Swine vesicular disease e
Activities in 2009
Swine vesicular disease
Emiliana Brocchi
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: emiliana.brocchi@izsler; website:
Summary of general activities related to the disease
1.Test(s) in use/or available for the specified disease
Test / For / Specificity / Surveillance& DiagnosisELISA 1) / Antibody / (screening) all antibodies / 144981a)
IgG-specific ELISA2) / Antibody / IgG class / 1760
IgM-specific ELISA2) / Antibody / IgM class / 1760
VNT / Antibody / neutralizing antibody / 1762
IB-RS2 cell culture3) / Virus isolation / SVD live virus / 285 b)
RT-PCR / Genome / SVDV 3D-fragment (all genotypes) / 2460
Sandwich ELISA2) / Antigen / SVDV (all antigenic variants) / 30 c)
Sequencing / Molecular analysis / 3D / 12
1) 5B7 Monoclonal antibody-based competitive ELISA (prescribed test for screening)
2) Sandwich ELISA with monoclonal antibodies of selected reactivity
a)In addition, approximately 300,000 further samples were analysed by other regional laboratories in compliance of the national surveillance programme using ELISA kit supplied by the National/OIE reference laboratory.
b) Mainly to monitor experimental infections
c) Applied forSVDV identification in cell cultures showing cytopatic effect
A total of18 SVD outbreaks, including primary and secondary outbreaks, were notified in the world during 2009, all of them in Italy.
2.Production and distribution of diagnostic reagents
All diagnostic reagents used to perform ELISA assays for antibody and antigen detection as described above were developed and produced within the laboratory.
Furthermore, during 2009, kits of 5B7-competitive ELISA (prescribed test for antibody screening) have been supplied on national basis to 10 Regional laboratories in sufficient amounts for testing of300,000 sera, to accomplish the national serosurveillance program for SVD.
Further diagnostic reagents for the 5B7-competitive ELISA were supplied to EstoniaandPoland.
Twenty-five SVDV-positive field samples (faeces homogenate) from different cases and years were supplied to The Netherlands, as reference materials for validation of in-house RT-PCR.
Thirty-two Italian isolates (collected from end of 2007 to end of 2008) have been provided to the Institute for Animal Health, Pirbright.
A sample of inactivated SVDV antigen, used in the 5B7-competitive ELISA, has been supplied to Cedi-Prionics to evaluate its suitability as antigen in the commercial kit.
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
As National Reference Centre for SVD, the laboratory organised the annual national inter-laboratory test, involving the participation of 12 Italian regional Institutes. .The proficiency panel was composed by 84 field sera. Objectives of 2009 ring-test were to evaluate the harmonisation/performance of the 5B7-competitive ELISA carried out in the different laboratories and to introduce the use of a single-dilution (1/7.5) instead of two dilutions (1/7.5 and 1/22.5). The use of single dilution in ELISA screening was adopted at the NRL during the laboratory emergency in2007 in order to increase the Laboratory throughput. The preliminary analysis of results indicated an adequate performance of all laboratories that enablestesting of a single dilution also in Regional Laboratories, maintaining accuracy of results.
The laboratory participated at the 2009Inter-laboratory proficiency testing exercise,that is annuallyorganised by the European Reference Laboratory (CRL) as one of its duty. Proficiency panels of samples, including sera, epithelium and faecal suspensions were examined by serological assays (VNT, 5B7-competitive ELISA, IgG/IgM specific ELISAs) and by virological assays (ELISA for antigen detection, virus isolation and PCR) respectively.
4.Preparation and supply of international reference standards for diagnostic tests or vaccines
Secondary Standards equivalent to EU Primary Standards for SVDV serology were provided to all laboratories that received kits or reagents for 5B7-competitive ELISA.
5.Research and development of new procedures for diagnosis and control
During 2009 a one-step RT-PCR, previously validated by comparison with the two-step RT-PCR described in the OIE Manual, replaced the two-step RT-PCR procedure for the routine testing of 2460 faeces homogenates, that were analysed in compliance with the SVD surveillance plan in progress in Italy. For both tests RNA extraction was obtained by a preliminary immune-purification of the SVD virus mediated by the monoclonal antibody 5B7. Studying analytical sensitivity, the one-step RT-PCR proved to be 1 log10 more sensitive than the two-step procedure, maintaining identical specificity.
A study to evaluate diagnostic performances of a recently described LAMP test (Loop mediated isothermal amplification) for SVDV genome amplification has been undertaken. This test is highly specific and efficient;the amplification is performed under isothermal conditions, it doesn’t need special reagents nor sophisticated equipments and is very fast (1 hour at 63°C).The adopted procedure uses three primers pairs, the target sequence is a portion of 163 bp of the 3D gene that is amplified as a long-stem loop; the product can be visualized by gel electrophoresis or by naked eye upon addiction of an intercalating dye. Detection limit of LAMP and one-step RT-PCR resulted similar, both tests easily detect 20 TCID50; however one-step PCR may be slightly more sensitive, since a weak positive result was obtained also with 2 TCID50 in one replicate.From preliminary results with faeces homogenates from experimental infections, the LAMP assay appeared to be moderately less sensitive in biological samples than the one step PCR, but consistently more sensitive than VI.
The screening tests used for SVD antibodies (5B7-competitve ELISA) is continuously submitted to Step 5 of the OIE validation process, being applied on a routine basis for the SVD surveillance plan in progress in Italy. The use of single dilution in ELISA screening was adopted at the NRL during the laboratory emergency in2006-2007 in order to increase the Laboratory throughput. Results of the 2009 national ring-test indicated an adequate performance of all participating laboratories, enabling the use of testing at single dilution also in Regional Laboratories, maintaining accuracy of results.
