Sensitivity of the standardized pseudorabies virus neutralization test varies with the test strain used.

The effect of altering the strain of the test virus used in the standardized pseudorabies virus neutralization (VN) test on the sensitivity of the assay was evaluated. Comparative VN tests were performed using 4 different strains: the avirulent Bartha parental, the avirulent recombinant Bartha gIIIKa, the moderately virulent Shope (currently used for the VN test at the National Veterinary Services Laboratory, Ames, IA), and the highly virulent P2208 (Funkhauser). A radioimmunoassay and a Western immunoblotting technique were employed to verify the presence of anti-pseudorabies virus (PrV) antibodies in sera. Statistical analysis indicated that replacement of the Shope strain by the Bartha gIIIKa or the P2208 strain resulted in VN titers that were 4.23- and 2.00-fold higher, respectively. Despite these differences, specificity with regard to PrV diagnosis was unaltered. This apparent enhancement of the sensitivity of the PrV VN test would be beneficial for the serologic identification of PrV-infected animals during an eradication effort.     

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.     

Transmissible gastroenteritis virus: plaques and a plaque neutralization test.

A plaquing system and plaque neutralization test in porcine thyroid cells were used to study different transmissible gastroenteritis isolates and hemagglutinating encephalomyelitis virus. Among transmissible gastroenteritis virus isolates, plaque size varied considerably and mixed size ranges sometimes occurred. The most recently isolated viruses produced smaller plaques than the laboratory viruses or hemagglutinating encephalomyelitis virus. All transmissible gastroenteritis virus isolates reacted in the plaque neutralization test with a transmissible gastroenteritis virus antiserum which showed no activity against hemagglutinating encephalomyelitis virus. Plaque neutralization results both from experimentally infected pigs and following a field outbreak demonstrated the reliability of this test and its greater sensitivity than the conventional tube test.     

Estimation of seroprevalence of encephalomyocarditis in Dutch sow herds using the virus neutralization test

Encephalomyocarditis virus (EMCV) has been found on pig farms worldwide and can cause myocarditis in young pigs and reproduction disorders in sows. So far, clinical signs of EMCV have not been reported in the Netherlands. The aim of this study was to estimate the seroprevalence of EMCV infection in Dutch sow herds. A total of 277 Dutch sow herds were randomly selected, from which 3237 serum samples were collected. These samples were tested for EMCV antibodies using the virus neutralization test (VN test). The apparent prevalence of EMCV antibodies was 9.3% in the total sow population, and the apparent herd prevalence was 58.8%. An exact determination of the prevalence of EMCV infections in the Dutch sow population was not possible because the characteristics of the VN test under field circumstances were not known. However, Dutch sow herds seem to be infected with EMCV because the distribution of positive blood samples in the tested sow population was significantly different from that expected if random false-positive reactions had occurred.     

A short incubation serum neutralization test for bovine viral diarrhea virus.

A three day serum neutralization (SN) test for the detection of antibodies to bovine viral diarrhea virus (BVDV), which is an improvement on the existing five day test, is described. The improved test results in a more rapid viral cytopathic effect and utilizes Madin Darby kidney (MDBK) cells, and horse serum as a medium supplement. A comparison of tests utilizing the NADL and the Singer strains of BVDV and the use of either secondary bovine kidney cells with calf serum (BKCS) or continuous MDBK cells with horse serum (MDHS) was performed. Analysis of the SN results of 685 serum samples from 445 Quebec and Ontario cattle showed that there was no difference, as expected, in the means of the SN antibody titers when the NADL strain was used in either the BKCS or MDHS system but SN antibody titers were elevated (p less than 0.01) when the Singer strain was used in the MDHS system. The SN test with the Singer strain also yielded significantly higher titers for sera from 200 Alberta cattle.     

Differentiation of feline coronavirus type I and II infections by virus neutralization test

Feline coronavirus (FCoV) is divided into two types I and II, based on their growth in vitro and antigenicity. In this study, virus neutralization (VN) test was applied for type differentiation of FCoV infections. Sera of cats which were clinically and serologically diagnosed as feline infectious peritonitis (FIP) possessed significantly higher VN titers to type I FCoV, and sera from cats experimentally infected with FIPV type II had high VN titers to type II but not type I viruses. A total of 79 cat sera collected in the years between 2004 and 2005 were examined to evaluate seroprevalence by the VN test, showing the following results: (1) 50 cats (63.3%) were sero-positive to FCoV; (2) of the 50 FCoV positive cat serum samples, 49 (98%) showed significantly higher titers to type I virus and only one (2%) for type II virus. These results indicate that the VN test described here can be used for serological differentiation of FCoV infections of cats, and that FCoV type I is a dominant type in recent years of Japan.     

