Appendix 76

Appendix 76

Post-vaccinal serosurveillance for FMD: a European perspective on progress and problems D J Paton1, K de Clercq2, A Dekker3 1FAO World Reference Laboratory for FMD, Institute for Animal Health, Pirbright, Surrey GU24 ONF, UK 2CODA-CERVA-VAR, Department of Virology, Section Epizootic Diseases, Groeselenberg 99, B-1180 Ukkel, Belgium 3Institute for Animal Science and Health, Department of Mammalian Virology, P.O. Box 65, NL 8200 AB

There has been much debate about the use of the so-called vaccinate-to-live policy for the control of FMD in Europe. According to this approach, spread of the FMD virus from future outbreaks could be controlled by a short period of “emergency” vaccination of surrounding herds, reducing the need for large-scale preemptive culling of at-risk animals. Since vaccinated ruminants may become subclinically and persistently infected with FMD virus following challenge exposure, it is necessary to either kill or slaughter under controlled conditions foreseen in the OIE Terrestrial Animal Health Code all vaccinates (vaccinate-to-kill) or to detect and kill or slaughter under controlled conditions all vaccinates that have become persistently infected (vaccinate-to-live), in order to rapidly regain the most favoured trading status of FMD-free without vaccination. The latter approach can be attempted by testing vaccinated animals for the presence of antibodies to certain non-structural proteins (NSP) of FMD virus, which are induced by FMD infection, but not by vaccination with purified vaccines. The numbers of herds and animals to be sampled and tested to be confident that infection has not been missed will depend upon the expected prevalence of subclinical infection amongst and within herds. This in turn will depend upon the manner in which infection is spread and on how vaccination is applied. The sensitivity of the tests used and the size of the herds will also influence the numbers of samples required to be collected and tested.

The new Council Directive 2003/85/EC on FMD takes account of these factors in its provisions for vaccination and for the use of post-vaccination serosurveillance to detect subclinical infection (Anon, 2003). According to the Directive, blood samples should be collected and tested from vaccinated animals and herds within a vaccination zone and from the unvaccinated offspring of vaccinated animals. Either all animals within vaccinated herds must be sampled and tested (Article 56, 3 (b)) or else sufficient numbers must be sampled and tested to enable a 5% prevalence of subclinical infection to be detected with 95% confidence (Annex III, point 2.2). Such sampling is not to take place until at least 30 days after the completion of emergency vaccination. EC legislation, taking into account that the necessary tests are regarded as herd tests and are not suited to verify the status of an individual animal, requires that herds within which at least one confirmed persistently infected animals has been detetcted must be slaughtered. However, difficulties in predicting the likely prevalence of post-vaccinal, subclinical infection and uncertainty over the performance of NSP tests has cast doubt over the suitability of these proposed sampling regimes.

There are now several commercially available NSP antibody ELISA tests. Under favourable sampling conditions, our estimates are that the sensitivity of these tests for detecting individual persistently infected vaccinated cattle can be as high as 90%, with a 99% specificity. This assumes that the vaccines used have been purified to remove traces of NSP and have been given in an emergency setting involving the application of a single vaccine dose. If all animals can be sampled, at least one animal should score positive to detect a herd. If a test with a 100% sensitivity is used in a herd of 100 animals a prevalence of 1% can be detected, but in a small herd of 10 animals 1 positive animal is equal to a prevalence of 10%. If the sensitivity is lower, e.g. 80%, the chance of missing positive animals is 0.2. To be sure we will detect a farm with 95% confidence the chance of missing animals (0.2n) should be lower than 0.05. This means the number of positive animals should be at least 2 (0.2log(0.05)=log(0.05)/log(0.2)=1.9). In this case on a large farm (100 animals) a prevalence of 2% can be detected (95% confidence), but on a small farm (10 animals) only a prevalence of 20%.

This therefore sets a limit on the degree of certainty that can be achieved for detecting low levels of persistently infected animals. It has been difficult to obtain reliable information on carrier prevalence under field conditions following vaccine breakdowns and even where available may not be relevant to regimes of husbandry and vaccination intensity that would prevail under European conditions. Sutmoller and Gagero (1965) reported a 50% prevalence at four months after a vaccine trial breakdown in Brazil.

