Rapid coagglutination test for the detection and typing of foot and mouth disease virus

Journal of Virological Methods ELSEVIER

Journal of Virological

Methods 50 (1994) 29-42

Rapid coagglutination test for the detection and typing of foot and mouth disease virus H.J.

Montassier, J.P. Arafijo Jr., A.A. Pinto *

Faculdade de CBncias Agrcirias e Veterincirias da Universidade Estadual Paulista, 14870-000, Jaboticabal-SP, Brazil Accepted

13 April 1994

Abstract Protein A containing Staphylococcus aureus was used to develop a coagglutination (COA) test for the detection and typing of foot and mouth disease virus (FMDV) 0, A and C serotypes in infected cells and tissues. Different batches and amounts of guinea pig anti-FMDV sera were assessed to optimize the preparation of COA conjugates. The sensitivity and specificity of the COA Test for the detection of FMDV 0, A and C serotypes and heterologous viruses was also characterized. Comparison between the COA Test and complement fixation test for the detection and typing of FMDV obtained from extracts of tongue epithelial tissues from infected cattle revealed high agreement in the results and indicated a potential application of the COA Test for the direct diagnosis of viruses. Keywords: Staphylococcal

coagglutination;

Foot-and-mouth

disease virus; Rapid diagnosis

1. Introduction Rapid diagnosis of foot and mouth disease (FMD) is of great importance in the prevention and control of FMD (Pereira, 1977; Crowther, 1977). Traditionally, the

complement fixation test (CFT) has been used for diagnosis of FMD in vesicular epithelial samples from suspected animals. More recently, ELISA procedures have been developed and applied successfully for this purpose (Hamblin et al., 1984; Roeder and Le Blanc Smith, 1987; Ferris and Dawson, 1988).

* Corresponding

author.

0166-0934/94/$07.00 0 1994 Elsevier Science B.V. All rights reserved .SsDI 0166-0934(94)00065-O

30

H.J. Montassier et al./Journal

of Virological Methods 50 (1994) 29-42

Other serological tests for use in primary FMD diagnosis, especially those based on passive agglutination, although approved for their efficiency in the detection of viruses, are not considered to be useful enough to replace the CFT. A possible difficulty is the coating of sheep erythrocytes which requires the purification of gamma globulin or IgG fractions from anti-FMDV hyperimmune serum. This procedure is as complex and arduous as the preparation of conjugates used in ELISA. Furthermore, the ELISA has some unquestionable advantages over passive hemagglutination and is superior to the agglutination test when used for FMDV detection and typing (Ouldridge et al., 1982). However, despite their efficiency and usefulness, these two serological techniques have some drawbacks such as requirements for relatively sophisticated instrumentation and technical training that are not always available (Voller and Bidwell, 1986; Hamblin et al., 1984; Roeder and Le Blanc Smith, 1987). Thus, alternatives can be considered to improve the diagnosis of FMD. The cell wall of Stu@zyZococc~s aureus containing protein A binds the Fc fragment of IgG from most mammalian species. This bacterium has been used as solid phase immunosorbent for the detection of bacterial and viral antigens in a form of agglutination assay called coagglutination test (COA Test) (Kronvall, 1973; Edwards and Larsen, 1974; Edwards and Hilderbrand, 1976; Suksanong and Dajani, 1977; Hebert et al., 1981; Pandya et al., 1981; Mogensen and Dishon, 1981; Skaug et al., 1983; Kang et al., 1984; Durigon et al., 1991). The coagglutination test has proved to be an efficient serological tool for the detection of rotaviruses in stool offering a fast, simple and inexpensive alternative method for the direct diagnosis of these viruses (Hebert et al., 1981; Skaug et al., 1983; Kang et al., 1984; Durigon et al., 1991). We describe the results of experiments to develop and standardize reagents, and characterize the sensitivity, specificity and potential application of the COA Test for the direct diagnosis of FMDV from infected cells and tissues.

