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Evidence Project Final Report

EVID4 Evidence Project Final Report (Rev. 06/11) Page 1 of 21

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Project identification

1. Defra Project code SE1125

2. Project title

Development and validation of confirmatory tests which identify FMD infection in vaccinated animals

3. Contractororganisation(s)

Institute for Animal HealthCompton LaboratoryComptonNewburyBerkshireRG20 7NN

54. Total Defra project costs £ 823,848(agreed fixed price)

5. Project: start date................ 01.01.2009

end date................. 30.06.2012


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Executive Summary7. The executive summary must not exceed 2 sides in total of A4 and should be understandable to the

intelligent non-scientist. It should cover the main objectives, methods and findings of the research, together with any other significant events and options for new work.

Vaccination can be a very effective measure to control the spread of foot-and-mouth disease (FMD) and to reduce reliance on preventive culling. Though cattle are the main target species for emergency vaccination in UK, vaccinating sheep may be required in certain circumstances. In either case, vaccination may mask infection without completely preventing it and this leads to (i) a requirement for post-vaccination testing to demonstrate freedom from infection, and (ii) a prolonged waiting period before the FMD-free status can be regained for international trade purposes. Therefore, under a "vaccinate-to-live" policy, it is intended that emergency-vaccinated animals should be subsequently tested to check that they had not been sub-clinically infected with FMD virus (FMDV) and then kept alive and free of restrictions other than that they could not be exported as live animals. Purified vaccines mainly stimulate antibodies against the structural proteins (SP) of the FMDV. In contrast, infection with live FMDV elicits antibodies to both structural and non-structural proteins (NSP). Therefore, NSP antibody tests can be used to differentiate infection in vaccinated animals (DIVA). In cattle, the best NSP tests (targeting the NSP 3ABC) can detect up to 90% of vaccinated animals that become carriers after exposure to infection, with a specificity of 98-99% (Brocchi et al., 2006). Due to insufficient sensitivity and specificity, detection of a low level of infection is difficult at the population level with a high degree of confidence (Paton et al., 2006). The low level non-1. Validation of NSP tests in small ruminants revealed that the Prionics NSP test is the best and can detect infection in vaccinated as well as in unvaccinated animals. As for cattle, the level of detection of infection in vaccinated flocks is comparatively less than for unvaccinated animals. Additionally, the Prionics NSP and the in-house Brescia 3ABC NSP tests were validated for Asian buffalo samples for the first time revealing similar sensitivity and specificity. Therefore, validations of 3ABC NSP antibody tests (3 commercial assays and one in-house assay) have been completed for cattle, buffalo and sheep and goats for detection of infection in vaccinated populations. Recently, we have validated two commercially available 3D NSP antibody tests using samples from 80 O1 Manisa vaccinated and O1 UKG challenged cattle. One of these assays showed a similar sensitivity to the Prionics 3ABC NSP test, although some animals showed high absorbance values during the post-vaccination period prior to challenge (i.e. non-specificity). 2. A 2B test that we had previously developed is being taken forward as a confirmatory test for the NSP 3ABC test. The test is very sensitive, but showed previously some non-specific detection of infection. We are now able to reduce the non-specificity of the test by introducing horse sera as a blocking reagent in each step of ELISA. Further, we have compared this 2B test with 3ABC Prionics tests for the detection of infection in unvaccinated infected and vaccinated sub-clinically infected cattle. We showed that the 2B test has a similar level of sensitivity and specificity and therefore could be used either as a screening or confirmatory test. This 2B test now needs to be validated for small ruminants. In addition, we have recently


developed a 3B peptide test which detects infection in vaccinated populations with higher sensitivity than 2B and 3ABC tests. However, this test needs further optimisation and validation. Furthermore, we have developed 3D, 3CD, 2C and 3ABC in-house recombinant indirect ELISAs. These ELISAs have been evaluated for a large number of samples from unvaccinated infected and vaccinated sub-clinically infected animals. However, the sensitivity of detection of infection by these in-house assays is lower than the 2B, 3B and 3ABC prionics assays. These difficulties may be overcome by simultaneously measuring the antibody response to a collection of different NSPs rather than a single NSP as is used in existing tests. As a proof of principle we had already shown the establishment of a multiplex assay using peptides (Perkins et al., 2007). Further in this project, utilising the recombinant antigens (3ABC, 3CD and 3D) available in our lab, we recently developed a multiplex ELISA to detect infection in vaccinated populations. The results are promising, but the tests need additional optimisation and validation with a larger number of samples along with evaluation of other recombinant antigens (2C) and peptides (2B and 3B).

3. As reported in annual reports, validation of our IgA test for serotype O using inactivated antigen has been completed and now validation has been extended to serotype A, by analysing samples that originated from 4 experimental as well as field vaccinated-and-infected cattle. Recently, we have obtained saliva samples from FLI Germany, originated from 7 serotype A vaccine challenge experiments in cattle. Samples from two animal experiments from Germany have already been analysed. The method showed sufficient sensitivity and specificity to detect FMDV carriers in vaccinated-and-infected cattle herds and therefore can be used either as an independent screening test or as a confirmatory test to NSP serology. Furthermore, our extensive analyses have illustrated for the first time an important qualitative difference between NSP serology and IgA tests on mucosal secretions. Whereas NSP serology detects past infection irrespective of whether there is virus persistence or not, IgA testing scores animals positive only if there has been prolonged and ongoing virus infection, and this makes the test the only antibody-based method for specific detection of carriers. Alternative virological tests (virus isolation and PCR) for carrier detection are both more cumbersome and much less sensitive (unless animals are sampled repeatedly). The IgA test therefore has important potential to determine the significance of findings of post vaccination sero-reactor animals. Further, we have extended the development of IgA assay to serotype Asia1 by analysing samples that originated from one experimental as well as field infected cattle. The assay detects the known carrier animals as in O and A serotypes, but the test needs to be optimised and validate further.

4. The current tests to detect FMDV-specific IgA antibodies rely on the use of structural protein (SP) antigens prepared from inactivated virus. In order to facilitate export of these tests to other laboratories it would be desirable to switch to the use of a recombinant antigen based on non-infectious empty viral capsids. Tests in this format reduce bio-security concerns since they are not reliant upon material derived from “live” FMDV. We have already developed such a test for serotype O and the assay revealed a similar sensitivity and specificity for the detection of FMDV carrier animals. At the moment, our work is focused on the replacement of the antigen for the serotype A test. We also plan to use a similar approach for the Asia 1 serotype test. We are able to express FMDV empty capsids for O, A and Asia 1 serotypes of FMDV in a Sendai virus vector expression system using reverse genetics approach.

