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Foot-and-mouth disease (FMD) is an acute, highly contagious, and economically devastating viral disease of domestic and wildlife species. For effective implementation of FMD control program, there is an imperative need for developing a rapid, sensitive, and specific diagnostics which help in the identification of serotypes involved in the outbreaks. The humoral immune response of the Camelidae is unique since in these animals 75% of circulating antibodies are constituted by heavy-chain antibodies and 25% are conventional immunoglobulin with two identical heavy chains. In the present study, we developed and characterized FMD virus-specific single-domain heavy-chain antibodies (VHHs) against inactivated whole-virus antigens of FMDV serotypes O (INDR2/1975), A (IND40/2000), and Asia 1 (IND63/1972) vaccine strains. After six rounds of panning and enrichment, these VHHs were stably expressed in Escherichia coli cells. The VHHs directed against outer capsid proteins of FMD virus were successfully utilized as the capture antibody in liquid-phase blocking ELISA (LPBE) thus replacing rabbit coating antibodies. Our study demonstrated the utility of FMD virus-specific VHHs as potential candidates in FMD research and diagnostic application.

The prophylactic use of vaccines against exotic viral infections in production animals is undertaken exclusively in regions where the disease concerned is endemic. In such areas, the infection pressure is very high and so, to assure optimal protection, the most efficient vaccines are used. However, in areas considered to be free from these diseases and in which there is the possibility of only limited outbreaks, the use of Differentiation of Infected from Vaccinated Animals (DIVA) or marker vaccines allows for vaccination while still retaining the possibility of serological surveillance for the presence of infection. This literature review describes the current knowledge on the use of DIVA diagnostic strategies for three important transboundary animal diseases: foot-and-mouth disease in cloven-hoofed animals, classical swine fever in pigs and avian influenza in poultry.

Aims: To determine the diagnostic specificities of two commercial screening ELISA, the Bommeli/IDEXX Chekit FMD 3ABC indirect ELISA (ELISA-1) and the Ceditest FMDV NS blocking ELISA (ELISA-2), for foot-and-mouth disease (FMD) in cattle, sheep and pigs in New Zealand, and to compare them with other published studies. To consider the implications for FMD surveillance of using two ELISA in series, a consideration arising from the absence of a gold standard virus neutralisation test (VNT) in New Zealand., Methods: Serum samples from non-infected cattle (n=1,015), sheep (n=1,185), and pigs (n=233) from New Zealand were tested in ELISA-1 and ELISA-2 for the detection of serum antibodies against non-structural proteins of the FMD virus. The ELISA were performed according to the manufacturers' instructions., Results: The diagnostic specificities for ELISA-1 for cattle, sheep and pigs were 99.9%, 99.7% and 99.6%, respectively, and for ELISA-2 were 99.5%, 99.7% and 99.6%, respectively. False-positive reactors in one ELISA were negative in the other ELISA, and vice versa. Using the diagnostic sensitivity data taken from international studies and the diagnostic specificities calculated in this study resulted in overall specificities of 100% in cattle and sheep using serial test interpretation, and 99.2% and 99.0%, respectively, using parallel test interpretation. Diagnostic sensitivities available in the literature varied considerably, and the associated overall serial sensitivity could be as low as 78.7% in cattle and 33.2% in sheep., Conclusions: Diagnostic specificities for both ELISA for the target population were comparable with those obtained in livestock populations elsewhere. In surveillance to re-establish New Zealand's freedom from FMD, the use of two ELISA in series would improve the overall specificity in individual animals. However, care would be required to ensure that herd sensitivity was sufficient to detect infection.

Aim: To use disease modelling to inform a response team about the number of animals per herd/flock to be examined, and the start date and duration of clinical surveillance required to be confident that foot-and-mouth disease (FMD) was not present on an island in New Zealand with a population of approximately 1,600 cattle, 10,000 sheep and a small number of pigs, goats and alpacas., Methods: Because the probability of detecting clinical disease in (the) primary case(s) in larger herds and flocks was extremely low, deterministic and stochastic mathematical SLIR (susceptible, latent, infectious, recovered) models for the transmission of infection were constructed to estimate the date when clinical lesions in herds and flocks would be detected with 95% confidence. Surveillance targeted the first wave of infections following a suspect index case., Results: If 70 cattle in herds of about 400 cattle were examined it was estimated it would take approximately 13 (90% stochastic range 9-19) days from first exposure before it would be possible to achieve 95% confidence for detecting clinical signs for a low-virulence virus, and 9 (7-14) days for a high-virulence virus. The duration of sufficiently accurate clinical detection was 17 (15-19) days and 13 (12-14) days for low- and high-virulence viruses, respectively. A sample of 70 sheep from flocks of >1,000 would be required to achieve clinical detection at about the same time but with a shorter period of detection than for cattle. The duration of effective detection could be increased by examining a larger sample in most sheep flocks, however the small size of many cattle herds in the study population limited the confidence of detecting group-level disease in cattle, therefore necessitating repeated herd inspections. The model suggested that group-level detection was not feasible if it was based on elevated body temperature alone because of short durations of fever in infected animals., Conclusion and Clinical Relevance: Simulation modelling is a useful and powerful tool for informing ongoing surveillance activities in the face of an exotic disease incursion. Results of modelling suggested to start clinical inspection activities at 4 days and to continue regular inspection twice a week for about 35 days after the date of first exposure, to satisfy the required 95% confidence threshold of clinical detection of FMD in cattle herds and sheep flocks.

