A comparative evaluation of two sensitive serum neutralization tests for bovine herpesvirus-1 antibodies.

Two sensitive serum neutralization (SN) tests for the detection of antibodies to bovine herpesvirus-1 (BHV-1) in bovine sera were evaluated. Both SN tests used a 24 h incubation of test sera with 100 CCID50 of BHV-1 before the addition of susceptible cells. The tests differed in the presence (C test) or absence (D test) of complement and were compared with a standard 1 h incubation SN test and the enzyme-linked immunosorbent assay (ELISA). Although the mean titer of the C test was twofold higher than the mean titer of the D test for 310 sera, the number of samples which were negative was not significantly different between tests. For 100 sera from herds with known reactors, which were negative in a 1 h incubation SN test, 32% tested positive in the C and D tests. Other investigations, including Western immunoblotting and radioimmune precipitation, suggest that the 24 h incubation tests produce some false positive results. In contrast, the 1 h incubation SN test and, to a much lesser extent, the ELISA appear to produce some false negative results. The C test was more sensitive than the D test for detecting an early immune response after experimental infection.     

Prevalence of serum antibodies to bovine herpesvirus-1 and bovine viral diarrhea virus in beef cattle in Uruguay

Our objective was to determine the prevalence of serum antibodies to bovine herpesvirus-1 (BHV-1) and bovine viral diarrhea (BVD) virus in beef cattle in Uruguay. A random sample of 230 herds selected with probability proportional to population size based on the number of cattle was chosen from a list frame of all registered livestock farms as of June 1999. Sera from up to 10 heifers, cows and bulls (up to 30 sera total per herd) were collected on selected farms between March 2000 and March 2001 and evaluated by means of enzyme-linked immunosorbent assays (ELISAs). Overall, 6358 serum samples were evaluated. We also collected data on previous diagnosis of BHV-1 or BVD infections and on the use of vaccines against these agents.

The estimated prevalence of exposure to BHV-1 and BVD at the herd level for the Uruguayan beef population was 99% and 100%, respectively. Approximately 37% of beef cattle in Uruguay have been exposed to BHV-1 and 69% to BVD virus. Only 3% of beef herds in Uruguay regularly (typically, annually) use vaccines against either of these agents.     

Infection of early bovine embryos with bovine herpesvirus-1

Recently hatched bovine embryos were exposed in vitro to 1 of 4 strains of bovine herpesvirus-1 to determine whether the viruses would replicate in these embryos and, if so, what pathologic consequences would ensue. Exposure to each of the viruses resulted in embryonic infection and death, and replication of the agents was demonstrated by electron microscopy and titration of progeny virus. There were no dramatic differences between virus strains in pathogenicity or in the ultrastructural pathologic findings of infection.     

Protective effect of inactivated bovine herpesvirus-1 in calves experimentally infected with bovine herpesvirus-1 and Pasteurella haemolytica.

The protective effect of an inactivated whole-virion bovine herpesvirus-1 (BHV-1) immunising inoculum, without adjuvant, against viral-bacterial respiratory disease was studied in three experimental treatment groups of five calves each. One group was boosted 14 days after the first vaccination and at this time the second group received their initial inoculation. Seven days later, calves were challenged with BHV-1 in aerosol and four days after this challenge all calves were exposed to Pasteurella haemolytica A1 in aerosol. Among the three groups, differences in rectal temperature responses four days after viral challenge (P less than 0.01) did not relate to protection. However the main response variable, viral-bacterial pneumonia, was reduced in boosted calves (P less than 0.05).     

Characteristics of live bovine herpesvirus-1 vaccines

The common disease caused by bovine herpes virus 1 infection is febrile rhinotracheitis (FRT) and under certain conditions the virus is strongly implicated in pre-disposing cattle to pneumonic pasteurellosis. These illnesses account for a significant economic loss in the cattle industry worldwide and vaccination is widely applied. In naive cattle, and for a short period of time, old and new vaccines significantly reduce clinical signs of FRT and also virus shedding. A single intranasal vaccination affords significant protection in face of maternally derived antibodies, and the protection can be significantly prolonged by a booster intramuscular vaccination. Current data recommend vaccination in face of an outbreak and vaccines appear safe for pregnant cattle.     

Risk factors for bovine herpesvirus-1 seropositivity

This paper reports the investigation of risk factors for bovine herpesvirus-1-seropositivity, based on a cluster-sample survey of the Belgian cattle population. This serosurvey was carried out in 1998 in 309 randomly selected unvaccinated herds of all types (dairy, mixed and beef) were all bovids (N = 11,284) were sampled.