5. We have developed an IgM assay and validated the test with 1000 negative sera each from cattle, sheep and pigs, 2000 sera from vaccinated and challenged cattle, sheep and pigs, a few thousand sera from the 2001 and 2007 outbreaks in the UK and 500 sheep sera from the 2007 Cyprus outbreaks. The kinetics of IgM development and persistence in animals with different status with respect to vaccination, infection and viral elimination and persistence were studied. Since high levels of IgM antibody do not seem to persist in vaccinated animals after 2-3 weeks of vaccination, its presence in animals known to have been vaccinated some time ago may be an indicator of infection and the assay may have DIVA potential. The assay is particularly helpful in sheep populations to detect infection where the clinical signs are not obvious.

6. Based on the NSP and IgA validation data collected in this and the previous projects, we have selected the best combination of tests to use for cattle to substantiate freedom from FMDV infection. We have calculated the sensitivity and specificity of the tests for detection of virus circulation and virus persistence (carriers). Finally, we have used mathematical modelling/ statistical approaches to identify the level of virus circulation or persistence with the chosen testing strategy. Acquisition of post-emergency-vacination fied data would have allowed for the development of more powerful models capable of determining the specific criteria any testing regime would need to satisfy in order to prove freedom from virus circulation or persistence with confidence, but such opportunity has not arisen during the tenure of this project.

Concluding summary:

1. Mucosal IgA test exclusively detects FMDV carrier cattle in vaccinated and subsequently infected as well as in unvaccinated infected populations and this test could be taken forward as an independent


screening test to detect the FMDV carrier animals or as a confirmatory test to the 3ABC Prionics test.

2. Prionics 3ABC NSP test should be considered as the test of choice in cattle and small ruminants for the detection of FMDV infection/virus circulation in vaccinated as well as unvaccinated infected populations However this test does not exclusively detect the FMDV carrier animals as it also detects recovered, non-carrier animals. A combination of 3ABC and IgA test increased the sensitivity of detection of infection.

3. We have shown our 2B test could be taken forward as a confirmatory test to 3ABC NSP test to detect infection in vaccinated and unvaccinated infected populations. 3B peptide test has potential to detect more infected animals in vaccinated population which needs to be further investigated.

4. Our IgM test has potential to identify early infection in infected populations, particularly when the clinical lesions are not clearly evident, e.g. in sheep flocks.

5. To overcome the low sensitivity of existing NSP antibody assays, we have developed the multiplex assay for the simultaneous measurement of the antibody response to a collection of different NSPs rather than a single NSP. The results are promising, but the tests need to be optimised and validated further.

6. Further development and validation of the IgA assay for Asia1 and SAT2 viruses are required as Asia1 and SAT2 viruses are circulating in Turkey and Egypt (Northern Africa), respectively. To reduce the bio-security concerns, replacement of FMDV inactivated antigen with non-infectious empty capsids in IgA assay would be beneficial.

Project Report to Defra8. As a guide this report should be no longer than 20 sides of A4. This report is to provide Defra with details of

the outputs of the research project for internal purposes; to meet the terms of the contract; and to allow Defra to publish details of the outputs to meet Environmental Information Regulation or Freedom of Information obligations. This short report to Defra does not preclude contractors from also seeking to publish a full, formal scientific report/paper in an appropriate scientific or other journal/publication. Indeed, Defra actively encourages such publications as part of the contract terms. The report to Defra should include: the objectives as set out in the contract; the extent to which the objectives set out in the contract have been met; details of methods used and the results obtained, including statistical analysis (if appropriate); a discussion of the results and their reliability; the main implications of the findings; possible future work; and any action resulting from the research (e.g. IP, Knowledge Exchange).

1a. Validation of NSP tests for small ruminants:

We have previously validated 3 commercial NSP tests (Prionics, Bommeli and UBI) in cattle showing up to a 90% sensitivity and 98-99% specificity for detection of infection in a vaccinated population (Brocchi et al., 2006).However, we could not validate these tests for samples originating from vaccinated and subsequently infected sheep and goats due to a lack of samples. As we reported earlier, we have collected more than 450 serum samples from vaccinated and subsequently infected (n=296) and unvaccinated and infected (n=157) sheep and goat flocks to validate the above-mentioned commercially available NSP tests. A Bayesian framework has now been used to make inferences about the prevalence of infection in each sub-population and the diagnostic characteristics for each test. The prevalence should be considered as true prevalence, since it has been calculated from both negative and positive populations. The predictive positive value (PPV), predictive negative value (PNV), positive likelihood ratio (LR+), and negative likelihood ratio(LR-) are calculated based on the Se, Sp, and prevalence from the survey data.

In the vaccinated and subsequently infected population (Table 1 and Figure 1), the sensitivity of detection of infection for PrioCHECK (Cedi), Chekit (Bommeli) and UBI are calculated as 47%, 32% and 12%, respectively. There is a low negative predictive value due to the low Se, so some results are classified as false negative. This affects largely the Chekit and UBI performance. The worst case found was for UBI that is not able to correctly classify almost the whole positive case (maximum 20% of prevalence), meaning that 80% of the cases are mis-classified as false negative.


Table.1 and Figure 1: Sheep-Goats Vaccinated/Infected strata (Bayesian posterior estimate. In Figure 1, PrioCHECK, solid black; Chekit, hashed green; UBI, hashed red).

Ceditest® Chekit UBI® Se [95% BPI] 0.478 [0.445 – 0.487] 0.329 [0.134 – 0.536] 0.124 [0.048 – 0.203] Sp [95% BPI] 0.907 [0.900 – 0.933] 0.854 [0.689 – 0.969] 0.942 [0.887 – 0.982]

Prevalence [95% BPI] 0.433 [0.392 – 0.443] PPV [95% BPI] 79.7% [75.1% - 84.3%] 63.2% [57.7% - 68.7%] 62.0% [56.5% - 67.5%] NPV [95% BPI] 69.5% [64.2% - 74.7%] 62.5% [60.0% - 68.0%] 58.5% [52.9% - 64.1%] LR+ [95% BPI] 5.14 [4.18 - 6.10] 2.25 [1.83 – 2.67] 2.14 [1.74 – 2.54] LR- [95% BPI] 0.57 [0.49 – 0.66] 0.79 [0.67 – 0.90] 0.93 [0.80 – 1.06]

In unvaccinated sheep and goat populations (Table 2 & Figure 2), the performance of the tests is better than the vaccinated-infected population. The sensitivity of detection of infection for PrioCHECK (Cedi), Chekit (Bommeli) and UBI are calculated as 94%, 77% and 67%, respectively. As in the case of vaccinated-infected sheep and goats, PrioCHECK is found as the test of choice for unvaccinated sheep and goats. The PPV and NPV are better related with the prevalence levels, the Chekit and UBI give less of an increment in the positive detection rate but a decrease in the negative prediction rate (due to the lower Sp).

Table.2 & Figure 2. Sheep-Goats Non-Vaccinated/Infected strata (Bayesian posterior estimate. In Figure 2, PrioCHECK, solid black; Chekit, hashed green; UBI, hashed red).


