Genetic variation in the response to vaccination.

Vaccines are the most powerful means to prevent and diminish the burden of infectious disease. However, there are limitations to their use: vaccines are not yet available for all infectious diseases (including human immunodeficiency virus and respiratory syncytial virus), they sometimes lack efficacy, the response to vaccination is limited by maternal antibodies in very young infants, and the response to vaccination is variable or may even be absent in some individuals. This review focuses on genetic factors that determine the variable response to vaccination. The highly polymorphic human leukocyte antigen system, which is involved in antigen presentation, has been researched most in this aspect, and clearly affects the response to vaccination. Other, but less polymorphic pathways involved are the Toll-like receptor pathway, which is involved in antigen recognition and stimulation of the immune system, and the cytokine immunoregulatory network. The heritability, or the proportion of total variance that is due to additive genetic factors, appears to be particularly large for vaccine-induced antibody responses in young infants compared with cell-mediated responses and antibody responses in older, immunologically more mature individuals. Both antibody and cell-mediated responses are not only affected by loci within, but also strongly by loci outside the human leukocyte antigen system. Because most genes that are important in influencing immune responses to vaccination are still unknown, clearly more work is required. A better understanding of the factors that determine an effective response to vaccination may lead to the identification of specific genes and pathways as targets for the development of novel more uniformly effective vaccines.

[Effect of genetic polymorphisms on the susceptibility to and course of infectious diseases]. / Invloed van genetische polymorfismen op de gevoeligheid voor en het beloop van infectieziekten.

The highly variable susceptibility to and course of infectious diseases are caused by variable environmental factors and by genetic differences in both the pathogens and the host. The genetic variability of the host is determined mainly by polymorphisms in genes that play a role in processes such as adhesion, specific and non-specific immunity, antigen presentation, and inflammation. These variations are important, for example, in infections with HIV or respiratory syncytial virus. It is important to combine genetic knowledge with knowledge about the functional properties of variant genes. Applications of knowledge about genetic variability can be found in the development of vaccines and therapeutic agents, prognostics, and the treatment of individual patients.

[Vaccination against pneumococci and hepatitis B in the Dutch National Immunisation Programme]. / Pneumokokken- en hepatitis B-vaccinatie in het Rijksvaccinatieprogramma.

All infants in the Netherlands, which are born after March 2006, receive additional vaccinations at the age of 2, 3, 4 and 11 months to protect them against pneumococcal infections. During the same visit to a consultation bureau, the children also receive a combination vaccine against diphtheria, pertussis, tetanus, poliomyelitis and Haemophilus influenzae (DTPa-IPV-Hib). Children of which at least one parent was born in a country where hepatitis B occurs relatively often are also vaccinated in the Netherlands against hepatitis B. This currently pertains to about 15% of all newborns. These children now receive a new combination vaccine in which a hepatitis B component has been added to the DTPa-IPV-Hib components. They will receive this combination vaccine 4 times. This combination vaccine is given during the same visit as the pneumococcal vaccination. Although pneumococcal vaccination may have a somewhat negative effect on the immune response to hepatitis B, it is expected that the new 4-fold vaccination schedule will induce good and long-lasting protection against hepatitis B in the vast majority of the children. About 700 children are born out of mothers infected with hepatitis B each year in the Netherlands. In the new vaccination schedule, they now receive 5 active vaccinations against hepatitis B and are examined serologically on an individual basis in order to detect breakthrough infections. This will also generate greater insight into the efficacy of the different vaccination schemes and intervention programmes to prevent vertical transmission of the virus.

In vivo recombination of pseudorabies virus strains in mice.