2. Material

and methods

2.1. Viruses and uirion subunits The FMDV strains 0, Campos, A,, Cruzeiro and C, lndaial and vesicular stomatitis virus (VSV) strain Indiana II were grown in monolayer cultures of BHK-21 cells. After BE1 inactivation (Bahnemann, 1975), the FMDV strains were purified by sucrose density gradient centrifugation to obtain whole virus (146s) (Ferris et al., 1984). They were quantified by absorbance at 259 nm (Bachrach et al., 1964) and stored at -70°C. FMDV subunits (12s) were prepared from purified FMDV strains by heating the 146s purified viral particles at 56°C for 2.5 h and stored at - 70°C (Have et al., 1984). The concentration of 12s antigen was extrapolated from the starting concentration of 146s antigen preparation (Smitsaart et al., 1990). The VSV and the non-infected BHK-cell cultures were used to determine the specificity of the COA Test. 2.2. Antisera Antisera against FMDV types O,, A,, both an infected BHK-cell suspensions

and C, were developed in guinea pigs using and purified (146s) suspension plus 12s

H.J. Montassier et al. /Journal

of Virological Methods 50 (1994) 29-42

31

subunits. Antigen preparations were emulsified with Freund’s incomplete adjuvant (v/v) and 1 ml of the preparation, containing approximately 1O7.3 TCID,, of crude viral BHK-cell suspension or 20 pg of 146s plus 20 pg of 12s antigens, was injected weekly for 5 weeks into 8 guinea pigs with each FMDV serotype. Animals were exsanguinated 45 days after the first immunization and the sera were pooled, inactivated at 56°C for 30 min and stored at -20°C. These two batches of antisera had a mean complement fixation titer of 2560. In addition, two other batches of anti-FMDV sera were prepared in guinea pigs using either the protocol of infection followed by hyperimmunization (Rivenson, 1956), which had a mean complement fixation titer of 640, or immunization with a single 10 pg dose of 146s FMD virus antigen emulsified with Freund’s complete adjuvant (Ferris et al., 19841, that had a mean complement fixation titer of 120. 2.3. Staphylococcus aureus suspension Staphylococcus aureus strain Cowan I was cultivated in Mi.iller-Hinton broth containing 0.5% yeast extract with agitation for 18 h at 37°C and the bacteria were then washed three times in PBS (0.14 M NaCl, 0.01 M PO,, pH 7.4) and inactivated with 0.5% formaldehyde in PBS at 4°C for 18 h. The bacteria were washed three times with PBS, heated at 80°C for 20 min, washed again in the same manner and resuspended in PBS with 0.5% Tween-20. The concentration of the bacterial suspension diluted 20 times was monitored by spectrophotometry at 525 nm, with absorbance of 0.69 (Goding, 1978). These bacteria corresponding to approximately 2.3 X lOlo cells/ml were stored subsequently at 4°C for a maximum of 3 months. 2.4. Antibody coating of staphylococci The COA-anti-FMDV conjugates were prepared by mixing 2 ml of the standardized S. aureus suspension, which was previously washed in PBS, with different volumes of each anti-FMDV antiserum. After 30 min at room temperature the mixture was centrifuged at 4000 X g for 10 min and washed three times with PBS. The last pellet of antiserum-coated bacteria was resuspended in 2 ml of PBS with 0.5% Tween-20 and 0.02% of sodium azide and kept at 4°C for a maximum of 4 weeks. A control COA conjugate was also prepared with normal inactivated guinea pig serum. 2.5. Coagglutinatination assay Twenty-five ~1 of the viral antigen suspension were mixed with an equal amount of COA anti-FMDV conjugates on a glass surface and the mixture was gently rocked for up to lo-15 min at room temperature. Each sample was tested in duplicate with the control and each of the anti-FMDV O,, anti-A,, or anti-C, COA conjugates. The agglutination pattern was read macroscopically, scoring the intensity and the time of the start of the reaction against a dark background with indirect light. In order to type the FMDV in epithelium samples by COA Test, the strongest and earliest reaction with one of the anti-FMDV COA conjugate was noted.

