Detection of bovine herpesvirus-1-specific IgM using a capture enzyme immune assay with isotype-specific monoclonal antibodies

The detection of virus-specific immunoglobulin M (IgM) antibodies in acute-phase serum samples offers the possibility of making an accurate and rapid serologic diagnosis. We have developed a solid-phase capture assay that uses murine monoclonal antibodies specific for bovine IgM to separate the whole IgM fraction of a bovine serum sample. The IgM specific for bovine herpesvirus-1 (BHV-1) is then detected by the addition of viral antigen, which in turn is detected by BHV-1-specific monoclonal antibodies conjugated to horseradish peroxidase. A BHV-1 IgM antibody response was detected during the early postinfection period (7-40 days PI). Bovine herpesvirus-1 IgM antibody was not detected in sera taken from 3 animals following dexamethasone-induced viral reactivation. This method compares favorably with viral isolation, antigen detection in the clinical samples, and paired serology in the diagnosis of BHV-1 infection at a herd level.     

Detection of bovine herpesvirus type 1 via an immunoperoxidase method, using monoclonal antibodies

The sensitivity and specificity of the immunoperoxidase technique, using monoclonal antibodies, for the detection of bovine herpesvirus type 1 (BHV-1) was assessed and compared with viral isolation methods. In this study, BHV-1 antigens were detected in impression smears of brain obtained from calves in which BHV-1 was isolated. False-positive results were not observed after double-blind examination. Preliminary identification of isolates as antigenic variants was possible by use of 3 monoclonal antibodies reactive with neurotropic and/or pneumotropic strains of BHV-1. Results were consistent with previous work in which characterization was performed by use of immunofluorescense and ELISA. The immunoperoxidase technique, using monoclonal antibodies, was determined to be specific and sensitive, compared with viral isolation, for the diagnosis of BHV-1 encephalitis. In addition, it has operative advantages in that the assay does not require tissue culture facilities, and results can be obtained within hours after specimens are obtained.     

Bovine T lymphocyte response to bovine herpesvirus-1: cell phenotypes, viral recognition and acid-labile interferon production

The phenotype of bovine cells that proliferate to bovine herpesvirus (BHV-1) were identified by peanut agglutinin (PNA) and monoclonal antibodies with specificity for Pan T cells (B29), a T cell subset (B24), and an MHC class II (Ia-like) antigen (R-1). PNA+ cells but not PNA- cells separated by fluorescent activated cell sorting responded to BHV-1. Virally-activated T cells expressed MHC class II antigens, and possibly antigen specific receptors for virus. Binding of radiolabeled virus increased six-fold beyond the expected value for activated cells suggesting specificity of the lymphocytes for the virus. Finally, cells that responded to BHV-1 produced high levels of acid sensitive interferon. Taken together these results characterize the phenotypes of bovine lymphocytes that interact with BHV-1.     

Study of bovine herpesvirus type 1 strains with monoclonal antibodies.

Fourteen strains of bovine herpesvirus type 1 (BHV-1, IBRV) representing all three groups of BHV-1 (BHV-1.1, BHV-1.2, BHV-1.3) were studied by ELISA using 106 monoclonal antibodies (Mabs) produced against BHV-1. On the basis of the ELISA, the Mabs could be divided into three groups. The first group (40 Mabs, 38%) reacted with all strains, the second group (43 Mabs, 41%) with the respiratory and genital strains (BHV-1.1 and BHV-1.2) while the third group (23 Mabs, 22%) only with the respiratory strains. Only 5 out of the antibodies neutralized respiratory and genital strains, and none of them neutralized the encephalitogenic strains (1.3). Three Mabs selected from each of the 3 groups, and the above five neutralizing strains were studied by Western blot. Antibodies of groups 1 and 3, and two neutralizing antibodies bound to a 90k protein (gpIII), whereas members of group 2 and 3 neutralizing antibodies reacted with a 74k and a 130k protein (both gpl). The results indicate that reactivity with monoclonal antibodies is as suitable for the classification of BHV-1 strains as is restriction endonuclease (RE) analysis but it cannot distinguish between subgroups within the groups.     

