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Table of Contents:
Taxonomy Information
  1. Species:
    1. Foot-and-mouth disease virus (Website 1):
      1. Common Name: Aftosa, Aphthous Fever
      2. GenBank Taxonomy No.: 12100
      3. Description: Foot-and-mouth disease virus (FMDV) is the prototype member of the Aphthovirus genus of the family Picornaviridae. Picornaviridae are nonenveloped viruses with single-stranded RNA genome of positive polarity. In addition, they are highly labile and rapidly lose infectivity at pH values of less than 7.0(Knipe et al., 2001). The virus exists in the form of seven different serotypes: A, O, C, Asia1, and South African Territories 1 (SAT1), SAT2, and SAT3, but a large number of subtypes have evolved within each serotype. Based upon the pioneering poliovirus genotyping studies of Rico-Hesse, FMDVs have been divided into genotypes based on comparisons of VP1 sequence data. For example, in a comprehensive study of FMD type O viruses they could be grouped into eight topotypes based on nucleotide differences of up to 15%. Similar studies are in progress for type A, C and Asia 1(Mason et al., 2003). Foot and Mouth Disease is endemic in parts of Asia, Africa, the Middle East and South America(Website 2). Outbreaks have occurred in every livestock-containing region of the world with the exception of New Zealand, and the disease is currently enzootic in all countries except Australia and North America. The disease affects domestic cloven-hoofed animals, including cattle, swine, sheep, and goats, as well as more than 70 species of wild animals, including deer. The recent outbreaks of foot-and-mouth disease (FMD) in a number of FMD-free countries, in particular Taiwan in 1997 and the United Kingdom in 2001, have significantly increased public awareness of this highly infectious disease of cloven-hoofed livestock. Furthermore, world concern following the terrorist attacks in the United States has raised the possibility that terrorist organizations or rogue states might target the $100 billion/year U.S. livestock industry by employing the etiologic agent of FMD. Although FMD does not result in high mortality in adult animals, the disease has debilitating effects, including weight loss, decrease in milk production, and loss of draught power, resulting in a loss in productivity for a considerable amount of time. Mortality, however, can be high in young animals, where the virus can affect the heart. In addition, cattle, sheep, and goats can become carriers, and cattle can harbor the virus for up to 2 to 3 years. FMD is one of the most highly contagious diseases of animals or humans, and FMDV rapidly replicates and spreads within the infected animal, among in-contact susceptible animals, and by aerosol(Grubman et al., 2004).
      4. Variant(s):
Lifecycle Information
  1. Foot-and-mouth disease virus lifecycle
    1. Stage Information:
      1. virion(Grubman et al., 2004):
        • Size: The FMD virion has a diameter of about 25 nm.
        • Shape: By electron microscopy, the FMD virion appears to be a round particle with a smooth surface. FMDV is distinguished from other picornaviruses by the lack of a surface canyon, or pit, which has been shown to be the receptor binding site for the entero-and cardioviruses. Another feature of the virion is the presence of a channel at the fivefold axis which permits the entry of small molecules, such as CsCl, into the capsid, resulting in FMDV having the highest buoyant density of the picornaviruses.
        • Description: FMDV, like other members of the Picornaviridae, has a relatively short infectious cycle in cultured cells. Depending on the multiplicity of infection, newly formed infectious virions begin to appear at between 4 and 6 hours after infection. The virus is cytocidal, and infected cells exhibit morphological alterations, commonly called cytopathic effects, which include cell rounding and alteration and redistribution of internal cellular membranes. The virus also causes biochemical alterations, including inhibition of host translation and transcription(Grubman et al., 2004).
    2. Picture(s):
      • Foot and Mouth disease virion



        Description: Aphthovirus: Molecular surface of Foot and Mouth Disease Virus, radially depth cued, as solved by X-ray crystallography(Website 135).
Genome Summary
  1. Genome of Foot-and-mouth disease virus
    1. Description: The virion is a 140S particle(Grubman et al., 2004). The genome of FMDV, which is over 8000 bases in length, is covalently bound at its 5-terminus to a 2324 amino acid residue genome-linked protein, 3B. In the mature virus, the genome is encapsidated in an icosahedral structure composed of 60 copies of four proteins (1A, 1B, 1C, and 1D). The genome contains a single long open reading frame (ORF), that has two alternative initiation sites, and the encoded polyprotein can be processed into over a dozen well-described mature polypeptides as well as a variety of partial cleavage intermediates. Most of the proteolytic events that produce these mature products are mediated by three viral proteinases, Lpro, 2A, and 3Cpro. The precise nature of the cleavage mechanisms utilized by 2A and the maturation cleavage of capsid protein 1AB into 1A and 1B remains unclear(Mason et al., 2003).
  2. Genome of Foot-and-mouth disease virus A
    1. Foot-and-mouth disease virus A(Website 106)
      1. GenBank Accession Number: NC_011450
      2. Size: 8161 bp(Website 106).
      3. Gene Count: The genome contains a single long open reading frame (ORF), that has two alternative initiation sites, and the encoded polyprotein can be processed into over a dozen well-described mature polypeptides as well as a variety of partial cleavage intermediates(Mason et al., 2003).
      4. Description: TEXT.
      5. Picture(s):
  3. Genome of Foot-and-mouth disease virus SAT 1
    1. Foot-and-mouth disease virus SAT 1(Website 108)
      1. GenBank Accession Number: NC_011451
      2. Size: 8173 bp(Website 108).
      3. Gene Count: The genome contains a single long open reading frame (ORF), that has two alternative initiation sites, and the encoded polyprotein can be processed into over a dozen well-described mature polypeptides as well as a variety of partial cleavage intermediates(Mason et al., 2003).
      4. Description: TEXT.
      5. Picture(s):
  4. Genome of Foot-and-mouth disease virus SAT 2
    1. Foot-and-mouth disease virus SAT 2(Website 110)
      1. GenBank Accession Number: NC_003992
      2. Size: 8203 bp(Website 110).
      3. Gene Count: The genome contains a single long open reading frame (ORF), that has two alternative initiation sites, and the encoded polyprotein can be processed into over a dozen well-described mature polypeptides as well as a variety of partial cleavage intermediates(Mason et al., 2003).
      4. Description: TEXT.
      5. Picture(s):
  5. Genome of Foot-and-mouth disease virus SAT 3
    1. Foot-and-mouth disease virus SAT 3(Website 112)
      1. GenBank Accession Number: NC_011452
      2. Size: 8170 bp(Website 112).
      3. Gene Count: The genome contains a single long open reading frame (ORF), that has two alternative initiation sites, and the encoded polyprotein can be processed into over a dozen well-described mature polypeptides as well as a variety of partial cleavage intermediates(Mason et al., 2003).
      4. Description: TEXT.
      5. Picture(s):
  6. Genome of Foot-and-mouth disease virus Asia 1
    1. Foot-and-mouth disease virus Asia 1(Website 114)
      1. GenBank Accession Number: NC_004915
      2. Size: 8167 bp(Website 100).
      3. Gene Count: The genome contains a single long open reading frame (ORF), that has two alternative initiation sites, and the encoded polyprotein can be processed into over a dozen well-described mature polypeptides as well as a variety of partial cleavage intermediates(Mason et al., 2003).
