Evaluation of an antigen-capture ELISA for the detection of bovine herpesvirus type 1 shedding from feedlot cattle

A total of 457 nasal swab specimens from cases of respiratory disease in 2 feed lots were evaluated for the detection of bovine herpesvirus Type 1 (BHV-1) by ELISA. Thirty-three were found to be positive for BHV-1 by the recovery of infectious virus and 21 of these were positive by ELISA, yielding a sensitivity of 64%. Fifteen other virus isolations were made and included bovine viral diarrhea viruses, rhinoviruses and parainfluenza Type 3 viruses; none of these cases were positive with the BHV-1 ELISA. Specificity of the ELISA was 100%. Eighty percent of the specimens with BHV-1 titers greater than 10(5) TCID50 were detected by ELISA; the median amount of virus in positive specimens that were detected by ELISA was 7 X 10(5) TCID50 and the median amount of virus in specimens not detected was 1.5 X 10(4) TCID50. BHV-1 infection was most frequently diagnosed in feedlot cattle that had been in the feedlot for 40-80 days. Approximately half of the infected cattle were carrying virus-neutralizing antibodies in their serum.     

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 (BHV-1), bovine leukemia virus (BLV) and bovine viral diarrhea virus (BVDV) infections in Algerian dromedary camels (<Emphasis Type="Italic">Camelus dromaderius</Emphasis>)

This study was performed to investigate the presence of bovine herpesvirus-1 (BHV-1), bovine leukemia virus (BLV) and bovine viral diarrhea virus (BVDV) infections in dromedary camels (Camelus dromaderius) kept in mixed herds with sheep and goats in Algeria, since the prevalence of BHV-1, BVDV, and BLV infections among dromedary camels in Algeria is unknown. Totally, 111 camel sera were collected from two provinces (Laghouat and Ghardaia) in Algeria. The sera were analyzed for BHV-1 specific antibodies, BVDV specific antibodies and BVDV antigen using the ELISA, and BLV nucleic acid using PCR. The seropositivity rate was 9.0% for BVDV-specific antibody, although 41.4% of camels tested were positive for BVDV antigen. Moreover, there was no evidence of BHV-1 and BLV infections. The results indicated that camels might represent an important source for BVDV infection in all ruminants, including cattle, sheep, and goats bred in mixed herds in Algeria, since they had a higher BVDV prevalence rates. Therefore, the prevention and control measures for BVDV infection should put in place in camel populations to limit the spread of BVDV infection to ruminant populations in Algeria.     

Specificity of the indirect enzyme-linked immunosorbent assay for detection of pseudorabies virus antibodies in pigs exposed to bovine herpesvirus-1

Six 5-week-old pigs were inoculated intranasally (IN) with 10(7.6) TCID50 of bovine herpesvirus-1 (BHV-1). Three of the pigs also were inoculated IV with a similar dose of BHV-1. Clinical responses were not observed in these 6 pigs before oronasal challenge exposure with 10(7.8) TCID50 of virulent pseudorabies virus (PRV) at postinoculation day 42. Two pigs inoculated IN with BHV-1 and challenge exposed with PRV remained healthy, whereas the remaining 4 pigs developed severe clinical signs of pseudorabies and were moribund at postinoculation day 50 (8 days after challenge exposure). Anti-BHV-1 antibodies were demonstrable by ELISA in all 6 pigs and by serum neutralization (SN) in 5 pigs before challenge exposure with PRV. Anti-PRV antibody was not detected by ELISA or SN before challenge exposure to PRV. After challenge exposure to PRV, pigs with humoral antibody to BHV-1 responded anamnestically, and anti-PRV antibody activity was demonstrable by ELISA and SN in the 2 surviving pigs.     

Antibody response to glycoprotein E after bovine herpesvirus type 1 infection in passively immunised, glycoprotein E-negative calves

This study was conducted to determine whether young calves with maternal antibodies against bovine herpesvirus type 1 (BHV-1) but without antibodies against glycoprotein E (gE) can produce an active antibody response to gE after a BHV-1 infection. Five calves received at birth colostrum from gE-seronegative cows which had been vaccinated two or three times with an inactivated BHV-1, gE-deleted marker vaccine. After inoculation with a wild-type virulent strain of BHV-1, all the passively immunised gE-negative calves shed virus in large amounts in their nasal secretions. All the calves seroconverted to gE within two to four weeks after inoculation and then had high levels of gE antibodies for at least four months. The development of an active cell-mediated immune response was also detected by in vitro BHV-1-specific interferon-gamma assays. All the calves were latently infected, because one of them re-excreted the virus spontaneously and the other four did so after being treated with dexamethasone. The results showed that under the conditions of this work the gE-negative marker could also distinguish between passively immunised and latently infected calves.     

