OIE Reference Laboratory Reports
Activities in 2011
Name of disease (or topic) for
which you are a designated OIE
Reference Laboratory:
Address of laboratory:
Rabbit haemorrhagic disease
Istituto Zooprofilattico Sperimentale della Lombardia ed Emilia
Romagna
Via Bianchi 9
25124 Brescia
ITALY
Tel.:
00390302290617
Fax:
00390302290559
e-mail address:
website:
Name (including Title and
Position) of Head of Laboratory
(Responsible Official):
Name(including Title and
Position) of OIE Reference
Expert:
Name (including Title and
Position) of writer of this report
(if different from above):
lorenzo.capucci@izsler.it
www.izsler.it/Referenza/CREMAVILA/CREMAVILA
Prof. Stefano Cinotti – IZSLER General
Director
Lorenzo Capucci DBR – Head of the
Proteomic division
Lorenzo Capucci
in collaboration with Antonio Lavazza
(responsible for Electron Microscopy
laboratory and National Reference
Centre for Lagomorphs Viral Diseases)
Annual reports of OIE Reference Laboratories and Collaborating Centres, 2010
1
Rabbit haemorrhagic 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
Virological Tests
Tests used for routine diagnostic work:
- Sandwich ELISA test using RHDV specific Monoclonal Antibody (MAb). The specificity of the test can be
enlarged including MAbs produced and specific for the subtype RHDVa. A similar test using specific EBHSV
MAbs is used for diagnosis of EBHS.
- Western Blot Analysis using RHDV-MAbs cross-reactive with EBHSV. It is usually performed on those few
samples giving doubtful results in ELISA test, i.e. in animals died due to the "chronic" form of the disease and
in which the presence of specific antibodies interfere with the ELISAs test. In order to gain in sensitivity, the
analysis is usually performed on samples previously concentrated 10-20 times by ultracentrifugation.
- Reverse transcription Polymerase Chain Reaction (RT-PCR): we have now introduced the reaction as routine
test on samples that resulted negative or not conclusive in ELISA test. The test is also used in those situations
characterized by the suspected presence of a non-pathogenic calicivirus related to RHDV. In order to
distinguish among the main different RHDV genetic variants, different primers have been designed and
validated (RHDV, RHDVa, the recently emerged RHDVFra10 and same minor variants identified in Italy
during 2011).
Additional test used for particular investigation:
- Negative staining ImmunoElectronMicroscopy (including ImmunoGold) and immunoperoxidase on thin
sections using both MAbs and rabbit and hare hyperimmune sera.
Serological Tests
Tests used for routine diagnostic work:
- Competition ELISA; two different tests, based on specific MAbs used as tracer, have been set up respectively
for RHDV and EBHSV.
- ELISAs developed using anti-isotype MAbs to test the sera for the presence of specific anti-RHDV IgM, IgA
and IgG isotypes.
Additional Tests used for particular investigations:
- Indirect ELISA with the purified RHDV adsorbed to the solid phase.
Test
2.
Specificity
Total
ELISA
Antibody
RHDV
891
ELISA
Antibody Isotypes (IgM-IgA-IgG)
RHDV
1052
ELISA
Antigen
RHDV
223
Western blotting
Antigen
RHDV
15
RT-PCR
Antigen
RHDV
153
ELISA
Antibody
EBHSV
1089
ELISA
Antigen
EBHSV
340
Western blotting
Antigen
EBHSV
16
RT-PCR
Antigen
EBHSV
17
Production and distribution of diagnostic reagents
-
2
For
RHDV MAbs,
EBHSV MAbs
RHDV as liver homogenate or semi-purified antigen “BS89 classical strain”
RHDV BS89 as antigen expressed in the baculovirus system
RHDV as liver homogenate or semi-purified antigen “RHDVa” variant strain
Anti-RHDV and anti-EBHSV hyperimmune sera
Annual reports of OIE Reference Laboratories and Collaborating Centres, 2010
Rabbit haemorrhagic disease
ELISA reagents for virological and serological diagnosis of RHD and European Brown Hare Syndrome (EBHS),
including internal ELISA controls (negative and positive) sent as “kit”. A total of 209 kits were supplied to various
Italian Istituti Zooprofilattici Sperimentali (Roma, Torino, Padova, Sassari, Perugia), to company that imports
hares from abroad, i.e. mainly Eastern European Countries (Poland, Hungary) and to other OIE Member Countries
(Germany, United States). Kit for the Isotype serological RHD ELISA were supplied to Australia as scientific
collaboration.
