Importance of cell mediated immunity for protection against Foot and Mouth Disease

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1 Appendix 34 Importance of cell mediated immunity for protection against Foot and Mouth Disease Yooni Oh 1*, ryan Charleston 1, David J. Paton 1, Jong-Hyun Park 2, Paul V. arnett 1, Yi-Seok Joo 2, Satya Parida 1 1 Pirbright Laboratory, Institute for Animal Health, Ash Road, Surrey, GU24 NF, UK. 2 FMD Research Laboratory, FADRD, NVRQS, Republic of Korea Abstract: Introduction: The humoral antibody titre is not always fully predictive of vaccine-induced protection against FMD. Therefore, this study has looked for a correlation between cell mediated immune responses and post-vaccination protection against FMDV infection. Another aim has been to evaluate the use of the FMDV whole blood IFN-γ assay to differentiate between vaccination and infection. Materials and Methods: Samples were collected from two vaccine potency challenge experiments (A May 97 and SAT 2) conducted at Pirbright, UK, according to the European Pharmacopoeia. Using a modified version of the OVIGAM assay for tuberculosis, whole blood samples from FMDV vaccinated, non-vaccinated and vaccinated-and-challenged cattle were re-stimulated overnight with inactivated FMDV antigen and the level of induced IFN-γ was measured. PMC from these animals were also used in ELIspot and cell proliferation assays. Humoral antibody levels were measured by virus neutralization test. Results: All 15 vaccinated cattle were clinically protected in the A May 97 study, whereas 4 out of 15 vaccinated cattle in the SAT 2 experiment developed generalized infection, even though similar levels of neutralizing antibody were detected in cattle from both experiments on the day of challenge. However, IFN-γ production in the re stimulated whole blood samples was much stronger in the A May 97 study than in the SAT 2 study both before and after challenge. A positive correlation was found between IFN-γ response and protection against the clinical disease. Therefore not only humoral antibody but also cell mediated immune responses might help to predict clinical protection from FMDV in vaccinated cattle and might contribute to a method for evaluation of vaccine efficacy without challenge. Introduction: Foot-and-mouth disease (FMD) affects cloven hoofed domestic and wildlife species, reducing productivity and severely constraining trade in animals and their products. Vaccination using inactivated and adjuvanted virions is an important method of FMD. Measuring the efficacy of FMD vaccines relies on either direct potency tests by challenge of vaccinated cattle or indirect assessment based on the serological response induced by vaccination. However, serological tests are not an absolutely reliable indicator of protection. Though immune defence against FMD has been related to the antibody mediated compartment and humoral antibody responses have been considered to be the most important factor in conferring protection against FMD (McCullough & Sobrino, 24), there are still instances where animals with medium or high neutralizing antibody titre are not protected upon challenge with virus (McCullough et al., 1992). Similarly, animals with low or no detectable virus neutralizing antibody on occasion do not succumb to disease (Sobrino et al., 21). However, there is limited knowledge on the role of cell mediated responses which might play a role against protection. As lymphocytes are responsible for humoral antibody production, but are dependant on the interaction with the Th lymphocytes (McCullough & Sobrino, 24), both humoral as well as cell mediated immunity are considered very important for protection. Recently we developed a whole blood IFN-γ assay and reported the existence of a correlation between IFN-γ and FMDV persistence (Parida et al., 26). Therefore, this present study looked at the correlation of both IFN-γ and neutralizing antibody with vaccine induced protection. Materials and Methods: Two vaccine potency experiments (A May 97 and SAT2) were carried out according to European Pharmacopoeia. 15 animals in each experiment, 5 in each group, were vaccinated with different doses of vaccine and challenged by homologous virus on 21 days post vaccination. Two more animals were included as unvaccinated s for each experiment. eside the 8 day monitoring 23