Bovine respiratory syncytial virus-specific monoclonal antibodies

Five hybridomas were produced which secreted monoclonal antibodies to bovine respiratory syncytial virus (BRSV). Two antibodies (8G12, 15C7) neutralized the virus and inhibited syncytia formation in vitro. These monoclonal antibodies also stained, by indirect fluorescent assay, an external envelope protein of living virus-infected cells, and recognized the 48k subunit of the viral fusion protein by Western blot analysis of bovine respiratory syncytial virus-infected cell lysates. Three other monoclonals (6A12, 14D3, 14E3) stained, by indirect fluorescent assay, acetone-fixed virus-infected cells but not living cells. Three hybridomas (6A12, 8G12, 15C7) secreted monoclonal antibody of isotype IgG1, k; two hybridomas secreted monoclonal antibody of isotype IgG2a, k. This apparently is the first report of monoclonal antibodies specific for BRSV glycoproteins.     

Production and characterization of monoclonal antibodies to bovid herpesvirus-4

Thirty-five hybridoma cell lines secreting monoclonal antibodies (Mabs) against bovid herpesvirus-4 (BHV-4) strain V. Test were produced. These hybrid cells resulted from the fusion of SP2/0 myeloma cells with splenocytes of BALB/c mice previously immunized with purified BHV-4. A modified indirect fluorescent antibody test (IFAT) was applied as a screening procedure and was compared with an indirect enzyme-linked immunosorbent assay. The selected Mabs were tested by the same IFAT against a panel of BHV-4 field isolates and against bovine herpesvirus-1, bovine herpesvirus-2 and alcelaphine herpesvirus-1 (AHV-1). Comparison of BHV-4 field isolates with Mabs confirmed their close antigenic relationships, but slight antigenic differences were observed between different isolates. One of the Mabs also reacted against AHV-1, indicating an antigenic relationship between BHV-4 and AHV-1. None of the Mabs reacting with BHV-4 possessed neutralizing activity against the strain used for immunization.     

Antigenic and molecular characterization of a herpesvirus isolated from a North American elk

OBJECTIVE: To determine whether a herpesvirus isolated from the semen of a North American elk was related to bovine herpesvirus 1 (BHV-1). SAMPLE POPULATION: Semen from 1 healthy bull elk and 2 subtypes of BHV-1 (BHV-1.1 and BHV-1.2). PROCEDURES: A virus with cytopathic and electron microscopic characteristics consistent with an alpha-herpesvirus was isolated from elk semen, using fetal bovine kidney cells. Cross-neutralization assays were performed with antisera against BHV-1 and the elk herpesvirus (EIkHV). Restriction endonuclease digests of EIkHV DNA were compared with digests of BHV-1.1 and BHV-1.2 DNA. A portion of the ElkHV DNA polymerase gene was amplified with consensus primers by use of the polymerase chain reaction and sequenced. Sequence was compared with known sequences of other herpesviruses. An immunoperoxidase monolayer assay was used to determine reactivities of 22 BHV-1-specific monoclonal antibodies (mAb) against ElkHV. In vitro neutralizing activities of the reactive mAb were determined by use of a microneutralization assay. RESULTS: Results of cross-neutralization assays indicated that ElkHV was serologically related to BHV-1. Endonuclease digestion of ElkHV DNA generated fragments that were distinct from those of BHV-1. Nucleotide sequencing confirmed that ElkHV is an alphaherpesvirus closely related to but distinct from BHV-1. Six of 22 BHV-1-specific mAb reacted against ElkHV; 2 of these 6 also neutralized in vitro infectivity of ElkHV. CONCLUSIONS AND CLINICAL RELEVANCE: ElkHV is antigenically and genetically distinguishable from BHV-1. However, the viruses are serologically related and share at least 6 antigenic determinants, one of which is a major neutralizing determinant.     