32

H_J. Montassier

et al. /Journal

of Yirological Methods 50 (1994) 29-42

2.6. FMDV coagg~~t~~ation ~iurn~~ng ~ltrastructural

analyst

A drop obtained 5 min after mixing 20 ~1 of the 1: 10 dilution of anti-146s plus 12s FMDV 0, Campos COA conjugate with an equal amount of 146s antigen FMDV homologous preparation (50 pg/ml) was examined by negative staining electron microscopy using 1% potassium phosphotungstate by the procedure of Brenner and Home 11959). 2.7. Complement

jikation

test (CFT)

CFTs were carried out in tubes using the 50% hemolysis technique described by Camargo et al. (1950) for the detection and typing of FMDV, The 50% complement fixation technique reported by Bier et al. (1968) was applied for the titration of guinea pig anti-FMDV sera. 2.8. Vesical epithelial

test samples

Thirty-seven tongue epithelial vesical samples from cattle naturally infected with PMDV, as confirmed by virus isolation and CFT, were mainta~ed in glycerol phosphate buffer (50% glycerol, v/v; 50% 0.04 M phosphate, v/v), pH 7.6, stored at - 20°C for different periods of time ranging from 1 to 6 years and assayed by the COA Test and again by the CIT. A 20% suspension of each sample (w/v) was prepared in PBS, pH 7.4, by grinding with a mortar pestle and sand and centrifuged, and the supematant was used for testing purposes.

3. Results 3.1. FMDV COA conjugates The results show the better pe~o~ance of the conjugate prepared with anti-146s plus 12s antigens serum (Table 1). This was due to the fact that this conjugate had both maximum sensitivity and maximum specificity, while the conjugate prepared with anti-FMDV crude antigenic BHK-cell suspension serum, though showing higher sensitivity, produced less specific reactions, including nonspecific coaggutination with control antigens, pa~ic~~ly when larger amounts of this serum were used. The optimum amounts of each antiserum for coating S. aureas were 80 ,ul and 40 ~1, respectively, for anti-146s plus 12s antiserum and for anti-FMDV infected BHK-cell suspension sera. Results quite similar to those described above were observed for the anti-FMDV A,, and C, COA conjugates prepared with analogous guinea pig sera. In contrast, the COA conjugates obtained from post-infection hyperimmune sera and from sera raised by single 146s antigen immunization by the methods of Rivenson (1956) or Ferris et al. (19841, respectively, showed a poor performance in the detection and typing of FMDV in infected BHK-cell cultures, requiring volumes of these antisera 3- to 4-times larger than those used for the former sera, and even that giving rise to a delayed and weak coagglutinatination.

lo/L1 2Oj&l 4O/.Ll 80~1 lO/.Ll 20/L] 4O/Ll 80~1

3+ 3+ 3+ 3+ 3+ 3+ 3+ 3+

2:20 1:30 0:45 0:42 0:58 0:45 0:35 0:26

3+ 2+ 2+ 3+ 3+ 3+ 3+ 3+

2:40 3:oo 2:13 2:07 1:58 1:56 1:07 0:30

l+ 1+ l+ 2+ 2+ 2+ 3+ 3+

i

i

i

t

l/4

_ 1+ 1+ 2+ 3+

3:04 4:48 3:03 1:50 0:51

i

l/8

_ _ _ 1+ 3+

_

5125 4:30 3:oo 1:20

_

i

2+

_

2:55

_ _ _ _ 2+

6130 2:30

_ _

_ _ _ _ _

_

_ _

_ _ _ _

i

t

3:31

_ _ _ _

t

Control antigens PBS

i

t

l/32

in order to use in COA test

l/16 t

for coating Staphylococci

590 5:20 4:20

t

necessary

l/2

t

antiserum

undiluted

FMDV 0, Campos *

of optimal amount of anti-FMDV

Serum 1 = Guinea pig serum anti 146s + 12s FMDV 0,Campos. Serum 2 = Guinea pig serum anti infected tissue culture with FMDV 0, Campos. i = intensity of coagglutination: (3 + 1, strong positive reaction; (2 + ), positive reaction; (1 + 1, weak positive reaction; (-), negative reaction. t = time in minutes:seconds from the beginning of coagglutination. * = FMDV 0, Campos undiluted BHK-21 cell suspension, containing approximately 1O7.35 TCID,, /ml.