2 period post challenge, all the experimental animals were also monitored a further 22 days post challenge to identify the carrier status. Clotted blood samples for serology, heparinized blood for cell mediated immune responses, such as whole blood IFN-γ re-stimulation assay, ELIspot, lymphocyte proliferation assay, FACS and T cell depletion assay, and probang samples for virus isolation and real time PCR were collected at different time points during pre- and post-challenge period until the end of the studies. Results: Clinical signs: Upon challenge with homologous virus, all the unvaccinated animals in both the experiments were infected and showed clinical lesions in all four feet and tongue as expected. All vaccinates in A May 97 potency test were clinically protected and vaccine passed with a PD 5 value 32, whereas three vaccinates from the 1/4 th dose group ( 87, 88 and 89) and one from the 1/16 th dose group (91) of the SAT2 potency experiment showed clinical lesions though the vaccine passed with a PD 5 value of 1. Virus isolation and real time RT-PCR : Live virus as well as genome copy was recovered from the oro-pharyngeal samples of all the four non-vaccinated animals of both the experiments. Out of these four, two from SAT 2 infected animals became carriers whereas virus could not be recovered after 28 days post challenge in A-May 97 infected animals. Though all vaccinates were clinically protected in the A May 97 experiment, live virus was isolated from two animals in the full dose vaccine group, three animals in the 1/4 dose group and from all five animals in the 1/16 dose group (Table 1). In contrast, live virus was isolated from all 15 vaccinates in the SAT 2 experiment (Table 2). Out of a total of 1 sub-clinically infected animals detected in the A May 97 experiment by virus isolation and PCR, one animal, each from the full dose and 1/4 dose group, and three animals from the 1/16 dose group became carriers (Table 1). Similarly, out of all 15 SAT2 vaccinates two from each of the full dose and 1/4 th dose group and four from the 1/16 th dose group were detected as carriers (Table 2). Detection of neutralising antibody: Mean virus neutralization antibody titre for each vaccinated group from both experiments was very similar (data not shown) before and after challenge. A vaccine dose dependent VN titre was observed pre and post challenge in the SAT 2 experiment. In the A May 97 experiment though a dose dependant response was seen in the pre-challenge period, and after infection the 1/16 dose group animals revealed much higher VN titres than the other two higher dose groups. However, in both experiments a minimum 1 times rise of VN antibody response was observed in all groups after infection. The virus neutralizing antibody responses on the day of challenge (21 days post vaccination) are shown in Figure 1 for both experiments. On the day of challenge, mean VN titre of the different vaccine dose groups of A May 97 experiment were compared with the corresponding groups of the Sat 2 experiment and no significant difference was observed (P>.593,.589 and.84 for full, 1/4 th, and 1/16 dose group respectively). However, when the mean titre VN value of different vaccine dose groups were compared within the experiment, a high statistical significance was observed. Whole blood IFN-γ re-stimulation assay: For the whole blood IFN-γ re-stimulation assay, heparinized blood samples from all the cattle of both experiments were collected at various time points and induced with inactivated vaccine antigen and positive/negative s were also included. The IFN-γ level was measured by ELISA with known standards and the results are presented in Figure 2. Within seven days of vaccination a considerable amount of IFN-γ production was observed (Fig. 2A) in all three vaccination groups in the A May 97 experiment whereas IFN-γ production was not obvious in the SAT 2 experiment (Fig. 2). Only in the full dose vaccinated animals of the SAT 2 experiment was an IFN-γ response apparent on the 2 nd week of vaccination whereas the other two groups only produced an IFN-γ response on or after the day of challenge (Fig. 2). Despite individual animal variation, analysis of the group mean data did not show a correlation between vaccine dose and IFN-γ production post-vaccination in the A May 97 experiment (Fig. 2A). In contrast, and throughout the SAT 2 experiment, a higher amount of IFN-γ response was observed in the full dose vaccine group than the other two groups. Further, this vaccine dose dependent IFN-γ response was not found when compared with the 1/4 th and 1/16 th dose groups of the SAT 2 experiment, and more IFN-γ response was observed in 1/16 th dose group than the 1/4 th dose group (Fig. 2). However, a significant difference (P<.5) in the IFN-γ response was not observed in-between all the 3 vaccination groups of the SAT 2 experiment at any of the time points for sample analysis was observed in the A May 97 trial. Unlike the A May 97 experiment, a rise of IFN-γ response was observed upon challenge, particularly on 1/4 th and 1/16 th dose groups. 231

