- Email: [email protected]
Comparison of two pseudorabies virus vaccines, that differ in capacity to reduce virus excretion after a challenge infection, in their capacity of reducing transmission of pseudorabies virus
veterinary microbiology ELSJSVIER
Veterinary
Microbiology 54 (1997) 113-122
Comparison of two pseudorabies virus vaccines, that differ in capacity to reduce virus excretion after a challenge infection, in their capacity of reducing transmission of pseudorabies virus A. Bouma a*b* *, M.C.M. De Jong a, T.G. Kimman a71 aDLO-l’nstituteof Animal Science and Health (ID-DLOJ, P.O. Box 65, 8200 AB Lelystad, The Netherlands b Deparhnent of Herd Health and Reproduction, University of Utrecht, P.O. Box 80151, 3508 TD Utrecht, The Netherlands Received 28 June 1996; accepted 1 October 1996
Abstract Pseudorabies virus (PRV) vaccines are often compared for their capacity to reduce virus excretion after a challenge infection. Vaccines, used for the eradication of PRV, however, should reduce transmission of PRV among pigs. The purpose of this study was to investigate whether the amount of virus excreted after a challenge infection is an accurate measure of the capacity of a vaccine to reduce transmission of PRV among pigs. Two experiments were carried out, each using two groups of 10 pigs. The pigs in group one were intramuscularly vaccinated once with the glycoprotein E (gE)-negative vaccine X, the pigs in group two with the gE-negative strain 783. Eight weeks later, 5 pigs in each group were inoculated with wild-type PRV. A gE-ELISA was used to detect PRV infection. The transmission of PRV was estimated from the number of contact infections and expressed as the reproduction ratio R. The inoculated pigs vaccinated with vaccine X shed significantly more virus than the inoculated pigs vaccinated with strain 783. However, despite the difference in virus excretion, the transmission of PRV between the two groups did not differ. We conclude that virus excretion is not an accurate measure for determining vaccine effectiveness. However, R of vaccine X (R = 0.98) was not significantly below one, whereas R
-* Corresponding author. Tel.: + 31-320-23823s; fax: + 31-320-238668; e-mail: [email protected]. I Present address: Research Laboratory for Infectious Diseases, National Institute of Public Health and the Environment,
P.O. Box 1, 3720 BA Bilthoven,
0378-l 135/97/$17.00 Copyright PII SO378-1135(96)01271-O
The Netherlands.
0 1997 Elsevier Science B.V. All rights reserved.
114
A. Bouma et al./ Veterinary Microbiology
54 (1997) 113-122
of vaccine 783 (R = 0) was significantly below one. Consequently, we cannot exclude possibility that major outbreaks of PRV occur among pigs vaccinated with vaccine X. Keywords:
the
Pseudorabies virus; Transmission; Vaccine; Virus excretion
1. Introduction Vaccines against pseudorabies virus (PRV) are often tested under laboratory conditions by measuring virus excretion and protection against the clinical signs of disease after a challenge infection with wild-type virus. These experiments showed that vaccination reduces the clinical signs of pseudorabies and the amount of excreted virus after a challenge infection and the duration of virus excretion. Moreover, vaccination increases the virus dose needed to establish an infection (Wittmann et al., 1982; De Smet et al., 1992). However, vaccination usually does not prevent the multiplication of virus, nor the establishment of an infection (Zuffa et al., 1982; De Leeuw and Van Oirschot, 1985; Pensaert et al., 1990; Vannier et al., 1991). Thus, usually vaccination against PRV induces only partial immunity. In the PRV eradication campaign, in which eradication of PRV should be achieved by means of vaccination, vaccines are tested for their capacity to induce immunity against a challenge infection, using the above mentioned parameters. Vaccines that induce a significantly lower immunity than the gold standard vaccine (PRV strain 783 in an oil-in-water (o/w) emulsion) are not allowed to be used in the eradication campaign. For example, a vaccine, here called vaccine X, did not reduce virus excretion comparable to the gold standard. As it did not fulfil the requirements, vaccine X was not allowed to be used in the eradication campaign. In the vaccination-challenge experiments, mentioned above, the direct effect of a vaccine, which is the protection of an individual animal against an infection or disease given a specific amount of exposure to infection (Halloran et al., 1991), is measured. It should be emphasized, however, that in eradication campaigns the aim of vaccination is not only to induce individual immunity, but primarily to stop transmission of infections within and between herds by inducing herd immunity (Anderson and May, 1991). Herd immunity is the reduced probability of an individual becoming infected when it is part of a vaccinated population (Halloran et al., 1991) and is the result of reduced virus transmission. Reduction of transmission is, in turn, the result of the combined effect of decreased infectivity of infected vaccinated individuals and decreased susceptibility of not yet infected vaccinated individuals. The efficacy of a vaccine refers to the capacity of a vaccine to induce individual immunity; the effectiveness of a vaccine refers to the capacity of a vaccine to induce herd immunity (Halloran et al., 1991). As herd immunity is an important benefit of vaccination (Anderson, 19921, vaccines should be evaluated for their capacity to induce sufficient herd immunity to stop transmission of PRV. The question is whether the measures for vaccine efficacy are indications for vaccine effectiveness. Clinical protection against an infection probably does not give information about possible transmission of PRV among vaccinated pigs, because factors that cause clinical signs differ from factors that cause transmission.
A. Bouma et al. / Veterinav
Microbiology
54 (1997) 113-122
115
Clinical signs are caused by virus replication within the pig, for example in neural tissues, whereas virus transmission occurs between pigs. Moreover, the onset of virus excretion starts before clinical signs become apparent (Pensaert and Kluge, 1989) and a PRV infection in vaccinated pigs often occurs subclinically. The amount of virus, excreted after a challenge infection, might be an indication for the transmission of PRV, since it can be considered a measure for infectivity of infected animals. However, whether or not an infection will spread in a population not only dependa on the infectivity of infectious animals, but also on the susceptibility of not yet infected animals (De Jong and Kimman, 1994). Thus, as the amount of excreted virus after a challenge infection is commonly used to measure vaccine efficacy, the question is whether the amount of virus excretion correlates with the transmission of PRV. The aim of this study was, therefore, to investigate whether vaccine X and vaccine 783, that differed in capacity to reduce virus excretion after a challenge infection, differed in capacity to reduce transmission of PRV. A stochastic epidemic model was used to interpret the results.