In recent studies in which cattle that had been vaccinated 21 days earlier were exposed to five days of direct contact with unvaccinated, infected cattle, nine out of 20 vaccinates became persistently infected with FMDV giving a prevalence of 45% (Cox et al., in press). On the one hand, this challenge was more even and severe than is likely under field conditions, but on the other hand field vaccination may be less reliable and in a field situation, challenge may occur before so much time has elapsed for the development of post-vaccination immunity. Therefore, scenarios can be envisaged in which the prevalence of carriers within a vaccinated population could be greater or smaller than 45%.

Measures can be taken to mitigate the risks from missing subclinical infections in vaccinated populations. If ring vaccination is used, then the animal population outside of the vaccination zone will have a higher susceptibility to FMD virus infection. Therefore, the EU Directive requires a buffer zone to be established around a vaccination zone and for animals to not be moved out of the vaccination zone until FMD-free status has been attained, which will be at least six months after the last outbreak or last vaccination, whichever is the longest. It would also be prudent to avoid the introduction of unvaccinated animals to vaccinated herds for the same time interval.

Another problem for post-vaccination serosurveillance is that testing large numbers of samples will lead to many false positive test results; on average at least one false positive result can be expected every time a herd of 100 or more animals is tested with a method that has a 99% specificity. No confirmatory tests have been introduced in European laboratories to verify whether or not such results are specific. However, recent findings suggest that some of the different 3ABC NSP ELISA tests do not score the same sera as false positive and therefore providing they are of sufficient sensitivity, they may be used to confirm each others results. Other solutions may be to analyse the test results on a herd-basis and to look for evidence of multiple seroconversions or sub-threshold increases in antibody levels that could be indicative of genuine infection. Another option is to retest all positive samples without changing the assay and then to resample and retest animals that continue to score positive, although this will marginally reduce the overall sensitivity of the assay. At the time of resampling reactors, additional blood collections should be made from neighbouring animals for delayed seroconversion that could have been missed at the first sampling. Herds testing negative at the second sampling would be considered uninfected, whilst herds showing an increased level of seroconversion would have to be culled. Individual reactors that remain seropositive would be culled. Based on the mathematics described above you need at least two positive animals to detect at least one with 95% confidence. If you cull only the one that reacted positive, you probably miss one carrier animal that was not NSP positive. The risk associated with carriers missed by NSP testing should therefore be quantified and compared to the risk related to the currently accepted non-vaccination policy in which clinically affected and at risk herds are destroyed along with those identified as infected by other means.

Evidently, the confidence with which available NSP tests can be expected to detect persistently infected vaccinated animals will be strongly linked to herd size. Since it is hard to predict the likely prevalence of infection in vaccinated herds, an approach for large herds could be to begin by sampling sufficient animals to detect a low prevalence of infection with 95% confidence and then to relax the stringency of sampling if warranted by the initial results. Small herds are more difficult to deal with. One could (1) accept the risk associated with a potentially inadequate level of sampling, (2) use only a vaccination-to kill policy in small herds, (3) have additional biosecurity restrictions on small herds after an outbreak is over or (4) avoid vaccinating such herds in the first place. This last solution is attractive on a number of grounds including the fact that small herds generally pose a relatively low risk to neighbours and are not therefore a high priority for vaccination. Secondly, it is extremely important to implement emergency vaccination as quickly as possible in order to reach a critical proportion of vaccinates in the susceptible population, whereas vaccinating small herds is slow on a per capita basis. However, it can be anticipated that such a policy would be unacceptable to the owners of small herds and therefore politically difficult to implement.

Vaccinate-to-live in pigs, potentially an attractive option following the adoption of the proposed amendments to the OIE Terrestrial Animal Health Code, may be less troublesome for NSP-based exit testing. Firstly carriers do not occur. Secondly, large herd sizes mean that low level virus circulation can be detected with confidence by NSP serosurveillance, even if test sensitivity is low. Specificity however becomes a greater problem, but since pigs are kept in pens, one could discriminate between false positives and true reactors by whether or not test positive results are spatially clustered.

