The general objective is to improve livestock production through effective control/eradication of livestock diseases. More specifically, the CRP aims to introduce molecular biological techniques for more effective diagnosis and surveillance of trypanosomosis.
Ten Research contracts and five Agreement holders have been awarded. The first Research Coordination Meeting was held in Antwerp, Belgium, 26-30 March, 2001.The second Meeting took place in Rio de Janeiro, Brazil from 7 to 11 April 2003 and the third Meeting was held in Hanoi Vietnam from 20 to 24 June 2005.
The developmental areas of the CRP include:
The general and more specific themes of the research and development are summarized below.
Standardization of methods and the role of PCR
In line with determination of 'fitness for purpose' by the OIE, emphasis on test development must be placed on an exact determination of the role of the PCR. Elements of relative cost,
sample collection and storage, sensitivity, specificity, the need for training, establishment of good laboratory facilities and contamination, should all be considered in the context of what exactly
is required in any study at the national level. It was generally agreed that the PCR should be a component of any testing and not aim to totally replace existing assays.
Methods and Protocols
It was agreed that each PCR protocol (based on methods and primers used), should be written and that a standard format should be used. Model protocols from Dr. Peter-Henning Clausen
have been received and passed on to participants. It was agreed that some basic background information should also be included with regard to reagent formulation, and Dr. G. Viljoen has
sent this material which has been distributed to participant.
DNA Bank
A reference bank of DNA was set up at the at the FAO/IAEA laboratory, Seibersdorf. The DNA is from trypanosomes, however it would be useful to ssend control DNA from various
livestock and insect species and DNA from relevant organisms likely to complicate the PCR diagnostic potential with regard to trypanosomosis. The strains selected were well characterized
and “reliable” in terms of their pedigree Approximately one milligram of DNA was sent by mosty participants for each sample. The DNA was characterized with respect to the PCR products
produced using standardized protocols an that the products would be sequenced to allow quality control.
GLP
The setting up of a PCR laboratory was fundamental. A good laboratory practice (GLP) document for PCR was developed.
Primers
Extensive discussions on which primers could be used to obtain products for use in various aspects of the study of trypanosomosis were made. Many papers presenting data are available
from the contract and agreement holders through RCM reports and publications to show this work.
Comparative testing and sensitivity/specificity
The diagnostic sensitivity (DSn) and diagnostic specificity (DSp) of all tests including PCR is paramount in evaluation studies. There was a need to define sensitivity in terms of both analytical
and diagnostic potential of the PCR. There are drawbacks to estimating analytical sensitivity from “spiked” samples by dilution of material. There is also a difficulty where field samples are
examined and the effect of the matrix on PCR analysis. The stages of sampling and nucleic acid extraction are dominating features in examining diagnostic features.
Antibody assays (ELISA)
The indirect ELISA developed at Seibersdorf was used in the first 2 years of the studies. outlined. This assay features the use of pre-coated plates with either T. vivax or T. congolense, the antigens
being totally denatured. The use of other ELISA systems to detect T. evansi was also noted.
PCR-ELISA
It was concluded that there was no direct benefits from the approach in most laboratories and that the advantages of high through put were not really required. There were discussions on the
sensitivity aspects of the methods.
More Specific Developments:
