The virus neutralization test (VNT) is currently considered as the “gold standard” for detection of antibodies to structural proteins of FMDV and is a prescribed test for import/export certification of animals/animal products. However, as various primary cells and cell lines with variable degrees of sensitivities are used in the VNTs, they are more prone to variability than other serological tests. Furthermore, VNT is slower, subject to contamination and requires restrictive biocontainment facilities in contrast to other serological tests which can use inactivated viruses as antigens.

The complement fixation test (CFT) was the test of choice for diagnosis of FMD and virus typing until the 1970s and is still used in some endemic areas. However, in order to overcome the problems of its low sensitivity and difficulty in interpretation of its results due to pro- and anti-complement activities, enzyme linked immunosorbent assays (ELISAs) for antigen detection and virus typing were desired. Roeder and Le Blanc Smith established suitable assays by using high titre antisera raised in rabbits and guinea pigs against purified 146S FMDV particles for antigen capture and detection, respectively. The assays were found to be 125 times more sensitive than the CFT and are still routinely used for the diagnosis of FMD and for virus typing. The ELISA, however, gives positive results with only about 70-80% of epithelial suspensions that contain virus due to a lack of sensitivity. Thus the virus may have to be propagated in tissue culture and subsequently tested in ELISA to detect the virus and ascertain the serotype.

Monoclonal antibody (MAb)-based ELISAs have also been developed for diagnosis of FMD and virus typing. Recently, a sandwich ELISA using recombinant integrin αvβ6 (a receptor for FMDV) for virus capture and serotype-specific monoclonal antibodies as detecting reagents was compared with the conventional polyclonal antibody-based sandwich ELISAs for the identification and serotyping of all the seven types of FMDV. The integrin/MAb ELISA recognized FMDVs of wide antigenic and molecular diversity from all seven serotypes. Although some FMDVs could not be detected, the assay showed greater specificity than the conventional polyclonal ELISA while retaining test sensitivity.

4.3. Virus isolation

As indicated above, the presence of relatively high levels of FMDV antigen in vesicular material can be detected by ELISA. However, when the virus concentration is too low to be detected by ELISA, then it has to be propagated in susceptible cell cultures. Primary cell cultures (such as bovine thyroid cells and porcine or ovine kidney cells) or cell lines (such as BHK or IBRS2) are considered to be generally suitable for isolation of FMDV. However, the production of consistent quality, ready-for-use primary cells is laborious, time-consuming and expensive. Furthermore, virus isolation requires the presence of infectious virus, which depends on sample quality. Up to 4 days may be required to demonstrate the presence of virus, especially when the levels of virus are low (thus it also takes 4 days to be confident, using this methodology, that no virus is present). Moreover, some FMDVs fail to grow in a specific cell type. Thus the absence of apparent growth does not guarantee absence of the virus and therefore samples collected from a suspected case of FMD should be subjected to further investigations, e.g. using another testing system. Additional disadvantages include the problems associated with obtaining and maintaining a regular supply of cells; possible contamination of cell cultures and the necessity to confirm any apparent virus growth by ELISA. These issues may delay the initiation of control measures to contain outbreaks.

The reverse transcription-polymerase chain reaction (RT-PCR) has been shown to be a useful tool for the diagnosis of FMD as it offers the advantages of fast, sensitive and reliable diagnosis. A variety of RT-PCR methods have been reported in recent years for the early detection of FMDV RNA in epithelium, cell culture isolates and other tissues using universal primers for all seven serotypes. Typing of FMDV by RT-PCR was first demonstrated by Rodriguez et al. for the differentiation of the serotypes O, A and C. Serotype specific primers have since been designed for the detection of all seven FMDV serotypes by RT-PCR.  Primers designed for these assays target various regions of the virus genome, including the 5′ UTR, the open reading frame and the 3′ UTR. However, evaluation of available sets of primers, designed for universal and serotype-specific diagnosis of FMDV, on a variety of field samples, representing all the seven serotypes of FMDV, has shown that no single primer set is capable of diagnosing the disease or typing of the virus. In order to improve the diagnostic sensitivity of RT-PCR, multiplex assays, incorporating more than one set of primers have been developed. However, differentiation/serotyping could only be made for certain groups of serotypes or individual isolates. Thus the conventional RT-PCR is not sufficiently sensitive and specific to replace methods using virus propagation in cell culture and ELISA.

Recently, real time/quantitative RT-PCR (rRT-PCR) methods have been developed which do not require post-PCR processing (e.g. gel analysis) and the signals can be monitored directly as the target cDNA is being amplified. Other advantages of the rRT-PCR include high throughput capability and the ability to quantify the genetic material in the starting sample. A TaqMan assay has been shown to be very robust and as effective for primary detection of FMDV as virus isolation in conjunction with antigen ELISA. Currently, two different rRT-PCR TaqMan assays are in common use, one targeting the internal ribosomal entry site (IRES) within the 5′ UTR and the second targeting the 3D (RNA polymerase) coding sequence.