Serum 2

Serum 1

Amount of serum used for coating s. allrells

Table 1 Determination

_ 3:31

_ 2+

_ _ _ _

t _ _ _

i

BHK-21

34

H.J. Montassier et al. /Journal

of Virological Methods 50 (1994) 29-42

Fig. 1. Agglutination pattern obtained from the mixture of COA-anti FMDV conjugate with the homologous virus present in infected BHK-21 cell culture extract, and pattern of the negative reaction with the same COA-conjugate and the noninfected BHK-21 cell culture extract.

Typical macroscopic patterns of positive and negative coagglutination reactions obtained by interaction of the anti-FMDV 0, COA conjugate with the homologous viral infected cell suspension and with the BHK cell negative control, respectively, are shown in Fig. 1.

3.2. Sensitivity

of the COA Test

The 146s and 12s antigen preparations from the 0, FMDV serotype were assayed by the COA Test with anti-146s + 12s FMDV homologous conjugates. The concentrations of these antigens ranged from 50 pg/ml to 0.31 pg/ml, as shown in Table 2. Positive coagglutinatinations were obtained with minimum detection levels of 1.25 pg/ml to 146s antigen, which is 4-fold lower than the detection limit of the corresponding 12s antigen. Consistently, the coagglutinatinations for 146s antigens occurred earlier and were stronger than those for 12s antigen.

3.3. Specificity

of the COA Test for the identification

of FMDV

The results presented in Tables 3 and 4 demonstrate only strong, 3 + scores, and homotypic reactions for the FMDV-infected cell suspension, which started 45 to 60 s after the test antigen suspension and the homologous anti-FMDV COA conjugate were mixed. Furthermore, there was no nonspecific coagglutination with VSV or the BHK-cell negative antigen control. With respect to the specificity of the coagglutination with 146s and 12s antigens, only homotypic reactions were observed for the former antigen and

aureu~ *

3+ 3+ 3+

i

1:20 0:25 0:21

t

3+ 3+ 3+

i

1:40 0:43 0:25

t

3+ 3+ 3+

i

5 /Ig/ml

2:25 0:52 0:29

t

2+ 3+

1+

i

t

vsv

2+ 2+ 3+

i

t

2:05 2:20 1:35

t

1+ 1+ 1+

2:45 4:12 3:lO

t

10 w8/ml i

3+ _

i

l:oo _ -

t

3+ _ -

i

i _ 3+

t _ 0:45 _ 0:50 -

_ -

_ _ -

i

_ -

_

_ _ -

i

_ _ _

t

BHK-21

FMDV A,,

i = intensity of coagglutination: same as Table 1. t = time from the beginning of coagglutination: same as Table 1. * = COA conjugates prepared with anti 146s + 12s FMDV guinea pig antisera in a volume of 80 ~1.

Anti 0, Anti A, Anti C, Control

_ -

_ -

from infected tissue culture

FMDV C,

of FMDV serotypes

t

i

12s FMDV 0, Campos

50 pg/ml

FMDV 0,

of COA test for the identification

_ 2139 0:57

t

0.62 pg/ml

FMDV subtype

Control antigens

*

Conjugates

1+ 2+

i

1.25 /kg/ml

of 0, Campos

Undiluted infected cell suspensions

of specificity

Evaluation

4:40 1:Ol 0:45

t

2.5 /.&g/ml

either 146s or 12s antigens

i = intensity of coagglutination: same as Table 1. t = time from the beginning of the coagglutination: same as Table 1. * = Guinea pig serum anti 146s + 12s FMDV 0, Campos.

20 pl 40 /Ll 80 ~1

S.