      4. Description: TEXT.
      5. Picture(s):
  7. Genome of Foot-and-mouth disease virus O
    1. Foot-and-mouth disease virus O(Website 116)
      1. GenBank Accession Number: NC_004004
      2. Size: 8134 bp(Website 116).
      3. Gene Count: The genome contains a single long open reading frame (ORF), that has two alternative initiation sites, and the encoded polyprotein can be processed into over a dozen well-described mature polypeptides as well as a variety of partial cleavage intermediates(Mason et al., 2003).
      4. Description: TEXT.
      5. Picture(s):
  8. Genome of Foot-and-mouth disease virus C
    1. Foot-and-mouth disease virus C(Website 118)
      1. GenBank Accession Number: NC_002554
      2. Size: 8115 bp(Website 118).
      3. Gene Count: The genome contains a single long open reading frame (ORF), that has two alternative initiation sites, and the encoded polyprotein can be processed into over a dozen well-described mature polypeptides as well as a variety of partial cleavage intermediates(Mason et al., 2003).
      4. Description: TEXT.
      5. Picture(s):
Biosafety Information
  1. General biosafety information
    1. Level: Due to the highly contagious nature and economic importance of FMD for many countries, the laboratory diagnosis and serotype identification of the virus should be done in a virus-secure laboratory. Countries lacking access to such a specialised national or regional laboratory should send specimens to the OIE/FAO World Reference Laboratory (WRL) for FMD(Website 132).
Culturing Information
  1. Primary culture (Website 132):
    1. Description: In animals with a history of vesicular disease, the detection of FMD virus in samples of vesicular fluid, epithelial tissue, milk, or blood is sufficient to establish a diagnosis. Diagnosis may also be established by the isolation of FMD virus from the blood, heart or other organs of fatal cases. Suspensions of field samples suspected to contain FMD virus once clarified are inoculated into cell cultures or unweaned mice. The cell cultures should be examined for cytopathic effect (CPE) for 48 hours. If no CPE is detected, the cells should be frozen and thawed, used to inoculate fresh cultures and examined for CPE for another 48 hours(Website 132).
    2. Medium: Primary culture of calf thyroid cells have been shown to be as sensitive for virus detection as intradermal inoculation in cattle, although primary pig, calf or lamb kidney cells can be used. But cryopreservation of the primary cells, after only one passage, result in less susceptibility and established cell lines such as IBR S2 or BHK 21 exhibit considerable inconsistency. In case of a vesicular condition in pigs, IBR-S2 cell line, susceptible to the SVD virus, permit the isolation of this virus, which grows only on porcine cells. IBR-S2 cells also prove to be useful to isolate porcinophilic strains of FMD virus(Remond et al., 2002).
    3. Upper Temperature: survival times at 37 degrees and 56 degrees celcius are 10 days and less than 30 minutes, respectively(Musser et al., 2004).
    4. Lower Temperature: The FMD virus is fairly stable at low temperatures, surviving for one year at 4 degrees celcius(Musser et al., 2004).
    5. Upper pH: 9.0(Musser et al., 2004).
    6. Lower pH: 6.0(Musser et al., 2004).
    7. Note: For laboratory diagnosis, the tissue of choice is epithelium. Ideally, at least 1 g of epithelial tissue should be collected from an unruptured or recently ruptured vesicle. Epithelial samples should be placed in a transport medium composed of equal amounts of glycerol and 0.04 M phosphate buffer pH 7.2-7.6, preferably with added antibiotics (penicillin [1000 international units (IU)], neomycin sulphate [100 IU], polymyxin B sulphate [50 IU], mycostatin [100 IU]). If 0.04 M phosphate buffer is not available, tissue culture medium or phosphate buffered saline (PBS) can be used instead, but it is important that the final pH of the glycerol/buffer mixture be in the range pH 7.2-7.6. Samples should be kept refrigerated or on ice until received by the laboratory. Where epithelial tissue is not available from ruminant animals, for example in advanced or convalescent cases, or where infection is suspected in the absence of clinical signs, samples of OP fluid can be collected by means of a probang (sputum) cup (or in pigs by swabbing the throat)(Website 132).
Epidemiology Information:
  1. Outbreak Locations:
    1. General: More than 50 of the 162 Member Countries of the Office International des Epizooties (OIE), the World Organisation for Animal Health, have obtained recognition from the OIE for freedom from foot and mouth disease (FMD) without vaccination. The virus continues to circulate in two-thirds of the remaining countries, thus dividing the globe into two zones. This has significant effects on international trade patterns in susceptible animals and animal products. Consequently, countries that do not have FMD-free status continue to suffer a severe handicap in terms of access to international markets. This situation was highlighted by the sudden and largely unexpected resurgence of FMD in Europe, South America and Asia at the beginning of the 21st Century. This endemic situation with respect to FMD in many parts of the world is a constant threat to countries that have acquired FMD-free status at considerable cost and effort. The threat has been exacerbated over the last decade by accelerated trade and movements of people due to globalization. At the same time, developed countries have either decreased or discontinued vaccination. The dangerous cocktail of globalization and non-immunised animals exploded in 2001, first in South America and then in the United Kingdom and other countries of the European Union(Vallat et al., 2003). In 1997 an FMD outbreak was reported in Taiwan, a country that had been free of the disease for 68 years. This devastating outbreak resulted in the slaughter of more than 4 million pigs, almost 38% of the entire pig population, at a cost of approximately U.S $6 billion and reminded the international animal health community of the severe economic consequences that a FMD outbreak could have for a previously disease-free country. Starting in late 1999 and 2000, a series of FMD outbreaks occurred in a number of countries in East Asia. This was followed by an outbreak in South Africa and culminated in the destructive outbreak in the United Kingdom, which then spread to the European continent. These outbreaks reemphasized the extreme virulence of the FMDV in a variety of animal species, the vulnerability of FMD-free countries as well as countries where FMD is enzootic to new viral strains, the efforts of globalization on increasing the risks of disease incursion, and hence the need for countries to more closely monitor for the presence of exotic disease(Grubman et al., 2004).
    2. Europe and Central Asia: In the past, the disease has ravaged European livestock, but has been gradually brought under control, at great cost, by preventive vaccination programmes, supplemented by the destruction of infected herds in most of the countries of continental Europe and, in the United Kingdom (UK) and Nordic countries, by destruction of infected herds alone. After careful evaluation of the two possible options for preventing the re-occurrence of the disease in Europe to either continue or discontinue mass vaccination the European Union decided to prohibit all vaccination after 1991. FMD remained and is still endemic in the Middle East, including Asian Turkey (Anatolia), and despite efforts of the Governments of Turkey and Europe, Anatolia appears to be a permanent source of sporadic outbreaks in the Balkans and a threat to Europe. In recent years, FMD was reported mainly in the Balkans. Despite these occasional incursions of FMD into south-east Europe, in all cases, the control measures were efficient and the disease never spread to such an extent as to become endemic. A major outbreak, which affected 2,030 farms occurred in the UK between February and September 2001. This was the first major epidemic of FMD in Europe since preventive vaccination had been abandoned in continental Europe in 1991. The disease also spread to Ireland, France and the Netherlands although the number of outbreaks was limited in these countries(Leforban et al., 2003).