We studied in vivo recombination of pseudorabies virus (PRV) by inoculating mice with non-lethal mutants that carry a small deletion or insertion in the thymidine kinase (TK) gene or the ribonucleotide reductase (RR) gene. After co-inoculation of mice with two different mutants, homologous recombination between the viral genomes resulted in the generation of wild-type PRV that was highly lethal for mice. Thus, recombination could easily be assessed by monitoring survival of inoculated animals. Our results demonstrated that recombination was only detectable when high doses of virus were used. Intragenic recombination was more efficient between mutations in the TK gene than between mutations in the RR gene. Efficient intragenic recombination in the TK gene occurred between mutations which were separated by as few as 266 nucleotides. When two mutants were inoculated with an interval of 2 h, recombination still occurred. No recombination could be detected when the viruses were inoculated at the same time but in separate parts of the body. When inoculated separately, none of the mutants tested could be isolated from the brains of mice. Virus could be recovered from the brain, however, after co-inoculation. Surprisingly, of these viruses 36-39% possessed the parental mutant genotype. This observation indicates that complementation enables these mutants to replicate in the brain and suggests that complementation may contribute to pathogenicity of PRV.

Vaccine-induced antibody responses as parameters of the influence of endogenous and environmental factors.

In laboratory animals, an adequate way to assess effects of environmental exposures on the immune system is to study effects on antigen-specific immune responses, such as after sensitization to T-cell-dependent antigens. This probably also applies to testing effects in the human population. It has thus been suggested that antibody responses to vaccination might be useful in this context. Vaccination responses may be influenced by a variety of factors other than environmental ones. One factor is the vaccine itself; a second is the vaccination procedure used. In addition, the intrinsic capacity of the recipient to respond to a vaccine, which is determined by sex, genetic factors, and age, is important. Psychological stress, nutrition, and (infectious) diseases are also likely to have an impact. We reviewed the literature on vaccine response. With regard to exogenous factors, there is good evidence that smoking, diet, psychological stress, and certain infectious diseases affect vaccination titers, although it is difficult to determine to what extent. Genetic factors render certain individuals nonresponsive to vaccination. In general, in epidemiologic studies of adverse effects of exposure to agents in the environment in which vaccination titers are used, these additional factors need to be taken into consideration. Provided that these factors are corrected for, a study that shows an association of exposure to a given agent with diminished vaccination responses may indicate suboptimal function of the immune system and clinically relevant diminished immune response. It is quite unlikely that environmental exposures that affect responses to vaccination may in fact abrogate protection to the specific pathogen for which vaccination was performed. Only in those cases where individuals have a poor response to the vaccine may exogenous factors perhaps have a clinically significant influence on resistance to the specific pathogen. An exposure-associated inhibition of a vaccination response may, however, signify a decreased host resistance to pathogens against which no vaccination had been performed.

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.

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 of vaccine 783 (R = 0) was significantly below one. Consequently, we cannot exclude the possibility that major outbreaks of PRV occur among pigs vaccinated with vaccine X.

Round table on control of Aujeszky's disease and vaccine development based on molecular biology.

A summary is given on the 4 topics which were discussed during the round table and which represent current knowledge on the molecular biology of Aujeszky's disease (pseudorabies) virus. They include a review on 1. the genome and gene products of the virus; 2. the viral genes associated with virulence; 3. the immunological role of the viral gene products and 4. studies intended to compare the efficacy of several commercially available vaccines and to establish a possible correlation between antibodies against individual structural viral proteins and degree of protection. It was concluded that gI deleted vaccines appear to be the best choice for use in intensive vaccination programmes towards eradication of Aujeszky's disease virus. However, there remains a need for development of more potent vaccines which induce strong humoral and cell mediated immune responses and afford complete protection, virological protection included. It is often observed that live vaccine strains which are completely avirulent lose much capacity to replicate and spread within the vaccinated animal. It is, however, not excluded that a certain degree of dissemination may be needed to be fully efficacious. Loss of virulence may thus be accompanied by too much loss of immunogenicity. An improved genetic stability of live vaccine strains when they are obtained by genetic manipulation, possibly justifies a more widespread dissemination of the vaccine strain in the body compared to that with conventionally developed strains or compared to what is presently allowed.

Immunohistological characterization of the local cellular response directed against pseudorabies virus in pigs.

