1.   

Identification of the agent
 

a)   

Virus isolation
 

The epithelium sample should be taken from the PBS/glycerol, blotted dry on absorbent paper to reduce the glycerol content, which is toxic for cell cultures, and weighed. A suspension should be prepared by grinding the sample in sterile sand in a sterile pestle and mortar with a small volume of tissue culture media and antibiotics. Further media should be added until a final volume of ten times that of the epithelial sample has been added, giving a 10% suspension. This is clarified on a bench centrifuge at 2000 g for 10 minutes. Such suspensions of field samples suspected to contain FMD virus once clarified are inoculated into cell cultures or unweaned mice. Sensitive cell culture systems include primary bovine thyroid cells and primary pig, calf or lamb kidney cells. Established cell lines, such as baby hamster kidney BHK-21 and IB-RS-2 cells, may be used but are less sensitive than primary cells for detecting low amounts of infectivity (9). The cell cultures should be examined for cytopathic effect (CPE) for 48 hours. If no CPE is detected, the cells should be frozen and thawed, used to inoculate fresh cultures and examined for CPE for another 48 hours. Unweaned mice are an alternative to cell cultures and should be 2-7 days of age and of selected inbred strains. Some field viruses may require several passages before they become adapted to mice (33).
 

b)   

Immunological methods
 

•   

Enzyme-linked immunosorbent assay
 

At the OIE/FAO WRL for FMD, the preferred procedure for the detection of FMD viral antigen and identification of viral serotype is the ELISA (20, 31). This is an indirect sandwich test in which different rows in multiwell plates are coated with rabbit antisera to each of the seven serotypes of FMD virus. These are the ‘capture’ sera. Test sample suspensions are added to each of the rows, and appropriate controls are also included. Guinea-pig antisera to each of the serotypes of FMD virus are added next, followed by rabbit anti-guinea-pig serum conjugated to an enzyme. Extensive washing is carried out between each stage to remove unbound reagents. A colour reaction on the addition of enzyme substrate, indicates a positive reaction. With strong positive reactions this will be evident to the naked eye, but results can also be read spectrophotometrically at an appropriate wavelength. In this case, an absorbance reading greater than 0.1 above background indicates a positive reaction; the serotype of FMD virus can also be identified. Values close to 0.1 should be confirmed by retesting or by amplification of the antigen by tissue culture passage and testing the supernatant once a CPE has developed. A suitable protocol is given below.
 

Depending on the species affected and the geographical origin of samples, it may be appropriate to simultaneously test for swine vesicular disease (SVD) virus or vesicular stomatitis (VS) virus. Ideally a complete differential diagnosis should be undertaken in all vesicular conditions.
 

Rabbit antiserum to the 146S antigen of each of the seven serotypes of FMD virus (plus SVD virus if required) is used as a trapping antibody at a predetermined optimal concentration in carbonate/bicarbonate buffer, pH 9.6.
 

Control antigens are prepared from selected strains of each of the seven types of FMD virus (plus SVD virus if appropriate) grown on monolayer cultures of BHK-21 cells (IB-RS-2 cells for SVD virus). The unpurified supernatants are used and pretitrated on ELISA plates. The final dilution chosen is that which gives an absorbance at the top of the linear region of the titration curve (optimal density approximately 2.0), so that the five-fold dilutions of the control antigens used in the test give two additional lower optimal density readings from which the titration curve can be derived. PBS containing 0.05% Tween 20 and phenol red indicator is used as a diluent (PBST).
 

Guinea-pig antisera prepared by inoculating guinea-pigs with 146S antigen of one of the seven serotypes of FMD virus (plus SVD virus if required) and preblocked with normal bovine serum (NBS) is used as the detecting antibody. Predetermined optimal concentrations are prepared in PBS containing 0.05% Tween 20, and 5% dried, nonfat skimmed milk (PBITM).
 

Rabbit (or sheep) anti-guinea-pig immunoglobulin conjugated to horseradish peroxidase and preblocked with NBS is used at a predetermined optimum concentration in PBITM.
 