The application of hybridoma technology to the study of bovine immunoglobulins

Studies are described in which hybridoma technology is used to produce a variety of reagents for the characterization and manipulation of the bovine humoral immune system. Selected members of a set of murine monoclonal antibodies (MAb) specific for each of four major isotypes of bovine Ig constant regions, one specific for anti-bovine Ig constant regions as well as one specific for anti-bovine light chains are discussed. Interspecific fusion of bovine lymphocytes with the established mouse cell line, SP2/0 was used to produce a collection of stable hybridomas among which were found secretors of bovine IgG1, IgG2, IgM, IgA and bovine light chain. Interspecific fusion of SP2/0 with lymphocytes from a multiparous Holstein four days post immunization with Streptococcus agalactiae yielded MAb with specificity for the immunizing antigen. One of these hybridomas, LHRB 19.17, which displayed a particularly stable secretory phenotype, was used as an immunogen for the production of a library of murine monoclonal anti-idiotype antibodies. Competitive antigen binding analysis showed that 15 of the 24 anti-LHRB 19.17 idiotype antibodies isolated blocked the binding of the idiotype to its nominal antigen and so were candidates for evaluation as antigen mimics. Some of the ways in which monoclonal anti-idiotypes in particular, and monoclonal in general, might be of use in problems of animal disease are discussed.     

Research on antibodies against BHV-1, BHV-2, BHV-4, BVD-MD virus, bovine adenovirus A and B, rotavirus and coronavirus in cattle in western Zaire: complementary results

Two-hundred bovine sera from western Zaire were screened for antibodies to 8 viruses: BHV-1, BHV-2, BHV-4, BVD-MD virus, bovine adenovirus A and B, bovine rotavirus and bovine coronavirus. Positive sera were found to all these viruses. For animals whose origin was undoubted, the main features were the high prevalence of infections by rotavirus and BHV-4 and the low prevalence of infections by coronavirus and BVD-MD virus.     

Humoral immune response and assessment of vaccine virus shedding in calves receiving modified live virus vaccines containing bovine herpesvirus-1 and bovine viral diarrhoea virus 1a

Susceptible calves were administered modified live virus (MLV) vaccines containing bovine herpesvirus-1 (BHV1) and bovine viral diarrhoea type 1 (BVDV1a) strains intramuscularly, with one vaccine containing both MLV and inactivated BHV-1 and inactivated BVDV1a. There was no evidence of transmission of vaccine (BHV-1 and BVDV1a) strains to susceptible non-vaccinated controls commingled with vaccinates. No vaccinates had detectable BHV-1 in peripheral blood leucocytes (PBL) after vaccination. Each of three vaccines containing an MLV BVDV1a strain caused a transient BVDV vaccine induced viremia in PBL after vaccination, which was cleared as the calves developed serum BVDV1 antibodies. The vaccine containing both MLV and inactivated BHV-1 induced serum BHV-1 antibodies more rapid than MLV BHV-1 vaccine. Two doses of MLV BHV-1 (days 0 and 28) in some cases induced serum BHV-1 antibodies to higher levels and greater duration than one dose.     

Characterization of monoclonal antibodies directed against the bovine herpesvirus-1 glycoprotein E and use for the differentiation between vaccinated and infected animals

A panel of seven monoclonal antibodies (MAbs) directed against the bovine herpesvirus-1 (BHV-1) glycoprotein E (gE) was obtained. For that purpose, mice were either tolerized to BHV-1 gE-negative virus and then immunized with wild type BHV-1 or immunized with plasmid DNA expressing the gE and gI glycoproteins. The MAbs were characterized by their reactivity with the gE protein or the gE/gI complex and by competition experiments. Results showed that the MAbs were directed against three antigenic domains, two located on the gE glycoprotein and one on the gE/gI complex. Blocking experiments were performed with sera from experimentally vaccinated and infected cattle. A competition was observed between gE-positive bovine sera and six of the seven MAbs. The bovine sera thus recognized two of the three antigenic sites. Field sera were then tested in blocking enzyme-linked immunosorbent assay using one horseradish peroxidase-conjugated MAb. A specificity of 98.2% and a sensitivity of 98.2% compared to the commercially available test were observed.     