Ceditest® Chekit UBI®Se [95% BPI] 0.941 [0.905 – 0.950] 0.774 [0.692 – 0.848] 0.677 [0.591 – 0.758]Sp [95% BPI] 0.928 [0.901 – 0.992] 0.895 [0.719 – 0.982] 0.883 [0.715 – 0.971]

Prevalence [95% BPI] 0.751 [0.710 – 0.761]PPV [95% BPI] 97.5% [95.1% - 99.9%] 95.7% [92.5% - 98.9%] 94.6% [91.0% - 98.1%]NPV [95% BPI] 83.9% [78.2% - 89.6%] 56.8% [49.0% - 64.5%] 47.5% [39.7% - 55.3%]LR+ [95% BPI] 13.07 [10.81 – 15.32] 7.37 [6.10 – 8.64] 5.79 [4.79 – 6.78]LR- [95% BPI] 0.06 [0.04 – 0.09] 0.25 [0.16 – 0.35] 0.37 [0.23 – 0.50]

1b. Validation of NSP tests for buffalo:

Further, the validation of 3ABC tests for 3 commercial tests as well as the in-house Brescia laboratory test has been carried out under this project at Pirbright for Asian buffalo samples. 620 negative buffalo samples from Italy were used to investigate the specificity. 616 buffalo sera from Myanmar and 339 buffalo sera from LaoPDR were used for the validation of NSP tests in buffaloes. As observed in cattle and small ruminants, the Prionics 3ABC NSP test scored highest among all the 4 tests (Table.3 & Figure 3).

Table 3 and Figure.3. Characterisation of test performances by bayesian framework in buffalo population from South East Asia

Se [95% BPI]

Sp [95% BPI] PPV [95% BPI] NPV [95% BPI] LR+ [95%

BPI] LR- [95% BPI]

PrioCHECK 0.803 [0.797-0.818]

0.995 [0.987-1]

99.65% [99.18%-100%]

72.64% [71.32%-74.28%]

9175.5 [63.68-3988]

0.1981 [0.1833-0.1997]

3ABC Italy 0.811 [0.798-0.839]

0.994 [0.987-1]

99.61% [99.19%-99.99%]

72.95% [70.11%-76.48%]

747.4 [63.21-2889]

0.1907 [0.1610.2039]

UBI 0.674 [0.673-679]

0.997 [0.990-1]

99.67% [99.13%-100%]

62.01% [61.52%-62.41%]

2047 [60.72-8660]

0.3266 [0.322-0.330]

Chekit 0.608 [0.524-0.716]

0.979 [0.960-0.999]

98.91% [97.39%-99.95%]

44.43% [27.80%-64.02%]

160.5 [14.34-574.6]

0.4001 [0.2882-0.4879]

IgA 0.800 [0.797-0.806]

0.996 [0.988-1]

99.74% [99.22%-100%]

72.57% [71.98%-73.27%]

5344 [68.21-6970]

0.2011 [0.1945-0.2044]

PrioCHECK + IgA

0.983 [0.894-0.999]

0.993 [0.987-

99.68% [99.36%-

96.33% [76.13%-

1707 [76.32-2953]

0.017 [0-0.1072]


0.999] 99.99%] 99.99%]

1c. Validation of 3D NSP tests in cattle:

During this project period, we have validated two commercially (Prionics) available 3D NSP antibody tests using 1000 samples from 4 cattle experiments involving O1 Manisa vaccination and subsequent O1 UKG challenge. One of these assays exhibited a similar sensitivity to the Prionics 3ABC NSP test although some animals showed a rise in absorbance value following vaccination even if this did not score above the normal cut off value. The 2nd 3D test has lower sensitivity than the 3ABC Prionics test.

1d. Development of new in-house NSP ELISAs:Recently, we have produced recombinant proteins 3D, 3CD, 2C and 3ABC and developed in-house indirect ELISAs that utilise these in a multiplex format. The results were compared with the Prionics 3ABC NSP test and revealed a similar sensitivity and specificity to the 3ABC Prionics test for detection of infection in unvaccinated and vaccinated clinically infected population. Although 3D, 3ABC and 3CD in-house assays had similar sensitivity to detect infection in serotype A, Asia and SAT2 vaccinated and needle challenged animals, they had lower sensitivity compared to Prionics tests for detecting infection in O1 Manisa vaccinated and subsequently contact challenged animals.

In conclusion, We have shown the prionics 3ABC NS test is the best commercially available test for the detection of infection or virus circulation in cattle, sheep and buffalo. Combining a 3ABC screening assay with another confirmatory test could further increase the sensitivity for detection of infection.

2.Development and validation of 2B peptide test as a confirmatory NSP test:

We have already shown the potential of the 2B peptide assay for detection of infection in vaccinated cattle herds (Inoue et al., 2006). Although this test had high sensitivity, potentially better than the 3ABC test, it had unacceptable specificity. As this is a peptide based assay, we next produced a series of mutated peptides to try to counteract the specificity problems without affecting test sensitivity. This has been partially successful. However, introducing horse serum into the blocking buffer in each step of the ELISA, has now increased the specificity to a similar level as for the Prionics 3ABC test. Now that the test has similar sensitivity and specificity as the 3ABC Prionics test, it could be taken forward as a confirmatory NSP test, with the advantage that it is based on a different non-structural protein (2B) than the screening (3ABC) assay (Paton et al., 2009). 827 naïve sera from Italian cattle were tested for both 2B and Prionics 3ABC test to detect the specificity. Several thousand sera from vaccinated infected cattle, unvaccinated infected cattle of experimental origin (needle challenged and contact challenged) and several hundred sera from known infected and uninfected animals from Turkey outbreaks were evaluated in both Prionics 3ABC and our 2B peptide test. An established NSP serum panel (n=36) (Parida et al., 2007) was also used to compare the tests. ROC analysis has been carried out to find out the area under the curve for both the tests to know the test suitability. As shown below (Table.4a, 4b & 4c) both the tests showed acceptable area under curve to be considered as good assays. A difference of detection of infection has been noticed for 2B tests in needle challenged and direct contact challenged animals.

Table 4a. ROC analysis for experimental samples

Test Obsevations AUC SE 95% CIOverall 2B 1278 0.837 0.014 0.815 – 0.856

Prionics 1283 0.883 0.012 0.864 – 0.900

Needle Challenge

2B 962 0.966 0.009 0.952 – 0.976Prionics 962 0.959 0.009 0.945 – 0.971

Contact Challenge 2B 1142 0.820 0.017 0.797-0.842


Prionics 1147 0.900 0.014 0.881 – 0.916

Table 4b. ROC analysis for field samples

Test Obs AUC SE 95% CI

2B 224 0.983 0.008 0.955 – 0.995

Prionics 224 0.994 0.004 0.975 – 0.999

Table 4c. ROC analysis for NSP panel samples

Test Obs AUC SE 95% CI

Overall 2B 862 0.972 0.019 0.959 – 0.982

Prionics 862 0.990 0.004 0.980 – 0.995

Vaccinated

2B 850 0.966 0.026 0.951 – 0.977

Prionics 850 0.992 0.004 0.983 – 0.997

Not Vaccinated

2B 838 0.999 0.001 0.993 – 1.000

Prionics 838 0.997 0.001 0.991 – 0.999

Needle Challenge

2B 845 0.960 0.037 0.945 – 0.973

Prionics 0.988 0.006 0.978 – 0.994

Contact Challenge

2B 843 0.990 0.006 0.980 – 0.995

Prionics 843 0.998 0.001 0.991 – 0.999

From the ROC analysis we have filtered a cut off point of 0.5 OD to optimise the sensitivity and specificity of the test. As shown in the Table 5, the 2B assay has a good sensitivity and specificity to detect infection in vaccinated population. However, for vaccinated and then contact challenged animals the detection rate was slightly lower, that may be due to either low replication of virus in these animals vaccinated with high PD50 vaccines or low level of virus challenge in comparison to needle challenge.