2. Materials
and methods
2.1. Animals Dutch Landrace pigs from the specific-pathogen-free herd of the DLO-Institute of Animal Science and Health were used. These pigs did not have neutralising antibodies against PRV. 2.2. Cells and uiruses Virus stocks for challenge inoculation were prepared in secondary porcine kidney cells as described by Kimman et al. (1992). Two glycoprotein E (gE) deleted vaccines were tested. Vaccine X was a commercially available vaccine that contained the conventionally attenuated PRV strain Norden. The vaccine was used according to the instructions of the manufacturer. The gold standard vaccine was strain 783 (Gielkens et al., 1989; Moormann et al., 1990). The vaccines were dissolved in an oil-in-water emulsion (o/w). The PRV field strain used for challenge inoculation was the second cell culture passage of the mildly virulent Sterksel strain, which expresses gE (Van Oirschot, 1988). 2.3. Experimental
design
Two experiments were conducted successively, each comparing the transmission of PRV in two groups of 10 pigs. In both experiments, pigs from two to three litters were randomly assigned to the two groups. Each group was housed in a separate unit (0.85 m2 per pig). All pigs were intramuscularly vaccinated once at the age of 10 weeks. Pigs in group ‘783 received 2 ml containing lo6 plaque forming units (pfu) of vaccine strain 783; pigs in group X received 2 ml containing lo6 pfu of vaccine X. At the start of each
116
A. Bouma et al./ Veterinary Microbiology 54 (1997) 113-122
experiment, 8 weeks after vaccination, five pigs from each group were removed from their group and were intranasally inoculated with 1 ml/pig containing lo5 pfu of strain Sterksel. After 24 h, the inoculated pigs were returned to their original groups, thus contact-exposing the remaining pigs in each group to PRV. The experiments lasted 6 weeks, during which various samples were collected as described below. 2.4. Sampling procedures
and tests
Oropharyngeal fluid (OPF) from all pigs was collected for 17 days after inoculation of half of each group. The samples were stored at -70°C and titrated for virus by plaque assay on monolayers of SK-6 cells as described (Kimman et al., 1992). The presence of PRV in the plaques of one positive OPF sample per group was confirmed by a neutralisation test using rabbit anti-PRV serum. Blood samples were collected from all pigs one day before vaccination and weekly after inoculation until the end of the experiment. Serum samples were stored at -20°C. They were then tested for the presence of antibodies against gE of PRV with an enzyme-linked immunosorbent assay (ELISA; Van Oirschot et al., 1988) and for the presence of virus neutralising antibodies (VN titers) as described (Kimman et al., 1992). VN titers are expressed as the reciprocal of the highest serum dilution that inhibited the cytopathic effect in 50% of the cell cultures. Detection of antibodies against gE was considered evidence of infection. In addition, we determined the lymphoproliferative response after in-vitro restimulation with PRV (Peeters et al., 1994; Kimman et al., 1995). Briefly, heparinised blood was collected weekly after inoculation. Peripheral blood mononuclear cells (PBMC) were isolated and were restimulated in-vitro with live PRV or tissue culture medium alone as control for 4 days. The proliferation of PBMC was measured by 3H-thymidine incorporation and was expressed as the number of delta counts, that is, the number of counts of PRV-stimulated PBMC minus the number of counts of medium-stimulated PBMC. 2.5. Statistical methods For the statistical analysis of the experiments a stochastic susceptible-infectious-removed (SIR) model was used (Becker, 1989). This model can be used to model chains of infection in small experimental populations and to derive estimates of transmission of PRV (De Jong and Kimman, 1994). A measure for the transmission of virus is the reproduction ratio R, which is defined as the average number of secondary cases caused by one typical infectious individual (Diekmann et al., 1990). This implies that an infection will fade out in a population when R is below one, but may spread when R is above one. The value of the reproduction ratio can be estimated from the outcome of the infection process in a group of pigs using a maximum likelihood estimator. To test whether transmission differed significantly in the two groups, the probability under the null hypothesis was calculated as the maximum probability over all possible values of R for the observed difference or more extreme (Kroese and De Jong, in preparation). The null hypothesis was rejected when this probability was less than 0.05.
A. Bouma et al. / Veterinary Microbiology
54 (1997) 113-122
117
The power of the experiments depends on the expected difference in R. Because of the threshold value of R, a high power is obtained if R in one group is below one and in the second group R is above one. This is also the biologically relevant difference in the comparison between the effectiveness of the two vaccines (Kroese and De Jong, in preparation). Kroese and De Jong (in preparation) calculated the power for several expected differences in R values. Power calculations are of interest when made before the start of the experiments. The efficacy of vaccine X, measured as reduction of virus excretion after a challenge infection, was significantly lower than the efficacy of vaccine 783. It could then be assumed that vaccine X is not effective in reducing transmission of PRV. If vaccine X will not reduce transmission, the a priori estimation of R in a group of pigs vaccinated with vaccine X is possibly comparable to R of an unvaccinated group, which was estimated to be 10 (De Jong and Kimman, 1994). A power > 75% can be expected when li! in one group is 0.5 and R in the second group is above 4.0 (Kroese and De Jong, in preparation). The power to detect a smaller difference in R values is much lower. For example, the power of an experiment in which the expected R in one group is 0.5 and R in the second group is above 2.0 is only 37%. Differences in virus excretion data were tested with the Mann-Whitney U-test (Siegel, 1956). Th e tes t s were applied to data of inoculated pigs that shed virus. The mean daily virus excretion is defined here as the total amount of virus shed by one pig divided by the total number of days that virus was shed by that pig. Differences in VN titers and lymphoproliferative responses were also tested with the Mann-Whitney U-test (Siegel, 1956).
3. Results 3.1. Experiment
1
In group X, all inoculated pigs seroconverted for gE and excreted virus. Two of the five contact-exposed pigs seroconverted for gE and one of these pigs shed virus. In group 783, all five inoculated pigs seroconverted for gE, but only two of these pigs excreted virus. None of the contact-exposed pigs shed virus nor seroconverted for gE (Table 1). Inoculated pigs vaccinated with vaccine X shed significantly more virus than the inoculated pigs vaccinated with 783 (p = 0.008). At the start of the experiment, 8 weeks after vaccination, the titer of neutralizing antibodies against PRV was significantly lower for pigs vaccinated with vaccine X than for pigs vaccinated with 783 ( p = 0.0001). No difference in proliferative response between pigs vaccinated with vaccine X and pigs vaccinated with vaccine 783 was observed ( p = 0.10) (data not shown). 3.2. Experiment
2
In group X, all inoculated pigs seroconverted for gE and excreted virus. Three of the five contact-exposed pigs seroconverted for gE and two of these pigs shed virus. In
118 Table 1 Comparison
Experiment
A. Bouma et al./ Veterina?
of virus excretion
in the OPF of gE-positive
54 (1997) 113-122
pigs
Number of pigs excreting virus
Mean daily virus excretion a (‘Olog pfu)
Duration of virus excretion b
vaccine X
vaccine 783
vaccine X
vaccine 783
vaccine X
vaccine 783
5 1
2 0
5.06 5.73
3.26 0
5.6 8.0
4.5 0
5 2
3 0
6.16 6.02
4.37 0
6.8 7.5
3.0 0
(days)
1
Inoculated Contact Experiment
Microbiology
2
Inoculated Contact
a Arithmetic titre averaged over those pigs that did excrete PRV. b Averaged over those days that virus was excreted.
group 783, all inoculated pigs seroconverted for gE, but only three of these pigs excreted virus. None of the contact-exposed pigs shed virus nor seroconverted for gE (Table I>. Inoculated pigs vaccinated with vaccine X shed more virus and during a longer period than the inoculated pigs vaccinated with 783 (p = 0.008). Pigs vaccinated with vaccine X developed a significantly lower VN titer (102.9> after vaccination than pigs vaccinated with vaccine 783 (104.‘> (p = 0.0001). No difference in proliferative response between pigs vaccinated with vaccine X and pigs vaccinated with vaccine 783 was observed ( p = 0.24) (data not shown). The transmission of PRV was estimated for both vaccine groups in both experiments. As no difference was observed between the two experiments for R of the same vaccine groups, a combined R was estimated for group X and for group 783: R for vaccine X was estimated to be 0.98; R for vaccine 783 was estimated to be 0. The difference in R between the two vaccine groups was not significant (p = 0.14). R for 783 was significantly below one ( p = 0.0171, whereas R for vaccine X was not significantly below one ( p = 0.65).