10 pg/ml

146s FMDV OtCampos

50 fig/ml

of COA Test for the detection

serum used for coating

sensitivity

Amountof

Comparative

Table 2

1+

_ _ _

i

PBS

5 kWm1 i

_ _ 5:50

t

_

_ _

t

-

-

t

2.5 pi/ml i

Y &

& 2 s g *

$ 3

s

%

3 3 fi.

ii 2. 9 P % \

36

HJ. Montassier

Table 4 Evaluation of specificity purified preparations COA Conjugates

Anti 0, Anti A, Anti C,

*

et al. /Journal

of Virological Methods 50 (I 994) 29-42

of COA test for the identification

146s FMDV a o

1

of FMDV serotypes

from either 146s or 12s

12s FMDV b

C3

44

0,

‘424

CT

i

t

i

t

i

t

i

t

i

t

i

t

3+ _ _

0:32

-

_ _

3+ _

0:21 _

_ -

_ -

3+ _

1:35 _

3+

0:30

-

-

3+ -

0:30 -

1+ 3+

2:50 0:35

i = intensity of coagglutination: same as Table 1. t = time from the beginning of coagglutination: same as Table 1. * = COA conjugates prepared with anti 146s + 12s FMDV guinea pig antisera in a volume of 80 yl. a 146s was assayed at a concentration of 50 pg/ml. ’ 12s was assayed at a concentration of 50 pg/ml.

Fig. 2. Electron micrograph of the mixture of COA-anti FMDV 0, conjugate with the homologl ous 146 S antigen preparation. The arrows show the 146 S particles closely attached to the S. aureus WIrface. The bacterial membrane (m) and cytoplasm (c) are also indicated.

H.J. Montassier et al. /Journal

Fig. 3. Electron micrograph of the clump produced homologous 146s antigen preparation.

of Virological Methods 50 (1994) 29-42

by the mixture of COA-anti

FMDV 0, conjugate

37

with the

both early and strong homotypic reactions, as well as delayed and weak heterotypic reactions, were observed for the 12s antigens. The ultrastructural analysis from the binding of 0, Campos FMDV COA conjugate and the homologous 146s viral particles show Figs. 2 and 3 a single cell or a clumping of bacterial cells, respectively, closely attached to the viral particles.

3.4. Comparison

of the COA Test with CFT

As summarized in Table 5, 37 originally positive infected FMDV tissue samples were submitted to the COA Test and CFT. The COA Test detected and typed FMDV in 27 samples (72.97%), and the CFI detected and typed FMDV in 26 (70.27%). Agreement of positive/positive results between COA and CFT was observed in 22 samples. In contrast, agreement of negative/negative results was observed in six samples. Disagreement between COA and CFI in terms of positive-COA Test/negative-CFI results was observed in five samples and disagreement in terms of negative-COA Test/positive-CFT results was observed in four samples. Seven samples among the 10 negative results recorded for the COA Test gave, in fact, positive coagglutinations which did not differ significantly in onset or intensity from any of the anti-FMDV COA-conjugates, indicating the presence of FMDV

H.J. Montassier

38

et al. /Journal

Table 5 Typing of FMDV from bovine epithelium Epithelium

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37

of Vimlogical Methods 50 (1994) 29-42

samples

COA conjugates

FMDV

FMDV

Anti 0,

type by COA

type by CFT

A A A A A

A A A _

Anti AZ4

i

t

_ _

_

_ _ _ _ _

_

1+ _

5:oo _

1+ _ _ _

7:oo _

1+ _

6:20 _

1+ _

6:00 _

3+ 3+ 2+ 1+ 1+ 3+ If _

3:oo 6:OO 3:oo 6:20 6:00 5:oo 8:OO _

1+ 1+ 2+ 3+ _

4:oo 5:20 3:oo l:oo _

2+ _

5:oo _

3f 3+ 1+

5:oo 2:30 9:40

Anti C, t

i

1+ 2+ 3+ 1+ 2+ _ _

9:00 6:OO 8:00 5:OO 5:OO

_ _ _

1+ 3+ 1+ 2+ 2+ 1+ 2+ 3+ 1+ 3+ 2+ 3+ 3+ 3+ 3+ 2+ 3+ 3+ 3+ 3+ 3+ _

9:00 3:oo 9:oo 6:00 9:oo 9:30 6:00 4:oo 8:00 5:oo 6:30 2:oo 4:oo 3:oo 4:30 3:30 7:oo 2:oo 4:oo 3:oo 4:oo _