Field experiments to evaluate the feasibility of eradication of Aujeszky's disease virus by different vaccination schedules.

In the present report, the extent of the reduction in Aujeszky's disease virus (ADV) dissemination achieved when pigs were intensively vaccinated with gI-deleted vaccines under field circumstances, was examined. On widely dispersed breeding-fattening farms, a gI-negative status was most rapidly obtained and the rate of new waves of infections was lowest when the attenuated Bartha strain was administered to both the sows and the fatteners. It was more difficult not only to reach but also to keep a gI-negative status on farms on which the sows were vaccinated with an inactivated vaccine and the fatteners with the attenuated Bartha strain or when the fattening pigs were not vaccinated at all. In a densely populated area, 9 of the 17 farms had gI-positive fatteners at the start of the intensive vaccination programme in which the attenuated Bartha strain was given to both the sows and the fatteners. Antibodies were not detected in the sera of the fatteners of each farm at some time during the experiments, but the fatteners on 7 of the 18 farms still showed antibodies against gI after 20 months of vaccination. At the end of the experiment, the percentage of fatteners with antibodies on these farms was markedly reduced compared with the percentage at the start of the experiment. Therefore, elimination of field virus may be feasible if intensive vaccination is carried out over a sufficiently long period of time. However, the high rate of reinfections experienced either due to reintroduction of the virus or to recrudescence should be a warning against too much optimism, particularly in regions with a dense swine population.     

The modification of fluorescent antibody virus neutralization (FAVN) test for the detection of antibodies to rabies virus

The fluorescent antibody virus neutralization test (FAVN) for the detection of antibodies against rabies virus was modified by using a monoclonal anti-rabies antibodies and peroxidase anti-mouse conjugate instead of a fluorescent anti-rabies conjugate. The results were read on an automatic multi-channel spectrophotometer. A total of 182 serum samples from dogs were tested by both the original and modified FAVN methods and the results were compared. Good correlation was found between the two tests. Practically, the modified FAVN test was quicker and could be used for a larger number of samples.     

Suitability of immune serums from different animal species for the preparation of conjugates against Aujeszky's disease virus]

A comparison was made of immune serum from rabbits, sheeps, pigs and cattle. Pure IgG was prepared by the Rivanol sulphate method and immune electrophoresis. Optimum protein quality, conjugation time under various conditions and using two preparations of fluorescein isothiocyanate were studied. Conjugate purified by DEAE-Sephadex A-50 and concentrated to 1% protein by ultrafiltration was freeze-dried and then tested for activity and the degree of non-specific fluorescence. Evaluation by yield of immuno-electrophoretically pure IgG, conjugation time, antibody content, degree of non-specific fluorescence and the intensity of specific fluorescence before and after dilution showed that pig serum was the best, followed by cattle and sheep serum; rabbit serum was the least suitable for conjugate preparation.     

Infectivity assay and neutralization test for equine influenza virus in microplate cell cultures

Several cell lines including Vero and MDCK cells were tested for susceptibility to the Miami and Prague strains of equine influenza virus to find cell cultures suited for study of these viruses. The viruses readily multiplied with cytopathic effects in these cell cultures. Of the cells tested, ESK cells derived from swine embryo kidney were the most susceptible to the viruses. Based on these results an infectivity assay of the viruses was worked out using ESK cell cultures prepared in microplates. The method is not only simple enough for routine use, but is also practically as sensitive as the egg inoculation method. The method was further adapted to a neutralization test.     

Improved sensitivity of the IPV-IBR virus-serum neutralization test

The serum neutralization (SN) test is still the most important method for the demonstration of infectious pustular vulvovaginitis/infectious bovine rhinotracheitis (IPV/IBR) antibodies. However, several authors have reported a relatively low sensitivity of the SN test, titers often being very low.     

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.