4. Discussion The purpose of this study was to determine whether virus excretion was an accurate measure of the effectiveness of two PRV vaccines: vaccine 783 and vaccine X. Confirming earlier results, inoculated pigs vaccinated with vaccine X shed significantly more virus after challenge infection with wild-type virus than inoculated pigs vaccinated with strain 783 in o/w. However, despite the difference in virus excretion, the transmission of PRV did not differ significantly in the two vaccine groups. Therefore, we conclude that virus excretion is not an accurate measure for determining vaccine effectiveness. The amount of virus excreted after a challenge infection in vaccination-challenge experiments is considered a measure of vaccine-induced immunity (De Leeuw and Van Oirschot, 1985; Pensaert et al., 1990; Vannier et al., 1991) and vaccines are evaluated for their capacity to reduce virus excretion. However, the significance of an observed
A. Bouma et al./ Veterinav
Microbiology
54 (1997) 113-122
119
difference in virus excretion between two vaccines with regard to transmission is unclear (De Jong and Kimman, 1994; Bouma et al., 1996) and it is unknown above which level of virus excretion a vaccine should be considered not sufficiently effective in reducing transmission of PRV. The amount of virus excreted after a challenge infection can be considered a measure for the infectivity of infected pigs. Infectivity, however, is only one of the factors that determines the spread of PRV. Another important factor is susceptibility of not yet infected pigs (De Jong and Kimman, 1994). Thus, both the infectivity of infected pigs and the susceptibility of not yet infected pigs determine whether PRV may spread, although the relative contribution of both factors is not known. Thus, although virus excretion might be an indication of the efficacy of a vaccine, this study demonstrated that measuring virus excretion in vaccination-challenge experiments is not sufficient to determine whether a vaccine will reduce transmission. As differences in virus excretion between the two vaccine groups were detected and differences in transmission were not, it can be argued that measuring virus excretion is the more sensitive method to detect differences between vaccines. However, if vaccines are applied in an eradication campaign in which PRV transmission should be prevented, the biologically relevant measure in the comparison of the effectiveness of two vaccines is transmission. Kroese and De Jong (in preparation) showed that, because of the threshold value of R, a high power can be obtained if R in one group is below one and in the second group R is considerably above one. If R of vaccine X would have been at least 4.0, which means that vaccine X would not be effective in reducing transmission of PRV, the power of these transmission experiments would have been sufficient to detect this difference. The power to detect a difference in R between two groups when R of both groups is either below or above one, or when R of the second group is slightly above one, is much lower. Increasing the number of experiments or increasing the group size per experiment will increase the power (Kroese and De Jong, in preparation). However, small differences in R between vaccines are not relevant, when R of both vaccines is either below or above one, because in either situation PRV will not spread or can spread, respectively. Whether a vaccine is effective in reducing transmission can be tested by comparing transmission in a vaccinated group with transmission in an unvaccinated control group (De Jong and Kimman, 1994). Another aspect of evaluating the effectiveness of vaccination in transmission experiments IISto test whether R of a vaccine is significantly below one. R for vaccine 783 was significantly below one (p = 0.0171, but R of vaccine X was not (p = 0.65). If R is below one, only minor outbreaks will occur and the infection will disappear. If R is not below one, there is a possibility that major outbreaks occur (De Jong and Diekman, 1992). As it is not certain that for vaccine X the R is below one, because it was not significant, we cannot exclude the possibility that major outbreaks of PRV could occur in groups of pigs vaccinated with vaccine X. Although the estimates of R did not differ between the two vaccines, vaccine strain 783 is probably a better vaccine than vaccine X, as A?for vaccine strain 783 was significantly below one. However, the two vaccines were tested under laboratory conditions using SPF pigs. Other factors might reduce the effectiveness of vaccines. Maternal immunity, for example, not only interferes with the induction of an active immune response (e.g. Vannier, 1985; Van Oirschot et al., 1991;
120
A. Bouma et al. / Veterinary Microbiology 54 (1997) 113-122
Vannier et al., 1995), but also interferes with the capacity of vaccine 783 to reduce transmission (Bouma et al., in press). Thus, the observation that a vaccine, tested under laboratory conditions in a transmission experiment, has an estimated R value significantly below one does not necessarily imply that under field conditions this vaccine will also have an estimated R value below one. It has been demonstrated that major outbreaks did occur under field conditions among pigs vaccinated with vaccine 783 (Stegeman et al., 1995). R for a single vaccination with 783 was estimated to be 3.5. Although it has not been demonstrated, it seems likely that under field conditions also the effectiveness of vaccine X will be reduced. Therefore, caution should be used in extrapolating these experimental results to the field and the effectiveness of vaccines should additionally be determined under field conditions. In the vaccine X groups in our experiments, all inoculated pigs seroconvert for gE and shed virus, but not all gE-seroconverted contact pigs shed virus. It is possible that these contact-infected pigs are not infectious. Then, R might have been overestimated. Possibly, the number of virus excreting contact pigs is a more suitable measure to use for the estimation of R. This would result in a lower estimation of R for the vaccine X groups. An explanation for the observation that gE-seroconverted pigs do not shed virus might be that these pigs have been in contact with low amounts of virus. De Smet et al. (1992), for example, showed that the localisation and extent of PRV replication depended on the inoculation dose. Arellano et al. (1992) showed that intramuscularly vaccinated pigs shed virus for a shorter time after inoculation with a low dose than after inoculation with a high dose. Wittmann et al. (1982) also observed that vaccinated pigs challenged with lo4 TCID,, did not shed virus, whereas pigs inoculated with lo9 TCID,, did. Mishkin et al. (1992) suggested that at low to moderate levels of PRV challenge, the vaccine elicited antibodies play a primary role in limiting the severity of infection. Thus, there might be a dose-dependent immune response. The virus dose for challenge-inoculation used in our experiments might have been too high to mimic a natural infection and might have overwhelmed the immune response. Possibly, to mimic a natural infection a lower dose than applied in vaccination-challenge experiments should be used (Miry and Pensaert, 1989; Pensaert et al., 1990; Vannier et al., 1991; Arellano et al., 1992). An alternative is to carry out an extended transmission experiment, as described (Bouma et al., in press). In such an experiment, transmission is estimated from contact-infected pigs to a second group of contact-exposed pigs. In the transmission experiments, not all contact-exposed pigs become gE-positive. It is possible that these pigs have not been in contact with PRV, or that an early immune response prevented the establishment of infection. It is also possible that gE-negative contact pigs have been latently infected, as latently infected pigs may lack detectable anti-PRV antibodies (Thawley et al., 1984). Kimman et al. (1995) demonstrated that after reinfection with a virulent PRV strain, pigs did develop an in-vitro lymphoproliferative response, whereas these pigs did not show a secondary B-cell response. It was suggested that this dichotomy indicated an effective T-cell memory response that could have quickly eliminated the challenge virus after infection, thus preventing a secondary B-cell response. In our experiments, we could not detect an increase in the in-vitro lymphoproliferative response in pigs that remained gE-negative. Nevertheless, we cannot exclude the possibility that gE-negative pigs have become infected. However, the
A. Boumn et al. / Veterinary Microbiology 54 (1997) 113-122
121
rate of reactivation is probably too low to be quantitatively important in pigs &nith and Grenfell, 1990; Van Nes et al., in press).