Table 5. ROC analysis for determination of Cut off, sensitivity and specificity for 2B assay

Test Type Cut point Sensitivity Specificity Msp Classified LR+ LR-2B needle 0.5 0.87 0.97 0.03 95.99% 26.4079 0.13112B Contact 0.5 0.46 0.95 0.05 85.90% 9.8141 0.57032B Field 0.5 0.96 1.00 0.00 96.88% 0.04432B Panel 0.5 0.85 0.98 0.02 98.03% 54.7152 0.1505

In conclusion, the 2B peptide assay has been validated for cattle and we showed a similar level of sensitivity and specificity as in Prionics 3ABC assay to detect infection in vaccinated cattle that were subsequently challenged or infected. However, this test now needs to be validated further for sheep. Recently we have also developed a 3B peptide test which detects infection in vaccinated population with promise of higher sensitivity than 3ABC and 2B assays. This 3B test needs further optimisation and validation. As both 2B and 3B tests use different proteins of FMDV to the 3ABC screening test, these confirmatory tests will provide more confidence of detection of FMDV


specific antibodies. Further, the 2B and 3B antigens used in these assays are synthetic peptides that are free of virus contamination and could be used in other labs without high containment, if required.

3. Development of NSP multiplex assay:

During previous defra projects (SE2918 and SE1122) and under the current one (SE1125), we have validated newly developed NSP ELISAs and existing NSP ELISAs for FMDV serology. Unfortunately, these results including the international validation results at Brescia show that the assay sensitivity and specificity are insufficient to detect small numbers of infected animals with a high degree of confidence. If used on a large-scale for post-vaccinal serosurveillance, there are likely to be problems in detecting a low prevalence of infection in small herds and in differentiating between true and false positive results. These difficulties may be overcome by simultaneously measuring the antibody response to a collection of different NSPs rather than a single NSP as is used in existing tests, a technique that has been widely employed in South America by means of western blotting. Based on this principle we had developed a multiplex assay using different synthetic peptides (Perkins et al., 2007). As our multiplex machine was broken down, we lost the techiniques for a couple of years. Under the current defra extended project (SE1125), we have been able to buy a new Biorad multiplex machine in early 2012. We have already expressed 3ABC,3D,3CD, 2C FMDV NS proteins and we have generated 2B and 3B immunogenic peptides which have been evaluated for immunogenicity using singleplex ELISAs in this project. So far, we have used 3ABC, 3CD and 3D peptides in our new multiplex ELISA with a small number of samples (Figure 4 ).The current set of results are promising with a high difference in MFI (median fluorence intencity) values between the negative and positive samples originated from cattle before and after vaccination and subsequently challenged. Currently, we are working on these recombinant proteins and peptides in the multiplex assay to increse the sensitivity and specificity of the current NSP assays .

Figure 4. Antibody response to 3ABC,3D and 3CD recombinant proteins in multiplex assay (dpv = days post vaccination; dpc = days post challenge).

In conclusion, to overcome the low sensitivity of existing NSP antibody assays, we are developing amultiplex assay for the simultaneous measurement of the antibody response to a collection of different NSPs rather than a single NSP. The results are promising, but need further work to optimise and validate the test(s) further.

4. Validation of the IgA test as a screening or confirmatory test:

Under SE1122, we had preliminarily validated a salivary IgA test for serotype O and showed that it could detect FMDV carrier animals. Under this project, we have evaluated anther two mucosal fluids (nasal and oro-pharyngeal fliuds) from these animals and found that the levels of IgA antibody are higher in nasal secretions than in saliva and oro-pharyngeal fluid. A comparative ROC analysis between these 3 fluids has been carried out which revealed that the nasal IgA test could detect more carriers than other fluids. Non-parametric ROC analysis for assessing the cut-off value of the nasal IgA after log-transformation of the percentage positive (PP) variable revealed that the sensitivity of the model is high, and is suited for the purpose and gives true parameter estimation. With a 35 PP value of cut-off, 79.7 % sensitivity and 98.6% specificity is observed. Further, Bayesian framework (Fig.4) was used for assessing the diagnostic characteristic of the nasal IgA at 35 PP cut off (three tests Bayesian model with conditional dependence between test and including probabilistic constraints). The data for the model were included from the ROC analysis, where the prevalence was calculated using the Prionics 3ABC test. The model was run for 20000 iterations with the


first 5000 discarded as burn-in to allow convergence. The sensitivity (Se) and specificity (Sp) parameterised by Bayesian analysis are increasing by 6% and 1% respectively. In this case, the odds of detecting a true positive case is 668 times higher than detecting a false positive. The probability of detecting a false negative is 0.142 times higher than detecting a true negative. Therefore, the assay is definitely considered as a good test.

Figure.5. Bayesian framework using probabilistic constraints

Median SD 95% BPI Se 0.854 0.0396 0.801 – 0.944 Sp 0.993 0.0038 0.987 – 1 LR+ 688.8 0.0399 65.83 – 2634 LR- 0.142 16770 0.056 – 0.201

Furthermore, we compared the carrier detection rates between the IgA and Prionics NSP 3ABC (PrioCHECK) tests in cattle (Table.6). We evaluated weekly samples (serum, mucosal and probang fluids) from 4 vaccine challenge experiments consisting of 80 vaccinated cattle followed for up to 6 months post challenge. 32 cattle were confirmed as carriers by repeated sampling and virological testing using virus isolation and real-time RT-PCR – this is taken as 100%. IgA and NSP tests both detect 29 (90.62%) carrier animals though only 27 (84.37%) animals were score concordantly positive in both the tests. Interestingly, the NSP test detected another 18 animals over and above the 32 carrier cattle. These 18 animals were transiently infected and recovered and never scored as virus positive after 28 days post challenge (time considered as carrier) in weekly tests of virus isolation and RT-PCR. Therefore, NSP tests detect the animals which became infected in the past and once sero-converted, but not exclusively the carrier animals (Parida S, 2009). If the strategy of post-outbreak serosurvellance is to detect the carrier, then the NSP test is not the ideal one. However, the IgA test detected exclusively carrier animals and with similar level of sensitivity and higher level of specificity than the NSP test. Therefore, the IgA test can be used as a screening test for detection of carriers. However, if the strategy is to identify the virus circulation in post-outbreak areas, then NSP test can be taken forwarded as a screening test as the test is not serotype specific and IgA test used to confirm the presence of carriers as a confirmatory test. If desired, both the tests could be conducted in parallel, thereby increasing the sensitivity from 91% to 97% as shown in the Table 6.

