strategic for validation of new diagnostic tests.

5. Research and development of new procedures for diagnosis and control

Phylogenetic analyses, based on 3D sequences of SVDV isolates, provided evidence that two sub-genomic lineages, one evolved in Italy and the other related to isolates from outbreaks occurred in Portugal in 2003 and 2007, are co-circulating in Italy. A recent study to compare diagnostic performances of SVDV genome amplification methodologies (described in literature but not validated with field samples) showed that only the in-

Annual reports of OIE Reference Laboratories and Collaborating Centres, 2011 3 Swine vesicular disease

house conventional one-step RT-PCR, carried out with primers described in the OIE Manual, reliably detects all SVDV genomic variants currently circulating. In contrast, RT-LAMP and the Real-Time PCR performed with 3- IR primers-probe sets were unable to amplify genomes of the Portuguese sub-lineage, while the 2B-IR Real-Time PCR missed some samples of the Italian sub-lineage. This observation has been confirmed with isolates collected in 2011. Since the most reasonable cause of this failure is mismatching within primer sequences and considering the potential advantages of Real-Time over conventional PCR, a new study has been undertaken for the development of a Real-Time PCR test able to amplify the different genomic variants with equal efficiency. The approaches adopted are focused both on improvement of the 2B-IR Real-Time PCR sensitivity by designing and evaluating degenerated primers, and on development of a new Real-Time PCR test based on more conserved target regions, like the 3D gene; 3D sequences produced from many recent and representative SVDV isolates will be useful for the design of the best suited primer-probe set.

A field study was conducted to evaluate the risk that products derived from pigs of a farm where an outbreak has been detected may be infected, becoming a potential source of disease spread. Samples of meat, as well as blood and faeces, were collected from animals during stamping out procedures in two outbreaks. Results showed that, although limited, the presence of animals with viraemia, harboring SVD virus in meat at the moment of the stamping out, cannot be excluded, as proven by the detection of infectious virus in meat samples of 2 out of 40 tested pigs, in one of the two outbreak-farms analyzed.

Finally, the OIE prescribed screening tests, respectively used for SVDV antibodies (5B7-competitve ELISA) and for genome detection in faeces (one-step RT-PCR) are submitted to a continuous validation, in compliance with the OIE validation process, being extensively and on a routine basis applied for the SVD surveillance plan ongoing in Italy. This led to implement tests modifications that improved throughput and/or performance: namely, the use of a single serum dilution instead of two in the serological ELISA screening and the replacement of the two-steps RT-PCR procedure with a one-step test that proved to be more sensitive and practicable. These modifications will be proposed for insertion into the specific chapter of the OIE diagnostic Manual.

6. Collection, analysis and dissemination of epizootiological data relevant to international disease control

The disease is officially reported only from Italy. During 2011 twenty-five SVD outbreaks were notified in Italy. Data and epidemiological information relevant to SVD outbreaks occurred in Italy and results of the national surveillance plan were regularly reported to international bodies (EU Commission, OIE) and presented at the annual meetings of the EU National Reference Laboratories for SVD. A dedicated Integrated Information System is maintained for the collection and analysis of data of the surveillance activities for SVD in Italy (www.cerves.it). It provides an archive of laboratory results since 2000.

Phylogenetic analyses, based on 3D sequences of SVDV isolates, provided evidence of the simultaneous circulation in Italy of two genomic sub-lineages (called “Italian” and “Portuguese”, due to their origin) up to 2010. However, all SVD viruses analyzed from 2011 belong to the same (Italian) sub-lineage. Molecular epidemiology, based on nucleotide sequence similarities between isolates, provided a useful tool to integrate traditional epidemiological investigations to trace outbreaks origin.

7. Maintenance of a system of quality assurance, biosafety and biosecurity relevant to the pathogen and the disease concerned

The OIE reference laboratory is accredited according to UNI/EN ISO/IEC 17025 provisions.

Laboratories structure where live SVDV is handled comply with the provisions of the “Minimum standards for laboratories working with FMDV in vitro/in vivo”, as adopted by the 38th General Session of the European Commission for the Control of Foot-and-mouth Disease (EuFMD), 2009. These laboratories correspond to OIE biosecurity level 4, with the exception of single HEPA filtration for exhaust air and not filtered input air. Diagnostic tests using inactivated antigens and safe reagents are conducted in dedicated BSL-2 laboratories.

8. Provision of consultant expertise to OIE or to OIE Member Countries

The OIE reference expert represented the OIE at the annual meeting of SVD EU National Reference Laboratories, on 5th May 2011 and provided the mission report to OIE.

4 Annual reports of OIE Reference Laboratories and Collaborating Centres, 2011 Swine vesicular disease

Consultancy was provided in occasion of an inspection visit in Italy by MAPA (Brasil) representatives to check the capability of early detection and control of SVD, assuring safe exportable products (July 2011). The laboratory participated with one member to an EFSA working group of experts, constituted in 2011 to provide a scientific opinion to the EU Commission on the current epidemiological situation with regard to SVD and its impact on susceptible livestock, with scientific advice for better targeted and modulated surveillance and proportionate protection measures.

9. Provision of scientific and technical training to personnel from other OIE Member Countries

Taking into consideration that SVD is notified only in Italy, technical training was requested and provided only within the country. Two workshops were co-organised with regional Institutes of Campania and Sicilia regions (24th February and 16th-17th November respectively) concerning “Field control and eradication of an SVD outbreak and implementation of biosecurity measures”.

10. Provision of diagnostic testing facilities to other OIE Member Countries

Confirmatory testing to clarify sporadic serological reactivities (singleton reactors), ended as false-positives, was performed upon request by the Switzerland NRL.

11. Organisation of international scientific meetings on behalf of OIE or other international bodies

Nothing to report.

12. Participation in international scientific collaborative studies

There is no formalized international project ongoing on SVD, however a standing collaboration is maintained with the other OIE Reference Laboratory (Institute for Animal Health, UK), with exchange of data and expertise regarding characterization of SVDV isolates (virulence, genomic and antigenic variation, molecular epidemiology and evolution) and development/validation of new diagnostic tools.

13. 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

Bellini S, Nassuato C, Grazioli S, Martinelli N, Bugnetti M, Eblé P, Dekker A, Lombardi G. Duration of passive immunity for swine vesicular disease virus in piglets born from experimentally infected pregnant. 5 th annual meeting Epizone : "Science on alert" : 11-14 April, 2011, Arnhem, The Netherlands. Abstract book, p 22.

Nassuato C, Boender GJ, Avisani D, Alborali G. Spatial transmission of Swine Vesicular Disease virus in 2006- 2007 epidemic in Lombardy. 5th annual meeting Epizone : "Science on alert" : 11-14 April, 2011, Arnhem, The Netherlands. Abstract book, p 30.

Grazioli S., Brocchi E. Swine Vesicular Disease in Italy during 2010: Epidemiology and Surveillance. Annual meeting of EU national SVD laboratories, Brussels, 5th May 2011.

Benedetti D., Pezzoni G., Grazioli S., Brocchi E. Comparative performance of three genome amplification assays for the detection of SVD virus in experimental and field samples”. Annual meeting of EU national SVD laboratories, Brussels, 5th May 2011.

Alborali L. “Biosecurity program and compartimentalization in Europe surveillance programs”. Biosecurity International Conference, Federation of Veterinarians of Europe, 2nd June 2011, Brescia, Italy.

______

Annual reports of OIE Reference Laboratories and Collaborating Centres, 2011 5