The speed and accuracy of detection of the rRT-PCR assay was further improved by coupling the assays with robotic methods for extraction of nucleic acid from the samples and for set up of the assays. This has made the assay highly suitable for the diagnosis of the primary index case and for use in an ongoing outbreak. The rRT-PCR assays are currently used as a routine test for FMD diagnosis and quantification of the virus in many developed countries. However, these assays are not designed to discriminate between serotypes of FMDV. Although these assays exploit highly conserved regions across all the seven serotypes of FMDV, serotype biases still exist within both of these assays. The 5′ UTR assay has been shown to be more sensitive in detecting serotype A viruses, whereas, the 3D assay has greater sensitivity for detecting the SAT viruses. Additionally each of the assays failed to detect a small number of FMDV isolates due to the presence of nucleotide mismatches within the region targeted by the probes. Thus, no single stand-alone assay is capable of detecting FMDV with 100% sensitivity. Recently, Tam and colleagues reported fluorescence-based multiplex rRT-PCR assays for the detection of FMDV and virus typing. The assay was found to have greater sensitivity for detection but some cross-reactivity between some serotypes was also noted. Further work is in progress to develop rRT-PCR assays for serotyping of the virus.

4.5. Reverse transcription loop-mediated isothermal amplification (RT-LAMP)

The development of portable equipment for rRT-PCR has enabled molecular diagnosis of FMD possible in the field. This approach, however, requires expensive and fragile instruments and relies on precision thermocycling. Thus other approaches, like loop-mediated amplification (LAMP), were developed, which enable the tests to be conducted in the field using inexpensive tools. LAMP amplifies specific nucleotide sequences at a constant temperature and thus does not require a thermocycler. The assay is based on the principle of DNA amplification by an autocycling strand displacement reaction. The assay is performed using a set of two specially designed inner primers and two outer primers and a DNA polymerase with high strand displacement activity. The primers recognize 6 independent target sequences in the initial stage and 4 independent sequences during the later stages of the LAMP reaction. The reaction is carried out in less than an hour using a standard water bath or heating block and the results can be visualized with the naked eye. The advantages of its simple operation, rapid reaction and potential for visual interpretation without instrumentation make the technique attractive for field use in endemic countries. The RT-LAMP assay that has been developed for FMDV detection can be used in a high throughput system. This assay has, however, not yet been extensively evaluated for its ability to replace or supplement the techniques currently in use.

Virus isolation combined with ELISA and RT-PCR assays are reliable and accurate for the diagnosis of FMD but the shipment of samples from the field to the laboratory and the poor quality/amount of submitted samples can result in hindrance of early diagnosis of the disease. A rapid and specific test for disease diagnosis at the site of a suspected outbreak may allow timely implementation of control measures. A MAb-based chromatographic strip test for FMD diagnosis was developed by Reid et al.. The test was found to be at least as sensitive as the conventional antigen ELISA for the detection of FMDV in epithelial suspensions tested and had an equivalent 100% sensitivity on the cell culture supernatants of FMDV serotypes O, A, C and Asia-1. Further research is underway to develop chromatographic strip tests capable of ascertaining the serotype of the virus.

Detection of animals that have been infected with FMDV is of considerable importance for the control of FMD especially in a previously FMD free country or in a country with sporadic outbreaks. Both previously infected and vaccinated animals can have neutralizing antibodies in their sera, but it is important for trade purposes to be able to distinguish previously infected animals from those that have just been vaccinated against the disease. This is because a high proportion (up to 50%) of animals infected with FMDV can become “carriers”, these are defined as animals which continue to have infectious virus present within the oropharynx more than 28 days post-infection. The animals are clinically normal and can maintain this state for a long period (ca. 2–3 years in cattle). It is possible that such animals can act as a source of infection for other animals, indeed evidence for this process exists for African buffalo, however it has not been demonstrated in experimental studies with cattle alone. Viral replication during infection results in the production of both structural (SP) and nonstructural (NSP) proteins. Like the SPs, some NSPs are immunogenic. Vaccines consisting of purified preparations of inactivated 146S virions induce antibodies almost exclusively against the SP of the virus (at least after a small number of vaccinations). Thus it can be possible to discriminate between infected and vaccinated animals based on the detection of antibodies to NSPs.

Differentiation of infection from vaccination based on the antibodies to NSP has been reported using either panels of proteins or the individual proteins 2C or 3ABC. Early assays for the detection of anti-NSP antibodies relied on radioimmunoprecipitation or enzyme linked immunoelectrotransfer blot assays. However, these assays are not suited for rapid examination of large numbers of sera and thus alternative techniques like ELISA were developed. Several ELISAs based on the detection of antibodies to various NSPs of FMDV have been established. However, these ELISAs used species-specific conjugates, making simultaneous examination of sera from different species difficult. Thus there was a need for an assay which enabled simultaneous testing of sera from different species. Sørensen et al. developed a blocking ELISA which was species independent; using baculovirus expressed FMDV NSPs as antigen and polyclonal antibodies produced in guinea pigs as capture and detecting antibodies. The polyclonal antibodies were later replaced with monoclonal antibodies for high throughput. As antibodies to NSP persist for long periods, positive animals are not necessarily still infected although they can be carriers (see above). However, tests for detection of NSP antibodies cannot be used for detection of carrier animals as some persistently infected animals do not show sero-conversion against NSPs , the carrier animal status may occur in previously vaccinated animals in which only limited virus replication occurs. Moreover, no serological tests are currently available that can differentiate between FMDV carriers and other animals that show a serological response to FMDV. Furthermore, it can be difficult to differentiate between vaccinated and previously-infected animals if the vaccine used has been prepared from cell culture supernatant (i.e. not purified) and/or contains varying degrees of contaminating viral NSPs especially if multiple vaccinations have occurred.