Older and male cattle had higher seroprevalence. Origin (homebred or purchased) and herd size interacted; for smaller herds (≤50 cattle on the premises), purchase status and larger herd size were risk factors, whereas these effects were not observed for larger herds.     

Prevalence of bovine herpesvirus-1 in the Belgian cattle population

The national bovine herpesvirus 1 (BHV-1) seroprevalence (apparent prevalence) in the Belgian cattle population was determined by a serological survey that was conducted from December 1997 to March 1998. In a random sample of herds (N=556), all cattle (N=28 478) were tested for the presence of antibodies to glycoprotein B of BHV-1. No differentiation could be made between vaccinated and infected animals, because the exclusive use of marker vaccines was imposed by law only in 1997 by the Belgian Veterinary Authorities. Twenty-one percent of the farmers vaccinated continuously against BHV-1.

In the unvaccinated group, the overall herd, individual-animal and median within-herd seroprevalences were estimated to be 67% (95% confidence interval (CI)=62–72), 35.9% (95% CI=35.0–36.8) and 33% (quartiles=14–62), respectively.

Assuming a test sensitivity and specificity of 99 and 99.7%, respectively, the true herd, individual-animal and median within-herd prevalence for the unvaccinated group of herds were estimated to be 65, 36 and 34%, respectively. The true herd prevalence for dairy, mixed and beef herds were respectively, 84, 89 and 53%; the true individual-animal prevalence for those types of herds were, respectively, 35, 43 and 31%; whereas, the true median within-herd prevalences were 36, 29 and 38%.     

Isolation of bovine herpesvirus-1 from vesicular lesions of the bovine udder

Bovine herpesvirus-1 was isolated from vesicular lesions on the udder and mammary papillae (teats) of a Charolais cow. Lesions on the animal consisted of papules and vesicles up to 10 mm in diameter. The virus was identified by fluorescent antibody and serum-neutralization tests.     

Experimental latent and recrudescent bovine herpesvirus-1 infections in calves

Latent bovine herpesvirus-1 (BHV-1) infection was established in 6 calves and was demonstrated by reinduction of virus shedding after administration of corticosteroids. Latently infected calves failed to transmit BHV-1 during 4 weeks' contact with sentinel calves. Infected calves were killed and necropsied during latency or induced recrudescence. The BHV-1 DNA was demonstrated intranuclearly in trigeminal ganglion neurons by in situ hybridization. The BHV-1 antigen was demonstrated by immunofluorescence in trigeminal ganglion neurons during recrudescence. By electron microscopy, changes in the appearance of the Nissl bodies and a high frequency of nuclear bodies were observed in trigeminal ganglion neurons.     

Characterization of anti-idiotype reagents to bovine herpesvirus-1 monoclonal antibody

A monoclonal antibody (MAb) to a neutralization epitope on the 97-kD glycoprotein of bovine herpesvirus-1 (BHV-1) was used to prepare an anti-idiotypic antibody in rabbits. Purified F(ab')2 fragments of the MAb were used to immunize the animals and the sera containing the greatest anti-idiotype activity were identified by ELISA. After digestion of the immunoglobulins with pepsin and purification by affinity chromatography, anti-idiotype F(ab')2 fragments reacted specifically with the MAb in ELISA. Binding of the anti-idiotypic (anti-id) antibody was inhibited by preincubation of the MAb with BHV-1. Using an ELISA inhibition assay with BHV-1, the anti-id reagent inhibited the binding of anti-BHV-1 MAb to BHV-1, suggesting that the anti-id mimics an epitope of the 97-kD glycoprotein by binding the antigen combining site of the MAb. Development and characterization of this anti-id and future studies of its immunomodulatory effects are discussed.     

Bovine herpesvirus-1 vaccination against experimental bovine herpesvirus-1 and Pasteurella haemolytica respiratory tract infection: onset of protection

The onset of protection offered by intranasal vaccination with attenuated bovine herpesvirus-1 (BHV-1) was studied in 18 calves given a virulent BHV-1 aerosol challenge inoculum and an aerosol challenge exposure to Pasteurella haemolytica. Calves challenge exposed with virus 3, 7, 11, 15, or 19 days after vaccination and challenge exposed 4 days later with Pasteurella haemolytica did not develop viral-bacterial pneumonia, whereas 2 of 3 control calves died of fibrinous bronchopneumonia 40 and 60 hours after the bacterial aerosol and the 3rd control calf had similar lesions. All vaccinated and control calves had detectable amounts of interferon at the time of viral challenge exposure. Protection was observed before detection of neutralizing antibodies to BHV-1 in nasal secretions or in serum. Protection was therefore present from day 3 through day 19 after vaccination, but the mechanism could not be explained completely by neutralizing antibody or interferon.     