•   

Test procedure
 

i)   

ELISA plates are coated with 50 µl/well rabbit antiviral sera in carbonate/bicarbonate buffer, pH 9.6. Rows A to H receive, respectively, antisera to serotypes O, A, C, SAT 1, SAT 2, SAT 3, Asia 1 and SVD virus (optional).
 

ii)   

Leave overnight at 4°C in a stationary position or place on an orbital shaker set at 100-120 revolutions per minute in a 37°C incubator for 1 hour.
 

iii)   

Prepare test sample suspension (with 10% original sample suspension or undiluted clarified cell culture supernatant fluid).
 

iv)   

The ELISA plates are washed five times in PBS.
 

v)   

On each plate, load wells of columns 4, 8 and 12 with 50 µl PBST. Additionally, add 50 µl of PBST to wells 2 and 3 of rows A to H on plate 1. To well 1 of row A of plate 1 add 50 µl of control antigen type O, and to well 2 of row A add 12.5 µl of control antigen type O. Mix antigen and diluent in well 2 and transfer 12.5 µl from well 2 to well 3 of row A. Mix and discard 12.5 µl from well 3 (this gives a five-fold dilution series of antigen O). Similarly repeat with antigen A, adding 50 µl of antigen type A to well 1 of row B, and 12.5 µl of antigen type A to well 2, and then mix and transfer 12.5 µl to well 3 (as done before with antigen type O), and continue for types C, SAT 1, SAT 2, SAT 3, Asia 1 and SVD (if appropriate). It is only necessary to change pipette tips on the micropipette between antigens. The remainder of the plate can be loaded with the test sample(s). Add 50 µl of sample one to wells 5, 6 and 7 of rows A to H, the second sample is placed similarly in columns 9, 10 and 11, rows A to H.
 

If more than two samples are to be tested at the same time, the other ELISA plates should be used as follows:
 

Dispense 50 µl of the PBST to the wells (rows A to H) of columns 4, 8 and 12 (buffer control columns). Note that the control antigens are not required on these plates. These test samples may be added in 50 µl volumes in rows A to H to columns 1, 2, 3; 5, 6, 7; 9, 10, 11, respectively.
 

vi)   

Cover with lids and place on an orbital shaker at 37°C for 1 hour.
 

vii)   

Wash the plates by flooding with PBS - wash three times as before and empty residual wash fluid. Blot the plates dry.
 

viii)   

Transfer 50 µl volumes of each guinea-pig serum dilution to each plate well in the appropriate order, e.g. rows A to H receive, respectively, antisera to serotypes O, A, C, SAT 1, SAT 2, SAT 3, Asia 1 and SVD virus (optional).
 

ix)   

Cover plates with lids and replace on the orbital shaker. Incubate at 37°C for 1 hour.
 

x)   

The plates are washed again three times and 50 µl of rabbit anti-guinea-pig immunoglobulin conjugated to horseradish peroxidase is added to each well. The plates are incubated at 37°C for 1 hour on a rotary shaker.
 

xi)   

The plates are washed again three times and 50 µl of orthophenylene diamine containing 0.05% H₂O₂ (30% w/v) is added to each well.
 

xii)   

The reaction is stopped after 15 minutes by the addition of 50 µl of 1.25 M sulphuric acid. The plates are read at 492 nm on a spectrophotometer linked to a computer.
 

•   

Complement fixation test
 

The ELISA is preferable to the complement fixation (CF) test because it is more sensitive and specific, and it is not affected by pro- or anti-complementary factors. If ELISA reagents are not available, however, the CF test may be performed as follows:
 

