FMD vaccines

Virus Research 91 (2003) 81
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Review

FMD vaccines T.R. Doel Merial Animal Health Ltd, Ash Road, Pirbright, Woking, Surrey, UK Keywords: Foot and mouth disease; Vaccination; Animal disease

1. Introduction During the height of the foot and mouth disease (FMD) epidemic in the UK in 2001, there was intense debate on the advantages and disadvantages of vaccination in terms of both the specific circumstances in the UK as well as in general. If nothing else, the debate served to highlight a number of misconceptions and to reveal, even now, the relatively low understanding of the prevalence and importance of the disease worldwide and its control by vaccination. This is somewhat disappointing considering that FMD has been with us for many centuries (Fracastorius, 1546) and the first attempts to develop vaccines started in the early 1900s.

2. Historical background The greatest advances in our knowledge of FMD and its control have been made during the last 100 years or so as demonstrated by the substantial international literature on all aspects of the disease. Regrettably, it will be possible to review here only a few of the most important findings. One of the first and most significant discoveries was made by Lo¨ffler and Frosch (1897) who demonstrated that the aetiological agent was a filterable particle, and, effectively, FMD was the first animal disease to be attributed to a virus. During the early part of the 20th century, the diverse antigenic nature of the virus was recognised and led to the description of the seven serotypes over the next 50 years (A and O, Valle´e and Carre´, 1922; C, Waldmann and Trautwein, 1926; SAT1, SAT2, SAT3, Galloway et al., 1948; Asia1, Brooksby and Rogers, 1957). The working definition of a serotype is that previous infection (and, by extrapolation, vaccination) with a given serotype does not confer protection against the other six serotypes. The profound importance of the variability of the virus in relation to

protection also extends to major antigenic variants within some serotypes such as the A serotype. Attempts to develop FMD vaccines also started in the early years of the 20th century when Belin (1927) described his experiments with attenuation of the virus. Later researchers also worked on attenuated FMD vaccines, including intensive studies in the 1960s, but major problems were encountered such as unpredictable virulence in the field. Effectively, this undermined any belief that a safe and stable attenuated product could be realised within a reasonable time frame. Given current knowledge and with the molecular biological tools at our disposal, it is conceivable that such a vaccine virus could now be developed. However, its use would most probably complicate the discrimination of naturally infected and vaccinated animals with the added potential problem of attenuated vaccine virus spreading to non-vaccinated livestock. The first practical inactivated vaccine was developed by Waldmann et al. (1937) using virus from the epithelium and vesicular fluid of tongues of deliberately infected cattle. Inactivation was with formaldehyde in the presence of aluminium hydroxide gel. The aluminium hydroxide functioned as an adjuvant as well as facilitating inactivation of the virus and providing a simple method of product concentration. Clearly, the need to deliberately infect cattle was undesirable and production of Waldmann type vaccines was greatly assisted by the work of Frenkel (1947) who used epithelium obtained from the tongues of recently slaughtered healthy cattle. Suspensions of the epithelial cells were prepared and maintained in vitro and subsequently infected in a manner similar to that used today with baby hamster kidney (BHK) cells. The Frenkel procedure became the corner stone of vaccine production for many years. Disadvantages with the use of bovine tongue epithelium included the logistics of collecting sufficient material as well as maintaining sterility throughout the process. This prompted research to find a cell line more

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appropriate to production needs. Mowat and Chapman (1962) adopted the BHK monolayer cell line, BHK-21, clone 13, previously developed by MacPherson and Stoker (1962) and showed that it could be used for the growth and titration of FMDV. While extremely labour intensive and having the disadvantage, in modern good manufacturing practice (GMP) terms, of multiple ‘open operations’, the monolayer BHK cells were quite widely used by various FMDV workers, including the Italian group at Brescia, for industrial scale virus and vaccine production. The most significant industrial developments in this area were made by Capstick et al. (1962) who adapted BHK monolayer cells to growth in suspension, and Telling and Elsworth (1965) who produced the suspension cells in large scale fermenters. Almost all modern FMD vaccines are now produced in this way. One very major regulatory advantage of using an immortalised cell line is that it is possible to test the Master Cell Stock (MCS) on a relatively infrequent basis for freedom from adventitious agents, whereas fresh cells recovered from cattle at the slaughterhouse would require full testing at each and every occasion. While formaldehyde inactivation proved an acceptable process for many years, it became increasingly apparent that its use carried a slight risk of residual contamination of inactivated vaccines with live virus. The use of an aziridine, acetylethyleneimine, as a first order inactivant of the virus was first described by Brown and Crick (1959) and the basic aziridine methodology grew in popularity because of the effectiveness and reliability of the process at the industrial scale. Acetylethyleneimine is no longer used and the most widely employed inactivation process is based on binary ethyleneimine (BEI) which is generated by the action of alkali on bromoethylamine hydrobromide shortly before it is required (Bahnemann, 1973). From a regulatory standpoint, BEI inactivation is invariably demanded by national and international authorities and formaldehyde inactivation is no longer acceptable. Using BEI in a two tank (vessel) system according to GMP it is possible to achieve the Ph.Eur requirement of less than 1 infectious particle per 10 000 l of FMD antigen preparation. Regardless of the cells used to grow the virus, FMD vaccine preparations often contain high concentrations of cell and media components unless the inactivated antigens have been subject to lengthy purification to remove these unwanted materials. Certainly, there is a considerable literature on the adverse responses of animals to crude vaccines (reviewed by Black and Pay, 1975) and this prompted work to concentrate and purify the inactivated antigens. Apart from the simple adsorption of antigen onto aluminium hydroxide gel, the earliest concentration methods to be used and subsequently adopted for industrial scale production were precipitation by polyethylene glycol and ultrafiltration

(reviewed by Morrow et al., 1974). Ultrafiltration has now largely replaced the PEG process for a number of reasons including the unquestionable advantage of ‘inline’ processing within closed systems. Purification of antigen from concentrates, such as those produced by ultrafiltration, was first reported by Adamowicz et al. (1974) who used adsorption and elution from polyethylene oxide at an industrial scale to achieve concentration factors of 1000-fold and substantial removal of the bulk of the extraneous proteins (95%). In our laboratory at Pirbright, we now use industrial scale chromatography to purify the antigens previously concentrated by ultrafiltration. Development of methods to concentrate and purify inactivated FMD antigens also had the advantage of allowing the production of higher quality vaccines as well as the storage of highly concentrated materials at very low temperature for long periods of time */the basis of emergency antigen banks (Doel and Pullen, 1990). The use of adjuvants with inactivated FMD antigen preparations is essential for satisfactory potency and aluminium hydroxide was eventually supplemented with a second adjuvant, saponin, which was first described by Espinet (1951). Both adjuvants are still routinely used in aqueous vaccines for ruminants. While oil based adjuvants had first been described by Freund in 1937 (Freund, 1956), they were not employed with FMD vaccines until 1961 when Michelsen reported the use of incomplete oil adjuvants for vaccination of pigs (Michelsen, 1961). Cunliffe and Graves (1963) reported similar studies in cattle. For historical reasons, this type of vaccine, which is somewhat analogous to the formulation of Freunds incomplete adjuvant, is the preferred product for ruminants and pigs in South America. Elsewhere in the world, oil emulsion vaccines of the so-called double oil emulsion (DOE) variety are now widely used for the immunisation of pigs and occasionally for cattle and other ruminants. In-process and final product testing of FMD vaccines are mandatory in the context of a full Quality Assurance system and a number of key observations and experiments deserve recognition. The important role of FMDV specific serum antibody in protection was demonstrated by Lo¨ffler and Frosch (1897) who showed that animals could be protected by passively administered convalescent serum. In fact, immune serum was quite widely used in FMD control campaigns in the early part of the 20th century by pioneers such as the late Charles Merieux and one record indicates that 12 825 animals were serum treated in France between October 1923 and December 1924 (Anonymous, 1968b). In the development of an assay for FMDV specific serum antibodies, Leunen (1959) used foetal calf kidney cells in an early version of the in vitro virus neutralisation test and Martin and Chapman (1961) reported

