Foot-and-mouth disease virus-infected but not vaccinated cattle develop antibodies against recombinant 3AB1 nonstructural protein

Summary  Foot-and-mouth disease (FMD) vaccines induce antibodies against structural and some nonstructural proteins present in vaccine preparations. To differentiate between FMDV-infected and vaccinated animals, we developed immunochemical assays capable of detecting antibodies against a FMDV nonstructural protein. Recombinant nonstructural 3AB1 protein was expressed in E. coli and in insect cells and used to detect anti-3AB1 antibodies. ELISA and Western blot analysis showed that sera from cattle infected with FMDV reacted with recombinant 3AB1 protein whereas sera from cattle which had been vaccinated against FMDV, mock-infected, or infected with different bovine viruses did not recognize the 3AB1 protein. In contrast, anti-virus infection associated antigen (VIAA) antibodies were present in both FMDV-infected and vaccinated animals. Detection of anti-3AB1 antibodies in sera of experimentally infected cattle obtained between 7 and 560 days postinfection indicated that immunological tests based on the detection of recombinant 3AB1 protein could be used for the diagnosis of FMDV infection. Received June 17, 1996 Accepted September 11, 1996     

High-level expression of recombinant 3AB1 non-structural protein from FMDV in insect larvae

For its potential usefulness in diagnosis, the non-structural protein 3AB1 from foot-and-mouth disease virus was expressed as a soluble protein by using Autographa californica nuclear polyhedrosis virus as a vector. The 3AB1 coding sequence was introduced into AcNPV genome via pBAcPAK3AB1 transfer vector to originate Ac3AB1 recombinant baculovirus of phenotype occ-. Rachiplusia nu larvae were injected with supernatants of Sf9 cells infected with Ac3AB1 and 5 days post-infection total protein extracts were obtained. An intense band of approximately 21.5 kDa was observed when total larvae extracts were SDS-PAGE resolved and the recombinant protein detected by an FMDV-infected guinea pig serum. ELISA tests and Western blot experiments were carried out using sera both from FMDV-infected cattle and from vaccinated animals. The recombinant protein was only recognized by sera from infected animals, suggesting that this method of production in insect larvae could be applied to an efficient mass production of proteins of diagnostic interest.     

Evaluation of the Immunogenicity of an Experimental Subunit Vaccine That Allows Differentiation between Infected and Vaccinated Animals against Bluetongue Virus Serotype 8 in Cattle

Bluetongue virus (BTV), the causative agent of bluetongue in ruminants, is an emerging virus in northern Europe. The 2006 outbreak of BTV serotype 8 (BTV-8) in Europe was marked by an unusual teratogenic effect and a high frequency of clinical signs in cattle. Conventional control strategies targeting small ruminants were therefore extended to include cattle. Since cattle were not routinely vaccinated before 2006, the immune responses to BTV have not been studied extensively in this species. With the aims of developing a subunit vaccine against BTV-8 for differentiation between infected and vaccinated animals based on viral protein 7 (VP7) antibody detection and of improving the current understanding of the immunogenicity of BTV proteins in cattle, the immune responses induced by recombinant VP2 (BTV-8) and nonstructural protein 1 (NS1) and NS2 (BTV-2) were studied. Cows were immunized twice (with a 3-week interval) with the experimental vaccine, a commercial inactivated vaccine, or a placebo. The two vaccines induced similar neutralizing antibody responses to BTV-8. Furthermore, the antibody responses detected against VP2, NS1, and NS2 were strongest in the animals immunized with the experimental vaccine, and for the first time, a serotype cross-reactive antibody response to NS2 was shown in cattle vaccinated with the commercial vaccine. The two vaccines evoked measurable T cell responses against NS1, thereby supporting a bovine cross-reactive T cell response. Finally, VP7 seroconversion was observed after vaccination with the commercial vaccine, as in natural infections, but not after vaccination with the experimental vaccine, indicating that the experimental vaccine may allow the differentiation of vaccinated animals from infected animals regardless of BTV serotype. The experimental vaccine will be further evaluated during a virulent challenge in a high-containment facility.     

