Appendix 68 3 ABC ELISA for the diagnosis of FMD in Egyptian sheep Laila E. El-Shehawy, Samira El-Kilany & A.M. Doaud Foot and Mouth Disease Department, Veterinary Serum and Vaccine Research Institute, Abbasia, P.O. 131, Cairo, Egypt Abstract Sheep play an important role in the epidemiology and transmission of foot and mouth disease (FMD). Moreover, FMD is suspected to have been transmitted to sheep in which infection is frequently sub-clinical. So, it is of importance to identify animals which have been exposed to the virus and have developed antibodies. Such animals may become carriers and thus be a potential source of new outbreaks. In addition to capsid proteins, certain non-structural proteins (NSP) are also produced during the process of infection by FMD virus (FMDV) and against which immunoglobulins may be formed. The most reliable single NSP indicator is the poly-protein 3 ABC, antibodies to which appear to provide conclusive evidence of previous infection, whether or not the animals have also been vaccinated. Three groups of sheep (4 animals in each group) were used in this study. The first group (Group1) were infected with FMDV O1/93/Aga-Egypt intradermolingually and the second group (Group 2) were in contact with Group 1 from day 1. The third group (Group 3) was vaccinated and revaccinated four weeks later with BEI-inactivated and aluminium hydroxide gel adjuvanted FMD-vaccine. Groups 1 and 2 were observed for clinical manifestations especially for vesicular lesions in the buccal cavity. Serum samples were collected from the three groups along with two sheep which were kept as controls. The serum neutralization test (SNT), a sandwich ELISA and an FMDV-3 ABC ELISA were used for the detection of antibodies to FMD virus. The results showed that the three groups of sheep produced serum-neutralizing (SN) antibodies. In Group 1 SN antibodies appeared as early as 2 days post infection (DPI), although protective levels were not evident until 7 DPI; these protective levels persisted until 40 weeks post infection (WPI). Antibodies were detected in Group 2 from 5 days post exposure (DPE) and protective levels from 8 DPE which persisted until 36 weeks post exposure (WPE). Group 3 had protective antibody levels from 12 days post vaccination (DPV) until 28 weeks post vaccination (WPV). Buccal lesions were observed in Groups 1 and 2; these differed in both severity and distribution but particularly affecting the tongue, lips and gums. Vesicular lesions progressed to become eroded areas and were subsequently healed; all lesions being completely healed by 12 days after either infection or exposure. Sheep in Groups 1 and 2 developed antibodies against FMDV-3 ABC antigen. NSP antibodies were detectable from 10 DPI until 40 WPI in Group 1 and from 15 DPE until 48 WPE in Group 2. Group 3 did not develop NSP antibodies during the course of the study. Therefore demonstrating the usefulness of the CHEKIT-FMD-3 ABC ELISA for the differentiation between infected and vaccinated sheep, which will be very important for FMD control in Egypt. Introduction FMD is a contagious disease of cloven hoofed animals which has a great potential to cause severe economic loss. FMD is endemic in two-thirds of OIE (Office Internationale des Epizooties) member countries where it is an economic problem and also provides a reservoir of virus which can spread into virus-free areas (Clavijo et al., 2004). Ganter et al. (2001) mentioned that goats and sheep might be carriers, so they play an important role in the epidemiology and transmission of FMD whilst Patil et al. (2002) calculated that sheep and goats constitute the majority of the world s FMD-susceptible