Address of laboratory: CIRAD-BIOS,
Control of Exotic and Emerging Animal Diseases
Campus International de Baillarguet TA A-15/G,
34398 Montpellier Cedex 5,
FRANCE
Tel.:
Fax: e-mail address: website:
33(0)4 67 59 37 98
33(0)4 67 59 38 50 genevieve.libeau@cirad.fr
http://www.cirad
.
Annual reports of OIE Reference Centres, 2011
1

Rinderpest and Peste des petits ruminants
1. Test(s) in use/or available for the specified disease/topic at your laboratory
List of tests available for the diseases.
Test
ELISA
Virus Titration
RT-PCR
QRT-PCR
Sequencing
Cell culture
For
Antibody
Virus
Genome
Genome
Genome
Virus isolation
Specificity
PPR-specific
PPR-specific
PPR-RP-specific
PPR-RP-specific
PPR
PPR
Surveillance
-
-
214
182
10
15
Research
820
50
25
65
10
10
Total
820
50
239
247
20
35
The competitive ELISA test (C-ELISA) for peste des petits ruminants (PPR) antibody detection is based on the anti- nucleoprotein (N) monoclonal antibody (Mab). The test has been validated under a new format based on precoated plates and a conjugated monoclonal antibody. It is an improved format in terms of ease-of-use. The assay is being put forward for accreditation to ISO17025. Now that rinderpest (RP) is eradicated, the implementation of an
RP Elisa test is not used as a routine test.
Serosurveillance is targeted on domestic ruminants and wildlife. The diagnostic activity is carried out in support to countries experiencing PPR.
RT-PCR and quantitative RT-PCR are used as routine tests and are adapted to different samples such as blood, tissue samples, tears and filter papers blotted with biological samples.
2. Production and distribution of diagnostic reagents
The N-PPR ELISA is prepared and supplied by IDVet, France. The immunocapture ELISA test (ICE) for the diagnosis of PPRV antigen is prepared by CIRAD and supplied through BDSL, UK.
Diagnostic reagents and ELISA kits for the detection of antigen and antibodies were supplied to the following countries: Germany, Algeria, Austria, Burkina Faso, Arab Emirates, United States, France, Guinea, Iraq, Italia,
Mongolia, United Kingdom, Senegal, Turkey etc...
Number, type of kits, vaccine, strains and of reagents:

Immunocapture ELISA: 26

NPPR ELISA: 60 (for 4000 sera) + 214 (for 320 sera)
Type of reagents:

RNA, cDNA,

Wild PPR strains, live or inactivated

Mabs, reference sera, cell lines,

Primers,

Plasmids containing PPR genes.
2
Annual reports of OIE Reference Centres, 2011

Rinderpest and Peste des petits ruminants
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
None 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

CIRAD experts are involved in a Coordinated Research Project with the FAO/IAEA Centre for ELISA and Molecular Techniques in Animal Disease Diagnosis. The project is entitled Early and Sensitive
Diagnosis of Peste des Petits Ruminants. The main objectives of this project are to standardize and validate new molecular-based (real time PCR, LAMP) PPR diagnostic tools and penside tests for early and sensitive diagnosis of PPR.

To enforce expertise of other laboratories from developing countries in PPR diagnosis, inter-laboratory ring trials for PPR antibody detection were organized (Ethiopia, Sudan). Reference materials and protocols were prepared and shared with the different participants.

CIRAD provided under an agreement with Galvmed the production capacity building and quality improvement of the Nigeria 75-1 PPR homologous vaccine at the National Veterinary Institute (NVI) laboratory in Ethiopia. This included the development of the technology using standard operating procedures, materials and methods developed by IBET (Portugal) in the frame of a previous EU project to improve the thermostability of the PPR vaccine. The transfer of the technology to NVI, materials and methods developed by CIRAD, the associated training and the necessary quality control, allowed NVI to successfully manufacture a stable PPR vaccine.
4. Preparation and supply of international reference standards for diagnostic tests or vaccines
International standards were widely distributed through diagnostic kits (see section 2) to different countries.
Positive and negative sera were produced and supplied to IDVet for the validation of the PPR ELISA under the improved format. In 2011, requests for vaccine quality control were satisfied.
5. Research and development of new procedures for diagnosis and control
In 2011 research projects were followed in order to (1) improve our approach to control the disease by the generation of biological antiviral, (2) improve diagnostic tools by the development of quantitative RT-PCR for
PPRV, (3) improve our knowledge of PPRV lineage distribution in collaboration with other national and international laboratories.
1) Development of antivirals. CIRAD developed new biological antivirals based on siRNA that target three regions on the nucleoprotein gene of PPRV but also RPV and MV and their in vitro effect on the viral replication and expression. Recombinant adenovirus and baculovirus vectors expressing short hairpin RNAs (shRNAs) against the PPR virus (PPRV) were also constructed and compared in vitro . The results show that baculoviral shRNA-expressing vectors have the potential for therapeutic use against morbillivirus infections (Nizamani et al.,
2011). In vitro potency of PPRV to escape siRNAs was studied. Except with the combination of three different siRNAs, the virus systematically escaped RNAi after 3 to 20 consecutive passages. The genetic modifications consisted of single or multiple point nucleotide mutations and a deletion (Holz et al., 2011).
2) Development of a quantitative RT-PCR (QRT-PCR) for PPRV.
The laboratory has developed a robotized method (BIOMEK FX) for the viral RNA extraction. Samples from naturally infected small ruminants were used
Annual reports of OIE Reference Centres, 2011
3