    3. South America: Since the signing in 1987 of the Hemispheric Plan for the Eradication of Foot-and-Mouth Disease by the countries of South America, clinical cases of foot and mouth disease have decreased significantly throughout the continent. During the early 1990s, national laboratories diagnosed an average of 766 cases per year in South America. By the late 1990s, this continent-wide average had fallen to 130. By the end of the 1990s, the international community recognized Argentina, Chile, Guyana, and Uruguay as free of FMD without vaccination. In 1999, clinical signs of FMD were absent in 60% of all cattle on the continent. These cattle represented 41% of all herds in South America and extended over 60% of the geographical area of the continent. However, in the spring of 2001, FMD re-appeared in certain countries of the Southern Cone. This wide-spread re-occurrence of the disease in Argentina, Uruguay and the State of the Rio Grande do Sul in Brazil called into question whether countries in South America can achieve and maintain FMD-free status, with or without vaccination(Melo et al., 2003).
    4. Middle East and North Africa: Only one country in the Middle East (Cyprus) is presently included in the OIE list of foot and mouth disease-free countries. The region is regarded as that most affected by FMD in the world. FMD has been recorded in all countries in the Middle East on numerous occasions between 1960 and 2000, serotype O being the most prevalent. In the past, exotic FMD viruses were the cause of panzootics, which spread to many areas of the region, even extending to the frontier of Europe. A remarkable example was the rapid dissemination of serotype SAT 1 virus, which occurred initially in Bahrain in December 1961. The virus spread north-westwards to reach Iraq, Jordan, Israel, and Syria by April 1962, continuing to Iran and Turkey. In September 1962, this serotype crossed the Bosporus to enter Europe for the first time, and in November, caused an outbreak further west, near the border between Turkey and Greece. Historically, epidemics mainly affected cattle and spread from east to the west in the Middle East. The slow spread of FMD from Tunisia in 1989 to Morocco in 1991 exemplifies the difficulty in controlling the disease since unregulated movements of herds of small ruminants may play an important role in spreading infection. The situation in the Middle East and North Africa constitutes a threat to other regions of the world, especially Europe(Aidaros et al., 2003).
    5. East Asia: Japan regained the status of freedom from foot and mouth disease without vaccination in September 2000 and the Republic of Korea likewise obtained this status in September 20001. However, new outbreaks of FMD caused by the Pan-Asian topotype have occurred in pigs in the Republic of Korea since May 20002. Taipei China has not experienced an outbreak of FMD since February 20001 and the country is currently implementing and eradication programme. These countries had been free from FMD for many decades when in 1997, the FMD virus once again invaded the region, particularly in 2000; this resulted in widespread occurrence of the disease. The types of FMDV were investigated by genome analysis, and in each case the virus concerned was found to be a member of the pan-Asian O lineage(Sakamoto et al., 2003).
    6. South-East Asia: Of the ten countries in South-East Asia, FMD is endemic is seven (Cambodia, Laos, Malaysia, Myanmar, the Philippines, Thailand, and Vietnam) and three are free of the disease (Brunei, Indonesia and Singapore). Part of the Philippines is also recognized internationally as being free of FMD. From 1996 to 2001, serotype O viruses caused outbreaks in all seven of the endemically infected countries. On the mainland, three different type O lineages have been recorded, namely: the South-East Asian topotype, the pig-adapted or Cathay topotype and the pan-Asian topotype. Prior to 1999, one group of SEA topotype viruses occurred in the eastern part of the region and another group in the western part. However, in 1999, the pan-Asian lineage was introduced to the region and has become widespread. The Cathay topotype was reported from Vietnam in 1997 and is the only FMD virus currently endemic I the Philippines. Type Asia 1 has never been reported from the Philippines but was reported from all countries on the mainland except Vietnam between 1996 and 2001. Type A virus has not been reported east of the Mekong River in the past six years and seems to be mainly confined to Thailand with occasional spillover into Malaysia. The distribution and movement of FMD in the region is a reflection of the trade-driven movement of livestock(Gleeson et al., 2003).
    7. Sub-Saharan Africa: Six of the seven serotypes of FMDV (i.e. all but Asia 1) are prevalent in Africa although there are marked regional differences in distribution. Three of these serotypes are unique in Africa -- the three SAT serotypes. Serotype C may also now be confined to Africa because it has not been reported elsewhere recently. In southern Africa at least, the SAT serotypes have an intimate and probably ancient association with African buffalo (Syncerus caffer) that is instrumental in their maintenance. Within each of the six prevalent serotypes, with the possible exception of C, there are a number of different lineages with more or less defined distributions (topotypes) that in some cases are sufficiently immunologically different from one another to require specific vaccines to ensure efficient control. This immunological diversity in prevalent serotypes and topotypes, in addition to the uncontrolled animal movement in most parts of the continent, render FMD difficult to control in present circumstances. This fact, together with poorly developed intercontinental trade in animals and animal products has resulted in the control of FMD being afforded a low priority in most parts of the continent, although the northern and southern regions of the continent are an exception. As a consequence, eradication of FMD from Africa as a whole is not a prospect within the foreseeable future(Vosloo et al., 2002A).
  2. Transmission Information:
    1. From: Artiodactyla , To: Humans (Sutmoller et al., 2003)
      Mechanism: The circumstances in which it does occur in humans are not well defined, though all reported cases have had close contact with infected animals. There is one report from 1834 of three veterinarians acquiring the disease from deliberately drinking raw milk from infected cows. There is no report of infection from pasteurized milk, and the Food Standards Agency considers that foot and mouth disease has no implications for the human food chain(Prempeh et al., 2001). People in contact with infected animals are exposed to enormous amounts of virus. Using large-volume air samplers, Sellers found that in a period of 30 minutes 10 million IU could be collected from the air of a stable housing infected pigs(Sutmoller et al., 2003). Sampling of human subjects, who had been in contact with diseased animals, showed that virus could be recovered from the nose, throat, and saliva of these people immediately after leaving the room. Nasal swabs of such persons usually contain 100-1000 IU, but some may contain as many as 10,000 IU(Sutmoller et al., 2003).
    2. From: Humans , To: (Musser et al., 2004). (Sutmoller et al., 2003)
      Mechanism: Nonsusceptible animals, such as horses, foxes, rats, birds, and humans, are a means of mechanical spread of the virus(Musser et al., 2004). During the FMD outbreak that took place in the UK in 2001, disease spread was reported to occur frequently by mechanical carriage of virus between flocks by humans or vehicles(Kitching et al., 2002B). People who work with infected animals or materials will carry FMD virus on their hair and skin and on clothes. If contaminating virus is not removed by showering and change of clothes there is a high probability that a susceptible animal will receive sufficient virus to become infected by fomites, aerosol or handling. In the 1967-68 epidemic in UK, veterinarians were incriminated in 6 of 51 outbreaks and in 4 other cases non-veterinary personnel were involved. Sellers reported that, under exceptional circumstances, FMD virus carried in the nose and throat could be transmitted from man to animals. Shortly after begin in contact with infected animals, these researchers discarded clothes, showered and moved to a different compound and succeeded, in transmitting and infecting one steer by examining the animals and at the same time sneezing, snorting, coughing and breathing a the muzzles of the animals. The exposure of each animal to this treatment lasted 30s for each person. However, in practice, such intimate contacts between people and susceptible cloven-hoofed animals is unlikely(Sutmoller et al., 2003).
    3. From: Artiodactyla , To: Cattle (Kitching et al., 2002C)
      Mechanism: Susceptible cattle coming into contact with an infected animal, whether sheep, goat, pig or wildlife species may be infected by the respiratory rout or through an abrasion on the skin or mucous membranes(Kitching et al., 2002C). Infection of cattle generally occurs via the respiratory route by aerosolized virus. Infection can also occur through abrasions on the skin or mucous membranes, but is very inefficient, requiring almost 10,000 times more virus. Virus is excreted into the milk of dairy cattle as well as in semen, urine and feces, and calves can become infected by inhaling milk droplets(Grubman et al., 2004). In 1981, cattle on the Isle of Wight in the United Kingdom were infected by windborne aerosol virus produced by infected pigs in Brittany, France and the virus was carried over 250 km across the English channel(Kitching et al., 2002C).