Within the past decade, changes in perceptions on the benefits of vaccination as an appropriate tool to achieve complete foot and mouth disease eradication have become evident. The former negative view was derived from misconceptions, resulting mainly from the belief that vaccines are not entirely effective and that vaccination masks asymptomatic viral circulation. The advent in the 1990s of vaccination policies implemented within a strategic eradication plan in South America, and during recurrence of the disease in disease-free regions contributed towards generating more reliable and visible outcomes of vaccination programs, paving the way towards a new perception. Particularly relevant was the development and application of novel serodiagnostic approaches to assess silent viral circulation, irrespective of vaccination. The use in South America of vaccination allied to serosurveys to accompany viral clarification during eradication campaigns and after emergencies clearly established the importance of this control tool to stop the spread of viral infection. This alliance gave input to break many myths associated with the use of vaccines, including the belief that immunized carrier animals pose an epidemiologic risk. This experience launched new concepts that supported the internationally recognized status of foot and mouth disease-free regions with vaccination and the 'vaccination to live' policy as an alternative to 'stamping out'.

This review describes current knowledge about persistent foot-and-mouth disease virus (FMDV) infections, the available methods to detect carrier animals, the properties of persisting virus, the immunological mechanisms, and the risk of transmission. In particular, knowledge about the carrier state, the period in which virus can be isolated from animals 28 days or longer post infection, is important, because the risk that animals may carry the virus will influence the diagnostic and preventive measures that need to be taken. Although many years of research have led to much knowledge about foot-and mouth disease and its causative agent, there are still numerous aspects of the virus and the disease that are not yet fully understood. Areas for further research on persistence of FMDV are discussed.

We had shown in preliminary studies with a small number of animals that antibodies against 2C could be detected in cattle and pigs which had been infected with FMDV but not in animals which had been vaccinated against the disease. To determine whether this test was generally applicable, sera from several hundred animals which had been vaccinated with different products in many countries have been tested in an ELISA using baculovirus expressed 2C. Our results show that only 1-2% of the sera gave a positive reaction by this method. In contrast, 100% of sera from convalescent animals gave a positive reaction. To be useful in differentiating between convalescent and vaccinated animals it is necessary to know how long these antibodies can be detected by our ELISA. We have determined the levels of antibodies against 2C and also other virus-specific proteins which are present in cattle and pigs following infection with FMDV. Our results show that levels of anti-3ABC antibodies could be detected by ELISA with baculovirus-expressed protein up to one year after infection. In contrast, the levels of anti-2C antibodies fell more rapidly than those against 3ABC indicating that the latter protein may be preferable for detecting convalescent animals. Nevertheless, we envisage that the final test format should include several virus-specific proteins to determine accurately the immune status of an animal.

Cattle which have been infected with foot-and-mouth disease (FMD) virus can be differentiated from those that have been vaccinated on the basis of the detection of antibody to one or more of the non-structural (NS) proteins of the virus. Cattle which have been protected by vaccination can become persistently infected with FMD virus (FMDV) without ever showing clinical signs. Vaccinated, protected cattle which are persistently infected cannot be distinguished from animals that merely have been vaccinated on the basis of serological tests for antibody to the structural proteins of FMDV. Sera were collected from groups of cattle for varying periods after exposure to infection under experimental conditions. On the basis of isolation of virus from probang samples collected during the course of the experiments it was possible to classify the cattle according to the following criteria; naive, infected and eliminated the virus (convalescent), infected and persistently infected with FMDV (carriers), vaccinated alone, vaccinated and either convalescent or carrier. Sera were examined for antibody to the NS proteins Lb, 2C, 3A, 3D, and 3ABC by an indirect profiling ELISA using E. coli-expressed fusion proteins as antigens. Considerable variation was observed in the antibody response to NS proteins of both naive and vaccinated animals following infection. The extent of individual variation was so great that convalescent animals could not be differentiated from carrier animals on the basis of their antibody response to any of the NS proteins examined. The majority of vaccinated, protected animals showed an antibody response to NS proteins, particularly 3ABC, following exposure to infection. However, the carrier state was demonstrated in some vaccinated, protected animals in which no antibody response to any of the NS proteins could be detected. The detection of antibody to NS proteins can therefore be used on a group, or herd, basis to detect circulation of virus in a vaccinated population but further investigations in the field are required to determine the sampling level necessary for statistical acceptance. On an individual animal basis, however, freedom from antibody to NS proteins in a vaccinated animal, or an animal of unknown history, does not necessarily imply that the animal is free from infection with FMD virus and, furthermore, the titre of antibody to NS proteins is not a useful predictive measure of whether or not an infected animal has successfully eliminated the virus.