Type of reagent
Amount supplied nationally
(including for own use)
Amount supplied
to other countries
RHDV serological kit (150 samples)
21
3
EBHSV serological kit (150 samples)
22
15
RHDV-EBHSV virological kits (70 samples)
45
4
RHDV Isotype ELISA (150 samples)
7
106
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
No particularly activity this year on this item.
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
The OIE Reference Laboratory for RHD is just one.
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
Actually we received just one request two years ago on this item. This is presumably due to the fact that
rabbit is considered a minor species and it has a relative commercial value only in a limited number of
countries. For 2012 we are planning to propose an inter-laboratory proficiency test for RHDV and EBHSV
serology to those laboratories that asked us diagnostic reagents in the previous years.
4.
Preparation and supply of international reference standards for diagnostic tests or vaccines
We have produced a panel of RHD reference sera (negative, low positive and positive) according to the OIE Guide
3 (International Reference Antibody Standards for Antibody Assays). However, due to some different reasons, we
still have to end the characterization of the sera and to prepare the documentation to be submitted for the approval
of the sera as “OIE reference sera for RHD” to the OIE Standards Commission. We have planned to end the
process within April-May 2012.
5.
Research and development of new procedures for diagnosis and control
We have worked on the following main areas:
1.
Going on with the standardization of RT-PCR for diagnosis of RHDV, particularly in relation to the
specificity toward the several RHDV variants now circulating in the field and to the RHDV-related not-
Annual reports of OIE Reference Laboratories and Collaborating Centres, 2010
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Rabbit haemorrhagic disease
2.
3.
4.
6.
pathogenic rabbits calicivirus. Towards the end of 2011 we also started the standardization of real time PRC
in order to be able to quantify RHDV and related virus in samples of different origin.
The main work in 2011 has been the characterization of the new RHDV variant Fra10 (Detection of a new
variant of rabbit haemorrhagic disease virus in France, Veterinary Record, February 5, 2011 pp.137-138,
doi: 10.1136/vr.d697). At first we worked on the strain RHDVFra10-1032 received from Dr. Ghislaine Le
Gall-Reculé, French Agency for Food, Environmental and Occupational Health & Safety (Anses), on the
basis of a scientific collaboration that is still going on. This collaboration started on the end of February, as
also communicated with a letter to Prof. Vallant – OIE Paris. After few months (in June) we had the first (and
unique) identified outbreak of RHDVFra10 in north-east Italy in two farms epidemiologically lnked. This
allowed to us to work directly on the field following the evolution of the case. In this contest we are studying
the degree of pathogenicity of RHDVFra11 and updating the diagnostic methods. Therefore we have
produced a new panel of MAbs using the Italian isolate of RHDVFra10 and we are characterising them in
order to use in ELISA for a first differential diagnosis and for a fine antigenic characterization. In addition,
considering the consistent genetic and antigenic distance between RHDVFra10 and other RHDV variants, we
hope to use MAbs also to develop a specific serological ELISA.
A work similar to that above described for RHDVFra has been done also for a minor variant identified in
Italy in 2011 (RHDVRome11). Also for this variant we have produced new MAbs. To underline that this
variant is related to the original RHDV and therefore the current available vaccine are able to protect rabbits
from RHDVRome11.
Using the RHDV MAb panel we have antigenically characterised several further RHDV isolates.