livestock. Kitching and Hughes (2002) indicated that sheep and goats are highly susceptible to infection with FMD by the aerosol route. The virus probably most often infects sheep and goats by direct contact. On the other hand, Kitching and Mackay (1994) found that the incubation period in sheep following infection with FMDV is usually between 3-8 days, but can be as short as 24 hours following experimental infection, or as long as 12 days. Davis (2002) reviewed that FMDV is excreted during viraemia for some days, thereafter as serum antibodies develops viraemia decreases and the animal ceases to be infectious as the lesions heal. DeClercq (2002) stated that the FMDV excretion starts 24-48 hours before the onset of clinical signs and declines with the appearance of FMD specific circulating antibody at around 4-5 days post infection. Hughes et al. (2002a) revealed that the optimal dose to infect sheep and for producing in-contact transmission is about 10 4 TCID 50. Moreover, Hughes et al. (2002b) mentioned that lesions may fail to develop in approximately 25% of infected sheep; a further 20% may develop only a single observable lesion. In this respect, Arafa (1980) stated that the vesicle appeared on the epithelium of the tongue at 1 DPI and on the gums at 3 to 4 DPI in sheep, while Blood et al. (1985) observed that characteristic the FMD lesions usually develop between 3 and 8 DPE. Amas et al. (2003) found that contact sheep with infected pigs had developed gross lesions consistent with FMD by 5 DPE. In 1964, Dellers 416
and Hyde detected virus neutralizing antibodies 60 hours post inoculation (which persisted for at least 147 days) in sheep. Peak antibody titres were detectable from 10 DPI. On the other hand, diagnosis of FMD is made by the demonstration of FMD viral antigen or nucleic acid in samples of tissue or fluid. Detection of specific humoral antibody can also be used for diagnosis. The detection of antibody to non-structural proteins (NSP) of FMDV has been used to identify past or present infection (DeDiego et al., 1997; Brocchi et al., 1998; Dekker et al., 1998 and Malirat et al., 1998). Sorensen et al. (1998) detected 3-ABC antibodies from day 10 after experimental infection of sheep. During the course of infection, immune responses to NSP appear later than responses to structural proteins of the virus. Following experimental infection antibodies to the 3-ABC antigen could not be detected earlier than 10 DPI in sheep (Chung et al., 2002). Perhaps the most reliable single NSP indicator is the poly-protein 3 ABC, antibodies to which appear to provide conclusive evidence of previous infection (Makay et al., 1998) whether or not the animals have also been vaccinated. Sorenson et al. (1998) stated that antibodies against 3 ABC have been detected up to 395 days post infection in both cattle and sheep whilst Kitching (2002) reported that the 3-ABC antibodies persist more than 12 months. The severity of infection may influence serum levels of NSP antibodies and also the duration for which this antibody is detectable. The aim of this work is to evaluate the usefulness of the CHEKIT-FMD-3-ABC ELISA as a diagnostic tool to differentiate between FMDV-vaccinated and FMDV-infected Egyptian sheep. Differentiating between vaccinated and infected sheep may help in the future FMD control strategy. Material and Methods Animals: Fourteen male sheep of local breed were used. These were all eight months of age and sero-negative against FMDV. Virus: Foot and mouth disease virus (FMDV) type O1/93/Aga-Egypt was used to infect sheep. The virus was titrated in baby mice (2-3 days old); the titre was 10 9 MID 50 /ml. The virus was injected