Bovine herpesvirus type 1 abortions detected by a semi-nested PCR in Brazilian cattle herds

Bovine herpesvirus type 1 (BHV-1) was investigated by a semi-nested polymerase chain reaction (SN-PCR) and by MDBK cell culture virus isolation in organ fragments from 55 aborted fetuses collected from beef and dairy cattle herds with history of reproductive problems in the North of Paraná State, Brazil. A 425 bp amplicon of the BHV-1 glycoprotein D gene was detected in 14 (25.4%) aborted fetuses. BHV-1 was isolated in MDBK cells from the tissue of 5 (9.1%) fetuses. The specificity of positive results was evaluated by Restriction Fragment Length Polymorphism (RFLP) with Bgl I restriction of DNA amplified by SN-PCR, and by virus-neutralization and immunofluorescence with rabbit anti-BHV-1 polyclonal antibodies for virus isolated in cell culture. The results of this work demonstrate the importance of using other diagnostic techniques, like SN-PCR, for BHV-1 detection in organ fragments from aborted fetuses and the high frequency of this virus in reproductive failures in Brazilian cattle herds.     

Immunological relationships between malignant catarrhal fever virus (alcelaphine herpesvirus 1) and bovine cytomegalovirus (bovine herpesvirus 3)

Strains of malignant catarrhal fever virus (alcelaphine herpesvirus 1 (AHV-1)) and bovine cytomegalovirus (bovine herpesvirus 3 (BHV-3)) were compared for serological relatedness by cross-titration in an indirect immunofluorescent (IIF) antibody assay. There was definite cross-reactivity between these 2 viruses, with heterologous sera staining intracellular and membrane antigens of infected cells. Heterologous antibody titres were approximately 50-fold lower than homologous titres and could be removed by absorption with either homologous or heterologous virus-infected cells, but not with uninfected cells. Regression analyses of IIF antibody titres to AHV-1 and BHV-3 virus in 3 groups of wild ungulate sera also indicated a serological relationship between these herpesviruses. In a cross-immunity trial, 2 of 3 cattle immunized with a BHV-3 virus and 2 of 3 cattle immunized with avirulent AHV-1 resisted challenge with virulent AHV-1-infected blood which killed 3 unimmunized controls. These results are discussed particularly with respect to the involvement of BHV-3 in malignant catarrhal fever.     

Characterization of monoclonal antibodies to bovine herpesvirus type I, Los Angeles strain.

We produced monoclonal antibodies (mAbs) against bovine herpesvirus type 1 (BHV-1), Los Angeles strain, and then evaluated them as potential candidates for preparing diagnostic reagents. Of the 318 cell lines expressing antibodies to the virus, 60% (192) secreted IgG and 40% (126) secreted IgM. Twenty-six mAbs were selected based on enzyme-linked immunosorbent assay (ELISA) and virus neutralization (VN) titers and characterized by immunoprecipitation, immunofluorescence and immunoblots. The selected mAbs were assigned to one of four groups based on their immunoprecipitation patterns. Group A (4 mAbs) precipitated a complex of three glycoproteins with molecular weight (MW) 130 kDa, 72 kDa and 55 kDa, which presumably represented gI of BHV-1. Monoclonal antibodies of this group were highly reactive in ELISA but had low VN titers. Group B (4 mAbs) precipitated a glycoprotein with MW of 71 kDa (gIV). This group of mAbs had high VN titers. Group C (16 mAbs) precipitated a 97 kDa glycoprotein (gIII). Monoclonal antibodies of this group had high ELISA but low VN titers. Group D (2 mAbs) precipitated a double band of non-glycosylated proteins with MW of 37/32 kDa; these proteins could not be assigned to any of the antigens of BHV-1 previously described. ELISA and VN titers of this group of mAbs were low. To test the antigenic variability of the antigenic determinants which were recognized by these 4 groups of mAbs, we adapted Madin Darby bovine kidney cell-propagated BHV-1 Los Angeles strain to Crandell's feline kidney cell line. After the tenth passage in feline kidney cells, the epitopes on the 37/32 kDa peptide recognized by the mAbs group D were no longer detectable. Additional changes were noted in the electrophoretic mobility of the 130 kDa and 71 kDa glycoproteins (gI) identified by mAb of group A shifted downward. The 71 kDa glycoprotein (gIV) reactive with mAb group B and the 97 kDa (gIII) reactive with mAb group C remained stable. Since clone No. 191 of group B mAb was potent in ELISA, VN, immunoblots and immunofluorescence, and recognized an epitope which did not change under selective pressure, we feel that the mAb produced by this clone are a good candidate for the production of diagnostic reagents.     