Collection, analysis and dissemination of epizootiological data relevant to international disease
control
2011 has been characterised by the identification of RHDVFra10, an RHDV variant that could be classified as
close to a new serotype. However, in spite the studies above described, doubtful persist on the degree of
pathogenicity of the variant that does not seem to be the same of the previous RHDV isolates. Scientific articles
are in preparation to communicate to the international contest the RHDVFra characteristics also for the
epizotological side.
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.
All the different types of laboratory activities involving RHDV and EBHSV (classified among the agents of Risk
Group 2), including the experimental infection of rabbits, are carried out by keeping at the minimum the risk to the
health of the staff (biosafety) and the environment (biocontainment). Actually all the indications listed in the OIE
Manual – Chapter 1.1.2 – Paragraph C are applied at laboratory level. Animal infections are carried out in isolated
rooms in BL3 facilities. Personnel involved in working with RHD is constantly updated on the correct protocols to
be used and on the risks related to the different operations. Personnel is also informed on the risks related to the
handling of specimens, especially when wild hares are involved, that may be positive for other agents than RHDV
or EVHSV (i.e. Tularemia).
8.
Provision of consultant expertise to OIE or to OIE Members
1.
2.
9.
We collaborated with the Health Italian Ministry, General Direction of Veterinary Service and Friuli Venezia
Giulia Regional Veterinary Service in occasion of the sanitary control of the first outbreks caused by the
RHDVFra10 variant in Italy, occurred on June-July 2011.
We collaborated with Carissa Embury-Hyatt DVM, MVSc, Head Pathology of the Canadian Food Inspection
Agency, National Centre for Foreign Animal Disease, Winnipeg, Manitoba and debated with her on the
occurrence of the first RHDV case occurred in Canada.
Provision of scientific and technical training to personnel from other OIE Members
We did not receive any request on this item during 2010.
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Annual reports of OIE Reference Laboratories and Collaborating Centres, 2010
Rabbit haemorrhagic disease
10. Provision of diagnostic testing facilities to other OIE Members
3.
4.
5.
6.
Statens Serum Institut SSI Diagnostica - Allerød DK-3450, Dr Erik Wedø sent us aproximately 30 rabbit sera
for RHDV serology.
Department of Virology, Immunbiology and Parasitology Laboratory - National Veterinary Institute Uppsala, Sweden sent us 4 rabbit sera for RHDV serology.
We received some organ samples (liver and spleen) from Malta for RHDV virological diagnosis
We tested a group of rabbit sera sent by the Laboratório de Doenças Infecciosas, ICBAS/UPR. Vairao,
Portugal for the detection of antibodies for RHDV (and Myxomatosis).
Some other diagnostic tests were also performed for those Institutes with whom we are collaborating (see point °
11)
11. Organisation of international scientific meetings on behalf of OIE or other international bodies
No activity on this item.
12. Participation in international scientific collaborative studies
We are collaborating with:
▪
▪
▪
▪
▪
▪
▪
the Instituto de Investigación en Recursos Cinegéticos, Ciudad Real, Spain in an epidemiological survey of the
hare wild population in the Navarra region. We analysed approximately one hundred sera with the EBHSV
ELISA.
the Elizabeth Macarthur Agriculture Institute, Woodbridge Rd, Menangle NSW – Australia in project that
characterizes several RHDV isolates from different countries in relation to their antigenic and genetic profile
and degree of pathogenesis. We are involved in the antigenic characterization using the MAb panel produced
and studied at our laboratory.
the Zoo and Wildlife Medicine Research Institute of Wildlife Ecology University of Veterinary Medicine of
Wien (Austria) for the detection of anti-EBHSV antibodies in captive and wild hare populations
the Office National de la Chasse et de la Faune Sauvage - Direction des études et de la recherche, Nantes,
France, for the serological detection of apathogenic RHD-like strains in wild rabbits in France
the ANSES (French agency for food, environmental, and occupational health safety) LERAPP (Laboratory for
studies and research on poultry, pig, and fish farming) UVIPAC (Unit of virology, immunology, and
parasitology in poultry and rabbits) Ploufragan France, for the serological diagnosis of RHDV infections in
wild and domestic rabbits and for the studies of antigenic characterization and biological properties of the new
antigenic Fra2010.
the IZW of Berlin for the analysis of EBHS seroprevalence in wild hares in Germany the diagnosis of EBHS in
captive hares and for vaccination studies
the Department of Water, Land and Biodiversity Conservation of the Australian Government (Adelaide). and
CSIRO Canberra (Australia), for studying the circulation of RHDV in wild populations.