    4. From: Cattle , To: Artiodactyla (Kitching et al., 2002C)
      Mechanism: The transmission of FMD virus within an unvaccinated herd is usually rapid, as was seen during the recent outbreak in the UK win which over 90% of a group could be showing clinical signs by the time the disease was first identified. Even within a vaccinated herd, the aerosol production of virus from a single infected animal can overcome the immunity of others in the herd resulting in a further increase in the level of challenge and the appearance of clinical disease(Kitching et al., 2002C). In all parts of the world with the exception of sub-Saharan Africa, FMD in free-ranging or captive wildlife appears to be an extension of the disease in lifestock. This has been documented for free-ranging moose, Alces alces, as well as in fallow, roe and red deer in Europe. In the former Soviet Union, FMD was described in free-ranging reindeer Rangifer tarandus and saiga Saiga tatarica, while in India severe clinical signs and mortality were reported in the blackbuck Antilope cervicapra. High ranging mortality also occurred in free-ranging mountain gazelles in Israel during epidemics in cattle. Similarly, outbreaks of FMD in zoological gardens in Paris, Zurich, and Buenos Aires coincided with outbreaks in FMD in domestic animals(Thomson et al., 2003). Infected cattle also aerosolize large amounts of virus, which can infect other cattle in addition to other species(Grubman et al., 2004). Infected cattle also produce up to log10 5.1 TCID50 of aerosol virus per day, and a large dairy herd could infect neighboring herds with their combined output of virus(Grubman et al., 2004). Cattle with FMD are usually the greatest producers of FMD virus of all species. It can be estimated that one infected cow, in addition to exhaled air, contaminates the environment with some 10 billion or more IU during the first week of disease with excretions (faeces, urine, milk), salivation, sloughed-off blister epithelium and vesicular fluid. The total amount of virus excreted by pigs and sheep is, in general, much smaller than cattle(Sutmoller et al., 2003).
    5. From: Artiodactyla , To: Pigs (Kitching et al., 2002D)
      Mechanism: Pigs usually become infected with the virus by eating FMDV-contaminated products, by direct contact with another infected animal, or by being placed in a heavily contaminated environment, for example a pen, an abattoir lairage or a transport lorry that has previously housed or transported infected animals. Pigs are considerably less susceptible to aerosol infection than ruminants, and recent studies using several virus strains indicated that a pig may require up to 6,000 50% tissue culture infective doses TCID50, possibly as much as 600 times more than the exposure to aerosol virus required by a bovine or an ovine, to cause infection. While this figure may vary with individual pigs and potentially could be different for certain FMDV strains, it was consistent with many field and experimental observations which described situations in which pigs were not infected when physically separated from infected animals. Once infection is established within a pig herd, transmission by direct contact between infected and susceptible animals can be very rapid, and many routes of viral entry bay be involved, i.e. aerosol, oral, mucosal, and through damaged epithelium which may play an important role under intensive conditions or other conditions (transportation and at abattoirs) where aggression among pigs may be increased(Kitching et al., 2002D). In the United States, feral pig populations are very large and widespread. Foot and mouth disease in feral pigs has been the subject of considerable research and modeling. For example, Australian scientists have estimated that FMD will spread among feral pigs at a rate of 2.8 km per day when pigs are at a fairly low population density (1 to 2/km2)(Leighton et al., 2002).
    6. From: Pigs , To: Artiodactyla (Kitching et al., 2002D)
      Mechanism: Pigs infected with FMDV do produce more aerosol virus than ruminants, and the same studies showed that the aerosol production from infected pigs infected with different stains also differed considerably. Maximum excretion of aerosol virus coincides with development of clinical disease and lesions on the snout, tongue and feet, and declines over the following 3 to 5 days as the antibody response develops(Kitching et al., 2002D). In 1981, cattle on the Isle of Wight in the United Kingdom were infected by windborne aerosol virus produced by infected pigs in Brittan, France and the virus was carried over 250 km across the English Channel(Kitching et al., 2002C).
    7. From: Artiodactyla , To: Sheep and Goats (Grubman et al., 2004)
      Mechanism: Sheep are highly susceptible to virus infection via aerosol and can excrete airborne virus; however during outbreaks they are most like infected by contact with other animals(Grubman et al., 2004). As is the case with other ruminants, sheep and goats are highly susceptible to infection with FMD virus by the aerosol route. Aerosol production by pigs can be as high as log10 8.6 TCID50 per day, theoretically sufficient to infect over 20 million sheep. But sheep are less likely to become infected by airborne virus than cattle because of their lower respiratory volume. Sheep and goats are probably most often infected by direct contact with infected animals. The virus may infect sheep and goats through abrasions on the skin or mucous membranes, through contaminated food, as well as by the respiratory route(Kitching et al., 2002B).
    8. From: Sheep and Goats , To: Artiodactyla (Kitching et al., 2002B)
      Mechanism: Aerosol production by infected sheep is considerably less. Aerosol transmission from infected sheep is unlikely to occur over distances greater than 100 meters. Sheep-to-sheep spread by contact appears to be restricted, to the extent that the rate of transmission within an affected flock is lower than that observed in infected pig or cattle herds. A good example of this phenomenon is illustrated by the outbreak of FMD that took place in Greece during 1994. Serological investigations showed that in many of the affected flocks not all individuals had sero-converted to the virus, indicating that the virus had not disseminated sufficiently to infect entire flocks. Similarly, evidence from the recent UK epidemic shows considerable variation in the level of intra-flock infection rates. On one farm visited, only 5% of 237 sheep that were blood tested were sero-positive, and 3% were virus-positive, whereas 91% of the 75 cattle present were clinically affected(Kitching et al., 2002B). The probability of transmission of FMD virus from infected sheep is highest during the viraemic phase and peaks at or just before the appearance of clinical signs. This period correlates well with the period of virus excretion, which ends at the point of sero-conversion. Levels of virus excretion are strain specific(Kitching et al., 2002B). Because it is very difficult to make a clinical diagnosis of FMD in sheep, the disease can be spread to other livestock prior to detection(Grubman et al., 2004). A recent study by Hughes has provided supportive evidence for the observed difference between the dynamics of FMD transmission in sheep populations as compared with cattle and pigs. The study showed that, using the 1994 Greek outbreak strain, there was significant reduction in the level of infection and estimated transmission rates over time during serial passage though groups of sheep. These results infer that some, possibly most, strains of FMD virus may die out if they are restricted to sheep. Infection of cattle and pigs may be sufficient to increase the level of circulating virus and consequently the probability of transmission of infection to in-contact sheep, thereby re-establishing the disease. This hypothesis requires further investigation using other strains of FMD virus(Kitching et al., 2002B).