The aim of this study was to investigate the kinetics of a primary and secondary immune response against pseudorabies virus (PRV). Pigs vaccinated with strain 783 and unvaccinated pigs were challenged with wild-type PRV by either intranasal or subcutaneous infection. Non-challenged pigs were used as controls. On days 1, 3 and 7 after challenge, tissues from the site of infection, and the tonsils of intranasally and the draining lymph nodes of subcutaneously challenged pigs were sampled. Immunohistological staining was used to characterize the various cell populations at the primary site of virus replication and in the lymphoid tissue. Tissue sections were stained for the T-cell markers CD2, CD3 gamma delta, CD4 and CD8, for the B-cell markers IgM, IgA and IgG, for a macrophage marker, and for PRV antigen. After challenge, PRV was detected during a shorter period in vaccinated pigs, and was less disseminated than in unvaccinated pigs. Cellular infiltrates were detected both in the nasal mucosa and the subcutaneous tissue of both unvaccinated and vaccinated pigs. Cell infiltrates, however, appeared earlier in vaccinated than in unvaccinated pigs, indicating a difference in kinetics of the primary and secondary immune response. The appearance of T-cells preceded the appearance of B-cells, but the proportion of the various subsets did not differ between unvaccinated and vaccinated pigs. These findings suggest that the early immune response in vaccinated pigs may contribute to the rapid clearance of virus at the primary site of infection. In addition, T-cells appear to have a more important role in the clearance of PRV than B-cells.

Role of different genes in the virulence and pathogenesis of Aujeszky's disease virus.

In this study the role of different genes located in the unique short region of the genome of Aujeszky's disease virus was examined. Inactivation of the genes encoding the protein kinase (PK), gp63, and gI reduced virulence of the virus for pigs, in contrast to inactivation of the genes encoding the 28 kDa protein, and gX. There was no correlation between virulence and virus multiplication in vitro or in the oropharynx in vivo. The morphogenesis of the PK mutant was altered. The gI mutant replicated to normal titres in the oropharynx and could be recovered from the trigeminal ganglia but not from other parts of the central nervous system, suggesting that gI facilitates the spread of the virus from neuron to neuron. All mutants induced neutralizing antibody and complete or partial protection against a challenge infection. PK and gp63 were required for the induction of complete protection, although these proteins are reportedly not targets for neutralizing antibody or cytotoxic T cells.

Evaluation of tests for detection of antibodies to Aujeszky's disease (pseudorabies) virus glycoprotein E in the target population.

Receiver operating characteristic (ROC) curves assess the quality of tests over the entire range of test signals. We compared the ability of an ELISA to detect antibodies to Aujeszky's disease (pseudorabies) virus gE in colostrum (test A) and in a single droplet of whole blood (test B) with the results obtained in serum (gold standard) in the target population by constructing and analyzing such curves. The area under the ROC curve, which is a quantitative measure of test performance, proved to be significantly (p < 0.01) smaller in test A than in test B or the gold standard. No significant differences in the area under the ROC curve were observed between test B and the gold standard.

No massive spread of pseudorabies virus in vaccinated sow herds.

In this study, we quantified transmission of PRV in 99 sow herds in which the sows were vaccinated three times a year with strain 783 O/W and found that the reproduction ratio R was 0.66, which is significantly below one. This implies that massive spread of PRV cannot occur in such herds.

Priming for local and systemic antibody memory responses to bovine respiratory syncytial virus: effect of amount of virus, virus replication, route of administration and maternal antibodies.

We studied the conditions under which calves can be primed for mucosal and serum antibody memory responses against bovine respiratory syncytial virus (BRSV), and the relationship between such responses and protection against the virus. Calves were primed via the respiratory tract with a low or high amount of live virus, with killed virus, or intramuscularly with live virus. Calves were challenged via the respiratory tract. Priming with live virus via the respiratory tract induced primary antibody responses in serum and on the mucosae, which were identical after the low and the high amount of virus. These responses were suppressed by maternal antibodies. Intramuscular priming of seronegative calves induced serum IgG1 and sometimes serum IgM and IgG2 responses, but no responses were detected on the mucosae. Sera of calves primed by the intramuscular or the respiratory route recognized the same viral proteins. No responses were observed after priming with killed virus, or after intramuscular priming of calves with maternal antibodies. After challenge, mucosal and serum antibody memory responses developed in calves that had been primed via the respiratory tract with live virus, whether they had maternal antibodies or not. One colostrum-fed calf showed a mucosal memory response, although serum responses were still suppressed by maternal antibodies. None of the calves thus primed shed virus after challenge. Intramuscular priming also primed for mucosal and serum memory responses after challenge, which however started perhaps slightly later and were not associated with protection against virus shedding. Priming with killed virus, or with live virus intramuscularly in the presence of maternal antibodies proved least effective in inducing memory and protection against virus shedding. Thus, protection against virus shedding was afforded by priming with live virus via the respiratory tract, both in calves with an without maternal antibodies. Protection was associated with a strong and rapid mucosal antibody memory response, but the reverse was not necessarily true. Protection against virus excretion had no relationship to titers of serum neutralizing or serum IgG1 or nasal IgA antibodies at the time of challenge.