Antisera to each of the seven types of FMD virus are diluted in veronal buffer diluent (VBD) in 1.5-fold dilution steps from an initial 1/16 dilution to leave 25 µl of successive antiserum dilutions in U-shaped wells across a microtitre plate or appropriate volumes in test tubes. To these are added 50 µl of 3 units of complement, followed by 25 µl of test sample suspension(s). The test system is incubated at 37°C for 1 hour prior to the addition of 25 µl of 1.4% standardised sheep red blood cells (SRBC) in VBD sensitised with 5 units of rabbit anti-SRBC. The reagents are incubated at 37°C for a further 30 minutes and the plates are subsequently centrifuged and read. Appropriate controls for the test suspension(s), antisera, cells and complement are included. CF titres are expressed as the reciprocal of the serum dilution producing 50% haemolysis. A CF titre ³36 is considered to be a positive reaction. Titre values of 24 should be confirmed by retesting an antigen that has been amplified through tissue culture passage.
 

c)   

Nucleic acid recognition methods
 

The polymerase chain reaction (PCR) can be used to amplify the genome fragments of FMD virus in diagnostic material (2, 7). Specific primers have been designed to distinguish between each of the seven serotypes. In situ hybridisation techniques have been developed for investigating the presence of FMD virus RNA in tissue samples (39). These techniques are only in use in specialised laboratories.
 

The molecular epidemiology of FMD is based on the comparison of genetic differences between virus isolates. Dendrograms showing the genomic relationship between vaccine and field strains for all seven serotypes have been published based on sequences derived from the 1D gene. Reverse-transcription polymerase chain reaction (RT-PCR) amplification of FMD virus RNA, followed by nucleotide sequencing, is the current preferred option for generating the sequence data to perform these comparisons. The WRL and other laboratories have developed techniques for performing these studies, and a database of over 2000 sequences is currently held.
 

The recommended method is to:
 

i)   

Extract FMD virus RNA directly from epithelial suspensions, or from a low cell culture passage.
 

ii)   

Perform an RT-PCR of the complete VP1 gene (or if only part of the VP1 gene, then the 3’ end of the gene is more useful).
 

iii)   

Determine the nucleotide sequence of the PCR product (or at least 170 nucleotides [preferably 420 for the SAT types] at the 3’ end of the gene).
 

A protocol, complete with primer sequences is available from the WRL on request or can be downloaded from the following World Wide Web URL:
 

Research on monoclonal antibodies (MAbs) that identify individual antigenic sites on the surface of the FMD virus may have future potential. Panels of neutralising antibodies are being developed for each serotype and characterised by nucleotide sequencing of escape mutant virus. The neutralising site against which each MAb acts can be deduced by identifying the sequence change in the mutant virus that allowed it to escape neutralisation.
 

2.   

Serological tests
 

FMD virus infection can be diagnosed by the detection of a specific antibody response. The tests generally used are virus neutralisation (VN) and ELISA (23, 24, 36). These are also the prescribed tests for trade. The VN test is serotype specific, requires cell culture facilities and takes 2-3 days to provide results. The ELISA is also serotype specific, sensitive and quantitative, and has the advantage that it is quicker to perform, is less variable, and is not dependent on tissue culture systems. Low titre false-positive reactions can be expected in a small proportion of the sera in either test. An approach combining screening by ELISA and confirming the positives by the VN test minimises the occurrence of false-positive results.
 

The detection of antibody to the nonstructural (NS) proteins of FMD virus has been used to identify past or present infection with any of the seven serotypes of the virus, whether or not the animal has also been vaccinated. Conventionally this has been carried out by measuring antibody to the virus infection-associated antigen (VIAA; the viral RNA polymerase protein 3D) using agar gel immunodiffusion (AGID) (28). Although relatively insensitive, the test is inexpensive, easy to perform and has been used extensively in South America to detect viral activity on a population basis during FMD eradication campaigns. The VIAA test has now largely been superseded by assays that measure antibody to FMD virus NS proteins produced by recombinant techniques in a variety of in-vitro expression systems. Antibody to the polyproteins 3AB or 3ABC are the single most reliable indicator of infection (10, 27, 34). In animals seropositive for antibody to 3AB or 3ABC, antibody to one or more of the other NS proteins including the L, 2C, 3A or 3D protein is further confirmation of infection (8, 27, 34). The test can be used on a herd basis to detect FMD virus infection in vaccinated and unvaccinated populations. Care must be taken when interpretating results from single animals as some repeatedly vaccinated animals produce antibody to 3ABC. It has not yet been established whether or not all animals that have been both vaccinated and infected seroconvert to this protein. A negative result for antibody to NS proteins cannot therefore be taken as definitive proof that an individual animal has not been exposed to FMD virus. This must be taken into account if NS protein antibody tests are used for assessment of risks for animals involved in international trade.
 