    9. From: African Buffalo , To: African Buffalo (Vosloo et al., 2002A)
      Mechanism: Most infections are believed to occur as a result of childhood epidemics within buffalo breeding herds when large numbers of juveniles (about 10% of each breeding herd) are recruited annually into the susceptible populations. They can become infected on the waning of maternally-derived immunity obtained from colostrum(Vosloo et al., 2002A). Infection of individual animals within the breeding herds of buffalo usually occurs when maternal immunity starts to wane at 2-4 months of age. Calves are not necessarily infected by their dams, and it is presumed that SAT viruses spread mainly during minor epidemics among animals in breeding herds, with carriers ensuring that the viruses survive interepidemic periods. Transmission of SAT type viruses between individual buffaloes appears to occur by two processes: (1) contact transmission between acutely infected and susceptible individuals, which is likely to account for most infections, and (2) occasional transmission between carrier buffaloes and susceptible individuals. However, the mechanism whereby carrier transmission occurs between buffaloes is obscure. A possibility, for which the evidence is still obscure, is sexual transmission(Thomson et al., 2003).
    10. From: African Buffalo , To: Cattle (Bastos et al., 2003)
      Mechanism: Transmission of SAT-type virus from persistently infected African buffalo to cattle under experimental and natural conditions has been unequivocally demonstrated(Bastos et al., 2003). Buffalo calves lose their maternal antibodies at 2-6 months of age and thereafter show seroconversion for one or more of the three types of SAT virus. Apparently during that period they acquire the infection from their dams. It has been quite difficult to show that the infection can pass from buffalo to domestic livestock species, but studies of Thomson in 1992 indicated that young buffalo in the acute stage of infection are likely to be the most infectious animals in the herd. Those contagious calves are responsible for maintaining FMD virus in the herd and the spread of FMD to other wildlife or domestic livestock species(Sutmoller et al., 2002). Buffalo bulls in the field have been observed by farmers to mount domestic cows on occasion and it is possible that sexual activity may be a way in which SAT-type viruses are transmitted form African buffaloes to cattle(Thomson et al., 2003).
    11. From: African Buffalo , To: Impala (Thomson et al., 2003)
      Mechanism: African buffaloes in the KNP in south Africa have been shown to be the usual source of infection for impala on the basis of sequencing studies(Thomson et al., 2003). Other susceptible species, principally impala, probably become exposed while infection is circulating among buffalo calves, possibly around permanent water points, where animals congregate(Thomson et al., 2003).
    12. From: Hedgehog , To: Artiodactyla (Thomson et al., 2003)
      Mechanism: There is evidence suggesting transmission in both directions between cattle and European hedgehogs Erinaceus europaeus, and for latent infections of hibernating hedgehogs. However, these reports should be viewed with caution, because there is not evidence that hedgehogs have participated in the propagation of FMD viruses in Europe or Africa in recent times(Thomson et al., 2003). During and outbreak in Norfolk in 1946 nine hedgehogs were found dying from FMD over an 11-week period. It was thought that there had been hedgehog to hedgehog transmission and that they had contributed to local spread of the disease. The authors considered that the outbreak could have originated in the hedgehogs as they had access to kitchen scraps containing imported meat products(Simpson et al., 2002).
    13. From: Cattle , To: Deer (Thomson et al., 2003)
      Mechanism: It might be considered that they could act as ideal hosts for the virus and that they might well acts as excellent reservoirs of infection. In the 1924 outbreak in California, over 20,000 deer were shot in order to control the spread of disease and over 2,000 had active or healed lesions. Yet in Europe, deer do not appear to act as disseminators of virus. In the UK during periodic outbreaks of FMD over the past fifty years, there has never been any suggestion that deer have directly involved. In an area such as the New Forest in the south of England, which is over 1000 square miles in extend, cattle and pigs share the forest grazings with at least four species of deer. Despite outbreaks in the farm livestock, no deer has ever been seen to be infected clinically. Many years ago, many deer were culled during an outbreak so that they could be examined by veterinary experts none were found with lesions(McDiarmid et al., 1975). Roe deer Capreolus capreolus, fallow deer Dama dama, sika deer Cervus Nippon, red deer Cervus elaphus and muntjac Muntiacus muntjac excreted FMD virus following experimental infection in approximately the same quantities as sheep and cattle. It has furthermore been shown that infection between deer and domestic livestock may occur in either direction(Thomson et al., 2003). White-tailed deer were shown to be susceptible to infection with FMD virus type O. The disease was transmitted by contact from deer to other deer, from deer to cattle, and from cattle to deer. White-tailed deer were clearly susceptible to infection from this strain of FMD virus both by intranasal inoculation and by contact exposure(McVicar et al., 1974).
    14. From: Deer , To: Artiodactyla (Thomson et al., 2003)
      Mechanism: Most deer, including white-tailed deer and mule deer like those found in North America, were assigned a high hazard category because of demonstration of FMDV transmission. At least one white-tailed deer remained a carrier of FMDV for 11 weeks after infection. Exotic deer, including red, sika, and fallow, have been gaining popularity for use on deer farms or game ranches. Such deer have been found to both acquire and transmit FMDV under natural conditions. There is no information on transmission from other cervids such as moose and elk. Accordingly, those cervids were placed in a moderate category(USDA et al., 1994). The opinion that FMD infected deer constitutes a low risk because sick animals hide and probably die, is not valid. Like cattle or sheep, susceptible deer are very infectious prior to the development of the lesions while they still actively move and graze. Also deer with sub-clinical or minor lesions will still roam around(Sutmoller et al., 2003). Roe deer Capreolus capreolus, fallow deer Dama dama, sika deer Cervus Nippon, red deer Cervus elaphus and muntjac Muntiacus muntjac excreted FMD virus following experimental infection in approximately the same quantities as sheep and cattle. It has furthermore been shown that infection between deer and domestic livestock may occur in either direction(Thomson et al., 2003).
    15. From: Artiodactyla , To: Llama. (Lubroth et al., 1990)
      Mechanism: Foot-and-mouth disease virus was shown to be transmitted from either cattle to llamas, llamas to swine or llamas to llamas(Lubroth et al., 1990). In a large experimental study where llamas were exposed to FMD infected pigs and cattle, the llamas were poorly susceptible to FMD and the few infected llamas only had virus in their pharyngeal mucosa for a short time. Moreover, recovered animals did not transmit virus to other susceptible species. Clearly, to become infected llamas need exceptional infection pressure. The lack of sero-conversion, when exposed to normal outbreak situations, indicates that llamas do not play a role in FMD epidemics(Sutmoller et al., 2003).
    16. From: Artiodactyla , To: Rodents. (Thomson et al., 2003)
      Mechanism: Capybaras (Hydrochaeris hydrochaeris) are susceptible to FMD and they may play a role in the epidemiology of FMD in cattle in South America(Thomson et al., 2003). The capybara, a large rodent that lives in groups in close contact with grazing livestock, has been shown to develop clinical disease. Capybaras were exposed to FMDV type O by the intramuscular route and virus was isolated from most of the organs collected from four animals slaughtered 24-48 hours post-inoculation. The remaining capybaras developed vesicular lesions on their feet between 72 and 96 hours post-infection and virus was shed in feces until at least 10 days post-infection. The susceptibility to capybaras to this strain of FMDV by intramuscular inoculation does not necessarily mean that they constitute an actual reservoir and the epidemiological significance of FMD in the species is unknown. Most likely cattle are the primary host and capybaras a dead-end host(Sutmoller et al., 2003).