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approximate correlations between neutralising antibody titres and protection of cattle against virulent challenge. While not the first publication in this area, Ahl et al. (1990) presented a comprehensive statistical analysis of data from cattle challenge experiments and calculated the relationship between neutralising antibody titres and protection with cattle. No such correlation exists with pig challenge studies due in part to the difficulty of carrying out meaningful challenge experiments with this species because of the possibility of overwhelming challenge conditions (de Leeuw et al., 1979). There is no evidence that cell mediated immune responses play any role in protection of livestock against FMD. Measurement of the potency (PD50) of FMD vaccines was first described by Henderson and Galloway (1953) who challenged cattle by the intradermolingual route. A version of this basic test is still prescribed by the European Pharmacopoeia Monograph on FMD vaccines for ruminants (1973). The work of many authors including Mussgay (1959), Bachrach (1960) and Wild and Brown (1967) demonstrated the labile nature of the virus to mild pH (:/6.5), heat (56 8C) and enzymes such as trypsin and led to the conclusion that the essential immunogenic component of FMD vaccines was the so-called 146S particle of which the VP1 protein appeared to be the most relevant constituent (Laporte et al., 1973). Measurement of 146S particle concentrations as originally described by Fayet et al. (1971) and refined by others such as Doel et al. (1982) remains a precise and invaluable procedure in FMD vaccine production and is the single most useful parameter for the formulation of effective FMD vaccines.

3. Economic consequences of the disease It has been suggested, quite erroneously, that FMD is not a particularly serious disease and that its relevance is largely in terms of international trade. While the trade consequences are certainly extremely damaging for some countries, FMD causes significant distress and suffering to animals and impacts on the livelihood of all farmers, regardless of the size and sophistication of their livestock unit. At the local level, infection of livestock invariably results in: (1) Substantial loss of milk yield of dairy cattle, averaging approximately 25% reduction per annum (Power and Harris, 1973; Hugh-Jones, 1979). (2) Reduction in the growth rate of meat animals. Estimates vary considerably but one study has indicated that cattle would require approximately 10
/20% longer to reach maturity (P. Ellis, personal communication). Power and Harris (1973) indicated a reduction of 20% per annum in pig meat production but this number

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embraced more factors than growth rate alone. However, the comparative severity of FMD in pigs would imply at least similar reductions to those given for cattle. (3) Temporary loss of draught power with working buffalo and cattle. This can amount to 60
/70% during the first month following infection (Hugh-Jones, 1979) and it is not uncommon for outbreaks to coincide with critical events such as harvesting of crops. (4) Reductions in fertility due to increased abortion rates (up to 10%) and delays in conception. (5) Death of very young animals. Very high mortality rates are observed with pigs and sheep in particular. In the case of pigs, Dunn and Donaldson (1997) gave a general rate of 40% for two outbreaks in Taiwan in 1997 and Chang et al. (1997) commented on the very high losses with piglets during this period. Geering (1967) cites mortality rates of 40, 45 and 94% of lambs in several outbreaks. Death of older animals is less frequently observed but may be significant with some virus strains. (6) The need to cull unproductive and chronically infected animals. (7) Gross disruption of farming practices including loss of income, loss of valuable breeding stock and disruption of livestock improvement programmes At the national level, notable effects of FMD are: (1) Disruption of livestock production and internal markets with consequential impacts on availability and prices of livestock products including the possible need to increase importation of the same or alternative foods. (2) Loss of export markets through embargoes imposed by trade partners. These losses often run into billions of US dollars as evidenced by countries such as Taiwan. Chen et al. (1999) cited losses of US$400 million for compensation payments, carcass disposal and other local costs and an additional US$3650 million for indirect costs. At the time of writing, Argentina, Uruguay and Brazil are also faced with large export losses until those countries are able to recover their export markets through control and eradication of the disease. (3) The many direct and indirect costs of an eradication policy including compensation to farmers, salaries of veterinarians and the many operational and support personnel, general business disruption, etc. Given the devastating consequences of an outbreak of FMD, it has been common practice for countries affected or threatened by the disease to carry out costbenefit analysis of different control policies for scenarios ranging from endemic disease in non-exporting countries to initial outbreaks in exporting countries. In the case of the former, it is clear that vaccination as part of an integrated control policy provides favourable costbenefit ratios and values of between 1:5 and 1:8 have been reported (James and Ellis, 1978). With disease-free countries or regions having a substantial export market

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for livestock and livestock products, cost-benefit analysis has usually favoured eradication over routine mass vaccination programmes (UK, Power and Harris, 1973; Denmark, Stougaard 1985; European Union (EU), Anonymous, 1989). A crucial component in the analysis of any non-vaccination scenario is the assumption made by epidemiologists on the future frequency and magnitude of FMD outbreaks in the country. Clearly, the threat to any one country or region can dramatically change due to circumstances beyond the control of the country and may invalidate the conclusions of the previous cost-benefit analysis.

4. Immunity to FMD It is clear from many studies that protection of individual animals vaccinated or recovered from infection is mediated by antibody of which serum neutralising antibody is the most relevant in terms of correlation with protection (e.g. Ahl et al., 1990). ELISA has also been used quite extensively in more recent years but it must be remembered that this assay almost certainly measures a wider spectrum of antibodies, an undefined proportion of which may not relate to antibody mediated protective mechanisms. Inactivated FMD vaccines given parenterally also appear to induce neutralising antibodies at mucosal surfaces after one dose of vaccine in pigs and two to three doses of vaccine in cattle (Francis et al., 1983; Francis and Black, 1983). FMD vaccines achieve the prime function of all vaccines, the prevention of the debilitating effects of the disease in the host. Like many vaccines, they do not induce sterilising immunity and may allow viral replication at epithelial surfaces including the development of the so-called carrier state in some vaccinated animals following live virus challenge. However, FMD vaccines have received something of a bad ‘press’ over the years and it is important to put their performance into proper context. Firstly, infection at epithelial surfaces by FMDV challenge, whether by inoculation or contact with infected animals, is a very rapid process and gives the immune system of the host very little time to respond to the invading virus. As an example, we have observed development of full blown FMD, including feet and snout lesions, in young pigs challenged little more than 24 h previously and one of the pigs died approximately 36 h post challenge as a result of cardiopathy. Against the often rapid development of disease in susceptible livestock, we have equally observed that a single vaccination with a high potency vaccine typical of those used for emergency antigen banks is capable of preventing the development of lesions in cattle tongues challenged with high titres of virus by the intradermolingual route. Boosting of the immune response by repeated vaccination, as used in many parts of the world,

dramatically increases both the magnitude and duration of neutralising antibody responses and would be expected to prevent even more effectively the local replication and spread of the virus at the point of infection. Effective immunity must also be considered on a herd basis and it is vital to vaccinate as many individuals within the population as possible. Protection of the herd as a whole reduces greatly the opportunity for virus to enter, replicate and challenge individuals which may not have been vaccinated or do not have sufficient immunity to resist. This is probably most important with pigs which, once infected, generate massive amounts of virus (of the order of 3000 times the quantity excreted by cattle) and where overwhelming challenge of otherwise immune cohorts is a real possibility. It is generally considered that vaccination of not less than approximately 80% of the herd is necessary to provide herd immunity (Lombard and Shermbrucker, 1994) and, certainly, EU countries did not start to make a significant impact on disease control until mass vaccination achieved this level of coverage (Fontaine, 1985). It must be mentioned however that mass vaccination in the European context related primarily to cattle and only relatively few sheep and pigs were routinely vaccinated (see later comments in this review).