a)   

Virus neutralisation (a prescribed test for international trade)
 

The quantitative VN microtest for FMD antibody is performed with IB-RS-2, BHK-21, lamb or pig kidney cells in flat-bottomed tissue-culture grade microtitre plates.
 

Stock virus is grown in cell monolayers and stored at -20°C after the addition of 50% glycerol. (Virus has been found to be stable under these conditions for at least 1 year.) The sera are inactivated at 56°C for 30 minutes before testing. The control standard serum is 21-day convalescent serum (usually pig). A suitable medium is Eagle’s complete medium/LYH (Hank’s balanced salt solution with yeast lactalbumin hydrolysate) with antibiotics.
 

The test is an equal volume test in 50 µl amounts.
 

•   

Test procedure
 

i)   

Starting from a 1/4 dilution, sera are diluted in a twofold dilution series across the plate, using at least two rows of wells per serum, preferably four rows, and a volume of 50 µl.
 

ii)   

Previously titrated virus is added; each 50 µl unit volume of virus suspension should contain about 100 TCID₅₀ (50% tissue culture infective dose) within an accepted range (e.g. 35-350 TCID₅₀).
 

iii)   

Controls include a standard antiserum of known titre, a negative serum, a cell control, a medium control, and a virus titration used to calculate the actual virus titre used in the test.
 

iv)   

Incubate at 37°C for 1 hour with the plates covered.
 

v)   

A cell suspension at 10⁶ cells/ml is made up in medium containing 10% bovine serum (specific antibody negative) for cell growth. A volume of 50 µl of cell suspension is added to each well.
 

vi)   

Plates are sealed with pressure-sensitive tape and incubated at 37°C for 2-3 days. Alternatively, the plates may be covered with loosely fitting lids and incubated in an atmosphere of 3-5% carbon dioxide at 37°C for 2-3 days.
 

vii)   

Microscope readings may be feasible after 48 hours, the plates are finally fixed and stained routinely on the third day. Fixation is effected with 10% formol/saline for 30 minutes. For staining, the plates are immersed in 0.05% methylene blue in 10% formalin for 30 minutes. An alternative fixative/stain solution is naphthalene blue black solution (0.4% [w/v] naphthalene blue black, 8% [w/v] citric acid in saline) (22). The plates are rinsed in tap water.
 

viii)   

Positive wells (where the virus has been neutralised and the cells remain intact) are seen to contain blue-stained cells sheets; the negative wells (where virus has not been neutralised) are empty. Titres are expressed as the final dilution of serum present in the serum/virus mixture at the 50% end-point, i.e. a well where there is an incomplete cell sheet (Kärber). The test is considered to be valid when the amount of virus used per well is in the range log₁₀ 1.5-2.5 TCID₅₀, and the positive standard serum is within twofold of its expected titre.
 

ix)   

Interpretation of tests can vary between laboratories in regard to end-points taken. Laboratories should establish their own criteria by reference to standard reagents that can be obtained from the OIE/FAO WRL for FMD. At the WRL, a titre of 1/45 or more of the final serum dilution in the serum/virus mixture is regarded as positive. Titres of 1/16 to 1/32 are considered to be doubtful, and further serum samples are requested for testing. Animals are considered to be positive if the second sample has a titre of 1/16 or greater. A titre of 1/8 or less is considered to be negative.
 

b)   

Liquid-phase blocking enzyme-linked immunosorbent assay (a prescribed test for international trade)
 

Rabbit antiserum to the 146S antigen of one of the seven types of FMD virus is used as the trapping antibody at a predetermined optimal concentration in carbonate/bicarbonate buffer, pH 9.6. (A chequerboard titration of the rabbit-trapping antiserum, the guinea-pig antiserum and the anti-guinea-pig antiserum is performed. Before using the antigen-trapping ELISA or the liquid-phase blocking ELISA, each of these reagents is titrated one against another, keeping the third reagent at a fixed concentration. In this way the optimal dilutions [for positive colour and low background colour] can be determined. These ‘predetermined’ dilutions are then used for all future tests using these particular batches of reagents.)
 