    17. From: Rodents. , To: Artiodactyla (Sutmoller et al., 2003)
      Mechanism: Rats, mice and birds might transmit the disease mechanically. FMDV has been found in rat feces and urine and in bird droppings. The maximum titer found in rat feces was 1000 ID50 per g. Sellers states that the feces from 160 rats would be required to attain sufficient virus to infect cattle by ingestion. However, the chance of infection will also depend on the numbers of animals contacting the infectious source, raising the likelihood of a transmission occurring with sources containing low viral loads. It has also been suggested that contamination of dust by rat feces or urine may lead to infection by inhalation. In this instance only a few IU would be required. It must be emphasized that the role of vermin such as rats is insignificant under conditions of extensive cattle management as occur in South America. Vermin might spread FMD from infected premises, particularly when cleaning and decontaminating have eliminated normally available feed sources(Sutmoller et al., 2003).
    18. From: Invertebrates. , To: Artiodactyla (USDA et al., 1994)
      Mechanism: Although the role of flies and ticks in the epizootiology of FMD is not usually large, it has been demonstrated that ticks and some species of biting flies can transmit the virus through bite. Tick, flies, and biting flies were categorized as high hazards, based either on transmission capability or long carrier status (whether mechanically or biologically). Houseflies can carry FMDV both externally and internally; whether they can transmit the virus is unknown. It is unlikely that the virus multiplies in the cells of invertebrates. However, experimental transovarial infection of a portion of a population of Dermacentor ticks has been reported(USDA et al., 1994).
  3. Environmental Reservoir:
    1. Artiodactyla(Sutmoller et al., 2003):
      1. Description: Currently, carrier animals are defined as those from which live virus can be isolated at 28 days, or later, after infection. The role of carrier animals in the spread of virus in the filed is still controversial. The mechanisms for the establishment and maintenance of the carrier state are not well understood, since persistence can occur in animals exposed to virus after either acute disease or vaccination. It does appear that the immune status of the animal probably controls the level of virus replication. Alexanderson and colleagues have proposed two mechanisms for the development of persistence in the pharynx. One suggests that FMDV can infect immune system cells, such as macrophages, or other immunologically privileged sites, leading to evasion of the immune response. The second mechanism proposes that the virus exploits the host response to provide favorable intracellular conditions for long-term persistence, possibly by utilizing cytokine signalling(Grubman et al., 2004).
    2. cattle(Sutmoller et al., 2003):
      1. Description: In the late 50s and early 60s it was shown that in countries with endemic FMD, virus could be isolated from the mucous and cell debris form oropharyngeal mucosa in as much as half of the cattle population. However, dependent on the virus strain, type of cattle and local circumstances figures may vary and individual cattle will show differences in duration and level of virus excretion. The long-term persistence of FMDV in the pharyngeal area of cattle is measured in years rather than in months(Sutmoller et al., 2003). More than 50% of cattle have recovered from infection with FMD virus and vaccinated cattle that have had contact with live virus become carriers. The FMD virus persists particularly in the basal epithelial cells of the pharynx and dorsal soft palate, and can be recovered from some animals for over three years, although the carrier state does not usually extend beyond a year(Kitching et al., 2002C).
      2. Survival: The FMD virus is pH sensitive, with an optimal pH between 7.2 and 7.6, and is inactivated at a pH less than 6.0 and greater than 9.0. The FMD virus is fairly stable at low temperatures, surviving for 1 year at 4 degrees C, but can survive for progressively shorter times as temperature increases. For instance, survival times at 37 degrees and 56 degrees C are 10 days and less than 30 minutes, respectively. However, the virus is not inactivated during pasteurization at 72 degrees C for 15 seconds. Milk from naturally infected cows must be heated to 100 degrees C for greater than 20 minutes for virus inactivation. The virus can be persistent in the environment and survives in the soil for 3 days in the summer and 28 days in the winter. In dry fecal material, the virus survives for 14 days in the summer, whereas it can survive for 6 months in manure slurry in winter conditions. The virus survives for up to 39 days in urine. To inactivate the FMD virus in slurry, the slurry must be heated to 67C for 3 minutes(Musser et al., 2004). Unlike those of other picornoviruses, the FMDV capsid is dissociated at pHs of below 6.5 into 12S pentameric subunits. The reason for this instability is thought to be a cluster of His residues at the interface between BP2 and VP3, which become protonated at low pH, weakening the capsid through electrostatic repulsion. This low-pH-induced instability of FMDV leads to difference in the mechanism of its uncoating upon infection of cells compared to that for other picornaviruses and also probably plays a role in the targeting of the virus to specific tissues and organs in susceptible hosts(Grubman et al., 2004). Preserved by refrigeration and freezing and progressively inactivated by temperatures above 50C. Inactivated by sodium hydroxide (2%), sodium carbonate (4%), and citric acid (0.2%). Resistant to iodophores, quaternary ammonium compounds, hypochlorite and phenol, especially in the presence of organic matter. Survives in lymph nodes and bone marrow at neutral pH, but destroyed in muscle when pH is less than 6.0 i.e. after rigor mortis. Can persist in contaminated fodder and the environment for up to 1 month, depending on the temperature and pH conditions(Website 121). When compared with viruses such as the smallpox virus, FMD virus is relatively fragile, but under the cool, moist, and often cloudy conditions (low ultraviolet light concentration) of winter and spring in the UK, it survives for several days and often longer(Gibbs et al., 2003).
    3. sheep and goats(Sutmoller et al., 2003):
      1. Description: Sheep and goats less frequently become a carrier and for shorter periods of than cattle often lasting for only 1-5 months. However, in some animals the carrier state may last up to 12 months. Unequivocal evidence of transmission form carrier sheep or goats has neither been demonstrated under experimental conditions or in the field(Sutmoller et al., 2003). A recent study by Hughes has provided supportive evidence for the observed difference between the dynamics of FMD transmission in sheep populations as compared with cattle and pigs. The study showed that, using the 1994 Greek outbreak strain, there was significant reduction in the level of infection and estimated transmission rates over time during serial passage through groups of sheep. These results infer that some, possibly most, strains of FMD virus may die out if they are restricted to sheep(Kitching et al., 2002B).
    4. Cervidae(Sutmoller et al., 2003):
      1. Description: FMDV was seldom recovered from the pharynx from red and roe deer beyond 14 days post-exposure. Fallow deer carried the virus for a minimum of 5 weeks. Two months after exposure 6 from the 12 deer were still positive. White tailed deer in the USA carried FMD virus regularly up to 5 weeks after exposure, but one deer had virus in the OP fluid as long as 11 weeks post-exposure(Sutmoller et al., 2003). Exotic deer, including red, sika, and fallow, have been gaining popularity for use on deer farms or game ranches. Such deer have been found to both acquire and transmit FMDV under natural conditions. There is no information from other cervids such as moose and elk(USDA et al., 1994). All of the deer tested 4 weeks after exposure had virus in the OPF and therefore could be classified as carriers. Generalization is not possible with such a small experimental group but he presence of virus in the OPF of one animal 11 weeks after exposure makes the existence of relatively long term carriers a distinct possibility(McVicar et al., 1974).
    5. African Buffalo(Sutmoller et al., 2003):
      1. Description: Individual animals may maintain the infection for periods of at least 5 years, but in most buffalo the rates peak in the 1-3 year age-group. Individual buffalo may be persistently infected with more than one type of FMDV in the pharyngeal region(Sutmoller et al., 2003). African buffalo are efficient maintenance hosts of the SAT type viruses, with individual animals maintaining the virus for up to 5 years, and isolated herds for up to 24 years although persistence in individual buffaloes is probably not lifelong(Vosloo et al., 2002A).
