Indirect ELISAs based on recombinant and affinity-purified glycoprotein E of Aujeszky's disease virus to differentiate between vaccinated and infected animals

Two indirect ELISAs for the detection of antibodies against glycoprotein E (gE) of Aujeszky's disease virus (ADV) in sera have been developed. The rec-gE-ELISA is based on the E. coli-expressed recombinant protein containing the N-terminal sequences of gE (aa 1-125) fused with the glutathione S-transferase from Schistosoma japonicum. The affi-gE-ELISA is based on native gE, which was purified from virions by affinity chromatography. The tests were optimised and compared with each other, as well as with the recently developed blocking gE-ELISA (Morenkov et al., 1997b), with respect to specificity and sensitivity. The rec-gE-ELISA was less sensitive in detecting ADV-infected animals than the affi-gE-ELISA (sensitivity 80% and 97%, respectively), which is probably due to the lack of conformation-dependent immunodominant epitopes on the recombinant protein expressed in E. coli. The specificity of the rec-gE-ELISA and affi-gE-ELISA was rather moderate (90% and 94%, respectively) because it was necessary to set such cut-off values in the tests that provided a maximum level of sensitivity, which obviously increased the incidence of false positive reactions. Though the indirect ELISAs detect antibodies against many epitopes of gE, the blocking gE-ELISA, which detects antibodies against only one immunodominant epitope of gE, showed a better test performance (specificity 99% and sensitivity 98%). This is most probably due to rather high dilutions of the sera used in the indirect gE-ELISAs (1:30) as compared to the serum dilution in the blocking gE-ELISA (1:2). We conclude that the indirect gE-ELISAs are sufficiently specific and sensitive to distinguish ADV-infected swine from those vaccinated with gE-negative vaccine and can be useful, in particularly affi-gE-ELISA, as additional tests for the detection of antibodies to gE.     

Comparable sensitivity and specificity in three commercially available ELISAs to differentiate between cattle infected with or vaccinated against foot-and-mouth disease virus

Three commercially available ELISAs for the detection of antibodies to the non-structural proteins of foot-and-mouth disease virus (FMDV) were evaluated, using sera from uninfected, vaccinated, infected, inoculated, first vaccinated and subsequently infected, and first vaccinated and subsequently inoculated cattle. We compared antibody kinetics to non-structural proteins, sensitivity, and specificity. One of the ELISAs had a higher sensitivity and much lower specificity than the other two, therefore we established standardised cutoff values for the compared assays using receiver operated characteristic (ROC) curves. Using the standardised cutoff values, all three ELISAs produced comparable results with respect to sensitivity and specificity. Antibody development to non-structural proteins after infection and after vaccination/infection was not significantly different. Development of antibodies, however, both neutralising and directed to non-structural proteins, was significantly delayed after intranasal inoculation as compared to intradermolingual infection. Based on results of sera obtained after vaccination and experimental infection all three assays can be used for testing sera collected between 4 weeks and 6 months after infection. More information is needed on the prevalence of positive reactors in a situation where emergency vaccination has been used and FMD transmission was still observed.     

Development and validation of a 3ABC indirect ELISA for differentiation of foot-and-mouth disease virus infected from vaccinated animals

Non-structural protein (NSP) 3ABC antibody is considered to be the most reliable indicator of present or past infection with foot-and-mouth disease virus (FMDV) in vaccinated animals. An indirect ELISA was established, using purified His-tagged 3ABC fusion protein as antigen, for detection of the antibody response to FMDV NSP 3ABC in different animal species. The method was validated by simultaneous detection of the early antibody responses to NSP and structural protein (SP) in FMDV Asia 1 infected animals. The performance of the method was also validated by detection of antibody in reference sera from the FMD World Reference Laboratory (WRL) in Pirbright, UK, and comparison with two commercial NSP ELISA kits. The results showed that the antibody response to SP developed more quickly than that to NSP 3ABC in FMDV infected animals. In contact-infected cattle, the antibody response to NSP 3ABC was significantly delayed compared with that to SP antibody. The early antibody responses to SP and NSP 3ABC in FMDV inoculated cattle and contact-infected or inoculated sheep and pigs were generally consistent. In pigs, 3ABC antibody was linked to the presence of clinical signs; however, in sheep, subclinical infection was detected by the development of 3ABC antibodies. Therefore, the antibody responses to 3ABC varied between host species. Eight out of 10 positive serum samples from FMD WRL were tested to be positive at cutoff value of 0.2. The rate of agreement with the ceditest FMDV-NS and the UBI NSP ELISA were 98.05% (302/308) and 93.2% (287/308), respectively. The prevalence of 3ABC antibodies reached 71.4% in some diseased cattle herds. The further work is required to evaluation the performance of this method in different animal species and different field situations.     