Bovine monoclonal antibodies specific for bovine herpesvirus-1 glycoprotein gIII

Spleen cells from a calf immunized with bovine herpesvirus-1 (BHV-1) were fused with the nonsecreting murine cell line SP2/0. Several bovine-murine hybridomas secreting bovine immunoglobulins were stabilized. Of these, 9 hybridomas secreted bovine monoclonal antibodies that specifically bound to BHV-1 in a radioimmunoassay. Two of these monoclonal antibodies reacted specifically with BHV-1 in an indirect fluorescent antibody test and immunoprecipitated a BHV-1 glycoprotein with molecular mass of 97 kilodaltons.     

Characterization of promoters integrated in the genome of bovine herpesvirus-1 (BHV-1).

Bovine herpesvirus-1 (BHV-1) has been used as a vector of live recombinant vaccines for cattle which express the genes of other pathogens. Because of the importance of the choice of the promoter which allows the efficient expression of the foreign genes in the BHV-1 vector, we compared the relative efficacy of various promoters integrated in the BHV-1 genome. The promoter sequences of the BHV-1 thymidine kinase (tk), gB, gC, SV40 early, and pseudorabies virus (PRV) immediate early (IE) genes were placed at the upstream of the open reading frame of the chloramphenycol acetyl transferase (CAT) gene and the promoter-CAT sequences were integrated into the tk gene of BHV-1 by homologous recombination. The promoter activity was assayed by measuring the CAT activity in the extracts of Madin Darby bovine kidney (MDBK) cells infected with the recombinant BHV-1. The PRV IE promoter was activated earlier and maintained at a higher level activity than the BHV-1 gB or gC promoters throughout the most of the growth phase of BHV-1. At the late phase, however, the activities of the BHV-1 gB and gC promoters reached the higher level. The BHV-1 tk promoter activity was low and the SV40 early promoter was hardly activated when integrated into the BHV-1 genome. promoter, recombinant BHV-1.     

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.     

Dynamics of natural bovine herpesvirus-1 (BoHV-1) infection in a dairy herd

In this study, natural cycling of BoHV-1 infection was investigated in two groups of dairy cattle containing 2120 head. Group 1 comprised 127 animals and they were monitored for BoHV-1 infection virologically and serologically in six consecutive sampling periods. It consisted of naive heifers between 6 and 8 months of age, while in group 2, age, sex and the BoHV-1 serostatus of the animals were disregarded. The animals in group 1 were found to have seroconverted at the second sampling. Results of the serological study showed slight antibody response after natural BoHV-1 infection in the herd and neutralizing titres fell below protective levels in the 6–8 months after the peak. During the 2-year study period, one recurrence was detected after primary infection. Virus isolation studies revealed a cytopathic effect indicative of BoHV-1 in two nasal swabs taken during the fifth sampling period from animals with mild upper respiratory tract symptoms. As the study was carried out under natural conditions, it is not known whether the viruses isolated were from recurrences or re-infections. Data from cross-neutralization tests with herd isolates showed higher antibody response than those with the reference virus. The dynamics of BoHV-1 in both groups were found to be statistically similar.     

Rationale and perspectives on the success of vaccination against bovine herpesvirus-1

Several characteristics of BHV-1 have contributed to the successful development of both conventional and marker vaccines. BHV-1 is a stable virus, which grows to high titers in vitro, has a limited host range and causes acute viremic infections. Furthermore, the protective antigens, as well as the antigens that are suitable as marker, are present in the predominant virus isolates and induce significant and long-lasting immune responses, both in na?ve and in previously vaccinated animals. In many parts of the world including North-America control of BHV-1 is achieved by vaccination with conventional attenuated or inactivated vaccines. With parts of Europe being BHV-1 free, the ability to differentiate infected from vaccinated animals has become critical as a trading tool. Live and killed gE-deleted marker vaccines are now widely used in Europe, in combination with gE-based diagnostic tests to monitor cattle. However, several issues remain to be resolved. BHV-1 causes latency, which creates a need for stringent management practices in case eradication is to be achieved. Since intramuscular delivery with a syringe and needle leads to considerable tissue damage, needle-free delivery methods should be adopted for beef cattle. Furthermore, conventional inactivated and attenuated vaccines are less efficacious in neonates, so alternative vaccine types such as CpG adjuvanted protein vaccines or DNA vaccines are required for effective vaccination of this age group.