3 Throughout the post challenge period in both experiments was the IFN-γ level significantly higher in the vaccinated animals compared to the group animals. A high IFN-γ response was detected after re-stimulation with PWM as a positive in all the animals whereas stimulation with PPDA as a negative and non-stimulated blood, as a baseline, induced no IFN-γ response throughout the experiments (data not shown). VNT titre A VNT titre full dose 1/4 dose 1/16 dose full dose 1/4 dose 1/16 dose Figure1. Virus neutralizing antibody titre in the serum of A May 97 (A) and SAT2 () cattle on the day of challenge. Full dose ( ), 1/4 dose ( ), 1/16 dose ( ) and ( ). A full dose 1/4 dose 1/16 dose full dose 1/4 dose 1/16 dose dpv 7dpv 14dpv 21dpv/dpc 1dpc 19dpc 31dpc days post vaccination/challenge dpv 14dpv 21dpv/dpc 1dpc 13dpc days post vaccination/challenge Figure2. Comparisons of group mean value of IFN-γ response in the whole blood re-stimulation assay for A May 97 (A) and SAT2 () cattle. Full dose ( ), 1/4 dose ( ), 1/16 dose ( ) and ( ). 19dpc 31dpc ELISpot assay: For the ELISpot assay, PMC were separated from all the animals in both experiments at 3 time points (19 th, 27 th and 31 st day post challenge) for A May 97 and 5 time points (14 th day post vaccination, 8 th, 13 th, 2 th and 31 st day post challenge) for the SAT 2 experiment. PMC were re-stimulated with specific vaccine antigen in vitro and IFN-γ producing cell spots were assessed by ELISpot. IFN-γ production was found to be vaccine dose dependant (Fig. 3A) for the A May 97 experiment though there was no significant difference (P>.5) between the different vaccine groups and the non-vaccinated group (ANOVA). In contrast, this dose dependant IFN-γ production was not observed in the SAT 2 experiment (Fig. 3). However, a significant difference (P<.5) of IFN-γ producing cell spots were observed between the vaccinated and non vaccinated groups apart from the 14 th day post vaccination (ANOVA). Since ELISpot is a well established assay to detect IFN-γ production, it was compared with the whole blood IFN-γ assay in both experiments, particularly between different vaccine doses and the animals. The mean value of ELISpot and whole blood IFN-γ ELISA for the 3 time points in the A May 97 (Fig. 4A) and the 5 time points in the SAT 2 trial (Fig. 4) were calculated for each vaccine dose group. These results from ELISpot assay (bar) were compared to IFN-γ ELISA results (line) and found to be comparable (Fig. 4). 232

4 A 35 3 full dose 1/4 dose 1/16 dose 35 3 full dose 1/4 dose 1/16 dose Spot number 2 15 Spot number dpc 27dpc 31dpc days post challenge 14dpv 1dpc 13dpc 19dpc 31dpc days post vaccination/challenge Figure3 Comparison of mean value IFN-γ responses in ELISpot assay in different vaccine dose groups following vaccination and subsequent challenge in A May 97 (A) and SAT2 () animals. Full dose ( ), 1/4 dose ( ), 1/16 dose ( ) and ( ). A 35 3 ELISPOT ELISA ELISPOT ELISA Spot number Spot number full dose 1/4 dose 1/16 dose Group full dose 1/4 dose 1/16 dose Figure4. Comparison of IFN-γ response in ELISpot assay (bar) and whole blood re-stimulation assay (line) in A May 97 (A) and SAT2 () animal. Lymphocyte proliferation responses: Lymphocyte proliferation assay was carried out only in full dose group and non-vaccinated group in both the potency experiments after re-stimulating PMC with vaccine antigen and the results were plotted in Figure 5. On the 7 th day post vaccination it was possible to differentiate the proliferative responses between vaccinates and non-vaccinates of A May 97 animals whereas it was only possible after 2 nd week of vaccination in the case of the SAT 2 animals. However, a significant difference (P<.5) in proliferative response between the vaccinated and non-vaccinated groups was only observed after the 14 th day post vaccination (2- Sample T-test) in the case of the A May 97 experiment and at the 21 st day post vaccination in case of the SAT2 experiment respectively. Interestingly, as in the whole blood IFN-γ assay, a significant difference (P<.5) in the proliferative response was observed between the full dose groups of both experiments starting from the first week post vaccination (2-sample t-test) apart from the 19 th and 28 th day post challenge. Group 233