A study of some biologic properties of Bovid herpesvirus-4.

This article summarizes the results of a study on several strains of Bovid herpesvirus-4 (BHV-4), isolated from cattle. The study had several objectives, namely, to verify (a) the disease-causing potential of the virus, (b) the possibility by BHV-4 to induce a latent infection in the natural host and (c) the entity of the relationships among strains of the virus isolated from different disease syndromes. The following data were obtained: (1) All strains tested were able to replicate in experimentally infected calves; however, only one strain (85/BH 16TV) caused an overt systemic disease. (2) The nervous system as well as the lymphoid structures appeared to be the target organs for replication of the virus. (3) BHV-4, like other herpesviruses, was able to establish latent infection in cattle. (4) When two strains of the virus, isolated from cattle affected by different disease syndromes, i.e. respiratory disease (strain DN-599) or vulvovaginitis (strain 85/BH 16TV), respectively, they resulted to be closely related to each other. In particular, they revealed a similar DNA pattern and both strains were able to cause respiratory disease in calves. Moreover, the two viral strains were mutually protective in that calves were generally found to be refractory to challenge inoculation with either the homologous or the heterologous virus. (5) All BHV-4 strains tested generally failed to evoke a significant production of neutralizing antibody in the experimental calves.     

Dynamics of bovine herpesvirus type 1 infection in Estonian dairy herds with and without a control programme

Bovine herpesvirus type 1 (BHV-1) is an important bovine pathogen, exacerbating poor health and the productivity of cattle. The aims of this study were to detect the efficacy of vaccination programmes in lowering the seroprevalence of BHV-1 gE within the dairy herd and to follow the dynamics of the infection in non-vaccinated herds with uninfected heifers. A two-year longitudinal study was carried out on seven herds that were vaccinated, and in five herds with uninfected heifers without applying a control programme. After the start of the vaccination programme, calves born remained free from the virus. However, in one herd, 7 per cent (95 per cent CI 2 to 18) of these animals showed antibodies to BHV-1 two years after the first vaccination. A decline in BHV-1 antibody prevalence was found in vaccinating herds. Among the five herds not under the control programme, one experienced active virus spread, although one herd experienced self-clearance of the virus. In the herds with high BHV-1 prevalence, vaccinating all cattle from three months of age twice a year with a commercial inactivated marker vaccine efficiently protected offspring from becoming infected, and lowered the prevalence of BHV-1 within the herd. A small proportion of herds may experience self-clearance of the virus.     

Detection of BHV-1 in a Naturally Infected Bovine Fetus by a Nested PCR assay

Bovine herpesvirus 1 (BHV-1) is frequently associated with abortion in naturally and experimentally infected cattle. Most of the virus isolation and immunofluorescent antibody protocols described in the literature for detecting BHV-1 in bovine foetuses are rather laborious, costly and time-consuming. The detection is described of BHV-1 in the tissues of a naturally aborted bovine foetus by a nested PCR assay with no further hybridization procedures.Optimal results were achieved by filtering the foetal tissues on a chromatography column before DNA extraction, by using two pairs of primers in a nested PCR and by evaluating the amplification products on silver-stained polyacrylamide gels.This nested PCR was faster and easier to perform than the virus isolation test. To our knowledge, this is the first time that BHV-1 has been detected in the tissues of a naturally infected bovine foetus by means of a nested PCR. The test seems to be a practical alternative for rapid detection of BHV-1 in bovine foetus.     

Bovine herpes virus expressing envelope protein (E2) of bovine viral diarrhea virus as a vaccine candidate.

The gene encoding the envelope protein (E2) of bovine viral diarrhea virus (BVDV) was expressed under the thymidine kinase (TK) promoter of Korean bovine herpesvirus 1 (BHV-1) isolate. Thymidine kinase negative (TK-) BHV-1 recombinants expressing E2 of BVDV were constructed and the expression of E2 was identified by immunofluorescence and Western blotting. Compared to wild type BHV-1, the recombinant BHV-1 had a delayed cytopathogenic effect in cells. The immunogenicity of the recombinant BHV-1 was examined in guinea pigs and cattle. Although an increase in body temperature was detected for a few days, the inoculated cattle returned to normal temperature with the development of neutralizing antibodies to BVDV.     