Taking account of technical progress and recognising the difficulty of proving the absence of a low level of persistently infected animals within a vaccinated population, the OIE, at its General Session in May 2004, has altered the requirements for countries using long-term vaccination programmes for FMD control. In this situation, vaccination takes place in the entire country or geographic zone and is applied continuously. Instead of being required to prove absence of persistently infected animals, the veterinary authorities in such countries will be required to show that virus is no longer circulating and will then gain the trading status of FMD-free with vaccination. Countries that are FMD-free with vaccination cannot easily export live ruminants due to the risk of some being persistently infected. So far the EU requires that meat derived from such animals must be de-boned, matured and pH-controlled before being exported, however the OIE recently modified its rules to allow such meat without restrictions..

To prove absence of virus circulation by serosurveillance is much easier than to prove absence of infection since no definite decision needs to be taken as to whether NSP reactors are genuine or not; it will suffice for herds with NSP seroreactors to be resampled and retested to show absence of seroconversion. The OIE is still finalising its FMD surveillance guidelines and an Epidemiology subgroup was formed to discuss this issue in June 2004. More detailed guidance has been provided on the followup procedures necessary when NSP reactors are discovered in vaccinated animals, but this guidance does not apply to the situation envisaged in Europe – i.e. emergency vaccination and then rapid recovery of the status of FMD-free without vaccination.

In conclusion, problems associated with the imperfect specificity of NSP tests may be overcome by a resampling and retesting programme, but the requirement to use serosurveillance to prove complete absence of any persistently infected cattle in a vaccinated population cannot be met. However, the risk associated with a low level of undetected carriers will be low. This risk needs to be better quantified and compared to the risks of other control policies, including the previously accepted one of non-vaccination. The revision of the OIE guidelines for serosurveillance requirements in countries wishing to attain the status of FMD-free with vaccination and the transposition of the new EC Directive on FMD with its effects also on imports might conceivably lead European countries to include in their contingency planning an approach to disease control in which emergency vaccination could be followed by serosurveillance to detect virus circulation rather than infection (including detection of all carriers).

However, there is currently no provision in the OIE code for a country that was previously FMD-free without vaccination to use emergency vaccination to help control an outbreak and then move rapidly to the status of FMD-free with vaccination; only countries that were FMD-free with vaccination in the first place can regain this status within 6 or 18 months, depending on the scenario.

Therefore, following an outbreak of FMD in a previously free-without vaccination country or zone there is the imperative need to regain free status followed by a decision whether this status will in future be maintained with or without regular prophylactic vaccination. While this situation may hypothetically become relevant also for certain peripheral parts of the Community where the FMD situation in a neighbour country deteriorates and intervention by the Community on the territory of that country would not be admitted, the disadvantages including difficulties in maintaining an adequate separation of livestock and their products within the two zones and possible trade disincentives for the zone which was free without vaccination (including the rest of the European Community) still make this a questionable option. Furthermore, such an approach is not in line with EU legislation.

References

Anon (2003). Council Directive 2003/85/EC on Community measures for the control of foot-and-mouth disease repealing Directive 85/511/EEC and Decisions 89/531/EEC and 96/665/EEC and amending Directive 92/46/EEC. Official Journal of the European Union L306, Volume 46, 22 November 2003.

Cox, SJ, Voyce, C, Parida, S, Reid, SM, Hamblin, PA, Paton, DJ & Barnett, PV (In press) Protection against direct contact challenge following emergency FMD vaccination of cattle and the effect on virus excretion from the oropharynx. Vaccine.

Sutmoller P & Gaggero A (1965) Foot and mouth disease virus carriers. Veterinary Record 77, 968969.

Authors Conclusions

• Problems associated with the imperfect specificity of NSP tests may be overcome by a resampling and retesting programme.

• It will not be possible to prove complete absence of infection (including detection of every carrier) by serosurveillance and this problem will be greatest in small herds.

Authors Recommendations

• More detailed guidelines are required for use of NSP serosurveillance in support of emergency vaccination-to-live policies in countries currently FMD-free without vaccination.

• Consideration could be given to use of an approach to disease control in which emergency vaccination in previously FMD-free countries could be followed by serosurveillance to detect virus circulation rather than infection (including detection of all carriers).