Activation of complement by bovine respiratory syncytial virus-infected cells.

Because complement activation is probably involved in the pathogenesis of as well as in recovery from the disease induced by bovine respiratory syncytial virus (BRSV), we studied the activation of complement by BRSV-infected cells in vitro in a homologous system. Binding of C3 on the surface of infected cells was measured in a biotin-streptavidin amplified ELISA, and complement-mediated lysis was measured in a 51Cr release assay. Without antibody, infected cells activated and bound more C3 than uninfected cells. C3 activation that occurred in the absence of antibody was largely mediated by the classical pathway and induced lysis inefficiently. BRSV-specific antibody enhanced complement activation as measured by both C3 ELISA and cytotoxicity assay. In the presence of antibody, C3 activation was largely dependent on the alternative pathway and efficiently induced lysis. Both IgG1 and IgM antibodies enhanced C3 activation, but IgG2 and IgA did not enhance C3 activation in our experiments. Preincubating cells with IgA or IgG2 did not inhibit C3 activation enhanced by IgG1 or IgM. Murine monoclonal IgG1 antibodies against epitopes on the Fusion protein of the virus also enhanced C3 binding, but differed in their capacity to induce complement-mediated lysis.

Time course of the porcine cellular and humoral immune responses in vivo against pseudorabies virus after inoculation and challenge: significance of in vitro antigenic restimulation.

We investigated the time course of porcine cellular and humoral immune responses against pseudorabies virus (PRV) after pigs were inoculated with PRV gE(-) mutant strain M141 and challenged with wild-type virus NIA-3. Peripheral blood mononuclear cells (PBMC) were isolated from blood samples; half were used directly and half were restimulated with PRV in vitro before use in a cytolytic assay. We determined time course and extent of PRV-specific lymphoproliferative and cytolytic response. In addition, serum samples were examined for neutralizing antibodies. After inoculation, the frequency of various lymphocyte subsets in peripheral blood was determined by FACScan. One week after inoculation, T-lymphocytes proliferated abundantly and a B-lymphocyte response was observed. When PBMC were used directly without restimulation, only 15% of the PRV-infected target cells were lysed, and about 15-20% of uninfected target cells were lysed. In contrast, when PBMC were restimulated with PRV, up to 50% of the PRV-infected target cells were lysed while only 30% of the uninfected target cells were lysed. The frequency of various T-lymphocyte subsets in the circulation did not change significantly after inoculation, which indicates that the number of PRV-specific lymphocytes in circulation was very small. After challenge, the T-lymphocyte response was enhanced, but the B-lymphocyte response was not. When PBMC were used directly, only 20% of the PRV-infected and uninfected target cells were lysed after challenge. In contrast, when PBMC were restimulated with PRV, they again lysed more PRV-infected target cells than uninfected target cells. Cytolytic cells were detected for a longer period after challenge than after inoculation. Since it was only possible to clearly detect cytolysis after lymphocytes were restimulated with PRV, it may be that they do not preferentially localize in blood or that they are too few in blood to be detected without further antigenic restimulation in vitro. These lymphocytes may instead localize in other tissues, such as mucosal tissues, tonsils and draining lymph nodes. Whether such a reservoir of PRV-specific cytolytic cells is important in clearing the virus is still unknown. In this study we demonstrated PRV-specific lymphocytes in circulation after they were restimulated in vitro with PRV.

Measurement of isotype-specific antibody responses to Aujeszky's disease virus in sera and mucosal secretions of pigs.