Antigens are prepared from selected strains of FMD virus grown on monolayers of BHK-21 cells. The unpurified supernatants are used and pretitrated according to the VN protocol but without serum. The final dilution chosen is that which, after addition of an equal volume of diluent (see below), gives an absorbance on the upper part of the linear region of the titration curve (optical density approximately 1.5). PBS containing 0.05% Tween 20 and phenol red indicator is used as a diluent (PBST).
 

Guinea-pig antisera prepared by inoculating guinea-pigs with 146S antigen of one of the seven serotypes and preblocked with NBS is used as the detecting antibody. Predetermined optimal concentrations are prepared in PBS containing 0.05% Tween 20, and 5% dried, nonfat skimmed milk (PBSTM).
 

Rabbit (or sheep) anti-guinea-pig immunoglobulin conjugated to horseradish peroxidase and preblocked with NBS is used at a predetermined optimum concentration in PBSTM.
 

Test sera are diluted in PBST.
 

•   

Test procedure
 

i)   

ELISA plates are coated with 50 µl/well rabbit antiviral sera and left overnight in a humid chamber at room temperature.
 

ii)   

The ELISA plates are washed five times with PBS.
 

iii)   

In U-bottomed multiwell plates (carrier plates) 50 µl of a duplicate, twofold series of each test serum are prepared, starting at 1/4. To each well, 50 µl of a constant dose of homologous viral antigen is added and the mixtures are left overnight at 4°C, or incubated at 37°C for 1 hour. The addition of the antigen increases the starting serum dilution to 1/8.
 

iv)   

Then 50 µl of serum/antigen mixtures are transferred from the carrier plates to the rabbit-serum coated ELISA plates and incubated at 37°C for 1 hour on a rotary shaker.
 

v)   

After washing, 50 µl of guinea-pig antiserum homologous to the viral antigen used in the previous step (iv) is added to each well. The plates are then incubated at 37°C for 1 hour on a rotary shaker.
 

vi)   

The plates are washed and 50 µl of rabbit anti-guinea-pig immunoglobulin conjugated to horseradish peroxidase is added to each well. The plates are incubated at 37°C for 1 hour on a rotary shaker.
 

vii)   

The plates are washed again and 50 µl of orthophenylene diamine containing 0.05% H₂0₂ (30%, w/v) is added to each well.
 

viii)   

The reaction is stopped after 15 minutes by the addition of 50 µl of 1.25 M sulphuric acid. The plates are read at 492 nm on a spectrophotometer linked to a microcomputer.
 

ix)   

Controls: A minimum of four wells each of strong positive, weak positive and negative bovine reference sera at a final dilution of 1/32 should be included on each plate together with an equivalent number of reaction (antigen) control wells containing antigen in diluent alone without serum. For end-point titration tests, duplicate twofold dilution series of positive and negative homologous bovine reference sera should be included on at least one plate of every run.
 

x)   

Interpretation of the results: Antibody titres are expressed as the 50% end-point titre, i.e. the dilution at which 50% of the wells show greater than 50% inhibition of the median OD of the reaction (antigen) control wells (Kärber). Titres greater than 1/40 are considered to be positive. Titres close to 1/40 should be retested using the VN test.
 

c)   

Nonstructural protein antibody tests
 

Antibody to VIAA is conventionally detected by AGID. The test is based on immunoprecipitation lines formed in the agar between the FMD antigen (concentrated cell-culture fluids rich in FMD virus RNA-dependent RNA polymerase or 3D) located in a centre well, and hexagonally arranged adjacent wells containing standard positive sera or unknown test sera. Precipitation lines forming between the test sera and the control antigen well that show identity with the lines of precipitation formed by the reference sera, confirm the specificity of the reactions (28).
 

