Appendix 12

Appendix 11

Proposals for Phase XIX

The main aim of this should be the organisation of two rounds of annual proficiency testing, one in 2005 and the other in 2006.

A strong positive sheep and a strong positive pig serum will be prepared from infected but unvaccinated animals along with a negative serum from each species. For each species, a titration series will be prepared and tested by different NSP methods. If acceptable dose response curves are obtained, aliquots of the strong and weak positive sera will be sent to laboratories, for use as NSP standards.

Efforts will be made to obtain bulk strong positive sera for the SAT serotypes. If necessary, these could be prepared at the WRLFMD, provided additional funding was made available. These strong positive sera could be distributed in the second year of the study for local titration and use as in-test control sera.

In addition, a position paper should be drafted on the purpose and use of secondary standards and intest control sera.

Proposal for 2005: 1. Concentrate on proficiency serum panel with serotypes O, A and Asia 1. 2. Aim to provide sheep and pig NSP strong positive sera. 3. Include more sera from vaccinated and infected animals, especially of serotype A. 4. Do not specify particular tests to be used, but rather invite labs to test sera with the methods that they would normally use. 5. Request that interpretation of results be given as if these samples were from animals being examined prior to import (i.e. when serotype/strain of infecting virus would be unknown). 6. Ask EUFMD Secretariat to identify participants and draw up agreement on terms of participation. 7. Make sera available to other participants on request, subject to payment for sera and shipping. 8. Send out serum panel in April 2005. 9. Prepare analysis of results in time for next closed meeting in September 2005.

Proposal for 2006: 1. To be finalised at closed meeting in September 2005. 2. Aim to include SAT serotype proficiency sera. 3. Send out serum panel in April 2006. 4. Prepare analysis of results in time for next open meeting in October 2006.

Appendix 12

Progress and future prospects for standardisation of FMD tests

D J Paton, FAO World Reference Laboratory for FMD, Institute for Animal Health, Pirbright, Surrey GU24 ONF, UK

Progress with serological standardisation

FAO has supported the FMD WRL in conducting a series of standardisation exercises for FMD serology known as the “Phase” studies. Currently, Phase XVIII is approaching completion. These studies have attempted to improve the reliability and consistency of serological testing for FMD carried out by EUFMD member states. Recent Phase exercises have combined the establishment of reference and proficiency sera both of which have been distributed to participating laboratories. The intention has been that the reference sera (RS) would be used to calibrate the various tests in use by member countries and then the proficiency sera would be tested to assess how effectively the tests performed.

At the end of Phase XVI, a set of RS were established and OIE adopted them as official standards. These RS were derived from cattle that had been experimentally infected with FMD virus of serotypes O (Manisa), A (A22 Iraq) or C (Noville). As well as a single negative RS, a strong positive, weak positive and cut-off serum was designated for each serotype. During Phase XVII additional RS were prepared by vaccinating or infecting cattle with FMDV O SKR, A Iran 96 and Asia 1 Shamir. This work was interrupted by the FMD outbreak in UK in 2001, but preliminary results were presented at the EUFMD Research Group meeting in Izmir in 2002. It was considered that the weak and cut-off RS were too weak, and it was proposed to strengthen them. A workplan was agreed for completion of Phase XVII, incorporating a six months extension to the project, until the end of June 2003.

Phase XVII had been largely completed by the time of the Gerzensee meeting of the EUFMD Research Group in September 2003, by which time a new set of candidate RS for strains O SKR, A Iran 96 and Asia 1 Shamir had been prepared, distributed and tested by 9 testing laboratories. The distribution included a new range of RS dilutions and two weak positive sera were selected for each serotype. Data sheets for the new RS were sent to the OIE Standards Commission in June 2004.

The aims of Phase XVIII were agreed at the EUFMD Research Group meetings in Izmir, 2002 and Gerzensee, 2003:

• Introduction of the solid phase competition ELISA (SPCE). • Preparation of secondary standards based on reference sera to O, A and Asia 1, including an analysis of what is done in this respect by participating laboratories. • Use of calibrated tests to examine local negative serum populations. • Use of calibrated tests to examine a proficiency panel. • Standardisation of internal quality control procedures. • Evaluation of NSP ELISAs (added in Gerzensee).

The workplan was to run from Jan 2003 to December 2004.