Table 6. Validation of IgA test for FMDV O serotype and comparison of test efficacy with NSP test for detection of carriers.


Animal Experiments

Clinically infected/

vaccinated challenged

animals

Vaccinated carriers

detected by VI+RT-PCR

Carriersdetected by Cedi-NSP test

NSP seroconver

sion by Cedi test

Carriers detected by

IgA test

Carriers concordantly detected by

both Cedi and IgA test

Carriers detected byeither Cedi

or IgA test or both

UV 0/20 9 7 10 8 7 9

UY 0/20 3 3 7 3 3 3

VH 5/20 9 9 18 9 9 9

VD/VE 6/20 11 10 12 9 8 10

11/80 32(100%)

29(90.62%)

47 29(90.62%)

27(84.37%)

31(96.87%)

We have also extended the validation of the IgA test to the FMDV A serotype and evaluated samples from four A serotype vaccine challenge potency tests as well as with 250 samples collected from an A serotype outbreak in Turkey. In experimental animals, the IgA test detected the carriers in a similar way as for the O serotype. We have analysed saliva samples of 4 heterologous cross-protection studies and two homologous challenge studies pertaining to A serotypes carried out at FLI, Germany (Brehm etal., 2008). The IgA ELISA performed well to detect the carrier animals in these experiments and comparable to VI and PCR results. More carrier animals were detected in heterologous challenged animals than the homologous challenge animals. Although these high potent vaccines could provide complete clinical protection in full dose vaccine groups upon heterologous challenge, IgA ELISA could detect sub-clinical infection in these animals. We have also analysed the saliva samples originated from a challenge study with A/Tur/14/98 carried out at CVI, The Netherlands. IgA ELISA could detect all the carrier animals detected by VI and PCR over a long period, up to 202 days post-challenge.

Evaluating serum, mucosal fluid and probang samples collected from 250 vaccinated and infected cattle from an A serotype outbreak of FMD in Turkey, we could confirm the field detection of carriers by the IgA test (Fig 6 and Table 7). Receiver Operating Characteristic (ROC) analysis has been carried out to compare the results of combined virus isolation + RT-PCR, IgA and NSP tests for detection of infection. Considering an ideal Gold Standard with 100% Se and 100% Sp, the test of equality reports the Area Under the Curve (AUC) values for the comparision of each test to the Gold Standard. As shown in the Figure 6 and Table 7, AUC for NSP Priocheck (Cedi), IgA and VI+RT-PCR cover 0.96, 0.82 and 0.75. VI and RT-PCR have not been considered as gold standard as sample has been taken only once and it is well known that one time sample collection underestimates the true number of carriers. Therefore, AUC for IgA is slightly more than AUC of VI+RT-PCR. If virological samples had been taken on several occasions, as in the above experimental animals, the AUC would have probably increased, as some more animals would have been detected as carriers. However, the proportion scored positive would probably not have reached as high as those scored in the PrioCHECK (AUC=0.969) test, as around 45-55% of animals are usually reported to become carriers in the total population, according to the literature. This would be consistent with our hypothesis that the NSP test not only detects carrier animals, but also those that have eliminated virus after infection. However, the joint test of PrioCHECK + IgA detect more infection than the individual ones (AUC=.975).


Fig 6 and Table7: ROC Analysis-Test of equality vs Gold Standard for Virus isolation, RT-PCR, Priochek (cedi) NSP and IgA test

ROC Area SE χ2 P value

Gold Standard 1 0.0000

PrioCHECK® 0.9690 0.0127 5.9291 0.015

IgA (A+O) 0.8291 0.0189 81.519 0.000

PCR+VI 0.7520 0.0265 87.653 0.000

PrioCHECK®+IgA 0.9754 0.0119 4.2570 0.039

During the second year of study, a comparison has been made between the IgA test and Prionics 3ABC NSP test for detection of carrier animals in field Asian buffalo. Saliva and serum samples from 898 buffalo from Myanmar and LAO PDR were tested in IgA and Prionics 3ABC test. Characterisation of test performances by bayesian framework in buffalo population at South East Asia showed that the IgA assay could detect 80% of infected buffalo which is similar to Prionics 3ABC test with a similar specificity (99%) (Fig 3 and Table 3). Combining both Prionics 3ABC test and IgA test we could increase the sensitivity of detection up to 98% with 99% specificity.

Further, we have extended the development of IgA assay to serotype Asia1 by analysing samples that originated from one PD50 experiment as well as from field infected cattle. The assay detects the known carrier animals as in O and A serotypes, but it needs to be further optimised and validated.

To address the question whether multiple vaccinations could induce the false detection of carrier animals, we have analysed nasal and serum samples in our IgA assay and Prionics assay, respectively. These samples were originated from 6 cattle which were vaccinated with O1 Manisa vaccine for 3 times with an interval of 3 weeks. As shown in Figure 7, 3 doses of high potency emergency vaccine did not increase the IgA level more than the cut-off value in the IgA assay where as two cattle after 3rd vaccination were detected as false positive in Prionics NSP assay. Therefore, IgA assay has potential to correctly identify carrier animals in endemic countries where mass vaccination is being practised on a repeated basis.


A B

Figure 7. Nasal (A) and serum (B) sample analysis from multiply vaccinated animals in IgA (A) and Prionics (B) 3ABC testsIn conclusion, we have shown that NSP antibody tests do not detect exclusively the FMDV carrier animals as they also detect animals that have eliminated FMDV infection. To avoid the problem of exclusive detection of FMDV carrier animals we have developed and validated a mucosal IgA test that has a similar level of sensitivity and specificity as for Prionics 3ABC test. The mucosal IgA test exclusively detects FMDV carrier cattle in vaccinated and subsequently infected as well as in unvaccinated infected populations. If the detection of carriers is crucial, we suggest to take forward the IgA test in the post-vaccination serosurveillance as an independent screening test. If the NSP 3ABC test is still taken forward to detect the carrier animals at a herd level, we recommend to take forward the IgA test as a confirmatory test to detect the FMD carrier.

4. Development and evaluation of IgA test using recombinant antigen:

Development and evaluation of an IgA test for serotype O using recombinant capsid antigen has been completed and the assay exhibited at least similar or more sensitivity and specificity for the detection of carrier animals as has been observed in case of cell culture prepared inactivated antigen (Fig 8). The recombinant capsid antigen for serotype O virus is produced either by using Vaccinia recombinant virus vector or Sendai virus vector and the antigen is purified by sucrose gradient fractionation. IgA ELISA using the empty capsids revealed a test specificity of 98.35% and sensitivity of 96.87%. This ELISA detected 31 carrier animals out of 32 known carrier cattle whereas when FMDV inactivated antigen was used, only 29 known carrier animals were detected. This high sensitivity and specificity may have been achieved due to the use of highly purified antigen, expressed as capsids with all the immunogenic sites available. Further, this IgA ELISA could be performed outside the containment facility by direct coating of the plates with capsid antigen without using the anti FMDV rabbit polyclonal antibody as a trapper.