3+ 3f _

2:oo 3:oo _

3+ 1+ 3+ 3+

4:00 7:oo 2:oo 3:oo

i

_

_

_ 1+ _

control t

_ _ 6:00

i

t

_ _ _ _ _ _ _ _

_

_ _

_ _ _ _ _ _

_

3+ 1+ 2+ If 1+ 3+ 1+ _

4:oo 9:oo 5:oo 6:20 6:00 7:oo 8:20 _

1+ _

5:oo _ _

2t _ _ _ _

3:oo _ _ _ _

2-k 1+

3:oo 9:50

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

_ _ _ _ _ _ _ _

_

A _* A A A A A A A A A A A NT NT NT A NT NT A A A A 0 NT A 0 A 0 NT A

A A A A A A A A A A _ A _ A _ A _ 0 A A A A 0 _ A 0 A 0 _ A

i = intensity of coagglutination: same as Table 1. t = time from the beginning of coagglutination: same as Table 1. NT = not typed. * Anticomplementary.

antigens, since no coagglutination with the control COA conjugate was observed for these samples. In spite of the cross-reactions in the COA Test, most of the epithelium samples were equally typed by the former test and CFT.

HJ. Montassier

et al. /Journal

of Virological Methods 50 (1994) 29-42

39

4. Discussion The COA Test is being used successfully in this laboratory to detect and type FMD viruses harvested from infected animal cells and tissues. The COA Test is also important for the preparation and standardization of the anti-FMDV COA conjugates. The performance of conjugates obtained from guinea pig serum previously immunized with FMDV BHK-cell suspension revealed a higher sensitivity in the COA Test. However, it showed some nonspecific reaction which were resolved by using a smaller amount of such serum for S. aureus coating. Although these guinea pig anti-FMDV sera might have antibodies to bovine Ig and BHK-cell proteins (Roeder and Le Blanc Smith, 1987), such antibodies did not react in the COA-Test, probably because the coagglutination is a secondary binding immunological test. Thus, before coagglutination takes place, an ideal number of antibody coated 5. aureus particles must combine with an ideal concentration of specific soluble antigens, cross-linking them and resulting in their clumping or coagglutinating. On the contrary, the ELISA, a primary serological test, detects directly the interaction between antibody and antigen, even when they are at very low concentrations (Tizard, 1982). The anti-FMDV guinea pig sera obtained by infection and hyperimmunization (Rivenson, 1956) or by immunization with a single 146s FMDV antigen dose (Ferris et al., 1984) showed a poor performance in the COA Test and they were not considered to be adequate for the preparation of anti-FMDV COA conjugates. The possible explanation is that they had lower anti-FMDV antibody titers than the former sera. The preparation of the anti-FMDV COA conjugate was simple, especially in the case of standardized Cowan I strain of S. aureus. The whole guinea pig anti FMDV sera were used for coating the staphylococci, instead of the serum gamma globulin or the IgG fractions which are necessary for the preparation of some reagents of the ELISA and the passive agglutination assays, using latex particles or sheep erythrocytes. Besides, the antiserum is used economically to prepare 2 ml of anti-FMDV COA conjugate and in amounts similar to those recommended in other viral COA Tests (Herbert and Caillert, 1981; Pandya et al., 1981; Skaug et al., 1983; Durigon et al., 1991). The minimum detection level found in the COA Test for the 146s FMDV antigen was similar to that recorded for the CFT (Garland et al., 1977) and higher than those reported for passive hemagglutination and trapping ELISA (Ouldridge et al., 1982; Roeder and IX Blanc Smith, 1987). This detection level for FMDV COA Test is also similar to the level found in the COA Test for the avian reticuloendotheliosis virus and for the Herpesvirus of turkeys (Pandya et al., 1981). The virion subunit 12s FMDV antigens were also detected in the COA Test at a level very much higher than that recorded by ELISA (Smitsaart et al., 1990). The specificity of the COA Test permitted the identification of a homologous FMDV serotype in infected animal cell and tissue culture, without the occurrence of nonspecific reactions with a heterologous virus such as VSV, and with heterologous FMDV serotypes. In fact, there were only heterotypic reactions with FMDV 12s subunits at a concentration of 50 pg/ml, which were weaker and more delayed than the homotypic reactions. Additionally, similar weak and delayed heterotypic reactions were recorded in the COA Test for some bovine epithelium vesical samples which did not preclude

