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.     

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.     

Generation and characterization of murine monoclonal antibodies specific for bovine immunoglobulin E

Monoclonal antibodies were produced against serum-derived bovine immunoglobulin E (IgE). Culture supernatants of hybridomas were initially screened by enzyme-linked immunosorbent assay (ELISA). Supernatant-derived antibodies were concentrated and further characterized using ELISA, reverse cutaneous anaphylaxis, immunohistochemical staining, and immunoblotting of IgE-containing samples separated by SDS-polyacrylamide gel electrophoresis (PAGE). Eight monoclonal antibodies showed specificity for bovine epsilon immunoglobulin heavy chain. Two antibodies (E2 and E32) reacted in immunoblots of SDS-PAGE of serum IgE under reducing conditions. Additionally, E2, E5, and E32 detected epsilon chain in serum separated by SDS-PAGE and then renatured. Antigen-specific IgE was detected in Western blots by E5 and E32. Immunoperoxidase staining of IgE-containing cells in mesenteric lymph node sections was detected with E5, E21 and E32. All eight antibodies produced positive reverse cutaneous anaphylaxis reactions in calf skin. All functioned well in ELISA as a plate-sensitizing reagent for quantitation of total IgE; E5 and E32 worked well as a primary antibody in antigen-specific IgE assays. These antibodies will be useful in research applications and in diagnostic assays.     

Production and characterization of monoclonal antibodies to cervine herpesvirus-1.

Fourteen hybridoma cell lines secreting monoclonal antibodies (Mab) to cervine herpesvirus-1 (CerHV-1) produced following the fusion of NSO myeloma cells with splenocytes of BALB/c mice previously immunized with gradient purified CerHV-1 were selected using an indirect enzyme-linked immunosorbent assay (ELISA) employing CerHV-1 antigen and tested by the ELISA against four other ruminant alphaherpesviruses from cattle (bovine herpesvirus type 1.1 and 1.2) goat (caprine herpesvirus-2) and reindeer (rangiferine herpesvirus-1). Comparison of all five ruminant alphaherpesviruses with these Mabs confirmed their close antigenic relationships, with two Mabs reacting against all viruses. Ten Mabs which were able to differentiate between the viruses reacted with a 64 kDa polypeptide in a western blot. Four Mabs including two specific only for CerHV-1 with neutralizing activity against the virus used for immunization were directed against a 74 kDa viral protein.     

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.     

Characterization of monoclonal antibodies to bovine IgG1

Monoclonal antibodies were developed to bovine IgG1. In addition, production of monoclonal antibodies to bovine light chain is also reported. Monoclonal antibody specificities were initially determined by solid-phase enzyme immunoassay. The monoclonal antibovine IgG1 was shown by a specificity-independent isotyping solid-phase enzyme immunoassay to be mouse IgG1 with kappa light chains. Ascites derived monoclonal antibovine IgG1 antibodies were linked to cyanogen bromide-activated Sepharose and used for affinity isolation of bovine IgG1. The bovine IgG1 eluted from the affinity column was characterized using immuno-electrophoresis, acrylamide gel electrophoresis, isoelectric focusing and solid-phase enzyme immunoassay. Affinity chromatography using monoclonal antibodies provided both a verification of monoclonal antibody specificity and a rapid technique for the isolation of bovine IgG1. This technique may also be employed to remove IgG1 contaminants during purification of bovine IgA.     

牛γ干扰素单克隆抗体的制备与抗原捕获ELISA检测方法的建立

为了建立一种简便、快速、可靠和经济的牛γ干扰素(BOIFN-γ)的免疫学检测方法,本研究通过杂交瘤技术建立了2株抗rBoIFN-γ单克隆抗体(MAb)2G6和5F9.Western blot、抗病毒活性阻断实验表明2株MAb与rBoIFN-γ有高亲和活性.相加EUSA表明2G6和5F9识别rBoIFN-γ不同的抗原表位.利用2G6作为捕获抗体,5F9-HEP作为检测抗体建立的抗原捕获ELlSA方法可特异性的检测不同系统表达的rBoIFN-γ,而且与rBoIFN-a、rBoIFN-β不发生交叉反应.该方法的建立为抗原捕获ELISA定量检测BoIFN-γ试剂盒的研制奠定了基础.     

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.     