References Anderson, R.M., 1992. The concept of herd immunity and the design of community-based immunization programmes. Vaccine, 10: 928-935. Anderson, R.M. and May, R.M., 1991. Infectious Diseases of Humans, Dynamics and Control. Oxford University Press, Oxford. Arellano, P.E., Morrison, R.B., Molitor, T.W. and Thawley, D.G., 1992. High- and low-challenge exposure doses used to compare intranasal and intramuscular administration of pseudo-rabies virus vaccine in passively immune pigs. J. Am. Vet. Med. Assoc., 5: 685-691. Becker, N.G., 1989. Analysis of Infectious Data. Chapman and Hall, London. Bouma, A., De Jong, M.C.M. and Kimman, T.G., 1996. Transmission of two pseudo-rabies virus strains, that differ in virulence and virus excretion, in groups of vaccinated pigs. Am. J. Vet. Res., 57: 43-47. Bouma, A., De Jong, M.C.M. and Kimman, T.G. The influence of maternal immunity on the transmission of pseudo-rabies virus and on the effectiveness of vaccination. Vaccine, in press. De Jong. M.C.M. and Diekman, O., 1992. A method to calculate, for computer-simulated infections, the threshold value, R,, that predicts whether or not the infection will spread. Prev. Vet. Med., 12: 269-285. De Jong, M.C.M. and Kimman, T.G., 1994. Experimental quantification of vaccine-induced reduction in virus transmission. Vaccine, 12: 761-766. De Leeuw, P.W. and Van Oirschot, J.T., 1985. Vaccines against Aujeszky’s disease: Evaluation of their efficacy under standardized laboratory conditions. Vet. Q., 7: 191-197. De Smet, K., De Waele, K. and Pensaert, M.B., 1992. Virulent Aujeszky’s disease virus infectious dose in vaccinated and non-vaccinated pigs. In: Proc. 12th Int. Pig Vet. Sot. Congr., The Hague, The Netherlands, p. 65. Diekmann, O., Heesterbeek, J.A.P. and Metz, J.A.J., 1990. On the definition and computation of the basic reproduction ratio R, in models for infectious diseases in heterogeneous populations. J. Math. Biol., 28: 365-382. Gielkens, A.L.J., Moormann, R.J.M., Van Oirschot, J.T. and Bems, A.J.M., 1989. Vaccine efficacy and innocuity of strain 783 of Aujeszky’s disease virus. In: Van Oirschot (Editor), Vaccination and Control of Aujeszky’s Disease. Kluwer Academic Publishers, Dordrecht, pp. 27-35. Halloran, M.E., Haber, M.L., Longini, I.M. and Stmchiner, C.J., 1991. Direct and indirect effects in vaccine efficacy and effectiveness. Am. J. Epidemiol., 133: 323-331. Kimman, T.G., De Wind, N., Oei-Lie, N., Pol, J.M.A., Bems, A.J.M. and Gielkens, A.L.J., 1992. Contribution of single genes within the unique short region of Aujeszky’s disease virus (suid herpesvirus type 1) to virulence, pathogenesis and immunogenicity. 3. Gen. Virol., 73: 243-251. Kimman, T.G., De Bruin, T.M.G., Voermans, J.J.M., Peeters, B.P.H. and Bianchi, A.T.J., 1995. Development and antigen specificity of the lymphoproliferation response of pigs to pseudo-rabies virus: Dichotomy between secondary B- and T-cell responses. Immunol., 86: 372-378. Kroese, A.H. and De Jong, M.C.M. Testing and estimation for experimental data on transmission, in preparation. Miry, C. and Peniaert, M., 1989. Aujeszky’s disease virus replication in tonsils and respiratory tract of non-immune and immune pigs. In: Van Oirschot (Editor), Vaccination and Control of Aujeszky’s Disease. Kluwer Academic Publishers, Dordrecht, The Netherlands, pp. 163-174. Mishkin, E.M., Blasiak, M., Giorgio, D. and Ishizaka, S.T., 1992. Effects of in-vivo depletion of immunocyte populations on Herpes simplex virus glycoprotein D vaccine-induced resistance to HSV2 challenge. Vir. Immunol., 5: 151-161. Moormann, R.J.M., De Rover, T., Briaire, J., Peeters, B.P.H., Gielkens, A.L.J and Van Oirschot, J.T., 1990. Inactivation of the thymidine kinase of a g1 deletion mutant of pseudo-rabies virus generates a safe but still highly immunogenic vaccine strain. J. Gen. Virol., 71: 1591-1595.
122
A. Bourn
et d/Veterinary
Microbiology
54 (1997) 113-122
Peeters, B., Bouma, A., De Bruin, T., Moormann, R., Gielkens, A. and Kimman, T., 1994. Non-transmissible pseudo-rabies virus gp50 mutants: A new generation of safe live vaccines. Vaccine, 12: 375-380. Pensaert. M.B. and Kluge, J.P., 1989. In: Pensaert (Editor), Virus Infections of Porcines. Elsevier Science Publishers, Amsterdam. Pensaert, M.B., De Smet, K. and De Waele, K., 1990. Extent and duration of virulent virus excretion upon challenge of pigs vaccinated with different glycoprotein-deleted Aujeszky’s disease vaccines. Vet. Microbiol., 22: 107-117. Siegel, S., 1956. In: Nonparametric Statistics for Behavioral Sciences. McGraw-Hill Book Co.. New York, pp. 271-213. Smith. G. and Grenfell. B.T., 1990. Population biology of pseudo-rabies in swine. Am. J. Vet. Res., 51: 148- 155. Stegeman, J.A.. Van Nes, A.. De Jong, M.C.M. and Bolder, W.M.M., 1995. Assessment of the effectivenss of vaccination against pseudo-rabies in finishing pigs. Am. J. Vet. Res., 56: 573-578. Thawley, D.G., Solorzano, R.F. and Johnson, M.E., 1984. Confirmation of pseudo-rabies virus infection, using virus recrudescence by dexamethasone treatment and in-vitro lymphocyte stimulation. Am. J. Vet. Res.. 5: 981-983. Van Nes, A.. Stegeman, J.A., De Jong, M.C.M., Loeffen, W.L.A., Kimman, T.G. and Verheijden, J.H.M. No major outbreaks of pseudo-rabies virus in well-immunised sow herds. Vaccine, in press. Vannier, P., 1985. Experimental infection of fattening pigs with pseudo-rabies (Aujeszky’s disease) virus: Efficacy of attenuated live- and inactivated-virus vaccines in pigs with or without passive immunity. Am. J. Vet. Res., 46: 1498-1502. Vannier, P.. Hutet, E. and Cariolet, R., 1991. Levels of virulent virus excreted by infected pigs previously vaccinated with different glycoprotein deleted Aujeszky’s disease vaccines. Vet. Microbial., 29: 213-223. Vannier, P.. Hutet, E. and Cariolet, R.. 1995. Influence of passive immunity on pig immunization with deleted Aujeszky’s disease vaccines measured by the amount of wild virus excretion after challenge. Vet. Microbial., 43: 53-63. Van Oirschot, J.T., 1988. Induction of antibodies to glycoprotein I in pigs exposed to different doses of a mildly virulent strain of Aujeszky’s disease virus. Vet. Rec., 122: 599-603. Van Oirschot, J.T., Houwers. D.J., Rziha. H.J. and Moonen, P.J.L.M., 1988. Development of an ELISA for the detection of antibodies to glycoprotein I of Aujeszky’s disease virus: A method for the serological differentiation between infected and vaccinated pigs. J. Virol. Methods, 22: 191-206. Van Oirschot. J.T.. Daus, F., Kimman, T.G. and Van Zaane, D., 1991. Antibody response to glycoprotein I in maternally immune pigs exposed to a mildly virulent strain of pseudo-rabies virus. Am. J. Vet. Res., 52: 1788-1793. Wittmann, G., Ohlinger, V. and Hahn, U., 1982. Die Vermehrung von Aujeszkyvirus (AV) in vakzinierten Schweinen nach experimenteller Infektion mit hohen und niederen Virusmengen. Zentralbl. Veterinaermed. Reihe B, 29: 24-30. Zuffa. A., Salaj, J. and Cernik, K., 1982. Immunity of pigs vaccinated by live or inactivated Aujeszky vaccines against experimental infection. Zentralbl Veterinaermed Reihe B, 29: 663-675.