Antibody to expressed, recombinant FMD virus NS proteins can be measured by ELISA or immunoblotting. No single test format has yet been conclusively demonstrated to be optimal. An MAb trapping (MAT) ELISA for detecting antibody to 3ABC (10) and blocking ELISAs for detecting antibody to 3AB or 3ABC (34) have been shown to be sensitive, specific and reliable in a number of laboratories. The simultaneous detection of antibody to several NS proteins in a single test by ELISA (27, 34) or by enzyme-linked immuno-electrotransfer blot (EITB), a type of Western blot, (8) is useful for confirmation of animals positive for antibody to 3AB or 3ABC. There are currently no internationally recognised standards for antibody to FMD virus NS proteins, but an application for these tests is described in detail below.
 

-   

Indirect enzyme-linked immunosorbent assay
 

-   

Test procedure
 

i)   

Microplates are coated overnight at 4°C with 1 µg/ml of the fusion antigen 3ABC in carbonate/ bicarbonate buffer, pH 9.6 (100 µl per well). Antigen 3ABC was expressed and purified as indicated for the EITB tests (30).
 

ii)   

The plates are washed six times with PBS, pH 7.2, supplemented with 0.05% Tween 20.
 

iii)   

Test sera (100 µl per well) are added in a 1/20 dilution in blocking buffer consisting of PBS, 0.05% Tween 20, 5% nonfat dry milk, 10% equine sera and 0.1% Escherichia coli lysate. Each plate includes a set of reference standards as defined for the EITB assay.
 

iv)   

The plates are incubated for 30 minutes at 37°C and washed six times in PBS/Tween.
 

v)   

Horseradish-peroxidase-conjugated rabbit anti-species IgG is diluted optimally in the blocking buffer, added at 100 µl per well and the plate incubated for 30 minutes at 37°C.
 

vi)   

After six washings, each well is filled with 100 µl of 3.3’, 5.5’-tetramethylbenzidine plus 0.004% (w/v) H₂O₂ in phosphate/citrate buffer, pH 5.5.
 

vii)   

The reaction is stopped after 15 minutes of incubation at room temperature by adding 100 µl of 0.5 M H₂SO₄. Absorbance is read at 450 nm and at 620 nm for background correction.
 

-   

Interpreting the results
 

For the test system to be valid the following performance criteria are applied: the absorbance of negative controls should be <0.10 after correction for absorbance of blank wells. The cut-off serum, obtained as described in the EITB test, should give absorbance values of 0.15-0.40. Results are expressed as an index derived by dividing the absorbance value of the serum tested by that of the cut-off control. The ratio of the weak positive/cut-off controls should be 2.5 with a coefficient of variation <20%. Test sera with ratios >0.8 are considered to be suspect or positive and are retested by EITB. Coated plates and secondary standards are available from the PANAFTOSA on request (Centre Panamericano de Fiebre Aftosa, Caixa Postal 589, 20001-970 Rio de Janeiro, Brazil. [dir@aftosa.ops-oms.org]).
 

-   

Enzyme-linked immunoelectrotransfer blot assay
 

The FMD situation in South America is complex and includes a variety of ecosystems and animal populations of varying immune status immunised with vaccines of different formulation. Tests for serological diagnosis therefore must accommodate many variables. To date, the EITB assay has been widely applied in South America for serosurveillance and risk assessment associated with animal movement. Currently, the procedure is to perform an initial screening test using an indirect ELISA for antibody to 3ABC, and to follow that by a confirmatory EITB assay if samples give positive or suspect results. This combination of tests is particularly recommended when serosurveillance involves a large number of samples. Further information is available from the OIE Reference Laboratory in Brazil (see Table given in Part 4 of this Manual).
 