intradermo-lingually, each sheep receiving 10 4 MID 50 of virus. Tissue culture adapted virus (on BHK21 cells) was used in SNT and preparation of sandwich ELISA antigen. Vaccine: BEI-inactivated, aluminium hydroxide gel and saponin-adjuvanted FMD vaccine, which was locally prepared, was administered subcutaneously at a dose of 1ml per sheep. Sample: Serum samples were collected 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15 (DPI or DPE or DPV) and at 4, 6, 8, 10, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48 and 52 (WPI or WPE or WPV). Serum neutralization test (SNT): The SNT was carried out according to OIE Manual (2000) based on the method described by Ferreira (1976). The titers expressed in log 10 were calculated according to Karber (1931). Sandwich ELISA: The Sandwich ELISA was carried out according to Voller et al. (1976) and Hamblin et al. (1986) for reagent preparation and test method, respectively. CHEKIT-FMD-3 ABC ELISA: The CHEKIT-FMD-3 ABC ELISA was provided by Bommeli Diagnostics, Liebefeold-Bern, Switzerland. The test was performed as described by the manufacturer using the following calculation formula: OD sample - OD negative Value % = ----------------------------------- X 100 OD positive - OD negative OD: Optical Density Negative : Negative control (OD = 0.063 = 0%) Positive : Positive control (OD = 1.215 = 100%) Results and Discussion Nowadays, the identification of sheep which are currently- or previously- infected with FMDV is very important to any strategy for the control and eradication of FMD, given that apparently healthy sheep may be the source of a new outbreak. The only way to efficiently identify carrier sheep is by detection of antibodies against non-structural proteins of FMDV, such as 3-ABC. NSP antibodies only develop following infection of animals with FMD virus and not after vaccination with purified, inactivated vaccine. To evaluate the use of an NSP antibody detection assay in differentiating between vaccination and infection, 417
an experiment was designed with three groups of sheep (with 4 animals in each group). The first group was infected with FMD O1/93/Aga-Egypt, the second group was exposed by contact with the first group from the day on which they were infected and the third group was vaccinated with an inactivated, adjuvanted vaccine (see the design of the experiment). Table 1 details the lesions observed in the buccal cavity of experimentally-infected and in-contact sheep. In Group 1 lesions on the tongue epithelium were apparent from 3 DPI whilst lesions appeared on the lips and gums at 4 DPI. All the experimentally-infected sheep developed buccal lesions which had healed completely by 8 DPI but no feet lesions were noticed in this group. These results are in agreement with Arafa (1980) who stated that the vesicles appeared on the epithelium of the tongue at 1 DPI and on the gum at 3 to 4 DPI and with Blood et al. (1985), they mentioned that the characteristic lesions usually developed after 3 to 8 DPE. From Table 1 it is also clear that in-contact sheep (Group 2) developed buccal vesicular lesions from 6 DPE and these lesions were found either on the lip or the gum or both; All lesions had healed by 12 DPE. The results also showed that only three of the four in-contact animals were infected. Kitching and Mackay (1994) found that the incubation period of FMDV in sheep is usually between 3 and 8 days. Also, Hughes et al. (2002b) mentioned that vesicular lesions may fail to develop in approximately 25% of infected sheep. Bouma et al. (2004) indicated that it may be possible that contact with a low amount of virus results in a quick immune response. This may prevent widespread