Differentiation of foot-and-mouth disease virus infected animals from vaccinated animals using a blocking ELISA based on baculovirus expressed FMDV 3ABC antigen and a 3ABC monoclonal antibody

Summary. A blocking ELISA that differentiated foot-and-mouth disease virus (FMDV) infected animals from vaccinated animals was developed which uses baculovirus expressed FMDV 3ABC non-structural protein as antigen and monoclonal antibody against FMDV 3ABC non-structural protein as capture and detector antibody. Sera from naive, vaccinated and infected cattle, sheep and pigs were examined. The specificity of the test was high. Non-specific reactions observed in particular in sera of cattle and sheep could be removed by filtration and inactivation. Positive reactions were obtained for sera from cattle infected with all seven serotypes of FMDV. The test detected antibodies from days 7 or 9 following experimental infection of non-vaccinated cattle and sheep, and in cattle strong positive reactions persisted for up to 395 days after infection. In vaccinated cattle that became carriers after challenge with homologous FMDV, positive reactions were obtained in all but one case. In some of these cattle the antibody response was detected late in comparison to the non-vaccinated infected cattle. The test gave results that compared favourably with two commercial ELISAs when used to test sera from cattle, pigs and sheep collected after experimental or natural infection. The blocking ELISA based on recombinant FMDV 3ABC antigen and a monoclonal antibody to 3ABC is a promising tool for FMD control and eradication campaigns, where vaccination has been carried out.     

Immune responses to structural proteins of avian infectious bronchitis virus.

Chickens were vaccinated with live and inactivated infectious bronchitis virus (IBV), and antibody responses to the individual structural proteins, S1, S2, M and N, followed by ELISA and western blotting. All four structural proteins elicited an antibody response in chicks vaccinated with either live or inactivated IBV. The S1, S2 and N proteins elicited similar titres of antibodies following vaccination with live IBV, whereas the M glycoprotein elicited significantly lower titres. Time of appearance and the course of development of the S1, S2 and N ELISA antibodies were similar, being first detected 2 weeks after vaccination and coincided with appearance of virus neutralizing antibodies. The M antibodies were first detected 4 weeks after vaccination. S1, S2, and N antibody titres were significantly higher in chicks vaccinated at 14 days of age than in chicks vaccinated at either 1 or 7 days of age, and reached maximum levels 4 weeks after the second vaccination. The S1, S2 and N proteins induced cross-reactive antibodies, whereas the M glycoprotein induced antibodies of limited cross-reactivity. Titres of cross-reactive N antibodies were higher than titres of cross-reactive S1 and S2 antibodies, which were similar. Epitopes on the N and S2 proteins that gave rise to cross-reactive antibodies showed the same degree of conservation, whereas the cross-reactive S1 epitopes were marginally less conserved. Vaccination with inactivated virus induced significantly lower antibody titres and at least three vaccinations were necessary for induction of S1, S2, N and M antibodies in all chicks. The S2 glycoprotein was the most immunogenic structural protein following vaccination with inactivated virus. All four proteins induced cell-mediated immune responses in chicks vaccinated with live IBV as determined by a delayed type hypersensitivity response.     

An inhibition enzyme-linked immunosorbent assay for the detection of antibody to Rift Valley fever virus in humans, domestic and wild ruminants

This paper describes the development and validation of an inhibition ELISA based on gamma-irradiated tissue culture-derived antigen for the detection of antibody to Rift Valley fever virus (RVFV) in humans, domestic and wild ruminants. Validation data sets derived from field-collected sera in Africa (humans=1367, cattle=649, goats=806, sheep=493, buffalo=258, camels=156) were categorized according to the results of a virus neutralisation test. In addition, individual sera from 93 laboratory workers immunized with inactivated RVF vaccine, 136 serial bleeds from eight sheep experimentally infected with wild-type of RVFV, and 200 serial bleeds from 10 sheep vaccinated with the live-attenuated strain of the virus, were used to study the kinetics of RVFV antibody production under controlled conditions. At cut-off values selected at 95% accuracy level by the two-graph receiver operating characteristic analysis the ELISA sensitivity ranged from 99.47% (humans) to 100% (sheep, buffalo, camels). The specificity ranged from 99.29% (sheep) to 100% (camels). Compared to virus neutralisation and haemagglutination-inhibition tests, the ELISA was more sensitive in detection of the earliest immunological responses in experimentally infected and vaccinated sheep. Our results demonstrate that the ELISA format reported here can be used as a safe, robust and highly accurate diagnostic tool in disease-surveillance and control programmes, import/export veterinary certification, and for monitoring of the immune response in vaccinees.     