13. Publication and dissemination of information relevant to the work of OIE (including list of
scientific publications, internet publishing activities, presentations at international conferences)

1.

1)
Presentations at international conferences and meetings
Lavazza A, Chiari M, Cavadini P, Gioia E, Barbieri I, Grilli G, Ferrazzi V, Zanoni M, Capucci L.
Surveillance program on the eastern cottontail (Sylvilagus floridanus) as reservoir of hares pathogens and
definition of its natural susceptibility to the European Brown Hare Syndrome. OIE Global Conference on
Wildlife: animal health and biodiversity: preparing for the future: Paris, France, 23 to 25 February 2011:
abstract book. Paris: World Organisation for Animal Health (OIE), 2011. - p 67
Scientific publications in peer-reviewed journals
Fernández E, Toledo JR, Chiong M, Parra F, Rodríguez E, Montero C, Méndez L, Capucci L, Farnós O.
Single dose adenovirus vectored vaccine induces a potent and long-lasting immune response against rabbit
hemorrhagic disease virus after parenteral or mucosal administration. Vet Immunol Immunopathol. 2011,
142, 179-188
Annual reports of OIE Reference Laboratories and Collaborating Centres, 2010
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Rabbit haemorrhagic disease
2)
3)
4)
5)



Le Gall-Recule G., Zwingelstein F., Fages M.P., Bertagnoli S., Gelfi J., Aubineau J., Roobrouck A., Botti G.,
Lavazza A. Marchandeu S. Characterisation of a non-pathogenic and non-protective infectious rabbit
calicivirus-like virus. Virology 2011, 410, 395–402
Paci G., Lavazza A, Ferretti M., Santilli F., Bagliacca M. Relationship between anti-european brown hare
syndrome serological titres and brown hare (Lepus europaeus Pallas) densities. International Journal of
Zoology 2011. Article ID 436193, 5 pages, 2011. doi:10.1155/2011/436193.
Drews B, Szentiks CA, Roellig K, Fickel J, Schroeder K, Duff JP, Lavazza A, Hildebrandt TB, Goeritz F.
Epidemiology, control and management of an EBHS outbreak in captive hares. Vet Microbiol. 2011. 154,
37-48.
Cerioli M., R. Brivio Ruggero, C. Tittarelli, G. Grilli, A. Lavazza Identification of health and welfare
parameters for rabbit production and definition of an evaluation score. Journal of Agricultural Science and
Technology A.2011, 1, 500-507.
Other communications
Müller A., Silva E., Capucci L., Thompson G. Case Report: Outbreak of rabbit haemorrhagic disease (RHD)
caused by an antigenic variant in meat rabbits. Congresso da Sociedade Portuguesa de Ciências Veterinárias
INRB-EZN, Fonte Boa, 13-15 de Outubro 2011
Capucci L. Le Gall-Reculé G., Scicluna M.T., Cavadini P., Barbieri I., Brocchi E., Botti G., Lavazza A.
Identificazione e caratterizzazione Preliminare di nuove varianti del Rabbit Haemorrhagic Disease Virus
Riassunti Workshop nazionale di virologia veterinaria. Brescia, 9-10 giugno 2011. A cura di Marina Monini,
Susan Babsa, Franco M. Ruggeri, Antonio Lavazza, Paolo Cordioli, Emiliana Brocchi 2011, v, 101 p.
ISTISAN Congressi 11/C3, p. 26.
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Annual reports of OIE Reference Laboratories and Collaborating Centres, 2010