Efficacy of an experimental BHV-1 subunit gIV vaccine in beef calves challenged with BHV-1 in aerosol.

Thirty-six beef calves were used to test the efficacy of an experimental truncated BHV-1 glycoprotein (tgIV) vaccine. Calves from 1 source and +/- 1 mo of age were randomly divided into 4 groups: 1) control (adjuvant VSA3), 2) vaccinated with tgIV at 3 and 4 mo of age, 3) vaccinated with tgIV at 3 and 7 mo of age, or 4) vaccinated with tgIV at 6 and 7 mo of age. Calves were challenged with BHV-1 in aerosol (strain 108) at 7 1/2 mo of age. Prior to challenge, serum neutralizing (SN) antibody titers to BHV-1 were significantly (P < 0.05) higher in all vaccinated calves than in controls. Calves vaccinated at 3 and 7, or 6 and 7, mo of age had significantly (P < 0.05) higher SN antibody and nasal antibody titers to BHV-1 and ELISA (enzyme linked immunosorbent assay) titers to gIV at prechallenge than those vaccinated at 3 and 4 mo of age or controls. Postchallenge nasal shedding of BHV-1 occurred only in controls and those vaccinated at 3 and 4 mo of age. Control calves lost significantly (P < 0.05) more weight and had higher sick scores after challenge than those vaccinated at 3 and 7, or at 6 and 7, mo of age. There were strong correlations (P < 0.001) between antibody titers, virus shedding, and sickness.     

Prevalence of bovid herpesvirus-4 and its antibody in cattle in Minnesota

Serologic analyses and virus isolation studies were carried out to determine the role of bovid herpesvirus-4 (BHV-4) in infections in cattle, principally those of the reproductive tract. Serologic analyses were performed, using an indirect fluorescent antibody test on thoracic fluid specimens from aborted fetuses and on sera from 3 sources of adult cattle. Virus isolation was attempted from field cases of abortion, early embryo death, and postpartum vulvovaginitis/metritis, using uterine discharge and buffy coat preparations obtained from cows and tissues obtained from aborted fetuses. Of 420 fetal thoracic fluid specimens examined, 5 were positive for BHV-4 antibodies. Seventeen percent of adult cattle from 2 sources ie, clinically normal herds and abattoir cattle, were seropositive for BHV-4 antibodies. Cattle from a third source, 4 herds with high incidence of reproductive tract disorders, had a seroprevalence rate between 36 and 88%. Two isolates of BHV-4 were also obtained from this group. The overall incidence of BHV-4 antibodies in clinically normal cattle was higher than previously recognized, with relatively higher prevalence in herds having reproductive problems (chi 2 = 156.5, P less than 0.005). At least 10% of the BHV-4 antibody-positive sera did not have neutralizing antibody against bovine viral diarrhea virus and/or bovid herpesvirus-1, both important causes of bovine reproductive tract disorders.     

Virological and serological evidence of bovine herpesvirus type 4 in cattle in Northern Ireland

Bovine herpesvirus type 4 (BHV-4), a member of the genus Rhadinovirus, subfamily Gammaherpesvirinae, within the family Herpesviridae, was isolated in fetal bovine lung cells from samples of vaginal discharge taken from a dairy herd in which approximately 50 per cent of the cattle developed metritis after calving. The identity of the isolate was confirmed by immunofluorescent staining with a BHV-4-specific monoclonal antibody and partial sequencing of a portion of the glycoprotein B gene. Serological testing failed to demonstrate a significant association between the exposure of the cattle to BHV-4 and the metritis, but several cattle seroconverted during the periparturient period, consistent with the recrudescence and shedding of virus associated with the stresses of parturition and the onset of lactation. Despite the previous failure to detect BHV-4 in Northern Ireland, a serological survey of 999 cattle in 49 dairy herds and 51 beef herds found widespread evidence of exposure: 29 of the dairy herds and 35 of the beef herds contained one or more seropositive cattle, and 33.3 per cent of the dairy cattle and 23.3 per cent of the beef cattle were positive.     

