The use of a 3ABC marker-test for FMD
22nd Conference of the OIE Regional Commission for Asia, the Far East and Oceania
Paul van Aarle DVM
Intervet International B.V.
The Netherlands
Introduction
Vaccination plays an important role in the control of FMD in Asia, Middle East, Africa and South America. In most FMD-free countries a non-vaccination policy is in place. Recent outbreaks in Europe clearly demonstrated the risk of this policy.
Using conventional diagnostic techniques, up to now it was not possible to distinguish FMD infected animals from purely vaccinated animals. In vaccinated areas disease control authorities had limited possibilities to monitor virus presence or circulation.
Vaccine composition
Modern, state of the art-vaccines are based upon highly purified antigens, which are free from Non-Structural Proteins (NSP) of the FMD virus. Other vaccines may be partly purified and contain a reduced amount of NSP. Animals, vaccinated with highly purified, NSP-free vaccines, produce antibodies against the Structural Proteins (SP) but not against NSP.
FMD virus infection induces antibodies against both SP as well as NSP.
NSP-free or NSP-reduced vaccines in combination with a NSP-test lead to a so-called marker-system.
The FMD marker principle
A test, which differentiates antibodies due to vaccination from antibodies due to infection, would be of great value in FMD control. Several tests, which are based on non-structural proteins (NSP) have been described (Berger et al., 1990, Neitzert et al., 1991, Bergmann et al., 1993, Lubroth et al., 1995).
The success of the principle has also been demonstrated for pigs (Rodriguez et al. 1994).
For the screening of large numbers of samples an ELISA would be highly preferable. An indirect-trapping ELISA for the detection of antibodies against 3ABC has been reported (De Diego et al., 1997). The sensitivity of the essay on experimental sera post-infection was reported to be 100%. The specificity was reported to be more than 99%.
From 1994 to 1997 the Commission of the European Communities sponsored a research program, Concerted Action CT93 0909, on the potential use of tests, based on NSP, for the differentiation between antibodies, induced by vaccination, from antibodies, induced by infection. The conclusions are reported in the Proceedings (Proceedings, 1998). It is concluded, that the polyprotein 3ABC is the single most reliable indicator of infection.
Bommeli Diagnostics, Switzerland, subsidiary of Intervet International, has developed Chekit-FMD-3ABC, an ELISA testkit, in collaboration with the World Reference Laboratory in Pirbright (U.K.) and the Instituto Zooprofilattico in Brescia (Italy). The test has been validated in three national reference laboratories.
Test development
The original test of the World Reference Laboratory in Pirbight (U.K.) and the Instituto Zooprofilattico in Brescia (Italy) was a trapping, indirect ELISA. The test was not ready for use and several sensitive steps had to be performed before the test could actually start. Because of this there have been problems with variable results in several laboratories with the NSP-test.

The new test, Chekit-FMD-3ABC, is a direct ELISA. The test-plates are coated with 3ABC. The test is ready to use. Compared to the original concept this has considerable advantages:

The marker-test principle
The conventional tests (VN, VP1-ELISA) demonstrate antibodies against FMD without differentiation between infection and vaccination. The VN-test has to be carried out in high-containment, because it is carried out with FDM-virus. Both tests are serotype specific. The test will only give a positive result, if the field strain is of the same serotype as the test-strain.
Chekit-FMD-3ABC only demonstrates antibodies after infection and not after vaccination. Chekit-FMD-3ABC is not serotype specific and will be positive for all FMD viruses, irrespective of the serotype.
Comparison serological results between the conventional and 3ABC marker-test:
|
Results serology |
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|
Conventional test |
3ABC-test |
||
| Vaccinated | Infected |
+ |
+ |
| Vaccinated | Not infected |
+ |
- |
| Not vaccinated | Infected |
+ |
+ |
| Not vaccinated | Not infected |
- |
- |
Validation of the test
Chekit-FMD-3ABC has been validated with the sera, which have been used for the validation of the original Brescia and Pirbright tests. The sera were from experiments as well as from the field, from well over 5.000 animals, cattle, sheep and swine. The sera involved all main FMDV-types. The sera have been supplied by the Instituto Zooprofilattica in Brescia (Italy), World Reference Laboratory Pirbright and the ID-Lelystad Institute from The Netherlands.
