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Fast solutions for foot and mouth testing

Researchers in many countries are looking for fast and reliable tests to detect either the foot and mouth disease virus (FMDV) or antibodies against the pathogen to help control outbreaks quickly. If such a test could also differentiate infected from vaccinated animals, it would help to limit the economic consequences of the disease. Since 1999, the Commission has funded a programme to help the key European research institutes in this area to exchange information and test data. Participants also carry out blind tests on the same samples. Some promising tests require further development, and the original three-year programme has been extended by a further year.

During the catastrophic outbreak of foot and mouth disease in the UK in 2001, three million serum samples were tested. The crisis highlighted the need for advanced diagnostic methods to identify infected animals. Such means would limit the economic consequences of the disease and help to contain it. Several institutes and laboratories around Europe have been working on the problem for some years, so in 1999 the Commission set up a Concerted Action project for joint evaluation of tests and exchange of information. Its objectives are to accelerate and coordinate the development and validation of new diagnostic methods.

The programme is coordinated by the German Federal Research Centre for the Virus Diseases of Animals and includes the leading veterinary institutes and animal virus research centres in Belgium, Denmark, Italy, the Netherlands, Spain and the UK. Three of them, the FRC, Germany, the Institute of Animal Health in Pirbright, UK and ID-Lelystad in the Netherlands, are equipped to keep infected animals in secure conditions for testing.

What testers look for

Foot and mouth viruses are very small and easily spread by the wind. FMDV genes are made up of RNA rather than DNA, so the virus readily mutates rapidly. The fact that there are many different strains grouped in seven serotypes complicates the diagnosis as well as the control of FMD. Laboratory diagnosis depends on detection of the virus itself - by assaying its effect on cultured cells or its components (antigens and nucleic acid) or the detection of antibodies produced in response to infection - in samples taken from suspect animals.

The position is complicated by the presence of antibodies in vaccinated animals as well as those that have contracted FMD. Established tests recognise antibodies to the structural proteins that are found in both vaccinated and infected animals. Non-structural proteins (NSP) are not present in vaccines, only in animals that have had contact with live FMD, so some researchers are concentrating on tests that detect NSP antibodies. A fast test of this type would have important benefits in controlling an FMD outbreak. By testing for NSP-antibodies, countries that used vaccination (without culling of all vaccinated animals) to control an outbreak could regain their 'FMD-free' status much faster.

Some vaccinated cattle that had contact with infectious virus may become virus carriers for up to three years without ever showing any symptoms. Whereas the probability that an individual carrier animal infects a susceptible animal is small, the dramatic consequences of this event require testing for hidden infection in vaccinated populations. With NSP-serology the testing of whole vaccinated populations becomes feasible. However, NSP serology will only work as a herd test, since some vaccinated and infected animals may not produce antibodies to NSPs.

Chicken gene library

Programme participants are working on a database of partially sequenced chicken DNA (cDNA), enriched by material from the cloning experiments. A procedure manual has been produced to ensure that work complies with the established principles of Good Laboratory Practice (GLP); it contains every step needed to produce of full-length sequences for the library, whether normalised or subtracted. Genes have been cloned from various tissues undergoing different kinds of immune responses in infected birds. This approach to identifying genes whose expression is involved in immune responses is likely to supplement more conventional approaches and make a major contribution to the identification of these genes in chickens. This data is available to all the programme participants, and will eventually be put into a public database for both academic and commercial researchers.

The basis of FMD tests

In full-blown FMD, samples taken from the characteristic mouth or feet blisters can be tested for virus or its components. The fastest, but least sensitive test is a technique known as ELISA (enzyme-linked immunosorbent assay). The ELISA detects antigens by an enzyme-triggered colour change in microwells of plastic plates. Other types of ELISAs detect the antibodies that are produced by infected or vaccinated animals after about one to three weeks.
In suspect cases of FMD, attempts are made to isolate the virus in cell culture, which is more sensitive than ELISA, but this takes several days to complete. In the absence of blisters, during the first two weeks after infection, nasal swabs can be used to look for virus. After the lesions have healed, virus can still be detected in samples taken from the pharynx, where the virus persists. However, virus isolation from those samples is laborious and time-consuming and is therefore not practicable for broad-scale screening.
Recognition of nucleic acid from the genetic material of the virus is the main alternative to virus isolation. The detection of viral genome has the advantage that it does not require viable virus or intact viral antigens. Genome fragments of FMD can be multiplied to give enough material for testing, through the polymerase chain reaction (PCR). This method is very sensitive, but the downside is that it can easily give 'false positive' results due to minute laboratory contaminations with specific nucleic acids . One goal of the current programme is to look at advanced PCR protocols in order to improve the reliability and throughput of this method.

Making progress

Participants meet every year at one of their institutes, when they discuss current work in the field, exchange results and up-date their programme goals. Four of the laboratories have set up a protocol for blind ring testing of 40 samples from FMDV infected animals, using virus identification, PCR and ELISA tests. The greatest sensitivity to virus serotypes prepared at Pirbright was achieved by the nested PCR test at Tübingen. These procedures will help to validate tests.

They are also working on improving several ELISA variants. One is based on the detection of antibodies to the '3ABC' NS polyprotein, which are considered to be the most reliable indicator of infection rather than vaccination. NS-Ab ELISAs for detecting antibodies are the tests currently offering the fastest throughput and the group is looking at their validation. A commercial kit based on this technique is being validated and the product will soon be launched. IgA antibodies in saliva are being investigated as an indicator of (vaccinated) carriers.

The ultimate aim is to have a good supply of accurate test kits ready and waiting for use in emergency. They need to be validated, simple to use and produced on an industrial scale.
In certain situations, portable tests that could be used on the farm (instead of taking samples to a laboratory) would be of advantage. One example recently published by the IAH, Pirbright, is a chromatographic strip test that could complement the antigen ELISA. Whereas cell culture will always have to be performed in a laboratory, it is possible that future PCR based methods may allow a district veterinarian to analyse samples on site, e.g. in a van with special equipment, for FMD specific nucleic acids. However, claims in the media that this is already practical are premature.

Project Coordinator: Dr Bernd Haas
E-mail: bernd.haas@tue.bfav.de
Telephone:+49 707 196 7252
Address: Federal Research Centre for Virus Diseases of Animals
Paul-Ehrlich-Strasse 28
D-72078 Tübingen
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