replication of the virus in the infected animal so that there are no visible signs of FMD. In general, the lesions in both groups 1 and 2 appeared as vesicles which progressed to become eroded areas and ended with complete healing. Serum samples were collected from the animals in the three groups at different times post infection, exposure and vaccination. Table 2 details the antibody titres against FMD. Two techniques were used to determine such titres (SNT and sandwich method ELISA) for vaccinated sheep in Group 3. Antibodies were first detected at 6 DPV, reaching protective levels (1.2 log 10 ) by 12 DPV. The titres continued to increase, reaching maximum levels 10 weeks after primary vaccination (which was 6 weeks after booster vaccination). Protective levels persisted from 12 DPV until 28 WPV; SN antibody titres ranging from 1.2 to 1.85 log 10. With ELISA detectable antibody levels were slightly greater, ranging from 1.2 to 2.15 log 10, a difference of about 0.2 log 10 in measured antibody levels between the two techniques used. These results are consistent with the statement of Hamblin et al. (1986) who explained that the SNT measures those antibodies which neutralize the infectivity of FMD virion, while ELISA probably measure all classes of antibodies even those produced against incomplete and non-infectious virus. In case of experimentally infected sheep a trace of neutralizing antibodies was detectable in the animal's sera from 2 DPI (0.35 log 10 ) and protective levels from 7 DPI. Protective levels persisted until 40 WPI reaching maximum titers of 1.80 to 1.90 log 10 between 4 and 28 WPI. When the sera were examined by ELISA a similar difference of about 0.2 log 10 was measured between antibody levels detected by ELISA and levels detected by SNT. In the case of contact sheep, SN antibodies were not detected until 5 DPE, protective levels were reached at 8 DPE, and these protective levels persisted until either 36 WPE (based on SNT results) or 40 WPE (when measured by ELISA). Once more, ELISA detected slightly higher levels than SNT. Dellers and Hyde (1964) detected virus neutralizing antibodies in sheep 60 hours after inoculation with FMDV, peak titers occurring by day 10 and detectable antibody persisting for at least 147 days. Davis (2002) indicated that FMDV is shed during viraemia for some days, thereafter as serum antibody develops viraemia decreases. Declercq (2002) showed the appearance of circulating FMD specific antibody at around 4 to 5 days post infection. Table 3 illustrates the detection of FMDV 3-ABC antibodies in sera collected from the three groups of sheep. Vaccinated sheep failed to produce any 3-ABC antibodies as for control sheep. Detectable 3-ABC antibodies started to appear at 10 DPI (about 1% positivity) in experimentally-infected sheep, increased gradually to a maximum (about 100% positivity) at 10 WPI and started to decrease from 24 WPI, reaching about 56% positivity at 36 WPI. The percentage positivity decreased dramatically from 40 WPI and antibody was no longer detectable by 44 WPI. In-contact sheep also developed antibodies against FMD non-structural proteins (3 ABC) by 15 DPE but detectable antibody disappeared completely after 44 WPE. Sorenson et al. (1998) stated that antibodies against 3 ABC have been detected up to 395 days post-infection in both cattle and sheep. They also showed that FMD-3 ABC antibodies could be detected from day 10 after experimental infection of susceptible sheep. In this study, the detection of the FMD 3-ABC antibodies were detected in both infected and in-contact sheep up until 44 weeks post-infection or post-exposure. The duration for which antibody is detectable may depend on the severity of the infection (Kitching, 2002). Moreover, Chung et al. (2002) found that antibodies to the 3 ABC antigen in sheep could not be detected earlier than 10 days after experimental infection. 418