There are 24 participating laboratories in Phase XVIII. During July 2004, each participant has been supplied with the ten RS prepared in Phase XVII, a proficiency panel of 12 sera of undisclosed characteristics, a set of reagents and instructions for SPCE for serotypes O, A and Asia 1, a request to test 500 naïve field sera by SPCE and a template for recording results. Participants were also requested to supply protocols for the tests used in their laboratories.

Results have so far been received from 13 countries (30th Sept). A summary of the results available to date will be sent by email to participants who have submitted results on 4th October and will be presented at the EUFMD Research Group Meeting along with provisional conclusions and recommendations from the study.

Future direction of serological standardisation work

Successful aspects of the Phase studies: 1. Proficiency aspect has been of great benefit ensuring regular assessment of serological competence for many laboratories.

2. RS have been established for serotypes O, A, C and Asia 1 and large stocks of these sera are available on request.

Problems with the Phase exercises: 1. It has proved difficult to keep to timetables for carrying out this work. Contributory factors have included the limited resource committed to this project, the time taken to obtain permits to send materials and the complex nature of the procedure for selection and processing of the sera. 2. The process of deriving the RS is complex and perhaps overly consensual. The main requirement for an OIE reference serum is quite simple in principle, being to produce a set of uniform test calibrators, i.e. reference standards that can be used to monitor the performance of a test over time and give comparability between laboratories. The most difficult issue is the decision on the threshold of positivity for weak positive sera and this issue is made much more complicated if the standard has to be used for different tests in which different cut-off points are applied depending on the purpose of testing. Generation of cut-off sera requires even greater precision and is inherently more difficult. 3. Preparation of the RS is enormously more difficult and time consuming than preparation of the proficiency panel, but actually there is little evidence that the RS are being used for their intended purpose, rather than as a de facto proficiency panel. How many laboratories are using the RS for routine test calibration via production of secondary RS? What use is actually being made of the RS? 4. Countries may have difficulty to prepare secondary standards. 5. Procedures for inactivation of sera have not been closely defined. OIE recommends gamma irradiation, but this is not very practical and potentially harmful to the quality of the RS. The WRL FMD prefers inocuity testing followed by BEI inactivation and if necessary further inocuity testing. 6. There is now uncertainty over the priority for introduction of the SPCE. On the one hand there is a shift towards the use of NSPE to detect infection and on the other hand, there is a lack of insight into interpretation of SPCE titres for post-vaccinal surveillance, creating a continued demand for LPBE test kits.

Solutions proposed and future objectives: 1. Simplify the process of creating RS. Establish strong positive and negative sera only and derive dose response curves for different tests by testing serial dilutions of the strong positive serum in the negative serum. For each test and test purpose a given dilution could be identified for use as a weak positive serum. This would hugely simplify the preparation, distribution, evaluation and re-evaluation of RS. It would give flexibility to readily derive further weak positive sera for new tests and test purposes. 2. Make provision for supply of alternative hyperimmune sera for creation of secondary standards to laboratories that cannot infect animals themselves. 3. Abandon the concept of cut-off sera for the time being until weak positive sera are established for all of the serotypes. 4. Completely separate the issue of establishing RS from proficiency testing. 5. Develop projects for further validation of SPCE

Progress with NSP serology standardisation

OIE set up an ad hoc group to look at progress with validation of NSP serological tests and this group has met three times, most recently in August 2004. The group recommended the adoption of the PANAFTOSA 3ABC ELISA as the index method. Data was supplied by PANAFTOSA on the performance of the test with sera from persistently infected cattle and this has been augmented by testing carried out at the Brescia workshop of the EU Improcon project in May 2004. This workshop also showed that some other tests were comparable to the index method in terms of their sensitivity and specificity. PANAFTOSA have supplied serial dilutions of two positive bovine sera as putative NSP RS. One of these serum samples is derived from a field case in an area where vaccination was practised and the other is from an experimentally infected animal. During the Brescia workshop the dilution series was tested with all of the available NSP ELISAs and dose response curves plotted. There were considerable differences in the shapes of the curves for some of the tests and there was no clear relationship between the diagnostic sensitivity of tests and their ability to detect higher dilutions of the RS. A specific dilution of one of the sera was chosen as the strong positive RS and a specific dilution of the other serum as the weak positive RS. It was agreed that the next priority was to establish NSP test RS from pigs and sheep since some of the NSP tests are species-specific. Some large stocks of sera are available at the WRL from unvaccinated, experimentally infected sheep and pigs and these will be examined for suitability. The WRL also agreed to establish a NSP proficiency panel with a limited availability for evaluation of new tests and batch control of existing tests.