5. The production process 5.1. The production environment Production of FMD antigens and vaccines must be carried out under conditions which fully comply with internationally recognised standards of GMP (within the EU, Directive 91/412/EEC), invariably within the framework of a Quality Assurance system, and a level of biocontainment which prevents any possibility of virus escape from the manufacturing facility. For the GMP conditions, these are well and extensively documented in publications such as the ‘orange guide’ (Medicines Control Agency, 1997) and include provision of a HEPA filtered air supply to the facility, full containment of the process within stainless steel vessels and other ‘in-line’ equipment, washable impervious walls, floors and ceilings and the avoidance of any working practice or process design which could result in contamination of the product either with a related product or extraneous agent(s). The principles of biocontainment with specific reference to FMDV have similar objectives and requirements to those of GMP and include the need to exhaust all air through double HEPA filters in sequence, the creation of minimum negative-to-ambient pressure gradients within areas where live FMDV is present (whether inside a sealed vessel or a safety cabinet), the enforce-

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ment of movement controls on staff within and outside of the facility (including bans on visiting farms and other susceptible livestock premises) and a range of equipment and processes at the boundary of a secure area including autoclaves, fumigation chambers and personnel showers so that materials and personnel can only leave the secure area following decontamination. For biosecurity, operational and cost reasons, it is desirable to retain the minimum essential amount of equipment and plant within a secure area. Thus, for example, ‘safe-change’ HEPA filter banks and HVAC equipment and air supply trunking can be located outside of the secure area and much of the electrical and electronic control equipment is also best sited in such areas where it can be readily accessed by qualified staff. This reduces unnecessary staff movements to and from secure areas. Essentially, the process line consists of a series of sealed stainless steel pressure vessels linked by stainless steel pipework and ancillary items such as in-line filters and concentration/purification equipment. Addition of anything to this in-line process is done in such a way as to prevent any possibility of virus escaping or contaminants entering the closed system. Thus, virus inoculum should be introduced into the cell culture vessel through a system of steam sterilisable ports and connectors. The production facility used for the blending of vaccine and filling of final product must be designed specifically to exclude any possibility of contamination of the final product with either live FMD virus or extraneous agents. Thus, the containment level relates exclusively to GMP and such controlled areas are supplied with HEPA filtered air under conditions of positive pressure to ambient. In Pirbright, staff working in these controlled areas are not permitted to visit or work in the secure areas where live virus is produced or tested. 5.2. The production process Fig. 1 shows a simple schematic of the production process used by the author’s company at Pirbright. BHK-21 suspension cells are grown in a nutrient medium until they reach a satisfactory titre on a scale of several thousand litres or more at which point the spent medium is decanted and the cells resuspended in fresh medium. The cells are then infected with the working seed virus and maintained for approximately 24 h, depending on the growth properties of the particular virus strain in the cell line. FMD viruses vary quite considerably in their ability to grow in BHK monolayer and suspension cells and development of vaccine strains is therefore something of an art, considering the need for rapid growth, good immunogenicity and high yield. Within the framework of a Quality Assurance system, the number of serial passages from

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the MCS before a fresh revival of cells is made is strictly limited to prevent significant changes in the properties of the BHK cells which otherwise might impact on quality and productivity. Similar but stricter constraints apply to master seed viruses (MSV) because of the theoretical possibility of antigenic drift from the MSV stock through unnecessary passages in tissue culture. A practical safeguard in this regard is to ensure that any serology such as comparisons between vaccine and field strains uses sera generated from final product and not from a virus isolate several passages removed. As soon as the majority of the cells are dead, the bulk virus is clarified by centrifugation or filtration to remove cell debris prior to the inactivation process. In our production unit, we follow exactly the GMP requirements of a two tank (vessel) system. The first addition of BEI is made to the virus with stirring and the contents of the vessel transferred to a second sterile vessel where the second addition of BEI is made. Samples are removed during the inactivation process for titration in a susceptible cell line in order to establish the rate of inactivation and determine by extrapolation the final titre at the end of the inactivation period. The Ph.Eur Monograph on FMD vaccines indicates that this must be less than one infectious particle per 10 000 l which equates to at least 2 million cattle doses depending on the serotype and strain of the virus. The importance of properly controlled and validated inactivation conditions cannot be stressed too much. Under our conditions of inactivation, we have never detected any residual infectivity in vitro nor are we aware of any evidence, circumstantial, alleged or otherwise, of residual infectivity in a target species. It remains quite common practice to formulate vaccines from this relatively crude, bulk inactivated antigen. However, in the author’s company, the antigen is subject to ultrafiltration and chromatography to obtain highly purified, highly concentrated, inactivated antigen which is usually stored in the gaseous phase of liquid nitrogen until it is required. This material is particularly suited to the needs of antigen banks as well as facilitating vaccine formulation when more than one strain is required in a vaccine. The inactivated antigens are processed in a separate production facility respecting the need to separate the virus production area from the antigen downstream processing area. Following purification, the concentrated antigens are subjected to a second innocuity test whereby not less than 200 cattle dose equivalents of antigen are used to inoculate susceptible cell monolayers which are inspected regularly to establish freedom from infectious virus. To maximise the sensitivity of this test, the procedure incorporates two sequential passages of fluid from the initial monolayers into fresh monolayers so as to be sure that there is absolutely no detectable residual infectivity.

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Fig. 1. Production of FMD antigen concentrates and vaccines.

The formulation of the final product depends on the strains required in the vaccine and the species to be vaccinated. FMD vaccines commonly contain more than one strain of the virus reflecting the epidemiological situation in the customer’s country. South American vaccines, for example, are commonly trivalent containing single isolates from the region of serotypes O, A and C. Other regions of the world may have more or less complex products. In the Arabian Peninsula, there is the

potential threat from serotypes prevalent in Africa, elsewhere in the Middle East, and India, and vaccines containing four serotypes (e.g. O, A, Asia1, Sat2), including several distinct strains within the O and A serotypes, are not uncommon. Probably the most common vaccine used in South East Asia is monovalent O and which may contain one or several O strains appropriate to the region. It is important to note that the O and A serotypes have a number of antigenically

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distinct strains of epidemiological importance (e.g. O Middle East/South East Asia of which O Manisa is the best known, O South America of which O Campos is the best known, A Middle East of which A22 Iraq, A Iran 1996 and A Iran 1999 are the best known and A South America of which A24 Cruzeiro is the best known) and the selection of the most appropriate strain(s) requires a sound knowledge of the FMD situation worldwide and the resources to determine and match the vaccine strain to the strains prevalent in a given region. Once the selection of strains is made, the antigen concentrates are removed from the liquid nitrogen freezer and diluted with buffers before blending either with an oily adjuvant or aluminium hydroxide and saponin. The oily adjuvant system produces an emulsion suitable for cattle, pigs, sheep and goats (at Merial, Pirbright, this is a DOE or water-in-oil-in-water emulsion) and the aluminium hydroxide/saponin adjuvant is prescribed for ruminants only. The payload of each antigen used depends on the antigenicity of the strain as well as the potency of vaccine required. In general, payloads vary from 1 to 10 mg of 146S per strain per vaccine dose and, for reasons which are still not entirely understood, more O serotype antigen is required than the A, C and Asia1 serotypes to achieve an equivalent potency. Because the relationship between 146S concentration and potency does not appear to be a simple linear function (Rweyemamu et al., 1982), payloads higher than approximately 10 mg of 146s of a given strain do not necessarily give proportionately higher potencies. 5.3. Raw material, intermediate and final product testing Although National and European legislation does not permit the use of FMD vaccines in Europe at this time, FMD vaccines intended for use in the EU must be licensed according to the provisions of EU Directive 81/ 851/EEC. In addition to specific quality tests made during the application for a marketing authorisation, certain routine batch tests are crucial to ensure the supply of safe and efficacious products. The following tests are among the more important. MSV are derived from field isolates by passage in BHK cells or another suitable cell line. The number of passages required to develop a high yielding efficacious MSV will depend very much on the strain of the virus and the skill of the scientist doing the work. While there are only seven serotypes of FMDV, it is common practice to develop a number of vaccine strains within each serotype in order to cover most eventualities. In our laboratory, we maintain approximately 25 MSVs covering the seven serotypes. MSVs and MCS are tested initially for freedom from adventitious agents i.e. mycoplasma spp, bacteria and fungi, viruses, using sensitive validated assays. For