Level of virulent virus excreted by infected pigs previously vaccinated with different glycoprotein deleted Aujeszky's disease vaccines.

Seven deleted Aujeszky's disease vaccines were compared for their ability to induce an immunity which suppresses virus excretion. For each vaccine, the levels of clinical protection and viral excretion were compared. Groups of eight pigs were vaccinated twice with attenuated deleted Aujeszky's disease vaccines (which do not express certain glycoproteins: gI, gX or gp63). Pigs were vaccinated at the beginning of the fattening period and challenge took place at the end of it when the pigs were 18-19 weeks old. Live virus vaccines were suspended in water or in an oil-in-water emulsion. The experiment was performed in three successive assays of two groups of eight pigs (except three groups for the first assay). At each assay, a control unvaccinated group of eight pigs was added to compare the effects of challenge between vaccinated and unvaccinated animals. In total, 80 pigs were involved in this experiment. All the vaccinated pigs excreted virus from 3 to 9 d after challenge. However the level of viral excretion and the duration of the period of excretion were reduced after vaccination and especially, when oil-in-water emulsion was used. There were obvious differences between vaccines. With some vaccines, when the level of viral excretion was low, the level of clinical protection was high. However, in other cases, the level of clinical protection could be good despite a higher level of viral excretion. The seroneutralizing titres were significantly and inversely related to a low level of viral excretion but not to the level of clinical protection.     

Antibody responses to inactivated vaccines and natural infection in cattle using bovine viral diarrhoea virus ELISA kits: Assessment of potential to differentiate infected and vaccinated animals

Bovine viral diarrhoea virus (BVDV) is one of the most common and economically important viral infections of cattle. As vaccination is common in most European countries, differentiation between infected and vaccinated animals is one of the key challenges facing BVDV eradication campaigns. This study was designed to compare the ability of commercial ELISA kits to differentiate antibodies generated following vaccination with four different commercial inactivated BVDV vaccines from antibodies generated following challenge with virulent BVDV. Although none of the tested vaccine–ELISA combinations was able to differentiate an infected from a vaccinated animal (DIVA) at the individual animal level, p80 blocking ELISAs, in combination with inactivated BVDV vaccines, may have some value under certain circumstances at herd level. In most cases, antibody responses to BVDV vaccines cannot be clearly distinguished from responses seen in the early phase of natural infection. No commercial BVD vaccine showed true marker qualities for DIVA using p80 blocking ELISAs.     

Serological response of pigs infected with Aujeszky's disease virus

The temporal development of antibody in four groups of pigs infected with different Aujeszky's disease virus isolates was examined. The enzyme-linked immunosorbent assay detected antibody by five to six days after infection and the antibody-dependent cell-mediated cytotoxicity assay detected antibody seven to nine days after infection. Neutralising antibody was first detected nine to 10 days after infection, whereas assays measuring complement mediated antibody lysis did not detect antibody until 10 days after infection. These results are discussed in terms of their importance to the diagnosis of and recovery from Aujeszky's disease.     

Protective efficacy of a Classical swine fever virus C-strain deletion mutant and ability to differentiate infected from vaccinated animals

Classical swine fever (CSF) continues to be the most economically damaging pig disease in the world. The disease can be effectively controlled by vaccination with the live C-strain vaccine. This vaccine, however, does not enable the serological differentiation between infected and vaccinated animals (DIVA) and its use can therefore impose severe trade restrictions. CSF-specific diagnostic ELISAs detect antibodies directed against the conserved and immunodominant A domain of the E2 structural glycoprotein. We previously reported the production of a C-strain virus in which the immunodominant TAVSPTTLR epitope of the A domain is stably mutated with the aim to render the virus suitable as a DIVA vaccine. We here report that a single vaccination with this vaccine virus protected pigs from a lethal challenge dose of the highly virulent Brescia strain. Analysis of the sera, however, demonstrated that a commercially available E2 ELISA was unsuitable as an accompanying DIVA test.     