Differentiation of infection from vaccination in foot-and-mouth disease by the detection of antibodies to the non-structural proteins 3D, 3AB and 3ABC in ELISA using antigens expressed in baculovirus

Summary.  The baculovirus expression system was found to be efficient at expressing the 3D, the 3AB and the 3ABC non-structural proteins (NSP) of foot-and-mouth disease virus (FMDV) as antigens recognised by immune sera in ELISA. ELISA’s using 3D, 3AB and 3ABC detected antibodies from day 8 and 10 after experimental infection of susceptible cattle and sheep and cattle remained seropositive for more than 395 days. The ELISA’s detected antibodies against any of the seven serotypes of FMDV. The 3D ELISA was specific and precise and as sensitive as established ELISA’s which measure antibody to structural proteins. The assay may be used as a resource saving alternative to established ELISA’s for the detection of antibodies against any of the seven serotypes. The 3AB and the 3ABC ELISA were also specific and precise. FMDV infected cattle could be differentiated from those that had been merely vaccinated as they gave a positive result in both the 3AB and the 3ABC ELISA’s. Two cattle that had been both vaccinated and infected also gave positive results in both tests, suggesting that the 3AB and 3ABC ELISA’s, but not the 3D ELISA might represent a reliable means of detecting infection in a vaccinated population. Received December 22, 1997 Accepted April 7, 1998     

A solid-phase competition ELISA for measuring antibody to foot-and-mouth disease virus

A solid-phase competition ELISA has been developed to measure antibodies to foot-and-mouth disease (FMD) virus and has been validated using an extensive range of sera from cattle. The assay uses polyclonal antisera and inactivated purified 146S antigens of FMD virus and was compared with the liquid-phase blocking ELISA and the virus neutralisation test on a range of serum sets. When examining test sera at a 1:5 dilution with a cut-off point of 30% inhibition of reaction, the solid-phase competition ELISA was as sensitive as the liquid-phase blocking ELISA for sera from infected or vaccinated animals. The limit of detection of the solid-phase ELISA was similar to that of the liquid-phase assay and both tests had lower limit of detection (i.e. were able to detect lower amounts of antibody) than the virus neutralisation test. The specificity of the solid-phase ELISA was considerably higher than that of the liquid-phase blocking ELISA and almost equivalent to that of the virus neutralisation test. The assay thus retains the sensitivity of the liquid-phase blocking ELISA whilst being easier to use, more robust and specific, and therefore offers an improvement for FMD virus antibody detection.     

Comparison of two 3ABC enzyme-linked immunosorbent assays for diagnosis of multiple-serotype foot-and-mouth disease in a cattle population in an area of endemicity

The development of a serological test for foot-and-mouth disease virus (FMDV) which is quick and easy to use, which can identify all seven serotypes, and which can differentiate vaccinated from convalescing or potential virus carriers would be a major advance in the epidemiological toolkit for FMDV. The nonstructural polyprotein 3ABC has recently been proposed as such an antigen, and a number of diagnostic tests are being developed. This paper evaluates the performance of two FMDV tests for antibodies to nonstructural proteins in an unvaccinated cattle population from a region of Cameroon with endemic multiple-serotype FMD. The CHEKIT-FMD-3ABC bo-ov (CHEKIT) enzyme-linked immunosorbent assay (ELISA) (Bommeli Diagnostics/Intervet) is a commercially available test that was compared with a competitive 3ABC ELISA (C-ELISA) developed in Denmark. The tests were compared with the virus neutralization test as the "gold standard." Diagnostic sensitivity and specificity were examined over a range of test cutoffs by using receiver operating characteristic curves, which allowed comparison of the overall performance of each test. The results indicated that the CHEKIT ELISA kit was 23% sensitive and 98% specific and the Danish C-ELISA was 71% sensitive and 90% specific at the recommended cutoff. These results have important implications if the tests are to be used to screen herds or individual cattle in surveillance programs, at border crossings for import-export clearance, or following emergency vaccination in an outbreak situation.     