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example, both EU regulations and 9 CFR (USDA Code of Federal Regulations) require that MSV and MCS do not show evidence of a wide range of potentially contaminating viruses using sequential passage through the production cell line, a sensitive cell of the species to be vaccinated and VERO cells. A number of starting materials of biological origin are routinely used in FMD vaccine production and are subject to particular attention. These include serum, peptone, and casein hydrolysate of ruminant origin and EU Directive 1999/104/EC requires that a risk assessment is carried out on each material by the supplier as well as the user of the material according to the EU guidelines on minimising the risk of contamination of medicinal products with transmissible spongiform encephalopathies. As far as Bovine spongiform encephalopathy is concerned, the most critical decisions of the vaccine manufacturer are the avoidance of materials sourced from countries with a history or even suspicion of having had BSE and from organs/tissues regarded as a theoretical risk in terms of transmission of the agent. From the Merial perspective, we elected in the late 1980s to use sera from New Zealand Ministry of Agriculture accredited farms and other biologicals from similar acceptable sources. In the routine production of FMD vaccines, it is common practice to test materials such as serum and peptone for absence of adventitious agents before they are accepted for use or to obtain them as gamma irradiated products (25 kGrays or greater). The importance of establishing innocuity (i.e. noninfectivity) of the antigen harvest has been stressed elsewhere and this is best done by both inactivation kinetics during the BEI inactivation process and by inoculation of a large number of cattle dose equivalents of inactivated antigen into a susceptible cell line. Innocuity is also determined in the final product safety test in cattle according to the Ph.Eur. FMD Monograph (1993). However, the route of inoculation prescribed, the bovine tongue, has little relevance to safety in terms of the conventional sites of inoculation of vaccine (subcutaneous or intramuscular routes in the region of the shoulder and neck) and owes more to historical data which reported that the bovine tongue was the most sensitive site to the virus. In fact, this traditional procedure is highly questionable (Doel et al., 1998), in part because of the reliability, reproducibility and greater sensitivity of tissue culture tests with tissue culture adapted virus (Anderson et al., 1970) and also because of the clear tropism of some strains of FMDV including the well known O Taiwan strains from 1997 which rarely produced disease even when injected into cattle (Dunn and Donaldson, 1997). It has been recognised for many years that the essential immunogenic component of FMD vaccines is the 146S particle of the virus. Any degradation of this particle greatly reduces the potency of the vaccine (Doel

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and Chong, 1982) and vaccines prepared from recombinant proteins and peptides based on the VP1 sequence of the virus have not proven as effective as conventional products. The concentration of 146S particles in a preparation is measured by sucrose gradient centrifugation and UV spectrophotometry of the continuously fractionated gradient at 254 or 260 nm (Doel et al., 1982). This simple method permits a rapid determination of the 146S concentration from the area of the virus peak on the spectrophotometer chart and is used throughout the production process to monitor virus and antigen yields as well as formulation of the final product. Clearly, a number of other important tests are made throughout the production process including identity and sterility, the former test being a confirmation that the strain in production has neither been wrongly selected in the first place nor contaminated with another strain during the process. The virus neutralising antibody test is valuable for both the assessment of vaccine potency of the final product as well as determining the relationship between a field isolate and a vaccine strain. In our virus identification laboratory at Pirbright, we routinely analyse field isolates obtained from the OIE and FAO World Reference Laboratory (WRL) for FMD by crossneutralisation studies in a susceptible cell line using a library of sera from single dose, single strain vaccinated target species. Potency testing of FMD vaccines is made by a number of different procedures. Within Europe, the official potency test as described by the Ph.Eur. (1993) and OIE Manual of Standards (2000) indicates the vaccination of three groups of cattle, each of at least 5 animals, using a different dose of vaccine for each group. The dose series used is conventionally 1 field dose, 1/4 of a dose and 1/16 of a dose by the prescribed route, using fractional volumes rather than dilution of the product. Three weeks after vaccination, the 15 cattle plus two controls are challenged with 10 000 ID50 by the intradermolingual route and all animals examined daily for 8 days. Protected animals may show lingual lesions but should not show vesicles elsewhere, with particular reference to the feet. Control animals should develop lesions on at least three feet for the test to be valid. The PD50 is calculated by a suitable method such as Spearman-Karber and should be 3 or greater (Ph.Eur). ReedMuench is not a suitable procedure unless the data set is very symmetrical with 0 and 100% endpoints. A truncated version of the test has been described in which the lowest dose is omitted and the assumption of zero protection is made (Barteling, 1998). While this saves animals, the statistical validity of the test is doubtful and only a minimum PD50 value can be given. Indeed the statistical validity of a test with even 15 animals has been questioned by a number of workers (Doel et al., 1998).

Another type of test, the PG test (Protection against Generalization) has been used in South and North America in which a group of 16 cattle is vaccinated with a single field dose and challenged after a suitable period of time (Vianna Filho et al., 1993). In this procedure, the pass requirement is usually 75% protection with the option to test in an additional group of 16 animals if the vaccine fails the first test. The data from both tests may then be pooled and the vaccine is satisfactory if 75% overall protection is achieved. According to Vianna Filho et al. (1993), 3 PD50 (Ph.Eur) corresponds to 78,78 and 79% in the PG test for A, O and C serotypes, respectively. This test is rarely used now, being replaced substantially by serological assays with vaccinated cattle. In Europe, a potency test based on virus neutralising antibody titres is acceptable provided a correlation with protection against challenge has been demonstrated (e.g. Ahl et al., 1990). Not only is this more acceptable from the animal welfare perspective, but the undoubted variability of the intradermolingual challenge procedure is avoided and the only inherent variation of any significance in the test is the variability of animals to vaccination, a characteristic which is common to vaccination with or without challenge. Serology offers a number of additional advantages including the opportunity to sample at different time points and assess multi-serotype vaccines with just one group of animals. The Ph.Eur does not describe a potency test in pigs by challenge. The reason for this is the very considerable difficulty in devising a meaningful and reliable procedure. Black et al. (1984) commented on the ‘between trials’ variability of susceptibility to clinical infection due to uncontrollable factors in the challenge environment. Thus, it is not a question of whether or not pigs respond to FMD vaccines or, indeed, are protected by vaccination (see later comments and van Bekkum et al., 1967) but the standardisation of the challenge procedure. Of special note is the concept of overwhelming challenge (de Leeuw et al., 1979) in which a non-immune pig (for example a control animal), having been challenged by needle injection of the coronary band or heel bulb, succumbs rapidly and presents a massive aerosol/contact challenge to the other pigs. Under these circumstances, it is difficult to determine a clear picture of the potency of the vaccine because of the dynamics of the population in question under the influence of factors such as the presence or absence of control pigs, the quantity of virus used to challenge, the size of the isolation box and almost certainly other variables such as number of animals, number of air changes in the box, virulence of the challenge virus, etc. While release of FMD vaccines within Europe would not normally occur until the final product has passed the sterility, safety and potency tests, the Ph.Eur. FMD Monograph (1993) has a special clause which permits

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early release before the completion of these tests provided the antigens have been shown previously to be satisfactory, a genuine emergency exists and the veterinary authorities of the receiving country officially request supply before completion of these tests.