Comparison of the herpesviruses of cattle by DNA restriction endonuclease analysis and serologic analysis

Reference strains and field isolates of herpesviruses recovered from cattle in the United States were compared by restriction endonuclease (RE) analysis and the indirect fluorescent antibody test. As a result of these comparisons, 5 major biotypes of bovine herpesvirus (BHV) were defined. These types were (i) infectious bovine rhinotracheitis virus (BHV-1), (ii) bovine herpes mammillitis virus (BHV-2), (iii) malignant catarrhal fever (MCF) virus (herpesvirus alcelaphinae), (iv) the group of slow-growth isolates represented by the prototype strain Movar 33/63 (bovine cytomegalovirus candidate), and (v) the syncytia-forming Pennsylvania 47 strain. Bovine herpesvirus-1 and BHV-2 did not cross-react serologically with any other type of BHV tested. A low, but consistent level of serologic cross-reactivity was detected among MCF virus, the Movar group, and Pennsylvania 47. Several nonsyncytial, slow-growth strains, which were recovered from dissimilar clinical syndromes and were serologically related to Movar 33/63, exhibited similar DNA RE cleavage patterns, confirming their identity as members of a single type. There was no isolate from American domestic cattle similar to the African MCF virus, which has been sporadically isolated from exotic ruminants in the United States. The African MCF virus isolated during a MCF epizootic in a United States zoo exhibited some DNA RE cleavage differences in comparison with the MCF virus world prototype strain WC 11, indicating that strain diversity exists within this biotype.     

Induction of immune responses in cattle with a DNA vaccine encoding glycoprotein C of bovine herpesvirus-1

A DNA vaccine expressing glycoprotein C (gC) of bovine herpesvirus-1 (BHV-1) was evaluated for inducing immunity in bovines. The plasmid encoding gC of BHV-1 was injected six times intramuscularly or intradermally into calves at monthly intervals. After immunization by both routes neutralizing antibody and lymphoproliferative responses developed. The responses in the intradermally immunized calves were better than those in calves immunized intramuscularly. However, the intradermal (i.d.) route was found to be less efficacious when protection against BHV-1 challenge was compared. Following intranasal BHV-1 challenge, all immunized calves demonstrated a rise in IgG antibody titre on day 3, indicating an anamnestic response. The control non-immunized calf developed a neutralizing antibody response on day 7 post-challenge. The immunized calves showed a slight rise in temperature and mild clinical symptoms after challenge. The intramuscularly immunized calves showed earlier clearance of challenge virus compared with intradermally immunized calves. These results indicate that DNA immunization with gC could induce neutralizing antibody and lymphoproliferative responses with BHV-1 responsive memory B cells in bovines. However, the immunity developed was not sufficient to protect calves completely from BHV-1 challenge.     

Detection of bovine herpesvirus-1 in peripheral blood mononuclear cells eight months postinfection.

Peripheral blood mononuclear cells (PBMCs) from 5 calves (3 controls and 2 vaccinates) used in a bovine herpesvirus 1 (BHV-1) vaccine study with a BHV-1 Cooper strain challenge were collected 6 months after challenge. The PBMCs from the control animals were positive by immunofluorescence for the BHV-1 glycoprotein D (gD) while the vaccinates were negative. The PBMC samples from 4 of the 5 animals were examined for BHV-1 DNA by polymerase chain reaction (PCR) and for gD immunofluorescence at 8 months after challenge. The BHV-1 DNA and viral antigen were detected in PBMC samples at 8 months postinfection, but no virus was isolated.     

A live bovine herpesvirus-1 marker vaccine is not shed after intramuscular vaccination

It should be established, whether animals vaccinated intramuscularly (IM) with a live Bovine herpesvirus type 1 (BHV-1) marker vaccine become viremic and/or excrete vaccine virus with nasal discharge. Five cattle, seronegative for BHV-1, were vaccinated with an overdose of the vaccine (Bovilis IBR marker live) via the IM route. Nasal swabs and blood samples were taken at regular intervals and tested for BHV-1 in a virus infectivity assay. In addition, a polymerase chain reaction (PCR) specific for BHV-1 DNA was performed on the blood samples. BHV-1 neutralizing antibody titres were determined in the sera taken prior to the vaccination and four weeks after immunisation. AIl animals were successfully vaccinated as judged by the development of BHV-1 neutralising antibodies. However, all nasal swab samples were tested negative for vaccine virus, and all blood samples were found negative for BHV-1 vaccine virus and BHV-1 specific DNA. From these data it can be concluded that the vaccine virus was not excreted with nasal discharge after IM vaccination and that the vaccinated animals did not have a detectable viremia. Therefore, it is recommended to apply the tested BHV-1 marker live vaccine by the IM route in situations where it is undesirable that the vaccine virus is excreted.