Veterinary
Microbiology 54 (1997) 113-122
Comparison of two pseudorabies virus vaccines, that differ in capacity to reduce virus excretion after a challenge infection, in their capacity of reducing transmission of pseudorabies virus A. Bouma a*b* *, M.C.M. De Jong a, T.G. Kimman a71 aDLO-l’nstituteof Animal Science and Health (ID-DLOJ, P.O. Box 65, 8200 AB Lelystad, The Netherlands b Deparhnent of Herd Health and Reproduction, University of Utrecht, P.O. Box 80151, 3508 TD Utrecht, The Netherlands Received 28 June 1996; accepted 1 October 1996
Abstract Pseudorabies virus (PRV) vaccines are often compared for their capacity to reduce virus excretion after a challenge infection. Vaccines, used for the eradication of PRV, however, should reduce transmission of PRV among pigs. The purpose of this study was to investigate whether the amount of virus excreted after a challenge infection is an accurate measure of the capacity of a vaccine to reduce transmission of PRV among pigs. Two experiments were carried out, each using two groups of 10 pigs. The pigs in group one were intramuscularly vaccinated once with the glycoprotein E (gE)-negative vaccine X, the pigs in group two with the gE-negative strain 783. Eight weeks later, 5 pigs in each group were inoculated with wild-type PRV. A gE-ELISA was used to detect PRV infection. The transmission of PRV was estimated from the number of contact infections and expressed as the reproduction ratio R. The inoculated pigs vaccinated with vaccine X shed significantly more virus than the inoculated pigs vaccinated with strain 783. However, despite the difference in virus excretion, the transmission of PRV between the two groups did not differ. We conclude that virus excretion is not an accurate measure for determining vaccine effectiveness. However, R of vaccine X (R = 0.98) was not significantly below one, whereas R
-* Corresponding author. Tel.: + 31-320-23823s; fax: + 31-320-238668; e-mail: [email protected]. I Present address: Research Laboratory for Infectious Diseases, National Institute of Public Health and the Environment,
P.O. Box 1, 3720 BA Bilthoven,
0378-l 135/97/$17.00 Copyright PII SO378-1135(96)01271-O
The Netherlands.
0 1997 Elsevier Science B.V. All rights reserved.
114
A. Bouma et al./ Veterinary Microbiology
54 (1997) 113-122
of vaccine 783 (R = 0) was significantly below one. Consequently, we cannot exclude possibility that major outbreaks of PRV occur among pigs vaccinated with vaccine X. Keywords:
the
Pseudorabies virus; Transmission; Vaccine; Virus excretion
1. Introduction Vaccines against pseudorabies virus (PRV) are often tested under laboratory conditions by measuring virus excretion and protection against the clinical signs of disease after a challenge infection with wild-type virus. These experiments showed that vaccination reduces the clinical signs of pseudorabies and the amount of excreted virus after a challenge infection and the duration of virus excretion. Moreover, vaccination increases the virus dose needed to establish an infection (Wittmann et al., 1982; De Smet et al., 1992). However, vaccination usually does not prevent the multiplication of virus, nor the establishment of an infection (Zuffa et al., 1982; De Leeuw and Van Oirschot, 1985; Pensaert et al., 1990; Vannier et al., 1991). Thus, usually vaccination against PRV induces only partial immunity. In the PRV eradication campaign, in which eradication of PRV should be achieved by means of vaccination, vaccines are tested for their capacity to induce immunity against a challenge infection, using the above mentioned parameters. Vaccines that induce a significantly lower immunity than the gold standard vaccine (PRV strain 783 in an oil-in-water (o/w) emulsion) are not allowed to be used in the eradication campaign. For example, a vaccine, here called vaccine X, did not reduce virus excretion comparable to the gold standard. As it did not fulfil the requirements, vaccine X was not allowed to be used in the eradication campaign. In the vaccination-challenge experiments, mentioned above, the direct effect of a vaccine, which is the protection of an individual animal against an infection or disease given a specific amount of exposure to infection (Halloran et al., 1991), is measured. It should be emphasized, however, that in eradication campaigns the aim of vaccination is not only to induce individual immunity, but primarily to stop transmission of infections within and between herds by inducing herd immunity (Anderson and May, 1991). Herd immunity is the reduced probability of an individual becoming infected when it is part of a vaccinated population (Halloran et al., 1991) and is the result of reduced virus transmission. Reduction of transmission is, in turn, the result of the combined effect of decreased infectivity of infected vaccinated individuals and decreased susceptibility of not yet infected vaccinated individuals. The efficacy of a vaccine refers to the capacity of a vaccine to induce individual immunity; the effectiveness of a vaccine refers to the capacity of a vaccine to induce herd immunity (Halloran et al., 1991). As herd immunity is an important benefit of vaccination (Anderson, 19921, vaccines should be evaluated for their capacity to induce sufficient herd immunity to stop transmission of PRV. The question is whether the measures for vaccine efficacy are indications for vaccine effectiveness. Clinical protection against an infection probably does not give information about possible transmission of PRV among vaccinated pigs, because factors that cause clinical signs differ from factors that cause transmission.
A. Bouma et al. / Veterinav
Microbiology
54 (1997) 113-122
115
Clinical signs are caused by virus replication within the pig, for example in neural tissues, whereas virus transmission occurs between pigs. Moreover, the onset of virus excretion starts before clinical signs become apparent (Pensaert and Kluge, 1989) and a PRV infection in vaccinated pigs often occurs subclinically. The amount of virus, excreted after a challenge infection, might be an indication for the transmission of PRV, since it can be considered a measure for infectivity of infected animals. However, whether or not an infection will spread in a population not only dependa on the infectivity of infectious animals, but also on the susceptibility of not yet infected animals (De Jong and Kimman, 1994). Thus, as the amount of excreted virus after a challenge infection is commonly used to measure vaccine efficacy, the question is whether the amount of virus excretion correlates with the transmission of PRV. The aim of this study was, therefore, to investigate whether vaccine X and vaccine 783, that differed in capacity to reduce virus excretion after a challenge infection, differed in capacity to reduce transmission of PRV. A stochastic epidemic model was used to interpret the results.