References Amas, S.F.; Pacheco, J.M.; Mason, P.W.; Schneider, J.L.; Alvarez, R.M.; Clark, L.K. and Ragland, D. (2003): Procedures for preventing the transmission of FMDV to pigs and sheep by personnel in contact with infected pigs. Vet. Rec., 153: 137-140. Arafa, M.H. (1980): Studies on the carrier state of goats exposed to FMDV. M.V.Sc. Thesis, Cairo Univ. Blood, D.C.; Radostitis, O.M. and Henderson, J.A. (1985): Veterinary Medicine: A textbook of the disease of cattle, sheep, goats and horses. 8th ed., ELBS and Bailliere Tindal. Brocchi, E.; DeDiego, M.I.; Berlinzani, A.; Gamba, D. and DeSimone, F. (1998): Diagnostic potential of Mab-based ELISAs for antibodies to non-structural proteins of FMDV to differentiate infection from vaccination. Proceedings of concerted action CT93-0909. Vet. Q., 20: 20-24. Bouma, A.; Dekker, A. and Mart, C.M. de Jong (2004): No foot and mouth disease virus transmission between individually housed calves. Vet. Microbiol., 98: 29-36. Chung, W.B.; Sorensen, K.J.; Liao, P.C. and Jong, M.H. (2002): Differentiating FMDV infected pigs from vaccinated pigs by blocking ELISA using non-structural protein 3 ABC as antigen and its application to an eradication program. J. Cl. Micro., 40: 2843-2848. Clavijo, A.; Wright, P. and Kitching, P. (2004): Developments in diagnostic techniques for differentiating infection from vaccination in FMD. Vet. J., 167: 9-22. Davis, G. (2002): Foot and mouth disease. Review Res. Vet. Sci., 73: 195-199. DeClercq, K. (2002): Overview on FMD diagnostic techniques. In FMD control strategies, Symposium Proceedings, 2-5 June 2002, Lyon, France, pp. 345-351. DeDiego, M.; Brocchi, E.; Makay, D. and DeSimone, F. (1997): The non-structural polyprotein 3 ABC of FMDV as a diagnostic antigen in ELISA to differentiate infected from vaccinated cattle. Archives of Virology, 142: 2021-2033. Dekker, A. and Gijsen, E. (1998): The possible use of native FMD non-structural protein 3A in a serological screening test. Vet. Q., 20: Suppl. 2. Dellers, R.W. and Hyde, J.I. (1964): Response of sheep to experimental infection with FMDV. Am. J. Vet. Res., 25: 105. Ferreira, M.E.V. (1976): Prubade microneutralization poraestudies de anticueropos de la fibre aftosa. Bltn. Cent. Panam. Fiebre Aftosa, 21 (22): 17-24. Ganter, M.; Graunke, W.D.; Steng, G. and Worbes, H. (2001): FMD in sheep and goats. Dtsch Tierarztl Wochenschr, 108 (12): 499-503. Hamblin, G.; Barnett, I.T.R. and Crowther, J.R. (1986): A new ELISA for detection of antibodies against FMDV. II. Application. J. Imm. Meth., 93: 123-129. Hughes, G.J.; Kitching, R.P. and Woolhouse, M.E.J. (2002a): Dose dependent response of sheep inoculated intranasally with a type O-FMDV. J. Comp. Path., 127: 22-29. Hughes, G.J.; Mioulet, V.; Kitching, R.P.; Woolhouse, M.E.J.; Andersen, S. and Donaldson, A.I. (2002b): FMD infection of sheep: implications for diagnosis and control. Vet. Rec., 150 (23): 724-727. Karber, G. (1931): 50% end point calculation. Archiv fur Experimentelle Pathologie und Pharmakologie, 162: 480-483. Kitching, R.P. (2002): Identification of FMDV carrier and subclinically infected animals and differentiation from vaccinated animals. Rev. Sci. Tech. Off. Int. Epiz., 21 (3): 531-538. Kitching, R.P. and Hughes, G.J. (2002): Clinical variation of FMD: sheep and goats. Rev. Sci. Tech. Off. Int. Epiz., 21 (3): 505-512. Kitching, R.P. and Makay, D.K. (1994): FMD. State Vet. J., 4: 7-10. Mackay, D.K.J.; Forsyth, M.A.; Davies, P.R.; Berlinzani, A.; Belsham, G.J.; Flint, M. and Rayon, M.D. (1998): Differentiating infection from vaccination in FMD using a panel of recombinant non-structural proteins in ELISA. Vaccine, 16: 446-459. Malirat, V.; Neitzert, E.; Bergmann, I.E.; Marade, E. and Beck, E. (1998): Detection of cattle exposed to FMDV by means of an indirect ELISA test using bioengineered non-structural polyprotein 3 ABC. Vet. Quarterly, 20 (Suppl. 