A solid-phase blocking ELISA for detection of type O foot-and-mouth disease virus antibodies suitable for mass serology

A simple solid-phase blocking ELISA for the detection of antibodies directed against type O foot-and-mouth disease virus (FMDV) was developed. The ELISA was validated using field sera collected from cattle, pigs and sheep originating from FMDV infected and non-infected Dutch farms, reference sera obtained from the World Reference Laboratory for foot-and-mouth disease at the Institute for Animal Health, Pirbright Laboratory, UK and sera from experimentally infected animals. Testing 2664 sera collected from non-infected cattle, pigs and sheep resulted in a specificity of 96%. A sensitivity relative to the virus neutralisation test (VNT) of >99% was achieved when testing 148 positive cattle, goat and sheep sera collected from FMDV-infected Dutch farms. All international reference sera scored consistently correct. The ELISA also correctly scored 398 of 409 positive experimentally derived sera. The sensitivity and specificity of this monoclonal antibody-based ELISA for detection of type O FMDV antibodies is sufficient for use as a screening ELISA. During the 2001 epidemic in the Netherlands, 8000 serum samples per day were regularly tested in this ELISA. The samples scoring positive were then tested by neutralisation for confirmation thus making optimum use of the neutralisation testing capacity.     

Impact of immunizations with porcine reproductive and respiratory syndrome virus on lymphoproliferative recall responses of CD8+ T cells

The objective of this study was to investigate the immune responses elicited by either a modified-live (MLV) or a killed virus (KV) porcine reproductive and respiratory syndrome virus (PRRSV) vaccine. Specifically, we investigated the effects of multiple vaccinations on antigen-specific cellular and antibody responses against PRRSV. Twelve sows were obtained from herds with either a history of repeated MLV or KV PRRSV vaccination and a non-vaccinated, PRRSV-negative herd. Within herd, sows were divided into three groups and vaccinated with MLV, KV, or injected with saline. On day 0, 27, and 38, recall responses of peripheral blood mononuclear cells (PBMC) to the parent strains of the vaccines (e.g., MLV-VR2332 or KV-ISUP) were examined. The concentrations of total PRRSV-specific and virus-neutralizing serum antibodies were determined by ELISA and serum neutralization assays. Following immunization, the antigen-specific proliferation of CD8alphabeta(+), CD4(+)CD8alphaalpha(+) T cells in the naive sows was greater than in sows repeatedly vaccinated with KV or MLV. This diminished lymphoproliferative responses of CD8alphabeta(+) and CD4(+)CD8alphaalpha(+) T cells could be partially overcome by heterologous immunization. However, B cell proliferation, PRRSV antibody concentrations and virus neutralizing antibody titers were not enhanced by heterologous immunization and only KV vaccination increased antibody levels in previously immunized (MLV or KV) sows.     

Production and application of recombinant antibodies to foot-and-mouth disease virus non-structural protein 3ABC

The stamping out of animals to control a foot-and-mouth disease (FMD) outbreak results in enormous livestock losses. The implementation of vaccination strategies can reduce these losses; however it complicates the process of establishing freedom from disease following an outbreak. The availability of quality diagnostic tests to differentiate infected from vaccinated animals (DIVA) is crucial to prove freedom from disease and allow for the resumption of trade in livestock products. All current foot-and-mouth disease virus (FMDV) DIVA tests rely on polyclonal or monoclonal hybridoma derived antibody reagents, which can be difficult to prepare and maintain in a quality-assured manner and in the quantities required for post-outbreak surveillance. Recombinant antibodies can be produced in large quantities at low cost in bacteria to guarantee the supply of a consistent and well-characterised reagent. The production of recombinant antibodies does not rely on animal immunisation and does not require the maintenance of viable hybridoma cell lines. In this study, phage display libraries of recombinant antibody single chain variable fragments (scFv) against FMDV were generated from chickens immunised with recombinant non-structural protein (NSP) 3ABC. A total of 32 positive clones were obtained that represented three distinctive genetic sequences, Chicken Recombinant Antibody-Foot-and-Mouth disease (CRAb-FM) 26, -FM27 and -FM29. Each was shown to bind the 3B region of the 3ABC protein. When evaluated in a C-ELISA format using sera derived from cattle, sheep and pigs representing na?ve, FMDV-vaccinated or FMDV-infected animals, CRAb-FM27 gave the best performance when paired with an E. coli-derived recombinant 3ABC, demonstrating the potential to be used as a species- and serotype-independent FMDV DIVA test. To our knowledge, this is the first FMDV DIVA test that uses both recombinant antibody and antigen derived from bacterial expression systems.     