2. Materials
and methods
2.1. Animals Dutch Landrace pigs from the specific-pathogen-free herd of the DLO-Institute of Animal Science and Health were used. These pigs did not have neutralising antibodies against PRV. 2.2. Cells and uiruses Virus stocks for challenge inoculation were prepared in secondary porcine kidney cells as described by Kimman et al. (1992). Two glycoprotein E (gE) deleted vaccines were tested. Vaccine X was a commercially available vaccine that contained the conventionally attenuated PRV strain Norden. The vaccine was used according to the instructions of the manufacturer. The gold standard vaccine was strain 783 (Gielkens et al., 1989; Moormann et al., 1990). The vaccines were dissolved in an oil-in-water emulsion (o/w). The PRV field strain used for challenge inoculation was the second cell culture passage of the mildly virulent Sterksel strain, which expresses gE (Van Oirschot, 1988). 2.3. Experimental
design
Two experiments were conducted successively, each comparing the transmission of PRV in two groups of 10 pigs. In both experiments, pigs from two to three litters were randomly assigned to the two groups. Each group was housed in a separate unit (0.85 m2 per pig). All pigs were intramuscularly vaccinated once at the age of 10 weeks. Pigs in group ‘783 received 2 ml containing lo6 plaque forming units (pfu) of vaccine strain 783; pigs in group X received 2 ml containing lo6 pfu of vaccine X. At the start of each
116
A. Bouma et al./ Veterinary Microbiology 54 (1997) 113-122
experiment, 8 weeks after vaccination, five pigs from each group were removed from their group and were intranasally inoculated with 1 ml/pig containing lo5 pfu of strain Sterksel. After 24 h, the inoculated pigs were returned to their original groups, thus contact-exposing the remaining pigs in each group to PRV. The experiments lasted 6 weeks, during which various samples were collected as described below. 2.4. Sampling procedures
and tests
Oropharyngeal fluid (OPF) from all pigs was collected for 17 days after inoculation of half of each group. The samples were stored at -70°C and titrated for virus by plaque assay on monolayers of SK-6 cells as described (Kimman et al., 1992). The presence of PRV in the plaques of one positive OPF sample per group was confirmed by a neutralisation test using rabbit anti-PRV serum. Blood samples were collected from all pigs one day before vaccination and weekly after inoculation until the end of the experiment. Serum samples were stored at -20°C. They were then tested for the presence of antibodies against gE of PRV with an enzyme-linked immunosorbent assay (ELISA; Van Oirschot et al., 1988) and for the presence of virus neutralising antibodies (VN titers) as described (Kimman et al., 1992). VN titers are expressed as the reciprocal of the highest serum dilution that inhibited the cytopathic effect in 50% of the cell cultures. Detection of antibodies against gE was considered evidence of infection. In addition, we determined the lymphoproliferative response after in-vitro restimulation with PRV (Peeters et al., 1994; Kimman et al., 1995). Briefly, heparinised blood was collected weekly after inoculation. Peripheral blood mononuclear cells (PBMC) were isolated and were restimulated in-vitro with live PRV or tissue culture medium alone as control for 4 days. The proliferation of PBMC was measured by 3H-thymidine incorporation and was expressed as the number of delta counts, that is, the number of counts of PRV-stimulated PBMC minus the number of counts of medium-stimulated PBMC. 2.5. Statistical methods For the statistical analysis of the experiments a stochastic susceptible-infectious-removed (SIR) model was used (Becker, 1989). This model can be used to model chains of infection in small experimental populations and to derive estimates of transmission of PRV (De Jong and Kimman, 1994). A measure for the transmission of virus is the reproduction ratio R, which is defined as the average number of secondary cases caused by one typical infectious individual (Diekmann et al., 1990). This implies that an infection will fade out in a population when R is below one, but may spread when R is above one. The value of the reproduction ratio can be estimated from the outcome of the infection process in a group of pigs using a maximum likelihood estimator. To test whether transmission differed significantly in the two groups, the probability under the null hypothesis was calculated as the maximum probability over all possible values of R for the observed difference or more extreme (Kroese and De Jong, in preparation). The null hypothesis was rejected when this probability was less than 0.05.
A. Bouma et al. / Veterinary Microbiology
54 (1997) 113-122
117
The power of the experiments depends on the expected difference in R. Because of the threshold value of R, a high power is obtained if R in one group is below one and in the second group R is above one. This is also the biologically relevant difference in the comparison between the effectiveness of the two vaccines (Kroese and De Jong, in preparation). Kroese and De Jong (in preparation) calculated the power for several expected differences in R values. Power calculations are of interest when made before the start of the experiments. The efficacy of vaccine X, measured as reduction of virus excretion after a challenge infection, was significantly lower than the efficacy of vaccine 783. It could then be assumed that vaccine X is not effective in reducing transmission of PRV. If vaccine X will not reduce transmission, the a priori estimation of R in a group of pigs vaccinated with vaccine X is possibly comparable to R of an unvaccinated group, which was estimated to be 10 (De Jong and Kimman, 1994). A power > 75% can be expected when li! in one group is 0.5 and R in the second group is above 4.0 (Kroese and De Jong, in preparation). The power to detect a smaller difference in R values is much lower. For example, the power of an experiment in which the expected R in one group is 0.5 and R in the second group is above 2.0 is only 37%. Differences in virus excretion data were tested with the Mann-Whitney U-test (Siegel, 1956). Th e tes t s were applied to data of inoculated pigs that shed virus. The mean daily virus excretion is defined here as the total amount of virus shed by one pig divided by the total number of days that virus was shed by that pig. Differences in VN titers and lymphoproliferative responses were also tested with the Mann-Whitney U-test (Siegel, 1956).
3. Results 3.1. Experiment
1
In group X, all inoculated pigs seroconverted for gE and excreted virus. Two of the five contact-exposed pigs seroconverted for gE and one of these pigs shed virus. In group 783, all five inoculated pigs seroconverted for gE, but only two of these pigs excreted virus. None of the contact-exposed pigs shed virus nor seroconverted for gE (Table 1). Inoculated pigs vaccinated with vaccine X shed significantly more virus than the inoculated pigs vaccinated with 783 (p = 0.008). At the start of the experiment, 8 weeks after vaccination, the titer of neutralizing antibodies against PRV was significantly lower for pigs vaccinated with vaccine X than for pigs vaccinated with 783 ( p = 0.0001). No difference in proliferative response between pigs vaccinated with vaccine X and pigs vaccinated with vaccine 783 was observed ( p = 0.10) (data not shown). 3.2. Experiment
2
In group X, all inoculated pigs seroconverted for gE and excreted virus. Three of the five contact-exposed pigs seroconverted for gE and two of these pigs shed virus. In
118 Table 1 Comparison
Experiment
A. Bouma et al./ Veterina?
of virus excretion
in the OPF of gE-positive
54 (1997) 113-122
pigs
Number of pigs excreting virus
Mean daily virus excretion a (‘Olog pfu)
Duration of virus excretion b
vaccine X
vaccine 783
vaccine X
vaccine 783
vaccine X
vaccine 783
5 1
2 0
5.06 5.73
3.26 0
5.6 8.0
4.5 0
5 2
3 0
6.16 6.02
4.37 0
6.8 7.5
3.0 0
(days)
1
Inoculated Contact Experiment
Microbiology
2
Inoculated Contact
a Arithmetic titre averaged over those pigs that did excrete PRV. b Averaged over those days that virus was excreted.
group 783, all inoculated pigs seroconverted for gE, but only three of these pigs excreted virus. None of the contact-exposed pigs shed virus nor seroconverted for gE (Table I>. Inoculated pigs vaccinated with vaccine X shed more virus and during a longer period than the inoculated pigs vaccinated with 783 (p = 0.008). Pigs vaccinated with vaccine X developed a significantly lower VN titer (102.9> after vaccination than pigs vaccinated with vaccine 783 (104.‘> (p = 0.0001). No difference in proliferative response between pigs vaccinated with vaccine X and pigs vaccinated with vaccine 783 was observed ( p = 0.24) (data not shown). The transmission of PRV was estimated for both vaccine groups in both experiments. As no difference was observed between the two experiments for R of the same vaccine groups, a combined R was estimated for group X and for group 783: R for vaccine X was estimated to be 0.98; R for vaccine 783 was estimated to be 0. The difference in R between the two vaccine groups was not significant (p = 0.14). R for 783 was significantly below one ( p = 0.0171, whereas R for vaccine X was not significantly below one ( p = 0.65).