Rinderpest and Peste des petits ruminants to validate the robotized extraction. This test assessed the delectability of PPRV by real time RT-PCR in different biological samples, including archived RNA on filter papers, and confirmed the validity of the robotized extraction.
3) Improve knowledge of PPRV lineage distribution in the world . PPR expansion is linked to international trade and animal movements. Data collected over the last decade illustrate the incursion from the Middle East of the Asiatic lineage IV into Africa. Using the relevant region of the N gene of PPRV samples from Sudan,
Morocco, the Central African Republic, Cameroun, and Bangladesh were sequenced and compared to the PPRV sequences available from GenBank. Two publications are devoted to the epidemiology of PPRV (Kwiatek et al.,
2011, Bhuiyan et al., Submitted).
6. Collection, analysis and dissemination of epizootiological data relevant to international disease control
All sequences analysed in 2011 are in the public domain (GenBank).
Regarding OIE Member Countries, the notifications of PPR to OIE, is the responsibility of the country itself, in case of positivity of their samples.
7. Maintenance of a system of quality assurance, biosafety and biosecurity relevant to the pathogen and the disease concerned
• To guaranty the quality of the reference activities and to satisfy the customers, the laboratory has implemented a quality system based on referential ISO 17025 “General Requirements for the Competence of Calibration and
Testing Laboratories”. Accreditation was obtained on February 2010 for a BT-ELISA and will be audited in 2012 for a PPR-ELISA. The laboratory also answers to the requirements related to the rights and duties of the National
Reference laboratories (Décret JORF n°0251 du 28 octobre 2011 page 18206 texte n° 48 (NOR:
AGRG1128680A).
•
The laboratory has a BSL2 and BSL3 plus. Biological samples that may contain pathogens and toxins for human are manipulated under biosafety cabinets in the BSL3. Dangerous products (when inhaled) are manipulated under cytotoxic security cabinets in the level 2 zone.
• The members of the staff must fill-in a risk sheet. The risk evaluation is made on one year. In case of frequent exposition to one or several risks during part of the year, the evaluation will be weighed on the year.
8. Provision of consultant expertise to OIE or to OIE Member Countries
Attendance to working groups for OIE and FAO:
• The Reference Laboratory expert provided advice directly to the OIE through Ad hoc group meeting for peste des petits ruminants, OIE HQ, Paris, June 2011;
• FAO/PANVAC Workshop on biosafety, sequestration and risk analysis for Laboratories holding Rinderpest
Virus. 4 – 8 July 2011, Ethiopia;
9. Provision of scientific and technical training to personnel from other OIE Member Countries

One PhD student from Brasil and a master were hosted by CIRAD. The subjects of the different students are:
- In vitro dynamic of relapse mutants of peste des petits ruminants virus (PPRV) under RNA interference pressure: implication for anti-morbillivirus therapy.
- Epidemiology of peste des petits ruminants in Sénégal.
4
Annual reports of OIE Reference Centres, 2011