    6. Animal-origin product, fomites or vehicles(USDA et al., 1994):
      1. Description: Many animal-origin products and other fomites or vehicles can serve as possible modes of FMDV transmission. A total of 76 products (15 nonfood and 61 food) and 21 fomites were identified by the USDA in this publication(USDA et al., 1994). Diseased animals excrete the virus in tremendous quantities. The most common way of dissemination is by infected live animal and contaminated animal products. Indirect transmission can be made by people, vehicles, equipment, hay or bedding contaminated with feces or urine of diseased animals. Over the years, illegal activities, have often been attributed to introductions of FFMD into non-infected countries, such as the importation of infected meat and feeding to pigs of non-heat treated swill(Sutmoller et al., 2003). The virus has a remarkable capacity for remaining viable in carcasses, in animal byproducts, in water, in such materials as straw and bedding, even in pastures. In 1994, USDA examined the source of all primary FMD outbreaks worldwide from 1870 through 1993. The study found that of the 558 outbreaks with a reported source, contaminated meat, meat products or garbage caused 66 percent of the outbreaks. For the latter 25 years under the study, the sources of most of the 69 primary FMD outbreaks were livestock importations, animal vaccines (including both contaminated vaccines and escapes of virus from vaccine production facilities), and contaminated meat, meat products or garbage(Federal et al., 2001). Primary infections in FMD free countries have frequently involved pigs, often on swill feeding holdings. Swill from ships and aircrafts forms a special risk in this respect. Therefore, swill feeding practices are not compatible with a FMD free status unless the swill is processed in officially validated plants that are well-controlled by the government(Sutmoller et al., 2003).
    7. Biologics(USDA et al., 1994):
      1. Description: The primary role of biologics in the transmission of FMDV has been through the use of improperly inactivated FMD vaccine. Outbreaks have occurred primarily in Europe due to the use of formalin-inactivated vaccines. In the early 1900's other biologics were found to be contaminated with FMDV. It is less likely that problems with inactivation or contamination of vaccines could occur today given the techniques now used by most manufacturers(USDA et al., 1994). During the past 20 years on at least at two occasions FMDV escaped from technically well-equipped high-containment laboratories causing outbreaks outside the facilities. Therefore, regular international inspection of FMD laboratories and vaccine production plants is needed. Inspection must be carried out on the status of facilities and equipment, on logistics, and on the execution of the internal control on bio-containment and biosafety. This is particularly important for such laboratories in countries with a FMD free status(Sutmoller et al., 2003).
    8. Semen(USDA et al., 1994):
      1. Description: FMDV was found in semen as early as 12 hours after inoculation of bulls and as long as 42 days after contact exposure. In addition, heifers artificially inseminated with infected semen have developed FMD. In swine, FMDV has not been transmitted through artificial insemination even though semen from infected swine contains FMDV. Consequently, although further transmissibility studies in swine may be warranted, porcine semen was categorized as a low hazard(USDA et al., 1994).
    9. Hides(USDA et al., 1994):
      1. Description: FMDV remained infective in hides preserved by 4 conventional methods for varying lengths of time, all over 14 days. The authors of the study noted that these experimentally observed time periods should not be considered maximum survival times. Further, imported hides were suspected of causing the 1914 outbreak in the United States, in which at least 22 states and the District of Columbia were affected. Untanned hides and skins are currently allowed into the United States if they are hard dried, pickled in a solution of salt containing mineral acid or treated with lime in such a manner and for such a period as to have become dehaired. No studies were found in which the effect of such processing on FMDV was examined(USDA et al., 1994).
    10. Other Animals(USDA et al., 1994):
      1. Description: Ninety-nine animals were identified as possible sources of FMDV Of those, 31 were characterized as high, 50 as moderate, and 18 as low. A complete listing of all 99 animals can be found in this publication(USDA et al., 1994).
  4. Intentional Releases:
    1. Intentional Release Information(Gibbs et al., 2003):
      1. Description: While there is no evidence to suggest that the recent epidemic of foot-and-mouth disease (FMD) in the UK and its subsequent spread to continental Europe were caused by bioterrorism, the extent of the epidemic shows that FMD could be a very powerful weapon for a bioterrorist wishing to cause widespread disease in livestock and economic disruption for the targeted country. A report by the National Academies has highlighted the vulnerability of the nations food supply. FMD was identified as the most important animal disease that the US must be prepared for. The potential use of FMD to physically cripple livestock and to economically cripple a country has been recognized for many years. Few know that in the 1970s the Irish Republican Army threatened to release FMD virus in the UK; in the 1980s Australia had to respond to an extortionist who similarly threatened to use FMD virus. Not surprisingly, there are unsubstantiated reports that al Qaeda has also studied the malevolent use of the virus(Gibbs et al., 2003).
      2. Emergency Contact: Due to FMDs highly infectious nature, any detection of the disease in the United States would warrant immediate activation of APHIS Emergency Operations Center. If FMD is found in the United States, the U.S. Department of Agricultures (USDA) Animal and Plant Health Inspection Service (APHIS) officials stationed in the Center would help to coordinate local, State, and Federal response and eradication efforts, coordinate inter-agency planning, and implement national communication and information-sharing strategies. APHIS has already established a toll-free telephone number that concerned citizens and cooperators can call to obtain information on FMD and APHIS response efforts. 1-800-6-1-9327(Website 120).
      3. Delivery Mechanism: Ways by which the virus or infectious RNA may escape or be carried out from laboratories include: Personnel, Air Effluent and other waste Equipment(Website 122). Compared to bio-terror, agro-terror is appallingly easy. Animal diseases of greatest concern are those that, by nature, are very infectious and spread rapidly through herds and flocks. FMD, for instance, is the most contagious disease known to exist, spreading from animal to animal with incredible rapidity and in a more efficient manner than even the most contagious of human diseases. Bringing FMDV into a naive area is surprisingly simple, and once introduced, it will spread quite readily, without any requirement for weaponization to facilitate spread(Brown et al., 2003). Were FMD to occur through bioterrorism, it is probable that terrorists would trigger several outbreaks in different parts of the country, possibly caused by several serotypes of the virus. Multiple routes of transmission demand complex disease control responses and disruption of society(Gibbs et al., 2003).
      4. Containment: FMD is one of the most contagious diseases known and manipulating the virus in the laboratory without adequate precautions is a hazard. The escape of a single infectious unit of FMDV from a laboratory could potentially cause an outbreak. The main sources of virus or infectious RNA (in increasing risk of hazard) are: infected tissue cultures, infected baby mice, guinea pigs, rabbits etc., physical and chemical processing of large quantities of virus outside closed vessels (e.g., concentration, purification, inactivation, etc.), infected pigs, cattle, sheep, goats and other susceptible animals. Ways by which the virus or infectious RNA may escape or be carried out from laboratories include: Personnel, Air Effluent and other waste Equipment. Therefore all laboratories manipulating FMD virus must work under high containment conditions. The safety precautions must preclude any escape of virus and special attention must be given to: the prevention of illegal entry into the restricted area, the presence of changing and showering facilities, the responsible behaviour of personnel within and when they leave the laboratory, application of rules for primary containment, the use of inactivated virus where possible, the maintenance of negative air pressure where virus is manipulated and decontamination of exhaust air, the decontamination of effluent, the disposal of carcasses in a safe manner, the decontamination of equipment and materials before removal from the restricted area. To achieve this containment a variety of technical installations and a comprehensive set of disease security regulations are required under the supervision of a Disease Security Officer(Website 122). The evidence that the virus can be transmitted by aerosol alerted workers for the need to operate under negative pressure, particularly when large amounts of the agent are involved. Circulating air should be filtered appropriately(Brown et al., 2001B). U.S. research and diagnostic work with live foot-and-mouth disease virus is permitted only in an island-based laboratory(USDA et al., 1994). The proven strategy for controlling an FMD outbreak includes several key actions: Quarantine and stop movement of animals and products. Disinfect vehicles and personnel. Slaughter infected and contact animals. Destroy infected carcasses. Assess the need for strategic vaccination of animals and implement this action as appropriate(Federal et al., 2001).