6. Selection and application of vaccines 6.1. Selection of vaccine strains While control of FMD by vaccination is complicated by the antigenic variability of the virus, the issue has been overplayed by some observers and represents few problems for the well organised vaccine producer. Provided the manufacturer monitors continuously the world situation and carries out timely testing of new field isolates to determine the appropriateness or otherwise of existing vaccines, emerging epidemiological situations can be recognised and new vaccine strains developed if necessary. In our laboratory, we routinely carry out two-way virus neutralisation tests using recent field isolates and a panel of vaccine strains, and field variants demonstrating a very weak relationship with antisera against a vaccine virus (r1 value B/0.4, Ferris and Donaldson, 1992) will often prompt the development of a new vaccine strain. Such was the case with A serotype field isolates from Iran in 1996 and 1999 which were found by the WRL to be significantly different from any of the A strains in its database and, equally, were not matched in our studies by any of the viruses within our vaccine strain library. Consequently, we were able to develop A Iran 96 and Iran 99 vaccine strains well in advance of the spread of the field isolates into Turkey in 1997 and 1999, respectively. In the development of a new vaccine strain, critical properties include productivity in the production cell line, stability of the virus and antigen, a close antigenic relationship to the field isolates, and sterility and freedom from adventitious agents. These properties can only be achieved against the background of a comprehensive vaccine strain development programme. In addition, immunodominance is a highly sought after characteristic and may be broadly described as a serological relationship between pairs of FMD virus strains where the relationship is not always symmetrical and one strain can be dominant over another. As an example, the vaccine strain O BFS 1860 used by our company has broad cross-reactivity (Rweyemamu, 1978) and although heterologous as far as the O Manisa family of viruses is concerned, would be expected to provide some degree of protection against the O UK isolates from 2001. Other immunodominant strains are mentioned below. Major antigenic variants do not occur with high frequency and it seems that the properties of each

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serotype are constrained within certain boundaries perhaps effectively defined by fundamental structural needs of the virus capsid (Samuel and Knowles, 2001). These authors cite the example of the Asia1 serotype which, on the basis of the predicted rate of evolution (10 2 substitutions per nucleotide position per year) would have been expected to generate significant antigenic variants since the serotype was first isolated in 1954. In fact, this is not the case and only one major Asia1 sub-group (in their terminology, a topotype) exists. On the basis of nucleic acid sequences of VP1 protein of the O serotype, these authors suggest that such constraints also apply to topotypes within a serotype and that such topotypes may represent evolutionary ‘cul-de-sacs’. Following from this, the antigenic ‘spectra’ of C and Asia1 serotypes appear to be quite narrow and only one or several vaccine strains are considered necessary. For example, the Asia1 Shamir vaccine strain appears to be immunodominant and is very effective in terms of crossneutralisation of a wide range of Asia1 field isolates. The same is very much the case for C serotype, added to which its occurrence is now very rare and has not been reported by the WRL since 1996. The SAT1 and SAT3 serotypes strains are confined to and relatively restricted within Africa although SAT1 made a major excursion into the Middle East in 1961 and spread from Bahrain to Saudi Arabia, Israel, Jordan, Iraq, Syria, Lebanon, Turkey, Iran, Greece and Bulgaria in less than 12 months (Anonymous, 1968a). The SAT2 serotype is very much the most problematical virus serotype in Africa and, in 2000, invaded Saudi Arabia where it caused major problems due in part to the fact that none of the vaccines in use in the country at that time contained a SAT2 component. SAT2 exhibits very broad antigenic characteristics as suggested by the higher sequence variability in the VP1 protein than that observed with A, O and C serotype viruses (Vosloo et al., 1992). This can be circumvented to a significant extent by the careful selection and use of immunodominant vaccine strains (Rweyemamu, 1978). Nevertheless and without diminishing the importance of other SAT serotypes, SAT2 would represent a very serious problem for world animal health if it were to escape from its current boundaries. Outside of Africa, the two most important serotypes in terms of prevalence are A and O. The A serotype is well known for its antigenic diversity and indeed, many isolates are effectively considered as separate serotypes and require closely matched vaccine strains. Particularly important vaccines include A24 Cruzeiro (South America), A22 Iraq (an old strain of general relevance to A strains outside of South America), and A isolates from Iran in 1996 and 1999. Fortunately, the prevalence of A serotype viruses is not as high as that of the O serotype which is distributed widely throughout the world.

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Broadly speaking, the O serotype does not show the antigenic diversity of the A serotype and isolates fall within one of two major sub-families. The first of these is O South America, of which O Campos is the best known vaccine strain. Related vaccine viruses include O Lausanne, OBFS 1860 (UK 1967) and O Kaufbeuren and these were extensively used in Europe prior to the cessation of vaccination during 1990
/1991. The second family of viruses effectively cover most countries/continents where FMD is present (North Africa, South Africa, Middle East, much of Asia, UK) and O Manisa (Turkey 1969) is the best known vaccine strain. Other vaccine strains which we have adapted to local needs include O Taiwan 98, O Philippines 98, O Geshur and O-3039. The choice of vaccine strain to be used will depend very much on circumstances. In an emergency situation it will not be feasible to immediately develop a vaccine strain from a field isolate but it may be possible to supply a closely matched strain if required. Within a few days of the report of the UK outbreak, we had identified several of our vaccine strains which matched closely the UK field isolates. Following an outbreak of A serotype in the Balkans in 1996, we carried out a similar exercise and matched a vaccine strain quite closely. However, the urgency of vaccine application was such that the EU supplied A22 Iraq 24/64 vaccine to the Balkans formulated from antigen held in the EU bank. While the A22 did not match very closely the field isolate, repeated vaccination was employed to boost heterologous antibodies. It is well established that two vaccinations can compensate for moderate antigenic differences between field and vaccine viruses (Fig. 2) although the degree of compensation cannot be relied upon to cover against very significant intraserotype variation. Dubourget et al. (1987) demonstrated that the homologous and hetero-

logous neutralising antibody profiles of repeatedly vaccinated cattle remained parallel and did not converge despite the increase in absolute antibody titres due to the booster vaccinations. Thus, it is not advisable or economic to attempt to compensate for very great antigenic differences between a field isolate and a vaccine strain but rather develop a new vaccine strain as soon as possible. In an established disease situation, where vaccine is routinely used and the differences between the field isolates and the vaccine strain are not very great it is quite common practice to develop a vaccine from a local field isolate to provide the maximum match possible. 6.2. Vaccine application Organisation of the vaccination programme is extremely important and particular attention must be paid to ensuring that adequate supplies of equipment are available, including cool boxes to maintain the temperature of the vaccine between 3 and 8 8C, restraints or crushes to allow safe application of the vaccine without undue distress to the animal (of special relevance to pregnant animals), ear tags, sterile syringes and needles. Also important is the provision of adequate protective clothing and disinfectants and adherence to strict zoosanitary measures so as to ensure that vaccination teams do not inadvertently carry disease between premises. It must be remembered that diseases other than FMD may be present on a farm (e.g. bovine leukosis) and the overuse of needles and syringes may spread these. It is also important to emphasise that only healthy animals should be vaccinated. Cattle, sheep, goats and pigs are routinely vaccinated with FMD vaccine. In the case of other animals and particularly wildlife, it is certain that many species

Fig. 2. Two doses of A22 vaccine in cattle stimulate sufficient cross-reactive neutralising antibody against the Saudi Arabian isolate A Sau 23/86 to confer greater than 85% protection. Adapted from C.G. Schermbrucker, unpublished results.

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respond or would be expected to respond to FMD vaccines but little or nothing is known of protection against disease. Hedger et al. (1980) reported virus neutralising antibody responses of African buffalo, eland and impala to FMD vaccination and while antibody titres were generally inferior to those of domestic cattle, it is difficult to imagine that such antibodies would not confer a substantial degree of protection against clinical disease. Unfortunately, such experiments are not feasible nor, arguably, ethical and, therefore, EU regulatory requirements do not permit a claim to be made for such species unless supported by repeated dose and overdose regulatory studies and, strictly speaking, challenge data. Aqueous based vaccines are administered to cattle, sheep and goats by the subcutaneous route, usually the upper neck or just in front of the shoulder. Dose volumes for large ruminants are of the order of 2
/3 ml and small ruminants usually receive one half to one third of a cattle dose. The dose given to young animals is the same as that for adult animals. Oil based vaccines are administered to cattle and pigs most commonly and by the intramuscular route. Once again, the upper neck is often used with cattle and the favoured site for pigs is behind the ear. A typical dose volume is 2 ml and once again there is no difference between young and adult animals. Provided vaccines are prepared from purified components (both antigen and adjuvant), reaction at the site of vaccination is mild and hypersensitivity reactions are very unlikely. There have been a number of claims that oil vaccines are greatly superior to aqueous (aluminium hydroxide/ saponin) vaccines. While this is certainly the case with pigs, which respond poorly to aqueous vaccines, the differences with ruminants, primarily cattle, seem to depend on the researcher. Thus our unpublished studies suggest little difference between the two adjuvants and this is supported by a number of publications (e.g. Kitching, 1997). It should also be said that there are many variations on the oil emulsion formulation (waterin-oil; water-in-oil-in-water (DOE); oil-in-water) and generalisations made by some authors are unwarranted at best and misleading at worse.