The following selected data give an impression on the performance of the testkit in negative and positive animals:



Antibodies against 3ABC after challenge in cattle:

Antibodies against 3ABC after challenge in sheep:

The specificity of sera from vaccinated animals is shown in Table I-III. Vaccination elicits virus neutralizing antibodies in cattle (Table I), in sheep (Table II) and in pigs (Table III). In contrast, none of these sera contain detectable amounts of anti-3ABC antibodies.
Table I. Testing of bovine vaccinated animalsa)
| Group 1 | Group 2 | |||||||
| ELISA (%)b) | VNT (log10c) | ELISA (%) | VNT (log10) | |||||
| pv weeks | x | SD | X | SD | x | SD | x | SD |
| 0 1 2 3 4 5 |
-0.7 -0.5 -2.8 -2.6 -1.8 2.1 |
2.65 3.69 0.44 1.32 0.08 3.73 |
<0.30 0.53 1.28 0.9 0.83 >2.55 |
NA 0.35 0.11 NA 0.11 NA |
-2.7 -2.1 -2.8 -1.3 -1.0 -2.4 |
0.60 0.68 0.24 1.40 0.84 0.20 |
<0.30 <0.30 <0.30 0.3 0.6 1.65 |
NA NA NA NA NA NA |
NA: not applicable
Table II. Testing of ovine vaccinated animalsa)
|
|
ELISA (%)b) | VNT (log10) | ||||||
| A22 Iraq | O1 Manisa | Asia1 Shamir | ||||||
| pv weeks | X | SD | X | SD | X | SD | x | SD |
| 0 1 2 3 |
-2.3 -2.5 -2.2 -2.5 |
0.53 0.72 0.41 0.28 |
<0.30 <0.30 0.63 0.57 |
NA NA 0.33 0.22 |
<0.30 0.75 1.29 1.53 |
NA 0.28 0.59 0.61 |
<0.30 0.99 1.98 1.62 |
NA 0.29 0.27 0.27 |
NA: not applicable
Table III. Testing of porcine vaccinated animalsa)
| ELISA (%)b) | VNT (log10) | |||||||
| A24 Cruzeiro | O1 Manisa | C1 Detmold | ||||||
| pv weeks | X | SD | X | SD | X | SD | x | SD |
| 0 1 2 3 4 |
5.2 7.8 5.2 5.0 4.4 |
2.69 1.42 4.78 1.19 1.92 |
0.09 0.42 0.60 1.11 1.44 |
0.20 0.27 0.21 0.38 0.29 |
0.09 0.99 1.23 1.80 2.01 |
0.20 0.39 0.33 0.35 0.39 |
0.09 0.45 0.84 1.35 1.74 |
0.20 0.50 0.23 0.32 0.31 |
NA; not applicable
Testing strategies
FMD disease control measures, whether used for control, eradication or prevention, have to be adapted to local requirements. It is therefore impossible to give precise ecommendations on the practical use of the test in a specific program. Below are possibilities for use of the 3ABC test and marker-system, which may function as a starting point for the development of the right control strategy.
Whilst setting the strategy for the test it is important to reckon with the main characteristics of Chekit-FMD-3ABC:
Chekit-FMD-3ABC has considerable advantages, also in situations where vaccination is not being practised. The test detects antibodies against the 3ABC polyprotein due to infection. The 3ABC protein is not serotype specific. A positive result of Chekit-FMD-3ABC indicates an infection with the FMD virus, irrespective of the serotype.
The 3ABC test is an ideal test for border control. Independently of which FMD virus serotype is circulating, Chekit-FMD-3ABC allows the detection of serologically positive animals. The results are known within hours, which prevents unnecessary waiting times before cross-border traffic.
Chekit-FMD-3ABC will confirm the diagnosis FMD within hours. In case of a positive result further work is necessary to establish the serotype. However zoosanitary measures can be taken before the serotype is established.
International trade intrinsically carries the risk on spread of diseases. Since vaccination may be practised in some areas the use of a marker test provides authorities with the real information on infection.
Although the sensitivity is more than 99%, there is a small risk that individual animals may be missed. Therefore it is recommended use Chekit-FMD-3ABC as a herd-check and to test all animals at the border.
Breeding farms and artificial insemination centres may decide (subject to Government approval) to opt for FMD vaccination, even if the country of origin is free from FMD. Through vaccination animals can be protected in countries, which have the risk on incidental outbreaks. Because of international traffic, the risk is realistic in most countries. In addition, in case of export of animals to FMD endemic areas it is highly attractive to vaccinate the animals well before the transport in order to deliver animals, which have full protection at the moment of arrival in the importing country.