Enzyme-linked immunosorbent assays (ELISAs) for the detection of porcine IgM, IgA, IgG1 and IgG2 antibodies directed against Aujeszky's disease virus (ADV) are described. ADV-specific IgA and IgM were detected in an antibody capture assay, and ADV-specific IgG1 and IgG2 were detected in an indirect double antibody sandwich assay. A selected set of samples was tested in the four ELISAs and in a 24 h virus neutralization assay. Comparison of the results showed that the ELISAs were isotype-specific, sensitive, and reproducible. Samples with ADV antibody of one isotype showed that ADV-specific IgG1, IgG2 and IgM were able to neutralize the virus in vitro. In vitro neutralization of virus can be enhanced by complement. ADV-specific IgA neutralized virus only weakly. ADV-infected cells activated complement in the absence of antibody. Specific IgG2 and IgM enhanced complement activation. Analysis of the time course of antibody responses after infection or vaccination revealed that the isotype-specific ELISAs are suitable to study the humoral antibody response of pigs to the virus in mucosal secretions. Wild-type virus (strain NIA-3) and an attenuated vaccine strain (Bartha) administered intranasally induced mucosal IgM and IgA responses to the virus. In contrast, a killed vaccine (Nobivac) administered intramuscularly induced only weak mucosal IgM responses. The attenuated vaccine strain primed for a mucosal IgA memory response evoked upon challenge infection with wild-type virus.

Establishment and characterization of porcine cytolytic cell lines and clones.

Although non-major-histocompatibility-complex-restricted cytolytic cells appear to significantly influence antiviral immunity in pigs, the phenotype and functional characteristics of these cells are not well defined. To allow a detailed analysis of these subsets, we established and characterized cell lines and clones of interleukin-2-activated (IL-2) cytolytic cells. Cell lines and clones were obtained from peripheral blood mononuclear cells of minipigs of the swine-leucocyte-antigen-complex (SLA) d/d haplotype. Cells were cultured in the presence of human recombinant IL-2 and cloned by double limiting dilution in the presence of gamma-irradiated L14 cells (a retrovirus immortalized B-lymphoblastoid cell line of the haplotype SLAd/d) or gamma-irradiated autologous peripheral blood mononuclear cells as feeder cells. Cytolytic cell lines and clones were characterized for their ability to kill different target cells and for their cell surface phenotype. All obtained clones expressed CD2 and CD8 and were negative for CD4. The following three subsets of cytolytic cells were identified: Subset 1) CD3- CD5- cells that killed K562 cells (a natural killer cell susceptible target cell line), as well as the pseudorabies virus (PRV)-infected or uninfected porcine kidney cells. These cells were considered to be typical natural killer cells. Subset 2) CD3 gamma/delta + CD5- T-cells that killed K562 cells and PRV virus-infected or uninfected porcine kidney cells, infected or uninfected L14 cells, and L14 cells constitutively expressing the PRV viral glycoprotein gB or gC. These cells were considered to be gamma/delta T-cells with natural killer activity. Subset 3) CD3 alpha/beta + CD5+ T-cells that killed L14 cells, PRV-infected L14 cells, and PRV gB- and gC-transfected L14 cells. These cells were possibly induced by the L14 feeder cells, used in the in vitro culture system. None of the cytolytic effector cells killed only MHC-matched viral infected cells. In conclusion, we describe a method to isolate, clone, and culture cytolytic cells from pigs. The clones could be cultured for 5 months, which allowed appropriate phenotypic and functional characterization of the various clones. Two of the subsets, CD3 gamma/delta T- and the natural killer cell subset may be involved in antiviral immunity in this species.

Interaction of pseudorabies virus with immortalized porcine B cells: influence on surface class I and II major histocompatibility complex and immunoglobulin M expression.