Similarly, serotype A and Asia1 recombinant antigens are produced using a Sendai virus vector expression system. Production of recombinant antigen should also help to achieve the goal of developing a penside IgA LFD test in future. Currently, by using the empty capsid of A 22 FMD virus in our nasal IgA test, we have detected the same number of carrier cattle in an A22 vaccine challenge experiment as detected by using inactivated A22 FMD antigen. Similarly, 276 nasal samples from unvaccinated, FMDV naïve cattle were analysed using this capsid antigen and no non-specificity has been recorded. However, capsid IgA assay needs final evaluation and validation with a larger number of animals.


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Figure 8. Comparative sensitivity between empty capsid-based IgA-ELISA (upper panel) and inactivated antigen-

based IgA-ELISA (lower panel) for the detection of vaccinated and subsequently infected carrier cattle on individual

sampling days. Percentage of positive outcome of each assay was represented in blue and percentage of negative

outcome of each assay was represented in red.

In conclusion, we have shown an achievement of high sensitivity and specificity of the IgA test by using highly purified capsid antigen. Further, this IgA ELISA using capsid antigen could be performed outside the containment facility that may reduce bio-security concerns.

5. Establishment and Validation of IgM antibody test for FMD:

In the absence of obvious clinical signs, to help identify the time of infection, a FMD-specific IgM assay was developed and validated. Experience from the FMD outbreaks in UK and Cyprus in 2007 had highlighted the problem of not being able to determine when seropositive animals without obvious lesions had been infected and whether this was recent or not. We have shown the development of an IgM assay in the final report of SE1122. Now, we have validated the test with 1000 negative sera each from cattle, sheep and pigs, including sera from the 2001 and 2007 outbreaks in the UK and sera from the 2007 Cyprus outbreaks. The assay showed 99 to 100% specificity in naïve cattle, sheep and pigs. Experimental as well as field infections with cattle, pig and sheep show that, as for other pathogens, IgM antibodies are the first to be produced (1st week) in response to infection or vaccination but do not persist beyond ~3 weeks following viral clearance. A high level of IgM antibody was evident on the 7th day after infection in unvaccinated animals (Fig 9, right hand panel) and on the 7th day after vaccination in uninfected animals. In vaccinated animals, IgM persisted until 2-3 weeks after vaccination. Vaccinated and subsequently contact-challenged, transiently-infected animals were positive in the IgM assay from 7 to 21 days after challenge.

In contrast, we have showed that FMDV carrier animals seem to produce a more sustained IgM response (Fig.9, left hand panel) Waheed et al., 2008). Recently, we have also evaluated samples from a long term (6 month) study of cattle vaccinated with A22 vaccine. This showed that although the level of IgM returns back to the base line by the end of the fourth week after vaccination, IgM reappears in many animals by from eight weeks post-vaccination and can then be sustained thereafter. Though the level of the reappearing IgM in vaccinated animals is higher than our cut off value (0.3 OD), it is lower than the level of IgM seen in carrier animals (Fig. 10). Another difference that we noticed in many carrier animals is that the IgM response is sustained beyond the fourth week after infection and reaches a high level. Having to rely on a qualitative rather than a quantitative difference in IgM response may cause difficulty with using the assay to distinguish between long-term vaccinated uninfected animals and FMDV carrier animals. However, the results obtained do suggest that the test may prove very useful in detailed investigations concerning the onset of infection in outbreaks, particularly in sheep populations where clinical infection is not clearly evident. This assay may also have potential to identify carriers during post-outbreak serosurveillance when


vaccination has not been used. Furthermore, the reasons for the differing IgM responses warrant investigation to gain a better understanding of immune mechanisms in FMD control.

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Figure. 9. High level of IgM antibody titre observed in vaccinated and unvaccinated cattle during1st to 2 nd week of vaccination and then continue in a high level in challenged cattle if the animals become FMDV carrier.

Samples and herd histories were traced from 13 farms that had been infected in the 2001 outbreak in which seropositive animals had been detected by SPCE tests in 2001, but where no clinical signs were seen at the time of sampling. These animals (n=1552), mainly sheep, have been tested on Cedi NS and IgM tests and NS results correlate very closely with the SPCE results from 2001. Of the 1552 sera tested, 96 were positive by Cedi NSP ELISA as compared to 102 that were originally positive by SPCE (4 of those positive on SPCE, but negative on NSP were VNT negative). All of these samples were negative in IgM test which is consistent with collection of these samples at least three weeks after infection and for the purposes of serosurveillance rather than because of clinical cases. Only 3 farms containing a total of 245 sheep could be traced that were submitted from flocks where signs of disease were present and these samples were tested recently by the NSP and IgM tests. Nine samples (5+2+2) were detected positive in IgM tests. However all samples were negative by Cedi NS test as seen by other SPCE test during the outbreak time. This shows the potential of IgM test to detect early infection.

Figure 10 IgM antibody level in A22 vaccinated cattle kept for 5.5 months.

A total of 479 ovine and bovine sera were examined from Cyprus. These were obtained in 2007 during investigations into the unexpected findings of FMD seropositive sheep and goats in a country that has the trading status of FMD free without vaccination. Examination of the sera by IgM assay found the vast majority to be IgM negative, which supports the conclusions of other investigations that the seropositive findings were a result of undiagnosed infection at least three years before (Paton et al., 2009). The new IgM assay was also used to carry out a retrospective examination of sera collected from IP5, one of the FMD affected herds from the UK 2007 FMD outbreaks. In this herd, clinical signs had been missed and the outbreak was detected late and only as a result of serological surveillance. Out of 22 IP5 cattle serum samples, only 5 samples showed a weak positive reaction in the new IgM


assay, whereas 7 out of 12 serum samples obtained from sheep on the same farm scored strongly positive in the same assay. Since, experimentally infected animals were found to remain IgM positive for about 3 weeks after infection, the IgM findings on IP5 are consistent with the estimates from lesion ageing that considered the farm had been infected 3-4 weeks before. The results also suggest that the cattle on IP5 were infected first and then the sheep afterwards, consistent with the fact that all of the other outbreaks involved only cattle, despite the presence of more sheep than cattle within the areas involved.

Further, we have analysed the serum samples in IgM assay that were originated from the O serotype multiple vaccination experiment in cattle. We did not observe any high level of IgM after 2 weeks of first vaccination except one animal in which IgM level has gone up again after 3rd vaccination (figure 11).

Figure 11. IgM antibody level in multiply vaccinated (O1 Manisa) cattle. Cattle are vaccinated 3 times at 3 week intervals. In conclusion, since high levels of IgM antibody do not seem to persist in vaccinated animals after 2-3 weeks of vaccination, its presence in animals known to have been vaccinated some time ago may be an indicator of infection and the assay may have DIVA potential. The assay is very helpful to estimate the time of infection where the clinical signs are not obvious or have been overlooked.