5 3 H TdR Incorporation 4x1 3 Full dose; A May 97 Control; A May 97 Full dose; SAT2 Control; SAT2 3x1 3 2x1 3 1x1 3 dpv 7dpv 14dpv 21dpv/dpc 1dpc 19dpc 28dpc days post vaccination/challenge Figure5. Comparison of lymphocyte proliferation response in A May 97 and SAT2 full dose and group animals after re-stimulation with FMD vaccine antigen and incorporation of 3 H thymidine. Full dose; A May 97 ( ), ; A May 97 ( ), Full dose; SAT2 ( ) and ; SAT2( ) Correlation of clinical protection with IFN-γ and VN antibody responses on the day of challenge : On the day of challenge, all the A May 97 vaccinated animals showed a considerable high IFN-γ response from the whole blood assay whereas only three of the full dose and one of the 1/16 th dose group of SAT2 animals conferred high IFN-γ response though significantly lower than A May 97 animals. All the 4 clinically unprotected animals in SAT2 experiment did not produce any, or very little IFN-γ on the day of challenge. However, the level of humoral antibody was not significantly different between the same vaccine dose treatment groups in the A May 97 and SAT 2 experiments. On the day of challenge, out of four infected animals (87, 88, 89 and 91), only 88 and 91 had low VN antibody titres, whereas the other two had high levels of VN antibody titres in comparison to other protected animals in the same experiment. To establish a correlation between clinical protection, IFN-γ and VN antibody responses on the day of challenge, all 3 vaccinated animals from both experiments were re-grouped into clinically unprotected, clinically protected, carrier and non-carrier, and their mean group value of IFN-γ and virus neutralization antibody titre were also compared (Fig. 6). Out of a total of 4 clinically unprotected animals, three became carriers whereas one did not. Out of 26 clinically protected animals, 1 became carriers whereas 16 were non-carriers. Therefore a total of 13 carriers and 17 non carriers were confirmed. A significant difference (P=.1) in the level of both IFN-γ and VN antibody was observed between clinically unprotected and clinically protected groups (Fig. 6A). The IFN-γ response was found to be significantly different (P=.17) when compared between the total carrier (n=13) and non-carrier (n=17) animals whereas for the VN antibody titre was not significant (P=.29) between these two groups. However, no significant difference was observed for the IFN-γ response and VN titre when compared between carrier and non carrier animals in the clinically protected and clinically unprotected animals separately (data not shown). 234

6 A 4 IFN-γ VNT IFN-γ VNT VNT titre 2 6 VNT titre clinically unprotected clinically protected carrier non-carrier Figure6. Comparison of IFN-γ (bar) and VNT (line) responses in-between 3 vaccinated animals of A May 97 and SAT2 on the day of challenge between clinically unprotected and clinically protected animals (A) and carrier animals and non-carrier animals (). Identification of IFN-γ producing T cell: Various T cell populations were depleted from PMC using each T cell surface marker to identify which cells are responsible for the IFN-γ production. IFN-γ production was measured before and after depletion of each T cell population. IFN-γ production was drastically decreased in the CD4 depleted population compared to whole PMC (Fig. 7A). Similarly γδ T cell (WC1-) depletion reduced a considerable amount of IFN-γ production (Fig. 7). However, depletion of CD8 and NK cells (difference between CD3 and CD2) resulted in very little IFN-γ reduction than observed with CD4 and γδ T cells (Fig. 7C and 7D). The purity of depleted cells was checked by FACS analysis which showed more than 95% purity. A IFNg (ng/ml) IFNg (ng/ml) PMC CD4- CD4/CD8- CD4/CD8/CD2- CD4/CD8/CD2/ WC1- PMC WC1- WC1/CD8- WC1/CD8/CD4- C D IFNg (ng/ml) PMC CD8- CD8/CD4- CD8/CD4/CD2- CD8/CD4/CD2/ WC1- IFNg (ng/ml) PMC CD3- PMC CD2- Figure7. IFN-γ production after re-stimulation of total PMC and depleted PMC. Discussion: The whole blood re-stimulation assay was developed for the diagnosis of tuberculosis as an alternative to the tuberculin skin test (Wood and Jones, 21). This assay measures IFN-γ production by ELISA after overnight exposure of heparinized whole blood to either M. bovis specific antigen or M. avium as a comparison. The whole blood IFN-γ assay is used as a measure of T- helper-1 activation of CD4+ and CD8+ T cells and can be readily used on a large number of samples (lack et al., 22). Parida et al, 26 showed this assay with some modifications could be used to differentiate between A Iran 96 vaccinated and vaccinated homologous challenged animals. Further they reported the existence of a correlation between IFN-γ production and the establishment of carrier status in cattle. These initial results prompted us to firstly, develop an easy 235