A study to evaluate the duration of maternal immunity has been carried out by experimental infection of pregnant saws and by monitoring the presence and the titre of antibodies in sequentially collected blood samples of born piglets. Data from the first group of experimental animals indicate that passive immunity persists up to six months.
6.Collection, analysis and dissemination of epizootiological data relevant to international disease control
Data and epidemiological information relevant to SVD outbreaks occurred in Italyand results of the 2009 surveillance program were regularly reported to the European Commission and OIE and presented at the annual meeting of the EU National Reference Laboratories for SVD.
Molecular epidemiology analysis, based on genomic sequencing of 2009 isolates,provided evidence of the simultaneous presence of two sub-genomic lineages, one evolved from previous Italian isolates and the other closely related to the isolates responsible for the outbreaks in Portugal in 2003, 2004 and 2007, the latter was firstly detected in Italy in 2004.
A dedicated Integrated Information System is maintained for the collection and analysisof data of the surveillance activities for SVDin Italy ( It provides an archive of laboratory results ofthe last five years.
7.Provision of consultant expertise to OIE or to OIE Members
Nothing to report
8.Provision of scientific and technical training to personnel from other OIE Members
A two-month training on SVD and FMD laboratory diagnosis was provided to Dr. Than Aye from Myanmar, supported by IAEA.
9.Provision of diagnostic testing facilities to other OIE Members
No request was received during 2009.
10.Organisation of international scientific meetings on behalf of OIE or other international bodies
The laboratory reported on the epidemiological situation in the country, on the control measures adopted, on diagnostic and research activity performed at the annual meeting of National SVD Reference Laboratories of EU.
11.Participation in international scientific collaborative studies
A standing collaboration is maintained with the Institute for Animal Health (UK) for the characterization of SVDV isolates (virulence, genomic and antigenic variation, molecular epidemiology and evolution).
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
Silvia Bellini, Loris Alborali, Vittorio Bonazza, Dominga Avisani, Giorgio Zanardi. Swine vesicular disease in Lombardy region: pattern of spread in a high density pig area. International Meeting on Emerging Diseases and Surveillance (IMED 2009). Vienna, 13-16 February 2009, p.96.
Grazioli S., E. Brocchi. Swine Vesicular Disease in Italy during 2009: Epidemiology and Surveillance, Laboratory and Research activity report. Annual meeting of EU national SVD laboratories, Brussels, 18January 2010.
Pezzoni G., Grazioli S., Bugnetti M., Brocchi E. Evaluation of a loop-mediated isothermalamplificationassay (LAMP) for detection of swine vesicular disease virus. Annual meeting of EU national SVD laboratories, Brussels, 18 January 2010.
Scientific publications in peer-reviewed journals
S. Bellini, L. Alborali, G. Zanardi, D. Avisani, V. Bonazza & E. Brocchi. Swine Vesicular Disease in northern Italy, diffusion in densely populated pig areas. Scientific and Technical Review, accepted
Martín-Acebes MA, González-Magaldi M, Rosas MF, Borrego B, Brocchi E, Armas-Portela R, Sobrino F.Subcellular distribution of swine vesicular disease virus proteins and alterations induced in infected cells: acomparative study with foot-and-mouth disease virus and vesicular stomatitis virus. Virology (2008); 374(2):432-43
Other communications (national journals/events)
Silvia Bellini, Paolo Cordioli e Stefano Cinotti. Malattia vescicolare del suino e criticita’ del comparto suinicolo.Summa Animali da Reddito, N° 3, 31 – 35, Aprile 2009.
C. Nassuato, D. Avisani, G. Zanardi, M. Cerioli, S. Bellini. Stima dei tassi di trasmissione della malattia vescicolare del suino durante l’epidemia 2006-2007.
Workshop nazionale di epidemiologia veterinaria. Abstract book pp 73. Torino, 10-11 Dicembre 2009.
Maresca C., Bartoccioni S., Bellini S., Costarelli1 S., De Curtis M., Faccenda L., Ferrarini N., Flamini A. R., Pauselli G. B., Scoccia E., Scorcelletti S., Cenci T. Swine vesicular disease, updates to December 31, 2008 - Webzine Sanità Pubblica Veterinaria: Numero 56, Ottobre 2009 [
Costarelli S,Biasini G, FaccendaL,Grazioli S, Mariotti C,Marchi S, Scoccia E, Sensi M,Maresca C.Malattia vescicolare del suino: epidemia in Umbria e Marche 2008-2009. XI Congresso Nazionale SIDILV, Parma 22-24 ottobre 2009, p. 122-123
Lessons on SVD laboratory diagnosis, SVD laboratory contingency plan, disease and diffusion routes, epidemiology and epidemiological investigation, etc. at specific courses for post-graduated students, field veterinarians, laboratory experts.
13.Inscription of diagnostic kits on the OIE Register
i)Did you participate in expert panels for the validation of candidate kits for inscription on the OIE Register? If yes, for which kits?
Yes, but not for SVDV diagnosis
ii)Did you submit to the OIE candidate kits for inscription on the OIE Register? If yes, for which kits?
No (ELISA for antibody detection and RT-PCR tests described in the Manual were developed at the reporting OIE laboratory)
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Annual reports of OIE Reference Laboratories and Collaborating Centres, 20091