Development of monoclonal antibody-linked ELISA for sero-diagnosis of vesicular stomatitis virus (VSV-IN) using baculovirus expressed glycoprotein

The gene encoding the envelope glycoprotein (GP) of vesicular stomatitis virus serotype, Indiana (VSV-IN), was expressed under the polyhedron promoter of baculovirus. The recombinant GP was applied as a diagnostic antigen for the detection of cattle and horse antibodies to VSV. In addition, the neutralizing monoclonal antibody (Mab) to GP of VSV-IN was used as trapping antibody in a Mab-linked indirect ELISA (MLI-ELISA) or detecting antibody in a Mab-linked competitive ELISA (MLC-ELISA). The diagnostic efficiencies of MLI-ELISA and MLC-ELISA were evaluated with currently available C-ELISA from OIE reference laboratory for vesicular stomatitis as a gold standard by using VSV-positive equine sera and negative bovine sera vaccinated against foot-and-mouth disease (FMD) in the field. When naturally infected equine sera and FMDV vaccinated bovine sera were tested, MLI-ELISA and MLC-ELISA showed relative sensitivities of 80% and 95% with relative specificity of 97% and 99%, respectively. However, both ELISAs cross-reacted with equine sera against New Jersey (VSV-NJ) serotype. The comparison of the two ELISAs revealed that MLC-ELISA was relatively more sensitive and specific than MLI-ELISA, indicating that MLC-ELISA can be applied to sero-diagnosis for VSV-IN infection.     

A new competitive enzyme-linked immunosorbent assay demonstrates adequate immune levels to rabies virus in compulsorily vaccinated Japanese domestic dogs.

A competitive enzyme-linked immunosorbent assay (c-ELISA) was developed as an alternative to the viral neutralization (VN) test for rapid and simple detection of antibodies to rabies virus. The competitor antibody in the c-ELISA was a biotinylated monoclonal antibody to the nucleoprotein of rabies virus. Initial comparisons showed a high correlation between titers obtained with the VN test and the c-ELISA (n = 88, r = 0.90), indicating that the c-ELISA could be used as a reliable substitute for the VN test. To evaluate the immune status of Japanese dogs to rabies virus, a total of 1,019 serum samples were collected from domestic dogs in 1994 and tested for antibodies with the c-ELISA. Overall, 84.8% of the dogs had antibodies against rabies virus, indicating that the vaccination strategy for preventing rabies outbreaks in domestic dogs is probably sufficient in Japan. Dogs receiving final vaccinations a year or more previously were 48.3 and 90.3% positive for antibodies when vaccinated once only or two or more times, respectively. This suggests that almost all dogs vaccinated twice or more remain seropositive for over 1 year in Japan.     

Effectiveness of annual booster inoculations against various influenza serotypes and the procedure for mass vaccination

The epidemiological observation during an outbreak of A (H3N2) influenza in February-March, 1983, showed that the third annual vaccination with killed influenza vaccine did not enhance the effectiveness of vaccinations in the populations under study. It was observed that 14 months after immunization, 55.9% of the subjects examined had antibody titres of 1:40 or higher to the A/Bangkok/1/79 strain antigenically related to the vaccine strain, and 41% of the subjects of this group had antibodies to the subsequent drift variant of influenza A (H3N2) virus. These values were significantly higher than those in the group of subjects given no influenza vaccine. It is suggested that after 2 years of vaccination with killed influenza vaccines with the maximum coverage of the entire population, vaccinations be given alternately to half of previously vaccinated subjects with a 2-year interval up to the emergence of a new shift variant of influenza A virus, when again vaccination of the entire population for two successive years will be required.     

Validation of the specific isotype assay to detect antibodies against foot-and-mouth disease virus in bovine milk

The specific isotype assay (SIA) detects IgG1 against foot-and-mouth disease (FMD) virus in bovine milk. A strong correlation was demonstrated between milk antibody titres, and those in serum as measured by the liquid phase blocking ELISA. Thus the SIA would be useful on a herd basis to monitor the milk of vaccinated cattle to determine when re-immunisation is advisable. The SIA titration ELISA was then simplified to a single dilution test and optimised to differentiate the reactions in the milk of FMD-naive cows from those in animals which had been infected with FMD or vaccinated against the disease. For milk from immunised cattle, the pH of the sample was important and borderline positive specimens with a pH of 6.0 or below gave negative results. For milk from naive animals, the optical density (OD) registered in the SIA varied according to the time of year that samples were collected which, in turn, influenced the OD above which milks might be considered positive. Studies showed that the pH of milk could be maintained within the range suitable for the SIA by either storing for up to 1 week at 4 degrees C or by freezing at -20 degrees C for an indefinite period.     