6.3. Primary responses and young animals Vaccinated livestock respond rapidly to the first dose of vaccine and produce peak antibody titres between 14 and 28 days depending on the vaccine composition. High potency vaccines made from antigens held in emergency antigen banks are capable of producing quite rapid immunity to challenge */within 3 or 4 days in the case of cattle */and are, therefore, potentially valuable for vaccination in the face of an approaching disease front (Doel et al., 1994). While it has been assumed and

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often implied that rapid protection is an exclusive property of very high potency vaccines, there are a number of reports where early protection has been demonstrated with conventional FMD vaccines (e.g. Donaldson and Kitching, 1989). Livestock from non-vaccinated dams appear to be fully immunocompetent and respond to FMD vaccination from an early age. Nicholls et al. (1985) concluded that calves as young as 1 week responded as effectively as adult cattle under experimental and field conditions and we have observed excellent immune responses within the first 2 weeks of life with cattle, sheep and pigs. These results contrast with those of Terpstra and Dekker (1997), who suggested that 2 week old calves may respond less effectively than adult cattle. In the absence of an obvious explanation, it is possible that the type (formulation) or purity of vaccines used in the different studies had a significant influence. In the case of routine vaccination programmes, the influence of maternally derived antibody (MDA) on the immune response of young animals from vaccinated dams is of considerable importance. The presence of MDA suppresses the response of the young animal to FMD vaccine and vaccination programmes need to be developed to take account of this limitation. Although the half-life of MDA in cattle and pigs is only 21 days approximately (cited in review of Kitching and Salt, 1995), the influence of MDA on FMD vaccine efficacy can extend for a number of months particularly if the colostral MDA has been elevated by repeated vaccination of the dam with high quality product. Less commonly, 5 or 6 months may elapse before an animal responds to vaccine (Kitching and Salt, 1995) and there may be no evidence of an anamnestic response, i.e. previous vaccinations in the face of MDA do not necessarily prime the immune system. A theoretical concern, although there is no evidence to the author’s knowledge, is the possibility that vaccination in the presence of MDA might influence or prevent the development of immunological memory, with obvious consequences for the immune status of long-lived animals. There have been reports that oil based FMD vaccines circumvent the problem of MDA. However, the evidence for this is not conclusive. In practical terms under endemic conditions, it is not realistic to hold young animals until all of the individual titres of MDA have completely waned otherwise the virus is presented with a ‘window of susceptibility’. Therefore, with both types of vaccine, we recommend vaccination of all animals from vaccinated dams at 2.5 months of age unless the epidemiological circumstances are very severe in which case 2.0 months is prescribed. In the case of animals free of MDA, we recommend the first vaccination at 14 days of age.

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6.4. Booster vaccinations and duration of immunity The application of booster vaccinations will depend on the value and life expectancy of the species as well as epidemiological circumstances and perceived risk of disease spread. Thus, with more valuable animals (notably cattle or animals kept for extended periods such as breeding stock), it is common practice to vaccinate a second time within approximately 1 month of the first vaccination followed by subsequent vaccinations every 4
/6 months or every year depending on the prevalence of the disease in the region. Vaccination practice in Europe prior to 1991 was largely restricted to cattle and booster vaccinations were made on an annual basis. We have examined the influence of the interval between the first and second vaccinations and, consistent with immunological theory, it appears to be preferable to wait as long as possible between the application of the first and second doses in terms of the magnitude of the anamnestic response. It is interesting to note that such a strategy used by the Dutch in the 1960s could explain the long duration of immunity reported by Fish et al. (1969). Unfortunately, prevailing field conditions may not permit the luxury of an excessive interval between the first and second doses and the booster vaccination may have to be made quite soon (i.e. 1 month) after the first dose of vaccine in order to minimise the ‘window of susceptibility’ of the animal. FMD vaccines are frequently criticised for their inability to provide long-term immunity and comparisons tend to be made with vaccines such as attenuated poliovirus. Such comparisons are unreasonable particularly when immunity following infection with FMD is considered. Duration of immunity in the vaccinated pig as measured by virus neutralising antibody and following a typical programme of two doses, a month apart, is not greatly different from that of convalescent animals */of the order of 6 months. It is not commonly known that pigs, recovered from infection, only remain resistant to reinfection with the same strain of virus for a period of about 3
/6 months (McKercher and Giordano, 1967). In cattle, it appears that convalescent cattle are probably able to resist reinfection with the same strain for up to 4.5 years although considerably shorter periods have been observed (Cunliffe, 1964). In contrast, vaccines confer about 6 months immunity following the initial two dose regime. However, the situation with cattle is more complex because of the prevalence of the carrier state in animals recovered from clinical disease. The presence of continuous or sporadic presence of virus in carrier animals obviously gives an opportunity for repeated stimulation of the immune response and hence maintenance of protective antibody titres. Despite the common perception of poor duration of immunity with FMD vaccines, there is good evidence of long-term immunity in animals under several of the

routine mass vaccination programmes employed by Europe up to the end of the 1980s. Fish et al. (1969) reported that Dutch calves receiving their first (single) dose of vaccine lost protective antibody titres within a few months of vaccination. However, annual booster vaccinations of young and older animals showed that antibody titres were fully sustained throughout the following 12 months until the next round of vaccination. Even more remarkable are several reports during the last decade in which European cattle were found to have significant titres of neutralising antibodies directly attributed to vaccination despite the fact that the European programme ceased 6 or 7 years previously (e.g. Remond et al., 1998). The most important conclusion is that well organised and extensive vaccination programmes such as those used in Europe before 1991 and countries such as Uruguay until 1994, not only confer high levels of protection to the individual animal and the herd but prevent establishment of the disease in the country as a whole and set the scene for eventual eradication. 6.5. Use with other vaccine FMD vaccines have been applied simultaneously with a wide range of other antigens/vaccines including Classical Swine Fever, Rinderpest, Haemorrhagic Septicaemia, Rabies, Brucella, Porcine Parvovirus and Anthrax (Joseph and Hedger, 1984; Hedger et al., 1986; Jera´bek et al., 1988; de Clercq et al., 1988) and there is little to indicate any problems either in terms of suppression of the immune response to FMD antigens or those of the other vaccine. In practice, it is difficult to make any regulatory claims in this regard. All of the examples quoted were not done according to modern regulatory standards and the regulatory hurdles are extremely daunting if a manufacturer wishes to make a new claim concerning the simultaneous or combined application of two different antigens. This is particularly the case with FMD vaccines where regulatory studies usually require the use of rare and costly biosecurity units for the animals. FMD vaccines are routinely formulated as mono- or poly-valent products depending on the epidemiological circumstances in a region or the perceived threat of virus introduction. Thus, vaccines which contain three or four serotypes, several of which may consist of two or three strains, are quite common and even heptavalent formulations exist. This raises the possibility of interference between different strains in terms of the immune response. In fact there is excellent evidence from both experimental studies (Black et al., 1986) and, indeed, many years of routine batch testing by our company that there is neither interference between serotypes nor within serotypes. However, it must be appreciated that some polyvalent vaccines, by virtue of their high pay-

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loads of FMD antigens, may contain high concentrations of BHK cell and media-derived extraneous proteins and there is the potential for adverse reactions to these vaccines unless all of the constituents have been purified.