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.     

Ultrastructural and immunologic characteristics of mouse x cattle xenogeneic hybridomas originating from bovine leukemia virus-infected cattle

Nine percent of xenogeneic hybridomas originating from a bovine leukemia virus (BLV)-infected cow secreted monoclonal IgM antibodies with multispecific reactivity. Similar reactivity was evident in some antibodies with an unusually long (> 50 amino acids) third complementarity-determining region of the heavy chain. Electron microscopy of hybridomas demonstrated the presence of c-type virus particles consistent with polymerase chain reaction detection of BLV env gene. Some hybridomas contained dilated rough endoplasmic reticulum and cisternae filled with moderately electron-dense granular substance compatible with plasma cells at presecretory stage. The number of chromosomes in xenogeneic hybridomas corresponded to the sum total of mouse and bovine chromosomes. None of the hybridomas showed polyploidy. The immunochemical and genetic analysis of stable bovine immunoglobulin-secreting xenogeneic hybridomas confirms that BLV infection causes polyclonal B cell activation regardless of antigen specificity. Presence of c-type particles in hybridomas suggests that T cell-derived cytokines are not required for sustained BLV expression.     

Occurrence of caprine herpesvirus (BHV-6) infection in goat population of the GDR

To clarify the occurrence of the caprine herpesvirus (BHV-6) infection in the goat population in the GDR, 175 sera, collected from the agricultural research station of Karl-Marx-Universit?t at Probstheida were tested for the presence of neutralizing antibodies against BHV-6. BHV-6 antibodies were present in 12% of the sera examined. The titer was low. Cross-neutralization of BHV-6 could not be determined with any other bovine herpesvirus. The goat sera tested were free of neutralizing antibodies against BHV-1 and BHV-3. The indirect immunofluorescence antibody test revealed cross-reactivity only between BHV-6 and BHV-1.     

Induction of antigen-specific T-cell subset activation to bovine respiratory disease viruses by a modified-live virus vaccine

OBJECTIVE: To determine the efficacy of a modified-live virus vaccine containing bovine herpes virus 1 (BHV-1), bovine respiratory syncytial virus (BRSV), parainfluenza virus 3, and bovine viral diarrhea virus (BVDV) types 1 and 2 to induce neutralizing antibodies and cell-mediated immunity in na?ve cattle and protect against BHV-1 challenge. ANIMALS: 17 calves. PROCEDURES: 8 calves were mock-vaccinated with saline (0.9% NaCl) solution (control calves), and 9 calves were vaccinated at 15 to 16 weeks of age. All calves were challenged with BHV-1 25 weeks after vaccination. Neutralizing antibodies and T-cell responsiveness were tested on the day of vaccination and periodically after vaccination and BHV-1 challenge. Specific T-cell responses were evaluated by comparing CD25 upregulation and intracellular interferon-gamma expression by 5-color flow cytometry. Titration of BHV-1 in nasal secretions was performed daily after challenge. Results-Vaccinated calves seroconverted by week 4 after vaccination. Antigen-specific cell-mediated immune responses, by CD25 expression index, were significantly higher in vaccinated calves than control calves. Compared with control calves, antigen-specific interferon-gamma expression was significantly higher in calves during weeks 4 to 8 after vaccination, declining by week 24. After BHV-1 challenge, both neutralizing antibodies and T-cell responses of vaccinated calves had anamnestic responses to BHV-1. Vaccinated calves shed virus in nasal secretions at significantly lower titers for a shorter period and had significantly lower rectal temperatures than control calves. CONCLUSION AND CLINICAL RELEVANCE: A single dose of vaccine effectively induced humoral and cellular immune responses against BHV-1, BRSV, and BVDV types 1 and 2 and protected calves after BHV-1 challenge for 6 months after vaccination.     

