AVIS - Foot and Mouth Disease

Laboratory Tests

OIE Manual

Summary

Text on foot-and-mouth disease from the Office International des Epizooties, Manual of Standards for Diagnostic Tests and Vaccines, (2000), Chapter 2.1.1.

This text is included in its entirety. The manual describes in general terms the 'prescribed' and 'alternative' diagnostic tests for FMD. 'Prescribed' tests are those that are required by the International Animal Health Code for the testing of animals before they are moved internationally. 'Alternative' tests are those that are suitable for the diagnosis of disease within a local setting, and can also be used in the import/export of animals after bilateral agreement.

More detailed descriptions of the actual procedures followed at the OIE/FAO WRL for FMD are given in the 'Laboratory Tests' section of this module.

FOOT AND MOUTH DISEASE

Summary

Foot-and-mouth disease (FMD) is the most contagious disease of animals and has a great potential for causing heavy loss in susceptible cloven-hoofed animals. There are seven serotypes of FMD virus, namely, O, A, C, SAT1, SAT2, SAT3 and Asia 1. Infection with one serotype does not confer immunity against another. FMD cannot be differentiated clinically from other vesicular diseases, including swine vesicular disease, vesicular stomatitis and vesicular exanthema. Laboratory diagnosis of any suspected FMD case is therefore a matter of urgency.

Typical cases of FMD are characterised by a vesicular condition of the feet, buccal mucosa and, in females, the mammary glands. Clinical signs can vary from mild to severe and fatalities may occur, especially in young animals. In some species the infection may usually be subclinical, e.g. African buffalo (Syncerus caffer). The preferred tissue for diagnosis is epithelium from unruptured or freshly ruptured vesicles. Where this is not possible, blood and/or oesophageal-pharyngeal fluid samples taken by probang cup in ruminants, or throat swabs from pigs provide an alternative source of virus. Myocardial tissue or blood can be submitted from fatal cases, but vesicles are again preferable if present.

It is vital that samples from suspected cases be transported under secure conditions and according to international regulations. They should only be dispatched to authorised laboratories.

Diagnosis of FMD is by the demonstration of FMD viral antigen or nucleic acid in samples of tissue or fluid. Detection of specific humoral antibody can also be used for diagnosis, especially in wildlife, but this requires the absence of any history of vaccination, as it is not always possible to differentiate a serological response to natural infection from that due to vaccination. Diagnosis based on serological response may also be problematical in endemic areas due to the possibility of previous infection.

Identification of the agent: The demonstration of FMD viral antigen is sufficient for a positive diagnosis.

Complement fixation (CF) has been the traditional test for diagnosis, but has been replaced in many laboratories by the enzyme-linked immunosorbent assay (ELISA), as this is more specific and sensitive and is not affected by pro- or anti-complementary factors. If the sample is inadequate or the test result inconclusive, it will be necessary to grow the virus in cell cultures or in 2-7-day old unweaned mice. The cultures should preferably be of primary bovine thyroid, but pig, lamb or calf kidney cells, or cell lines of comparable sensitivity may be used. When a cytopathic effect (CPE) appears in the cultures, the fluids can be used in CF tests or ELISAs. Similar tests can be performed on homogenised suspensions of the dissected musculo-skeletal tissues of any mice that die. In the absence of CPE or any dead mice, a further passage should be made at a 48-hour interval, with freeze/thawing of the cells, before the sample is declared to be negative.

Nucleic acid recognition tests, such as the polymerase chain reaction and in situ hybridisation, are being used increasingly as rapid and sensitive diagnostic methods. Electron microscopic examination of lesion material is sometimes useful to differentiate FMD from disease caused by pox or other viruses.

Serological tests: The demonstration of specific antibody titres in non-vaccinated animals, where a vesicular condition is present, is sufficient for a positive diagnosis. This is useful in mild cases or where epithelial tissue cannot be collected.

Virus neutralisation (VN) tests and ELISA are used as serotype-specific serological tests. VN tests depend on tissue cultures and are, therefore, more prone to variability than ELISA; they are also slower and subject to contamination. ELISAs for antibodies have the advantage of being faster and are not dependent on cell cultures. The ELISA might be performed with inactivated antigens, thus requiring less restrictive biocontainment facilities.

Requirements for vaccines and diagnostic biologicals: Inactivated virus vaccines of varying composition are available commercially. Typically, virus is used to infect a suspension or monolayer cell culture and the resulting preparation is clarified, inactivated with ethyleneimine and blended with adjuvant. Many FMD vaccines are multivalent to provide cover against the different serotypes likely to be encountered in a given field situation.

The finished vaccine must be shown to be free from residual live virus. This is usually done using a combination of in-vitro tests on the inactivated virus preparation and in-vivo tests on the finished vaccine. Challenge tests are also conducted in vaccinated cattle to establish a PD₅₀ (50% protective dose) value, although a serological test is considered to be satisfactory where the vaccine producer has established a statistically significant correlation between protection and specific antibody response.

Diagnostic and reference reagents are available from the OIE/FAO (Food and Agriculture Organization of the United Nations)

World Reference Laboratory for FMD
Pirbright Laboratory
Ash Road
Pirbright
Woking
Surrey
GU24 ONF
United Kingdom

or from Regional Reference Laboratories for FMD.