7. Disease control strategy and vaccination This is a controversial and complex issue and there is no simple deductive process to arrive at a definitive strategy for FMD control. The overall policy of a country or region affected by the disease will be based on a broad range of factors including the cost and feasibility of different control or eradication measures, the impact on domestic and export markets (if any), the loss of animal productivity, the consequences for other elements of the economy such as tourism, and social implications including animal welfare, cultural/religious considerations and environmental issues. To some extent, this complexity may be simplified by the division of countries and regions into those which have a significant dependence on revenue from export of livestock and livestock products and those which are primarily concerned with domestic productivity. The first category usually adopts a slaughter policy in the case of the first outbreak of disease until it is clear that its spread can only be checked by additional measures, notably vaccination. The second category will turn more readily to vaccination to protect the health and productivity of the national livestock. 7.1. Vaccination as a tool in integrated control programmes It is very clear that routine mass vaccination programmes, properly and extensively applied, can help in reducing disease to the point of eradication. This may be achieved even without vaccination of all susceptible livestock. Donaldson (2000) cites the example of Uruguay where the sheep:cattle ratio was 2.6:1 and both species graze together. Prior to the cessation of vaccination in 1994, sheep vaccination was abandoned as impractical and uneconomical and the vaccination coverage of cattle was increased. The strategy was successful and cattle outbreaks not only declined to zero but there was no evidence of the continued circulation of virus in the sheep population. During the mass vaccination programmes in Europe before 1991, it was also the common practice to concentrate on the cattle, partly because of their high value, and relatively few sheep and pigs were immunised unless there was an outbreak. Clearly, this strategy was very successful during this period although it would be prudent to point out the dangers of extrapolating too far from the specific circumstances of Europe prior to

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1991 (field strains, animal husbandry, more restricted animal movement, etc.). If routine vaccination is not applied to all of the susceptible livestock population, it may be necessary to use emergency vaccination measures for outbreaks in the non-vaccinated animals. An example is that of the Netherlands in the mid 1960s which also serves to demonstrate the practical value of vaccines in the control of FMD in pigs. Typically, the Dutch authorities only applied routine mass vaccination to cattle and sporadic outbreaks of disease in pigs were sometimes controlled by a combination of vaccination and other measures such as slaughter of clinically diseased animals. van Bekkum et al. (1967) reported on the control and stamping out of an outbreak of C serotype FMD in Dutch pigs in 1965 and 1966 where vaccine was used for the first time. In two heavily infected areas with a total pig population of 70 000 animals, a single dose of vaccine was applied to all animals within 1 week and proved effective to bring the disease under control within a fortnight. In total, 1289 farms were covered by vaccination and clinical disease was subsequently found on only 46 premises. In other areas of the Netherlands, using a programme of two separate doses of less potent vaccine with approximately 80% coverage of the pig population, the disease disappeared about 10 days after the first dose of vaccine. Field experience of FMD control by vaccine is supported by experimental transmission studies which have demonstrated that vaccination of cattle, sheep and pigs substantially reduces excretion of virus by these species, when subsequently challenged. Equally importantly, fully susceptible control animals did not develop clinical disease when placed in contact with the vaccinated/challenged animals provided there was a sufficient interval between the vaccination and the challenge dates (Donaldson and Kitching, 1989; Salt et al., 1998; Cox et al., 1999). The interval required, such that excretion, and consequently transmission, was effectively blocked, depended on the species and was between 5 and 21 days. Presumably the potency of the vaccine would be expected to have some bearing on this. Interestingly, when the interval was 4 or 7 days in the case of the cattle experiment, the in-contact control animals became clinically diseased and overwhelmed the developing immunity of the vaccinated/challenged cattle (Donaldson and Kitching, 1989). 7.2. Strategic use of vaccination in an emergency The cessation of vaccination in Europe following the eradication of FMD and the growing awareness by other countries of the vulnerability of their fully susceptible livestock to virus introduction has led, over the last three decades, to the establishment of national and international reserves of inactivated FMD vaccines

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or antigens. One of the first such reserves was set up by the UK in the 1970s, largely in response to the views and recommendations of the committee charged with the official enquiry into the 1967/68 outbreak in the UK (the ‘Northumberland’ Report, 1969). While most members of the committee concluded that vaccination would not have assisted the control of the disease, the report indicated. . . ‘we think that in certain conditions ring vaccination could be a useful adjunct to slaughter’. . .and. . . ‘stocks of suitable vaccine prepared from the predicted types or sub-types would have to be maintained’. . . In the case of vaccines, reserves are still maintained by a number of countries and fresh product is purchased once the existing product has reached the end of its shelf life. Antigen reserves or ‘banks’ are now extremely popular and many countries have access to such banks either through a single contract with a vaccine manufacturer or as a member country of one of the three international banks (Garland, 1997). The two key advantages of any form of these banks is that they are able to implement emergency vaccination regardless of the level of demand in the world (and this is often very unpredictable) and the supply is almost immediate following the placing of the order. The international banks are the EU bank representing all EU countries, the North American FMD Vaccine Bank, representing Canada, Mexico and the USA, and the International Vaccine Bank, representing Australia, Finland, the Republic of Ireland, New Zealand, Norway, Sweden and the UK with Malta as an associate member (Garland, 1997). The quantities and types of antigens held in the various banks reflect the perceived threat(s) to a given country or group of countries. The largest bank is probably the EU bank which holds up to 5 million cattle dose equivalents of most of the major vaccine strains including the SAT serotypes. As with most banks, the EU bank antigens are stored as highly concentrated inactivated preparations in the gaseous phase of liquid nitrogen and the process of emergency vaccine production entails the relatively quick thawing and dilution of the antigens in bulk adjuvant followed by filling into final product containers. Typically, vaccines made from bank antigens are 6 PD50 or greater rather than the 3 PD50 stipulated by the Ph.Eur. (1993). To some extent this reflects the perception that higher potency vaccines will be more effective in stimulating early immunity although studies with conventional potency vaccines have also demonstrated protection after only a few days (Sellers and Herniman, 1974). The EU bank is certainly the most active and vaccines prepared from some of its antigens have been used successfully in a number of outbreaks in regions outside of the EU. Perhaps the best known is the supply of A22 vaccine to the Balkans in 1996 (Leforban, 1997) when vaccine application was considered to play a valuable

role in the control and eradication of the disease from the region. Before discussing some of the issues in the application of emergency vaccines, it is perhaps useful if the terminology is explained. Emergency vaccination essentially falls into three categories and the names used for the categories are numerous. Ring vaccination or barrier vaccination involves the development of an immune barrier by vaccinating livestock between endemic or epidemic areas and disease-free areas. An example of barrier vaccination is the Western Buffer Zone in Turkey, funded by the FAO and EU, to prevent the spread of FMD into Greece and Bulgaria. Thus ring or barrier vaccination may either be used only at the time of an outbreak or represent a relatively permanent component of the regional control policy. Dampening down or suppressive vaccination is used as close as possible to the outbreak to ensure that animals which may become infected despite vaccination do not excrete as prolifically as if they had not been vaccinated at all. Thus the general levels of virus in the environment will be greatly reduced as will the probability of spread of the virus within and outside of the region. Obviously, both basic strategies will have common elements and may very well be used together. Finally, mass vaccination is the blanket coverage of all of the animals in the region and, while it could be employed just once, tends to be used on a more routine basis. Given the immense pressures on national veterinary authorities during a FMD crisis, it is very valuable to develop a preparedness plan during ‘peacetime’ covering as many disease scenarios and control options as possible. Such a plan should at least facilitate the decision making process if an outbreak occurs. In relation to emergency ring vaccination, Donaldson and Have (1997) reviewed the many considerations and listed the key criteria supporting a decision to vaccinate. It is important to stress that their paper related primarily to the use of emergency vaccination by a country wishing to regain rapidly its FMD-free status and some of the criteria given below would be heavily weighted against vaccination on the basis of loss of export markets. Nevertheless, many of the criteria would need to be considered regardless of whether or not an affected country had a significant export trade in livestock or livestock products: 1)

2)

Quality of veterinary (and social) infrastructure including speed, accuracy and comprehensiveness of disease reporting. Without this, the definition of a vaccine zone will be unreliable at best. Susceptible livestock density, herd size and locations, species composition. In particular, the presence of pigs with other species because of their propensity to excrete massive quantities of virus.