6. Selection of testing strategies:

Based on the NSP and IgA validation data collected in this and the previous projects, we have selected the best combination of tests to use for cattle, sheep and buffalo to substantiate freedom from FMDV infection. We have calculated the sensitivity and specificity of the tests for detection of virus circulation and virus persistence (carriers). Finally, we have used bayesian model/ statistical approaches to indicate the level of virus circulation or persistence with the chosen testing strategy.

In summary, we have shown the prionics 3ABC NS test is the best one for the detection of infection or virus circulation in cattle, sheep and buffalo. In addtion, we have dveloped and validated a 2B peptide NSP assay and showed that the assay has similar sensitivity and specificity to Prionics NS test and could be used as a confirmatory NSP antibody test to help detect virus circulation or infection. However, we have shown that these NSP antibody tests do not detect exclusively the FMDV carrier animals as they also detect fully recovered animals. For exclusive detection of FMDV carrier animals we have developed and validated mucosal IgA test that has similar level of sensitivity and specificity as for Prionics 3ABC test. If the detection of carriers is crucial, we suggest to take forward the IgA test in the post-vaccination serosurveillance as an independent screening test. If the NSP 3ABC test is still taken forward to detect the carrier animals in herd level, we recommend to take forward the IgA test as a confirmatory test to detect the individual FMD carrier.

Acquisition of post-emergency-vacination field data would have allowed for the development of more powerful models capable of determining the specific criteria any testing regime would need to satisfy in order to prove freedom from virus circulation or persistence with confidence, but such opportunity has not arised during the tenure of this project.



References to published material9. This section should be used to record links (hypertext links where possible) or references to other

published material generated by, or relating to this project.

Oral presentations:

1. Blesilda Verin, John Edwards, Aravindh Babu, S. Grazioli, E. Brocchi, David J Paton, Carolyn Benigno,Keith Sumption and Satya Parida. Detection of FMDV in carrier buffalo in South East Asia. EU FMD Reports: Vienna, Austria, 28th Dept-3rd October 2010.

2. Jitendra K Biswal, Can Cokcaliskan, Sahara Rai, Helen Ambrose, Philippa Grainger, Katja Ebert, Antonello Di Nardo, Unal Parlak, Musa Alkan, Fuat Ozyoruk, Geraldine Taylor, David J Paton and Satya Parida. Comparison of tests that detect Persistent FMDV Infection in Cattle. EU FMD Reports: Vienna, Austria, 28th Dept-3rd October 2010.

3. Jitendra K Biswal, David J Paton, Geraldine Taylor and Satya Parida. Detection of Persistently FMDV Infected Cattle by mucosal IgA Test. Modern Mucosal Vaccines, Adjuvants and Microbicides (MMVAM), April 28-30th,2010, Dublin, Republic of Ireland.

4. Can Cokcaliskan, Philippa Grainger, Musa Alkan, David Paton, Katja Ebert, Fuat Ozyoruk and Satya Parida. Detection of FMD Infection in Vaccinated Animals (DIVA) in Turkey:Comparison of tests to detect FMDV carrier animals. EPIZONE, Antalya, Turkey, May13th-15th, 2009.

5. D. Paton & S. Parida. Requirements and plans for vaccine strain selection in East Africa. CIDLID meeting, Arusha, Tanzania, 12 – 14 December 2011.

6.Lembo T. etal., Epidemiological patterns of foot-and-mouth disease in livestock-wildlife interface areas of northern Tanzania. 2012 GFRA Scientific Workshop, Hazyview, South Africa,17 - 19 April 2012.

7. Bari F., Satya Parida, David J. Paton, and Mana Mahapatra. Antigenic and Genetic Characterization of Foot-and-Mouth Disease Virus (FMDV) Serotype A Circulating in East Africa. Europic, St. Raphael, France, 3-7 June 2012.

8.Lembo T. (speaker), Auty, H., Fyumagwa, R., Haydon, D., Hoare, R., Kamani, E., Kasanga, C., Kazwala, R., King, D., Knight-Jones, T., Knowles, N., Marsh, T., Mshanga, D., Parekh, K., Parida, S., Paton, D., Reeve, R., Yoder, J., Cleaveland, S. Patterns and household implications of foot-and-mouth disease in rural Tanzanian communities. Conference abstract for International Society for Veterinary Epidemiology and Economics, Maastricht, 2012

9.Lembo T. (speaker), Parida S., Mshanga D., Kamani E.,Fyumagwa R.,Parekh K., Reeve R., Kazwala R., King D., Haydon D. and Cleaveland S. Understanding patterns of foot-and-mouth disease infection in livestock and wildlife populations in Africa to develop approaches for its control. Conference abstract for 3rd European Congress of Conservation Biology, Glasgow, 2012

10. Verin Bicbic, John Edwards and Satya Paridal., 2012. Detection of carrier buffaloes in South East Asia swamp buffaloes.EU-India FMD meeting, February 8-10th,2012, New Delhi India.

11. Verin Bicbic, John Edwards and Satya Paridal., 2011. Comparison of tests for the detection of carrier buffaloes in South East Asia swamp buffaloes.OIE SEACFMD meeting, Bali March, 2011.

Publications:

1.Uttenthal A, Parida S, Rasmussen TB, Paton DJ, Haas B, Dundon WG.(2010) Strategies for differentiating infection in vaccinated animals (DIVA) for foot-and-mouth disease, classical swine fever and avian influenza. Expert Rev Vaccines. 2010 Jan;9(1):73-87.

2. Sarah J. Cox, B. Veronica Carrb, Satya Parida, Pip A. Hamblina, Helen Prenticeb,Bryan Charleston B, David J. Patona, Paul V. Barnett(2010) Longevity of protection in cattle following immunisation with emergency FMD A22 serotype vaccine from the UK strategic reserve. 2010, 8;28(11):2318-22.

3. Paton DJ, Ferris NP, Hutchings GH, Li Y, Swabey K, Keel P, Hamblin P, King DP, Reid SM, Ebert K, Parida S, Savva S, Georgiou K, Kakoyiannis C.(2009) Investigations into the cause of foot-and-mouth disease virus seropositive small ruminants in Cyprus during 2007.Transbound Emerg Dis. 2009


http://www.ncbi.nlm.nih.gov/pubmed/19744234?itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=7

http://www.ncbi.nlm.nih.gov/pubmed/19744234?itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=7

Oct;56(8):321-328.

4. Satya Parida (2009) Vaccination against foot-and-mouth disease virus: strategies and effectiveness. “Expert Review Vaccine”, 8 (3), 347-365.

5. D. Schley, D.J. Paton, S.J. Cox, S. Parida & S. Gubbins. The effect of vaccination on undetected persistence of foot-and-mouth disease virus in cattle herds and sheep flocks. Epidemiology and Infection. Epidemiol. Infect. (2009), 137, 1494–1504.

6.Parida S, Anderson J, Cox S, Barnet PV and Paton DJ. 2006. Secretory Ig A as an indicator of oropharyngeal FMDV replication. Vaccine 24: 1107-1116.

7. Ko YJ, Lee HS, Park JH, Lee KN, Kim SM, Cho IS, Joo HD, Paik SG, Paton DJ, Parida S.Field application of a recombinant protein-based ELISA during the 2010 outbreak of foot-and-mouth disease type A in South Korea.J Virol Methods. 2012 Jan;179(1):265-8. Epub 2011 Oct 5

8.Yooni Oh, Lucy Fleming, Bob Statham,David Paton, Park JH and satya Parida.2012. Interferon gamma response produced by CD4+ cells correlate with FMD vaccine induced protection. Accepted for publication in PLoS One.9. Fowler V, Robinson L, Bankowski B, Cox S, Parida S, Lawlor C, Gibson D, O'Brien F, Ellefsen B, Hannaman D, Takamatsu HH, Barnett PV.A DNA vaccination regime including protein boost and electroporation protects cattle against foot-and-mouth disease. Antiviral Research 94 (2012), pp. 25-34 DOI information: 10.1016/j.antiviral.2012.02.002

Cited publications

1. Brocchi EI, Bergmann A, Dekker D et al : 2006. Comparative performance of six ELISAs for antibodies to the non-structural proteins of foot-and-mouth disease. Vaccine, 24: 6503-6512.

2. Paton DJ, Clercq KD, Greiner M, et al.: 2006, Application of non-structural protein antibody tests in substantiating freedom from foot-and-mouth disease virus infection after emergency vaccination of cattle. Vaccine 24: 6966-6979.

93. Inoue T, Parida S, Paton DJ, etal T. 2006.Development and evaluation of an indirect ELISA to detect Foot-and-Mouth disease virus non-structural protein antibody using a chemically synthesized 2B peptide as antigen. Journal of Veterinary Diagnostic Investigation 18: 545-552.

4. Arnold ME, Paton DJ, et al., (2008). Modelling studies to estimate the prevalence of foot-and-mouth disease carriers after reactive vaccination. J Royal Soc B Proc Biol Sci. 7; 275(1630):107-15.

5 . Bronsvoort B.M.deC., Parida S., et al.,(2008) Serological survey for foot-and-mouth disease in wildlife in East Africa and parameter estimate of the Ceditest NSP ELISA for buffalo. Clin Vaccine Immunol. 2008 Jun;15(6):1003-11.

6.. Parida S, Fleming L et al., (2007a).Bovine serum panel for evaluation of FMDV non structural protein antibody tests. In press". J. Vet. Diagn. Invest. Vol. 19, 2007a

7.. Parida S, L Fleming et al, 2007b. Reduction of foot-and-mouth disease (FMD) virus load in nasal excretions, saliva and exhaled air of vaccinated pigs following direct contact challenge. Accepted in vaccine. On line available.

8. Parida S, L Fleming, et al. (2008)Emergency vaccination of sheep against foot-and-mouth disease: significance and detection of subsequent sub-clinical infection. Vaccine 26, 3469-3479.

9. Parida S, OH Yooni et al.,.( 2006b). Interferon-γ production in vitro from whole blood of foot-and-mouth disease virus (FMDV) vaccinated and infected cattle after incubation with inactivated FMDV. Vaccine 24:964-969.

10. Paton DJ, Clercq KD, Greiner M, et al.: 2006, Application of non-structural protein antibody tests in substantiating freedom from foot-and-mouth disease virus infection after emergency vaccination of cattle. Vaccine 24: 6966-6979.


http://www.ncbi.nlm.nih.gov/pubmed/22001272



11. Paton D, Sammin D, Dyrting K, Chow M, Fleming L, Verin B, Ferris N, McDonagh G, O’Connor M, Hamblin P, Gibson D and Parida S. (2006).Comparative evaluation of serological tests for the detection of foot-and-mouth disease virus infection in vaccinated pigs. Full paper published in the open session of the research group of the standing technical committee of the European Commission for the control of Foot and mouth Disease, October 16th to 20th, 2006, Paphos, Cyprus.

15. Perkins J, Parida S, Clavijo A. 2007. Evaluation of Multiplexed Foot-and-Mouth Disease Nonstructural Protein Antibody Assay Against Standardized Bovine Serum Panel. Clinical vaccine immunology. 14(11),1472-1482.

12. Sammin D.J., D. J. Paton, S. Parida et al., (2007).Evaluation of laboratory tests for SAT serotypes of foot-and-mouth disease virus withspecimens collected from convalescent cattle in Zimbabwe. Vet Record. 160: 647-654.

13. Sarah Cox, Satya Parida et al., (2007). Further evaluation of higher potency vaccines for early protection of cattle against FMDV direct contact challenge. Vaccine, 25 (44):7687-7695.

14.Schley D, D.J. Paton, S.J. Cox, S. Parida & S. Gubbins. The effect of vaccination on undetected persistence of foot-and-mouth disease virus in cattle herds and sheep flocks. Revised Manuscript ID HYG-OM-1815-Nov-08, Epidemiology and Infection.

15. Vannier P., Capua I., Le Potier M.F., Mackay D., Muylkens B., Parida S., Paton D.J., Thiry E. (2007). Marker vaccines and impact of their use on diagnostic and prophylactic measures (Review). Rev.Sci.tech.off.int.Epiz.,26(2)351-372..

16. Waheed U., D. Gibson, D. J Paton, Q. M Khan and S. Parida*.DEVELOPMENT AND EVALUATION OF IGM ELISA FOR THE DETECTION OF FMDV SPECIFIC. IGM ANTIBODIES IN BOVINE AND OVINE SERA. Tools, ideas and ideals – Erice, Italy 14-17 October 2008, PP 383-386.

17. Biswal D J. K, D. Paton, G. Taylor and S. Parida*. DETECTION OF PERSISTENTLY FOOT-AND-MOUTH DISEASE INFECTED CATTLE BY SALIVARY IGA TEST. The Global control of FMD - Tools, ideas and ideals – Erice, Italy 14-17 October 2008, PP 377-382.

18.Amareen S, P. Grainger, L. Fleming, M. Mahapatra, H. Khalil, I. Bani Younis, A. Tahaineh, D. Paton, F. Aldomy and S. Parida* SERO-SURVEILLANCE AGAINST FOOT-AND-MOUTH DISEASE VIRUS (FMDV) NONSTRUCTURALPROTEIN ANTIBODIES IN SHEEP, GOATS AND CATTLE IN JORDAN AFTER 2006 OUTBREAK The Global control of FMD - Tools, ideas and ideals – Erice, Italy 14-17 October 2008, PP 271-274.

19. Brehm KE, Kumar N, Thulke HH, Haas B. High potency vaccines induce protection against heterologous challenge with foot-and-mouth disease virus.Vaccine. 2008 Mar 20;26(13):1681-7.




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