2): S24-S26. OIE (2000): FMD, Chapter 2.1.1. In Manual of Standard for Diagnostic Test and Vaccine, 4th ed., 2000, Paris, pp. 77-92. Sorensen, K.J.; Brocchi, E.; Mackay, D. and DeSimone, F. (1997): FMD, detection of antibodies in cattle sera by blocking ELISA. Vet. Microbiol., 23 (3-4): 253-265. Voller, A.; Bidwell, D.E. and Ann Bartlett (1976): ELISA in medicine. Theory and Practice. Bul. WHO, 53: 55-56. 419
Table 1 Design of the experiments Experimental Groups Group 1 (4 sheep) Group 2 (4 sheep) Group 3 (4 sheep) Group 4 (2 sheep) Time of sera collection Days Weeks 2 3 4 5 6 7 8 9 10 12 15 4 6 8 10 12 16 20 24 28 32 36 40 44 48 52 Infected intradermo-lingually with the virulent FMD O1/93/Aga-Egypt in a dose of 10 4 MID 50 /ml In-contact with the experimentally infected group of sheep (Group 1) from the first day of infection Vaccinated subcutaneously with 1ml of BEI-inactivated, aluminium hydroxide gel-adjuvanted vaccine containing 1-2 µg 140S/dose; revaccinated with the same vaccine dose four weeks after primary vaccination Negative control sheep (Unvaccinated and uninfected) 420
Table 2: Follow-up of antibodies against FMD in different groups of sheep by SNT and sandwich method ELISA expressed in log 10 Time of sera Group (1) Group (2) Group (3) collection SNT ELISA SNT ELISA SNT ELISA 2 0.35 0.55 0 0 0 0 3 0.40 0.65 0 0 0 0 4 0.65 0.75 0 0 0 0 5 0.65 0.85 0.25 0.25 0 0 6 0.85 1.20 0.35 0.55 0.35 0.35 Days 7 1.20 1.35 0.85 1.35 0.35 0.35 8 1.35 1.50 1.25 1.35 0.65 0.45 9 1.36 1.50 1.20 1.40 1.00 1.20 10 1.35 1.65 1.45 1.55 1.00 1.25 12 1.40 1.65 1.60 1.75 1.20 1.50 15 1.65 1.75 1.65 1.85 1.25 1.65 4 1.85 2.10 1.65 1.85 1.35 1.65 6 1.85 2.10 1.70 1.85 1.85 2.15 8 1.80 2.15 1.75 1.90 1.80 2.10 10 1.82 1.95 1.90 1.95 1.80 1.95 12 1.80 1.95 1.85 2.15 1.55 1.95 16 1.85 1.95 1.75 2.15 1.45 1.75 20 1.90 1.90 1.75 1.95 1.35 1.45 Weeks 24 1.85 1.95 1.65 1.90 1.25 1.25 28 1.80 1.95 1.35 1.65 1.25 1.25 32 1.50 1.75 1.20 1.40 0.85 1.25 36 1.45 1.65 1.25 1.45 0.55 0.85 40 1.25 1.35 1.00 1.20 0.35 0.55 44 0.80 1.20 0.95 1.00 0 0 48 0.35 0.60 0.25 0.30 0 0 52 0.25 0.45 0 0 0 0 Two negative control sheep were seronegative allover the experiment. The titers of antibodies are the mean of the animals in each group. 421
Table 3: Tracing of FMD-3 ABC antibodies in different groups of sheep calculated in percent Time of sera Group (1) Group (2) Group (3) collection OD % OD % OD % 2 0.062 0 0.057 0 0.058 0 3 0.063 0 0.061 0 0.071 0 4 0.058 0 0.071 0 0.063 0 5 0.061 0 0.063 0 0.062 0 6 0.073 0 0.061 0 0.062 0 Days 7 0.063 0 0.065 0 0.058 0 8 0.059 0 0.071 0 0.058 0 9 0.062 0 0.063 0 0.081 0 10 0.131 10 0.059 0 0.062 0 12 0.725 57 0.062 0 0.061 0 15 0.855 60 0.685 56 0.054 0 4 0.950 70 0.721 57 0.062 0 6 1.130 90 0.760 58 0.063 0 8 1.211 90 0.853 60 0.062 0 10 1.321 100 0.850 60 0.073 0 12 1.411 100 0.933 70 0.071 0 16 1.400 100 0.980 85 0.065 0 20 1.393 100 1.123 90 0.061 0 Weeks 24 0.882 80 1.125 90 0.061 0 28 0.860 60 1.332 100 0.063 0 32 0.879 60 1.323 100 0.061 0 36 0.721 56 1.410 100 0.073 0 40 0.136 10 0.933 70 0.063 0 44 0.065 0 0.625 55 0.058 0 48 0.071 0 0.125 10 0.063 0 52 0.056 0 0.059 0 0.066 0 Two negative control sheep were seronegative allover the experiment. The OD readings are the mean of the animals in each group. 422