40

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of~iro~ogi~a~Methods

50 (1994) 29-42

FMDV typing, at least for most of them, because the homotypic reactions were always stronger and earlier than the heterotypic ones, except for seven samples which produced reactions of similar strength and onset for anti 0, A and C COA conjugates. An alternative explanation may be that the COA Test and CFT have similar minimum detection levels for the 146s FMDV antigens and are able to detect the same amount of 12s FMDV subunits, so that the performance of these tests revealed high agreement between their results. The failures in FMD diagnosis observed for the COA Test and the CFT could be explained by the long length of storage of the vesical FMDV-infected samples before testing (unpublished results). In conclusion, the COA Test, because of its simplicity and rapidity of performance and its low cost, has a great potential for use for direct detection and identification of FMDV as a screening strategy at the sites of FMD outbreaks.

Acknowledgements We would like to thank the Funda@o de Amparo h Pesquisa do Estado de SBo Paulo {FAPESP), Ssio Paulo, Brazil for financial support (Proc. no. 88/2353-4) and Dra. H. Tanaka, P.P. Joazeiro and J.J. Kisielius from the Instituto Adolf0 Lutz, SIo Paulo, Brazil for examining samples in the electron microscope. We are indebted to A.E.G. Lima and M.L.F. Tamanini for technical assistance and also M. Pittuco of the Instituto Biologico, SBo Paulo, Brazil for her help in providing bovine epithelial samples.

References Abu Elzein, E.M.E. and Crowther, J.R. (1979) The specific detection of foot and mouth disease virus whole particle antigen (140s) of enzime labelled-immunosorbent assay. J. Hyg. Camb. 83, 127-133. Bachrach, H.L., Trautman, R. and Breese, S.S. (1964) Chemical and physical properties of virtually pure foot-and-mouth disease virus. Am. J. Vet. Res. 25, 333-342. Bahnemann, H.G. (1975) Binary ethylenemine as an inactivant for foot-and-mouth disease virus and its application for vaccine production. Arch. Viral. 47, 45-55. Bremrer, S. and Home, R.W. (1959) A negative staining method for high resolution eletron microscopy of viruses. B&him. Biophys. Acta 34, 103-110. Camargo, N.F., Eichorn, E.A., Levine, J.M. and Tellez-Giron, A.A. (19.50) A complement fixation technique for foot and mouth disease and vesicular stomatitis. Proc. Annu. Meet. Am. Med. Vet. Assoc. 87, 207-211. Crowther, J.R. (1977) Examination of differences between foot and mouth disease virus strain using a radioimmunoassay technique. Biol. Standard. 35, 185-193. Crowther, J.R. and Abu Elzein, E.M.E. (1979) Application of the enzyme-linked immunosorbent assay to the detection and identification of foot and mouth disease viruses. J. Hyg. Camb. 83, 513-519. Durigon, E.L., Candeias, J.A.N., Jerez, J.A., Bittencourt, M.J. and Ortolani, E.L. (1991) Comparison of Staphylococcat co-agglutination with other assays for rapid diagnosis of rotavirus infection in humans, calves and piglets. J. Virol. Methods 3.5, 73-79. Edwards, E.A. and Hilderbrand, R.L. (1976) Method for identifying Salmonella and Shigella directly from the primary isolation peak by coa~utination of protein A-cont~ning staphyloco~i sensitized with specific antibody. J. Clin. Microbial. 3, 339-343.

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of Virological Methods 50 (1994) 29-42

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