An ELISA detecting antibody to conserved pestivirus epitopes.

A monoclonal antibody based competition-ELISA is described for the detection of pestivirus antibodies directed against conserved epitopes on the p80 viral protein. The ELISA detected increases in serum antibody following experimentally induced infections of pigs, cattle and sheep with a wide range of pestiviruses, although the sensitivity of the test was not uniform for the different viruses studied. The ELISA was compared with virus neutralization tests for the assessment of porcine, bovine and ovine field sera. At a cut-off value of 50% inhibition, the ELISA showed a high specificity relative to virus neutralization tests, but appeared less sensitive for the detection of some weakly positive samples from pigs. Sera from both ruminants and pigs could be assessed without any modification of the test.     

Chaperonin 10 of Mycobacterium tuberculosis induces a protective immune response to foot-and-mouth disease virus

Summary.  Chaperonin 10 of M. tuberculosis conferred partial or total protection against generalized foot-and-mouth disease (FMD) in guinea-pigs challenged with O₁ Lausanne FMD virus. Chaperonin 10-immunized animals mounted an antibody response to the protein, one epitope of which was found in the C-terminal half. A similar recognition pattern was observed in FMD-convalescent guinea-pigs, swine and cattle. Anti-chaperonin 10 sera showed antiviral activity against FMDV-infected BHK-21 cells. There was strong evidence that early after infection these cells actively secrete their histones and that antisera to the chaperonin recognize them. The same antisera reacted with purified histones in immunoblotting. Most important, exogenously added histones abrogated the anti-viral activity of the antiserum and an anti-histone monoclonal antibody had strong antiviral activity against FMDV-infected BHK-21 cells. These results are consistent with previous reports on displacement of histones from the nuclear compartment and immune recognition of self-histones after viral infections. On the whole, they * indicate that M. tuberculosis chaperonin 10 enables the immune system to react against early abnormalities of virus-infected cells; this is accomplished by antibody cross-reacting with histones released during virus infection. Received May 18, 1998 Accepted December 18, 1998     

Effect of vaccination on transmission characteristics of highly virulent Newcastle disease virus in experimentally infected chickens

An experimental study was conducted to evaluate the effect of vaccines produced in Ethiopia from vaccine strains used worldwide on the transmission characteristics of velogenic Newcastle disease virus field strain after different vaccination schemes. Chickens were vaccinated with Hitchner B1, La Sota or I-2 via the intraocular and intranasal routes. Vaccine and challenge viruses induced high antibody levels, both in inoculated and contact birds. Prime-boost vaccination protected birds against morbidity and mortality and significantly reduced the incidence of viral shedding from chickens compared with single vaccinated and unvaccinated birds. Protection from disease and mortality was correlated with the presence of positive antibody titres (>4 log₂) at day of challenge. Most of the unvaccinated and in-contact birds excreted the virus and showed a high level of antibody titres, indicating the high infectivity of the challenge virus. The detection of the challenge virus in most of vaccinated birds demonstrated that the tested vaccination protocols cannot fully protect birds from viral infection, replication and shedding, and vaccinated–infected birds can act as a source of infection for susceptible flocks. The high mortality observed in unvaccinated birds and their contacts confirmed the virulence of the challenge virus and indicated that this field virus strain can easily spread in an unvaccinated poultry population and cause major outbreaks. Progressive vaccinations supported by biosecurity measures should therefore be implemented to control the disease and introduction of the virus to the poultry farms.     

The Foot and Mouth Disease Virus Type O Outbreak of 1992 Is Not Related to Vaccine Strain (O/R2/75)

Vaccination is the only pragmatic approach to control foot and mouth disease in India. Strict quality control measures are essential to supply potent vaccine to the field application, in addition to monitoring the performance of the vaccine in the field. During the process of monitoring, an outbreak of FMD in vaccinated animals caused by type “O” virus in Tanjavur district of Tamil Nadu and a type “O” virus from unvaccinated herd of Karnataka were studied. Field isolates and vaccine virus were sequenced and analyzed. Data indicated that the virus from the outbreak in vaccinated cattle was a variant which could escape neutralization by antibodies against vaccine virus. This revised version was published online in August 2006 with corrections to the Cover Date.