7 assay to correlate the cell-mediated immune response and protection and secondly, to develop an assay which could be used as a diagnostic assay for differentiation of subsequent FMDV infection in vaccinated animals. In this study, blood samples were taken from two vaccine potency tests and analysed by the whole blood IFN-γ assay to measure the cell mediated immune response in FMD vaccinated and infected animals. The IFN-γ response was higher in vaccinated animals in comparison to non-vaccinated animals throughout the period of study which confirmed earlier findings (Parida et al., 26). The quantity of IFN-γ production was found to be further elevated after challenge. This IFNγ production may be triggered by vaccination and then boosted by infection. Therefore, this whole blood assay may have potential as a supporting assay to the established serological assays in confirming infection in a vaccinated herd. However, in the present A May 97 potency study, an IFNγ response was observed at a high level by 7 days post vaccination and remained constant throughout the experiment, even after challenge, which makes it difficult in its use to differentiate between vaccinated and vaccinated subsequently challenged animals. Similarly, in the SAT 2 experiment it was difficult to differentiate the vaccinated animals before and after challenge as there was not much difference in IFN-γ response on the day of challenge and post challenge. However, in all the animal experiments the non-vaccinated infected animals produced less IFN-γ, which might be due to an immunosuppressive effect by the FMDV (Parida et al., 26). To evaluate the newly developed whole blood IFN-γ assay, the ELISA result was compared to the well established ELISpot assay. The IFN-γ responses from both assays are very comparable in both experiments. However, the whole blood IFN-γ assay is easier, less time consuming and more feasible for large scale surveillance. The lymphocyte proliferation assay was conducted on the full dose vaccine group and nonvaccinated s in both potency experiments after re-stimulating the PMC with FMD specific vaccine antigen. As in the whole blood IFN-γ assay, a significant difference (P<.5) in the proliferative response was observed between the full dose groups in both experiments as from the first week post vaccination. On the day of challenge, there was no considerable difference in the neutralizing antibody titre of the different groups from both the experiments. However, four out of 15 cattle were clinically unprotected in the SAT 2 potency test whereas all the animals in A Malaysia 97 experiment were clinically protected. Apart from the neutralizing antibody titre, the cell mediated immune responses detected by many of the assays (whole blood IFN-γ assay, ELISpot assay and Lympho proliferation assay) were considerably higher for the A May 97 vaccinated animals compared to the SAT 2 animals. This may indirectly be the reason for all the animals in A May 97 being clinically protected. Though it is well established that more antigen pay load is required in vaccine formulations of other serotypes than the A serotype (personal communication with Tim Doel, Merial, Pirbright), the SAT2 animals were nevertheless not protected fully. Another reason for the poorer protection results may be the instability of the SAT 2 antigen following immunization in the target host. Though humoral antibody response is very important factor in conferring protection against FMD infection (McCullough & Sobrino, 24), still animals with high or medium neutralizing antibody titre were not clinically protected (McCullough et al., 1992). Our study supports the fact that though similar level of VN titre was observed for both the experiments, four of the SAT 2 animals were infected which showed none or very little IFN-γ response. Further this has been supported by the fact that the clinically protected group of animals had high IFN-γ and VN antibody titre than the clinically unprotected animals. Thus not only humoral antibody but also cell-mediated immune response plays a key role in FMD vaccine induced protection. y measuring the mean value of IFN-γ production on the day of challenge in the A May 97 and SAT 2 animals, a significant difference was observed between carrier and non-carrier animals as was seen in A Iran 96 experiment (Parida et al, 26) though such a difference was not observed for the VN titres. However, when a comparison had been made between carrier and non-carrier animals separately within the clinically protected and clinically unprotected, a significant difference in IFN-γ response was not observed though always a high amount of IFN-γ level was seen in non carrier animals than the carrier animals. Different doses of vaccine (full dose, 1/4 th and 1/16 th dose) were used in both animal experiments conducted. A correlation between the vaccine dose and clinical protection was demonstrated in this study which corroborates well with the findings of Cox et al. (25). Animals from 1/16 th dose group and from 1/4 th dose group of SAT2 experiment were not protected from the development of lesions. However, all the animals from the full dose group of SAT 2 study and all the 15 vaccinates 236