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3)

4)

5)

6) 7)

8) 9) 10) 11)

Prediction of high risk of diffusion of airborne virus. Other factors which could impact on the risk of virus diffusion should also be considered e.g. human population density and difficulty of enforcing movement controls. Vaccination zone can be clearly defined. This is a particularly complex problem and, apart from the quality of epidemiological information, factors would include the topography, meteorology, and locations of farms, markets, etc. Operational problems prevent or indicate a delay in the completion of stamping out. This is a very considerable potential problem in areas such as Belgium and the Netherlands where susceptible livestock numbers and densities are very high (Donaldson and Doel, 1992). Contingency plans for emergency vaccination are in place. Logistic/operational needs can be met. This includes quantity and quality of suitable vaccine, personnel, support services. Economic consequences of ring vaccination are acceptable. Primarily, the loss of export trade. Vaccinated animals can be permanently identified and the vaccination zone can be monitored. Contingency plans in place to manage continued productivity and/or slaughter of animals. Legislation in place to support all control measures.

In fact, many of these criteria also relate to emergency suppressive vaccination, an example of which is given above (van Bekkum et al., 1967), and even routine mass vaccination where it is clear that an effective programme requires both proper planning and an adequate control infrastructure.

8. Implications of vaccination Within the scope of this subject, the main issues include the safety of FMD vaccines in the target species, vaccination in relation to the carrier state and disease transmission, and the export implications which are of particular importance to a number of FMD-free countries. The latter topic will not be considered here and is fully covered in the International Animal Health Code of the Office International des Epizooties (OIE). 8.1. Innocuity and safety Certainly there were reports many years ago (Beck and Strohmaier, 1987) that the handling of the virus by production or research laboratories or the use of vaccine was very occasionally associated with outbreaks of the disease in Europe. While these authors were undoubt-

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edly correct in their general conclusions, the frequency of O and A serotype outbreaks appeared very much higher than with the C serotype, even though many routine vaccines employed in Europe were trivalent, suggesting that the incidence of natural introduction of disease may have been underestimated. Nevertheless, since that time there have been very substantial improvements in the biosecurity standards of all facilities and major changes made to the methodology of inactivation of virus (notably replacement of formaldehyde with BEI) and innocuity testing of the intermediate products. Consequently, FMD vaccines made according to current internationally recognised standards of GMP and tested stringently by procedures such as those described in the FMD Monograph in Ph.Eur. (1993) can be considered completely innocuous and do not present a risk of introduction of live virus into a population. Related to innocuity, is the safety of the vaccine and potential adverse reactions in livestock. There were numerous reports by workers in the 1970s of allergic type reactions in vaccinated animals, sometimes serious or even fatal (Black and Pay, 1975). Extensive research during this period highlighted a number of potential causes including the use of formaldehyde which was believed to modify extraneous proteins in the crude antigen harvests, the quality of the saponin, and the general protein burden within the final product. Undoubtedly such reactions continue to occur and are occasionally reported but there is no longer any justification for these to happen if the appropriate measures are taken. In the case of our FMD vaccines, we use both purified antigens and purified saponin and inactivate with BEI. The combination of these approaches results in only minimal reactions at the site of inoculation and never the extreme allergic reactions seen many years ago with some vaccines. In this way the welfare and productivity of the vaccinated animal is ensured. The importance of food quality standards is increasingly recognised and the acceptability for human consumption of meat and milk products from vaccinated animals seems to be routinely questioned at the time of a new outbreak (UK, 2001; Taiwan, 1997; Philippines, 1995) with often serious implications for the domestic market prices of the products concerned. Considering the fact that many billions of doses of FMD vaccines have been sold worldwide since vaccination became a widely adopted practice, it is clear that there is no evidence whatsoever that consumption of these products has any implications for human health. 8.2. The carrier state and transmission of carrier virus Apart from the immediate impact of FMD on livestock and the farming community, the existence of

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the carrier state with cattle and, to some extent sheep, has significant implications for control and eradication programmes. In these species, exposure to the virus can give rise to the development of the carrier state in which, by definition, virus can be recovered from the oesophageal/pharyngeal tissue and fluid of the animal at least 28 days after the initial challenge. The carrier state in cattle appears to last up to several years in extreme cases and up to 6
/9 months in sheep and goats. However, the majority of cattle and sheep appear to lose their carrier status within a relatively short period of time. Hedger (1970) gave values of 5.4% carrier animals 12 months after an outbreak in a partially immune cattle population in Botswana compared with 68% during the outbreak. The most important questions are whether carrier cattle or sheep are able to transmit disease to susceptible animals and whether vaccination prevents or otherwise the carrier state. The first question is still unresolved although the ‘negative’ data is, in the author’s opinion, more convincing than the ‘positive’. There have been a number of reviews of the subject including those of Mohler cited by Singh (1969) in the reference list and Wittman, (1990) and several notable experiments do not support a significant role for carrier cattle in the maintenance of disease (van Bekkum et al., 1959; Mohler, 1924 cited by Singh). These included a large experiment with cattle recovered from an outbreak in the National Dairy Show in Chicago in 1914 which were used in an unsuccessful attempt to transmit to 50 susceptible cattle and 50 susceptible pigs. Wittman, (1990) was sufficiently convinced by the evidence of his review to comment ‘from all these experiments, it can be concluded that FMDV carrier cattle are of no epizootiologic importance’. Similar conclusions have been made about sheep (Wittman, 1990) and the carrier state does not exist in pigs. The ‘positive’ experiments are relatively few, aside from anecdotal observations, and have involved carrier buffalo in Africa (Hedger and Condy, 1985). While transmission from carrier buffalo to domestic cattle may be a mechanism for maintenance of the disease in the African environment, it must be said that carrier experiments are particularly difficult to conduct under even controlled field situations because of the absolute need to prevent transmission other than by the carrier virus and the often long-term nature of the study. A single dose of FMD vaccine does not prevent the development of the carrier state in cattle (and probably sheep) although potent preparations typical of those used for emergency ‘vaccine’ banks appear to reduce the frequency of virus isolation or the virus titre provided the interval between vaccination and challenge is sufficient (:/21 days, Doel et al., 1994; Salt et al., 1995). While the prevalence of the carrier state in vaccinated cattle in these experiments was not greatly different from the data of previous workers such as

McVicar and Sutmoller (1969), there are, nevertheless, indications that good quality vaccines could have a significant benefit, particularly if routinely and repeatedly applied. This claim is supported by a field study in which the incidence of carriers within repeatedly vaccinated cattle herds was substantially reduced compared to non-vaccinated animals (Anderson et al., 1974). However, the possibility remains that repeated vaccination worked in this situation by preventing outbreaks of clinical disease and consequent levels of virus circulating within the cattle population rather than directly suppressing the development of the carrier state. Considering that repeated vaccination of cattle eventually stimulates mucosal immunity whereas only a single dose of vaccine is needed to stimulate mucosal immunity in pigs, which do not become carriers (Francis et al., 1983; Francis and Black, 1983), it would be valuable to examine whether repeated application of high quality vaccines followed by challenge prevented completely the development of the carrier state in cattle. Because of the perceived importance of the carrier state, considerable effort has been made to develop a test which would effectively discriminate a vaccinated animal from an animal which was either subclinically infected or a carrier. A number of tests, including several described within the OIE Manual of Standards (2000) have been developed and these all rely on the measurement of antibodies against the non-structural (NS) proteins of the virus. NS proteins are required for replication of the virus in the host cells and antibodies against them (3AB, 3ABC, 2C, 3A, 3B, 3D) are considered to be the most reliable serological indicators of infection (OIE Manual of Standards, 2000). Unfortunately, the same NS proteins are synthesised during industrial scale virus production and remain associated with the vaccine in the case of final product prepared from non-purified viral antigens. Thus, such vaccines will induce the production of antibodies to NS proteins in the same way as natural infection and confuse the interpretation of the assays described by the OIE. The remedy to this problem, and to allow greater meaningfulness of the NS antibody assays, is to remove as much as possible the contaminating NS proteins by purification of the industrial scale FMD antigens. In our company, all inactivated antigens are concentrated and purified to a very high degree such that repeated vaccination does not induce significant antibody titres against the NS proteins.

Acknowledgements I would like to thank my colleagues Joa˜o Amaral, Duncan Fawthrop, Bruno Saint-Marc and, particularly, Michel Lombard for their helpful discussions and comments.

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