In countries, where FMD is endemic, individual farms (e.g. high standard farms, breeding farms, A.I. centres, experimental stations), may decide to aim for FMD status with vaccination.
Special attention needs to be given as to a testing scheme and sample size.
Although endemic, FMD virus may be circulating in livestock-intensive areas. Usually in less intensive areas virus circulation is less and it may be possible to aim for regional FMD eradication. Initially vaccination can be intensified in the region and if the number of 3ABC positive animals is close to zero, eradication may be continued without vaccination.
A FMD regional eradication is important as it may provide a source of FMD-free animals for restocking on depopulated farms.
In FMD endemic countries, a conventional VP1 Elisa or VN test may be used to check for vaccination discipline. The 3ABC marker test provides information on the frequency of FMD infection in the same (vaccinated) animals and additional information on vaccination discipline. Animals, which show high titres in a conventional test are frequently considered to be well-vaccinated. With Chekit-FMD-3ABC it can be decided, whether high VN-titres are due to efficient vaccination or due to a field infection.
In the past the status of "FMD free with vaccination" was obtained, based on absence of clinical disease and absence of FMD infection in sentinel animals. Some FMD strains show very few clinical symptoms. The use of sentinel animals is therefore not very reliable. Besides, an infection of sentinel animals may well hamper earlier results in reduction of virus circulation.
The 3ABC test is a reliable herd-test to trace infected farms. While using Chekit-FMD-3ABC, the use of sentinel animals is no longer a prerequisite to trace virus circulation.
If the status "FMD free with vaccination" has been obtained with the use of a 3ABC marker test, the absence of circulating virus is sure and the country may safely take the necessary steps to stop vaccination.
Most FMD-free countries adopted the non-vaccination policy. The vulnerability of the livestock industry (under the non-vaccination policy) became clear in the recent outbreaks in Europe.
In case of an outbreak in a (formerly) FMD-free country, vaccination remains an important tool in FMD-control. This has a.o. been demonstrated in The Netherlands, where vaccination has been used in combination with a very stringent stamping-out policy. Within 2 weeks after vaccination, there were no new outbreaks of FMD.
Vaccination however was only allowed on the condition, that all vaccinated animals would be identified and killed and destroyed within 2 months.
Because of the possibility to trace infection in vaccinated animals, Chekit-FMD-3ABC enables authorities to start emergency vaccination whilst monitoring the spread of the virus. Vaccination prevents the spread of the disease. Because infection can be traced in vaccinated animals, it is no longer necessary to slaughter all vaccinated animals. Massive (preventive) culling procedures have met strong criticism from the public because of environmental, ethical and emotional reasons and are economically unattractive.
Vaccinated animals from farms without 3ABC positive animals can safely be offered for human consumption and will not pose a risk on spreading of FMD. If considered necessary, it is still possible to restrict slaughter of vaccinated animals from 3ABC negative farms to slaughterhouses within the vaccinated area.
With the 3ABC marker-test and –vaccine authorities have the full choice to decide for protective or suppressive vaccination in one of the following geographical scenario’s:
Last but not least, success in FMD control depends on many control mechanisms, of which vaccination and testing is only one. Stringent zoosanitary measures remain essential to success.
Practical issues of Chekit-FMD-3ABC
Virus spread within a group of vaccinated animals.
comments from Dr Keith Sumption
We asked Dr Keith Sumption to comment on the implications of continued active infection within a vaccinated group.
6th August 2001
The spread of virus within a vaccinated population is low; although it is proven that
vaccinates will shed virus to in-contact animals. This means that it is not safe to
:
1) send a vaccinated animal into the general population while any risk of continued virus challenge continues, since the vaccinated animal could unknowingly be infected, and
2) do not mix unvaccinated
and vaccinated stock, i.e. aim for 100% cover in sheep (since cattle would act as
indicators) or 100% in all species. Vaccinating cattle alone would be a risky
strategy since sheep would not show many signs and could by weight of challenge
overcome the immunity in some of the cattle. Ditto pigs, if unvaccinated pigs
are kept on a holding these again might shed virus into sheep/cattle, even though
first infecting pigs is not easy by the aerosol route.
The arguments for these scenarios are what I am working on, essentially since we
know vaccinates can shed virus (when challenged), the sensible use of vaccination is in the context of aiming at 100%
cover in target areas, and maintaining movement restrictions until acute
infections can be expected to be extinguished and evidence of continued virus
circulation is not found. The latter could be achieved by a programme of blood
sampling and virus testing (probang samples). The evidence of limited dispersion
of virus under vaccination comes from the Balkans, and Korea , where testing
after vaccination revealed a few animals which had been both vaccinated and met
live virus in villages and owners around those villages where disease had been
seen, but not more widely in the vaccination zone and not outside. i.e. the sub clinical
continued circulation of virus may have occurred for can be identified by
testing.