Serological response of chickens either vaccinated or artificially infected with Haemophilus paragallinarum

The serological response of chickens either vaccinated or artificially infected with Haemophilus paragallinarum (Hpg) serovar A or C was investigated using both a specific hemagglutinin (HA) antigen and a common HA antigen. With Hpg serovar A, both vaccinated and artificially infected chickens produced hemagglutination-inhibition (HI) antibodies to Hpg serovar-specific and Hpg common HA antigens. Most chickens vaccinated with Hpg serovar C had detectable HI antibodies to both types of HA antigen by 3 weeks postvaccination, after which titers gradually declined. In contrast, most chickens artificially infected with serovar C produced HI antibodies to only the common HA antigen; very few of these chickens produced HI antibodies to the serovar-specific HA antigen.     

Sensitive glycoprotein gIII blocking ELISA to distinguish between pseudorabies (Aujeszky's disease)-infected and vaccinated pigs.

A blocking enzyme-linked immunosorbent assay (ELISA) test has been developed to distinguish pseudorabies virus (PRV) (Aujeszky's disease virus) -infected pigs from those immunized with a glycoprotein g92 (gIII) deletion mutant, PRV (dlg92dltk) [OMNIMARK-PRV]. This blocking ELISA test utilizes an anti-PRV gIII monoclonal antibody (mAbgIII)-horseradish peroxidase (HRPO) conjugate, TMB for color development and a cloned PRVg92 (gIII) antigen to coat wells of microtiter test plates. Undiluted sera are used to block the binding of the mAbgIII-HRPO conjugate to the antigen. The gIII blocking ELISA is specific and has a sensitivity comparable to screening ELISA and latex agglutination tests. PRV-negative sera and sera from pigs vaccinated once, twice, or four times with the gIII-negative vaccine all showed negative S/N values of greater than 0.70 (S/N defined as the optical density at 630 nm of test sera/optical density at 630 nm of negative control sera). Sera from PRV-infected herds, sera from pigs experimentally infected with virulent PRV, and sera from pigs vaccinated with modified-live or inactivated gIII+ vaccines were positive for gIII antibodies (S/N less than 0.7). Sera from pigs experimentally infected with 200 PFU virulent PRV seroconverted to gIII+ antibodies 7-10 days postinfection. Sera from pigs vaccinated with gpX- and gI- vaccines seroconverted to gIII+ antibodies 7-8 days after vaccination. The gIII antibodies persisted after gIII+ vaccinated for at least 376 days postvaccination. Sera from pigs protected by vaccination with PRV (dlg92dltk) and then challenge exposed to virulent PRV at 21 days postvaccination showed gIII+ antibodies by 14 days postchallenge. The specificity and sensitivity of the gIII blocking ELISA assay was further demonstrated on the United States Department of Agriculture-National Veterinary Services Laboratory (USDA-NVSL) sera from the 1988 PRV check set and the 1989 gIII PRV check set by comparing the gIII blocking ELISA assay with virus neutralization, screening/verification ELISA and latex agglutination assays.     

Air sampling procedure for evaluation of viral excretion level by vaccinated pigs infected with Aujeszky's disease (pseudorabies) virus.

Five groups of eight fattening pigs were vaccinated and then infected with Aujeszky's disease virus. Viral excretion was evaluated by two means: deep nasal swabbing and air sampling. It appeared that infectious airborne virus could be recovered from day 1 to day 6 after infection in the isolated units where control animals were raised. In vaccinated animals, airborne particles were also detected but the amount and duration varied in relation to their immune status at the day of virulent challenge: viral excretion was significantly lower in pigs presenting a high antibody level (1/16 to 1/64) just before infection. Results obtained with nasal swabs and with air samples were closely related. Despite its low sensitivity, the air sampling procedure could be considered as an efficient tool for reflecting infectious viral pressure in a confined atmosphere.     

Functional antibody responses in pigs vaccinated with live and inactivated Aujeszky's disease virus

Functional antibody tests, including virus neutralising activity of serum, antibody dependent cellular cytotoxicity and complement mediated lysis, were used to measure the response of pigs given either live or inactivated Aujeszky's disease virus vaccines. Pigs were then challenged with virulent Aujeszky's disease virus and antibody responses were analysed and found not to correlate with protection. Reasons for this lack of correlation are discussed and it is suggested that these results indicate that more emphasis should be placed on measuring the local immune response.     

Survival of Aujeszky's disease virus in infected pig slurry

It has been demonstrated that after experimental infection of pig slurry from the space under the slatted floor (infection dose of 10(6)PFU per ml), the Aujeszky's disease virus (ADV) survived for 72 hours at the temperature of 15 degrees C and at pH 6.5, but was inactivated after 96 hours. When technologically treated pig slurry from the storage tanks was saturated with water and infected with ADV at the dose of 10(5)PFU per ml, the virus survived for 23 days when kept at 15 degrees C and 4 degrees C and at pH 6.8, but was inactivated under the same conditions after 30 days. When the infective ADV dose in the technologically treated pig slurry in the storage tanks was reduced to 10(4)PFU per ml, the virus survived 16 days at +4 degrees C and pH 7.0 and 8.0 but was inactivated within 23 days after infection.     

Studies on maternally derived antibodies to Aujeszky's disease virus in piglets born to naturally or experimentally infected sows

The concentration of maternally derived antibodies to Aujeszky's disease virus was compared in 10 litters born to infected sows. Four sows had been naturally infected and six were experimentally infected. Both IgG and IgM were present in piglet sera during the first days of life. IgM antibodies went rapidly to undetectable levels; IgG persisted much longer. The duration was dependent upon the original concentration. There was a clear relationship between the clinical disease experienced by the sow and the titre of antibodies in serum and colostrum; sows which had been naturally infected or experimentally infected with a highly virulent strain passed onto their litters a high amount of specific antibodies while sows which after experimental infection presented only mild clinical signs had very low titres of neutralizing antibodies and their litters were virtually devoid of specific antibodies at 3 weeks after birth. A return to detectable levels of IgM was noticed in some litters at around 9 weeks postcolostrum intake suggesting a development of a specific immune response.     

A simple technique to differentiate between animals infected with Yersinia enterocolitica IX and those infected with Brucella abortus.

While both Brucella abortus and Yersinia enterocolitica IX have O antigens in common, they differ significantly with respect to motility. Thus Br abortus is always non-motile while Y enterocolitica is motile when grown at room temperature. The presence of yersinia H agglutinins in serum can be shown to be evidence of previous exposure to Y enterocolitica. These agglutinins are not generated by brucella infection. A rapid H agglutination test will serve to provide this differentiation without interference from cross-reacting O antigens.     

Establishment of an antibody avidity test to differentiate vaccinated cattle from those naturally infected with Mycoplasma bovis

Mycoplasma bovis is a major pathogen of bovine respiratory disease (BRD) in China and a live attenuated vaccine has recently been developed. This study aimed to establish an IgG avidity test to differentiate between naturally infected and vaccinated animals. An indirect ELISA (iELISA) was first established in the laboratory to detect antibodies specific to M. bovis using whole cell proteins as coating antigens and serum samples from experimentally infected cattle. The specificity and sensitivity of the iELISA was confirmed using a commercial ELISA kit as a reference standard. Both tests showed substantial agreement as indicated by a κ value of 0.78 (95% confidence interval, CI, 0.62, 0.93), and an overall 92.0% (80/87) agreement between the two tests. Based on the laboratory iELISA, a sodium thiocyanate (NaSCN) competitive iELISA was then developed for the detection of IgG avidity, expressed as relative avidity index (AI).Two-hundred and one experimentally immunised and naturally infected animals were used. These comprised 36 immunised calves, 38 negative control calves, 37 naturally infected calves, 87 calves of unknown status, and an additional three immunised calves that were used for a time trial. By testing true positive and negative antisera from either naturally infected or immunised calves, the AI cut-off value was defined as 70.4%. The diagnostic accuracy of the in-house NaSCN competitive iELISA was determined using serum samples collected from the experimental animals. The IgG avidity test demonstrated 96.0% sensitivity (95% CI 80.5%, 99.3%) and 95.8% specificity (95% CI 79.8%, 99.3%), and was successfully established as a valuable first test for differentiating vaccinated animals from those infected with M. bovis. This test may be a useful tool for clarifying the magnitude of M. bovis infection and in assessing the efficacy of vaccination in exposed animal populations.