8 from A May 97 experiment were fully protected. Out of a total of 13 carriers observed in both SAT 2 and A May 97 experiments, three were from the full dose group, three from 1/4 th dose group and the remaining 7 from the 1owest dose (1/16 th ) group. Further, during the pre-challenge and post challenge period a dose dependant IFN-γ response was observed in the SAT 2 animals where the full dose group produced more IFN-γ than the other two groups. Some times 1/16 th dose group animals produced more and/or same amount of IFN-γ as the 1/16 th dose group. This may be due to the difficulty of measurement of accurate volume of 1/16 th dose of vaccine which used to be carried out according to the European Pharmacopoeia. A correlation between IFN-γ production post vaccination and protection has also been reported by Parida et al. (26). The present study mainly concentrates on Th1 response (IFN-γ). However it might be useful to check the other FMD specific cytokine profile for both Th1 and Th2 responses as suggested by previous researchers (arnard et al., 25 and Eble et al., 26). Conclusions: A positive correlation of IFN-γ and VN antibody responses with vaccine induced protection has been observed. The IFN-γ re-stimulation assay may be useful for vaccine potency evaluation. The use of IFN-γ re-stimulation assay as a DIVA test for FMD may not be possible when high potency vaccines are used. Recommendations: T cell stimulation assays such as the whole blood IFN-γ assay along with VNT are potential candidates for vaccine evaluation and could reduce the need for in vivo challenge in the future. Acknowledgements: We would like to acknowledge NVRQS, Korea and Defra, UK for the funding through PhD165 and SE1122 research grants respectively. References: arnard, A. L., Arriens, A., Cox, S., arnett, P., Kristensen,., Summerfield, A. & McCullough, K. C. 25. Immune response characteristics following emergency vaccination of pigs against foot-and-mouth disease. Vaccine. 23, lack, G. F., Weir, R. E., Floyd, S., liss, L., Warndorff, D. K., Crampin, A. C., Ngwira,., Sichali, L., Nazareth,., lackwell, J. M., ranson, K., Chaguluka, S. D., Donovan, L., Jarman, E., King, E., Fine, P. E. & Dockrell, H. M. 22. CG-induced increase in interferongamma response to mycobacterial antigens and efficacy of CG vaccination in Malawi and the UK: two randomised led studies. Lancet, 359, Cox, S. J., Voyce, C., Parida, S., Reid, S. M., Hamblin, P. A., Paton, D. J. & arnett, P. V. 25. Protection against direct-contact challenge following emergency FMD vaccination of cattle and the effect on virus excretion from the oropharynx. Vaccine, 23, Eble, P. L., de ruin, M. G., ouma, A., van Hemert-Kluitenberg, F. & Dekker, A. 26. Comparison of immune responses after intra-typic heterologous and homologous vaccination against foot-and-mouth disease virus infection in pigs. Vaccine 24, Hohlich,. J., Wiesmuller, K. H., Haas,., Gerner, W., Correa, R., Hehnen, H. R., Schlapp, T., Pfaff, E. & Saalmuller, A. 23. Induction of an antigen-specific immune response and partial protection of cattle against challenge infection with foot-and-mouth disease virus (FMDV) after lipopeptide vaccination with FMDV-specific -cell epitopes. J Gen Virol, 84, McCullough, K., De Simone, F., rocchi, E., Capucci, L., Crowther, J. R. & Kihm, U Protective immune response against foot-and-mouth disease. J Virol, 66, McCullough, K. & Sobrino, F. 24. Immunology of Foot-and-Mouth Disease. In Foot-and-Mouth Disease, pp

9 Parida, S., Oh, Y., Reid, S. M., Cox, S. J., Statham, R. J., Mahapatra, M., Anderson, J., arnett, P. V., Charleston,. & Paton, D. J. 26. Interferon-gamma production in vitro from whole blood of foot-and-mouth disease virus (FMDV) vaccinated and infected cattle after incubation with inactivated FMDV. Vaccine, 24, Sobrino, F., Saiz, M., Jimenez-Clavero, M. A., Nunez, J. I., Rosas, M. F., aranowski, E. & Ley, V. 21. Foot-and-mouth disease virus: a long known virus, but a current threat. Vet Res, 32, 1-3. Wood, P.R.& Jones, S. L. 21. OVIGAM: an in vitro cellular diagnostic test for bovine tuberculosis. Tuberculosis (Edinb), 81,

10 Table1. Virus isolation and real time RT-PCR results for A May 97 potency test C/S 1dpc 12dpc 19dpc 28dpc 31dpc * + * - - full dose /4th dose * * +* * * * - 1/16th dose * +* +* 75 - * - +* +* * C/S refers to clinical sign generalized animals, + indicates virus isolation positive, * indicates PCR positive and indicates virus isolation and PCR negative. Table2. Virus isolation result for SAT2 potency test C/S 8dpc 13dpc 16dpc 2dpc 23dpc 27dpc 3dpc 34dpc full dose NS /4th dose /16th dose C/S refers to clinical sign generalized animals, NS refers to no sample, + indicates virus isolation positive and indicates virus isolation negative. 239