Diagnostic Tests Information
  1. Immunoassay Test:
    1. ELISA :
      1. Time to Perform: 1-hour-to-1-day
      2. Description: At the OIE/FAO WRL for FMD, the preferred procedure for the detection of FMD viral antigen and identification of viral serotype is the ELISA. This is an indirect sandwich test in which different rows in multi-well plates are coated with rabbit antisera to each of the seven serotypes of FMD virus(Website 132).
    2. Complement Fixation (Vangrysperre et al., 1996):
      1. Time to Perform: 1-hour-to-1-day
      2. Description: The ELISA is preferable to the complement fixation (CF) test because it is more sensitive and specific, and it is not affected by pro- or anti-complementary factors. If ELISA reagents are not available, however, the CF test may be performed. Antisera to each of the seven types of FMD virus are diluted in veronal buffer diluent (VBD) in 1.5-fold dilution steps from an initial 1/16 dilution to leave 25 l of successive antiserum dilutions in U-shaped wells across a microtitre plate or appropriate volumes in test tubes. To these are added 50 l of 3 units of complement, followed by 25 l of test sample suspension(s). The test system is incubated at 37C for 1 hour prior to the addition of 25 l of 1.4% standardised sheep red blood cells (SRBC) in VBD sensitised with 5 units of rabbit anti-SRBC. The reagents are incubated at 37C for a further 30 minutes and the plates are subsequently centrifuged and read. Appropriate controls for the test suspension(s), antisera, cells and complement are included. CF titres are expressed as the reciprocal of the serum dilution producing 50% haemolysis. A CF titre greater than or equal to 36 is considered to be a positive reaction. Titre values of 24 should be confirmed by retesting an antigen that has been amplified through tissue culture passage(Website 132).
    3. Virus Neutralization (Website 132):
      1. Time to Perform: 2-to-7-days
      2. Description: FMD virus infection can be diagnosed by the detection of a specific antibody response. The tests generally used are virus neutralisation (VN) and ELISA. VN test is serotype specific, requires cell culture facilities and takes 2 to 3 days to provide results. The ELISA is a blocking- or competitive-based assay that uses serotype-specific polyclonal or monoclonal antibodies. It is therefore serotype specific, sensitive and quantitative, and has the advantage that it is quicker to perform, is less variable, and is not dependent on tissue culture systems. Low titre false-positive reactions can be expected in a small proportion of the sera in either test. An approach combining screening by ELISA and confirming the positives by the VN test minimises the occurrence of false-positive results. The quantitative VN microtest for FMD antibody is performed with IB-RS-2, BHK-21, lamb or pig kidney cells in flat-bottomed tissue-culture grade microtitre plates. Stock virus is grown in cell monolayers and stored at -20C after the addition of 50% glycerol. (Virus has been found to be stable under these conditions for at least 1 year.) The sera are inactivated at 56C for 30 minutes before testing. The control standard serum is 21-day convalescent serum (usually pig). A suitable medium is Eagles complete medium/LYH (Hanks balanced salt solution with yeast lactalbumin hydrolysate) with antibiotics(Website 132).
      3. False Positive: Low titre false-positive reactions can be expected in a small proportion of the sera in either test(Website 132).
    4. Liquid-phase blocking ELISA (Vangrysperre et al., 1996):
      1. Time to Perform: 1-hour-to-1-day
      2. Description: FMD virus infection can be diagnosed by the detection of a specific antibody response. The tests generally used are virus neutralisation (VN) and ELISA. VN test is serotype specific, requires cell culture facilities and takes 2 to 3 days to provide results. The ELISA is a blocking- or competitive-based assay that uses serotype-specific polyclonal or monoclonal antibodies. It is therefore serotype specific, sensitive and quantitative, and has the advantage that it is quicker to perform, is less variable, and is not dependent on tissue culture systems. Low titre false-positive reactions can be expected in a small proportion of the sera in either test. Antigens are prepared from selected strains of FMD virus grown on monolayers of BHK-21 cells. The unpurified supernatants are used and pretitrated according to the VN protocol but without serum. The final dilution chosen is that which, after addition of an equal volume of diluent (see below), gives an absorbance on the upper part of the linear region of the titration curve (optical density approximately 1.5). PBS containing 0.05% Tween 20 and phenol red indicator is used as a diluent (PBST). Guinea-pig antisera prepared by inoculating guinea-pigs with 146S antigen of one of the seven serotypes and preblocked with NBS is used as the detecting antibody. Predetermined optimal concentrations are prepared in PBS containing 0.05% Tween 20, and 5% dried, nonfat skimmed milk (PBSTM). Rabbit (or sheep) anti-guinea-pig immunoglobulin conjugated to horseradish peroxidase and preblocked with NBS is used at a predetermined optimum concentration in PBSTM. Test sera are diluted in PBST(Website 132).
      3. False Positive: Low titre false-positive reactions can be expected in a small proportion of the sera in either VN or ELISA(Website 132).
      4. Antigen:
        • 146S
    5. Nonstructural Protein Antibody Test (Website 132):
      1. Time to Perform: unknown
      2. Description: Antibody to VIAA is conventionally detected by AGID. The test is based on immunoprecipitation lines formed in the agar between the FMD antigen (concentrated cell-culture fluids rich in FMD virus RNA-dependent RNA polymerase or 3D) located in a centre well, and hexagonally arranged adjacent wells containing standard positive sera or unknown test sera. Precipitation lines forming between the test sera and the control antigen well that show identity with the lines of precipitation formed by the reference sera, confirm the specificity of the reactions. Antibody to expressed, recombinant FMD virus NS proteins can be measured by ELISA or immunoblotting. No single test format has yet been conclusively demonstrated to be optimal. A MAb trapping (MAT) ELISA for detecting antibody to 3ABC and blocking ELISAs for detecting antibody to 3AB or 3ABC have been shown to be sensitive, specific and reliable in a number of laboratories. The simultaneous detection of antibody to several NS proteins in a single test by ELISA or by enzyme-linked immuno-electrotransfer blot (EITB), a type of Western blot, (8) is useful for confirmation of animals positive for antibody to 3AB or 3ABC. There are currently no internationally recognized standards for antibody to FMD virus NS proteins(Website 132). Although it is recognized that measuring antibody to the RNA polymerase or 3D protein alone cannot differentiate infection from vaccination, this antigen is still useful as an indicator of previous exposure to FMD antigen which is not serotype specific. The leader protease (L protein) is the less immunogenic NS protein and therefore cannot be recommended. NS proteins 2C, 3AB and 3ABC have the potential to discriminate infected from vaccinated or naive. Out of them, 3ABC is the most immunogenic and has been extensively studied. To avoid non-specific reactions against antigen from host cell expression system, which can co-purify with the recombinant products, mapping of continuous immunodominant site was undertaken with overlapping peptides from 2C and 3ABC. One synthetic peptide was selected in 3B protein to set up a peptide based ELISA with promising specificity(Remond et al., 2002).
  2. Nucleic Acid Detection Test: