Vaccine 21 (2003) 32403248

Foot-and-mouth disease vaccine potency testing: determination and statistical validation of a model using a serological approach
Paul V. Barnett a, , Robert J. Statham a , Wilna Vosloo b , Daniel T. Haydon c
b a Institute for Animal Health, Pirbright Laboratory, Ash Road, Pirbright, Woking, Surrey GU24 0NF, UK ARC-Onderstepoort Veterinary Institute, Exotic Diseases Division, Private Bag X5, Onderstepoort 0110, South Africa c Department of Zoology, University of Guelph, Guelph, Ont., Canada N1G 2W1

Received 7 October 2002; received in revised form 13 February 2003; accepted 11 March 2003

Abstract European foot-and-mouth disease vaccine manufacturers are required to quantify the efcacy of their product in accordance with the European Pharmacopoeia (EP). The method used most often to establish the potency of foot-and-mouth disease vaccines requires viral challenge of vaccinated cattle. Alternative approaches, such as challenge-free serological assessments have many advantages over existing methods and could be used if robust statistical models could be developed that related antibody titres to protection from challenge. Logistic regression analysis of data from two independent research laboratories, representing six of the seven main serotypes of FMD, permitted the parameterisation of these models and indicated that a signicant relationship existed between antibody titre and probability of protection. Furthermore, no signicant differences were observed in the parameters of logistic models tted to different strains within the serotypes A, O, and SAT-3, or when strains from serotypes A, O, and Asia-1, or SAT-1 and SAT-3, were combined. However, signicant differences in the model parameters did exist between different laboratories. Using these models a bootstrap analysis suggested that for vaccines that induced consistently high titres, as few as six to eight individual animals could be used to establish with condence the minimum protective doses that would protect 50% of vaccinated animals. We conclude that a serologically evaluated truncated test that eliminates the need to virus challenge cattle is a credible alternative for quantifying vaccine potency. 2003 Elsevier Science Ltd. All rights reserved.
Keywords: Foot-and-mouth disease; Vaccine; Potency; Challenge test; Logistic regression; Antibody titre

1. Introduction Inactivated foot-and-mouth disease virus (FMDV) is used in vaccine preparations to control foot-and-mouth disease (FMD), one of the most economically important diseases affecting livestock. These vaccines are used in many parts of the world, particularly where the disease is endemic, including South America, Africa, the Middle East and the Far East. In disease free countries, concentrated inactivated FMD virus antigens are also kept in strategic reserves, so-called antigen banks, which can be rapidly formulated into vaccine during an emergency should an outbreak require additional control measures. In order to assess the quality of the vaccine, all FMD vaccine producers are obliged to instigate a series of tests to establish the safety and efcacy of their product. These tests include a measurement of potency which in accordance
Corresponding author. Tel.: +44-1483-232441; fax: +44-1483-232448. E-mail address: paul.barnett@bbsrc.ac.uk (P.V. Barnett).

with the European Pharmacopoiea (EP) requires that vaccine batches be tested in groups of at least ve cattle inoculated with reduced dose volumes of vaccine so that potency can be expressed in terms of 50% protective doses [1] (PD50 , dened as the factor by which the concentrate may be diluted such that 50% of vaccinated animals are protected). This method has practical and logistical problems, and disadvantages from the perspective of animal welfare. For example, in any extinction point test, approximately 50% of the animals that are not protected will suffer the painful clinical manifestations of the disease and even some protected animals may show primary lesions at the site of challenge. Also, only one valency can be tested in any given trial. Clinically infected animals represent a disease security hazard requiring expensive high security housing. Finally, given the nancial commitment of purchasing these animals and maintaining them during the trial period the smallest permitted group sizes are often used leading to lack of statistical power and imprecision [2]. However, the European Pharmacopoiea has supported the use of alternative testing methods provided a correlation

0264-410X/03/$ see front matter 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0264-410X(03)00219-6

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can be established between them and the challenge test. Serologically-based methods have certain advantages, not least the relatively insignicant discomfort caused to the animals as a result of vaccination and subsequent blood sampling. They allow several FMD vaccines to be tested simultaneously and/or a number of serotypes to be examined in a single polyvalent vaccine and can signicantly improve the accuracy of the result. For this reason many research workers developed and evaluated a variety of serological tests and assessed the correlation of the results with protection. These have included the virus neutralisation test, plaque reduction test, the metabolic inhibition test, mouse protection test and different forms of the enzyme linked immunosorbent assay (ELISA). Although some disadvantages have been noted with these tests, it is generally accepted by FMD research workers that there is an excellent correlation between the virus neutralisation antibody titres of primo-vaccinated cattle and their protection from virus challenge at 21 days post-vaccination [36]. Stellman [7] developed this approach further by showing how such antibody titres could be used to statistically assess vaccine and Pay and Parker [8] described a method for relating log virus neutralisation titres of cattle sera to the potency of a vaccine in antigen PD50 units. A correlation was also established between the antigen dose in the vaccine, the virus neutralising antibody response and the level of induced protection [9]. Certainly, there are strong arguments for replacing the challenge test by in vitro serological assays, however, any form of test would have to meet certain general criteria. The test system must be consistent and reproducible, it must be standardised and include standard reference reagents and parameterised using substantial data sets from which the models relating titre to protection can be reliably established. Two methods have primarily been used to calculate the degree of protection in a group of vaccinated animals. Pay and Hingley [2] and Ahl et al. [10] used mean serum antibody titres to calculate a PD50 or a percentage protection. Bengelsdorff [11] and van Maanen and Terpstra [12] used a second method in which an individual titre or index was assigned to protection and non-protection and the assessment of a group was calculated from the passed and not-passed individuals. In this paper, we have accrued data from two laboratories: The Institute for Animal Health, based at Pirbright in the United Kingdom and the ARC-Onderstepoort Veterinary Institute in South Africa, in which cattle challenge tests and serology have been performed to establish the potency of many different batches of FMD vaccine. At the Pirbright Laboratory these tests were performed to either substantiate that a given antigen could be formulated into a high potency (10 PD50 ) vaccine for acceptance into the International FMD Vaccine Bank [13], or to show stability of the antigen during ultra-low temperature storage. Similarly, by using a truncated version of that specied in the OIE manual [14], involving just a single vaccine dilution group, the

laboratory at Onderstepoort performed cattle challenge tests to ensure that their vaccines had potency values of 8 PD50 . Encompassing six different serotypes, the objective of this analysis was to: determine the relationship between virus neutralising titre and protection; quantify any variation in this relationship between laboratories or as a result of the use of different vaccine strains or serotypes; provide a model that could be used to calculate the probability that vaccines achieve potency levels at or in excess of a pre-specied requirement. 2. Methods 2.1. Data The data comprised the results of vaccine potency trials conducted in the two laboratories (Pirbright and Onderstepoort) on six different serotypes (O, A, Asia-1, SAT-1, SAT-2, and SAT-3). The data is summarised in Table 1 and Fig. 1. Animals were vaccinated (except control individuals) with various dilutions of vaccine of varying valency, and the neutralising antibody titre (log 10 SN50 /100 TCID50 ) to the vaccine virus strains estimated from serum samples taken at 21 days post-vaccination using the virus neutralisation test (VNT). At this time animals were challenged with homologous virus to the vaccine strain by intradermal inoculation of 10,000 ID50 into the tongue. Animals were observed closely for the subsequent reading period, normally 810 days, and the occurrence of generalised lesions on any one or more feet taken as evidence that the animal was not protected by the vaccination. 2.2. Analysis The data for each vaccinated animal thus constitutes: laboratory, serotype, strain, log antibody titre from the VNT, and protected or unprotected status. Logistic regression implemented within MINITAB was used to determine the signicance of differences between strains within serotypes, and between laboratories on the relationship between log antibody titre and protection. The signicance of all two-way interactions was examined and those revealed to be insignificant at the 5% level were omitted from subsequent analysis. When no signicant differences between strains or serotypes were revealed the data were combined and parameters for a single model estimated. Appropriate minimal models were determined, and characterised by their intercepts and slopes (with accompanying standard errors (S.E.)), the covariance between the estimated slope and intercept, T50 (dened as the titre at which animals are protected with probability 50%), the 95% condence intervals (95% CI) on T50 as determined using Fiellers theorem [15] and T95 (dened as the titre at which animals are protected with probability 95%).

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Table 1 Summary of the data (P: Pirbright, O: Onderstepoort) Serotype Asia-1 O A Challenge strains P: India P: Manisa P: Lausanne P: Cruzeiro P: Thailand P: Iraq O: Sar/9/81 O: Bot/1/77 O: Zim/7/83 O: KNP/19/89 O: Bec/1/65 O: KNP/10/90 No. of animals challenged 46 36 26 102 36 26 77 12 77 7 14 33 No. of animals protected (%) 37 (80) 31 (86) 19 (73) 53 (52) 32 (88) 22 (85) 49 (64) 7 (58) 44 (57) 1 (14) 2 (14) 22 (67) Average neutralising Ab titre of vaccinated animals 2.44 (40) 2.34 (32) 2.36 (24) 1.67 (92) 2.25 (32) 2.29 (24) 1.68 (55) 1.79 (10) 1.79 (55) 1.72 (5) 0.66 (10) 1.70 (25)

SAT-1 SAT-2 SAT-3

The number of animals challenged includes control unvaccinated animals.

The 95% condence regions (95% CR) for adopted models were also determined using methods described in Sokal and Rolf [16]. The expected probability of protection (i ) for an individual animal with titre ti is given by: i = exp(a0 + a1 ti ) 1 + exp(a0 + a1 ti ) (1)

where a0 and a1 are the intercept and slope of the associated logistic regression model relating protection to titre. The expected proportion of protected cattle, , in a group comprising n individuals, with log titres t1 , t2 , t3 , . . . tn , is given by: = 1/n n i=1 i . An estimate of whether or not this differs signicantly from 50% of the group can be approximated n n/2 by inspecting the quantity p = i=0 i (1 )(ni) i which constitutes a (one-tailed) test of the hypothesis that 0.5. If n cattle in a potency trial are vaccinated with vaccine diluted X-fold and the hypothesis that 0.5 is

rejected in favour of the hypothesis that > 0.5 then it can be concluded that the PD50 of the vaccine is greater than X. The test is conservative because the variance of a mixed binomial process (in which each trial succeeds or fails with a different probability) is n i=1 i (1 i ), which is always greater than the variance for a homogenous process with the same mean (in which each trial succeeds or fails with the same probability)which is n (1 ) [17]. Thus, assuming the process to be homogenous when it is in fact mixed should lead to an overestimate of p. We can, in principle, use a bootstrap analysis to investigate the relationship between the number of animals in such challenge-free trials, and the probability that signicantly more than half the animals in such trials would be protected. We can randomly assign all vaccinated individuals in a trial of n animals a titre from the set of observed titres for that serotype, and use the model combined over all strains for that serotype reported in Table 2 to calculate the mean and variance of the expected number of animals

Table 2 Parameters corresponding to logistic regression models tted to subsets of the data in which no signicant heterogeneity could be detected at the 5% level Model no. Virus/lab Vaccine/challenge strain n a0 S.E. a0 a1 S.E. a1 Covariance (a0 , a1 ) T50 T50 (95% CI) T95

Onderstepoort 1 SAT-1 2 SAT-1 3 SAT-1 4 SAT-2 5 SAT-2 6 SAT-2 7 SAT-3 Pirbright 8 9 10 11 A O Asia-1 All 3 combined

Sar/9/81 Bot/1/77 Sar/9/81 + Bot/1/77 Zim/7/83 KNP/19/89 Zim/7/83 + KNP/19/89 Bec/1/65 + KNP/10/90 Cruzeiro + Thailand + Iraq Manisa + Lausanne India Cruzeiro + Thailand + Iraq + Manisa + Lausanne + India

77 12 89 77 7 84 47 164 62 46 272

5.155 15.200 4.780 6.002 5.760 5.000 6.920 8.889 10.123 6.923

1.18 12.96 1.02 1.30 1.18 1.41 1.14 3.43 5.31 0.957

4.726 9.780 4.157 4.482 4.065 4.310 4.773 5.673 5.803 4.658

1.190 102.020 0.820 1.162 Insufcient data 0.805 0.912 1.287 1.740 1.054 8.008 0.840 0.934 0.728 1.967 2.634 0.581 0.807 6.668 13.860 0.540

1.091 1.554 1.150 1.339 1.417 1.160 1.450 1.567 1.744 1.486

0.932 1.258 Insufcient data 0.988 1.307 1.164 1.517 1.243 0.965 1.326 1.116 0.409 1.377 1.594 1.469 1.560 1.749 1.977 1.581

1.714 1.855 1.858 1.996 2.141 1.843 2.067 2.086 2.252 2.118

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Fig. 1. Histograms showing the frequency distribution of titres induced by vaccines of different FMD serotypes.

protected, and the P-value indicative of whether signicantly more than half the animals were protected. Repeating this process 500 times each for a range of different possible sizes of trials, allows us to compute the probability that signicantly more than half the animals in a trial would be protected as a function of the number of animals in the trial.

3. Results 3.1. The relationship between virus neutralising titre and protection No signicant interactions between neutralising antibody titre and serotype, or antibody titre and strain were

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detected in any of the analyses described below. Analysis indicated a highly signicant effect of laboratory on the relationship between titre and protection, we therefore proceeded to analyse results from the different laboratories separately. The parameter estimates, with standard errors, covariances and T50 and T95 values are summarised in Table 2. 3.2. ARC-Onderstepoort Veterinary Institute Of the two strains of SAT-1 examined, signicant differences in model intercept were established between Sar/9/81 and Bot/1/77 (P < 0.05): a signicant relationship existed between titre and probability of protection against Sar/9/81 (P < 0.001) but not against Bot/1/77 (presumably a consequence of the small number of potency results available for this strain). Similarly, a signicant relationship between titre and protection existed for the SAT-2 strain Zim/7/83 (P < 0.001) but insufcient data existed to establish such a relationship for the other SAT-2 strain, KNP/19/89. No signicant differences between titre and protection could be established for the SAT-3 strains Bec/1/65 and KNP/10/90 so these were combined and a signicant relationship between titre and protection was apparent (P < 0.001). When strains Sar/9/81, Zim/7/83, and (KNP/10/90 + Bec/1/65) representing the three SAT strains are compared, the Zim/7/83 was found to be modelled by a lower intercept to the SAT-1 and SAT-3 strains (which were not distinguishable) indicating that this SAT-2 strain requires a higher titre to achieve the same level of protection compared to Sar/9/81, KNP/10/90 and Bec/1/65. This is reected in the T95 values for SAT-1, SAT-2, and SAT-3 vaccines which were 1.858, 2.141, and 1.843, respectively (for models with strains combined within serotypes).

3.3. Institute for Animal Health, Pirbright Signicant relationships existed between antibody titre and probability of protection for all strains for which data were available for serotypes Asia-1, O, and A. In addition, no signicant differences were revealed in the intercepts of logistic models tted to the three strains of the A serotype, namely, A15 Thailand, A22 Iraq and A24 Cruzeiro, or the two strains, Manisa and Lausanne, of the O strain. Analysis of all three serotypes combined also revealed no signicant differences in intercepts between serotypes. T95 values for serotypes A, O, and Asia-1 vaccines were 2.067, 2.086, and 2.252, respectively (once again for models with strains combined within serotypes). An observation previously reported using independent data from tests involving other O, A, and C vaccine strains [18]. The probabilities of protection with different titres of antibody for different subsets of the data sets that are statistically indistinguishable are indicated in Fig. 2. The estimates of slope and intercept for the logistic regressions exhibit negative covariance (see Table 2). This means that uncertainty in the estimated value of the intercept is negatively correlated with uncertainty in the estimate of the slope. Therefore, the condence regions in parameter space which encapsulate the true parameters governing the relationship between titre and protection with 95% probability, are angled ellipses (see Fig. 3a and b). 3.4. Determining the probability that a particular test vaccine has a specied/required PD50 Suppose that a vaccine concentrate of serotype Asia-1 from Pirbright was diluted X-fold and used to vaccinate eight test animals. At 21 days post-vaccination, these animals were

Fig. 2. The best tting models to four different subsets of the data.

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Fig. 3. The condence regions for the models tted to the data from vaccine potency trials for serotypes from the two laboratories. The condence regions are for models 3, 6, and 7 and 8, 9, and 10 (see Table 2). The angle in the orientation of these ellipses arises from the negative covariance between estimates of slope and intercepts reported in Table 2.

bled, and log antibody titre determined. Suppose the eight log titres, ti , were: 2.70, 2.15, 2.85, 2.85, 2.25, 3.15, 3.15, and 1.65. Using these titres and the estimated model (a0 and a1 model number 10) this laboratory from Table 2, we would estimate that the expected proportion of animals protected would be = (1/8) 8 i=1 (exp(a0 + a1 ti ))/(1 + exp(a0 + a1 ti )) = 0.90260 (in reality seven of eight (87.5%) of these animals with these titres were protected from challenge). n n/2 The quantity i=0 i (1 )(ni) yields a P-value of i 0.0046. If the PD50 of the test vaccine was X then we would expect 50% of the animals to be protected, but from these results we anticipate that over 90% would be protected, so

the probability that the PD50 really is X or less is in this case very small and we could be 99.54% condent that the PD50 of the stored vaccine concentrate was greater than X. As a second example suppose that 10 test animals had been vaccinated with a SAT-1 vaccine from Onderstepoort, diluted Y-fold. Suppose the 21-day log titres were: 1.80, 1.90, 1.40, 1.70, 2.10, 2.40, 2.10, 1.50, 1.30, and 1.60. Using these titres and the estimated model (number 3) for the combined SAT-1 results from this laboratory from Table 2, we would estimate that the expected proportion of animals protected would be = (1/10) 10 i=1 (exp(a0 + a1 ti ))/(1 + exp(a0 + a1 ti )) = 0.882 (in reality 7 of the 10 (70%) animals with these titres were protected from challenge).

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Fig. 4. The probability of being able to show that the PD50 is signicantly greater than that used in these vaccine trials as a function of the number of individuals used in the trial. Each point is the proportion of 500 re-sampled sets of titre values (from the indicated serotype) in which signicantly more than 50% of individuals in the set would be protected. The number of individuals (the number of re-sampled titres) in each trial is indicated on the x-axis. Parameter values are those for the combined strains model for each serotype (numbers 3, 610).

If the PD50 of the test vaccine was Y then we would expect 50% of the animals to be protected, but from these results we anticipate that 88.2% would be protected, so the probability that the PD50 really is Y or less is in this case n n/2 i (1 )(ni) = 0.0034 and in this case, we i=0 i could be 99.66% condent that the PD50 of the vaccine tested was greater than Y. The number of animals required in a trial to establish with 95% condence that the vaccine protected 50% of vaccinated animals obviously depends on the data used but some examples using data analysed in this study are shown in Fig. 4. If the titres from each serotype analysed here were representative generally of those obtainable from vaccines prepared from these serotypes then only modest numbers of animals (as few as six) are required to establish that the most effective and consistent vaccines (those of serotype O and Asia-1) protect half the animals when diluted as they were in these trials. For the more variable titres obtained from SAT-1 and SAT-3 vaccines, even using as many as 18 animals would not necessarily indicate 50% protection with any degree of condence.

4. Discussion This analysis reveals that the relationships documented between vaccinally induced antibody titre and protection against clinical foot-and-mouth disease differs between two different laboratories. Whether this is because of methodological differences in the adopted procedures, or because

different relationships hold for the different serotypes studied in each of the different laboratories cannot be determined from the data available. However, previous studies have also documented differences in the test results between different laboratories [19]. In general, it appears that so long as sample sizes were not too small, results from different strains of the same serotype usually conformed to a single logistic model (this was the case for three serotypes represented by more than one strain: A, O, and SAT-3). The parameterisation of these models for each serotype enables the use of challenge-free trials to estimate the effectiveness of these vaccines. We developed a simple statistical framework for analysis of challenge-free trials for which existing data suggests that for some serotypes, six to eight animals might provide sufcient statistical power to condently establish required PD50 values. This is fewer animals than that currently stipulated by the European Pharmacopoiea. Usually potency testing of inactivated vaccines is performed on the target species. This takes into account not only the active substance, i.e. antigen, but other components such as the adjuvants which are incorporated to further enhance the immunity of the vaccine. This is true for foot-and-mouth disease vaccine as this vaccine always incorporates either aluminium hydroxide/saponin or mineral oil-based adjuvants. This in vivo potency assessment usually involves cattle, which makes the test very costly and involves the examination of virus challenged animals to establish the protective ability of the vaccines. The extremely infectious nature of FMDV means that high level containment facilities are required to perform such tests, which further limits accessibility and adds to the overall cost. The results are then

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based on whether generalisation of disease has occurred at sites other than that of challenge. This of course means that animals not protected will suffer the painful clinical manifestations of FMD and even some protected animals may undergo some pain due to the development of primary lesions at the site of challenge. Results are, therefore, either black or white, with no intermediary level in which an animal could be perceived as being partially protected. These results are then extrapolated into percentages of protected animals within the vaccine dose groups and converted to a PD50 value by the Krber method [20] or some similar procedure, to establish the lowest dilution of vaccine that would protect 50% of the animals. Such tests are often based on relatively small numbers of animals. For example, the European Pharmacopoeia monograph for foot-and-mouth disease vaccines requires three vaccine dilution groups of ve animals per group. This test has been viewed as both statistically weak and inaccurate with a 90% condence interval for the PD50 value lying anywhere between 45 and 220% of the potency [21]. A further important consideration is that often FMD vaccines can be multivalent containing three or four different strains of virus and it is not possible to undertake a challenge test with more than a single strain of virus at any one time. A test approach that relies on large numbers of dened sera that have an established correlation with potency and protection in the target species therefore seems an attractive alternative. By far the most explored area of investigation has been the correlation between protection and serological parameters such as neutralising antibody [2,7,2224]. The data of this sort collected at Onderstepoort and Pirbright permits a feasibility study of this approach. In order to develop logistic models it is important that the data span a wide range of titres. Data for serotype C from Pirbright could not be used in this analysis because no records of vaccinated but unprotected animals existedthe vaccine always protected vaccinates, and thus a logistic model could not be tted. The bootstrap analysis of the number of animals required to conclude with reasonable condence that signicantly more than half the animals in a group are protected (Fig. 4) is only intended for illustratory purposes. Specically, we do not claim that the titres included in the analysis here are representative of those that would be expected from eld use of market ready product. Data for some strains and serotypes (very usefully) includes vaccine used at higher dilutions, and thus induce a wider range of titres than would be expected from non-experimental use. However, it does show, rst, that its very straightforward to use this kind of data to suggest the number of animals required in such trials, and second, that if a vaccine regularly induces high levels of protection then this number could be quite modestpossibly as low as 68. In summary, statistical analysis of serological and protection data accrued from challenge/potency tests performed at two independent research laboratories and encompassing vaccine strains representing six of the seven serotypes of FMD indicates a signicant effect of laboratory on the rela-

tionship between neutralising antibody titre and protection. However, individual analysis of the data from each laboratory showed that, provided reasonable sample sizes are available, a signicant relationship can be established between antibody titre and probability of protection. Furthermore, no signicant differences were observed in the parameters of logistic models tted to combined strains within serotypes A, O, and SAT-3, or when the strains from serotypes A, O, and Asia-1 or SAT-1 and SAT-3 are combined. These models permit the development of a truncated test, that avoids the use of virus challenge and requires only a single group of cattle administered with a vaccine diluted to a level equal to or greater than the required potency value (PD50 ) potency, providing an alternative method for evaluating the vaccine potency required.

Acknowledgements Daniel T. Haydon was supported by the Wellcome Trust. We thank Victoria Edge for helpful discussions. References
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