-   

Preparation of test strips containing the recombinant antigens
 

The five bioengineered FMD virus NS proteins 3A, 3B, 2C, 3D and 3ABC are expressed in E. coli C600 by thermo-induction. The 3D polypeptide is expressed in its complete form (30), whereas the rest of the proteins are obtained as fusions to the N-terminal part of the MS-2 polymerase gene (35).
 

The expressed polymerase is purified over phosphocellulose, followed by poly(U) Sepharose columns. The fused proteins 3A, 3B, 2C and 3ABC are purified by sequential extraction of the bacterial extracts with increasing concentrations of urea. The 7M fraction containing the fusion proteins is further purified on a preparative 10% SDS/PAGE (sodium dodecyl sulphate/polyacrylamide gel electrophoresis). The fusion protein band is excised from the gel and electroeluted (30).
 

A mixture containing 20 ng/ml of each one of the purified recombinant polypeptides is separated on 12.5% SDS/PAGE and electrophoretically transferred to nitrocellulose (30).
 

-   

Test procedure
 

i)   

The required amount of test strips should be assessed, taking into account that for each nitrocellulose sheet, which defines one transferred gel, a positive, a weakly positive, a cut-off and a negative control serum should be assayed. In general, 24 nitrocellulose strips, each 3 mm wide, should result from a gel.
 

ii)   

A volume of 0.8 ml of saturation buffer (50 mM Tris-HCl, pH 7.5; 150 mM NaCl; 0.2% Tween 20; 5% nonfat dry milk; and 0.05% bacterial E. coli lysate) is added to each well. The antigen-coated strips are blocked by placing the trays on a rocker and agitating for 30 minutes at room temperature (20-22°C).
 

iii)   

A dilution of 1/200 of test sera and of each of the controls is added to the appropriate trough. The strips must be completely submerged and facing upwards, and maintained in that position during the whole process.
 

iv)   

Strips are incubated for 60 minutes on a rocker at room temperature.
 

v)   

Liquid is removed from the trays, and each test strip is washed three times with washing solution (50 mM Tris-HCl, pH 7.5; 150 mM NaCl; and 0.2% Tween 20) by agitation for 5 minutes.
 

vi)   

The alkaline-phosphatase-conjugated rabbit anti-bovine solution is added to each test well, and the strips are incubated with shaking for 60 minutes at room temperature.
 

vii)   

The liquid is removed from the trays and each test strip is washed three times with washing solution as above.
 

viii)   

Substrate solution (0.015% bromochloroindolylphosphate/0.03% nitroblue tetrazolium) is prepared in substrate buffer (100 mM NaCl; 5 mM MgCl₂; and 100 mM Tris-HCl, pH 9.3), and is added to each test well.
 

ix)   

Strips are incubated by placing the test tray on the orbital mixer and agitating until the cut-off control shows five distinct, discernible bands. Strips are washed with running deionised water and air-dried.
 

-   

Reading the results
 

A sample is nonreactive if all the bands are below the reactivity of the cut-off control, or a maximum of two bands are above the reactivity of the cut-off control. A sample is reactive if all four antigens (3ABC, 3A, 3B and 3D) +/- 2C have a reactivity equal to or higher than the cut-off control. A sample has indeterminate reactivity if the above-mentioned criteria for reactive or nonreactive samples are not met. Densitometric reading is possible, but visual reading is recommended not only because it is less expensive, but also because borderline reactions rarely occur. The cut-off control serum is derived from a pool of sera and/or dilution of positive sera, representing background EITB reactivities observed individually for the different antigens in nonvaccinated animals from FMD-free regions. These controls are secondary standards, validated against a primary standard corresponding to a serum from an animal at 714 days after experimental infection from which no virus had been recovered from OP fluid for the past 364 days (8) This primary standard is diluted 1/2 when the test is used as an input for evaluation of risk analysis during selection of animals for import/export testing.
 

Sensitised strips and secondary standards are available on request from the PANAFTOSA.