Dr Keith J. Sumption
PhD MA VetMB MRCVS
Lecturer in International Animal Health
Centre for Tropical Veterinary Medicine, University of Edinburgh
Distinguishing infected from vaccinated animals.
One of the objections raised to vaccination is the problem of identification of infected animals. Several tests are already commercially available claiming to be able to do this effectively.
We are at present gathering information about the validity of these claims. If you have any information on this subject please email us.
One such manufacturer is United Biomedical, Inc. They have perfected a method to distinguish FMD serum antibodies in a vaccinated animal from thise following natural infection and thus likely to be a carrier. This research outfit also has a synthetic vaccine. More information is available on : http://www.unitedbiomedical.com/faqs.htm (NB you don't need to download the Chinese character set)
The following has been mirrored from their site:
| Are UBI FMDV diagnostic kits commercially available now? | |||||||||||||
| Yes, the UBI diagnostic kits have been vigorously tested and validated, and they are manufactured in a GMP facility with strict quality assurance and quality control. The kits have been commercially available since 1999, and are currently sold worldwide through a USDA export license (except US domestic market).<top> | |||||||||||||
| How many tests can a technician perform in one day? | |||||||||||||
| In a clinical/reference laboratory such as a blood bank's viral antibody screening operation where ELISA plate washer, reader and automated sampling equipment are available, about 2,000 samples (i.e. about 20 plates) can be performed by one technician in a day. This output can be increased by more sophisticated automation. | |||||||||||||
| What approach can a country adopt to scientifically manage and control FMD after the initial outbreak? | |||||||||||||
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| Why don't most of the world's industrialized countries who hold an FMD-free status vaccinate their livestock routinely with the current killed virus vaccine for FMD? | |||||||||||||
The
short answers are that:
Once a nation has eradicated the disease from an area, thus gaining FMD-free status, not vaccinating the animals with the currently available killed virus vaccine allows the country to maintain a sensitive means of detecting whether the virus has crept back in again, as any accidental infection will lead to a full-blown outbreak. However, this strategy of "non-vaccination" is a gamble. It assumes that the nation will be able to contain the outbreak by stamping out the infected herds quickly enough to prevent real damage and thereafter, in the absence of further outbreaks and vaccination, quickly regain its FMD-free status. Furthermore, the non-vaccination policies are based on the calculation that such outbreaks will happen rarely and be detected quickly so that this calculated vulnerability will be cheaper than the method of routine mass vaccination. Another concern for vaccination is that there was one case report which stated that in a few animals vaccinated with the killed virus vaccines, FMDV can still cause subclinical infection. It is not clear whether this can infect other animals, or how long such subclinical infection persists. The theoretical risk that this might have happened means meat from a vaccinated animal might be carrying the virus. Until now, standard tests could not distinguish whether an animal has antibodies to infection or to the vaccine. Other regions trying to remain FMD-free without vaccination will not buy meat from countries that vaccinate because some vaccinated animals may harbor such subclinical virus. For the above reasons, the "FMD-free, non-vaccination" policy has been adopted by most of the industrialized nations in order to maintain their FMD-free status. From the world trade point of view, there is another benefit to the non-vaccination calculation. If a country does not vaccinate for FMD and other livestock diseases, that country can refuse imports from countries that have the disease, or that vaccinate. These vaccinating countries may be poorer countries with lower production costs, who may offer lower prices. Therefore, this "FMD-free, non-vaccination" policy can constitute an import barrier in today's world trade. The official statement from EU regarding why it does not vaccinate is that it must protect its exports to FMD-free countries. However, such exports only amounted to 5 per cent of total EU meat sales in 1999 (which reached $17 billion), prior to some import bans due to BSE. More than 78 per cent of meat sales in 1999 were domestic. If the "FMD-free, non- vaccinating" policy did not constitute an "IMPORT BARRIER", some of the domestic meat sales might have been lost to foreign competitors who vaccinate for FMD. <top> |
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| The old practice in FMD control for the industrialized nations has run into challenges. In light of the current situation, what is the risk-benefit calculation for non-vaccination? | |||||||||||||
In
this era of the global village, the risks of non-vaccination are
escalating as described below:
The current outbreak in Europe suggests not vaccinating may not be cheaper after all. <top>
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| Why is it time for countries to eradicate FMD, then vaccinate and conduct active surveillance for infection? | |||||||||||||
|
This
alternative strategy has only now become available. It is certainly the
wisest policy for any government to adopt. In the short run it would be
more expensive than not vaccinating and simply watching for outbreaks, but
surely cheaper than Europe's current FMD disaster, both in terms of money
and public opinion. In addition, surveillance technology could become
cheaper and more reliable with modest investment. It is time for the
international community to seriously weigh the new technological options
for vaccinating and differential diagnosis versus the increased risks of
not vaccinating.
