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Early and sensitive diagnosis and control of Peste des Petits Ruminants (PPR)
Overall objective:
The overall objective is to develop, validate and transfer to Member States sensitive, specific and rapid tests for the diagnosis of Peste des petits ruminants (PPR) to help them better manage and control this trans-boundary animal disease.
Specific objectives:
- Evaluate and validate current Reverse Transcriptase-PCR (RT-PCR) methods in use for the diagnosis of PPR. There are different methodologies for RNA extraction and different sets of primers, target different regions of the genome, etc. These methodologies will be standardised, compared and validated and SOPs produced.
- Evaluate and validate real-time PCR procedures. This quantitative method does not require several handling procedures but can be run as a one-step procedure. In comparison to conventional PCR, real-time PCR approaches offer increased sensitivity and specificity in a rapid format that is also less prone to contamination.
- Development and evaluation of iso-thermal amplification procedures (e.g. loop-mediated isothermal amplification (LAMP PCR). This is a novel PCR approach performed at one temperature, thereby circumventing the involvement of costly temperature ramping equipment but still allows for high sensitivity. The LAMP PCR is very specific because it is based on six primers (i.e. 3 primer pairs) recognizing the target sequences. In addition, this procedure is in general faster than temperature ramping procedures and quantitative as well and the amplification products can be detected by visual inspection or in real-time by the addition of a DNA intercalating fluorescent dye.
- Evaluate and validate a penside test currently under development for on-site identification of PPR virus in the field. PPRV specific monoclonal antibodies produced by APU at Seibersdorf will serve as the basis of this test.
- Evaluate and validate the use of several ELISA-formats in epidemiological studies and for the differentiation of infected from vaccinated (DIVA) animals. Control of PPR can be achieved by effective vaccination strategies. The successful implementation of such vaccination campaigns can be monitored by sero-surveillance as the example of Rinderpest eradication by PARC and PACE demonstrated. The existing tests will therefore be applied in the field and results compared to define the fitness for purpose and its diagnostic characteristics.
- Contribute to the build up of a PPRV gene sequence data bank for molecular epidemiological analysis. PCR amplified products that will be obtained from new PPR cases during the CRP will be sequenced and the data were compared with information already available.
Activities:
First RCM of the CRP was held from 31 March to 04 April 2008 at the IAEA, Vienna. The RCM focused on the evaluation and validation of molecular-based diagnostic tests in light of the OIE template for test certification. An update was made on new technologies applicable to early and rapid diagnosis of trans-boundary animal diseases.
Programme of Work following the first RCM (2 years)
Research Contract holders will work according to their work plans with the support of the CRP scientific officer. Ring tests involving all participants will be organised. Conventional PCR will be validated according to the OIE guidelines and compared to the immunocapture test for PPRV detection in pathological samples. Eventual PPR outbreaks in countries should be attended by the counterpart for samples collection and testing. Different methodologies of sample collection (filter paper sample collection/versus classical collection and storage in cold conditions) will be compared. Positive PCR products were sequenced in a partner country or by Animal Production Unit at Seibersdorf. The immunocapture test will be compared with the penside test which is expected to be available in 2009. ELISA’s for sero-monitoring will be compared in outbreak and vaccination situations.
Second RCM
The second RCM will be held in 2009 to review the results from the first phase and to consider proposing agreed standardised PPRV RT-PCR protocol to the OIE. Detailed work plans for the next 24 months will be made.
Second 24 months Programme of Work following second RCM
It is expected that during this second phase of the CRP, activities will focus more on the validation of the newly developed amplification technologies and extensive comparison with conventional PCR and other PPRV detection methodologies. Ring tests will be organised and PPR outbreaks attended to.
Final RCM
The final RCM should be held in the middle of 2011 to review the results from the CRP with the SOPs to be published in 2011 as an Agency TECDOC or external publication. Nineteen Research and Agreement Holder Contracts have now been awarded to Member State organizations in Austria, Bangladesh, Burkina Faso, Cameroon, China, France Ghana, India, Côte d'Ivoire, Malí, Nigeria, Pakistán, Sudan, Sweden, Turkey, Uganda and UK.