The use of a 3ABC marker-test for FMD

 

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:

7         Test is based on a validated and established system.

7         The test is ready for use.

7         Monophasic assay.

7         Established large scale production.

7         Expertise from IAH & IZP for the 3ABC-ELISA system.

 


 

 

 


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

 

 

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

 

a)       Cattle (n=2), were immunized with O1 Manisa (Group 1) or served as control (Group 2) Both Groups were challenged with the homologous strain.

b)       CHEKIT-FMD-3ABC

c)       Strain: O1 Manisa

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

 

a)       Sheep (n=5), were immunized with a vaccine comprising strains A22 Iraq, O1 Manisa, O1 Marocco, and Asia1 Shamir

b)       CHEKIT-FMD-3ABC

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

 

a)       Pigs (n=5), were immunized with a vaccine comprising strains A24 Cruzeiro, O1 Manisa, and C1 Detmold

b)       CHEKIT-FMD-3ABC

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:

'         The test is serotype aspecific.

'         Antibodies against 3ABC will be demonstrated as from 10-14 days after infection.

'         The test does not contain any infectious material and can be run in every laboratory, which is equipped for ELISA.

'         The test provides results within hours.

1.      General screening

 

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.

 

1.1.   Screening border control

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.

 

1.2.   Screening suspected infections

 

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.

 

2.      Marker-system: Import / border control

 

2.1.   Border control

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.

 

2.2.   Export

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.

 

3.      Marker-system: Endemic situations:

 

3.1.   Farm-use:

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.