4. Discussion The purpose of this study was to determine whether virus excretion was an accurate measure of the effectiveness of two PRV vaccines: vaccine 783 and vaccine X. Confirming earlier results, inoculated pigs vaccinated with vaccine X shed significantly more virus after challenge infection with wild-type virus than inoculated pigs vaccinated with strain 783 in o/w. However, despite the difference in virus excretion, the transmission of PRV did not differ significantly in the two vaccine groups. Therefore, we conclude that virus excretion is not an accurate measure for determining vaccine effectiveness. The amount of virus excreted after a challenge infection in vaccination-challenge experiments is considered a measure of vaccine-induced immunity (De Leeuw and Van Oirschot, 1985; Pensaert et al., 1990; Vannier et al., 1991) and vaccines are evaluated for their capacity to reduce virus excretion. However, the significance of an observed
A. Bouma et al./ Veterinav
Microbiology
54 (1997) 113-122
119
difference in virus excretion between two vaccines with regard to transmission is unclear (De Jong and Kimman, 1994; Bouma et al., 1996) and it is unknown above which level of virus excretion a vaccine should be considered not sufficiently effective in reducing transmission of PRV. The amount of virus excreted after a challenge infection can be considered a measure for the infectivity of infected pigs. Infectivity, however, is only one of the factors that determines the spread of PRV. Another important factor is susceptibility of not yet infected pigs (De Jong and Kimman, 1994). Thus, both the infectivity of infected pigs and the susceptibility of not yet infected pigs determine whether PRV may spread, although the relative contribution of both factors is not known. Thus, although virus excretion might be an indication of the efficacy of a vaccine, this study demonstrated that measuring virus excretion in vaccination-challenge experiments is not sufficient to determine whether a vaccine will reduce transmission. As differences in virus excretion between the two vaccine groups were detected and differences in transmission were not, it can be argued that measuring virus excretion is the more sensitive method to detect differences between vaccines. However, if vaccines are applied in an eradication campaign in which PRV transmission should be prevented, the biologically relevant measure in the comparison of the effectiveness of two vaccines is transmission. Kroese and De Jong (in preparation) showed that, because of the threshold value of R, a high power can be obtained if R in one group is below one and in the second group R is considerably above one. If R of vaccine X would have been at least 4.0, which means that vaccine X would not be effective in reducing transmission of PRV, the power of these transmission experiments would have been sufficient to detect this difference. The power to detect a difference in R between two groups when R of both groups is either below or above one, or when R of the second group is slightly above one, is much lower. Increasing the number of experiments or increasing the group size per experiment will increase the power (Kroese and De Jong, in preparation). However, small differences in R between vaccines are not relevant, when R of both vaccines is either below or above one, because in either situation PRV will not spread or can spread, respectively. Whether a vaccine is effective in reducing transmission can be tested by comparing transmission in a vaccinated group with transmission in an unvaccinated control group (De Jong and Kimman, 1994). Another aspect of evaluating the effectiveness of vaccination in transmission experiments IISto test whether R of a vaccine is significantly below one. R for vaccine 783 was significantly below one (p = 0.0171, but R of vaccine X was not (p = 0.65). If R is below one, only minor outbreaks will occur and the infection will disappear. If R is not below one, there is a possibility that major outbreaks occur (De Jong and Diekman, 1992). As it is not certain that for vaccine X the R is below one, because it was not significant, we cannot exclude the possibility that major outbreaks of PRV could occur in groups of pigs vaccinated with vaccine X. Although the estimates of R did not differ between the two vaccines, vaccine strain 783 is probably a better vaccine than vaccine X, as A?for vaccine strain 783 was significantly below one. However, the two vaccines were tested under laboratory conditions using SPF pigs. Other factors might reduce the effectiveness of vaccines. Maternal immunity, for example, not only interferes with the induction of an active immune response (e.g. Vannier, 1985; Van Oirschot et al., 1991;
120
A. Bouma et al. / Veterinary Microbiology 54 (1997) 113-122
Vannier et al., 1995), but also interferes with the capacity of vaccine 783 to reduce transmission (Bouma et al., in press). Thus, the observation that a vaccine, tested under laboratory conditions in a transmission experiment, has an estimated R value significantly below one does not necessarily imply that under field conditions this vaccine will also have an estimated R value below one. It has been demonstrated that major outbreaks did occur under field conditions among pigs vaccinated with vaccine 783 (Stegeman et al., 1995). R for a single vaccination with 783 was estimated to be 3.5. Although it has not been demonstrated, it seems likely that under field conditions also the effectiveness of vaccine X will be reduced. Therefore, caution should be used in extrapolating these experimental results to the field and the effectiveness of vaccines should additionally be determined under field conditions. In the vaccine X groups in our experiments, all inoculated pigs seroconvert for gE and shed virus, but not all gE-seroconverted contact pigs shed virus. It is possible that these contact-infected pigs are not infectious. Then, R might have been overestimated. Possibly, the number of virus excreting contact pigs is a more suitable measure to use for the estimation of R. This would result in a lower estimation of R for the vaccine X groups. An explanation for the observation that gE-seroconverted pigs do not shed virus might be that these pigs have been in contact with low amounts of virus. De Smet et al. (1992), for example, showed that the localisation and extent of PRV replication depended on the inoculation dose. Arellano et al. (1992) showed that intramuscularly vaccinated pigs shed virus for a shorter time after inoculation with a low dose than after inoculation with a high dose. Wittmann et al. (1982) also observed that vaccinated pigs challenged with lo4 TCID,, did not shed virus, whereas pigs inoculated with lo9 TCID,, did. Mishkin et al. (1992) suggested that at low to moderate levels of PRV challenge, the vaccine elicited antibodies play a primary role in limiting the severity of infection. Thus, there might be a dose-dependent immune response. The virus dose for challenge-inoculation used in our experiments might have been too high to mimic a natural infection and might have overwhelmed the immune response. Possibly, to mimic a natural infection a lower dose than applied in vaccination-challenge experiments should be used (Miry and Pensaert, 1989; Pensaert et al., 1990; Vannier et al., 1991; Arellano et al., 1992). An alternative is to carry out an extended transmission experiment, as described (Bouma et al., in press). In such an experiment, transmission is estimated from contact-infected pigs to a second group of contact-exposed pigs. In the transmission experiments, not all contact-exposed pigs become gE-positive. It is possible that these pigs have not been in contact with PRV, or that an early immune response prevented the establishment of infection. It is also possible that gE-negative contact pigs have been latently infected, as latently infected pigs may lack detectable anti-PRV antibodies (Thawley et al., 1984). Kimman et al. (1995) demonstrated that after reinfection with a virulent PRV strain, pigs did develop an in-vitro lymphoproliferative response, whereas these pigs did not show a secondary B-cell response. It was suggested that this dichotomy indicated an effective T-cell memory response that could have quickly eliminated the challenge virus after infection, thus preventing a secondary B-cell response. In our experiments, we could not detect an increase in the in-vitro lymphoproliferative response in pigs that remained gE-negative. Nevertheless, we cannot exclude the possibility that gE-negative pigs have become infected. However, the
A. Boumn et al. / Veterinary Microbiology 54 (1997) 113-122
121
rate of reactivation is probably too low to be quantitatively important in pigs &nith and Grenfell, 1990; Van Nes et al., in press).