Two non-vaccinated sheep were experimentally infected with FMDV and one day later 4 other sheep were brought in contact. Although the contact sheep showed no clinical signs, serology indicated that all sheep became infected. Various secretion samples, taken over a period of at least one month, and various tissue samples were examined for the presence of FMDV by RT-PCR and by virus isolation. FMDV was most often found in saliva (mouth swabs), followed by nasal secretion and sera. Faecal material, wool and milk were less suitable. The period of detection with the highest frequency of positive isolations was between 2 to 4 days pi for the infected sheep and between 5 to 10 days pc for the contact animals. It was established that in subclinically infected sheep, with a very low amount of virus present, FMD viral RNA could be detected by a sensitive RT-PCR-ELISA although virus isolation and standard RT-PCR remained negative. Moreover there was some evidence of active spreading of FMDV from the contact sheep to two sentinel pigs. This indicates that serologically positive contact sheep without clinical signs may be considered as a danger for the transmission of FMDV.

Four groups of ten nine-month-old Nelore heifers were used for this study. Each group received one of four foot-and-mouth disease (FMD) trivalent vaccines for the duration of the experiment. The four vaccine formulations (Normal, 2X, 4X and 8X) differed in 140S content to determine the serological reactivities to FMD virus (FMDV) nonstructural proteins 2C, 3ABC and 3D. Vaccination was by the intramuscular administration of vaccine on day 0, 180 and 360. Bleedings were done at 30 days post vaccination (dpv), 90 dpv, 30 days post revaccination (dpr), 90 dpr, and 30 days post third administration (dprr). There was a general tendency to have higher mean 3D responses with increased vaccine application but not with increased concentration of antigen. With 2C and 3ABC this tendency was not seen, neither with repeated application of vaccine nor with increased antigen concentration. All individual animal observations to 2C and 3ABC remained within three standard deviations of the average observed for naive bovids. Percent of positive (PP) reactions was determined using an ELISA for nonstructural proteins 2C, 3ABC and 3D expressed in baculovirus as previously described. A value of > 25 PP to 2C or 3ABC could be considered as an indication of previous infection or of the presence of viral activity. PP results between 18 and 25 PP suggest viral activity and animals should be retested. Those responses below 15 PP are suggestive of vaccination or naive status. As diagnosis in the laboratory is not divorced from the field epidemiological scene, the intermediate zone between 10 and 20 PP should be considered and acted upon according to the overall zoosanitary situation of that country or region and the purposes of the ongoing FMD control efforts.

A RT-PCR assay for the specific detection of RNA sequences from foot-and-mouth disease virus (FMDV) has been developed. The procedure permits also the detection of sequences that correlate with established FMDV serotypes. A computer program that allows selection of genotype-specific primers for RT-PCR amplification was used for the identification of FMDV specific sequences for PCR amplification on RNA replicase (3D) gene regions. Specific, rapid and highly sensitive detection was achieved for a wide collection of RNA samples from FMDV types C, A and O, either purified from tissue culture or extracted from lesions of infected animals. Similarly, serotype-specific primers were designed to amplify the carboxy-terminal end of the VP1 gene of FMDV types either C, A or O. The results of PCR amplification of different FMDV RNAs using type-specific primers are in agreement with the serological typing of the corresponding viruses. A combination of this approach with a simplified sample processing, carried out following direct adsorption of viral suspensions to microtiter plates, provides a rapid, reliable method of viral diagnosis.

ELISA's for antibodies to non-structural proteins of foot-and-mouth disease developed to date use recombinant proteins as antigens. To compare the antibody response to recombinant antigen and native antigen we developed an antigen capture ELISA for foot-and-mouth protein 3A. The concentration of 3A protein in virus cultures was significantly higher in the cell debris than in the supernatant, which made it possible to use proteins directly eluted from cells separated from virus culture using Filteraid. The antigen was trapped between one monoclonal antibody coated to the plate, and a second monoclonal antibody conjugated with horseradish peroxidase. The reaction of the second (conjugated) monoclonal antibody could be blocked by several post-infection sera. Further research has to be performed to determine whether or not this method can result in a reproducible serological test.

This paper summarises the development of monoclonal antibody (Mab)-based immunoassays measuring antibodies to non-structural proteins of FMDV to differentiate infection from vaccination. Of the three non-structural proteins 2C, 3C and 3ABC evaluated in this study, the polypeptide 3ABC was the most immunogenic. Three ELISAs for the detection of antibodies to 3ABC were developed. Two assays rely on the competition of test sera against either a anti-3A Mab or against antisera to 3ABC raised in rabbits and guinea-pigs. The third, 3ABC Mat-ELISA, based on the direct binding of antibodies to the 3ABC trapped by a specific Mab, provided the best combination of specificity and sensitivity. The 3ABC Mat-ELISA was extensively validated for cattle, either in experimental and in field conditions, showing specificity of 99% in vaccinated and in naive cattle and the capacity to detect silent infections in FMD-vaccinated populations. The test showed similar specificity and sensitivity in experimentally vaccinated and infected sheep.