We examined whether the L14 cell line, an immortalized B cell line originating from inbred miniature pigs of the MHC haplotype d/d, could be useful to study T cell responses of pigs to pseudorabies virus (PRV). Compared with porcine kidney cells, the replication of PRV in L14 cells was slower and yielded lower quantities of infectious virus, which agrees with the reported poor replication of PRV in peripheral blood lymphocytes of swine. The virus yield and the number of L14 cells expressing the viral glycoprotein gE were both maximal at 48 h postinfection, when approximately 90% of all viable L14 cells expressed gE. Morphologically detectable effects of PRV replication in L14 cells were not obvious, but the number of viable cells at 72 h postinfection was lower in infected cultures than in uninfected cultures. Major histocompatibility complex (MHC) class I and II antigen expression was significantly higher at different time points postinfection on infected than on uninfected L14 cells. In contrast, expression of IgM appeared very slightly reduced on infected L14 cells, indicating a selective influence of PRV on cellular protein expression. PRV-infected L14 cells were lysed by lymphocytes from PRV-immune minipigs of MHC haplotype d/d, indicating their usefulness in in vitro cytolytic assays.

The influence of maternal immunity on the development of the in vitro lymphocyte proliferation response against pseudorabies virus in pigs.

In this study, the influence of maternal immunity against pseudorabies virus (PRV) on the development of a PRV-specific T-cell response in pigs was investigated. Pigs with or without maternal immunity were challenged by inoculation with wild-type PRV, or were vaccinated with 783 and subsequently inoculated. Peripheral blood lymphocytes, collected after vaccination and/or inoculation, were used for PRV-induced lymphocyte proliferation assays in vitro as a measure of a PRV-specific T-cell response. In unvaccinated pigs, the presence of maternal immunity did not inhibit the development of a T-cell response after inoculation with PRV. In contrast, maternal immunity did inhibit the development of a PRV-induced response after intramuscular vaccination. Moreover, vaccinated pigs without maternal immunity shed virus for a shorter period after inoculation than vaccinated pigs with maternal immunity. The greater T-cell response induced by the vaccine might have contributed to the more rapid clearance of PRV in these pigs.

Isotype-specific ELISAs for the detection of antibodies to bovine respiratory syncytial virus.

Isotype-specific ELISAs for the detection of antibodies to bovine respiratory syncytial virus (BRSV) are described. BRSV-specific IgG1 and IgG2 were determined in indirect double antibody sandwich assays. For IgA and IgM antibody capture assays were used. The isotype specificity of the assays was confirmed by the observation that samples with a high titre of BRSV-specific antibodies of particular isotype were negative in the assays for the other isotypes and vice versa. Comparison of the results obtained in the ELISAs and in the virus neutralisation test showed that acute phase antibodies were more efficiently detected in the latter. It also showed that the presence of BRSV-specific IgA was not correlated with neutralising activity in vitro. The serum antibody response of BRSV-infected seronegative calves from the field consisted of a nearly simultaneous increase of IgM, IgA and IgG1-antibodies in the acute phase of the disease, while the IgG2-response followed at various intervals thereafter. In young animals with maternal antibodies a different pattern was found. There was no increase in IgG1 and IgG2, but six of eight animals showed a weak IgM response and two of these six calves also showed a weak and short lasting IgA response. Because maternal antibodies are insufficiently effective in protecting calves against BRSV, the presence of such antibodies at mucosal surfaces was investigated. Maternal immunity was found to be restricted to IgG1 antibodies in serum. This agrees with the failure of maternal antibodies to protect mucosal surfaces against BRSV infection.

Assessment of the quality of tests for the detection of antibodies to Aujeszky's disease virus glycoprotein gE in a target population by the use of receiver operating characteristic curves.

The performance of tests for the detection of antibodies to Aujeszky's disease virus glycoprotein E (gE) in a target population was evaluated by constructing and analysing receiver operating characteristic (ROC) curves. These curves assess the discriminating ability of a test over the entire range of test signals. The advantages of applying the analysis to a sample of the target population (all commercial pigs in the Netherlands), as compared to using a panel of test sera, are that the estimates of sensitivity and specificity, the comparisons between tests and the choices of the cut-off values are all relevant for the target population. The results of a gE-ELISA in colostrum (test A) and in a single droplet of whole blood (test B) were compared with the results obtained with the same ELISA in serum (gold standard). The area under the ROC curve, which is a quantitative measure of test performance, was significantly (P < 0.01) smaller with test A than test B or the gold standard, indicating that test B performed better than test A. No significant difference was observed between test B and the gold standard.