Despite the EU non-vaccination policy, Belgium and the Netherlands have formally asked other EU countries to consider vaccinating routinely for FMD again. They have not said how this would be coupled with monitoring for subclinical infection. (Such routine prophylaxis must be clearly distinguished from the emergency vaccination permitted and now planned in the EU to contain outbreaks. All such emergency vaccinated animals must be destroyed before the region regains its disease-free status under current rules.) Answers to the last few questions have incorporated the essay written by Debora MacKenzie, a European correspondent for New Scientist magazine. <top> |
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| What would the US do if FMD outbreaks occur there? Culling? No way! | |||||||||||||
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There
are several arguments against mass culling which, when taken together, add
up to making it an impossible task for the USA, a country with a very high
density of livestock in many states.
First, mass culling policy dictates the killing of many adult animals which would otherwise have survived infection, albeit with perhaps a 10 percent loss in productivity. Despite this mass culling policy, a legacy of surviving but infectious animals are left behind, which would perhaps take years to eliminate in order for the USA to return to FMD-free status. Japan and South Korea account for over 60 percent of US beef and pork exports, and will ban imports of both from the US at the first report of a US outbreak. Australia and New Zealand will be happy to fill the gap. If US adopts a ring vaccination and selective culling policy, the outbreak will only result in a 10 percent loss in meat production as a result of an outbreak. This slight decrease in meat production will not affect the capacity to export to markets other than Japan and Korea. Second, the logistics of slaughtering and disposing of possibly millions of head of livestock quickly overwhelms capacity. During the FMD outbreak in Taiwan in 1997, 4 million pigs were culled at a peak rate of 130,000 per day after the army got up to speed. Assuming the US Army could do the same on the larger US feed lots, it will still take 30 days to kill and depose of 4 million animals; 30 days in which the disease is still spreading. It is questionable, however, whether the public would stand for the spectacle of the US Army or National Guard shooting livestock. Third, the risk of delays due to litigation are great. Farmers and national associations of breeders would seek injunctions. Animal rights and environmental groups would do the same. Slaughtering could be held up for weeks. Also the substantial financial resources necessary for a big emergency would exceed existing provisions and require additional legislation, preceded by much debate, before being approved. Even emergency funds from the White House would take time to arrive. The general experience with public health authorities has been that they will always hope that the outbreak will be contained with minimum expenditure, inevitably leading to too little funding too late. There is no reason to believe animal health authorities will react differently. Models exist that purport to show US conditions under the most optimistic circumstances. If culling of 95 percent of latently infected plus infectious herds can be achieved beginning in the second week after recognition of the first outbreak, "only" 20 percent of an affected region's herds need be destroyed to achieve control. But if culling to that level is delayed by just one week, the eventual destruction will have to be more than 90 percent. It is unlikely that any state agency will be able to move that fast, or be able to cull to that level. Finally, a recent analysis of the potential cost of FMD in California projects direct costs, plus production losses, plus trade losses would sum to around US$8000 per head culled, or US$ 8 billion per million head -- and this may be a conservative estimate. Twenty percent of the cattle in Texas alone is 1.6 million head. References: Ekboir J 1999. Potential impact of Foot and Mouth Disease in California. Agricultural Issues Center, Div Ag & Nat Res, Univ. Cal. Davis. Yang PC, Chu RM, Chung WB, Sung HT 1999. Epidemiological characteristics and financial costs of the 1997 Foot and Mouth Disease epidemic in Taiwan. Vet Rec 145 (25): 731?734. The above discussion on "why mass culling?as opposed to selective culling- although being used in the UK for the current outbreak is out of the question for the USA" is adapted from the contribution by Dr. Jack Woodall woodall@bioqmed.ufrj.br, originally appeared in www.promedmail.org on April 4, 2001. Dr. Jack Woodall's official affiliation is at: Dept. of Medical Biochemistry, Institute of Biomedical Sciences, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil.<top>
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