Rinderpest and Peste des petits ruminants
Country/Diploma Type of training Starting date length
Brasil/PhD Research 01/01/11 11 months
Côte d’Ivoire/PhD Research 01/12/11 11 months
• A training course was initiated in 2011 and will be annually renewed at CIRAD to provide knowledge on different methods classically implemented for serological diagnosis and molecular diagnosis of peste des petits ruminants. This training includes harmonized protocols and procedures as well as equipment and diagnostic guidelines. The specific objectives are the following:
•
Training in serological diagnosis of PPRV
• Knowledge on OIE reference protocols for serology (i.e. virus neutralisation and CELISA)
• Training in molecular diagnosis of PPRV
• Knowledge on different procedures of RT and rRT-PCR
• Information about molecular sequencing and phylogenetic analysis of PPRV.
A technology transfer of serological and molecular methods beneficial to the Maghreb countries (Algeria,
Morocco, Mauritania and Tunisia) was implemented in the frame of the FAO project « Assistance for the prevention and control of peste des petits ruminants (PPR) in Maghreb » (TCP/RAB/3302), Rabat, Morocco.
A proposal for twinning between Morocco and CIRAD for PPR was submitted to the Biological Standards
Commission meeting in September 2011 and the scientific content of the project accepted.
10. Provision of diagnostic testing facilities to other OIE Member Countries
Country Species Sample
No. tests
No. positive samples/dubious
Test
Nature of the test
Confirmatory or tentative
RPV 2 /PPRV 1
Senegal Caprine/ovine
Nasal swabs
71
26
15 rtRT-PCR
RT-PCR
Confirmatory testing
Vaccine/Cell/Serum Contaminants
Ethiopia
Turkey
Spain
Spain
-
-
-
Vaccine
Vaccine
Vaccine
3
3
2
-
-
-
QualityControl 4 Confirmatory testing
QualityControl 4 Confirmatory testing
QualityControl 4 Confirmatory testing
QualityControl 4 Confirmatory testing - Vaccine 1 -
1 PPRV : Peste des Petits Ruminants Virus.
2 RPV: Rinderpest Virus (all samples tested remained negative).
3 RT-PCR: Reverse Transcription and amplification.
4 Sterility test + PCR (RP, PPR, BVD, mycoplasma) + titration (CPE visualized by immunoflorescence test using an anti-PPRV
Mab) + sequencing.
11. Organisation of international scientific meetings on behalf of OIE or other international bodies
None
12. Participation in international scientific collaborative studies
Participation to Epizone project (Network of Excellence for Epizootic Disease Diagnosis and Control) which objective is to improve research on preparedness, prevention, detection, and control of epizootic diseases within Europe.
Annual reports of OIE Reference Centres, 2011
5

Rinderpest and Peste des petits ruminants
13. Publication and dissemination of information relevant to the work of OIE (including list of scientific publications, internet publishing activities, presentations at international conferences)

Libeau, G., Lancelot, R., Martinez, D. (2011). The CIRAD contribution to the rinderpest eradication. EMPRES
Transboundary Animal Disease Bulletin 38, 54.
Libeau, G., Kwiatek, O., Albina, E. (2011) La peste des petits ruminants: incidence croissante dans le monde.
Bulletin de l’OIE. 2, 52.
Nizamani ZA, Keil GM, Albina E, Holz C, Minet C, Kwiatek O, Libeau G, Servan de Almeida R. Potential of adenovirus and baculovirus vectors for the delivery of shRNA against morbilliviruses. Antiviral Res. 2011 Apr;
90(1):98-101. Epub 2011 Feb 26.
Holz CL, Albina E, Minet C, Lancelot R, Kwiatek O, Libeau G, Servan de Almeida R. RNA Interference against
Animal Viruses: How Morbilliviruses Generate Extended Diversity To Escape Small Interfering RNA Control. J
Virol. 2012 Jan;86(2):786-95. Epub 2011 Nov 9.
A. R. Bhuiyan, E. H. Chowdhury, O. Kwiatek, R. Parvin, M. M. Rahman, E. Albina, M. R. Islam and G. Libeau.
PPRV diagnosis, load testing and genotyping from blotted filter papers: a powerful approach for understanding peste des petits ruminants epidemiology. Submitted.

Libeau, G. (2011). CIRAD & Twinning. Fifth annual meeting of the Regional Animal Health Centres for West and Central Africa (RESOLAB). Bamako, Mali 12-16 décembre 2011 (presentation).
Libeau, G. (2011). PPR virology and the disease status in a rinderpest free world”. 5ème réunion annuelle de coordination du réseau régional d’Afrique de l’Ouest et du Centre des laboratoires nationaux vétérinaires
(RESOLAB). Bamako, Mali 12-16 décembre 2011 (presentation).
Libeau, G. Rinderpest and PPR: How to make sure we can discriminate. FAO/PANVAC Workshop on biosafety, sequestration and risk analysis for Laboratories holding Rinderpest Virus. 4 – 8 July 2011, Debre Zeit, (Ethiopia),
(presentation).
Libeau, G. Opportunities for research with morbilliviruses, RPV and PPRV. FAO/PANVAC Workshop on biosafety, sequestration and risk analysis for Laboratories holding Rinderpest Virus. 4 – 8 July 2011, Debre Zeit,
(Ethiopia), (presentation).
_______________
6
Annual reports of OIE Reference Centres, 2011