The immunogenicity and efficacy of replication-defective and replication-competent bovine adenovirus-3 expressing bovine herpesvirus-1 glycoprotein gD in cattle

Replication-competent and replication-defective bovine adenovirus type 3 recombinants expressing the bovine herpesvirus type 1 (BHV-1) glycoprotein D (gD) were tested for induction of gD specific immune responses in calves using intratracheal (1st and 2nd immunization) and sub-cutaneous (3rd immunization) route of immunization. The replication-defective recombinant BAV501 induced systemic immune responses against gD as low titers of anti gD-IgG were detected in the serum. However, the efficacy of the replication-competent BAV3.E3gD to induce gD-specific antibodies in the serum and the nasal secretions was superior to that of replication-defective BAV501 when both viruses were given at the same dosage. Partial protection from challenge was induced in calves immunized with replication-competent BAV3.E3gD. A dramatic increase in the titers of anti-gD IgG and IgA levels, both in serum and nasal secretions, following BHV-1 challenge (anamnestic response) suggested that the animals immunized with replication-defective BAV501 had been primed for gD-specific antibody responses.     

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.     

Characterization of monoclonal antibodies against canine P-selectin glycoprotein ligand-1 (PSGL-1)

Thirteen different monoclonal antibodies against canine P-selectin glycoprotein ligand-1 (cPSGL-1) were obtained by immunization of rats with cells of a canine lymphoma cell line (Ema). O-sialoglycoprotein endopeptidase treatment of Ema cells showed that all of these antibodies recognized O-glycosylated peptides of canine PSGL-1. Experiments using deletion or point mutants of cPSGL-1 indicated that these antibodies could be categorized into several groups based on their cPSGL-1 recognition characteristics. These anti-cPSGL-1 monoclonal antibodies will be useful for analysis of the canine P-selectin and PSGL-1 system.     

Immunohistology of workshop monoclonal antibodies to the bovine homologue of CD1.

Six monoclonal antibodies putatively to the BoCD1 antigen were compared by immunohistology on cryostat sections from a range of tissues. The different staining patterns observed allowed the mAbs to be placed in three groups (a) 20-27, (b) CC13, CC14, TH97A and (c) CC20, CC40. An ovine mAb VPM5 did not stain bovine tissues sufficiently strongly to enable a comparison with the other CD1 mAbs.     

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%.     

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.     

Generation of monoclonal antibodies specific for ORF68 of koi herpesvirus

Outbreaks of koi herpesvirus (KHV) infection in carp are still a serious problem worldwide. KHV is closely related to other two cyprinid herpesviruses, pox herpesvirus (CHV) and haematopoietic necrosis herpesvirus (CyHV-2) in goldfish. In this study, two major KHV antigenic proteins (ORF62 and ORF68) were identified by immunoscreening using a KHV-specific polyclonal antibody, and then monoclonal antibodies were generated for immunodiagnostic studies. After screening hybridoma cells, one mAb against ORF68 (mAb-7C6) was obtained but no mAbs against ORF62. mAb-7C6 specifically reacted with a lysate of KHV-infected koi fin cells (KF-1 cells) but not with lysates of CHV- or CyHV-2-infected KF-1 cells in an immuno-blotting analysis. Similar results were shown in the following tests: (1) a indirect fluorescent antibody test using infected KF-1 cells and (2) an immunohistochemical investigation by fast red stain (infected liver) or FITC detection (infected spleen). These results suggested that mAb-7C6 specifically reacts with KHV ORF68 protein.     

Production and characterisation of monoclonal antibodies specific for chicken interleukin-12

Using genetic immunisation of mice, we produced antibodies against chicken interleukin-12p40 (chIL-12p40), also known as IL-12β. After a final injection with a recombinant chIL-12p40 protein, several stable hybridoma cell lines were established which secreted monoclonal antibodies (mAbs) to this component of the heterodimeric IL-12 cytokine. Specific binding of three of the mAbs to COS-7 cell-derived recombinant chIL-12p40 and the chIL-12p70 heterodimer was demonstrated in an indirect ELISA, and in dot blots. Two of the mAbs were used to develop a capture ELISA, suitable for detecting both recombinant protein (chIL-12p40 and the heterodimeric p70 protein) and native chIL-12. The mAbs were further characterised to show utility in immunocytochemistry.     

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.