References Anderson, R.M., 1992. The concept of herd immunity and the design of community-based immunization programmes. Vaccine, 10: 928-935. Anderson, R.M. and May, R.M., 1991. Infectious Diseases of Humans, Dynamics and Control. Oxford University Press, Oxford. Arellano, P.E., Morrison, R.B., Molitor, T.W. and Thawley, D.G., 1992. High- and low-challenge exposure doses used to compare intranasal and intramuscular administration of pseudo-rabies virus vaccine in passively immune pigs. J. Am. Vet. Med. Assoc., 5: 685-691. Becker, N.G., 1989. Analysis of Infectious Data. Chapman and Hall, London. Bouma, A., De Jong, M.C.M. and Kimman, T.G., 1996. Transmission of two pseudo-rabies virus strains, that differ in virulence and virus excretion, in groups of vaccinated pigs. Am. J. Vet. Res., 57: 43-47. Bouma, A., De Jong, M.C.M. and Kimman, T.G. The influence of maternal immunity on the transmission of pseudo-rabies virus and on the effectiveness of vaccination. Vaccine, in press. De Jong. M.C.M. and Diekman, O., 1992. A method to calculate, for computer-simulated infections, the threshold value, R,, that predicts whether or not the infection will spread. Prev. Vet. Med., 12: 269-285. De Jong, M.C.M. and Kimman, T.G., 1994. Experimental quantification of vaccine-induced reduction in virus transmission. Vaccine, 12: 761-766. De Leeuw, P.W. and Van Oirschot, J.T., 1985. Vaccines against Aujeszky’s disease: Evaluation of their efficacy under standardized laboratory conditions. Vet. Q., 7: 191-197. De Smet, K., De Waele, K. and Pensaert, M.B., 1992. Virulent Aujeszky’s disease virus infectious dose in vaccinated and non-vaccinated pigs. In: Proc. 12th Int. Pig Vet. Sot. Congr., The Hague, The Netherlands, p. 65. Diekmann, O., Heesterbeek, J.A.P. and Metz, J.A.J., 1990. On the definition and computation of the basic reproduction ratio R, in models for infectious diseases in heterogeneous populations. J. Math. Biol., 28: 365-382. Gielkens, A.L.J., Moormann, R.J.M., Van Oirschot, J.T. and Bems, A.J.M., 1989. Vaccine efficacy and innocuity of strain 783 of Aujeszky’s disease virus. In: Van Oirschot (Editor), Vaccination and Control of Aujeszky’s Disease. Kluwer Academic Publishers, Dordrecht, pp. 27-35. Halloran, M.E., Haber, M.L., Longini, I.M. and Stmchiner, C.J., 1991. Direct and indirect effects in vaccine efficacy and effectiveness. Am. J. Epidemiol., 133: 323-331. Kimman, T.G., De Wind, N., Oei-Lie, N., Pol, J.M.A., Bems, A.J.M. and Gielkens, A.L.J., 1992. Contribution of single genes within the unique short region of Aujeszky’s disease virus (suid herpesvirus type 1) to virulence, pathogenesis and immunogenicity. 3. Gen. Virol., 73: 243-251. Kimman, T.G., De Bruin, T.M.G., Voermans, J.J.M., Peeters, B.P.H. and Bianchi, A.T.J., 1995. Development and antigen specificity of the lymphoproliferation response of pigs to pseudo-rabies virus: Dichotomy between secondary B- and T-cell responses. Immunol., 86: 372-378. Kroese, A.H. and De Jong, M.C.M. Testing and estimation for experimental data on transmission, in preparation. Miry, C. and Peniaert, M., 1989. Aujeszky’s disease virus replication in tonsils and respiratory tract of non-immune and immune pigs. In: Van Oirschot (Editor), Vaccination and Control of Aujeszky’s Disease. Kluwer Academic Publishers, Dordrecht, The Netherlands, pp. 163-174. Mishkin, E.M., Blasiak, M., Giorgio, D. and Ishizaka, S.T., 1992. Effects of in-vivo depletion of immunocyte populations on Herpes simplex virus glycoprotein D vaccine-induced resistance to HSV2 challenge. Vir. Immunol., 5: 151-161. Moormann, R.J.M., De Rover, T., Briaire, J., Peeters, B.P.H., Gielkens, A.L.J and Van Oirschot, J.T., 1990. Inactivation of the thymidine kinase of a g1 deletion mutant of pseudo-rabies virus generates a safe but still highly immunogenic vaccine strain. J. Gen. Virol., 71: 1591-1595.
122
A. Bourn
et d/Veterinary
Microbiology
54 (1997) 113-122
Peeters, B., Bouma, A., De Bruin, T., Moormann, R., Gielkens, A. and Kimman, T., 1994. Non-transmissible pseudo-rabies virus gp50 mutants: A new generation of safe live vaccines. Vaccine, 12: 375-380. Pensaert. M.B. and Kluge, J.P., 1989. In: Pensaert (Editor), Virus Infections of Porcines. Elsevier Science Publishers, Amsterdam. Pensaert, M.B., De Smet, K. and De Waele, K., 1990. Extent and duration of virulent virus excretion upon challenge of pigs vaccinated with different glycoprotein-deleted Aujeszky’s disease vaccines. Vet. Microbiol., 22: 107-117. Siegel, S., 1956. In: Nonparametric Statistics for Behavioral Sciences. McGraw-Hill Book Co.. New York, pp. 271-213. Smith. G. and Grenfell. B.T., 1990. Population biology of pseudo-rabies in swine. Am. J. Vet. Res., 51: 148- 155. Stegeman, J.A.. Van Nes, A.. De Jong, M.C.M. and Bolder, W.M.M., 1995. Assessment of the effectivenss of vaccination against pseudo-rabies in finishing pigs. Am. J. Vet. Res., 56: 573-578. Thawley, D.G., Solorzano, R.F. and Johnson, M.E., 1984. Confirmation of pseudo-rabies virus infection, using virus recrudescence by dexamethasone treatment and in-vitro lymphocyte stimulation. Am. J. Vet. Res.. 5: 981-983. Van Nes, A.. Stegeman, J.A., De Jong, M.C.M., Loeffen, W.L.A., Kimman, T.G. and Verheijden, J.H.M. No major outbreaks of pseudo-rabies virus in well-immunised sow herds. Vaccine, in press. Vannier, P., 1985. Experimental infection of fattening pigs with pseudo-rabies (Aujeszky’s disease) virus: Efficacy of attenuated live- and inactivated-virus vaccines in pigs with or without passive immunity. Am. J. Vet. Res., 46: 1498-1502. Vannier, P.. Hutet, E. and Cariolet, R., 1991. Levels of virulent virus excreted by infected pigs previously vaccinated with different glycoprotein deleted Aujeszky’s disease vaccines. Vet. Microbial., 29: 213-223. Vannier, P.. Hutet, E. and Cariolet, R.. 1995. Influence of passive immunity on pig immunization with deleted Aujeszky’s disease vaccines measured by the amount of wild virus excretion after challenge. Vet. Microbial., 43: 53-63. Van Oirschot, J.T., 1988. Induction of antibodies to glycoprotein I in pigs exposed to different doses of a mildly virulent strain of Aujeszky’s disease virus. Vet. Rec., 122: 599-603. Van Oirschot, J.T., Houwers. D.J., Rziha. H.J. and Moonen, P.J.L.M., 1988. Development of an ELISA for the detection of antibodies to glycoprotein I of Aujeszky’s disease virus: A method for the serological differentiation between infected and vaccinated pigs. J. Virol. Methods, 22: 191-206. Van Oirschot. J.T.. Daus, F., Kimman, T.G. and Van Zaane, D., 1991. Antibody response to glycoprotein I in maternally immune pigs exposed to a mildly virulent strain of pseudo-rabies virus. Am. J. Vet. Res., 52: 1788-1793. Wittmann, G., Ohlinger, V. and Hahn, U., 1982. Die Vermehrung von Aujeszkyvirus (AV) in vakzinierten Schweinen nach experimenteller Infektion mit hohen und niederen Virusmengen. Zentralbl. Veterinaermed. Reihe B, 29: 24-30. Zuffa. A., Salaj, J. and Cernik, K., 1982. Immunity of pigs vaccinated by live or inactivated Aujeszky vaccines against experimental infection. Zentralbl Veterinaermed Reihe B, 29: 663-675.