Bulk milk testing for antibody seroprevalences to BVDV and BHV-1 in a rural region of Peru

Bulk milk from 60 herds of dairy cattle in a rural region in the central highlands of Peru was tested for antibodies to bovine viral-diarrhoea virus (BVDV) and bovine herpesvirus type 1 (BHV-1). None of the herds had been vaccinated against BVDV or BHV-1. Commercially available indirect ELISA-kits were used for antibody detection. True prevalences of BVDV and BHV-1 antibody-positive herds were 96 and 51%, respectively. A relatively low proportion of strongly positive herds suggests, however, a low prevalence of active BVDV infection. BVDV optical densities (ODs) in bulk milk increased with herd size--indicating a higher within-herd prevalence in the larger herds (probably, in part a consequence of a higher rate of animal movement into these herds). For BHV-1, this pattern was not found; a relatively high proportion of the herds was free from BHV-1 infection in each size category. This could indicate a low rate of reactivation of latent BHV-1 infection.     

Encephalitis in aborted bovine fetuses associated with Bovine herpesvirus 1 infection.

Brain tissue from 12 aborted bovine fetuses submitted to the Wisconsin Veterinary Diagnostic Laboratory revealed histologic lesions that consisted of glial nodules and variable degrees of mononuclear inflammation, microhemorrhage, neuronal necrosis, and cerebral cortical cavitation. A diagnosis of Bovine herpesvirus 1 (BHV-1) abortion had been made in all of these cases through multiple testing modalities. Brain tissue from 8 of the 12 fetuses was immunohistochemically stained with a monoclonal antibody specific to BHV-1, and, in 5 fetuses, there was positive intralesional staining of neurons, glial cells, and endothelial cells. This preliminary data suggested that herpesviral infection of brain tissue led to the described neurologic lesions. BHV-1 was then amplified from brain tissue in all 12 of the fetuses and was confirmed by partial sequencing of the thymidine kinase and glycoprotein C genes. To the authors' knowledge, neurologic lesions have not previously been described in BHV-1-infected fetuses, nor has BHV-1 previously been identified in bovine fetal brain tissue. The neurologic histopathology attributed to BHV-1 infection in these cases overlaps with the neurologic lesions produced by Neospora caninum, a common etiologic agent of bovine abortion. Therefore, when bovine fetal neurologic lesions are found, both etiologies should be considered and then distinguished by using additional diagnostic tools.     

Preparation of monoclonal antibodies against cell surface antigens in cattle

The BALB/c mice were immunized three times with bovine lymphocytes and thrombocytes in vivo. Besides, dissociated spleens of the mice were immunized with bovine lymphocytes in vitro. Hybridomas producing monoclonal antibodies were formed as a result of the fusion of the spleen cells with the murine myeloma cells. On the whole, four fusions were performed after immunization in vivo and three fusions after immunization in vitro, and 70 stable hybridoma clones were obtained. Five monoclonal antibodies exhibited an identical specific reaction only with bovine thrombocytes, two antibodies reacted only with a certain limited population of bovine spleen cells. The remaining monoclonal antibodies exhibited no tissue specificity and were bound to the lymphocytes of peripheral blood, lymph nodes, thymus, and spleen, to thrombocytes, liver cells, and spermatozoa, but never to erythrocytes. As for the amount of the obtained hybridomas and specific antibodies, no significant difference was observed in the effectiveness of in vivo and in vitro immunization.     

抗A型产气荚膜梭菌单克隆抗体的研制和鉴定

本试验用NSo细胞和产气荚膜梭菌肠毒素免疫的BALB／C小鼠脾细胞融合，获得了2株能分泌抗A型产气荚膜梭菌肠毒索单克隆抗体的杂交瘤细胞。经鉴定．这2株杂交瘤细胞的平均染色体数目为96．8条，单抗亚类为IgG1，杂交瘤细胞培养上清及腹水单克隆抗体的效价均较高，而且与其他几种常见的毒素SE、LT、S—LT、产气荚膜梭菌α毒素等不存在交叉反应。1D5能完全中和NCTC8239、S020718所产生的CPE的生物学活性，而284的中和活性则较差。     

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.